Donald L. Sodora - US grants

Oral transmission of HIV is one of the factors fueling the current AIDS pandemic. Here we utilize the simian immunodeficiency virus (SIV)/macaque AIDS animal model to investigate the early events following oral transmission. We hypothesize that the very early events in mucosal transmission are crucial to determining whether or not an infection occurs. By investigating these earliest events, a clear picture will emerge as to how infection via the oral route occurs and why a successful HIV/SIV infection is only observed after some oral inoculations. A fluorescently labeled SIV (SIVmac239gfp) will be used to enable us to identify the cell infected very early after the virus inoculation (Aim 2). Low doses of virus will be used to assess the role of an inflamed gingivitis mucosa in SIV transmission (Aim 2). The role of gingivitis will be assessed in juvenile macaques and the role of inflamed gums associated with the cutting of teeth will be assessed in infants. Transient infections represent an important disease outcome in which the virus is detected several weeks post-inoculation but can not be detected at later time points. We will investigate both virologic and immunologic aspects of transient infections (Aim 3). This study will include an assessment of the number of SIV responsive T-cells in macaques utilizing a new assay developed by Dr. Louis Picket at the University of Texas Southwestern Medical Center. PBMC's will be obtained from SIV vaccinated, SIV infected and uninfected macaques and assessed for the ability to produce cytokines in the presence of SIV antigens through a flow cytometric analysis. In total these studies are designed to assess the entry of virus via the oral mucosa, follow the virus as it disseminates throughout the macaque and to assess the factors which influence a successful or abortive oral transmission.

Description (Adapted from the Investigator's Application): In this competing renewal the experimental design has been adjusted so that maximum information on the pathogenesis of early infection from patients who are treated with potent antiretroviral therapy can be derived. The applicants have preliminary results that lead to hypothesize that the immune response during acute HIV-1 infection is being driven by excess antigen. There are several possible consequences of this strong antigen driven response. First, CTL will rapidly expand and may, under certain situations, either become clonally exhausted or they may cause the selection of CTL escape variants. The CTL response by removing that antigen drive through the introduction of antiretroviral therapy during acute infection will be addressed. Early intervention may result in reversion of CTL from an activated to a memory phenotype, and that the immune system may be able to respond appropriately if the patient is non- adherent to the therapy and viral replication resumes. Second, the applicants hypothesize that the proliferation of CD4+ T cells in response to HIV-1 antigen supplies the virus with its major source of target cells for replication during acute infection. The consequence of this scenario is that at the completion of the acute phase of injection, most of the HIV-1-responsive CD4+ T cells have been clonally deleted. If HIV-1- specific (or other antigen-specific) CD4+ T cell responses are severely depleted at the end of the acute phase of injection, and whether early intervention results in retention of these responses will be determined. Finally, the applicants believe that low levels of antigen within lymphoid tissues may serve as a nidus to maintain an active immune response to HIV- 1 and they will specifically determine if immunologic responses to HIV-1 are maintained in lymphoid tissues after they have been lost from peripheral blood.

DESCRIPTION: (Adapted from Applicant's Abstract) MHC Class I-restricted CD8+ T cells are an important component of the immune response to HIV as well as the simian immunodeficiency virus (SIV). It is generally accepted that a functional vaccine against HIV will need to elicit an effective CD8+ T cell response. This proposal will develop techniques to be used in the Rhesus macaque AIDS vaccine animal model to assess functional and quantitative aspects of the CD8+ T cell response to SIV. We will use established techniques including the intracellular cytokine assay along with a novel method we are developing to quantify SIV specific CD8+ T cell clonotypes in macaques. These techniques will assess the magnitude (percent of CD8+ cells), breadth (number of SIV epitopes), and the clonality (number of T cell receptors (TCRs)) of the CD8+ T-cell immune response to SIV vaccination and compare these to a natural infection. The specific aims for this proposal are as follows: 1. To identify and quantify by intracellular cytokine staining SIV specific CD8+ T cells in Rhesus macaques using peptide panels which encompass entire SIV proteins The innovative aspect of Aim 1 is that it permits an in depth analysis of the CD8+ T cell response to an entire protein but does not rely on any assumptions of immunodominance of one epitope over another and can be used in any macaque regardless of MHC haplotype. 2. To develop a TCR Molecular Clonotype Tracking Assay to identify and monitor the TCR genotypes of SIV specific CD8+ T cells in Rhesus macaques. The innovative aspect of Aim 2 is that it utilizes a combination of SIV peptide stimulation and cytokine secretion permitting an analysis of the TCR clonotypic repertoire and tracking of the responding CD8+ T cells. This will permit an in depth analysis of the clonality of SIV-specific CD8+ genotypes elicited during vaccination and a determination of whether these clonotypes expand or are eliminated after viral challenge. Utilizing these techniques requires no prior knowledge of either the epitope specificities of SIV-specific CD8+ T cells or the macaque's MHC haplotype. We can therefore proceed with these studies in macaques with the reagents we currently possess. Development of the techniques described above will provide in depth information with regard to SIV-specific CD8+ T cells elicited during vaccination or infection which cannot be obtained with current technology.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The association between CD4+ T-cell depletion and an accelerated rate of disease progression is well established for HIV infection in humans and simian immunodeficiency virus (SIV) infection in macaques. However, SIV infection in its natural primate host (i.e. sooty mangabeys) results in near normal levels of CD4+ T-cells despite similar plasma viral loads. Following transfer of plasma from an SIV+ mangabey in the Yerkes colony, two mangabeys exhibited a decline in CD4+ T cell levels to below 100 cells/ul of blood which is also be observed in lymph nodes. These levels have remained between 18 and 100 cells/ul of blood for the past 8 years. The presence of AIDS defining CD4+ T-cell levels in SIV+ mangabeys is an uncommon occurrence within the Yerkes colony (only four of the 105 SIV+ mangabeys screened are CD4-low). The goal of these experiments was to characterize the mechanisms behind the observation of CD4 depletion as well as assess the ability of these mangabeys to mount immune responses in a CD4-low mileu. To undertake this study, three additional mangabeys were infected with plasma virus from a CD4-low mangabey and the CD4 levels in these three mangabeys declined to below 100 CD4 cells/ul of blood by 21 days post-infection. We recently tested the ability of the now 5 CD4-low SIV+ mangabeys to respond to an influenza vaccination. We vaccinated them twice and sent the plasma to the CDC for analysis to quantify the levels of influenza specific antibodies. In this way we will be able to determine if mangabeys are able to elicit effective immune responses even when their CD4+ T cell levels are low.

DESCRIPTION (provided by applicant): The transmission of an infectious agent from one host to another is critical to the success of any pathogen. In the case of HIV, a successful transmission is dependent on the ability of the virus to transverse a mucosal membrane (rectal, penile, vaginal or oral) of a new host and establish a productive infection. This proposal builds on findings we obtained while undertaking a previous grant funded by the NIH (NIDCR) (1999 to 2003) to assess the earliest events following oral transmission of SIV in the macaque model. Oral transmission of HIV represents a major route of transmission from mother to infant, through ingesting the virus during breast-feeding;as well as in adults during oral genital transmission (virus in semen). One major finding from our previous studies was the identification of the oral mucosa, esophagus and tonsils as sites of SIV entry following a nontraumatic oral application of the virus and the assessment of the innate and adaptive immune responses that correlate with the rates of disease progression. Here we expand upon our previous findings by assessing the impact of immune activation on the transmission of SIV administered into the oral cavity. We hypothesize that higher levels of immune activation will increase the potential for SIV to elicit a productive infection when administered to the oral cavity of macaques. These studies will utilize a low dose regimen of viral inoculations to mimic the viral levels that are present in breast milk and semen. The goal of specific aim 1 is to determine the appropriate viral inoculum to undertake aim two, and to investigate the correlates of viral transmission in the absence of any immune modifications. Aim 2 will assess transmission of SIV while the mucosa is experiencing a state of immune activation, gingivitis. Gingivitis is a common inflammation of the oral mucosa in humans and therefore may be impacting oral transmission in humans exposed to HIV containing semen (through receptive oral intercourse). These studies assessing the impact of the immune response on oral transmission will likely be applicable to other sites of mucosal transmission, and will be important for determining the immune mechanisms that impact the success of SIV/HIV mucosal transmission. Our long term goal is to utilize these findings to aid in the design of SIV/HIV vaccines that are able to elicit sterilizing immunity by preventing the infection of the first target cell when mucosal sites are exposed to HIV/SIV.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. These experiments will provide important preliminary data for the development of the SIV foreskin/penile macaque challenge model. Based on the finding from the Merck STEP trial, in which vaccinated men with a foreskin were more likely to become HIV+, we propose that the foreskin challenge route should be utilized as a mucosal challenge route in future SIV vaccines. Optimizing the foreskin/penile challenge route will be important to test vaccines via this important mucosal route in the SIV/monkey model. These experiments utilize a coinfection model with the bacteria Haemophilus Ducreyi (causative agent of chancroid) to evaluate the factors that influence SIV infection via the foreskin. The most recent experiment is one in which foreskin/penile SIV transmission is assessed following the induction of H. Ducreyi infection, with the H. Ducreyi being administered to the foreskin area of the macaques via two different infection routes (intradermal and subcutaneously). Assessment of these experiments is currently underway.

DESCRIPTION (provided by applicant): The association between CD4+ T cell depletion and accelerated rate of disease progression is well established for HIV in humans and simian immunodeficiency virus (SIV) infection of Rhesus macaques. In contrast to pathogenic infections, SIV infection of certain African primate species (i.e. sooty mangabeys) does not result in progression to simian AIDS despite high levels of viral replication, initially this absence of disease progression was attributed to both lower immune activation and preserved CD4+ T cell levels. More recent studies have challenged the idea that maintenance of normal CD4+ T cell levels is a key factor that protects natural SIV hosts from simian AIDS. In 2007 the Sodora laboratory published a study in which plasma from a naturally SIV-infected mangabey with normal CD4+ T cell counts (above 600 cells/ul) was inoculated to six SIV-uninfected mangabeys in October 2000. Two of these experimentally infected mangabeys exhibited a dramatic CD4+ T cell decline in blood (<80 cells/ul) as well as lymph nodes and GALT. These SIV+ CD4-low mangabeys show no evidence of systemic immune activation during chronic infection. We determined that this dramatic CD4+ T cell depletion is virally mediated, as replicating virus has an expanded co-receptor utilization profile and passage of this virus to three mangabeys in October 2006 again resulted in dramatic CD4+ T cell depletion. To date, none of the SIV+ CD4-low mangabeys have exhibited any clinical signs of simian AIDS even though they have been CD4-low for between 3 to 8 years. The goal of this proposal is to ascertain the mechanisms by which SIV+ CD4-low mangabeys are able to maintain immunologic health. We hypothesize that the preserved immune system is due in part to the presence of double-negative (CD3+/CD4-/CD8-) T cells that are able to provide the necessary T helper function. The first aim will characterize the similarities between double-negative and CD4+ T cells in mangabeys, macaques, and humans, assessing both phenotype and function. The second aim will functionally assess the double-negative T cells from these three species with regard to T helper function;both B cell antibody class switching and regulatory T cell activity. The third aim will evaluate the ability of SIV-infected CD4-low mangabeys to elicit effective innate and adaptive immune responses to diverse vaccine challenges and to assess the role of double-negative T cells the vaccine specific immune responses. This unique cohort of animals allows for the innovative functional characterization of T cell populations that do not express CD4, have the potential to perform the function of helper T cells and are refractory to SIV infection. Importantly, our studies will also be assessing the double-negative T cells in Rhesus macaques and humans permitting a determination if these cells have potential to perform a similar task in HIV+ patients. Our findings could potentially lead to novel strategies for development of immune therapies or vaccines that induce functional activity from the double-negative T cell subset which is refractory to the HIV infection.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The association between CD4+ T-cell depletion and an accelerated rate of disease progression is well established for HIV infection in humans and simian immunodeficiency virus (SIV) infection in macaques. Prior to our studies, SIV infection in its natural primate host (i.e. sooty mangabeys) was observed to result in near normal levels of CD4+ T-cells despite similar plasma viral loads. Following transfer of plasma from an SIV+ mangabeys in 2000, and 2006, five mangabeys exhibited a decline in CD4+ T cell levels to below 100 cells/ul of blood which is also be observed in lymph nodes. These levels have remained between 18 and 100 cells/ul of blood for the past 9 years. The presence of what should be AIDS defining CD4+ T-cell levels in SIV+ mangabeys is an uncommon occurrence within the Yerkes colony (only four of the 105 SIV+ mangabeys screened are CD4-low). We recently tested the ability of these five (5) CD4-low SIV+ mangabeys to respond to an influenza vaccination. We vaccinated them twice and sent the plasma to the CDC for analysis to quantify the levels of influenza specific antibodies. Through microneutralization assays and hemaglutination inhibition it was determined that all five mangabeys were able to mount effective antibody responses to the influenza vaccination, even with the very low levels of CD4+ T cells. We are now assessing the various T cell subsets in these mangabeys to determine which of these subsets could potentially be fulfilling the T helper cell role in these SIV+ CD4-low mangabeys. Efforts thus far have identified the CD3+/CD4-/CD8- (double-negative) T cells as the most likely candidate.

DESCRIPTION (provided by applicant): Numerous African non-human primates, including the sooty mangabeys, are natural hosts of SIV and have co- evolved with their respective SIVs for at least 30,000 years. Studies of SIV infections in natural host species have dramatically influenced the way we think about AIDS pathogenesis in two fundamental ways. First, prevention of disease progression does not require suppression of virus replication, as natural SIV hosts remain free of clinical signs despite high levels of viremia. Second, the absence of AIDS correlates with low levels of systemic immune activation during chronic infection. The Sodora laboratory has established a cohort of SIV-infected mangabeys that unexpectedly developed severe CD4+ T cell depletion (CD4 blood levels are <80 cells/ul) but still do not progress to AIDS. These CD4-low animals challenge the current paradigm that very low CD4+ T cell counts are not compatible with long term survival. The key unanswered question that the SIV-infected CD4-low mangabey cohort invokes is: How do these sooty mangabeys maintain ostensibly normal T helper cell function with so few CD4+ T cells? In this proposal we will test the hypothesis that CD3+CD4-CD8- double-negative (DN) T cells provide basic T helper cell functions in CD4-low SIV-infected sooty mangabeys, allowing for maintenance of a normal immune system, whereas DN T cell function is sub- optimal during the CD4+ T cell depletion that follows pathogenic HIV/SIV infections in humans/macaques. DN T cells are inherently resistant to SIV infection due to the absence of the CD4 receptor. In addition to studying the function of these cells in sooty mangabeys, we will investigate the potential for DN T cells to function as helper cells during pathogenic HIV and SIV infections of humans and macaques. In previous studies, we have demonstrated that in CD4-low SIV-infected sooty mangabeys, DN T cells: (i) Are present at relatively high levels in peripheral blood both prior to and following an SIV infection; (ii) Can be SIV specific and produce cytokines in response to SIV peptide stimulation; (iii) Principally exhibit a central memory T cell phenotype; and (iv) Produce Th1, Th2 and Th17 cytokines. These preliminary data indicate that the DN T cells provide helper T cell like function that could assist in immune responses against both SIV and opportunistic pathogens in CD4-low mangabeys. The specific aims of the proposal will assess phenotype and function of DN T cells in SIV-infected mangabeys as well as in two pathogenic infections, SIV-infected macaques and HIV infected humans (Aim 1), as well as the ability of DN T cells to make antigen specific responses in immunized mangabeys (Aim 2). Ultimately, this work will provide the basic evaluations necessary to determine if a future immune therapeutic intervention to increase function of DN T cells during pathogenic HIV/SIV infections is possible. We envision that immune therapeutic approaches improving DN T cell function may provide a 'Functional Cure' in which HIV-infected individuals will achieve a non-pathogenic state that is similar to what we have documented in SIV-infected natural hosts.

DESCRIPTION (provided by applicant): HIV causes distinct pathology and immunologic dysregulation at multiple tissue sites throughout the body. While HIV virus itself initiates these events the prolonged state of systemic immune activation seen in HIV- infected individuals likely drives much of the observed immune dysfunction. Within gut associated lymphoid tissue (GALT), HIV mediates the loss/dysfunction of key immune cells leading to increased levels of bacterial products in the blood, which contribute to the sustained immune hyper-activation. The key role of macrophages for detecting microbial products via toll-like receptors (TLRs) and their ability to produce cytokines involved in activation of immune responses make these cells likely key players in this immune pathology. In particular, Kupffer cells are liver macrophages that will be the first to encounter any bacterial products that traverse the gut mucosa (via the portal vein), and the major focus of this proposal will be to investigate the potential for Kupffer cell dysfunction to contribute to the elevated levels of bacterial products and inflammatory cytokines. The simian immunodeficiency virus (SIV)-macaque model has been utilized to provide insights into Kupffer cell function through the identification of viral proteins associated with hyperplasia hypertrophy, multinuclear giant cell formation and inflammatory cytokine production of Kupffer cells. In this proposal we combine the individual strengths of two laboratories, the Sodora laboratory with expertise in SIV non-human primate models and the Crispe laboratory with expertise in liver immunology. We will focus on the Kupffer cell bed located in the sinusoids of the liver, as these are the first population of innate immune cells to see portal vein bacterial products, and produce inflammatory cytokines. We hypothesize that systemic immune activation results in part from the production of inflammatory cytokines by liver macrophages (Kupffer cells). And that therapeutic control of the inflammatory response of Kupffer cells can reduce the level of systemic immune activation and signs of SIV disease progression in pigtail macaques. Our first aim will assess the activation state of liver Kupffer cells obtained at necropsy from uninfected versus SIV infected pigtail macaques. Our second aim will mechanistically assess the role of the NF-kB pathway (stimulated by viral and bacterial products) by suppressing this inflammatory response in Kupffer cells of pigtail macaques utilizing an NF-kB inhibitor. CAPE (Caffeic Acid Phenethylester) prevents the translocation of NF-kB to the nucleus and CAPE infused in polyketal particles are preferentially taken up by Kupffer cells. These studies build on work by Dr. Crispe's laboratory utilizing CAPE-polyketal particles to assess Kupffer cell function of mice. This exploratory R21 is designed to provide insights into the understudied field of liver and Kupffer cell biology with regard to SIV pathogenesis. Through an inhibition of liver macrophage activation in the SIV- macaque model, we will determine if targeting liver macrophages is a potential strategy for immunotherapy in HIV-infected patients.

DESCRIPTION (provided by applicant): Current candidate HIV-1 vaccines induce T cells that (i) possess restricted breadth; i.e. recognize only a few different epitopes; and (ii) predominantly target variable regions of HIV-1. Furthermore, during acute HIV-1 infection individuals who possess T cells targeting variable HIV-1 epitopes progress to disease faster than individuals possessing T cells targeting conserved HIV-1 epitopes. These data support the views that an effective T cell-based immunogen will contain only conserved regions of HIV-1 (the conserved immunogen approach). An alternative approach (the mosaic immunogen approach) has been to design immunogens to include multiple variants. This approach is based upon the theory that inclusion of multiple variants would enable responses to a broader range of circulating variants and could prime the immune system against common escape mutants. This proposal seeks to test the hypothesis that the conservation (as measured by conservation score) rather than the diversity of response against non-conserved epitopes correlates with effectiveness of vaccine-induced T cell responses. This hypothesis will be addressed in the following three Specific Aims: 1) To assess the effectiveness of T cells targeting conserved vs. variable HIV-1 epitopes to suppress viral replication in vitro, 2) To determine if T cells induced using conserved or mosaic immunogens will be activated in the context of natural HIV-1 infection and assess their ability to control viral replication; and 3) To determine if fragmentation of HIV immunogens will increase breadth of induced T cell response by increasing the pool of epitopes derived from defective ribosomal products (DRiPs). This proposal will utilize a novel in vitro priming assay to predict the epitope specificities that will be recognized by induced T cells upon vaccination with candidate vaccines. This assay could be used as a first screen of other candidate T cell based immunogens allowing only those candidates that show the most promise to move forward into more costly human clinical trials. The proposed studies have far reaching implications for design of T cell-based HIV vaccines. Determining whether effective anti-viral T cell responses induced by vaccination are due to (i) increasing the number of HIV-1 variants that are recognized or (ii) increasing specificity such that only conserved regions of HIV-1 are targeted (or both) will also have implications for the development of vaccines for other infectious organisms with high mutation rates e.g. Hepatitis C Virus (HCV).

DESCRIPTION (provided by applicant): Vaccinations are the most successful and cost-effective interventions in modern medicine. An HIV vaccine could save millions of lives and would be the centerpiece of HIV eradication efforts. However, numerous challenges remain in developing a vaccine that shows substantial efficacy in humans. Infants are one group likely to benefit from vaccine protection by preventing the nearly 400,000 new Mother-to-child transmissions (MTCT) of HIV worldwide each year. Access to anti-retroviral treatment (ART) has dramatically reduced MTCT in developed countries; however postpartum HIV transmission persists at unacceptably high levels in resource-poor settings. The reasons for this include: formula feeding not being affordable or safe (water contamination), lack of compliance to taking the ART, benefits of breastfeeding for the health of the infant and access of ART to all pregnant HIV-infected woman and their babies. In South Africa (where we have an ongoing study to assess impact of BCG vaccination in infants), and many other developing countries, the two vaccines given at birth are Bacillus Calmette-Guerin (BCG) and Oral Polio Vaccine (OPV). While these vaccines have clear protective benefits against disseminated tuberculosis and polio infection, there is also a potential downside. The very immune activation that elicits these beneficial responses may simultaneously increase an infant's susceptibility to HIV. Sub-analyses from the STEP as well as subsequent macaque SIV vaccine studies have implicated specific vaccine-induced immune responses in promoting HIV infection in vaccine recipients. If vaccines with anti-HIV/SIV activity can enhance HIV/SIV susceptibility, it is critical to assess if current immunization strategies can impact HIV transmission in HIV-exposed infants. The goals of this proposal are to: 1) Assess the immunologic impact of administration of BCG and OPV vaccines on HIV/SIV target cell recruitment and activation in infant rhesus macaques, and 2) Define the mechanism of BCG/OPV-mediated immune modulation through ex-vivo stimulation of macaque and human cells. This proposal will test the hypothesis that early administration (within days of birth) of the neonatally-administered vaccines BCG/OPV increases the risk of oral SIV acquisition in infants. To address this hypothesis, the Sodora laboratory will draw on our extensive experience in innate immunology and macaque models of SIV-infection while collaborating with Dr. Deborah Fuller, an expert in vaccination studies and adaptive immunity, to develop a thorough understanding of BCG/OPV-induced immune responses. This proposal will define the impact of neonatal vaccinations on recruitment and activation of HIV/SIV target cells as well as SIV transmission, to provide insights into the role of BCG/OPV neonatal vaccinations on oral MTCT SIV/HIV transmission. The investigation of vaccines currently administered to HIV-exposed infants makes these studies particularly relevant for current clinical practice while also facilitating the development of an effective HIV vaccine with optimal safety for HIV-exposed individuals.

Developing an effective vaccine against HIV/AIDS remains an important global health target to inhibit and reverse the high level of HIV-1 transmission currently ongoing around the world. To date, there have been four large HIV-1 vaccine trials, of these only RV144 exhibited a limited, but significant protection (31.2%) from HIV- 1 acquisition. The assessment of the results from this study has revealed a number of key correlates of protection, including antibodies that target a conformational epitope in the V1V2 region of Env. It is clear that new vaccination methodologies are needed to improve upon the findings in the RV144 trial by generating high titers of V1-V2 and functional antibodies, creating long lived B cell memory, and driving antibody maturation toward broadly neutralizing activity. The oral mucosal tissues provide a unique environment in which to vaccinate, owing to its accessible lymphoid organs, high density of immune cells and increased potential for generating a mucosal response that is able to prevent HIV-1 acquisition at a mucosal surface. We propose a vaccination strategy to deliver recombinant HIV-1 trimeric Envelope (Env) protein vaccines directly to the oral mucosa (buccal mucosa of the cheek) by intra-epithelial (IEp) vaccination. Vaccines will be tested with two adjuvant formulations designed to stimulate strong innate induction through TLR4 and TLR7/8 pathways. The goal of this proposal is to optimize oral mucosal vaccination of the HIV-1 Env protein in order to enhance Env- specific antibody responses, and to characterize the effect of oral vaccination with Env by conducting a thorough multi-parametric assessment of vaccine-elicited immunity. The first Specific Aim will assess the innate immune response to the vaccine, with a focus on the influence of the vaccine at the buccal mucosa where the vaccine is administered by IEp injection. Innate immune changes are interesting for their potential to reveal a biomolecular signature that we believe will correspond to superior HIV-1 ENV antibody responses. The second and third Specific Aims will evaluate whether supplying Env to the oral mucosa drives superior antibody responses at mucosal sites and systemically. An assessment of the neutralization and ADCVI activity will provide a functional assessment that can be correlated with the findings from studies of the innate immune response. Aims 2 and 3 will also involve a mechanistic assessment of the anti-Env antibody response through an assessment of both CD4 T Follicular Helper cell responses and antibody maturation in these macaques. In addition to the optimization of oral mucosal IEp vaccination and the exhaustive multi-parametric analyses, the combined expertise of the Sodora and Sather laboratories is one of the major strengths of this application. The experiments outlined in this proposal utilize an oral mucosal IEp delivery of next-generation HIV-1 Env antigens, as well as an assessment of both innate and adaptive immune responses. This comprehensive approach results in a high degree of confidence that undertaking these studies will make a significant contribution to HIV-1 vaccine development and advance efforts toward reversing the ongoing HIV-1 pandemic.

Gene expression is precisely controlled at all levels and modulation of mRNA stability is an important regulatory step that dictates the ultimate accumulation and functionality of mRNA. Indeed, mRNA molecules have distinct intrinsic half-lives that can span from minutes to several days and can vary in response to different physiological states. The goal of this RFA (DA-18-009) is to identify RNA modifications involved in HIV function to lay the foundation for development of novel therapeutics to treat people dealing with HIV infection and substances of abuse. Here we focus on evaluating a newly identified 5' cap, Nicotinamide Adenine Dinucleotide (NAD+), for its ability to influence both HIV and host RNA expression in the background of HIV, antiretroviral drugs (ART) and cigarette smoke condensate (CSC). The first 5' mRNA cap to be identified was N7- methyl guanosine (m7G) which among other functions imparts stability onto mRNA molecules. Additional caps have subsequently been reported with the most recent identified by Dr. Kiledjian and colleagues as a 5' end NAD+ cap in eukaryotes that can promote RNA decay. Building on this work, the first goal of this proposal is to evaluate the NAD+ 5' cap mRNA modification as it impacts the decay of both host and HIV-specific mRNAs during an HIV infection of CD4+ T cells. In addition, it is important to consider that inflammation may influence RNA modifications, and drugs of abuse in HIV-infected patients are particularly problematic in this regard. This proposal will focus on how cigarette smoke can influence the NAD+ 5' cap RNA modification. Smoking is particularly relevant as an estimated 40-70% of HIV+ patients smoke, representing a prevalence rate 2-3-fold higher than in the general population. Therefore, the second goal of this proposal is to evaluate the impact of CSC on the ability of both host and HIV-specific mRNAs to be NAD+ 5' cap modified. The two aims of the proposal are: 1. Evaluate the ability of ART treatment and CSC to influence NAD+-5' capping of HIV-specific RNA (Aim 1), and 2. Quantify NAD+-5' capping of cellular RNA following HIV infection, ART treatment and in presence of cigarette smoke condensate (Aim 2). This proposal presents an experimental plan that will utilize the expertise that exists between two laboratories to address the experimental objectives; Dr. Sodora's laboratory (HIV/SIV) and Dr. Kiledjian's laboratory (mRNA stability and decay). This R21 will undertake a series of vitro/ex vivo experiments to provide insights into the modifications to host and HIV- specific mRNAs in presence of both cART and cigarette smoke. Our long-term goal is to identify if there is a role for the NAD+ 5'cap on HIV specific and/or cellular RNAs following both HIV infection and exposure to cigarette smoke. These studies have the potential to identify the role of this novel 5' cap to impact RNA decay and to identify a mechanism to explain HIV latency and/or how the HIV virus alters cellular RNA to maximize viral production.

Liver disease is currently the most common cause of non-AIDS morbidity and mortality in developed countries amongst HIV infected people. Indeed, non-alcoholic fatty liver disease (NAFLD) is more prevalent during HIV infection compared to the uninfected population occurring in 30-40% of HIV-infected individuals. Critically, fatty liver disease is becoming an increasingly recognized precursor to non-alcoholic steatohepatitis (NASH), which can further develop into cirrhosis and liver failure. Progression toward NAFLD and steatohepatitis is multifactorial, and includes metabolic changes, cytokine release associated with TLR stimulation and oxidative stress. With regards to HIV infection, the precise drivers and mechanisms of liver disease are not well defined. This proposal will utilize the pathogenic SIV infection of rhesus macaques and in vitro human cell cultures to delineate the early mediators that drive liver disease during SIV/HIV infection. Our previous study assessing livers from SIV-infected and SIV-infected-cART-treated macaques (assessed at necropsy) identified increased levels of bacterial 16s DNA in the livers of both groups. Importantly, an unexpected finding from this study was the enrichment of Mycobacterial 16s DNA in the liver of infected macaques, which we have subsequently identified as Mycobacteria smegmatis, a commensal or potentially opportunistic pathogen. These data, as well as published findings, have led to the hypothesis that translocation of bacteria and bacterial products to the liver (including Mycobacteria-associated dysbiosis) are key mediators of liver inflammation during cART-treated HIV/SIV-infection and can initiate the early events that trigger fatty liver disease. This hypothesis will be tested through three specific aims the first two Aims assess immune and microbiome changes within the liver, lymph node and blood in SIV-infected-cART-treated macaques. Aim 3 will evaluate the mechanisms underlying changes observed in human macrophages or hepatocytes utilizing in vitro experiments following exposure to HIV, cART and bacteria/PAMPs. Our goal is to delineate the role of bacterial translocation and microbiome dysbiosis in HIV/SIV-associated liver inflammation, with particular focus on mycobacteria. To undertake these aims, this study will be led by Dr. Sodora, who has 18 years of experience evaluating immune inflammation during HIV/SIV disease including previous studies assessing liver inflammation during SIV-infection and cART-treatment. In addition, the team consists of Drs. Burwitz, Sacha and Smedley at the Oregon National Primate Research Center who have the necessary expertise to successfully undertake the outlined experiments. Collectively, these approaches will allow us to undertake a mechanistic assessment of the precise contributions of HIV/SIV virus, cART drugs and gut-derived microbes in liver inflammation as well as identify potential synergistic effects of these mediators when combined in a macaque or in vitro. Our long-term goal is to identify an immune therapeutic strategy to reduce incidence and/or severity of liver disease in HIV-infected and cART-treated individuals.

Despite significant reductions in mother-to-child HIV transmission (MTCT), HIV infection during breastfeeding still occurs at unacceptably high rates, contributing to 150,000 new infections annually. Children are more susceptible to AIDS-related illnesses than adults, with those under two years of age being more likely to succumb to rapid disease progression than any other age group. Rapid progression in infants is characterized by immune dysfunction that can include CD4 T cell depletion, B cell dysfunction and hypo-gammaglobulinemia (low plasma levels of IgM/IgG). To investigate different rates of disease progression in infants, SIV-infected (SIV+) infant rhesus macaques have proven to be a valuable model system, recapitulating several aspects of pediatric HIV infection. Using this model, the Sodora laboratory identified a rapid progressor (RP) phenotype encompassing high SIV plasma viremia and low or undetectable levels of SIV-specific antibodies. Additional analyses revealed that RP infant macaques exhibit elevated and sustained type-1 Interferon (IFN-1) levels following the acute stage of the infection, IFN-induced protein expression within B cell follicles (BCF), and that these changes were associated with germinal center dysfunction within lymphoid tissues. These differences raise important questions about the mechanism by which sustained IFN-1 signaling potentially contributes to rapid HIV/SIV disease progression, as well as the relationship between progression rate and response to combination antiretroviral therapy (cART), immune recovery following treatment, and establishment and maintenance of the latent viral reservoir. Previous studies from Dr. Chahroudi?s laboratory (proposal co-investigator) revealed differences in the latent SIV reservoir in infant compared to adult macaques, including a bias toward naïve CD4 T cells in harboring the majority of latently infected cells in infants. These findings lead to our central hypotheses: 1. Rapid progression in SIV+ infant macaques results from an elevated and prolonged type-1 IFN response that inhibits formation of effective germinal centers in lymph nodes as well as an insufficient anti-SIV humoral immune response. 2. Rapid progression in infants results in delayed immune recovery and a larger latent reservoir during administration of combination antiretroviral therapy. Aim 1 will assess the ability of transiently administered IFN- 1 receptor antagonist, during the post-acute infection period to influence disease progression and immune outcome in infant macaques. Aims 2 and 3 will evaluate the effectiveness of antiretroviral therapy on immune recovery and latent viral reservoirs in both the RP and typically progressing infant macaques. Over the last 20 years, the Sodora laboratory has investigated immune factors that modulate SIV oral transmission and disease progression in the rhesus macaque model, and the experiments outlined here expand upon these previous studies. Undertaking experiments outlined in this proposal has the potential to identify therapeutic targets for HIV+ infants with distinct disease trajectories and will provide insights into immune therapeutic approaches that to aid in immune recovery and reservoir reduction.