Phase I/II feasibility study evaluating the generation of leukemia-reactive cytotoxic T lymphocyte lines for treatment of patients with relapsed leukemia after allogeneic stem cell transplantation

Background and Objectives Graft-versus-host-disease may be avoided and the likelihood of a graft-versus-leukemia reaction increased by infusion of in vitro-generated, leukemia-reactive, cytotoxic T lymphocyte (CTL) lines as treatment for patients with relapsed leukemia after allogeneic stem cell transplantation, instead of donor lymphocyte infusion. The aim of this phase I/II study was to assess the feasibility of large-scale in vitro generation of leukemia-reactive CTL for clinical use.Design and Methods Using a modified limiting dilution culture system donor T cells were stimulated with HLA-identical leukemic antigen-presenting cells. Feasibility experiments demonstrated that in 16 of 27 donor-recipient pairs tested a CTL line could be generated. Twelve of these 16 patients developed a relapse and for 11 of these 12 patients a CTL line was generated under Good Manufacturing Practice conditions.Results The CTL lines showed moderate to high cytotoxic activity against original recipient leukemic cells in vitro. Eight patients with a relapse received from one to seven CTL lines. One patient entered a complete remission after CTL infusion only, one entered a complete remission after combined CTL infusion and donor lymphocyte infusion, two patients had temporarily stable disease, and in four patients no response was observed.Interpretation and Conclusions Although the current procedure to generate these CTL lines is feasible, the strategy is logistically complex and time-consuming, and needs further improvement

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

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

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

PI4K is a prophylactic, but not radical curative target in Plasmodium vivax-type malaria parasites

Two Plasmodium PI4 kinase inhibitors, KDU691 and LMV599, were selected for in vivo testing as causal prophylactic and radical cure agents of P. 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 KDU691 and LMV599 compounds were fully protective when administered prophylactically and the more potent compound LMV599 achieved protection as a single oral 25 mg/kg dose. 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 were 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 compounds activity on established hypnozoites and predict radical cure in vivo

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