4 Discussion

This study utilized multiple approaches to demonstrate the relative safety of the DDVax vaccine candidate from the perspective of relevant mosquito species transmissibility and regulations regarding potential environmental impacts following field-use. These experiments were designed as part of a series of safety studies required prior to human clinical trials. DDVax showed favorable environmental safety profiles (e.g., low mosquito dissemination, and impaired transmission from inoculated livestock) compared to MP-12 vaccine and the wild-type parental virus, ZH501. Mosquitoes in two epidemiologically-relevant genera were challenged with viral titers up to 2 to 5 log10 PFU/ml higher than mosquitoes would be expected to encounter in the field from vaccinated animals, and there was only one questionably positive transmission event. In a previous study, sheep vaccinated with DDVax did not develop any detectable vaccine-associated viremia following inoculation, suggesting that the overall burden of DDVax in animals is very low (Bird et al., 2011). Additionally, DDVax viral RNA copy numbers in bodies and legs/wings were significantly reduced in both Aedes and Culex compared to those infected with either MP-12 or ZH501 (Figure 2). This result is consistent with previously observed impaired viral dissemination phenotype in mosquitoes due to the deletion of the NSm coding region (Crabtree et al., 2012; Kading et al., 2014). Only one of 140 mosquito saliva samples contained live DDVax virus (Table 2), which was also consistent with previous experiments(Crabtree et al., 2012). This single positive saliva sample showed a single plaque, which may not have been infectious and for which we cannot rule out the possibility that it represents low-level contamination. Expected virus infection rates in these mosquito species have previously ranged between 63-84% for virulent recombinant ZH501 (rZH501) for Ae. aegypti (Crabtree et al., 2012; Kading et al., 2014), 58 – 95% for Cx. tarsalis (Bergren, Borland, Hartman, & Kading, 2021; Turell, Wilson, & Bennett, 2010) with midgut titers of over 6 log10 PFU in actively infected Cx. tarsalis (Bergren et al., 2021). Similarly, up to 100% infection occurred MP-12 infected Cx. pipiens (Turell & Rossi, 1991). In contrast, we expected 0% infection with DDVax infectedAe. aegypti (Crabtree et al., 2012). Overall, we observed similar results between ZH501 and MP-12 strains, with a significant reduction in infection of mosquitoes with DDVax.
While DDVax RNA was detectable in multiple body compartments of the mosquito, infectivity was very reduced or nil given the low RNA copy number detected in mosquitoes 14 days post in vitro infection (Figure 1B). For example, if mosquitoes imbibed a 5 µl blood meal of 8 log10 PFU/ml, then 5.7 log10 PFU would have been acquired. In our study, after two weeks incubation, 2.9 log10 mean RNA copies were detected in Culexbodies, 1.8 log10 RNA copies in legs/wings and 1.5 log10 RNA copies saliva (Figure 1B), suggesting that the virus may have somehow disseminated and persisted at a low level, but was not actively replicating in the mosquitoes. By comparison, mosquitoes of each species exposed to MP-12 and ZH501 had RVFV RNA copy numbers between 7-8 log10 by 14 days post-exposure (Figure 1B) after exposure to a blood meal containing greater than an order of magnitude less virus than that of DDVax (Figure 1A). This pattern was consistent with the results of the dose response experiment, in which the RNA copy number in different tissue compartments seems to be relatively stable after 14 days across all three exposure doses (Figure S3). It is not clear at this time how this spread would be occurring. Further, it is expected that RNA copy numbers would exceed infectious titers rendering the truly infectious virus population even lower(Wichgers Schreur et al., 2021).
Consistent with these findings, Kading et al.(Kading et al., 2014) reported 80% infection and 60% dissemination rates of rZH501 byAe. aegypti mosquitoes, compared with 0% infection and 0% dissemination rates of the rZH501-delNSm (NSm deletion) strain, by plaque assay. Nevertheless, in rZH501-delNSm infections, viral protein was detected in most mosquitoes by immunofluorescence assay (IFA), consistent with viral protein translation with defective packaging or dissemination. Moreover, IFA foci in the midguts of mosquitoes infected with rZH501-delNSm were also very small compared with extensive midgut foci characteristic of rZH501(Kading et al., 2014). Therefore, detection of viral RNA (this study) and antigen (Kading et al., 2014) outside the midgut, in the absence of infectious virus, warrants further study.
Viral RNA detected in Culex saliva could be the result of cell-to-cell spread of DDVax through tissues in the absence of efficient viral assembly, or possibly “leakage” of virions from the alimentary tract in the absence of viral replication. Romoser and colleagues reported the particular affinity of virulent ZH501 RVFV for the cardia, intussuscepted foregut, fat body and salivary glands in Culex pipiens mosquitoes(Lerdthusnee, Romoser, Faran, & Dohm, 1995; Romoser, Faran, Bailey, & Lerdthusnee, 1992). The cardia and intussuscepted foregut are transitional tissues between the esophagus and the anterior midgut in the mosquito digestive tract(Romoser et al., 1992). Salivary glands are proximal to this region, embedded in fat body. One possible explanation is that DDVax retained similar tissue affinity in the absence of NSs and NSm, and, when combined with presumed less efficient viral assembly, led to detection of viral RNA but no infectious virus (Table 2, Figure 1B, S1 Table). In addition, Romoser et al. reported that, in Culex , RVFV ZH501 was able to escape to peripheral tissues as early as 1 day following an infectious blood meal (Romoser et al., 1992), making it particularly rapid in disseminating compared to other arboviruses, eg., flaviviruses, which often require at least a week to reach the salivary glands(Sanchez-Vargas et al., 2009), depending on extrinsic incubation temperature. RVFV affinity for salivary glands was substantiated by the DDVax dose response experiment, in which nearly 19% of mosquitoes showed viral RNA in salivary expectorants at the lowest bloodmeal titer of 3.5 log10PFU/ml (Table S2). This level approached that of the presence of viral RNA in legs/wings.
To address concern about the one possible transmission event, Cx. tarsalis mosquitoes were subsequently challenged with artificial blood meals containing a range of viral titers. As expected, the percentage of mosquitoes that became infected, as determined by RNA genome copy number, decreased proportionally with the titer of DDVax in the artificial blood meal, but did not reach zero. The stable persistence of DDVax RNA in different tissue compartments was evident in all dosing groups (Figure S3). As experimentally predicted, the higher the blood meal titer, the higher the percentage of mosquitoes had detectable RNA, although infectious virus was not assayed in mosquitoes challenged with lower titer blood meals.
These results were further confirmed and placed into a realistic epidemiological context by feeding mosquitoes on inoculated goats. Infection of goats with wild-type ZH501 was not possible in this study due to biosafety considerations. Mosquitoes were fed on goats on days 1-3 post-inoculation with DDVax or MP-12. As expected, goats did not develop any detectable viremia, as determined by plaque assay. However, small ruminants, e.g. sheep, would be expected to develop a viremia ranging from ~5-6 log10TCID50/ml titers between 1-3 days post infection with a wild-type strain(Wichgers Schreur et al., 2021). Similarly, neither Wilson et al.(Wilson et al., 2014) nor Nyundo et al.(Nyundo et al., 2019) observed any detectable viremia in ruminants following vaccination with MP-12 strain. Morrill et al.(Morrill et al., 1991) noted a transient, low-titer viremia in lambs vaccinated with MP-12 strain. Sheep inoculated with DDVax failed to develop any detectable viremia (Bird et al., 2011). Therefore, it was surprising to observe that mosquitoes fed on these inoculated goats and held for seven days post-feeding developed infections (Figure 2, S3 Table).
Analysis on goat serum samples showed very low (<10 RNA copies /ml) RNA levels of RVFV in goat serum (Figure S4), which we interpreted to represent residual, circulating virus as opposed to actively replicating virus. The sensitivity of mosquito feeding was able to pick up this residual viral inoculum, however none of these mosquitoes developed a disseminated infection by 7 days post-exposure. For infection with ZH501 strain, dissemination has previously been documented to occur as early as 3 days post exposure(Romoser et al., 1992), with all mosquitoes having developed a disseminated infection by 10 days post-exposure(Kading et al., 2014).
Mosquito infectivity also becomes a function of volumetric constraints of mosquito blood meal size. While the probability of one mosquito imbibing infectious virions is lower at low virus titers, many mosquitoes imbibing a blood meal simultaneously would draw a larger collective volume of blood that could result in one or more mosquitoes picking up infectious virions. For example, detection of virus in a single mosquito blood meal is limited to titers >3 log10 PFU/ml serum, (approximately one PFU in one microliter of serum in a blood meal) (Kading et al., 2014). For a 25% probability of detecting virus in a single 2 µl mosquito blood meal, the serum titer needs to be 2.72 log10 PFU/ml (95% CI 2.19–3.27), while for a 50% probability of detection, the titer needs to be 3.64 log10 PFU/ml (95% CI 3.20–4.08)(Kading et al., 2014). Corresponding titers for 75% and 90% probabilities of detection were 4.56 log10 PFU/ml (95% CI 4.02–5.10) and 5.48 log10 PFU/ml (95%CI 4.71–6.24), respectively(Kading et al., 2014).
Wichgers Schreur et al.(Wichgers Schreur et al., 2021) documented the extraordinary efficiency of RVFV transmission between lambs andAe. aegypti mosquitoes when using an animal model as opposed to an artificial system. Approximately 30% more RVFV saliva-positive mosquitoes resulted from feeding on viremic lambs than from feeding on a membrane system(Wichgers Schreur et al., 2021) testifying to the value of conducting these experiments with an in vivo model system to more realistically represent vertebrate infectiousness to mosquitoes. While dissemination of DDVax after our 7-day time point cannot be ruled out, our collective results suggest that transmission risk would be very low because any disseminated virions would not be infectious. In addition, based on previous reports, we expected a low combined probability of a single mosquito imbibing an infectious virion precisely after inoculated and an extremely low imbibed virus titer. Moreover, impaired dissemination was due to the deletion of the NSm gene. Finally, we saw the lack of infectious DDVax expectorated in mosquito saliva even after a high titer virus challenge. These combined features combine to give DDVax a safe environmental profile.
4.2 Conclusion
Due to the double gene deletion of NSs and NSm, DDVax has less efficient viral replication in mosquitoes than a previous vaccine strain, MP-12 or wild-type ZH501. Mosquitoes were able to imbibe and harbor infectious DDVax following a high titer challenge in the lab or feeding on inoculated goats. However, DDVax replication and dissemination was impaired in mosquitoes, and only one individual mosquito had one DDVax plaque in its saliva after a high titer challenge. Given the combined probability of a single mosquito imbibing an infectious virion precisely after inoculation, the extremely low imbibed virus titer, the impaired dissemination in mosquitoes due to the deletion of the NSm gene, and the lack of infectious DDVax expectorated in mosquito saliva even after a high titer virus challenge, the transmission and dissemination of DDVax by mosquitoes from vaccinated individuals in an epidemiologically relevant scenario is highly unlikely.