Diversity and evolution of computationally predicted T cell epitopes against human respiratory syncytial virus.

Human respiratory syncytial virus (RSV) is a major cause of lower respiratory infection. Despite more than 60 years of research, there is no licensed vaccine. While B cell response is a major focus for vaccine design, the T cell epitope profile of RSV is also important for vaccine development. Here, we computationally predicted putative T cell epitopes in the Fusion protein (F) and Glycoprotein (G) of RSV wild circulating strains by predicting Major Histocompatibility Complex (MHC) class I and class II binding affinity. We limited our inferences to conserved epitopes in both F and G proteins that have been experimentally validated. We applied multidimensional scaling (MDS) to construct T cell epitope landscapes to investigate the diversity and evolution of T cell profiles across different RSV strains. We find the RSV strains are clustered into three RSV-A groups and two RSV-B groups on this T epitope landscape. These clusters represent divergent RSV strains with potentially different immunogenic profiles. In addition, our results show a greater proportion of F protein T cell epitope content conservation among recent epidemic strains, whereas the G protein T cell epitope content was decreased. Importantly, our results suggest that RSV-A and RSV-B have different patterns of epitope drift and replacement and that RSV-B vaccines may need more frequent updates. Our study provides a novel framework to study RSV T cell epitope evolution. Understanding the patterns of T cell epitope conservation and change may be valuable for vaccine design and assessment.

Self-Replicating RNAs Drive Protective Anti-tumor T Cell Responses to Neoantigen Vaccine Targets in a Combinatorial Approach.

Historically poor clinical results of tumor vaccines have been attributed to weakly immunogenic antigen targets, limited specificity, and vaccine platforms that fail to induce high-quality polyfunctional T cells, central to mediating cellular immunity. We show here that the combination of antigen selection, construct design, and a robust vaccine platform based on the Synthetically Modified Alpha Replicon RNA Technology (SMARRT), a self-replicating RNA, leads to control of tumor growth in mice. Therapeutic immunization with SMARRT replicon-based vaccines expressing tumor-specific neoantigens or tumor-associated antigen were able to generate polyfunctional CD4+ and CD8+ T cell responses in mice. Additionally, checkpoint inhibitors, or co-administration of cytokine also expressed from the SMARRT platform, synergized to enhance responses further. Lastly, SMARRT-based immunization of non-human primates was able to elicit high-quality T cell responses, demonstrating translatability and clinical feasibility of synthetic replicon technology for therapeutic oncology vaccines.

Coxiella burnetii Epitope-Specific T-Cell Responses in Patients with Chronic Q Fever.

Infection with Coxiella burnetii, the causative agent of Q fever, can result in life-threatening persistent infection. Reactogenicity hinders worldwide implementation of the only licensed human Q fever vaccine. We previously demonstrated long-lived immunoreactivity in individuals with past symptomatic and asymptomatic Coxiella infection (convalescents) to promiscuous HLA class II C. burnetii epitopes, providing the basis for a novel T-cell targeted subunit vaccine. In this study, we investigated in a cohort of 22 individuals treated for persistent infection (chronic Q fever) whether they recognize the same set of epitopes or distinct epitopes that could be candidates for a therapeutic vaccine or aid in the diagnosis of persistent infection. In cultured enzyme-linked immunosorbent spot (ELISpot) assays, individuals with chronic Q fever showed strong class II epitope-specific responses that were largely overlapping with the peptide repertoire identified previously for convalescents. Five additional peptides were recognized more frequently by chronic subjects, but there was no combination of epitopes uniquely recognized by or nonreactive in subjects with chronic Q fever. Consistent with more recent/prolonged exposure, we found, however, stronger ex vivo responses by direct ELISpot to both whole-cell C. burnetii and individual peptides in chronic patients than in convalescents. In conclusion, we have validated and expanded a previously published set of candidate epitopes for a novel T-cell targeted subunit Q fever vaccine in treated patients with chronic Q fever and demonstrated that they successfully mounted a T-cell response comparable to that of convalescents. Finally, we demonstrated that individuals treated for chronic Q fever mount a broader ex vivo response to class II epitopes than convalescents, which could be explored for diagnostic purposes.

Development and validation of an epitope prediction tool for swine (PigMatrix) based on the pocket profile method.

BACKGROUND: T cell epitope prediction tools and associated vaccine design algorithms have accelerated the development of vaccines for humans. Predictive tools for swine and other food animals are not as well developed, primarily because the data required to develop the tools are lacking. Here, we overcome a lack of T cell epitope data to construct swine epitope predictors by systematically leveraging available human information. Applying the "pocket profile method", we use sequence and structural similarities in the binding pockets of human and swine major histocompatibility complex proteins to infer Swine Leukocyte Antigen (SLA) peptide binding preferences. We developed epitope-prediction matrices (PigMatrices), for three SLA class I alleles (SLA-1*0401, 2*0401 and 3*0401) and one class II allele (SLA-DRB1*0201), based on the binding preferences of the best-matched Human Leukocyte Antigen (HLA) pocket for each SLA pocket. The contact residues involved in the binding pockets were defined for class I based on crystal structures of either SLA (SLA-specific contacts, Ssc) or HLA supertype alleles (HLA contacts, Hc); for class II, only Hc was possible. Different substitution matrices were evaluated (PAM and BLOSUM) for scoring pocket similarity and identifying the best human match. The accuracy of the PigMatrices was compared to available online swine epitope prediction tools such as PickPocket and NetMHCpan. RESULTS: PigMatrices that used Ssc to define the pocket sequences and PAM30 to score pocket similarity demonstrated the best predictive performance and were able to accurately separate binders from random peptides. For SLA-1*0401 and 2*0401, PigMatrix achieved area under the receiver operating characteristic curves (AUC) of 0.78 and 0.73, respectively, which were equivalent or better than PickPocket (0.76 and 0.54) and NetMHCpan version 2.4 (0.41 and 0.51) and version 2.8 (0.72 and 0.71). In addition, we developed the first predictive SLA class II matrix, obtaining an AUC of 0.73 for existing SLA-DRB1*0201 epitopes. Notably, PigMatrix achieved this level of predictive power without training on SLA binding data. CONCLUSION: Overall, the pocket profile method combined with binding preferences from HLA binding data shows significant promise for developing T cell epitope prediction tools for pigs. When combined with existing vaccine design algorithms, PigMatrix will be useful for developing genome-derived vaccines for a range of pig pathogens for which no effective vaccines currently exist (e.g. porcine reproductive and respiratory syndrome, influenza and porcine epidemic diarrhea).

C3d adjuvant effects are mediated through the activation of C3d-specific autoreactive T cells.

Complement fragment C3d covalently attached to antigens enhances immune responses, particularly for antigens lacking T-cell epitopes. Enhancement has been attributed to receptor cross-linking between complement receptor CR2 (CD21) and polysaccharide antigen to surface IgM on naïve B cells. Paradoxically, C3d has still been shown to increase immune responses in CD21 knockout mice, suggesting that an auxiliary activation pathway exists. In prior studies, we demonstrated the CD21-independent C3d adjuvant effect might be due to T-cell recognition of C3d T-helper epitopes processed and presented by major histocompatibility complex class II on the B-cell surface. C3d peptide sequences containing concentrated clusters of putative human C3 T-cell epitopes were identified using the epitope-mapping algorithm, EpiMatrix. These peptide sequences were synthesized and shown in vitro to bind multiple human leukocyte antigen (HLA)-DR alleles with high affinity, and induce interferon-γ responses in healthy donor peripheral blood mononuclear cells. In the present studies, we establish further correlations between HLA binding and HLA-specific lymphocyte reactions with select epitope clusters. In addition, we show that the T-cell phenotype of C3d-specific reactive T cells is CD4(+)CD45RO(+) memory T cells. Finally, mutation of a single T-cell epitope residing within the P28 peptide segment of C3d resulted in significantly diminished adjuvant activity in BALB/c mice. Collectively, these studies support the hypothesis that the paradoxical enhancement of immune responses by C3d in the absence of CD21 is due to internalization and processing of C3d into peptides that activate autoreactive CD4(+) T-helper cells in the context of HLA class II.

CHOPPI: a web tool for the analysis of immunogenicity risk from host cell proteins in CHO-based protein production.

Despite high quality standards and continual process improvements in manufacturing, host cell protein (HCP) process impurities remain a substantial risk for biological products. Even at low levels, residual HCPs can induce a detrimental immune response compromising the safety and efficacy of a biologic. Consequently, advanced-stage clinical trials have been cancelled due to the identification of antibodies against HCPs. To enable earlier and rapid assessment of the risks in Chinese Hamster Ovary (CHO)-based protein production of residual CHO protein impurities (CHOPs), we have developed a web tool called CHOPPI, for CHO Protein Predicted Immunogenicity. CHOPPI integrates information regarding the possible presence of CHOPs (expression and secretion) with characterizations of their immunogenicity (T cell epitope count and density, and relative conservation with human counterparts). CHOPPI can generate a report for a specified CHO protein (e.g., identified from proteomics or immunoassays) or characterize an entire specified subset of the CHO genome (e.g., filtered based on confidence in transcription and similarity to human proteins). The ability to analyze potential CHOPs at a genomic scale provides a baseline to evaluate relative risk. We show here that CHOPPI can identify clear differences in immunogenicity risk among previously validated CHOPs, as well as identify additional "risky" CHO proteins that may be expressed during production and induce a detrimental immune response upon delivery. We conclude that CHOPPI is a powerful tool that provides a valuable computational complement to existing experimental approaches for CHOP risk assessment and can focus experimental efforts in the most important directions. Biotechnol. Bioeng. 2014;111: 2170-2182. © 2014 Wiley Periodicals, Inc.

In vitro and in vivo studies of IgG-derived Treg epitopes (Tregitopes): a promising new tool for tolerance induction and treatment of autoimmunity.

Tregitopes are regulatory T cell epitopes derived from immunoglobulin G (IgG) that stimulate CD25(+) FoxP3(+) T cells to expand. In conjunction with these Tregs, Tregitopes can prevent, treat, and even cure autoimmune disease in mouse models, suppress allo-specific responses in murine transplant models, inhibit CD8(+) T cell responses to recombinant adeno-associated virus (AAV) gene transfer vectors, and induce adaptive Tregs in DO11.10 mice. In this review of recent Tregitope studies, we summarize their effects in vitro and describe recent comparisons between intravenous IgG (IVIG) and Tregitopes in standard in vivo immune tolerance models. Further investigations of the mechanism of action of Tregitopes in the preclinical models described here will lead to clinical trials where Tregitopes may have the potential to alter the treatment of autoimmune disease, transplantation, and allergy, and to improve the efficiency of gene and protein replacement therapies.

Identification of immunodominant T cell epitopes induced by natural Zika virus infection.

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.

Effect of HLA DR epitope de-immunization of Factor VIII in vitro and in vivo.

T cell-dependent development of anti-Factor VIII (FVIII) antibodies that neutralize FVIII activity is a major obstacle to replacement therapy in hemophilia A. To create a less immunogenic therapeutic protein, recombinant FVIII can be modified to reduce HLA binding of epitopes based on predicted anchoring residues. Here, we used immunoinformatic tools to identify C2 domain HLA DR epitopes and predict site-specific mutations that reduce immunogenicity. Epitope peptides corresponding to original and modified sequences were validated in HLA binding assays and in immunizations of hemophilic E16 mice, DR3 and DR4 mice and DR3×E16 mice. Consistent with immunoinformatic predictions, original epitopes are immunogenic. Immunization with selected modified sequences lowered immunogenicity for particular peptides and revealed residual immunogenicity of incompletely de-immunized modified peptides. The stepwise approach to reduce protein immunogenicity by epitope modification illustrated here is being used to design and produce a functional full-length modified FVIII for clinical use.

Novel H7N9 influenza immunogen design enhances mobilization of seasonal influenza T cell memory in H3N2 pre-immune mice.

Strategies that improve influenza vaccine immunogenicity are critical for the development of vaccines for pandemic preparedness. Hemagglutinin (HA)-specific CD4+ T cell epitopes support protective B cell responses against seasonal influenza. However, in the case of avian H7N9, which poses a pandemic threat, HA elicits only weak neutralizing antibody responses in infection and vaccination without adjuvant. We hypothesized that an immune-engineered H7N9 HA incorporating a broadly reactive H3N2 HA-specific memory CD4+ T cell epitope that replaces a regulatory T cell-inducing epitope at the corresponding position in H7N9 HA could harness preexisting influenza T cell immunity to increase CD4+ T cells that are needed for protective antibody development. We designed and produced a virus-like particle (VLP) vaccine that carries the epitope augmented H7N9 HA (OPT1) and immunized HLA-DR3 transgenic mice with established H3N2 immunity. OPT1-VLPs stimulated higher stem cell, central, and effector memory CD4+ T cell levels over wild type VLP immunization. In addition, activated, IL-21-producing follicular helper T cell frequencies were enhanced. This novel immunogen design strategy illustrates that site-specific modifications aimed to augment T cell epitope content enhance CD4+ T cell responses among critical subpopulations capable of aiding protective immune responses upon antigen re-encounter and that mobilization of immune memory can be used to overcome the poor immunogenicity of avian influenza viruses.

Evaluation of a Human T Cell-Targeted Multi-Epitope Vaccine for Q Fever in Animal Models of Coxiella burnetii Immunity.

T cell-mediated immunity plays a central role in the control and clearance of intracellular Coxiella burnetii infection, which can cause Q fever. Therefore, we aimed to develop a novel T cell-targeted vaccine that induces pathogen-specific cell-mediated immunity to protect against Q fever in humans while avoiding the reactogenicity of the current inactivated whole cell vaccine. Human HLA class II T cell epitopes from C. burnetii were previously identified and selected by immunoinformatic predictions of HLA binding, conservation in multiple C. burnetii isolates, and low potential for cross-reactivity with the human proteome or microbiome. Epitopes were selected for vaccine inclusion based on long-lived human T cell recall responses to corresponding peptides in individuals that had been naturally exposed to the bacterium during a 2007-2010 Q fever outbreak in the Netherlands. Multiple viral vector-based candidate vaccines were generated that express concatemers of selected epitope sequences arranged to minimize potential junctional neo-epitopes. The vaccine candidates caused no antigen-specific reactogenicity in a sensitized guinea pig model. A subset of the vaccine epitope peptides elicited antigenic recall responses in splenocytes from C57BL/6 mice previously infected with C. burnetii. However, immunogenicity of the vaccine candidates in C57BL/6 mice was dominated by a single epitope and this was insufficient to confer protection against an infection challenge, highlighting the limitations of assessing human-targeted vaccine candidates in murine models. The viral vector-based vaccine candidates induced antigen-specific T cell responses to a broader array of epitopes in cynomolgus macaques, establishing a foundation for future vaccine efficacy studies in this large animal model of C. burnetii infection.

H. pylori vaccines: why we still don't have any.

Helicobacter pylori was appreciated as the major cause of peptic ulcers about 30 years ago and the most significant etiological agent in gastric cancer in the mid-1990s. Since that time, progress in the development of a preventive or therapeutic H. pylori vaccine has been relatively slow. The impediments to rapid advances in the field include a luke-warm enthusiasm among clinicians, research scientists, and public health authorities concerning the need for a vaccine, rudimentary understanding of the correlates of gastric immunity to H. pylori and of gastric mucosal immunology in general, the geographical heterogeneity of the H. pylori genome and insufficient pharmaceutical industry support. Recent enhancements in our understanding of the gastric immune response together with advances in H. pylori genomics now provide the potential to accelerate progress in H. pylori vaccine development. Whether an H. pylori vaccine becomes a reality will likely depend upon our ability to appropriately target the populations at highest risk of the adverse sequelae of infection.

Quantifying the Persistence of Vaccine-Related T Cell Epitopes in Circulating Swine Influenza A Strains from 2013-2017.

When swine flu vaccines and circulating influenza A virus (IAV) strains are poorly matched, vaccine-induced antibodies may not protect from infection. Highly conserved T cell epitopes may, however, have a disease-mitigating effect. The degree of T cell epitope conservation among circulating strains and vaccine strains can vary, which may also explain differences in vaccine efficacy. Here, we evaluate a previously developed conserved T cell epitope-based vaccine and determine the persistence of T cell epitope conservation over time. We used a pair-wise homology score to define the conservation between the vaccine's swine leukocyte antigen (SLA) class I and II-restricted epitopes and T cell epitopes found in 1272 swine IAV strains sequenced between 2013 and 2017. Twenty-four of the 48 total T cell epitopes included in the epitope-based vaccine were highly conserved and found in >1000 circulating swine IAV strains over the 5-year period. In contrast, commercial swine IAV vaccines developed in 2013 exhibited a declining conservation with the circulating IAV strains over the same 5-year period. Conserved T cell epitope vaccines may be a useful adjunct for commercial swine flu vaccines and to improve protection against influenza when antibodies are not cross-reactive.

Highly conserved, non-human-like, and cross-reactive SARS-CoV-2 T cell epitopes for COVID-19 vaccine design and validation.

Natural and vaccine-induced SARS-CoV-2 immunity in humans has been described but correlates of protection are not yet defined. T cells support the SARS-CoV-2 antibody response, clear virus-infected cells, and may be required to block transmission. In this study, we identified peptide epitopes associated with SARS-CoV-2 T-cell immunity. Using immunoinformatic methods, T-cell epitopes from spike, membrane, and envelope were selected for maximal HLA-binding potential, coverage of HLA diversity, coverage of circulating virus, and minimal potential cross-reactivity with self. Direct restimulation of PBMCs collected from SARS-CoV-2 convalescents confirmed 66% of predicted epitopes, whereas only 9% were confirmed in naive individuals. However, following a brief period of epitope-specific T-cell expansion, both cohorts demonstrated robust T-cell responses to 97% of epitopes. HLA-DR3 transgenic mouse immunization with peptides co-formulated with poly-ICLC generated a potent Th1-skewed, epitope-specific memory response, alleviating safety concerns of enhanced respiratory disease associated with Th2 induction. Taken together, these epitopes may be used to improve our understanding of natural and vaccine-induced immunity, and to facilitate the development of T-cell-targeted vaccines that harness pre-existing SARS-CoV-2 immunity.

Identification and Immune Assessment of T Cell Epitopes in Five Plasmodium falciparum Blood Stage Antigens to Facilitate Vaccine Candidate Selection and Optimization.

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.

Bridging Computational Vaccinology and Vaccine Development Through Systematic Identification, Characterization, and Downselection of Conserved and Variable Circumsporozoite Protein CD4 T Cell Epitopes From Diverse Plasmodium falciparum Strains.

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.

Identification, Selection and Immune Assessment of Liver Stage CD8 T Cell Epitopes From Plasmodium falciparum.

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.

Activation of natural regulatory T cells by IgG Fc-derived peptide "Tregitopes".

We have identified at least 2 highly promiscuous major histocompatibility complex class II T-cell epitopes in the Fc fragment of IgG that are capable of specifically activating CD4(+)CD25(Hi)FoxP3(+) natural regulatory T cells (nT(Regs)). Coincubation of these regulatory T-cell epitopes or "Tregitopes" and antigens with peripheral blood mononuclear cells led to a suppression of effector cytokine secretion, reduced proliferation of effector T cells, and caused an increase in cell surface markers associated with T(Regs) such as FoxP3. In vivo administration of the murine homologue of the Fc region Tregitope resulted in suppression of immune response to a known immunogen. These data suggest that one mechanism for the immunosuppressive activity of IgG, such as with IVIG, may be related to the activity of regulatory T cells. In this model, regulatory T-cell epitopes in IgG activate a subset of nT(Regs) that tips the resulting immune response toward tolerance rather than immunogenicity.

A method for individualizing the prediction of immunogenicity of protein vaccines and biologic therapeutics: individualized T cell epitope measure (iTEM).

The promise of pharmacogenomics depends on advancing predictive medicine. To address this need in the area of immunology, we developed the individualized T cell epitope measure (iTEM) tool to estimate an individual's T cell response to a protein antigen based on HLA binding predictions. In this study, we validated prospective iTEM predictions using data from in vitro and in vivo studies. We used a mathematical formula that converts DRB1* allele binding predictions generated by EpiMatrix, an epitope-mapping tool, into an allele-specific scoring system. We then demonstrated that iTEM can be used to define an HLA binding threshold above which immune response is likely and below which immune response is likely to be absent. iTEM's predictive power was strongest when the immune response is focused, such as in subunit vaccination and administration of protein therapeutics. iTEM may be a useful tool for clinical trial design and preclinical evaluation of vaccines and protein therapeutics.

Immune escape and immune camouflage may reduce the efficacy of RTS,S vaccine in Malawi.

The RTS,S/AS01 malaria vaccine will undergo a pilot vaccination study in sub-Saharan Africa beginning in 2019. RTS,S/AS01 Phase III trials reported an efficacy of 28.3% (children 5-17 months) and 18.3% (infants 6-12 weeks), with substantial variability across study sites. We postulated that the relatively low efficacy of the RTS,S vaccine and variability across sites may be due to lack of T-cell epitopes in the vaccine antigen, and due to the HLA distribution of the vaccinated population, and/or due to 'immune camouflage', an immune escape mechanism. To examine these hypotheses, we used immunoinformatics tools to compare T helper epitopes contained in RTS,S vaccine antigens with Plasmodium falciparum circumsporozoite protein (CSP) variants isolated from infected individuals in Malawi. The prevalence of epitopes restricted by specific HLA-DRB1 alleles was inversely associated with prevalence of the HLA-DRB1 allele in the Malawi study population, suggesting immune escape. In addition, T-cell epitopes in the CSP of strains circulating in Malawi were more often restricted by low-frequency HLA-DRB1 alleles in the population. Furthermore, T-cell epitopes that were highly conserved across CSP variants in Malawi possessed TCR-facing residues that were highly conserved in the human proteome, potentially reducing T-cell help through tolerance. The CSP component of the RTS,S vaccine also exhibited a low degree of T-cell epitope relatedness to circulating variants. These results suggest that RTS,S vaccine efficacy may be impacted by low T-cell epitope content, reduced presentation of T-cell epitopes by prevalent HLA-DRB1, high potential for human-cross-reactivity, and limited conservation with the CSP of circulating malaria strains.