Bovine Respiratory Syncytial Virus Challenge Research Articles

The titers, duration, and residual clinical protection of passively transferred nasal and serum antibodies are similar among beef calves that nursed colostrum from vaccinated or unvaccinated dams and were challenged experimentally with bovine respiratory syncytial virus at three months of age.

To compare initial titers, duration, and residual clinical protection of passively transferred bovine respiratory syncytial virus (BRSV) nasal immunoglobulin (Ig) G-1 and IgA, and serum neutralizing (SN) antibodies. 40 three-month-old beef steers born either to unvaccinated or vaccinated cows. During the last trimester of gestation, cows were assigned randomly to either vaccinated or unvaccinated groups. Calves were grouped on the basis of whether they nursed colostrum from unvaccinated dams (NO-VACC group; n = 20) versus dams vaccinated with 2 doses of an inactivated BRSV vaccine (VACC group; n = 20). At 3 months of age, calves were challenged with BRSV. Respiratory signs were scored. Nasal BRSV IgG-1 and IgA and SN antibodies were compared before and after the challenge. The presence of BRSV in nasal secretions was evaluated by reverse transcription-PCR assays. Respiratory scores after BRSV challenge were similar between treatment groups. Nasal BRSV IgG-1 and SN antibodies were significantly greater in VACC calves at 48 hours of life; however, by 3 months of age, titers had decayed in both groups. Nasal BRSV IgA titers were minimal after colostrum intake and before the BRSV challenge, and increased in both groups after the challenge. The NO-VACC group had a significantly greater probability of shedding BRSV compared with VACC calves. At 3 months of age, titers of passively transferred BRSV antibodies in VACC and NO-VACC calves had decayed to nonprotective levels. Calves born to vaccinated dams had a decreased probability of BRSV shedding; however, this was not related to differences in SN or nasal BRSV antibody titers.

Single-Shot Vaccines against Bovine Respiratory Syncytial Virus (BRSV): Comparative Evaluation of Long-Term Protection after Immunization in the Presence of BRSV-Specific Maternal Antibodies.

The induction of long-lasting clinical and virological protection is needed for a successful vaccination program against the bovine respiratory syncytial virus (BRSV). In this study, calves with BRSV-specific maternally derived antibodies were vaccinated once, either with (i) a BRSV pre-fusion protein (PreF) and MontanideTM ISA61 VG (ISA61, n = 6), (ii) BRSV lacking the SH gene (ΔSHrBRSV, n = 6), (iii) a commercial vaccine (CV, n = 6), or were injected with ISA61 alone (n = 6). All calves were challenged with BRSV 92 days later and were euthanized 13 days post-infection. Based on clinical, pathological, and proteomic data, all vaccines appeared safe. Compared to the controls, PreF induced the most significant clinical and virological protection post-challenge, followed by ΔSHrBRSV and CV, whereas the protection of PreF-vaccinated calves was correlated with BRSV-specific serum immunoglobulin (Ig)G antibody responses 84 days post-vaccination, and the IgG antibody titers of ΔSHrBRSV- and CV-vaccinated calves did not differ from the controls on this day. Nevertheless, strong anamnestic BRSV- and PreF-specific IgG responses occurred in calves vaccinated with either of the vaccines, following a BRSV challenge. In conclusion, PreF and ΔSHrBRSV are two efficient one-shot candidate vaccines. By inducing a protection for at least three months, they could potentially improve the control of BRSV in calves.

Demonstration of Protection Provided by a Single Dose Modified Live BRSV Vaccine against Virulent BRSV Challenge

Bovine respiratory syncytial virus (BRSV) is an economically important cause of respiratory disease in cattle worldwide. The virus is an important respiratory pathogen in naïve cattle of all ages, but as with other respiratory syncytial viruses, it primarily affects young calves. The virus has also been implicated as one of the viral agents involved in the inducing phase of bovine respiratory disease complex. Efficacy of vaccines developed to protect against BRSV related respiratory disease must be demonstrated in models that exhibit clinically relevant respiratory disease in susceptible cattle. The model used in this trial involves experimental infection with a virulent field isolate of BRSV, resulting in induction of severe clinical disease and associated pneumonic lesions.

Efficacy of a live intranasal vaccine against parainfluenza type 3 and bovine respiratory syncytial virus in young calves with maternally derived antibodies

Trial designTwo randomised controlled vaccination trials with artificial challenges were carried out in addition to a serological survey of levels of maternally derived antibodies (MDA) to parainfluenza type 3 virus...

Vitamin A deficiency impairs the immune response to intranasal vaccination and RSV infection in neonatal calves

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.

Vaccine safety and efficacy evaluation of a recombinant bovine respiratory syncytial virus (BRSV) with deletion of the SH gene and subunit vaccines based on recombinant human RSV proteins: N-nanorings, P and M2-1, in calves with maternal antibodies.

The development of safe and effective vaccines against both bovine and human respiratory syncytial viruses (BRSV, HRSV) to be used in the presence of RSV-specific maternally-derived antibodies (MDA) remains a high priority in human and veterinary medicine. Herein, we present safety and efficacy results from a virulent BRSV challenge of calves with MDA, which were immunized with one of three vaccine candidates that allow serological differentiation of infected from vaccinated animals (DIVA): an SH gene-deleted recombinant BRSV (ΔSHrBRSV), and two subunit (SU) formulations based on HRSV-P, -M2-1, and -N recombinant proteins displaying BRSV-F and -G epitopes, adjuvanted by either oil emulsion (Montanide ISA71VG, SUMont) or immunostimulating complex matrices (AbISCO-300, SUAbis). Whereas all control animals developed severe respiratory disease and shed high levels of virus following BRSV challenge, ΔSHrBRSV-immunized calves demonstrated almost complete clinical and virological protection five weeks after a single intranasal vaccination. Although mucosal vaccination with ΔSHrBRSV failed to induce a detectable immunological response, there was a rapid and strong anamnestic mucosal BRSV-specific IgA, virus neutralizing antibody and local T cell response following challenge with virulent BRSV. Calves immunized twice intramuscularly, three weeks apart with SUMont were also well protected two weeks after boost. The protection was not as pronounced as that in ΔSHrBRSV-immunized animals, but superior to those immunized twice subcutaneously three weeks apart with SUAbis. Antibody responses induced by the subunit vaccines were non-neutralizing and not directed against BRSV F or G proteins. When formulated as SUMont but not as SUAbis, the HRSV N, P and M2-1 proteins induced strong systemic cross-protective cell-mediated immune responses detectable already after priming. ΔSHrBRSV and SUMont are two promising DIVA-compatible vaccines, apparently inducing protection by different immune responses that were influenced by vaccine-composition, immunization route and regimen.

Duration of immunity to experimental infection with bovine respiratory syncytial virus following intranasal vaccination of young passively immune calves

To determine whether a combination modified-live bovine respiratory syncytial virus (BRSV) vaccine could stimulate protective immunity in young BRSV-seropositive calves following intranasal administration and determine the duration of clinical immunity. Controlled challenge study. Animals-84 dairy calves (3 to 11 days old). Responses to BRSV challenge of seronegative calves vaccinated under licensing trial conditions were compared with those of seropositive calves 2 times after vaccination. In experiment 1, young BRSV-seronegative calves were vaccinated intranasally with a minimum immunizing dose of BRSV and challenged with BRSV approximately 7 weeks later. In experiments 2 and 3, young BRSV-seropositive calves were vaccinated intranasally with a commercially available combination modified-live virus vaccine containing the commercial dose of the BRSV fraction and challenged with BRSV 9 weeks or approximately 14 weeks later, respectively. In experiments 1 and 2, BRSV-vaccinated calves had significantly higher Pao2, significantly fewer lung lesions, and significantly lower mortality rate than did unvaccinated calves subsequent to BRSV challenge. In contrast, in experiment 3, there were no differences in Pao2, lung lesions, or mortality rate between vaccinated and control calves after BRSV challenge approximately 14 weeks after vaccination. Protected calves in experiment 1 consistently had significant anamnestic mucosal and systemic antibody responses after challenge, whereas in experiments 2 and 3, antibody responses after challenge were more variable. A combination BRSV vaccine administered intranasally to young calves induced protective immunity in the presence of maternal antibodies. The duration of immune responses induced by intranasal vaccination was short (≤ 4 months). Boosting immunity iatrogenically, or by natural exposure, is probably required to obtain optimal responses to neonatal intranasal vaccination.

Local and systemic immune responses in mice to intranasal delivery of peptides representing bovine respiratory syncytial virus epitopes encapsulated in poly (dl-lactide-co-glycolide) microparticles

The potential of a microparticulate vaccine delivery system in eliciting a specific mucosal antibody response in the respiratory tract of mice was evaluated. Two vaccine candidate peptides representing epitopes from the G attachment and F fusion antigens from bovine respiratory syncytial virus (BRSV) were encapsulated into poly(dl- lactide co-glycolide) biodegradable microparticles. The encapsulation process did not denature the entrapped peptides as verified by detection of peptide-specific antibodies in mucosal secretions by ELISA using peptide as antigen. Following intranasal immunisation, the encapsulated peptides induced stronger upper and lower respiratory tract specific-IgA responses, respectively, than the soluble peptide forms. Moreover, a strong peptide-specific cell-mediated immune response was measured in splenocytes in vitro from the mice inoculated with the encapsulated peptides compared to their soluble form alone indicating that migration of primed T cells had taken place from the site of mucosal stimulation in the upper respiratory tract to the spleen. These results act as a foundation for vaccine efficacy studies in large animal BRSV challenge models.

Response to experimentally induced infection with bovine respiratory syncytial virus following intranasal vaccination of seropositive and seronegative calves

To determine whether a combination modified-live bovine respiratory syncytial virus (BRSV) vaccine can stimulate protective immunity in young BRSV-seropositive calves following intranasal (IN) administration. Controlled challenge study. 66 Holstein bull calves, 3 to 8 days old. In experiment 1, BRSV-seropositive and -seronegative calves were vaccinated IN with a commercially available combination modified-live virus vaccine formulated for SC administration; calves underwent BRSV challenge 4.5 months later. In experiment 2, BRSV-seronegative calves were vaccinated IN or SC (to examine the effect of route of administration) with the same combination vaccine that instead had a 1/100 dose of BRSV (to examine the effect of dose); calves underwent BRSV challenge 21 days later. In experiment 1, BRSV challenge resulted in severe respiratory tract disease with low arterial partial pressures of oxygen and lung lesions in most calves from all groups. Maximum change in rectal temperature was significantly greater in seropositive IN vaccinated calves, compared with seronegative IN vaccinated and seropositive control calves. Number of days of BRSV shedding was significantly lower in seronegative IN vaccinated calves than in seropositive IN vaccinated and seropositive control calves. In experiment 2, maximum change in rectal temperature was significantly greater in seronegative control calves, compared with seronegative IN and SC vaccinated calves. Shedding of BRSV was significantly reduced in seronegative IN and SC vaccinated calves, compared with control calves; also, lung lesions were reduced in seronegative IN and SC vaccinated calves. Maternal antibodies may inhibit priming of protective responses by IN delivered BRSV vaccines.

Formulation of bovine respiratory syncytial virus fusion protein with CpG oligodeoxynucleotide, cationic host defence peptide and polyphosphazene enhances humoral and cellular responses and induces a protective type 1 immune response in mice

Respiratory syncytial virus (RSV) is the leading cause of serious respiratory tract disease in children and calves; however, RSV vaccine development has been slow due to early observations that formalin-inactivated vaccines induced Th2-type immune responses and led to disease enhancement upon subsequent exposure. Hence, there is a need for novel adjuvants that will promote a protective Th1-type or balanced immune response against RSV. CpG oligodeoxynucleotides (ODNs), indolicidin, and polyphosphazene were examined for their ability to enhance antigen-specific immune responses and influence the Th-bias when co-formulated with a recombinant truncated bovine RSV (BRSV) fusion protein (DeltaF). Mice immunized with DeltaF co-formulated with CpG ODN, indolicidin, and polyphosphazene (DeltaF/CpG/indol/PP) developed higher levels of DeltaF-specific serum IgG, IgG1 and IgG2a antibodies when compared with DeltaF alone, and displayed an increase in the frequency of gamma interferon-secreting cells and decreased interleukin (IL)-5 production by in vitro restimulated splenocytes, characteristic of a Th1 immune response. These results were observed in both C57BL/6 and BALB/c strains of mice. When evaluated in a BRSV challenge model, mice immunized with DeltaF/CpG/indol/PP developed significantly higher levels of BRSV-neutralizing serum antibodies than mice immunized with the DeltaF protein alone, and displayed significantly less pulmonary IL-4, IL-5, IL-13 and eotaxin and reduced eosinophilia after challenge. These results suggest that co-formulation of DeltaF with CpG ODN, host defence peptide and polyphosphazene may result in a safe and effective vaccine for the prevention of BRSV and may have implications for the development of novel human RSV vaccines.

Response of calves to challenge exposure with virulent bovine respiratory syncytial virus following intranasal administration of vaccines formulated for parenteral administration

To determine whether single-fraction and combination modified-live bovine respiratory syncytial virus (BRSV) vaccines commercially licensed for parenteral administration could stimulate protective immunity in calves after intranasal administration. Randomized controlled trial. 39 calves. Calves were separated from dams at birth, fed colostrum with a minimal concentration of antibodies against BRSV, and maintained in isolation. In 2 preliminary experiments, 9-week-old calves received 1 (n = 3) or 2 (3) doses of a single-component, modified-live BRSV vaccine or no vaccine (8 control calves in each experiment), and were challenged with BRSV 21 days after vaccination. In a third experiment, 2-week-old calves received combination modified-live virus (MLV) vaccines with or without BRSV and calves were challenged with BRSV 8 days later. Calves were euthanized, and lung lesions were measured. Immune responses, including serum and nasal antibody and nasal interferon-alpha concentrations, were assessed. BRSV challenge induced signs of severe clinical respiratory tract disease, including death and pulmonary lesions in unvaccinated calves and in calves that received a combination viral vaccine without BRSV. Pulmonary lesions were significantly less severe in BRSV-challenged calves that received single or combination BRSV vaccines. The proportion of calves that shed virus and the peak virus titer was decreased, compared with control calves. Protection was associated with mucosal IgA antibody responses after challenge. Single and combination BRSV vaccines administered intranasally provided clinical protection and sparing of pulmonary tissue similar to that detected in response to parenteral delivery of combination MLV and inactivated BRSV vaccines previously assessed in the same challenge model.

Protection against bovine respiratory syncytial virus challenge following a single dose of vaccine in young calves with maternal antibody

Twenty-one young calves with maternally derived antibody to bovine respiratory syncytial virus (BRSV) were divided into three groups of seven, each group balanced for BRSV antibody titre. The calves had...

Duration of immunity of a quadrivalent vaccine against respiratory diseases caused by BHV-1, PI 3V, BVDV, and BRSV in experimentally infected calves

Several laboratory studies assessed the duration of immunity of a quadrivalent vaccine (Rispoval™4, Pfizer Animal Health) against bovine respiratory diseases (BRD) caused by bovine herpes-virus type-1 (BHV-1), parainfluenza type-3 virus (PI 3V), bovine viral-diarrhoea virus type 1 (BVDV), or bovine respiratory syncytial virus (BRSV). Calves between 7 weeks and 6 months of age were allocated to treatment and then were injected with two doses of either the vaccine or the placebo 3 weeks apart. Six to 12 months after the second injection, animals were challenged with BHV-1 ( n = 16), PI 3V ( n = 31), BVDV ( n = 16), or BRSV ( n = 20) and the course of viral infection was monitored by serological, haematological (in the BVDV study only), clinical, and virological means for ≥2 weeks. Infection induced mild clinical signs of respiratory disease and elevated rectal temperature in both vaccinated and control animals and was followed by a dramatic rise in neutralising antibodies in all treatment groups. Titres reached higher levels in vaccinated calves than in control calves after challenge with BHV-1, BVDV, or BRSV. On day 3 after PI 3V challenge, virus shedding was reduced from 3.64 log 10 TCID 50 in control animals to 2.59 log 10 TCID 50 in vaccinated animals. On days 6 and 8 after BRSV challenge, there were fewer vaccinated animals ( n = 2/10 and 0/10, respectively) shedding the virus than control animals ( n = 8/10 and 3/10, respectively). Moreover, after challenge, the mean duration of virus shedding was reduced from 3.8 days in control animals to 1 day in vaccinated animals in the BVDV study and from 3.4 days in control animals to 1.2 days in vaccinated animals in the BRSV study. The duration of immunity of ≥6 months for PI 3V, BHV-1 and BVDV, and 12 months for BRSV, after vaccination with Rispoval™4, was associated mainly with enhanced post-challenge antibody response to all four viruses and reduction of the amount or duration of virus shedding or both.

DNA vaccination against respiratory syncytial virus in young calves

A DNA vaccine encoding the fusion (F) gene (DNA-F) of bovine respiratory syncytial virus (BRSV) induced significant protection against BRSV infection in young calves. However, serum antibody to RSV developed more slowly in animals vaccinated with DNA-F when compared with those previously infected with BRSV. Furthermore, protection against BRSV infection was not as great as that induced by prior BRSV infection. Although there was little difference in the level of protection induced in calves vaccinated with DNA-F by either the intramuscular (i.m.) or intradermal (i.d.) routes, only the i.m. route primed for a rapid BRSV-specific IgA response after BRSV challenge. These results indicate that a DNA vaccination may be effective against RSV infection even in very young infants and calves.

Bovine respiratory syncytial virus ISCOMs—protection in the presence of maternal antibodies

The protection induced by immunostimulating complexes (ISCOMs) against bovine respiratory syncytial virus (BRSV) was evaluated and compared to that of a commercial inactivated vaccine (CV) in calves with BRSV-specific maternal antibodies. Following experimental challenge, controls ( n = 4) and animals immunized with CV ( n = 5) developed moderate to severe respiratory disease, whereas calves immunized with ISCOMs ( n = 5) remained clinically healthy. BRSV was re-isolated from the nasopharynx of all controls and from all calves immunized with CV, but from none of the calves immunized with ISCOMs. BRSV-RNA was detected by real-time PCR from a single animal in this group. Significantly higher BRSV-specific nasal IgG, serum IgG 1 and IgG 2 titers were detected before and after challenge in animals immunized with ISCOMs versus CV. In conclusion, the ISCOMs overcame the suppressive effect of maternal antibodies in calves and induced strong clinical and virological protection against a BRSV challenge.

Mucosal immunization with live recombinant bovine respiratory syncytial virus (BRSV) and recombinant BRSV lacking the envelope glycoprotein G protects against challenge with wild-type BRSV.

Recombinant bovine respiratory syncytial virus (rBRSV) and an rBRSV deletion mutant lacking the G gene (rBRSVDeltaG) were characterized in calves with respect to replication competence, attenuation, and protective efficacy as live-attenuated BRSV vaccines. Both recombinant viruses were safe and induced protection against a BRSV challenge infection. rBRSV replicated efficiently in the upper respiratory tract. Intranasal immunization with rBRSVDeltaG led to infection but not to mucosal virus replication. Neutralizing antibodies were induced by rBRSV and rBRSVDeltaG. Thus, the BRSV attachment glycoprotein G seems to be dispensable in vaccinating calves against BRSV.

Efficacy of an Inactivated BRSV Vaccine Against a Virulent BRSV Experimental Challenge in Young Calves

The objective of this study was to determine the efficacy of a vaccine containing inactivated Bovine Respiratory Syncytial Virus (BRSV) against a highly virulent BRSV challenge in calves.

Interferon gamma production during bovine respiratory syncytial virus (BRSV) infection is diminished in calves vaccinated with formalin-inactivated BRSV

Formalin-inactivated respiratory syncytial virus (FI-RSV) vaccination has been associated with severe disease in humans. Research in mice suggests that FI-RSV may prime for decreased interferon gamma (IFN-γ) production at subsequent infection. Interferon-γ production by peripheral blood mononuclear cells (PBMC) was measured following challenge of calves vaccinated with FI-BRSV to determine whether a similar mechanism is operative in a host naturally susceptible to RSV. Eight-week old male Holstein calves were administered FI-BRSV and mock challenge (V/M, n=6); mock vaccination and BRSV challenge (M/C, n=6) or FI-BRSV and BRSV challenge (V/C, n=7). Vaccine was administered twice at a 2-week interval; challenge followed one month later. On days 0, 5 and 10 postchallenge (PC), PBMC were stimulated in vitro for 24 h with live BRSV, concanavalin A (positive control) or spent media (negative control). Supernatants were assayed for IFN-γ using ELISA. Interferon-γ production by BRSV-stimulated PBMC was increased in M/C and V/C calves as compared to V/M calves on day 5 PC ( p<0.015); and increased in M/C calves compared to V/C and V/M calves on day 10 PC ( p<0.015). Over time postchallenge, a significant increase in IFN-γ production by BRSV-stimulated PBMC was seen in M/C calves ( p<0.025) but not in V/C calves. FI-BRSV vaccination of calves led to diminished IFN-γ production postchallenge. Decreased IFN-γ production may have contributed to impaired viral clearance and enhanced disease in FI-BRSV vaccinated calves.

Resistance to bovine respiratory syncytial virus (BRSV) induced in calves by a recombinant bovine herpesvirus-1 expressing the attachment glycoprotein of BRSV.

The ability of a bovine herpesvirus-1 (BHV-1) recombinant expressing the G protein of bovine respiratory syncytial virus (BRSV) to protect against BRSV infection was examined in calves. A synthetic G gene was inserted behind the gE promoter of BHV-1 to give a gE-negative, BHV-1/G recombinant. Gnotobiotic calves, vaccinated intranasally and intratracheally with BHV-1/G were challenged 6 weeks later with the Snook strain of BRSV. As controls, calves were vaccinated with a gE-negative mutant of BHV-1 which contains a frame-shift (BHV-1/gEfs). Whereas infection with BHV-1/gEfs induced only mild clinical signs, infection with BHV-1/G resulted in more severe clinical disease and higher titres of BHV-1/G were isolated from the lungs when compared with BHV-1/gEfs. Thus, expression of the G protein of BRSV increased the virulence of BHV-1 for calves. Vaccination with BHV-1/G induced BRSV-specific antibody in serum and respiratory secretions. However, only one calf developed low levels of BRSV complement-dependent neutralizing antibody. Although BHV-1/G primed calves for BRSV-specific lymphocyte proliferative responses, there was no evidence for priming of BRSV-specific cytotoxic T cells. After challenge with BRSV, there was a significant reduction in nasopharyngeal excretion of BRSV in BHV-1/G-vaccinated calves compared with controls and BRSV was isolated from the lung of only one of five vaccinated calves compared with all four control animals. In addition, the extent of gross pneumonic lesions 7 days after BRSV challenge was significantly reduced in calves vaccinated with BHV-1/G compared with controls given BHV-1/gEfs.

Comparison of DNA application methods to reduce BRSV shedding in cattle

We compared the protection afforded by three different DNA application methods against bovine respiratory syncytial virus (BRSV) infection in cattle. A synthetic gene that codes for the G protein of BRSV was inserted into a eukaryotic vector and was used in the vaccine. Intradermal (i.d.) application with a needleless injecyor (NI), the Pigjet™, reduced BRSV excretion significantly better after BRSV challenge than intramuscular (i.m.) or i.d. vaccination with a needle. Serum antibodies against the G protein were consistently the highest and showed less variation in calves vaccinated with the NI compared with those in i.m. and i.d. vaccinated calves. After BRSV challenge, secondary serum and mucosal antibody responses were also the highest in NI vaccinated calves. We conclude that DNA application with the needleless injector is substantially better than i.m. or i.d. application, and is capable to prime the immune response at the respiratory mucosa.