In the current study, we used the licensed influenza vaccine against the H1 and H3 serotypes as a model system to study the immunogenicity of an inactivated influenza virus vaccine in animals that were sero-negative to influenza virus antigens. Then, we compared the relative levels of protective antibodies between homologous (DNA + DNA or TIV + TIV) and heterologous (DNA + TIV) prime-boost vaccination strategies. In this study, New Zealand White rabbits were used to study the immunogenicity of various vaccination regimens. In many previous studies, mouse models were frequently used with the potential benefit to observe the protective effect of vaccines when immunized mice are subject to a lethal influenza virus challenge. On the other hand, achieving protection in mouse models is relatively easy even when the levels of protective antibodies were below detection prior to challenge [22
]. At the same time, each mouse can only provide a limited amount of blood which does not allow for the extensive array of antibody assays used to characterize different antibody responses. Rabbit is a widely used animal model for the study of vaccine immunogenicity due to the fact that rabbits are highly immunogenic in that they produce high-titer antigen-specific antibodies and a large amount of blood can be obtained for various assays, as we have recently demonstrated [18
]. While there is limited experience in using rabbit model for challenge studies, functional antibody analyses provide surrogate markers for the protection efficacy of influenza vaccines. For example, the levels of increase in HI antibody responses are used as part of the licensing criteria for human influenza vaccines. Using rabbit immune sera in the current study, we were able to conduct various binding and functional antibody assays to compare the relative immunogenicity among different influenza vaccination regimens.
In current report, we have shown that the clinically licensed trivalent inactivated influenza virus vaccine (TIV) has poor immunogenicity in naïve animal hosts. Furthermore, HA-specific antibodies and functional antibodies, as measured by HI and MN assays, were even lower than in HA DNA vaccinated animals when the same two-dose immunization schedule at Weeks 0 and 4 was employed. Interestingly, the heterologous DNA prime and TIV boost elicited significantly higher levels of both binding and protective antibodies when compared to homologous prime-boost immunization regimens that used either the TIV or DNA vaccines alone. The effect of this heterologous prime-boost approach is long-lasting and was still the most effective approach when the immunized hosts received another TIV boost vaccination approximately 6 months later. On the other hand, one time co-delivery of DNA and TIV, with the same amount of two vaccines as that used in the regular sequential DNA-TIV regimen, could not achieve the same levels of antibody responses elicited by the DNA-TIV. Even with two immunizations, the co-delivery approach was not more effective in eliciting antibody responses when compared to the sequential prime-boost regimen despite the fact that the co-delivery approach resulted in the administration of 2 times the dose given to animals in the sequential prime-boost group. Putting these data together, the sequential approach has clear immunologic advantage that can not be replaced by the homologous prime-boost or co-delivery of both types of vaccines.
New mutant strains of seasonal influenza H1 and H3 serotypes continue to emerge and the worldwide spread of the highly pathogenic avian influenza H5 serotype has raised concern over the next influenza pandemic. Administration of the currently licensed TIV has been effective in reducing the morbidity and mortality associated with seasonal influenza virus infections in humans with only one injection [23
]. This was accomplished most likely due to a priming effect on the immune system as a result of previous vaccination and/or to previous exposure to related influenza virus serotypes. However, in the event of a pandemic influenza virus of the H5N1 serotype, a pre-existing immunity would be lacking in the majority of the population as would any previous vaccination. This, in conjunction with recent literature suggesting that two immunizations are needed for an inactivated H5 avian influenza vaccine to be effective in eliciting protective levels of antibody responses [13
], would require that an unprecedented number of vaccine doses be produced. While a number of strategies have been tested to increase the immunogenicity of inactivated H5 avian influenza vaccines, including higher antigen doses and the inclusion of an adjuvant [16
], this H5 inactivated vaccine still requires two immunizations in order to be protective. Furthermore, the need for higher doses and/or additional components (i.e., adjuvants) will add an additional burden to a complicated influenza vaccine production process and will further impact an already insufficient influenza vaccine production capacity throughout the world. Therefore, additional novel strategies are needed to prepare the world against a potential pandemic avian influenza virus infection.
While the current study was not designed to conduct an in-depth immunological analysis on the mechanism(s) of why a heterologous prime-boost regimen would be more effective than a homologous prime-boost, our results indicate that the DNA prime may be more effective than the TIV in priming the immune system. This hypothesis is partially supported by our current results showing that a prime-boost regimen with the DNA vaccine alone is more immunogenic than the TIV alone prime-boost approach. It is possible that DNA immunization is more effective in eliciting better and potentially longer lasting HA-specific B cell memory. Well established literature on B cell immunology suggests that low dose antigen delivery is more effective in eliciting better antibody responses and B cell memory [24
]. If a DNA vaccine is truly effective in priming a longer lasting B cell memory, then this technology can be very useful for the overall strategy of avian influenza vaccine development against a potential pandemic.
A key challenge facing avian influenza vaccine developers is the lack of sufficient manufacturing facilities in the world. With such limited capacity, the ability to produce enough doses of an avian influenza vaccine at the time of outbreak of a pandemic within a narrow window of time would be extremely difficult, if not impossible. Stockpiling of vaccine can relieve some of this pressure but the world’s stockpiling capacity is also limited as the shelf life of past seasonal influenza vaccines has been short, partially due to the need to modify influenza vaccine formulations to correspond to the predicted circulating viruses for that particular season. In the case of avian influenza, it is already known that H5N1 avian influenza viruses already have several different genetic clades based on its evolution in the last 10 years. At this point, nobody can predict which strain of H5 virus will be the source of a potential pandemic which makes stockpiling vaccines very difficult.
Results from the current report provide a more attractive solution. DNA vaccines expressing different H5 HA antigens can be produced and administered to the population in order to prime the immune system before an influenza pandemic. This will allow for a strong recall protective antibody response upon the administration of an inactivated H5 avian influenza vaccine boost which can be produced at the time of pandemic outbreak. Since the currently licensed inactivated avian influenza vaccine requires two doses in order to elicit an immune response, DNA immunization will reduce the number of inactivated avian influenza virus vaccine doses by at least half. Production of a DNA vaccine is simple and has a relatively low cost, particularly if the DNA vaccine can be delivered by a highly effective delivery method as was recently shown using a gene gun device to elicit protective antibody responses in humans [26
]. Multiple DNA vaccines expressing several different HA antigens from the H5 serotype can be injected at the same time to serve as a prime for broad protection once boosted with an inactivated influenza virus vaccine matching with the pandemic strain. However, practical issues need to be considered for including a DNA prime. For example, how to clearly label the prime and boost vaccines so there will be no confusion on which one should be used first. However, this should not be a problem for pre-pandemic avian flu immunization since the DNA prime immunization will be given once and the boost is only needed at the time of pandemic outbreak. Future studies should analyze the immunogenicity and protection potential a DNA prime-inactivated vaccine boost strategy against H5N1 viruses in both animal model and human populations.