Comparative Efficacy of Monoclonal Antibodies That Bind to Different Epitopes of the 2009 Pandemic H1N1 Influenza Virus Neuraminidase

Abstract

Antibodies against the neuraminidase (NA) of influenza virus correlate with resistance against disease, but the effectiveness of antibodies against different NA epitopes has not been compared. In the present study, we evaluated the in vitro and in vivo efficacies of four monoclonal antibodies (MAbs): HF5 and CD6, which are specific to two different epitopes in the NA of 2009 pandemic H1N1 (pH1N1) virus, and 4E9 and 1H5, which are specific to a conserved epitope in the NA of both H1N1 and H5N1 viruses. In the in vitro assays, HF5 and CD6 inhibited virus spread and growth more effectively than 4E9 and 1H5, with HF5 being the most effective inhibitor. When administered prophylactically at 5 mg/kg of body weight, HF5 and CD6 protected ϳ90 to 100% of DBA/2 mice against lethal wild-type pH1N1 virus challenge; however, at a lower dose (1 mg/kg), HF5 protected ϳ90% of mice, whereas CD6 protected only 25% of mice. 4E9 and 1H5 were less effective than HF5 and CD6, as indicated by the partial protection achieved even at doses as high as 15 mg/kg. When administered therapeutically, HF5 protected a greater proportion of mice against lethal pH1N1 challenge than CD6. However, HF5 quickly selected pH1N1 virus escape mutants in both prophylactic and therapeutic treatments, while CD6 did not. Our findings confirm the important role of NA-specific antibodies in immunity to influenza virus and provide insight into the properties of NA antibodies that may serve as good candidates for therapeutics against influenza. IMPORTANCENeuraminidase (NA) is one of the major surface proteins of influenza virus, serving as an important target for antivirals and therapeutic antibodies. The impact of NA-specific antibodies on NA activity and virus replication is likely to depend on where the antibody binds. Using in vitro assays and the mouse model, we compared the inhibitory/protective efficacy of four mouse monoclonal antibodies (MAbs) that bind to different sites within the 2009 pandemic H1N1 (pH1N1) virus NA. The ability of each MAb to protect mice against lethal pH1N1 infection corresponded to its ability to inhibit NA activity in vitro; however, the MAb that was the most effective inhibitor of NA activity selected pH1N1 escape variants in vivo. One of the tested MAbs, which binds to a conserved region in the NA of pH1N1 virus, inhibited NA activity but did not result in escape variants, highlighting its suitability for development as a therapeutic agent.

Substrate Binding by the Second Sialic Acid-Binding Site of Influenza A Virus N1 Neuraminidase Contributes to Enzymatic Activity

“…The NA protein is essential for release of virus particles from infected cells and decoy receptors (e.g., in mucus) and for preventing virion aggregation. The NA protein is also an important target of the immune response and antiviral drugs (1)(2)(3). The IAV NA protein appears, however, understudied compared to the HA protein.…”

Antibody neutralization of an influenza virus that uses neuraminidase for receptor binding

“…These antibodies all bind to Cal09 NA and inhibit NA catalytic activity to varying degrees. HF5 and CD6 are strain-specific antibodies that each target distinct epitopes, and strongly inhibit NA activity as measured by ELLA [5]. Antibodies 1H5 and 4E9 recognize a similar epitope on the lateral surface of the NA head that is conserved across the N1 subtype, but these antibodies only weakly inhibit NA activity in ELLA assays [5].…”

“…NA also plays an important role in vivo by helping the virus penetrate mucus barriers to reach target cells [2,3]. Because only HA is needed for viral entry, anti-NA antibodies are not strongly neutralizing in infection assays where viruses are only allowed to undergo a single cycle of growth [4,5]. However, many studies have shown that anti-NA antibodies are associated with reduced disease severity in humans [6][7][8].…”

“…Here, we utilize the G147R NA in conjunction with a binding-deficient HA to develop a sensitive neutralization assay for anti-NA antibodies. Specifically, we test the susceptibility of this NAbinding-dependent virus to neutralization by four monoclonal antibodies targeting distinct epitopes of N1 NA [5], and find that some of these antibodies neutralize the NA-binding-dependent virus much better than they neutralize virus that can bind cells via HA. We then leverage this discovery to select an in vitro escape mutation to one of the antibodies, demonstrating the value of these viruses for antigenic mapping.…”

An influenza A virus (H7N9) anti-neuraminidase monoclonal antibody protects mice from morbidity without interfering with the development of protective immunity to subsequent homologous challenge

“…Six of these mAbs are directed against various epitopes contained within the HA surface protein while one targets the matrix protein 2 (M2). In addition to the HA and M2, anti-neuraminidase (NA) mAb have also proven to be protective in the mouse model but, to our knowledge, have yet to enter clinical development (Doyle et al, 2013; Jiang et al, 2015; Shoji et al, 2011; Wan et al, 2013; Wohlbold et al, 2016; DiLillo et al, 2016). One potential concern regarding mAb as antivirals include the possibility that their use may drive the evolution of viral escape mutations which could be resistant not only to the mAb but to natural or vaccine-induced immunity.…”