abstract: Elucidation of Antigen-Antibody (Ag-Ab) interactions is critical to the understanding of humoral immune responses to pathogenic infection. B cells are crucial components of the immune system that generate highly specific antibodies, such as IgG, towards epitopes on antigens. Serum IgG molecules carry specific molecular recognition information concerning the antigens that initiated their production. If one could read it, this information can be used to predict B cell epitopes on target antigens in order to design effective epitope driven vaccines, therapies and serological assays. Immunosignature technology captures the specific information content of serum IgG from infected and uninfected individuals on high density microarrays containing ~105 nearly random peptide sequences. Although the sequences of the peptides are chosen to evenly cover amino acid sequence space, the pattern of serum IgG binding to the array contains a consistent signature associated with each specific disease (e.g., Valley fever, influenza) among many individuals. Here, the disease specific but agnostic behavior of the technology has been explored by profiling molecular recognition information for five pathogens causing life threatening infectious diseases (e.g. DENV, WNV, HCV, HBV, and T.cruzi). This was done by models developed using a machine learning algorithm to model the sequence dependence of the humoral immune responses as measured by the peptide arrays. It was shown that the disease specific binding information could be accurately related to the peptide sequences used on the array by the machine learning (ML) models. Importantly, it was demonstrated that the ML models could identify or predict known linear epitopes on antigens of the four viruses. Moreover, the models identified potential novel linear epitopes on antigens of the four viruses (each has 4-10 proteins in the proteome) and of T.cruzi (a eukaryotic parasite which has over 12,000 proteins in its proteome). Finally, the predicted epitopes were tested in serum IgG binding assays such as ELISAs. Unfortunately, the assay results were inconsistent due to problems with peptide/surface interactions. In a separate study for the development of antibody recruiting molecules (ARMs) to combat microbial infections, 10 peptides from the high density peptide arrays were tested in IgG binding assays using sera of healthy individuals to find a set of antibody binding termini (ABT, a ligand that binds to a variable region of the IgG). It was concluded that one peptide (peptide 7) may be used as a potential ABT. Overall, these findings demonstrate the applications of the immunosignature technology ranging from developing tools to predict linear epitopes on pathogens of small to large proteomes to the identification of an ABT for ARMs. / Dissertation/Thesis / Doctoral Dissertation Biochemistry 2020

INTRODUCTION: Investigating mechanisms of how intracellular bacterial pathogens such as Mycobacterium. avium (M. avium) evade the host immune response and replicate within macrophages is crucial to devising rational targets for host-directed therapies (HDT) against these associated diseases. This studied utilized the congenic mouse strain B6.Sst1S, which contains the super-susceptibility to tuberculosis (TB) allele. Among murine models of TB, this strain uniquely replicates human disease because mice develop granulomas with central caseous necrosis. Utilizing a susceptible model for M. avium infection, this study investigated the effect of mycobacterial pathogenesis on altering macrophage phenotypes and T cells distribution in areas of pulmonary granulomatous inflammation. METHODS:12 formalin fixed paraffin embedded (FFPE) lung sections from M. avium infected B6.Sst1S and B6 mice were examined microscopically (12 weeks post infection (wpi) n=5, 16 wpi=7). A targeted histology approach was initiated by using MRI coordinates to dictate the depths at which formalin fixed paraffin embedded (FFPE) lung samples were sectioned. Since interpretation of MRI images displayed no evidence of 2 discrete necrotizing granulomas, lungs were cut at sections representative of diffuse pathology at 2 mm into FFPE blocks. Using the Opal MethodTM (Akoya Biosciences), 6- plex immunohistochemical staining was performed with Arginase-1 (Arg1), inducible nitric oxide synthase (iNOS), CD68, CD3, M. tuberculosis antigen (cross-reacts with M. avium) and DAPI to segment nuclei. Slides were digitized by a Vectra PolarisTM fluorescent whole slide scanner. Autofluorescence was removed by InFormTM, and image analysis (IA) was conducted using HaloTM IA software. Statistical analysis was conducted using GraphPad PrismTM 8.0. RESULTS: Sst1 mediated susceptibility was statistically evident at 16 wpi but not at 12 wpi. B6.Sst1S mice showed a statistically significant (P <0.05) increase in M. avium+ cell expression in the non-inoculated lung lobes, but not the inoculated lung lobes. Pulmonary lesions within the inoculated and non-inoculated lung lobes contain different immune signatures. The predominately primary lesions of the inoculated lung lobes were associated with increased CD3+, M. avium+, and iNOS+ cell levels. When controlling for level of infection, there was lower levels of CD3+ cells within granulomatous lesions of B6.Sst1S mice, especially in the non-inoculated lung lobe. Controlling for level of infection also revealed elevated iNOS+ M. avium- cell expression in B6 mice. We observed elevated Arg1+ cell expression near iNOS+ M. avium+ cells, and, qualitatively, around larger lesions. T cell proximity analysis was contradictory and offers lessons for future the development of future IA modules. CONCLUSIONS: Sst1 mediated susceptibility was evident at 16 wpi and predominately mediated through secondary, metastatic lesions. Sst1 mediated susceptibility was also associated with fewer supportive cells (T cells and iNOS+ M. avium- cells) within granulomatous lesions. Future studies are necessary to evaluate to what degree granulomatous lesion Arg1+ cell expression and CD3+ proximity correlate to susceptibility.

Acute viral infections induce CD8 memory T cells that play an important role in the protection of the host upon re-infection with the same pathogen. These virus epitope-specific memory CD8 T cells develop complex TCR repertoires that are specific for that epitope. As individuals age virus-specific immunity appears to wane. Older people have difficulty controlling infection with common viruses such as influenza A (IAV), a RNA virus which causes recurrent infections due to a high rate of genetic mutation, and Epstein Barr virus (EBV), a DNA virus which persists in B cells for life in the 95% of people that become acutely infected. Many factors may contribute to this waning immunity including changes in virus-specific TCR repertoires. We hypothesize that epitope-specific memory CD8 TCR repertoires to these two common viruses change with increasing age and that CD8 T cell cross-reactivity may be one of the mechanisms mediating these changes. To address this hypothesis in our first study, we compared epitope-specific CD8 memory TRBV repertoires directly ex vivo for these two common human viruses. In cross-sectional and longitudinal studies of EBV seropositive, HLA-A2+, young (18-22 years), middle age (25-59 years), and older (>60 years) donors, we demonstrated that CD8 memory TCR repertoires to three immunodominant epitopes, known to have cross-reactive responses, IAV-M158-66, EBV-BM280-288, and EBV-BR109-117 co-evolve as individuals age. Cross-sectional studies showed that IAV-M1-and both EBV-specific repertoires narrowed their TRBV usage by middle-age. In fact, narrowing of EBV-BM and EBV-BR-specific TRBV usage correlated with increasing age. Although narrowing of IAV-M1-specific TRBV did not directly correlate with increasing age there was clear evidence that the TRBV usage was changing with age. The dominant TRBV19 usage appeared to become bimodal in the older age group and interestingly TRBV30 usage did directly correlate with age. For the EBV epitope-specific responses there was preferential usage of particular TRBV and changes in the hierarchy of TRBV usage in the different age groups. Longitudinal studies tracking 3 donors over 10-15 years (middle age to older) showed that there were changes in the TCR repertoire of IAV-M1, EBV-BM and -BR-specific responses over time. In two of the donors who experienced acute IAV infection there was evidence these repertoire changes may be influenced by TCR cross-reactivity, which is enhanced during acute IAV infection. The results of this first ex vivo study are consistent with our hypothesis. They suggest that virus-specific TCR repertoires change over time as an individual ages leading to narrowing of the repertoire and may co-evolve in the presence of CD8 T cell cross-reactivity. To further test our hypothesis in a second study we compared CD8 memory TRAV and TRBV repertoires to the three immunodominant epitopes IAV-M1, EBV-BM, and EBV-BR in the two extreme age groups, young donors (YSP) (18-22 years) and older donors (OSP) (>60 years) using the same donors as in the first study. Since these three epitopes are known to generate cross-reactive CD8 T cell responses and humans during their lifetime are frequently infected with both viruses at the same time these studies were also designed to more closely examine if TCR cross-reactivity could contribute to changes in TCR repertoire with increasing age. We examined the differences in both TRAV and TRBV in these two age groups by monoclonal antibody (mAb) staining and by deep sequencing and single cell sequencing in tetramer positive sorted cells from short-term cultures. Our initial studies showed that there were strong correlations in TRBV usage between short-term cultured and ex vivo antigen-specific responses; functional differences as well as differences in TRBV usage and diversity as measured by mAb staining particularly for the EBV epitope-specific responses between YSP and OSP donors. The TCR deep sequencing data also showed significant differences in TRBV usage between YSP and OSP. However, there were many more differences in TRAV and TRAJ usage than TRBV between the age groups suggesting that TRAV may play a greater role in evolution of the TCR repertoire. With increasing age, there was a preferential selection or retention of TCR for all three epitopes that have features in their CDR3a and b that increase their ease of generation, such as greater usage of convergent recombinant amino acids, and increase cross-reactive potential, such as multiple glycines. YSP and OSP differed in the patterns of TRAV/AJ and TRBV/BJ pairings and usage of dominant CDR3 motifs in all three epitope responses. Both YSP and OSP had cross-reactive responses between these 3 epitopes which were unique and differed from the cognate responses. Analyses of single cell sequencing data suggested that unique combinations of TRAV and TRBV are occurring, where one chain has features consistent with interaction with antigen one and the other chain had features consistent with interaction with antigen two. Interestingly, both the deep sequencing and single cell data show an increased tendency for the classic IAV-M1 specific clone BV19-IRSS-BJ2.7/AV27-CAGGGSQGNLIF-AJ42 to appear among the cross-reactive clones, suggesting that the dominance of this highly public clone may relate to its cross-reactivity with EBV. These results suggest that although OSP and YSP retain some of the classic TCR features for each epitope the TCR repertoire is gradually changing with age retaining TCR that are cross-reactive between these two common human viruses that we are exposed to frequently, one with recurrent infections and the other a persistent virus which frequently reactivates. These results are highly supportive our hypothesis and their importance in relation to viral immune-pathogenesis and potential novel immunotherapies will be discussed. These studies further emphasize the complexity and potential importance of human virus-specific T cell responses and TCR repertoires as people age and the need for a better understanding of TCR cross-reactivity between different viruses. For instance, at the present time these studies are highly relevant to better understanding the immune-pathogenesis observed during the COVID19 pandemic.

Crosspresentation allows antigen presenting cells to present peptides from exogenously derived antigens onto MHC Class I for presentation to CD8+ T cells. Though this pathway shares key players with the Classical Class I and Class II pathways, several questions remain. A genomewide siRNA screen was performed to look for genes that selectively affected the crosspresentation or the Class II pathways. Among the genes identified in the screen was the Rab GTPase Rab39a. Rab39a was required for efficient crosspresentation but was dispensable for the presentation of endogenously expressed antigen. Both TAP-dependent and independent antigen required Rab39a for efficient presentation. Rab39a localized to late endosomes and phagosomes, though interestingly it was not required for the Class II pathway. Analysis of phagosomes from Rab39a KO or rescued cells has shown that in the presence of Rab39a, phagosomes were enriched for the open form of MHC Class I as well as TAP1, a member of the peptide loading complex. The enriched open form of MHC-I was peptide receptive, suggesting that it could contribute to crosspresentation. Phagosomes from Rab39a positive cells had reduced degradative capability and had increased levels of Sec22b, a SNARE protein reported to deliver ER-golgi sourced cargo to phagosomes. Furthermore, inhibition of ER-golgi transport via brefeldin A abolished the phenotype conferred by Rab39a. Thus, we hypothesize that Rab39a mediates the delivery of ER-golgi derived cargo to the antigen containing phagosome. This delivery allows peptide receptive MHC-I, as well as the peptide loading complex to reach the antigen, thereby facilitating crosspresentation.

Malarial infection in naïve individuals induces a robust innate immune response, but our understanding of the mechanisms by which the innate immune system recognizes malaria and regulates its response remain incomplete. Our group previously showed that stimulation of macrophages with Plasmodium falciparum genomic DNA (gDNA) and AT-rich oligodeoxynucleotides (ODNs) derived from this gDNA induces the production of type I interferons (IFN-I) through a STING/TBK1/IRF3-dependent pathway; however, the identity of the upstream cytosolic DNA receptor remained elusive. Here, we demonstrate that this IFN-I response is dependent on cyclic GMP-AMP synthase (cGAS). cGAS produced the cyclic dinucleotide 2’3’-cGAMP in response to P. falciparum gDNA and AT-rich ODNs, inducing IRF3 phosphorylation and IFNB transcription. In the recently described model of innate immune memory, an initial stimulus primes the innate immune system to either hyperrespond (termed “training”) or hyporespond (“tolerance”) to subsequent immune challenge. Previous work in mice and humans demonstrated that infection with malaria can both serve as a priming stimulus and promote tolerance to subsequent infection. In this study, we demonstrate that initial stimulation with P. falciparum-infected red blood cells (iRBCs) or the malaria crystal hemozoin (Hz) induced human adherent peripheral blood mononuclear cells (PBMCs) to hyperrespond to subsequent Toll-like receptor (TLR) challenge. This hyperresponsiveness correlated with increased H3K4me3 at important immunometabolic promoters, and these epigenetic modifications were also seen in monocytes from Kenyan children naturally infected with malaria. However, the use of epigenetic and metabolic inhibitors indicated that malaria-induced trained immunity may occur via previously unrecognized mechanism(s).

Necroptosis, a type of regulated necrotic cell death, involves cell membrane permeabilization and has been implicated in various acute and chronic pro-inflammatory diseases, including ischemia-reperfusion injury and neurodegenerative diseases. By using in vitro reconstitution studies and a chemical inhibitor, the kinase activity of the serine/threonine kinase RIPK1 had been shown to regulate necroptotic signaling downstream of TNF and Toll-like receptors (TLRs). To investigate the contribution of RIPK1 kinase activity to inflammation and necroptosis in vivo, we generated kinase inactive RIPK1 knock-in mice. Utilizing fibroblasts and macrophages from these mice, we demonstrate that RIPK1 kinase activity is required for necroptotic complex formation and death induction downstream of TNFR1 and TLRs 3 and 4. We show that RIPK1 kinase inactive mice are resistant to TNF-induced shock and exhibit impaired upregulation of TNF-induced cytokines and chemokines in vitro and in vivo. By using bone marrow reconstitution experiments, we demonstrate that RIPK1 kinase activity in a non-hematopoietic lineage drives TNF-induced lethality. We establish that RIPK1 kinase activity is required for TNF-induced increases in intestinal and vascular permeability and clotting, and implicate endothelial cell necroptosis as an underlying factor contributing to TNF/zVAD-induced shock. Thus, work in this thesis reveals that RIPK1 kinase inhibitors may have promise in treating shock and sepsis.

Mycobacterium tuberculosis (Mtb) causes human tuberculosis, and more people die of it than of any other pathogen in the world. Immunodominant antigens elicit the large majority of T cells during an infection, making them logical vaccine candidates. Yet, it is still unknown whether these immunodominant antigen-specific T cells recognize Mtb-infected cells. Two immunodominant antigens, TB10.4 and Ag85b, have been incorporated into vaccine strategies. Surprisingly, mice vaccinated with TB10.4 generate TB10.4-specific memory CD8+ T cells but do not lead to additional protection compared to unvaccinated mice during TB. Ag85b-specific CD4+ T cells are also generated during vaccination, but the literature on whether these cells recognize Mtb-infected cells is also inconsistent. We demonstrate that TB10.4-specific CD8+ T cells do not recognize Mtb-infected cells. However, under the same conditions, Ag85b-specific CD4+ T cells recognize Mtb-infected macrophages and inhibit bacterial growth. In contrast, polyclonal CD4+ and CD8+ T cells from the lungs of infected mice can specifically recognize Mtb-infected macrophages, suggesting macrophages present antigens other than the immunodominant TB10.4. The antigen location may also be critical for presentation to CD8+ T cells, and live Mtb may inhibit antigen presentation of TB10.4. Finally, we propose that TB10.4 is a decoy antigen as it elicits a robust CD8+ T cell response that poorly recognizes Mtb-infected macrophages, allowing Mtb to evade host immunity.

Sepsis is dysregulated host immune response to infection causing life-threatening organ dysfunction. Endothelial cell dysfunction and uncontrolled inflammatory responses are two contributors for sepsis-induced mortality. The crosstalk between endothelial and immune cells plays a critical role in the pathophysiology of sepsis. Therefore, understanding the mechanism of interaction between endothelial and immune cells will provide novel information to develop therapeutic strategies for sepsis. Pathogen associated moleculear patterns (PAMPs) and/or damage associated molecular patterns (DAMPs) produced during sepsis, activate endothelial cells to increase the expression of adhesion molecules, attracting immune cell infiltration into the tissues. Uncontrolled inflammatory responses during the early phase of sepsis contribute to organ failure and lethality. Over 100 clinical trials, targeting inflammatory responses in sepsis, have failed in the past three decades. Thereby, developing novel therapeutic strategies for sepsis are urgent. Heat shock protein A12B (HSPA12B), as one member of HSP70 family, predominately expressed in the endothelial cells, plays important roles in many pathophysiological processes. Currently, we observed endothelial cell specific HSPA12B deficiency (HSPA12B-/-) exacerbates mortality in sepsis induced by cecal ligation puncture (CLP). HSPA12B-/- septic mice exhibits increased expressions of adhesion molecule and infiltrated macrophages in the myocardium and activated macrophages in the peritoneal cavity. In vitro studies show that HSPA12B could be secreted from endothelial cells via exosome. HSPA12B carried by exosomes can be uptaken by macrophages to downregulate macrophage NF-kB activation and pro-inflammatory cytokine production. Trained immunity, induced by β-glucan, causes immune memory in innate immune cells, with an altered response towards another challenge. We have found that mice received β-glucan seven days before CLP sepsis exhibit attenuated mortality with decreased pro-inflammatory responses. We found that β-glucan significantly increased the levels of HSPA12B in endothelial cells and endothelial exosomes. β-glucan induced endothelial exosomes markedly suppress macrophage NF-kB activation and pro-inflammatory responses. The current data suggests that HSPA12B plays a novel role in the regulation of immune and inflammatory responses and that HSPA12B could be an important mediator for the crosstalk between endothelial cells and macrophages during sepsis. β-glucan regulates endothelial cell functions and immune/inflammatory responses, thus improving survival outcome in CLP sepsis.

No description available.

INTRODUCTION: SARS-CoV-2 is a novel virus from the coronavirus family that emerged in the Hubei province of China in December 2019 and rapidly spread throughout the world. On March 11, 2020, the World Health Organization declared a global pandemic. Infection with SARS-CoV-2 causes coronavirus disease 19 (COVID19) which can be fatal. There is an obvious and pressing need for research surrounding SARS-CoV-2 that will aid in eradication of this pandemic. OBJECTIVE: The goal of this study was to absolve the dire need for small animal models of human disease that demonstrate hallmark pathological features of infection. Due to ethical and financial obstacles, the use of animals that closely resemble human immunity, such as non-human primates, is often not a viable option. For this reason, there is a push to develop small animal models that can mimic human disease responses, particularly those in viral infections that have a narrow species tropism. To achieve this in the context of the novel coronavirus, SARS-CoV-2, we studied various mouse models and their capacity to become infected with and mount an immune response to SARS-CoV-2. Our goal was to identify a model that sufficiently mimics severe COVID19 seen in humans as well as provide molecular insight into pathways that prevent the development of severe disease. METHODS: NRG-L and HIS-NRGF-L mice were subcutaneously implanted with human fetal lung xenografts and infected with SARS-CoV-2. Tissues were stained with H&E for histopathological scoring. NRG-L and HIS-NRGF-L tissues were fluorescently labeled using 2 different multiplex immunohistochemistry panels. Slides were digitized by a Vectra Polaris™ fluorescent whole slide scanner and digital analysis was completed using HALO™. Statistical analysis was conducted using GraphPad Prism™ 9.0.1. RESULTS: Infected NRG-L mice present extensive histopathological manifestations when compared to uninfected controls. Cumulative histology scores at both 2 and 7DPI were increased when compared to uninoculated fLX. Neutrophil influx, intra-airspace necrosis, capillary fibrin thrombi, and presence of syncytial cells were the most significant independent observations that contributed to the increased cumulative score. Together these findings indicate that fLX inoculated with SARS-CoV-2 faithfully recapitulate several features of diffuse alveolar damage (DAD) described in severe COVID-19. HIS-NRGF-L mice displayed decreased influx of neutrophils, intra-airway necrosis, and syncytial cells when compared to NRG-L fLX. Hemorrhage was decreased at both 2 and 7 DPI for HIS-NRGF-L fLX compared to NRG-L fLX. Cumulative histology scores were decreased in HIS-NRGF-L fLX at 7 DPI when compared to NRG-L fLX. Taken together these findings support the hypothesis that human myeloid and lymphoid infiltrates suppress or prevent the disparate host response observed in NRGL-L fLX that manifested in pronounced diffuse alveolar damage. CONCLUSION: Using digital image analysis of multiplex immunohistochemistry paired with semi-quantitative histopathological scoring, this study characterized important hallmark lesions observed in severe COVID19 as seen in small animal models. NRG-L and HIS-NRGF-L mice that are subcutaneously implanted with human fetal lung xenografts are susceptible to infection with SARS-CoV-2 and can produce severe and moderate disease phenotypes respectively. Co-engraftment with human fetal lung tissue and human immune system components in HIS-NRGF-L mice suppresses the divergent host response that is observed in NRG-L mice. For this reason, NRG-L mice engrafted with fLX are an ideal small animal model of severe COVID19, whereas HIS-NRGF-L mice severe as a valuable and informative model for deciphering molecular mechanisms driving severe COVID-19 that will serve as targets for therapeutic development.