RU2159128C1 - Oral chemical vaccine for protecting against cholera - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: vaccine contains cholerogenous anatoxin and O- antigen from Inab V. Cholrae 569B strain and immunogenic antigen complex. The immunogenic antigen complex is obtained from culturing liquid medium of V. cholerae strain O139 serovar. It consists of the basic somatic O- antigen of lipopolysaccharide nature of O139 serovar with molecular mass of 1000 kD, glycoprotein capsule of O139 serovar with molecular mass of 600 kD and protein exoenzymes of O139 serovar with molecular mass of 100-200 kD. It contains neuraminidase, phospholipase, lipase, and protease as exoenzymes. When required, the vaccine contains filler for making tablets and capsules (starch, sugar, calcium stearate or talc). EFFECT: enhanced effectiveness in protection against cholera vibrio strain O139. 1 dwg, 3 tbl

Description

 The invention relates to the field of biotechnology, in particular to the production of bacterial preparations for the specific prophylaxis of cholera caused by epidemically significant strains of NAH vibrios 0139 serovar.

 In 1992-93, for the first time in the history of the cholera pandemic in India and Bangladesh, and then in other countries of South Asia, a major epidemic of cholera occurred, covering tens of thousands of cases of diseases with significant mortality, the causative agent of which was a new strain of Vibrio cholerae, not 01 serovar, but designated 0139. This strain produced an in vivo toxin similar to Vibrio cholerae 01 serovar and caused clinically, pathogenetically and epidemiologically typical cholera (Lomov Yu.M., Mazrukho B.L., Mishankin B.N. et al. territory of Russia cholera caused by new sulfur aroma // ZhMEI. -1995.-N2 (Appendix).-S. 97-101).

 The strains of V. cholerae 0139 of the serovar also produce a polysaccharide capsule, which contributes to the cases of septicemia described with this serogroup. It is known that the capsular polysaccharide helps adhesion to epithelial cells, plays a role in immunogenesis (Hisatsune K., Kondo S., Issuki Y., lguchi T., Kavamota Y., Shimada T. / Biochem.-Biophys.-Res.-Commun . 1993, N 196 (3). P. 1309-15; Knirel YA, Paredes L., Jansson PE, Weintraub A., Widmalm G., Albert MG / Eur. J. Biochem., 1995, N 233 (2). P. 391-6).

 At the same time, cross-immunity with typical pathogens of serovar cholera 01 is not created, and therefore, WHO has concluded that the existing commercial cholera vaccines made from V. cholerae 01 serovar strains are unsuitable for protection against infection with V. cholerae 0139 serovar strains and the importance of creating oral vaccine against strains of this serovar (Weekly epidemiological record, N 24, 1998).

 There are reports of the design of potentially vaccine strains to create a live vaccine against 0139 serovar strains: parenteral immunization of rabbits with a recombinant strain induced high titers of antibodies against LPS and capsular polysaccharide (Infection and Immunity, 1996. Vol. 64, No. 9).

 The closest analogue to the claimed vaccine is a chemical vaccine consisting of a cholerogen toxoid along with O-antigen of Serovar Inaba. The method of its production consists in sequential fractionation of ammonium sulfate from the culture fluid of the Inaba strain of the Inaba somatic O-antigen between 0.2 and 0.35 saturation and cholerogen toxoid between 0.35-0.8 saturation, followed by dialysis, lyophilization and construction of the tablet preparation with by applying an acid-resistant coating to tablets containing 100,000 EC toxoid and 1000 conv. units Inaba O-antigen (reverse titer of RPHA) (Russian patent N 2076734, A 61 K 39/00).

 This vaccine provides specific protection against infection with serovar cholera vibrios 01, however, it does not have a protective effect against 0139 serovar strains, like all other known cholera vaccines.

 The objective of the invention is the creation of an oral chemical vaccine that protects against an epidemically dangerous strain of cholera vibrio 0139 serovar.

 The essence of the invention lies in the fact that the composition of the oral chemical vaccine against cholera containing the cholerogen toxoid and O-antigen from strain V. cholerae 569B Inaba additionally introduced antigenic immunogenic complex obtained from the culture fluid of strain V. cholerae 0139 serovar, consisting of the main somatic O-antigen of lipopolysaccharide nature 0139 serovar with mol. weighing 1000 kD, glycoprotein capsule 0139 serovar with mol. weighing 600 kD, protein exoenzymes 0139 of serovar (neuraminidase, phospholipase, lipase, protease) with mol. weighing 100-200 kD. If necessary, the inventive vaccine contains an excipient for tablets or capsules.

 The inventive vaccine is a bivalent vaccine consisting of a cholerogen toxoid, which is a common antigen for serovars 01 and 0139 and O-antigen of strain 0139 of serovar, specific for representatives of 0139 serovar. In addition, the claimed vaccine incorporates the Inaba O-antigen, which increases the immunological activity of the vaccine, is adsorbed on the cholerogen toxoid and its removal reduces the yield and activity of the drug.

 The invention will be presented using a drawing with a graph illustrating the infrared spectrum of the O-antigen included in the inventive vaccine, and tables 1-3, where in table. 1 presents comparative data on the composition of the culture fluid of cholera vibrio strains 01 and 0139 of serovars by ultrafiltration through amicon membranes; in table 2 presents the results of the isolation of O-antigens of producer strains 01 and 0139 of serovars from the culture fluid; in table 3 shows the chemical composition and serological properties of the fraction isolated from strain 0139 of serovar.

 Signs that distinguish the claimed vaccine are in particular the structure of the O-antigen of strain 0139 of serovar, its lower molecular weight and the presence of a glycoprotein capsule. In the table. 1 for comparison, the composition of the culture fluid of the strains of cholera vibrio 01 of serovar (the closest analogue) and 0139 of serovar is presented. As can be seen, 100% of O-antigen in strain 01 of serovar was retained on high molecular weight membranes (300XM and 100XM), i.e. had a pier. a mass of more than 100 kD, while the strain 0139 of the serovar, the bulk of the O-antigen was retained on the membrane 50RM, i.e. pier mass is lower. To obtain the O-antigen of strain 0139, a different precipitation path was used, the fractionation interval was 20-80%, and not 50-80%, as for 01 serovar (Table 2).

 As a result, the biomedical properties of the claimed vaccine differ from the properties of the closest analogue.

 1. To obtain the claimed vaccine, fractionation of O-antigen is carried out in the range of 0.2-0.8 saturation, since the molecular weight of the drug is taken into account.

 2. The resulting vaccine component contains a significant amount of 3,6-dideoxyhexose, which indicates the presence of a capsule and is not typical for strains 01 of the serovar cholera vibrio.

 A method of obtaining the inventive vaccine.

 As a producer of the oral chemical cholera vaccine, strains of the cholera vibrio 0139 of serovar are used (for example, M045, P16064 and the like). For the second component of the vaccine containing the cholerogen toxoid and Inaba O-antigen, the toxigenic strain of serovar cholera vibrio 01 (for example, 569B and others) is used as a producer and this vaccine component is obtained in accordance with the method described in Russian patent N 2076734, A 61 K 39/00.

Cholera vibrio 0139 serovar is grown at a temperature of 37 ^o C in a reactor on a medium from casein enzymatic hydrolyzate (pH 8.0, 200 mg% amine nitrogen, Na ₂ HPO ₄ - 0.05%, NaCl - 0.5%) under conditions of deep cultivation with fertilizing glucose and ammonia. In the stationary phase of growth (10-11 hours of cultivation), when the concentration of microbial bodies, reaching 70-80 billion per ml, remained constant for 3 hours, the cultivation was stopped by adding 0.6% - 0.2% formalin. The content of O-antigen dissolved in the culture fluid at this time by the reaction of diffusion precipitation with O-serum is 4-8 conv. units (reverse titer of the diffusion precipitation reaction in the gel). Killed microbial bodies after 12 hours are separated by centrifugation at 15,000 rpm, and the supernatant is kept for 30 days at a temperature of (10-12) ^o C to reduce the toxicity of O-antigen. After that, they begin to isolate O-antigen from the culture fluid. At the first stage, ammonium sulfate is added to the precipitation reactor to 0.2 saturation (152 g of salt per 1 liter), stirred, left for 18-20 hours at 18-22 ^o C and centrifuged on SGO-100 at 8000 rpm. The resulting precipitate of the ballast fraction is discarded and ammonium sulfate is again added to 0.8 saturation (456 g of salt per 1 liter). After centrifugation, the pellet is dialyzed against water. The solution of O-antigen is diluted with distilled water (on average, to a volume of 4-5 L), poured into sterile containers of 250 ml and sterilized by fluid steam for 30 minutes or by adding sodium thiolate at a concentration of 1: 10000 - 1: 20,000. Subsequently, the liquid preparation is freeze-dried and used to construct a tablet vaccine.

 The chemical composition and serological properties of the fraction isolated from strain 0139 are presented in table. 3.

Analysis of the infrared spectrum of the drug 0139, the registration of which was carried out within the boundaries of 400-4000 cm ^-1 (Fig. 1), made it possible to identify absorption bands characteristic of polysaccharides in the preparations (spectral range from 700 to 1450 cm ^-1 ). There is also an absorption band of 1540 cm ^-1 , which belongs to the deformation vibrations of NH bonds in proteins. The drug has an absorption band of 1740 cm ^-1 , located on the shoulder of an intense band of 1670 cm ^-1 , associated with the presence of lipids. In the region of 2800-3000 cm ^-1 , the bands of stretching vibrations of CH ₂ groups of fatty acids are clearly distinguished. Thus, the data of IR spectroscopy of the preparations correlated with the results of chemical analysis and confirm the lipopolysaccharide nature of the obtained preparation.

 The monosaccharide composition of this preparation, studied by thin-layer chromatography, was represented by galactose, glucose, mannose, ribose and 3,6-dideoxyhexose, which is supposedly a component of the capsular polysaccharide.

The content of exoenzymes in the fraction was: 32 units / mg proteinase and 410 units. / mg phospholipase. The toxicity of the drug according to LD ₅₀ for white mice, which averaged 1.2 mg subcutaneously, was half that of a commercial oral vaccine from strain 01 of serovar. The immunogenicity of this preparation, its ED ₅₀ for white mice, when infected with 100 dcl of strain 0139, was 3-5 μg, which corresponded to the protective ability of a commercial vaccine against infection with serovar cholera vibrio 01 strains.

 The vaccine contains in 1 tablet 100,000 EC cholera toxoid, 1000 srvc. units O-antigen 139 serovar, 1000 conv. units Inaba (reverse titers of RPHA and RID), filler (starch, sugar, calcium stearate, talc), is coated with an acid-resistant coating (capsules can be used).

Claims (1)

 Oral chemical vaccine against cholera containing cholerogen toxoid and O-antigen from Inaba strain Vibrio cholerae 569B, characterized in that it additionally contains an antigenic immunogenic complex obtained by culturing serovar strain Vibrio holerae 0139, consisting of the main somatic O-antigen 01 lipopolysaccharide nature serovar with a molecular weight of 1000 kD, glycoprotein capsules 0139 serovar with a molecular weight of 600 kD, protein exoenzymes 0139 serovar with a molecular weight of 100 - 200 kD, and, if necessary, filler d I have a tablet or capsule.

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