Different Antibody Response against the Coxsackievirus A16 VP1 Capsid Protein: Specific or Non-Specific

<div><p>Coxsackievirus A16 (CA16) is one of the major causative agents of hand, foot, and mouth disease worldwide. The non-neutralizing antibody response that targets CA16 VP1 remains poorly elucidated. In the present study, antibody responses against CA16 VP1 in Shanghai blood donors and Shanxi individuals were analyzed by ELISA and inhibitory ELISA using five CA16 VP1 antigens: VP1_1-297, VP1_41-297, VP1_1-60, VP1_45-58 and VP1_61-297. The correlation coefficients for most of the reactions against each of the five antigens and the inhibition of the anti-CA16 VP1 antibody response produced by the various antigens were higher in Shanghai blood donors compared to those in Shanxi individuals. VP1_1-297 and VP1_41-297 strongly inhibited the anti-CA16 VP1 response in serum samples from both populations, while VP1_45-58 and VP1_61-297 intermediately and weakly inhibited the anti-CA16 VP1 response, respectively, in only Shanghai group. A specific type of inhibition (anti-CA16 VP1 was completely inhibited by both VP1_1-60 and VP1_41-297) characterized by high neutralizing antibody titers was identified and accounted for 71.4% of the strongly reactive samples from the Shanghai group. These results indicate that the Shanghai blood donors exhibited a consistent and specific antibody response, while the Shanxi individuals showed an inconsistent and non-specific antibody response. These findings may improve the understanding of host humoral immunity against CA16 and help to identify an effective approach for seroepidemiological surveillance and specific diagnosis of CA16 infection based on normal and competitive ELISA.</p></div

The design of the four truncated CA16 VP1 proteins.

<p>In the present study, four CECRS-based antigens VP1_1-297 (full antigen), VP1_1-60 (N antigen), VP1_45-58 (core antigen), VP1_41-297 (C antigen), and a control antigen (VP1_61-297) without the CECRS were designed and constructed.</p

Comparison of the inhibition activities of five proteins in the Shanghai blood donors (a) and Shanxi individuals (b).

<p>The percentage of inhibition based on the competitive ELISA was plotted on the y-axis and six inhibitor proteins were plotted on the x-axis (CA16 VP1, VP1_41-297, VP1_1-60, VP1_45-58, VP1_61-297 and pET32a). Each symbol represents an individual sample, and the lines indicate Q1, Q2 and Q3 value of the group from the bottom to the top, respectively. Statistical significance was tested using the Nemenyi non-parametric test. * indicates p<0.05, ** indicates p<0.001. # represents p<0.05 comparing all other groups.</p

Characterization of antibody reactivity against CA16 VP1 and the anti-CA16 neutralizing titres of 70 and 43 serum samples strongly reactive against VP1 from the Shanghai blood donors (a and b) and Shanxi individuals (c and d).

<p>The samples strongly reactive against VP1 were divided into two groups: the specific inhibition type (the anti-CA16 VP1 reaction were completely inhibited by VP1_1-60 and VP1_41-297) and the other types. The specific inhibition type and the other types include 48 and 12 serum samples in the Shanghai blood donors and 3 and 40 serum samples in the Shanxi individuals, respectively. Statistical significance was tested using the independent samples t test and Wilcoxon rank sum test. * indicates p<0.05, ** indicates p<0.001, “NS” represents no significant difference (p>0.05).</p

Analysis of the correlation between the reactivity and inhibition of anti-CA16 VP1 reaction between complete CA16 VP1 and the four truncated VP1 proteins in the Shanghai blood donors and Shanxi individuals.

<p>Analysis of the correlation between the reactivity and inhibition of anti-CA16 VP1 reaction between complete CA16 VP1 and the four truncated VP1 proteins in the Shanghai blood donors and Shanxi individuals.</p

Characterization of different types of inhibition profiles against anti-CA16 antibody response by five antigens between the Shanghai blood donors (a) and Shanxi individuals (b).

<p>The inhibition profiles against anti-CA16 reaction were sorted based on the combination of inhibition degrees by VP1_1-60 and VP1_41-297. Eleven types of inhibition profiles were defined in both groups. Individual values are shown in colour as indicated beneath Fig 4b, with yellow representing the strongest inhibition degree. The constituent ratios of the types of inhibition profiles in the Shanghai blood donors (c) and Shanxi individuals (d) are shown.</p

IgM confirmation detection of five cases of HRP-LD5 assay positive alone serum samples^*.

<p>*Serum samples treated without (−) or with (+) β-mercaptoethanol (BME) were used to test anti-HCV IgM antibodies. Data shown are the A₄₅₀ values, and the cut-off values of the ELISA were either 0.200 (HRP-LD5 based assay and established anti-HCV IgM assay) or 0.150 (Chang Zheng kit).</p

Anti-HCV detection in 195 serum specimens from hemodialysis patients by different ELISA assays.

<p>, A₄₅₀ values, with ELISA cut-off values of either 0.150 indicated in solid line (established anti-HCV IgG assay and Chang Zheng anti-HCV diagnostic ELISA kit) or 0.200 indicated in dotted line (established anti-HCV IgM assay and HRP-LD5 based assay).</p

Comparison of anti-HCV detection against the national reference panel of HRP-LD5 assay with three commercial diagnostic kits.

1<p>95% CI, 95% confidence interval.</p

Comparison of anti-HCV antibody detection in serum samples from 195 hemodialysis patients of HRP-LD5 based assay with Chang Zheng anti-HCV kit.

1<p>Data shown are the number of cases with each test result. Data shown in parentheses are the percentages of detected cases in the 195 total serum samples.</p>2<p>95% CI, 95% confidence interval.</p