ABSTRACT
An effective vaccine is needed for the prevention and elimination of malaria. The only immunogens that have been shown to have a protective efficacy of more than 90% against human malaria are Plasmodium falciparum (Pf) sporozoites (PfSPZ) manufactured in mosquitoes (mPfSPZ)1-7. The ability to produce PfSPZ in vitro (iPfSPZ) without mosquitoes would substantially enhance the production of PfSPZ vaccines and mosquito-stage malaria research, but this ability is lacking. Here we report the production of hundreds of millions of iPfSPZ. iPfSPZ invaded human hepatocytes in culture and developed to mature liver-stage schizonts expressing P. falciparum merozoite surface protein 1 (PfMSP1) in numbers comparable to mPfSPZ. When injected into FRGhuHep mice containing humanized livers, iPfSPZ invaded the human hepatocytes and developed to PfMSP1-expressing late liver stage parasites at 45% the quantity of cryopreserved mPfSPZ. Human blood from FRGhuHep mice infected with iPfSPZ produced asexual and sexual erythrocytic-stage parasites in culture, and gametocytes developed to PfSPZ when fed to mosquitoes, completing the P. falciparum life cycle from infectious gametocyte to infectious gametocyte without mosquitoes or primates.
Subject(s)
Plasmodium falciparum , Sporozoites , Animals , Humans , Mice , Culicidae/parasitology , Malaria/parasitology , Malaria/prevention & control , Malaria Vaccines/biosynthesis , Malaria Vaccines/chemistry , Malaria, Falciparum/parasitology , Plasmodium falciparum/growth & development , Sporozoites/growth & development , Sporozoites/pathogenicity , Hepatocytes/parasitology , Liver/parasitology , Merozoite Surface Protein 1 , Erythrocytes/parasitology , In Vitro TechniquesABSTRACT
Staphylococcal extracellular polymeric substances (EPS) such as extracellular DNA (eDNA) and poly-N-acetylglucosamine surface polysaccharide (PNAG) mediate numerous virulence traits including host colonization and antimicrobial resistance. Previous studies showed that EPS-degrading enzymes increase staphylococcal biocide susceptibility in vitro and in vivo, and decrease virulence in animal models. In the present study we tested the effect of EPS-degrading enzymes on staphylococcal skin colonization and povidone iodine susceptibility using a novel in vivo pig model that enabled us to colonize and treat 96 isolated areas of skin on a single animal in vivo. To quantitate skin colonization, punch biopsies of colonized areas were homogenized, diluted, and plated on agar for colony forming unit enumeration. Skin was colonized with either Staphylococcus epidermidis or Staphylococcus aureus. Two EPS-degrading enzymes, DNase I and the PNAG-degrading enzyme dispersin B, were employed. Enzymes were tested for their ability to inhibit skin colonization and detach preattached bacteria. The effect of enzymes on the susceptibility of preattached S. aureus to killing by povidone iodine was also measured. We found that dispersin B significantly inhibited skin colonization by S. epidermidis and detached preattached S. epidermidis cells from skin. A cocktail of dispersin B and DNase I detached preattached S. aureus cells from skin and increased their susceptibility to killing by povidone iodine. These findings suggest that staphylococcal EPS components such as eDNA and PNAG contribute to skin colonization and biocide resistance in vivo. EPS-degrading enzymes may be a useful adjunct to conventional skin antisepsis procedures in order to further reduce skin bioburden.
