The research efforts of our laboratory focus on understanding the cellular and molecular regulation of viral gene expression, HIV replication, entry into the cell, development of improved HIV envelope immunogens, optimization of immune responses to gene-based vaccination, and correlates of immune protection, with the goal of developing rationally designed vaccines against HIV, Ebola/Marburg viral hemorrhagic fevers, influenza and other emerging and re-emerging infectious diseases. Various elements of vaccine design and delivery, including optimization of vector and protein design, adjuvants, dosing, and methods and routes of delivery, are studied. Other areas of research address mechanisms of viral gene regulation, assembly of viruses, viral cell interactions, and insight into the regulation of eukaryotic gene expression.
An effective vaccine against HIV poses unique challenges. HIV infects CD4 cells, which play a major role in the stimulation of both cellular and T-cell dependent humoral immune responses. Because there are few examples of natural immunity to this virus, we do not have a clear understanding of immune correlates and mechanisms of protection against primary HIV infection. To address these problems and to develop effective vaccines, we are exploring a number of approaches, including the identification of immunogens that elicit broadly neutralizing antibodies, understanding the molecular and cellular basis for immune responses to components of HIV, identification of relevant forms of viral proteins for antigen presentation, stimulation of relevant T cell types, and enhancement of antigen-presenting, dendritic cell function. A high priority for effective vaccine development is the quantitation of immune responses in animals and in humans, identification of surrogate markers of immune protection, streamlined vaccine production, and rapid evaluation of candidate vaccines for testing in clinical trials. The technologic advancements in genomics also offer potential to facilitate vaccine design and allow identification of genetic determinants of immunogenicity for vaccine-induced responses. Though the immune correlates of protection against HIV remain unknown, there is evidence that cell-mediated immunity confers protection from viral replication. At the same time, evidence from other successful vaccine approaches indicates that the antibody response plays a critical role in immune protection. The challenge for HIV disease is to develop a vaccine that can elicit a broadly reactive T cell response that is long lasting and to identify antigens that will elicit a broadly neutralizing antibody response to conserved regions of the virus.
CTL-based vaccines: The development of immunogens that generate cellular immunity will be guided by research that defines cell-mediated immune responses which control viremia and prevent clinical consequences of infection. In this setting, clinical studies of preventive vaccine candidates and therapeutic vaccination models can provide specific information on the role of HIV-specific CD4+ and CD8+ T cells in the control of HIV infection. This information will improve our understanding of the composition of protective immune responses that can be elicited by preventive vaccines. Until recently, it was difficult to measure cellular immune responses to HIV in a reproducible and quantitative manner. However, we can now characterize, precisely and quantitatively, the fundamental aspects of the HIV-specific CD4+ T cell memory response and functional CD8+ T cell responses, and correlate these with clinical and virologic parameters of HIV disease.
A major goal of the lab is to advance these efforts through the design of effective immunogens that induce CTL. To facilitate the development of CTL-based vaccine candidates for evaluation in phase I trials, we have prepared numerous gene-based immunogens by inserting HIV cDNAs into relevant plasmids. Various cDNAs have been tested using plasmid-based gene delivery and selected candidates that express either Gag, Pol, various Gag-Pol fusion proteins and mutants, as well as Env and Nef cDNAs, have also been inserted into viral vectors. These vectors include replication-defective forms of adenoviruses and poxviruses. The viral genes include both clade B and non-clade B viruses. Gene-based immunogens are modified to improve protein expression and immunogenicity. Also, mutagenesis to synthesize alternative proteins with enhanced immunogenicity will be developed. These approaches provide great flexibility in identifying immunogens that can induce broad and potent CTL immune responses.
Neutralizing antibody-based vaccines: Because CTL responses alone are unlikely to provide protection, it is important that an HIV vaccine elicit broadly neutralizing antibodies to the virus. Such antibodies are dependent on memory B cells, a long-lived cell population that can divide and differentiate into the antibody-producing plasma cell upon re-exposure to antigen, thus conferring long-term protection. Another advantage of antibodies is that they have the potential to inactivate virus before it has a chance to infect the cells of the host. Antibodies may also mobilize the inflammatory system, including the complement system, neutrophils, and monocytes. Thus, even when an antibody does not directly neutralize the virus, there is potential for amplification through the inflammatory system.
A major hurdle for a highly effective HIV vaccine has been the development of antigens that elicit broadly neutralizing antibodies. To develop vaccines which elicit broadly neutralizing antibodies, a rational method of deletion and mutational analysis of conserved and nonconserved viral envelope structures is being used in the lab. Systematic deletion and/or mutation of constant and variable regions has been applied using plasmid-based, as well as prime-boost approaches (e.g., DNA/ADV or DNA/MVA), and these structures are being characterized.
Several mutants of gp160 have been developed that remain highly active in their ability to elicit cytolytic T cell responses and are also able to stimulate enhanced antibody responses. To improve the immune response to native gp160 and to expose the core protein for optimal antigen presentation and recognition, we have analyzed the immune response to modified forms of the protein. The role of conserved N-linked glycosylation sites has been studied, and analogues of fusion intermediates have been developed. An expression vector with deletions in the cleavage site, the fusion peptide, and the interspace between the two heptad repeats was shown to elicit a more potent humoral immune response while retaining its ability to stimulate Env-specific CTL.
Another approach which is being initiated to identify broadly neutralizing antibodies includes screening of recombinant immunogens. This effort will include the immunization of small animals (guinea pigs and/or rabbits) using various envelope genes delivered with DNA priming and ADV boosting. Sera from these immunized animals are analyzed in neutralization assays. If broadly neutralizing antibodies are detected by this approach, pools will be deconvoluted, and individual immunogens will be identified. Promising candidates are evaluated in non-human primate models.
The rapid spread of HIV as an infectious pathogen has prompted us to study emerging viruses and explore potentially common mechanisms of interactions with host cells. The molecular basis of the pathogenicity of Ebola virus has been studied more recently and provides important lessons for the development of AIDS vaccines.
The Ebola virus has been identified as the cause of several highly lethal outbreaks of hemorrhagic fever, but the molecular basis for its pathogenicity is unknown. Further, the failure to document immunity to the virus, together with the difficulty in identifying its natural host reservoir and lack of antiviral drugs, stimulated our efforts to characterize this virus and its host interactions further. To determine whether it was possible to obtain immunity to these viruses, we developed a DNA vaccination approach and have recently shown that it is possible to generate protective immunity against Ebola virus infection (Xu et al., 1998). Immunity was achieved most effectively with plasmid expression vectors encoding the viral glycoprotein or secreted glycoprotein in a guinea pig model of disease, whose pathology is similar to the human infection.
This approach was further evaluated for its applicability to humans by testing in primate models. We developed and tested a highly effective vaccine strategy for Ebola virus infection in non-human primates (Sullivan et al., 2000). A combination of DNA immunization and boosting with adenoviral vectors generated cellular and humoral immunity in cynomolgus macaques. Challenge with a lethal dose of the highly pathogenic, wild type, 1976 Mayinga strain of Ebola Zaire virus resulted in uniform infection in controls, who progressed to a moribund state and death in less than one week. In contrast, all vaccinated animals were asymptomatic for more than six months, with no detectable virus after the initial challenge. These findings demonstrate that it is possible to develop a preventive vaccine against Ebola virus infection in primates.
The availability of eukaryotic expression vectors which encode these genes has allowed further characterization of these glycoproteins and their interactions that could not otherwise be studied with a highly pathogenic virus. To characterize the interactions of Ebola glycoproteins with different cell types, the full-length glycoprotein, which arises from the same open reading frame by post-transcriptional editing, was used to pseudotype retroviral vectors, and infection of a variety of cell types was analyzed. Although it can infect different cell types, the virus showed preferential infection of endothelial cells (Yang et al., 1998). Expression of this viral gene product also induces cytopathicity in this cell type which raised the possibility that this viral glycoprotein may also directly contribute to the pathogenicity of the disease (Yang et al., 2000). Interestingly, a second form of the viral glycoprotein, the secreted glycoprotein, does not bind to endothelial cells but instead interacts with neutrophils and inhibits early events in neutrophil activation. Thus, this viral gene is used to generate two gene products, one that appears to inhibit the host inflammatory response to the virus, and a second that directs the virus to relevant target cells where it induces cellular damage that is the likely cause of the lethal effects of Ebola virus infection.
When HIV infects T lymphocytes, specific interactions between viral and cellular gene products are required for productive viral replication. HIV gene expression is enhanced in T lymphocytes upon cellular activation by cytokines or specific signal transduction pathways. In earlier studies, we demonstrated that an inducible cellular transcription factor, NF-kB, was activated after cellular stimulation and provided a mechanism to increase HIV transcription (Nabel and Baltimore, 1987). This effect was mediated by binding of the transcription factor to cis-acting regulatory sequences in the viral long-terminal repeat. Additional studies defined cytokines, tumor necrosis factor-k?and interleukin-1, which activate NF-kB and the HIV enhancer (Osborn et al., 1989) and established that stimulation of HIV expression by induction of NF-kB also occurred during monocyte differentiation (Griffin et al., 1989). We subsequently reported the isolation of a cDNA that encoded a previously unknown NF-kB/Rel family member (NF-kB2) and increased HIV gene expression cooperatively with RelA (Schmid et al., 1991). A novel subunit of IkB, termed IkB-e, was also identified from a yeast two-hybrid screen in our laboratory (Li and Nabel, 1997) which is expressed constitutively in lymphoid cells and inhibits the induction of kB-dependent genes. The role of this family member, in contrast to previously defined IkB's, was characterized, and it was found to be differentially regulated from IkB-a and IkB-a. Our efforts have most recently focused on the regulation of NF-kB in the nucleus, where we have learned that alternative mechanisms of regulation, independent of IkB, have profound effects on NF-kB regulation and HIV transcription.
Our previous studies established a relationship between regulation of cell cycle progression and induction of NF-kB through a mechanism involving nuclear transcriptional coactivators, p300 and CBP (Perkins et al., 1997). These studies arose from the observation that NF-kB induction, either in response to specific cytokines or stress, is associated with growth arrest. Our studies therefore began to focus on links between cell activation, regulated by transcription factors, and cell proliferation, under the control of cellular kinases and proteases. Progression through the eukaryotic cell cycle is controlled by the assembly and activation of specific cyclin dependent kinase (CDK) complexes, a process regulated, in part, through their interaction with CDK inhibitory proteins (CKIs). We recognized that stimuli which induce the CKI, p21, such as DNA damage, serum growth factors, phorbol esters, and okadaic acid, also activate NF-kB and showed that p21 stimulated kB-dependent gene expression in the absence of a direct increase in NF-kB DNA binding activity. We have since shown that this effect is mediated by the interaction of RelA(p65) with either the p300 or CBP transcriptional coactivators. Specifically, the COOH-terminal transcriptional activation domain of RelA(p65) interacts with an NH2-terminal region of p300 at the same time that the CDK, largely comprised of the cyclin E-Cdk2 complex, was able to bind to a distinct COOH-terminal region of p300. The interaction of NF-kB and CDKs through the p300 and CBP coactivators provides a mechanism for the coordination of transcriptional activation with cell cycle progression (Perkins et al., 1997).
This mechanism of transcriptional regulation is relevant to HIV gene expression and replication. For example, several groups have shown the accessory protein, Vpr, causes arrest of cell cycle progression at G2/M, presumably through its effect on Cyclin B1/Cdc2 activity. Vpr also displays a transcriptional activation function. We have recently shown that the ability of Vpr to activate HIV transcription correlates with its ability to induce G2/M growth arrest, and this effect is mediated by the p300 transcriptional coactivator, which promotes cooperative interactions between the RelA subunit of NF-kB and Cyclin B1/Cdc2 (Felzien et al., 1998). Vpr cooperated with p300, which regulates NF-kB and the basal transcriptional machinery, to increase HIV gene expression and viral replication. These data suggested that p300, through its interactions with NF-kB, basal transcriptional components, and CDKs, is modulated by Vpr and regulates HIV replication. The regulation of p300 by Vpr provides a mechanism to enhance viral replication in proliferating cells after growth arrest by increasing viral transcription. In addition, we have also found an interaction between HIV-1 Tat and p300/CBP. Tat transactivation was inhibited by the 12S form of the adenoviral E1A gene product which inhibits p300 function, and this inhibition was independent of its effect on NF-kB transcription. A biochemical interaction of p300 with Tat was demonstrated in vitro and in vivo by co-immunoprecipitation. The COOH-terminal region of p300, which binds to E1A, was shown to bind specifically to the highly conserved basic domain of Tat that also mediates binding to the TAR RNA stem loop structure. The ability of Tat to interact physically and functionally with p300 or CBP provides a mechanism to assemble a basal transcription complex which may subsequently respond to the effect of Tat on transcriptional elongation and represents a novel interaction between an RNA binding protein and a transcriptional coactivator. It may also provide a mechanism by which to modify chromatin structure of the provirus through the histone acetylase activity associated with these coactivators.
We have also conducted studies on the regulation of HIV replication with regard to host cell apoptosis. The possible role of apoptosis had been previously suggested as a mechanism to account for T cell depletion in AIDS. We determined that PBMCs or T leukemia cells exposed to HIV-1 undergo enhanced viral replication in the presence of the cell death inhibitor, z-VAD-fmk. z-VAD-fmk, which targets specific pro-apoptotic caspases, stimulated endogenous virus production in activated PBMCs derived from HIV-1-infected asymptomatic individuals. These data suggested that programmed cell death may serve as a beneficial host defense to limit HIV spread in infected individuals (Chinnaiyan et al., 1997).
Another area explored by our laboratory is the cellular and molecular basis of immune suppression. One mechanism associated with inhibition of immune function and induction of lymphoid apoptosis involves the Fas-Fas ligand (also termed CD95-CD95L) system. Our findings suggest that gene transfer of CD95L generates apoptotic and proinflammatory responses which can induce regression of both CD95+ and CD95- tumors (Arai et al., 1997). More recently, we have begun to define the molecular basis for the suppression in immune-privileged sites and in CD95L-induced inflammation. Our data indicate that TGF-b suppresses the proinflammatory effects of CD95L. Because both CD95L and TGF-b1 inhibit T cell function, these cytokines are together likely to contribute to the development of immunologic tolerance and may inhibit immune responses to tumors (Chen et al., 1998). Advances in the understanding of molecular immunology and gene delivery have provided alternative molecular genetic strategies that may improve our understanding of AIDS in addition to having application to the treatment of cancer.
Wei CJ, Boyington JC, McTamney PM, Kong WP, Pearce MB, Xu L, Andersen H, Rao S, Tumpey TM, Yang ZY, Nabel GJ. Induction of broadly neutralizing H1N1 influenza antibodies by vaccination. Science. 2010 Aug 27;329(5995):1060-4. Epub 2010 Jul 15.
Wu X, Yang ZY, Li Y, Hogerkorp CM, Schief WR, Seaman MS, Zhou T, Schmidt SD, Wu L, Xu L, Longo NS, McKee K, O'Dell S, Louder MK, Wycuff DL, Feng Y, Nason M, Doria-Rose N, Connors M, Kwong PD, Roederer M, Wyatt RT, Nabel GJ, Mascola JR. Rational design of envelope identifies broadly neutralizing human monoclonal antibodies to HIV-1. Science. 2010 Aug 13;329(5993):856-61. Epub 2010 Jul 8.
Zhou T, Georgiev I, Wu X, Yang ZY, Dai K, Finzi A, Kwon YD, Scheid JF, Shi W, Xu L, Yang Y, Zhu J, Nussenzweig MC, Sodroski J, Shapiro L, Nabel GJ, Mascola JR, Kwong PD. Structural basis for broad and potent neutralization of HIV-1 by antibody VRC01. Science. 2010 Aug 13;329(5993):811-7. Epub 2010 Jul 8.
Cen S, Niu M, Saadatmand J, Guo F, Huang Y, Nabel GJ, Kleiman L (2004). Incorporation of pol into human immunodeficiency virus type 1 Gag virus-like particles occurs independently of the upstream Gag domain in Gag-pol. J Virol. 2004 Jan;78(2):1042-9.
Burstein E, Ganesh L, Dick RD, Van De Sluis B, Wilkinson JC, Klomp LW, Wijmenga C, Brewer GJ, Nabel GJ, Duckett CS (2004).
A novel role for XIAP in copper homeostasis through regulation of MURR1. EMBO J. 2004 Jan 14;23(1):244-54. Epub 2003 Dec 18.
Ganesh L, Burstein E, Guha-Niyogi A, Louder MK, Mascola JR, Klomp LW, Wijmenga C, Duckett CS, Nabel GJ (2003). The gene product Murr1 restricts HIV-1 replication in resting CD4+ lymphocytes. Nature. 2003 Dec 18;426(6968):853-7.
Kong WP, Huang Y, Yang ZY, Chakrabarti BK, Moodie Z, Nabel GJ (2003). Immunogenicity of multiple gene and clade human immunodeficiency virus type 1 DNA vaccines. J Virol. 2003 Dec;77(23):12764-72.
Lemiale F, Kong WP, Akyurek LM, Ling X, Huang Y, Chakrabarti BK, Eckhaus M, Nabel GJ (2003). Enhanced mucosal immunoglobulin A response of intranasal adenoviral vector human immunodeficiency virus vaccine and localization in the central nervous system. J Virol. 2003 Sep;77(18):10078-87.
Sullivan N, Yang ZY, Nabel GJ (2003). Ebola virus pathogenesis: implications for vaccines and therapies. J Virol. 2003 Sep;77(18):9733-7.
Tritel M, Stoddard AM, Flynn BJ, Darrah PA, Wu CY, Wille U, Shah JA, Huang Y, Xu L, Betts MR, Nabel GJ, Seder RA (2003). Prime-boost vaccination with HIV-1 Gag protein and cytosine phosphate guanosine oligodeoxynucleotide, followed by adenovirus, induces sustained and robust humoral and cellular immune responses. J Immunol. 2003 Sep 1;171(5):2538-47.
Sullivan NJ, Geisbert TW, Geisbert JB, Xu L, Yang ZY, Roederer M, Koup RA, Jahrling PB, Nabel GJ (2003). Accelerated vaccination for Ebola virus haemorrhagic fever in non-human primates. Nature. 2003 Aug 7;424(6949):681-4.
Nabel GJ (2003). Cancer gene therapy: present status and future directions. Ernst Schering Res Found Workshop. 2003;(43):81-8. Review. No abstract available.
Nabel GJ (2003). The future of gene therapy. Ernst Schering Res Found Workshop. 2003;(43):1-16. Review. No abstract available.
Barouch DH, McKay PF, Sumida SM, Santra S, Jackson SS, Gorgone DA, Lifton MA, Chakrabarti BK, Xu L, Nabel GJ, Letvin NL (2003). Plasmid chemokines and colony-stimulating factors enhance the immunogenicity of DNA priming-viral vector boosting human immunodeficiency virus type 1 vaccines. J Virol. 2003 Aug;77(16):8729-35.
Klausner RD, Fauci AS, Corey L, Nabel GJ, Gayle H, Berkley S, Haynes BF, Baltimore D, Collins C, Douglas RG, Esparza J, Francis DP, Ganguly NK, Gerberding JL, Johnston MI, Kazatchkine MD, McMichael AJ, Makgoba MW, Pantaleo G, Piot P, Shao Y, Tramont E, Varmus H, Wasserheit JN (2003). Medicine. The need for a global HIV vaccine enterprise. Science. 2003 Jun 27;300(5628):2036-9.
Nabel GJ. Vaccine for AIDS and Ebola virus infection. Virus Res. 2003 Apr;92(2):213-7. Review.
Yang ZY, Wyatt LS, Kong WP, Moodie Z, Moss B, Nabel GJ (2003). Overcoming immunity to a viral vaccine by DNA priming before vector boosting. J Virol. 2003 Jan;77(1):799-803.
Sullivan NJ, Sanchez A, Rollin PE, Yang Z-Y, Nabel GJ. (2000). Development of a preventive vaccine for Ebola virus infection in primates. Nature. 408, 605-609.
Yang Z-Y, Duckers HJ, Sullivan NJ, Sanchez A, Nabel EG, Nabel GJ. (2000). Identification of the Ebola virus glycoprotein as the main viral determinant of vascular cell cytotoxicity and injury. Nat Med. 6, 886-889.
Chen J-J, Sun Y, Nabel GJ. (1998). Regulation of the proinflammatory effects of Fas ligand (CD95L). Science. 282, 1714-1717.
Felzien, L.K., Woffendin, C., Hottiger, M.O., Subbramanian, R.A., Cohen, E.A., and Nabel, G.J. (1998). HIV transcriptional activation by the accessory protein, VPR, is mediated by the p300 co-activator. Proc Natl Acad Sci USA. 95, 5281-5286.
Xu L, Sanchez A, Yang Z, Zaki SR, Nabel EG, Nichol ST, Nabel GJ. (1998). Immunization for Ebola virus infection. Nat Med. 4, 37-42.
Yang, Z., Delgado, R., Xu, L., Todd, R.F., Nabel, E.G., Sanchez, A., and Nabel, G.J. (1998). Distinct cellular interactions of secreted and transmembrane Ebola virus glycoproteins. Science. 279, 1034-1037.
Arai H, Gordon D, Nabel EG, Nabel GJ. (1997). Gene transfer of Fas ligand induces tumor regression in vivo. Proc Natl Acad Sci USA. 94, 13862-13867.
Chinnaiyan AM, Woffendin C, Dixit VM, Nabel GJ. (1997). The inhibition of pro-apoptotic ICE-like proteases enhances HIV replication. Nat Med. 3, 333-337.
Li Z, Nabel GJ. (1997). A new member of the IkB protein family, IkBe, inhibits RelA (p65)-mediated NF-kB transcription. Mol Cell Biol. 17, 6184-6190.
Perkins ND, Felzien LK, Betts JC, Leung K, Beach DH, Nabel GJ. (1997). Regulation of NF-kB by cyclin-dependent kinases associated with the p300 co-activator. Science. 275, 523-527.
Schmid RM, Perkins ND, Duckett CS, Andrews PC, Nabel GJ. (1991). Cloning of an NF-kB subunit which stimulates HIV transcription in synergy with p65. Nature. 352, 733-736.
Griffin GE, Leung K, Folks TM, Kunkel S, Nabel GJ. (1989). Activation of HIV gene expression during monocyte differentiation by induction of NF-kB. Nature. 339, 70-73.
Osborn L, Kunkel S, Nabel GJ. (1989). Tumor necrosis factor a and interleukin-1 stimulate the human immunodeficiency virus enhancer by activation of the nuclear factor kB. Proc Natl Acad Sci USA. 86, 2336-2340.
Nabel G, Baltimore D. (1987). An inducible transcription factor activates expression of human immunodeficiency virus in T cells. Nature. 326, 711-713.
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Last Updated February 07, 2011