Results
Relationship between the TLR3.rs3775290 (c.1377C/T) genotype and the HCV-specific IFN-γ ELISpot response
All subjects enrolled into the study were tested for IFN-γ production by PBMCs using ELISpot assay to evaluate CMI response against nine HCV genotype 4a isolate ED43 peptide antigen pools composed of 15 (15mer) and overlapping by 10 amino acids. Only 140 (53%), 20 (80%), 35 (40%) and 70 (41.4%) samples had valid CMI data among the four study groups (seronegative, aviraemic subjects; seronegative, viraemicsubjects; spontaneously resolved subjects and chronic HCV patients groups, respectively).
There was no relationship between the outcome of the HCV-specific immune response and the TLR3.rs3775290 genotype among the 263 HCWs with valid CMI responses and TLR3.rs3775290 genotyping tests (p= 0.222). Also, there was a significantly (p =0.004) higher proportion of the CC genotype (73.3%) in responding subjects than in non-responding seronegative, aviraemic subjects (65.1%). In addition, there was a differential distribution of the three TLR3.rs3775290 genotypes among the total subjects with and without HCV-specific CMI (Table 1).
The average totals (±SEM) of IFN-γ responses measured in SFC per million PBMCs in the responding total subjects were 455±90, 507±122, and 317±45 with the TLR3.rs3775290 genotype CC (n=37), CT (n=16) and TT (n=6). On the other hand, among the non-responding subjects, the average totals (±SEM) of IFN-γ SFC per million PBMCs in total subjects were 29±5, 23±5, and 49±19 with the TLR3.rs3775290 genotype CC (n=130), CT (n=65) and TT (n=9), respectively. There was no significant difference between the responding total subjects with different TLR3.rs3775290 genotypes (p =0.89; Figure 1A).
For the seronegative, aviraemic subjects, the average totals of HCV-specific IFN-γ SFC/106 PBMCs (±SEM) of the responding subjects were 404±97.7, 1058±807.5, and 249±21.9, among those with the TLR3.rs3775290 CC genotype (n=22), CT (n=4) and TT (n=4) genotypes, respectively. Differences were not statistically significant (p =0.88). On the other hand, the average totals of IFN-γ SFC/106 PBMCs (± SEM) of the non-responding subjects were 26.7±5.7, 16.7±4, and 13.3±13.3, among those with the TLR3.rs3775290 CC (n=71), CT (n=36) and TT (n=2) genotypes, respectively (Figure 1B).
Regarding seronegative , viraemic subjects, the average totals (±SEM) of HCV-specific IFN-γ SFC/106 PBMCs forresponding subjects were 657±443, and 163, among those with the TLR3.rs3775290 genotype CC (n=3) and CT (n=1) genotypes, respectively. There were no responding subjects with the TT genotype. Differences were not statistically significant (p =0.18). On the other hand, the average totals of IFN-γ SFC/106 PBMCs (±SEM) of the non-responding subjects were 42±22, 30±11 and 8±8, among those with the TLR3.rs3775290 CC (n=9), CT (n=5) and TT (n=2) genotypes, respectively (Figure 1C).
For spontaneously resolved subjects, the average totals of HCV-specific IFN-γ SFC/106 PBMCs (±SEM) of theresponding subjects were 793±605.8, and 325±121, among those with the TLR3.rs3775290 CC (n=2), CT (n=5) genotypes, respectively. No TT genotype was recorded in this category. Differences were not statistically significant (p =0.698). On the other hand, the average totals of IFN-γ SFC/106 PBMCs (±SEM) of the non-responding subjects were 51±21, 15±12 and 58, among those with the TLR3.rs3775290 CC (n=14), CT (n=12) and TT (n=1) genotypes, respectively (Figure 1D).
For chronic HCV patients , the average totals of HCV-specific IFN-γ SFC/106 PBMCs (±SEM) of the respondingsubjects were 439±214, 349 ±181, and 453±9, among those with the TLR3.rs3775290 CC (n=10), CT (n=6) and TT (n=2) genotypes, respectively. Differences were not statistically significant (p =0.228). On the other hand, the average totals of IFN-γ SFC/106 PBMCs (±SEM) of the non-responding chronic HCV patients were 26±8, 38±13, and 84±36, among those with the TLR3.rs3775290 CC (n=36), CT (n=13) and TT (n=4) genotypes, respectively (Figure 1E).