We have investigated detailed immune responses induced by influenza VLPs, including antibody isotypes, neutralizing activity, cellular immune responses, and induction of memory responses, which have not been investigated previously. In addition, our findings provide evidence that influenza VLPs containing HA, but not HA-negative M1 VLPs, can induce protective immunity against a lethal virus challenge with homologous as well as heterologous virus strains.
Immunization with recombinant influenza virus HA proteins was previously demonstrated to afford protection in chickens against challenge infection (5
). However, preparing HA proteins with high purity as a vaccine candidate on a large scale may require a high-cost manufacturing process. In this regard, the production and purification processes of influenza VLPs in insect cells can be relatively simple and easily scalable. Insect cells do not add sialic acids to the N-glycans during posttranslational modifications (16
), which explains why VLPs with HA are effectively released from the insect cell surfaces in this and other studies (8
). Nonetheless, it will be interesting to determine the effect of neuraminidase coexpression on VLP budding and yield in the insect cells. In addition, incorporating an additional component, neuraminidase, into VLPs would be an advantage for an influenza virus vaccine, although VLPs containing influenza virus HA and M were found to be effective in inducing protective immune responses in the absence of adjuvants.
Maintaining the VLP structure and functionality of HA are expected to be important for inducing protective immunity. It is likely that the HA molecules on the surfaces of the VLPs maintain the native-like conformation as evidenced by hemagglutination activity and cleavability of HA in VLPs. Disrupting the intact VLP structure and inactivating the hemagglutination activity of HA abrogated the humoral immune responses against A/PR8 virus and did not induce protective immunity. Therefore, the particulate nature and intactness of VLPs are critically important in inducing protective immunity and may be necessary in facilitating interaction with antigen-presenting cells leading to strong immune responses. In support of this notion, HIV VLPs were found to preferentially interact with CD11b+ monocyte/macrophage and B220+ B-cell populations in vitro (present study).
The current, parenterally administered influenza virus vaccine is considered to provide protective immunity against circulating viruses by inducing neutralizing antibodies directed against HA, although it is relatively less effective against antigenic variants within a subtype (1
). Serum antibodies induced by intranasal immunization with VLPs were found to have the capability to neutralize virus infectivity in vitro. We demonstrated that intranasal immunization with PR8 VLPs can confer 100% protection against PR8 as well as WSN strains using a 10× LD50
dose without any clinical symptoms, and VLP-immunized mice also survived a lethal dose of both strains as high as 200× LD50
with some weight loss (data not shown). The PR8 HA has approximately 91% amino acid homology with WSN HA on the basis of sequence analysis (GenBank accession numbers NC_004521 and ABF47955 for PR8 HA and WSN HA, respectively). Reflecting serological differences between A/PR8 and A/WSN, in addition to differences in lung viral titers, hemagglutination inhibition titers and neutralizing activity of PR8 VLP immune sera against A/WSN were two- to threefold less than those against A/PR8. Also, sera of mice infected with sublethal doses of WSN showed four- to eightfold differences in binding antibody titers against PR8 compared to those of the homologous antigen WSN, and this serologic difference was similarly observed when sera of mice infected with sublethal doses of PR8 were tested (data not shown). Nonetheless, we observed cross-reactive binding antibodies against A/WSN in VLP immune sera, and humoral and cellular immune responses were rapidly expanded upon lethal virus challenge with PR8 or WSN. Therefore, our studies demonstrate that influenza VLPs can be developed as a candidate vaccine. It will be of interest to determine whether the immune responses induced by influenza VLPs are cross-reactive with more distantly related strains within the same subtype. Influenza M1 VLPs can incorporate different subtype HAs, resulting in mixed influenza VLPs, and experiments to determine whether such phenotypically mixed VLPs can provide protection against influenza viruses of different subtypes are in progress.
An important goal of vaccination is to induce memory immune responses, which can provide long-term protective immunity. The cells responsible for memory response are T and B lymphocytes that can persist for long periods of time and can quickly be reactivated following infection. Induction of memory cells has been mostly investigated following live virus infection (30
), but not much is known about memory responses after immunization with nonreplicating VLP vaccines. A fraction of memory B lymphocytes developed in the secondary lymphoid organs is routed to the bone marrow, resides there as long-lived plasma cells, and secretes antibodies, maintaining long-term serum antibody levels. We observed the presence of influenza virus-specific antibody-secreting plasma cells in the bone marrow of the VLP-immunized mice and found that VLP-immunized mice were protected equally well 4 weeks or 5 months after the final immunization. In addition, naïve mice that received intranasal administration of heat-treated immune sera collected 5 months postvaccination were completely protected from lethal virus challenge with either homologous or heterologous strains (Table ), demonstrating the protective role of antibodies induced by VLPs. Taken together, these results suggest that influenza VLPs can induce the differentiation of B cells to long-lived plasma cells secreting antibodies, which may play a role in maintaining long-term protective immunity.
Lung cytokine-mediated immunoinflammatory reactions as well as infiltration of activated lymphocytes may be a cause of the morbidity and mortality associated with influenza virus infections (21
). We observed that high levels of IL-6 and IFN-γ were detected in naïve or HA-negative M1 VLP-immunized mouse lungs after challenge, whereas little or no proinflammatory cytokines were present in the lungs of the influenza HA VLP-immunized mice. Also, there seems to be a correlation between lung viral titers and the levels of inflammatory cytokines. This is consistent with a previous study demonstrating that high levels of lung viral titers and proinflammatory cytokines (IFN-α and IL-6) were found in the lungs of pigs with swine influenza virus infection (35
). Also, lymphocytes expressing CD69, an activation marker, were lower in influenza HA VLP-immunized mice than in naïve mice after challenge (data not shown). Thus, influenza VLP immunization can prevent immunopathologic lung inflammation upon influenza virus infection.
In summary, our results demonstrate that influenza VLPs can induce neutralizing antibodies and cellular immune responses, which can confer protection against lethal virus infection by homologous or heterologous strains within the same subtype. In addition, mucosal antibody and cellular immune responses induced by influenza VLPs were rapidly expanded upon challenge virus infection, inhibiting viral replication and lung inflammatory cytokine production. These results provide insight for developing effective prophylactic vaccines based on VLPs to fight pathogenic influenza viruses that pose a pandemic threat.