Llama-Derived Single-Chain Antibody Fragments Directed to Rotavirus VP6 Protein Possess Broad Neutralizing Activity In Vitro and Confer Protection against Diarrhea in Mice

Abstract

Group A rotavirus is one of the most common causes of severe diarrhea in human infants and newborn animals. Rotavirus virions are triple-layered particles. The outer capsid proteins VP4 and VP7 are highly variable and represent the major neutralizing antigens. The inner capsid protein VP6 is conserved among group A rotaviruses, is highly immunogenic, and is the target antigen of most immunodiagnosis tests. Llama-derived single-chain antibody fragments (VHH) are the smallest molecules with antigen-binding capacity and can therefore be expected to have properties different from conventional antibodies. In this study a library containing the VHH genes of a llama immunized with recombinant inner capsid protein VP6 was generated. Binders directed to VP6, in its native conformation within the viral particle, were selected and characterized. Four selected VHH directed to conformational epitopes of VP6 recognized all human and animal rotavirus strains tested and could be engineered for their use in immunodiagnostic tests for group A rotavirus detection. Three of the four VHH neutralized rotavirus in vivo independently of the strain serotype. Furthermore, this result was confirmed by in vivo partial protection against rotavirus challenge in a neonatal mouse model. The present study demonstrates for the first time a broad neutralization activity of VP6 specific VHH in vitro and in vivo. Neutralizing VHH directed to VP6 promise to become an essential tool for the prevention and treatment of rotavirus diarrhea.Group A rotavirus (RV) is the leading cause of acute gastroenteritis in human infants less than 5 years old, causing 611,000 deaths per year (41). It is also the main cause of severe diarrhea in the neonates of many animal species of economic interest (43,47). RV virions are triple-layered particles composed by a core (protein VP2), an inner capsid (protein VP6), and an outer capsid (proteins VP7 and VP4) (16,29). The inner capsid protein, VP6, is a trimer representing 51% of the virion mass. According to the antigenic variation of VP6, RVs are classified into seven groups (A to G) (16). Depending on the presence or absence of two different epitopes in the VP6 protein, group A RV strains are further divided into subgroups (Sb) I, II, IϩII, and no I no II. Despite the different subgroups mentioned, VP6 is a strongly conserved protein among all group A RVs (Ͼ90% amino acid homology). It is highly immunogenic and constitutes the target antigen of most immunodiagnosis tests for group A RV detection. In contrast, the outer capsid proteins VP7 (glycoprotein) and VP4 (protease sensitive) are highly variable and constitute the major neutralizing antigens. Based on the variation of VP7 and VP4, group A RVs are further classified into G and P types, respectively.

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

“…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). The neutralizing capacity associated with the recognition of crossreactive epitopes within VP6 of these antibodies may also relate to their small size, as bivalent VHH antibodies showed a much reduced neutralizing activity compared with the monovalent VHH, suggesting that these may have no or limited access to the exposed VP6 of an infectious triple-layered RV particle (50).…”

“…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).…”

Llama Antibody Fragments with Cross-Subtype Human Immunodeficiency Virus Type 1 (HIV-1)-Neutralizing Properties and High Affinity for HIV-1 gp120

“…The VHH domain can be easily cloned and expressed to high levels in bacteria and yeast (26,27). This notion, together with advantageous characteristics in terms of stability and solubility (20,64,81), has led to successful development of camelid VHH in a number of applications against a range of biological targets (2,13,14,19,21,57,58,69,83,84), including neutralization of rotavirus (28,60). We hypothesized that the small size of VHH in combination with their protruding CDR3 loops and their preference for cleft recognition may allow them to recognize conserved motifs on gp120 that are occluded from conventional antibodies.…”

Immunogenicity and antigenicity of a recombinant chimeric protein containing epitopes of poliovirus type 1

“…4h). VP6 possess broad neutralizing activity in vitro and confer protection against diarrhea in mice (Garaicoechea et al, 2008) and neonatal gnotobiotic piglets (Vega et al, 2013).…”

“…For the last few years, studies have shown that VP6 could stimulate a protective immune response (Bugli et al, 2014;Li et al, 2014;Marashi et al, 2014;Pastor et al, 2014;Shoja et al, 2015) and a short fragment of VP6 could provide significant reduction in virus infectivity in vitro (El-Senousy et al, 2013). Previous experiments demonstrated that anti-VP6 llama-derived single-chain antibody fragments (VHH) had neutralizing activity against VP6 in vitro (Garaicoechea et al, 2008). Using RV VP6 as a vector (VP6F), the chimeric proteins carrying epitopes derived from the VP4 of RV were constructed and demonstrated that these chimeric proteins had good antigenic reactivity and immunogenicity (Teng et al, 2014).…”