Elsevier

Vaccine

Volume 29, Issue 38, 2 September 2011, Pages 6532-6539
Vaccine

Intraperitoneal immunization of recombinant HSP70 (DnaK) of Salmonella Typhi induces a predominant Th2 response and protective immunity in mice against lethal Salmonella infection

https://doi.org/10.1016/j.vaccine.2011.07.005Get rights and content

Abstract

Heat shock proteins serve as important antigens in defense against infectious diseases. Members of HSP70 family, particularly microbial HSP70s have acquired special significance in immunity. In the present study, we evaluated the immunogenicity and protective efficacy of HSP70 of Salmonella enterica serovar Typhi against lethal infection by Salmonella in mice with or without adjuvants. The HSP70 gene was cloned and expressed in Escherichia coli BL21 and purified by affinity chromatography. Immunization of mice with HSP70 either alone or in combination with complete Freund's adjuvant (CFA) resulted in a significant increase in antibody titers as compared to control. Antibody isotyping revealed that HSP70 immunization induces both IgG1 and IgG2a antibodies to a significant extent but a higher IgG1/IgG2a ratio indicates a predominant Th2 response. There was a significant increase in lymphocyte proliferation, and levels of both Th2 and Th1 cytokines in cells isolated from immunized mice as compared to control. Immunization of mice with recombinant HSP70 either alone or in combination with CFA conferred 70–90% protection against lethal infections by Salmonella Typhi Ty2 or Salmonella Typhimurium. However, passive immunization with anti-HSP70 sera induced only partial protection in the immunized mice.

Highlights

► We investigated the protective efficacy of rHSP70 against lethal Salmonella infection. ► Results revealed that HSP70 immunization significantly increased IgG1 and IgG2a. ► Lymphocyte proliferation, Th2 and Th1 cytokines were also increased significantly. ► HSP70 was found to be immunogenic and protective even without adjuvants. ► Immunization of rHSP70 conferred 70–90% protection against Salmonella infection.

Introduction

Vaccines have been highly successful in preventing several infections, but still there is an unmet need for development of novel vaccine strategies targeted to infections of global importance. Since heat shock proteins (HSPs) can be early targets in the immune response against a variety of pathogens, they are being exploited as antigens for vaccine development [1]. HSPs are highly conserved, ubiquitous and intracellular molecules, which contribute to cell survival under stress by facilitating the proper folding of denatured proteins [2], [3]. HSPs are known to play a dual role in vaccine development against infectious diseases; first, pathogen derived HSPs are being exploited as vaccine antigens [1]; second, HSPs are also being exploited as adjuvants to enhance the induction of antigen peptide-specific cellular immunity. Interestingly, HSPs induce potent innate and adaptive immune response, serving as ‘danger signals’ that prime multiple host defense pathways, making them a suitable target for cancer and infections including intracellular and extracellular pathogens [4].

Heat shock proteins, particularly those of HSP60 and HSP70 families are immunodominant molecules and a significant element of the immune response to pathogenic microorganisms is directed towards peptides derived from heat shock proteins [5], [6]. The occurrence of immunodominant immune responses to HSP70 is likely to result from several factors that are related to their function. First, because HSPs play important role in cell survival, their sequences are highly conserved between different species of bacteria. So, each time the immune system encounters bacteria, it first targets the strongly conserved regions of the bacterial HSP that are recognized by memory cells arising from previous infections with related or unrelated bacteria. Further, during infection, the invading microorganism also experiences a type of stress, and therefore it up-regulates expression of its own HSPs, making them easily available targets for the host immune cells. Moreover, HSP specific responses might be immunodominant because, as molecular chaperones, HSPs associate with other unfolded or newly synthesized antigenic microbial proteins or with microbial proteins that are denatured or partially unfolded as a result of stress. As a consequence, B cells with cell-surface B-cell receptors specific for a particular microbial antigen can internalize the associated microbial HSP together with the microbial antigen, process them both, and present peptides derived from the two proteins in the context of MHC class II molecules for recognition by T helper (TH) cells.

Non-availability of relevant drugs and rapid development of microbial drug resistance have led to the need of efficacious and affordable vaccines to control typhoid fever, a systemic infection, caused by Gram negative bacterium Salmonella enterica serotype Typhi. Two currently available vaccine, Typhim Vi® (Pasteur Merieux) and Vivotif®, have deficiencies that limit their universal use. Typhim Vi® is composed of purified Vi antigen from S. Typhi and it provide only 55–75% protection against typhoid fever. Moreover, no booster effect is observed by repeated injections, indicating the lack of immunological memory [7] and also this vaccine is ineffective in infants less than 2 years of age [8]. Vivotif®, consisting of the attenuated live S. Typhi Ty21a strain, was generated by chemical mutagenesis of the parental virulent S. Typhi Ty2 [9], but this vaccine is also restricted to adults and children over 6 years of age [10] and is 60% protective [11].

The research has also been focused on outer membrane proteins (OMPs) of Salmonella and they have been considered possible candidates for conferring protection against typhoid. Studies have shown that S. Typhi porin based candidate vaccine protected the immunized mice and also found safe and immunogenic in healthy humans [12], [13]. A major nonporin OMP with a molecular mass of 49 kDa from Salmonella enterica serovar Typhimurium was found to be highly immunogenic, evoked humoral and cell mediated immune responses and conferred complete protection to immunized animals against lethal challenge by S. Typhimurium [14]. Efforts are also being made to fuse Vi polysaccharide with porins [15], iron-regulated outer membrane proteins (IROMPs) of S. Typhi [16] or with recombinant, nontoxic Pseudomonas aeruginosa exotoxin A (Vi-rEPA). A field trial conducted in Vietnam showed a two dose immunization schedule of Vi-rEPA resulted in 92% protection of children 2–5 years of age [17]. A recent field trial of Vi-rEPA revealed that Vi conjugate typhoid vaccine is safe, immunogenic and is compatible with routine infant vaccines [18]. In addition, Vi from a BSL1 organism, Citrobacter freundii strain WR7011, was conjugated to CRM197, a nontoxic mutant to diphtheria toxin and this Vi-CRM197 appears to be a suitable candidate for development of a commercially viable, effective typhoid vaccine [19].

Researchers are also attempting to develop several live attenuated typhoid strains that may serve as a single dose, oral typhoid vaccines and out of these some of the attenuated strains have been successfully tested in clinical trials. These vaccine strains include CVD 908-htrA, Ty800 and M01ZH09 [20], [21]. A single oral immunization of CVD 908-htrA, was shown to induce strong antibody and cell mediated immune responses in systemic and mucosal tissues that were comparable or even higher to those elicited by multiple doses of Ty21a [22], [23], [24]. Similar to Ty21a, none of these vaccines reliably stimulate serum Vi antibodies that might contribute to protection [24]. CVD 908-htrA was further improved and engineered to constitutively express S. Typhi Vi, resulting in a new vaccine strain CVD 909 [25]. This strain was found to be safe and immunogenic in Phase I clinical trial [26]. Although, unexpectedly it failed to raise significant level of serum Vi antibodies. The vaccine recipients, however, developed mucosally primed Vi-specific IgA antibody secreting cells and robust cell mediated immunity which include memory T cell response similar to those induced by parent strain CVD 908-htrA [26], [27].

Considering the limitations of currently licensed vaccines and the emergence of multidrug resistant strains [28], [29], [30], still there is a need to find out the immunodominant and more importantly protective antigen against typhoid fever. As already discussed, HSPs promise to be a suitable vaccine candidate for use in humans because of their ability to induce a memory T-cell response and having the ability to induce strong immune response even in the absence of adjuvants. Another important feature that HSPs share is that they can directly activate cytotoxic T-lymphocytes without the assistance of Th cells [31], which might be very useful in eliciting immune response even in immunocompromised individuals.

Based on this concept, the work was carried out to evaluate the immunogenicity of HSP70 (DnaK) of S. Typhi by cloning and expressing HSP70 in the heterologous host E. coli BL21(DE3). An effort was also made to determine the efficacy of HSP70 in protecting the mice against lethal challenge by S. Typhi and S. Typhimurium.

Section snippets

Mice

All the experiments were carried out by using six to eight week old female BALB/c mice, which were maintained in the Experimental Animal Facility of the institute under standard laboratory conditions with 12 h light and 12 h dark cycle. All the animals were given sterile water and feed ad libitum throughout. Mice were handled and disposed off according to the guidelines of Institutional Ethical Committee.

Bacterial strains, vectors and reagents

S. Typhi MTCC 733 procured from the Institute of Microbial Technology, Chandigarh, India, was

Cloning, expression and purification of HSP70

PCR amplified hsp70 was cloned into BamHI and HindIII sites of pQE30 expression vector. The majority of protein was expressed in soluble fraction and was purified twice by metal chelate affinity chromatography under native conditions. SDS-PAGE analysis of affinity purified protein revealed a single band of approximately 68 kDa (data not shown). The protein was dialyzed extensively and used for immunization of animals. The yield of purified protein was approximately 20 mg/l.

The concentration of

Discussion

In this study, we show that HSP70 of Salmonella Typhi is highly immunogenic and gives almost complete protection against lethal Salmonella infections. HSPs of microbial origin appear to play an important role in pathogenesis and host immune response. Stewart et al. have shown that over expression of heat shock proteins reduces survival of Mycobacterium tuberculosis in chronic phase of infection [32]. They constructed a mutant strain of M. tuberculosis that constitutively over expresses HSP70

Acknowledgements

We thank Dr. Rameshwar Singh and Mr. Bhagwat Singh of Experimental Animal Facility for their valuable support and technical assistance with animal experiments. This work was supported by Defence Research and Development Organization, Ministry of Defence, Government of India.

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