Two doses of influenza vaccine given four weeks apart are recommended to induce protective immunity particularly for children, who are more likely to be immunologically naïve. The majority of the population is also immunologically naïve to the HA glycoprotein antigens from pandemic potential H5N1 influenza viruses. It would therefore be highly desirable to develop an effective single dose vaccination. In this study, for the first time, we have investigated protective immune correlates including the detailed kinetics of antibody induction, protective potential, and the generation of long-lived plasma and memory B cell immune responses after a single dose intramuscular vaccination with H5 VLPs containing the wild type or mutant H5 HA, or a subunit vaccine consisting of recombinant H5 HA protein. H5 HA VLPs were found to be superior to the recombinant H5 HA protein in inducing binding and functional antibodies, and in providing protective immunity as well as in generating plasma and memory B cell responses.
Many influenza vaccine strains especially for vaccines against highly pathogenic avian influenza viruses are generated as reassortants using reverse genetics technology (Govorkova et al., 2006
; Lipatov et al., 2006
; Nicolson et al., 2005
; Subbarao et al., 2003
; Webby et al., 2004
). The HA genes in the HPAI vaccines are modified to remove the highly cleavable multibasic amino acid motif, a known virulence determinant. However, the effect of the presence of this multibasic amino acid motif in the WT H5 HA on inducing protective immune responses has not been well studied. We produced VLPs containing either WT HA or a deletion mutant HA, lacking the multibasic sequence, and compared their immunogenicities. The mutant form of HA presented in an uncleaved precursor conformation on VLPs induced comparable immune responses as the WT form of HA presented in an processed conformation on VLPs and possibly provided superior protection as evidenced by effective virus clearance. Thus, this study provides first evidence supporting the rationale for generating pandemic vaccines with a mutated HA cleavage site.
Vaccination consisting of formalin-inactivated whole virus particles that were first licensed in 1945 in the U.S. was often associated with a relatively high incidence of adverse events particularly among children (Nicholson et al., 1979
; Wright et al., 1977
). Since then, detergent split or ether-disrupted vaccines or purified viral subunit HA and NA proteins have been more commonly used for intramuscular vaccination. Influenza VLPs have been suggested as a promising alternative candidate to influenza vaccines against highly pathogenic avian strains (Bright et al., 2008
; Haynes et al., 2009
; Kang et al., 2009
). Also, Bright et al demonstrated that seasonal influenza VLPs induced broader immune responses compared to inactivated whole influenza virus vaccine (Bright et al., 2007
). In these previous studies, prime and boost immunizations were applied for inducing protective immune responses against avian influenza viruses, which is a common vaccination regimen in using animal models. Considering that most individuals in the population are naïve to avian or pandemic potential strains, protection with a single dose vaccination in a naïve immune condition would have significant implications for public health. The present study provides insight into the kinetics and types of immune responses contributing to protection after a single dose VLP vaccination as well as potential mechanisms explaining low immunogenicity of soluble subunit vaccine.
Compared to the recombinant H5 HA vaccine, the H5 VLPs induced significantly higher levels of IgG antibody responses with faster kinetics. Vaccination with the recombinant H5 HA protein showed a delayed type of antibody responses until day 28 post vaccination. Also, the IgG1 antibody was found to be a major isotype, which was observed in the high dose H5 HA group (2 μg HA) only at a later time point. In contrast, both low and high doses of H5 VLP vaccines showed induction of IgG2a and IgG2b isotypes as early as day 8 post-vaccination. These observations suggest that the VLP vaccines stimulate the host immune system to initiate adaptive immunity in a different mechanism compared to that of the recombinant antigens. In supporting this idea, we recently observed that influenza VLPs were immunogenic even in CD4 T cell deficient mice, whereas, the recombinant HA protein was not able to induce antibody responses in the absence of CD4 T cells (Kang et al., unpublished data). Therefore, vaccines designed to mimic the virion structure as is the case with VLPs provide an approach to improve vaccine efficacy while retaining the safety of a recombinant protein vaccine.
The type of the immune response induced by vaccines is also important for protection. Infection by influenza virus or other viruses induces IgG2a dominantly in mice (IgG2c in C57BL/6 mice) (Coutelier et al., 1987
; Fazekas et al., 1994
). IgG2a antibody interacts efficiently with complement and Fc receptors by virtue of its Fc domain properties (Gessner et al., 1998
; Heusser, Anderson, and Grey, 1977
; Huber et al., 2006
; Neuberger and Rajewsky, 1981
). Therefore, induction of IgG2a antibody by VLP vaccination is considered to significantly contribute to viral clearance, probably via activation of the complement system, stimulation of antibody-dependent cellular cytoxicity and clearance of opsonized virus by macrophages (Huber et al., 2001
; Jayasekera, Moseman, and Carroll, 2007
; Mozdzanowska et al., 2006
). The protective role of IgG2a antibody is further supported by a recent study demonstrating that IgG2a induced by vaccination with viral replicon particles was significantly more effective in protection with enhanced viral clearance against lethal challenge than IgG1 antibodies induced by DNA vaccination (Huber et al., 2006
). Consistent with these previous studies, the recombinant H5 HA vaccine group which induced predominantly an IgG1 isotype response at a similar level to the high dose VLP group was not effective in controlling viral replication as this group showed lung viral titers as high as the naïve control challenge group. In contrast, H5 VLP immunized mice that were capable of inducing mainly IgG2a isotype had significantly lower lung viral titers by more than one hundred fold to below the detection limit.
Regarding the T cell responses, previous studies provided evidence that Th1 cells are superior to Th2 cells in providing protection against viral infection by secreting IFN- γ, stimulating B cells, and directing CD8+ T cell mediated cytolysis (Bot, Bot, and Bona, 1998
; Moran et al., 1999
; Swain et al., 2006
; Thomas et al., 2006
). VLP vaccination was found to be more effective in generating T cells secreting INF- γ indicating Th1 responses. In contrast, recombinant HA vaccination induced T cells secreting IL-4 cytokine representing Th2 type responses, which was not a protective response since all mice in this group died after challenge. One or two immunizations with the H5 HA protein (3 μg HA, A/Indonesia/05/2005) were reported to induce protection against lethal infection although mice showed a loss in body weight and protective immune correlates were not identified (Bright et al., 2008
). There are many factors which could be involved in different results including the vaccine strain (A/Vietnam vs A/Indonesia) and dose, challenge virus strain (wild type vs reassortant) and dose (50LD50
), and timing of challenge infection (6 months vs 5 weeks post vaccination). Nevertheless, the current study clearly indicates that influenza VLPs are an advantageous format of vaccine inducing predominantly IgG2a isotype antibodies as well as Th1 type cytokine such as IFN- γ, both of which are contributing to inducing protective immunity.
Long-term serum antibodies are maintained by plasma cells residing in the bone marrow and spontaneously secreting antibodies into the bloodstream (Slifka and Ahmed, 1996
; Slifka et al., 1998
; Slifka, Matloubian, and Ahmed, 1995
). During the differentiation and development of B cells in the germinal centers upon antigen exposure, memory B cells are generated (Crotty et al., 2003
; Kalia et al., 2006
). Previously, prime and boost intranasal immunizations with H5 VLPs were shown to induce plasma and memory B cells in mice (Kang et al., 2009
). In the present study to better understand the long-lasting protective immunity after a single intramuscular vaccination, the recombinant HA protein and VLP vaccines were compared. We observed that the recombinant H5 protein vaccine exhibited a significant limitation in generating both plasma cells in bone marrow and rapidly responding memory B cells in spleen compared to the VLP vaccine. In clinical studies, high dose and prime-boost immunizations with recombinant or conventional H5 vaccines were required, indicating their low immunogenicity (Bresson et al., 2006
; Lin et al., 2006
; Nicholson et al., 2001
; Treanor et al., 2006
; Treanor et al., 2001
). Our study provides evidence for superior immunogenicity of influenza VLPs to recombinant H5 protein based subunit vaccine. It is speculated that influenza VLPs are an effective immunogen in stimulating innate immune cells particularly dendritic cells, a potent antigen presenting cells. A vaccine form capable of strongly activating pathogen recognition receptors such as Toll-like receptors on dendritic cells has been suggested to effectively induce adaptive immunity including Th1 type immune responses (Pulendran, 2004
; Pulendran and Ahmed, 2006
). Viruses themselves are known to induce strong B cell and cytotoxic T cell responses, while purified recombinant antigens require an adjuvant to induce cellular immune responses (Bachmann, Zinkernagel, and Oxenius, 1998
). Thus, alternatively, VLPs mimic viruses in structure and morphology, and present HA molecules in a highly structured repetitive array, which might allow efficient cross-linking of B cell receptors (Bachmann and Zinkernagel, 1996
) However, the underlying mechanisms remain to be determined. Therefore, VLPs appear to be a promising approach for developing a vaccine that effectively generates memory B cells that rapidly differentiate into antibody secreting plasma cells upon antigenic stimulation, thus, contributing to long-term protective immunity.
In conclusion, a single intramuscular administration of H5 VLPs but not of a recombinant H5 HA protein, provided complete protection against lethal challenge infection. The H5 VLP vaccine was found to be more effective in inducing Th1 type immune responses including IgG2a and IFN- γ cytokine-secreting T cells as well as in generating plasma and memory B cells contributing to protection after a single intramuscular vaccination. Therefore, influenza VLPs will offer significant advantages for vaccination particularly if a pandemic occurs. Considering the current platforms of approved vaccines, it is important to investigate detailed comparative immunogenicity and protective efficacy of H5 VLPs in comparison with inactivated whole virus and attenuated live H5N1 vaccines.