Antibody responses to non-immunogenic synthetic peptides induced by co-immunization with immunogenic peptides.

Chimeric peptides comprising B- and T-helper cell epitopes from the proteins of infectious agents represent immunogens with potential for use as new vaccines. However, it has become clear that the orientation of the epitopes, the presence of spacer residues and the number of copies of the epitopes influence the specificity, levels and affinity of the antibody produced following immunization with such constructs. Furthermore, the response to peptides is under genetic control leading to major histocompatibility complex (MHC)-linked non-responsiveness. In this study, we have investigated the potential of co-immunization of immunogenic peptides (to provide T-cell help) with non-immunogenic peptides (representing B-cell epitopes) to overcome the non-response to the latter. For this purpose, we have employed peptides representing T- and B-cell epitopes derived from the sequences of the fusion and haemagglutinin glycoproteins of measles virus. The results obtained show that simple co-immunization of a B-cell epitope with a T-cell epitope results in the production of antibody to the B-cell epitope without the requirement for covalent linkage of the two peptides. This approach could thus be used to overcome the problem of poor immunogenicity of peptides and will be of potential value in the design of immunization strategies using synthetic immunogens.     

Estimating design space available for polyepitopes through consideration of major histocompatibility complex binding motifs

Major histocompatibility complex (MHC ) epitope presentation is needed for robust adaptive immune responses. Core peptide binding motifs for class I and class II MHC are 8–10 amino acids long, containing two or more “anchor” residues. These binding motifs define epitope anchor amino acid content and spacing, and knowledge of them has facilitated emergence of polyepitope vaccines. However, polyepitopes can exhibit “junctional epitopes” (neoepitopes interfering with vaccine function) resulting from juxtaposition of authentic epitopes. We have developed an algorithm for consideration of polyepitope sequence in light of MHC motifs to exhaustively identify all junctional-free polyepitope designs for any given set of authentic epitopes, and in so doing discovered that the number of such variants of any given polyepitope can be astronomically high. Our approach designs polyepitopes of any length, considers multiple MHC class I or class II motifs simultaneously and can be adapted to design variants of existing proteins with pre-selected epitope contents. We have also implemented the algorithm as a computer-based tool (CANVAC II), which we make available to interested parties. The vast diversity of junctional-free polyepitopes suggests that the number of potential T-helper epitope free protein variants may also be large, which may have implications for discovery of bioactive but non-immunogenic therapeutics.     

Efficient MHC class I-peptide binding is required but does not ensure MHC class I-restricted immunogenicity

Cytotoxic T lymphocyte (CTL) epitopes are naturally processed peptides bound and presented by major histocompatibility (MHC) class I molecules. Since they are expressed at the cell surface in sufficient amounts to be recognized by CTL, it is generally believed, and in some cases demonstrated, that they bind efficiently to MHC class I molecules in vivo. Based on this knowledge, candidate CTL epitopes are now searched for by identifying peptides that efficiently bind to MHC class I molecules in vitro. We analysed whether this approach is valid by systematically investigating the relationship between MHC class I-peptide binding and peptide immunogenicity. Fifteen peptides that represent known CTL epitopes were tested for their MHC class I binding ability. In a comparative study with 83 peptides that bear the appropriate MHC class I allele-specific motifs but do not contain known CTL epitopes, the CTL epitope-bearing peptides showed the highest binding affinity for MHC class I. This was true for two MHC class I alleles in two different assay systems that monitor peptide-MHC class I binding. Furthermore, selected motif-bearing K^b binding peptides were used to induce peptide-specific CTL responses in mice. Only a subset of the high affinity K^b binding peptides induced reproducible peptide-specific CTL responses, whereas none of the low affinity K^b binding peptides induced a response. Taken together, these results indicate that efficient peptide-MHC class I binding is required for immunogenicity. Vice versa, immunogenicity is not guaranteed by efficient peptide-MHC class I binding, implying that additional factors are involved. Nevertheless, selection of candidate CTL epitopes on the basis of MHC class I binding seems valid. Our data indicate that, although an excess of peptides might be selected, the chance of missing immunogenic peptides is minimal.     

Signal peptides and trans-membrane regions are broadly immunogenic and have high CD8+ T cell epitope densities: Implications for vaccine development

Cell mediated immune response has a major role in controlling the elimination of infectious agents. The rational design of sub-unit peptide vaccines against intracellular pathogens or cancer requires the use of antigenic sequence/s that can induce highly potent, long lasting and antigen-specific responses in the majority of the population. A promising peptide selection strategy is the detection of multi-epitope peptide sequences with an ability to bind multiple MHC alleles. While past research sought the best epitopes based on their specific antigenicity, we ask whether specific defined domains have high epitope densities. Signal peptides and trans-membrane domains were found to have exceptionally high epitope densities. The improved MHC binding of these domains relies on their hydrophobic nature and, in signal peptides, also on their specific sequence. The high epitope density of SP was computed using in-silico methods and corroborated by the high percentage of identified SP epitope in the IEDB (immune epitope database). The enhanced immunogenicity of SP was then experimentally confirmed using a panel of nine peptides derived from Mycobacterium tuberculosis (MTb) proteins used in human PBMC proliferation assays and T cell lines functional assays. Our results show the exceptionally high antigen specific response rates and population coverage to SP sequences compared with non-SP peptide antigens derived from the same proteins. The results suggest a novel scheme for the rational design of T cell vaccines using a domain based rather than an epitope based approach.     

A strategy for rational design of fully synthetic glycopeptide conjugate vaccines.

The present study describes a strategy to rationally design fully synthetic glycopeptide conjugate vaccines. Glycopeptide immunogens were constructed by coupling synthetic oligosaccharides comprising repeating units of synthetic 3-beta-D-ribose-(1-1)-D-ribitol-5-phosphate (sPRP) to synthetic peptides containing potent T-helper cell determinants and B-cell epitopes of the Haemophilus influenzae type b (Hib) outer membrane proteins (OMPs) P1, P2, and P6. Rabbit immunogenicity studies revealed that some of these fully synthetic glycoconjugates were capable of eliciting high titers of both anti-PRP and anti-OMP immunoglobulin G antibodies. In addition, we systematically investigated the factors which could influence their immunogenicity. We observed that the magnitude of the anti-PRP antibody response markedly depended on the relative spatial orientation of sPRP and T-cell epitopes, the anti-PRP antibody response was enhanced when a multiple antigenic peptide was used as a carrier, the anti-PRP antibody response was optimal for three PRP repeating units, and lipidation of peptide-PRP conjugates had a minimal effect on the magnitude of the anti-PRP antibody response. The results of this study clearly demonstrate that coupling a carbohydrate hapten to a peptide can provide T-cell help and convert it into a T-cell-dependent antigen. The antisera raised against these conjugates were also found to be protective against Hib infection in the infant rat model of bacteremia.     

Prediction of cancer neoepitopes needs new rules

Tumors are immunogenic and the non-synonymous point mutations harbored by tumors are a source of their immunogenicity. Immunologists have long been enamored by the idea of synthetic peptides corresponding to mutated epitopes (neoepitopes) as specific “vaccines” against tumors presenting those neoepitopes in context of MHC I. Tumors may harbor hundreds of point mutations and it would require effective prediction algorithms to identify candidate neoepitopes capable of eliciting potent tumor-specific CD8⁺ T cell responses. Our current understanding of MHC I-restricted epitopes come from the observance of CD8⁺ T cell responses against viral (vaccinia, lymphocytic choriomeningitis etc.) and model (chicken ovalbumin, hen egg lysozyme etc.) antigens. Measurable CD8⁺ T cell responses elicited by model or viral antigens are always directed against epitopes possessing strong binding affinity for the restricting MHC I alleles. Immense collective effort to develop methodologies combining genomic sequencing, bioinformatics and traditional immunological techniques to identify neoepitopes with strong binding affinity to MHC I has only yielded inaccurate prediction algorithms. Additionally, new evidence has emerged suggesting that neoepitopes, which unlike the epitopes of viral or model antigens have closely resembling wild-type counterparts, may not necessarily demonstrate strong affinity to MHC I. Our bearing need recalibration.     

Does Binding of Complement Factor H to the Meningococcal Vaccine Antigen,Factor H Binding Protein,Decrease Protective Serum Antibody Responses?

Factor H binding protein (fHbp) is a principal antigen in a multicomponent meningococcal vaccine recently licensed in Europe for prevention of serogroup B diseases. The protein recruits the complement downregulator, factor H (fH), to the bacterial surface, which enables the organism to resist complement-mediated bacteriolysis. Binding is specific for human fH. In preclinical studies, mice and rabbits immunized with fHbp vaccines developed serum bactericidal antibody responses, which in humans predict protection against developing meningococcal disease. These studies, however, were in animals whose fH did not bind to the vaccine antigen. Here we review the immunogenicity of fHbp vaccines in human fH transgenic mice. The data suggest that animals with high serum human fH concentrations have impaired protective antibody responses. Further, mutant fHbp vaccines with single amino acid substitutions that decrease fH binding are superior immunogens, possibly by unmasking epitopes in the fH binding site that are important for eliciting serum bactericidal antibody responses. Humans immunized with fHbp vaccines develop serum bactericidal antibody, but achieving broad coverage in infants required incorporation of additional antigens, including outer membrane vesicles, which increased rates of fever and local reactions at the injection site. The experimental results in transgenic mice predict that fHbp immunogenicity can be improved in humans by using mutant fHbp vaccines with decreased fH binding. These results have important public health implications for developing improved fHbp vaccines for control of serogroup B meningococcal disease and for development of vaccines against other microbes that bind host molecules.     

Computer‐aided design of T‐cell epitope‐based vaccines: addressing population coverage

Epitope‐based vaccines (EVs) make use of short antigen‐derived peptides corresponding to immune epitopes, which are administered to trigger a protective humoral and/or cellular immune response. EVs potentially allow for precise control over the immune response activation by focusing on the most relevant – immunogenic and conserved – antigen regions. Experimental screening of large sets of peptides is time‐consuming and costly; therefore, in silico methods that facilitate T‐cell epitope mapping of protein antigens are paramount for EV development. The prediction of T‐cell epitopes focuses on the peptide presentation process by proteins encoded by the major histocompatibility complex (MHC). Because different MHCs have different specificities and T‐cell epitope repertoires, individuals are likely to respond to a different set of peptides from a given pathogen in genetically heterogeneous human populations. In addition, protective immune responses are only expected if T‐cell epitopes are restricted by MHC proteins expressed at high frequencies in the target population. Therefore, without careful consideration of the specificity and prevalence of the MHC proteins, EVs could fail to adequately cover the target population. This article reviews state‐of‐the‐art algorithms and computational tools to guide EV design through all the stages of the process: epitope prediction, epitope selection and vaccine assembly, while optimizing vaccine immunogenicity and coping with genetic variation in humans and pathogens.     

A design strategy to generate a SARS-CoV-2 RBD vaccine that abrogates ACE2 binding and improves neutralizing antibody responses

The structure-based design of antigens holds promise for developing vaccines with higher efficacy and improved safety profiles. We postulate that abrogation of host receptor interaction bears potential for the improvement of vaccines by preventing antigen-induced modification of receptor function as well as the displacement or masking of the immunogen. Antigen modifications may yet destroy epitopes crucial for antibody neutralization. Here, we present a methodology that integrates deep mutational scans to identify and score SARS-CoV-2 receptor binding domain variants that maintain immunogenicity, but lack interaction with the widely expressed host receptor. Single point mutations were scored in silico, validated in vitro, and applied in vivo. Our top-scoring variant receptor binding domain-G502E prevented spike-induced cell-to-cell fusion, receptor internalization, and improved neutralizing antibody responses by 3.3-fold in rabbit immunizations. We name our strategy BIBAX for body-inert, B-cell-activating vaccines, which in the future may be applied beyond SARS-CoV-2 for the improvement of vaccines by design.     

Breaking the non-responsiveness of C57BL/6 mice to the malarial antigen EB200--the role of carrier and adjuvant molecules

Poor immunogenicity and major histocompatibility complex (MHC) restriction of immune responses to certain recombinant proteins or synthetic peptides impose problems in developing effective vaccines. EB200 is one of the vaccine candidate antigens from Plasmodium falciparum, which induces MHC-restricted immune responses in mice of different haplotypes. A way of overcoming this problem is to conjugate the antigen to an immunogenic protein carrier and to use optimal adjuvant substances. We have investigated the carrier effect of glutathione-S-transferase (GST) in CBA and C57BL/6 mice which are high and low responder to EB200, respectively. Our results reveal that the MHC restriction in C57BL/6 mice was broken by the use of GST as a carrier. Studies on the B-cell repertoires in both strains of mice immunized with GST-EB200 by preparing hybridoma cell lines indicate that the B-cell repertoires were similar in both CBA and C57BL/6 mice. However, the antibody affinity and the magnitude of the response were still lower in the low-responder C57BL/6 mice compared with that in CBA even when cholera toxin (CT) was used as adjuvant. To improve the response, the efficacy of various adjuvant substances like alum and Hsp 70 from Trypanosoma cruzi and the combination of various adjuvants was analysed. CT and Hsp 70 together act synergistically and markedly improve the immunogenicity of EB200 by increasing antibody affinity and the magnitude of the responses in C57BL/6 mice, which may be explained by the complementary effect of adjuvants. These results are of importance in the design of efficient vaccines.     

Immunodominance of cytotoxic T lymphocyte epitopes co-injected in vivo and modulation by interleukin-12

Immunodominance (ID) of T cell epitopes is a well-documented phenomenon that might have profound significance in the evolution of T cell responses to pathogens, tumors, autoantigens and vaccines. With the intention of developing vaccines composed of several cytotoxic T cell (CTL) epitopes, we injected mice with peptide mixtures containing two to five CTL epitopes and observed clear patterns of ID. In a first case, ID strictly correlated with the competitor activity of the individual peptides for H-2K^d, whereas in a second case, the absence of correlation between ID and competitor activity, binding affinity, half-life of the peptides in serum, induction of proliferation in vitro and the individual immunogenicity of the peptides, suggested to us that ID of co-injected CTL epitopes can be determined both at the peptide level (binding affinity to H-2K^d) and at the T cell level. This hypothesis is supported by our finding that interleukin-12 strongly modulates ID when it is not correlated with MHC binding.     

Identification of human CD4 T-cell epitopes on the VP1 capsid protein of enterovirus 71

The identification of human CD4 T-cell epitopes within a protein vaccine candidate is of great interest,as it provides a better understanding of the mechanisms involved in protective immunity and may therefore help in the design of effective vaccines and diagnostic tools. The entire amino acid sequence of the VP1 capsid protein from enterovirus 71 (EV 71) strain 41 was submitted to analysis by the ProPred algorithm for the identification of potential promiscuous human CD4 T-cellepitopes. Three regions spanning amino acids 66-77, 145-159, and 247-261 of VP1 were predicted to bind more than 25 HLA-DR alleles. The corresponding synthetic peptides (SP1 to SP3) were then tested for their abilities to induce proliferation of CD4 T cells isolated from five human volunteers screened positive for previous EV 71 exposure and one EV 71-negative volunteer. Upon stimulation with either peptide, CD4 T-cell proliferative responses were observed for all EV 71-positive volunteers,indicating the presence of EV 71-specific memory CD4 T cells. The amplitude of the proliferative responses was peptide- and HLA-DR-dependent, and correlated well with the ProPredpredicted binding efficiencies. Moreover, CD4 T cells from EV 71-positive volunteers produced significant levels of IL-2 and IFN- upon stimulation, indicative of a T-cell differentiation into Th-1-type subset. Among the three peptides, SP2 induced the highest proliferative response and cytokine production. Moreover, SP2-induced proliferative response could be inhibited with anti-major histocompatibility complex (MHC) class II antibody, indicating that SP2 represents a MHC class II-restricted CD4 T-cell epitope. This study demonstrates that the ProPred algorithm can accurately predict the presence of human CD4 T-cell epitopes within the VP1 capsid protein of EV 71, and therefore represents a useful tool for the design of subunit vaccines against EV 71.     

Immunoinformatics-aided identification of T cell and B cell epitopes in the surface glycoprotein of 2019-nCoV

The 2019 novel coronavirus (2019-nCoV) outbreak has caused a large number of deaths with thousands of confirmed cases worldwide, especially in East Asia. This study took an immunoinformatics approach to identify significant cytotoxic T lymphocyte (CTL) and B cell epitopes in the 2019-nCoV surface glycoprotein. Also, interactions between identified CTL epitopes and their corresponding major histocompatibility complex (MHC) class I supertype representatives prevalent in China were studied by molecular dynamics simulations. We identified five CTL epitopes, three sequential B cell epitopes and five discontinuous B cell epitopes in the viral surface glycoprotein. Also, during simulations, the CTL epitopes were observed to be binding MHC class I peptide-binding grooves via multiple contacts, with continuous hydrogen bonds and salt bridge anchors, indicating their potential in generating immune responses. Some of these identified epitopes can be potential candidates for the development of 2019-nCoV vaccines.     

The influence of MHC polymorphism on the selection of T-cell determinants of FMDV in cattle.

There is a quest for the development of a new generation of vaccines consisting of well-defined subunit antigens. For a number of practical reasons it is attractive to develop vaccines on the basis of synthetic peptides. However, their efficacy may be limited by genetic restrictions imposed on T-cell recognition via major histocompatibility complex (MHC) polymorphism, as shown by many studies using inbred animal species. To study the effect of MHC polymorphism in an outbred species, we selected four cattle homozygous for different A-DR-DQ haplotypes, and another four cattle which shared one haplotype in combination with a haplotype of one of the MHC homozygous animals. We analysed responses to synthetic peptides comprising defined T-cell epitopes of foot-and-mouth disease virus (FMDV) in this selected group of FMDV-vaccinated cattle. This analysis shows that even in outbred animals. MHC polymorphism influences the responses to synthetic peptides. Interestingly, one of the peptides, VP4[20-34], was recognized in association with at least four different MHC haplotypes. Fine specificity analysis of this peptide revealed subtle shifts in the core epitope recognized. All peptides that induced lymphocyte proliferation in vitro were found to induce a T-helper type-1 (Th1) type of response, irrespective of the MHC haplotype involved. Together, these data support the notion that individuals carrying distinct MHC types can be vaccinated successfully by vaccines that include only a limited number of peptides. In the design of a peptide vaccine against FMDV we suggest inclusion of the highly conserved VP4 sequence 20-34.     

Effect of pre-existing cytotoxic T lymphocytes on therapeutic vaccines

Therapeutic vaccines which aim to induce CD8(+) cytotoxic T lymphocyte (CTL) responses will often be required to perform in the presence of pre-existing CTL which recognize epitopes within the vaccine. Here we explore the ability of a viral vaccine vector presenting several co-dominant CTL epitopes to prime CTL responses in animals that have a pre-existing CTL response to one of the epitopes in the vaccine. The vaccine was usually capable of inducing multiple new responses, suggesting that immunodomination effects of pre-existing CTL may generally be minimal following vaccination. However, when large numbers of pre-existing CTL were present, a novel type of immune modulation was observed whereby (1) the vaccine failed to prime efficiently new CTL responses that were restricted by the same MHC gene as the pre-existing responses, and (2) vaccine-induced CTL responses restricted by other MHC genes were enhanced. These results may have implications for therapeutic multi-epitope vaccines for diseases like HIV and melanoma, which aim to broaden CTL responses.     

CD40-mediated enhancement of immune responses against three forms of influenza vaccine

There is potential for influenza A infections to cause massive morbidity and mortality. Vaccination may be the primary defence against pandemic influenza, and potential pandemic'flu vaccines may be produced conventionally, in embryonated eggs, or as recombinant protein or synthetic peptide vaccines. However the vaccines are produced, the supply may be limiting, and it will be important to enhance the immunogenicity of the vaccines as much as possible. We have shown that conjugation to CD40 binding antibody is a very efficient way of enhancing immune responses against model antigens, but were interested in assessing the effectiveness of this system using influenza vaccines. We produced conjugates of CD40 monoclonal antibody (mAb) and isotype control with three potential influenza vaccines: a peptide-based vaccine containing T- and B-cell epitopes from virus haemagglutinin; a whole, killed virus vaccine; and a commercially produced split virus vaccine. CD40 mAb conjugates in each case were more immunogenic, but the adjuvant effect of CD40 conjugation was greatest with the split vaccine, where antibody responses were enhanced by several hundred-fold after a single immunization, and lymphocyte proliferation in response to antigen in vitro was also strongly enhanced.     

T-helper epitopes identified within the E6 transforming protein of cervical cancer-associated human papillomavirus type 16

The E6 oncoprotein of human papillomavirus type 16 (HPV16 E6) produced by tumor cells of HPV16-associated cervical carcinoma is poorly immunogenic in patients, but nonetheless is a tumor-specific antigen to which therapeutic vaccine strategies may be directed. To investigate the subunit immunogenicity of E6 protein at the T-helper cell level, we immunized mice with overlapping peptides spanning the entire 158 amino acid sequence. Two peptides recalled a proliferative response in lymph node cells (LNC) from C57BL/6 (H-2b)-immunized mice. One of these peptides also recalled proliferative responses in the context of 5/5 other major histocompatibility complex (MHC) class II haplotypes, indicating a "promiscuous" T-epitope. Minimal consensus motif analysis identified the epitopes as 60VYRDGNPYA68 and 98GYNKPLCDLL107. LNC from mice immunized with T-epitope proliferated in response to challenge with whole E6 protein. Immunization with E6 T-epitopes linked to B-epitopes of HPV16 E7 protein elicited specific antibody indicating that T-cells recognizing the T-epitopes provided cognate "help" for B-cells. LNC from mice co-immunized with E6 T-epitope and the major T-helper epitope of HPV16 E7 (48DRAHYNI54) proliferated comparably when challenged with the peptides individually indicating co-dominance of the two T-epitopes. The findings have implications for incorporation of E6 into a therapeutic vaccine.     

Contemplating immunopeptidomes to better predict them

The identification of T-cell epitopes is key for a complete molecular understanding of immune recognition mechanisms in infectious diseases, autoimmunity and cancer. T-cell epitopes further provide targets for personalized vaccines and T-cell therapy, with several therapeutic applications in cancer immunotherapy and elsewhere. T-cell epitopes consist of short peptides displayed on Major Histocompatibility Complex (MHC) molecules. The recent advances in mass spectrometry (MS) based technologies to profile the ensemble of peptides displayed on MHC molecules – the so-called immunopeptidome – had a major impact on our understanding of antigen presentation and MHC ligands. On the one hand, these techniques enabled researchers to directly identify hundreds of thousands of peptides presented on MHC molecules, including some that elicited T-cell recognition. On the other hand, the data collected in these experiments revealed fundamental properties of antigen presentation pathways and significantly improved our ability to predict naturally presented MHC ligands and T-cell epitopes across the wide spectrum of MHC alleles found in human and other organisms. Here we review recent computational developments to analyze experimentally determined immunopeptidomes and harness these data to improve our understanding of antigen presentation and MHC binding specificities, as well as our ability to predict MHC ligands. We further discuss the strengths and limitations of the latest approaches to move beyond predictions of antigen presentation and tackle the challenges of predicting TCR recognition and immunogenicity.     

Lipopeptide immunization without adjuvant induces potent and long-lasting B,T helper,and cytotoxic T lymphocyte responses against a malaria liver stage antigen in mice and chimpanzees

We have employed a 26-amino-acid synthetic peptide based on Plasmodium falciparum liver stage antigen-3 to evaluate improvements in immunogenicity mediated by the inclusion of a simple lipid-conjugated amino acid during peptide synthesis. Comparative immunization by the peptide in Freund's adjuvant or by the lipopeptide in saline shows that the addition of a palmitoyl chain can dramatically increase T helper (Th) cell responses in a wide range of major histocompatibility complex (MHC) class II haplotypes, to the extent that responses were induced in mice otherwise unable to respond to the non-modified peptide injected with Freund's adjuvant, and that the increased immunogenicity of the lipopeptide led to high and longer lasting antibody production (studied up to 8 months). B and T cell responses induced by the lipopeptide were reactive with native parasite protein epitopes, and a lipopeptide longer than ten amino acids was endogenously processed to associate with MHC class I and elicit cytotoxic T lymphocyte (CTL) responses. Finally, the lipopeptide was safe and highly immunogenic in chimpanzees, whose immune system is very similar to that of humans. Our results suggest that relatively large synthetic peptides, carefully chosen from pertinent areas of proteins and incorporating a simple palmitoyllysine, can induce not only CTL, but also strong Th and antibody responses in genetically diverse populations. Lipopeptides engineered in this way are simple to produce and purify under GMP conditions, they are well tolerated by apes, and with the enhanced immunogenicity without the need for adjuvant that we report here, they offer a quick and relatively low-cost route to provide material for human malaria vaccination trials.     

人类肝素酶B细胞表位的预测和免疫原性鉴定

目的 预测并鉴定肝素酶(heparanase)蛋白B细胞表位免疫原性.方法 以肝素酶蛋白的氨基酸序列为基础,采用DNAStar分析软件以及Bcepred在线二级结构分析工具分析其蛋白二级结构并预测B细胞表位.根据预测结果 ,采用8分支多抗原肽结构合成针对该表位的抗原肽,将后者与通用型T辅助表位人IL-1β线性短肽(VQGEESNDK,氨基酸163～171)联合免疫日本白毛黑眼兔,检测免疫血清效价,鉴定其特异性和免疫原性.结果 软件预测显示,肝素酶蛋白大亚基的第1～15位(MAP1)、第279～293位(MAP2)及175～189位(MAP3)氨基酸序列最可能为其优势B细胞表位.间接酶联免疫吸附试验、免疫印迹及免疫组化分析,证实MAP1、MAP2及MAP3均能诱导机体产生高滴度抗体,但仅MAP1、MAP2抗体具有高特异性,MAP2抗体与肝癌组织的结合力最强.结论 肝素酶大亚基的第1～15位、第279～293位氨基酸为其优势B细胞表位,其中第279～293位氨基酸的免疫原性最强,这为肝素酶多肽抗体及B细胞优势短肽疫苗研制提供了理论依据.