Simultaneous detection and subtyping of porcine endogenous retroviruses proviral DNA using the dual priming oligonucleotide system

The purpose of this study was to develop a multiplex PCR that can detect porcine endogenous retrovirus (PERV) proviral genes (pol, envA, envB, envC) and porcine mitochondrial DNA, using a dual priming oligonucleotide (DPO) system. The primer specifically detected the PERV proviral genes pol, envA, envB, envC, and porcine mitochondrial DNA only in samples of pig origin. The sensitivity of the primer was demonstrated by simultaneous amplification of all 5 target genes in as little as 10 pg of pig DNA containing PERV proviral genes and mitochondrial DNA. The multiplex PCR, when applied to field samples, simultaneously and successfully amplified PERV proviral genes from liver, blood and hair root samples. Thus, the multiplex PCR developed in the current study using DPO-based primers is a rapid, sensitive and specific assay for the detection and subtyping of PERV proviral genes

Cross-Protective Shigella Whole-Cell Vaccine With a Truncated O-Polysaccharide Chain

Shigella is a highly prevalent bacterium causing acute diarrhea and dysentery in developing countries. Shigella infections are treated with antibiotics but Shigellae are increasingly resistant to these drugs. Vaccination can be a countermeasure against emerging antibiotic-resistant shigellosis. Because of the structural variability in Shigellae O-antigen polysaccharides (Oag), cross-protective Shigella vaccines cannot be derived from single serotype-specific Oag. We created an attenuated Shigella flexneri 2a strain with one rather than multiple Oag units by disrupting the Oag polymerase gene (Δwzy), which broadened protective immunogenicity by exposing conserved surface proteins. Inactivated Δwzy mutant cells combined with Escherichia coli double mutant LT(R192G/L211A) as adjuvant, induced potent antibody responses to outer membrane protein PSSP-1, and type III secretion system proteins IpaB and IpaC. Intranasal immunization with the vaccine preparation elicited cross-protective immunity against S. flexneri 2a, S. flexneri 3a, S. flexneri 6, and Shigella sonnei in a mouse pneumonia model. Thus, S. flexneri 2a Δwzy represents a promising candidate strain for a universal Shigella vaccine

An ultralong CDRH2 in HCV neutralizing antibody demonstrates structural plasticity of antibodies against E2 glycoprotein.

A vaccine protective against diverse HCV variants is needed to control the HCV epidemic. Structures of E2 complexes with front layer-specific broadly neutralizing antibodies (bNAbs) isolated from HCV-infected individuals, revealed a disulfide bond-containing CDRH3 that adopts straight (individuals who clear infection) or bent (individuals with chronic infection) conformation. To investigate whether a straight versus bent disulfide bond-containing CDRH3 is specific to particular HCV-infected individuals, we solved a crystal structure of the HCV E2 ectodomain in complex with AR3X, a bNAb with an unusually long CDRH2 that was isolated from the chronically-infected individual from whom the bent CDRH3 bNAbs were derived. The structure revealed that AR3X utilizes both its ultralong CDRH2 and a disulfide motif-containing straight CDRH3 to recognize the E2 front layer. These results demonstrate that both the straight and bent CDRH3 classes of HCV bNAb can be elicited in a single individual, revealing a structural plasticity of VH1-69-derived bNAbs

Natural killer T cell sensitization during neonatal respiratory syncytial virus infection induces eosinophilic lung disease in re-infected adult mice

<div><p>Respiratory syncytial virus (RSV) is a major viral pathogen that causes severe lower respiratory tract infections in infants and the elderly worldwide. Infants with severe RSV bronchiolitis tend to experience more wheezing and asthma in later childhood. Because invariant natural killer T (iNKT) cells are associated with the asthma pathology, we investigated whether neonatal iNKT cells are involved in the aggravation of pulmonary diseases following RSV infection in mice. Intranasal exposure to the iNKT cell ligand α-galactosylceramide (α-GC) with RSV primary infection in neonatal mice elicited neither cytokine production (except for a slight increase of IL-5) nor pulmonary eosinophilia, despite the presence of both CD1d⁺ cells and NKT cells. Interestingly, in adult mice re-infected with RSV, neonatal iNKT cell sensitization by α-GC during RSV primary infection resulted in much higher levels of pulmonary Th2 cytokines and elevated eosinophilia with airway hyperresponsiveness, whereas this was not observed in <i>cd1d</i> knockout mice. In contrast, α-GC priming of adults during RSV re-infection did not induce more severe airway symptoms than RSV re-infection in the absence of α-GC. α-GC co-administration during RSV primary infection facilitated RSV clearance regardless of age, but viral clearance following re-infection was not iNKT cell-dependent. This study clearly demonstrates that RSV-induced immune responses can be altered by iNKT cells, suggesting that neonatal iNKT cell sensitization during RSV primary infection is associated with exacerbation of pulmonary diseases following RSV re-infection in adulthood.</p></div

Effects of neonatal α-GC sensitization with RSV primary infection on pulmonary immune responses following re-infection in adulthood.

<p>Wild-type or CD1d KO neonatal mice were i.n. administered PBS, α-GC, RSV, or α-GC+ RSV and challenged at 8 weeks of age with RSV. Lung tissue was formalin-fixed, paraffin-embedded, and stained with hematoxylin-eosin (H&E) (A) or periodic acid–Schiff (PAS) (B) on day4 after RSVA2 challenge. (C) Cytokine levels in BAL fluid were determined by luminex assay. Data are expressed as mean ± SEM of 5 or 6 mice per group. *p<0.05.</p

Effects of neonatal α-GC sensitization with RSV primary infection on eosinophils following re-infection in adulthood.

<p>Wild-type or CD1d KO neonatal mice were i.n. administered PBS, α-GC, RSV, or α-GC+ RSV and challenged at 8 weeks of age with RSV. Four days after challenge, cells were isolated from the BAL fluid, and the ratio of eosinophils to granulocytes was determined as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176940#pone.0176940.g001" target="_blank">Fig 1</a> (A). The total cell count and absolute number of eosinophils are shown (B). Data are mean ± SEM with 2–4 mice per group and representative of three independent experiments. **p<0.01.</p

Effects of neonatal α-GC sensitization with RSV primary infection on airway hyperresponsiveness and viral clearance following re-infection in adulthood.

<p>(A) Airway hyperresponsiveness measured by methacholine test (Penh). Wild-type (wt) or CD1d knockout neonatal mice were infected with vehicle (Veh), α-GC, RSV, or α-GC with RSV and at age 8 weeks they were challenged with RSV. Four days after challenge, mice were given nebulized methacholine; 0 or 10 mg/mL. Data are expressed as mean ± SEM of 6 mice given 10 mg/mL of methacholine. **p<0.01. (B) Lungs were harvested and viral titers assessed by plaque assay on day 4 after challenge. Results are representative of two independent experiments. Data are expressed as mean ± SEM of 4–6 mice per group. **p<0.01; ***p<0.001; ****p<0.0001.</p

Effects of adult NKT cell priming by α-GC during RSV re-infection.

<p>Neonate mice (7 days old) were infected with RSV and at age 6 weeks i.n. administered vehicle (Veh), α-GC, RSV, or α-GC with RSV. (A) Four days after infection, cells were isolated from BAL fluid and the ratio of eosinophils per granulocyte determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176940#pone.0176940.g001" target="_blank">Fig 1</a>. Data are mean ± SEM with 5 or 6 mice per group. **p<0.01; ***p<0.001. (B) Histologic examination of lungs on day 4 after infection. Lung tissue was formalin-fixed, paraffin-embedded, and stained with hematoxylin-eosin (H&E) or periodic acid–Schiff (PAS). (C) Cytokine levels in BAL fluid were determined by luminex assay. Data are mean ± SEM of 5 or 6 mice per group. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. (D) Airway hyperresponsiveness was measured by methacholine test (Penh) 4 days after infection when mice were given nebulized methacholine; 0 and 10 mg/mL. Data are expressed as mean ± SEM of 5 or 6 mice at 10 mg/mL of methacholine in each group.</p

Cytokine production in BAL fluid in response to RSV infection with or without α-GC.

<p>(A) Mice were i.n. administered vehicle (Veh), α-GC, RSV, or α-GC+RSV at ages 7 days and 6 weeks. BAL fluid was collected from lungs 4 days after infection. Cytokine levels in BAL fluid were determined by luminex and cytometric bead assay. Data are expressed as mean ± SEM of 9–12 mice in each group of neonates and of 3 or 4 mice in each group of adults. *p<0.05</p