Introduction
Vaccine adjuvants are used because they affect antigen absorption, thereby increasing the longevity of disease protection, which leads to increased antibody titres (Hassan et al., Reference HASSAN, KHALIQUE, PASHA, AKRAM, MAHMOOD, SAHOTA, IMRAN and SALEEM2017). Adjuvants increase the seroconversion rate in the population and allow the use of smaller doses of the antigen in the vaccine, which ultimately reduces cost (Coffman et al., Reference COFFMAN, SHER and SEDER2010). There are a wide variety of adjuvants used in vaccines, e.g. mineral or other oils and immunostimulating complexes (ISCOMs) (Abbas et al., Reference ABBAS, IQBAL, ABBAS, KHAN and KHAN2017). Oligonucleotides containing CpG dinucleotides (CpG ODNs) were among the first molecular structures explicitly designed to model a pathogen associated molecular pattern (PAMP) (Krieg et al., Reference KRIEG, YI, MATSON, WALDSCHMIDT, BISHOP, TEASDALE, KORETZKY and KLINMAN1995). These molecules stimulate certain immune cells, like plasma dendritic cells (pDC) and lymphocytes (Shirota et al., Reference SHIROTA, TROSS and KLINMAN2015; Gul et al., Reference GUL, KHAN, FAROOQ, NIAZ, AHMAD, KHATOON, HUSSAIN, SALEEMI and HASSAN2017). These molecules stimulate immune cells of the host only when they are unmethylated. However, methylation, as well as inversion of CpG sequence to GpC, leads to the stoppage of their immunostimulatory effect (Häcker et al., Reference HÄCKER, REDECKE and HÄCKER2002). The CpG ODNs are acknowledged as the PAMPs most frequently recognised by TLR 9, which are present on B-cells and DCs in vertebrates (Weiner et al., Reference WEINER, LIU, WOOLDRIDGE, CHRISTOPHER and ARTHUR1997). Different CpG DNAs have inhibitory immune effects on monocytes of various species, which prevent many pathological problems associated with TLR-9 activation (Li et al., Reference LI, CAO, WANG, XIANG, LU, ZHAO, ZHENG and ZHOU2011).
Overall, there are four types of CpG ODNs, which are available synthetically. These include oligonucleotide type K, which is also known as ODN type B and oligonucleotide type K, which helps in the differentiation of pDC resulting in the production and proliferation of immunoglobulin M (IgM) antibodies by B cells and the production of tumour necrosis factor alpha (TNF-alpha). A third type is ODN-C, which is found in both early and late endosomes hence why it resembles both type K and type D. Finally, type A ODNs produce INF-alpha from pDC and secrete IL-6 by the activation of B cells (Bode et al., Reference BODE, ZHAO, STEINHAGEN, KINJO and KLINMAN2011). Cellular immune response (the ‘Th1 immune response’) is valuable in countering many pathogens (Petrovsky and Aguilar, Reference PETROVSKY and AGUILAR2004; Khan et al., Reference KHAN, EL-ASHRAM, LIU, KHAN, AYAN, LIU, WANG, TANG, SUO and HASSAN2016). CpG ODNs are considered to be immune stimulants because they trigger the Th1 immune response, causing the activation of macrophages and natural killer cells (Oxenius et al., 1999). This results in the release of cytokines such as IL and TNF (Chu et al., Reference CHU, TARGONI, KRIEG, LEHMANN and HARDING1997), which are responsible for the pro-inflammatory response (Romagnani, Reference ROMAGNANI2000).
With the help of an effective adjuvant, mean titres of antibodies can be achieved by using lower doses of vaccine (Coffman et al., Reference COFFMAN, SHER and SEDER2010). When CpG ODNs are used they magnify cellular as well as humoral immune responses against different tumours, pathogens and allergens (Shirota et al., Reference SHIROTA, TROSS and KLINMAN2015). Immunostimulatory ODN containing CpG motifs are potent molecules, which enhance the adaptive as well as innate immune system because they mimic bacterial DNA (Zaheer et al., Reference ZAHEER, HUSSAIN, RAHMAN, YOUNAS, ZAHEER, ABBAS and NASIR2017). As a result, they provide protection against tumours, infectious diseases and allergies (Zhang and Gao, Reference ZHANG and GAO2017). CpG ODNs are preferred over other adjuvants as they have advantages over the others because these induce antigenic specific immunity against co-administered vaccines and are well tolerated in healthy individuals. The phosphorotioate backbone of the compound increases half-life and is resistant to nuclease digestion with little or no tissue reactions, which may not be easily differentiated from lesions of aflatoxicosis (Hussain et al., Reference HUSSAIN, KHAN, SALEEMI, KHAN and RAFIQUE2016). The CpG ODNs stimulate TLR-9, resulting in rapid activation of innate immunity (Scheiermann and Klinman, Reference SCHEIERMANN and KLINMAN2014). In chickens, immunostimulatory CpG ODNs are ligands of TLR21 (Singh et al., Reference SINGH, ALKIE, HODGINS, NAGY, SHOJADOOST and SHARIF2015). The TLR21 from chickens and geese are similar to each other and both can be stimulated by the CpG ODNs (Qi et al., Reference QI, YAN, CHEN, CHEN, WANG, JIA, ZHU, LIU, LIU, YANG, SUN, WU, CHEN, JING and CHENG2016), being equivalent to mammalian TLR9 (He et al., Reference HE, GENOVESE, SWAGGERTY, MACKINNON and KOGUT2012).
CpG ODNS in Avian influenza vaccines
Vaccines for avian influenza virus are generally administered through mucosal routes, namely oral, ocular and intranasal routes. The CpG ODNs may be used as an effective and safe mucosal vaccine adjuvant and provide protection against infections in the upper respiratory tract. It provides both local and systemic immune response. When administered through the mucosal route the use of CpG ODNs in influenza vaccine prevents tissue damage and the risk of inflammation, even with high dose applications which induce an elevated level of CD8+ T-cells in nasal mucosa and the number of B-cells in the spleen (Kodama et al., Reference KODAMA, ABE, HIRANO and SUZUKI2006). For avian influenza virus (AIV) CpG ODN-F3 is more safe, potent and cost-effective compared to other adjuvants because of the presence of the partial phosphoro-thioate backbone. This means that CpG ODN-F3 can be used for inactivated AIV vaccine of chickens as a reasonable nasal adjuvant compared to others, including CpG ODN-F1 (Fu et al., Reference FU, LIANG, KANG, LIN, YU and YANG2013).
The immunostimulatory effect of plasmids along with different copy numbers of CpG ODNs was studied in chickens immunised with avian influenza vaccine. The plasmid containing 64 copies of CpG motif (64CpG plasmid) considerably increased mRNA expression of TLR-7, TLR-3 and IFN-gamma in splenocytes of chickens. Chickens administered with H5N2 avian influenza vaccine (V52) along with 64CpG plasmid showed elevated haemaglutination inhibition (HI) titres, peripheral blood mononuclear cell multiplication and mRNA expression of TLR-3, TLR-7, TLR-21, IFN- and IFN- in chicken splenocytes compared to those administered with V52 alone (Wibowo et al., Reference WIBOWO, ANGGORO, AMANU, WAHYUNI, UNTARI, ARTANTO and ASMARA2017). Additionally, a significantly greater survival rate of around 100% against a highly virulent strain H5N1was present in chickens vaccinated with the V52 + 64CpG ODNs plasmid (Hung et al., Reference HUNG, TSAI, WANG, LI, HUANG, LIEN and CHAUNG2011).
Vaccination against AIV with virosomes adjuvant plus CpG ODNs produced an enhanced haemaglutination inhibition antibody titre and IgG and IgA serum antibody response. These types of responses can be improved by addition of CpG ODNs along with virosomes (Mallick et al., Reference MALLICK, PARVIZI, READ, NAGY, BEHBOUDI and SHARIF2011; Fatima et al., Reference FATIMA, KHAN, AHMAD, MUHAMMAD, KHWAJA, KHAN, ANWAR, AHAD and MAHMOOD2017). Addition of CpG ODNs in an intranasal vaccine against H5N2 AIV increased local immunostimulatory cells in the respiratory tract and enhanced immune responses in relation to lymphocytes, particularly the T cells and IgA and IgG (Xiaowen et al., Reference XIAOWEN, QINGHUA, XIAOFEI and QIAN2009). Significant increases in IgG and IgA antibodies levels in the tissues of the trachea and throat and elevated haemaglutination inhibition (HI) titres in the serum of birds were obtained between the 3rd and 7th weeks of inoculation. This was observed in 10-day old ducks vaccinated through the intra nasal route with inactivated AIV in combination with CpG-ODNs (Kang et al., Reference KANG, WANG, YU and YANG2012).
Use of CpG ODNs combined with the AIV vaccine against H9N2 showed an increase in antibody titres and TLR21 pathways, which are involved in the maturation of Ig (Singh et al., Reference SINGH, ALKIE, ABDELAZIZ, HODGINS, NOVY, NAGY and SHARIF2016). CpG ODNs mixed with oil emulsion vaccine before administration produced greater IgG titre, HI and interferon-C concentrations, but lowered the production of interleukin-10.
The addition of CpG ODNs prior to inoculation may enhance the humoral as well as Th-1 type immune response as compared to separate incorporation of the adjuvant (Wang et al., Reference WANG, SHAN, MING, LIU, DU and JIANG2009). By the deliverance of the H5N1 inactivated avian influenza virus (IAIV) via the nasal route in vivo, tissues of the upper respiratory tract (URT) exhibited some enhanced mRNA expression of interleukin-6, interleukin -12, interferon-gamma and TLR-21 (Fu et al., Reference FU, LIANG, KANG, LIN, YU and YANG2013). CpG ODNs increased the transfer of H9N2 whole inactivated virus (WIV) via trans-epithelial dendrites (TEDs) of nasal mucosa DCs; a fundamental mechanism for a favourable adaptive immunostimulatory responses. CpG ODNs assisted H9N2 WIV in recruiting DCs to the cells of the nasal epithelium and formed TEDs to catch luminal viruses. The CD-103 dendritic cells participated in this procedure, and chemokine CCL-20 from the nasal epithelia played a vital role in driving DCs staffing and trans-epithelial cells formation. Virus-containing DCs swiftly migrated towards the draining cervical lymph-node for antigen presentation (Qin et al., Reference QIN, YIN, YU, HUANG, WANG, LIN and YANG2015).
ODN induced different types of responses through different routes of administration along with AIV vaccine. Intramuscular administration of non-capsulated CpG ODNs in conjunction with AIV vaccine elicited an elevated antibody response compared to the encapsulated CpG ODNs via the same route (Singh et al., Reference SINGH, ALKIE, ABDELAZIZ, HODGINS, NOVY, NAGY and SHARIF2016). For AIV, CpG-ODNs significantly minimised virus shedding. Birds treated with CpG-ODNs 1826 in conjunction with a TLR ligand, were highly efficient for reducing oral shedding of AIV (Barjesteh et al., Reference BARJESTEH, SHOJADOOST, BRISBIN, EMAM, HODGINS, NAGY and SHARIF2015).
Specially synthesised CpG-ODNs can inhibit gene replication and hence produce a significant antiviral response against AIV. Such structure is called antisense oligonucleotide (AS-ODNs), which are specific for the RNA binding site of nucleoprotein (NP) present on the virus. AS-ODN treatments caused a reduction in replication of AIV virus in MDCK-cells and provided protection against the highly lethal H5N1 strain and provided good responses in RT-PCR, titres and indirect immuno-fluorescence assays (IFA) in comparison with a viral control. By targeting AIV NP-genes, AS-ODNs can potentially inhibit the replication of H5N1 (Zhang et al., Reference ZHANG, WANG, ZHAO, LIANG, GAO, YANG, QIN, WANG and XIA2011). Ducks immunised with inactivated AIV H9N2 combined with CpG-ODNs and a high glucose dose showed enhanced local and systemic immune responses after challenge by specific virus strains (Kang et al., Reference KANG, WANG, YU and YANG2013).
Effect of CPG ODNS against Newcastle disease
The proliferation of immune cells and peripheral blood mononuclear cells (PBMCs) is higher in chickens receiving CpG ODNs with Newcastle disease (ND) vaccine given via the nasal route. It has been reported that CpG ODNs has greater adjuvanticity than guanosine phosphate cytosine oligodeoxynucleotide (GpC ODNs) delivered by the same route, i.e. intranasally (Zhang et al., Reference ZHANG, ZHANG, LI, CAO, TIAN and ZHOU2008; Han et al., Reference HAN, GAO, WU, XIAO, WANG, LIU, TONG, HAO, ZHANG, DANG and YANG2017). IgG antibodies in serum and lymphocyte proliferation in serum were significantly higher in chickens which received ODNs as compared to those receiving only vaccine against ND (Linghua et al., Reference LINGHUA, XINGSHAN and FENGZHEN2007). Even at low concentration, CpG ODNs induced immunostimulatory effects. Two groups of birds were compared. The birds which received ND virus plus CpG ODNs had higher titres and protection than those which received just CpG ODNs after challenge with a virulent strain of ND, which caused the disease.
CpG ODNs are potent adjuvants, which can protect chickens from ND and increase IgG antibodies by three-fold as compared to commercial vaccine (Bode et al., Reference BODE, ZHAO, STEINHAGEN, KINJO and KLINMAN2011). CpG ODNs protect the chickens from Newcastle disease by producing pDCs, which ultimately produce interferons (Zhang and Wang, Reference ZHANG and WANG2005). PDCs can be produced by CpG ODNs, which ultimately produces INF-alpha and controls ND virus, and are effective against tumours (Kemp et al., Reference KEMP, ELZEY and GRIFFITH2003).
Effect of CPG ODNS against infectious bursal disease
The use of CpG ODNs combined with DNA vaccines against infectious bursal disease virus (IBDV) is not always effective (Roh et al., Reference ROH, SUNG and KWON2006). CpG ODNs and LPS are stimulants of TLRs and stimulate innate immunity, resulting in the expression of interferon regulatory factor 7 (IRF7) and INF type 1 (Alkie et al., Reference ALKIE, ST. PAUL, BARJESTEH, BRISBIN and SHARIF2015). The inoculation of CpG ODNs along with the cationic microparticles and DNA vaccines against IBDV result in the influx of T cells and a reduction in antigen load into the bursa of Fabricious (Negash et al., Reference NEGASH, LIMAN and RAUTENSCHLEIN2013). The up regulation of INF-gamma and IL-10 can be enhanced by CpG ODNs and this can increase antigen presentation through MHC class 2 and CD80 in the cells of bursa of Fabricious (Paul et al., Reference PAUL, PAOLUCCI, READ and SHARIF2012).
Effect of CPG ODNS against avian leucosis virus
Avian leucosis virus type E (ALV-E) is characterised by emaciation in chickens, especially in laying hens (Koo et al., Reference KOO, JEON, BAE, MO, KIM and MO2016). Immunosuppressive lesions have been significantly reduced in CpG ODNs adjuvanted vaccinated birds as compared to those receiving Freund's adjuvant vaccine (Dou et al., Reference DOU, LI, CHENG, ZHAO, LIU, CUI, LIU, JING and GUO2013; Yu et al., Reference YU, GUO, ZHANG, LUO, TIAN and LI2017). When CpG ODNs are used against the gp85 protein antigen of ALV-A, better antibody titres and enhanced protection was observed. There were fewer cases of cloacal detoxification, viremia and death of birds (Zhang et al., Reference ZHANG, LI, ZHANG, SUN, CHENG, LIU, ZHAO, REN and GUO2015) in CpG ODN adjuvanted treated chickens.
Effect of CPG ODNS against infectious laryngotracheitis
In ovo delivery of CpG ODNs protects chickens against the threat of infectious laryngotracheitis virus (ILTV) due to expression of IL-1 beta, leading to macrophage recruitment in the lungs (Thapa et al., Reference THAPA, CADER, MURUGANANTHAN, NAGY, SHARIF, CZUB and ABDUL-CAREEM2015).
Effect of CPG ODNS against infectious bronchitis
Prevention of the expression of the gene of cytokine IL-6 and increases in IL-8, INF-gamma and macrophage inflammatory protein (MIP-1 beta) have been associated with the inhibition of viral replication in the lungs in CpG ODNs treated birds when compared to PBS-treated groups (Dar et al., Reference DAR, TIKOO, POTTER, BABIUK, TOWNSEND, GERDTS and MUTWIRI2014; Aslam et al., Reference ASLAM, SHAHZAD, PARVEEN, ASHRAF, NAZ, ZEHRA, KAMRAN, QAYYUM and MUKHTAR2016).
CPG ODNS against herpes simplex virus
CpG ODNs were injected along with herpes simplex virus 2 (HSV-2) and antigen recombinant glycoprotein B (rgB) as adjuvants (Kwant et al., 2004). Immunisation via the genital tract proved helpful in eradication and protection against many sexually transmitted diseases (STDs). CpG ODNs adjuvanted antigens induced immunity for the protection of female genital organs from HSV antigenic challenge. High levels of IgA titres and IgG titres in the vagina and serum were produced, as compared to mice immunised only with rgB antigen. Mice immunised with CpG ODNs plus antigen had higher survival rates, low disease scores and reduced viral titres in the vagina, as compared to others challenged with HSV-2. With an appropriate adjuvant like CpG ODNs, the protection of the genital tract may be achieved.
Effect of CPG ODNs against viral enteritis virus
Oligonucleotide provides a ligand for TLR21 and ultimately produces pro-inflammatory cytokines, like INFs and ILs, which are ultimately detected in peripheral blood. Thus, the protection against new type gosling viral enteritis virus (NGVEV; Qi et al., Reference QI, YAN, CHEN, CHEN, WANG, JIA, ZHU, LIU, LIU, YANG, SUN, WU, CHEN, JING and CHENG2016) may be explored by the use of CpG ODNs adjuvanted vaccines because these possess the capability to elicit INFs and ILs directly without having any immunosuppressive effects on the host.
CPG ODNS against Marek's disease virus
Delaying the onset is one of the major tasks for the prevention of Marek's disease infection. The agonist of TLR-21, i.e. CpG ODNs, have been reported to be heavily involved in delaying disease onset and enhance cytokine production against MDV in chickens. CpG ODNs can modify the immune system by producing cytokines (Parvizi et al., Reference PARVIZI, ABDUL-CAREEM, MALLICK, HAQ, HAGHIGHI, OROUJI, HEIDARI, BEHBOUDI and SHARIF2014). However, there is a blank period after immunisation in which birds can be vulnerable to MDV (Cui et al., Reference CUI, LI, SU, CUI, DING, KANG, SUN and ZHU2016).
CPG ODNS against egg lysozyme
CpG ODNs are reported to stimulate macrophages, which then secrete interleukin-12 (IL-12; Chu et al., Reference CHU, TARGONI, KRIEG, LEHMANN and HARDING1997). These further stimulate interferon gamma secretions, influencing natural killer cells. In an experiment when incomplete Fraund's adjuvant (IFA) plus hen egg lysozyme (HEL) was injected, macrophages secreted interferon gamma rather interleukin 5 (IL-5) against HEL, representing a Th-2 response. However, when CpG ODNs were injected with the IFA-HEL, this lead to the production of high levels of interferon gamma, IL-2 and a lesser production of interleukin 5, specific to HEL. It was observed that IgG2a against HEL was only induced in the CpG ODNs group, while this response may not be induced in its absence. Thus, it was clear that only the CpG ODNs group induced Th-1 type immune responses.
Effect of CPG ODNS against Escherichia coli
There was a reduction in the lesions of E. coli-induced cellulitis in birds which received CpG ODNs with both a systemic as well as local enhancement of the immune response (Gomis et al., Reference GOMIS, BABIUK, GODSON, ALLAN, THRUSH, TOWNSEND, WILLSON, WATERS, HECKER and POTTER2003). Survival rate was near to zero in the chickens which received only saline water as a control, whereas significant survival rates were reported in those which were treated with CpG ODNs alone or in combination with another effective adjuvant, i.e. polyphosphazene (Taghavi et al., Reference TAGHAVI, ALLAN, MUTWIRI, FOLDVARI, VAN KESSEL, WILLSON, BABIUK, POTTER and GOMIS2009). When the chickens were exposed to a virulent E. coli strain by injection into subcutaneous tissue of the neck, survival rate was significantly high at 60-70% in birds which received CpG ODNs as compared to other groups which received other treatments like saline or carbon nanotube (CNT) or lipid surfactant (LSC), where survival was poor (Gunawardana et al., Reference GUNAWARDANA, FOLDVARI, ZACHAR, POPOWICH, CHOW-LOCKERBIE, IVANOVA, TIKOO, KURUKULASURIYA, WILSON and GOMIS2015). By administering CpG ODNs in neonatal chickens via the intra-muscular route it is possible to eliminate diseases by releasing some cytokines (Patel et al., Reference PATEL, GOMIS, DAR, WILLSON, BABIUK, POTTER, MUTWIRI and TIKOO2008).
The above experiments support the fact that CpG ODNs are not only immunostimulators when administered along with a vaccine, but, even if they are injected alone, the innate immune system can be boosted to protect the host against infections, regardless of the type of pathogens.
CPG ODNS against Salmonella spp.
A CpG ODNs treated group of heterophils showed significant bactericidal activity against Salmonella enteritidis through the generation of reactive oxygen species and degranulation when compared to non-CpG ODNs and PBS treated groups (He et al., Reference HE, GENOVESE, SWAGGERTY, NISBET and KOGUT2007). In neonatal chickens, CpG ODNs enhanced immunity against S. enteritidis and protected neonates from infection (Shahrokhi et al., Reference SHAHROKHI, RAD and KALIDARI2013). CpG motifs present in the ODNs enhanced vaccine efficacy against salmonellosis especially S. enterica subtype Enteritidis and S. enterica subtype Galinarum (Hartley et al., Reference HARTLEY, SALISBURY and WIGLEY2012).
Conclusions
Using CpG ODNs protect chickens from various diseases including ND, AIV H9N2, AIV H5N1, salmonellosis and E. coli. Both cellular and humoral immune responses are produced when CpG ODNs is administered with vaccines against different diseases. CpG ODNs is an effective adjuvant which can be used alongside vaccines to protect chickens from viral, parasitic and bacterial diseases.
