Jenna E. Ryan

Correlations among measles virus‐specific antibody, lymphoproliferation and Th1/Th2 cytokine responses following measles–mumps–rubella‐II (MMR‐II) vaccination

Immunity to measles is conferred by the interplay of humoral and cellular immune responses, the latter being critical in maintaining long‐term recall response. Therefore, it is important to evaluate measles‐specific humoral and cellular immunity in populations several years after vaccination and understand the correlations among these measures of immunity. We examined measles‐specific antibodies, lymphoproliferation and the Th1/Th2 signature cytokines, interferon (IFN)‐γ and interleukin (IL)‐4, in a population‐based cohort of healthy children from Olmsted County, Minnesota after two doses of measles–mumps–rubella‐II (MMR‐II) vaccine. We detected positive measures of measles‐specific cellular and humoral immunity in the majority of our study population. However, a small proportion of subjects demonstrated an immune response skewed towards the Th2 type, characterized by the presence of either IL‐4 and/or measles‐specific antibodies and a lack of IFN‐γ production. Further, we observed a significant positive correlation between lymphoproliferation and secretion of IFN‐γ (r = 0·20, P = 0·0002) and IL‐4 (r = 0·15, P = 0·005). Measles antibody levels were correlated with lymphoproliferation (r = 0·12, P = 0·03), but lacked correlation to either cytokine type. In conclusion, we demonstrated the presence of both long‐term cellular and humoral responses after MMR‐II vaccination in a significant proportion of study subjects. Further, a positive correlation between lymphoproliferation and IL‐4 and IFN‐γ suggests that immunity to measles may be maintained by both Th1 and Th2 cells. We speculate that the Th2 biased response observed in a subset of our subjects may be insufficient to provide long‐term immunity against measles. Further examination of the determinants of Th1 versus Th2 skewing of the immune response and long‐term follow‐up is needed.

Relationship between HLA polymorphisms and gamma interferon and interleukin-10 cytokine production in healthy individuals after rubella vaccination.

ABSTRACT We studied the association between HLA alleles and rubella-specific gamma interferon (IFN-γ) (Th1) and interleukin-10 (IL-10) (Th2) cytokine responses among 106 healthy children (ages, 14 to 17 years) previously immunized with two doses of rubella vaccine. Antibody titers and cytokine responses to rubella vaccination were not sex or age dependent. Several class I HLA-A (*0201, *2402, *6801) alleles were significantly associated with rubella vaccine-induced IFN-γ secretion. Several class II HLA-DRB1 (*0101) and HLA-DQB1 (*0501) alleles were also suggestive of an association with IFN-γ secretion. Alleles with potential associations with rubella-specific IL-10 production included HLA-A (*0201, *6801), HLA-B (*4901), and HLA-DRB1 (*1302). The class I A*0201 and A*6801 alleles were associated with both IFN-γ and IL-10 secretion. These tentative associations need to be validated in larger studies with subjects of differing ethnicities. These results provide additional evidence that HLA genes may influence Th1- and Th2-specific cytokine response(s) following rubella immunization, which in turn can influence both cellular and humoral immune responses to rubella vaccination.

Influence of host genetic variation on rubella-specific T cell cytokine responses following rubella vaccination

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-gamma, IL-2, and IL-12p40), Th2 (IL-4, IL-5, and IL-10), and innate/proinflammatory (TNF-alpha, 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-alpha 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.

Inter-operator variation in ELISPOT analysis of measles virus-specific IFN-γ-secreting T cells

The ELISPOT assay is a highly sensitive technique used for the detection of individual cytokine releasing cells. We have developed an IFN‐γ ELISPOT assay utilizing unfractionated frozen peripheral blood mononuclear cells (PBMC) to quantify the frequency of measles virus (MV)‐specific IFN‐γ‐secreting T cells in 117 healthy children who had been previously immunized with two doses of the measles‐mumps‐rubella vaccine. We have also estimated the variability associated with the quantification of ELISPOT plates and compared the number of MV‐specific IFN‐γ‐secreting T cells for each subject as determined by two different operators of an ELISPOT reader. The median frequency of MV‐specific IFN‐γ‐producing memory T cells detected by this assay was 0.005 % and 0.01 % as determined by an in‐house and commercial operator, respectively. Although we found a significant correlation (r = 0.83, p<0.0001) between the number of spots counted by the commercial and in‐house operators of an ELISPOT reader, the median number of spots counted by the commercial operator was twice the number of spots counted by an in‐house operator (p<0.001). This demonstrates the importance of using a common ELISPOT reader and operator, among other parameters, to quantify the number of spots when a large volume of plates are being scanned and analyzed.

Response surface methodology to determine optimal cytokine responses in human peripheral blood mononuclear cells after smallpox vaccination

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.

Detection of Measles Virus-Specific Interferon-γ-Secreting T-Cells by ELISPOT

The enzyme-linked immunospot (ELISPOT) assay is a highly sensitive tool used to measure the frequency of antigen-specific T-cells in vitro. Among its many applications, this assay is useful for the characterization of cellular immune responses after immunization against measles and other viral pathogens. A description of the measles virus-specific interferon (IFN)-gamma ELISPOT assay optimized in our laboratory is described in detail in this chapter. Procedures for the preparation of measles virus, infection of peripheral blood mononuclear cells with measles virus, and the IFN-gamma ELISPOT assay are also outlined. These methods can also be broadly adapted to measure activated T-cells to other viral pathogens and/or pathogen-derived peptides. Therefore, the ELISPOT assay can be used for the design, development, and evaluation of new vaccines.

Optimizing high dimensional gene expression studies for immune response following smallpox vaccination using Taqman® Low density immune arrays

INTRODUCTION We sought to determine the time and vaccinia virus dose combination that would maximize the number of acute immune response changes in response to vaccinia stimulation in preparation for a large gene expression microarray experiment. METHODS PBMCs from ten subjects were exposed to five vaccinia virus doses for three lengths of time. Gene expression was measured for 90 immune response genes via Taqman® Low Density Immune Arrays. Expression data were normalized via model-based non-linear normalization. Linear mixed effects model results were used to standardize changes across genes and determine the time/multiplicity of infection (MOI) combination with the largest number of changes. RESULTS The greatest number of changes occurred with a MOI of 5.0 and exposure time of 48 h. Further inspection revealed that most changes had occurred earlier and faded at this combination. The second highest number of changes was found at a MOI of 0.5 PFU/cell and time of 18 h. CONCLUSIONS We conclude a time of 18 h with a MOI of 0.5 PFU/cell is the optimal time/MOI combination for the full scale gene expression study. The strategy described herein is a general and resource efficient way to make critical decisions regarding experimental parameters for studies utilizing expensive assays that interrogate a large number of variables.