Phase I trial of an alhydrogel adjuvanted hepatitis B core virus-like particle containing epitopes of Plasmodium falciparum circumsporozoite protein

The objectives of this non-randomized, non-blinded, dose-escalating Phase I clinical trial were to assess the safety, reactogenicity and immunogenicity of ICC-1132 formulated with Alhydrogel (aluminum hydroxide) in 51 healthy, malaria-naive adults aged 18 to 45 years. ICC-1132 (Malariavax) is a recombinant, virus-like particle malaria vaccine comprised of hepatitis core antigen engineered to express the central repeat regions from Plasmodium falciparum circumsporozoite protein containing an immunodominant B [(NANP)(3)] epitope, an HLA-restricted CD4 (NANPNVDPNANP) epitope and a universal T cell epitope (T*) (amino acids 326-345, NF54 isolate). We assessed an Alhydrogel (aluminum hydroxide)-adjuvanted vaccine formulation at three ICC-1132 dose levels, each injected intramuscularly (1.0 mL) on study days 0, 56 and 168. A saline vaccine formulation was found to be unstable after prolonged storage and this formulation was subsequently removed from the study. Thirty-two volunteers were followed for one year. Local and systemic adverse clinical events were measured and immune responses to P. falciparum and hepatitis B virus core antigens were determined utilizing the following assays: IgG and IgM ELISA, indirect immunofluorescence against P. falciparum sporozoites, circumsporozoite precipitin (CSP) and transgenic sporozoite neutralization assays. Cellular responses were measured by proliferation and IL-2 assays. Local and systemic reactions were similarly mild and well tolerated between dose cohorts. Depending on the ICC-1132 vaccine concentration, 95 to 100% of volunteers developed antibody responses to the ICC-1132 immunogen and HBc after two injections; however, only 29-75% and 29-63% of volunteers, respectively, developed malaria-specific responses measured by the malaria repeat synthetic peptide ELISA and IFA; 2 of 8 volunteers had positive reactions in the CSP assay. Maximal transgenic sporozoite neutralization assay inhibition was 54%. Forty-seven to seventy-five percent demonstrated T cell proliferation in response to ICC-1132 or to recombinant circumsporozoite protein (rCS) NF-54 isolate. This candidate malaria vaccine was well tolerated, but the vaccine formulation was poorly immunogenic. The vaccine may benefit from a more powerful adjuvant to improve immunogenicity. TRIAL REGISTRATION: ClinicalTrials.gov NCT00587249.     

Chemoselective assembly and immunological evaluation of multiepitopic glycoconjugates bearing clustered Tn antigen as synthetic anticancer vaccines

In this paper we investigated the use of regioselectively addressable functionalized templates (RAFTs) as new scaffolds for the design of anticancer vaccine candidates. We report the synthesis of well-defined multiepitopic RAFT scaffolds and their immunological evaluation. These conjugates exhibit clustered Tn analogue as tumor-associated carbohydrate antigen (TACA, B-cell epitope) and the CD4+ helper T-cell peptide from the type 1 poliovirus. The saccharidic and peptidic epitopes were both synthesized separately and combined regioselectively to the RAFT core using a sequential oxime bond formation strategy. B- and T-antigenicity and immunogenicity of the vaccine candidates were investigated in vitro and in vivo. These studies clearly demonstrate that the saccharidic part of the conjugates is recognized by Tn-specific monoclonal antibodies. Moreover, the antibodies elicited by immunization of mice with our vaccine candidates recognize the native form of Tn epitope expressed on human tumor cells. Together with oxime ligation technique, these results suggest that the RAFT scaffold provides a promising and suitable tool for engineering potent synthetic anticancer vaccine.     

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.     

Immunoinformatics and the prediction of immunogenicity

Immunoinformatics is the application of informatics techniques to molecules of the immune system. One of the key goals of immunoinformatics is the development of computer aided vaccine design (CAVD), or computational vaccinology, and its application to the search for new vaccines. Key to solving this challenge is the prediction of immunogenicity, be that at the level of epitope, subunit vaccine or attenuated pathogen. This paper reviews the current state of play in the prediction of immunogenicity and focuses on well developed methods for the prediction of peptide binding affinity to major histocompatibility complexes, which are the necessary preliminary to the in silico identification of T cell epitopes.     

Structural Analysis of the Synthetic Duffy Binding Protein (DBP) Antigen DEKnull Relevant for Plasmodium vivax Malaria Vaccine Design

The Plasmodium vivax vaccine candidate Duffy Binding Protein (DBP) is a protein necessary for P. vivax invasion of reticulocytes. The polymorphic nature of DBP induces strain-specific immune responses that pose unique challenges for vaccine development. DEKnull is a synthetic DBP based antigen that has been engineered through mutation to enhance induction of blocking inhibitory antibodies. We determined the x-ray crystal structure of DEKnull to identify if any conformational changes had occurred upon mutation. Computational and experimental analyses assessed immunogenicity differences between DBP and DEKnull epitopes. Functional binding assays with monoclonal antibodies were used to interrogate the available epitopes in DEKnull. We demonstrate that DEKnull is structurally similar to the parental Sal1 DBP. The DEKnull mutations do not cause peptide backbone shifts within the polymorphic loop, or at either the DBP dimerization interface or DARC receptor binding pockets, two important structurally conserved protective epitope motifs. All B-cell epitopes, except for the mutated DEK motif, are conserved between DEKnull and DBP. The DEKnull protein retains binding to conformationally dependent inhibitory antibodies. DEKnull is an iterative improvement of DBP as a vaccine candidate. DEKnull has reduced immunogenicity to polymorphic regions responsible for strain-specific immunity while retaining conserved protein folds necessary for induction of strain-transcending blocking inhibitory antibodies.     

A totally synthetic polyoxime malaria vaccine containing Plasmodium falciparum B cell and universal T cell epitopes elicits immune responses in volunteers of diverse HLA types

This open-labeled phase I study provides the first demonstration of the immunogenicity of a precisely defined synthetic polyoxime malaria vaccine in volunteers of diverse HLA types. The polyoxime, designated (T1BT(*))(4)-P3C, was constructed by chemoselective ligation, via oxime bonds, of a tetrabranched core with a peptide module containing B cell epitopes and a universal T cell epitope of the Plasmodium falciparum circumsporozoite protein. The triepitope polyoxime malaria vaccine was immunogenic in the absence of any exogenous adjuvant, using instead a core modified with the lipopeptide P3C as an endogenous adjuvant. This totally synthetic vaccine formulation can be characterized by mass spectroscopy, thus enabling the reproducible production of precisely defined vaccines for human use. The majority of the polyoxime-immunized volunteers (7/10) developed high levels of anti-repeat Abs that reacted with the native circumsporozoite on P. falciparum sporozoites. In addition, these seven volunteers all developed T cells specific for the universal epitope, termed T(*), which was originally defined using CD4(+) T cells from protected volunteers immunized with irradiated P. falciparum sporozoites. The excellent correlation of T(*)-specific cellular responses with high anti-repeat Ab titers suggests that the T(*) epitope functioned as a universal Th cell epitope, as predicted by previous peptide/HLA binding assays and by immunogenicity studies in mice of diverse H-2 haplotypes. The current phase I trial suggests that polyoximes may prove useful for the development of highly immunogenic, multicomponent synthetic vaccines for malaria, as well as for other pathogens.     

Pooled-Peptide Epitope Mapping Strategies Are Efficient and Highly Sensitive: An Evaluation of Methods for Identifying Human T Cell Epitope Specificities in Large-Scale HIV Vaccine Efficacy Trials

The interferon gamma, enzyme-linked immunospot (IFN-γ ELISpot) assay is widely used to identify viral antigen-specific T cells is frequently employed to quantify T cell responses in HIV vaccine studies. It can be used to define T cell epitope specificities using panels of peptide antigens, but with sample and cost constraints there is a critical need to improve the efficiency of epitope mapping for large and variable pathogens. We evaluated two epitope mapping strategies, based on group testing, for their ability to identify vaccine-induced T-cells from participants in the Step HIV-1 vaccine efficacy trial, and compared the findings to an approach of assaying each peptide individually. The group testing strategies reduced the number of assays required by >7-fold without significantly altering the accuracy of T-cell breadth estimates. Assays of small pools containing 7–30 peptides were highly sensitive and effective at detecting single positive peptides as well as summating responses to multiple peptides. Also, assays with a single 15-mer peptide, containing an identified epitope, did not always elicit a response providing validation that 15-mer peptides are not optimal antigens for detecting CD8+ T cells. Our findings further validate pooling-based epitope mapping strategies, which are critical for characterizing vaccine-induced T-cell responses and more broadly for informing iterative vaccine design. We also show ways to improve their application with computational peptide:MHC binding predictors that can accurately identify the optimal epitope within a 15-mer peptide and within a pool of 15-mer peptides.     

T cell determinants incorporating beta-amino acid residues are protease resistant and remain immunogenic in vivo

A major hurdle in designing successful epitope-based vaccines resides in the delivery, stability, and immunogenicity of the peptide immunogen. The short-lived nature of unmodified peptide-based vaccines in vivo limits their therapeutic application in the immunotherapy of cancers and chronic viral infections as well as their use in generating prophylactic immunity. The incorporation of beta-amino acids into peptides decreases proteolysis, yet its potential application in the rational design of T cell mimotopes is poorly understood. To address this, we have replaced each residue of the SIINFEKL epitope individually with the corresponding beta-amino acid and examined the resultant efficacy of these mimotopes. Some analogs displayed similar MHC binding and superior protease stability compared with the native epitope. Importantly, these analogs were able to generate cross-reactive CTLs in vivo that were capable of lysing tumor cells that expressed the unmodified epitope as a surrogate tumor Ag. Structural analysis of peptides in which anchor residues were substituted with beta-amino acids revealed the basis for enhanced MHC binding and retention of immunogenicity observed for these analogs and paves the way for future vaccine design using beta-amino acids. We conclude that the rational incorporation of beta-amino acids into T cell determinants is a powerful alternative to the traditional homologous substitution of randomly chosen naturally occurring alpha-amino acids, and these mimotopes may prove particularly useful for inclusion in epitope-based vaccines.     

Deletion of fusion peptide or destabilization of fusion core of HIV gp41 enhances antigenicity and immunogenicity of 4E10 epitope

The human monoclonal antibody 4E10 against the membrane-proximal external region (MPER) of HIV-1 gp41 demonstrates broad neutralizing activity across various strains, and makes its epitope an attractive target for HIV-1 vaccine development. Although the contiguous epitope of 4E10 has been identified, attempts to re-elicit 4E10-like antibodies have failed, possibly due to the lack of proper conformation of the 4E10 epitope. Here we used pIg-tail expression system to construct a panel of eukaryotic cell-surface expression plasmids encoding the extracellular domain of gp41 with deletion of fusion peptide and/or introduction of L568P mutation that may disrupt the gp41 six-helix bundle core conformation as DNA vaccines for immunization of mice. We found that these changes resulted in significant increase of the antigenicity and immunogenicity of 4E10 epitope. This information is thus useful for rational design of vaccines targeting the HIV-1 gp41 MPER.     

CD133表位联合热休克蛋白佐剂疫苗引发抗白血病的免疫应答

肿瘤干细胞具有肿瘤形成、自我更新和抗化疗的特性,因而受到广泛关注和研究。CD133作为重要的肿瘤干细胞标志物与肿瘤细胞的干性与恶性密切相关。本研究筛选并且鉴定了CD133的3个H2-Kd限制性的细胞毒性T细胞 (CTL) 的表位,分别是CD133419–428、CD133702–710和CD133760–769。将重组热休克蛋白gp96为佐剂联合CD133表位制备表位疫苗,将疫苗免疫CD133+白血病移植的小鼠后引发抗肿瘤的特异性T细胞免疫应答。转输CD133表位特异的T细胞同样可以抑制小鼠淋巴瘤的生长。该研究为设计抗CD133+的白血病和其他肿瘤的表位疫苗提供了依据。     

Robust immune responses elicited by a fully synthetic three-component vaccine

The overexpression of saccharides such as Globo-H, Lewis(Y) and Tn antigen is a common feature of oncogenic transformed cells. Endeavors to exploit this aberrant glycosylation for cancer vaccine development have been complicated by difficulties in eliciting high titers of IgG antibodies against classical conjugates of tumor-associated carbohydrates to carrier proteins. We have designed, chemically synthesized and immunologically evaluated a number of fully synthetic vaccine candidates to establish strategies to overcome the poor immunogenicity of tumor-associated carbohydrates and glycopeptides. We have found that a three-component vaccine composed of a TLR2 agonist, a promiscuous peptide T-helper epitope and a tumor-associated glycopeptide can elicit in mice exceptionally high titers of IgG antibodies that can recognize cancer cells expressing the tumor-associated carbohydrate. The superior properties of the vaccine candidate are attributed to the local production of cytokines, upregulation of co-stimulatory proteins, enhanced uptake by macrophages and dendritic cells and avoidance of epitope suppression.     

Identification and validation of T-cell epitopes in outer membrane protein (OMP) of Salmonella typhi

This study aims to design epitope-based peptides for the utility of vaccine development by targeting outer membrane protein F (Omp F), because two available licensed vaccines, live oral Ty21a and injectable polysaccharide, are 50% to 80% protective with a higher rate of side effects. Conventional vaccines take longer time for development and have less differentiation power between vaccinated and infected cells. On the other hand, Peptide-based vaccines present few advantages over other vaccines, such as stability of peptide, ease to manufacture, better storage, avoidance of infectious agents during manufacture, and different molecules can be linked with peptides to enhance their immunogenicity. Omp F is highly conserved and facilitates attachment and fusion of Salmonella typhi with host cells. Using various databases and tools, immune parameters of conserved sequences from Omp F of different isolates of Salmonella typhi were tested to predict probable epitopes. Binding analysis of the peptides with MHC molecules, epitopes conservancy, population coverage, and linear B cell epitope prediction were analyzed. Among all those predicted peptides, ESYTDMAPY epitope interacted with six MHC alleles and it shows highest amount of interaction compared to others. The cumulative population coverage for these epitopes as vaccine candidates was approximately 70%. Structural analysis suggested that epitope ESYTDMAPY fitted well into the epitope-binding groove of HLA-C*12:03, as this HLA molecule was common which interact with each and every predicted epitopes. So, this potential epitope may be linked with other molecules to enhance its immunogenicity and used for vaccine development.     

Analysis of Human RSV Immunity at the Molecular Level: Learning from the Past and Present

Human RSV is one of the most prevalent viral pathogens of early childhood for which no vaccine is available. Herein we provide an analysis of RSV epitope data to examine its application to vaccine design and development. Our objective was to provide an overview of antigenic coverage, identify critical antibody and T cell determinants, and then analyze the cumulative RSV epitope data from the standpoint of functional responses using a combinational approach to characterize antigenic structure and epitope location. A review of the cumulative data revealed, not surprisingly, that the vast majority of epitopes have been defined for the two major surface antigens, F and G. Antibody and T cell determinants have been reported from multiple hosts, including those from human subjects following natural infection, however human data represent a minority of the data. A structural analysis of the major surface antigen, F, showed that the majority of epitopes defined for functional antibodies (neutralizing and/or protective) were either shown to bind pre-F or to be accessible in both pre- and post-F forms. This finding may have has implications for on-going vaccine design and development. These interpretations are in agreement with previous work and can be applied in the larger context of functional epitopes on the F protein. It is our hope that this work will provide the basis for further RSV-specific epitope discovery and investigation into the nature of antigen conformation in immunogenicity.     

Enhancing immune responses against SARS-CoV nucleocapsid DNA vaccine by co-inoculating interleukin-2 expressing vector in mice

The immunogenicity of SARS-CoV nucleocapsid DNA vaccine and the immunoregulatory activity of interleukin-2 (IL-2) were investigated. DNA vaccine plasmids, pcDNA-N and pcDNA-IL2, were constructed and inoculated into BALB/c mice with or without pcDNA-IL2 by intramuscular injection. Cellular and humoral immune responses were assessed by indirect ELISA, lymphocyte proliferation assays, ELISPOT and FACS. The nucleocapsid DNA vaccine had good immunogenicity and can induce specific humoral and cellular immunity in BALB/c mice, while IL-2 plays an immunoadjuvant role and enhances specific immune responses. This study provides a frame of reference for the design of DNA vaccines against SARS-CoV.     

Modelling the spread of HIV immune escape mutants in a vaccinated population

Because cytotoxic T-lymphocytes (CTLs) have been shown to play a role in controlling human immunodeficiency virus (HIV) infection and because CTL-based simian immunodeficiency virus (SIV) vaccines have proved effective in non-human primates, one goal of HIV vaccine design is to elicit effective CTL responses in humans. Such a vaccine could improve viral control in patients who later become infected, thereby reducing onwards transmission and enhancing life expectancy in the absence of treatment. The ability of HIV to evolve mutations that evade CTLs and the ability of these 'escape mutants' to spread amongst the population poses a challenge to the development of an effective and robust vaccine. We present a mathematical model of within-host evolution and between-host transmission of CTL escape mutants amongst a population receiving a vaccine that elicits CTL responses to multiple epitopes. Within-host evolution at each epitope is represented by the outgrowth of escape mutants in hosts who restrict the epitope and their reversion in hosts who do not restrict the epitope. We use this model to investigate how the evolution and spread of escape mutants could affect the impact of a vaccine. We show that in the absence of escape, such a vaccine could markedly reduce the prevalence of both infection and disease in the population. However the impact of such a vaccine could be significantly abated by CTL escape mutants, especially if their selection in hosts who restrict the epitope is rapid and their reversion in hosts who do not restrict the epitope is slow. We also use the model to address whether a vaccine should span a broad or narrow range of CTL epitopes and target epitopes restricted by rare or common HLA types. We discuss the implications and limitations of our findings.     

The position of heterologous epitopes inserted in hepatitis B virus core particles determines their immunogenicity.

The nucleocapsid (HBcAg) of the hepatitis B virus (HBV) has been suggested as a carrier moiety for vaccine purposes. We investigated the influence of the position of the inserted epitope within hybrid HBcAg particles on antigenicity and immunogenicity. For this purpose, genes coding for neutralizing epitopes of the pre-S region of the HBV envelope proteins were inserted at the amino terminus, the amino terminus through a precore linker sequence, the truncated carboxy terminus, or an internal site of HBcAg by genetic engineering and were expressed in Escherichia coli. All purified hybrid HBc/pre-S polyproteins were particulate. Amino- and carboxy-terminal-modified hybrid HBc particles retained HBcAg antigenicity and immunogenicity. In contrast, insertion of a pre-S(1) sequence between HBcAg residues 75 and 83 abrogated recognition of HBcAg by 5 of 6 anti-HBc monoclonal antibodies and diminished recognition by human polyclonal anti-HBc. Predictably, HBcAg-specific immunogenicity was also reduced. With respect to the inserted epitopes, a pre-S(1) epitope linked to the amino terminus of HBcAg was not surface accessible and not immunogenic. A pre-S(1) epitope fused to the amino terminus through a precore linker sequence was surface accessible and highly immunogenic. A carboxy-terminal-fused pre-S(2) sequence was also surface accessible but weakly immunogenic. Insertion of a pre-S(1) epitope at the internal site resulted in the most efficient anti-pre-S(1) antibody response. Furthermore, immunization with hybrid HBc/pre-S particles exclusively primed T-helper cells specific for HBcAg and not the inserted epitope. These results indicate that the position of the inserted B-cell epitope within HBcAg is critical to its immunogenicity.     

Self-assembly polymerization enhances the immunogenicity of influenza M2e peptide

The extracellular domain of Influenza M2 protein (M2e) was considered as a promising target for universal influenza vaccine development. Several M2e-based influenza vaccines have been developed and many of them used a mutant M2e peptide, in which the two conserved cysteine residues were substituted by serine residues. In this paper, we compared the antigenicity and immunogenicity of wild type and cysteine-mutant M2e peptides. We found that the cysteine substitution slightly affected the antigenicity of M2e epitope, but greatly reduced the immunogenicity of M2e peptide. The cysteine substitution also disabled the M2e peptide from inducing protection against influenza virus challenge in mice. Further analysis revealed that the immunogenicity of M2e peptide was enhanced by the self-assembly of the peptide through inter-peptide disulfide bonds. These results provide new information to improve the design of M2e-based vaccines against potential influenza pandemics.     

Vaccine-induced CD8+ T lymphocytes of rhesus monkeys recognize variant forms of an HIV epitope but do not mediate optimal functional activity

The sequence diversity of HIV-1 presents a challenge for the development of an effective HIV-1 vaccine, because such a vaccine must confer protection against diverse forms of the virus. The present studies were initiated to explore how vaccine-induced clonal populations of CD8(+) T lymphocytes of rhesus monkeys recognize variants of an HIV-1 envelope epitope sequence. Evaluating a subset of variants of a selected epitope peptide that retain their binding to the MHC class I molecule of rhesus monkeys that presents this epitope peptide, we show that vaccine-elicited CD8(+) T lymphocytes comparably recognize the wild-type and a number of variant epitope peptides as determined by tetramer binding assays. In fact, the same clonal populations of CD8(+) T lymphocytes recognize the wild-type and variant epitope peptides. However, functional assays show that many of these variant epitope peptides stimulate suboptimal cytokine production by the vaccine-elicited CD8(+) T lymphocytes. These findings suggest that vaccine-induced CD8(+) T lymphocyte populations may recognize diverse forms of a viral epitope, but may not function optimally to confer protection against viruses expressing many of those variant sequences.     

Compositional bias and mimicry toward the nonself proteome in immunodominant T cell epitopes of self and nonself antigens.

We investigated whether and how molecular mimicry affects the shaping of the helper T cell repertoire. We implemented an algorithm that measures the probability of mimicry between epitopes of known immunogenicity and self or nonself proteomes. This algorithm yields 'similarity profiles', which represent the probability of matching between all contiguous overlapping peptides of the antigen under examination and those in the proteome(s) considered. Similarity profiles between helper T cell epitopes (of self or microbial antigens and allergens) and human or microbial SWISSPROT collections were produced. For each antigen, both collections yielded largely overlapping profiles, demonstrating that self-nonself discrimination does not rely on qualitative features that distinguish human from microbial peptides. However, epitopes whose probability of mimicry with self or nonself prevails are, respectively, tolerated or immunodominant and coexist within the same (auto-)antigen regardless of its self/nonself nature. Epitopes (on self and nonself antigens) can cross-stimulate T cells at increasing potency as their similarity with nonself augments. Mimicry, rather than complicating self-nonself discrimination, assists in the shaping of the immune repertoire and helps define the defensive or autoreactive potential of a T cell. Being a predictor of epitope immunogenicity, it bears relevance to vaccine design.