Protective efficacy of a recombinant adenovirus expressing novel dual F and HN proteins of bovine parainfluenza virus type 3

doi:10.21203/rs.3.rs-4243219/v1

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Protective efficacy of a recombinant adenovirus expressing novel dual F and HN proteins of bovine parainfluenza virus type 3

https://doi.org/10.21203/rs.3.rs-4243219/v1

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Bovine parainfluenza virus type 3 (BPIV3) is a viral respiratory pathogen in cattle that cause significant economic losses. We generated a recombinant adenovirus expressing both the fusion (F) and hemagglutinin-neuraminidase (HN) glycoprotein of BPIV3 based on human adenovirus serotype 5 (rHAd5), named rHAd5-F + HN. Immunization with rHAd5-F + HN induced a notable humoral immune response specific to BPIV3 in both mice and calves. Serum antibodies responses were evaluated by ELISA, hemagglutination inhibition (HI), and neutralizing antibodies (Nab). After boosting immunity with rHAd5-F + HN, mice were able to produce higher levels of antibodies against the BPIV3 genotype A and genotype C strains, significantly exceeding those in the rHAd5-F and rHAd5-HN. The mice splenic CD3⁺/CD8⁺T lymphocytes and IL-4⁺ cytokine percentages were more significant in the rHAd5-F + HN group. The protective efficacy of rHAd5-F + HN was evaluated using a mouse model challenged with BPIV3. Mice immunized with rHAd5-F + HN exhibited significantly lower viral loads in the lungs and tracheas compared to the control group. Furthermore, no significant histopathological changes were observed in the lungs of mice vaccinated with rHAd5-F + HN. Also, the rHAd5-F + HN elicited a humoral immune response in calves, following the booster intramuscular injection with the rHAd5-F + HN, the serum antibodies levels against BPIV3 genotype C strain were 1:20,452, 1:1,024 and 1:426 in calves, as detected by ELISA, HI and Nab, respectively. The HI antibodies and Nab levels against BPIV3 genotype A strain were 1:213 and 1:85 in calves, respectively. These results indicated that rHAd5-F + HN effectively induced robust immunity against BPIV3 infection.

Bovine parainfluenza virus type 3

Adenovirus vector vaccine

F and HN proteins

Protection efficacy

Bovine parainfluenza virus type 3 (BPIV3) is a respiratory tract pathogen belonging to the genus Respirovirus within the family Paramyxoviridae [1]. BPIV3 commonly coexists with bovine coronavirus (BCoV), bovine herpesvirus type 1 (BHV-1), bovine respiratory syncytial virus (BRSV), bovine viral diarrhea virus (BVDV), and Mycoplasma [2]. These co-infections, coupled with the potential for secondary bacterial infections, played a significant role in the development of Bovine Respiratory Disease Complex (BRDC) in cattle, leading to substantial economic losses in the cattle industry annually [3].

The genome of BPIV3 spans approximately 15,000 nucleotides, containing genes for six structural proteins (N, P, L, M, F, and HN) and three non-structural proteins (C, D, and V) [4]. BPIV3 is classified into three genotypes: A, B, and C [5]. The novel BPIV3 genotype C was initially detected in China and spreading globally [2–4, 6–8]. Epidemiological investigations revealed that genotype C was the predominant strain in China [8]; however, there was no commercial vaccine available in China [9]. Previous studies demonstrated the high transmissibility of BPIV3 genotype C and a reduced cross-reactivity of genotype C sera to genotypes A and B viruses [10, 11], which suggested a potential weakening in the cross-protective efficacy against these other genotypes [4, 7, 12, 13]. Therefore, there is an urgent requirement for a safe and effective vaccine to manage epidemics caused by the BPIV3 genotype C strain.

As protective antigen in the paramyxovirus family, the fusion (F) protein and hemagglutinin-neuraminidase (HN) protein can stimulate the production of neutralizing antibodies, making them ideal targets for the development of genetic engineering vaccines [14–20]. The self-cleaving peptide P2A, consisting of 22 amino acids, is located between two proteins of the porcine teschovirus [21]. P2A is capable of efficiently cleaving adjacent proteins through ribosomal skipping, with a high efficiency rate of 99%. This technique was commonly employed in the construction of multi-gene expression vectors [22], which had been successfully utilized in the construction of genetic engineering vaccines [23, 24].

The replicating-defective recombinant human adenovirus type 5 (rHAd5) was widely used as a vector in vaccine development due to its ability to induce strong cellular, humoral, and mucosal immune responses in animals [25, 26]. Therefore, rHAd5 has been extensively utilized in vaccine development for a wide range of diseases [27, 28]. In a previous study, we successfully developed a recombinant adenovirus (rHAd5-F) expressing the F protein of BPIV3 genotype C, which exhibited excellent immunogenicity and demonstrated significant protective potential in mice [20]. Therefore, we developed a novel vaccine with enhanced immunogenicity, a recombinant adenovirus based on rHAd5 in this study, and P2A was added to enable the co-expression of the F and HN proteins, aiming to develop a vaccine against BPIV3.

2.1 Experimental animals

Specific pathogen-free (SPF) female BALB/c mice were sourced from Chengdu Dossy Experimental Animals Co, Ltd. All the calves used in this experiment were sourced from Sichuan Xuebao Dairy Group Company.

2.2 Cells lines and viruses

The HEK293 and MDBK cell lines are preserved in our laboratory. BPIV3 genotype C (SMU-SC20) strain (GenBank: OM621819.1) [29] and BPIV3 genotype A (B15) strain (GenBank: PP025529.1) were isolated from yaks and water buffaloes suffering from BRDC, respectively. This viruses titer were 1×10^6.5 TCID₅₀/mL and 1×10^6.45 TCID₅₀/mL, respectively [29].

2.3 Construction and characterization of recombinant adenoviruses expressing BPIV3 F and HN proteins

The F and HN genes of BPIV3, derived from the SMU-SC20 strain, were codon-optimized using UpGene software. The self-cleaving peptide P2A was strategically inserted between the F and HN genes, facilitating the simultaneous expression of both genes segments in a concatenated fashion. After sequencing validation, the adenoviral shuttle plasmid containing the target gene were co-transfected with the adenoviral backbone plasmid (pBHGloxΔE1,3Cre) into HEK293 cells for further analysis.

Western blot and indirect immunofluorescence (IFA) techniques were performed to confirm the expression of the F and HN proteins in this recombinant adenoviruses. The specific methods were as previously described [20].

The rHAd5-HN, and rHAd5-F + HN were each consecutively passaged 12 times. Passages 1, 3, 6, 9, and 12 were utilized to assess the stability of the plasmid. The HN and F + HN genes were amplified using pDC316 universal primers, followed by sequence alignment.

2.4 Chromatographic purification of recombinant adenoviruses

This recombinant adenoviruses were purified using Diamond Layer 700 BA, which employs anionic and hydrophobic interactions along with molecular sieving, and Diamond Q Mustang, which involves ion exchange interactions. The HN, and F + HN genes were amplified using the universal primers of pDC316. Following purification, the viral titer (TCID₅₀ mL^− 1) of this recombinant adenoviruses were determined in HEK293 cells using the Reed-Muench method.

To determine the viral growth kinetics of this recombinant adenoviruses, we collected cell culture supernatants every 12 h post-infection with this recombinant adenoviruses to determine the TCID₅₀ at each time point.

2.5 Immunization and challenge

SPF BALB/c mice were randomly assigned to four groups, with five mice per group. To initiate immunization, this recombinant adenoviruses were segregated into high and low titer groups, and then intramuscularly injected into the thigh muscles of the mice. The intramuscular (IM) injection volume was 200 µL, with a titer of (1×10⁶ TCID₅₀ mL^− 1) for the high titer group and (1×10⁵ TCID₅₀ mL^− 1) for the low titer group. Additionally, a group were included in which mice received intramuscular injections of rHAd5-WT, which does not contain any foreign genes. The inactivated BPIV3 were administered intramuscularly at 200 µL (1×10^6.5 TCID₅₀ mL^− 1), additionally, a phosphate-buffered saline (PBS) control group was included.

To assess the vaccine's protective efficacy, mice were challenged with 100 µL of the SMU-SC20 strain at a titer of 1×10^6.5 TCID₅₀ mL^− 1. On day 7 post-infection (dpi), all mice were euthanized, and necropsy was performed. The tracheas and lungs of the mice were collected to measure the mRNA levels of BPIV3. Additionally, histopathological analysis was conducted on the lung tissues.

Nine commercially raised calves, aged 2–3 months, were randomly divided into three groups, with three calves in each group. Before vaccination, nasal swabs were collected from the calves, and BVDV, BPIV3, IBRV, and BCoV tests were performed. The results showed that all tests were negative. The rHAd5-F + HN was administered via IM injection for immunization, with a total volume of 2 mL (1×10^6.4 TCID₅₀ mL^− 1). The inactivated BPIV3 was administered intramuscularly at 2 mL (1×10^6.5 TCID₅₀ mL^− 1), and the control calves were intramuscularly injected with PBS 2 mL.

2.6 Indirect enzyme-linked immunosorbent assay (ELISA)

For the BPIV3-specific IgG antibodies in mice and calves serum samples, The 96-well polyethylene microtiter plate was coated with inactivated SMU-SC20 strain and then incubated overnight at 4°C. The secondary antibodies used were anti-mouse-HRP or anti-bovine-HRP (Millipore Sigma) at a dilution of 1:5,000, respectively. The specific methods were as previously described [20].

2.7 BPIV3 hemagglutination inhibition (HI) assay

HI antibodies were measured using methods previously described [17]. Serum samples, serially diluted two-fold (50 µL each), were incubated at 37°C for 1 hour with an equal volume of diluent containing the hemagglutination units of either SMU-SC20 or B15 strains. After that, 50 µL of 1.0% chicken red blood cells was added, and the mixture was incubated at 37°C for 1 hour. The HI antibody titer was determined as the highest serum dilution that completely inhibited hemagglutination.

2.8 BPIV3 neutralization assay

Nab titers against SMU-SC20 and B15 strains were determined in the serum of mice and calves using the serum neutralization test, as previously described [20]. The serum was diluted two-fold, and 100 µL of the diluted sample was mixed with an equal volume of viral supernatant containing 200 TCID₅₀/0.1 mL of either SMU-SC20 or B15 strains, and then incubated. The highest serum dilution showing a cytopathic effect of more than 50% was chosen as the titer for virus-neutralizing antibodies.

2.9 Flow cytometry assays

Three weeks after the immunization boost, splenic lymphocytes were isolated from immunized mice (n = 5 per group) and resuspended at a concentration of 5×10⁶ cells/mL in RPMI-1640 containing 10% fetal bovine serum. Subsequently, the cells were seeded into 6-well plates at 2 mL per well. Then, the cells were stimulated with the inactivated SMU-SC20 strain (60 µg/mL) and ionomycin (1 µg/mL) for 12 hours. After the incubation, the cells were stained with anti-CD3-FITC, anti-CD4-FITC, anti-CD8-PE/Cy7, anti-IFN-γ, and anti-IL-4 antibodies (Biolegend, USA) at 4°C for 2 hours. The percentage of positive cells was measured using the CytoFLEX Cell Analyzer (Beckman, CA, USA).

2.10 Quantitative reverse-transcription polymerase chain reaction (RT-PCR)

As previously mentioned, our laboratory established a real-time RT-PCR method to quantify the viral loads of BPIV3 in mice tissues [20]. The assay sensitivity of the RT-PCR method was 1.0×10² copies/µL and the virus loads were calculated as y = − 3.203x + 45.76.

2.11 Histopathological examination

After euthanizing the mice, lung tissues were collected for histopathological examination. Hematoxylin and eosin staining was performed using an optical microscope (Leica DM4000B, Wetzlar, Germany) to observe pathological changes.

2.12 Statistical analysis

The data were analyzed and plotted using GraphPad Prism software (version 10.1.2). Differences were analyzed using one-way analysis of variance (ANOVA).

3.1. Construction and identification of rHAd5-HN and rHAd5-F + HN

This recombinant adenoviruses rHAd5-F and rHAd5-WT were previously constructed, identified, and preserved in our laboratory [20]. To ensure experimental consistency, we simultaneously identified and purified three types of adenoviruses. The F genes spans a length of 1,623 base pairs (bp), encoding a total of 540 amino acids (aa). The entire coding sequence of the HN genes was 1,719 bp in length, which corresponded to 573 aa. The P2A gene was 84 bp, encoding a protein with 28 aa. It was inserted between the F and HN genes, facilitating the translation of a protein that could be cleaved into two distinct and independent proteins.

The schematic diagram of the recombinant adenovirus construct was shown in Fig. 1a. The infection of recombinant adenoviruses resulted in cytopathic effects were associated with the formation of adenoviruses within the cells (Figure. 1b). The results of the Western blot experiment indicated the expression of F and HN proteins (Figure. 1c). The expression of F and HN proteins were validated through immunofluorescence assay, where specific green fluorescence were observed under a fluorescence microscope (Figure. 1d). Based on these findings, the recombinant adenoviruses successfully expressed the F and HN proteins in HEK293 cells.

PCR analysis of the serially passaged recombinant adenoviruses revealed detectable presence of HN and F + HN genes at every passage. Sequencing confirmed the accuracy of the results (Fig. 1e), indicating the genetic stability of the recombinant adenoviruses.

3.2 Recombinant adenoviruses purified by chromatography

To enhance the purity of this viruses, the recombinant adenoviruses in this experiment were subjected to purification by anion exchange chromatography. The purification process involved chromatography on a Diamond Layer 700 BA, which utilizes anionic interactions and molecular sieving.

The purified liquid, which contained the recombinant adenoviruses, were collected at the point when a peak appeared on the UV curve (Figure. 2a). The purified recombinant adenoviruses were then utilized to infect HEK293 cells in order to assess its remaining infectivity (Figure. 2b). PCR analysis were performed to verify the presence of exogenous genes in the purified recombinant adenoviruses (Figure. 2c). The TCID₅₀ values of the purified recombinant adenoviruses, namely rHAd5-F, rHAd5-HN, rHAd5-F + HN and rHAd5-WT, were calculated using the Reed-Muench method and were 1×10^6.06 TCID₅₀/0.2 mL^− 1, 1×10^7.12 TCID₅₀/0.2 mL^− 1, 1×10^6.40 TCID₅₀/0.2 mL^− 1, and 1×10^5.49 TCID₅₀/0.2 mL^− 1, respectively (Figure. 2d). These results indicated that the recombinant adenoviruses were purified.

3.3 Recombinant adenoviruses induced humoral immune responses in mice

To assess the humoral immune response of the recombinant adenoviruses in mice, serum samples were collected from immunized mice every weeks (Figure. 3a). The variations in IgG levels, HI antibodies titers and Nab titers were dynamically monitored.

The specific IgG antibodies titers against BPIV3 in mice serum were determined using an indirect ELISA method. During the peak period of 14 days after the second immunization, the comparison of antibodies titers among different groups showed that the high dose groups (rHAd5-F, rHAd5-HN, and rHAd5-F + HN) of recombinant adenoviruses demonstrated significantly higher antibodies titers compared to the low dose groups rHAd5-F (p < 0.05), rHAd5-HN (p < 0.05), and rHAd5-F + HN (p < 0.001) (Figure. 3b). The average antibodies titer of the high-titer rHAd5-F + HN group reached as high as 1:40,906, significantly higher than the titer of 1:28,924 in the high-titer rHAd5-F group (p < 0.05), and the titer of 1:17,198 in the high-titer rHAd5-HN group (p < 0.05). The average antibodies titer in the BPIV3 inactivated vaccine group was 1:28,526, which was lower than the high dose group rHAd5-F + HN, but the statistical analysis showed no significant difference (ns, p ≥ 0.05).

Serum samples were subjected to a HI assay to detect the presence of HI antibodies against the SMU-SC20 and B15 strains (Figure. 3c). In the high dose rHAd5-F + HN group, the HI titers in the serum were 1:813 (genotype C) and 1:160 (genotype A), which were significantly higher than that of the high dose rHAd5-F group (1:384) (genotype C) (p<0.05) / (1:96) (genotype A) (p<0.01) and the high dose rHAd5-HN group (1:192) (genotype C) (p<0.05) / (1:112) (genotype A) (p<0.05). The average HI antibodies titer in the BPIV3 inactivated vaccine group were 1:1,792 (genotype C) and 1:320 (genotype A), which were higher than that of the high dose rHAd5-F + HN group. However, the statistical analysis indicated no significant difference between the two groups (ns, p ≥ 0.05).

Serum samples were analyzed using a virus-neutralization assay to ascertain the presence of Nab against the SMU-SC20 and B15 strains (Figure. 3d). In the high dose rHAd5-F + HN group, the Nab titers in the serum were 1: 640 (genotype C) and 1:384 (genotype A), which were significantly higher than that of the high dose rHAd5-F group (1:320) (genotype C) (p<0.05)/(1:247) (genotype A) (p<0.05) and the high dose rHAd5-HN group (1:301) (genotype C) (p<0.01)/(1:96.5) (genotype A) (p<0.01). The average Nab titer in the BPIV3 inactivated vaccine group were 1:488 (genotype C) and 1:288 (genotype A), which was lower than that of the high dose rHAd5-F + HN group. However, the statistical analysis indicated no significant difference between the two groups (ns, p ≥ 0.05).

The results suggested that the rHAd5-F + HN group could generate higher IgG antibodies titers and robust humoral immune responses compared to the rHAd5-F group, rHAd5-HN group, and inactivated BPIV3 group. The rHAd5-F + HN could induce the production of high titer HI and Nab in mice, and it has a neutralizing effect on different genotypes of BPIV3 strains. Additionally, there were no significant differences in HI and Nab levels between the low-dose and high-dose groups of recombinant adenoviruses, indicated that even a low dose of adenoviruses also could induce high titers of HI and Nab against BPIV3.

3.4 Recombinant adenovirus induced cellular immune responses in mice

The cellular immune response of rHAd5-F + HN in mice was depicted in Fig. 3e, the percentages of CD3⁺ T and CD8⁺ T lymphocytes and the IL-4 of cytokine in the rHAd5-F + HN group were significantly higher compared to the PBS group (p < 0.01)/(p < 0.05)/(p<0.05).

These results indicated that rHAd5-F + HN had a potent immunogenic effect, as it induced a greater number of mature DCs in the spleen and enhances T lymphocyte proliferation. Furthermore, these results demonstrated the potential of rHAd5-F + HN as a promising vaccine candidate for viral infections.

3.5 Recombinant adenoviruses protected mice against BPIV3 infection

On day 7 after challenge infection, mice were euthanized, and their lungs and tracheas were collected for measuring viral loads and conducting histopathological examination (Fig. 4a).

The high dose rHAd5-F + HN group demonstrated the lowest viral loads in the tracheas and lungs of mice, with levels almost reaching the detection limit of the assay (Figs. 4c,d). The viral loads in the tracheas of the mice injected with the high dose rHAd5-F + HN group was significantly lower than that in the high dose rHAd5-HN group (p < 0.05). However, there were no significant differences in viral loads between the high-dose and low-dose groups in the tracheas and lungs (p > 0.05), indicated that these varying administration doses exhibited similar efficacy in controlling SMU-SC20 strain infection.

Visual assessment and histopathological analysis of the lungs were conducted following challenge with the SMU-SC20 strain (Figs. 4b). The findings of microscopy and visual assessment showed no pathological changes in the lungs of mice in the high-dose groups (such as rHAd5-F, rHAd5-HN and rHAd5-F + HN), uninfected groups and inactivated BPIV3 groups. However, various pathological changes such as swelling and bleeding were visibly apparent, and microscopic analysis revealed varying degrees of alveolar epithelial hyperplasia, interalveolar capillary hyperemia, and infiltration of lymphocytes and red blood cells in the alveolar septum and bronchioles in the lungs of mice from both the PBS and rHAd5-WT groups. In the low-dose groups of rHAd5-F and rHAd5-HN, the lungs also showed varying degrees of mild hemorrhage, congestion, thickening of alveolar septa, and infiltration of red blood cells. These findings suggested that immunizing mice with the low-dose of rHAd5-F and rHAd5-HN led to different levels of pathological damage in lung tissues, suggesting that rHAd5-F + HN provided enhanced immune protection against BPIV3C in mice.

3.6 Recombinant adenovirus induced humoral immune responses in calves

In order to evaluate the humoral immune response of the rHAd5-F + HN in calves, serum samples were collected from immunized calves (Figure. 5a). The changes in IgG levels, HI antibodies titers, and Nab titers were dynamically monitored.

To evaluate the levels of specific IgG antibodies against BPIV3 in the serum of immunized calves groups, the levels of IgG antibodies in both the rHAd5-F + HN immunization group and the BPIV3 inactivated vaccine group started to increase after 21 days of primary immunization, the rHAd5-F + HN immunization group exhibited an average IgG antibodies titer of 1:20,452 at 26 days after the second immunization, which was significantly higher than that in the PBS group (p < 0.01). However, there were no significant difference observed between the rHAd5-F + HN immunization group and the BPIV3 inactivated vaccine group (ns, p ≥ 0.05).

Serum samples were analyzed using a HI assay to detect the presence of HI antibodies against the SMU-SC20 and B15 strains (Figure. 5c,e). In the rHAd5-F + HN group, the average HI titers in the serum were 1:1,024 (genotype C) and 1:213 (genotype A), which was significantly higher than that of the PBS group (p<0.05). The average HI titer in the BPIV3 inactivated vaccine group were 1:1,365 (genotype C) and 1:314 (genotype A), which was higher than that of the rHAd5-F + HN group. However, that were no significant difference between the two groups (ns, p ≥ 0.05).

Serum samples were subjected to a virus-neutralization assay to determine the presence of Nab against the SMU-SC20 and B15 strains (Figure. 5d,f). In the rHAd5-F + HN group, the Nab titers in the serum were 1:426 (genotype C) and 1:85 (genotype A), which was significantly higher than that of the PBS group (p<0.01). The average Nab titer in the BPIV3 inactivated vaccine group were 1:341 (genotype C) and 1:37 (genotype A), which were lower than that of the rHAd5-F + HN group. However, that were no significant difference between the two groups (ns, p ≥ 0.05).

These results suggested that the recombinant adenovirus rHAd5-F + HN could induce the production of high titer IgG, Nab and HI antibodies in calves, and it had a neutralizing effect on different genotypes of BPIV3 strains.

BPIV3 is one of important respiratory pathogens in development of BRDC, which causes significant economic losses in the cattle industry, and vaccination was widely recognized as the most effective means to prevent this disease [30]. However, there were currently no commercial vaccines targeting this pathogen available in China [1]. In a previous study, we found that the recombinant adenovirus rHAd5-F, expressing the F protein of BPIV3, demonstrated significant protective potential in mice [20]. To further enhance its immunogenicity, this study successfully constructed a recombinant adenovirus, rHAd5-F + HN, using the P2A peptide to link the F and HN proteins of BPIV3. The self-cleaving peptide P2A was extensively utilized in the construction of multi-gene expression vectors due to its remarkable efficiency in protein cleavage [31]. In this study, a candidate vaccine was developed using the rHAd5 system, enabling the concurrent expression of the F and HN proteins of BPIV3 by utilizing the P2A peptide. According to the Western blot analysis, the F and HN proteins were completely cleaved and were demonstrated strong biological activity. This study further confirmed the feasibility and advantages of P2A in constructing polygene expression adenovirus vector, and provided a practical reference for the design of BRDC multi-gene engineering vaccine.

The F and HN glycoproteins were protective antigens of the virus to induce neutralizing antibodies [14, 19, 32]. rHAd5 have gained extensive usage in the design of veterinary vaccines due to its capability to stimulate humoral, cellular, and mucosal immune responses [25–28]. A lot of adenovirus vectors have been developed based on the F and HN proteins within the paramyxoviridae family, showcasing potent humoral and cellular immune responses [15, 18, 33, 34]. Studies have demonstrated that the utilization of recombinant adenovirus through different immunization routes could have varying degrees of impact on the immune response [35]. In a previous study, we immunized mice with the rHAd5-F through two different routes: intramuscular injection and intranasal administration. The results showed that the intramuscular immunization route was superior to intranasal immunization [20]. Therefore, in this study, we consistently employed the intramuscular injection route to assess the optimal immunization efficacy of the constructed recombinant adenovirus.

Interestingly, in a study targeting the respiratory syncytial virus (RSV), a member of the paramyxoviridae family, it was discovered that virus-like particles (VLPs) containing both glycoproteins exhibited superior immunogenicity compared to VLPs containing only the F protein or G protein in a cotton rat model [36]. Moreover, the results suggested that the presence of the G protein in VLPs influenced the conformation of the F protein, resulting in a significant elevation in Nab levels and offering enhanced protection for cotton rats against RSV infection [36]. The G protein in RSV and the HN protein in BPIV3 are both primary transmembrane surface glycoproteins of this viruses, located in the same coding region of the viral genome, and play similar roles in viral infection and entry [37, 38]. Furthermore, this study produced comparable findings, suggesting that the HN protein and F protein could synergistically boost antibody levels and protective efficacy. Although previous studies have demonstrated that the two proteins could synergistically enhance their immunogenic effects [18], it was remains unclear that these proteins collaborate to boost their immunogenicity.

Recombinant adenovirus in this study induced a robust humoral immune response in mice. The rHAd5-F + HN induced a significantly higher titer of IgG antibodies in mice than the rHAd5-F and rHAd5-HN; similar results were obtained in previous studies [18, 19]. Nab and HI antibodies were utilized to assess an individual’s immune status with regard to controlling infectious diseases, and were often considered indicators of vaccine protection [17]. We chose the SMU-SC20 and B15 strains to determine the titers of Nab and HI. Previous studies demonstrated that antibodies induced by rHAd5-F and rHAd5-HN in cotton mice are effective against the BPIV3 genotype A sf4 strain with an average HI and Nab titer of 1:363 and 1:239, respectively [18]. These findings suggested that the rHAd5-F + HN vaccine induced cross-neutralizing titers in BALB/c mice against two different genotypes of BPIV3 with high titers being recorded, which was superior to findings in previous studies [18]. The reason might be the influence of preexisting immunity [39, 40], previous preparations involved recombinant adenoviruses expressing F and HN proteins separately, requiring separate immunizations [18]. An increased number of immunizations would significantly affect the immune efficacy of the adenovirus vector vaccine [39]. Other investigators found the vaccines induced a cellular immune response that were critical for defending against respiratory viruses [41–43]. We observed that rHAd5-F + HN significantly enhanced the proliferation of CD3⁺/CD8⁺ T lymphocytes and increased IL-4 production in mice. These findings indicated that rHAd5-F + HN effectively induced a cellular immune response.

BALB/c mice were widely acknowledged as an optimal animal model for studying respiratory tract infections caused by BPIV3. Employing this model for virus challenge studies was essential in evaluating the protective efficacy of vaccines [17]. Therefore, in this study, we employed BALB/c mice as a representative model to assess the immune protection conferred by the recombinant adenovirus against SMU-SC20 strain infection. These results indicated that the rHAd5-F + HN could protect mice from BPIV3 genotype C infection.

The immunization of recombinant adenovirus candidate vaccine in cattle and the evaluation of its safety and immunological effect are the key indicators of whether the vaccine can be commercialized. Currently, there was limited research on genetic engineering vaccines developed for BPIV3, both domestically and internationally, and on their immunological studies in calves. Haanes et al (1997) utilized an insect cell baculovirus expression system to develop a subunit vaccine, HN-Baculo, incorporating the BPIV3 HN protein. The study revealed that calves immunized with HN-Baculo demonstrated elevated levels of specific serum Nab and HI antibodies. The peak Nab titer reached 1:96, while the highest HI antibodies titer reached 1:48. The levels of Nab and HI antibodies induced in calves by the rHAd5-F + HN in this study were significantly higher than those observed in other studies. Furthermore, these results indicated that the rHAd5-F + HN vaccine candidate exhibited a high level of safety in calves and effectively triggered high titers of Nab and HI antibodies against BPIV3 genotypes A and C. These findings had critical implications for developing effective vaccines against BPIV3 infection in cattle.

In conclusion, we successfully constructed a BPIV3 vaccine candidate rHAd5-F + HN and tested its humoral and cellular immunity in mice. The mice and calves immunized with rHAd5-F + HN generated robust immune responses against BPIV3 genotypes A and C. After a challenge, the mice immunized with the rHAd5-F + HN showed reduced lungs pathological damage.

Acknowledgments

This study were supported by 14th Five-Year Plan National Key Research and Development Project (2021YFD1600203), and Sichuan Transfer Payment Technology Project (220022), and Fundamental Research Funds for the Central Universities, Southwest Minzu University (ZYN2023111).

Author contribution

ZB designed and initiated the study. ZJQ participated in the design and conducted most of the experiments in the study and drafted the manuscript. ZCX, DGN, AAJ and MYZ took the animal experiment. HCS, GR and LL analyzed the data. All authors read and approved the final manuscript.

Availability of data and materials

All the data generated or analysed during this study were included in this published article.

Ethics approval and consent to participate

The animal experiment was approved by the Sichuan Laboratory Animal Welfare and Ethics Committee of the Sichuan Administration Committee of Laboratory Animals, and received approval from the Research Ethics Committee of Southwest Minzu University (reference number SMU-202401073).

Competing interests

The authors declare that they have no competing interests.

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Protective efficacy of a recombinant adenovirus expressing novel dual F and HN proteins of bovine parainfluenza virus type 3

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Abstract

Figures

1. Introduction

2. Materials and methods

2.1 Experimental animals

2.2 Cells lines and viruses

2.3 Construction and characterization of recombinant adenoviruses expressing BPIV3 F and HN proteins

2.4 Chromatographic purification of recombinant adenoviruses

2.5 Immunization and challenge

2.6 Indirect enzyme-linked immunosorbent assay (ELISA)

2.7 BPIV3 hemagglutination inhibition (HI) assay

2.8 BPIV3 neutralization assay

2.9 Flow cytometry assays

2.10 Quantitative reverse-transcription polymerase chain reaction (RT-PCR)

2.11 Histopathological examination

2.12 Statistical analysis

3. Results

3.1. Construction and identification of rHAd5-HN and rHAd5-F + HN

3.2 Recombinant adenoviruses purified by chromatography

3.3 Recombinant adenoviruses induced humoral immune responses in mice

3.4 Recombinant adenovirus induced cellular immune responses in mice

3.5 Recombinant adenoviruses protected mice against BPIV3 infection

3.6 Recombinant adenovirus induced humoral immune responses in calves

4. Discussion

Declarations

References

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