Statistical evaluation for detection of peptide specific interferon-gamma secreting T-cells induced by HIV vaccine determined by ELISPOT assay.

Interferon-gamma (IFN-gamma) secreting T-cells play an important role in immunodeficiency virus (HIV) disease pathogenesis. A common technique to determine the efficacy of the HIV vaccines in murine models is to compare the number of IFN-gamma secreting T-cells induced by HIV vaccine to a control group. The measurement of IFN-gamma secreting T-cells relies on an ELISPOT assay. This assay is carried out in triplicate wells from the same mouse on the same ELISPOT plate. The numbers of spot forming cells (SFC) from these wells are correlated counts. Traditionally, simple statistical methods, such as ANOVA or Kruskall-Wallis tests, are performed on means by mouse. These approaches ignore the fact that the data are correlated counts. Count data are usually assumed to follow the Poisson distribution. However, some count data exhibit overdispersion that can affect the test statistics. The negative binomial distribution is an alternative to the Poisson distribution in the presence of overdispersion. Hence, negative binomial regression is a more suitable approach for overdispersed count data. In this study, we used a negative binomial regression to determine that IL-12 was a good adjuvant. The results of the study using a negative binomial regression are discussed.

A modified cholera holotoxin CT-E29H enhances systemic and mucosal immune responses to recombinant Norwalk virus-virus like particle vaccine.

In this study, we evaluated the potential of a genetically modified cholera toxin, CT-E29H as an adjuvant for recombinant Norwalk virus like particle (NV-VLP) vaccine. This detoxified mutant, containing E to H substitution at amino acid 29 of the CT-A1 subunit, was administered with a recombinant Norwalk virus like particle vaccine to Balb/c mice by mucosal routes to monitor the induction of mucosal, humoral and cellular responses. We observed that a low dose of NV-VLP (5 microg) with the adjuvant delivered by the intranasal route (IN) was more effective than the highest dose (200 microg) delivered by oral route at inducing both cellular and NV-VLP specific IgG and IgA responses. Higher counts of antigen specific IgA secreting cells were observed in the Peyer's Patches (PP) following delivery of the vaccine with CT-E29H as compared to delivery of vaccine by mucosal routes without CT-E29H. Furthermore, there was an increase in antigen specific cells producing IL-4 from animals that received the vaccine with the adjuvant. Delivery of the vaccine by the oral route results in antigen specific CD4(+) and CD8(+) T cells in PP and spleen. Addition of CT-E29H results in an increase of antigen specific CD4(+) cell population in PP and both CD4(+) and CD8(+) populations in the spleen. These cellular and cytokine responses suggest that combining the vaccine with CT-E29H results in a stronger Th2 type response. Collectively, these results indicate that immune responses to NV-VLP vaccine are qualitatively and quantitatively improved when the vaccine is delivered along with CT-E29H, and thus merits its further consideration as a mucosal adjuvant.