JPH07505613A - Major histocompatibility complex class 2 antigen as a vaccine against immunodeficiency virus - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Major histocompatibility complex class as a vaccine against immunodeficiency virus ■ Antigens This invention relates to vaccines against immunodeficiency viruses.

Hopes for a promising vaccine against AIDS are pessimistic. The obstacle is It often seemed insurmountable. Effective vaccines destroy CD4+ cells This is a virus that can penetrate into the host DNA and exhibit rapid antigenic changes. must be protected from infection by viruses. Furthermore, defense is the primary part of an intrusion. on the mucosal surface, as well as cell-free and cell-bound viruses. must be valid for both sources. UK MRC AIDS Management Plan (the [Jnited Kingdom MRCAIDS DirectedPro Gramme) shows that vaccination is feasible and that these obstacles can be overcome. Simian immunodeficiency virus (SIV) was developed in the United States with the primary goal of )-Maker Modelt 2 was adopted.

We now show that the major histocompatibility complex (MHC) class ■ antigen is demonstrated that it could protect animals in the Kirk model. Therefore, the present invention provides In particular, immunodeficient horses for use in methods of treatment of human or animal bodies by therapy. Class I antigens for use as vaccines against viruses.

The present invention also provides a pharmaceutically acceptable carrier or diluent, and MHC as an active ingredient. Pharmaceutical compositions comprising class II antigens are provided. The present invention further provides immunodeficiency viruses. MHC class ■Antigens when preparing pharmaceuticals for use as vaccines against Provide use.

The antigen is preferably a human class II antigen. Therefore, the antigen is HLA - with HLA-DR antigens such as DP, HLA-DQ, or HLA-DR4; It's possible. These are known antigens and can be obtained in purified form. these can be prepared as a recombinant protein.

Alternatively, class II antigens can be used in transfected cells, i.e., those encoding the antigen. obtained from cells that have been transfected with a gene that encodes the antigen and as a result express the antigen. You can also do that. Transfected cells that can be administered to humans are human diploid cells. It may be a cell-based transfected cell. Such cell lines can be used for the production of human vaccines. Tests are being conducted to ensure safety. A suitable cell line is MRC5 cells. It's a line.

Allogeneic lymphocytes presenting class ■ antigens may be administered to the patient.

The lymphocytes can be given as live cells, eg, as a blood transfusion. or They can be provided as fixed or inactivated cells. The lymphocytes are e.g. class by stimulating it with mitogens or gamma-interferon, for example. It may be one in which antigen expression is enhanced.

The antigen can be used to vaccinate a host against an immunodeficiency virus. Ru. The host may be human or animal, but typically contains human immunodeficiency virus (H It would be desirable to vaccinate humans against IV). that will The virus is HIV-1 or HIV-2. Therefore, infection with immunodeficiency virus Prophylactic treatment of medical conditions caused by can be provided. Class ■antigens are, in particular, AID can function as a S vaccine.

An effective amount of antigen is administered to the host to be vaccinated.

The antigen can be administered parenterally in any form, e.g. subcutaneously, intramuscularly or intravenously. can be given within. The amount of antigen per dose depends on the age and age of the subjects involved. It depends on various factors such as health and health status. Parenteral dosage is typically 20μ gx1 mg of antigen, for example 50-500 μg of antigen. As the number of doses can be administered, for example, 2 to 4 times over a period of up to 6 months. Each dose is 1-2 It is held every other month.

The result is a drug for use as a vaccine against immunodeficiency viruses. be done. It also formulates pharmaceutical compositions containing an acceptable carrier or diluent. You can also do that. Therefore, the composition is sterile and pyrogen-free. Ru. The composition may also contain an adjuvant such as AI(Oll)h or saponin. May contain.

Compositions for intramuscular or subcutaneous injection may be formulated using an acceptable carrier, such as sterile water, Olive oil, ethyl oleate, glycols such as propylene glycol, and If desired, an appropriate amount of lidocaine hydrochloride may be included together with the antigen. Intravenous injection Solutions for injection or instillation can, for example, contain sterile water as a carrier, and are preferably Alternatively, it may be in the form of sterile saline.

MHC class ■ antigens have negligible toxicity and can be used safely.

The present invention will be explained below with reference to Examples.

Example I In the initial experiments, a relatively crude, inactivated vaccine was used cautiously (Table 1). . Virus-infected C8166 cells (Virology, 129.51-64.1 983. Here the cells are called C63/CR11-2 cells) or partially spermatozoa. The prepared virus was inactivated with either aldehyde or β-propiolactone. and administered to 3 or 4 cynomolgus monkeys per group. Vaccine administered 4 times, 3 times There was a rest period of at least 6 months between the first and last dose. Quil-a (purified saponin), 5AF-1 (synthin containing threonylmuramyl dipeptide) Available in three different varieties, either Syntex Emulsion or Freund's Juvant was used. Each group of vaccinated monkeys was 2 weeks after the last dose of the vaccine, along with a control group. Challenged intravenously with 32H isolate of SI Vmac251. all contrasts group was infected. The virus was repeatedly isolated and the polymerase chain After amplification by reaction, proviral DNA was detected in peripheral blood lymphocytes. Ta. Furthermore, a significant antibody response against SIV was detected. On the other hand, Waku None of the monkeys vaccinated with Chin had any evidence of viral infection by these criteria. . These experiments using inactivated virus vaccines have changed the schedule of immunization. This was extended to demonstrate that the administration could be reduced to three doses given one month apart.

4 to 6 months after the last dose of vaccine, the monkeys are challenged again to obtain protection. The duration of control was evaluated. Five out of eight cynomolgus monkeys were protected. other public announcements These results, together with data 2, 34, and 5, indicate that the inactivated vaccine is effective against crab eaters. It elicits strong protection against SIV infection in monkeys, and this protection The results indicate that the virus was still detectable for at least 6 months after the completion of inoculation.

cross defense Formalin-inactivated SIV using 5AF-1 as an adjuvant was administered to eight animals. The SIV vaccine was tested by inoculating rhesus monkeys and eight cynomolgus monkeys. We investigated the range of defenses induced by Two weeks after the fourth vaccination, the four animals A rhesus monkey and four cynomolgus monkeys were challenged with the same virus. all 8 The monkeys were completely protected from infection. 10M ID for the remaining 4 rhesus monkeys SO's S I Vaezamsto (M, to Dr. Murphy-Corb Therefore, I took up the challenge with the following (which was kindly provided to me). These monkeys also thwart infection. It stopped. The remaining four cynomolgus monkeys were treated with IOM ID so HIV25aLs. ss* (to Dr. P. Putkonen and Dr. G. Biberfield) Therefore, I took up the challenge with the following (which was kindly provided to me). These cynomolgus monkeys are all sensual. dyed. Analysis of the viruses involved in these cross-protection experiments was carried out by SI Vmac. 251 and SIV, . . . , both have amino acids of their envelope proteins. It was shown that the amino acid sequences had 83% homology. On the other hand, S Vmac251 and HIV 2s++t are simply found in envelope proteins. Only 73% are homologous.

30 monochromes created for the envelope protein of SI Vmac251 A panel of null antibodies was used to establish the antigenic diversity of these viruses. child All of these antibodies reacted with the vaccine virus in the ELI SA assay. However, 11 antibodies did not react with SIV, and 20 antibodies did not react with HIV 2. Did not react with SBL. These results are consistent with the inactivated wax prepared from SIVmac. Cutin is an antigenically distinct strain of SIV. 1. , complete against the challenge using This cross-protection does not extend to the more distantly related HIV-2 virus. Indicates that there is no Therefore, the antigenic variability of immunodeficiency viruses is not as great as originally feared. , may not pose a major obstacle to a promising vaccine. however , this conclusion requires re-explanation in terms of anti-cellular responses discussed below. May.

mucosal immunity The issue of inducing protection at mucosal surfaces was investigated using the intrarectal route of challenge. . The standard challenge virus pool of 32H isolate of SI Vmac251 was Although used for all previous intravenous challenges, the intrarectal route was used to challenge the challenge. It was first titrated in rhesus monkeys. This route is used to infect monkeys. A thousand times more viruses were needed. However, the subsequent course of the infection was stagnant. The course of infection after intravenous administration was virtually indistinguishable. Next, add 5AF-1 to the horse mackerel. Formalin-inactivated SIV was inoculated subcutaneously into four rhesus macaques using it as a rejuvant. did. Two weeks after the fifth vaccination, the monkeys were tested based on intrarectal titration (I OMID,. I did an intrarectal challenge. 4 vaccines challenged at the same time All uninoculated control monkeys became infected. Against this, the vaccinated 4 All monkeys remained uninfected for at least 6 months. this Experiments have shown that immunity can be induced against challenge via mucosal surfaces. Karu.

Challenge with cell-bound virus The cell-bound virus material for challenge was prepared by incubating SIVmac251 for 10 weeks. was prepared from the pancreas of cynomolgus monkey J82, which had been infected with a 32H isolate of cynomolgus monkey J82. cell By cryopreserving an aliquot of and then co-culturing with C8166 cells, Titration was performed in vitro (Table 2). The infectivity titers of cells and their supernatants are log,. They were 4.5 and 2.5. Therefore, 99% of infections bind to cells. Ori, IIDI.

corresponded to 72 viable cells. Next, in vivo titration of monkey lung cells log, giving an end point of o3.0, corresponding to lID5o of 2.300 cells.

Prepare a suitable cell-bound viral challenge and perform an intravenous titration prior to Inactivated SIV was inoculated subcutaneously for intravenous challenge with cell-free virus. Four cynomolgus monkeys were selected that were shown to be protected. after the first challenge These monkeys, which remained virus-free for 12 months, were revaccinated and tested at 2 weeks. Thereafter, cells were challenged intravenously with cell-bound virus (Table 3). Vaccine administered 4 All monkeys were infected, along with four unvaccinated controls. Will Viral and proviral DNA was repeatedly detected in peripheral blood lymphocytes. this As described above, intravenous challenge using cell-free virus grown in a human T cell line Vaccines that protected against SIV also did not protect against lung cells of monkeys infected with SIV. I couldn't come.

Next, protection is provided by immunization with various recombinant proteins derived from the SIV gene. We searched for specific compounds in inactivated vaccines that are involved in. on yeast virus-like particles p27 expressed in 100% and mixed with aluminum hydroxide, or recombinant vaccinia wi purified gp160 from Rus, or gp130 expressed by CHO cells, or 4 animals per group using either gp140 expressed by baculovirus. of monkeys were immunized. Each envelope protein was was administered with the adjuvant formulation. The vaccine is administered in 4 doses, 2 weeks after the final dose. Later, IOM ID soL along with a non-vaccinated control group of 4 animals. :1') I challenged the monkey with SIV. gp14 from baculovirus All of these monkeys were infected except for one vaccinated with 0. Therefore, The recombinant protein can induce high titers of antibodies against the SIV envelope. but they failed to protect monkeys against intravenous challenge. Ta.

Immune correlates of protection Obtained on the day of challenge from 55 vaccinated monkeys used in these studies. By measuring the antibody titer in the serum, we analyzed the immune response that correlates with protection.

43 monkeys were administered the inactivated vaccine and 12 were administered the recombinant envelope protein. was administered (Table 4).

SI Vmac251 proliferated as a persistent infection in HUT-78 cells. Neutralizing antibodies against were measured. Thirty-two animals were protected after receiving the inactivated vaccine. In the two groups, the average neutralizing antibody titer was log+J, 0±0.5. defense The same mean value was observed in the group of 11 monkeys that did not. Furthermore, recombinant 11 monkeys vaccinated with envelope protein and not protected had a higher mean antibody titer of log+o2.9±0.5. Therefore , there was no clear correlation between neutralizing antibody titers and protection in these monkeys. Ta. These blood against recombinant envelope gp140 by EL I SA Titration of the serum also failed to show any correlation with protection. Similarly, this These vaccines induce a strong T helper cell proliferative response against SIV; and in some cases induced MHC class-restricted cytotoxic cells. However, there was no clear correlation between cellular responses and protection against SIV. . The strong protection we observed following vaccination and the immune response we measured. It is an unfortunate result that we were unable to find any correlation between . However, the immunoassay we used may have been inappropriate. Ru.

Reactions to cellular components At this point, the results of vaccine experiments are clear that provide an explanation for our observations. (Table 5). This experiment demonstrated that two doses of vaccine were administered with cell-free virus. to examine whether it is sufficient to protect against intravenous challenge with It was originally planned for. 5IV using QuiiA as an adjuvant - Infected C8166 cells were vaccinated into 4 cynomolgus monkeys at 0 and 4 weeks. Ta. A control group of four monkeys was similarly vaccinated with uninfected C8166 cells. I planted a seed. Two weeks after the second dose of vaccine, both groups showed 10MID5a virus. The challenge was made using 5IV-4 monkeys vaccinated with infected cells One of the animals became infected, but surprisingly, four animals vaccinated with uninfected cells Of these, only two became infected. To confirm these surprising results, For this purpose, the protected monkeys were further vaccinated at 26 weeks, and 2 weeks later, 4 vaccinated monkeys were vaccinated. The monkeys were re-challenged with control monkeys that had not been vaccinated. Non-infectious C Partial protection was again observed in monkeys immunized with 8166 cells, whereas All four control monkeys that were not inoculated with cutin were infected. These vaccine cells antibodies against the components, using detergent lysates of C8166 cells as antigens, Measured by ELISA (Table 6). Average antibody in 8 protected monkeys The titer was 10 g + o3.5, and in 5 unprotected monkeys it was lo The glo was 2.4. The difference between these two groups was highly significant. Inactivation work Analysis of anti-cell antibody levels in all monkeys treated with Chin showed that protected monkeys Similar differences were observed between monkeys that were tested and those that were not tested. Therefore, in C8166 cells There was a statistical correlation between antibody titers and protection in these monkeys.

conclusion These studies have shown that three different inactivated vaccines were used in a cell-free vaccine of the same origin. It has been shown to protect against SIV. In vaccinated monkeys, challenge Since neither virus nor proviral DNA is detected for a long time after infection, the cause is The defense provided is powerful. 5 different adjuvants and various immunization techniques order is effective. Inactivated vaccines are heterogeneous vaccines using SIV,... Protects against range, but not against HIV-2. for the challenge Immunity declines 4 and 8 months after vaccination, but is still detectable. be. Parenteral vaccination using inactivated virus is a direct method of cell-free virus vaccination. Intravenous monkey tile cells that protect against intestinal challenge but are infected with SIV Does not defend against internal challenges. Three different types of SIV envelope proteins Although the product was found to be highly immunogenic, live intravenous challenge could not defend against it. That observed protection is based on the measured S There was no correlation with immune response to IV. However, defense and C8166 There was a correlation between antibody levels against the cells. From these results, the observation The protection provided is due, at least in part, to the cellular components present in the inactivated vaccine. It is suggested that this is mediated by an immune response to

(Margins below this page) Table 1 Effective inactivated SIV vaccines +5IV-infected cynomolgus monkey J82 tile cells Table 3: Challenge with cell-bound SIV This antibody titer is log. Closed display Table 5 Vaccination using C8166 cells (Test 22) Table 6 C8166 cells Correlation between antibodies and protection against this antibody titer is expressed as IOg+o Example 2 A second experiment was designed to confirm the protection exerted by uninfected human T cells. (Table 6). C8166 cells (human T cell cellular vaccines using either RK-13 cells (rabbit kidney fibroblasts) or RK-13 cells (rabbit kidney fibroblasts). Inoculated. Group 3 was used as a non-vaccinated control. The cells were 0.075 % of glutaraldehyde, and Quil as an adjuvant. It was mixed with A (purified saponin). Each dose consisted of 2XlO” cells and 10 μg Qu It consisted of il A. The vaccine was administered subcutaneously at 0, 4.8 and 16 weeks. Ta. Two weeks after the final dose of vaccine, all 12 monkeys were grown in C8166 cells. Infectious dose of simian immunodeficiency virus (SI Vmac32H) grown in 10 monkeys I took on the challenge with (MIDs.). A control group of monkeys and a vaccine using RK-13 cells were used. Viral and proviral DNA was detected in all of the monkeys inoculated with C. In monkeys inoculated with 08166 cells, it was detected in only 2 out of 4 animals. There wasn't.

To confirm and further extend this observation, we tested the two protected monkeys for 30 weeks. The eyes were given another dose of C8166 cells. Two weeks later, these monkeys and the vaccine A control group of 4 animals that had not received any vaccinations was added to the control group of 4 animals that had not been vaccinated S, an antigenically distinct virus grown in peripheral blood mononuclear cells (PBMCs). I took on the challenge with IVsm3's IOM ID io. The control group were all infected, but , two vaccinated animals maintained protection.

Finally, the protected monkeys were revaccinated at 44 weeks along with four control animals. 10M IDs of SI Vmac251 grown in monkey PBMC. . I took on the challenge. All monkeys were infected.

From this experiment, the uninfected human T cells are the same as the cells used as the vaccine. At least two antigenically different strains of SIV grown in unnecessary human T cells It was confirmed that it protects against strains. This defense is developed by growing inside monkey cells. It was not as good as SIV.

Table 6 Non-infected cell vaccines +Number of infected monkeys/Number of challenged monkeys Example 3 The major antigen present on the surface of allogeneic or xenogeneic T cells is the major histocompatibility antigen (MH C) Class I and Class ■. whether these are involved in the observed protection. In order to investigate whether a) normal mouse fibroblasts (L cells), b) MHC clusters transfected with the human gene for SLAB7+β2 microglobulin. c) MHC class n (HLA-DR4) Using either L cells (8115 line) transfected with human genes A group of four cynomolgus monkeys were immunized. 8024 and 81 by fluorescent antibody staining. More than 90% of the 15 cells expressed class I or class II antigens, respectively. Applicable Cells were carefully fixed with 0.075% glutaraldehyde and adjuvanted. and 10 μg of Quil A as an agent (Table 7). 0.4.8 and 16 2X10' cells were administered subcutaneously to monkeys four times a week. Two weeks after the last dose, C81 In IOM ID s of SI Vmac32H grown in 66 cells, All 12 monkeys were challenged intravenously. All monkeys in groups a) and b) infected, but only 2 out of 4 animals given cells expressing class ■ were infected. did.

From this result, human MHC class Ⅰ, that is, HLA-DR4, is found in human T cells. It can be seen that monkeys can be protected from grown SIV.

beast 1. Desrosiers RC, Ringler DJ, rAIDs search Use of Simian Immunodeficiency Virus for J Int Virol 30.301 7312 (1989).

2, Desrosiers RC, Wyand MS, Kodama T et Niji, vaccine protection against rsIV infection J Proc Natl 8ca dSci, New York, 86.6353-57 (1989).

3. Murphy-Corb M, Martin LN, Davison -Fairburn B et Rainbow.

Formalin-inactivated whole simian immunodeficiency virus vaccine J 5CienCe 246.1293-97 (1989).

4, 5tott EJ, Chan WL, Mills KHG et al ,　Preliminary report 2 Simian immunodeficiency virus number by fixed infected cell vaccine Defense of lunar-eating monkeys against two J Lancet 336.1538-41 (1 990).

5. Carlson JR, McGraw TP, Keddie E et al, r Vaccine protection of rhesus macaques from simian immunodeficiency virus infection J AIDS SR Antibody J Nature 353.393 (1991).

Continuation of front page (72) Inventor Kitchen, Beater Anthony UK EN63 Cu - Hertfordshire, Botters Bar, South Mimms, Blanche Leigh National Institute for Biological Standards And control (no address) (72) Inventors Mills, Kickston Henry Gorton United Kingdom EN63Q Hertfordshire, Potter's Bar, South Mims, Plancheley National Institute for Biological Standards And control (no address) (72) Inventor: Chang, Wuling, United Kingdom, EN63Q Hertfordshire, Botters Bar, South Mimms, Blanche Leigh National Institute for Biological Standards And control (no address) (72) Inventor Page, Mark United Kingdom EN63Q Hertfordshire, Potter's Bar, South Mims, Plancheley National Institute for Biological Standards And control (no address) (72) Inventor Tuffs, Leslie Frank UK EN63Q G Hertfordshire, Potter's Bar, South Mims, Plancheley National Institute for Biological Standards And control (no address)

Claims (11)

[Claims] 1. Primary histocompatibility for use in therapeutic methods of treatment of human or animal bodies Gene complex class II antigen. 2. The anti-inflammatory agent according to claim 1 for use as a vaccine against immunodeficiency virus. original. 3. 3. The antigen according to claim 2, wherein the virus is human immunodeficiency virus (HIV). 4. 4. The antigen according to claim 3, wherein the virus is HIV-1. 5. 4. The antigen according to claim 3, wherein the virus is HIV-2. 6. The antigen according to any one of claims 1 to 5, which is a human class II antigen. 7. The anti-inflammatory agent according to claim 6, which is an HLA-DP, HLA-DQ or HLA-DR antigen. original. 8. A pharmaceutically acceptable carrier or diluent and major histocompatibility compounds as active ingredients. A pharmaceutical composition comprising a gene complex class II antigen. 9. In the production of medicines for use as vaccines against immunodeficiency viruses Use of major histocompatibility complex class II antigens. 10. administering an effective amount of a major histocompatibility complex class II antigen to a host; A method of vaccinating a host against an immunodeficiency virus, including. 11. Contains major histocompatibility complex class II antigens and is effective against immunodeficiency viruses. A drug useful as a vaccine.