First-in-human assessment of safety and immunogenicity of low and high doses of Plasmodium falciparum malaria protein 013 (FMP013) administered intramuscularly with ALFQ adjuvant in healthy malaria-naïve adults

McSPI-Europe Research Group*.*See Appendix 1 for participating European centers and investigators, and Appendix 2 for other analysis investigators.Received from the Departments of Anesthesiology, Cardiology, Surgery, Medicine, and Epidemiology and Biostatistics of the member centers of the McSPI-Europe Research Group, the Ischemia Research and Education Foundation (IREF), the San Francisco Veterans Affairs Medical Center, and the University of California, San Francisco, and Stanford University, Palo Alto, California. Submitted for publication December 8, 1995. Accepted for publication November 1, 1996. Supported by grants from UCB Pharma, Belgium, and the Ischemia Research and Education Foundation.Dr. Mangano, as director of McSPI, assumes full responsibility for the integrity of the data and analyses.Address reprint requests to Dr. Mangano: c/o McSPI-Europe, 250 Executive Park Boulevard, Suite 3400, San Francisco, California 94134.Of the 60 million patients a year who undergo noncardiac surgery in the United States, Canada, Europe, Australia, New Zealand, and Japan, [1,2]approximately 30% have, or are at risk of, coronary artery disease (CAD). Of these, 3 million have a serious adverse perioperative cardiac outcome-myocardial infarction (MI), cardiac death, unstable angina, heart failure, or life-threatening dysrhythmias. [1]These complications add approximately $40 billion annually to worldwide surgical health care costs. [3]Mangano DT, and colleagues [4]demonstrated that the single most important predictor of such adverse cardiac outcome is the occurrence of myocardial ischemia soon after surgery (i.e., within 48 h). Occurring in 41% of patients undergoing elective noncardiac surgery, ischemia is associated with a ninefold increase in the odds of having in-hospital cardiac death, nonfatal MI, or unstable angina. In addition, the risk of long-term (up to 2 yr) adverse cardiac outcomes increases twofold in patients with postoperative ischemia alone, and 14- to 20-fold in patients with postoperative MI or unstable angina. [5]The entire perioperative period is known to be stressful, characterized by complex and rapidly changing physiologic responses that may be poorly tolerated by patients with compromised circulation or poor left ventricular function. During, and particularly after, anesthesia and surgery, hypertension, tachycardia, and increased sympathetic activity are common occurrences. [1,6]These hyperdynamic cardiovascular responses adversely affect the balance between myocardial oxygen supply and demand, predisposing the vulnerable myocardium to ischemia.Compounds with alpha-2 adrenoceptor agonist activity are reported to have sympatholytic, sedative, analgesic, and anxiolytic effects and attenuate the catecholamine response to perioperative stress. Therefore, alpha2-adrenoceptor agonists may prevent perioperative hyperdynamic changes, mitigating imbalances between myocardial oxygen supply and demand, and possibly reduce the incidence of myocardial ischemia. [7–9]Mivazerol (2-hydroxy-3 [1H-imidazole-4-methyl] benzamide hydrochloride) is a new alpha2-adrenoceptor agonist with a high affinity and a marked specificity for alpha2-adrenergic receptors. In animal studies, mivazerol blunts the surge in heart rate during emergence from halothane anesthesia, decreases basal norepinephrine concentrations, and reduces ischemia in animal models of coronary occlusion.* In clinical pharmacology studies, mivazerol decreases basal plasma levels of norepinephrine and, at higher doses, produces mild bradycardia, negative inotropic effects, and an early and transient increase in afterload.** In patients with stable angina undergoing a treadmill exercise tolerance test, mivazerol reduced ischemia and angina. [10]Most recently, in high-risk patients undergoing noncardiac surgery, a 24-patient dose-response trial demonstrated safety and tolerability.***Based on the above, we conducted a multicenter phase II trial to investigate whether mivazerol could reduce the incidence of, and treatment for, tachycardia and hypertension in patients with, or at risk for, CAD who were undergoing vascular surgery and general anesthesia. We also determined the effect of mivazerol on the incidence and severity of perioperative myocardial ischemia, adverse clinical events, and anesthetic and analgesic requirements.After institutional approval and informed consent were obtained, we enrolled 317 patients from 23 medical centers in 7 European countries (Table 8Appendix 1) between March and December 1993; analysis for the current study was completed in March 1996. The study was placebo-controlled, double-blind, and randomized with parallel groups using block randomization within each center to assign placebo or one of two doses of mivazerol to patients. For inclusion in the study, patients were required to be at least 21 yr old, to have CAD and normal renal function, and to be undergoing vascular surgery (excluding aortic surgery) that required general anesthesia for at least 1 h. Coronary artery disease was confirmed by the existence of any of the following conditions:(1) a history of either typical angina pectoris (as defined by the Canadian Heart Classification system [11]) or atypical angina with an ischemic (electrocardiographic, echocardiographic) response to exercise testing, or with scintigraphic evidence of myocardial perfusion defect;(2) a history of MI;(3) new Q wave on electrocardiogram typical of MI, with no history of MI; or (4) angiographic evidence of CAD. Patients were excluded from study if they:(1) had been taking alpha-methyl dopa, alpha2-agonists, or tricyclic antidepressants;(2) were in cardiogenic shock and had clinical signs of congestive heart failure or required chronic inotropic support for ventricular dysfunction;(3) had unstable angina or treated, uncontrolled hypertension;(4) had conduction defects that precluded electrocardiographic analysis of the ST segment;(5) were pregnant or of childbearing potential but not using reliable contraception; or (6) were American Society of Anesthesiologists physical status V.The primary measures used to judge the efficacy of mivazerol and placebo were hemodynamic instability (tachycardia, hypertension) and the need for cardiovascular medications to treat instability in the first 24 h after surgery, and secondarily for the intraoperative and 24- to 72-h postoperative periods. Other secondary measures consisted of the incidence and severity of myocardial ischemia and the need for medications to treat ischemia. Safety was assessed by the occurrence of adverse clinical events and hemodynamic abnormalities (bradycardia, hypotension), and by the use of cardiovascular medications to treat these hemodynamic abnormalities.The investigators gathered demographic and clinical data by taking a complete history and physical examination that included a neurologic examination and gathering information on previous and concurrent medications. Research data were obtained from measurement of hemodynamic variables and cardiac enzymes, and from ambulatory (Holter) and 12-lead electrocardiography. Preoperative cardiac medications were continued until the day of surgery. Premedication consisted of 5 mg diazepam given orally on the day of surgery.Anesthesia was induced by intravenous administration of sodium thiopental (as much as 5 mg/kg) and fentanyl (as much as 2 micro gram/kg). Anesthesia was maintained by continuous intravenous infusion of fentanyl (1 micro gram [center dot] kg-1[center dot] h-1) and isoflurane (end-tidal concentration of up to 2.0%). Vecuronium provided muscle relaxation. Systolic blood pressure (SBP) and heart rate (HR) were kept within 20% of preoperative baseline values by the use of cardiovascular drugs and prespecified changes in the anesthetic. The anesthetic concentration was first altered if necessary to treat hemodynamic abnormalities. After this, tachycardia with concomitant hypertension was to be treated with a beta blocker. Hypertension was to be treated with hydralazine or sodium nitroprusside. Bradycardia was to be treated with atropine or, when necessary, isoproterenol. Hypotension was treated with phenylephrine, methoxamine, or norepinephrine. Intravenous fluids were used, as was dopamine, when necessary. Prophylactic use of nitrates, calcium-channel blockers, or beta-adrenergic blocking drugs to prevent ischemia was specifically prohibited; nitroglycerin was used only to treat documented myocardial ischemia (as demonstrated by ST-segment changes on the clinical monitor).The “low-dose” group was given 2 micro gram/kg mivazerol for 20 min before induction of anesthesia, followed by 0.75 micro gram [center dot] kg-1[center dot] h-1intraoperatively and for as long as 72 h postoperatively. The “high-dose” group was given 4 micro gram/kg and then 1.5 micro gram [center dot] kg-1[center dot] h-1, following the same protocol. These doses were selected to produce plasma levels of mivazerol of approximately 1.0 ng/ml in the low-dose group and 2.0 ng/ml in the high-dose group during surgery and for as long as 72 h after surgery.Heart rate was to be kept between 40 and 100 beats/min, and SBP between 90 and 180 mmHg, during the 96-h postoperative period, using prespecified analgesic, sedative, and cardiac adjuvant therapies. Morphine sulfate was administered intravenously or by patient-controlled analgesia, and midazolam was given for anxiety.Routine clinical monitoring included continuous five-lead electrocardiography, measurement of radial artery pressure and arterial blood oxygen saturation, and assessment of inspired concentrations of isoflurane and oxygen. In parallel with clinical monitors, research monitors included three-channel, seven-lead Holter electrocardiography, continuous measurement of HR and blood pressure, 12-lead electrocardiography, and assays of cardiac enzymes. Clinical decisions were not controlled by study protocol, and clinicians caring for the patients had no knowledge of any research data.Baseline HR and blood pressure were determined by averaging 3 readings obtained 5 min apart before induction of anesthesia. Intraoperatively and for as long as 96 h after surgery, HR, SBP, diastolic blood pressure, and mean arterial pressure were measured every 10 s and stored by portable patient monitor (McSPI/Ischemia Research and Education Foundation-(IREF) Patient Monitor System) until the arterial line was removed, and then an automated blood pressure cuff recorded pressures, which also were stored in the computer. Heart rate continued to be recorded every 10 s throughout all periods. The HR and blood pressure data were compressed into 1-min data samples and were then reviewed to eliminate artifacts (such as those associated with drawing of blood or flushing of the catheter). Median values for these data were calculated, and each value was evaluated for out-of-bounds conditions, which were annotated. The occurrence of episodes of hemodynamic instability was determined, and the frequency distribution and characteristics of these episodes were derived for the intraoperative and postoperative periods. (Table 9)For the intraoperative period, a hemodynamic episode was said to have occurred if:(1) HR increased at least 20% from baseline or was 40 beats/min or less;(2) SBP increased or decreased 20% or more from baseline; and (3) the episode lasted at least 5 min. For the postoperative period, episodes were said to have occurred if:(1) HR decreased to 40 beats/min or less, or increased to 100 beats/min or more;(2) SBP decreased to 90 mmHg or less, or increased to 180 mmHg or more. In addition, treatment for hemodynamic changes (tachycardia, bradycardia, hypertension, hypotension) were recorded and characterized. To evaluate hemodynamic rebound, the incidence of, and treatment for, either tachycardia or hypertension during the 12-h post-infusion period (> 72 h) was compared between the placebo and mivazerol groups.Two independent, blinded clinicians individually scanned the annotated hemodynamic files, located the out-of-bounds conditions, and reviewed the annotated data to determine whether the hemodynamic abnormalities were real or artifact. A computerized report that described all episodes identified by the two clinicians and any discrepancies between the two clinicians was generated after both primary readers completed their analyses. A third independent reviewer reviewed the generated report and validated all previous readings and resolved any discrepancies. To assess concordance of the hemodynamic analysis, a concordance plan was pre-specified, and 20% of the patient population was randomly selected and reanalyzed for hemodynamic events. All analysis individuals (IREF) were blinded to previous results, patient identification, clinical events, and other outcome events. The results of this analysis, which occurred after completion of the main analysis, were compared statistically as described earlier.Patients were monitored with a three-channel AM Holter electrocardiogram recorder (series 8500; Marquette Electronics, Milwaukee, WI), commencing at least 8 h before surgery and continuing up to the 96th postoperative hour. Bipolar leads (CC5, CM5, ML) were used. Each complete electrocardiographic recording was scanned using an electrocardiographic analysis system (Marquette Series Laser Holter SXP); all abnormal QRS complexes (i.e., ventricular ectopic beats, conduction abnormalities) were excluded, and a continuous three-lead ST-segment trend was generated, as described elsewhere. [4]ST deviation values were determined at 60 msec past the J point, in each of the three channels, unless that point fell within the T wave, in which case the measurement point of ST segment depression was shortened to a minimum of J + 40 msec. Electrocardiographic episodes of ischemia were defined as:(1) horizontal or downsloping ST segment depression from baseline of greater or equal to 1 mm lasting at least 1 min and separated from other episodes by greater or equal to 1 min;(2) ST segment evaluation from baseline greater or equal to 2 mm measured at the J point lasting at least 1 min and separated from other episodes by greater or equal to 1 min. The reversal of an ischemic episode was defined by the return of the ST segment to baseline for at least 1 min. Each episode was assessed for duration, magnitude, severity (area under the curve), as well as for “ischemic burden”(minutes of ischemia/hours monitored). Results of Holter scanner interpretation were recorded on data sheets by the first electrocardiographer. A second electrocardiographer reviewed these results. A third-level electrocardiographer also reviewed all the results and resolved all differences.The intraoperative results also were analyzed by dividing the period into the nonemergence period and the emergence from anesthesia period, with emergence being defined as the 30-min period before leaving the operating room. The period definitions were arbitrary, but were specified before unblinding.Twelve-lead electrocardiograms were obtained at the time of patient screening, on arrival at the intensive care unit, daily on postoperative days 1 to 5, on postoperative day 7, and at hospital discharge. Creatine kinase myocardial band (CK-MB) isoenzyme levels were obtained preoperatively; on arrival at the intensive care unit; and at 4, 8, 12, 16, 20, 24, 32, 40, 48, 60, 72, 84, and 96 h after surgery. The occurrence of perioperative MI was assessed for the period from induction of anesthesia to hospital discharge. Myocardial infarction was diagnosed if any of the following occurred:(1) new Q wave on a postoperative 12-lead electrocardiogram, as determined by application of the Minnesota Code criteria [12](1–1 to 1–3) and analysis panel validation; or (2) an elevation in CK-MB levels to greater or equal to 100 ng/ml at any time after surgery, or to greater or equal to 70 ng/ml within 12 h after surgery; or (3) diagnosis of acute MI made during autopsy.Two blinded investigators applied the Minnesota Code criteria, as modified by Chaitman et al., [13]to code the 12-lead electrocardiograms. If disagreements arose between the two investigators regarding coding or diagnosis, a third investigator reviewed the set of electrocardiograms, and, along with the original two investigators, a final diagnosis was achieved by consensus.Determinations of CK-MB levels were performed centrally by the Bioanalytical Research Corporation (BARC, Gent, Belgium) using an immunoenzymetric assay (Hybritech Tandem-E CK-MB). Diagnosis of MI by autopsy was made by the pathologist at the participating center.Blood samples for clinical chemistry and for determination of plasma mivazerol concentrations were performed at predetermined times.Adverse events and serious adverse events were ascertained by site investigators and reported on Case Report Forms using the system organ classification of the World Health Organization. [14]All research data (Holter electrocardiogram, hemodynamic data, 12-lead electrocardiographic data) were analyzed at the coordinating center (IREF, San Francisco, CA) in a blinded fashion to ensure that uniform criteria for data analysis were used. Because block randomization was performed at each of the 23 centers, the analyses presented herein include an adjustment for any effect associated with individual centers.For the analysis of the incidence of hemodynamic or ischemic episodes, when the outcome variable was binary and the explanatory variable was treatment, the two-by-three contingency Table analysiswas controlled for center effect using the Cochran-Mantel-Haenszel general association chi-square statistic. For the high-dose versus placebo comparison, the same technique was performed using data for the high-dose and placebo groups. These analyses were performed using PROC FREQ of the Statistical Analysis System (SAS, SAS Institute, Cary, NC). For a continuous response variable, a general linear model was used and included center and treatment-by-center effects, to derive the adjustment treatment effect.The treatment-by-center effect was included in all models, regardless of whether the effect was statistically significant (at the 5% level), because the sample size per center was not sufficiently large to assess adequately whether there was treatment-by-center effect, and because randomization was carried out at each center. In addition, in most of the models, the center effect was significant, suggesting a high level of heterogeneity in the least-squares estimated means of the response variable across the 23 centers. PROC GLM (General Linear Model) was used to fit these models and to obtain the adjusted estimated treatment effect. The comparison between high-dose mivazerol and placebo was carried out using data from the high-dose and placebo groups, and the same technique as described was used.The secondary efficacy variables included myocardial ischemia, anesthetic and analgesic requirements, and adverse clinical outcomes. For these endpoints, incidence was compared using either chi-square or Fisher's exact test. Analysis of variance or the Kruskal-Wallis test was used to analyze characteristics of hemodynamic and ischemic abnormalities. For analysis of area under the CK-MB curve and the maximum CK-MB, the data window was taken to be 4–96 h after surgery, a period that encompassed 14 measurements of for area under the CK-MB and maximum CK-MB were compared across treatment groups using analysis of variance data were and excluded from because the values were at across study groups, the were to be for the sample for the concordance analysis of the primary outcome variable, hemodynamic the and severity of hemodynamic (tachycardia, bradycardia, hypertension, or hypotension) during each of the study periods. were selected randomly from the and were reanalyzed using the same as for the original analysis, with all patient blinded to the between the original analysis results and the results was first determined for the incidence of each of of hemodynamic during the primary outcome period, as well as all other time periods. The concordance was evaluated using the an prespecified to equal which is to second of the analysis the concordance of the frequency and severity of hemodynamic episodes during each of the study periods. was defined as the of hemodynamic episodes (tachycardia, bradycardia, hypertension, or hypotension) per patient for each severity were defined as:(1) the mean of all for each and (2) the mean of all area under the curve for each The concordance for frequency and severity was evaluated by the described by the in which the of as the for the of this analysis, a greater or equal to was prespecified before analysis, and is patient case report were into a SAS by the The data were up daily a computer. A of the that consisted of all the information from the case report was every to the coordinating and analysis center in San Francisco in a The data that included the Holter and 12-lead electrocardiographic data were into a at the analysis center in San Francisco and data were individually by research investigators using SAS The continuous hemodynamic data were by the centers to and were to SAS The data were from Belgium) to on a in a and then were to All data were carried out using the The final was then to SAS and all data analyses were performed using this the 317 patients enrolled in the study, were not to a study because of patient or Of the were given low-dose and high-dose Table that demographic data and preoperative cardiac medications were for the three groups. patients were and and were taking a of cardiovascular medications. to the study not for the three groups all the HR and SBP for time periods. For all time HR was with high-dose mivazerol with Systolic blood pressure was in the high-dose group at 8, 16, and 24 h after leaving the operating the incidence of hemodynamic changes, and Table the to treat those administration of the incidence of tachycardia to be higher in the high-dose during administration (i.e., during the early and postoperative the incidence of tachycardia was in the high-dose group (Table For the low-dose the incidence of tachycardia during the intraoperative period was versus for placebo and was reduced during the postoperative period The incidence of hypertension was decreased with low-dose or high-dose mivazerol for the intraoperative period only (Table For all time the incidence of either tachycardia or hypertension was with mivazerol (at either with the for the three groups during the time were as for the intraoperative period, and versus for the early postoperative period, and versus and for the postoperative period, and versus effect also was after of the infusion of and versus need for treatment of tachycardia was with high-dose mivazerol for the early and postoperative (Table The need for treatment of hypertension was in the and low-dose groups (Table The low-dose group not from the placebo group regarding treatment for tachycardia or hypertension, for the intraoperative period After of treatment for tachycardia or hypertension not between of occurred in both mivazerol groups during all time of administration and after of the the incidence was higher that with placebo (Table treatment for not across groups during any The groups not in the incidence of during the period, at which time the low-dose group had a higher incidence the placebo group was no in the incidence of either or for any of the postoperative periods. for the intraoperative period, the low-dose group had a higher incidence of either or for not between the groups during or after administration (Table the incidence of changes in the ST segment on Holter incidence was for the high-dose group for the placebo group during the intraoperative analysis, derived by the intraoperative period into the period (i.e., the min before of the patient from the operating and the nonemergence period, that the incidence was in the emergence For other these were not significant (Table the characteristics of ST segment The high-dose group had a of ischemia and area under the and ischemic for the postoperative treatment of ischemia, the need for nitrates, calcium-channel blockers, or was in the high-dose group for the postoperative period, being with placebo individual cardiac the use of was in the high-dose group in the postoperative period for patients had an adverse as defined by the World Health System The incidence of serious adverse events also was not across study and for the and placebo groups, patients during the in the high-dose group from after surgery, one from and one from MI and cardiogenic and one in the placebo group by and cardiogenic of these was reported by the had no knowledge of study group as or being associated with a analysis, there was no in the incidence of hypertension between study groups during the acute infusion period before anesthetic induction was there any in the incidence of hypertension before infusion infarction occurred in of patients given in 1 of patients given low-dose and in 2 of patients given high-dose The sample size the to these results. The of patients in the and placebo groups who had other adverse cardiac outcomes as and 1 patient had unstable 1, and 3 patients had congestive heart and and 1 patient had and patients had and 1, and 1 patient had cardiac was for all groups. For the and placebo groups, the mean of fentanyl required to anesthesia and The mean induction of thiopental was and and the mean concentration of isoflurane required was and All patients required sulfate during the first 48 h after surgery.

Introduction
\nMalaria is caused by intracellular organisms that belong to the genus Plasmodium. In 2015, there were an estimated 438,000 deaths and 214 million clinical illnesses due to malaria infection, of which the majority were in sub-Saharan African children below five years of age. Amongst the five species that are known to infect humans, Plasmodium falciparum causes the most severe disease, mostly in children and pregnant women in sub-Saharan Africa. Despite malaria control programs being operational for many years, malaria elimination in most endemic regions is far from being achieved. Vaccination is considered the most cost effective method of preventing infectious diseases. To date, there are no effective vaccines available for parasitic infections, despite the existence of strong evidence of acquired immunity in most parasitic infections studied. It is therefore highly likely that the addition of an effective tool such as a vaccine to the current malaria control strategy would have a strong positive impact on our ability to control this disease. In the first part of this thesis, we aimed to investigate the vaccine efficacy as well as the cellular and humoral immunity of African paediatric volunteers vaccinated with the most clinically advanced malaria vaccine; the RTS, S/AS01.
\n Meanwhile, novel vaccination and testing approaches are being pursued to improve or replace the recombinant subunit malaria vaccine approach to meet the goals formulated in the Malaria Vaccine Roadmap of WHO (http://www.who.int/immunization/topics/malaria/ vaccine_roadmap/en). These goals strategized that by 2030, licensed vaccines targeting Plasmodium falciparum and Plasmodium vivax should encompass the following two objectives, for use by the international public health community:
\ni)\tFirst, it should have a protective efficacy of at least 75 percent against clinical malaria and be suitable for administration to appropriate at-risk groups in malaria- endemic areas.
\nii)\tSecondly, it should reduce transmission of the parasite and thereby substantially reduce the incidence of human malaria infection; enable elimination in multiple settings and be suitable for administration in mass campaigns.
\nCurrently, the most promising candidate seems to be the whole malaria sporozoite approach, which is formed of cryopreserved, purified whole live-attenuated (either by irradiation or genetic attenuation) sporozoites. One of the novel tools used to analyze induced vaccine efficacy in sub-Saharan Africa experimentally vaccinated volunteers is controlled human malaria infection (CHMI). Many CHMIs using infectious mosquito bites or purified sporozoites have been successfully conducted in the USA and Europe over many years, but this approach had not been employed in sub-Saharan Africa until 2012. The aim of the second part of this thesis was to describe the potential of using CHMI as a tool to accelerate malaria vaccine development in sub-Saharan Africa and to dissect malaria- specific immunity induced by CHMI based on our trial conducted in 2012 in Bagamoyo.
\n
\nMethods and findings
\nIn the first part of this thesis (Chapter 4), the aim was to investigate safety, efficacy, cellular and humoral immunity in RTS,S/AS01 vaccinated Tanzanian paediatric populations. Adverse events were used to determine the safety of the RTS,S/AS01 vaccine in this age group (paper I), ELISA to measure the vaccine-induced CS-specific antibodies and Luminex to measure vaccine-induced cytokine responses (paper II and III). Furthermore, flow cytometry was used to investigate vaccine-induced cellular immune responses (paper III). We also looked into the implications and practicalities of immunological sampling in the African paediatric population. We did community sensitization and collected blood samples from 400 children for immunological study (paper IV). We showed that in 6-12 week old infants, vaccine efficacy against clinical malaria 14 months after first vaccination was 30.1% (95% CI, 23.6 to 36.1) in the intention-to-treat (ITT) and 31.3% (97.5% CI, 23.6 to 38.3) in the per-protocol (PP) population. Furthermore, the vaccine efficacy against severe malaria was 26.0% (95% CI, −7.4 to 48.6) and 36.6% (95% CI, 4.6 to 57.7) in the ITT and PP populations, respectively. The safety of the vaccine in terms of serious adverse events showed similar trends in both study groups. We identified two main RTS,S/AS01 vaccine induced cellular immune mechanisms:- (i) Th1-related responses such as CS-specific IFN-g, GM-CSF and IL-15 are associated with protection and (ii) Th2-related responses mediated by CS-specific IL5 and RANTES are associated with increased odds of malaria. Moreover, antibody avidity alone did not predict protective efficacy in the current study. The induction of RTS, S/AS01 protective Th1 and pro-inflammatory responses was lower in infants compared to children; a scenario that might explain the lower efficacy observed in the infant cohort. Furthermore, we also showed that immunology studies in the paediatric population can feasibly be conducted in African research institutions.
\nIn the second part of this thesis (Chapter 5), we conducted in 2012 the first CHMI using cryo-preserved purified non-attenuated sporozoites in Tanzanian adult volunteers with previous malaria exposure (paper V). In this study, the humoral and cellular immune responses elicited following CHMI were evaluated (paper VI and VII). We used adverse events to determine the safety of the CHMI model in malaria pre-exposed volunteers. We also used blood slide microscopy to define sporozoite infectivity rates, Luminex assays to examine the sporozoite-induced antibodies, B-cell Elispot analysis, single cell RNA sequencing, flow cytometry and cell sorting followed by in vitro stimulation assays to investigate and define the affected innate and adaptive immune responses following CHMI (paper VIII). Our studies showed that: (i) CHMI is safe, tolerable and infective when used in malaria endemic regions, (ii) a single dose of intradermal sporozoite (PfSPZ) challenge elicited long-lived merozoite-opsonizing antibodies and long-lasting innate and innate-like lymphocyte populations, (iii) When we compared Dutch (malaria naïve) and Tanzanian (malaria exposed) subjects undergoing the same challenge study, Dutch subjects responded differently to PfSPZ challenge compared to Tanzanian (malaria pre-exposed) subjects.
\nConclusion
\nSubstantial investment in research and development is needed to develop a highly efficacious malaria vaccine. To date, the recombinant subunit vaccines are yet to give the desired levels of protection for malaria elimination but seem to prevent malaria disease in high transmission settings. Large scale manufacturing, storage and distribution of live whole malaria sporozoite-based vaccines for mass administration need further development. So far, data generated from the PfSPZ vaccine trials conducted in the USA, Europe and in African research institutions imply that malaria naive individuals respond better to malaria vaccines than malaria pre-exposed individuals. The question remains to be, “what exactly constitutes the reason for lack of durable protection against malaria infection in endemic areas?” The most important factor in accelerating future vaccine development is a better understanding of the biology and nature of acquired immunity, which will lead to improved vaccine design. We have established the foundation for using CHMI to assess efficacy of new interventions against malaria and to study the mechanisms of the lack of protection conferred by different malaria vaccines in endemic settings. This study has opened new doors in the field of malaria intervention, whereby malaria vaccine and drug efficacy can be easily tested using CHMI in the target population.
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Liposomes containing monophosphoryl lipid A and QS-21 serve as an effective adjuvant for soluble circumsporozoite protein malaria vaccine FMP013

Orexin Antagonist Shows Promise for Insomnia

A Pilot Trial of Two Dose Levels of Rabbit Antithymocyte Globulin, (rATG), Thymoglobulin, as Part of a Myeloablative-Conditioning for a HLA Identical Matched Related Donor Stem Cell Transplant (SCT) with Cyclosporine (CsA) as Graft Versus Host Disease (GvHD) Prophylaxis.

Taking a Bite out of Malaria: Controlled Human Malaria Infection by Needle and Syringe

This study aimed to determine the effect of fish oil on bone mineral density (BMD). There were no differences in the 2-year BMD measures between high and low dose groups after adjusting for baseline BMD. This randomized controlled trial did not demonstrate any efficacy of omega-3 fatty acids on bone loss in adults. The purpose of this study is to investigate whether supplementation with high dose omega-3 fish oil could have an impact on BMD. In a multicentre, double-blind randomized controlled trial (RCT) (ACTRN 12607000415404), 202 Australian participants aged ≥40 with knee osteoarthritis (mean age, 61.0 ± 10.0years; 49% female) were randomized to receive either high dose (4.5g eicosapentaenoic acid and docosahexaenoic acid daily) or low dose (0.45g/day) omega-3 fish oil for 2years. BMD was assessed at baseline and 2years by dual energy X-ray absorptiometry. In subjects with baseline and 2-year assessments, mean standardized BMD at baseline for low or high dose group was 1198 ± 198 and 1157 ± 169mg/cm(2), respectively, for the lumbar spine and was 1035 ± 165 and 1017 ± 174mg/cm(2), respectively, for the femoral neck. There were no differences in the 2-year BMD measures between high and low dose groups after adjusting for baseline BMD in the complete case regression analyses (lumbar spine 3.7, 95% confidence interval (CI) -7.9 to 15.3mg/cm(2) and femoral neck -5.5, 95% CI -14.9 to 3.9mg/cm(2)). The findings did not change with additional adjustments of age, gender, study centre and uses of bone-related drugs during the study period as well as using the intention-to-treat analysis or limiting to older participants (≥55years at the baseline) (all P ≥ 0.25). Mild adverse events such as headache and gastrointestinal intolerance were common but did not occur more frequently in either group. There were no serious adverse events related to the intervention. A 2-year supplementation with high-dose omega-3 fish oil did not alter bone loss among men and women with knee osteoarthritis.

Effects of different Chinese herbal prescriptions on cytokines in autoimmune prostatitis rats

Objective To observe the effect of different doses of mivacurium chloride on muscle relaxation time-course and hemodynamics in children with different ages. Methods One hundred children of selective inguinal hernia repair under general anesthesia with endotracheal intubation from January 2016 to January 2017 were enrolled, and the age was 0.5 to 6.0 years. The children were divided into low age group (0.5 to 3.0 years) and high age group (3.1 to 6.0 years) according to the age, then the children were divided into low dose group (mivacurium chloride 0.20 mg/kg) and high dose group (mivacurium chloride 0.25 mg/kg) according to the doses of mivacurium chloride. Therefore, the children were divided into low age low dose group, low age high dose group, high age low dose group and high age high dose group with 25 cases each. The mean arterial pressure (MAP) and heart rates before anesthesia (T0) and 1 min (T1), 3 min (T2), 5 min (T3), 10 min (T4) after intravenous injection of mivacurium chloride were recorded. The times of first intravenous injection of mivacurium chloride to neuromuscular block 75% (ThD75), 90% (ThD90) and maximum (ThDmax) were recorded. The recovery index (RI) was recorded. The times of last intravenous injection of mivacurium chloride to onset of muscle convulsions (Th) and muscle convulsions recovery 10% (ThR10), 25% (ThR25), 75% (ThR75), 90% (ThR90) were recorded. The times of ratio of the fourth muscle twitch to the first muscle twitch (TOFR) recovery 75% (TOFR75) and 90% (TOFR90) were recorded. Results There were no statistical difference in MAP and heart rate among 4 groups (P>0.05). The ThD75, ThD90 and ThDmax in low age low dose group were (126 ± 40), (163 ± 59) and (192 ± 49) s, those in low age high dose group were (73 ± 15), (115 ± 41) and (142 ± 37) s, those in high age low dose group were (149 ± 38), (193 ± 44) and (221 ± 47) s, and those in high age high dose group were (105 ± 32), (138 ± 35) and (167 ± 44) s. The ThD75, ThD90 and ThDmax in low age high dose group were significantly shorter than those in low age low dose group, those in high age high dose group were significantly shorter than those in high age low dose group, those in high age low dose group were significantly longer than those in low age low dose group, those in high age high dose group were significantly longer than those in low age high dose group, and there were statistical differences (P 0.05). Conclusions In the children of 0.5 to 3.0 years, the effect of mivacurium chloride is significantly faster than that in the children of aged 3.1 to 6.0 years. Compared with 0.20 mg/kg of mivacurium chloride, 0.25 mg/kg of mivacurium chloride has less time to display muscle relaxation effect. The recovery time is not affected by age and induction dose. Mivacurium chloride has no significant effect on hemodynamics. Key words: Anesthesia; Child; Muscle relaxation; Hemodynamics; Mivacurium chloride

Yunchang Capsule in treatment of functional constipation: a randomized, double-blinded controlled, multicenter trial

BackgroundA DNA-prime/human adenovirus serotype 5 (HuAd5) boost vaccine encoding Plasmodium falciparum (Pf) circumsporozoite protein (PfCSP) and Pf apical membrane antigen-1 (PfAMA1), elicited protection in 4/15 (27%) of subjects against controlled human malaria infection (CHMI) that was statistically associated with CD8+ T cell responses. Subjects with high level pre-existing immunity to HuAd5 were not protected, suggesting an adverse effect on vaccine efficacy (VE). We replaced HuAd5 with chimpanzee adenovirus 63 (ChAd63), and repeated the study, assessing both the two-antigen (CSP, AMA1 = CA) vaccine, and a novel three-antigen (CSP, AMA1, ME-TRAP = CAT) vaccine that included a third pre-erythrocytic stage antigen [malaria multiple epitopes (ME) fused to the Pf thrombospondin-related adhesive protein (TRAP)] to potentially enhance protection.MethodologyThis was an open label, randomized Phase 1 trial, assessing safety, tolerability, and VE against CHMI in healthy, malaria naïve adults. Forty subjects (20 each group) were to receive three monthly CA or CAT DNA priming immunizations, followed by corresponding ChAd63 boost four months later. Four weeks after the boost, immunized subjects and 12 infectivity controls underwent CHMI by mosquito bite using the Pf3D7 strain. VE was assessed by determining the differences in time to parasitemia as detected by thick blood smears up to 28-days post CHMI and utilizing the log rank test, and by calculating the risk ratio of each treatment group and subtracting from 1, with significance calculated by the Cochran-Mantel-Haenszel method.ResultsIn both groups, systemic adverse events (AEs) were significantly higher after the ChAd63 boost than DNA immunizations. Eleven of 12 infectivity controls developed parasitemia (mean 11.7 days). In the CA group, 15 of 16 (93.8%) immunized subjects developed parasitemia (mean 12.0 days). In the CAT group, 11 of 16 (63.8%) immunized subjects developed parasitemia (mean 13.0 days), indicating significant protection by log rank test compared to infectivity controls (p = 0.0406) and the CA group (p = 0.0229). VE (1 minus the risk ratio) in the CAT group was 25% compared to -2% in the CA group. The CA and CAT vaccines induced robust humoral (ELISA antibodies against CSP, AMA1 and TRAP, and IFA responses against sporozoites and Pf3D7 blood stages), and cellular responses (IFN-γ FluoroSpot responses to CSP, AMA1 and TRAP) that were not associated with protection.ConclusionsThis study demonstrated that the ChAd63 CAT vaccine exhibited significant protective efficacy, and confirmed protection was afforded by adding a third antigen (T) to a two-antigen (CA) formulation to achieve increased VE. Although the ChAd63-CAT vaccine was associated with increased frequencies of systemic AEs compared to the CA vaccine and, historically, compared to the HuAd5 vectored malaria vaccine encoding CSP and AMA1, they were transient and associated with increased vector dosing.

Antibodies to Circumsporozoite protein (CSP) confer protection against controlled human malaria infection (CHMI) caused by the parasite Plasmodium falciparum. Although CSP is highly immunogenic, it does not induce long lasting protection and efforts to improve CSP-specific immunological memory and duration of protection are underway. We have previously reported that the clinical grade CSP vaccine FMP013 was immunogenic and protective against malaria challenge in mice when combined with the Army Liposomal Formulation adjuvant containing immune modulators 3D-PHAD™ and QS21 (ALFQ). To move forward with clinical evaluation, we now report the safety, toxicity and immunogenicity of clinical grade FMP013 and ALFQ in Rhesus macaques. Three groups of Rhesus (n = 6) received half or full human dose of FMP013 + ALFQ on a 0-1-2 month schedule, which showed mild local site reactions with no hematologic derangements in red blood cell homeostasis, liver function or kidney function. Immunization induced a transient systemic inflammatory response, including elevated white blood cell counts, mild fever, and a few incidences of elevated creatine kinase, receding to normal range by day 7 post vaccination. Optimal immunogenicity in Rhesus was observed using a 1 mL ALFQ + 20 µg FMP013 dose. Doubling the FMP013 antigen dose to 40 µg had no effect while halving the ALFQ adjuvant dose to 0.5 mL lowered immunogenicity. Similar to data generated in mice, FMP013 + ALFQ induced serum antibodies that reacted to all regions of the CSP molecule and a Th1-biased cytokine response in Rhesus. Rhesus antibody response to FMP013 + ALFQ was found to be non-inferior to historical benchmarks including that of RTS,S + AS01 in humans. A four-dose GLP toxicity study in rabbits confirmed no local site reactions and transient systemic inflammation associated with ALFQ adjuvant administration. These safety and immunogenicity data support the clinical progression and testing of FMP013 + ALFQ in a CHMI trial in the near future.

Safety and immunogenicity of an AS03-adjuvanted SARS-CoV-2 recombinant protein vaccine (CoV2 preS dTM) in healthy adults: interim findings from a phase 2, randomised, dose-finding, multicentre study

Safety, tolerability, pharmacokinetics, and clinical outcomes following treatment of painful knee osteoarthritis with senolytic molecule UBX0101

A randomized study was designed in dogs with spontaneous soft tissue sarcomas to gain information about the relationship between hyperthermia dose and outcome. The study compared two levels of thermal dose applied to dogs with heatable tumours, so it was necessary to deliver either a low (2-5 CEM 43°C T90) or high (20-50 CEM 43°C T90) thermal dose as precisely as possible. It was also desirable to have similar numbers of hyperthermia treatments in each thermal dose group. Identification of heatable tumours and randomization to high or low heat dose group was done during the first hyperthermia treatment. This was readily accomplished using mapping of temperatures in thermometry catheters, manual recording of thermal data, and visual inspection of raw thermal data with subsequent adjustment of the duration of the hyperthermia treatment. An analysis of precision of thermal dose delivery was conducted after approximately 50% of projected accrual had been met in a randomized phase III assessment of thermal dose effect. Fifty-four dogs were eligible for randomization; in 48 dogs the tumour was deemed heatable according to predetermined temperature criteria applied during the first heat treatment. Twenty-four dogs were randomized to the high heat dose group, and 24 to the low heat dose group. Median (range) total thermal dose for dogs in the high dose group was 43.5 CEM 43°C T90 (16.4-66.6) compared to 3.2 CEM 43°C T90 (2.1-4.6) for dogs in the low dose group. There was no overlap of thermal doses between groups. Thus, thermal dose could be delivered accurately, being within the predetermined range in 47 of the 48 dogs. Thermal dose quantified as CEM 43°C T50, however, did overlap between groups and the clinical significance of this finding will not be known until outcome data are analysed. Most dogs in both groups received five hyperthermia treatments. Median (range) treatment duration for dogs in the high dose group was 300min (147-692) compared to 111min (51-381) for dogs in the low dose group. Relatively simple but accurate methods of delivering prescribed thermal dose as described herein will aid the translation of clinical hyperthermia from the research setting into more general practice once the characteristics of the relationship between hyperthermia dose and outcome are understood.