William D. Martin

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.

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.

The two-faced T cell epitope: examining the host-microbe interface with JanusMatrix.

Advances in the field of T cell immunology have contributed to the understanding that cross-reactivity is an intrinsic characteristic of the T cell receptor (TCR), and that each TCR can potentially interact with many different T cell epitopes. To better define the potential for TCR cross-reactivity between epitopes derived from the human genome, the human microbiome, and human pathogens, we developed a new immunoinformatics tool, JanusMatrix, that represents an extension of the validated T cell epitope mapping tool, EpiMatrix. Initial explorations, summarized in this synopsis, have uncovered what appear to be important differences in the TCR cross-reactivity of selected regulatory and effector T cell epitopes with other epitopes in the human genome, human microbiome, and selected human pathogens. In addition to exploring the T cell epitope relationships between human self, commensal and pathogen, JanusMatrix may also be useful to explore some aspects of heterologous immunity and to examine T cell epitope relatedness between pathogens to which humans are exposed (Dengue serotypes, or HCV and Influenza, for example). In Hand-Foot-Mouth disease (HFMD) for example, extensive enterovirus and human microbiome cross-reactivity (and limited cross-reactivity with the human genome) seemingly predicts immunodominance. In contrast, more extensive cross-reactivity with proteins contained in the human genome as compared to the human microbiome was observed for selected Treg epitopes. While it may be impossible to predict all immune response influences, the availability of sequence data from the human genome, the human microbiome, and an array of human pathogens and vaccines has made computationally–driven exploration of the effects of T cell epitope cross-reactivity now possible. This is the first description of JanusMatrix, an algorithm that assesses TCR cross-reactivity that may contribute to a means of predicting the phenotype of T cells responding to selected T cell epitopes. Whether used for explorations of T cell phenotype or for evaluating cross-conservation between related viral strains at the TCR face of viral epitopes, further JanusMatrix studies may contribute to developing safer, more effective vaccines.

In Silico-Accelerated Identification of Conserved and Immunogenic Variola/Vaccinia T-Cell Epitopes

Epitopes shared by the vaccinia and variola viruses underlie the protective effect of vaccinia immunization against variola infection. We set out to identify a subset of cross-reactive epitopes using bioinformatics and immunological methods. Putative T-cell epitopes were computationally predicted from highly conserved open reading frames from seven complete vaccinia and variola genomes using EpiMatrix. Over 100 epitopes bearing low human sequence homology were selected and assessed in HLA binding assays and in T-cell antigenicity assays using PBMCs isolated from Dryvax-immunized subjects. This experimental validation of computational predictions illustrates the potential for immunoinformatics methods to identify candidate immunogens for a new, safer smallpox vaccine.

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.

Identification of genome-derived vaccine candidates conserved between human and mouse-adapted strains of H. pylori

Computational methods accelerate vaccine development by rapid identification of potential vaccine candidates. We screened the Helicobacter pylori J99 and 26695 genomes for T-cell epitopes using the epitope mapping algorithm EpiMatrix and selected 150 sequences for experimental validation in a pre-clinical mouse model. Because strains of H. pylori that infect humans do not generally infect mice, and the sequence of the mouse-adapted “Sydney” strain (SS1) is not publicly available, we used targeted PCR to confirm that epitopes we computationally predicted from the human H. pylori isolates J99 and 26695 are conserved in SS1. Epitopes conserved in SS1 were further analyzed for binding to MHC in vitro and for antigenicity in infected mice to select candidates for an epitope-based vaccine.

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.

Peptide-pulsed dendritic cells induce the hepatitis C viral epitope-specific responses of naïve human T cells

Hepatitis C virus (HCV) is a major cause of liver disease. Spontaneous resolution of infection is associated with broad, MHC class I- (CD8(+)) and class II-restricted (CD4(+)) T cell responses to multiple viral epitopes. Only 20% of patients clear infection spontaneously, however, most develop chronic disease. The response to chemotherapy varies; therapeutic vaccination offers an additional treatment strategy. To date, therapeutic vaccines have demonstrated only limited success in clinical trials. Vector-mediated vaccination with multi-epitope-expressing DNA constructs provides an improved approach. Highly-conserved, HLA-A2-restricted HCV epitopes and HLA-DRB1-restricted immunogenic consensus sequences (ICS, each composed of multiple overlapping and highly conserved epitopes) were predicted using bioinformatics tools and synthesized as peptides. HLA binding activity was determined in competitive binding assays. Immunogenicity and the ability of each peptide to stimulate naïve human T cell recognition and IFN-γ production were assessed in cultures of total PBMCs and in co-cultures composed of peptide-pulsed dendritic cells (DCs) and purified T lymphocytes, cell populations derived from normal blood donors. Essentially all predicted HLA-A2-restricted epitopes and HLA-DRB1-restricted ICS exhibited HLA binding activity and the ability to elicit immune recognition and IFN-γ production by naïve human T cells. The ability of DCs pulsed with these highly-conserved HLA-A2- and -DRB1-restricted peptides to induce naïve human T cell reactivity and IFN-γ production ex vivo demonstrates the potential efficacy of a multi-epitope-based HCV vaccine targeted to dendritic cells.

H7N9 T-cell epitopes that mimic human sequences are less immunogenic and may induce Treg-mediated tolerance

Avian-origin H7N9 influenza is a novel influenza A virus (IAV) that emerged in humans in China in 2013. Using immunoinformatics tools, we identified several H7N9 T cell epitopes with T cell receptor (TCR)-facing residues identical to those of multiple epitopes from human proteins. We hypothesized that host tolerance to these peptides may impair T helper response and contribute to the low titer, weak hemagglutination inhibiting (HI) antibody responses and diminished seroconversion rates that have been observed in human H7N9 infections and vaccine trials. We found that the magnitude of human T effector responses to individual H7N9 peptides was inversely correlated with the peptides resemblance to self. Furthermore, a promiscuous T cell epitope from the hemagglutinin (HA) protein suppressed responses to other H7N9 peptides when co-administered in vitro. Along with other highly ‘human-like’ peptides from H7N9, this peptide was also shown to expand FoxP3+ regulatory T cells (Tregs). Thus, H7N9 may be camouflaged from effective human immune response by T cell epitope sequences that avert or regulate effector T cell responses through host tolerance.

Universal H1N1 influenza vaccine development

Immune responses to cross-conserved T cell epitopes in novel H1N1 influenza may explain reports of diminished influenza-like illnesses and confirmed infection among older adults, in the absence of cross-reactive humoral immunity, during the 2009 pandemic. These cross-conserved epitopes may prove useful for the development of a universal H1N1 influenza vaccine, therefore, we set out to identify and characterize cross-conserved H1N1 T cell epitopes. An immunoinformatic analysis was conducted using all available pandemic and pre-pandemic HA-H1 and NA-N1 sequences dating back to 1980. Using an approach that balances potential for immunogenicity with conservation, we derived 13 HA and four NA immunogenic consensus sequences (ICS) from a comprehensive analysis of 5 738 HA-H1 and 5 396 NA-N1 sequences. These epitopes were selected because their combined epitope content is representative of greater than 84% of pre-pandemic and pandemic H1N1 influenza strains, their predicted immunogenicity (EpiMatrix) scores were greater than or equal to the 95th percentile of all comparable epitopes, and they were also predicted to be presented by more than four HLA class II archetypal alleles. We confirmed the ability of these peptides to bind in HLA binding assays and to stimulate interferon-γ production in human peripheral blood mononuclear cell cultures. These studies support the selection of the ICS as components of potential group-common H1N1 vaccine candidates and the application of this universal influenza vaccine development approach to other influenza subtypes.