Transmission-reducing and -enhancing monoclonal antibodies against Plasmodium vivax gamete surface protein Pvs48/45

Abstract

Gamete surface protein P48/45 has been shown to be important for male gamete fertility and a strong candidate for the development of a malaria transmission-blocking vaccine (TBV). However, TBV development for Plasmodium vivax homolog Pvs48/45 has been slow because of a number of challenges: availability of conformationally suitable recombinant protein; the lack of an in vivo challenge model; and the inability to produce P. vivax gametocytes in culture to test transmission-blocking activity of antibodies. To support ongoing efforts to develop Pvs48/45 as a potential vaccine candidate, we initiated efforts to develop much needed reagents to move the field forward. We generated monoclonal antibodies (mAbs) directed against Pvs48/45 and characterized putative functional domains in Pvs48/45 using recombinant fragments corresponding to domains D1-D3 and their biological functionality through ex vivo direct membrane feeding assays (DMFAs) using P. vivax parasites from patients in a field setting in Brazil. While some mAbs partially blocked oocyst development in the DMFA, one mAb caused a significant enhancement of the infectivity of gametocytes in the mosquitoes. Individual mAbs exhibiting blocking and enhancing activities recognized non-overlapping epitopes in Pvs48/45. Further characterization of precise epitopes recognized by transmission-reducing and -enhancing antibodies will be crucial to design an effective immunogen with optimum transmission-reducing potential.

Keywords: Anopheles darlingi; Plasmodium vivax; Pvs48/45; monoclonal antibodies; transmission-blocking vaccine; transmission-enhancing activity; transmission-reducing activity.

Fig 1

Immunofluorescence reactivity of representative mAbs. Parasites were fixed with 4% paraformaldehyde (not permeabilized) prior to incubation with various mAbs. Purified IgG from NMS was used as a negative control. Cells were counter stained with Hoechst 33342 prior to fluorescent examination. Shown are corresponding representative panels (Hoechst, Alexa Fluor 488, and Merged). The inset in the NMS Alexa Fluor panel also shows a bright field image of the parasite.

Fig 2

Schematic representation of single and double domains of Pvs48/45. All the cysteine residues were predicted to form disulfide bonds, except for one unpaired cysteine in D1 and D1D2 fragments. Signal sequence (SS, residues 1–27) and glycosylphosphatidylinositol (GPI) membrane anchor (residues 438–450) are shown in full-length Pvs48/45 scheme.

Fig 3

DMFA with mAbs revealing mixed minimal effects. All the mAbs (+) and NMS IgG (−) were tested at 0.5 mg/mL final concentration using blood from different donors identified by D number above each set. The total number of mosquitoes dissected varied between 29 and 31. The values below each data panel also present mean number of oocysts and percent prevalence of infected mosquitoes. Results were analyzed using Kruskal-Wallis test followed by Dunn’s multiple comparison test, and P-values are indicated.

Fig 4

DMFA with mAbs 47 and 5.55.5 exhibiting enhancing and reducing activities. The mAbs (+) and NMS IgG (−) were tested at 0.5 mg/mL final concentration using blood from different donors identified by D number above each set. The total number of mosquitoes dissected varied between 29 and 31 for each feed. The values below each data panel also present mean number of oocysts and percent prevalence of infected mosquitoes. Results were analyzed using Kruskal-Wallis test followed by Dunn’s multiple comparison test, and P-values are indicated.

Fig 5

Competition between mAb 47 and mAb 5.55.5 for binding to Pvs48/45 in an ELISA. Panel (A) shows binding of HRP-conjugated mAb 5.55.5 in the presence of increasing ratio of unlabeled mAb 47 (dotted line) or unlabeled mAb 5.55.5 (solid line). Panel (B) shows binding of HRP-conjugated mAb 47 in the presence of increasing molar ratio of unlabeled mAb 5.55.5 (dotted line) or unlabeled mAb 47 (solid line). The assays were done in duplicate wells and repeated two times and the panels show data from a representative experiment.

Fig 6

Binding of HRP-mAb 47 and HRP-mAb 5.55.5 to Pvs48/45 in an ELISA in the presence of excess of all the mAbs. All the competing unlabeled mAbs identified on x-axis were tested at 25- to 50-fold excess concentration. Panel (A) shows binding of HRP-conjugated mAb 5.55.5 in the presence of unlabeled competing mAbs. Panel (B) shows binding of HRP-conjugated mAb 47 in the presence of competing unlabeled mAbs. Figure shows results with 50-fold excess of competing mAbs with similar pattern revealed when competing mAbs were tested at 25-fold molar excess.