Gain-of-Function Research: Background And Alternatives - NCBI

TYPES OF GAIN-OF-FUNCTION (GOF) RESEARCH

Subbarao explained that routine virological methods involve experiments that aim to produce a gain of a desired function, such as higher yields for vaccine strains, but often also lead to loss of function, such as loss of the ability for a virus to replicate well, as a consequence. In other words, any selection process involving an alteration of genotypes and their resulting phenotypes is considered a type of Gain-of-Function (GoF) research, even if the U.S. policy is intended to apply to only a small subset of such work.

Subbarao emphasized that such experiments in virology are fundamental to understanding the biology, ecology, and pathogenesis of viruses and added that much basic knowledge is still lacking for SARS-CoV and MERS-CoV. Subbarao introduced the key questions that virologists ask at all stages of research on the emergence or re-emergence of a virus and specifically adapted these general questions to the three viruses of interest in the symposium (see Box 3-1). To answer these questions, virologists use gain- and loss-of-function experiments to understand the genetic makeup of viruses and the specifics of virus-host interaction. For instance, researchers now have advanced molecular technologies, such as reverse genetics, which allow them to produce de novo recombinant viruses from cloned cDNA, and deep sequencing that are critical for studying how viruses escape the host immune system and antiviral controls. Researchers also use targeted host or viral genome modification using small interfering RNA or the bacterial CRISPR-associated protein-9 nuclease as an editing tool.

BOX 3-1

General Virology Questions and Questions Specific to Influenza, SARS, and MERS Research. Why/how does the virus infect and kill mammals? What are the critical host range and virulence determinants of MERS-CoV?

During Session 3 of the symposium, Dr. Yoshihiro Kawaoka, from the University of Wisconsin-Madison, classified types of GoF research depending on the outcome of the experiments. The first category, which he called “gain of function research of concern,” includes the generation of viruses with properties that do not exist in nature. The now famous example he gave is the production of H5N1 influenza A viruses that are airborne-transmissible among ferrets, compared to the non-airborne transmissible wild type. The second category deals with the generation of viruses that may be more pathogenic and/or transmissible than the wild type viruses but are still comparable to or less problematic than those existing in nature. Kawaoka argued that the majority of strains studied have low pathogenicity, but mutations found in natural isolates will improve their replication in mammalian cells. Finally, the third category, which is somewhere in between the two first categories, includes the generation of highly pathogenic and/or transmissible viruses in animal models that nevertheless do not appear to be a major public health concern. An example is the high-growth A/PR/8/34 influenza strain found to have increased pathogenicity in mice but not in humans. During the discussion, Dr. Thomas Briese, Columbia University, further described GoF research done in the laboratory as being a “proactive” approach to understand what will eventually happen in nature.

In Session 8 of the symposium, Dr. Ralph Baric, University of North Carolina and a member of the symposium planning committee, explained that GoF experiments for CoV research encompass a very diverse set of experiments that are critical to the development of broad-based vaccines and therapeutics. Like Subbarao and Kawaoka, Baric listed experiments important for the identification of determinants of pathogenesis and virulence, defined the virus-host interaction networks, and described the alleles responsible for susceptibility and the host response patterns that drive a pathogenic or protective responses. However, he specifically noted that transmissibility studies for SARS and MERS-CoV actually fall in a different category than influenza research because of fundamental biological differences between these viruses. He first explained that the SARS-CoV has evolved over the past ~800 years to efficiently infect human cells that expressed the ACE2 viral receptor. To illustrate this, he shared sequencing results obtained from the Chinese during the 2003 SARS-CoV pandemic that show the gradual changes in the amino acid sequence across the genome associated with the expending epidemic. Among the 16 mutations found at the end of the pandemic, two were associated with the increased efficiency of the civets' strains to use the ACE2 receptor to invade human cells. In vitro experiments on human airway epithelial (HAE) cells and in vivo experiments on transgenic mice showed that while the human strain can efficiently infect and replicate in cells expressing the human, bat, and civet ACE2 receptor, the civet strain cannot use the human ACE2 receptor. This demonstrates the human SARS-CoV strain evolved to maintain its capacity to replicate and cause expanding epidemics while keeping its capacity to cycle through civets and most likely retreat into the bat reservoir following the control of the epidemic. In most instances, GoF experiments looking at receptor interactions with SARS-CoV and MERS-CoV showed that in in vitro or in vivo models with a civet strain gain human ACE2 receptors but also lose the civet ACE2 receptor. Cell receptors for influenza viruses are relatively similar across different species, and this prompts a concern about possible increased transmission in humans from an influenza virus that is adapted for readier transmission in other mammals. By contrast, the ACE2 orthologue receptor interface for coronaviruses varies more markedly across different species.