Toll-like receptors (TLRs) represent the critical "bridge" between innate and adaptive immunity to viral pathogens. We hypothesized that single nucleotide polymorphisms (SNPs) that potentially influence the expression/function of TLRs and their associated intracellular signaling molecules contribute to variations in humoral and cellular immunity to measles vaccine. We genotyped 190 randomly selected subjects (12-18 years old), previously vaccinated with two doses of measles, for known SNPs in TLR 2, 3, 4, 5, 6, 7, 8 and 9, and their associated intracellular signaling genes. Specific SNPs in the TLR 2, 3, 4, 5, 6, MyD88 and MD2 genes were associated with measles-specific humoral and cellular immunity. Heterozygous variants for rs3775291 (Phe412Leu) and rs5743305 (−926bp in promoter region) of the TLR3 gene were associated with low antibody and lymphoproliferative responses (p ≤ 0.02) to measles vaccination. Heterozygous variants for rs4986790 (Gly299Asp) and rs4986791 (Ile399Thr) in the TLR4 gene demonstrated higher levels of (p ≤ 0.02) IL-4 secretion. Heterozygous variants for SNPs in TLR5 (rs5744174) and TLR6 (rs5743818) were associated with higher levels of (p ≤ 0.02) IFN-γ secretion. In addition, SNPs in MyD88 and MD2, intracellular molecules that associate with TLRs, also demonstrated associations with variations in antibody and IL-10 production (p ≤ 0.03). Thus, we identified specific SNP associations between TLRs and their associated signaling molecules that have a known role in viral immunity and variations in both humoral and cellular immunity following measles vaccination. These data contribute to understanding the immunogenetic mechanisms underlying variations in the immune response to measles vaccine.

Measles virus-specific T cells and the production of cytokines play a critical role in the immune response following measles immunization. To understand the genetic factors that influence variation in IFN-gamma and IL-4 responses following measles immunization and to provide insight into the factors influencing both cellular and humoral immunity to measles, we assessed associations between human leukocyte antigen (HLA) class II genes and measles-specific Th1 and Th2-type cytokine responses in peripheral blood lymphocytes from 339 children previously vaccinated with two doses of measles-mumps-rubella vaccine (MMR-II). Median values for measles-specific IFN-gamma and IL-4 secretion levels were 40.73 and 9.71 pg/ml, respectively. The global tests suggested associations between measles-specific IFN-gamma response and alleles of the DRB1 and DQB1 loci (P=0.07 and P=0.02, respectively). Specifically, DRB1*0301, *0901, and *1501 alleles were significantly associated with IFN-gamma secretion. The alleles that suggested evidence of an HLA association with IL-4 secretion were DRB1*0103, *0701, and *1101. Th1 cytokine responses and DQB1 allele associations revealed that the alleles with the strongest association with IFN-gamma secretion were DQB1*0201, *0303, *0402, and *0602. Specific alleles with a suggestive association with low measles-specific Th2 cytokine responses were DQB1*0202 and *0503. In addition, DPB1*0101, *0201, and *0601 alleles provided suggestive evidence of an HLA association with measles-induced IFN-gamma response, while DPB1*0501 was associated with an IL-4 response. These data suggest that IFN-gamma and IL-4 cytokine responses to measles may be genetically restricted in part by HLA class II genes, which in turn can restrict the cellular immune response to measles vaccine.

The variability of immune response modulated by immune response gene polymorphisms is a significant factor in the protective effect of vaccines. We studied the association between cellular (cytokine) immunity and HLA genes among 738 schoolchildren (396 males and 342 females) between the ages of 11 and 19 years, who received two doses of rubella vaccine (Merck). Cytokine secretion levels in response to rubella virus stimulation were determined in PBMC cultures by ELISA. Cell supernatants were assayed for Th1 (IFN-γ, IL-2, and IL-12p40), Th2 (IL-4, IL-5, and IL-10), and innate/proinflammatory (TNF-α, GM-CSF, and IL-6) cytokines. We found a strong association between multiple alleles of the HLA-DQA1 (global p-value 0.022) and HLA-DQB1 (global p-value 0.007) loci and variations in rubella-specific IL-2 cytokine secretion. Additionally, the relationships between alleles of the HLA-A (global p-value 0.058), HLA-B (global p-value 0.035), and HLA-C (global p-value 0.023) loci and TNF-α secretion suggest the importance of HLA class I molecules in innate/inflammatory immune response. Better characterization of these genetic profiles could help to predict immune responses at the individual and population level, provide data on mechanisms of immune response development, and further inform vaccine development and vaccination policies.

Feasibility, amount of sample aliquots, processing time and cost are critical considerations for optimizing and conducting assays for large-population based studies. Well designed statistical approaches that quickly identify optimal conditions for a given assay could assist efficient completion of the laboratory assays for such studies.For example, assessment of the profile of secreted cytokines is important in understanding the immune response after vaccination. To characterize the cytokine immune response following smallpox vaccination, PBMC obtained from recently vaccinated subjects were stimulated with varying doses of live or UV-inactivated vaccinia virus and cultured for up to 8 days.In this paper, we describe a novel statistical method to identify optimal operating conditions for length in culture and virus MOI in order to measure a panel of secreted Th1, Th2, and inflammatory cytokines. This statistical method is comprised of two components. It first identifies a subset of the possible time in culture by virus MOI combinations to be studied. It then utilizes response surface analysis techniques to predict the optimal operating conditions for the measurement of each secreted cytokine. This method was applied, and the predicted optimal combinations of length in culture and virus MOI for maximum vaccinia-specific cytokine secretion were identified. The use of the response surface methodology can be applied to the optimization of other laboratory assays; especially when the number of PBMC available limits the testing of all possible combinations of parameters.

The variability of immune responses modulated by human leukocyte antigen (HLA) genes and secreted cytokines is a significant factor in the development of a protective effect of measles vaccine. We studied the association between type 1 helper T cells (Th1)- and Th2-like cytokine immune responses and HLA class I alleles among 339 schoolchildren who previously received two doses of the measles vaccine. Median values for measles-specific interferon gamma (IFN-gamma) and interleukin-4 (IL-4) cytokines were 40.7 pg/ml [interquartile range (IQR) 8.1-176.7] and 9.7 pg/ml (IQR 2.8-24.3), respectively. Class I HLA-A (*0101 and *3101) and HLA-Cw (*0303 and *0501) alleles were significantly associated with measles-virus-induced IFN-gamma secretion. HLA-A*3101 and Cw*0303 were associated with a higher median IFN-gamma response, while A*0101 and Cw*0501 were associated with lower measles-specific IFN-gamma response. We found limited associations between HLA class I gene polymorphisms and Th2-like (IL-4) immune responses after measles vaccination, indicating that HLA class I molecules may have a limited effect on measles-vaccine-induced IL-4 secretion. Understanding the genetic factors that influence variations in cytokine secretion following measles vaccination will provide insight into the factors that influence both cell-mediated and humoral immunity to measles.