Search

Efforts to eradicate Plasmodium vivax malaria are hampered by the presence of hypnozoites, persisting stages in the liver that can reactivate after prolonged periods of time enabling further transmission and causing renewed disease. Large-scale drug screening is needed to identify compounds with antihypnozoite activity, but current platforms rely on time-consuming high-content fluorescence imaging as read-out, limiting assay throughput. We here report an ultrafast and sensitive dual-luciferase-based method to differentiate hypnozoites from liver stage schizonts using a transgenic P. cynomolgi parasite line that contains Nanoluc driven by the constitutive hsp70 promoter, as well as firefly luciferase driven by the schizont-specific lisp2 promoter. The transgenic parasite line showed similar fitness and drug sensitivity profiles of selected compounds to wild type. We demonstrate robust bioluminescence-based detection of hypnozoites in 96-well and 384-well plate formats, setting the stage for implementation in large scale drug screens.

Two Plasmodium PI4 kinase (PI4K) inhibitors, KDU691 and LMV599, were selected for in vivo testing as causal prophylactic and radical-cure agents for Plasmodium cynomolgi sporozoite-infected rhesus macaques, based on their in vitro activity against liver stages. Animals were infected with P. cynomolgi sporozoites, and compounds were dosed orally. Both the KDU691 and LMV599 compounds were fully protective when administered prophylactically, and the more potent compound LMV599 achieved protection as a single oral dose of 25 mg/kg of body weight. In contrast, when tested for radical cure, five daily doses of 20 mg/kg of KDU691 or 25 mg/kg of LMV599 did not prevent relapse, as all animals experienced a secondary infection due to the reactivation of hypnozoites in the liver. Pharmacokinetic data show that LMV599 achieved plasma exposure that was sufficient to achieve efficacy based on our in vitro data. These findings indicate that Plasmodium PI4K is a potential drug target for malaria prophylaxis but not radical cure. Longer in vitro culture systems will be required to assess these compounds' activity on established hypnozoites and predict radical cure in vivo .

Relapses of Plasmodium dormant liver hypnozoites compromise malaria eradication efforts. New radical cure drugs are urgently needed, yet the vast gap in knowledge of hypnozoite biology impedes drug discovery. We previously unraveled the transcriptome of 6 to 7 day-old P. cynomolgi liver stages, highlighting pathways associated with hypnozoite dormancy (Voorberg-van der Wel et al., 2017). We now extend these findings by transcriptome profiling of 9 to 10 day-old liver stage parasites, thus revealing for the first time the maturation of the dormant stage over time. Although progression of dormancy leads to a 10-fold decrease in transcription and expression of only 840 genes, including genes associated with housekeeping functions, we show that pathways involved in quiescence, energy metabolism and maintenance of genome integrity remain the prevalent pathways active in mature hypnozoites.

To overcome polymorphism in the malaria vaccine candidate Plasmodium falciparum apical membrane antigen 1 (PfAMA1), fusion protein chimeras comprised of three diversity-covering (DiCo) PfAMA1 molecules (D1, D2, and D3) and two allelic variants of the C-terminal 19-kDa region of merozoite surface protein 1 (MSP1 19 ) (variants M1 and M2) were generated. A mixture of fusion proteins (D1M1/D2M2D3) and the D1M1D2M2D3 fusion were compared to a single-unit mixture (D1/D2/D3/M1) in an immunological study in groups of rabbits. Following immunization, titers of antibodies (Abs) against four naturally occurring PfAMA1 alleles were high for all groups, as were growth inhibition assay (GIA) levels against two antigenically distinct laboratory parasite strains. Fusion of AMA1 to MSP1 19 did not suppress levels of antibodies against the AMA1 component. In addition, the breadth of antibody responses was unaffected. Anti-AMA1 antibodies were largely responsible for parasite growth inhibition, as shown in reversal-of-inhibition experiments by adding competing AMA1 antigen. For all groups, titration of the MSP1 19 antigen into the GIA led to only a small decrease in parasite inhibition, although titers of antibodies against MSP1 19 were increased 15-fold for the groups immunized with fusion proteins. GIA with affinity-purified anti-MSP1 19 antibodies showed that the 50% inhibitory concentrations of the anti-MSP1 19 antibody preparations were in the same order of magnitude for all animals tested, leading to the conclusion that fusing MSP1 19 to PfAMA1 leads to a small but significant increase in functional antibody levels. This study shows that combination of multiple vaccine candidates in fusion proteins may lead to improved characteristics of the vaccine.

Plasmodium falciparum apical membrane antigen 1 (PfAMA1) is a leading asexual blood stage vaccine candidate for malaria. In preparation for clinical trials, three Diversity Covering (DiCo) PfAMA1 ectodomain proteins, designed to overcome the intrinsic polymorphism that is present in PfAMA1, were produced under Good Manufacturing Practice (GMP) in Pichia pastoris. Using identical methodology, the 3 strains were cultivated in 70-L scale fed-batch fermentations and PfAMA1-DiCos were purified by two chromatography steps, an ultrafiltration/diafiltration procedure and size exclusion chromatography, resulting in highly pure (>95%) PfAMA1-DiCo1, PfAMA1 DiCo2 and PfAMA1 DiCo3, with final yields of 1.8, 1.9 and 1.3 gram, respectively. N-terminal determinations showed that approximately 50% of each of the proteins lost 12 residues from their N-terminus, in accordance with SDS-PAGE (2 main bands) and MS-data. Under reducing conditions a site of limited proteolytic cleavage within a disulphide bonded region became evident. The three proteins quantitatively bound to the mAb 4G2 that recognizes a conformational epitope, suggesting proper folding of the proteins. The lyophilized Drug Product (1:1:1 mixture of PfAMA1-DiCo1, DiCo2, DiCo3) fulfilled all pre-set release criteria (appearance, dissolution rate, identity, purity, protein content, moisture content, sub-visible particles, immuno-potency (after reconstitution with adjuvant), abnormal toxicity, sterility and endotoxin), was stable in accelerated and real-time stability studies at -20°C for over 24 months. When formulated with adjuvants selected for clinical phase I evaluation, the Drug Product did not show adverse effect in a repeated-dose toxicity study in rabbits. The Drug Product has entered a phase Ia/Ib clinical trial.

Plasmodium falciparum apical membrane antigen 1 (PfAMA1) is a leading blood stage vaccine candidate. Plasmodium knowlesi AMA1 (PkAMA1) was produced and purified using similar methodology as for clinical grade PfAMA1 yielding a pure, conformational intact protein. Combined with the adjuvant CoVaccine HT™, PkAMA1 was found to be highly immunogenic in rabbits and the efficacy of the PkAMA1 was subsequently tested in a rhesus macaque blood-stage challenge model. Six rhesus monkeys were vaccinated with PkAMA1 and a control group of 6 were vaccinated with PfAMA1. A total of 50 µg AMA1 was administered intramuscularly three times at 4 week intervals. One of six rhesus monkeys vaccinated with PkAMA1 was able to control parasitaemia, upon blood stage challenge with P. knowlesi H-strain. Four out of the remaining five showed a delay in parasite onset that correlated with functional antibody titres. In the PfAMA1 vaccinated control group, five out of six animals had to be treated with antimalarials 8 days after challenge; one animal did not become patent during the challenge period. Following a rest period, animals were boosted and challenged again. Four of the six rhesus monkeys vaccinated with PkAMA1 were able to control the parasitaemia, one had a delayed onset of parasitaemia and one animal was not protected, while all control animals required treatment. To confirm that the control of parasitaemia was AMA1-related, animals were allowed to recover, boosted and re-challenged with P. knowlesi Nuri strain. All control animals had to be treated with antimalarials by day 8, while five out of six PkAMA1 vaccinated animals were able to control parasitaemia. This study shows that: i) Yeast-expressed PkAMA1 can protect against blood stage challenge; ii) Functional antibody levels as measured by GIA correlated inversely with the day of onset and iii) GIA IC(50) values correlated with estimated in vivo growth rates.

Both Plasmodium and Babesia species 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 with Babesia microti was able to control infection with Plasmodium cynomolgi (a parasite of macaques with characteristics very similar to those of Plasmodium vivax ) better than naïve monkeys. To confirm this and to investigate the underlying immunopathology, six naïve rhesus monkeys were infected with B. microti . After 24 days, four of these monkeys and four naïve rhesus monkeys were challenged with P. cynomolgi blood-stage parasites. B. microti persisted at low levels in all monkeys, and the clinical parameters were comparable to those of noninfected controls. There was a significant decrease in P. cynomolgi parasitemia in animals coinfected with B. microti compared to the parasitemia in animals infected with P. cynomolgi alone. This decrease in P. cynomolgi parasitemia correlated with increases in the levels of proinflammatory monocytes at the time of P. cynomolgi infection and with higher C-reactive protein (CRP) serum levels 1 week after malaria infection. Therefore, we conclude that ongoing infection with B. microti parasites leads to suppression of malaria infection.

BACKGROUND: Continuous high global tuberculosis (TB) mortality rates and variable vaccine efficacy of Mycobacterium bovis Bacille Calmette-Guérin (BCG) motivate the search for better vaccine regimes. Relevant models are required to downselect the most promising vaccines entering clinical efficacy testing and to identify correlates of protection. METHODS AND FINDINGS: Here, we evaluated immunogenicity and protection against Mycobacterium tuberculosis in rhesus monkeys with two novel strategies: BCG boosted by modified vaccinia virus Ankara expressing antigen 85A (MVA.85A), and attenuated M. tuberculosis with a disrupted phoP gene (SO2) as a single-dose vaccine. Both strategies were well tolerated, and immunogenic as evidenced by induction of specific IFNgamma responses. Antigen 85A-specific IFNgamma secretion was specifically increased by MVA.85A boosting. Importantly, both MVA.85A and SO2 treatment significantly reduced pathology and chest X-ray scores upon infectious challenge with M. tuberculosis Erdman strain. MVA.85A and SO2 treatment also showed reduced average lung bacterial counts (1.0 and 1.2 log respectively, compared with 0.4 log for BCG) and significant protective effect by reduction in C-reactive protein levels, body weight loss, and decrease of erythrocyte-associated hematologic parameters (MCV, MCH, Hb, Ht) as markers of inflammatory infection, all relative to non-vaccinated controls. Lymphocyte stimulation revealed Ag85A-induced IFNgamma levels post-infection as the strongest immunocorrelate for protection (spearman's rho: -0.60). CONCLUSIONS: Both the BCG/MVA.85A prime-boost regime and the novel live attenuated, phoP deficient TB vaccine candidate SO2 showed significant protective efficacy by various parameters in rhesus macaques. Considering the phylogenetic relationship between macaque and man and the similarity in manifestations of TB disease, these data support further development of these primary and combination TB vaccine candidates.