Recombinant protein

P. falciparum FVO strain AMA1 allele sequence was used in the vaccine. Expression and purification of recombinant FVO AMA1 full-length ectodomain (residues 25-546) in P. pastoris is described elsewhere.34 The recombinant protein was confirmed and validated to be of good quality as determined by various analytical methods described previously.34 SDS-PAGE and western blotting with a conformation-specific mAb (4G2) were used to verify correct protein folding.

Peptide synthesis

Peptides were synthesized at Lifetein LLC (New Jersey, USA). Quality control performed include mass spectrometry for mass accuracy and high performance liquid chromatography for purity. Peptides used were >95% pure as determined using these methods.

Surface plasmon resonance (SPR)

SPR measurements were made with a BIAcore T100 instrument at 25 °C according to the manufacturer’s instructions. Sensor CM5, amine coupling reagents, and buffers were purchased from GE Healthcare, Piscataway, NJ. The CM5 sensor chip was activated with N-hydroxysuccinimide and 3-(dimethylamino) propyl carbodiimide (EDC) for 7 min. Then, 40 µg mL−1 recombinant FVO AMA1-10 mM sodium acetate (pH 5.0) was injected for 7 min followed by blocking with 1 M ethanolamine (pH 8.5) for 7 min. A flow rate of 30 µL min−1 was used for all steps. For the binding assay, RON2L peptide was dissolved at different concentrations in 10 mM HEPES (pH 7.5)–0.5 mM EDTA–1 mM MgCl2–0.2% Tween 20. Binding at each concentration was done with an exposure of 2 min followed by 10 min for the dissociation phase. Regeneration was done with a 30-s pulse of 10 mM glycine HCl (pH 2.5). The kinetic data for RON2L binding were fitted to a two-step binding kinetic model. BIAcore T100 evaluation software was used for kinetic analysis.

Animals

Aotus monkeys used in the study were housed-in female pairs and veterinary care was provided by NAMRU-6 attending veterinarian (MAJ Luis Lugo-Roman) and were monitored twice daily by animal caretakers. The monkeys were fed twice a day (morning and afternoon) with Iquitos Primate Center biscuits plus fresh fruits (bananas, apples). Room temperature (RT) was maintained at 24–27 °C, relative humidity between 30–70%, and a minimum of 10–15 air changes per hour and a light cycle consisting of a 12-h illumination followed by 12 h of dim red illumination. Nest boxes and perches (PVC tube pipes) were placed in the cages so the monkeys may “scent mark” and sit comfortably above the floor of the cage. Cleaning of the nest boxes and the cages were alternated on a weekly basis to maintain a “scent-marked” area in their cages at all times and additional toys were placed in the cages on a rotating basis for enrichment.

Vaccines, animals, vaccinations and sample collection

AMA1–RON2L complex was prepared by mixing AMA1 and RON2L peptide in a 1: 3 gram ratio in PBS (pH 7.4). The mixture was incubated in the dark at RT for 30 min. All vaccinations were performed at the US Naval Medical Research Unit No. 6 (NAMRU-6), a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care. A total of 22 malaria naïve, captive-bred, adult owl monkeys (Aotus nancymaae) were obtained from the Center for the Breeding and Conservation of Primates of Iquitos. They were randomized by sex and pretrial weight into three groups, Group1: PBS control (n = 6), Group 2: AMA1 alone (n = 8) and Group 3: AMA1–RON2L complex (n = 8). PBS, AMA1 alone or AMA1–RON2L complex were emulsified 1:1 in Freund’s complete (first immunization) or incomplete adjuvant (second and third immunizations) using two 5 mL syringes connected by double female luer lock (Smiths Medical# MX494) to form a thick emulsion. Three doses of the vaccine each containing 40 µg of AMA1 (Group 2) and 40 µg AMA1 + 120 µg RON2L (Group 3) in 0.5 mL were given subcutaneously (into the interscapular area) on study days 0, 21 and 42. Vaccination sites were monitored for adverse local reaction, and the hematocrit and weight of the animals was monitored at 3-week intervals. Plasma was collected under ketamine anesthesia from 2 mL EDTA-anticoagulated blood, 3 weeks after every vaccination. The study protocol was approved by NAMRU-6’s Institutional Animal Care and Use Committee (protocol number: NAMRU-6 14-01/NRD891), the Department of the Navy Bureau of Medicine and Surgery (NRD-748) and the Institut Nacional de Recursos Naturales (INRENA) at the Peruvian Ministry of Agriculture. (Resolucion Directoral No. 067-2014-SERFOR-DGGSPFFS). The experiments reported herein were conducted in compliance with the Animal Welfare Act and in accordance with the principles set forth in the “Guide for the Care and Use of Laboratory Animals”, Institute of Laboratory Animal Resources, National Research Council, National Academy Press, 2011.

Parasite challenge

Four weeks after the final vaccination (study day 70), animals were challenged intravenously with 104 FVO-strain P. falciparum-infected RBC collected freshly from a donor monkey. Parasitemia was measured by daily thin-film blood smears and hematocrit measurements were conducted on alternate days. Animals were treated with mefloquine (Roche Laboratories) when (i) parasite density reached ≥200,000 μL−1, (ii) when Hct fell to ≤25%, (iii) if SP upon reaching 40 days after challenge (study day 110). Animals that self-cured were monitored for 3 days for continued absence of parasite and were treated with mefloquine. Based on this criteria all 6 animals in Group 1 were mefloquine-treated for high parasitema. In Group 2, animal T3169 was treated for high parasitemia while T3042 and T3121 were treated due to anemia. T3095, T3118, T3171 and T3173 were treated 3 days after self-curing parasitemia. In Group 3, T3123 was treated due to anemia while T3160 died during self-curing parasitemia possibly due to anemia. Such occasional deaths have been recorded in this Aotus model of human malaria.22, 35, 36 T3174 developed anemia 2 days after self-curing and was also treated with mefloquine.

ELISA

The assay was performed as described elsewhere.37 Briefly, ELISA plates were coated overnight with 1 µg mL−1 recombinant AMA1. PfAMA1 allele-specific ELISA units were determined by first generating a standard curve using serially diluted IgG mixture containing anti-AMA1 antibodies. The reciprocal of the dilution giving an OD 405 = 1 was used to assign ELISA units to standards and all samples were tested against the same standard as described.37

Competition ELISA

The levels of AMA1-RON2 blocking antibodies were determined as follows. ELISA plates were coated overnight at 4 °C with 1 μg mL−1 RON2L peptide and blocked with 0.5% bovine serum albumin, 0.1% tween-20 in PBS (blocking buffer). Serial dilutions of plasma or purified IgG containing known antibody titers (AMA1 ELISA units, see above) prepared in blocking buffer were mixed with 2.5 μg mL−1 of recombinant FVO AMA1 and incubated for 30 min at RT. This mixture was applied to RON2L coated plates for 1 h at RT. After washing the plates, rabbit polyclonal AMA1 IgG was added to the plates at a 5 μg mL−1. The levels of bound AMA1 were measured using alkaline phosphatase-conjugated anti-rabbit secondary antibody at 1:3000 dilution. OD 405 was measured and IC 50 (antibody level, which inhibits 50% of AMA1 binding to RON2) for each sample was calculated using Graphpad Prism 5 software. Spearman correlation (r s ) between AMA1-RON2 blocking antibodies and GIA (for IgG) and time to patency (for plasma) was analyzed by plotting of the IC 50 (in Log10[EU] scale) using Graphpad Prism 5 software. Levels of blocking antibodies in the IgG (n = 8 per group) was tested once and the levels of blocking antibodies in the plasma samples (n = 7 per group) was tested in four independent experiments and the mean ± SEM of all experiments are shown.

IgG antibody avidity ELISA

IgG antibody avidity was assessed by measuring the urea displacement method.38 ELISA was performed as described above. Individual IgG from animals in Group 2 (n = 5) and Group 3 (n = 8) were analyzed in duplicate. Following incubation and washing of IgG, an ascending concentration of urea (0 to 5 M) was added in duplicate wells. Plates were incubated for 15 min at RT and the concentration of urea needed to cause 50% reduction of the OD405 compared to the urea-free wells for each sample (i.e., the concentration of urea required to reduce the OD405 to 50% of that without urea = IC 50 ) was used as a measure of avidity.

P. falciparum parasite culture

All parasites strains were grown in RPMI 1640 supplemented with 25 mM HEPES and 50 μg mL−1 hypoxanthine (KD Medical), 0.5% Albumax (Invitrogen), 0.23% sodium bicarbonate (Gibco) using O + RBCs (Interstate Blood Bank, Jackson, TN) and monitored daily by Giemsa-stained blood smears as described.39

Growth inhibition assay (GIA)

All assays for GIA were performed at the GIA reference center, NIAID, NIH. Plasma was heat inactivated for 20 min at 56 °C and pre-adsorbed against uninfected RBC. IgG was purified from plasma using Protein G sepharose (GE life sciences) using the low pH elution method and were neutralized immediately. Eluted IgG were dialyzed against RPMI 1640 (KD Medical) and concentrated to 10 mg mL−1. For performing GIA, IgGs at the desired concentrations were incubated with infected RBCs (0.3% parasitemia at 1% hematocrit) in a final volume of 40 µL for 40 h at 37 °C. A biochemical measurement using a Pf lactate dehydrogenase assay, as described previously, was used to quantify parasitemia.23

Competition GIA

The GIA assay was performed as described above with some minor modifications. The ability of the various recombinant proteins (FVO AMA1, 3D7 AMA or reduced and alkylated FVO AMA1) to adsorb the inhibitory activity of IgG was measured by pre-incubating the IgG with saturating concentration (2 µM) of recombinant proteins for 30 min at RT as determined previously.21

Statistical analysis

Primary end point for vaccine efficacy was analyzed by Mantel-Cox test of time-to-patency of animals in Group 2 and Group 3. Secondary efficacy outcome was measured by comparing log cumulative parasitemia between Groups 2 and 3 by Mann–Whitney test. Comparison of the levels of anti-AMA1 antibodies in the plasma, purified IgG and comparison of GIA between the two groups were also performed by Mann-Whitney test. Association between immunological parameters, in vitro and in vivo outcome202s were assumed to be non-Gaussian distribution and were analyzed by Spearman’s rank correlation. To determine whether there was a quality difference between Group 2 and 3 IgGs in GIA, a multiple regression analysis was performed. GIA level was used as a response variable, and AMA1 antibody level and Group as explanatory variables. For the relationship between levels AMA1–RON2L blocking antibodies and in vitro GIA or in vivo time-to-patency, IC 50 was calculated using a non-linear fit of normalized dose response curves for each sample containing a known amount (ELISA unit) of anti-AMA1 antibodies and analyzed by Spearman’s rank correlation.

The attending veterinarian and technicians who assisted or performed vaccination, parasite challenge, parasitemia counts, GIA, ELISA were all blinded to the vaccination groups. Data were unblinded during analysis by the primary investigators.

Data availability

All relevant data are available from the authors

Disclaimer

The views expressed in this article are those of the authors and do not necessarily reflect the views of USAID nor the official policy or position of the Department of the Navy, Department of Defense or the US Government.

Copyright statement

Several authors are employees of the U.S. Government and this work was prepared as part of official duties. Title 17 U.S.C. §105 provides that ‘Copyright protection under this title is not available for any work of the United States Government.’ Title 17 U.S.C. §101 defines a U.S. Government work as a work prepared by an employee of the U.S. Government as part of that person’s official duties.