Bacterial Virus Lambda Gpd-Fusions to Cathelicidins, α- and ß-Defensins, and Disease-Specific Epitopes Evaluated for Antimicrobial Toxicity and Ability to Support Phage Display.

We showed that antimicrobial polypeptides, when translated as gene fusions to the bacteriophage lambda capsid decoration protein gpD, formed highly toxic molecules within E. coli, suggesting that they can retain their antimicrobial activity conformation when fused to gpD. These include gpD-fusions to human and porcine cathelicidins LL37 and PR39, ß-defensins HBD3 and DEFB126-Δ (deleted for its many COOH-terminal glycosylation sites), and α-defensin HD5. Antimicrobial toxicity was only observed when the peptides were displayed from the COOH-terminal, and not the NH2-terminal end, of gpD. This suggests that COOH-terminal displayed polypeptides of gpD-fusions can more readily form an active-state conformation than when they are displayed from the NH2-terminal end of gpD. The high toxicity of the COOH-displayed gpD-defensins suggests either that the fused defensin peptides can be oxidized, forming three correct intramolecular disulfide bonds within the cytosol of bacterial cells, or that the versions without disulfide bonds are highly toxigenic. We showed the high efficiency of displaying single epitope 17 amino-acid fusions to gpD on LDP (lambda display particles), even when the gpD-fusion protein was toxic. The efficient formation of high display density LDP, displaying a single disease specific epitope (DSE), suggests the utility of LDP-DSE constructs for use as single epitope vaccines (SEV).

Lambda display phage as a mucosal vaccine delivery vehicle for peptide antigens.

Bacteriophage are structurally stable in the gastro-intestinal tract and have favorable traits of safety, stability, ease of production, and immunogenicity. These attributes make them potential candidates as oral vaccine delivery vehicles but little is known about their capacity to induce mucosal immune responses in the small intestine. Whole body imaging of mice confirmed lambda bacteriophage (LP) were distributed throughout the gastro-intestinal tract 24 h after oral delivery. In newborn calves, targeted delivery of LP within the small intestine confirmed LP were immunogenic in a dose-dependent manner and were taken up by Peyer's patches. LP-specific IgA responses were induced within both Peyer's patches and draining mesenteric lymph nodes. A lambda display phage (LDP) was constructed to present three immunogenic disease specific epitopes (DSE) from cervid prion protein (amino acids 130-140 [YML]; 163-170 [YRR]; and 171-178[YRR]) fused to phage capsid head protein D (LDP-DSE). Targeted delivery of purified LDP-DSE to intestinal segments induced IgA responses to all three peptide epitopes. Further, delivery of bacteria expressing soluble D-DSE also induced epitope-specific IgA responses in the targeted Peyer's patches. These are the first studies to report use of LDP to induce epitope-specific IgA responses in the small intestine andconfirm Peyer's patchesfunction as a site for LP uptake. Furthermore, IgA responses to peptide epitopes on LDP were observed in the absence of a mucosal adjuvant. These observations confirm LDP have the capacity to function as a mucosal delivery vehicle with protein D as an effective carrier for peptide epitopes.

Dual expression system for assembling phage lambda display particle (LDP) vaccine to porcine Circovirus 2 (PCV2).

The bacteriophage lambda small capsid protein D forms trimers on the phage head. D-fusion polypeptides can be expressed from plasmids in E. coli and remain soluble without aggregation. We report a dual expression system for the display of four immunodominant regions of porcine Circovirus 2 (PCV2) capsid protein (CAP) as D-CAP fusions on lambda display particles (LDP). The LDP-D-CAP preparation proved an effective vaccine in pigs, eliciting both cellular and humoral immune responses and PCV2 neutralizing antibodies. In our dual system wild type D expression was encoded by a heteroimmune infecting phage. The D-fusion protein expression in the infected cells was from an inducible plasmid, enabling the deferral of D-fusion expression until needed. The effective vaccine preparation depended upon the gradient purification of very high concentration, essentially tail-less display particles, not previously described.

Blocking the T4 lysis inhibition phenotype.

Nonlysogenic Escherichia coli K cells exhibit a delay in lysis when infected by T4rII phage termed lysis inhibition (LIN). E. coli K cells expressing lambda rexB from either a prophage defective for rexA, or a multicopy plasmid supported T4rII infection, but prevented the establishment of LIN. In addition, E. coli null mutations in either the periplasmic "tail-specific protease" tsp, or the 10Sa RNA ssrA, completely blocked the establishment of LIN following T4 infections. The expression of rexB in the absence of rexA resulted in several cellular phenotypes, including aberrant cell surface morphology, the partial to near complete suppression of mutations of lambda S and T4t holin genes, and lysis by cells aging on plates or growing with high rexB expression at elevated temperatures. These activities of RexB were impeded in the presence of RexA.