Evaluation of CD4+/CD8 + T-cell expression and IFN-γ, perforin secretion for B–T constructs of F1 and V antigens of Yersinia pestis

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Summary

Yersinia pestis is a facultative bacterium that can survive and proliferate inside host macrophages and cause bubonic, pneumonic and systemic infection. Understanding the immune response generated by epitopes recognized by CD4 + and CD8 + T cells is important for the development of safe and effective vaccines designed to promote protective cellular immunity. Apart from humoral response, CD4 + T cells have shown to have a major role in combating the pneumonic form of the disease. In the present study, the secretion of IFN-γ and IL-4 by splenocytes, stimulated by different constructs of B and T cell epitopes of F1 and V antigens, was measured by ELISpot assay. We also measured perforin and IFN-γ expression as a function of cell mediated immunity by flow cytometry. Three B–T constructs of F1 and seven B–T constructs of V antigens produced a high number of IFN-γ secreting cells as compared to native antigen and a low number of IL-4 secreting cells. B–T conjugates of F1 and V antigens showed significantly high (p < 0.001) percentage of CD4 + IFN-γ+ cells as compared to CD8 + IFN-γ+ cells. Thus, the study highlights the importance of Th1 cytokine and existence of high proportion of CD4 + T cells probably contributing protection in the host. This study proposes a new perspective for the development of vaccination strategies for Y. pestis that trigger T cell immune response.

Highlights

► F1 and V antigens are putative plague vaccine candidates. ► Subunit/peptide based vaccines can be made after identifying protective B and T cell epitopes of protein. ► Different B–T constructs of F1 and V antigens were synthesized. ► Cellular responses at systemic level and cytokine profile were studied in murine model using internasal immunization. ► FACS analysis proved expansion of CD4 + T cells and CD8 + T cells with IFN-γ, perforin and granzine were studied.

Introduction

Plague, caused by the Gram-negative bacterium Yersinia pestis, is a disease that had catastrophic effects on human society from ancient times [1]. There are three recorded plague pandemics and several regional outbreaks, which are thought to have caused 200 million human deaths worldwide [2]. Although outbreaks today are very few and isolated, there is still an average of about 2000 cases reported every year. In its natural cycle, Y. pestis infection is endemic in rodent populations. It is usually transmitted to other animals and humans by the bite of infected fleas. This mode of transmission causes bubonic plague in humans, which can progress to pneumonic plague. Humans also contract the infection by direct contact or inhalation of infected air droplets, which leads to the fatal and highly contagious pneumonic form of plague [3]. Although bubonic plague is often successfully treated with antibiotics, antibiotic therapy is rarely successful in pneumonic plague because of the rapid onset of the disease. The recurring incidence of plague still continues to pose a threat to public health worldwide, and there is growing concern over possible use of aerosolized Y. pestis as a biological weapon. The effectiveness of currently licensed killed whole cell plague vaccine is questionable against pneumonic plague and cause local and systemic side effects. The limited applicability of this vaccine necessitated the development of an acellular vaccine against plague consisted of immunogenic subunits derived from the parent organism. Plague vaccines comprising both F1 and V antigens are at the forefront of current development [4], [5]. A subunit vaccine combining F1 and V antigens or a recombinant fusion F1–V protein have been shown to confer protection against both bubonic and pneumonic plague in mice, guinea pigs, and non-human primates [6], [7], [8]. A Phase I clinical trial demonstrated that intramuscular immunization with a vaccine containing F1 and V antigens is immunogenic in humans with highly variable immune response [9]. F1 antigen is a capsular protein associated with anti-phagocytic property of Y. pestis [10]. F1 elicits strong antibody response and passive transfer of anti-F1 antibodies provided protection [11], [12], [13]. V antigen has been shown to play a key role in regulating the type III secretion system (TTSS) in all the Yersinia species, and is intrinsic in forming a channel between the bacterium and the host cell, through which the Yersinia outer proteins (Yops) are translocated inside the cell [14], [15]. Purified V antigen has been shown to suppress the inflammatory response in host by down regulating the expression of TNF-α and IFN-γ. It also caused the production of IL-10 by macrophages and inhibited neutrophil chemotaxis [16], [17], [18], [19]. Anti-V antibodies administered to infected mice restored the production of TNF- α and IFN-γ and shown to block partially the delivery of Yops into macrophages [20]. Newer vaccines that induce long lived immunity against bubonic and pneumonic plague however may need to be developed [21].

Y. pestis can replicate within the cells during initial stages of infection in vivo [22]. Cell-mediated immunity (CMI) to Y. pestis is important for vaccine-mediated protection against plague, however, the exact nature and role of CMI in protection is not clear, probably it could be through the activation of CD4 + cells. Peptides generated from V antigen are recognized by murine T cells and vaccination with V peptides induced IFN-γ production by CD4 + T cells and was sufficient to protect in vivo against challenge with Y. enterolitica [23]. Role of IL-17 has been identified in host defense that enhances protection against pulmonary Y. pestis challenge [24]. Vaccine-primed cytokine production help in protection, but T cells also contribute protection via direct lysis of the infected cells [25]. In addition, CD4 + T cells can exert cytolytic activity on MHC class II bearing targets [26]. Perforin-mediated cytotoxicity is well documented in CTL-mediated defense against host cells infected with viruses, bacteria, tumor, and parasites, although the expression of perforin by CD4 + CTLs is gaining importance in some of the intracellular pathogens. Some studies demonstrated that CD4 + CTLs express perforin [27], [28], [29], [30], [31]. Since intracellular phase sequesters Y. pestis from antibody contact, CTL response and enhanced innate immunity may help in achieving sterile immunity, particularly in the naive host. Several new studies have raised concern that full-length V antigen was immunosuppressive and caused predominantly polarization of Th2 cytokine [32], [33]. V and F1 antigens induced both IgG1/IgG2a which is consistent with a mixed type immune response [34]. The region of V antigen spanned from amino acid sequence 135 to 275 contained a major protective region, although other regions of V antigen also contribute protection [41]. rV10, a mutant lacking amino acids 271 to 300 of V antigen, displayed a significant decrease in its ability to induce IL-10 and suppress TNF-α or IFN-γ release [32]. These observations strongly suggested that cytokine-mediated cellular immunity is very important to combat Y. pestis infection. In our laboratory, liposome/microsphere delivery of different constructs of B and T cell epitopes of F1 antigen induced high antibody titres at systemic and mucosal sites, and a few conjugates showed in vivo protection [36], [37]. Further we delineated protective B and T cell epitopes on V antigen and studied the involvement of CD4 + T cells in inducing lymphocyte proliferation and cytokine release profile of different peptides [38]. Moreover, three epitopes were identified within the Y. pestis V antigen, that activated CD4 T cells in C57BL/6 mice [23]. Two out of the three sequences were in the same region of V antigen as identified in our laboratory. When different B–T constructs of V antigen were tested, four constructs induced strong humoral, and mucosal immune response and provided in vivo protection in murine model. In a recent study we showed that different B–T constructs of F1 and V antigens were able to show strong T cell immune response in mucosal and systemic compartments after intranasal immunization in two strains of mice. Cytokine release profile of these B–T constructs were shown to be Th1 polarized. Since some of the B–T constructs of F1 and V antigens showed in vivo protection during challenge experiment [35], [39] the role of CD4 + T cell in releasing perforin and IFN-γ as a measure of cytolytic activity as a marker of in vivo protection was evaluated in detail. The aim of the current study was to evaluate the cytokine profile and its contribution to immune response by measuring perforin, IFN-γ expressing T cells using prominent B–T constructs of F1 and V antigens of Y. pestis.

In the present study, these B–T constructs (conjugated B and T-cell epitopes) of F1 and V antigens produced a high number of IFN-γ secreting cells compared to native antigen and a low number of IL-4 secreting cells. Furthermore, we observed a significantly high percentage of CD4 + IFN-γ+ cells compared to CD8 + IFN-γ+ cells as well as a significantly high percentage of CD4 + perforin cells as compared to CD8 + perforin cells.

Section snippets

Peptide sequences

All the peptide conjugates of B and T cell epitopes of F1 and V antigens with Glycine–Glycine (GG) spacer were synthesized on Wang resin using Fmoc chemistry. After purification, all the peptides were > 95% pure as confirmed by HPLC and amino acid analysis. B1, B2 and B5 are B cell epitopes and T1 is a T cell epitope of F1 antigen [37]. Peptides d and k are T cell epitopes, peptide a is B cell epitope and peptides b, f and i are both B- and T-cell epitopes of V antigen [38].

Peptides and B–T

Enumeration of cytokine secreting cell

We herein measured IFN-γ and IL-4 production by ELISpot assay, a method that quantifies the actual number of T cells producing specific cytokine, parallel with the ELISA based measurements of total cytokine production [39], [40] As with the ELISA results, ELISpot assay also detected specific IFN-γ and IL-4 production during in vitro stimulation of splenocytes. Inbred (H-2d) and outbred mice were used to evaluate the IFN-γ and IL-4 production on days 20 and 60 after intranasal delivery of

Discussion

Humoral, cellular and mucosal immune responses potentially contribute to vaccine efficacy [40]. Humoral immunity relies upon B-cell production of antibodies and effectively neutralizes extracellular pathogens and toxins, while cellular immunity relies upon the cytolytic and cytokine-producing capacities of T cells and is particularly effective in eradicating intracellular pathogens [23]. Cell-mediated protection against intracellular bacteria often relies upon the development of Th1 type immune

Acknowledgments

F1 antigen was kindly provided by Dr. A. Friedlander, USA and V antigen by E. D. Williamson, UK. The authors are grateful to Department of Biotechnology, New Delhi for financial assistance to carry out the study and Indian Council of Medical Research, New Delhi for providing fellowship to Ms. Geetanjali Gupta. Special thanks to Dr. Rajni Rani of National Institute of Immunology, New Delhi for providing ELISpot image analyzer.

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