Predicting tolerance in vaccine antigens: Application to influenza, HCV and HIV

JanusMatrix, a newly developed algorithm by EpiVax, Inc, identifies cross-reactive T cell epitopes by matching the T cell receptor (TCR)-accessible amino acids (the TCR face) of peptides that bind the same human leukocyte antigen (HLA) [1]. Taking into both HLA binding and TCR contact account, JanusMatrix is uniquely able to compare T cell epitope conservation between protein sequences from bacterial and viral organisms that make up the human gut microbiome, autologous proteins from the human genome, and human viral and bacterial pathogens. We recently discovered that pathogens might escape human immune response by mutating their epitopes to present “human-like” amino acid sequences to TCR when displayed on antigen-presenting cells [2]. We hypothesized that such “human-like” T cell epitopes contained in pathogens may trigger autologous regulatory T cells (Tregs), actively suppress immune response to themselves, thus improving their ability to survive in the host. We have used JanusMatrix to identify such “human-like” pathogen sequences that both bind HLA and share the same TCR-face patterns as human proteins. In a large scale analysis of viruses that infect humans, we found that chronic viruses that establish persistent infection in human (such as Epstein-Barr, Herpes Simplex Virus and Cytomegalovirus) contain a significantly higher number of T cell epitopes that are cross-conserved (at the TCR face) with human proteins than viruses that do not establish chronic infection (such as Ebola and Marburg) [3]. Using JanusMatrix, we identified human-like T cell epioteps in H7N9 influenza hemagglutinin (HA) protein [4]. A promiscuous T cell epitope from H7N9 HA expanded Tregs and suppressed responses to other H7N9 peptides.This may explain the low titer of H7N9 HA inhibiting antibody responses and diminished seroconversion rates. Depletion the Treg-activating epitope increased antibody titers by 5 fold and B cell response by 20 fold (Unpublished). We also identified a “human-like” HCV T cell epitope, HCV_G1_p7_794, induced a marked increase of Tregs in PBMC derived from HCV-infected patients [5]. In HIV-1 Env, we found a human-like epitope that shares a TCR-face with a large number of human leukocyte antigen (HLA) class I molecule sequences. This highly conserved epitope is found in both the HIV-1 E and B Env antigens used in the ‘moderately effective’ HIV RV144 trial. It was found that this HIV Env-derived T cell epitope trigger functional Treg responses in HIV negative individuals. All these results suggest that JanusMatrix tool opens up a new window on the networks of cross-reactive T cell epitopes in human immune response, which may lead to significant improvements in the efficacy of vaccines. References: 1. Moise L, Gutierrez AH, Bailey-kellogg C, et al. The two-faced T cell epitope: examining the host-microbe interface with JanusMatrix. Hum Vaccin Immunother. 2013;9(7):1577-86. 2. Moise L, Liu R, Gutierrez AH, Tassone R, Bailey-Kellogg C, et al. Immune Camouflage: Relevance to Vaccine Design and Human Immunology. Hum Vaccin. Immunother. 2014;1:e36134. 3. He L, De Groot AS, Gutierrez AH, Martin WD, Moise L, et al. Integrated assessment of predicted MHC binding and cross-conservation with self reveals patterns of viral camouflage. BMC Bioinformatics. 2014; 15:S1. 4. Liu R, Moise L, Tassone R, Gutierrez AH, Terry FE, et al. H7N9 T-cell epitopes that mimic human sequences are less immunogenic and may induce Treg-mediated tolerance. Hum Vaccin Immunother. 2015;11(9):2241-2252. 5. Losikoff PT, Mishra S, Terry F, et al. HCV epitope, homologous to multiple human protein sequences, induces a regulatory T cell response in infected patients. J Hepatol. 2015;62(1):48-55

Concomitant occurrence of FXTAS and clinically defined sporadic inclusion body myositis: report of two cases.

This report describes unique presentations of inclusion body myositis (IBM) in two unrelated patients, one male and one female, with genetically and histologically confirmed fragile X-associated tremor/ataxia syndrome (FXTAS). We summarize overlapping symptoms between two disorders, clinical course, and histopathological analyses of the two patients with FXTAS and sporadic IBM, clinically defined per diagnostic criteria of the European Neuromuscular Centre. In case 1, a post-mortem analysis of available brain and muscle tissues is also described. Histopathological features (rimmed vacuoles) consistent with clinically defined IBM were detected in both presented cases. Postmortem testing in case 1 revealed the presence of an FMR1 premutation allele of 60 CGG repeats in both brain and skeletal muscle samples. Case 2 was a premutation carrier with 71 CGG repeats who had a son with FXS. Given that FXTAS is associated with immune-mediated disorders among premutation carriers, it is likely that the pathogeneses of IBM and FXTAS are linked. This is, to our knowledge, the first report of these two conditions presenting together, which expands our understanding of clinical symptoms and unusual presentations in patients with FXTAS. Following detection of a premutation allele of the FMR1 gene, FXTAS patients with severe muscle pain should be assessed for IBM

Highly cross-conserved burkholderia T cell epitopes generate effector T cell responses in vitro

Burkholderia pseudomallei and Burkholderia mallei cause glanders and melioidosis, respectively. Both of them are classified as Category B biothreat agents due to their high infectivity and potential use as a bioweapon. The related species Burkholderia cepaciae causes fatal \u27cepacia syndrome\u27 in cystic fibrosis patients, which is characterized by rapid deterioration, bacteremia and necrotizing pneumonia. Clinical eradication of Burkholderia infection often fails due to antimicrobial resistance. Effective vaccination against Burkholderia infection is critically important to protect populations living in endemic areas worldwide and against bioterror threats. No vaccines or other prophylactics for these pathogens are available. Vaccines against Burkholderia should target cell-mediated immune response, which is believed to be essential to successfully clear Burkholderia infection. We hypothesize that a single vaccine comprising highly cross-conserved Burkholderia T cell epitopes might generate protective cell-mediated immune response against all the three species. Immunoinformatics tools were used to identify immunogenic consensus sequences (ICS) that are enriched for promiscuous and highly conserved CD4+ T cell epitopes in all three Burkholderia species. The ICS peptides were validated in peripheral blood mononuclear cells (PBMCs) derived from healthy donors [1]. All of the peptides (100%) bound to at least two HLA alleles, 98% bound to at least three HLA alleles, 98% bound to at least four HLA alleles and 92% bound to all seven HLA alleles. The overall predictive accuracy was 81% (both positive and negative) [2]. Significant IFNg response was induced by all peptides in at least one human donor as measured by IFNg ELISpot assay. 86% of the peptide-specific IFNg ELISpot responses were completely inhibited by antibody block of HLA-DR, indicating that these peptides are HLA-DR-restricted. Significant peptide-specific proliferation and Th1 cytokine production (IFNg, TNFa and IL-2) in CD4+ T cells from healthy donors were observed in flow cytometry analysis. Immunoinformatics predictions, coupled with in vitro validation, can accelerate the selection of highly conserved T cell epitopes from genome sequence databases. The approach can be used for rapid selection of vaccine candidates for a wide array of emerging infectious diseases and biodefense targets. References: 1. Wullner D, Zhou L, Bramhall E, Kuck A, Goletz TJ, Swanson S, Chirmule N and Jawa V. Considerations for optimization and validation of an in vitro PBMC derived T cell assay for immunogenicity prediction of biotherapeutics. Clinical Immunology. 2010,137, 5-14. 2. De Groot AS, Ardito M, Moise L, Gustafson EA, Spero D, Tejada G and Martin W. Immunogenic Consensus Sequence T helper Epitopes for a Pan-Burkholderia Biodefense Vaccine. Immunome Res. 2011, 7(2). pii: e7

T Cell Epitope Redundancy: Cross-conservation of the TCR face between Pathogens and Self and its Implications for Vaccines and Auto-immunity

T cells are extensively trained on ‘self’ in the thymus and then move to the periphery, where they seek out and destroy infections and regulate immune response to self-antigens. T cell receptors (TCR) on T cells’ surface recognize T cell epitopes, short linear strings of amino acids presented by antigen-presenting cells. Some of these epitopes activate T effectors, while others activate regulatory T cells. It was recently discovered that T cell epitopes that are highly conserved on their TCR face with human genome sequences are often associated with T cells that regulate immune response. These TCR-cross-conserved or ‘redundant epitopes’ are more common in proteins found in pathogens that have co-evolved with humans than in other non-commensal pathogens. Epitope redundancy might be the link between pathogens and autoimmune disease. This article reviews recently published data and addresses epitope redundancy, the “elephant in the room” for vaccine developers and T cell immunologists

Immune Camouflage: Relevance to Vaccine Design and Human Immunology

High strain sequence variability, interference with innate immune mechanisms, and epitope deletion are all examples of strategies that pathogens have evolved to subvert host defenses. To this list we would add another strategy: immune camouflage. Pathogens whose epitope sequences are cross-conserved with multiple human proteins at the TCR-facing residues may be exploiting ignorance and tolerance, which are mechanisms by which mature T cells avoid immune responses to self-antigens. By adopting amino acid configurations that may be recognized by autologous regulatory T cells, pathogens may be actively suppressing protective immunity. Using the new JanusMatrix TCR-homology-mapping tool, we have identified several such \u27camouflaged\u27 tolerizing epitopes that are present in the viral genomes of pathogens such as emerging H7N9 influenza. Thus in addition to the overall low number of T helper epitopes that is present in H7 hemaglutinin (as described previously, see http://dx.doi.org/10.4161/hv.24939), the presence of such tolerizing epitopes in H7N9 could explain why, in recent vaccine trials, whole H7N9-HA was poorly immunogenic and associated with low seroconversion rates (see http://dx.doi.org/10.4161/hv.28135). In this commentary, we provide an overview of the immunoinformatics process leading to the discovery of tolerizing epitopes in pathogen genomic sequences, provide a brief summary of laboratory data that validates the discovery, and point the way forward. Removal of viral, bacterial and parasite tolerizing epitopes may permit researchers to develop more effective vaccines and immunotherapeutics in the future

Universal H1N1 influenza vaccine development

Immune responses to cross-conserved T cell epitopes in novel H1N1 influenza may explain reports of diminished influenza-like illnesses and confirmed infection among older adults, in the absence of cross-reactive humoral immunity, during the 2009 pandemic. These cross-conserved epitopes may prove useful for the development of a universal H1N1 influenza vaccine, therefore, we set out to identify and characterize cross-conserved H1N1 T cell epitopes. An immunoinformatic analysis was conducted using all available pandemic and pre-pandemic HA-H1 and NA-N1 sequences dating back to 1980. Using an approach that balances potential for immunogenicity with conservation, we derived 13 HA and four NA immunogenic consensus sequences (ICS) from a comprehensive analysis of 5 738 HA-H1 and 5 396 NA-N1 sequences. These epitopes were selected because their combined epitope content is representative of greater than 84% of pre-pandemic and pandemic H1N1 influenza strains, their predicted immunogenicity (EpiMatrix) scores were greater than or equal to the 95th percentile of all comparable epitopes, and they were also predicted to be presented by more than four HLA class II archetypal alleles. We confirmed the ability of these peptides to bind in HLA binding assays and to stimulate interferon-γ production in human peripheral blood mononuclear cell cultures. These studies support the selection of the ICS as components of potential group-common H1N1 vaccine candidates and the application of this universal influenza vaccine development approach to other influenza subtypes

iVax: An integrated toolkit for the selection and optimization of antigens and the design of epitope-driven vaccines

Computational vaccine design, also known as computational vaccinology, encompasses epitope mapping, antigen selection and immunogen design using computational tools. The iVAX toolkit is an integrated set of tools that has been in development since 1998 by De Groot and Martin. It comprises a suite of immunoinformatics algorithms for triaging candidate antigens, selecting immunogenic and conserved T cell epitopes, eliminating regulatory T cell epitopes, and optimizing antigens for immunogenicity and protection against disease. iVAX has been applied to vaccine development programs for emerging infectious diseases, cancer antigens and biodefense targets. Several iVAX vaccine design projects have had success in pre-clinical studies in animal models and are progressing toward clinical studies. The toolkit now incorporates a range of immunoinformatics tools for infectious disease and cancer immunotherapy vaccine design. This article will provide a guide to the iVAX approach to computational vaccinology

Epitope Recognition in HLA-DR3 Transgenic Mice Immunized to TSH-R Protein or Peptides

Development of Graves\u27 disease is related to HLA-DR3. The extracellular domain (ECD) of human TSH receptor (hTSH-R) is a crucial antigen in Graves\u27 disease. hTSH-R peptide 37 (amino acids 78–94) is an important immunogenic peptide in DR3 transgenic mice immunized to hTSH-R. This study examined the epitope recognition in DR3 transgenic mice immunized to hTSH-R protein and evaluated the ability of a mutant hTSH-R peptide to attenuate the immunogenicity of hTSH-R peptide 37. DR3 transgenic mice were immunized to recombinant hTSH-R-ECD protein or peptides. A mutant hTSH-R 37 peptide (ISRIYVSIDATLSQLES: 37m), in which DR3 binding motif position 5 was mutated V\u3eA, and position 8 Q\u3eS, was synthesized. 37m should bind to HLA-DR3 but not bind T cell receptors. DR3 transgenic mice were immunized to hTSH-R 37 and 37m. Mice immunized to hTSH-R-ECD protein developed strong anti-hTSH-R antibody, and antisera reacted strongly with hTSH-R peptides 1–5 (20–94), 21 (258–277), 41 (283–297), 36 (376–389), and 31 (399–418). Strikingly, antisera raised to hTSH-R peptide 37 bound to hTSH-R peptides 1–7 (20–112), 10 (132–50), 33 (137–150), 41, 23 (286–305), 24 (301–320), 36, and 31 as well as to hTSH-R-ECD protein. Both antibody titers to hTSH-R 37 and reaction of splenocytes to hTSH-R 37 were significantly reduced in mice immunized to hTSH-R 37 plus 37m, compared with mice immunized to hTSH-R 37 alone. The ability of immunization to a single peptide to induce antibodies that bind hTSH-R-ECD protein, and multiple unrelated peptides, is a unique observation. Immunogenic reaction to hTSH-R peptide 37 was partially suppressed by 37m, and this may contribute to immunotherapy of autoimmune thyroid disease

An Integrated Genomic and Immunoinformatic Approach to \u3cem\u3eH. pylori\u3c/em\u3e Vaccine Design

Background: One useful application of pattern matching algorithms is identification of major histocom-patability complex (MHC) ligands and T-cell epitopes. Peptides that bind to MHC molecules and interact with T cell receptors to stimulate the immune system are critical antigens for protection against infectious pathogens. We describe a genomes-to-vaccine approach to H. pylori vaccine design that takes advantage of immunoinformatics algorithms to rapidly identify T-cell epitope sequences from large genomic datasets. Results: To design a globally relevant vaccine, we used computational methods to identify a core genome comprised of 676 open reading frames (ORFs) from amongst seven genetically and phenotypically diverse H. pylori strains from around the world. Of the 1,241,153 9-mer sequences encoded by these ORFs, 106,791 were identical amongst all seven genomes and 23,654 scored in the top 5% of predicted HLA ligands for at least one of eight archetypal Class II HLA alleles when evaluated by EpiMatrix. To maximize the number of epitopes that can be assessed experimentally, we used a computational algorithm to in-crease epitope density in 20-25 amino acid stretches by assembling potentially immunogenic 9-mers to be identically positioned as they are in the native protein antigen. 1,805 immunogenic consensus sequences (ICS) were generated. 79% of selected ICS epitopes bound to a panel of 6 HLA Class II haplotypes, repre-senting \u3e90% of the global human population. Conclusions: The breadth of H. pylori genome datasets was computationally assessed to rapidly and care-fully determine a core set of genes. Application of immunoinformatics tools to this gene set accurately pre-dicted epitopes with promising properties for T cell-based vaccine development

H7N9 T-cell epitopes that mimic human sequences are less immunogenic and may induce Treg-mediated tolerance

Avian-origin H7N9 influenza is a novel influenza A virus (IAV) that emerged in humans in China in 2013. Using immunoinformatics tools, we identified several H7N9 T cell epitopes with T cell receptor (TCR)-facing residues identical to those of multiple epitopes from human proteins. We hypothesized that host tolerance to these peptides may impair T helper response and contribute to the low titer, weak hemagglutination inhibiting (HI) antibody responses and diminished seroconversion rates that have been observed in human H7N9 infections and vaccine trials. We found that the magnitude of human T effector responses to individual H7N9 peptides was inversely correlated with the peptide\u27s resemblance to self. Furthermore, a promiscuous T cell epitope from the hemagglutinin (HA) protein suppressed responses to other H7N9 peptides when co-administered in vitro. Along with other highly ‘human-like’ peptides from H7N9, this peptide was also shown to expand FoxP3+ regulatory T cells (Tregs). Thus, H7N9 may be camouflaged from effective human immune response by T cell epitope sequences that avert or regulate effector T cell responses through host tolerance