Universal T-cell Epitope Research Articles

MRNA-Based Vaccines Are Highly Immunogenic and Confer Protection in the Gnotobiotic Pig Model of Human Rotavirus Diarrhea.

Human rotavirus (HRV) is still a leading cause of severe dehydrating gastroenteritis globally, particularly in infants and children. Previously, we demonstrated the immunogenicity of mRNA-based HRV vaccine candidates expressing the viral spike protein VP8* in rodent models. In the present study, we assessed the immunogenicity and protective efficacy of two mRNA-based HRV trivalent vaccine candidates, encoding VP8* of the genotypes P[8], P[6], or P[4], in the gnotobiotic (Gn) pig model of Wa (G1P[8]) HRV infection and diarrhea. Vaccines either encoded VP8* alone fused to the universal T-cell epitope P2 (P2-VP8*) or expressed P2-VP8* as a fusion protein with lumazine synthase (LS-P2-VP8*) to allow the formation and secretion of protein particles that present VP8* on their surface. Gn pigs were randomly assigned into groups and immunized three times with either P2-VP8* (30 µg) or LS-P2-VP8* (30 µg or 12 µg). A trivalent alum-adjuvanted P2-VP8* protein vaccine or an LNP-formulated irrelevant mRNA vaccine served as the positive and negative control, respectively. Upon challenge with virulent Wa HRV, a significantly shortened duration and decreased severity of diarrhea and significant protection from virus shedding was induced by both mRNA vaccine candidates compared to the negative control. Both LS-P2-VP8* doses induced significantly higher VP8*-specific IgG antibody titers in the serum after immunizations than the negative as well as the protein control. The P[8] VP8*-specific IgG antibody-secreting cells in the ileum, spleen, and blood seven days post-challenge, as well as VP8*-specific IFN-γ-producing T-cell numbers increased in all three mRNA-vaccinated pig groups compared to the negative control. Overall, there was a clear tendency towards improved responses in LS-P2-VP8* compared to the P2-VP8*mRNA vaccine. The demonstrated strong humoral immune responses, priming for effector T cells, and the significant reduction of viral shedding and duration of diarrhea in Gn pigs provide a promising proof of concept and may provide guidance for the further development of mRNA-based rotavirus vaccines.

The next generation virus-like particle platform for the treatment of peanut allergy.

Allergy to peanut is one of the leading causes of anaphylactic reactions among food allergic patients. Immunization against peanut allergy with a safe and protective vaccine holds a promise to induce durable protection against anaphylaxis caused by exposure to peanut. A novel vaccine candidate (VLP Peanut), based on virus-like particles (VLPs), is described here for the treatment of peanut allergy. VLP Peanut consist of two proteins: a capsid subunit derived from Cucumber mosaic virus engineered with a universal T cell epitope (CuMVTT ) and a CuMVTT subunit fused with peanut allergen Ara h 2 (CuMVTT -Ara h 2), forming mosaic VLPs. Immunizations with VLP Peanut in both naïve and peanut-sensitised mice resulted in a significant anti-Ara h 2 IgG response. Local and systemic protection induced by VLP Peanut were established in mouse models for peanut allergy following prophylactic, therapeutic and passive immunizations. Inhibition of FcγRIIb function resulted in a loss of protection, confirming the crucial role of the receptor in conferring cross protection against peanut allergens other than Ara h 2. VLP Peanut can be delivered to peanut-sensitized mice without triggering allergic reactions, whilst remaining highly immunogenic and offering protection against all peanut allergens. In addition, vaccination ablates allergic symptoms upon allergen challenge. Moreover, the prophylactic immunization setting conferred the protection against subsequent peanut-induced anaphylaxis, showing the potential for preventive vaccination. This highlights the effectiveness of VLP Peanut as a prospective break-through immunotherapy vaccine candidate towards peanut allergy. VLP Peanut has now entered clinical development with the study PROTECT.

Combining Well-Tempered Metadynamics Simulation and SPR Assays to Characterize the Binding Mechanism of the Universal T-Lymphocyte Tetanus Toxin Epitope TT830-843.

Peptide TT830-843 from the tetanus toxin is a universal T-cell epitope. It helps in vaccination and induces T-cell activation. However, the fine molecular interaction between this antigen and the major histocompatibility complex (MHC) remains unknown. Molecular analysis of its interaction with murine MHC (H-2) was proposed to explore its immune response efficiency. Molecular dynamics simulations are important mechanisms for understanding the basis of protein-ligand interactions, and metadynamics is a useful technique for enhancing sampling in molecular dynamics. SPR (surface plasmon resonance) assays were used to validate whether the metadynamics results are in accordance with the experimental results. The peptide TT830-843 unbinding process was simulated, and the free energy surface reconstruction revealed a detailed conformational landscape. The simulation described the exiting path as a stepwise mechanism between progressive detachment states. We pointed out how the terminus regions act as anchors for binding and how the detachment mechanism includes the opening of α-helices to permit the peptide's central region dissociation. The results indicated the peptide/H-2 receptor encounter occurs within a distance lesser than 27.5 Å, and the encounter can evolve to form a stable complex. SPR assays confirmed the complex peptide/H-2 as a thermodynamically stable system, exhibiting enough free energy to interact with TCR on the antigen-presenting cell surface. Therefore, combining in silico and in vitro assays provided significant evidence to support the peptide/H-2 complex formation.

K-Ras Peptide Mimotope Induces Antigen Specific Th1 and B-Cell Immune Responses against G12A-Mutated K-Ras Antigen in Balb/c Mice.

KRAS G12A somatic point mutation in adenocarcinomas is categorized clinically as ineligibility criteria for anti-epidermal growth factor receptor (EGFR) monoclonal antibody therapies. In this study, a modified G12A-K-ras epitope (139A) with sequence-specific modifications to improve immunogenicity was developed as a potential vaccine against G12A-mutant KRAS cancers. Additionally, coupling of the 139A epitope with a tetanus toxoid (TTD) universal T-cell epitope to improve antigenicity was also reported. To facilitate convenient oral administration, Lactococcus lactis, which possesses innate immunomodulatory properties, was chosen as a live gastrointestinal delivery vehicle. Recombinant L. lactis strains secreting a G12A mutated K-ras control and 139A with and without TTD fusion were generated for comparative immunogenicity assessment. BALB/c mice were immunized orally, and high survivability of L. lactis passage through the gastrointestinal tract was observed. Elevations in B-cell count with a concomitant titre of antigen-specific IgG and interferon-γ secreting T-cells were observed in the 139A treated mice group. Interestingly, an even higher antigen-specific IgA response and interferon-γ secreting T-cell counts were observed in 139A-TTD mice group upon re-stimulation with the G12A mutated K-ras antigen. Collectively, these results indicated that an antigen-specific immune response was successfully stimulated by 139A-TTD vaccine, and a TTD fusion was successful in further enhancing the immune responses.

Immunogenicity assessment of Clostridium perfringens type D epsilon toxin epitope-based chimeric construct in mice and rabbit.

Epsilon toxin (Etx) belongs to family of pore-forming toxin and is produced by Clostridium perfringens type D. The Etx toxin is responsible for the pathogenesis of enterotoxaemia in sheep and goats, and occasionally in other livestock animals. The present study aimed to develop a Clostridium perfringens epsilon toxin-based chimeric epitope construct having immunodominant B-cell epitope and universal T-cell epitope and its immunogenicity was evaluated in mice and rabbit. An artificial chimeric epitope construct (CEC) was prepared by joining tandem repeats of a peptide containing amino acids (aa) 134–145 of epsilon toxin B-cell epitope and universal T-cell epitopes. The CEC was expressed in the Escherichia coli following codon optimization for efficient translational efficiency and purified by affinity chromatography. The antigenic reactivity of r-CEC proteins was confirmed by western blot with rabbit anti-r-Etox hyperimmune sera. The immunogenicity of the recombinant single CEC was examined in mice and rabbit by indirect ELISA. It was found that r-CEC yielded high titers of neutralizing antibodies (≥ 1.035 IU/ml) in immunized mice and rabbit. The potency of chimeric protein immunized serum was observed to be higher than the recommended level (0.1–0.3 IU/ml) for protection in sheep and goats. This indicated the potential ability of the chimeric protein as a vaccine candidate. This further requires studying the immune response in targeted host species (sheep and goat).

Multifactorial Design of a Supramolecular Peptide Anti-IL-17 Vaccine Toward the Treatment of Psoriasis.

Current treatments for chronic immune-mediated diseases such as psoriasis, rheumatoid arthritis, or Crohn's disease commonly rely on cytokine neutralization using monoclonal antibodies; however, such approaches have drawbacks. Frequent repeated dosing can lead to the formation of anti-drug antibodies and patient compliance issues, and it is difficult to identify a single antibody that is broadly efficacious across diverse patient populations. As an alternative to monoclonal antibody therapy, anti-cytokine immunization is a potential means for long-term therapeutic control of chronic inflammatory diseases. Here we report a supramolecular peptide-based approach for raising antibodies against IL-17 and demonstrate its efficacy in a murine model of psoriasis. B-cell epitopes from IL-17 were co-assembled with the universal T-cell epitope PADRE using the Q11 self-assembling peptide nanofiber system. These materials, with or without adjuvants, raised antibody responses against IL-17. Exploiting the modularity of the system, multifactorial experimental designs were used to select formulations maximizing titer and avidity. In a mouse model of psoriasis induced by imiquimod, unadjuvanted nanofibers had therapeutic efficacy, which could be enhanced with alum adjuvant but reversed with CpG adjuvant. Measurements of antibody subclass induced by adjuvanted and unadjuvanted formulations revealed strong correlations between therapeutic efficacy and titers of IgG1 (improved efficacy) or IgG2b (worsened efficacy). These findings have important implications for the development of anti-cytokine active immunotherapies and suggest that immune phenotype is an important metric for eliciting therapeutic anti-cytokine antibody responses.

Update on group A streptococcal vaccine development.

There is a global need for well tolerated, effective, and affordable vaccines to prevent group A streptococcal infections and their most serious complications. The aim of this review is to highlight the recent progress in the identification of promising vaccine antigens and new approaches to vaccine design that address the complexities of group A streptococcal pathogenesis and epidemiology. Combination vaccines containing multiple shared, cross-protective antigens have proven efficacious in mouse and nonhuman primate models of infection. The development of complex multivalent M protein-based vaccines is continuing and several have progressed through early-stage human clinical trials. Formulations of vaccines containing universal T-cell epitopes, toll-like receptor agonists, and other adjuvants more potent than alum have been shown to enhance protective immunogenicity. Although the group A streptococcal vaccine antigen landscape is populated with a number of potential candidates, the clinical development of vaccines has been impeded by a number of factors. There are now concerted global efforts to raise awareness about the need for group A streptococcal vaccines and to support progress toward eventual commercialization and licensure. Preclinical antigen discovery, vaccine formulation, and efficacy studies in animal models have progressed significantly in recent years. There is now a need to move promising candidates through the clinical development pathway to establish their efficacy in preventing group A streptococcal infections and their complications.

Incorporation of T-cell epitopes from tetanus and diphtheria toxoids into in-silico-designed hypoallergenic vaccine may enhance the protective immune response against allergens.

Objective(s):New generation of allergy vaccines is capable of promoting the development of protective IgG and blocking the functionality of allergen-specific IgE. We incorporated universal and powerful T-cell epitopes from tetanus and diphtheria toxoids (TD epitope) into recombinant Che a 2, the well-known allergic profilin of Chenopodium album, to determine its immunological properties.Materials and Methods:The sequence and accordingly the structure of the recombinant Che a 2 was altered to generate a hypoallergenic variant (rChe a 2.rs). Moreover, TD epitope was incorporated to produce a novel vaccine that was nominated as rChe a 2.rsT.D. The effect of treatment with these variants was evaluated on the generation of allergen-specific IgG class, as well as lymphocyte proliferation in mice. Moreover, IgE-binding characteristics of the allergic patients’ sera were determined by ELISA and proliferation and cytokine production was measured in T-cells. Results:ELISA and dot blot revealed strong reduction of the IgE-reactivity of human sera to the variants of Che a 2 as compared to the wild-type molecule. Furthermore, Che a 2.rs and Che a 2.rsT.D induced much lower levels of IL5 and IL13 secretion from allergic patients’ PBMCs in comparison to wild-type Che a 2 protein. In mice, rChe a 2.rsT.D induced high titers of Che a 2-specific IgG antibody capable of blocking IgE-binding to rChe a 2 and induced lymphocyte proliferation more potently than rChe a 2.rs. Conclusion:Collectively, incorporation of T-cell epitopes of tetanus and diphtheria into hypoallergenic vaccines can dramatically enhance anti-allergic immune mechanisms, particularly in poor responders.

Immunization of cats to induce neutralizing antibodies against Fel d 1, the major feline allergen in human subjects

Cat allergy in human subjects is usually caused by the major cat allergen Fel d 1 and is found in approximately 10% of the Western population. Currently, there is no efficient and safe therapy for cat allergy available. Allergic patients usually try to avoid cats or treat their allergy symptoms. We developed a new strategy to treat Fel d 1-induced allergy in human subjects by immunizing cats against their own major allergen, Fel d 1. A conjugate vaccine consisting of recombinant Fel d 1 and a virus-like particle derived from the cucumber mosaic virus containing the tetanus toxin-derived universal T-cell epitope tt830-843 (CuMVTT) was used to immunize cats. Afirst tolerability and immunogenicity study, including a boost injection, was conducted by using the Fel-CuMVTT vaccine alone or in combination with an adjuvant. The vaccine was well tolerated and had no overt toxic effect. All cats induced a strong and sustained specific IgG antibody response. The induced anti-Fel d 1 antibodies were of high affinity and exhibited a strong neutralization ability tested both invitro and invivo. Areduction in the endogenous allergen level and a reduced allergenicity of tear samples, were observed. Vaccination of cats with Fel-CuMVTT induces neutralizing antibodies and might result in reduced symptoms of allergic cat owners. Both human subjects and animals could profit from this treatment because allergic cat owners would reduce their risk of developing chronic diseases, such as asthma, and become more tolerant of their cats, which therefore could stay in the households and not need to be relinquished to animal shelters.

Treating insect-bite hypersensitivity in horses with active vaccination against IL-5

Insect-bite hypersensitivity is the most common allergic dermatitis in horses. Excoriated skin lesions are typical symptoms of this seasonal and refractory chronic disease. On a cellular level, the skin lesions are characterized by massive eosinophil infiltration caused by an underlying allergic response. To target these cells and treat disease, we developed a therapeutic vaccine against equine IL-5 (eIL-5), the master regulator of eosinophils. The vaccine consisted of eIL-5 covalently linked to a virus-like particle derived from cucumber mosaic virus containing the tetanus toxoid universal T-cell epitope tt830-843 (CMVTT). Thirty-four Icelandic horses were recruited and immunized with 400μg of eIL-5-CMVTT formulated in PBS without adjuvant (19 horses) or PBS alone (15 horses). The vaccine was well tolerated and did not reveal any safety concerns but was able to induce anti-eIL-5 autoantibody titers in 17 of 19 horses. This resulted in a statistically significant reduction in clinical lesion scores when compared with previous season levels, as well as levels in placebo-treated horses. Protection required a minimal threshold of anti-eIL-5 antibodies. Clinical improvement by disease scoring showed that 47% and 21% of vaccinated horses reached 50% and 75% improvement, respectively. In the placebo group no horse reached 75% improvement, and only 13% reached 50% improvement. Our therapeutic vaccine inducing autoantibodies against self IL-5 brings biologics to horses, is the first successful immunotherapeutic approach targeting a chronic disease in horses, and might facilitate development of a similar vaccine against IL-5 in human subjects.

Experimental and Theoretical Approaches To Investigate the Immunogenicity of Taenia solium-Derived KE7 Antigen.

Taenia solium cysticercosis, a parasitic disease that affects human health in various regions of the world, is preventable by vaccination. Both the 97-amino-acid-long KETc7 peptide and its carboxyl-terminal, 18-amino-acid-long sequence (GK-1) are found in Taenia crassiceps Both peptides have proven protective capacity against cysticercosis and are part of the highly conserved, cestode-native, 264-amino-acid long protein KE7. KE7 belongs to a ubiquitously distributed family of proteins associated with membrane processes and may participate in several vital cell pathways. The aim of this study was to identify the T. solium KE7 (TsKE7) full-length protein and to determine its immunogenic properties. Recombinant TsKE7 (rTsKE7) was expressed in Escherichia coli Rosetta2 cells and used to obtain mouse polyclonal antibodies. Anti-rTsKE7 antibodies detected the expected native protein among the 350 spots developed from T. solium cyst vesicular fluid in a mass spectrometry-coupled immune proteomic analysis. These antibodies were then used to screen a phage-displayed 7-random-peptide library to map B-cell epitopes. The recognized phages displayed 9 peptides, with the consensus motif Y(F/Y)PS sequence, which includes YYYPS (named GK-1M, for being a GK-1 mimotope), exactly matching a part of GK-1. GK-1M was recognized by 58% of serum samples from cysticercotic pigs with 100% specificity but induced weak protection against murine cysticercosis. In silico analysis revealed a universal T-cell epitope(s) in native TsKE7 potentially capable of stimulating cytotoxic T lymphocytes and helper T lymphocytes under different major histocompatibility complex class I and class II mouse haplotypes. Altogether, these results provide a rationale for the efficacy of the KETc7, rTsKE7, and GK-1 peptides as vaccines.

Virus-Like Particle (VLP) Plus Microcrystalline Tyrosine (MCT) Adjuvants Enhance Vaccine Efficacy Improving T and B Cell Immunogenicity and Protection against Plasmodium berghei/vivax

Vaccination is the most effective prophylactic tool against infectious diseases. Despite continued efforts to control malaria, the disease still generally represents a significant unmet medical need. Microcrystalline tyrosine (MCT) is a well described depot used in licensed allergy immunotherapy products and in clinical development. However, its proof of concept in prophylactic vaccines has only recently been explored. MCT has never been used in combination with virus-like particles (VLPs), which are considered to be one of the most potent inducers of cellular and humoral immune responses in mice and humans. In the current study we assessed the potential of MCT to serve as an adjuvant in the development of a vaccine against malaria either alone or combined with VLP using Plasmodium vivax thrombospondin-related adhesive protein (TRAP) as a target antigen. We chemically coupled PvTRAP to VLPs derived from the cucumber mosaic virus fused to a universal T-cell epitope of the tetanus toxin (CMVtt), formulated with MCT and compared the induced immune responses to PvTRAP formulated in PBS or Alum. The protective capacity of the various formulations was assessed using Plasmodium berghei expressing PvTRAP. All vaccine formulations using adjuvants and/or VLP increased humoral immunogenicity for PvTRAP compared to the antigen alone. The most proficient responder was the group of mice immunized with the vaccine formulated with PvTRAP-VLP + MCT. The VLP-based vaccine formulated in MCT also induced the strongest T cell response and conferred best protection against challenge with recombinant Plasmodium berghei. Thus, the combination of VLP with MCT may take advantage of the properties of each component and appears to be an alternative biodegradable depot adjuvant for development of novel prophylactic vaccines.

A Targeted and Adjuvanted Nanoparticle for Immunochemotherapy of Leishmania Infections

Targeted drug delivery is critical for minimizing off-target toxicity by chemotherapeutic drugs. Amphotericin-B is an effective anti-leishmaniasis agent that induces significant nephrotoxicity. Complexing amphotericin-B with liposomes (AmBisome, LAmB) reduces nephrotoxicity and improves LAmB efficacy in treating leishmaniasis in humans. However, complicated dosing regimens are required to minimize side effects. Given that Leishmania species are obligate parasites in phagocytes, selectively targeting LAmB to infected phagocytes would likely improve efficacy, decrease systemic toxicity, and simplify dosing regimens. To target LAmB to phagocytes, we developed a PADRE-derivatized-dendrimer (PDD)-LAmB nanocarrier platform by functionalizing dendrimers that complex LAmB with a peptide that targets major histocompatibility complex (MHC) class II receptors. MHC class II receptor expression increases on phagocytes during the course of leishmaniasis, further enhancing infected cells as targets for PDD-LAmB. In a murine model for Leishmania major infection, PDD-LAmB efficiently targeted infected phagocytic cells, reducing the LAmB effective dose by 80 %, with improved pharmacokinetics. Moreover, the PDD MHC class II-targeting peptide is a universal helper T-cell epitope, allowing PDD-LAmB complexes to elicit parasite-specific T-cell responses. Thus, PDD-LAmB complex is a promising candidate for further development and human clinical trials, reducing and simplifying LAmB dosing, reducing side effects, and stimulating T-cell responses.

Immunological Evaluation of Lipopeptide Group A Streptococcus (GAS) Vaccine: Structure-Activity Relationship

Streptococcus pyogenes (group A streptococcus, GAS) is a Gram-positive bacterial pathogen responsible for a wide variety of diseases. To date, GAS vaccine development has focused primarily on the M-protein. The M-protein is highly variable at the amino (N)-terminus (determining serotype) but is conserved at the carboxyl (C)-terminus. Previously a 29 amino acid peptide (named J14) from the conserved region of the M-protein was identified as a potential vaccine candidate. J14 was capable of eliciting protective antibodies that recognized many GAS serotypes when co-administered with immuno-stimulants. This minimal epitope however showed no immunogenicity when administered alone. In an attempt overcome this immunological non-responsiveness, we developed a self-adjuvanting vaccine candidate composed of three components: the B-cell epitope (J14), a universal helper T-cell epitope (P25) and a lipid moiety consisting of lipoamino acids (Laas) which target Toll-like receptor 2 (TLR2). Immunological evaluation in B10.BR (H-2k) mice demonstrated that the epitope attachment to the point of lipid moiety, and the length of the Laa alkyl chain have a profound effect on vaccine immunogenicity after intranasal administration. It was demonstrated that a vaccine featuring C-terminal lipid moiety containing alkyl chains of 16 carbons, with P25 located at the N-terminus, and J14 attached to the side chain of a central lysine residue was capable of inducing optimal antibody response. These findings have considerable relevance to the development of a broad spectrum J14-based GAS vaccine and in particular provided a rational basis for peptide vaccine design based on this self-adjuvanting lipopeptide technology.

Structure–activity relationship of lipopeptide Group A streptococcus (GAS) vaccine candidates on toll-like receptor 2

Incorporation of lipoamino acids (LAAs) into peptide structures effectively imparts self-adjuvanting activity onto otherwise ineffective immunogens. Our fully synthetic lipopeptide vaccine candidates against group A streptococcus (GAS) were composed of J14 as a target GAS B-cell epitope alongside a universal helper T-cell epitope (P25) and a LAA-based lipid moiety. In the current study, we investigated the ability of our lipopeptides to activate nuclear factor-κB (NF-κB) in a toll-like receptor-2 (TLR2)-dependent manner as the possible mode of action and reported the structure–function requirements for novel TLR2 targeting lipopeptides based on LAAs. The NF-κB activation was dependent on the dose and the length of the alkyl chains of the incorporated lipid moieties with the hierarchy LAA 3 (16 carbons)>LAA 2 (14 carbons)>LAA 1 (12 carbons). The position of the lipid moiety (C-terminus vs. Nɛ-terminus of the central lysine residue) does not significantly affect NF-κB activation. Lipopeptides containing different copies of LAA 3 were synthesized and the di-lipidated analogue was the most effective in NFκB activation.

Structure–Activity Relationship of a Series of Synthetic Lipopeptide Self-Adjuvanting Group A Streptococcal Vaccine Candidates

The development of 16 self-adjuvanting group A streptococcal vaccine candidates, composed of (i) a universal helper T-cell epitope (P25), (ii) a target GAS B-cell epitope (J14), and (iii) a lipid moiety, is described. Systemic J14-specific IgG antibodies were detected following subcutaneous immunization of BALB/c (H-2 (d)) mice with each construct without the need for an additional adjuvant. The effect of changing the order of P25, J14, and lipid moiety attachment or incorporation of P25 and J14 into a lipid-core peptide system on antibody titers was assessed. The point of lipid moiety attachment had the greatest influence on systemic J14-specific IgG antibody titers. Overall, the best vaccines featured a C-terminal lipid moiety, conjugated through a lysine residue to P25 at the N-terminus, and J14 on the lysine side chain.

Intranasal vaccination with a lipopeptide containing a minimal, conserved M-protein derived peptide and a self-adjuvanting lipid induces both systemic and mucosal immune responses in mice

Group A streptococcus (GAS) is a global health problem. Infection with GAS may result in a number of human diseases ranging from the relatively benign pharyngitis to the life-threatening sequelae of rheumatic fever (RF) and rheumatic heart disease (RHD), therefore highlighting the need for a prophylactic vaccine. We demonstrate that a peptide from the M-protein when incorporated into a lipopeptide construct containing a universal T-cell epitope and a self-adjuvanting lipid moiety is capable of inducing peptide-specific serum IgG and peptide-specific mucosal IgA.

Characterization of T-cell reactive epitopes in glycoprotein G of herpes simplex virus type 2 using synthetic peptides

We have previously shown that the CD4+ T-cell response to herpes simplex virus type 2 glycoprotein G-2 is type-specific and can thus be used to evaluate herpes simplex virus type 2-specific T-cell responses in individuals with a concomitant herpes simplex virus type 1 infection. In this study we have followed the glycoprotein G-2-specific T-cell responses over time, and also tried to identify T-cell epitopes in the membrane bound portion and the secreted portion of glycoprotein G-2 using synthetic peptides spanning the whole amino acid sequence of glycoprotein G-2. We found that the magnitude of the glycoprotein G-2-specific response varied considerably in infected individuals over time, even though all patients responded to at least one of the two glycoproteins at all time-points examined. We could also document strong T-cell responses to synthetic peptides from the secreted glycoprotein G-2 but only low responses to synthetic peptides corresponding to sequences from the heavily glycosylated membrane-bound glycoprotein G-2. We were able to map an immunogenic region (amino acid 31-125) within the secreted glycoprotein G-2. This region of the glycoprotein induced proliferative responses in 47% of the herpes simplex virus type 2-infected individuals. However, we were not able to identify any universal T-cell epitope.

Use of long synthetic peptides to study the antigenicity and immunogenicity of the Plasmodium vivax circumsporozoite protein

Three long synthetic peptides corresponding to amino (N), repeat (R) and carboxyl (C) regions of the Plasmodium vivax circumsporozoite (CS) protein were synthesised and used to assess their potential as vaccine candidates. Antigenicity studies were carried out using human blood samples from residents of a malaria-endemic area of Colombia, and immunogenicity was tested in Aotus monkeys. The N and C peptides spanned the total native amino and carboxyl flanking regions, whereas the R peptide corresponded to a construct based on the first central nona-peptide repeated in tandem three times and colinearly linked to a universal T-cell epitope (ptt-30) derived from tetanus toxin. All three peptides had been shown previously to contain several B-, T-helper (Th) and Cytotoxic T Lymphocytes (CTL) epitopes. Sixty-one percent of the human sera reacted with the R region, whereas 35 and 39% of the samples had antibodies against the N and C peptides, respectively. Human Peripheral Blood Mononuclear Cells (PBMC) showed higher levels of IFN-γ than IL-4 when stimulated with peptides containing Th epitopes. Aotus monkeys immunised with the peptides formulated in either Montanide ISA720 or Freund's adjuvants produced strong antibody responses that recognised the peptide immunogens and the native circumsporozoite protein on sporozoites. Additionally, high IFN-γ production was induced when Aotus lymphocytes were stimulated in vitro with each of the three peptides. We observed boosting of antibody responses and IFN-γ production by exposure to live sporozoites. These results confirm the high antigenicity and immunogenicity of such synthetic polypeptides and underline their vaccine potential.

Differential antibody responses to Plasmodium falciparum-derived B-cell epitopes induced by diepitope multiple antigen peptides (MAP) containing different T-cell epitopes

Epitopes of universal character are needed when designing subunit vaccines against infectious diseases such as malaria. We have compared the immunogenicity of B-cell epitopes from the Plasmodium falciparum antigen repeats DPNANPNV (PfCS protein) and VTEEI (Pf332) when assembled with four different universal T-cell epitopes in diepitope multiple antigen peptides (MAP). T-epitopes employed were from P. falciparum antigens (CS.T3, [T *] 4 and EBP3) or from the Clostridium tetani toxin (P2). In association with either of the T-epitopes, the genetic unresponsiveness to the B-epitopes was successfully bypassed. Our results show that the immunogenicity of a T-epitope alone does not necessarily predict the ability of the T-epitope to provide T-cell help when combined with other epitopes in an immunogen. Further, the nature of the immune responses in terms of total IgG antibodies and their subclass distribution, T-cell proliferation and IFN-γ production, varied with the T-epitope and mouse strain, which may indicate the need for inclusion of a combination of different universal T-epitopes in a future malaria subunit vaccine.