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Research Laboratories
Virology Laboratory
Vector Core Laboratory
Gary J. Nabel, M.D., Ph.D.
The
research efforts of our laboratory center on understanding the cellular
and molecular regulation of viral gene expression, entry into the
cell, and correlates of immune protection, with the goal of developing
safe and effective AIDS vaccines. The major areas of investigation
involve the human immunodeficiency virus (HIV) and emerging viruses.
Other areas of study address mechanisms of viral gene regulation
and provide insight into the regulation of eukaryotic gene expression.
HIV Vaccines
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.
Emerging viruses
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.
Regulation of HIV Gene Expression
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.
NF-kB, Cell Cycle Progression, and Transcriptional
Coactivation
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).
Immune Suppression
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.
Selected Relevant Publications
- 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,N.J., Sanchez,A., Rollin,P.E., Yang,Z.-Y.,
and Nabel,G.J. (2000). Development
of a preventive vaccine for Ebola virus infection in primates.
Nature 408, 605-609.
- Yang,Z.-Y., Duckers,H.J., Sullivan,N.J.,
Sanchez,A., Nabel,E.G., and Nabel,G.J. (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., and Nabel,G.J. (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,S.R.,
Nabel,E.G., Nichol,S.T., and Nabel,G.J. (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,E.G., and Nabel,G.J.
(1997). Gene
transfer of Fas ligand induces tumor regression in vivo. Proc.
Natl. Acad. Sci. USA 94, 13862-13867.
- Chinnaiyan,A.M., Woffendin,C., Dixit,V.M.,
and Nabel,G.J. (1997). The
inhibition of pro-apoptotic ICE-like proteases enhances HIV replication.
Nat. Med. 3, 333-337.
- Li,Z. and Nabel,G.J. (1997). A
new member of the IkB protein family, IkBe, inhibits RelA (p65)-mediated
NF-kB transcription. Mol. Cell. Biol. 17, 6184-6190.
- Perkins,N.D., Felzien,L.K., Betts,J.C.,
Leung,K., Beach,D.H., and Nabel,G.J. (1997). Regulation
of NF-kB by cyclin-dependent kinases associated with the p300
co-activator. Science 275, 523-527.
- Schmid,R.M., Perkins,N.D., Duckett,C.S.,
Andrews,P.C., and Nabel,G.J. (1991). Cloning
of an NF-kB subunit which stimulates HIV transcription in synergy
with p65. Nature 352, 733-736.
- Griffin,G.E., Leung,K., Folks,T.M., Kunkel,S.,
and Nabel,G.J. (1989). Activation
of HIV gene expression during monocyte differentiation by induction
of NF-kB. Nature 339, 70-73.
- Osborn,L., Kunkel,S., and Nabel,G.J. (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. and Baltimore,D. (1987).
An
inducible transcription factor activates expression of human immunodeficiency
virus in T cells. Nature 326, 711-713.
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