Lymphoblastoid cell lines (LCLs) are created by culturing lymphocytes from the peripheral blood and adding Epstein-Barr virus (EBV), a ubiquitous human herpesvirus which infects, activates, and transforms B cells. These cell lines are used for genotyping, as targets for cytotoxic cells, and as models for EBV immortalization of B cells, particularly post-transplant lymphoproliferative disease (PTLD) in which EBV-immortalized cells proliferate in the absence of a cytotoxic T-cell response. Studies have shown more diversity in LCLs than would be expected from cell lines that are often treated as interchangeable. It is not known how their diversity in factors like morphology, growth factor production, or cellular gene expression influences the EBV life cycle. In this study I investigated connections between LCLs cellular and viral phenotypes, categorizing them as either low in EBV copy number or fluctuating within a high range. As measured by lytic EBV replication and viral gene expression, LCLs showed high or low lytic permissivity, with permissivity defined as the likelihood that a cell will switch from stable latent infection into the lytic EBV life cycle. Permissivity was not affected by blocking the late events of the lytic cycle. I used flow cytometry to characterize 19 aspects of LCL surface phenotype, but found little association with lytic permissivity. Microarrays and PCR were used to identify genes expressed at higher levels in non-permissive LCLs, including transcription factors that maintain B cell lineage. Unfolded protein response (UPR) genes and the UPR protein Grp94 were expressed at higher levels in permissive LCLs. A drug was used to investigate effects of the UPR on permissive and non-permissive LCLs that had been maintained for short or long periods of time. The UPR enhanced permissivity, causing more cells to enter the lytic cycle, but this did not lead to lytic replication. This study enhances our knowledge about EBV life cycles by giving us new information about host factors that contribute to the lytic switch. This data about LCL diversity has public health relevance to the diversity of PTLD cases, since identifying risk factors for PTLD is a significant part of care for EBV-positive transplant recipients.

The chemokine CCL20 is thought to be an integral part of the communication between the innate and adaptive arms of the immune system, due to expression of the cognate receptor, CCR6, on immature dendritic cells and on memory T cells and B cells. Interest in this particular chemokine/chemokine receptor interaction has grown over time and more recently due to roles in SIV infection, mucosal immunology, and vaccinology. The need to further study the CCL20/CCR6 interactions is bolstered by our laboratorys previous findings of increased expression of CCL20 in acutely SIV infected lymph nodes and the increased expression of CCL20 in response to PAMPs in cells of lymphatic vessels. This thesis aims to develop and improve a system for studying the interaction between CCL20 and CCR6. I have found that the recombinant expression system utilized to obtain macaque chemokines provided highly pure fusion proteins. However, cleavage of the fusion protein into macaque CCL20 has been inefficient. Rhesus macaque CCL20 chemically synthesized using regioselective cyclization was highly biologically active using the chemotaxis assay and stable cell lines expressing CCR6. Chemotactic inhibition studies identified five compounds that inhibited CCL20 induced chemotaxis. The surfactant, GML, did not inhibit CCL20 induced migration. The anti-inflammatory botanicals, EGCG and gallotannin, both inhibited CCL20-driven migration at high concentrations. The three CCR6 extracellular loop mimetic peptides also partially inhibited CCL20 induced migration at high concentrations. In conclusion, I have utilized both a recombinant protein expression system and regioselective cyclization peptide synthesis to obtain bioactive, nonhuman primate chemokines. I have also successfully developed an in vitro system to study CCL20 induced migration, and have identified a number of botanical and biochemical elements that inhibit CCL20-induced migration. The public health significance of this study is related to the fact that vaccine efficacy may be affected by anti-inflammatory compounds that inhibit CCL20 mediated chemotaxis. Another way in which public health could be affected by this study is in using the anti-inflammatory compounds studied to treat chronic inflammatory conditions in which the pathology of the disease is related to up-regulation of CCL20 and CCR6.

Inhibition of reverse transcriptase (RT) is a vital tactic in the prevention of human immunodeficiency virus 1 (HIV-1). Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are a class of compounds demonstrated to act as allosteric inhibitors of RT DNA polymerization. However, several lines of evidence suggest that polymerization may not be the main mechanism of inhibition of reverse transcription. It has been demonstrated that NNRTIs also have the ability to modulate RT ribonuclease (RNase) H cleavage. Additionally, recent evidence suggests that resistance to chain-terminating nucleoside reverse transcriptase inhibitors (NRTIs) is dependent on a balance between the polymerase and RNase H activities of the enzyme. In light of this, I hypothesize that NNRTIs block reverse transcription by exerting effects on both the DNA polymerase and RNase H active sites of the enzyme, significantly disrupting the equilibrium between these two enzymatic activities. Therefore, the ability for NNRTIs to be combined with other classes of RT inhibitors in antiretroviral therapies will depend on how these compounds respond to the NNRTI-induced shift in the polymerase/RNase H activity equilibrium. This study demonstrates that NNRTIs cause the accelerated appearance of secondary RNase H cleavage products that have decreased RNA/DNA hybrid structures. As a result, these template/primers(T/Ps) are not sufficient substrates for NRTI removal and therefore, excision is less efficient in the presence of NNRTIs. Additionally, fluorescent resonance energy transfer experiments demonstrate that NNRTIs cause a shift in the binding of RT and T/P such that the RNase H domain is moved away from the 5end of the primer. Finally, subunit-specific analysis shows that resistance to RTI combination therapy facilitated by the N348I mutation is a result of effects from the p51 subunit. I propose that the binding of NNRTIs cause RT to bind to T/P in a polymerase-incompetent mode, resulting in decreased polymerization and shorted RNase H cleavage products. Additionally, N348I can facilitate dual resistance by favoring the polymerase-competent binding mode. This work is of public health significance because it lays the foundation for the development of new reverse transcriptase inhibitors and highlights the importance of resistance in the connection domain of HIV-1 RT.

Severe anemia is the primary outcome of childhood malaria in holoendemic malaria transmission regions such as western Kenya. HIV-1 and bacteremia are equally important diseases in Kenyan children. Anemia is also the hallmark trait of pediatric HIV-1 infection, and despite our previous reports of exacerbated anemia in malaria/HIV-1 co-infected childrena, we also observed significantly lower parasitemias without worsening anemia in malaria/bacteremia co-infected children. Abundant production of pro-inflammatory cytokines is known to adversely affect erythropoiesis and is common to malaria, HIV-1, and bacteremia. As such, we performed a comprehensive bead-based 25-plex Multiplex assay to identify cytokines patterns and profiles associated with negative and positive hematolgic outcomes in co-infected children. Children in Aims 1 and 2 infected with P. falciparum malaria (Pf[+], aged 3-36 mos., n=542) were stratified into three groups: HIV-1 negative (HIV-1[-]/Pf[+]); HIV-1 exposed (HIV-1[exp]/Pf[+]); and HIV-1 infected (HIV-1[+]/Pf[+]). In Aim 3, malaria and bacteremia co-infected children (n=192) were divided into three categories: malaria alone, Pf[+]; Gram negative bacteremia/malaria co-infected, G[-]/Pf[+]; and Gram positive bacteremia/malaria co-infected, G[+]/Pf[+]. Univariate, correlational, and hierarchical regression analyses were used to determine differences among the groups and to define predictors of worsening anemia. Aim 1 analyses revealed HIV-1[+]/Pf[+] children had significantly more malarial pigment-containing neutrophils (PCN), monocytosis, increased severe anemia (Hb<6.0g/dL), and ~10-fold greater mortality. Hierarchical multiple regression revealed that worsening anemia was associated with elevated pigment-containing monocytes, younger age, and increasing HIV-1 status (HIV-1[-]→HIV-1[exp]→HIV-1[+]). Aim 2, addressing the inflammatory milieu, demonstrated that exacerbated anemia was associated with inflammatory mediator (IM) dysregulation, but not parasitemic or erythropoietic indices. A principal component analysis revealed that IL-12 was the most influential variable on Hb levels in HIV-1[+]/Pf[+] children, while the IL-1β:IL-10 ratio was most influenced by PCN. In Aim 3, both bacteremia co-infected groups had lower parasitemia compared to the Pf[+] group. A multiple mediation model examining IMs responsible for decreased parasitemia in the bacteremia co-infected groups identified IL-4, IL-10, IL-12, and IFN-γ as the key molecules in decreasing parasitemia. Thus, malaria/HIV-1 co-infection is defined by significantly enhanced anemia that is associated with unique IM profiles known to exacerbate anemia, while enhanced immune activation in malaria/bacteremia co-infected children appears to promote reduced parasitemia without adversely affecting anemia outcomes. By defining the inflammatory milieu associated with severe anemia, therapies can be developed to mitigate detrimental immune responses, thereby lessening the pediatric public health burden in western Kenya.

Dengue virus (DENV) is a reemerging infectious agent causing an estimated 50-100 million cases annually and is endemic in more than 100 countries. Recently there has been an increase in the geographic spread, the overall number, and the severity of disease cases, making DENV an increasing threat to public health. The most prevalent of the arboviruses, DENV is introduced into human hosts during a vector mosquitos blood meal; here, virus is deposited into the epidermal and dermal layers of the skin. Therefore, elucidating the cutaneous immune response to DENV is critical to our understanding of the immune mechanisms involved in protection and pathogenesis of DENV and to the development of effective treatments and vaccines. In addition to resident T- and B-cells, the skin immune system includes three major populations of innate immune cells, dermal dendritic cells (dDCs), dermal macrophages (dMØs), and epidermal Langerhans cells (LCs). These immune cells are potential targets of DENV infection. The receptor profile of dDCs and dMØs indicates that they are susceptible to DENV infection, however evidence of this is lacking. LCs are infected following ex vivo inoculation of human skin explants, yet their role in DENV pathogenesis remains unclear. Upon exposure to a pathogen, cutaneous immune cells mature and migrate to the cutaneous draining lymph node via the draining lymphatics; these functions are critical to the induction of adaptive immune responses and to pathogen clearance. It remains unclear how DENV infection affects the maturation, migration and survival of cutaneous immune cells. Herein, we use a combination of in vitro studies and ex vivo inoculation of healthy human skin explants to explore the role of cutaneous immune cells in DENV infection. We investigated the degree to which cutaneous immune cells become infected with DENV and measured upregulation of activation markers, migration out of the skin, and the life span of infected cells. We discovered that populations of cutaneous DCs, as well as dMØs, become infected with DENV upon scarification of skin with virus. In vitro, we confirmed that DENV infection partially inhibits maturation of DCs and potentially MØs. Additionally, DENV infection induces DC apoptosis in a caspase-dependent manner. These results help to clarify the role of cutaneous immune cells in the immunopathogenesis of DENV.

Current antiretroviral therapy (ART) has reduced morbidity and mortality from HIV-1 infection, but the long-term efficacy of ART is limited by selection of HIV-1 drug-resistant variants. Most HIV-1 drug resistance mutations that have been studied are located in the polymerase domain of HIV-1 reverse transcriptase (RT) and this region of RT is sequenced in genotyping tests used clinically to guide ART. Recently, attention has focused on the connection and RNase H domains of RT as locations of drug resistance mutations, but the prevalence, molecular mechanisms, and impact of such mutations on response to ART are uncertain. We therefore performed a series of studies to address this uncertainty, including in vitro selection of HIV-1 resistant to 3-azidothymidine (AZT), drug susceptibility studies, biochemical assays and genotype analysis of clinical samples to identify and characterize resistance mutations in the RT connection and RNase H domains. From this work, we provide several lines of evidence that connection and RNase H domain mutations emerge with ART and impact nucleoside/nucleotide reverse transcriptase inhibitor (NRTI) susceptibility. First, the connection domain mutation A371V and the RNase H domain mutation Q509L are selected in vitro with AZT and confer > 50-fold AZT resistance and low-level cross resistance to lamivudine, abacavir and tenofovir when in the context of thymidine analog mutations (TAMs) in the polymerase domain of RT. Second, we show that mutation Q509L in the RNase H domain promotes dissociation of RT from RNA/DNA template/primer bound in an RNase H competent mode, thereby decreasing secondary RNase H cleavage and destruction of the template/primer. As a consequence, template/primer binds in a polymerase competent mode allowing AZT-monophosphate excision, DNA polymerization and AZT resistance. Third, the connection domain mutation A360V emerges in patients after prolonged exposure to AZT monotherapy and increases resistance to AZT in the context of 3 or more TAMs. Fourth, connection and RNase H domain mutations are not more frequent at virologic failure in HIV-1 subtype B infected patients treated with 2 NRTI plus efavirenz when failure is defined as a small increase in plasma HIV-1 RNA. However, the connection domain mutation N348I emerges frequently at virologic failure in HIV-1 subtype C infected patients in South Africa who were treated with efavirenz/lamivudine/stavudine or nevirapine/lamivudine/stavudine when virologic failure is defined as confirmed plasma HIV-1 RNA > 1,000 copies/mL. This work provides strong evidence that RT connection and RNase H domain mutations emerge in HIV-1 infected patients treated with ART and these mutations are missed with currently available genotype tests. Mutations missed by routine genotyping tests pose a potential public health threat if left undetected and transmitted to others.

Rift Valley Fever virus (RVFV) is an arthropod-borne bunyavirus that causes a zoonotic disease associated with abortion storms, neonatal mortality in livestock and hemorrhagic fever with a high case/fatality ratio in humans. To date, vaccine developments against RVF have been based on inactivated or attenuated strains but their widespread use has been hampered due to deleterious effects or incomplete protection, justifying further studies to improve the existing vaccines or to develop others. To address this, DNA plasmid and alphavirus replicon vector (VEEV) expressing RVFV Gn glycoprotein were constructed and evaluated for their ability to induce protective immune responses in mice against RVFV. An experimental live-attenuated vaccine (MP12) and its inactivated counterpart (WIV MP12) were developed to serve as benchmarks for comparison. Test vaccine candidates efficiently expressed the RVFV glycoprotein in vitro and elicited anti-RVFV antibody responses in immunized mice, as determined by RVFV specific ELISA, IgG isotype ELISA, and virus neutralization. Interestingly, these vaccine strategies elicited cellular immune responses as determined by Gn specific ELISPOT. More importantly these vaccines not only protected immunized mice from virulent RVFV when challenged via intraperitoneal route, but also conferred protection when challenged via aerosol route. This work is of public health significance as it describes the development of safe and effective vaccine candidates that have the ability to protect both livestock and humans against possible routes of exposure to this zoonotic threat.

It has been shown that HIV/SIV preferentially replicates in the gut associated lymphoid tissue (GALT) which leads to immune activation and disease progression. Different sections of the gastrointestinal (GI) tract have unique biological functions with specialized lymphoid tissue composition and distribution, which may contribute differently to viral pathogenesis. We hypothesize that the GALT serves as an active viral replication site during the acute and AIDS stages of HIV/SIV infection and as a viral reservoir in long-term non-progressors (LTNP) and antiretroviral therapy (ART) treated individuals; this viral replication results in local immune activation which contributes to disease progression. The RNA and DNA were isolated from tissues along the GI tract of uninfected and SIV infected rhesus macaques in different disease stages with or without ART. Real-time PCR was utilized to measure viral RNA and DNA, and mRNAs of CD4, TNF-α, IL-6, IL-1β, and MyD88. Our results showed that SIV DNA/RNA were detected in all GI tissues from infected monkeys regardless of the disease stage and drug intervention. However, the viral load distribution profile in the GI tract varied from monkey to monkey. Despite the undetectable viral load in peripheral blood, both viral RNA and DNA were detected in GI tissues of ART treated monkeys. Compared to the uninfected monkeys, low levels of CD4 mRNA were detected in SIV infected monkeys, particularly LTNP. There is a positive association between viral load and mRNA levels of TNF-α, IL-6, and MyD88 in the stomach and duodenum. However, no association was observed between viral loads and IL-1β mRNA levels in any of the GI tissues examined. Data from this study indicates that the entire GI tract serves as a SIV replication site in all stages of infection, which leads to pro-inflammatory cytokine production and local inflammation. This study reveals the importance of the entire GI tract in HIV/SIV pathogenesis. Especially, in ART treated individuals with undetectable viral loads in blood, active viral replication in gut tissue may lead to development of drug resistant variants and faster progression to AIDS. This will have a profound impact on clinical intervention and public health as a whole.

Highly active anti-retroviral treatment (HAART) targets less than a third of the proteins produced during HIV-1 infection. Testing the effectiveness of an anti-retroviral drug requires assays specific for the individual target that take into account its mechanism of action. Most HIV-1 proteins need to undergo dimerization in order to become functional in the viral life cycle. Historically, it has been difficult to visualize and quantify changes in a protein-protein interaction, which has left this characteristic of proteins unexplored as potential antiviral targets. In this study, a bimolecular fluorescence complementation based screening assay is developed that can quantify a change in dimerization, using the HIV-1 accessory protein Vpr as a proof of concept. Results demonstrated that bimolecular fluorescence complementation of Vpr could be competed off in a dose-dependent manner using untagged, full length Vpr as a competitor molecule. The change in signal intensity was measured quantitatively through flow cytometry and fluorescence microscopy in a high content screening assay. High content imaging was used to screen a library of peptides and a library of small molecules for an effect on Vpr dimerization. None of the Vpr peptides were shown to have an effect; however, one of the small molecules was shown to interfere with Vpr dimerization in a dose-dependent manner. Statement of Public Health relevance: Dimerization is a unique property of many HIV-1 viral proteins and is necessary to complete the viral life cycle, thus it has been identified as a potential drug target. By developing an assay that screens for inhibition of HIV-1 protein dimerization, high throughput screening can be performed to detect inhibitors of a new target in HIV-1 replication. Small molecules identified using this screening method could be developed into a novel anti-retroviral drug.

Objectives/Research questions: Epigallocatechin gallate (EGCG) is a major catechin (antioxidant) component of green tea. Recent studies have shown that it possesses many health benefits, including antimicrobial activity against some bacteria and viruses. Due to its known antimicrobial activity, this study focused on exploring the effects of EGCG on the growth of Mycobacterium tuberculosis, the causative agent of tuberculosis, as it remains a major public health concern. Through the use of a model mycobacterial species, Mycobacterium smegmatis, the experiments investigated the effects of varying concentrations of EGCG on mycobacteria. Methods: The effects of EGCG on the growth of M. tuberculosis and M. smegmatis were explored by setting up serial dilutions and carrying out cell counts over certain time periods. High Pressure Liquid Chromatography (HPLC) was used to fractionate the EGCG in order to better understand its behavior in the media. Results: The study has found that medium containing EGCG has anti-mycobacterial activity, but it is not EGCG itself that carries it out but a degradant molecule. This observation was achieved following experiments showing that a 3-day pre-incubation of EGCG in the medium strengthens its anti-mycobacterial activity. However, the HPLC showed that EGCG is fully degraded by day 1. Therefore, by day 3, there are only degradant molecules in the medium. Findings were consistent with an experiment which found that a pre-incubated green tea extract has more activity against the cells than a fresh one. Conclusions: A three day pre-incubation of green tea extract at 37°C enhances anti-mycobacterial activity, which may have implications in using green tea as a prophylactic agent. Implications for public health: If EGCG has antimicrobial activity green tea could be used as a prophylactic agent against Mycobacterium tuberculosis.