A C-terminal Pfs48/45 malaria transmission-blocking vaccine candidate produced in the baculovirus expression system

Shwu-Maan Lee,Jordan L Plieskatt,C Richter King,John M Hickey,Sangeeta B Joshi,David B Volkin,Kazutoyo Miura

doi:10.1038/s41598-019-57384-w

Abstract

The Plasmodium falciparum gametocyte surface protein, Pfs48/45, is a potential target for malaria transmission-blocking vaccines. However, due to its size and complexity, expression of the full-length protein has been difficult, leading to focus on the C-terminal six cysteine domain (6C) with the use of fusion proteins to facilitate expression and folding. In this study, we utilized the baculovirus system to evaluate the expression of three Pfs48/45 proteins including the full-length protein, the 6C domain fragment and the 6C domain mutant to prevent glycosylation. Expression of the recombinant Pfs48/45 proteins was conducted in super Sf9 cells combined with the use of tunicamycin to prevent N-glycosylation. The proteins were then evaluated as immunogens in mice to demonstrate the induction of functionally active polyclonal antibody responses as measured in the standard membrane feeding assay (SMFA). Only the 6C protein was found to exhibit significant transmission-reducing activity. Further characterization of the biologically active 6C protein demonstrated it was homogeneous in terms of size, charge, conformation, absence of glycosylation, and containing proper disulfide bond pairings. This study presents an alternative expression system, without the need of a fusion protein partner, for the Pfs48/45 6C protein fragment including further evaluation as a potential transmission-blocking vaccine candidate.

Highlights

The Plasmodium falciparum gametocyte surface protein, Pfs48/45, is a potential target for malaria transmission-blocking vaccines
Development attention has shifted to the transmission-blocking vaccines (TBVs) candidates Pfs[230] and Pfs48/45 in view of the poor outcomes associated with Pfs25-based constructs and the fact that sera from Plasmodium infected individuals are associated with transmission-blocking antibodies attributable to these gametocyte surface antigens[7]
The insect cells used in the baculovirus system have active glycosylation machinery while the evidence to date is that P. falciparum parasites do not[21]

Summary

Introduction

The Plasmodium falciparum gametocyte surface protein, Pfs48/45, is a potential target for malaria transmission-blocking vaccines. Affinity purified anti-Pfs48/45 and anti-Pfs[230] antibodies from naturally exposed individuals can prevent the transmission of cultured P. falciparum gametocytes[8], when concentrated nine times the physiological concentration, thereby demonstrating the functionality of these natural antibody responses and the potential for these antigens in TBV development. To design a recombinant TBV, Outchkourov et al analyzed the transmission-blocking (TB) epitopes on the Pfs48/45 protein and determined the C-terminal six-cysteine module termed 6C was recognized by the 85RF45.1 monoclonal antibody[17]. Biochemical characterization has been well reported for R0.6C, with a final purified yield of 25 mg/L culture as well as the ability to elicit functional antibodies in rats[20] While this approach is promising, we sought to generate Pfs48/45 antigens that might focus the immune response onto the 6C region alone without fusion partners

Methods

Results

Conclusion