Protection against hydatid disease induced with the EG95 vaccine is associated with conformational epitopes

This paper describes attempts to map the location of host-protective epitopes of a recombinant vaccine antigen by assessing the ability of truncated regions of the antigen to elicit protective immune responses in sheep. Sheep were immunised with three truncated regions (EG95-1, EG95-2 and EG95-3) of the hydatid vaccine antigen, EG95. These regions overlapped each other and corresponded to amino acids 1-70 (EG95-1), 51-106 (EG95-2) and 89-153 (EG95-3) of the full length recombinant protein. Each region elicited antibody which reacted with the parent antigen, although these reactivities were a small proportion of the level of reactivity generated by immunisation with the full length antigen. Antisera raised against each of the truncated proteins reacted with the native parasite antigen. In vaccination and parasite challenge trials in sheep, none of the truncated regions elicited significant protection against challenge infection or antibody which was lethal to the parasite in vitro. Antibodies from sheep immunised with the combination of all three overlapping truncations elicited a comparatively low but significant level of lysis of the parasite in vitro. These antigens did not inhibit anti-EG95 antibody reactivity with EG95 nor did they inhibit in vitro oncosphere killing induced by anti-EG95 antibodies. These results indicate that the major part of the immune response induced by EG95 vaccination is directed against conformational epitopes and that the host-protective epitope(s) is/are conformational.     

Isolation of antibodies specific to a single conformation-dependant antigenic determinant on the EG95 hydatid vaccine

EG95 is a recombinant vaccine protein that elicits protection against hydatid disease in sheep. Previous studies have shown that the host-protective epitopes on EG95 depend on correct conformation and cannot be represented by simple “linear” peptides. By screening random peptide phage display libraries with polyclonal antibodies directed against conformation-dependant epitopes of EG95, we have selected a number of peptides that mimic these epitopes. The selected peptides did not show sequence homology to EG95. Antigen binding assays involving these peptides have provided evidence of at least four conformationally-dependant epitope regions on EG95. One of the selected peptides, E100, has been used to purify antibodies from anti-sera raised in sheep vaccinated with EG95. This yielded monospecific antibodies capable of recognizing recombinant EG95 in ELISA and native EG95 in Western blot assays. This antibody was demonstrated to be effective in antibody-dependant complement-mediated in vitro killing of Echinococcus granulosus oncospheres. Peptide E100 may represent the basis for a quality control assay for EG95 production, and has the potential to become a component of a synthetic peptide-based vaccine against E. granulosus.     

Synthetic peptide antigens induce antibodies to Taenia ovis oncospheres

Sheep immunised with the Taenia ovis recombinant 45W antigen are protected from infection with the parasite. Two peptides were synthesised corresponding to putative host-protective regions at the N- and C-termini of 45W. Sera from sheep immunised with 45W or related recombinant proteins reacted strongly with the N-terminal peptide. Approximately 40% of the antibody directed against 45WB/X, a truncated form of 45W, was found to be directed against the N-terminal peptide sequence. Sheep were immunised with the N- and C-terminal peptides alone or conjugated to a carrier protein. The N-terminal peptide was found to be highly immunogenic whereas the C-terminal peptide required conjugation to a carrier protein to be immunogenic. Antibodies raised against each of these immunogens crossreacted with the parent protein, 45WB/X, however, only antibodies specific for the N-terminal peptide were found to bind to antigens from the T. ovis oncosphere.     

Engineering filamentous phage carriers to improve focusing of antibody responses against peptides

The filamentous bacteriophage are highly immunogenic particles that can be used as carrier proteins for peptides and presumably other haptens and antigens. Our previous work demonstrated that the antibody response was better focused against a synthetic peptide if it was conjugated to phage as compared to the classical carrier, ovalbumin. We speculated that this was due, in part, to the relatively low surface complexity of the phage. Here, we further investigate the phage as an immunogenic carrier, and the effect reducing its surface complexity has on the antibody response against peptides that are either displayed as recombinant fusions to the phage coat or are chemically conjugated to it. Immunodominant regions of the minor coat protein, pIII, were removed from the phage surface by excising its N1 and N2 domains (Δ3 phage variant), whereas immunodominant epitopes of the major coat protein, pVIII, were altered by reducing the charge of its surface-exposed N-terminal residues (Δ8 phage variant). Immunization of mice revealed that the Δ3 variant was less immunogenic than wild-type (WT) phage, whereas the Δ8 variant was more immunogenic. The immunogenicity of two different peptides was tested in the context of the WT and Δ3 phage in two different forms: (i) as recombinant peptides fused to pVIII, and (ii) as synthetic peptides conjugated to the phage surface. One peptide (MD10) in its recombinant form produced a stronger anti-peptide antibody response fused to the WT carrier compared to the Δ3 phage carrier, and did not elicit a detectable anti-peptide response in its synthetic form conjugated to either phage carrier. This trend was reversed for a different peptide (4E10_L), which did not produce a detectable anti-peptide antibody response as a recombinant fusion; yet, as a chemical conjugate to Δ3 phage, but not WT phage, it elicited a highly focused anti-peptide antibody response that exceeded the anti-carrier response by ∼65-fold. The results suggest that focusing of the antibody response against synthetic peptides can be improved by decreasing the antigenic complexity of the phage surface.     

Filamentous phage as an immunogenic carrier to elicit focused antibody responses against a synthetic peptide

Filamentous bacteriophage are widely used as immunogenic carriers for "phage-displayed" recombinant peptides. Here we report that they are an effective immunogenic carrier for synthetic peptides. The f1.K phage was engineered to have an additional Lys residue near the N-terminus of the major coat protein, pVIII, so as to enhance access to chemical cross-linking agents. The dimeric synthetic peptide, B2.1, was conjugated to f1.K (f1.K/B2.1) in high copy number and compared as an immunogen to B2.1 conjugated to ovalbumin (OVA/B2.1) and to phage-displayed, recombinant B2.1 peptide. All immunogens were administered without adjuvant. The serum antibody titers were measured against: the peptide, the carrier, and, if appropriate, the cross-linker. All immunogens elicited anti-peptide antibody titers, with those elicited by OVA/B2.1 exceeding those by f1.K/B2.1; both titers were greater than that elicited by recombinant B2.1 phage. Comparison of the anti-peptide and anti-carrier antibody responses showed that f1.K/B2.1 elicited a more focused anti-peptide antibody response than OVA/B2.1. The anti-peptide antibody response against f1.K/B2.1 was optimized for the injection route, dose and adjuvant. Dose and adjuvant did not have a significant effect on anti-peptide antibody titers, but a change in injection route from intraperitoneal (IP) to subcutaneous (SC) enhanced anti-peptide antibody titers after seven immunizations. The optimized anti-peptide antibody response exceeded the anti-carrier one by 21-fold, compared to 0.07-fold elicited by OVA/B2.1. This indicates that phage as a carrier can focus the antibody response against the peptide. The results are discussed with respect to the advantages of phage as an alternative to traditional carrier proteins for synthetic peptides, carbohydrates and haptens, and to further improvements in phage as immunogenic carriers.     

Recognition by the human immune system of candidate vaccine epitopes of Toxoplasma gondii measured by a competitive ELISA

A competitive ELISA for the detection of human antibody to specific candidate vaccine epitopes of Toxoplasma gondii is described. The method is based on competition between antibody in human serum and murine monoclonal antibody to bind to a crude sonicated antigen of T. gondii. A total of 301 normal human sera were tested against a panel of four anti-T. gondii murine monoclonal antibodies. The results obtained with two of the monoclonal antibodies (FMC 22 and FMC 23) gave good agreement with those of an indirect ELISA for human IgG to T. gondii, a measure of previous exposure to the parasite. The epitopes against which these two monoclonal antibodies are directed were recognized by the immune system of all patients previously exposed to the parasite, while greater than 92 and greater than 96% respectively of these patients recognized the epitopes which the other two monoclonal antibodies (FMC 18 and FMC 19) are directed against. An ELISA double antibody binding system was used to ascertain relationships in the epitope binding sites of the four monoclonal antibodies. The data obtained demonstrated that FMC 19 and FMC 22 bound to related epitopes. FMC 18, FMC 22 and FMC 23 also bound to related epitopes, but the binding of FMC 19 was found to be independent of the binding of FMC 18 and FMC 23 to their epitope binding sites.     

Recognition by the human immune system of candidate vaccine epitopes of Toxoplasma gondii measured by a competitive ELISA

A competitive ELISA for the detection of human antibody to specific candidate vaccine epitopes of Toxoplasma gondii is described. The method is based on competition between antibody in human serum and murine monoclonal antibody to bind to a crude sonicated antigen of T. gondii. A total of 301 normal human sera were tested against a panel of four anti-T. gondii murine monoclonal antibodies. The results obtained with two of the monoclonal antibodies (FMC 22 and FMC 23) gave good agreement with those of an indirect ELISA for human IgG to T. gondii, a measure of previous exposure to the parasite. The epitopes against which these two monoclonal antibodies are directed were recognized by the immune system of all patients previously exposed to the parasite, while greater than 92 and greater than 96% respectively of these patients recognized the epitopes which the other two monoclonal antibodies (FMC 18 and FMC 19) are directed against. An ELISA double antibody binding system was used to ascertain relationships in the epitope binding sites of the four monoclonal antibodies. The data obtained demonstrated that FMC 19 and FMC 22 bound to related epitopes. FMC 18, FMC 22 and FMC 23 also bound to related epitopes, but the binding of FMC 19 was found to be independent of the binding of FMC 18 and FMC 23 to their epitope binding sites.     

幽门螺杆菌基因克隆表达及其抗原表位分析

目的 克隆表达幽门螺杆菌基因hp0410,构建12肽噬菌体展示库,对hp0410的抗原表位进行筛选和分析,为构建多表位嵌合疫苗提供理论依据。方法 从幽门螺杆菌NCTC11639基因组DNA中扩增出hp0410,并克隆入pGEX-4T-1中表达。利用纯化重组蛋白筛选出特异性单抗E018,利用该单抗和M13噬菌体12肽随机肽库,构建HP0410抗原表位的抗原展示库。竞争-抑制实验验证获得的阳性噬菌体并测序,生物信息学分析HP0410的抗原表位。结果 成功构建可高水平分泌型表达HP0410的原核表达系统。对13株阳性噬菌体测序后获得8个表位,其中候选表位1个。HP0410可有效抑制展示该表位的噬菌体与单抗E018结合。分析表明,获得的8个表位与HP0410同源性不高,但处于生物信息学预测的区域。结论 获得8个HP0410高抗原性区域的模拟表位,其中1个为模拟单抗E018特异性结合的抗原决定基的候选表位。     

Polyvalent synthetic vaccines: relationship between T epitopes and immunogenicity.

Three different synthetic polyvalent vaccines have been constructed by conjugating four synthetic peptides without any carrier protein. The peptides were copy fragments of two bacterial antigens (Streptococcus pyogenes M protein and diphtheria toxin), two parasitic antigens (circumsporozoite protein of Plasmodium falciparum and Plasmodium knowlesi), and one viral antigen (hepatitis B surface antigen). Outbred guinea-pigs immunized with polyvalent vaccine containing streptococcal, diphtheric, P. knowlesi and hepatitis peptides raised high specific antibody response against the four specificities. Individual T cell analysis demonstrated that hepatitis peptide bears T dominant epitope. A similar immune response was obtained with a second polyvalent vaccine where the P. knowlesi peptide had been replaced by the P. falciparum peptide. In both experiments the malarial peptides behave like pure B epitopes. Prediction of immunodominant helper T-cell antigenic sites were performed with the five peptides using computer algorithm. Hepatitis and diphtheric peptides were selected whereas the streptococcal peptide was rejected although it can experimentally contain a T epitope. To confirm this result animals were immunized with a third polyvalent vaccine which does not contain the hepatitis peptide. No T cell proliferation or antipeptide antibodies were detected. These results demonstrate that the cooperative immune response requires a certain degree of antigenic complexity for the induction of antibody response.     

Peptide mimics as surrogate immunogens of mosquito midgut carbohydrate malaria transmission blocking targets

Transmission blocking vaccines (TBV) against mosquito midgut carbohydrate epitopes is a promising approach to curbing the spread of malaria. However, carbohydrates as immunogens can be problematic. Via the malaria transmission blocking monoclonal antibody, MG96, we isolated dodecapeptide mimics of the conserved, nominal mosquito carbohydrate epitope from a peptide-display library. Two peptide clones, bearing a constrained, consensus motif competitively inhibited MG96 reactivity with its nominal midgut microvillar antigen. However, rabbit polyclonal antisera against these synthetic peptides recognized heterologous mosquito midgut carbohydrate and protein epitopes along the midgut basal lamina. Consequently, antisera did not block parasite development within the mosquito vector. Therefore, it is imperative that peptides not only need to be functional mimics but also complete mimotopes to effectively direct the vertebrate immune response towards the nominal, protective carbohydrate epitope on mosquito microvilli.     

Specificity of antibodies reactive with hepatitis B surface antigen following immunization with synthetic peptides

The amino acid sequence 139–147 from hepatitis B surface antigen (HBsAg) has previously been shown to represent a B-cell epitope with potential as a component of a synthetic peptide vaccine against hepatitis B. In this paper, two regions of HBsAg which act as T-cell epitopes in inbred mice have been identified (residues 23–34 and residues 160–171). The ability of synthetic peptides representing these epitopes to provide help for the production of antibody against the 139–147 epitope has been assessed following their co-linear synthesis with the B-cell epitope and following co-immunization of the peptides in an uncoupled form. Both these strategies result in the induction of anti-peptide antibodies which specifically react with recombinant HBsAg. The results presented give further support to the concept that synthetic peptides representing appropriately chosen B- and T-cell epitopes from HBsAg could form the basis of a synthetic vaccine against hepatitis B.     

Physicochemical and immunological characterization of recombinant host-protective antigen (VP2) of infectious bursal disease virus.

Small fusions to the N-terminal end of the host-protective antigen (VP2) of infectious bursal disease virus lead to stable expression of VP2 in Escherichia coli and yeast, and reduce the levels of inclusion body formation in E. coli in comparison to VP2 constructs with larger N-terminal fusions. VP2 produced with small N-terminal fusions, like native viral VP2, can be fractionated into a high molecular weight 'multimeric' form and a monomeric form. A virus-neutralizing monoclonal antibody that only recognizes undenatured VP2 preferentially reacts with multimeric forms of recombinant VP2. Both native and recombinant monomeric forms of VP2 are non-immunogenic. The multimeric forms of viral and yeast-derived VP2 are highly immunogenic, while those produced in E. coli are not.     

Immunodominant T cell peptides from four candidate malarial antigens as biomarkers of protective immunity against malaria

A malaria vaccine with high efficacy and capable of inducing sterile immunity against malaria within genetically diverse populations is urgently needed to complement ongoing disease control and elimination efforts. Parasite-specific IFN-γ and granzyme B-secreting CD8 + T cells have been identified as key mediators of protection and the rapid identification of malaria antigen targets that elicit these responses will fast-track the development of simpler, cost-effective interventions. This study extends our previous work which used peripheral blood mononuclear cells (PBMCs) from adults with life-long exposure to malaria parasites to identify immunodominant antigen-specific peptide pools composed of overlapping 15mer sequences spanning full length proteins of four malarial antigens. Our current study aimed to identify CD8 + T cell epitopes within these previously identified positive peptide pools. Cryopreserved PBMCs from 109 HLA-typed subjects were stimulated with predicted 9-11mer CD8 + T cell epitopes from P. falciparum circumsporozoite protein (CSP), apical membrane antigen 1 (AMA1), thrombospondin related anonymous protein (TRAP) and cell traversal for ookinetes and sporozoites (CelTOS) in FluoroSpot assays. A total of 135 epitopes out of 297 tested peptides from the four antigens were experimentally identified as positive for IFN-γ and/or granzyme B production in 65 of the 109 subjects. Forty-three of 135 epitopes (32 %) were promiscuous for HLA binding, with 31 of these promiscuous epitopes (72 %) being presented by HLA alleles that fall within at least two different HLA supertypes. Furthermore, about 52 % of identified epitopes were conserved when the respective sequences were aligned with those from 16 highly diverse P. falciparum parasite strains. In summary, we have identified a number of conserved epitopes, immune responses to which could be effective against multiple P. falciparum parasite strains in genetically diverse populations.     

日本血吸虫童虫早期诊断抗原模拟表位的初步筛选研究

目的：利用噬菌体随机12肽库技术筛选日本血吸虫感染早期诊断抗原。方法：用日本血吸虫感染后21d兔血清从噬菌体12肽库中经3轮筛选,有效地富集与早期感染兔血清有亲和力的噬菌体克隆。随机挑取11个单克隆分别纯化、扩增,随后采用ELISA、DOT-ELISA等检测方法,挑选出与早期感染兔血清有高亲和力的噬菌体克隆。结果：挑选出3个与感染后21d兔血清具有高亲和力的噬菌体单克隆。结论：用感染后21d兔血清从噬菌体随机12肽库中能筛选到日本血吸虫童虫早期诊断抗原模拟表位。     

Synthetic peptides coupled to the surface of liposomes effectively induce SARS coronavirus-specific cytotoxic T lymphocytes and viral clearance in HLA-A*0201 transgenic mice

We investigated whether the surface-linked liposomal peptide was applicable to a vaccine based on cytotoxic T lymphocytes (CTLs) against severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV). We first identified four HLA-A*0201-restricted CTL epitopes derived from SARS-CoV using HLA-A*0201 transgenic mice and recombinant adenovirus expressing predicted epitopes. These peptides were coupled to the surface of liposomes, and inoculated into mice. Two of the liposomal peptides were effective for peptide-specific CTL induction, and one of them was efficient for the clearance of vaccinia virus expressing epitopes of SARS-CoV, suggesting that the surface-linked liposomal peptide might offer an effective CTL-based vaccine against SARS.     

Promiscuous T-cell epitopes of Plasmodium merozoite surface protein 9 (PvMSP9) induces IFN-γ and IL-4 responses in individuals naturally exposed to malaria in the Brazilian Amazon

Plasmodium vivax merozoite surface protein (PvMSP9) stimulates both cellular and humoral immune responses in individuals who are naturally infected by this parasite species. To identify immunodominant human T-cell epitopes in PvMSP9, we used the MHC class II binding peptide prediction algorithm ProPred. Eleven synthetic peptides representing predicted putative promiscuous T-cell epitopes were tested in IFN-γ and IL-4 ELISPOT assays using peripheral blood mononuclear cells (PBMC) derived from 142 individuals from Rondonia State, Brazil who had been naturally exposed to P. vivax infections. To determine whether the predicted epitopes are preferentially recognized in the context of multiple alleles, MHC Class II typing of the cohort was also performed. Five synthetic peptides elicited robust cellular responses, and the overall frequencies of IFN-γ and IL-4 responders to at least one of the promiscuous peptides were 62% and 46%, respectively. The frequencies of IFN-γ and IL-4 responders to each peptide were not associated with a particular HLA-DRB1 allelic group since most of the peptides induced a response in individuals of 12 out of 13 studied allelic groups. The prediction of promiscuous epitopes using ProPred led to the identification of immunodominant epitopes recognized by PBMC from a significant proportion of a genetically heterogeneous population exposed to malaria infections. The combination of several such T-cell epitopes in a vaccine construct may increase the frequency of responders and the overall efficacy of subunit vaccines in genetically distinct populations.     

Comparison of antibody responses to the circumsporozoite protein repeat region and to intact sporozoites during acute falciparum malaria

Most acute falciparum malaria patients mount an antibody response to the circumsporozoite (CS) protein which contains a dominant B-cell epitope. In order to investigate whether antibodies against other epitopes on the sporozoite surface may be important during a particular phase of infection or convalescence, we longitudinally studied the antibody responses of 13 Thai patients with acute falciparum malaria. Antibody comparisons were made using intact Plasmodium falciparum sporozoites in an indirect fluorescent antibody test and the recombinant peptide, R32tet32, as capture antigen in an enzyme-linked immunosorbent assay. Antibody response curves derived using the 2 methods were similar, and adsorption with R32tet32 greatly (greater than 95%) diminished anti-sporozoite activity in sera. Thus, peptide constructs containing the CS repeat region epitope, (NANP)n, can be used with confidence to assay anti-sporozoite antibodies, independent of both the time of infection and prior malaria history.     

Malaria vaccines: immunogen selection and epitope mapping

In recent years major efforts have been made to characterize parasite antigens thought to be suitable candidates for malaria vaccines. Many of the relevant plasmodial antigens have been found to contain extensive areas of short amino acid sequences organized in tandem repeats. These are usually strongly antigenic, forming linear epitopes seen by antibodies of the infected host. Several such epitopes have been identified and subunit vaccines are being designed in which synthetic peptides or gene constructs serve as immunogens. However, as an efficient malaria vaccine should give rise to anamnestic T-dependent antibody responses following reinfection after vaccination as well as to antibody independent cell-mediated immunity, efforts are now also being made to identify T-cell epitopes on the vaccine candidate antigens. In this paper the current Plasmodium falciparum sporozoite vaccines and the merozoite antigen Pf155/RESA, a possible candidate for a P. falciparum blood stage vaccine, serve as examples to illustrate recent advances made in this area as well as some of the problems remaining to be resolved.     

Identification of T helper cell-recognized epitopes in the chitinase of the filarial nematode Onchocerca volvulus

T helper cell-recognized epitopes were determined in chitinase of Onchocerca volvulus, a vaccine candidate protein. The proliferation of splenic T cells of mice immunized with recombinant protein was tested with a library of chitinase-peptides of 16 amino acids with termini overlapping by 12 amino acids, and a library of "designer peptides", i.e. sequences identified with three epitope-predicting algorithms. Fourteen epitope-bearing stretches were identified with the peptides of the overlapping library. Testing of the designer peptides partially confirmed these data and revealed additional epitopes. Five clusters of epitopes were identified for the creation of peptide or minigene DNA vaccines with good potency and potential range of MHC allele presentation.     

麻疹病毒血凝素蛋白抗原表位的预测与分析

目的 探讨麻疹病毒(MeV)流行株血凝素蛋白(H)抗原表位上氨基酸(aa)变异对病毒抗原性的可能影响。方法 利用生物信息学软件预测MeV H蛋白上B细胞线性表位,设计并合成来源于疫苗株和流行株表位以及同一区域非表位上的多肽对。间接ELISA法检测合成多肽的免疫原性,并制备多肽免疫血清。采用交叉ELISA法分析两条多肽间的抗原性差异,计算抗原比。结果 合成的多肽均能与MeV免疫血清结合,其中设计在表位区的多肽对CW23/CW22(273～282 aa)结合能力最强,而非表位区多肽对CW150/CW151(418～427 aa)结合能力最弱。多肽对中来源不同两条多肽间抗原性差异较大,其中CW23(疫苗株来源)与CW22(流行株来源)间抗原比为16,CW123(疫苗株来源)与CW124(流行株来源)(236～246 aa)间的抗原比为2.877±0.583。非表位多肽对中,CW125与CW126(356～364 aa)间抗原比为1.631±0.481,而CW150与CW151间抗原比为10.367±1.617。结论 麻疹流行株上仍存在保守的抗原表位,但预测的抗原表位及非表位区上的部分aa变异导致疫苗株与流行株间抗原性存在差异。