Samuel E. Moretz

Phase Ia clinical evaluation of the Plasmodium falciparum blood-stage antigen MSP1 in ChAd63 and MVA vaccine vectors.

Efficacy trials of antibody-inducing protein-in-adjuvant vaccines targeting the blood-stage Plasmodium falciparum malaria parasite have so far shown disappointing results. The induction of cell-mediated responses in conjunction with antibody responses is thought to be one alternative strategy that could achieve protective efficacy in humans. Here, we prepared chimpanzee adenovirus 63 (ChAd63) and modified vaccinia virus Ankara (MVA) replication-deficient vectors encoding the well-studied P. falciparum blood-stage malaria antigen merozoite surface protein 1 (MSP1). A phase Ia clinical trial was conducted in healthy adults of a ChAd63-MVA MSP1 heterologous prime-boost immunization regime. The vaccine was safe and generally well tolerated. Fewer systemic adverse events (AEs) were observed following ChAd63 MSP1 than MVA MSP1 administration. Exceptionally strong T-cell responses were induced, and these displayed a mixed of CD4(+) and CD8(+) phenotype. Substantial MSP1-specific serum immunoglobulin G (IgG) antibody responses were also induced, which were capable of recognizing native parasite antigen, but these did not reach titers sufficient to neutralize P. falciparum parasites in vitro. This viral vectored vaccine regime is thus a leading approach for the induction of strong cellular and humoral immunogenicity against difficult disease targets in humans. Further studies are required to assess whether this strategy can achieve protective efficacy against blood-stage malaria infection.

Phase 1 trial of AMA1-C1/Alhydrogel plus CPG 7909: an asexual blood-stage vaccine for Plasmodium falciparum malaria

Background Apical Membrane Antigen 1 (AMA1), a polymorphic merozoite surface protein, is a leading blood-stage malaria vaccine candidate. This is the first reported use in humans of an investigational vaccine, AMA1-C1/Alhydrogel, with the novel adjuvant CPG 7909. Methods A phase 1 trial was conducted at the University of Rochester with 75 malaria-naive volunteers to assess the safety and immunogenicity of the AMA1-C1/Alhydrogel+CPG 7909 malaria vaccine. Participants were sequentially enrolled and randomized within dose escalating cohorts to receive three vaccinations on days 0, 28 and 56 of either 20 µg of AMA1-C1/Alhydrogel®+564 µg CPG 7909 (n = 15), 80 µg of AMA1-C1/Alhydrogel® (n = 30), or 80 µg of AMA1-C1/Alhydrogel+564 µg CPG 7909 (n = 30). Results Local and systemic adverse events were significantly more likely to be of higher severity with the addition of CPG 7909. Anti-AMA1 immunoglobulin G (IgG) were detected by enzyme-linked immunosorbent assay (ELISA), and the immune sera of volunteers that received 20 µg or 80 µg of AMA1-C1/Alhydrogel+CPG 7909 had up to 14 fold significant increases in anti-AMA1 antibody concentration compared to 80 µg of AMA1-C1/Alhydrogel alone. The addition of CPG 7909 to the AMA1-C1/Alhydrogel vaccine in humans also elicited AMA1 specific immune IgG that significantly and dramatically increased the in vitro growth inhibition of homologous parasites to levels as high as 96% inhibition. Conclusion/Significance The safety profile of the AMA1-C1/Alhydrogel+CPG 7909 malaria vaccine is acceptable, given the significant increase in immunogenicity observed. Further clinical development is ongoing. Trial Registration ClinicalTrials.gov NCT00344539

Phase 1 Study of Two Merozoite Surface Protein 1 (MSP142) Vaccines for Plasmodium falciparum Malaria

Objectives: To assess the safety and immunogenicity of two vaccines, MSP142-FVO/Alhydrogel and MSP142-3D7/Alhydrogel, targeting blood-stage Plasmodium falciparum parasites. Design: A Phase 1 open-label, dose-escalating study. Setting: Quintiles Phase 1 Services, Lenexa, Kansas between July 2004 and November 2005. Participants: Sixty healthy malaria-naïve volunteers 18–48 y of age. Interventions: The C-terminal 42-kDa region of merozoite surface protein 1 (MSP142) corresponding to the two allelic forms present in FVO and 3D7 P. falciparum lines were expressed in Escherichia coli, refolded, purified, and formulated on Alhydrogel (aluminum hydroxide). For each vaccine, volunteers in each of three dose cohorts (5, 20, and 80 μg) were vaccinated at 0, 28, and 180 d. Volunteers were followed for 1 y. Outcome Measures: The safety of MSP142-FVO/Alhydrogel and MSP142-3D7/Alhydrogel was assessed. The antibody response to each vaccine was measured by reactivity to homologous and heterologous MSP142, MSP119, and MSP133 recombinant proteins and recognition of FVO and 3D7 parasites. Results: Anti-MSP142 antibodies were detected by ELISA in 20/27 (74%) and 22/27 (81%) volunteers receiving three vaccinations of MSP142-FVO/Alhydrogel or MSP142-3D7/Alhydrogel, respectively. Regardless of the vaccine, the antibodies were cross-reactive to both MSP142-FVO and MSP142-3D7 proteins. The majority of the antibody response targeted the C-terminal 19-kDa domain of MSP142, although low-level antibodies to the N-terminal 33-kDa domain of MSP142 were also detected. Immunofluorescence microscopy of sera from the volunteers demonstrated reactivity with both FVO and 3D7 P. falciparum schizonts and free merozoites. Minimal in vitro growth inhibition of FVO or 3D7 parasites by purified IgG from the sera of the vaccinees was observed. Conclusions: The MSP142/Alhydrogel vaccines were safe and well tolerated but not sufficiently immunogenic to generate a biologic effect in vitro. Addition of immunostimulants to the Alhydrogel formulation to elicit higher vaccine-induced responses in humans may be required for an effective vaccine.

Anti-Apical-Membrane-Antigen-1 Antibody Is More Effective than Anti-42-Kilodalton-Merozoite-Surface-Protein-1 Antibody in Inhibiting Plasmodium falciparum Growth, as Determined by the In Vitro Growth Inhibition Assay

ABSTRACT Apical membrane antigen 1 (AMA1) and the 42-kDa merozoite surface protein 1 (MSP142) are leading malaria vaccine candidates. Several preclinical and clinical trials have been conducted, and an in vitro parasite growth inhibition assay has been used to evaluate the biological activities of the resulting antibodies. In a U.S. phase 1 trial with AMA1-C1/Alhydrogel plus CPG 7909, the vaccination elicited anti-AMA1 immunoglobulin G (IgG) which showed up to 96% inhibition. However, antibodies induced by MSP142-C1/Alhydrogel plus CPG 7909 vaccine showed less than 32% inhibition in vitro. To determine whether anti-MSP142 IgG had less growth-inhibitory activity than anti-AMA1 IgG in vitro, the amounts of IgG that produced 50% inhibition of parasite growth (Ab50) were compared for rabbit and human antibodies. The Ab50s of rabbit and human anti-MSP142 IgGs were significantly higher (0.21 and 0.62 mg/ml, respectively) than those of anti-AMA1 IgGs (0.07 and 0.10 mg/ml, respectively) against 3D7 parasites. Ab50 data against FVO parasites also demonstrated significant differences. We further investigated the Ab50s of mouse and monkey anti-AMA1 IgGs and showed that there were significant differences between the species (mouse, 0.28 mg/ml, and monkey, 0.14 mg/ml, against 3D7 parasites). Although it is unknown whether growth-inhibitory activity in vitro reflects protective immunity in vivo, this study showed that the Ab50 varies with both antigen and species. Our data provide a benchmark for antibody levels for future AMA1- or MSP142-based vaccine development efforts in preclinical and clinical trials.

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.

Comparison of Biological Activity of Human Anti-Apical Membrane Antigen-1 Antibodies Induced by Natural Infection and Vaccination

Vaccines represent a significant potential means of decreasing global morbidity and mortality due to malaria. Clinical trials in the United States with Plasmodium falciparum Apical Membrane Antigen 1 (AMA1) showed that the vaccine induced biologically active Abs judged by an in vitro parasite growth inhibition assay (GIA). However, the same vaccine in Malian adults did not increase biological activity, although it elevated ELISA titers. Because GIA has been used to evaluate the biological activity of Abs induced by blood stage malarial vaccine candidates, we explored this discrepancy in this study. We affinity purified AMA1-specific Abs from both U.S. vaccinees and nonvaccinated individuals living in a malaria-endemic area of Mali and performed ELISA and GIA. Both AMA1-specifc Abs induced by vaccination (U.S.) and by natural infection (Mali) have comparable biological activity in GIA when the ELISA titer is normalized. However, a fraction of Malians’ IgG that did not bind to AMA1 protein (Mali-non-AMA1 IgG) reduced the biological activity of the AMA1 Abs from U.S. vaccinees; in contrast, U.S.-non-AMA1 IgGs did not show a reduction of the biological activity. Further investigation revealed that the reduction was due to malaria-specific IgGs in the Mali-non-AMA1 IgGs. The fact that both U.S.- and Mali-AMA1-specific Abs showed comparable biological activity supports further development of AMA1-based vaccines. However, the reduction of biological activity of AMA1-specific Ab by other malaria-specific IgGs likely explains the limited effect on growth-inhibitory activity of Abs induced by AMA1 vaccination in Malian adults and may complicate efforts to develop a blood stage malaria vaccine.

Functional Comparison of Plasmodium falciparum Transmission-Blocking Vaccine Candidates by the Standard Membrane-Feeding Assay

ABSTRACT Recently, there has been a renewed interest in the development of transmission-blocking vaccines (TBV) against Plasmodium falciparum malaria. While several candidate TBVs have been reported, studies directly comparing them in functional assays are limited. To this end, recombinant proteins of TBV candidates Pfs25, Pfs230, and PfHAP2 were expressed in the wheat germ cell-free expression system. Outbred CD-1 mice were immunized twice with the antigens. Two weeks after the second immunization, IgG levels were measured by enzyme-linked immunosorbent assay (ELISA), and IgG functionality was assessed by the standard membrane-feeding assay (SMFA) using cultured P. falciparum NF54 gametocytes and Anopheles stephensi mosquitoes. All three recombinant proteins elicited similar levels of antigen-specific IgG judged by ELISA. When IgGs purified from pools of immune serum were tested at 0.75 mg/ml in the SMFA, all three IgGs showed 97 to 100% inhibition in oocyst intensity compared to control IgG. In two additional independent SMFA evaluations, anti-Pfs25, anti-Pfs230, and anti-PfHAP2 IgGs inhibited oocyst intensity in a dose-dependent manner. When all three data sets were analyzed, anti-Pfs25 antibody showed significantly higher inhibition than the other two antibodies (P < 0.001 for both), while there was no significant difference between the other two (P = 0.15). A proportion of plasma samples collected from adults living in an area of malaria endemicity in Mali recognized Pfs230 and PfHAP2. This is the first study showing that the HAP2 protein of P. falciparum can induce transmission-blocking antibody. The current study supports the possibility of using this system for a comparative study with multiple TBV candidates.

Engineering the Chloroplast Targeted Malarial Vaccine Antigens in Chlamydomonas Starch Granules

Background Malaria, an Anopheles-borne parasitic disease, remains a major global health problem causing illness and death that disproportionately affects developing countries. Despite the incidence of malaria, which remains one of the most severe infections of human populations, there is no licensed vaccine against this life-threatening disease. In this context, we decided to explore the expression of Plasmodium vaccine antigens fused to the granule bound starch synthase (GBSS), the major protein associated to the starch matrix in all starch-accumulating plants and algae such as Chlamydomonas reinhardtii. Methods and Findings We describe the development of genetically engineered starch granules containing plasmodial vaccine candidate antigens produced in the unicellular green algae Chlamydomonas reinhardtii. We show that the C-terminal domains of proteins from the rodent Plasmodium species, Plasmodium berghei Apical Major Antigen AMA1, or Major Surface Protein MSP1 fused to the algal granule bound starch synthase (GBSS) are efficiently expressed and bound to the polysaccharide matrix. Mice were either immunized intraperitoneally with the engineered starch particles and Freund adjuvant, or fed with the engineered particles co-delivered with the mucosal adjuvant, and challenged intraperitoneally with a lethal inoculum of P. Berghei. Both experimental strategies led to a significantly reduced parasitemia with an extension of life span including complete cure for intraperitoneal delivery as assessed by negative blood thin smears. In the case of the starch bound P. falciparum GBSS-MSP1 fusion protein, the immune sera or purified immunoglobulin G of mice immunized with the corresponding starch strongly inhibited in vitro the intra-erythrocytic asexual development of the most human deadly plasmodial species. Conclusion This novel system paves the way for the production of clinically relevant plasmodial antigens as algal starch-based particles designated herein as amylosomes, demonstrating that efficient production of edible vaccines can be genetically produced in Chlamydomonas.

The IC50 of anti-Pfs25 antibody in membrane-feeding assay varies among species

Plasmodium falciparum surface protein 25 (Pfs25) is a candidate for transmission-blocking vaccines (TBVs). Anti-Pfs25 antibodies block the development of oocysts in membrane-feeding assays and we have shown the activity correlates with antibody titer. In this study, we purified Pfs25-specific IgGs to convert antibody titer to microg/mL and determined the amount of antibody required to inhibit 50% of oocyst development (IC(50)). The IC(50) were, 15.9, 4.2, 41.2, and 85.6microg/mL for mouse, rabbit, monkey and human, respectively, and the differences among species were significant. Anti-Pfs25 sera from rabbit, monkey and human showed different patterns of competition against 6 mouse monoclonal antibodies, and the avidity of antibodies among four species were also different. These data suggests that information obtained from animal studies which assess efficacy of TBV candidates may be difficult to translate to human immunization.

Effect of red blood cell variants on childhood malaria in Mali: a prospective cohort study

BACKGROUND Red blood cell variants protect African children from severe falciparum malaria. However, their individual and interactive effects on mild disease and parasite density, and their modification by age-dependent immunity, are poorly understood. In this study, we address these knowledge gaps in a prospective cohort study of malaria risk and Plasmodium falciparum densities in Malian children. METHODS The Kenieroba Innate Defense Study for Malaria (KIDS-Malaria) was a 4-year prospective cohort study of children aged 6 months to 17 years undertaken in Mali between 2008 and 2011. Red blood cell variants were haemoglobin S (HbS), haemoglobin C (HbC), α thalassaemia, ABO blood groups, and glucose-6-phosphate dehydrogenase (G6PD) deficiency encoded by the X-linked A- allele. The primary outcome was malaria incidence, measured as the number of uncomplicated or severe malaria episodes over time. The secondary outcome was parasite density at the time of a malaria episode. We modelled incidence rate ratios with quasi-Poisson regression and we analysed parasite densities using generalised estimating equations. This study is registered with ClinicalTrials.gov, number NCT00669084. FINDINGS Between May 1, 2008, and Dec 29, 2011, we enrolled 1586 children into the study. We successfully typed all five red blood cell variants for 1543 of these children, who therefore constituted the evaluable population and in whom we diagnosed 4091 malaria episodes over 2656 child-years of follow-up. In these 1543 children, red blood cell variants were common, and occurred at the following frequencies: sickle cell trait (HbAS) 220 (14%), HbC heterozygosity (HbAC) 103 (7%), α thalassaemia 438 (28%), type O blood group 621 (40%), and G6PD deficiency 72 (9%) in 767 boys and 158 (20%) in 776 girls. The overall incidence of malaria was 1.54 episodes per child-year of follow-up, ranging from 2.78 episodes per child-year at age 3 years to 0.40 episodes per child-year at age 17 years. The malaria incidence was lower in HbAS children than in HbAA children with normal haemoglobin (adjusted incidence rate ratio [aIRR] 0.66 [95% CI 0.59-0.75], p<0.0001) and lower in G6PD A-/A- homozygous girls than in G6PD A+/A+ girls (0.51 [0.29-0.90], p=0.020), but was higher in HbAC children than in HbAA children (1.15 [1.01-1.32], p=0.039). Parasite density was lower in HbAS children (median 10,550 parasites per μL [IQR 1350-26,250]) than in HbAA children (15,150 parasites per μL [4250-31,050]; p=0.0004). The HbAS-associated reductions in malaria risk and parasite density were greatest in early childhood. INTERPRETATION The individual and interactive effects of HbAS, HbAC, and G6PD A-/A- genotypes on malaria risk and parasite density define clinical and cellular correlates of protection. Further identification of the molecular mechanisms of these protective effects might uncover new targets for intervention. FUNDING Intramural Research Program, National Institute of Allergy and Infectious Diseases, National Institutes of Health.