The aim of this study was to raise antibodies with broad cross-reactivity by using antigens that were either highly conserved (F88 peptide) or had highly divergent sequences deleted (Sudan GP with mucin-like domain deleted). The observation that rabbit polyclonal antibodies (R.F88-2 and R.GP-S-1) and mouse monoclonal antibodies (M.F88-H3D5, M.F88-H3C8) could be raised to detect recombinant or Filovirus produced GP1,2
representing all known ebolaviruses in selected assays suggests that this strategy was effective. However, the rabbit monoclonals derived from splenocytes of the GP-S-mucin-deleted-immunized rabbit, showed differences in specificity, in that those are specific only for SUDV GP1,2
(, , and ; ), perhaps due to clonal bias. Interestingly, in a prior study by Yu et al.
, use of SUDV-GP1,2
-derived peptides generated polyclonal antibodies with more restricted ability to detect ebolaviruses (i.e., only SUDV or SUDV plus one other ebolavirus) (Yu et al., 2006
). Lucht et al.
, isolated antibodies with more restricted reactivity when full-length EBOV GP1,2
was used as the immunogen (Lucht et al., 2004
), suggesting again the use of the mucin-deleted form of GP1,2
as an improved strategy to induce broadly reactive antibodies.
Reactivity of anti-ebolavirus GP1,2 antibodies assessed by different assays
In the initial analysis, the heavy chain of the rabbit monoclonal R.GP-S-55-1 was difficult to reduce and/or denature (). Even when the concentration of DTT was increased to 100 mM during the preparation of the sample, the heavy chain was observed to form a smear, rather than a distinct band. Scientists at Epitomics, Inc (Burlingame, CA) also observed a similar phenomenon for other rabbit monoclonal antibodies, but regarding light chains, not heavy chains (personal communications). The smear is likely to be derived from the rabbit immunoglobulin heavy chain for three reasons. First, the hybridoma cells were grown in serum-free medium for production and the antibody was purified using protein A beads. Second, the purified antibody can detect SUDV GP1,2 in different assays, which involved the use of horseradish peroxidae/Alexa647-conjugated anti-rabbit IgG. Third, the light chain size was comparable to that of others. Similarly, the bands for R.GP-S-46-12 light chain and R.GP-S-53-7 heavy chain were not sharp either.
Both the rabbit polyclonal and the mouse monoclonal anti-F88 antibodies were generally broadly reactive with the ebolavirus GP1,2 tested with the exception of SUDV GP1,2 (). In both ELISA () and FACS () assays, but not in WB () assay, binding to SUDV GP1,2 was lower compared to the other ebolavirus GP1,2 tested (EBOV, TAFV, RESTV in ELISA and BDBV, EBOV, and TAFV in FACS). Interestingly, alignment of the F88 38-mer peptide sequences from these five ebolaviruses reveals a striking diversity within the N-terminal eight amino acid residues (). In the case of SUDV GP1,2, three of these eight residues are distinct, whereas the BDBV, EBOV, and TAFV residues are identical and the RESTV peptide differs from those in only a single residue. This observation suggests indirectly that the anti-F88 antibodies may be directed primarily towards the N-terminal region or that this portion of the peptide influences the conformation resulting in a different tertiary conformation of this region in SUDV GP1,2. Notably, the N-terminal region of MARV in this 38 amino acid region is almost completely distinct from the corresponding region in EBOV GP1,2, sharing only a single glycine residue at the N-terminus. Therefore, perhaps not surprisingly, none of the anti-F88 antibodies used could detect MARV GP1,2.
Of the detection assays used, the FACS analysis gave the most variability compared to ELISA and WB. When comparing across the different antibodies, there is sufficient data to suggest that each of the GP1,2 was expressed at comparable levels based on the percentage of positive cells detected. While 5% of pVR1012-SudanGP-transfected cells were detected as positive when evaluated using the anti-F88 panel of antibodies, 25% or more of the same cells were detected as positive when evaluated with the anti-GP-S panel of antibodies, demonstrating that the reduced detection level with the anti-F88 antibodies was due to reduced reactivity as opposed to poor expression levels (). Reduced detection of the cells expressing EBOV and BDBVGP1,2 by the rabbit polyclonal anti-GP-S antibody (R.GP-S-1) was also due to reduced reactivity to these native proteins (). Notably, in the sandwich ELISA or WB assays, detection of EBOV and BDBV GP1,2 by this same polyclonal antibody was not decreased, suggesting that the epitopes that this polyclonal antibody recognizes are presented in a different manner in the ELISA or WB assay compared to cell surface-expressed GP1,2. The ability of the R.GP-S-1, R-GP-S-46-12, and M.F88-H3D5 antibodies to recognize authentic ebolavirus-infected cells by immunofluorescence assay supports the assertion that these antibodies recognize native GP1,2.
R.GP-S-1 neutralized weakly SUDV GP1,2-pseudotyped MoMLV, whereas none of the other antibodies demonstrated neutralization activity against the tested pseudotypes (). Of note, the affinity study of the two monoclonal anti-F88 antibodies exhibited a fairly weak binding to EBOV GP-Fc with KD values greater than 100 nM (148 nM and 361 nM for H3C8 and H3D5, respectively). This low affinity to native GP1,2 may provide one explanation for the lack of neutralization activity of these antibodies.
Neutralization of gammaretroviral pseudotypes bearing various ebolavirus GP1,2
All anti-F88 antibodies (R.F88-2, M.F88-H3D5, and M.F88-H3C8) could detect RESTV sGP by WB (). Given that the F88 peptide sequence is identical in both sGP and full-length GP1,2
and that these antibodies could detect EBOV, SUDV and TAFV full-length GP1,2
, these antibodies should detect the sGP of EBOV, SUDV and TAFV as well. The ability to detect sGP is significant as sGP accounts for about 80% of the ebolavirus GP
gene transcripts in natural infections. Consequently, these cross-reactive antibodies may be useful for detection and diagnosis of ebolavirus infections (Sanchez et al., 1996
; Volchkova et al., 1998
). While the polyclonal R,GP-S-1 was also able to detect RESTV sGP by WB, it’s notable that the rabbit anti-GP-S monoclonal antibodies (R.GP-S-46-12, 53-4, 53-7, 55-1) were specific for SUDV GP1,2
only. The specificity of the rabbit monoclonals may provide useful reagents for ebolavirus differentiation with respect to SUDV only (, and ). The ELISA results with sera from mice injected with mouse-adapted Ebola virus demonstrated that the R.GP-S-46.12 antibody could not detect this virus specifically. However, analysis using either the R.GP-S polyclonal or the M.F88-H3D5 antibodies yielded specific responses above levels detected for control sera. These results support the further development of these antibodies in combinations as described here. Alternatively, pairing with other antibodies proven useful for detection of ebolaviruses by ELISA (Lucht et al., 2004
) (Yu et al., 2006
) may provide tools for detection and potentially differential diagnosis of ebolavirus infections in animal studies and/or clinical samples.
In summary, a panel of cross-reactive or ebolavirus-specific rabbit and mouse antibodies was developed and analyzed in a variety of different assay formats. The development of antibodies with similar specificity but different host origins may allow for more versatile pairing in the development of diagnostic assays, such as sandwich ELISA, using one for capture and the other one for detection. Using such sandwich ELISA for detecting ebolavirus GP1,2, may allow for increased assay specificity and sensitivity. In addition, these antibodies could also be useful for research of GP1,2 structure and function.