Cytokine adjuvants for avian viral vaccines

Cytokines are small soluble proteins secreted mainly by immune cells. They play pivotal roles in cell-to-cell signaling and thus control immune responses to pathogenic infections and vaccination (BAROUCH et al. 2004). Recently, several avian cytokines have been cloned and their biological activities have been investigated in vitro and in vivo. Over 23 interleukins, eight type I IFNs, IFN-γ, IFN-λ, and GM-CSF are characterized in chickens (KAISER et al. 2005; KARPALA et al.

2008; KAISER 2010). The functional domains of most of the avian cytokines (GU et al. 2010a; GU et al. 2010b; KIM et al. 2012), which interact with the receptors on the surface of the immune cells are sufficiently characterized (GU et al. 2010c; JEONG et al. 2012; KIM et al. 2012). The intracellular signalling cascades (JAK/STAT) that result in the modulation of the immune response have been well determined (KAMPA u. BURNSIDE 2002; OSINALDE et al. 2011).

The multifunctional effects of these cytokines on the innate and adaptive immune system of the host include the activation and differentiation of APCs, B- and T-cell proliferation, and induction of the expression of costimulatory molecules (HILTON et al. 2002). Chickens inoculated with recombinant chIL-2 (HILTON et al. 2002) or plasmid encoded chIL-2/IL-15 (LILLEHOJ et al. 2001) developed an increase in the numbers of peripheral blood CD4+- and CD8+ T-cells. These cells expressed higher levels of the IL-2 receptor (IL-2R) (HILTON et al. 2002). Potent therapeutics and vaccine adjuvant effects of several avian cytokines were described for poultry

(LILLEHOJ et al. 2001; KAISER 2010; KUMAR et al. 2010). Cytokine adjuvants can be classified as Th1 promoting and include IL-2, IFN-γ, IL-12, IL-15, and IL-18. These cytokines can augment CMI. Th2 promoting cytokines like granulocyte-macrophage cell stimulating factor (GM-CSF) and IL-4 enhance humoral immunity (RAHMAN u.

EO 2012). Simultaneous application of Th1 and Th2 cytokine adjuvants may result in balanced Th1/Th2 immune responses to an Ag.

The major advantages of cytokine adjuvants are their suitability to be administered with DNA, killed, and live viral vaccines (Table 5 and 6). They may show synergistic effects when two or more of them are administered simultaneously. They can be delivered as recombinant cytokines (XIAOWEN et al. 2009), and have been shown to induce significant humoral immune responses when co-administered for example together with inactivated Newcastle disease (ND) and infectious bronchitis (IB) vaccines (DEGEN et al. 2005; WANG et al. 2006; HUNG et al. 2010). However, the major limitations of these recombinant cytokines as vaccine adjuvants are their extraordinary short in vivo half-life time. To circumvent this problem, cytokine-encoding plasmids are utilized and co-administered with Ags (BAROUCH et al.

2004). Large numbers of plasmid encoded cytokines have been evaluated as DNA or conventional vaccine adjuvants for several viral Ags in chickens (Table 5). They may be cloned into a bicistronic eukaryotic expression vector together with the immunogenic target gene to enhance the protective levels of both humoral and cellular immunity (KUMAR et al. 2009).

Alternatively, cytokine genes encoded by live viral vectors such as HVT, fowlpox (FPV) and adenovirus (Ad) vectors may enhance neutralizing Ab production of the co-administered vaccines (Table 6). Orally administered live attenuated S.

Typhimurium that expressed IL-18 modulated both humoral and Th1-biased CMI against the co-delivered inactivated avian influenza vaccine (RAHMAN et al. 2012).

Expression of chIL-2 as fusion protein with influenza neuraminidase VLPs has been shown to induce strong humoral immunity when applied together with inactivated influenza A (H3N2) vaccine (YANG et al. 2009).

Although the adjuvant properties are well recognized, none of the cytokine-based adjuvants are licensed so far. Appropriate methods of cytokine delivery still need to be developed.

Table 5: Improvement of the protective efficacy of avian viral vaccines after co-administration of plasmid encoded avian cytokines

Cytokines Vaccines Evaluation of immunity/protection after challenge References IFN-γ Live cell-free HVT ↓tumor incidence after vMDV challenge (HAQ et al. 2011)

NDV-DNA ↑HI-Ab titer, ↑T-cell proliferation (SAWANT et al. 2011)

IL-18 Live ND 44% of the live chicks had IgM after vaccination (DILAVERIS et al. 2007) Inactivated ND ↑HI-Ab, ↑↑IFN-γ, ↑↑CD8+ T-cells, 100% protection

against NDV

(HUNG et al. 2010)

ILTV-DNA ↑CD8+ T-cells, ↑IFN-γ, 80% protection against ILTV (CHEN et al. 2011b)

AIV-DNA ↑HI-Ab, ↑CD4+ T-cells (LIM et al. 2012)

IL -15 AIV-DNA ↑↑HI-Ab, ↑↑CD4+ T-cells (LIM et al. 2012)

IL-4 NDV-DNA ↑↑↑HI-Ab, 40% survival after challenge with vNDV

(SAWANT et al. 2011)

GM-CSF IBV-DNA ↑ELISA-Ab, ↑T-cell proliferation, 86% protection (TAN et al. 2009) IL-18 IBDV-DNA ↑ELISA-Ab, ↑T-cell proliferation, ↑IL-4 & IFN-γ mRNA expression, 93%

protection from clinical signs & mortality, milder bursal lesions

(LI et al. 2013a)

IL-6 IBDV-DNA ↑ELISA-Ab, 60% protection, milder bursal lesions (SUN et al. 2005) IBDV-DNA* ↑ELISA-Ab, 90% protection, milder bursal lesions (SUN et al. 2005)

NDV-DNA is a bicistronic plasmid encoding the HN & F gene of NDV; ILTV-DNA represents a plasmid encoding glycoprotein B (gB) of ILTV; AIV-DNA encodes the HA of H5N1; IBV-DNA encodes S1 gene of IBV; IBDV-DNA represents a plasmid encoding VP2 of IBDV & IBDV-DNA* encodes the PP gene. IBDV= infectious bursal disease virus; AIV= avian influenza virus; ILTV= infectious laryngotracheitis virus; HVT= herpesvirus of Turkeys; NDV= Newcastle disease virus. ↑ denotes increase and ↓ denotes decrease.

Table 6: Examples for avian cytokines delivered by live viral vectors as adjuvants for avian viral vaccines

Cytokine vectors Vaccines/target immunogen Evaluation of immunity/protection after challenge References

rHVT-IL-2 HVT ↑VN-Ab, 43% protection against vMDV (TARPEY et al. 2007a)

Live IBV ↑ELISA-Ab, 83% protection against vIBV, ↓ciliostasis (TARPEY et al. 2007b) rFPV-HN-IL-12 HN-NDV ↑↑HI-Ab, 100% protection against vNDV (SU et al. 2011) rFPV-IL-12 rFPV-HN-NDV ↑HI-Ab, ↑IFN-γ, 83% protection against vNDV (SU et al. 2011) rFPV-HA-IL-6 HA-H5N1 ↑HI-Ab, ↑T-cell proliferation, 80% protection against H5N1,

↓virus shedding

(QIAN et al. 2012)

rFPV-gB-IL-18 gB-ILTV ↑CD4: CD8+ T-cell ratio, 100% protection against vILTV (CHEN et al. 2011a) rFPV-HA-IL-18 rFPV-HA (H9N2) ↑HI-Ab,↑↑ T-cell proliferation, no viral shedding (CHEN et al. 2011a) rAd-GM-CSF-S1 S1-IBV ↑HI-Ab, ↑↑IFN-γ, 100% protection, mild renal lesions (ZESHAN et al. 2011) rAd-GM-CSF rAd-S1-IBV ↑HI-Ab, ↑CMI, 100% protection against IBV, mild renal

lesions

(ZESHAN et al. 2011)

rFPV-IL-12 rFPV-VP2 ↑VN-Ab, 100% protection from challenge, some birds with bursal lesions

(SU et al. 2011)

rHVT-IL-2 represents recombinant HVT expressing IL-2; rFPV-HN-IL-12 coexpresses HN of NDV & IL-12; rFPV-HA-IL-6 coexpresses HA of H5N1 & IL-6; rFPV-gB-IL-18 represents FPV co-expressing gB of ILTV & IL-rFPV-gB-IL-18; rAd-GM-CSF-S1 co-expresses GM-CSF & S1of IBV; rFPV-IL-12 expresses IL-12; rFPV-HN-NDV expresses the HN gene of NDV; rFPV-HA (H9N2) expresses HA of H9N2; rAd-S1-IBV expresses S1 gene of IBDV; rFPV-VP2 expresses VP2 of IBDV. rFPV= recombinant fowlpox virus; ILTV= infectious laryngotracheitis virus; rHVT= recombinant herpesvirus of Turkeys; rAd= recombinant Adenovirus. ↑ denotes increase and ↓ denotes decrease.

2.8.1.1. Chicken IL-2 and IFN-γ as adjuvants for IBDV-DNA vaccines

IL-2 is mainly secreted by T helper cells. Apart from its proliferative effects on T-cells and APCs (HILTON et al. 2002), IL-2 promotes growth of chickens (FORD et al.

2002). Co-administration of plasmid encoded chIL-2 together with plasmid encoded IBDV-VP2 or PP genes promoted IBD-specific neutralizing Ab production and T-cell responses. The vaccinated SPF chickens were partially protected against lethal virus challenge (HULSE u. ROMERO 2004; LI et al. 2004; KUMAR et al. 2009). The immune potentiating effect of IL-2 was found to be significant when cloned together with the VP2 gene into a bicistronic eukaryotic expression vector (KUMAR et al.

2009). In contrast to these findings, lack of adjuvant effects of plasmid-encoded chIL-2 was demonstrated after in ovo application with an IBDV DNA vaccine (PARK et al.

2009). The co-administration of plasmids encoding chIFN-γ and IBDV-PP did not enhance the immune response and protection against IBDV (ROH et al. 2006).

Repeated IM co-administration of chIFN-γ and PP encoding plasmids at the same injection sites was found to be immunosuppressive and challenged chickens developed more severe bursal lesions compared to the administration of the two plasmids at different sites (HSIEH et al. 2006).