Integrated assessment of predicted MHC binding and cross-conservation with self reveals patterns of viral camouflage.

BACKGROUND: Immune recognition of foreign proteins by T cells hinges on the formation of a ternary complex sandwiching a constituent peptide of the protein between a major histocompatibility complex (MHC) molecule and a T cell receptor (TCR). Viruses have evolved means of "camouflaging" themselves, avoiding immune recognition by reducing the MHC and/or TCR binding of their constituent peptides. Computer-driven T cell epitope mapping tools have been used to evaluate the degree to which particular viruses have used this means of avoiding immune response, but most such analyses focus on MHC-facing 'agretopes'. Here we set out a new means of evaluating the TCR faces of viral peptides in addition to their agretopes, integrating evaluations of both sides of the ternary complex in a single analysis. METHODS: This paper develops what we call the Janus Immunogenicity Score (JIS), bringing together a well-established method for predicting MHC binding, with a novel assessment of the potential for TCR binding based on similarity with self. Intuitively, both good MHC binding and poor self-similarity are required for high immunogenicity (i.e., a robust T effector response). RESULTS: Focusing on the class II antigen-processing pathway, we show that the JIS of T effector epitopes and null or regulatory epitopes deposited in a large database of epitopes (Immune Epitope Database) are significantly different. We then show that different types of viruses display significantly different patterns of scores over their constituent peptides, with viruses causing chronic infection (Epstein-Barr and cytomegalovirus) strongly shifted to lower scores relative to those causing acute infection (Ebola and Marburg). Similarly we find distinct patterns among influenza proteins in H1N1 (a strain against which human populations rapidly developed immunity) and H5N1 and H7N9 (highly pathogenic avian flu strains, with significantly greater case mortality rates). CONCLUSION: The Janus Immunogenicity Score, which integrates MHC binding and TCR cross-reactivity, provides a new tool for studying immunogenicity of pathogens and may improve the selection and optimization of antigenic elements for vaccine design.

From bench to bedside via bytes: Multi-omic immunoprofiling and integration using machine learning and network approaches.

A significant surge in research endeavors leverages the vast potential of high-throughput omic technology platforms for broad profiling of biological responses to vaccines and cutting-edge immunotherapies and stem-cell therapies under development. These profiles capture different aspects of core regulatory and functional processes at different scales of resolution from molecular and cellular to organismal. Systems approaches capture the complex and intricate interplay between these layers and scales. Here, we summarize experimental data modalities, for characterizing the genome, epigenome, transcriptome, proteome, metabolome, and antibody-ome, that enable us to generate large-scale immune profiles. We also discuss machine learning and network approaches that are commonly used to analyze and integrate these modalities, to gain insights into correlates and mechanisms of natural and vaccine-mediated immunity as well as therapy-induced immunomodulation.

H1N1 G4 swine influenza T cell epitope analysis in swine and human vaccines and circulating strains uncovers potential risk to swine and humans.

BACKGROUND: Pandemic influenza viruses may emerge from animal reservoirs and spread among humans in the absence of cross-reactive antibodies in the human population. Immune response to highly conserved T cell epitopes in vaccines may still reduce morbidity and limit the spread of the new virus even when cross-protective antibody responses are lacking. METHODS: We used an established epitope content prediction and comparison tool, Epitope Content Comparison (EpiCC), to assess the potential for emergent H1N1 G4 swine influenza A virus (G4) to impact swine and human populations. We identified and computed the total cross-conserved T cell epitope content in HA sequences of human seasonal and experimental influenza vaccines, swine influenza vaccines from Europe and the United States (US) against G4. RESULTS: The overall T cell epitope content of US commercial swine vaccines was poorly conserved with G4, with an average T cell epitope coverage of 35.7%. EpiCC scores for the comparison between current human influenza vaccines and circulating human influenza strains were also very low. In contrast, the T cell epitope coverage of a recent European swine influenza vaccine (HL03) was 65.8% against G4. CONCLUSIONS: Poor T cell epitope cross-conservation between emergent G4 and swine and human influenza vaccines in the US may enable G4 to spread in swine and spillover to human populations in the absence of protective antibody response. One European influenza vaccine, HL03, may protect against emergent G4. This study illustrates the use of the EpiCC tool for prospective assessment of existing vaccine strains against emergent viruses in swine and human populations.

A SARS-CoV-2 NSP7 homolog of a Treg epitope suppresses CD4+ and CD8+ T cell memory responses.

Pathogens escape host defenses by T-cell epitope mutation or deletion (immune escape) and by simulating the appearance of human T cell epitopes (immune camouflage). We identified a highly conserved, human-like T cell epitope in non-structural protein 7 (NSP7) of SARS-CoV-2, RNA-dependent RNA polymerase (RdRp) hetero-tetramer complex. Remarkably, this T cell epitope has significant homology to a T regulatory cell epitope (Tregitope) previously identified in the Fc region of human immunoglobulin G (IgG) (Tregitope 289). We hypothesized that the SARS-CoV-2 NSP7 epitope (NSP7-289) may induce suppressive responses by engaging and activating pre-existing regulatory T cells. We therefore compared NSP7-289 and IgG Tregitopes (289 and 289z, a shorter version of 289 that isolates the shared NSP7 epitope) in vitro. Tregitope peptides 289, 289z and NSP7-289 bound to multiple HLA-DRB1 alleles in vitro and suppressed CD4+ and CD8+ T cell memory responses. Identification and in vitro validation of SARS-CoV-2 NSP7-289 provides further evidence of immune camouflage and suggests that pathogens can use human-like epitopes to evade immune response and potentially enhance host tolerance. Further exploration of the role of cross-conserved Tregs in human immune responses to pathogens such as SARS-CoV-2 is warranted.

Immune-engineered H7N9 influenza hemagglutinin improves protection against viral influenza virus challenge.

The influenza hemagglutinin (HA) isolated from avian H7N9 influenza virus strains elicit weak immune responses. This low immunogenicity may be due to a regulatory T cell (Treg)-stimulating epitopes in HA from the H7N9 isolate A/Anhui/1/2013 (Anh/13). In this report, this Treg stimulating sequence was removed from the wild-type (WT) H7 HA amino acid sequence and replaced with a conserved CD4 + T cell stimulating sequences from human seasonal H3N2 strains and designed OPT1 H7 HA. The effectiveness of this optimized H7 HA protein was determined using a humanized mouse (HLA-DR3) expressing the human leukocyte antigen (HLA) DR3 allele. HLA-DR3 mice were pre-immunized by infecting with H3N2 influenza virus, A/Hong Kong/4108/2014 and then vaccinated intramuscularly with either the WT H7 HA from Anh/13 or the OPT1 H7 HA antigen without adjuvant. The OPT1 H7 HA vaccination group elicited higher H7 HA-specific IgG titers that resulted in a lower mortality, weight loss, and lung viral titer following lethal challenge with the H7N9 Anh/13 influenza virus compared to WT-vaccinated mice. Overall, T-cell epitope-engineered vaccines can improve the immunogenicity of H7 HA antigens resulting in enhanced survival and lower morbidity against H7N9 influenza virus challenge.

New Immunoinformatics Tools for Swine: Designing Epitope-Driven Vaccines, Predicting Vaccine Efficacy, and Making Vaccines on Demand.

Novel computational tools for swine vaccine development can expand the range of immunization approaches available to prevent economically devastating swine diseases and spillover events between pigs and humans. PigMatrix and EpiCC are two new tools for swine T cell epitope identification and vaccine efficacy analysis that have been integrated into an existing computational vaccine design platform named iVAX. The iVAX platform is already in use for the development of human vaccines, thus integration of these tools into iVAX improves and expands the utility of the platform overall by making previously validated immunoinformatics tools, developed for humans, available for use in the design and analysis of swine vaccines. PigMatrix predicts T cell epitopes for a broad array of class I and class II swine leukocyte antigen (SLA) using matrices that enable the scoring of sequences for likelihood of binding to SLA. PigMatrix facilitates the prospective selection of T cell epitopes from the sequences of swine pathogens for vaccines and permits the comparison of those predicted epitopes with "self" (the swine proteome) and with sequences from other strains. Use of PigMatrix with additional tools in the iVAX toolkit also enables the computational design of vaccines in silico, for testing in vivo. EpiCC uses PigMatrix to analyze existing or proposed vaccines for their potential to protect, based on a comparison between T cell epitopes in the vaccine and circulating strains of the same pathogen. Performing an analysis of T cell epitope relatedness analysis using EpiCC may facilitate vaccine selection when a novel strain emerges in a herd and also permits analysis of evolutionary drift as a means of immune escape. This review of novel computational immunology tools for swine describes the application of PigMatrix and EpiCC in case studies, such as the design of cross-conserved T cell epitopes for swine influenza vaccine or for African Swine Fever. We also describe the application of EpiCC for determination of the best vaccine strains to use against circulating viral variants of swine influenza, swine rotavirus, and porcine circovirus type 2. The availability of these computational tools accelerates infectious disease research for swine and enable swine vaccine developers to strategically advance their vaccines to market.

The immune vulnerability landscape of the 2019 Novel Coronavirus, SARS-CoV-2.

The outbreak of the 2019 Novel Coronavirus (SARS-CoV-2) rapidly spread from Wuhan, China to more than 150 countries, areas, or territories, causing staggering numbers of infections and deaths. In this study, bioinformatics analyses were performed on 5,568 complete genomes of SARS-CoV-2 virus to predict the T cell and B cell immunogenic epitopes of all viral proteins, which formed a systematic immune vulnerability landscape of SARS-CoV-2. The immune vulnerability and genetic variation profiles of SARS-CoV were compared with those of SARS-CoV and MERS-CoV. In addition, a web portal was developed to broadly share the data and results as a resource for the research community. Using this resource, we showed that genetic variations in SARS-CoV-2 are associated with loss of B cell immunogenicity, an increase in CD4+ T cell immunogenicity, and a minimum loss in CD8+ T cell immunogenicity, indicating the existence of a curious correlation between SARS-CoV-2 genetic evolutions and the immunity pressure from the host. Overall, we present an immunological resource for SARS-CoV-2 that could promote both therapeutic/vaccine development and mechanistic research.

Prediction of immunogenicity: in silico paradigms, ex vivo and in vivo correlates.

Immunogenicity can be a major obstacle to successful protein drug therapy. Antidrug antibodies may neutralize therapeutic function, influence pharmacokinetics and, in some cases, lead to severe adverse effects. These effects depend on factors including dose, regimen, delivery route and contaminants, among others. Importantly, immunogenicity is a consideration that is better addressed during preclinical development before complications arise in clinical trials or following licensure. This article will address the development and application of computational tools for immunogenicity assessment of protein therapeutics, and validation of those predictions using peripheral blood from exposed subjects or alternative in vivo methods.

Multi-antigen Vaccination With Simultaneous Engagement of the OX40 Receptor Delays Malignant Mesothelioma Growth and Increases Survival in Animal Models.

Malignant Mesothelioma (MM) is a rare and highly aggressive cancer that develops from mesothelial cells lining the pleura and other internal cavities, and is often associated with asbestos exposure. To date, no effective treatments have been made available for this pathology. Herein, we propose a novel immunotherapeutic approach based on a unique vaccine targeting a series of antigens that we found expressed in different MM tumors, but largely undetectable in normal tissues. This vaccine, that we term p-Tvax, is comprised of a series of immunogenic peptides presented by both MHC-I and -II to generate robust immune responses. The peptides were designed using in silico algorithms that discriminate between highly immunogenic T cell epitopes and other harmful epitopes, such as suppressive regulatory T cell epitopes and autoimmune epitopes. Vaccination of mice with p-Tvax led to antigen-specific immune responses that involved both CD8+ and CD4+ T cells, which exhibited cytolytic activity against MM cells in vitro. In mice carrying MM tumors, p-Tvax increased tumor infiltration of CD4+ T cells. Moreover, combining p-Tvax with an OX40 agonist led to decreased tumor growth and increased survival. Mice treated with this combination immunotherapy displayed higher numbers of tumor-infiltrating CD8+ and CD4+ T cells and reduced T regulatory cells in tumors. Collectively, these data suggest that the combination of p-Tvax with an OX40 agonist could be an effective strategy for MM treatment.

Preclinical development of HIvax: Human survivin highly immunogenic vaccines.

Our previous work involved the development of a recombinant fowlpox virus encoding survivin (FP-surv) vaccine that was evaluated for efficacy in mesothelioma mouse models. Results showed that FP-surv vaccination generated significant immune responses, which led to delayed tumor growth and improved animal survival. We have extended those previous findings in the current study, which involves the pre-clinical development of an optimized version of FP-surv designed for human immunization (HIvax). Survivin-derived peptides for the most common haplotypes in the human population were identified and their immunogenicity confirmed in co-culture experiments using dendritic cells and T cells isolated from healthy donors. Peptides confirmed to induce CD8(+) and CD4(+) T cells activation in humans were then included in 2 transgenes optimized for presentation of processed peptides on MHC-I (HIvax1) and MHC-II (HIvax2). Fowlpox vectors expressing the HIvax transgenes were then generated and their efficacy was evaluated with subsequent co-culture experiments to measure interferon-γ and granzyme B secretion. In these experiments, both antigen specific CD4(+) and CD8(+) T cells were activated by HIvax vaccines with resultant cytotoxic activity against survivin-overexpressing mesothelioma cancer cells. These results provide a rationale for clinical testing of HIvax1 and HIvax2 vaccines in patients with survivin-expressing cancers.

Cross-conservation of T-cell epitopes: now even more relevant to (H7N9) influenza vaccine design.

A novel avian-origin H7N9 influenza strain emerged in China in April 2013. Since its re-emergence in October-November 2013, the number of reported cases has accelerated; more than 220 laboratory-confirmed cases and 112 deaths (case fatality rate of 20-30%) have been reported. The resurgence of H7N9 has re-emphasized the importance of making faster and more effective influenza vaccines than those that are currently available. Recombinant H7 hemagglutinin (H7-HA) vaccines have been produced, addressing the first problem. Unfortunately, these recombinant subunit vaccine products appear to have failed to address the second problem, influenza vaccine efficacy. Reported unadjuvanted H7N9 vaccine seroconversion rates were between 6% and 16%, nearly 10-fold lower than rates for unadjuvanted vaccine seroconversion to standard H1N1 monovalent (recombinant) vaccine (89% to pandemic H1N1). Could this state of affairs have been predicted? As it turns out, yes, and it was. In that previous analysis of available H7-HA sequences, we found fewer T-cell epitopes per protein than expected, and predicted that H7-HA-based vaccines would be much less antigenic than recent seasonal vaccines. Novel approaches to developing a more immunogenic HA were offered for consideration at the time, and now, as the low immunogenicity of H7N9 vaccines appears to indicate, they appear to be even more relevant. More effective H7N9 influenza vaccines can be produced, provided that the role of T-cell epitopes is carefully considered, and accumulated knowledge about the importance of cross-conserved epitopes between viral subtypes is applied to the design of those vaccines.

Immune camouflage: relevance to vaccines 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 'camouflaged' 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.

T cell epitope: friend or foe? Immunogenicity of biologics in context.

Like vaccines, biologic proteins can be very immunogenic for reasons including route of administration, dose frequency and the underlying antigenicity of the therapeutic protein. Because the impact of immunogenicity can be quite severe, regulatory agencies are developing risk-based guidelines for immunogenicity screening. T cell epitopes are at the root of the immunogenicity issue. Through their presentation to T cells, they activate the process of anti-drug antibody development. Preclinical screening for T cell epitopes can be performed in silico, followed by in vitro and in vivo validation. Importantly, screening for immunogenicity is complicated by the discovery of regulatory T cell epitopes, which suggests that immunogenicity testing must now take regulatory T cells into consideration. In this review, we address the application of computational tools for preclinical immunogenicity assessment, the implication of the discovery of regulatory T cell epitopes, and experimental validation of those assessments.