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J Virol. 2011 January; 85(1): 448–455.
Published online 2010 October 27. doi:  10.1128/JVI.01812-10
PMCID: PMC3014175

T Cell-Mediated Protection against Lethal 2009 Pandemic H1N1 Influenza Virus Infection in a Mouse Model [down-pointing small open triangle]


Genetic mutation and reassortment of influenza virus gene segments, in particular those of hemagglutinin (HA) and neuraminidase (NA), that lead to antigenic drift and shift are the major strategies for influenza virus to escape preexisting immunity. The most recent example of such phenomena is the first pandemic of H1N1 influenza of the 21st century, which started in 2009. Cross-reactive antibodies raised against H1N1 viruses circulating before 1930 show protective activity against the 2009 pandemic virus. Cross-reactive T-cell responses can also contribute to protection, but in vivo support of this view is lacking. To explore the protection mechanisms in vivo, we primed mice with H1 and H3 influenza virus isolates and rechallenged them with a virus derived from the 2009 H1N1 A/CA/04/09 virus, named CA/E3/09. We found that priming with influenza viruses of both H1 and H3 homo- and heterosubtypes protected against lethal CA/E3/09 virus challenge. Convalescent-phase sera from these primed mice conferred no neutralization activity in vitro and no protection in vivo. However, T-cell depletion studies suggested that both CD4 and CD8 T cells contributed to the protection. Taken together, these results indicate that cross-reactive T cells established after initial priming with distally related viruses can be a vital component for prevention of disease and control of pandemic H1N1 influenza virus infection. Our results highlight the importance of establishing cross-reactive T-cell responses for protecting against existing or newly emerging pandemic influenza viruses.

Influenza A virus remains an important pathogen that causes respiratory diseases in humans and animals. The virus has eight separate gene segments that encode two surface glycoproteins (hemagglutinin [HA] and neuraminidase [NA]) and six internal proteins (PA, PB1, PB2, NP, M1, and M2) in mature virus particles. There are also nonstructural proteins, including NS1 and NS2 (NEP), produced during replication, although NS1 is not assembled into virions and only a small amount of NEP is found in mature virus (23, 29, 43). Influenza A virus has been classified into different subtypes based on the cross-reactivity of HA and NA. So far, there are 16 HA (H1 to H16) and 9 NA (N1 to N9) subtypes that have been identified in viruses from humans and animals (23). There are three major subtypes (H1N1, H2N2, and H3N2) circulating in the human population that have caused epidemic and pandemic occurrences. Although other subtypes, including avian H5, H7, and H9 viruses, have also been transmitted to humans, efficient human-to-human transmission of these subtypes has not been observed to date (23, 28). Neutralizing antibody raised by vaccination or infection targets primarily surface HA to prevent infection and transmission, while processing and presentation of the internal and nonstructural proteins generate adaptive T-cell responses that help to eliminate virus (37). Current trivalent influenza vaccines focus on eliciting anti-HA neutralizing antibody to the vaccine strains and to closely related isolates within the same HA subtypes, but such vaccination strategies are usually ineffective in the prevention of epidemics or pandemics of other influenza virus subtypes.

Genetic mutation and reassortment of influenza virus gene segments, in particular those of HA and NA, that lead to antigenic drift and shift are the major strategies used by new strains to escape existing immunity. Excellent examples of such phenomena are the three major influenza pandemics (1918 H1N1 Spanish influenza, 1957 H2N2 Asian influenza, and 1968 H3N2 Hong Kong influenza) that occurred in the 20th century and killed millions of people. Shortly after the beginning of the 21st century, in April 2009, a novel influenza virus strain with a distinctive combination of gene segments from both North American and Eurasian swine lineages (17, 35) emerged in Mexico, the United States, and several other countries. On 11 June 2009, the World Health Organization officially declared this outbreak to be a global pandemic (9). Seasonal influenza virus infection usually causes high morbidity and mortality in populations of the very young, the elderly, and immunocompromised individuals. However, epidemiological data suggest that aged groups (>60 years old [those born before 1949]) have a very low rate of infection with this 2009 pandemic H1N1 influenza virus (1, 4).

Studies have detected high levels of cross-reactive antibody to this new pandemic virus in the aged population (2, 22), suggesting an association of the antibody-based preexisting immunity with protection. It is also known that CD4 and CD8 T-cell-mediated immunity could provide heterologous protection against different influenza virus subtypes or variants of the same strains (13, 15, 21, 26). In addition, published data have shown that conserved CD4 and CD8 T-cell epitopes against the 2009 pandemic H1N1 virus exist in the general population (12, 18, 20, 42), but in vivo support for whether T cells contribute to the control of the 2009 pandemic H1N1 virus is lacking. To explore the protection mechanism in vivo, mice were primed with old and contemporary influenza virus isolates and rechallenged with a virus derived from a 2009 H1N1 virus isolated in California. We found that T-cell immunity established after initial priming is a vital component for the prevention and control of pandemic 2009 H1N1 influenza virus infection, while convalescent-phase sera from mice primed with heterologous viruses provided no protection.



Female C57BL/6 mice were obtained from The Jackson Laboratory and used between the ages of 8 and 10 weeks. All animals were housed in the University of Rochester Vivarium facilities under specific-pathogen-free conditions, using microisolator technology. All animal experiments were approved by the University Committee of Animal Resources and complied with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Research Council (28a).

Viruses and infection.

Influenza A/PR/8/34 (PR8; H1N1), influenza A/HKx31 (X31; H3N2), and influenza A/New Caledonia/20/99 (NC/99; H1N1) viruses were prepared in eggs as described previously (30, 33). Influenza virus X31 is a recombinant virus containing the HA and NA segments from a 1968 Hong Kong influenza virus but sharing the internal viral proteins of the PR8 virus. Influenza A/California/04/2009 (CA/09; H1N1) virus was acquired through the NIH Centers of Excellence in Influenza Research and Surveillance and was propagated in eggs to generate a laboratory stock (A/CA/4_NYICE_E3/2009 [H1N1]; also called CA/E3/09). Virus titers were determined as 50% egg infective doses (EID50s) or PFU/ml. Mice were inoculated intranasally with 30 μl of different indicated doses of viruses (PR8, X31, NC/99, and CA/E3/09). For rechallenge study, mice were primed with nonlethal doses of PR8 (5 PFU), X31 (3 × 105 EID50), NC/99 (9 × 104 EID50), and CA/E3/09 (30 PFU) viruses and rested for 42 days before challenge with a lethal dose of CA/E3/09 virus (3,000 PFU per mouse).

Virus sequencing.

Viral RNAs were isolated from the CA/E3/09 stock virus grown in embryonated eggs by use of an RNAspin Mini apparatus (GE Healthcare) and were used as templates for reverse transcription-PCR (RT-PCR). cDNAs of viral genes were synthesized using SuperScript III One-Step RT-PCR Platinum Taq HiFi (Invitrogen) and primers which hybridize to the noncoding region of each gene. The cDNAs were purified after agarose gel electrophoresis and used directly for sequencing.

Plaque assay.

Virus titers in the collected lung samples were analyzed by MDCK cell-based plaque assay as described previously (10).

HAI assay.

A standard hemagglutination inhibition (HAI) assay was carried out using 4 hemagglutination units (HAU) of individual influenza viruses. Briefly, virus was diluted to 4 HAU, mixed with an equal volume of heat-inactivated serially diluted sera obtained from immunized or infected animals, and incubated for 1 h at room temperature. An equal volume of 1% chicken red blood cells (CRBC) was added and incubated for 45 min. Plates were then tilted and wells observed for agglutination. The HAI titer was determined to be the inverse of the last dilution where CRBC were not agglutinated.

Passive serum transfer.

Individual serum pools were obtained from mice 3 weeks or 5 weeks after infection with the respective influenza virus. Each serum pool was then injected intraperitoneally in a volume of 200 μl into naive mice. Twelve hours after injection, the mice were anesthetized and challenged intranasally with a lethal dose of CA/E3/09 virus (3,000 PFU per mouse). Mice were monitored daily for weight loss and survival.

MN assay.

Neutralizing antibodies for CA/E3/09 virus in sera from infected mice were detected using a microneutralization (MN) assay. Briefly, heat-inactivated serum samples were serially diluted 2-fold in incomplete minimal essential medium (MEM) and incubated at 37°C with 100 50% tissue culture infectious doses (TCID50) of CA/E3/09 virus for 1 h. The serum-virus mixture was then transferred into wells containing confluent MDCK cells and incubated for an additional 1 h. After a wash, 200 μl of incomplete medium containing tosylsulfonyl phenylalanyl chloromethyl ketone (TPCK)-trypsin (0.5 μg/ml) was added, and the plates were incubated for 2 days before MDCK cells were fixed. The neutralizing titer was determined as the reciprocal of the highest dilution at which the serum provided complete protection from viral cytopathic effects.

T-cell depletion.

For T-cell depletion study, mice were primed with X31 virus and rested for 42 days. Groups of mice were then injected intraperitoneally with 100 μg anti-CD4 antibody (clone GK1.5; eBioscience), 100 μg anti-CD8 antibody (clone 2.43; a gift from Paul Thomas, St. Jude Children's Research Hospital), both 100 μg CD4 and 100 μg CD8 antibodies, or 100 μg rat IgG2b isotype controls (eBioscience). Antibodies were injected every other day three times before and once at day 3 after rechallenge with a lethal dose of CA/E3/09 virus (3,000 PFU per mouse). After infection, mice were monitored daily for weight loss and survival.

Statistical analysis.

Statistical significance was evaluated using the two-tailed unpaired Student t test to compare appropriate groups. A P value of <0.05 was considered statistically significant.


Production of A/CA/4_NYICE_E3/2009 (H1N1) virus working stocks in eggs.

Samples of the prototype 2009 H1N1 influenza A/CA/04/09 (CA/09) virus were distributed shortly after emergence of the pandemic strain. The initial growth characteristics of this virus in our hands, both in tissue culture and after inoculation into mice, were poor relative to those of other laboratory strains of virus. These characteristics suggested that the virus stocks were poorly adapted to the growth conditions in culture or in mice or that the stock contained a high concentration of defective particles that were interfering with replication. Consequently, we performed a limiting dilution inoculation of the virus into embryonated hen eggs and collected allantoic fluid from eggs at the highest dilution (10−3) that was positive by hemagglutination assay for virus. Allantoic fluid from a single HA-positive egg (designated E2) was titrated in a separate set of eggs, resulting in a titer of 108. This was then used to inoculate a large number of eggs at 100 EID50 per egg, producing uniform infection of all eggs. Allantoic fluids from these eggs were pooled, clarified by low-speed centrifugation, and designated an E3 working stock of the virus. The EID50 titer and MDCK cell PFU titer of this stock were 108.5/ml and 1 × 107/ml, respectively. This E3 stock of the CA/09 virus was named A/CA/4_NYICE_E3/2009 (H1N1), abbreviated CA/E3/09. Although sequencing of the CA/E3/09 viral genes revealed four mutations compared to the A/CA/04/09 reference virus that result in amino acid substitutions, including three mutations in the HA protein (K136N, S200P, and D239G) and one in NP (D107G), the antigenic conservation of the original stock was verified using polyclonal ferret antiserum against CA/09 or a seasonal H1N1 virus (Table (Table1).1). The HAI titer of the ferret antiserum was slightly reduced for our CA/E3/09 virus compared to that of the CA/09 parent, suggesting that the 3 HA mutations affected but did not eliminate antigenic similarity to the parent. The CA/E3/09 virus was then used to inoculate mice at a range of doses to determine its pathogenic potential and the optimal dose for the challenge studies.

Comparison of HA antigenicity between CA/09, CA/E3/09, and A/Brisbane/59/07 influenza viruses

Dose response of the CA/E3/09 H1N1 stock in mice.

To determine the in vivo pathogenesis of the CA/E3/09 stock virus generated, groups of mice were infected with different doses of virus, and weight loss and survival were monitored. All mice inoculated with 3,000 PFU of virus displayed rapid weight loss and succumbed to infection before day 12 (Fig. 1A and B). Mice infected with 600 PFU of virus also showed rapid weight loss, and 80% of the mice in this group died between day 8 and day 14 after infection (Fig. 1A and B). The mice surviving at day 14 regained body weight and finally recovered from the illness. The groups of 5 mice given 300 PFU and 60 PFU of virus also gradually lost body weight after infection, with survival rates of 40% and 80%, respectively (Fig. 1A and B). The mice given 30 PFU of virus did not show clear weight loss in the first few days after inoculation, but after day 5, continued weight loss was observed until day 10 (Fig. (Fig.1A).1A). None of the mice in this group suffered death (Fig. (Fig.1B).1B). Hence, as reported by others (6, 27, 32), the results we show here suggest that the 2009 pandemic H1N1 stock we produced is pathogenic in mice and can cause lethal infection without rounds of adaptations.

FIG. 1.
Dose-dependent infection of mice with CA/E3/09 virus. As indicated in the figure, different doses of CA/E3/09 virus were inoculated intranasally into C57BL/6 mice. Body weight loss (A) and survival (B) were monitored daily. Data presented are average ...

Protection against CA/E3/09 infection by prior priming with different strains.

One of the characteristics of 2009 pandemic H1N1 influenza virus infection is a low rate of disease in the aged population (>60 years old [born before 1949]), suggesting the possibility that infection with H1N1 virus circulating during the 1930s and 1940s was protective (1, 4). Preexisting T-cell immunity might also provide protection and reduce the severity of 2009 pandemic H1N1 infection, as common CD4 and CD8 T-cell epitopes shared between seasonal influenza viruses and the 2009 pandemic H1N1 virus have been predicted or mapped (12, 18, 20, 42). To test this, we primed naïve mice with H1N1 PR8 virus that was originally isolated from human sputum specimens in 1934 (14). Infection was confirmed by body weight loss (Fig. (Fig.2A).2A). Six weeks later, mice were rechallenged with a lethal dose of CA/E3/09 virus generated from the CA/09 virus, a representative pandemic strain isolated shortly after the spread of the novel influenza epidemic in Mexico in 2009 (3). We found that PR8-primed mice displayed weight loss in the first 6 days after CA/E3/09 virus infection, similar to infected mice with no priming (Fig. (Fig.2B).2B). However, after day 6, PR8-primed mice had rapidly regained body weight, while nonprimed mice continued to suffer weight loss (Fig. (Fig.2B).2B). Not surprisingly, all of the PR8-primed mice survived the lethal challenge of CA/E3/09 virus, in contrast to the nonprimed controls, which all died before day 12 (Fig. (Fig.2C).2C). As a positive control, mice primed with a nonlethal dose of CA/E3/09 virus had only mild reductions of body weight, but no death, after a lethal dose of the homologous virus (Fig. 2A, B, and C).

FIG. 2.
Prior priming with different influenza virus strains provides protection against lethal infection with CA/E3/09. (A) Cohorts of C57BL/6 mice were primed with PR8 (5 PFU/mouse), X31 (3 × 105 EID50/mouse), NC/99 (9 × 104 EID50/mouse), or ...

X31 virus differs from PR8 virus only in the surface HA and NA proteins. Cross-reactive neutralizing antibody responses are avoided when prime and challenge experiments are performed with these two viruses, and cross-reactive T-cell responses against the common internal proteins are responsible for protection during rechallenge with either of these viruses (37). We reasoned that if cross-reactive T-cell immunity contributed to the protection conferred by PR8 priming in our study, priming of mice with X31 virus would also provide a certain level of protection against CA/E3/09 virus rechallenge. Mice were infected with X31 virus, which caused body weight loss (Fig. (Fig.2A).2A). X31-primed mice were then administered a lethal dose of the CA/E3/09 virus after 42 days of resting. Similar to the case for PR8-primed mice, weight loss of X31-primed animals was observed in the first 6 days of reinfection, but again, after day 6, X31 virus-primed mice gradually regained body weight, and none of them died from the CA/E3/09 infection (Fig. 2B and C). In addition, compared to the nonprimed controls, mice primed with either PR8 or X31 virus had significantly reduced virus titers in the lungs on day 3 after lethal CA/E3/09 infection, although the difference was not more than 10-fold (Fig. (Fig.2D).2D). These results suggested that T-cell immunity established after priming could be crucial in controlling lethal pandemic infection in mice. This conclusion was further strengthened by the finding that mice primed with the contemporary seasonal H1N1 New Caledonia virus (NC/99) could also be protected from lethal challenge with CA/E3/09 virus, as evidenced by reduced weight loss, death, and virus replication (Fig. 2B, C, and D) and the fact that sera from children immunized with seasonal influenza vaccines containing the NC/99 isolate have no microneutralization activity against 2009 pandemic H1N1 virus (22).

Sera from primed mice do not provide protection against CA/09 virus challenge.

Although our data clearly indicated the potential involvement of memory T cells in response to pandemic H1N1 infection, the potential contribution of antibodies induced after priming could not be excluded completely. To evaluate the neutralization ability of sera obtained from either PR8, X31, or NC/99 virus-infected mice, an MN assay was performed. As shown in Table Table2,2, 3- or 5-week sera from CA/E3/09 virus-primed mice showed a high MN titer of 1,024, while none of the sera from PR8-, X31-, or NC/99-primed mice had detectable neutralization activity against the CA/E3/09 virus (MN titer of <8). We also did not observe detectable HAI titers against CA/E3/09 virus in the sera from PR8, X31 or NC/99 virus-primed mice (data not shown). To further evaluate the potential in vivo activity of these convalescent-phase sera, a passive transfer study was performed. Twelve hours after passive transfer of immune sera by injection, mice were infected with lethal CA/E3/09 virus. As shown in Fig. Fig.3A,3A, mice administered 3-week sera from CA/E3/09 virus-infected animals did not show substantial weight loss after lethal CA/E3/09 virus infection. However, animals receiving 3-week sera from PR8, X31, or NC/99 virus-primed mice showed continuous weight loss after infection, similar to the mice that received normal control sera. In addition to severe weight loss, all animals that received sera primed with heterologous virus or normal control sera died by day 12 after infection (Fig. (Fig.3C).3C). Similar results were observed when mice were given 5-week-primed sera (Fig. 3B and D). Taken together, these results suggested that mice primed with the heterologous viruses did not generate protective cross-reactive antibodies against the novel pandemic H1N1 virus.

FIG. 3.
Immune sera from primed mice were not protective. Mice were infected with PR8 (5 PFU), X31 (3 × 105 EID50), NC/99 (9 × 104 EID50), or CA/E3/09 (30 PFU) virus, and sera were collected 3 and 5 weeks after infection. Serum samples from each ...
Immune sera from primed mice lack neutralization activity against CA/E3/09 virusa

Depletion of T cells dramatically impairs protection.

To further confirm the role of T cells in protection against lethal CA/E3/09 virus infection, we performed CD4, CD8, and CD4-plus-CD8 depletion studies as described in Materials and Methods. To examine the depletion effect, we used CD4 (clone RM4-5) and CD8 (clone 53-6.7) antibodies that are different from the clones used for depletion to avoid potential competing effects. In both the lung and spleen samples examined, 99% of CD8 cells were wiped out, while more than 95% of CD4 cells were depleted (data not shown). Mice primed with X31 virus or without priming were infected with 3,000 PFU of CA/E3/09 virus. As shown in Fig. Fig.2B,2B, without priming, naïve mice continuously lost weight after infection with CA/E3/09 virus, and no mice in this group survived beyond day 12 (Fig. 4A and B). X31-primed mice that received isotype control antibody injection also suffered weight loss, but after day 6 they recovered rapidly, and all the mice survived (Fig. 4A and B). However, protection was severely impaired upon depletion of either CD4 or CD8 T cells, as weight loss of the mice in these two groups was greater than that in the isotype control group and animals did not show signs of recovery until day 8 (Fig. (Fig.4A).4A). After day 7, mice in the CD4 or CD8 depletion group started to gain weight in a similar fashion, but compared to the isotype antibody-treated mice, their recovery was significantly delayed (P < 0.01 for both the CD4 and CD8 groups at days 7, 8, and 9) (Fig. (Fig.4A).4A). Protection was significantly impaired upon depletion of both CD4 and CD8 T cells before challenge. All of the mice in this group continued to lose weight after day 6, and 2 of 5 mice died by day 12 (Fig. 4A and B). Thus, CD4 and CD8 T cells together provided better protection against lethal CA/E3/09 virus infection.

FIG. 4.
CD4 and CD8 cells contributed to protection against CA/E3/09 virus infection. Mice were primed with X31 virus (3 × 105 EID50) and rested for 42 days. Anti-CD4 (GK1.5), anti-CD8 (2.43), both CD4 and CD8 antibodies, or isotype control antibodies ...


The novel pandemic H1N1 virus has emerged and spread rapidly since early 2009. Although a number of cases of severe pulmonary diseases in children and adults with underlying clinical illness were reported, only mild symptoms are experienced by the majority of people, and the overall case fatality rate is not higher than that of regular seasonal influenza virus infection (5, 19).

The observation of a lower infection rate for the 2009 pandemic H1N1 virus in people aged 60 years or older (1, 4) and the prediction and mapping of cross-reactive CD4 and CD8 T-cell epitopes against this pandemic virus in the general healthy population (12, 18, 20, 42) prompted us to establish an animal model of preexisting T-cell immunity against 2009 pandemic H1N1 virus infection.

Heterosubtypic T-cell immunity against influenza is a well-established phenomenon in mice. The prototypic priming and challenge models involve the use of the laboratory strain PR8 H1N1 virus and the reassortant X31 virus, which carries the same six internal proteins as PR8 virus and the HA and NA proteins from the 1968 Hong Kong strain (H3N2) of influenza virus (37). We initially used PR8 H1N1 virus to prime the mice to establish T-cell memory for two major reasons. First, the virus is among the earliest isolated from human influenza virus infections and the protection of aged people from 2009 H1N1 pandemic infection could be due to cross-reactive immunity established after infection with this or a closely related virus circulating in the early 1930s. Second, the PR8 virus has been used widely as a backbone to produce some recombinant human influenza vaccines through reverse genetic techniques, which usually involves the replacement of HA and NA of PR8 virus with those of target viruses that cannot grow well in eggs or cell cultures (41). Vaccination with these recombinant viruses could thus potentially elicit some level of T-cell response that could cross-react with the 2009 H1N1 virus. Establishing an animal model using the PR8 virus for evaluating cross-protection against 2009 pandemic H1N1 virus would provide direct evidence for the assumptions proposed by earlier studies (12, 18, 20, 42).

Previous analysis of human serum samples found neutralizing antibodies against the 2009 pandemic H1N1 virus in people born around 1910, indicating that the 1918 pandemic H1N1 virus is antigenically related to this recent pandemic H1N1 virus (24). Recently, using an animal model, Garcia-Sastre's lab excellently confirmed that antibodies generated after vaccination with 1918-like virus or 1976 swine H1N1 virus could provide complete protection from lethal infection with 2009 H1N1 virus (27). Furthermore, cross-reactive monoclonal antibodies against the HA protein of either 1918 virus or CA/09 virus could offer full protection from death by 2009 pandemic virus infection. The importance of eliciting neutralizing antibody for protection from 2009 pandemic H1N1 virus infection has also been indicated in several other animal studies (6, 31, 32). However, none of these studies carefully examined whether T-cell responses could contribute to the control of 2009 H1N1 influenza virus infection.

Our data reported here also showed that infection with CA/E3/09 virus, but not PR8, X31, or NC/99 virus, could induce potent neutralizing antibody, as demonstrated in vitro and in vivo. Our in vitro microneutralization assay data suggest that neutralizing antibodies are not likely responsible for protection, especially in animals infected with X31 virus, which is a subtype of H3N2. Recent studies indicated that nonneutralizing antibodies, for example, anti-NP antibodies, could also be protective (8). However, mice that received sera from homosubtypic and heterosubtypic virus-infected mice did not show any signs of protection. Very recently, Suphaphiphat et al. also reported that immune sera collected from ferrets infected with their A/CA/04/09 virus stock had undetectable HAI titers against PR8 virus (36). In addition, Skountzou et al. reported that sera of mice infected with different H1N1 viruses isolated between 1930 and 1960 had low levels of neutralization activity (HAI titers) against their A/CA/04/09 virus (34). Thus, our inability to detect cross-reactive HAI activity against CA/E3/09 in PR8-infected sera is essentially comparable with these studies.

It should be pointed out that there are likely some slight variations in antigenicity among the CA/09 stock viruses used in studies from different labs. For example, Skountzou et al. used a CA/09 stock that was mouse adapted after 5 continuous passages, raising the possibility of mutations from the adapting process. Sequencing of our CA/E3/09 virus revealed three mutations in HA (K136N, S200P, and D239G). Due to differences in the numbering system chosen, the S200P mutation corresponds to the S186P mutation in the plasmid F8 genetic mutant CA/09 virus created by Suphaphiphat et al., which has been shown to increase the growth of the virus in either eggs or MDCK cells (36). The single mutation of S to P at position 186 does not change the antigenicity of the virus (36). In addition, the K136N mutation in our stock virus is in the same position as the K119N mutation found in an MDCK cell-adapted CA/07/09 virus (11). The D239G mutation observed in our stock is also the same as the D222G mutation, which results in impaired α2,6-sialic acid (SA) binding and increased recognition of α2,3-SA (11). To our knowledge, these three mutations occurring together has not been described. Given the low-passage history of our stock, it is likely that this variant may have been present in the seed stock. These results indicate that the HA mutations in our stock contribute to the efficient growth of the virus in eggs and improve its pathogenicity in mice, possibly by altering receptor specificity and SA-binding preference, though antigenicity is preserved, as tested by ferret antisera.

Our CD4 and CD8 T-cell depletion experiments with X31-primed mice indicated that T cells are crucial components of heterosubtypic immunity against the 2009 pandemic influenza virus. Furthermore, CD4 and CD8 T cells contributed equally to protection against 2009 pandemic H1N1 infection. These data are in line with our previous studies and other earlier studies showing that both CD4 and CD8 cells can provide protection against influenza virus infection (7, 26, 38-40). Cross-reactive CD8 protection was also illustrated by Skountzou et al. (34) by the use of priming with their CA/09 virus and rechallenge with A/FM/1/47 or A/Aichi/2/68 virus. We did not perform a T-cell depletion study with PR8-primed mice, but since PR8 and X31 share the same six internal proteins that are major targets of T-cell responses, we believe that the protective effect of CD4 and CD8 cells seen in the X31-primed mice should also be operational in PR8-primed mice after rechallenge.

The protection of NC/99 (H1N1)-primed mice from lethal challenge with CA/E3/09 virus is interesting. The NC/99 virus was continuously used in seasonal influenza vaccines for several years. Recent reports of surveillance data indicated that at least partial levels of protection against the recent pandemic H1N1 virus are associated with prior vaccination with seasonal influenza vaccines (16, 25). Since in our hands there was no observed neutralization activity or protection by sera from NC/99-primed mice against CA/E3/09 virus, we speculate that the general population having previously received seasonal influenza vaccines containing NC/99 virus could possibly harbor some level of T-cell immunity that might help to reduce disease severity during 2009 pandemic influenza infection.

In summary, our study provides direct evidence that preexisting T-cell immunity could contribute to protection from 2009 pandemic H1N1 infection. Seasonal influenza vaccination that can induce T-cell immunity would be beneficial for current and future pandemic occurrences.


This work was supported by NIH grants N01-AI50020 and HHSN266200700008C.


[down-pointing small open triangle]Published ahead of print on 27 October 2010.


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