w/o, without. Epitope mapping of group I anti-L1 MAb. CDR2 of the heavy chain, which are highly conserved among antibodies realizing the epitope. These antibodies, however, experienced divergent light-chain and heavy-chain CDR3 sequences. Our study demonstrates that this conformational L1 epitope with Asp35 is usually a common site of vulnerability for potent neutralization by a divergent group of antibodies. IMPORTANCE Vaccinia computer virus, the live vaccine for smallpox, is one of the most successful vaccines in human history, but it presents a level of risk that has become unacceptable for the current populace. Studying the immune protection mechanism of smallpox vaccine is usually important for understanding the basic principle of successful vaccines and the development of next-generation, safer vaccines for highly pathogenic orthopoxviruses. We analyzed antibody targets in smallpox vaccine by developing potent neutralizing antibodies against vaccinia computer virus and comprehensively characterizing their epitopes. We found a site in vaccinia computer virus L1 protein as the target of a group of highly potent murine neutralizing antibodies. The analysis of antibody-antigen complex structure and the sequences of the antibody genes shed light on how these potent neutralizing antibodies are elicited from immunized mice. INTRODUCTION Variola computer virus and monkeypox computer virus are orthopoxviruses that are highly pathogenic to humans, are considered to be potential bioterrorism brokers (1), and are emerging pathogens (2). A related orthopoxvirus, vaccinia computer virus AN2728 (VACV), serves as the vaccine against these pathogens. Live VACV immunization is usually capable of eliciting neutralizing antibodies against a variety of targets on two antigenically unique forms of virions, the intracellular mature virions (MV) and the extracellular enveloped virions (EV) (3, 4). Vaccinia vaccine is usually arguably the most successful vaccine in human history, having led to the eradication AN2728 of smallpox (5). However, it was also associated with a relatively high rate of adverse events (6). Consequently, safer multicomponent DNA or protein vaccines that include a subset of MV and EV antigens (Ag) have been developed, and they showed protection against orthopoxvirus difficulties in mice and nonhuman primates (7,C10). While many MV antigens have been shown to be neutralization targets (11, 12), the MV antigen that is invariably included in these subunit vaccines is usually L1. L1 is an immunodominant neutralizing antibody target in mice, although it is usually a less common target in humans (13). It is a 250-amino-acid myristoylated protein with a C-terminal transmembrane domain name that spans residues 186 to 204 (14, 15). L1 associates with the virus-encoded multiprotein entry-fusion complex (EFC) and plays an essential role in viral access (16). Despite the importance of L1 as a neutralizing target and AN2728 subunit vaccine component, relatively little is known about its neutralizing epitopes Rabbit Polyclonal to MEF2C and the corresponding paratopes. A conformational epitope with Asp35 as the key residue is usually recognized by several murine monoclonal antibodies (MAbs) (17), which potently neutralize MV. The sequence of one of the MAbs, 7D11, has been reported, and a structure of the Fab domain name of 7D11 bound to L1 has been decided (18). A linear epitope (residues 118 to 128) of L1 is usually recognized by several antibodies, which neutralized MV with reduced potency compared to 7D11 (19). In an effort to gain a more comprehensive understanding of neutralizing epitopes on L1 and the neutralizing mechanism of anti-L1 antibodies, we developed additional MAbs against L1, examined their neutralizing abilities and studies. Monoclonal antibodies used in the study are the following: anti-B5, B126 (21); anti-A10, BG3.1 (22); anti-H3, JH4 (22) and 41 (23); anti-A27, 1G6 and 12G2 (unpublished data); anti-D8, JE10 (22) and LA5 (24). Hybridoma generation and characterization. The generation and characterization of the AN2728 hybridomas were performed as explained previously (22), except for some changes in the immunization process explained below. BALB/c mice in the beginning were infected intranasally with 5 103 PFU of VACVWR. Subsequently, the mice were boosted twice with intraperitoneal (i.p.).