Elicitation of Anti-1918 Influenza Virus Immunity Early in Life
Prevents Morbidity and Lower Levels of Lung Infection by 2009
Pandemic H1N1 Influenza Virus in Aged Mice
Brendan M. Giles,a,bStephanie J. Bissel,cJodi K. Craigo,a,dDilhari R. DeAlmeida,aClayton A. Wiley,cTerrence M. Tumpey,e
and Ted M. Rossa,b,d
Center for Vaccine Research,aGraduate Program in Immunology,bDivision of Neuropathology, Department of Pathology,cDepartment of Microbiology and Molecular
Genetics,dUniversity of Pittsburgh, Pittsburgh, Pennsylvania, USA, and Centers for Disease Control and Prevention, Influenza Division, Atlanta, Georgia, USAe
In 2009, a novel strain of H1N1 influenza virus emerged from
swine and quickly spread among humans, resulting in the World
Health Organization declaring the first pandemic of the 21st cen-
tury (13). The 1918 Spanish influenza virus pandemic was the
mortality (675,000 total deaths) in the United States (64) and
killed up to 50 million people worldwide (33). In comparison to
with the majority of cases being uncomplicated (4). The most
common feature of fatal disease was various degrees of alveolar
infection and damage (25, 42, 58). This differed from seasonal
influenza virus, as fatal cases rarely involve alveolar cells, with
virus located primarily in the major airways, such as the trachea
and bronchioles (27, 37). Interestingly, the majority of severe
cases from the 2009 H1N1 pandemic were reported in children
and young adults, while the elderly population was relatively pro-
tected from infection and severe disease (4). This pattern of sus-
observed during seasonal influenza virus epidemics but is similar
to what was reported for the 1918 pandemic (1). Although the
1918 pandemic is believed to have emerged from avian species
into both swine and humans nearly simultaneously, the human
and swine lineages quickly diverged. Sequence analysis indicated
that the 2009 H1N1 pandemic virus is related to the 1918 H1N1
virus, and it has been proposed that the swine population has
maintained an “antigenically frozen” H1N1 lineage (39). Struc-
tions and estimated annual averages of approximately 36,000
of 1918 and 2009 pandemic hemagglutinin (HA) proteins that is
not present in contemporary seasonal H1N1 viruses (73, 77).
Antigenic similarities and serologic evidence of cross-reactive
antibody in older adults have led to the hypothesis that exposure
to 1918-like viruses confer cross-protective immune responses
(29, 32). Several studies have identified cross-reactive antibodies
(15, 78), with monoclonal antibodies derived from survivors of
the 1918 pandemic cross-neutralizing the 2009 pandemic viruses
(36). Humans repeatedly experience different influenza virus an-
multiple drifted antigens likely broadens the circulating antibody
repertoire, the unique history for any one individual or age group
ally, direct evidence of the cross-protective efficacy elicited by ex-
animal models (39, 59), but the duration of this cross-protective
immune response(s) has not been evaluated. We therefore hy-
pothesized that cross-protection elicited by a single antigen early
Aging is associated with the decreasing ability of the immune
Received 16 August 2011 Accepted 12 November 2011
Published ahead of print 30 November 2011
Address correspondence to Ted M. Ross, firstname.lastname@example.org.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
jvi.asm.org 0022-538X/12/$12.00Journal of Virologyp. 1500–1513
system to respond to new antigens (7). Although elderly individ-
uals are impaired in their immune responses, immunological
memory responses to antigens experienced prior to the onset of
decreased immune function can be retained and offer protection
against reexposure to similar pathogens. Indeed, successful vac-
cines are usually antibody based, and memory B cell responses, at
of long-lived cells: memory B cells (MBC) and long-lived plasma
cells (LLPC) (74). Both subsets generally develop in the germinal
center and go through differential degrees of affinity maturation:
MBC differentiate earlier and have fewer somatic mutations,
while LLPC emerge later with higher affinity (19, 60, 74). Both
and MBC respond to a second infection that escapes circulating
derived from the LLPC arm of the B cell memory response. Inter-
estingly, infection with the 2009 pandemic virus in humans led to
development of broadly neutralizing antibody-producing cells,
and it has been suggested that these cells were derived from pre-
the reason for the cross-protection have been largely antibody
cellular component (39, 59).
cines against a variety of viral pathogens (8, 9, 24, 43, 44, 75).
These vaccines are composed of noninfectious, nonreplicating
VLPs that present functional HA and NA on the surface of a viral
particle. VLPs efficiently elicit high-titer immune responses that
protect small animals against lethal viral challenge (8, 24). In this
study, HA and NA from the 1918 influenza virus, A/South Caro-
lina/1/1918, were expressed on the surface of a VLP that was ex-
at a young age (8 to 12 weeks) and allowed to age to elderly status
(20 months). Antibody titer to the 1918 antigens persisted for the
lifetime of the animals, indicating that the nonreplicating VLP
vaccine elicits enduring antibody titers. The 1918 VLPs efficiently
protected the aged mice from 2009 pandemic challenge and pre-
vented alveolar infection. Additionally, the protection involved
not only the previously described cross-reactive antibodies but
also a robust cellular recall response.
MATERIALS AND METHODS
Purification of viruslike particles. Human embryonic kidney (HEK)
Plasmids expressing the HIV-1NL4-3Gag gene products only, pGagp24,
were derived from codon-optimized sequences (phGag), as previously
described (31). pGagp24encodes for an immature, unprocessed HIV-1
(A/Brevig Mission/1/1918) genes were kindly provided by A. Garcia-
Sastre. Supernatants were collected and cell debris removed by low-speed
centrifugation followed by vacuum filtration through a 0.22-?m sterile
filter. VLPs were purified via ultracentrifugation (100,000 ? g through
20% [wt/vol] glycerol) for 4 h at 4°C. The pellets were subsequently re-
suspended in phosphate-buffered saline (PBS) and stored at ?80°C until
use. Protein concentration was determined by the Micro BCA protein
assay reagent kit (Pierce Biotechnology, Rockford, IL).
Animals and vaccinations. BALB/c mice (Mus musculis, females, 6 to
housed in microisolator units, allowed free access to food and water, and
cared for under USDA guidelines for laboratory animals. Mice were vac-
cular injection at weeks 0 and 3. Vaccination experiments were initially
prepared with and without 10 ?g CpG oligonucleotides (Sigma-Aldrich,
St. Louis, MO). Due to no observed effect of the adjuvant, subsequent
vaccinations investigating longevity and cross-reactivity were performed
blood was collected from anesthetized mice via the retro-orbital plexus
and transferred to a microcentrifuge tube. Tubes were centrifuged and
sera were removed and frozen at ?20°C. A subset of vaccinated mice was
allowed to age to a final age of 20 months (?17 months after final vacci-
nation), with blood collected at 10 months and 20 months of age. All
procedures were in accordance with the NRC Guide for the Care and Use
of Laboratory Animals, the Animal Welfare Act, and CDC/NIH Biosafety
in Microbiological and Biomedical Laboratories.
ELISA. The enzyme-linked immunosorbent assay (ELISA) was used
to assess total antibody titer and IgG isotype titer to the 1918 HA. High-
binding, 96-well polystyrene plates (Costar, Lowell, MA) were coated
overnight with 50 ng/well of recombinant 1918 HA. Plates were blocked
with 5% milk diluted in PBS with 0.05% Tween 20. Serum samples were
diluted in blocking buffer and added to plates. Serum was 2-fold serially
diluted and allowed to incubate for 1 h at room temperature. Plates were
IgG3 and linked to horseradish peroxidase (HRP) (Southern Biotech,
Birmingham, AL) were diluted in blocking buffer and added to plates.
Plates were incubated for 1 h at room temperature. Plates were washed,
and HRP was developed with substrate (Sigma-Aldrich, St. Louis, MO).
Plates were incubated in the dark for 30 min, and then the reaction was
stopped with 2 N H2SO4. Optical densities at a wavelength of 450 nm
(OD450) were read by a spectrophotometer (BioTek, Winooski, VT), and
endpoint dilution titers were determined. Endpoint titers were deter-
the mean OD450plus two standard deviations of naïve animal serum.
Hemagglutination inhibition assay. The hemagglutination inhibi-
inhibit agglutination of turkey erythrocytes. The protocol was adapted
To inactivate nonspecific inhibitors, sera were treated with receptor-
destroying enzyme (RDE) prior to being tested (10–12, 46, 55). Briefly,
three parts RDE was added to one part serum and incubated overnight at
37°C. RDE was inactivated by incubation at 56°C for ?30 min. RDE-
treated serum was 2-fold serially diluted in V-bottom microtiter plates.
Equal volumes of either 1918 VLP or wild-type pandemic H1N1 virus,
adjusted to approximately 4 hemagglutinating units (HAU)/25 ?l, were
added to each well. 1918 VLPs were produced as described above. Wild-
type viruses were propagated in eggs and included the strains A/Califor-
erythrocytes (RBC) (Lampire Biologicals, Pipersville, PA) in PBS for a
final concentration of 0.5% RBC. Red blood cells were stored at 4°C and
used within 72 h of preparation. The plates were mixed by agitation and
covered, and the RBCs were allowed to settle for 30 min at room temper-
ature (2). The HAI titer was determined by the reciprocal dilution of the
last well which contained nonagglutinated RBC. Positive and negative
serum controls were included for each plate. All mice were negative
(HAI ? 10) for preexisting antibodies to currently circulating human
influenza viruses prior to vaccination.
Virus microneutralization. To assess the ability of mouse immune
ization assay was used as previously described (47). Briefly, sera were
Influenza Virus Abs Elicited in Young Mice Protect Aged Mice
February 2012 Volume 86 Number 3jvi.asm.org 1501
2-fold serially diluted and then incubated with 100 50% tissue culture
infective doses (TCID50) of 1918 virus for 60 min at room temperature.
The serum-virus mixture was then added to Madin-Darby canine kidney
(MDCK) cells and allowed to incubate for 2 days at 37°C. Specific neu-
tralizing activity was calculated as the lowest concentration of serum that
displayed neutralizing activity.
SPR. To assess the binding properties of serum antibodies, surface
(GE/Biacore AB, Uppsala, Sweden). Protein A (Pierce, Rockford, IL) was
immobilized to the surface of a CM5 sensor chip (GE/Biacore, Inc., Pis-
chip was activated using a 1:1 mixture of N-hydroxysuccinimide and
1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC)
(Biacore, Inc.). Protein A (75 ?g/ml) was immobilized on experimental
and reference (adjacent) flow cells at a high level of density (approxi-
inactivated with an injection of ethanolamine. Pooled polyclonal IgG
from vaccinated mice was diluted in HBS-EP buffer (GE Healthcare/Bia-
core, Inc., Piscataway, NJ) and captured at approximately 300 RU. After
capture of IgG, various concentrations (0.8 to 66 nM, series of 3-fold
ware (Biacore AB). Polyclonal serum appeared to yield monospecific
binding, and hence a 1:1 Langmuir fit was utilized for kinetic determina-
tions. However, since kinetic rates returned using these binding models
for polyclonal serum represent only apparent rates of binding due to the
multiple specificities inherent to a polyclonal response, we referred to the
results as “relative association” and “relative dissociation.” The percent-
age ([RL/RU captured serum] ? 100) of HA-specific antibody captured
Rmaxfrom the BIAevaluation parameters of the run.
Viral challenge. To determine the homologous efficacy of the 1918
vaccine, mice were challenged with the reconstructed 1918 virus (51, 69).
Briefly, 2 weeks after the final vaccination, adult animals were challenged
intranasally with 50 LD50of 1918 virus in a volume of 50 ?l. Mice were
monitored daily for disease signs and death for 16 days postinfection
(dpi). Body weights were recorded for individual mice at various days
postinoculation. All virus challenge experiments were performed under
the guidance of the U.S. National Select Agent Program in negative-
pressure HEPA-filtered biosafety level 3? (BSL3?) enhanced laborato-
ries with the use of a battery-powered Racal HEPA filter respirator and
To determine the cross-protective efficacy of the 1918 vaccine, mice
were infected with a 2009 pandemic H1N1 isolate: A/Mexico/4108/2009.
10 to 20% weight loss and development of clinical illness (data not
A/Mexico/4108/2009 virus in a volume of 50 ?l. Mice were monitored
daily for disease signs and death for 14 days postinfection. Body weights
and sickness scores were recorded for individual mice at various days
normal, 1 ? reduced, 2 ? severely reduced), hunched back (0 ? absent,
1 ? present), and ruffled fur (0 ? absent, 1 ? present) (66). Any animal
reaching ?20% weight loss was humanely euthanized. All experiments
using 2009 pandemic H1N1 virus were performed under biosafety level 2
Virus titrations. On day 4 after 1918 virus challenge, four mice per
for virus titration. Lungs were homogenized in 1 ml of sterile PBS, and
clarified homogenate virus titers were determined using a 50% egg infec-
method of Reed and Muench (54). The limit of virus detection was 101.5
For 2009 pandemic H1N1 virus infections, lung virus titers were de-
termined using a plaque assay (67, 68). Briefly, lungs from infected mice
were thawed and weighed, and single-cell suspensions were prepared via
Cell suspensions were centrifuged at 2,000 rpm for 5 min, and the super-
natants were collected. MDCK cells were plated (5 ? 105) in each well of
to 1 ? 106) and overlaid onto the cells in 100 ?l of Dulbecco’s modified
Eagle’s medium (DMEM) supplemented with penicillin-streptomycin
and incubated for 1 h. Virus-containing medium was removed and re-
erford, NJ) and incubated for 96 h at 37°C with 5% CO2. Agarose was
removed and discarded. Cells were fixed with 10% buffered formalin and
then stained with 1% crystal violet for 15 min. After being thoroughly
washed in distilled water (dH2O) to remove excess crystal violet, plates
were allowed to dry, plaques were counted, and PFU/g was calculated.
Histopathological analysis. Left lobes of lungs from infected mice
were collected 4 days postinfection and placed into 10% buffered forma-
prepared for histopathological analysis. Tissue sections were stained with
hematoxylin and eosin and examined for bronchial inflammation and
denudation and alveolar infiltration.
Immunohistochemistry was performed as described before (6). Sec-
tions containing lung were stained using antibodies against influenza A
virus (1:500; Maine Biotechnology Services, Portland, ME), Iba1 (1:500;
Wako Pure Chemical Industries, Osaka, Japan), CD3 (1:500; Dako,
species-appropriate secondary antibodies (Jackson ImmunoResearch
Laboratories, West Grove, PA, and Rockland Immunochemicals, Gil-
of influenza virus-infected cells and abundance of CD3?T cells, Iba1-
positive macrophages, myeloperoxidase-labeled neutrophils, and IgA ex-
For in situ hybridization (ISH), vectors containing 760 bp of
Influenza/California/04/2009 matrix protein and full-length murine
interferon-? (Open Biosystems, Huntsville, AL) were linearized to create
antisense and sense templates.35S-labeled riboprobes were generated us-
performed as described before (5). Control riboprobes did not hybridize
to lung tissue at any time point postinfection, and noninfected tissue did
and scored for abundance of foci.
Cellular assays. The number of anti-influenza virus-specific cells se-
munospot (ELISpot) assay (R&D systems, Minneapolis, MN) by follow-
precoated anti-IFN-? plates were blocked with RPMI plus 10% fetal calf
serum (FCS) and antibiotics (cRPMI) for 30 min at room temperature.
Medium was removed from wells, and 105cells were added to each well.
Cells were stimulated with 1918 recombinant HA (truncated at residue
530; 1 ?g/well), inactivated A/Mexico/4108/2009 virus (1:100 dilution of
inactivated stock; 100 ?l/well), or the immunodominant H2-KdCD8?T
cell epitope in H1 HA: HA533(IYSTVASSL; 1 ?g/well) (Pepscan Presto,
Leystad, Netherlands). Additional wells were stimulated with phorbol
myristate acetate (PMA) (50 ng/well) and ionomycin (500 ng/well) as
positive controls or Ova257(SIINFEKL; 1 ?g/well) (Pepscan Presto, Ley-
(10 U/ml) was added to each well. Plates were incubated at 37°C for 48 h.
Giles et al.
jvi.asm.orgJournal of Virology
After incubation, plates were washed four times with R&D wash buffer
and incubated at 4°C overnight with biotinylated anti-mouse IFN-?.
Plates were washed as described before and incubated at room tempera-
ture for 2 h with streptavidin conjugated to alkaline phosphatase. Plates
at room temperature for 1 h in the dark with BCIP (5-bromo-4-chloro-
3-indolylphosphate)/NBT chromogen substrate. The plates were washed
were sacrificed at 6 dpi, and spleens and lungs were harvested and pre-
pared in single-cell suspensions. Briefly, membrane plates with 0.45 ?m
polyvinylidene difluoride (PVDF) (Millipore, Billerica, MA) were coated
each well. Plates were incubated at 37°C for 48 h. After incubation, plates
h with horseradish peroxidase-conjugated anti-mouse IgG (Southern
Biotech, Birmingham, AL). Plates were washed as described before, and
spots were developed at room temperature for 1 h in the dark with detec-
washed extensively with DI H2O and allowed to dry overnight prior to
spots being counted using an ImmunoSpot ELISpot reader (Cellular
Technology Ltd., Cleveland, OH).
Passive transfer of sera. Sera from adult and aged vaccinated mice
9-week-old recipient BALB/c mice (n ? 5/group). Equal amounts of se-
rum from each mouse in a particular vaccine/age group were pooled and
heat inactivated for 30 min at 56°C. A total of 200 ?l of pooled and
inactivated serum was transferred to recipient mice via intraperitoneal
(i.p.) injection. Twenty-four hours posttransfer, mice were infected with
2009 pandemic H1N1 virus as described above.
Statistical analysis. Statistical analyses of immune responses were
performed using a one-way analysis of variance (ANOVA) with Dunn’s
posttest to compare each group. A P value of ?0.05 was considered sig-
statistical analyses were performed using GraphPad Prism software.
Homologous immunogenicity of 1918 VLP vaccines in mice.
BALB/c mice (6 to 8 weeks) were vaccinated twice at weeks 0 and
plus CpG adjuvant. Two weeks after the final vaccination, serum
was analyzed for antibody responses. All mice vaccinated with
1918 VLPs had HAI antibodies to the homologous test antigen
any HAI antibodies to the 1918 VLPs (Fig. 1A). To evaluate the
inhibition (HAI) serum antibody titers were determined for each vaccine group using 1918 VLPs as the test antigen (A). Bars represent the log2-transformed
1918 virus. (D) Four days postinfection, lung virus titers were determined. Bars represent the log10-transformed mean virus titers (?standard deviation). A P
value of less than 0.05 was considered significant (?, P ? 0.05; ??, P ? 0.01; ???, P ? 0.001).
Influenza Virus Abs Elicited in Young Mice Protect Aged Mice
February 2012 Volume 86 Number 3jvi.asm.org 1503
ability of the elicited antibody response to block live virus infec-
tion, serum was tested in a live virus neutralization assay. Similar
to the 1918 VLP HAI results, all mice vaccinated with 1918 VLPs
had neutralizing antibodies with a GMT of 1:208, while mice re-
ceiving Gag VLPs or CpG alone failed to generate any detectable
neutralizing antibodies (Fig. 1B). Additional vaccines adminis-
tered without the CpG adjuvant elicited similar HAI titers, and
therefore CpG adjuvant was removed from subsequent vaccine
preparations (data not shown).
after the final vaccination, mice were challenged intranasally with
the vaccines. 1918 VLP-vaccinated mice were protected from
weight loss (maximum, 94.8% at 2 dpi), while Gag VLP- and
more, mice receiving the 1918 VLP vaccine were completely pro-
tected from death, while Gag VLP- and CpG-vaccinated animals
completely succumbed to infection by 7 dpi (Fig. 1C). To deter-
mine the ability of the vaccines to control viral replication in the
dpi and analyzed for viral titers (Fig. 1D). Mice vaccinated with
1918 VLPs did not have detectable virus, while mice receiving the
Gag VLPs or CpG alone had significantly higher viral loads (P ?
Longevity of the antibody response. To determine the dura-
tion of the antibody response elicited by the 1918 VLP, mice (n ?
adjuvant and allowed to age. Serum was collected at 3 months
(two weeks postfinal vaccination), 10 months, and 20 months of
age and analyzed for antibody responses to the 1918 VLPs (Fig.
mice over the lifetime of the animals. Although the HAI GMT
decreased from 1:127 to 1:88 to 1:54 at 3, 10, and 20 months,
respectively, the observed differences were not significant (P ?
0.05), indicating that the 1918 VLPs elicited lifelong homologous
demic viruses, sera from 1918 VLP-vaccinated adult (3 to 4
months; n ? 13) and aged (20 months; n ? 14) animals were
analyzed for heterologous antibody responses. Sera from adult
mice were collected 2 weeks after the final vaccination, while sera
from aged mice were collected 16 months (?64 weeks) after the
cross-reactive antibodies, and no significant differences between
the age groups were detected (P ? 0.05; Fig. 2B). Although the
2009 pandemic HAI titers in the 1918 VLP-vaccinated animals
failed to achieve significance compared to the titer of the mock-
vaccinated animals, 100% of adult animals and 50% of the aged
animals receiving the 1918 VLP vaccine had detectable HAI titers
(range of 1:20 to 1:160 and 1:10 to 1:320 for adult and aged mice,
and similar titers were found between strains. Additionally, adult
homologous 1918 test antigen (P ? 0.05), and these titers were
vaccinated controls (P ? 0.05).
To evaluate the relative binding kinetic profile of the antibody
elicited by the 1918 VLPs, sera from adult and aged mice were
novel H1N1. Serum samples were diluted, polyclonal IgG was
described with minor modifications, detailed in Materials and
Methods (8, 61, 62). Sensograms demonstrating the specifics of
in Fig. 2C and D. Sera from adult mice collected at 3 months
postvaccination had an apparent single population of antibody
of 1.84 ? 105(Fig. 2E). Similarly, the apparently monospecific
relative association rate for sera collected at 20 months postvacci-
log10difference in relative dissociation rates (Kd) was observed
between sera collected from adult and aged mice. Sera collected
from adult mice had a relative dissociation rate of 3.03 ? 10?4,
and sera from aged mice had a dissociation rate of 6.33 ? 10?5.
These differences in binding resulted in approximately a log10
lower affinity for HA of the adult sera than that of the aged sera,
1.65 ? 10?9and 2.12 ? 10?10, respectively. Although similar RU
levels were captured from the adult and aged mouse serum sam-
ples, the percentage of HA-specific antibody collected from aged
mice vaccinated with the 1918 VLPs (8.5%) was greater than
2-fold higher than sera collected from 1918 VLP-vaccinated adult
mice (3.8%) (Fig. 2F). Therefore, the aged mouse sera compared
to the adult mouse sera had higher concentrations of HA-specific
antibody that demonstrated a log10lower dissociation rate, which
resulted in a log10higher affinity for the HA protein.
Protection against 2009 pandemic virus challenge. Adult (2
cination) vaccinated mice were then challenged intranasally with
1 ? 106PFU of the 2009 pandemic virus A/Mexico/4108/2009.
This isolate is not mouse adapted and does not cause a lethal
adult and aged animals receiving the 1918 VLPs were protected
from development of clinical sickness and weight loss, while
mock-vaccinated animals became ill and rapidly lost weight (Fig.
dpi and 3 to 13 dpi for adult and aged mice, respectively) and
mice, respectively) compared to the age-matched mock-
between adult and aged animals within the 1918 VLP or mock
vaccine groups. To evaluate viral burden, lungs were harvested
from infected mice at 4 dpi (n ? 3/group), and viral replication
was determined (Fig. 3C). Adult 1918 VLP-vaccinated mice had
significantly decreased lung viral titers compared to those of the
adult mock-vaccinated animals (P ? 0.0071). In contrast, aged
1918 VLP- and mock-vaccinated mice had equivalent viral titers
despite the differences observed for disease signs and weight loss
(P ? 0.1579).
logical features after the A/Mexico/4108/2009 challenge could ex-
plain the dichotomy observed in aged 1918 VLP-vaccinated
tions were assessed for histopathological changes, presence of in-
fluenza virus, and differences in immune response. At 4 dpi, the
Giles et al.
jvi.asm.orgJournal of Virology
lungs of adult 1918 VLP-vaccinated mice had minimal bronchial
and alveolar inflammation compared to those of adult mock-
vaccinated mice, which showed bronchial epithelial intracellular
edema and necrosis with moderate inflammatory infiltration and
areas of intraalveolar exudate and cellular consolidation (Fig. 4A
and B). Similar to adult 1918 VLP-vaccinated mice, aged 1918
VLP-vaccinated mice showed minor alveolar involvement; how-
ever, bronchial epithelium had moderate intracellular edema,
20 months. (A) Hemagglutination inhibition (HAI) serum antibody titers were evaluated at each time point using 1918 VLPs as the test antigen. (B) Sera from
Binding of rHA to pooled serum samples from vaccinated adult and aged mice was analyzed utilizing SPR as described in Materials and Methods. Gray lines in
the sensograms demonstrate binding (RU) to rHA over time for adult (C) and aged (D) mouse sera. HA protein concentrations were run in duplicate from 66
the diagonal lines of the graph. (F) Calculated rHA-specific antibody percentages were plotted as a function of vaccination group.
Influenza Virus Abs Elicited in Young Mice Protect Aged Mice
February 2012 Volume 86 Number 3jvi.asm.org 1505
with necrotic epithelium sloughing into airway spaces (Fig. 4C).
Aged mock-vaccinated mice showed severe bronchial inflamma-
tion and epithelial necrosis, but alveolar spaces were less involved
than adult mock-vaccinated mice (Fig. 4D).
To evaluate the location and severity of influenza viral antigen
scored on 4-dpi lung sections. Adult 1918 VLP-vaccinated ani-
mals had occasional bronchial epithelium infection and viral rep-
to P). This was in contrast to significant bronchial epithelium
infection and replication observed in adult mock-vaccinated and
aged animals regardless of vaccination (Fig. 4F to H, J to M, and
O). Alveolar spaces in adult and aged mock-vaccinated animals
vaccinated mice showed less alveolar infection than mock-
vaccinated animals (Fig. 4F to H, J to L, N, and P).
from challenged mice were scored for the presence of macro-
phages, T cells, neutrophils, IgA-secreting cells, and IFN-? tran-
scription. Macrophage and T cell infiltrates were less abundant in
adult 1918 VLP-vaccinated mice than in adult mock-vaccinated
and aged animals, regardless of vaccination (Fig. 5A to J). Similar
results were observed with neutrophils (data not shown). Total
numbers of IgA-positive cells (not influenza virus specific) were
higher in aged animals than in adult mice, regardless of vaccina-
tion status (Fig. 5K to O). Immunohistochemistry for IgG and
IgM was attempted, but the level of background staining pre-
cluded analysis. Adult 1918 VLP-vaccinated mice showed few
IFN-? RNA foci compared to adult and aged mock-vaccinated
and aged 1918 VLP-vaccinated mice (Fig. 5P to T).
Postinfection cellular responses. To determine the magni-
tude of influenza virus-specific cellular responses postinfection,
spleens and lungs from vaccinated animals (n ? 3/group) were
?-producing cells were analyzed by ELISpot assay. Although the
the spleens of any animals, but 1918 VLP-specific ASC were de-
tected in the lungs of 1918 VLP-vaccinated mice regardless of age
(Fig. 6A). Importantly, both adult and aged 1918 VLP-vaccinated
animals had equivalent numbers of lung ASC, which were signif-
icantly increased compared to the age-matched controls (P ?
Additionally, 1918 VLP vaccine-primed influenza virus-
specific IFN-?-producing cells were analyzed (Fig. 6B). IFN-?
production in the spleen was low to undetectable, and no signifi-
cant differences were found between any of the groups regardless
of stimulating antigen. Adult mice receiving the 1918 VLPs had
significantly more lung IFN-?-producing cells responding to the
immunodominant peptide HA533than adult mock-vaccinated
animals (P ? 0.05). Aged mice vaccinated with 1918 VLPs and
stimulated with HA533peptide had increased numbers of IFN-?-
producing cells compared to those of aged mock-vaccinated ani-
1918 VLP-vaccinated animals had equivalent numbers of IFN-?-
producing cells after infection. Although IFN-?-producing cells
were detected in the lung using truncated 1918 HA protein or
intact virus as stimulating antigens, no significant differences be-
tween groups were observed.
5/group) were administered pooled sera via i.p. injection from
adult (2 weeks postvaccination) and aged (16 months postvacci-
nation) 1918 VLP-vaccinated animals and aged mock-vaccinated
animals. At 24 h after serum transfer, mice were challenged intra-
nasally with the 2009 pandemic virus A/Mexico/4108/2009 (1 ?
FIG 3 Protection from 2009 pandemic virus challenge. BALB/c mice were
4108/2009) 2 weeks (adult) or 16 months (aged) after final vaccination. Mice
were evaluated daily to monitor sickness (A) and weight loss (B). Sickness
score was determined by evaluating activity (0 ? normal, 1 ? reduced, 2 ?
severely reduced) and hunched appearance (0 ? absent, 1 ? present). Lung
virus titers were determined 4 days postinfection. Bars represent mean virus
titer (?standard deviation). A P value of less than 0.05 was considered signif-
icant (?, P ? 0.05).
Giles et al.
jvi.asm.orgJournal of Virology
is more severe and, as such, the young mice are a more sensitive
model for evaluating protective efficacy (unpublished observa-
tions). Pooled serum from adult and aged 1918 VLP-vaccinated
animals was confirmed to have equivalent levels of anti-1918 an-
tibody titers prior to transfer (total IgG endpoint dilution of
1:3,200 and HAI of 1:80 for each VLP group). Mice receiving
either adult or aged 1918 VLP-vaccinated serum developed mild
clinical illness, while mice receiving mock-vaccinated serum de-
with the 2009 pandemic virus (A/Mexico/4108/2009) 2 weeks (adult) or 16 months (aged) after final vaccination. Lungs were collected 4 days postinfection,
formalin fixed, and paraffin embedded. Representative images from hematoxylin-and-eosin-stained sections: adult 1918 VLP-vaccinated mice (A), adult
mock-vaccinated mice (B), aged 1918 VLP-vaccinated mice (C), and aged mock-vaccinated mice (D). Immunohistochemistry for influenza virus antigen (E to
3 ? more than one focus per field.
Influenza Virus Abs Elicited in Young Mice Protect Aged Mice
February 2012 Volume 86 Number 3jvi.asm.org 1507
veloped more severe disease (P ? 0.05; 4 to 8 dpi) (Fig. 7A). In-
terestingly, adult 1918 VLP serum recipients resolved the clinical
(P ? 0.05; 7 to 8 dpi). Mice receiving adult 1918 VLP serum had
ients (P ? 0.05; 4 to 8 dpi) and aged 1918 VLP serum recipients
(P ? 0.05; 6 to 8 dpi) (Fig. 7B). Aged 1918 VLP serum recipients
had equivalent weight loss compared to the mock serum recipi-
ents at every time point except for 8 dpi. Although the aged 1918
VLP serum recipient mice developed longer-lasting disease and
lost more weight than the adult 1918 VLP serum recipients, both
adult and aged 1918 VLP recipients were completely protected
from death, while 80% of the mock recipients had reached exper-
imental endpoint by 8 dpi (P ? 0.01; Fig. 7C). Interestingly, both
adult and aged 1918 VLP transferred serum had similar levels of
IgG1, IgG2a, and IgG2b, but only the adult 1918 VLP serum had
detectable IgG3(Fig. 7D).
In this study, we evaluated the efficacy of preimmunity to 1918-
in aged mice. Sequence analysis indicated that the HA protein
1918 virus than the H1N1 strains that reemerged in humans in
1977 (39). Indeed, structural similarities between 1918 HA and
2009 pandemic HA have been elegantly described and indicate
conservation within antigenic sites that is not present in contem-
tibodies derived from human survivors of the 1918 pandemic
groups, epidemiological evidence has indicated that elderly pop-
ulations were unusually protected from severe infections during
FIG 5 Analysis of immune response by immunohistochemistry and ISH after 2009 pandemic challenge. BALB/c mice were vaccinated with 1918 VLPs and
formalin fixed, and paraffin embedded. Fluorescent immunohistochemistry (red) for Iba1-positive macrophages (B to E), CD3?T cells (G to J), and total
IgA-positive cells (L to O) and ISH for IFN-? (Q to T) were performed on sections from paraffin-embedded lung tissue. Sections were scored for severity of
infiltrate: Iba1-postive macrophages (A), CD3?T cells (F), IgA? cells (K), and IFN-? foci (P). Iba1 scoring: 0 ? scattered throughout section; 1 ? collected
section; 2 ? abundant throughout section; 3 ? abundant with collections surrounding bronchi. Total IgA scoring (not influenza virus specific): average of the
number of positive cells in 25 microscopic fields (40?). IFN-? scoring: number of foci in section.
Giles et al.
jvi.asm.orgJournal of Virology
levels of cross-reactive antibodies in the older populations, and it
is hypothesized that this is due to prior exposure(s) to antigeni-
cally similar influenza virus (29, 32, 78). Furthermore, there is
direct evidence of the protective efficacy in mice with prior expo-
sure to 1918-like or classical swine H1N1 influenza virus to 2009
pandemic infection (39, 59). Cross-protective efficacy of older
mechanistic evidence for the observed phenomenon of decreased
evaluated the duration of the cross-reactive immune responses.
We sought to confirm and expand these findings to aged animals
in order to more closely mimic the findings in elderly humans.
Although our model is unable to account for multiple exposures
FIG 6 Postinfection antigen-specific cellular immune response. BALB/c mice were vaccinated with 1918 VLPs and infected with 2009 pandemic virus (A/
prepared, and the numbers of anti-1918 HA antibody-secreting cells (A) and influenza virus-specific IFN-?-producing cells (B) were determined by ELISpot
assay. Values represent the mean spots (?standard deviation) for each group.
FIG 7 Passive transfer protection from 2009 pandemic challenge. BALB/c mice were vaccinated with 1918 VLPs and blood collected 2 weeks (adult) or 16
months (aged) after final vaccination. Serum was pooled for each age group, heat inactivated, and transferred via i.p. injection to naïve recipient mice. One day
pooled transferred serum was determined via ELISA. A P value of less than 0.05 was considered significant (??, P ? 0.01).
Influenza Virus Abs Elicited in Young Mice Protect Aged Mice
February 2012 Volume 86 Number 3jvi.asm.org 1509
to drifted influenza virus antigens, the results indicate that anti-
1918 influenza virus immunity acquired early in life can indeed
retain its cross-protective efficacy against a 2009 pandemic chal-
lenge in later stages of life.
Age-associated defects in immune responses generally lead to
increased susceptibility to infectious disease and decreased re-
virus vaccine in elderly humans (22). Although several studies
have established a defect in immune responses to vaccination in
the elderly (18, 22, 23, 49), we sought to evaluate the durability
and efficacy of immune responses initially elicited in young ani-
mals. We found that vaccine responses were produced efficiently
in adult mice (Fig. 1A and B) and were robust enough to protect
against the highly lethal reconstructed 1918 virus challenge (Fig.
1C and D). The duration of homologous receptor-blocking anti-
body was evaluated, and titers elicited as adults were maintained
throughout the lifespan of the mouse (Fig. 2A). Although the an-
tibody titers tended to decrease with age, these differences were
only that aged animals maintained equivalent levels of cross-
reactive antibody titers compared to adult mice but also that sera
from aged vaccinated animals had increased relative antibody
avidity to the novel H1N1 HA protein compared to that of sera
from adult vaccinated mice (Fig. 2). Shortly after vaccination, the
antibody response is dominated by low-affinity responses pro-
duced by short-lived plasma cells that may have the benefit of
being more cross-reactive due to reduced somatic mutation in
response to a specific antigen (60). Prior studies have evaluated
only antibody responses at 2 to 4 weeks after antigen exposure,
and as such the observed cross-reactivity may be due to the kinet-
ics of the antibody response and not completely indicative of the
long-lasting antibody repertoire. Our results indicate that the
cross-reactive antibodies observed in adult animals are long last-
ing and therefore probably not only produced by short-lived
also maintained by LLPC that continue to produce high-affinity
antibody for an entire lifetime.
An important caveat of this study is that we evaluated a single
realistic scenario is one that includes multiple exposures, via in-
fection or vaccination, of antigenically distinct viruses over a life-
be unable to respond to the new antigens, an accumulation of
diverse LLPC and resulting high-affinity serum antibodies could
lead to even more robust cross-reactivity. In support of this idea,
serologic data from humans suggest that those individuals who
to be positive for other historic viruses (78). A second, but not
mutually exclusive, possibility is that memory B cells (MBC) may
be specifically boosted by sequential exposure. Indeed, the in-
creased numbers of broadly neutralizing antibody-secreting cells
in response to 2009 pandemic infection in humans supports the
notion that the MBC specific for a cross-reactive epitope can be
activated during heterologous infection (71). The increased avid-
ity in the aged mice supports the hypothesis of LLPC-derived an-
tibody mediating cross-protection. Additional antigen exposures
and therefore drive an even more cross-reactive antibody profile.
Therefore, the use of a single antigen in our model provides a
protective efficacy against 2009 pandemic challenge.
ease in mice, except for highly pathogenic viruses, including 1918
and H5N1 isolates (3). The 2009 pandemic virus also readily in-
comparing multiple virus isolates (39). The strain used for chal-
lenge infections in these studies (A/Mexico/4108/2009) is not le-
thal to adult mice but does cause significant morbidity, with in-
fected cells being detected by in situ hybridization at 3 dpi or
earlier in both bronchial and alveolar spaces. The predominant
bronchiolar infection peaks at 3 dpi, while infection in alveolar
spaces peaks at 3 to 5 dpi. By 10 dpi, rare infected cells are ob-
served, and virus is cleared by 14 dpi (unpublished observations).
We found that naïve aged and adult animals had morbidity pro-
in the aged animals at both the initiation and contraction stages,
leading to prolonged inflammation in the lungs (66). Vaccinated
animals from both age groups did not develop any signs of mor-
were recovered from lungs of aged vaccinated animals 4 dpi (Fig.
3C). Although the differences were not significant between the
tochemistry for influenza virus antigen and in situ hybridization
for influenza virus RNA. In situ analysis of pathological responses
and viral replication revealed that adult vaccinated animals were
protected from infection of both bronchial and alveolar spaces,
while aged vaccinated animals were protected only from alveolar
infection (Fig. 4). One possible explanation for why virus was
detected in the bronchial epithelium in aged vaccinated animals,
but not in adult vaccinated animals, is that the initial immune
response is delayed in the aged cohort (66). Similar numbers of
than was evaluated in these studies. Alternatively, the time from
vaccination could also contribute to the differences in viral repli-
cation between adult and aged vaccinated mice. Adult vaccinated
mice were challenged only 2 weeks after the final vaccination,
while aged mice were challenged 16 months after final vaccina-
cells in adult mice could be available at the time of infection and
therefore more efficiently control virus replication.
Overall in the histopathological analysis of the immune re-
sponse, we observed greater infiltrates of macrophages, T cells,
neutrophils, and IFN-? RNA-expressing cells in the aged and
adult mock-vaccinated mice than in the adult vaccinated mice.
This suggests that the intensity of the observed immune response
is a reflection of the level of viral replication in the lungs. Indeed,
aged vaccinated mice had viral burdens and immune infiltrate
similar to those of unvaccinated mice, but surprisingly they did
not lose weight or display any signs of disease. Therefore, restric-
tion of viral replication to the bronchial spaces in aged vaccinated
mice likely contributed to the less severe disease than that ob-
prechallenge effector cells in the aged vaccinated animals permits
bronchial infection and inflammation, but the high-affinity anti-
body efficiently restricts the virus from reaching the alveolar
spaces. Furthermore, the high-affinity nature of the antibody
Giles et al.
jvi.asm.org Journal of Virology
from aged mice is likely critical in this scenario to overcome the
initial bronchial infection and prevent alveolar spread due to the
inflammation and/or infection play a greater contribution to the
for the clearance of virus-infected cells after influenza virus infec-
tion and are detectable after primary infection by day 5 (45). One
defect that is associated with the aging immune response is the
reduction in CD8?T cell function (28). Our results indicate that
IFN-?-producing T cells specific to a class I immunodominant
peptide are recruited to the site of infection as efficiently in vacci-
nated aged animals as in adult animals (Fig. 6). Consistent with
aging-associated T cell defects, a reduced, albeit not significant,
number of IFN-?-producing cells was found in naïve aged ani-
mals compared to that found in the adult controls. In addition to
T cell-related age defects, B cells are also impaired (14, 21). Vac-
antibody prechallenge and similar numbers of antigen-specific
antibody-secreting cells detectable in the lungs 6 days postinfec-
tion, while unvaccinated animals did not have any detectable
antigen-specific antibody-secreting cells (Fig. 6). Interestingly,
antigen-specific antibody-secreting cells were not detectable in
the adaptive immune response that was primed in young animals
antigens might be impaired.
Serum surveillance of humans has indicated that the elderly
population has an increased frequency of 2009 pandemic cross-
reactive antibodies (29, 32, 78). To determine if cross-reactive
systemic antibody is sufficient to protect from 2009 pandemic
challenge, we passively transferred immune serum from both
adult and aged mice into naïve recipient mice prior to challenge.
are highly susceptible to 2009 pandemic challenge and therefore
provide a sensitive model for evaluating protective efficacy (63).
Serum from vaccinated mice, regardless of age group, protected
mice from death, while naïve animal serum did not (Fig. 7C).
Interestingly, adult serum recipients lost less weight and had re-
due to differences in administered serum antibody titer, as adult
and aged serum had equivalent levels of both total anti-HA anti-
serum from aged mice for HA had a slower dissociation rate than
the serum from adult mice, demonstrating that the antibody re-
mained bound longer to HA protein. The IgG subclass profile
the aged mice (Fig. 7D). IgG3is a minor fraction of antibody to
T-cell-dependent antigens and is the major isotype to T cell-
of the classical complement pathway, likely due to the properties
of cooperative binding (17, 26). Enhanced complement fixation
mediated by the HA-specific IgG3in adult sera could be a mech-
anism responsible for the decreased morbidity observed in the
aged mice is due to LLPC being more efficiently generated in the
presence of T cell help, whereas it is retained in the adult mice
to IgG3variability, non-HA antibodies could also explain the ob-
served morbidity differences. Antibodies to the NA protein of the
protective role of anti-NA immunity cannot be excluded in this
any signs of morbidity implies a critical protective role for the
cellular component of the immune response in addition to the
the presence of protective antibodies is also more predictive of
protection in the context of highly pathogenic 1918 influenza vi-
rus challenge (52). Therefore, although serum antibody is suffi-
cient to protect from severe disease and death, cellular immune
to protection from the morbidity associated with 2009 pandemic
infection in both aged and adult mice.
Viruslike particles are an intriguing platform for developing
new influenza virus vaccines (9, 30, 38, 41). The VLPs are self-
assembling and completely nonpathogenic particles similar in
morphology to intact virions (72). For the vaccines used in this
study, the influenza virus proteins HA and NA were pseudotyped
onto the surface of HIV Gag particles. This strategy has been used
for multiple applications and takes advantage of the robust bud-
ding properties of the Gag protein (30, 48). Lifelong antibody
nonreplicating antigens (16). We found that vaccination with
1918 influenza VLPs elicited robust lifelong immunity that was
effective at protecting against heterologous 2009 pandemic virus
challenge. Importantly, the cohort of vaccinated animals allowed
to age was not vaccinated in the presence of adjuvant, indicating
that the duration of the elicited immune response was not a func-
tion of an adjuvant. These studies were performed in mice and
here indicate that VLP-based vaccinations are capable of eliciting
lifelong immunity, as measured by both serum antibody (Fig. 2)
and cellular recall to infection (Fig. 6).
This is the first evaluation of cross-reactive immunity to 2009
pandemic influenza virus in an aged-animal model. Antigenic
similarities between the pandemic influenza virus strains of 1918
by human data and confirmed in adult-animal models (29, 39,
73). Here, we show that animals that experience 1918 influenza
virus antigens during adulthood maintain the cross-protective
immunity to 2009 pandemic H1N1 influenza virus late into life.
The aged animals were not protected from viral replication but
restricted the virus to larger airways and did not show signs of
alveolar infection, which is the most common feature of fatal hu-
established by vaccination with a nonreplicating VLP rather than
by infection and included B and T cell cross-reactive responses in
addition to serum antibody. The studies reported here confirm
prior work by others that 1918 influenza virus can elicit cross-
expands those findings to aged animals, further validating the hy-
ulation observed during the 2009 H1N1 pandemic may be due to
prior exposure to antigenically similar viruses.
Influenza Virus Abs Elicited in Young Mice Protect Aged Mice
February 2012 Volume 86 Number 3jvi.asm.org 1511
This work was supported by an NIH training grant award, T32AI060525,
for Vaccine Research. In addition, T.M.R. was partially supported by a
cifically disclaims responsibility for any analyses, interpretations, or con-
We thank Brooke Pierce for technical assistance and Hermancia Eu-
gene and Nitin Bhardwaj for helpful discussions.
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