Chimeric Filoviruses for Identification and Characterization of Monoclonal Antibodies

Abstract

Recent experiments suggest that some glycoprotein (GP)-specific monoclonal antibodies (MAbs) can protect experimental animals against the filovirus Ebola virus (EBOV). There is a need for isolation of MAbs capable of neutralizing multiple filoviruses.Antibody neutralization assays for filoviruses frequently use surrogate systems such as the rhabdovirus vesicular stomatitis Indiana virus (VSV), lentiviruses or gammaretroviruses with their envelope proteins replaced with EBOV GP or pseudotyped with EBOV GP. It is optimal for both screening and in-depth characterization of newly identified neutralizing MAbs to generate recombinant filoviruses that express a reporter fluorescent protein in order to more easily monitor and quantify the infection. Our study showed that unlike neutralization-sensitive chimeric VSV, authentic filoviruses are highly resistant to neutralization by MAbs. We used reverse genetics techniques to replace EBOV GP with its counterpart from the heterologous filoviruses Bundibugyo virus (BDBV), Sudan virus, and even Marburg virus and Lloviu virus, which belong to the heterologous genera in the filovirus family. This work resulted in generation of multiple chimeric filoviruses, demonstrating the ability of filoviruses to tolerate swapping of the envelope protein. The sensitivity of chimeric filoviruses to neutralizing MAbs was similar to that of authentic biologically derived filoviruses with the same GP. Moreover, disabling the expression of the secreted GP (sGP) resulted in an increased susceptibility of an engineered virus to the BDBV52 MAb isolated from a BDBV survivor, suggesting a role for sGP in evasion of antibody neutralization in the context of a human filovirus infection. IMPORTANCEThe study demonstrated that chimeric rhabdoviruses in which G protein is replaced with filovirus GP, widely used as surrogate targets for characterization of filovirus neutralizing antibodies, do not accurately predict the ability of antibodies to neutralize authentic filoviruses, which appeared to be resistant to neutralization. However, a recombinant EBOV expressing a fluorescent protein tolerated swapping of GP with counterparts from heterologous filoviruses, allowing high-throughput screening of B cell lines to isolate MAbs of any filovirus specificity. Human MAb BDBV52, which was isolated from a survivor of BDBV infection, was capable of partially neutralizing a chimeric EBOV carrying BDBV GP in which expression of sGP was disabled. In contrast, the parental virus expressing sGP was resistant to the MAb. Thus, the ability of filoviruses to tolerate swapping of GP can be used for identification of neutralizing MAbs specific to any filovirus and for the characterization of MAb specificity and mechanism of action.T he family Filoviridae is composed of the genus Ebolavirus, which includes Ebola (EBOV), Sudan (SUDV), Taï Forest (TAFV), Reston (RESTV), and Bundibugyo (BDBV) viruses, the genus Marburgvirus, which includes Marburg (MARV) and Ravn (RAVV) viruses, and the putative genus Cuevavirus, which i...

Systematic Analysis of Monoclonal Antibodies against Ebola Virus GP Defines Features that Contribute to Protection

“…In natural infection, abundant sGP could limit effective neutralization of circulating virus by sGP-cross-reactive antibodies (Ilinykh et al, 2016; Mohan et al, 2012) (Figure 2). However, as mentioned above, we typically saw equivalent or weaker relative neutralization in rVSV relative to the sGP-containing ΔVP30 and EBOV assays.…”

Analysis of a Therapeutic Antibody Cocktail Reveals Determinants for Cooperative and Broad Ebolavirus Neutralization

“…The authentic EBOV-eGFP, mouse-adapted EBOV Mayinga (EBOV-MA, GenBank: AF49101) and SUDV strain Gulu were described previously (Bray et al, 1998;Sanchez and Rollin, 2005;Towner et al, 2005). The chimeric infectious EBOV/BDBV-GP and EBOV/ SUDV-GP viruses expressing eGFP were obtained by replacing the gene encoding EBOV GP with that of BDBV (GenBank: KU174137) or SUDV (GenBank: KU174142), respectively (Ilinykh et al, 2016).…”

“…Virus neutralization assays were performed in a high-throughput format using the recombinant EBOV-eGFP or chimeric EBOV viruses in which GP was replaced with its counterpart from BDBV or SUDV, as described previously (Ilinykh et al, 2016). Briefly, four-fold dilutions of the respective mAb starting at 200 mg/mL were mixed in triplicate with 400 PFU of the virus in U-bottom 96-well plates and incubated for 1 h at 37 C. Mixtures were applied on Vero-E6 cell monolayer cultures in 96-well plates and incubated for four days at 37 C. In the absence of mAb neutralizing activity, the infection resulted in uniform eGFP fluorescence from the monolayer of cells that was readily detected by fluorescence microscopy.…”

Development of a micro-neutralization assay for ebolaviruses using a replication-competent vesicular stomatitis hybrid virus and a quantitative PCR readout

“…It has been found that neutralization titers obtained with surrogate neutralization assays do not always correspond to those obtained using the native virus [45]. Unfortunately, we were unable to perform PRNT with the infectious ebolaviruses to compare the antibody neutralizing titers measured from our microneutralization assay.…”