Identification of Human Monoclonal Antibodies That Neutralize Pandemic GII.4 Noroviruses

Human monoclonal antibodies able to neutralize live human norovirus GII.4 Sydney 2012—the predominant strain responsible for recent outbreaks—are reported in the December issue of Gastroenterology. Researchers analyzed these antibodies and identified the neutralizing epitopes, providing insight into the human immune response to this deadly virus.

Inhibition of replication of GII.4 Sydney 2012 virus using selected IgGs, IgAs, and a nonspecific mAb, tested in the human intestinal enteroid system. Controlsfor each experiment were virus incubated without mAb. The IC50 value for each mAb is indicated on each graph. Data presented are an average of 2 independent experiments for NORO-263, -250B, -320, -273A, and -318 and a Dengue virus–specific control antibody 2D22.

Human noroviruses are the major cause of epidemic and sporadic acute gastroenteritis, due to their low infectious dose, environmental stability, high levels of shedding, and prolonged shedding even after symptoms have resolved. Noroviruses cause an average 19–21 million cases of infection and 570–800 deaths in children younger than 5 years each year in the United States, although they infect people of all ages. Even though infection is usually acute and self-limited, disease can become life threatening in young, old and immunocompromised people.

The extreme antigenic diversity of strains poses challenges to developing antibodies or vaccines to prevent noroviruses-associated disease. Noroviruses are classified into 7 different genogroups (genogroups [G] I–VII), and at least 41 different genotypes. Viruses from G1 and the rapidly evolving GII account for nearly all human infections. The human norovirus genome contains 3 open-reading frames (ORFs). ORF1 encodes nonstructural proteins, whereas ORF2 and ORF3 encode the major and minor capsid proteins, respectively.

Noroviruses could not previously be cultivated in cell culture, but the VP1 and VP2 protein sequences could be expressed using a baculovirus expression system to produce human noroviruses virus-like particles (VLPs). These VLP reagents have facilitated the study of norovirus evolution, antigenicity, and the emergence of strains.

In the journal Clinical Gastroenterology and Hepatology, Shintaro Sato et al report propagation of human norovirus (the the GII.3, GII.4, GII.6, and GII.17 genotypes) in human induced pluripotent stem cell-derived intestinal epithelial cells.

Since the mid-1990s, viruses from GII genotype 4 (GII.4) have caused most outbreaks, with new strains emerging every 2–3 years. In 2012, the GII.4 Sydney strain emerged and began spreading globally—it has continued to predominate among circulating strains. However, the molecular basis for antibody-mediated recognition of these strains and their mechanisms of action are not well characterized.

Gabriela Alvarado et al isolated and characterized a panel of human monoclonal antibodies that bind to GII.4 Sydney 2012 VLPs. They found that most of these antibodies also blocked receptor binding, based on their ability to inhibit hemagglutination of human O Rh+ red blood cells or the interaction between GII.4 Sydney 2012 VLPs and porcine gastric mucin (PGM). These assays are surrogate systems for testing norovirus neutralization.

There have been previous attempts to culture human noroviruses, but none could establish a robust, reproducible system of viral growth. The authors’ development of an in vitro replication system, using human intestinal organoid technology, made it possible to cultivate human noroviruses and test their inhibition (see figure).

Alvarado et al used a human jejunal monolayer culture system to identify antibodies that neutralize live GII.4 Sydney 2012 HuNoV. They identified neutralizing human monoclonal antibodies against norovirus, as well as a panel of human anti-GII.4 Sydney 2012 VLP-binding IgGs and anti-GII.4 human IgA molecules.

Twenty-five human monoclonal antibodies specific to pandemic strain GII.4 Sydney 2012 VLPs were isolated. Using stem cell-derived human enteroids and live virus, the authors identified the monoclonal antibodies that inhibited virus replication. Almost 70% of the monoclonal antibodies isolated had high level of potency, inhibiting GII.4 Sydney 2012 VLPs from binding to PGM at half-maximal effective concentrations (EC50) below 24 μg/mL.

The authors used this panel of antibodies to identify major antigenic sites on the GII.4 Sydney 2012 major capsid protein. In competition binding studies, most antibodies recognized 3 major antigenic sites on the GII.4 Sydney 2012 protruding (P) domain. Definition of neutralization epitopes in high resolution with neutralizing antibodies could be used in rational structure-based vaccine design.

Of the 25 human antibodies isolated in this study, 17 blocked GII.4 Sydney 2012 VLPs from binding to PGM at concentrations as low as 2.4 μg/mL. Eighteen monoclonal antibodies that blocked GII.4 Houston 2002 from binding to PGM at concentrations as low as 2 μg/mL also either blocked GII.4 Sydney 2012 VLPs from binding to PGM or inhibited hemagglutination at the concentrations tested. Interestingly, 13 of the 14 IgGs that blocked GII.4 Houston 2002 VLPs from binding to PGM did so at a lower EC50 values than those for GII.4 Sydney 2012. This might mean that the donors included in this study had prior exposure to an earlier norovirus variant similar to GII.4 Houston 2002.

Alvarado et al conclude that these findings provide important information about the human humoral immune response to norovirus and identify monoclonal antibodies that might be used in diagnosis and treatment of these infections.

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