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Oral immunisation with live aroA attenuated Salmonella enterica serovar Typhimurium expressing the Yersinia pestis V antigen protects mice against plague
Vaccine 21 (2003) 3051–3057
Oral immunisation with live aroA attenuated Salmonella enterica serovar Typhimurium expressing the Yersinia pestis V antigen protects mice against plague Helen S. Garmory a,∗ , Kate F. Griffin a , Katherine A. Brown b , Richard W. Titball a,c a Dstl Chemical and Biological Sciences, Porton Down, Salisbury SP4 0JQ, UK Department of Biological Sciences, Centre for Molecular Microbiology and Infection, Imperial College of Science, Technology and Medicine, London SW7 2AY, UK Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London WC1E 7HT, UK b
c
Received 27 September 2002; received in revised form 24 October 2002; accepted 21 January 2003
Abstract Bubonic and pneumonic plague are caused by the bacterium Yersinia pestis. The V antigen of Y. pestis is a protective antigen against plague. In this study, an aroA attenuated strain of Salmonella enterica serovar Typhimurium (SL3261) has been used to deliver the Y. pestis V antigen as a candidate oral plague vaccine. SL3261 was transformed with the expression plasmid pTrc-LcrV, containing the lcrV gene encoding V antigen. Immunoblot analysis showed V antigen expression in SL3261 in vitro and intragastric immunisation of mice with the recombinant Salmonella resulted in the induction of V antigen-specific serum antibody responses and afforded protection against Y. pestis challenge. However, the antibody responses induced by the recombinant Salmonella did not correlate with the protection afforded, indicating that immune responses other than antibody may play a role in the protection afforded against plague by this candidate vaccine. Crown Copyright © 2003 Published by Elsevier Science Ltd. All rights reserved. Keywords: Plague; V antigen; Salmonella enterica serovar Typhimurium
1. Introduction Plague has caused an estimated 200 million deaths in three world pandemics [1] and is still a public health problem, especially in regions of Africa, Asia and South America [2]. The disease is caused by the bacterium Yersinia pestis, an obligate pathogen that is transmitted among natural animal reservoirs (primarily rats) and usually by a flea vector. Following the bite of an infected flea, man is an occasional host in the cycle of the disease, most commonly presented as bubonic plague. However, the haematogenous spread of Y. pestis from the infected lymph nodes (‘buboes’) to the lungs can lead to outbreaks of primary pneumonic plague. Various vaccines against plague have been developed. In recent years, a number of killed whole-cell vaccines have been available. The current plague vaccine is the Commonwealth Serum Laboratories (CSL) vaccine, although it is only licensed for use in Australia. Killed whole-cell vaccines ∗ Corresponding author. Tel.: +44-1980-614755; fax: +44-1980-614307. E-mail address: [email protected] (H.S. Garmory).
are believed to be effective in providing protection against bubonic plague, primarily on the basis of the low incidence of the disease in vaccinated US troops exposed to plague in Vietnam [3], even though cases of disease occurred in the civilian population. However, killed whole-cell vaccines are relatively ineffective against pneumonic plague in mice [4], and cases of pneumonic disease have been reported in vaccinated individuals [5]. A further problem with this vaccine is the high incidence of side effects, where a range of local reactions (11–24%) and systemic effects (4–10%) have been reported in vaccinated individuals [6]. The V antigen of Y. pestis has been identified as a major protective antigen against plague [7,8] and, along with the Y. pestis F1 capsule antigen, forms part of the improved protein sub-unit vaccine [9,10] that is currently in clinical trials. V antigen is a 37 kDa secreted protein encoded by the lcrV gene on the 70–75 kb virulence plasmid and is believed to have a multifunctional role in Y. pestis virulence. First, V antigen has been shown to be a positive regulator of expression of low calcium response virulence genes [11]. Secondly, it is thought that V antigen plays a direct role in the translocation of effector proteins into eukaryotic cells via the type III
0264-410X/03/$ – see front matter. Crown Copyright © 2003 Published by Elsevier Science Ltd. All rights reserved. doi:10.1016/S0264-410X(03)00112-9
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secretion system, and that it is exposed on the Y. pestis cell surface prior to contact with host cells [12,13]. V antigen has also been attributed with various direct anti-host effects involving immunomodulation [14–17]. An improved plague vaccine would ideally be an orally-delivered vaccine giving long-lasting protective immunity without side effects. Attenuated Salmonella enterica serovar Typhimurium strains have been well characterised in their use as carriers of heterologous antigens in the murine model [18–21]. They may be administered orally and are capable of eliciting both systemic and mucosal immune responses, which may be important in protecting against bubonic and pneumonic plague. A Salmonella vaccine strain expressing the V antigen as a fusion to the F1 antigen of Y. pestis has previously been reported [22]. This study demonstrated the feasibility of expressing V antigen in Salmonella. However, co-synthesis of V antigen caused a significant reduction in the yield of F1 antigen by Salmonella, and although protective immunity against plague was afforded, this was only achieved following intravenous (rather than oral) administration of the recombinant Salmonella strain. In contrast, in this report we describe the use of an aroA attenuated strain of S. enterica serovar Typhimurium for delivery of the Y. pestis V antigen as a candidate orally-delivered plague vaccine. 2. Materials and methods 2.1. Enzymes and reagents Materials for the preparation of growth media were obtained from Oxoid Ltd. or Difco Laboratories. Enzymes used in the manipulation of DNA were obtained from Roche Diagnostics Ltd. and used according to manufacturer’s instructions. All other chemicals were obtained from Sigma-Aldrich, unless described otherwise. Polyclonal anti-V antigen serum was obtained from rabbits that had been inoculated with recombinant V antigen in Alhydrogel. The anti-V antigen serum, monoclonal antibody mAb7.3 and biotinylated mAb7.3 were kindly provided by Jim Hill. Recombinant V antigen was prepared as described previously [7,9].
Pharmacia Biotech), was kindly provided by D. Lawton, Imperial College of Science, Technology and Medicine, London. pTrc-LcrV and pTrc99A were passaged through S. enterica serovar Typhimurium LB5010 to ensure methylation, and then electroporated into S. enterica serovar Typhimurium SL3261. Since the plasmids both contained a -lactamase gene conferring resistance to ampicillin, recombinant Salmonella were cultured in medium supplemented with 50 g/ml ampicillin. To produce inocula, S. enterica serovar Typhimurium was cultured statically overnight, then centrifuged (6000×g, 20 min, 4 ◦ C), washed once in phosphate buffered saline (PBS) and re-centrifuged, and re-suspended in PBS to a final cell density of approximately 1–5 × 1010 cfu/ml. Viable bacteria were enumerated on LB agar plates containing ampicillin where appropriate. 2.3. Expression of V antigen in SL3261 Cultures of recombinant S. enterica serovar Typhimurium SL3261 were grown in LB broth containing ampicillin until OD260 0.3–0.4. If required, isopropyl-d-thiogalactopyranoside (IPTG) was then added to a final concentration of 1 mM and growth was continued for a total of 6 h. Cell lysates were prepared as described by Sambrook et al. [23] and normalised so that the number of viable cells used to prepare each sample was similar. Expression of V antigen was examined by SDS-PAGE on 12.5% gradient polyacrylamide gels (PhastSystemTM , Amersham Pharmacia Biotech Ltd., Bucks, UK) and Western Blotting [23] using rabbit polyclonal anti-V antigen serum at a dilution of 1:1000. Protein bands were visualised using ECL Western Blotting Detection Reagents (Amersham Pharmacia Biotech). 2.4. In vitro stability of plasmids in SL3261 in LB broth The in vitro stability of plasmids in SL3261 was determined by culturing the recombinant bacteria in LB broth without ampicillin selection and enumerating on LB agar with and without ampicillin at 0, 8 and 24 h.
2.2. Bacterial strains and growth conditions
2.5. Colonisation and in vivo stability of pTrc-LcrV in SL3261
S. enterica serovar Typhimurium SL3261 (aroA) and LB5010 (r− m+ galE) were kindly provided by B.A.D. Stocker, Stanford University, and G. Dougan, Imperial College, London, respectively. S. enterica serovar Typhimurium strains were routinely cultured in LB broth or on LB agar [23], supplemented with 1% (v/v) histidine, 1% (v/v) ‘aromix’ (4 mg/ml tyrosine, 4 mg/ml phenylalanine, 1 mg/ml p-aminobenzoic acid, 1 mg/ml dihydroxybenzoic acid). Plasmid pTrc-LcrV [24], produced by cloning the lcrV gene into the pTrc99A expression vector (Amersham
Groups of six 8- to 10-week old female BALB/c mice (Charles-River Laboratories, Kent, UK) were inoculated intragastrically with approximately 1–5 × 109 cfu of SL3261/pTrc-LcrV or SL3261/pTrc99A re-suspended in 100 l PBS using a gavage needle. On days 8, 12 and 16, mice were culled by cervical dislocation and spleens were removed. Tissues were homogenised in 2 ml PBS using 50 m cell strainers (Becton Dickinson Labware) and suitable dilutions were plated onto LB agar or LB agar containing ampicillin for enumeration.
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2.6. Intragastric immunisation of mice and in vivo protection assay Groups of 20 mice were immunised intragastrically using a gavage needle with approximately 1–5 × 109 cfu of SL3261/pTrc-LcrV, SL3261/pTrc99A or SL3261 resuspended in 100 l LB broth. Mice were immunised on days 0, 14, 28, 42 and 56. Tail vein serum samples were collected on days 13, 27, 41, 55, 69 and 83. On day 97 inoculated mice and naive controls were challenged subcutaneously (s.c.) with 97 cfu of Y. pestis strain GB. This strain of Y. pestis has a LD50 of approximately 1 cfu in BALB/c mice by the s.c. route [26]. The mice were observed for 24 days and the time to death was recorded where appropriate. At the end of this period the experiment was terminated.
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as follows: binding (%) test serum sample OD414 nm − average 0% OD414 nm = average 100% OD414 nm 2.8. Statistical analysis Values for the standard error of the mean were calculated for data. The Student’s t-test was used to test for the significance of differences.
3. Results 3.1. In vitro analysis of recombinant SL3261 expressing V antigen
2.7. Measurement of serum antibody titre Serum antibody responses against V antigen were assessed by using a modified enzyme linked immunosorbant assay (ELISA) [27]. Briefly, 96-well microtitre plates were coated overnight at 4 ◦ C with purified recombinant V antigen (5 g/ml in PBS) and then blocked for 1 h at 37 ◦ C with 1% (w/v) skimmed milk powder in PBS (BLOTTO). The test sera were serially diluted in BLOTTO in duplicate on the plates and incubated for 1 h at 37 ◦ C. Washes between steps were performed three times with PBS containing 0.05% (v/v) Tween-20. Bound antibody was detected by addition of HRP-conjugated secondary antibody against mouse IgG, diluted 1:4000 in BLOTTO, for 1 h at 37 ◦ C. 2,2 -Azino bis(3-ethylbenzthiazoline-6-sulfonic acid) substrate (ABTS) was added and the plates were incubated at room temperature for 20 min. Serum antibody concentrations were calculated in ng/ml using a standard curve. Negative controls (sera from mice immunised with SL3261 alone or naive mice, as appropriate) were included. Concentrations were calculated using RevelationTM (Dynex Technologies Ltd., West Sussex, UK). A competition ELISA was used to measure serum antibody competing with a protective monoclonal antibody, mAb 7.3. This monoclonal recognises a conformational epitope between amino acids 135 and 275 of V antigen and protects mice against plague [28]. The 96-well microtitre plates were coated, blocked and washed as described above. Biotinylated mAb 7.3 and serially diluted serum samples (in BLOTTO), were added together to the plate and incubated at 37 ◦ C for 1 h. Bound antibody was detected by addition of 100 l per well of streptavidin-HRP-conjugated secondary antibody against mouse IgG diluted 1:1000 in BLOTTO for 1 h at 37 ◦ C. One hundred microliters of ABTS was added to each well, and the plates were incubated at room temperature for 20 min before measuring OD414 nm using a plate reader (MRX microplate reader, Dynex Technologies Ltd., West Sussex, UK). Inhibition of mAb 7.3 binding to rV by serum at a concentration of 1/25 was calculated
Plasmids pTrc-LcrV and pTrc99A were passaged through S. enterica serovar Typhimurium LB5010 for methylation, then transformed into S. enterica serovar Typhimurium SL3261. The level of expression of V antigen was compared in SL3261/pTrc-LcrV cultured in LB broth with or without IPTG supplementation. Cells were lysed by boiling in SDS-PAGE sample buffer and the lysates were examined by SDS-PAGE and Western Blotting. Polyclonal anti-V antigen sera recognised a protein of between 34.8 kDa and 49.5 kDa in both IPTG-induced and uninduced cultures of SL3261/pTrc-LcrV (Fig. 1), consistent with the estimated molecular mass of V antigen (37 kDa). The lysates were prepared from cultures containing similar numbers of viable cells, and the intensities of the bands on the Western blot suggested that there was little difference between the
Fig. 1. Western blot of S. enterica serovar Typhimurium SL3261/ pTrc-LcrV cell lysates separated by SDS-PAGE and probed with polyclonal anti-V serum. Cultures of SL3261/pTrc-LcrV were grown for 6 h in the presence or absence of IPTG, and harvested cells were normalised and lysed in SDS-PAGE sample buffer. Lane M: broad range biotinylated molecular weight marker (Bio-Rad); lane 1: purified rV antigen; lane 2: SL3261/pTrc99A (−IPTG); lane 3: SL3261/pTrc-LcrV (−IPTG); lane 4: SL3261/pTrc-LcrV (+IPTG).
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In some mice, colonisation of the bacterial strains was undetectable. However, in colonised tissues, there was no significant difference between splenic colonisation by SL3261/pTrc-LcrV and SL3261/pTrc99A (Fig. 2), indicating that cloning of the lcrV gene into pTrc99A did not affect colonisation of the bacteria. In addition, enumeration of bacteria in these homogenised tissues showed that pTrc-LcrV was stable in SL3261 in vivo (100%). 3.3. Immunogenicity and protective efficacy of SL3261/pTrc-LcrV Fig. 2. Colonisation of spleens following inoculation of mice (n = 5) with S. enterica serovar Typhimurium SL3261 carrying plasmids pTrc99A (triangles) or pTrc-LcrV (circles). Organs were removed 8, 12 or 16 days after inoculation and homogenised in PBS before plating onto LB agar (closed symbols) or LB agar containing ampicillin (open symbols) for enumeration of bacteria.
level of expression of V antigen in induced and uninduced cultures of SL3261/pTrc-LcrV cultures. 3.2. In vitro and in vivo stability of SL3261 derivatives The stability of pTrc-LcrV and pTrc99A in S. enterica serovar Typhimurium SL3261 in vitro was assessed. Enumeration of the bacteria on agar indicated that pTrc-LcrV and pTrc99A were retained by 100% and 96%, respectively, of SL3261 that had been cultured in L-broth without antibiotic selection for 24 h. The in vivo stability of SL3261/pTrc-LcrV was assessed by determining the colonisation of the Salmonella strain in murine tissues. Following intragastric inoculation of BALB/c mice with 1–5 × 109 cfu of the SL3261 recombinants, it was shown that SL3261/pTrc99A and SL3261/pTrc-LcrV colonised spleens at low levels (Fig. 2).
The induction of V antigen-specific antibodies in mice inoculated orally with S. enterica serovar Typhimurium SL3261/pTrc-LcrV was measured. Groups of 20 BALB/c mice were intragastrically immunised five times at 14-day intervals with 1–5 × 109 cfu of SL3261/pTrc-LcrV, SL3261/pTrc99A or SL3261. V antigen-specific IgG1 and IgG2a concentrations in serum taken from individual mice throughout the study are shown in Fig. 3. On days 69 and 83, the V antigen-specific IgG2a concentration was significantly higher than the IgG1 concentration (P < 0.05). Animals immunised with SL3261/pTrc-LcrV were challenged subcutaneously with 97 LD50 s of a virulent strain of Y. pestis to determine whether immunisation with the Salmonella vaccine strain provided protection against plague. All mice inoculated with Salmonella developed strong Salmonella-specific serum IgG responses, as determined by ELISA (data not shown). Similarly, all mice inoculated with SL3261/pTrc-LcrV developed V antigen-specific serum IgG responses. However, the amount of specific antibody induced varied between individual mice, with IgG1 and IgG2a concentrations of 2470 ± 750 ng/ml and 27600 ± 8340 ng/ml, respectively. Following challenge with Y. pestis GB, 6 out of 20 mice
Fig. 3. IgG1 and IgG2a responses to V antigen determined by ELISA of serum samples taken at various times following intragastric immunisation of mice (n = 20) with S. enterica serovar Typhimurium SL3261/pTrc-LcrV. Results show mean values, with S.E.M. values shown as bars.
H.S. Garmory et al. / Vaccine 21 (2003) 3051–3057 Table 1 Survival of immunised mice against challenge with Y. pestis Group
Number of survivors/total number
SL3261/pTrc-LcrV SL3261/pTrc99A SL3261 Control (naive)
6/20 0/20 1/20 0/5
Groups of mice (n = 20) were inoculated with SL3261/pTrc-LcrV, SL3261/pTrc99A or SL3261 (five doses at 2-week intervals). On day 97, immunised mice and naive control mice (n = 5) were challenged subcutaneously with 97 LD50 s of Y. pestis GB and observed for 24 days.
immunised with SL3261/pTrc-LcrV were protected against plague (Table 1). In comparison, 44 out of the 45 control mice died within 9 days. The surviving control mouse had been inoculated with SL3261 alone and showed symptoms of infection following the Y. pestis challenge. Thus, immunisation of mice with SL3261/pTrc-LcrV afforded significantly greater protection against plague when compared to SL3261/pTrc99A controls (P < 0.05) or naive controls (P < 0.005). However, the protected mice did not show significantly higher V antigen-specific IgG1 or IgG2a responses than non-survivors. The capability of the induced V antigen-specific antibody to compete with a monoclonal antibody, mAb7.3, in binding to recombinant V antigen was evaluated in a competition ELISA. mAb7.3 is a protective monoclonal antibody against plague and is specific for the Y. pestis V antigen. All mice immunised with SL3261/pTrc-LcrV developed anti-V antigen antibody that was able to compete with mAb7.3 (with binding ranging between 13.4% and 52.1%). However, there was no significant difference between binding of serum antibody developed in survivors and non-survivors of plague challenge.
4. Discussion Salmonella vaccines are attractive as carriers of V antigen for a number of reasons. They can be administered orally and, particularly, they are capable of eliciting both systemic and mucosal immune responses, which may be important in protecting against bubonic and pneumonic plague. In this study, the viability of expressing V antigen in Salmonella as a candidate orally-delivered vaccine against plague has been examined. Plasmid pTrc-LcrV [24] was transformed into S. enterica serovar Typhimurium SL3261 and V antigen expression was studied in vitro and in vivo. pTrc-LcrV contains the lcrV gene cloned downstream of the trc promoter and also the lacIq repressor [25]. Although an ideal construct for use in an oral Salmonella vaccine would not contain the lacIq repressor (since co-dosing with IPTG would be required for induction of expression of antigen in vivo), Salmonella containing pTrc-LcrV with 60% of the lacIq gene deleted expressed similar levels of V anti-
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gen as SL3261/pTrc-LcrV as determined by immunoblot analysis (H.S. Garmory, unpublished data). Unexpectedly, no difference in V antigen expression was seen between IPTG-induced and uninduced cultures of SL3261/pTrc-LcrV (Fig. 2). The reason for this is unclear, although ‘leaky’ regulation of protein expression is known to occur when using the pTrc99A expression vector. Expression of V antigen in uninduced cultures of Yersinia pseudotuberculosis containing pTrc-LcrV has also been seen [29]. Therefore, it is possible that maximal levels of expression of V antigen were occurring in uninduced cultures. In our experiments studying the stability of pTrc-LcrV in SL3261 we found that the construct was stable when cultured in L-broth in vitro. Similarly, in Salmonella that colonised murine tissues in vivo, pTrc-LcrV was stably maintained. Although the SL3261 recombinants colonised murine tissues at only low levels, SL3261/pTrc-LcrV colonised murine spleens at a level similar to that of SL3261/pTrc99A (Fig. 2), indicating that V antigen expression did not affect colonisation of the bacteria. SL3261 colonisation was shown to be affected by harbouring the pTrc99A plasmid alone [30]. Following immunisation with SL3261/pTrc-LcrV, V antigen-specific IgG was detected in the serum of all mice. Furthermore, significant protection was afforded against challenge with 97 LD50 s of Y. pestis (Table 1). This is the first time that oral administration of Salmonella expressing V antigen has resulted in protection against plague. However, the concentrations of V antigen-specific antibody induced in mice protected against plague challenge were not significantly different to some antibody concentrations induced in non-protected mice. Similarly, the amount of anti-V antigen antibody that was able to compete with mAb7.3 in binding to recombinant V antigen was not significantly different in survivors and non-survivors. These results suggest that the protection afforded by SL3261/pTrc-LcrV involves an immune mechanism other than antibody against V antigen. The protection afforded against plague in one control mouse inoculated with SL3261 alone raises the interesting possibility of protection afforded by non-specific immunity. It is known that during the initial stages of infection with Salmonella innate immunity is stimulated [31]. Macrophages phagocytose the bacteria and cell wall components of the bacteria, such as lipopolysaccharide and lipoproteins, and induce an inflammatory response in the surrounding tissues. This results in the expression of inflammatory cytokines and various chemokines that recruit cells of the immune system to these sites. Thus, it is clear that the innate immune responses induced by Salmonella vaccination may be sufficient to protect against other non-related pathogens. Immune responses induced by immunisation with purified recombinant V antigen have been well characterised. A sub-unit vaccine consisting of the V antigen and the F1 capsular antigen is currently in clinical trials. IgG1 has been shown to be a correlate of protection for this vaccine in mice [27], indicative of a cytokine profile associated
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with Th2 -type responses. However, Salmonella vaccines are known to elicit Th1 -type responses, indicated by a bias towards an IgG2a isotype in mice. Indeed, in this study immunisation with SL3261/pTrc-LcrV induced predominantly IgG2a against V antigen (Fig. 3), indicating a Th1 -type response. We have previously shown that splenic T cells isolated from mice vaccinated with Salmonella expressing a V antigen-F1 capsular antigen fusion protein proliferated extensively when re-exposed to V antigen in vitro [22]. Furthermore, putative CD4+ T cell epitopes in V antigen have been identified (J. Robinson, personal communication). These findings support the idea that following immunisation, Salmonella expressing V antigen may be able to induce cell-mediated immune responses which are involved in protection against plague. Earlier studies have shown that mice immunised with Salmonella expressing the F1 capsular antigen of Y. pestis can be highly protected against plague [32]. However, it has also been shown that mice vaccinated with F1 capsular antigen alone were not protected against naturally occurring or genetically mutated F1 capsular antigen-negative strains [33]. Hence, the development of a Salmonella vaccine against plague should ideally include expression of V antigen. Co-expression of an F1-V antigen fusion protein confirmed the importance of immune responses to V antigen in protection against plague [22], although the immunity induced was limited and required immunisation via the intravenous route. This study is the first report of protection against plague afforded by oral immunisation of Salmonella expressing V antigen alone. The results from this study should contribute to the development of a highly protective orally-delivered vaccine against plague.
Acknowledgements We thank Sophie Leary and Diane Williamson for their advice and gratefully acknowledge Tony Stagg, Debbie Rogers and Petra Oyston for their expert assistance in this work. We also thank Julie Miller for providing purified recombinant V antigen and Jim Hill for providing anti-V antigen antibodies.
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Oral immunisation with live aroA attenuated Salmonella enterica serovar Typhimurium expressing the Yersinia pestis V antigen protects mice against plague Helen S. Garmory a,∗ , Kate F. Griffin a , Katherine A. Brown b , Richard W. Titball a,c a Dstl Chemical and Biological Sciences, Porton Down, Salisbury SP4 0JQ, UK Department of Biological Sciences, Centre for Molecular Microbiology and Infection, Imperial College of Science, Technology and Medicine, London SW7 2AY, UK Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London WC1E 7HT, UK b
c
Received 27 September 2002; received in revised form 24 October 2002; accepted 21 January 2003
Abstract Bubonic and pneumonic plague are caused by the bacterium Yersinia pestis. The V antigen of Y. pestis is a protective antigen against plague. In this study, an aroA attenuated strain of Salmonella enterica serovar Typhimurium (SL3261) has been used to deliver the Y. pestis V antigen as a candidate oral plague vaccine. SL3261 was transformed with the expression plasmid pTrc-LcrV, containing the lcrV gene encoding V antigen. Immunoblot analysis showed V antigen expression in SL3261 in vitro and intragastric immunisation of mice with the recombinant Salmonella resulted in the induction of V antigen-specific serum antibody responses and afforded protection against Y. pestis challenge. However, the antibody responses induced by the recombinant Salmonella did not correlate with the protection afforded, indicating that immune responses other than antibody may play a role in the protection afforded against plague by this candidate vaccine. Crown Copyright © 2003 Published by Elsevier Science Ltd. All rights reserved. Keywords: Plague; V antigen; Salmonella enterica serovar Typhimurium
1. Introduction Plague has caused an estimated 200 million deaths in three world pandemics [1] and is still a public health problem, especially in regions of Africa, Asia and South America [2]. The disease is caused by the bacterium Yersinia pestis, an obligate pathogen that is transmitted among natural animal reservoirs (primarily rats) and usually by a flea vector. Following the bite of an infected flea, man is an occasional host in the cycle of the disease, most commonly presented as bubonic plague. However, the haematogenous spread of Y. pestis from the infected lymph nodes (‘buboes’) to the lungs can lead to outbreaks of primary pneumonic plague. Various vaccines against plague have been developed. In recent years, a number of killed whole-cell vaccines have been available. The current plague vaccine is the Commonwealth Serum Laboratories (CSL) vaccine, although it is only licensed for use in Australia. Killed whole-cell vaccines ∗ Corresponding author. Tel.: +44-1980-614755; fax: +44-1980-614307. E-mail address: [email protected] (H.S. Garmory).
are believed to be effective in providing protection against bubonic plague, primarily on the basis of the low incidence of the disease in vaccinated US troops exposed to plague in Vietnam [3], even though cases of disease occurred in the civilian population. However, killed whole-cell vaccines are relatively ineffective against pneumonic plague in mice [4], and cases of pneumonic disease have been reported in vaccinated individuals [5]. A further problem with this vaccine is the high incidence of side effects, where a range of local reactions (11–24%) and systemic effects (4–10%) have been reported in vaccinated individuals [6]. The V antigen of Y. pestis has been identified as a major protective antigen against plague [7,8] and, along with the Y. pestis F1 capsule antigen, forms part of the improved protein sub-unit vaccine [9,10] that is currently in clinical trials. V antigen is a 37 kDa secreted protein encoded by the lcrV gene on the 70–75 kb virulence plasmid and is believed to have a multifunctional role in Y. pestis virulence. First, V antigen has been shown to be a positive regulator of expression of low calcium response virulence genes [11]. Secondly, it is thought that V antigen plays a direct role in the translocation of effector proteins into eukaryotic cells via the type III
0264-410X/03/$ – see front matter. Crown Copyright © 2003 Published by Elsevier Science Ltd. All rights reserved. doi:10.1016/S0264-410X(03)00112-9
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secretion system, and that it is exposed on the Y. pestis cell surface prior to contact with host cells [12,13]. V antigen has also been attributed with various direct anti-host effects involving immunomodulation [14–17]. An improved plague vaccine would ideally be an orally-delivered vaccine giving long-lasting protective immunity without side effects. Attenuated Salmonella enterica serovar Typhimurium strains have been well characterised in their use as carriers of heterologous antigens in the murine model [18–21]. They may be administered orally and are capable of eliciting both systemic and mucosal immune responses, which may be important in protecting against bubonic and pneumonic plague. A Salmonella vaccine strain expressing the V antigen as a fusion to the F1 antigen of Y. pestis has previously been reported [22]. This study demonstrated the feasibility of expressing V antigen in Salmonella. However, co-synthesis of V antigen caused a significant reduction in the yield of F1 antigen by Salmonella, and although protective immunity against plague was afforded, this was only achieved following intravenous (rather than oral) administration of the recombinant Salmonella strain. In contrast, in this report we describe the use of an aroA attenuated strain of S. enterica serovar Typhimurium for delivery of the Y. pestis V antigen as a candidate orally-delivered plague vaccine. 2. Materials and methods 2.1. Enzymes and reagents Materials for the preparation of growth media were obtained from Oxoid Ltd. or Difco Laboratories. Enzymes used in the manipulation of DNA were obtained from Roche Diagnostics Ltd. and used according to manufacturer’s instructions. All other chemicals were obtained from Sigma-Aldrich, unless described otherwise. Polyclonal anti-V antigen serum was obtained from rabbits that had been inoculated with recombinant V antigen in Alhydrogel. The anti-V antigen serum, monoclonal antibody mAb7.3 and biotinylated mAb7.3 were kindly provided by Jim Hill. Recombinant V antigen was prepared as described previously [7,9].
Pharmacia Biotech), was kindly provided by D. Lawton, Imperial College of Science, Technology and Medicine, London. pTrc-LcrV and pTrc99A were passaged through S. enterica serovar Typhimurium LB5010 to ensure methylation, and then electroporated into S. enterica serovar Typhimurium SL3261. Since the plasmids both contained a -lactamase gene conferring resistance to ampicillin, recombinant Salmonella were cultured in medium supplemented with 50 g/ml ampicillin. To produce inocula, S. enterica serovar Typhimurium was cultured statically overnight, then centrifuged (6000×g, 20 min, 4 ◦ C), washed once in phosphate buffered saline (PBS) and re-centrifuged, and re-suspended in PBS to a final cell density of approximately 1–5 × 1010 cfu/ml. Viable bacteria were enumerated on LB agar plates containing ampicillin where appropriate. 2.3. Expression of V antigen in SL3261 Cultures of recombinant S. enterica serovar Typhimurium SL3261 were grown in LB broth containing ampicillin until OD260 0.3–0.4. If required, isopropyl-d-thiogalactopyranoside (IPTG) was then added to a final concentration of 1 mM and growth was continued for a total of 6 h. Cell lysates were prepared as described by Sambrook et al. [23] and normalised so that the number of viable cells used to prepare each sample was similar. Expression of V antigen was examined by SDS-PAGE on 12.5% gradient polyacrylamide gels (PhastSystemTM , Amersham Pharmacia Biotech Ltd., Bucks, UK) and Western Blotting [23] using rabbit polyclonal anti-V antigen serum at a dilution of 1:1000. Protein bands were visualised using ECL Western Blotting Detection Reagents (Amersham Pharmacia Biotech). 2.4. In vitro stability of plasmids in SL3261 in LB broth The in vitro stability of plasmids in SL3261 was determined by culturing the recombinant bacteria in LB broth without ampicillin selection and enumerating on LB agar with and without ampicillin at 0, 8 and 24 h.
2.2. Bacterial strains and growth conditions
2.5. Colonisation and in vivo stability of pTrc-LcrV in SL3261
S. enterica serovar Typhimurium SL3261 (aroA) and LB5010 (r− m+ galE) were kindly provided by B.A.D. Stocker, Stanford University, and G. Dougan, Imperial College, London, respectively. S. enterica serovar Typhimurium strains were routinely cultured in LB broth or on LB agar [23], supplemented with 1% (v/v) histidine, 1% (v/v) ‘aromix’ (4 mg/ml tyrosine, 4 mg/ml phenylalanine, 1 mg/ml p-aminobenzoic acid, 1 mg/ml dihydroxybenzoic acid). Plasmid pTrc-LcrV [24], produced by cloning the lcrV gene into the pTrc99A expression vector (Amersham
Groups of six 8- to 10-week old female BALB/c mice (Charles-River Laboratories, Kent, UK) were inoculated intragastrically with approximately 1–5 × 109 cfu of SL3261/pTrc-LcrV or SL3261/pTrc99A re-suspended in 100 l PBS using a gavage needle. On days 8, 12 and 16, mice were culled by cervical dislocation and spleens were removed. Tissues were homogenised in 2 ml PBS using 50 m cell strainers (Becton Dickinson Labware) and suitable dilutions were plated onto LB agar or LB agar containing ampicillin for enumeration.
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2.6. Intragastric immunisation of mice and in vivo protection assay Groups of 20 mice were immunised intragastrically using a gavage needle with approximately 1–5 × 109 cfu of SL3261/pTrc-LcrV, SL3261/pTrc99A or SL3261 resuspended in 100 l LB broth. Mice were immunised on days 0, 14, 28, 42 and 56. Tail vein serum samples were collected on days 13, 27, 41, 55, 69 and 83. On day 97 inoculated mice and naive controls were challenged subcutaneously (s.c.) with 97 cfu of Y. pestis strain GB. This strain of Y. pestis has a LD50 of approximately 1 cfu in BALB/c mice by the s.c. route [26]. The mice were observed for 24 days and the time to death was recorded where appropriate. At the end of this period the experiment was terminated.
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as follows: binding (%) test serum sample OD414 nm − average 0% OD414 nm = average 100% OD414 nm 2.8. Statistical analysis Values for the standard error of the mean were calculated for data. The Student’s t-test was used to test for the significance of differences.
3. Results 3.1. In vitro analysis of recombinant SL3261 expressing V antigen
2.7. Measurement of serum antibody titre Serum antibody responses against V antigen were assessed by using a modified enzyme linked immunosorbant assay (ELISA) [27]. Briefly, 96-well microtitre plates were coated overnight at 4 ◦ C with purified recombinant V antigen (5 g/ml in PBS) and then blocked for 1 h at 37 ◦ C with 1% (w/v) skimmed milk powder in PBS (BLOTTO). The test sera were serially diluted in BLOTTO in duplicate on the plates and incubated for 1 h at 37 ◦ C. Washes between steps were performed three times with PBS containing 0.05% (v/v) Tween-20. Bound antibody was detected by addition of HRP-conjugated secondary antibody against mouse IgG, diluted 1:4000 in BLOTTO, for 1 h at 37 ◦ C. 2,2 -Azino bis(3-ethylbenzthiazoline-6-sulfonic acid) substrate (ABTS) was added and the plates were incubated at room temperature for 20 min. Serum antibody concentrations were calculated in ng/ml using a standard curve. Negative controls (sera from mice immunised with SL3261 alone or naive mice, as appropriate) were included. Concentrations were calculated using RevelationTM (Dynex Technologies Ltd., West Sussex, UK). A competition ELISA was used to measure serum antibody competing with a protective monoclonal antibody, mAb 7.3. This monoclonal recognises a conformational epitope between amino acids 135 and 275 of V antigen and protects mice against plague [28]. The 96-well microtitre plates were coated, blocked and washed as described above. Biotinylated mAb 7.3 and serially diluted serum samples (in BLOTTO), were added together to the plate and incubated at 37 ◦ C for 1 h. Bound antibody was detected by addition of 100 l per well of streptavidin-HRP-conjugated secondary antibody against mouse IgG diluted 1:1000 in BLOTTO for 1 h at 37 ◦ C. One hundred microliters of ABTS was added to each well, and the plates were incubated at room temperature for 20 min before measuring OD414 nm using a plate reader (MRX microplate reader, Dynex Technologies Ltd., West Sussex, UK). Inhibition of mAb 7.3 binding to rV by serum at a concentration of 1/25 was calculated
Plasmids pTrc-LcrV and pTrc99A were passaged through S. enterica serovar Typhimurium LB5010 for methylation, then transformed into S. enterica serovar Typhimurium SL3261. The level of expression of V antigen was compared in SL3261/pTrc-LcrV cultured in LB broth with or without IPTG supplementation. Cells were lysed by boiling in SDS-PAGE sample buffer and the lysates were examined by SDS-PAGE and Western Blotting. Polyclonal anti-V antigen sera recognised a protein of between 34.8 kDa and 49.5 kDa in both IPTG-induced and uninduced cultures of SL3261/pTrc-LcrV (Fig. 1), consistent with the estimated molecular mass of V antigen (37 kDa). The lysates were prepared from cultures containing similar numbers of viable cells, and the intensities of the bands on the Western blot suggested that there was little difference between the
Fig. 1. Western blot of S. enterica serovar Typhimurium SL3261/ pTrc-LcrV cell lysates separated by SDS-PAGE and probed with polyclonal anti-V serum. Cultures of SL3261/pTrc-LcrV were grown for 6 h in the presence or absence of IPTG, and harvested cells were normalised and lysed in SDS-PAGE sample buffer. Lane M: broad range biotinylated molecular weight marker (Bio-Rad); lane 1: purified rV antigen; lane 2: SL3261/pTrc99A (−IPTG); lane 3: SL3261/pTrc-LcrV (−IPTG); lane 4: SL3261/pTrc-LcrV (+IPTG).
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In some mice, colonisation of the bacterial strains was undetectable. However, in colonised tissues, there was no significant difference between splenic colonisation by SL3261/pTrc-LcrV and SL3261/pTrc99A (Fig. 2), indicating that cloning of the lcrV gene into pTrc99A did not affect colonisation of the bacteria. In addition, enumeration of bacteria in these homogenised tissues showed that pTrc-LcrV was stable in SL3261 in vivo (100%). 3.3. Immunogenicity and protective efficacy of SL3261/pTrc-LcrV Fig. 2. Colonisation of spleens following inoculation of mice (n = 5) with S. enterica serovar Typhimurium SL3261 carrying plasmids pTrc99A (triangles) or pTrc-LcrV (circles). Organs were removed 8, 12 or 16 days after inoculation and homogenised in PBS before plating onto LB agar (closed symbols) or LB agar containing ampicillin (open symbols) for enumeration of bacteria.
level of expression of V antigen in induced and uninduced cultures of SL3261/pTrc-LcrV cultures. 3.2. In vitro and in vivo stability of SL3261 derivatives The stability of pTrc-LcrV and pTrc99A in S. enterica serovar Typhimurium SL3261 in vitro was assessed. Enumeration of the bacteria on agar indicated that pTrc-LcrV and pTrc99A were retained by 100% and 96%, respectively, of SL3261 that had been cultured in L-broth without antibiotic selection for 24 h. The in vivo stability of SL3261/pTrc-LcrV was assessed by determining the colonisation of the Salmonella strain in murine tissues. Following intragastric inoculation of BALB/c mice with 1–5 × 109 cfu of the SL3261 recombinants, it was shown that SL3261/pTrc99A and SL3261/pTrc-LcrV colonised spleens at low levels (Fig. 2).
The induction of V antigen-specific antibodies in mice inoculated orally with S. enterica serovar Typhimurium SL3261/pTrc-LcrV was measured. Groups of 20 BALB/c mice were intragastrically immunised five times at 14-day intervals with 1–5 × 109 cfu of SL3261/pTrc-LcrV, SL3261/pTrc99A or SL3261. V antigen-specific IgG1 and IgG2a concentrations in serum taken from individual mice throughout the study are shown in Fig. 3. On days 69 and 83, the V antigen-specific IgG2a concentration was significantly higher than the IgG1 concentration (P < 0.05). Animals immunised with SL3261/pTrc-LcrV were challenged subcutaneously with 97 LD50 s of a virulent strain of Y. pestis to determine whether immunisation with the Salmonella vaccine strain provided protection against plague. All mice inoculated with Salmonella developed strong Salmonella-specific serum IgG responses, as determined by ELISA (data not shown). Similarly, all mice inoculated with SL3261/pTrc-LcrV developed V antigen-specific serum IgG responses. However, the amount of specific antibody induced varied between individual mice, with IgG1 and IgG2a concentrations of 2470 ± 750 ng/ml and 27600 ± 8340 ng/ml, respectively. Following challenge with Y. pestis GB, 6 out of 20 mice
Fig. 3. IgG1 and IgG2a responses to V antigen determined by ELISA of serum samples taken at various times following intragastric immunisation of mice (n = 20) with S. enterica serovar Typhimurium SL3261/pTrc-LcrV. Results show mean values, with S.E.M. values shown as bars.
H.S. Garmory et al. / Vaccine 21 (2003) 3051–3057 Table 1 Survival of immunised mice against challenge with Y. pestis Group
Number of survivors/total number
SL3261/pTrc-LcrV SL3261/pTrc99A SL3261 Control (naive)
6/20 0/20 1/20 0/5
Groups of mice (n = 20) were inoculated with SL3261/pTrc-LcrV, SL3261/pTrc99A or SL3261 (five doses at 2-week intervals). On day 97, immunised mice and naive control mice (n = 5) were challenged subcutaneously with 97 LD50 s of Y. pestis GB and observed for 24 days.
immunised with SL3261/pTrc-LcrV were protected against plague (Table 1). In comparison, 44 out of the 45 control mice died within 9 days. The surviving control mouse had been inoculated with SL3261 alone and showed symptoms of infection following the Y. pestis challenge. Thus, immunisation of mice with SL3261/pTrc-LcrV afforded significantly greater protection against plague when compared to SL3261/pTrc99A controls (P < 0.05) or naive controls (P < 0.005). However, the protected mice did not show significantly higher V antigen-specific IgG1 or IgG2a responses than non-survivors. The capability of the induced V antigen-specific antibody to compete with a monoclonal antibody, mAb7.3, in binding to recombinant V antigen was evaluated in a competition ELISA. mAb7.3 is a protective monoclonal antibody against plague and is specific for the Y. pestis V antigen. All mice immunised with SL3261/pTrc-LcrV developed anti-V antigen antibody that was able to compete with mAb7.3 (with binding ranging between 13.4% and 52.1%). However, there was no significant difference between binding of serum antibody developed in survivors and non-survivors of plague challenge.
4. Discussion Salmonella vaccines are attractive as carriers of V antigen for a number of reasons. They can be administered orally and, particularly, they are capable of eliciting both systemic and mucosal immune responses, which may be important in protecting against bubonic and pneumonic plague. In this study, the viability of expressing V antigen in Salmonella as a candidate orally-delivered vaccine against plague has been examined. Plasmid pTrc-LcrV [24] was transformed into S. enterica serovar Typhimurium SL3261 and V antigen expression was studied in vitro and in vivo. pTrc-LcrV contains the lcrV gene cloned downstream of the trc promoter and also the lacIq repressor [25]. Although an ideal construct for use in an oral Salmonella vaccine would not contain the lacIq repressor (since co-dosing with IPTG would be required for induction of expression of antigen in vivo), Salmonella containing pTrc-LcrV with 60% of the lacIq gene deleted expressed similar levels of V anti-
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gen as SL3261/pTrc-LcrV as determined by immunoblot analysis (H.S. Garmory, unpublished data). Unexpectedly, no difference in V antigen expression was seen between IPTG-induced and uninduced cultures of SL3261/pTrc-LcrV (Fig. 2). The reason for this is unclear, although ‘leaky’ regulation of protein expression is known to occur when using the pTrc99A expression vector. Expression of V antigen in uninduced cultures of Yersinia pseudotuberculosis containing pTrc-LcrV has also been seen [29]. Therefore, it is possible that maximal levels of expression of V antigen were occurring in uninduced cultures. In our experiments studying the stability of pTrc-LcrV in SL3261 we found that the construct was stable when cultured in L-broth in vitro. Similarly, in Salmonella that colonised murine tissues in vivo, pTrc-LcrV was stably maintained. Although the SL3261 recombinants colonised murine tissues at only low levels, SL3261/pTrc-LcrV colonised murine spleens at a level similar to that of SL3261/pTrc99A (Fig. 2), indicating that V antigen expression did not affect colonisation of the bacteria. SL3261 colonisation was shown to be affected by harbouring the pTrc99A plasmid alone [30]. Following immunisation with SL3261/pTrc-LcrV, V antigen-specific IgG was detected in the serum of all mice. Furthermore, significant protection was afforded against challenge with 97 LD50 s of Y. pestis (Table 1). This is the first time that oral administration of Salmonella expressing V antigen has resulted in protection against plague. However, the concentrations of V antigen-specific antibody induced in mice protected against plague challenge were not significantly different to some antibody concentrations induced in non-protected mice. Similarly, the amount of anti-V antigen antibody that was able to compete with mAb7.3 in binding to recombinant V antigen was not significantly different in survivors and non-survivors. These results suggest that the protection afforded by SL3261/pTrc-LcrV involves an immune mechanism other than antibody against V antigen. The protection afforded against plague in one control mouse inoculated with SL3261 alone raises the interesting possibility of protection afforded by non-specific immunity. It is known that during the initial stages of infection with Salmonella innate immunity is stimulated [31]. Macrophages phagocytose the bacteria and cell wall components of the bacteria, such as lipopolysaccharide and lipoproteins, and induce an inflammatory response in the surrounding tissues. This results in the expression of inflammatory cytokines and various chemokines that recruit cells of the immune system to these sites. Thus, it is clear that the innate immune responses induced by Salmonella vaccination may be sufficient to protect against other non-related pathogens. Immune responses induced by immunisation with purified recombinant V antigen have been well characterised. A sub-unit vaccine consisting of the V antigen and the F1 capsular antigen is currently in clinical trials. IgG1 has been shown to be a correlate of protection for this vaccine in mice [27], indicative of a cytokine profile associated
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with Th2 -type responses. However, Salmonella vaccines are known to elicit Th1 -type responses, indicated by a bias towards an IgG2a isotype in mice. Indeed, in this study immunisation with SL3261/pTrc-LcrV induced predominantly IgG2a against V antigen (Fig. 3), indicating a Th1 -type response. We have previously shown that splenic T cells isolated from mice vaccinated with Salmonella expressing a V antigen-F1 capsular antigen fusion protein proliferated extensively when re-exposed to V antigen in vitro [22]. Furthermore, putative CD4+ T cell epitopes in V antigen have been identified (J. Robinson, personal communication). These findings support the idea that following immunisation, Salmonella expressing V antigen may be able to induce cell-mediated immune responses which are involved in protection against plague. Earlier studies have shown that mice immunised with Salmonella expressing the F1 capsular antigen of Y. pestis can be highly protected against plague [32]. However, it has also been shown that mice vaccinated with F1 capsular antigen alone were not protected against naturally occurring or genetically mutated F1 capsular antigen-negative strains [33]. Hence, the development of a Salmonella vaccine against plague should ideally include expression of V antigen. Co-expression of an F1-V antigen fusion protein confirmed the importance of immune responses to V antigen in protection against plague [22], although the immunity induced was limited and required immunisation via the intravenous route. This study is the first report of protection against plague afforded by oral immunisation of Salmonella expressing V antigen alone. The results from this study should contribute to the development of a highly protective orally-delivered vaccine against plague.
Acknowledgements We thank Sophie Leary and Diane Williamson for their advice and gratefully acknowledge Tony Stagg, Debbie Rogers and Petra Oyston for their expert assistance in this work. We also thank Julie Miller for providing purified recombinant V antigen and Jim Hill for providing anti-V antigen antibodies.
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