Computational Analysis of African Swine Fever Virus Protein Space for the Design of an Epitope-Based Vaccine Ensemble

African swine fever virus is the etiological agent of African swine fever, a transmissible severe hemorrhagic disease that affects pigs, causing massive economic losses. There is neither a treatment nor a vaccine available, and the only method to control its spread is by extensive culling of pigs. So far, classical vaccine development approaches have not yielded sufficiently good results in terms of concomitant safety and efficacy. Nowadays, thanks to advances in genomic and proteomic techniques, a reverse vaccinology strategy can be explored to design alternative vaccine formulations. In this study, ASFV protein sequences were analyzed using an in-house pipeline based on publicly available immunoinformatic tools to identify epitopes of interest for a prospective vaccine ensemble. These included experimentally validated sequences from the Immune Epitope Database, as well as de novo predicted sequences. Experimentally validated and predicted epitopes were prioritized following a series of criteria that included evolutionary conservation, presence in the virulent and currently circulating variant Georgia 2007/1, and lack of identity to either the pig proteome or putative proteins from pig gut microbiota. Following this strategy, 29 B-cell, 14 CD4+ T-cell and 6 CD8+ T-cell epitopes were selected, which represent a starting point to investigating the protective capacity of ASFV epitope-based vaccines.info:eu-repo/semantics/publishedVersio

T-Cell Immunogenicity and Dysfunction in Cancer and Viral Diseases

abstract: CD8+ T-lymphocytes (CTLs) are central to the immunologic control of infections and are currently at the forefront of strategies that enhance immune based treatment of a variety of tumors. Effective T-cell based vaccines and immunotherapies fundamentally rely on the interaction of CTLs with peptide-human leukocyte antigen class I (HLA-I) complexes on the infected/malignant cell surface. However, how CTLs are able to respond to antigenic peptides with high specificity is largely unknown. Also unknown, are the different mechanisms underlying tumor immune evasion from CTL-mediated cytotoxicity. In this dissertation, I investigate the immunogenicity and dysfunction of CTLs for the development of novel T-cell therapies. Project 1 explores the biochemical hallmarks associated with HLA-I binding peptides that result in a CTL-immune response. The results reveal amino acid hydrophobicity of T-cell receptor (TCR) contact residues within immunogenic CTL-epitopes as a critical parameter for CTL-self/nonself discrimination. Project 2 develops a bioinformatic and experimental methodology for the identification of CTL-epitopes from low frequency T-cells against tumor antigens and chronic viruses. This methodology is employed in Project 3 to identify novel immunogenic CTL-epitopes from human papillomavirus (HPV)-associated head and neck cancer patients. In Project 3, I further study the mechanisms of HPV-specific T-cell dysfunction, and I demonstrate that combination inhibition of Indoleamine 2, 3-dioxygenase (IDO-1) and programmed cell death protein (PD-1) can be a potential immunotherapy against HPV+ head and neck cancers. Lastly, in Project 4, I develop a single-cell assay for high-throughput identification of antigens targeted by CTLs from whole pathogenome libraries. Thus, this dissertation contributes to fundamental T-cell immunobiology by identifying rules of T-cell immunogenicity and dysfunction, as well as to translational immunology by identifying novel CTL-epitopes, and therapeutic targets for T-cell immunotherapy.Dissertation/ThesisDoctoral Dissertation Biological Design 201

Design and Humoral Analysis of Two Epitope-Based Brucella abortus DNA Vaccines

Brucella is a genus of Gram negative, facultative intracellular pathogens which cause brucellosis, one of the most wide spread zoonotic diseases. Brucellosis causes a severe economic burden due to reproductive loss in animals and human infection. Vaccination of animals has proven to be the most effective means of controlling brucellosis; however the current live-attenuated vaccines are not considered ideal. The live-attenuated vaccines Brucella abortus Strain 19 and RB51 remain pathogenic to humans, and the former interferes with diagnostic tests due to induction of antibodies against the O-polysaccharide. DNA vaccination with single antigens has proven to be successful at protecting mice against B. abortus challenge, however this method is less effective in large animals. Immunization with a combination of antigens has been shown to provide more protection than single antigens. In an attempt to develop a better DNA vaccine, two multivalent multi-epitope plasmids were constructed using known protective antigens and bioinformatics technologies. Epitopes predicted to induce cell-mediated immunity were selected from Cu/Zn superoxide dismutase, outer membrane protein (Omp) 16, Omp19, ribosomal subunit L7/L12, BP26, ribosomal subunit L9, and Omp25. The plasmids were transfected into Chinese hamster ovary (CHO K1) cells, and PCR was used to confirm presence of the sequences in the genome. The transcription of the BabV1 and BabV2 genes to RNA was confirmed using RT-PCR. Finally, Western blots using sera from Strain 19 infected goats suggest the protein is not recognized by the humoral response of vaccinated animals. Further research is required to determine if the p425/BabV1 and p425/BabV2 vaccines are recognized by the cell-mediated immune response of infected or vaccinated animals

A Meeting of Minds: In Recognition of the Contributions of Randall J. Cohrs

A Special Issue in memory of Randall J. Cohrs, Ph.D. Topics include original research reports on a variety of viruses as well as reviews and commentaries on Randy’s contributions to many investigations

Antigen presentation and the boundary between self and non-self: applications in computational immunology

This thesis focuses on the applications of computational prediction methods in the areas of antigen presentation and recognition close to the boundary between self and non-self. Publicly available MHC binding prediction tools are combined with data about the frequencies of different HLA alleles within global and regional populations. Additionally, a new in silico method called proteome scanning is presented that assesses whether, in the light of central tolerance mechanisms that remove self-reactive T cells, an individual is likely to have T cells capable of binding to a given peptide-MHC surface. These methods are applied in three main application areas. Firstly, predictions are made concerning individuals with missense mutation haemophilia A and their risk of developing inhibitors against the replacement Factor VIII used to treat the condition. Inhibitor formation is known to be a CD4+ T cell-dependent process; the analysis presented here demonstrates that understanding the risk of inhibitor formation generally requires knowledge of an individual’s HLA types. Secondly, predictions are made concerning the risk of transplant rejection and suggest that the proteome scanning approach can be used to predict whether a given HLA mismatch between donor and recipient is likely to increase rejection risk. Thirdly, predictions are made concerning candidate peptide biomarkers (derived from known tumour antigens) for hepatocellular carcinoma, with the selection of peptide pools optimised in terms of MHC binding affinity and global population coverage. These are currently undergoing laboratory evaluation at the Institute of Hepatology, with promising early results. Taken together, these applications illustrate the breadth of potential contributions to clinical practice afforded by the computational prediction of antigen presentation and recognition close to the self/non-self boundary, including the novel proteome scanning methodology

FC gamma receptors: genetic variation and role in HIV-1 infection

Low affinity Fcγ receptors (FcγR) mediate key immune effector mechanisms through the engagement of the Fc portion of immunoglobulin G (IgG). These receptors are involved in multiple biological processes, including clearance of antigen/antibody immune complexes, enhancement of antigen presentation, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, regulation of antibody production, and activation of inflammatory cells. FcγR phenotypic variability modulates these processes through altering receptor IgG subclass binding affinity (FcγRIIa-H131R and FcγRIIIa-F158V), subcellular localization (FcγRIIb-I232T), post-translational modification (FcγRIIIb-HNA1a/b/c), expression of an otherwise pseudogene (FcγRIIc), and receptor surface density (gene copy number variability and promoter haplotypes). Accumulating data suggest that FcγR-mediated effector functions play a significant role in HIV-1 protective immunity, which is substantiated by the association of FcγR phenotypic variants with HIV-1 disease outcome. This study set out to characterize FcγR functional variability in the South African population, and to investigate the potential role thereof in HIV-1 transmission and disease progression in South African Black individuals. Since the only known determinant of FcγRIIIa surface density – FCGR3A gene copy number – is rare, this study investigated novel genetic determinants of FcγRIIIa expression by flow cytometry and nucleotide sequencing. FcγRIIIa expression on peripheral blood mononuclear cells was characterized for 32 South African Caucasian individuals and 22 South African Black individuals (Chapter 3). Significant differences in the proportion of FcγRIIIa-positive monocytes and FcγRIIIa expression levels on natural killer (NK) cells were observed between the population groups. A novel four-variant FCGR3A intragenic haplotype that associated with increased surface expression of FcγRIIIa on NK cells was detectable in Caucasian individuals, but not Black individuals and may account for the observed population differences. Further exploration of genetic diversity at the low affinity FCGR gene locus was extended to include all currently known functional variants of FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb using a commercial multiplex ligation-dependent probe amplification assay (Chapter 4). Thirty-two South African Caucasian individuals and 131 South African Blac

Synthetic Biology

Synthetic biology gives us a new hope because it combines various disciplines, such as genetics, chemistry, biology, molecular sciences, and other disciplines, and gives rise to a novel interdisciplinary science. We can foresee the creation of the new world of vegetation, animals, and humans with the interdisciplinary system of biological sciences. These articles are contributed by renowned experts in their fields. The field of synthetic biology is growing exponentially and opening up new avenues in multidisciplinary approaches by bringing together theoretical and applied aspects of science

Immunopathological signatures in multisystem inflammatory syndrome in children and pediatric COVID-19

: Pediatric Coronavirus Disease 2019 (pCOVID-19) is rarely severe; however, a minority of children infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) might develop multisystem inflammatory syndrome in children (MIS-C), with substantial morbidity. In this longitudinal multi-institutional study, we applied multi-omics (analysis of soluble biomarkers, proteomics, single-cell gene expression and immune repertoire analysis) to profile children with COVID-19 (n = 110) and MIS-C (n = 76), along with pediatric healthy controls (pHCs; n = 76). pCOVID-19 was characterized by robust type I interferon (IFN) responses, whereas prominent type II IFN-dependent and NF-κB-dependent signatures, matrisome activation and increased levels of circulating spike protein were detected in MIS-C, with no correlation with SARS-CoV-2 PCR status around the time of admission. Transient expansion of TRBV11-2 T cell clonotypes in MIS-C was associated with signatures of inflammation and T cell activation. The association of MIS-C with the combination of HLA A*02, B*35 and C*04 alleles suggests genetic susceptibility. MIS-C B cells showed higher mutation load than pCOVID-19 and pHC. These results identify distinct immunopathological signatures in pCOVID-19 and MIS-C that might help better define the pathophysiology of these disorders and guide therapy

Viral Gene Therapy

The development of technologies that allow targeting of specific cells has progressed substantially in recent years for several types of vectors, particularly viral vectors, which have been used in 70% of gene therapy clinical trials. Particular viruses have been selected as gene delivery vehicles because of their capacities to carry foreign genes and their ability to efficiently deliver these genes associated with efficient gene expression. This book is designed to present the most recent advances in viral gene therap

ELUCIDATION OF THE MECHANISM OF ACTION OF A RESPIRATORY SYNCYTIAL VIRUS SUBUNIT VACCINE CANDIDATE CONTAINING A POLYMER-BASED COMBINATION ADJUVANT

Human respiratory syncytial virus (RSV) is the primary cause of respiratory illnesses in infants, young children, elderly and immunocompromised individuals. Supportive care is the mainstay of RSV treatment. Currently no licensed vaccine against RSV is available. We have developed a subunit RSV vaccine candidate (ΔF/TriAdj) consisting of a truncated version of the RSV fusion protein (ΔF) formulated with a combination adjuvant (TriAdj) comprised of low molecular weight (LMW) polyinosinic:polycytidylic acid [poly(I:C)], an innate defense regulator (IDR) peptide and poly[di(sodium carboxylatoethylphenoxy)]-phosphazene (PCEP). We previously demonstrated the safety and protective efficacy of ΔF/TriAdj in several animal models. The overall objective of this thesis was to elucidate the mechanism of action of ΔF/TriAdj in BALB/c mice. First, we determined that ΔF/TriAdj when delivered intranasally plays a crucial role in stimulating innate immune responses in both upper and lower respiratory tracts of immunized mice as demonstrated by local production of cytokines, chemokines and interferons, as well as infiltration and activation of immune cells. Innate activation subsequently led to robust adaptive immunity and protection against RSV. Next, we elucidated the mechanisms of action of ΔF/TriAdj at the cell-signaling level in macrophages. Macrophages responded directly to in vitro stimulation with ΔF/TriAdj with induction of both endosomal and cytosolic pattern recognition receptors (PRRs). Based on inhibition studies, we determined that multiple signal transduction pathways are involved in ΔF/TriAdj-mediated activation of macrophages. Finally, we conducted a comprehensive chemical isotope labeling liquid chromatography-mass spectrometry (CIL LC-MS) analysis of the lung tissues from vaccinated and unvaccinated, RSV-infected mice as well as healthy controls, to understand the underlying mechanisms of action of ΔF/TriAdj at the further downstream metabolomic level. Metabolomic profiling revealed alterations of tryptophan metabolism (including kynurenine pathway), biosynthesis of amino acids (including arginine biosynthesis), urea cycle and tyrosine metabolism due to RSV infection. Interestingly, ΔF/TriAdj was found to a play a critical role in modulating alterations in the concentrations of the metabolites of the above-mentioned pathways in response to RSV infection. Ultimately, information on the mechanism of action of this RSV vaccine candidate may serve to identify potential biomarkers for immunogenicity and protective efficacy of ΔF/TriAdj in future