A humanized monoclonal antibody neutralizes yellow fever virus strain 17D-204 in vitro but does not protect a mouse model from disease

Highlights

• Murine-human chimeric MAb 864-cIgG was developed for therapy of severe adverse effects (SAEs) from YFV vaccination.

• MAb 864-cIgG bound YFV vaccine strain 17D-204 in ELISA and neutralized 17D-204 infectivity for cell culture.

• MAb 864-cIgG had no protective capacity in the IFN α/β and γ receptor-deficient AG129 mouse model of 17D-204 infection.

• MAb 864-cIgG therapy appears to have no potential for therapy of SAEs associated with YFV vaccination.

Abstract

The yellow fever virus (YFV) vaccine 17D-204 is considered safe and effective, yet rare severe adverse events (SAEs), some resulting in death, have been documented following vaccination. Individuals exhibiting post-vaccinal SAEs are ideal candidates for antiviral monoclonal antibody (MAb) therapy; the time until appearance of clinical signs post-exposure is usually short and patients are quickly hospitalized. We previously developed a murine-human chimeric monoclonal antibody (cMAb), 2C9-cIgG, reactive with both virulent YFV and 17D-204, and demonstrated its ability to prevent and treat YF disease in both AG129 mouse and hamster models of infection. To counteract possible selection of 17D-204 variants that escape neutralization by treatment with a single MAb (2C9-cIgG), we developed a second cMAb, 864-cIgG, for use in combination with 2C9-cIgG in post-vaccinal therapy. MAb 864-cIgG recognizes/neutralizes only YFV 17D-204 vaccine substrain and binds to domain III (DIII) of the viral envelope protein, which is different from the YFV type-specific binding site of 2C9-cIgG in DII. Although it neutralized 17D-204 in vitro, administration of 864-cIgG had no protective capacity in the interferon receptor-deficient AG129 mouse model of 17D-204 infection. The data presented here show that although DIII-specific 864-cIgG neutralizes virus infectivity in vitro, it does not have the ability to abrogate disease in vivo. Therefore, combination of 864-cIgG with 2C9-cIgG for treatment of YF vaccination SAEs does not appear to provide an improvement on 2C9-cIgG therapy alone.

Introduction

Despite the availability of a vaccine considered generally safe and very effective, yellow fever (YF) remains an important mosquito-borne disease in tropical regions of South America and Africa. It is estimated that there are 180,000 human infections and 78,000 deaths annually due to YF (Garske et al., 2014). Yellow fever virus (YFV) is the prototype flavivirus and has a positive sense, single-stranded RNA genome that encodes three structural proteins (C, prM/M, and E) and seven nonstructural proteins (Chambers et al., 1990, Hahn et al., 1987, Rice et al., 1985). The virion is composed of a nucleocapsid surrounded by an envelope derived from infected cell membranes that contains the E and M proteins. The E or envelope glycoprotein is the major flaviviral surface protein, and is arranged as a scaffold of 90 homodimers on the mature virion surface (Kaufmann et al., 2010, Kuhn et al., 2002, Zhang et al., 2013a). The E protein is a class II fusion protein that contains the major determinants for cell attachment, cell entry of virions, and elicitation of virus-neutralizing antibodies (Crill and Roehrig, 2001, Heinz, 1986, Heinz and Allison, 2003, Roehrig, 2003, Stiasny et al., 2007). The crystal structure of the E protein has been solved for several flaviviruses, although not for YFV (Luca et al., 2012, Luca et al., 2013, Modis et al., 2003, Modis et al., 2005, Nybakken et al., 2006, Rey et al., 1995). The flaviviral E protein can be divided into three structural domains: DI, DII, and DIII. Epitopes in DII and DIII elicit virus-neutralizing monoclonal antibodies (MAbs) and anti-DIII MAbs usually have more potent in vitro neutralization titers than anti-DII MAbs (Roehrig, 2003). Certain anti-E MAbs possess antiviral prophylactic and therapeutic activity in animal models of flavivirus infection (Brandriss et al., 1986, Engle and Diamond, 2003, Gould et al., 1986, Hawkes et al., 1988, Johnson and Roehrig, 1999, Julander et al., 2014, Kimura-Kuroda and Yasui, 1988, Mathews and Roehrig, 1984, Thibodeaux et al., 2012b), and the humanized anti-West Nile virus (WNV) EDIII MAb MGAWN1 has demonstrated efficacy in animal models and undergone Phase I clinical trials to demonstrate safety in humans (Beigel et al., 2010). We recently developed a YFVtype-specific chimeric murine-human MAb, 2C9-cIgG, and demonstrated its prophylactic and therapeutic activity in two animal models of infection (Julander et al., 2014, Thibodeaux et al., 2012b). MAb 2C9-cIgG reacts with both virulent and vaccine YFV, binding to an epitope in DII of the E protein (Lobigs et al., 1987). Interferon receptor-deficient AG129 mice were protected from or successfully treated after challenge with 17D-204 when the cMAb was inoculated 24 h prior to or 24 h after viral infection (Thibodeaux et al., 2012b). MAb 2C9-cIgG was more effective in an immunocompetent hamster model challenged with virulent YFV Jimenez strain (Julander et al., 2014). Hamsters were protected from disease when 2C9-cIgG was administered 24 h before and up to 72 h post-infection (PI).

Yellow fever vaccination with live-attenuated 17D-204 is generally considered safe and effective; however, rare severe adverse events (SAEs), some resulting in death, have been documented following vaccination, particularly in individuals with innate immunity defects or >60 years of age (Wkly Epidemiol Rec, CDC, 2001, Martin et al., 2001, Monath, 2010, Vasconcelos et al., 2001). SAEs are not due to mutations in the vaccine virus but rather to as yet undetermined host-specified factors (Monath, 2010). There are no specific therapies for YFV infection (Julander, 2013, Monath, 2008). Because the timing of viral exposure is known for vacinees, individuals experiencing post-vaccinal SAEs are likely candidates for anti-YF antibody therapy. One theoretical limitation of single MAb therapy for flaviruses is the high mutation rate of flaviviral ssRNA genomes, which could result in generation of MAb escape mutants (Ryman et al., 1998). Neutralization escape variants of WNV have been selected both in vitro and in vivo following single dose MAb treatment (Zhang et al., 2009, Zhang et al., 2010). To reduce this possibility, cocktails of MAbs reactive with different E protein epitopes might be more effective for therapy.

We report here the generation of a second YFV-reactive cMAb, 864-cIgG, and its in vitro and in vivo activity. The parent murine MAb 864 (m864) was isolated following immunization of mice with 17D-204 (Buckley and Gould, 1985, Cammack and Gould, 1986, Gould et al., 1985, Gould et al., 1986). MAb 864 is substrain specific and reacts only with YFV 17D-204 vaccine, neutralizes virus infectivity, and has been shown to protect mice from virus challenge when administered to 3–4 week-old immunocompetent mice as mouse ascitic fluid 24 h before YF-17D challenge via the intracerebral route (Cammack and Gould, 1986, Gould et al., 1986). Unlike mMAb 2C9, mMAb 864 identifies a neutralization epitope in DIII of the E protein (Ryman et al., 1998); thus we predicted that combined therapy using 864-cIgG and 2C9-cIgG should increase therapeutic efficacy compared to 2C9-cIgG alone.