An immunogenic complex, essentially consisting of neisserial outer membrane protein proteosomes hydrophobically complexed to native purified bacterial lipopolysaccaride and formulated in accordance with the current invention for mucosal delivery such as via the oral or intranasal route is used as a vaccine. Specifically, a vaccine using shigella lipopolysaccharides complexed to proteosomes for such mucosal administration induces IgG and IgA antibodies in sera and in respiratory and intestinal fluids. Furthermore, such antibodies are associated with protection against shigella infection and these vaccines are herein demonstrated to protect against mucosal infection with shigella.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a novel vaccine delivery system using proteosomes hydrophobically complexed to lipopolysaccharides and formulated for oral or intranasal administration to induce protective antibodies in sera and/or respiratory and/or intestinal secretions that are associated with protection against disease.

In accordance with the invention, vaccines using Shigella flexneri 2a or Shigella sonnei lipopolysaccharide (LPS) induce anti-LPS antibodies in the aformentioned fluids and protect against homologous shigella infection in a well-known animal model of shigellosis.

DETAILED DESCRIPTION OF THE INVENTION

LPS Preparation

LPS was extracted by hot phenol from single isolate of Shigella flexneri 2a or Shigella sonnei (Westphal, O. and K. Jann. 1965. Carbohydr. Chem. 5:83.11). The LPS extraction protocol was generally as follows:

Grow bacteria in LB for 2 3 hrs at 37.degree. C. Incubate 3 ml starter in rho-bottles with nutrient agar (+2% agar), overnight at 37.degree. C. Harvest bacteria with 15 ml saline/bottle (+glass beads) and filter the suspension through gauze pad. Centrifuge the filtrate at 5000 RPM, 4.degree. C., (Sorval RC5B, 250ml tubes, cat. 03937 in GSA rotor) for 15 minutes and resuspend the pellet in 1/45 ml saline/Tube. Add acetone to the resuspended bacteria (1.5 L/10 ml resuspended bacteria) and left for two days. Remove the Acetone by filtration through Whatman paper under vacuum and dry the bacteria completely under vacuum to form white, fine powder. Add dH.sub.2O to the powder (25 ml/1.4 gr dried bacteria) and gently steer until all powder is hydrated. Add phenol (at 37.degree. C.) to the mixture (1:1 w/w) and agitate gently in 68.degree. C. preheated water bath for 10 min. Cool the mixture in ice and centrifuge at 3000 RPM, 4.degree. C., (30 ml tubes, cat. 03163 in GSA rotor) for 30 min. Gently collect the upper phase (water phase) and add cold dH.sub.2O (0.5 volume of the collected upper phase) to the lower phase and mix well. Centrifuge as in #9, collect upper phase and combine the two upper phases. Reextract the combined upper phases as in #8 #11. Dialyze against H.sub.2O for two days (15 K dialyzis bag) with at least two water exchanges. Centrifuge at 10000 RPM, 4.degree. C. (30 ml tubes, cat. 03163 in GSA rotor) for 30 minutes and collect supernatant. Add absolute ethanol (ethanol/supernatant 10:1 v/v) and leave for two days at 4.degree. C. Centrifuge at 10000 RPM, 4.degree. C. (250 ml tubes, cat. 03937 in GSA rotor) for 2 hrs, discard supernatant and resuspend pellet in 1/410 ml dH.sub.2O Dialyze against dH.sub.2O in 15 K dialyzis bag for two days. Freeze dry.

Alkaline detoxified LPS (LPSad) was prepared using mild alkaline treatment (Skidmore, B. J. et al. 1975. J. Immunol. 114:770). This alkaline-detoxified LPS was ineffective in the system.

Proteosome Preparation

Outer membrane proteins from group B serotype 2b N. meningitides were extracted with detergent as follows: 1.0 PREPARATION OF BACTERIAL PASTE Dissolve lyophilized ampule of group C type 2 Meningococci in d.w. Plate on petri plates of Neisseria selective media overnight in CO.sub.2 incubator or candle jar at 37.degree. C. Suspend swab full of isolated colonies in 150 ml Modified Watson-Sherp liquid media in a 1000 ml ehrlenmeyer. For 30 liters final culture, need to start with eighteen (18) 1000 ml ehrlenmeyers each containing 150 ml media so that total media volume in 18 1000 ml ehrlenmeyers is 2.7 liters. Grow 4 hrs at 37.degree. C. in a shaking incubator. Transfer each 150 ml of this 4 hr growth to 1.5 liter media (i.e. 1:11 dilution) in a 5 liter fernbach (or ehrlenmyer) so that each 5 liter vessel contains a total 1.65 liters. Eighteen vessels have a total of 30 liters. Therefore, alternatively, at this point, can use 30-liter fermenter. Expect 0.8 1.0 gms final material (outer membrane protein proteosome vesicles) per 30 liters. Four such runs or one 120 liter fermenter would therefore yield 3.2 4.0 gms protein. Grow 24 hrs with good aeration at 37.degree. C. in a shaking incubator to late log phase but not stationary phase. Sample growth to verify purity of culture by: a) plating on non-selective plates (Mueller-Hinton) for 24 hr growth and b) gram stain. Add 0.5% phenol (5 cc/liter of 90%). Let sit for 1 hr. to kill bacteria. Centrifuge as fast as possible (4 10,000 g) for 40 min (9,000 RPM sorval GS3). Wash x1 in NS in 1/5culture volume and spin in weighed culture bottles. Record lot number and amount of paste per bottle and total weight of paste: Freeze at -20.degree. C. until ready for extraction. 2.0 EXTRACTION OF OUTER MEMBRANE VESICLES FROM KILLED CELLS: ETHANOL PRECIPITATION 2.1 Area cleaned, cleared and ready for use. 2.2 Thaw cells at room temperature (if frozen) and Record lot nos. and amount of killed cell paste used: 2.3 Suspend cells in 1 M Sodium Acetate buffer, pH 5.0 using 1 ml per gram of cells. 2.4 Mix the suspension in a Waring type blender for 15 sec. at low speed at 22 25.degree. C. 2.5 Add d.w. at 22 25.degree. C.: 1.5 ml d.w. per ml of suspension from step 2.3. 2.55 Mix: form an even suspension; measure volume in a graduated cylinder: 2.6 To the combined volume from step 2.55 in a beaker, add an equal volume of the extraction solution 1.0 M Calcium Chloride/Empigen 6.0%. 2.65 Stir mixture at 22 25.degree. C. for 1 hour on a magnetic mixer. 2.7 FIRST ETHANOL ADDITION calculation (rounded to nearest 0.1 ml) to yield final concentration of 20% ethanol vol/vol): Amount of Ethanol added=(combined total volume from step 2.6)(0.25): 2.8 Add calculated volume from step 2.7 of absolute Ethanol to the suspension from step 2.65 and stir on magnetic stirrer for 15 min. at 22 25.degree. C. 2.9 Centrifuge the suspension from step 2.8 at 17,800.times.g for 10 min. at 1 5.degree. C. (10,000 rpm using the Beckman JA-10 rotor or equivalent) 2.95 Pour the supernatant thru clean cheesecloth and recover filtrate. 2.10 SECOND ETHANOL ADDITION calculation (rounded to nearest 0.1 ml) to yield final concentration of 45% ethanol vol/vol): Amount of Ethanol added=(combined total volume from step 2.95)(0.45): 2.11 Add calculated volume from step 2.1 of absolute Ethanol to the suspension from step 2.95 and stir on magnetic stirrer for 15 min. at 22 25.degree. C. 2.12 Centrifuge suspension from step 2.11 at 17,800.times.g for 10 min. at 1 5.degree. C. (e.g. using the Beckman JA-10 rotor at 10,000 rpm or equivalent) as in step 2.9; pour off and discard supernatnant. 3.0 PREPARATION OF OUTER MEMBRANE VESICLES (OMV): EXTRACTION WITH TEEN 1.0%: 3.1 FIRST TEEN 1.0% EXTRACTION: Resuspend the pellets from step 2.12 with 1.5 ml TEEN 1.0% buffer per gram extracted cell paste used in step 2.2. 3.2 BREAK UP pellets with a spatula. Transfer aliquots of suspension to Dounce Teflon Homogenizer and gently crush pellets until all material is a fine suspension. 3.3 With a sterile 21 gauge needle on a 50 cc syringe, aspirate and release suspension into beaker to create finest suspension and then stir on magnetic stirrer for 15 min. at 22 25.degree. C. 3.4 SONICATE the suspension in a Branson Sonication Bath model 5200 starting at 19 25.degree. C. using 200 ml solution per batch in a 600 ml beaker for 15 min. or 500 ml solution in a 1000 ml beaker as needed. 3.5 Centrifuge the sonicated solution from step 3.4 at 27,500.times.g (13,000 rpm) for 25 min. in Beckman JA-14 rotor or equivalent at 1 5.degree. C. Carefully recover supernatants, pool and store at 1 5.degree. C. SAVE pellets for second extraction. 3.6 SECOND TEEN 1.0% EXTRACTION: Resuspend the pellets from step 3.5 using one-half the volume used in step 3.1. 3.7 BREAK UP pellets using spatula, Dounce HOMOGENIZATION, and SONICATION as in steps 3.2 3.4. CENTRIFUGE as in step 3.5; RECOVER SUPERNATANTS and SAVE PELLETS for Third extraction. 3.8 THIRD TEEN 1.0% EXTRACTION: Resuspend the pellets from step 3.7 using one-Third the volume used in step 3.6. 3.9 BREAK UP pellets using spatula, Dounce homogenization, and sonication as in steps 3.2 3.4. CENTRIFUGE as in step 3.5, RECOVER SUPERNATANTS and SAVE PELLETS (if substantial) for Extra extraction to repeat as in step 3.8. 3.10 COMBINE the first, second and third pooled supernatants from steps 3.6, 3.8 and 3.10, and Remove two 3 ml samples for analyis including Average protein determinations (by Lowry and Bradford). 3.11 CONCENTRATE to about 50% of volume to yield protein of at least 4.0 mg/ml using Amicon filtration system (3 atmos.). Remove two 3 ml samples for analysis. Concentrated protein determinations: 3.12 Material is stored at 1 5.degree. C. if further processing to start within 24 hrs. If not, store at -20.degree. C. 4.0 REMOVAL OF ENDOTOXIN FROM OMV: PART A: AMMONIUM SULFATE PRECIPITATION 4.1 Obtain and, if necessary, thaw, material from step 3.13. 4.2 FIRST AMMONIUM SULFATE addition added at 500 g per liter solution. 4.3 STIR to dissolve at 19 25.degree. C. and let stand at same temperature for 15 minutes. 4.4 CENTRIFUGE in plastic centrifuge bottles at 20,000.times.g (11,100 rpm in sorval GSA rotor or 11,500 in Beckman JA 14 rotor) or equivalent for 20 min. at 1 5.degree. C.; discard supernatants. 4.5 DISSOLVE the pellets in TEEN 1.0% buffer using one-quarter the volume used in step 4.2 magnetically stirring at 1 5.degree. C. 4.55 MEASURE Protein content: Adjust to 2 3 mg/ml: add TEEN 1.0% solution (if turbid, LPS not removed; if <2.0 mg/ml protein lost). 4.6 STIR at 19 25.degree. C. with magnetic stiring bar for one hr. 4.65 SONICATE in a Branson Sonication Bath model 5200 or equivalent for 15 min. at 19 25.degree. C. using 200 ml solution per batch in a 600 ml beaker or 500 ml solution in a 1000 ml beaker as appropriate. 4.7 SECOND AMMONIUM SULFATE addition: to combined sonicated batches, ADD 500 g per liter of solution as in step 4.2: 4.8 STIR and CENTRIFUGE at 20,000.times.g as in steps 4.3 and 4.4. 4.9 DISSOLVE pellets using TEEN 1.0% buffer using 85% of "New volume" used in step 4.55 stirring at 1 5.degree. C. 4.10 STIR and SONICATE as in steps 4.6 and 4.65. 4.11 THIRD AMMONIUM SULFATE addition: to combined sonicated batches, ADD 500 g per liter of solution as in step 4.2: 4.12 STIR and CENTRIFUGE at 20,000.times.g as in steps 4.3 and 4.4. 5.0 REMOVAL OF ENDOTOXIN FROM OMV: PART B: TEEN 1.0% EXTRACTION 5.1 FIRST TEEN 1.0% EXTRACTION: Dissolve pellets in TEEN 1.0% buffer using 60% of the "New volume" used in step 4.55 stirring at 1 5.degree. C. 5.2 SONICATE as in step 4.65. 5.3 CENTRIFUGE the sonicated solution from step 5.2 at 27,500.times.g for 25 min. at 1 5.degree. C. (13,000 rpm in a Beckman JA-14 rotor or equivalent). Carefully RECOVER SUPERNATANTS, pool and store at 1 5.degree. C. SAVE pellets for second extraction. 5.4 SECOND TEEN 1.0% EXTRACTION: Dissolve the pellets from step 5.3 using 30 ml of TEEN 1.0% buffer per 250 ml centrifuge bottle stirring at 1 5.degree. C. 5.5 BREAK UP pellets using spatula, Dounce homogenizer, syringe and sonication as in steps 3.2 3.4. 5.6 CENTRIFUGE as in step 5.3. Recover supernatants and SAVE pellets for Third extraction. 5.7 THIRD TEEN 1.0% EXTRACTION: Resuspend the pellets from step 5.6 using 25 ml of TEEN 1.0% buffer per 250 ml centrifuge bottle stirring at 1 5.degree. C. 5.8 BREAK UP pellet using spatula, Dounce homogenizer, syringe and sonication as in steps 3.2 3.4. CENTRIFUGE as in step 5.3, RECOVER SUPERNATANTS, pool and store at 1 5.degree. C. 5.9 COMBINE the FIRST, SECOND and THIRD pooled supernatants from steps 5.3, 5.7 and 5.8, and measure the total volume: 5.10 DIALYZE this solution vs. TEEN 0.1% buffer with Spectrapor #3787-D42 dialysis tubing (12,000 MWCO) for 40 44 hrs. at 1 5.degree. C. with two buffer changes. Buffer/to solution volume ratios mus be at least 10:1. 5.11 RECOVER THE RETENTATE and SAVE; measure and record the volume. Remove two 2.5 ml samples for analyis including Average protein determinations (by Lowry and Bradford). 5.12 ALIQUOT into 5 ml vials, label and store at -70.degree. C. Label "N. mening. Gp. B tp 2 OMP PROTEOSOMES" with lot no. and date. Remove two 2.5 ml samples for analyses: SDS-PAGE, LPS and nucleic acid.

Vaccine Preparation

LP-S or LPSad from Shigella flexneri 2a or S. sonnei and group B serotype 2b Neisseria meningitides outer membrane proteins were mixed at 1:1 ratio (w/w) in 1% Empigen (2 mg/ml final concentration of LPS/LPSad and protein). The mixture was dialyzed in 1000 DALTON molecular weight cutoff (MWCO) dialyzis bags against PBS at 4.degree. C. for 10 days with daily buffer exchanges versus PBS or Tris saline buffer or their equivelant. Since both the proteosomes and the LPS are of very high molecular weight, it is obviously likely that dialysis membrane with MWCO of far greater than 1000 daltons can reasonably be used and similarly, the length of the dialysis time would be considerably shortened. The vaccine preparations, prot-LPS or prot-LPSad were kept at 4.degree. C. and diluted to the specific concentration with PBS just prior to vaccination.
 

Claim 1 of 20 Claims

1. An oral or intranasal subunit nonliving vaccine comprising a carrier and an effective amount of an immunogenic hydrophobic complex essentially consisting of proteosomes and at least one non-detoxified antigenic lipopolysaccharide, wherein the effective amount is sufficient, upon being administered orally or intranasally, to protect against infectious or toxic diseases entering at mucosal surfaces.

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