Development of a real-time RT-PCR for quantification of bovine TLR4 mRNA and evaluation of its use during a BRSV vaccine challenge

The Bovine respiratory syncytial virus (BRSV) causes bronchiolitis and interstitial pneumonia, predominantly in calves, and is a major cause of bovine respiratory disease worldwide. In humans, BRSV is paralleled by the closely related Human respiratory syncytial virus (HRSV), an important cause of respiratory disease, most severe in infants. The clinical signs and pathology during RSV infection is caused, not only by the direct effects of viral replication, but also by the response of the host immune system. The immunopathology of RSV has long obfuscated our understanding of the disease, and development of effective treatment and vaccines will be very difficult until greater knowledge is gained. One of the components of the immune system that has come into focus in RSV research the last few years, is the Toll-like receptor 4 (TLR4). The TLR4 receptor is well known as the receptor that binds lipopolysaccaride (LPS), and initiates the host response to bacterial infection. Recently, it has been shown that the fusion protein of RSV also interacts with, and up-regulates the expression of, the TLR4 receptor. Whether this has a predominantly protective effect, as would be expected from an immune response, or if it is mainly detrimental to the host, remains to be determined. The objective of this work was to develop an assay for quantification of TLR4 mRNA in clinical samples and to determine if TLR4 mRNA in bronchoalveolar lavage (BAL) cell samples could be used as a marker of protection during experimental BRSV infection. Two one-step quantitative real-time PCR systems were developed and optimized in this work. One for detection of TLR4 mRNA, and the other for detection of the housekeeping gene product 28S rRNA. The assays showed good efficiency as well as intra- and inter-assay reproducibility. Furthermore, BAL cell samples collected during an experimental vaccinal challenge were used to evaluate the level of TLR4 mRNA expression in relation to detected BRSV RNA. When the results were analyzed, it appeared that TLR4 mRNA quantification can not be used as a marker of protection against BRSV infection after previous vaccination.Bovint respiratoriskt syncytialt virus (BRSV) orsakar bronkiolit och interstitiell pneumoni, framförallt hos kalvar, och är en betydande orsak till respiratoriskt lidande hos nötkreatur över hela världen. Lika betydelsefullt på humansidan är det nära besläktade Humant respiratoriskt syncytialt virus (HRSV), som orsakar sjukdom liknande BRSV, framförallt hos spädbarn. Kliniska symtom och patologi vid RSV-infektion är inte bara en direkt följd av virusets replikation, utan även en effekt av värdens immunsvar. Denna immunopatologi har länge höljt patogenesen för RSV i ett dunkel, och utvecklandet av effektiva behandlingar och vacciner kommer att bli svårt tills en djupare förståelse erhållits. En av de komponenter i immunförsvaret som kommit i blickfånget de senaste åren inom RSV-forskning, är Toll-like receptor 4 (TLR4). TLR4 är välkänd för att binda lipopolysackarid (LPS) från Gram-negativa bakterier, och därmed initiera immunförsvaret vid bakteriella infektioner. Nya studier har visat att även RS-virusets fusionsprotein interagerar med TLR4, och uppreglerar uttrycket av denna receptor på infekterade cellers yta. Hurvida detta har övervägande skyddande effekt, som man skulle förvänta sig av ett immunsvar, eller om denna uppreglering har huvudsakligen negativa effekter för värden, är inte klarlagt. Syftet med denna studie var att utveckla en analysmetod för att kvantifiera uttrycket av TLR4 i kliniska prover, samt att avgöra om nivån av TLR4 mRNA i cellprover från bronchoalveolar lavage (BAL) kunde användas som en markör för hur väl skyddat djuret är mot experimentell BRSV infektion. Två kvantitativa realtids PCR-metoder (qPCR) utvecklades och optimerades i detta projekt. En av dessa qPCR-metoder kvantifierar uttrycket av TLR4 mRNA, den andra 28S rRNA. De uppmätta värdena av 28S rRNA användes för att standardisera resultaten för TLR4 mRNA och BRSV-titer. Båda dessa qPCR-metoder uppvisade god effektivitet och reproducerbarhet. Dessutom analyserades BAL-prover, som samlats in från kalvar som deltog i ett vaccinförsök, för att utvärdera uttrycket av TLR4 mRNA i förhållande till BRSV-titer. När dessa resultat analyserades verkade det som om nivån TLR4 mRNA inte utgör någon bra markör för graden av skydd mot BRSV-infektion efter tidigare vaccination

Proteome analysis of bronchoalveolar lavage from calves infected with bovine respiratory syncytial virus-Insights in pathogenesis and perspectives for new treatments

Human and bovine respiratory syncytial viruses (HRSV/BRSV) are major causes of severe lower respiratory tract infections in children and calves, respectively. Shared epidemiological, clinical, pathological and genetic characteristics of these viruses make comparative research highly relevant. To characterise the host response against BRSV infection, bronchoalveolar lavage supernatant (BAL) from i) non-vaccinated, BRSV-infected ii) vaccinated, BRSV-infected and iii) non-infected calves was analysed by tandem mass spectrometry. Proteins were semi-quantified and protein expression was validated by immunoblotting. Correlations between selected proteins and pathology, clinical signs and virus shedding were investigated. Calves with BRSV-induced disease had increased total protein concentrations and a decreased number of proteins identified in BAL. The protein profile was characterised by neutrophil activation and a reduction in identified antioxidant enzymes. The presence of neutrophils in alveolar septa, the expression level of neutrophil-related or antioxidant proteins and LZTFL1 correlated significantly with disease. Citrullinated histone 3, an indicator of extracellular traps (ETs), was only detected in non-vaccinated, BRSV-infected animals. By bringing disequilibrium in the release and detoxification of reactive oxygen species, generating ETs and causing elastine degradation, exaggerated neutrophil responses might exacerbate RSV-induced disease. Neutrophil-mitigating or antioxidant treatments should be further explored

Development and evaluation of new generation vaccines against bovine respiratory syncytial virus

Bovine respiratory syncytial virus (BRSV) is a major cause of respiratory disease in cattle worldwide. Improved BRSV vaccines are needed, with better efficacy and longer duration of protection, in particular when used in calves with specific maternally derived antibodies (MDA). New generation BRSV vaccines should also be DIVA compliant, and thus allow continued seromonitoring in vaccinated herds, including continuous evaluation of vaccine safety and efficacy. In this thesis work, classic BRSV immunostimulating complexes (BRSV-ISCOMs) were shown to overcome inhibition by specific MDA, and induce a high level of protection, probably mediated by strong humoral and Th1 type T cell responses. Characterization of proteins in BRSV-ISCOMs was performed to facilitate future standardization or improvement of BRSV-ISCOMs, and to serve as a basis to design efficient subunit vaccines (SU). SU consisted of recombinant human RSV (HRSV) proteins P, M2-1 and N nanorings with epitopes from BRSV proteins F and G. Three DIVA-compatible vaccines, all omitting the SH protein, were evaluated: two vaccines formulated using SU, adjuvanted by either Montanide ISA71VG (SUMont) or AbISCO-300 (SUAbis); and ΔSHrBRSV, a live recombinant BRSV with deleted SH gene. The safety, immunogenicity and protective efficacy of SUMont, SUAbis and ΔSHrBRSV were compared in calves with specific MDA in a BRSV infection model with high clinical expression, which was also developed in this thesis work. Both ΔSHrBRSV and SUMont induced protection against BRSV infection, seemingly by activating different immunological pathways. ΔSHrBRSV induced almost complete clinical and virological protection, which appeared to rely mainly on mucosal IgA and systemic neutralizing antibodies directed against viral surface proteins, and T cell priming in the airways. SUMont induced a good level of protection, which appeared to be mediated by HRSV/BRSV cross-reactive specific T cells. SUAbis induced limited protection from BRSV challenge. The three vaccines BRSV-ISCOMs, ΔSHrBRSV and SUMont, individually identified as promising vaccine candidates, are described and discussed in this thesis, including possibilities to improve their immunogenicity and protective efficacy, and to further investigate induced immunity and DIVA compliancy

Detection of citrullinated histone 3 and myeloperoxidase in serially diluted BAL.

<p>Detection of citrullinated histone 3 (a) and myeloperoxidase (b) by dotblot in bronchoalveolar lavage (BAL) from BRSV-infected calves with clinical signs of disease and high levels of virus shedding (k-o and D1-D5) compared to that in BRSV-ISCOM-vaccinated, BRSV-infected calves with no or little clinical signs of disease and no or low levels of virus shedding (a-e, Experiment I), or non-vaccinated, non-infected calves (E1-E5, Experiment II). The proteins were selected based on being present in neutrophil extracellular traps, incriminated to be important in the pathogenesis of RSV [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186594#pone.0186594.ref030" target="_blank">30</a>].</p

Animals and experimental design.

<p>Animals and experimental design.</p

Scores of neutrophilic infiltration of alveolar septa in BRSV-infected calves.

<p>HE stained sections. a) score 0 (vaccinated calf a, experiment I), b) score 1 (vaccinated calf b, experiment I), c) score 2 (non-vaccinated, BRSV-infected calf l, experiment I) and d) score 3 (non-vaccinated, BRSV infected calf D5, experiment II). Lesions are examples of neutrophil scores and not representative for the overall inflammation regarding exudate and consolidation.</p

Correlations (Spearman’s rho) between relative expression level of selected proteins in bronchoalveolar lavage from calves six days post BRSV infection, BRSV-induced disease and presence of neutrophils in alveolar septa.

<p>Correlations (Spearman’s rho) between relative expression level of selected proteins in bronchoalveolar lavage from calves six days post BRSV infection, BRSV-induced disease and presence of neutrophils in alveolar septa.</p

Semi-quantification of selected proteins involved in processes that were affected by BRSV-infection.

<p>Proteins were detected in bronchoalveolar lavage from non-vaccinated, BRSV-infected calves with clinical signs of disease and high levels of virus shedding (black bars) and/or in BRSV-ISCOM-vaccinated, BRSV-infected calves with no or little clinical signs of disease and no or low levels of virus shedding (a, experiment I, grey bars), and/or non-vaccinated, non-infected calves (b, experiment II, grey bars). Proteins were identified by LC-MS/MS and semi-quantified by label-free analysis. Statistically significant differences are indicated by asterisks; p≤0.05(*); p≤0.01 (**); p≤0.001 (***). Proteins were selected based on being related to neutrophil activation and chemotaxis or detoxification of reactive oxygen species: biological processes identified by protein pathway analysis.</p

Proteins associated with BRSV-induced disease.

<p>Proteins associated with BRSV-induced disease.</p