Iana H. Haralambieva

Fine Mapping Causal Variants with an Approximate Bayesian Method Using Marginal Test Statistics

Two recently developed fine-mapping methods, CAVIAR and PAINTOR, demonstrate better performance over other fine-mapping methods. They also have the advantage of using only the marginal test statistics and the correlation among SNPs. Both methods leverage the fact that the marginal test statistics asymptotically follow a multivariate normal distribution and are likelihood based. However, their relationship with Bayesian fine mapping, such as BIMBAM, is not clear. In this study, we first show that CAVIAR and BIMBAM are actually approximately equivalent to each other. This leads to a fine-mapping method using marginal test statistics in the Bayesian framework, which we call CAVIAR Bayes factor (CAVIARBF). Another advantage of the Bayesian framework is that it can answer both association and fine-mapping questions. We also used simulations to compare CAVIARBF with other methods under different numbers of causal variants. The results showed that both CAVIARBF and BIMBAM have better performance than PAINTOR and other methods. Compared to BIMBAM, CAVIARBF has the advantage of using only marginal test statistics and takes about one-quarter to one-fifth of the running time. We applied different methods on two independent cohorts of the same phenotype. Results showed that CAVIARBF, BIMBAM, and PAINTOR selected the same top 3 SNPs; however, CAVIARBF and BIMBAM had better consistency in selecting the top 10 ranked SNPs between the two cohorts. Software is available at https://bitbucket.org/Wenan/caviarbf.

The genetic basis for interindividual immune response variation to measles vaccine: new understanding and new vaccine approaches

The live-attenuated measles vaccine is effective, but measles outbreaks still occur in vaccinated populations. This warrants elucidation of the determinants of measles vaccine-induced protective immunity. Interindividual variability in markers of measles vaccine-induced immunity, including neutralizing antibody levels, is regulated in part by host genetic factor variations. This review summarizes recent advances in our understanding of measles vaccine immunogenetics relative to the perspective of developing better measles vaccines. Important genetic regulators of measles vaccine-induced immunity, such as HLA class I and HLA class II genotypes, single nucleotide polymorphisms in cytokine/cytokine receptor genes (IL12B, IL12RB1, IL2, IL10) and the cell surface measles virus receptor CD46 gene, have been identified and independently replicated. New technologies present many opportunities for identification of novel genetic signatures and genetic architectures. These findings help explain a variety of immune response-related phenotypes and promote a new paradigm of ‘vaccinomics’ for novel vaccine development.

Associations between single nucleotide polymorphisms and haplotypes in cytokine and cytokine receptor genes and immunity to measles vaccination

Identification of host genetic determinants of measles vaccine-induced immunity can be used to design better vaccines and ultimately predict immune responses to vaccination. We performed a comprehensive candidate gene association study across 801 genetic markers in 56 cytokine/cytokine receptor genes, in a racially diverse cohort of 745 schoolchildren after two doses of MMR vaccine. Using linear regression methodologies we examined associations between SNPs/haplotypes and measles virus-specific immunity. Forty-eight significant SNP associations with variations in neutralizing antibodies and measles-specific IFNγ Elispot responses were identified (p<0.05). Our study replicated an important previously found association of a functional IL12B genetic variant rs3212227 with variations in measles-specific humoral immunity (p=0.037). Similarly, two previously reported promoter IL10 and IL2 polymorphisms (rs1800890 and rs2069762) demonstrated associations with measles-specific cellular immunity in Caucasians (p≤0.034). Multiple IL7R polymorphisms, including a non-synonymous functional SNP (rs6897932/Thr244Ile), were associated with humoral (p≤0.024) and/or cellular (IFNγ Elispot, p≤0.023) measles-specific immune responses in Caucasians, but not African-Americans. Haplotype level analysis confirmed the association of IL7R genetic variants with measles vaccine-induced immunity in the Caucasian group (global p-value=0.003). Our results validate previous findings and identify new plausible genetic determinants, including IL7R polymorphisms, regulating measles vaccine-induced immunity in a race-specific manner.

Genome-wide analysis of polymorphisms associated with cytokine responses in smallpox vaccine recipients

The role that genetics play in response to infection or disease is becoming increasingly clear as we learn more about immunogenetics and host–pathogen interactions. Here we report a genome-wide analysis of the effects of host genetic variation on cytokine responses to vaccinia virus stimulation in smallpox vaccine recipients. Our data show that vaccinia stimulation of immune individuals results in secretion of inflammatory and Th1 cytokines. We identified multiple SNPs significantly associated with variations in cytokine secretion. These SNPs are found in genes with known immune function, as well as in genes encoding for proteins involved in signal transduction, cytoskeleton, membrane channels and ion transport, as well as others with no previously identified connection to immune responses. The large number of significant SNP associations implies that cytokine secretion in response to vaccinia virus is a complex process controlled by multiple genes and gene families. Follow-up studies to replicate these findings and then pursue mechanistic studies will provide a greater understanding of how genetic variation influences vaccine responses.

A large observational study to concurrently assess persistence of measles specific B-cell and T-cell immunity in individuals following two doses of MMR vaccine.

The measurement of measles-specific neutralizing antibodies, directed against the surface measles virus hemagglutinin and fusion proteins, is considered the gold standard in measles serology. We assessed functional measles-specific neutralizing antibody levels in a racially diverse cohort of 763 young healthy adolescents after receipt of two doses of measles-mumps-rubella vaccine, by the use of an automated plaque reduction microneutralization (PRMN) assay, and evaluated their relevance to protective antibody levels, as well as their associations with demographic and clinical variables. We also concurrently assessed measles-specific IFNγ Elispot responses and their relation to the observed antibody concentrations. The geometric mean titer for our cohort was 832mIU/mL (95% CIs: 776; 891). Sixty-eight subjects (8.9%) had antibody concentrations of less than the protective threshold of 210mIU/mL (corresponding to PRMN titer of 120; suggesting protection against symptomatic disease), and 177 subjects (23.2%) demonstrated persisting antibody concentrations above 1841mIU/mL (corresponding to PRMN titer of 1052; suggesting total protection against viral infection), 7.4 years after vaccination, in the absence of wild-type virus boosting. The mean measles-specific IFNγ Elispot response for our cohort was 46 (95% CIs: 43; 49) IFNγ-positive spots per 200,000 cells with no relation of cellular immunity measures to the observed antibody concentrations. No significant associations between antibody titers and demographic and clinical variables, including gender and race, were observed in our study. In conclusion, in a large observational study of measles immunity, we used an automated high-throughput measles virus-specific neutralization assay to measure humoral immunity, and concurrently determined measles-specific cellular immunity to aid the assessment of potential susceptibility to measles in vaccinated populations.

Genetic polymorphisms in host antiviral genes: associations with humoral and cellular immunity to measles vaccine.

Abstract Host antiviral genes are important regulators of antiviral immunity and plausible genetic determinants of immune response heterogeneity after vaccination. We genotyped and analyzed 307 common candidate tagSNPs from 12 antiviral genes in a cohort of 745 schoolchildren immunized with two doses of measles–mumps–rubella (MMR) vaccine. Associations between SNPs/haplotypes and measles virus-specific immune outcomes were assessed using linear regression methodologies in Caucasians and African-Americans. Genetic variants within the DDX58/RIG-I gene, including a coding polymorphism (rs3205166/Val800Val), were associated as single-SNPs (p ≤0.017; although these SNPs did not remain significant after correction for false discovery rate/FDR) and in haplotype-level analysis, with measles-specific antibody variations in Caucasians (haplotype allele p-value=0.021; haplotype global p-value=0.076). Four DDX58 polymorphisms, in high LD, demonstrated also associations (after correction for FDR) with variations in both measles-specific IFN-γ and IL-2 secretion in Caucasians (p ≤0.001, q =0.193). Two intronic OAS1 polymorphisms, including the functional OAS1 SNP rs10774671 (p =0.003), demonstrated evidence of association with a significant allele-dose-related increase in neutralizing antibody levels in African-Americans. Genotype and haplotype-level associations demonstrated the role of ADAR genetic variants, including a non-synonymous SNP (rs2229857/Arg384Lys; p =0.01), in regulating measles virus-specific IFN-γ Elispot responses in Caucasians (haplotype global p-value=0.017). After correction for FDR, 15 single-SNP associations (11 SNPs in Caucasians and 4 SNPs in African-Americans) still remained significant at the q-value<0.20. In conclusion, our findings strongly point to genetic variants/genes, involved in antiviral sensing and antiviral control, as critical determinants, differentially modulating the adaptive immune responses to live attenuated measles vaccine in Caucasians and African-Americans.

The Association of CD46, SLAM and CD209 Cellular Receptor Gene SNPs with Variations in Measles Vaccine-Induced Immune Responses: A Replication Study and Examination of Novel Polymorphisms

Background: The measles virus (MV) interacts with two known cellular receptors: CD46 and SLAM. The transmembrane receptor CD209 interacts with MV and augments dendritic cell infection. Methods: 764 subjects previously immunized with measles-mumps-rubella vaccine were genotyped for 66 candidate SNPs in the CD46, SLAM and CD209 genes as part of a larger study. Results: A previously detected association of the CD46 SNP rs2724384 with measles-specific antibodies was successfully replicated in this study. Increased representation of the minor allele G for an intronic CD46 SNP was associated with an allele dose-related decrease (978 vs. 522 mIU/ml, p = 0.0007) in antibody levels. This polymorphism rs2724384 also demonstrated associations with IL-6 (p = 0.02), IFN-α (p = 0.007) and TNF-α (p = 0.0007) responses. Two polymorphisms (coding rs164288 and intronic rs11265452) in the SLAM gene that were associated with measles antibody levels in our previous study were associated with IFN-γ Elispot (p = 0.04) and IL-10 responses (p = 0.0008), respectively, in this study. We found associations between haplotypes, AACGGAATGGAAAG (p = 0.009) and GGCCGAGAGGAGAG (p < 0.001), in the CD46 gene and TNF-α secretion. Conclusion: Understanding the functional and mechanistic consequences of these genetic polymorphisms on immune response variations could assist in directing new measles and potentially other viral vaccine design, and in better understanding measles immunogenetics.

2′-5′-Oligoadenylate synthetase single-nucleotide polymorphisms and haplotypes are associated with variations in immune responses to rubella vaccine.

Abstract Interferon-induced antiviral genes are crucial players in innate antiviral defense and potential determinants of immune response heterogeneity. We selected 114 candidate single-nucleotide polymorphisms (SNPs) from 12 antiviral genes using an LD tagSNP selection approach and genotyped them in a cohort of 738 school children immunized with two doses of rubella vaccine. Associations between SNPs/haplotypes and rubella virus-specific immune measures were assessed using linear regression methodologies. We identified 23 significant associations (p < 0.05) between polymorphisms within the 2′-5′-oligoadenylate synthetase (OAS) gene cluster, and rubella virus-specific IL-2, IL-10, IL-6 secretion, and antibody levels. The minor allele variants of three OAS1 SNPs (rs3741981/Ser162Gly, rs1051042/Thr361Arg, rs2660), located in a linkage disequilibrium block of functional importance, were significantly associated with an increase in rubella virus-specific IL-2/Th1 response (p ≤ 0.024). Seven OAS1 and OAS3 promoter/regulatory SNPs were similarly associated with IL-2 secretion. Importantly, two SNPs (rs3741981 and rs10774670) independently cross-regulated rubella virus-specific IL-10 secretion levels (p ≤ 0.031). Furthermore, both global tests and individual haplotype analyses revealed significant associations between OAS1 haplotypes and rubella virus-specific cytokine secretion. Our results suggest that innate immunity and OAS genetic variations are likely involved in modulating the magnitude and quality of the adaptive immune responses to live attenuated rubella vaccine.

Development of a novel efficient fluorescence-based plaque reduction microneutralization assay for measles virus immunity.

ABSTRACT The measurement of functional measles virus-specific neutralizing antibodies is of considerable interest for vaccine-related research. In this study, we developed and standardized a simple, rapid, highly sensitive, and reproducible fluorescence-based plaque reduction microneutralization (PRMN) assay with visual and automated readout, using a recombinant measles virus engineered to express enhanced green fluorescent protein. The assay is performed in micro format, requires less time to complete (2 versus 4 to 7 days), and is less labor-intensive and less costly than the classical plaque reduction neutralization (PRN) test, widely accepted as the “gold standard” in measles serology. Two available WHO international anti-measles virus standards and one in-house reference serum were used to develop and standardize the new assay. The mean PRMN values from repeated assays were found to be similar to those reported in the literature or assigned to the WHO standards by the classical PRN assay. For validation, we used three groups of low, moderate, and high measles virus vaccine responders’ sera with moderate values of correlation in antibody levels (mIU/ml) between PRMN and the Dade Behring immunoglobulin G enzyme immunoassay (EIA). The PRMN assay was more sensitive at low antibody levels and more informative in terms of protection than this commercial EIA. In conclusion, we have developed and validated a sensitive and high-throughput measles virus-specific PRMN that can be readily used in large population-based measles studies.

Associations between demographic variables and multiple measles-specific innate and cell-mediated immune responses after measles vaccination.

Measles remains a public health concern due to a lack of vaccine use and vaccine failure. A better understanding of the factors that influence variations in immune responses, including innate/inflammatory and adaptive cellular immune responses, following measles-mumps-rubella (MMR) vaccination could increase our knowledge of measles vaccine-induced immunity and potentially lead to better vaccines. Measles-specific innate/inflammatory and adaptive cell-mediated immune (CMI) responses were characterized using enzyme-linked immunosorbent assays to quantify the levels of secreted IL-2, IL-6, IL-10, IFN-α, IFN-γ, IFN-λ1, and TNF-α in PBMC cultures following in vitro stimulation with measles virus (MV) in a cohort of 764 school-aged children. IFN-γ ELISPOT assays were performed to ascertain the number of measles-specific IFN-γ-secreting cells. Cytokine responses were then tested for associations with self-declared demographic data, including gender, race, and ethnicity. Females secreted significantly more TNF-α, IL-6, and IFN-α (p<0.001, p<0.002, p<0.04, respectively) compared to males. Caucasians secreted significantly more IFN-λ1, IL-10, IL-2, TNF-α, IL-6, and IFN-α (p<0.001, p<0.001, p<0.001, p<0.003, p<0.01, and p<0.02, respectively) compared to the other racial groups combined. Additionally, Caucasians had a greater number of IFN-γ-secreting cells compared to other racial groups (p<0.001). Ethnicity was not significantly correlated with variations in measles-specific CMI measures. Our data suggest that innate/inflammatory and CMI cytokine responses to measles vaccine vary significantly by gender and race. These data further advance our understanding regarding inter-individual and subgroup variations in immune responses to measles vaccination.