Ad26.RSV.preF completely protects calves from severe respiratory disease induced by bovine RSV challenge

Abstract

Vaccination with Ad26.RSV.preF, an Adenoviral serotype 26 vector encoding RSV F protein stabilized in its prefusion conformation, has previously shown to be immunogenic and protective in RSV seropositive adults and immunogenic in seropositive infants. Human and bovine RSV (bRSV) are genetically highly related and share many aspects of pathogenesis, epidemiology and clinical manifestations at young age. As such, infection of calves with bRSV represents a clinically relevant model with high translational value, enabling preclinical evaluation of Ad26.RSV.preF vaccine efficacy in seronegative young animals. Immunization of young calves with Ad26.RSV.preF induced antibodies neutralizing both human and bovine RSV as well as RSV-specific cellular responses. After bRSV challenge, placebo immunized calves showed viral replication in the respiratory tract, and developed fever and lethargy accompanied with severe respiratory distress, resulting in pre-termination of 7/8 calves. In contrast, all Ad26.RSV.preF immunized calves completed the study with only mild clinical symptoms, strongly and significantly diminished viral loads in nasopharynx and lungs, and only minimal lung pathology. Thus, Ad26.RSV.preF is immunogenic in young calves and efficacious in a stringent heterologous bRSV challenge model, demonstrating induction of broadly protective immunity against severe disease.

Fig. 1. Ad26.RSV.preF induces humoral and cellular immune responses in young calves.

a hRSV preF binding antibody response in serum as measured by ELISA. Neutralizing antibody responses in serum against (b) hRSV strain CL57 and (c) bRSV strain RB94. (d) RSV F specific IFNγ responses in PBMCs, as measured by ELISpot. For all graphs, n = 8 animals for the Ad26.RSV.preF group and n = 9 for the placebo group, except for panel d, where various animals were excluded from graphing and analysis based on predefined ELISpot exclusion criteria. Color coding per animal is consistently applied in Fig. 1, Figs. 2b, c, 3a, b, 5, 6. Dotted lines in panel a, c indicate assay lower limit of detection. Dotted lines in panel b and d indicate background used for graphical purposes and statistical analysis. Mean and 95% confidence intervals are indicated for panels a, b and c, whereas the median is indicated with a horizontal line in d. P-values were calculated via a Tobit model. h/bRSV Human/Bovine Respiratory Syncytial Virus. VNA Virus Neutralization Assay. SFU Spot Forming Units. PBMC peripheral blood mononuclear cells. NS not significant.

Fig. 2. Ad26.RSV.preF vaccination protects from general illness and pre-termination or death upon bRSV challenge.

a Overview on bRSV challenge study timeline and sampling events. b Pre-termination or death after bRSV challenge, with open symbols indicating individual placebo animals. c General illness score for the Ad26.RSV.preF vaccinated animals (right panel) and placebo animals (left panel). d Body temperature measured daily and e relative bodyweight as measured on -2 (pre), 2, 5, 7, 9 and 12 days post infection. For all graphs, n = 8 animals per group. Color coding per animal is consistently applied in Figs. 1, 2b, c, 3a, b, 5, 6.

Fig. 3. Ad26.RSV.preF vaccination protects from bRSV-induced upper and lower respiratory tract disease.

a Upper respiratory disease score for the Ad26.RSV.preF vaccinated animals (right panel) and placebo animals (left panel). b Lower respiratory disease score for the Ad26.RSV.preF vaccinated animals (right panel) and placebo animals (left panel). c Respiratory rate and d oxygen saturation along the course of the infection. For all graphs, n = 8 animals per group. Color coding per animal is consistently applied in Figs. 1, 2b, c, 3a, b, 5 and 6.

Fig. 4. Reduction of airway bRSV viral load after Ad26.RSV.preF vaccination.

Viral load of nasopharyngeal brush samples taken at several timepoints prior and after bRSV infection was determined by (a) TCID50 assay and (b) qPCR. Viral load of bronchoalveolar lavage samples taken at several timepoints prior and after bRSV infection was as determined by (c) TCID50 assay and (d) qPCR (pre = 5 days prior to infection). For all graphs, n = 8 animals per group. TCID50: 50% tissue culture infectious dose. qPCR: quantitative Polymerase Chain Reaction.

Fig. 5. Protection against bRSV-induced lung pathology by Ad26.RSV.preF without signs of enhanced respiratory disease.

a Percentage consolidated lung area with the respective days of termination of the animals. b–e Sum score of histopathological assessment of lung tissue of 3 sample sites collected on 6, 7, 9 or 13 days post infection for the presence of (b) alveolitis, (c) (endo)bronchitis, (d) peribronchitis and (e) interstitial cell infiltration. Maximal score is indicated with dotted line. Median score per group is indicated with a horizontal line. For all graphs, n = 8 animals per group. Color coding per animal is consistently applied in Figs. 1, 2b, c, 3a,b, 5 and 6.

Fig. 6. Boosting of Ad26.RSV.preF-induced immune responses by bRSV challenge.

a hRSV preF binding antibody response in serum as measured by ELISA. Neutralizing antibody responses in serum against (b) hRSV strain CL57 and c) bRSV strain RB94. d hRSV F specific IFNγ responses in PBMCs, as measured by ELISpot. Color coding per animal is consistently applied in Figs. 1, 2b, c, 3a, b, 5 and 6. Dotted lines in panels indicate assay limit of detection. Mean and 95% confidence intervals are indicated for panels a–c, whereas the median is indicated with a horizontal line in d. P-values were calculated using a Tobit model. h/bRSV Human/Bovine Respiratory Syncytial Virus. VNA Virus Neutralization Assay. SFU Spot Forming Units. PBMC peripheral blood mononuclear cells. NS not significant.