Characterization of the in situ immunological responses to vaccine adjuvants

Adjuvants are included with many inactivated and some modified live vaccines to enhance immune responses to specific antigens. While early vaccines relied exclusively upon aluminum salts, still the major adjuvant used in human vaccines, other adjuvant products are used in veterinary medicine. In addition to enhancing antigen presentation, adjuvants can also enhance the development of specific immune responses. Thus, alum adjuvants often preferentially stimulate humoral immune responses. By contrast, lipid-based adjuvants are often more effective at stimulating cell-mediated immune responses. Metastim® is a lipid-based adjuvant reported to elicit both humoral and cellular immune responses, though the mechanism responsible for this activity remains unclear. In this study, we compared the ability of equine influenza virus vaccines containing either saline or Metastim® or an aluminum phosphate adjuvant to stimulate antigen presenting cell function in vivo. Six ponies were intradermally inoculated with inactivated equine influenza (KY97) mixed with either adjuvant or saline. Multiple sites were injected so that biopsies could be collected at different times post injection. The 4 mm punch biopsies were formalin-fixed, paraffin-embedded, and stained with hematoxylin and eosin (H&E). Total RNA was isolated from 2 mm punch biopsies for the determination of gene expression by real-time PCR. H&E staining revealed a variety of cells recruited to the injection sites, including lymphocytes, neutrophils, eosinophils and macrophages. Real-time PCR analysis of the injection site confirmed this cellular infiltration and identified increased expression of activation markers. Both vaccines also stimulated gene expressions of pro-inflammatory cytokines. The vaccine containing Metastim® elicited significantly higher gene expression of interferon-γ, IL-12, CD4and CD83 compared to alum (p < 0.05). While the greater induction of IFNγ-related gene expression indicates that Metastim® can elicit Th-1 immune responses more effectively than the aluminum salt, there was also evidence of Th2 cytokine induction.