An adjuvant is an agent that stimulates the immune system and increases the host response to an antigen without itself conferring a specific antigenic effect (Bowersock and Martin, 1999). Effective adjuvants utilize multiple compounds and mechanisms to achieve the desired immunological enhancement such as long lasting antigen depots, immunological presentation of vaccine antigens, and induction of T lymphocyte responses (Reed et al., 2009). As of present, much progress has been made to develop novel adjuvants that augment humoral and cell-mediated immunity by enhancing efficacy of vaccines (Bowersock and Martin, 1999; Newman and Powell, 1995), which is particularly crucial for commercial poultry industries in tackling economically important diseases such as Eimeria protozoa-induced avian coccidiosis (Shirley and Lillehoj, 2012).

Examples of adjuvants used with variety of vaccines include Montanide incomplete Seppic adjuvant (ISA) series, ISA 70 VG (ISA 70) and ISA 71 VG (ISA 71), and Quil A, cholesterol, dimethyl dioctadecyl ammonium bromide, and Carbopol (QCDC) adjuvant complex, whereby the former is a water-in-oil emulsion and the latter is composed of Quil A, cholesterol, dimethyl dioctadecyl ammonium bromide (DDA), and Carbopol (Aucouturier et al., 2006; Cox et al., 2003; Dominowski et al., 2009).

ISA 70 and ISA 71 have been successfully applied to enhance immune response against pathogens of poultry, cattle, and small ruminants (Dupuis et al., 2006). Previous studies have shown that either ISA 70 or ISA 71 in conjunction with the recombinant profilin, which is an Eimeria specific antigen, enhances protective immunity against experimental avian coccidiosis in chicken (Jang et al., 2010; Jang et al., 2011; Lee et al., 2011). Also, other than the Montanide ISA series, improvement in vaccine responses against a variety of veterinary pathogens has been demonstrated in the use of QCDC adjuvants (Dominowski et al., 2009). It has been understood that Carbopol, such as dextran, polyethelyne, glycol, or polyacrylic acid that has been added to the QCDC, improves the solubility of DDA and thus makes the final formulation a highly effective adjuvant. Further incorporation of Bay R1005, a synthetic glycolipid analogue, endows the complex with the ability to stimulate both Th1-and Th2-type immunity, giving the QCDCR adjuvant a broad range of desirable immune enhancing characteristics (Dominowski et al., 2009).

Chickens infected with Eimeria spp. commonly develop protective immunity against reinfection by the homologous parasite, which makes immunization with parasite vaccines a viable method to control coccidiosis (Lillehoj et al., 2000a). Basically, profilin in Eimeria stimulates cell-mediated immunity against experimental avian coccidiosis, which is what makes it a promising vaccine candidate (Lillehoj et al., 2000b; Yarovinsky et al., 2005). In addition, the evidence of profilin shown as a potential immunogenic protein has been published in two studies that, when Montanide ISA series and QCDC formulations were used, efficacy of the profilin vaccine was improved and thus lead to more protective immunity against coccidiosis (Jang et al., 2013). However, molecular signatures related to immune-stimulatory activities of these adjuvants have not been analyzed comparatively.

Therefore, in this study, we attempted to comparatively analyze integrated microarray data from two vaccine adjuvants in experimental coccidiosis model, i.e. ISAs and QCDCs, which confer protective immunity in combination with parasitic antigen, and sought to identify common gene ontology (GO) and pathways that are targeted by these adjuvants. The idea of integrating datasets from independent, but related, sources for a comparative analysis for this study stemmed from the increasing amount of research that has used microarray technology based datasets from publicly available repositories such as Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo), ArrayExpress (http://www.ebi.ac.uk/arrayexpress), and Stanford Microarray Database (http://smd.princeton.edu).

For the comparative analysis of the effects of two different adjuvant types, we collected microarray datasets from the independent trials for each adjuvant groups, ISAs and QCDCs, and identified GO terms and pathways modified by the DEGs.

In the present study, the most enriched categories of BP affected by two adjuvants, showing 6 categories that are commonly affected by two adjuvants. But, further detailed analysis revealed that the categories consisting of the same BPs in both adjuvants are quite specific, rather than common. For example, in the term of “Response to stimulus”, no BPs was identified simultaneously in the two different adjuvant groups (Table 3). Generally, the QCDC adjuvants showed effects on innate immune response and immune response to external stimulus, such as “Response to toxin” and “Response to bacterium”. For the immune response to toxins, a good humoral immune response is required in the adjuvants. Most adjuvants on the market today mainly activate the humoral immune response. Nevertheless, it is obvious that the introduction of cell-mediated immune response by adjuvants is beneficial to control bacterial infection, where cytotoxic T cells and Th1 cells mediate these responses (Leclerc, 2003). In this sense, ISCOM, a component of QCDC adjuvants, has been known to generate both strong humoral and cellular immune responses in an extensive range of animal species (Drane et al., 2007). The ISCOMATRIX adjuvant has also shown to be safe and well tolerated as well as immunogenic, generating both antibody (Ab) and CD4⁺ and CD8⁺ T cell responses (Drane et al., 2007). Also, DDA is reported to promote both strong cell mediated immune responses and humoral immune responses, which are essential for the induction of protective immunity against most diseases (Davidsen et al., 2005).

As for the adjuvants of ISA series, these modified the BPs that regulate cell movement such as cell locomotion, chemotaxis, and cell migration. Although ISA and QCDC adjuvants modified biological functions related to the regulation of cell death and apoptosis, they showed a different manner in mode of action. The effects of ISA adjuvants on cell death were both of positive and negative regulation while the QCDC adjuvants had no function in regulating cell death negatively. Such apoptotic and necrotic effects are reported to be associated with emulsion-type vaccine adjuvants such as the ISA series and QCDC whereby both are composed of various oils and surfactants (Yang et al., 2004). The mechanism behind the surfactants is that their amphiphilic nature adsorbs not only the oil/water interface in the emulsion, but also biological membranes, resulting in an increase in surface pressure that thus lead to apoptosis and necrosis of the cells (Yang et al., 2004). This suggests that the surfactants in ISA adjuvants and Quil A in QCDC adjuvants could induce apoptosis or cell death during immunization. In the results of pathway analysis, “Adherens junctions” and “Focal adhesion” pathways were the two main types of junction that were modified by ISA adjuvants, which are known to mediate adhesion in epithelial cells (Yeatman, 2004). Cell-cell adherens junctions are important for maintaining tissue architecture and cell polarity and can limit cell movement and proliferation. Cell-matrix adhesions, also called focal adhesions, play essential roles in important BPs including cell motility, cell proliferation, cell differentiation, regulation of gene expression and cell survival. Collectively, the ISA adjuvants may have effects on cell mobility and mitigate cytotoxicity through the cellular junction pathways.

In addition, lysosomal and endocytic reactions were also modified by ISA adjuvants. In the case of lysosomes, the lysosomal enzymes’ important role in the inflammatory process has been documented in previous studies in arthritic condition induced by injection of Freund’s complete adjuvant (FCA) in rats (Anderson, 1970; Reddy and Dhar, 1988; Geetha and Varalakshmi, 1999). When endocytosis takes place, adjuvants first exert their targeting mechanism by binding to antigens and the adjuvant–antigen complex is delivered to antigen-presenting cells (APCs) to form aggregates that are then engulfed by APCs to form endosomes (Cox and Coulter, 1997; Zamze et al., 2002). More effective targeting can be achieved by using adjuvants with residues that are recognized by receptors on APCs such as the mannose receptor that belongs to the endocytic pattern recognition receptors (PRRs) that bind compounds containing mannose, N-acetylglucosamine, or fucose residues and sulfated oligosaccharides (Stahl and Ezekowitz, 1998).

The ISA adjuvants’ functions related with cellular response to oxidative stress shown from our result can be used to partly explain the immune-stimulatory properties of adjuvants that induce inflammation and oxidative stress in the host animal (Kumar and Roy, 2007). An example can be drawn from previous studies that showed that intra-articular injection of a well-known adjuvant, i.e., FCA, induced inflammation as well as immune response and produced features that resembled rheumatoid arthritis in humans. Such acute inflammatory response induced by FCA is associated with leukocyte infiltration, mast cell activation, and release of cytokines and free radicals (Nigrovic and Lee, 2005; Yamada et al., 2006). Despite the shared similarity both ISA adjuvants and FCA and FIA (Freund’s incomplete adjuvant) have with respect to antibody responses, the fact that less inflammatory response is induced by ISA adjuvants relative to FCA and FIA needs to be taken into account (Johnston et al., 1991; Leenaars et al., 1994; Leenaars et al., 1995; Leenaars et al., 1998).

In the pathway analysis, QCDC adjuvants altered genes involved in “CAMs” pathway. Inflammatory and immune responses involve adhesive interactions that mediate migration of cells to sites of inflammation and the effector functions of cell within the lesions. Therefore, CAMs, as complex proteins expressed on the cell surface and precisely regulated by cytokines and other biologic response modifiers, are versatile mediators of the complex dynamics of cell interactions in the inflammatory/immune response (Crawford and Watanabe, 1994).

In conclusion, the integrative analysis of microarray datasets from the treatment of two different types of adjuvants, ISA series and QCDC formulations, has provided discriminative and common molecular signatures as well as aided the distinction of immunological functions of the two adjuvant groups in chicken. Although much progress has been undertaken in the formulation of novel adjuvants that augment the immunogenicity of protein vaccines, very little information is available in poultry (Baldridge and Ward, 1997; Gupta and Siber, 1995; Richards et al., 1996). Therefore, this study could contribute to the selection of appropriate adjuvants according to the types of vaccines or diseases as well as the development of efficient vaccine adjuvants in poultry industry. Furthermore, the potent immune adjuvants might promise another usefulness of vaccine as therapeutic agents rather than prophylactic agents.

Limitations such as small sample size and limited case numbers are weaknesses of this study; however, to our knowledge, this is the first integrative approach to identify molecular signatures impacted by adjuvant in livestock, especially in chicken. Therefore, this study could serve as an informative framework to integrated microarray data to identify molecular signatures that can be used to develop precise adjuvants based on their immunological functions.