Immunization of cattle with recombinant Newcastle disease virus expressing bovine herpesvirus-1 (BHV-1) glycoprotein D induces mucosal and serum antibody responses and provides partial protection against BHV-1

Abstract

Bovine herpesvirus-1 (BHV-1) is a major cause of respiratory tract diseases in cattle. Vaccination of cattle against BHV-1 is a high priority. A major concern of currently modified live BHV-1 vaccines is their ability to cause latent infection and subsequent reactivation resulting in many outbreaks. Thus, there is a need for alternative strategies. We generated two recombinant Newcastle disease viruses (NDVs) expressing the glycoprotein D (gD) of BHV-1 from an added gene. One recombinant, rLaSota/gDFL, expressed gD without any modification. The other recombinant, rLaSota/gDF, expressed a chimeric gD in which the ectodomain of gD was fused with the transmembrane domain and cytoplasmic tail of the NDV fusion F glycoprotein. Remarkably, the native gD expressed by rLaSota/gDFL virus was incorporated into the NDV virion 2.5-fold more efficiently than the native NDV proteins, whereas the chimeric gD was not detectably incorporated even though it was abundantly expressed on the infected cell surface. The expression of gD did not increase the virulence of the rNDV vectors in chickens. A single intranasal and intratracheal inoculation of calves with either recombinant NDV elicited mucosal and systemic antibodies specific to BHV-1, with the responses to rLaSota/gDFL being higher than those to rLaSota/gDF. Following challenge with BHV-1, calves immunized with the recombinant NDVs had lower titers and earlier clearance of challenge virus compared to the empty vector control, and reduced disease was observed with rLaSota/gDFL. Following challenge, the titers of serum antibodies specific to BHV-1 were higher in the animals immunized with the rNDV vaccines compared to the rNDV parent virus, indicating that the vaccines primed for secondary responses. Our data suggest that NDV can be used as a vaccine vector in bovines and that BHV-1 gD may be useful in mucosal vaccine against BHV-1 infection, but might require augmentation by a second dose or the inclusion of additional BHV-1 antigens.

Fig. 1

Genome maps of recombinant NDV LaSota bearing an insert encoding unmodified or chimeric versions of BHV-1 gD. (A) A transcription cassette encoding either unmodified or chimeric gD was cloned into the PmeI (italicized) site at the junction of the P and M genes of the NDV LaSota antigenomic cDNA. The gD ORF (ATG initiation and TGA termination signals in bold) was flanked by an NDV gene end (GE) transcription signal [boxed], an intergenic T nucleotide, and a gene start (GS) transcription signal [boxed]. (B) Top diagram: the pLaSota/gDFL vector bearing an insert encoding unmodified gD (filled box). Middle diagram: the pLaSota/gDF vector bearing a gD insert with the coding sequence for the gD ectodomain (filled portion of box) fused with the transmembrane (TM) domain and cytoplasmic tail (CT) of the NDV fusion protein (open portion of box). Bottom diagram: the parent pLaSota vector. NDV genes are shown as open boxes.

Fig. 2

Intracellular localization of BHV-1 gD. MDBK (panels a–c) and DF1 (panels d and e) cells were infected with rLaSota (panels a and d), rLaSota/gDFL (panels b and e) rLaSota/gDF (panels c and f) viruses at an MOI of 0.1. At 24 h post-infection, the infected cells were fixed, permeabilized, probed with a pool of gD-specific monoclonal antibodies followed by incubation with Alexa Fluor 488-conjugated goat anti-mouse IgG antibodies, and analyzed by immunofluorescence. The cells were visualized under Nikon Eclipse TE fluorescent microscope. Arrows indicate localization of gD.

Fig. 3

Surface expression of BHV-1 gD protein. (A) MDBK (panels a–c) and DF1 (panels d and e) cells were infected with rLaSota (panels a and d), rLaSota/gDFL (panels b and e) rLaSota/gDF (panels c and f) viruses at an MOI of 0.1. At 24 h post-infection, the infected cells were fixed and analyzed by indirect immunofluorescence as described in the legend to Fig. 2. Arrows indicate localization of gD. (B) Flow cytometry analysis of the surface expression of BHV-1 gD. DF1 cells were infected with the rLaSota/gDFL (panel a) or rLaSota/gDF (panel b) viruses at an MOI of 0.1, in parallel with cells that were mock-infected or infected with the LaSota empty vector. At 24 h post-infection, the cells were probed with the pool of gD-specific monoclonal antibodies followed by incubation with Alexa Fluor 488-conjugated goat anti-mouse IgG antibodies and analyzed by Flowjo program of FACSRIA II flow cytometer.

Fig. 4

Incorporation of BHV-1 gD into recombinant NDV virions. Nine-day-old embryonated SPF chicken eggs were infected with recombinant viruses. The allantoic fluid of infected eggs was harvested 48 h post-infection and clarified by low-speed centrifugation, and NDV virions were purified from the harvested medium by sucrose gradient centrifugation. The purified virus was subjected to 10% SDS/PAGE under denaturing and reducing conditions and stained with Coomassie blue R-250. The positions of the BHV-1 gD dimer and monomer are indicated by arrows in the right margin. The positions of the NDV HN, NP, P and M proteins are indicated in the right margin. Molecular masses of the marker proteins (in kilodaltons) are shown in the left margin.

Fig. 5

Western blot detection of gD-specific antibodies in sera from chickens following inoculation with NDV recombinants by the oculo-nasal route. BHV-1 that was grown in cell culture and purified by sucrose gradient centrifugation was subjected to 10% SDS/PAGE under reducing conditions and transferred to nitrocellulose. This was cut into strips and incubated with 1:100 dilutions of sera from representative chickens that had been inoculated, as described in the text and in Table 2, with (a) rLaSota, (b) rLaSota/gDFL, or (c) rLaSota/gDF viruses. The position of the BHV-1 gD monomer (71 kDa) is indicated by the arrow in the right margin.

Fig. 6

Induction of mucosal and serum antibodies specific to BHV-1 gD following IN and IT immunization of calves with recombinant NDVs. Calves in groups of 3 were immunized with the rLaSota, rLaSota/gDF, or rLaSota/gDFL virus. Calves Y83, R67 and R74 belong to the rLaSota control group; W181, R34 and R60 belong to the rLaSota/gDF group; R32, R42 and R45 belong to the rLaSota/gDFL group. (A) BHV-1 specific serum IgG titers, (B) BHV-1 specific IgA titers. The S/P ratio was calculated by subtracting the average normal control absorbance from each sample absorbance, then dividing the difference by the corrected positive control, which is the difference between average positive absorbance and average normal control absorbance. According to manufacturer's protocol, a sample was considered to be positive for BHV-1 antibodies if the S/P ratio was ≥0.3.

Fig. 7

Mean rectal temperatures of rNDV-immunized calves after challenge with BHV-1 strain Copper. Calves in groups of 3 were immunized with the rLaSota, rLaSota/gDF, or rLaSota/gDFL virus and challenged on day 28 with 10⁷ PFU/calf of BHV-1 strain Cooper. Calves Y83, R67 and R74 belong to the rLaSota control group; W181, R34 and R60 belong to the rLaSota/gDF group; R32, R42 and R45 belong to the rLaSota/gDFL group.

Fig. 8

Shedding of BHV-1 in nasal swabs following IN challenge of calves that had been immunized once IN/IT with recombinant NDVs. The immunizations were described in the legend to Fig. 6. All animals were challenged on day 28 post-immunization with 10⁷ PFU/calf of BHV-1 strain Cooper. The titers of BHV-1 were measured by plaque assay from nasal swabs soaked in 1 ml of cold minimum essential medium.

Fig. 9

BHV-1-specific serum IgG response induced in rNDV-immunized calves 12 days after challenge with BHV-1 strain Cooper. Sera were analyzed with a commercial ELISA kit using purified BHV-1 as antigen. Positive responses were determined at S/P ratio of 0.3 or higher as described in the legend to Fig. 6. Calves Y83, R67 and R74 belong to the rLaSota control group; W181, R34 and R60 belong to the rLaSota/gDF group; R32, R42 and R45 belong to the rLaSota/gDFL group.