SK377992A3 - Bivalent immunogenes and method of their preparation - Google Patents

Abstract

Bivalent immunogens effective against diseases caused by viruses from the Orthomyxoviridae or Paramyxo-viridae tribe and gram-negative bacteria or spirochete Borrelia Burgdorferi consist of virus glycoproteins connected via their C- or N- endings with bacterial (spirochete) proteoses. The immunogens are prepared by a compound dialysis containing a detergent, virus glycoproteins and bacterial (spirochete) proteoses with a physiological medium at progressive pH decrease. After detergent's disposal, there is a mutual bonding between virus and bacterial molecules. Purified preparation is dried in lyophilisation.

Description

The invention relates to bivalent immunogens active against diseases caused by viruses from the family Orthomyxoviridae or Paramyxoviridae on the one hand and gram-negative bacteria or spirochete Borrelia Burgdorferi (Lyme disease) on the other, as well as a process for their preparation.

BACKGROUND OF THE INVENTION

For prophylactic influenza vaccines, vaccines consisting of partially purified and formaldehyde-killed virus are currently used. Their disadvantage is the low degree of purity of the preparations, denaturation of the envelope glycoprotein ncuraininidase - responsible for inducing surface mucosal immunity by formaldehyde, and the presence of glycolipids of the viral envelope in the preparation. This means the possibility of an increased frequency of post-vaccination reactions, a lower immunogenicity of the preparations, as well as the possibility of inducing autoimmune processes by glycolipids in the body.

According to the present results on various animal species, it is believed that the presence of antibodies against osp A / outcr fever protein A / Borrelia Burgdorferi spirochete in the body prior to infection can prevent the development of Lyme disease; in the course of already developed chronic disease, antibody-forming antibodies are no longer capable of protection (Edelman, Vaccine, 9, 531, (1991)).

SUMMARY OF THE INVENTION

These disadvantages of vaccines consisting of whole killed pathogens (viruses or bacteria) eliminate bivalent immunogens against influenza and Lyme disease, or other bacterial or paramyxovirus diseases of the invention, consisting of native and only immunorelevant polypeptides. The principle of the invention is that immunogens consisting e.g. from the influenza virus glycoproteins hemagglutinin and neuraminidase with a molecular weight of 76, respectively. 65 kDa, which are bound by their C- and C- respectively. N-termini with bacterial proteosomes. The spatial orientation of the viral glycoproteins in the preparation is the same as in virion particles - virions, which allows the presentation of their epitopes to immunocompetent cells of the organism. The immunogens thus prepared are preparations of high purity and do not contain unwanted viral or bacterial substances (except for the aforementioned virus glycolipids or e.g. lipopolysaccharides / LPS / bacteria, and the like).

Bivalent immunogens against influenza and Lyme disease or diseases induced by paramyxoviruses or gram-negative bacteria can be prepared by the method of the invention by mixing a mixture of glycoproteins e.g. influenza virus - haemagglutinin (HA) and neuraminidase (NA) and bacterial proteosomes dialyzed for 4 to 7 days against saline at pH 8.0 to 7.2 using first tris / hydroxymethyl / methylammonium chloride, followed by a mixture of primary and secondary phosphate sodium for buffering. Further removal of detergent, which is used to isolate both viral glycoproteins and bacterial proteosomes, is achieved by repeated filtration on the membrane in a mixing chamber and replenishment with sterile saline buffered to pH 7.2, complete removal of detergent and other substances by gel chromatography. on a granular agarose column. The resulting preparations of bivalent immunogens containing bound molecules of the influenza virus envelope glycoproteins and the proteosomes of the bacteria are dried, preferably by lyophilization. They are stored in vacuum sealed glass ampoules after addition of aluminum hydroxide as adjuvant.

The preparations thus prepared are highly immunogenic when administered with or without an adjuvant.

The viral and bacterial polypeptide molecules present in the resulting preparation exhibit a maximum degree of purity, which ensures that undesirable post-vaccination reactions of the organism to the vaccine are minimized. In the case of viral glycoproteins, this is a triple purification, that is to say in the purification of the virus, subsequently in the isolation of the envelope glycoproteins, and finally in the purification of the complexed material by gel chromatography. Gel chromatography removes e.g. high and low molecular weight substances of bacterial origin, such as fragments of cytoplasmic cylinders q. Burgdorferi and the like.

The following abbreviations are used below: TBS - tris / hydroxymethyl / methylammonium chloride buffered saline; PBS - a mixture of primary and secondary sodium phosphate buffered saline; EDTA - ethylenediaminetetraacetate disodium salt; PAGE - polyacrylamide gel electrophoresis.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

Isolation of influenza virus and paramyxovirus glycoproteins

The virus is propagated on the chick embryo chorionalantoic membrane and purified from alentoic fluid by differential centrifugation and subsequent sucrose density gradient electrophoresis (Rosenberg et al., Acta Virologica, 25, 7B, 1981). The purified virus is treated for 1 hour at 36-38 ° C with a detergent-N-dodecyl-N, N-dimethylglycine (Empigen BB) at a concentration of 1% at a concentration of 300 µg protein per ml and layered in a volume of 1 ml. to 3-15% sucrose density gradients of 4 ml and containing 0.5% detergent. Centrifuge for 4.5 hours at 45,000 rpm. and 4 ° C in the SW 55.Ti rotor of the L-8 centrifuge. Polypeptide M sediments minimally and remains in the volume of liquid approximately as before centrifugation, the viral envelope glycoproteins are at the interface between the upper and lower half of the centrifuge tube volume, and the virus nucleocapsid sedimentes into the sediment below the sucrose density gradient column. The content of viral polypeptides in each fraction is determined by PAGE. Fractions containing virus glycoproteins are pooled and either stored frozen at -18 ° C after protein concentration determination or used for complexing with bacterial proteosomes immediately after isolation.

Paramyxovirus glycoproteins are isolated in the same manner

Example 2

Preparation of bacterial proteosomes from B. Burgdorferi spirochete

Spirochetes are suspended in TBS pH 7.4 containing 10 mM EDTA at a protein concentration of 1-2 mg per ml and treated with a detergent.

Empigen BB under the same conditions as the virus (Example 1) Spirochete proteins are precipitated by the addition of crystalline ammonium sulfate in an amount of 5 g per 10 ml of liquid. After dissolution of the salt, the suspension is stored with occasional shaking for 1 hour at 4 ° C and the precipitate obtained by centrifugation. The deposition is formed in the form of a blank floating over the fluid, since osp A and osp B spirochetes are low density lipoproteins. The blank is separated from the liquid and dissolved in T BS-EDTA with 1% detergent content. The precipitation is repeated with ammonium sulfate and the residue is dissolved and precipitated a third time. The final deposit, after dissolution, first clarifies at 3000 g for 10 minutes and then at 18 000 g for the same time. The clarified supernatant is layered in a volume of 1 ml per 4 ml of a 5-20% sucrose density gradient containing 0.3% detergent and centrifuged for 3 hours at 36-0.0 rpm. and 4 ° C in the SW-50.1 rotor of the L-8 fusion center. The proteosomal spirochete material is located in the upper 40-45% of the centrifuge tube volume at the end of centrifugation. Aliquots of individual fractions are analyzed by PAGE for polypeptide content. Proteosome-containing fractions are pooled and either stored frozen at -18 ° C after determination of protein concentration or used for complexing with viral envelope glycoproteins immediately after preparation.

Example 3

Preparation of bivalent immunogens

Influenza virus glycoproteins are mixed with proteasomes of gram-negative bacteria in a hesitant 1: 1 protein ratio or with B. Burgdorferi spirochete proteosomes 1: 1.5 in the presence of 1% Empigen BB. The mixtures are dialyzed at 4 ° C for 4-7 days, especially initially with frequent dialysis buffer changes. First, TBS-EDTA pH 8.0 was used, followed by PBS pH 7.2. Further removal of detergent from the material is achieved by triple ultrafiltration on an XM-300 membrane in a mixing chamber. The material is concentrated to a protein concentration of about 2 mg per ml and applied to a Seph rose 6 B column; For a 1 ml sample, approximately a 26-30 ml column is sufficient. Gel chromatography is performed using PBS pH 7.2. If there are fragments of so-called material in the material. cytoplasmic cylinders of spirochete; void volume / V / columns. Beyond this peak, the elution of virus-bacterial complexes begins, and just before the total column volume (V *), detergent residues elute at the symmetrical peak. The molecular weight corresponding to the elution volume (V) of the detergent, i.e. about 70 kDa, indicates its presence in solution in micellar form. The simultaneous presence of viral and bacterial polypeptides in the individual fractions obtained during gel chromatography is again determined by PAGE.

Fractions containing the bivalent immunogen preparation thus prepared are pooled and lyophilized such that 20 µg of protein of the complex of viral glycoproteins with gram-negative bacterial proteosomes or 25 µg of the protein of complexes of viral glycoproteins with the proteosome of spirochete B are contained in one vial. 20 mg of aluminum hydroxide is added to each vial prior to lyophilization. After lyophilization, the vials are stopped under vacuum and stored at -18 ° C. _x Resuspension is made immediately before application by shaking the contents of the vial in 1 ml of sterile PBS pH 7.2. Ten vaccine doses of 0.1 ml containing 1 [mu] g of viral glycoprotein protein, 1 [mu] g of proteosome protein of gram-negative bacteria, and preferably 1.5 [mu] g of protein spirochete protein and finally 2 mg of aluminum hydroxide are thus obtained.

Influenza virus envelope glycoproteins were administered subcutaneously to adult white mice, either as a bivalent immunogen with the B. Burgdorferi proteosomes (HANA-PBB) or formaldehyde-killed purified virus (FV), at 2-week intervals. The preparations were administered either with or without aluminum hydroxide (Al). Protein weights of administered viral glycoproteins were:

= 1.0 / µg HANA-PBB; 2 = idem-A1; 3 = 100 ng HANA-PBB; 4 = idem-A1; Δ = 1.0 µg FV; 6 = idem-A1; 7 = 100 ng FV; 8 = idem-A1;

In the sera of vaccinated mice obtained two weeks after the second dose, hemagglutination-inhibiting antibodies against influenza virus were found in reciprocal titers:

1 = 512; 2 = 2.048; 3 = 256; 4 = 2.04B; Δ = 256; Δ = 512; 7 = 64 and 8 = 12B.

Virus-neutralizing antibodies in the same sera showed reciprocal titers:

1 = 128; = 1.024; 3 = 32; 4 = 512; 5 = 32; 6 = 128; 7 = 8 and 8 = 16.

The protection test was performed by inducing so-called " toxic pneumonia by administering a massive dose of about 10? ID µg of live virus instilled in intranasally vaccinated mice. 48 hours after challenge, virus was isolated from the lungs of mice in infectious titers expressed per ml of a 10¾ lung suspension:

l = 10 ³ ; 2 = <10 °; 3 = 10 ⁵ ; 4 = <10 °; 5 = 10 ⁵ ; 6 = 10 ³ ; 7 = 10 ⁷ ; 8 = 10 ⁶ ;

control unvaccinated mice were found to have a titer of 10 ⁸ ID / ml lung suspension.

The macroscopic appearance of the lung lobes of vaccinated mice 48 hours after induction of toxic pneumonia was as follows:

1) lungs of normal size, pink color, in the peritracheal regions smaller red edematous lesions; 2) general slight edematous (spongy) enlargement of the lung lobes, without local lesions, the lungs are pink in color; 3) generally edematous enlarged spongy lobes of a gray color, with medium-sized red local edematous lesions; 4) greyish, sponge-enlarged lung with large red edematous lesions;

5) dark violet edematous lesions over the entire surface area of the lung lobes; 6) sponge-enlarged lung lobes with extensive dark violet local edematous lesions; 7) gray lung lobes covered over the entire surface with dark violet edematous lesions; 8) gray-pink lung lobes with extensive dark violet edematous lesions. In control unvaccinated mice, retracted lung lobes with red edematous lesions predominating in the peritracheal regions were found.

The detection of influenza virus in the lung tissue of FV-vaccinated mice corresponds to the inactivation of the virus neuraminidase by formaldehyde, thereby preventing the formation of superficial mucosal IgA antibodies.

In observing the persistence of hemagglutinating-inhibiting antibodies in the organism of mice, we found two resp. a tube of lower titers than the sera obtained two weeks after the second vaccine dose.

Influenza virus glycoproteins complexed with Ncisse ria meningitidis protcosones elicited antibodies in mice at the same titers as they did with their Borrelia Burgdorferi proteosome complexes. B. Burgdorferi proteosomes impart comparable adjuvantic, i.e., B-cell mitogenic and T-helper cell activities to complexed preparations, as predicted for Neisseria meningitidis proteosomes (Lowell et al., J. Exp. Med., 167, 658, 1988). In the protection test, vaccinated mice failed to cultivate live B. burgdorferi from their spleens and bladders, whereas in control unvaccinated animals such culture was successful.

The degree of purity of the preparations complexed from influenza virus glycoproteins and B. burgdorferi proteosomes was verified by Western blotting. The sera of mice obtained two weeks after the second vaccination dose contained antibodies only against influenza virus glycoproteins (HA, HA1, HA2, NA) and against osp A and partially osp B spirochete polypeptides. By inducing antibodies against osp A, a preparation containing the B. burgdorferi proteosomes complexed with viral envelope polypeptides provides the preconditions for protecting the organism against the development of Lyme disease (Edelman, Vaccine, 1991, supra).

All these investigations document the potency of complexed preparations compared to other types of chemovaccines.

Industrial usability

The invention can be used in the field of biochemistry and immunology.

Claims (3)

Bivalent immunogens against diseases caused by viruses of the family Ortho - or Paramyxoviridae and gram-negative bacteria or spirochete Borrelia Burgdorferi, characterized in that they consist of glycoproteins e.g. influenza virus hemagglutinin and neuraminidase with a molecular weight of 76, respectively. 65 kDa, for paramyxoviruses with mol. weight 79 resp. 61 kDa for H and F glycoprotein, which are bound by their C- and C- respectively. Ends with bacterial or spirochetal proteosomes. I 2. A process for the preparation of divalent immunogens according to claim 1, wherein the mixture of viral glycoproteins and bacterial resp. Spirochetal proteosomes are dialyzed for 4 to 7 days against saline at a pH of from 8.0 to 7.2 using first tris / hydroxymethyl / methylammonium chloride and then a suitable buffer to remove residual detergent from the preparation by first repeated filtration on a membrane in a mixing chamber and replenishing with sterile saline buffered to pH 7.2 and finally by gel chromatography on a granular agarose column and the obtained bivalent immunogens containing bound molecules of viral glycoproteins and proteosomes of bacteria or spirochetes are preferably lyophilized. 3. A process according to claim 2, characterized in that a mixture of primary and secondary sodium phosphate is used for buffering.