A new subunit vaccine based on nucleoprotein nanoparticles confers partial clinical and virological protection in calves against bovine respiratory syncytial virus

Abstract

Human and bovine respiratory syncytial viruses (HRSV and BRSV) are two closely related, worldwide prevalent viruses that are the leading cause of severe airway disease in children and calves, respectively. Efficacy of commercial bovine vaccines needs improvement and no human vaccine is licensed yet. We reported that nasal vaccination with the HRSV nucleoprotein produced as recombinant ring-shaped nanoparticles (N(SRS)) protects mice against a viral challenge with HRSV. The aim of this work was to evaluate this new vaccine that uses a conserved viral antigen, in calves, natural hosts for BRSV. Calves, free of colostral or natural anti-BRSV antibodies, were vaccinated with N(SRS) either intramuscularly, or both intramuscularly and intranasally using Montanide ISA71 and IMS4132 as adjuvants and challenged with BRSV. All vaccinated calves developed anti-N antibodies in blood and nasal secretions and N-specific cellular immunity in local lymph nodes. Clinical monitoring post-challenge demonstrated moderate respiratory pathology with local lung tissue consolidations for the non-vaccinated calves that were significantly reduced in the vaccinated calves. Vaccinated calves had lower viral loads than the non-vaccinated control calves. Thus N(SRS) vaccination in calves provided cross-protective immunity against BRSV infection without adverse inflammatory reaction.

Fig. 1

Production of soluble N from HRSV or BRSV origin. (a) Coomassie blue-stained SDS-PAGE analysis of GST-PCT and N proteins from HRSV (strain Long) and BRSV (strain A2Gelfi) expressed in E. coli. Cell lysates (L) were centrifuged and the soluble (S) or unsoluble (P) fractions were run on a 12% polyacrylamide gel. Proteins were purified by glutathione–Sepharose affinity from the cell lysates and the proteins pulled-down with the sepharose beads (B) were analyzed on the same gel. GST-PCT from HRSV or BRSV together with the HRSV N protein were soluble, while BRSV N was only found in the unsoluble fraction. The HRSV N protein was efficiently purified by the BRSV PCT fragment. (b) N protein sequence comparison between HRSV Long strain and BRSV 3761 strain with the ClustalW2 sequence alignment program. Stars and points indicate amino acid identities and similarities (two dots indicate strong similarity, one dot weak similarity), respectively.

Fig. 2

N-specific antibody responses elicited in serum and nasal secretion upon N^SRS vaccination and BRSV challenge. Calves were vaccinated twice with N^SRS i.m. or i.m. + i.n. (day 0 and 21) followed by challenge with BRSV (day 42). (a) Serum Ig(H + L) and (b) nasal Ig(H + L) titers to N were measured by an ELISA endpoint assay. N-specific IgG1 and IgA Ab were quantified in nasal secretions (c and d). Data are expressed as mean ± SEM and plotted with a logarithmic scale. Stars indicate significant differences between the two vaccinated group (N^SRS i.m. and i.m. + i.n.) and the non-vaccinated one.

Fig. 3

Clinical scores following BRSV challenge. Respiratory rhythm, anorexia, presence of nasal discharge, lung sounds, cough and demeanour were recorded daily after challenge and clinical scores were calculated. Data represent means ± SEM (n = 6) in each group from day 0 (challenge) to day 19 after challenge. Stars indicate significant differences between the two vaccinated group (N^SRS i.m. and i.m. + i.n.) and the non-vaccinated one.

Fig. 4

Macroscopic and microscopic lung lesions following BRSV challenge. On day 6 post-BRSV challenge (peak of clinical scores), two calves per group were euthanized and their lungs dissected out for macroscopic analysis of lesions (a and b). Lung pieces were sampled in the right cranial lobe at the border between red atelectatic collapsed pulmonary areas and healthy tissue, fixed in formalin and embedded in paraffin. Histological examination of sections counterstained with hematoxylin/eosin/saffran showed areas of bronchointerstitial pneumonia with proliferative alveolitis in non-vaccinated calves. This marked infiltration of inflammatory cells was observed in the alveolar, peribronchiolar and bronchiolar areas (c) and was associated to a necrotizing bronchiolitis (e). Bronchiolar lumen contained sloughed necrotic epithelial cells and sometimes multinucleate syncytial cells closely associated with the bronchiolar epithelium, and few inflammatory cells infiltrating the bronchiolar epithelium. Similar sections in vaccinated calves showed alveolar functional areas with minimal thickening of alveolar septa (d) and bronchiolar lumen clear of cellular debris (f). The same lung tissue sections were stained for BRSV antigens with an anti-F monoclonal antibody (brown staining) and counterstained with hematoxylin (pale blue staining). The control immunohistochemical reaction with an isotype-matched irrelevant mouse IgG was negative (data not shown). Immunohistochemical staining of BRSV-F revealed virus-infected bronchiolar epithelial cells (g and h) with viral antigens among the necrotic cells sloughed the bronchiole lumen (g). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Fig. 5

BRSV RNA detection and viral load following challenge. The viral loads were examined by performing quantitative real time RT-PCR on total RNA extracted from BAL cells and lung pieces collected on two calves per group euthanized on day 6 after challenge (a) and from the nasal swabs sampled daily from the day of challenge up to 19 days after (b and c). Viral load is expressed as the log of BRSV copies per 10⁷ GAPDH cDNA (mean ± SEM, n = 6). Stars indicate significant differences between the two vaccinated group (N^SRS i.m. and i.m. + i.n.) and the non-vaccinated one. The daily percentage of positive calves per group is shown (c).

Fig. 6

Cell subsets recruited to the BAL following BRSV infection. Two calves per group were euthanized 6 days after BRSV challenge and the other calves were euthanized 20 days after challenge. Lungs were dissected out of the thoracic cage and lavaged with 500 ml of medium. The cells present in BAL were collected by cyto-centrifugation. The cellular composition of the BAL was established after May-Grünwald-Giemsa coloration and numeration of macrophages, lymphocytes, neutrophils and eosinophils (a). BAL cells were labeled with anti-CD45RO, CD4 and CD8 antibodies and analyzed by flow cytometry to determine which T lymphocyte subsets were recruited to the lung upon infection (b). 200,000 events were acquired, gated on lymphocytes according to FSC/SSC and CD45RO⁺ criteria (at least 5000 events were gated). Data are mean ± SEM, n = 2 at day 6 and n = 6 at day 20.

Fig. 7

N-specific memory T cell responses following vaccination and challenge. The lymph nodes draining the site of i.m. vaccination (prescapular) and the upper and lower respiratory tract (tracheo-bronchial and mediastinal, respectively) were dissected out on day 20 after BRSV challenge and processed to isolate lymph node cells. (a) In vitro lymphocyte proliferation was evaluated by measuring [³H]thymidine incorporation after N^SRS or mock antigenic restimulation for 96 h and values were expressed as stimulation index (SI). Individual SI is plotted for each group (square, circle, triangle) and the mean is shown next (black line). (b) IFN-γ was measured in the supernatant of lymph node cells cultivated for 72 h with N^SRS or with medium only (mock). Results are expressed as SI and data displayed individually as in (a).