HK1151554A - Hiv preventive vaccine based on hiv specific antibodies - Google Patents
Hiv preventive vaccine based on hiv specific antibodies Download PDFInfo
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- HK1151554A HK1151554A HK11105455.4A HK11105455A HK1151554A HK 1151554 A HK1151554 A HK 1151554A HK 11105455 A HK11105455 A HK 11105455A HK 1151554 A HK1151554 A HK 1151554A
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The present invention relates to methods of making an HIV vaccine, preventing HIV infection, and/or preventing the pathogenesis of HIV infection in an individual. In particular, the present invention provides HIV-specific antibodies that can be generated as an immune response in an individual that can bind to existing HIV subtypes and mutants selected after antiretroviral therapy. The present invention also relates to HIV-specific antibodies that recognize and bind to substantially all HIV subtypes.
Technical Field
Human immunodeficiency virus class 1 (HIV-1) is characterized by significant genetic variability, caused by the accumulation of mutations that arise during viral replication, and which may also be formed by recombination [1, 18, 24 ]. This high mutability of HIV-1 strains has led to the failure of chemotherapeutic approaches to HIV therapy [8 ]. Previous studies have shown that resistant viral variants develop rapidly in patients after different courses of antiretroviral therapy, even after combination therapy (highly active antiretroviral therapy (HAART)). These resistant viruses have specific changes in their protein conformation and structure. Mutations that allow HIV-1 to evade existing therapies are typically conserved and accumulated under selection of treatment conditions.
Treatment with anti-HIV-1 drugs does not completely prevent viral replication, allowing pre-existing drug resistant mutations to be selected and accumulated, and new mutations to be generated and accumulated, thereby bringing new vitality to the survival of the virus. Thus, all existing antiretroviral drug formulations (nucleoside reverse transcriptase inhibitors (NRTI), non-nucleoside reverse transcriptase inhibitors (NNRTI), protease inhibitors, fusion inhibitors and even mixtures of different drugs, such as HAART) slow down HIV-1 replication only for more or less extended periods of time, [7] until the emergence and expansion of resistant viral strains. The widespread spread of HIV-1 resistant variants, resistant to commonly used anti-HIV therapies, has become a serious problem, particularly in economically developed countries, since HIV-infected patients in developed countries often receive antiretroviral therapy [8 ].
In the history of 25 years of research on HIV, various vaccine development strategies for HIV immunotherapy have been proposed and their practical application has been investigated. These protocols can be classified as follows, depending on the active components of the vaccine, the mechanism of action, and the method of preparation of the vaccine:
class 1: HIV/AIDS vaccine based on HIV specific monoclonal antibody;
class 2: a vaccine based on disrupted HIV virions;
class 3: HIV peptide-based vaccines; and
class 4: DNA plasmid or viral vector vaccines (adenovirus, adeno-associated virus, fowlpox, vaccinia, etc.) of genes encoding HIV peptides.
Class 1: HIV/AIDS therapeutic vaccines based on HIV specific monoclonal antibodies, including neutralizing antibodies such as monoclonal antibodies (mAbs) or cocktail combinations of 2-3 HIV-neutralizing monoclonal antibodies (cocktails) [5, 14, 28 ].
The first mechanism discovered for HIV infection was the entry of the virus into lymphocytes or other host cells via the CD4 receptor and the CCR5 and CXCR4 co-receptors. The structure of the HIV envelope protein was then studied (fig. 10a-b) and established the variability of gp120 loop 3D structure and the principle that formation of gp120-gp41 complex plays a critical role in recognition and adhesion of CD4 and co-receptors. Monoclonal antibodies capable of finding the viral env protein and binding to epitopes responsible for entry of HIV into cells, or capable of binding to the CD4 receptor and to co-receptor related domains or epitopes and thereby understandably capable of blocking the HIV infection process or cell binding, are called HIV neutralizing antibodies.
A major problem in antibody-based vaccine development is that recombinant antibodies elicited by certain HIV antigens are unable to neutralize different HIV-1 isolates, also due to HIV genetic variability. Most anti-HIV-1 monoclonal antibodies elicited by immunization have no or only poor cross-neutralizing activity and usually the determinants to which they bind are either virally different due to mutation or insufficiently exposed on the surface of the infectious virion. Although a number of different neutralizing monoclonal antibodies have been made, subsequent clinical trials have demonstrated that vaccines based on neutralizing antibodies against gp120 and gp41 are no longer effective within 1-2 months (in few cases they are no longer effective from the very beginning of their effect), due to the same-variations and changes in the surface epitopes of the HIV proteins of interest.
The drawbacks of the aforementioned vaccine development protocols reside in the method of monoclonal selection of antibodies to viral antigens for immunization of animals. Even if a series of neutralizing antibodies are made that specifically target different variants of the viral target protein, each monoclonal antibody is produced in a bacterial system in a recombinant monoclonal manner. Furthermore, the antibodies recombinantly produced by prokaryotes have at least 10-fold lower affinity for their antigen compared to the natural antibodies in animal or human serum. Polyclonal HIV-specific immunoglobulins produced in animals are often immunotoxic to other organisms such as humans. They can be used for diagnostic purposes, but they are likely to produce allergic reactions, which is a natural limitation of their application in immunotherapy. Hybridoma monoclonal antibody production techniques do not account for differences in immunoglobulins in biological samples. The techniques for producing humanized or chimeric monoclonal antibodies are laborious, time-consuming and expensive. Thus, using this technique, it is not possible to produce variants of the ten or hundred monoclonal antibodies for use in anti-HIV immunotherapy.
Class 2: vaccines based on disrupted HIV virions [9, 20 ]. The idea of using natural HIV virions and HIV peptides has been proposed 15 years ago and is reequipped in a variety of forms. One such approach is the use of beta-propiolactone, psoralen, or similar substances that are recognized as lethal to small viruses but less destructive to peptide bonds and protein conformations, and which retain the infectious activity of HIV viral particles. It was soon discovered that the concentration of native virus obtained from the patient's blood stream by ultrafiltration did not provide a useful quantity of virus for immunization nor did it provide a material for research and analysis. Thus, practical alternatives to such vaccines are in vitro infection-culture of the experimental strain, or infection with a primary isolate and culture in donor lymphocytes. In both cases large scale production in hundred liter fermenters is required to provide the large number of virus particles necessary to generate the immune response to the HIV proteins following immunization.
This idea is not entirely miscalculated itself, and it has advantages even over the other three vaccines. First, such asThe safety of using inactivated virus particles for immunization becomes even more evident if the RNA copy number of HIV is quantified in real time after ultrafiltration in a Sucrose Gradient (Sucrose Pilot Gradient). The viral RNA is mostly broken down into small fragments down to a level 10 lower than the actual concentration of HIV virions or their proteins obtained after sucrose gradient concentration4-105. Second, the access to native viral proteins appears to have a greater opportunity to cover the diversity of existing epitopes of the HIV env protein. The latter is the true reason why such vaccines have never been effective.
The development of a vaccine based on disrupted HIV virions is a best example and can illustrate how the in vitro conditions of genetic mutation selection differ from the same process range (bounds) in animal or human organisms. Analysis of viral peptides has shown that not only are different virus subtypes highly variable in antigenic epitopes, but even multiple virus variants isolated from the same patient. However, all laboratory strains, including the highly infectious BIII, a455, have constant and more uniform components of the env peptide sequence. Mass spectrometry or 3D structural method analysis showed that the diversity of env peptide libraries of laboratory HIV strains was only 5% of that of equivalent material obtained from one patient. The same trend was observed for primary HIV isolates co-cultured in vitro with donor blood lymphocytes, or with human cell cultures carrying CD4, CCR5, or CXCR 4. This means that the conditions for selection of in vitro viral infection are very different from the natural replication and virion formation of the virus in the organism, and that the survival pathway of the virus in the human organism is 95% wider than that during in vitro culture. Thus, all attempts to produce virions for use in the preparation of anti-HIV vaccines by large scale in vitro production followed by inactivation have failed, as have peptide-based vaccines derived from laboratory HIV strains.
Class 3: HIV peptide-based vaccines [3, 6, 13, 15, 27, 33, 36 ]. This modern vaccine class includes small HIV peptides, a variety of small 15-20 amino acid fragments of larger HIV proteins that mimic the epitopes of viral proteins responsible for receptor recognition and infection activity, as well as combinations of these small peptides. As one of the members of the small lentivirus family, HIV consists of a small number of peptides (18 peptides in total), the main HIV peptide vaccine includes the gp120 fragment (gp40, gp160) or two env proteins gp120 and gp41, the others include a small easily maintained matrix peptide and the p24 fragment. Other parts of this class are the full-length env peptides produced in yeast or large fragments thereof, or the so-called carbohydrate-based HIV vaccines, which carry glycosylation naturally occurring in the HIV life cycle. Some HIV peptide vaccines are intended for therapeutic immunization, and some claim to have prophylactic effects.
However, to date, neither cocktail combinations of recombinant HIV peptides nor synthetic 15-20 amino acid peptides have been resistant to viral infection and replication, the main reason for which can be revealed by the principles obtained by analysis of these peptides. The recombinant peptide sequence is obtained by automated DNA sequencing techniques, by RT-PCR of viral material from the patient to obtain samples and sequencing, including the steps of PCR amplification with long Taq polymerase (usually 1000-3000bp) to obtain HIV genomic fragments, or by PCR post-sequencing of DNA from the patient's lymphocytes with HIV specific primers and selection of transformed E.coli clones. The prior art is based on single cloning of HIV genotypes in a random manner (regime), with a frequency of selection from 105-106From the value at which the average infectious virus titer was 1%, the infectious virus was 10 in number3-104And (4) copying. It is well known to researchers who have performed HIV genome preparation and analysis themselves that the complete HIV genome data may vary widely between two sequences obtained by this technique from the same blood sample from the same patient. Thus, using these recombinant peptides, or even using cocktail combinations of 3-4 recombinant peptides that are properly glycosylated (carbohydrate) in eukaryotic expression systems, does not create an immune response to specifically inactivate currently-encountered viral variants.
The method of making small HIV peptides of synthetic amino acids [37] is controversial-hundreds of variants are prepared as mixtures in an automated peptide synthesizer and a mixture of many amino acids, if any, in the known HIV sequence is added in each cyclic step of peptide bond formation. A plurality of variants of the variable region of the env protein can be obtained by a peptide synthesizer. However, the size of these peptides is limited to 15-20, up to 30 amino acids, and longer peptides are only possible using recombinant systems. In practice, HIV immune responses can be boosted high enough with small synthetic peptides and their cocktail combinations, but with no or only low specificity. In addition, even if attempts were made to immunize animals (rhesus monkeys) with synthetic HIV peptides, the results were not satisfactory and no HIV-specific antibodies were found in their blood using standard ELISPOT procedures. There may be some hope that existing peptide-based HIV vaccines can be used in combination with HAART as a composition for treatment. However, to date, none of the peptide vaccines have shown efficacy in preventing HIV infection after immunization.
Class 4: DNA plasmid or viral vector vaccines (adenovirus, adeno-associated virus, fowlpox, vaccinia, etc.) of genes encoding HIV peptides [11, 12, 16, 21, 26, 29, 30 ]. Most of the 55 anti-HIV vaccines that have been licensed for 99 clinical studies worldwide belong to the DNA-based class. However, only one candidate vaccine passed the phase IIb clinical study and had some potential for entry into the phase III clinical study [37, 42 ]. The idea of using such vaccines has a healthy background, i.e. DNA immunization does not lead to immediate side effects such as autoimmune complications and allergic reactions, and therefore its clinical use is safe and simple. Despite this advantage, all viral and non-viral DNA vaccines suffer from a number of drawbacks, making them potentially very poor hope for anti-HIV effects.
Since DNA itself does not cause any immune response, the effectiveness of a vaccine is mainly reflected by three conditions, each of which is of equal importance:
1) transfection/infection efficiency, or how many cells can get genetic material from a single application of quantitative DNA;
2) expression level, or how much protein is expressed in the cell from which one or more gene copies are obtained;
3) the duration of the immune response, or the duration of time that the MHC elicits monoclonal antibodies that recognize the pathogen of interest.
In vitro transfection/infection efficiency measurements refer to the percentage of cells expressing the fluorescent protein or LacZ transformed simultaneously under the same conditions that are contained in cells expressing the existing protein counted 24 hours after gene transfer until the cells can enter the next division cycle. For non-viral plasmid vectors, efficiencies of 40-90% in vitro are possible, but the same vectors are only 1-5% efficient at best when administered intravenously in vivo. Of these 40-90% (1-5% in vivo) efficiencies, 98-99% are transient or episomal (episomal) expression that disappears after 2 weeks, while only 1-2% of the transfected genetic material can insert into the cell genome, providing long-term expression. Dosage of plasmid DNA vaccine [16]Limited to the maximum tolerated dose of the receptor for the delivery substance, which is cationic lipids and liposomes made therefrom, cationic polymers (polyethyleneimine, polylysine), pluronics (pluronic), and various combinations thereof. Virtually all cationic species that can bind to and carry negatively charged DNA are at 105-104The concentration of M has higher toxicity. The expression level of the non-viral vector is relatively high compared to the expression of the viral vector.
The infection efficiency of viral DNA vectors varies, but generally does not exceed 10-20% in vitro experiments. But are attractive because viral vectors can deliver genetic material directly to the genome. Therefore, although the infection efficiency of viral vectors is only 2-5% on average when administered in vivo, the expression of the target protein is mainly long-term, not transient. Thus, the viral DNA vector is considered to have sufficient persistence of immune response required for therapeutic or prophylactic action, and anti-HIV activity.
However, limitations in their expected activity can be observed by studying the components of viral DNA vaccines and their mechanism of action step by step. The first type of DNA vector to enter clinical studies is the adenovirus construct. Although their modern versions have shown infection efficiencies of less than 0 and the titers of monoclonal antibodies induced following their immunization are detectable by all immunochemical methods, they have never been used alone. The problem is that adenovirus-ADV 11 or AAV 29 produces only a relatively low level of expression of the transfer protein and is generally recognized only after two weeks of immunization using ELISA, INF-gamma ELISPOT or Western blot. If these ADV and AAV data are compared to the antibody titer at two weeks after standard immunization with any recombinant protein or protein mixture, it is evident that the absolute values for ADV and AAV vaccination are 5-10 fold lower. The researchers see these figures to draw some conclusions about the possible time of the immune response.
The only one vaccine composition that entered clinical stage III and was applied in thailand to 16000 uninfected individuals since 10 months 2003 was a combined (lined-up) immunization based on a plasmid DNA-gag-pol-env vaccine (aidvax B/E), followed by two vaccinations of Vaccinia Virus (Vaccinia Virus) -HIV vaccine (ALVAC-HIV). The test data of this patent show that in blood samples from vaccinated rhesus monkeys, the elicited antibody titers increased from one to three weeks after each immunization, while the remaining time of the one year period of immunization was slightly biased towards the positive plot (Plus plot) of the control values [12 ]. How to assess the persistence of the immune response in this case is a problem. It should also be noted that adenovirus and vaccinia virus are one of the largest viral families, exposing hundreds of their own proteins on their surfaces and in the viral matrix. This means that the immune response which is enhanced within a short period of time (one to two weeks) after administration is high, but most of them are nonspecific, and nonspecific causes side effects such as immunotoxicity.
The only exception to the effectiveness of viral vaccines is the approach based on retroviral (lentiviral) vectors [26 ]. HIV itself is a good representative from the lentivirus family. The infection efficiency of lentiviral vectors in vivo is sufficiently high (up to 5%), the gene proteins delivered by expression are sufficiently abundant and expressed for a long period of time, even if the expression is not stable, since it can infect the genome of the cell. As a cancer treatment vaccine, the retrovirus vector shows the antitumor activity in clinical research to be obviously superior to that of any other genetic construct. Only one feature of all retroviruses, including HIV, makes their therapeutic use questionable, while prophylactic use is not considered-that is, they can enter the human genome as mobile genetic elements, driving a variety of genetic mutations, a cascade of events that can become uncontrollable over time, leading to a variety of cancers in different cells and tissues.
The basic deficiency of DNA-based vaccines is derived from their original nucleotide sequence, which is obtained in the same way as the recombinant HIV peptide composition described above, e.g.by standard DNA sequencing after PCR and monoclonality, the average number of HIV genetic variations contained in the bloodstream of a patient can be understood as 105-106Variants, which are close to the true values. In this way one or more sequences are obtained in a random manner, and the genetic constructs thus obtained are in principle not effective against most HIV variants, even if they originate from the same patient. All plasmid DNA-based and any viral vector-based HIV vaccines are based on the sequence of individual env, pol, gag, and their pooled regions. Before these constructs consisted of monoclonal nucleotide sequences of HIV genomic regions, HIV vaccine development was a culprit. To combat HIV genetic variability and mutability, it is necessary to maintain a quantitative analysis of its existing variations and to prepare new vaccines against more frequently present variants.
As mentioned above, another major limitation of DNA-based HIV vaccines is the weaker immune response due to the imperfection of known methods of in vivo delivery of viral and non-viral gene therapy vectors. Described below is a suitable comparison that helps the academic scientist understand that DNA-based vaccine species are difficult to use to provide immunity against infection of any species. Please imagine hypothetical monoclonal antibodies (mAbs) to any protein or antigen, and their recombinantly linked L-H IgG chains produced in prokaryotic E.coli systems. We now compare the affinity of these two monoclonal antibody species for binding to antigen using all possible laboratory immunoreactive assays-ELISA, ELISPOT, immunodot blot, Western blot, flow cytometer, fluorescence microscopy, etc. In each picture we will see that the position of these species in one experiment-the affinity of the recombinant monoclonal antibody is always at least 10 times lower than that of the native animal monoclonal, and that the difference in minimal binding activity during titration can reach 100 to 200 times. If researchers analyze the activity of DNA-based and protein-based compositions for immunization of animals, the same will be found as in vivo evaluation of vaccine immunogenicity. The effectiveness of specific immune responses is determined by measuring the titer of monoclonal antibodies against existing antigens in the blood of immunized animals, and the effectiveness of specific immune responses of antigens delivered by genetic vectors is many times lower than that of the original protein antigens. DNA variants are typically 5 to 20 times less potent than "positive control" -protein variants in specific antigen immune responses.
There is also a small class of compositions described as potential HIV vaccine candidates-they are also known as dendritic vaccines. They were developed based on stem cell science and dendritic vaccines can be used in combination with chemotherapy or radiotherapy to treat a wide variety of tumors, albeit at a relatively high cost (an average cost of treatment for one patient of $ 4.5 to $ 6 ten thousand) but with modest therapeutic benefit. However, since dendritic cell-macrophage precursors are taught to identify and kill certain specific pathogens or microorganisms in situ in vivo, they can only be applied to the blood of the same patient by themselves, and their effectiveness for HIV treatment, and further for preventing infection, is highly questionable. The same rationale exists as to where to obtain viral peptides for "teaching" macrophages, where the sequence of recombinant viral peptides has been unchanged for many years, while native viral peptides require extremely high concentrations that cannot be isolated. Therefore, the use of dendritic cells cannot be considered as a reliable candidate for an anti-HIV vaccine.
The only possible approach to HIV-1 epidemic control is to create a vaccine that can prevent HIV-1 infection and/or prevent its pathogenesis by immunizing uninfected individuals, particularly representative populations of high risk groups. Such a vaccine must contain a mixture of epitopes of a single native HIV-1 peptide, namely the major HIV 1-1 envelope protein gp120, which is the only extrinsic protein of the viral surface, an epitope of a fragment thereof, and the gp41 peptide, which is the material in the env gp120-gp41 tetramer, with appropriate outer portions and/or epitopes that can be recognized by the immune system of the vaccinated individual. For the reasons mentioned above, these peptides cannot be natural peptides derived from viruses (pages 3-4, lines 13-30, 1-20). For recombinant polypeptides, correct sequence information should be provided. We have developed an alternative approach to HIV vaccine development, the specific details of which are described in this patent application, including the study of env sequences based on:
1) collecting and purifying natural virus peptides by using a phage display reverse panning technique (reverse panning technique);
2) subsequent quantitative and sequencing analysis of the native viral peptides using a panel of liquid chromatography-mass spectrometry (LC-MS) methods provides sequence information about gp120 and fragments thereof, which are present in most variants of the current viral cohort of HIV-infected individuals;
3) reconstitution of native env peptide epitopes with leishmania systems for recombinant production of env peptides with the same glycosylation as HIV and eukaryotic cells;
4) an HIV prophylactic vaccine composition is made using a method of stereostable liposome packaging or virosomes (virosomes) with immunogenic env peptides, providing a) an extension of the necessary immune response potentiation period, and b) control of immunotoxicity.
Proteomic analysis of gp120, which has been done so far, is rare and incomplete because of the lack of variants of the native peptide purified from cocktail combinations of other viral peptides and cellular proteins. This problem can be solved by affinity adsorption of the viral env peptide on a column with sufficient absorption capacity by reverse panning techniques. Before making a vaccine composition for immunization against HIV infection, it is necessary to select the isoforms of the env peptide that are predominantly present in the current viral group of HIV-infected individuals.
Although the genetic variants of individual patients have a mean variability of up to 105However, the most adapted and more infectious viability variants will be selected in each individual infected. Epidemiological variability data demonstrate that transmission of HIV variants has regional limitations, sexual transmission, or dependence on exposure to drugs Injected (IDU) transmitted individuals due to the presence of genetic sequences. The number of dominant viral peptide variants is much smaller than the number of genetic variants, although the number of dominant variants may change sufficiently rapidly. Moreover, the nucleotide sequence does not give information about which variants are dominant and susceptible to infection, and can only be shown by quantitative and sequence analysis in proteomics. The method we tried was liquid chromatography ion electrospray mass spectrometry.
The native gp120HIV peptide is highly immunogenic, but in order for the recombinant variant to also have the same level of immunogenicity without loss of epitopes, it is necessary to use a recombinant system with similar glycosylation. Cell culture, yeast culture, and leishmania systems can be used to address this problem. Recombinant peptides produced in eukaryotic cell culture are low in yield because they themselves have a large amount of cellular proteins-on average tens of millions of times that of E.coli. Yeast culture provides sufficient yield but the carbohydrate compounds in yeast are not as similar to eukaryotes and HIV as previously thought. Therefore, we selected Leishmania system with inducible high expression and with glycosylation pattern specific to eukaryotes. The recombinant gp120 variants produced in Leishmania provide a 100% HIV-specific high immune response, and the next stage is to prolong this response and thus prevent the development of infection.
Sterically stabilized liposomes can be used as peptide vaccine vectors in two possible ways: one is encapsulation of the peptide into the aqueous component of the liposome carrier, and the other is conjugation of the peptide to the distal end of activated polyethylene glycol (PEG) and presentation on the liposome surface. In both cases, the env peptide is protected from rapid cleavage and degradation by proteases, so that the immune activation phase is prolonged. The sterically stabilized liposomes are themselves non-toxic and harmless. These vectors retain the immunogenic peptide encapsulated over a period of weeks or months, and release their contents gradually over a sufficiently long period of time, rather than once. This allows the use of a greater amount of protein in a single vaccination. When HCC is provided with longer permanent contact with foreign proteins, a stronger and longer lasting immune response can be developed. This may be critical to the prevention of HIV infection and the successful development of vaccines.
Finally, it is important to keep in mind with respect to HIV vaccine candidates that no existing in vivo model is sufficient to evaluate its efficacy in preclinical evaluations. All attempts to use gorilla as a model for HIV infection and further to treat with developed antiretroviral chemotherapeutic drugs are convincing and effective, but it is not possible to evaluate the immune response against HIV in orangutan or rhesus monkeys. The immunogenic response elicited in apes and monkeys is very different in response spectrum from the immune response generated by immunization in humans with the same antigen. Moreover, gorilla, for example, can be infected by any HIV subtype, and can survive for many years without incident and without the symptoms of disease onset following infection with viral loads lethal to humans, and the same is true for monkey virus infections. Thus, for any test of an anti-HIV immunogenic composition, normal laboratory mice are no worse than simians, but can be used in statistically desirable quantities and there are more frequently used blood immunization experiments. Clinical studies can only prove whether the current new HIV vaccines can provide immunoprotective effects.
Drawings
FIG. 1: analysis of B lymphocytes from HIV-infected individuals using CD-45 monoclonal antibody and, confocal microscopy:
a, b) isolating a "good" source of HIV-specific monoclonal antibody RNA;
c, d) a very "bad" source in patients in the advanced stages of disease development (AIDS);
e) t and B lymphocytes from infected human blood, transparency scans (transplency scanning);
FIG. 2: according to a preferred embodiment of the method of the invention, a schematic representation of the process of obtaining a phage DNA library;
FIG. 3: according to a preferred embodiment of the present invention, there is shown a schematic diagram of a protocol for selecting clones producing positive antibodies by ELISA technique;
FIG. 4: forming and panning a recombinant phage library;
FIGS. 5 a-b: the structure of recombinant helper M13 phage, the "head" of which presents an enriched HIV env peptide-specific antibody library. Contact mode analysis by scanning probe microscopy (SPM or AFM) was performed using a Nano Wizard (JPK Instruments, Germany) based on Nikon Eclipse 2000U using a CSC 17/noA1 cantilever (sting cantilever) with a resonance frequency of 12 kilohertz (MicroMash, Irania).
The average length of the phage was 800nm, the thickness was 40-50nm, and the HIV-specific ScFv library was presented with 2-10 antibody molecules per phage particle; the average size of this "head" was measured to be 200-250 nm.
a) Recombinant M13 phage and its "head" presented with HIV-specific antibody libraries;
b) m13Ko7 helper phage control.
FIGS. 6 a-b: the structure of ultra-large pore epoxy activated monolithic affinity column (affinity macroreticular adsorbed column) used in the reverse elutriation technique. Scanning Probe Microscopy (SPM) contact mode analysis was performed using a Nano Wizard contact pattern, with CSC 17/no A1 spiking the cantilever.
a) Ultra-large pore epoxy activated monolithic adsorbents prior to recombinant phage insertion
b) Ultra-large pore epoxy-activated monolithic adsorbents after M13 monoclonal antibody intercalation, with HIV-specific ScFv libraries of displayed recombinant phage;
FIGS. 7 a-b: reverse panning technique for collection of HIV env peptides:
a) case of fractions eluted from RP affinity column (subtype A pool isolate, concentrated by PEG precipitation followed by ultracentrifugation at 100000g in sucrose gradient). Detecting whether the peaks A and B contain specific env peptides by using a polyclonal antibody against HIV by using a Western blotting method;
b) profile of fractions eluted from RP affinity column (subtype a pool isolate, supernatant concentrated by ultrafiltration). Peaks were detected by Western blotting using polyclonal antibodies against HIV.
FIGS. 8 a-b: fractions of the pool of env peptides of the HIVA subtype eluted from the reverse-panning column were analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting (ECL detection).
a) 1-high range marker; 2-4 th component, 3-5 th component, 4-6 th component, 5-7 th component, 6-8 th component, 7-11 th component, 8-9 th component, all experiments were made with beta mercaptoethanol (beta-ME).
b) 1-1 st component comprising β -ME; 2-2 nd component containing beta-ME; 3-HIV-PEG, containing β -ME; 4-HIV precipitate, containing β -ME; 5-HIV supernatant, containing β -ME; 6-high range marker; 7-fraction 1, no beta-ME; 8-2 nd component, no beta-ME; 9-6 th component, no beta-ME; 10-HIV-PEG, without beta-ME; 11-HIV precipitation, without β -ME; 12-HIV supernatant, free of β -ME;
FIGS. 9 a-c: reconstruction of the env signal peptide gp120 structure, using sequencing and 2D analysis:
a)#Al.RU.03.03RU20_06_13_AY500393
MKAKGMQRNYQHLWRWGXMLFWXIIM
b)B.RU.04.04RU128005_AY682547
MRARGIRKNYQGLLRWGTLLLGILMI
c)#B.RU.04.04RU129005_AY751406
MRAKGTRKNYQRLWRWGIMLLGMLMI
FIGS. 10 a-d: schematic 3D structure of HIV-1 envelope peptide.
a) Schematic 3D structure of gp120 core [40, 41 ];
b) 3D structural schematic of CD4-CCR5 binding epitope of gp120 [24 ];
c) 3D structural schematic of gp120 deformation in the formation of CD4 binding loops [22 ];
d) the structure and variability of the gp41 ectodomain (ectodomain) [34 ].
FIGS. 11 a-b: DNA fragments of HIV env peptide were amplified by PCR, these fragments encoding:
a) the entire gp120, the gp120 medial and lateral domains and the V2, V3, and V4 loops;
b) the entire gp41 and gp41 ectodomains.
FIG. 12: production of HIV env peptides and fragments thereof in different expression systems:
a) inducible expression of the gp120 medial domain, the gp41 ectodomain, SD-PAGE
b) Permanent expression of gp120, gp41, SDS-PAGE and ECL Western blot detection
FIG. 13: the N-glycosylation process of proteins in Leishmania tarentolae species (Leishmania tarentolae) cells (LEXSY expression system) is shown in comparison with the glycosylation of other protein expression systems. The glycosylation pattern obtained in mammalian cells and in Leishmania tarentolae species differs only in that the former has N-acetylneuraminic acid at the end of the sugar chain (Jena Bioscience GmbH).
FIG. 14: schematic representation of the pLEXSY I-2 vector family, containing cloning sites to replace a 1kb stuffer fragment with the target gene. The 5 'odc and 3' odc are regions of homologous recombination, and enter the host chromosome by homologous recombination after the expression plasmid is linearized with SwaI. Utr1 derived from 0.4k-IR of aprt gene of Leishmania tarentolae species, utr2 derived from 0.4k-IR of camcB gene, utr3 derived from 1.7k-IR, all of which are optimized adjacent to the untranslated region of the gene, providing splicing signals for post-transcriptional processing of mRNA for expression of the target gene and marker gene in LEXSY host T7-TR. SP represents the signal peptide of the secreted acid phosphatase LMSAP1(7) of Leishmania mexicana, and the H6 hexahistidine stretch. Other cloning strategies result in either cytoplasmic (c) or secretory(s) expression of the protein of interest. The 5' insertion site for cytoplasmic expression is BglII, NcoI, or SlaI, and for secretory expression SalI or XbaI. At the 3' end of the stuffer sequence, fusion to the C-terminal hexahistidine (His6) stretch can be achieved by restriction sites NheI, MspCI, or KpnI, while fusion to the His6 stretch can be avoided by using a NotI cloning site. Usable marker genes are the ble gene (bleomycin-resistant) and the neo gene (aminoglycoside phosphotransferase) (Jena Bioscience GmbH).
FIGS. 15 a-d: chromatographic purification step of HIV env recombinant peptide:
a) 6Hisp120id1 was expressed in E.coli (SDS-PAGE 5-20%);
b) purifying 6Hisp120id1 on a nickel-nitrilotriacetic acid (Ni-NTA) column;
c) 6Hisp120id1 was purified on a 10 μm 4.6X 50mm column of Q-PEEK (Waters, USA);
d) 6Hisp120id1 was purified by gel filtration chromatography on Superose 1210/300 GL before and after purification.
FIGS. 16 a-b: classes of liposomal adjuvants for HIV env recombinant peptides to enhance immune responses:
a)150nm, schematic representation of a PEG-400 stereostabilized liposome carrying recombinant HIVenv peptide in the aqueous portion of the vector;
b)200nm, schematic representation of PEG-2000 sterically stabilized liposomes conjugated with recombinant HIV env peptide at the PEG activated distal end.
FIG. 17: gaussian and Nicomp particle size distribution of SSL vaccine components: the average diameter of the support was 155 nm.
Detailed description of the invention
The present invention provides prophylactic vaccines for HIV, preferably HIV-1 subtype A or B, that elicit a specific immune response in individuals immunized with the vaccines provided herein, thereby providing protection. Thus, the active substance is a recombinant polypeptide/peptide mixture, which is prepared and selected according to the complex techniques described in detail below. The basic vaccine components are virus surface protein and envelope protein and fragments thereof, and according to a preferred embodiment, the basic vaccine components comprise HIV envelope proteins gp120, gp140, gp160 (figure 1) and gp41 with different glycosylation states, conserved structural domains of V1-V3 loop structures of gp120, antibodies of variable parts related to drug resistance of V1-V5 loops of gp120, and glycosylation variants of gp 41; the epitope of the envelope proteins gp120, gp140, gp160 of the virus that binds to CD4, with its adjacent loops V1/V2 and V3 undergoing conformational changes upon interaction of the CD4 receptor with the HIV-1 envelope spike (spike) and the outer portion of the gp41 protein; CXCR5 and CCR4 co-receptor binding sites in viral envelope proteins; different epitopes of p24 viral peptides.
These recombinant polypeptides and mixtures thereof are collected, identified, and cloned using recombinant phage display antibody libraries generated from mRNA from B lymphocytes from different donors. Each generated phage antibody library specifically binds to a different epitope of recombinant gp120, gp41 and native HIV polypeptides, and preferably also to epitopes present on recombinant gp140, gp160 and p24HIV-1 subtype A.
These recombinant phage antibody libraries can be used for different applications, such as detection, analysis and/or purification applications [23 ]. Applications using the above antibody libraries include, but are not limited to, immunoassays, immunoblots, chromatography, and the like.
The antibodies provided by the invention can also be used for developing novel medicaments for treating and/or preventing HIV.
In a preferred embodiment of the invention, recombinant antibodies displayed on the M13KO7 phage are used to develop HIV prophylactic vaccines.
Since the phagemid library displays antibody fragments that bind to essentially conserved conformational epitopes of HIV proteins, the antibodies can be targeted as vaccines against HIV infection because upon immunization, the individual's immune system can develop specific immune responses against these epitopes, e.g., mature B and T cells, which can eventually become memory cells, present in the individual to confer immunity.
An HIV vaccine according to the invention comprises recombinant gp41 and p24HIV-1 subtype A proteins and fragments of gp120, gp140 and gp160, which fragments (Table 9 in example 3) bind to the antibodies produced by the method of the invention, together with conventional carriers and adjuvants and optionally an immunostimulant.
The vaccine can prevent HIV infection and the further development of HIV infection because it provides the immune system of an individual with memory cells specific for an epitope that may be present on any HIV virus, but also on mutant HIV viruses.
The recombinant proteins and/or fragments are based on sequence information obtained by binding and analyzing native HIV-1 epitope proteins, which can be selected using HIV-specific antibodies obtained by the methods of the invention.
In particular, proteins, such as envelope proteins, are obtained from disrupted viral particles by suitable methods, such as by ultrafiltration and lysis of the viral particles.
Suitable proteins may be screened using any suitable screening method known to the skilled person. In a preferred embodiment, the screening may be performed by: i) performing phage panning using recombinant phage displaying antibodies that can collect viral envelope proteins; and/or ii) affinity adsorption with a plastic surface for culture to which HIV-specific antibodies are adhered; and/or iii) screening for viral envelope proteins by affinity chromatography using columns embedded with HIV-specific antibodies.
In the next step, the 3D conformation of the sequences and/or subtypes of the native viral proteins obtained and screened can be identified. In doing so, a mixture of variants of highly specific variable and/or constant fragments of viral proteins are available, such as gp120, gp41 and p24, which circulate in the bloodstream of individuals infected with HIV-1, as well as those individuals who have received antiretroviral therapy in different treatment regimens such as NRTI, NNRTI and HAART.
Based on the sequence, recombinant polypeptides and/or fragments of the viral protein are produced. These sequences may be obtained by any method suitable for producing polypeptides which are recognized by the immune system and which therefore elicit an appropriate immune response.
The recombinant polypeptide may be obtained by any suitable expression system, such as eukaryotic expression systems, e.g., leishmania inducible expression systems and yeast with eukaryotic-like glycosylation.
An exemplary and general technique for preparing a prophylactic vaccine for different variants of HIV-1 subtypes A and B of the present invention includes steps 1-9, described in more detail below:
1. establishing a human recombinant IgG phagemid library which contains an HIV-specific ScFv antibody fragment (phage display technology);
2. enrichment of recombinant phagemid libraries presenting ScFv fragments with HIV-specific antibodies (biopanning);
3. optionally, viral material not receiving antiretroviral therapy is amplified in situ using the PBMC-MT infection method;
4. concentrating the HIV particles and peptides; inactivating and destroying viruses;
5. collecting natural HIV env peptide by a phagemid library of recombinant ScFv showing HIV specificity through a reverse panning technology;
6. optionally, quantifying and sequence analyzing the variability and frequency of env peptides using a liquid chromatography-mass spectrometry (LC-MS) method;
7. optionally, cloning the major HIV env peptide, producing recombinant peptide for vaccine development in leishmania tarentolae species;
8. optionally, chromatographic purification and 3D structural analysis of the recombinant HIV env peptide;
9. compositions for enhancing immune responses in prophylactic vaccines for HIV are prepared, preferably using sterically stabilized liposomes or virosomes as carriers for vaccine delivery.
1. Establishing a human recombinant IgG phagemid library which contains an HIV-specific ScFv antibody fragment (phage display technology);
thus, in the method of the invention, a phagemid library can be established in step 1) according to stages i) to iii), comprising:
i) preparing DNA fragments amplified from RNA encoding light chain variable regions and heavy chain variable regions of IgGs expressed in B lymphocytes obtained from a plurality of individuals infected with HIV, respectively;
ii) combining the two DNA fragments of the light and heavy chains obtained in i) into a structure comprising nucleotides encoding the variable region of an IgG light chain in combination with nucleotides encoding the variable region of an IgG heavy chain;
iii) transformation into pCANTAB phagemid vector.
Specifically, B and/or T lymphocytes, which are known to be infected with HIV, and in which antibodies specific for HIV are believed to be present, are first isolated from a plurality of individuals. Individuals with drug resistant HIV variants may also be included. Isolation of B cells can be performed using any known technique, e.g., leukapheresis (Leukapherese) and subsequent isolation of B/T cells in a lymphocyte population [19 ]. Subsequently, RNA is isolated from B/T lymphocytes using techniques well known in the art, e.g., as described in [23 ].
Preferably, to create an ScFv library, mRNA sequences containing HIV-specific immunoglobulins are isolated. In this connection, the number of B lymphocytes is assessed prior to RNA isolation, for example, in a CD45 monoclonal antibody immunization experiment using confocal microscopy in the blood of HIV-infected persons. The data shown in figure 1 indicate that in some patients in the advanced stages of the disease and presenting symptoms of aids, the proportion of B lymphocytes in total isolated lymphocytes is low (figure 1c, d), and generally low CD45 immunostaining is associated with high viral load and a very low CD4: CD8 status. Unlike others (fig. 1a, b), these patients are a poor source of the virus cluster established by HIV-specific phagemid libraries. High viral loads or previous courses of antiretroviral therapy and their frequency do not limit the chances of obtaining HIV-specific ScFv libraries.
The total RNA thus obtained can be transcribed into cDNA by, for example, using oligothymidine (dT), or, according to a preferred embodiment, oligonucleotides as primers which specifically bind to the constant regions of the heavy and light chains of immunoglobulins. The sequences of the different constant regions of immunoglobulin heavy and light chains are well known in the art, and therefore appropriate primers for transcription into cDNA can be readily designed. In this way, a first round of selection of immunoglobulin transcripts can be performed in the RNA pool and different RNA samples from different donors will be easier to handle, since unwanted material can be excluded in the first step. In addition, it is foreseeable and preferred to combine pools of RNA from different donors before transcription of mRNA into cDNA, since a wider diversity can be obtained (cf. The complementary strand of the cDNA thus prepared can be synthesized according to a technique known in the art.
To prepare a sufficient amount of DNA fragments, mRNA, cDNA obtained from B/T lymphocytes, or double-stranded DNA prepared from cDNA can be directly used as a template to amplify a region of interest.
For the PCR reaction, suitable primers which bind to the 5 'and 3' ends of the nucleotide sequence to be amplified, which are typically oligonucleotides of 10 to 40, preferably 15 to 30, more preferably 20 to 30 nucleotides in length, may be used.
Reverse primer oligonucleotides may be used, the sequence of which is derived from the constant region of an immunoglobulin. Preferably, the reverse oligonucleotide primer hybridizes to the CH1 region of the heavy chain, or to the C λ or ck region of the light chains λ and κ, respectively. The forward primers used hybridized to the opposite ends of the heavy chain variable region and the light chain variable region.
In a preferred embodiment, the forward and reverse primers used for the initial PCR amplification are selected from the nucleic acid sequences shown in tables 1 to 3, which are taken from the V BASE database (http:// vbase. mrc-cpe. cam. ac. uk). The PCR reaction generally results in a fragment of about 750 a in length.
Table 1:oligonucleotide primer List for PCR amplification of human immunoglobulin light chain kappa
| # | Primer name and orientation | Nucleotide sequence (5 '-3') |
| 1 | V κ 1a Forward | RAC ATC CAG ATG ACC CAG |
| 2 | V.kappa.1b Forward | GMC ATC CAG TTG ACC CAG |
| 3 | V κ 1c Forward | GCC ATC CRG ATG ACC CAG |
| 4 | V κ 1d Forward | GTC ATC TGG ATG ACC CAG |
| 5 | V kappa 2a forward direction | GAT ATT GTG ATG ACC CAG |
| 6 | V kappa 2b Forward | GAT RTT GTG ATG ACT CAG |
| 7 | V kappa 3a Forward | GAA ATT GTG TTG ACR CAG |
| 8 | V kappa 3b Forward | GAA ATA GTG ATG ACG CAG |
| 9 | V kappa 3c Forward | GAA ATT GTA ATG ACA CAG |
| 10 | V kappa 4a Forward | GAC ATC GTG ATG ACC CAG |
| 11 | V kappa 4b Forward | GAT ATT GTG ATG ACC CAC ACT CC |
| 12 | V kappa 5a Forward | GAA ACG ACA CTC ACG CAG |
| 13 | V kappa 6a Forward | GAA ATT GTG CTG ACT CAG |
| 14 | V kappa 6b Forward | GAT GTT GTG ATG ACA CAG |
| 15 | C.kappa.1' reverse | ACA CTC TCC CCT GTT GAA GCT C |
Table 2:forOligonucleotide primer List for PCR amplification of human immunoglobulin light chain Lambda
| # | Primer name and orientation | Nucleotide sequence (5 '-3') |
| 1 | V lambda 1 a' forward direction | CAG TCT GTG CTG ACT CAG CCA CC |
| 2 | V lambda 1 b' forward direction | CAG TCT GTG YTG ACG CAG CCG CC |
| 3 | V lambda 1 c' forward direction | CAG TCT GTC GTG ACG CAG CCG CC |
| 4 | V lambda 2 forward direction | CAG TCT GCC CTG ACT CAG |
| 5 | V lambda 3a forward direction | TCC TAT GWG CTG ACT CAG |
| 6 | V lambda 3b forward direction | TCC TAT GAG CTG ACA CAG |
| 7 | V lambda 3c forward direction | TCT TCT GAG CTG ACT CAG |
| 8 | V lambda 3d forward direction | TCC TAT GAG CTG ATG CAG |
| 9 | V lambda 4 forward direction | CAG CYT GTG CTG ACT CAA |
| 10 | V lambda 5 forward direction | CAG SCT GTG CTG ACT CAG |
| 11 | V lambda 6 forward direction | AAT TTT ATG CTG ACT CAG |
| 12 | V lambda 7 forward direction | CAG RCT GTG GTG ACT CAG |
| 13 | V lambda 8 forward direction | CAG ACT GTG GTG ACC CAG |
| 14 | V lambda 4/9 forward direction | CWG CCT GTG CTG ACT CAG |
| 15 | V lambda 10 forward direction | CAG GCA GGG CTG ACT CAG |
| 16 | C lambda 2' reverse | TGA ACA TTC TGT AGG GGC CAC TG |
| 17 | C7' reverse direction | AGA GCA TTC TGC AGG GGC CAC TG |
Table 3:oligonucleotide primer List for PCR amplification of human immunoglobulin heavy chains (IgM, IgG, IgA)
In a next step, the two DNA fragments of the light and heavy chains obtained in i) are ligated into a structure comprising nucleotides encoding the variable region of the IgG light chain, which nucleotides are bound to the nucleotides encoding the variable region of the IgG heavy chain, thereby allowing the expression of a polypeptide comprising the variable region ScFv of each of the IgG light and heavy chains.
According to a preferred embodiment, to obtain specific linkages between the DNA fragments encoding the variable light and heavy chains, a quantity of the sample obtained in step i) is aliquoted, for example, in two portions and optionally diluted. The DNA fragments, either cDNA amplified from mRNA, cDNA or double-stranded DNA derived from cDNA, can be contacted individually with linkers specific for either the light chain or the heavy chain, respectively, such that the linkers bind only the corresponding DNA fragments of each sample fraction, i.e.a fraction of light chain-only linkers and a fraction of heavy chain-only linkers. The linkers used may be allowed to hybridize to each other under appropriate conditions, thereby obtaining a DNA fragment containing the light chain variable region and the heavy chain variable region. In addition, the linkage of the two DNA segments remains in the same reading frame (in frame), such that the combination of the two DNA segments will produce a polypeptide comprising the amino acid sequences of the light chain variable region and the heavy chain variable region. If desired, two heavy chains and two light chains can be obtained in the same manner.
Preferred primers are listed in tables 4 and 5.
Table 4:reverse oligonucleotide primer List for second round PCR amplification of human lambda and kappa light chain variable fragments
| # | Primer name and orientation | Nucleotide sequence (5 '-3') |
| 1 | J λ 235 reverse | TAG GAC GGT CAG CTY GGT CCC |
| 2 | J λ 7 reverse direction | GAG GRC GGT CAG CTG GGT GCC |
| 3 | J lambda 1 reversal | TAG GAC GGT GAC CTT GGT CCC |
| 4 | J lambda 6 reversal | GAG GAC GGT CAC CTT GGT GCC |
| 5 | J4 inverse | ACC TAA AAT GAT CAG CTG GGT TCC |
| 6 | J kappa 2 inversion | TCG TTT GAT CTC CAG CTT GGT CCC |
| 7 | J kappa 3 inversion | TCG TTT GAT ATC CAC TTT GGT CCC |
| 8 | J kappa 14 reverse | TCG TTT GAT YTC CAC CTT GGT CCC |
| 9 | J kappa 5 reverse | TCG TTT AAT CTC CAG TCG TGT CCC |
Table 5:oligonucleotide primer lists for PCR amplification and assembly of human immunoglobulin light and heavy chains
In a more preferred embodiment of the invention, the code ((Gly)4Ser)3A linker fragment of the polypeptide linker is added to the nucleotide sequences encoding the variable heavy and variable light chains of the immunoglobulin. The linker portions of the heavy and light chains bind to each other and the fill-in reaction (a fill-in reaction) is initiated in the presence of TaqSE DNA polymerase. Finally, the heavy and light chains are assembled into a single gene by their DNA linker fragment portions.
In doing so, a large number of artificially produced antibodies can be obtained by randomly linking nucleotides encoding a light chain variable region or a heavy chain variable region of one immunoglobulin to nucleotides encoding a light chain variable region or a heavy chain variable region of another immunoglobulin, respectively, which also contain a combination of light and heavy chains for accumulating antigen binding sites that were not present in the initially obtained RNA pool. It can be shown that antibodies with higher and constant affinity for HIV proteins than those naturally occurring in HIV-infected individuals can also be produced by combining, in a random fashion, already naturally preformed portions of the antigen binding sites on the immunoglobulin variable regions.
Additional restriction sites may also be introduced into the resulting DNA fragment, which may be useful in subsequent applications, for example, in cloning procedures. In principle, any suitable restriction enzyme site may be used as desired, without the appropriate site for CHO-Klose being outside the knowledge of the skilled person. The restriction enzyme site may be introduced by any suitable method known in the art, for example, using oligonucleotide primers containing the nucleotide sequence of the restriction enzyme site, or using linker molecules containing the nucleotide sequence of the restriction enzyme site, which are attached at the 5 'and/or 3' ends, respectively.
In a preferred embodiment of the invention, Sfi I and Not I restriction sites are introduced at the ends of the ligated nucleotide fragments, which according to a preferred embodiment may contain a light chain and heavy chain nucleotide sequence, wherein the restriction sites are used for further cloning steps into the vector. Sfi I and Not I restriction sites were added to the 5 'and 3' ends of the ligated fragment (ScFv gene), respectively. These specific restriction sites occur very infrequently in the antibody gene, allowing the majority of the resulting ligated fragments, e.g., nucleotide sequences containing light and heavy chains, to be cloned as a single Sfi I/Not I fragment. In a more preferred embodiment of the invention, the Sfi I and Not I restriction sites are introduced by oligonucleotide primers. Preferred oligonucleotide primers containing Sfi I site and Not I site can be designed based on the primer sequences in article [21 ]. Primers useful for introducing Sfi I and Not I restriction sites at the ends of the resulting ligated fragments containing light and heavy chain nucleotide sequences are shown in Table 6.
Table 6:list of oligonucleotide primers that introduce Sfi I and Not I restriction sites at the end of the assembled ScFv genes.
For cloning and expressing the obtained ligated fragments comprising the light and heavy chain nucleotide sequences, any suitable cloning and/or expression vector known to the person skilled in the art may be used. In a preferred embodiment, a phagemid vector is used, which includes, for example, pCANTAB 5 E.coli phagemid vector.
The phagemid pCANTAB 5E carries the replication origin of the M13 and ColE1 plasmids and can therefore be conveniently amplified either as plasmids or packaged into recombinant M13 phages with the aid of helper phages, such as M13KO 7. The antibody variable region genes after Sfi I and Not I digestion were cloned between the main body and leader sequence of M13 gene 3 of pCANTAB 5E phagemid vector. The obtained fusion protein retains the functions of the two parent proteins. The g3p leader sequence directs protein transport to the inner membrane/periplasm of E.coli, where the major domain of g3p attaches the fusion protein to the top of the phage under assembly. pCANTAB 5E also contains an amber stop codon at the interval between the cloned ScFv and the g3p sequence. The resulting pool of pCANTAB 5E plasmid derivatives containing the ScFv fragment was used to transform a supE strain of E.coli, e.g., TG 1. In the E.coli supE strain, translation continued through the amber stop codon in pCANTAB 5E, resulting in the expression of ScFv-g3p fusion protein at the phage tip.
Table 7:oligonucleotide primers for re-amplification of scFv fragment mixtures
| Primer and method for producing the same | 5 '-3' nucleotide sequence |
| VH12467SfiIReampl | TGC GGC CCA GCC GGC CSA G |
| VH35SfiIReampl | TGC GGC CCA GCC GGC CGA RG |
| JL1235NotIReampl | GAC TTG CGG CCG CTA GGA CG |
| JL4NotIReampl | GAC TTG CGG CCG CAC CTA AAA TG |
| JL67NotIReampl | GAC TTG CGG CCG CGA GGR C |
| JK1234NotIReampl | GAC TTG CGG CCG CTC GTT TG |
| JK5NotIReampl | GAC TTG CGG CCG CTC GTT TAA TC |
The recognition site for Not I restriction enzyme is labeled with blue; the recognition site of the Sfi I restriction endonuclease is marked with green.
In non-suppressor strains, such as HB2151, the stop codon can be recognized, so protein synthesis terminates at the end of the scFv gene, and the g3p fusion protein fails to be synthesized. In this case, the resulting ScFv protein is transported into the periplasmic space but cannot be assembled into phage particles because it lacks the gene 3 domain. However, soluble antibody fragments accumulate in the periplasm, and leak into the culture medium when the culture time is prolonged. Therefore HB2151 and similar E.coli strains were used to produce soluble antibodies after infection with selected antigen-positive phages, but could not be used for the purposes herein. The procedure for the establishment of the ScFv library is described in example 1.
2. Enrichment of recombinant phagemid libraries presenting ScFv fragments with HIV-specific antibodies (biopanning);
expression of the antibody can be carried out in a suitable host to obtain a polypeptide capable of binding the antigen, and thus the polypeptide is obtained using recombinant gp120, gp41 and native HIV polypeptides isolated from different donors. Thus, the expressed polypeptide may be more advantageous, although not required, if presented on the surface of the host. Suitable hosts for expressing the antibodies include viral systems, prokaryotic and eukaryotic cells and/or cell cultures.
In a more preferred embodiment, fragments of the antibodies are expressed in bacteriophage M13, forming a phage display library that allows a large number of different constructs to be displayed, each represented by a different phage, for use in phage display technology. The phage display method is a powerful tool for cloning immunoglobulin genes and expressing and detecting functional antibodies. It allows the heavy and light chain variable fragments of antibodies to be displayed as fusion proteins on the phage surface, forming a pool or library of HIV-specific antibodies, without going through the monoclonal antibody selection stage. This method allows for the rapid discovery of antibodies to any antigen and, if desired, the production of soluble variants with or without glycosylation in other expression systems.
Phagemid library panning is an in vitro technique that allows rapid screening of large numbers of clones, where phage displaying antibodies on their surface exhibit binding affinity for a selected HIV polypeptide, can be identified and used to maintain recombinant phagemids and produce new phage for further screening steps. Cross-and-cycle use of SDS-PAGE, Western blotting and ELISA screening methods in the art allows the analysis of binding affinities of phage-displayed antibody libraries to identify antigen-positive clones.
Since the displayed ScFv antibody fragments retain their antigen binding ability, recombinant phages expressing specific antibodies can be enriched by means of affinity screening. In this way, antibodies with defined specificity and affinity can be rapidly screened from a population. The obtained antibody gene library is screened to improve the antigen binding capacity. This technique, called panning, involves subsequent binding and harvesting of phage displaying an ScFv fragment specific for HIV using the following polypeptides:
i) recombinant env peptides of gp120, gp140, gp160, gp41 HIV-1 subtype A and B;
ii) native HIV env polypeptides isolated from different donors infected with HIV.
In a preferred embodiment, the number of displayed antibodies is from 10, due to the selection of a phage library that exhibits binding affinity for all of the above listed polypeptides7-1012Reduced to 102-103. Since the polypeptides are contacted with specific recombinant HIV polypeptides in separate cycles, wherein the sequences of those polypeptides are i) known and ii) constant, and with native HIV polypeptides isolated from different donors, wherein these polypeptides may have undergone mutation, it is possible to select antibodies that bind to essentially all known HIV mutants, indicating that the antibodies may recognize the HIV polypeptidesIn a substantially constant conformation.
Proceeding in this way, a library of recombinant antibodies specific for HIV, derived from a large pool of infected organisms, exhibiting binding affinity for the selected HIV polypeptide even when these polypeptides in the pool are mutated, can be selected using two different approaches:
i) standard biological elutriation process [4]
ii) solid state immobilized native HIV peptide is embedded in nitrocellulose membrane.
According to the first method i), to produce recombinant phages, 4X 10 were used10pfu of M13KO7 helper phage was added to a prepared logarithmic growth phase of transformed TG1 E.coli culture, pre-cultured for 1 hour and cultured in the presence of 100. mu.g/ml ampicillin and 50. mu.g/ml kanamycin at 37 ℃ for 12 hours with rotation at 250rpm (typical phage yield was 10 per ml)10To 1011Ampicillin transduction unit). The use of polypropylene tubing is recommended because phages may non-specifically adsorb on other plastic surfaces.
PEG precipitation was then performed. The bacterial cultures were centrifuged at 1000g for 10 minutes and the supernatants were collected and cooled. 1/5 volume ratio of 20% PEG/2.5M NaCl cold solution is added to the supernatant, at 0 degrees C were cultured for 60 minutes, then in Beckmann JA-20 rotor at 10000g, 4 degrees C centrifugal 20 minutes. The supernatant was discarded. The pellet (which may not be readily visible) was resuspended in 16ml of 2 XYT medium containing 0.01% thimerosal (chronosal). We recommend that, if storage is desired (at 4 ℃), the supernatant is filtered through a 0.45 μm filter. The solution containing the recombinant phage will be used for panning.
PEG precipitation and phage panning cycles should be performed as soon as possible after rescue (rescue) because some phage-displayed recombinant antibody preparations may be unstable. Logarithmic phase TG1 cell colonies in the basal medium plate were transferred to 5ml of 2 XYT medium and cultured overnight at 37 ℃ under shaking at 250 rpm. Then, 100. mu.l of overnight culture was inoculated in 10ml of fresh 2 XYT medium and cultured at 37 ℃ under shaking at 250rpm until the A600 of the culture reached 0.3.
Using a suitable buffer, e.g. PBS or 0.05M NaCO3(pH9.6), 5ml of the antigen solution was diluted to 10. mu.g/ml with it and coated to 25cm2The tissue culture flask of (1). The coating of the antigen can be performed at room temperature for 1-2 hours, or at 4 ℃ overnight. The conditions under which the culture dish is coated, such as buffer, culture temperature and time, depend on the antigen and should be similar to the conditions used in the immunoassay for the original polyclonal or monoclonal antibody from which the new recombinant antibody was derived. The concentration of antigen coating can vary depending on the desired affinity (antigen binding capacity) of the recombinant phage antibody. Antibodies with high affinity require a smaller amount of antigen than those with low affinity. However, for the isolation of antibodies with specific affinity, solution-based screening may be preferred over solid-phase screening because the amount of antigen used in the screening can be more precisely controlled.
Flasks were washed three times with PBS and emptied completely after each wash. The flask was then filled with blocking buffer to block any remaining sites on the surface of the flask, and then incubated at room temperature for 1 hour. The flasks were washed three more times with PBS and emptied completely after each wash.
The blocking buffer was freshly prepared and contained 0.01% thimerosal or 0.01% sodium azide as preservative. 16ml of PEG-precipitated recombinant phage were diluted with 14ml of blocking buffer (containing preservative) and incubated for 10-15 minutes at room temperature. During the panning step, non-specific hydrophobic protein-protein interactions may occur between the native protein of the M13 bacteriophage and certain antigens. This interaction can be reduced if Triton X-100 is added to the diluted phage supernatant to a final concentration of 0.1%. Alternatively, elution of specifically bound phage can be performed with glycine or tyrosine solution. 20ml of the diluted recombinant phage was poured into a culture flask and cultured at 37 ℃ for 2 hours. The flask was then emptied and washed 20 times with 30-50ml PBS and 20 times with PBS containing 0.1% Tween20 (the flasks were useful in dispensing the elution solution). The flasks were emptied completely each time.
10ml of log-phase TG1 cells (see step 1) were added in their entirety to a culture flask or sieve and incubated at 37 ℃ for 1 hour. After 1 hour, 100. mu.l of the cell suspension was removed from 10ml and prepared into 10-fold dilutions (1: 10, 1: 100, 1: 1000) in 2 XYT medium. Mu.l of undiluted cells and 100. mu.l of each dilution were placed in separate SOBAG plates using a sterile glass spreader (spreader). When dry, the plate should be inverted and incubated overnight at 30 ℃. If the colonies are too small to pick up after incubation, the plates can be left at 30 ℃ for an additional 4-8 hours. These SOBAG plates were treated as follows: a) stock cultures were prepared by scraping cells from the plates. 5ml of 2 XYT medium was poured into a plate, and the cells were scraped into the medium using a sterile glass spreader. Adding glycerol to make final concentration 15-30%, and storing at-70 deg.C. B) The plates were sealed and stored at 4 ℃ for up to 2 weeks for later rescue experiments.
According to a second method, modified from [25 ]]Ii) running the mixture of native HIV peptides on a 10% SDS-PAGE gel and then electrotransfering to 7X 30mM in Western transfer buffer (25mM Tris, 193mM glycine, and 20% methanol)2Is cut through with 10% porcine gelatin and 5X 1011CFU/ml helper phage were blocked by incubation at 4 ℃ overnight. After blocking, the membrane was transferred to binding buffer (5% gelatin, 3 × 10)11CFU/ml helper phage, 0.5M NaCl) and 10 added12A library of CFU scFv phagemid antibodies. The phage library was incubated with the membrane at 4 ℃ for 4 hours and shaken gently. The membranes were washed 6 times with PBS, 0.1% Tween20 (100 ml solution washes each time) and 6 times with PBS (100 ml solution washes each time). Alternatively, 3 washes with PBS (PBST) containing 0.1% Tween20 for 5 minutes, 5 washes with 10% MPBS containing 25% glycerol for 20 minutes, and finally 3 washes with PBS for 5 minutes. The membrane containing the protein band was cut with a razor blade and the phage was eluted with 100mM TEA for 10 minutes at room temperature. Neutralize toThereafter, the eluted phage particles were incubated with gelatin-blocked membranes or gelatin-coated immune tubes for 30 minutes at room temperature. The supernatant was then used to infect TG 1. Phage were prepared from E.coli for the next round of screening as described above.
3. Amplifying virus-isolated material from a patient not receiving antiretroviral therapy by the PBMC-MT method;
HIV is known to be successfully amplified in cell culture rich in the CD4-CCR5-CXCR4 receptor, however, this approach has many practical limitations. First, when natural viral material or laboratory strains derived from patients are infected in vitro, the infectious titer never exceeds 1-2% of the total viral concentration determined by different methods (real-time RT-PCR, p24 ELISA, etc.). This means, for example, if the number of virus copies in the infected material is 105Then the number of initial copies that the investigator can analyze by amplifying in vitro is only 103The remaining 102The variability that was possible in the original HIV was lost and could not be analyzed. Second, the number of HIV variants selected by in vitro infection is at best derived from original 103And thus the laboratory virus strain is not representative of the true aspects of HIV genetic and peptide variability. Third, HIV from HAART or other antiretroviral therapy treated patients loses its ability to expand in vitro, and thus resistant HIV variants cannot be cultured in vitro. Secondly, our experience in the culture of native viruses has shown that the best results are obtained in the following cases:
i) lymphocytes from HIV-infected patients were co-cultured with lymphocytes from healthy donors isolated from heparinized fresh blood using Ficoll-paque solution as described [19 ]. It is worth mentioning that the HIV-1 subtype A is widely spread in the territory of the Russian Federal, and that infection is successful in most cases if the HIV-infected lymphocytes are co-cultured with monocytes isolated from fresh blood of healthy donors as described [19 ].
ii) MT-2 or MT-4 or any other cell line for HIV culture (CCR5F-CEM, PM-1, HeLa, U937 etc.) was made 0.25X 106At a concentration of/ml, and then co-cultured with harvested equal amounts of HIV-propagated monocytes, which were harvested by adding RPMI-1640 medium to a total volume of 50ml, followed by centrifugation at 425g for 10 minutes, repeated twice. The cell mixture was resuspended in CL medium, 10. mu.l/ml IL-2 was added, and 25cm was used2The tissue culture flasks of (3) were cultured in an upright position at 37 ℃. Virus-containing culture broth was collected every 3-4 days, half of the culture broth was removed and replaced with the same volume of fresh culture broth (RPMI + 10% FCS).
The effect of viral infection was monitored by microscopic analysis of cell death and syncytial formation, as well as p24 ELISA experiments. The harvested culture broth was centrifuged at 3000rpm (1000g) for 15 minutes to remove cells, and stored at-80 ℃.
4. Concentration of HIV particles (by ultrafiltration, ultracentrifugation), inactivation and destruction of virus;
stock solutions containing approximately 20% by weight of viral particles were prepared from plasma or culture supernatant. The supernatant was first centrifuged at 3000rpm (1000g) for 15 minutes, and the resulting supernatant was then centrifuged again at 13200rpm (16000g) for 15 minutes. Placing 20% sucrose in the bottom of an ultracentrifuge tube (stirred) in a volume of about half of the total volume of the sample (the sucrose solution has a density of 1.16-1.18 g/sm)3) The supernatant containing the retroviral particles is then poured from above the tube. The tubes were centrifuged in MLS-50 rotor Optima MAX, Beckmann at 38000rpm (160000g) for 1 hour 35 min [19]. The precipitate is dissolved in a small volume of culture medium (e.g., RPMI-1640).
Inactivation of HIV
Cleavage of HIV precipitate and obtaining of HIV protein
The first method is carried out as described in [1 ]. The HIV lysis buffer (radioimmunoprecipitation buffer) comprises 20mM Tris-Cl, pH8.0, 120mM NaCl, 2mM EDTA, 0.5% deoxycholate, 0.5% NP-40, 2. mu.g PMSF, 10. mu.g/ml apolipoprotein and 10. mu.g/ml pepsin inhibitor A. After addition of the detergent, the mixture was gently stirred with a magnetic stirrer and heated at low temperature (50 ℃).
The second method is a standard preparation of peptide mixtures for mass spectrometry and crystal analysis. The pH of the resulting HIV-1 protein mixture was adjusted to 2.5 with 2N HCl and incubated with 0.15% (w/v) of porcine pepsin (Sigma Chemical Co., St. Louis, Mo.) for 4 hours at 37 ℃. Hydrolysis was terminated by heating to 80 ℃ for 15 minutes, and the pH was then adjusted to 7.5-8 by the addition of 2M NaOH. The hydrolyzed protein mixture was then ultrafiltered through a 10kDa hydrolysis membrane to remove pepsin and other unhydrolyzed proteins. The filtered hydrolyzed protein mixture was lyophilized and stored at-80 ℃.
5. Native HIV env peptides were collected by reverse panning techniques using a phagemid library exhibiting a recombinant ScFv specific for HIV.
In [35 ]]The method of vaccine development using phage display technology using antibody-displaying phage libraries is described, fuzzily. Before starting the operation of the recombinant phage ScFv library, the column embedded with the library presented by M13 was checked for specificity by a modified Western blot method. Running the probe on a gradient SDS-PAGE, then electrotransfering it to a nitrocellulose membrane [25 ]]. The antigen spots were first soaked in PBS containing 1% Tween20 for 1 hour to renature the proteins at the spots. The membrane was further incubated with 4% gelatin solution in PBS at 37 ℃ for 2 hours to block, and then incubated with 10 ℃12The CFU/ml phage (pre-incubated for 30 minutes at room temperature with 4% gelatin solution containing 1.5% BSA) were incubated for 1 hour at room temperature. The membranes were then washed three times with PBS, 0.1% Tween20, three times with PBS, and phage binding was detected by incubation with HRP-conjugated anti-M-13 antibody diluted 1: 8000 in 5% skim milk powder/PBS for 1 hour at room temperature. After three washes with PBS/0.1% Tween20 and three washes with PBS, the bands were visualized with ECL detector (Amersham). After continued washing with TPBS blot, membranes were incubated in ECL reagent for 1 min. Each film was then glued with Hyperfilm-ECL glueAnd (5) culturing and developing the sheet.
Recombinant monoclonal antibodies typically exhibit only 10-30% affinity compared to native antibodies isolated from the organism. However, the collection of individual (patient) HIV-specific monoclonal antibodies (phagemid library) generated by phage display technology per virus variant cohort was sufficient to select most HIV env and other peptides and proteins for the development of a prophylactic vaccine against HIV-1 (fig. 7a, b, 8a, b).
To prepare a phagemid library exhibiting recombinant phages, M13KO7 helper phage was added to an overnight culture of TG1 E.coli for a pre-incubation period of 1 hour and cultured at 37 ℃ for 12 hours in the presence of 100. mu.g/ml ampicillin and 50. mu.g/ml kanamycin (usually, the phage yield was 10 per ml)10To 1011One ampicillin transduction unit). The culture was centrifuged at 1000g for 10 minutes and the supernatant was collected and cooled. Then, 1/5 volume ratio of PEG8000/NaCl (20% PEG/2.5M NaCl) solution was added to the supernatant, and the mixture was incubated on ice for 1 hour, and then centrifuged at 4 ℃ at 10000g for 20 minutes to perform precipitation. The precipitate was dissolved with LB or 10mM TrisHCl pH8.0 and filtered through a 0.45 μm membrane. If 0.01% thimerosal is added, the recombinant phage may be stored at 4 ℃.
i) An immobilized M13-specific monoclonal antibody was embedded in an ultra-large pore epoxy activated monolithic cryogel (cryogel) (Protista Biotechnology) chromatography column. A monoclonal antibody specific for M13 was embedded in an ultra-large pore epoxy activated whole cryogel (protista biotechnology). For this purpose, the dry adsorbent was resuspended in 0.1M NaHCO containing 0.5M NaCl3pH8.3 buffer solution. The monoclonal antibody specific to M13 was dissolved in the same buffer to a concentration of 10mg/ml, added to the adsorbent, and incubated at room temperature for 1 hour under mechanical agitation. After incubation, the adsorbent was loaded with 5 volumes of the same 0.1M NaHCO3pH8.3/0.5M NaCl buffer washing. To block non-specific reactive groups, the adsorbent was incubated with 0.1M Tris-HCl buffer pH8.0, or 1M ethanolamine pH8.0 for 2 hours at room temperature and then adjusted to a 5ml column.
For both methods, M13 phage particles specific for gp120, gp140, gp160 and fragments thereof, gp41, p24 were first incubated with the (stage 4) hydrolyzed HIV-1 peptide mixture obtained above for 40 minutes at 37C. The phage particles were then embedded with the help of immobilized M13 phage-specific antibodies:
the prepared ultra-large pore epoxy-activated monolithic cryogel column with the inserted M13-specific monoclonal antibody was equilibrated with 0.05M Tris-HCl pH8.0 buffer, and then recombinant M13 dissolved in the same buffer was adjusted at a rate of 0.5ml/min for 5 hours using a liquid chromatography system, Actaprime Plus (GE Healthcare). The column was then washed with 5 volumes of the same 0.05M Tris-HCl pH8.0 buffer.
The embedding of recombinant phages was investigated by means of scanning probe microscopy (atomic force microscopy). Fig. 6b shows a cryogel successfully embedded in the HIV-specific ScFv phage library and fig. 6a shows the structure of a control ultra-large pore epoxy-activated monolithic cryogel column.
The HIV-1 peptide mixture, hydrolyzed in 0.05M Tris-HCl pH8.0 buffer, was injected into the previously embedded affinity column at a rate of 0.5ml/min for 5 hours. The column was then washed with 5 volumes of the same 0.05M Tris-HCl, pH8.0 buffer.
Phage that bound HIV peptide were eluted with 0.1M glycine in a gradient of pH 2.2. The resulting fractions were incubated in glycine elution buffer containing 0.001MPMSF for 5 hours at room temperature until the phage antigen complex was completely readjusted.
HIV peptides were analyzed [2] and purified by high performance liquid chromatography (HPLC, Waters). Analytical reverse phase HPLC was performed using a Waters 1525HPLC system equipped with a Symmetry C18 column (5 μm, 4.6 mm. times.150 mm, flow rate 0.5 ml/min). Preliminary reverse phase HPLC was performed using a Waters 1525HPLC system with a Symetric C-18 column (10 μm, 5.0 cm. times.25 cm) and a Waters UV detector. Bound peptide was eluted with a linear gradient of acetonitrile in water/0.1% trifluoroacetic acid (TFA).
6. Quantifying and sequencing the variability and frequency of env peptides using a liquid chromatography-mass spectrometry (LC-MS) method;
native HIV-1env peptide was collected from the reverse panned HIV-specific phage library as a sample source. Quantitative selection, mass distribution and characterization of env peptides were performed by one-dimensional liquid chromatography-mass spectrometry (LC-MS analysis).
Analysis of the SDS-PAGE gels by the Esquire 6000plus device (Bruker Daltonics, Bremen, Germany) by the ion electrospray quaternary mass analysis trapping method appeared to be similar to [10 ]]Similar protein bands, and single points in 2D that were not identified as peptide mass fingerprints. Samples were taken via an online protocol of a low pressure chromatography system Ultimate LC Packing and a sample selector Famos LC Packing (Dionex, ca, usa). The chromatographic section consists of two columns connected in series with a solenoid valve between them. The first column (100. mu. m.times.3 sm) had a hydrophobic polymer phase Poros R2, macropore diameter, analogue C8It is used for concentration and desalination of samples. The second column (75 μm.times.25 sm) had a Phenomex adsorbent with a hard particle size of 5 μm and a pore diameter ofAnalog C18Which is used to separate a mixture of desalted tri (triptic) peptides. The chromatographic conditions were as follows: 200 mul/min, the actual consumption rate before the separator was 900nl/min, 200nl/min during the separation. Peptide separation was performed for 48 min using a linear gradient with 5% to 60% solution B (75% acetonitrile, 25% isopropanol, 0.1% formic acid).
All tests were measured between 300 and 2500m/z, with capture quality optimization equal to 700. Ions with a charge number of 2 or more, and ions with a density exceeding a threshold value were used for the tandem experiment. The obtained quality prints (mass-prints) were sent to the MASCOT search system. The results were verified using the software complex backbone Scaffold 01-07-00(http:// www.proteomesoftware.com) for peptide identification validation by searching in the proteome database. Peptides identified with expectation values over 95% are listed in the final number table. All observed peptide masses were within 0.5Da of the calculated average mass match.
The tables represent the hydrophobic (positive Y-axis) and hydrophilic (negative) segments of the polypeptide sequence of the env protein molecule (fig. 3).
onf: confidence (0 ═ low, 9 ═ high)
pred: predicted secondary structure (H ═ helix, E ═ chain, C ═ coil)
AA: target sequence
Hydrophobic amino acid correlation value:
Ala：1.800Arg：-4.500Asn：-3.500Asp：-3.500Cys：2.500Gln：-3.500Glu：-3.500Gly：-0.400His：-3.200Ile：4.500Leu：3.800Lys：-3.900Met：1.900Phe：2.800Pro：-1.600Ser-0.800Thr：-0.700Trp：-0.900Tyr：-1.300Val：4.200Asx：-3.500GIx：-3.500Xaa：-0.490
a)#Al.RU.03.03RU20_06_13_AY500393
gp120 medial domain
CKAAENLWVTVYYGVPVWRDAETTLFCASDAKAYDKEVHNVWATHACVPTDPNPQEIALE
CPKVTFEPIPIHYCAPAGFAILKCKDTNFTGTGPCKNVSTVQCTHGIKPV
gp120 outer domain
VSTOLLLNGSLAEKEVMXRSENITDNGKXIIVOLTEPVNITCIRPGNNTRTSIRIGPGQT
FYATXDVIGDIRKAYCXVSRAAWXSTLQKISTQLRKYFNNKTIXFKNSSGGDLEVTTHSF
GN
gp120 medial domain
GGNMRDNWRSELYKYKVVKIEPIGVAPTRAKRRVVEREKR
b)B.RU.04.04RU128005AY682547
gp120 medial domain
CSAAGNLWVTVYYGVPVWKEADTTLFCASDAKGXSTEVHNVWATHACVPTDPNPQEIDLE
NVTENFNMWQNNMVEQMHEDIISLLDQSLKPCVKLTPLCVTLNKXNMVEQMHEDIISLWD
NGTGPCKNVSTVQCTHGIRPV
gp120 outer domain
VSTOLLLNGSLAEEEVVVRSRNFSDNAKNIIVQLKDPVQINCTRPSNNTRKSISIGPGXA
FYATGDIIGDIRXAHCNLSGADWTKTLEQIVKKLXEQYNKTIVFKQSSGGDPEIXMHSFN
KGQIKCSSNITGLLLTRDGGSNSTNNETFRPAGGD
gp120 medial domain
GGDIRDNWRSELYKYKVVKIEPLGVAPTMAKRRVVQREKR
c)#B.RU.04.04RU129005_AY751406
gp120 medial domain
SSXAEQLWVTVYYGVPVWKEATTTLFCASDARALNTEXHNVWATHACVPTDPNPQEXLLE
GTGPCTNVSTVXCTHGIRPV
gp120 outer domain
VSTQLLLNGSLAEEEVVIRSANFTNNAKTIIVQLNESXVINCTRPXNNTRKSIPIGPGRA
FYTTGDIIGDIRQAHCXLSSTKWNDTLRQIVEKLREQFGNKTIKFNQSSGGDPEIVMHSF
PIRGQISCSSNITGLLLTRDGGANNSTTEVFRPGGGX
gp120 medial domain
GGXMRDNXRSELYKYXVVKIEPLGVXPTKAKRRVVQREKX
7. Cloning the major HIV env peptide and producing recombinant peptides for vaccine development in leishmania tarentolae species;
the life cycle of HIV occurs in humans, monkeys, or rodents, and glycosylation of its proteins is closer to mammalian metabolism. Eukaryotic expression systems include not only yeast systems, filamentous fungi, but also cell culture systems derived from insects, mammals and/or plants. gp120 and gp41 are highly glycosylated in their outer domains. If the expressed fragments or proteins are to have glycosylation, they should be expressed in eukaryotic systems, such as in yeast, in mammalian cell culture, in Leishmania cell culture, in vaccinia virus expression culture. Expression can be carried out in mammalian cells, such as CHO-K1 (Chinese hamster cell) or Cos-7 (African green monkey kidney epidermal cell), but since mammalian cells have millions of proteins in the cellular metabolism, expression of recombinant proteins is low and the produced recombinant proteins are difficult to separate by chromatography. Therefore, we chose to use Leishmania tarentolae species as production system for env peptide.
After analysis by quantitative mass spectrometry, gp120 variants representing the overwhelming majority of pools were sequenced and cloned. As shown in a number of documents, the variation of the gp41 sequence is not important for HIV-specific immune responses (fig. 10d, example 4). The level of glycosylation and conjugation to gp41 is more important for the production of HIV-specific antibodies than the sequence variation of gp41 [31], so we consider taking only the gene from one variant of the patient population as a standard component for cloning. On the basis of the gp120 protein sequence obtained, the corresponding proviral DNA fragment encoding the gp120env peptide was amplified by two rounds of nested PCR from the cDNA set of the patient's lymphocytes, using a specific primer pair (Table 8). The primers themselves and their pairs can vary depending on the results of the LC-MS analysis.
DNA fragments encoding the entire gp120 and gp41 peptides, the gp120 internal and gp120 external domains and the gp41 external domain (see gp120 structure in FIGS. 10a, b, c) can be cloned. The PCR amplification scheme of the DNA fragments of HIV-1 encoding gp120, gp41 and their major domains is shown in FIGS. 11a, b. Primer pairs for amplification of envgp120, gp41 and their domains are shown in table 8. Restriction sites were selected based on the variant of the cloning vector, NcoI marker pink, XbaI-blue, NotI-orange and NheI-green. In each case, the region which is the most suitable for cloning for optimal immunization results is selected by the researcher on the basis of his technical knowledge or experience.
TABLE 8 oligonucleotide primers used for the amplification of HIV-1gp120, gp41 and the DNA regions encoding the major domains thereof
The 120 forward primer refers to a forward primer for amplifying the gp120 internal domain; 41 Forward primer refers to the forward primer used to amplify the gp41 ectodomain.
Several features are of paramount importance in selecting expression systems for recombinant protein production in vaccine development. Their expression should be: i) (ii) inducible; ii) have similar glycosylation or mammalian post-translational modifications.
Inducible expression is necessary to obtain reasonable quantities and concentrations of recombinant peptide. As shown in FIG. 12, in the inducible system, the expression of the recombinant protein was visible in the SDS-PA gel electrophoresis scan (FIG. 12 a). If the cells were transfected with a non-inducible expression vector, Western blotting is usually required for detection, as it is not evident enough on SDS-PAGE (FIG. 12 b).
The glycosylation of recombinant peptides produced for vaccination should resemble as much as possible the natural typical form of the viral host-eukaryotic lymphocytes. In eukaryotic cell cultures, it is difficult and very expensive to obtain expression of any sufficient amount of recombinant protein in their own millions of proteins. Thus, production of HIV-1 envelope proteins (gp120, gp41 and whole gp160) can be carried out in yeast strains, insect cells or eukaryotic cellular parasite systems. The choice we consider is the host leishmania tarentolae species of trypanosomatid protozoa (trypanosomatid protozoan) which combines the expression/folding/modification patterns of eukaryotic proteins with ease of manipulation and is not pathogenic to mammals. The main advantage of this expression system is that mammalian-type post-translational modifications, such as glycosylation, phosphorylation or prenylation, can be performed on the protein of interest (fig. 13).
The most convenient method is to clone HIV-1 envelope protein by using pLEXSY vector in LEXSYcon2 expression kit and LEXSinduce2 expression kit designed by Jena bioscience GmbH company. In trypanosomatids, the mRNA is transcribed into polycistronic precursors, which can be processed post-transcriptionally into a single mRNA by reverse splicing and polyadenylation in the intergenic region. Protein expression regulation in these species occurs primarily at the RNA level, possibly influenced by the structure of the intergenic region. In the pLEXSY vector, the intergenic region used was optimized for heterologous protein expression in Leishmania tarentolae species (Jena bioscience GmbH).
The pLEXSY-2 vector allows constitutive expression of the protein of interest, with or without a secretion signal peptide (SP in FIG. 14), followed by integration of the expression cassette into the chromosomal 18S rRNA locus (ssu). The same vector can therefore be used to clone an Open Reading Frame (ORF) for cytoplasmic or secretory expression. The LmSAP signal peptide encoded on these vectors was derived from the secreted acid phosphatase gene of Leishmania mexicana (LmSAP 1). Fusion of the ORF of the target HIV-1 protein with this signal peptide in an expression cassette allows for secretory expression in the LEXSY host, whereas the cloning of any restriction sites at the 5' end of the sequence encoding the signal peptide results in cytoplasmic expression.
Inserting target gene into pLEXSY expression vector
The pLEXSY-2 vector allows direct insertion of the target gene expression cassette by replacing the 1kb filler fragment it contains. The ligation mixture obtained is used to transform competent cells of E.coli, which are tolerant to Leishmania sequences (Stbl2, Stbl4, XL-1, XL-10, SURE, etc.). Ampicillin was used to select for recombinant E.coli clones. After construction in E.coli, the expression plasmid was linearized by SwaI complete digestion, after which the expression cassette containing the gene of interest was integrated by means of homologous recombination into the chromosomal 18S rRNA ssu locus of LEXSY host P10. In E.coli, there is no transcription and/or translation signal before the insertion point of the target gene, and therefore, gene expression that cannot be performed in E.coli is advantageous for producing a construct of a protein toxic to E.coli.
For constitutive cytoplasmic expression or constitutive secretory expression supported by the HIV-1 envelope signal peptide, the HIV-1 envelope genes (gp120, gp41 and the entire env gene encoding the signal peptide, gp120 and gp 41) were amplified with primers containing NcoI (forward) and NheI (reverse) sites (Table 8), digested with NcoI/NheI and cloned into the pLEXSY-2 vector. In this case, the target HIV-1 protein is fused to a hexahistidine stretch with a C-terminus. Alternatively, the HIV-1 envelope gene was amplified with primers containing NcoI (forward) and NotI (reverse) sites, digested with NcoI/NheI and cloned into pLEXSY-2 vector. In this case, the target HIV-1 protein obtained lacks the C-terminal hexahistidine stretch.
For constitutive secretory expression ensured by the LmSAP signal peptide from the pLEXSY-2 vector, the HIV-1 envelope genes (gp120, gp41 and the entire env gene lacking the signal peptide portion) were amplified with primers containing XbaI (forward) and NheI (reverse), digested with XbaI/NheI and cloned into the pLEXSY-2 vector. In this case, the target HIV-1 protein is fused to a hexahistidine stretch with a C-terminus. Alternatively, the HIV-1 envelope gene was amplified with primers containing XbaI (forward) and NotI (reverse) sites, digested with XbaI/NotI and cloned into pLEXSY-2 vector. In this case, the target HIV-1 protein was obtained without the C-terminal hexahistidine stretch.
Note that: restriction sites such as XbaI, NcoI, NheI and NotI are less common in env genes derived from the HIV-1A 1 subtype of the former Soviet Union. A schematic representation of the pLEXSY-2 vector is shown in FIG. 14. The LEXSinduce2 expression kit contains pLEXSY _ I-neo2 (encoding a glucosamine phosphotransferase) suitable for tetracycline-induced bacteriophage T7 polymerase-driven expression in the LEXSY host T7-TR.
Expression of recombinant proteins
The pLEXSY _ I-2 vector allows inducible expression of the protein of interest with or without a secretion signal peptide. Thus, the same vector can be used to clone an open reading frame for inducible cytoplasmic expression or inducible secretory expression. The LmSAP signal peptide encoded in these vectors is derived from the gene for the secreted acid phosphatase of Leishmania mexicana (LmSAP 1). Fusion of the ORF of the protein of interest with this signal peptide in the expression cassette allows for secretory expression in the LEXSY host, while cloning any restriction sites at the 5' end of the sequence encoding the signal peptide results in cytoplasmic expression (fig. 5). The pLEXSY _ I-2 vector family ensures inducible expression of the protein of interest after integration of the expression cassette into the ornithine decarboxylase (odc) locus in the chromosome of a T7-TR recipient strain of the species T7-TR of the Leishmania tarentolae. Leishmania T7-TR recipient strain constitutively expresses bacteriophage T7 RNA polymerase and the TET repressor under the control of host RNA polymerase I. In an initial cloning step, the target gene is provided with a linker sequence containing a restriction enzyme site so that it can be inserted downstream of the T7 promoter/TET operon structure of the pLEXSY _ I-2 vector. These vectors contain optimized untranslated regions located in the vicinity of the insertion site of the target gene, which provide splicing signals for post-transcriptional mRNA processing. After construction in E.coli, the expression plasmid was linearized and then integrated by homologous recombination into the odc locus of the LEXSY host T7-TR.
For tetracycline-inducible cytoplasmic expression or tetracycline-inducible secretory expression ensured by the HIV-1 envelope signal peptide, the HIV-1 envelope genes (gp120, gp41 and the entire env genes encoding the signal peptides, gp120 and gp 41) were amplified with primers containing NcoI (forward) and NheI (reverse) sites, digested with NcoI/NheI and cloned into the pLEXSY-2 vector. In this case, the target HIV-1 protein is fused to a hexahistidine stretch with a C-terminus. Alternatively, the HIV-1 envelope gene was amplified with primers containing NcoI (forward) and NotI (reverse) sites, digested with NcoI/NotI and cloned into pLEXSY-2 vector. In this case, the target HIV-1 protein was obtained without the C-terminal hexahistidine stretch.
For tetracycline-inducible secretory expression ensured by the LmSAP signal peptide from the vector, the HIV-1 envelope gene (gp120, gp41 and the entire env gene lacking the signal peptide portion) was amplified with primers containing XbaI (forward) and NheI (reverse), digested with XbaI/NheI, and cloned into the pLEXSY-2 vector. In this case, the target HIV-1 protein is fused to a hexahistidine stretch with a C-terminus. Alternatively, the HIV-1 envelope gene was amplified with primers containing XbaI (forward) and NotI (reverse) sites, digested with XbaI/NotI and cloned into pLEXSY-2 vector. In this case, the target HIV-1 protein was obtained without the C-terminal hexahistidine stretch.
The entire HIV-1env gene cloned with NcoI/NheI or NcoI/NotI sites in the pLEXSY vector family, or the HIV-1env gene without the intrinsic signal peptide, and, or the HIV-1env gene fused with the LmSAP signal peptide from the pLEXSY vector (cloned with XbaI/NheI or XbaI/NotI in the pLEXSY vector) can be used to construct plasmid constructs allowing rapid replacement of specific gp120 sequences with other gp120 sequence variants obtained from different HIV-1 strains. For this purpose, a further XbaI site was introduced by site-directed mutagenesis between the gp120 end of the env gene sequence and the gp41 start. After this, pLEXSY:: HIV-1env plasmid construct was digested with NcoI/XbaI (when the entire env gene was cloned in NcoI/NheI or NcoI/NotI sites) or only XbaI (when the env gene of HIV-1 without intrinsic signal peptide was cloned in XbaI/NheI or XbaI/NotI sites) and gp120 sequence was removed. The resulting plasmid derivatives are suitable for cloning gp120 sequences which were amplified from other HIV-1 virus variants by PCR using primers containing NcoI (forward) or XbaI (forward) and XbaI (reverse) sites.
Culture of LEXSY-2 host and expression strain
Leishmania are grown aerobically in two stages: flagellated promastigotes (wild-type in insect hosts) and amastigotes in vertebrate hosts. In the T7-TR LEXSY-2 host, both stages were cultured in vitro, cultured in the dark at 26 ℃, using complete media (LEXSY BHI or LEXSY YS), or chemically prepared media (synthetic LEXSY media), prepared with 37g/l LEXSY BHI powder, autoclaved (amber color), and stored for up to 6 months. Before use, 5. mu.g/ml heme, 100. mu.g/ml penicillin and 50. mu.g/ml streptomycin were added to the culture to prevent bacterial contamination. The culture medium can be stored at 4 deg.C in dark, and can be used within 2 weeks after addition. Serum was not added to the complete medium because fetal calf serum did not promote the growth of Leishmania tarentolae species. In the case of growth inhibition of the host or LEXSY strain, the cells should be centrifuged at 2000g for 5 minutes, carefully resuspended in fresh medium, and then cultured. Cell lines can be maintained by routine dilution at a ratio of 1: 10 to 1: 50, and continued suspension culture. In the middle-late growth phase (OD 2-3; 8X 10)7-1.4×108Individual cells/ml) can be used for optimal results. For the maintenance of cell lines, it is convenient to dilute 10ml of culture 1: 20 fold on Monday and Friday and culture it upright in TC flasks. The cells that survive under the microscope are motile promastigotes with motile flagella; dead cells are round or broken in shape and they do not move.
Recombinant protein expression cultures can be performed in gas-permeable Tissue Culture (TC) flasks for suspension culture in a volume of 10-200ml, or in triangular flasks in an incubator with shaking at about 140rpm in a standard bioreactor in a volume of 50ml to 1L, up to 1L. Recombinants of the pLEXSY-neo2 vector were selected in the presence of 50. mu.g/ml neomycin.
LEXSY hosts and LEXSY expressing strains can be stored in 20% glycerol at-80 ℃ for at least 1 year. A15 ml Falcon tube was charged with 1/4 volumes of autoclaved glycerol (80%) and 3/4 volumes of a mixture containing 4-8X 10 cells in mid-growth phase7Cells/ml (OD1.2-1.8) LEXSY BHI medium, mixed with glycerol and dispensed into sterile cryovials. The tubes were left at room temperature for 10 minutes, then on wet ice for 1 hour, at-20 ℃ for a period of time, and transferred to-80 ℃ for long-term storage. To reactivate the glycerol stock, the frozen tubes were thawed on ice, the contents poured into 10-fold supplemented medium, and incubated for 2 days at 26 ℃ in an upright, air-permeable flask until the culture became cloudy.
Preparation of expression plasmid for LEXSY host transfection
1-5. mu.g of an expression plasmid containing the target gene obtained from E.coli was completely digested with SwaI. The resulting 2.9kbp fragment represents a component of E.coli, and a larger fragment represents the linearized expression cassette containing the gene of interest to be integrated into the chromosomal ssu locus of the LEXSY host, and these fragments were run on an agarose gel. The larger expression cassette fragment was isolated using agarose gel extraction kit. Enzymes and buffer salts can be removed using a PCR purification kit. Alternatively, digests were ethanol precipitated, washed with 70% ethanol, and re-solubilized with up to 50 μ l of sterile double distilled water or 10mM Tris pH8 for each transfection.
Electroporation transfection of LEXSY host strains
For efficient transfection, a pre-culture of Leishmania tarentolae species was inoculated into 10ml of LEXSY BHI medium at a ratio of 1: 20 and cultured upright at 26 ℃ in a Tissue Culture (TC) flask, and after two days, the pre-culture was diluted 1: 10 in 10ml of medium and cultured overnight under the same conditions. Grow intoShould contain 6X 107Individual cells/ml (OD1.4, wavelength between 550 and 600nm, 3% formalin); microscopic examination was used to ensure that the cells were viable and in the shape of water droplets. Cells were centrifuged at 2000g for 5 minutes at room temperature and 1/2 volumes of supernatant were removed. The pellet was resuspended in the remaining culture medium (10)8Individual cells/ml), left on wet ice for 10 minutes. In parallel tubes 0.1-5. mu.g of the transforming DNA in up to 50. mu.l of water or Tris buffer, prepared on wet ice. Mu.l of pre-cooled cells were added to the tubes with DNA and transferred to electroporation cuvettes with a diameter of 2mm on wet ice, avoiding the formation of air bubbles. Electroporation parameters were 450V, 450. mu.F, pulses 5-6 ms. After electroporation, the electroporation cup was placed back on ice for exactly 10 minutes. The electroporated cells were then transferred to 10ml LEXSY BHI using a capillary and incubated overnight (ca.20 hours, OD0.3-0.4) at 26 ℃.
Selection of transgenic LEXSY strains
To establish the expression strain, two conventional methods described in parallel below can be used. Linearized expression cassettes designed for chromosomal integration can be selected after transfection, either cloned or uncloneally, and when comparing cultures derived from these two selections, they were found to always have similar expression levels. However, transfection of circular expression plasmids requires clonal selection, since the episome is more easily amplified and eventually integrated into the genome in a heterologous manner. Non-clonal selection in suspension culture after transfection with circular DNA often results in reduced expression levels.
Plating on solid medium for clone selection
LEXSY host cells were selected on freshly prepared agarose plates. From the 10ml o/n cultures after transfection, 1-4 batches of 2ml samples were taken and the remaining cultures were used in parallel for non-clonal selection. The cells were centrifuged at 2000g at 20 ℃ for 5 minutes, the pellet resuspended in 50-100. mu.l of the remaining culture medium, and the resuspended cells were spread onto freshly prepared LEXSY BHI agarose base supplemented with 50. mu.g/ml neomycin by streaking the cells onto nitrocellulose filters placed on the agarose surface. Plating on these membranes is easier than plating directly on a 1% agarose base, and can also reduce clumping of cells. In addition, plating on the membrane may allow for lifting of the membrane for expression profiling of clonal populations, e.g., by fluorescence scanning or specific detection methods for a given protein of interest. The plates were sealed with a sealing film and cultured in an inverted state at 26 ℃.
Small, clear colonies began to appear 5-7 days after plating, and when colonies grew to 1-2mm diameter, they were transferred to 0.2ml selective growth medium in 96-well plates using a pipette tip, and after 1 day of culture, to 1ml selective medium in 24-well plates 7-9 days after plating. After a further 24-48 hours incubation at 26 ℃, the culture can be expanded to 10ml of selective medium in TC bottles, which can be used for evaluation and cryopreservation.
Screening of suspension cultures
10ml o/n cultures obtained from transfection experiments (see 4.4) become slightly turbid once they start (OD 6000.4, ca.10)7Individual cells/ml; usually about 20 hours after electroporation), 50. mu.g/ml neomycin was added and incubation continued at 26 ℃ for 7 days. Under the microscope it was seen that the recombinant cells were mobile, in the shape of a drop of water and grown into a "fog" suspension culture, while the negative control cells began to die during the screening and appeared spherical or irregular under the microscope, with no flagella. It is generally sufficient to obtain turbid cultures of antibiotic-resistant recombinant cell lines on the 7 th day of selection followed by inoculation of the cells in fresh medium containing neomycin in a ratio of 1: 10.
Confirmation of genomic integration and recombinant env peptide expression
Integration of the expression cassette at the ssu locus can be confirmed by diagnostic PCR or sequencing using genomic DNA of the transgenic strain as template. For the pLEXSY I-2 vector, diagnostic PCR (annealing temperature 55 ℃) was performed using the forward primer of the antibiotic resistance expression cassette and odc reverse primer P1510 (Table 9). Integration of the expression cassette into the odc locus resulted in a characteristic fragment (1.9 or 2.0kbp respectively) which was not found in the control reaction. In addition, diagnostic PCR (annealing temperature 60 ℃) can also be performed using odc forward primer A1304 and aprt reverse primer A1715 (hybridized in the 5' untranslated region of the target gene). Integration of the expression cassette into the odc locus resulted in a characteristic 1.1kbp fragment which could not be obtained in reactions using the control group of expression plasmids or genomic DNA of the LEXSY host strain.
Expression of the protein of interest in the recombinant LEXSY strain was assessed by analyzing cell extracts, or aliquots of the supernatant if secreted, by SDS-PAGE and Western blot. For optimal expression, modulation of tetracycline-induced expression, culture conditions and harvest time, averaged 1. mu.g/ml in different tetracycline concentrations, can be selected for each individual protein.
Table 9: primer sequences available for LEXSinduce2 kit (Jena Bioscience).
| Primer and method for producing the same | 5 '-3' nucleotide sequence | |
| Insert sequencing Forward P1442 | CCG ACT GCA ACA AGG TGT AG | All "AP" expression vectors with 5' untranslated region of aprt |
| Insert sequencing reverse A264 | CAT CTA TAG AGA AGT ACA CGT AAAAG | All LEXSI expressionCarrier |
| Odc Forward primer A1304 | TCC GCC ATT CAT GGC TGG TG | Diagnostic integration of all odc expression vectors |
| Aprt reverse primer A1715 | TAT TCG TTG TCA GAT GGC GCA C | Diagnostic integration of all aprt expression vectors with the 5' untranslated region of aprt |
| Neo forward primer a1432 | GCA TGG CGA TGC CTG CTT GC | Diagnostic integration of all odc expression vectors |
| Odc reverse primer P1510 | GTG CAC CCA TAG TAG AGG TGC | Diagnostic integration of all SSU integration vectors |
8. Production and chromatographic purification of recombinant HIV env peptides
For protein production, the recombinant strains were grown in complete LEXSY broth BHI (Jena bioscience) to OD600D2 (10)8Individual cells/ml). 1 hour after the cells were transferred into fresh medium, 5mg/ml tetracycline was added to induce protein production, and the culture was incubated at 26 ℃ on a multitronII incubator shaker (Infors AG, Switzerland) at 130rpm for 24-72 hours until the OD reached ca.1.8. Recombinant gp120 present in the culture supernatant and the cells was detected by polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting with sodium dodecyl sulfate. To verify N-sugarsIn the presence of the substrate, we treated the culture supernatant or cells with N-glycosylase and analyzed the electrophoretic migration of the treated proteins.
Leishmania cells expressing the protein were centrifuged at 2500g for 10 min and the pellet resuspended in 20mM Tris, pH8.0, 150mM NaCl, 5mM EDTA, 1mM PMSF. Cell lysis was performed with a 19mm probe at 2OkHz using a sonicator, with 10 pulses for 1 minute on ice, with 2 minute intervals. The clear supernatant was collected, filtered through a 0.45 μm pore size filter and affinity purified for recombinant gp120 in a column containing immobilized metal ions using nickel-loaded nitrilotriacetic acid (Ni-NTA) (GE Healthcare) coupled to agarose. Briefly, the Ni-NTA column was washed with a triple volume of buffer (LC Akta Prime Plus, GE Healthcare) containing 20mM Tris, pH8.0, 150mM NaCl, 5mM EDTA, 1mM PMSF at a flow rate of 1 ml/min. The filtered supernatant containing recombinant gp120(r-gp120) was then applied to the column at a flow rate of 0.25 ml/min. After loading, the column was washed with three volumes of washing buffer (20mM Tris-HCl, 500mM NaCl, 5mM imidazole, pH 7.4). r-gp120 is cleaved by enterokinase in the column, thereby removing the polyhistidine tail. To do this, the column is filled with a solution containing 10mM Tris-HCl, 10mM CaCl2pH8.0 buffer was introduced with 1 International Unit (IU) of enterokinase (Ek) and the cleavage reaction was allowed to proceed for 18 hours at 25 ℃. Alternatively, the target protein with a polyhistidine tail was eluted in an imidazole gradient (0-0.5M imidazole in 100mM Tris-HCl pH8.0, containing 150mM NaCl). The protein-containing fractions were combined and concentrated by ultrafiltration. These fractions were analyzed by SDS-PAGE/silver staining and Western blotting with anti-human gp120 antibody. Fractions containing r-gp120 were pooled, dialyzed against 0.1M Tris-EDTA buffer, pH8.0, and loaded onto a column according to standard protocol [2]]Anion exchange column (Q-PEEK 10 μm AXC Biosuite, Waters) equilibrated with the same buffer. The protein was eluted with a 0-1M NaCl gradient. The fraction containing r-gp120 was finally purified by gel filtration using Sephacryl S-200HR (GE healthcare).
The N-terminus of purified rhEPO was sequenced by automated Edman degradation. The purified protein was assayed for concentration using the BCA assay. Fractions obtained and purified at different stages of protein expression were analyzed by SDS-PAGE. Protein bands were visualized by Coomassie brilliant blue R-250 or silver staining.
a. Compositions for enhancing immune responses in prophylactic HIV vaccines are prepared using sterically stabilized liposomes or virosomes as carriers for vaccine delivery.
Any drug administered orally, subcutaneously, intramuscularly or intravenously for providing a specific immune response to certain bacterial or viral infectious diseases upon future exposure to these diseases, which is commonly referred to as a "vaccine", must meet a number of requirements. These requirements are mainly:
iii) the immune response is highly specific for a defined pathogenic microorganism or infection;
iv) the immune response is sufficient to counteract the development of this particular infection, preventing the onset of disease symptoms;
v) the immune response can last for a long period of time, months or years;
vi) in addition to the above mentioned requirements, the vaccine is not reactive (immunotoxic) to the human organism.
According to the present disclosure, the effect of an HIV prophylactic vaccine can be enhanced by combining it with an immunostimulant or an immunogenic carrier such as an adjuvant. The mixture of carbohydrate-based recombinant gp120 and the native HIV env protein is strongly immunogenic, is not well tolerated when vaccinated subcutaneously and provides a strong immune response by itself (example 4). However, biodegradation in the organism is rapid for all pure proteins, and if the peptides are not immobilized with any preservative or protease inhibitor adjuvant, the immune response will be exhausted within two to three weeks. Our first idea was to protect the env peptides from degradation, packaging them in Sterically Stabilized Liposomes (SSL) that are not visible to the reticuloendothelial system. However, in initial mouse experiments, SSL was able to maintain the peptide in an endogenous (loaded) or bound state for a period of time long enough to reduce its acute immune toxicity. For peptides, the stereostabilized liposome platform combines the advantages of low overall immunotoxicity and better efficacy (timely release of drug) as has been demonstrated in liposomal drug forms of anthracyclines. The liposomal peptide can be released slowly and accurately from the SSL as can cell stabilizers (cytostatics) or other small molecular weight agents. Inhibition of the immediate immune response allows for increased peptide doses in a single administration and prolonged association (junction) contact time of the viral env peptide with the MHC to generate a sufficiently long enhanced immune response. By this approach, SSL is used both as an adjuvant to enhance the immune response and as a vaccine delivery system.
The specific formulation form of the effective composition of the present invention can be prepared by any suitable method such that the adjuvant has biodegradability, safety and effectiveness in the subject to which the formulation is administered when the formulation is administered. Two approaches are further described below:
i) the env peptide mixture was loaded into a stereostabilised liposome (SSL);
ii) a PEG activating group covalently attached to SSL for env peptide;
iii) the env peptide is presented on virions with possible constructs (pNL3-4, IRIV, etc.).
i) Preparation of sterically stabilized liposomes and loading of peptides.
The sterically stabilized liposomes are prepared by reacting a mixture of phospholipids containing about 7: 3: a method in which a mixture of cholesterol and 0.2-0.5 Mol/% of polyethylene glycol-distearoyl (phosphatidyl) ethanolamine (PEG-DSPE) is vacuum-dried to remove chloroform and formed into vesicles in a nitrogen stream [40 ]. The lipid mixture used was: DOPC/Chol/DSPE-PEG350, DOPC/Chol/DSPE-PEG400, etc. (Avanti Polar Lipids, Burminghan, Alabama). The main component of the liposome is Dioleoylphosphatidylcholine (DOPC), which can be extracted from natural sources such as egg yolk, brain tissue or soybean, or prepared synthetically. Cholesterol is essential to stabilize bilayer phospholipids in liposome membranes, and PE-PEG stabilizes and strengthens the membranes, prevents fusion or degradation of suspended liposomes, and enables them to maintain their particle size distribution and in-formulation loading without leakage for months. The ideal molecular weight of PEG in SSL is 400-700, and the use of longer PEG chain 1000-2000 in SSL design is not advantageous because the liposome membrane has increased firmness beyond that required for delivery of its contents, and the long PEG SSL composition does not meet the requirement for self-biodegradation. The percentage of DSPE-PEG is a major tuning factor in order to obtain liposomes with the desired characteristics.
The dried lipid was mixed with an organic solvent-chloroform or ethanol-chloroform-and then evaporated in a rotary evaporator (BuchiR-200) to form a thin lipid film. The liposomal suspension is prepared in a further hydration process, the hydration process being carried out in the presence of a dissolved substance (e.g., 50mM NaH)2PO4400mM NaCl, pH 8.0), at a temperature of +45 ℃ for 30 minutes at a speed of 300-400 rpm. A mixture of large multilamellar vesicles (MLV, 300nm-1 μm) and small unilamellar vesicles (SUV, 80-250nm) was prepared. For delivery of any water soluble substance, such as peptides, small unilamellar vesicles are necessary, therefore, sonication (600mV, Avanti Polar Lipids) is performed and several rounds of filtered extrusion are performed with 0.4-0.2-0.1 μm polycarbonate membranes (Avanti Polar Lipids). In addition, formulations useful for immunization were prepared by extrusion through 0.2-0.1 μm membranes under sterile conditions (layered sterile syringe, membrane and flask). The particle size distribution and stability of liposomes in aqueous suspension was determined by dynamic light scattering laser submicron particle size analysis method using DLS Nicomp-380 instrument (fig. 17).
Recombinant peptide mixtures for immunization are introduced into the liposome composition during the hydration phase of the lipid membrane-the peptide mixtures are dissolved in phosphate buffer and loaded into the internal aqueous phase of the liposome vesicles [40 ]. After the extrusion process, SSL was transferred by size exclusion gel filtration chromatography using Sephacryl S-200HR and Akta Prime LC system (GE Healthcare), and extra peptides outside the vesicles were separated and retained in the column. If necessary, SSL suspensions, containing vaccine compositions useful for immunization, can be concentrated by dialysis.
Subcutaneous administration of SSL vaccine compositions may suppress the immune effect because this approach slows the exit of env recombinant peptides from neutral to MHC liposomes. This process can be regulated by thermosensitive liposome-tSSL. TSSL differs from other liposomes in that the membrane components have a specific quantitative combination or have some additional phospholipid components that allow the liposome membrane to melt immediately when the temperature reaches a certain degree, typically 40-45 ℃. When local heating occurs, thermosensitive liposomes are destroyed and their contents-peptides-are put into the tissue. For example, a common sterically stabilized liposome has a melting temperature of about 54-58 ℃, and the dry weight mixture used to form the lipid membrane consists of phosphatidylcholine: cholesterol: distearoyl-phosphatidylethanolamine-PEG in the ratio: for PC: Chol: DSPE-PEG400 6.85: 2.75: 0.4 (up to 0.5) Mol/%, for longer PEG chains PC: Chol: DSPE-PEG2000 6.9: 2.95: 0.15 (up to 0.25) Mol/%. To prepare thermosensitive liposomes, researchers can vary certain parameters, first the proportion of lipids in the mixture: the amount of cholesterol was increased from 27-29 to 30-35 Mol/%, and the percentage of PE-PEG was decreased from 2-5 Mol/% to 1.5-2 Mol/%, respectively. Other methods of softening the liposome membrane and lowering its melting point are to use phospholipids with shorter fatty acid tails: dimyristoylphosphatidylcholine (DMPC, C-14), distearoylphosphatidylcholine (DSPC, C-16), or 30-40 Mol/% DMPC or DSPC instead of an equivalent amount of DOPC.
ii) env peptide coupled to PEG activating group of SSL
The second liposome carrier for the lipid mixture of env recombinant peptides is represented by the longer DSPE-PEG2000 activated for peptide conjugation: PDP-PEG2000-DSPE/Chol/DOPC, maleimide (phenylbutyrate) -PEG2000-DSPE/Chol/DOPC, p-nitrophenyl (carbonyl) -PEG 2000-DSPE/Chol/DOPC. The concentration of PEG-2000 in these lipid mixtures should not exceed 1.5-2 Mol/%, since longer polyethylene glycols are more effective in increasing the stability of the liposomes than shorter polyethylene glycols, and the same concentration can make the liposome membrane too strong to release the vaccine and hinder harmless biodegradation of the lipids.
The first method of peptide and PEG activated distal conjugation [38] was to react p-nitrobenzene (carbonyl) -PEG-2000-DSPE with the amino group of the peptide in a liposomal suspension at a ratio of 1mg peptide to 25-40mg lipid in 0.1M citrate buffer at pH 4.0-5.0 (total suspension volume 5.5-9 ml). The reaction was terminated by adding NaOH to raise the pH to 7.5-8.5 without any special peptide treatment.
Method Using Maleimide-PEG-2000-DSPE [39]The peptide needs to be pre-thiolated with Trautt reagent (2-iminothiolane). Dissolving 1mg of ICO-25 in Na-containing solution3BO3And EDTA in borate buffer, then 50-70. mu.g of dried Trautt reagent was added, the mixture was incubated at room temperature for 1 hour, and then excess protein was washed off by ultrafiltration while the buffer was changed to PBS at pH 8.0. The liposome fraction with uniform size and uniform concentration of the cell-loading stabilizer was extracted by Sepharose CL-6B (GE Healthcare, Sweden) liquid chromatography.
ODN-HIV env peptides conjugated to liposomes containing PDP-PEG-PE
To prepare the PDP-peptide derivatives, the peptide was dissolved in 25mM HEPES, 140mM NaCl, pH7.4 solution at a concentration of 10mg/ml, and then 25mM succinimidyl-4-MPB (SMPB) solution in DMF was slowly added to the peptide solution to a molar ratio of 20: 1 (SMPB: peptide) and incubated at room temperature for 30 minutes. Unbound SMPB was removed at low pH by gel filtration using a Sephacryl S-200HR chromatography column (GE Healthcare) in 25mM HEPES, 25mM MES, 140mM NaCl, pH 6.7 buffer.
Dithiothreitol (DTT) was added to a final concentration of 20mM and incubated at room temperature for 30 minutes to reduce the pyridine dithiol group at the distal end of the PEG chain. The liposomes were passed through a Sephadex G-25 column at elevated pH, eluting with 25mM HEPES, 25mM MES, 140mM NaCl, pH 6.7 solution, to isolate DTT. Thiolated liposomes were incubated with MPB-peptide derivatives overnight at room temperature, with a peptide/lipid ratio of about 1: 1000. The liposomes were passed through a Sephacryl S-200HR chromatography column with a solution of 25mM HEPES, 140mM NaCl, pH7.4 to remove unbound peptides by gel filtration.
ODN-HIV env peptides coupled to COOH-PEG-PE-liposomes
To 300. mu.l suspension of liposomes containing HO2C-PEG-PE in MES buffer at pH 4-5.5 (3. mu. mol total lipids) were added 120. mu.l of a 0.25M aqueous solution of 1-ethyl-3- (3-dimethyl-aminopropyl) carbodiimide and 120. mu.l of a 0.25M aqueous solution of N-hydroxysulfosuccinimide. The mixture was incubated at room temperature for 10 minutes and neutralized to pH 7.5 with 1M NaOH. mu.M HIV env peptide was added to the activated liposomes and the reaction mixture was incubated at 4 ℃ for 8 hours with gentle agitation. Peptide-bound liposomes were separated from unbound peptides using Sephacryl S-200HR chromatography column (GE Healthcare) pre-equilibrated with PBS. The peak fractions of peptide-bound liposomes that elute in empty volume (void volume) are collected, pooled, and diluted with saline to the desired volume if necessary.
The env peptide may also be conjugated to a nickel modified phospholipid located between the PC tail and the PEG-DSPE tail of the liposome composition DOPC/DOGS-NTA-Ni/Chol/DSPE-PEG-2000. However, while DOGS-NTA is known to stimulate mucosal and other B-lymphocyte immune responses, the env peptide is buried in the PEG position, impairing its specificity against HIV. The method is as follows.
In the binding reaction, recombinant (His) 6-peptide (10-80. mu.g) was mixed with liposomes (1. mu.M) in a total volume of 50. mu.l phosphate buffer (50mM NaH)2PO4400mM NaCl, pH 8), at 37 ℃ or room temperature for 30 minutes [17 ]]. The amount of liposome-bound protein was indirectly quantified by measuring the amount of free protein at the end of the reaction. Unbound protein is used as a carrierThe resulting mixture was centrifuged using a Microcon-100 centrifuge. Before centrifugation, the liposome-peptide mixture was diluted to a final volume of 250 μ l in phosphate buffer. After centrifugation at 12000g for 13 min, 20. mu.l of the filtrate was analyzed for the content of free protein using a micro-BCA assay. The amount of peptide bound to the liposomes was determined by subtracting the amount of free protein from the total amount used. Comparing this indirect quantification of His-protein binding to liposomes with the direct method, which involves size exclusion gel filtration chromatography on Sephacryl S-200HR gel arrays (GE Healthcare) to isolate free proteins, followed by direct quantification of peptide binding to liposomes using the micro-BCA assay, gives the same results as the direct method.
iii) env peptides presented on virions with possible constructs (pNL3-4, IRIV, etc.)
The small viruses and vectors composed thereof can be used to express and present env peptide HIV vaccines. Virosomes are unlikely to be suitable for use in prophylactic vaccine technology and their HIV-specific immune response enhancing effect is lower than that provided by SSL technology for vaccine delivery. The vaccine composition contained only the HIVenv peptide expressed on small virus surface-virions (virosomes) -rather than the gene for the env peptide delivered by the viral vector. Large viruses such as adenovirus, adeno-associated virus, vaccinia virus, etc. are not as good as virion candidates because they have hundreds of peptides expressed on their capsid and they enhance immune responses more non-specifically than specifically after administration. Virosome vectors include the defective HIV derivative pNL3-4, the influenza virus vector IRIV with confirmed efficacy against malaria, and hepatitis a vaccines, measles virus derivatives, alphaviruses of different encephalitis pathogens, yellow fever virus vectors and other possible variants.
Host animals to which the adjuvants and adjuvant-containing vaccine compositions of the invention can be effectively applied include primates and rodents or other mammals. BalbC mice were used to confirm the first immune enhancing response. The two loaded liposome adjuvants can be used separately or mixed in different ratios.
BalbC mice, 3 weeks old, were immunized subcutaneously at a dose of 20-50 μ g of pure animal peptide, and the adjuvant concentration in a suspension of dried lipid MW was 5 mg/ml. 7-8 or more mice were taken per group. Animals weighing 11-14g, starting to eat hard food, were immunized, the first at 3 weeks of age, the second at 2 weeks later when they were 5 weeks of age, and the third at 1 month later when they were 9 weeks of age. Recombinant gp120 and its domains and recombinant gp41 and its ectodomains are used separately or together to make compositions. The titer of HIV env peptide antibodies was determined by ELISA using r-gp120(gp110, gp160) variants, r-gp41, and the native HIV protein mix previously used in the bioleach of the phagemid libraries. ELISA tests were performed on the third, fourteenth and twenty-eighth days after the last subcutaneous dose. Some results are shown in example 4.
Two main conclusions can be drawn from the experiments in mice:
gp120 and all its derivatives, recombinants and native peptides are highly immunogenic after subcutaneous injection into BalbC mice, eliciting HIV-specific, sustained, strong immune responses. Recombinant gp41 and its ectodomain variants inoculated by the same method elicited specific monoclonal and polyclonal antibodies with titers several fold lower. The same was observed for antibody titers in the serum of HIV-infected humans, as well as in the presentation of the antibody library on phage M13. This is believed to be due to the internal location of gp41 in the patient, which is below gp120 in the viral envelope. However, the same phenomenon of recombinant protein immunization supports our notion that in the development of HIV prophylactic vaccines, correct recognition of gp120 sequences and their recombination is important, and gp41 should be used as a combinatorial peptide "material", but the variation in its sequence is of little importance.
2. The liposomal adjuvant composition provides an enhanced immune response for a longer period of time than immunization with the peptide alone, while reducing acute immunotoxic responses, thereby allowing an increase in peptide dosage to generate a protective response against HIV.
The dosage rates and appropriate dosage forms of the adjuvant and vaccine compositions of the present invention can be readily determined by one of ordinary skill in the art without undue experimentation using conventional antibody titer measurement techniques and conventional bioeffective/biocompatible methods, depending on the desired therapeutic effect for the particular species of adjuvant, and the length of desired biological activity. The mode of administration of the vaccine and its components may include parenteral methods such as: subcutaneous injection, transdermal administration, intranasal administration, and intramuscular administration.
The HIV antibody library of the infected group is used for the selection and generation of HIV prophylactic vaccines developed to inhibit infections transmitted by HIV variants of said infected group, and in this regard, the development of HIV prophylactic vaccines is a step forward on the road of personalized medicine. The vaccine cannot be used as a single disposable composition as a universal weapon against the transmission of HIV infection. However, the full epidemiological knowledge of HIV collected over the last 25 years in HIV research and in AIDS fight against it will provide a strong support for its practical development.
Examples
Example 1: the following electrophoretic data show the various stages of the human recombinant IgG phagemid library (phage display technology) containing HIV-specific ScFv antibody fragments.
PCR-I results-variable chains of kappa and lambda with corresponding partial C kappa or CL fragments.
The bands of interest were cut from the gel, as indicated by the arrows.
Abbreviations: 1 VL-Lambda variable chain with partial CL segments
2 VL-Lambda variable chain with partial CL segments
1 VK-kappa variable chain with partial C.kappa.fragment
1 a-10-different primer pairs
VK-PCR negative control
100bp-GeneRulerTM100bp DNA ladder (Fermentas) GeneRulerTM 100bp
Ladder-shaped DNA (deoxyribonucleic acid) strip
PCR-I results-kappa-variable chain with partial C.kappa.fragment, band of interest excised from the gel, indicated by arrow.
1 a-10-different primer pairs 1VK (2VK, 3VK, 4VK)
Kappa-variable chains with partial C kappa fragments
100bp-GeneRulerTMLadder-shaped strip of 100bp DNA (Fermentas)
PCR-I results-variable lambda chains with partial CL fragment and variable IgM and IgG heavy chain (H) chains with partial CHl fragment, bands of interest excised from the gel, indicated by arrows.
1 VL-library 1, lambda-variable chain with partial CL fragment
1VHM (2VHM, 3VHM, 4VHM) -heavy chain variable chain (IgM) with partial CHl fragments.
1VHG (2VHM, 3VHM, 4VHM) -libraries 1, 2, 3, 4, heavy chain (H) variable chain (IgG) with partial CH1 fragment.
1 a-10-different primer pairs
-a VL; -VHG; -VHM-PCR negative control group
100bp-GeneRulerTMLadder-shaped strip of 100bp DNA (Fermentas)
PCR-II results-variable heavy (H) chain with added linker fragment, encoding ((Gly)4Ser)3. The bands of interest were cut from the gel, as indicated by the arrows.
1HG (2HG, 3HG, 4HG) -libraries 1, 2, 3, 4, from heavy chain variable chains in IgG cDNA pools;
1HM (2HM, 3HM, 4HM) -libraries 1, 2, 3, 4 from the variable chain of the heavy chain in the IgM cDNA pool
1a-7 a-different primer pairs containing a linker
-H-PCR negative control;
PCR-II results-kappa-and lambda-variable chains with added linker fragment, coding ((Gly)4Ser)3. The bands of interest were cut from the gel, as indicated by the arrows.
1K (2K, 3K) -library 1, 2, 3, kappa-variable chains
1L (2L, 3L, 4L) -library 1, 2, 3, 4, Lambda-variable chains
1 a-8-different primer pairs containing a linker
Negative control of-K and-L-PCR
100bp-GeneRulerTMLadder-shaped strip of 100bp DNA (Fermentas)
PCR-II results-kappa-, lambda-and heavy chain (H) variable chains with added linker fragments, encoding ((Gly)4Ser)3. The bands of interest cut from the gel are indicated by arrows.
1K (2K, 3K, 4K) -, kappa-variable chain
1L-library 1, lambda-variable chains
2HG (3HG, 4HG) -libraries 2, 3 and 4, heavy chain variable chains from IgG cDNA pools;
3HM (4HM) -libraries 3 and 4, heavy chain variable chains from IgM cDNA pool
1 a-8-different primer pairs containing a linker
Negative control of-K and-L-PCR
100bp-GeneRulerTMLadder-shaped strip of 100bp DNA (Fermentas)
Gel quantification of the collected ScFv fragments. Different VHConnecting fragment-VkappaScFv variants (library 4).
Amplify V againHConnecting sheetsegment-VkappaScFv mix (from library 4). The bands of interest were cut from the gel, as indicated by the arrows.
K- 100bp VHConnecting fragment-VkappaScFv mixtures
Example 2: specific quantitation of recombinant HIV-specific libraries
a) ELISA detection result of recombinant monoclonal antibody cloning by using HIV-1 positive serum
b) ELISA results from 38 phage monoclonals in a phage library selected from the viral peptide A455 (SigmaPlut 10.0 statistical analysis)
c) ELISA results for 26 phage clones from phage libraries selected from recombinant gp110-, gp160 (SigmaPlut 10.0 statistical analysis)
Example 3: HIV Env peptide variability
HIV differs from other pathogenic viruses in that its peptide sequence has a very high heterogeneity. The three-dimensional structure of peptides with sequence changes also becomes different, and in many cases these changes are caused by the same mutation after the appearance of the phenotype of the resistant virus. Thus, using a library of monoclonal antibodies, frequently encountered variants can be collected and recombinant forms of surface viral proteins obtained. Common variants of certain subtype A and B HIV env peptide sequences are listed below. Variable amino acids are labeled with blue and conserved amino acids are labeled with red. Some of these sequences were previously obtained in our laboratory.
Example 4: preliminary results of animal immunization (SigmaPlut 10.0 statistical analysis)
BalbC mice weighing 11-14g at 3 weeks of age were immunized subcutaneously at a dose of 20-50 μ g of pure animal peptide and a lipid concentration MW of 5 mg/ml. Mice were immunized at 3 weeks of age, 2 weeks later when they were 5 weeks of age and a third month later when mice were 9 weeks of age. Recombinant gp120 elicited an average 5-fold higher immune response than the recombinant gp41 ectodomain. The same difference in specific antibody titers was observed when human polyclonal antibodies isolated from patient sera were used for ELISA staining of recombinant gp120 and gp41 at the same concentration.
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Claims (18)
1. A method for preparing an HIV vaccine for preventing infection comprising the steps of:
i) establishing a phagemid library of monoclonal antibodies expressed on B lymphocytes obtained from a plurality of individuals infected with HIV-1 subtype A or B;
ii) panning with native or recombinant HIV-1 peptide to enrich the antibody phagemid library;
iii) collecting HIV-1 peptide/polypeptide/protein material using a reverse panning method using an HIV-1 phagemid library bound to a support;
iv) identifying and analyzing the peptide material obtained in step iii);
v) using the results in step iv), producing a glycosylated recombinant HIV-1env peptide in an expression system;
vi) purifying the recombinant HIV-1env peptide and producing a vaccine composition.
2. The method of claim 1, wherein the individual, who is the source of the acquired HIV material, is infected with the same or a different HIV subtype.
3. The method of any preceding claim, wherein the individual, who is the source of the acquired HIV material, is a patient who has not received antiretroviral therapy or a patient who has received antiretroviral therapy.
4. The method of any preceding claim, wherein the obtained viral material is amplified prior to contacting with the support.
5. The method of any preceding claim, wherein the support carrying a plurality of different HIV-specific antibodies and/or antibody fragments is selected from a phagemid library, a peptide chip, or a bacterial library.
6. The method of claim 5, wherein the phagemid library is prepared by:
a) preparing DNA fragments derived from nucleotides encoding a light chain variable region and a heavy chain variable region, respectively, of an immunoglobulin expressed in B lymphocytes obtained from a plurality of individuals infected with HIV;
b) connecting DNA segments encoding light and heavy chain of immunoglobulin to express the polypeptides containing light chain variable region and heavy chain variable region of immunoglobulin separately to obtain great amount of different specificities;
c) the ligated fragments are cloned in a phagemid vector, and the bacterial strain is transformed to express them on the surface of a bacteriophage.
7. The method of claim 6, wherein the preparing of the DNA fragments in step a) comprises preparing cDNA from a pool of RNA obtained from B lymphocytes of a patient infected with HIV-1 subtypes A and B, said patient having and/or not having been treated with antiretroviral therapy, and amplifying the light chain variable region and the heavy chain variable region.
8. The method of claim 7, wherein amplification is performed with any of the primer combinations listed in tables 1-7.
9. The method of any one of claims 6 to 8, wherein the obtained scFv phagemid recombinant antibodies are specific for a resistant HIV strain from a patient who has received HAART or any other antiretroviral therapy.
10. The method of claims 6 to 9, wherein step i) further comprises enriching a phagemid library presenting antibody ScFv fragments during panning in which HIV specific antibodies bind to recombinant gp120, gp41 and native HIV polypeptides isolated from different donors.
11. The method according to claim 1, wherein in step iii) the isolation of HIV-1env peptide/polypeptide/protein is performed using the reverse panning technique of the invention.
12. The method of claim 1, wherein in step iv) quantitative analysis, identification and sequencing of HIV-1gp120 and its standard and variable fragments is performed using liquid phase mass spectrometry.
13. The method according to any preceding claim, wherein step v) further comprises producing the recombinant HIV-1 peptide/polypeptide/protein in a suitable host having eukaryotic glycosylation capabilities.
14. The method of any preceding claim, which comprises preparing an HIV prophylactic vaccine composition by adding/incorporating an optional immunogenic stimulant, adjuvant or carrier.
15. The HIV vaccine according to claim 13, comprising recombinant gp41 and gp120HIV-1 subtype a and B env peptides/polypeptides/proteins.
16. The HIV vaccine of claims 10-15, further comprising a stereostabilized liposome (SSL) or virosome and possible variants thereof as adjuvant-carrier.
17. An HIV prophylactic vaccine obtainable according to the method of any preceding claim.
18. The HIV vaccine of claim 16, for use in immunizing an uninfected individual against acquisition and development of HIV infection and aids.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US60/978,536 | 2007-10-09 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| HK1151554A true HK1151554A (en) | 2012-02-03 |
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