Humanized Antibodies Derived from Chimpanzee Fabs that Neutralize Flaviviruses

National Institute Of Allergy And Infectious Diseases

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Abstract

The four dengue virus serotypes (DENV-1 to DENV-4) cause more morbidity in humans than any other arthropod-borne flaviviruses. Up to 100 million DENV infections occur every year, mostly in tropical and subtropical areas. Antibody-dependent enhancement (ADE) has been proposed as an underlying pathogenic mechanism of severe dengue, characterized by dengue hemorrhagic fever and dengue shock syndrome. We have recovered monoclonal antibodies (MAbs) from chimpanzees infected with multiple dengue virus serotypes. These MAbs are valuable for studies of functional and structural specificities and their role in protection in a primate model relevant to human dengue virus infection. Chimpanzee MAb 5H2 is specific for DENV-4 and neutralizes the virus at a high titer in vitro. The epitope detected by the antibody was mapped by sequencing neutralization-escape variants of the virus. One variant contained a Lys174-Glu substitution and another contained a Pro176-Leu substitution in domain I of the DENV-4 envelope protein (E). These mutations reduced binding affinity to the antibody by 18 to >100 fold. In the course of constructing the full-length humanized MAb 5H2, we isolated a variant that had a 9-amino-acid deletion in the Fc region, resulting from an alternative splicing due to a nucleotide substitution in the expression plasmid. The variant antibody, designated as MAb 5H2ΔD, and its full-length MAb 5H2 neutralized DENV-4 equally efficiently. We also demonstrated that MAb 5H2ΔD abrogated ADE of DENV infection. Such an antibody is particularly attractive for further exploring its potential for clinical application. Passive transfer of MAb 5H2ΔD at 20 micro g/mouse afforded 50% protection of suckling mice against challenge. Passive transfer of antibody to monkeys was conducted to demonstrate proof of concept for protection against DENV challenge with 25 lethal dose50 of mouse neurovirulent DENV-4 strain H241. Monkeys that received 2 mg/kg of MAb 5H2ΔD were completely protected against 100 monkey infectious dose50 of DENV-4, as indicated by the absence of viremia and sero-conversion. These studies have important implications for antibody-mediated prevention of dengue virus infection. [unreadable] Several other arthropod-borne flaviviruses, including West Nile virus, tick-borne encephalitis virus and Japanese encephalitis virus (JEV), are also important human pathogens. JEV is widely distributed in South Asia, Southeast Asia, and the Asian Pacific Rim. It is estimated that JEV causes 35,000-50,000 cases of encephalitis, including 10,000 deaths and as many neurologic sequelae each year. As an extension of the studies on antibodies against DENV, we recovered chimpanzee antibodies against JEV for analysis of the E antigenic structure, mechanisms of neutralization in vitro and in vivo and for possible use in immunoprophylaxis and immunotherapy. JEV-specific Fab antibodies were recovered by repertoire cloning of a phage library constructed from samples of chimpanzees that were initially immunized with inactivated JE-VAX and then boosted with attenuated JEV strain SA14-14-2. Three highly-neutralizing antibodies, termed Fabs A3, B2 and E3, which recognized spatially separated regions on the virion, were identified. These antibodies reacted with epitopes in different domains: the major determinant for Fab A3 was Lys179 (domain I), for Fab B2 was Ile126 (domain II) and for Fab E3 was Gly302 (domain III) in the envelope protein, suggesting that these antibodies neutralize the virus by different mechanisms. Binding affinity analysis showed that potent neutralizing antibodies reacted with a low number of binding sites available on the virion. These three Fabs and derived humanized MAbs exhibited high neutralizing activities against a broad spectrum of JEV genotype strains. Demonstration of antibody-mediated protection of JEV infection in vivo is provided using the mouse encephalitis model. MAb B2 was most potent, with a 50% protective dose (ED50) of 0.32 micro g, followed by MAb A3 (ED50 of 5.8 micro g) and then MAb E3 (ED50 of 24.7 micro g) for a 4-week-old mouse. Administration of 200 g/mouse of MAb B2 one day after otherwise lethal JEV infection protected 50% of mice and significantly prolonged the average survival time compared to mice in the unprotected group, suggesting a therapeutic potential for use of MAb B2 in humans.