Introduction

Numerous viruses have the potential for transmission from animals to humans, including but not limited to the influenza virus (IV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Japanese encephalitis virus (JEV), and dengue virus (DENV). Within the spectrum of zoonotic pathogens, avian species often serve as either intermediate or natural hosts. Among these, several flaviviruses, such as Usutu virus (USUV), West Nile virus (WNV), tick-borne encephalitis virus (TBEV), and JEV, have established birds as their reservoir hosts1,2,3,4. Bird trade and human movements may be a possible mode for flaviviruses spread5. Vaccination is a pivotal strategy for prevention and containment of virus transmission. DNA vaccines, characterized by their safety profile, ease of production, and transportability, have emerged as promising candidates for the prevention of emerging infectious diseases in birds.

Tembusu virus (TMUV), containing three structural proteins (E, prM/M, C) and seven nonstructural proteins (NS1-NS5), is classified as an avian pathogenic flavivirus. It becomes one major pathogen in ducks. The virus demonstrates rapid transmission dynamics, disseminating not only through mosquitoes but also via fecal contamination and aerosolized routes6. Until now, the TMUV exhibits the capacity to infect a range of bird species, including chickens, ducks, geese, and sparrows. It has caused huge economic losses in the poultry industry in China. Furthermore, it has been observed that TMUV antibodies are detectable in duck farm workers7. The virus has demonstrated the ability to replicate within human cell types, including nerve cells, liver cells, and kidney cells8. Further tests showed that TMUV was sensitive to the mammal type I interferon system9. Collectively, TMUV may pose a pathogenic risk to immunocompromised individuals. Consequently, it is imperative to implement robust and efficacious strategies to prevent TMUV infection and transmission. Despite the availability of commercial attenuated and inactivated vaccines, they fail to provide comprehensive protection against TMUV infection. Attenuated vaccines pose the risk of virulence regression and may create new strains. The immunogenicity of inactivated vaccines can be affected by the transport device and the duration of transport, resulting in its unstable immunological efficacy. In the end, the inactivated vaccines cannot completely protect the host against TMUV. Therefore, new TMUV vaccines with efficiency and easy transportation are needed to be further developed. Given that the prM-E proteins constitutes the envelope of TMUV, a DNA vaccine expressing the envelope proteins of TMUV has been developed in the current study.

CD40L, classified as a type II transmembrane glycoprotein, belongs to the ligand member of the tumor necrosis factor (TNF) superfamily and plays a pivotal role in the development and regulation of adaptive immunity. CD40L is prominently expressed on activated antigen presentation cells (APCs), CD4+ T cells, and CD8+ T cells, emphasizing the inseparable connection between cellular immune responses and CD40L signaling10,11. It exhibits the capacity to activate monocytes/macrophages, leading to the production of cytokines such as TNF-α, IL-1, IL-6, and IL-812. These cytokines play a pivotal role in activation between T cells and APCs, profoundly influencing the subsequent quality of cellular immune responses. Mouse CD40L (mCD40L) serves to enhance the activation and proliferation of tumor-specific cytotoxic CD8+ T cells, resulting in the inhibition of tumor growth13. Additionally, as its receptor CD40 is expressed on B cells, CD40L plays a pivotal role in regulating humoral immune responses. Through the CD40L-CD40 signaling axis, it is possible to enhance the antigen-presenting capacity of B cells, promote B cell proliferation and plasma cell generation14. The addition of equine CD40L (eCD40L) to the WNV E domain III subunit (WNV EDIII) vaccine results in a notable enhancement of neutralizing antibody titers produced by horses against WNV15. Furthermore, Daniel Graf and colleagues identified a natural 18 kDa fragment in human CD40L (hCD40L) that constitutes the extracellular domain of hCD40L, termed soluble CD40L (sCD40L)16. Despite the absence of certain extracellular and transmembrane regions, this domain retains similar biological functionality to the full-length CD40L. Gupta et al. harnessed mouse sCD40L (msCD40L) in conjunction with a gp100 DNA vaccine, resulting in the promotion of dendritic cell activation and the inhibition of B16-F10 tumor growth17.

However, the role of avian CD40L in the context of vaccination has not been fully elucidated. As duck-derived sCD40L (dusCD40L) is conserved in birds, it was aimed to further assess the adjuvant activity of duCD40L. It was found that dusCD40L possesses the capacity to activate immune cells from both duck and chicken. When dusCD40L was employed in conjunction with the TMUV prM-E DNA vaccine, it significantly enhanced both the humoral and cellular immune responses elicited by the vaccine. Furthermore, dusCD40L could strengthen virus clearance of the prM-E DNA vaccine in ducks post-TMUV challenge. This study provides a new efficient adjuvant for the development of TMUV DNA vaccine in ducks. Moreover, it underlines the importance of duCD40L as an adjuvant for vaccines against zoonotic infection diseases in birds.

Results

Molecular characterization of duCD40L

To elucidate the evolutionary relationship of duCD40L across various species, an evolutionary tree was constructed employing the amino acid (AA) sequence of CD40L (Supplementary Fig. 1a). The phylogenetic analysis underscored a notably robust evolutionary affinity among bird-derived CD40L, duCD40L, black swan CD40L (bsCD40L), and chicken CD40L (chCD40L) (Supplementary Fig. 1a). The duCD40L protein possesses a length of 272 amino acids (AA), which mirrors the identical length observed in both bsCD40L and chCD40L (Supplementary Fig. 1b). This length exceeds that of hCD40L and mCD40L(Supplementary Fig. 1b). Furthermore, duCD40L encompasses a structurally homologous domain akin to TNF (Supplementary Fig. 1b). These observations signify the conservation of CD40L domains across diverse species. In-depth molecular characterization of duCD40L involved aligning the amino acid sequence of dusCD40L. The analysis revealed that dusCD40L shares a notably high sequence identity with black swan sCD40L (bssCD40L) and chicken sCD40L (chsCD40L), registering at 92.75% and 82.61%, respectively (Supplementary Fig. 1b). Following this, the sequence similarity decreases, with hsCD40L and msCD40L exhibiting identity levels of 46.48% and 40.85%, respectively (Supplementary Fig. 1b). Notably, rainbow trout sCD40L (rtsCD40L) displayed a relatively lower sequence identity of 36.00% when compared to dusCD40L (Supplementary Fig. 1b). Taken together, we hypothesized that the function of dusCD40L is conserved in birds.

DusCD40L promotes avian peripheral blood mononuclear cell (PBMC) proliferation

CD40L naturally exists in two distinct forms: the membrane-bound form and the soluble form. As the latter one contains the entire TNF homology domain and can bind to CD40, it has biological activity16. Therefore, dusCD40L (spanning from amino acids 110 to 272, Supplementary Fig. 1b) was selected for investigating the biological functions of duCD40L. The findings revealed the successful expression of dusCD40L in supernatants (Fig. 1a) and its capacity to upregulate duCD40 expression (Fig. 1b). As dusCD40L has a high sequence identity among birds (Supplementary Fig. 1), its ability to promote the proliferation of duck PBMCs (duPBMCs) and chicken PBMCs (chPBMCs) was detected in the present study. It was shown that dusCD40L could stimulate duPBMCs and chPBMCs proliferation (Fig. 1c, Supplementary Fig. 2). Moreover, incubation of duPBMCs with dusCD40L resulted in a dose-dependent stimulation of cell proliferation (Fig. 1c). To discern the specific cell types responsive to dusCD40L, we conducted a detailed analysis in duPBMCs. Due to the lack of antibodies to detect APCs and B cells, we employed a quantitative real-time PCR (qRT-PCR) assay to quantify the expression of marker genes associated with the various cell types. In this context, duCD80, duMHCI, and duMHCII were chosen as markers representative of APCs. The outcomes demonstrated a noteworthy enhancement of APC proliferation induced by dusCD40L (Fig. 1d). Avian genomes encompass only two-thirds of the immunity genes present in mammals. For instance, genes such as IRF3 and CD20 are notably absent in avian species. To evaluate B cell proliferation, the expression of the duCD21 gene, which serves as a marker for mature B cells, was quantified. The results revealed that dusCD40L led to an approximately 2.5-fold increase in duCD21 gene expression (Fig. 1e). Intriguingly, despite CD40L typically being expressed by T cells, this study also uncovered the proliferation of T cells by qRT-PCR, including both CD4+ and CD8+ T cells (Fig. 1f). Additionally, the proliferation of T cells was confirmed by flow cytometry. The gating strategy was shown in Supplementary Fig. 3. As shown in Fig. 1g, the dusCD40L induced an increase in CD4+ T cell counts and CD8+ T cell counts. This observation suggests a potential autocrine role for duCD40L in T cell proliferation. Overall, these results proved that dusCD40L could stimulate the proliferation of avian PBMC, including APC, B cell, and T cell.

Fig. 1: dusCD40L could induce duPBMC proliferation.
figure 1

a dusCD40L expression was detected by WB in DEFs at 48 h post pVAX-TPA (control) or pVAX-TPA-dusCD40L (dusCD40L) transfection. b duCD40 gene expression levels in duPBMCs were measured by qRT-PCR. DuPBMCs were inoculated in a 12-well plate, 5×106 cells/well. Supernatants (1:8 dilution) of DEFs at 48 h post pVAX-TPA (control) or pVAX-TPA-dusCD40L (dusCD40L) transfection were added into the duPBMCs for 48 h. c The proliferation of duPBMCs was performed by CCK-8 assay. DuPBMCs were inoculated in a 96-well plate, 5×105 cells/well. They were stimulated with dusCD40L, DMEM (mock), control, or LPS (5 μg/mL) with 1:8 dilution or 1:16 dilution for 48 h. d–f duPBMCs were inoculated in a 12-well plate, 5×106 cells/well. They were stimulated with dusCD40L or control at 1:8 dilution for 48 h. duCD80 (d), duMHCI (d), duMHCII (d), duCD21 (e), duCD3 (f), duCD4 (f), duCD8 (f) were measured by qRT-PCR. g The percentages of CD4+ and CD8+ T cells in suspended duPBMCs at 48 h after ConA (5 μg/mL), mock, control, or dusCD40L at 1:8 dilution stimulation were measured by flow cytometry. Data were presented as means ± s.d. (n = 3). Statistical significance was assessed using an unpaired Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001.

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dusCD40L enhances T/B cell responses

The interaction between CD40 and CD40L serves as the secondary signal crucial for T cell activation. The specific cytokine milieu in which cells are situated plays a pivotal role in determining immune cell differentiation18. Notably, Th1 cytokines such as IFNγ and IL-12 enhance cytotoxic T cell (CTL) responses, contributing to the suppression of intracellular pathogen infections. Conversely, prominent Th2 cytokines, IL-6 and IL-10, play a pivotal role in supporting B cell responses. It was observed that dusCD40L significantly elevated the expression of duIl-10 (Fig. 2c). Remarkably, dusCD40L induced a dramatic increase, by thousands of-fold, in the expression of duIfnγ, duIl-12, and duIl-6 (Fig. 2a–c). Moreover, the secretion levels of duIFNγ also could be enhanced post dusCD40L stimulation (Fig. 2a). The results demonstrated that dusCD40L can activate duck T cells.

Fig. 2: dusCD40L promotes T/B cell activation.
figure 2

The plasmids pVAX-TPA (control) and pVAX-TPA-dusCD40L (dusCD40L) were transfected into DEFs, respectively. The supernatants were obtained at 48 h post-transfection. DuPBMCs were seeded into a 12-well plate. And then the DEF transfected supernatants were added to the duPBMCs at a 1:8 dilution, respectively. The cells (for qRT-PCR) and supernatant (for ELISA) were collected at 48 h post-stimulation. a The titers of duIfnγ and duIFNγ were detected by qRT-PCR and ELISA, respectively. b–d The mRNA expression levels of duIl-21 (b), duIl-6 (c), duIl-10 (c), and duBAFF (d) were measured by qRT-PCR. The expression levels of mRNAs were calculated by 2-ΔΔCt. Data were presented as means ± s.d. (n = 3). Statistical significance was assessed using an unpaired Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001.

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B cell activating factor (BAFF) holds a vital role in immunomodulation, influencing B cell survival, antibody isotype switching, and the maintenance of germinal centers19,20. To assess the impact of dusCD40L on B cell activation, BAFF expression levels were measured both before and after dusCD40L treatment. The findings revealed that dusCD40L substantially upregulated duBAFF expression by four-fold (Fig. 2d).

Collectively, these results suggest that dusCD40L possesses the capability to bolster the activation of T cells, encompassing Th1 and Th2 subsets, as well as B cells.

Construction of a novel TMUV DNA vaccine containing a dusCD40L gene fragment

The verification of dusCD40L’s multifaceted role in modulating immune cell responses in vitro paved the way for an investigation into its influence on humoral and cellular immune responses in ducks. The genome of TMUV encodes a total of three structural proteins, namely, C, prM/M, and E, along with seven non-structural proteins designated as NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5. The envelope of TMUV consists of prM/M and E proteins. Notably, the prM protein undergoes cleavage by furin within the trans-Golgi network, yielding pr and M proteins21. Consequently, prM and E proteins serve as the primary targets in the development of TMUV vaccines. In this study, we have engineered the TMUV DNA vaccine to co-express prM-E along with dusCD40L (Fig. 3a). To enhance the expression of prM-E, we employed the signal peptide of prM originating from the TMUV C protein, specifically the C-terminal 22 amino acid residues of TMUV CQW1 C (Fig. 3a). Additionally, to ensure the preservation of the spatial arrangement between prM-E and dusCD40L, we introduced a porcine tescherovirus 2A (P2A) proteolytic cleavage site22 (Fig. 3a). The research findings by Arvia E and colleagues have indicated that the inclusion of an isoleucine zipper (ILZ) segment can induce the formation of CD40L trimers, thereby augmenting its biological activity22,23. Accordingly, we introduced an ILZ fragment at the N-terminus of dusCD40L, linked with Leu-Leu, to enhance the functionality of dusCD40L. Subsequently, the E with or without dusCD40L proteins were successfully expressed in duck embryo fibroblasts (DEFs) following transfection with the DNA vaccine plasmids pVAX-prM-E-dusCD40L and pVAX-prM-E, respectively (Fig. 3b, c). One week after intramuscular injection in ducks, the two plasmids were found to enable the expression of the E protein in the spleen (Fig. 3d). These results collectively demonstrate the successful construction of TMUV prM-E-based DNA vaccines, incorporating or not with the biological adjuvant dusCD40L.

Fig. 3: The construction of a novel TMUV DNA vaccine expressing prM-E together with dusCD40L.
figure 3

a Schematic diagram of the TMUV prM-E DNA vaccine containing dusCD40L. The DEFs were transfected with the prM-E-based three plasmids, respectively. The expression of E and dusCD40L were measured by IFA (b) and WB (c) assays at 48 h post the plasmids transfection. Scale bars, 40 μm. d The expression of E protein was measured in vivo. Ducks were immunized with 200 μg of pVAX1, pVAX-prM-E, or pVAX-prM-E-dusCD40L by intramuscular injection. They were sacrificed at 7 days post-immunization. The spleens were collected and the E protein was detected by immunohistochemistry assay. The black arrows indicate the E proteins. Scale bars, 20 μm.

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Enhancement of TMUV-specific humoral immune responses by dusCD40L

To evaluate the adjuvant effects of dusCD40L in vivo, Pekin ducks were intramuscularly (i.m) immunized with the TMUV DNA vaccines. The immunization and sampling procedures are illustrated in Fig. 4a. The anti-TMUV IgG titers in the serum of ducks vaccinated with pVAX-prM-E-dusCD40L were significantly higher than those in ducks vaccinated with pVAX-prM-E from 7 w to 10 w (Fig. 4b). Furthermore, the TMUV prM-E DNA vaccine incorporating dusCD40L exhibited the capacity to elevate neutralizing antibodies titers in comparison to the TMUV prM-E DNA vaccine (Fig. 4c). While there were no discernible differences in antigen-specific IgG titers between ducks immunized with DNA vaccine containing or lacking dusCD40L, the presence of dusCD40L significantly boosted neutralizing antibodies levels in comparison to the DNA vaccine lacking dusCD40L at the 5 w (Fig. 4b, c). These findings underscore dusCD40L’s ability to reinforce the humoral immune responses triggered by the TMUV prM-E DNA vaccine.

Fig. 4: TMUV-specific B cell responses post-vaccination.
figure 4

a Schematic diagram of the vaccination and sampling procedure, as indicated by arrows. b The anti-TMUV IgG in different groups was measured by ELISA at 5 w to 10 w post first vaccination. c Neutralizing antibodies against TMUV in sera were detected at 5 w and 8 w after prime immunization. Data were presented as means ± s.d. (n = 3). Statistical significance was assessed using an unpaired Student’s t-test. ns > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001.

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Augmentation of cellular immune responses with dusCD40L incorporated into the TMUV prM-E DNA vaccine

To evaluate the cellular immunogenicity of the DNA vaccine, ducks were immunized and sampled at various time points, as depicted in Fig. 5a. To gauge the adjuvant effect of dusCD40L on elicited cellular immunity, a TMUV-specific duPBMC proliferation assay was conducted at 5 w following vaccination. As shown in Fig. 5b, the proliferation rates of the TMUV prM-E DNA vaccine and the TMUV prM-E-dusCD40L DNA vaccine were around 20% and 30%, respectively; the proliferation rate of the TMUV inactivated vaccine was around 8%. Therefore, the TMUV DNA vaccines notably stimulated duPBMC proliferation more efficiently than the inactivated vaccine did (Fig. 5b). Furthermore, dusCD40L as an adjuvant exhibited a significant enhancement in TMUV-specific duPBMC proliferation induced by the TMUV prM-E DNA vaccine (Fig. 5b). The levels of IFNγ in sera were quantified using ELISA, revealing that the DNA vaccines were capable of eliciting stronger IFNγ responses compared to the TMUV inactivated vaccine (Fig. 5c). Moreover, dusCD40L significantly augmented IFNγ secretion when incorporated into the prM-E DNA vaccine (Fig. 5c). These findings collectively indicate that dusCD40L, as an adjuvant, holds the potential to bolster cellular immunity in the context of the TMUV prM-E DNA vaccine.

Fig. 5: TMUV-specific T cell responses post-vaccination.
figure 5

a Schematic diagram of the vaccination and sampling procedure. b The proliferation of duPBMCs was detected by CCK-8 assay. DuPBMCs were seeded into a 96-well plate, 5×105 cells/well, and stimulated with 100 μg/mL inactivated TMUV. The relative proliferation rate = vaccine (OD450 TMUV stimulation - OD450 mock) / OD450 mock × 100%. c The IFNγ titers were detected by ELISA. Data were presented as means ± s.d. (n = 3). Statistical significance was assessed using an unpaired Student’s t-test. ns > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001.

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DusCD40L mitigated viral load and enhanced post-infection viral control

To assess the immunoprotective efficacy of dusCD40L in the DNA vaccine, ducks were subjected to a challenge with 108 median tissue culture infectious dose (TCID50) TMUV through intramuscular injection at 11 w post the initial vaccination (Fig. 6a). There were no fatalities throughout the experiment. In the PBS group, some ducks showed depression and reduced feed intake. In the immunized groups (prM-E DNA vaccine group, prM-E-dusCD40L DNA vaccine group, and inactivated TMUV vaccine group), the ducks did not show significant clinical signs. Next, the virus titers in the serum, spleen, and brain from the four groups were quantified using TCID50 at 7 days post-TMUV infection (dpi). As depicted in Fig. 6b, the TMUV titers in all three vaccinated groups were notably lower than those in the PBS group. Additionally, the viral titers in ducks from the prM-E-dusCD40L DNA vaccine group were significantly lower in comparison to ducks from the prM-E DNA vaccine group (Fig. 6b). These results collectively demonstrate that the dusCD40L adjuvant can effectively mitigate viral load and enhance post-infection viral control.

Fig. 6: Effect of dusCD40L adjuvant on efficacy for virus control by TMUV DNA vaccine.
figure 6

a Ducks were immunized with the vaccines and were challenged with 108 TCID50 TMUV by i.m. The ducks were sacrificed at 7 dpi. b The viral load in serum, spleen, and brain were detected. Data were presented as means ± s.d. (n = 3). Statistical significance was assessed using an unpaired Student’s t-test. ns > 0.05, *P < 0.05, **P < 0.01.

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Discussion

CD40L, by binding to the receptor CD40 on APCs, induces the expression of MHC, co-stimulatory molecules (e.g., CD80), and cytokines (e.g., IL-12). This interaction provides a crucial second signal for the activation of both T lymphocytes and B lymphocytes, thereby facilitating both cellular and humoral immune responses. Numerous investigations have focused on harnessing sCD40L and incorporating it into vaccines to enhance their immune efficacy24,25. However, the function and adjuvant activity of dusCD40L is unknown. To address this, our study delved into the homogeneity of dusCD40L across different species and explored the in vitro biological activity of dusCD40L. We found that dusCD40L exhibits a high degree of homology in birds, and, importantly, this protein possesses the potential to stimulate both duPBMC and chPBMC responses. By constructing a TMUV prM-E DNA vaccine incorporating dusCD40L, we have substantiated that dusCD40L can serve as a biological adjuvant to enhance the immune response of the DNA vaccine and elevate the vaccine’s immunoprotection.

The immunostimulatory properties of CD40L have been harnessed to bolster B- and T-cell responses across various vaccination strategies in mammalian species. The interaction between CD40L on activated CD4+ T cells and CD40 plays a pivotal role in providing a vital co-stimulatory signal. This signal is instrumental in immunoglobulin class switching, enhancing antibody affinity maturation, and priming CD8+ T cells26,27,28. Upon employing dusCD40L to stimulate avian PBMCs, we observed its capability to activate the expression of CD40, and promote duPBMC and chPBMC proliferation. These findings align with the functional characteristics of duCD40L as reported by Gares et al.29. Our results suggest that the region spanning amino acids 110 to 272 of duCD40L (dusCD40L) constitutes its functional structural domain. Furthermore, this study conducted further investigations into which cell types’ proliferation dusCD40L promoted. The findings revealed that dusCD40L significantly enhances the proliferation of APCs, B cells, and T cells. Therefore, we speculated that dusCD40L, much like its mammalian counterpart CD40L, possesses the capability to enhance adaptive immune responses.

Since the antigens encoded by DNA vaccines are synthesized de novo in the transfected cells, these antigens are processed primarily by the proteasome and are presented to CD8+ T cells primarily through MHC I30. However, it is essential to note that somatic cells, which are the primary transfectants in DNA vaccination, do not express MHCII. Furthermore, DNA-encoded antigens exhibit limited release from transfected somatic cells, making them less accessible for uptake by APCs31. Consequently, for effective vaccine outcomes, it is imperative to prime and expand MHC II-restricted CD4+ T cells. CD4+ T cells play a pivotal role in several crucial aspects of immune responses, including the generation of high-affinity neutralizing antibodies, immunoglobulin class switching, and the optimal expansion of CD8+ cytotoxic T lymphocytes32,33,34. Therefore, the identification of modifications for DNA vaccines that enhance MHC II presentation and stimulate CD4+ T cell responses is of fundamental importance in optimizing vaccine-induced immunity against a diverse spectrum of microbial pathogens. To delve deeper into the function of dusCD40L, this study conducted comprehensive investigations, confirming that this protein was capable of promoting the activation of both CD4+ and CD8+ T cells. Additionally, our findings unveiled that dusCD40L could simultaneously enhance the expression of pro-inflammatory cytokine IL-6 and anti-inflammatory cytokine IL-10. This dual regulatory effect may be attributed to the role played by the CD40-CD40L signaling pathway in B cell differentiation and functional maturation35. It is also plausible that dusCD40L possesses the capability to suppress autoimmune and inflammatory responses36. In conclusion, dusCD40L demonstrates the potential to provide an appropriate cytokine milieu for both humoral and cellular immune responses, thereby serving as a promising candidate for utilization as a DNA vaccine adjuvant.

As it is well-known, mammalian sCD40L possesses adjuvant activity. The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein fused with msCD40L could enhance the humoral immunogenicity of RBD37. Cows were co-immunized with bovine sCD40L (bosCD40L) and heat-killed Staphylococcus aureus (HKSA), significantly increasing the number of HKSA-specific CD4+ T and CD8+ T cells in draining lymph nodes38. BosCD40L as a molecular adjuvant was incorporated into bovine herpesvirus 1 (BoHV-1) glycoprotein D DNA vaccine, improving the efficacy of the DNA vaccine against BoHV-139. Gares SL et al. found that dusCD40L significantly improved the core-specific antibody response of the duck hepatitis B virus (DHBV) DNA vaccine either via plus or fusion with the viral antigen40. It was shown that the DHBV DNA vaccine expressing dusCD40L fusion with core could induce stronger antigen-specific antibody response than dusCD40L DNA plus with the DHBV core DNA vaccine40. However, the ability of dusCD40L to assist DNA vaccine to promote neutralizing antibodies was unknown. Neutralizing antibodies are necessary to prevent antigens from invading the host. The production of neutralizing antibodies is associated with viral protein structure41. The prM-E constitutes the envelope of TMUV and can form virus-like particles. According to those, dusCD40L was incorporated into the TMUV prM-E DNA vaccine with P2A22. The results demonstrated that dusCD40L increased the levels of TMUV-specific IgG and neutralizing antibodies induced by the prM-E DNA vaccine. This enhancement could be attributed to the role of the CD40-CD40L signaling pathway in promoting B cell proliferation, differentiation, activation, and antibody production19,35,42. Consistent with the results of Gares SL et al.40, dusCD40L also improved the ability of the TMUV prM-E DNA vaccine to induce antigen-specific duPBMC proliferation. It is generally recognized that IFNγ dependent Th1, besides neutralizing antibodies, responses are necessary to prevent Flavivirus infection43. Gao et al. employed duck IL-2 (duIL-2) as an adjuvant, which could increase the antibody level of inactivated TMUV vaccine, but could not promote the IFNγ yield of TMUV inactivated vaccine44. Therefore, there is a need to further explore adjuvants that stimulate better IFNγ-dependent cellular immunity. Previous research has shown that using anti-CD40 agonistic antibodies in mice to interact with CD40 expressed on APCs can stimulate the production of IL-12 via TRAF6-dependent signaling pathways12. Consequently, the increased IL-12 production leads to upregulation of IFNγ and promotes Th1 polarization, which, in turn, mediates the cytotoxic activity of CTLs12. The dusCD40L could bind and promote duCD40 expression. However, the ability of dusCD40L to promote IFNγ generation is unknown in the DNA vaccine study of Gares et al.40. In this study, the analysis of IFNγ levels among different immunization groups indicated that dusCD40L can enhance IFNγ expression induced by the TMUV prM-E DNA vaccine. Our work further demonstrates that, similar to other DNA vaccines, duCD40L can augment the adaptive immune responses in avian species.

Interestingly, the inactivated TMUV vaccine induced a weak cellular immune response, characterized by limited duPBMC proliferation and low levels of IFNγ expression. The DNA vaccine’s capacity to induce a cellular immune response is significantly superior to that of the inactivated vaccine. This discrepancy may be attributed to the fact that the inactivated vaccine primarily processes its antigens through the MHC II pathway for presentation to CD4+ T cells, thereby assisting B lymphocytes in generating a humoral immune response45. Only a small fraction of the inactivated vaccine’s antigens are presented via the MHC I pathway to activate CD8+ T cells, leading to a cellular immune response45. However, due to the limited cross-presentation of antigens and the absence of an immune microenvironment that promotes cellular immune responses, such as Th1 cell assistance and the expression of related cytokines like IFNγ, the cellular immune responses induced by the inactivated vaccine are not prominent. In contrast, the prM-E DNA vaccine incorporated with dusCD40L could present and stimulate both MHC I and MHC II molecules. This capability can induce antibody production, T lymphocyte proliferation, and CTL cytotoxic activity46,47.

While dusCD40L could enhance the humoral immune responses of the prM-E DNA vaccine, the levels of TMUV-specific IgG and neutralizing antibodies in this DNA vaccine immunization group were lower than those in the inactivated vaccine group. This outcome might be caused by two potential factors. First, it could be due to the low expression levels, resulting in insufficient immune response intensity. The strength of immune responses induced by vaccines is closely associated with the antigens available to APCs. To acquire a larger and more persistent amount of antigen, strategies like constructing a dual-promoter (e.g., CMV-nirB) system to increase antigen production and subsequently augment the immune responses were developed48. The second reason could be the direct intramuscular injection of naked plasmids using a syringe. The biggest obstacle to DNA vaccination is low immunogenicity due to the difficulty of delivering DNA plasmids into the host cell via needle injection. Which deposits the DNA in the intracellular space rather than inside the cell. To overcome the major problem of low immunogenicity of DNA vaccines, effective delivery of plasmids to preferred tissues or cells is a key area of research, including codelivery with nanoparticles and bacterial vectors49,50. Compared to parenteral routes, oral administration in birds offers advantages such as ease of mass application and reduced need for highly trained personnel. Therefore, in future research, it is possible to increase antigen expression levels by modifying the promoter of the expression vector. Additionally, using a suitable oral vaccine delivery vector can facilitate the entry of the target plasmid into host cells, thereby enhancing the expression and presentation efficiency of the target antigen.

The current investigation centered on assessing the biological efficacy of dusCD40L and its impact on enhancing the immunogenicity of the TMUV prM-E DNA vaccine. The dusCD40L was capable of augmenting both humoral and cellular immune responses, thereby bolstering the immune protection of the vaccine. Consequently, it represents a promising adjuvant for the development of TMUV DNA vaccines with enhanced efficacy. Additionally, the findings in the present study revealed that dusCD40L may be a universal adjuvant for birds.

Methods

Ethics statement

All experimental procedures involving animals received approval from the Institutional Animal Care and Use Committee of Sichuan Agricultural University, China, under Permit Number SYXK(川)2019-187.

Cells, animals, and virus

BHK-21 cells were maintained in Dulbecco’s modified Eagle medium (DMEM, Gibco, USA) supplemented with 10% new bovine serum (NBS, Gibco, USA). Nine-day-old duck embryos were sourced from the duck industry in Ya’an, China. Duck embryo fibroblasts (DEFs) were cultured in a minimum essential medium (MEM, Gibco, USA) supplemented with 10% NBS (Gibco, USA). One-day-old Peking ducks were obtained from the duck industry in Chengdu, China. The 20-day-old chickens were kindly provided by Dr. Xia, Sichuan Agricultural University. The TMUV CQW1 strain (GenBank: KM233707.1) was propagated in DEFs as previously described51.

Bio-information analysis of duCD40L

Nine ORF sequences of CD40L retrieved from GenBank were compiled. Phylogenetic relationships among these sequences were assessed utilizing the neighbor-joining method within MEGA software version 7.0. Protein domains were identified using the Simple Modular Architecture Research Tool (SMART, http://smart.embl-heidelberg.de/). Alignment of the amino acid sequences of the dusCD40L from various species was performed either through DNAMAN or an online BLAST tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi).

Plasmids construction

DusCD40L with codon optimization for duck protein expression was synthesized by Wuhan Jinkairui Bioengineering Co., China. Then, the fragment was integrated into the pVAX1 vector from Invitrogen, which includes the signal sequence of tissue plasminogen activator (TPA) (MDAMKRGLCCVLLLCGAVFVS), resulting in the construction of the dusCD40L eukaryotic expression plasmid designated as pVAX-TPA-dusCD40L. The fragments dusCD40L, P2A, ILZ, and Leu-Leu were introduced into the pVAX-prM-E plasmid as described by Huang et al.51 using specific primers (refer to Supplementary Table 1). A schematic representation of pVAX-prM-E-dusCD40L can be observed in Fig. 4a.

Western blot (WB) assay

DEFs were seeded at a density of 2 × 106 cells per well in a six-well plate and maintained at 37 °C in an atmosphere containing 5% CO2. The following plasmids, pVAX1, pVAX-TPA, pVAX-TPA-dusCD40L, pVAX-prM-E, or pVAX-prM-E-dusCD40L, were individually transfected into DEFs when the cells reached a confluence of 90%. Transfection was carried out using TransIntroTM EL Transfection Reagent (TransGen Biotech, China) following the manufacturer’s instructions. After 48 h post-transfection, both cell supernatants and cell pellets were harvested. The pellets were lysed using 100 μL of radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, China) containing 1% phenylmethanesulfonyl fluoride (PMSF, Beyotime, China). Protein separation was accomplished by performing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a 15% acrylamide gel for resolving cell supernatants and lysates. The samples were separately loaded into the gels, 20 μL/lane. Subsequently, proteins were transferred onto a PVDF membrane (Bio-Rad, USA) at a current of 220 mA for 80 minutes. The membrane was blocked with 5% nonfat milk for 2 h at room temperature. Primary antibodies used included rabbit anti-E polyclonal antibody (prepared in this study, 1:1000) and rabbit anti-duCD40L polyclonal antibody (prepared in this study, 1:500), both diluted accordingly. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (H + L) (Cat# RS0002, Immunoway, China) at a dilution of 1:5000 served as the secondary antibody for the detection of E protein and dusCD40L expression. Membrane imaging was achieved using Clarity™ Western ECL Substrate (Bio-Rad, USA). Uncropped and unprocessed scans of the blots are in the Supplementary Figs. 4 and 5. All blots derive from the same experiment and that they were processed in parallel.

Indirect immunofluorescence assay (IFA)

When DEFs reached approximately 90% confluence, they were individually transfected with the following plasmids, pVAX1, pVAX-prM-E, and pVAX-prM-E-dusCD40L. Subsequently, the cells were fixed by incubating them with 0.5% paraformaldehyde for an overnight period at 4 °C at 48 h post-transfection. To block nonspecific binding sites, a 5% nonfat milk solution was applied to the cells for 30 min at room temperature. Following this, the cells were subjected to incubation with rabbit anti-duCD40L polyclonal antibody (prepared in this study, 1:250) or rabbit anti-E polyclonal antibody (prepared in this study, 1:500) at 4 °C. After overnight incubation, a secondary antibody, PE-conjugated goat anti-rabbit IgG (Cat# HS121-01, TransGen, China) at a dilution of 1:1000, was utilized. The visualization of DEFs was accomplished using a fluorescence microscope (Nikon, Japan).

qRT-PCR assay

DEFs were subjected to transfection with either pVAX-TPA (control) or pVAX-TPA-dusCD40L (dusCD40L) using TransIntroTM EL Transfection Reagent (TransGen Biotech, China) as per the manufacturer’s instructions. At 48 h post-transfection, cell culture supernatants were collected for the following assays.

Ducks around 30-day-old were used for blood collection. DuPBMCs were isolated using a Peripheral Blood Mononuclear Cell Separation Kit (Tianjin Hao Yang Biological Products Technology Co., Ltd., Tianjin, China) following the manufacturer’s protocols. DuPBMCs were then treated with the collected supernatants at dilutions of 1:8 or 1:16 for a 48-hour incubation period. Total RNA was extracted from the treated duPBMCs using RNAiso (Lablead, China), and this RNA was reverse-transcribed into cDNA utilizing a PrimeScript RT Reagent kit with gDNA Eraser (TaKaRa, Japan).

QRT-PCR was employed to analyze the relative mRNA expression levels of various genes, including MHCI, MHCII, CD3, CD4, CD8, CD21, CD40, CD80, Il-6, Il-10, Il-12, Ifnγ, BAFF, and GAPDH. The primers for qRT-PCR are listed in Supplementary Table 1. The qRT-PCR was conducted following the manufacturer’s instructions for TB Green® Premix Ex TaqTM (Tli RNaseH Plus) (TaKaRa, Japan), comprising an initial denaturation step at 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 5 s and annealing at 60 °C for 30 s. The relative gene expression levels were calculated using the 2-ΔΔCt method.

Flow cytometry

DuPBMCs were isolated as mentioned above. CD4+ and CD8+ T cells were measured by the flow cytometry assay. It was carried out as described previously52. Briefly, duPBMCs were adjusted to 1 × 107 cells per mL and seeded into 96 well plates, 100 μL/well. They were stimulated with dusCD40L (1:8), control (1:8), ConA (5 μg/mL), or DMEM (mock) for 48 h. Then, the suspended cells were collected. The anti-duck CD4 monoclonal antibody (Cat# MCA2478, Bio-rad, USA) at 1:200 dilution and the anti-duck CD8 monoclonal antibody (Cat# MCA2479, Bio-rad, USA) at 1:200 dilution were added into the cells, respectively. The cells were separately incubated with the antibodies at 4 °C, 30 min. After washing with PBS, Alexa Flour 488-labeled goat anti-mouse IgG (Cat# A32723, ThermoFisher, USA) as the secondary antibody at 1:500 dilution was added and the cells were incubated in the dark. Flow cytometry was performed on an CytoFLEX™ (Beckman counlter, USA). Data was analyzed using CytExpert software.

Immunization and challenge

A total of forty 1-day-old ducklings were reared for 1 w and subsequently randomly allocated into four groups, each consisting of ten ducks. Vaccination was administered to the ducks when they were 7 days old. One group of ducks received an intramuscular injection of pVAX1 at a dose of 200 μg in 0.5 mL of PBS. Two other groups of ducks were intramuscularly injected with either the vaccine pVAX-prM-E or pVAX-prM-E-dusCD40L at a dose of 200 μg in 0.5 mL of PBS. The final group of ducks received an intramuscular injection of 0.5 mL of inactivated TMUV vaccine (Yangzhou Ubang Biopharmaceutical Co., China) in the lateral thigh. Ducks in the prM-E-based groups were subjected to intramuscular injections in the lateral thigh three times at 3-week intervals, while ducks in the inactivated vaccine group received two intramuscular injections in the lateral thigh at 3-week intervals. At 7 days after the first immunization, three ducks from DNA-vaccinated groups were randomly selected and euthanized for spleen collection. At 5 w, 6 w, 7 w, 8 w, and 10 w post the first immunization, three ducks from each group were randomly selected for blood collection. Subsequently, ducks from each group were challenged with 108 TCID50 TMUV CQW1 strain per duck via intramuscular injection at 5 w after the third immunization. The clinical symptoms were recorded for continuous 7 days. At 7 days post-challenge, three ducks from each group were randomly selected and euthanized for serum, spleen, and brain sampling.

Immunohistochemical analysis

In each DNA-vaccinated group, ducks were euthanized at 7 days after the first immunization (n = 3). They were anesthetized by sodium pentobarbital (100 mg/kg) and were cut open for bloodletting bled. Then the spleens were collected. The presence of the E protein was confirmed through immunohistochemistry, following established protocols51. Briefly, the collected tissues were sectioned at a thickness of 4 μm using a Leica RM2128 microtome (Germany). These sections were initially fixed with 4% paraformaldehyde and then embedded in paraffin. Subsequently, the sections underwent dewaxing with xylene, rehydration with a gradient of ethanol, and were subsequently treated with 0.3% hydrogen peroxide (H2O2). Antigen retrieval was performed using a citrate buffer solution (CBS, 0.01 M, pH 6.0). The sections were incubated with a 10% normal goat serum to block nonspecific antigens for 1 h at 37 °C. Afterward, the sections were stained with a rabbit polyclonal antibody targeting TMUV E protein (prepared in this study) at a 1:200 dilution, followed by an incubation overnight at 4 °C. An HRP-conjugated goat anti-rabbit antibody (Cat# HS101-01, Transgen Biotech, China) at a 1:1000 dilution was used for a 2 h incubation at room temperature. The visualization of E protein expression was achieved using DAB (Solarbio, China), and the images were captured using microscopy (Olympus, Japan).

Cell counting kit-8 (CCK-8) assay

DuPBMCs were obtained from 30-day-old ducks or ducks that had been vaccinated at 5 w post the initial vaccination using the peripheral blood mononuclear cell separation kit (Tianjin Hao Yang Biological Products Technology Co., Ltd., Tianjin, China) following the manufacturer’s protocols. ChPBMCs were isolated from 20-day-old chickens by the same method as duPBMCs. The freshly isolated duPBMCs and chPBMCs were adjusted to a concentration of 5 × 106 cells/mL and were then seeded into 96-well plates at a volume of 100 μL per well, respectively. Subsequently, they were stimulated with dusCD40L at dilutions of 1:8 or 1:16 (prepared in this study), LPS (5 μg/mL, Solarbio, China), ConA (5 μg/mL, Solarbio, China), or 100 µg/mL of inactivated TMUV (prepared in this study) for a duration of 48 h at 37 °C in an atmosphere containing 5% CO2. Following stimulation, lymphocyte proliferation was assessed by measuring the optical density at 450 nm (OD450) using a CCK-8 kit (Solarbio, China), following the manufacturer’s instructions.

ELISA assay

For the detection of antibodies against inactivated TMUV, a 96-well ELISA plate was coated with inactivated TMUV (3.2 μg/well) in a coating buffer (pH 9.6) overnight at 4 °C. Following this, the plates were washed three times with PBS (pH 7.4) containing 0.1% Tween-20 (PBS-T). After blocking with PBS-T containing 5% nonfat milk, the plates were incubated with serum samples (diluted 1:400, 200 μL/well) collected earlier for 2 h at 37 °C. Subsequently, the plates were washed five times with PBS-T. HRP-conjugated goat anti-duck IgG (Cat# 5220-0296, KPL, USA), diluted at 1:2000, was added to the plates (100 μL/well) for 1 h at 37 °C. Following this incubation, the plates were washed, and 3, 3′, 5, 5′-tetramethylbenzidine (TMB) was added (100 μL/well) for 10 min at room temperature in the dark. The reaction was halted by adding 2 M H2SO4 (50 μL/well), and the OD450 was measured using a spectrophotometer (Bio-Rad, USA).

To determine IFNγ titers, a 96-well ELISA plate was coated with rabbit anti-duIFNγ polyclonal antibody (prepared in this study, 1:80 dilution) in coating buffer (pH 9.6) overnight at 4 °C. The plates were then blocked with PBS-T containing 5% nonfat milk at 37 °C for 1 h. DuPBMCs were seeded into 96-well plates with 1 × 107 cell/well in 100 μL. They were obtained and treated as mentioned above. The supernatants of the duPBMCs were separately collected at 48 h post dusCD40L stimulation. They and serum samples (1:30 dilution) from vaccinated ducks collected at 5 w and 8 w post-primary immunization were added to the plates and incubated at 37 °C for 1.5 h. Mouse anti-duIFN-γ polyclonal antibody (prepared in this study), at a 1:160 dilution, was subsequently added to the plates, and the plates were incubated at 37 °C for 2 h. HRP-conjugated goat anti-mouse IgG antibody (Cat# HS201-01, TransGen, China) at a 1:8000 dilution was added to the plates and incubated at 37 °C for 1 h. After the incubation, the plates were washed, and TMB (100 μL/well) was added for 10 min at room temperature in the dark. The reaction was stopped with 2 M H2SO4 (50 μL/well), and the OD450 values were measured using a spectrophotometer (Bio-Rad, USA).

Neutralizing antibodies measurement

Sera were collected at 5 w and 8 w post the initial immunization with TMUV vaccines, as previously described. The collected sera were subjected to inactivation at 56 °C for 30 min and then were serially diluted in twofold increments (2−1–2−8) using DMEM. Each dilution was mixed with an equal volume of TMUV CQW1 at a concentration of 200 TCID50. The resulting mixture was added to BHK-21 cells cultured in 96-well plates with approximately 90% confluence, at a volume of 100 μL per well. After 2 h, the supernatant was removed, and the plates were washed three times with PBS. Following the washes, the cells were cultured in DMEM supplemented with 2% NBS, at a volume of 100 μL per well, and maintained at 37 °C in an atmosphere containing 5% CO2 for 5 days. Each dilution was replicated in 10 wells. The cytopathic effect (CPE) was observed and recorded during this period. Neutralizing activity was subsequently analyzed using the Reed-Muench method.

TCID50 assay for viral titration

Ducks from each group were anesthetized and euthanized as mentioned above at 7 days post-challenge (n = 3). The sera, spleens and brains were collected at 7 dpi. A total of 100 mg/spleen or 100 mg/brain was added to 1 mL DMEM with 1% penicillin and 1 μg/mL streptomycin for homogenization. The homogenized samples were centrifuged at 12,000 r/min for 10 min at 4 °C. Then the supernatants were collected. They and the sera were diluted in serial 10-fold increments (10−1–10−9) in DMEM containing 2% FBS. When the BHK-21 cells grown in 96-well plates reached approximately 90% confluence, the samples were added to the plates individually at 100 µL/well. The plates were incubated at 37 °C with 5% CO2 for 5 days. Then, the CPE was recorded and TCID50 was calculated by the Reed-Muench method.

Statistical analysis

Data were analyzed by GraphPad Prism (version 8.0) and presented as the means ± standard deviation (s.d.). Statistical significance was assessed using an unpaired Student’s t-test. The significant differences are annotated as *P < 0.05, **P < 0.01, ***P < 0.001.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.