Cell Mol Immunol. 2015 Jan;12(1):96-106.

Adjuvant efficacy of IL22 in modulating the immune response and protection accorded by S. Typhi rGroEL against multiple pathogens in mice


Gurpreet Kaur, Chitradevi, Charu, Anju Bansal

Division of Experimental Biology, Defense Institute of Physiology and Allied Sciences, Defence Research and Development Organization, Lucknow Road, Timarpur, Delhi, India-110054.

Email: gurpreet.dipas@gmail.com (Gurpreet Kaur), anjubansal_dipas@rediffmail.com (Anju Bansal)



Salmonella infection, ranging from mild, self-limiting diarrhea to severe gastrointestinal, septicemic disease and enteric fever, is a global health problem both in humans and animals. Rapid development of microbial drug resistance has led to a need for efficacious and affordable vaccines against Salmonella. Microbial heat shock proteins (HSPs), including HSP60 and HSP70, are the dominant antigens that promote the host immune response. Co-administration of these antigens with cytokines, such as IL-22, which plays an important role in antimicrobial defense, can enhance the immune response and protection against pathogens. Therefore, the aim of the present study was to determine the immunogenicity of rGroEL (Hsp60) of S. Typhi, alone or administered in combination with murine rIL-22 and its protective efficacy against lethal infection with Salmonella in mice. There was appreciable stimulation of the humoral and cell-mediated immune responses in mice immunized with rGroEL alone. However, co-administration of rGroEL with rIL-22 further boosted the antibody titers (IgG, IgG1 and IgG2a), T-cell proliferative responses and the secretion of both Th1 and Th2 cytokines. Additionally, rGroEL alone accorded 65%–70% protection against lethal challenge with S. Typhi and S. Typhimurium, which increased to 90% when co-administered with rIL-22. There exists a high sequence homology between HSPs of different life forms. We, therefore, also evaluated the cross protective efficacy of GroEL alongwith IL22 against different bacterial pathogens. Significant increase in protection was observed in co-immunization group against K.pneumoniae and E.coli infections (50-70%).



The development and widespread use of effective vaccines had an extraordinary impact on global health but still there are many infectious diseases for which vaccines are not available or are not optimal. Many of the newer vaccine candidates are based on protective antigens, which are inherently less immunogenic than the whole cell inactivated or live attenuated vaccines or multicomponent conjugate vaccines that were developed in the past. Therefore, adjuvants have become an increasingly important component in novel vaccines being developed today. Adjuvant is a term derived from the Latin word adjuvare, which means to aid or to help and was first coined by Ramon in 1926, who observed that horses that developed abscess at the site of an injection of diphtheria toxoid produced higher antitoxin titers than animals without abscess. Although Freund’s complete adjuvant (FCA) is one of the most potent adjuvants known (usually referred to as the gold standard adjuvant), it is relatively toxic, frequently inducing keloid formation and abscess at the site of inoculation, which precludes it from being used in human vaccines. Freund’s incomplete adjuvant (FIA), which is the water-in-oil emulsion without added mycobacteria, is less toxic and has been used in some human vaccine formulations but had certain limitations. Currently, the aluminum salt/gel-based (alum) adjuvants are the only ones licensed for human use in the United States. Alum can enhance the ability of the immune system to respond to non-self antigens after vaccination but its ability to polarize the immune system is quite poor and has several side effects [1].


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Fig.1: A Schematic Diagram showing work strategy involved in the study.


Cytokines play important roles in cell proliferation, differentiation and mobility, as well as in defence against pathogens and tumours. Co-immunization with cytokines has been reported to enhance the immune response and protection against pathogens. Cytokines have been added to vaccine formulations either as recombinant proteins or as cytokine-encoding plasmids [2] to either tilt the immune response phenotype; to influence the quality of memory cells; or just simply as classical adjuvant to strengthen the immune response elicited upon vaccination.

IL-22, a cytokine belonging to IL-10 family of cytokines, was originally identified as a cytokine induced in CD4 T cells after IL-9 stimulation, with 22% homology to IL-10. Several studies have suggested that IL-22, a cytokine secreted by Th17 cells can orchestrate the host defense against various bacterial pathogens [3, 4]. IL22 plays critical role in regulating host defense, tissue homeostasis and inflammation, including chronic inflammatory and infectious diseases. We have earlier reported that S.Typhi GroEL alone conferred 65-70% protection against lethal infection by S.Typhi and S.Typhimurium in mice while 80-90% protection was observed when immunized along with adjuvant CFA [5]. We have also reported the immunomodulatory effect of IL-22 expressing plasmid either by co-delivery or by fusion with the GroEL gene of S. Typhi in mice [6]. In the present study, we investigated the potential of codelivery of GroEL and IL-22 recombinant proteins to modulate the immune response in mice and protective efficacy against Salmonella infections without the need for conventional adjuvants. The strategy followed in the study is shown in Fig.1.


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Fig. 2: PCR amplification of rIL-22 and rGroEL. Lane M: 1Kb DNA Ladder Lane 1: amplified IL-22 gene (540bps); Lane 2: amplified GroEL gene (1.6Kb)


GroEL was isolated from genomic DNA of S. Typhi, cloned into expression vector followed by expression and purification. IL-22 was isolated from mouse splenocytes by RT-PCR [Fig.2]. The recombinant proteins were purified from transformed E.coli BL21 cells. SDS-PAGE and western blot confirmed the expression of recombinant proteins. Biological activity of IL-22 was checked by cell proliferation studies using MTT assay. The ability of rIL22 to induce proliferation in T-cells in vitro confirmed that it was biologically active. Further, GroEL was also purified from recombinant strain by affinity chromatography under denaturing conditions and refolded, dialyzed before immunizing in mice.

Recombinant purified proteins of GroEL and IL22 were injected i.p. either individually or co-delivered in mice. Antibody responses have been reported to play an important role in protection against Salmonella infection. There was maximum increase in the IgG antibody level in co-immunized group that received both rGroEL and rIL-22 proteins as compared to control or GroEL or IL-22 alone group. To determine the type of immune response, we measured IgG subtypes. The production of both IgG1 and IgG2a subclass indicates the induction of both Th1 and Th2 immune response in all groups, though the level of IgG1 was higher than IgG2a. The ratio of IgG1/IgG2a was >1 in all immunized groups, indicating a predominant Th2 response. But the ratio was highest in GroEL immunized group followed by decrease in other immunized groups, showing shift towards Th1 response [Fig.3].


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Fig. 3: Effect of co-immunization of rproteins on antibody titres(IgG) in mice. Groups of mice were immunized with rproteins and sacrificed after one week of last booster injection. Sera samples were assayed to determine IgG by ELISA


To further characterize the phenotype, lymphocyte proliferation studies were done. The results revealed a significant (p>0.01) increase in lymphocyte proliferation in splenocytes isolated from co-immunized mice as compared to GroEL or IL-22 alone. Significant increase was observed in the levels of both Th1 and Th2 cytokines in all the immunized groups as compared to control, maximum in co-immunized group. Hence, the development of protective immunity against Salmonella infection is bidirectional, linking the cellular and humoral immune responses. A balanced Th1–Th2 response induced by IL-22 might be an important factor for the generation of protective immunity against pathogens.

Murine infection with the natural mouse pathogen S. Typhimurium is the most widely accepted model for the study of immunity in typhoid fever. The model closely resembles the pathogenesis of human typhoid and also helps in determining the efficacy of the vaccine against Salmonellosis in animals. S.Typhi has been reported to kill mice when injected at high doses; therefore we determined LD of strains and determined that S.Typhi was able to kill mice at 107 CFU and that of S.Typhimurium was found to be 105 CFU. The results revealed that immunization with GroEL alone conferred 65-70% protection and IL-22 alone elicited 30-40% protection. However, co-immunization with IL22 and GroEL provides 80-90% protection against S. Typhi and S. Typhimurium. [Fig.4]. Induction of anti-microbial proteins and maintenance of epithelial barriers by IL-22 allow for reduced dissemination and increased bactericidal activity, both leading to reduced pathogen burden in the host and ultimately clearance. The organ burden studies further revealed the reduction in the bacterial load in different tissues of mice immunized with GroEL/IL-22 alone. The bacterial count was significantly decreased in the intestine, spleen and liver (p<0.001 vs control) of animals co-administered with GroEL+IL-22. Histopathological analysis also revealed the similar level of protection, normal morphology was observed in co-administered group. The highly conserved nature of HSPs makes them a common antigen providing some degree of cross-protection against different infections. Therefore, we determined the efficacy of recombinant proteins in eliciting cross- protective efficacy against other enteric infections. A separate set of female BALB/c mice were immunized i.p. with rproteins and fifteen days after last immunization, animals were challenged with a lethal dose of K.pneumonia and E.coli. Interestingly, we observed that co-administration of GroEL and IL-22 significantly enhanced the protective efficacy. In K.pneumonia infection, GroEL alone was able to provide 40% protection while IL-22 alone protected 50% of immunized mice. Co-immunization increased the protective efficacy to 70% (Fig 5a). Fig. 5b depicts the graph showing percentage protection elicited by recombinant proteins against E.coli challenge. Co-immunization of rproteins elicited 50-55% protection. IL22 and GroEL provided 30% and 40% protection respectively. The results implicate the potential use of microbial HSPs as single vaccine candidate molecules against different infections.

Overall, the results demonstrated that co-administration of rGroEL antigen with rIL-22 as an effective adjuvant can enhance its protective efficacy against Salmonella and other bacterial pathogens, signifying the prospective of IL-22 as a potent immunopotentiator in recombinant vaccines against different bacterial infections.


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Fig 4: Effect of co-immunization of recombinant proteins on survival of mice against lethal challenge of (a) S. Typhi (b) S. Typhimurium. Graph shows percent survival of mice in different groups.


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Fig 5: Cross-protective efficacy of rproteins on survival of mice against lethal challenge of (a) K.pneumonia (b) E.coli. Graph shows percent survival of mice in different groups.



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