Treatment of urinary schistosomiasis by chemotherapy remains challenging due to rapid re-infection and possibly to limited susceptibility to praziquantel treatment. Therefore, therapeutic vaccines represent an attractive alternative control strategy. The objectives of this study were to assess the safety and tolerability profile of the recombinant 28 kDa glutathione S-transferase of Schistosoma haematobium (rSh28GST) in healthy volunteers, and to determine its immunogenicity. Volunteers randomly received 100 µg rSh28GST together with aluminium hydroxide (Alum) as adjuvant (n = 8), or Alum alone as a comparator (n = 8), twice with a 28-day interval between doses. A third dose of rSh28GST or Alum alone was administered to this group at day 150. In view of the results obtained, another group of healthy volunteers (n = 8) received two doses of 300 µg of rSh28GST, again with a 28-day interval. A six-month follow-up was performed with both clinical and biological evaluations. Immunogenicity of the vaccine candidate was evaluated in terms of specific antibody production, the capacity of sera to inhibit enzymatic activity of the antigen, and in vitro cytokine production. Among the 24 healthy male participants no serious adverse events were reported in the days or weeks after administration. Four subjects under rSh28GST reported mild reactions at the injection site while a clinically insignificant increase in bilirubin was observed in 8/24 subjects. No hematological nor biochemical evidence of toxicity was detected. Immunological analysis showed that rSh28GST was immunogenic. The induced Th2-type response was characterized by antibodies capable of inhibiting the enzymatic activity of rSh28GST. rSh28GST in Alum did not induce any significant toxicity in healthy adults and generated a Th2-type immune response. Together with previous preclinical results, the data of safety, tolerability and quality of the specific immune response provide evidence that clinical trials with rSh28GST could be continued in humans as a potential vaccine candidate against urinary schistosomiasis.
Schistosomiasis, the second major human parasitic infection after malaria, remains a major health problem in many developing countries, mainly among children and it is estimated that this chronic disease is responsible for 300 000 deaths per year. During Schistosoma haematobium infection, morbidity and mortality are associated with worm fecundity and the deposition of schistosome eggs in tissues, especially in the genital and urinary tracts. More than 20 years after its introduction, the most effective intervention for the control of schistosomiasis remains the use of chemotherapy by praziquantel (PZQ) [1] but, it is generally agreed that this shows numerous limits. Indeed, rapid re-infection following treatment is commonly observed in most endemic areas [2]. Thus, efficient drug delivery requires a substantial infrastructure to regularly cover endemic areas, which makes chemotherapy an expensive approach [3]. In addition, although there is not yet clear evidence of the existence of PZQ-resistant strains, a decreased susceptibility to the drug has been suspected in several countries [4], [5]. The lack of efficient treatment emphasizes the need for more specific and long-term approaches against schistosomiasis. A vaccine strategy may therefore play a crucial role in the control of this parasitic disease. Among several vaccine candidates [6], the 28 kDa glutathione S-transferase antigen (28ShGST) has been well characterized (from molecular cloning to crystallisation) [7], [8]. Immunization of rodents [9], [10], monkeys [11], [12] or cattle [13], [14] with 28GST, followed by experimental infection led to a reduction in worm burden and/or a significant decrease in parasite fecundity, establishing this antigen as a promising vaccine candidate [15], [16]. In addition, field studies have shown that resistance to re-infection in humans could be associated with the presence of acquired anti-28GST antibodies able to inhibit the parasite GST enzymatic activity [17][18]. Moreover, studies have demonstrated the role of Th2-type responses in anti-schistosome protective immunity in human infection [19]. Toxicological studies were conducted in animal models to support the clinical use of the recombinant Sh28GST (CERB, France and Pasteur Institute of Lille, France; unpublished results). In compliance with Good Laboratory Practice standards, a rabbit model was selected because it produces immune responses to the protein, and the full dose intended for human use can be subcutaneously administered to rabbits. In these conditions, a 4-week study was performed where each rabbit was tested with 9 injections distributed over the lumbar area (2 with aluminium hydroxide (Alum), 2 at a 100-µg dose of rSh28GST in Alum, 2 at a 300-µg dose, 2 at a 1000-µg dose and a final mock injection). A transient local inflammatory reaction after injection was common (erythema, swelling or small induration at the site of injection) and comparable to that seen with Alum alone. In another toxicity study using 3 subcutaneous injections in rats (40-µg dose of rSh28GST in Alum, a 200-µg dose in Alum or Alum alone), animals were monitored for morbidity, mortality, general health and signs of toxicity, food consumption, body weight, clinical observations and also for respiratory, circulatory, dermatological, autonomic and central nervous system parameters. Hematology, blood chemistry, and immunological analyses were also performed. Major tissues and organs were histologically examined on necropsy. In these conditions, no significant clinical, anatomical or biological alterations were found. Finally, a lack of significant alterations was also demonstrated in toxicity studies performed in dogs. All these findings taken together justify the decision to initiate a phase 1 clinical trial to evaluate the safety and tolerability of the rSh28GST in healthy adult male volunteers. The secondary objective of this randomized phase 1 study was to assess the immunogenicity of rSh28GST.
According to requirements of the French Health authorities regarding first-in-human clinical trials that should avoid the inclusion of children and women with childbearing potential except under specific conditions, the present phase 1 study was conducted in young healthy Caucasian male adults. All subjects had to meet the study inclusion criteria within 21 days prior to treatment, which included written informed consent, age from 18 to 30, absence of any medical history of schistosomiasis, biological parameters (haematological, biochemical, renal and hepatic) within the normal range as well as a normal clinical examination. Moreover, subjects were not smokers and were exempt of any inflammatory or immunological pathology such as atopic diseases, evidence of inflammation or acute infection (including positive serology to viral hepatitis B and C or HIV), any immunological deficiency, any clinically relevant alcohol or drug use (cannabis, opiates, cocaine, amphetamines, benzodiazepines, nicotine, barbiturates, meprobamates or antidepressant drugs according to a urinary drug and metabolites screen) or any other medication use within 2 weeks before the study, any vaccination within the last 6 months and had no antibodies against the Sh28GST protein. This phase 1 randomized controlled study was designed to investigate the safety and tolerability of rSh28GST antigen injected subcutaneously (deltoid muscle) with Alum as adjuvant. In a first step, the study was performed on healthy male volunteers according to a double-blind design. Subjects selected at random received either two 100 µg doses of rSh28GST together with Alum as vaccine adjuvant (n = 8) or two doses of Alum in a 0. 5 ml solution as a comparator (n = 8) with a 28-day interval (Day 0 [D0] and Day 28 [D28]) between doses in both groups (figure 1). These subjects also received a third administration of 100 µg Sh28GST or Alum alone on day 150 [D150]. As a dose escalation trial, a third group of healthy volunteers received two 300 µg doses of rSh28GST at a 28-day interval according to an open-label design (figure 1). All biological analyses were carried out blind to treatment conditions. Batches of rSh28GST were produced and purified from recombinant Saccharomyces cerevisiae culture (TGY73. 4 - pTG8889 strain) under Good Manufacturing Practice (GMP) conditions by Eurogentec S. A. (Belgium). The rSh28GST clinical batch (# B98H11) was conserved lyophilized (124 µg per vial for the administrated dose of 100 µg; 352 µg per vial for the administered dose of 300 µg) by Sterilyo (France) under GMP conditions. The lyophilized preparation was re-suspended extemporaneously using 0. 6 ml of apyrogenic and sterile aluminium hydroxide solution 0,2% (Al2O3 0. 2%; Al (OH) 3 3%; NaCl 9 g/L; ammonium carbonate buffer 10 mM, pH7. 8) (Alum from Superfos, Denmark; batch #14093) and administered in a volume of 0. 5 ml. Following each administration, participants were kept under constant observation during 4 hours and further evaluated at D1, D21, D28, D29, D49, D120, D150, D165 and D180. Clinical evaluations consisted of measurement of vital signs and assessment of the local injection site and general signs or symptoms. Local signs and symptoms included pain or tenderness, swelling, induration and erythema at the site of injection. Assessment of general tolerability was obtained through a complete physical examination including an examination of general appearance, body weight and rectal temperature, head, eyes, ears, nose and throat, neck, skin, cardiovascular and respiratory system, abdominal system, nervous system, lymphatic area, blood pressure, pulse and respiratory rates. Recordings of 12-lead ECG were also obtained allowing analysis of ventricular rate, rhythm, PR interval, QRS duration and QT/QTc. A number of general signs and symptoms were specially considered including fever (rectal temperature ≥38°C), headache, nausea, vomiting, myalgia, arthralgia, irritability/fussiness and drowsiness, loss of appetite and sleep disturbances. In addition to these predefined signs and symptoms, all adverse events were reported throughout the study. Adverse events were graded by severity following FDA guidance [22] and judged for potential association with vaccination. Briefly, grade 1 adverse events were easily tolerated, causing minimal discomfort and not interfering with daily activities; grade 2 adverse events were sufficiently discomforting to interfere with normal activities, grade 3 adverse events prevented normal daily activities while grade 4 were potentially life threatening [22]. Biological tolerability was assessed at baseline and at D1, D21, D28, D29, D49, D120, D150, D165 and D180 using clinical laboratory tests including haemoglobin, haematocrit, packed cell volume, mean corpuscular volume, mean cell haemoglobin, mean cell haemoglobin concentration, white blood cells and platelet counts; coagulation tests such as activated partial thromboplastin time (PTT), prothrombin time and International Normalized Ratio (INR); serum biochemistry such as sodium, potassium, creatinine, urea, alanine amino transaminase (ALT), aspartate amino transaminase (AST), gamma glutamine transferase (gamma-GT), bilirubin, uric acid, total protein, alkaline phosphatases, calcium, C-reactive Protein (CRP) and fasting glucose; urinalysis including pH, protein, glucose, ketones, urobilirubin, blood and leucocytes. For all these laboratory tests, toxicity grading was assigned using local laboratory values or previously published normal reference ranges [23]. Specific antibody titers in individual sera were determined by ELISA. rSh28GST (10 µg/ml) was coated on 96-well plates (Nunc, Roskilde, Denmark) 2h30 at 37°C. Plates were blocked with phosphate-buffered saline containing 0. 5% gelatin (Merck, Darmstadt, Germany). Then, six serial dilutions of individual sera (first dilution for IgG1 1/100; IgG2, IgG3 and IgA 1/20; IgG4 and IgE 1/10) were added and incubated. Each corresponding biotinylated mAbs to human Ig isotype (Southern Biotechnology, Birmingham, AL, USA) was incubated (2 h at 37°C) at a 1/1000 dilution for IgG, IgG1, IgG2, IgG3, IgG4, IgE and IgA, and 1/1500 for IgG1 detection. Peroxidase-conjugated streptavidin (1/1500; 30 min at 37°C) was then added (Amersham, Les Ulis, France). Colorimetric development was performed with ABTS (Sigma, St. Louis, MO, USA) in 50 mM citrate buffer (pH = 4) and absorbance (OD) was measured at 405 nm. Titers were defined as the highest dilution yielding an absorbance value three times above the background (wells containing buffer instead of serum in the same plate). Vaccinated individuals were considered as positive responders when their titer was greater than three fold the standard deviation above the mean titer value of all individuals at D0 (vaccine and comparator group). Cytokine production was evaluated in whole blood diluted in RPMI 1640 medium (Gibco, Courbevoie, France) to obtain 2×106 mononuclear cells per ml. The blood preparation (1 ml) was then plated in duplicate culture in 24 flat-bottomed plates (Nunc) and stimulated with 1 ml of rSh28GST antigen (20 µg/ml). Supernatants were harvested on day 3 for analysis of cytokine production. Quantitative ELISA for human IL-2, IL-4, IL-5, IL-10, IL-12, IL-13 and IFN-γ detection were performed in supernatants using EASIA Kits (Immunotech, Marseille, France) and human TGF-β1 by ELISA System Kit (Promega, Madison, WI, USA). Cytokine concentrations were expressed as pg/ml or IU/ml (for IFN-gamma) after subtracting the amount detected in unstimulated control cultures. Cytokine production was considered stimulated when the value was more than three times the standard deviation above the mean value. To evaluate the capacity of induced antibodies to inhibit GST activity, 20 µl of rSh28GST solution (4 µg/ml in 50 mM potassium phosphate at pH 6. 5) was incubated with 20 µl of human serum for 1 h at 37°C in Immulon 3 Plates (Nunc, Roskilde, Denmark). The enzymatic reaction was carried out using 1-chloro-2,4, dinitrobenzene (Sigma, St. Louis, MO) substrate as described elsewhere [18]. Enzymatic reaction intensity was measured by OD at 340 nm. Every enzyme-inhibition test was performed with appropriate controls (enzyme without serum and tested serum alone). The percentage of inhibition was calculated by the ratio of GST activity after serum incubation to the GST activity control. Percentage inhibition above 10% was considered significant. The sample size for this first-in-human study was not powered on the basis of statistical hypothesis testing since as is customary for phase 1 studies, hypothesis testing is not part of the primary objective. The target number of subjects was expected to minimize the risk by not exposing too large a population to treatment, while providing an ample size to gain information on safety and tolerability. The sample size of 8 subjects for each group (both rSh28GST and control) ensured at least an 85% probability of observing ≥1 subject with a specific adverse event if the background incidence rate of that adverse event was ≥10%. This sample size also ensured a ≥80% probability of observing common placebo-associated adverse events, defined as those events with a population incidence ≥15%. Descriptive statistics are presented using mean and standard deviation or median and range as required. Baseline laboratory values were defined as the pre-treatment values obtained during the baseline screening visit. Statistical analysis for the immunological studies was performed using the Wilcoxon signed rank test to assess the difference in immunological parameters between days using SPSS v15 for Windows. Differences were considered significant at p<0. 05.
A total of 31 healthy male volunteers underwent initial evaluation for the study. Among these, 7 were eliminated using exclusion criteria such as allergy or hypersensitivity, tobacco use or elevated immunoglobulins. Finally, 24 volunteers were randomized. They were 18 to 29 years old (23±3 years), height from 170 to 191 cm (178±6 cm) and weight from 52 to 82 kg (71±8 kg). All other clinical and routine laboratory data were considered in normal ranges although 1 subject in the comparator group and 3 subjects in the 300 µg group had a slight increase in bilirubin according to local laboratory values (main baseline biological data in table 1). All volunteers were followed until the end of the study. All local, general and biological adverse events reported in the present study are detailed in table 2.
This phase 1 study demonstrates the safety, tolerability and immunogenicity of rSh28GST in Alum as a first vaccine candidate against schistosomiasis. These results allowed us to conduct further clinical trials in endemic regions (manuscripts in preparation). The objectives of these studies were again to assess the safety, tolerability and immunogenic profile of the recombinant rSh28GST in Alum in healthy school children, and also in adults and children infected with S. haematobium. A clinical trial evaluating the efficacy of the vaccine candidate is currently in progress in a large cohort of infected schoolchildren.
Therapeutic vaccines represent an attractive tool in the fight against schistosomiasis. Pre-clinical immunization studies with the schistosome enzyme 28 kDa glutathione S-transferase (28GST) has been shown to significantly reduce schistosome egg production and subsequent pathology. The objective of this study was to assess the safety and immunogenicity of the recombinant 28GST of Schistosoma haematobium (rSh28GST) in healthy adult volunteers. After three administrations of 100 µg or two of 300 µg, no serious adverse events were reported in the days or weeks after each administration. Some mild adverse events were noted, including minor reactions at the injection site reported for four subjects receiving rSh28GST, but there was no hematological or biochemical evidence of toxicity. Immunological analysis showed that rSh28GST induced a consistent immune response characterized by antibodies endowed with the capacity to inhibit 28GST enzymatic activity. Present data provide evidence that clinical trials with rSh28GST could be continued in humans as a potential vaccine candidate against urinary schistosomiasis.
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Safety and Immunogenicity of rSh28GST Antigen in Humans: Phase 1 Randomized Clinical Study of a Vaccine Candidate against Urinary Schistosomiasis