Natural feeding of Culex
spp. and Aedes
spp. mosquitoes on flavivirus-susceptible C3H/HeJ mice has been shown to down-regulate Th1-type cytokines and up-regulate Th2 cytokines 
, which are not favorable for an effective immune defense against infection by arthropod-borne viruses 
. Our prior studies showed that mice vaccinated with Cx. tarsalis
salivary gland extract and subsequently challenged with WNV by mosquito bite had decreased viral titers in the brain early in infection (Machain-Williams et al., submitted). These results suggested that development of and immunization with a MSP vaccine could potentially neutralize saliva-induced immunomodulation and protect the host from mosquito-transmitted WNV infection. D7 family proteins are specifically expressed in the salivary glands of adult female hematophagous diptera 
, are immunogenic 
and are among the most abundant proteins in the saliva of female mosquitoes 
, comprising between 5 and 20% of total salivary protein 
. These characteristics led us to choose a D7 family protein as our vaccine candidate. As we began our study, Cx. tarsalis
D7 salivary proteins had neither been isolated nor characterized. We identified 36 kDa and 40 kDa proteins by mass spectrometry that have significant similarity to two Cx. pipiens
D7 salivary proteins. We chose to focus on the 36 kDa protein, which had 85% amino acid sequence identity to a Cx. pipiens
D7 salivary protein. The cDNA encoding the 36 kDa protein was cloned, sequenced and used to express recombinant protein by an alphavirus transducing system in cultured Aedes albopictus
cells. This system has the advantage of abundant expression in mosquito cells, which should yield authentic native protein.
Vaccinated mice exhibited slightly increased expression of IFN-γ and significantly reduced expression of IL-10 shortly after uninfected mosquito bite. These results, along with our observation of more inflammatory cell infiltration at the bite site upon non-infected mosquito feeding suggested that rD7 protein immunization might favorably alter the immune response to WNV infection delivered by mosquito bite.
To our surprise, rD7 vaccinated mice that were subsequently bitten by WNV-infected mosquitoes exhibited higher mortality rates than mock- immunized mice. Passive immunization of mice with sera from rD7-vaccinated mice also resulted in increased mortality due to mosquito-delivered WNV infection compared to mice that received serum from non-immunized mice or were passively immunized with sera of naturally mosquito-exposed mice. These results indicate that vaccine-induced antibodies specific to D7 protein or other serum components play a predominant role in viral pathogenesis in immunized mice. Schneider et al
demonstrated that prior exposure to Aedes aegypti
mosquito bites or passive transfer of serum from mosquito-exposed mice increased the proportion of animals that succumbed to Ae. aegypti
-transmitted WNV infection. One possible reason for these observations is that high levels of circulating antibodies to MSPs could impede mosquito blood-feeding by neutralization of vasodilator and platelet inhibitor functions. This could lead to increased probing time and thus more virus-containing saliva being deposited, resulting in an increased initial infectious virus dose. Differences in effects of passive transfer of serum from mosquito-exposed mice on outcome of subsequent WNV infection seen in our study could be due to differences in doses, routes and duration of exposure to MSP as well as to the mosquito genera and species. In our study, although rD7-immunized mice had an anti-D7 IgG response equivalent to that of mice repeatedly exposed to mosquito bites, they produced only IgG1 and not IgG2a anti-D7 antibodies, as did naturally-exposed mice. IgG1 and IgG2a subtypes are normally made during Th2- and Th1-type immune responses, respectively. Th1-type CD4+ T-cells have been shown to produce IL-12p70 and IFNγ, which promote class switching of antibodies from IgM to IgG2a. In contrast, Th2-type T-cells characteristically produce IL-4 that directly inhibits the Th1 response and induces class switching to IgG1. A Th2-dominated response is usually seen after mosquito bite; however, administration of a MSP vaccine with complete Freund's adjuvant (CFA) was expected to result in a Th1 type response 
and generation of IgG2a antibodies. The D7 protein may drive the development of IgG1 subtype antibodies regardless of the use of CFA, as has been reported before 
. In fact, we observed a larger proportion of IL-10-producing WNV-specific CD4+
T-cells from rD7-vaccinated infected mice and significant decreases in pro-inflammatory cytokine production by CD4+ T cells. Pro-inflammatory cytokines are essential to effectively respond to WNV infection 
, and IL-10 is known to be involved in WNV pathogenesis 
Histological examination at the site of an uninfected Cx. tarsalis
bite showed that rD7-vaccinated mice had moderately severe inflammation and PMN and mononuclear cell infiltration. Monocytes and dendritic cells are susceptible to WNV infection 
. A local increase in susceptible cells at the site of WNV injection could make more cells available for immediate infection and migration to regional lymph nodes and thereby result in more virus spread in the periphery. Overall, immunization of mice with rD7 protein induced exclusively IgG1 anti-D7 responses, more inflammatory cell infiltration at the site of mosquito feeding and did not result in a shift to Th1-type cytokine production after WNV infection, which together could contribute to enhanced host susceptibility to virus infection and disease. This response failed to favorably alter the immunomodulatory effect of MSP on WNV infection.
Our prior studies showed that mice vaccinated with Cx. tarsalis salivary gland extract and subsequently challenged with WNV initially had decreased viral titers in the brain (Machain-Williams et al., submitted), suggesting that a MSP vaccine was potentially protective against WNV infection. Nevertheless, our studies presented here showed that immunization of mice with the 36 kDa rD7 protein resulted in enhanced mortality upon WNV infection. It appears that the 36 kDa D7 protein is not the right candidate for a MSP vaccine and its selection on the bases of abundance and immunogenicity did not predict efficacy. Future studies should focus on other MSPs. These findings illustrate the complex nature of the immune response to salivary proteins and effects this may have on arbovirus transmission and infection and provide important information to direct future studies in the field of immunomodulatory effects of mosquito saliva.