Malaria is still a disease of immense public health importance due to its prevalence and high rates of mortality in the tropics and developing countries, especially in children under five [
1]. Global efforts to eliminate the disease have led to an increase in the search for transmission reducing interventions that target the parasite stage required for transmission, called gametocytes, as well as the mosquito vectors. Currently, the main focus of elimination and pre-elimination control measures is to decrease the mosquito vector population as well as control parasitaemia by prompt treatment with effective anti-malarial. Unfortunately, most commonly used anti-malarials, including artemisinin-based combination therapy (ACT), do not effectively eliminate gametocytes and in endemic countries there are also many individuals with asymptomatic infections that never seek treatment and serve as an infectious reservoir. Control efforts would be greatly enhanced by the development of effective vaccines, but to date these efforts have been complicated by antigenic diversity both within and between the distinct stages of the parasite that develop in the human host.
One current strategy is to develop a multi-component vaccine that targets antigens found at different parasite stages, including the stages required for transmission [
1].
Pfs48/45 and
Pfs230 are sexual stage antigens that are expressed on the surface of the intraerythrocytic,
Plasmodium falciparum gametocyte and become exposed on the surface of extracellular gametes in the mosquito midgut [
2]. Within 10 min after a gametocyte-infected erythrocyte is taken up in a blood meal by a mosquito, the parasite emerges as an extracellular gamete. Emergence exposes surface-antigens, such as
Pfs48/45 and
Pfs230, to antibodies also present in the human blood meal. Both antigens elicit an immune response during a natural infection [
3] and
Pfs48/45 or
Pfs230-specific antibodies have been shown to block transmission to mosquitoes in a standard membrane feeding assay (SMFA) [
4‐
7]. However, despite their potentially important role in malaria transmission, these antigens have not been included in many field studies due the lack of properly-folded recombinant proteins. Therefore, relatively little is known about the epidemiology of the host response against these antigens. Prior studies have used two-site or competition ELISAs to capture antigen from gametocyte extract using mAb then assaying for the ability of antibodies in human serum or plasma samples to bind or to compete with the binding of a second antibody, respectively [
8‐
10]. These are elegant assays to test for antibody responses to defined epitopes, but require access to in vitro cultured parasites and monoclonal antibodies (mAb) making them difficult to include in most field studies. Additionally, the epitope recognized by the capture mAb is blocked and not accessible to the test antibodies. An alternative is to generate properly folded recombinant protein and test directly for immunoreactivity; however
Pfs48/45 and
Pfs230 both contain multiple 6-cysteine (6-cys) domain motifs which complicates the expression of correctly-folded recombinant proteins in heterologous systems [
11‐
13]. The inclusion of a fusion partner such as maltose binding protein (MBP) [
14] or
P. falciparum glutamate rich protein (GLURP.R0) [
11,
15] facilitates folding and expression of the 6-cys component. However, both GLURP.R0 and MBP are themselves immunogenic, requiring the antibody responses generated in an ELISA against the fusion partner to be subtracted from the overall response to the fusion protein [
16].
Pfs230 has also been expressed using plant or cell-free wheat germ agglutinin expression systems, but both these methods require specialized techniques that are difficult to scale up [
17,
18]. As an economical alternative to these methods, recombinant proteins that were specific for
Pfs48/45 or
Pfs230 were designed and produced using the
Lactococcus lactis (
L. lactis) expression system. This expression system has recently been used to produce properly folded chimeric GMZ2.48/45, which includes
Pfs48/45 amino acid residues (aa 291–428), that induces transmission blocking antibodies in rats and is being developed as a vaccine candidate [
11,
13]. A Tobacco Etch Virus (TEV) protease site was introduced between GMZ2 and
Pfs48/45-6C components of the chimeric GMZ2.48/45 to allow the efficient removal of the fusion partner. The
L. lactis expression system was also used to produce the N-terminal region (amino acid residues 443–590) of the processed form of
Pfs230 that is retained on the gamete surface after erythrocyte emergence [
17]. Previous work demonstrated that when this region of
Pfs230, called C0, was produced in the wheat germ cell-free system the C0 recombinant protein induced antisera in mice that reduced the infectivity of
P. falciparum to
Anopheles stephensi mosquitoes in the presence of complement [
17].