M A J O R A R T I C L E
CD4+T-Cell Expansion Predicts Neutralizing
Antibody Responses to Monovalent, Inactivated
2009 Pandemic Influenza A(H1N1) Virus
Subtype H1N1 Vaccine
Jennifer L. Nayak,1Theresa F. Fitzgerald,2Katherine A. Richards,3Hongmei Yang,4John J. Treanor,2and
Andrea J. Sant3
1Department of Pediatrics,2Department of Medicine, Division of Infectious Diseases,3David H. Smith Center for Vaccine Biology and Immunology,
Department of Microbiology and Immunology, and4Department of Biostatistics and Computational Biology, University of Rochester Medical Center,
CD4+T cells and vaccine-induced neutralizing antibody responses has been controversial. The emergence of
swine-origin 2009 pandemic influenza A virus subtype H1N1 (A[H1N1]pdm09) provided a unique opportunity
to examine responses to an influenza vaccine composed of both novel and previously encountered antigens and to
probe the relationship between B-cell and T-cell responses to vaccination.
Methods. We tracked CD4+T-cell and antibody responses of human subjects vaccinated with monovalent
subunit A(H1N1)pdm09 vaccine. The specificity and magnitude of the CD4+T-cell response was evaluated using
cytokine enzyme-linked immunosorbent spot assays in conjugation with peptide pools representing distinct influ-
enza virus proteins.
Results. Our studies revealed that vaccination induced readily detectable CD4+T cells specific for conserved
portions of hemagglutinin (HA) and the internal viral proteins. Interestingly, expansion of HA-specific CD4+T
cells was most tightly correlated with the antibody response.
Conclusions.These results indicate that CD4+T-cell expansion may be a limiting factor in development of
neutralizing antibody responses to pandemic influenza vaccines and suggest that approaches to facilitate CD4+T-
cell recruitment may increase the neutralizing antibody produced in response to vaccines against novel influenza
The ability of influenza vaccines to elicit CD4+T cells and the relationship between induction of
response; immunodominance; epitopes.
influenza vaccines; pandemic H1N1 influenza; influenza virus; CD4+T cells; cellular immune
Influenza A viruses can evade protective immune re-
sponses through both gradual antigenic drift of viral
surface proteins and sporadic reassortment that can
result in antigenic shift. Such reassortment occurred
in 2009, when the novel, swine-origin pandemic influ-
emerged and spread globally, resulting in the first in-
fluenza pandemic of the 21st century [1–4]. Character-
ization of immunity to this virus revealed little
antigenic seroreactivity with contemporary seasonal
influenza A virus subtype H1N1 (A[H1N1]) , rela-
tively few conserved B-cell epitopes within the hemag-
glutinin (HA) protein , and little or no preexisting
neutralizing antibody in unexposed children or adults
aged <60 years [7–9]. In contrast, preexisting memory
CD4+T cells, including cells directed against epitopes
within the HA protein, were detected in peripheral
blood mononuclear cells (PBMCs) of subjects not pre-
viously exposed to A(H1N1)pdm09 [6, 10–13]. Thus,
Received 29 June 2012; accepted 23 August 2012; electronically published 12
Presented in part: 6th Annual NIAID Centers for Excellence in Influenza
Research and Surveillance Meeting, New York, New York, July 2012.
Correspondence: Jennifer L. Nayak, MD, Department of Pediatrics, Division of
Infectious Diseases, University of Rochester Medical Center, 601 Elmwood Ave,
Box 690 Rochester, NY 14642 (firstname.lastname@example.org).
The Journal of Infectious Diseases2013;207:297–305
© 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:
CD4+T Cells and Ab Response to A(H1N1)pdm09 Vaccine • JID 2013:207 (15 January) • 297
by guest on February 2, 2016
this pandemic offered a unique opportunity to study CD4+T-
cell responses with an influenza virus strain that would simul-
taneously induce naive and memory CD4+T cells without
large amounts of coexisting B-cell–mediated immunity.
Individuals are repeatedly exposed to influenza virus anti-
gens through both vaccination and infection, resulting in com-
petition between memory and naive lymphocytes over time.
How this competition will affect the specificity of CD4+T-cell
responses is unknown. While the typical seasonal trivalent in-
activated vaccine (TIV) is enriched for HA, it also contains
the more conserved internal viral proteins, such as nucleopro-
tein (NP) and matrix protein (M1) [14, 15]. Thus, both repeat-
ed infections and vaccinations may lead to a dominance of T
cells specific for peptide epitopes conserved among virus
strains. While recent studies examining the T-cell repertoire in
humans have shown broad reactivity to diverse viral proteins
[10, 16, 17], few studies have examined the distribution of re-
activity among conserved and novel epitopes.
Neutralizing antibody is considered the major correlate of
protection following influenza vaccination [18, 19], while the
protective role of CD4+T cells remains more poorly under-
stood. Whether CD4+T-cell responses even develop after vac-
cination with TIV remains controversial [12, 20–28]. Lack of
consensus may be due to inadequate subject numbers, variable
levels of baseline anti-influenza immunity in study popula-
tions, or the use of recall antigens that do not elicit the full
repertoire of influenza-reactive cells. Knowledge of the rela-
tionship between CD4+T cells and the development of a neu-
tralizing antibody response following administration of TIV is
even more limited, with the few studies addressing this question
failing to find a correlation between these parameters [12, 20],
except when an adjuvanted influenza A virus subtype H5N1
vaccine was used .
In this study, we used an experimental approach designed
to maximally detect antigen-specific CD4+T cells by using cy-
tokine enzyme-linked immunosorbent spot (ELISPOT) assays
with pools of overlapping synthetic peptides as recall antigens
to quantify responses to conserved and novel epitopes follow-
ing vaccination of adults with monovalent inactivated A
(H1N1)pdm09 vaccine. We also examined the relationship
between CD4+T-cell responses and neutralizing antibody
titers. These analyses revealed a readily detectable increase in
numbers of CD4+T cells directed against conserved portions
of HA and the NP and M1 proteins. Further, CD4+T-cell ex-
pansion was correlated with the development of a neutralizing
antibody response against this pandemic virus.
MATERIALS AND METHODS
Study Population and Procedures
Forty-nine healthy subjects were enrolled in 2 age groups
(18–32 years and ≥60 years) between March and October
2010. Subjects with a history of previous laboratory-
documented infection or vaccination with A(H1N1)pdm09,
vaccination with A/New Jersey/76, egg allergy, immunosup-
pression, or active neoplastic disease were excluded. Younger
subjects were excluded if they had a baseline A/California/07/
09 hemagglutinin inhibition (HAI) titer of >10, but this was
not an exclusion criterion for older adults because of greater
levels of expected preexisting immunity. Blood was obtained
before and at days 7, 14, and 28 after administration of inacti-
vated subunit A/California/07/09 monovalent vaccine (Novar-
tis, East Hanover, NJ). PBMCs were purified using Accuspin
tubes with Histopaque-1077 cell separation media (Sigma-
Aldrich, St. Louis, MO) and were frozen at a controlled rate in
fetal bovine serum (FBS) containing 10% DMSO.
All sera were tested using the microtiter technique for neutral-
ization of an egg-grown virus derived from the 2009 monova-
MedImmune, Gaithersburg, MD). Sera were heat inactivated
prior to testing, starting at a 1:10 dilution. Viral growth was
determined by enzyme-linked immunosorbent assay following
fixation with acetone, using NP-specific monoclonal anti-
bodies (WHO reagent kit). The antibody titer was defined as
the reciprocal of the highest dilution that resulted in 50% inhi-
bition of signal as compared to control wells. An end point
titer was determined on all sera with an initial neutralizing
antibody titer of ≥1280. Sera without detectable neutralization
activity were assigned a titer of 5, and values >40 000 were
assigned a titer of 40000 for calculation purposes.
HAI testing was performed on all sera in microtiter format,
using turkey red blood cells with 4 HA units of egg-grown
virus derived from the 2009 monovalent A(H1N1)pdm09 live
attenuated influenza vaccine (lot 500914P; MedImmune,
Gaithersburg, MD) as antigen. Sera were treated with receptor-
destroying enzyme (Denka Seiken, Tokyo, Japan) and heat inac-
tivated prior to testing, starting at a 1:10 dilution. The antibody
titer was defined as the reciprocal of the highest dilution that
resulted in inhibition of hemagglutination.
Quantification of CD4+T-Cell Responses
Cryopreserved PBMCs were thawed and then rested overnight
at 37°C and 5% CO2in Roswell Park Memorial Institute 1640
medium containing 10% FBS and gentamicin (Life Technolo-
gies, Carlsbad, CA), with a typical yield of >85% viable cells
after thawing. After rest, PBMCs were depleted of CD8+and
CD56+cells, using MACS positive selection with LD separa-
tion columns as per manufacturer’s instructions (Miltenyi
Biotec, Auburn, CA). ELISPOT assays were performed as pre-
viously described , with 400 000 or 200 000 CD8- and
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