Vaccination with Plasmodium knowlesi AMA1 Formulated in the Novel Adjuvant Co-Vaccine HT™ Protects against Blood-Stage Challenge in Rhesus Macaques

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 IC50 values correlated with estimated in vivo growth rates

MVA.85A Boosting of BCG and an Attenuated, phoP Deficient M. tuberculosis Vaccine Both Show Protective Efficacy Against Tuberculosis in Rhesus Macaques

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

Suppression of Plasmodium cynomolgi in Rhesus Macaques by Coinfection with Babesia microti▿ †

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

A dual fluorescent Plasmodium cynomolgi reporter line reveals in vitro malaria hypnozoite reactivation

Plasmodium vivax malaria is characterized by repeated episodes of blood stage infection (relapses) resulting from activation of dormant stages in the liver, so-called hypnozoites. Transition of hypnozoites into developing schizonts has never been observed. A barrier for studying this has been the lack of a system in which to monitor growth of liver stages. Here, exploiting the unique strengths of the simian hypnozoite model P. cynomolgi, we have developed green-fluorescent (GFP) hypnozoites that turn on red-fluorescent (mCherry) upon activation. The transgenic parasites show full liver stage development, including merozoite release and red blood cell infection. We demonstrate that individual hypnozoites actually can activate and resume development after prolonged culture, providing the last missing evidence of the hypnozoite theory of relapse. The few events identified indicate that hypnozoite activation in vitro is infrequent. This system will further our understanding of the mechanisms of hypnozoite activation and may facilitate drug discovery approaches

The effect of the interventions on hallmarks and complications of T2DM.

<p>Body weight, epidydimal adipose tissue weight, fasting plasma concentrations of glucose, insulin, cholesterol and triglycerides (TG) of the experimental groups are shown together with the intrahepatic TG concentrations, the urinary albumin/creatinine ratio and the atherosclerotic lesion area. Data represent the effects of drug intervention or dietary lifestyle intervention (DLI) at 16 weeks, <i>i.e.</i> the end of study. One group was sacrificed earlier and prior to the start of the interventions at 9 weeks (HFD 9w).</p>*<p>P<0.05 compared to HFD 16w,</p>#<p>P<0.05 for within subject change from 9 weeks to 16 weeks compared to HFD. Data are shown as mean ± SEM.</p

Risk factors of developing T2DM induced by 9 weeks of HFD in LDLr−/− mice.

*<p>P<0.05 compared to T = 0.</p

Molecular network of genes related to atherosclerosis signaling and hepatic fibrosis signaling pathway.

<p>Genes differentially expressed in dietary lifestyle intervention (DLI), compared to HFD were subjected to network analysis (Ingenuity Pathway Analysis). The network of genes associated with processes “Cellular Growth and Proliferation”, “Connective Tissue Development and Function” and “Hepatic System Development and Function” (network score 32) is represented in the figure. Genes or gene products are represented as nodes, and the biological relationships between two nodes are represented as edges (lines). The nodes of the network are colored according to log₂ gene expression changes in the DLI vs. HFD comparison (red: upregulation, green: downregulation). The bar graph associated with each node represents log₂ expression changes in chow (1st bar) and DLI groups (2nd bar) vs. HFD group, highlighting that all represented genes change in equivalent direction in chow and DLI conditions. The function “Overlay: Canonical Pathway” was used to highlight network genes associated with “atherosclerosis signaling” (11 genes, top enriched pathway) and “hepatic fibrosis/hepatic stellate cell activation” (9 genes, 3rd enriched pathway). All genes associated with these pathways, as well as majority of genes in the network are downregulated in DLI group, indicating withdrawal of pathogenic signals upon dietary lifestyle intervention.</p

Effect of drug and dietary lifestyle interventions on hepatic transcriptome and metabolite concentration changes.

<p>The changes in expression (transcripts, A) or concentration (metabolites, B) that are significantly different in at least one of 12 experimental conditions compared to HFD group are plotted in a heatmap (log₂ ratios vs. mean of HFD group). The number of significantly different transcripts or metabolites in each experimental condition is provided above the heatmap. The cluster tree (Pearson correlation, complete linkage) is based on average log₂ ratios vs. HFD values per intervention group. The upper part of the heatmap (1) represents transcripts and metabolites that are significantly changed in chow control vs. HFD control and defines changes that are associated with developing disease. The lower part of the heatmap (2) represents transcripts/metabolites that are significantly changed in at least one of interventions (dietary lifestyle (DLI) or one of drug interventions) compared to HFD. The transcript/metabolite profiles demonstrate high similarity between molecular signatures of chow and DLI groups and pronounced effects of fenofibrate and T0901317. (A) The expression changes of 4286 transcripts that are significantly different in at least one of 12 experimental conditions, compared to HFD group. (B) The concentration changes of 75 metabolites that are significantly different in at least one of 12 experimental conditions, compared to HFD group.</p

Effect of drug and dietary lifestyle interventions on protein and metabolite profiles in plasma.

<p>Shown are 67 proteins and metabolites with significantly different concentrations compared to HFD control group in at least one of the experimental conditions. The numbers of parameters (metabolites/proteins) with significantly different concentration compared to HFD control group in each experimental condition are provided above the heatmap. Log₂ ratios of a particular group vs. mean of HFD control group are plotted in a heatmap. Each vertical lane within a treatment group represents a response of one mouse in that treatment group. The cluster tree (Pearson correlation, complete linkage) is based on average log₂ ratios of a particular group vs. HFD. The upper part of the heatmap (1) represents proteins and metabolites that are significantly changed in the comparison chow control vs. HFD control group and defines thus changes that are associated with developing disease. The lower part of the heatmap (2) represents proteins and metabolites that are significantly changed in at least one of the interventions (i.e. dietary lifestyle (DLI) or one of the drug interventions) compared to HFD. The protein/metabolite profiles demonstrate a remarkable similarity between molecular signatures of chow control and DLI.</p

The effect of rosiglitazone, fenofibrate and T090131 on key hepatic processes required for the reversal of HFD-induced disorders.

<p>The Gene Ontology biological processes “glucose metabolic process”, “fatty acid metabolic process”, “oxidation reduction”, “immune response”, “apoptosis”, “cell cycle” and “wound healing” were selected for a detailed analysis to investigate whether the hepatic transcriptome changes induced by the drugs with marked hepatic effects (fenofibrate, T090131 and rosiglitazone) are in line with the changes induced by DLI, i.e. a “return to healthy” profile. The heatmaps show the mean log₂ expression ratio of each treatment vs. HFD group for genes involved in these processes (red: upregulation, blue: downregulation). Both fenofibrate and T090131 show many additionally and oppositely changing genes compared to DLI and/or chow group. Rosiglitazone has minor effects all of which were in line with the changes in chow/DLI groups.</p