Genetically Modified Mosquitos Neutralize Dengue Virus

Researchers at the University of California San Diego have identified a target gene in mosquitos that renders the insects completely refractory to all 4 serotypes of the dengue virus and thus, incapable of transmitting the virus to humans, according to study results published in PLoS Pathogens.¹

Dengue fever is caused by infection with 1 of 4 distinct serotypes of the dengue virus, and is most commonly transmitted to humans via a bite from an infected Aedes aegypti mosquito. Characteristics of dengue fever include myalgias, headache, and severe fever; progression to potentially fatal hemorrhagic fever and dengue shock syndrome is common in endemic regions, and disproportionately affects children and people who are immunocompromised. According to the Pan American Health Organization, cases of dengue are at a record high, with >50% of the world’s population at risk for infection.² However, there are currently no treatment or prophylaxis protocols for treating the infection.

Previous research has identified a dengue virus-targeting monoclonal antibody, 1C19, which is present and naturally occurring in humans who have been vaccinated for or have survived infection with the dengue virus. In vitro studies have shown that this antibody can neutralize all 4 dengue virus serotypes. Therefore, researchers in the current study engineered Ae aegypti to express 1C19-based, broadly neutralizing, single-chain variable fragments, rendering these mosquitos unable to be infected with or transmit the dengue virus.

Researchers used reverse-transcriptase polymerase chain reaction amplification of the 1C19 antibody gene from complementary DNA sequences, which were identified using Sanger sequence analyses to extract RNA. The RNA was then cloned into a plasmid that was subsequently primed and then amplified with a carboypeptidase promoter, designed to induce gene expression following a blood meal and thus, generate a gene vector for the 1C19 antibody: OA984. This gene vector, or transgene, was subsequently tagged with a hemagglutinin epitope to facilitate downstream gene analysis. Ae aegypti embryos were injected with a combination of tagged and untagged vectors. Researchers mated 1 transgenic male mosquito with 10 wildtype females to create independent lines and inbred them for >20 generations to generate a homozygous population; a heterozygous population was also generated via crossing out homozygous mosquitos to wildtype mosquitos.

Wildtype and transgenic mosquitos were exposed to dengue virus strains 1, 2, 3, and 4. Salivary samples were used to determine subsequent viral titers. Researchers also obtained a population of Ae aegypti infected with the gram-negative bacteria Wolbachia. Previous studies have established that infection with this bacteria blocks viral replication (of dengue, Zika, and Chikungunya viruses) within mosquito hosts and catalyzes an inheritable genomic shift.

Results demonstrated several key findings. The anti-dengue virus transgene was stably integrated into Ae aegypti genome, and the 1C19 antibody was successfully and appropriately expressed such that the dengue virus was suppressed. None of the mosquitoes homozygous for the transgene with the strongest phenotype were positive for strain 2 of dengue virus or viral replication compared with 85.4% and 86.6%, respectively, of mosquitoes that were heterozygous for the transgene. Of note, the heterozygous mosquitos had significantly lower viral levels compared with wildtype mosquitos (P <.001). Further, no salivary samples from mosquitos homozygous for the strongest phenotype of the transgene contained strain 2 of dengue virus, while the strain was found in 83.3% of samples from heterozygous mosquitos.

Tests for infection, viral replication, and transmission parameters were conducted for strains 1, 3, and 4 of dengue virus. Results demonstrated a similar pattern of findings.

Direct comparison of mosquitos homozygous for the strongest phenotype of the transgene with mosquitos infected with Wolbachia demonstrated the transgene to be significantly more effective at repelling dengue infection. The heterozygous transgene demonstrated similar effectiveness to Wolbachia infection.

With regard to mosquito survivorship, results demonstrated no significant differences between male mosquitos homozygous for the transgene and male wildtype mosquitos; female mosquitos homozygous for the transgene lived for, on average, 4.5 days less than their wildtype counterparts.

Researchers concluded that “results show that heterozygous 1C19 [single-chain variable fragment] antibody-expressing transgenic mosquitoes are just as efficient at viral suppression as—and homozygous mosquitoes are significantly more efficient than—Wolbachia-infected mosquitoes, which are currently being released for [dengue virus] control because they are known to be refractory to [the virus].”

Reference

1. Buchman A, Gamez S, Li M, et al. Broad dengue neutralization in mosquitioes expressing an engineered antibody. PLoS Pathog. doi:10.1371/journal.ppat.1008103

2. Mosquitoes Engineered to Repel Dengue Virus [news release]. San Diego, CA: University of California San Diego; January 13, 2020. https://www.newswise.com/articles/mosquitoes-engineered-to-repel-dengue-virus?sc=dwhr&xy=10020744. Accessed January 16, 2020.