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.