Evaluation of an Engineered Zika Virus-Like Particle Vaccine Candidate in a Mosquito-Mouse Transmission Model

Abstract

The primary route of Zika virus (ZIKV) transmission is through the bite of an infected Aedes mosquito, when it probes the skin of a vertebrate host during a blood meal. Viral particles are injected into the bite site together with mosquito saliva and a complex mixture of other components. Some of them are known to play a key role in the augmentation of the arbovirus infection in the host, with increased viremia and/or morbidity. This vector-derived contribution to the infection is not usually considered when vaccine candidates are tested in preclinical animal models. In this study, we performed a preclinical validation of a promising ZIKV vaccine candidate in a mosquito-mouse transmission model using both Asian and African ZIKV lineages. Mice were immunized with engineered ZIKV virus-like particles and subsequently infected through the bite of ZIKV-infected Aedes aegypti mosquitoes. Despite a mild increase in viremia in mosquito-infected mice compared to those infected through traditional needle injection, the vaccine protected the animals from developing the disease and strongly reduced viremia. In addition, during peak viremia, naive mosquitoes were allowed to feed on infected vaccinated and nonvaccinated mice. Our analysis of viral titers in mosquitos showed that the vaccine was able to inhibit virus transmission from the host to the vector. IMPORTANCE Zika is a mosquito-borne viral disease, causing acute debilitating symptoms and complications in infected individuals and irreversible neuronal abnormalities in newborn children. The primary vectors of ZIKV are Aedes aegypti mosquitoes. Despite representing a significant public health burden with a widespread transmission in many regions of the world, Zika remains a neglected disease with no effective antiviral therapies or approved vaccines. It is known that components of the mosquito bite lead to an enhancement of viral infection and spread, but this aspect is often overlooked when vaccine candidates undergo preclinical validation. In this study, we included mosquitoes as viral vectors, demonstrating the ability of a promising vaccine candidate to protect animals against ZIKV infections after the bite of an infected mosquito and to also prevent its further transmission. These findings represent an additional crucial step for the development of an effective prevention tool for clinical use.

Keywords: Aedes aegypti; Zika virus; arbovirus; flavivirus; mosquito; vaccine.

FIG 1

Evaluation of VLP-cvD vaccine efficacy against ZIKV PRVAB59 in a mosquito-mouse transmission model. (A) Schematic representation of the immunization and challenge procedure. Each (n = 6) 4-week-old A129 mouse received 3 doses of VLP-cvD or PBS mixed with AddaVax adjuvant. Seven days prior to bite-mediated challenge, mosquitoes were infected with ZIKV by intrathorax microinjection. (B) Anti-dimeric E antibody titers of sera collected from animals immunized with VLP-cvD (blue) or PBS (gray). Antibody titers were determined using ELISA plates coated with mono-biotinylated dimeric E. The titer was defined as the maximum dilution that gives a value higher than three times the value given by the preimmune sera. Control sera were negative at the lowest dilution (1:900), and their titer was calculated as one-third of that dilution (300). Data are geometric means with geometric standard deviations (SD) from three independent experiments. (C) Neutralization of PRVABC59 ZIKV infection. Serially diluted samples of mouse sera were incubated with ZIKV for 1 h before infecting Vero-furin cells. At 72 h postinfection, the intracellular levels of E were determined by capture sandwich ELISA, and the percentage infectivity relative to that of the virus alone was calculated. The results were plotted as MN₅₀ values. Data are geometric means with geometric SD from three independent experiments. (D) Number of fed mosquitoes per mouse at the end of the feeding procedure. Mice were anesthetized and put on cardboard cups containing 10 infected mosquitoes each. After 20 min, mice were removed, mosquitoes were anesthetized by exposure to low temperature, and the engorged mosquitoes were counted. (E) Relative quantification of ZIKV genome copies normalized on the host genome in mosquito carcasses. (F and G) Animals were weighed (F) and scored for clinical signs daily postchallenge (G). The scoring system used to monitor animal health following ZIKV challenge was as follows: 0 (green) for no signs of distress or disease, 1 (yellow) for one sign of distress, 2 (orange) for two signs of distress or mild disease, and 3 (red; humane endpoint) for more than two signs of severe disease or loss of 15% of body weight. (H) Viral titers in challenged animals. The levels of ZIKV in the serum at days 2, 3, 4, and 7 postinfection were quantified by RT-qPCR, and the results were plotted as equivalent PFU per milliliter. The limit of quantification was estimated to be 100 PFU/mL, indicated by the dotted line. Data are geometric means from all mice with geometric SD. Assays were performed in triplicate. **, P = 0.0021; ****, P < 0.0001.

FIG 2

Evaluation of VLP-cvD vaccine efficacy against ZIKV MP1751 in a mouse-mosquito transmission model. (A) Anti-dimeric E antibody titers of sera collected from A129 mice immunized with VLP-cvD (blue and pink) or PBS (gray and brown). Data are geometric means with geometric SD from three independent experiments. (B) Neutralization of MP1751 ZIKV infection. The results were plotted as MN₅₀ values. Data are geometric means with geometric SD from three independent experiments. (C) Number of fed mosquitoes per mouse at the end of the feeding procedure. (D) Relative quantification of ZIKV genome copies normalized to the host genome in mosquito carcasses. (E) Survival of mice in the course of the 14-day challenge. (F) Mouse scoring for signs of infection. (G) Animal body weight variations, calculated as a percentage of the initial weight. The red line indicates the day of the transmission feeding procedure (day 4). (H) Viral titers in challenged animals at days 3 and 5. Data are geometric means from all mice with geometric SD. Quantification was performed in triplicate. The dotted line indicates the limit of quantification. Red arrowheads indicate data for control-mosquito mouse 5 and orange arrowheads indicate data for vaccinated-needle mouse 1 throughout the figure. *, P = 0.0332; **, P = 0.0021; ***, P = 0.0002; ****, P < 0.0001.

FIG 3

Reverse transmission from mammalian host to invertebrate vector. (A) Schematic of A129 mouse immunization, virus challenge, and subsequent postfeeding mosquito analysis. Animals correspond to the needle groups whose data are shown in Fig. 2. (B) Viral titer in challenged animals at days 3 (solid bars) and 5 (hatched bars). The x axis indicates individual mice. Quantification was performed in triplicate. (C) Number of mosquitoes that completed a blood meal on VLP-cvD (pink)- or PBS (brown)-injected mice. Data are the number of engorged mosquitoes at the end of the feeding procedure (day 0) and the individual surviving the 2 weeks incubation period (day 14). (D) Relative quantification of ZIKV genome copies normalized to the host genome from mosquito carcasses. (E) Quantification of viral titer in homogenized mosquito salivary glands by FFA. The data are shown as box plots with minimum and maximum values indicated; dots represent individual data points. The orange arrowhead indicates vaccinated mouse 1. (F) Percentage of infected (red) and noninfected (green) mosquitoes 14 days after feeding in vaccinated or control mice. Data are percentages of the total analyzed individuals. ****, P < 0.0001.