Performance of Detecting IgM Antibodies against Enterovirus 71 for Early Diagnosis

Enterovirus 71 (EV71) infection is more likely to induce severe complications and mortality than other enteroviruses. Methods for detection of IgM antibody against EV71 had been established for years, however, the performance of the methods in the very early diagnosis of EV71 infection had not been fully evaluated, which is especially meaningful because of the short incubation period of EV71 infection. In this report, the performance of an IgM anti-EV71 assay was evaluated using acute sera collected from 165 EV71 infected patients, 165 patients infected with other enteroviruses, and more than 2,000 sera from healthy children or children with other infected diseases. The results showed a 90% sensitivity in 20 patients who were in their first illness day, and similar sensitivity remained till 4 days after onset. After then the sensitivity increased to 95% to 100% for more than one month. The specificity of the assay in non-HFMD children is 99.1% (95% CI: 98.6–99.4), similar as the 99.9% specificity in healthy adults. The cross-reaction rate in patients infected with other non-EV71 enteroviruses was 11.4%. In conclusion, the data here presented show that the detection of IgM anti-EV71 by ELISA affords a reliable, convenient, and prompt diagnosis of EV71 infection

In vivo time-related evaluation of a therapeutic neutralization monoclonal antibody against lethal enterovirus 71 infection in a mouse model.

Enterovirus 71 (EV71) is a neurotropic virus capable of inducing severe neurological symptoms and death. No direct targeting antivirals are useful in the treatment of severe EV71 infection. Because of low toxicity and good specificity, monoclonal antibodies (MAb) are a potential candidate for the treatment of viral infections. Therefore, we developed an EV71-specific conformational MAb with high in vitro cross-neutralization activity to heterologous EV71 subgenotypes. The in vivo treatment experiment at different days post-infection indicated that a single treatment of MAb CT11F9 within day 3 post-infection fully protected mice from morbidity and mortality (0% PBS vs. 100% at 10 µg/g per body weight ***P<0.0001). Immunohistochemical and histological analysis confirmed that CT11F9 significantly prohibited EV71 VP1 expression in various tissues and prevented EV71-induced myonecrosis. Moreover, thrice-treatment at day 4, 5, 6 post-infection was associated with an increased survival rate (18.2% single vs. 50% thrice at 20 µg/g per body weight), and the mice recovered from limb paralysis. Competitive ELISA also confirmed that CT11F9-recognized epitopes were immunodominant in humans. In conclusion, MAb CT11F9 is an ideal candidate to be humanized and used in severe EV71 infection

Antigenic analysis of divergent genotypes human Enterovirus 71 viruses by a panel of neutralizing monoclonal antibodies: Current genotyping of EV71 does not reflect their antigenicity

In recent year, Enterovirus 71 (EV71)-associated hand, foot and mouth disease (HFMD) has become an important public health issue in China. EV71 has been classified into genotypes A, B1–B5 and C1–C5. With such genetic diversity, whether the convalescent or recovery antibody responses can cross-protect infections from other genotypes remains a question. Understanding of the antigenicity of such diverse genetic EV71 isolates is crucial for the EV71 vaccine development. Here, a total of 186 clones anti-EV71 MAbs was generated and characterized with Western blot and cell-based neutralization assay. Forty neutralizing anti-EV71 MAbs were further used to analyze the antigenic properties of 18 recent EV71 isolates representing seven genotypes in neutralization assay. We found that most neutralizing anti-EV71 MAbs are specific to conformational epitopes. We also classified the 40 neutralizing anti-EV71 MAbs into two classes according to their reactivity patterns with 18 EV71 isolates. Class I MAb can neutralize all isolates, suggesting conserved epitopes are present among EV71. Class II MAb includes four subclasses (IIa–IId) and neutralizes only subgroups of EV71 strains. Conversely, 18 EV71 strains were grouped into antigenic types 1 and four antigenic subtypes (2.1–2.4). These results suggest that the current genotyping of EV71 does not reflect their antigenicity which may be important in the selection of EV71 vaccine strains. This panel of neutralizing anti-EV71 MAbs may be useful for the recognition of emerging antigenic variants of EV71 and vaccine development

Antigenic analysis of divergent genotypes human Enterovirus 71 viruses by a panel of neutralizing monoclonal antibodies: Current genotyping of EV71 does not reflect their antigenicity

In recent year, Enterovirus 71 (EV71)-associated hand, foot and mouth disease (HFMD) has become an important public health issue in China. EV71 has been classified into genotypes A, B1–B5 and C1–C5. With such genetic diversity, whether the convalescent or recovery antibody responses can cross-protect infections from other genotypes remains a question. Understanding of the antigenicity of such diverse genetic EV71 isolates is crucial for the EV71 vaccine development. Here, a total of 186 clones anti-EV71 MAbs was generated and characterized with Western blot and cell-based neutralization assay. Forty neutralizing anti-EV71 MAbs were further used to analyze the antigenic properties of 18 recent EV71 isolates representing seven genotypes in neutralization assay. We found that most neutralizing anti-EV71 MAbs are specific to conformational epitopes. We also classified the 40 neutralizing anti-EV71 MAbs into two classes according to their reactivity patterns with 18 EV71 isolates. Class I MAb can neutralize all isolates, suggesting conserved epitopes are present among EV71. Class II MAb includes four subclasses (IIa–IId) and neutralizes only subgroups of EV71 strains. Conversely, 18 EV71 strains were grouped into antigenic types 1 and four antigenic subtypes (2.1–2.4). These results suggest that the current genotyping of EV71 does not reflect their antigenicity which may be important in the selection of EV71 vaccine strains. This panel of neutralizing anti-EV71 MAbs may be useful for the recognition of emerging antigenic variants of EV71 and vaccine development

Development of an IgM-capture ELISA for Coxsackievirus A16 infection

National Key Technology Research and Development program of China [2008BAI70B00]; Science and Technology Plan Program of Xiamen City [3502Z20093010]; National Natural Science Foundation of China [30972826]Diagnosis of Coxsackievirus A16 (CA16) infection in China relies mainly on reverse transcription-polymerase chain reaction (RT-PCR) that require expensive equipment and special trained personnel, thus making its wide application in health care settings unlikely. In this study, a novel IgM anti-CA16 assay was developed for the detection of IgM antibodies to CA16 in serum. The responses and diagnostic value of IgM for the CA16 infection were:assessed by testing 1970 serum samples. The results showed that sensitivity of IgM test was 84.6% (259/306, 95% CI: 80.1-88.5), and specificity in control subjects and patients with CA16 HFMD was 99.2% (1508/1520, 95% CI: 98.6-99.6) and 90.3% (14/144, 95% CI: 84.2-94.6), respectively. The IgM positive rate reached 56.3% in the sera collected within the first day after onset, increased continuously to 95.3% at day 5 to day 7 after onset, and then reached 100% after more than 8 days. The cross-reaction rate in patients infected with other non-CA16 enteroviruses was 9.7% (14/144). These results suggest that the IgM anti-CA16 assay offers a rapid, convenient, and reliable method to detect acute CA16 infections. (C) 2010 Elsevier B.V. All rights reserved

<i>In vivo</i> single-treatment of CT11F9 at different times after infection with EV71.

<p>(<b>A</b>) Kaplan-Meier survival curve and health scores of newborn BALB/c mice i.p. challenged with 10⁷ TCID₅₀ pSVA-MP4 and then treated with MAb CT11F9 at different days post-infection (DPI). Deaths were scored as 5 until the end of the experiment; the treatment time is indicated by the black arrow. (<b>B</b>) Brain, limb muscle and intestine were analyzed by IHC; limb muscle was also analyzed by HE staining. Positive reactions are indicated by the black arrow. Representative images are shown at a magnification of 200X. The asterisks indicates significant differences of ***P<0.0001.</p

Analysis of CT11F9 by immunofluorescence and an <i>in vitro</i> neutralization test.

<p>(<b>A</b>) Immunofluorescence images of CT11F9 against EV71 Jiangsu-infected RD cells or uninfected RD cells are shown as DAPI, GFP and a merged image of DAPI and GFP, respectively. (<b>B</b>) Neutralization titers of MAb CT11F9 against seven EV71 subgenotypes and CA16.</p

Competitive ELISA of CT11F9 against human serum.

<p>The MAb CT11F9 (10 µg/g) competed with eight human serum samples for specifically binding to EV71 Jiangsu coated on the well. The irrelevant MAb control was a CB3-VP1 targeted MAb. The MAb CT11F9 blocked 63% of EV71-infected human sera from binding EV71 Jiangsu. The asterisks indicates significant differences of ***P<0.0005.</p