Interleukin-17B (IL-17B) is a protective cytokine of the IL-17 family and plays an essential role in the regulation of mucosal inflammation. However, little is known about the role of IL-17B in the control of viral infections. In this study, a recombinant Lactobacillus plantarum, designated as NC8-ChIL17B, was constructed to express the chicken IL-17B (ChIL-17B) gene. The recombinant ChIL17B (rChIL17B) protein was about 14 kDa and was anchored to the surface of NC8 cells. In vitro, it was found that the rChIL17B protein inhibited the proliferation of the infectious bronchitis virus (IBV) through activation of nuclear factor kappa B (NF-κB) and the JAK (Janus kinase)-STAT (signal transducers and activators of transcription) signaling. Moreover, to evaluate the immunoadjuvant activities of NC8-ChIL17B, 40 three-day-old specific pathogen-free (SPF) chickens were divided into four groups. Three groups were orally vaccinated with fresh NC8, NC8-ChIL17B, and phosphate buffered saline (PBS), along with the infectious bronchitis virus vaccine, and the other group was the PBS-negative control. The results of the IBV-specific antibody titer and the concentration of the cytokines IL-2, IL-4, IL-6, and interferon gamma (IFN-γ) in sera, as well as the concentration of secretory immunoglobulin A (sIgA) in the tracheal and small intestinal mucosa, the number of cluster of differentiation 4 positive (CD4⁺) and cluster of differentiation 8 positive (CD8⁺) T cells in the blood, and the expression of immune-related genes all indicated that NC8-ChIL17B efficiently enhanced the humoral and cellular immune responses to IBV vaccine. Moreover, the viral loads in the NC8-ChIL17B- and IBV-vaccinated group were significantly lower than in the control groups, suggesting a significant promotion of the immunoprotection of IBV vaccination against the virulent IBV strain. Therefore, ChIL-17B is a promising, effective adjuvant candidate for chicken virus vaccines.

In order to develop an effective and safe immunomodulator to enhance the antimicrobial bioactivity and immunity of animals against infectious bacterial diseases, a recombinant plasmid pGAPZαA-IL2-B co-expressing pig interleukin-2 (PIL-2) and fused bovine cathelicidin (FBC) genes were constructed using the 2A self-cleavage technique. After being expressed in Pichia pastoris strain SMD1168, the recombinant yeast was administered orally to 5-week-old female ICR mice. The control mice were similarly dosed with P. pastoris with a blank plasmid or FBC recombinant plasmid alone. At 28 days post-treatment, the mice were challenged intraperitoneally with virulent strains of either E. coli or S. aureus. Compared with the control groups, the mice that received recombinant yeast co-expressing PIL-2/FBC manifested significant increases in the number of leukocytes, CD4+ and CD8+ T cells, IgG, and the gene expressions of TLRs(TLR1,4,6,9), antimicrobial peptides(CRP4 and CRAMP) and cytokines (IL-2, 4, 6, 7, 12, 15, 23, IFN-γ, and TNF-α) in the blood. Furthermore, the treated mice displayed significantly higher survival than the other two control groups after the challenge. These results suggest that the antimicrobial activity and immunity of animals can be effectively enhanced by the in vivo co-expression of IL-2 and the FBS gene, which can facilitate the development of new immunopotentiation molecules to overcome the infection of antibiotic-resistant bacteria.

In order to develop a novel and effective immunoregulator to enhance both the immune response and antimicrobial function, a recombinant eukaryotic expression plasmid-pVAX1 co-expressing fusion cathelicidin antimicrobial peptides (CAMPs) and fusion porcine interleukin-4/6 gene (IL-4/6) was constructed and encapsulated in chitosan nanoparticles (CS-VAP4/6), prepared by the ionotropic gelation method. Four-week-old female Kunming mice were divided into three groups and intramuscularly injected, respectively, with CS-VAP, CS-VAP4/6, and CS-pVAX1. On 28 days post-inoculation, the mice were challenged by intraperitoneal injection with Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 25922); IgG, IgG1 and IgG2a, CD4+, and CD8+ T cells increased significantly in the VAP- and VAP4/6- treated mice, detected by ELISA and flow cytometry, correspondingly (p < 0.05). As analyzed by qPCR, expression levels of Toll-like receptor (TLR) 1, TLR4, TLR6, TLR9, IL-1, IL-2, IL-4, IL-6, IL-7, IL-12, IL-15, IL-23, Tumor Necrosis Factor (TNF)-α, and Interferon-gamma (IFN-γ) genes were also significantly up-regulated in comparison with those of the control mice (p < 0.05). Their immunological markers were elevated significantly to different degrees in CS-VAP4/6-treated mice compared with CS-VAP in different days post-inoculation (p < 0.05). After challenge with E. coli and Staphylococcus aureus, most of the VAP- and VAP4/6- treated mice survived, and no symptoms of bacterial infection were observed. In contrast, 80% of control mice died of infection. Among the treated groups, VAP4/6 had a stronger resistance against challenge with E. coli infection. These results demonstrated that the fusion gene of antimicrobial peptide and interleukin-4/6 has the promising potential as a safe and effective immunomodulator for the control of bacterial infections.

Feline calicivirus, herpesvirus, and panleukopenia virus are major infectious viruses to cause serious diseases in cats, yet their current trivalent vaccines have limitations in immune efficacy and duration of protection. This study assessed the immune-enhancing effects of novel adjuvants (feline IL-15, IL-23, and metabolic modulators) on vaccine responses. Forty mice were randomly assigned to four groups: Group A (composite adjuvants), Group B (metabolic regulatory molecules and Mn adjuvant), Group C1 (Mn adjuvant), and Group C2 (blank commercial vaccine). Results showed that Group A had significantly higher neutralizing antibody titers against calicivirus post-booster immunization, while both Group A and B exhibited enhanced antibody responses against herpesvirus and parvovirus. Notably, Group A displayed increased proportions of memory T cells, follicular B cells, and activated B cells. These findings suggest that the combination of feline IL-15, IL-23, and metabolic modulators are safe and effective immunoadjuvants for the trivalent feline vaccines to promote immune cell differentiation and antibody production, thus representing a promising strategy to optimize vaccine efficacy.

Drug resistance in economic animals to pathogens is a matter of widespread concern due to abuse of antibiotics. In order to develop a safe and economical immunopotentiator to raise the immunity and antibacterial response as a replacement for antibiotics, a recombinant yeast co-expressing pig interleukin-17 (IL-17) and IL-22 was constructed and designated as Po1h-pINA1297-IL-17/22. To evaluate the immunoregulator activities of Po1h-pINA1297-IL-17/22, two experiment groups (oral inoculation with Po1h-pINA1297 or Po1h-pINA1297-IL-17/22) and a negative control group (PBS) were set up using 4-week-old female BALB/c mice (10/group). The level of cytokines, including IL-2, IL-4, IL-10, and IFN-γ, were detected by ELISA, and the circulating CD4+ and CD8+ lymphocytes were quantified by flow cytometry. The IgG and secretory IgA (SIgA) levels in both small intestine and fecal matter were also measured by ELISA. The results indicated that the IgG antibody titer and SIgA concentration increased significantly in the Po1h-pINA1297-IL17/22 group in comparison with the controls (p < 0.05) and so did the cytokine levels in the serum (IL-2, IL-4, IL-10, and IFN-γ). In addition, CD4+ and CD8+ T cells were also obviously elevated in the Po1h-pINA1297-IL17/22 group on 35th day (p < 0.05). After challenge with pathogenic Salmonella typhimurium, the Po1h-pINA1297-IL17/22 group showed a relatively higher survival rate without obvious infectious symptoms. On the contrary, the mortality of control group reached 80% due to bacterial infection. As for the piglet experiment, 30 healthy 7-day piglets were similarly attributed into three groups. The oral inoculation of piglets with Po1h-pINA1297-IL17/22 also markedly improved the growth performance and systemic immunity (up-regulations of IL-4, IL-6, IL-15, IL-17, IL-22, and IL-23). Overall, the results indicated that Po1h-pINA1297-IL17/22 effectively promoted the humoral and cellular immunity against bacterial infection. These proved the promising potential of Po1h-pINA1297-IL-17/22 to be a potent immunopotentiator for the prevention of microbial pathogen infections.

The feline calicivirus, herpesvirus, and panleukopenia viruses are major infections that cause serious diseases in cats; however, current trivalent vaccines have limitations in immune efficacy and their duration of protection. This study assesses the immune-enhancing effects of novel adjuvants (feline IL-15, IL-23, and metabolic modulators) on vaccine responses. Forty mice were randomly assigned to four groups: Group A (composite adjuvants), Group B (metabolic regulatory molecules and Mn adjuvant), Group C1 (Mn adjuvant), and Group C2 (a blank commercial vaccine). The results showed that Group A had significantly higher neutralizing antibody titers against calicivirus post-booster immunization, while both Groups A and B exhibited enhanced antibody responses against the herpesvirus and panleukopenia viruses. Notably, Group A displayed increased proportions of memory T cells, follicular B cells, and activated B cells. These findings suggest that the combination of feline IL-15, IL-23, and metabolic modulators are safe and effective immunoadjuvants for trivalent feline vaccines to promote immune cell differentiation and antibody production, thus representing a promising strategy to optimize vaccine efficacy.