A Neonatal Mouse Model of Coxsackievirus A16 for Vaccine Evaluation

Abstract

cTo evaluate vaccine efficacy in protecting against coxsackievirus A16 (CA16), which causes human hand, foot, and mouth disease (HFMD), we established the first neonatal mouse model. In this article, we report data concerning CA16-induced pathological changes, and we demonstrate that anti-CA16 antibody can protect mice against lethal challenge and that the neonatal mouse model could be used to evaluate vaccine efficacy. To establish a mouse model, a BJCA08/CA16 strain (at 260 50% lethal doses [LD 50 ]) was isolated from a patient and used to intracerebrally (i.c.) inoculate neonatal mice. The infection resulted in wasting, hind-limb paralysis, and even death. Pathological examination and immunohistochemistry (IHC) staining indicated that BJCA08 had a strong tropism to muscle and caused severe necrosis in skeletal and cardiac muscles. We then found that BJCA08 pretreated with goat anti-G10/CA16 serum could significantly lose its lethal effect in neonatal mice. When the anti-G10 serum was intraperitoneally (i.p.) injected into the neonatal mice and, within 1 h, the same mice were intracerebrally inoculated with BJCA08, there was significant passive immunization protection. In a separate experiment, female mice were immunized with formaldehyde-inactivated G10/CA16 and BJCA08/CA16 and then allowed to mate 1 h after the first immunization. We found that there was significant protection against BJCA08 for neonatal mice born to the immunized dams. These data demonstrated that anti-CA16 antibody may block virus invasion and protect mice against lethal challenge, and that the neonatal mouse model was a viable tool for evaluating vaccine efficacy. C oxsackievirus A16 (CA16) belongs to the Enterovirus genus of the Picornaviridae family and is one of the major pathogens associated with human hand, foot, and mouth disease (HFMD) (4,16,19). CA16 was first isolated in 1951 (43). It is a single positive-stranded RNA virus and has an icosahedral symmetry structure. Its genome has approximately 7,410 nucleotides with one predominant serotype. Based on the VP4 nucleotide sequences, CA16 is classified into three distinct genetic lineages: A, B, and C. Before the 1990s, lineages A and B were the major epidemic strains in Asia (predominantly the B strain). After that, the CA16 gene gradually mutated to form lineage C, which replaced the B strain as the predominant epidemic strain (22).Epidemics of HFMD have been reported in England, Australia, Japan, Germany, Malaysia, Singapore, mainland China, and Taiwan (2,4,11,19,23,27,32,38,41,48). Recently, HFMD was highly epidemical in the west Pacific region, resulting in severe illness and fatalities (15, 23). The HFMD epidemics were mainly caused by CA16 and human enterovirus 71 (EV71), which circulated alternatively or together in the epidemic area (19,22,23,25,37). Because the most severe or fatal cases were caused by EV71, studies have mainly focused on EV71 but not CA16. However, in England, the largest HFMD outbreak (in 1994) was caused by CA16 (2). Similarly, in Taiwan the leading cau...

Evaluation of monovalent and bivalent vaccines against lethalEnterovirus71 andCoxsackievirusA16 infection in newborn mice

“…Many research groups have reported that inactivated virus and VLP vaccines retain their antigenicity and immunogenicity. 24,26,35 A formalin-inactivated EV71 vaccine was recently shown to induce either low or no cross-neutralization activity against CVA16 and vice versa. 35 Therefore, a bivalent EV71 and CVA16 vaccine should be considered as a candidate for HFMD.…”

“…VLPs have a greater safety profile than inactivated vaccines since they do not contain the virus genome. Currently, several experimental CVA16 and EV71 vaccines are under development, including inactivated CVA16, [23][24][25] and CVA16 VLPs derived from insect cells 26 and Saccharomyces cerevisiae. 27 Among these, the EV71 inactivated vaccine has been evaluated and passed through a phase III clinical trial, while the development of EV71 VLPs in insect cells and S. cerevisiae is still ongoing.…”

Is a multivalent hand, foot, and mouth disease vaccine feasible?

“…186 Mouse-adapted strains, neonatal suckling mice and immunodeficient animals have been widely used to evaluate the protective efficacy of EV-A71 and CV-A16 vaccine candidates, but they do not mimic human infections. 168,186,187 In contrast, cardiac pathogenesis in Balb/c and SWR mice infected with CV-B3 is very similar to that of human patients. 177 Macaques develop antibody responses to EV-A71 vaccines similar to those observed in human; however they are not suitable to study neurovirulence and pulmonary edema complications and their use is limited by ethical and economic considerations.…”

Comparative study of the immunogenicity in mice and monkeys of an inactivated CA16 vaccine made from a human diploid cell line

“…(B) G10 challenge group: 400 eU × 2n (n = 12-13), 200 eU × 2n (n = 10-11), 100 eU × 2n (n = 10-11), 50 eU × 2n (n = 12-15), 25 eU × 2n (n = 11-16), 12.5 eU × 2n (n = 13-15), n.c × 2n (n = 10-12). (C) Ma154 challenge group: 400 eU × 2n (n = 10-11), 200 eU × 2n (n = 12-13), 100 eU × 2n (n = 10-13), 50 eU × 2n (n = 12-13), 25 eU × 2n (n = 11-15), 12.5 eU × 2n (n = 10-13), n.c × 2n (n = [12][13][14] monkeys, the considerably higher antibody GMTs most likely exhibited a cross-neutralization ability to different genotype viral strains rather than the lower antibody GMTs; this is an essential issue to consider when determining the antigen dosage for the development of candidate inactivated CA16 vaccines.…”

“…12 Although some studies report that mice could be a candidate model for HFMD, the characterization of this immune response in different animal models (especially in primates, who share a close relationship to humans) would provide more support for these studies. 13 In this study, we characterized the immune response of mice and rhesus monkeys induced by an experimental inactivated vaccine prepared from CA16 virus that was adapted to grow in a human diploid cell line. The results showed that the immune response induced by this candidate vaccine could produce a specific neutralizing antibody and a specific IFN-γ-secreting cellular response against viral strains that were of different or similar genotypes; thus, this inactivated CA16 is a candidate vaccine.…”