In this study we report that without adjuvant, H5 HA proteins are poorly immunogenic in H5 naïve C57BL/6 mice. In our system, substantial antibody responses can be mounted when the response is adequately boosted. The need for a booster shot is not unlike the recommendations for seasonal vaccination of infants receiving their first influenza vaccination or adults receiving a pandemic vaccination [31
]. However, the immunogenicity of avian H5 HA in humans and other animal models has been reported to be weak in comparison to other serotypes of influenza HA proteins [23
]. The reasons for this are not clear, but baculovirus-derived rHA have been shown to be effective immunogens in human subjects [24
]. These differences in immunogenicity between subtypes of rHAs in human subjects could reflect a past history of infection, or lack of thereof in the case of pandemic strains, with the various serotypes of influenza virus. However, the IgG titers generated following our homologous vaccination strategy are comparable to what has been previously reported in other adjuvanted vaccine-boost models with rH5 and rH3 [26
]. Interestingly, the dose of rHA use in our system is between 3 and 20 fold lower than that administered by other groups. However, a direct and comprehensive comparison of the immunogenicity of baculovirus produced full-length rHAs of various subtypes in mice is not currently available. This makes it difficult to determine if rH5 HAs are intrinsically less immunogenic than other rHAs or if other factors such as virus-priming and/or previous vaccination can influence the immunogenicity of rHAs upon vaccination. Indeed, the data presented here suggests that prior vaccination with H5IN05 primes the humoral arm of the immune system to respond to closely related drifted HA proteins in a magnitude that is comparable to homologous vaccination. However, this result is in the context of vaccination with the drifted variant two weeks after boosting with H5IN05. The factors involved in this type of response could very well be time dependent, thus the length of time between priming with H5IN05 and the drifted variant administration merits further investigation. It is worth noting that human subjects retain the ability to respond to a drifted variant with “boost-like” antibody and cellular response years after priming [20
], with recent evidence that longer intervals between immunizations may enhance the response [37
Evaluation of the antibody response in mice vaccinated with 3 doses of H5VN04 revealed antibody cross-reactivity to H5IN05, but not rH1 or rH3 HAs, in an ELISA based assay. However, very little cross-reactivity to H5IN05 was observed when testing the same serum in a competitive ELISA. This phenomenon can be replicated with serum from mice receiving a homologous vaccine containing H5IN05. One possible explanation is that the serum generated by H5VN04 vaccination is indeed cross-reactive to the drift variant HA, but it has a higher affinity to the immunizing antigen. Thus, it is possible that the antibodies bound to H5IN05 dissociate in the presence of H5VN04 resulting in binding to the homologous protein rather that the drifted variant.
Administration of the drifted variant strategy elicited a subpopulation of H5IN05 specific IgG that cross-reacted with the clade 1 H5VN04 variant. More importantly, this increase in reactivity against the clade 1 variant was not at the expense of reactivity to H5IN05 (compare H5VN04 results in ). However, whether this increased reactivity to the drifted translates to broadened antibody specificity against other 3rd
party rH5 HAs and /or H5 avian virus is unknown. In addition, despite the high H5VN04 antibody titers, we observed low incidence of neutralizing antibodies against the ΔH5xPR8 virus generated following one dose of H5VN04 in mice previously immunized with H5IN05 (). Indeed, other groups have reported low antibody neutralizing activity following administration of one dose of experimental H5N1 vaccines in mice [28
]. Nevertheless, protection from viral challenge in the absence of a detectable neutralizing antibody response has been observed. [28
]. However, while the efficiency of influenza vaccination in humans is measured by the ability of the vaccine to induce a neutralizing antibody titer equal or greater than 1:40 [29
], it is not clear how appropriate this measurement is as a correlate for protection in animal models. In our studies in which immune serum was diluted below 1:40, protection was diminished, suggesting this correlate is sound. Nevertheless, previous vaccination with H5IN05 provides little to no advantage in the induction of neutralizing antibodies following one dose of the drifted H5VN04. Moreover, because our experimental system is currently limited to measuring MN activity to ΔH5xPR8 we do not know if this cross-clade antibody reactivity comes at the expense of the neutralizing antibodies to H5IN05. Indeed, recently published work [39
] has shown that prime-boost vaccination with inactivated viruses, containing drifted H5 HAs, increases cross-clade neutralizing antibody reactivity at the expense of the magnitude of the neutralizing response to the priming virus. It is worth noting that, contrary to the observations made by Sabarth et al. [39
], we observed minimal neutralizing activity against a virus containing the drifted variant H5VN04. Neuraminidase dependent enhancement of the HA-specific response has been recently reported by Bosch et al. [40
]. It is possible that the different results on virus neutralization relate to the presence, or absence, of the viral neuraminidase in the vaccine preparation. We are currently investigating how these results relate to rH5 HA drift variant vaccination.
As previously stated, protection from viral challenge in the absence of neutralizing antibodies has been observed following vaccination [38
]. Here we observe that both homologous and drift variant vaccination generates an H5 HA specific immune response that is capable of reducing morbidity upon viral challenge even in the absence of high neutralizing antibody titers. The observation of substantial immune protection in the absence of neutralizing antibody titers suggests the possibility of other immune mechanisms operating to control infection. More importantly, these mechanisms of control of infection potentially appear to play an important role early (>7 days) after challenge. It is possible that protection from morbidity is correlated to the total circulating H5VN04-specific antibody titers. Indeed, passive transfer of immune sera confirmed that the antibodies generated by drift variant vaccination are sufficient for protection, from virus induced morbidity and death, regardless of the antibody MN activity (). Interestingly, serum containing high MN activity failed to protect mice from morbidity and death when diluted 8-fold ( open squares). A possible explanation for this result is that because the dilution of the MN activity also results in the dilution of the total non-neutralizing H5VN04 specific antibody it effectively eliminates the protective activity provided by these antibodies. Mechanisms for protection by non-neutralizing antibodies have been previously described for influenza virus [41
]. However, the possibility of a threshold or balance between antibodies, with and without MN activity, which promotes protection is both novel and intriguing. These data supports the notion that influenza vaccines that do not induce high titers of neutralizing antibodies can provide protection. However, the degree of protection offered by these vaccines may vary in comparison to those that induce neutralizing antibodies. Another possibility is that H5 HA specific memory T cell responses play a role in reducing virus induced morbidity effects in vaccinated mice. However, this route of vaccine administration is not associated with the induction of lung-resident memory T cells. Regardless, in spite of the poor incidence of neutralizing antibodies, prior vaccination may provide a clinical benefit.
These studies support the idea of pre-vaccination as a viable pandemic control strategy. However, one major concern to this strategy is that vaccination with antigenically distinct variants will result in a misdirected antibody response primarily focused against the priming antigen [17
]. This phenomenon, termed “Original Antigenic Sin”, has been demonstrated in the context of live influenza infection, but its role in experimental influenza vaccines is still under investigation. Kim et al. [19
] reported that sequential vaccination with inactivated virus resulted in a significant decreased in the development of protective immunity and recall responses to the virus containing the drifted HA with minimal effects of OAS. In addition, the group reported that sequential vaccination with DNA encoding drifted HAs induced significant responses directed to the priming antigen at the expense of the response to the drifted HA. However, in vivo
protection was not accessed after DNA vaccination. In contrast, following our vaccination strategy we did not observe differences in the recall responses between mice receiving a homologous vaccine and those receiving a drift variant vaccine. The difference in results may be attributed to different vaccination strategies (DNA vs. rHA), the correlate of protection measured, the subtype of HA used (H1 vs. H5), and/or the antigenic distance between hemagglutinin pairs. We believe that our observation lends support to the idea that the Original Antigenic Sin may not apply to protein vaccination [44
]. However, it is currently unknown how vaccination with drifted variants affects the response to an infection with a 3rd
party virus containing an HA related to those used for immunization.
This study further supports the observations reported in experimental human vaccine trials where B cell responses and serum HAI and MN activity [20
] suggest advantages to pre-pandemic vaccination. More importantly, the advantages of H5 pre-vaccination are not restricted to the H5 HA pair previously described [20
]. Pre-vaccination with stockpiled H5 vaccines can potentially induce baseline immunity to H5 that, in the event of a pandemic, could lower the dose requirement of vaccination while providing significant protection against the emerging drifted H5 strain.
- H5VN04 and H5IN05 rHA are used in drifted variant vaccination strategy.
- Vaccine tunes reactivity to drifted variant without loss of reactivity to priming HA.
- Low incidence of neutralizing Ig generated to a virus containing the drifted HA.
- HA-specific cross-reactive, but not neutralizing, Ig are sufficient for protection.