Bovine respiratory syncytial virus ISCOMs—Immunity, protection and safety in young conventional calves

Abstract

Bovine respiratory syncytial virus (BRSV) is a major cause of bronchiolitis and pneumonia in cattle and causes yearly outbreaks with high morbidity in Europe. Commercial vaccines against this virus needs improvement of efficacy, especially in calves with BRSV-specific maternally derived antibodies (MDA). We previously reported that an experimental BRSV-ISCOM vaccine, but not a commercial vaccine, induced strong clinical and virological protection in calves with MDA, immunized at 7-15 weeks of age. The aim of the present study was to characterize the immune responses, as well as to investigate the efficacy and safety in younger animals, representing the target population for vaccination. Four groups of five 3-8 week old calves with variable levels of BRSV-specific MDA were immunized s.c. twice at a 3 weeks interval with (i) BRSV immunostimulating complexes (BRSV-ISCOMs), (ii) BRSV-protein, (iii) adjuvant, or (iv) PBS. All calves were challenged with virulent BRSV by aerosol 2 weeks later and euthanized on day 6 after infection. The cellular and humoral responses were monitored as well as the clinical signs, the viral excretion and the pathology following challenge. Despite presence of MDA at the time of the immunization, only a minimum of clinical signs were observed in the BRSV-ISCOM group after challenge. In contrast, in all control groups, clinical signs of disease were observed in most of the animals (respiratory rates up to 76min(-1) and rectal temperatures up to 41°C). The clinical protection was associated to a highly significant reduction of virus replication in the upper and lower respiratory tract of calves, rapid systemic and local antibody responses and T helper cell responses dominated by IFNγ production. Animals that did not shed virus detectable by PCR or cell culture following challenge possessed particularly high levels of pulmonary IgA. The protective immunological responses to BRSV proteins and the ability to overcome the inhibiting effect of MDA were dependent on ISCOM borne antigen presentation.

Purification, Stability, and Immunogenicity Analyses of Five Bluetongue Virus Proteins for Use in Development of a Subunit Vaccine That Allows Differentiation of Infected from Vaccinated Animals

“…This is mediated by efficient and prolonged uptake of associated antigens by antigen-presenting cells, by facilitating quick entry into the draining lymphatic system, by supporting antigen transport and availability to class I and II major histocompatibility complex pathways, by inducing relevant cytokine production, and by presenting antigens as particulates that mimic native virus (57)(58)(59)(60). Here, the specific lymphocyte proliferative responses support previous research demonstrating the induction of cellular immunity following immunization with proteins or virus combined with ISCOM-based adjuvants (61)(62)(63).…”

Characterization of an Experimental Vaccine for Bovine Respiratory Syncytial Virus

“…Briefly, BRSV was purified by (i) centrifugation of frozen and thawed infected cell cultures at 200 ϫ g and 5°C for 5 min; (ii) the resulting supernatants were centrifuged at ϳ53,900 ϫ g and 5°C for 5 h; (iii) the resulting pellets were dissolved in phosphate-buffered saline (PBS) and centrifuged at 200 ϫ g and 5°C for 5 min, and the new pellets were treated in a similar manner five times; (iv) those supernatants were centrifuged over 20% sucrose at ϳ50,000 ϫ g and 5°C for 20 h; and (v) the resulting pellets were dissolved as described in iii. The proteins in the supernatants collected at this stage were solubilized and separated over a sucrose gradient by centrifugation, and one sample fraction was selected for further inclusion in the ISCOMs, as described previously (19). The ISCOMs were prepared through the addition of cholesterol, phosphatidylcholine, and Quil A (QPUF300; Desert King, Chile), dialysis, and purification, as described previously (19).…”

“…The proteins in the supernatants collected at this stage were solubilized and separated over a sucrose gradient by centrifugation, and one sample fraction was selected for further inclusion in the ISCOMs, as described previously (19). The ISCOMs were prepared through the addition of cholesterol, phosphatidylcholine, and Quil A (QPUF300; Desert King, Chile), dialysis, and purification, as described previously (19). Control ISCOMs (Vero-ISCOMs) were similarly prepared from uninfected Vero cells that underwent the virus purification and solubilization protocols.…”

“…The presence of live BRSV in the BRSVISCOMs was analyzed by attempting virus isolation in cell culture. In total, 50 l BRSV-ISCOM preparation was diluted 1:20 to dilute the toxic effects of Quil A on cell culture and inoculated onto 25-cm 2 sections of fetal bovine turbinate (FBT) cells, as described previously (19).…”

“…We previously produced and evaluated an experimental vaccine, BRSV-immunostimulating complexes (BRSV-ISCOMs), which induced rapid protection against virulent challenge in calves with MDA, likely partly mediated through mucosal IgA, and that seemed safe with regard to any toxic and disease-enhancing effects (18,19). ISCOMs are spherical particles that contain proteins from purified solubilized virus as well as lipids and Quillaja saponin (Quil A) (20).…”