A Single Shot Pre-fusion-Stabilized Bovine RSV F Vaccine is Safe and Effective in Newborn Calves with Maternally Derived Antibodies

Abstract

Achieving safe and protective vaccination against respiratory syncytial virus (RSV) in infants and in calves has proven a challenging task. The design of recombinant antigens with a conformation close to their native form in virus particles is a major breakthrough. We compared two subunit vaccines, the bovine RSV (BRSV) pre-fusion F (preF) alone or with nanorings formed by the RSV nucleoprotein (preF+N). PreF and N proteins are potent antigenic targets for neutralizing antibodies and T cell responses, respectively. To tackle the challenges of neonatal immunization, three groups of six one-month-old calves with maternally derived serum antibodies (MDA) to BRSV received a single intramuscular injection of PreF, preF+N with Montanide^TM ISA61 VG (ISA61) as adjuvant or only ISA61 (control). One month later, all calves were challenged with BRSV and monitored for virus replication in the upper respiratory tract and for clinical signs of disease over one week, and then post-mortem examinations of their lungs were performed. Both preF and preF+N vaccines afforded safe, clinical, and virological protection against BRSV, with little difference between the two subunit vaccines. Analysis of immune parameters pointed to neutralizing antibodies and antibodies to preF as being significant correlates of protection. Thus, a single shot vaccination with preF appears sufficient to reduce the burden of BRSV disease in calves with MDA.

Keywords: bovine; neonate; pre-fusion conformation; respiratory syncytial virus; subunit vaccine.

Figure 1

Vaccination with preF or preF+N provides strong clinical protection against BRSV challenge. (a) Study design. D0 is the day of challenge (4 weeks after a single vaccination). Calves were sacrificed 7 days after challenge (D7), 5 weeks after vaccination. (b) Monitoring of clinical protection against BRSV infection of vaccinated calves (3 groups of 5–6 animals). The clinical scores were calculated as described in [30] based on daily clinical examinations of calves. (c) Macroscopic lung lesions are expressed as a % of total lung surface calculated from photographs of the lungs made post mortem (d) (ImageJ v1.52a free software, https://imagej.nih.gov/ij/index.html). Each spot represents one calf (controls/blue; preF/red; preF+N/green). Mean values with standard error of the mean are indicated by the black line. (d) Ventral and dorsal aspects of the lung were photographed. Ventral and dorsal lesions were also drawn onto a lung scheme. Statistics are Mann–Whitney two-tailed (b,c), day by day comparison of groups (b), * indicates significant difference between controls and vaccinated calves (black stars: mock versus both preF and preF+N, red stars: mock versus preF), ^# indicates a significant difference between the two vaccinated groups (preF versus preF+N). (* p < 0.05, ** p < 0.01).

Figure 2

Safety and efficacy of preF vaccines according to their inflammation profiles in BAL and lungs. (a) % of granulocytes counted in BAL upon May–Grünwald–Giemsa staining. Each spot represents one calf (controls/blue; preF/red; preF+N/green). Mean values with standard error of the mean are indicated by black line. Statistics are Mann–Whitney two-tailed values (* p < 0.05, ** p < 0.01). (b) Biopsies were taken from the cranial lobe, and lung sections were stained with H&E. (c) Broncho-alveolar lavage (BAL) supernatants from vaccinated calves and controls, obtained on D7 after BRSV challenge, were analysed by liquid chromatography coupled to tandem mass spectrometry (the shotgun method, LC-MS/MS, Orbitrap, Thermo Scientific, Bremen, Germany). Proteins were identified, and a total label-free intensity analysis was performed using MaxQuant 1.5.1.2 software. Twenty-five proteins were selected based on being related to neutrophils, and the relative quantities of these are presented as label-free quantification (LFQ) intensities. Statistical tests are Wilcoxon, with corrected p-values (Benjamini–Hochberg). Comparisons between groups were done for each of the 25 proteins (mock versus preF or mock versus preF+N). (* p<0.05, ** p<0.01).

Figure 3

Vaccination with preF provides strong virological protection against BRSV in upper and lower respiratory tracts. Nasal secretions were collected daily after challenge, from D0 to D7 (before autopsy). BAL cells were collected post-mortem. Total RNA was extracted, and BRSV-RNA detected by real time PCR, expressed as TCID50eq. The cut off of the RT-PCR assay is 0.7 TCID 50eq. (a) Kinetics of virus replication in the nasal passages. Mean values with standard deviation are plotted (controls/blue; preF/red; preF+N/green). (b) Area under the curve (AUC) for total virus RNA D0-D7. (c) BRSV-RNA in BAL cells collected post mortem. (b–c) Each spot represents one calf (controls/blue; preF/red; preF+N/green). Mean values with standard error of the mean are indicated by black lines. Statistics are Mann–Whitney two-tailed, day by day comparison of groups (a), * indicates a significant difference between controls and vaccinated calves (black stars: mock versus both preF and preF+N, red stars: mock versus preF, green stars: mock versus preF+N). (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 4

Systemic antibody and T cell responses elicited by pre-F or pre-F and N. Calves were vaccinated IM with preF or preF+N in ISA61 adjuvant or mock (ISA61 alone) and were challenged 4 weeks later with BRSV. (a) BRSV-specific IgG1, IgG2, IgA titers in sera at weeks 4 and 5 after vaccination. (b) pre-F-specific Ab titers in sera at weeks 0, 4, and 5 after vaccination. (c) N-specific Ab titers in sera at weeks 0, 4, and 5 after vaccination. (d) BRSV-neutralizing Ab titers in sera at weeks 0, 4, and 5 after vaccination. (e) IFNγ BRSV-specific T cells among PBMC depleted from γδ⁺ T-cells at weeks 4 and 5 after vaccination counted by ELISPOT. Each spot represents one calf (controls/blue; preF/red; preF+N/green). Mean values with standard error of the mean are indicated by the black line. Statistical analysis was performed by the non-parametric test Mann–Whitney, two-tailed p value. * indicates a significant difference between controls and the preF- or preF+N-vaccinated calves, # indicates a significant difference between the two vaccinated groups (preF and preF+N). (* # p < 0.05, ** ## p < 0.01). Limits of detection are log10 0.5 for N-specific IgG, log10 1.4 for BRSV-specific IgG2, log10 1.7 for BRSV-specific IgA and IgG1, and log10 1.0 for BRSV neutralising antibodies.

Figure 5

Mucosal antibody responses elicited by pre-F or pre-F and N. Calves were vaccinated IM with preF or preF+N in ISA61 adjuvant or mock (ISA61 alone) and were challenged 4 weeks later with BRSV. (a) BRSV-specific IgA in nasal secretions sampled on the day of challenge (week 4) and one week after challenge (week 5). (b) BRSV-, preF-, and N-specific IgA in BAL supernatant was collected post-mortem (week 5). Each spot represents one calf (controls/blue; preF/red; preF+N/green). Mean values with standard error of the mean are indicated by the black line. Statistical analysis was performed by the non-parametric test Mann–Whitney, two-tailed p value. * indicates a significant difference between controls and the preF- or preF+N-vaccinated calves, # indicates a significant difference between the two vaccinated groups (preF and preF+N). (* # p < 0.05, ** ## p < 0.01).

Figure 6

Principal component analysis (PCA) and correlation analysis of the clinical, virological, and immunological response data of the calves vaccinated with preF, preF+N or mock-vaccinated. Calves were vaccinated IM with preF or preF+N in ISA61 adjuvant or mock (ISA61 alone) and were challenged 4 weeks later with BRSV and sacrificed one week after challenge. (a) The PCA plot of the clinical, virological, and immunological response data (21 PCA variables) of vaccinated and non-vaccinated calves is depicted, with each calf represented as a dot plus ID number in a specific color according to its group assignment: Dim1 explained 54.65% of the total variation of the data between calves, and Dim 2 explained a further 12.42% of the variation. The vaccination regimen is indicated by a distinct color (blue for controls, red for preF, and green for preF+N). (b) 357 individual input values were loaded (21 parameters measured on 17 calves, see Materials and methods), and the most relevant ones are indicated on the figure for clarity (R > 0.5). In red the virological, inflammatory & clinical scores; in black the immunological parameters measured at week 4 (pre-challenge), and in blue the immunological parameters measured at week 5 (post-mortem). (c) Same as (a) but considering only the 252 individual data collected from the 12 vaccinated calves (preF and preF+N). (d) Same as (b) but considering only the 252 individual data collected from the 12 vaccinated calves (preF and preF+N). (e) Spearman Correlation analyses, two-tailed, between the immune response data collected pre-challenge (week 4) versus the clinical and virological data of the 12 vaccinated calves. Only significant/nearly significant correlations are shown, in green where negative and in red where positive.