Transmission-blocking activity of antibodies to Plasmodium falciparum GLURP.10C chimeric protein formulated in different adjuvants

Construction of the recombinant R0.10C hybrid molecule, a fusion protein containing the regions GLURP_27–500 and Pfs48/45_{159– 428} and a stretch of six histidines in the C-terminus, has been described in detail elsewhere [19]. The theoretical molecular weight of the protein is 89.8 kDa of which 30.7 kDa originates for the 10C fragment.

All fermentations at the 40L scale were done at the cGMP-facility of Gennova, India, using the standard proposed medium composition as described earlier [19]. Briefly, a fermentor containing reconstituted medium with the final pH 7.0, adjusted with sterile 2 N NaOH, was inoculated with overnight culture of inoculum. The fermenter was programmed to maintain pH 6.5, temperature 30 °C at the low agitation rate (100 rpm) without aeration, maintaining anaerobic conditions. At the stationary phase when no more alkali (2 N NaOH) uptake was observed to maintain pH 6.5, the culture was harvested from the fermenter. Following clarification of the fermentation broth by centrifugation, the culture supernatant was concentrated five to six times using tangential flow filtration (TFF) and diafiltered using 2.5 sq m membrane with pore size cut-off of 10 kDa with buffer-1 (50 mM phosphate buffer, pH 7.0, 6 diafiltration volumes (DV) followed by buffer-2 (50 mM phosphate buffer, pH 7.0 and 0.25 M NaCl, 6 DV). The diafiltrate, now defined as ‘affinity-input’ containing 10 mM imidazole and 5 mM β-ME, was loaded on the Ni2+-Sepharose column pre-equilibrated with 4CV of equilibration buffer (50 mM phosphate buffer, pH 7.0, 0.25 M NaCl, 20 mM Imidazole). After loading affinity-input, the column was washed with 4 column volumes (CV) of equilibration buffer, followed by washing with redox-buffer of glutathione exists in both 4 mM reduced (GSH) and 0.4 mM oxidized (GSSG) states for 60 min. Following redox-wash, the recombinant R0.10C was eluted isocratically with the elution buffer containing 50 mM phosphate buffer, 0.25 M NaCl, 200 mM Imidazole, pH 7.0. The peak fraction was finally purified on anti-Pfs48/45 mAb 45.1 (epitope 1) coupled to agarose using the AminoLink^® Plus Immobilization Kit (Pierce: 44894) according to the instructions of the manufacturers. The eluate contained immune purified R0.10C (correctly folded) whereas the run-through contained <1 % of correctly folded R0.10C. Eluted samples were concentrated to at least 1 mg/ml against 1× phosphate buffered saline (PBS) on a Vivaspin20, 30-kDa molecular mass cut-off ultra-filtration unit (Vivascience, Germany). Protein concentrations were measured on a NanoDrop 2000 (Thermo Scientific, USA) using a molar extinction coefficient of 0.3 and Bradford’s method.

Experimental animals, including female Wistar Hanover rats and female CB6F1 (BALB/c × C57BL/6 F1 hybrid) mice, were selected for the study. The animals were purchased from Charles River and kept in the Laboratory Animal Facility Centre of the Radboud University Medical Centre (RUMC). All procedures regarding animal immunization complied with European and national regulations.

Immunizations were performed by the subcutaneous route three times, 2 weeks apart with 20 µg of R0.10C adjuvanted with different formulations in a total volume of 100 µl for mice and 200 µl for rats.

Rats receiving adjuvant formulations aluminium hydroxide [(Alhydrogel, Brenntag (Alum: SSI (expiry date June 2012)], Alum/GLA, SE/GLA and AbISCO-100 and for mice complete Freund’s adjuvant (CFA)/incomplete Freund’s adjuvant (IFA) (Sigma-Aldrich), Alum/GLA, SE/GLA and AbISCO-100 (ISCONOVA). The Freund’s group was used for reasons of comparison with the former MBP-PF10C immunizations previously described [6]. For the Alum-based formulation, groups of five animals were immunized with R0.10C adsorbed to Alum with or without a supplement of 5 µg of an aqueous formulation of GLA (IDRI:AQ014). For the SE-based immunization, groups of five animals were immunized with R0.10C and SE (IDRI:EM030) supplemented with 5 µg of GLA in SE (IDRI:EM031). The components of the Alum and SE-based formulations were mixed gently for 30 min and allowed to rest on ice for 1 h before injection. Sera were collected from peripheral blood samples on days 0, 14, 28, and 42 separated by centrifugation of blood at 1500×g for 10 min and stored at −20 °C until further analysis.

Antibody responses in immunized animals were measured by ELISA. All experimental steps, except for coating, were performed at room temperature (RT). Briefly, ELISA MaxiSorp microtitre plates (Sterilin^® ELISA plates, The Netherlands) were coated at 4 °C overnight with 50 μl of the R0.10C (2 µg/ml), GLURP.RO (1.4 µg/ml) or MBP.10C (2 µg/ml) in PBS pH 7.4. Wells were then blocked with 5 % skimmed milk powder in PBS for 1 h followed by three washings with PBS containing 0.05 % Tween-20 (PBST). Plates were incubated with 50 μL two-fold serial dilutions of sera starting with 1:100 in PBST for 4 h at RT. After washing, the plates were incubated with 50 μL of 1:3000 diluted rabbit-anti rat IgG-HRP (H + L) (DAKO, 1:3,000 diluted in PBST) or rabbit anti-mouse IgG-HRP (DAKO, 1:4,000 diluted in PBST) for 2 h. Wells were washed with PBS and subsequently incubated with tetramethyl benzidine (TMB) substrate solution for 20 min. The colour reaction was stopped with 0.2 N H₂SO₄, and the optical density (OD) was read at 450 nm in an Anthos 2001 Microplate Reader (Labtec BV). Midpoint (EC50) values were calculated using GraphPad Prism, (GraphPad Software, USA). MBP.10C is used for comparison of results with the data as described by Outchkourov et al. [6]. The discrepancy between the antibody titer measured in the R0.10C and MBP.10C ELISAs is due to antibodies against the R0 region of R0.10C.

Antisera obtained from mice and rats immunized with R0.10C were assessed for TB activity by SMFA as previously described [29, 30]. Briefly, 30 μl of rat or mouse serum was mixed with 90 μl of naïve human serum and 150 μl of in vitro gametocyte cultures of the P. falciparum (NF54) and for comparison (NF135.C10 Cambodian line [31]) laboratory lines. The mixture was fed to Anopheles stephensi mosquitoes through a membrane feeding apparatus. Pre-immune sera served as the controls. Fully engorged mosquitoes were separated and held at 26 °C. Seven days later, midguts of 20 mosquitoes were examined for oocysts. The observed TB activity of serum (TRA) was determined as the percentage reduction in the arithmetic mean oocyst number in test samples compared to that in controls. The experiment was considered valid when at least 85 % of the mosquitoes feeding on control sera were infected. Data were analysed by a non-parametric test by comparing the medians of two groups using the Mann–Whitney test, or by comparing three or more groups using the Kruskal–Wallis test, followed by post-tests. If significance was indicated, Dunn’s analysis was used for comparison with the control group.