Proteome-wide screening of the European porcine reproductive and respiratory syndrome virus reveals a broad range of T cell antigen reactivity.

The porcine reproductive and respiratory syndrome virus (PRRSV) is a rapidly evolving and diversifying pathogen necessitating the development of improved vaccines. Immunity to PRRSV is not well understood although there are data suggesting that virus-specific T cell IFN-γ responses play an important role. We therefore aimed to better characterise the T cell response to genotype 1 (European) PRRSV by utilising a synthetic peptide library spanning the entire proteome and a small cohort of pigs rendered immune to PRRSV-1 Olot/91 by repeated experimental infection. Using an IFN-γ ELISpot assay as a read-out, we were able to identify 9 antigenic regions on 5 of the viral proteins and determine the corresponding responder T cell phenotype. The diversity of the IFN-γ response to PRRSV proteins suggests that antigenic regions are scattered throughout the proteome and no one single antigen dominates the T cell response. To address the identification of well-conserved T cell antigens, we subsequently screened groups of pigs infected with a closely related avirulent PRRSV-1 strain (Lelystad) and a divergent virulent subtype 3 strain (SU1-Bel). Whilst T cell responses from both groups were observed against many of the antigens identified in the first study, animals infected with the SU1-Bel strain showed the greatest response against peptides representing the non-structural protein 5. The proteome-wide peptide library screening method used here, as well as the antigens identified, warrant further evaluation in the context of next generation vaccine development.

Fatty acid and peptide profiles in plasma membrane and membrane rafts of PUFA supplemented RAW264.7 macrophages

The eukaryotic cell membrane possesses numerous complex functions, which are essential for life. At this, the composition and the structure of the lipid bilayer are of particular importance. Polyunsaturated fatty acids may modulate the physical properties of biological membranes via alteration of membrane lipid composition affecting numerous physiological processes, e.g. in the immune system. In this systematic study we present fatty acid and peptide profiles of cell membrane and membrane rafts of murine macrophages that have been supplemented with saturated fatty acids as well as PUFAs from the n-3, the n-6 and the n-9 family. Using fatty acid composition analysis and mass spectrometry-based peptidome profiling we found that PUFAs from both the n-3 and the n-6 family have an impact on lipid and protein composition of plasma membrane and membrane rafts in a similar manner. In addition, we found a relation between the number of bis-allyl-methylene positions of the PUFA added and the unsaturation index of plasma membrane as well as membrane rafts of supplemented cells. With regard to the proposed significance of lipid microdomains for disease development and treatment our study will help to achieve a targeted dietary modulation of immune cell lipid bilayers.

Serodiagnosis of Echinococcus spp. infection: explorative selection of diagnostic antigens by peptide microarray

BACKGROUND: Production of native antigens for serodiagnosis of helminthic infections is laborious and hampered by batch-to-batch variation. For serodiagnosis of echinococcosis, especially cystic disease, most screening tests rely on crude or purified Echinococcus granulosus hydatid cyst fluid. To resolve limitations associated with native antigens in serological tests, the use of standardized and highly pure antigens produced by chemical synthesis offers considerable advantages, provided appropriate diagnostic sensitivity and specificity is achieved. METHODOLOGY/PRINCIPAL FINDINGS: Making use of the growing collection of genomic and proteomic data, we applied a set of bioinformatic selection criteria to a collection of protein sequences including conceptually translated nucleotide sequence data of two related tapeworms, Echinococcus multilocularis and Echinococcus granulosus. Our approach targeted alpha-helical coiled-coils and intrinsically unstructured regions of parasite proteins potentially exposed to the host immune system. From 6 proteins of E. multilocularis and 5 proteins of E. granulosus, 45 peptides between 24 and 30 amino acids in length were designed. These peptides were chemically synthesized, spotted on microarrays and screened for reactivity with sera from infected humans. Peptides reacting above the cut-off were validated in enzyme-linked immunosorbent assays (ELISA). Peptides identified failed to differentiate between E. multilocularis and E. granulosus infection. The peptide performing best reached 57% sensitivity and 94% specificity. This candidate derived from Echinococcus multilocularis antigen B8/1 and showed strong reactivity to sera from patients infected either with E. multilocularis or E. granulosus. CONCLUSIONS/SIGNIFICANCE: This study provides proof of principle for the discovery of diagnostically relevant peptides by bioinformatic selection complemented with screening on a high-throughput microarray platform. Our data showed that a single peptide cannot provide sufficient diagnostic sensitivity whereas pooling several peptide antigens improved sensitivity; thus combinations of several peptides may lead the way to new diagnostic tests that replace, or at least complement conventional immunodiagnosis of echinococcosis. Our strategy could prove useful for diagnostic developments in other pathogens.

Membrane disrupting antimicrobial peptide dendrimers with multiple amino termini

Antimicrobial peptide dendrimer H1 Leu8(Lys-Leu)4(Lys-Phe)2Lys-LysNH2 (Lys = branching lysine) was identified by screening a 6750-membered combinatorial library by the bead-diffusion assay. Sequence variations also revealed dendrimer bH1 Leu8(Dap-Leu)4(Dap-Phe)2Dap-LysNH2 (Dap = branching 2,3-diaminopropanoic acid) as a more potent analog. H1 and bH1 showed good antimicrobial activities mediated by membrane disruption (MIC = 2–4 μg mL−1 on Bacillus subtilis and Escherichia coli) but low hemolytic activity (MHC = 310 μg mL−1 respectively >2000 μg mL−1).