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Research Laboratories
Viral Pathogenesis Laboratory
Barney S. Graham, M.D., Ph.D.
The goal of the Viral Pathogenesis Laboratory (VPL) is to better understand basic aspects of viral pathogenesis and apply that knowledge towards development of safer and more effective vaccines. Defining how viruses cause disease requires investigation of functional and structural features of the viral pathogen, as well as studies to determine mechanisms for regulating the composition and timing of the host immune response. The studies encompass in vitro systems, animal models, and clinical trials. The major project areas include: 1) respiratory syncytial virus pathogenesis, 2) vaccine delivery - focused on immunization with plasmid DNA, and 3) vaccine evaluation - both preclinical studies and endpoint analysis for candidate vaccines in clinical trials.
Respiratory syncytial virus pathogenesis. RSV is an important cause of respiratory disease that has received high priority for vaccine development. Previous vaccine candidates have failed in clinical trials, and a formalin-inactivated whole virus preparation (FI-RSV) was associated with vaccine-enhanced illness. Our laboratory has contributed original discoveries and observations in the following areas with the aim of better understanding RSV pathogenesis and identifying new strategies for vaccine development and therapy:
| 1988-1991 |
Development and characterization of a murine model for RSV and defining the role of antibody and T cells in viral clearance and disease.
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| 1992-1995 |
RSV infection of neonatal lambs
can induce apnea, prolong reflex apnea, and diminish arousal. |
| 1993-1999 |
The original observation was that priming with inactivated RSV induces a type 2 T helper lymphocyte (Th2) response (dominant IL-4 expression), whereas priming with live RSV induces a Th1-like response (dominant IFN-γ expression) in mice following RSV challenge. This is now the leading hypothesis for why FI-RSV caused enhanced illness. Subsequently a series of studies defined the cytokines and cells responsible for regulating this process and identified vaccine approaches that could potentiate or mitigate the aberrant immune responses associated with the RSV vaccine-enhanced illness. This work included the finding that IL-12 is a potent vaccine adjuvant for RSV.
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| 1994-1995 |
Vitamin A levels are reduced during
acute RSV infection and can be normalized by supplementation.
This was demonstrated in both murine studies and clinical trials. |
| 1998-2004 |
The RSV F glycoprotein interacts with RhoA, and a RhoA-derived peptide can neutralize RSV, PIV-3, and HIV-1. The mechanism of peptide inhibition was found to be based on its structure and charge. The peptide blocks attachment and membrane fusion similar to other polyanions. |
| 1999-2005 |
RSV infection activates RhoA and RhoA activation is required for RSV-induced cell-to-cell fusion. Inhibitors of RhoA isoprenylation can inhibit RSV and PIV-3 replication. This work also led to the finding that RSV assembles in cholesterol-rich membrane microdomains, and that the site of assembly determines virion morphology. We then showed that disruption of lipid microdomains with methyl-b-cyclodextrin inhibits RSV infection in vitro and in mice, suggesting a new approach for topical prophylaxis. |
| 1999- ongoing |
Allergic airway inflammation potentiates and prolongs RSV-induced airway hyperresponsiveness (AHR), while prior RSV infection can diminish allergen-induced AHR. This work has led to a number of findings defining the interactions of IL-4, IL-5, IL-13, IL-17, and IL-23, and their signaling pathways in eosinophilia, mucus production, and AHR. In addition, these studies have opened a new area of research that explores the influence of prostanoids on the regulation of T cell responses and airway inflammation in the setting of RSV infection. This work continues through collaboration with Dr. Stokes Peebles at Vanderbilt University School of Medicine. |
| 1994- ongoing |
The secreted form of the RSV G glycoprotein plays a distinct role in the induction of IL-13, IL-5, and eosinophilia, and thereby modulates the composition of subsequent immune responses to RSV. This work led to other studies that demonstrated the formulation of antigen and the pattern of antigen processing played a larger role than specific G epitopes in the altered immune response induced by FI-RSV. |
| 1994- ongoing |
IL-4 modulates RSV-specific CD8+ CTL function. One possible basis for this was shown to be diminished perforin-mediated and increased FasL-mediated target cell lysis. The altered T cell response is associated with increased TNF-a production and consequently more immunopathology. These studies have been extended to demonstrate the role of various cytokines and T cell ligands on modulating CD8+ effector phenotype. |
There are two major projects in the VPL focused on RSV pathogenesis. They are derived from the last two projects listed above and have direct implications for vaccine development. One addresses the structural and antigenic properties of G, and the impact of G on modulating immune response and airway physiology. The second is to define the functional properties of CD8+ T cells associated with efficient virus clearance and those associated with immunopathology. In particular, finding vaccine delivery approaches that can selectively induce distinct functional subsets of CD8+ CTL is a priority. These studies will inform our work on vaccine delivery as described below, and are relevant to vaccine development in general.
Vaccine delivery. Immunization by
gene delivery is a major VRC platform technology. Using DNA plasmids
for gene delivery is particularly attractive because they are easy
to construct, modify, and manufacture. A major advantage of vaccination
with DNA is that there is no preexisting immunity to the vector.
In addition, there is no antigen produced other than the intended
vaccine antigen, so there is no competition for antigen processing
and presentation that may occur with more complex vector systems.
Finally, in clinical trials DNA vaccines have caused no serious
adverse reactions that threaten their clinical utility. However,
DNA vaccination has never achieved its potential, especially in
humans. While clinical trials using VRC plasmids have been well
tolerated and shown promising immunogenicity, the dose is still
high and not all subjects respond. Therefore, the VPL is exploring
new approaches for DNA vaccine delivery that may improve entry,
expression, processing, and immunogenicity.
Vaccine evaluation. Another project
area for the VPL is the support of clinical vaccine studies either
through preclinical evaluation of candidate vaccines or by developing
assays for endpoint analysis on clinical trial samples. These studies
are intended to enhance the design of clinical trials and to improve
the interpretation of results.
An example of this work is the murine studies performed
prior to the initiation of clinical trials evaluating MVA as a candidate
smallpox vaccine. These studies not only showed that MVA was immunogenic
and could induce protective immunity against challenge with vaccinia,
but began defining the correlates of immunity and demonstrated protection
even against a molecularly-modified IL-4 recombinant vaccinia virus
with enhanced virulence. In addition, all the vaccinia cultures
for the clinical trial were performed in the VPL, and plaque-reduction
neutralization assays were performed on sera from the study participants.
Finally, a novel kinetic ELISA was developed to measure antibody
responses against selected proteins from different forms of vaccinia
virus.
Another example in this project area is the evaluation
of T cell responses in persons infected with non-clade B HIV. Cryopreserved
PBMCs isolated from African immigrants infected with non-clade B
HIV are stimulated with peptide pools based on sequences contained
within the VRC candidate HIV vaccine, and evaluated for activation
of CD4+ and CD8+ T cells by intracellular cytokine staining. In
combination with full length sequencing these studies will demonstrate
the potential for cross-reactive T cell responses elicited by the
VRC multi-clade HIV vaccine to be relevant against HIV strains from
diverse epidemics.
Selected references
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Graham BS, Bunton LA, Wright
PF, Karzon DT. Respiratory syncytial virus in anti-µ treated
mice. J Virol 1991; 65:4936-4942.
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Graham BS, Bunton LA, Wright
PF, Karzon DT. The role of T cell subsets in the pathogenesis
of primary infection and reinfection with respiratory syncytial
virus in mice. J Clin Invest 1991; 88:1026-1033.
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Lindgren C, Jing L, Graham BS,
Grogard J, Sundell H. Respiratory syncytial virus infection
reinforces reflex apnea in young lambs. Pediatric Research 1992;31:381-385.
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Graham BS, Henderson GS, Tang
YW, Lu X, Neuzil KM, Colley DG. Priming immunization determines
cytokine mRNA expression patterns in lungs of mice challenged
with respiratory syncytial virus. J Immunol 1993; 151:2032-2040.
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Tang YW, Graham BS. Anti-IL-4
treatment at immunization modulates cytokine expression, reduces
illness, and increases cytotoxic T lymphocyte activity in mice
challenged with respiratory syncytial virus. J Clin Invest 1994;
94:1953-1958.
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Tang YW, Graham BS. Interleukin
12 treatment during immunization elicits a Th1-like immune response
in mice challenged with respiratory syncytial virus and improves
vaccine immunogenicity. J Infect Dis 1995; 172:734-738.
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Lindgren C, Lin J, Graham BS,
Gray ME, Parker RA, Sundell HW. Respiratory syncytial virus
infection enhances the response to laryngeal chemostimulation
and inhibits arousal from sleep in young lambs. Acta Pediatrica
1996;85:789-797.
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Tang YW, Graham BS. T cell source
of type 1 cytokines determines illness pattern in respiratory
syncytial virus-infected mice. J Clin Invest 1997; 99:2183-2191.
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Fischer JE, Johnson JE, Kuli-Zade R, Johnson
TR, Aung S, Parker RA, Graham BS. Overexpression
of IL-4 delays virus clearance in mice infected with RSV. J
Virol 1997;71:8672-8677.
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Johnson TR, Johnson JE, Roberts SR, Wertz GW,
Parker RA, Graham BS. Priming with secreted
glycoprotein G of respiratory syncytial virus (RSV) augments
interleukin-5 production and tissue eosinophilia after RSV challenge.
J Virol 1998;72:2871-2880.
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Peebles RS, Jr., Sheller JR, Johnson JE, Mitchell
DB, Graham BS. Respiratory syncytial virus
infection prolongs methacholine-induced airway hyperresponsiveness
in ovalbumin-sensitized mice. J Med Virol 1999; 57:186-192.
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Pastey MK, Crowe JE, Jr., Graham BS.
RhoA interacts with the fusion glycoprotein of respiratory syncytial
virus and facilitates virus-induced syncytium formation. J Virol
1999; 73:7262-7270.
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Johnson TR, Graham BS. Secreted
respiratory syncytial virus (RSV) G protein induces IL-5, IL-13,
and eosinophilia by an IL-4-independent mechanism. J Virol 1999;
73:8485-8495.
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Aung S, Tang YW, Graham BS.
IL-4 inhibits induction of cytotoxic T lymphocyte activity in
mice infected with recombinant vaccinia virus expressing respiratory
syncytial virus M2 protein. J Virol 1999; 73:8944-8949.
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Pastey MK, Gower TL, Spearman PW, Crowe JE, Jr.,
Graham BS. A RhoA-derived peptide inhibits
syncytium formation induced by respiratory syncytial virus and
parainfluenza virus type 3. Nature Med 2000; 6:35-40.
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Aung S, Graham BS. Differential
regulation of perforin- and FasL-mediated cytotoxicity by IL-4.
J Immunol 2000;164:3487-3493.
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Gower TL, Graham BS. Anti-viral
activity of lovastatin against respiratory syncytial virus in
vivo and in vitro. Antimicrob Agents Chemother 2001; 45:1231-1237.
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Gower TL, Peeples ME, Collins PL, Graham
BS. RhoA is activated during respiratory syncytial
virus infection. Virology 2001; 283:188-196. (Cover Photomicrograph)
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Aung S, Rutigliano JA, Graham BS.
Alternative mechanisms of respiratory syncytial virus clearance
in perforin knockout mice lead to enhanced disease. J Virol
2001; 75:9918-9924.
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Johnson TR, Hong S, Van Kaer L, Altman JD, Koezuka
Y, Graham BS. NK T cells contribute to expansion
of CD8(+) T cells and amplification of antiviral immune responses
to respiratory syncytial virus. J Virol 2002; 76:4294-4303.
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McCurdy LH, Graham BS. The role
of the plasma membrane lipid microdomain in respiratory syncytial
virus filament formation. J Virol 2003; 77:1747-1756.
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Johnson TR, Parker RA, Johnson JE, Graham
BS. IL-13 in sufficient for respiratory syncytial virus
(RSV) G glycoprotein-induced eosinophilia following RSV challenge.
J Immunol 2003; 170:2037-2045.
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Budge P, Graham BS. Antiviral
activity of RhoA-derived peptides against respiratory syncytial
virus is dependent on the formation of peptide dimers. Antimicrob
Agents Chemother 2003; 47:3470-3477.
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Rutigliano J, Hollinger T, Fischer JE, Johnson
TR, Aung S, Johnson JE, Graham BS. Anti-LFA-1
treatment prevents respiratory syncytial virus-induced illness
and delays virus clearance. J Virol 2004; 78:3014-3023.
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Budge PJ, Li Y, Beeler JA, Graham BS.
RhoA-derived peptide dimers share mechanistic properties with
other polyanionic inhibitors of respiratory syncytial virus,
including disruption of viral attachment and dependence on RSV
G. J Virol 2004; 78:5015-5022.
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Johnson TR, Teng MN, Collins PL, Graham
BS. Respiratory syncytial virus (RSV) G glycoprotein
is not necessary for vaccine-enhanced disease induced by immunization
with formalin-inactivated RSV (FI-RSV). J Virol 2004; 78:6024-6032.
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Johnson TR, Varga SM, Braciale TJ, Graham
BS. Vß14+ T cells do not mediate the vaccine-enhanced
disease induced by immunization with formalin-inactivated RSV
(FI-RSV). J Virol 2004; 178:8753-8760.
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Rutigliano JA, Graham BS. Prolonged
production of tumor necrosis factor alpha exacerbates illness
during respiratory syncytial virus infection. J Immunol 2004;
173:3408-3417.
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McCurdy LH, Rutigliano JA, Johnson TR, Chen M,
Graham BS. Modified vaccinia Ankara immunization
protects against lethal challenge with recombinant vaccinia
expressing murine IL-4. J Virol 2004; 78:12471-12479.
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Hashimoto K, Durbin J, Zhou W, Collins RD, Ho
SB, Sheller JR, Goleniewska K, O’Neal JF, Olson SJ, Mitchell
D, Graham BS, Peebles RS. Respiratory syncytial
virus infection in the absence of STAT1 results in airway dysfunction,
induction of airway mucus, and augmented IL-17 production. J
Allergy Clin Immunol 2005; (in press).
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Gower TL, Pastey MK, Peeples ME, Collins PL,
Hart TK, Guth A, Johnson TR, Graham BS. RhoA
signaling is required for RSV-induced syncytia formation and
filamentous virion morphology. J Virol 2005; 79:5326-5336.
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Rutigliano JA, Rock MT, Johnson AK, Crowe JE
Jr, Graham BS. Identification of an H-2Db-restricted
CD8+ cytotoxic T lymphocyte epitope in the matrix protein of
respiratory syncytial virus. Virology 2005; (in press).
Press bibliography
for a complete list of references and visit the Clinical
Trials Core (CTC) page to learn more about VRC clinical studies,
and press here
to see a list of current trials.
If you are interested in a Research Fellowship,
please send your CV to:
Barney S. Graham, M.D., Ph.D.
VRC/NIAID/NIH
Building 40, Room 2502
40 Convent Drive, MSC 3017
Bethesda, MD 20892-3017 |
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