Direct Interrogation of Viral Peptides Presented by the Class I HLA of HIV-Infected T Cells

Identification of CD8+ cytotoxic T lymphocyte (CTL) epitopes has traditionally relied upon testing of overlapping peptide libraries for their reactivity with T cells in vitro. Here, we pursued deep ligand sequencing (DLS) as an alternative method of directly identifying those ligands that are epitopes presented to CTLs by the class I human leukocyte antigens (HLA) of infected cells. Soluble class I HLA-A*11:01 (sHLA) was gathered from HIV-1 NL4-3-infected human CD4+ SUP-T1 cells. HLA-A*11:01 harvested from infected cells was immunoaffinity purified and acid boiled to release heavy and light chains from peptide ligands that were then recovered by size-exclusion filtration. The ligands were first fractionated by high-pH high-pressure liquid chromatography and then subjected to separation by nano-liquid chromatography (nano-LC)–mass spectrometry (MS) at low pH. Approximately 10 million ions were selected for sequencing by tandem mass spectrometry (MS/MS). HLA-A*11:01 ligand sequences were determined with PEAKS software and confirmed by comparison to spectra generated from synthetic peptides. DLS identified 42 viral ligands presented by HLA-A*11:01, and 37 of these were previously undetected. These data demonstrate that (i) HIV-1 Gag and Nef are extensively sampled, (ii) ligand length variants are prevalent, particularly within Gag and Nef hot spots where ligand sequences overlap, (iii) noncanonical ligands are T cell reactive, and (iv) HIV-1 ligands are derived from de novo synthesis rather than endocytic sampling. Next-generation immunotherapies must factor these nascent HIV-1 ligand length variants and the finding that CTL-reactive epitopes may be absent during infection of CD4+ T cells into strategies designed to enhance T cell immunity

A versatile computational pipeline for bacterial genome annotation improvement and comparative analysis, with Brucella as a use case

We present a bacterial genome computational analysis pipeline, called GenVar. The pipeline, based on the program GeneWise, is designed to analyze an annotated genome and automatically identify missed gene calls and sequence variants such as genes with disrupted reading frames (split genes) and those with insertions and deletions (indels). For a given genome to be analyzed, GenVar relies on a database containing closely related genomes (such as other species or strains) as well as a few additional reference genomes. GenVar also helps identify gene disruptions probably caused by sequencing errors. We exemplify GenVar's capabilities by presenting results from the analysis of four Brucella genomes. Brucella is an important human pathogen and zoonotic agent. The analysis revealed hundreds of missed gene calls, new split genes and indels, several of which are species specific and hence provide valuable clues to the understanding of the genome basis of Brucella pathogenicity and host specificity

Unique CRF01_AE Gag CTL Epitopes Associated with Lower HIV-Viral Load and Delayed Disease Progression in a Cohort of HIV-Infected Thais

Cytotoxic T Lymphocytes (CTLs) play a central role in controlling HIV-replication. Although numerous CTL epitopes have been described, most are in subtype B or C infection. Little is known about CTL responses in CRF01_AE infection. Gag CTL responses were investigated in a cohort of 137 treatment-naïve HIV-1 infected Thai patients with high CD4+ T cell counts, using gIFN Enzyme-Linked Immunospot (ELISpot) assays with 15-mer overlapping peptides (OLPs) derived from locally dominant CRF01_AE Gag sequences. 44 OLPs were recognized in 112 (81.8%) individuals. Both the breadth and magnitude of the CTL response, particularly against the p24 region, positively correlated with CD4+ T cell count and inversely correlated with HIV viral load. The breadth of OLP response was also associated with slower progression to antiretroviral therapy initiation. Statistical analysis and single peptide ELISpot assay identified at least 17 significant associations between reactive OLP and HLA in 12 OLP regions; 6 OLP-HLA associations (35.3%) were not compatible with previously reported CTL epitopes, suggesting that these contained new CTL Gag epitopes. A substantial proportion of CTL epitopes in CRF01_AE infection differ from subtype B or C. However, the pattern of protective CTL responses is similar; Gag CTL responses, particularly against p24, control viral replication and slow clinical progression

Prior mucosal exposure to heterologous cells alters the pathogenesis of cell-associated mucosal feline immunodeficiency virus challenge

<p>Abstract</p> <p>Background</p> <p>Several lines of research suggest that exposure to cellular material can alter the susceptibility to infection by HIV-1. Because sexual contact often includes exposure to cellular material, we hypothesized that repeated mucosal exposure to heterologous cells would induce an immune response that would alter the susceptibility to mucosal infection. Using the feline immunodeficiency virus (FIV) model of HIV-1 mucosal transmission, the cervicovaginal mucosa was exposed once weekly for 12 weeks to 5,000 heterologous cells or media (control) and then cats were vaginally challenged with cell-associated or cell-free FIV.</p> <p>Results</p> <p>Exposure to heterologous cells decreased the percentage of lymphocytes in the mucosal and systemic lymph nodes (LN) expressing L-selectin as well as the percentage of CD4+ CD25+ T cells. These shifts were associated with enhanced ex-vivo proliferative responses to heterologous cells. Following mucosal challenge with cell-associated, but not cell-free, FIV, proviral burden was reduced by 64% in cats previously exposed to heterologous cells as compared to media exposed controls.</p> <p>Conclusions</p> <p>The pathogenesis and/or the threshold for mucosal infection by infected cells (but not cell-free virus) can be modulated by mucosal exposure to uninfected heterologous cells.</p

Proteus mirabilis urease: histidine 320 of UreC is essential for urea hydrolysis and nickel ion binding within the native enzyme

Proteus mirabilis urease, a nickel-containing enzyme, has been established as a critical virulence determinant in urinary tract infection. An amino acid sequence (residues 308 to 327: TVDEHLDMLMVCHHLDPSIP) within the large urease subunit, UreC, is highly conserved for every urease examined thus far and has been suggested to reside within the enzyme active site. Histidine residues have been postulated to play a role in catalysis by coordinating Ni2+ ions. To test this hypothesis, oligonucleotide-directed mutagenesis was used to change amino acid His-320 to Leu-320 within UreC. The base change (CAT for His-320 to CTT for Leu-320) was confirmed by DNA sequencing. The recombinant and mutant proteins were expressed at similar levels in Escherichia coli as detected by Western blotting (immunoblotting) of denaturing and nondenaturing gels. Specific activities of the enzymes were quantitated after partial purification. Strains expressing the mutant enzyme showed no detectable activity, whereas strains expressing the recombinant enzyme hydrolyzed urea at 149 mumol of NH3 per min per mg of protein. In addition, the mutant enzyme was able to incorporate only about one-half (58%) of the amount of 63Ni2+ incorporated by the active recombinant enzyme. While the mutation of His-320 to Leu-320 within UreC does not affect expression or assembly of urease polypeptide subunits UreA, UreB, and UreC His-320 of UreC is required for urea hydrolysis and proper incorporation of Ni2+ into apoenzyme.</jats:p

Single-step purification of Proteus mirabilis urease accessory protein UreE, a protein with a naturally occurring histidine tail, by nickel chelate affinity chromatography.

Proteus mirabilis urease, a nickel metalloenzyme, is essential for the virulence of this species in the urinary tract. Escherichia coli containing cloned structural genes ureA, ureB, and ureC and accessory genes ureD, ureE, ureF, and ureG displays urease activity when cultured in M9 minimal medium. To study the involvement of one of these accessory genes in the synthesis of active urease, deletion mutations were constructed. Cultures of a ureE deletion mutant did not produce an active urease in minimal medium. Urease activity, however, was partially restored by the addition of 5 microM NiCl2 to the medium. The predicted amino acid sequence of UreE, which concludes with seven histidine residues among the last eight C-terminal residues (His-His-His-His-Asp-His-His-His), suggested that UreE may act as a Ni2+ chelator for the urease operon. To exploit this potential metal-binding motif, we attempted to purify UreE from cytoplasmic extracts of E. coli containing cloned urease genes. Soluble protein was loaded onto a nickel-nitrilotriacetic acid column, a metal chelate resin with high affinity for polyhistidine tails, and bound protein was eluted with a 0 to 0.5 M imidazole gradient. A single polypeptide of 20-kDa apparent molecular size, as shown by sodium dodecyl sulfate-10 to 20% polyacrylamide gel electrophoresis, was eluted between 0.25 and 0.4 M imidazole. The N-terminal 10 amino acids of the eluted polypeptide exactly matched the deduced amino acid sequence of P. mirabilis UreE. The molecular size of the native protein was estimated on a Superdex 75 column to be 36 kDa, suggesting that the protein is a dimer. These data suggest that UreE is a Ni(2)+-binding protein that is necessary for synthesis of a catalytically active urease at low Ni(2+) concentrations

Engineering of the LukS-PV and LukF-PV subunits of Staphylococcus aureus Panton-Valentine leukocidin for Diagnostic and Therapeutic Applications

Abstract Background: Staphylococcus aureus produces several toxins, including Panton-Valentine leukocidin (PVL). The involvement of PVL in primary skin infections, necrotizing pneumonia, musculoskeletal disorders, brain abscess, and other diseases, some of which are life-threatening, has been reported. Following expert opinion, we aimed to provide the tools for establishment of sequence-based diagnostics and therapeutics for those conditions. We engineered the synergistic S and F (LukS-PV and LukF-PV respectively) pro-toxin subunits from Staphylococcus aureus USA400 into separate expression E. coli BL21(DE3)-pLysS hosts. Results: Following Nickel affinity chromatography (NAC), the F subunit came out without bands of impurity. The S sub-unit did not come off very pure after NAC thus necessitating further purification by size exclusion and ion-exchange chromatography. The purification plots showed that the BioLogic-LP and AKTA systems are reliable for following the progress of the chromatographic purification in real-time. Computer predicted Mw for the 6His-LukF-PV and 6His-LukS-PV were 35645.41 Da and 33530.04 Da respectively, while the mass spectrometry results were 35643.57 Da and 33528.34 Da respectively. Conclusion: The BioLogic-LP and AKTA systems are commendable for reliability and user-friendliness. As a recent work elsewhere also reported that a second round of chromatography was necessary to purify the S subunit after the first attempt, we speculate that the S subunit might contain yet unidentified motif(s) requiring further treatment. The purified S and F sub-units of PVL were supplied to the Nottingham Cancer Immunotherapy group who used them to establish sequence-based monoclonal antibodies for diagnostic and therapeutic uses targeting PVL

Effect of inosiplex on the humoral and cell-mediated immune responses to intradermal human diploid cell rabies vaccine.

Antigen-stimulated lymphocyte transformation was studied in recipients of intradermal human diploid cell rabies vaccine (HDCV). HDCV was administered intradermally at 8 different anatomical sites, 0.1 ml each, on day 0; followed by another 4-site injection on day 7. Rabies antigen-stimulated in vitro proliferative response was evident as early as 7 days after starting immunization. It reached a peak on day 14 and had declined by day 28. The cellular proliferative response preceded and roughly correlated with the antirabies antibody response. Simultaneous administration of inosiplex, an antiviral and immunopotentiating drug, during the first 10 days of intradermal HDCV immunization did not result in heightened antibody titres or cell-mediated immune response to the vaccine. The number of T cells and the lymphocyte proliferative response to phytohaemagglutinin in inosiplex-treated vaccinees were similarly not significantly different from untreated controls. Our results confirm other previous findings that a specific cell-mediated immune response can be consistently and rapidly induced by an intradermal regimen of HDCV immunization. The addition of inosiplex to this regimen did not enhance the humoral or cell-mediated immune responses to the vaccine. The apparent lack of immunostimulating effect of inosiplex in this setting may be the result of several factors such as the immunization schedule and the immunologic parameters examined