Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 90 (1), 279-91

Macaque Monoclonal Antibodies Targeting Novel Conserved Epitopes Within Filovirus Glycoprotein

Affiliations

Macaque Monoclonal Antibodies Targeting Novel Conserved Epitopes Within Filovirus Glycoprotein

Zhen-Yong Keck et al. J Virol.

Abstract

Filoviruses cause highly lethal viral hemorrhagic fever in humans and nonhuman primates. Current immunotherapeutic options for filoviruses are mostly specific to Ebola virus (EBOV), although other members of Filoviridae such as Sudan virus (SUDV), Bundibugyo virus (BDBV), and Marburg virus (MARV) have also caused sizeable human outbreaks. Here we report a set of pan-ebolavirus and pan-filovirus monoclonal antibodies (MAbs) derived from cynomolgus macaques immunized repeatedly with a mixture of engineered glycoproteins (GPs) and virus-like particles (VLPs) for three different filovirus species. The antibodies recognize novel neutralizing and nonneutralizing epitopes on the filovirus glycoprotein, including conserved conformational epitopes within the core regions of the GP1 subunit and a novel linear epitope within the glycan cap. We further report the first filovirus antibody binding to a highly conserved epitope within the fusion loop of ebolavirus and marburgvirus species. One of the antibodies binding to the core GP1 region of all ebolavirus species and with lower affinity to MARV GP cross neutralized both SUDV and EBOV, the most divergent ebolavirus species. In a mouse model of EBOV infection, this antibody provided 100% protection when administered in two doses and partial, but significant, protection when given once at the peak of viremia 3 days postinfection. Furthermore, we describe novel cocktails of antibodies with enhanced protective efficacy compared to individual MAbs. In summary, the present work describes multiple novel, cross-reactive filovirus epitopes and innovative combination concepts that challenge the current therapeutic models.

Importance: Filoviruses are among the most deadly human pathogens. The 2014-2015 outbreak of Ebola virus disease (EVD) led to more than 27,000 cases and 11,000 fatalities. While there are five species of Ebolavirus and several strains of marburgvirus, the current immunotherapeutics primarily target Ebola virus. Since the nature of future outbreaks cannot be predicted, there is an urgent need for therapeutics with broad protective efficacy against multiple filoviruses. Here we describe a set of monoclonal antibodies cross-reactive with multiple filovirus species. These antibodies target novel conserved epitopes within the envelope glycoprotein and exhibit protective efficacy in mice. We further present novel concepts for combination of cross-reactive antibodies against multiple epitopes that show enhanced efficacy compared to monotherapy and provide complete protection in mice. These findings set the stage for further evaluation of these antibodies in nonhuman primates and development of effective pan-filovirus immunotherapeutics for use in future outbreaks.

Figures

FIG 1
FIG 1
Immunization study design and antibody response. (A) Immunization and bleed schedule for rhesus macaques. (B) Anti-GP antibody titers (EC50s) in sera from immunized macaques determined in an ELISA with GPΔTM for the indicated virus species as the antigen. (C) Neutralization titer in macaque sera from day 112 determined using VSV-pseudotyped viruses with EBOV, SUDV, and MARV GP, respectively.
FIG 2
FIG 2
Reactivity of macaque-human chimeric antibodies to filovirus glycoproteins and dose-response binding of the indicated antibodies to GPΔTM of EBOV (A), SUDV (B), BDBV (C), RESTV (D), and MARV (E). Values are optical density at 650 nm (OD650) values from three to five ELISA experiments performed over the indicated range of antibody concentrations. The EC50s (in micrograms per milliliter) for binding of each antibody to the respective antigen are shown in each panel.
FIG 3
FIG 3
Binding region of pan-ebolavirus antibodies. (A) The domain structure of the EBOV glycoprotein (GenBank accession no. Q05320) is shown. Cathepsin and furin cleavage sites are indicated by arrows. SP, signal peptide; TM, transmembrane. (B) Structure of the MLD-deleted GP (GPΔmuc) (top) and binding of the antibodies to this protein (bottom). (C) Structural representation of EBOV GP after cleavage with thermolysin (top) and reactivity of each antibody to GPcl (bottom). (D) Structure of the EBOV soluble GP (sGP) (top) and its reactivity with the chimeric antibodies (bottom). The N-terminal tail that forms part of the GP base is shown in cyan, and the receptor binding region is shown in green. The glycan cap is shown in blue, and GP2 is shown in red. The EC50s (in micrograms per milliliter) for binding of each antibody to the respective antigen are shown in each panel.
FIG 4
FIG 4
Epitope mapping of FVM02p and FVM09. Epitopes for FVM02p and FVM09 were determined by competition ELISA using overlapping peptides spanning the full EBOV GP sequence. Peptides were preincubated at 100-fold molar excess with FVM02p or FVM09, and binding of the antibodies in the presence and absence of peptide was determined by ELISAs. (A) Sequences of the five overlapping peptides (top) surrounding the core sequence (boxed) that showed competition with FVM09 binding in an ELISA (bottom). (B) Location of the core FVM09 epitope (yellow circles) within a disordered loop connecting β17 and β18 within GP structure (PDB accession no. 3CSY). (C) Sequence identity of the FVM09 epitope and surrounding regions among ebolavirus species. (D) Sequences of the five overlapping peptides (top) surrounding the core sequence (boxed) that showed competition with FVM02p binding in an ELISA (bottom). (E) Position of the core FVM02p epitope within GP fusion loop (PDB accession no. 3CSY). The body of the fusion loop is shown in yellow with its tip containing FVM02p epitope in red. (F) Sequence identity of FVM02p epitope and surrounding regions among ebolavirus species as well as RAVV and MARV strains.
FIG 5
FIG 5
Neutralizing activity of the chimeric antibodies. The neutralizing activity of FVM04, FVM02p, FVM01p, FVM09, and FVM20 were determined for authentic SUDV (A) and EBOV (B) using a high-content imaging assay as described in Materials and Methods.
FIG 6
FIG 6
Efficacy of the chimeric antibodies in mouse model of EBOV infection. Mice were infected with 1,000 PFU of MA-EBOV and treated 2 h after infection (day 0) and on day 3 or treated only once on day 3 postinfection as indicated in the panels. (A) Protective efficacy of individual MAbs shown as a percentage of survival. Statistical differences were assessed for each treatment group compared to the values for the negative-control group using Mantel-Cox log rank test (P values of <0.3536 for FVM01p, 0.0003 for FVM02p, <0.0001 for FVM04 [days 0 and 3], 0.0060 for FVM04 [day 3 only], and 0.0060 for FVM09 and FVM20). (B) Percent weight change (group average of surviving animals) after infection and treatment with individual animals from the study shown in panel A. (C) Efficacy of the antibody cocktails shown in the panel. Statistical differences were assessed for each treatment group compared to the negative-control group using the Mantel-Cox log rank test (P values of <0.0001 for FVM02p plus FVM09 and <0.0001 for FVM09 plus m8C4). (D) Percent weight change in animals treated with antibody cocktails shown in panel C. The number of animals (n), antibody dose, and treatment regimen in each group are shown for each study.
FIG 7
FIG 7
Efficacy of FVM02p in mouse model of MARV. Mice were infected with 1,000 PFU of MA-MARV and treated either 2 h after infection (day 0) and on day 3 or at 2 h and 3 days as indicated in the panels. (A) Percent survival of challenged mice. (B) Percent weight change (group average of surviving animals) after infection and treatment with individual animals from the study shown in panel A.

Similar articles

See all similar articles

Cited by 28 articles

See all "Cited by" articles

Publication types

MeSH terms

Associated data

LinkOut - more resources

Feedback