M A J O R A R T I C L E
High-Dimensional Gene Expression Profiling
Studies in High and Low Responders to Primary
Iana H. Haralambieva,1,2Ann L. Oberg,3Neelam Dhiman,1,2,aInna G. Ovsyannikova,1,2Richard B. Kennedy,1,2
Diane E. Grill,3Robert M. Jacobson,1,2,4and Gregory A. Poland1,2,4
1Mayo Clinic Vaccine Research Group,2Program in Translational Immunovirology and Biodefense,3Department of Health Sciences Research, Division
of Biomedical Statistics and Informatics, and4Department of Pediatric and Adolescent Medicine, Mayo Clinic, Rochester, Minnesota
well understood, but are of considerable interest to a deeper understanding of poxvirus immunity and correlates
Methods.We assessed transcriptional messenger RNA expression changes in 197 recipients of primary small-
pox vaccination representing the extremes of humoral and cellular immune responses.
Results. The 20 most significant differentially expressed genes include a tumor necrosis factor–receptor super-
family member, an interferon (IFN) gene, a chemokine gene, zinc finger protein genes, nuclear factors, and histones
(P≤1.06E−20, q≤2.64E−17). A pathway analysis identified 4 enriched pathways with cytokine production by the
T-helper 17 subset of CD4+ T cells being the most significant pathway (P=3.42E−05). Two pathways (antiviral
actions of IFNs, P=8.95E−05; and IFN-α/β signaling pathway, P=2.92E−04), integral to innate immunity, were
enriched when comparing high with low antibody responders (false discovery rate,<0.05). Genes related to immune
function and transcription (TLR8, P=.0002; DAPP1, P=.0003; LAMP3, P=9.96E−05; NR4A2, P≤.0002; EGR3,
P=4.52E−05), and other genes with a possible impact on immunity (LNPEP, P=3.72E−05; CAPRIN1, P=.0001;
XRN1, P=.0001), were found to be expressed differentially in high versus low antibody responders.
Conclusion.We identified novel and known immunity-related genes and pathways that may account for differ-
ences in immune response to smallpox vaccination.
The mechanisms underlying smallpox vaccine-induced variations in immune responses are not
Despite the availability of effective smallpox vaccines
and the eradication of smallpox in 1980, orthopoxvi-
ruses still remain legitimate public health threats,
given the ongoing concerns of emerging orthopoxvi-
rus diseases and the possible use of poxviruses as bio-
weapons [1–3]. There is a continued need for poxvirus
research in virus biology, pathogenesis, and host re-
sponse, including vaccine-induced immunity and cor-
relates of protection [1, 3–8].
The duration and magnitude of protective immuni-
ty following smallpox immunization is still a topic of
debate ranging from either lifelong protective immuni-
ty to only several years of persistence . Even in pop-
ulations with a documented vaccine vesicular “take,”
or development of a pustule at the vaccination site, a
small proportion (up to 2%) fail to mount a strong
neutralizing antibody response . The mechanisms
underlying protection and the observed wide range of
humoral and cellular immune host responses follow-
ing smallpox vaccination and/or infection are still not
understood, although they are of considerable interest
to a deeper and more comprehensive understanding of
poxvirus immunity and correlates of protection, as
well as for the development of safer and more effective
vaccines and antiviral therapies.
In this study, we assessed changes in messenger
RNA (mRNA) expression using high-throughput mi-
croarray transcriptional profiling of 197 recipients of
Received 30 March 2012; accepted 18 June 2012; electronically published 4
aPresent affiliation: Summa Health System, Akron, Ohio.
Correspondence: Gregory A. Poland, MD, Mayo Clinic Vaccine Research Group,
Mayo Clinic, Guggenheim 611C, 200 First St SW, Rochester, MN 55905 (poland.
The Journal of InfectiousDiseases2012;206:1512–20
© The Author 2012. Published by Oxford University Press on behalf of the Infectious
Diseases Society of America. All rights reserved. For Permissions, please e-mail:
1512 • JID 2012:206 (15 November) • Haralambieva et al
by guest on February 2, 2016
primary smallpox Dryvax vaccination with humoral and cellu-
lar immune responses at the extremes of the biological spec-
trum (out of 1076 healthy vaccinated subjects). We identified
specific transcriptome signatures and pathways that might
account for differences in immune response to smallpox
MATERIALS AND METHODS
For gene expression profiling, we selected 200 subjects from
the extremes (high and low) of the humoral (neutralizing anti-
body) and cellular (interferon γ [IFN-γ] Elispot) responses to
primary smallpox vaccination out of 1076 healthy, eligible in-
dividuals (ages 18–40 years) whose demographic and immune
variables were previously described . As previously report-
ed, these subjects were recruited as participants in a smallpox
immunization program at the Naval Health Research Center
(NHRC) in San Diego, CA, and the Department of Health
and Human Services civilian healthcare worker smallpox im-
munization program at Mayo Clinic (Rochester, MN) [10, 11].
All study subjects were immunized with a single dose of
Dryvax smallpox vaccine (Wyeth Laboratories, Marietta, PA)
within 4 years prior to recruitment and had a documented
vaccine vesicular “take.” Specifically, 4 sex-specific quadrants
were defined on the basis of sex-specific medians to define
high (above the median) and low (below the median) respons-
es as demonstrated in Figure 1. For each of the humoral and
cellular responses, the squared difference from the median
was calculated per subject and then scaled to range from 0 to
1 for all 1076 subjects. For each subject, the product of the
humoral and cellular scaled, squared deviations was then com-
puted. Twenty-five males and 25 females having the largest of
these values in each quadrant were chosen for study. The in-
stitutional review boards of the Mayo Clinic and NHRC
granted approval to the study, and written informed consent
was obtained from each study participant.
Viral Stocks and Immune Assays
For Elispot and cytokine secretion assays, we used vaccinia
virus grown from Dryvax vaccine (a multiclonal vaccine con-
taining a mixture of closely related vaccinia virus strains), and
for measuring vaccinia-specific neutralizing antibody titers, we
used the recombinant vaccinia virus vSC56, expressing
β-galactosidase (a gift from B. Moss, National Institute of
Allergy and Infectious Diseases). We propagated both viruses
in HeLa S3 cells (ATCC) and purified, titered, and inactivated
(when needed) the stocks as described previously .
Vaccinia-Specific Neutralizing Antibody Assay
We quantified neutralizing antibodies to vaccinia virus using a
neutralization assay as previously described [10, 12]. Assay
results were defined as the serum dilution that inhibits 50% of
virus activity (ID50), as previously described . The mean
coefficient of variation (CV), based on 3 measurements, was
6.9% in our laboratory [10, 12].
IFN-γ Elispot Assay and Cytokine Measurements
We measured the frequencies of IFN-γ–producing cells in
peripheral blood mononuclear cell (PBMC) cultures using
total and CD8+ human IFN-γ Elispot kits (R & D Systems,
Minneapolis, MN) as previously described and following the
manufacturer’s protocol . The intraclass correlation coeffi-
cients comparing multiple observations per subject were 0.94
for stimulated values and 0.85 for unstimulated values, indi-
cating good measurement reproducibility. We quantified
based on their vaccinia virus–specific humoral and cellular immune re-
sponses. Subjects selected for microarray studies from the overall cohort
of 1076 subjects were dichotomized based on the magnitude of their
cellular (differences in interferon γ [IFN-γ] Elispot counts in stimulated vs
unstimulated cells; vertical axis) and humoral (log neutralizing antibody
titer that inhibits 50% of virus activity [ID50; horizontal axis) responses.
Fifty subjects (25 male and 25 female) were selected from each of 4
quadrants. The quadrants are defined as HH (high humoral and cellular
responses), HL (high humoral and low cellular response), and LH (low
humoral and high cellular response), and LL (low responses for both) ac-
cording to assay medians. Vertical and horizontal dashed lines represent
the median for males, overall, and for females, respectively, from left-to-
right for vertical lines and from top-to-bottom for horizontal lines.
Subjects with the extremes of the humoral and cellular response distri-
butions were chosen for microarray analysis as described in the Study
Subjects subsection of Materials and Methods. The dark colored filled
symbols represent patients used in the microarray study (vs open
symbols), while the circles and triangles represent males and females,
Scatterplot of subjects selected for microarray studies,
Gene Expression Profiling • JID 2012:206 (15 November) • 1513
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