Porcine epidemic diarrhea virus (PEDV) causes severe diarrhea in piglets. The ideal route of protection against PEDV for piglets is through passive (lactogenic) immunity, which is not provided by current inactivated and subunit vaccines on the market. In this study, we investigated whether a DNA vaccine encoding the full PEDV spike protein adjuvanted with cyclo-peptide nanotubes (cPNTs) can provide protection against PEDV through active and passive immunity. For the active immunization experiment, piglets were vaccinated, and the immune response was analyzed, followed by a PEDV challenge test. In a separate experiment, to evaluate the passive (lactogenic) immunity elicited by the cPNTs-adjuvanted DNA vaccine, pregnant sows in a local farm were immunized, and the survival of farrowed piglets was examined. The results showed that, in the active immunization experiment, the DNA vaccine elicited IFN-γ and IL-12 production in piglets. IgA antibodies were detected in the serum, and the expansion of CD4 + and CD8 + T cells was observed. Upon virus challenge, vaccinated piglets remained healthy, gained weight, and showed only mild signs of diarrhea, with minimal virus shedding (Ct value of 33, compared with 16 for the saline-vaccinated control group). For the passive immunity experiment, results show that the DNA vaccine administered orally induced higher levels of IgA in the colostrum of vaccinated sows compared to mock vaccination. The survival rate of the farrowed piglets was higher at 84% for the DNA-oral group compared to that of the mock vaccination group (68%). In conclusion, the cPNTs-adjuvanted DNA vaccine can not only generate protective immunity through direct immunization of piglets but also induce lactogenic immunity in pregnant sows to protect farrowed piglets from PEDV infection.

Porcine Epidemic Diarrhea Virus (PEDV) and Porcine Deltacoronavirus (PDCoV) are considered the greatest threats to the world swine industry since they cause a very high morbidity rate in piglets. Although vaccines with various formulations have been tested and validated in different settings, they still cannot be considered to provide long-lasting, comprehensive protection against these pathogens under real-world conditions. This review provides a comprehensive and critical overview of parallel efforts to develop vaccines against PEDV and PDCoV, with particular emphasis on vaccine formulation strategies, virus strains used in challenge studies, and the geographical applicability of these vaccines. Recent advances in vaccine development are discussed, highlighting the use of newly developed adjuvants and advanced delivery systems to enhance vaccine efficacy, especially in terms of inducing mucosal immune responses. Rather than merely summarizing progress in vaccine development to date, this review presents challenges, including viral diversity, lack of cross-protection, and maternal antibody interference, which reduce vaccine efficacy. Additionally, we discuss novel directions for future research on broadly protective next-generation antigen-detection systems and integrated approaches targeting different porcine coronaviruses. Thus, this review offers an up-to-date, in-depth overview of the current state of PEDV and PDCoV vaccine research, while also outlining future perspectives to drive innovation toward practical and sustainable disease control.

Introduction Bovine respiratory disease (BRD) complex is a leading cause of economic losses in the beef and dairy cattle industries. Mannheimia haemolytica is recognised as the primary pathogen associated with this disease. While antibiotics and vaccines are widely used against it, antimicrobial resistance and limited vaccine efficacy remain obstacles. Mannheimia haemolytica leukotoxin A (LktA) has been identified as a promising candidate for subunit vaccine development against BRD. However, the low expression and biological instability of the full-length LktA complicate its production. This study evaluated the immunogenic potential of the truncated LktA protein for subunit vaccine development.Material and Methods Truncated proteins of LktA N-terminal (nLktA) and C-terminal (cLktA) were expressed in E. coli which were small enough for stable expression yet large enough to function as effective immunogens. The immunogenicity of the recombinant truncated LktA proteins was evaluated in mouse and goat models against a phosphate-buffered saline (PBS) negative-control group. Recombinant cLktA was emulsified with oil adjuvant and used to immunise mice and goats.Results The cLktA group had significantly higher antibody levels at four weeks post-immunisation (wpi) than the PBS group. In goats, cLktA elicited high antibody responses up to six wpi. A single administration of cLktA conferred 80% and 100% survival against a M. haemolytica challenge.Conclusion These findings show the C-terminal region of Mannheimia haemolytica LktA to be a highly immunogenic and protective antigen and suggest its potential as a candidate for subunit vaccine development.

Streptococcus suis is a bacterial pathogen that can cause significant economic losses in the swine industry due to high morbidity and mortality rates in infected animals. Vaccination with bacterins, which consist of inactivated bacteria and adjuvants to enhance the pig's immune response, is an effective approach to control S. suis infections in piglets. Here we provide a description of S. suis bacterins and the methods for vaccine preparation. Moreover, this chapter also describes the addition of recombinant Sao (rSao-L) protein to the S. suis bacterin, aiming to enhance the efficacy of the bacterins against S. suis in piglets. Furthermore, the methods for evaluating the immune response elicited by the bacterins are also covered in this chapter.

Bovine herpesvirus 1 (BoHV-1), a significant pathogen in the alpha-herpesvirus subfamily, primarily infects cattle and causes the upper respiratory disease known as infectious bovine rhinotracheitis (IBR). In silico studies evaluated the BoHV-1 D protein to be non-allergenic, non-toxic, and highly antigenic, highlighting its potential as an antigen for vaccine development. Therefore, this study aimed to evaluate the efficacy of a subunit vaccine using the ectodomain of glycoprotein D (gD34-380) as an antigen. The truncated gD was successfully cloned and expressed in both Escherichia coli (E. coli, termed EgD) and baculovirus (termed BgD) systems, with expected molecular weights of 65kDa and 50kDa, respectively. For the vaccine formulation, the gD proteins were used either alone or in combination with in-house inactivated BoHV-1. Vaccination of mice and bovines showed that baculovirus-expressed gD34-380 accelerated the antibody response. Moreover, the BgD-vaccinated group also showed significantly higher neutralizing antibody levels against BoHV-1 than the control group (p<0.0001). In conclusion, our study found that BgD from BoHV-1 can increase the immune response and enhance vaccine efficacy.

Fowl cholera, caused by Pasteurella multocida infection, poses challenges for prevention because of its many serotypes. Bacterins are currently widely used for vaccination against fowl cholera, but protection is limited to homologous strains. Live attenuated vaccines of P. multocida provide some heterologous protection, but side effects are considerable. More recently, protein-based antigens are promising subunit vaccines when their low immunogenicity has been addressed with effective adjuvants. Bacterial flagellin has been widely considered a promising adjuvant for vaccines. In this study, we tested the adjutancy of flagellin in a subunit vaccine against P. multocida in a mice and chicken models. For vaccine formulation, the antigen fPlpE (P. multocida liporotein E) was combined with fFliC (Salmonella Typhimurium flagellin). The recombinant proteins of fPlpE and fFliC were successfully expressed using the Escherichia coli system as the expected sizes of 55 kDa and 70 kDa, respectively. The fFliC elicited strong expression levels of proinflammatory cytokine (IL-1β, IL-8, and IL-6) when stimulated in native chicken peripheral blood mononuclear cells. Immunization of mice and chickens with the subunit vaccines containing fFliC accelerated the antibody response. In the challenge tests, fFliC increased vaccine protective efficacy against the heterologous strain P. multocida A1 and highly virulent strain Chu01 in mice and chickens, respectively. These data indicated potential possibilities of using fFliC as an immunostimulant adjuvant in developing a subunit vaccine against fowl cholera.

Streptococcus suis ( S. suis ) and Pasteurella multocida ( P. multocida ) are pathogens that can cause zoonotic diseases. P . multocida toxin (PMT) is an important virulence factor that causes atrophic rhinitis in pigs. Suilysin (Sly) is an extracellular protein of S. suis and has been shown to be a potential adjuvant. Previous studies have indicated that subunit vaccines containing several fragments of PMT as antigens are safer than traditional inactivated or live-attenuated vaccines. However, protein-based vaccines need strong adjuvants to enhance their immunogenicity. In this study, recombinant PMT-NC (rPMT-NC) protein antigen was formulated with either recombinant Sly (rSly) or CpG oligodeoxynucleotides (CpG) as the adjuvant. The immune responses elicited by these vaccines and the protective efficacy after challenge with live P. multocida were evaluated in piglets. In the dose-dependent test, piglets immunized with the low dose (100 µg) of rSly had increased antigen-specific total IgG, interferon (IFN)-γ gene expression, and CD4 + and CD8 + T-cell populations. Compared to piglets in the commercial (Al-gel) adjuvant and the control groups ( p < 0.05), piglets in the biological adjuvant groups showed significantly reduced turbinate atrophy, nasal distortion, and lung lesion scores after challenge with P. multocida serotype A. Vaccines containing rSly or CpG adjuvant enhanced humoral and cellular immune responses and protection against P. multocida . This combination of a protein-based antigen formulated with a biological adjuvant showed synergistic and protective effects against atrophic rhinitis and has potential to be developed as part of a bivalent vaccine.

Background Cyclic peptide nanotubes (cPNTs) formed from the spontaneous beta-sheet stacking of peptide rings may serve as a safe and effective oral delivery vehicle/adjuvant for DNA vaccines. Aim In this study, we sought to determine if a DNA vaccine expressing the VP2 protein of goose parvovirus, adjuvanted with cPNTs, may elicit virus-specific antibody response through oral vaccination. Material and methods Forty 20-day-old Muscovy ducks were randomly assigned to two groups of 20 ducks each and vaccinated. Ducks were orally vaccinated (Day 0) and boosted (Day 1 and Day 2) or were mock-vaccinated with saline as the negative control. For immunohistochemical staining, the primary antibody used comprised a rabbit anti-GPV antibody, and the secondary antibody was a goat anti-rabbit antibody. Goat-anti-mouse-IgG was used as a tertiary antibody. IgG and IgA antibody titers in serum were analyzed by the GPV virus-coated ELISA. For IgA antibody analysis, intestine lavage was harvested too. Results A DNA vaccine, coated with cPNTs, can induce a significant antibody response in ducklings. Immunohistochemical staining of tissues from vaccinated ducklings showed that VP2 proteins can be detected in the intestines and livers for up to six weeks, confirming the antigen expression by the DNA vaccine. Antibody analysis found that this vaccine formulation was very efficient at inducing IgA antibodies in the serum and the intestinal tract. Conclusion A DNA vaccine adjuvanted with cPNTs can effectively express the antigen and can significantly induce an antibody response against goose parvovirus through oral vaccination.

Recombinant Pasterurella multocida lipoprotein E (PlpE) has been shown to protect against fowl cholera. This study aimed to determine if the signal sequence may contribute to the antigenicity and protec-tive efficacy of recombinant PlpE. A small antigenic domain of PlpE (termed truncated PlpE, tPlpE) was constructed with (SP-tPlpE) or without (tPlpE) the signal sequence and evaluated in vitro and in vivo. In vitro, the HEK-Bule hTLR2 Cells were used to evaluate the activation of NF-kB in the test associated with the stimulation of the SP-tPlpE and tPlpE proteins. When chickens were immunized, compared to the tPlpE vac-cine group, the SP-tPlpE group showed higher antibody levels and enhanced CD4+ T cell response. In a challenge test, the SP-tPlpE group showed a survival rate of 87.5% (n = 8), compared to 25% for the tPlpE group. It is confirmed that the inclusion of the native signal sequence enhanced protective efficacy against fowl chol-era and may act as a vaccine adjuvant. The short SP-tPlpE construct is amenable to further vaccine engineer-ing and has potential to be developed as a fowl cholera vaccine.

The hemolytic/leukolytic toxins ApxI, II, and III play a central role in the pathogenicity of Actinobacillus pleuropneumoniae and may serve as effective vaccine antigens. However, since these toxins are large in size, we aimed to determine whether only the pore-forming domain of the toxins may be sufficient for protective immunity. Mice were vaccinated with the pore-forming domains for immune response analysis and challenge test. Significant antibody response was observed for the ApxI and II vaccine groups. For cellular immune response, CD4+ and CD8+ T cell expansion was observed for ApxI. Pro-inflammatory cytokine (IL-1b, IL-6) and TH2-type cytokine (IL-4, IL-10) gene expression was detected for the ApxI and II groups. Finally, in a challenge test with a A. pleuropneumoniae serotype 1 strain, the pore-forming domains of ApxI, II, III and three of pore-forming domains conferred 100%, 40%, 0% and 100% protection, respectively. The pore-forming domain of the Apx toxins shows promise as vaccine antigen against A. pleuropneumoniae.

Abstract Introduction Bovine adenovirus (BAdV) type 3 causes respiratory and gastroenteric diseases of varying severity in cattle, particularly newborn calves. Trials have been conducted of a vaccination against the diseases caused by BAdV using both modified live-virus and inactivated-virus preparations in cattle, but no commercial BAdV-3 vaccine has yet reached the market. Therefore, there is an urgent need to develop new, safe, and effective vaccines against BAdV-3. Material and Methods Recombinant hexon protein (rhexon) of BAdV-3 was expressed in the E. coli system to evaluate immune response in mice and goats. Antibody responses and cytokine levels were analysed and the effects of administrations of different amounts of recombinant protein compared. Long-term antibody production was evaluated by indirect ELISA, and the total immunoglobulin G secreted by goats and mice immunised with the purified rhexon protein was determined. Results The immunised mice had a stronger antibody response than the control group at eight weeks post vaccination. The immunised groups also showed significantly higher (P ˂ 0.05) expression of interferon-γ, interleukin 2 (in mice), and interleukin 21 (in goats) at four weeks. Furthermore, vaccination with rhexon was able to induce long-term antibody production for at least 16 weeks in mice and goats. Conclusion The rhexon protein induced immune responses, especially long-term antibody production and T helper 1 cell cytokine production in mice and goats. The immunogenic properties of this protein make it a promising subunit vaccine antigen.

Porcine circovirus (PCV) comprises four distinct species, namely, PCV1–4, with considerable differences between them, resulting in limited crossprotection. PCV2 inflicts substantial economic losses on the swine industry. PCV3 was found to have been circulating before its discovery in 2015. PCV3 is suspected of having a comparable impact to PCV2; however, its characterization studies are still underway. The symptoms of PCV3 infection are similar to those of PCV2 infection. However, PCV2 and PCV3 share a maximum amino acid identity of only 37%, which partially explains the inadequacy of the PCV2 vaccine in protecting against PCV3 infection. Swift measures must be taken to control PCV3, including vaccine development, as it also poses a threat to swine populations. This review synthesizes the recent findings on PCV3 in comparison with PCV2 and highlights the prevailing challenges encountered in vaccine development. Various strategies and innovative approaches to producing PCV3 vaccines, such as using RNA particle technology and baculovirus vectors, are also discussed. Addressing research gaps in diagnostic methods, viral isolation, and vaccine development is crucial to controlling this virus, which poses a threat to swine populations.

Background Flagellin elicits potent immune response and may serve as a vaccine adjuvant. We previously reported that the N-terminus of flagellin (residues 1–99, n FliC) is sufficient for vaccine efficacy enhancement against Pasteurella multocida challenge in chickens. In this study, we futher tested the adjuvancy of n FliC in a subunit vaccine against the pig pathogen Actinobacillus pleuropneumoniae in a mice model. For vaccine formulation, the antigen ApxIIPF (the pore-forming region of the exotoxin ApxII) was combined with n FliC, either through genetic fusion or simple admixture. Results Immune analysis showed that n FliC, introduced through genetic fusion or admixture, enhanced both humoral (antibody levels) and cellular (T cell response and cytokine production) immunity. In a challenge test, n FliC increased vaccine protective efficacy to 60–80%, vs. 20% for the antigen-only group. Further analysis showed that, even without a supplemental adjuvant such as mineral salt or oil emulsion, genetically linked n FliC still provided significant immune enhancement. Conclusions We conclude that n FliC is a versatile and potent adjuvant for vaccine formulation.

Suilysin (Sly) from Streptococcus suis has been shown to elicit strong immune responses and may act as a vaccine adjuvant. In the present study, we tested the adjuvant effect of Sly using an engineered Pasteurella multocida toxin, rPMT-NC, as the antigen. The antigen was also formulated with other conventional adjuvants (aluminum hydroxide, water-in-oil-in-water) for comparison. The efficacy of these vaccine formulations were evaluated in mice. The optimal dosage of purified rSly for enhancing immune responses in mice was first determined to be 40 μg/ml based on significantly (p < 0.05) increased serum antibody titers, expression of cytokines, including interleukin (IL)-4, IL-12, and interferon (IFN)-γ and the survival rate after challenge with P. multocida. Mice immunized with rPMT-NC + rSly had augmented antibody production and cellular immunity compare to those immunized with rPMT-NC plus other adjuvants. In addition, the survival rate of mice immunized with rPMT-NC + rSly was the highest (70% v.s. 30% of mice immunized with rPMT-NC alone) among all groups. In conclusion, rSly has the potential to be used as a biological adjuvant to enhance immune responses and protective efficacy of protein-based vaccines.

Pasteurella multocida (P. multocida) infects the swine respiratory tract and mainly causes atrophic rhinitis (AR). Recently, many commercially inactivated and subunit vaccines have been used as preventive strategies. However, the best antigenic protein portion has not been selected, and the aluminum gel was used as the adjuvant, which may not induce full protection. P. multocida toxin (PMT) is the major virulence factor responsible for AR. PMT is a monomeric 146 kDa protein (approximately 1285 amino acids) encoded by the tox A gene. In this study, we expressed different fragments of recombinant PMT proteins, combined them with a water-in-oil-in-water adjuvant, and evaluated mice's immune response. The results indicated that the rPMT-C-immunized group showed significantly higher levels (p < 0.05) of IgG, IgG2a antibody and interferon-γ, IL-12 cytokine expression than other groups. Furthermore, vaccination with rPMT-C recombinant protein can provide homologous and heterologous protection against P. multocida challenge. In conclusion, our approach may be feasible for developing an effective subunit vaccine against atrophic rhinitis with a cost-down simple ingredient.

AIMS:Vaccines for bovine ephemeral fever virus (BEFV) are available but are difficult to produce, expensive or suffer from genetic instability. Therefore, we designed constructs encoding C-terminally truncated forms (transmembrane anchoring region deleted) of glycoproteins G and GNS such that they were secreted from the cell into the media to achieve high-level antigen expression, correct glycosylation pattern and enable further simple purification with the V5 epitope tag.METHODS AND RESULTS:In this study, synthetic biology was employed to create membrane-bound and secreted forms of G and GNS glycoprotein. Mammalian cell culture was employed as an antigen expression platform, and the secreted forms of G and GNS protein were easily purified from media using a highly effective, single-step method. The V5 epitope tag was genetically fused to the C-termini of the proteins, enabling detection of the antigen through immunoblotting and immunomicroscopy. Our data demonstrated that the C-terminally truncated form of the G glycoprotein was efficiently secreted from cells into the cell media. Moreover the immunogenicity was confirmed in mice test.CONCLUSIONS:The immuno-dot blots showed that the truncated G glycoprotein was present in the total cell extract, and was clearly secreted into the media, consistent with the western blotting data and live-cell images. Our strategy presented the expression of secreted, epitope-tagged, forms of the BEFV glycoproteins such that appropriately glycosylated forms of BEFV G protein was secreted from the BHK-21 cells. This indicates that high-level expression of secreted G glycoprotein is a feasible strategy for large-scale production of vaccines and improving vaccine efficacy.SIGNIFICANCE AND IMPACT OF THE STUDY:The antigen expression strategy designed in this study can produce high-quality recombinant protein and reduce the amount of antigen used in the vaccine.

During industrial-scale production of viruses for vaccine manufacturing, anti-viral response of host cells can dampen maximal viral antigen yield. In addition to interferon responses, many other cellular responses, such as the AMPK signaling pathway or senescence-like response may inhibit or slow down virus amplification in the cell culture system. In this study, we first performed a Gene Set Enrichment Analysis of the whole-genome mRNA transcriptome and found a senescence-like cellular response in BHK-21 cells when infected with bovine ephemeral fever virus (BEFV). To demonstrate that this senescence-like state may reduce virus growth, BHK-21 subclones showing varying degrees of a senescence-like state were infected with BEFV. The results showed that the BHK-21 subclones showing high senescence staining could inhibit BEFV replication while low senescence-staining subclones are permissive to virus replication. Using a different approach, a senescence-like state was induced in BHK-21 using a small molecule, camptothecin (CPT), and BEFV susceptibility were examined. The results showed that CPT-treated BHK-21 is more resistant to virus infection. Overall, these results indicate that a senescence-like response may be at play in BHK-21 upon virus infection. Furthermore, cell clone selection and modulating treatments using small molecules may be tools in countering anti-viral responses.

Aims Riemerella anatipestiferinfections of goslings and ducklings can result in high mortality. Since there are at least 21 serotypes ofR. anatipestifer, cross-protection is an important goal for vaccine development. Methods and Results In this study, we evaluated the immunostimulatory effect of different immunization regimens - the traditional inactivated vaccinevsprime-boost regimens using DNA and protein subunit vaccines (DNA+subunit, subunit+subunit, subunit+inactivated and DNA+DNA). Results showed that, when compared to the inactivated vaccine, prime-boost regimens induced higher and up to 16-week longer lasting levels of antibody responses, significantly elevated the percentage of the cytotoxic CD8(+)T cell and higher expression levels of IFN-gamma, IL-6 and IL-12 mRNAs. Furthermore, as an indication of cross-protection, sera from prime-boost regimens were able to recognize lysates ofR. anatipestiferserotypes 1, 2 and 6. Conclusions Prime-boost regimens especially DNA-prime and protein-boost, induce strong long-term immune response and may prove protective for breeder ducks requiring long-term protection. Significance and Impact of the Study It is worth mentioning that the subunit+inactivated regimen group also elicited strong immune response. The cost of this regimen may only be half of the other prime-boost regimens, making this subunit + inactivated combination an attractive option.

Background Bovine ephemeral fever virus (BEFV) causes fever and muscle stiffness in cattle, resulting in negative economic impact for cattle and dairy farms. During the manufacturing process of inactivated vaccine for virus control, it is important to determine the virus titer, but traditional methods such as plaque assay and TCID50 assay require days of waiting time. We sought to develop a quick dot blot assay for BEFV titering. Results Three different kinds of BEFV antigens were prepared to raise primary antibodies for BEFV detection in dot blot assays: 1) purified BEFV particles, 2) Escherichia coli (E. coli)-expressed BEFV G1 region, and 3) E. coli-expressed BEFV N protein. Results showed that antibodies raised against purified BEFV particles can detect BEFV particles, but it also showed a high background level from the proteins of BHK-21 cells. Antibodies raised against E.coli-expressed BEFV G1 region could not detect BEFV particles in dot blot assays. Finally, antibodies raised against E.coli-expressed BEFV N protein detected BEFV particles with a high signal-to-noise ratio in dot blot assays. Conclusions E.coli-expressed N protein is a suitable antigen for the production of antiserum that can detect BEFV particles with a high signal-to-noise ratio. A dot blot assay kit using this antiserum can be developed as a quick and economical way for BEFV titering.

Abstract Introduction Riemerella anatipestifer (RA) infections can lead to high mortality in ducklings. Inactivated vaccines against RA are commercially available, but they fail to provide cross-protection against various serotypes. We have previously demonstrated that a subunit vaccine containing recombinant outer membrane protein A (rOmpA) antigen of serotype 2 formulated with CpG oligodeoxynucleotides (ODN) as the adjuvant was able to stimulate both humoral and cellular immunities. Material and Methods In the present study, thirty healthy 7-day-old Pekin ducks were randomly assigned to three equal treatment groups: rOmpA-vaccinated, rOmpA + CpG-vaccinated, and control. Vaccine was injected intramuscularly and a booster dose of the same vaccine was given two weeks after primary immunisation. The long-term antibody response and cross-serotype reaction of this vaccine were evaluated in ducks. Results Compared to ducks immunised with rOmpA alone, ducks immunised with rOmpA + CpG ODN had significantly (p < 0.05) increased serum antibody titre from two weeks until nine months after primary immunisation. In addition, expression of cytokines including interferon (IFN)-α, IFN-γ, interleukin (IL)-6, and IL-12 was significantly (p < 0.05) enhanced in PBMC of ducks immunised with rOmpA + CpG ODN two weeks after primary immunisation. Antibodies from ducks immunised with the rOmpA + CpG ODN vaccine could also detect RA serotypes 1 and 6 in Western blot analysis. Conclusion Combination of rOmpA and CpG ODN could be a feasible strategy for developing a subunit RA vaccine with long term and broader-ranging protection.