The malaria vaccine candidate GMZ2 elicits functional antibodies in individuals from malaria endemic and non-endemic areas.

BACKGROUND: GMZ2 is a hybrid protein consisting of the N-terminal region of the glutamate-rich protein fused in frame to the C-terminal region of merozoite surface protein 3 (MSP3). GMZ2 formulated in Al(OH)3 has been tested in 3 published phase 1 clinical trials. The GMZ2/alum formulation showed good safety, tolerability, and immunogenicity, but whether antibodies elicited by vaccination are functional is not known. METHODS: Serum samples prior to vaccination and 4 weeks after the last vaccination from the 3 clinical trials were used to perform a comparative assessment of biological activity against Plasmodium falciparum. RESULTS: We showed that the maximum level of immunoglobulin G (IgG) antibodies obtained by GMZ2 vaccination is independent of ethnicity, time under malaria-exposure, and vaccine dose and that GMZ2 elicits high levels of functionally active IgG antibodies. Both, malaria-naive adults and malaria-exposed preschool children elicit vaccine-specific antibodies with broad inhibitory activity against geographically diverse P. falciparum isolates. Peptide-mapping studies of IgG subclass responses identified IgG3 against a peptide derived from MSP3 as the strongest predictor of antibody-dependent cellular inhibition. CONCLUSIONS: These findings suggest that GMZ2 adjuvanted in Al(OH)3 elicits high levels of specific and functional antibodies with the capacity to control parasite multiplication.

Flow cytometric readout based on Mitotracker Red CMXRos staining of live asexual blood stage malarial parasites reliably assesses antibody dependent cellular inhibition.

BACKGROUND: Functional in vitro assays could provide insights into the efficacy of malaria vaccine candidates. For estimating the anti-parasite effect induced by a vaccine candidate, an accurate determination of live parasite count is an essential component of most in vitro bioassays. Although traditionally parasites are counted microscopically, a faster, more accurate and less subjective method for counting parasites is desirable. In this study mitochondrial dye (Mitotracker Red CMXRos) was used for obtaining reliable live parasite counts through flow cytometry. METHODS: Both asynchronous and tightly synchronized asexual blood stage cultures of Plasmodium falciparum were stained with CMXRos and subjected to detection by flow cytometry and fluorescence microscopy. The parasite counts obtained by flow cytometry were compared to standard microscopic counts obtained through examination of Giemsa-stained thin smears. A comparison of the ability of CMXRos to stain live and compromised parasites (induced by either medium starvation or by anti-malarial drug treatment) was carried out. Finally, parasite counts obtained by CMXRos staining through flow cytometry were used to determine specific growth inhibition index (SGI) in an antibody-dependent cellular inhibition (ADCI) assay. RESULTS: Mitotracker Red CMXRos can reliably detect live intra-erythrocytic stages of P. falciparum. Comparison between staining of live with compromised parasites shows that CMXRos predominantly stains live parasites with functional mitochondria. Parasite counts obtained by CMXRos staining and flow cytometry were highly reproducible and can reliably determine the ability of IgG from hyper-immune individuals to inhibit parasite growth in presence of monocytes in ADCI assay. Further, a dose-dependent parasite growth inhibitory effect could be detected for both total IgG purified from hyper-immune sera and affinity purified IgGs against the N-terminal non-repeat region of GLURP in ADCI assays coupled with determination of parasite counts through CMXRos staining and flow cytometry. CONCLUSIONS: A flow cytometry method based on CMXRos staining for detection of live parasite populations has been optimized. This is a rapid and sensitive method with high inter-assay reproducibility which can reliably determine the anti-parasite effect mediated by antibodies in functional in vitro assays such as ADCI assay.

Distinct patterns of blood-stage parasite antigens detected by plasma IgG subclasses from individuals with different level of exposure to Plasmodium falciparum infections.

BACKGROUND: In endemic regions naturally acquired immunity against Plasmodium falciparum develops as a function of age and exposure to parasite infections and is known to be mediated by IgG. The targets of protective antibodies remain to be fully defined. Several immunoepidemiological studies have indicated an association of cytophilic anti-parasite IgG with protection against malaria. It has been hypothesized that the initial antibody responses against parasite antigens upon first few Plasmodium falciparum infections is dominated by non-protective IgG2/IgG4 and IgM antibodies, which then gradually develop into protective response dominated by cytophilic IgG1 and IgG3 antibodies. METHODS: Naturally occurring IgG antibodies against P. falciparum blood-stage antigens were analysed from plasma samples collected from four groups of individuals differing in age and level of exposure to P. falciparum infections. Western Blot profiling of blood-stage parasite antigens displaying reactivity with individual plasma samples in terms of their subclass specificities was conducted. Parasite antigens detected by IgG were grouped based on their apparent molecular sizes resolved by SDS-PAGE as high molecular weight (≥ 70 kDa) or low molecular weight (< 70 kDa). The number of discernable low molecular weight parasite antigens detected by different IgG subclass antibodies from each plasma sample was recorded. Using Wilcoxons rank sum test these reactivities were compared amongst groups of individuals with different levels of exposure to P. falciparum infections. RESULTS: IgG4 and IgM antibodies in plasma samples from all groups detected very few parasite antigens. IgG2 antibodies from all groups detected a common pattern of high molecular weight parasite antigens. Cytophilic IgG subclasses in plasma samples from individuals with higher levels of exposure to P. falciparum infections distinctly detected higher numbers of low molecular weight parasite antigens. CONCLUSIONS: In the present study, there was no evidence for switching of antibody responses from non-cytophilic to cytophilic subclasses against blood-stage parasite antigens as a likely mechanism for induction of protective immunity against malaria.

A synthetic TLR4 agonist formulated in an emulsion enhances humoral and Type 1 cellular immune responses against GMZ2--a GLURP-MSP3 fusion protein malaria vaccine candidate.

GMZ2 adjuvanted by aluminum hydroxide is a candidate malaria vaccine that has successfully passed phase 1 clinical testing in adult German and Gabonese volunteers and Gabonese children under five. Here we report a preclinical study screening a series of adjuvant vehicles and Toll-like receptor (TLR) agonists in CB6F1 mice to identify an improved formulation of GMZ2 suitable for further human clinical studies. GMZ2 formulated in an oil-in-water emulsion plus the synthetic TLR4 agonist GLA elicits the highest (a) vaccine-specific IgG2a and total IgG titers, (b) parasite-specific IFA titers, (c) levels of Type 1 cytokine responses (IFN-γ), and (d) number of long-lived-plasma cells (LLPC) secreting antibodies against both the GMZ2 fusion and its two components. Thus, GLA helps to elicit a vaccine-specific Type 1 antibody profile together with high levels of LLPC, both of which are thought to be essential for the development of long-term protective immunity against clinical malaria.