Immunol Cell Biol. 2013 Nov-Dec;91(10):642-51.

Co-expression of S. Typhi GroEL and IL-22 gene augments immune responses against Salmonella infection.

Kaur G, STS C, Nimker C, Singh M, Saraswat D, Saxena S, Bansal A.

Division of Experimental Biology, Defence Institute of Physiology and Allied Sciences, Defence Research and Development Organization, Timarpur, Delhi, India.

 

Abstract

Recombinant DNA vaccines represent a novel method for generating in situ expression of vaccine antigens. Intramuscular injections of naked DNA are able to elicit potent humoral and cellular immune responses but still numerous factors limit the immunogenicity of DNA vaccines. Co-expression of cytokines with antigen encoding genes in DNA vectors can improve the immune responses and modify Th1/Th2 balance. In this study, the immunomodulatory effect of Interleukin 22 (IL-22) as an adjuvant was studied by DNA vaccination with S. Typhi Heat shock protein 60 (HSP60/GroEL) in mice. Further, DNA construct of IL-22 gene fused with GroEL was developed and immunization studies were carried out in mice. DNA vaccination with GroEL alone stimulated humoral and cell-mediated immune responses. Co-immunization (IL-22+GroEL) further resulted in increase in T-cell proliferative responses, antibody titres (IgG, IgG1, IgG2a) and secretion of IFNγ (Th1), IL-1β and Th2 (IL-4, IL-6) cytokines. Co-expression (IL-22-GroEL DNA) also promoted antibody titres and cytokine levels were significantly higher as compared to co-immunized group. A reduction in bacterial load in spleen, liver and intestine was seen in all the immunized groups as compared to control, with least organ burden in fusion DNA construct group (co-expression). Improved protective efficacy (90%) against lethal challenge by Salmonella was observed with IL-22-GroEL co-expressing DNA vector as compared with plasmid encoding GroEL only (50-60%) or co-immunization group (75-80%). This study thus shows that co-expression of IL-22 and GroEL genes enhances the immune responses and protective efficacy, circumventing the need of any adjuvant.

PMID: 24145856

 

SUPPLEMENT

Infectious diseases represent a major cause of mortality worldwide and continue to threaten global health.Typhoid fever caused by the bacterium S. Typhi remains an important health concern worldwide, mainly in developing countries. Typhoid strikes about 21 million people around the world and kills 216,000-600,000 predominantly children of school-age or younger, making it comparable in mortality to several other diseases, including rotavirus and HPV.The emergence of multidrug-resistant strains of Salmonella with increased virulence, communicability and survivability has further complicated its management. Moreover, neonates infected with multidrug resistant organisms pose a great epidemiologic hazard to the family and community because of prolonged carrier state that is typical of this age group. Strategies to control these infections by therapeutic or preventive means become a prerequisite in developing countries. Immunization strategies are an effective and economical approach to the prevention and control of infectious diseases. Currently available vaccines for typhoid fever have less-than-desired efficacy and certain unacceptable side effects, making it pertinent to search for new immunogen suitable for vaccine formulation.

Heat shock proteins (HSPs) are the most phylogenetically conserved molecules present in prokaryotes and eukaryotes that play an important role in protein folding, assembly and in translocation of proteins between different compartments. HSPs represent major antigens in wide spectrum of infections as they are abundant proteins in pathogens, especially under stressful conditions and have been shown to provide significant protection against various pathogens in different disease models.The ability of HSPs to elicit memory T cell response and stimulate strong immune responses even in the absence of CD4 cells makes them ideal vaccine candidate molecules.Strategies to more effectively induce immunity with HSPs include the use of DNA vaccines.

DNA vaccines are third generation vaccines, made up of small circular pieces of bacterial DNA (plasmid) that has been genetically engineered to produce one or two specific proteins (antigens) from a pathogen.The use of naked DNA as vaccine to elicit the immune system against disease provides a variety of practical benefits for large scale vaccine production that are not as easily manageable with other forms of vaccine including recombinant protein or whole tumour cells. The major advantage of DNA immunization is that both cellular (including CD4+ and CD8+ T cells) and humoral immune responses can be induced because the encoded antigen is processed through both endogenous and exogenous pathways and peptide epitopes are presented by major histocompatibility complexes (MHC) class I and II. The uptake of plasmid DNA containing the gene of interest by host cells results in in vivo synthesis of the encoded protein. A large number of clinical trials have shown the immunogenicity of DNA vaccines in humans, although their potency is limited for reasons that are still unclear.

Research on protective immunity and adjuvants that generate vigorous immune responses may help generate effective vaccines against such pathogens for which vaccines are unavailable or noneffective. The incorporation of adjuvants into vaccine formulations is aimed at enhancing, accelerating and prolonging the specific immune response to vaccine antigens. However, most effective adjuvants are unsuitable for human use due to their toxicity.

The inclusion of cytokines or other immuno-modulatory molecules in vaccine formulations (recombinant subunit/DNA vaccines) can customize vaccine-induced immune responses, with the aim of eliciting protective immune-effector mechanisms avoiding pathological immune responses.Numerous cytokines have been shown to significantly modulate the inflammatory process. They can act as pro-inflammatory or anti-inflammatory mediators. Cytokines assist the immune response in determining the ideal response to pathogens and the magnitude of the response, by signalling immune cells, T cells and macrophages, to travel to sites of infection and by stimulating them to produce more cytokines. Co-expression/co-administration of cytokine encoding genes along with antigen can augment and modulate the immunogenicity and efficacy of plasmid DNA vaccines. Cytokines fused to antigen can influence APC or responding T- cells to drive the response toward a Thl or Th2 direction and/or potentiate it in an antigen-specific manner.

Fig_1

With the identification of new TH subset, it is now well established that TH17 cells promote acute inflammatory responses, with the key cytokine IL-22 being a potential anti-microbial therapeutic molecule.The maintenance of barrier function at exposed surfaces of the body is necessary for regulating contact with environmental stimuli preventing bacterial dissemination of pathogenic microbes and retaining homeostasis of the entire body. It induces a variety of proliferative, anti-apoptotic and anti-microbial effects, plays an important role in the innate immune defense and is crucial in maintaining local control of gram negative pathogens.

Fig_2

In order to explore the role of cytokines in modulating the immune response and protective efficacy of recombinant DNA vaccine against microbial infections, we developed and studied the adjuvant potential of IL-22 in a DNA vaccination vector co-administered or co-expressed with GroEL of S. Typhi against lethal infection of Salmonella in mice. We cloned both the genes individually in the pVAX1 eukaryotic DNA vector as well as developed fusion DNA construct co-expressing both the genes as a single fusion protein with an aim to improve the immune responses [Fig. 2]. Strong humoral responses were observed after immunization with different DNA constructs as was evident by significant boost in total IgG antibody titres as compared to control [Fig.3a]. Very low antibody titre was seen in serum of immunized animals after 1st immunization which increased steadily by the end of immune duration. However, the maximum increase in IgG levels was seen in mice immunised with fusion DNA and co-immunized group as compared to GroEL antigen or IL22 DNA alone. Elevated IgG1 and IgG2a antibody titres in all the four immunized groups as compared to control, indicates induction of mixed immune response (Th0). The fusion DNA immunized group showed maximum increase in both subtypes followed by co-immunization group. Positive IgG1/IgG2a ratio shows predominance of Th2 type of immune response in all the groups [Fig.3b]. Cellular responses as determined by lymphocyte proliferation, secretion of Th1 and Th2 cytokines and Nitric oxide (NO) level were also highest in animals immunised with fusion gene (IL22-GroEL). NO is a major immune mediator and is an important part of the host defense against various infections. It mediates much of the antimicrobial activity of mouse macrophages against some fungal, helminthic, protozoal and bacterial pathogens. Intracellular parasites like Salmonella require stimulation of both cell mediated and humoral immunity, therefore it is important that both arms of immunity are effectively generated by DNA vaccination.

Fig_3

To study the effect of DNA immunization in eliciting protective immunity in mice against Salmonella Typhi and S. Typhimurium infection, challenge studies were done two weeks after last immunization. Improved protective efficacy (90%) against lethal challenge by Salmonella was observed with IL22-GroEL co-expressing DNA vector as compared with plasmid encoding GroEL only (50-60%). 75-80% protection was seen in co-immunized group. Interestingly, we also found 40-45% protection in IL22 DNA alone group [Fig. 4]. A reduction in bacterial load in spleen, liver, intestine was seen in all the immunized groups as compared to control. IL22–DNA also showed reduced number of bacterial counts in tissues compared to control mice. Co-immunization led to a further decrease in organ load. The protection elicited by fusion DNA plasmid was maximal as seen by maximum decrease in bacterial load in fusion DNA construct group. Overall, the results demonstrated that IL22-GroEL DNA vaccine construct effectively enhanced the protective efficacy against Salmonella infection. Efficacy was more than that achieved by co-administration of two genes and efficiently higher than IL22 or GroEL DNA alone. Moreover,IL-22 is critical for limiting bacterial replication and dissemination, probably in part by inducing the expression of antimicrobial peptides from epithelial cells at these barrier surfaces, thus leading to reduced pathogen burden in the host and ultimately enhanced protection from infection.

Fig_4

Further, the protection accorded by the DNA immunization confirmed that LPS did not have any role in eliciting protection against Salmonella because the plasmid DNA used for immunizing the animals was purified and endotoxin free.

Our results thus show the potential of co-administration of IL22 and GroEL or fusion gene as recombinant DNA vaccine constructs in enhancing the immune responses and protective efficacy, circumventing the need of any adjuvant. Our study also forms the basis for the development of therapeutics with cytokine genes as a better and improved alternative strategy in vaccine development for the treatment of infectious diseases.While additional studies are needed to further improve the potency and consistency of these approaches in primates, these results may have important implications for the development of clinically relevant vaccines and immunotherapy approaches using DNA platform.

 

References:

  1. Kaur G, STS Chitra, Nimker C, Singh M, Saraswat D, Saxena S, Bansal A. Co-expression of S. Typhi GroEL and IL-22 gene augments immune responses against Salmonella infection. Immunology and Cell Biology 2013, 91: 642-651.
  2. Paliwal PK, Bansal A, Sagi SS, Mustoori S, Govindaswamy I. Cloning, expression and characterization of heat shock protein 60 (GroEL) of Salmonella enterica serovar typhi and its role in protective immunity against lethal Salmonella infection in mice. ClinImmunol 2008; 126: 89–96.
  3. Bansal A, Paliwal PK, Sagi SS, Sairam M. Effect of adjuvants on immune response and protective immunity elicited by recombinant hsp60 (GroEL) of Salmonella typhi against S. Typhi infection. Mol Cell Biochem 2010; 337: 213–221.

 

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