The Mycobacterium bovis Bacillus Calmette-Guerin (BCG) vaccine is administered parenterally to infants and young children to prevent tuberculosis (TB) infection. However, the protection induced by BCG is highly variable and the vaccine does not prevent pulmonary TB, the most common form of the illness. Until improved TB vaccines are available, it is crucial to use BCG in a manner which ensures optimal vaccine performance. Immunization directly to the respiratory mucosa has been shown to promote greater protection from TB in animal models. γδ T cells play a major role in host defense at mucosal sites and are known to respond robustly to mycobacterial infection. Their positioning in the respiratory mucosa ensures their engagement in the response to aerosolized TB vaccination. However, our understanding of the effect of respiratory BCG vaccination on γδ T cell responses in the lung is unknown. In this study, we used a calf model to investigate the immunogenicity of aerosol BCG vaccination, and the phenotypic profile of peripheral and mucosal γδ T cells responding to vaccination. We observed robust local and systemic M. bovis-specific IFN-γ and IL-17 production by both γδ and CD4 T cells. Importantly, BCG vaccination induced effector and memory cell differentiation of γδ T cells in both the lower airways and peripheral blood, with accumulation of a large proportion of effector memory γδ T cells in both compartments. Our results demonstrate the potential of the neonatal calf model to evaluate TB vaccine candidates that are to be administered via the respiratory tract, and suggest that aerosol immunization is a promising strategy for engaging γδ T cells in vaccine-induced immunity against TB.

Respiratory syncytial virus (RSV) infection is a leading cause of severe acute lower respiratory tract infection in infants and children worldwide. Vitamin A deficiency (VAD) is one of the most prevalent nutrition-related health problems in the world and is a significant risk factor in the development of severe respiratory infections in infants and young children. Bovine RSV (BRSV) is a primary cause of lower respiratory tract disease in young cattle. The calf model of BRSV infection is useful to understand the immune response to human RSV infection. We have previously developed an amphiphilic polyanhydride nanoparticle (NP)-based vaccine (i.e., nanovaccine) encapsulating the fusion and attachment proteins from BRSV (BRSV-NP). Calves receiving a single, intranasal dose of the BRSV-NP vaccine are partially protected from BRSV challenge. Here, we evaluated the impact of VAD on the immune response to the BRSV-NP vaccine and subsequent challenge with BRSV. Our results show that VAD calves are unable to respond to the mucosal BRSV-NP vaccine, are afforded no protection from BRSV challenge and have significant abnormalities in the inflammatory response in the infected lung. We further show that acute BRSV infection negatively impacts serum and liver retinol, rendering even well-nourished individuals susceptible to VAD. Our results support the use of the calf model for elucidating the impact of nutritional status on mucosal immunity and respiratory viral infection in infants and underline the importance of VA in regulating immunity in the respiratory mucosa.

Respiratory syncytial virus (RSV) is a leading cause of morbidity and mortality in human infants. Bovine RSV infection of neonatal calves is pathologically and immunologically similar to RSV infection in infants, and is therefore a useful preclinical model for testing novel therapeutics. Treatment of severe RSV bronchiolitis relies on supportive care and may include use of bronchodilators and inhaled or systemic corticosteroids. Interleukin-17A (IL-17) is an inflammatory cytokine that plays an important role in neutrophil recruitment and activation. IL-17 is increased in children and rodents with severe RSV infection; and in calves with severe BRSV infection. It is currently unclear if IL-17 and Th17 immunity is beneficial or detrimental to the host during RSV infection. Digoxin was recently identified to selectively inhibit IL-17 production by antagonizing its transcription factor, retinoid-related orphan receptor γ t (RORγt). Digoxin inhibits RORγt binding to IL-17 and Th17 associated genes, and suppresses IL-17 production in vitro in human and murine leukocytes and in vivo in rodent models of autoimmune disease. We demonstrate here that in vitro and in vivo digoxin treatment also inhibits IL-17 production by bovine leukocytes. To determine the role of IL-17 in primary RSV infection, calves were treated prophylactically with digoxin and infected with BRSV. Digoxin treated calves demonstrated reduced signs of clinical illness after BRSV infection, and reduced lung pathology compared to untreated control calves. Digoxin treatment did not adversely affect virus shedding or lung viral burden, but had a significant impact on pulmonary inflammatory cytokine expression on day 10 post infection. Together, our results suggest that exacerbated expression of IL-17 has a negative impact on RSV disease, and that development of specific therapies targeting Th17 immunity may be a promising strategy to improve disease outcome during severe RSV infection.