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While conventional in silico immunogenicity risk assessments focus on measuring immunogenicity based on the potential of therapeutic proteins to be processed and presented by a global population-wide set of human leukocyte antigen (HLA) alleles to T cells, future refinements might adjust for HLA allele frequencies in different geographic regions or populations, as well for as individuals in those populations. Adjustment by HLA allele distribution may reveal risk patterns that are specific to population groups or individuals, which current methods that rely on global-population HLA prevalence may obscure. This analysis uses HLA frequency-weighted binding predictions to define immunogenicity risk for global and sub-global populations. A comparison of assessments tuned for North American/European versus Japanese/Asian populations suggests that the potential for anti-therapeutic responses (anti-therapeutic antibodies or ATA) for several commonly prescribed Rheumatoid Arthritis (RA) therapeutic biologics may differ, significantly, between the Caucasian and Japanese populations. This appears to align with reports of differing product-related immunogenicity that is observed in different populations. Further definition of population-level (regional) and individual patient-specific immunogenic risk profiles may enable prescription of the RA therapeutic with the highest probability of success to each patient, depending on their population of origin and/or their individual HLA background. Furthermore, HLA-specific immunogenicity outcomes data are limited, thus there is a need to expand HLA-association studies that examine the relationship between HLA haplotype and ATA in the clinic.

Antibody maturation in the lymphoid follicle produces antibodies with improved binding affinity. This process requires iterative rounds of mutation and B cell expansion, supported by T cells that recognize epitopes presented on the B cell's MHC-II. In this comprehensive antibody repertoire analysis, we find that established regulatory T cell epitopes (Tregitopes) decline in maturing antibody sequences as somatic hypermutation (SHM) increases, but potential T effector epitopes do not decline. A previous analysis of B cell receptor (BCR)-derived HLA-DR epitopes present in memory antibody repertoires from seven healthy human donors revealed a decrease in donor-specific epitope content with SHM. Moreover, T cell epitope depletion was associated with class-switching and long-term secretion of antibody into serum. Significant depletion of high-affinity germline-encoded epitopes in high SHM sequences was also observed, but the predicted phenotype of T cells responding to the BCR-derived epitopes (regulatory vs. effector) was not previously evaluated. In this follow-on study, we screened a different set of four donor repertoires to investigate the dynamics of donor-specific HLA-DR T cell epitopes and three subsets of T cell epitope content: previously validated T cell epitopes recognized by thymus-derived Tregs (Tregitopes), potentially tolerated T cell epitopes, and potential effector T cell epitopes. Our results show that Tregitope content reduction is correlated with SHM, suggesting that Tregitopes are removed during maturation. Moreover, T cell epitopes that are likely to be tolerated or tolerogenic were also removed with SHM progression. In contrast, potential T effector epitope content increased with SHM. Tregitope depletion occurred in multiple V-gene pair combinations and was the most frequent T cell epitope change. Furthermore, Tregitope content in IgA and IgG sequences was lower and had greater negative correlation with SHM than IgM, indicating that Tregitope removal is likely associated with class-switching. Tregitope depletion was also associated with maturation to plasmablasts. In vitro, representative Tregitopes inhibited CD4+ T cell proliferation. Mutations introduced by SHM altered Tregitope HLA-DR binding affinities. The correlation of Tregitope depletion with increasing SHM implies that the activity of thymus-derived Treg cells in immune responses to antibodies is diminished with SHM, maturation, and isotype switching, supporting the generation of anti-idiotype responses.

Zika virus (ZIKV) is a flavivirus primarily transmitted by Aedes species mosquitoes, first discovered in Africa in 1947, that disseminated through Southeast Asia and the Pacific Islands in the 2000s. The first ZIKV infections in the Americas were identified in 2014, and infections exploded through populations in Brazil and other countries in 2015/16. ZIKV infection during pregnancy can cause severe brain and eye defects in offspring, and infection in adults has been associated with higher risks of Guillain-Barré syndrome. We initiated a study to describe the natural history of Zika (the disease) and the immune response to infection, for which some results have been reported. In this paper, we identify ZIKV-specific CD4+ and CD8+ T cell epitopes that induce responses during infection. Two screening approaches were utilized: an untargeted approach with overlapping peptide arrays spanning the entire viral genome, and a targeted approach utilizing peptides predicted to bind human MHC molecules. Immunoinformatic tools were used to identify conserved MHC class I supertype binders and promiscuous class II binding peptide clusters predicted to bind 9 common class II alleles. T cell responses were evaluated in overnight IFN-γ ELISPOT assays. We found that MHC supertype binding predictions outperformed the bulk overlapping peptide approach. Diverse CD4+ T cell responses were observed in most ZIKV-infected participants, while responses to CD8+ T cell epitopes were more limited. Most individuals developed a robust T cell response against epitopes restricted to a single MHC class I supertype and only a single or few CD8+ T cell epitopes overall, suggesting a strong immunodominance phenomenon. Noteworthy is that many epitopes were commonly immunodominant across persons expressing the same class I supertype. Nearly all of the identified epitopes are unique to ZIKV and are not present in Dengue viruses. Collectively, we identified 31 immunogenic peptides restricted by the 6 major class I supertypes and 27 promiscuous class II epitopes. These sequences are highly relevant for design of T cell-targeted ZIKV vaccines and monitoring T cell responses to Zika virus infection and vaccination.

Swine influenza is a highly contagious respiratory viral infection in pigs that is responsible for significant financial losses to pig farmers annually. Current measures to protect herds from infection include: inactivated whole-virus vaccines, subunit vaccines, and alpha replicon-based vaccines. As is true for influenza vaccines for humans, these strategies do not provide broad protection against the diverse strains of influenza A virus (IAV) currently circulating in U.S. swine. Improved approaches to developing swine influenza vaccines are needed. Here, we used immunoinformatics tools to identify class I and II T cell epitopes highly conserved in seven representative strains of IAV in U.S. swine and predicted to bind to Swine Leukocyte Antigen (SLA) alleles prevalent in commercial swine. Epitope-specific interferon-gamma (IFNγ) recall responses to pooled peptides and whole virus were detected in pigs immunized with multi-epitope plasmid DNA vaccines encoding strings of class I and II putative epitopes. In a retrospective analysis of the IFNγ responses to individual peptides compared to predictions specific to the SLA alleles of cohort pigs, we evaluated the predictive performance of PigMatrix and demonstrated its ability to distinguish non-immunogenic from immunogenic peptides and to identify promiscuous class II epitopes. Overall, this study confirms the capacity of PigMatrix to predict immunogenic T cell epitopes and demonstrate its potential for use in the design of epitope-driven vaccines for swine. Additional studies that match the SLA haplotype of animals with the study epitopes will be required to evaluate the degree of immune protection conferred by epitope-driven DNA vaccines in pigs.

Infantile-onset Pompe disease (IOPD) is a glycogen storage disease caused by a deficiency of acid alpha-glucosidase (GAA). Treatment with recombinant human GAA (rhGAA, alglucosidase alfa) enzyme replacement therapy (ERT) significantly improves clinical outcomes; however, many IOPD children treated with rhGAA develop anti-drug antibodies (ADA) that render the therapy ineffective. Antibodies to rhGAA are driven by T cell responses to sequences in rhGAA that differ from the individuals’ native GAA (nGAA). The goal of this study was to develop a tool for p ersonalized im munogenicity risk a ssessment (PIMA) that quantifies T cell epitopes that differ between nGAA and rhGAA using information about an individual’s native GAA gene and their HLA DR haplotype, and to use this information to predict the risk of developing ADA. Four versions of PIMA have been developed. They use EpiMatrix, a computational tool for T cell epitope identification, combined with an HLA-restricted epitope-specific scoring feature (iTEM), to assess ADA risk. One version of PIMA also integrates JanusMatrix, a Treg epitope prediction tool to identify putative immunomodulatory (regulatory) T cell epitopes in self-proteins. Using the JanusMatrix-adjusted version of PIMA in a logistic regression model with data from 48 cross-reactive immunological material (CRIM)-positive IOPD subjects, those with scores greater than 10 were 4-fold more likely to develop ADA (p<0.03) than those that had scores less than 10. We also confirmed the hypothesis that some GAA epitopes are immunomodulatory. Twenty-one epitopes were tested, of which four were determined to have an immunomodulatory effect on T effector response in vitro . The implementation of PIMA V3J on a secure-access website would allow clinicians to input the individual HLA DR haplotype of their IOPD patient and the GAA pathogenic variants associated with each GAA allele to calculate the patient’s relative risk of developing ADA, enhancing clinical decision-making prior to initiating treatment with ERT. A better understanding of immunogenicity risk will allow the implementation of targeted immunomodulatory approaches in ERT-naïve settings, especially in CRIM-positive patients, which may in turn improve the overall clinical outcomes by minimizing the development of ADA. The PIMA approach may also be useful for other types of enzyme or factor replacement therapies.

The hurdles to effective blood stage malaria vaccine design include immune evasion tactics used by the parasite such as redundant invasion pathways and antigen variation among circulating parasite strains. While blood stage malaria vaccine development primarily focuses on eliciting optimal humoral responses capable of blocking erythrocyte invasion, clinically-tested Plasmodium falciparum (Pf) vaccines have not elicited sterile protection, in part due to the dramatically high levels of antibody needed. Recent development efforts with non-redundant, conserved blood stage antigens suggest both high antibody titer and rapid antibody binding kinetics are important efficacy factors. Based on the central role of helper CD4 T cells in development of strong, protective immune responses, we systematically analyzed the class II epitope content in five leading Pf blood stage antigens (RH5, CyRPA, RIPR, AMA1 and EBA175) using in silico , in vitro , and ex vivo methodologies. We employed in silico T cell epitope analysis to enable identification of 67 HLA-restricted class II epitope clusters predicted to bind a panel of nine HLA-DRB1 alleles. We assessed a subset of these for HLA-DRB1 allele binding in vitro , to verify the in silico predictions. All clusters assessed (40 clusters represented by 46 peptides) bound at least two HLA-DR alleles in vitro . The overall epitope prediction to in vitro HLA-DRB1 allele binding accuracy was 71%. Utilizing the set of RH5 class II epitope clusters (10 clusters represented by 12 peptides), we assessed stimulation of T cells collected from HLA-matched RH5 vaccinees using an IFN-γ T cell recall assay. All clusters demonstrated positive recall responses, with the highest responses – by percentage of responders and response magnitude – associated with clusters located in the N-terminal region of RH5. Finally, a statistically significant correlation between in silico epitope predictions and ex vivo IFN-γ recall response was found when accounting for HLA-DR matches between the epitope predictions and donor HLA phenotypes. This is the first comprehensive analysis of class II epitope content in RH5, CyRPA, RIPR, AMA1 and EBA175 accompanied by in vitro HLA binding validation for all five proteins and ex vivo T cell response confirmation for RH5.

Immunization with radiation-attenuated sporozoites (RAS) has been shown to protect against malaria infection, primarily through CD8 T cell responses, but protection is limited based on parasite strain. Therefore, while CD8 T cells are an ideal effector population target for liver stage malaria vaccine development strategies, such strategies must incorporate conserved epitopes that cover a large range of class I human leukocyte antigen (HLA) supertypes to elicit cross-strain immunity across the target population. This approach requires identifying and characterizing a wide range of CD8 T cell epitopes for incorporation into a vaccine such that coverage across a large range of class I HLA alleles is attained. Accordingly, we devised an experimental framework to identify CD8 T cell epitopes from novel and minimally characterized antigens found at the pre-erythrocytic stage of parasite development. Through in silico analysis we selected conserved P. falciparum proteins, using P. vivax orthologues to establish stringent conservation parameters, predicted to have a high number of T cell epitopes across a set of six class I HLA alleles representative of major supertypes. Using the decision framework, five proteins were selected based on the density and number of predicted epitopes. Selected epitopes were synthesized as peptides and evaluated for binding to the class I HLA alleles in vitro to verify in silico binding predictions, and subsequently for stimulation of human T cells using the Modular IMmune In-vitro Construct (MIMIC ® ) technology to verify immunogenicity. By combining the in silico tools with the ex vivo high throughput MIMIC platform, we identified 15 novel CD8 T cell epitopes capable of stimulating an immune response in alleles across the class I HLA panel. We recommend these epitopes should be evaluated in appropriate in vivo humanized immune system models to determine their protective efficacy for potential inclusion in future vaccines.

An effective malaria vaccine must prevent disease in a range of populations living in regions with vastly different transmission rates and protect against genetically-diverse Plasmodium falciparum (Pf) strains. The protective efficacy afforded by the currently licensed malaria vaccine, Mosquirix™, promotes strong humoral responses to Pf circumsporozoite protein (CSP) 3D7 but protection is limited in duration and by strain variation. Helper CD4 T cells are central to development of protective immune responses, playing roles in B cell activation and maturation processes, cytokine production, and stimulation of effector T cells. Therefore, we took advantage of recent in silico modeling advances to predict and analyze human leukocyte antigen (HLA)-restricted class II epitopes from PfCSP – across the entire PfCSP 3D7 sequence as well as in 539 PfCSP sequence variants – with the goal of improving PfCSP-based malaria vaccines. Specifically, we developed a systematic workflow to identify peptide sequences capable of binding HLA-DR in a context relevant to achieving broad human population coverage utilizing cognate T cell help and with limited T regulatory cell activation triggers. Through this workflow, we identified seven predicted class II epitope clusters in the N- and C-terminal regions of PfCSP 3D7 and an additional eight clusters through comparative analysis of 539 PfCSP sequence variants. A subset of these predicted class II epitope clusters was synthesized as peptides and assessed for HLA-DR binding in vitro . Further, we characterized the functional capacity of these peptides to prime and activate human peripheral blood mononuclear cells (PBMCs), by monitoring cytokine response profiles using MIMIC ® technology (Modular IMmune In vitro Construct). Utilizing this decision framework, we found sufficient differential cellular activation and cytokine profiles among HLA-DR-matched PBMC donors to downselect class II epitope clusters for inclusion in a vaccine targeting PfCSP. Importantly, the downselected clusters are not highly conserved across PfCSP variants but rather, they overlap a hypervariable region (TH2R) in the C-terminus of the protein. We recommend assessing these class II epitope clusters within the context of a PfCSP vaccine, employing a test system capable of measuring immunogenicity across a broad set of HLA-DR alleles.

•Approach used here can be expanded to generate flu vaccine with universal relevance.•Identified T cell epitopes induced heterologous protection in HLA transgenic mice.•Conserved flu putative immunogenic sequences were identified via immunoinformatics.•Predicted HLA-A2 and pan-DR-restricted epitopes immunogenic in HLA-transgenic mice.•Predicted CD4 and CD8 epitopes induced effector function in human T cells. Influenza world-wide causes significant morbidity and mortality annually, and more severe pandemics when novel strains evolve to which humans are immunologically naïve. Because of the high viral mutation rate, new vaccines must be generated based on the prevalence of circulating strains every year. New approaches to induce more broadly protective immunity are urgently needed. Previous research has demonstrated that influenza-specific T cells can provide broadly heterotypic protective immunity in both mice and humans, supporting the rationale for developing a T cell-targeted universal influenza vaccine. We used state-of-the art immunoinformatic tools to identify putative pan-HLA-DR and HLA-A2 supertype-restricted T cell epitopes highly conserved among > 50 widely diverse influenza A strains (representing hemagglutinin types 1, 2, 3, 5, 7 and 9). We found influenza peptides that are highly conserved across influenza subtypes that were also predicted to be class I epitopes restricted by HLA-A2. These peptides were found to be immunoreactive in HLA-A2 positive but not HLA-A2 negative individuals. Class II-restricted T cell epitopes that were highly conserved across influenza subtypes were identified. Human CD4+ T cells were reactive with these conserved CD4 epitopes, and epitope expanded T cells were responsive to both H1N1 and H3N2 viruses. Dendritic cell vaccines pulsed with conserved epitopes and DNA vaccines encoding these epitopes were developed and tested in HLA transgenic mice. These vaccines were highly immunogenic, and more importantly, vaccine-induced immunity was protective against both H1N1 and H3N2 influenza challenges. These results demonstrate proof-of-principle that conserved T cell epitopes expressed by widely diverse influenza strains can induce broadly protective, heterotypic influenza immunity, providing strong support for further development of universally relevant multi-epitope T cell-targeting influenza vaccines.

Background Predicting vaccine efficacy against emerging pathogen strains is a significant problem in human and animal vaccine design. T‐cell epitope cross‐conservation may play an important role in cross‐strain vaccine efficacy. While influenza A virus (IAV) hemagglutination inhibition (HI) antibody titers are widely used to predict protective efficacy of 1 IAV vaccine against new strains, no similar correlate of protection has been identified for T‐cell epitopes. Objective We developed a computational method (EpiCC) that facilitates pairwise comparison of protein sequences based on an immunological property—T‐cell epitope content—rather than sequence identity, and evaluated its ability to classify swine IAV strain relatedness to estimate cross‐protective potential of a vaccine strain for circulating viruses. Methods T‐cell epitope relatedness scores were assessed for 23 IAV HA sequences representing the major H1 swine IAV phylo‐clusters circulating in North American swine and HA sequences in a commercial inactivated vaccine (FluSure XP®). Scores were compared to experimental data from previous efficacy studies. Results Higher EpiCC scores were associated with greater protection by the vaccine against strains for 23 field IAV strain vaccine comparisons. A threshold for EpiCC relatedness associated with full or partial protection in the absence of cross‐reactive HI antibodies was identified. EpiCC scores for field strains for which FluSure protective efficacy is not yet available were also calculated. Conclusion EpiCC thresholds can be evaluated for predictive accuracy of protection in future efficacy studies. EpiCC may also complement HI cross‐reactivity and phylogeny for selection of influenza strains in vaccine development.