Remember the 2016 Olympics in Brazil? It was the year Brazil defeated Germany in the final game of the men's football (aka soccer) tournament, but it was also the year when Zika virus made its entry into the Americas.
Zika virus is a mosquito-transmitted virus and member of the flaviviridae family, which also includes dengue virus. Zika virus has only one serotype, whereas dengue virus has four. Both Zika and dengue viruses are structurally similar and are prevalent in many overlapping regions of the world infecting millions of people every year. Here is the problem: If you are infected with one of these serotypes and raise a successful immune response, you may be at greater risk of becoming seriously ill if you get a second infection with a different serotype. This is due to antibody-dependent enhancement of infection, wherein cells expressing Fc-gamma receptor uptake viral particles that are bound – but not neutralized – by antibodies. This mechanism is the major explanation for increased disease severity in during a person’s second lifetime infection.
Currently, there are no vaccines available that elicit protection against all four dengue virus serotypes and Zika virus. Thus, there is immense interest in isolating so-called broadly neutralizing antibodies and studying their characteristics. Neutralizing antibodies against Zika and dengue viruses typically target the surface envelope (E) protein, preventing them from infecting cells. Previous efforts have isolated many Zika virus-specific (i.e., ‘targeted’) antibodies and a handful of broadly neutralizing antibodies (which neutralize both dengue virus and Zika virus). However, the effect of viral mutations in the E protein, and the potential for these mutations to lead to escape from antibody neutralization, remains undefined.
In a recent study, led by Caroline Kikawa, an MD-PhD student co-mentored by Dr. Leslie Goo, an assistant professor, and Dr. Jesse Bloom, a professor at Fred Hutch, used deep mutational scanning to identify potential single mutations on the Zika virus E protein that led to escape from antibody-mediated neutralization. “Infections can result in serious, sometimes fatal, hemorrhagic fever (in the case of dengue virus) or neuroinvasive disease (in the case of Zika virus). As these co-circulating flaviviruses are responsible for potentially hundreds of millions of infections each year, there is substantial interest in designing a vaccine that would simultaneously protect against all these viruses,” said Kikawa. “Broadly neutralizing antibodies have previously been isolated from humans and their structures in complex with viral proteins have been solved. However, the effect of viral adaptation and variation – in its simplest form of single amino acid mutations – on escape from broadly neutralizing antibodies had never been measured,” she added.
Deep mutational scanning of viral entry proteins is a major interest in the Bloom lab. This approach allows for the mapping of mutations within a target protein, in this case the Zika virus E protein, that allow the virus to escape antibody neutralization. A deep mutational scanning library for Zika virus E protein was created wherein each virus expressed a unique E protein sequence containing a single amino acid mutation. Once the viruses were generated, the researchers performed neutralization assays with candidate antibodies. They then used next-generation sequencing to identify the viral mutations that escaped neutralization. The team tested a panel of four antibodies, including broad and targeted neutralizing antibodies. The antibodies EDE1-C10, EDE1-C8 and SlgN-3C all neutralize both Zika virus and the four dengue virus serotypes, whereas the pseudo-broad antibody MZ4 neutralizes only Zika virus and two serotypes of dengue virus. The researchers also studied ZV-67, which neutralizes only Zika virus.
Using the deep mutational scanning library of the Zika virus E protein, the team found that “broad antibodies [EDE1 and SlgN-3C] are quantifiably more difficult to escape than targeted, Zika-specific antibodies [ZV-67].” Additionally, the authors found that “mutations that confer escape sometimes do so across viral lineages.” “Furthermore, we show that antibodies with highly similar structural protein-protein interactions are escaped by different amino acid mutations,” said Kikawa. “This indicates that functional requirements for neutralization cannot be inferred from structural antibody studies alone.”
Deep mutational scanning is a powerful tool to examine the functional and antigenic effects of viral protein mutations. “As the endemic ranges of Zika and dengue viruses spread with climate change, so too will the infection rates of global populations,” commented Kikawa. “It will therefore be useful to know how these viral proteins might adapt and evolve.” Going forward, the team is interested in “using these methods to measure functional tolerance and antigenicity of other flavivirus envelope proteins.” Kikawa concluded by stating, “our paper measures escape from monoclonal antibodies, but these methods could also be used to examine the escape profiles from human immune serum samples containing a complex, polyclonal antibody repertoire. Measuring effects of single mutations on escape from broadly neutralizing human sera would be a natural extension of our work.”
This spotlighted research was supported by the Viral Pathogenesis and Evolution Training Grant, the Disease of Public Health Importance Training Grant, and the Fred Hutchinson Cancer Center Diverse Trainee Fund.
Kikawa C, Cartwright-Acar CH, Stuart JB, Contreras M, Levoir LM, Evans MJ, Bloom JD, Goo L. 2023. The effect of single mutations in Zika virus envelope on escape from broadly neutralizing antibodies. J Virol. 97(11):e0141423.