Neutralizing Antibodies of Inactivated Thai Enterovirus A71 Strain in Mice for Development of Enterovirus A71 Vaccine

Abstract

Since enterovirus A71 is known as a pathogen which may cause severe complications as critical neurological manifestations, pulmonary edema, cardio respiratory failure and even death to infected children, therefore, the vaccine against EV-A71 infection has been expected to prevent such serious problems even in Thailand. In this study, we developed a vaccine candidate from a sub genotype C4 EV-A71 strain collected from a Thai fatal case. The target virus was firstly compared VP1 nucleotide and amino acid identities with other 13 Thai strains of one C4, three C5 and nine B5 sub genotypes. For nucleotide homologies, the virus shared 96.3%, 91.5%, and 90.3%, respectively, while it contained amino acid identities as 99.9%, 100% and 97.2%, with C4, C5 and B5 strains, respectively. Before vaccine development, the target virus was initially confirmed to be a single strain by inoculation of a single plaque serially from first cell culture to another, and the passage 9 still showed positive to the monoclonal antibody against EV-A71 by IFA. For production of the virus, EV-A71 could be cultured very well in Vero cells using roller bottles which the yield was 4.4 4.5 x 109 pfu/ml at day 3 4 post infection. By purification, total proteins left were monitored after 100 kDa tangential flow filtration and 10% – 50% sucrose density gradient centrifugation as 46.2% and 1.0% mean, respectively. Immunogenicity of the inactivated EV-A71 produced was tested in mice. After a single injection of 1 or 2.5 μg purified total proteins, it induced neutralizing antibodies against the homologous virus especially when with alum, as compared to placebo groups. After 2nd immunization, both 1 and 2.5 μg with alum induced many antibodies than those without alum and the groups of a single immunization. The 3rd immunization of the vaccines gave very much titer in all immunized groups even without alum; however, highest was 24,525 TCID50/ml after 4 weeks by 1 μg with alum. All titers seemed to maintain after 6 weeks studied. This study confirmed that an inactivated EV-A71 vaccine with triple injections is a good choice for further development.