lipopolysaccharide — protein complex as potent vaccine against salmonellosis

LIPOPOLYSACCHARIDE - PROTEIN COMPLEX AS POTENT VACCINE AGAINST SALMONELLOSIS

RAMESH CHANDER and NORMAN F. LEWIS

Food Technology & Enzyme Engineering Division Bhabha Atomic Research Centre

Bombay 400 085 India

Accepted for Publication November 3, 1986

ABSTRACT

Lipopolysaccharide-protein (US-P) vaccine prepared from cells of S . typhimurium contains 11 % protein. Immunization of mice with LPS-P in- trapentoneally at a dose level of 50 pglmouse conferred 100% protection against subsequent challenge with S . typhimurium and seven other strains of Salmonella each possessing typical 0-antigen determinants. The immunity lasts for six months and the vaccine is equally effective in all six strains of mice studied. The lyophilized LPS-P preparation is stable for eighteen months, thus providing op- portunity for large scale field trial experiments.

INTRODUCTION

Salmonellosis and other enteric bacterial infections are major health problems all over the world (Ashcroft 1964; Black et al. 1982; Black et al. 1981; Edelman and Levine 1980). Chloramphenicol and ampicillin are generally prescribed for the control of these diseases, however, these enteropathogens develop resistance to antibiotics.

Typhoid vaccines are made from whole Salmonella cells which are heat killed, phenol treated or acetone killed or alcohol killed (Joo 1971). A polyvalent radiovaccine consisting of radiation killed Salmonella cells was described by Chander et al. (1984). A major limitation of using whole cell vaccine is the presence of nonessential immunogens. This may cause antigenic competetion and adversely affect the efficacy of whole cell vaccines. This study was under- taken to isolate effective immunogens from Salmonella cells which could be used as vaccines against salmonellosis.

Journal of Food Safety 8(1987) 89-99. All Rights Reserved. @Copyright 1987 by Food & Nutrition Press, Inc., Wesport, Connecticut. 89

90 RAMESH CHANDER AND NORMAN F. LEWIS

MATERIALS AND METHODS

Bacterial Cultures

The eight strains of Salmonella used in these studies were S. typhimurium (H.391) S. paratyphi A (13) S. paratyphi B (5705), S. thompson (5740), S. Virginia (6947) S. gallinarum (5775), S. anatum (6680) and S. senfrenberg J.T. (1502). S. typhimurium was obtained from the Halfkine Institute, Bombay (In- dia), the other seven Salmonella strains were obtained from the Central Research Institute, Kasauli (India). All Salmonella strains were maintained on nutrient agar (Difco) slants at refrigerated temperatures.

Isolation of Lipopolysaccharide-Protein (LPS-P) Complex

LPS-P was extracted from the cells of S. typhimurium (H391) by the method of Staub (1965) and lipopolysaccharide (LPS) was isolated by the phenol water extraction method of Westphal et al. (1952).

Protein Determination

ing bovine serum albumin (Sigma) as a standard. Protein of the LPS-P was estimated by the method of Lowry et al. (1951) us-

Gel Electrophoresis

Proteins of the LPS-P were determined by the polyacrylamide gel electrophoresis method of Davis (1964). Disc electrophoresis was carried out at pH 8.3. The protein bands were stained with Coomassie blue (Sigma) was destained in 7% acetic acid.

Fatty Acid Estimation

Fatty acids of LPS-P and LPS of S. typhimurium were determined by GLC analysis of their methyl esters prepared by the method of Hancock and Meadow (1969). The methyl ester extracts were taken up in 100 pl of ether and 5 pl was used for injection into a Shimadzu GC model 7A Gas chromatograph fitted with a flame ionization detector. Fatty acid methyl esters were separated on a 10% Silar 7C stainless steel column (5M X 3 mm) operated isothermally at 180°C with nitrogen carrier gas (50 mL/min). Fatty acids were tentatively identified by comparing their retention time with those of reference standards obtained from Sigma Chemicals.

Experimental Animals

Swiss inbred male mice weighing 20-25 g and 7-8 weeks of age were usually

VACCINE AGAINST SALMONELLOSIS 91

used. Some experiments were also performed with AKR, CBA, C57B16, C,H and DBA strains of mice. All animals were fed stock diet and water ad libitum.

Immunization

Lypophilized LPS-P was suspended at a concentration of 50 pg/mL of sterile saline and 0.5 mL was administered intraperitoneally on days 0 and 7. Mice were subsequently challenged on day 22.

Challenge

Exponential phase cells of S. t y ~ h ~ ~ u ~ ~ ~ in nutrient broth (Difco) were harvested by centrifugation at 3000xg at room temperature. The cells were washed twice wtih sterile saline and resuspended in sterile saline to give desired cell numbers. Cell numbers were assessed from OD measurements at 600 nm using a Bausch and Lomb Spectronic 20 spectrophotometer and a previously developed standard curve. Normally mice were challenged with 1 X lo* cells 22 days after the first vaccination dose.

Toxicity of LPS-P Complex

a period of one month. LPS-P was administered ip at various doses and mortality was recorded during

Mortality

number of mice challenged. Mortality was defined as the ratio of the number of mice dead to the total

RESULTS

Composition of LPS-P Complex

The protein content of the LPS-P complex was 1 % . Polyacrylamide gel electrophoresis of the protein complex showed the presence of three major and two minor proteins. GLC analysis of the fatty acid components of LPS-P from S. typhimurium showed eight major and five minor peaks as depicted in Fig. 1. Some of the fatty acids identified had carbon chains of Clz, CI4, CI6, Cl, and Czo. The fatty acids of the vaccine were comparable to those of LPS from S. typhimurium (Fig. 1).

Toxicity of LPS-P Complex

The toxicity of the complex was determined by ip administration of various doses of this complex into groups of mice and then observing the mortality over


w v, z v, 0 lJl W

a W 0 a

w

a

a

8 a

TIME IN MIN

FIG. 1. GAS CHROMATOGRAMS OF METHYL ESTERS OF FATTY ACIDS OF

LPS-P ( ) AND LPS (-------) OF S. TYPHIMURIUM(H-391)

a period of one month. The complex was toxic to some mice at a dose level of 100 pg/mouse and was toxic to all mice at a dose of 250 pg/mouse.

Optimization of Dose of LPS-P for Vaccination

The efficacy of the vaccine was evaluated at three dose levels. The results presented in Table 1, show that all three dose levels offered protection to mice challenged with S. typhimurium. The mortality observed with the 100 p g dose of vaccine is apparently due the toxicity of LPS-P. In subsequent studies, a dose of 50 pg/mouse was administered intraperitoneally in two doses of 25 pg/mouse on days 0 and 7.


Table 1 . Optimization of dose of lipopolysaccharide-protein vaccine

Dose o f v o r t a l i t y a T o t a l on vaccine Day Number 15th day

1 2 6 15

o dose 1.e. cont ro l 10/10 - - - 10/10

20 ug/mouse 0/11 0/11 0/11 0/11 0/11 p4 i n j c t ions of l o ug e tch on aaye 0 4 & 7

Td in jec t ione o f 23/ug each on dsye 0 and 7

1 OO/ug/mous e 0/12 6/12 2/12 0/12 8/12 TWO i n j ec t ions o f 50 ug each on aaye 0 And 7.

50 ug/mousa 0/12 0/12 0/12 0/12 0/12

Vhallenge consisted of 1 x lo* cells of S. typhirnurium given ip on day 22.

Efficacy of Various Routes of Vaccination

The route of vaccination is an important factor in determining the immunogenicity of a vaccine. The results shown in Table 2 indicate that only the intraperitoneal (ip) administration of vaccine offered protection. Subcutaneous or oral administration routes were ineffective.

Route of Challenge

Immunized mice were challenged with S. typhimurium through various routes. The results in Table 2 indicate that the mice were fully protected irrespective of the challenge route.

Duration of Immunity

The duration of immunity conferred by LPS-P vaccine was examined. Groups of mice were immunized and subsequently challenged at intervals of time up to six months. The results recorded in Table 3 indicate that the mice were protected even when challenged six months after immunization.


Table 2. Efficacy of various routes of vaccination and challenge

- e Route of ooute of F'ortalA t y v a c c i n a t i ona cha l lenge Cont ro l group Vaccinsted g r o u p

Oral Jnt rape ri t oneal 20/20 20/20

Subcutaneous I n t r z p e r i t o n e a l 20/20 20/20

I n t r a p e r i t o n e a l Oral 22/2 2 0/20

Subcutaneous 20/20 o j 2 0

I n t r a p e r i t o n e a l 20 /20 0 /20

a. LPS-protein was suspended in sterile saline. This was administered on days 0 and 7 by the

b. Challenge comprised 1 X lo8 cells of S. typhirnun'urn administered on day 22. c. Mortality = Number of animals dead to the total number of animals challenged. d. Control group received saline in place of vaccine.

various routes indicated. Each mouse received 50/pg of the vaccine.

Table 3. Duration of immunity conferred by LPS-protein vaccine

d nay o f a FJor ta l i ty c h a l l e n g e a f t e r c o n t r o l groupc Vaccinated group 1st dose of Day Number Day Number v a c c i n a t i o n 1 7 15 30 1 7 15 30

15 10/10 - - - 0/10 0/10 0/10 0/10

30 10/10 - - - 0/10 0/10 0/10 0/10

60 10/10 - - - 2/10 0/10 0/10 0/10

90 8/10 2/10 - - 0/10 0/10 0/10 0/10

120 10/10 - - - 0/10 0/10 0/10 0/10

180 8/10 2/10 - I 0/10 0/10 0/10 0/10

150 10/10 - - - 0 / 8 0/8 0/8 0/8

ryphimuriurn cells were harvested in the logarithmic phase washed twice with saline and resuspended to give 2 X lo8 cellslml saline. Of this 0.5 mL was then injected ip.

bMortality = Number of animals deadltotal number of animals challenged. 'Control group received saline. dVaccinated group was administered ip with 0.5 mL of 50 pglmL LPS-protein vaccine on days

Oand 7.


Immunogenicity of LPS-P Vaccine to Different Strains of Salmonella

The efficacy of LPS-P vaccine to protect mice against other strains of Salmonella was examined. Groups of mice immunized with LPS-P vaccine on day 0 and 7 were challenged on day 22 with the cells of eight strains individually as well as with mixed cultures of these strains. The results compiled in Table 4 show that immunized mice were completely protected against challenge with these eight strains.

Table 4. Immunogenicity of LPS-protein vaccine to heterologous strains of Salmonella

b C h a l l e n p a organism c o n t r o l groupd v a c c i n a t e d group‘

fir0 rt a1 i ty

Day Number Day Number 1 2 7 30 1 30

S,typhimuriurn(H39?] 12/72 - - - 0/12 0/12 - - 0/10 0/10

- O/li 0/12

- S . p a r a t y p h i B ( 5 1 0 5 ) 6/8 2/8 - - S . p a r a t y p h i A ( 1 5 ) 0/12 12/12 - S.thompson (5’140) 6/72 - 4/12 - O/l0 - S . v i r g i n i a (0947) 4/10 6/10 - - 0/12 0/12

- 7 m m 4 s . g a l l i n a r u m ( 5775) 8/12 - 2/12 - 0/12 0/12

ynixed c u l t u r e a o f 12/12 - - - 0/10 0/10

- - 0/6 ‘0/6 s I s e n f t e n b e r 6/6 - -

- s. anaturn (6680) 12/12 - - - 0/12 0/12

above strains

- -

aMice were challenged ip with 1 X los cells per mouse on day 22 bMofidity ,Number of animals dead

‘Vaccinated mice were immunized as described previously ‘Control mice were given saline injections

Total number of animals challenged

Stability of Vaccine

Lyophilized vaccine which remains effective is desirable for field trials. The lyophilized preparations of LPS-P, stored at low temperature, wre stable and no change in immugenicity was observed over a period of 18 months.


Efficacy of LPS-P Vaccine in Six Strains of Mice

The efficacy of LPS-P vaccine was evaluated in six different strains of mice. The results are presented in Table 5 . The natural resistance to Salmonella varied from one strain of mouse to another as evident from the differences in survival times for the control groups of mice challenged with same number of cells. Swiss mice were most susceptible. The CBA strain of mouse was highly resis- tant to S. fyphimurium (H391) infection. The vaccinated groups were completely protected and no mice died during the 30 days observation period after which the mice were sacrificed.

DISCUSSION

The results demonstrate that LPS-P is an effective immunogen of S. ryphimurium that can be used as a vaccine to protect mice against a variety of salmonellae and retains activity after lyophylization and months of room temperature storage.

The primary structural difference between LPS and LPS-P is that the latter possesses a greater amount of protein (Hepper ef at. 1979). The protein apparently contributes to the immunological potential. LPS alone is a weak immunogen and does not confer any protection against challenge with Salmonella (Angerman and Eisenstein 1980). The observed long lasting immunity against Salmonella infection resulting from LPS-P vaccine is similar to the results reported by Hodges et al. (1982), that LPS-P from Fusobucterium necrophomm had the ability to induce immunologic memory. In their experiments, antibody forming splenocytes were detectable at 120 days after the first injection. The removal of the protein component from LPS-P by enzyme digestion or extraction with phenol water resulted in its failure to induce immunological response. The protein moiety induces the helper T-cells that participate in the formation of IgG synthesizing B lymphocytes (Hepper et al. 1979; Sultzer and Goodman 1976; Hodges ef al. 1982). The LPS-P molecule can be visualized as a built-in adjuvant-antigen system with LPS portion acting to stimulate B-cells and the protein moiety serving as a T-cell stimulant. The LPS-P vaccine could enhance both humoral (B-cell mediated) and cellular (T-cell mediated) responses involv- ed in the defense against infections.

The LPS-P isolated from one strain of S. ryphimurium conferred protection against seven other Salmonella strains. This multispecificity of the vaccine may be attributable to the enhanced immunogenicity against the porin (Salmonella surface protein) associated with it. Salmonella porins are not subspecies specific but are universal for the Salmonella group (Svenson et al. 1979).


The vaccine is equally effective in six strains of mice with varying genetic constitution and thus with different natural resistance to Salmonella infection. Although the efficacy of the LPS-P vaccine has been observed in mice, its response in other animals needs to be tested.

REFERENCES

ANGERMAN, C.R. and EISENSTEIN, T.K. 1980. Correlation of the duration and magnitude of protection against Salmonella infection afforded by various vaccines with antibody titers. Infect. Immun. 27, 435-443.

ASHCROFT, M.T. 1964. Typhoid and paratyphoid fevers in the tropics. J. Trop. Med. Hyg. 67, 185-189.

BLACK, R.E., BROWN, K.M., BECKER, S. , ABDUL, A.R.M.A. and HUQ, I. 1982. Longitudinal studies of infections diseases and physical growth of children in Bangladesh. II. Incidence of diarrhea and association with known pathogens. Am. J. Epidemiol. 115, 315-324.

BLACK, R.E., MERSON, M.M., HUQ, I., ABDUL, A.R.M.A. and YUNUS, M. 1981. Incidence and severity of rotavirus and Escherichia coli diarrhoel in rural Bangladesh. Lancet i, 141-143.

CHANDER, R., NERKAR, D.P., BANDEKAR, J.R. and LEWIS, N.F. 1984. A note: Polyvalent radiovaccine against Salmonella. J. Food Safety, 7, 31.

DAVIS, B.J. 1964. Disc electrophoresis. 11. Method and application to human serum proteins. Ann. N.Y. Acad. Sci. 121, 404-427.

EDELMAN, R. and LEVINE, M.M. 1980. Acute diarrheal infections in in- fants. 11. Bacterial and viral causes. Hosp. Pract. 15, 97-104.

HANCOCK, I.C. and MEADOW, P.M. 1969. The extractable lipids of Pseudomonas aeroginosa. Biochem. Biophys. Acta. 187, 366-371.

HEPPER, K.P., GARMAN, R.D., LYONS, M.F. 1979. Plaque forming cell response in BAL B/C mice to two preparations of LPS extracted from Salmonella enteritidis. J. Immunol. 122, 1290-1293.

duction of immunological memory by a lipopolysaccharide-protein complex isolated from Fusobacterium necrophorum. Cellular response. Am. J. Vet. Res. 43, 117-121.

JOO, I. 1971. Present status and perspectives of vaccination against typhoid fever. Pan. Am. Health Organ. Sci. Publ. No. 226, 329-341.

LOWRY, O.H., ROSEBROUGH, N.J., FARR, A.L. and RANDALL, R.J. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem.

HODGES, G.F., REGAN, K.M., FOSS, C.L. and TERESA, G.W. 1982. In-

193, 265-275.


STAUB, A.M. 1965. Bacterial lipid protein polysaccharide (0-somatic an- tigens) extracted with trichloracetic acid. In Methods in Carbohydrate Chemistry, (R.L. Whistler, ed.) 5, pp. 92-93, Academic Press, New York.

SULTZER, B.M. and GOODMAN, G.W. 1976. Endotoxin protein: A B-cell mitogen and polyclonal activator of C3HIHeJ lymphocytes. J. Exp. Med. 144, 821-827.

SVENSON, S.B., NURMIMEN, M. and LINDERBERG, A.A. 1979. Ar- tificial Salmonella vaccines: 0-antigenic oligosaccharide-protein conjugates induce protection against infection with Salmonella typhimurium. Infect. Im- mun. 25, 863-872.

WESTPHAL, O., LUDERITZ, 0. and BISTER, F. 1952. Ubes die extraktion von bacterien mit phenol/wasser. Z. Naturforsch 7, 148.

lipopolysaccharide — protein complex as potent vaccine against salmonellosis

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