Protective porcine influenza virus-specific monoclonal antibodies recognize similar haemagglutinin epitopes as humans.

Pigs are natural hosts for the same subtypes of influenza A viruses as humans and integrally involved in virus evolution with frequent interspecies transmissions in both directions. The emergence of the 2009 pandemic H1N1 virus illustrates the importance of pigs in evolution of zoonotic strains. Here we generated pig influenza-specific monoclonal antibodies (mAbs) from H1N1pdm09 infected pigs. The mAbs recognized the same two major immunodominant haemagglutinin (HA) epitopes targeted by humans, one of which is not recognized by post-infection ferret antisera that are commonly used to monitor virus evolution. Neutralizing activity of the pig mAbs was comparable to that of potent human anti-HA mAbs. Further, prophylactic administration of a selected porcine mAb to pigs abolished lung viral load and greatly reduced lung pathology but did not eliminate nasal shedding of virus after H1N1pdm09 challenge. Hence mAbs from pigs, which target HA can significantly reduce disease severity. These results, together with the comparable sizes of pigs and humans, indicate that the pig is a valuable model for understanding how best to apply mAbs as therapy in humans and for monitoring antigenic drift of influenza viruses in humans, thereby providing information highly relevant to making influenza vaccine recommendations.

Correction: Protective porcine influenza virus-specific monoclonal antibodies recognize similar haemagglutinin epitopes as humans.

[This corrects the article DOI: 10.1371/journal.ppat.1009330.].

Timelines of infection and transmission dynamics of H1N1pdm09 in swine.

Influenza is a major cause of mortality and morbidity worldwide. Despite numerous studies of the pathogenesis of influenza in humans and animal models the dynamics of infection and transmission in individual hosts remain poorly characterized. In this study, we experimentally modelled transmission using the H1N1pdm09 influenza A virus in pigs, which are considered a good model for influenza infection in humans. Using an experimental design that allowed us to observe individual transmission events occurring within an 18-hr period, we quantified the relationships between infectiousness, shed virus titre and antibody titre. Transmission event was observed on 60% of occasions when virus was detected in donor pig nasal swabs and transmission was more likely when donor pigs shed more virus. This led to the true infectious period (mean 3.9 days) being slightly shorter than that predicted by detection of virus (mean 4.5 days). The generation time of infection (which determines the rate of epidemic spread) was estimated for the first time in pigs at a mean of 4.6 days. We also found that the latent period of the contact pig was longer when they had been exposed to smaller amount of shed virus. Our study provides quantitative information on the time lines of infection and the dynamics of transmission that are key parts of the evidence base needed to understand the spread of influenza viruses though animal populations and, potentially, in humans.

Establishment of a Pig Influenza Challenge Model for Evaluation of Monoclonal Antibody Delivery Platforms.

mAbs are a possible adjunct to vaccination and drugs in treatment of influenza virus infection. However, questions remain whether small animal models accurately predict efficacy in humans. We have established the pig, a large natural host animal for influenza, with many physiological similarities to humans, as a robust model for testing mAbs. We show that a strongly neutralizing mAb (2-12C) against the hemagglutinin head administered prophylactically at 15 mg/kg reduced viral load and lung pathology after pandemic H1N1 influenza challenge. A lower dose of 1 mg/kg of 2-12C or a DNA plasmid-encoded version of 2-12C reduced pathology and viral load in the lungs but not viral shedding in nasal swabs. We propose that the pig influenza model will be useful for testing candidate mAbs and emerging delivery platforms prior to human trials.

Simultaneous co-infection with swine influenza A and porcine reproductive and respiratory syndrome viruses potentiates adaptive immune responses.

Porcine respiratory disease is multifactorial and most commonly involves pathogen co-infections. Major contributors include swine influenza A (swIAV) and porcine reproductive and respiratory syndrome (PRRSV) viruses. Experimental co-infection studies with these two viruses have shown that clinical outcomes can be exacerbated, but how innate and adaptive immune responses contribute to pathogenesis and pathogen control has not been thoroughly evaluated. We investigated immune responses following experimental simultaneous co-infection of pigs with swIAV H3N2 and PRRSV-2. Our results indicated that clinical disease was not significantly exacerbated, and swIAV H3N2 viral load was reduced in the lung of the co-infected animals. PRRSV-2/swIAV H3N2 co-infection did not impair the development of virus-specific adaptive immune responses. swIAV H3N2-specific IgG serum titers and PRRSV-2-specific CD8ß+ T-cell responses in blood were enhanced. Higher proportions of polyfunctional CD8ß+ T-cell subset in both blood and lung washes were found in PRRSV-2/swIAV H3N2 co-infected animals compared to the single-infected groups. Our findings provide evidence that systemic and local host immune responses are not negatively affected by simultaneous swIAV H3N2/PRRSV-2 co-infection, raising questions as to the mechanisms involved in disease modulation.

Effectiveness of semantic therapy for word-finding difficulties in pupils with persistent language impairments: a randomized control trial.

BACKGROUND: Word-finding difficulties (WFDs) in children have been hypothesized to be caused at least partly by poor semantic knowledge. Therefore, improving semantic knowledge should decrease word-finding errors. Previous studies of semantic therapy for WFDs are inconclusive. AIMS: To investigate the effectiveness of semantic therapy for secondary school-aged pupils with WFDs using a randomized control trial with blind assessment. METHODS & PROCEDURES: Fifteen participants with language impairments and WFDs (aged 9;11-15;11) were randomly assigned to a therapy versus waiting control group. In Phase 1 the therapy group received two 15-min semantic therapy sessions per week for 8 weeks with their usual speech and language therapist. Therapy for each participant targeted words from one of three semantic categories (animals, food, clothes). All participants were tested pre- and post-phase 1 therapy on the brief version of the Test of Adolescent Word Finding (TAWF), semantic fluency and the Test of Word Finding in Discourse (TWFD). In Phase 2 the waiting control group received the same therapy as the original therapy group, which received therapy targeted at other language areas. Testing after Phase 2 aimed to establish whether the waiting control group made similar progress to the original therapy group and whether the original therapy group maintained any gains. OUTCOMES & RESULTS: The original therapy group made significant progress in standard scores on the TAWF (d= 0.94), which was maintained 5 months later. However, they made no progress on the semantic fluency or discourse tests. Participants in the waiting control group did not make significant progress on the TAWF in Phase 1 when they received no word-finding therapy. However, after Phase 2, when they received the therapy, they also made significant progress (d= 0.81). The combined effect of therapy over the two groups was d= 1.2. The mean standard scores on the TAWF were 67 pre-therapy and 77 post-therapy. CONCLUSIONS & IMPLICATIONS: Four hours of semantic therapy on discrete semantic categories led to significant gains on a general standardized test of word finding, enabling the participants to begin to close the gap between their performance and that of their typically developing peers. These gains were maintained after 5 months. A small amount of therapy can lead to significant gains even with secondary aged pupils with severe language difficulties. However, further studies are needed to find ways of improving word-finding abilities in discourse.

Immunogenicity and Protective Efficacy of Seasonal Human Live Attenuated Cold-Adapted Influenza Virus Vaccine in Pigs.

Influenza A virus infection is a global health threat to livestock and humans, causing substantial mortality and morbidity. As both pigs and humans are readily infected with influenza viruses of similar subtype, the pig is a robust and appropriate model for investigating swine and human disease. We evaluated the efficacy of the human cold-adapted 2017-2018 quadrivalent seasonal LAIV in pigs against H1N1pdm09 challenge. LAIV immunized animals showed significantly reduced viral load in nasal swabs. There was limited replication of the H1N1 component of the vaccine in the nose, a limited response to H1N1 in the lung lymph nodes and a low H1N1 serum neutralizing titer. In contrast there was better replication of the H3N2 component of the LAIV, accompanied by a stronger response to H3N2 in the tracheobronchial lymph nodes (TBLN). Our data demonstrates that a single administration of human quadrivalent LAIV shows limited replication in the nose and induces detectable responses to the H1N1 and H3N2 components. These data suggest that pigs may be a useful model for assessing LAIV against influenza A viruses.

Electrochemical sensor array for glucose monitoring fabricated by rapid immobilization of active glucose oxidase within photochemically polymerized hydrogels.

BACKGROUND: Currently, monitoring blood glucose levels for diabetic patients is invasive and painful, involving pricking the finger to obtain a blood sample three to four times daily. The need for frequent tests and pain involved with testing leads to poor compliance. In order to raise compliance, we propose to create an implantable electrochemical sensor array that would monitor glucose levels continuously. METHODS: Glucose sensor arrays were fabricated on gold electrodes on flexible polyimide sheets by photopolymerization of the biocompatible polymer poly(ethylene glycol) diacrylate (PEG-DA) to develop hydrogels and encapsulate the sensing elements. Using conventional silicon fabrication methods, arrays of five gold microdisk electrodes were fabricated using lift-off photolithography and sputtering techniques. A redox polymer was then attached electrostatically to the electrode, and glucose oxidase was entrapped inside the hydrogel on the array of electrodes by ultraviolet-initiated photopolymerization of PEG-DA. RESULTS: When the array of fabricated sensors was sampled together the elements behaved like one large electrode with peak current equivalent to the sum of individual array elements. The enzyme, glucose oxidase, catalyzed the oxidation of glucose and then exchanged electrons with the redox polymer in the hydrogel. The entrapped glucose oxidase was found to respond linearly to increasing glucose concentrations (0-360 mg/dl), as determined using cyclic voltammetry. CONCLUSION: The fabricated microarray sensors were individually addressable and showed no cross talk between adjacent array elements as assessed using cyclic voltammetry. We have fabricated an array of glucose sensors on flexible polyimide sheets that exhibits the desired linear response in the biological range.