The effects of gene-based vaccination on immune responses are becoming increasingly understood. For example, DNA priming followed by adenoviral boosting for several different antigens can elicit strong CD4 and CD8 cellular immune responses (9
). In contrast, protein vaccines typically utilize adjuvants that elicit strong CD4 responses and minimal CD8 responses (21
). More recently, Toll receptor agonists, particularly CpGs, have been shown to improve the cellular immune responses elicited by proteins, particularly in murine models and in some nonhuman primate systems (11
). Because we have found previously that the increase in SARS S antibody titers generated by DNA/rAd or rAd immunization do not broaden protection against civet and recent SARS-CoV isolates (73
), we have focused on gene-based vaccination and protein adjuvant combinations that increase the antibody response at the same time they maintain or enhance balanced T-cell immunity.
Genetic vaccination in combination with inactivated virus boosting showed that boosting with adjuvanted proteins induces a qualitatively different immune response, with increased CD4 responses relative to CD8 and strong antibody responses; gene-based responses, particularly those using rAd vector boosting, elicit strong CD8 responses and less marked CD4 responses, although the immunoglobulin response is substantial. These disparate effects are likely due to the nature of the interaction of the various vaccines with dendritic cells. rAd is thought to infect immature dendritic cells, which upon maturation can present synthesized antigens within class I major histocompatibility complex proteins to stimulate CD8 immunity, while alum and other protein adjuvants typically boost CD4 and humoral responses through class II major histocompatibility complex pathways. When inactivated virus with adjuvant was used to boost animals primed with rAd vectors, an antigen-specific increase in the CD4 response was observed (Fig. ). In contrast, the CD8 response was not affected either by the antigen or by the adjuvant and was also similar in magnitude to the stimulation seen without boosting (Fig. , middle panel, lane 5). We interpret this finding to indicate that the adjuvant effect after rAd priming, which induces a CD8 response, is CD4 specific. This finding is consistent with its ability to boost the T-dependent antibody response.
These results suggest that the final boost of a gene-based vaccine with adjuvanted protein can lead to a CD4-enhanced response and diminish the CD8 response, although the antibody responses in many instances can be comparable. In contrast, DNA priming followed by rAd boosting causes stronger antigen-specific CD8 expansion. Though the mechanism of this selective activation is not completely understood, this effect is most likely due to the ability of rAd to synthesize the gene product within dendritic cells that promote better class I antigen presentation. In the case of the inactivated virus, such a response would require uptake of preformed protein antigens. Cross-presentation would therefore be required to induce a CD8 response, which is less efficient. These responses are typically Th1 responses, based on the isotypes of the antibodies and the pattern of cytokine secretion, and we have not observed changes in isotype ratios that indicate a shift to a Th2 response with any of the alternative immunizations.
The ability to stimulate the CD4 responses may be desirable if the immune response is largely dependent on neutralizing antibodies; however, in the case of SARS, several S glycoproteins have been identified that are resistant to neutralization. In some instances, it appears that these S glycoproteins either emerged more recently or have been derived from palm civet viruses. The S glycoprotein recognizes a cellular receptor, the human ACE-2 protein (35
). The region sensitive to neutralization maps to the receptor binding domain (36
). Recent data suggest that the antibody-resistant strains preferentially recognize the civet ACE-2 and may not have adapted as effectively as the isolates that were identified during the outbreaks in late 2003 and 2004 (36
). The ability to induce a strong humoral response would be desirable if it is possible to generate antibodies to these new strains; however, it is not possible to elicit such antibodies using homologous immunization in animals with this same strain. Therefore, it would appear desirable presently to elicit cellular immunity by vaccination. Whether there are alternative means to combine the protein with different Toll receptor agonists, including novel CpGs or small molecule Toll receptor agonists such as imiquimod or resiquimod (6
), is yet unknown but would be highly desirable. Though the coupling of CpGs directly to inactivated virus or proteins might enhance cellular immunity similar to gene-based vaccination, the present data suggest that the simple addition of the CpG to the adjuvant does not provide the type of cross-priming in a murine model that would be desirable for improvement of CD8 responses. In this study, inactivated virus rather than purified S protein was used as an immunogen. Though inactivated virus contains additional virus proteins and contains more epitopes than purified S protein, the inactivated virus was studied here for two reasons. First, the inactivated viral particles contain native SARS S protein, the most relevant antigenic structure with respect to neutralizing antibodies. Previous studies have shown that this antigen is capable of inducing such neutralizing antibody responses (13
), and the structure found on the virion presents the relevant native conformation. Second, similar vaccines are being tested or are under development for human clinical studies (2
). Therefore, the ability to perform a prime-boost immunization with clinically relevant products and to analyze their immunogenicity affords an opportunity to examine synergy between these different vaccine candidates that have clinical implications. The neutralization of the prototypic human SARS coronavirus is encouraging, and the neutralization titer has also correlated with ELISA titers for the Urbani strain (73
). It may be tempting to pursue a vaccine strategy based on antibody neutralization, but it is important to recognize that vaccines for animal coronaviruses which rely on humoral immunity have not proven efficacious (16
). Current animal models of SARS infection that faithfully replicate human disease do not exist. Moreover, it is not possible to propagate viruses which preferentially utilize the palm civet ACE-2 receptor that are insensitive to antibody neutralization (36
). It is presently not possible to evaluate relative vaccine efficacy; however, a variety of novel approaches, including adaptation of virus to different species, the development of transgenic animals expressing the human ACE-2 receptor, and the use of aged animals in challenge models, may assist in this effort in the future. Further analysis of vaccine candidates with alternative immunologic profiles in relevant animal models will assist in the selection of vaccines that will be most appropriate for development in humans.