RU1837890C - Method of preparing of immunogen stimulating human immune system with hiv-infection - Google Patents

Abstract

Field of use: virologists. SUMMARY OF THE INVENTION: A non-infectious immunogen is provided comprising retroviral particles lacking outer coat proteins or containing selected antigens isolated from retrovirus. A vaccine effective against HIV is also provided. In one embodiment of the invention, the immunogen is useful for immunizing an individual previously infected with a retrovirus, including HIV. with that. to induce immunoprotective factors protective against the development of infection. The immunogen can also be used to produce antibodies for passive immunotherapy, as such or in combination with active immunotherapy, in individuals infected with retrovirus, including HIV, preferably those individuals that show low levels of antibodies to retroviral gene products other than the outer coat. 3 ill., 1 tab. (L C

Description

The invention relates to the control of retroviruses, and more particularly, to a method for radiating an immunogen that stimulates the immune system of a person infected with HIV.

The purpose of the invention is to provide a highly effective agent for controlling retro-iri, primarily HIV retrovirus.

The problem is solved by the proposed method for producing a person stimulating the immune system infected with HIV, namely, HIV-infected cells are grown in a ReJAPM 1640 in the presence of cattle embryonic serum, the 5-supernatant is filtered, / 3-propiolactone is added to the filtrate and incubated for 5 hours at 37 ° C and a pH of 7.2-7.4, after which they are frozen, irradiated with gamma rays, thawed, concentrated. They are passed through a millipore polysulfone filter, first purified by centrifugation with 30% sucrose, then centrifuged in a gradient of 30-45% sucrose, and the product is obtained by resuspending the resulting precipitate in PBS buffer at a concentration of 1 ml per 10 l of the starting material.

In FIG. 1 is a schematic model of HIV.

As shown in Figure 2, the HIV RNA genome encodes three major structural genes: gag, pol and env, which are flanked at both ends by long terminal duplications (LTR). The gag gene encodes specific proteins of the core, p55, p39, p24, p17 and p15. The pol genes encode p65 / p51 reverse transcriptase and p31 protease. Env genes encode outer membrane glycoprotein dr 120 and

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its predecessor gp 160, as well as the transmembrane glycoprotein dr41. Some of the genes are extremely variable, in particular env genes.

In addition, there are five other genes not present in other retroviruses that are either involved in transcriptional or translational regulation or encode other structural proteins. The structure of the HIV gene is known. It is described by Ratner et al. Nature, 313, p. 277 (1985).

HIV binds to host cells by the interaction of membrane glycoproteins with a cell surface receptor. It appears that upon contact of HIV with a T4 cell, the dr120 protein interacts with the CD4 receptor. The viral envelope then fuses with the cell membrane, and the inner core of the virus enters the infected cell, where transcription of RNA into the DNA virus is catalyzed by reverse transcriptase. Provirus can remain latent in the cell for several months or years. and all this time, the infected individual is asymptomatic. However, if the virus is later activated, causing viral replication and immunosuppression, the individual will then be susceptible to opportunistic infections, including cancer, associated with AIDS.

In FIG. Figure 3 shows a schematic reproduction of the level of certain antibodies and antigens present in development from an asymptomatic state to the state of ARC (aids-related complex) and to SAGDM in HIV-seropositive HIV-infected patients.

The results of the Western immunogen assay of Example 1 show that it is free of outer envelope proteins when screened for homologous and heterologous sera that contain high antibody titers against outer envelope proteins.

As shown schematically in FIG. 3, high levels of anti-gp 160/120 antibody (outer sheath), which are present in the asymptomatic phase of HIV infection, also continue to exist in the symptomatic phase. The level of p24 antibody in the asymptomatic phase is high, but appears to decrease in the symptomatic phase. Similarly, HIV seropositive sera contain an antibody that inhibits reverse transcriptase function. In patients in which the anti-reverse transcriptase antibody is present at high levels, attempts are relatively

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virus secretions are less often positive than those in which it is absent. Therefore, it follows that a decrease in cellular and humoral immunoprotective factors such as T4 cells and anti-GAG antibodies, including anti-p24, and anti-pol antibodies, including anti-reverse transcriptase antibody, are associated with the development of AIDS.

The term HIV as used includes types 1 and 2 and is synonymous with HTLV-III, LAV-1 and LAV-2. HIV refers to the virus in a general sense and includes all forms, subtypes and variations.

As used herein, the outer sheath protein refers to that portion of the retrovirus membrane glycoprotein that extends beyond the membrane, as opposed to the transmembrane protein, dr41. The outer envelope protein of HIV is synonymous with dr120 and its precursor dr160.

The term dr120 or dr160 / 120, as used, refers to glycoproteins having either antigenic specificity or the biological function of the outer coat protein; dr160 is believed to be a precursor to dr120.

As used herein, a gene product refers to a polypeptide or protein that is encoded by a gene. The term is intended to include protein derivatives such as glycoproteins. It is understood that limited modifications can be made to the amino acid sequence of a gene product without impairing the biological function or immunogenicity of the gene product, and only a portion of the primary sequence may be required for immunogenicity.

The identification of HIV specific genes and gene products is based on HIV type 1 terminology shown in Fig. 1. However, it is contemplated that reference to a specific HIVTwna 1 gene or gene product, based on its molecular weight, will also include the corresponding H1V gene or gene product. type 2 and, when a homologous gene is present, other retroviruses. Gene products of other types and genera may have slightly different molecular weights. For instance. gp41 H1V type 1 is equivalent to drzb type 2, while d p120 type 1 corresponds to dr130 type 2.

HIV can be cultured from a peripheral blood sample of infected individuals. For example, mononuclear cells from peripheral blood, such as a lymphocyte, can be obtained by layering

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n Eoba heparinized venous blood 1 according to the density gradient Ficoll-Hypaque and centrifugation of the sample. Single nuclear cells are then harvested, activated, for example, with phytohemagglutinin, for two days and cultured in a suitable seed, preferably supplemented with int-fleukin 2. The virus can be detected either by reverse t-enanscriptase analysis, analysis with p24 antigen capture, immunofluorescence, or electron microscopy to detect the presence of viral particles in the cells. All of these methods are well known to those skilled in the art. Once isolated, / ipyc can be incorporated into other cells.

It is important to use a non-infectious vaccine in order to avoid infection from entering the host. Various methods are well known to make a pathogen non-infectious. E virus can be inactivated or replication deficient. However, it is preferred to treat it with a combination of beta-propiolactone and gamma radiation. In this case, / 3-propiolactene must be in contact with the virus for at least 2.5 hours. In order to remove any residual Beta-propiolactone by bareness, beta-propiolactone must remain in solution for at least five hours at a temperature of 37 ° C.

The isolated .virus is then treated to remove the outer envelope proteins. Such removal is preferably accomplished by repeatedly freezing and thawing the virus in combination with physical methods that cause swelling and contraction of the viral particles, although other physical and nonphysical methods, such as ultrasound disruption, individually or in combination, can also be used. together.

For immunization of a human or animal, the t mmunogen obtained by the proposed method is used together with excipients. But it can also be used in its aqueous form without the Excipient. In this case, the dose of E is chosen so that it is immunologically highly effective, and as a rule, it is from 1 to 100 μg of protein, g, preferably about 30 μg of protein.

Active immunization is carried out ti, preferably repeated once with a minimum interval of at least 90 /, although additional booster doses may be appropriate in accordance with changes in the level of immune activity based on, for example, a decrease in the number of

antibodies against HIV-gene products other than the outer shell proteins. Such immunization is preferably carried out initially by intramuscular injection of 5 followed by intradermal injection, although any combination of intramuscular and intramuscular injection can be used.

Preferably, the immunocompetence or immune activity of the seropositive individual is determined prior to immunization in order to determine the appropriate treatment modality. As a method for this determination of patient serum

15 are screened for the presence of anti-p24 antibodies (for example, by enzyme-linked immunosorbent assay), antibodies against reverse transcriptase and / or T4 cell level using well

0 known methods. Patients showing low immunocompetence indicators, e.g., low p24 titers or antibodies against reverse transcriptase or low numbers of T4 cells, are

5 suitable candidates for passive immunotherapy, preferably in combination (prior to or in conjunction) with active immunization.

Seronegative individuals may

0 to be vaccinated in order to induce immunoprotective factors to prevent infection. Preferably, the vaccine is administered initially by intramuscular injection followed by

5 by the introduction of a booster, carried out either intramuscularly or intradermally. A physiologically effective dose preferably contains from 1 to 100 μg, and more preferably about 30 μg of the immunogen.

0 The vaccine is preferably administered in combination with an adjuvant, i.e. an adjuvant, most preferably an adjuvant, to provide a preparation of the type water. in oil. Various

5 suitable adjuvants are well known in the art.

In addition, since anti-dr160 / 120 antibodies can promote viral absorption by cells, these specific antibodies can be removed from a patient infected with HIV before immunotherapy. Immunosorbent columns made of hollow cellulose fibers are modified to covalently bind ligands that are reactive with anti-dp160 / 120 antibodies. Such ligands include dr160 / 120 antigens derived from purified glycoproteins, which in turn are derived from freshly assembled viral particles or from the culture supernatant of H Y-producing T4 lymphocytes. Alternatively, such ligands can be obtained by recombinant DNA methods using transfected cells. Alternatively, idiotypes that are reactive with antibodies against dr160 / 120 can be used for this purpose.

Such anti-idiotypic antibodies can be obtained by methods well known in the art.

Example 1. Obtaining viral particles free of outer shell proteins.

Cells infected with HIV strain No. Hz 321 (isolated from the plasma of a 26-year-old African woman from Saira) were grown in medium A consisting of RPMI 1640 with 10% fetal bovine serum, 25 mM HEPES buffer, 50 μg / ml gentamicin and 100 mgk / ml streptomycin.

Cultures are expanded in volume by feeding the original cells into centrifuge tubes and adjusting the volume to approximately 8 L with the addition of pre-heated medium A. The dilution ratio is approximately 1: 5. A second dilution of 1: 3 is then carried out. The supernatant from a 5-7-day suspension of HIV-infected cells is filtered through a 0.45 μm filter. A freshly prepared 1:40 beta-propiolactone solution was added to the supernatant to a final concentration of 1: 4000. The solution was incubated for five hours at a temperature of 37 ° C and the pH was maintained at 7.2-7.4.

After 5 hours, 20 ml of the supernatant solution was removed in order to determine the level of infectivity and the amount of remaining beta-propiolactone. Then the supernatant is frozen at a temperature of -70 C. The frozen supernatant is then subjected to gamma irradiation with cobalt 60 with a power of 4.5 MR.

The supernatant solution is then thawed and concentrated by 40-fold filtration through 100,000 filters made with a molar mass. The concentrate is fed into T-21 flasks equipped with a mixer, pretreated with 70% isopropanol, and centrifuged at 28,000 rpm for one hour. The supernatant was removed and the pellet was resuspended in ETUK-containing sodium buffer to a total final volume of approximately 24 ml. 4 ml of this suspension is layered over 8 ml of 30% sucrose in pre-treated polyallomeric tubes

70% isopropanol, and centrifuged for one hour at 28,000 rpm. The supernatant was drained from 30% sucrose and the precipitate was resuspended in the indicated sodium buffer.

The gradient in ultralight centrifuge tubes pretreated with 7% isopropanol is set by adding 3 ml

0 45% sucrose in vitro and topcoated with 6 ml of 30% sucrose. 3 ml of the above suspension layered on the solution. The tubes are centrifuged for 60 minutes at 28,000 rpm.

5 The solution above the layers at a 35% transition is aspirated and pipetted. The layers from all tubes are combined in one conical tube and diluted 1:10 with a phosphate buffer

0 saline. The raster is centrifuged in polyallomeric tubes pretreated with 70% isopropanol for one hour at 28,000 rpm. The supernatant was removed and the pellets in the tubes were resuspended in the indicated phosphate-containing buffer solution at a concentration of 1 ml per 10 L of the starting material.

Alternatively, especially when the immunogen is obtained in large quantities, the irradiation step can be carried out after the layers are combined. Then the virus is stratified on a gradient of 15-50% sucrose. Viral layers are combined and resuspended

5 in the above phosphate-containing buffer solution. The virus is centrifuged at 28,000 rpm for one hour and resuspended in said buffer solution to a final concentration of 1.0 mg / ml.

The amount of protein available is determined according to the Bradford method (see Ap. Blochem. 72, p. 248, 1976). The protein is diluted with the above phosphate-containing buffer solution to a concentration of 1.0 mg / ml,

The level of residual beta-propiolactone in the immunogen is determined using capillary gas-liquid

0 chromatograph. In this case, standard solutions of beta-propiolactone and butyric acid are prepared having concentrations between 141,500 ppm. Samples of 2 μm volume were introduced into a capillary chromatograph according to the manufacturer's recommendation. The detection limit is 0.1 h / ml. The immunogen contains less than 0.05 h / ml beta propiolactone.

Eight immunogen samples were analyzed for beta-propiopectone by capillary gas chromatography using

malic acid as an internal andart. The following chromatographic conditions are used: column: 30 mx 0.25

m | l, quartz glass, open, tubular; stationary phase: 100% cyano-1l silicon; sample inlet temperature

1 0 ° C; operating temperature: 70 ° C / 1min, ° C / min to 130 ° C; sample injection procedure: fissile. In the above fishing, the retention times of beta-propioctone and butyric acid correspond to 52 minutes and 6.70 minutes, respectively. Entry 2

mCl 1 h / ml, a solution of beta-propiolactone leads to a peak, which can be measured quantitatively. A concentra- / 1 1 h / ml of a solution up to 1/10 of its volume by arivation of the solvent at a temperature of -40 ° C under reduced pressure

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ta-propiolactone. The limit detected by the concentration is 0.1 h / ml / 0.1 m | kg / ml. With that. in order to confirm that omunogen is free of outer scaffold proteins, the immunogen is first divided into 1.0% polyacrylamide gels using sodium dodecyl sulfate according to the Laemmli method, see Nature 7, p. 680, 1970) . The purified material is then transferred to nitrocellulose paper in accordance with the method proposed by Taubin et al. (See Progos, Nature. Acad, i., 76, p. 4350, 1979) and immunostained according to the method, proposed-) M Tsang et al. (cM.Meth. Enzymalogy, 92, p. 377, 1983). The spots of the immunogen thus obtained do not have a band corresponding to dr 120/160, as indicated in the control tests, when interacting with sera containing high anti-dr160 / 120 titers.

Commercial strips with the available HIV proteins screened with heterologous serum (chimpanzee / 86-C and A-3) and containing high titers of nibble against outer membrane proteins, with p160 / 120, are negative (unreacted, ionic) on the strips obtained by the I-th in the analysis of the obtained E as described above immunogen, sv {one from the outer shell. Homologous human sera (003 and 010) containing high-titers of antibodies against (outer codules, dr160 / 120, are reactive with commercial strips, but negative with strips obtained from an immunogen free of outer shell.Homologous as well as heterologous serum interact with

other gene products HIV. The absence of outer shell proteins on the proposed immunogen is confirmed by electron microscopy. 2.5% glutaraldehyde and osmium anhydride are successively added to the immunogen preparation, dehydrated with various concentrations of ethanol solutions and poured into EPON-812. Thin sections

0 is obtained using an LKB Ml crotome instrument (LKB, Uppisala, Sweden) using a diamond scalpel. The thickness of the sections is 60 nm. Sections stain uranyl acetate

5 with lead citric acid, observed and photographed using an Zeiss 109 electron microscope. Serving as a control, cells infected with HIV were prepared using the same technique. In this case, it is found that the immunogen does not have a dark-colored outer shell, which is inherent in the control samples and which reflects other 160/120 on the viral surface.

5 Example 2. Immunotherapy of seropositive individuals. Nine HIV-positive patients are treated with immunotherapy to administer the immunogen of Example 1. In this case, the immunogen is emulsified in a 1: 1 ratio in an incomplete Freund adjuvant in a Spex 8000 Mixer Mill brand foreign company Spex Industries Inc., USA. 1.0 ml of a solution containing 100 µg of protein is administered intramuscularly. A bead of 5 g of 100 µg of protein without adjuvant was administered after 90 days by intradermal injection.

The presence of HIV virus in patients' peripheral blood lymphocytes is determined both before and after immunization by co-cultivation with freshly prepared peripheral blood lymphocytes stimulated with phytohemagglutin and interleukin-2 in accordance with

5 by the method of Gallo et al. (CM.J. CIIn. Microb., 25, p. 1291.1987). Equipment for detecting the presence of HIV antigens, such as p24, is commercially available (e.g., from a foreign firm, E.I. Du-Pont & De-Nemours Co., Inc.,

0 United States). Each patient is examined three times before immunization and after 2,4,6.8,12 and 14.week after immunization.

The table shows the results of studies. Patients 010 and 003, of which

5 HIV is isolated before immunization; the virus does not show up to 12 weeks after immunization. Patients 008 and 009 are not carriers of the virus until 8 weeks after immunization. All four of these patients show high

anti-p24 titers (1: 5000) and anti-reverse transcriptase antibodies (1: 1000) before immunization. The remaining five patients who show low anti-p24 titers and anti-reverse transcriptase antibodies before immunization continue to show the secreted virus after immunization.

Claims (1)

SUMMARY OF THE INVENTION A method for producing an immunogen stimulating the immune system of a person infected with HIV, wherein the HIV infected cells are grown in RPMI 1640 medium in the presence of fetal 5 cattle serum, a 5-7-day supernatant is filtered, / -propiolactone is added to the filtrate and incubated for 5 hours at 37 ° C and a pH of 7.2-7.4, then frozen, irradiated with y-rays, thawed, concentrated, passing through millipore polysulfone filters, first by centrifugation with 30% sucrose, then centrifuged in a gradient of 30-45% sucrose, and the target product is obtained by resuspending the resulting precipitate in PBS buffer at a concentration of 1 ml per 10 l starting material. IjAHmreno against reverse trans. 2} Neutralizing antibody UPID Membrane dr120 DR41 Squirrels dra oo so -d oe so o Outer shell proteins | about "J se t and and w to diuj.