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In silico immunogenicity risk assessment has been an important step in the development path for many biologic therapeutics, including monoclonal antibodies. Even if the source of a given biologic is ‘fully human’, T cell epitopes that are contained in the sequences of the biologic may activate the immune system, enabling the development of anti-drug antibodies that can reduce drug efficacy and may contribute to adverse events. Computational tools that identify T cell epitopes from primary amino acid sequences have been used to assess the immunogenic potential of therapeutic candidates for several decades. To facilitate larger scale analyses and accelerate preclinical immunogenicity risk assessment, our group developed an integrated web-based platform called ISPRI, (Immunogenicity Screening and Protein Re-engineering Interface) that provides hands-on access through a secure web-based interface for scientists working in large and mid-sized biotech companies in the US, Europe, and Japan. This toolkit has evolved and now contains an array of algorithms that can be used individually and/or consecutively for immunogenicity assessment and protein engineering. Most analyses start with the advanced epitope mapping tool (EpiMatrix), then proceed to identify epitope clusters using ClustiMer, and then use a tool called JanusMatrix to define whether any of the T cell epitope clusters may generate a regulatory T cell response which may diminish or eliminate anti-drug antibody formation. Candidates can be compared to similar products on a normalized immunogenicity scale. Should modifications to the biologic sequence be an option, a tool for moderating putative immunogenicity by editing T cell epitopes out of the sequence is available (OptiMatrix). Although this perspective discusses the in-silico immunogenicity risk assessment for monoclonal antibodies, bi-specifics, multi-specifics, and antibody-drug conjugates, the analysis of additional therapeutic modalities such as enzyme replacement proteins, blood factor proteins, CAR-T, gene therapy products, and peptide drugs is also made available on the ISPRI platform.

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-Barre 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-g 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. [ABSTRACT FROM AUTHOR]

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.

The induction of immunological tolerance is a promising strategy for managing autoimmune diseases, allergies, and transplant rejection. Tregitopes, a class of peptides, have emerged as potential agents for this purpose. They activate regulatory T cells, which are pivotal in reducing inflammation and promoting tolerance, by binding to MHC II molecules and facilitating their processing and presentation to Treg cells, thereby encouraging their proliferation. Moreover, Tregitopes influence the phenotype of antigen-presenting cells by attenuating the expression of CD80, CD86, and MHC class II while enhancing ILT3, resulting in the inhibition of NF-kappa B signaling pathways. Various techniques, including in vitro and in silico methods, are applied to identify Tregitope candidates. Currently, Tregitopes play a vital role in balancing immune activation and tolerance in clinical applications such as Pompe disease, diabetes-related antigens, and the prevention of spontaneous abortions in autoimmune diseases. Similarly, Tregitopes can induce antigen-specific regulatory T cells. Their anti-inflammatory effects are significant in conditions such as autoimmune encephalomyelitis, inflammatory bowel disease, and Guillain-Barré syndrome. Additionally, Tregitopes have been leveraged to enhance vaccine design and efficacy. Recent advancements in understanding the potential benefits and drawbacks of IVIG and the discovery of the function and mechanism of Tregitopes have introduced Tregitopes as a popular option for immune system modulation. It is expected that they will bring about a significant revolution in the management and treatment of autoimmune and immunological diseases. This article is a comprehensive review of Tregitopes, concluding with the potential of these epitopes as a therapeutic avenue for immunological disorders. [Display omitted] [ABSTRACT FROM AUTHOR]

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.

The resurgence of whooping cough since the introduction of acellular (protein) vaccines has led to a renewed interest in the development of improved pertussis vaccines; Outer Membrane Vesicles (OMVs) carrying pertussis antigens have emerged as viable candidates. An in silico immunogenicity screen was carried out on 49 well-known Bordetella pertussis proteins in order to better understand their potential role toward the efficacy of pertussis OMVs for vaccine design; seven proteins were identified as being good candidates for including in optimized cellular and acellular pertussis vaccines. We then screened these antigens for putative tolerance-inducing sequences, as proteins with reduced tolerogenicity have improved vaccine potency in preclinical models. We used specialized homology tools (JanusMatrix) to identify peptides in the proteins that were cross-reactive with human sequences. Four of the 19 identified cross-reactive peptides were detolerized in silico using a separate tool, OptiMatrix, which disrupted the potential of these peptides to bind to human HLA and murine MHC. Four selected cross-reactive peptides and their detolerized variants were synthesized and their binding to a set of eight common HLA class II alleles was assessed in vitro. Reduced binding affinity to HLA class II was observed for the detolerized variants compared to the wild-type peptides, highlighting the potential of this approach for designing more efficacious pertussis vaccines.

Computational vaccinology includes epitope mapping, antigen selection, and immunogen design using computational tools. Tools that facilitate the in silico prediction of immune response to biothreats, emerging infectious diseases, and cancers can accelerate the design of novel and next generation vaccines and their delivery to the clinic. Over the past 20 years, vaccinologists, bioinformatics experts, and advanced programmers based in Providence, Rhode Island, USA have advanced the development of an integrated toolkit for vaccine design called iVAX, that is secure and user-accessible by internet. This integrated set of immunoinformatic tools comprises algorithms for scoring and triaging candidate antigens, selecting immunogenic and conserved T cell epitopes, re-engineering or eliminating regulatory T cell epitopes, and re-designing antigens to induce immunogenicity and protection against disease for humans and livestock. Commercial and academic applications of iVAX have included identifying immunogenic T cell epitopes in the development of a T-cell based human multi-epitope Q fever vaccine, designing novel influenza vaccines, identifying cross-conserved T cell epitopes for a malaria vaccine, and analyzing immune responses in clinical vaccine studies. Animal vaccine applications to date have included viral infections of pigs such as swine influenza A, PCV2, and African Swine Fever. “Rapid-Fire” applications for biodefense have included a demonstration project for Lassa Fever and Q fever. As recent infectious disease outbreaks underscore the significance of vaccine-driven preparedness, the integrated set of tools available on the iVAX toolkit stand ready to help vaccine developers deliver genome-derived, epitope-driven vaccines.

Computational vaccinology includes epitope mapping, antigen selection, and immunogen design using computational tools. Tools that facilitate the in silico prediction of immune response to biothreats, emerging infectious diseases, and cancers can accelerate the design of novel and next generation vaccines and their delivery to the clinic. Over the past 20 years, vaccinologists, bioinformatics experts, and advanced programmers based in Providence, Rhode Island, USA have advanced the development of an integrated toolkit for vaccine design called iVAX, that is secure and user-accessible by internet. This integrated set of immunoinformatic tools comprises algorithms for scoring and triaging candidate antigens, selecting immunogenic and conserved T cell epitopes, re-engineering or eliminating regulatory T cell epitopes, and re-designing antigens to induce immunogenicity and protection against disease for humans and livestock. Commercial and academic applications of iVAX have included identifying immunogenic T cell epitopes in the development of a T-cell based human multi-epitope Q fever vaccine, designing novel influenza vaccines, identifying cross-conserved T cell epitopes for a malaria vaccine, and analyzing immune responses in clinical vaccine studies. Animal vaccine applications to date have included viral infections of pigs such as swine influenza A, PCV2, and African Swine Fever. "Rapid-Fire" applications for biodefense have included a demonstration project for Lassa Fever and Q fever. As recent infectious disease outbreaks underscore the significance of vaccine-driven preparedness, the integrated set of tools available on the iVAX toolkit stand ready to help vaccine developers deliver genome-derived, epitope-driven vaccines. [ABSTRACT FROM AUTHOR]

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.

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. [ABSTRACT FROM AUTHOR]

Highlights • A multiepitopic protein against Zaire ebolavirus (Zerola) was designed. • Zerola comprises Th and B cell epitopes. • Epitopes were selected by immunoinformatic approaches and experimental evidence. • Zerola antigen was successfully expressed in tobacco plants. Abstract The recent Ebola virus disease (EVD) outbreaks make the development of efficacious and low cost vaccines against Ebola virus (EBOV) an urgent goal. Multiepitopic vaccines allow a rational design rendering vaccines able to induce proper immune responses in terms of polarization and potency. In addition, the pathogen variants can be easily covered by including epitopes conserved among relevant isolates. Other important aspects to consider in vaccination are the costs associated to production, distribution, and administration of the vaccine. Plants provide an advantageous platform for this purpose, since they yield biomass at very low costs and some species can be used to formulate purification-free oral vaccines. In the present study, a multiepitopic protein called Zerola, which carries epitopes from the EBOV glycoprotein (GP), was designed based on immunoinformatic approaches and current experimental evidence on B cell protective GP epitopes. Moreover, expression studies performed in nuclear-transformed tobacco lines confirmed the capacity of the plant cell to synthetize the Zerola antigenic protein. The generation of this plant-based candidate vaccine is a step forward in the development of highly efficient and low cost EBOV vaccines. [ABSTRACT FROM AUTHOR]

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). [ABSTRACT FROM AUTHOR]

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.

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.

Ticks are notorious vectors of disease for humans, and many species of ticks transmit multiple pathogens, sometimes in the same tick bite. Accordingly, a broad-spectrum vaccine that targets vector ticks and pathogen transmission at the tick/host interface, rather than multiple vaccines against every possible tickborne pathogen, could become an important tool for resolving an emerging public health crisis. The concept for such a tick protective vaccine comes from observations of an acquired tick resistance (ATR) that can develop in non-natural hosts of ticks following sensitization to tick salivary components. Mice are commonly used as models to study immune responses to human pathogens but normal mice are natural hosts for many species of ticks and fail to develop ATR. We evaluated HLA DR3 transgenic (tg) "humanized" mice as a potential model of ATR and assessed the possibility of using this animal model for tick protective vaccine discovery studies. Serial tick infestations with pathogen-free Ixodes scapularis ticks were used to tick-bite sensitize HLA DR3 tg mice. Sensitization resulted in a cytokine skew favoring a Th2 bias as well as partial (57%) protection to infection with Lyme disease spirochetes (Borrelia burgdorferi) following infected tick challenge when compared to tick naïve counterparts. I. scapularis salivary gland homogenate (SGH) and a group of immunoinformatic-predicted T cell epitopes identified from the I. scapularis salivary transcriptome were used separately to vaccinate HLA DR3 tg mice, and these mice also were assessed for both pathogen protection and epitope recognition. Reduced pathogen transmission along with a Th2 skew resulted from SGH vaccination, while no significant protection and a possible T regulatory bias was seen in epitope-vaccinated mice. This study provides the first proof-of-concept for using HLA DR tg "humanized" mice for studying the potential tick protective effects of immunoinformatic- or otherwise-derived tick salivary components as tickborne disease vaccines.

Visceral leishmaniasis (VL) is a serious parasitic disease for which control measures are limited and drug resistance is increasing. First and second generation vaccine candidates have not been successful. The goal of the present study was to select possibly immunogenic L. donovani donovani GP63 peptides using immunoinformatics tools and to test their immunogenicity in vitro. The amino acid sequence of L. donovani donovani GP63 [GenBank accession: ACT31401] was screened using the EpiMatrix algorithm for putative T cell epitopes that would bind to the most common HLA class II alleles (DRB1*1101 and DRB1*0804) among at-risk populations. Four T cell epitopes were selected from nine potential candidates. Stimulation of whole blood from healthy volunteers using the peptides separately produced mean IFN-γ and IL-4 levels that were not significantly different from negative controls, while the pooled peptides produced a moderate IFN-γ increase in some volunteers. However, mean IL-10 levels were significantly reduced for all individuals compared with controls. The immunogenicity of these epitopes may be harnessed most effectively in a vaccine delivered in combination with immune-modulating adjuvants.