Emily Locke

Mosquito Feeding Assays to Determine the Infectiousness of Naturally Infected Plasmodium falciparum Gametocyte Carriers

Introduction In the era of malaria elimination and eradication, drug-based and vaccine-based approaches to reduce malaria transmission are receiving greater attention. Such interventions require assays that reliably measure the transmission of Plasmodium from humans to Anopheles mosquitoes. Methods We compared two commonly used mosquito feeding assay procedures: direct skin feeding assays and membrane feeding assays. Three conditions under which membrane feeding assays are performed were examined: assays with i) whole blood, ii) blood pellets resuspended with autologous plasma of the gametocyte carrier, and iii) blood pellets resuspended with heterologous control serum. Results 930 transmission experiments from Cameroon, The Gambia, Mali and Senegal were included in the analyses. Direct skin feeding assays resulted in higher mosquito infection rates compared to membrane feeding assays (odds ratio 2.39, 95% confidence interval 1.94–2.95) with evident heterogeneity between studies. Mosquito infection rates in membrane feeding assays and direct skin feeding assays were strongly correlated (p<0.0001). Replacing the plasma of the gametocyte donor with malaria naïve control serum resulted in higher mosquito infection rates compared to own plasma (OR 1.92, 95% CI 1.68–2.19) while the infectiousness of gametocytes may be reduced during the replacement procedure (OR 0.60, 95% CI 0.52–0.70). Conclusions Despite a higher efficiency of direct skin feeding assays, membrane feeding assays appear suitable tools to compare the infectiousness between individuals and to evaluate transmission-reducing interventions. Several aspects of membrane feeding procedures currently lack standardization; this variability makes comparisons between laboratories challenging and should be addressed to facilitate future testing of transmission-reducing interventions.

Quantitative Detection of Plasmodium falciparum DNA in Saliva, Blood, and Urine

BACKGROUND Current methods for detecting malaria parasites are invasive and associated with poor compliance when repeated sampling is required. New methods to detect and quantify parasites in a less-invasive manner would greatly enhance the potential for longitudinal surveillance in clinical trials. METHODS Saliva, urine, and blood samples from 386 Gambian outpatients with suspected malaria infections were analyzed by nested polymerase chain reaction (nPCR) to detect infection and to evaluate diagnostic accuracy in comparison to expert microscopy. The amount of parasite DNA in malaria-positive samples was estimated using real-time quantitative PCR (qPCR). RESULTS Blood parasite density as estimated by qPCR correlated well with parasite counts established by microscopy (p = 0.94; P < .001). qPCR results for saliva had a significant correlation with microscopy counts (p = 0.58; P < .001), whereas qPCR results for urine had a positive but poor correlation with microscopy counts (p = 0.20; P = .117). The mean amounts of parasite DNA quantified in blood were greater than the mean amounts quantified in saliva and urine samples obtained concurrently from the same individual, by approximately 600-fold and approximately 2500-fold, respectively. When nPCR results were compared with microscopy results, nPCR of saliva had a sensitivity of 73% and a specificity of 97%; its sensitivity increased to 82% in samples with a parasite density of > or = 1000 parasites/microL. nPCR of urine had a sensitivity of 32% and a specificity of 98%. CONCLUSION Saliva sampling is a promising less-invasive approach for detecting malaria infection.

Viral vectors for malaria vaccine development

Abstract A workshop on viral vectors for malaria vaccine development, organized by the PATH Malaria Vaccine Initiative, was held in Bethesda, MD on October 20, 2005. Recent advancements in viral-vectored malaria vaccine development and emerging vector technologies were presented and discussed. Classic viral vectors such as poxvirus, adenovirus and alphavirus vectors have been successfully used to deliver malaria antigens. Some of the vaccine candidates have demonstrated their potential in inducing malaria-specific immunity in animal models and human trials. In addition, emerging viral-vector technologies, such as measles virus (MV), vesicular stomatitis virus (VSV) and yellow fever (YF) virus, may also be useful for malaria vaccine development. Studies in animal models suggest that each viral vector is unique in its ability to induce humoral and/or cellular immune responses. Those studies have also revealed that optimization of Plasmodium genes for mammalian expression is an important aspect of vaccine design. Codon-optimization, surface-trafficking, de-glycosylation and removal of toxic domains can lead to improved immunogenicity. Understanding the vectors ability to induce an immune response and the expression of malaria antigens in mammalian cells will be critical in designing the next generation of viral-vectored malaria vaccines.

Qualification of standard membrane-feeding assay with Plasmodium falciparum malaria and potential improvements for future assays.

Vaccines that interrupt malaria transmission are of increasing interest and a robust functional assay to measure this activity would promote their development by providing a biologically relevant means of evaluating potential vaccine candidates. Therefore, we aimed to qualify the standard membrane-feeding assay (SMFA). The assay measures the transmission-blocking activity of antibodies by feeding cultured P. falciparum gametocytes to Anopheles mosquitoes in the presence of the test antibodies and measuring subsequent mosquito infection. The International Conference on Harmonisation (ICH) Harmonised Tripartite Guideline Q2(R1) details characteristics considered in assay validation. Of these characteristics, we decided to qualify the SMFA for Precision, Linearity, Range and Specificity. The transmission-blocking 4B7 monoclonal antibody was tested over 6 feeding experiments at several concentrations to determine four suitable concentrations that were tested in triplicate in the qualification experiments (3 additional feeds) to evaluate Precision, Linearity and Range. For Specificity, 4B7 was tested in the presence of normal mouse IgG. We determined intra- and inter-assay variability of % inhibition of mean oocyst intensity at each concentration of 4B7 (lower concentrations showed higher variability). We also showed that % inhibition was dependent on 4B7 concentration and the activity is specific to 4B7. Since obtaining empirical data is time-consuming, we generated a model using data from all 9 feeds and simulated the effects of different parameters on final readouts to improve the assay procedure and analytical methods for future studies. For example, we estimated the effect of number of mosquitoes dissected on variability of % inhibition, and simulated the relationship between % inhibition in oocyst intensity and % inhibition of prevalence of infected mosquitos at different mean oocysts in the control. SMFA is one of the few biological assays used in preclinical and early clinical development of transmission-blocking vaccines, and this study strongly supports its further development and application.

Proteomic identification of host and parasite biomarkers in saliva from patients with uncomplicated Plasmodium falciparum malaria

BackgroundMalaria cases attributed to Plasmodium falciparum account for approximately 600,000 deaths yearly, mainly in African children. The gold standard method to diagnose malaria requires the visualization of the parasite in blood. The role of non-invasive diagnostic methods to diagnose malaria remains unclear.MethodsA protocol was optimized to deplete highly abundant proteins from saliva to improve the dynamic range of the proteins identified and assess their suitability as candidate biomarkers of malaria infection. A starch-based amylase depletion strategy was used in combination with four different lectins to deplete glycoproteins (Concanavalin A and Aleuria aurantia for N-linked glycoproteins; jacalin and peanut agglutinin for O-linked glycoproteins). A proteomic analysis of depleted saliva samples was performed in 17 children with fever and a positive–malaria slide and compared with that of 17 malaria-negative children with fever.ResultsThe proteomic signature of malaria-positive patients revealed a strong up-regulation of erythrocyte-derived and inflammatory proteins. Three P. falciparum proteins, PFL0480w, PF08_0054 and PFI0875w, were identified in malaria patients and not in controls. Aleuria aurantia and jacalin showed the best results for parasite protein identification.ConclusionsThis study shows that saliva is a suitable clinical specimen for biomarker discovery. Parasite proteins and several potential biomarkers were identified in patients with malaria but not in patients with other causes of fever. The diagnostic performance of these markers should be addressed prospectively.

A chemiluminescent-western blot assay for quantitative detection of Plasmodium falciparum circumsporozoite protein.

Highly sensitive and reliable assays based on the quantitation of immunologically relevant component(s) in recombinant or whole parasite-based vaccines would facilitate pre-clinical and clinical phases and the monitoring of malaria vaccine deployment. Here we report a laboratory-grade Western Blot assay for quantitative detection of Plasmodium falciparum circumsporozoite protein (PfCSP) in P. falciparum sporozoite (PfSPZ) and in recombinant (rPfCSP) product. This assay is based on the immuno-reactivity of an anti-P. falciparum CSP monoclonal antibody (mAb 2A10) with the NANP-repeat units on PfCSP. The antigen-antibody complex is detected by reaction with a commercially obtained chemiluminescence-linked Immunodetection system. The linear range for detecting the recombinant P. falciparum CSP (rPfCSP) in this assay is 3-12pg (R(2)=0.9399). The range for detecting the day 15 salivary-gland PfSPZ is between 0.0625 and 1 parasite (R(2)=0.9448) and approximately 10.0pg of PfCSP was detected on each sporozoite. The assay was highly reproducible in measuring the PfCSP on PfSPZ. The inter-assay Coefficient of Variation (CV%) was 10.31% while the intra-assay CV% on three different days was 6.05%, 2.03% and 1.42% respectively. These results suggest that this ECL-WB assay is highly sensitive and robust with a low degree of inter-assay and intra-assay variations. To our knowledge, this is the most sensitive immunoassay for the detection of a recombinant or native malarial protein and may have a wider range of applications including the quantification of immunological component(s) in a vaccine formulation, determination of the antigenic integrity in adjuvanted-vaccine and in stability studies. In addition, this assay can be applied to measure the mosquito infectivity in malaria transmission areas and to determine the effects of intervention measures on malaria transmission.

A Slot Blot Immunoassay for Quantitative Detection of Plasmodium falciparum Circumsporozoite Protein in Mosquito Midgut Oocyst

There is still a need for sensitive and reproducible immunoassays for quantitative detection of malarial antigens in preclinical and clinical phases of vaccine development and in epidemiology and surveillance studies, particularly in the vector host. Here we report the results of sensitivity and reproducibility studies for a research-grade, quantitative enhanced chemiluminescent-based slot blot assay (ECL-SB) for detection of both recombinant Plasmodium falciparum circumsporozoite protein (rPfCSP) and native PfCSP from Oocysts (Pf Oocyst) developing in the midguts of Anopheles stephensi mosquitoes. The ECL-SB detects as little as 1.25 pg of rPfCSP (linear range of quantitation 2.5–20 pg; R2 = 0.9505). We also find the earliest detectable expression of native PfCSP in Pf Oocyst by ECL-SB occurs on day 7 post feeding with infected blood meal. The ECL-SB was able to detect approximately as few as 0.5 day 8 Pf Oocysts (linear quantitation range 1–4, R2 = 0.9795) and determined that one Pf Oocyst expressed approximately 2.0 pg (0.5–3 pg) of native PfCSP, suggesting a similar range of detection for recombinant and native forms of Pf CSP. The ECL-SB is highly reproducible; the Coefficient of Variation (CV) for inter-assay variability for rPf CSP and native PfCSP were 1.74% and 1.32%, respectively. The CVs for intra-assay variability performed on three days for rPf CSP were 2.41%, 0.82% and 2% and for native Pf CSP 1.52%, 0.57%, and 1.86%, respectively. In addition, the ECL-SB was comparable to microscopy in determining the P. falciparum prevalence in mosquito populations that distinctly contained either high and low midgut Pf Oocyst burden. In whole mosquito samples, estimations of positivity for P. falciparum in the high and low burden groups were 83.3% and 23.3% by ECL-SB and 85.7% and 27.6% by microscopy. Based on its performance characteristics, ECL-SB could be valuable in vaccine development and to measure the parasite prevalence in mosquitoes and transmission-blocking interventions in endemic areas.

Robust antibody and CD8 + T-cell responses induced by P. falciparum CSP adsorbed to cationic liposomal adjuvant CAF09 confer sterilizing immunity against experimental rodent malaria infection

Despite several decades of extensive research, the development of a highly efficacious malaria vaccine has yet to be accomplished. While the RTS,S malaria vaccine candidate shows the potential to prevent a substantial number of clinical malaria cases, significant improvements in protective efficacy are still needed. Multiple studies have shown that RTS,S induces protective antibody and CD4+ T-cell responses, but limited or negligible CD8+ T cells. In this study, we evaluated the immunogenicity and protective capacity of full-length recombinant Plasmodium falciparum circumsporozoite protein administered with the novel cationic liposomal adjuvant system CAF09. Using newly developed transgenic rodent malaria parasites expressing the full-length Plasmodium falciparum circumsporozoite protein, we demonstrate that this liposome-based protein-in-adjuvant formulation is capable of inducing robust antibody and CD8+ T-cell responses that strongly inhibit parasite infection and development of liver stages, conferring durable sterilizing immunity. These findings underscore the potential of liposome-based adjuvants for inducing robust humoral and CD8+ T-cell responses and warrant further studies toward the development of novel subunit vaccine formulations with this adjuvant system.Malaria: A more effective vaccineA vaccine consisting of parasitic proteins enveloped by fatty molecules provides comprehensive protection against malaria in a rodent model, Previous and current malaria vaccines concentrate on priming antibodies to recognize malarial infection, despite evidence that, by activating ‘killer’ CD8+ T cells, greater protection is conferred against the disease. Fidel Zavala, of the Johns Hopkins University, United States, and an international group of researchers developed their vaccine by encapsulating proteins from the malaria-causing parasite Plasmodium falciparum in fat-based carriers called liposomes. In past experiments, killer T cells recruited via this vaccine-type have effectively protected against other diseases. In this study, the vaccine induced both CD8+ T cell and antibody responses and provided significant immunity against P. falciparum-instigated malaria. As a highly efficacious vaccine against malaria is not yet available, this research will likely prove invaluable in guiding further studies.

Western Blot Assay for Quantitative and Qualitative Antigen Detection in Vaccine Development

Immunological methods for quantitative measurement, antigenic characterization, and monitoring the stability of active immunogenic component(s) are a critical need in the vaccine development process. This unit describes an enhanced chemiluminescence‐based western blot for quantitative detection of Plasmodium falciparum circumsporozoite protein (PfCSP), a major malaria candidate vaccine antigen. The most salient features of this assay are its high sensitivity and reproducibility; it can reliably detect ∼5 to 10 pg PfCSP expressed on native parasites or recombinantly expressed in Escherichia coli. Although described for a specific vaccine antigen, this assay should be applicable for any antigen‐antibody combination for which relevant detection reagents are available. Detailed stepwise experimental procedures and methods for data acquisition and analysis are described. Curr. Protoc. Microbiol. 33:18.4.1‐18.4.11.

Transcriptome analysis based detection of Plasmodium falciparum development in Anopheles stephensi mosquitoes

The Plasmodium life cycle within the mosquito involves the gamete, zygote, motile ookinete, and the oocyst stage that supports sporogony and sporozoite formation. We mapped the P. falciparum transcriptome as the parasite progresses through the oocyst stage of development on days 2, 4, 6, and 8 post-P. falciparum infectious blood meal. Through these genomic studies, we identified 212 novel transmission stage biomarkers including genes that are developmentally expressed at a single time point and genes that are pan-developmentally expressed at all four time points in P. falciparum oocysts. Validation of a small subset of genes at the transcriptional and translational level resulted in identification of a signature of genes/proteins that can detect parasites within the mosquito as early as day 2 post-infectious blood meal and can be used to distinguish early versus late stage P. falciparum oocyst development in the mosquito. Currently, circumsporozoite protein (CSP), which is detectable only after day 7 post-infection, is the only marker used for detection of P. falciparum infection in mosquitoes. Our results open the prospect to develop a non-CSP based detection assay for assessment of P. falciparum infection in mosquitoes and evaluate the effect of intervention measures on malaria transmission in an endemic setting.