We evaluated a novel multiple-clade reverse genetics–derived inactivated whole-virus H5N1 influenza vaccine and three single-clade vaccines in the ferret model by comparing their immunogenicity, cross reactivity, and protective efficacy. The multiple-clade adjuvanted vaccine could be useful in allowing timely initiation of vaccination against an unknown pandemic virus. All vaccines protected ferrets against lethal challenge with A/VN/1203/04 virus and markedly reduced clinical disease signs and virus replication in the upper respiratory tract after non-lethal challenge with A/JWE/HK/1038/06 or A/WS/MG/244/05 virus. Two doses of vaccine were required to substantially increase HI and virus neutralizing antibody titers to both homologous and heterologous H5N1 viruses. Inclusion of MF59 adjuvant increased antibody titers to both homologous and heterologous viruses. Importantly, we found that two doses of the adjuvanted single-component rg-A/VN/1203/04 vaccine, the H5N1 vaccine that is stockpiled in various parts of the world, was most effective in reducing the upper respiratory tract virus load after both homologous and cross-clade challenge. Our results strongly support the previous observation of greater antibody induction by two doses of inactivated whole-virus rg-A/HK/213/03 (H5N1) vaccine in ferrets [19
] and the results of clinical studies of adjuvanted, inactivated subunit, or split-virion vaccines (rg-A/VN/1203/04 or rg-A/VN/1194/04) in adults [11
A feature highly desirable in a pandemic influenza vaccine is the ability to induce cross-reactive immune responses sufficient to protect against variants that have undergone antigenic drift. We assessed the immunogenicity and cross reactivity of H5N1 influenza vaccines representing different HA clades/subclades as well as the immune response induced by a multiple-clade H5N1 vaccine. Interestingly, the highest serum antibody titers induced by the vaccines were those against the A/HK/213/03 HA antigen, suggesting that A/HK/213/03 may be a more immunogenic antigen than other H5N1 viruses. Alternatively, the sensitivity of the HI assay may be enhanced by the arginine found at residue 223 of the A/HK/213/03 HA1 subunit [28
]. The immune correlates of protection remain poorly defined for H5N1 influenza vaccines and may differ from those established for seasonal influenza vaccines. Concerning overall cross reactivity, the multiple-clade vaccine induced serum antibody titers to homologous viruses that were almost as high as those induced by single-clade vaccines (in the case of rg-A/JWE/HK/1038/06 vaccine, higher). Three single-clade vaccines also induced cross-reactive immunity to all four antigens, but antibody titers to heterologous viruses were lower. It is worthy of note that the addition of MF59 adjuvant induced a stronger cross-reactive immunity as compared to nonadjuvanted vaccines. Interestingly, titers against the A/HK/213/03 and A/JWE/HK/1038/06 antigens were not proportional to the lower dose in ferrets that received the multiple-clade vaccine vs. the single-clade vaccines, possibly reflecting a reaction between antigens that increased the vaccine’s immunogenicity.
Preclinical animal studies are useful for evaluation of the protective efficacy of vaccines against highly pathogenic influenza A (H5N1) viruses. Importantly, all vaccinated animals in the present study were protected against death after challenge with a high dose (106 EID50) of lethal A/VN/1203/04 (H5N1) virus and were protected against morbidity after a similar dose of non-lethal A/WS/MG/244/05 or A/JWE/HK/1038/06 H5N1 virus (ours is the first study to our knowledge to examine A/JWE/HK/1038/06 virus infection in the ferret model). Rg-A/VN/1203/04 vaccine offered the best protective efficacy and provided substantial cross reactivity and immunogenicity, although it induced lower serum antibody titers than the multiple-clade vaccine. Further, nonadjuvanted rg-A/VN/1203/04 vaccine protected against lethal challenge with homologous virus, despite inducing a relatively low antibody titer to that virus; conversely, we must consider that rg-A/VN/1203/04 was the only vaccine evaluated that was homologous to lethal challenge virus.
In some instances, ferrets were protected from morbidity or lethality of challenge despite relatively low serum antibody titers (i.e. ferrets receiving nonadjuvanted vaccines then challenged with A/VN/1203/04), which raises the question of whether HI and VN assays, both of which primarily detect antibodies to influenza virus HA, are adequate measures of a protective antibody response. We must comment that the use of CRBC for the estimation of serum antibody responses against avian H5N1 influenza viruses may be a limitation of the current study, and the use of horse RBC may increase the level of sensitivity. However, the possibility that both humoral and cellular immune responses play a role in protection of ferrets against influenza cannot be ruled out. This possibility is particularly valid in the case of whole-virion vaccines, which (unlike subunit vaccines) contain internal conserved proteins targeted by cell-mediated immune responses. Little is currently known about the cellular immune response in ferrets, as the necessary reagents are unavailable. In the mouse model, in which the role of T-cell immunity to influenza virus is known [29
], MF59 adjuvant enhanced T-cell responses to a trivalent influenza vaccine significantly more than numerous other adjuvants [30
]. In humans, the level of cellular immune response is positively correlated with protection from influenza [31
]. Clinical trials have showed that adjuvanted vaccines for hepatitis B and malaria induce cellular immune responses that may contribute to protection [33
Whole-virus H5N1 vaccines can reduce the amount of antigen required to induce an adequate immune response [35
], as can adjuvants. These considerations are important in view of the likely gap between vaccine production and demand during an influenza pandemic. Because of safety concerns, MF59 and aluminum salts are the only vaccine adjuvants approved for use in humans [21
]. Aluminum salts do not always enhance immunity to split H5N1 influenza vaccines in humans [36
], whereas MF59 had an excellent safety profile and enhanced the immunogenicity of split H5 vaccines in clinical trials [23
]. In our study, one dose of whole-virus H5N1 vaccine with MF59 adjuvant increased HI titers to homologous virus, and two doses increased virus neutralizing titers as much as 30 times more than nonadjuvanted vaccine. Additionally, MF59-adjuvanted vaccines induced cross-reactivity, prevented fever, and reduced URT viral load to a greater extent than nonadjuvanted vaccines. Similarly, in a recent preclinical study by Baras et al [39
], two doses of an H5N1 split vaccine with a proprietary oil-in-water emulsion–based adjuvant increased cross- reactivity in ferrets and decreased URT virus shedding. In our study, rg-A/VN/1203/04 was the vaccine that most effectively reduced the URT viral load and URT shedding of all challenge viruses. This information is noteworthy, as URT shedding is the main method of transmission of influenza virus, and a decrease in the URT viral load may suppress transmission. Reduction of virus shedding appeared to depend on the overall immunogenicity of the vaccine and the homology of vaccine to the challenge virus. MF59 adjuvant helped to reduce the severity of clinical signs after non-homologous challenge, but it remains unclear whether adjuvant reduces the URT viral load in ferrets when used with naturally highly immunogenic vaccines. The rg-A/VN/1203/04 vaccine, which is currently the H5N1 vaccine approved for human use, was also the vaccine that most effectively reduced the incidence of infection as determined by seroconversion.
At least 6 months are likely to elapse between the start of an influenza pandemic and the initial availability of a strain-specific vaccine. Stockpiling of a broadly cross-reactive vaccine could help to ensure the interim availability of an alternative vaccine until an antigenically matched vaccine becomes available. Further, after receiving a cross-reactive H5N1 influenza vaccine, individuals may require only a single dose of the strain-specific vaccine, as the result of cross-clade priming [4
]. Clinical trials have shown that a protective antibody response to H5N1 viruses can be induced by priming and boosting with A/DK/Singapore/97 (H5N3) vaccine [23
] or by boosting with A/VN/1203/04 after priming with a heterologous H5N1 virus [2
In summary, our results showed that a multiple-clade H5N1 influenza vaccine can provide protective cross-clade immunity. Such a vaccine may elicit protection against a broader range of viruses than a single-clade vaccine. If single vaccines of different clades are stockpiled, it may be possible in the event of a pandemic to mix various single vaccines to create a cross-clade protective vaccine for use until an antigenically matched vaccine is available. Our findings also suggest that a heterologous vaccine can offer protection, mainly if adjuvanted with MF59. Importantly, the rg-A/VN/1203/04 vaccine administered as described provided immunogenicity, cross reactivity, and protective efficacy comparable to that of the multiple-clade vaccine. In the future, it would beneficial to compare single- and multiple-clade vaccines based on other H5N1 vaccine strains and challenge viruses to determine if multiple-clade H5N1 vaccines repeatedly offer advantages comparable to a single-clade vaccine. Use of the MF59 adjuvant considerably increased clade-specific and cross-clade antibody responses to all vaccines, an effect that may help to spare antigen in the event of a pandemic. The fact that in some instances, such as with the A/VN/1203/04 challenge/nonadjuvanted vaccine, protection from mortality was provided despite relatively low serum antibody titers, suggests that the role of cellular immunity in this model should also be investigated.