Introduction
Immune enhancers are substances that are unrelated to antigens and can augment the body´s immune response when used alone or simultaneously with antigens, whereas immune adjuvants are substances that can be used in combination with vaccines to improve the immune response to vaccine antigens [1]. Studies have shown that several substances exist with immuneenhancing effects, such as oil-water emulsions, microorganisms and their metabolites, nucleic acids and their analogs, cytokines, propolis, immunostimulatory complexes, liposomes, and artificial chemically synthesized molecules. However, these substances face many challenges, such as the induction of disorders, allergic reactions, hypersensitization, and cytotoxicity [2, 3]. In recent years, biosafety and green food safety have placed high demands on future biologics, especially on the biosafety of preventive biological agents. Therefore, research has focused on natural peptides and proteins derived from animals as immune adjuvants or enhancers. Natural immune peptides, which are mostly peptides with natural immunomodulatory activity obtained from animals or food proteins [4], can regulate specific and non-specific immune functions by affecting the growth and development of immune organs (tissues), proliferation and transformation of lymphocytes, and/or release of immune molecules (cytokines and antibodies). Natural immune peptides play important roles in maintaining immune function and health [5].
As they are natural proteins derived from animals, no biosafety or food safety concerns exist. Thymic peptides are a class of peptides secreted by the epithelial cells of the thymus and are involved in immune development and regulation. Among them, thymosin Tα1 is a short peptide consisting of 28 amino acids and is highly conserved among different animal species [6, 7]. Thymosin Tα1 has important immunomodulatory activities [8]: the peptide can promote T cell proliferation, and secretion of various cytokines by natural killer (NK) cells, such as interleukin-2 (IL-2) and interferon-α (IFNα). Moreover, thymosin Tα1 is an immune enhancer against CD3 +-induced apoptosis and enhances NK cell function, inhibits tumor growth, and performs a series of other immunomodulatory functions [9]. At present, the thymus peptides used in veterinary clinical practice are mostly extracted from bovine thymus tissue and are mostly a mixture of small peptides, which have various concerns, such as large content of impurities, low Tα1 content (about 0.1%), poor treatment effect, requirement for large dosage, serious side reactions, and other problems; furthermore, the extraction of peptides from the thymus is challenging due to low yield [5]. Purified synthetic Tα1 products are also available; although they are expensive. Therefore, exploring the use of genetic engineering methods to obtain highly active Tα1 at low cost has become the first choice. IFN, discovered in 1957 as a substance that interferes with viral reproduction, is a natural protein produced by the immune system of most vertebrates when resisting viruses, bacteria, parasites, tumor cells, and other foreign substances. IFN-γ is mainly produced by activated T cells and natural killer cells and is involved in immune regulation and inflammatory responses, with critical immunomodulatory and antiviral effects. In recent years, some studies have confirmed that the combined application of interferon and thymosin synergistically promotes T cell growth, proliferation, differentiation, and activation of NK cell activity, which can enhance the body´s immune capacity [10, 11]. The combination of IFNs and thymosin as natural immune enhancers or immune adjuvants has brought new hope for the treatment of viral and neoplastic diseases [12].
However, natural peptides obtained from animal tissues face many difficulties, such as difficulties in extracting sufficient amounts and purification. Moreover, the use of genetic engineering to express natural proteins is challenging. Many scientists have used Escherichia coli and yeast to express natural proteins; however, their applications are limited by the presence of substances such as endotoxins. Food-grade lactic acid bacteria genetically engineered to express natural proteins have become the first choice because they do not contain endotoxins. Therefore, they can completely replace E. coli as live vectors for delivering proteins of interest in food or in vivo. They are the most commonly used gram-positive host bacteria and gene expression systems. Lactic acid bacteria have been widely and successfully used in food fermentation for thousands of years [13].
Most lactic acid bacteria species are beneficial to animals, plants, and humans; as probiotics, they are not only endotoxinfree, but their cell wall and nucleic acid and other components possess immune adjuvant activity [14]. Among the many benefits of probiotics, their ability to modulate the immune system occupies an important position [15], and several studies have provided clear evidence of the modulatory properties of certain probiotics on the immune system [16]. Furthermore, some probiotics can enhance the non-specific immune functions of the organism, including the phagocytic activity of innate immune cells and the cytotoxicity of NK cells [16], and also enhance the specific immune response function of the organism, promoting the stimulation of B cells to secrete specific IgA and IgG and activating helper T lymphocytes and macrophages [17, 18].
Amidst the current critical scenario of food safety and biosafety, utilizing food-grade lactic acid bacteria, with their inherent safety features, becomes the preferred choice for developing immune adjuvants and enhancers. This choice aligns with their safety and probiotic functions. In the present study, recombinant Lactococcus lactis (r-L. lactis-Tα1-IFN) expressing chicken thymosin Tα1 and chicken IFN-γ fusion protein was constructed using food-grade Lactococcus lactis NZ3900 and an antibiotic-free labeled expression vector. In vitro and in vivo experiments demonstrated that the lysis mixture of r-L. lactis-Tα1-IFN had good immune booster and immune adjuvant activities. The findings of this study provide a strong foundation for the research and application of green food-safe grade vaccine adjuvants or immune enhancers.