In Vitro Neutralisation of Rotavirus Infection by Two Broadly Specific Recombinant Monovalent Llama-Derived Antibody Fragments

Abstract

Rotavirus is the main cause of viral gastroenteritis in young children. Therefore, the development of inexpensive antiviral products for the prevention and/or treatment of rotavirus disease remains a priority. Previously we have shown that a recombinant monovalent antibody fragment (referred to as Anti-Rotavirus Proteins or ARP1) derived from a heavy chain antibody of a llama immunised with rotavirus was able to neutralise rotavirus infection in a mouse model system. In the present work we investigated the specificity and neutralising activity of two llama antibody fragments, ARP1 and ARP3, against 13 cell culture adapted rotavirus strains of diverse genotypes. In addition, immunocapture electron microscopy (IEM) was performed to determine binding of ARP1 to clinical isolates and cell culture adapted strains. ARP1 and ARP3 were able to neutralise a broad variety of rotavirus serotypes/genotypes in vitro, and in addition, IEM showed specific binding to a variety of cell adapted strains as well as strains from clinical specimens. These results indicated that these molecules could potentially be used as immunoprophylactic and/or immunotherapeutic products for the prevention and/or treatment of infection of a broad range of clinically relevant rotavirus strains.

Rice-based oral antibody fragment prophylaxis and therapy against rotavirus infection

“…A large number of serotypes have been isolated from infected individuals, although the most common serotypes worldwide are G1P [8], G2P [4], G4P [8], and G9P [8] (31). Moreover, it could also bind the recently emerged G8, G10, and G12 strains (31).…”

“…The outer layer proteins of RV, VP7 and VP4, both contain neutralizing epitopes, although these tend to be serotype specific (31). The middle layer protein, VP6, is the most abundant and immunodominant viral protein and contains the group and subgroup determining epitopes (31).…”

“…Recently, the ARP1 llama-derived antibody that binds specifically to RV VP6 has been shown to bind to RV in ELISA and immune electron microscopy and to neutralize infection with a variety of RV genotypes in vitro using classical neutralization assays and in an in vivo mouse pup model, which suggests that these antibodies are likely to neutralize RV by immune exclusion (31,(50)(51)(52)(53). Although the mechanism by which the llama antibodies neutralize infection is not yet understood, it is possible that the VP6-specific VHH could block a conformational change in the outer layer proteins of the RV particle, VP7 and/or VP4, preventing attachment and/or entry of the virus particle, as previously suggested (31,50). This is supported by existing evidence that the phenotype of the outer layer VP4 can be altered by interactions with different VP7 proteins and of the marked impact of the protein-protein interactions among the constituent proteins of the RV inner, middle and outer layers (54).…”

“…Frenken., unpublished observations). Western blots using a variety of RV strains and recombinant VP6 suggest that ARP1 binds to polymeric VP6 (31). VP6 is the most conserved protein in the RV capsid and contains the group and subgroup determining epitopes (31).…”

An Exopolysaccharide-Deficient Mutant of Lactobacillus rhamnosus GG Efficiently Displays a Protective Llama Antibody Fragment against Rotavirus on Its Surface

“…The ARP1 antibody fragment neutralizes a wide variety of rotavirus serotypes in vitro (15) and was shown to be safe and effective in reducing the severity of rotavirus-induced diarrhea in children (16).…”