In silico methods for immunogenicity risk assessment and human homology screening for therapeutic antibodies.

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.

ISPRI (Interactive Screening and Protein Reengineering Interface): Integrated, cloud-based, comprehensive toolkit for Immunogenicity Risk Assessment.EpiMatrix Immunogenicity Score: Combined T effector and Treg Epitope Content per unit protein.Tregitopes: Treg Epitopes found in IgG Framework that have been shown to modulate antigen-specific effector T cell responses.ClustiMer: Tool for identifying epitope rich polypeptides from within a given protein sequence.JanusMatrix: Tool for Predicting Tolerance, Putative Treg Epitopes, and Anti-self-immune responses.OptiMatrix: Tool for modifying T cell epitope sequences to reduce (or enhance) MHC binding.

Immune Tolerance-Adjusted Personalized Immunogenicity Prediction for Pompe Disease.

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 personalized immunogenicity risk assessment (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.

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.

Better Epitope Discovery, Precision Immune Engineering, and Accelerated Vaccine Design Using Immunoinformatics Tools.

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.

HCV epitope, homologous to multiple human protein sequences, induces a regulatory T cell response in infected patients.

BACKGROUND & AIMS: Spontaneous resolution of hepatitis C virus (HCV) infection depends upon a broad T cell response to multiple viral epitopes. However, most patients fail to clear infections spontaneously and develop chronic disease. The elevated number and function of CD3(+)CD4(+)CD25(+)FoxP3(+) regulatory T cells (T(reg)) in HCV-infected patients suggest a role of Treg cells in impaired viral clearance. The factors contributing to increased Treg cell activity in chronic hepatitis C cases remain to be delineated. METHODS: Immunoinformatics tools were used to predict promiscuous, highly-conserved HLA-DRB1-restricted immunogenic consensus sequences (ICS), each composed of multiple T cell epitopes. These sequences were synthesized and added to cultures of peripheral blood mononuclear cells (PBMCs), derived from patients who resolved HCV infection spontaneously, patients with persistent infection, and non-infected individuals. The cells were collected and following 5days incubation, quantified and characterized by flow cytometry. RESULTS: One immunogenic consensus sequence (ICS), HCV_G1_p7_794, induced a marked increase in Treg cells in PBMC cultures derived from infected patients, but not in patients who spontaneously cleared HCV or in non-infected individuals. An analogous human peptide (p7_794), on the other hand, induced a significant increase in Treg cells among PBMCs derived from both HCV-infected and non-infected individuals. JanusMatrix analyses determined that HCV_G1_p7_794 is comprised of Treg cell epitopes that exhibit extensive cross-reactivity with the human proteome. CONCLUSIONS: A virus-encoded peptide (HCV_G1_p7_794) with extensive human homology activates cross-reactive CD3(+)CD4(+)CD25(+)FoxP3(+) natural Treg cells, which potentially contributes to immunosuppression and to the development of chronic hepatitis C.

Dendritic cell-mediated, DNA-based vaccination against hepatitis C induces the multi-epitope-specific response of humanized, HLA transgenic mice.

Hepatitis C virus (HCV) is the etiologic agent of chronic liver disease, hepatitis C. Spontaneous resolution of viral infection is associated with vigorous HLA class I- and class II-restricted T cell responses to multiple viral epitopes. Unfortunately, only 20% of patients clear infection spontaneously, most develop chronic disease and require therapy. The response to chemotherapy varies, however; therapeutic vaccination offers an additional treatment strategy. To date, therapeutic vaccines have demonstrated only limited success. Vector-mediated vaccination with multi-epitope-expressing DNA constructs alone or in combination with chemotherapy offers an additional treatment approach. Gene sequences encoding validated HLA-A2- and HLA-DRB1-restricted epitopes were synthesized and cloned into an expression vector. Dendritic cells (DCs) derived from humanized, HLA-A2/DRB1 transgenic (donor) mice were transfected with these multi-epitope-expressing DNA constructs. Recipient HLA-A2/DRB1 mice were vaccinated s.c. with transfected DCs; control mice received non-transfected DCs. Peptide-specific IFN-γ production by splenic T cells obtained at 5 weeks post-immunization was quantified by ELISpot assay; additionally, the production of IL-4, IL-10 and TNF-α were quantified by cytokine bead array. Splenocytes derived from vaccinated HLA-A2/DRB1 transgenic mice exhibited peptide-specific cytokine production to the vast majority of the vaccine-encoded HLA class I- and class II-restricted T cell epitopes. A multi-epitope-based HCV vaccine that targets DCs offers an effective approach to inducing a broad immune response and viral clearance in chronic, HCV-infected patients.

Coupling sensitive in vitro and in silico techniques to assess cross-reactive CD4(+) T cells against the swine-origin H1N1 influenza virus.

The outbreak of the novel swine-origin H1N1 influenza in the spring of 2009 took epidemiologists, immunologists, and vaccinologists by surprise and galvanized a massive worldwide effort to produce millions of vaccine doses to protect against this single virus strain. Of particular concern was the apparent lack of pre-existing antibody capable of eliciting cross-protective immunity against this novel virus, which fueled fears this strain would trigger a particularly far-reaching and lethal pandemic. Given that disease caused by the swine-origin virus was far less severe than expected, we hypothesized cellular immunity to cross-conserved T cell epitopes might have played a significant role in protecting against the pandemic H1N1 in the absence of cross-reactive humoral immunity. In a published study, we used an immunoinformatics approach to predict a number of CD4(+) T cell epitopes are conserved between the 2008-2009 seasonal H1N1 vaccine strain and pandemic H1N1 (A/California/04/2009) hemagglutinin proteins. Here, we provide results from biological studies using PBMCs from human donors not exposed to the pandemic virus to demonstrate that pre-existing CD4(+) T cells can elicit cross-reactive effector responses against the pandemic H1N1 virus. As well, we show our computational tools were 80-90% accurate in predicting CD4(+) T cell epitopes and their HLA-DRB1-dependent response profiles in donors that were chosen at random for HLA haplotype. Combined, these results confirm the power of coupling immunoinformatics to define broadly reactive CD4(+) T cell epitopes with highly sensitive in vitro biological assays to verify these in silico predictions as a means to understand human cellular immunity, including cross-protective responses, and to define CD4(+) T cell epitopes for potential vaccination efforts against future influenza viruses and other pathogens.

The role of glutamic or aspartic acid in position four of the epitope binding motif and thyrotropin receptor-extracellular domain epitope selection in Graves' disease.

CONTEXT: Development of Graves' disease (GD) is related to HLA-DRB1*0301 (DR3),and more specifically to arginine at position 74 of the DRB1 molecule. The extracellular domain (ECD) of human TSH receptor (hTSH-R) contains the target antigen. OBJECTIVE AND DESIGN: We analyzed the relation between hTSH-R-ECD peptides and DR molecules to determine whether aspartic acid (D) or glutamic acid (E) at position four in the binding motif influenced selection of functional epitopes. RESULTS: Peptide epitopes from TSH-R-ECD with D or E in position four (D/E+) had higher affinity for binding to DR3 than peptides without D/E (D/E-) (IC(50) 29.3 vs. 61.4, P = 0.0024). HLA-DR7, negatively correlated with GD, and DRB1*0302 (HLA-DR18), not associated with GD, had different profiles of epitope binding. Toxic GD patients who are DR3+ had higher responses to D/E+ peptides than D/E- peptides (stimulation index 1.42 vs. 1.22, P = 0.028). All DR3+ GD patients (toxic + euthyroid) had higher responses, with borderline significance (Sl; 1.32 vs. 1.18, P = 0.051). Splenocytes of DR3 transgenic mice immunized to TSH-R-ECD responded to D/E+ peptides more than D/E- peptides (stimulation index 1.95 vs. 1.69, P = 0.036). Seven of nine hTSH-R-ECD peptide epitopes reported to be reactive with GD patients' peripheral blood mononuclear cells contain binding motifs with D/E at position four. CONCLUSIONS: TSH-R-ECD epitopes with D/E in position four of the binding motif bind more strongly to DRB1*0301 than epitopes that are D/E- and are more stimulatory to GD patients' peripheral blood mononuclear cells and to splenocytes from mice immunized to hTSH-R. These epitopes appear important in immunogenicity to TSH-R due to their favored binding to HLA-DR3, thus increasing presentation to T cells.

Immunoinformatic comparison of T-cell epitopes contained in novel swine-origin influenza A (H1N1) virus with epitopes in 2008-2009 conventional influenza vaccine.

In March 2009 a novel swine-origin influenza A (H1N1) virus (S-OIV) emerged in Mexico and the Western United States. Vaccination with conventional influenza vaccine (CIV) does not result in cross-reactive antibodies, however, the disproportionate number of cases (37%) occurring among persons younger than 50 years old suggested that adaptive immune memory might be responsible for the relative lack of virulence in older, healthy adults. Using EpiMatrix, a T-cell epitope prediction and comparison tool, we compared the sequences of the three hemagglutinin (HA) and neuraminidase (NA) proteins contained in 2008-2009 CIV to their counterparts in A/California/04/2009 (H1N1) looking for cross-conserved T-cell epitope sequences. We found greater than 50% conservation of T helper and CTL epitopes between novel S-OIV and CIV HA for selected HLA. Conservation was lower among NA epitopes. Sixteen promiscuous helper T-cell epitopes are contained in the S-OIV H1N1 HA sequence, of which nine (56%) were 100% conserved in the 2008-2009 influenza vaccine strain; 81% were either identical or had one conservative amino acid substitution. Fifty percent of predicted CTL epitopes found in S-OIV H1N1 HA were also found in CIV HA sequences. Based on historical performance, we expect these epitope predictions to be 93-99% accurate. This in silico analysis supports the proposition that T-cell response to cross-reactive T-cell epitopes, due to vaccination or exposure, may have the capacity to attenuate the course of S-OIV H1N1 induced disease-in the absence of cross-reactive antibody response. The value of the CIV or live-attenuated influenza vaccine containing the 2008-2009 vaccine strains, as defense against H1N1, could be further tested by evaluating human immune responses to the conserved T-cell epitopes using PBMC from individuals infected with H1N1 and from CIV vaccinees.

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.