Immunization of Mice with Recombinant Mosquito Salivary Protein D7 Enhances Mortality from Subsequent West Nile Virus Infection via Mosquito Bite

<div><h3>Background</h3><p>Mosquito salivary proteins (MSPs) modulate the host immune response, leading to enhancement of arboviral infections. Identification of proteins in saliva responsible for immunomodulation and counteracting their effects on host immune response is a potential strategy to protect against arboviral disease. We selected a member of the D7 protein family, which are among the most abundant and immunogenic in mosquito saliva, as a vaccine candidate with the aim of neutralizing effects on the mammalian immune response normally elicited by mosquito saliva components during arbovirus transmission.</p> <h3>Methodology/Principal Findings</h3><p>We identified D7 salivary proteins of <em>Culex tarsalis</em>, a West Nile virus (WNV) vector in North America, and expressed 36 kDa recombinant D7 (rD7) protein for use as a vaccine. Vaccinated mice exhibited enhanced interferon-γ and decreased interleukin-10 expression after uninfected mosquito bite; however, we found unexpectedly that rD7 vaccination resulted in enhanced pathogenesis from mosquito-transmitted WNV infection. Passive transfer of vaccinated mice sera to naïve mice also resulted in increased mortality rates from subsequent mosquito-transmitted WNV infection, implicating the humoral immune response to the vaccine in enhancement of viral pathogenesis. Vaccinated mice showed decreases in interferon-γ and increases in splenocytes producing the regulatory cytokine IL-10 after WNV infection by mosquito bite.</p> <h3>Conclusions/Significance</h3><p>Vector saliva vaccines have successfully protected against other blood-feeding arthropod-transmitted diseases. Nevertheless, the rD7 salivary protein vaccine was not a good candidate for protection against WNV disease since immunized mice infected <em>via</em> an infected mosquito bite exhibited enhanced mortality. Selection of salivary protein vaccines on the bases of abundance and immunogenicity does not predict efficacy.</p> </div

Outcomes of WNV infection by mosquito bite of rD7-vaccinated mice.

<p>A. Survival curves comparing WNV-infected rD7-vaccinated mice (solid line, n = 13) with mock-vaccinated mice (dotted line, n = 12). <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001935#s3" target="_blank">Results</a> are combined from four experiments (p = 0.0347). B. Survival curves after passive serum transfer and infection with WNV. Mice were injected IP with pooled sera from rD7-vaccinated mice (solid line, n = 4), non-vaccinated mice (dotted line, n = 5), or mice repeatedly bitten by <i>Cx. tarsalis</i> (dashed line, n = 5) and 30 h later infected with WNV <i>via</i> bites of infected <i>Cx. tarsalis.</i> Mice passively immunized with rD7-vaccinee serum exhibited significantly higher mortality rates than either control group (p = 0.002). Mortality rates of mice passively immunized with mosquito-exposed mouse serum were not different from those that received untreated mouse serum. C. Proportion of total splenocytes from rD7- and mock-vaccinated mice that were CD4+ T-lymphocytes staining positive for intracellular IL-10 two days post-WNV infection; rD7-vaccinated animals (filled bar) had significantly higher IL-10 levels than mock-vaccinated animals (p<0.05, n = 3 per group). D. CBA assay of medium from WNV peptide-stimulated splenocytes that were collected at 2 days post-WNV infection; all cytokine levels from rD7-immunized mice (filled bars) were significantly lower than those from mock-immunized mice (open bars) (p<0.05, n = 3 per group).</p

Humoral immune response in rD7-vaccinated mice.

<p>Measurement of serum anti-rD7 IgG by ELISA. A. Total IgG specific for rD7. B. IgG1 subtype rD7 antibodies. C.IgG2a subtype rD7 antibodies. Dashed lines: pooled sera of mice repeatedly exposed to the bites of <i>Cx. tarsalis</i> (n = 2). Solid lines: sera of mice vaccinated with rD7 (n = 5).</p

Characterization of <i>Cx. tarsalis</i> D7 salivary protein.

<p>A. Saliva from adult female <i>Cx. tarsalis</i> mosquitoes was collected, proteins were concentrated, PAGE-fractionated and visualized with silver stain (lane 2). Four major proteins, indicated by arrows at right, were present in the saliva with approximate molecular weights of 14 kDa, 36 kDa, 40 kDa, and 60 kDa. Sizes of protein molecular markers (lane 1) are shown at left. B. Amino acid sequence alignment of <i>Cx. tarsalis</i> and <i>Cx. pipiens</i> D7 proteins. The <i>Cx. tarsalis</i> sequence includes the six-histidine tag used for identification and purification of recombinant protein. The amino acid sequences are 85% identical; non-identical residues are shaded. C. Immunoblot using his-tag-HRP antibody or serum from <i>Cx. tarsalis</i>-exposed mice to show antibodies made to native protein also bind rD7. Lane 1: Protein size markers. Lane 2: rD7 detected with his-tag HRP antibody. Lane 3: rD7 detected with mouse serum.</p

Models of flavivirus 3’UTR secondary RNA structures.

<p><b>(A)</b> Comparative analysis of predicted RNA structures of members of the Flavivirus genus. Mosquito-borne, tick-borne, no-know vector and insect-specific flaviviruses are shown. Pseudoknots are indicated with red lines. Common SLs and DB structures are labeled with red and green lines, respectively. Specific yellow fever repeats are indicated with yellow lines and repeats in tick-borne flaviviruses with black and grey lines. See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004604#ppat.1004604.s006" target="_blank">S1 Table</a> for information on sources of sequences used for this analysis. The distance tree was drawn using neighbor joining method and jukes-cantor substitution model. <b>(B)</b> Conserved sequence of SL and DB structures common to all MBFVs are shown.</p

Fitness advantage of RNA structure duplication during DENV host switching.

<p><b>(A)</b> Normalized luciferase levels expressed by reporter DENV with or without mutations resembling adaptations in mosquito or mammalian cells, respectively, carrying one or two SLs are shown for mammalian cells. The luciferase values are the mean +/- SD, n = 4. <b>(B)</b> The same as in <b>(A)</b> using mosquito cells. <b>(C)</b> Schematic representation of adaptable RNA structures emulating host switching of viruses with single or double SLs. Viruses with a single SL encounter a fitness barrier in the transition from mosquito to mammalian cells due to accumulation of mosquito adaptive mutations, while viruses with double SLs show robustness during host switching.</p

Structural organization of the 3’UTR of dengue virus.

<p><b>(A)</b> Secondary structure model predicted by conservation and stability. Different DENV type 2 genotypes were analyzed using RNAalifold and RNAz softwares. A plot with the base pairing probability calculated for the A1, A2, A3 and A4 domains (top) and the most stable conserved RNA structures (bottom) are shown. <b>(B)</b> Secondary and tertiary structures assessed by chemical probing. SHAPE reactivity plot (top) and predicted RNA structure model for DENV 3’UTR (bottom) are shown. An unstructured non-conserved region of 23 nucleotides is followed by two SLs (SLI and SLII) and two dumbbell (DB1 and DB2) structures. Four pseudoknots are predicted as indicated (PKI, PKII, PKIII and PKIV). <b>(C)</b> Comparison between structure conservation and SHAPE prediction of DEN-SLI and DEN-SLII. Predicted pseudoknots and additional hybridization at the base of the stem-loops are shown with red and green lines, respectively; indicating the existence of two alternative conformations. <b>(D)</b> Regions with identical sequences in DEN-SLI and DEN-SLII are shown (blue boxes), suggesting a common origin of these two RNA structures.</p

Sequence variations at the DENV 3’UTR during experimental host adaptation.

<p><b>(A)</b> Deep sequencing of viral populations after 10 and 20 passages (P10 and P20) in mosquito and mammalian cells. The area and color of circles represent the frequency and number of mutations of the viral variants, respectively. A schematic fan dendrogram indicating the distance between the variants for each population is also shown. For nucleotide sequences and frequencies of each viral variant also see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004604#ppat.1004604.s001" target="_blank">S1 Fig</a>. <b>(B)</b> Sequence of cloned amplicons corresponding to the complete 3’UTR. For each P10 population obtained, 20 individual clones were sequenced. Cells lines used for adaptation experiments are indicated (mosquito C6/36, mosquito U4.4, and human A549 cells). Mutations are indicated in red and deletions in grey. A conservation plot is presented at the bottom. The three independent experiments in C6/36 cells are indicated on the left (I, II and III).</p

Fitness parameters and evolution of viral populations after host switch indicate that the DENV 3’UTR is under host-specific selective pressure.

<p><b>(A)</b> Immunofluorescence and growth kinetics of recombinant virus carrying the 3’UTR of a variant selected in mosquito cells (Mut1) compared with the parental virus (WT) performed in C6/36 cells. Cytopathic effect (CPE) is indicated. Inset shows the accumulation of specific viral RNA. <b>(B)</b> Fitness studies in BHK cells for the two viruses shown in A. <b>(C)</b> and <b>(D)</b> Deep sequencing of viral populations passaged 10 successive times in mammalian or mosquito cells obtained after host switch as indicated. For nucleotide sequences and frequencies of each viral variant also see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004604#ppat.1004604.s004" target="_blank">S4 Fig</a>.</p

Nucleotide variations detected in experimentally adapted viruses correlate with mutations found in natural isolates.

<p><b>(A)</b> Adaptive mutations and deletions were mapped on the RNA structure of the variable region of the viral 3’UTR. Deletions and point mutations rescued from mosquito cell-adapted populations are indicated in grey and red, respectively. Mutation selected in mammalian cells is shown in blue. <b>(B)</b> Conservation plots comparing variable regions of cell adapted viruses and natural isolates. <b>(C)</b> Representation of SL-II RNA structures of DENV genomes from natural isolates corresponding to different serotypes. Sequence alignment plots and secondary RNA structure models are shown for DENV isolates from humans (top) and mosquitoes (bottom).</p