Role of antibody to lipopolysaccharide in protection against low- and high-virulence strains of Francisella tularensis

Vaccine 19 (2001) 4465– 4472 www.elsevier.com/locate/vaccine

Role of antibody to lipopolysaccharide in protection against lowand high-virulence strains of Francisella tularensis Mark Fulop a, Pietro Mastroeni b, Michael Green a, Richard W. Titball a,* b

a Defence E6aluation and Research Agency, CBD, Porton Down, Salisbury, Wiltshire SP4 0JQ, UK Centre for Veterinary Science, Department of Clinical Veterinary Medicine, Uni6ersity of Cambridge, Madingley Road, Cambridge CB3 0ES, UK

Received 5 February 2001; received in revised form 7 May 2001; accepted 22 May 2001

Abstract Mice immunised with lipopolysaccharide (LPS) from Francisella tularensis were protected against challenge with the live vaccine strain (LVS). However, when similarly immunised mice were challenged using the fully virulent F. tularensis strain Schu4, only an increase in the time to death was observed. Passive transfer of serum from LPS-immunised mice to naive mice afforded protection against F. tularensis LVS. LPS-immunised mice depleted of either CD4 + or CD8+ T-cells survived a F. tularensis LVS challenge although the rate of clearance of bacteria from the spleen was significantly reduced in the CD8 + depleted group. LPS-immunised mice boosted with F. tularensis LVS were re-challenged with F. tularensis Schu4. This cohort was significantly protected (LD50 increased from B 1 to \1000 CFU). However, passive transfer of serum did not confer protection and mice depleted of CD4 + or CD8+ T-cells did not survive. Crown Copyright © 2001 Published by Elsevier Science Ltd. All rights reserved. Keywords: Francisella tularensis; Lipopolysaccharide; Live vaccine strain

1. Introduction Francisella tularensis is the causative agent of tularemia in man and animals. The glandular, ulceroglandular, ocular, oropharyngeal or pneumonic forms of the disease are the result of the different routes of entry of the pathogen into the host [1]. The high infectivity in man of fully virulent strains of F. tularensis has limited studies with these strains in recent years. However, F. tularensis live vaccine strain (LVS) is virulent in mice [2,3] especially when given by the intraperitoneal (i.p.) or intravenous (i.v.) routes. In addition, sub-lethal doses of F. tularensis LVS induce an immune response, which provides protection against a subsequent i.p. or i.v. challenge with an otherwise lethal dose of F. tularensis LVS. This murine model has been used to explore mechanisms of protection against tularemia [3 – 7].

* Corresponding author. Tel.: + 44-1980-613-301; fax: +44-1980614-307. E-mail address: [email protected] (R.W. Titball).

In humans, F. tularensis LVS is administered by scarification [8–10] and this vaccine has significantly reduced, but not eliminated, tularemia in laboratory personnel exposed to F. tularensis [1,9]. Therefore, the vaccine is generally considered to induce protective immune responses in humans [8,9]. However, the basis of attenuation of the LVS strain is not known [9] and the conditions used to culture the bacterium can influence the degree of attenuation [11]. Against this background, several groups have explored the possibility that a defined sub-unit vaccine could be developed. A range of outer membrane proteins have been shown to be recognised in humans naturally infected with F. tularensis [1,12]. However, immunisation studies with these proteins, even when delivered using systems which stimulate T-cell responses, have failed to elicit protective immune responses against tularemia [13 –15]. In several Gram-negative pathogens, including Escherichia coli [16], Shigella flexneri [17], Brucella spp. [18], Pasteurella multocida [19] and Pseudomonas aeruginosa [20] lipopolysaccharide (LPS) has been shown to be an important protective antigen. Immunisation with purified LPS extracted from F. tularensis LVS protects

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mice from a subsequent challenge with this strain [13]. In this paper we analyse the immune responses that contribute to this protection and compare the protection afforded against a fully virulent strain of F. tularensis.

2. Materials and methods

2.1. Bacterial strains and culture Two strains of F. tularensis were used in this study; F. tularensis LVS and strain Schu4 were obtained from the US Army Medical Research Institute for Infectious Diseases, Maryland, USA. F. tularensis strain Schu4 was originally isolated from a human case of tularemia [21]. F. tularensis LVS was isolated from a mixture of attenuated strains transferred to the USA from the former USSR during the 1950s [1]. Vaccine lots of F. tularensis LVS have been produced in the USA for human use since the 1950s. Clonal master cultures of these strains were stored at −70 °C in modified cysteine partial hydrolysate broth (MCPH) broth supplemented with 10% glycerol. Sub-cultures from these master cultures had been used in our previous studies [22] which had established that in BALB/c mice the i.p. LD50 dose of F. tularensis LVS was 71CFU and the LD50 dose of F. tularensis Schu4 was 1CFU. F. tularensis LVS and strain Schu4 were cultured on blood cysteine glucose agar (BCGA) or in MCPH broth [23,24]. Bacteria used for animal challenge were washed three times in phosphate buffered saline (PBS), re-suspended in MCPH broth supplemented with 10% glycerol, and stored in 0.5 ml volumes at − 70 °C.

when given by the i.p. route, we were unable to immunise mice by this route with this strain. Therefore we considered that immunisation with purified LPS and then with F. tularensis LVS would allow us to investigate whether protection against high virulence strains of F. tularensis required immune responses additional to those induced by purified LPS. These mice were immunised with LPS and boosted i.p. with 1×106 CFU F. tularensis LVS eight weeks after the final LPS immunisation. Unless otherwise indicated, groups of 5 BALB/c female mice (6–8 weeks old) were used throughout this investigation.

2.4. CD4+ and CD8+ T-cell depletions Rat anti-mouse CD4+ (clone YTS191.1) and CD8 + (clone YTS169.4.2) antibody producing hybridomas [27,28] were used to produce the depleting monoclonal antibodies (MAbs). These hybridomas were a kind gift from A.A. Nash, University of Edinburgh, Scotland. Total globulins from ascitic fluid were obtained after 40% ammonium sulphate precipitation and dialysis against PBS. Three days before challenge mice received 0.5 mg MAb i.p. One day before challenge and at 5 day intervals after challenge mice received 0.25 mg MAb i.p.

2.5. Clearance of F. tularensis Mice were killed at various intervals post challenge and their spleens aseptically removed. The spleens were homogenised in 10 ml PBS using a Lab Blender 80 stomacher and the number of viable bacteria determined after the culture of diluted homogenates on BCGA agar. Results are expressed as CFU/spleen.

2.2. Lipopolysaccharide preparation 2.6. Fluorescence acti6ated cell sorting analysis LPS from F. tularensis LVS was extracted by the hot phenol method [25] and purified using ultracentrifugation and treatment with nucleases and proteases as described by Fulop et al. [23]. The protein content of the LPS preparation was determined by FarbstoffKonzenrat reagent (BioRad, Watford, UK) and the nucleic acid content was determined by addition of ethidium bromide and measurement of the UV-induced fluorescence as described by Sambrook et al. [26].

2.3. Immunisations To investigate the role of antibody against LPS we immunised mice i.p. with LPS isolated from F. tularensis LVS. Mice were given three 50 mg doses of LPS at 7 day intervals. However, we also considered that additional vaccine antigens of F. tularensis LVS might be required for protection against high virulence strains of F. tularensis. Because the LVS strain is virulent in mice

Spleens from T-cell depleted mice and control mice were removed aseptically. Splenocytes were washed in Dulbecco’s modified eagle’s media supplemented with 2 mM glutamine, 10% fetal calf serum, 100 U/ml penicillin, and 0.1 mg/ml streptomycin. 2× 105 cells were added to 3 ml of PAF (PBS supplemented with 0.1% sodium azide and 10% foetal calf serum) and were washed in this solution. For each animal duplicate tubes of the following MAb-conjugate combinations (obtained from Pharminigen) were set up: anti-CD3phycoerythrin (PE) labelled MAb with anti-CD4fluorescin isothiocyanate (FITC) labelled MAb, anti-CD3-PE labelled MAb with anti-CD8-FITC labelled, and control-MAb-PE labelled with controlMAb-FITC labelled. The MAb/cell suspension was incubated for 30 min at room temperature in the dark before 2 ml of lysing fluid (Becton Dickinson UK Ltd., Oxford, UK) was added to each tube to selectively lyse

M. Fulop et al. / Vaccine 19 (2001) 4465–4472

erythrocytes. Cells were incubated at room temperature for 20 min then washed and resuspended in 0.3 ml PAF. Two colour analysis performed by FACScan (Becton Dickinson UK Ltd.) was completed on 4000 cells from each sample.

2.7. Passi6e serum transfer Serum was obtained from LPS-immunised mice and F. tularensis LVS-boosted mice by cardiac puncture 8 weeks after the last immunisation dose. Naive mice received 0.4 ml serum i.p. 2 h before challenge. Control mice received 0.4 ml of normal mouse serum i.p.

2.8. Enzyme-linked immunosorbent assay The quantity of F. tularensis LPS-specific immunoglobulin (Ig) M, IgG and its subclasses was determined by ELISA following the method of Gupta and Siber [29] with these modifications. In all ELISAs the antibodies were diluted in PBS supplemented with 1% (w/v) skimmed milk powder. Briefly, LPS was coated onto the wells in a microtitre plate (Dynatech) in antigen binding buffer, pH 9.6 (Sigma). After washing, diluted mouse serum was added. Serum antibody which bound to the LPS was detected using horseradish peroxidase-labelled antibodies specific for murine IgM, IgG, IgG1 or IgG2a (Sera-lab). To generate a standard curve for the quantitation of the bound antibody, other wells in the microtitre tray were first coated with antimurine FaB antibody. After washing known amounts of murine IgM, IgG, IgG1 or IgG2a (Sigma) were added to the wells. Bound antibody was detected using the horseradish peroxidase-labelled antibodies specific for murine IgM, IgG, IgG1 or IgG2a (Sera-lab) which were used for the LPS-based ELISA. The enzyme substrate used was 2,2%-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) in 0.056 M citric acid, 0.088 M Na2HPO4, pH 5.5 buffer supplemented with 0.003% H2O2. Preliminary experiments showed that the anti-IgG subclass horseradish peroxidase conjugates and the IgM horseradish peroxidase conjugate did not react with LPS or the anti-murine FaB antibody. The results are expressed as the amount of antibody per ml of serum.

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method of Reed and Muench [30]. Ninety five percent confidence limits were determined by probit analysis [31]. To determine significant difference in the mean time to death the Student’s t-test was used. Spleens from dead mice were aseptically removed, smeared onto BCGA supplemented with 55 mg ml − 1 ampicillin, and incubated at 37 °C. The identity of colonies as F. tularensis was confirmed by Gram-staining and by using the polymerase chain reaction [24].

3. Results

3.1. Passi6ely transferred antibodies to LPS protect against challenge with F. tularensis LVS Previously we showed that mice immunised with LPS from F. tularensis LVS survived a subsequent i.p. challenge with F. tularensis LVS (LD50 increased from : 100 CFU to greater than 106 CFU) [13]. Our initial experiments were designed to identify the immune responses responsible for protecting LPS-immunised mice from a F. tularensis LVS challenge. Sera were taken from mice immunised with LPS and analysed using an ELISA. The concentrations of IgM antibody to LPS in these sera were 3.4 times the concentrations of total IgG antibody to LPS (Fig. 1). The predominant IgG subclass to LPS was IgG1 (66% of the total IgG). To examine the role of humoral immunity in protection, serum obtained from LPS-immunised mice was transferred to naive mice which were subsequently challenged with F. tularensis LVS ( : 1000 LD50 doses). Mice receiving serum containing anti-LPS antibodies all survived the challenge whereas the control mice, which received normal mouse serum, all died (Table 1).

2.9. Challenge Mice were challenged i.p. with either F. tularensis LVS (1 ×105 CFU) or with fully virulent F. tularensis strain Schu4 (100 CFU) 8 weeks after their final immunisation, and were observed for 14 days. These doses were selected because the time to death in naive mice is 4 days and represents an equal level of challenge. Statistical significance was determined using chi square analysis with Yates’ correction. For some experiments the median lethal dose (LD50) was determined by the

Fig. 1. Serum concentration of IgM, IgG and IgG subclass antibodies against F. tularensis LPS, in mice which had been immunised with LPS, or immunised with LPS and then boosted with F. tularensis LVS. Error bars are the standard error of the mean of four replicates.

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Table 1 Experiment to determine the roles of cell mediated immunity and humoral immunity in LPS immunised mice Immunological status

Survivors

Naive mice+serum from LPS-immunised mice Naive mice+normal mouse serum LPS-immunised mice, CD4+ T-cell depleted LPS-immunised mice, CD8+ T-cell depleted LPS-immunised mice, CD4+ and CD8+ T-cell depleted LPS-immunised mice, control MAb Naive mice, CD4+ T-cell depleted Naive mice, CD8+ T-cell depleted

5/5 0/5 5/5 5/5 5/5 5/5 0/5 0/5

Survival in LPS-immunised mice depleted of CD4+ or CD8+ T-cells, naive mice transferred with serum from LPS-immunised mice was measured for 14 days after a challenge with F. tularensis LVS (1000 LD50).

3.2. CD8+ T-cells contribute to the clearance of F. tularensis LVS in LPS-immunised mice To determine the role of T-cells in protective immunity we depleted LPS-immunised mice of CD4+ or CD8 + or CD4 + and CD8 + T-cells immediately prior to and during an F. tularensis LVS challenge. To show that mice receiving anti-CD4+ or anti-CD8 + MAbs were depleted of these cell types fluorescence activated cell sorting (FACS) analyses were completed on splenocytes obtained from mice receiving antiCD4 + MAb, and anti-CD8 + MAb, and a control MAb. Approximately 65% of CD3 + cells were CD4+ and 35% were CD8+ in mice receiving the control MAb. Less than 1% of CD3 + cells were CD8+ when anti-CD8 + MAb was administered and less than 2% were CD4 + when anti-CD4 + MAb was administered to mice. All of the mice which had been immunised with LPS and then depleted of CD4+ or CD8 + or CD4 + and CD8 + T-cells survived i.p. challenge with 1× 105 CFU (:103 LD50 doses) of F. tularensis LVS (Table 1). To investigate whether similarly treated mice showed different abilities to clear the challenge dose, the number of bacteria present in splenic tissue was determined at intervals after challenge (Fig. 2). The results showed similar bacterial loads in the spleens of control and T-cell depleted mice which had been immunised with LPS on day 3 of the infection. In control mice which had not been immunised, the bacterial numbers in the spleen were 100-fold higher. These mice died on day 4 of the experiment whereas the number of bacteria in the spleens of LPS-immunised mice declined until the termination of the experiment (day 14). Later in the infection, the rate of bacterial clearance from spleens was reduced in CD8+ T-cell depleted mice as compared to controls and CD4+ T-cell depleted animals (Fig. 2). We concluded that anti-LPS antibodies were

sufficient to limit an infection caused by F. tularensis LVS. In mice passively immunised with anti-LPS antibodies, CD4+ T-cells were not required for bacterial clearance and CD8+ T-cells played a role in enhancing the clearance of bacteria.

3.3. Immunisation with LPS increases the time to death of mice challenged with F. tularensis Schu4 We examined the immune responses that would protect mice from a virulent F. tularensis Schu4 challenge. LPS-immunised mice were challenged with increasing doses of F. tularensis Schu4. Control mice which were not immunised died within 4 days of challenge. No significant increase in the LD50 was observed compared to naive mice although there was a significant increase in the mean time to death of : 24 h at each dose (Table 2).

3.4. Mice immunised with LPS then F. tularensis LVS are protected against challenge with F. tularensis Schu4 Mice which had been immunised with LPS were subsequently boosted by i.p. inoculation of F. tularensis LVS (1 ×106 CFU/ dose). Sera from these mice showed a similar profile of antibodies to LPS, as sera taken from mice which had been immunised only with LPS. However, the concentration of antibodies was higher in the group which were boosted with F. tularensis LVS. Eight weeks later these mice were challenged with F. tularensis Schu4 (Table 2). These mice were significantly protected (LD50 \ 1000 CFU) compared to control

Fig. 2. Clearance of F. tularensis LVS from spleens from mice which had been immunised with LPS. Also shown is the pattern of clearance from mice which had been immunised with LPS and then depleted of CD4+ or CD8+ T-cells before challenge. Naive mice were not immunised with LPS before challenge. Error bars represent the standard error of the mean of six. Statistical significance was determined using the Student’s t-test.

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Table 2 Experiment to determine the immunological status required for BALB/c mice to survive an i.p. challenge with F. tularensis Schu4

Naive mice LPS-immunised mice LPS-immunised F. tularensis LVS-boosted mice

Challenge dose of F. tularensis schu4 (CFU) 1000 100

10

LD50 (CFU)

Survivors

mean ttd

Survivors

mean ttd

Survivors

mean ttd

0/5 0/5 4/5

96 h ( 9 0) 115 h ( 9 11) 240 h

0/5 0/5 4/5

96 h ( 90) 120 h ( 90) 168 h

0/5 0/5 5/5

100 h ( 911) 134 h ( 913) NA

B10 B10* \1000

ttd, time to death ( 9 SD); NA, not applicable; Mean time to death was : 24 h longer than control group. * PB0.01 as determined by Student’s t-test.

mice (LD50 B 10 CFU). To evaluate the role humoral immunity played in the observed protection, serum from F. tularensis LVS boosted mice was transferred into five naive mice. None of these mice survived when subsequently challenged with 100 CFU of F. tularensis Schu4. To determine the role of T-cell immunity in the observed protection, we immunised 15 mice with LPS then boosted them with F. tularensis LVS. Groups of five mice were depleted of CD4+ or CD8 + or CD4 + and CD8 + T-cells and challenged with 100 LD50 doses of F. tularensis Schu4. All of these mice died.

4. Discussion The LVS strain of F. tularensis is the only vaccine available against tularemia, and has been used widely in man as an investigational new drug. We have set out to investigate the immune responses to the LPS from F. tularensis LVS and to determine the potential use of LPS as a vaccine against the low and high-virulence strains (LVS and Schu4) of F. tularensis. Previous workers have shown that the virulence of F. tularensis LVS can vary according to the cultural conditions [11]. The LD50 doses of the LVS and Schu4 strains were not measured in the work reported here. However, we have previously shown that the same isolates of F. tularensis derived from the same master cultures, and cultured in the same manner, were of low and highly virulence respectively in the murine model of disease [22]. Following immunisation of humans with F. tularensis LVS, responses have been demonstrated against a range of protein antigens and LPS [1,10,12]. F. tularensis is an intracellular pathogen and the nature of the protective response is generally assumed to be T-cell mediated [1,12,32,33]. Although several T-cell reactive outer membrane proteins have been identified [12], studies to date have failed to identify the protective antigen(s) expressed by the LVS strain. Although most of the recent studies have focused on the role of T-cells in protection against tularemia, there

is evidence that antibody also plays a role in protection at least against some strains of F. tularensis. Humans who had been infected with F. tularensis possessed serum antibodies against LPS [5] and against outer membrane proteins [38]. Sera obtained from humans or mice which had been immunised with F. tularensis LVS, protected naive mice against a subsequent i.p. challenge with LVS [4– 6]. Early attempts to treat tularemia in humans with immune sera provided conflicting results [1]. Some workers showed that immune serum could successfully be used to treat individuals suffering from tularemia [34] whilst others reported it was of no value for the treatment of tularemia in humans or in animals [35– 37]. The identity of the protective antibody which was passively transferred in these previously reported studies was not determined. It is suggested that the conflicting results in humans might have reflected differences in the virulence of the infecting strains [1] and it may be significant that all of the recent studies demonstrating protection with immune sera have used mice challenged with the low-virulence LVS strain. We have previously shown that mice immunised with purified LPS isolated from F. tularensis LVS were protected against i.p. challenge with F. tularensis LVS [13]. The high levels of IgM and IgG3 following immunisation with LPS are indicative of a T-independent response [39]. The high level of IgG1 (an IL-4 dependent antibody) to LPS suggests either some form of T-cell bystander help or some involvement of other cytokine producing cells such as NK cells. In this study we have shown that serum taken from LPS-immunised mice, and transferred into naive mice, provides protection against subsequent i.p. challenge with 105 CFU of F. tularensis LVS ( : 103 LD50 doses). This result contrasts with the finding of Dreisbach et al. [40] who reported that antisera containing antibodies to LPS from F. tularensis LVS could not protect mice against 102 CFU of F. tularensis LVS given by the i.p. route. However, these workers used only a single immunising dose of 10 mg of LPS whereas we have immunised mice with three 50 mg doses of LPS to obtain immune serum. Conlan et al. [32] have previously shown that mice which had

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been immunised i.v. with a sublethal dose of F. tularensis LVS were subsequently protected against challenge with : 10 LD50 doses of F. tularensis LVS given by the same route. Mice which were depleted of CD4+ and/ or CD8 + T-cells immediately before and during challenge were also protected. We observed a similar pattern of protection in our experiments using mice immunised with purified LPS and then depleted of CD4 + and/ or CD8 + T-cells. Therefore, we believe that the protection which was reported by Conlan et al. may have been in part due to antibodies against LPS, which developed after immunisation with F. tularensis LVS. Although mice depleted of CD4+ and/ or CD8 + T-cells were able to control an infection with F. tularensis LVS, our results indicate that CD8+ T-cells do play a role in the clearance of bacteria. These results are similar to the findings of Rhinehart-Jones et al. [4] who showed that immune serum raised against F. tularensis LVS was not able to protect nu/nu mice or mice depleted of interferon-g (IFN-g). In other studies IFN-g and tumour necrosis factor-a have been shown to play a key role in controlling the growth of F. tularensis LVS in vivo in both non-immune mice [41] and in mice previously immunised with a sub-lethal dose of F. tularensis LVS [42]. Overall, our results are consistent with the hypothesis proposed by Rhienhart-Jones et al. [4] that antibodies to F. tularensis, which we believe to be directed mainly against LPS, provide sufficient protection to allow the host to clear the challenge. Although we have shown that CD8 + T-cells play a role in clearance, other cells such as NK cells may also contribute to this protective response [7]. Previous studies have shown that passive immunisation of mice with serum from rabbits immunised with F. tularensis LVS did not provide protection against high virulence strains of the bacterium [35]. In this study we have shown that immunisation with LPS isolated from F. tularensis LVS did not protect mice against death after challenge with 102 LD50 doses of the fully virulent F. tularensis strain Schu4. However, the time to death of these mice was significantly increased. This increase in time to death for LPS-immunised mice indicated that the disease process had been modulated. This is the first time significant protection has been obtained to a fully virulent F. tularensis challenge in BALB/c mice. However, clearly antibodies against LPS alone were not sufficient to alter the host-pathogen relationship sufficiently to provide solid protection against high virulence strains. It is possible that the LPS produced by F. tularensis strains LVS and Schu4 is not immunologically identical. However, we have previously shown that a monoclonal antibodies against the O-antigen and core polysaccharide of the LPS from F. tularensis LVS reacted equally with a wide range of F. tularensis strains, including strain Schu4 [23]. In addi-

tion, sera from humans immunised with F. tularensis LVS has been shown to recognise LPS from both F. tularensis LVS and strain Schu4 [5]. These findings suggest that the LPS from these strains are immunologically similar. However, we cannot discount the possibility that there are some antigenic differences between the LPS isolated from these strains which were not identified using these approaches. Our findings therefore support the suggestion that antibody is only effective in controlling tularemia caused by low virulence strains of F. tularensis [1]. The nature of the differences in the mechanisms of clearance of low and high virulence strains of F. tularensis require further investigation. However, in preliminary studies we have shown that F. tularensis Schu4 grows unrestrictedly in murine macrophages leading to cell lysis whereas F. tularensis LVS persists but does not grow [43]. This may explain why cell mediated responses are more important for the clearance of F. tularensis Schu4 in vivo. When subsequently boosted with F. tularensis LVS, LPS-immunized mice were protected against lethal challenge with F. tularensis Schu4. Because we could not transfer immunity with serum, and depletion of either CD4+ or CD8 + T-cells abolished this protection, we believe that cell mediated immunity involving CD4 + and CD8 + T-cells is required to protect mice against fully virulent strains of F. tularensis. Clearly the exposure of LPS-immunised mice to an otherwise lethal challenge of F. tularensis LVS allowed the development of a protective immune response. It is likely that antigens in addition to LPS were recognised after immunisation with F. tularensis LVS. These additional antigens may have contributed to the induction of a protective response and may have been responsible for the protective CD4+ and CD8 + T-cell response in these mice. Our findings suggest that the immunogenic moiety of LPS from F. tularensis should be considered as one component of a sub-unit vaccine to protect against tularemia. This is likely to be the O-antigen, but further work is required to investigate this possibility. Removal of the lipid A moiety might also be necessary to detoxity the LPS sufficiently for use in a human vaccine. In addition, the suitability of LPS from F. tularensis LVS as a component of a sub-unit vaccine is dependent on the demonstration that LPSs from a range of F. tularensis strains are immunologically similar. Our findings also suggest that other antigens would be required in a sub-unit vaccine to protect against all high virulence strains of F. tularensis. Additional protein antigens could serve as carriers for the immunogenic polysaccharide region of the LPS. Finally, a sub-unit vaccine for tularemia would need to induce both humoral and cellular responses to provide protection against high-virulence strains of F. tularensis.

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