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.

HIV infects and depletes CD4+ T cells in Gut Associated Lymphoid Tissues (GALT) of the Gastrointestinal (GI) tract at a very early stage of infection. Furthermore, GALT are the major reservoirs of HIV-1 and may constantly shed virus and CD4+ T cells into the intestinal lumen throughout the entire course of infection. We hypothesize that the dynamic changes of HIV-1 and CD4+ T cell quantities in feces are linked to disease progression and can be used to predict disease prognosis. The aims of this study are to establish sensitive methods for detection and quantitation of HIV-1 and CD4 mRNA in feces, and to use the methods to monitor the amount of HIV-1 RNA/DNA and CD4 mRNA in feces samples of HIV infected patients and to correlate the findings with disease progression. In addition, since urine may potentially serve as a vehicle for HIV-1 transmission we have also measured HIV-1 RNA/DNA in the urine samples from the same population used for the feces study. Our results showed that using normal feces spiked with known copies of DNA and RNA, as low as 2.5 copies of HIV-1 DNA and 40 copies of HIV-1 RNA were detected per input in both nested PCR and RT-nested PCR reactions respectively. Human CD4 mRNA was also detected in feces. From HIV-1 infected volunteers of the Multicenter AIDS Cohort Study (MACS), HIV-1 DNA, RNA and human CD4 mRNA was detected in 8%, 19% and 31%, respectively, in the feces samples from patients with detectable viral load in blood. In the urine samples from the same study population, HIV-1 DNA was detected in 26% of HIV-1 infected donors and this detection is not always correlated with the presence of detectable viral load in blood. This study has major Public health significance as it demonstrates that HIV-1 RNA/DNA could be detected in feces and urine samples, which may lead to the development of a future non-invasive approach to evaluate disease progression and prognosis. In addition, our study demonstrated, for the first time, the presence of human CD4 mRNA in fecal specimens of infected donors, which could be used as a valuable tool in the future to assess the pathogenesis of Gut Associated Lymphoid Tissue over the course of HIV-1 infection.

CD4+T-cells have a central role in induction and homeostasis of the immune response, and are also the major target cells for HIV. HIV has devised mechanisms to subvert the immune system and further its cause of survival and dissemination. vpr is one of the accessory genes, which is essential for the virus survival in vivo. Being a soluble protein with an ability to transduce across cell membranes, Vpr can potentially affect bystander cells. We hypothesize that HIV-1 Vpr alters the functions of both infected and bystander T lymphocytes, utilizing direct and indirect mechanisms, and these Vpr-mediated effects contribute in part to the immune dysregulation, and aid in viral dissemination. The Specific Aims of this proposal are to: (1) Assess the immune modulatory effects of Vpr in infected and bystander T-lymphocytes; (2) Understand the role of Vpr in T lymphocytes, natural killer (NK) cells and dendritic cells (DC) interactions; and (3) Analyze the structure-function role of Vpr in immunopathogenesis. We utilized HIV-1wt and HIV-1ΔVpr viruses and compared the difference in the effects of these viruses under controlled simulated invitro conditions. The differences observed in the effect of these two viruses can be attributed to Vpr provided that the infections in both the experimental sets are similar. In some studies, to clearly identify infected cells, we employed EGFP reporter viruses. The effects in infected cells and bystander cells were evaluated. Results indicate that HIV-1 Vpr has a role in dysregulation of immune cells during HIV infection. Vpr differentially regulates the surface expression of T cell costimulatory molecules, CD28 and CTLA-4, and inhibits IFN-γ production in infected T cells. Vpr also inhibits NK cell function by augmenting TGF-β production and inducing altered expression of NK receptor ligands. Further, oligomerization of Vpr has a role in gag incorporation of Vpr and in viral pathogenesis. The findings presented in this study are significant for public health because mechanistic understanding of the pathogenesis will aid in development of novel anti-viral therapeutics.

Bacterial and viral meningitis are public health concerns as they are contagious and highly fatal without treatment. Newborn infants are at high risk for bacterial and viral meningitis. The onset of the infection is rapid and without quick diagnosis and treatment, many infants will die. Diagnosis requires the positive identification of the causative agent through culture or the use of polymerase chain reaction (PCR) from specimens of cerebrospinal fluid (CSF). Because these tests can take hours or days to perform, it is important identify children who have a higher likelihood of serious bacterial or viral infection so that empiric therapy can be initiated while awaiting further results. Previous studies have indicated that CSF characteristics can be accurate early predictors of viral and bacterial meningitis. Although CSF characteristics have been established for infection, normal values for infants less than 60 days of age are still not clear. To improve characterization of CSF values for infants, this study set to answer three questions: Is there a temporal relationship for CSF WBC, glucose, and protein? What are the means and confidence intervals for the means for each of these variables? What is the range of normal values that a physician could expect to find in infants less than two months of age? This study involved three independent retrospective chart reviews over a 15-year period to identify infants less than two months of age who presented to The Childrens Hospital of Pittsburgh emergency department with fever and had lumbar punctures performed but were not found to have bacterial or viral meningitis. For CSF WBC and protein, the data from the three cohorts were pooled and a single set of reference values was generated for infants less than two months of age. CSF glucose values were not pooled due to differences that existed between the cohorts and reference values were calculated for the cohorts individually. CSF white blood cell (WBC), glucose, and protein values were analyzed to answer our three study objectives. A temporal trend was found for CSF WBC and protein with values being highest during the first weeks of life. CSF glucose values did not change with time. These values will be potentially valuable reference tools in emergency departments for physicians who are faced with decisions regarding care and treatment of febrile infants.

The gut mucosa is an important portal for HIV-1 transmission and infection. Therefore, a vaccine which can prevent virus transmission at mucosal surfaces would be an ideal HIV-1 vaccine candidate. Clostridium perfringens has been used as a vehicle to deliver SIV proteins in large quantity to the terminal ileum. A mucosal immunization strategy using C. perfringens should be able to induce potent mucosal immune responses against HIV-1. A recombinant C. perfringens expressing large amount of HIV-1 Gag protein (Cp-Gag) was constructed. Under in vitro conditions, Cp-Gag was found to induce bone marrow derived dendrite cell (BMDC) to mature and stimulate HIV-1 Gag specific T cell responses. Then in vivo experiments were performed in mice to demonstrate orally delivered Cp-Gag ability to prime gut mucosal T cell responses. Since oral tolerance is a major obstacle for orally delivered immunization approaches, a combination of mutated heat-labile enterotoxin of E. coli (mLT) and CpG containing oligodeoxynucleotides (CpG-ODN) were used as adjuvants for oral administration with Cp-Gag. Orally delivered Cp-Gag was tested for induction of HIV-1 Gag specific T cell responses in a prime-boost model with intranasal inoculation of HIV-1 virus like particles (VLP). HIV-1 specific cellular immune responses in both the effector (Lamina propria) and inductive sites (Peyers patches) of the gastrointestinal (GI) tract were significantly higher in mice immunized using Cp-Gag and VLPs in prime-boost approaches compared to mice immunized with either Cp-Gag or VLPs alone. Such cellular immune response was found to be mediated by both CD8+ and CD4+ T cells. These groups of mice also seemed to have HIV-1 specific multifunctional T cells in PPs and LP of the GI tract. In summary, mucosal immunization of mice with a Cp-Gag and VLPs in a prime-boost mode led to strong HIV-1 Gag specific cellular immune responses in both mucosal and systemic immune compartments. Such strong mucosal immune response could be very important to control HIV-1 infection at mucosal surfaces. The proposed vaccine strategy has great public health significance for developing a practical vaccine against HIV due to its safety, low production cost and easy administration.