Isolation of Neutralizing Monoclonal Antibodies to Human Astrovirus and Characterization of Virus Variants That Escape Neutralization

Abstract

Human astroviruses (HAstVs) cause severe diarrhea and represent an important health problem in children under two years of age. Despite their medical importance, the study of these pathogens has been neglected. To better understand the astrovirus antigenic structure and the basis of protective immunity, in this work we produced a panel of neutralizing monoclonal antibodies (Nt-MAbs) to HAstV serotypes 1, 2, and 8 and identified the mutations that allow the viruses to escape neutralization. We first tested the capacity of the recombinant HAstV capsid core and spike domains to elicit Nt-Abs. Hyperimmunization of animals with the two domains showed that although both induced a potent immune response, only the spike was able to elicit antibodies with neutralizing activity. Based on this finding, we used a mixture of the recombinant spike domains belonging to the three HAstV serotypes to immunize mice. Five Nt-MAbs were isolated and characterized; all of them were serotype specific, two were directed to HAstV-1, one was directed to HAstV-2, and two were directed to HAstV-8. These antibodies were used to select single and double neutralization escape variant viruses, and determination of the amino acid changes that allow the viruses to escape neutralization permitted us to define the existence of four potentially independent neutralization epitopes on the HAstV capsid. These studies provide the basis for development of subunit vaccines that induce neutralizing antibodies and tools to explore the possibility of developing a specific antibody therapy for astrovirus disease. Our results also establish a platform to advance our knowledge on HAstV cell binding and entry. IMPORTANCE Human astroviruses (HAstVs) are common etiological agents of acute gastroenteritis in children, the elderly, and immunocompromised patients; some virus strains have also been associated with neurological disease. Despite their medical importance, the study of these pathogens has advanced at a slow pace. In this work, we produced neutralizing antibodies to the virus and mapped the epitopes they recognize on the virus capsid. These studies provide the basis for development of subunit vaccines that induce neutralizing antibodies, as well as tools to explore the development of a specific antibody therapy for astrovirus disease. Our results also establish a platform to advance our knowledge on HAstV cell binding and entry.

A simplified workflow for monoclonal antibody sequencing

“…Next, we tested the ability of the recombinant chimeric mAb 2D9 to bind its antigen, Spike 8, the recombinant capsid spike domain from human astrovirus serotype 8, against which mouse mAb 2D9 was raised. [25] By indirect ELISA, we found that both chimeric mAb 2D9 and mouse mAb 2D9 bind Spike 8 (Fig 4B and 4C). The other four chimeric mAbs also bind the human astrovirus spike against which they were raised.…”

“…To sequence the variable regions of five mouse monoclonal IgG1 antibodies (2D9, 3B4, 3E8, 3H4, and 4B6),[25] we extracted total RNA from the hybridoma cell lines expressing these antibodies and applied a modified RT-PCR (reverse transcription polymerase chain reaction) using SMART (switching mechanism at 5' end of RNA transcript) technology. [26, 27] This technology is based on the intrinsic features of the reverse transcriptase from the Moloney murine leukemia virus (MMLV) and the application of a custom-sequence template-switch oligonucleotide (template-switch oligo) forward primer containing 3 riboguanines (rGrGrG) at its 3' end.…”

“…The hybridoma cells producing monoclonal antibodies were generated as described in [25]. Total RNA was extracted from the hybridoma cells using TRIzol Reagent (Invitrogen, 15596026) according to the manufacturer’s instructions.…”

Protein Disulfide Isomerase A4 Is Involved in Genome Uncoating during Human Astrovirus Cell Entry

“…The capsid of the mature, infectious virus has an icosahedral morphology of T = 3 and is composed of two proteins: VP34, which forms the shell of the virus particle (core protein), and VP27, a protein that constitutes the 30 dimeric globular spikes that protrude from the virion (spike protein) [ 3 ]. The spike protein, but not the core protein, has been shown to induce neutralizing antibodies [ 4 ] and several neutralizing antigenic determinants have been mapped on the spike, defined either by X-ray crystallography [ 5 ] or by sequencing HAstV-1, -2, and -8 mutants that escape neutralization by monoclonal neutralizing antibodies [ 4 ]. In addition, the HAstV spike has been shown to specifically bind to the surface of Caco-2 cells and to contain the receptor-binding domain [ 5 ].…”

“…HAstV has been shown to enter cells by clathrin-mediated endocytosis [ 6 ]; however, very little is known about the viral receptor or other cell factors that may determine virus entry. In this work we took advantage to the fact that the astrovirus capsid core and spike proteins can be successfully produced in Escherichia coli while maintaining their native structure [ 4 , 7 , 8 ] to identify interacting cellular proteins relevant for HAstV infection using a far-Western blotting approach. We found protein disulfide isomerase A4 (PDIA4) is an entry factor associated with genome uncoating of HAstV serotypes 1 and 8.…”