falciparumsporozoites were metabolically labeled in Dulbecco’s modied Eagle’s medium (DMEM) without Cys/Met, 1% BSA and 400 Ci/mL L-[35S]-Cys/Met for 1 hour at 28C and then kept on ice or chased at 28C for 2 hours in DMEM with Cys/Met and 1% BSA in the presence of the indicated concentrations of mAb5D5, in the presence of the isotype control mAb3D11 (specific against theP. againstPlasmodiumshould include the CSP N-terminal region. Keywords:antibodies, CSP, malaria,P. falciparum, sporozoites, vaccine The circumsporozoite protein (CSP) is the major surface protein ofPlasmodiumsporozoites, forming a dense coat on the parasite’s surface. This protein plays a critical role in the journey ofPlasmodiumparasites from the mosquito to the mammalian host and it is expressed in sporozoites of rodent, primate, and humanPlasmodiumspecies. The basic structure of the CSP consists of a central repeat domain flanked by an N-terminal portion containing a conserved proteolytic cleavage site, and a C-terminal flanking region containing a type I thrombospondin repeat (TSR) motif. As the parasites migrate from the mosquito salivary glands to the liver of the mammalian host, the CSP undergoes major conformational changes [1,2]. Sporozoites injected into the skin display a folded CSP, such that the N-terminal region masks the C-terminal TSR. Upon reaching the liver, this protein undergoes proteolytic cleavage of the N-terminal domain, exposing the C-terminus region, in what appears to be a critical requirement for hepatocyte invasion by sporozoites [1,2]. CSP has been intensively studied as a vaccine candidate, as it is well established that antibodies and T-cell responses against this antigen inhibit infection in mammalian hosts and provide protective immunity in experimental animal models and humans [3,4]. The malaria vaccine candidate RTS,S is a virus-like particle consisting of the central repeat and C-terminal regions of theP. falciparumCSP fused to a hepatitis B virus surface antigen (HBVsAg). A number of phase 2 and 3 vaccine trials showed that 30%50% of children were protected after vaccination [59]. These studies also indicated that anti-CSP antibody titers specific for the repeat domain strongly correlate with the protection observed among vaccinees. The protective efficacy of the RTS,S vaccine candidate is encouraging and suggests that further improvements of this subunit vaccine may provide increased protection. Importantly, the RTS,S vaccine does not include the CSP N-terminal region, which contains important functional domains, such as a ligand-binding domain [10] and a proteolytic processing site [2], and may be an important target for protective antibodies. Here we describe the characterization of a monoclonal antibody, mAb5D5, which binds to a linear epitope located in the N-terminal region of theP. falciparumCSP and prevents its enzymatic processing. UsingP. bergheitransgenic parasite strains expressing a chimeric CSP that contains the N-terminal region ofP. falciparum[11], we demonstrate that mAb5D5 strongly inhibits sporozoite infection in vivo. Moreover, we show that combining mAb5D5 with antibodies against the repeat domain of theP. falciparumCSP greatly enhances the in vivo neutralization of sporozoites. Rabbit Polyclonal to ERI1 Taken together, these data demonstrate that the CSP N-terminus represents Picaridin a promising target for antibody-based protection against parasite infection of hepatocytes, and indicates that this CSP region should be included as part of a CSP-based, pre-erythrocytic vaccine construct. == MATERIALS AND METHODS == == Antibody Development == Antibodies were prepared by Precision Antibody, a division of A&G Pharmaceutical Inc (Columbia, Maryland) using proprietary technology and mouse immunization protocols. Mice were immunized with full-length recombinant CSP (rCSP) [12] and serum titers determined by enzyme-linked immunosorbent assay (ELISA). Splenocytes were harvested Picaridin and fused once titers exceeded 1:50 000. Hybridoma supernatants were screened for reactivity to rCSP by ELISA. A total of 14 clones were selected for monoclonal antibody (mAb) production Picaridin and immunoglobin G (IgG) was purified using a Protein G column followed by a buffer change to phosphate-buffered saline (PBS). == Epitope Specificity Analysis == The fine epitope specificity of mAb5D5 was determined using 2 types of ELISAs. Direct binding to shortP. falciparumCSP peptides. An ELISA with CSP 15-mer peptides overlapping.