Association of IL1RN VNTR polymorphism with chikungunya infection: A study from Western India.

Chikungunya, caused by the chikungunya virus (CHIKV) mostly presents as acute and chronic articular inflammatory manifestations. Interleukin 1 receptor antagonist (IL-1RN) is a potent endogenous competitive inhibitor of IL-1α and 1ß and has an anti-inflammatory role. The present study evaluated the possible association of IL1RN variable number tandem-repeat (VNTR) alleles and genotypes, and CHIKV stimulated IL-1RN cytokine production with resistance and/or susceptibility to chikungunya infection and disease state in 224 patients with chikungunya (61 patients with acute chikungunya and 163 patients with chronic chikungunya) and 355 healthy controls. Polymerase chain reaction, CHIKV stimulated cytokine assay and luminex platform were used for assessing polymorphism and protein levels respectively. The study revealed a significant association of IL1RN*1/*1 genotype under recessive genetic model with the risk of developing chikungunya infection. Our findings also indicated that IL1RN *2 allele under dominant mode was associated with protection to chronic chikungunya. The results also revealed a higher production of IL-1 RN protein in patients with chronic chikungunya. To conclude, the results suggest the association of ILRN VNTR polymorphism and IL-RN protein levels with chronic chikungunya.

Regulation of the chikungunya-virus-induced innate inflammatory response by protein tyrosine phosphatase non-receptor 6 in muscle cells.

Chikungunya virus (CHIKV)-induced myositis is an emerging affliction with high incidence globally. Given the essential regulatory role of protein tyrosine phosphatase non-receptor 6 (PTPN6) in virus-induced myositis, the expression of the PTPN6 and TNF-α genes in a CHIKV-infected muscle cell line was examined by quantitative PCR, and the expression of PTPN6 and STAT 3 was examined by immunoblotting. In addition, the effect of PTPN6 siRNA treatment on TNF-α gene expression was assessed. Increased higher expression of PTPN6 and TNF-α, and significant upregulation of TNF-α upon PTPN6 siRNA treatment were observed, suggesting that CHIKV has the ability to induce host PTPN6 gene expression, which may lead to a decreased pro-inflammatory immune response in the host.

Differential inhibitory and activating NK cell receptor levels and NK/NKT-like cell functionality in chronic and recovered stages of chikungunya.

The role of natural killer (NK; CD3-CD56+)/NKT (CD3+CD56+)-like cells in chikungunya virus (CHIKV) disease progression/recovery remains unclear. Here, we investigated the expression profiles and function of NK and NKT-like cells from 35 chronic chikungunya patients, 30 recovered individuals, and 69 controls. Percentage of NKT-like cells was low in chronic chikungunya patients. NKp30+, CD244+, DNAM-1+, and NKG2D+ NK cell percentages were also lower (MFI and/or percentage), while those of CD94+ and NKG2A+ NKT-like cells were higher (MFI and/or percentage) in chronic patients than in recovered subjects. IFN-γ and TNF-α expression on NKT-like cells was high in the chronic patients, while only IFN-γ expression on NK cells was high in the recovered individuals. Furthermore, percentage of perforin+NK cells was low in the chronic patients. Lower cytotoxic activity was observed in the chronic patients than in the controls. CD107a expression on NK and NKT-like cells post anti-CD94/anti-NKG2A blocking was comparable among the patients and controls. Upregulated inhibitory and downregulated activating NK receptor expressions on NK/NKT-like cells, lower perforin+ and CD107a+NK cells are likely responsible for inhibiting the NK and NKT-like cell function in the chronic stage of chikungunya. Therefore, deregulation of NKR expression might underlie CHIKV-induced chronicity.

Impaired NK cell functionality and increased TNF-α production as biomarkers of chronic chikungunya arthritis and rheumatoid arthritis.

The chronic chikungunya arthritis symptoms closely mimic the rheumatoid arthritis (RA) symptoms, thus making it difficult to distinguish between these two clinical entities. The current comparative study characterizes NK (CD3-CD56+) and NK-like T (CD3+CD56+) cell responses in patients with chronic chikungunya arthritis and RA. Phenotype and functions of NK and NK-like T cells repertoire were assessed in 56 chronic chikungunya arthritis, 26 RA patients and 82 controls using flow cytometry. TNF-α and IFN-γ-secreting NK-like T cells were high in both chronic arthritis patients than in controls. Percentage of TNF-α+ NK cells was higher in RA patients than in controls. Percentage of perforin+ NK cells was low in both chronic arthritis patient groups. Among the patient groups, expressions of perforin+ and IFN-γ+ NK-like T cells were higher in RA. Overall, our data show reduced frequency of NK-like T cells, lower expression of perforin+ NK, higher expression of TNF-α+ NK-like T and IFN-γ+ NK-like T cells as the markers of chronic arthritic diseases. In the absence of any specific treatment for chronic chikungunya induced arthritis and promising results of anti-TNF-α therapy against RA, current data may form the basis for future in vivo studies and has scope as possible therapeutics against chikungunya.

Specific memory B cell response and participation of CD4+ central and effector memory T cells in mice immunized with liposome encapsulated recombinant NE protein based Hepatitis E vaccine candidate.

BACKGROUND: Liposome encapsulated neutralizing epitope protein of Hepatitis E virus (HEV), rNEp, our Hepatitis E vaccine candidate, was shown to be immunogenic and safe in pregnant and non-pregnant mice and yielded sterilizing immunity in rhesus monkeys. METHODS: The current study in Balb/c mice assessed the levels and persistence of anti-HEV IgG antibodies by ELISA, frequencies of B, memory B, T and memory T cells by flow cytometry and HEV-specific IgG secreting memory B cells by ELISPOT till 420days post immunization (PI) with 5?g rNEp encapsulated in liposome based adjuvant (2 doses, 4weeks apart). Mice immunized with a lower dose (1?g) were assessed only for anamnestic response post booster dose. RESULTS: Vaccine candidate immunized mice (5?g dose) elicited strong anti-HEV IgG response that was estimated to persist for lifetime. At day 120 PI, frequency of memory B cells was higher in immunized mice than those receiving adjuvant alone. Anti-HEV IgG titers were lower in mice immunized with 1?g dose. A booster dose yielded a heightened antibody response in mice with both high (>800GMT, 5?g) and low (?100GMT, 1?g) anti-HEV IgG titers. At day 6th post booster dose, HEV-specific antibody secreting plasma cells (ASCs) were detected in 100% and 50% of mice with high and low anti-HEV IgG titers, respectively, whereas the frequencies of CD4+ central and effector memory T cells were high in mice with high anti-HEV IgG titers only. CONCLUSIONS: Taken together, the vaccine candidate effectively generates persistent and anamnestic antibody response, elicits participation of CD4+ memory T cells and triggers memory B cells to differentiate into ASCs upon boosting. This approach of assessing the immunogenicity of vaccine candidate could be useful to explore the longevity of HEV-specific memory response in future HEV vaccine trials in human.

Phenotypic and functional analyses of NK and NKT-like populations during the early stages of chikungunya infection.

The aim of this study was to characterize NK (CD56(+)CD3(-)) and NKT-like cell (CD56(+)CD3(+)) responses early after chikungunya infection. Expression profiling and functional analysis of T/NK/NKT-like cells were performed on samples from 56 acute and 31 convalescent chikungunya patients and 56 control individuals. The percentages of NK cells were high in both patient groups, whereas NKT-like cell percentages were high only in the convalescent group. The percentages of NKp30(+)CD3(-)CD56(+), NKp30(+)CD3(+)CD56(+), CD244(+)CD3(-)CD56(+), and CD244(+)CD3(+)CD56(+)cells were high, whereas the percentages of NKG2D(+)CD3(-)CD56(+) and NKG2D(+)CD3(+)CD56(+)cells were low in both patient groups. The percentages of NKp44(+)CD3(-)CD56(+) cells were high in both patient groups, whereas the percentages of NKp44(+)CD3(+)CD56(+) cells were higher in the acute group than in convalescent and control groups. The percentages of NKp46(+)CD3(-)CD56(+) cells were high in both patient groups. Higher percentages of perforin(+)CD3(-)CD56(+) and perforin(+)CD3(+)CD56(+) cells were observed in acute and convalescent patients, respectively. Higher cytotoxic activity was observed in acute patients than in controls. IFN-γ expression on NK cells of convalescent patients and on NKT-like cells of both patient groups was indicative of the regulatory role of NK and NKT-like cells. Collectively, these data showed that higher expression of activating receptors on NK/NKT-like cells and perforin(+) NK cells in acute patients could be responsible for increased cytotoxicity. The observed expression of perforin(+) NK cells in the acute phase and IFN-γ(+) NKT-like cells in the subsequent convalescent stage showed that NK/NKT-like cells mount an early and efficient response to chikungunya virus. Further study of the molecular mechanisms that limit viral dissemination/establishment of chronic disease will aid in understanding how NK/NKT-like cells control chikungunya infection.

Association of human leukocyte antigen class II allele and haplotypes in chikungunya viral infection in a western Indian population.

BACKGROUND: Genes coding for human leukocyte antigen (HLA) class II molecules are polymorphic and have been shown to influence susceptibility to viral diseases. METHODS: One hundred patients with acute chikungunya with and without viral load and 250 chikungunya negative controls from western India were studied for the distribution of HLA class II alleles by PCR with sequence-specific primer (SSP) method. RESULTS: Frequency of DRB1*11 allele group (patients vs controls: p=0.002, Pc=0.036, OR=0.21) and haplotype DRB1*11/DQB1*03 (patients vs controls: p=0.007, OR=0.15) were significantly low, while haplotype DRB1*04/DQB1*03 (patients vs controls: p=0.042, OR=1.94) was significantly high in the patient population. HLA DQB1*04 allele was found only in the patient group with viral load (n=17), suggesting possible involvement of the same with chikungunya virus (CHIKV) replication. CONCLUSIONS: Association of HLA-DRB1*11 and the emergence of DRB1*11/DQB1*03 & DRB1*04/DQB1*03 as resistant and susceptible haplotypes towards CHIKV infection is being reported for the first time. Our results suggest that genetic susceptibility and/or resistance to chikungunya infection may be modulated by HLA class II alleles.