32 research outputs found

    Pathogen-specific deep sequence-coupled biopanning: A method for surveying human antibody responses

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    <div><p>Identifying the targets of antibody responses during infection is important for designing vaccines, developing diagnostic and prognostic tools, and understanding pathogenesis. We developed a novel deep sequence-coupled biopanning approach capable of identifying the protein epitopes of antibodies present in human polyclonal serum. Here, we report the adaptation of this approach for the identification of pathogen-specific epitopes recognized by antibodies elicited during acute infection. As a proof-of-principle, we applied this approach to assessing antibodies to Dengue virus (DENV). Using a panel of sera from patients with acute secondary DENV infection, we panned a DENV antigen fragment library displayed on the surface of bacteriophage MS2 virus-like particles and characterized the population of affinity-selected peptide epitopes by deep sequence analysis. Although there was considerable variation in the responses of individuals, we found several epitopes within the Envelope glycoprotein and Non-Structural Protein 1 that were commonly enriched. This report establishes a novel approach for characterizing pathogen-specific antibody responses in human sera, and has future utility in identifying novel diagnostic and vaccine targets.</p></div

    Generation of MS2-VLP DENV-3 antigen fragment library.

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    <p>(A) The DENV-3 polyprotein was used to identify all possible 10 amino acid peptides, overlapping by 9 amino acids. (B) <i>E</i>. <i>coli</i> codon optimized coding sequences were synthesized by a massively parallel microchip-based synthesis-on-chip technique (LC Sciences), and (C) then used to generate a corresponding plasmid library in the pDSP62 vector. (D) The library was then expressed as VLPs in <i>E</i>. <i>coli</i> and used in subsequent deep sequence-coupled biopanning experiments.</p

    Deep sequencing results from two rounds of deep sequence-coupled iterative biopanning with serum sample 1536.

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    <p>(A) Fold % enrichment of each peptide is presented for the entire DENV-3 polyprotein. (B and C) E and NS1 protein regions are shown enlarged from (A). Note the change in y-axis scale.</p

    DENV-3 plasmid starting library.

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    <p>DENV-3 antigen fragment plasmid library was assessed by Ion Torrent Deep Sequencing to determine coverage at each amino acid position. (A) Deduced peptide sequences were aligned to the DENV-3 polyprotein with NCBI BLAST and used to determine the coverage at each amino acid position. Each peak represents the % total population of reads representing peptides that align starting at that amino acid position. The red and blue regions represents the E protein and NS1 protein, respectively. (B and C) The E protein and NS1 protein coverage is enlarged from (A) in order to show the coverage of the plasmid library for these regions.</p

    NS1 and E enriched epitopes identified by deep sequence-coupled biopanning with patient serum after two rounds of biopanning.

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    <p>NS1 conservation (A) is shown for the highly enriched overlapping peptides from patient 1536 after two rounds of biopanning. Bold letters indicate the flanking amino acids that are solved in the crystal structure (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171511#pone.0171511.g005" target="_blank">Fig 5F</a> for structure). E conservation (C) is shown for the most enriched overlapping peptides from patient 1536 after two rounds of biopanning. ELISAs against synthetic peptides representing the NS1 epitope (B) and the E epitope (D) are shown. Open circles represent the data point for patient 1536 (the sample used to identify the epitopes). Primary serum samples (n = 31) and secondary serum samples (n = 30) isolated from patients 7 days post onset of fever were used.</p

    Commonly targeted E- and NS1 peptides.

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    <p>(A and D) The number of patients with a peptide enriched (10-fold or greater enrichment above starting library) is indicated at each amino acid position for E (A) and NS1 (D). Bars are indicated with letters are peptides enriched in 5 or more patients to highlight the commonly targeted epitopes (A and D). Individual E (B) and NS1 (E) peptides targeted by at least 5 patients are shown and colors correspond to the trimer (fusion) conformation of E (C) or NS1 (F). Dotted black lines in (F) represent the putative disordered region in which the LKYSWKTWGKAK epitope is located and is shown in (D) with an arrow. Colors of residues in (A) correspond to the colors of residues in the structure shown in (C). The junction of domain III and the stem region of E is indicated by an open arrow.</p

    Table1_Norovirus in children under 2 years of age: an epidemiological study in Panama during the COVID-19 pandemic.docx

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    IntroductionNorovirus infection is a common cause of acute gastroenteritis (AGE). Surveillance activities are important to aid investigation into effective norovirus control strategies, including vaccination. Here, we report ancillary findings related to the incidence, prevalence, and etiology of AGE caused by norovirus in Panama after adjustment of study methodology to comply with national coronavirus disease 2019 (COVID-19) mandates.MethodsIn January 2020, children aged ResultsIn the longitudinal surveillance cohort [N = 400 (Chiriquí, n = 239; Panama, n = 161)], a total of 185 AGE episodes were documented (Chiriquí, n = 85; Panama, n = 100) resulting in an overall AGE incidence of 11.6 (95% CI: 9.99–13.4) episodes per 100 child-months. The norovirus-related AGE incidence was 0.3 (95% CI: 0.10–0.73) episodes per 100 child-months (5/185 AGE episodes) and the prevalence of norovirus was 4.6% (13/282 stool samples collected). In the hospital-based surveillance cohort, at least one pathogen was detected in 50% of samples (44/88 stool samples collected) and norovirus prevalence was 6.8% (6/88 stool samples collected).DiscussionThis report demonstrates how the occurrence of the COVID-19 pandemic hindered the conduct of clinical trials. However, this also created unique research opportunities to investigate the potential impact of pandemic control measures on the etiology of infectious diarrheal disease.</p

    Epidemiology of Emergent Madariaga Encephalitis in a Region with Endemic Venezuelan Equine Encephalitis: Initial Host Studies and Human Cross-Sectional Study in Darien, Panama

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    <div><p>Background</p><p>Neurotropic arboviral infections are an important cause of encephalitis. A zoonotic, vector-borne alphavirus, Madariaga virus (MADV; formerly known as South American eastern equine encephalitis virus), caused its first documented human outbreak in 2010 in Darien, Panama, where the genetically similar Venezuelan equine encephalitis virus (VEEV) is endemic. We report the results of a seroprevalence survey of animals and humans, illustrating contrasting features of MADV and VEEV ecology and epidemiology.</p><p>Methods</p><p>Small mammals were trapped in 42 sites in Darien, Panama, using Sherman traps, Tomahawk traps, and mist nets for bats. Blood was tested for the presence of neutralizing antibodies to MADV and VEEV. In addition, bird sera collected in 2007 in Chagres, Panama, were tested for MADV and VEEV neutralizing antibodies. Viremia was ascertained by RT-PCR. Human exposure to these two viruses was determined by IgG ELISA, followed by plaque reduction neutralization tests. To identify relevant risk factors for MADV or VEEV exposure, logistic regression analysis was performed, and the most parsimonious model was selected based on the Akaike information criterion.</p><p>Results</p><p>The animal survey yielded 32 bats (16 species), 556 rodents (12 species), and 20 opossums (4 species). The short-tailed cane mouse (<i>Zygodontomys brevicauda</i>) found abundantly in pasture and farms, had the highest MADV seroprevalence (8.3%). For VEEV, the shrub and forest-dwelling long-whiskered rice rat (<i>Transandinomys bolivaris</i>) had the highest seroprevalence (19.0%). Viremia was detected in one animal (<i>Z</i>. <i>brevicauda</i>). Of the 159 bird sera (50 species) tested, none were positive for either virus. In humans (n = 770), neutralizing antibodies to MADV and VEEV were present in 4.8% and 31.5%, respectively. MADV seropositivity was positively associated with cattle ranching, farming, and fishing. Having VEEV antibodies and shrubs near the house diminished risk. Age, forest work, farming and fishing were risk factors for VEEV, while having MADV antibodies, glazed windows, waste pick-up and piped water were protective.</p><p>Conclusion</p><p>Our findings suggest that the short-tailed cane mouse and the long-whiskered rice rat serve as hosts for MADV and VEEV, respectively. The preferred habitat of these rodent species coincides with areas associated with human infection risk. Our findings also indicate that MADV emerged recently in humans, and that the transmission cycles of these two sympatric alphaviruses differ spatially and in host utilization.</p></div
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