In this study, we evaluated the efficacy of preimmunity to 1918-derived antigens in protecting against 2009 pandemic H1N1 virus in aged mice. Sequence analysis indicated that the HA protein from the 2009 H1N1 pandemic virus is more closely related to the 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 contemporary seasonal HA molecules (73
). Additionally, neutralizing antibodies derived from human survivors of the 1918 pandemic were able to cross-neutralize the 2009 pandemic virus, confirming the antigenic predictions (36
). Although severe seasonal influenza virus infections usually occur in the very young and older age groups, epidemiological evidence has indicated that elderly populations were unusually protected from severe infections during the 2009 H1N1 pandemic. Our group and others have found high levels of cross-reactive antibodies in the older populations, and it is hypothesized that this is due to prior exposure(s) to antigenically similar influenza virus (29
). Furthermore, there is direct evidence of the protective efficacy in mice with prior exposure to 1918-like or classical swine H1N1 influenza virus to 2009 pandemic infection (39
). Cross-protective efficacy of older viruses to 2009 pandemic infection in animal models has provided mechanistic evidence for the observed phenomenon of decreased disease severity in the older human population but has not directly 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 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 challenge in later stages of life.
Age-associated defects in immune responses generally lead to increased susceptibility to infectious disease and decreased responsiveness to vaccines (7
). Furthermore, intrinsic defects within the B cell response directly cause decreased responses to influenza virus vaccine in elderly humans (22
). Although several studies have established a defect in immune responses to vaccination in the elderly (18
), we sought to evaluate the durability and efficacy of immune responses initially elicited in young animals. We found that vaccine responses were produced efficiently in adult mice (A and B) and were robust enough to protect against the highly lethal reconstructed 1918 virus challenge (C and D). The duration of homologous receptor-blocking antibody was evaluated, and titers elicited as adults were maintained throughout the lifespan of the mouse (A). Although the antibody titers tended to decrease with age, these differences were not statistically significant (P
> 0.05). Furthermore, we found not 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 (). Shortly after vaccination, the antibody response is dominated by low-affinity responses produced 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 kinetics 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 lasting and therefore probably not only produced by short-lived plasma cells generated immediately in response to vaccination but 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 antigen and its role in eliciting cross-protective immunity. A more realistic scenario is one that includes multiple exposures, via infection or vaccination, of antigenically distinct viruses over a lifetime, as happens in the human population. Although LLPC would 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 have anti-2009 pandemic cross-reactive antibodies are more likely to be positive for other historic viruses (78
). A second, but not mutually exclusive, possibility is that memory B cells (MBC) may respond to the new antigens, and a cross-reactive epitope(s) could be specifically boosted by sequential exposure. Indeed, the increased 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 avidity in the aged mice supports the hypothesis of LLPC-derived antibody mediating cross-protection. Additional antigen exposures would be predicted to increase the numbers and diversity of LLPC and therefore drive an even more cross-reactive antibody profile. Therefore, the use of a single antigen in our model provides a stringent evaluation of the duration of anti-1918 immunity and its protective efficacy against 2009 pandemic challenge.
Most human influenza viruses require adaptation to cause disease in mice, except for highly pathogenic viruses, including 1918 and H5N1 isolates (3
). The 2009 pandemic virus also readily infects mice, although lethality differences have been reported when comparing multiple virus isolates (39
). The strain used for challenge infections in these studies (A/Mexico/4108/2009) is not lethal to adult mice but does cause significant morbidity, with infected 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 observed, and virus is cleared by 14 dpi (unpublished observations). We found that naïve aged and adult animals had morbidity profiles similar to those of aged animals displaying prolonged signs of disease (A). This could be due to a delayed immune response 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 morbidity in response to the infection even though high levels of virus were recovered from lungs of aged vaccinated animals 4 dpi (C). Although the differences were not significant between the vaccinated groups, the observation was confirmed by immunohistochemistry 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 (). 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 innate immune cells were found in aged and adult animals at 4 dpi, but it is possible that the differential occurs earlier in the infection than was evaluated in these studies. Alternatively, the time from vaccination could also contribute to the differences in viral replication 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 vaccination. Because of the shorter time frame, larger numbers of effector 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 response, we observed greater infiltrates of macrophages, T cells, neutrophils, and IFN-β 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, restriction of viral replication to the bronchial spaces in aged vaccinated mice likely contributed to the less severe disease than that observed in the naïve adult or aged mice. We propose that the lack of prechallenge effector cells in the aged vaccinated animals permits bronchial infection and inflammation, but the high-affinity antibody efficiently restricts the virus from reaching the alveolar spaces. Furthermore, the high-affinity nature of the antibody from aged mice is likely critical in this scenario to overcome the initial bronchial infection and prevent alveolar spread due to the high ratio of virus to antibody. These findings suggest that despite the predominance of bronchiolar infection in this model, alveolar inflammation and/or infection play a greater contribution to the development of morbidity than bronchial infection, which is consistent with postmortem analysis of fatal human cases (25
Influenza virus-specific CD8+
T cells are primarily responsible for the clearance of virus-infected cells after influenza virus infection 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-γ-producing T cells specific to a class I immunodominant peptide are recruited to the site of infection as efficiently in vaccinated aged animals as in adult animals (). Consistent with aging-associated T cell defects, a reduced, albeit not significant, number of IFN-γ-producing cells was found in naïve aged animals compared to that found in the adult controls. In addition to T cell-related age defects, B cells are also impaired (14
). Vaccinated animals in both age groups had equivalent levels of serum antibody prechallenge and similar numbers of antigen-specific antibody-secreting cells detectable in the lungs 6 days postinfection, while unvaccinated animals did not have any detectable antigen-specific antibody-secreting cells (). Interestingly, antigen-specific antibody-secreting cells were not detectable in any group prior to challenge (data not shown). The rapid recall of both T and B cells in the lungs of vaccinated animals indicates that the adaptive immune response that was primed in young animals is maintained late in life, even though the ability to respond to new 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
). 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. Consistent with published results, young mice (<10 weeks of age) 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 (C). Interestingly, adult serum recipients lost less weight and had reduced morbidity compared to aged serum recipients. This was not due to differences in administered serum antibody titer, as adult and aged serum had equivalent levels of both total anti-HA antibody and receptor-blocking antibody. Furthermore, the avidity of serum from aged mice for HA had a slower dissociation rate than the serum from adult mice, demonstrating that the antibody remained bound longer to HA protein. The IgG subclass profile indicated that although the adult and aged animals had equivalent levels of the dominant isotypes (IgG1, IgG2a
, and IgG2b
), the adult mice had low but increased amounts of IgG3
compared to those of the aged mice (D). IgG3
is a minor fraction of antibody to T-cell-dependent antigens and is the major isotype to T cell-independent antigens (50
). Additionally, IgG3
is a potent activator of the classical complement pathway, likely due to the properties of cooperative binding (17
). Enhanced complement fixation mediated by the HA-specific IgG3
in adult sera could be a mechanism responsible for the decreased morbidity observed in the recipient mice. It is interesting to speculate that the loss of IgG3
in 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 because of the shorter kinetics of the vaccine regimen. In addition to IgG3
variability, non-HA antibodies could also explain the observed morbidity differences. Antibodies to the NA protein of the 2009 pandemic virus are increased in elderly humans (40
), and the protective role of anti-NA immunity cannot be excluded in this study. The finding that directly vaccinated animals did not display any signs of morbidity implies a critical protective role for the cellular component of the immune response in addition to the cross-reactive receptor-blocking antibodies. Cellular immunity in the presence of protective antibodies is also more predictive of protection in the context of highly pathogenic 1918 influenza virus challenge (52
). Therefore, although serum antibody is sufficient to protect from severe disease and death, cellular immune responses in both the T and B cell compartments likely contribute 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
). 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 budding properties of the Gag protein (30
). Lifelong antibody responses are more efficiently produced by virus infection than by 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 function of an adjuvant. These studies were performed in mice and evaluated only a single HA antigen; however, the findings reported here indicate that VLP-based vaccinations are capable of eliciting lifelong immunity, as measured by both serum antibody () and cellular recall to infection ().
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 and 2009 have been demonstrated at the structural level, indicated by human data and confirmed in adult-animal models (29
). 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 human disease. The lifelong immunity evaluated in these studies was 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-reactive antibody responses to 2009 pandemic influenza virus and expands those findings to aged animals, further validating the hypothesis that decreased disease severity in the elderly human population observed during the 2009 H1N1 pandemic may be due to prior exposure to antigenically similar viruses.