Twenty-nine throat swab samples from healthy children were collected under the permission of their parents or guardians in 2010. All the specimens collected from healthy children in this study were confirmed as negative for EV71 infection(EV71-) and had no clinical EV71-infection hospital record(detailed information not shown).
The time of samples collection span three years from 2010 to 2012. One hundred throat swab specimens were enrolled from young outpatients, which had been diagnosed clinically as suspected EV71 infection by physician and then further confirmed by molecular assay realtime PCR(EV71+). Eighty-six among one hundred patients were under 5 years old. Numbers of male and female patients are totally 69 and 31 respectively. Ninetysix patients went to a hospital within 3 days once clinical symptoms appeared. The detailed patient demographic information is summarized in Table 1.
Table 1. Characteristics of 100 children with clinically confirmed EV71 infection
In order to identify the possibility of existence of EV71-specific antibody IgG in clinically confirmed spec-imen, we examined them by ELISA coating VP1, VP2, 3D as well as purified inactivated EV71 virus particle. The specimens were four fold diluted and determined the IgG response for VP1, VP2, 3D and EV71. Value of OD405 by ELISA was used to reflect the level of IgG antibody. And for the same clinical sample, different IgG antibody profiles appeared when detected with distinct proteins or virus. Basically, antibody in most specimens could not be detected when thirty-two or more fold diluted, which therefore mean a relatively low-level of IgG titer for EV71 in mucosal specimen.
To discover the possible relationship of IgG titers between VP1, VP2, 3D as well as purified EV71 for each sample, we analysed the correlation of the IgG titers detected by different coating proteins by a Spearman linear regression. Resultantly, IgG response against any coating protein(VP1, VP2, 3D or EV71) significantly correlated with that of one another(p < 0.0001)(Figure 1). The results released that low-level of mucosal IgG antibody against EV71 broadly existed in children population, and IgG response against viral capsid protein significantly correlate with that against non-structure protein, e.g. 3D.
Figure 1. Relationships between IgG titers against different components of EV71 in the specimens with viral infection. The specimens were four fold diluted and determined the IgG response for VP1, VP2, 3D and EV71 by ELISA. Value of OD405 by ELISA was used to reflect the level of IgG antibody. And then the correlations of the IgG titers detected by different coating proteins were determined by a Spearman linear regression. The IgG titer of VP2 significantly correlated with that of VP1 (A) and EV71 (B) (p < 0.0001). The IgG titer of VP1 significantly correlated with that of EV71 (C) (p < 0.0001). The IgG titer of 3D significantly correlated with that of VP2 (D), VP1 (E) as well as EV71 (F) (p < 0.0001).
To assess the EV71-specific IgG profile in virus infected clinical sample, we first determined the cut-off value of IgG response against VP2, VP1, 3D, or EV71 in healthy control by ELISA. Throat swab specimens from twenty-nine healthy children were collected and measured for VP2 and 3D responses, in which RNA copy number was under 100 copies of virus/mL by using realtime PCR. Therefore, virus-or protein-specific IgG were reported as absorbance at 405 nm, and values exceeding [Mean(seronegative control)+ 2 standard deviations] were considered positive. Based on this formula, the cutoff values of IgG VP2, 3D, VP1 and EV71 were calculated to be 0.113, 0.142, 0.094, and 0.092 respectively(Figure 2A–D).
Figure 2. Relationships between IgG antibody titers and RNA copy numbers in EV71-infected clinical samples. IgG responses for VP2 and 3D of throat swab specimens from healthy children and EV71-infected patients by ELISA. Pharyngo-laryngeal mucosal specimen giving OD405 values greater than the mean OD405 plus two standard deviations for healthy children controls were considered antibody response positive. The cut-off value for VP2 IgG is 0.113 (discontinuity line). The mean RNA copy number of VP2 IgG positive group is strikingly higher than that of VP2 IgG negative group (p = 0.0009) (A). The cut-off value for 3D IgG is calculated 0.142 (discontinuity line), and the mean RNA copy number in 3D IgG positive group is prominently higher than that of 3D IgG negative (p = 0.0026) (B). Likewise, VP1 and EV71 antibody positive samples present significant EV71 RNA genome number than that of IgG negative samples (p = 0.0007 and p = 0.0004) (C, D).
Combing the information above mentioned that 96% patients went to a hospital within a few days once clinical symptoms appeared, we speculated that the occurrence of detected low-level of antibody must be induced due to the former EV71 infection rather than the ongoing sickness. In order to determine whether the preexisting viral antibody IgG correlate with the viral infection in vivo, we here divided the whole infected samples into two parts based on the antibody response against EV71 VP2: first group with IgG OD405 value < 0.113(without former VP2 IgG) and second group with OD405 value ≥ 0.113(with former VP2 IgG)(Figure 2A). The mean RNA copy number of VP2 IgG positive group is strikingly higher than that of VP2 IgG negative group(p = 0.0009)(Figure 2A). Meanwhile, the OD405 value under 0.142 was considered 3D IgG response negative, thus thirty specimens were negative(without preexisting 3D IgG) and seventy specimens were positive(with preexisting 3D IgG). Likewise, the mean RNA copy number in 3D IgG positive group is prominently higher than that of 3D IgG negative(p = 0.0026)(Figure 2B). Furthermore, VP1 and EV71 antibody positive samples present significant EV71 RNA genome number than that of IgG negative samples(p = 0.0007 and p = 0.0004). It therefore indicates that the children population with mucosal preexsiting EV71 IgG were prone to be infected.
In order to further evaluate the effect of preexsiting antibody on the EV71 infection and replication in vivo, we further analysed the relationship of EV71-specific antibody titer and viral copy number in each sample by a Spearman linear regression. Subsequently, the mean RNA copy number strikingly correlated with the VP2-IgG titer(p = 0.0101)(Figure 3A). Likewise, the mean RNA copy number prominently associated with 3D-IgG(p = 0.0042), VP1-IgG(p = 0.0189) and EV71-IgG(p = 0.0029) titer(Figure 3B–D). However, the difference of protein contents in each sample were not significant, which excluded the possibility that samples containing high amount of total material(cells, secretions) contain automatically higher amounts of virus and higher amounts of antibodies(data not shown). Overall, it further elucidated that the viral acquisition and replication increased accompanying the raise of mucosal preexsiting IgG response against EV71 structure and non-structure proteins, which suggested a former viral IgG mediated enhancement of viral infection in vivo.
Figure 3. Correlation between IgG titer and RNA copy number in infected specimens. The mean RNA copy number strikingly increases accompanied with the elevation of VP2-specific IgG titer (p = 0.0101) (A). Likewise, the mean RNA copy number strikingly increases accompanied with the elevation of 3D-specific (p = 0.0042) (B), VP1-specific (p = 0.0189) (C) and EV71-specific (p = 0.0029) (D) IgG titer. Each correlation was analyzed using a Spearman nonparametric test with GraphPad Prism software version 5.0.