A glucan-particle based tularemia subunit vaccine induces T-cell immunity and affords partial protection in an inhalation rat infection model.

Tularemia is a zoonotic disease caused by the facultative intracellular gram-negative bacterium Francisella tularensis. F. tularensis has a very low infection dose by the aerosol route which can result in an acute, and potentially lethal, infection in humans. Consequently, it is classified as a Category A bioterrorism agent by the US Centers for Disease Control (CDC) and is a pathogen of concern for the International Biodefence community. There are currently no licenced tularemia vaccines. In this study we report on the continued assessment of a tularemia subunit vaccine utilising ß-glucan particles (GPs) as a vaccine delivery platform for immunogenic F. tularensis antigens. Using a Fischer 344 rat infection model, we demonstrate that a GP based vaccine comprising the F. tularensis lipopolysaccharide antigen together with the protein antigen FTT0814 provided partial protection of F344 rats against an aerosol challenge with a high virulence strain of F. tularensis, SCHU S4. Inclusion of imiquimod as an adjuvant failed to enhance protective efficacy. Moreover, the level of protection afforded was dependant on the challenge dose. Immunological characterisation of this vaccine demonstrated that it induced strong antibody immunoglobulin responses to both polysaccharide and protein antigens. Furthermore, we demonstrate that the FTT0814 component of the GP vaccine primed CD4+ and CD8+ T-cells from immunised F344 rats to express interferon-γ, and CD4+ cells to express interleukin-17, in an antigen specific manner. These data demonstrate the development potential of this tularemia subunit vaccine and builds on a body of work highlighting GPs as a promising vaccine platform for difficult to treat pathogens including those of concern to the bio-defence community.

BacScan: a novel genome-wide strategy for uncovering broadly immunogenic proteins in bacteria.

In response to the global threat posed by bacterial pathogens, which are the second leading cause of death worldwide, vaccine development is challenged by the diversity of bacterial serotypes and the lack of immunoprotection across serotypes. To address this, we introduce BacScan, a novel genome-wide technology for the rapid discovery of conserved highly immunogenic proteins (HIPs) across serotypes. Using bacterial-specific serum, BacScan combines phage display, immunoprecipitation, and next-generation sequencing to comprehensively identify all the HIPs in a single assay, thereby paving the way for the development of universally protective vaccines. Our validation of this technique with Streptococcus suis, a major pathogenic threat, led to the identification of 19 HIPs, eight of which conferred 20-100% protection against S. suis challenge in animal models. Remarkably, HIP 8455 induced complete immunity, making it an exemplary vaccine target. BacScan's adaptability to any bacterial pathogen positions it as a revolutionary tool that can expedite the development of vaccines with broad efficacy, thus playing a critical role in curbing bacterial transmission and slowing the march of antimicrobial resistance.

Outer membrane vesicles generated by an exogenous bacteriophage lysin and protection against Acinetobacter baumannii infection.

BACKGROUND: The outer membrane vesicles (OMVs) produced by Gram-negative bacteria can modulate the immune system and have great potentials for bacterial vaccine development. RESULTS: A highly active Acinetobacter baumannii phage lysin, LysP53, can stimulate the production of OMVs after interacting with A. baumannii, Escherichia coli, and Salmonella. The OMVs prepared by the lysin (LOMVs) from A. baumannii showed better homogeneity, higher protein yield, lower endotoxin content, and lower cytotoxicity compared to the naturally produced OMVs (nOMVs). The LOMVs contain a significantly higher number of cytoplasmic and cytoplasmic membrane proteins but a smaller number of periplasmic and extracellular proteins compared to nOMVs. Intramuscular immunization with either LOMVs or nOMVs three times provided robust protection against A. baumannii infections in both pneumonia and bacteremia mouse models. Intranasal immunization offered good protection in the pneumonia model but weaker protection (20-40%) in the bacteremia model. However, with a single immunization, LOMVs demonstrated better protection than the nOMVs in the pneumonia mouse model. CONCLUSIONS: The novel lysin approach provides a superior choice compared to current methods for OMV production, especially for vaccine development.

Method for quantifying the Pasteurella multocida antigen adsorbed on aluminum hydroxide adjuvant in swine atrophic rhinitis vaccine.

Swine atrophic rhinitis is a disease caused by Pasteurella multocida and Bordetella bronchiseptica that affects pigs. Inactivated vaccines containing the toxins produced by Pasteurella multocida and Bordetella bronchiseptica have been widely used for the prevention of swine atrophic rhinitis. The efficacy of a vaccine is correlated with the amount of antigen present; however, the protective toxin of P. multocida bound to aluminum hydroxide, which is used as an adjuvant, can hinder the monitoring of the antigen concentration in the vaccine. This study assessed the applicability of a dot immunoassay as an antigen quantification method using monoclonal antibodies. This quantification method was able to detect the antigen with high specificity and sensitivity even when the antigen was bound to the adjuvant, and its application to vaccine products revealed a correlation between the amount of antigen present in the vaccine and the neutralizing antibody titers induced in pigs. The antigen quantification method presented in this study is a simple and sensitive assay capable of quantifying the amount of antigen present in a vaccine that can be used as an alternative quality control measure.

Protective immunity induced through two calving seasons following administration of live epizootic bovine abortion agent (EBAA) vaccine.

A live, infectious vaccine candidate for epizootic bovine abortion, designated EBAA Vaccine, USDA-APHIS Product code #1544.00, has been reported to be both safe and effective. Previous studies established that a single dose of EBAA vaccine administered to cows at potencies of either 2000 or 500 live P. abortibovis-infected murine spleen cells (P.a.-LIC) induced protective immunity for a minimum of 5 months. The current study employed 19 pregnant cows that were challenged with P. abortibovis in their 2nd trimester of gestation; 9 were vaccinated 17.2-months earlier as 1-year-olds with 2000 P.a.-LIC and 10 served as negative controls. Eighty-nine percent of the vaccinates gave birth to healthy calves as compared to 10% of challenge controls. Vaccine efficacy was significant when analyzed by prevented fractions (87.7%; 95% CI=0.4945-0.9781). Serologic data supports previous findings that pregnant cows with detectable P. abortibovis antibodies are immune to P. abortibovis challenge as demonstrated by the birth of healthy calves.

Campylobacteriosis and Control Strategies against Campylobacters in Poultry Farms.

Campylobacteriosis is a significant foodborne illness caused by Campylobacter bacteria. It is one of the most common bacterial causes of gastroenteritis worldwide, with poultry being a major reservoir and source of infection in humans. In poultry farms, Campylobacters colonize the intestinal tract of chickens and contaminate meat during processing. Vaccines under development against Campylobacters in poultry showed partial or no protection against their cecal colonization. Therefore, this review will elaborate on campylobacteriosis and emphasize the control strategies and recent vaccine trials against Campylobacters in poultry farms. The epidemiology, diagnosis, and treatment of Campylobacter infection, along with specific mention of poultry Campylobacter contamination events in Malaysia, will also be discussed.

Evaluation of the safety and efficacy of the novel Mycoplasma gallisepticum vaccine, Vaxsafe MG304, after spray-vaccination of 1-day-old specific pathogen-free chicks.

Mycoplasma gallisepticum causes chronic respiratory disease in poultry. A novel vaccine, Vaxsafe MG304 (the ts-304 strain), has greater protective efficacy in chickens than the Vaxsafe MG (strain ts-11) vaccine when delivered by eye drop at 3 weeks of age. Applying this vaccine in the hatchery to 1-day-old birds, using mass administration methods, would improve animal welfare and reduce labour costs associated with handling individual birds. This study assessed the protection provided by vaccination with Vaxsafe MG304 after administration to 1-day-old chicks. Chicks were administered a single dose of the vaccine to assess the efficacy of either a high dose (107.0 colour changing units, CCU) or a low dose (105.7 CCU) after eye drop or spray (in water or gel) administration against experimental challenge with virulent M. gallisepticum strain Ap3AS at 7 weeks of age. The vaccine was able to colonise the palatine cleft of chicks after vaccination by eye drop (at both doses) or by spray (in water or gel) (at the high dose). The high dose of vaccine, when delivered by eye drop or spray, was shown to be safe and induced a serological response and protective immunity (as measured by tracheal mucosal thickness and air sac lesion scores) against challenge. Vaccination of 1-day-old chicks with Vaxsafe MG304 by eye drop induced protective immunity equivalent to vaccination at 3 weeks of age. Vaxsafe MG304 was also protective when applied by both coarse- and gel spray methods at the higher dose and is therefore a suitable live attenuated vaccine for use in 1-day-old chicks.

A novel chimeric vaccine containing multiple epitopes for simulating robust immune activation against Klebsiella pneumoniae.

BACKGROUND: Due to antibiotic resistance, the Klebsiella genus is linked to morbidity and death, necessitating the development of a universally protective vaccine against Klebsiella pathogens. METHODS: Core sequence analysis prioritized non-redundant host molecules and expected lipid bilayer peptides from fully sequenced Klebsiella genomes. These proteins were refined to identify epitopes, examining their immunogenicity, toxicity, solubility, and interaction with MHC alleles. Epitopes were linked to CPG ODN C274 via EAAAK, HEYGAEALERAG, and GGGS linkers to enhance immunological responses. The vaccine's tertiary structure was modelled and docked with MHC-I and MHC-II. RESULTS: Fifty-five proteins were recognized in the Vaxign collection as having remarkable features. Twenty-three proteins with potential pathogenicity were then identified. Eight options for vaccines emerged after the immunogenicity of proteins was examined. The best antigens were three proteins: MrkD, Iron-regulated lipid membrane polypeptides, and RmpA. These compounds were selected for their sensitivity. The structural protein sequences of K. pneumoniae were utilized to identify seven CTL epitopes, seven HTL epitopes, and seven LBL epitopes, respectively. The produced immunization displayed a stable contact with the receptors, based on molecular dynamic simulations lasting 250 nanoseconds. Intermolecular binding free energies also indicated the dominance of the van der Waals and electrostatic energies. CONCLUSION: In summary, the results of this study might help scientists develop a novel vaccine to prevent K. pneumoniae infections.

Longitudinal analysis within one hospital in sub-Saharan Africa over 20 years reveals repeated replacements of dominant clones of Klebsiella pneumoniae and stresses the importance to include temporal patterns for vaccine design considerations.

BACKGROUND: Infections caused by multidrug-resistant gram-negative bacteria present a severe threat to global public health. The WHO defines drug-resistant Klebsiella pneumoniae as a priority pathogen for which alternative treatments are needed given the limited treatment options and the rapid acquisition of novel resistance mechanisms by this species. Longitudinal descriptions of genomic epidemiology of Klebsiella pneumoniae can inform management strategies but data from sub-Saharan Africa are lacking. METHODS: We present a longitudinal analysis of all invasive K. pneumoniae isolates from a single hospital in Blantyre, Malawi, southern Africa, from 1998 to 2020, combining clinical data with genome sequence analysis of the isolates. RESULTS: We show that after a dramatic increase in the number of infections from 2016 K. pneumoniae becomes hyperendemic, driven by an increase in neonatal infections. Genomic data show repeated waves of clonal expansion of different, often ward-restricted, lineages, suggestive of hospital-associated transmission. We describe temporal trends in resistance and surface antigens, of relevance for vaccine development. CONCLUSIONS: Our data highlight a clear need for new interventions to prevent rather than treat K. pneumoniae infections in our setting. Whilst one option may be a vaccine, the majority of cases could be avoided by an increased focus on and investment in infection prevention and control measures, which would reduce all healthcare-associated infections and not just one.

Vaccines and Monoclonal Antibodies as Alternative Strategies to Antibiotics to Fight Antimicrobial Resistance.

Antimicrobial resistance (AMR) is one of the most critical threats to global public health in the 21st century, causing a large number of deaths every year in both high-income and low- and middle-income countries. Vaccines and monoclonal antibodies can be exploited to prevent and treat diseases caused by AMR pathogens, thereby reducing antibiotic use and decreasing selective pressure that favors the emergence of resistant strains. Here, differences in the mechanism of action and resistance of vaccines and monoclonal antibodies compared to antibiotics are discussed. The state of the art for vaccine technologies and monoclonal antibodies are reviewed, with a particular focus on approaches validated in clinical studies. By underscoring the scope and limitations of the different emerging technologies, this review points out the complementary of vaccines and monoclonal antibodies in fighting AMR. Gaps in antigen discovery for some pathogens, as well as challenges associated with the clinical development of these therapies against AMR pathogens, are highlighted.

Immunization of turkeys with Clostridium septicum alpha toxin-based recombinant subunit proteins can confer protection against experimental Clostridial dermatitis.

Clostridial dermatitis (CD), caused by Clostridium septicum, is an emerging disease of increasing economic importance in turkeys. Currently, there are no effective vaccines for CD control. Here, two non-toxic domains of C. septicum alpha toxin, namely ntATX-D1 and ntATX-D2, were identified, cloned, and expressed in Escherichia coli as recombinant subunit proteins to investigate their use as potential vaccine candidates. Experimental groups consisted of a Negative control (NCx) that did not receive C. septicum challenge, while the adjuvant-only Positive control (PCx), ntATX-D1 immunization (D1) and ntATX-D2 immunization (D2) groups received C. septicum challenge. Turkeys were immunized subcutaneously with 100 µg of protein at 7, 8 and 9 weeks of age along with an oil-in-water nano-emulsion adjuvant, followed by C. septicum challenge at 11 weeks of age. Results showed that while 46.2% of birds in the PCx group died post-challenge, the rate of mortality in D1- or D2-immunization groups was 13.3%. The gross and histopathological lesions in the skin, muscle and spleen showed that the disease severity was highest in PCx group, while the D2-immunized birds had significantly lower lesion scores when compared to PCx. Gene expression analysis revealed that PCx birds had significantly higher expression of pro-inflammatory cytokine genes in the skin, muscle and spleen than the NCx group, while the D2 group had significantly lower expression of these genes compared to PCx. Peripheral blood cellular analysis showed increased frequencies of activated CD4+ and/or CD8+ cells in the D1 and D2-immunized groups. Additionally, the immunized turkeys developed antigen-specific serum IgY antibodies. Collectively, these findings indicate that ntATX proteins, specifically the ntATX-D2 can be a promising vaccine candidate for protecting turkeys against CD and that the protection mechanisms may include downregulation of C. septicum-induced inflammation and increased CD4+ and CD8+ cellular activation.

Co-immunization with DNA vaccines encoding yidR and IL-17 augments host immune response against Klebsiella pneumoniae infection in mouse model.

Klebsiella pneumoniae is an important gram-negative bacterium that causes severe respiratory and healthcare-associated infections. Although antibiotic therapy is applied to treat severe infections caused by K. pneumoniae, drug-resistant isolates pose a huge challenge to clinical practices owing to adverse reactions and the mismanagement of antibiotics. Several studies have attempted to develop vaccines against K. pneumoniae, but there are no licensed vaccines available for the control of K. pneumoniae infection. In the current study, we constructed a novel DNA vaccine, pVAX1-YidR, which encodes a highly conserved virulence factor YidR and a recombinant expression plasmid pVAX1-IL-17 encoding Interleukin-17 (IL-17) as a molecular adjuvant. Adaptive immune responses were assessed in immunized mice to compare the immunogenicity of the different vaccine schemes. The results showed that the targeted antigen gene was expressed in HEK293T cells using an immunofluorescence assay. Mice immunized with pVAX1-YidR elicited a high level of antibodies, induced strong cellular immune responses, and protected mice from K. pneumoniae challenge. Notably, co-immunization with pVAX1-YidR and pVAX1-IL-17 significantly augmented host adaptive immune responses and provided better protection against K. pneumoniae infections in vaccinated mice. Our study demonstrates that combined DNA vaccines and molecular adjuvants is a promising strategy to develop efficacious antibacterial vaccines against K. pneumoniae infections.

A novel vaccination strategy against Vibrio harveyi infection in Asian seabass (Lates calcarifer) with the aid of oxygen nanobubbles and chitosan.

Immersion vaccination, albeit easier to administer than immunization by injection, sometimes has challenges with antigen uptake, resulting in sub-optimal protection. In this research, a new strategy to enhance antigen uptake of a heat-inactivated Vibrio harveyi vaccine in Asian seabass (Lates calcarifer) using oxygen nanobubble-enriched water (ONB) and positively charged chitosan (CS) was explored. Antigen uptake in fish gills was assessed, as was the antibody response and vaccine efficacy of four different combinations of vaccine with ONB and CS, and two control groups. Pre-mixing of ONB and CS before introducing the vaccine, referred to as (ONB + CS) + Vac, resulted in superior antigen uptake and anti-V. harveyi antibody (IgM) production in both serum and mucus compared to other formulas. The integration of an oral booster (4.22 × 108 CFU/g, at day 21-25) within a vaccine trial experiment set out to further evaluate how survival rates post exposure to V. harveyi might be improved. Antibody responses were measured over 42 days, and vaccine efficacy was assessed through an experimental challenge with V. harveyi. The expression of immune-related genes IL1ß, TNFα, CD4, CD8, IgT and antibody levels were assessed at 1, 3, and 7-day(s) post challenge (dpc). The results revealed that antibody levels in the group (ONB + CS) + Vac were consistently higher than the other groups post immersion immunization and oral booster, along with elevated expression of immune-related genes after challenge with V. harveyi. Ultimately, this group demonstrated a significantly higher relative percent survival (RPS) of 63 % ± 10.5 %, showcasing the potential of the ONB-CS-Vac complex as a promising immersion vaccination strategy for enhancing antigen uptake, stimulating immunological responses, and improving survival of Asian seabass against vibriosis.

Recombinant lactococcal-based oral vaccine for protection against Streptococcus agalactiae infections in tilapia (Oreochromis niloticus).

Streptococcosis outbreaks caused by Streptococcus agalactiae infection in tilapia aquaculture have been consistently reported and associated with high mortality and morbidity leading to significant economic losses. Existing vaccine candidates against Streptococcus spp. are designed for intraperitoneal injections that are not practical and labor-intensive which have prompted farmers to protect aquatic animals with antibiotics, thus encouraging the emergence of multidrug resistant bacteria. In this study, a live recombinant L. lactis vaccine expressing a 1403 bp surface immunogenic protein (SIP) and a 1100 bp truncated SIP (tSIP) gene was developed and evaluated against S. agalactiae infection in tilapia. Both SIP and tSIP sequences were cloned and transformed into L. lactis. The recombinant L.lactis vaccine was orally administered to juvenile tilapia for a month. Detection of SIP-specific serum IgM in vaccinated groups compared to control groups indicated that recombinant proteins expressed from L. lactis could elicit immunogenic reactions in tilapia. Fish immunized with the tSIP vaccine also showed the highest level of protection compared to other test groups, and the mortality rate was significantly reduced compared to both control groups. The relative percentage of survival (RPS) against S. agalactiae for both SIP and tSIP-vaccinated groups was 50 % and 89 %, respectively, at 14 days post-challenge. Significant up-regulation of IgM, IL-1ß, IL-10, TNF-α and IFN-γ were observed at day 34 between the vaccinated and control groups. These results indicated that the recombinant lactococcal tSIP vaccine can elicit both cell-mediated and humoral responses and is recommended as a potential oral vaccine against S. agalactiae infection. Future work will include further in vivo challenge assessments of this vaccine candidate fused with adjuvants to boost immunogenicity levels in tilapia.

In silico identification of drug targets and vaccine candidates against Bartonella quintana: a subtractive proteomics approach.

BACKGROUND: The availability of genes and protein sequences for parasites has provided valuable information for drug target identification and vaccine development. One such parasite is Bartonella quintana, a Gram-negative, intracellular pathogen that causes bartonellosis in mammalian hosts. OBJECTIVE: Despite progress in understanding its pathogenesis, limited knowledge exists about the virulence factors and regulatory mechanisms specific to B. quintana. METHODS AND FINDINGS: To explore these aspects, we have adopted a subtractive proteomics approach to analyse the proteome of B. quintana. By subtractive proteins between the host and parasite proteome, a set of proteins that are likely unique to the parasite but absent in the host were identified. This analysis revealed that out of the 1197 protein sequences of the parasite, 660 proteins are non-homologous to the human host. Further analysis using the Database of Essential Genes predicted 159 essential proteins, with 28 of these being unique to the pathogen and predicted as potential putative targets. Subcellular localisation of the predicted targets revealed 13 cytoplasmic, eight membranes, one periplasmic, and multiple location proteins. The three-dimensional structure and B cell epitopes of the six membrane antigenic protein were predicted. Four B cell epitopes in KdtA and mraY proteins, three in lpxB and BQ09550, whereas the ftsl and yidC proteins were located with eleven and six B cell epitopes, respectively. MAINS CONCLUSIONS: This insight prioritises such proteins as novel putative targets for further investigations on their potential as drug and vaccine candidates.

A rational designed multi-epitope vaccine elicited robust protective efficacy against Klebsiella pneumoniae lung infection.

BACKGROUND: The emergence of drug-resistant strains of Klebsiella pneumoniae (K. pneumoniae) has become a significant challenge in the field of infectious diseases, posing an urgent need for the development of highly protective vaccines against this pathogen. METHODS AND RESULTS: In this study, we identified three immunogenic extracellular loops based on the structure of five candidate antigens using sera from K. pneumoniae infected mice. The sequences of these loops were linked to the C-terminal of an alpha-hemolysin mutant (mHla) from Staphylococcus aureus to generate a heptamer, termed mHla-EpiVac. In vivo studies confirmed that fusion with mHla significantly augmented the immunogenicity of EpiVac, and it elicited both humoral and cellular immune responses in mice, which could be further enhanced by formulation with aluminum adjuvant. Furthermore, immunization with mHla-EpiVac demonstrated enhanced protective efficacy against K. pneumoniae channeling compared to EpiVac alone, resulting in reduced bacterial burden, secretion of inflammatory factors, histopathology and lung injury. Moreover, mHla fusion facilitated antigen uptake by mouse bone marrow-derived cells (BMDCs) and provided sustained activation of these cells. CONCLUSIONS: These findings suggest that mHla-EpiVac is a promising vaccine candidate against K. pneumoniae, and further validate the potential of mHla as a versatile carrier protein and adjuvant for antigen design.

Multicomponent Pathogen-Mimicking Nanoparticles Induce Intestinal Immune Responses against Paratuberculosis.

Given the worldwide problem posed by enteric pathogens, the discovery of safe and efficient intestinal adjuvants combined with novel antigen delivery techniques is essential to the design of mucosal vaccines. In this work, we designed poly (lactic-co-glycolic acid) (PLGA)-based nanoparticles (NPs) to codeliver all-trans retinoic acid (atRA), novel antigens, and CpG. To address the insolubility of the intestinal adjuvant atRA, we utilized PLGA to encapsulate atRA and form a "nanocapsid" with polydopamine. By leveraging polydopamine, we adsorbed the water-soluble antigens and the TLR9 agonist CpG onto the NPs' surface, resulting in the pathogen-mimicking PLPCa NPs. In this study, the novel fusion protein (HBf), consisting of the Mycobacterium avium subspecies paratuberculosis antigens HBHA, Ag85B, and Bfra, was coloaded onto the NPs. In vitro, PLPCa NPs were shown to promote the activation and maturation of bone marrow-derived dendritic cells. Additionally, we found that PLPCa NPs created an immune-rich microenvironment at the injection site following intramuscular administration. From the results, the PLPCa NPs induced strong IgA levels in the gut in addition to enhancing powerful systemic immune responses. Consequently, significant declines in the bacterial burden and inflammatory score were noted in PLPCa NPs-treated mice. In summary, PLPCa can serve as a novel and safe vaccine delivery platform against gut pathogens, such as paratuberculosis, capable of activating both systemic and intestinal immunity.

Lactobacilli-derived adjuvants combined with immunoinformatics-driven multi-epitope antigens based approach protects against Clostridium perfringens in a mouse model.

Clostridium perfringens is ubiquitously distributed and capable of secreting toxins, posing a significant threat to animal health. Infections caused by Clostridium perfringens, such as Necrotic Enteritis (NE), result in substantial economic losses to the livestock industry annually. However, there is no effective commercial vaccine available. Hence, we set out to propose an effective approach for multi-epitope subunit vaccine construction utilizing biomolecules. We utilized immunoinformatics to design a novel multi-epitope antigen against C. perfringens (CPMEA). Furthermore, we innovated novel bacterium-like particles (BLPs) through thermal acid treatment of various Lactobacillus strains and selected BLP23017 among them. Then, we detailed the structure of CPMEA and BLPs and utilized them to prepare a multi-epitope vaccine. Here, we showed that our vaccine provided full protection against C. perfringens infection after a single dose in a mouse model. Additionally, BLP23017 notably augmented the secretion of secretory immunoglobulin A (sIgA) and enhanced antibody production. We conclude that our vaccine possess safety and high efficacy, making it an excellent candidate for preventing C. perfringens infection. Moreover, we demonstrate our approach to vaccine construction and the preparation of BLP23017 with distinct advantages may contribute to the prevention of a wider array of diseases and the novel vaccine development.

Planting a chemical flag on antigens.

Next-generation live vaccines are created by autonomous production of nitrated antigens.

Investigation of the safety and protective efficacy of an attenuated and marker M. bovis-BoHV-1 combined vaccine in bovines.

Bovine respiratory disease (BRD) is one of the most common diseases in the cattle industry worldwide; it is caused by multiple bacterial or viral coinfections, of which Mycoplasma bovis (M. bovis) and bovine herpesvirus type 1 (BoHV-1) are the most notable pathogens. Although live vaccines have demonstrated better efficacy against BRD induced by both pathogens, there are no combined live and marker vaccines. Therefore, we developed an attenuated and marker M. bovis-BoHV-1 combined vaccine based on the M. bovis HB150 and BoHV-1 gG-/tk- strain previously constructed in our lab and evaluated in rabbits. This study aimed to further evaluate its safety and protective efficacy in cattle using different antigen ratios. After immunization, all vaccinated cattle had a normal rectal temperature and mental status without respiratory symptoms. CD4+, CD8+, and CD19+ cells significantly increased in immunized cattle and induced higher humoral and cellular immune responses, and the expression of key cytokines such as IL-4, IL-12, TNF-α, and IFN-γ can be promoted after vaccination. The 1.0 × 108 CFU of M. bovis HB150 and 1.0 × 106 TCID50 BoHV-1 gG-/tk- combined strain elicited the most antibodies while significantly increasing IgG and cellular immunity after challenge. In conclusion, the M. bovis HB150 and BoHV-1 gG-/tk- combined strain was clinically safe and protective in calves; the mix of 1.0 × 108 CFU of M. bovis HB150 and 1.0 × 106 TCID50 BoHV-1 gG-/tk- strain was most promising due to its low amount of shedding and highest humoral and cellular immune responses compared with others. This study introduces an M. bovis-BoHV-1 combined vaccine for application in the cattle industry.