Most anautogenous female mosquitoes ingest plant carbohydrates for flight energy and survival, and they imbibe vertebrate blood for egg development. We evaluated the effect of different sucrose meals following a blood meal containing West Nile virus (WNV) on Culex pipiens pipiens survival, nutritional status, and susceptibility to viral infection and transmission. Ten days after blood feeding, no mosquitoes survived on distilled water, 55% survived on 2% sucrose, 61% on 10 and 20% sucrose meals, and over 70% survived on 40% sucrose. There was a positive correlation between sucrose meal concentration and detectable sugars, glycogen, and lipid in whole-body homogenates. Average sugar values increased from 0 microg per starved mosquito (range 0-1.0 microg) to an average of 392 microg per mosquito fed on 40% sucrose (85-1088 microg). Average glycogen values increased from 0 microg (0-5.7 microg) to an average of 620 microg (118-1421 microg). Average lipid values were identical for mosquitoes in the starved and 2% sucrose series (38 microg) and increased to 172 microg per mosquito fed on 40% sucrose (92-266 microg). Mosquitoes in all sucrose series were equally susceptible to WNV infection (p > 0.5), but mosquitoes with lower nutrient reserves as a result of lower sucrose meals were more likely to orally transmit virus (p < 0.05). We discuss how mosquito nutritional status influences probability of daily survival, susceptibility to infection, and vectorial capacity. We conclude that maintaining C. p. pipiens on standard 10% sucrose is justified in light of these results.

Coccidioides DNA was amplified from serum by a PCR using coccidioid-specific primers. A 239-bp product was visualized when 10 fg of exogenous coccidioidal DNA was subjected to amplification. This product was demonstrated in some human and mouse sera prior to the detection of coccidioidal antibodies

Mannose is the predominant monosaccharide in the coccidioidal antigen preparation T27K. Mannan and anti-CD206 antibody significantly decreased the surface expression of mannose receptor (MR) on adherent peripheral blood mononuclear cells and reduced the interleukin-2 (IL-2) release induced by T27K. These data suggest that MR mediates IL-2 release by T27K.The mannose receptor (MR) is found on the surface of macrophages and dendritic cells, can bind terminal mannoses found on fungi and other pathogens (6), and has been shown to mediate the in vitro cellular immune response to fungal antigens (7). We used mannan, an MR ligand (11), and anti-CD206 (␣CD206), a monoclonal antibody directed against MR (4), to block the surface expression of MR on adherent peripheral blood mononuclear cells (PBMC) and examined the effect of this on the cellular immune response induced by T27K, a glycosylated coccidioidal antigen preparation (1-3).PBMC, derived from the blood of healthy human donors of known coccidioidal immunity, were resuspended in RPMI 1640 (GIBCO, Grand Island, Mich.) with 10% autologous serum, added to 35-mm flat-bottom wells (Falcon, Becton Dickinson Labware), and incubated at 37°C in 95% air-5% CO 2 . For the first 30 min, mannan (from Sacchyromyces cerevisiae; Sigma Chemical Company, St. Louis, Mo.) or ␣CD206 (from clone 19.2; BD Biosciences Pharmingen, San Diego, Calif.) was added to wells. In some experiments, immunoglobulin G1 (IgG1) (no. 555748; BD Biosciences Pharmingen), the isotype of ␣CD206, was used. Control wells received nothing. After 30 min, 10-g/ml T27K was added to cells and further incubated for 72 h. Adherent cells were removed and incubated with fluorescein isothiocyanate (FITC)-labeled ␣CD206 or FITC-labeled IgG1 for 30 min at 22°C in the dark. Cell viability just prior to flow cytometry was Ͼ90%, as determined by trypan blue exclusion. A gate was set around viable nonlymphocytes and 4,000 events were collected. MR surface expression was measured as the ratio of the geometric mean fluorescent intensity (MFI) of samples stained with FITC-labeled ␣CD206 divided by the geometric MFI of cells stained with labeled isotype. Interleukin-2 (IL-2) and gamma interferon (IFN-␥) concentrations in harvested supernatant were measured using a flow cytometry-based immunoassay (CBA; BD Immunosciences, San Jose, Calif.) or by enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, Minn.).Monosaccharide analysis of T27K was performed by the Glycotechnology Core Resource of the University of California at San Diego by using high-pH anion-exchange chromatography with pulsed amperometric detection after protein denaturization and desalting (A. Datta, personal communication). The Wilcoxon signed-rank test for paired data was employed for statistical analysis. All work was approved by the Human Subjects Protection Program of the University of Arizona.To assess mannan blocking of MR, 3.0 ϫ 10 6 PBMC in 2 ml of RPMI 1640 with 10% autologous serum were incubated for 72 h with or without 3 mg of mannan/ml. Subsequent...

Assessment of the cellular immune response in coccidioidomycosis has epidemiologic and prognostic importance. Measurement of delayed-type hypersensitivity to skin testing has been used in the past to determine cellular immunity in coccidioidomycosis. However, no skin tests are currently available in the United States. Assay of gamma interferon (IFN-␥) release in whole blood in response to incubation with antigen has been used to assess cellular immunity in tuberculosis. We used a similar assay using the coccidioidal antigen preparation T27K to measure the in vitro cellular immune responses among a cohort of 69 subjects with active coccidioidomycosis. IFN-␥ release was bimodal, with concentrations above and below 5 IU/ml. Using multivariate logistic regression, underlying disease and disseminated or chronic pulmonary coccidioidomycosis was significantly associated with the release of IFN-␥ at a concentration of <5 IU/ml (P ‫؍‬ 0.02 or 0.05, respectively). In addition, the release IFN-␥ concentration was <5 IU/ml in all subjects with a clinical severity score of >6 (P ‫؍‬ 0.02). The release IFN-␥ concentration correlated with expression of CD69 on T lymphocytes in an in vitro assay using T27K as the antigen (Spearman's rho ‫؍‬ 0.59; P < 0.01). These results suggest that the IFN-␥ release assay with T27K as the antigen may be a useful clinical test for assessing cellular immunity in patients with active coccidioidomycosis.

The coccidioidal T27K vaccine is protective in mice against respiratory challenge with Coccidioides posadasii (C. posadasii) arthroconidia. The vaccine is a subcellular multicomponent preparation that has not been fully characterized. To identify potential protective antigens in the heterogeneous mixture, the vaccine has been separated by two-dimensional gel electrophoresis and then analyzed for seroreactive proteins using immunoblot analysis with pooled sera from patients with coccidioidomycosis. Two seroreactive spots of identical apparent molecular weight were identified and sequenced using tandem mass spectrometry. Three peptides were generated, two of which matched a tentative consensus sequence in the TIGR C. posadasii 2.0 gene index database that is similar to fungal 1,2-alpha-mannosidases. The 5' and 3' ends of the mannosidase cDNA were mapped using rapid amplification of cDNA ends (RACE) polymerase chain reaction (PCR), and a full-length cDNA was then obtained using reverse-transcription (RT) PCR. The cDNA was cloned and sequenced and expressed as a recombinant protein. The predicted protein consists of 519 amino acids, has a theoretical molecular weight and pI of 56,918 Da and 4.84, respectively, and is very similar (>60%) to other fungal 1,2-alpha-mannosidases. Class I 1,2-alpha-mannosidase enzyme activity was also detected in the T27K vaccine using the substrate, Man-alpha-1,2-Man-alpha-OCH(3) in a spectrophotometric assay.

Previous studies have demonstrated that the coccidioidal T27K vaccine preparation is protective in mice against respiratory challenge using Coccidioides posadasii (C. posadasii) arthroconidia. Proteomic methods have been employed to define the molecular components within the vaccine. This method has led to the identification of novel and previously uncharacterized coccidioidal proteins including a Cu,Zn superoxide dismutase. A two-dimensional gel of the T27K vaccine was run and spots were excised for mass spectrometric analysis. One peptide was obtained from the T27K gel that matched a TIGR C. posadasii 2.0 gene index tentative consensus sequence, TC1072, which is similar to fungal Cu,Zn superoxide dismutase. Activity assays performed with native PAGE gels of the T27K vaccine showed that the vaccine contains superoxide dismutase. The cDNA encoding the enzyme has been cloned and sequenced and expressed as a recombinant protein.

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