Correctly folded Pfs48/45 protein of Plasmodium falciparum elicits malaria transmission-blocking immunity in mice

Abstract

Malaria kills >1 million people each year, in particular in subSaharan Africa. Although asexual forms are directly responsible for disease and death, sexual stages account for the transmission of Plasmodium parasites from human to the mosquito vector and therefore the spread of the parasite in the population. Development of a malaria vaccine is urgently needed to reduce morbidity and mortality. Vaccines against sexual stages of Plasmodium falciparum are meant to decrease the force of transmission and consequently reduce malaria burden. Pfs48/45 is specifically expressed in sexual stages and is a well established transmissionblocking (TB) vaccine candidate. However, production of correctly folded recombinant Pfs48/45 protein with display of its TB epitopes has been a major challenge. Here, we show the production of a properly folded Pfs48/45 C-terminal fragment by simultaneous coexpression with four periplasmic folding catalysts in Escherichia coli. This C-terminal fragment fused to maltose binding protein was produced at medium scale with >90% purity and a stability over at least a 9-month period. It induces uniform and high antibody titers in mice and elicits functional TB antibodies in standard membrane feeding assays in 90% of the immunized mice. Our data provide a clear perspective on the clinical development of a Pfs48/45-based TB malaria vaccine. M alaria parasites are spread in the population by Plasmodium-infected Anopheles mosquitoes. Successful transmission of malarial parasites from humans to mosquitoes depends on the presence and infectiousness of gametocytes in the peripheral blood and the number of Anopheles mosquitoes in the area. Transmission of Plasmodium falciparum can be blocked inside the mosquito by antibodies that have been ingested together with the gametocytes as part of a blood meal, interrupting the sporogonic cycle inside the mosquito (1).Pfs48/45 is a transmission-blocking (TB) target protein expressed by gametocytes (2-4) and present on the surface of the sporogonic (macrogametes) stages of the malaria parasites. Pfs48/45 plays a key role in parasite fertilization (5) and antibodies that exclusively target conformational epitopes of Pfs48/45 protein prevent fertilization (6, 7). Furthermore, antiPfs48/45 antibodies are present in human sera from endemic areas (8) and correlate with TB activity (8-10). The induction of antibodies after natural infection as observed in the field creates the highly beneficial potential of vaccine boosting in the endemic setting. TB vaccines might be applied alone or more likely as part of a combination vaccine or package of control measures depending on the intensity of malaria transmission (11).A strategy for vaccine development requires the production of correctly folded recombinant Pfs48/45 protein. Proper folding of many cysteine-rich proteins, including Pfs48/45, depends on correct formation of disulphide bridges. In eukaryotes the oxidizing environment of the endoplasmic reticulum (ER) provides a milieu for disulphide bonds formation. Plasmodium p...

Structure of thePlasmodium6-cysteine s48/45 domain

“…To date, no experimentally determined molecular structure for any s48/45 domain has been reported, mainly because of proteinexpression challenges (29)(30)(31)(32)(33)(34). However, considering the importance of structural information in the effective design of subunit vaccine candidates, we present the solution three-dimensional structure of the s48/45 domain obtained by NMR spectroscopy of the C-terminal domain of Pf12 expressed in Escherichia coli.…”

Fertilization Mechanisms of the Rodent Malarial Parasite Plasmodium berghei

“…Such a requirement for correct folding of recombinant TBV proteins for effective antibody production has been observed in cases in which the TBV candidate is produced in yeast and algal systems (Kaslow et al 1994 ;Gregory et al 2013 ). In addition to this problem, the immunization protocol needs to be improved to generate higher antibody titers (Kubler-Kielb et al 2007 ;Outchkourov et al 2008 ).…”

Transgenesis, Paratransgenesis and Transmission Blocking Vaccines to Prevent Insect-Borne Diseases

“…Immunization of mice with this recombinant protein led to production of antibody titers that were capable of reducing P. falciparum oocyst intensity in An. stephensi by at least 88% in 11 out of 12 assays (Outchkourov et al 2008). …”

“…P. falciparum proteins Pfs25, Pfs28, Pfs48/45, and Pfs230, and their orthologs in Plasmodium vivax, have been tested in transmission-blocking assays (Quakyi et al 1987;Kaslow et al 1988;Duffy & Kaslow 1997;Hisaeda et al 2000;Sattabongkot et al 2003;Malkin et al 2005;Outchkourov et al 2008). …”