HIV-1 Vpr is known to regulate both viral and host cellular promoters resulting in transcriptional regulation of various cellular factors in host immune cells, such as T cells, macrophages and dendritic cells. It has been shown that Vpr has a role in the upregulation of proinflammatory cytokine TNF-á, which affects immune regulation during infection. However, the mechanisms by which TNF-á is regulated by HIV-1 Vpr are not well understood. A goal of this project is to determine the effects of Vpr in its biologically relevant forms and identify the domains of Vpr involved in TNF-á production. Additionally, we also sought to determine whether TNF-á is up-regulated in infected/exposed cells and/or bystander cells. From our experiments, we conclude that HIV-1 Vpr increases TNF-a production in the context of infection as well as exposure in the absence of other viral proteins. Furthermore, HIV-1 Vpr has multiple domains capable of inducing TNF-á production. However, the increase in TNF-alpha production in DC is dependent on LPS stimulation. We were unable to conclusively determine the cell type that is responsible for this observed phenotype however the results from our studies indicate that infected/exposed cells could be the dominant producers. Due to the association of Vpr with transcriptional regulation of various cellular factors, we investigated the domains of the TNF-á promoter involved in Vpr-mediated TNF-á regulation. Using the HeLa T4 cell line, TNF-á promoter mediated transactivation was increased by two fold when exposed to HIV-1 Vpr(+) as opposed to HIV-1 Vpr(-) as detected by luciferase reporter assay. A six fold increase was observed in the transactivation of full length and mutant TNF-á promoter in macrophage-derived microglia cell line in the presence of Vpr expression. Results from mapping studies indicate that HIV-1 Vpr can regulate TNF-á production via multiple domains of the TNF-á promoter, however for maximum transactivation, the full-length promoter is required. Statement of Public Health Significance: By determining the details of HIV-1 Vpr and TNF-á interaction and the mechanisms for which they interact could reveal novel targets for the development of HIV-1 therapeutics in the fight against this epidemic.

Neisseria meningitidis is a leading cause of invasive meningococcal disease and humans are the only known host. The administration of meningococcal vaccines has reduced the number of meningococcal cases and carriage rates in humans. Current vaccine strategies target important immunological determinants. Insertion sequence 1301 (IS1301) has been shown to facilitate evasion of the host immune response by disrupting antigen expression. The public health importance of this study is in the design of future vaccines against N. meningitidis and in understanding the emergence of new clones. In the 1990s there was an increase in serogroup C meningococcal disease in Maryland that was associated with antigenic shift at the fetA gene. The isolates were characterized as either an early clone or late clone based on the outer membrane protein sequence profiles. The 2:P1.5,2:F.1-30 sequence profile is classified as an early clone while the 2:P1.5,2:F.3-6 sequence profile is classified as a late clone. Previous studies determined that the late clone contained IS1301, while the early clone did not. The goal of this present study is to characterize the IS1301 insertion sites in the late clone to determine if this genetic element contributed to clonal emergence. Early and late clone isolates were characterized by DNA sequence analysis of the housekeeping gene, fumC. A single nucleotide polymorphism characteristic of the hypervirulent ET-15 clone was identified in the late clone isolates. Southern blot analysis using an IS1301 probe revealed a heterogeneous population with multiple insertion sites, ranging from five to ten insertions, within the serogroup C late clone genomes. Of note was a high molecular weight triplet banding pattern common to the majority of isolates. Several different IS1301 specific, PCR-based strategies were performed in an attempt to clone the IS1301 elements corresponding to the bands of the IS1301 triplet. In addition, whole genome sequence analysis was performed on one of the late clone isolates. Initial whole genome analysis demonstrates IS1301 integration within an opacity-associated protein (Opa), which promotes adherence to host cells. Further investigations are necessary to determine the effect of IS1301 insertion on antigenic variation.

Studies of viral accessory genes have progressed in order to understand pathogenesis and develop effective therapeutics and vaccines. For human immunodeficiency virus type-1 (HIV-1), one such gene receiving special focus is vpr. Vpr has been implicated in dysregulation of host cellular events (including cell cycle arrest and apoptosis), infection of non-dividing cells, and increased viral replication in infected T cells. In simian immunodeficiency virus (SIV), a similar gene is seen in a slightly different form, including vpr as well as a duplicate, vpx. In SIV, these two genes have been shown to split the functions of HIV-1 vpr. In order to use SIV as a model for HIV-1, it must be determined which SIV gene is responsible for mediation of different functional effects. HIV-1 vpr has been shown to downmodulate surface markers on dendritic cells and alter cytokine environments in vivo. Studies have shown that HIV-1 vpr pushes natural killer cells into anergy, rendering them non-functional. Results presented in this study indicate SIV infection also results in these effects, but responsibility for these effects is split between SIV vpr and vpx. The vpx gene appears to play a role in downmodulation of surface receptors on dendritic cells and changes the cytokine environment within the dendritic cells. The vpr gene, however, appears to be responsible for decreased functionality of NK cells, leading to a non-functional anergic state. These findings suggest SIV vpx and vpr cause similar effects compared to HIV-1 vpr and, as expected, the SIV genes split the functions of their HIV-1 homolog. Statement of Public Health Relevance: HIV infection and disease is a growing epidemic and it has become increasingly apparent that in vitro studies are not sufficient to provide the data needed to create an effective vaccine. Because vaccine research cannot be performed on human subjects, the best mode for transition would be a shift to in vivo studies on non-human primates using SIV as a model for HIV-1 infection and disease. Before this can be adopted, it will be necessary to show HIV-1 and SIV have similar effects in vitro on immune cells and can be used interchangeably.

Dendritic cells (DCs) are a heterogeneous population of immune cells that influence a wide variety of immune responses, including immunity to infectious diseases and malignant tumors, and in the generation of tolerance. In their immature state, DCs are highly specialized at capturing and internalizing exogenous antigens. Cell-associated antigens are of special interest because they play a role in both the induction of immunity and tolerance. This study aimed to add to the field of DC biology by further describing how DCs handle cell-associated proteins from both live and apoptotic cells. We hypothesized that the DCs ability to capture, internalize, and process integral membrane proteins would vary based on the target cells viability and that the DCs ability to capture cell-associated protein would vary based on the proteins intracellular localization. To quantitatively and qualitatively characterize uptake, we created a biologically relevant system using the Epstein Barr virus latent membrane protein 2 and the melanoma protein gp100, each fused to the enhanced green fluorescent protein (EGFP) and expressed at the outer plasma membrane of a tumor cell line, along with a cell line expressing EGFP in the cytoplasm. We found (1) DCs captured integral membrane proteins but not cytoplasmic protein from live cells; (2) DCs captured membrane and cytoplasmic proteins from apoptotic cells more efficiently and at a faster rate than from live cells; (3) during direct physical interactions DCs transiently surveyed live cells capturing small quantities of membrane, but stayed in prolonged contact with apoptotic cells while continuously internalizing membrane fragments; (4) DC internalization of membrane protein from live cells was clathrin-dependent while uptake from apoptotic cells was clathrin- and caveolae-dependent; and (5) internalized membrane protein from both live and apoptotic cells was found in early endosomes, late endosomes, and lysosomes. This work has potential broad public health implications as it is important to understand all aspects of DC biology when developing vaccines for both chronic and acute diseases. We hope that by uncovering the intricacies of DC handling of cell-associated proteins we will gain a better understanding of how to possibly manipulate DCs in order to influence the immune response.

Hypersusceptibility (HS) to non-nucleoside reverse transcriptase inhibitors (NNRTI) improves virological response to NNRTI-containing regimens. NNRTI HS is associated with nucleoside RT inhibitor (NRTI) mutations, especially those conferring resistance to AZT. Recent logistic regression analyses of a large genotype-phenotype dataset showed the NRTI mutations most strongly associated with NNRTI HS are T215Y, H208Y and V118I. We hypothesized that NRTI mutations V118I, H208Y, and T215Y in combination cause NNRTI HS and that this phenotype is due to multiple mechanisms including a decrease in enzyme activity and/or deficient viral replication due to decreased virion packaged RT. Therefore we sought to: (1) determine the phenotypic effects of these mutations alone and in combination on the susceptibilityof infectious molecular clones and recombinant reverse transcriptase proteins to efavirenz, delavirdine, and nevirapine; and (2) elucidate differences in viral replication, protein production and packaging for virus containing mutations V118I, H208Y, and/or T215Y. We established different patterns of NNRTI HS and replication capacity depending on the combination of mutations present. HIV-1 virus containing H208Y + T215Y were HS to all NNRTI; whereas the V118I/T215Y virus was only HS to delavirdine and nevirapine. H208Y + T215Y viruses exhibited reduced replication capacity compared to wildtype HIV-1. In comparison, the V118I/T215Y virus replicated as efficiently as wildtype virus. Upon further investigation we found the amount of HIV-1 RT incorporated into the H208Y + T215Y viruses was significantly reduced compared with wildtype virus due to decreased viral packaging of GagPol precursors. Interestingly, the RT content in the V118I/T215Y virus was similar to wildtype virus. Furthermore, purified recombinant RT containing the H208Y+ T215Y mutations were not NNRTI HS. In contrast, the V118I/T215Y mutant RT showed five-fold increased susceptibility to NNRTI. Our work highlights the complexity of the HS phenotype and provides an in-depth understanding of how NRTI mutations V118I, H208Y, and T215Y contribute to increased NNRTI susceptibility.

The female genital tract is the main route for heterosexual transmission of HIV-1. Studies with SIV in macaque model indicate that after crossing the mucosa of cervix and vagina, SIV infected cells migrate to draining lymph nodes where the virus expands before it gets disseminated into the gut and other organs of the body. We have developed a combined organ culture system composed of cervical tissue together with tonsil tissue (surrogate lymphoid tissue for lymph node) to study HIV-1 transmission across the mucosal barrier and to evaluate the fate of HIV-1 and HIV-1 infected cells after crossing cervical mucosa and migrating to regional lymph nodes. To study the transmission of virus, a defined amount of pretitered cell-free or cell-associated HIV-1BAL or HIV-1IIIB was added to the top of the cervix tissue and incubated at 37°C in a CO2 incubator. The top well was removed after 3-4 days and tonsil tissue or tonsil mononuclear cells in the bottom well were cultured for an additional 12 days. Replication of cell-free and cell-associated HIV-1BAL and HIV-1IIIB in tonsillar cells after crossing the cervix mucosa was confirmed by the presence of HIV-1 p24 in culture supernatant, the detection of HIV-1 DNA by real-time PCR, presence of p24 antigen by immunofluorescence assay and presence of HIV-1 RNA by simultaneous immunophenotyping and ultrasensitive fluorescence in situ hybridization. We have also characterized by immunofluorescence assay HIV-1 infected migratory cells exiting from the cervix tissue. CD4+ (T cells), CD11c+ (dendritic cells) and CD68+ (macrophages) cells were found to migrate from cervix tissue and were positive for HIV-1. The public health relevance of this model is that we may use the combined cervix and tonsil tissue/cell model to determine the mechanism of sexual transmission of HIV-1 in women at the cellular and molecular level and to evaluate anti-HIV microbicides.

Dendritic cells (DC) are a heterogenous population of antigen presenting cells important in both innate and adaptive immune responses. The two major subsets of DC, CD11c+ myeloid DC (mDC) and CD123+ plasmacytoid DC (pDC), are depleted in the blood of human-immunodeficiency virus (HIV) 1 infected individuals. It has been proposed that DC loss may be due to lymph node recruitment, direct viral infection, bone marrow suppression or death, although this has not been directly addressed. Using the highly relevant, rhesus macaque/simian immunodeficiency virus (SIV) model of HIV infection, we investigated DC dynamics during acute pathogenic SIV infection and simian AIDS. We hypothesized that SIV infection causes a dysregulation of DC trafficking and death not solely dependent upon direct viral infection. The specific aims of this project are to: 1) determine the phenotypic heterogeneity of DC in blood from healthy macaques and develop a rapid assay for frequent longitudinal quantitation of absolute DC numbers; 2) determine whether mDC and pDC are recruited to lymphoid tissues in simian AIDS; 3) determine the dynamics and possible mechanisms of pDC loss and redistribution to lymphoid tissue during acute SIV infection. We found that rhesus macaque DC were more phenotypically homogeneous than their human counterparts and could be accurately quantified in small volumes of blood. In monkeys with simian AIDS, DC were depleted in both blood and secondary lymphoid tissues associated with increased spontaneous apoptosis. However, the remaining DC were phenotypically normal. During acute SIV infection, pDC responded to infection in a biphasic manner, with rapid mobilization into blood followed by depletion in both blood and lymphoid tissue. However, pDC production from bone marrow was normal and BrdU-labeling indicated increased pDC mobilization and recruitment to lymphoid tissues despite net loss of pDC. In lymph nodes, pDC were directly infected with virus, activated, and undergoing increased levels of apoptosis but retained functional TLR7 signaling. The findings in this study are significant to public health because defining the mechanisms leading to DC loss will offer new opportunities for therapeutic interventions to augment immune responses in HIV-infected individuals.

The public health significance of this work reflects the HIV/AIDS epidemic and growing concerns of long-term consequences of antiretroviral therapy. The advent of highly active antiretroviral therapy (HAART) has greatly improved survival among those with HIV-infection. As a corollary, clinicians and researchers face a range of long-term complications previously of little importance to HIV-infected patients. HIV-associated lipodystrophy syndrome (HIV-LS) was first described in 1998 and involves a constellation of metabolic and morphologic abnormalities. Whereas AIDS wasting syndrome has been associated with immunosuppression and high viral burden, HIV-LS has been documented with immunocompetence and suppressed viral concentration. Participants of the local site of the Multicenter AIDS Cohort Study (MACS) consented to photography of their lipodystrophic body habitus changes during routine clinic appointments. The compilation of these photographs was used to develop a manual for clinicians at all four of the MACS sites in order to accurately document the syndrome, and permitted initial classification of HIV-LS into two phenotypes. A third phenotype was identified following a preliminary observation of lipoaccumulation extending bilaterally and symmetrically from the breasts laterally into the axilla. Additional cases were subsequently identified within the MACS; all subjects had pre-existing lipoaccumulation of at least one other anatomical site. It was speculated as to whether this represented a previously unrecognized evolution of HIV-LS. Our next project involved studying the health related quality of life (HRQL) of men with HIV-LS. We found HIV-LS does not negatively affect HRQL or exacerbate depressive symptoms above and beyond the diagnosis of HIV-infection. The metabolic abnormalities of HIV-LS include insulin resistance and dyslipidemia, both of which are considered pro-atherogenic risks. The final segment of this project involved detecting coronary artery calcification via electron beam computed tomography among HIV-infected men treated with HAART. In this male population with well controlled HIV-infection, chronic use of HAART did not impact the progression of subclinical coronary atherosclerosis. In contrast, traditional atherosclerosis risk factors of smoking and advancing age were predictive of coronary atherosclerosis. HIV-infection requires life-long combination treatment. Clinicians, researchers and patients recognize dyslipidemia, peripheral lipoatrophy and central lipohypertrophy as significant consequences of this combination therapy, and hope that concerns regarding increasing cardiac risk are not warranted.

Human herpesvirus-8 (HHV-8, also known as Kaposi's sarcoma associated herpesvirus, KSHV) is a gamma-2 herpesvirus and is the etiological agent of Kaposi's sarcoma, primary effusion lymphoma and a subset of Multicentric Castleman's disease. We have previously shown that HHV-8 uses DC-SIGN (CD209) for entry into susceptible cell types, including immature dendritic cells. In the present study, we demonstrate that DC-SIGN expression renders previously non-permissive cells permissive to HHV-8 infection. Also, we have demonstrated that HHV-8 infection of dendritic cells and endothelial cells results in the expression of some viral lytic proteins initially but subsequently switches to only latent protein expression. However, infection appears to be non-productive as the infected cells maintain viral DNA copies at a low level but this level does not increase over time, nor is encapsidated viral DNA found in the supernatant. Secondly, we demonstrate that the glycoprotein B homologue of HHV-8 binds to DC-SIGN in a dose-responsive manner and that DC-SIGN binds HHV-8 in a region of the carbohydrate recognition domain that is unique, though overlapping, with the HIV-1 gp120 and ICAM-2/3 binding sites. Lastly, we demonstrate that infection of immature DC results in the expression of IL-6, TNF-a, MIP-1a, MIP-1b, and IL-12p40, but not bioactive IL-12p70. This cytokine release occurs quickly after infection and is maintained for up to 72 hours post-infection, suggesting that virus binding is sufficient for at least some of the cytokine release and that the virus may be active in skewing infected cells to illicit a TH2 response. The significance of these findings from a public health standpoint centers on the fact that while HHV-8-related cancers have decreased in incidence in the United States, they still represent a serious global health concern in other countries. Our findings give insight into the initial interactions of HHV-8 and its target cells and as a result, can be used for the design of targeted therapies to prevent viral infection and spread.

A vaccine capable of preventing or therapeutically limiting human immunodeficiency virus (HIV) pathogenesis is urgently needed to contain the acquired immunodeficiency syndrome (AIDS) pandemic. Recombinant adenovirus (Ad)-based vectors are being explored as vaccine candidates due to their potent induction of cell-mediated immunity. To circumvent the limitations of vector-specific humoral immunity, novel Ad serotypes impervious to pre-existing immunity against conventional vectors have been developed. Utilizing the nonhuman primate model of HIV infection, we evaluated the immunogenicity of conventional Ad serotype 5- (Ad5) and novel serotype 35- (Ad35) based vaccinations against simian immunodeficiency virus (SIV) infection. In a preventative, proof-of-concept vaccination regimen, immunization against the SIV Gag protein proved highly efficacious, demonstrating robust boosting of Ad5-based vaccine-induced cellular immunity by Ad35-based vectors. Ad5/Ad35-based vaccination induced durable, high-frequency effector T cell responses that were later recalled upon heterologous SIV challenge. Vaccination resulted in modest reductions in SIV viremia, notable given the limited scope of immunization. We then tested the capacity of Ad5/Ad35-based vaccination targeting the SIV Gag, Env, and Nef proteins, with or without IL-15 augmentation, to promote cellular immunity during antiretroviral-treated chronic SIV infection with the goal of limiting rebound viremia following cessation of antiretroviral therapy (ART). Vaccination enhanced both systemic and mucosal antigen-specific cell-mediated immunity, increasing the breadth and strength over innate response to infection. Ad-induced immunity consisted of CD4+ and CD8+ T lymphocyte TH1 cytokine production of a predominantly monofunctional nature. Furthermore, vaccination enhanced both central and effector memory CD4+ and CD8+ T cell populations without augmenting niave T cell responses. Although Ad-based immunotherapy transiently restored the systemic central memory CD4+ T cell compartment, vaccination failed to salvage effector memory or mucosal CD4+ T cells. Therapeutic intervention was associated with transient containment of rebound viremia upon ART cessation which vaccination failed to augment. An effective vaccination against HIV represents the most efficient method to end the AIDS pandemic, and is of considerable public health significance. The findings presented herein provide evidence to support the continued evaluation of Ad-based vectors in novel treatment strategies against HIV infection, representing an incremental advancement in the field of HIV vaccine development.