Pan-ebolavirus and Pan-filovirus Mouse Monoclonal Antibodies: Protection against Ebola and Sudan Viruses

Abstract

The unprecedented 2014-2015 Ebola virus disease (EVD) outbreak in West Africa has highlighted the need for effective therapeutics against filoviruses. Monoclonal antibody (MAb) cocktails have shown great potential as EVD therapeutics; however, the existing protective MAbs are virus species specific. Here we report the development of pan-ebolavirus and pan-filovirus antibodies generated by repeated immunization of mice with filovirus glycoproteins engineered to drive the B cell responses toward conserved epitopes. Multiple pan-ebolavirus antibodies were identified that react to the Ebola, Sudan, Bundibugyo, and Reston viruses. A pan-filovirus antibody that was reactive to the receptor binding regions of all filovirus glycoproteins was also identified. Significant postexposure efficacy of several MAbs, including a novel antibody cocktail, was demonstrated. For the first time, we report cross-neutralization and in vivo protection against two highly divergent filovirus species, i.e., Ebola virus and Sudan virus, with a single antibody. Competition studies indicate that this antibody targets a previously unrecognized conserved neutralizing epitope that involves the glycan cap. Mechanistic studies indicated that, besides neutralization, innate immune cell effector functions may play a role in the antiviral activity of the antibodies. Our findings further suggest critical novel epitopes that can be utilized to design effective cocktails for broad protection against multiple filovirus species. IMPORTANCEFiloviruses represent a major public health threat in Africa and an emerging global concern. Largely driven by the U.S. biodefense funding programs and reinforced by the 2014 outbreaks, current immunotherapeutics are primarily focused on a single filovirus species called Ebola virus (EBOV) (formerly Zaire Ebola virus). However, other filoviruses including Sudan, Bundibugyo, and Marburg viruses have caused human outbreaks with mortality rates as high as 90%. Thus, cross-protective immunotherapeutics are urgently needed. Here, we describe monoclonal antibodies with cross-reactivity to several filoviruses, including the first report of a cross-neutralizing antibody that exhibits protection against Ebola virus and Sudan virus in mice. Our results further describe a novel combination of antibodies with enhanced protective efficacy. These results form a basis for further development of effective immunotherapeutics against filoviruses for human use. Understanding the cross-protective epitopes are also important for rational design of pan-ebolavirus and pan-filovirus vaccines.

Macaque Monoclonal Antibodies Targeting Novel Conserved Epitopes within Filovirus Glycoprotein

“…FVM04 binds to a conformational epitope within the core of GP1 (residues 31 to 200), a region that encompasses the RBR and the base of GP1 (22). While previous reports of antibodies targeting the GP1 core indicated that epitopes in this region are likely concealed (16,19; also see the accompanying article by Holtsberg et al [42]), the FVM04 binding profile was not significantly changed upon removal of the MLD, suggesting that the epitope of FVM04 is well exposed and likely on the top of the trimeric GP chalice. Interestingly, FVM01p, another MAb that strongly bound the GP1 core, failed to protect mice, indicating that the specificity of the epitope is critical for protection.…”

“…Thus, we sought to determine whether combination of our pan-ebolavirus antibodies that bind to novel epitopes will lead to enhanced efficacy compared to individual MAbs. To this end, we tested two different combinations: (i) FVM02p and FVM09, targeting the fusion loop and glycan cap, respectively; and (ii) FVM09 and m8C4, a mouse pan-ebolavirus antibody (see the accompanying article by Holtsberg et al [42]), which binds to a conformational epitope involving the glycan cap. Both of these combinations provided 100% protection against lethal challenge although the antibodies were administered at a lower dose compared to the study with individual antibodies (Fig.…”

“…Partial competition was also observed between FVM04 and m13C6. The competition was also tested for a series of mouse pan-ebolavirus antibodies m16G8, m8C4, m17C6, and m4B8 (the "m" prefix indicates the murine origin of the antibody) that we have recently identified (see the accompanying article by Holtsberg et al [42]). Except for FVM04 that strongly competed with m17C6, suggesting overlap in the epitope targeted by these two antibodies, no other competition was observed between the mouse and macaque pan-ebolavirus MAbs.…”

Ferrets Infected with Bundibugyo Virus or Ebola Virus Recapitulate Important Aspects of Human Filovirus Disease

“…Recently, pan-Ebolavirus monoclonal antibodies with demonstrated in vitro activity have been identified (32)(33)(34), and these experimental candidates could be screened in ferrets in vivo. Other advantages with using ferrets as a potential animal model include better availability, smaller body sizes, less stringent housing requirements, and reduced costs.…”

Vesicular Stomatitis Virus Pseudotyped with Ebola Virus Glycoprotein Serves as a Protective, Noninfectious Vaccine against Ebola Virus Challenge in Mice

“…In comparisons of the vaccine efficacy of wild-type and N-linked-glycan-deficient glycoproteins, the amount of the different pseudovirions administered in each vaccine study was adjusted for GP equivalents. To quantify this, purified pseudovirions were serially diluted, and proteins were separated by SDS-PAGE, transferred to nitrocellulose, and probed with anti-EBOV GP monoclonal antibody 21D10 (72) and VSV matrix (M) 23H12 (73). 21D10 recognizes filovirus GP residues 81 to 89, a region of GP that does not contain N-linked glycosylation and so should recognize the denatured GPs equivalently.…”