Short Popers Active protection of mice against Salmonella typhi by immunization with strain-specific porins A r m a n d o Isibasi *°°, V i a n n e y O r t i z - N a v a r r e t e t, J o r g e P a n i a g u a * , R o s a n a P e l a y o * , C 6 s a r R. Gonzfilez*, Jos6 A. G a r c i a * a n d Jesfls K u m a t e ~
N I H mice were immunized with between 2.5 and 30 Ilg o f two highly purtfied porins, 34 kDa and 36 kDa, isolated from the virulent strain Salmonella typhi 9,12, Vi.'d. O f mice immunized with 10 itg o f porins, 90% were protected against a challenge with up to 500 LDso (50% lethal doses) o f S. typhi 9,12, Vi:d and only 30% protection was observed in mice immunized with the same dose o f porins but challenged with the heterologous strain Salmonella typhimurium. These results demonstrate the utilio' o f porins for the induction o f a protective status against S. typhi in mice. Keywords:Salmonellatypht, mice, strata-specificporms
Owing to the localization of the outer membrane proteins (OMPs) on the bacterial surface i 2, OMPs have been recently considered as important antigens in the induction of specific protective immune response against infection by gram-negative bacteria 3 9. We have previously demonstrated in a mouse model that immunization with a crude preparation of OMPs, which also contained 4% LPS, isolated from a wild-type strain of Salmonella typht is capable of inducing a high level of protection against S. o,phi, and that the OMPs known as porins are the main target of the humorai immune response 1°. Moreover, patients with typhoid fever in the convalescence phase generate immunoglobulin G antibodies that recognize mainly S. o'phl porlns 1~. These results suggest that humoral immumty induced by porins m~ght play an important role in the immunity against S typhi. In order to develop a vaccine free of endotoxin side effects lz we evaluated the ability of purified porins to confer immunity against S. typht
Bacterial strains The virulent strain Sahnonella t),pht 9,12,Vi:d was isolated from a patient with typhoid fever ~°, and Salmonella typhlmurium 1,4,5,12 was kindly donated by the Instltuto Nacional de Hlglene in Mexico City, Mexico. Purification and characterization of porins Porlns were isolated from peptidoglycan-assoclated materials and purified by size exclusion chromatography on a Sephacryl S-200 column (Pharmacla, Uppsala, Sweden) according to a modification of the method of Nikaido 13 as described elsewhere 14. The protein concentration was determined by the method of Lowry et al. ~5. LPS contamination of the porin samples was determined by measuring the concentration of flhydroxy-myrlsuc acid by reverse phase high-performance hquid chromatography (HLPC) 16. Sodium dodecyl sulphate-polyacrylamide slab gel electrophoresis ( SDS PAGE) was carried out on 11.5% gels as described by Laemmll x7
MATERIALS AND METHODS Animals
Immunization and challenge
Female outbred NIH strain mice were obtained from the National Institutes of Health (Bethesda, MD, USA ).
Groups of 10 mace, 8 weeks old and 13 15 g in weight, were immunized intraperltoneally (l.p) on day 0 and day 15 with porms dissolved in 200 pl TE buffer (50 mM Tns, pH 7 2, 5 mM EDTA). Ten days after the second immunization, they were challenged i.p. with 20 or 500 LDso of bacteria suspended in 500/~l TE containing 5% gastric mucin (Sigma Chemical Co, St Louis, MO, USA ). Protection was defined as the percentage survival during the 10 days following the challenge. The LD5o for each Salmonella strain was calcuated as described by Reed and Muench t8 and were 1.5 x 105 for S. typht and 1.2 × 102 for S. typhimurtum.
*Laboratorro de Inmmunoquimica, Unldad de Investlgaclones Biom6dlcas, Inshtuto Mexlcano del Seguro Social, PO Box 73-032, Mexico DF 03020. tDepartamento de Investlgacl6n, Instituto Naclonal de Higlene, Mexico DF 11400 tSecretaria de Salud, Mexico DF 06600 ~To whom correspondence should be addressed. (Recewed 26 September 1991; rewsed 1 April 1992; accepted 7 Aprd 1992) 0264-410X/92/120811-03 1992 Butterworth-HememannLtd
Vaccine, Vol 10, Issue 12, 1992 811
Short paper A Istbast et al
RESULTS
Porin preparation Figure I shows the porto homotrlmers purified to homogeneity by chromatography of a crude porin preparation over a Sephacryl S-200 column m a high salt buffer. The tnmers eluted as a sharp peak with an estimated molecular weight of 120kDa. When this fracUon was analysed on S D S - P A G E (reset) under reducing conditions, only the two porto monomers of 34 kDa and 36 kDa were observed and contamination w~th other proteins was not seen. The ~soelectnc points of the two porms were 4.5 for the 34 kDa and 5.0 for the 36 kDa monomer and the LPS present as contaminant in the purified ponns preparation was 0.04% (data not shown ).
Induction of protective immunity by porins The capacity of the S. typhl purified porin trimers to reduce protection was analysed in m~ce that had been immunized twice with doses ranging from 2.5 to 30 k~g
Table 1
Protection of porln-lmmunized mice ~
S typht
S typhtmunum
Porlns administered (Izg)
20
500
20
500
25 50 10 0 30 0
60 100 90 90
10 80 90 90
ND 20 30 70
ND 0 30 40
"Protection is expressed as percentage survival after 10 days observation period ND, not done
of porms. It can be seen in Table I that 100% of the mice immunized with 5/~g of porms surwved the challenge with 20 LD5o of S. typht and 80% survived the challenge with 500 LDso. Higher doses of porin (10 or 30~g) induced 90% protection for both low and high S. typh~ challenge. Protection against S. typhlmurium was observed in mice immunized with 30/lg of porins, 70% surviving 20 LDso and 40% surviving 500 LDso. Control mice injected with TE buffer all died the day after the challenge with 20 LDso of either strain. DISCUSSION In a prewous report we showed induction of active immunity against S. typhi in mice using a crude preparation consisting of 10-12 different O M P s with molecular ratios ranging from 17 to 70 kDa, and which also contained 4% LPS t°. From this and other studies ~,~9 it was postulated (but not proven) that the anugens responsible for the induction of the protection observed may be the porms. In order to test this assumptxon we punfied porins from S. typhl as high molecular weight homotrimers by gel filtration to a degree where two proteins were observed in S D S - P A G E and lsoelectrofocusing. Therefore, ~t ~s not likely that additional protein contaminants are present in the purified porin preparataon but we cannot exclude the poss~bihty of an extremely low level of contammaUon that is not detectable on our gels. LPS is present m a very low amount (0.04%) as determined by the concentration of/~-hydroxy-mirisUc acid; another well defined Salmonella antigen, flagellar H, was not detected. A major finding in this report is that a very low concentration (5/ag) of the two purified porins protects N I H mice against a challenge of up to 500 LDso of the
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N I H mice were immunized with between 2.5 and 30 Ilg o f two highly purtfied porins, 34 kDa and 36 kDa, isolated from the virulent strain Salmonella typhi 9,12, Vi.'d. O f mice immunized with 10 itg o f porins, 90% were protected against a challenge with up to 500 LDso (50% lethal doses) o f S. typhi 9,12, Vi:d and only 30% protection was observed in mice immunized with the same dose o f porins but challenged with the heterologous strain Salmonella typhimurium. These results demonstrate the utilio' o f porins for the induction o f a protective status against S. typhi in mice. Keywords:Salmonellatypht, mice, strata-specificporms
Owing to the localization of the outer membrane proteins (OMPs) on the bacterial surface i 2, OMPs have been recently considered as important antigens in the induction of specific protective immune response against infection by gram-negative bacteria 3 9. We have previously demonstrated in a mouse model that immunization with a crude preparation of OMPs, which also contained 4% LPS, isolated from a wild-type strain of Salmonella typht is capable of inducing a high level of protection against S. o,phi, and that the OMPs known as porins are the main target of the humorai immune response 1°. Moreover, patients with typhoid fever in the convalescence phase generate immunoglobulin G antibodies that recognize mainly S. o'phl porlns 1~. These results suggest that humoral immumty induced by porins m~ght play an important role in the immunity against S typhi. In order to develop a vaccine free of endotoxin side effects lz we evaluated the ability of purified porins to confer immunity against S. typht
Bacterial strains The virulent strain Sahnonella t),pht 9,12,Vi:d was isolated from a patient with typhoid fever ~°, and Salmonella typhlmurium 1,4,5,12 was kindly donated by the Instltuto Nacional de Hlglene in Mexico City, Mexico. Purification and characterization of porins Porlns were isolated from peptidoglycan-assoclated materials and purified by size exclusion chromatography on a Sephacryl S-200 column (Pharmacla, Uppsala, Sweden) according to a modification of the method of Nikaido 13 as described elsewhere 14. The protein concentration was determined by the method of Lowry et al. ~5. LPS contamination of the porin samples was determined by measuring the concentration of flhydroxy-myrlsuc acid by reverse phase high-performance hquid chromatography (HLPC) 16. Sodium dodecyl sulphate-polyacrylamide slab gel electrophoresis ( SDS PAGE) was carried out on 11.5% gels as described by Laemmll x7
MATERIALS AND METHODS Animals
Immunization and challenge
Female outbred NIH strain mice were obtained from the National Institutes of Health (Bethesda, MD, USA ).
Groups of 10 mace, 8 weeks old and 13 15 g in weight, were immunized intraperltoneally (l.p) on day 0 and day 15 with porms dissolved in 200 pl TE buffer (50 mM Tns, pH 7 2, 5 mM EDTA). Ten days after the second immunization, they were challenged i.p. with 20 or 500 LDso of bacteria suspended in 500/~l TE containing 5% gastric mucin (Sigma Chemical Co, St Louis, MO, USA ). Protection was defined as the percentage survival during the 10 days following the challenge. The LD5o for each Salmonella strain was calcuated as described by Reed and Muench t8 and were 1.5 x 105 for S. typht and 1.2 × 102 for S. typhimurtum.
*Laboratorro de Inmmunoquimica, Unldad de Investlgaclones Biom6dlcas, Inshtuto Mexlcano del Seguro Social, PO Box 73-032, Mexico DF 03020. tDepartamento de Investlgacl6n, Instituto Naclonal de Higlene, Mexico DF 11400 tSecretaria de Salud, Mexico DF 06600 ~To whom correspondence should be addressed. (Recewed 26 September 1991; rewsed 1 April 1992; accepted 7 Aprd 1992) 0264-410X/92/120811-03 1992 Butterworth-HememannLtd
Vaccine, Vol 10, Issue 12, 1992 811
Short paper A Istbast et al
RESULTS
Porin preparation Figure I shows the porto homotrlmers purified to homogeneity by chromatography of a crude porin preparation over a Sephacryl S-200 column m a high salt buffer. The tnmers eluted as a sharp peak with an estimated molecular weight of 120kDa. When this fracUon was analysed on S D S - P A G E (reset) under reducing conditions, only the two porto monomers of 34 kDa and 36 kDa were observed and contamination w~th other proteins was not seen. The ~soelectnc points of the two porms were 4.5 for the 34 kDa and 5.0 for the 36 kDa monomer and the LPS present as contaminant in the purified ponns preparation was 0.04% (data not shown ).
Induction of protective immunity by porins The capacity of the S. typhl purified porin trimers to reduce protection was analysed in m~ce that had been immunized twice with doses ranging from 2.5 to 30 k~g
Table 1
Protection of porln-lmmunized mice ~
S typht
S typhtmunum
Porlns administered (Izg)
20
500
20
500
25 50 10 0 30 0
60 100 90 90
10 80 90 90
ND 20 30 70
ND 0 30 40
"Protection is expressed as percentage survival after 10 days observation period ND, not done
of porms. It can be seen in Table I that 100% of the mice immunized with 5/~g of porms surwved the challenge with 20 LD5o of S. typht and 80% survived the challenge with 500 LDso. Higher doses of porin (10 or 30~g) induced 90% protection for both low and high S. typh~ challenge. Protection against S. typhlmurium was observed in mice immunized with 30/lg of porins, 70% surviving 20 LDso and 40% surviving 500 LDso. Control mice injected with TE buffer all died the day after the challenge with 20 LDso of either strain. DISCUSSION In a prewous report we showed induction of active immunity against S. typhi in mice using a crude preparation consisting of 10-12 different O M P s with molecular ratios ranging from 17 to 70 kDa, and which also contained 4% LPS t°. From this and other studies ~,~9 it was postulated (but not proven) that the anugens responsible for the induction of the protection observed may be the porms. In order to test this assumptxon we punfied porins from S. typhl as high molecular weight homotrimers by gel filtration to a degree where two proteins were observed in S D S - P A G E and lsoelectrofocusing. Therefore, ~t ~s not likely that additional protein contaminants are present in the purified porin preparataon but we cannot exclude the poss~bihty of an extremely low level of contammaUon that is not detectable on our gels. LPS is present m a very low amount (0.04%) as determined by the concentration of/~-hydroxy-mirisUc acid; another well defined Salmonella antigen, flagellar H, was not detected. A major finding in this report is that a very low concentration (5/ag) of the two purified porins protects N I H mice against a challenge of up to 500 LDso of the
1.0 Vo
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0.5
eL
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