Alan W. Thomas

The safety and immunogenicity of PfAMA1, adjuvanted with Alhydrogel® was assessed in malaria–experienced Malian adults. The malaria vaccine, PfAMA1-FVO [25-545] is a recombinant protein Pichia pastoris-expressed AMA-1 from Plasmodium falciparum FVO clone adsorbed to Alhydrogel®, the control vaccine was tetanus toxoid produced from formaldehyde detoxified and purified tetanus toxin. A double blind randomized controlled phase 1 study enrolled and followed 40 healthy adults aged 18–55 years in Bandiagara, Mali, West Africa, a rural setting with intense seasonal transmission of P. falciparum malaria. Volunteers were randomized to receive either 50 µg of malaria vaccine or the control vaccine. Three doses of vaccine were given on Days 0, 28 and 56, and participants were followed for 1 year. Solicited symptoms were assessed for seven days and unsolicited symptoms for 28 days after each vaccination. Serious adverse events were assessed throughout the study. The titres of anti-AMA-1 antibodies were measured by ELISA and P. falciparum growth inhibition assays were performed. Commonest local solicited adverse events were the injection site pain and swelling more frequent in the PfAMA1 group. No vaccine related serious adverse events were reported. A significant 3.5-fold increase of anti-AMA-1 IgG antibodies was observed in malaria vaccine recipients four weeks after the third immunization compared to the control group. The PfAMA1 showed a good safety profile. Most adverse events reported were of mild to moderate intensity. In addition, the vaccine induced a significant though short-lived increase in the anti-AMA1 IgG titres. Registered on www.clinicaltrials.gov with the number NCT00431808

Background: Increasing the breadth of the functional antibody response through immunization with Plasmodium falciparum apical membrane antigen 1 (Pf AMA1) multi-allele vaccine formulations has been demonstrated in several rodent and rabbit studies. This study assesses the safety and immunogenicity of three Pf AMA1 Diversity-Covering (DiCo) vaccine candidates formulated as an equimolar mixture (DiCo mix) in CoVaccine HT™ or Montanide ISA 51, as well as that of a Pf AMA1-MSP1₁₉ fusion protein formulated in Montanide ISA 51. Methods: Vaccine safety in rhesus macaques was monitored by animal behaviour observation and assessment of organ and systemic functions through clinical chemistry and haematology measurements. The immunogenicity of vaccine formulations was assessed by enzyme-linked immunosorbent assays and in vitro parasite growth inhibition assays with three culture-adapted P. falciparum strains. Results: These data show that both adjuvants were well tolerated with only transient changes in a few of the chemical and haematological parameters measured. DiCo mix formulated in CoVaccine HT™ proved immunologically and functionally superior to the same candidate formulated in Montanide ISA 51. Immunological data from the fusion protein candidate was however difficult to interpret as four out of six immunized animals were non-responsive for unknown reasons. Conclusions: The study highlights the safety and immunological benefits of DiCo mix as a potential human vaccine against blood stage malaria, especially when formulated in CoVaccine HT™, and adds to the accumulating data on the specificity broadening effects of DiCo mix.

Background: Antibodies to key Plasmodium falciparum surface antigens have been shown to be important effectors that mediate clinical immunity to malaria. The cross-strain fraction of anti-malarial antibodies may however be required to achieve strain-transcending immunity. Such antibody responses against Plasmodium falciparum apical membrane antigen 1 (Pf AMA1), a vaccine target molecule that is expressed in both liver and blood stages of the parasite, can be elicited through immunization with a mixture of allelic variants of the parasite molecule. Cross-strain antibodies are most likely elicited against epitopes that are shared by the allelic antigens in the vaccine cocktail. Methods: A standard competition ELISA was used to address whether the antibody response can be further focused on shared epitopes by exclusively boosting these common determinants through immunization of rabbits with different Pf AMA1 alleles in sequence. The in vitro parasite growth inhibition assay was used to further evaluate the functional effects of the broadened antibody response that is characteristic of multi-allele vaccine strategies. Results: A mixed antigen immunization protocol elicited humoral responses that were functionally similar to those elicited by a sequential immunization protocol (p > 0.05). Sequential exposure to the different Pf AMA1 allelic variants induced immunological recall of responses to previous alleles and yielded functional cross-strain antibodies that would be capable of optimal growth inhibition of variant parasites at high enough concentrations. Conclusions: These findings may have implications for the current understanding of the natural acquisition of clinical immunity to malaria as well as for rational vaccine design.

Background: A DNA prime, poxvirus (COPAK) boost vaccination regime with four antigens, i.e. a combination of two Plasmodium knowlesi sporozoite (csp/ssp2) and two blood stage (ama1/msp1 ₄₂ ) genes, leads to self-limited parasitaemia in 60% of rhesus monkeys and survival from an otherwise lethal infection with P. knowlesi. In the present study, the role of the blood stage antigens in protection was studied in depth, focusing on antibody formation against the blood stage antigens and the functionality thereof. Methods: Rhesus macaques were immunized with the four-component vaccine and subsequently challenged i.v. with 100 P. knowlesi sporozoites. During immunization and challenge, antibody titres against the two blood stage antigens were determined, as well as the in vitro growth inhibition capacity of those antibodies. Antigen reversal experiments were performed to determine the relative contribution of antibodies against each of the two blood stage antigens to the inhibition. Results: After vaccination, PkAMA1 and PkMSP1₁₉ antibody titres in vaccinated animals were low, which was reflected in low levels of inhibition by these antibodies as determined by in vitro inhibition assays. Interestingly, after sporozoite challenge antibody titres against blood stage antigens were boosted over 30-fold in both protected and not protected animals. The in vitro inhibition levels increased to high levels (median inhibitions of 59% and 56% at 6 mg/mL total IgG, respectively). As growth inhibition levels were not significantly different between protected and not protected animals, the ability to control infection appeared cannot be explained by GIA levels. Judged by in vitro antigen reversal growth inhibition assays, over 85% of the inhibitory activity of these antibodies was directed against PkAMA1. Conclusions: This is the first report that demonstrates that a DNA prime/poxvirus boost vaccination regimen induces low levels of malaria parasite growth inhibitory antibodies, which are boosted to high levels upon challenge. No association could, however, be established between the levels of inhibitory capacity in vitro and protection, either after vaccination or after challenge.

BothPlasmodiumandBabesiaspecies are intraerythrocytic protozoans that infect a wide range of hosts, including humans, and they elicit similar inflammatory responses and clinical manifestations that differ markedly in severity. We recently reported that a rhesus macaque that was chronically infected withBabesia microtiwas able to control infection withPlasmodium cynomolgi(a parasite of macaques with characteristics very similar to those ofPlasmodium vivax) better than naïve monkeys. To confirm this and to investigate the underlying immunopathology, six naïve rhesus monkeys were infected withB. microti. After 24 days, four of these monkeys and four naïve rhesus monkeys were challenged withP. cynomolgiblood-stage parasites.B. microtipersisted at low levels in all monkeys, and the clinical parameters were comparable to those of noninfected controls. There was a significant decrease inP. cynomolgiparasitemia in animals coinfected withB. microticompared to the parasitemia in animals infected withP. cynomolgialone. This decrease inP. cynomolgiparasitemia correlated with increases in the levels of proinflammatory monocytes at the time ofP. cynomolgiinfection and with higher C-reactive protein (CRP) serum levels 1 week after malaria infection. Therefore, we conclude that ongoing infection withB. microtiparasites leads to suppression of malaria infection.

Macaques are the closest evolutionary relatives of humans routinely used in basic and applied biomedical research. Their genetic, physiological, immunological and metabolic similarity to humans, second only to that of the great apes, makes them invaluable models of human disease. These similarities also mean that macaques are often the only experimental models available for evaluating increasingly specific drugs in development, and as a proof-of-concept bridge can help reduce the numbers of compounds that fail in clinical pharmaceutical research. In vertebrates, red blood cells (RBCs) diseases are frequently severe as their role as sole gas transporter makes them indispensable to survival; much research has therefore focused on an in-depth understanding of the functioning of the RBC. RBCs also host malaria, babesia and other parasites. Recently, we presented an in-depth proteome for the human RBC and a comparative human/mouse RBC proteome. Here, we present directly comparable data for the human, mouse and rhesus RBC proteomes. All proteins were identified, validated and categorized in terms of sub-cellular localization, protein family and function and, in comparison with the human and mouse RBC, were classified as orthologues, family-related or unique. Splice isoforms were identified and polypeptides migrating with anomalous apparent molecular weights were grouped into putatively ubiquitinylated or partially degraded complexes. Overall there was close concordance between mouse, human and rhesus proteomes, confirming the unexpected RBC complexity. Several novel findings in the human and mouse proteomes have been confirmed here. This comparison sheds light on several open issues in RBC biology and provides a departure point for more comprehensive understanding of RBC function.

Apical Membrane Antigen 1 (AMA1), a merozoite protein essential for red cell invasion, is a candidate malaria vaccine component. Immune responses to AMA1 can protect in experimental animal models and antibodies isolated from AMA1-vaccinated or malaria-exposed humans can inhibit parasite multiplication in vitro. The parasite is haploid in the vertebrate host and the genome contains a single copy of AMA1, yet on a population basis a number of AMA1 molecular surface residues are polymorphic, a property thought to be primarily as a result of selective immune pressure. After immunisation with AMA1, antibodies more effectively inhibit strains carrying homologous AMA1 genes, suggesting that polymorphism may compromise vaccine efficacy. Here, we analyse induction of broad strain inhibitory antibodies with a multi-allele Plasmodium falciparum AMA1 (PfAMA1) vaccine, and determine the relative importance of cross-reactive and strain-specific IgG fractions by competition ELISA and in vitro parasite growth inhibition assays. Immunisation of rabbits with a PfAMA1 allele mixture yielded an increased proportion of antibodies to epitopes common to all vaccine alleles, compared to single allele immunisation. Competition ELISA with the anti-PfAMA1 antibody fraction that is cross-reactive between FVO and 3D7 AMA1 alleles showed that over 80% of these common antibodies were shared with other PfAMA1 alleles. Furthermore, growth inhibition assays revealed that for any PfAMA1 allele (FVO or 3D7), the cross-reactive fraction alone, on basis of weight, had the same functional capacity on homologous parasites as the total affinity-purified IgGs (cross-reactive+strain-specific). By contrast, the strain-specific IgG fraction of either PfAMA1 allele showed slightly less inhibition of red cell invasion by homologous strains. Thus multi-allele immunisation relatively increases the levels of antibodies to common allele epitopes. This explains the broadened cross inhibition of diverse malaria parasites, and suggests multi-allele approaches warrant further clinical investigation.

Preclinical animal models informing antimalarial drug development are scarce. We have used asexual erythrocytic Plasmodium cynomolgi infections of rhesus macaques to model Plasmodium vivax during preclinical development of compounds targeting parasite phospholipid synthesis. Using this malaria model, we accumulated data confirming highly reproducible infection patterns, with self-curing parasite peaks reproducibly preceding recrudescence peaks. We applied nonlinear mixed-effect (NLME) models, estimating treatment effects in three drug studies: G25 (injected) and the bisthiazolium prodrugs TE4gt and TE3 (oral). All compounds fully cured P. cynomolgi -infected macaques, with significant effects on parasitemia height and time of peak. Although all three TE3 doses tested were fully curative, NLME models discriminated dose-dependent differential pharmacological antimalarial activity. By applying NLME modeling treatment effects are readily quantified. Such drug development studies are more informative and contribute to reduction and refinement in animal experimentation.