Pathobiology

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Her Mucosa, Her Rules: Regulation of Memory T Cells in the Female Genital Tract
(2026-04-20) Cruz Talavera, Irene; Lund, Jennifer M
The lower female genital tract (FGT) is a mucosal barrier and site of exposure to various pathogens, including fungi, parasites, bacteria, and viruses. The FGT faces the unique challenge of maintaining tolerance to a semi-allogenic fetus and the optimal vaginal microbiome, in addition to preventing infection through cell-mediated surveillance for pathogens and subsequent measured immune activation without excessive damage to host tissues. The lower FGT (vagina and ectocervix) is covered in multi-layered squamous epithelial cells, whereas the upper FGT (endocervix, uterus and fallopian tubes) is covered by a single layer of columnar epithelial cells. Beneath the epithelium is a layer of stromal fibroblasts providing structure to the tissue. Dynamic populations of immune cells are distributed throughout the stroma, with T lymphocytes being the most abundant immune cell subset within the lower FGT. Mechanisms involved in immune responses within the FGT are further complicated by changes in sex-hormones throughout an individual’s life. In addition to regulating changes in epithelial and stromal structural reorganization and repair, hormonal fluctuations throughout the menstrual cycle can also participate in modulation immune cell function to maintain tolerance to innocuous antigens and protection against pathogens. Tissue-resident memory T cells (Trm) mediate protection within tissue sites of prior or persisting infection. Upon pathogen re-exposure and antigen recognition, Trm elicit robust immunity locally within the tissue and orchestrate the recruitment of other T cells and innate immune cells. To avoid excessive tissue damage associated with this robust tissue immune reaction, we predicted that tissue recall T cell responses must be subject to regulation. We previously demonstrated that regulatory T cells (Treg) accumulate within infected tissue and coordinate early immune responses and T cell priming during primary infection, though their role in shaping tissue memory T cell responses remains unclear. Using transient Treg depletion in mice, we show a requirement for Treg in limiting tissue pathology following vaginal HSV-2 challenge, despite normal viral clearance. While Treg-depleted mice exhibit an elevated frequency and number of vaginal CD44+Ki67+ T cells and granzyme-B+CD8+ T cells by day 3 post-challenge, there is no difference in the magnitude of the antigen-specific CD8+ T cell response. Using adoptive transfer of TCR-transgenic OT-1 CD8 T cells, we show that Treg-depleted mice have a significantly stronger vaginal bystander-activated cytotoxic T cell (BA-CTL) response upon challenge. In vivo antibody blocking demonstrates that Treg limit cytotoxicity of both HSV-2-specific CD8 and BA-CTL responses via an IL-2-dependant mechanism that additionally restricts IL-15 trans-presentation by antigen presenting cells. Our findings highlight Treg’ role in selectively restraining cytotoxic function during a tissue recall response, while preserving the pathogen-specific tissue T cell response, to balance viral clearance with restriction of immunopathology. In addition to regulation by Treg, immunity can also be modulated by fluctuations in sex hormones. Characterization local FGT and systemic T cell responses throughout the menstrual cycle is paramount to better understanding mechanisms underlying susceptibility to sexually transmitted infections and correlates of protection for improved design of vaccines and therapeutics. We sought to characterize the impact of menstrual phase on local cervicovaginal and systemic T cell responses in Kenyan women. Using high-parameter, high-throughput flow cytometry and a large panel we compared follicular vs luteal phase phenotypes in T cell populations isolated from vaginal tract (VT) and ectocervix (CX) mucosal tissue biopsies and PBMC samples. Given the abundance of T cells in the lower FGT, cervicovaginal tract (CVT) biopsies may better capture the mucosal immune environment than other minimally invasive sampling methods such as cervicovaginal lavage, vaginal/cervical swabs, or cytobrushes. Additionally, we evaluated 71 immunomodulatory molecules in serum and cervicovaginal secretions in follicular vs luteal phase. Overall, we did not find changes in the frequencies of T cell subsets (CD4+, CD8+, Treg) or increases in HIV susceptibility markers on CD4+ T cells (CCR5, HLA-DR, CD38) between follicular vs luteal phase in any of the tissue sample types. Interestingly, all phenotypic differences were observed in CD8+ T cells within CX and VT. Specifically, CCR7+ CD45RA- central memory CD8 T cells were significantly elevated during the luteal phase in both the CX and PBMCs, whereas CCR7- CD45RA+ terminally differentiated effector memory T cells CD8 T cells were more prevalent in the CX during the follicular phase. Additionally, CD8 T cells in the CX during the luteal phase were more activated, exhibiting higher frequencies of HLA-DR, CD38, and the exhaustion marker CD39. We also found altered expression of soluble factors in follicular phase, with increased concentrations of CCL15 and IL-2 in cervicovaginal secretions and increased CXCL12 in serum samples. These findings suggest that menstrual phase modulates CD8 T cell memory subsets and intrinsic regulation of activation, providing new insights into how hormonal fluctuations influence FGT immunity to infection.
Distinct protective roles of CD4 and CD8 T cells in vaccine-mediated immunity against Mycobacterium tuberculosis
(2025-01-23) Barrett, Holly Wei-Ling; Urdahl, Kevin B
Despite widespread vaccination with Bacille Calmette-Guerin (BCG) globally, tuberculosis (TB) remains a major global health concern. A new vaccine is urgently needed, but our limited understanding of immunity against Mycobacterium tuberculosis (Mtb) infection is a major barrier to designing more effective vaccines. Mice are commonly used to study TB immunity but deficiencies in the current conventional dose (CD) (50-100 CFU) mouse model have left little confidence in its translation to humans. Our lab has overcome these limitations by developing a physiologic ultra-low dose (ULD) (1-3 CFUs) Mtb infection, which resembles several aspects of human Mtb infection. Here, we assess vaccine-mediated protection using the ULD model and identify three parameters of BCG-mediated protection: 1. Durable reduction in overall lung burden, 2. Prevention of dissemination to the contralateral lung, and 3. Prevention of detectable infection in a subset of BCG vaccinated mice. Importantly, this is the first evidence that BCG vaccination can mediate sterilizing immunity in any mouse model of TB. Next, we sought to determine the lymphocyte subsets responsible for BCG-mediated immunity. We investigated the impact of BCG vaccination on early Mtb immunity before confounding differences in bacterial burden occurred. We found that BCG induced a robust effector CD4 and CD8 T cell response in the lung during early Mtb infection while changes in lung B cells were less pronounced. Furthermore, T cell depletion reversed the protection conferred by BCG, whereas B cell deficiency had no effect. Finally, we assessed the relative roles of CD4 and CD8 T cells in BCG-mediated immunity and found that CD4 and CD8 T cells promoted distinct aspects of immunity. CD4 T cells played larger roles in control of lung burden and prevention of dissemination to the contralateral lung, whereas CD8 T cells were more important for prevention of infection and played a minor role in control of lung burden. These newfound roles of CD8 T cells in BCG-mediated protection were masked using the supraphysiologic conventional dose challenge and could only be detected in the ULD model. Together, these studies further our understanding of vaccine-mediated protection against Mtb and highlight the ULD model as a superior platform for pre-clinical vaccine testing. Additionally, our work demonstrates distinct roles for CD4 and CD8 T cells in vaccine-mediated immunity. Targeting both aspects of immunity could be key to designing a more effective TB vaccine.
Determining the Major Genetic Regulators of Mycobacterium tuberculosis-induced Cytokine Expression in Monocytes and Macrophages with a Cellular GWAS
(2024-04-26) Ivie, Joshua; Hawn, Thomas
Tuberculosis (TB), largely due to infection with the causative pathogen Mycobacterium tuberculosis (Mtb), has claimed over 1 billion lives in the past 200 years, including 1.3 million lives in 2022 alone. Efforts to eradicate TB have been complicated by heterogeneity in infection outcomes after Mtb exposure. Host genetics may contribute as much as half of an individual’s susceptibility to certain infection outcomes, however, the causal variants responsible remain unknown. 70,000 years of human infection has led to intricate pathogen and host adaptations that fight for control of crucial infection responses. In particular, the macrophage cytokine response is heavily modulated by both pathogen and host and requires a fine-tuned balance for successful control of infection. Prior studies attempting to define genetic regulators of Mtb infection have often focused on genome-wide association studies (GWAS) measuring clinical phenotypes. Acellular GWAS, which measures genetic regulation of the cytokine response to live Mtb infection, in vitro, may identify important biology, and has not been assessed previously. This dissertation aimed to assess if Mtb-induced human monocyte and macrophage cytokine responses are genetically controlled and if a cellular GWAS approach can be used to identify genetic regulators that are important in modulating immune and clinical TB response outcomes. Using Mtb-induced cytokine expression profiles from individuals in a Ugandan cohort, we performed WGCNA, heritability, and cellular GWAS analyses. These findings were further assessed in independent cohorts from South Africa and Seattle, and in subsequent in vitro validation and mechanistic investigation. Initial expression profiling identified 35 hub genes which were central to the Mtb-induced cytokine response, and determined a heritable component of Mtb-induced IL1B and IL6 expression. Our cellular GWAS in Ugandan individuals identified several SNPs surpassing suggestive significance and a significant enrichment of Mtb-induced TNF association in the genes of two pathways. Further investigation revealed that one of these pathways, alpha-linolenic acid metabolism, was validated in vitro using PLA2 inhibitors. We further identified multiple SNPs which showed population spanning effect when assessed in the Seattle population and validated cytokine effect for two of these SNPs which mapped to the genes SLIT3 and SLC1A1. SLIT3 was additionally found to enhance Mtb intracellular replication. Finally, SNPs from both of our in vitro validated loci, associated with SLC1A1 and SLIT3, were associated with the clinical TB phenotypes, susceptibility to tuberculous meningitis (TBM), and TBM survival, respectively. Taken together, this research identified multiple genetic regulators of the Mtb-induced myeloid cell cytokine response which were validated in vitro, and shows the promise of the cellular GWAS approach for identifying novel host response factors with clinical relevance.
Immunogenicity and protection mediated by next-generation EBV gH/gL vaccines in non-human primate and humanized mouse models of infection
(2024-04-26) Edwards, Kristina; McGuire, Andrew T
Epstein Barr Virus (EBV) is an orally transmitted, γ-herpesvirus infecting 90% of adults worldwide. Infection is associated with various cancers and development of multiple sclerosis. A vaccine that prevents infection and reduce the global burden of EBV-associated disease is an urgent, yet unmet need. EBV infects epithelial cells and B cells, therefore an effective vaccine would likely have to block infection of both these cell types. The viral gH/gL glycoprotein complex is essential for entry, making it an attractive vaccine target. Additionally, antibodies against gH/gL have been shown to be protective against infection in vitro and in vivo. In this work we evaluate two different next-generation vaccine platforms to immunize animals with EBV gH/gL. In one project we evaluated the immunogenicity of a multimeric gH/gL nanoparticle vaccine administered with two different adjuvants, Sigma Adjuvant System (SAS) and Saponin/MPLA nanoparticles (SMNP), in rhesus macaques. Formulation with SMNP elicited higher titers of binding and neutralizing antibodies, and higher frequency of vaccine-specific IFNɣ+IL2+ CD4+ T cells. After oral challenge with the EBV orthologue rhesus lymphocryptovirus (rhLCV), all macaques in the control and SAS groups became infected, while one of four animals in the SMNP group remained aviremic and were seronegative against non-vaccine antigens, indicating protection. Further study revealed considerable antigenic disparity between the rhLCV gH/gL and EBV gH/gL. We found that our protected animal displayed a broader pattern of epitope recognition, as visualized by electron microscopy polyclonal epitope mapping, which may be linked to protection. The observed prevention of rhLCV infection in one macaque, despite the substantial antigenic disparity between the vaccine and challenge strain, supports the further pursuit of gH/gL nanoparticle vaccines against EBV. In a second project, we have developed and optimized an alphavirus-derived self-amplifying mRNA vaccine platform (repRNA) to immunize mice with EBV gH/gL. The lead candidate tested was able to elicit a vaccine-specific CD8+ T cell response as well as high-titers of neutralizing antibodies that persisted for at least 8 months post immunization. Transfer of vaccine-elicited IgG protected humanized mice from EBV-driven tumor formation and death following high-dose viral challenge. These data demonstrate that repRNA-gH/gL formulated with a localizing cationic nanocarrier (LION) is a promising candidate vaccine for preventing EBV infection and/or related malignancies in humans and encourage clinical development of vaccines targeting EBV gH/gL. A third work-in-progress section describes a potential route of Fc-mediated EBV infection. In sum, this work describes the generation of immunogenic next-generation vaccines targeting EBV gH/gL and highlights some of the challenges and considerations of using the two most common animal models for EBV to evaluate vaccine-elicited protection.
TRIM34 and TRIM5a co-operatively restrict primate lentiviruses
(2024-02-12) Twentyman, Joy; Emerman, Michael
Human immunodeficiency virus (HIV) and other lentiviruses adapt to new hosts by evolving to evade host-specific innate immune proteins that differ in sequence and often viral recognition between host species. Understanding how these host antiviral proteins, called restriction factors, constrain lentivirus replication and transmission is key to understanding the emergence of pandemic viruses like HIV-1. One such restriction factor is TRIM5α, which blocks replication by multimerizing onto the HIV core, inducing aberrant capsid uncoating. TRIM5α-mediated restriction requires multimerization of TRIM5α monomers. Human TRIM34, a paralogue of the well-characterized lentiviral restriction factor TRIM5α, was previously identified by our lab via CRISPR-Cas9 screening as a restriction factor of certain HIV and SIV capsids. Notably, TRIM34-mediated restriction requires TRIM5α. Thus, we propose that TRIM34 requires multimerization with TRIM5α to restrict lentiviral capsids. Here, we show that diverse primate TRIM34 orthologues from non-human primates can restrict a range of Simian Immunodeficiency Virus (SIV) capsids including SIVAGM-SAB, SIVAGM-TAN and SIVMAC capsids, which infect sabaeus monkeys, tantalus monkeys, and rhesus macaques, respectively. All primate TRIM34 orthologues tested, regardless of species of origin, were able to restrict this same subset of viral capsids. We demonstrate that TRIM5α is necessary, but not sufficient, for TRIM34-mediated restriction of these capsids, and that human TRIM5α functionally interacts with TRIM34 from different species. Finally, we find that both the TRIM5α SPRY domain, in particular the v1 loop, and the TRIM34 SPRY domain are essential for TRIM34-mediated restriction. These data suggest that TRIM34 is a conserved primate lentiviral restriction factor and that TRIM34 and TRIM5α interact with each other and capsids. This supports a model in which TRIM34 is a broadly conserved primate lentiviral restriction factor that acts in tandem with TRIM5α, such that together, these proteins can restrict capsids that neither can restrict alone. The goals of this work are to define the range of primate lentiviruses against which TRIM34 is active, identify determinants of antiviral specificity for TRIM34-mediated restriction, to identify domains of TRIM34 and TRIM5α that are required for lentiviral restriction. Ultimately, these studies can help lead to a better understanding of TRIM34’s role in host-pathogen evolutionary history.
The Role of Enteric RORγt+ Regulatory T Cells in Altering Systemic Vaccine Responses Following Oral Antigen Exposure
(2023-09-27) Potchen, Nicole; Kublin, James G; Lund, Jennifer M
Many promising vaccine candidates and licensed vaccines lead to variable immune responses within humans and can fail to elicit protection against critical pathogens. Recent studies suggest that environmental exposures in the gastrointestinal (GI) tract could contribute to a reduction in vaccine efficacy via immune tolerance at this site. Tolerogenic mechanisms in the GI tract are partly achieved by the characteristic high abundance of regulatory T cells (Tregs). Subsets of regulatory T cells have been recently described; specifically, RORγt+ FoxP3+ Tregs could contribute to tolerating orally acquired antigens. This subset relies on the presence of commensal bacteria for development and has been described in regulating Type 2 associated immune responses. Currently, it is unclear which Treg subsets control systemic vaccine responses following oral antigen pre-exposure. In this study, we implemented a conditional RORγt+ Tregs knock-out (cKO) mouse model to examine the role of this subset in the suppression of systemic antibody titers after oral exposure to vaccine antigen. Following oral exposure to the model antigen ovalbumin (OVA) prior to immunization, we found similar induction of vaccine-induced antibody responses in mice lacking RORγt+ Tregs compared to sufficient controls. cKO mice exhibit higher frequencies of IL-33R+ and GATA3+ Tregs at baseline in the gut, suggesting that there is possible compensation from other subsets upon loss of expression of RORγt+ in Tregs. By utilizing a variety of adjuvants in our model, we have determined that vaccine-specific IgG1 is suppressed following oral antigen exposure, but not IgG2c, regardless of adjuvant strength or skew. In RORγt+ Treg cKO mice, use of different adjuvants led to similar findings; no differences were observed in the maintenance of tolerance to systemic OVA vaccination in cKO mice upon use of various adjuvants. Methods were explored to understand antigen-specific CD4+ response upon oral exposure prior to systemic vaccination; adoptive transfer of OVA-specific OT-II cells will continue to be explored in future studies. Taken together, our data suggest that RORγt+ Tregs are not solely responsible for controlling tolerance to oral antigens in a murine model of tolerance to a systemic vaccine.
Multiple Receptor Tyrosine Kinases Regulate Dengue Infection of Hepatocytes
(2023-09-27) Bourgeois, Natasha Marie; Kaushansky, Alexis
Currently, there is no way to constrain dengue virus (DENV) infections in the clinic. My dissertation work explored the hypothesis that host kinase regulators of DENV infection have the potential to be therapeutically disrupted. I performed a thorough review of literature on host factors regulating dengue and summarized the growing body of evidence supporting kinase-targeted interventions for DENV infection and disease. I also conducted experimental work to elucidate kinases regulating DENV infection that can be targeted by existing drugs. Specifically, I performed kinase regression (KiR), an innovative tool that predicts kinase regulators of infection using existing drug-target information and a small drug screen. From this, thirty-six kinases were predicted to have a functional role in DENV infection. I investigated the role of the predicted receptor tyrosine kinases (RTKs) – EPH receptor A4 (EPHA4), EPH receptor B3 (EPHB3), EPH receptor B4 (EPHB4), erb-b2 receptor tyrosine kinase 2 (ERBB2), fibroblast growth factor receptor 2 (FGFR2), Insulin like growth factor 1 receptor (IGF1R), and ret proto-oncogene (RET) – because there is already an existing repertoire of drugs against RTKs in the clinic. I found that predicted RTKs are expressed at higher levels in DENV infected cells, and that the activity of ERBB2 and IGF1R is induced following infection. I also demonstrated that knockdown of ERBB2, FGFR2 and IGF1R reduces DENV infection in some cases. I discuss therapeutic strategies that my work suggests could block dengue infection in the clinic and highlight further research that should be done to bolster these strategies. In addition to my primary dissertation research, I collaborated with colleagues to publish a research article uncovering how DENV interacts with mammalian target of rapamycin (MTOR) to support infection, as well as a review article highlighting how identification of host regulators across viral infections, malaria, and cancer have informed therapeutic strategies.
Preclinical optimization of a prime-and-trap malaria vaccine
(2023-08-14) Watson, Felicia; Murphy, Sean C
Malaria is a deadly parasitic disease that disproportionally impacts infants and children under five years old. A more effective vaccine is urgently needed to reduce the global burden of malaria. The liver stage (LS) is a critical bottleneck in the parasite lifecycle that if blocked, would prevent blood stage infection, clinical disease, and transmission. Radiation-attenuated sporozoite (RAS) vaccines are critical for sterile LS protection in mice, non-human primates (NHP), and humans by inducing antibodies and CD8+ T cells, including liver resident memory CD8+ T cells (Trm) that are critical for long-term protection. Such T cells can also be induced by a novel two-step vaccine strategy called prime-and-trap that was designed to simplify and improve upon RAS-only vaccination. Prime-and-trap combines DNA priming against theimmunodominant circumsporozoite protein (CSP) with a subsequent intravenous (IV) dose of liver-homing RAS to “trap” the activated and expanding T cells in the liver. This strategy induces durable protective CSP-specific CD8+ liver Trm cells in mice, and efforts are underway to translate this vaccine strategy to NHPs and humans. Reducing the RAS dose and/or determining if RAS must be strictly administered by the IV route would further enhance the translational potential of the prime-and-trap malaria vaccine. This dissertation provides preclinical research aimed at enhancing the efficacy of the RAS trapping component of the prime-and-trap vaccine. This dissertation aimed to optimize the translational potential of RAS trapping by co-administration with the potent glycolipid adjuvant 7DW8-5 to 1) reduce the dose, and 2) improve the administration route to be more suitable for clinical use. Additionally, since these studies revealed drastic differences between protection in male and female mice, this dissertation also explored if the addition of the 7DW8-5 adjuvant could enhance protection in male mice. First, this study found that in female mice, IV-administered freshly-dissected RAS could be replaced with the more desired but less immunogenic cryopreserved RAS and that the dose of IV-RAS could be reduced four-fold by co-administration with 7DW8-5. Next, the study found intradermal (ID) co-administration of RAS and 7DW8-5 in ultra-low volumes (2.5 μL) was completely protective and dose sparing compared to standard ID volumes (10-50 μL) and induced protective levels of CSP-specific CD8+ T cells in the liver. The finding that adjuvants and ultra-low volumes are required for ID-RAS efficacy may explain why prior reports about higher volumes of unadjuvanted ID-RAS proved less effective. Finally, the study found that male mice were not protected from any prime-and-trap vaccine regimen (IV-RAS or ID-RAS) with or without 7DW8-5. Further, the final studies found significant sex differences in cytokine expression induced by 7DW8-5 in mice. Additional studies are required to understand the mechanism of protection induced by IV- or ID-RAS and 7DW8-5 and to understand the dramatic protection differences observed between male and female mice. The ID route may offer significant translational advantages over the IV route and may improve RAS vaccine development. Taken together, this research found that the translational potential of the prime-and-trap malaria vaccine could be improved by the co-administration of 7DW8-5. These studies pave the way for further evaluation of prime-and-trap in NHPs and humans and future studies should continue to include sex as a biological variable.
ZIKV Protein Accumulation is a Major Regulator of Innate Immunity, Controlling Viral Replication and Spread
(2023-04-17) Lu, Amy; Gale, Jr., Michael
Asian lineage Zika virus (ZIKV) strains emerged globally, causing outbreaks linked with critical clinical disease outcomes unless ZIKV is effectively restricted by host immunity. We have previously shown that retinoic acid-inducible gene-I (RIG-I) senses ZIKV to trigger innate immunity to direct interferon (IFN) production and antiviral responses that can control ZIKV infection. However, ZIKV proteins have been demonstrated to antagonize IFN. Here, we conducted in vitro analyses to assess how divergent prototypic ZIKV variants differ in virologic properties, innate immune regulation, and infection outcome. We comparatively assessed African lineage ZIKV/Dakar/1984/ArD41519 (ZIKV/Dakar) and Asian lineage ZIKV/Malaysia/1966/P6740 (ZIKV/Malaysia) in a human epithelial cell infection model. De novo viral sequence determination identified amino acid changes within the ZIKV/Dakar genome compared to ZIKV/Malaysia. Viral growth analyses revealed that ZIKV/Malaysia accumulated viral proteins and genome earlier and to higher levels than ZIKV/Dakar. Both ZIKV strains activated RIG-I/interferon regulatory factor (IRF) 3 and nuclear factor kappa B (NFκB) pathways to induce inflammatory cytokine expression and types I and III IFNs. However, ZIKV/Malaysia, but not ZIKV/Dakar, potently blocked downstream IFN signaling. Remarkably, ZIKV/Dakar protein accumulation and genome replication were rescued in RIG-I knockout (KO) cells late in acute infection, resulting in ZIKV/Dakar-mediated antagonism of IFN signaling. We found that RIG-I signaling specifically restricts viral protein accumulation late in acute infection where early accumulation of viral proteins in infected cells confers enhanced ability to limit IFN signaling, promoting viral replication and spread. Our results reveal a novel function of RIG-I-mediated innate immune signaling in restricting ZIKV protein accumulation, which permits IFN signaling and antiviral actions that control ZIKV infection.
Impact of HIV Drug Resistance on Maternal Treatment Outcome & Vertical Transmission
(2023-01-21) Boyce, Ceejay; Frenkel, Lisa M.
HIV pretreatment drug resistance – defined as resistance detected prior to the initiation of antiretroviral therapy (ART) – has increased in prevalence over time in parallel with the increased global utilization of ART. The prevalence of pretreatment drug resistance has surpassed 10% to non-nucleoside reverse transcriptase inhibitors (NNRTI) in multiple countries, potentially threatening the efficacy of NNRTI-containing regimens which have been recommended for first-line ART in resource-limited settings for nearly two decades. In 2019, the WHO updated the recommended first-line regimen to integrase inhibitor-based ART to address the high levels of NNRTI pretreatment resistance; however, NNRTIs are still recommended in the alternative first-line regimen and for postnatal prophylaxis in infants to prevent vertical HIV transmission. The work presented in this dissertation sought to better understand the impact of HIV drug resistance on maternal treatment outcome and vertical transmission. We performed a series of clinical trial sub-studies that investigated genotypic HIV drug resistance – using Sanger and/or next-generation sequencing assays – among women living with HIV in resource-limited settings. Salient observations from the three studies include that (1) NNRTI resistance was prevalent among women prior to efavirenz-based ART initiation, but NNRTI resistance alone was not associated with virologic failure by 12 months of treatment; (2) maternal drug resistance was a risk factor for vertical transmission during breastfeeding, and drug resistance was prevalent among infants and accumulated during breastfeeding; and (3) pretreatment drug resistance among pregnant women living with HIV did not significantly contribute to virologic non-suppression at term; however, higher pretreatment HIV RNA load, randomization to efavirenz- versus integrase inhibitor-based ART, and shorter duration of ART were associated with non-suppression. Taken together, these results support WHO recommendations of early prenatal care engagement and initiation of integrase inhibitor-based ART for pregnant women living with HIV for optimal maternal outcomes and to prevent vertical transmission. However, for individuals who are unable to take integrase inhibitor-based ART, our data and others suggest the utility of TLE could be extended despite the high prevalence of NNRTI pretreatment resistance in resource-limited settings. Lastly, our findings suggest that NNRTI infant prophylaxis may be insufficient against drug-resistant variants in maternal breast milk and support the recommendation to replace NNRTI infant prophylaxis with regimens that have a greater barrier to drug resistance to prevent vertical transmission and retain NNRTI susceptibility in infected infants.
An innate-like role of memory T cells in HSV-2 immunity
(2023-01-21) Arkatkar, Tanvi; Lund, Jennifer M
Mucosal infections pose a significant global health burden and remain a top cause of mortality worldwide. Herpes Simplex Virus type-2 (HSV-2) infection is one of the most prevalent sexually transmitted infections, yet no vaccine is currently available. An understanding of the cellular composition of tissue-resident immune cells at the primary site of infection is essential for effective vaccine design for targeted immune response. Antigen-specific tissue-resident memory T cells or TRMs are critical to maintaining barrier immunity. TRMs are non-circulating memory CD8 T cells which remain poised at the barrier sites and possess an increased cytotoxic potential to antigen-reencounter, can proliferate in-situ. We previously observed that systemic immunization with an HSV glycoprotein B (gB) epitope activates mucosal CD8 TRM and confers partial protection against HSV-2 challenge. However, recent studies reveal that the sensing and alarming function of CD8 TRMs is not restricted to cognate antigen interaction, but CD8 TRM can mediate protection against antigenically unrelated pathogens, termed “bystander activation.” Previous studies in the context of systemic infection suggest that memory CD8 T cells may also provide innate-like protection against antigenically unrelated pathogens independent of TCR engagement. Whether "bystander T cell activation" is also an important defense mechanism in the mucosa is poorly understood. In chapter 2, we investigated if innate-like memory CD8 T cells could protect against a model mucosal virus infection, genital HSV-2. We found that immunization with an irrelevant antigen delayed disease progression from a lethal HSV-2 challenge, suggesting that memory CD8 T cells may mediate protection despite the lack of antigen-specificity. Upon HSV-2 infection, we observed an early infiltration, rather than substantial local proliferation of antigen-non-specific CD8 T cells, which became bystander-activated upon entering the vaginal mucosa from circulation. Further, local cytokine cues within the tissue microenvironment after infection were sufficient for bystander activation of mucosal tissue memory CD8 T cells from mice and humans. Finally, we show that bystander-activated CD8 T cells are sufficient to delay disease progression and reduce viral burden after HSV-2 infection. Altogether, our findings suggest that local bystander activation of infiltrating CD8 memory T cells contributes a fast and effective innate-like response in mucosal tissue. Consistent antigen exposure in a systemic chronic infection such as LCMV leads to T cell exhaustion; however, whether episodic mucosal localized infection results in T cell exhaustion has not been well studied. Detailed analysis of HSV-infected human tissue reveals that TRMs limit the duration and severity of HSV-2 shedding episodes. Since CD8 TRMs are subjected to recurrent antigen exposure due to the episodic nature of HSV-2 infection, it is important to assess the cellular kinetics and functionality of the HSV-specific and non-specific population within the genital mucosa upon episodic infection. We investigated the T cell phenotype and function within HSV-2 lesion in HSV-2 positive patients and within the genital mucosa in HSV infected mice at the acute phase, effector phase and the lesion resolution phase in chapter 3. Our findings indicate that T cells maintain their functionality in response to recurrent antigen exposure rather than becoming functionally exhausted.
Identification of novel inhibitors of intracellular M. tuberculosis and their mechanisms of action
(2022-04-19) Ahmed, Sara Ahmed Labib; Parish, Tanya
Tuberculosis (TB) is the second leading cause of death from infectious disease worldwide, accounting for 1.5 million deaths per year. The emergence of drug-resistant TB strains further highlights the urgent need for new drugs and new drug targets. TB is caused by the bacterium Mycobacterium tuberculosis, a facultative intracellular pathogen that can survive and replicate inside macrophages. Thus, drug screening in the macrophage mimics the physiological context of the disease, which makes it possible to identify inhibitors of targets/pathways that are involved in the M. tuberculosis adaptation to the intracellular environments and virulence. Our lab has developed and validated a fluorescence-based, live-cell, high-content analysis (HCA) assay to examine drug efficacy against intracellular M. tuberculosis. Here, we describe a high throughput screening where 10,000 diverse small molecules were screened to identify novel inhibitors of M. tuberculosis growth within macrophage-like cells using high content analysis. We selected a number of series for follow-up based on their physicochemical properties and the novelty of the chemotypes and identified five chemical classes of interest. We tested available analogs for each series to complete a limited structure-activity relationship study. A benzene amide ether (BAE) series was identified from the screen. BAE compounds exhibited good activity against intracellular M. tuberculosis as well as low cytotoxicity against the murine RAW 264.7 and human THP-1 cell lines. Compounds had minimal inhibitory activity against M. tuberculosis cultured axenically but did show bactericidal activity against nutrient-starved M. tuberculosis, suggesting that the compounds primarily target the bacteria. Understanding the target and mechanism of action of compounds identified from phenotypic screens is important for downstream drug development to optimize their potency better, identify combination therapies more rationally, and better predict toxic side effects. Therefore, we conducted a series of assays to identify the mechanism of action of the BAE series. An increase in reactive oxygen species (ROS) was observed in M. tuberculosis treated with BAE compounds. In addition, we demonstrated that BAE compounds depleted ATP levels of treated M. tuberculosis culture under both replicating and starved culture.Additionally, BAE compounds increased the oxygen consumption rate. Finally, we examined the transcriptomic changes induced by BAE compound treatment in replicating and nutrient-starved wild-type strains of M. tuberculosis. BAE compound treatment induced changes in energy metabolism under both conditions. Notably, the mechanism of action of one of the BAE compounds was predicted as targeting respiration using the PROSPECT screen. These results suggest that this novel BAE series targets respiration in M. tuberculosis. We also conducted a series of assays to investigate the mode of action of the Phenylthiourea series. We were able to exclude common targets which suggest that the PTU compounds exert antitubercular activity through a distinct or novel target and/or mechanism. Future work will focus on target identification for both series.
The Mycobacterium tuberculosis protein O-phosphorylation landscape
(2022-04-19) Frando, Andrew; Grundner, Christoph
Protein phosphorylation is a main mechanism for translating extracellular signals into cellular adaptations. In bacteria, the two-component system has been the paradigm of protein phosphorylation. Increasingly, however, protein serine/threonine and tyrosine phosphorylation (O-phosphorylation) mediated by serine/threonine protein kinases (STPKs) is identified. Mycobacterium tuberculosis (Mtb) in particular has a larger repertoire of both STPKs and O-phosphoproteins than most bacteria, suggesting a more prevalent role of STPKs in Mtb. Many studies have identified individual STPK functions and substrates, but a systems-level understanding and the full scope of O-phosphorylation in bacteria in general and Mtb in particular remains unknown. In this dissertation, we aimed to establish a systems-wide understanding of Mtb O-phosphorylation. To this end, we combined kinase loss-of-function (LoF) and gain-of-function (GoF) mutants with mass spectrometry (MS)-based phosphoproteomics. We found that the Mtb phosphoproteome is 5x larger than was previously reported. By identifying hypo-phosphorylated proteins in the LoF mutants and hyper-phosphorylated protein in the GoF mutants, we comprehensively identified over 1,400 STPK-substrates in the bacterium, assigning putative STPK substrates and functions. We showed that the O-phosphoproteome is organized as a higher-order network of a complexity that has previously only been associated with eukaryotes. Transcription factors (TFs) were highly phosphorylated, suggesting a large functional interface between O-phosphorylation and transcription. We further characterized this interface by using transcriptomics and discovered large transcriptional effects of individual STPKs. We used these data to predict functional phosphorylation events and validated these predictions for the putative zinc regulator Zur. These data provide the deepest bacterial O-phosphoproteome to date, identify a complex O-phosphorylation network, and provide a resource to assign thousands of specific phosphorylation events to individual STPKs and their transcriptional effects. Together, these data challenge the paradigm of TCSs as the main bacterial phosphosignaling mechanism and suggest that Mtb regulates a large swath of physiology through O-phosphorylation.
Chlamydia trachomatis overcomes human cell-autonomous immunity through secretion of a novel inclusion membrane protein IncU
(2022-01-26) Fernandez, Mark Christopher; Hybiske, Kevin
Chlamydia is the most common sexually transmitted bacterial infection in the world. In the United States, over 100 million people are infected each year, and from 2015 to 2019 incidence of chlamydia rose by 19%. Of extreme concern, this increase has occurred despite readily available effective antibiotics and national screening recommendations for young women at highest risk of contracting chlamydia. A chlamydia vaccine is needed, however our near-complete lack of knowledge of protective immune responses that could prevent infection is a major barrier for vaccine development. Research to unveil correlates of protection is a significant challenge—studies in humans carry major ethical considerations, and there is no animal model that faithfully recapitulates the natural history of infection as it occurs in humans. Careful design of in vitro research using human cells and tissues and work to identify the utility and limitations of various animal models are critical research priorities. To date, interferon gamma (IFNγ) secreting T-cells represent the strongest plausible correlate of protection against chlamydia. Because the etiological agent of chlamydia, the bacterium Chlamydia trachomatis, is an obligate intracellular pathogen that primarily infects human epithelial cells, an important aspect to the protective capacity of IFNγ lies in the ability of this cytokine to stimulate innate immunological responses in the cells that harbor and support chlamydial growth. The ability of these classically non-immune cells to mount innate immune responses to protect themselves against intracellular pathogens is often termed cell-autonomous immunity. IFNγ-stimulated cell-autonomous immunity against intracellular pathogens is a topic of ongoing investigation in the Chlamydia field and other systems, and its importance as a potent anti-Chlamydia response is only recently appreciated. While murine cell-autonomous immunity renders epithelial cells able to mark C. trachomatis-containing vacuoles for destruction, this pathogen readily evades detection and growth restriction in its native human host cells. I have dedicated my dissertation work to understanding the relationship between human cell-autonomous immunity and Chlamydia trachomatis and, importantly, identifying how this pathogen resists this potent immune pathway. By screening a novel library of chimeric interspecies Chlamydia mutant strains, I identified a genetic locus required for resistance. Follow up analysis of mutant C. trachomatis strains aided my identification of a single virulence gene, incU. Human cells infected with incU-mutants can recognize chlamydial vacuoles, termed inclusions, leading to the recruitment of downstream cell-autonomous immunity proteins. Ultimately, this leads to severe growth restriction of these C. trachomatis mutants. With contributions from collaborators at the University of Washington and Oregon State University, I found that incU mutants failed to replicate in vivo in the nonhuman primate model. In vitro infection of primary NHP fibroblasts revealed that NHP cells exhibit an IFNγ-inducible cell-autonomous immune response that is mechanistically similar to the human pathway, providing supporting evidence that in vivo growth defects were due to incU mutants’ susceptibility to IFNγ-stimulated cell-autonomous immunity. Taken together, these data shed new light on the relationship between IFNγ-stimulated immunity and C. trachomatis, while identifying a relevant animal model in which to further explore the protective capacity of IFNγ in humans.
Vaginal regulatory T cells in health and infection
(2022-01-26) Traxinger, Brianna R; Lund, Jennifer M; Prlic, Martin
Regulatory T cells (Tregs) mediate immune homeostasis, yet also facilitate nuanced immune responses during infection, balancing pathogen control while limiting host inflammation. Recent studies have identified Treg populations in non-lymphoid tissues that are phenotypically distinct from Tregs in lymphoid tissues (LT), including performance of location-dependent roles. Mucosal tissues serve as critical barriers to microbes while performing unique physiologic functions, so we sought to identify distinct phenotypical and functional aspects of mucosal Tregs in the female reproductive tract. In healthy human and mouse vaginal mucosa, we found that Tregs are highly activated compared to blood or LT Tregs. To determine if this phenotype reflects acute activation or a general signature of vaginal tract (VT)-residency, we infected mice with HSV-2 to discover that VT Tregs express granzyme-B (GzmB) and acquire a VT Treg signature distinct from baseline. To determine the mechanisms that drive GzmB expression, we performed ex vivo assays to reveal that a combination of type-I interferons and interleukin-2 is sufficient for GzmB expression. Together, we highlight that VT Tregs are activated at steady state and become further activated in response to infection; thus, they may exert robust control of local immune responses, which could have implications for mucosal vaccine design. Additionally, little is known about the role of Tregs in recurrent HSV-2 outbreaks or secondary exposure to HSV-2. Tissue-resident memory T cells (Trm), which reside in tissue sites without recirculation, have been shown to be crucial for protection from HSV-2 in mouse models and HSV-2 control. However, HSV-2 symptoms and viral load are highly variable between infected individuals, despite the presence of vaginal Trm. Therefore, we predicted that Tregs prevent immune-mediated vaginal tissue damage during HSV-2 challenge by restraining CD4+ and CD8+ Trm responses, possibly at the expense of impaired viral clearance. Additionally, we hypothesized that Tregs may directly contribute to the repair of vaginal HSV-2 lesions through production of amphiregulin (Areg), a growth factor known to facilitate tissue repair in other tissue Tregs. We used immunohistochemistry to assess vaginal pathology in mice lacking Tregs at the time of HSV-2 challenge and found that Treg-depleted mice experienced delayed tissue healing by day 7 post-challenge, compared to Treg-replete controls. However, PCR revealed that vaginal HSV-2 viral titers were comparable in both groups, suggesting that tissue damage was immune-mediated. We also found that vaginal Tregs in mice express Areg transcript and Areg protein by day 7 post-HSV-2 primary infection, suggesting that Tregs may directly contribute to vaginal tissue repair of HSV-2 lesions, but further studies are necessary to functionally link Treg-produced Areg to vaginal mucosa healing and confirm Treg production of Areg in human tissues. Experiments are ongoing to assess the role of Tregs on Trm abundance, activation, and cytotoxic activity after HSV-2 challenge, as well as to define the role of Treg-produced Areg in tissue pathology using conditional knockout mice. Together, these findings suggest that vaginal Tregs may have vagina-specific roles in tissue protection and immune control during viral infection, further evidencing the need to consider Tregs in mucosal vaccine design. Likewise, Treg-based therapeutics could be an important tool for reversing pathology in mucosal infection or autoimmunity.
Comprehensively Defining the Determinants of Neutralization by Broadly Cross-Reactive Flavivirus Antibodies
(2022-01-26) Stuart, Jackson; Goo, Leslie
Zika virus (ZIKV) and dengue virus (DENV) are two highly related mosquito-borne members of the genus Flavivirus that cause significant human morbidity and mortality. There is no licensed vaccine available for ZIKV and the sole vaccine available for DENV is suboptimal both in terms of its efficacy and safety. There is much interest in the development of a single vaccine effective against ZIKV and DENV as both viruses co-circulate due to shared vectors and vertebrate hosts, and due to the risk of antibody-dependent enhancement of subsequent infection by either virus. Two classes of broadly neutralizing antibodies (bnAbs) capable of potently neutralizing both viruses have recently been isolated: EDE-class and MZ4-class bnAbs. Through traditional epitope mapping methods, EDE bnAbs were found to bind to a conserved quaternary epitope found on the flavivirus envelope protein (E) while MZ4 bnAbs bind to a linker region found between domain I and domain III of E protein. However, these mapping methods are only capable of providing data on binding and structural footprints. In order to better understand the determinants of neutralization for these bnAbs, I utilized a high-throughput approach called deep mutational scanning (DMS) that is capable of providing amino acid-resolution data on the determinants of neutralization – data that will be useful for further investigation of these bnAbs as potential therapeutics and as the basis for a cross-protective vaccine. I demonstrate the successful rescue of a ZIKV E protein virus library, the identification of an inhibitory concentration of 99% for both bnAbs, and the initial attempts at performing a DMS selection assay.
Characterizing host immune responses to Group B Streptococcus hemolysin during invasive infection
(2022-01-26) Furuta, Anna M; Rajagopal, Lakshmi
Group B Streptococcus (GBS) is a gram-positive, ꞵ-hemolytic bacteria that asymptomatically colonizes the vaginal tract. However, during pregnancy maternal GBS colonization greatly predisposes the neonate to a wide range of adverse outcomes, including preterm birth (PTB), stillbirth, and fetal injury. Perinatal transmission results in life-threatening GBS disease including pneumonia, meningitis, and sepsis. Additionally, rates of invasive GBS disease in nonpregnant adults have been on the rise, especially those with advanced age and underlying medical conditions. Reports of fatal GBS infection in healthy adults and the steadily increasing rates of antibiotic resistance in GBS clinical isolates are concerning. Given that there are no FDA-approved GBS vaccines, the development of a safe and effective therapeutic is an important global health objective and requires a better understanding of virulence factors implicated in multiple facets of GBS disease. GBS encodes an arsenal of virulence factors that facilitates invasive disease in vulnerable hosts niches. However, one of the most important virulence factors that GBS deploys is hemolysin, also known as the hemolytic pigment or granadaene. GBS hemolysin is a surface-associated toxin that is responsible for the trademark hemolytic activity of GBS. Notably, hyperhemolytic GBS strains have been isolated from various disease manifestations of invasive GBS infection in neonates, pregnant women, and nonpregnant adults. However, unlike many other bacterial hemolytic or cytolytic toxins which are proteinaceous in nature, the GBS hemolysin is an ornithine rhamnopolyene. Here, we characterize interactions between hemolysin and host immune responses during invasive GBS infection in adults and neonates. In previous work, we synthesized a non-toxic analog of hemolysin and showed that immunization provided protection against systemic GBS infection in nonpregnant adults. In this thesis, we have further characterized the adaptive immune responses to the analog by demonstrating the importance of T cell and antibody responses to generating protection against GBS infection. Additionally, given that the lung serves as a portal of entry for GBS, we optimized an intranasal neonatal murine model to show that GBS utilizes hemolysin to disseminate from the lung and causes increased neonatal morbidity. Furthermore, we show that hemolytic membrane vesicles (MVs) released from GBS exacerbate invasive lung disease and sepsis in neonates. Collectively, these studies presented in this dissertation provide new insights critical for our understanding of GBS hemolysin and for the development of therapeutic strategies that target hemolysin.
T cell immunity in the female genital tract
(2021-03-19) DavÃ©, Veronica Anjali; Lund, Jennifer; Prlic, Martin
Many pathogens of global health significance are sexually transmitted, highlighting the importance of studying immune cells in genital barrier tissues. T cells are a critical component of the immune response to viruses, yet their role in protecting the female genital tract against viral infection is incompletely understood. Here, we combined studies of the mouse and human female genital tract to characterize T cells isolated from female genital barrier tissues. In human cervicovaginal tissue, CD8 T cells resembled effector T cells, raising the question of whether they were recently activated. By employing mouse models, we found that systemic immunization resulted in a cervicovaginal CD8 T cell compartment that was poorly maintained and underwent progressive differentiation that was not observed in spleen, lymph nodes, or small intestine lamina propria. This progressive differentiation occurred over five months, after which CD8 T cells gained an effector-like phenotype, ultimately resembling human cervicovaginal CD8 T cells. Vaginal inflammation combined with vaginal antigen exposure accelerated this differentiation process. The CD8 T cell compartment induced by systemic immunization mediated partial protection against vaginal infection with herpes simplex virus type 2 (HSV-2). This protection waned over time as the loss and differentiation of the CD8 T cell compartment progressed. Together, these results demonstrate that tissue-intrinsic factors interact with environmental cues to dictate the final longevity and phenotype of the cervicovaginal CD8 T cell compartment.Women living with HSV-2 often experience recurring genital herpes lesions. We explored the T cell compartment in human genital skin during these localized HSV-2 reactivation events. Upon HSV-2 lesion formation, we found that CD4 and CD8 T cells expanded in the infected skin site and upregulated markers of proliferation and activation. Some of the expanded T cells resembled circulating memory T cells, suggesting influx of immune cells from the blood. Cell-intrinsic and cell-extrinsic regulatory mechanisms were also upregulated in HSV-2 lesions, indicating that the immune system acted to control the virus as well as limit immunopathology. Meanwhile, T cells isolated from a nearby unaffected region distinct from the HSV-2 lesion were quiescent, instead resembling the T cell isolate from genital biopsies from HSV seronegative participants. Despite the frequency of HSV-2 reactivation in people with chronic genital herpes, we did not observe evidence of T cell exhaustion or loss of functionality. After lesion resolution, the T cell infiltrate in HSV-2 lesions returned to quiescence, again resembling the T cell compartment in contralateral and seronegative control biopsies. Given these results, we conclude that T cell responses to HSV-2 reactivation events in genital skin are highly localized, include both resident T cells and circulating memory T cells, and are rapidly constrained by regulatory T cells and intrinsic regulatory mechanisms. Taken together, our data highlight unique features of T cells in the female genital tract and their roles in vaccination and viral immunity.
Eliciting VRC01-class antibodies against HIV-1 through immunization
(2020-10-26) parks, katherine rachael; Stamatatos, Leonidas
With more that 37 million people currently infected with HIV-1, there is a need for a vaccine to prevent HIV-1 infection. One goal of an effective HIV-1 vaccine is to elicit broadly neutralizing antibodies (bnAbs). These antibodies (Abs) neutralize the majority of HIV-1 viruses in circulation. bnAbs have been isolated from chronically infected individuals and were shown, in studies of passive administration in non-human primates and humanized mice, to be protective against SHIV and HIV respectively. Thus, it is thought that if bnAbs can be elicited through vaccination, they will be protective.VRC01-class Abs are amongst the most broad and potent neutralizing and therefore desirable to elicit during vaccination. VRC01-class Abs target the CD4-binding site (CD4-BS) on the HIV-1 Env. All members of this class have high levels of somatic hypermutation (SHM); share similar gene ontogenies, all utilizing the VH1-2*02 allele in the heavy chain paired with one of the following light chains: k3-20, k1-33, l2-14, k3-15, k1-5; and use the same mode of recognition to engage the CD4-BS. A major roadblock to eliciting such Abs is the fact that the unmutated or germline precursors which gives rise to these bnAbs fail to bind known recombinant Env. This has led to the development of immunogens that engage the germline (gl)VRC01 precursor, thus named germline-targeting immunogens. This thesis examines germline-targeting immunogens in vivo. The germline-targeting immunogens evaluated are Env-based and also anti-idiotypic antibodies (aiAbs). In Chapter II we evaluated Env-based germline-targeting immunogens. In these studies, we utilized a mouse model expressing B cells with the glVRC01 heavy chain (glH-VRC01 mouse, Table 1.3). Mice were immunized with one of the following germline-targeting immunogens: 426c Core, eOD- GT8, or 426cOD. These immunogens differ in their affinity for glVRC01, the structural presentation of the glVRC01 epitope, and genetically. We found that while all of the glVRC01- targeting immunogens effectively activated VRC01-like B cells in vivo, the immunogens selected for B cells with distinct genetic characteristics that influence their ability to recognize diverse Env proteins. These results will inform HIV-1 vaccine design, as both 426c Core and eOD-GT8 are being evaluated in human clinical trials. These results are also more broadly applicable to vaccine design, by highlighting the importance of structure and biophysical and biochemical properties of the immunogen on B cell activation. While, these germline-targeting immunogens are able to activate VRC01-like B cells in vivo, the Abs they produce are non-neutralizing. Therefore in the work reported in Chapter III, we employed a boosting immunogen in an effort to guide the maturation of these B cells to their neutralizing form. We found that priming glH-VRC01 mice with the germline-targeting immunogen 426c Core and boosting with the HxB2 wild type (WT) Core protein increased SHM in the VRC01-like B cell receptors, improved Ab binding to diverse Env proteins and increasedthe Abs ability to neutralize the autologous WT virus. This work is the first to identify a prime- boost immunization scheme that can elicit VRC01-class Abs capable of neutralizing a WT virus. We also evaluated the aiAb, iv8, as a glVRC01-class germline-targeting immunogen in Chapter IV. In an adoptive transfer experiment, we found that iv8 is able to activate and expand B cells expressing the mature 3BNC60 heavy chain and gl3BNC60 light chain (SI-3BNC60, Table 1.3), as well as decrease the non-VRC01-epitope targeted plasma Ab response in comparison to an Env-based immunogen. Iv8 was also able to expand B cells expressing VRC01-class characteristics in the glH-3BNC60 mouse model. Therefore, we have demonstrated that iv8 can activate glVRC01-class B cells in vivo and should be considered for use in combination or as an alternative to Env-based immunogens in vaccination studies designed to elicit VRC01-class bnAbs.
Targeting tumor tight junctions: The Junction Opener protein
(2020-10-26) Kim, Jiho; Carter, Darrick A
With the advent of new approaches - including cellular therapy and immunotherapies - cancer treatment has entered a new era. However, solid epithelial tumors have yet to benefit from these new modalities, and new tools for diagnosis and treatment are sorely needed. Desmoglein-2 (DSG2) is a desmosomal protein involved in cellular structure integrity and junction formation, contributing to the formation of tight physical barriers. Several cancers have been observed to upregulate its expression to resist therapies and the immune system, representing its potential as a biomarker and target. This dissertation explores the identification of DSG2 as a biomarker and prognostic indicator for ovarian cancer, which commonly manifests as an epithelial solid tumor. DSG2 overexpression was linked to survival, chemoresistance, and prognosis. A recombinant, engineered adenoviral protein, Junction Opener (JO) targets DSG2 and causes the transient opening of tight junction allowing easier passage of chemotherapeutics. JO’s ability to home to overexpressed DSG2 and to compromise one of a tumor’s defense mechanisms makes it an attractive co-therapeutic with monoclonal antibodies or chemotherapeutics. A conjugatable form of JO, JOC-x, was developed and confirmed to retain JO’s properties and binding characteristics. JOC-x was then successfully conjugated to PEGylated liposomal doxorubicin (PLD) and was shown to have enhanced efficacy over the separate combination, thus indicating its potential as a chemotherapeutic candidate. Finally, we explored the pharmacokinetic and immunogenic properties of JO and PLD in Macaca fascicularis to ascertain parameters for future use in humans. Macaque studies indicated induction of anti-JO antibodies and inflammatory cytokines, but these could be suppressed with an immunosuppressive regimen commonly found in the clinic. In short, this dissertation explores the establishment of DSG2 as an ovarian cancer biomarker and therapeutic target for the JO protein as tools to use in ovarian cancer diagnosis and treatment.

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