West Nile virus (WNV), a positive-sense RNA virus and a member of the Flaviviridae family, recently became endemic in North America, with annual outbreaks of severe encephalitis occurring mostly in immunocompromised or elderly individuals. There is currently no vaccine approved for human use, and treatment is primarily supportive. The WNV genome encodes three structural proteins (C, prM/M, and E) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). During the course of WNV infection, antibodies are raised against prM/M and E as well as NS1, NS3, and NS5, with a majority of the protective antibody response against the E protein (12, 63).The crystal structure of the ectodomain of the E protein has been determined for dengue virus (DENV), tick-borne encephalitis virus (TBEV), and WNV (43,45,48,56,65). Flavivirus E proteins have three separate domains and form headto-tail homodimers on the surface of the virion. Domain I (DI) is the central structural domain and consists of a 10-stranded ␤-barrel. DII is formed from two extended loops that project from DI. At the end of DII is a highly conserved loop, amino acid residues 98 to 110, that has been implicated in the acidcatalyzed type II fusion event (1,7,44). In the E dimer, the fusion loop lies in a pocket at the DI-DIII interface of the adjacent E protein. DIII, located on the opposite side of DI, forms a seven-stranded immunoglobulin-like fold and has been implicated in receptor binding (5, 10, 14). Short, flexible linker regions connect the domains and allow for the conformational changes associated with virus maturation and fusion (65).The structure of the WNV virion has been defined by cryoelectron microscopy (36, 47). The mature WNV is ϳ500 Å in diameter and has a relatively smooth surface with no apparent spikes or large projections. The 180 E monomers lay flat along the virion surface as sets of three parallel dimers. The arrangement of the 180 E monomers has quasi-icosahedral symmetry such that there are three E monomers in the asymmetric unit and three distinct chemical environments available for antibody or ligand binding (47). The reduced pH in the endosome causes the E protein to convert from a homodimer to a homotrimer and exposes the fusion loop (44).Antibodies are critical for the control of flavivirus infection in vivo (4,17,18,20,23,50,59), and this protection has been correlated with neutralizing activity in vitro (32,53,58). However, there have been reports of strong and weak in vivo protection with nonneutralizing (6,11,29,31,34,58) and neutralizing (30, 32, 41) monoclonal antibodies (MAbs), respectively. Several recent studies suggest that specific epitopes elicit flavivirus-reactive MAbs with particular functional activities (3,37,38,50,57,60). Most type-specific neutralizing antibodies map to DIII of the E protein. Cross-reactive, neutralizing MAbs bind to regions outside DIII and have been mapped to

The neuropathogenicity of West Nile virus (WNV) and two derived attenuated strains WN25 and WN25A, was studied in young adult ICR mice and in severe combined immunodeficient (SCID) mice. Similarity in serology and RNA fingerprints were found between WNV and WN25. The viral envelope proteins of the attenuates differed from WNV in their slower mobility in SDS-PAGE due probably to the presence of N-linked glycan. The three strains were lethal to ICR mice by intracerebral (IC) inoculation, but when inoculated intraperitoneally (IP), WNV caused viremia, invaded the CNS and was lethal, whereas the attenuates showed no viremia or invasion of the CNS. The attenuates elicited antibodies to comparable levels as WNV in IP-infected mice, conferring upon them immunity to IC challenge with the wild type. In IP-inoculated SCID mice the three strains exhibited similar high viremiae that lasted until death of the animals. All strains invaded the CNS and proliferated in the mouse brain to similar high titers, but differed largely in the time of invasion: WNV invaded the CNS of SCID mice (and two other mouse strains) much earlier than the attenuates, which showed large intervals in their time of invasion into individual mouse brains within the group. The data presented for SCID mice indicate that WN25 and WN25A have truly lost the neuroinvasive property, and that this property materialized by a prescribed, active process specific for WNV.

This article reviews the development of two attenuated West Nile virus (WNV) variants, WNI-25 and WNI-25A. These variants have lost the neuroinvasion trait of the parental virus. Attenuation was achieved through serial passages in mosquito cells and neutralization escape from WNV-specific monoclonal antibody. Genetic analysis reveals amino acid changes between the parental and each of the variants. The attenuated variants preserve the ability to replicate in mice and geese and to induce a protective immune response. WNI-25A was found to be a genetically stable virus. This variant was successfully used as a live vaccine to protect geese against a wild-type virulent WNV field isolate that closely resembles the WNV isolated during the 1999 New York epidemic.

Botulinum neurotoxins (BoNT) are considered some of the most lethal known substances. There are seven botulinum serotypes, of which types A, B and E cause most human botulism cases. Anti-botulinum polyclonal antibodies (PAbs) are currently used for both detection and treatment of the disease. However, significant improvements in immunoassay specificity and treatment safety may be made using monoclonal antibodies (MAbs). In this study, we present an approach for the simultaneous generation of highly specific and neutralizing MAbs against botulinum serotypes A, B, and E in a single process. The approach relies on immunization of mice with a trivalent mixture of recombinant C-terminal fragment (Hc) of each of the three neurotoxins, followed by a parallel differential robotic hybridoma screening. This strategy enabled the cloning of seven to nine MAbs against each serotype. The majority of the MAbs possessed higher anti-botulinum ELISA titers than anti-botulinum PAbs and had up to five orders of magnitude greater specificity. When tested for their potency in mice, neutralizing MAbs were obtained for all three serotypes and protected against toxin doses of 10 MsLD50–500 MsLD50. A strong synergistic effect of up to 400-fold enhancement in the neutralizing activity was observed when serotype-specific MAbs were combined. Furthermore, the highly protective oligoclonal combinations were as potent as a horse-derived PAb pharmaceutical preparation. Interestingly, MAbs that failed to demonstrate individual neutralizing activity were observed to make a significant contribution to the synergistic effect in the oligoclonal preparation. Together, the trivalent immunization strategy and differential screening approach enabled us to generate highly specific MAbs against each of the A, B, and E BoNTs. These new MAbs may possess diagnostic and therapeutic potential.

Several studies have demonstrated that the passive transfer of protective antigen (PA)-neutralizing antibodies can protect animals against Bacillus anthracis infection. The standard protocol for the isolation of PA-neutralizing monoclonal antibodies is based upon a primary selection of the highest PA-binders by ELISA, and usually yields only few candidates antibodies. We demonstrated that by applying a PA-neutralization functionality-based screen as the primary criterion for positive clones, it was possible to isolate more than 100 PA-neutralizing antibodies, some of which exhibited no measurable anti-PA titers in ELISA. Among the large panel of neutralizing antibodies identified, mAb 29 demonstrated the most potent activity, and was therefore chimerized. The variable region genes of the mAb 29 were fused to human constant region genes, to form the chimeric 29 antibody (cAb 29). Guinea pigs were fully protected against infection by 40LD50 B. anthracis spores following two separate administrations with 10 mg/kg of cAb 29: the first administration was given before the challenge, and a second dose was administered on day 4 following exposure. Moreover, animals that survived the challenge and developed endogenous PA-neutralizing antibodies with neutralizing titers above 100 were fully protected against repeat challenges with 40LD50 of B. anthracis spores. The data presented here emphasize the importance of toxin neutralization-based screens for the efficient isolation of protective antibodies that were probably overlooked in the standard screening protocol. The protective activity of the chimeric cAb 29 demonstrated in this study suggest that it may serve as an effective immunotherapeutic agent against anthrax.