Salmonella typhi O-polysaccharide-tetanus toxoid conjugated vaccine

Manoj Saxena and Jose L. Di Fabio*

A Salmonel la typhi conjugated vaccine was prepared by covalently linking the antigenic O-polysaccharide, selectively activated by periodate oxidation, to tetanus toxoid via reductive amination. The immunogenicity of the conjugate (O-TT) was examined by injecting Balb/c mice with 5129 of the conjugate and Alhydrogel as adjuvant, boosting 14 and 28 days alter the primary immunization, and quantification of the development of anti-polysaccharide and anti-tetanus toxoid antibodies by enzyme-linked immunosorbent assay. Mean anti-O-chain titres after the first and second boost were 129 and 502, respectively, while anti-tetanus toxoid titres were 159 and 1000, respectively. Anti-O-polysaccharide antibodies exhibited complement-mediated bactericidal activity against S. typhi. Immunized mice were fully protected against challenge with 10 LDso of S. typhi Ty2 (p<O.O01) and partially protected against challenge with IOOLDso of S. typhi Ty2 (p < 0.04).

Keywords: O-polysaccharide; tetanus toxoid; conjugate vaccines; Salmonella typhi

Typhoid fever continues to be an important public health problem, especially in the developing world, with an estimated 12.5 million cases per year 1. The worldwide case fatality rate is reported to be about 1% 2 . The isolation of multiple-drug-resistant Salmonella typhi from typhoid cases 3'4 and the high incidence of adverse reactions associated with the traditional whole-cell killed vaccines 5 have led to the development of a new generation of typhoid vaccines. These include the galE mutant Ty21a 6 and purified Vi capsular polysaccharide alone or conjugated to protein carriers 7'8. Experimental evidence of the contribution of Salmonella lipopoly- saccharide (LPS) to bacterial pathogenicity 9, as well as seroepidemiological observations of the appearance of elevated anti-LPS antibodies in convalescent and immune sera 1° 13, highlights the potential of using LPS or its components in the formulation of a vaccine against typhoid fever. While the innate toxicity of the LPS molecule 14 precludes its use in a parenteral vaccine, the non-toxic antigenic O-chain polysaccharide may be a

Bacterial Products Division, Bureau of Biologics, Tunney's Pasture, Ottawa, Ontario, Canada K1A 0L2. *To whom correspondence should be addressed at: Pan American Health Organization, 525 Twenty Third Street, NW, Washington, DC 20037-2895, USA. (Received 21 April 1993; revised 2 November 1993; accepted 8 November 1993)

Presented in part at the NATO Advanced Research Workshop on 'Biology of Salmonella', Sicily, Italy, 10-15 May 1992

possible alternative. Antibodies to O-polysaccharide are expected to opsonize LPS and whole bacteria and thus would provide an additional bactericidal effect on Gram-negative bacteria.

Improvement in the efficacy of polysaccharide-based vaccines has largely been possible due to the development of techniques for the covalent linking of oligo- or polysaccharides to protein carriers 15. The present study was undertaken to synthesize a S. typhi O-chain tetanus toxoid conjugate and to evaluate its immunogenicity in mice.

M A T E R I A L S A N D M E T H O D S

Isolation and oxidation of O-chains from S. typhi LPS

S. typhi LPS (Sigma, St Louis, MO, USA), purified by enzyme treatment and ultracentrifugation 16, was dissolved in 1% acetic acid and heated for 1 h at 100°C. Precipitated lipid A was removed by low-speed centrifugation. The resultant supernatant was chromatographed on Sephadex G-50 (Pharmacia, Piscataway, N J, USA) using 0.05 M pyridinium acetate buffer, pH 5.4, as the eluent. The high-molecular-weight glycose-positive fractions were pooled and lyophilized, representing native O-chains. Native O-chains (30rag), dissolved in 3.0ml of water, were oxidized by treatment with sodium metaperiodate (0.3 hal, 0.1 N), followed by boiling in 1% acetic acid to perform a Smith hydrolysis 17. Purified oxidized O-chains were obtained by re-chromatography on Sephadex G-50 as described above for native O-chains.

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Salmonella typhi conjugated vaccine: M. Saxena and J.L. Di Fabio

Conjugation of S. typhi O-chain polysaccharide to tetanus toxoid

Concentrated tetanus toxoid (TT) was obtained from Connaught Labs, Willowdale, Ontario, Canada. Mono- meric TT was isolated by chromatography on BioGel A 0.5m (BioRad Lab., Richmond, CA, USA), using 0.01 M phosphate-buffered saline (PBS), pH 7.2, as the eluent. Conjugation of oxidized O-chains to monomeric TT was performed via reductive amination TM as adapted by Aron eta[. 19. In brief, 5 mg of oxidized O-chain was dissolved in 0.1 y sodium bicarbonate buffer, pH 8.1 (200 #1). TT {4 rag) was added to the O-chain solution, followed by 10rag of sodium cyanoborohydride. The reaction mixture was left at room temperature for 7 days. Successful conjugation was monitored by chromato- graphing small aliquots of the reaction mixture on Superose 12 (Pharmacia: 0.01M PBS, pH7.2) at different time intervals. At the end of the incubation period, the conjugate was purified by chromatography on BioGel A 0.5m (0.01 M PBS, pH 7.2). The high- molecular-weight fractions containing carbohydrate and protein were pooled, dialysed and freeze-dried yielding 3.1 mg of purified tetanus toxoid O-chain conjugate (O-TT}.

Conjugation of S. typhi O-chain to bovine serum albumin

The conjugation of S. typhi O-polysaccharide to bovine serum albumin (BSA) was performed using a method similar to that used for the tetanus toxoid conjugate, yielding 4.2 mg of BSA O-chain conjugate (O-BSA). BSA fraction V was procured from Sigma, USA and used as supplied. O-BSA conjugate was used as coating antigen in the anti-O-chain enzyme-linked immunosorbent assay (ELISA).

Chemical characterization of conjugates

The protein content of O TT and O BSA was quantified by the Bradford assay 2°. Carbohydrate content was measured by the phenol sulfuric acid assay 2 using purified O-chain as the standard.

Immunization schedule

Female Balb/c mice weighing 18 22 g each were used for the study. Three groups of 20 mice each were used and were injected subcutaneously with the following preparations:

• group I: 5#g O TT mixed in 50#1 of Alhydrogel (Cedarlane, Hornby, Ontario, Canada)

• group II: 5#g O - T T in 50#1 of physiological saline

• group III: saline control.

Mice in all three groups were boosted with the same preparation 14 and 28 days after the first immunization. Mice were bled via the retro-orbital plexus 21 and 35 days after the first injection. Sera were collected and stored at - 2 0 ° C until further analysis.

Determination of anti-polysaccharide antibodies

S. typhi O-chain-specific mouse antibodies were quantified by ELISA with goat anti-mouse IgG conjugated to horseradish peroxidase (HRPO) (Kirke- gaard & Perry Labs, Gaithersburg, MD, USA) at a 1/1000 dilution. ELISA plates (96 wells; Nunc Immuno

Polysorp, Roskilde, Denmark) were coated by adding 100#1 per well of an O-BSA conjugate solution (10#1m1-1) in 0.01 M PBS, pH 7.0, and incubating for 2 h at 37°C. Plates were blocked by addition of 1% (w/v) skimmed milk solution (Difco, Detroit, MI, USA). Mouse sera to be tested were added at different dilutions, followed by the addition of enzyme-labelled goat anti-mouse IgG. Enzyme activity was measured by the addition of tetramethyl benzidine (TMB: Kirkegaard & Perry Labs) as substrate, and the colour reaction was stopped by the addition of 0.1 M phosphoric acid. Plates were read at 450nm on an automated ELISA reader (Molecular Devices UVMax, Menlo Park, CA, USA). Antibody titres were defined as the highest dilution of serum that gave an absorbance value of 0.5 under standardized conditions.

In another experiment, a modification of the above ELISA method was used to determine the specificity of antibodies of the O-chains. S. o'phosa smooth LPS (S-LPS, Sigma, USA), S. minnesota Ra LPS (Ra-LPS, List Biologicals, Campbell, CA, USA) and O BSA were used as coating antigens at concentrations of 1 and 0.1 #g/well, The quantification of antibodies to the three antigens was similar to the method described above.

Determination of anti-tetanus antibodies

Anti-tetanus (anti-TT) antibodies were quantified by ELISA using plain tetanus toxoid at a concentration of 0.25 flocculation units {L D ml-1 as the coating antigen. Blocking was performed with 1% BSA in PBS Tween. The addition of test sera, HRPO-labelled goat anti-mouse IgG and substrate (TMB) was similar to that described above. Reactions were stopped by addition of 0.1 M phosphoric acid and plates were read at 450 nm. Anti-TT titres were defined as the highest dilution of serum that gave an absorbance value of 0.5 under standardized conditions.

Determination of in v#ro bactericidal activity

To determine whether antibody to O-polysaccharide could interact with complement to kill S. typhi Ty2, a bactericidal assay based on a modification of standard assays w a s used 22.

S. o'phi Ty2 was grown to mid-logarithmic phase in brain heart infusion broth (BHI), pelleted by centri- fugation, washed twice with physiological saline and finally resuspended to 3 x 103 colony-forming units (c.f.u.)m1-1 in Hanks buffered salt solution (HBSS) containing Ca 2 +, Mg 2 + and 3% BSA. Normal guinea-pig sera were adsorbed with S. typhi Ty2 at 4°C to remove any antibacterial activity from the normal guinea-pig sera. Different dilutions of test mouse sera (50 #1) were mixed with bacterial cell suspension (30 #1) and adsorbed guinea-pig complement (20 #1). Reaction mixtures were incubated at 37~C with continuous agitation. After 1 h incubation, suspensions were plated onto BHI agar to determine the number of surviving bacteria in each case. The bactericidal titre was calculated as the maximum dilution of mouse serum required to kill 50% of the initial inoculum. To rule out non-specific killing due to complement, control wells containing HBSS (50#1), complement (20 #1) and 30/A of bacterial cell suspension were used. The absence of bacterial killing in these wells was used to check for complete adsorption of sera.

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Protection studies

The LDso of S. typhi Ty2 was determined by intraperitoneally injecting graded doses of the test organism into groups of ten Balb/c mice (102-107 c.f.u, ml --1 in 0.5 ml of 5% hog gastric mucin (Sigma, USA). Challenged mice were observed for 7 days for mortality, and the number of deaths was recorded for each group. The test dose resulting in death in 50% of the animals was calculated by standard methods and found to be 3 × 103 c.f.u, m1-1. The immunoprotective ability of the conjugate was studied as follows. One day after the second bleed, i.e. on day 36, immunized mice were divided into groups of ten mice each and challenged intraperitoneally with 10 and 100 LDso of S. typhi Ty2 (0.5 ml in 5% hog gastric mucin). Mice were observed for 7 days postinfection and the number of deaths was recorded for each group. Non-immunized mice, similarly challenged, served as controls. The data were analysed for statistical significance using the X 2 test.

Table 1 Immunogenicity of S. typhi O-chain-tetanus toxoid conjugate for Balb/c mice. Anti-polysaccharide antibodies in mouse sera after one and two boosts

Mean anti-polysaccharide titre ( _+ s.d.)*

Immunizing Test group preparation a b c

Group 1 5/~g O -TT+ <20 129_+34 - 502-+48 50 pl Alhydrogel

Group 2 5/~g O -TT+ <20 <20 63+ 13 50/~1 saline

Group 3 Saline control <20 <20 <20

*a, prebleed titres; b, sera collected 2 weeks after first boost; c, sera collected 2 weeks after second boost

Table 2 Immunogenicity of O-chain-tetanus toxoid conjugate for Balb/c mice. Anti-tetanus antibodies in mouse sera after one and two boosts

Mean anti-TT titre (+s.d.)* Immunizing

Test group preparation a b c

RESULTS

Synthesis of O-chain-protein conjugates

Purified and selectively oxidized O-chain polysac- charide from S. typhi was covalently linked to tetanus toxoid and to bovine serum albumin by reductive amination. The conjugation was monitored by analysing aliquots on a fast protein liquid chromatography (FPLC) system, using a Superose 12 column. An increase in the molecular weight (Kav) of the protein was observed during the conjugation process, which was considered complete when no further shift of the protein peak was recorded. The conjugate was purified by gel permeation chroma- tography on BioGel A 0.5m, and the high-molecular- weight fractions containing both carbohydrate and protein were pooled and freeze-dried after exhaustive dialysis. Fractionation was done in such a way that no unbound O-polysaccharide could be present. The conjugation reactions yielded 3.1 mg of purified O-TT and 4.2 mg of purified O-BSA conjugate.

Chemical analysis of the conjugates indicated 32% O-polysaccharide and 68% protein for the TT conjugate, and 37.5% O-polysaccharide and 62.5% protein for the BSA conjugate. These percentages assume protein and carbohydrate to be the sole components of the preparations. In terms of molar composition, the tetanus conjugate thus consists of 3 or 4 O-chains attached to each TT molecule, and the BSA conjugate consists of 2 or 3 O-chains attached to each BSA molecule, assuming that the molecular weight of the O-chain is between 15 000 and 20 000.

lmmunogenicity of the O-chain-TT conjugate

The immunogenicity of the O-TT conjugate was examined by subcutaneously injecting 5/zg of the conjugate into Balb/c mice with and without Alhydrogel. Animals were boosted twice at two-weekly intervals with the same preparation. Groups of animals were bled prior to the experiment and then 2 weeks after each boost. The development of anti-polysaccharide antibodies was quantified by an ELISA assay directed to the O-chain. The results in Table I show that detectable antibody responses were seen in the group receiving conjugate with Alhydrogel as early as 2 weeks after the first boost.

Group 1 5 #g O-TT+ <20 159_+23 1000_+109 50 #1 Alhydrogel

Group 2 5 pg O-TT+ <20 120_+18 630_+37 50/~1 saline

Group 3 Saline control <20 <20 <20

*a, prebleed titres; b, sera collected 2 weeks after first boost; c, sera collected 2 weeks after second boost

E

,<

0.9

0.6

0.3

0.0 3 4

l O-BSA

log serum dilution

l S-LPS ,t Ra-LPS

Figure 1 ELISA of sera from mice immunized with S. typhi O-chain-tetanus toxod conjugate using S. typhi O-chain-BSA, S. typhi S-LPS and S. minnesota Ra-LPS as coating antigens

In addition, the animals exhibited a fourfold increase in titre after the second boost. However, animals injected with the conjugate alone did not show anti-O-chain antibodies after the first boost. Further, after the second boost in this group, sera were positive for anti O-chains in the ELISA but had titres which were quantitatively lower than those from the adjuvant group after one boost.

The O-chain specificity of the sera was investigated by ELISA. Nunc Polysorp plates were coated with 0.1 or l#g/well of three antigens, namely O-chain-BSA conjugate, S-LPS from S. typhi and Ra-LPS. Mouse immune sera were then added to different wells at fixed dilutions ranging from 10 2 to 10 5. The results in Figure 1 show that the antibodies bind to the BSA conjugate and the S-LPS and that they do not bind to the Ra-LPS

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Salmonella typhi conjugated vaccine: M. Saxena and J.L. Di Fabio

which corresponds to Salmonella rough LPS with complete core.

Conjugation of the polysaccharide did not affect the immunogenicity of the protein carrier. The results in Table 2 show that the antibodies against TT were induced in conjugates with and without Alhydrogel. As expected, reinjection of the vaccine had a booster effect and anti-TT titres were 5-6-fold higher after the second boost compared with the first.

O-chain Core Lipid A

' I ; ' l

Core Lipid A

S - L P S

Bactericidal assay

The functional activity of the anti-O-chain antibodies was determined in vitro by quantifying complement- mediated bactericidal activity towards S. typhi Ty2. The sera were bactericidal for the target organism and the mean bactericidal titre after two boosts was found to be 60 (Figure 2). The bactericidal activity after one boost was low and was not significantly different from background activity.

Mouse protection studies

The ability of anti-O-chain antibodies to confer active immunity against typhoid was studied in the mucin-mouse model. After immunization, 100% (10/10) and 50% (5/10) of the mice survived intraperitoneal challenge with l0 and 100 LDso of S. typhi Ty2 (in 5% hog gastric mucin), respectively.

DISCUSSION

LPS is considered to be a major virulence factor in Salmonellae. A number of researchers have reported that LPS has multiple roles in the pathogenesis of Salmonella infections, and, besides the toxicity of the lipid A component, the O-antigen chains play an important role in resistance to serum bactericidal killing and phago- cytosis23 -25. In addition, Mroczenski-Widley et al. 9 have shown the role of smooth LPS in adhesion and invasion of S. typhi, processes which are critical for the pathogenic process. Jimenez-Lucho and Foulds 26 have documented that both the amount and composition of LPS have a bearing on the virulence of S. typhi isolates. The LPS of

1 O0

80

o) 60

E

e 40

20

\

\ \

\

\

\

L 1 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0

Iog2serum di lut ion

Figure 2 In vitro bactericidal activity of mouse immune sera towards S. typhi Ty2

R - L P S

Figure 3 Schematic representation of smooth and rough Salmonella lipopolysaccharides. The blocks represent oligosaccharides, r.u. is the oligosaccharide repeating unit of the O-chain

Salmonella, like those of Gram-negative bacteria, are constructed according to a common general architecture. They consist of a polysaccharide region and a hydrophobic lipid component (lipid A). The poly- saccharide region can be subdivided into the O-specific polysaccharide (O-chain) and the core oligosaccharide (Figure3). Lipopolysaccharides having this complete structural organization are called smooth LPS (S-LPS) while those lacking the O-chain are called rough LPS (R-LPS).

While it is increasingly being recognized that both humoral and cell-mediated immunity are important in typhoid fever 27'28, the role of O-antigen in immuno- protection remains controversial 29. Johnson et al. 3° have reported that the immune response directed to S. o'phi O-antigens is important in preventing death in experimental models of typhoid. Significantly, field studies with killed and live oral typhoid vaccines, while not establishing a protective role, have indicated that development of anti-O-chain antibodies correlates with the level of protection in vaccinees< Further, it has also been suggested that protection in mice immunized with S. typhi porins may partly be due to contaminating LPS 31. While the chemistry and immunology of O-antigen-specific glycoconjugates have been studied extensively in non-typhoidal Salmonellae 32 ~4, not much information is available on the use of S. typhi O-chain protein conjugates as a vaccine for typhoid fever.

The O-chain of S. typhi is composed of a polymer of the following pentasaccharide repeating unit:

o~- D- Tyvl! c~-D- Glcp_2 -OAc i I

i i 3 4

-,2) -~-D-Manp- (1-,4) -c~-L-Rhap_- (1-->3) -o~-D-Gal]o- (i-~

Mild acid hydrolysis with acetic acid releases the O-chain (with core oligosaccharides attached) from lipid A, thus removing the toxic activity of the lipopoly- saccharide. Purification can be achieved by centrifugation, to remove the lipid A, followed by gel permeation chromatography. The latter process separates the high-molecular-weight material corresponding to O- chains of various lengths attached to the core oligosaccharide region, derived from the smooth lipopolysaccharides, from those attached to the core oligosaccharides derived from the rough lipopoly- saccharides. Both are present in the majority of smooth lipopolysaccharide preparations.

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With the removal of lipid A, the lipopolysaccharides lose their immunogenic activity. It is well known that conjugating polysaccharides to proteins enhances and modifies their immunological properties 35. In the present study, periodate oxidation and reductive amination procedures were used to covalently link the O- polysaccharide obtained from S. typhi to proteins. Periodate oxidizes vicinal diols. The rhamnosyl and glucosyl residues in the repeating unit are susceptible to oxidation. Several sugar residues in the core oligo- saccharide can undergo similar oxidation with periodate. The kinetics of the oxidation are dependent on the conformation of the vicinal diols, i.e. cis-diols are oxidized much faster than trans-diols and exocyclic diols are oxidized much faster than cyclic diols. The oxidation process can thus be controlled by the amount of periodiate added as well as by the reaction time. This principle was followed in order to oxidize selectively the free exocyclic diols from the heptoses and 3-deoxy-2- keto-D-manno-octulosonic acid present in the core oligosaccharide region, without affecting the other susceptible residues. The oxidation was followed by a Smith-type hydrolysis and the high-molecular-weight material was separated by gel permeation chromato- graphy. In case undesired oxidation had occurred in the repeating units, depolymerization would have occurred during the acid treatment which would have been noticed during the gel permeation chromatography step.

Conjugation was performed by mixing the two components, selectively oxidized polysaccharide and protein, in 0.1 n bicarbonate buffer followed by the addition of sodium cyanoborohydride. Tetanus toxoid was chosen as a carrier protein because of its medical usefulness and long history as a safe and effective vaccine component. The conjugation process involves the formation of Schiff bases between the aldehydes from the polysaccharide and the free amino groups of the protein. The reducing agent reduces the bases to stable secondary amino groups.

Covalent coupling of the polysaccharide to tetanus toxoid enhanced the immunogenicity of the O-chain in animals. It is significant to note that the memory immune response, as evidenced by the booster reaction, is indicative of a T-cell-dependent antigen, an observation similar to that for other conjugate vaccines 36. As reported elsewhere 3v, the isotype pattern of primary and booster response in mice immunized with the O-TT conjugate with Freund's adjuvant showed that the primary response was an IgM type response, while the booster response was IgG type, again characteristic of a T-cell-dependent reaction. In summary, in preparation of the conjugated antigen, the O-chain fraction of the LPS was used selectively as the polysaccharide component, consisting of the high-molecular-weight O-chains attached to their respective core regions. These core regions were partially modified by the periodate treatment and used as linkers to the carrier proteins. Antibodies induced were thus directed exclusively to epitopes on the O-chain of the S. typhi LPS as indicated by their binding to the BSA conjugate and the S-LPS, and not binding to the Ra-LPS, which corresponds to Salmonella R-LPS with the complete core.

The results of this study also show that antibodies to O-chain were bactericidal for S. typhi in vitro. For more meaningful analysis of the protective behaviour of anti-polysaccharide antibodies in typhoid fever, their

role in conferring active immunity was examined in mice. Protection studies in the mouse mucin model of typhoid 3s showed that 100% of O-TT-immunized mice survived challenge with 10 LDso of S. typhi Ty2, while 50% of animals were protected against challenge with 100LDs0 of the challenge strain. This interesting observation warrants more research to elucidate the exact contribution of anti-O-chain antibodies in protection against typhoid fever, both in a better challenge system and with other immunizing doses of the vaccine.

A C K N O W L E D G E M E N T S

The authors are grateful to Dr P.B. Percheson and Dr F.C. Cabello for scientific discussions and to Nicole Beausoleil for help in the animal experiments. M.S. was supported by a Visiting Biotechnology Fellowship from the Natural Sciences and Engineering Research Council of Canada.

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