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Mol Ther. Dec 2010; 18(12): 2182–2189.
Published online Sep 28, 2010. doi:  10.1038/mt.2010.202
PMCID: PMC2997593

A Universal Influenza A Vaccine Based on Adenovirus Expressing Matrix-2 Ectodomain and Nucleoprotein Protects Mice From Lethal Challenge

Abstract

A universal influenza vaccine, designed to induce broadly cross-reactive immunity against current and future influenza A virus strains, is in critical demand to reduce the need for annual vaccinations with vaccines chosen upon predicting the predominant circulating viral strains, and to ameliorate the threat of cyclically occurring pandemics that have, in the past, killed tens of millions. Here, we describe a vaccine regimen based on sequential immunization with two serologically distinct chimpanzee-derived replication-defective adenovirus (Ad) vectors expressing the matrix-2 protein ectodomain (M2e) from three divergent strains of influenza A virus fused to the influenza virus nucleoprotein (NP) for induction of antibodies to M2e and virus-specific CD8+ T cells to NP. In preclinical mouse models, the Ad vaccines expressing M2e and NP elicit robust NP-specific CD8+ T-cell responses and moderate antibody responses to all three M2e sequences. Most importantly, vaccinated mice are protected against morbidity and mortality following challenge with high doses of different influenza virus strains. Protection requires both antibodies to M2e and cellular immune responses to NP.

Introduction

Each year seasonal influenza infections cause severe illness in 3–5 million people worldwide and kill 250,000–500,000 humans.1 The currently licensed influenza (flu) vaccines are annual vaccines that induce subtype-specific virus neutralizing antibodies, which do not protect against new subtypes or antigenic variants.2 Due to the unpredictability of evolving influenza viruses, the advanced design of vaccines against new strains using current strategies is unfeasible. A universal flu vaccine to induce broadly cross-reactive immunity against current and future influenza viruses is thus in critical demand. Such a vaccine would also ameliorate the need for annual vaccination and would be expected to increase overall vaccine coverage.

Influenza A viruses are negative-sense, single-stranded, segmented RNA viruses, which contain eight RNA segments, encoding for 11 proteins (HA, NA, NP, M1, M2, NS1, NEP/NS2, PA, PB1, PB1-F2, PB2). Matrix protein 2 (M2) is a tetrameric transmembrane protein of influenza A virus. Its ectodomain (M2e) shows conservation among human influenza A virus strains. M2e-specific antibodies, although not neutralizing, reduce in animals the severity of infection with a wide range of influenza A virus strains.3,4 Influenza A nucleoprotein (NP), the major protein component of ribonucleoprotein (RNP) complexes, is also relatively conserved making it an attractive candidate for a universal flu vaccine. Although the NP protein induces an antibody response, the role of such antibodies in providing protection remains controversial.5,6 The NP induces a vigorous CD8+ T-cell response both in mice and men7,8 that, as epidemiological studies suggest, may contribute to resistance against severe disease following influenza A virus infection.9

Influenza vaccines based on M2e, NP, or both have been tested extensively in animal models, where they have shown sufficient promise that some of them advanced to clinical trials (http://clinicaltrials.gov/ct2/show/NCT00993083).3,4,10,11,12,13 However, results from efficacy trials, which have commonly only shown limited efficacy even for licensed vaccines,14,15 are not yet available.

Here, we introduce a different vaccine platform that is uniquely suited to induce potent and sustained immune responses. Specifically, we generated E1-deleted adenovirus (Ad) vectors from chimpanzee serotypes C68 (AdC68) or C6 (AdC6, ref. 16) expressing, in tandem, three M2e sequences from diverse strains of influenza A virus (H1N1, H5N1, and H7N2) fused to H1N1 NP. Ad vaccines expressing M2e and NP elicit robust NP-specific CD8+ T-cell responses and moderate antibody responses to the three M2e sequences in mice. Most importantly, vaccinated young mice are protected against mortality following challenge with high doses of different influenza viruses. This protection is dependent upon both antibodies to M2e and cellular immune responses to NP. Old mice develop a robust immune response upon vaccination but fail to be protected.

Results

Transgene product expression

The M2e(3)-NP chimeric gene encodes the M2e of A/PR/8, an H1N1 virus, a pathogenic H5N1 virus that evolved in 1997, and an avian H7N2 strain isolated in 2007 (Figure 1a). The three M2e sequences were combined with the full-length NP sequence. Linker sequences, encoding three alanine residues, were inserted between each gene and a signal sequence from HSV-1 glycoprotein D was placed upstream of the chimeric gene (Figure 1b). Western blotting was conducted with a monoclonal antibody to M2e termed 14C2-S1-4.2.17 Results show that AdC68M2e(3)-NP and AdC6M2e(3)-NP express comparable levels of the chimeric protein in vitro (Figure 1c). Similar results were obtained upon blotting with an antibody to NP (data not shown).

Figure 1
M2e(3)-NP fusion protein. (a) Amino acid sequences of three M2e in the construct and of A/Fort Monmouth/1/47 virus. (b) Schematic representation of the chimeric M2e(3)-NP gene. (c) Expression of M2e(3)-NP protein by different vectors in infected cells ...

Antibody responses to M2e

Groups of young C57Bl/6 mice were vaccinated with 1 × 1010 virus particles (vp) of AdC68M2e(3)-NP; some of them were boosted 2 months later with 1 × 1010 vp of AdC6M2e(3)-NP. Sera were harvested from individual mice 5 weeks after the boost, and together with naive control sera or, in separate experiments, sera from mice vaccinated with vectors expressing the rabies virus glycoprotein (rab.gp), tested for antibodies to M2e on the different M2-transfected or sham-transfected HeLa cell lines (Figure 2a). Antibody titers were comparable upon testing on the three cell lines and increased after the boost. Sera from mice vaccinated with the control vectors showed background reactivity similar to that of sera from naive mice (e.g., average antibody titers to M2e in naive ICR mice, 1.2 µg/ml; average antibody titers in ICR mice after an AdCrab.gp prime boost regimen: 0.99 µg/ml). To ensure that the vaccine induced a response in genetically distinct strains of mice, outbred ICR mice were tested using the same vaccine regimens. Antibody titers achieved after priming were lower than those in C57Bl/6 mice, but comparable after the boost (Figure 2b).

Figure 2
Humoral responses in C57Bl/6 mice or ICR mice. (a) Three cell lines expressing M2 of H1N1, H5N1, or H7N2 were used in cellular enzyme-linked immunosorbent assays to measure M2e-specific antibody titers in sera of C57Bl/6 mice (n = 10) harvested 3 months ...

NP-specific CD8+ T-cell responses

Vaccine-induced CD8+ T-cell responses to NP were tested at different time points after vaccination by intracellular cytokine staining for interferon-γ (Figure 3a). After priming with AdC68M2e(3)-NP, all of the mice developed detectable frequencies of NP-specific CD8+ T cells in blood, which gradually declined. A booster immunization with AdC6M2e(3)-NP given 2 months after priming affected an increase in circulating NP-specific CD8+ T cells. Mice were killed 4 months after priming and frequencies of NP-specific CD8+ T cells were determined from lymphocytes isolated from blood, spleens, and lungs of individual mice (Figure 3b). Frequencies were higher in mice that had received the prime-boost regimens; frequencies were highest in lungs and lowest in spleens. Higher frequencies in peripheral tissues that primary attract effector/effector memory cells than lymphatic tissues such as spleen is typical for Ad vector–induced T-cell responses as has been described previously.18

Figure 3
NP-specific CD8+ T-cell responses in C57Bl/6 mice (a) Intracellular IFN-γstaining of NP-specific CD8+ T cells was carried out on peripheral blood mononuclear cells from mice (n = 10) at weeks 2, 5, 8, 10, and 12, after vaccination. ...

Protective immunity

C57Bl/6 mice were vaccinated and then infected with 10 mean lethal dose (LD50) of A/PR/8 virus. Lung virus titers were measured from the right inferior lobe of the lungs of individual mice 5 days after challenge (Figure 4a). This time point was chosen for the following reasons. Influenza virus replication can be detected rapidly within 48 hours in lungs of mice. Depending on the dose of challenge virus, mice that are able to fend off the infection then show a decline in titers around day 7.19 Vaccines that induce neutralizing antibodies would be expected to decrease virus titers from the onset while vaccine such as ours that induces immune mechanisms directed against infected cells would be expected to act with a delay. Mice that had been primed with AdC68M2e(3)-NP showed a reduction in mean titers this did not reach significance compared to control mice (P = 0.2). A significant reduction in lung viral titers was achieved upon prime-boosting (P = 0.0003). Lungs of individual mice from a given group showed a rather surprisingly large range of virus titers; PCR reactions rather a neutralization assays were used to measure viral titers. Although PCR-based assays have the advantage of higher sensitivity, they not only measure infectious virus but also defective genome-containing particles. Another factor that may have contributed to the high variability in virus titers was the use of a single time point that may have missed peak virus titers in some of the mice.

Figure 4
Protection against challenge. (a) C57Bl/6 mice were challenged with 10LD50 of A/PR/8 virus 4 months after priming or 2 months after boosting. Five days later lung virus titers were determined. Graph shows titers of viral genomes (vgs) per gram of tissue ...

To ensure that the reduction in viral titers resulted in a clinical benefit, the experiment was repeated with the prime-boost regimen and mice were challenged with 10LD50 of A/PR/8 or A/Fort Monmouth virus. Vaccinated, naïve, or sham-vaccinated mice lost weight after challenge. Weight loss of vaccinated mice peaked by days 6–8 after challenge and then mice began to gain weight and by 21 days after challenge most mice had returned to their prechallenge weight. Naive or sham-vaccinated control mice continued to lose weight after challenge till they died or required euthanasia (Figure 4b,c). Upon challenge with either virus strain, 90% of the vaccinated mice survived while all of the control mice died (Figure 4d,e). Lung lobes harvested 5 days after challenge were stained with hematoxylin and eosin and analyzed for signs of inflammation using the scoring system described in the Materials and Methods section. Most of the unvaccinated mice had perivascular infiltrates in their lungs and half of them had interstitial infiltrates with an average pathology score of 2.35 (Figure 4f). Pathology was less pronounced in mice that had received the prime-boost vaccination, and the average pathology score of their lungs was 1.85 (P = 0.04). Mice were in infected at 4 months after priming. Those that received a second dose of vaccine were boosted at 2 months after priming and then challenged 2 months later. This protocol allowed us to prime and challenge mice together thus reducing experimental variability. One could make the argument that differences in the time interval between vaccination and challenge may have biased the results. We, therefore, in additional experiments, tested mice that only received one dose of the AdC68M2(e)3-NP vaccine at 2 months after vaccination. Protection was comparable to that observed in mice challenged at 4 months after immunization.

The experiment was repeated with ICR and BALB/c mice. Mice were challenged 4 months after priming or 2 months after the boost with 10LD50 of A/PR/8 virus. By day 5 after challenge, lung virus titers were significantly reduced in ICR mice that were primed (P = 0.0002) or primed and boosted (P = 0.0003; Figure 5a). In 30% of primed mice and 50% of mice that received the prime-boost regimen, virus had been cleared completely from their lungs while all of the mice of the two control groups had titers in excess of 105 genome copies. Similar results were obtained with BALB/c mice (Figure 5b).

Figure 5
Protection of ICR and BALB/c mice. (a) ICR mice were challenged with 10LD50 A/PR/8 4 months after priming or 2 months after the boost. Five days after challenge, lung virus titers were determined. Graph shows titers of vgs per gram of tissue of individual ...

Next, vaccinated ICR mice were challenged with an increased dose of 150LD50 of A/PR/8 virus (Figure 5c,d). In spite of this very severe challenge, which killed 90% of the control mice, 70% of the vaccinated mice survived. Histological analyses (Figure 5e) of lung section of ICR mice conducted 5 days after challenge revealed a significant reduction in inflammation in vaccinated mice with an average pathology score of 2.7 in naive mice and 1.9 in vaccinated mice (P = 0.009).

Influenza viruses cause death mainly in the aged and commercially available vaccines are commonly poorly immunogenic in this population.20 To test whether the AdM2(e)3-NP vectors induce protection in aged mice, we immunized a group of 20-month-old C57Bl/6 mice with AdC68M2(e)3-NP and boosted them 2 months later with AdC6M2(e)3-NP. Mice were challenged 3 months after the boost with 3LD50 of A/Fort Monmouth virus and viral titers were determined from lungs 5 days later. Groups of young mice were tested in parallel. Antibody responses to M2e were comparable in old and young vaccinated mice although old naive mice had slightly higher background titers (average antibody titers to M2e in old mice: 17 µg/ml; average background titers in old mice: 6 µg/ml). Frequencies of NP-specific CD8+ T cells were higher in aged mice at the time of challenge (% NP-specific CD8+ T cells/all CD8+ T cells: old mice: 10.2%; young mice: 5.9%, P = 0.03). By 5 days after challenge, lung virus titers in young vaccinated mice were significantly below those of young naive mice, while such a difference was not seen in aged mice, although they had on average lower titers compared to the young (Figure 6). In addition, young vaccinated mice showed reduced weight loss following challenge compared to young naive mice, which again was not seen for the aged mice indicating that the vaccines, although they induced immune responses in aged mice, nevertheless, lacked efficacy in this population.

Figure 6
Protection of aged mice. Old and young C67BL/6 mice were primed with AdC68M2e(3)-NP and 2 months later boosted with AdC6M2e(3)-NP. Three months later vaccinated and age-matched naive control mice were challenged with 3LD50 A/Fort Monmouth virus. Five ...

Immune correlates of protection

To elucidate the immune mechanisms that contribute to protection in vaccinated mice, the vaccines were tested in β2-microglobin knockout mice, which lack CD8+ T cells. Mice received a prime-boost regimen or were left naive. Antibody titers measured at 5 weeks after the boost were comparable to those achieved in wild-type C57Bl/6 mice (mean titer of 10.7 µg of antibodies to M2e/ml). Upon challenge of mice with 10LD50 A/PR/8, only 33.3% (2/6) of vaccinated mice survived, while all of the naive mice succumbed the infection (Figure 7a). This difference was not significant suggesting that antibodies alone failed to provide protection against severe challenge.

Figure 7
Correlates of protection. (a) Protection of β2-microglobin knockout mice. The graph shows the survival curves of naive and vaccinated (prime/boost regimen) mice upon challenge with 10LD50 of A/PR/8 virus. Vaccinated mice (n = 6) versus naive mice ...

In a second experiment, the role of antibodies was assessed by adoptive transfer studies. Donor C57Bl/6 mice received the prime-boost regimen with the AdM2e(3)-NP vectors and 1 ml of their pooled sera, which at the time of harvest contained 14.3 µg of M2e-specific antibodies/ml, was transferred to naive C57Bl/6 recipients, which were challenged 24 hours later with 10LD50 of A/PR/8. Control mice received sera from naive donors. Transfer of M2e immune sera protected 50% of the recipients, while all of the mice injected with the control sera died following challenge (Figure 7b).

To further assess correlates of protection, mice were vaccinated with an AdC68 vector expressing NP only (AdC68NP) or they were primed with AdC68NP and then boosted 2 months later with AdC6NP. Frequencies of NP-specific CD8+ T cells were measured from blood 2 months later (Figure 7c) and were found to be comparable to those achieved with the AdC68M2e(3)-NP vaccine or a prime-boost regimen with the two heterologous Ad vectors. It should also be noted that AdNP-vaccinated mice developed antibodies to NP. Two months after vaccination, one group of AdC68NP-vaccinated mice received 1.0 ml of sera obtained from C57Bl/6 mice that had received the prime-boost regimen with the AdM2e(3)-NP vectors, the other group received 1.0 ml of sera from naive mice. Mice were challenged 24 hours later with 10LD50 of A/PR/8 virus. All of the control animals died, while 33.3% of AdC68NP-vaccinated mice transferred with naive serum survived (Figure 7d). This degree of survival did not reach statistical significance. Survival was significant at 55.6% in AdC68NP-vaccinated mice that received the immune serum again suggesting that vaccine-induced protection against high-dose challenge requires both antibodies to M2e and CD8+ T cells to NP. Enhancing NP-specific CD8+ T cells by booster immunization did not result in increased protection, but mice rather showed a trend toward accelerated death potentially suggesting that a potent NP-specific CD8+ T-cell response may exacerbate disease. In addition, these results show that a combination of T cells and antibodies to NP does not suffice for protection but that antibodies to M2e are essential.

Discussion

Novel and more efficacious flu vaccines that induce broad and sustained protection against a wide range of influenza A viruses are necessary to reduce the need for annual vaccination campaigns with vaccines based upon viral strains predicted to be the predominant circulating strains and to ameliorate the threat of future pandemics that can potentially kill millions.

Various strategies have been tested to develop a universal flu vaccine. Most of these strategies focused on vaccines expressing the M1, M2, or NP of influenza virus (http://clinicaltrials.gov/ct2/show/NCT00993083),3,4,10,11,12,13,21 which are relatively conserved between different viral strains. Both the M1 and the NP are dominant targets of CD8+ T cells in humans, while the M2e binds non-neutralizing antibodies that provide some, albeit limited, protection. Although epidemiological evidence supports the development of CD8+ T-cell vaccines to influenza A viruses,9 in animal models, the effectiveness of CD8+ T cells in protecting against influenza A viruses remains controversial; while some investigators reported induction of protection,21 others reported lack of efficacy22 or even exacerbation of disease following viral challenge.23 In our hands, vaccines that induce very high frequencies of NP-specific CD8+ T cells such as Ad vectors expressing the NP provided only marginal protection.

M2e is poorly immunogenic during a natural influenza A virus infection due to the paucity of its expression on virions.24 Humans develop M2e-specific antibodies following infection but titers are low and not sustained.25 Several studies conducted in mice and ferrets have shown that M2e-specific antibodies can restrict subsequent virus replication and reduce morbidity and mortality to a broad range of influenza A virus strains.3,4 Although M2e is relatively conserved, variability between different strains has been described26 and vaccines that express only one version of M2e may thus lack efficacy against strains with M2e mutations. By the same token it was shown that antibodies to M2e select for escape mutants.27 A number of vaccine platforms have been tested (http://clinicaltrials.gov/ct2/show/NCT00993083)3,4,10,11,12,13 and several of those such as the M2e-hepatitis B core protein vaccine subsequently underwent early clinical testing, which confirmed their safety and immunogenicity.3

We decided to explore a universal flu virus vaccine based on Ad vectors for the following reasons. Production, purification, and quality control procedures for Ad vectors are well established.16 Ad vectors induce innate immune responses ameliorating the need for addition of adjuvants. They also induce very potent B and CD8+ T-cell responses, which, due to low-level persistence of the vectors, are remarkably sustained.28 Pre-existing neutralizing antibodies to common human serotypes of Ad viruses such as serotype 5, which impact vaccine efficacy,16 can readily be avoided by the use of serotypes from other species such as chimpanzees, which typically neither circulate in humans nor cross-react with human serotypes.29 In cases where prime-boost regimens are needed to achieve immune responses of sufficient potency, vectors based on distinct Ad serotypes are available.16 Ad viruses and Ad vectors have been used extensively in the clinic where they were well tolerated. They can be applied through a variety of routes including mucosal routes such as the airways30 or even orally upon encapsidation as was shown with vaccine to Ad viruses 4 and 7 used by the US military.31

In order to develop vaccines that induce broadly cross-reactive immunity, we expressed a fusion protein composed of three divergent versions of M2e for induction of antibodies and the NP for stimulation of CD8+ T cells in two serologically distinct Ad vectors derived from chimpanzee isolates. The vaccines induce modest titers of antibodies to M2e in all mouse strains tested. Antibody titers were very similar to all three versions of M2e including that derived from an H7N2 virus, which, but for one amino acid (serine), is identical to that of the currently pandemic H1N1 virus. Whether this reflects that all three sequences are equally immunogenic, or whether most antibodies cross-react, remains to be tested. The vaccines also induced a potent CD8+ T-cell response that could be detected in blood as well as in lungs. Although the vaccines apparently failed to induce sterilizing immunity against high-dose viral challenge, they resulted in lowering of viral loads by day 5 following infection, reduction of infection-associated lung pathology, and they prevented influenza A virus–associated mortality. Protection was achieved against A/PR/8/34 virus from which the NP as well as one of the M2e sequences originated as well as against A/Fort Monmouth/1/47, which carries an M2e sequence that is divergent from those expressed by the vaccines. Solid vaccine-induced protection against challenge required both antibodies and CD8+ T cells. Either of these two immune mechanisms could provide some protection as was shows by passive transfer of vaccine-induced immune sera, by the use of mice lacking CD8+ T cells, or by the AdNP vaccines, but the level of protection by only one of the immune mechanisms was well below that achieved with both.

Not surprisingly our studies show that a preventative vaccine that aims to induce protective immunity to a rapidly mutating pathogen such as influenza virus may require a comprehensive approach in which multiple antigens are provided for stimulation of different arms of the immune system rather than the reductionist approach of single epitope vaccines.

Materials and Methods

Ad vectors. AdC68 and AdC6 vectors expressing the M2e(3)-NP chimeric protein were generated as follows: the 3 M2e-encoding sequences with a signal peptide was synthesized by Integrated DNA Technologies (Coraville, IA) and cloned into pShuttle (Clonetech, Mountain View, CA). The NP gene, upon deletion of the start codon, was cloned in frame downstream of the M2e sequences. Upon digestion with I-CeuI and PI-SceI, the fusion gene was cloned from pShuttle into the E1 domain of the molecular clones of AdC68 and AdC6, respectively. Recombinant Ad vectors (AdC68M2e(3)-NP and AdC6M2e(3)-NP) were rescued by transfection of plasmid DNA into HEK 293 cells. The Ad vectors were purified by cesium chloride density-gradient centrifugation and virus particle content was determined by spectrophotometry at 260 nm. Vectors were titrated to determine infectious units and vector batches had vp to infectious unit ratios below 200 and were cleared for endotoxin contamination. Other vectors encoding NP only or the glycoprotein of rabies virus (rab.gp) were generated and quality controlled using the same methods.

Expression of the vaccine antigen. HEK 293 cells were infected with 10–1,000 vp/cell. At 24 hours after infection, western blots were performed and membranes were blotted with a monoclonal antibody to M2e (14C2-S1-4.2).

Influenza virus. Influenza viruses A/PR/8/34 and A/Fort Monmouth/1/47 were grown in the chorioallantoic fluid of embryonated chicken eggs and titrated in adult mice upon their intranasal infection to determine the LD50.

Mice. Female C57Bl/6, BALB/c, and ICR mice were purchased at 6–8 weeks of age from ACE Animals (Boyertown, PA). Female C57Bl/6J mice (β2M−/−, strain B6.129P-B2mtm1 Unc) were purchased at 6–8 weeks of age from Jackson Laboratory (Bar Harbor, ME). All mice were housed in the Wistar Institute Animal Facility. C57Bl/6 mice were aged at the Animal Facility of the Wistar Institute and used once they were >20 months old. Animal procedures in this study were conducted in accordance with Institutional Animal Care and Use Committee guidelines.

Immunization of mice. Groups of 6–15 mice were vaccinated with 1 × 1010 vp of the AdC68M2e(3)-NP vector given intramuscularly. Two months later, some groups of mice were boosted with 1 × 1010 vp of the AdC6M2e(3)-NP vector given intramuscularly.

Challenge of mice. Two months after vaccination, mice were anesthetized and then challenged intranasally with either 10 or 150 LD50 of influenza A/PR/8/34 virus or 3 or 10 LD50 of influenza A/Fort Monmouth/1/47 virus, diluted in 30 µl phosphate-buffered saline. Mice were weighed daily. They were killed if they lost in excess of 30% of their prechallenge weight. In some experiments, mice were euthanized 5 days after challenge.

Virus titration. The assay was adopted from a previously published method32 that was validated against the standard plaque assay. Lung tissue samples were excised from experimental mice, and their weight was recorded. After tissue samples were mechanically homogenized, RNAs were isolated by using TRIzol reagent (Invitrogen, Carlsbad, CA) and were resuspended in 50 µl of diethylpyrocarbonate-treated water (Ambion, Austin, TX). The RNA concentration of each sample was determined spectrophotometrically at an absorbance of 260 nm. From the entire 50 µl RNA solutions, complementary DNA (cDNA)was obtained using 100 µl reaction volumes with the manufacturer-specified component proportions of a High-Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA). Reactions were run on thermal cycler (Eppendorf Mastercycler; Hamburg, Germany) in one cycle at 25 °C for 10 minutes, 37 °C for 120 minutes, and 85 °C for 5 minutes. Concentrations of cDNA were standardized to 50 ng/5 ml, and influenza A/PR8 cDNA was used to create a standard curve by serial dilution, ranging from 4 ng/5 ml to 0.0064 ng/5 ml. Viral cDNA was quantified using a TaqMan real-time PCR assay on an ABI Prism 7000 Sequence Detector (Applied Biosystems). The primers for viral cDNA quantification were specific to the influenza A matrix protein gene (MP), which were MP sense (5′-AAGACCAATCCTGTCACCTCTGA-3′) and MP antisense (5′-CAAAGCGTCTACGCTGCAGTCC-3′). The reporter probe was a TaqMan TAMARA (Applied Biosystems) of sequence [6-FAM]-5′-TTTGTGTTCACGCTCACCGTT-3′-[TAMARA]. The cDNA samples were quantified in triplicate. Each reaction totaled 25 µl, and included 12.5 µl TaqMan Universal PCR Master Mix, 5 pmol/l reporter probe, 22 pmol/l MP sense primer, 22 pmol/l MP antisense primer, and 5µl (50 ng) cDNA sample template. Reactions were run at 50 °C for 2 minutes, 95 °C for 10 minutes, and then cycled 40 times between 95 °C for 15 seconds and 60 °C for 1 minute. In analyzing the spectral curves, the cycle threshold was defined just above the emission baseline to stay within the exponential amplification phase of the PCR. Viral copy numbers were normalized with the original tissue sample masses, and calculated based on the molar mass of influenza A/PR8 genome.

Antibody titers to M2e. Antibody responses specific to M2e were measured from sera of individual mice by a cellular enzyme-linked immunosorbent assay that was modified from a previously published procedure.17 We cloned the three full-length M2 sequences from which the M2e sequences of the vaccines had originated, into lentivirus vectors. Lentivirus was rescued in 293T cells and used to infect HeLa cells to generate stable cell lines that express full-length M2. A control cell line was generated by infection of 293T cells with empty lentivirus. These cell lines were used as immunosorbents in an enzyme-linked immunosorbent assay as described.17 The assay was standardized with a purified antibody that recognizes all of the three M2e sequences (unpublished results).

Intracellular cytokine staining. Frequencies of NP-specific interferon-γ-producing CD8+ T cells were determined following vaccination at different time points from blood as described.33 Samples were analyzed using an EPICS XL (Beckman-Coulter, Brea, CA). FlowJo 7.1.1 software (Tree Star, Ashland, OR) was used for postacquisition analysis. Frequencies of NP-specific CD8+ T cells are shown as interferon-γ+ CD8+ over all CD8+ T cells.

Tetramer staining. Lymphocytes were isolated from lung, blood, and spleen of individual mice before or 5 days after challenge. Cells were stained with an allophycocyanin-labeled major histocompatibility complex class I NP peptide tetramer (ASNENTETM, Tetramer Core Facility, Emory University, GA), in combination with an anti-CD8a-PerCP-Cy5.5 antibody (BD Biosciences, San Jose, CA) for 1 hour at 4 °C. Flow cytometry was performed with the Beckman-Coulter XL (Beckman-Coulter) at The Wistar Institute Flow Cytometry Core Facility and data were analyzed with FlowJo 7.1.1 (Tree Star).

Histology. Lungs were perfused with 1% fetal bovine serum–supplemented phosphate-buffered saline and the lobes were gently inflated with 200 µl of a 10% formalin solution through a 30g needle. The inflated lung samples were submerged in 10% formalin for tissue fixation for 24 hours at 4 °C. Formalin-fixed lung samples were paraffin-embedded and sectioned at 4 µm and mounted on glass slides. Sections were stained with hematoxylin and eosin and two random sections of each lung sample were examined. Histopathological changes were examined by an investigator, who was unaware of the samples' origin. Lung pathology was scored as follows: 1, no observable pathology; 2, perivascular infiltrates, 3, perivascular and interstitial infiltrates affecting <20% of the lobe section; 4, perivascular and interstitial infiltrates affecting 20–50% of the lobe section; 5, perivascular and interstitial infiltrates affecting >50% of the lobe section.

Statistical analyses. Immune responses, pathology grade, as well as virus titers were analyzed using samples from individual mice. Results are shows as mean ± SD. Significance of differences between groups was determined by one-tailed Student's t-test or analysis of variance. Differences between pathology scores were analyzed by Wilcoxon's two-sample test. The statistical significance of protection of vaccinated groups compared to the control group was determined using Fisher's exact test.

Acknowledgments

This work was funded by a grant from NIH/NIAID (HHSN266200500030C), by a grant from the State of Pennsylvania, and the Wistar Cancer Center Support Grant (NCI—P30 CA 010815). We thank Walter Gerhard for advise and sharing of reagents and Christina Cole for help in preparation of the manuscript.

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