JP2001507004A - Chlamydia vaccine - Google Patents

Abstract

  (57) [Summary] A vaccine formulation is provided comprising a major outer membrane protein from the vaccine Chlamydia and a mucosal adjuvant such as cholera toxin or heat labile enterotoxin. Such formulations provide protection from chlamydia-induced fertility.

Description

Detailed Description of the Invention Chlamydia Vaccine The present invention relates to a vaccine formulation for preventing Chlamydia infection. In particular, E. Formulation containing recombinant or purified major outer membrane protein (MOMP) from Chlamydia trachomatis in combination with mutated heat-labile enterotoxin (mLT) or cholera toxin (CT) from E. coli About. The gram-negative bacterium Trachoma chlamydia is a common human pathogen; it is transmitted from person to person and causes eye and genital infections that can have long-lasting sequelae. Genital chlamydial infection, the target of these vaccine formulations, is the most common bacterial sexually transmitted disease (STD) in the United States. The most detrimental consequences of the infection are exerted in women and the most common site of infection is the cervix, but ascending infections can lead to infertility and endometriosis leading to ectopic pregnancy, pelvic inflammatory diseases ( Serious complications such as PID) and salpingitis can occur. It has been shown that a single PID episode could cause a 6.1% infertility rate, while more than three episodes caused a 54% infertility rate (20). Chlamydia trachomatis is divided into 15 subgroups by serotype, and STDs are caused by serovars D to K encompassing three different serogroups (19); It should have a protective effect on several blood group subtypes that are somewhat related. In human and non-human primates, vaccine tests performed with whole organisms as immunogens have yielded subgroup-specific protection, but some vaccines were more severe during reinfection. A severe reaction was developed (6). Several studies have demonstrated that the pathology associated with Chlamydia infection is mediated by immunity (7); furthermore, purified Chlamydia 57 kDa protein (Hsp60) is found in previously infected animals. (13, 14). These observations lead to the conclusion that protection against Chlamydia infection can be achieved with subunit vaccines. Much interest has been focused on the 40 kDa major outer membrane protein (MOMP) for subunit vaccine design. This protein, which has been shown to function as a porin in vitro (4), exists throughout the life cycle of the bacterium (8) and has high immunogenicity in humans and animals. MOMPs exhibit four variable domains (VD) surrounded by five constants that are highly conserved among serovars (15,22). In vitro and in vivo neutralizing B cell epitopes have been mapped on these VDs (2, 23), whereas T cell epitopes have been identified in both variable and constant domains (1, 16). . The protein is produced by a signal sequence that is cleaved to produce a mature protein. Immunization with recombinant or purified MOMP followed by homotypic or heterotypic challenge with chlamydia was performed in different animal models, with varying effects on infection parameters (3, 17, 18). In an atypical challenge experiment, Tuff Frey et al. Reported that parenteral and mucosal immunization with rMOMP adsorbed on alhydrogel increased the severity of salpingitis and colonization of the lower reproductive tract. Decrease the duration of each. However, the preparation did not provide protection against infertility due to infection. In a recent study, using the same mouse model, we found that immunization with a vaccine containing 3D-MPL and QS21 or DQ and MOMP from serovar L2 could result in infertility due to heterologous chlamydial infection. (12). In this particular case, the increased ratio of MOMP-specific IgG2a in the sera of the immunized mice, and that the immune spleen cells express interferon (IFN) -gamma upon restimulation in vitro. Secretion confirmed that protection was associated with an antigen-specific Th1-like immune response (Th1-like immune response). Similarly, others have shown that adoptive transfer of MoPn-specific Th1 clones can elucidate the infection of nude mice whose genitals have been infected with MoPN. Activation of a predominantly Th1-like subset is also consistent with a protective immune response against other intracellular pathogens, such as Leishmania (9) and Mycobacterium (21). The present invention provides vaccine compositions effective at the mucosal level to provide protection against infertility due to Chlamydia infection. Advantageously, the vaccine is effective in mucous membranes where chlamydial infections are primarily associated. The vaccine may be administered by any known route, but is advantageously useful as an oral or intranasal vaccine. Accordingly, the present invention provides a vaccine formulation comprising a recombinant or purified major outer layer protein (rMOMP) and a mucosal adjuvant. In particular, the vaccine comprises MOMPs from blood group variants L2, F, D or E, but may further comprise MOMPs from other blood group variants. Combination vaccines containing MOMPs from two or more blood group variants may be utilized. Preferred combinations include MOMPs from D and E subtypes. In a preferred composition of the invention, the mucosal adjuvant is E. coli. E. coli mutant LT (eg, LT R192G) or cholera toxin. Mutant LT R192G is no. It can be obtained as described in IPA PCT / US95 / 09005 published under 96/06627. Cholera toxin can be purchased from Swiss Serum, Bern. The protein content in each vaccine is chosen as the amount that elicits an immunoprotective response in a typical vaccine without significant adverse side effects. Generally, each dose will be expected to contain between 1-1000 μg, preferably 2-100 μg, typically between 4-40 μg of protein. The optimal amount of a particular vaccine can be ascertained by standard tests involving observation of appropriate immune responses in patients. Following the initial vaccination, the patient may receive one or several boosters at appropriate intervals. The formulations of the present invention may be used for both prophylactic and therapeutic purposes. Accordingly, in one aspect, the invention provides a method of treatment comprising administering to a patient an effective amount of a vaccine of the invention. In another embodiment of the invention, the vaccine may be administered intranasally. Materials and Methods Production and Formulation of Purified rMOMP The acquisition and use of the DNA construct pET15-MOMP for antigen production is described in PCT No. It is described in GB 9550663 published as 96/31236. Purification of the protein was performed using a histidine-binding resin (His. Bind. Resin) [Novagen] under denaturing conditions as disclosed in that patent; in the final product, LPS and protein concentrations were Each was measured using the Limulus amoeba cell lysis test (Coatest, Chromogenix) and the BCA method (BCA kit, Pierce). One dose of the vaccine used for intranasal immunization was 10 μg mL T in a final volume of 20 μl PBS [obtained from SmithKlein Beecham Biologicals] or CT [Swiss Searam] (Swiss Serum, Bren)] was prepared by mixing with 10 μg of rMOMP P serovar F (rMOMPF) or L2 (rMOMPL2). Vaccination, Fertility, Sampling and Immunological Follow-Up for Salpingitis in a Mouse Model A group of 10 female C3H mice (6 weeks old, Ifa Credo) was administered at weeks 0 and 2 Immunization was performed by intranasal administration of 20 μl of a vaccine formulation containing CT or mLT under anesthesia, Hypnprm [Janssen-Cilag] and Dormicum [Roche]. The experimental challenge was performed as follows: At week 5, mice received 2.5 mg progesterone intraperitoneally [Depo-Provera, Upjohn], at week 6, 100 μl. 5x10 in sucrose phosphate glutamate buffer (SPG) ^Five Infections were made by bilateral intrauterine inoculation with inclusion body forming units (IFU) trachomatis chlamydia NI1 (serovar F) or, for the fertility-positive control group, 100 μl of McCoy cell extract. At 10 weeks, treated mice were caged with males for 3 months for fertility assessment (1 male for every 2 females per cage, males every week in each group). The parameters used to evaluate the fertility of the groups were: F (number of mice that gave birth at least once 総 数 total number of mice), M (number of mice born (whether live or dead) ÷ number of births) ) And N (number of mice born (whether dead or not) divided by total number of mice). Measurement of MOMP-Specific Humoral Response In a somewhat modified version of the method disclosed in patent GB9506863.1, supra, sampling and quantification of antibody (Ab) response by ELISA on individual mice was performed. Vaginal secretions were collected at weekly intervals from week 3 to week 7 by repeated washing and aspiration with 50 μl PBS, diluted 1: 4 in PBS containing 0.5% BSA and 0.1% Tween20. And rMOMP-specific secretory IgA or IgG antibodies. Total IgA was also weighed because specific antibody concentrations could be affected by fluctuations in liquid recovery during washing, but only in the first experiment. Subsequent vaginal lavage was performed only for MOMP-specific IgA analysis, as we detected little or no variation in total antibody concentration between the mice analyzed (not shown). CT-specific IgA and IgG in the first experimental sample were also measured to evaluate the efficacy of intranasal immunization. The titer is arbitrarily determined as the reciprocal of the sample dilution fold corresponding to an optical density of 1 at 492 nm, and mice showing at least one titer equal to or higher than 4 are considered antigen-specific IgA positive Was done. Blood samples were collected at week 6 (the week of challenge) and analyzed for the presence of rMOMP-specific IgG in serum. In the first experiment, CT-specific IgG in serum was also measured to confirm the efficacy of intranasal immunization; therefore, microtiter plates contained 0.5 μg of CT / well [ Swiss Serum, Bern] and then treated as described in patent GB9506863.1. Measurement of MOMP-specific Cell Response Five groups of 5 female C3H mice (6 weeks old, Ifa Credo) were treated at week 0 and week 2 with Hypnprm [Janssen-Slag. Cilag) and Dormicum [Roche] Under anesthesia, immunization was performed by intranasal administration of 20 μl of a vaccine formulation containing mLT; sham-immunize). Animals in groups 1 and 2 and corresponding control animals were bled for serologic analysis and sacrificed 9 and 19 days after the second boost, respectively; spleens were aseptically removed and pooled And a single cell suspension was prepared for restimulation with 1 μg / ml rMOMP serovar L2, or 4 μg / ml Concanavalin A (Boerchinger Mannheim) as a positive control; not restimulated The culture was used as a negative control for cell activation. To measure cell proliferation, 5x10 in 200 μl RPMI with 10% fetal calf serum [FCS, Gibco-BRL] added per well to a 96-well round bottom culture plate. ^Four A triplicate culture was prepared using the responder cells; _Two After incubating at 37 ° C. for 72 hours in the medium, the supernatant is re-cold, while the cells are pulsed with 1 μCi of tritiated thymidine (Amersham) per well for 18 hours, and glass fiber [Scatron]. (Skatron)], air-dried and beta-radiation counted by standard liquid scintillation. Stimulation index (SI) divided by the mean of tritiated thymidine uptake by antigen or Concanavalin A (ConA) -stimulated T cells per triplicate well by the mean of unstimulated T cell uptake per triplicate well. Was calculated for each group. IFN gamma in the culture supernatant was measured using a commercially available ELISA kit (Duoset, Genzyme). For cells obtained on day 9 post boost, the 72 hr culture supernatant of the lymphocyte proliferation assay pooled for triplicate cultures was used, while for cells obtained on day 19, A 24 well plate specially prepared for that purpose (5 × 10 5 / ml of RPMI 1640 containing 10% FCS) ⁶ 48 hours of culture supernatant derived from cells). Results Evidence that mucosal immunization with rMOMP in combination with CT or mLT may provide protection against infertility caused by chlamydia challenge is provided by the first two experiments described below. Because these experiments were designed primarily for the assessment of systemic immunization (not shown), the negative and positive control groups received subcutaneous adjuvants other than CT or mLT; rMOMP-sensitive animals (negative control group) Animals infected to confirm the effect of the challenge on fertility, while immunized with rMOMP (positive control group) take into account fertility variability that may be due to handling of animals during in utero inoculation. Because of this, they were pseudo-infected. A third experiment was set up to characterize cell activation induced by rMOMP with mLT adjuvant, in which the negative control group consisted of mice sham-immunized intranasally with mLT alone. Experiment 1 In the first experiment (Table 1), intranasal immunization with rMOMP + CT was evaluated for its protective effect against infertility caused by Chlamydia infection (homologous challenge). Analysis of the humoral immune response immediately prior to challenge shows that all mice show CT-specific IgG in serum and CT-specific IgG and IgA in vaginal secretions, but in the same pre-elevations. It was found that there was no detectable rMOMP-specific IgG or rMOMP-specific IgA response. However, after challenge, this group exhibited F and N fertility parameters that reached 77 and 66% of the positive control group, respectively, while the negative control group was almost completely infertile (14% of the F value recorded in the positive control group). And 13% of the N value). Experiment 2 In a second experiment (Tables 2 and 3), groups of mice were immunized intranasally with rMOMPF in combination with CT, or rMOMPF in combination with CT or mLT; in addition to the negative and positive control groups described above. In addition, a sham-immunized control group receiving CT alone intranasally was included in the experiment. As observed in the first experiment, intranasal administration of rMOM PF + CT resulted in serum collected immediately prior to challenge (IgG response) and vaginal secretions collected weekly from boost to challenge (IgA response). Nor did it elicit a detectable humoral rMOMPF specific response. In contrast, intranasal administration of rMOMPFL2 in combination with CT or mLT elicited an antigen-specific humoral response in some animals; 1 and 3 out of 10 mice, respectively, were collected immediately prior to challenge The sera were analyzed to be IgG positive, while 5 and 7 out of 10 mice were IgA positive in at least one of the vaginal lavages taken weekly from boost to challenge, respectively. It has been found. Infection did not enhance the MOMP-specific IgA response, as indicated by analysis performed one week after challenge. Compared with the positive control mock-infected group, the fertility of the negative control group was almost completely abolished, suggesting a specific effect of Chlamydia infection. Fertility of the mucosa-administered group revealed that immunization with rMOMPF or rMOMPFL2 in combination with CT provided an equivalent level of protection (63 and 75 F-values respectively recorded in the positive control group). %, And 81 and 58% of the N value). Immunization with rMOMPL2 in combination with mLT provided the highest level of protection, with comparable F values and higher N values (150%) compared to the values recorded in the positive control group. Administration of CT alone also appeared to reduce infertility levels, but to a lesser extent than the rMOMP + CT formulation, with F values 40% of those recorded in the positive control group and N values 35%. Experiment 3 Cell activation induced by antigen formulated with mLT was analyzed through cell proliferation and IFN gamma secretion upon antigen-specific restimulation. When examined 9 and 19 days after the boost, spleen cells from the group immunized with the antigen developed a strong specific proliferative immune response (38% and 108% of the positive control, respectively), while the mLT Those from control animals sham-immunized alone did not respond to restimulation in vitro (Tables 4 and 5). Spleen cells harvested at both time points and restimulated with antigen showed in the culture supernatant IFN gamma at concentrations ranging from those restimulated with 4 μg / ml ConA at the same time. On the other hand, cells isolated from mock-vaccinated animals and cultured with antigen produced relatively lower levels of INF gamma compared to cell counterparts cultured with ConA (Tables 4 and 5). When looking at the humoral response, we were unable to detect rMOMP-specific IgG in the pool and individual sera, nor in the pool and individual vaginal lavage fluid and in the harvest at both time points. Was. These data indicate that mucosal administration of rMOMP, when combined with CT or mLT, provides protection (homogeneous and atypical) against infertility caused by chlamydial challenge. The fact that protection is not associated with local rMOMP-specific IgA demonstrates the existence of an immunoprotective mechanism distinct from specific secreted antigen responses. Later experiments suggest that in mice, intranasal administration of rMOMP in combination with mLT induces a specific Th1 T cell immune response that may be responsible for the observed protection. Reference 1. Allen, JE, R.M.Loksley and R.S.Stephens. 1991. A single peptide from the major outer membrane protein of Chlamydia trachomatis induces T cell assistance for antibody production against protective determinants. 8. Journal of Immunology (J. Immunol.) 147: 674-67 2. Baehr, W., XX. Zhang, T. Joseph, T. Joseph, H. Su, F. Nano. E. Nano), EK Everett and HD Calwell. 1988. Mapping of the antigenic domain expressed by the trachomatis chlamydia major outer membrane protein gene. Proceedings in National Academic Science (Proc. Natl. Acad. Sci.). USA (USA). 85: 4000-4004. 3. Batteiger, BE, RG Rank, PM Bavoil and LSF Soderberg ( LSF Soderberg). 1993. Partial protection against re-infection of the genital tract by immunization of guinea pigs using isolated outer membrane proteins of the chlamydial pathogen of guinea pig inclusion body conjunctivitis. The Journal of General Microbiology (J. Gen. Microbiol.). 139: 2965-2972. 4. Bavoil, P., A., Ohlin and J. Schachter. Role of disulfide bonds in the structure and permeability of outer membranes in Chlamydia trachomatis. Infection and immunity (Infect. Immun.). 44: 479-485. 5. Dickinson, B.L. and J.D. Clements. Dissociation of the heat-labile enterotoxin of Escherichia coli from the adjuvant ADP-ribosyltransferase activity. Infection and immunity (Infect. Immun.). 63: 1617-1623. 6. Grayston, J.T. and SP Wang. New knowledge of chlamydia and the diseases caused by them. Journal of Infection Diseases (J. Infect. Dis.). 132: 87-105. 7. Grayston J.T., SP Wang, L.J.Yeh, and CC C.O. Kuo). 1985. Importance of reinfection in the pathogenesis of trachoma. Reviews of Infections Diss. (Rev. Infect. Dis.) 7: 717-725. 8. Hatch, TP, M. Micelli, J. E. Sublett. 1986. Synthesis of Disulfide Bonded Outer Membrane Proteins During the Developmental Cycle of Chlamydia sictus and Chlamydia trachomatis. Journal of Bacteriology (J. Bacteriol.). 165: 379-385. 9. Heinzel, FP, MD Sadick, BJ Hol dayy, R.L.Koffman (RL) Coffman) and RM Loksley. Mutant expression of interferon gamma and interleukin-4 during resolution and progression of murine leishmania. Journal of Experimental Medicine (J. Exp. Med.) 169: 59-726. 10. Holmgren, J., N. LYcke and C. Czerkinsky. 1993. Cholera toxin and cholera toxin B subunit as oral mucosal adjuvant and antigen vector system. Vaccine 11: 1179-1184 11. Igietsem, JU, KH Ramsey, DM Magee, DM Williams Williams), TJ Kincy and RG Rank. 1993. Resolution of murine chlamydial genital infection by adoptive transfer of TH1 lymphocyte clones, subspecies-specific. Regional Immunology (Reg. Immunol.) 5: 317-324. 12. Maisonneuve, J.F., C. Charlier, P. Momin, N. Garson, N. Garland and V. Verlant . 1995. Chlamydia trachomatis recombinant major outer membrane protein (rMOMP) in combination with MPL + QS21 induces partial protection against infertility due to atypical challenge in mice. Abstract monograph of the eleventh meeting of STD International Conference. 1995, New Orleans, Louisiana. 13. Morrison, R.P., RJ Bellland, K.Ling, and H.D. Caldwell. Etiology of chlamydial diseases. The 57-kD chlamydial hypersensitivity antigen is a stress response protein. Journal of Experimental Medicine (J. Exp. Med.) 170: 1271-1283. 14． Patton, DL, YT Cosgrove Sweeney and CC Kuo. 1994. Demonstration of delayed-type hypersensitivity in tracheoma chlamydia salpingitis in monkeys: Etiological mechanism of fallopian tube injury Journal of Infections Dis. 169: 680-683. 15. Stephens, R.S., R. Sanchez-Pscador, R.A. Wager, C. Inouye and MS Urdie (MS Urdea). Diversity of major outer membrane proteins of Chlamydia trachomatis. Journal of Bacteriology (J. Bacteriol.) 169: 3879-3885. 16. Su, H., R.P. Morrison, NG Watkins, and HG Caldwell . Identification and characterization of T helper cell epitopes of the major outer membrane protein of Chlamydia trachomatis. Journal of Experimental Medicine (J. Exp. Med.) 172: 203-212 17. Taylor, HR, J. Whitum-Hudson, J. Schachter, HD Caldwell And R. A. Plendergast. 1988. Oral immunization with Chlamydia major outer membrane protein (MOMP). Investigation Ophthalmology and Visual Science (Invest. Ophthamol. Visual. Sci.) 29: 1847-1852. 18. Tufrey, M., F. Alexander, W. Conlan, C. Woods, and M. Ward. 1992. Atypical protection of Chlamydia trachomatis by a human serum atypical F isolate against chlamydial salpingitis and colonization of the lower reproductive tract by pre-immunization with recombinant L1 major outer membrane protein. J. Gen. Microbiol. 138: 1707-1715. 19. Wang, SP, CC Kuo, R.C. Barnes, R.S.Stephens (R.S. Stephens) and JT Grayston. 1985. Immunotyping of Chlamydia trachomatis by monoclonal antibodies. Journal of Infection Diseases (J. Infect. Dis) 152: 791-800. 20. Westrom, L. and Mardh PA. 1983. Chlamydial salpingitis. British Medical Bulletin 39: 145-150. 21. Yamaura, M., K. Uye mura, Earl. Jay. RJ Deans, K. Weinberg, TH Rea, BR Bloom and R L Modlin (RL Modlin). 1991. Definition of the protective response to pathogens: the nature of cytokines in leprosy lesions. Science 254: 277-279. 22. Yuan, Y., YXZ hang, Watkin NG, and HD Caldwell. . 1989. Amino acid sequence deduced from nucleotides and four variable domains of 15 major outer membrane proteins of Chlamydia trachomatis serovar. Infection and Immunity (Infect. Immun.) 57: 1040-1049. 23. Zhang, YX, S. Stewart, T. Joseph, H.R. Taylor and H.D. Cold Well (HD Caldwell). Protected monoclonal antibodies recognize epitopes located on the major outer membrane proteins of Chlamydia trachomatis. Journal of Immunology (J. Immunol.) 138: 575-5 81.

────────────────────────────────────────────────── ─── Continuation of front page    (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, L U, MC, NL, PT, SE), OA (BF, BJ, CF) , CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, M W, SD, SZ, UG, ZW), EA (AM, AZ, BY) , KG, KZ, MD, RU, TJ, TM), AL, AM , AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, E S, FI, GB, GE, GH, GM, GW, HU, ID , IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, M G, MK, MN, MW, MX, NO, NZ, PL, PT , RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, V N, YU, ZW (72) Inventors Verlan, Vincent Georges Qu             Listian Louis             Belgium, base-1330 Rixenzarth, Li             Du du Lansituit 89, Sumi             Skrain Beecham Biological             Su Societe Anonym

Claims (1)

[Claims] 1. Major outer membrane protein (MOMP) of Chlamydia trachomatis and mucosal adjuvant Vant-containing vaccines 2. The vaccine according to claim 1, wherein the adjuvant is selected from mLT or CT. 3. 2. The outer membrane protein is selected from blood group variants D to K or L. Vaccine. 4. The vaccine according to claim 3, wherein the outer membrane is selected from F, L2, D or E. 5. The vaccine according to the preceding claim, comprising MOMPs from two or more blood group variants. 6. The outer membrane protein is E. coli by recombinant DNA technology. E. coli produced in E. coli The vaccine according to claim. 7. A mucosal adjuvant replaces arginine at position 192 with glycine. A vaccine as claimed herein according to the preceding claim, which is a LT holotoxin. 8. Claims wherein the MOMP is a full-length mature protein lacking a signal sequence. The vaccine according to claim 1. 9. Claims herein as claimed in the preceding claims adapted for oral or intranasal administration. Vaccine. 10. Mixing MOMP from Chlamydia trachomatis with mucosal adjuvant And a method for producing a vaccine. 11. Administering a safe and effective amount of the vaccine according to the claim. To treat patients with or susceptible to Chlamydia trachomatis infection Treatment method. 12. For the manufacture of a vaccine for the treatment or prevention of Chlamydia trachomatis infection , Use of MOMPs from Mucosal Adjuvants and Chlamydia trachomatis.