Preventive vaccine
HCV is similar to animal plague virus and human flavivirus in biological classification. It is known that flavivirus nonstructural (NS) protein and plague virus gp33/gp55 can stimulate the body to produce neutralizing antibodies, suggesting that HCV envelope protein which is very similar to its gene and hydrophobic map may be the target of immune attack. Choo et al. [1] once inoculated seven black chimpanzees with HCV envelope proteins E 1 and E2 expressed by recombinant vaccinia virus in HeLa cells, and confirmed that the antibody induced by HCV e protein is a neutralizing antibody, which can prevent/terminate the persistent infection of the virus, so it can be used as a preventive vaccine. Farci et al. [2] inactivated the plasma of a chronic HCV-infected person of different ages, mixed it with the virus and inoculated it into chimpanzees to study whether there are specific neutralizing antibodies in HCV-infected people. The results showed that chimpanzees inoculated with plasma and virus mixture infected with HCV 14 were all infected, but chimpanzees inoculated with plasma and virus mixture infected with HCV V2 did not show HCV infection. Therefore, the authors believe that HCV quasispecies and specific neutralizing antibodies exist in chronic HCV infected people. Subsequently, the author [3] immunized rabbits with two peptides, 390 ~ 4 10/and 384 ~ 41,synthesized by HCVHH77/ hypervariable region1,and then used them to obtain anti-HVR 1 hyperimmune serum. The results showed that anti-HVR 1 had immune defense effect on homologous H77 strain, but had no immune defense effect on mixed HCV quasispecies. Therefore, it is considered that the main target of neutralizing antibody attack is HVR 1 of E2/NS 1, and the neutralizing effect against HVR 1 has virus strain specificity.
Weiner[4]F found two HVRs in E2, and determined that HVRs contained epitopes that induced neutralizing antibodies. Rosa et al. [5] established a neutralization quantitative detection system of HCVE2 protein binding to target cells by combining cell fluorescence labeling and flow cytometry. The results show that there are at least two neutralizing epitopes, one of which is variable in height and the other exists in other regions of E2. Zibert et al. [6] used fibroblasts (VH3) to study the blocking of HCV infection in vitro, and also found that there were a few cross-reactions when neutralizing anti-HVR 1, and not all anti-HVR were strain-specific; They also observed that the anti-HVR 1 antibody on the envelope glycoprotein can lead to self-limiting infection in the early stage of HCV infection, indicating that anti-HVR 1 has a protective effect and can block the development of HCV infection [7]. All these indicate that the target of neutralizing antibody is HVR 1 of E2/NS 1, and it is feasible to prepare preventive vaccine based on HCV envelope glycoprotein HVR 1.
However, the protein epitopes recognized by neutralizing antibodies are mostly discontinuous, that is, neutralizing epitopes are mostly conformations, and antibodies recognize configurations rather than their sequences, which has been confirmed in experiments such as X-ray crystal molecular diffraction analysis of peptides with neutralizing antibodies induced by FMDV [8]; Recent studies have also shown that the formation of conformational epitopes on E 1E2 complex plays an important role in the protective immunity induced by envelope glycoprotein [9]. This requires us not only to pay attention to the immunogenicity of the selected peptide, but also to consider the conformational limitations involved in the interaction between immunogenic proteins and other proteins and nucleic acids when synthesizing HCV envelope glycoprotein polypeptide vaccine. Therefore, studying and simulating the configuration of E protein epitope and its exposure state on cell membrane is the basis of finally preparing effective vaccine.
Multi-antigenic peptide (MAP) takes the core matrix as cross-linking body or dendritic arm, and couples several monomer peptides with the same or different antigenic epitopes to form a dendritic structure, which can not only overcome the shortcomings of the above methods, but also simulate the conformation of natural epitopes well, and has obvious advantages. MAP has been used to analyze the protective antigens of many pathogens, such as human immunodeficiency virus (HIV), FMDV, hepatitis B virus (HBV) and plasmodium, and to design molecular vaccines. MAP skeleton only contains multiple lysine, which can bind four or eight polypeptide chains in the exact position, and the molecular weight of polypeptide chains can account for 95% of the whole vaccine. Because the number and position of peptide chains bound on MAP are accurate, the prepared vaccine has uniform structure and high purity, and the structures of MAP and polypeptide have strong immunogenicity, and antibodies with high titer and high affinity can be induced without coupling carrier protein, while MAP itself has no immunogenicity, which not only improves the quality of vaccine, but also removes the defects of carrier protein.
Therapeutic vaccine
At present, some scholars believe that HCV infection can not produce effective protective antibodies, so the role of cellular immunity in anti-HCV infection has been paid more and more attention. Cytotoxic T cells (CTL) belong to CD8+T cells, and are the main effector cells that perform cellular immune response. When their surface receptors (TCR) bind to HCV antigens of target cells, they can destroy the virus by dissolving the infected target cells under the mediation of MHC class I antigens, but at the same time, they will also cause damage to infected tissues. Studies have shown that the pathological changes of liver in patients with hepatitis C are mainly manifested as lymphocyte aggregation and follicular formation in portal area, CD4+ cells mainly infiltrate in the necrotic area of liver, and CD8+ cells mostly infiltrate in the lobular area of liver. Specific CTL in peripheral blood and liver of patients with acute and chronic HCV infection can recognize one or more epitopes in HCV coding protein. Synthetic HCV polypeptide can stimulate the proliferation of CTL in vitro and enhance its ability to dissolve HCV-infected target cells, so CTL may play an important role in clearing HCV infection.
Kita et al. [10] found that CD8+ specific anti-HCV cTL could be induced by repeatedly stimulating patients' peripheral blood mononuclear cells (PBMC) with synthetic peptide of HCV core region, which not only recognized the synthetic peptide of nucleocapsid protein (NP) 81-kloc-0/00 combined with HLA-B44+ molecules, but also. The author [1 1] repeatedly stimulated PBMC of 3 patients with chronic hepatitis C with recombinant HCVNP and synthetic overlapping peptide as antigens, and found that all of them could produce CTL with specificity and HLA-B44+ restriction, and could recognize NP8 1- 100 sequence. Peripheral blood lymphocytes which can recognize NP 1-120 sequence were observed in1patients, and proved to be CD4+T cells, which mainly recognized the epitope of NP2 1-40 and the epitope of 81-kloc-0/00, while NP The author noticed that this patient's blood HCV was negative and the level of alanine aminotransferase (ALT) was normal, while the other two patients with NP but no CTL activity were positive and the level of ALT increased. The author thinks that when HCV continues to be infected, although CTL is involved in immunopathological injury, which leads to severe liver injury, it can accelerate the clearance of HCV in infected body. The existence of Th epitope can not only enhance the reactivity of HCVNP-specific CTL, but also improve the antibody level through the internal interaction with B epitope, thus increasing the body's immunity to HCV infection.
Lechmann et al [12] studied humoral immunity and cellular immunity of patients with different prognosis after HCV infection. * * * The serum antibody levels of 10 asymptomatic blood donors with positive anti-HCVA serum (group A) and 29 patients with chronic hepatitis C (group B) were observed, and similar phenomena were found: (1)A but the B cell reaction in group B was more common. (2) Through the proliferation reaction induced by the same peptide in each group, it can be determined that there are at least five different CTL epitopes in the core region (20 ~ 44, 39 ~ 63, 79 ~ 103, 1 18 ~ 152 and152). The author thinks that the low antibody titer of asymptomatic patients with positive serum anti -HCV may be due to the strong T cell effect of such patients inhibiting virus replication, which leads to the weakening or disappearance of virus antigen stimulation.
It should be noted that although there are specific CTL in infected people, most HCV infected people will still turn chronic. It is speculated that the reasons may be as follows: (1) Immunoselective pressure causes CTL epitope mutation, which leads to the loss of specific CTL function; (2) A large number of lymphocytes in the body are infected by HCV, which damages the immunity of the body; (3) The drop of HCV in the body is too low, which leads to the weak CTL response. At present, it tends to think that the high mutation of HCV gene leads to immune escape, thus making the infection persist. Therefore, it is undoubtedly of great significance to explore the cross-protection effect of CTL vaccine.
Shirai et al. [13] found that the synthetic peptide containing the th epitope with more than 60 epitopes of HIV GP/kloc-0 is similar to the universal Th epitope, and its selectivity to MHC is low. In order to study the influence of endogenous and exogenous th epitopes on CTL effect, the N-terminal of CTL epitope peptide in NS5 region (P 17) was coupled with the above-mentioned HIV universal Th epitope to form a chimeric peptide (PCLUS3- 17), which was combined with the full-length CTL epitope peptide in the core region (C7) and some fragments containing CTL epitopes in the core region (C7 A65438). The results showed that the proliferation effect of CTL induced by P 17 peptide alone was significantly weaker than that of PCLUS3- 17, suggesting that P 17 peptide lacked Th epitope, while C 17 containing Th epitope could induce stronger CTL effect. Similarly, the CTL effect induced by C7A 10 peptide, which may lack Th epitope, is also weak. The authors believe that non-immunogenic peptides can be transformed into immunogenic peptides by embedding suitable multi-Th cell epitopes. CTL epitope peptide lacking endogenous Th epitope inducing activity; It should be a broad-spectrum Th epitope with valence of * * *; For CTL epitope peptides that already contain endogenous Th epitopes, adding broad-spectrum Th epitopes can expand MHC recognition types and make vaccine protection more effective. This undoubtedly provides a good idea for constructing CTL synthetic peptide vaccine with strong immunogenicity and cross-protection.
Obviously, there are both enhancement epitopes and suppression epitopes in virus antigens. In order to improve the immune protection, efforts should be made to enhance the role of epitopes and reduce and eliminate the role of epitopes. As far as HCV infection is concerned, there are many reports on CTL enhancement epitopes, but there is no study on the influence of inhibitory epitopes or antibodies against enhanced epitopes on CTL function, that is, the positive effect of viral epitopes on CTL function is emphasized, while the negative effect of viral epitopes and their antibodies on CTL function is ignored, which is one-sided in expounding CTL function defects during HCV infection. Therefore, the study of anti-HCVA epitope suppression or enhancement of epitope antibody inhibition on CTL function can undoubtedly reveal the causes of CTL dysfunction caused by HCV infection in a deeper level, which is of great significance for the formation of immune tolerance during HCV infection and guiding vaccine research. In addition, the successful application of MAP in the analysis of HIV, FMDV, HBV and other protective antigens and molecular vaccines is a useful inspiration for the design and preparation of HCV vaccines. Macromolecular chimeric peptides with certain conformation composed of Th epitope, neutralization epitope and/or CTL epitope may eventually become effective vaccines.