Safety and immunogenicity of a recombinant Plasmodium falciparum AMA1 malaria vaccine adjuvanted with Alhydrogel, Montanide ISA 720 or AS02

Abstract

Background: Plasmodium falciparum Apical Membrane Antigen 1 (PfAMA1) is a candidate vaccine antigen expressed by merozoites and sporozoites. It plays a key role in red blood cell and hepatocyte invasion that can be blocked by antibodies.

Methodology/principal findings: We assessed the safety and immunogenicity of recombinant PfAMA1 in a dose-escalating, phase Ia trial. PfAMA1 FVO strain, produced in Pichia pastoris, was reconstituted at 10 microg and 50 microg doses with three different adjuvants, Alhydrogel, Montanide ISA720 and AS02 Adjuvant System. Six randomised groups of healthy male volunteers, 8-10 volunteers each, were scheduled to receive three immunisations at 4-week intervals. Safety and immunogenicity data were collected over one year. Transient pain was the predominant injection site reaction (80-100%). Induration occurred in the Montanide 50 microg group, resulting in a sterile abscess in two volunteers. Systemic adverse events occurred mainly in the AS02 groups lasting for 1-2 days. Erythema was observed in 22% of Montanide and 59% of AS02 group volunteers. After the second dose, six volunteers in the AS02 group and one in the Montanide group who reported grade 3 erythema (>50 mm) were withdrawn as they met the stopping criteria. All adverse events resolved. There were no vaccine-related serious adverse events. Humoral responses were highest in the AS02 groups. Antibodies showed activity in an in vitro growth inhibition assay up to 80%. Upon stimulation with the vaccine, peripheral mononuclear cells from all groups proliferated and secreted IFNgamma and IL-5 cytokines.

Conclusions/significance: All formulations showed distinct reactogenicity profiles. All formulations with PfAMA1 were immunogenic and induced functional antibodies.

Trial registration: (Clinicaltrials.gov) NCT00730782.

Figure 1. Study flow chart showing number of volunteers randomised, withdrawn and completing follow-up.

Coding for adjuvant as follows: Alum = Alhydrogel™, Mon = Montanide. Reasons for withdrawal are given: “rash” = allergic rash unrelated to study procedure, “erythema” = grade 3 injection site erythema leading to withdrawal, “other vac.” = concomitant Hepatitis B vaccination leading to exclusion.

Figure 2. Mean log anti-AMA-1 titers with standard error of the mean for low and high dose per adjuvant group.

Anti-AMA-1 titers were determined by ELISA for the six different groups, immunized with Alhydrogel™, Montanide and AS02 adjuvanted PfAMA1 vaccine. Dashed lines represent high dose of PfAMA1 (50 µg), continuous lines represent low dose groups (10 µg). Measurements were performed at baseline, 28 days after the first, second and third immunisation (day 28, 56 and 84 respectively) and day 140 and 365.

Figure 3. Representative immunofluorescent microscopy picture, showing recognition of native antigen on merozoites by induced anti-AMA1 antibodies.

Immunofluorescence picture of merozoites incubated with 40× diluted anti-AMA1 plasma from an immunized volunteer one month after final immunisation stained with Rabbit anti-human immunoglobulin FITC (A) and DAPI (B). Photo was taken at magnification 400×. Incubation with monoclonal antibody 4G2, a pan-specific anti-AMA1 antibody, confirmed the surface staining pattern (not shown).

Figure 4. ELISPOT assay for IFNγ (A) and IL-5 (B) after stimulation with 6 µg PfAMA1.

Peripheral Blood Mononuclear Cells from immunised volunteers 28 days after the second immunisation and 28 days after the third immunisation (day 56 and 84 respectively) were stimulated with 6 µg of PfAMA1 vaccine. Production of IFNγ and IL-5 was measured by counting spots in ELISPOT plates. Box plots and whiskers show the range and the 25^th, 50^th and 75^th percentile of spots per 2×10⁵ cells. Circles represent outliers.

Figure 5. Stimulation indices in response to 6 µg/ml PfAMA1 presented as box plots and whiskers.

Peripheral Blood Mononuclear Cells from immunised volunteers 28 days after the second immunisation and 28 days after the third immunisation (Day 56 and 84 respectively) were stimulated with 6 µg of PfAMA1 vaccine. Cell proliferation was measured by adding ³H thymidine and calculated relative to control wells. Box plots and whiskers show the range and the 25^th, 50^th and 75^th percentile. Circles represent outliers. Measurements were performed at baseline, 28 days after the second immunisation and 28 days after the third immunisation (day 56 and 84 respectively).

Figure 6. Percentage of growth inhibition of FVO-strain P. falciparum parasites after addition of 5 or 10 mg/ml IgG.

Serum samples from volunteers immunised with PfAMA1 were obtained four weeks after the final immunisation by per protocol analysis and included in a merozoite growth inhibition assay. Growth inhibition is expressed as a percentage to control.Boxes show 25^th, 50^th and 75^th percentile growth inhibition, whiskers show the range, circles are outliers.