Opendra "Bill" Narayan, D.V.M., Ph.D. - US grants

Visna of sheep and HIV encephalopathy are chronic neurological diseases caused by ovine and human lentiviruses. The viruses are species-specific and cause infection host to host and in the brain by invasion of infected cells across mucosal surfaces and across the blood-brain-barrier, respectively. Neuropathogenesis is associated with virus replication in macrophages in brain but the mechanism for destruction of the neuropil is not understood. Experiments outlined in this proposal will determine the feasibility of inducing protective immunity in sheep against virus invasion across mucosal surfaces and across the blood-brain-barrier. Inactivated virus particles will be compared for efficacy with recently developed transgenic sheep PBMC which express the virus envelope protein as well as allotypic MHC antigens. Experiments will determine whether immunization of sheep via the intratracheal and the GI routes, respectively, will induce protection at both sites against infected cells and whether protective, group-specific immunity (against heterologous virus) can be induced. Because of the similarity between the two lentiviral systems in the mucosal mode of infection, the sheep experiments will be of value in identifying possible strategies for controlling heterosexual spread of HIV. Various factors (identified in preliminary experiments) that contribute to neuropathogenesis will be explored. The effects of rupture of the vascular endothelium of the brain by physical and pathogenic autoimmune (EAE) processes and the effects of cytokine-induced expression of adhesion and MHC II antigens in the cultured vascular endothelial cells will be explored. Infection in the bone marrow will be evaluated to confirm that this event precedes traffic of infected cells across the BBB and that the severity of infection in precursor cells in the marrow influences the rate of entry of virus-infected cells into the brain. Antibodies to viral, leukocyte and other cellular antigens will be delivered into brains of infected sheep via ventricular access devices to determine whether modulation of trafficking of specific mononuclear cells or expression of specific cellular antigens will affect CNS lesions and virus replication. Finally, the viral genetic basis of tissue- specific virulence will be determined using an infectious molecular clone of visna virus which will have mutated and become tropic for a particular tissue during virus passage through he specific tissue, in sequentially inoculated sheep.

This is an application to continue studies on the delineation of pathogenic mechanisms of SIVmac in infected rhesus macaques. Our earlier studies had suggested that the ability of the virus to cause disease in the lymphoid or the nervous system correlated with nucleotide sequences in the viral DNA that conferred tropism of the virus for CD4+ T cells (L) or (brain-specific?) macrophages (M), respectively; that mild disease was associated with early development of virus-inhibitory CD8+ cells; that severe disease correlated with an intense and prolonged early phase of immunological activation of infected CD4+ T cells; and that resurgence of viral replication occurred in infected animals that had developed immunological control over their infection. Experiments proposed in this application will test these hypotheses. 1. Whereas disease in the lymphoid system may be caused by both L- and M-tropic viruses, dementia can be caused only by a subclass of M-tropic viruses (AIM 1,3) and requires only incomplete or defective virus replication in CNS cells (AIM 1). Full-blown encephalopathy is a severe complication of dementia and occurs only when infected macrophages in the brain become immunologically activated, a state that renders these cells permissive for full expression of the virus life cycle (AIM 3). 2. If antiviral CD8+ cells are the main host defense against replication of L- and M-tropic viruses, then specific interference with the function of these cells should result in severe disease (AIDS or neurological) (AIM 1). 3. If severe disease and dissemination of infected cells to the CNS ("neuroinvasion") are a direct function of immunological activation of CD4+ T cells (which both L and M-tropic viruses infect), then immuno- intervention aimed at reducing the level of cellular activation should result in only mild disease and sparing of the CNS (AIM 2). 4. If resurgence of virus replication in infected immune animals is caused by CTL-escape variants, then such viruses, but not the original virus, should be able to replicate selectively in cultured autologous CD4+ T cells in the presence of CD8+ cells that inhibit the original virus. Our study on molecular immunopathogenesis of CNS disease will also be evaluated physiologically with assessment of cognitive and motor functions of the animals and also with a study to determine whether neurovirulence of the viruses correlates with non-neutralizing antibodies that enhance infection in macrophages.

The human immunodeficiency virus (HIV) causes persistent infection and chronic progressive disease involving multiple organ systems but the pathogenesis of the lesions is not understood. Development of lesions correlates with a high degree of virus gene expression of macrophages in pathologically affected tissues. Among non-human primates, different species of African monkeys are naturally infected with indigenous lentiviruses, but these agents are apathogenic in these animals. However, inoculation of Asian macaques with the viruses results in disease that resembles the HIV disease complex. Lesions in diseased macaque tissues are accompanied by extensive virus replication in tissue macrophages, similar to that seen in human tissues. In this application, we ask if there are cell-culture correlates that can predict pathogenicity of these viruses. Our central hypothesis is that host macrophages, in addition to T4 lymphocytes, must be susceptible to productive replication of viruses that cause disease. This predicts that host species of animals that do not develop disease will have T4 lymphocytes permissive for virus replication but macrophages will be non- permissive. On the other hand, species that do develop disease will have permissive lymphocytes and permissive macrophages. We will test the hypothesis in established model systems of apathogenic infection in African monkeys and fulminant disease in macaques. Lymphocytes and macrophages from uninfected animals will be tested for permissiveness of virus replication and similar cells from infected animals will be examined for evidence of productive virus replication. Multiple parameters of infection in the cell cultures will include tests for viral RNA, viral proteins, virions, and virus infectivity. We will then use this strategy to test permissiveness of cells of different macaque species to infection with lentiviruses of unknown pathogenicity. Fully permissive systems will be tested for confirmation of pathogenicity in limited experiments in macaques at the Yerkes Primate Center. The project will combine expertise in lentivirus-macrophage biology, ultrastructural analysis and existing expertise of lentivirus pathogenesis in macaques at the Yerkes Primate Center and access to exotic species of non-human primates. These experiments will provide a valuable, relatively inexpensive test system for identification of new models of AIDS and contribute to the further understanding of lentivirus disease.

This application addresses the great need for an animal model that reflects the pathogenic potential of HIV-1. Building upon our experience with the SIVmac and ruminant lentiviruses, we passaged SHIV through macaques and in pigtail macaques, and derived a virulent SHIV swarm that caused persistent, productive infection with a severe precipitous loss of CD4+ T cells. In this proposal, we will examine the pathogenesis of infection in pigtail macaques with this SHIV warm, addressing the hypothesis that loss of CD4+ T cells is caused by viral lysis of the cells, phenomenon evident by apoptotic changes in the lymphoid tissues. We hypothesize two periods of lysis of the T cells which are matched by a decline in CD4+ T cell count in peripheral blood. The first period of decline occurs early after infection and the second after the immune responses to the virus break down. We will determine whether particular sequences in the HIV-1 env gene are associated with the pathogenic effects of the virus and whether such genotypes are selected in particular tissues. Cloning and sequencing of env genes obtained by PCR from tissues, and examination of biological properties of viruses isolated from tissues, and examination of chimeric viruses constructed with the env genes from tissues will be used to answer these questions. We will derive pathogenic molecular clones of SHIV directly from affected tissues. This will provide information on the molecular basis of SHIV pathogenicity and supersede use of limited tissue material for reproducing disease. Finally, we will infect pigtail macaques with SHIV-delta-nef as a vaccine and challenge them with virulent SHIV. Prevention of disease, rather than infection by the virulent virus, is the goal of this experiment. We expect that pigtail macaques infected with the virulent SHIV swarm will develop AIDS, with loss of CD4+ T cells (less than 200/microliter) and opportunistic infections; that apoptosis will be demonstrable in lymphoid issues during the periods of CD4+ loss, and that some macaques will also develop disease in non-lymphoid issues, enabling us to link specific genotypes with particular tissues. We also expect that macaque infected with the SHIV-delta-nef will develop persistent non-pathogenic infection and will not develop disease hen challenged with the virulent SHIV virus swarm.

This is an application to continue studies on the delineation of pathogenic mechanisms of SIVmac in infected rhesus macaques. Our earlier studies had suggested that the ability of the virus to cause disease in the lymphoid or the nervous system correlated with nucleotide sequences in the viral DNA that conferred tropism of the virus for CD4+ T cells (L) or (brain-specific?) macrophages (M), respectively; that mild disease was associated with early development of virus-inhibitory CD8+ cells; that severe disease correlated with an intense and prolonged early phase of immunological activation of infected CD4+ T cells; and that resurgence of viral replication occurred in infected animals that had developed immunological control over their infection. Experiments proposed in this application will test these hypotheses. 1. Whereas disease in the lymphoid system may be caused by both L- and M-tropic viruses, dementia can be caused only by a subclass of M-tropic viruses (AIM 1,3) and requires only incomplete or defective virus replication in CNS cells (AIM 1). Full-blown encephalopathy is a severe complication of dementia and occurs only when infected macrophages in the brain become immunologically activated, a state that renders these cells permissive for full expression of the virus life cycle (AIM 3). 2. If antiviral CD8+ cells are the main host defense against replication of L- and M-tropic viruses, then specific interference with the function of these cells should result in severe disease (AIDS or neurological) (AIM 1). 3. If severe disease and dissemination of infected cells to the CNS ("neuroinvasion") are a direct function of immunological activation of CD4+ T cells (which both L and M-tropic viruses infect), then immuno- intervention aimed at reducing the level of cellular activation should result in only mild disease and sparing of the CNS (AIM 2). 4. If resurgence of virus replication in infected immune animals is caused by CTL-escape variants, then such viruses, but not the original virus, should be able to replicate selectively in cultured autologous CD4+ T cells in the presence of CD8+ cells that inhibit the original virus. Our study on molecular immunopathogenesis of CNS disease will also be evaluated physiologically with assessment of cognitive and motor functions of the animals and also with a study to determine whether neurovirulence of the viruses correlates with non-neutralizing antibodies that enhance infection in macrophages.

This project proposes to extend studies on efficacy of an attenuated SHIV vaccine to protect macaques against SHIV/KU-induced AIDS following inoculation of this highly virulent virus on mucosal surfaces. The model is relevant to biological properties of patient isolates of HIV-1. Following a traumatic intravaginal deposition of SHIV/KU into non- immunized sexually mature macaques, the virus caused infection in large cells in the mucosa and within two days, infected cells were present in the pelvic and mesenteric lymph nodes. This was followed by explosive replication in the lymphoid system, resulting in severe global loss of CD4+ T cells within 1 month and AIDS within 8 months in 6/6 macaques. In collaboration with Dr. Harold McClure at Yerkes, we initiated vaccine experiments using attenuated gene-deleted SHIV's in hopes of protecting macaques against SHIV/KU-induced STD. These studies showed that 5/6 macaques injected subcutaneously with deltavpu deltanefSHIV-4 and 6/6 immunized orally with deltavpuSHIV-PPc resisted productive systemic infection and AIDS caused by intravaginally-inoculated SHIV/KU, given 6 months later. In this application, the PI and Dr. McClure plan to further utilize the resources of Yerkes Regional Primate Center to pursue new studies on the vaccine efficacy against SHIV/KU disease using orally inoculated deltavpuSHIV-PPc vaccine. We will collaborate with Dr. J. Sastry at the University of Texas, Bastrop, to study CMI correlates of protection. In five Specific Aims, we will determine 1) whether vaccine protection will last as long as two years; 2) whether vaccination is effective against challenge virus SHIV/KU given orally and rectally in addition to vaginally; 3) whether immunized animals will resist heterologous pathogenic viruses that are not neutralized by antibodies induced by the vaccine virus; 4) whether fetuses become infected during vaccination or challenge of pregnant animals and, 5) whether new born animals exposed to vaccine virus at birth will resist heterologous pathogenic virus later. We will use animals in Aims 1, 2 and 3 to explore immunological correlates of vaccine-induced protection. 70 animals will be used in the five-year period and they will be house at Yerkes.

The principal investigator proposes to employ a pathogenic chimeric simian-human immunodeficiency virus (SHIV) to determine whether passive immunization can protect against mucosa (vaginal) or needle stick exposure to HIV. Two sources of SHIV/HIV-neutralizing antibody will be evaluated: a monoclonal anti-HIV gp120 (mAb b12) or polyconal anti-SHIV pooled sera from infected rhesus macaques with low virus burdens. Passive immunotherapy will be initiated either 12 hours pre- or 12 hours post intravaginal SHIV exposure and 10 pre- or 1 or 12 hours post subcutaneous injection. The establishment of infection and disease progression will then be monitored over a 12 month period and compared with animals not receiving passive immunization. The results of these studies are designed to establish whether passive immunization with virus neutralizing antibody can prevent or abort SHIV infection or influence the progression of SHIV disease.

SHIVKU containing the envelope of HIV-1 is a recently derived virus that causes AIDS and neurological disease in Rh macaques after severe depletion of CD4+ T cells. In previous studies, we used this virus to characterize the histological nature of the neuropathological changes and the nature of the altered systemic infection during and after withdrawal of anti-retroviral therapy. We also established that it causes a low-grade systemic infection after given as a challenge to vaccinated animals. In this application, in Aim 1, we will use the model to obtain more detailed information on the chronology of development of CNS disease in order to better understand the nature of virus-host responses associated with this complication. This information will be used as a backdrop for intervention strategies focused on the nature of the infection in the CNS of animals placed on drug therapy and in vaccinated animals. Questions that cannot be answered in humans can now be approached. In Aim 2 we will determine the effects of anti-retroviral therapy on the progress of infection in the CNS and on neuropathological processes already in progress. In Aim 3 we will determine whether live vaccine virus causes infection in the CNS. Although vaccination prevented AIDS, SHIVKU given as challenge still caused systemic infection, a result that can be expected in immunized humans exposed to HIV-1. As part of Aim 3 we will determine also whether this low-grade infection by SHIVKU also becomes established in the CNS. Do these viruses which cause persistent CNS infection mutate along divergent pathways from virus in lymph nodes? In Aim 4 we will extend in vitro findings that anti-IL-4 strategies causes reduction of virus replication in macaque macrophages. We will use gene therapy with antisense IL-4 DNA to treat SHIV encephalitis in Rh macaques in attempts to cause reduction in virus replication in brain macrophages and thus cure the encephalitis.

cytotoxic T lymphocyte; disease /disorder model; human immunodeficiency virus; interleukin 10; interleukin 4; simian immunodeficiency virus

SHIVKU containing the envelope of HIV-1 is a recently derived virus that causes AIDS and neurological disease in Rh macaques after severe depletion of CD4+ T cells. In previous studies, we used this virus to characterize the histological nature of the neuropathological changes and the nature of the altered systemic infection during and after withdrawal of anti-retroviral therapy. We also established that it causes a low-grade systemic infection after given as a challenge to vaccinated animals. In this application, in Aim 1, we will use the model to obtain more detailed information on the chronology of development of CNS disease in order to better understand the nature of virus-host responses associated with this complication. This information will be used as a backdrop for intervention strategies focused on the nature of the infection in the CNS of animals placed on drug therapy and in vaccinated animals. Questions that cannot be answered in humans can now be approached. In Aim 2 we will determine the effects of anti-retroviral therapy on the progress of infection in the CNS and on neuropathological processes already in progress. In Aim 3 we will determine whether live vaccine virus causes infection in the CNS. Although vaccination prevented AIDS, SHIVKU given as challenge still caused systemic infection, a result that can be expected in immunized humans exposed to HIV-1. As part of Aim 3 we will determine also whether this low-grade infection by SHIVKU also becomes established in the CNS. Do these viruses which cause persistent CNS infection mutate along divergent pathways from virus in lymph nodes? In Aim 4 we will extend in vitro findings that anti-IL-4 strategies causes reduction of virus replication in macaque macrophages. We will use gene therapy with antisense IL-4 DNA to treat SHIV encephalitis in Rh macaques in attempts to cause reduction in virus replication in brain macrophages and thus cure the encephalitis.

The major effect of HIV-1 infection is elimination of the CD4+T cell population, the event that correlates with the profound immunosuppression caused by the virus. Antiretroviral therapy of the infection causes dramatic curtailment of virus replication, evidenced by virtual elimination of virus particles from plasma and recovery of CD4+T cell numbers. However, the virus persists in lymphoid tissues, and upon withdrawal of therapy (for whatever reason), virus replication begins again in most of these individuals, with renewed attack on the CD4+T cell population. The question arises whether a vaccine given during the period of drug therapy could induce protective immune responses that would prevent viral resurgence when the therapy is withdrawn. Problems with this concept are that immunological correlates of protection against HIV are not known, and a prophylactic vaccine, on which a therapeutic vaccine should be based, has not been developed yet. Even if an effective prophylactic vaccine were on hand, proof of concept for therapeutic immunization would still be required. In this application, we will use the SHIVKU/macaque model of HIV infection-disease to examine the feasibility of therapeutic immunization. SHIV has the env of HIV-1 on a background of SIV and is the closest genetically related agent to HIV-1 that will replicate productively in macaques. We adapted SHIV to macaques and derived a highly virulent strain, SHIVKU, which causes a very rapid type of HIV-like disease, with sub-total elimination of CD4+T cells and AIDS in 6 months. Using genetic engineering, we also recently developed a live virus vaccine that replicated transiently in macaques and induced immune responses that prevented replication of SHIVKU, given as challenge, for 2.5 years. Having established its efficacy prophylactically, we will now examine the potential of this vaccine for therapeutic efficacy. Macaques will be inoculated with SHIVKU then placed on temporary antiretroviral therapy, which will suppress replication of this virus. A portion of the animals will then be inoculated with drug-resistant vaccine virus. Following withdrawal of therapy, we will determine whether vaccine -induced immune responses inhibited resurgence of SHIVKU from reservoirs of latently infected cells in lymphoid tissues. In anticipation of success, we will then determine whether a more conservative therapeutic immunogen, inactivated virus particles, could also function therapeutically. These studies will establish proof-of- concept for the feasibility of performing this type of immunization to effectively prevent HIV resurgence in drug- treated infected individuals who come off therapy. This vaccine approach would provide a clear alternative to the dreary prospect of permanent post infection drug therapy.

DESCRIPTION (provided by applicant): Five years of support are requested to create a faculty development program that will enable junior faculty involved in research at the three PhD granting institutions in Kansas, (KU at Lawrence and the KU School of Medicine in Kansas City, and K State University) to become more competitive for securing NIH grant support. This will be achieved by creation of a strong mentoring body (comprised of Dr. Narayan and other established, NIH-funded senior faculty at the three institutions), establishment of committees of internal and outside advisors to the COBRE program, institutionalization of monthly seminar and data presentation meetings, and an annual meeting dedicated to factors important in preparation of NIH grant applications. Three cores, one administrative, and two technical will be established to expedite the research program. The COBRE will receive extensive institutional support in the form of FTEs, new research equipment, and space. The research theme is centered on mechanisms inhibiting replication of pathogenic microbes, with the long-term goal of controlling infectious diseases important in human health. Studies will be focused on identification of genes and gene products that have a seminal role in microbial replication. To find inhibitors for these proteins, we will link up with the COBRE program at KU/Lawrence to exploit resources in combinatorial chemistry and high-throughput screens for inhibitors of the proteins in question. We will also exploit more biological approaches using core support with phage display and aptamer technology, which will be shared among investigators. Further, we will establish core facilities in X-ray crystallography and fermentation for the isolation and determination of the 3-D structures of the proteins, which will be useful for development of potential drugs. Six junior faculty members, two of whom are experienced X-ray crystallographers, have been selected for support for two to three years. The projects of currently selected faculty were based on scientific merit, potential for complementary interaction with other projects, and potential for exploiting the core resources of both COBRE programs. Projects of new junior faculty will be selected and awards made upon recommendation of the internal advisory committee. A portion of the COBRE fund will be used to enhance faculty recruitment packages.

DESCRIPTION: (Provided by Applicant) This is an application to continue longitudinal studies in macaques that have been used to explore mechanisms of safety of a SHIV vaccine, and the remarkable effectiveness of this vaccine not only in conferring long term protection against disease caused by pathogenic SIV and SHIVs, but also elimination of these pathogens given as challenge. Earlier studies established that the vaccine is safe for at least 4 years after inoculation into newborns and after serial passage in young macaques. Studies on efficacy showed that following transient replication, virus could not be re-isolated from the animals but viral DNA persisted for more than 4 years in lymphoid tissues. Immunized animals challenged with pathogenic viruses between 7 and 18 months later became infected but replication of these agents was dramatically curtailed, and in many animals, both the challenge viruses and their DNA became progressively undetectable during three years of post-challenge study. Infectious DNA of the vaccine virus is effective in causing the immunizing infection, and because it induces immune responses to some HIV proteins, it has been proposed to the FDA as a therapeutic vaccine for HIV-infected persons. Data from studies proposed in this application will be used as preclinical support for an IND application to the FDA. New proposed studies will address the following devil's advocate hypothesis: 1. Challenge viruses that are thought to have been eliminated are still present in lymphoid tissues and can be induced by treatment of the challenged animals with anti-CD8+T cell mAb. 2. The vaccine-induced long-term efficacy that correlates with persistence of memory antiviral CD8+T cells, is maintained by antigen provided by persistently-replicating vaccine virus in lymphoid tissues. This will be tested in studies in which some immunized animals will be treated with anti CD8+T cell mAbs to induce the virus. Others will be treated with PMPA to suppress putative virus replication so that immune responses will decay in the absence of antigen. Further, new animals will be inoculated with lymph node cells from virus-negative, immune animals and tested for infection. 3. Pathogenicity of the vaccine virus could be demonstrated by further serial passage of the virus in new animals, and by injection of the agent into macaques that are immunosuppressed by treatment with anti-CD8+T cell mAbs.

DESCRIPTION (provided by applicant): Currently, use of macaques to evaluate efficacy of HIV vaccines has shown that although numerous types of vaccines can blunt acute infections by pathogenic challenge viruses, no vaccine can predictably prevent infection and the inevitable establishment of viral latency that accompanies the infection. The burden on the vaccine therefore is to maintain protective responses to prevent rebound of the virus from latently infected cells. The increasing incidence of virus breakthroughs after years of protection suggests that it may be necessary to administer post-exposure boosts at regular intervals in order to indefinitely maintain prevention of virus rebound. With this in mind, we investigated the feasibility of using a new type of DNA vaccine that could be used prophylactically and continued after exposure to pathogenic virus. We chose SHIVku2 DNA as a lentiviral vector that expresses several HIV genes, among which are the env and gag that can be tailored to match the genes of any particular subtype of HIV. The vaccine backbone consists of SIV promoter/enhancer sequences driving expression of the high-replication-competent SHIVku2 genome from which the rt, integrase, vif, and 3 'LTR were deleted (delta4), and the rev and tat retained. Proof of concept has shown that the delta4 DNA expressing SIV gag and X4 HIV env induced protection against heterologous X4 SHIV without the benefit of viral protein boosts and that immunization could be continued following challenge. However, proof of efficacy against R5 viruses of different subtypes would require availability of pathogenic SHIVs expressing the env/gag of these viruses. In Aim 1 of this proposal, we will develop new pathogenic SHIVs that express the env/gag of patient isolates of subtypes B and C by incorporating these genes into the genome of highly pathogenic SHIVku2. These viruses will then be used in Aim 3 as challenge to test the efficacy of new delta4 SHIV DNA vaccines expressing env and gag of HIV subtypes B and C. We will use DNAs of cytokines GM-CSF and IL-15 as adjuvants to boost the magnitude and duration of long term immunity induced by the already successful DNA vaccine, depending on results of studies in Aim 2, in which mice will be used to assess these potential adjuvanting effects. We will then extend the study parameters in Aim 4, where we will determine whether the DNA vaccine, possibly strengthened with the cytokine adjuvants, can be used to immunize chronically infected animals under the cover of antiretroviral therapy, to re-induce immunity that would have waned during therapy. Vaccine boosts will continue after drug therapy had been withdrawn. These studies will be applicable to HIV infected persons under HAART.

[unreadable] DESCRIPTION (provided by applicant): Five years of support are requested to create a faculty development program that will enable junior faculty involved in research at the three Ph.D. granting institutions in Kansas (KU at Lawrence and the KU School of Medicine in Kansas City, and Kansas State University) to become more competitive for securing NIH grant support and to recruit senior faculty who could develop new research programs. A strong mentoring program comprised of Dr. Narayan and individual Project mentors, all of whom are established investigators, a writing program designed to assist COBRE members in preparation of manuscripts and grants, and an external advisory committee who will review progress of all Projects twice annually will be established. Five Cores, one administrative, one focused on writing, and three technical, have already been established in the final year of the present COBRE to expedite the new research program. The COBRE has received extensive new institutional support in the form of FTEs and new research equipment in the three technical Cores. The research theme is centered on basic and translational mechanisms aimed at inhibiting replication of pathogenic microbes. Studies will be focused on identification of microbial genes and gene products that have a seminal role in their replication and use of these as targets for therapy. The translational portion of the COBRE will be dedicated to use of gene therapy using nanoparticles as vectors for delivery in vivo. The COBRE support will be used to support three-year Projects of four junior faculty and one senior faculty, funds for enhancement of recruitment packages of new faculty at the three institutions, and funds for further recruitment of senior faculty who could contribute to development of new research programs. Funds will also be used to operate the three technical Cores comprised of state-of-the-art flow cytometry, signal transduction, and Luminex technology, which will facilitate rapid analysis of host response gene products resulting from infection. The Projects of currently selected faculty were based on scientific merit, review by External Advisory Committee members, with a focus for complementary interaction with other Projects, potential for development into new research programs headed by senior faculty, and likelihood for receiving NIH grant support. Two of the technical Cores will be directed by two faculty members who were previously supported by the COBRE and who have each secured two NIH grants. The new program was developed in response to the new RFA that suggested recruitment of senior faculty who could help develop and head new research programs. One such senior faculty has already been recruited. Finally, the new program has been based on our track record in which eleven investigators supported by the previous COBRE have secured a total of 16 extramural grants. Eight of the eleven have secured 12 NIH grants. [unreadable] [unreadable] [unreadable]