M A J O R A R T I C L E
A West Nile Virus DNA Vaccine Utilizing
a Modified Promoter Induces Neutralizing
Antibody in Younger and Older Healthy Adults
in a Phase I Clinical Trial
Julie E. Ledgerwood,1Theodore C. Pierson,2Sarah A. Hubka,1Niraj Desai,1Steve Rucker,1Ingelise J. Gordon,1Mary
E. Enama,1Steevenson Nelson,2Martha Nason,3Wenjuan Gu,4Nikkida Bundrant,1Richard A. Koup,1Robert T. Bailer,1
John R. Mascola,1Gary J. Nabel,1Barney S. Graham,1and the VRC 303 Study Team*,1
1Vaccine Research Center,2Viral Pathogenesis Section, Laboratory of Viral Diseases, and3Biostatistics Research Branch, Division of Clinical Research,
National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, and4Biostatistics Research Branch, SAIC-Frederick, Inc,
National Cancer Institute at Frederick, Maryland
licensed vaccines to prevent WNV in humans. The safety and immunogenicity of a first-generation WNV DNA
vaccine was demonstrated in a clinical trial and a similar DNA vaccine has been licensed for use in horses.
Methods. A DNA vaccine encoding the protein premembrane and the E glycoproteins of the NY99 strain of
WNV under the transcriptional control of the CMV/R promoter was evaluated in an open-label study in 30 healthy
adults. Half of the subjects were age 18–50 years and half were age 51–65 years. Immune responses were assessed by
enzyme-linked immunosorbent assay, neutralization assays, intracellular cytokine staining, and ELISpot.
Results. The 3-dose vaccine regimen was safe and well tolerated. Vaccine-induced T cell and neutralizing
antibody responses were detected in the majority of subjects. The antibody responses seen in the older age group
were of similar frequency, magnitude, and duration as those seen in the younger cohort.
Conclusions. Neutralizing antibody responses to WNV were elicited by DNA vaccination in humans, including
in older individuals, where responses to traditional vaccine approaches are often diminished. This DNA vaccine
elicited T cell responses of greater magnitude when compared with an earlier-generation construct utilizing a CMV
Clinical Trials Registration. NCT00300417.
West Nile virus (WNV) is a flavivirus that causes meningitis and encephalitis. There are no
West Nile virus (WNV) is a flavivirus transmitted pri-
marily by mosquitoes to a variety of vertebrate hosts.
Flaviviruses are positive-stranded RNA viruses and in-
clude important human pathogens such as yellow fever
virus, St Louis encephalitis virus, dengue virus, and
Japanese encephalitis virus (JEV). WNV was initially
isolated from a human residing in the West Nile district
of Uganda in 1937 . The virus is present throughout
Africa, Asia, the Middle East, and the Americas. WNV
was first recognized in the United States in 1999 when it
caused an epidemic in New York state. Since 1999,
WNV has spread throughout the Americas [2–4]. The
incidence in the United States peaked at 9862 cases in
2003. The infection is now considered endemic in the
United States and in 2009 there were 720 reported cases
The mature WNV virioniscomposedof180 copiesof
the envelope protein (E) arranged with pseudo T 5 3
icosahedral symmetry. The nucleocapsid core contains
copies of RNA encoding for genome and capsid
Received 3 November 2010; accepted 22 December 2010.
Potential conflicts of interest: none reported.
*The VRC 303 Study Team includes Brenda Larkin, LaSonji Holman, Laura Novik,
Cynthia Starr Hendel, LaChonne Stanford, Tiffany Alley, Sandra Sitar, Yesenia
Merino, Joseph Casazza, Trishna Goswami, Phillip Gomez, Charla Andrews, and
Correspondence: Julie E. Ledgerwood, DO, Deputy Chief, Clinical Trials Core,
VRC, NIAID, 9000 Rockville Pike, CRC Bldg 10, Room 5-2440, Bethesda, MD 20892
The Journal of Infectious Diseases
Published by Oxford University Press on behalf of the Infectious Diseases Society of
0022-1899 (print)/1537-6613 (online)/2011/20310-0001$14.00
d JID 2011:203 (15 May)
d Ledgerwood et al
proteins, and the general arrangement of WNV is similar to that
of dengue virus . The major surface protein (E) mediates
interactions with the cell surface and facilitates fusion between
the virus and cell membranes. Virions also incorporate the
protein premembrane (prM), which is cleaved into a smaller
virion-associated membrane (M) peptide during virion matu-
ration. Surface envelope proteins are the primary target for the
humoral response against flavivirus infection.
WNV is an enzootic infection and is maintained in a mos-
quito–bird transmission cycle; incidental hosts have been iden-
tified, including humans, horses, and alligators [3, 8]. The
principal form of transmission to humans is from the bite of
including blood transfusion, organ transplantation, breastfeed-
ing, and transplacental or laboratory acquisition [2, 9]. Human
illness peaks in late summer or early autumn, reflecting peak
viral amplification within the bird–mosquito–bird cycle .
WNV infection of humans has been associated with a varie-
ty of symptoms from asymptomatic to severe encephalitis.
Central nervous system involvement occurs in 1 in 150 pa-
tients [10, 11]. Care is supportive but intravenous immuno-
globulin, alpha interferon, and ribavirin have been investigated
for severe cases [12, 13]. One investigational therapy with
potential for benefit is a humanized monoclonal antibody,
Hu-E16, which binds to the envelope protein of WNV and has
shown efficacy in preclinical testing and safety in clinical
As vaccines are developed, consideration for those at greatest
risk is a priority. For WNV, advanced age is a risk factor for
severe disease ; however, the mechanism for increased sus-
ceptibility in the elderly and immunocompromised remains
unknown. Published data suggest a role for antibody in pro-
tection and clearance of flavivirus infections [18, 19]. In vitro
data also implicate dysregulation of toll-like receptor 3 (TLR3)
in macrophages in the elderly, leading to higher cytokine (in-
terleukin [IL]-6, interferon [IFN]-b, tumor necrosis factor
[TNF]-a) levels, which are associated with higher viral burdens
in macrophages and facilitation of WNV entry into the cere-
brospinal fluid secondary to blood-brain barrier disruption. In
contrast, in young adults, TLR3 expression declines during
WNV infection, diminishing WNV entry and cytokine release
. In general, vaccines induce decreased immunity in the
elderly [21–23]. Taken together, these data describe im-
munosenescence, an age-related change in immunity, which
may impact the predilection of the aged to become seriously
affected by WNV and is a possible reason for the generalized
decreased vaccine efficacy seen in older adults [21, 23].
WNV infection is a veterinary health concern, and infection
in horses carries a 30%–40% mortality rate [24, 25]. Equine
vaccine development provides an animal model for the de-
velopment of a human WNV vaccine. The equine DNA vaccine,
pCBWN (Fort Dodge Animal Healthwith the Centers for
Disease Control and Prevention), encodes for the prM and
E proteins from WNV in a similar configuration as the DNA
vaccine described here. It elicits neutralizing antibody and
protects mice and horses from WNV . That vaccine was
and represents the first license issued for a veterinary DNA
Investigational WNV vaccines for humans have been evalu-
ated in preclinical and clinical studies, and candidate plat-
forms include gene-based vaccines and viral-like particles .
A candidate DNA vaccine for WNV has previously been eval-
uated in a phase I clinical trial (VRC 302) and was shown to be
safe and immunogenic. That study provided evidence that
a DNA vaccine, based on the equine vaccine, elicited neutral-
izing antibody in humans .
In the current study (VRC 303), a nearly identical recombi-
nant DNA vaccine encoding WNV prM and E proteins was
used. This newer-generation DNA plasmid construct differs
from the previously tested vaccine construct in that a modified
promoter, CMV/R, was utilized rather than the original CMV
promoter. The CMV/R promoter includes the regulatory R re-
gion from the 5# long terminal repeat of human T cell leukemia
virus type (HTLV-1), which serves as a transcriptional and
posttranscriptional enhancer. The CMV/R promoter has im-
proved protein expression of transduced genes, which has been
associated with greater immunogenicity following DNA im-
munization of animals . The CMV/R promoter has been
utilized in vaccines in other phase I and II clinical trials [30–32],
and although a direct comparison of these promoters in DNA
vaccines encoding identical antigens has not been conducted in
a randomized clinical trial, the CMV/R promoter has been
shown to enhance the immunogenicity of DNA vaccines in both
mice and nonhuman primates . The results of the clinical
trial reported here allow for a direct comparison of the safety
and immunogenicity of a DNA vaccine in 2 age groups (VRC
303) as well as an indirect comparison of this newer-generation
WNV vaccine encoding the CMV/R promoter (VRC 303) to the
previously published clinical study (VRC 302)  results as-
sessing an earlier-generation WNV DNA vaccine utilizing the
MATERIALS AND METHODS
Protocol VRC 303 was a single-site, phase I, open-label study to
examine the safety, tolerability, and immune response to an
investigational recombinant DNA WNV vaccine. Healthy adult
subjects in 2 age groups(18–50 years and 51–65 years)who were
negative for WNV immunoglobulin G (IgG) by a commercial
assay (Focus Technologies) at baseline were enrolled at the
Vaccine Research Center (VRC), National Institute of Allergy
and Infectious Diseases (NIAID), National Institutes of Health
WNV DNA Vaccine is Immunogenic in Older Adults
d JID 2011:203 (15 May)
21. Targonski PV, Jacobson RM, Poland GA. Immunosenescence: role and
measurement in influenza vaccine response among the elderly. Vaccine
22. Andersson CR, Vene S, Insulander M, Lindquist L, Lundkvist A,
Gunther G. Vaccine failures after active immunisation against tick-
borne encephalitis. Vaccine 2010; 28:2827–31.
23. Kumar R, Burns E. Age-related decline in immunity: implications for
vaccine responsiveness. Expert Rev Vaccines 2008; 7:467–79.
24. United States Department of Agriculture. USDA issues license for West
Nile Virus DNA vaccine for horses. http://www.aphis.usda.gov/lpa/
news/2005/07/wnvdna_vs.html. Accessed 1 October 2010.
25. Salazar P, Traub-Dargatz JL, Morley PS, et al. Outcome of equids with
clinical signs of West Nile virus infection and factors associated with
death. J Am Vet Med Assoc 2004; 225:267–74.
26. Davis BS, Chang GJ, Cropp B, et al. West Nile virus recombinant DNA
vaccine protects mouse and horse from virus challenge and expresses in
vitro a noninfectious recombinant antigen that can be used in enzyme-
linked immunosorbent assays. J Virol 2001; 75:4040–7.
27. Spohn G, Jennings GT, Martina BE, et al. A VLP-based vaccine tar-
geting domain III of the West Nile virus E protein protects from lethal
infection in mice. Virol J 2010; 7:146.
28. Martin JE, Pierson TC, Hubka S, et al. A West Nile virus DNA vaccine
inducesneutralizing antibodyinhealthyadultsduringa phase1 clinical
trial. J Infect Dis 2007; 196:1732–40.
29. Barouch DH, Yang ZY, Kong WP, et al. A human T-cell leukemia virus
type 1 regulatory element enhances the immunogenicity of human
immunodeficiency virus type 1 DNA vaccines in mice and nonhuman
primates. J Virol 2005; 79:8828–34.
30. Catanzaro AT, Roederer M, Koup RA, et al. Phase I clinical evaluation
of a six-plasmid multiclade HIV-1 DNA candidate vaccine. Vaccine
31. Martin JE, Sullivan NJ, Enama ME, et al. A DNA vaccine for Ebola
virus is safe and immunogenic in a phase I clinical trial. Clin Vaccine
Immunol 2006; 13:1267–77.
32. Kibuuka H, Kimutai R, Maboko L, et al. A phase 1/2 study of a mul-
ticlade HIV-1 DNA plasmid prime and recombinant adenovirus
serotype 5 boost vaccine in HIV-uninfected East Africans (RV 172).
J Infect Dis 2010; 201:600–7.
33. Pierson TC, Sanchez MD, Puffer BA, et al. A rapid and quantitative
assay for measuring antibody-mediated neutralization of West Nile
virus infection. Virology 2006; 346:53–65.
34. Davis CW, Nguyen HY, Hanna SL, Sanchez MD, Doms RW,
Pierson TC. West Nile virus discriminates between DC-SIGN and
DC-SIGNR for cellular attachment and infection. J Virol 2006;
35. Graham BS, Koup RA, Roederer M, et al. Phase 1 safety and immu-
nogenicity evaluation of a multiclade HIV-1 DNA candidate vaccine.
J Infect Dis 2006; 194:1650–60.
36. Catanzaro AT, Koup RA, Roederer M, et al. Phase 1 safety and im-
munogenicity evaluation of a multiclade HIV-1 candidate vaccine
delivered by a replication-defective recombinant adenovirus vector.
J Infect Dis 2006; 194:1638–49.
37. Martin JE, Louder MK, Holman LA, et al. A SARS DNA vaccine in-
duces neutralizing antibody and cellular immune responses in healthy
adults in a phase I clinical trial. Vaccine 2008; 26:6338–43.
38. Martin JE, Pierson TC, Graham BS. Reply to Rottinghaus and Poland.
J Infect Dis 2008; 197:1628–9.
39. McElhaney JE. The unmet need in the elderly: designing new influenza
vaccines for older adults. Vaccine 2005; 23(Suppl 1):S10–25.
40. Giudice EL, Campbell JD. Needle-free vaccine delivery. Adv Drug Deliv
Rev 2006; 58:68–89.
41. Holland D, Booy R, De Looze F, et al. Intradermal influenza
vaccine administered using a new microinjection system produces
superior immunogenicity in elderly adults: a randomized controlled
trial. J Infect Dis 2008; 198:650–8.
d JID 2011:203 (15 May)
d Ledgerwood et al