Isolation of Human Monoclonal Antibodies That Neutralize Human Rotavirus

Abstract

A human antibody library constructed by utilizing a phage display system was used for the isolation of human antibodies with neutralizing activity specific for human rotavirus. In the library, the Fab form of an antibody fused to truncated cp3 is expressed on the phage surface. Purified virions of strain KU (G1 serotype and P[8] genotype) were used as antigen. Twelve different clones were isolated. Based on their amino acid sequences, they were classified into three groups. Three representative clones-1-2H, 2-3E, and 2-11G-were characterized. Enzyme-linked immunosorbent assay with virus-like particles (VLP-VP2/6 and VLP-VP2/6/7) and recombinant VP4 protein produced from baculovirus recombinants indicated that 1-2H and 2-3E bind to VP4 and that 2-11G binds to VP7. The neutralization epitope recognized by each of the three human antibodies might be human specific, since all of the antigenic mutants resistant to mouse monoclonal neutralizing antibodies previously prepared were neutralized by the human antibodies obtained here. After conversion from the Fab form of an antibody into immunoglobulin G1, the neutralizing activities of these three clones toward various human rotavirus strains were examined. The 1-2H antibody exhibited neutralizing activity toward human rotaviruses with either the P[4] or P[8] genotype. Similarly, the 2-3E antibody showed cross-reactivity against HRVs with the P[6], as well as the P[8] genotype. In contrast, the 2-11G antibody neutralized only human rotaviruses with the G1 serotype. The concentration of antibodies required for 50% neutralization ranged from 0.8 to 20 g/ml.Rotavirus is the major cause of severe acute gastroenteritis among infants and young children. Rotavirus infection is lifethreatening in developing countries, resulting in 500,000 to 600,000 deaths annually (33). In developed countries, rotavirus infections lead to a high disease burden with considerable medical expense due to the high morbidity. Furthermore, adults, particularly the elderly, are also affected by rotavirus infection (34, 39), and immunocompromised children and adults develop persistent rotavirus diarrhea (12,42,43). Thus, vaccination is thought to be the best way to reduce severe rotavirus gastroenteritis worldwide. Tetravalent rhesus rotavirus (RRV) human reassortant vaccine comprising RRV and three RRV-based monoreassortants carrying the VP7 genes from G1, G2, and G4 human rotaviruses (HRVs) was developed (25), and 1.5 million doses of this vaccine had been administered to infants by the end of May 1999 in the United States. However, the vaccine was withdrawn due to the occurrence of gut intussusception, which appeared to be epidemiologically linked to vaccine application (5, 38). Moreover, even if a safe and effective rotavirus vaccine is developed, vaccination would be less effective in immunocompromised patients.Rotaviruses have two outer capsid proteins, viral protein 4 (VP4) and VP7, encoded on RNA segment 4 and RNA segment 7, 8, or 9, depending on the strain, respectively (19). VP4 and VP7 are kn...

Isolation of cross‐reactive human monoclonal antibodies that prevent binding of human noroviruses to histo‐blood group antigens

“…But immunoglobulin preparations from human sera possibly contain contaminants including infectious agents, and have the problem of quantitative limitation. The Fab-cp3 or scFv-cp3 form of antibodies described here can be easily converted to the human IgG form of the antibodies, and can be produced on a large scale with little risk of contamination by using mammalian cell culture systems [Higo-Moriguchi et al, 2004]. Among the eight representative clones that were assessed for their blocking activities against binding of homotypic VLPs to histo-blood group antigens, six exhibited sufficient blocking activities, suggesting their neutralizing activity.…”

“…To screen anti-HuNoV antibodies, a panning method was performed as described previously [Higo-Moriguchi et al, 2004]. In brief, immunotubes (Nunc-Immunomodules Polysorp) were coated with 3 ml of a VLP suspension (100 mg/ml) of purified r104 (GII.4) or rCV (GI.4) in phosphate buffered saline (pH7.5) containing 100 mg of CaCl 2 ·2H 2 O and MgCl 2 ·6H 2 O/ml [PBS(þ)] overnight at 4˚C.…”

“…After a blocking step with 2% (w/v) skim milk in PBS(þ) [PBS(þ)-SM 2% ], a suspension of phages, 1 Â 10 13 colony forming unit (CFU), in PBS(þ)-SM 2% was added to the tube, followed by incubation at room temperature for 2 hr. The procedures for washing the tube or elution of phages, infection of E. coli DH12S cells with the eluate, superinfection with helper phages, and the culture conditions were the same as described previously [Higo-Moriguchi et al, 2004]. Panning cycles were repeated until the output/input values exhibited an increase.…”

“…The availability of VLPs made it possible to study the antigenic properties of HuNoV, and several reports on immune responses against HuNoV VLPs in rodent models have appeared [Ball et al, 1998;Hale et al, 2000;Kitamoto et al, 2002;Hansman et al, 2006;Shiota et al, 2007;Parra et al, 2012]. Anti-rotavirus human neutralizing monoclonal antibodies were previously isolated from a phage-displayed antibody library that should reflect the antibody repertoires produced in naturally infected persons, and demonstrated that epitopes recognized by these human neutralizing antibodies were essentially different from those recognized by mouse neutralizing antibodies produced in immunized animals [Higo-Moriguchi et al, 2004;Monnier et al, 2006], suggesting the value of examining the antibody repertories for other pathogens produced in the naturally infected human immune system. Because NoV infection is very common worldwide and dominant strains have changed over several years [Lindesmith et al, 2008[Lindesmith et al, , 2010, it is expected that various antibodies should be produced by and persist in the healthy human immune system.…”

Identification of one peptide which inhibited infectivity of avian infectious bronchitis virus in vitro

“…Phage display has been widely utilized to screen antigen epitope [10] or monoclonal antibodies [11] , and study interaction between viruses and receptors. Virus surface glycoprotein displayed on phage is often used to pan against cells to screen new virus receptor or functional domain of receptors, for instance, in adenovirus type 5 (Ad5) [12] , hepatitis C virus [13] , HIV [14] , rotavirus [15] , etc.…”