MSP1 chimeric recombinant protein vaccine for Plasmodium falciparum malaria

Vaccine 26 (2008) 6864–6873

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A Phase 1 trial of PfCP2.9: An AMA1/MSP1 chimeric recombinant protein vaccine for Plasmodium falciparum malaria Elissa Malkin c,∗ , Jinhong Hu a , Zhen Li a , Zhihui Chen a , Xinling Bi a , Zarifah Reed d , Filip Dubovsky e , Jian Liu f , Qiang Wang f , Xuegong Pan f , Tom Chen g , Birgitte Giersing c , Yu Xu a , Xin Kang a , Jun Gu a , Qian Shen a , Kathryn Tucker h , Eveline Tierney c , Weiqing Pan b , Carole Long i , Zhifang Cao f a

Second Military Medical University, National Medicine Clinical Trial Institution, Shanghai Changhai Hospital, Shanghai, China Second Military Medical University, Department of Pathogenic Biology, Shanghai, China c PATH Malaria Vaccine Initiative, Bethesda, MD, USA d World Health Organization, Initiative for Vaccine Research, Special Programme for Research and Training in Tropical Diseases, Geneva-27, Switzerland e MedImmune, Gaithersburg, MD, USA f Shanghai Wanxing Bio-Pharmaceuticals, Shanghai, China g ProMetic BioTherapeutics, Inc., Rockville, MD, USA h Statistics Collaborative, Inc., Washington DC, USA i Laboratory of Malaria & Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD, USA b

a r t i c l e

i n f o

Article history: Received 28 May 2008 Received in revised form 18 September 2008 Accepted 26 September 2008 Available online 16 October 2008 Keywords: Malaria vaccine Clinical trial Malaria

a b s t r a c t Apical Membrane Antigen 1 (AMA1) and Merozoite Surface Protein 1 (MSP1) were produced as a recombinant fusion protein and formulated with the adjuvant Montanide ISA 720 with the aim of replicating the structure present in the parasite protein. A previous trial with this construct demonstrated the vaccine was safe and immunogenic but was associated with injection site reactogenicity. This Phase 1a dose-escalating, double blind, randomized, controlled trial of PfCP2.9/Montanide ISA 720 was conducted to evaluate alternative dose levels and vaccination schedules, with a pre-formulated vaccine that had undergone more in-depth and frequent quality control and stability analysis. The trial was conducted in seventy healthy Chinese malaria-naïve volunteers between January 2006 and January 2007. The objective was to assess the safety, reactogenicity and immunogenicity of 5, 20 and 50 ␮g of PfCP2.9/ISA 720 under 2 different schedules. The most common adverse event was injection site tenderness (53%). The frequency and severity of adverse events was similar in both vaccination schedules. Antibody responses were induced and remained elevated throughout the study in volunteers receiving vaccine (p < 0.001). Although high antibody titers as measured by ELISA to the PfCP2.9 immunogen were observed, biological function of these antibodies was not reflected by the in vitro inhibition of parasite growth, and there was limited recognition of fixed parasites in an immunofluorescence assay. At all three dose levels and both schedules, this formulation of PfCP2.9/ISA 720 is well tolerated, safe and immunogenic; however no functional activity against the parasite was observed. © 2008 Elsevier Ltd. All rights reserved.

1. Introduction Plasmodium falciparum parasites caused an estimated 311 million cases of malaria in 2004 [1], resulting in over one million deaths [2]. Sub-Saharan Africa suffers the greatest mortality due to P. falciparum as more than 80% of these deaths occur in this region [3]. Further, the World Health Organization observed that malaria’s

∗ Corresponding author at: PATH Malaria Vaccine Initiative, 7500 Old Georgetown Road, 12th floor, Bethesda, MD 20814, USA Tel.: +1 240 395 2700; fax: +1 240 395 2591. E-mail address: [email protected] (E. Malkin). 0264-410X/$ – see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2008.09.081

burden of disease increased in Africa during the 1980s and 1990s [3]. Upon departure from the liver, the P. falciparum parasite begins a cycle of erythrocyte invasion, replication and cell lysis, which results in the signs and symptoms of clinical malaria. Inhabitants of endemic areas develop immunity to P. falciparum due to repeated exposure [4,5], which is thought to be partly facilitated by parasitespecific antibodies directed towards blood stage antigens [6,7]. The introduction of a blood stage vaccine that could impact this cycle of infection might reduce P. falciparum morbidity and mortality in infants and young children who experience the greatest burden of disease. PfCP2.9, a recombinant fusion protein expressed from the yeast Pichia pastoris, consists of domain III of Apical Membrane Antigen

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1 [AMA1 (III)] and the 19 kDa portion of the Merozoite Surface Protein 1 (MSP119 ) from the 3D7 and K1/FVO P. falciparum strains, respectively. The portions of AMA1 and MSP1 are fused via a hinge region encoding a Gly-Pro-Gly-Pro repeat [8]. AMA1 and MSP1 are promising malaria vaccine antigens because they are believed to be essential for merozoite invasion of erythrocytes. While MSP1 is found on the merozoite surface, AMA1 is localized to the apical organelles and is discharged onto the merozoite surface during or just prior to invasion of erythrocytes [9]. Data in rodent and nonhuman primate model systems have provided supporting evidence that both of these molecules produced as recombinant proteins can elicit protective responses to parasite infection [10–15]. The construction of a chimera derived from these two prominent blood stage antigens was attractive for development because it potentially combines putative functional components of each. Both domain III of AMA1 and the 19 kDa C-terminus of MSP1 have been shown to be targets of parasite inhibitory antibodies or associated with protection from clinical disease in sera isolated from individuals living in malaria-endemic regions [16–18]. The fusion of the AMA1 (III) and MSP119 components was found to enhance both the yield of the product and the immunogenicity of the individual components [8]. Development of several candidate vaccines composed of or derived from individual recombinant MSP1 and AMA1 proteins has been underway over the past several years [9,19–24]. The PfCP2.9/ISA 720 vaccine candidate was evaluated previously in a Phase 1a trial between August 2003 and October 2004 [25]. The trial showed the vaccine to be generally well tolerated and immunogenic. However, 9 of 52 volunteers experienced mild to moderate induration at the injection site occurring 2 to 4 weeks after vaccination. This delayed reactogenicity was not observed in the lowest antigen dose group or in placebo controls. The immunogenicity response profiles were similar for the four escalating doses (20, 50, 100 and 200 ␮g), with no significant dose–response relationship. Based on the reactogenicity and lack of dose response, the formulation conditions for PfCP2.9 antigen and this waterin-oil (w/o) adjuvant were re-evaluated. In addition, more robust integrity, potency and stability assays were developed and implemented to ensure the quality of the vaccine formulations at the time of vaccination. This current trial was designed to evaluate whether the safety and immunogenicity of the PfCP2.9/ISA 720 candidate vaccine could be improved by assessing lower dosage levels (5, 20 and 50 ␮g) given at two different immunization schedules (0, 2, 6 months vs. 0, 3, 6 months), using a vaccine formulation with an established potency and stability profile. Two different vaccination schedules were tested to assess whether a delay of the second vaccination would increase the magnitude and longevity of the immune response at lower doses, in addition to enhancing the safety profile.

2. Materials and methods 2.1. Participants The trial was conducted at the National Medicine Clinical Trial Institution within the Shanghai Changhai Hospital. Seventy healthy volunteers, 18–45 years of age, were recruited from the Shanghai area in China. Volunteers were excluded if they had evidence of clinically significant systemic disease; were pregnant or breast feeding; had serologic evidence of chronic hepatitis B or C infection or HIV; were receiving corticosteroids or immunosuppressive drugs; had a history of malaria or positive markers for antibodies to P. falciparum cultured parasites by IFA and/or ELISA; or had a history of residing in a malaria endemic region or malaria exposure (travel) within the last two years.

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2.2. Ethics This study was performed under the approval of the State Food and Drug Administration, China (SFDA), and adhered to Good Clinical Practice guidelines of the SFDA, ICH and World Health Organization (WHO). The protocol, amendments to the protocol, informed consent form, advertisements and other study-related documents were reviewed and approved by the Changhai Hospital Independent Ethics Committee, the WHO Ethical Committee and the Western Institutional Review Board (the IRB for the PATH Malaria Vaccine Initiative). Volunteers were recruited and consented using a protocol presentation and consent form approved by the 3 participating IRBs. Written informed consent was obtained before enrollment. 2.3. Vaccine The construction, fermentation and purification of Pichia pastoris produced PfCP2.9 bulk antigen, and the emulsification with Montanide ISA 720 adjuvant for this clinical trial were all performed at Shanghai Wanxing Bio-Pharmaceuticals Co., Ltd in its current Good Manufacturing Practices (cGMP) SFDA qualified facility (manuscript describing antigen production, vaccine formulation and charcterization in preparation). PfCP2.9 is a highly purified (>98%), stable recombinant protein of 26.9 kDa. The bulk antigen PfCP2.9 was formulated with the adjuvant Montanide ISA 720 (Seppic) at a ratio of 30% protein:70% adjuvant based on volume. The adjuvant was sterile filtered according to the manufacturer’s recommendations. Optimal emulsification conditions for the antigen/adjuvant mixture were identified by screening a range of parameters (temperature, homogenization speed, time), to attain recommended specifications for the formulation (particle size 0.1 ␮M) that remained stable. A Silverson L4RT Homogenizer was used to emulsify the antigen/adjuvant mixture and the vaccine emulsion particle size was measured by a Mastersizer 2000 laser diffraction-based particle size analyzer attached to a Hydro 2000S sample dispersion unit (Malvern Instruments, Inc., Southborough, MA). A single phase water-in-oil emulsion resulted from homogenization at 4000 rpm for 3 min at room temperature. The vaccine emulsion was filled into autoclaved 2 ml vials to a final volume of 1 ml, at vaccine antigen concentrations of 10, 40, and 100 ␮g/ml. The control emulsion was prepared by homogenization of citrated-saline buffer (20 mM Citrate, 150 mM NaCl, pH 6.0) and Montanide ISA 720 at a ratio of 30%:70% by volume. Each vaccine dose lot was stored at 2–8 ◦ C. Each vaccine dose lot underwent comprehensive quality control analysis to ensure purity and stability by antigen recovery and reduced and non-reduced SDS-PAGE, integrity by particle size measurement as previously described [26], identity by reactivity with conformational, MSP1 specific monoclonal antibodies as previously described [8], and potency analysis by immunogenicity studies in mice (ED50 ) to meet the standards for clinical use (manuscript in preparation). Real time stability monitoring of each PfCP2.9/ISA 720 clinical dosage lot was initiated at the time of formulation and 3 months prior to initiation of the trial. These characterization assays were performed at 0, 1, 2, 3, 6, 9 and 12 months as part of the stability program, and each specification was met prior to release of the clinical trial material and within two weeks of its use in a clinical trial. In addition, the stability and potency of a pre-clinical batch of each dose of vaccine was prepared and analyzed 5 months ahead of the clinical batch, according to the schedule above. Regular analysis of the preclinical batch enabled identification of the time point at which a particular dose would fall out-of specification.

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Table 1 Randomization scheme. Randomization 1 (following screening for eligibility) Dose cohort 1 (5 ␮g), n = 28

Dose cohort 2 (20 ␮g), n = 28

Dose cohort 3 (50 ␮g), n = 14

Schedule A*

Schedule B*

Schedule A*

Schedule B*

Schedule B*

10 4 14 28 70

10 4 14

10 4 14 28

10 4 14

10 4 14 14

Randomization 2 (prior to Day 0)

# of vaccinees # of controls Total by schedule Total by dose cohort Total by study *

Vaccination days for Schedule A: 0, 60 and 180; vaccination days for Schedule B: 0, 90 and 180.

A second batch of the 20 ␮g and 50 ␮g clinical formulations for the third vaccinations (administered at 6 months) were required since these vaccine lots were out-of-specification at this time point. All clinical trial material was formulated under the same conditions and specifications. The ability of the clinical material to elicit antibodies with biological activity was assessed by GIA in sera from immunized rabbits according to the methods reported previously [27]. 2.4. Trial design Volunteers meeting all inclusion and no exclusion criteria received either 5 ␮g, 20 ␮g or 50 ␮g of PfCP2.9 malaria vaccine or ISA 720 alone as a control, as shown in Table 1. Volunteers were vaccinated three times with a 0.5 mL intramuscular injection in alternate upper arms. Schedule A was administered on days 0, 60 and 180. Schedule B was administered on days 0, 90 and 180. The clinical trial design did not include a 50 ␮g/Schedule A dose cohort based on the clinical data accumulated from the first Phase 1 trial [25]. A control group was felt to be necessary to serve as a baseline by which to compare safety and immune responses of the candidate vaccine. Dose escalation proceeded to the next higher dose because no pre-defined stopping criteria were met. Stopping criteria for this study were: at least one volunteer experiences a serious adverse event (SAE) that is judged to be possibly, probably or definitely related to the vaccine; or at least one volunteer experiences a hypersensitivity reaction (a Grade 3 allergic reaction), that is probably or definitely related to the vaccine; or three or more volunteers in a single dose group experience Grade 3 or higher injection site induration; or one volunteer experiences abscess formation at the injection site requiring drainage for diagnosis and treatment. An independent local safety monitor monitored the safety of the study participants throughout the trial. A data and safety monitoring board reviewed interim safety reports prior to dose escalation and as needed. The study was conducted from January 2006 to January 2007. 2.5. Safety The primary objective was to assess the safety and reactogenicity of PfCP2.9/ISA 720 vaccine in healthy adult volunteers when given at different doses and on two different vaccination schedules. Following each vaccination, volunteers were observed for 4 h and then evaluated for local and systemic reactogenicity on days 1, 2, 7, 14 and 30 after each vaccination. An abbreviated history and physical examination were performed at each follow-up visit. Injection sites were examined for tenderness, erythema, swelling, induration and nodules. Solicited systemic adverse events included fever (>37.5 ◦ C), headache, nausea, malaise, myalgia, arthralgia and urticaria. Volunteers were asked to keep diary cards to record axil-

lary temperature and any local or systemic adverse events daily for 30 days following each vaccination. All abnormal signs and symptoms were considered adverse events. Adverse events were graded as mild (easily tolerated), moderate (interfered with activities of daily living), or severe (prevented activities of daily living) and assigned causality relative to the study vaccine using the following terms: definite, probable, possible, unlikely or unrelated. Injection site erythema and swelling were graded as mild (>0 and ≤20 mm), moderate (>20 to ≤50 mm), or severe (>50 mm). A complete blood count, serum creatinine, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were performed immediately before each vaccination, as well as 2, 7, 14 and 30 days after each vaccination. 2.6. Immunogenicity The secondary objective was to assess the immunogenicity by evaluating antigen-specific antibody responses (anti-PfCP2.9 ELISA). Exploratory analyses included measurement of the inhibition of parasite growth by vaccine-induced antibodies as measured by the in vitro parasite GIA and antibody recognition of the parasite by IFA. Operators were blinded to treatment assignment and subject identification number, but not to study day or cohort. 2.6.1. ELISA A standardized ELISA protocol was employed to measure antiPfCP2.9 antibodies in sera from vaccinees. Serum samples were collected to measure antibody responses from volunteers in Schedule A on days 0, 30, 60, 74, 90, 120, 194 and 240 and from volunteers in Schedule B on days 0, 30, 60, 90, 104, 120, 180, 194 and 240. The positive human control for the ELISA was derived from a pool of sera from malaria-infected patients living in YunNan in the South of China. The negative control was derived from pooled pre-immune sera from the volunteers. Ninety-six well, flat-bottomed microtiter plates (Costar) were coated with 100 ␮L per well of PfCP2.9 antigen (2 ␮g/mL in carbonate buffer, pH 9.6) for 1 h at 37 ◦ C. The coating solution was removed and the plate was washed 6 times with wash buffer solution (PBS containing 0.01% Tween 20, pH 7.4). The plates were blocked with 200 ␮L per well of wash buffer containing 3% skimmed milk powder for 2 h at 37 ◦ C. Following removal of the blocking buffer and plate washing, the samples were added to the plate at a 1:200 dilution. Serial dilutions were performed to yield a linear range for each sample that could be analyzed with curve-fitting software to calculate the theoretical dilution needed to give an optical density of twice the mean value of the control (adjuvant alone) group. Secondary antibody (goat anti-human IgGHRP, 1:20,000 dilution, Sigma–Aldrich) was added for 1 h at 37 ◦ C, and detected with TMB (3,3 ,5,5 -Tetramethyl benzidine). The reaction was stopped after 10 min by the addition of 50 ␮L H2 SO4 . The plates were read at dual wavelengths of 450 nm/630 nm, using a

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microplate reader (MK3, Thermoelectric). A dilution curve of the positive standard was run on each plate to assess intra- and interassay consistency and reproducibility. All samples and controls were run in triplicate. Titers were reported as the mean of individual triplicate values, and a volunteer was considered to be a responder if the anti-PfCP2.9 titer was greater than 1:200. 2.6.2. Growth inhibition assay and immunofluorescence assay The exploratory analyses for this trial included GIA, which was performed at the MVI GIA reference center, NIAID, NIH, to measure the functional immune responses and IFA to measure direct reactivity with parasites was performed at the Department of Pathogenic Biology, Second Military Medical University. The GIA assay measures the ability of purified IgG from vaccinees to inhibit merozoite invasion or subsequent growth of parasites in uninfected erythrocytes. For the GIA analysis, serum was collected from volunteers before the first immunization, and 2 weeks after the second and third vaccinations, i.e., days 0, 74 and 194 in Schedule A and days 0, 104 and 194 in Schedule B. Total IgGs were first isolated from sera of each volunteer in the 50 ␮g dose cohort (Schedule B) from day 194 sera. Samples were run in triplicate with two sets of positive controls and negative controls are included on each test plate and analyzed by standardized GIA against both FVO and 3D7 parasite strains, at a final concentration of 10 mg/ml, approximately that of normal human IgG [21,24]. The FVO strain was selected because it has the same MSP119 sequence as the PfCP2.9 immunogen and the 3D7 strain was also tested since the AMA1 portion of the chimeric protein was derived from this strain [8]. Specificity of the inhibition was assessed by GIA reversal using the full length AMA1 and MSP119 polypeptides produced by the Malaria Vaccine Development Branch, NIAID, NIH. In conjunction with the GIA assay, a standardized ELISA was also performed on the purified IgGs and the trial sera at the NIH and results of unknown samples were compared with a standard curve generated by serial dilutions of a reference antibody standard run on each test plate [28]. To examine the specificity of the antibody responses, antibodies were tested by this ELISA on the fusion protein as well as its components—full length AMA1 and MSP142 , kindly provided by the Malaria Vaccine Development Branch, NIAID, NIH. Results were expressed as NIH ELISA units, which are the reciprocal of the dilution required to attain an O.D. = 1 in this standardized assay. IFA was performed using cultured parasites of Pf FCC1/HN isolate as antigens on samples collected on the screening visit, days 0, 30, 60, 74 (Schedule A), 90, 104 (Schedule B), 120, 180, 194 and 240. This isolate has been adapted to rabbit sera for cultivation. IFA slides were prepared using culture material with 5–10% parasitemia. The slides were blocked with 3% nonfat powdered milk in phosphate buffered saline (PBS) before adding serum samples diluted 2-fold starting at 1:20 and incubated for 1 h at room temperature. After extensive washing with PBS, the slides were incubated with fluorescein isothiocyanate-labeled secondary anti-human IgG at 1:100 with blocking buffer for 1 h at room temperature. The bound secondary antibodies were examined by fluorescence microscopy. End-point titers of immune sera were determined as the last dilution beyond that observed with pre-immune sera tested on the same slide. To demonstrate whether or not the clinical vaccine formulation was able to elicit antibody with growth inhibitory activity, a group of five rabbits was immunized 3 times at 30 day intervals with 70 ␮g of the 100 ␮g/mL clinical vaccine formulation adjuvanted with ISA 720. The experiment was designed to test a 100 ␮g dose, however only 0.7 mL was able to be drawn up from the vial. Due to an error in the protocol, sera were collected on day 63 instead of day 90 for GIA testing. Therefore the interpretation of the results is based on the immune response after two immunizations.

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2.7. Statistical methods Differences in the proportion of adverse events between the volunteers receiving vaccine vs. the group of controls (n = 20) within each dose and schedule assignment were analyzed using Fisher’s Exact Test. Dose effects on adverse events were also tested using Fisher’s Exact Test. P-values of less than or equal to 0.05 were considered significant. No adjustment for multiple tests was made. Differences in the antibody levels were compared descriptively between dose cohorts and control and between vaccination schedules within a dose cohort. Wilcoxon Rank Sum Tests were used to compare antibody responses between active and control groups and to compare different timepoints within the same vaccine group. The SAS (Version 9.1; SAS) software package was used for analyses. 2.7.1. Sample size The sample size for this study was determined by the requirement to judge the safety of each dosing regimen. The study design was not designed to determine statistically significant differences in immunogenicity between dose cohorts or regimens. Although not powered to detect differences in the incidence of specific adverse events or immune responses between the vaccine candidate and control, a group size of 10 per dose level of PfCP2.9/ISA 720 was chosen to give a reasonable probability of detecting one or more serious or severe vaccine-related adverse events. Ten subjects at each vaccine dose constituted a reasonable sample size to provide preliminary estimates of the frequency of commonly occurring adverse events (AEs). Each dose cohort of PfCP2.9/ISA 720 was compared to the group of controls (n = 20), which served as a comparator for both safety and immunologic assessments. 2.7.2. Randomization and blinding Seventy healthy adult volunteers meeting all inclusion and no exclusion criteria received the first randomization for assignment to one of the three dose cohorts: 28 volunteers to Dose Cohort 1 (5 ␮g), 28 volunteers to Dose Cohort 2 (20 ␮g), and 14 volunteers to Dose Cohort 3 (50 ␮g), as outlined in Table 1. Each dose cohort commenced vaccination on a different calendar day; therefore randomization of volunteers to a specific vaccine date attempted to prevent bias when assigning volunteers to specific dose cohorts. On the day of the first vaccination, volunteers in Dose Cohorts 1 and 2 underwent a second randomization for assignment to vaccination Schedule A (0, 60, and 180 days) or Schedule B (0, 90, and 180 days). Within each schedule group, 10 volunteers received PfCP2.9/ISA 720 and four received ISA 720 control. All volunteers in Dose Cohort 3 received vaccinations under Schedule B, with 10 volunteers randomly assigned to PfCP2.9/ISA 720 and four to ISA 720 control. Study participants and investigators who assessed safety were blinded to vaccine assignment. Access to randomization codes was limited to the study pharmacists. The PfCP2.9/ISA 720 vaccine and the control had identical color, appearance, and packaging and therefore were indistinguishable. As a result, the investigators and lab staff who assessed AEs and reactogenicity were not aware of the treatment allocation. 3. Results Of the 118 adults screened and consented, 80 were deemed eligible for enrollment (70 volunteers with 10 alternates), and 38 were considered screening failures (Fig. 1). A total of 70 volunteers (43 male and 27 female), all Chinese, were randomized to one of 3 dose cohorts and received a first vaccination. The age range was

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Fig. 1. Trial flow .

21–43 years. Sixty-seven volunteers received all 3 vaccinations as scheduled. Three volunteers did not receive all vaccinations, as they withdrew consent. One volunteer in the 5 ␮g/Schedule B group developed a delayed local reaction following the first vaccination and experienced several adverse events that were not related to vaccination. Because of these events, the study team and volunteer agreed to discontinue additional vaccinations. One volunteer each in the 20 ␮g/Schedule A group and the 20 ␮g/Schedule B group moved away from Shanghai following the second vaccination and could not attend subsequent follow-up visits. Because each volunteer received at least 1 vaccination, the safety analyses include all 70 volunteers. The immunogenicity analyses presented here exclude these 3 volunteers. New batches of vaccine formulation were prepared for the 6 month time point in the 20 and 50 ␮g dose cohorts as these vaccines

were out of specification; the 5 ␮g dose cohort remained within specification throughout the trial. 3.1. Safety PfCP2.9/ISA720 recombinant malaria vaccine was safe and well tolerated (Table 2). The most common reported adverse events were tenderness (53%) and pain (23%) at the injection site. Ninetyfour percent of local reactions were graded as mild, and none was graded as greater than moderate. Swelling, nodule, erythema and pruritus also occurred at low frequencies (1.4–7.1%) following each vaccination. Two volunteers in the 5 ␮g dose cohort developed nodules (4 cm and 1 cm) accompanied by pruritus or tenderness at the injection site on day 14 and day 21 following the first vaccination lasting 35 and 9 days, respectively. One volunteer in the 20 ␮g dose

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Fig. 2. Longitudinal anti-PfCP2.9 antibody responses in study participants from the first (blue) and second (red) cohorts compared to controls (black), by schedule A (closed circles) and B (open circles). Antibody titers were measured by ELISA in sera collected on: Day 0 (baseline), 30, 60, 74 (schedule A groups only), 90, 104 (schedule B groups only), 120, 180, 194 and 240. Points represent the geometric mean antibody titer against PfCP2.9, error bars are ±1 standard error of the geometric mean titer and arrows represent the vaccination time points. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

cohort developed pain and tenderness at the injection site 17 days after the first vaccination. The pain lasted for a total of 3 days and the tenderness for a total of 5 days. The most common solicited systemic adverse events were headache and nausea (2.9% for each). No clinically significant deviations were observed in the laboratory tests. No immediate hypersensitivity reaction was observed and no serious adverse events occurred during the trial.

The incidence of adverse events did not differ significantly between PfCP2.9/ISA 720 vaccinees and controls. (Fisher’s Exact Test p-values ≥0.25 for all by-group comparisons of local, general, and overall solicited AEs; no adjustment made for multiple tests.) Neither the dose levels nor the vaccination schedules were found to have any relationship with the occurrence or severity of the adverse events. The incidence of symptoms decreased with subsequent

Fig. 3. Longitudinal anti-PfCP2.9 antibody responses in study participants from the first (blue), second (red) and third (green) cohorts compared to controls (black), by schedule B. Antibody titers were measured by ELISA in sera collected on: Day 0 (baseline), 30, 60, 90, 104, 120, 180, 194 and 240. Points represent the geometric mean antibody titer against PfCP2.9, error bars are ±1 standard error of the geometric mean titer and arrows represent the vaccination time points. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Note: Column header counts are the number of randomized volunteers who have received at least one vaccination. Denominators are the number of volunteers who received the corresponding vaccination. A volunteer is counted at most once within each event type, event class (local or systemic), and vaccination group; only the greatest reported intensity is presented for each level. AEs with onset before the indicated vaccination or with an unknown intensity level are not presented. a Fever was defined as Mild ≥37.5–38.0 ◦ C; Moderate: >38.0–39 ◦ C; Severe: >39 ◦ C.

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 (10) 0 0 0 2 (10) 0 0 0 0 1 (5) 0 0 0 0 0 1 (5) 0 0 3 (15) 0 0 0 2 (10) 0 1 (5) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 (10) 0 1 (5) 1 (5) 1 (5) 1 (5) 1 (5) 0 0 2 (10) 0 0 2 (10) 0 0 0 0 0 Systemic Fevera Rash Nausea Headache Malaise Myalgia Arthralgia Urticaria

2 (10) 0 0 2 (10) 0 0 0 0 0

0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0

2 (10) 0 1 (5) 1 (5) 1 (5) 1 (5) 1 (5) 0 0

Severe (%) Moderate (%)

0 0 0 0 0 0 0 0 4 (40) 2 (20) 4 (40) 2 (20) 0 0 0 0

Mild (%) Any (%)

4 (40) 2 (20) 4 (40) 2 (20) 0 0 0 0 0 0 0 0 0 0 0 0

Severe (%) Moderate (%)

2 (10) 1 (5) 0 1 (5) 0 0 0 0 9 (45) 6 (30) 11 (55) 0 0 1 (5) 0 0

Mild (%) Any (%)

11 (55) 7 (35) 11 (55) 1 (5) 0 1 (5) 0 0 0 0 0 0 0 0 0 0

Severe (%) Moderate (%)

2 (10) 0 0 1 (5) 0 0 1 (5) 0 10 (50) 2 (10) 11 (55) 1 (5) 0 0 1 (5) 1 (5)

Mild (%) Any (%)

12 (60) 2 (10) 11 (55) 2 (10) 0 0 2 (10) 1 (5)

Severe (%)

0 0 0 0 0 0 0 0

Moderate (%)

1 (5) 0 1 (5) 0 0 0 0 0

Mild (%)

10 (50) 5 (25) 10 (50) 0 0 0 0 0 11 (55) 5 (25) 11 (55) 0 0 0 0 0

Any (%)

Local Pain Tenderness Swelling Induration Erythema Nodule Itching

Cohort 2 PfCP-2.9/ISA 720 (20 ␮g) (N = 20) Cohort 1 PfCP-2.9/ISA 720 (5 ␮g) (N = 20) ISA 720 Control (N = 20)

Table 2 Frequency of adverse events in volunteers by grade and dose cohort.

0 0 0 0 0 0 0 0

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Cohort 3 PfCP-2.9/ISA 720 (50 ␮g) (N = 10)

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vaccinations; the frequency of symptoms was highest after the first vaccination (40% overall), followed by vaccination 2 (33% overall), and vaccination 3 (12% overall). Seven volunteers (10%) experienced at least one general symptom after the first vaccination, and no volunteer experienced a general solicited symptom after receiving vaccination 3. 3.2. Immunogenicity 3.2.1. Antibody assessment by ELISA All but two volunteers who received the ISA 720 control showed no immune response (response defined as titer >200) after vaccination. All volunteers from each active dose cohort (5, 20, and 50 ␮g) and dosing schedule (Schedules A and B) demonstrated an antigenspecific IgG response to the fusion protein (Wilcoxon p < 0.001) and experienced boosting following the second vaccination (Wilcoxon p < 0.05). Fig. 2 shows the immune responses for controls and the 5 and 20 ␮g dose groups from both dosing schedules. All subjects’ baseline antibody levels were below the limit of detection. Several recipients of the PfCP2.9 vaccine showed an immune response by 30 days after the first vaccination. The geometric mean titer (GMT) of each active group (5 and 20 ␮g) increased approximately 10-fold from day 30 until 14 days after the second immunization (day 74 for Schedule A and day 104 for Schedule B), at which time volunteers from both dosing schedules had comparable GMTs (5 ␮g cohort: Schedule A GMT = 2900, Schedule B GMT = 5400; 20 ␮g cohort: Schedule A GMT = 11,000, Schedule B GMT = 13,000). At 14 days after the third vaccination (day 194 for both schedules) and day 240, both dosing schedules showed similar GMTs and error bounds (day 240 results: 5 ␮g cohort: Schedule A GMT = 15,000, Schedule B GMT = 16,000; 20 ␮g cohort: Schedule A GMT = 24,000, Schedule B GMT = 21,000). Fig. 3 summarizes GMTs over time for volunteers vaccinated using Schedule B. Volunteers in the 50 ␮g dose group experienced a higher initial response following the first vaccination in antibody levels than those in the lower dose groups. By day 60, GMTs increased for all dosing groups (5 ␮g GMT = 500; 20 ␮g GMT = 700; 50 ␮g GMT = 5200). Between days 60 and 90, the GMTs for the 5 and 20 ␮g groups continued to increase (GMTs = 700 and 900, respectively), whereas the GMT for the 50 ␮g group decreased slightly (GMT = 3400). The second immunization for Schedule B occurred on day 90 and resulted in a notable increase in antibody titer for all groups. These antibody levels remained high for the remainder of the trial and were not markedly increased as a result of the third immunization given on day 180. The final serum sample was obtained on day 240 and at that time point the GMTs for the 5, 20, and 50 ␮g groups were indistinguishable (GMTs = 16,000, 21,000, and 17,000, respectively). All vaccine doses clearly elicited an immune response. All dosage levels appeared to produce comparable antibody levels by day 240, regardless of which dosing schedule was followed. 3.2.2. Fine specificity of antibody response To dissect whether the antibodies were directed to the fusion protein or the individual components of the fusion protein, an ELISA was performed on day 194 sera from vaccinees in the 50 ␮g dose cohort and tested against the PfCP2.9, MSP119 FVO, MSP142 FVO, and AMA1 3D7 (See Table 3). This ELISA was performed at the NIH GIA Reference Center using a different protocol from Figs. 2 and 3. The four control vaccinees did not show any antibody response. In contrast, the 10 vaccinees developed significant titers to the PfCP2.9 immunogen. Most responders made detectable levels of antibody to both the AMA1 and MSP1 components of the fusion protein, but the titers were considerably lower than to the PfCP2.9 protein.

E. Malkin et al. / Vaccine 26 (2008) 6864–6873

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Table 3 50 ␮g Dose Cohort Immunology Results—study day 194. Volunteer 009 012 018 (control) 027 046 (control) 050 056 059 (control) 062 067 (control) 083 113 117 118

PfCP-2.9 Unitsa

AMA1-3D7 Unitsa

MSP119 -FVO Unitsa

3,747 615 8 5,278

204 8 2 358

165 46 0 385

0 215 5,998 10 3,730 0 3,899 3,286 2,527 10,950

2 9 428 3 187 1 328 252 29 620

0 18 442 0 214 0 361 286 175 771

MSP142 -FVO Unitsa

% inhibition b (para-3D7)

IFAc

591 130 0 1,338

8 0 −4 4

160 80 <20 320

0 44 1,551 1 950 0 1,036 903 655 2,771

-1 0 7 -3 6 -5 9 5 5 13

<20 20 160 <20 80 <20 160 320 160 640

a Sera taken on day 194 were tested by ELISA against several different plate antigens to assess fine specificity of the antibody responses. ELISA unit values shown represent the reciprocal of the dilution which gives an O.D. = 1 in a standardized assay against the immunogen (PfCP2.9) or its components (AMA1-3D7, MSP119 -FVO or the larger MSP142 -FVO). Individual polypeptides were produced by the Malaria Vaccine Development Branch/NIAID/NIH. b The GIA assay results are presented as % inhibition of growth of P. falciparum 3D7 parasites compared to wells with normal human serum. Samples tested were IgG fractions isolated from the sera and adjusted to 10 mg/ml. Growth of parasites after 40 h of incubation was assessed by biochemical measurement of parasite lactate dehydrogenase. c IFA was performed by serial dilution of antisera against P. falciparum FCC1/HN parasites. Data are presented as the reciprocal of the last serial dilution giving a positive fluorescence in parasitized cells.

3.2.3. Functional antibody activity by GIA IgGs were purified from human sera and adjusted to 10 mg/ml, approximately the concentration of human sera. These samples were tested against both 3D7 and FVO parasites, which were selected because they have the same AMA1 and MSP119 sequences as the components of the PfCP2.9 polypeptide. Despite the high levels of antibody to the fusion protein observed by ELISA, only one out of ten samples from day 194 in the 50 ␮g dose cohort displayed more than 10% inhibitory activity to either parasite strain. This sample (#118) showed 13% and 12% inhibition of 3D7 and FVO parasites, respectively (Table 3 for 3D7 results; FVO strain data not shown), values which are slightly above the 10% error level which has been established for the GIA assay. This sample also had the highest ELISA titer to the PfCP2.9 fusion protein. Day 194 samples from the 5 and 20 ␮g cohorts were held until after the 50 ␮g results were obtained and a decision was made not to run the assay on these samples. In contrast to the human data, preclinical rabbit sera derived from animals that were immunized with the clinical formulation did demonstrate inhibition of parasite growth. Following immunization 3 times at 30 day intervals with 70 ␮g of PfCP2.9/ISA 720, sera were collected from 5 rabbits on day 63 for GIA testing. At a four-fold lower concentration of purified total IgG as compared to the human immunoglobulin, the levels of GIA in the 5 rabbits ranged from 47 to 88% (average 69%) against the 3D7 strain of parasites (data not shown). Reversal of the GIA by the addition of specific AMA1 or MSP1 antigen indicated that most of the inhibitory activity was due to antibodies directed to MSP1. By ELISA, rabbit antibody titers to PfCP2.9 were higher (approximately 10-fold) than those seen in the human trial. In addition, rabbit antibody titers to the fusion protein were higher than to the AMA1 (III) and MSP119 components, but only by approximately 4–5 fold (data not shown). 3.2.4. Recognition of intact parasites by IFA To investigate whether antibodies generated to the fusion protein recognized native parasites, sera from the vaccinated volunteers were tested by IFA against the FCC1/HN strain of P. falciparum. The result showed that all the volunteers that received the PfCP2.9/ISA 720 formulation generated low to moderate levels of detectable antibody that recognized the cultured parasite (range: 20–640; data not shown). Table 3 includes a comparison of the IFA titers with the ELISA titers from the 50 ␮g cohort at day 194.

4. Discussion This trial was designed to evaluate an optimized formulation of the PfCP2.9/ISA 720 candidate vaccine at three dose levels (5, 20, 50 ␮g), with the primary objective to assess the safety and reactogenicity of PfCP2.9/ISA 720 vaccine and the secondary objective to assess immunogenicity by evaluating antigen-specific antibody responses (anti-PfCP2.9 ELISA). Exploratory analyses included measurement of the inhibition of parasite growth by vaccine-induced antibodies as measured by the in vitro GIA and antibody recognition of the parasite by IFA. The first Phase 1a study with this vaccine candidate evaluated 4 doses (20, 50, 100 and 200 ␮g) of PfCP2.9/ISA 720 at a 0, 60 and 180 day schedule, in fifty-two volunteers from the Shanghai area [25]. Although the vaccine was generally well tolerated and immunogenic, local reactogenicity was observed, which appeared to increase with increasing dose of antigen. Additionally, no significant dose-response relationship was observed when comparing the immunogenicity of the four doses. Post-trial analysis of the formulation raised concerns as to the integrity, stability and potency of the PfCP2.9 antigen in the vaccine formulation. Such compromise of vaccine quality has been reported previously for other preformulated ISA 720 based vaccine candidates [29], and is thought to be the result of inter- and intramolecular cross-linking of antigen in the presence of the adjuvant [26]. This modification has been shown to occur for numerous antigens, and can be reduced and delayed by the addition of glycine or glycylglycine to the antigen prior to emulsification with ISA 720. Delayed local injection site reactogenicity, eg., nodule formation/induration, pain and tenderness, was seen in the previous trial with higher doses of this vaccine formulation. From this study and other studies, it has been speculated that a possible cause may be due to quantity of antigen load, quality of the formulation and the mechanistic action of ISA 720 to establish a depot at the site of injection. Interestingly, the 2 volunteers to experience a nodule formation in the present trial were administered the 5 ␮g dosage. In this recent trial, an attempt was made to reduce reactogenicity with an extended schedule and lower dosages. In addition, robust procedures for the evaluation of the integrity, stability and potency of the pre-formulated vaccine were implemented, and each clinical dose was assayed within 2 weeks of use to ensure that the

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formulation was in specification at the time of vaccination. There was no difference in the incidence of other adverse events in the groups receiving PfCP2.9/ISA 720 or ISA 720 alone. In addition, the dose levels or schedules were not found to have any relationship with the occurrence or severity of the adverse events, which may be due to the quality of the formulations. Antibody responses were induced in all volunteers receiving three vaccinations of PfCP2.9/ISA 720 in all dose groups; these responses remained elevated through day 240. Boosting was observed with both schedules, with titers increasing almost 10-fold from after the first to after the third vaccination. With the exception of the rapid rise in antibody titer in the 50 ␮g group following the 1st vaccination, the kinetics of the responses did not differ by dose or schedule. The IFA was performed to measure antibody recognition of native antigen on the parasite surface and the GIA was used to assess the levels of functional antibody response in vaccinees. Virtually all individuals receiving vaccine developed low levels of antibody that recognized cultured parasites by IFA. Volunteers with the highest IFA titers corresponded to those with the highest anti-PfCP2.9 ELISA titers. In addition to the IFA, a standardized GIA measured the ability of vaccine-induced antibodies to inhibit growth of both FVO and 3D7 parasite strains. At IgG concentrations that approximate physiological levels (10 mg/ml), only one volunteer in the highest dose cohort (50 ␮g) displayed any level of GIA which was above assay background, and this individual also had the highest antibody titers by ELISA. In this clinical trial, volunteers developed high ELISA antibody responses to PfCP2.9 across the three dose ranges but lower IFA antibody responses to native AMA1 and MSP1 in parasites. There was inconsistency in this study between the apparent high levels of antibody to the fusion protein by ELISA and the GIA response. When responses to the individual components of this fusion protein were tested, purified IgGs from at least four individuals in the 50 ␮g cohort had significant anti-AMA1-3D7 antibody titers (>2000) on day 194; such IgG titers have been shown to elicit moderate levels of GIA to 3D7 parasites in a Phase 1 study with full length AMA1/Alhydrogel [24]. This raises the question as to whether antibodies to domain III of AMA1 have the same level of GIA activity as antibodies to other regions of the protein [8,16,30], however it should also be noted that the correct conformation of domain III in the fusion molecule could not be confirmed due to lack of available reagents such as conformational monoclonal antibodies and it may be that this does not mimic native AMA1 structure and is consequently less effective in eliciting antibodies with GIA activity. Moreover, when ELISA units to the subunits are compared to reactivity to the fusion protein, antibodies to the fusion protein appear to be much more abundant. The nature of these epitopes on the PfCP2.9 antigen is unknown, but that these specificities did not appear to be functional in the GIA. In contrast to the clinical trial results, rabbits immunized with the clinical formulation of the fusion protein displayed levels of growth inhibition ranging from 47 to 88% against 3D7 parasites at a four-fold lower concentration of total IgG, and the antigen reversal results indicated that most of this effector function was due to antibodies directed to MSP1. In addition, a higher proportion of rabbit antibodies recognized the AMA1 and MSP1 components when compared with the antibody of the vaccinees. As in the human clinical trial, many rabbits displayed anti-AMA1 ELISA titers, which should have contributed significantly to the functional activity but apparently did not. This also suggests that the antibodies to domain III may not be as functional as antibodies to the intact protein. These results also contrast with recent results with another fusion protein using all three domains of AMA1 fused to MSP119 ; rabbits immunized with that construct gave higher GIA activity than

either component alone or a mixture of the two proteins [31]. These observations raise the possibility of differences in epitope specificity between humans and rabbits and underscore the potential pitfalls of relying on animal data for designing vaccines. Previous formulations of ISA 720 with different antigens have proven to be reactogenic, which was not the case with this particular study [23,29,32–38]. The observations from this trial contribute to the previous experience with this adjuvant, and show optimization of the formulation to achieve a well characterized product that remains stable and potent can improve the tolerability profile of the product. It underscores the importance of formulation characterization and optimization to ensure stability and potency of the vaccine early in this development phase so that the critical attributes of a potential candidate, namely safety and efficacy, can be reliably assessed. The results of this trial extend the evidence from the prior trial on the safety and immunogenicity of PfCP2.9/ISA 720 and highlight several important findings. This is the first malaria vaccine candidate based on a recombinant chimeric protein to be developed and tested in the clinic, and this trial demonstrates the feasibility of producing and testing constructs derived from the fusion of multiple antigenic components [31,39]. Even though the GIA has not been shown to correlate with protection in those living in malaria endemic areas, the lack of biological activity of the human antibodies elicited to PfCP2.9, as measured by the GIA, raises the question as to the potential impact on efficacy for this particular chimeric protein. Overall, the safety and reactogenicity profile of this optimized Montanide ISA 720 formulation was improved over the previous Phase 1 trial [25], as well as by comparison with the majority of trials using this adjuvant [23,29,32–38]. Considering that public sector research and development into novel vaccines are restricted by a lack of accessible adjuvants, these experiences are valuable additions to the existing database on a commercially available (although not licensed) adjuvant for clinical evaluation.

Acknowledgements The Phase 1 trial was conducted at Shanghai Changhai Hospital, Shanghai, China. We are grateful to all trial site personnel for the recruitment of volunteers and their execution of the clinical trial. We also give our special thanks to the volunteers who participated in the trial. We thank Xiangyang Xue for his technical assistance with the IFA and IgG purification and we thank Hong Zhou, Sam Moretz, and Ababacar Diouf for the GIA studies. We thank Chad Lipton for his useful comments and editing. Funding: This work was supported by the PATH Malaria Vaccine Initiative and the Intramural Program of NIAID/NIH. The PATH Malaria Vaccine Initiative and WHO reviewed the study protocol and contributed to its design, the writing of the paper and the decision to submit it for publication. WHO provided support for on-site training on GCP and ethics, as well as oversight for safety monitoring.

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