The mAb 4G2dc1 only recognizes conformational epitopes of AMA1, suggesting that AMA1 remained correctly folded after conjugation

The mAb 4G2dc1 only recognizes conformational epitopes of AMA1, suggesting that AMA1 remained correctly folded after conjugation. mice for the poor immunogen (Pfs25) and for the larger protein (AMA1). These conjugates now need to be tested in humans to determine if mice are predictive of the response in humans. Introduction While there are several potent experimental adjuvants in the literature many have a limited safety profile in humans, are not of suitable quality for clinical use or access to the clinical grade material is restricted [1C3]. However, carrier proteins, such as Diphtheria Toxoid, CRM197 (a nontoxic mutant of diphtheria toxin), OMPC (outer membrane protein complex of serogroup B), and Tetanus Toxoid, have been shown as part of effective licensed vaccines to be safe in humans [4C7]. Conjugating bacterial polysaccharides to carrier proteins has dramatically improved the immunogenicity and efficacy especially in children under the age of two [8]. However, the ease of manufacture and access to these clinical grade carrier proteins is often difficult and limited. The mutant, nontoxic carrier protein, recombinant ExoProtein A (rEPA), from is easily expressed and purified in high yields as a soluble protein from [9] Difloxacin HCl and is in the public domain. Recombinant EPA has been conjugated to the capsular polysaccharide, Vi, of serovar Typhi and shown to be more than 90 % efficacy of typhoid fever reduction in 2C5 year old children Mouse monoclonal to Myostatin [10, 11]. While immunologists for many years have conjugated poorly immunogenic peptides and some proteins to carrier proteins for research [12C17], little is known in vaccinology about the ability to conjugate large recombinant antigens covalently to carrier proteins to improve immunogenicity and to elicit functional antigen-specific antibodies apical membrane antigen 1 (AMA1), which is an integral membrane protein with a molecular weight of 83 kDa [24, 25]. The AMA1 protein is synthesized by parasites late in schizogony, and it is initially located in an apical organelle of merozoites (micronemes). During invasion, the protein is processed to a 66 kDa product and relocated onto the surface of merozoites. The other model protein is the surface protein 25 (Pfs25) which is a 25 kDa protein expressed by parasites at the onset Difloxacin HCl of zygote formation in the mosquito midgut [26]. Here we demonstrate that AMA1 and Pfs25 can be conjugated Difloxacin HCl to rEPA successfully. AMA1-rEPA and Pfs25-rEPA, when formulated on Alhydrogel, elicited significantly higher antibody responses compared to when unconjugated AMA1 and Pfs25 were formulated on Alhydrogel alone. Furthermore, the conjugation process did not destroy critical epitopes required to elicit a functional immune response. Purified IgG from mice immunized with AMA1-rEPA inhibited invasion of into red cells. The Pfs25-rEPA mouse immune sera inhibited oocyst formation in the midgut of the mosquito. Materials and Methods Expression and purification of recombinant mutant of ExoProtein A The rEPA expression plasmid, pVC45D/PE553D, was provided by Joseph Shiloach (NIDDK, NIH) [9, 27]. For bench scale expression and production of the lot used in the mouse immunogenicity studies, cells BL21(DE3) were transformed, and overnight colonies were used to inoculate 1 L of LB Broth which was then grown at 37C Difloxacin HCl in a rotating shaker at 250 rpm until the A600 reached an OD of 0.6. Isopropyl -D-1-thiogalactopyranoside was added to the culture to a final concentration of 1 1 mM to induce the expression of the recombinant rEPA. After 3 h induction, the supernatant of the culture was harvested by centrifugation, from which the rEPA was purified using one step of hydrophobic interaction chromatography (Phenyl Sepharose 6 Fast Flow, GE Healthcare, Piscataway, NJ) and two steps of anion exchange (DEAE Sepharose Fast Flow and SOURCE 30Q, GE Healthcare, Piscataway, NJ). For scale up and large scale production of rEPA the modified rEPA gene was cloned into the kanamycin resistant expression plasmid pET-24a(+) (Novagen, San Diego, CA). The gene was amplified using pVC45D/PE553D as template and exoA-NF (5-GGGCAACATATGAAAAAGACAGCTATCGCG-3) and exoA-SR (5-CCGGTCGGAGCTCTTACTTCAGG-3) as primers. The production clone was fermented with defined media in 5L bioreactors as.