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,
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Center for Immunology and Microbial Disease, Albany Medical College, Albany, NY 12208;
Laboratory of Immunology, Wadsworth Center, Albany, NY 12201;
Department of Biomedical Sciences, School of Public Health, State University of New York, Albany, NY 12201; and
§
Genetics Institute, Cambridge, MA 01810
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-10, and IL-15 mRNA in
the spleens of newborn mice compared with animals exposed to vaccine
only. In addition, these animals showed dramatic increases in IFN--,
IL-2-, and IL-4-secreting cells, and in IgG2a Ab levels upon adult
challenge compared with mice primed with vaccine alone. Most
importantly, animals vaccinated and simultaneously treated with IL-12
at birth displayed enhanced survival after lethal challenge with
infectious influenza virus as adults compared with infected animals
that had been primed with vaccine alone. This augmented protection
required B cells and could be transferred to naive mice by immune
serum. Collectively, these results provide evidence that administration
of IL-12 to neonates induces a Th1-like response in newborns and
elicits protective antiviral immune memory. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recent evidence indicates that the neonatal immune system can be primed to recognize foreign Ags under certain conditions. Matzinger and colleagues (6) showed that the nature of Ag presentation in newborn animals determines the state of immune responsiveness. Specifically, they found that the transfer of adult dendritic cells to newborn mice allowed the animals to be primed rather than tolerized to allogenic spleen cells. Neonatal B cells that are unresponsive to polysaccharide Ags at birth were also recently shown to become responsive in vitro by incubation with recombinant cytokines such as IL-1 and IL-6 (7). Forsthuber et al. (8) demonstrated that injection of neonatal animals with protein Ags in IFA actually induces immune deviation rather than tolerance and stimulates preferential expression of Th cell type 2 (Th2) cytokines and Ab isotypes. Similarly, Siegriest and colleagues (9, 10) found that neonatal vaccine immunization results in biased Th2-type responses that still remain after adult boosting. Collectively, these findings have significant implications for neonatal vaccination strategies and suggest that redirection of the neonatal immune system to a Th1 pathway might increase responsiveness to vaccine Ags.
IL-12 is a 70-kDa heterodimeric protein that is produced by APCs and has pleiotrophic effects on innate and adaptive immune responses (11, 12, 13, 14). We (15, 16, 17) and others (18, 19, 20, 21, 22) have demonstrated the effectiveness of IL-12 as an adjuvant for humoral immunity in adult animals. In addition, we have found that neonatal animals have reduced IL-12 expression in the periphery and that IL-12 delivered at birth can serve as an adjuvant to induce a Th1-like cytokine response and cause priming for enhanced Ab responses to DNP-OVA and hen eggwhite lysozyme (HEL)3 (23). Similarly, Cairo and colleagues (24) showed that human cord blood cells have reduced expression of IL-12 and IL-15 compared with adult peripheral blood. The direct therapeutic efficacy of IL-12 treatment in young animals has been demonstrated during neonatal cryptosporidial and streptococcal B infections (25, 26). Therefore, immunoregulatory molecules such as IL-12 could contribute significantly in shaping the immune responses of neonates to a variety of infectious organisms.
In the present study, we have examined for the first time whether administration of IL-12 with an influenza subunit vaccine at birth enhances the protective efficacy of antiviral vaccination. The immune response of mice to influenza virus is especially well characterized and provides an attractive model to analyze the adjuvant properties of IL-12 in neonates. Our results show that IL-12 coadministered with an subunit influenza vaccine to newborn animals strongly influences protection against lethal virus challenge as adults.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BALB/c mice were obtained from the National Cancer Institute (Bethesda, MD). C57BL/6 IgM deficient (µMT) and C57BL/6 wild-type mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice were housed and bred at the Albany Medical College and provided food and water ad libitum. Animal care and experimental procedures were performed in compliance with the Institutional Animal Care and Use Committee guidelines.
Neonatal immunization procedures
One-day-old mice were injected i.p with 1 µg of subunit influenza vaccine, which consisted of soluble hemagglutinin subtype 1 (H1) and neuraminidase subtype 1 (N1) purified from influenza virus A/PR8/34 (provided by Dr. Doris Bucher, New York Medical College, New York, NY). The Ag was mixed with 2 mg/ml of alum (Rehydrogel Low Viscosity Gel; Reheis, Berkeley Heights, NJ). Mice were also injected i.p. on day 1 with 1 µg of recombinant murine IL-12 that was diluted in PBS containing 1% normal BALB/c mouse serum (PBS-NMS) or, in the case of control mice, with PBS-NMS only. The mice were allowed to mature to adulthood, and, at 5–6 wk of age, they were boosted with the same amount of Ag mixed in alum. No toxicity was observed with this treatment regimen. Sera were prepared by bleeding mice from the orbital plexus 7 days after the boost.
RNA isolation and RT-PCR
Total RNA isolation from snap-frozen spleens was performed with
the Ambion Total RNA Isolation Kit (Austin, TX) according to the
manufacturer’s instructions. Briefly, the frozen tissues were
homogenized with a mortar and pestle and immediately transferred into
tubes containing 1.0 ml of denaturation solution. Following
phenol-chloroform extraction, the homogenized samples were centrifuged
at 10,000 x g for 10 min at 4°C. The supernatants
were subjected to another round of phenol-chloroform extraction, and
the resulting RNA was precipitated with isopropanol, washed twice with
75% ethanol, and solubilized in diethyl pyrocarbonate-treated water.
The concentration of total RNA was determined by spectrophotometric
analysis at 260 nm. Three micrograms of total RNA were reverse
transcribed into cDNA using a reverse transcription kit (Life
Technologies, Gaithersburg, MD) using
oligo(dT)16–18 primers. The resulting cDNA was
amplified using specific primers for IFN- and IL-10 with
hypoxanthine phosphoribosyl transferase (HPRT) primers as a control.
The sense and antisense primers had the following sequences: IFN-,
5'-TGAACGCTACACACTGCATCTTGG-3' and 5'-CGACTCCTTTTCCGCTTCCTGAG- 3';
IL-10, 5'-ATGCAGGACTTTAAGGGTTACTTGGGTT-3' and
5'-ATTTCGGAGAGAGGTACAAACGAGGTTT-3'; and HPRT,
5'-GTTGGATACAGGCCAGACTTTGTTG-3' and
5'-GATTCAACTTGCGCTCATCTTAGGC-3'. PCR amplification was performed by
mixing 2 µl of cDNA, 0.25 mM dNTPs (Invitrogen, San Diego, CA), 0.8
µM primer, and 2.5 U of Taq DNA Polymerase (Life
Technologies) in a final volume of 50 µl in 60 mM Tris-HCl, pH 8.5,
15 mM (NH4)2
SO4, and 0.4 mM MgCl2. The
mixtures were incubated at 95°C for 5 min and then subjected to
the following amplification profile: 1 min at 95°C, 1 min at
56°C, and 1 min at 72°C for a duration of 35 cycles. This was
followed by a final extension for 10 min at 72°C. The PCR products
were separated on a 2.5% agarose gel, stained with ethidium
bromide, and visualized by UV transillumination.
RNase protection assay
Cytokine mRNA levels were determined using the RiboQuant multiprobe RNase protection assay system (PharMingen, San Diego, CA) according to the manufacturer’s instructions. Briefly, 10 µg of total RNA were hybridized to 32P-labeled RNA probes overnight at 56°C. The nucleic acid was treated with RNase for 45 min at 30°C to digest single-stranded RNA, and the remaining nucleic acid was subjected to phenol-chloroform extraction and resolved on a 6% denaturing polyacrylamide gel. Transcript levels were quantified on a Storm 840 PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Total RNA was normalized to the housekeeping gene GAPDH, and relative cytokine mRNA levels were expressed as arbitrary values.
Enzyme-linked immunospot (ELISPOT) analysis
ELISPOT assays were used to enumerate the frequency of
cytokine-secreting cells in spleens of adult mice primed at birth with
1 µg of H1N1 subunit influenza vaccine ± IL-12 and boosted in
vitro with H1N1. Nitrocellulose-backed microtiter plates (Millipore,
Bedford, MA) were coated overnight with 100 µl of anti-IL-2 (2
µg/ml; clone JES6-1A12, PharMingen), anti-IFN- (4 µg/ml;
clone R4-6A2, PharMingen), or anti-IL-4 (3 µg/ml; clone 11B11)
capture Ab. Following washing, the wells were blocked with 1% BSA in
PBS. Spleen cells were incubated (106 cells/well)
with 0.1 µg of H1N1 at 37°C in 5% CO2 for
24 h (IL-2 and IFN- assays) or 48 h (IL-4 assays). The
plates were subsequently washed and incubated overnight at 4°C with
100 µl of biotinylated detection Ab (0.5 µg/ml anti-IL-2 (clone
JES6-5H4, PharMingen), 0.5 µg/ml anti-IFN- (clone XMG1.2,
PharMingen), or 1 µg/ml anti-IL-4 (clone BVD6–24G2)). The plates
were then washed and incubated with streptavidin-alkaline phosphatase
(Life Technologies) for 2 h at room temperature. Spots were
visualized by the addition of nitroblue tetrazolium
5-bromo-4-chloro-3-indolyl phosphate substrate and counted with the aid
of a dissecting microscope. Statistical significance was determined
using a two-tailed Student’s t test.
Detection of Ab and isotype levels by ELISA
Anti-H1N1 levels in serum were determined by ELISA essentially as described (15, 27) with minor modifications. Briefly, microtiter plates (Nalge Nunc International, Rochester, NY) were coated overnight with 1 µg/ml of H1N1 in PBS. The plates were washed with PBS containing 0.3% Brij-35 (Sigma, St. Louis, MO) and blocked for 1 h at room temperature with PBS containing 5% FCS (HyClone Laboratories, Logan, UT) and 0.1% Brij-35. Serial dilutions of serum were added, and the plates were incubated for 2 h at room temperature. The plates were washed and incubated with goat anti-mouse whole Ig, IgM, IgG1, or IgG2a conjugated to alkaline phosphatase (Southern Biotechnology Associates, Birmingham, AL). After incubation for 1 h, the plates were washed and p-nitrophenyl phosphatase substrate was added to obtain color development. Plates were read at 405 nm with an ELISA microplate reader (Bio-Tek Instruments, Winooski, VT). In all cases, appropriate working dilutions and isotype specificities of the secondary Ab conjugates were determined using purified myeloma proteins of known isotypes (Sigma). Statistical analyses were performed using the Mann-Whitney U test. Data were considered statistically significant if values of p < 0.05 were obtained using 50% end point titers.
Virus challenge
For protection studies, 1-day-old mice were immunized i.p. with
1 µg of H1N1 subunit influenza vaccine mixed with alum. The mice also
received 1 µg of IL-12 in PBS-NMS or PBS-NMS only. Some mice received
only PBS-NMS (no H1N1 subunit vaccine). Approximately 4–5 wk after
primary immunization, viral challenge was performed using infectious
A/PR8/34 influenza virus (provided by Dr. Doris Bucher) administered
intranasally to anesthetized mice in 40 µl of sterile PBS. The mice
were then weighed daily and monitored for morbidity and mortality.
Survival data were analyzed using a log-rank test and compared using
2 analysis.
Passive transfer of sera
For passive transfer experiments, sera were obtained from adult mice that had been primed at birth with PBS-NMS only, vaccine only, or vaccine plus IL-12. Normal adult recipients were injected i.p. with 100 µl of a 1:5 dilution of pooled serum and challenged 6 h later with infectious influenza virus intranasally.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IL-12 has strong potentiating effects on the immune system through
its ability to preferentially activate Th1 and NK cells and induce
IFN- production (13, 14). To determine the effects of
vaccination and IL-12 administration on newborn mice, we analyzed
cytokine gene expression in the spleens of 3-day-old mice that had been
immunized 2 days previously with H1N1 subunit vaccine mixed with alum.
It was found that neonatal mice that received PBS-NMS or Ag 24 h
after birth did not express appreciable levels of splenic IFN- mRNA
(Fig. 1
). However, neonates that were
immunized with H1N1 subunit vaccine and simultaneously treated with
IL-12 exhibited significant induction of IFN- expression.
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To quantify and further examine cytokine profiles after neonatal
immunization with influenza vaccine, levels of cytokine transcripts
were measured by a multiplex RNase protection assay (Fig. 2). It was found that IFN- mRNA levels
were increased 25-fold in animals treated at birth with H1N1 plus IL-12
compared with mice treated with the vaccine alone (Table I). Furthermore, IL-10 mRNA expression
was enhanced 50-fold after IL-12 treatment. Coadministration of the
influenza vaccine plus IL-12 also caused an 18-fold increase in
expression of IL-15 mRNA levels. There was no detectable expression of
other cytokines such as IL-4 or IL-5 after neonatal vaccination.
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Based on the RT-PCR and RNase protection analyses performed soon
after birth, we determined whether the Th1 cytokine response was still
maintained in adult mice. Isolated spleen cells from mice primed at
birth were tested with an ELISPOT assay for cytokine production to the
recall Ag. Neonatal mice vaccinated and treated with IL-12 at birth had
increased numbers of IL-2- and IFN--secreting cells compared with
animals receiving the vaccine alone (Fig. 3). Interestingly, we also observed an
enhancement of IL-4-producing spots after neonatal IL-12 exposure. The
ratio of IFN-- to IL-4-producing cells was 7-fold greater with IL-12
treatment compared with mice receiving the vaccine alone.
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Our laboratory has previously demonstrated that IL-12 administered
to adult mice i.p. can alter isotype-restricted Ab responses to HEL
(15, 16). Furthermore, we recently showed that IL-12 given
to neonates enhances serum Ab levels to the DNP hapten and HEL
(23). We have now found that IL-12 delivered to newborn
animals has similar effects on anti-H1N1 Ab expression. Mice that
were immunized at birth with H1N1 showed elevated titers of total
anti-H1N1 serum Ab after boosting with homologous Ag as adults
compared with animals exposed to Ag only as adults (Fig. 4). IgM, IgG1, and IgG2a Ab levels were
all significantly enhanced in mice exposed to the vaccine at birth,
confirming the results of our previous findings in model protein and
hapten-carrier systems (23). Importantly, treatment with
H1N1 plus IL-12 at birth resulted in an even greater enhancement of
IgG2a Ab expression upon adult Ag challenge. However, there were no
significant differences in IgM and IgG1 Ab levels between mice
inoculated at birth with H1N1 and IL-12 and those inoculated with only
H1N1. There was no IgA detected in the sera of any of the animals (data
not shown).
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The effects of coadministrating IL-12 and H1N1 at birth on
survival and clinical outcome after challenge with influenza virus were
next assessed by treating 1-day-old BALB/c mice with H1N1 vaccine and 1
µg of IL-12 or PBS vehicle. Some mice received only PBS vehicle. Four
to 5 wk after immunization, the mice were inoculated intranasally with
A/PR8/34 influenza virus and monitored daily for morbidity and
mortality. It was found that all mice that were pretreated with only
PBS-NMS at birth (no H1N1 subunit vaccine) displayed progressive weight
loss and died within 13 days after virus challenge (Fig. 5).
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Augmented protection against influenza infection after vaccination with H1N1 plus IL-12 at birth is Ab-mediated
To evaluate the contribution of humoral immunity in protection
from influenza virus infection, we examined the host immune responses
of B cell deficient C57BL/6 (µMT) mice (30). Mice were
vaccinated at birth as described and challenged as adults with lethal
influenza virus infection intranasally. It was found that µMT mice
pretreated at birth with PBS-NMS alone succumbed to infection by day 9
compared with wild-type mice, which survived significantly longer (Fig. 6). All the µMT mice that received the
H1N1 vaccine alone also died by day 9 compared with 67% survival in
wild-type littermates. Among µMT mice that were vaccinated and
treated with IL-12, all but one succumbed to infection. In contrast,
every wild-type mouse that was similarly treated survived. Thus, B
cells rather than cytotoxic T cells play a crucial role in the enhanced
protection conferred by IL-12 treatment at birth.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We previously demonstrated that neonates have a reduction in IL-12
expression that could be responsible for poor responsiveness and a bias
toward Th2 immune responses (23). IL-12 is a pivotal
cytokine that regulates the biological functions of T cells and NK
cells and enhances the production of IFN- and other cytokines
(13, 14). In addition, IFN- stimulates production of
opsonizing Abs such as IgG2a (15, 17, 31). In the present
study, we found that coadministration of IL-12 and influenza vaccine at
birth resulted in significant increases in neonatal IFN- mRNA
levels. Expression of IL-15, which has similar biological activities as
IL-2 and induces proliferative responses in T cells, B cells, and NK
cells (32, 33, 34), was also increased by neonatal IL-12
treatment, as was IL-10. IL-10 is an important regulator of T cells and
is suggested to be involved in a feedback mechanism to modulate the Th1
pathway and down-regulate the effects of IFN-. While enhancing
Th1-type cytokines, IL-12 had no effects on Th2-type cytokines such as
IL-4 or IL-5 in neonates. The impaired cytokine responses of newborns
and young infants is an important consideration in any neonatal vaccine
strategy. Cairo and colleagues (24) have shown that IL-12
and IL-15 are expressed at lower levels in human cord blood, and this
reduction in cytokine expression was speculated to contribute to the
impairment of neonatal cell-mediated immunity. In addition, Barrios et
al. (9, 10) demonstrated that neonatally immunized animals
secrete higher levels of IL-5 and lower levels of IFN- compared with
adults. These findings have crucial implications for neonatal vaccine
strategies that require robust Th1-type immune responses, particularly
against viral diseases such as influenza, respiratory syncytial virus,
and HIV. Our results establish the ability of IL-12 administered at
birth to direct the generation of Th1 immunity and serve as a potent
adjuvant for viral vaccines.
Interestingly, animals vaccinated and treated with IL-12 at birth had
elevated levels of IL-2, IFN-, and IL-4 production upon adult
challenge. The augmented IL-4 production observed after IL-12 treatment
could be related to the use of alum during vaccination, which primarily
induces a Th2 immune response. In addition, IL-12 priming at birth may
have a differential effect on the development of neonatal vs adult T
cells. In vitro treatment of human neonatal T cells with IL-12 has been
shown to induce both IFN- and IL-4 secretion, a phenotype associated
with maturation of neonatal T cells (35).
It was found that inclusion of IL-12 in the vaccination regimen also resulted in enhancement of Th1-type Ab responses. Specifically, mice primed at birth with the influenza subunit vaccine and IL-12, and boosted as adults with vaccine alone, exhibited a significant increase in serum IgG2a Ab titers compared with animals primed with vaccine alone at birth. These results confirm and extend to a clinically relevant Ag our previous findings showing that IL-12 delivered at birth significantly enhances neonatal B cell memory responses to model T-dependent Ags (23). Animals primed at birth with IL-12 and a protein or hapten carrier, and boosted as adults with Ag only, were found to display elevated IgG1, IgG2a, and IgG2b Ab levels compared with mice injected with Ag alone (23). In addition, we have shown the ability of IL-12 to induce significant amounts of IgG2a Abs in adult animals responding to HEL (15, 16). Murine Abs of the IgG2a isotype are known to be efficient at opsonization and complement fixation and could be beneficial in neonatal vaccination strategies. IgG2a is the dominant Ab isotype elicited by viral infections (36), and, in fact, Graham and colleagues have shown the importance of serum IgG2a in protection against respiratory syncytial virus infections (37). Therefore, the ability of IL-12 to enhance newborn IgG2a Ab levels should have major implications in the development of infant viral vaccines.
The major finding of the present study is that administration of IL-12 at birth together with the influenza subunit vaccine increased the protective effects of the vaccine as shown by enhanced survival after influenza virus challenge. Our laboratory has recently demonstrated that IL-12 administered intransally to adult mice serves as a potent mucosal adjuvant for anti-viral vaccination (38). In addition, we have now demonstrated in preliminary studies that endogenous IL-12 expression is a crucial component of host defense against influenza virus infection. Specifically, adult IL-12 p35-deficient mice vaccinated intransally with H1N1 subunit vaccine alone are much more susceptible to subsequent influenza virus challenge compared with wild-type littermates (V. C. Huber and D. W. Metzger, unpublished observations). Inclusion of IL-12 during immunization augmented the protective efficacy of vaccination in IL-12 p35-deficient mice. Similarly, the importance of endogenous IL-12 in the immune response to influenza virus infection has been reported by Trinchieri and colleagues (39).
Use of B cell-deficient mice and passive transfer of serum demonstrated that the protection induced by IL-12 is mediated by Ab. It is believed that cell-mediated immunity is important in recovery from viral infections, whereas humoral responses are pivotal for protection against viral challenge. There is also evidence indicating that CD8 T cells provide protective immunity against influenza B virus infections in adult mice lacking Ig and mature B cells (40). Nevertheless, it is becoming clear that Abs play a major role in recovery from viral infections (41). In fact, several independent groups have shown that B cell-deficient animals are highly susceptible to lethal influenza virus infection compared with wild-type littermates (41, 42, 43, 44). The ability of IL-12 to augment protective neonatal B cell memory provides strong evidence for its use as powerful vaccine adjuvant.
Effective immunization of newborns and young infants against a variety
of infectious agents represents a serious challenge to both
industrialized and developing countries (45, 46). Due to
the preferential Th2 bias of the neonate and increased susceptibility
to infections, there has been continued interest in identifying safe
adjuvants that stimulate Th1 immunity. Currently approved adjuvants
such as alum are poor stimulators of Th1 immunity, which is believed to
be a crucial component of host defense against viruses
(47). There is interest in the use of DNA vaccination to
induce protective immunity in neonatal animals. Siegrist and colleagues
(48) reported that immunization of newborn animals with
DNA plasmids encoding measles virus hemagglutinin resulted in
enhancement of IFN- and IgG2a production. In addition, Bot et al.
(49) demonstrated that DNA vaccination of newborn mice
with a plasmid encoding the influenza hemagglutinin gene conferred
protective immunity against lethal challenge of adults with influenza
virus. There is evidence to suggest that the augmented effects seen
with DNA vaccination are due to IL-12, because it has been found that
treatment of adult mice with synthetic oligodeoxynucleotides containing
CpG motifs stimulates IL-12 production (50). In a recent
study, the incorporation of CpG DNA as an adjuvant for hepatitis B
virus surface Ag was evaluated in young mice (51). CpG
coadministered with Ag at birth had some effects on humoral and
cell-mediated responses, but augmented responses were only observed in
7-day-old immunized mice. We have now shown that IL-12 administered at
birth serves as a safe and powerful viral vaccine adjuvant to induce
protective Th1 immunity.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Dennis W. Metzger, Center for Immunology and Microbial Disease, Mail Code 151, Albany Medical College, 47 New Scotland Avenue, Albany, NY 12208. E-mail address:
3 Abbreviations used in this paper: HEL, hen eggwhite lysozyme; HPRT, hypoxanthine phosphoribosyl transferase; H1, hemagglutinin subtype 1; N1, neuraminadase subtype 1, NMS, normal mouse serum; ELISPOT, enzyme-linked immunospot.
Received for publication February 22, 1999. Accepted for publication January 21, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-dependent and -independent mechanisms. Eur. J. Immunol. 27:1958.[Medline]
stimulates IgG2a secretion by murine B cells stimulated with bacterial lipopolysaccharide. J. Immunol. 140:2121.[Abstract]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), H1N1 in alum plus PBS vehicle (
), or PBS vehicle
only without Ag (
). All mice were then challenged at 5–6 wk of age
with H1N1 in alum, and the sera were tested 7 days later by
isotype-specific ELISA using H1N1-coated microtiter plates. Titers
correspond to the serum dilutions giving 50% maximal binding. Each
symbol represents the results from an individual mouse. The differences
in binding between mice primed at birth with H1N1 ± IL-12 and
those primed with PBS vehicle only were significant at
p < 0.05 for total Ab, IgM, IgG1, and IgG2a. The
differences in binding between mice primed at birth with H1N1 and IL-12
and those primed with H1N1 and PBS vehicle were significant at
p < 0.05 for IgG2a.
) mice were
immunized at birth with H1N1 subunit vaccine in alum plus IL-12,
vaccine plus PBS vehicle, or PBS vehicle only. All mice (6–11 per
group) were then challenged intranasally 4–5 wk later with
103 pfu of A/PR/8/34 influenza virus. The mice were
monitored daily for mortality and weight loss. The differences in
survival between C57BL/6 µMT and C57BL/6 wild-type mice were
significant at p < 0.05.