JPH10505668A - Estimation test for resistance to hepatitis B - Google Patents

Abstract

  (57) [Summary] Disclosed are methods for identifying resistance to hepatitis B infection, as well as peptides capable of modifying the immune response and methods for treating hepatitis B.

Description

DETAILED DESCRIPTION OF THE INVENTION                     Estimation test for resistance to hepatitis B   The present invention relates to a novel test for estimating resistance to hepatitis B, Peptides useful for modifying response, vaccines against hepatitis B and hepatitis B It relates to a treatment method.   Hepatitis B virus (HVB) infection is asymptomatic from fulminant hepatitis due to liver failure Causes a number of medical conditions, up to sexual persistence (carriage). Most patients Suffer acute hepatitis while killing the virus. In North America and Europe In about 5% of patients cannot clear the virus, but in West Africa up to 15% Stained patients cannot clear HVB (Ryder, R. W.) et al., Lancet ii (8400): 449-52, 1984). . Persistent HBV infection predisposes the host to chronic liver disease and hepatocellular carcinoma (Bee Ray, Beasley, R, et al., Lancet, ii, 1159-63, 1 981), which is a common cause of death of working-age adults in West Africa (La Ryder, R.W. et al., Am J. Epidemioru (Am.J. Epidemiol.), 136 (9): 1122-31, 1992).   The outcome of HBV infection is determined by the variation in viral virulence. And the course of the disease is influenced by the host's immune response. Persistence Some immunocompromised conditions such as HIV (cross guard, Kro sgaard, K.) et al., Hepatology, 7: 37-41, 1987) Is γ-globulinemia (Hermaczewski, R.A.) Ra, Cue J Med (Q.J. Med.), 86 (1): 31-42, 1993), but mostly immune Not defenseless.   In West Africa, HBV is a young child infected by older brothers and playmates. Conveyed via a horizontal route by a child (up to 10 years old) (Marin ier, E.) et al., West Africa J. Pediat r.), 106 (5): 843-9, 1985; and Bota, Jeff (Botha, J.F.) et al., Lancet i (Lmcet, i), 1210-2, 1984). Seen in the Far East Because vertical infections are rare, immature immune system and transplacental HBV'e ' Tolerogenic effects of antigen (HBeAg) transmission do not appear to affect outcome of infection Can be Epidemiological studies show that about 80% of the adult population is infected with HBV, Persistent retention, as measured by HBV surface antigen retention, is around 12-15%. (Ryder et al., Supra). Death from HBV-related hepatocellular carcinoma Are the leading cause of mortality among working-age adults in the Gambia (Rider (R yder) et al., Am. J. Epidemiol., supra; Kiire, C.F., Vaccine, 8: 5107-112, 199. 0 years).   Recognition of foreign antigens by T lymphocytes is located in the groove of the MLA-encoded HLA molecule. This is accomplished through the presentation of the antigenic peptide in the antigen. Such immunity Response is MHC restricted, which indicates that foreign antigens are Meant to be recognized only when presented by a Class I or Class II molecule. To taste. In patients with acute hepatitis B, cytotoxic T lymphocytes restricted by class I ( CTL) recognizes HBV nucleocapsid and envelope antigens in peripheral blood (Bertoletti, A., et al., Plock Nachol)   Akasai, USA (Proc. Natl. Acad. Sci, USA), 88 (23) : 10445-9, 1991). In addition, potent against nucleocapsid antigens There is a proliferative response restricted by the class II (Ferrari, C.) J. Clin. Invest., 88: 214-22, 1. 991). CD4 against viral nucleocapsid and envelope antigen + T helper cell response supports the development of anti-HBe and anti-HBs antibodies (Milich, D., et al., Nature, 31). 9: 547-9, 1987). CTL is detected in patients with chronic HBV infection. No proliferative response is present or significantly suppressed (Ferrari, C ri, C.) et al., J. Immunol., 145 (10): 3442-9, 1990; and Tsai, S. et al., Jacqueline Invest ( J. Clin. Invest.), 89: 87-96, 1992).   Studies have shown that the outcome of HBV infection can be The role of the phenotype of MHC was examined (Vm-Hattum J.) Et al., Hepatology, 7 (1): 11-14, 1987; D. Forzani, B. et al., Hepatology, 4: 1107-10, 1 984; Lepage, L. et al., Tissue Antigens.  Antigens), 18: 105-7, 1981; and Kaslow, Earl. R.) and Shaw, S., Epidemiol. Rev. 3: 9 0-114, 1981). However, most of these studies are based on MHC tables. A size study that only detected a very strong association between genotype and medical condition. Sa Furthermore, the sensitivity of these studies indicates that many alleles are assigned the same serological specificity. By using a serologically defined MHC class II typing method It was decided.   DRB1 * 1302 reduces the risk of cerebral malaria in Gambian children Associated with a reduction in the number of DRB1 * 1302-DRB3 * in this population. 0301-DQA1 * 0102-DQB1 * 0501 and DRB1 * 1302- Haplotype of DRB3 * 0301-DQA1 * 0102-DQB1 * 0604 (Lepage, L. et al., Supra). Northern Europe In Gambia, the frequency of human haplotypes is 4.4% 16.4% (Hm, A. et al., Nature, 352 (63 36): 595-600, 1991). Malaria and HBV are both in West Africa It is an important cause of premature mortality in Rica. However, severe mala Although rear mortality is high in children under the age of five, mortality associated with HBV does not work. It is common in all ages. Recovery from HBV infection is due to DRB in addition to malaria If related to 1 * 1302, HBV is DRB1 * 1302 in Gambia There are selective benefits for people with haplotypes.   Recent studies have shown that DRB1 * 1302 and DRB1 * 1301 A protective effect was demonstrated against topapilloma virus (HPV) -related cervical cancer (A Apple, R.J., et al., Nature Genetics (Nature)  Genetics), 6 (2): 157-162, 1994).   Serological supertypes of DRB1 * 1302 and DRB1 * 1301 (sup ertype), DRw6, is potentially protective against persistent HBV infection. Have been reported in two previous studies. Van Hattum et al. HBV-removed patients in Northern Europe who did not remove the virus DRw6 was found twice as frequently as compared to that of Van-Hat J tum J.) et al., supra). Of chronic HBV infection to treatment with interferon Affect response By studying the contributing factors, we conclude that DRw6 is associated with a favorable response (Scully, L. et al., Hepatology, 1 2: 1111-17, 1990). However, both of these studies It was too small to pick a statistical advantage.   Here, the presence of certain HLA molecules confers resistance to infection by hepatitis B. Was discovered.   Thus, the present invention identifies the presence of HLA-DRB1 * 1302 (ident ify) stage, which provides a method for identifying resistance to hepatitis B infection. . Preferably, the identification is performed on a blood sample. Therefore, the present invention It is intended to provide a simple method for estimating the resistance to hepatitis B in a given person. Therefore, it is possible to make predictions about the outcome of hepatitis B infection in individual patients. You.   According to one embodiment, the method of the present invention further comprises HLA-DRB1 * 1301 Identifying the presence of Again, this analyzes the blood sample It is preferred that this be done by:   Additionally, HLA-DRB1 * 1302 and / or HLA-DRB1 * 13 01, which is related to resistance to hepatitis B, Modification of human immune responses by peptides that bind to one or both offspring It is possible. Thus, according to another concept, the present invention also provides HLA-DRB1 * 1302 and / or one or more pairs capable of binding to HLA-DRB1 * 1301. It provides a peptide. Such a peptide is HLA-DRB1 * 13 02 and / or the ability of HLA-DRB1 * 1301 to induce an immune response And can be used for. Such methods are useful in immunizing against HBV infection And in those with persistent infection, DRB1 * 1302 and * 1301, (Therapeutic immunization). According to a preferred embodiment Then, the immune response is suppressed by the binding of one or more modified peptides. This is Particularly, it is preferable for treatment of symptoms such as fulminant hepatitis B.   More generally, the invention relates to the immunization of HLA in response to hepatitis B antigen. An object of the present invention is to provide a peptide capable of modifying the ability to induce a reaction.   Preferably, the peptide is derived from a hepatitis B antigen. For example, peptide Consists only of specific regions known to bind to specific HLA molecules You may. Alternatively, the synthetic peptide is composed of a binding region and other non-binding regions. May be constructed as follows.   The peptide can be administered in the form of a pharmaceutical formulation, for example, an intravenous formulation. Thus, according to another concept, the present invention relates to HLA-DRB1 * 1302 and And / or one or more peptides capable of binding to HLA-DRB1 * 1301, and 1 Provide a pharmaceutical formulation comprising one or more pharmaceutically acceptable carriers and / or excipients Is what you do.   In addition, such peptides can be used in the manufacture of a vaccine against hepatitis B. You. Therefore, the present invention also provides HLA-DRB1 * 1302 and / or HLA-DRB1 * 1302. Hepatitis B consisting of one or more peptides that can bind to LA-DRB1 * 1301 Vaccine against the disease.   The peptide of the present invention also provides another method for treating hepatitis B. An example For example, antigen-specific lymphocytes can be obtained by using the peptide of the present invention in vitro. Can be formed. These can be administered to patients suffering from hepatitis B. Accordingly Thus, the present invention provides a method for preparing a A composition comprising a sphere is provided. Administering such a composition to a patient Also provided is a method of treating hepatitis B comprising a floor.   Usually, the lymphocytes are derived from the subject to be treated or have a similar HLA type. May be derived from another subject having   The present invention will be described with reference to the following examples. There is no restriction.   The examples refer to the following drawings.   FIG. 1. Exposure of hepatitis B and HBsAg protection by age among children. Indicates an increase in prevalence;   FIG. 2 shows the frequency of HLA class I serotypes for each group of children and adults. Is;   FIG. 3 shows the class II haplotype of a child;   Figure 4 shows adult class II haplotypes; and   FIG. 5 shows a differential analysis of the risk of individual DRB1 * 1302 haplotypes. It is.Example 1 Research population   All subjects were located in the western coastal area, around the capital, Banjul. He was a Gambian living. Two different groups between 1988 and 1990 Were recruited in two stages of the study. In the first phase, children up to the age of 10 are linked to HBV Various symptoms were already seen in Banjul's Royal Of Victoria Hospital (Royal Victoria Hospital) and Fajara , Medical Research Council Hospital Hospital). The adult population was recruited from healthy male blood donors. Twin The other population was previously studied as part of a patient-control study of susceptibility to malaria. (Hill, A. et al., Supra).   At both stages, subjects were divided into groups according to serologic tests for HBV . Group A, not exposed to HBV, is anti-HBV core antibody (anti-HBc) negative. Was. Group B spontaneously recovered from HBV infection Was anti-HBc positive and HBV surface antigen (HBsAg) negative. Lasting Group C, which had been infected with HBV, was anti-HBc positive and HBsAg positive. Was. Group C patients with IgM antibodies to the HBV core antigen may be subject to this analysis. Excluded. Persons vaccinated against HBV (<10%) were placed in group A Therefore, they were not included in the analysis of HLA frequency. There were a total of 1,344 children Group A had 891 people, group B had 218 people, and group C had 185 people; Was. In the adult population, there were a total of 260 subjects; Group A had 25 subjects and B There were 195 groups and 41 groups. Very few (<1%) H Subjects with IV antibodies were excluded.Serological test   Plasma samples were taken from all subjects and stored at -20 <0> C. Anti-HBc, anti HBc (IgM) and HBsAg status and anti-HBs antibody concentration were determined by the manufacturer. (Boehringer Mannheim, Munich, Germany) Measured by ELISA according to instructions. Welcozyme EL for anti-HIV status ISA (Wellcozyme ELISA) (Welcome, Beckenham, UK) and positive results are confirmed by Western blotting. I accepted.By HLA type   Class I serotyping was performed on approximately half of the subjects in each population. Serological MHC class I type was used for 180 cells on fresh or cryopreserved cells. Standard microlin with 180 well chracterised antisera Determined by microlymphocytotoxicity assay (Rood, J., Van), Manual of Tissue   Typing Techniques (Manual of Tissue Typing Techniques), Bethesda, Maryland (Bethesda, Maryland): National Institute of Health (National Institute of Health), 104-105, 1979). MH Classification of C class II was performed using restriction enzymes TaqI and BamHI as described above. And by restriction fragment length polymorphism analysis (Hill et al., Supra; and Hill, A. et al., Plock Naschol Akadesai, USA ( Proc. Natl. Acad. Sci, USA), 89: 227-81, 1992). To this If amplified by polymerase chain reaction and sequence specific oligonucleotides Supplemented blotting (Hill et al., Supra; and Hill, A.) Proc.Natl.Acad.Sci, US A), 89: 227-8L 1992). All patients were of type II Was.Statistical analysis   Group B and Group C phenotypes (haplotypes defined in RFLP for class II) ）) Frequency comparison,^Two23 HLA class I antigens and 10 using test Performed with a Class II haplotype. Obviously, when performing multiple comparisons simultaneously Significant associations may occur by chance. Prevent these types of mistakes To do this, we used the following two-step strategy: Multiple comparisons, and then test the second population (adults) for the only hypothesis. (Hill, A., J. RCP (J.RCP), 26 (1): 11-16, 1992). Calculate relative danger with approximate probability And obtained with a 95% confidence interval. The Gambia has a mixed ethnicity (Hill, A (H ill, A.) et al., Proc. Natl. Acad. Sci, USA), supra), the Mantel-Haenszel test Confused with possible ethnic sources The effect was evaluated. The anti-HBs concentration was determined using the unpaired t test. Adult between subject with LA-DRB1 * 1302 and subject without Comparisons were made in populations.result   Of the 1,344 recruited children, 453 (33.7%) were anti-HBc positive And 185 (13.8%) of these were HBsAg positive and anti-H Bc (IgM) was negative. 218 (16.2%) children had anti-HBc (total Part) positive, anti-HBc (IgM) negative and HBsAg negative. Anti-HBc and The age-dependent increase in HBsAg and HBsAg prevalence is shown in FIG. Book 260 adults Recruited for study, of these, 235 (90.4%) were anti-HBc positive Yes, 41 (15.8%) were HBsAg positive. HL for each child group The frequencies of A class I serotypes and class II haplotypes are shown in Tables 1 and 2, respectively. And 3.   HLA class I antigens, HLA-B50 and HLA-Cw1 and persistent HBsAg Potential associations with retention were suggested by data from child studies ( Table 1). However, these findings may be inconsistent with the adult data studied. Not suggested (Table 1). Thus, the class I phenotype is HBV clearance Was not significantly related.   The HLA class II haplotype was first identified as a restriction fragment length polymorphism (RFLP) haplotype. Determined using the restriction enzyme TaqI which defines the rotype (Hill, A.) Proc.Natl.Acad.Sci, US A), above). Class II haplotype, DRB1 * 1302-DRB3 * 030 1-DQA1 * 0102 -DQB1 * 0501 (DRw13 / DQw5) and D RB1 * 1302-DRB3 * 0301-DQA1 * 0102-DQB1 * 06 04 (DRw 13 / DQw6), RFLP pattern 25-1 eliminates HBV infection In 26.6% of affected children (Group B) and persistently infected with HBV (Group C) At 16.2% (relative risk 0.53 [95% CI 0.32- 0.90], p = 0.012), which is protective against persistent HBV retention Related to Class II haplotype, DRB1 * 1301-DBR3 * 010 RFLP haplotype corresponding to 1-DQA1 * 0103-DQB1 * 0603 Group 13-2 is also found at a significantly reduced frequency in persistently infected children (P = 0.037). Are other haplotypes showing significant frequency changes? Was. Therefore, we analyze the above two possible associations in the adult population. I decided to. HLA-DRB1 * 1302 haplotypes are group B ) Of 195 adult subjects (25.6%) and persistent infections Found in 3 (7.5%) of 40 patients (relative risk 0.24 [9 5% confidence interval 0.04-0.80], p = 0.012). Classified ethnic sources Later, the biased relative risk from Mantel-Haenszel was zero. . 23 [95% CI 0.05-0.83], p = 0.022; Thus, any significant confounding effects of ethnic sources were eliminated. HLA-DRB1 * Possible association with 1301 not confirmed by adult population data Was.   Subjects with a 25-1 haplotype from both populations were additionally treated with DQw5 or Were divided into DQw6 haplotypes. Relative risk and 95% confidence interval for both Haplotypes were calculated and compared in Table 4. HBV elimination works with both haplotypes Relevant, protection from persistent HBV possession has DRB1 * 1302 It is suggested to be related to the HLA-DR molecule.   In 25 adults (average age 28.6 years) with DRB1 * 1302, The average anti-HBs antibody concentration was 15.5 iu / L (sd 41.5) and DRB In 25 adults without 1 * 1302 (mean age 32.2 years), the average anti-HB The s antibody concentration was 22.3 iu / L (sd 48.5); p = 0.598 .discussion   This study is the first large HLA of HBV infection that introduced class II analysis of molecules. This is a study showing relevance. In addition, this study shows that the haplotype It is clearly shown that step 25-1 is associated with the ability to clear HBV after infection. 25 -1 haplotypes are two MHC class II haplotypes, DRB1 * 13 02-DRB3 * 0301-DQA1 * 0102-DQB1 * 0501 and DR B1 * 1302-DRB3 * 0301-DQA1 * 0102-DQB1 * 060 4 is represented. Further analysis shows that the two DRB1 * 1302 haplotypes are both HL were shown to be related to HBV clearance It is believed that one or both of the A-DR alleles can respond to the above association You. Only HLA-DRB1 * 1302 found on 25-1 RFLP haplotype HLA-DRB1 * 1301 was found on other RFLP haplotypes in Gambia Be discovered. Furthermore, the product of DRB1 * 1302 is lower than the product of the DRB3 locus. Because they are all expressed at higher levels, they are of greater functional importance. we, In the 25-1 haplotype, DRB1 * 1302 is involved in virus clearance. And seemed to be important. Class II haplotype, DRB1 * 1301-DBR3 * 0101-DQA1 * 0103-DQB1 * 0603 The corresponding RFLP haplotype 13-2 is also available in persistently infected children. Data suggest that HBV persistence in the population It is thought to be related to the degree of resistance to The haplotype, DRB1 * 1 The allele of DRB1 at 301 is HLA-DRB1 * with a single amino acid substitution. 1302.   HBV-specific, as determined by the proliferative response to HBcAg and HBsAg Differential CD4 + helper activity is associated with acute self limiting infecti on), there is a marked decrease in patients with persistent infection (Ferrari, (Ferrari, C.) et al., J. Immunol., Supra). In antigen presentation Through its role, class II molecules are involved in the development of CD4 + helper T cell responses. Because of its importance, the ability of class II molecules to present differential antigen 4+ Exhibit as variability in help level. This is HBV clearer Consistent with a special class II association detection with We have sustained HBV infection Patients who do not express anti-HBe and anti-HBs and have a sufficient number of active HBV- Non-expression of specific CTLs is due, at least in part, to those with relatively low antigen-presenting ability. Infer that it is related to the lack of CD4 + help mediated by the SII molecule.   No reasonable expression of anti-HBs after vaccine treatment Associated with HLA B8, SC01, DR3 (Egea, E. et al., J. Exp. Med., 173 (3): 531-8, 1991 Year). The relationship between HLA-DRB1 * 1302 and HBV clearance is described in HLA above. May be reflected in higher titers of anti-HBs antibodies in people with different alleles It is thought that there is. However, anti-HBs in adults who have excluded HBV Antibody levels are higher in people with the HLA-DRB1 * 1302 allele. Extra “help” for the formation of the above antibody reaction The result is an HB with an improved carrier of HLA-DRB1 * 1302 It was suggested that this is not an important mechanism in V clearance.   HBV-infected hepatocytes are recognized and recognized by HLA class I restricted CTLs. (Mondelli, M., et al., J. Immunol. 129 (6): 2773-78, 1982; and Pignateliem. attelli, M.) et al., J. Hepatol. 4: 15-21, 1987. ). The absence of a strong HLA class I association with HBV clearance indicates that HBV Antigens may be present by all class I molecules frequently found in Gambia. Means to be presented more effectively. In chronic HBV infection, HBV-specific CTLs are not easily detectable, which indicates that their numbers are low or CTLs are inactive Is suggested to be sexual. Association of HLA class II with HBV clearance One interpretation of our findings is that CTL activity is a potent CD4 + T helper cell. Need help and its level is measured by HLA class II polymorphisms ( Nonax, R (Nonacs, R.) et al., J. Exp.Med. 176: 519-29, 1992).   DRB1 * 1302 is associated with effective protective effects against three important infectious agents. It seems that there are reams, but it is unclear how the above effect is exhibited . Malaria, HBV and HPV have hundreds of potentials with various MHC restriction elements It is a complex pathogen in which a T cell epitope must be present. For the above diseases Association with MHC indicates that only a few epitopes are present Be inspired. Therefore, polymorphisms at the MHC class I and II loci are It is believed that this is due to the variability in outcomes from HBV infection.

────────────────────────────────────────────────── ─── Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI G01N 33/50 G01N 33/50 T 33/576 33/576 // A61K 38/00 ACS A61K 37/02 ACS (81) Designated country EP (AT, BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (KE, MW, SD, SZ, UG), AM, AT, AU, BB, BG, BR, BY, CA, CH, CN , CZ, DE, DK, EE, ES, FI, GB, GE, HU, IS, JP, KE, KG, KP, KR, KZ, LK, LR, LT, LU, LV, MD, MG, N, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, TJ, TM, TT, UA, UG, US, UZ, VN (72) Mark, Earl United Kingdom, London Double 21 P.G., St. Mary's Hospital Medical School, Academic Department of Medicine, Hepatology Unit (No Address) (72) Inventor Thomas, Howard, Christopher United Kingdom, London Double 21 P.G., St. Mary's Hospital Medical School, Academic Department of Medicine, Hepatology Unit (no address)

Claims (1)

[Claims] 1. Hepatitis B sensation, comprising the step of identifying the presence of HLA-DRB1 * 1302 A method for identifying resistance to staining. 2. Further comprising the step of identifying the presence of HLA-DRB1 * 1301. The method of claim 1, wherein 3. 3. The method according to claim 1, wherein the identification is performed on a blood sample. The method described. 4. Estimating the outcome of a patient's hepatitis B infection, comprising identifying the patient's tissue type Law. 5. HLA-DRB1 * 1302 and / or HLA-DRB1 * 1301 Peptides that can bind. 6. HLA-DRB1 * 1302 and / or HLA-DRB1 * 1301 The peptide according to claim 5, which is capable of modifying the ability to induce an immune response. 7. Can modify the ability of HLA to induce an immune response in response to hepatitis B antigen peptide. 8. The pep according to any one of claims 5 to 7, which is derived from a hepatitis B antigen. Chid. 9. A peptide according to any of claims 5 to 8, and one or more pharmaceutical agents. A pharmaceutical formulation comprising a pharmaceutically acceptable carrier and / or excipient. 10. 10. The pharmaceutical formulation according to claim 9, which is for intravenous administration. B comprising the peptide according to any one of claims 5 to 8, which is 11.1 or more. Vaccine against hepatitis B. 12. The peptide according to any one of claims 5 to 8, which is 12.1 or more. A composition comprising previously treated lymphocytes. 13. 9. Injecting one or more peptides according to any of claims 5 to 8 into a patient. A method of modifying a patient's immune response to hepatitis B comprising the step of administering. 14． 14. The method according to claim 13, wherein the immune response is suppressed. 15. 15. The method of claim 14, which is used to treat fulminant hepatitis B. Law. 16. 9. Injecting one or more peptides according to any of claims 5 to 8 into a patient. Administering or preventing hepatitis B. 17． A step of administering to a patient the pharmaceutical composition according to claim 9 or 10. A method for treating or preventing hepatitis B, comprising: 18. B comprising the step of administering to the patient the vaccine of claim 11. For treating or preventing hepatitis B. 19. 13. Type B, comprising the step of administering to the patient the composition of claim 12. How to treat or prevent hepatitis.