The protocol for this trial and supporting CONSORT checklist are available as supporting information; see Checklist S1
and Protocol S1
Participants were healthy adults age 18–50 enrolled at the Center for Immunization Research in Washington, DC. Exclusion criteria included prior malaria infection, recent or planned travel to a malaria-endemic area, recent use of malaria prophylaxis, and pre-existing autoimmune disease. Participants were required to be in good general health, without known significant medical conditions or significant medical history, and were required to have normal results for screening laboratories: complete blood count, alanine aminotransferase (ALT), and creatinine; no serologic evidence of hepatitis B, hepatitis C, or human immunodeficiency virus infection; and negative anti-double stranded DNA (dsDNA) as a marker for autoimmune disease. Serum pregnancy testing was performed at screening and urine pregnancy testing was performed prior to each vaccination for females.
The study protocol and consent documents were reviewed and approved by the Institutional Review Board (IRB) of the National Institute of Allergy and Infectious Diseases (NIAID) and the Western IRB. The study protocol was also reviewed and approved by the FMPOS Ethics Committee in Bamako, Mali, since the protocol combined Phase 1 trials; one in malaria-naïve adults in the U.S. and the other in malaria-exposed adults in Mali, the results of which are being published separately. Individual written informed consent was obtained from all participants prior to any study procedures.
BSAM2 is a combination of the FVO and 3D7 alleles of AMA1 and MSP142
, with equal amounts by weight of each of the four recombinant proteins mixed, bound to Alhydrogel®, and administered by mixing with CPG 7909 at the time of injection. The AMA1 and MSP142
combination vaccines (AMA1-C1/Alhydrogel®+/−CPG 7909 and MSP142
-C1/Alhydrogel®+/−CPG 7909) have been previously evaluated in Phase 1 trials, and production and characterization of the recombinant proteins and the formulated vaccines have also been previously described 
. The AMA1 protein used in this study was made using a modified procedure which will be reported elsewhere. In brief, the purification procedure as described in 
, was modified to increase the quality and scalability of the recombinant AMA-1 drug substances. The order and type of column chromatography procedures were revised to include capture by Ni-Sepharose-FF (GE Healthcare), purification using a Butyl-Sepharose 4 Fast Flow (GE Healthcare) hydrophobic interaction column and Q-Sepharose-HP (GE Healthcare) anion exchange column, followed by size exclusion chromatography (Superdex 75 (GE Healthcare)), sterile filtration using a 0.22 µm filter prior to bulk vialing and storage at <−70°C. All column chromatography procedures and solutions were in accordance with cGMP. Final GMP manufacture and fill of the vialed CPG 7909 and BSAM2/Alhydrogel® was conducted at the Biopharmaceutical Development Program, National Cancer Institute, Science Applications International Corporation, Frederick, Maryland.
A GLP toxicology study using a higher dose product (BSAM1/Alhydrogel® +/− CPG 7909, containing 160 µg MSP142 and 80 µg AMA1 for a total of 240 µg antigenic proteins) was conducted in rabbits prior to study initiation (CRL Preclinical Services, Horsham, Pennsylvania), with no safety issues identified, and with induction of antibodies which inhibited growth in an in vitro parasite growth inhibition assay (GIA). BSAM2/Alhydrogel® and CPG 7909 were also separately evaluated in rabbit pyrogenicity tests using a cGLP compliant procedure, and none of the vaccine components were pyrogenic. Since the CPG 7909 used in the current study was manufactured in a new production facility (Avecia Biotechnology Inc., Milford Massachusetts), potency of the new CPG 7909 (without antigens) in comparison with the lot used in previous clinical trials was confirmed in a mouse study.
The components of the vaccine, BSAM-2/Alhydrogel® (low and high dose) and CPG 7909, were supplied separately in single-use vials to the study site pharmacist where a point of injection formulation was prepared. Vaccine components were stored at 2–8°C. Shortly before vaccination, 0.7 mL of BSAM-2/Alhydrogel® was withdrawn and added to a single dose vial containing 0.08 mL CPG 7909. The calculated total dose of antigen in the 0.55 mL injected volume was 39.5 µg (low dose) and 158.0 µg (high dose) of protein, shown as 40 µg or 160 µg for simplicity, with 564 µg of CPG 7909 administered with both the high and low doses. The stability of BSAM2/Alhydrogel® and CPG 7909 was assessed annually during the course of the study, and potency of BSAM2/Alhydrogel® in mice was assessed every six months. All components remained stable and potent during the course of the clinical trial.
Vaccines were given by intramuscular injection into the deltoid muscle in alternating arms when possible at study Days 0, 56, and 180. An independent Safety Monitoring Committee (SMC) reviewed adverse event data at scheduled safety reviews and as needed as the study progressed.
The primary objective of this study was to assess safety and reactogenicity of BSAM2/Alhydrogel®+CPG 7909 in malaria-naïve US adults. The secondary objective was to determine the antibody response of the combination vaccine to the AMA1 and MSP142 proteins, as measured by antibody levels and parasite growth inhibition assay. Exploratory objectives included determination of the extent to which the antibody responses to the individual antigens (AMA1 and MSP142) were correlated with each other.
Volunteers were observed for 30 minutes after each vaccination to evaluate immediate adverse events and were given diary cards to record events occurring during the first week after each vaccination. The diary cards were used as a memory prompt and were reviewed with volunteers at follow up visits, when adverse events were recorded. Participants were seen at 3, 7, 14, and 28 days after each vaccination, and then approximately monthly for a total of 12 months (to study Day 360), with an additional follow up phone call scheduled at study Day 720. All adverse events were graded for severity and relationship to study product. Solicited injection site adverse events were pain, erythema, and induration. Solicited systemic adverse events were fever, headache, nausea, myalgia, arthralgia, and rash. Fever was graded as 1
100.4°F–101.5°F (38.0°C–38.6°C), 2
101.6°F–102.4°F (38.7°C–39.1°C), 3
≥102.5°F (≥39.2°C). Pain and other solicited adverse events were graded as follows: 0
easily tolerated, 2
interferes with daily activity or treatment given, 3
prevents daily activity. Unless otherwise specified, non-solicited adverse events were graded as 0
no effect on activities of daily living and no treatment given, 2
partial limitation in activities of daily living or treatment given, 3
activities of daily living limited to <50% of baseline or medical evaluation required. Injection site erythema, swelling, and induration were graded based on the maximum diameter as follows: mild
>0 to 20 mm, moderate
21–50 mm, and severe
>50 mm. Hematological (hemoglobin, white blood cell counts, and platelets) and biochemical (ALT and creatinine) laboratory parameters were measured at screening, on days of immunization, and 3 and 14 days after each vaccination; hematological parameters were also checked 7 days after each vaccination. Anti-dsDNA was checked as a marker for autoimmunity at screening, 14 days after the second and third vaccination, and at Day 360. Serious adverse events (SAEs) followed the FDA Code of Federal Regulation, and were defined as any adverse event that resulted in death; was life-threatening; required hospitalization; resulted in disability, incapacity, congenital anomaly, or birth defect; or any other event that required intervention to prevent such outcomes.
The standardized methodology for performing the enzyme-linked immunosorbent assay (ELISA) and the growth inhibition assay (GIA) have been described previously 
. For both AMA1 and MSP142
, FVO and 3D7 allele specific IgG antibody levels were assessed by ELISA at baseline for each vaccination, two weeks after each vaccination, and at Days 270 and 360. The minimum detection level of the assay was 33 ELISA units; for all antigens results less than these values were assigned a value of 17 ELISA units for statistical analysis. ELISA results were converted to ng/mL by multiplying the ELISA units by protein and parasite specific conversion factors (AMA1-FVO
24.39). GIA was performed using purified IgG from serum collected two weeks post-second vaccination to assess biologic activity of the induced antibody against FVO and 3D7 parasites. In this assay, purified antibody was added to the parasite cultures at approximately the same concentration as present in the corresponding serum sample (10 mg/mL in GIA well).
The study was powered to provide sufficient safety data before proceeding to a clinical trial in malaria-exposed adults. A sample size of 12 in each dose group gave a probability of 0.80 for detecting 1 or more adverse events that occurred with a frequency of 0.125 per participant. Fifteen volunteers per dose group were included in case of withdrawals or loss to follow-up.
Participants were sequentially enrolled in the two dose escalating groups. Within each group enrollment was staggered for safety purposes. Five volunteers were planned to be vaccinated at each dose level followed by vaccination of the remaining 10 volunteers in the group a minimum of 2 weeks later. Enrollment was further staggered after the occurrence of severe related adverse events, as described in the Results
This was an open label dose-escalating study, with both participants and those conducting clinical and laboratory assessments aware of the interventions received.
The safety analysis was descriptive, with the frequency of local and systemic adverse events presented by dose group (40 µg low dose, and 160 µg high dose), and by vaccination (first, second, third). Data from all participants who received one or more vaccination are shown. For the immunological analysis only subjects who received all 3 vaccines and did not meet criteria for exclusion (i.e., received H1N1 vaccine close to the time of study vaccine administration) were used. Antibody results were analyzed as follows: concordance for allelic responses (FVO and 3D7) to the AMA1 and MSP142
antigens was calculated using the random marginal agreement coefficient with the squared error loss function 
. FVO and 3D7 responses at each time point were averaged, since responses for each allele were highly concordant, consistent with previous studies 
. Average FVO and 3D7 responses were plotted over time. Confidence intervals on geometric means used the t-distribution on log transformed responses. To test for a dose response (low versus high) a Wilcoxon Mann Whitney (WMW) test with Hodges-Lehmann confidence intervals was used to compare log transformed AMA1 and MSP142
average antibody levels. To test for a further increase in antibody after third vaccination (Day 70 versus Day 194) a Wilcoxon signed rank test was used. GIA results were plotted against ELISA antibody levels in the GIA well for the homologous AMA1 and MSP142
antigens. The correlation between antibody level and growth-inhibitory activity was tested by a Spearman rank test with confidence intervals calculated using Fisher's Z transformation on the correlation of the ranks. Calculations were done using R 2.14.2.