Shwu‐Maan Lee

Single-chain antigen-binding proteins are novel recombinant polypeptides, composed of an antibody variable light-chain amino acid sequence (VL) tethered to a variable heavy-chain sequence (VH) by a designed peptide that links the carboxyl terminus of the VL sequence to the amino terminus of the VH sequence. These proteins have the same specificities and affinities for their antigens as the monoclonal antibodies whose VL and VH sequences were used to construct the recombinant genes that were expressed in Escherichia coli. Three of these proteins, one derived from the sequence for a monoclonal antibody to growth hormone and two derived from the sequences of two different monoclonal antibodies to fluorescein, were designed, constructed, synthesized, purified, and assayed. These proteins are expected to have significant advantages over monoclonal antibodies in a number of applications.

The DOPA-rich polyphenolic protein secreted by the marine mussel Mytilus edulis establishes key chemical linkages in a water-resistant adhesive. Molecular cloning of the gene for this remarkable protein reveals its primary structure as one of the most repetitive proteins identified in the animal kingdom. Expression and purification of polyphenolic proteins from recombinant yeast have provided sufficient material to demonstrate adhesivity of these polypeptides in the laboratory. Adhesive tests reveal a water-resistant bonding capacity of the protein that is dependent on in vitro modification of tyrosine residues to DOPA and the subsequent oxidation to quinone.

gametocytes. A recombinant, his-tagged Pfs230 fragment (Pfs230C1; amino acids 443-731) formed serum-stable particles upon incubation with liposomes containing cobalt-porphyrin-phospholipid (CoPoP). In mice, immunization with Pfs230C1, admixed with the adjuvants Alum, Montanide ISA720 or CoPoP liposomes (also containing synthetic monophosphoryl lipid A; PHAD), resulted in elicitation of IgG antibodies, but only those induced with CoPoP/PHAD or ISA720 strongly reduced parasite transmission. Immunization with micrograms of Pfs230C1 adjuvanted with identical liposomes lacking cobalt (that did not induce particle formation) or Alum was less effective than immunization with nanograms of Pfs230C1 with CoPoP/PHAD. CoPoP/PHAD and ISA720 adjuvants induced antibodies with similar Pfs230C1 avidity but higher IgG2-to-IgG1 ratios than Alum, which likely contributed to enhanced functional activity. Unlike prior work with another transmission-blocking antigen (Pfs25), Pfs230C1 was found to be effectively taken up by antigen-presenting cells without particle formation. The anti-Pfs230C1 IgG response was durable in mice for 250 days following immunization with CoPoP/PHAD, as were antibody avidity and elevated IgG2-to-IgG1 ratios. Immunization of rabbits with 20 µg Pfs230C1 admixed with CoPoP/PHAD elicited antibodies that inhibited parasite transmission. Taken together, these results show that liposomes containing CoPoP and PHAD are an effective vaccine adjuvant platform for recombinant malaria transmission blocking antigens.