Identification by Phage Display and Characterization of Two Neutralizing Chimpanzee Monoclonal Antibodies to the Hepatitis E Virus Capsid Protein

Abstract

Two monoclonal antibodies (MAbs) against the ORF2 protein of the SAR-55 strain of hepatitis E virus (HEV) were isolated by phage display from a cDNA library of chimpanzee (Pan troglodytes) ␥1/ antibody genes. Both MAbs, HEV#4 and HEV#31, bound to reduced, denatured open reading frame 2 (ORF2) protein in a Western blot, suggesting that they recognize linear epitopes. The affinities (equilibrium dissociation constants, K d ) for the SAR-55 ORF2 protein were 1.7 nM for HEV#4 and 5.4 nM for HEV#31. The two MAbs also reacted in an enzyme-linked immunosorbent assay with recombinant ORF2 protein from a heterologous HEV, the Meng strain. Each MAb blocked the subsequent binding of the other MAb to homologous ORF2 protein in indirect competition assays, suggesting that they recognize the same or overlapping epitopes. Radioimmunoprecipitation assays suggested that at least part of the linear epitope(s) recognized by the two MAbs is located between amino acids 578 and 607. MAbs were mixed with homologous HEV in vitro and then inoculated into rhesus monkeys (Macaca mulatta) to determine their neutralizing ability. Whereas all control animals developed hepatitis (elevated liver enzyme levels in serum) and seroconverted to HEV, those receiving an inoculum incubated with either HEV#4 or HEV#31 were not infected. Therefore, each MAb neutralized the SAR-55 strain of HEV in vitro.Hepatitis E is an acute disease endemic in many countries throughout developing parts of the world, in particular on the continents of Africa and Asia, where epidemics have also occurred. The causative agent, hepatitis E virus (HEV), is transmitted via the fecal-oral route, predominantly through contaminated water (45). HEV is an RNA virus with a positive-sense genome approximately 7.5 kb in length. The genome contains three open reading frames (ORFs); ORF2 encodes the putative capsid protein (45). Hepatitis E has a low mortality rate in young adults (38). However, mortality rates can reach 20% in women in the third trimester of pregnancy (32, 58). Surprisingly, in industrialized countries such as the United States, where hepatitis E is not endemic, a significant proportion of healthy individuals within the general population are seropositive (over 20% in some areas; 39, 50). However, clinical hepatitis E is rare in these countries and usually is seen in individuals who acquired the infection during travel to a region in which HEV is endemic or epidemic.It has been suggested that animals serve as reservoirs for HEV and that human infections may, in part, be zoonoses. There have been several reports of HEV-specific (anti-HEV) antibody in animals (1, 9, 28, 29). Furthermore, an HEV-like virus was recently isolated from naturally infected swine in the United States (40). Although four genotypes of HEV have been identified, to date, only one serotype of HEV has been recognized. Therefore, it may be possible to produce a broadly protective vaccine. Passively transferred anti-HEV serum significantly reduced virus shedding in feces and abrogated disease in nonhu...

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