Antibody Responses against Enterovirus Proteases are Potential Markers for an Acute Infection

Figures

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  Figure 1. Viral 3C and VP1 proteins are expressed in murine pancreata following infection with Coxsackievirus B3 (CVB)3. C57BL/6J mice were mock-infected with PBS (A,C,E,G) or infected with CVB3 (102 PFU/mouse) (B,D,F,H) for 3 days. Pancreas sections were stained with either an antibody detecting 3C (A,B) or a directly biotinylated antibody detecting VP1 (E,F). As control for the 3C staining, sections from mock-infected and CVB3 infected animals were stained with a biotinylated secondary antibody alone (C,D) prior to visualization of antibody binding by DAB as described in Materials and Methods section. For the VP1 controls, sections were incubated without secondary antibody prior to visualization of antibody binding (G,H). Representative sections are shown (n = 3–4 animals per condition). Positivity is indicated with the brown staining. Original magnification ×10.

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  Figure 2. Antibody reactivity towards enterovirus structural and non-structural proteins in serum from 6-month old children, adults and CVB infected mice. (A) 2A, 3C, and CVB3 VP1 antibodies in sera pooled from 6 month-old humans (1:1000 dilution, white) and from Nanogam® serum (1:10,000 dilution, gray bars) and (B) 2A, 3C, VP1, and CVB3 antibodies in pooled naïve (white bars) and CVB3 infected (21 dpi; grey bars) mouse sera (both 1:1000 dilution) as detected by ELISA. Each bar represents a pool of sera (n = 20 for humans, n = 18 for infected mice and n = 4 for naïve mice). Data is presented as means ± S.E.M.

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  Figure 3. CVB-infection induces a short-lived antibody response towards CVB3 proteases in mice. Antibody responses towards the protease antigens (A) 2A and (B) 3C (at 1:1000 dilutions) and to the (C) structural proteins in whole virus and (D) the capsid protein VP1 (1: 64,000) in CVB3 infected mice measured using ELISA. The data is shown as the fold change compared to antibody responses from pre-infection serum samples. Each data point is an average of three parallel samples. Stat: Mann–Whitney test, means and standard errors of means shown as gray bars, * p < 0.05 and ** p < 0.001.

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  Figure 4. Antibody response towards viral proteins in acutely infected patients. Serum samples obtained from acutely infected humans with confirmed enteroviral infections (IgM positive, n = 22) and healthy controls (IgM negative, n = 20) were analyzed for immunoreactivity towards the indicated viruses or viral proteins. (A) Boxplot diagrams show the antibody responses towards different antigens analyzed using ELISA (serum diluted 1:1000) for acutely infected human patients (gray) and controls (black). ** p < 0.001 and *** p < 0.0001, two-tailed Wilcoxon signed-rank test analyzed in R. (B) The data set in A was used for creating a correlation matrix with pairwise comparisons. The top-right diagonal in panel (B) shows the Spearman correlation coefficients of antigen pairs and the bottom-left diagonal shows pairwise scatterplots with linear regression trend lines. Abbreviations: HPIV, human Parainfluenzavirus; ADV, Adenovirus; 2A, CVB3 2A protease; 3C, CVB3 3C protease; VP1, CVB3 VP1; CVB3, heat inactivated whole Coxsackie B3 virus.

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  Figure 5. Acute enterovirus infection criteria and test descriptors based on ELISA results. (A) Different cutoff criteria based on 2A and 3C signals (based on 6-month children average + 3 × SD (n = 20) or the adjusted thresholds) and IgM positivity. In the plot (A), IgM positive data points are colored red and control data points are cyan. (B) Confusion matrix and test descriptors calculated from the traditional cutoff values based on results from 6-month children comparing the new protease-ELISA test to the IgM ELISA by analysis in R. Sensitivity is the proportion of true positives and specificity is the proportion of true negatives correctly categorized with respect to IgM ELISA by the protease ELISA. LR+ and LR– stand for positive and negative likelihood ratios respectively and DOR the overall diagnostic odds ratio. (C) Confusion matrix and test descriptors based on the adjusted diagnosis criteria.