Development and Characterization of Neutralizing Antibodies Against Zaire Ebolavirus Glycoprotein and Protein 40

Abstract

Background/Aims: Monoclonal antibodies (mAbs) are presently the most promising treatment against Ebola virus disease (EVD), and cocktail of two or more antibodies likely confers protection through complementary mechanisms. Zaire Ebolavirus (EBOV) glycoprotein (GP) and viral protein 40 (VP40) are targets for designing neutralizing antibodies. Currently, the antiviral therapeutics of mAb-cocktails are still limited solely to anti-GP antibodies，there is no Abs cocktail against Zaire EBOV GP and VP40, which both have important interactions with host cellular membrane. Methods: We used hybridoma technology to produce anti-Zaire EBOV GP mAb against GP receptor binding domain, and anti-Zaire EBOV VP40 mAbs against the N-terminal domain, the C-terminal domain, respectively; synthesized Zaire EBOV transcription and replication competent virus like particles (trVLPs), which model even all aspects of the EBOV life cycles in order to evaluate the anti-viral effect of mAbs. Then, we characterized the anti- Zaire EBOV trVLPs effect of anti-GP and VP40 mAbs in vitro by real time-PCR, immunofluorescence assay and western blot analysis. Results: Our results demonstrate that anti-GP or anti-VP40 mAbs effectively inhibit trVLPs replication. The cocktails of anti-GP and anti-VP40 mAbs, or between anti-VP40 mAbs, had synergistic anti-trVLPs effect. Meanwhile, the detailed DNA and amino acid sequences of the mAbs were checked. Conclusion: The study verifies neutralizing efficacy of anti-GP or anti-VP40 mAb, report promising cocktail of anti-GP and anti-VP40 mAb, or cocktail of two anti-VP40 mAbs. To our knowledge, this is the first account to report the important anti-viral effect of cocktails of anti-GP and anti-VP40 mAbs in vitro.

Therapeutic Strategies against Ebola Virus Infection

“…For instance, combinations of PMOs (targeting VP24, VP35, and L) [ 97 ], positively charged PMOs (PMOplus AVI-6002 targeting VP24, VP35, and L) [ 98 ], or SNALPs in a cocktail containing different siRNAs (also targeting VP24, VP35, and L) [ 131 ] have been reported, and the SNALP cocktail was shown to protect rhesus macaques from lethal EBOV challenge [ 131 ]. A combination of mAbs targeting different viral proteins (e.g., anti-GP and anti-VP40) [ 101 ] or combinations of IFNs and nucleoside analogs [ 102 ] have also been shown to inhibit the replication of EBOV VLP in vitro. In addition, another study showed that adenovirus-vectored IFN-α (Ad-IFN-α) was able to enhance the protection of adenovirus-based vaccine expressing EBOV GP (Ad-CAGoptZGP) in rodents [ 99 ] and ZMAb in NHP [ 100 ].…”

Filovirus Neutralising Antibodies: Mechanisms of Action and Therapeutic Application

“…For example, anti-VP40 mAbs have been shown to neutralise EBOV by preventing the formation of new progeny ( Figure 2 ). This is bolstered when administrated in cocktails with mAbs targeting different sites on the VP40 protein or with GP-specific antibodies [ 143 , 144 ]. Antibodies, particularly polymeric IgM and IgA, can also agglutinate virus particles, forming aggregates that reduce the contact between the virus and the cell and affect subsequent entry, in addition to signalling, for antibody-dependent phagocytosis ( Figure 2 ) [ 145 , 146 , 147 ].…”

Generation of neutralizing and non-neutralizing monoclonal antibodies against H7N9 influenza virus

“…Briefly, 6-week-old, female, BALB/c mice (Shanghai Laboratory Animal Center) were immunized with two intramuscular injection of H7N9 virus vaccine in Quick Antibody adjuvant (Biodragon, China) with 2week interval. The mice then received an additional intravenous injection of the same viral antigen 3 days without adjuvant before the fusion of splenocytes with SP2/0 myeloma cells [29]. After cell fusion, hybridoma culture supernatants were screened by enzymelinked immunosorbent assay (ELISA) [30].…”

“…Through limiting dilution, positive hybridoma lines were cloned, expanded and further sub-cultured. Hybridoma cell clones were expanded significantly, harvested, and injected intraperitoneally in pristane-primed BALB/c mice [29]. The ascites was collected and purified by protein-G column (GE) according to the manufacturer's instructions.…”

“…The IFA was performed as described previously [29]. Monolayers of MDCK cells were infected at a multiplicity of infection (MOI) of 0.5 with virus.…”