DK175959B1 - Human HIV-2 retrovirus that can cause AIDS, and method and means for detecting this retrovirus in vitro - Google Patents
Human HIV-2 retrovirus that can cause AIDS, and method and means for detecting this retrovirus in vitro Download PDFInfo
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- DK175959B1 DK175959B1 DK198704934A DK493487A DK175959B1 DK 175959 B1 DK175959 B1 DK 175959B1 DK 198704934 A DK198704934 A DK 198704934A DK 493487 A DK493487 A DK 493487A DK 175959 B1 DK175959 B1 DK 175959B1
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- hiv
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Description
DK 175959 B1DK 175959 B1
Opfindelsen angår et human retrovirus, der har evne til at inficere humane T4-lymphocyter og er i stand til at forårsage lymphadenopatier, der herefter kan gå over til det erhvervede immunmanglende syndrom (AIDS) hos mennesket. Opfindelsen angår også antigener, der kan genkendes af antistoffer 5 frembragt hos et menneske af denne vims. Herudover angår opfindelsen ari-- tistoffer induceret af antigener opnået fra disse vira.The invention relates to a human retrovirus which has the ability to infect human T4 lymphocytes and is capable of causing lymphadenopathies which can then pass on to the acquired immune deficiency syndrome (AIDS) in man. The invention also relates to antigens which can be recognized by antibodies 5 produced in a human by this virus. In addition, the invention relates to antibodies induced by antigens obtained from these viruses.
Opfindelsen angår ligeledes klonede DNA-sekvenser, der er i besiddelse af sekvensanalogi eller komplementaritet med mindst en del af det genome . 10 RNA fra. ovennævnte vims. Desuden angår den fremgangsmåder til fremstil-, ling af disse klonede DNA-sekvenser.The invention also relates to cloned DNA sequences possessing sequence analogy or complementarity with at least a portion of the genome. 10 RNA from. the above vims. In addition, it relates to methods for producing these cloned DNA sequences.
Opfindelsen tilvejebringer desuden et præparat indeholdende et antigen med aminosyresekvenser kodet af de klonede DNA-sekvenser eller et antigen, 15 der har immunologiske egenskaber for HIV-2 p12, p16, p26, p36, p42 og gp140.The invention further provides a composition containing an antigen with amino acid sequences encoded by the cloned DNA sequences or an antigen having immunological properties of HIV-2 p12, p16, p26, p36, p42 and gp140.
Opfindelsen angår ligelédes anvendelsen af disse antigener i en fremgangsmåde til in vitro diagnose hos.mennesker af muligheden for visse former af 20 AIDS og med hensyn til visse af disse antigener fremstillingen af immunogene præparater og vacciner over for dette retrovirus. Derudover angår opfindelsen anvendelsen af ovennævnte antistoffer til nævnte formål samt for visse af dem, deres anvendelse ved fremstillingen af aktive lægemiddelprincipper over for disse former af human AIDS.The invention also relates to the use of these antigens in a method of in vitro diagnosis in humans of the possibility of certain forms of AIDS and with regard to certain of these antigens the preparation of immunogenic preparations and vaccines against this retrovirus. In addition, the invention relates to the use of the aforementioned antibodies for said purpose and to some of them, their use in the manufacture of active drug principles against these forms of human AIDS.
2525
Endelig angår opfindelsen anvendelsen af .de klonede DNA-sekvenser og af polypeptider opnået fra disse sekvenser som prober i diagnostiske kits.Finally, the invention relates to the use of the cloned DNA sequences and of polypeptides obtained from these sequences as probes in diagnostic kits.
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Isoleringen og karakteriseringen af et første retrovirus, betegnet LAV, der 30 anses som ansvarlig for udviklingen af AIDS, er omtalt i en artikel af F. Barre-Sinoussi et al. allerede i 1983 (Science, vol. 220, nr. 45-99, 20, p. 868-871). . Anvendelsen visse ekstrakter af dette vims og mere specielt af visse af proteinerne heraf til diagnose af tilstedeværelsen af antistoffer over for viruset er 2 DK 175959 B1 mere specielt beskrevet i europæisk patentansøgning nr. 138 667. Siden da er lignende stammer og visse varianter af LAV blev isoleret. Eksempler, der kan nævnes, er de der betegnes HTLV-III og ARV.The isolation and characterization of a first retrovirus, designated LAV, which is considered responsible for the development of AIDS, is discussed in an article by F. Barre-Sinoussi et al. as early as 1983 (Science, vol. 220, no. 45-99, 20, p. 868-871). . The use of certain extracts of this yeast and more particularly of some of its proteins for the diagnosis of the presence of antibodies to the virus is more specifically described in European Patent Application No. 138 667. Since then, similar strains and certain variants of LAV were isolated. Examples which may be mentioned are those designated HTLV-III and ARV.
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5 For at anvende de nye regler for nomenklatur publiceret i Nature i Maj 1986 kaldes retrovira, der er i stand til hos mennesket at inducere, ovennævnte lymphadenopatier og AIDS, den generelle betegnelse "HIV", eh forkortelse for udtrykket "humant immunodeficient virus". Undergruppen af retrovira dannet af LAV og dets varianter betegnedes oprindeligt "LAV type P eller.5 To apply the new rules for nomenclature published in Nature in May 1986, retroviruses capable of inducing in humans the aforementioned lymphadenopathies and AIDS are commonly referred to as the term "HIV" or the abbreviation for the term "human immunodeficiency virus". The subset of retroviruses formed by LAV and its variants was initially designated "LAV type P or.
10 "LAV-I". Sidstnævnte undergruppe vil i det efterfølgende blive betegnet HIV-1, idet det skal forstås, at udtrykket LAV stadig vil blive bibeholdt for atbe-, tegne den stamme, blandt stammer af retrovirus (i specielt LAAV, IDAV-4 og IDAV-2) hørende til HIV-1 virusgruppen, der er beskrevet i ovennævnte europæiske patentansøgning nr. 138 667, der anvendtes til sammenligningsfor-15 søg beskrevet senere, nemlig LAVbru. der deponeredes i Collection Nationale des Cultures de Micro-organismes (CNCM). (National Collection of Microorganism Cultures) i Pasteurinstituttet i Paris, Frankrig den 15. juli 1983 under nr. I-232.10 "LAV-I". The latter subgroup will hereafter be referred to as HIV-1, it being understood that the term LAV will still be retained to designate, draw the strain of retrovirus strains (in particular LAAV, IDAV-4 and IDAV-2) belonging to to the HIV-1 virus group described in the above-mentioned European Patent Application No. 138,667 used for comparative experiments described later, namely LAV use. deposited in the Collection Nationale des Cultures de Microorganisms (CNCM). (National Collection of Microorganism Cultures) of the Pasteur Institute in Paris, France on July 15, 1983 under No. I-232.
20 Det omhandlede hidtil ukendte retrovirus, og virusstammer, der er i slægt hermed, og som ligesom dette er i stand til at multiplicere i humane lympho-cyter, er tidligere betegnet som "LAV type II" eller "LAV-IP og betegnes i det efterfølgende "HIV-2", idet det skal forstås, at betegnelserne for visse HIV-2 isolater beskrevet neden for vil blive efterfulgt af tre bogstaver, der henfører 25 til de patienter, hvorfra de er isoleret.The aforementioned novel retrovirus, and related strains of viruses which, like this, are capable of multiplying in human lymphocytes, have previously been referred to as "LAV type II" or "LAV IP and are designated therein. hereinafter "HIV-2", it should be understood that the names of certain HIV-2 isolates described below will be followed by three letters referring 25 to the patients from which they are isolated.
"HIV-2" gruppen kan defineres som en gruppe vira, der udviser in vitro tropisme over for humane T4 lymphocyter, og som har en cytopathogen virk- ‘ ning over for disse lymphocyter, når de formerer sig heri, og som derefter en-30 ten forårsager generelle og persistente polyadenopatier eller en af formerne af AIDS. HIV-2 retrovira har vist sig at være forskellig fra HIV-1 type vira under betingelser beskrevet nærmere nedenfor. Ligesom disse sidstnævnte 3 DK 175959 B1 vira er de forskellige fra andre humane retrovira, der allerede er kendt (HTLV-I og HTLV-II).The "HIV-2" group can be defined as a group of viruses that exhibit in vitro tropism against human T4 lymphocytes and which have a cytopathogenic action against these lymphocytes as they proliferate therein, and then It causes general and persistent polyadenopathies or one of the forms of AIDS. HIV-2 retroviruses have been found to be different from HIV-1 type viruses under conditions described further below. Like these latter viruses, they are different from other human retroviruses already known (HTLV-I and HTLV-II).
Skønt der er en forholdsvis vid genetisk variabilitet i viruset, har forskellige | * · 5 HIV-1 stammer isoleret indtil nu fra amerikanske, europæiske, afrikanske og patienter fra haiti antigene pladser fælles bevaret på deres vigtigste proteiner dvs. kemeprotein p25, omhyldningsglycoprotein gp110 og transmembranproteinet gp41-43. Disse forhold gør det muligt f.eks. for prototype LAV stammen at anvendes som en stamme for antigener til at afsløre antistoffer over .Although there is a relatively wide genetic variability in the virus, different | * · 5 HIV-1 strains isolated so far from American, European, African and Haitian antigenic sites are jointly conserved on their major proteins ie. nuclear protein p25, the envelope glycoprotein gp110 and the transmembrane protein gp41-43. These conditions make it possible e.g. for the prototype LAV strain to be used as a strain for antigens to reveal antibodies above.
10 for alle HIV-1 gruppevirus, hos alle mennesker der bærer dem, uafhængig af deres oprindelse. Denne stamme er indtil for nylig anvendt til at afsløre anti-, HIV-1 antistoffer hos bloddonorer og hos patienter speciélt ved hjælp af im-munofluorescens og specielt ved hjælp af metoden betegnet som ELISA, "Western Blot" (eller immuno-aftryk) og "RIPA" en forkortelse for radioimmu-15 noprecipitationsforsøg.10 for all HIV-1 group viruses, in all people carrying them, regardless of their origin. This strain has until recently been used to detect anti-, HIV-1 antibodies in blood donors and in patients especially by immunofluorescence and especially by the method referred to as ELISA, "Western Blot" (or immuno-imprinting) and "RIPA" is an abbreviation for radioimmunoprecipitation experiments.
I en serologisk undersøgelse udført med et HIV-1 lysat på patienter, der stammer fra Vestafrika, er det imidlertid blevet observeret, at visse af dem gav seronegative eller meget svage positive reaktioner, medens de viste kli-20 niske og immunologiske tegn på AIDS.However, in a serological study conducted with an HIV-1 lysate on patients originating from West Africa, it has been observed that some of them gave seronegative or very weak positive reactions while showing clinical and immunological signs of AIDS.
Dyrkede lymphocyter fra en af disse patienter var kilden ifor et første HIV-2 isoleret retrovirus, hvis opbygning i elektronmikrocop og hvis proteinprofil på SDS gelelektrophorese viste en lighed med egenskaberne hos HIV-1. Dette 25 nye retrovirus HIV-2 besad imidlertid kun et ringe slægtskab med HIV-1 med hensyn til både antigenhomologi af dets proteiner og med hensyn til homolo-gien af dets genetiske materiale.Cultured lymphocytes from one of these patients were the source of a first HIV-2 isolated retrovirus, whose structure in the electron microscope and whose protein profile on SDS gel electrophoresis showed a similarity to the characteristics of HIV-1. However, this new HIV-2 retrovirus had only a minor relationship with HIV-1 in terms of both antigen homology of its proteins and the homology of its genetic material.
Dette nye retrovirus eller retrovira med ækvivalente antigene og immunologi-30 ske egenskaber kan derfor udgøre kilder for antigener til diagnose af infektion med dette virus og varianter heraf, der frembringer en AIDS form af den type, der er observeret i de oprindelige tilfælde hos afrikanske patienter eller hos folk, der har tilbragt en tid i Afrika.This new retrovirus or retrovirus with equivalent antigenic and immunological properties may therefore constitute sources of antigens for the diagnosis of infection with this virus and variants thereof, producing an AIDS form of the type observed in the original cases in African patients. or in people who have spent time in Africa.
4 DK 175959 B14 DK 175959 B1
Dette virus isoleredes f.eks. fra blod udtaget i nærværelse af heparin fra en 28 årig heteroseksuel patient, der aldrig havde modtaget blodtransfusion og som ikke var narkoman. Siden 1983 havde han haft næsten kronisk diarre ogThis virus was isolated, e.g. from blood taken in the presence of heparin from a 28-year-old heterosexual patient who had never received blood transfusion and who was not a drug addict. Since 1983 he had had almost chronic diarrhea and
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5 væsentligt vægttab (17 kg) med af og til opdukkende feber. For nylig havde han haft Candida og serratia infektioner, herunder en oseophageal candidiasis typisk for AIDS.5 significant weight loss (17 kg) with occasional fever. Recently, he had had Candida and serratia infections, including an oseophageal candidiasis typical of AIDS.
Denne patient udviste også anæmi, cutan anergi, lymphopeni og et T4 10 lymphocyt-/T8 lymphocytforhold på 0,15 med et T4 lymphocytniveau på min-, dre end 100 pr. mm3 serum. Hans lymphocyter svarede ved dyrkning ikke på stimulering med phytohæmagglutinin og concanavalin A. Denne patient blev også diagnostiseret som lidende af gentagen bakteriæmi på grund af S. ente-riditis, cryptosporidiose, infektioner på grund af Isopora belli og cerebrale ιοί 5 xoplasmoser.This patient also exhibited anemia, cutaneous anergy, lymphopenia, and a T4 lymphocyte / T8 lymphocyte ratio of 0.15 with a T4 lymphocyte level of less than 100 per day. mm3 serum. His lymphocytes, when cultured, did not respond to stimulation with phytohaemagglutinin and concanavalin A. This patient was also diagnosed as suffering from recurrent bacteremia due to S. enteriditis, cryptosporidiosis, infections due to Isopora belli and cerebral ιοί 5 xoplasmosis.
Denne kombination af kliniske tegn var bevis på "komplekse symptomer i forbindelse med AIDS” eller "ARC" (forkortelse for "AlDS-relateret kompleks") af den type, der er forårsaget af HIV-1 virus. Disse forskellige observartioner 20 var også i overensstemmelse med de kriterier, der anvendes af centeret for sygdomskontrol (CDC) i Atlanta, USA.This combination of clinical signs was evidence of "complex symptoms associated with AIDS" or "ARC" (abbreviation for "AlDS-related complex") of the type caused by the HIV-1 virus. in accordance with the criteria used by the Center for Disease Control (CDC) in Atlanta, USA.
Dyrkningen af lymphocyter fra disse patienter og isoleringen af retroviruset udførtes i overensstemmelse med metoder, der allerede er beskrevet for iso-25 lering af HIV-1 i artiklen af Barre-Sinoussi et al. og i europæisk patentansøgning nr. 84/401 834 - 0 138 667. De omtales alle kort nedenfor.The culture of lymphocytes from these patients and the isolation of the retrovirus were performed according to methods already described for isolation of HIV-1 in the article by Barre-Sinoussi et al. and in European Patent Application No. 84/401 834 - 0 138 667. They are all discussed briefly below.
Lymphocyter stimuleret i 3 dage med phytohæmagglutinin (PHA) dyrkedes i RPMI 1640 dyrkningssubstrat, hvortil 10% føtalt kalveserum og 10'5 M β-mer-captoethanol, interleukin-2 og anti-(human interferon a) serum var tilsat.Lymphocytes stimulated for 3 days with phytohemagglutinin (PHA) were grown in RPMI 1640 culture medium to which 10% fetal calf serum and 10'5 M β-mer-captoethanol, interleukin-2 and anti (human interferon a) serum were added.
3030
Produktionen af virus blev fulgt ved hjælp af dens omvendte transcriptaseak-tivitet. I dyrkningssupernatanten forekom den maksimale virale aktivitet mellem dag 14 og dag 22, hvorefter den aftog. Nedgangen og uddøen af celle 5 DK 175959 B1 kulturen skete herefter. Som med HIV-1 viste sektioner af lymphocyter inficeret med HIV-2 ved elektronmikroskopi virioner, der havde nået modenhed, og virale partikler, der stak ud fra overfladen af de inficerede celler. Cellelinierne i . anvendt til frembringelse af kulturerne af disse isolerede vira kan, afhængigt 5 af tilfældet, være cellelinier af HUT, CEM eller MOLT typen eller én hvilken som helst immortaliseret lymphocytlinie indeholdende T4 receptorer.The production of virus was monitored by its reverse transcriptase activity. In the culture supernatant, the maximum viral activity occurred between day 14 and day 22, after which it decreased. The decline and extinction of cell 5 subsequently occurred. As with HIV-1, sections of lymphocytes infected with HIV-2 by electron microscopy showed virions that had reached maturity and viral particles protruding from the surface of the infected cells. The cell lines i. used to produce the cultures of these isolated viruses may, depending on the case, be cell lines of the HUT, CEM or MOLT type or any immortalized lymphocyte line containing T4 receptors.
Viruset propageredes herefter i kulturer af lymphocyter fra bloddonorer, og . derefter i kontinuerte linier af leukæmisk oprindelse som f.eks. HUT 78. Det 10 karakteriseredes herefter ved hjælp af dets antigener og dets nucleinsyre som værende i det væsentlige forskellig fra HIV-1. Viruset rensedes som be-, skrevet i de allerede nævnte tidligere artikler. Et første isolat af dette virus deponeredes med CNCM den 19. december 1985 under nr. I-502. Det betegnedes herefter med navnet LAV-II MIR. Et andet isolat deponeredes med 15 CNCM den 21. februar 1986 under nummeret I-532, Dette andet isolat fik referencenavnet LAV-II ROD..Disse isolater vil af og til senere simpelt hen blive omtalt som MIR eller ROD.The virus was then propagated in cultures of blood donor lymphocytes, and. then in continuous lines of leukemic origin, e.g. HUT 78. It was then characterized by its antigens and its nucleic acid as substantially different from HIV-1. The virus was purified as described in the previously mentioned articles. A first isolate of this virus was deposited with CNCM on December 19, 1985 under No. I-502. It was then named LAV-II MIR. Another isolate was deposited with CNCM on February 21, 1986 under the number I-532, This second isolate was given the reference name LAV-II ROD. These isolates will sometimes be referred to simply as MIR or ROD.
Generelt angår opfindelsen en hvilken som helst variant af ovennævnte.vira 20 eller et hvilken som helst ækvivalent virus (f.eks. HIV-2-IRMO og HIV-2-EHO deponeret i CNCM den 19. december 1986 under nummrene henholdsvis I-642 og I-643), der indeholder strukturelle proteiner, der har sammen immunologiske egenskaber som HIV-2 vira deponeret i CNCM under numrene I-502 eller I-532. Med hensyn til definitionen af disse kriteriers ækvivalens skal .25 dette nærmere omtales nedenfor.In general, the invention relates to any variant of the above virus 20 or any equivalent virus (e.g., HIV-2-IRMO and HIV-2-EHO deposited in CNCM on December 19, 1986 under Nos. I-642, respectively). and I-643) containing structural proteins that together have immunological properties such as HIV-2 viruses deposited in CNCM under numbers I-502 or I-532. With regard to the definition of the equivalence of these criteria, .25 this should be discussed in more detail below.
Opfindelsen angår også en fremgangsmåde til at frembringe HIV-2 viruset eller varianter af dette i permanente cellelinier stammende fra T4 lymphocyter eller lymphocyter, der indeholder T4 phenotypen, idet denne metode be-30 står i at dyrke disse linier, der forud er inficeret med HIV-2 virus, og at man udvinder, specielt når niveauet for omvendt transcriptaseaktivitet har nået en særlig tærskelværdi, visse mængder af viruset frigjort i dyrkningssubstratet.The invention also relates to a method for producing the HIV-2 virus or variants thereof in permanent cell lines derived from T4 lymphocytes or lymphocytes containing the T4 phenotype, this method being intended to grow these lines previously infected with HIV -2 virus, and to recover, especially when the level of reverse transcriptase activity has reached a particular threshold, certain amounts of the virus released into the culture substrate.
6 DK 175959 B1 I et første aspekt angår opfindelsen et humant retrovirus, der har evne til at inficere humane T4-lymphocyter og er i stand at forårsage AIDS hos mennesker, og som er ejendommelig ved, at retroviruset udgøres af et vilkårligt af de i CNCM deponerede retrovirus med numrene I-502, I-532, I-642 og I-643 5 eller i ECACC deponerede retrovirus med numrene 87.011001 og 87.011002 (svarende til henholdsvis I-502 og I-532) eller en variant af en af disse retrovirus omfattende alle de.antigener, der genkendes af antistofferne, der fremkaldes mod de tilsvarende antigener af et vilkårligt af de nævnte, i CNCM eller i ECACC deponerede retrovirus.In a first aspect, the invention relates to a human retrovirus which is capable of infecting human T4 lymphocytes and is capable of causing AIDS in humans, and which is peculiar to the retrovirus being any of those in CNCM. deposited retroviruses with numbers I-502, I-532, I-642 and I-643 5 or in ECACC deposited retroviruses with numbers 87.011001 and 87.011002 (corresponding to I-502 and I-532, respectively) or a variant of one of these retroviruses comprising all the antigens recognized by the antibodies raised against the corresponding antigens by any of the aforementioned retroviruses deposited in CNCM or in ECACC.
10 I et andet aspekt angår opfindelsen et renset antigen af retrovirus HIV-2, der er ejendommeligt ved, at det har de immunologiske egenskaber for henholdsvis HIV-2 p12, p16, p26, p36, p42 og gp 140, der genkendes immunologisk af antistoffer fremkaldt mod ovennævnte retrovirus og har en moleky- 15 lærvægt i størrelsesordenen henholdsvis 12000 dalton, 16000 dalton, 26000 dalton, 36000 dalton, 42000-45000 dalton og 130000-140000 dalton.In another aspect, the invention relates to a purified antigen of retrovirus HIV-2, characterized in that it has the immunological properties of HIV-2 p12, p16, p26, p36, p42 and gp 140, which are immunologically recognized by antibodies. induced against the aforementioned retrovirus and having a molecular weight of the order of 12000 daltons, 16000 daltons, 26000 daltons, 36000 daltons, 42000-45000 daltons and 130000-140000 daltons, respectively.
I et tredie aspekt angår opfindelsen et antigen, der er ejendommeligt ved, at det har en af de følgende aminosyresekvenser eller en del af sekvenserne, 20 idet de fremkalder en specifik immunologisk reaktion med henholdsvis anti-p12-antistoffer, anti-plé-antistoffer,. anti-p26-antistoffer og anti-gp140-antistoffer mod det ovenfor beskrevne HIV-2 retrovirus, specielt når antigenet bringes i kontakt med serum fra en patient, der er inficeret med HIV2: 25 for anti-p12-antistoffer 30 7 DK 175959 B1 ftrgLysAUFheLy· # · * C y sTrpABiiCy sdlyty eC'IuG.lyHa.s S erAl aArgClnCys'Arg * · · · ·. 1200 .In a third aspect, the invention relates to an antigen which is characterized in that it has one of the following amino acid sequences or part of the sequences, in that it elicits a specific immunological reaction with anti-p12 antibodies, anti-plé antibodies, respectively. . anti-p26 antibodies and anti-gp140 antibodies to the above-described HIV-2 retrovirus, especially when the antigen is contacted with serum from a patient infected with HIV2: 25 for anti-p12 antibodies 30 7 DK 175959 B1 ftrgLysAUFheLy · # · * C y sTrpABiiCy sdlyty eC'IuG.lyHa.s S erAl aArgClnCys'Arg * · · · ·. 1200.
AlaProArgArgGlaGI jCyelrpLy sCy-sGlyLysProGlyHxit • · · · I leMetTbrAfinCysProAspArgClnAl aGlyPheLeuClyI.ee . . 1300AlaProArgArgGlaGI jCyelrpLy sCy-sGlyLysProGlyHxit • · · · In leMetTbrAfinCysProAspArgClnAl aGlyPheLeuClyI.ee. . 1300
GlyProTrpGlyLysLysProArg AsnPbeProValålaGlnVal • · · · · P x oG IbG lyLeuTbrP roThrAl aP r o? roV&lAs pPfoA^aVal . * · \ *.·.· ·*·«GlyProTrpGlyLysLysProArg AsnPbeProValålaGlnVal • · · · · P x and IbG lyLeuTbrP roThrAl aP r o? roV & lAs pPfoA ^ aVal. * · \ *.
AspLeuLeuGluLy sTyrHe tG InG lnG lyLysArgGlnArgGlu 1*00 . · .AspLeuLeuGluLy sTyrHe tG InG lnG lyLysArgGlnArgGlu 1 * 00. ·.
G InArgGluAr gPtoTyrLy sG lu ValThrCluAspLeuLeuHi* · . · ·G InArgGluAr gPtoTyrLy sG lu ValThrCluAspLeuLeuHi * ·. · ·
Le uG 1 uG 1 nCl yGluThrProTyr ArgC luPro Pr oTbrCliiAsp * · Γ300 · · · ·Le uG 1 uG 1 nCl yGluThrProTyr ArgC luPro Pr oTbrCliiAsp * · Γ300 · · · ·
LeuLe.unisLeuAsnSerLeuPbeClyLysXspGla m · · · 8 DK 175959 B1 for anti-p16-antistofferLeuLe.unisLeuAsnSerLeuPbeClyLysXspGla m · · · 8 DK 175959 B1 for anti-p16 antibodies
Me tCl yAlaArgAenSerPalLeuArgGlyLysLy sAlaAapGlo .1 . * · i . * ·. *Me tCl yAlaArgAenSerPalLeuArgGlyLysLy sAlaAapGlo .1. * · I. * ·. *
LeuGluArgIleAxjLéuAxgPxoGl.jGlyLyeLj*LysTyxArg' • · * · ·LeuGluArgIleAxjLéuAxgPxoGl.jGlyLyeLj * LysTyxArg '• · * · ·
LetiLy sHis Ile ValTrpAl aAlaÅsnXy sLeuAe pArgPbeGly 1 DO · · ·. . . » .LeuAlaGluSerLevLeoGluSeTLyeGXcGlyCysCIsLysIle- • m eLetiLy sHis Ile ValTrpAl aAlaÅsnXy sLeuAe pArgPbeGly 1 DO · · ·. . . ».LeuAlaGluSerLevLeoGluSeTLyeGXcGlyCysCIsLysIle- • m e
LeuThxValLeuAspPxoMet ValPxoTbxGlySerC luAsoLeo 200 . . .LeuThxValLeuAspPxoMet ValPxoTbxGlySerC luAsoLeo 200. . .
Ly s SexLeuPheAanThxValCy s Val I lelrp-Cys I leEisAla .· · ·· · ' pLy s SexLeuPheAanThxValCy s Val I lelrp-Cys I leEisAla. · · ·· · 'p
GluGluLy s ValLysAspTbxGluGlyAlaLy s Glnlle ValArg ' . . 300 ·GluGluLy's ValLysAspTbxGluGlyAlaLy's Glnlle ValArg '. . 300 ·
AxgRisLeuPalAlaGliiTbxGlyThxAloCluLysHetPxoSer • · ·· · ··AxgRisLeuPalAlaGliiTbxGlyThxAloCluLysHetPxoSer • · ·· · ··
TbrSe rAr gPr oTbxAlaProSe r S erG lu Ly sGly G lyAenTyr.TbrSe rAr gPr oTbxAlaProSe r S erG lu Ly sGly G lyAenTyr.
. 400 i ' 9 DK 175959 B1 for anti-p26-antistoffer ; ProValGlnBia ValClyClyAenTyrThrHialleProLeuSer. 400 in '9 for anti-p26 antibodies; ProValGlnBia ValClyClyAenTyrThrHialleProLeuSer
Vi " * . · ·'*..· ·* · ··.·We "*. · · '* .. · · * · ··. ·
Fr oArgTbTLenAsnAlaTrp ValLy eLeu Val GluGluLysLyi* * * . · »Fr oArgTbTLenAsnAlaTrp ValLy eLeu Val GluGluLysLyi * * *. · »
PheG lyAl aGlu ValValProGlyPheGlnAlaLeuSerG luGly 500' . · . CysThrProTyrAspIleAsaGlaMetLeuAsaCys ValGlyAspPheG lyAl aGlu ValValProGlyPheGlnAlaLeuSerG luGly 500 '. ·. CysThrProTyrAspIleAsaGlaMetLeuAsaCys ValGlyAsp
BisGlnAlaAlaUetGlnllelleAr^.CluIlelleAEDGluGlo % ♦·*··· % « 600 · .j · ·BisGlnAlaAlaUetGlnllelleAr ^ .CluIlelleAEDGluGlo% ♦ · * ···% «600 · .j · ·
AlaA-laCl'uTrpAspValGlnHisProTlePToClyProLeuPro·.· • · · · ' .·» . · AlaGlyGlDLeaArgGluProArgGlySerAspIleAlaGlyTbr: • - ·# . . 700 . ·AlaA-laCl'uTrpAspValGlnHisProTlePToClyProLeuPro ·. · • · · · '. · ». · AlaGlyGlDLeaArgGluProArgGlySerAspIleAlaGlyTbr: • - · #. . 700. ·
TbrSerThrVa lGlnGluGlnlleGlnTrpHetPbeArgProGln ♦♦TbrSerThrVa lGlnGluGlnlleGlnTrpHetPbeArgProGln ♦♦
Aa&FroValP.ro7a 1ClyA«alleTyzArgArgTrpl'leGlalle .Aa & FroValP.ro7a 1ClyA «alleTyzArgArgTrpl'leGlalle.
• . * a • · . SOO ·.·•. * a • ·. SOO ·.
ClyLeuGlnLysCyeValArgMetTyrAsnProThrÅsnlleLea . ” ** . “ r • · ·’ ’ · ·_ClyLeuGlnLysCyeValArgMetTyrAsnProThrÅsnlleLea. ”**. “R • · ·’ ’· · _
AspIleLyaGlaClyProlyaGliiProFkeClaSerTyrValAsp • " . 900AspIleLyaGlaClyProlyaGliiProFkeClaSerTyrValAsp • ". 900
ArgFkeTyrLysSerLeuArgAlaGluG InThxAspProAlaVal ·· · · ·ArgfkeTyrLysSerLeuArgAlaGluG InThxAspProAlaVal ·· · · ·
LysAsaTrpUe tTbrGlaTbrLeuLea Va lClaAsaA'laAsnPrOLysAsaTrpUe tTbrGlaTbrLeuLea Va lClaAsaA'laAsnPrO
• ·.*'· · ·• ·. * '· · ·
AspCyaLysLeoVaILeuLysGlyLeuGlyMetAsaProTbrLea 1000 ·- . * .AspCyaLysLeoVaILeuLysGlyLeuGlyMetAsaProTbrLea 1000 · -. *.
GluGluHetLeuTbrAlaCy sGlaG ly ValGlyGlyProGlyCla • * · »’ ··GluGluHETLeuTbrAlaCy sGlaG ly ValGlyGlyProGlyCla • * · »'··
XysAlaArgLeuHe t AlaGlaAlaLeuLy sGluValll eGlyPro noo * ...XysAlaArgLeuHe t AlaGlaAlaLeuLy sGluValll eGlyPro noo * ...
AlaProlleFroPbeAlaAlaAlaGlnC la 10 DK 175959 B1 for anti-gp140-antistoffer:AlaProleProbeAlaAlaAlaGlnC la 10 DK 175959 B1 for anti-gp140 antibodies:
ENVRHENVRH
Ile tHe tAsnGlnLeuLeuX 1 eÅl al 1 eLeuLeuAl aSexAlaCys • · · ·- .Ile tHe tAsnGlnLeuLeuX 1 eÅl al 1 eLeuLeuAl aSexAlaCys • · · · -.
LeuValTyrCysTbrGlnTyrValThrValPheTyrGlyValPrOLeuValTyrCysTbrGlnTyrValThrValPheTyrGlyValPrO
,·' · · · . ·, · '· · ·. ·
ThrTrpLysAsnAlaTbr 11eF roLe uPheCysAlaThrArgAsn 100 . . .ThrTrpLysAsnAlaTbr 11eF roll uPheCysAlaThrArgAsn 100. . .
ArgAspTbrTrpClyXhrl1eGl&CysLeuProAspAsnAspAsp.ArgAspTbrTrpClyXhrl1eGl & CysLeuProAspAsnAspAsp.
TyrGlnGlulleThrLeuAs nValThrGluAlaPheAspAlaTrp • 200 . .TyrGlnGlulleThrLeuAs nValThrGluAlaPheAspAlaTrp • 200. .
AsnAenThiVa lTbrGluGlnAlall eGluAsp ValTrpHisLeu • · · _ · P b eG 1 uThrSe ri le ly sPx o Cys Va lLysLeuTbrProLeuCy* .AsnAenThiVa lTbrGluGlnAlall eGluAsp ValTrpHisLeu • · · _ · P b eG 1 uThrSe ri le ly sPx o Cys Va lLysLeuTbrProLeuCy *.
300.300th
Val Al allet Lys CysSerS er TbrGluSerSer-TbrGlyAsnAsn • · · · ♦ ·Val All All Light CysSerS is TbrGluSerSer-TbrGlyAsnAsn • · · · ♦ ·
TbrTbr SerLy a.Ser Tbr S e r TbrThrThrTbr TbrPr oTbr As p . . > 400 C1nGl u GIn Glu11e S e r G1u λ s p Tb r P r o C y sA1aÅrgA1aA c p • · · · · ·«TbrTbr SerLy a.Ser Tbr S e r TbrThrThrTbr TbrPr oTbr As p. . > 400 C1nGl u GIn Glu11e S e r G1u λ s p Tb r P r o C y sA1aÅrgA1aA c p • · · · · · «
AsnCysSerGlyLeuGlyGluGluGluThxIleAsoCvsGInPae • · · «AsnCysSerGlyLeuGlyGluGluGluThxIleAsoCvsGInPae • · · «
AsnrletThrGlyLeuGluArgAspLysLysLysGlnTyxAsnGlu 500 . .. .AsnrletThrGlyLeuGluArgAspLysLysLysGlnTyxAsnGlu 500. ...
TbrTrpTyrSerLysAspValValCysGluThrAsnAstSerTnr • · · ·TbrTrpTyrserLysAspValValCysGluThrAsnAstSerTnr • · · ·
AsnGlnThrGlnCysTyrMetAsnEisCysAsnTbrSerVallle • 600 · t »AsnGlnThrGlnCysTyrMetAsnEisCysAsnTbrSerVallle • 600 · t »
TbrGluSerCyEAspLysEisTyrTrpAspAlalleAxgPheArg • · · ·TbrGluSerCyEAspLysEisTyrTrpAspAlalleAxgPheArg • · · ·
TyxCysAlaProPxoG lyTyrAlaLeuLeuArgCy sAsnA.spThr - 700TyxCysAlaProPxoG lyTyrAlaLeuLeuArgCy sAsnA.spThr - 700
AssTyrSerGlyPbeAlaProAsnCysSerLysValValAlaSer i 11 DK 175959 B1AssTyrsGlyPbAlaProAsnCysSerLysValValAlAs i 11 DK 175959 B1
TbrCy sThrAr gile tMe tCluTbrG InTbrSerThrTrpPheC ly • «. · 8 00 ·TbrCy sThrAr gile tMe tCluTbrG InTbrSerThrTrpPheC ly • «. · 8 00 ·
Pb.eAsnGlyThrArgAlaCluAsnArgTbrTyrlleTyrTrpBi·-- « · '· ·Pb.eAsnGlyThrArgAlaCluAsnArgTbrTyrlleTyrTrpBi · - «· '· ·
ClyArgAsyAsnArgTbrllel1eSerLeuAsbLysTyrTyrAan • % · « 900ClyArgAsyAsnArgTbrllel1eSerLeuAsbLysTyrTyrAan •% · «900
LeuSerLeuH-isCy sLysArgProG 1 yA s nLysThrVaILysGln • « · ·LeuSerLeuH-isCy sLysArgProG 1 yA s nLysThrVaILysGln • «· ·
IleHetLeuMetSerGlyBisValPheHisSerHisTyrGJnPro i • .· · ·· y · 11eAsnLysArgProArgClnAlaTrpCysTrpPbeLysG lyLy» 1000 * .. .IleHetLeuMetSerGlyBisValPheHisSerHisTyrGJnPro i •. · · ·· y · 11eAsnLysArgProArgClnAlaTrpCysTrpPbeLysG lyLy »1000 * ...
TrpLysAapAlaMe tClnGluValLyaTbrLeuAlaLysHisPre ' · ' · ·♦.’··· AJtgTyz AxgGlyThrAsnAspThrAr gAsnll éS erPbeAl a A la 1100 . .TrpLysAapAlaMe tClnGluValLyaTbrLeuAlaLysHisPre '·' · · ♦. '··· AJtgTyz AxgGlyThrAsnAspThrAr gAsnll éS erPbeAl a A la 1100. .
FroGlyLysGlySerAspProGluValAlaTyrKe tTrpTbrAan • - · · · ·FroGlyLysGlySerAspProGluValAlaTyrKe tTrpTbrAan • - · · · ·
Cy s ArgGlyGluPheLe'uTyrCy sAsnllé tTbr TrpPheLeuAan . · 12u0Cy s ArgGlyGluPheLe'uTyrCy sAsnllé tTbr TrpPheLeuAan. · 12h0
TrplleGluAsnLyaTbrHisArgAsnTyrAlaProCysEialle • · · · -··· ^ «TrplleGluAsnLyaTbrHisArgAsnTyrAlaProCysEialle • · · · - ··· ^ «
LysGlnllelleAsnThrTrpEiaLysTalClyArgAsnValTyr . . 1300LysGlnllelleAsnThrTrpEiaLysTalClyArgAsnValTyr. . 1300
Leu?ToPxoArgGluGlyGluLeuSerCysAscS.erThrValThr • · · · · ·Leu? ToPxoArgGluGlyGluLeuSerCysAscS.erThrValThr • · · · · ·
Seril elleAlaAsnlleAspTrpGlnAsnAscAsbGlnThrAsn • » · · 11eThrPheSerA1aG1uVa ΙΑ 1 aG1uLeuTyrArgLeuGluLeb 1*00 . . . .Seril elleAlaAsnlleAspTrpGlnAsnAscAsbGlnThrAsn • »· · 11eThrPheSerA1aG1uVa ΙΑ 1 aG1uLeuTyrArgLeuGluLeb 1 * 00. . . .
• ClyAspTyrLysLeuTaldulleThrProIleGlyPbeAlaPre 12 DK 175959 B1• ClyAspTyrLysLeuTaldulleThrProIleGlyPbeAlaPre 12 DK 175959 B1
ThrLjsCluLysArgtyrSexSerAlaHisClyArsHifThrArg 1500 . . . i C lyVslPheVaILeuGlyPh eLeuG iyPheLeuAlaTbrAlaGly ‘ j * · · * ' ^ThrLjsCluLysArgtyrSexSerAlaHisClyArsHifThrArg 1500. . . i C lyVslPheVaILeuGlyPh eLeuG iyPheLeuAlaTbrAlaGly 'j * · · *' ^
SerAlalietG1yAlaArgA1 sSérLeuThrVa1SerAlaCInSer i 9 9 1600 ·. ·SerAlalietG1yAlaArgA1 sSérLeuThrVa1SerAlaCInSer i 9 9 1600 ·. ·
ArgThrLeuLeuAlaGlylleValGlnGloGlsGInGInLeuLeu • · · ·ArgThrLeuLeuAlaGlylleValGlnGloGlsGInGInLeuLeu • · · ·
AspVa1ValLysArgCIdGInGluLeuLeuArgLéuThrValTrp > ..·*·' · · 1 • · · 17 00AspVa1ValLysArgCIdGInGluLeuLeuArgLéuThrValTrp> .. · * · '· · 1 • · · 17 00
ClyTbrLysAsnLeuGl&AlaArgValThrAlaXleGluLysTyr '.· • · ·'ClyTbrLysAsnLeuGl & AlaArgValThrAlaXleGluLysTyr '. · • · ·'
LeuGlnA s pGlaAlaArgLeuAscS e rTrpG1yCyεAlaPbeArg . . . 1800 GInVaICysHisThrThrValProTrpValAsnAspSerLéuAia • · · · · P roAspTrpAspAsnKetThrTrpGlnG luTrpGloLysGInVal ♦ ·* *'·'LeuGlnA s pGlaAlaArgLeuAscS e rTrpG1yCyεAlaPbeArg. . . 1800 GInVaICysHisThrThrValProTrpValAsnAspSerLéuAia • · · · · P roAspTrpAspAsnKetThrTrpGlnG luTrpGloLysGInVal ♦ · * * '·'
Ar gTy rLe υC luA 1 aAs ήΐ 1 é S e rLy s S e r LYu G ioClaA1a G in 1900 . . . .Ar gTy rLe υC luA 1 aAs ήΐ 1 é S e rLy s S e r LYu G ioClaA1a G in 1900. . . .
1leGloGInGluLysAsntie tTyxGluLeuGlnLysLeuAsnSer • · · · ·1leGloGInGluLysAsntie tTyxGluLeuGlnLysLeuAsnSer • · · · ·
TrpAspllePbeG1yAsnTrpPheAspLeuThrSerTrpValLys . 2000 .TrpAspllePbeG1yAsnTrpPheAspLeuThrSerTrpValLys. 2000.
TyrlleGlnTyrGlyValLeuIlelleValAlaVallleAlaLeu • · · · ·TyrlleGlnTyrGlyValLeuIlelleValAlaVallleAlaLeu • · · · ·
Argil e.VallleTyrValVelGlniietLeuSerArgLeuArg Lys 2100Argil e.VallleTyrValVelGlniietLeuSerArgLeuArg List 2100
ClyTyrArgProVaIPbeSerSerProProGlyTyTlleGlnGln 13 DK 175959 B1 lleEisIleEisLysAspArgG lyGlnProAIaAs&C luGluXhr . . * . 2200 GluGluAspGlyGlySerAsoG lyGlyAapArg TyrTrpProTj^ ·*.·.· · · « ·* ProIleAlaTyrlleEisPheLeuIleArgClnLeulleArgLeu • · m· *· ·ClyTyrArgProVaIPBeSerSProProGlyTyLeLlNGlN 13 DK 175959 B1 lleEisIleEisLysAspArgG lyGlnProAIaAs & C luGluXhr. . *. 2200 GluGluAspGlyGlySerAsoG lyGlyAapArg TyrTrpProTj ^ · *. ·. · · · «· * ProIleAlaTyrlleEisPheLeuIleArgClnLeulleArgLeu • · m · * · ·
LeuTbrArg LeuTyrSerlleCy sArgAspLeoLeuSerArgSer 2300 . . ... .LeuTbrAr LeuTyrSerlleCy sArgAspLeoLeuSerArgSer 2300. . ....
PbeLeuThrLeuGInLeuIleTyrGInAsnXenArgAipTrpLau • · * ·* .· ‘ ✓PbeLeuThrLeuGInLeuIleTyrGInAsnXenArgAipTrpLau • · * · *. · '✓
ArgLéuArgTbrAlaPbeLeuG InTy rG ly'CysG luTrpIleGln . ‘ 2400 . . .ArgUlArgTbrAlaPbeLeuG InTy rG ly'CysG luTrpIleGln. '2400. . .
GluAlaPheGlnAlaAlsAlaArgAlaThrArgGluTbrLeuAla • · · ♦ ·GluAlaPheGlnAlaAlsAlaArgAlaThrArgGluTbrLeuAla • · · ♦ ·
GlyAlaCysArgGlyLeuTrpArgValLéuG ltiArgl 1 eG lyATg 2500 . . .GlyAlaCysArgGlyLeuTrpArgValLéuG ltiArgl 1 eG lyATg 2500. . .
G 1yl1eLeuA1aValProArgArgIleArgG1dGIyAlaGluIleG 1yl1eLeuA1aValProArgArgIleArgG1dGIyAlaGluIle
AlaLcubeu 14 DK 175959 B1 I et fjerde aspekt angår opfindelsen et præparat til in vitro påvisning af tilstedeværelsen i en biologisk prøve af antistoffer mod et humant HIV-2 virus, der er i stand til at have cytopatogen virkning på T4 lymfocytter, der er ejendommeligt ved, at det indeholder mindst et antigen fra et HIV-2 retrovirus ifølge et' 5 vilkårligt af kravene 1-9 eller mindst et antigen ifølge et vilkårligt af kravene 10-19.AlaLcubeu 14 DK 175959 B1 In a fourth aspect, the invention relates to a composition for in vitro detection of the presence in a biological sample of antibodies against a human HIV-2 virus capable of cytopathogenic action on T4 lymphocytes peculiar to It contains at least one antigen from an HIV-2 retrovirus according to any one of claims 1-9 or at least one antigen according to any one of claims 10-19.
I et femte aspekt angår opfindelsen en fremgangsmåde til in vitro påvisning i en human biologisk prøve, f.eks. serum, af tilstedeværelsen af antistoffer i 10 mennesker, som er inficeret med et humant HIV-2 retrovirus, specielt til in vitro diagnose af en potentiel eller eksisterende LAS eller AIDS, forårsaget af HIV-2 retrovirus, og som er ejendommelig ved, at man bringer den biologiske prøve fra den person, der skal diagnosticeres, i kontakt med et antigen, der genkendes af et antistof, som er fremkaldt i den inficerede person af et hu-15 man HIV-2 retrovirus som defineret i et vilkårligt af. kravene 1-9, under betingelser, der tillader dannelse af et immunologisk konjugat mellem antigenet og antistoffet, og at man påviser det muligt dannede immunologiske konjugat mellem antistof og antigen.In a fifth aspect, the invention relates to a method for in vitro detection in a human biological sample, e.g. serum, from the presence of antibodies in 10 people infected with a human HIV-2 retrovirus, specifically for the in vitro diagnosis of a potential or existing LAS or AIDS, caused by HIV-2 retroviruses and which are peculiar to brings the biological sample of the person to be diagnosed into contact with an antigen recognized by an antibody induced in the infected person by a human HIV-2 retrovirus as defined in any of the above. claims 1-9, under conditions which allow for the formation of an immunological conjugate between the antigen and antibody, and to detect the possible immunological conjugate between antibody and antigen.
20 I ét sjette aspekt angår opfindelsen et kit til påvisning af antistoffer mod et retrovirus af HIV-2 typen, der er i stand til at have cytopatogen virkning på . mennesker, i en biologisk væske, specielt hos en person, der muligvis bærer disse antistoffer, og som er ejendommelig ved, at det omfatter mindst et antigen som defineret i et vilkårligt af kravene 10-19, eller et præparat som de-25 fineret i et vilkårligt af kravene 20-24; og midler til påvisning af det immunologiske konjugat, der dannes ved den immunologiske reaktion mellem antigenet og den biologiske prøve.In one sixth aspect, the invention relates to a kit for detecting antibodies to a HIV-2 retrovirus capable of cytopathogenic action. human, in a biological fluid, especially in a person possibly carrying these antibodies, characterized in that it comprises at least one antigen as defined in any one of claims 10-19, or a composition as defined in any one of claims 20-24; and means for detecting the immunological conjugate formed by the immunological reaction between the antigen and the biological sample.
I et syvende aspekt angår opfindelsen et immunogent præparat, der er ejen-30 dommeligt ved, at det indeholder et omhylningsglycoprotein af det humane HIV-2 retrovirus ifølge et vilkårligt af kravene 1-9, såsom gp140 fra retrovi-ruset, eller en del af glycoproteinet, der er i stand til at fremkalde antistoffer DK 175959 B1 mod HIV-2. sammen med en farmaceutisk acceptabel bærer, der er egnet til at danne en vaccine, der virker mod HIV-2.In a seventh aspect, the invention relates to an immunogenic composition, characterized in that it contains a envelope glycoprotein of the human HIV-2 retrovirus according to any one of claims 1-9, such as gp140 from the retrovirus, or a portion of the glycoprotein capable of inducing antibodies DK 175959 B1 against HIV-2. together with a pharmaceutically acceptable carrier suitable for forming an anti-HIV-2 vaccine.
I et ottende aspekt angår opfindelsen et antistof, der er ejendommeligt ved, 5 at det specifikt genkender et antigen af et HIV-2 retrovirus ifølge et vilkårligt af kravene 1-9.In an eighth aspect, the invention relates to an antibody characterized in that it specifically recognizes an antigen of an HIV-2 retrovirus according to any one of claims 1-9.
I et niende aspekt angår opfindelsen en nukleinsyre, der eventuelt er mærket . og er afledt i det mindste delvist af RNA fra HIV-2 virus eller fra en af dets 10 varianter som defineret i ét vilkårligt af kravene 1-9, og som er ejendommelig ved, at nukleinsyren er specifik for detektering af mindst en del af RNA af HIV-2 ifølge et vilkårligt af kravene 1-9, ved en specifik hybridise-ringskapacitet med RNA fra HIV-2 viruset og ved fravær af hybridisering under kraftige betingelser med probeme 1-4, der omfatter henholdsvis nukleo-15 tiderne 990-1070, 990-1260, 2170-2240 og 3370-3640 af DNA fra HIV-1 ge-nomet.In a ninth aspect, the invention relates to a nucleic acid optionally labeled. and is derived at least in part from RNA from HIV-2 virus or from one of its 10 variants as defined in any one of claims 1-9, characterized in that the nucleic acid is specific for detecting at least a portion of RNA of HIV-2 according to any one of claims 1-9, by a specific hybridization capacity with RNA from the HIV-2 virus and in the absence of hybridization under strong conditions with probes 1-4 comprising nucleotides 990-1070, respectively. , 990-1260, 2170-2240 and 3370-3640 of DNA from the HIV-1 genome.
I et tiende aspekt angår opfindelsen en rekombinant nukleinsyre, der er ejendommelig ved, at den omfatter et cDNA eller en del heraf ifølge et vilkårligt af 20 kravene 35-42 og/eller et RNA eller en del heraf, der svarer til cDNA’et, og er indført i en nukleinsyre fra en vektor.In a tenth aspect, the invention relates to a recombinant nucleic acid, characterized in that it comprises a cDNA or a portion thereof according to any one of claims 35-42 and / or an RNA or a portion thereof corresponding to the cDNA. and is introduced into a nucleic acid from a vector.
I et ellevte aspekt angår opfindelsen en fremgangsmåde til påvisning af en retrovirus af HIV-2 typen ifølge et vilkårligt af kravene 1-9, der er i stand til at 25 have cytopatogen virkning hos mennesker, eller af dets RNA i en biologisk væske eller væv, specielt til in vitro diagnosticering hos mennesker af muligheden for eller eksistensen af LAS eller AIDS, og som er ejendommelig ved, at man bringer nukleinsyren indeholdt i den biologiske væske eller væv i kontakt med en probe indeholdende en nukleinsyre ifølge et vilkårligt af 30 kravene 38-42 under kraftige hybridiseringsbetingelser.In an eleventh aspect, the invention relates to a method for detecting a retrovirus of the HIV-2 type according to any one of claims 1-9 capable of having cytopathogenic effect in humans, or of its RNA in a biological fluid or tissue. , in particular for in vitro diagnosis in humans of the possibility or existence of LAS or AIDS, characterized by contacting the nucleic acid contained in the biological fluid or tissue with a probe containing a nucleic acid according to any of claims 38 -42 under strong hybridization conditions.
I et tolvte aspekt angår opfindelsen en fremgangsmåde til fremstilling af et retrovirus af HIV-2 typen ifølge et vilkårligt af kravene 1-9, der er i stand til at 16 DK 175959 B1 have cytopatogen virkning hos mennesker, og som er ejendommelig ved, at man dyrker humane T4 lymfocyter eller permanente cellelinier afledt fra T4 lymfocyter, som bærer T4-phenotypen, hvorved lymfocyteme eller cellelinierne i forvejen er blevet inficeret med et isolat af HIV-2 virus som de-5 fineret i et vilkårligt af kravene 1-9, og at man, specielt når niveauet af omvendt transkriptase aktivitet har nået en forudbestemt værdi, udvinder og renser de i dyrkningmediet af lymfocyter eller cellelinier frigjorte virusmængder, specielt ved differentialcentrifugering i en sucrosegradient eller en metri-zamid-gradient.In a twelfth aspect, the invention relates to a method of producing a retrovirus of the HIV-2 type according to any one of claims 1-9, capable of having cytopathogenic effect in humans and which is characterized in that cultivating human T4 lymphocytes or permanent cell lines derived from T4 lymphocytes bearing the T4 phenotype, whereby the lymphocytes or cell lines have already been infected with an isolate of HIV-2 virus as defined in any of claims 1-9, and that, especially when the level of reverse transcriptase activity has reached a predetermined value, the amounts of virus released in the culture medium by lymphocytes or cell lines, especially by differential centrifugation in a sucrose gradient or a metrazamide gradient, are recovered and purified.
10 I et trettende aspekt angår opfindelsen en fremgangmåde til fremstilling af specifikke antigener af HIV-2 retrovirus ifølge et vilkårligt af kravene 1-9, der er ejendommelig ved, at man lyserer det rensede virus, der opnås ved fremgangsmåden ifølge krav 57, specielt ved hjælp af et detergent, såsom natri-15 umdodecylsulfat (SDS), f.eks. 0,1 % SDS i en RIPA-buffer, og at man udvinder lysatet indeholdende antigenerne.In a thirteenth aspect, the invention relates to a method for producing specific antigens of HIV-2 retroviruses according to any one of claims 1-9, characterized by lysing the purified virus obtained by the method of claim 57, in particular by using a detergent such as sodium dodecyl sulfate (SDS), e.g. 0.1% SDS in a RIPA buffer and recovering the lysate containing the antigens.
. I et fjortende aspekt angår opfindelsen en fremgangsmåde til fremstilling af en hybridiseringsprobe til påvisning af RNA fra HIV-2 retrovirus, som er ejen-20 dommelig ved, at man indsætter en DNA sekvens, fortrinsvis ifølge et vilkårligt af kravene 35-42, i en kloningsvektor ved in vitro rekombination, kloner den opnåede, modificerede vektor i en egnet cellevært og udvinder de opnåede DNA-rekombinanter.. In a fourteenth aspect, the invention relates to a method for producing a hybridization probe for detection of RNA from HIV-2 retroviruses, which is useful in inserting a DNA sequence, preferably according to any of claims 35-42, into a DNA sequence. cloning vector by in vitro recombination, clones the obtained modified vector into a suitable cell host and extracts the obtained DNA recombinants.
25 I et femtende aspekt angår opfindelsen et hybridom, som er ejendommeligt ved, at det udskiller det monoklonale antistof ifølge krav 34.In a fifteenth aspect, the invention relates to a hybridoma which is characterized in that it secretes the monoclonal antibody of claim 34.
I et sekstende aspekt angår opfindelsen anvendelsen af polyklonale antistoffer ifølge krav 33 eller monoklonale antistoffer ifølge krav 34 til påvisning af 30 HlV-2 retrovirus som defineret i et vilkårligt af kravene 1-9 i en biologisk præparation.In a sixteenth aspect, the invention relates to the use of polyclonal antibodies according to claim 33 or monoclonal antibodies according to claim 34 for the detection of 30 HIV-2 retroviruses as defined in any one of claims 1-9 in a biological preparation.
17 DK 175959 B1 I et syttende aspekt angår opfindelsen en fremgangsmåde til fremstilling af et protein eller et glycoprotein eller en del af et protein eller glycoprotein bestående af ekspressionsproduktet af en nukleinsyresekvens, der er afledt fra genomet af et retrovirus, og som er ejendommelig ved, at man i en vektor, ' 5 som er i stand til at transformere en udvalgt værtscelle og tillade udtrykkelse i værten af et indskud i vektoren, indsætter nukleinsyresékvensen fra et humant HIV-2 retrovirus, der har evne til at inficere humane T4-lymphocyter og er i stand til at forårsage AIDS hos mennesker, hvor HIV-2 retroviruset udgøres af et af de i CNCM deponerede retrovirus med numrene I-502, Ι-ΙΟ 532, i-642 og 1-643 eller de i ECACC deponerede retrovirus med numrene 87.011001 og 87.011002, eller indsætter nukleinsyresekvensen fra en variant af en af disse retrovirus, hvis hovedomhylningsglycoprotein genkendes af antistoffer dannet mod hovedomhylningsglycoproteinet gp140 af et HIV-2 retrovirus, indfører vektoren indeholdende nukleinsyresekvensen i den valgte 15 vært, dyrker den transformerede værtscelle, og udvinder og renser det af nukleotidsekvensen udtrykte protein eller del af protein, idet proteinet eller proteindelen er i stand til at frembringe en specifik immunologisk reaktion med antistoffer, der er rettet mod HIV-2 retrovirus.In a seventeenth aspect, the invention relates to a process for producing a protein or a glycoprotein or a portion of a protein or glycoprotein consisting of the expression product of a nucleic acid sequence derived from the genome of a retrovirus, which is characterized by inserting into a vector, 5 capable of transforming a selected host cell and allowing expression in the host of a insert into the vector, the nucleic acid sequence of a human HIV-2 retrovirus capable of infecting human T4 lymphocytes and is capable of causing AIDS in humans where the HIV-2 retrovirus is one of the retroviruses deposited in CNCM with numbers I-502, Ι-ΙΟ 532, i-642 and 1-643 or the retroviruses deposited in ECACC 87.011001 and 87.011002, or inserts the nucleic acid sequence from a variant of one of these retroviruses whose major envelope glycoprotein is recognized by antibodies formed against the main envelope glycoprotein gp140 of an HIV-2 retrovirus, introduces the vector containing the nucleic acid sequence into the selected host, cultivates the transformed host cell, and extracts and purifies the protein or portion of protein expressed by the nucleotide sequence, the protein or protein moiety being capable of producing a specific immunological reaction with antibodies directed against HIV-2 retroviruses.
20 I et attende aspekt angår opfindelsen en cellekultur, der er ejendommelig ved, at den er transformeret med et rekombinånt DNA indeholdende en nukleinsyre afledt fra i det mindste en del af RNA fra det humane HIV-2 retrovirus eller en af dets varianter under betingelser, der tillader fremstilling af et protein eller del af et protein ved fremgangsmåden ifølge krav 53, hvilken 25 nukleinsyre er i stand til at hybridisere med RNA fra det humane HIV-2 retrovirus og ikke hybridisere under kraftige betingelser med probeme 1-4, der omfatter henholdsvis nukleotideme 990-1070, 990-1260, 2170-2240 og 3370:3640 af DNA fra HIV-1 genomet.In an eighteenth aspect, the invention relates to a cell culture characterized by being transformed with a recombinant DNA containing a nucleic acid derived from at least a portion of RNA from the human HIV-2 retrovirus or one of its variants under conditions. permitting production of a protein or portion of a protein by the method of claim 53, which is capable of hybridizing with RNA from the human HIV-2 retrovirus and not hybridizing under strong conditions with probes 1-4 comprising, respectively, nucleotides 990-1070, 990-1260, 2170-2240 and 3370: 3640 of DNA from the HIV-1 genome.
30 I et nittende aspekt angår opfindelsen anvendelsen af en fremgangsmåde ifølge et vilkårligt af kravene 53-63 til fremstilling af proteiner eller dele af proteiner, der kan anvendes i reaktioner til påvisning af antistoffer mod HIV-2 retrovirus.In a nineteenth aspect, the invention relates to the use of a method according to any of claims 53-63 for the production of proteins or portions of proteins that can be used in reactions to detect antibodies against HIV-2 retroviruses.
18 DK 175959 B118 DK 175959 B1
En foretrukken permanent cellelinie til dyrkning af HIV-2 er f.eks. HUT 78 celletypen. En HUT 78 linie inficeret med HIV-2 deponeredes 6. februar 1986 i CNCM under nummeret 1-519. Dyrkning udføres f.eks. på følgende måde:· 5 HUT. 78 celler (106/ml) dyrkes sammen med inficerede normale humane lymphocyter (106/ml). Dyrkningssubstratet er RPMI 1640 med 10% føtalt kalveserum. Efter 15-21 dage ses en, cytopathogen virkning i HUT 78 cellerne. Omvendt transcriptase undersøges 1 uge efter denne observation i 10 den supematante del af dyrkningsmediet. Det er herefter muligt at begynde at udvinde viruset fra den supematante del.A preferred permanent cell line for growing HIV-2 is e.g. HUT 78 cell types. A HUT 78 line infected with HIV-2 was deposited in CNCM on February 6, 1986 under number 1-519. Cultivation is carried out e.g. as follows: · 5 HUT. 78 cells (106 / ml) are cultured together with infected normal human lymphocytes (106 / ml). The culture medium is RPMI 1640 with 10% fetal calf serum. After 15-21 days, a cytopathogenic effect is seen in the HUT 78 cells. Reverse transcriptase is examined 1 week after this observation in the supernatant portion of the culture medium. It is then possible to start extracting the virus from the supernatant part.
En anden foretrukken linie til dyrkning hører til de linier, der er betegnet CEM.Another preferred line for cultivation is one of the lines designated CEM.
15 Infektion og herefter dyrkning af de inficerede CEM-celler kan udføres specielt som beskrevet nedenfor.Infection and then culture of the infected CEM cells can be performed specifically as described below.
T4 lymphocyter inficeret forud med HIV-2 virus og uinficerede celler af CEM linien dyrkes sammen i et tidsrum, der er nødvendig for infektion af CEM.T4 lymphocytes pre-infected with HIV-2 virus and uninfected cells of the CEM lineage are cultured together for a period of time necessary for CEM infection.
20 Dyrkningen fortsættes herefter yderligere i et passende substrat f.eks. det, der er beskrevet nedenfor, og når den omvendte transcriptaseaktivitet i de inficerede celler har nået et tilstrækkeligt niveau, udvindes det frembragte virus fra dyrkningssubstratet.Cultivation is then further continued in a suitable substrate e.g. that described below, and when the reverse transcriptase activity in the infected cells has reached a sufficient level, the virus generated is recovered from the culture substrate.
25 Specielt udføres den samtidige dyrkning under betingelser, der er beskrevet nedenfor for humane T4 lymphocyter, der 5 dage forud er inficeret med en stamme af HIV-2 virus, der stammer fra en patient, i det efterfølgende betegnet "ROD" i et tilfælde og CEM i et andet tilfælde.Specifically, concomitant culture under conditions described below for human T4 lymphocytes infected with a strain of HIV-2 virus originating from a patient is hereinafter referred to hereinafter as "ROD" in one case and CEM in another case.
30 De inficerede T4 lymphocyter aktiveret forud med phytohæmagglutinin viste sig at være i besiddelse af en omvendt transcriptaseaktivitet på 5.000 cpm/106 normale T.lymphocyter 3 dage efter infektionens begyndelse. Dyrkningen fortsatte indtil den målte omvendte transcriptaseaktivitet nåede 19 DK 175959 B1 100.000 cpm i den supematante del. Disse T4 lymphocyter anbragtes herefter i kontakt med CEM celler (3x10® inficerede normale T lymphocyter) og inkuberedes atter i følgende dyrkningssubstrat: RPMI 1640 indholdende 2,92 mg/ml L-glutamin, 10% dekomplementeret føtalt kalveserum, 2 pg/ml po-5 lybren, 0,05% anti-inteferon-a-sérum, 100.000 pg/ml penicillin, 10 pg/ml streptomycin og 10.000 pg/ml neomycin.The T4 infected lymphocytes pre-activated with phytohemagglutinin were found to have a reverse transcriptase activity of 5,000 cpm / 106 normal T. lymphocytes 3 days after the onset of infection. Cultivation continued until the measured reverse transcriptase activity reached 100,000 cpm in the supernatant portion. These T4 lymphocytes were then contacted with CEM cells (3x10® infected normal T lymphocytes) and again incubated in the following culture medium: RPMI 1640 containing 2.92 mg / ml L-glutamine, 10% decomposed fetal calf serum, 2 pg / ml 5 lybren, 0.05% anti-interferon-α-semen, 100,000 pg / ml penicillin, 10 pg / ml streptomycin and 10,000 pg / ml neomycin.
Dyrkningssubstratet udskiftedes to gange om ugen.The culture medium was replaced twice a week.
10 Målingerne af den omvendte transcriptaseaktivitet i den supematante del var følgende: på dag 0 1.000 (baggrund) på dag 15 20.000 15 på dag 21 200.000 på dag 35 1.000.000The measurements of the reverse transcriptase activity in the supernatant were as follows: on day 0 1,000 (background) on day 15 20,000 15 on day 21 200,000 on day 35 1,000,000
En CEM kultur inficeret med HIV-2 vims deponeredes i Collection Nationale de Cultures de Micro-organismes (CNCM) i Institut Pasteur under nummer I-20 537 den 24. marts 1986.A CEM culture infected with HIV-2 vims was deposited in the Collection National de Cultures de Microorganisms (CNCM) at the Institut Pasteur under number I-20 537 on March 24, 1986.
Nogle enkelte egenskaber ved antigenerne og nukleinsyreme i forbindelse med oplysningen af HIV-2 blev fundet ud fra de eksperimenter, der udførtes under de neden for beskrevne betingelser. De vil i mange tilfælde bedre kun-25 ne undersøges ved sammenligning af den samme type egenskaber i forbindelse med andre typer retrovirus specielt HIV-1 og SIV.Some individual properties of the antigens and nucleic acids associated with the disclosure of HIV-2 were found from the experiments carried out under the conditions described below. In many cases, they will be better investigated by comparing the same type of properties with other types of retroviruses, especially HIV-1 and SIV.
Idet der henvises til tegningen viser 30 fig. 1a, 1b og 1c krydsede immuno-præcipitationsforsøg mellem sera henholdsvis fra patienter inficeret med HIV-1 og HIV-2 og en rhesusabe inficeret med STLV-IH på den ene side og med virale ekstrakter af HIV-1 på den anden side; 20 DK 175959 B1 ftg. 2a og 2b viser sammenlignende resultater med hensyn til de elektropho-retiske vandringen af proteinerne fra henholdsvis HIV-1, HIV-2 og STLV-III i SDS-polyacrylamidgeler; 5 fig. 3 viser resultaterne af.krydset hybridisering mellem genome sekvenser af HIV-1, HIV-2 og STLV-III på den ene side og prober indeholdende forskellige subgenome sekvenser af HIV-1 virus på den anden side; 10 fig. 4 er et restriktionskort over cDNA stammende fra RNA af HIV-2 ROD; fig. 5 er et restrjktionskort af et E2 fragment af cDNA stammende fra HIV-2, idet dette fragment indeholder en region svarende til 3' LTR regionen af HIV-2; 15 fig. 6 er en nucleotidsekvens af en del af E2, idet denne sekvens svarer til U3/R regionen af HIV-2; fig. 7 viser: 20 på den ene side skematisk strukturelle elementer af HIV-1 (fig.Referring to the drawing, FIG. 1a, 1b and 1c crossed immunoprecipitation experiments between sera, respectively, from patients infected with HIV-1 and HIV-2 and a rhesus monkey infected with STLV-IH on the one hand and with viral extracts of HIV-1 on the other; 20 DK 175959 B1 ftg. 2a and 2b show comparative results with respect to the electrophoretic migration of the proteins of HIV-1, HIV-2 and STLV-III in SDS-polyacrylamide gels, respectively; 5 FIG. 3 shows the results of cross-hybridization between genomic sequences of HIV-1, HIV-2 and STLV-III, on the one hand, and probes containing different subgenome sequences of HIV-1 virus, on the other hand; 10 FIG. 4 is a restriction map of cDNA derived from RNA of HIV-2 ROD; FIG. 5 is a restriction map of an E2 fragment of cDNA derived from HIV-2, containing this region corresponding to the 3 'LTR region of HIV-2; FIG. 6 is a nucleotide sequence of a portion of E2, this sequence corresponding to the U3 / R region of HIV-2; FIG. 7 shows: 20, on the one hand, schematic structural elements of HIV-1 (fig.
7A) og på linie méd en region indeholdende HIV-1 3’ LTR, sekvensen stammende fra E2 regionen af HIV-2 cDNA og, 25 på den anden side de fælles nucleotider til stede henholdsvis i sekvensen stammende fra E2 regionen af HIV-2 og den tilsvarende sekvens af HIV-1 placeret på linie på bekostning af et antal deletioner og indskud (fig. 7B); 30 fig. 8 viser skematisk opbygningen af adskillige kloner af en fag modificeret ved adskillige indskud, der stammer fra cDNA stammende fra HIV-2 (kloneme ROD4, ROD27 og ROD35); sekvenserne stammer for deres vedkommende fra ROD4, ROD27 og ROD35 21 DK 175959 B1 subklonet i et plasmid pUC18, der ogsåer vist skematisk på denne tegning, idet disse sidstnævnte sekvenser er anbragt således at de korresponderer med regionerne fra ROD4, ROD27 og ROD35, som de henholdsvis stammer fra; 5 fig. 9 viser de relative intensiteter af hybridisering mellem a/11 fragmenter fjernet fra fprskellige regioner af det komplette HlV-1-genom (idet fragmenterne er vist skematisk for neden på 10 figuren), på den ene side og HIV-2 cDNA, der findes i ROD4, på den anden side, og b/ fragmenter stammende fra HIV-2 med det samme cDNA.7A) and aligned with a region containing HIV-1 3 'LTR, the sequence originating from the E2 region of HIV-2 cDNA and, on the other hand, the common nucleotides present respectively in the sequence originating from the E2 region of HIV-2 and the corresponding sequence of HIV-1 aligned at the expense of a number of deletions and insertions (Fig. 7B); FIG. Figure 8 shows schematically the construction of several clones of a phage modified by several insertions derived from cDNA derived from HIV-2 (clones ROD4, ROD27 and ROD35); the sequences are derived from ROD4, ROD27 and ROD35 B1 subcloned into a plasmid pUC18, also schematically shown in this drawing, these latter sequences being arranged to correspond to the regions of ROD4, ROD27 and ROD35 originate respectively; 5 FIG. Figure 9 shows the relative intensities of hybridization between α / 11 fragments removed from different regions of the complete HIV-1 genome (the fragments are schematically shown below in Figure 10), on the one hand, and the HIV-2 cDNA found in ROD4, on the other hand, and b / fragments derived from HIV-2 with the same cDNA.
15 Generelt stammer HIV-2 antigenerne anvendt i de sammenlignende forsøg, hvor beskrivelsen følger senere, fra HIV-2 MIR stammen deponeret i CNCM under nummer I-502, og DNA sekvenserne stammer fra det genome DNA af HIV-2, der stammer fra stammen HIV-2 ROD deponeret under nummer I-532 i CNCM.Generally, the HIV-2 antigens used in the comparative experiments where the description follows later are from the HIV-2 MIR strain deposited in CNCM under number I-502, and the DNA sequences are from the genome DNA of HIV-2 derived from the strain. HIV-2 ROD deposited under number I-532 in CNCM.
20 I - Antigener, specielt proteiner 6a alvcooroteinerI - Antigens, especially proteins 6a alpha proteins
Viruset oprindeligt dyrket i HUT 78 mærkedes metabolisk med [35S]cystein og [35S]methionin, de inficerede celler inkuberedes i nærvær af disse radioaktive 25 aminosyrer i dyrkningssubstrat uden de tilsvarende umærkede aminosyrer i 14-16 timer specielt således som skrevet i artiklen betegnet med referencenummeret 21 i litteraturlisten, der findes efter selve beskrivelsen, med hensyn til mærkning med [35S]cystein. Den supernatante del klares herefter, og viruset ultracentrifugeres i 1 time ved 100.000 g på en pude af 20% saccha-30 rose. Hovedantigeneme af viruset skilte ad ved elektrophorese i en poly-acrylamidgel (12,5%) under denaturerende betingelser (SDS), eller i en gel bestående af polyacrylamid (10%) + bisacrylamid (0,13%) med SDS (0,1% 22 DK 175959 B1 slutkoncentration). Følgende farvede markører anvendtes som molekylvægtsreferencer: myosin 200 kd 5 phosphorylase B 97,4 kd BSA 68 kd ovalbumin 43 kd a-chymotrypsin 25,7 kd β-lactoglobulin 18,4 kd 10 lysozym 14,3 kdThe virus originally cultured in HUT 78 was metabolically labeled with [35S] cysteine and [35S] methionine, the infected cells were incubated in the presence of these radioactive 25 amino acids in culture substrate without the corresponding unlabeled amino acids for 14-16 hours specifically as written in the article designated by the reference number 21 in the literature list found after the description itself, for labeling with [35S] cysteine. The supernatant portion is then cleared and the virus is ultracentrifuged for 1 hour at 100,000 g on a pad of 20% sucrose. The major antigens of the virus were separated by electrophoresis in a polyacrylamide gel (12.5%) under denaturing conditions (SDS), or in a gel consisting of polyacrylamide (10%) + bisacrylamide (0.13%) with SDS (0.1 % 22 DK 175959 B1 final concentration). The following colored markers were used as molecular weight references: myosin 200 kd 5 phosphorylase B 97.4 kd BSA 68 kd ovalbumin 43 kd α-chymotrypsin 25.7 kd β-lactoglobulin 18.4 kd 10 lysozyme 14.3 kd
Andre molekylvægtmarkører anvendtes i andre forsøg. Dette gælder specielt for fig. 1a, 1 b og 1c, der henviser til andre kendte molekylvægtmarkører (under bogstavet M i disse figurer). Antigenerne kan endnu lettere adskilles efter 15 immunopræcipitation (RIPA) eller ved immunoaftryk (Western blot) under anvendelse af antistoffer, der forefindes i patientens serum: deres tilsyneladende molekylvægte bestemt ved deres tilsyneladende vandring er meget tæt på HIV-1 antigenerne.Other molecular weight markers were used in other experiments. This is especially true of FIG. 1a, 1b and 1c, referring to other known molecular weight markers (under the letter M of these figures). The antigens can be even more readily separated after immunoprecipitation (RIPA) or by immunoprinting (Western blot) using antibodies present in the patient's serum: their apparent molecular weights determined by their apparent migration are very close to the HIV-1 antigens.
20 Det skal specielt bemærkes, at i den efterfølgende tekst, svarer tallene, der følger efter betegnelserne "p" og/eller "gp” til de omtrentlige molekylvægte af de tilsvarende proteiner og/eller glycoproteiner divideret med 1000. F.eks. har p36 en molekylvægt af størrelsesordenen 36.000. Det skal imidlertid forstås således, at disse molekylvægtsværdier kan variere inden for et område, 25 der kan nå 5%, 10% eller endog mere afhængig af den metode, der er anvendt til bestemmelse af disse molekylvægte.In particular, it should be noted that in the following text, the numbers following the terms "p" and / or "gp" correspond to the approximate molecular weights of the corresponding proteins and / or glycoproteins divided by 1000. For example, p36 It is to be understood, however, that these molecular weight values may vary within a range 25 which may reach 5%, 10% or even more depending on the method used to determine these molecular weights.
Gentagelsen af forsøgene muliggjorde at de tilsyneladende molekylvægte af HIV-2 antigenerne kunne bestemmes mere nøjagtigt. Det blev således fun-30 det, at molekylvægtene af de tre kemeproteiner, der oprindeligt var tildelte molekylvægtene af størrelsesordenen henholdsvis 13.000, 18.000 og 25.000 faktisk havde tilsyneladende molekylvægte nærmere følgende værdier hen- _;______ 23 DK 175959 B1 holdsvis 12.000,16.000 og 26.000. Disse proteiner er i det efterfølgende be-. . tegnet med forkortelserne p12, p16 og p26.The repetition of the experiments allowed the apparent molecular weights of the HIV-2 antigens to be determined more accurately. Thus, it was found that the molecular weights of the three core proteins originally assigned to the molecular weights of the order of 13,000, 18,000 and 25,000, respectively, actually had apparent molecular weights of the following values, respectively: 12,000, 16,000 and 26,000 . These proteins are hereinafter described. . drawn with the abbreviations p12, p16 and p26.
De samme betragtninger gælder for eksistensen af protein eller glycoprotein-5 bånd, hvis tilsyneladende molekylvægte blev fastlagt ved værdier, der kunne ligge fra 32.000 til 42.000-45.000. Gentagelse af målingerne muliggør til slut et bånd, der svarede til en molekylvægt på nøjagtigt 36.000. I den efterfølgende tekst er dette bånd betegnet med forkortelsen p36. Et andet bånd ved 42.000-45.000 (p42) er i overensstemmelse hermed også fastlagt således.The same considerations apply to the existence of protein or glycoprotein-5 bands, whose apparent molecular weights were determined at values ranging from 32,000 to 42,000-45,000. Repeating the measurements finally allows a band corresponding to a molecular weight of exactly 36,000. In the following text this band is denoted by the abbreviation p36. A second band at 42,000-45,000 (p42) is accordingly also determined thus.
10 Den ene eller den anden af p36 eller p42 udgør sandsynligvis et transmem-branglycoprotein af viruset.One or the other of p36 or p42 probably constitutes a transmembrane glycoprotein of the virus.
Et hovedomhylriingsglycoprotein med en molekylvægt af størrelsesordenen 130-140 kd er blevet observeret: dette glycoprotein er i det efterfølgende be-15 tegnet gp140.A major envelope glycoprotein having a molecular weight of the order of 130-140 kd has been observed: this glycoprotein is hereinafter designated gp140.
Det skal bemærkes, at sædvanligvis fastlægges molekylvægtene med en • nøjagtighed på + 5%, idet denne nøjagtighed endog kan være lidt lavere for antigener med høje molekylvægte, således som det er fundet for gp140 (mo-20 lekylvægt på 140 + 10%). Denne gruppe af antigener, (dersom de er mærket med [35S]cystein) genkendes klin svagt om overhovedet, af patientsera indeholdende anti-HIV-1-antistoffer i de systemer til afsløring, der anvendes i laboratoriet, eller under anvendelsen af tests, der anvender HIV-1-lysater, som f.eks. de, der markedesføres af Diagnostics Pasteur under navnet "ELAVIA".It should be noted that usually the molecular weights are determined with an accuracy of + 5%, this accuracy may even be slightly lower for high molecular weight antigens, as found for gp140 (molecular weight of 140 + 10%). This group of antigens (if labeled with [35S] cysteine) is clinically poorly recognized at all, by patient sera containing anti-HIV-1 antibodies in the detection systems used in the laboratory, or during the use of tests that uses HIV-1 lysates, such as those marketed by Diagnostics Pasteur under the name "ELAVIA".
25 Kun p26 proteinet immunopræcipiteredes svagt med disse sera. Omhyldningsproteinet udfældedes ikke. Serumet fra patienten inficeret med det omhandlede virus (HIV-2) genkender svagt et p34 protein af HIV-1. De anvendte systemer til afsløring genkendtes andre HIV-1 proteiner ikke.25 Only the p26 protein was weakly immunoprecipitated with these sera. The envelope protein did not precipitate. The patient's serum infected with the subject virus (HIV-2) weakly recognizes a p34 protein of HIV-1. Other detection systems used did not recognize other HIV-1 proteins.
30 I modsætning hertil er HIV-2 i besiddelse af nogle proteiner, der viser en vis immunologisk lighed med lignende strukturelle proteiner eller glycoproteiner, der er fraskilt under lignende betingelser fra et retrovirus, der for nylig er isoleret fra tamme macaq-aber af rhesusarten, medens dette immunologiske 24 DK 175959 B1 forhold har en tendens til at blive udvisket for andre proteiner eller gly-coproteiner. Dette sidstnævnte retrovirus, der antages at være det ætiologiske agens for AIDS hos aber,.betegnedes af forskerne, der isolerede det (litteraturreferencerne 16-18 nedenfor) med navnet "STLV-lllmac". Af bekvem-5 melighedshensyn vil det i det efterfølgende simpelt hen blive betegnet med "STLV-IIIagm" (eller alternativt med udtrykket SIV, en forkortelse for "Abe im-munodeficient virus").In contrast, HIV-2 possesses some proteins that show some immunological similarity to similar structural proteins or glycoproteins secreted under similar conditions from a recent retrovirus isolated from domestic macaq monkeys of the rhesus species. whereas this immunological relationship tends to be eradicated for other proteins or glycoproteins. This latter retrovirus, believed to be the aetiological agent of AIDS in monkeys, was designated by the researchers who isolated it (literature references 16-18 below) named "STLV-11mac". For convenience, hereinafter it will simply be designated "STLV-IIIagm" (or, alternatively, the term SIV, an abbreviation for "Abe immunodeficient virus").
Et andet retrovirus betegnet "STLV-IIIagm" eller "SIVagm" isoleredes fra vilde 10 grønne aber. I modsætning til viruset, der fandtes i macaq-rhesusaber, synes tilstedeværelsen af "STLV-IIIagm" ikke at inducere en AlDS-type sygdom hos. afrikanske grønne aber.Another retrovirus designated "STLV-IIIagm" or "SIVagm" was isolated from wild 10 green monkeys. In contrast to the virus found in macaq rhesus monkeys, the presence of "STLV-IIIagm" does not appear to induce an AlDS type of disease. African green monkeys.
Det immunologiske slægtsskab mellem de strukturelle proteiner og glycopro-15 teiner fra HIV-2 på den ene side og STLV-lllmac og STLV-IIIagm retrovira på den anden side og som et konsekvens heraf slægtsskabet mellem deres nu-kleinsyresekvenser forbliver imidlertid begrænset. Forsøg har muliggjort en første skelnen mellem de retrovira, der er i stand til at inficere mennesker eller aber, hvor følgende er blevet klarlagt: 20 HIV-2 virus formerer sig ikke på kronisk måde i lymphocyter fra rhesusaber, dersom det er blevet injiceret in vivo og under arbejdsbetingelser, der muliggør udvikling af STLV-lllmac vira således som beskrevet af N. L. Letvin et al., Science (1985), vol. 230, 71-75.However, the immunological relationship between the structural proteins and glycoproteins from HIV-2 on the one hand and STLV-IIImac and STLV-IIIagm retroviruses on the other, and as a consequence, the relationship between their nucleic acid sequences remains limited. Trials have allowed a first distinction between the retroviruses capable of infecting humans or monkeys where the following has been elucidated: 20 HIV-2 virus does not multiply chronically in lymphocytes from rhesus monkeys if injected in vivo and under operating conditions that allow the development of STLV-IIImac viruses as described by NL Letvin et al., Science (1985), Vol. 230, 71-75.
2525
Denne manglende evne af HIV-2 til af udvikle sig hos aber under naturlige betingelser muliggør på den anden side at HIV-2 vira og STLV-lll virusisola-ter er biologisk differentiel.This inability of HIV-2 to develop in monkeys under natural conditions, on the other hand, allows HIV-2 viruses and STLV-III virus isolates to be biologically differential.
30 Under anvendelse af samme metoder som nævnt ovenfor viste det sig, at det også var muligt at opnå følgende fra STLV-lll: 25 DK 175959 B1 et hoved p27 kemeprotein, der havde en molekylvægt af størrelsesordenen 27 kd, et hovedomhylningsglycoprotein, gp140, 5 et p32 protein, sandsynligvis transmembran, der Ikke observeres IRIPA, dersom viruset er forud mærket med [35S]cystein, men som kan ses ved immu-noaftryksforsøg (Western blot) i form af brede bånd.Using the same methods as mentioned above, it was found that it was also possible to obtain the following from STLV-III: a major p27 nuclear protein having a molecular weight of the order of 27 kd, a major envelope glycoprotein, gp140, 5 a p32 protein, probably transmembrane, that IRIPA is not observed if the virus is pre-labeled with [35S] cysteine, but which can be seen by immunoassay experiments (Western blot) in the form of broad bands.
10 Hovedomhylningsglycoproteinet af HIV-2 har vist sig at være immunologisk nærmere ved hovedomhylningsglycoproteinet af STLV-lll end ved hovedomhylningsglycoproteinet af HIV-1.10 The main envelope glycoprotein of HIV-2 has been found to be immunologically closer to the main envelope glycoprotein of STLV-III than to the envelope glycoprotein of HIV-1.
Disse fund gælder ikke alene med hensyn til molekylvægtene, 130-140 kd for 15 hovedglycoproteineme af HIV-2 og STLVrlll i forhold til omtrent 110 for hovedomhylningsglycoproteinet af HIV-1, men også med hensyn til de immu-nolgiske egenskaber, da sera udtaget fra patienter inficeret med HIV-2 og mere specielt antistoffer dannet over for HIV-2 gp140 genkender STLV-lll gp140 medens, i sammenlignende forsøg, de samme sera og de samme an-20 tistoffer over for HIV-2 ikke genkender HIV-1 gp110. Imidlertid genkender anti-HIV-2 ikke HIV-1 gp110. Anti-HIV-1-sera, der aldrig har reageret med HIV-2 gp140, udfælder imidlertid et [35S]cysteinmærket 26 kd protein, der findes i ekstrakter af HIV-2.These findings apply not only to the molecular weights, 130-140 kd, for the 15 major glycoproteins of HIV-2 and STLVr11l relative to about 110 for the major envelope glycoprotein of HIV-1, but also with respect to the immunologic properties, since sera taken from patients infected with HIV-2 and more specifically antibodies to HIV-2 gp140 recognize STLV-III gp140 while, in comparative trials, the same sera and antibodies to HIV-2 do not recognize HIV-1 gp110. However, anti-HIV-2 does not recognize HIV-1 gp110. However, anti-HIV-1 sera that have never reacted with HIV-2 gp140 precipitate a [35S] cysteine-labeled 26 kd protein found in extracts of HIV-2.
25 Hovedkerneproteinet af HIV-2 synes at have en gennemsnitsmolekylvægt (omtrent 26.000) midt imellem molekylvægten af HIV-1 p25 og p27 af STLV- lll.The major core protein of HIV-2 appears to have an average molecular weight (approximately 26,000) between the molecular weights of HIV-1 p25 and p27 of STLV-III.
Disse observationer stammer fra forsøg udført med virale ekstrakter opnået 30 fra HIV-2 isoleret fra en af de ovennævnte patienter. Lignende resultater er opnået med virale ekstrakter af HIV-2 isoleret fra en anden patient.These observations stem from trials conducted with viral extracts obtained from HIV-2 isolated from one of the above patients. Similar results have been obtained with viral extracts of HIV-2 isolated from another patient.
26 DK 175959 B126 DK 175959 B1
Celler Inficeret henholdsvis med HIV-1, HIV-2 og STLV-III inkuberedes i et substrat indeholdende 200 pCi/ml [35S]cystein i et substrat μden umærket cystein i 16 timer. De klarede supernatante dele centrifugeredes ved 60.000 . g i 90 min. Pellen lyseredes i en RIPA puffer (1), immunopræcipiteredes med 5 forskellige sera og underkastedes herefter elektrophorese på polyacrylamid-gel tilsat natriumdodecylsulfat (SDS-PAGE).Cells infected with HIV-1, HIV-2, and STLV-III, respectively, were incubated in a substrate containing 200 µCi / ml [35 S] cysteine in a substrate of unlabeled cysteine for 16 hours. The clarified supernatant portions were centrifuged at 60,000. g for 90 min. The pellet was lysed in a RIPA buffer (1), immunoprecipitated with 5 different sera and then subjected to electrophoresis on polyacrylamide gel with sodium dodecyl sulfate (SDS-PAGE).
Resultaterne er angivet i 1a, 1b og 1c.The results are given in 1a, 1b and 1c.
10 Fig. 1a viser de observerede resultater af immunopræcipitation mellem et viral ekstrakt af HIV-1 opnået fra en OEM C1.13 cellelinie og henholdsvis følgende sera: anti-HIV-1-positiv serum (bånd 1), 15 serum opnået fra den først omtalte patient (bånd 2), serum fra en rask afrikansk bærer af anti-HIV-1-antistoffer (bånd 3), 20 serum fra en macaq-abe inficeret med STLV-III (bånd 4), og serum fra den anden omtalte patient (bånd 5).FIG. Figure 1a shows the observed immunoprecipitation results between a viral extract of HIV-1 obtained from an OEM C1.13 cell line and the following sera, respectively: anti-HIV-1 positive serum (lane 1), serum obtained from the first mentioned patient ( band 2), serum from a healthy African carrier of anti-HIV-1 antibodies (band 3), 20 serum from a macaq monkey infected with STLV-III (band 4), and serum from the other patient mentioned (band 5) ).
I fig. 1b er optegnet de observerede resultater af immunopræcipitation mel-25 lem HIV-2 antigenerne opnået fra den første patient efter forudgående dyrkning med HUT-78 celler, og forskellige sera, mere specielt serumet fra førstnævnte patient (bånd 1), det anti-HIV-1-positive serum (bånd 2), serumet fra macaq-aben inficeret med STLV-III (bånd 3) og serumet fra ovennævnte anden patient (bånd 4).In FIG. Figure 1b shows the observed results of immunoprecipitation between the HIV-2 antigens obtained from the first patient after prior culture with HUT-78 cells, and various sera, more particularly the serum of the first patient (band 1), the anti-HIV virus. 1 positive serum (band 2), the macaq monkey infected with STLV-III (band 3) and the serum of the above second patient (band 4).
Til slut viser fig. 1c de observerede resultater for immunopræcipitation mellem antigenerne af et STLV-III isolat opnået fra en macaq-abe med en abe 30 27 DK 175959 B1 AIDS. Det anvendte sera, hvortil båndene 1-5 henfører, er de samme som de, der er omtalt i forbindelse med fig. 1a.Finally, FIG. 1c the observed results for immunoprecipitation between the antigens of an STLV-III isolate obtained from a macaq monkey with a monkey 30 AIDS. The sera used to which the bands 1-5 refer are the same as those mentioned in connection with FIGS. 1a.
M betyder markørerne myosin (200 kd), galactosidase (130 kd), bovint se-5 rumalbumin (69 kd), phosphorylase B (93 kd), ovalbumin (46 kd) og carbon-syreanhydrase (30 kd).M means the markers myosin (200 kd), galactosidase (130 kd), bovine serum albumin (69 kd), phosphorylase B (93 kd), ovalbumin (46 kd) and hydrochloric acid anhydrase (30 kd).
Fig. 2a og 2b viser lignende resultater for de elektrophoretiske vandringsegenskaber af proteinerne fra HIV-1, HIV-2 og STLV-III.FIG. 2a and 2b show similar results for the electrophoretic migration properties of the proteins from HIV-1, HIV-2 and STLV-III.
1010
Fig. 2a angår forsøgene udført med ekstrakter af virus mærket med [35S]cystein efter immunopræcipitation på SDS-PAGE. De forskellige bånd henfører til følgende virusekstrakter: virus opnået fra patient 1 og immuno-præcipiteret med serum stammende fra samme patient (bånd 1), ekstrakt af 15 det samme virus immunopræcipiteret med et negativt kontrolserum stam- i mende fra en person, der ikke bærer anti-HIV-1 eller anti-HIV-2-antistoffer (bånd 2), ekstrakt af STLV-III virus immunopræcipiteret med et serum, der stammer fra en macaq-abe inficeret med STLV-III (bånd 3), immunopræcipitation observeret mellem ekstrakter af det samme virus og et negativt kon-20 trolserum (bånd 4), og ekstrakt af HIV-1 immunopræcipiteret med serumet fra en europæisk patient inficeret med AIDS.FIG. 2a relates to the experiments performed with extracts of virus labeled with [35S] cysteine after immunoprecipitation on SDS-PAGE. The different bands refer to the following virus extracts: virus obtained from patient 1 and immunoprecipitated with serum derived from the same patient (band 1), extract of the same virus immunoprecipitated with a negative control serum derived from a person not carrying anti-HIV-1 or anti-HIV-2 antibodies (band 2), extract of STLV-III virus immunoprecipitated with a serum derived from a macaq monkey infected with STLV-III (band 3), immunoprecipitation observed between extracts of the same virus and a negative control serum (band 4), and extract of HIV-1 immunoprecipitated with the serum of a European patient infected with AIDS.
Fig. 1b viser resultater opnået ved Western blot (immuno-aftryk) forsøg. Cel-lelysater stammende fra uinficerede eller inficerede HUT-78 celler underka-25 stedes elektrophorese på SDS-PAGE og overførtes herefter elektrophoretisk til et nitrocellulosefilter, inden der omsattes med serumet fra ovennævnte første patient (serum fortyndet 1/100). Nitrocellulosefilteret vaskedes herefter og tilstedeværelse af de bundne antistoffer visualiseredes med 125l-mærket gede anti-humant IgG.FIG. Figure 1b shows results obtained by Western blot (immunoprint) experiments. Cell lysates derived from uninfected or infected HUT-78 cells were subjected to electrophoresis on SDS-PAGE and then electrophoretically transferred to a nitrocellulose filter before reacting with the serum of the above first patient (serum diluted 1/100). The nitrocellulose filter was then washed and the presence of the bound antibodies visualized with 125 I-labeled goat anti-human IgG.
3030
Pletterne observeret i båndene 1, 2 og 3 angår henholdsvis agglutinations-forsøgene mellem det ovennævnte serum og ekstrakterne af uinficerede HUT-78 celler (bånd 1), ekstrakter af HUT-78 celler inficeret med et HIV-2 28 DK 175959 B1 virus (bånd 2) og ekstrakter af HUT-78 celler inficeret med STLV-III (bånd 3). Tallene, der forekommer i margenen ved sidene af båndene, svarer til de omtrentlige molekylvægte af de mest repræsentative virale proteiner (molekylvægte i kilodalton).The spots observed in bands 1, 2 and 3 relate to the agglutination assays between the above serum and the extracts of uninfected HUT-78 cells (band 1), respectively, extracts of HUT-78 cells infected with an HIV-2 virus (band 1). 2) and extracts of HUT-78 cells infected with STLV-III (band 3). The numbers appearing in the margin at the sides of the bands correspond to the approximate molecular weights of the most representative viral proteins (molecular weights in kilodaltons).
5 II - Nukleinsyrer 1/ RNA fra HIV-2 retrovirus 10 RNA af viruset aflejret på et filter således som det foretages ved "spot blot" metoden hybridicerede ikke under kraftige betingelser med DNA fra HIV-1.II - Nucleic Acids 1 / RNA from HIV-2 Retrovirus 10 The RNA of the virus deposited on a filter as performed by the spot blot method did not hybridize under strong conditions with HIV from HIV-1.
Ved "kraftige betingelser" skal forstås de betingelser ved hvilke hybridise-ringsreaktionen mellem RNA fra HIV-2 og den udvalgte probe, radioaktivt 15 mærket med 32P (eller mærket på anden måde) efterfulgt af vask af prøven udføres. Hybridiseringen, på en membran, udføres ved 42 °C i nærvær af en vandig opløsning specielt af 50% formamid (rumfang/rumfang) i 0,1% SDS/5X SSC i 18 timer. Membranen, hvorpå hybridiseringsreaktionen udføres, vaskes herefter ved 65 °C i en puffer indeholdende 0,15% SDS og 0,1 X 20 SSC.By "strong conditions" is meant the conditions under which the hybridization reaction between RNA from HIV-2 and the selected probe, radioactively labeled with 32 P (or otherwise labeled) followed by washing the sample is carried out. The hybridization, on a membrane, is performed at 42 ° C in the presence of an aqueous solution especially of 50% formamide (vol / vol) in 0.1% SDS / 5X SSC for 18 hours. The membrane on which the hybridization reaction is carried out is then washed at 65 ° C in a buffer containing 0.15% SDS and 0.1 X 20 SSC.
Ved "ikke-kraftige betingelser" skal forstås de betingelser ved hvilke hybridiseringen og vasken udføres. Hybridiseringen udføres ved kontakt med den den udvalgte probe, mærket med (eller mærket på anden måde) nemlig 25 ved 42 °C i en 5 x SSC puffer, 0,1% SDS indeholdende 30% formamid i 18 timer. Vask af membranen udføres ved 50 °C med en puffer indeholdende 0,1% SDS og 2 x SSC.By "non-vigorous conditions" is meant the conditions under which the hybridization and washing are performed. The hybridization is carried out by contact with the selected probe, labeled (or otherwise labeled), namely 25 at 42 ° C in a 5 x SSC buffer, 0.1% SDS containing 30% formamide for 18 hours. Washing of the membrane is performed at 50 ° C with a buffer containing 0.1% SDS and 2 x SSC.
Hybridiseringsforsøgene udførtes også med en hybridiseringsprobe beståen-30 de af et rekombinant plasmid pBT 1 opnået ved kloning af DNA fra HIV-1 stammende fra AJ19 (Cell 1985, vol. 40, p. 9) i vektoren pUC18. Under ikke-kraftige betingelser observeredes kun meget svag hybridisering mellem RNA’en fra HIV-2 og det klonede DNA stammende fra HIV-1.The hybridization experiments were also performed with a hybridization probe consisting of a recombinant plasmid pBT 1 obtained by cloning HIV-1 DNA derived from AJ19 (Cell 1985, vol. 40, p. 9) in the vector pUC18. Under non-potent conditions, only very weak hybridization was observed between the HIV-2 RNA and the cloned DNA derived from HIV-1.
29 DK 175959 B129 DK 175959 B1
Andre prober indeholdende klonede sekvenser af HIV-1 anvendtes: \ a/ enkeltstrengede prober fra subgenomt DNA fra HIV-1 produceret fra sub-5 kloner af HIV-1-genomet og indført i fag M13. De klonede regioner står i forbindelse med proteasegenet eller "endonuclease" genet.Other probes containing cloned sequences of HIV-1 were used: µ / single-stranded probes from HIV-1 sub-genome DNA produced from sub-5 clones of the HIV-1 genome and introduced into phage M13. The cloned regions are associated with the protease gene or "endonuclease" gene.
Kun en probe fra endonuleaseregionen af HIV-1 (nucleotidsekvensen mellem base nr. 3760 og 4130) gav svag hybridisering under ikke-kraftige betingelser 10 med HIV-2. "Protease" proben (HIV-1 nucleotidsekvensen mellem baserne nr. 1680 og 1804) hybridiserede ikke selv under ikke-kraftige betingelser med HIV-2.Only one probe from the endonulease region of HIV-1 (nucleotide sequence between base nos. 3760 and 4130) gave weak hybridization under non-potent conditions 10 with HIV-2. The "protease" probe (HIV-1 nucleotide sequence between bases # 1680 and 1804) did not hybridize even under non-potent conditions with HIV-2.
b/ En probe pRS3 bestående af sekvensen, der koder for "omhylning" regio-15 nen af HIV-1 (subkloning i pUC18) hybridiserede ikke under ikke-kraftige betingelser med HIV-2.b / A probe pRS3 consisting of the sequence encoding the "envelope" region of HIV-1 (subcloning in pUC18) did not hybridize under non-potent conditions with HIV-2.
"Spot blot" teknikken er også betegnet "dot blot" (overførsel ved hjælp af pletter).The "spot blot" technique is also referred to as "dot blot" (stain transfer).
2020
Yderligere hybridiseringsresultater mellem genome RNA fra HIV-1, HIV-2 og STLV-III på den ene side og prober indeholdende forskellige subgenome sekvenser af HIV-1 virus på den anden side er vist i fig. 3.Further hybridization results between genomic RNAs of HIV-1, HIV-2 and STLV-III, on the one hand, and probes containing different subgenome sequences of HIV-1 virus, on the other hand, are shown in Figs. Third
25 De supematante dele af de forskellige dyrkningssubstrater (i mængder på 0,5 til 1 ml pr. plet) centrifugeredes i 5 minutter ved 45.000 omdrejninger pr. minut; pellen blev genopslæmmet i en NTE puffer indeholdende 0,1% SDS og anbragtes på et nitrocellulosefilter. Dette var forud dyppet i et 2 x SSC substrat (0,3 M NaCI, 0,03 M natriumnitrat). Efter bagning (i 2 timer ved 80 30 °C) hybridiseredes filtrene med forskellige prober indeholdende genome sub- fragmenter af HIV-1 under ikke-kraftige betingelser (30% formamid, 5 x SSC,The supernatant portions of the various culture substrates (in amounts of 0.5 to 1 ml per spot) were centrifuged for 5 minutes at 45,000 rpm. minute; the pellet was resuspended in an NTE buffer containing 0.1% SDS and placed on a nitrocellulose filter. This was pre-dipped in a 2 x SSC substrate (0.3 M NaCl, 0.03 M sodium nitrate). After baking (for 2 hours at 80 ° C), the filters were hybridized with various probes containing genomic sub-fragments of HIV-1 under non-vigorous conditions (30% formamide, 5 x SSC,
40 °C), vaskedes ved 50 °C med 2 x SSC opløsning indeholdende 0,1% SDS40 ° C), washed at 50 ° C with 2 x SSC solution containing 0.1% SDS
30 DK 175959 B1 og autoradiograferedes herefter i 48 timer ved -70 °C med en forstørrelsesskærm.And was then radiographed for 48 hours at -70 ° C with a magnifying screen.
Proberne 1-4 er enkeltstrengede probér opnået ved "prime cut" metoden, 5 således som beskrevet i (25). Kort fortalt ligeredes de enkeltstrengede fragmenter stammende fra M13 viruset og indeholdende subgenome HIV-1 indskud (30) til oligomere fragmenter (17 nucleotider) stammende fra M13 (Biolabs). Den komplementære streng syntetiseredes herefter med Klenow enzym i en TM puffer (10 mM Tris, pH 7,5, 10 mMMgCI2) i nærværelse af 10 dATP, dGTP, dTTP og dCTB mærket med 32p ved α-stillingen (Amersham, 3000 Ci/mmol). Herefter blev DNA nedbrudt med de passende restriktionsenzymer, varmedenatureret og underkastet elektrophorese på en denaturerende polyacrylamidgel (indeholdende 6% acrylamid, 8 M urinstof i en TDE puffer). Herefter autoradiograferedes gelen i 5 minutter. Proben blev 15 herefter afskåret og elueret i en 300 mmol NaCI, 0,1% SDS puffer. Specifik aktivitet (SA) af de enkeltstrengede prober bestemtes ved 5 x 108-109 henfald pr. minut/mikrogram (dpm/pg).Probes 1-4 are single-stranded probes obtained by the prime cut method, as described in (25). Briefly, the single-stranded fragments derived from the M13 virus and containing subgenome HIV-1 insert (30) were ligated to oligomeric fragments (17 nucleotides) derived from M13 (Biolabs). The complementary strand was then synthesized with Klenow enzyme in a TM buffer (10 mM Tris, pH 7.5, 10 mMMgCl 2) in the presence of 10 dATP, dGTP, dTTP and dCTB labeled with 32p at the α position (Amersham, 3000 Ci / mmol ). Then, the DNA was digested with the appropriate restriction enzymes, heat-denatured, and subjected to electrophoresis on a denaturing polyacrylamide gel (containing 6% acrylamide, 8 M urea in a TDE buffer). The gel was then autoradiographed for 5 minutes. The probe was then cut off and eluted in a 300 mmol NaCl, 0.1% SDS buffer. Specific activity (SA) of the single-stranded probes was determined at 5 x 108-109 decays per minute / microgram (ppm / pg).
De karakteristiske sekvenser til stede i de forskellige prober var følgende: 20The characteristic sequences present in the various probes were as follows:
Probe 1 .nucleotider 990-1070,Probe 1. Nucleotides 990-1070,
Probe 2 : nucleotider 980-1260,Probe 2: Nucleotides 980-1260,
Probe 3 : nucleotider 2170-2240,Probe 3: Nucleotides 2170-2240,
Probe 4 : nucleotider 3370-3640.Probe 4: Nucleotides 3370-3640.
2525
Numrene på de ovennævnte nucleotider er de, der er anført under litteraturhenvisningen (30).The numbers of the above nucleotides are those listed in the literature reference (30).
Endelig består probe 5 af et plasmid pUC18 der bærer EcoR1-Sac1 fragmen- 30 tet af HIV-klonen i AJ19 (31), der underkastedes nicktranslation til opnåelse af en SA på omtrent 108 dpm/pg.Finally, probe 5 consists of a plasmid pUC18 carrying the EcoR1-Sac1 fragment of the HIV clone in AJ19 (31), which was subjected to nick translation to obtain an SA of approximately 108 ppm / pg.
31 DK 175959 B131 DK 175959 B1
De relative beliggenheder af de subgenome fragmenter i probeme med hensyn til hele HIV-1 genomet er angivet skematisk i fig. 3. De forskellige pletter svarer henholdsvis til følgende: 5 Plet A: et virus er opnået fra en kultur af CEM C1.13 celler inficeret med HIV-1,The relative locations of the subgenome fragments in the probes with respect to the entire HIV-1 genome are indicated schematically in FIG. 3. The different spots correspond to the following, respectively: 5 Spot A: a virus is obtained from a culture of CEM C1.13 cells infected with HIV-1,
Plet B: et virus er opnået fra HUT-78 celler inficeret med STLV-III, 10 Plet C og D: isolater opnået henholdvis fra vira af de ovennævnte to afrikanske patienter,Stain B: a virus obtained from HUT-78 cells infected with STLV-III, Stain C and D: isolates obtained respectively from viruses by the aforementioned two African patients,
Plet E: negativ kontrolcelleekstrakt opnået fra uinficerede HUT-78 celler, 15 Plet F: virus opnået fra en patient fra Zaire der lider af AIDS, der er dyrket i normale T lymphocyter i nærvær af TCGF.Stain E: negative control cell extract obtained from uninfected HUT-78 cells, Stain F: virus obtained from a Zaire patient suffering from AIDS grown in normal T lymphocytes in the presence of TCGF.
Alle pletterne opnåedes med en mængde virus svarende til 25.000 dpm omvendt transcriptaseaktivitet, bortset fra plet C: 15.000 dpm.All of the spots were obtained with an amount of virus corresponding to 25,000 ppm reverse transcriptase activity, except spot C: 15,000 ppm.
20 Følgende observationer blev foretaget:The following observations were made:
De genome RNA fra de to HIV-2 isolater opnået fra rensede virale partikler hybridiserede ikke med nogen af proberne under de ovenfor beskrevne kraf-25 tige betingelser, skønt de virale partikler isoleredes og rensedes fra dyrk-ningssupematantdelene af højt inficerede celler, der viste tegn på høj omvendt transcriptaseaktivitet.The genomic RNA of the two HIV-2 isolates obtained from purified viral particles did not hybridize with any of the probes under the above-described powerful conditions, although the viral particles were isolated and purified from the culture supernatant portions of highly infected cells showing signs on high reverse transcriptase activity.
Under de ovenfor beskrevne milde betingelser blev følgende observationer 30 gjort: Alle probeme hybridiserede kraftigt med de genome RNA opnået fra kontrol HIV-1 præparaterne og fra et andet isolat opnået fra en patient fra Zaire, der lider af AIDS.Under the mild conditions described above, the following observations were made: All probes hybridized strongly with the genomic RNA obtained from the control HIV-1 preparations and from another isolate obtained from a Zaire patient suffering from AIDS.
32 DK 175959 B1 !32 DK 175959 B1!
To af de opnåede probér (nucleotider 990-1070 og 990-1260, der begge stammede fra gag regionen af HIV-1) hybridiserede lidt med pletterne fra ekstrakter af HIV-2 retrovira; kun en af disse to probér (nucleotider 990-1260) viste også ringe hybridisering med STLV-III pletten (fig. 3). Hvad angår pro-5 ben indeholdende et fragment af pol regionen (nucleotideme 2170-2240) observeredes hybridisering med STLV-III og, skønt betydelig svagere med RNA af HIV-2. Den anden probe af pol regionen (nucleotider 3370-3640) hybridiserede ikke med nogen af HIV-2 og STLV-III pletterne.Two of the probes obtained (nucleotides 990-1070 and 990-1260, both originating from the gag region of HIV-1) hybridized slightly with the spots from extracts of HIV-2 retroviruses; only one of these two probes (nucleotides 990-1260) also showed poor hybridization with the STLV-III stain (Fig. 3). As for probes containing a fragment of the pol region (nucleotides 2170-2240), hybridization with STLV-III was observed and, although significantly weaker with HIV-2 RNA. The second probe of the pol region (nucleotides 3370-3640) did not hybridize with any of the HIV-2 and STLV-III spots.
10 Endelig hybridiserede proben modificeret ved nicktranslation og indeholdende hele env genet og LTR (nucleotideme 5290-9130) af HIV-2 ikke hverken med RNA af STLV-III eller med RNA af HIV-2.Finally, the probe modified by nick translation and containing the entire env gene and LTR (nucleotides 5290-9130) of HIV-2 did not hybridize with either RNA of STLV-III or with RNA of HIV-2.
Det skal også bemærkes, at en anden probe, der ved 5-enden af pol aflæs-15 ningsrammen af HIV-1 (svarende til proteaseregionen) ikke hybridiserede hverken med RNA af HIV-2 eller med RNA af STLV-III.It should also be noted that another probe that at the 5-end of the pole reading frame of HIV-1 (corresponding to the protease region) did not hybridize with either RNA of HIV-2 or with RNA of STLV-III.
Det fremgår således også af det ovenstående, at HIV-2 viruset synes at være fjernere, fra et strukturelt standpunkt, fra HIV-1 viruset, end det er fra STLV-20 III. HIV-2 er imidlertid signifikant forskellig fra STLV-III, hvilket viser sig i de forskellige resultater opnået med hensyn til de infektiøse evner af HIV-2 vira, der er bogstavelig talt nul i aber, i forhold til de klare infektiøse egenskaber af STLV-III vira i de samme abearter.Thus, it is also apparent from the above that the HIV-2 virus appears to be more distant, from a structural standpoint, from the HIV-1 virus than it is from the STLV-20 III. However, HIV-2 is significantly different from STLV-III, as evidenced by the different results obtained with respect to the infectious ability of HIV-2 viruses, which are literally zero in monkeys, relative to the clear infectious properties of STLV III viruses in the same monkeys.
25 Restriktionskortene og de genome RNA sekvenser af HIV-2 eller af cDNA opnået fra disse genome RNA'er er tilgængelige for fagmanden, da stammerne af HIV-2 deponeret i CNCM efter passende multiplicering kan udstyre fagmanden med det genetiske udstyr, der er nødvendig for bestemmelsen af disse restriktionskort og nucleotidsekvenser. De betingelser, under hvilke 30 restriktionskortet af genomet af den ene af de omhandlede HIV-2 isolater blev fastlagt og betingelserne under hvilke visse dele af cDNA stammende fra disse genomer blev sekvensbed ømt, er beskrevet nedenfor.The restriction maps and genomic RNA sequences of HIV-2 or of cDNA obtained from these genomic RNAs are available to those skilled in the art, as the strains of HIV-2 deposited in the CNCM, after appropriate multiplication, can equip the skilled person with the genetic equipment necessary for the determination of these restriction maps and nucleotide sequences. The conditions under which the restriction map of the genome of one of the subject HIV-2 isolates were determined and the conditions under which certain parts of the cDNA derived from these genomes were sequenced are described below.
33 DK 175959 B133 DK 175959 B1
Restriktionskortet af genomet af et retrovirus, der er repræsentativ for HIV-2 retrovira, er angivet i fig. 4. Restriktionskortet af et væsentligt fragment af dette cDNA er vist i fig. 5. Endelig er en del af dette sidstnævnte fragment blevet sekvensbedømt.The restriction map of the genome of a retrovirus representative of HIV-2 retroviruses is given in FIG. 4. The restriction map of a substantial fragment of this cDNA is shown in FIG. 5. Finally, part of this latter fragment has been sequenced.
55
Denne sekvens og et antal af restriktionspladseme, som den indeholder, er angivet i fig. 6. Det klonede hele cDNA - eller klonede fragmenter af dette cDNA-, kan i sig selv anvendes som specifikke hybridiseringsprober.This sequence and a number of the restriction sites it contains are given in FIG. 6. The cloned whole cDNA - or cloned fragments of this cDNA - can itself be used as specific hybridization probes.
10 2/ cDNA og fragmenter af dette cDNA stammer fra RNA af HIV-210 2 / cDNA and fragments of this cDNA are derived from the RNA of HIV-2
De betingelser, under hvilket ovenfor beskrevne cDNA blev opnået, er beskrevet nedenfor.The conditions under which the cDNA described above was obtained are described below.
15 Første trin i fremstillingen af dette cDNA omfattede produktionen af et oll·· go(dT), der tjener som en primer eller en initiator cDNA streng, ved at udføre en endogen reaktion aktiveret med et detergent, under anvendelse af den omvendte transcriptase af HIV-2 på rensede virioner opnået fra de superna-tante dele af inficerede CEM-celler. CEM-cellelinien var en lymphoblastoid 20 CD4 + cellelinien beskrevet af G. E. Goley et al. i Cancer 18: 522-529 (1965), som der herved henvises til. Disse anvendte CEM celler inficeres med et ROD isolat, der havde vist sig at frembringe væsentlige mængder HIV-2 kontinuert.The first step in the preparation of this cDNA involved the production of an oll · go (dT) serving as a primer or an initiator cDNA strand, by performing an endogenous reaction activated with a detergent, using the reverse transcriptase of HIV. -2 on purified virions obtained from the supernatant portions of infected CEM cells. The CEM cell line was a lymphoblastoid 20 CD4 + cell line described by G. E. Goley et al. in Cancer 18: 522-529 (1965), to which reference is hereby made. These CEM cells used were infected with a ROD isolate that was found to produce significant amounts of HIV-2 continuously.
25 Efter syntesen af den anden streng (i nærværelse af nucleotider og en bakteriel DNA polymerase) indførtes de dobbelstrengede cDNA i en bakteriel fag vektor M13 TG130. Et fagbibliotek på 104 rekombinante M13 fager opnåedes og underkastedes en in situ afsøgning med en HIV-1 probe. Denne indeholder et 1,5 kb fragment stammende fra 3-enden af cDNA stammende fra RNAAfter the synthesis of the second strand (in the presence of nucleotides and a bacterial DNA polymerase), the double-stranded cDNA was introduced into a bacterial phage vector M13 TG130. A phage library of 104 recombinant M13 phages was obtained and subjected to an in situ scan with an HIV-1 probe. This contains a 1.5 kb fragment derived from the 3-end of cDNA derived from RNA
30 af LAV isolatet (vist i fig. 7A). Omtrent 50 positive plaques afsløredes, rensedes og karakteriseredes ved krydshybridisering af indskudene og sekvensbedømmelse af enderne.30 of the LAV isolate (shown in Fig. 7A). About 50 positive plaques were revealed, purified and characterized by cross-hybridization of the deposits and sequence assessment of the ends.
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Denne fremgangsmåde muliggjorde, at forskellige kloner kunne isoleres, indeholdende sekvenser omtrent komplementære med 3-enden af det polya-denylerede RNA af LTR [forkortelse for "long terminal repeat" af HIV-1, beskrevet af S. Wain Hobson et al. i Cell 40: 9-17 (1985)] regionen, som der 5 herved henvises til.This approach allowed different clones to be isolated, containing sequences approximately complementary to the 3-end of the polyhedronylated RNA of LTR [short-term repeat of HIV-1, described by S. Wain Hobson et al. in the Cell 40: 9-17 (1985)] region to which reference is hereby made.
Det største af indskudene af gruppen af de pågældende M13 kloner, der hy-bridiserer med 3' LTR regionen af HIV-1, er en omtrentlig 2 kb klon betegnet E2. Ligesom 3’ LTR regionen af HIV-1 indeholder klonen E2 et AATAAA sig-10 nal anbragt omtrent 20 nucleotider oven for en poly(A) endestilling, og en 3' LTR region svarende til regionen af HIV-2. Efter delvis sekvensbedømmelse viste denne 3' LTR region af HIV-2 sig at være i besiddelse af et svagt slægtsskab med den homologe region af HIV-1.The largest of the deposits of the group of M13 clones in question hybridizing with the 3 'LTR region of HIV-1 is an approximately 2 kb clone designated E2. Like the 3 'LTR region of HIV-1, clone E2 contains an AATAAA signal located approximately 20 nucleotides above a poly (A) terminus, and a 3' LTR region corresponding to the region of HIV-2. After partial sequence assessment, this 3 'LTR region of HIV-2 was found to possess a weak kinship with the homologous region of HIV-1.
15 Fig. 5 er et restriktionskort af fragmentet af E2 (aflangt rektangulært areal) indarbejdet i plasmid pSPE2, der indeholder det. Det omfatter en del af R regionen og U3 regionen af HIV-2. Tegningen viser ikke grænserne af R og U3 regionerne.FIG. 5 is a restriction map of the fragment of E2 (oblong rectangular area) incorporated into plasmid pSPE2 containing it. It includes part of the R region and the U3 region of HIV-2. The drawing does not show the boundaries of the R and U3 regions.
20 Sekvensen af en del af E2 er vist I fig. 6. Stillingerne for de specifikke restriktionspladser er angivet heri. Den lille grad af slægtskab mellem 3' LTR regionerne af HIV-1 og HIV-2 er angivet i fig. 7. Faktisk kan kun omtrent 50% af nucleotiderne af de to LTR sekvenser anbringes på linie (omtrent 50% se-kvenshomologi) på bekostning af visse indskud eller deletioner. Til sammen-25 ligning hermed er sekvenshomologien af de tilsvarende områder af de forskellige amerikanske og afrikanske isolater af HIV-1 større end 95% uden indskud eiler deletion.The sequence of a portion of E2 is shown in FIG. 6. The posts for the specific restriction sites are set out herein. The small degree of kinship between the 3 'LTR regions of HIV-1 and HIV-2 is indicated in FIG. 7. In fact, only about 50% of the nucleotides of the two LTR sequences can be aligned (approximately 50% sequence homology) at the expense of certain insertions or deletions. By comparison, the sequence homology of the corresponding regions of the various US and African isolates of HIV-1 is greater than 95% without insertion or deletion.
Klonen E2 anvendtes som en specifik probe for HIV-2, til identificering på et 30 hybridiseringsfilter af sekvenserne stammende fra HIV-2 og til stede i andre kloner.The clone E2 was used as a specific probe for HIV-2, for identification on a hybridization filter of the sequences derived from HIV-2 and present in other clones.
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Denne probe afslører også det genome RNA af HIV-2 under kraftige betingelser. Den muliggør ligeledes afsløring ved hjælp af den såkaldte "Southern blot” metode på DNA fra CEM eller lignende celler inficeret med et ROD iso-lat eller med andre HIV-2 isolater. Intet signal afsløres under de samme be-5 lastningsbetingelser i forsøg med hybridisering af denne probe med cDNA stammende fra uinficerede celler eller fra celler inficeret med HIV-1. Disse resultater bekræfter den exogene natur af HIV-2 med hensyn til HIV-1. En omtrentlig 10 kb art, sandsynligvis svarende til det ikke-integrerede virale DNA, afsløredes som en principiel bestanddel i det ikke-nedbrudte DNA fra 10 celler inficeret med HIV-2. En anden DNA med en omtrentlig størrelse på 6 kb, muligvis svarende den cirkulære form af det virale DNA, afsløredes også.This probe also reveals the genomic RNA of HIV-2 under strong conditions. It also enables detection by the so-called "Southern blot" method of DNA from CEM or similar cells infected with a ROD iso- late or with other HIV-2 isolates. No signal is detected under the same loading conditions in hybridization experiments. of this probe with cDNA originating from uninfected cells or from cells infected with HIV-1. These results confirm the exogenous nature of HIV-2 with respect to HIV-1. An approximately 10 kb species, probably similar to the non-integrated viral DNA , was revealed as a principal constituent of the non-degraded DNA from 10 cells infected with HIV-2. Another DNA with an approximate size of 6 kb, possibly corresponding to the circular shape of the viral DNA, was also revealed.
De andre dele af HIV-2 genomet identificeredes også. Til dette formål opbyggedes et genombibliotek i fag AL47. Fag AL47.1 er beskrevet af W.A.M.The other parts of the HIV-2 genome were also identified. To this end, a genome library was built in subject AL47. Subjects AL47.1 are described by W.A.M.
15 Loenen et al. i Gene 10 249-259 (1980), som der herved henvises til.Loenen et al. in Gene 10, 249-259 (1980), to which reference is made.
Det genome bibliotek opbygges med fragmenter opnået ved nedbrydning af DNA stammende fra CEM cellelinien inficeret med HIV-2 ROD efter nedbrydning med enzymet Sau3AI.The genomic library is built up with fragments obtained by degradation of DNA derived from the CEM cell line infected with HIV-2 ROD after degradation with the enzyme Sau3AI.
2020
Omtrent 2 x 106 rekombinante plaques blev afsøgt in situ med en klon indeholdende det mærkede E2 indskud fra HIV-2 cDNA. 10 rekombinante fager afsløredes på plaques og rensedes. Restriktionskortene over tre af disse fager, karakteriseret ved hjælp af deres evne til "Southern blot" hybridisering 25 med E2 indskud under kraftige betingelser såvel som med subgenome probér fra HIV-1 under milde betingelser er angivet.Approximately 2 x 10 6 recombinant plaques were screened in situ with a clone containing the labeled E2 insert from HIV-2 cDNA. Ten recombinant phages were revealed on plaques and purified. The restriction maps of three of these phages, characterized by their ability to "Southern blot" hybridization with E2 insert under severe conditions as well as with subgenome probes from HIV-1 under mild conditions, are indicated.
En klone indeholdende et 9,5 kb indskud og stammende fra hele det cirkulære virale DNA indeholdende det komplette HIV-2 genom identificeredes. Den 30 betegnedes "AROD 4". De andre to kloner AROD27 og AROD35 stammende fra integrerede provira bærer LTR sekvenser af de virale kodende sekvenser og nabostillede celle DNA sekvenser. De forskellige sekvenser er angivet i fig. 8.A clone containing a 9.5 kb insert and originating from the entire circular viral DNA containing the complete HIV-2 genome was identified. The 30 was designated "AROD 4". The other two clones AROD27 and AROD35 originate from integrated proviruses carry LTR sequences of the viral coding sequences and adjacent cell DNA sequences. The various sequences are given in FIG. 8th
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Fragmenter af λ-kloneme subklonedes i plasmid vektor pUC18. Fragmenterne stammende fra AROD4, AROD27 og AROD35 og respektive subkloner i ovennævnte plasmidvektor ses også i fig. 8. Følgende subkloner blev opnå-5 et: pROD 27-5, stammende fra AROD27, indeholder en 5,2 kb region af HIV-2 genomet og nabostillede cellesekvenser (5' LTR og 5' kodende viralsekvens omkring en EcoRI plads); 10 pROD 4.8, stammende fra AROD4, indeholdende et omtrentligt 5 kb Hindlll fragment. Dette fragment svarer til den centrale del af HIV-2 genomet; pROD 27-5’ og pROD 4.8 indeholder HIV-2 indskud der overlapper hinan-15 den; pROD 4.7 indeholder et 1,8 kb Hindlll fragment af AROD4; dette fragment er anbragt i 3-retningen med hensyn til det subklonede fragment i pROD 4.8 og indeholder omtrent 0,8 kb kodende virale sekvenser og en del beliggende 20 mellem BamHI og Hindlll kloningspladseme af den venstre arm af fag λ (AL47.1).Fragments of the λ clones were subcloned into plasmid vector pUC18. The fragments originating from AROD4, AROD27 and AROD35 and respective subclones of the above plasmid vector are also seen in FIG. 8. The following subclones were obtained: pROD 27-5, derived from AROD27, contains a 5.2 kb region of the HIV-2 genome and neighboring cell sequences (5 'LTR and 5' coding viral sequence around an EcoRI site); 10 pROD 4.8, derived from AROD4, containing an approximately 5 kb HindIII fragment. This fragment corresponds to the central portion of the HIV-2 genome; pROD 27-5 'and pROD 4.8 contain HIV-2 deposits that overlap; pROD 4.7 contains a 1.8 kb HindIII fragment of AROD4; this fragment is positioned in the 3-direction with respect to the subcloned fragment of pROD 4.8 and contains approximately 0.8 kb of coding viral sequences and a portion located between the Bam HI and HindIII cloning sites of the left arm of phage λ (AL47.1).
pROD 35 indeholder alle HIV-2 kodesekvenserne i 3-retningen i forhold til EcoRI pladsen, 3’ LTR enden og omtrent 4 kb nabostillede nucleotidsekven-25 ser af cellulær oprindelse; pROD 27-5’ og pROD 35 til stede i E. coli HB 101 deponeredes den 21. november 1986 hos CNCM under betegnelsen I-626 og I-633; 30 Det komplette HIV-2 ROD genom, hvis restriktionskort er angivet i fig. 4, blev genopbygget fra pROD 35, forud lineariseret med EcoRI og pROD 27-5’.pROD 35 contains all the 3-way HIV-2 coding sequences relative to the Eco RI site, the 3 'LTR end, and approximately 4 kb neighboring nucleotide sequences of cellular origin; pROD 27-5 'and pROD 35 present in E. coli HB 101 were deposited on November 21, 1986 with CNCM under designations I-626 and I-633; 30 The complete HIV-2 ROD genome, whose restriction map is indicated in FIG. 4, was rebuilt from pROD 35, pre-linearized with EcoRI and pROD 27-5 '.
EcoRI indskuddet af pROD 27-5 ligeredes i den tilsvarende retning i EcoRI pladsen af pROD 35.The EcoRI insert of pROD 27-5 was equated in the corresponding direction in the EcoRI slot of pROD 35.
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Graden af slægtskab mellem HIV-2 og de andre humane eller aberetrovira blev undersøgt ved gensidige hybridiseringsforsøg. Den relative homologi mellem de forskellige områder af HIV-1 og HIV-2 genomeme bestemtes ved 5 hybridiseringsforsøg af fragmenter stammende henholdsvis fra klonet HIV-1 genom og fra radioaktivitetsmærket AROD4. De relative beliggenheder af disse fragmenter (nummereret fra 1-11) i forhold til HIV-1 genomet er angivet forneden på fig. 9.The degree of kinship between HIV-2 and the other human or aberetroviruses was investigated by reciprocal hybridization experiments. The relative homology between the different regions of the HIV-1 and HIV-2 genomes was determined by 5 hybridization experiments of fragments derived from the cloned HIV-1 genome and from the radioactivity label AROD4, respectively. The relative locations of these fragments (numbered from 1 to 11) relative to the HIV-1 genome are given below in FIG. 9th
10 Selv under meget milde betingelser (Tm 42 °C) hybridiserer HIV-1 og HIV-2 genomeme kun på niveauet i forhold til deres respektive gag gener (plet 1 og 2), omvendte transcriptaseregioner i pol (plet 3) og regioner af pol, Q (eller sor) gener (plet 5) og F (eller 3' orf) gener og 3' LTR (plet 11). HIV-1 fragmentet anvendt til afsløring af de første cDNA kloner af HIV-2 svarer til sub-15 klonen fra plet 11, der hybridiserer forholdsvis godt med HIV-2 under milde betingelser. Et signal stammende fra plet 5 er det eneste, der bliver tilbage efter kraftig afvaskning. Omhylningsgenet, tat området og en del af pol viser sig at være højt divergent. Disse resultater såvel som sekvensen opnået med LTR (fig. 33) viser at HIV-2 ikke er (under alle omstændigheder med hensyn 20 til dens omhylning) en variant af HIV-1.Even under very mild conditions (Tm 42 ° C), HIV-1 and HIV-2 genomes hybridize only at the level of their respective gag genes (spots 1 and 2), reverse transcriptase regions in pole (spot 3), and regions of pole , Q (or sor) genes (spot 5) and F (or 3 'orf) genes and 3' LTR (spot 11). The HIV-1 fragment used to detect the first cDNA clones of HIV-2 corresponds to the sub-clone from spot 11, which hybridizes relatively well with HIV-2 under mild conditions. A signal emanating from spot 5 is the only one that remains after heavy washing. The envelope gene, tat area and part of the pole turn out to be highly divergent. These results, as well as the sequence obtained with LTR (Fig. 33), show that HIV-2 is not (in any case with respect to its envelope) a variant of HIV-1.
Det blev observeret, at HIV-2 er nærmere beslægtet med SIV [beskrevet af M. D. Daniel et al. i Science 228: 1201-1204 (1985)], som der herved henvises til, end den er med HIV-1.It was observed that HIV-2 is more closely related to SIV [described by M. D. Daniel et al. in Science 228: 1201-1204 (1985)], which is hereby referred to as having HIV-1.
2525
Alle proteinerne fra SIV, herunder også omhylningsproteinet, immunopræci-piteres af sera fra patienter inficeret med HIV-2, medens den serologiske krydsreaktivitet af HIV-1 og HIV-2 er begrænset til kerneproteineme. Imidlertid kan SIV og HIV-2 skelnes på grund af de nævnte forskelle med hensyn til 30 molekylvægten af deres proteiner.All of the SIV proteins, including the envelope protein, are immunoprecipitated by sera from patients infected with HIV-2, whereas the serological cross-reactivity of HIV-1 and HIV-2 is limited to the core proteins. However, SIV and HIV-2 can be distinguished due to the differences mentioned with respect to the molecular weight of their proteins.
Med hensyn til nucleotidsekvenserne skal det også bemærkes, at HIV-2 er beslægtet med SIV.Regarding the nucleotide sequences, it should also be noted that HIV-2 is related to SIV.
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Herudover gør karakteriseringen af HIV-2 det også muligt at afmærke regio-! nen for omhylningsglycoproteinet, der er ansvarlig for at binde viruset til over- i fladen af target cellerne og den efterfølgende internalisering af viruset. Den 5 gensidige indvirkning sker via selve CD4 molekylet, og det synes, at HIV-1 og HIV-2 anvender den samme receptor.In addition, the characterization of HIV-2 also makes it possible to mark the region! the envelope glycoprotein responsible for binding the virus to the surface of the target cells and the subsequent internalization of the virus. The mutual interaction occurs via the CD4 molecule itself, and it appears that HIV-1 and HIV-2 use the same receptor.
Skønt der således er store forskelle mellem env generne af HIV-1 og 2, kan de afgrænsede homologe regioner af omhylningeme af de to former af HIV 10 betragtes som værende bestanddele af binding til en fælles receptor fra T4 lymphocyter. Disse pladser er nødvendige for at danne epitoper, der bærer immunogenisiteten af peptider, der kan anvendes til hos mennesker at fremkalde et beskyttende immunrespons over for HIV-vira.Thus, although there are large differences between the env genes of HIV-1 and 2, the delimited homologous regions of the envelopes of the two forms of HIV 10 can be considered as constituents of binding to a common T4 lymphocyte receptor. These sites are needed to form epitopes that carry the immunogenicity of peptides that can be used in humans to elicit a protective immune response to HIV viruses.
15 Fordelagtige sekvenser til at dannelse af prober ved hybridiseringsreaktioner med det genetiske materiale fra patienter, der bærer vira eller provira, specielt til afsløring af tilstedeværelsen af HIV-2 virus RNA i deres lymphocyter, indeholder en nucleotidsekvens, der er et resultat af kombinationen af 5 kb Hindi 11 fragmenter af ROD4 og E2 cDNA. Forsøgene kan udføres ved alle 20 metoder, specielt ved hjælp af "Northern blot", "Southern blot" og "dot blot" teknikker.Advantageous sequences for generating probes in hybridization reactions with the genetic material of patients carrying viruses or proviruses, specifically for detecting the presence of HIV-2 virus RNA in their lymphocytes, contain a nucleotide sequence resulting from the combination of 5 kb Hindi 11 fragments of ROD4 and E2 cDNA. The tests can be performed by all 20 methods, especially by "Northern blot", "Southern blot" and "dot blot" techniques.
Yderligere beskrivelser af opfindelsen vil fremgå af de efterfølgende eksempler til identifikation af visse dele af det retrovirale genom og af fremstillingen 25 af et antal rekombinante DNA, der indeholder forskellige dele af et cDNA, der stammer fra det retrovirale genom af HIV-2.Further descriptions of the invention will be apparent from the following examples for the identification of certain portions of the retroviral genome and of the preparation of a number of recombinant DNAs containing various portions of a cDNA derived from the retroviral genome of HIV-2.
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EKSEMPLEREXAMPLES
EKSEMPEL IEXAMPLE I
5 DNA probe til anvendelse i diaanoseudstvr for HIV-25 DNA probes for use in diaanosis equipment for HIV-2
Et cDNA komplementært til det genome RNA opnået fra rensede virioner fremstilledes på følgende måde: 10 Den supematante del opnået efter 48 timers dyrkning af CEM celler inficeret • med et HIV-2 ROD isolat af HIV-2 ultracentrifugeredes. Centrifugepellen in deholdende virionen centrifugeredes på en sacchanosegradient til dannelse af en ny centrifugepelle, i det væsentlige således som beskrevet i europæisk patentansøgning nr. 84/401.234 - 0 138 667, der allerede er omtalt.A cDNA complementary to the genomic RNA obtained from purified virions was prepared as follows: The supernatant portion obtained after 48 hours of culture of CEM cells infected with an HIV-2 ROD isolate of HIV-2 was ultracentrifuged. The centrifuge pellet containing the virion was centrifuged on a sucrose gradient to form a new centrifuge pellet, essentially as described in European Patent Application No. 84 / 401,234 - 0 138 667 already disclosed.
1515
Det rensede HIV-2 præparat anvendtes til syntese af cDNA, idet man anvendte en endogen reaktion aktiveret med et detergent.The purified HIV-2 preparation was used for cDNA synthesis using an endogenous reaction activated with a detergent.
Kort fortalt sattes virionpræparatet til en reaktionsblandingen indeholdende 20 50 mM Tris-HCI, 5 mM MgCfe, 10 mM DTT, 0,025% af detergentet markeds ført under navnet ’Triton” og 50 μΜ af hver af de 4 deoxynucleosidtriphos-phater og en oligo(dT) initiator. Omsætningen udførtes ved 37 °C i 90 minutter.Briefly, the virion preparation was added to a reaction mixture containing 20 50 mM Tris-HCl, 5 mM MgCfe, 10 mM DTT, 0.025% of the detergent market under the name 'Triton' and 50 μΜ of each of the 4 deoxynucleoside triphosphates and one oligo (dT ) initiator. The reaction was carried out at 37 ° C for 90 minutes.
25 Efter ekstraktion med phenol af de tilstedeværende proteiner i det første reaktionsmedium syntetiseredes den anden cDNA kæde i nærværelse af RNA-se, E. coli DNA polymerase 1 og de 4 deoxynucleotider i 1 time ved 15 °C og 1 time ved 22 °C. Der frembragtes stumpe ender på dette dobbeltstrengede cDNA ved indvirkning af T4 DNA polymerase. Alle reagenser ved denne me-30 tode er kommercielt tilgængelige (Amersham cDNA kit) og anvendtes ifølge fabrikantens anbefalinger.After extraction with phenol of the proteins present in the first reaction medium, the second cDNA chain was synthesized in the presence of RNAse, E. coli DNA polymerase 1 and the 4 deoxynucleotides for 1 hour at 15 ° C and 1 hour at 22 ° C. Blunt ends were produced on this double-stranded cDNA by the action of T4 DNA polymerase. All reagents of this method are commercially available (Amersham cDNA kit) and used according to the manufacturer's recommendations.
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Efter (1) ligation af adaptorer (linkere) indeholdende en EcoRI plads (leveret af Pharmacia) til de stumpe ender af cDNA i tilstedeværelse af en T4 DNA ligase (markedsført af Biolabs), (2), nedbrydning af disse linkere med restrik-tionsendonucleasen EcoRI, og (3) fjernelse af linkerfragmenteme ved gel-5 filtrering (gelsøjle markedsført under navnet "UltrogeO på AcA 34 (LKB-IBF) indføres cDNA i en M 13 TG 130 vektor spaltet med EcoRI. Et cDNA-bibliotek opnåedes efter transformation af E. coli stamme TG1. Der opnåedes omtrent 104 rekombinante M13 plaques.Following (1) ligation of adapters (linkers) containing an EcoRI site (provided by Pharmacia) to the blunt ends of cDNA in the presence of a T4 DNA ligase (marketed by Biolabs), (2), degrading these linkers with the restriction endonuclease EcoRI, and (3) removal of the linker fragments by gel-5 filtration (gel column marketed under the name "UltrogeO on AcA 34 (LKB-IBF) cDNA is inserted into an M13 TG130 vector digested with EcoRI. A cDNA library was obtained after transformation of E. coli strain TG1 Approximately 104 recombinant M13 plaques were obtained.
10 For i cDNA-biblioteket at selektere de rekombinante M13 kloner, der indeholder HIV-2 cDNA, anvendtes plaqueshybridiseringsmetoden. DNA fra M13 plaquene overførtes til nitrocellulosefiltre og hybridiserdes med subgenome HIV-1-prober stammende fra "AJ19" klonen af et LAV (eller HIV) virus beskrevet i den europæiske patentansøgning. Denne probe indeholdt et ind-15 skud bestående af en del med en omtrentlig længde på 1500 basepar (bp) HIV-1 DNA. Dette indskud blev bundet ved hjælp af 2 Hindlll restriktionspladser henholdsvis inden for den åbne aflæsningsramme af "env" genet og i R-segmentet af 3' LTR enden af HIV-1. Denne probe indeholdt 3' enden af env genet, hele F-genet, U3-segmentet og en del af R-segmentet af 20 LTR med en omtrentlig længde på 1500 basepar (bp).To select in the cDNA library the recombinant M13 clones containing HIV-2 cDNA, the plaque hybridization method was used. DNA from the M13 plaques were transferred to nitrocellulose filters and hybridized with subgenome HIV-1 probes derived from the "AJ19" clone of a LAV (or HIV) virus described in the European patent application. This probe contained an insert of a portion with an approximate length of 1500 base pairs (bp) of HIV-1 DNA. This insert was bound by 2 HindIII restriction sites, respectively, within the open reading frame of the "env" gene and in the R segment of the 3 'LTR end of HIV-1. This probe contained the 3 'end of the env gene, the entire F gene, the U3 segment, and a portion of the R segment of 20 LTRs with an approximate length of 1500 base pairs (bp).
Proben indeholdende 1,5 kg Hindlll indskudet mærkedes med [32P]-dCTP og -dTTP (32000 Ci x 103 mol) ved at inkubere proben i nærværelse af initiato-rer og Klenow DNA polymerase I i 4 timer ved 15 °C (under anvendelse af et 25 Amersham kit). Hybridiseringsforsøgene over cDNA klonerne fra biblioteket udførtes natten over under milde betingelser i en opløsning af et hybridise-ringssubstrat indeholdende 5 x SSC, 5 x Denhart, 25% formamid, 100 pg/ml denatureret laksesperma DNA og en mærket probe (2 x 107 cpm med en i specificitet på 109 cpm/pg) ved 37 °C. Trinnene underkastedes tre vaskebe- i j 30 handlinger efter hinanden i tilstedeværelse af tre opløsninger, hvis sammen- i sætning er angivet nedenfor:The probe containing 1.5 kg of HindIII insert was labeled with [32 P] -dCTP and -dTTP (32000 Ci x 103 mol) by incubating the probe in the presence of initiators and Klenow DNA polymerase I for 4 hours at 15 ° C (using of a 25 Amersham kit). The hybridization experiments on the cDNA clones from the library were performed overnight under mild conditions in a solution of a hybridization substrate containing 5 x SSC, 5 x Denhart, 25% formamide, 100 µg / ml denatured salmon sperm DNA and a labeled probe (2 x 107 cpm with one in specificity of 109 cpm / pg) at 37 ° C. The steps were subjected to three washing operations in 30 consecutive operations in the presence of three solutions, the composition of which is given below:
Vask nr. 1: 5 x SSC, 0,1 % SDS, ved 25 °C i 4 x 15 minutter | j j 41 DK 175959 B1Wash # 1: 5 x SSC, 0.1% SDS, at 25 ° C for 4 x 15 minutes | j j 41 DK 175959 B1
Vask nr. 2: 2 x SSC, 0,1% SDS, ved 42 °C i 2 x 30 minutterWash # 2: 2 x SSC, 0.1% SDS, at 42 ° C for 2 x 30 minutes
Vask nr. 3: 0,1 x SSC, 0,1% SDS, ved 65 °C i 2 x 30 minutter 5Wash # 3: 0.1 x SSC, 0.1% SDS, at 65 ° C for 2 x 30 minutes 5
Hver vask blev efterfulgt af autoradiografi af filtrene.Each wash was followed by autoradiography of the filters.
Adskillige positive kloner afsløredes efter vask nr. 1 og kunne stadig ses efter vask nr. 2. Imidlertid forsvandt alle signalerne efter vask nr. 3. Dette indice-10 rer, at de positive kloner kun havde svagt slægtsskab med HIV-1 genomet, der ikke desto mindre var tilstrækkelig til at udføre ovennævnte selektion. De positive kloner dyrkedes videre, anbragtes atter på plaques og hybri-diseredes med den samme probe under betingelser, der svarende til vask nr.Several positive clones were revealed after wash # 1 and could still be seen after wash # 2. However, all the signals after wash # 3 disappeared, indicating that the positive clones had only weak kinship with the HIV-1 genome, which nonetheless was sufficient to carry out the above selection. The positive clones were further cultured, placed again on plaques, and hybridized with the same probe under conditions similar to wash no.
1. De fleste af dem var stadig positive.1. Most of them were still positive.
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Klonerne anvendtes også til at udvælge en total human DNA probe under moderate betingelser og ved hybridisering i 5 x SSC, 5 x Denhart og 40% formamid efterfulgt af vask i 1 x SSC, 0,1% SDS ved 50 °C. Ingen af de tidligere positive kloner afsløredes og som en konsekvens heraf svarer ikke til 20 specifik gentaget DNA eller til cDNA fra det ribosomale RNA.The clones were also used to select a total human DNA probe under moderate conditions and by hybridization in 5 x SSC, 5 x Denhart and 40% formamide followed by washing in 1 x SSC, 0.1% SDS at 50 ° C. None of the previously positive clones were revealed and, as a consequence, do not correspond to 20 specific repeat DNA or to cDNA from the ribosomal RNA.
De positive M13 rekombinantkloner dyrkedes i et flydende substrat og karakteriseredes på følgende måde: 25 Ml Størrelse af deres indskud:The positive M13 recombinant clones were grown in a liquid substrate and characterized as follows: 25 ml Size of their insert:
En M13 enkeltstrenget type DNA opnåedes fra hver enkelt klon, og syntese af den anden streng udførtes med en M13 17-mer initiatorsekvens og Klenow enzymet. Indskudene blev udskåret under anvendelse af EcoRI (Boehringer) 30 og analyseret ved hjælp af agarosegelelektrophorese. Hoveddelen af indskudene indeholdt fra 200 til 600 og 200 bp bortset fra klonen betegnet E2.1, der havde en omtrentlig længde på 2 kbp.An M13 single-stranded type of DNA was obtained from each clone and synthesis of the other strand was performed with an M13 17-mer initiator sequence and the Klenow enzyme. The deposits were cut using Eco RI (Boehringer) 30 and analyzed by agarose gel electrophoresis. The bulk of the deposits contained from 200 to 600 and 200 bp except for the clone designated E2.1, which had an approximate length of 2 kbp.
42 DK 175959 B1 (2) Analyse af nucleotidsekvensen:42 DK 175959 B1 (2) Analysis of the nucleotide sequence:
Adskillige kloner blev delvis sekvensbedømt under anvendelse af dideoxy-metoden beskrevet af Sanger et al., i.Proc. Natl. Acad. Sci. 74:5463-7 5 (1977), som der herved henvises til. Forskellige uafhængige kloner indeholdt lignende nucleotidsekvenser bortset fra poly(A) kæderne ved deres 3' ender, hvis længde var forskellig. Disse resultater viser, at disse cDNA kloner stammede fra RNA skabelonen. Detaljeret sekvensanalyse af disse cDNA kloner, herunder 3' enden af HIV-2 genomet, viste et begrænset slægtsskab 10 medHIV-1.Several clones were partially sequenced using the dideoxy method described by Sanger et al., I.Proc. Natl. Acad. Sci. 74: 5463-7 (1977), to which reference is hereby made. Different independent clones contained similar nucleotide sequences except for the poly (A) chains at their 3 'ends, whose length was different. These results show that these cDNA clones originated from the RNA template. Detailed sequence analysis of these cDNA clones, including the 3 'end of the HIV-2 genome, showed a limited lineage 10 with HIV-1.
(3) Hvbridiserinq med det genome RNA og DNA af HIV-2: (a) Produktion af det genome RNA og HIV-2: 15(3) Hybridization with the Genome RNA and DNA of HIV-2: (a) Production of the Genome RNA and HIV-2: 15
En inficeret supernatant del centrifugeredes (50.000 omdrejninger, 30 minutter). Det aflejrede bundfald genopslæmmedes i 10 mm Tris pH 7,5, 1 mM EDTA, 0,1% SDS. En af indskudskloneme, F1.1, mærkedes og anvendtes som en probe til hybridisering med det genome RNA fra forskellige virale iso-20 later, i overensstemmelse med "dot-blot" metoden.An infected supernatant portion was centrifuged (50,000 rpm, 30 minutes). The deposited precipitate was resuspended in 10 mm Tris pH 7.5, 1 mM EDTA, 0.1% SDS. One of the insertion clones, F1.1, was labeled and used as a probe for hybridization with the genomic RNA from various viral islets, according to the "dot blot" method.
"Dot-blot" metoden omfattedes følgende trin: (i) Anbringelse af prøven (HIV-2 lysat) i pletter på en nitrocellulosemembran 25 forud vædet i 20 x SSC (3 M NaCI, 0,3 M natriumcitrat) og lufttørret, (ii) bagning af membranen i 2 timer ved 80 °C og (iii) udførsel af hybridiseringen.The "dot blot" method included the following steps: (i) Placing the sample (HIV-2 lysate) in spots on a nitrocellulose membrane 25 pre-wetted in 20 x SSC (3 M NaCl, 0.3 M sodium citrate) and air dried, (ii baking the membrane for 2 hours at 80 ° C and (iii) performing the hybridization.
Hybridiseringen udførtes under kraftige betingelser (5 x SSC, 5 x Denhart, 50% formamid ved 42 °C). Den efterfulgtes af vask i 0,1 x SSc, 0,1% SDS 30 ved 65 °C. Under disse betingelser hybridiseredes proben kraftigt til pletterne, der stammede fra to uafhængige isolater af HIV-2, herunder LAV-III ROD, hvorfra det klonede cDNA stammede. Et svagt hybridiseringssignal afsløredes med pletten dannet af STLV-III mac [abe T-lymphotropi virus (og- ! 43 DK 175959 B1 så betegnet "SIV"), type III macaque-abe] og ingen hybridisering afsløredes med HIV-1 isolater.The hybridization was carried out under strong conditions (5 x SSC, 5 x Denhart, 50% formamide at 42 ° C). It was followed by washing in 0.1 x SSc, 0.1% SDS 30 at 65 ° C. Under these conditions, the probe hybridized strongly to the stains derived from two independent isolates of HIV-2, including the LAV-III ROD from which the cloned cDNA originated. A weak hybridization signal was detected with the stain formed by STLV-III mac [monkey T-lymphotropy virus (also called "SIV"), type III macaque monkey] and no hybridization was detected with HIV-1 isolates.
"Southern blot" forsøgene under anvendelse af klonen E2.1 indeholdende 2 5 kb indskuddet som en 32P-mærket probe afslørede ikke nogen som helst hybridisering med DNA fra uinficerede celler, men afslørede bånd i frigjorte celler inficeret med HIV-2 under kraftige betingelser. HIV-2 udviste poly-morphisme på det sted af restriktionskortet, der er ækvivalent med restriktionskortet over HIV-1. Med det komplette cellulære DNA fra inficerede celler, 10 afsløres to typer signal ved hjælp af "Southern blot" metoden: (1) i DNA fraktioner med molekylvægte på omtrent 20 kb og mere i det tilfælde det drejer sig om integrerede former af viruset, og (2) i fraktioner med lavere molekylvægt (9,10 kb) når det drejer sig om viruset ikke integreret i genomet.The "Southern blot" experiments using the clone E2.1 containing the 2.5 kb insert as a 32P-labeled probe did not reveal any hybridization with DNA from uninfected cells, but revealed bands in released cells infected with HIV-2 under severe conditions. HIV-2 exhibited polymorphism at the site of the restriction card equivalent to the HIV-1 restriction card. With the complete cellular DNA from infected cells, 10 types of signal are revealed by the "Southern blot" method: (1) in DNA fractions of about 20 kb molecular weight and more in the case of integrated forms of the virus, and (2) in lower molecular weight fractions (9.10 kb) in the case of the virus not integrated into the genome.
15 Disse egenskaber er højt specifikke for et retrovirus.These properties are highly specific to a retrovirus.
Visse forsøg udført med STLV-III (SIV-3) fra inficerede celler muliggjorde at påvise, at abe-retroviruset er relativt langt fra HIV-2 (signalet afsløres udelukkende under milde betingelser). Disse forsøg viser, at ovennævnte prober 20 muliggør specifik afsløring af HIV-2.Certain experiments performed with STLV-III (SIV-3) from infected cells made it possible to demonstrate that the monkey retrovirus is relatively far from HIV-2 (the signal is revealed only under mild conditions). These experiments show that the above probes 20 enable specific detection of HIV-2.
(4) Subkloning af cDNA'et af HIV-2 i en bakteriel plasmidvektor(4) Subcloning the cDNA of HIV-2 into a bacterial plasmid vector
Den positive M13 klon, E2.1, selekteredes og subklonedes i en plasmidvek-25 tor. DNA fra den rekombinante M13 (TG130) fag E2 rensedes i form af en-keltstrenget DNA (M-13-ROD-E2) indeholdende 2 kb indskuddet indeholdende 3’ delen af HIV-2 genomet (opnået fra HIV-2 ROD). Dette indskud overførtes til et plasmid pSP65 beskrevet af Melton, D.A., i 357 Nucleic Acid Res. 12:035-8056(1984).The positive M13 clone, E2.1, was selected and subcloned into a plasmid vector. DNA from the recombinant M13 (TG130) phage E2 was purified in the form of single-stranded DNA (M-13-ROD-E2) containing the 2 kb insert containing the 3 'portion of the HIV-2 genome (obtained from HIV-2 ROD). This deposit was transferred to a plasmid pSP65 described by Melton, D.A., in 357 Nucleic Acid Res. 12: 035-8056 (1984).
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En anden kæde opbyggedes in vitro i nærværelse af den 17-mer initiatorse- ; kvens (Amersham), de fire nucleotider A, C, T, G og DNA polymerase I (Kle-now). EcoRI indskuddet blev udskåret ved hjælp af EcoRI kløvning og renset 44 DK 175959 B1 på agarosegel og herefter ligeret til pSP65, der for sit vedkommende forud var kløvet med EcoRI. Ligationsblandingen anvendtes til at transformere E. coli stammen DH1, og rekombinanter selekteredes på basis af deres evne til resistens over for ampicillin. De identificerede rekombinanter dyrkedes på LB 5 substrat (Luria substrat) indeholdende 50 pg/ml ampicillin. Disse rekombinan-te plasmider rensedes og undersøgtes for tilstedeværelsen af det korrekte indskudte fragment.Another chain was built in vitro in the presence of the 17-mer initiator; sequence (Amersham), the four nucleotides A, C, T, G and DNA polymerase I (Kle-now). The EcoRI deposit was excised by EcoRI cleavage and purified on agarose gel and then ligated to pSP65, which had been previously cleaved with EcoRI. The ligation mixture was used to transform E. coli strain DH1, and recombinants were selected based on their ability to resist ampicillin. The identified recombinants were grown on LB 5 substrate (Luria substrate) containing 50 µg / ml ampicillin. These recombinant plasmids were purified and assayed for the presence of the correctly inserted fragment.
En af de opnåede kloner, betegnet med referencen pSPE2, deponeredes i 10 CNCM i Paris, Frankrig under registreringsnummeret I-595 den 5. september 1986.One of the clones obtained, designated by reference pSPE2, was deposited in 10 CNCMs in Paris, France under registration number I-595 on September 5, 1986.
Indskuddene stammende fra cDNA af HIV-2, og som forefandtes indført i ovennævnte probe, indeholdt nucleotidsekvensen, der er beskrevet ovenfor, i 15 overensstemmelse med en del af E2.The inserts derived from cDNA of HIV-2, which were inserted into the above probe, contained the nucleotide sequence described above in accordance with a portion of E2.
EKSEMPEL 11EXAMPLE 11
Kloning af et cDNA komplementært med DNA'et komplementært med det 20 genome RNA fra HIV-2 virioner HIV-2 virioner rensedes fra 5 liter af en dyrkningssupernatant del fra en CEM linie inficeret med et ROD isolat. En første strenget cDNA syntetiseredes i kontakt med sedimenteret renset virus i nærværelse af en oligo (dT) initiator 25 og under anvendelse af en endogen reaktion aktiveret med en detergent således som beskrevet af Alizon et al., Nature 312. 757-760 (1984). RNA/cDNA hybriderne rensedes ved ekstraktion med en phenol/chloroformblanding og ved udfældning med ethanol. Den anden streng af cDNA frembragtes i nærværelse af DNA polymerase l/RNAse H, således som beskrevet af Gubier og 30 Hoffman. Der henvises herved til denne artikel.Cloning of a cDNA complementary to the DNA complementary to the 20 genome RNA from HIV-2 virions HIV-2 virions were purified from 5 liters of a culture supernatant portion from a CEM line infected with a ROD isolate. A first stranded cDNA was synthesized in contact with sediment purified virus in the presence of an oligo (dT) initiator 25 and using an endogenous reaction activated with a detergent as described by Alizon et al., Nature 312. 757-760 (1984) . The RNA / cDNA hybrids were purified by extraction with a phenol / chloroform mixture and by precipitation with ethanol. The second strand of cDNA was produced in the presence of DNA polymerase I / RNAse H, as described by Gubier and Hoffman. Please refer to this article.
45 DK 175959 B145 DK 175959 B1
Det dobbeltstrengede cDNA blev udstyret med stumpe ender i nærværelse af DNA polymease T4 under anvendelse af bestanddele fra et cDNA synteseudstyr markedsført af Amersham.The double-stranded cDNA was fitted with blunt ends in the presence of DNA polymease T4 using components from a cDNA synthesis equipment marketed by Amersham.
5 EcoRI adaptorer (linkere) markedsført af Pharmacia, blev fastgjort til enden af cDNA; det på denne måde modificerede cDNA blev efter kløvning i nærværelse af EcoRI indført i en dephosphoryleret fag vektor M13tg130, derfor sit vedkommende var kløvet med EcoRI, også markedsført af Amersham.5 EcoRI adapters (linkers) marketed by Pharmacia were attached to the end of the cDNA; the cDNA modified in this way, after cleavage in the presence of EcoRI, was introduced into a dephosphorylated phage vector M13tg130, which is why it was cleaved with EcoRI, also marketed by Amersham.
Der opnåedes et cDNA bånd efter transformation af E. coli stamme TG1. Re-10 kombinante plaques (104) afsøgtes in situ på filtre, hvilket muliggjorde aftryk ved hybridisering med klonen J19 indeholdende 1,5 kb Hindlll fragmentet nævnt oven for stammende fra HIV-1.A cDNA band was obtained after transformation of E. coli strain TG1. Re-10 combinative plaques (104) were searched for in situ on filters, allowing imprinting by hybridization with the clone J19 containing the 1.5 kb HindIII fragment mentioned above derived from HIV-1.
Filtrene præhybridiseredes i nærværelse af et substrat indeholdende 5 x 15 SSC, 5 x Denhart opløsning, 25% formaldehyd og denatureret laksesperma DNA (100 pg/ml) ved 37 °C i 4 timer og herefter hybridiseredes 16 timer i samme puffer (Tm-42 °C) i nærvær af en yderligere mærket probe (4 x 107 cpm) til opnåelse af en sluthybridiseringspufferopløsning indeholdende 106 cpm/ml.The filters were prehybridized in the presence of a substrate containing 5 x 15 SSC, 5 x Denhart solution, 25% formaldehyde and denatured salmon sperm DNA (100 µg / ml) at 37 ° C for 4 hours and then hybridized for 16 hours in the same buffer (Tm-42 ° C) in the presence of a further labeled probe (4 x 10 7 cpm) to obtain a final hybridization buffer solution containing 106 cpm / ml.
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Vask udførtes med en 5 x SSC, 0,1% SDS opløsning ved 25 °C i 2 timer (idet det skal bemærkes, at 20 x SSC svarer til en 3 M NaCI og 0,3 M natriumcit-ratopløsning). Plaques, der svarede positivt, rensedes, og de M13 enkeltstrengede DNA blev fremstillet og deres ender sekvensbedømt således som 25 beskrevet af Sanger et al.Washing was performed with a 5 x SSC, 0.1% SDS solution at 25 ° C for 2 hours (it should be noted that 20 x SSC corresponds to a 3 M NaCl and 0.3 M sodium citrate solution). Positive-responding plaques were purified and the M13 single-stranded DNAs were prepared and their ends sequenced as described by Sanger et al.
Hvbridiserina af et DNA fra celler inficeret med henholdsvis HIV-1 og HIV-2 og med RNA fra HIV-1. HIV-2 oo fra SIV virioner med en probe stammende fra et klonet cDNA af HIV-2 30 DNA blev ekstraheret fra inficerede CEM celler, der kontinuert frembragte henholdsvis HIV-1 og HIV-2. DNA prøver af disse to retrovira, kløvet i visse tilfælde med 20 pg Pstl og ikke kløvet i andre tilfælde, underkastedes elek- 46 DK 175959 B1 trophorese på 0,8% agarosegel og overførtes ved hjælp af "Southern" metoden til en nylonmembran. Smårumfang inficeret supematantdel optaget i en NTE puffer indeholdende 0,1% SDS og med den samme omvendte transcrip-taseaktivitet anbragtes på nitrocellulose, der forud var vædet i en 2 x SSC 5 opløsning.Hybridization of a DNA from cells infected with HIV-1 and HIV-2 and with RNA from HIV-1, respectively. HIV-2 oo from SIV virions with a probe derived from a cloned cDNA of HIV-2 DNA was extracted from infected CEM cells that continuously produced HIV-1 and HIV-2, respectively. DNA samples of these two retroviruses, cleaved in some cases with 20 µg of PstI and not cleaved in other cases, were subjected to electrophoresis on 0.8% agarose gel and transferred by the Southern method to a nylon membrane. Small volume infected supernatant moiety taken in an NTE buffer containing 0.1% SDS and with the same reverse transcriptase activity was applied to nitrocellulose previously wetted in a 2 x SSC 5 solution.
En præhybridisering udførtes i en opløsning indeholdende 50% formamid, 5 x SSC, 5 x Denhart og 100 mg/ml denatureret laksesperma DNA i 4 timer ved 42 °C. En hybridisering udførtes i den samme puffer, hvortil 10% dex-10 transulfat og 106 cpm/ml E2 mærket indskud (specifik aktivitet 109 cpm/pg) var tilsat i 16 timer ved 42 °C. To vask blev herefter udført med 0,1 x SSC, 0,1% SDS opløsning i 30 minutter hver. Efter eksponering i 16 timer for en forstørrelsesskærm dehybridiseredes Southern pletten i 0,4 N NaOH, neutraliseredes og hybridiseredes atter under de samme betingelser med HIV-1 15 proben mærket med 109cpm/pg.A prehybridization was carried out in a solution containing 50% formamide, 5 x SSC, 5 x Denhart and 100 mg / ml denatured salmon sperm DNA for 4 hours at 42 ° C. Hybridization was performed in the same buffer to which 10% dex-10 transulfate and 106 cpm / ml E2 labeled insert (specific activity 109 cpm / pg) were added for 16 hours at 42 ° C. Two washes were then performed with 0.1 x SSC, 0.1% SDS solution for 30 minutes each. After exposure for 16 hours to a magnifying screen, the Southern blot was dehybridized in 0.4 N NaOH, neutralized and again hybridized under the same conditions with the HIV-1 probe labeled at 109cpm / µg.
EKSEMPEL IIIEXAMPLE III
Kloning i fag λ af det komplette DNA fra HIV-2 proviruset 20 DNA fra CEM celler inficeret med HIV-2 ROD (fig. 2, bånd a og c) spaltes delvist med Sau3AI. 9-15 kb fraktionen selekteredes på en 5-40% saccharo-segradient og ligeredes til BamHI armen af λ L47.1 vektoren. Plaques (2 x 106) opnået efter in vitro pakning og anbringelse på E. coli stammen LA 101 25 afsøgtes in situ ved hybridisering med indskuddet fra E2 klonet cDNA. Omtrent 10 positive kloner rensedes på plaques og propageredes i E. coli C600 recBC. Kloneme λ ROD 4, ROD 27 og ROD 35 blev opformeret, og deres DNA karakteriseredes ved at optegne deres restriktionskort og ved hybridisering ved hjælp af Southern metoden med cDNA klonen af HIV-2 under krafti-30 ge betingelser og med gag-pol proberne fra HIV-1 under milde betingelser.Cloning in phage λ of the complete DNA from HIV-2 provirus 20 DNA from CEM cells infected with HIV-2 ROD (Fig. 2, bands a and c) is partially cleaved with Sau3AI. The 9-15 kb fraction was selected on a 5-40% sucrose gradient and ligated to the BamHI arm of the λ L47.1 vector. Plaques (2 x 10 6) obtained after in vitro packaging and application to E. coli strain LA 101 25 were scanned in situ by hybridization with the insert from the E2 cloned cDNA. About 10 positive clones were purified on plaques and propagated in E. coli C600 recBC. Clones λ ROD 4, ROD 27 and ROD 35 were amplified and their DNA characterized by recording their restriction maps and by hybridization by the Southern method with the cDNA clone of HIV-2 under strong conditions and with the gag-pol probes of HIV-1 under mild conditions.
Fig. 8 viser skematisk opbygningerne af 3 af de rekombinante opnåede fager ROD 4, ROD 27 og ROD 35.FIG. 8 schematically shows the structures of 3 of the recombinantly obtained phages ROD 4, ROD 27 and ROD 35.
i 47 DK 175959 B1in 47 DK 175959 B1
De aflange rektangulære dele af disse diagrammer svarer til provirale sekvenser, der stammer fra DNA fra de oprindelige inficerede CEM celler, idet de klare dele svarer til de retrovirale sekvenser, de afskyggede dele til delene 5 af cellulære DNA og de sorte dele til LTR i de virale sekvenser.The elongated rectangular portions of these diagrams correspond to proviral sequences derived from the DNA of the initially infected CEM cells, the clear portions corresponding to the retroviral sequences, the detached portions to portions 5 of cellular DNA, and the black portions to LTRs in the viral sequences.
De tynde linier betegnet med bogstaverne L og R svarer til armene stammende fra λ L47.1 fag vektoren, der anvendtes til kloningen.The thin lines denoted by the letters L and R correspond to the arms derived from the λ L47.1 phage vector used for the cloning.
10 Visse af restriktionspladseme er også angivet: disse er mere specielt følgende pladser: B: BamHI; E: EcoRI; H: Hindlll; K: Kpnl; Ps: Pstl; Pv: Pvull; S:10 Some of the restriction sites are also listed: these are more specifically the following sites: B: BamHI; E: EcoRI; H: HindIII; K: Kpnl; Ps: Pstl; Pv: Pvull; S:
Sag I; X: Xbal.Case I; X: Xbal.
Disse virale sekvenser har dele fælles med E2 sekvensen. De relative belig-15 genheder af disse dele bestemt ved hybridisering med E2 er også angivet i figurerne.These viral sequences have parts in common with the E2 sequence. The relative locations of these moieties determined by hybridization with E2 are also indicated in the figures.
ROD 4 stammer fra et cirkulært viralt DNA. ROD 27 og ROD 35 stammer fra provira integreret i en cellulær DNA opbygning.ROD 4 is derived from a circular viral DNA. ROD 27 and ROD 35 are derived from proviruses integrated into a cellular DNA structure.
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Til sidst subklonedes indskudene under ovennævnte betingelser, og deres relative positioner i forhold til de tilsvarende ROD 4, ROD 27 og ROD 35 sekvenser er angivet i disse figurer.Finally, the deposits were subcloned under the above conditions and their relative positions relative to the corresponding ROD 4, ROD 27 and ROD 35 sequences are given in these figures.
25 Disse er mere specielt indskudene af plasmideme pROD 27-5', pROD 35-3’, pROD 4.6, pROD 4.8 og pROD 4.7.More specifically, these are the insertions of the plasmids pROD 27-5 ', pROD 35-3', pROD 4.6, pROD 4.8 and pROD 4.7.
Fig. 9 er en afbildning af de relative intensiteter af hybridiseringspletterne, der blev frembragt mellem ROD-4 og subfragmenterne 1-11, der stammer hen-30 holdsvis fra de forskellige dele af det enkeltstrengede DNA, der stammer fra en M13 subklon indeholdende en nukleinsyre, der stammer fra hele LAV ge-nomet. De relative beliggenheder af disse forskellige fragmenter i forhold til hele LAV genomet (bestemt ved sekvensbedømmelse) er angivet for neden 48 DK 175959 B1 på tegningen. Punkt 12 svarer til en kontrolplet frembragt under anvendelse af et kontrol DNA fra fag λ.FIG. 9 is a representation of the relative intensities of the hybridization spots generated between ROD-4 and the sub-fragments 1-11, respectively, originating from the various parts of the single stranded DNA derived from an M13 subclone containing a nucleic acid containing derives from the entire LAV genome. The relative locations of these various fragments relative to the entire LAV genome (determined by sequence assessment) are indicated below 48 in the drawing. Point 12 corresponds to a control spot generated using a control DNA from phage λ.
Hybridiseringsforsøgene i pletoverførsel (dot blot) metoden udførtes under de 5 milde betingelser fra eksempel II under anvendelse med hensyn til en probe, af λ ROD 4 rekombinanten indeholdende det totale cDNA fra HIV-2. Afvaskningerne udførtes herefter efter hinanden under følgende betingelser: 2 x SSC, 0,1% SDS ved 25 °C (Tm 42 °C), 1 x SSC, 0,1% SDS ved 60 °C (Tm-20 °C) og 0,1% x SSC, 0,1% SDS ved 60 °C (Tm-3 °C).The hybridization experiments in the blot transfer (dot blot) method were performed under the 5 mild conditions of Example II using a probe, of the λ ROD 4 recombinant containing the total cDNA from HIV-2. The washings were then performed consecutively under the following conditions: 2 x SSC, 0.1% SDS at 25 ° C (Tm 42 ° C), 1 x SSC, 0.1% SDS at 60 ° C (Tm-20 ° C), and 0.1% x SSC, 0.1% SDS at 60 ° C (Tm-3 ° C).
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De angivne pletter opnåedes efter radiografisk eksponering natten over.The indicated spots were obtained after radiographic exposure overnight.
EKSEMPEL IVEXAMPLE IV
15 In vitro diagnostisk undersøgelse for tilstedeværelsen af HIV-2 virus i et biologisk medium i i15 In vitro diagnostic study for the presence of HIV-2 virus in a biological medium in i
Materialer og metoder 20 Patienter HIV-2 inficerede patienter udvalgtes blandt personer, der besøgte Egas Mo-niz hospitalet i Lissabon enten for indlæggelse eller til konsultation mellem september 1985 og september 1986. Dette udvalg, der alle var personer af 25 afrikansk oprindelse eller havde levet i Afrika, fik foretaget serumprøve for antistoffer over for bånde HIV-1 (immunofluorescenc -IFA- og/eller ELISA) og HIV-2 (IFA). Kun de patienter, der viste sig serologisk at være inficeret med HIV-2, blev taget med i undersøgelsen.Materials and Methods 20 Patients HIV-2 infected patients were selected from people who visited Egas Mo-niz hospital in Lisbon either for hospitalization or for consultation between September 1985 and September 1986. This committee, all of whom were 25 African people or had lived in Africa, serum test was performed for antibodies to both HIV-1 (immunofluorescence-IFA- and / or ELISA) and HIV-2 (IFA). Only those patients who were serologically infected with HIV-2 were included in the study.
30 Isolering af virus På 12 patienter var HIV isolering forsøgt således som tidligere beskrevet.Virus isolation In 12 patients, HIV isolation was attempted as previously described.
Kort fortalt stimuleredes patienternes perifere blodlymphocyter (PBL) med 49 DK 175959 B1 PHA, dyrkedes med normale humane PHA-stimulerede PBL'er og vedligeholdtes i tilstedeværelse af interleukin-2 (IL-2). Kulturerne kontrolleredes for tilstedeværelsen af cytopatisk virkning (CPE) og for omvendt transcriptase (RT) aktivitet i den supernatante del.Briefly, patients' peripheral blood lymphocytes (PBL) were stimulated with 49 PH 175, cultured with normal human PHA-stimulated PBLs, and maintained in the presence of interleukin-2 (IL-2). The cultures were checked for the presence of cytopathic effect (CPE) and for reverse transcriptase (RT) activity in the supernatant moiety.
55
Immunofluorescensforsøa (IFA) IFA objektglas fremstilledes på følgende måde: HIV-2-inficeret MOLT-4 celler vaskedes 2 gange i PBS og anbragtes på IFA glasplader (104 celler/brønd), 10 lufttørredes og fikseredes med kold acetone. For undersøgelse for IFA omsattes disse celler med 1/10 fortynding af forsøgsserumet i 45 minutter ved 37 °C, vaskedes, tørredes og omsattes med et fluorescein-konjugeret gede- antihumant IgG, A, M (1/100 fortyndet) i 30 minutter ved 37 °C. Efter vask i modfarvedes cellerne i 0,006% Evans blåt, anbragtes i 90% glycerol, 10% ^ 15 PBS og undersøgtes under fluorescens mikroskop.Immunofluorescence Assay (IFA) IFA slides were prepared as follows: HIV-2-infected MOLT-4 cells were washed twice in PBS and placed on IFA glass plates (104 cells / well), air dried, and fixed with cold acetone. For testing for IFA, these cells were reacted with 1/10 dilution of the test serum for 45 minutes at 37 ° C, washed, dried and reacted with a fluorescein-conjugated goat anti-human IgG, A, M (1/100 diluted) for 30 minutes at 37 ° C. After washing in the counterstained cells, the 0.006% Evans blue was placed in 90% glycerol, 10% PBS and examined under fluorescence microscope.
ELISAELISA
Visse patienters sera undersøgtes for antistoffer over for HIV-1 under anven-20 delse af de kommercielt tilgængelige serumforsøg ELAVIA (Pasteur Diagnostics) eller Abbott.Some patients' sera were examined for antibodies to HIV-1 using the commercially available serum tests ELAVIA (Pasteur Diagnostics) or Abbott.
RadioimmunoDræcipitationsforsøq (RIPAl 25 HIV-1 eller HIV-2 Inficerede CEM celler dyrkedes i tilstedeværelsen af 35s cystein (200 pCi/ml) i 16 timer. Den supernatante del opsamledes, virale partikler pelleteredes og lyseredes i RIPA puffer (Tris-HCL 50 mM, pH 7,5, NaCI 150 mM, EDTA 1 mM, 1% Triton x 100, natriumdeoxycholat 0,1%, SDS 0,1%). Til hver reaktion omsattes 50 pi af en fortynding af lysat svarende til 30 105 cpm med 5 μΙ forsøgsserum i 18 timer ved 4 °C. Immunkomplekser blev bundet til sepharose-protein A (Pharmacia), vasket og elueret ved kogning i 2 minutter. Eluerede antigener analyseredes herefter ved hjælp af SDS-polyacrylamidgelelektrophorese og autoradiografi.Radioimmunoprecipitation experiments (RIPAl 25 HIV-1 or HIV-2 Infected CEM cells were cultured in the presence of 35s cysteine (200 µCi / ml) for 16 hours. The supernatant was collected, viral particles were pelleted and lysed in RIPA buffer (Tris-HCL 50 mM, pH 7.5, NaCl 150 mM, EDTA 1 mM, 1% Triton x 100, sodium deoxycholate 0.1%, SDS 0.1%) For each reaction 50 µl of a dilution of lysate corresponding to 30 105 cpm was reacted with 5 μΙ experimental serum for 18 hours at 4 ° C. Immunocomplexes were bound to Sepharose Protein A (Pharmacia), washed and eluted by boiling for 2 minutes, then eluted antigens were analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography.
50 DK 175959 B150 DK 175959 B1
Pot-blot hvbridiserinaPot-blot hybridization
Vims isoleret fra patienters PBL pelleteredes og lyseredes i Tris-HCL 10 mM 5 pH 7,5, NaCI 10 mM, EDTA 1 mM, SDS 0,5%. En mikroliter af hvert lysat svarende til 50.000 spm RT-aktivitet anbragtes på nitrocellulose. Hy-bridisering og vask udførtes under kraftige betingelser: hybridisering i 6 x SSC, 5 x Denhart, 50 % formamid i 18 timer ved 42 °C; og vask i 0,1 x SSC, 0,1% SDS ved 65 °C. Der anvendtes HIV-1 og HIV-2 prober, 32P mærket til 10 en specifik aktivitet på 108 cpm/pg. HIV-1 proben svarer til det komplette genom af LAVbru isolatet, og HIV-2 proben stammede fra en 2 kilobase cDNA klon fra LAV-2ROd isolat.Vims isolated from patients' PBL were pelleted and lysed in Tris-HCL 10 mM pH 7.5, NaCl 10 mM, EDTA 1 mM, SDS 0.5%. One microlitre of each lysate corresponding to 50,000 ppm RT activity was placed on nitrocellulose. Hybridization and washing were performed under strong conditions: hybridization in 6 x SSC, 5 x Denhart, 50% formamide for 18 hours at 42 ° C; and wash in 0.1 x SSC, 0.1% SDS at 65 ° C. HIV-1 and HIV-2 probes, 32P labeled for 10, had a specific activity of 108 cpm / pg. The HIV-1 probe corresponds to the complete genome of the LAV-use isolate, and the HIV-2 probe was derived from a 2-kilobase cDNA clone from the LAV-2ROd isolate.
Resultater 15Results 15
Fordeling af patienter 30 patienter med serologisk og/eller virologisk tegn påHIV-2 infektion undersøgtes. Det var 12 mænd og 18 kvinder. Gennemsnitsalderen var 35, stræk-20 kende fra 11-55 år. Alle patienter bortset fra en havde boet adskillige år i Vestafrika; 26 var født og havde levet i Guniea-Bissau og 2 stammede fra Cape Verde øerne. En patient var en 11 år gammel dreng fra Angola, der havde boet på Cape Verde i adskillige år. Den eneste europæer i undersøgelsen var en 40 år gammel portugiser, der havde levet 8 år i Zaire og som 25 nægtede at have boet i Vestafrika.Distribution of patients 30 patients with serologic and / or virologic signs of HIV-2 infection were studied. It was 12 men and 18 women. The average age was 35, ranging from 11-55 years. All but one patient had lived in West Africa for several years; 26 were born and had lived in Guniea-Bissau and 2 were from the Cape Verde Islands. One patient was an 11-year-old boy from Angola who had lived on Cape Verde for several years. The only European in the study was a 40-year-old Portuguese, who had lived for 8 years in Zaire and who 25 refused to live in West Africa.
Kliniske kendetegnClinical characteristics
Blandt de 30 patienter havde 17 AIDS i overensstemmelse med CDC kriteri-30 erne. Hovedsymptomet hos disse patienter var kronisk diarre sammen med i de fleste tilfælde vægttab på mere end 10 kg i løbet af 1 år. Hos 10 af patienterne viste diarreen sig at være i forbindelse med tilstedeværelsen af en tarm opportunistisk infektion; de 7 tilfælde var pathogenen Isosporabelli alene, en 51 DK 175959 B1 patient havde Cryptosporidium alene og 2 havde begge pathogener. I 3 tilfælde fandtes ingen opportunistisk tarmpathogen. Blandt alle 17 AIDS tilfælde diagnostiseredes hos 8 oseophageal candidiasis. 6 AIDS patienter havde respirationssymptomer. Pulmonær tuberculose diagnostiseredes hos 2 og en 5 anden uidentificeret mycobakterium fandtes hos en patient. To patienter havde pulmonær aspergillosis, én efter en tuberculose. To andre AIDS patienter havde gentagne anfald af pneumonier uden identificerede pathogener, og en patient havde Pneumocystis carinii pneumonia, der først diagnostiseredes post-mortem. Fire af de 17 AIDS patienter havde Kaposis sarcom: i 3 tilfælde 10 var sygdommen begrænset til huden, og hos en patient afslørede postmortem undersøgelse udbredte viscerale angreb. Centralnervesystemsygdomme fandtes hos to AIDS patienter: en havde cerebral lymphoma, og den anden havde subakut encephalitis af ukendt oprindelse.Among the 30 patients, 17 had AIDS according to the CDC criteria. The main symptom of these patients was chronic diarrhea along with in most cases weight loss of more than 10 kg over 1 year. In 10 of the patients, the diarrhea was found to be associated with the presence of a gut opportunistic infection; the 7 cases were the pathogen Isosporabelli alone, one patient had Cryptosporidium alone and 2 had both pathogens. In 3 cases, no opportunistic gut pathogen was found. Of all 17 AIDS cases, 8 were diagnosed with 8 oseophageal candidiasis. 6 AIDS patients had respiratory symptoms. Pulmonary tuberculosis was diagnosed in 2 and 5 other unidentified mycobacteria were found in one patient. Two patients had pulmonary aspergillosis, one after a tuberculosis. Two other AIDS patients had recurrent seizures of pneumonias without identified pathogens, and one patient had Pneumocystis carinii pneumonia, first diagnosed post-mortem. Four of the 17 AIDS patients had Kaposi's sarcoma: in 3 cases 10, the disease was limited to the skin and in one patient, postmortem examination revealed widespread visceral attacks. Central nervous system disorders were found in two AIDS patients: one had cerebral lymphoma and the other had subacute encephalitis of unknown origin.
15 Fire patienter blev fundet med symptomer på det AlDS-relaterede kompleks (ARC): to udviste diffus lymphadenopati med vedvarende feber, en havde kronisk diarre med vægttab, og en havde gentagne anfald af broncopneumo-ni og multiple lymphadenopatier.15 Four patients were found with symptoms of the AlDS-related complex (ARC): two had diffuse lymphadenopathy with persistent fever, one had chronic diarrhea with weight loss, and one had recurrent attacks of bronchopneumonia and multiple lymphadenopathies.
20 Blandt de øvrige 9 patienter havde 6 ingen symptomer, der kunne betragtes som relateret med HIV infektion, en havde udelukkende pulmonær tubercu- ; lose, en havde udelukkende persistens diffus lymphadenopati, og en udviste neurologisk syphillis. I løbet af den 12 måneders observationsperiode døde 7 patienter, der alle udviste AIDS.20 Of the other 9 patients, 6 had no symptoms that could be considered related to HIV infection, one had only pulmonary tuberculosis; lose, one had persistent diffuse lymphadenopathy persistently, and one exhibited neurological syphillis. During the 12-month observation period, 7 patients all died of AIDS.
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Serologisk undersøgelseSerological examination
Hver patient undersøgtes mindst en gang for serumindhold af antistoffer over for både HIV-1 og HIV-2. Alle patienters sera undersøgtes ved hjælp af IFAEach patient was examined at least once for serum antibody levels against both HIV-1 and HIV-2. All patients' sera were examined using IFA
30 for antistoffer over for HIV-2, og alle var positive. Blandt disse undersøgtes 21 også for antistoffer over for HIV-2 ved hjælp af RIPA: alle udfældede klart det højmolekylære omhylningsglycoprotein af viruset betegnet gp 140, og 16 52 DK 175959 B1 af disse reagerede også med hovedkemeproteinet p26, mens kun en reagerede med det andet virale kemeprotein, betegnet p16.30 for antibodies to HIV-2 and all were positive. Of these, 21 were also tested for antibodies to HIV-2 by RIPA: all clearly precipitated the high molecular weight glycoprotein of the virus designated gp 140, and 16 of these also reacted with the main germ protein p26, while only one reacted with the other viral core protein, designated p16.
Sera undersøgtes for tilstedeværelsen af krydsreagerende antistoffer over for 5 HIV-1 under anvendelse af en anden undersøgelsesmetode. En IFA undersøgelse anvendtes til 24 sera: 12 var negative, 10 var svagt reaktive og 2 var positive. Med ELISA undersøgtes 21: 16 var negative og 5 var positive. Til slut undersøgtes 11 sera for antistoffer over for HIV-1 proteiner ved hjælp RIPA. Tre var ikke i stand til at reagere med noget som helst viralt protein, 2 10 udfældede kun pol gen produktet p34 og 5 reagerede med hovedkemeproteinet p25. To sera reagerede kun svagt med ophylningsglycoproteinet gp 110 af HIV-1. Disse to sera, og alle sera med positive IFA eller ELISA forsøg for antistoffer over for HIV-1, udviste stærk reaktivitet med gp 140 fra HIV-2 ved RIPA, hvilket snarere indicerede infektion med HIV-2 end med HIV-1. Kun en 15 patient, som ikke deltog i undersøgelsen, viste sig serologisk at være inficeret med HIV-1 og ikke med HIV-2. Denne patient var en 21 årig kvinde fra Centralafrika (Sao Tome Islands) med AIDS.Sera were examined for the presence of cross-reacting antibodies to 5 HIV-1 using a different assay method. An IFA study was used for 24 sera: 12 were negative, 10 were weakly reactive and 2 were positive. With ELISA examined, 21: 16 were negative and 5 were positive. Finally, 11 sera for antibodies to HIV-1 proteins were examined by RIPA. Three were unable to react with any viral protein, 210 precipitated only the pol gene product p34, and 5 reacted with the main germ protein p25. Two sera reacted only weakly with the envelope glycoprotein gp 110 of HIV-1. These two sera, and all sera with positive IFA or ELISA tests for antibodies to HIV-1, showed strong reactivity with gp 140 from HIV-2 by RIPA, indicating infection with HIV-2 rather than with HIV-1. Only 15 patients who did not participate in the study were serologically infected with HIV-1 and not with HIV-2. This patient was a 21 year old woman from Central Africa (Sao Tome Islands) with AIDS.
Isolering af virus 20Virus isolation 20
Isolering af retrovira fra perifere blodlymphocyter forsøgtes hos 12 patienter.Isolation of retroviruses from peripheral blood lymphocytes was attempted in 12 patients.
HIV isoleredes hos 11 i overensstemmelse med tilstedeværelsen af en typisk cytopatisk virkning og af en top i form af partikelassociseret omvendt transcriptaseaktivitet i de supernatante dele fra dyrkningen.HIV was isolated at 11 according to the presence of a typical cytopathic effect and of a peak in the form of particle-associated reverse transcriptase activity in the supernatant parts of the culture.
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Alle 11 isolater identificeredes som HIV-2 under anvendelse af en dot-blot hybridiseringsteknik. Virale pletter fra 10 isolater viste sig at hybridisere kraftigt under stærke hybridiseringsbetingelser og vask med en HIV-2 probe stammende fra et klonet HIV-2 cDNA, medens ingen af disse hybridiserede 30 med en HIV-1 probe under samme betingelser. Et isolat hybridiserede kun svagt med HIV-2 proben, men var ikke i stand til at hybridisere med HIV-1 proben.All 11 isolates were identified as HIV-2 using a dot-blot hybridization technique. Viral spots from 10 isolates were found to hybridize vigorously under strong hybridization conditions and wash with an HIV-2 probe derived from a cloned HIV-2 cDNA, while none of these hybridized with an HIV-1 probe under the same conditions. An isolate hybridized only weakly with the HIV-2 probe but was unable to hybridize with the HIV-1 probe.
53 DK 175959 B153 DK 175959 B1
Immunologisk bedømmelse 13 patienter undersøgtes for antallet af cirkulerende lymphocyter identificeret som hjælper T celler (CD4+) og for forholdet mellem hjælper:supressor T 5 celler. Blandt disse patienter havde 11 AIDS: deres gennemsnitlige absolutte hjælper T celletal var 243 + 300/μΙ og deres gennemsnitlige hjælpen supres-sorforhold var 0,25 + 0,15. En af patienterne, der klinisk udviste ARC, havde et hjælper T lymphocyttal på 240/μΙ og et forhold på 0,18. En anden patient med neurologisk syphillis og intet tegn på HIV-relaterede symptomer havde 10 et hjælper T lymphocyttal på 690/μΙ og et forhold på 0,82.Immunological assessment 13 patients were examined for the number of circulating lymphocytes identified as helper T cells (CD4 +) and for the ratio of helper: supressor T 5 cells. Among these patients, 11 had AIDS: their mean absolute helper T cell count was 243 + 300 / μΙ and their mean helper supressor ratio was 0.25 + 0.15. One of the patients clinically showing ARC had a helper T lymphocyte count of 240 / μΙ and a ratio of 0.18. Another patient with neurologic syphillis and no evidence of HIV-related symptoms had a helper T lymphocyte count of 690 / μΙ and a ratio of 0.82.
I denne undersøgelse er påvist HIV-2 infektion hos 30 vestafrikanske patienter, der udviste enten AIDS, ARC eller som ikke havde nogen åbenlyse HIV-relaterede symptomer. Resultaterne muliggør imidlertid den konklusion, at 15 HIV-2 må betragtes som værende et hovedætiologisk agens for AIDS hos vestafrikanske patienter. De serologiske og virologiske profiler man fandt, indicerer, at HIV-2 infektion ikke ofte fandtes i forbindelse med HIV-1 infektion hos de pågældende patienter. På trods af vigtige antige og genetiske forskelle udviser HIV-1 og HIV-2 ens tropisme over for CD4+ T lymphocyter, 20 ens cytopatiske virkninger, ens morphologi og deler fælles immunoreaktive epitoper i visse af deres konstitutive proteiner. Da alle vestafrikanske patienter med HIV infektion i den nærværende undersøgelse viste sig at være inficeret med HIV-2 og ingen af dem med HIV-1, må dette hidtil ukendte virus HIV-2 være hovedgrunden til AIDS i Vestafrika.In this study, HIV-2 infection was detected in 30 West African patients who exhibited either AIDS, ARC or who had no obvious HIV-related symptoms. However, the results allow the conclusion that 15 HIV-2 must be considered as a major etiologic agent for AIDS in West African patients. The serological and virological profiles found indicate that HIV-2 infection was not often found in association with HIV-1 infection in the patients concerned. Despite important antigenic and genetic differences, HIV-1 and HIV-2 exhibit similar tropism to CD4 + T lymphocytes, 20 similar cytopathic effects, similar morphology, and share common immunoreactive epitopes in some of their constitutive proteins. Since all West African patients with HIV infection in the present study were found to be infected with HIV-2 and none of them with HIV-1, this novel virus HIV-2 must be the main cause of AIDS in West Africa.
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Symptomerne på HIV-2-relateret AIDS var ikke forskellige fra symptomerne på HIV-1-relateret AIDS i Centralafrika: det mest almindelige symptom var kronisk diarre med kraftig vægttab, for det meste på grund af Isospora belli og/eller Crytopsporidium. Hyppigheden af andre opportunistiske infektioner 30 som f.eks. candidiasis, mycobacterier (herunder M. tuberculosis) og to-xoplasmose var som for HIV-1-relateret afrikansk AIDS. Pneumocystis carinii pneumonia, en meget almindelig komplikation i forbindelse med AIDS i USAThe symptoms of HIV-2-related AIDS did not differ from the symptoms of HIV-1-related AIDS in Central Africa: the most common symptom was chronic diarrhea with heavy weight loss, mostly due to Isospora belli and / or Crytopsporidium. The frequency of other opportunistic infections 30, e.g. candidiasis, mycobacteria (including M. tuberculosis) and toxoplasmosis were as for HIV-1-related African AIDS. Pneumocystis carinii pneumonia, a very common complication of AIDS in the United States
54 DK 175959 B1 og Europa, fandtes kun en enkelt gang i denne undersøgelse, og der sås ikke cryptococcal meningitis.54 DK 175959 B1 and Europe, was found only once in this study and no cryptococcal meningitis was seen.
Ikke desto mindre er de immunologiske abnormaliteter fundet hos HIV-2-5 inficerede AIDS patienter identiske med de, der findes og er beskrevet i forbindelse med ΗIV-1-relateret AIDS.Nevertheless, the immunological abnormalities found in HIV-2-5 infected AIDS patients are identical to those found and described in association with ΗIV-1-related AIDS.
Blandt de 30 patienter, der alle havde serumantistoffer reaktive med HIV-2 antigener, havde kun 7 HIV-1 specifikke antistoffer, der kunne afsløres under 10 anvendelse af IFA eller ELISA. Ved RIPA havde alle disse 7 patienter antistoffer reaktive med de andre hovedkemeproteiner p25 (HIV-1) eller p26 (HIV-2), der har stærk immunoreaktive epitoper til fælles. 5 patienter manglede disse antistoffer: alle 5 havde en negativ HIV-1 ELISA. IFA var på grænsen hos 3 og negative hos 2. Skønt visse af disse ikke var fuldstændig be-15 dømt, fandt man imidlertid 9 patienter med serumantistoffer over for det virale kerneprotein p26 af HIV-2, der udviste en svag reaktiv eller negativ H1V-1-specifik IFA og/eller ELISA. Disse fund peger på vigtigheden af at indbefatte HIV-2 antigener i HIV serumundersøgelserne, der anvendes i Afrika, og måske også i andre områder.Among the 30 patients who all had serum antibodies reactive with HIV-2 antigens, only 7 had HIV-1 specific antibodies that could be detected using 10 IFA or ELISA. At RIPA, all these 7 patients had antibodies reactive with the other major nuclear proteins p25 (HIV-1) or p26 (HIV-2), which have strong immunoreactive epitopes in common. 5 patients lacked these antibodies: all 5 had a negative HIV-1 ELISA. However, IFA was at the borderline of 3 and negative at 2. Although some of these were not completely assessed, however, 9 patients with serum antibodies to the viral core protein p26 of HIV-2 were found to exhibit a weak reactive or negative H1V gene. 1-specific IFA and / or ELISA. These findings point to the importance of including HIV-2 antigens in the HIV serum studies used in Africa, and perhaps in other areas as well.
2020
Et retrovirus isoleredes fra de perifere lymphocyter hos 11 patienter. I alle tilfælde opåedes viral vækst i løbet af 2 uger, der var karakteriseret ved tilstedeværelsen af omvendt transcriptaseaktivitet i dyrkningens supematante del og cytopatisk virkning. Denne cytopatiske virkning synes imidlertid at va-25 riere fra det ene isolat til det andet: visse isolater gav adskillige store syncy-tier sammen med kraftig cellelyse, medens andre kun udviste få syncytier af begrænset størrelse, og bevirkende nedsat levedygtighed af kulturen.A retrovirus was isolated from the peripheral lymphocytes in 11 patients. In all cases, viral growth was achieved over 2 weeks characterized by the presence of reverse transcriptase activity in the supernatant part of the culture and cytopathic effect. However, this cytopathic effect seems to vary from one isolate to another: some isolates conferred several large syncytia along with vigorous cell lysis, while others exhibited only limited syncytia and impaired culture viability.
RNA fra alle bortset fra et isolat viste sig klart at hybridisere under meget 30 kraftige betingelser med en probe stammende fra en HIV-2 cDNA klon, der repræsenterer 3' enden af genomet. Ingen hybridiserede med en HIV-1 probe under samme betingelser. Dette viser, at isolaterne, der inficerer disse patienter, alle hører til samme type virus. Kun et isolat hybridiserede dårligt 55 DK 175959 B1 med HIV-1. Dette virus var imidlertid isoleret fra en patient med serumantistoffer, der reagerede med alle antigenerne fra HIV-2 ved RIPA.RNA from all but an isolate was clearly found to hybridize under very strong conditions with a probe derived from an HIV-2 cDNA clone representing the 3 'end of the genome. No one hybridized with an HIV-1 probe under the same conditions. This shows that the isolates that infect these patients all belong to the same type of virus. Only one isolate poorly hybridized with HIV-1. However, this virus was isolated from a patient with serum antibodies that reacted with all the HIV-2 antigens by RIPA.
Opfindelsen beskriver generelt ud over HIV-2-virus varianter heraf, et hvilket 5 som helst ækvivalent virus, der kan inficere ét menneske, og som er i besiddelse af immunologiske egenskaber, der er specifikke for HIV-2. Opfindelsen angår generelt et vilkårligt virus der ud over egenskaberne, der findes i forbindelse med HIV-2 vira deponeret i CNCM, også er i besiddelse af neden for beskrevne egenskaber.The invention generally describes, in addition to HIV-2 virus variants thereof, any equivalent virus that can infect one human being and possess immunological properties specific to HIV-2. The invention generally relates to any virus which, in addition to the properties found in connection with HIV-2 viruses deposited in CNCM, also possesses the properties described below.
1010
Det foretrukne mål for HIV-2 viruset er humane Leu 3 celler (eller T4. lymphocyter) og "immortaliserede" celler stammende fra disse T4 lymphocyter som f.eks. celler af HUT 78 linierne, der er omtalt i forbindelse med nærværende patentansøgning. Med andre ord det har specifik tropisme for disse celler.The preferred target of the HIV-2 virus is human Leu 3 cells (or T4 lymphocytes) and "immortalized" cells derived from these T4 lymphocytes such as cells of the HUT 78 lines disclosed in the present patent application. In other words, it has specific tropism for these cells.
15 Det kan dyrkes i permanente linier af HUT, CÉM, MOLT eller lignende type, der udtrykker T4 proteinet. Det er ikke infektiøst for T8 lymphocyter. Det er cytotoxisk over for humane T4 lymphocyter. Den cytopathogene natur af HIV-2 vira med hensyn til T4 lymphocyter manifesterer sig specielt ved tilstedeværelsen af muftinucleare celler. Det udviser Omvendt trans-20 criptaseaktivitet, der kræver tilstedeværelsen af Mg2+ ioner og har en stærk affinitet over for polyadenylat oligodeoxythymidylat (poly(A)-oligo(dT) 12-18).It can be grown in permanent lines of HUT, CÉM, MOLT or similar type expressing the T4 protein. It is not infectious for T8 lymphocytes. It is cytotoxic to human T4 lymphocytes. The cytopathogenic nature of HIV-2 viruses with respect to T4 lymphocytes is manifested especially in the presence of muftinuclear cells. It exhibits reverse trans-criptase activity requiring the presence of Mg2 + ions and has a strong affinity for polyadenylate oligodeoxythymidylate (poly (A) -oligo (dT) 12-18).
Det har en massefylde på omtrent 1,16 i en saccharosegradient. Det har en gennemsnitsdiameter på 140 nanometer og en kerne med en gennemsnitsdiameter på 41 nanometer. Lysateme af dette virus indeholder et p26 prote-25 in, der ikke krydser immunologisk med p24 proteinet fra HTLV-1 virus eller HTLV-II virus. Disse p26 proteiner har derfor en molekylvægt, der er lidt højere (omtrent 1000) end de tilsvarende p25 proteiner fra HIV-1 og lidt lavere (igen af størrelsesordenen 1000 lavere) end de tilsvarende p27 proteiner fra SIV. Lysateme af HIV-2 indeholder herudover et p16 protein, der ikke gen-30 kendes immunologisk af p19 proteinet fra HTLV-1 eller fra HTLV-II i RIPA (forkortelse for radioimmunopræcipitationsforsøget). De indeholder herudover et omhylningsglycoprotein med en molekylvægt af størrelsesordenen 130.000-140.000, der ikke krydser immunologisk med gp110 fra HIV-1, men 56 DK 175959 B1 som, på den anden side, krydser immunologisk med gp140 om-hylningsglycoproteinet fra STLV-III. Disse lysater indeholder også proteiner eller glycoproteiner, der kan mærkes med (35S)cystein. med molekylvægte henholdsvis af størrelsesordenen 36.000 og 42.000-45.000. Det genome 5 RNA fra HIV-2 hybridiserer ikke med det genome RNA fra HIV-1 under kraftige betingelser. Under milde betingelser hybridiserer det ikke med nucleotid-sekvensen stammende på HIV-1 og indeholdende env genet og LTR nabostillet hertil. Specielt hybridiserer det ikke med nucleotidsekvensen 5290-9130 fra HIV-1, heller ikke med sekvenserne fra pol regionen af HIV-1 geno-10 met, specielt med nucleotidsekvensen 2170-2240. Under milde bétingelser hybridiserer det svagt med nucleotidsekvenser fra HIV-1 regionen, specielt nucleotidsekvenserne i 990-1070 og 990-1260.It has a density of about 1.16 in a sucrose gradient. It has an average diameter of 140 nanometers and a core with an average diameter of 41 nanometers. The lysates of this virus contain a p26 protein that does not cross immunologically with the p24 protein of HTLV-1 virus or HTLV-II virus. Therefore, these p26 proteins have a molecular weight that is slightly higher (about 1000) than the corresponding p25 proteins from HIV-1 and slightly lower (again on the order of 1000 lower) than the corresponding p27 proteins from SIV. In addition, the lysates of HIV-2 contain a p16 protein that is not immunologically recognized by the p19 protein from HTLV-1 or from HTLV-II in RIPA (Abbreviation for the Radioimmunoprecipitation Trial). In addition, they contain a envelope glycoprotein having a molecular weight of the order of 130,000-140,000 which does not cross immunologically with gp110 from HIV-1 but, on the other hand, crosses immunologically with the gp140 envelope glycoprotein from STLV-III. These lysates also contain proteins or glycoproteins that can be labeled with (35S) cysteine. with molecular weights of the order of 36,000 and 42,000-45,000 respectively. The HIV-2 genomic RNA does not hybridize to the HIV-1 genomic RNA under strong conditions. Under mild conditions, it does not hybridize with the nucleotide sequence derived from HIV-1 and containing the env gene and LTR adjacent to it. In particular, it does not hybridize with the nucleotide sequence 5290-9130 of HIV-1, nor with the sequences from the pol region of the HIV-1 genome, especially with the nucleotide sequence 2170-2240. Under mild conditions, it weakly hybridizes with nucleotide sequences from the HIV-1 region, especially the nucleotide sequences of 990-1070 and 990-1260.
Det skal bemærkes, at et hvilket som helst retrovirus, der er infektiøs for 15 mennesker, der er i stand til at fremkalde en af formerne for AIDS, der udviser ovennævnte væsentlige egenskaber, og hvis genome RNA er i stand til at hybridisere under kraftige betingelser med de HIV-2 viralé stammer deponeret i CNCM (eller med et cDNA eller et cDNA fragment stammende, fra disse genome RNA-forbindelser) skal bestragtes som værende ækvivalente 20 med HIV-2.It should be noted that any retrovirus that is infectious to 15 people capable of inducing any of the forms of AIDS exhibiting the above essential characteristics and whose genomic RNA is capable of hybridizing under strong conditions with the HIV-2 viral strains deposited in the CNCM (or with a cDNA or a cDNA fragment derived from these genomic RNAs) are deemed to be equivalent to HIV-2.
Opfindelsen angår også enhver af de antigener, specielt proteiner og glycoproteiner i renset tilstand, der kan opnås fra HIV-2. Omtale af "rensede" proteiner eller glycoproteiner betyder, at disse proteiner eller, glycoproteiner 25 fører til henholdsvis kun enkelte bånd ved polyacrylamidgeielektrophorese, specielt under de forsøgsbetingelser, der er beskrevet ovenfor. En hvilken som helst velegnet metode til adskillelse og/eller rensning til at opnå disse kan anvendes. F.eks. kan de metoder anvendes, der er beskrevet af R. C. Montelaro et al., J. of Virology, Juni 1982, p. 1029-1038.The invention also relates to any of the antigens, especially proteins and glycoproteins in purified state, obtainable from HIV-2. Mention of "purified" proteins or glycoproteins means that these proteins or, glycoproteins, 25, respectively, only lead to single bands by polyacrylamide gel electrophoresis, especially under the experimental conditions described above. Any suitable method of separation and / or purification to achieve these may be used. Eg. For example, the methods described by R. C. Montelaro et al., J. of Virology, June 1982, pp. 1029-1038.
3030
Opfindelsen angår generelt alle antigener, specielt proteiner, glycoproteiner eller polypeptider, der stammer fra en HIV-2, og som udviser immunologiske egenskaber svarende til egenskaberne fra de antigene forbindelser HIV-2. To 57 DK 175959 B1 antigener betegnes som værende "ækvivalente" i den forstand, at .de genkendes af de samme antistoffer, specielt af antistoffer, der kan isoleres fra et serum fra en patient, der er inficeret med en HIV-2, eller så længe som de tilfredsstiller betingelserne for "immunologisk ækvivalens" som beskrevet 5 ovenfor.The invention generally relates to all antigens, especially proteins, glycoproteins or polypeptides derived from an HIV-2, which exhibit immunological properties similar to those of the antigenic compounds HIV-2. Two 57 antigens are designated as "equivalent" in the sense that they are recognized by the same antibodies, especially by antibodies which can be isolated from a serum of a patient infected with an HIV-2, or so as long as they satisfy the conditions of "immunological equivalence" as described above.
Blandt ækvivalente polypeptider, proteiner eller glycoproteiner skal indbefattes fragmenter af ovennævnte antigener (eller peptider opbygget ved kemisk syntese) så længe som de forårsager immunologiske krydsreaktioner med 10 antigenerne, hvorfra de stammer; Med andre ord opfindelsen angår et hvilket som helst polypeptid, der fælles med ovennævnte antigener har identiske eller lignende epitoper, der er i stand til at genkendes af de samme antistoffer. Hørende til denne sidstnævnte type polypeptider er udtrykkel-sesprodukteme af tilsvarende sekvenser af DNA-forbindelser, der koder for 15 de tilsvarende polypeptidsekvenser.Equivalent polypeptides, proteins or glycoproteins must include fragments of the above antigens (or peptides built by chemical synthesis) as long as they cause immunological cross-reactions with the 10 antigens from which they originate; In other words, the invention relates to any polypeptide in common with the above antigens having identical or similar epitopes capable of being recognized by the same antibodies. Associated with this latter type of polypeptides are the expression products of corresponding sequences of DNA compounds encoding the corresponding polypeptide sequences.
HIV-2 viruset har vist sig at kunne anvendes som en kilde for antigener til afsløring af antistoffer hos alle personer, der har været i kontakt med HIV-2 viruset.The HIV-2 virus has been found to be useful as a source of antigens for the detection of antibodies in all persons who have been in contact with the HIV-2 virus.
2020
Opfindelsen béskriver generelt et hvilket som helst præparat, der kan anvendes til diagnose af tilstedeværelsen i en biologisk væskeserum, specielt fra mennesker, der har været i kontakt med HIV-2, af antistoffer over for mindst en af antigenerne fra HIV-2. Præparatet kan anvendes til selektiv diagnose af 25 den tilsvarende varietet af AIDS, idet der anvendes diagnosemetoder som de, der f.eks. er beskrevet i den ovennævnte europæiske patentbeskrivelse, bortset.fra at ekstrakter, lysater eller rensede antigener af HIV-2 anvendes i stedet for sådanne af HIV-1.1 denne forbindelse beskriver opfindelsen endvidere præparater indeholdende mindst en af proteinerne p12, p16, p26, der er 30 indre proteiner, eller p36 eller gp140. Som eksempel kan nævnes præparater, der samtidig indeholder følgende: 58 DK 175959 B1 p26 og gp 36 p26, p36 og gp 140, p12, p16 og p26, p16, p26 og gp 140, osv.The invention generally prescribes any composition that can be used to diagnose the presence in a biological fluid serum, especially from humans who have been in contact with HIV-2, of antibodies to at least one of the antigens of HIV-2. The composition can be used for selective diagnosis of the corresponding variety of AIDS, using diagnostic methods such as those used in e.g. is disclosed in the aforementioned European patent specification, except that extracts, lysates or purified antigens of HIV-2 are used in place of those of HIV-1.1. This invention further discloses compositions containing at least one of the proteins p12, p16, p26 which are 30 internal proteins, or p36 or gp140. Examples include preparations containing at the same time: B1 p26 and gp 36 p26, p36 and gp 140, p12, p16 and p26, p16, p26 and gp 140, etc.
55
Det er klart, at disse præparater kun er eksempler. Specielt beskriver opfindelsen virale ekstrakter eller lysater indeholdende denne gruppe proteiner og/eller glycoproteiner eller alle fraktioner, fra hvilket en eller flere af ovennævnte proteiner eller glycoproteiner forud er skilt.It is clear that these preparations are merely examples. In particular, the invention discloses viral extracts or lysates containing this group of proteins and / or glycoproteins or all fractions from which one or more of the aforementioned proteins or glycoproteins are previously separated.
1010
Her omtales endvidere præparater indeholdende en kombination af proteiner og/eller glycoproteiner af et HIV-2 med proteiner og/eller glycoproteiner af et HIV-1 som f.eks: 15 enten kerneproteinér af HIV-1 og HIV-2, specielt p25 fra et HIV-1 og p26 fra et HIV-2, eller alternativt p18 fra et HIV-1 og p16 fra et HIV-2, eller omhylningsglycoproteiner fra et HIV-1 og omhylningsproteiner fra et HIV-2, specielt gp 110 fra HIV-1 og gp 140 fra HIV-2, eller alternativt p42 fra 20 HIV-1 og p36 eller p42-45 fra HIV-2, eller naturligvis blandinger af proteiner og/eller glycoproteiner fra et HIV-1 og proteiner og/eller glycoproteiner fra et HIV-2.Also disclosed herein are compositions containing a combination of proteins and / or glycoproteins of an HIV-2 with proteins and / or glycoproteins of an HIV-1 such as: either core proteins of HIV-1 and HIV-2, especially p25 from a HIV-1 and p26 from an HIV-2, or alternatively p18 from an HIV-1 and p16 from an HIV-2, or envelope glycoproteins from an HIV-1 and envelope proteins from an HIV-2, especially gp 110 from HIV-1 and gp 140 from HIV-2, or alternatively p42 from 20 HIV-1 and p36 or p42-45 from HIV-2, or of course mixtures of proteins and / or glycoproteins from an HIV-1 and proteins and / or glycoproteins from an HIV 2nd
25 Disse præparater gør det, når de anvendes ved diagnose muligt at diagnosti-sere AIDS eller symptomer, der står i forbindelse hermed, idet de strækker sig over et bredere spektrum af de ætiologiske agens, der er ansvarlige herfor. Anvendelsen af præparater til diagnostiske metoder, der kun indeholder proteiner og/eller glycoproteiner af HIV-2, er ikke desto mindre anvendelige til 30 mere selektiv diagnose af den katagori af retrovirus, der er ansvarlig for denne sygdom.These preparations, when used in diagnosis, make it possible to diagnose AIDS or associated symptoms, extending over a wider range of the etiologic agents responsible for this. However, the use of preparations for diagnostic methods containing only proteins and / or glycoproteins of HIV-2 is useful for 30 more selective diagnosis of the category of retrovirus responsible for this disease.
59 DK 175959 B159 DK 175959 B1
Opfindelsen beskriver også DNA eller DNA fragmenter, mere specielt klonede DNA og DNA fragmenter opnået ud fra RNA og cDNA stammende fra RNA af HIV-2 retroviruset. Opfindelsen beskriver specielt alle ækvivalente DNA-forbindelser, specielt et hvilket som helst DNA, der er i besiddelse af 5 sekvenshomologi med DNA fra HIV-2, specielt med de sekvenser, der koder for env og pol regionerne af stammen HIV-2 deponeret i CNCM, lig hermed indtil mindst 50%, fortrinsvis indtil 70% og foretrukkent indtil 90%. Det skal også bemærkes, at opfindelsen beskriver et hvilket som helst ækvivalent DNA (eller RNA), der er i stand til at hybridisere med DNA eller RNA fra HIV-10 2 ved "spot blot" metoden, under de ovenfor definerede milde betingelser.The invention also discloses DNA or DNA fragments, more particularly cloned DNA and DNA fragments obtained from RNA and cDNA derived from RNA of the HIV-2 retrovirus. In particular, the invention discloses all equivalent DNA compounds, especially any DNA possessing 5 sequence homology with HIV-2 DNA, especially with the sequences encoding the env and pol regions of the strain HIV-2 deposited in CNCM. , equal to at least 50%, preferably up to 70%, and preferably up to 90%. It should also be noted that the invention discloses any equivalent DNA (or RNA) capable of hybridizing with HIV or DNA DNA or RNA by the "spot blot" method, under the mild conditions defined above.
Opfindelsen beskriver ligeledes sera, der kan frembringes i dyr ved at inocu-lere disse med HIV-2. Derfor beskriver opfindelsen mere specielt polyklonale antistoffer rettet mere specifikt over for hver af antigenerne, specielt proteiner 15 eller glycoproteiner, fra viruset. Herudover beskriver opfindelsen monoklona-le antistoffer, der kan frembringes på traditionel måde, idet disse monoklona-le antistoffer er rettet henholdsvis mere specifikt mod de forskellige proteiner fra HIV-2.The invention also describes sera that can be produced in animals by inoculating them with HIV-2. Therefore, the invention more particularly describes polyclonal antibodies directed more specifically to each of the antigens, especially proteins or glycoproteins, of the virus. In addition, the invention discloses monoclonal antibodies which can be produced in the conventional manner, these monoclonal antibodies being directed more specifically to the various proteins of HIV-2, respectively.
20 Disse polyklonale eller monoklonale antistoffer kan anvendes til forskellige formål. Deres anvendes til at neutralisere tilsvarende proteiner eller endog hæmme den infektiøse evne af hele virus skal specielt nævnes. De kan også anvendes til f.eks. at demonstrere virale antigener i biologiske præparater eller til at udføre fremgangsmåder til rensning åf de tilsvarende proteiner og/-25 . eller glycoproteiner f.eks. ved at anvende disse i affinitetskromatografisøjler.These polyclonal or monoclonal antibodies can be used for various purposes. Their use to neutralize similar proteins or even inhibit the infectious ability of the whole virus must be specifically mentioned. They can also be used for e.g. to demonstrate viral antigens in biological compositions or to perform purification procedures on the corresponding proteins and / -25. or glycoproteins e.g. by applying these in affinity chromatography columns.
Det skal bemærkes, at generel tilgængelige teknisk litteratur (specielt den, der er angivet som litteraturliste i forbindelse med nærværende beskrivelse) med hensyn til HIV-1 og virus betegnet HTLV-III skal betragtes som værende 30 en del af opfindelsen, idet der henvises til disse, idet metoder beskrevet i disse artikler anvendes under lignende betingelser til isolering af HIV-2 virus eller af ækvivalente vira, og angår produktionen ud fra disse vira af deres forskellige bestanddele (specielt proteiner, glycoproteiner, polypeptider og 60 DK 175959 B1 nukleinsyrer). Man kan også anvende, hvad der er beskrevet i den tekniske litteratur med hensyn til anvendelse af forskellige bestanddele specielt med hensyn til diagnosemetoder for de tilsvarende former af LAS eller AIDS.It should be noted that generally available technical literature (especially that listed as a reference list for the purposes of this specification) with regard to HIV-1 and virus designated HTLV-III should be considered as part of the invention, with reference to these, the methods described in these articles being used under similar conditions to isolate HIV-2 viruses or equivalent viruses, and relate to the production from these viruses of their various constituents (especially proteins, glycoproteins, polypeptides and nucleic acids). It is also possible to use what has been described in the technical literature regarding the use of various components, especially with regard to diagnostic methods for the corresponding forms of LAS or AIDS.
5 Den foreliggende opfindelse beskriver mere specielt en fremgangsmåde til in vitro diagnose af AIDS, der er ejendommelig ved, at man bringer serum eller et andet biologisk medium stammende fra en patient, der skal underkastes diagnose, i kontakt med et præparat indeholdende mindst en af proteinerne eller glycoproteineme af HIV-2, eller alternativt et ekstrakt eller iysat af viru-10 set, og at den immunologiske reaktion afsløres. Eksempler på sådanne præparater er angivet ovenfor.More particularly, the present invention discloses a method for in vitro diagnosis of AIDS, characterized by contacting serum or other biological medium derived from a subject to be diagnosed with a composition containing at least one of the proteins or the glycoproteins of HIV-2, or alternatively an extract or lysate of virus, and that the immunological reaction is revealed. Examples of such compositions are given above.
Foretrukne fremgangsmåder indebærer f.eks. immunoenzymatiske reaktioner af ELISA eller immunofluorescenstypen. Titreringer kan være målinger 15 ved direkte eller indirekte immunofluorescens, ved direkte eller indirekte immunoenzymatiske forsøg.Preferred methods involve e.g. immunoenzymatic reactions of ELISA or immunofluorescence type. Titrations can be measurements by direct or indirect immunofluorescence, by direct or indirect immunoenzymatic experiments.
Den foreliggende opfindelse beskriver også ekstrakter af virus (eller et ekstrakt af et eller flere HIV-2 vira alene eller en blanding af ekstrakter stam-20 mende fra en eller flere HIV-2 vira på den ene side og en eller flere HlV-1 vira på den anden side), idet disse ekstrakter er mærket. En hvilken som helst type velegnet mærke kan anvendes: enzymatisk mærkning, fluorescensmærkning, radioaktivitetsmærkning og lignende.The present invention also discloses extracts of viruses (or an extract of one or more HIV-2 viruses alone or a mixture of extracts derived from one or more HIV-2 viruses on one side and one or more HIV-1 viruses on the other hand), with these extracts labeled. Any type of suitable label can be used: enzymatic labeling, fluorescence labeling, radioactivity labeling and the like.
25 Sådanne titreringer omfatter f.eks.: anbringelse af afmålte mængder af det omhandlede ekstrakt eller omhandlede præparat i brøndene på en mikrotitreringsplade; 30 indførsel i disse brønde af stigende fortyndinger af serum principielt indeholdende de antistoffer, hvis tilstedeværelse skal afsløres in vitro; inkubering af mikrotitreringspladen; 61 DK 175959 B1 omhyggelig vask af mikrotitreringspladen med en passende puffer; indførsel i brøndene på en mikrotitreringsplade af mærkede antistoffer speci-5 fikke for humane immunoglobuliner, idet mærkning udføres med et enzym valgt blandt sådanne, der er i stand til at hyrolysere et substrat på en sådan måde, at sidstnævnte herefter undergår en forandring af dets strålingsabsorption, i det mindste i et specielt bølgelængdebånd, og 10 afsløring, fortrinsvis ved sammenligning med en kontrol, af hydrblysegraden af substratet som et udtryk for den potentielle risiko eller som en egentlig'tilstedeværelse af sygdommen.Such titrations include, for example: placing metered amounts of the subject extract or composition in the wells of a microtitration plate; 30 introduction into these wells of increasing dilutions of serum containing, in principle, the antibodies whose presence is to be revealed in vitro; incubating the microtitration plate; 61 GB 175959 B1 careful washing of the microtitration plate with a suitable buffer; introducing into the wells a microtiter plate of labeled antibodies specific for human immunoglobulins, labeling is carried out with an enzyme selected from those capable of hydrolyzing a substrate in such a way that the latter then undergoes a change in its radiation absorption. , at least in a particular wavelength band, and detection, preferably by comparison with a control, of the degree of hydration of the substrate as an expression of the potential risk or as an actual presence of the disease.
Den foreliggende opfindelse beskriver også udstyr eller kits til ovennævnte 15 diagnose, der er ejendommelig ved: et ekstrakt eller en mere kraftigt renset fraktion af de ovennævnte virustyper, idet denne ekstrakt eller fraktioner er mærket f.eks. radioaktivt, enzymatisk eller ved immunofluorescens; 20 anti-(human immunoglobuliner) eller et protein A (fordelagtig bundet på et understøttelseselement, der er uopløseligt i vand, som f.eks. agarosekugler); i i et ekstrakt af lymphocyter opnået fra en rask person; 25 pufre og eventuelt substrater til visualisering af mærkningen.The present invention also discloses equipment or kits for the above diagnosis which are characterized by: an extract or a more heavily purified fraction of the aforementioned virus types, this extract or fractions being labeled e.g. radioactive, enzymatic or by immunofluorescence; 20 anti- (human immunoglobulins) or a protein A (advantageously attached to a water-insoluble support element, such as agarose beads); in an extract of lymphocytes obtained from a healthy person; 25 buffers and optionally substrates for visualization of the label.
Af ovenstående vil det fremgå, at opfindelsen beskriver diagnosen af HIV-2 virus eller af en variant som et resultat af anvendelsen åf de ovennævnte 30 beskrevne prober ved en fremgangsmåde, der anvender forskellige nedenfor anførte trin, idet disse trin er således arrangeret, at de blotlægger de karakteristiske egenskaber ved HIV-2 virus.From the above, it will be understood that the invention discloses the diagnosis of HIV-2 virus or of a variant as a result of the use of the above-described probes by a method using various steps listed below, these steps being arranged so that reveals the characteristic features of HIV-2 virus.
62 DK 175959 B162 DK 175959 B1
Opfindelsen beskriver naturligvis også anvendelsen af cDNA-forbindelser eller deres fragmenter (eller rekombinanter indeholdende disse) som prober ved diagnosen for tilstedeværelsen eller mangel på tilstedeværelse af HIV-2 5 virus i serumprøver eller andre biologiske væsker eller væv opnået fra patienter, der er mistænkte for at være bærere af HIV-2 virus. Sådanne prober er fortrinsvis også mærkede (radioaktive, enzymatiske, fluorescensmærket og lignende). Særligt velegnede probér til udførsel af fremgangsmåden til diagnostisering af HIV-2 virus eller en variant af HIV-2 kan karakteriseres 10 ved, at de omfatter alt eller en fraktion af cDNA'en komplementært til geno-met af HIV-2 viruset, eller alternativt, specielt fragmenter, der findes i de forskellige ovenfor beskrevne kloner. Mere specielt skal nævnes en fraktion af cDNA af HIV-2, der findes i klonen E2, mere specielt sekvensen af 3’ enden (LTR) og/eller af 5' enden af HIV-sékvensen af ovennævnte klon E2, eller 15 alternativt cDNA indeholdende env regionen af cDNA fra HIV-2 viruset.Of course, the invention also discloses the use of cDNAs or their fragments (or recombinants containing them) as probes for the diagnosis of the presence or absence of HIV-2 virus in serum samples or other biological fluids or tissues obtained from patients suspected of being carriers of HIV-2 virus. Such probes are also preferably labeled (radioactive, enzymatic, fluorescent labeled and the like). Particularly suitable probes for performing the method for diagnosing HIV-2 virus or a variant of HIV-2 may be characterized by comprising all or a fraction of the cDNA complementary to the genome of the HIV-2 virus, or alternatively , especially fragments found in the various clones described above. More particularly, mention should be made of a fraction of cDNA of HIV-2 found in clone E2, more particularly the sequence of the 3 'end (LTR) and / or of the 5' end of the HIV sequence of the above clone E2, or alternatively cDNA containing env region of cDNA from HIV-2 virus.
De anvendte prober ved denne diagnosemetode for HIV-2 virus og i de diagnostiske kits er ikke på nogen måde begrænset til de oven for beskrevne prober. De omfatter tværtimod alle nucleotidsekvenser, der stammer fra ge-20 nomet af HIV-2 viruset, af en variant af HIV-2 eller af strukturel beslægtet vims, så længe som de muliggør at antistoffer rettet mod HIV-2, kan afsløres i biologiske væsker hos personer, der kan udvikle en af formerne for AIDS. Naturligvis er anvendelsen af nucleotidsekvenser stammende fra et HIV-2, der oprindeligt er infektiøs for mennesket, ikke desto mindre at foretrække.The probes used in this diagnostic method for HIV-2 virus and in the diagnostic kits are by no means limited to the probes described above. On the contrary, they include all nucleotide sequences derived from the genome of the HIV-2 virus, a variant of HIV-2 or of structurally related vims, as long as they allow antibodies directed against HIV-2 to be detected in biological fluids. in people who can develop one of the forms of AIDS. Of course, the use of nucleotide sequences derived from an HIV-2 that is initially infectious to humans is nevertheless preferred.
2525
Afsløringen kan udføres på alle i og for sig kendte måder specielt ved at bringe disse prober i kontakt enten med nukleinsyrerne opnået fra celler til stede i disse sera eller andre biologiske medier f.eks. cerebrospinalvæsker, spyt og lignende, eller med disse medier som sådan, så længe som deres 30 nukleinsyrer er blevet gjort tilgængelige for hybridisering med disse probér, idet dette skal være under betingelser, der muliggør hybridisering mellem disse probér og disse nukleinsyrer, og ved afsløring af hybridiseringen, som frembringes. Ovennævnte diagnose, der indebærer hybridiseringsreaktioner, 63 DK 175959 B1 kan også udføres under anvendelse af blandinger af probér, stammende henholdsvis fra en HIV-1 og en HIV-2, al den stund det er unødvendigt at | differentiere mellem den søgte type HIV virus.The detection can be carried out in all known ways, especially by contacting these probes either with the nucleic acids obtained from cells present in these sera or other biological media, e.g. cerebrospinal fluids, saliva and the like, or with these media as such, as long as their 30 nucleic acids have been made available for hybridization with these probes, this being subject to conditions allowing hybridization between these probes and these nucleic acids and upon detection of the hybridization produced. The above diagnosis involving hybridization reactions may also be performed using mixtures of probes derived from HIV-1 and HIV-2, respectively, while it is unnecessary to | Differentiate between the type of HIV virus searched.
5 Sædvanligvis indebærer diagnosemetoden for tilstedeværelsen eller mangel på tilstedeværelse af HIV-viruset eller variant heraf i serumprøver eller andre vævs- eller væskeprøver opnået fra patienter, der mistænktes for at være bærere af HIV-2 viruset, følgende trin: 10 1) fremstilling af en mærket probe, 2) mindst et hybridiseringstrin udført under kraftige betingelser, ved at bringe DNA fra celler i prøven fra den mistænkte patient i kontakt med den mærkede probe på en passende membran, 15 3) vask af membranen med en opløsning, der medfører bibeholdelse af disse kraftige betingelser for hybridisering, og 4) afsløring af tilstedeværelsen eller mangel på tilstedeværelsen af HIV-2 20 viruset ved en immunoafsløringsmetode.Usually, the method of diagnosis of the presence or absence of the HIV virus or variant thereof in serum or other tissue or fluid samples obtained from patients suspected to be carriers of the HIV-2 involves the following steps: 2) at least one hybridization step performed under severe conditions, by contacting DNA of cells in the sample of the suspected patient with the labeled probe on a suitable membrane; 3) washing the membrane with a solution which causes retention of these potent conditions for hybridization; and 4) detection of the presence or absence of the presence of the HIV-2 virus by an immuno-detection method.
I en anden udførelsesform for den omhandlede fremgangsmåde udføres nævnte hybridisering under milde betingelser, og. vask af membranen udføres under betingelser passende for sådan hybridisering.In another embodiment of the present process, said hybridization is carried out under mild conditions, and. washing of the membrane is performed under conditions suitable for such hybridization.
25 '25 '
Opfindelsen angår specielt HIV-2 vira, der er ejendommelige ved at deres virale RNA svarer til et cDNA, hvis GAG og ENV gener henholdsvis omfatter de efterfølgende nucleotidsekvenser.In particular, the invention relates to HIV-2 viruses, which are characterized in that their viral RNA corresponds to a cDNA, whose GAG and ENV genes comprise the subsequent nucleotide sequences, respectively.
30 De fremkommer ved sekvensbedømmelse af de tilsvarende regioner af cDNA svarende til genomet HIV-2 ROD. De er i overensstemmelse med de aminosyrer, som de koder.They arise by sequencing the corresponding regions of cDNA corresponding to the HIV-2 ROD genome. They are consistent with the amino acids they encode.
64 DK 175959 B164 DK 175959 B1
CAGRODHCAGRODH
MetGlyAlaArgAenSerVaILeuArgGlyLysLysAlaAspGlu atgggcgcgagaaactccgtcttcagaggcaaaaaagcagatgaa i J % · · ...... ·MetGlyAlaArgAenSerVaILeuArgGlyLysLysAlaAspGlu atgggcgcgagaaactccgtcttcagaggcaaaaaagcagatgaa i J% · · ...... ·
LeuGluArgl leArgLeuArgPr oGlyClyLy s Ly s Ly sTyrAr g TTAGAAAGAATCAGGTTACGGCCCGGCGGAAAGAAAAAGTACAGG .LeuGluArgl leArgLeuArgPr oGlyClyLy s Ly s Ly sTyrAr g TTAGAAAGAATCAGGTTACGGCCCGGCGGAAAGAAAAAGTACAGG.
LeuLysBisIleValTrpAlsAlaAsnXys LeuAs pArgPheG1y CTAAAACATATTGTGTGGGCAGCCAATAAATTGCACAGATTCGGALeuLysBisIleValTrpAlsAlaAsnXys LeuAs pArgPheG1y CTAAAACATATTGTGTGGGCAGCCAATAAATTGCACAGATTCGGA
loo . ;loo. ;
LeuAlaGluSerLeuLeuCluSerLyeGluGlyCysGlnLysIle 10 TTAGCAGAGAGCCTGTTGGAGT CAA AAGAGGGXTGTCAA AAAATILeuAlaGluSerLeuLeuCluSerLyeGluGlyCysGlnLysIle 10 TTAGCAGAGAGCCCCTGTTGGAGT CAA AAGAGGGXTGTCAA AAAATI
• .» ^ ♦ ·•. »^ ♦ ·
LeuThrVa1LeuAs pFroKetValProThrGlySerGluAsuLeu CTTACAGTITTAGATCCAATGGTACCGACAGCTTCAGAAAATTTALeuThrVa1LeuAs pFroKetValProThrGlySerGluAsuLeu CTTACAGTITTAGATCCAATGGTACCGACAGCTTCAGAAAATTTA
200 ;200;
LysSerLeuPheAsnlhrValCysVa111eTrpCys 11eE i sAla AAAAGTCTITTTAATACTGTCTGCGTCATTTGGTGCATACACGCALysSerLeuPheAsnlhrValCysVa111eTrpCys 11eE in all AAAAGTCTITTTAATACTGTCTGCGTCATTTGGTGCATACACGCA
15 · · · .·" ·*·.· .· · *15 · · · · · · · · · · · · ·
GluGluLysValLysAspThrGluGlyAlaLysGlnlleValAxg gaagagaaagtgaaagatactgaacgagcaaaacaaatagtgccg ' * . '300 -GluGluLysValLysAspThrGluGlyAlaLysGlnlleValAxg gaagagaaagtgaaagatactgaacgagcaaaacaaatagtgccg '*. '300 -
ArgHisLeuValAlaGluThrGlyTarAlaGluLysMecProSer AGACATCTAGTGGCAGAAACAGGAACTGCAGAGAAAATGCCAAGC * · ♦♦···.*'· «ArgHisLeuValAlaGluThrGlyTarAlaGluLysMecProSer AGACATCTAGTGGCAGAAACAGGAACTGCAGAGAAAATGCCAAGC * · ♦♦ ···. * '· «
TbrSerArgProThrAlaProSerSerGluLysGlyGlyAsnTyr 0 ACAAGTAGACCAACAGCACCATCTAGCGAGAAGGGAGGAAATTACTbrSerArgProThrAlaProSerSerGluLysGlyGlyAsnTyr 0 ACAAGTAGACCAACAGCACCATCTAGCGAGAAGGGAGGAAATTAC
40 040 0
ProValGlnRisVaIGlyGlyAsoTyrThrHisIleProLeuSer ccacxccaacatgtAggcgccaactacacccatatacccctcaci • · · · · · ProAtgTbrLeuAsnAlaTrpValLysLeuVaiGluCluLysLys CCCCGAACCCIAAATCCCTGGCTAAAATTAGTACAGGAAAAAAAG 25 · · . .-· ·ProValGlnRisVaIGlyGlyAsoTyrThrHisIleProLeuSer ccacxccaacatgtAggcgccaactacacccatatacccctcaci • · · · · · ProAtgTbrLeuAsnAlaTrpValLysLeuVaiGluCluLysLys CCCCGAAC .- · ·
PbeGlyAlaGluValValProGlyPheGlnAlaLeuSerGluGly XTCGGGGCAGAAGTAGTGCCAGGATXTCAGGCACTCTCAGAAGGC 500 ...PbeGlyAlaGluValValProGlyPheGlnAlaLeuSerGluGly XTCGGGGCAGAAGTAGTGCCAGGATXTCAGGCACTCTCAGAAGGC 500 ...
CysThrProTyrAspIleAscCloMe tLeuAsoCys Va1GlyAsp TGCACGCCCTATGATATCAACCAAATGCTTAATTGTGTCCGCGACCysThrProTyrAspIleAscCloMe tLeuAsoCys Va1GlyAsp TGCACGCCCTATGATATCAACCAAATGCTTAATTGTGTCCGCGAC
♦ ♦ « . - •-•m · 30 HisGlnAlaAlaMe cGloIlelleArgGlullelleAsnGluGlu catcaaccagccatgcagataatcaccgacAttatcaatgagcaa » 600 ( · ♦ · . AlaAlaGIuTrpAsp Va lGlnHLsProIleProC 1 yPr oLeuPro GCAGCAGAATCGGATGTCCAACATCCAATACCAGGCCCCTTACCA ' · . . · -· · AlaGlyGlnLeuArgGluProArgGlySerAspIleAlaGlyThr GCGGCGCAGCTTAGAGACCCAAGGGGATCTGACATAGCAGGCACA i 35 . . 700 . . . .♦ ♦ «. - • - • m · 30 HisGlnAlaAlaMe cGloIlelleArgGlullelleAsnGluGlu catcaaccagccatgcagataatcaccgacAttatcaatgagcaa '600 (· ♦ ·. AlaAlaGIuTrpAsp Va lGlnHLsProIleProC 1 YPR oLeuPro GCAGCAGAATCGGATGTCCAACATCCAATACCAGGCCCCTTACCA' ·.. · - · · AlaGlyGlnLeuArgGluProArgGlySerAspIleAlaGlyThr GCGGCGCAGCTTAGAGACCCAAGGGGATCTGACATAGCAGGCACA in the 35th. 700th...
ThrSerThcValCluCiuCloIleCInTrpMe tPh eArgProG ta ACAAGCACAGTAGAAGAACACATCCACTCGATCTTTACGCCACAAThrSerThcValCluCiuCloIleCInTrpMe tPh eArgProG to ACAAGCACAGTAGAAGAACACATCCACTCGATCTTTACGCCACAA
65 DK 175959 B165 DK 175959 B1
AsoProValProValGlyAenlleTyrArgArglrpIleGlnHé AATCCTGTACCAGTAGGAAACATCTATAGAAGATGCAICCAGATA . · . . . 8.00 CiyLeuClnLysCysValArgMetTyrAsnProThrAsnlleLeu CGATTGCAGAACTGTGTCAGGATGTACAACCCGACCAACATCCTAAsoProValProValGlyAenlleTyrArgArglrpIleGlnHe AATCCTGTACCAGTAGGAAACATCTATAGAAGATGCAICCAGATA. ·. . . 8.00 CiyLeuClnLysCysValArgMetTyrAsnProThrAsnlleLeu CGATTGCAGAACTGTGTCAGGATGTACAACCCGACCAACATCCTA
J · · · ·J · · · ·
AspIleLysCInGlyProLysGluProPbeGlnSerTyrVa lAsp CACATAAAACAGGGACCAAAGGAGCCGTTCCAAAGCTATCTACAT ... 900AspIleLysCInGlyProLysGluProPbLinSerTyrVa lAsp CACATAAAACAGGGACCAAAGGAGCCGTTCCAAAGCTATCTACAT ... 900
ArgPheTyr LysSerLeuArgA laGluGlnThrAspProAlaVal AGATTCTACAAAAGCT7GAGCCCAGAACAAACACATCCAGCAGTG • · · ♦·ArgPheTyr LysSerLeuArgA laGluGlnThrAspProAlaVal AGATTCTACAAAAGCT7GAGCCCAGAACAAACACATCCAGCAGTG • · · ♦ ·
Ly s AsnTrplle tTbrGlnThrLeuLeu Va lGlnAsnAlaAeoPro AAGAATTCGATGACCCAAACACTGCTAGTACAAAATGCCAACCCA .Ly s AsnTrplle tTbrGlnThrLeuLeu Va lGlnAsnAlaAeoPro AAGAATTCGATGACCCAAACACTGCTAGTACAAAATGCCAACCCA.
• · ·'*.·"» » AspCysLysLeuValLeuLysGlyLeuGlyMetAsnProThrLeu GACTGTAAATTÅGTGCTAAAAGGACTAGGGATCAACCCTACCTTA 1000 · . ...• · · '*. · "» »AspCysLysLeuValLeuLysGlyLeuGlyMetAsnProThrLeu GACTGTAAATTÅGTGCTAAAAGGACTAGGGATCAACCCTACCTTA 1000 ·. ...
GluGluMetLeuThrAlaCysGlnGlyValGlyGlyProGlyGln GAACAGATGCTGACCGCCTGTCACCCGGTACGTGCGCCAGGCCAG 15 · * . · .GluGluMetLeuThrAlaCysGlnGlyValGlyGlyProGlyGln GAACAGATGCTGACCGCCTGTCACCCGGTACGTGCGCCAGGCCAG 15 · *. ·.
LysAlaArgLeulIetAlaGluAlaLeuLysGluVallleGlyPro AAAGCTACATTAATGGCAGAGGCCCTGAAAGAGGTCATAGGACCT . 1100LysAlaArgLeulIetAlaGluAlaLeuLysGluVallleGlyPro AAAGCTACATTAATGGCAGAGGCCCTGAAAGAGGTCATAGGACCT. 1100
AlaProZleFroPheAlaAlaA1 aG1 oGInArgLysAlaPheLye GCCCCTATCCCATTCGCAGCAGCCCAGCAGAGAAACGCATTTAAA . . . * .·'*’ 20 CysTrpAsnCysGlyLysGluGlyHisSerAl’aArgGlnCysArgAlaProZleFroPheAlaAlaA1 aG1 oGInArgLysAlaPheLye GCCCCTATCCCATTCGCAGCAGCCCAGCAGAGAAACGCATTTAAA. . . 20 CysTrpAsnCysGlyLysGluGlyHisSerAl'aArgGlnCysArg
U TGCTCGAACTCTCGAAAGGAAGGGCACTCGGCAACACAATGCCGAU TGCTCGAACTCTCGAAAGGAAGGGCACTCGGCAACACAATGCCGA
1200 .1200.
AlaProArgArgGlnClyCysTrpLysCysGlyLysProGlyHis CCACCTAGAAGGCAGCGCTGCTGGAAGTGTGCTAAGCCAGGACAC • · · ·' · 11eMe tTb rAsnCysProAs pArgClnAl aG lyPbeLeuG lyLeu ATCATGACAAACTGCCCAGATAGACAGGCACGTTTTTTAGCACTG 25 . ... 1300AlaProArgArgGlnClyCysTrpLysCysGlyLysProGlyHis CCACCTAGAAGGCAGCGCTGCTGGAAGTGTGCTAAGCCAGGACAC • · · · 11eMe tTb rAsGGGLGLLGPLAlgPlAl ... 1300
ClyProTrpClyLysLysProArgAsnPbeProValAlaGlnVal CGCCCTTGGGGAAAGAAGCCCCGCAACTTCCCCCTGGCCCAAGTT * · · · · ·ClyProTrpClyLysLysProArgAsnPbeProValAlaGlnVal CGCCCTTGGGGAAAGAAGCCCCGCAACTTCCCCCTGGCCCAAGTT * · · · · ·
ProGlnGlyLeuThrProThrAlaProProValAspFroAlaVal CCGCAGGGGCTGACACCAACAGCACCCCCAGTGGATCCACCAGTG . · · · ♦ 30 AspLeuLeuGluLysTyritetGInGInGlyLysArgG InArgGlu.ProGlnGlyLeuThrProThrAlaProProValAspFroAlaVal CCGCAGGGGCTGACACCAACAGCACCCCCAGTGGATCCACCAGTG. · · · ♦ 30 AspLeuLeuGluLysTyrityGInGInGlyLysArgG InArgGlu.
GATCTACTGGACAAATATATGCAGCAAGGGAAAAGACAGAGAGAG 1400 ...GATCTACTGGACAAATATATGCAGCAAGGGAAAAGACAGAGAGAGAG 1400 ...
G InArgG lu Ar gProTyrLy.sG luValThrCluAspLeuLeuHis CAGAC/.GAGAGACCATACAAGGAAGTGACACAGGACTTACTGCAC • · · ·G InArgG lu Ar gProTyrLy.sG luValThrCluAspLeuLeuHis CAGAC / .GAGAGACCATACAAGGAAGTGACACAGGACTTACTGCAC • · · ·
LeuGluGlnGlyGluTbrProTy rArgCluProProThrCluAsp CTCCAGC/.CGCCCACACACCATACACGGAGCCACCAACACACGAC 35 * . 1500LeuGluGlnGlyGluTbrProTy rArgCluProProThrCluAsp CTCCAGC / .CGCCCACACACCATACACGGAGCCACCAACACACGAC 35 *. 1500
LeuLeuHisLeuAsoSerLeuPheClyLysAspGln TTCCTGCACCTCAATTCTCTCTTTGGAAAAGACCAG • · · i 66 DK 175959 B1 EN van .. MecMetAsnGlnLeuLeuIleAlalleLeuLeuAlaSerAlaCys! ATCATGAATCAGCTGCTTATTGCCATTTTATTAGCTAGTGCTTGCl i • · · . ·LeuLeuHisLeuAsoSerLeuPheClyLysAspGln TTCCTGCACCTCAATTCTCTCTTTGGAAAAGACCAG • · · i 66 DK 175959 B1 EN van .. MecMetAsnGlnLeuLeuIleAlalleLeuLeuAlaSerAlaCys! ATCATGAATCAGCTGCTTATTGCCATTTTATTAGCTAGTGCTTGCl i • · ·. ·
LeuValTyrCysThrGlnTyrValThrValPheTyrGlyValPro 5 TTAGTATATT GCACCCAATAXGTAACTGTTTTCTATGGCGTACCCLeuValTyrCysThrGlnTyrValThrValPheTyrGlyValPro 5 TTAGTATATT GCACCCAATAXGTAACTGTTTTCTATGGCGTACCC
• · · · « ThrTrpLysAsnAlaThrlleProLéuPheCysAlaThrArgAsa ACGTGGAAAAATGCAACCATTCCCCTCTTTTGTGCAACCAGAAAT 100 ...• · · · «ThrTrpLysAsnAlaThrlleProLéuPheCysAlaThrArgAsa ACGTGGAAAAATGCAACCATTCCCCTCTTTTGTGCAACCAGAAAT 100 ...
ArgAs pTbrTrpGlyTh rIlcGloCysLeuProAspAsnAspAsp AGGGATACTTGGGGAACCATACAGTGCTTGCCTGACAATGATGATArgAs pTbrTrpGlyTh rIlcGloCysLeuProAspAsnAspAsp AGGGATACTTGGGGAACCATACAGTGCTTGCCTGACAATGATGAT
10 · . · ♦10 ·. · ♦
TyrGlnGluIleThrLeuAsnValThrGluAlaPbeAspAlaTrp TAT CAGGAAATAACTTTGAATGTAA CAGAGG CTTTTCATCCATGG . 200TyrGlnGluIleThrLeuAsnValThrGluAlaPbeAspAlaTrp TAT CAGGAAATAACTTTGAATGTAA CAGAGG CTTTTCATCCATGG. 200
AsnAsoTbrVaIThrGluGInA1 al 1 eG luAsp Va lTrpH i s Leu AATAATACAGTAACAGAACAAG CAATAGAAGATCTCTGCCATCTA ♦ · · ·AsnAsoTbrVaIThrGluGInA1 al 1 eG luAsp Va lTrpH i s Leu AATAATACAGTAACAGAACAAG CAATAGAAGATCTCTGCCATCTA ♦ · · ·
PbeGluTbrSerrieLysProCysValLysLeuTbrProLeuCys TTCCAGACATCAATAAAACCATGTGTCAAACTAACACCTTTATCTPbeGluTbrSerrieLysProCysValLysLeuTbrProLeuCys TTCCAGACATCAATAAAACCATGTGTCAAACTAACACCTTTATCT
300300
ValAlaMetLysCysSerSerTbrGluSerSerTbrClyAsnAsn CTAGCAATGAAATGCAGCAGCACAGAGAGCAGCACAGCGAACAAC • · . · ·ValAlaMetLysCysSerSerTbrGluSerSerTbrClyAsnAsn CTAGCAATGAAATGCAGCAGCACAGAGAGCAGCACAGCGAACAAC • ·. · ·
TbrThrSerLysSerThrSerThrThrThrThrTbrProTbrAsp ACAACCTCAAAGAGCACAAGCACAACCACAACCACACCCACAGAC 20 ... .400TbrThrSerLysSerThrSerThrThrThrThrTbrProTbrAsp ACAACCTCAAAGAGCACAAGCACAACCACAACCACACCCACAGAC 20 ... .400
GlnGluGloGluIleSerGluAspTbrProCysAlaArgAlaAsp CAGCAGCAAGAGATAAGTGAGGATACTCCATGCGCACGCGCAGAC ♦ · · · ·GlnGluGloGluIleSerGluAspTbrProCysAlaArgAlaAsp CAGCAGCAAGAGATAAGTGAGGATACTCCATGCGCACGCGCAGAC ♦ · · · ·
AsnCysSerClyLeuGlyGluGluCluThrlleAsnCysCloPhe AACTGCTCAGGATTGGGAGAGGAAGAAACGATCAATTGCCAGTTC « · · ♦ 25 Asnlie tThrClyleuGluAr gAs pLy s Ly sLysGlnTyrAsnGlu AATATGACAGGATTAGAAAGAGATAAGAAAAAACAGTATAATGAA 500 ...AsnCysSerClyLeuGlyGluGluCluThrlleAsnCysCloPhe AACTGCTCAGGATTGGGAGAGGAAGAAACGATCAATTGCCAGTTC «· ♦ 25
TbrTrpTyrSerLysAspVaIValCysGluThrAsoAsoSerThr ACATCGTACTCAAAAGATGTGGTTTGTGAGACAAATAATAGCACATbrTrpTyrSerLysAspVaIValCysGluThrAsoAsoSerThr ACATCGTACTCAAAAGATGTGGTTTGTGAGACAAATAATAGCACA
· ·· ·
AsnGlnTbrGloCysTyrMe tAsnE i sCysAsoTbrSerVal 11 e AATCAGACCCAGTGTTACATCAACCATTCCAACACATCAGTCATC 30 . . 600 ...AsnGlnTbrGloCysTyrMe tAsnE i sCysAsoTbrSerVal 11 e AATCAGACCCAGTGTTACATCAACCATTCCAACACATCAGTCATC 30. . 600 ...
ThrGluSerCysAspLysHisTyrTrpAspAlalleArgPheArg ACAGAATCATCTCACAACCACTATTCCCATCCTATAAGGTTTAGA % . · · ·ThrGluSerCysAspLysHisTyrTrpAspAlalleArgPheArg ACAGAATCATCTCACAACCACTATTCCCATCCTATAAGGTTTAGA%. · · ·
TyrCysAlaProProGlyTyrAlaLeuLeuArgCysAsnAspThr TACIGTGCACCACCGGCTTATGCCCTATTAAGATCTAATGATACC . 700 35 AsnTyrSerClyPheAlaProAsnCysSerLysValValAlaSerTyrCysAlaProProGlyTyrAlaLeuLeuArgCysAsnAspThr TACIGTGCACCACCGGCTTATGCCCTATTAAGATCTAATGATACC. 700 35 AsnTyrSerClyPheAlaProAsnCysSerLysValValAlAs
AATTATTCACGCTTTGCACCCAACTCTTCTAAACTAGTAGCTTCTAATTATTCACGCTTTGCACCCAACTCTTCTAAACTAGTAGCTTCT
67 DK 175959 B167 DK 175959 B1
ThrCysTbrArgMetMetGluTbrGlnTbrSerTbrTrpPbeGlyThrCysTbrArgMetMetGluTbrGlnTbrSerTbrTrpPbeGly
ACATGCACCACGATCATCCAAACGCAAACTTCCACATGGTTTCGCACATGCACCACGATCATCCAAACGCAAACTTCCACATGGTTTCGC
800800
PheAsnGlyThrArgAlaGluAsnArgThrTyrlleTyrTrpHia TTTAATGGCACTAGAGCAGAGAATAGAACAIATATCTATTGGCAT: 5 « · · «PheAsnGlyThrArgAlaGluAsnArgThrTyrlleTyrTrpHia TTTAATGGCACTAGAGCAGAGAATAGAACAIATATCTATTGGCAT: 5 «· ·«
C 1yArgAspAsnArgThrllel1eSerLeuAsnLysTyrTyrAsn . CGCAGACATAATACAACTATCATCAGCTTAAACAAATATTATAAT . . . . 900; LeuSerieuEisCysLysArgProGlyAsnLysTbrVaILysGln' CTCAGTTTGCATTGTAAGAGCCCAGGGAATAAGA CAGTGAAACAAC 1yArgAspAsnArgThrllel1eSerLeuAsnLysTyrTyrAsn. CGCAGACATAATACAACTATCATCAGCTTAAACAAATATTATAAT. . . . 900; LeuSerieuEisCysLysArgProGlyAsnLysTbrVaILysGln 'CTCAGTTTGCATTGTAAGAGCCCAGGGAATAAGA CAGTGAAACAA
• · · · in 11eHetLeuMetSerGlyBie7alPbeHisSerHi s TyrGInPro• · · · in 11eHetLeuMetSerGlyBie7alPbeHisSerHi s TyrGInPro
ATAATGCTTATGTCAGGACATGTGTTTCACTCCCAC TACCAGCCGATAATGCTTATGTCAGGACATGTGTTTCACTCCCAC TACCAGCCG
♦ « · · · • i 11eAsnLysArgProArgGInAlaTrpCysTrpPbeLysGlyLys! ATCAATAAAAGACCCAGACAAGCATGGTGCTGGTTCAAAGGCAAA 1000 · . .·♦ «· · · • i 11eAsnLysArgProArgGInAlaTrpCysTrpPbeLysGlyLys! ATCAATAAAAGACCCAGACAAGCATGGTGCTGGTTCAAAGGCAAA 1000 ·. . ·
TrpLysAspAlaMetGlnG luVa lLysT.brLsuAlaLysHi$Pro 1GGAAAGACGCCATGCAGGAGGTGAAGACCC TTGCAAAACATCCC 15 • · · . · · ·TrpLysAspAlaMetGlnG luVa lLysT.brLsuAlaLysHi $ Pro 1GGAAAGACGCCATGCAGGAGGTGAAGACCC TTGCAAAACATCCC 15 • · ·. · · ·
ÆrgTyrArgGlyThrAsnA s pTb rArgA sni1e S erPbeAlaAla AGGTATAGAGGAACCAATGACACAAGGAATATTAGCTTTGCAGCGYEAR TIRE ARGGYTHRAsnA s pTb rArgA cut S isPePaAlaAla AGGTATAGAGGAACCAATGACACAAGGAATATTAGCTTTGCAGCG
1100 . .1100. .
ProClyLysGlySerAspProGlu7alAlaTyrMetTrpTbrAsn CCAGCAAAAGCCTCACACCCAGAAGTAGCATACATGTGGACTAAC • . « · · · 20 CysATgGlyGluPheLeuTyrCysAsnMetThrTrpPbeLeuAsnProClyLysGlySerAspProGlu7alAlaTyrMetTrpTbrAsn CCAGCAAAAGCCTCACACCCAGAAGTAGCATACATGTGGACTAAC •. «· · · 20 CysATgGlyGluPheLeuTyrCysAsnMetThrTrpPbeLeuAsn
ICCAGAGGAGAGTTXCTC7ACTGCAACATGACTTGCTTCCTCAATICCAGAGGAGAGTTXCTC7ACTGCAACATGACTTGCTTCCTCAAT
12001200
TrpIleGluAsnLysTbrHisArgAsnTyrAlaProCysHisIle TCCATAGAGAATAAGACACACCGCAATTATCCACCGTGCCATATA • ' ♦ · · *TrpIleGluAsnLysTbrHisArgAsnTyrAlaProCysHisIle TCCATAGAGAATAAGACACACCGCAATTATCCACCGTGCCATATA • '♦ · · *
LysGlnllelleÅsnTbrfrpHisLysValClyArgAsnValTyr AAGCAAATAATTAACACATCGCATAAGGTAGGGAGAAATGTATAT 3 .... 1300LysGlnllelleAsnTbrfrpHisLysValClyArgAsnValTyr AAGCAAATAATTAACACATCGCATAAGGTAGGGAGAAATGTATAT 3 .... 1300
LeuProProArgGluGlyGluLeuSerCysAsnSerThrValThr' TTCCCTCCCACGGAAGGGGAGCTGTCCTGCAACTCAACAGTAACC * · · · · ·LeuProProArgGluGlyGluLeuSerCysAsnSerThrValThr 'TTCCCTCCCACGGAAGGGGAGCTGTCCTGCAACTCAACAGTAACC * · · · · ·
SerilelleAlaAsnlleAspTrpGInAsnAsnAsnGInThrAsn! ACCATAATTGCTAACATTGACTCGCAAAACAATAATCACACAAAC! • . . .SerilelleAlaAsnlleAspTrpGInAsnAsnAsnGInThrAsn! ACCATAATTGCTAACATTGACTCGCAAAACAATAATCACACAAAC! •. . .
30 IleThrPheSerAlaCluVaΙΑ 1aGluLeuTyrArgLeuGluLeu ATTACCTTTAGTGCAGAGCTGGCAGAACTATACAGATTGGAGTTG j 1400 . .. . .!30 IleThrPheSerAlaCluVaΙΑ 1aGluLeuTyrArgLeuGluLeu ATTACCTTTAGTGCAGAGCTGGCAGAACTATACAGATTGGAGTTG j 1400. ... .!
GlyAspTyrLysLeuVa1Glul 1eThrProl1eGlyPheAlaProI ' GGAGATTATAAATTCGTACAAATAACACCAATTCCCTTCGCACCTl 35 DK 175959 B1 68GlyAspTyrLysLeuVa1Glul 1eThrProl1eGlyPheAlaProI 'GGAGATTATAAATTCGTACAAATAACACCAATTCCCTTCGCACCTl 35 DK 175959 B1 68
ThrLysGluLysArgTyrSerSerAIaHisGlyArgHisThrArgThrLysGluLysArgTyrSerSerAIaHisGlyArgHisThrArg
ACAAAAGAAAAAAGATACTCCTCTGCTCACGGGAGACATACAAGAACAAAAGAAAAAAGATACTCCTCTGCTCACGGGAGACATACAAGA
. 1500 i. 1500 i
ClyValPheValLeuGlyPheLenGlyPheLeuAlaThrAlaCly . CGTGTGTTCGTGCTAGGGTTCTTGGGTTTTCTCGCAACAGCACGTClyValPheValLeuGlyPheLenGlyPheLeuAlaThrAlaCly. CGTGTGTTCGTGCTAGGGTTCTTGGGTTTTCTCGCAACAGCACGT
c · " · · * 3 SerAlaKetGlyAlaArgAlaSerLeuTbrValSerAlaGInSerc · "· · * 3 SerAlaKetGlyAlaArgAlaSerLeuTbrValSerAlaGinSer
TCTGCAATGGGCGCTCGAG CGTCCCTGACCGTGTCGGCTCAGTCCTCTGCAATGGGCGCTCGAG CGTCCCTGACCGTGTCGGCTCAGTCC
• · · 16 00 m · i• · · 16 00 m · i
ArgThrLeuLeuAlaGlylleValGlnGlDGlnGlnGlnLeuLeu CGCACTTTACTCGCCGGGATAGTGCAG CAACACCAACAG CTGTTG • · · · AspValValLysArgGlnGluGluLeuLeuArgLeuThrValTrp 10 CACGTCGTCAAGAGACAACAAGAACTGTTGCGACTGACCGTCTGGArgThrLeuLeuAlaGlylleValGlnGlDGlnGlnGlnLeuLeu CGCACTTTACTCGCCGGGATAGTGCAG CACACCAACAG CTGTTG
17001700
ClyThrLysAsnLeuGlnAiaArgValThrAlalleCluLysTyr' CGAACGAAAAACCTCCACGCAAGAGTCACTCCTATACACAAGTAC.ClyThrLysAsnLeuGlnAiaArgValThrAlalleCluLysTyr 'CGAACGAAAAACCTCCACGCAAGAGTCACTCCTATACACAAGTAC.
• % » · LeuGlnAspGlnAlaArgLeuAscSerTrpGlyCysAlaPbeArg CTACAGGACCÅGGCGCGCCTAAATTCATCGGGATGTGCGTTTAGA•% »· LeuGlnAspGlnAlaArgLeuAscSerTrpGlyCysAlaPbeArg CTACAGGACCÅGGCGCGCCTAAATTCATCGGGATGTGCGTTTAGA
· · * . « 1800· · *. «1800
GloTaICysBisTbrThrValProT rpValAsoAs pSerLeuAla CAAGTCTGCCACACTACTGTACCATGGGTTAATGATTCCTTAGCAGloTaICysBisTbrThrValProT rpValAsoAs pSerLeuAla CAAGTCTGCCACACTACTGTACCATGGGTTAATGATTCCTTAGCA
• · · · ·,• · · · ·
ProAspTrpAs pAs nHetTbrTrpGlnGluTrpGluLysGlcVal CCTGACTGGGACAATATGACGTCGCAGGAATGGGAAAAACAAGTCProAspTrpAs pAs nHetTbrTrpGlnGluTrpGluLysGlcVal CCTGACTGGGACAATATGACGTCGCAGGAATGGGAAAAACAAGTC
# 9 · · ·# 9 · · ·
XrgTyrLeuGluA laAs ni1eS erLy s S e rLeuGluGlnÅlaG In 20 CGCIACCTGGAGGCAAATATCAGTAAAAGTTTAGAACACGCACAAXrgTyrLeuGluA laAs ni1eS erLy s S e rLeuGluGlNaL In 20 CGCIACCTGGAGGCAAATATCAGTAAAAGTTTAGAACACGCACAA
1900 .1900.
IleGlnGlnGlulysAsnMetTyrGluLeuGlnLysLeuAsnSerIleGlnGlnGlulysAsnMetTyrGluLeuGlnLysLeuAsnSer
ATTCAGCAAGACAAAAATATGTATGAACTACAAAAATTAAATAGCATTCAGCAAGACAAAAATATGTATGAACTACAAAAATTAAATAGC
• i · · «• i · · «
TrpAspIlePbeGlyAsnTrpPbeAspLeuThrSerTrpValLya TCGGATATTTTTGGCAATTGGTTTGACTTAACCTCCTCGGTCAAC 25 · 2000 lyrlleGinTyrGlyValLeuIlelleValAlaVallleAlaLeuTrpAspIlePbeGlyAsnTrpPbeAspLeuThrSerTrpValLya TCGGATATTTTTGGCAATTGGTTTGACTTAACCTCCTCGGTCAAC 25 · 2000 lyrlleGinTyrGlyValLeuIlelleValAlaVallleAlaLeu
IATATTCAATATGGAGTGCTTATAATAGTAGCAGTAATAGCTTTAIATATTCAATATGGAGTGCTTATAATAGTAGCAGTAATAGCTTTA
* · I t ·* · I t ·
ArglleVallleTyrValValGlnHetLeuSerArgLeuArgLys ACAATAGTGATATATGTAGTACAAATCTTAAGTAGGCTTAGAAAG . . 2100 . .ArglleVallleTyrValValGlnHetLeuSerArgLeuArgLys ACAATAGTGATATATGTAGTACAAATCTTAAGTAGGCTTAGAAAG. . 2100. .
dlTytArgProValPheSerSerProProClyTyrlleGloGln CCCTATAGCCCTGTTTTCTCTTCCCCCCCCGGTTATATCCAACACdlTytArgProValPheSerSerProProClyTyrlleGloGln CCCTATAGCCCTGTTTTCTCTTCCCCCCCCGGTTATATCCAACAC
35 DK 175959 B1 69 • t35 DK 175959 B1 69 • t
. 11 eE is 11 en isLy sA spArgG lyGloPr oAl aAstiG luGluThr ATCCATATCCACAAGGACCCCGGACAG.CCAGCCAACGAAGAAACA. 11 eE is 11 and isLy sA spArGG lyGloPr oAl aSTiG luGluThr ATCCATATCCACAAGGACCCCGGACAG.CCAGCCAACGAAGAAACA
2 2 0 0Λ2 2 0 0Λ
GluGluAspGlyClySerAsr.GlyGlyAspArgTyrTrpProTrpGluGluAspGlyClySerAsr.GlyGlyAspArgTyrTrpProTrp
GAACAAGACGGTGGAAGCAACCGTCGACACAGATACTGCCCCTCGGAACAAGACGGTGGAAGCAACCGTCGACACAGATACTGCCCCTCG
C « · · ‘ · · !· ProIleAlaTyrlleRisPheLeuIleArgGlnLeuIleArgLeuC «· · '· ·! · ProIleAlaTyrlleRisPheLeuIleArgGlnLeuIleArgLeu
CCCATAGCATATA.TACATTT CCTGATCCGCCACCTGATTCGCCTCCCCATAGCATATA.TACATTT CCTGATCCGCCACCTGATTCGCCTC
• t · . ’ · ' t · J• t ·. 'T
LeuThrArgLeuTyrSerlleCysArgAspLeuLeuSerArgSer TTCACCAGAC7ATACAGCATCTGCAGCCACTTACTATCCAGGAGC 2300LeuThrArgLeuTyrSerlleCysArgAspLeuLeuSerArgSer TTCACCAGAC7ATACAGCATCTGCAGCCACTTACTATCCAGGAGC 2300
PheLeuThrLeuGlnLeuI1eTyrCInAsnLeuArgA spTrpLeu 10 TTCCTGACCCTCCAAC VCATCTACCAGAATCTCAGAGACTGGCTGPheLeuThrLeuGlnLeuI1eTyrCInAsnLeuArgA spTrpLeu 10 TTCCTGACCCTCCAAC VCATCTACCAGAATCTCAGAGACTGGCTG
• · « «• · ««
ArgLeuArgThrAlaPheLeuGlalyrGlyCysGluTrpZleGla AGACTTAGAACAGCCTTCTTGCAATATGGGTGCCAGTGGATCCAA· 2400 . . .ArgLeuArgThrAlaPheLeuGlalyrGlyCysGluTrpZleGla AGACTTAGAACAGCCTTCTTGCAATATGGGTGCCAGTGGATCCAA · 2400. . .
GluAlaPtieG InAl aA I aAlaArgA 1 aTfcrAr gG LuThrLeuAla GAAGCATTCCAGGCCGCCGCGAGGGCTACAAGAGAGACTCTTGCGGluAlaPtieG InAl aA I aAlaArgA 1 aTfcrAr gG LuThrLeuAla GAAGCATTCCAGGCCGCCGCGAGGGCTACAAGAGAGACTCTTGCG
15 · · ♦ ·15 · · ♦ ·
GlyAlaCysArgGlyLeuTrpArgValLeuGluArglleGlyArg GGCGCGTGCAGGGGCTTGTGGAGGGTATTGGAACGAATCGGGAGGGlyAlaCysArgGlyLeuTrpArgValLeuGluArglleGlyArg GGCGCGTGCAGGGGCTTGTGGAGGGTATTGGAACGAATCGGGAGG
25002500
GlyI1eLeuA1aVaIProArgArgI1eArgCloG1yAlaGlulle GGAATACTCGCGGTTCCAAGAAGGATCAGACAGGGAGCAGAAAXC • * · 2Q AlaLeuLeu*'**GlyThrAlaVa ISerAlaGlyArgLeuTyrGlu GCCCTCCTGTCAGGGACGCCACTATCAGCACGGAGACTTTATGåA . . . 2600 Tyr SerMe cG luGiyProSe r Se T.ArgLysG lyGluLysPheVa 1 TACTCCATGGAAGGACCCAGCAGCAGAAAGGGAGAAAAATTTGTA; • ·GlyI1eLeuA1aVaIProArgArgI1eArgCloG1yAlaGlulle GGAATACTCGCGGTTCCAAGAAGGATCAGACAGGGAGCAGAAAXC • * · 2Q AlaLeuLeu * '** GlyThrAlaVa ISerAlaGlyArgLeuTyrGlu GCCCTCCTGTCAGGGACGCCACTATCAGCACGGAGACTTTATGåA. . . 2600 Tyr SerMe cG luGiyProSe r See T.ArgLysG lyGluLysPheVa 1 TACTCCATGGAAGGACCCAGCAGCAGAAAGGGAGAAAAATTTGTA; • ·
GlnA.laThrLysTyrG lyGlnA.laThrLysTyrG ly
CACGCAACAAAATATGCACACGCAACAAAATATGCA
25 30 35 70 DK 175959 B125 30 35 70 DK 175959 B1
Som allerede angivet oven for beskriver opfindelsen naturligvis alle HIV-2 vira, hvis RNA er i besiddelse af lignende egenskaber, specielt GAG og ENV regionerne, der omfatter sekvenser med nucleotidsekvenshomologi på mindst 50%, fortrinsvis 70% og foretrukkent 90% med.de tilsvarende GAG og 5 ENV sekvenser af HIV-2 ROD.Of course, as already stated above, the invention discloses all HIV-2 viruses whose RNA possesses similar properties, particularly the GAG and ENV regions, comprising sequences with nucleotide sequence homology of at least 50%, preferably 70%, and preferably 90%, with the corresponding GAG and 5 ENV sequences of HIV-2 ROD.
Specielt beskriver opfindelsen cDNA fragmenter der koder henholdsvis for p16, p26 og p12, hvis opbygninger også er inkluderet i GAGRODN. Specielt beskriver den sekvenserne, der strækker sig: 10 fra nucleotid 1 til nucleotid 405 (kodende for p16); fra nucleotid 406 til nucleotid 1155 (kodende for p26); og 15 fra nucleotid 1156 til nucleotid 1566 (kodende for p12).In particular, the invention discloses cDNA fragments encoding p16, p26 and p12, respectively, whose structures are also included in GAGRODN. Specifically, it describes the sequences that extend: 10 from nucleotide 1 to nucleotide 405 (encoding p16); from nucleotide 406 to nucleotide 1155 (encoding p26); and 15 from nucleotide 1156 to nucleotide 1566 (coding for p12).
Specielt beskriver opfindelsen også cDNA fragmentet, der koder for gp140 indbefattet i ENVR, og som strækker fra nucleotid 1 til nucleotid 2574.Specifically, the invention also discloses the cDNA fragment encoding gp140 included in ENVR and extending from nucleotide 1 to nucleotide 2574.
i .i.
20 Opfindelsen beskriver ligeledes nucleotidsekvenser, der adskiller sig fra de foregående ved nucleotidsubstitutioner, idet der drages fordel af.degenerationen af den genetiske kode, så længe som substitutionerne ikke indebærer en modifikation af aminosyresekvenserne kodet af disse nucleotidsekvenser.The invention also discloses nucleotide sequences which differ from the foregoing by nucleotide substitutions, taking advantage of the generation of the genetic code as long as the substitutions do not involve a modification of the amino acid sequences encoded by these nucleotide sequences.
25 Herudover beskriver opfindelsen proteiner eller glycoproteiner, hvis aminosy-resekvenser svarer til de, der er angivet på de foranstående sider, samt ækvivalent peptider dvs. péptider, der fremkommer fra deforegåénde sider ved addition, substitution eller fjernelse af aminosyrer, som ikke påvirker de totale immunologiske egenskaber ved nævnte peptider.In addition, the invention discloses proteins or glycoproteins whose amino acid sequences correspond to those indicated on the foregoing pages, as well as equivalent peptides ie. peptides arising from defective sites upon addition, substitution or removal of amino acids which do not affect the overall immunological properties of said peptides.
3030
Specielt beskriver opfindelse omhylningsglycoproteinet, der udviser aminosy-resekvensen kodet af ENVRN.Specifically, the invention discloses the envelope glycoprotein which exhibits the amino acid sequence encoded by ENVRN.
71 DK 175959 B171 DK 175959 B1
Opfindelsen angår blandt andet et immunogent præparat, der er ejendommelig ved, at det omfatter dosisenheder af omhylningsantigen, specielt gp140 af HIV-2 viruset, således at det er muligt at administrere dosisenheder på fra 10 til 500, specielt fra 50 til 100 mg/kg legemsvægt 5The invention relates, inter alia, to an immunogenic composition, characterized in that it comprises dose units of envelope antigen, in particular gp140 of the HIV-2 virus, so that it is possible to administer dosage units of from 10 to 500, especially from 50 to 100 mg / kg. body weight 5
Endelig beskriver opfindelsen en fremgangsmåde til at frembringe en vilkårlig af ovennævnte angivne proteiner (p12, p16 eller p26) eller af proteinet, der udviser opbygningen gp140 eller en hvilken som helst forud bestemt del af disse proteiner, idet fremgangsmåden omfatter indførsel af den tilsvarende 10 nukleinsyresekvens i en vektor, der er i stand til at transformere en passende udvalgt værtscelle og at muliggøre udtrykkelse af det indførte i denne vektor, at den udvalgte vært transformeres af vektoren, der indeholder nukleinsyren, at den transformerede cellevært med den modificerede vektor dyrkes, og at man udvinder og renset det udtrykte protein.Finally, the invention discloses a method for producing any of the aforementioned proteins (p12, p16 or p26) or of the protein exhibiting the structure gp140 or any predetermined portion of these proteins, the method comprising introducing the corresponding 10 nucleic acid sequence in a vector capable of transforming an appropriately selected host cell and enabling expression of the introduced into this vector, that the selected host is transformed by the vector containing the nucleic acid, that the transformed cell host with the modified vector is grown, and that the expressed protein is extracted and purified.
1515
Metoder omhandlet i europæisk patentansøgning nr. 85 905513.9 indleveret 18. oktober 1985 til fremstilling af peptider eller proteiner bestående af eks-. pressionsprodukter af nukleinsyresekvenser stammende fra genomet af HIV-1 er også velegnede til produktion af nævnte peptider eller proteiner stam-20 mende fra HIV-2. Der henvises herved til denne europæiske patentansøgning som en speciel metode.Methods disclosed in European Patent Application No. 85 905513.9 filed October 18, 1985 for the preparation of peptides or proteins consisting of ex. expression products of nucleic acid sequences derived from the genome of HIV-1 are also suitable for the production of said peptides or proteins derived from HIV-2. This European patent application is hereby referred to as a special method.
Nedenfor er angivet molekylvægte af HIV-2 proteiner i forhold til molekylvægte af HIV-1.Below are molecular weights of HIV-2 proteins relative to molecular weights of HIV-1.
25 72 DK 175959 B125 72 DK 175959 B1
Molekylvægt af Molekylvægt af HIV-2 proteiner HIV-1 proteiner kd kd 5 hele gag 58,3 hele gag 55,8.Molecular weight of Molecular weight of HIV-2 proteins HIV-1 proteins kd kd 5 whole gag 58.3 whole gag 55.8.
p 16 15 p 18 14,9 p 26 27,6 p 12 15,8 env 98,6 env 97,4 10 ydre env 57,4p 16 15 p 18 14.9 p 26 27.6 p 12 15.8 env 98.6 env 97.4 10 outer env 57.4
Transmembran env 41,2 HIV-2 Mir og HIV-2 ROD er også deponeret i "National Collection of Animal 15 Cell Cultures" (ECACC) i Salisbury (England) den 9. januar 1987 henholdsvis under registreringsnumrene 87 011001 og 87 011002.Transmembrane env 41.2 HIV-2 Mir and HIV-2 ROD are also deposited in the "National Collection of Animal 15 Cell Cultures" (ECACC) in Salisbury (England) on January 9, 1987 under registration numbers 87 011001 and 87 011002 respectively.
Yderligere er plasmideme pROD35 og pROD27.5 deponeret i "National Collection of Industrial Bacteria (NCIB) i Aberdeen (England) den 9. januar 1987 20 henholdsvis under registreringsnumrene 12 398 og 12 399.In addition, plasmids pROD35 and pROD27.5 are deposited in the National Collection of Industrial Bacteria (NCIB) in Aberdeen (England) on January 9, 1987 20 under registration numbers 12 398 and 12 399 respectively.
Der henvises herved til de i denne ansøgning angivne litteraturstedér.Reference is made to the literature sites listed in this application.
LITTERATURLISTEBIBLIOGRAPHY
25 1. F. Barre-Sinoussi et al., Science 220, 868 (1983).1. F. Barre-Sinoussi et al., Science 220, 868 (1983).
2. L. Montagnier et al., Human Tcell leukemia Orlym-phoma viruses (Gallo, R.C., Essex, M.E., Gross, L., eds.) Cold Spring Harbor Laboratory, 30 New York, 363(1984).2. L. Montagnier et al., Human Cell Leukemia Orlymphoma viruses (Gallo, R.C., Essex, M.E., Gross, L., eds.) Cold Spring Harbor Laboratory, New York, 363 (1984).
3. M. Popovic, M. G. Sarngadharan, E. Read, R. C. Gallo, Science 224, 497(1984).3. M. Popovic, M. G. Sarngadharan, E. Read, R. C. Gallo, Science 224, 497 (1984).
73 DK 175959 B1 4. J. Levy et al., Science 225, 840 (1984).73 Levy et al., Science 225, 840 (1984).
5. P. Sonigo et al., Cell 42, 369(1985).5. P. Sonigo et al., Cell 42, 369 (1985).
5 7. J.W. Curran et al., Science 229, 1352 (1985).7. J.W. Curran et al., Science 229, 1352 (1985).
8. A. Ellrodt et al., Lancet i, 1383 (1984).8. A. Ellrodt et al., Lancet, 1383 (1984).
10 9. P. Piot et al., Lancet ii, 65 (1984).9. P. Piot et al., Lancet ii, 65 (1984).
10. F. Brun-Vezinet et al., Science 226, 453 (1984).10. F. Brun-Vezinet et al., Science 226, 453 (1984).
11. N. Clumeck et al., N. Engl. J. Med. 313,182 (1985).11. N. Clumeck et al., N. Engl. J. Med. 313, 182 (1985).
15 12. A. B. Rabson og Μ. A. Martin, Cell 40, 477 (1985).15 12. A. B. Rabson and Μ. A. Martin, Cell 40, 477 (1985).
13. S. Benn et al., Science 230, 949 (1985).13. S. Benn et al., Science 230, 949 (1985).
20 14. M. Alizon, Manuskript under udarbejdelse.20 14. M. Alizon, Manuscript in preparation.
' 15. F. Brun-Vezinet, ikke publiceret.'15. F. Brun-Vezinet, unpublished.
16. M.D. Daniel et al., Science 228,1201 (1985).16. M.D. Daniel et al., Science 228, 1201 (1985).
25 17. P. J. Kanki et al., Science 228,1199 (1985).17. P. J. Kanki et al., Science 228, 1199 (1985).
18. N. L. Letwin et al., Science 230, 71 (1985).18. N. L. Letwin et al., Science 230, 71 (1985).
30 19. A. Gatzar et al., Blood 55, 409(1980).19. A. Gatzar et al., Blood 55, 409 (1980).
20. D. Klatzmann et al., Science 225, 59 (1984).20. D. Klatzmann et al., Science 225, 59 (1984).
74 DK 175959 B1 21. L Montagnier et al., Virology 144, 283 (1985).21. L Montagnier et al., Virology 144, 283 (1985).
22. J. S. Allan et al., Science 228,1091 (1985).22. J. S. Allan et al., Science 228.1091 (1985).
5 23. F. CHIVel, manuskript under udarbejdelse.5 23. F. CHIVel, manuscript in preparation.
24. F. diMarzo Veronese et al., Science 229,1402 (1985).24. F. diMarzo Veronese et al., Science 229, 1402 (1985).
25; V. S. Kalyanaraman et al., Science 218, 571 (1982).25; V. S. Kalyanaraman et al., Science 218, 571 (1982).
10 26. I.S.Y. Chen, J. McLaughlin, J. C. Gasson, S.C. Clark og D.W. Golde,10 26. I.S.Y. Chen, J. McLaughlin, J.C. Gasson, S.C. Clark and D.W. Golde,
Nature 305, 502 (1983).Nature 305, 502 (1983).
27. F. Barin et al., Lancet ii, 1387 (1985).27. F. Barin et al., Lancet ii, 1387 (1985).
15 28. P. J. Kanki, J. Alroy, M. Essex, Science 230, 951 (1985).28. P. J. Kanki, J. Alroy, M. Essex, Science 230, 951 (1985).
29. H. Towbin et al., Proc. Natl. Acad. Sci. USA 76, 4350 (1979).29. H. Towbin et al., Proc. Natl. Acad. Sci. USA 76, 4350 (1979).
20 30. S. Wain-Hobson, P. Sonigo, O. Danos, S. Cole og M. Alizon, Cell 40, 9 (1985).20 30. S. Wain-Hobson, P. Sonigo, O. Danos, S. Cole, and M. Alizon, Cell 40, 9 (1985).
31. M. Alizon et al., Nature 312, 757 (1984).31. M. Alizon et al., Nature 312, 757 (1984).
Claims (77)
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|---|---|---|---|
| DK200500170A DK176253B1 (en) | 1986-01-22 | 2005-02-04 | New type of human immuno-deficiency virus, infections for T4 cells - and derived antigens, immunogens, monoclonal antibodies and nucleic acid sequences, e.g. for diagnosis of AIDS |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FR8600910 | 1986-01-22 | ||
| FR8600911 | 1986-01-22 | ||
| FR8600910A FR2593189B1 (en) | 1986-01-22 | 1986-01-22 | NEW RETROVIRUS THAT MAY CAUSE AIDS, ANTIGENS OBTAINED FROM THIS RETROVIRUS AND CORRESPONDING ANTIBODIES AND THEIR APPLICATIONS TO AIDS DIAGNOSIS |
| FR8600911A FR2593190B1 (en) | 1986-01-22 | 1986-01-22 | NEW RETROVIRUS THAT MAY CAUSE AIDS, ANTIGENS OBTAINED FROM THIS RETROVIRUS AND CORRESPONDING ANTIBODIES AND THEIR APPLICATIONS TO AIDS DIAGNOSIS |
| FR8601635A FR2593922B1 (en) | 1986-02-06 | 1986-02-06 | NEW RETROVIRUS THAT MAY CAUSE AIDS, ANTIGENS OBTAINED FROM THIS RETROVIRUS AND CORRESPONDING ANTIBODIES AND THEIR APPLICATIONS TO AIDS DIAGNOSIS |
| FR8601635 | 1986-02-06 | ||
| FR8601985A FR2594229B1 (en) | 1986-02-13 | 1986-02-13 | NEW LAV-II TYPE RETROVIRUS LIKELY TO CAUSE AIDS, ANTIGENS OBTAINED THEREFROM AND CORRESPONDING ANTIBODIES AND THEIR APPLICATIONS TO AIDS DIAGNOSIS |
| FR8601985 | 1986-02-13 | ||
| US83522886 | 1986-03-03 | ||
| US06/835,228 US4839288A (en) | 1986-01-22 | 1986-03-03 | Retrovirus capable of causing AIDS, antigens obtained from this retrovirus and corresponding antibodies and their application for diagnostic purposes |
| FR8603881A FR2596063B1 (en) | 1986-03-18 | 1986-03-18 | NEW RETROVIRUS THAT MAY CAUSE AIDS, ANTIGENS OBTAINED FROM THIS RETROVIRUS AND CORRESPONDING ANTIBODIES AND THEIR APPLICATIONS TO AIDS DIAGNOSIS |
| FR8603881 | 1986-03-18 | ||
| FR8604215A FR2597500B1 (en) | 1986-03-24 | 1986-03-24 | NEW RETROVIRUS THAT MAY CAUSE AIDS, ANTIGENS OBTAINED FROM THIS RETROVIRUS AND CORRESPONDING ANTIBODIES AND THEIR APPLICATIONS TO AIDS DIAGNOSIS |
| FR8604215 | 1986-03-24 | ||
| US91608086A | 1986-10-06 | 1986-10-06 | |
| US91608086 | 1986-10-06 | ||
| US93318486A | 1986-11-21 | 1986-11-21 | |
| US93318486 | 1986-11-21 | ||
| FR8700025 | 1987-01-22 | ||
| PCT/FR1987/000025 WO1987004459A1 (en) | 1986-01-22 | 1987-01-22 | Retrovirus of the hiv-2 type susceptible of inducing aids, and its antigenic and nucleic constituents |
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| DK493487D0 DK493487D0 (en) | 1987-09-21 |
| DK493487A DK493487A (en) | 1987-11-17 |
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| DK200500170A DK176253B1 (en) | 1986-01-22 | 2005-02-04 | New type of human immuno-deficiency virus, infections for T4 cells - and derived antigens, immunogens, monoclonal antibodies and nucleic acid sequences, e.g. for diagnosis of AIDS |
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| EP (2) | EP0320495B1 (en) |
| JP (7) | JP2611106B2 (en) |
| AR (1) | AR243931A1 (en) |
| AT (2) | ATE47725T1 (en) |
| AU (1) | AU601397B2 (en) |
| DE (3) | DE3752319T2 (en) |
| DK (2) | DK175959B1 (en) |
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| WO1987002892A1 (en) * | 1985-11-14 | 1987-05-21 | President And Fellows Of Harvard College | T-lymphotrophic virus |
| WO1988005440A1 (en) * | 1987-01-16 | 1988-07-28 | Institut Pasteur | Peptides having immunological properties 2-hiv-2 |
| DE3856595D1 (en) * | 1987-01-16 | 2009-01-22 | Pasteur Institut | HIV-2 and SIV-1 nucleotide sequences |
| WO1988007375A1 (en) * | 1987-03-23 | 1988-10-06 | Hiver Limited | Novel vaccines |
| SE8701765L (en) * | 1987-04-28 | 1988-10-29 | Statens Bakteriologiska Lab | METHOD OF ANALYSIS AND AGENTS FOR THIS |
| US4812556A (en) * | 1987-05-18 | 1989-03-14 | Virovahl | Synthetic peptide antigen for the detection of HIV-2 infection |
| FR2620030B1 (en) * | 1987-09-07 | 1990-03-23 | Transgene Sa | VECTOR FOR THE EXPRESSION OF PROTEINS OF HIV-2 VIRUS, A CAUSAL AGENT FOR AIDS, CELL CULTURE INFECTED OR TRANSFORMED THROUGH THIS VECTOR, PROTEINS OBTAINED, VACCINE AND ANTIBODIES OBTAINED |
| US5780038A (en) * | 1987-11-16 | 1998-07-14 | Roche Diagnostic Systems, Inc. | HIV-2 envelope polypeptides |
| DE3879881D1 (en) * | 1987-11-16 | 1993-05-06 | Hoffmann La Roche | RECOMBINANT HIV-2 POLYPEPTIDE. |
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| US4652599A (en) * | 1984-04-23 | 1987-03-24 | The United States Of America As Represented By The Department Of Health And Human Services | Method of continuous production of retroviruses (HTLV-III) from patients with AIDS and pre-AIDS using permissive cells |
| EP0201540B2 (en) * | 1984-10-18 | 2001-10-31 | Institut Pasteur | Envelope antigens of lymphadenopathy associated virus and their applications |
| US4600413A (en) * | 1984-12-10 | 1986-07-15 | Sundstrand Corporation | Centrifugal deaerator and pump |
| JP3105581B2 (en) * | 1991-07-03 | 2000-11-06 | 内橋エステック株式会社 | Planar temperature fuse |
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