RU2014845C1 - Method for preparing aids vaccine - Google Patents

Abstract

FIELD: virology. SUBSTANCE: for preparation of AIDS vaccine, antigen that is sequence 735-752 of viral capsid protein gp 41 is injected to animal. The antibodies produced are isolated and injected to another animal from which antibody- producing splenocytes are isolated to produce hybridomas. After incubation of the hybridomas, there is obtained an anti- idiotypic antibody that is capable of attaching to CD 4-cell marker-related epitope and inhibiting the binding between CD 4 cell marker and any of indicator antibodies Leu AAA and OKT AAAA. EFFECT: increased efficacy. 14 tbl

Description

 The invention relates to vaccines against acquired immune deficiency syndrome (AIDS) containing antibodies or their equivalents, and the treatment and prevention of AIDS using such vaccines.

 AIDS was first discovered in the United States in the late 70s, when it was believed that this disease is quite rare. However, since then it has been rapidly spreading and now the pace of its development has reached enormous proportions. This virus infects mainly cells of the immune system, in particular cells of leukocytes carrying a CD4 or T4 receptor molecule; moreover, it has recently been discovered that it also infects cells of the nervous system and cells of a cell sequence of a monocyte macrophage.

 If AIDS develops after infection with this virus (human immune deficiency virus HIV), the subject will first become susceptible to opportunistic infections due to a virus that renders the immune system unusable due to infection of CD4 helper cells. Four stages of infection were recognized. Progressive generalized lymphadenopathy (PGL) is characterized by general malaise and chronic diseases caused by other mild infections. The AIDS-related kit (ARS) is associated with general debilitation and chronic diseases, while the condition known as AIDS is characterized by at least one attack of pneumonia Pneumolystis carynii and usually Koposi sarcoma. Although victims usually die in a severe form of conditionally pathogenic (pathogenic) infection (s), it is impossible for them to die simply from the Human Immunodeficiency Virus (HIV) virus, since this virus can infect the central nervous system (CNS), which leads to fatal from neurological attrition.

 Currently, HIV is known as Type I Human Immunodeficiency Virus (HIV-I) and is a lentivirus-related retrovirus. The “I” index is necessary because since the source isolates were obtained in West Africa, other related viruses have been found. These new viruses were isolated from patients with AIDS (LAV-2) disease or from healthy individuals whose sera cross-interact with the monkey immune deficiency virus (SIV) antigens (HTLY-IV).

 Both of these viruses are closely related to the SIV virus, isolates were obtained from rhesus monkeys, African green monkeys and black mangobes. This small cross-reactivity, as measured by ELISA, Western Spot and immunoprecipitation, occurs between sera from patients with HIV-1 and HIV-2 infection or SIV antigens with their glycoprotein coatings and similarly between sera from subjects with glycoprotein coat HIV-2 and HIV-1. Sera from subjects infected with HIV-2 actually show weak cross-neutralization of the HIV-1 virus, which is consistent with the fact that there are relatively few protected sequence homology zones between the HIV-I and HIV-2 sheath zones. However, despite the general lack of cross-reactivity, the HIV-1, HIV-2, and STV viruses are tropical for the same lymphocyte family.

 Many attempts have been made to find a cure or to find an AIDS vaccine. An obstacle to identifying a treatment method is the fact that HIV is a retrovirus, and after an infection has occurred, it is impossible to completely remove the virus from the body. Although efforts to search in this direction are still ongoing, it is important that a vaccine has been found to help prevent infection. An obstacle to vaccine development is the ability of this virus to alter the immunogenic determinants of its protein coat.

 Recently, a whole series of works was devoted to a subgroup of vaccines using the main glycoprotein of the HIV virus envelope encoded by the env gene, either in a form purified from a congenital virus, or in the form of a recombinant protein from bacteria, vaccines, SV40 viruses, or a mammalian animal cell line, or form of synthetic peptides. This approach has yielded different results. Immunization of animals with some of these products revealed a specific antibody reaction, which in some cases contains a neutralizing effect. However, the observed neutralizing effect was relatively weak and was specific for isolates due to the indicated variability of envelope proteins between isolates.

 All of the above, together with the fact that the reaction of a neutralizing antibody in humans is also weak, in all likelihood does not provide protection and, in addition, is somewhat specific for isolates, indicates the inability to create a sufficiently protective immune response against the envelope protein presented either in its natural form, or as a recombinant or synthesized product.

 The possibility of using anti-idiotype antibodies (anti-Id), i.e. antibodies having a binding point that recognizes the binding point of a natural antibody, or an idiotype (Id) acting against an antigen (Ag). This approach to solving the problem also has a disadvantage associated with the variability of the env product. The advantage of using an anti-idiotype antibody in a vaccine is that it can take the form of a virus receptor or virus envelope protein. However, the production of anti-idiotype antibodies (Ab-2), which are an exact mirror image of the binding points of conventional antibodies (Ab-1), is even more difficult than the production of Ab-1, since this will not only produce the original antibody, which in itself it’s useful, which is not yet possible, but the anti-idiotype should only recognize the connecting point or idiot of the idiot.

 Attempts have been made to find an anti-idiotype antibody that recognizes the HIV virus epitope, but such studies have been unsuccessful. Attempts to find the Ab-1 antibody recognizing the HIV virus through the use of env have had only limited success in that only certain strains of the HIV virus are neutralized.

 It has recently been discovered that HIV binds to any cell expressing the CD4 signal gene, and that antibodies against epitopes of a given signal gene block the binding of this virus. The production of suitable antibodies in the required amounts outside the host organism is virtually impossible and attempts to produce a vaccine that stimulates the generation of such antibodies have not been successful. The vaccines studied so far included synthetic peptides in the form of the CD4 epitope recognized by the HIV virus. These vaccines were unstable due to the fact that they did not accept the correct configuration directly when using them. Recently, using CD4 monoclonal antibodies, a map of epitopes on CD4 was constructed, which are very important in the binding of HIV-1. It has been shown that the CD4 epitopes involved in the binding of very different isolates of the HIV virus are very close. It was found that HIV, HIV-2 and some SIV isolates use the CD4 antigen as a cell receptor in human cells, that epitopes are very close in binding and that expression of this receptor reduces subsequent infection.

 A thorough study of immunoglobulin idiotypes (Id) was carried out since they were first recognized on induced antibodies in humans and rabbits. The net theory of immunoregulation encompasses concepts regarding idiotype and postulates that Id networks due to Id-anti-Id reactions can be included in the regulation of the immune system. According to the definition of terms, Id is an antigenic determinant associated with the variable (V) region of an antibody molecule. Thus, Id can be associated with points involved in antibody binding (optionally formed) and / or points within zone V in which the antigen is unbound (bridging). Id may be surrounded by serological counterpairs related to anti-Id. According to another possibility, Id and anti-Id relate to Ab-1 (first antibody) and Ab-2 (second antibody), respectively.

 Together with retroviruses (like other enveloped viruses), these antigenic determinants associated with the induction of neutralizing antibodies are found on the surface of the glycoproteins of the envelope.

 The gene product of the HIV virus envelope is synthesized as a polyprotein precursor, and then it undergoes glycosylation in infectious infected cells. This glycosylated protein with a mol.v. 160 k Dalton (dr 160) is processed by transformation into a submolecular structure with an amino terminal group (dr 120) and into a trans-membrane submolecular structure with a carboxyl group, dr 41.

Previous studies with anti-Id and hepatitis B virus showed that an internal image of anti-Id (Ab-2b), which takes the form of the original antigen, can induce an immune response Id ⁺ anti-HBs ⁺ , which contains 1d together with mouse anti-HBs ⁺ produced by HBsAg. In contrast, anti-HBs induced by AB-2 expresses Id, which is usually not present during the immune response to HBsAg. Similar results were obtained for the TMU Id system, where Ab-2α induced an anti-TMU reaction in BALB / c mice, which contained Id together with the original rabbit anti-TMU used to generate anti-Id.

 Thus, two different classes of anti-Id could induce antiviral responses without exposure to viral antigens or without immunization with viral antigens.

 Similar results were obtained with Ab-2α and Ab-2 β, inducing an antiphosphorylcholine immune response in mice.

 The present invention provides a vaccine suitable for use to create immunity against an AIDS virus and comprising an antibody or antibodies, or idiotypic fragments thereof, or their equivalent, directed either against an AIDS virus or its cellular receptor, and a non-toxic pharmaceutically acceptable carrier.

 A method is provided for creating effective immunity against viruses such as AIDS in humans, which consists in administering the vaccine described above.

 The vaccines of this invention are particularly suitable for the treatment and prevention of diseases such as AIDS. Previously, this was not possible for the reasons stated. The use of vaccines eliminates the need for vaccination against each individual type of AIDS-related virus and provides universal protection against such viruses, while solving the problem of the lack of immunogenicity of natural viral proteins.

 In addition, the invention provides a method of treating people from infection with the HIV virus, which comprises administering to the body an effective amount of an anti-AIDS vaccine.

 By the term “anti-AIDS quantity” is meant that amount of a substance or mixture of substances that can substantially prevent or eliminate the infection of HIV or other retroviruses that cause at least one symptom such as AIDS in humans or other mammalian animals. The term “retroviral” refers to those retroviruses that are responsible for immune deficiency, and especially those that cause one or more symptoms commonly associated with AIDS in humans or other mammalian animals.

 AIDS viruses are viruses that are based on recognition of the CD4 signaling gene for inefficiency. It should be noted that when these vaccines contain antibodies, etc., which take the form of or mirror the recognition epitope of the CD4 gene, then one vaccine can create immunity to all viruses such as AIDS. These type of AIDS viruses are preferably viruses that infect humans, in particular HIV-1, LAV-2 and HTLY-IV.

By the term “mAb” is meant an antibody, its diotypic fragment or its equivalent, as well as polyclonal antibodies, fragments and their equivalents and, in any case, recognition of an AIDS virus or its cellular receptor. The term also includes those mAbs that are created by grafting with a CDR (optionally determining zone), for example those that are obtained by grafting a CDR from a murine antibody to a human antibody. The mAb fragments are, respectively, Fab and Fab ₂ ¹ fragments, or their articulated or synthetic sequences.

 Some CD4 epitopes (as recognized by CD4 mAbs) play a more important role in the binding and infection of viruses such as AIDS than others. This is determined by the effectiveness of blocking syncytia (colectation) under the action of mAbs and by the ability to cross-compete with each other. This pattern of suppression is highly reproducible, for example, with four distinct HIV-I virus isolates.

 In the examples, CD4 mAbs are divided into three groups: 1) those that are effective in blocking the HIV-CD4 interaction and thus act independently of the viral isolate or its type, 2) those 2 mAbs that are clearly unable to block the formation of syncytium (cochlement) with any from viruses or their isolators, 3) those 4 mAbs that have a weak effect when blocking syncytium and have an unequal ability to block various isolates and types. These results indicate that very close CD4 epitopes are included in env binding in all tested viruses.

The binding point of immune deficiency viruses is also a highly conserved area. Usually, the sequence homology in the env gene of various isolates of the HIV-I virus changes by up to 30%. An even greater dissimilarity occurs between HIV-I and LAY-2 with only 41.7% of the conserved sequence throughout the env gene. Data from a study in which a sample containing the LAY-1 env gene interacted with the LAY-2 and STLV-III _mac genome (SIC _mac ) showed no apparent hybridization between these zones.

Similar results were obtained when the STLV-III genome was selected together with the env LTR zone of HIV-I virus, in which only a very mild reaction was observed. When comparing the HTL V-IV restriction map with the STLV-III _mac and STLV-III _aym restriction maps, only very slight differences were found. These data correspond to the insignificant reactivity observed between the major glycoprotein of the HIV-I virus envelope and the reactive HIV-2 or SIV antisera or vice versa between the env product of the HIV-2 or SIV virus and the antivirus of the HIV-1 virus 3.4.

 The importance of these discoveries lies in the fact that no conserved zones of the env product were detected, in relation to which a significant immune response is manifested. Given that the binding point of the CD4 gene virus is highly preserved, it becomes clear that this binding point is not immunogenic enough to elicit an antibody response.

 This may be the result of a “hidden” epitope, which appears only when close to the CD4 gene, masking the epitope by glycosylation or under the influence of salicylic acid, taking the form of an autoantigen, such as MHC-II, or a non-conformational epitope to which B cells are unable to respond.

 A particularly important advantage of the present invention is that Id, anti-Id and their equivalents are provided that recognize a generally inaccessible epitope on the HIV virus, thus providing protection.

 A further advantage of the present invention is that antagonistic (or antagonistic) Id, anti-Id and their equivalents are provided that can induce those described above.

 Modulation of expression of the CD4 antigen after expression of cells by the HIV-I virus occurred in pathogenetic processes in diseases associated with this virus. In addition, infection of cells bearing CD4 caused by human or monkey immune deficiency viruses leads to a significant reduction in CD4.

 All known viruses such as AIDS, with the possible exception of HTLV-IV, are known to cause AIDS type syndrome in natural hosts; it is possible that the effect of an excessive decrease in the normal expression of CD4 is significant during the pathogenesis of this viral infection.

 The combination of dr 120 HIV-I virus with CD4 suppresses T-cell activation in response to PHA in the final cells, which suggests that the loss of functional epitopes or internalization of CD4 leads to the loss of some immune functions. Thus, according to the present invention, it is desirable that the vaccines are not directed to the active binding of the CD4 signal gene.

 Mimotypes of these antibodies can be used according to the invention. By the term mimotype is meant a peptide or peptide derivative, in particular synthesized to bind the paratope of a given antibody. Mimotypes can be obtained according to the methods of Geysen H.M. and etc..

 Rabbit antiserum acting against the natural antigenic determinant associated with dr41 neutralizes the efficacy of the HIV virus in vitro. This epitope was identified by the production of antiserum in experimental animals, acting against a synthetic peptide (mimotype), a similar sequence of amino acid groups 735-752 of the HIV HIV isolate HTLY-IIIB.

 The present invention provides for the reproduction of an anti-Id reagent that recognizes Id on antibodies that act against HIV, especially dr 41, a synthetic piptid.

 Anti-Id immunization induces an antipeptide reaction in BALB / c mice that binds recombinant drug dr 160. Serologically, the anti-Id-induced murine anti-HIV reaction suppresses the Id-anti-Id response and expresses an Id different from the murine anti-HIV produced by immunization with a synthetic peptide dr41. These results indicate that Id networks are involved in modulating the immune response to HIV.

The production of anti-idiotype antibodies (anti-Id) ³ against chimpanzee antibodies directed against a synthetic peptide corresponding to a natural epitope associated with another 41 glycoprotein coat of human immune deficiency virus (HIV) in rabbits has been demonstrated. This peptide is similar to the sequence of amino acid groups from 735 to 752 from the HTLY-IIIB isolate of the HIV virus.

 Immunization of BAL B / c anti-Id mice induces an antipeptide reaction that binds the recombinant dr160 preparation without subsequent exposure of the peptide or dr160.

 The described examples show that it is possible to induce a population of Id + with anti-HIV specificity by immunization with anti-Id. In addition, when these vaccines contain antibodies or their equivalent, for example, mimotypes that take the form or mirror the recognition of the CD4 recognition epitope, a recombinant env (for example, obtained from benentech Ine. California, USA) is revaccinated in the form of a vaccine. Although seeding with recombinant env was not carried out, a revaccination was performed after the reaction was established.

 The antibodies used could be either idiotypic, or anti-idiotypic, or antibodies reproduced from them directly or indirectly. Any type of antibody can be used in the vaccine, providing either active or passive immunity. Passive immunity is achieved by injecting the antibody in a large enough dose to provide protection against AIDS per se. Active immunity can be created by vaccination with an immunogenic amount of an antibody that can induce the production of an anti-idiotype for the first antibody.

 Some monoclonal antibodies (mAbs) act against the CD4 signaling gene, including OKT4a and Leu 3a. They can be used in the vaccines of this invention. So, for example, the generated anti-idiotype binds to the envelope of the HIV virus and especially to the product 120 kD env (dr120 or dr41). However, not all anti-idiotypes acting against mAbs that recognize the CD4 signaling gene must necessarily bind to HIV, since HIV binds only one or some of the epitopes present in the CD4 molecule.

Anti-idiotypes for mAbs such as Leu 3a and OKT4a, used as components of these vaccines, create anti-idiotypes (Ab-3, also known as Ab-I ¹ ) that recognize CD4 and can mask HIV binding by blocking the receptor.

 Although any antibody can be used according to the present invention, it is desirable that the generated immunity is directed against viruses, and not against a cellular receptor. So, in the case of using anti-CD4 mAbs, it is desirable to prepare vaccines in such a way that an anti-idiotype reaction is triggered in the patient, and when the anti-idiotype anti-CD4 mAb is administered, it is desirable to prepare the vaccine so that it gives the patient passive immunity.

 In one aspect, the invention primarily relates to anti-idiotype antibodies. When using an anti-idiotype in a vaccine, there is a need to induce an anti-idiotype reaction. When using an anti-idiotype, it can serve either to trigger an anti-anti-idiotype reaction, or as a passive vaccine.

 According to another aspect, the present invention relates to anti-idiotypes, but using products of genetic engineering or antibodies capable of masking the described actions. It is usually desirable that the vaccines inducing active immunity contain an antibody component that mirrors the CD4 signal gene, and passive vaccines contain antibody components that take the form of a CD4 signal gene. The administration of the vaccines of this invention varies depending on a number of circumstances, including factors such as age, weight and general condition of the patient. The vaccine can be introduced into the body as a single sufficient dose or as a series of doses over a period of time. Repeated dosing of the drug for active immunization or to maintain immunity is usually also desirable in the subsequent period, usually in the next three months, but such increased dosage can be carried out at an earlier period or at any time during the patient’s remaining life and the same amount repetitions.

 For ease of use of the vaccine, pharmaceutical saline can be used as a carrier of an antibody or antibodies. However, since the reaction of the anti-idiotype to such a formulation is usually not very strong, it is advisable to use an adjuvant synergist.

 Particularly valuable adjuvant synergist and carrier proteins used in this invention are fissurel hemocyanin (KLH) and alum preparations, examples of which are given below. It was determined that the use of these substances leads to a significant increase in the reaction of Ab-2. Antigens not associated with the carrier protein may be weakly immunogenic or even completely non-immunogenic.

 When passive immunity is desired, no adjuvant synergist is required. However, if an anti-idiotic reaction is created, then the adjuvant synergist along with an immunostimulating amount of mAb is especially useful.

 In the case where it is desirable to use the proposed vaccine for the purpose of prevention, it is usually preferable to use those vaccines that are suitable to stimulate active immunity. Vaccines that give active immunity are still finding prophylactic use where there is a risk of direct infection with HIV or where it is assumed that active immunity cannot be created or cannot be created quickly enough.

 When vaccines are intended to treat diseases caused by an infectious HIV infection, it is usually desirable to use those vaccines that are capable of imparting passive immunity to provide rapid anti-AIDS effects. Vaccines suitable for stimulating active immunity can be used in these conditions, providing a long-term effective effect. When using the adjuvant synergist, it can be administered together with mab in the same or different preparations, or separately at a point in time other than when the vaccine was administered.

 The vaccines of this invention are usually administered to the body in a conventional manner, for example, they can be administered by intraperitoneal, intramuscular or subcutaneous injection, by skin incision, or orally. Such vaccines usually contain a pharmaceutically acceptable carrier and, if desired, an adjuvant synergist, substances that make the vaccine isotonic with body fluids, and, if necessary, flavoring agents, emulsifiers and other components.

 Passive immunity vaccines usually contain a large dose of mAb sufficient to create passive immunity in the patient. Immune response vaccines usually contain an immunostimulating amount of Ab, which is usually less than the amount required for passive immunity. Such vaccines can be subdivided for separate administration, either simultaneously with each other, or with a time gap, preferably after a week.

 PRI me R 1. The production and screening of antiserum antidiotype in mice.

 A. Conjugate binding of a monoclonal antibody (mAb) to fissurelli hemocyanin (KLH).

mAB in the form of purified immunoglobulin (1 g) in Dulbecco's “A” phosphate buffered saline (PBS “A”) was further diluted in PBS “A” to a concentration of 1 mg / ml. Fissurel hemocyanin (KLH) was dissolved in PBS "A" to a concentration of 1 mg per ml and dialyzed overnight at 100 vol. PBS 'A' at ⁴ ° C for 1. mAB solution was mixed with 1 vol. KLH solution, the reagents were conjugated by introducing 1 vol.% solution of 28% glutaraldehyde. After the reaction for 1 h at room temperature, the solution was dialyzed with 100 vol. PBS "A" overnight at 4 ° ^C.

 B. Immunization of mice.

1.1. Seeding of mice with mAB / KLH complex
The 1 mg / ml mAB / KLH complex was emulsified with an equal volume of Fred's complete adjuvant synergist (FCA) to form a water-in-oil emulsion. 200 ml of an emulsion containing 100 μg of the mAB / KLH complex was inoculated subcutaneously (SC) by injecting it under the skin of the abdomen of each of the Balb / C Imbred Cancer Research Foundation (ICRF) mice.

 1.2. Revaccination of mice.

 28 days after seed inoculation of mice, 200 μl of the water-in-oil emulsion, 100 μg of the mAB / KLH complex in equal volumes of the incomplete adjuvant synergist Friend (FIA) were inoculated under the skin of the abdomen and 200 μl were injected into the femoral muscle of the right hind leg.

 After 10 days, mice were bled from the tail of the mice (obtaining approximately 100 to 200 μl of blood) and the mice were left alone. 14 days after the first revaccination, each mouse was subjected to subsequent revaccination by injecting 200 μl of a mAb / KLH solution containing 200 μg of this complex into the abdominal cavity. After 10 days, bloodletting from the tail of the mice was carried out, as before, and another reactive inoculation was performed. After another 10 days, the tail was bled again and then the mice were left alone.

 C. Test of serum for anti-idiotype activity (i). Radioimmunoassay of binding on plates.

A monoclonal-Id antibody with a concentration of 50 μg per ml in PBS "A" was injected into a 96-cell Cook plate (Coke) in an amount of 50 μl per cell. After incubation at ⁴ ° C overnight, the plate was washed three times with PBS 'A' / 1% BSA, then incubated further with 50 ug PBS cell "A" / 1% BSA for 1 hour at 37 ^C. Then, PBS 'A "replaced with antisera from immune mice at dilutions of 1/10 or more in PBS" A "/ 1% BSA. The negative control groups were only PBS A / BCA, diluted serum solutions of preimmune or normal mice, the positive control group consisted of 20 μg / ml multivalent rabbit anti-mouse serum or its diluted solutions. The plate was incubated for 1 hour at ³⁷ ° C, washed three times as described, and injected into each cell from 2000 to 4000 in particular units of minute (CPM) ¹²⁵ I-labeled Id in 50 ul PBS 'A' / BSA. After an hour, the plate was washed again, the cells were filled with wax, and the CPM for each cell was determined on an LKB ultrasound.

The analysis results were expressed as a percentage of ¹²⁵ I-labeled linkage with transverse linkage in the test samples compared to the maximum CPM value for the positive control group.

 (ii) Assay for inhibition of binding.

From 2000 to 5000 CPM specific idiotype antibodies labeled with ¹²⁵ I, was mixed with antiserum from immune mice with various dilutions and incubated at ⁴ ° C for 0.5 h in a 96-mesh plate Cook. Then, CD4-bearing CEM cells were introduced with a concentration of 10 ⁵ cells per cell, after which incubation was performed as described above. Cells were washed three times in PBS "A" / 1% BSA by centrifugation, the cells were filled with wax and the CPM bound to the cells was measured using an LKB ultragram.

 D. Screening antiserum for virus-specific reactivity.

Indirect immunoluminescent staining of virus infected cells
Both uninfected CEM cells and virus-infected NIV, incubated at a concentration of 10 ⁵ cells in 50 ul of MEM with 2% FCS and 0.1% sodium azide per well of 96-mesh plate for 0.5 hours at 4 ^{° C} with 50 μl of both immune antiserum and normal mouse serum with a dilution of 1/10. Cells were washed three times by centrifugation with MEM / 2% FCS / 0.1% sodium azide, then resuspended in rabbit multivalent anti-mouse 1 g FITC conjugate (Sigma) diluted 1/100 and incubated as described above. The cells were again washed three times, resuspended in 200 .mu.l of MEM / 2% FCS / 0,1% sodium azide at ⁴ ° C and the percentage fluorescence was determined by FACS analysis in the analyzer Becton Dickinson.

 E. Test antiserum on the neutralizing effect of the virus.

Suppression of virus-induced syncytium formation
Virus-induced syncytium formation by mixing NIV producing cell line (H9) with CD4 sheer line (YM or C8166) in a ratio of 1: 5 in RPMI 1640 + 10% FCS, incubation overnight at 37 ° ^C. It is possible to determine the inhibition of syncytium formation by blocking the interaction of CD4 - HIV, either by means of CD4 mAbs, or by antibodies directed against the glycoprotein of the envelope of this virus. Specific inhibition of syncytium formation through a reagent that interacts with the virus rather than with the receptor can be demonstrated by a two-step analysis. The antiserum with various degrees of dilution was added at 10 ⁵ indicator cells in 50 ul of MEM / FCS and incubated for 0.5 h at ⁴ C. Cells were washed three times and mixed with NIV producing cell line at a ratio of 5: 1. An equal number of CD4-bearing indicator cells were incubated with antiserum and mixed with virus-producing cells without any washing. Blocking of syncytium in the second, but not in the first analysis showed inhibition of the virus-receptor interaction on the virus, and not on the receptor.

 The test antiserum had an anti-Id activity, detectable at dilutions of at least 1: 250 or 1: 200 (Table 1). This activity was specific for immunization with Id mAb (the antiserum did not bind to irrelevant guys or to OKT4, mAB recognizing another CD4 epitope) and could be blocked by introducing Id mab into the antiserum in this assay.

 Staining live cells from CEM cells with antiserum from mice immunized with any mABs, or staining CEM / CBL I cells with antiserum from mice immunized with MT151 was equivalent to staining with normal mouse serum. However, the serum from mice immunized with Leu 3a stained CEM / CBLI cells at a 1:10 dilution. To confirm that the sera from Leu Za mice interacted with a virus-specific agent, they were used for immunoprecipitation from 35S-methionine-labeled CEM cells of uninfected or infected with HIV isolates of CBLI or HTLV-IIIRF. Areas with a molecular weight of 120 KDaltons were specifically precipitated from HEM cells infected with HEM. No regions with a given molecular weight were detected in the control precipitate from uncharged CEM cells.

 Antiserum data were also evaluated in a syncytium suppression assay. Neither the serum from immunized MTIS I (MAb, recognizing the CD4 epitope different from the epitope recognized by Leu 3a or OKT4a) mice, nor the serum from normal mice are able to suppress syncytium induction. The data in the table. 1 show that sera from mice immunized with Leu 3a are able to suppress all test viral isolates. H9-ARY-2 neutralized more effectively than H9 / IIIB, and H9 PUT and RF required higher antiserum concentrations to suppress syncytium.

 PRI me R 2.

 A. Generation of monoclonal anti-idiotype antibodies.

 BAL B / C mice aged 3 to 5 weeks were immunized by intravenous administration with a dose of 30 μg / mouse of purified Leu-3a Mab (Becten-Dickinsen, Mounatain Ceiw, CA) prepared according to the following procedures (i) or (ii). 6 injections were made at weekly intervals. 3 days after the last injection, the mice were sacrificed and their spleen cells were fused to the NS-1 myeloma cell line.

 (i) Coupled clutch with KLH.

Calculate the number of moles of antibody, EDAC and KLH that should be used based on the number of antibodies that must undergo conjugation:
Ab = x moles mol. at. 1g G = 150,000
EDACx = 10000 (x) mol Igm = 900000
KLH + = x / 50 mol EDAC = 191
KLH = 1,000,000
1. Ab is diluted in the minimum volume of BBC or PBS, EDAK is diluted in dH ₂ O, KLH is diluted in BBC or PBS; 2. Cool the antibody to 4 ^° C; 3. Add EDAC. Mix for 30 seconds at ⁴ ° C;
4. Add KLH. Stir for 2 hours at 4 ° ^C.

Stir overnight at 25 ° ^C.

5. Carry out an analysis on the BBS;
6. Precipitation by alum. Protein concentrate = Ab + KLH, ^* EDAC [1-ethyl-3- (3-dimethylaminopropyl) carbodiimide], Cat. # E-7750 sigma + KLH (fissurel hemocyanin) Cat # 374817 Calbiochem.

 (ii) Precipitation by alum.

Solutions: 10% AIKSO ₄ (potassium aluminum sulfate, cat. # A-601 Fisher) in 0.005 mol. PBS, pH 6.2, filtered (gives 5.6 mg / ml alumina).

Procedure:
1) dropwise add 0.4 mg of aluminum oxide / 50 μg of protein to the protein solution while stirring at room temperature. Bring the volume to 10 ml by adding PBS;
2) Bring the pH to 6.8-7.3. The solution becomes very cloudy;
3) maintain the solution for 2 hours at room temperature so that the alum is absorbed with slow stirring;
4) centrifuge the solution at a speed of 3000 rpm for 10 min;
5) check the optical density of the surface layer containing unabsorbed protein;
6) rinse granules in 0.85% saline solution, centrifuge;
7) re-suspend the granules in PBS to a concentration of 100 μg / ml.

 B. Assay of bound immunosorbent enzymes.

 Analyzes of the HILV-III ELISA (Electro-Nucleonics nc, silver spring, MD) and LAY EIA (Genetics System, WA) were performed according to the manufacturers specifications.

 The goat anti-human IgG enzyme conjugate was replaced with goat anti-mouse radish peroxidase antibodies (Vector Laboratories, Burlingame, CA). ELISA analysis using psoralen and inactivated with ultraviolet rays of the virus HIV was carried out in a known manner. The surface layer containing a culture of H-9 cells infectious with 7-day HIV (HTLV-III3) was concentrated 10 times and inactivated by exposure to (three times) psoralen (Calbiochem, San Diego, California) at a final concentration of 10 μg / ml and by irradiation with ultraviolet rays (360 nm) for 15 minutes Inactivated HIV was then passed through a 4B Sepharose column. The free mass was collected and concentrated for use. ELISA was performed using microtiter plates coated with 50 μl of antigens, the concentration of which was established by using an extinction coefficient of 14 for a 1% solution at 280 nm.

To determine the binding of HF 1.7 to Leu-3a, ascitic fluids from HF 1.7 or from a control monoclonal anti-Id (GB-2) recognizing the idiotype associated with mab specific for hepatitis B surface antigen were fractionated with 50% saturated ammonium sulfate. The resulting immunoglobulin-containing precipitate was again suspended in saline containing a borate buffer solution, and the antibody concentration was determined using an extinction coefficient of 14 for a 1% solution at 280 nm. By changing the concentration of anti-Id, mAbs were absorbed three times in the cells of the microtiter plates. After blocking nonspecific points with 10% goat serum (NGts), the cells interacted with biotinylated Leu-3a or with a control mAb specific for the large tumor antigen of monkey virus 40 (SV40 antigen). These antibodies were biotinylated using a concentration of 7 mg / ml; a 1: 1000 dilution in 10% NGtS was used in the analysis. After incubation for 1 h at ³⁷ ° C unbound antibodies were removed by washing and the specific binding was determined using Avidin - horseradish peroxidase and ABTS with H ₂ O _2.

 C. Suppression of the binding of HF1.7 mAb to Leu-3a.

 Microtiter plates were coated with 500 ng / Hf cell 1.7 mAb purified by adsorption on Protein A-Sepharose 4B (Pharmacia, Piscataway) and elution from this adsorbent. After blocking non-specific points, 5 μg of various inhibitors were injected into the anti-Id-coated plate cells for 1 h. After incubation and washing to remove unbound antibodies, biotinylated Leu-3a was introduced with a dilution of 1: 1000 and ELISA analysis was performed as described above.

Ascitic fluids from TC-1 or from a control monoclonal anti-Id that recognizes the idiotype associated with the antibody specific for hepatitis B surface antigen were fractionated with 50% saturated ammonium sulfate. The resulting precipitate containing immunoglobulin was again suspended in saline containing a borate buffer solution, and the antibody concentration was determined using an extinction coefficient of 14 for a 1% solution at 280 nm. By changing the concentration of anti-Id, mABs was adsorbed in the cells of the microtiter plates. After blocking non-specific points with 10% NGts, the cells interacted with biotinylated Leu-3a (A) or with a control mAb specific for the SV40 antigen (B). These antibodies were biotinylated at a concentration of 7 mg / ml and a dilution of 1: 1000 in 10% NGts was used in the analysis. After incubation for 1 h at ³⁷ ° C unbound antibodies were removed by washing, specific binding was determined using Avidin - horseradish peroxidase and ABTS with H ₂ O _2.

 D. Immunofluorescence staining.

Infected and non-infected viable H-9 cells were obtained by centrifugation using Ficoll-Hypaque gradients, they were washed twice with staining buffer solution (PBS with 5% FCS and 0.02% NaN3 added to it). One million cells reacted with anti-Id NF1,7 or with negative control group of the same isotype antibodies for 30 min at 4 ° ^C. Then the cells were washed with the staining buffer, and reacted with anti-mouse IgG (Suppel Laboratories) goat FITC for another 30 minutes at 4 ° ^C. After incubation, the cells were washed, immobilized in -th 0.37% formaldehyde and analyzed using analyzer Becton- Dickinson FACS, connected to a BD Consort 30 (Becton-Dickinson, Mountain View, CA). To determine the inhibition of the binding of Leu-3a to CD4 ⁺ cells via HF 1.7, the human T cell line CEM A3.0I was used. FITC-Leu-3a incubated with PBS or with 10 ug of purified HF 1.7, or from 10 mcg control anti-Id mAb for 1 hour at ⁴ ° C and injected them into 5x10 ⁵ A3.01 cells. These cells were incubated for 30 min at ⁴ ° C and washed twice and analyzed on the analyzer FACS.

 E. Neutralization of HIV infection in vitro.

One hundred thousand or NIV TCID ₅₀ virus in 100 ul were incubated with 100 mD HF 1.7, or control anti-Id GB-2, or culture medium for 1 hour at 37 ° ^C. The concentrations mAbS adjusted so that the final concentration was 0.5 mg / ml. After incubation NIV-treated virus was administered in June ¹⁰ A3.0I cells and incubated at ³⁷ ° C for 2 hours in the presence of polybrene concentration of 10 ug / ml. The cells were then washed and resuspended in 10% FCS RPMI 1640 with a density of 10 ⁶ / ml. At the indicated time intervals, aliquots of the liquid culture were removed and the activity of the reversible transcriptase was determined. HIV-free cells were harvested from chronically infectious A3.01 cell culture, titrated on uninfected A3.0I cells, and the titer was expressed as an infectious dose of 50% tissue culture (TCID ₅₀ ).

Hybrids were selected by ELISA using plates coated with HIV antigen (Table 2). Of the 389 tested hybrids, one was found that reacted in all three analyzes. This mAb, designated HF 1.7, was cloned twice by limiting dilution. Its isotope has been identified as Ig C _2b . To determine the specificity of HF binding, 1.7 microtiter plates were coated with HF1.7 taken at different concentrations and control mАb, GB-2; they interacted with biotinylated Leu-3a or biotinylated control mАb of the same isotope as Leu-3a, but recognizing antigen SV40 T. HF1.7, anti-Id mab was specifically associated with biotinylated Leu-3a and control anti-Id. Neither HF 1.7 nor the control anti-Id mAB were associated with a biotinylated control mab specific for SV 40 antigen. HF 1.7, anti-Id did not react with a group of irrelevant murine mABs, which include Leu-I, Leu- 2a, Leu-5b, Leu-8, Leu-M1 and IgG of normal mice.

 At a concentration of 5 μg, irrelevant mABs were not able to significantly inhibit the binding of Leu-3a to their anti-Id, the suppression limit was 0-5%, (Table 3). Leu-3a and two other mABs that recognize the CD4 molecule (OCT-4 and T4) were effective inhibitors of the Id-anti-Id reaction. These data show that HF 1.7 recognize the determinant of Id on Leu-3a. This determinant Id was the fraction of other mAbS that recognized CD4 but was not present on mABs that recognized phenotypic signal genes of other lymphocytes. Leu-3a, OCT-4A, and T4 block in vitro infection with the HIV virus, so the ability to suppress the Id-anti-Id response is interconnected with the ability to block the infection of HIV in vitro.

 The binding of HF 1.7 mAb to Leu-3a was confirmed in another cytometric binding suppression experiment. In the analysis with immunofluorescence staining with Leu-3a, the human T cell line AZ-01 was used, approximately 95% of which expressed surface CD4. Incubation of Leu-3a with HF 1.7 anti-Id resulted in a partial, but rather significant decrease in the intensity of Leu-3a fluorescence staining. Leu-3a staining of AZ.01 cells was not significantly affected by previous incubation with control anti-Id mAb. These data confirm that anti-Id binds Leu-3a and partially inhibits the binding of Leu-3a to surface CD4 present on human T cells. Therefore, anti-Id must recognize at least a portion of the antibody binding point on Leu-3a. These characteristics also confirm that HF 1.7 recognizes the determinant Id associated with the antibody-binding point on Leu-3a.

 To determine the expression of the antigen recognized by H 1.7 on the surface of HIV-infected cells by anti-Id, an indirect immunofluorescence assay was performed on uninfected and continuously infected H-9 cells. Anti-Id staining of infected H-9 cells resulted in a clear increase in fluorescence intensity, while uninfected H-9 cells did not stain.

 Approximately 25% of infected HIV H-9 cells were stained with HFI.7 anti-Id.

 In order to determine the kinetics of the surface expression of the antigen recognized by anti-Id on cells infected with HIV in vitro, the human T cell line AZ-01 was infected with the HIV isolate designated NY-5, and on day 1-7 the infection was carried out indirect immunofluorescence analysis of a viable cell membrane using anti-Id mab. The antigen recognized by HF 1.7 was not detected until 4 days of infection, the moment at which approximately 10-15% of the AZ.01 cells were stained. Thus, it was found that anti-Id recognizes the determinant (determinants) present on infected HIV T. cells.

To identify the characteristics of an antigen reactive with respect to HF 1.7 anti-Id, strips of nitrocellulose paper (Bio-Red immunoassay), onto which HIV antigens were applied by an electric dot method, were exposed to HF 1.7 or a negative control anti-Id. The collected human AIDS sera were used as a positive control group at a dilution of 1: 100. The human antiserum recognized the characteristic proteins of the HIV virus p18 and p24 and the gag of the p55 precursor along with the glycoprotein of the gp120 and gp41 membrane. HF 1.7 anti-Id with a molecular weight M _I in the range of about 110-120 KDaltons. No reactivity was detected to the negative control mAb. Anti-Id recognized the glycoprotein of the HIV membrane, gp120, the region in which HIV binds the CD4 molecule.

The ability of HF 1.7 to inactivate HIV was determined in an in vitro assay to determine neutralization. The results are presented in table. 3. The activity of reversible transcriptase was suppressed in cultures treated with HF 1.7 anti-Id, a method determined by the dose of the virus. The most pronounced suppression of viral replication was observed on the 7th day of culture growth, in which 58 and 90% of the reverse transcriptase activity was suppressed at 1000 TCID ₅₀ and at 100 TCID ₅₀ of the HIV virus, respectively. On the 9th day, the suppression of the activity of reversible transcriptase in HF-treated 1.7 cultures decreased to 44 and 80% at 1000 TCID ₅₀ and 100 TCID ₅₀ of the HIV virus, respectively. In contrast, the treated GB-2 cultures gave about the same reversible transcriptase activity as was determined in the medium treated cultures. The increased activity of reversible transcriptase in cultures treated with TCI mAB on the 9th day of culture growth was probably the result of HIV replication, which eliminated inactivation.

 PRI me R 3. Preparation of antibodies anti-idiotype OKT4a.

The mice were immunized, as described above, by OKT4a obtained by the method described in Example 2, A, i, as well as the method described in Example 2 A, ic followed by conjugation to KLH by using glutaraldehyde. The resulting sera were tested on HTLE-III by ELISA (Electronuclcinics) at OD ₄₉₂ . At dilutions of 1: 400, only positive results were observed for mice immunized with OKT4A without treatment with glutaraldehyde. Two mAB cultures were obtained from successfully immunized mice that gave ELISA readings at OD ₄₉₂ > 0.5 to bind both HIV and gp160 recombinants.

These two mAbs also suppressed the binding of OKT4A (OD ₄₁₀ = 0.24), but not OKT4B or Leu-2a (OD ₄₁₀ 0.04 and 0.03, respectively). The tests were carried out as described previously.

 PRI me R 4. Immunization of baboons OKT4a.

Two baboons were immunized with precipitated alum OKT4a 3 times in 2 weeks, followed by immunization more than 2 times a month. Analysis of an HTLV-III ELISA (Vigro) at OD ₄₉₂ gave a reading of 0.14 and 0.19. For recombinant DR 160 at OD _{410, the} corresponding readings were 0.10 and 0.14. These data have an advantage over comparable data for the negative control and pre-immune serum OD ₄₉₂ 0.02 and 0.04, OD ₄₁₀ 0.02 and 0.03.

 Immune serum of baboons suppressed staining of anti-CD4 T cell lines, including Leu-3a and OKT4A, but not OKT4, as expected, and stained infected HIV cells.

 PRI me R 5. The study of binding.

 Neutralization by suppressing infectivity or suppressing syncytium showed that it occurs in all isolators with a dilution of about 1/200. The results are presented in table 4.

 Weak binding in mouse 2 increased with revaccination.

 Therefore, these anti-Leu 3a idiotypes recognize infected cells (see Table 5) and neutralize viral infectivity (see Table 6).

 PRI me R 6. Viral cross-reactivity.

 With 4D 4m Ab (T419TNU5D 7), which has been shown to effectively suppress the interaction between HIV-1 m CD4 (12), was selected to compare the inhibition of peripheral blood lymphocyte infections with the HIV-1 and HIV-2 viruses, followed by the production of activity reversible transcriptase (RT).

The relative inhibition of RT (CPM x x ^{10 3} ) when the cells were treated prior to infection [HIV (CPM reversible transcriptase) administered on day 0] mAb is approximately the same in cultures infected with both HIV-1 and HIV-2 from day 7 to day 16 after infection.

Peripheral blood leukocytes (PBL) were cultured for 3 days in a complete nutrient medium (10% fetal calf serum RPMI 1640, 0.2 u / ml anti-interferon serum and 2.02% polybreno) with phytohemagglutinin A (PHA) injected into it. with a dilution ratio of 1/300. Cells were divided into two aliquots. One was incubated for 1 hour at ⁴ ° C with CD4 mAb 19TNU5D7 concentration of 0.05 g / ml, and the other was incubated without mAb. Surface layers of sludge containing NIV-1 or HIV-2 at various concentrations were injected in both portions of the cells that were incubated for 1 hour at 37 ^C. After washing three times, cells were cultured in complete medium and those aliquots are preheated with a mAb, incubated in 0.1 μg mAb to obtain a 6-day culture. Reversible transcriptase activity was measured in the surface layer of sludge containing the culture at various time points after infection.

 Syncytium was suppressed at higher mAb concentrations and compared after incubation overnight with CD4-bearing cell lines stably infected with various types of viruses and viral isolates (Table 7). Most of the mAbs that were tested were divided into two main clusters of epitopes, both of which block the interaction of HIV-CD4, and they make up group 1. All these mAbs block all tested viruses except for the Muson-Pfizer D-type monkey virus (MRMU). Group 2 included 2 mAbs, which were unable to stably suppress the formation of syncytium with any of the tested viruses. Four other mAbS had different ability to block the interaction between HIV-1, HIV-2 and CD4 and at best only partially suppressed the formation of syncytium. These mAbs were unable to block any of the monkey viruses. Pseudotypes of the vesicular stomatitis virus (VSV) virus carrying antigens of the HIV-2 and HIV-2 virus envelopes were also tested for the dependence of viral binding and penetration on CD4 receptors.

The results shown in table 7 were obtained as follows. 24 CD4 mAbS were used to suppress the formation of syncytium between HIV-producing T9 cell lines H9, HIT78 or C8166 and CD4-bearing C8166 or MT4 cell lines. 90 μl of a suspension of the indicator cell line bearing CD4 in RPMI 1640 nutrient medium (Gibco), including 10% fetal calf serum with a concentration of 2 x 10 ⁵ cells per ml, was injected into 10 μl of CD4 mAbS in each cell of the microtiter plate. CD4 AbS was either a purified immunoglobulin with an initial concentration of 100 g per ml or ascites liquid with a dilution of 1/10 with an initial titer of at least 1/1000 with immunofluorescence treatment. To each well of the plate was added an additional 100 .mu.l of a suspension of virus producing cells (5 x 10 ⁴ per ml), the plate incubated overnight at 37 ^° C Rating Scale plates were as follows: - = no syncytia suppression (as in cells incubated without mAb or irrelevant mAb); + = partial suppression of syncytium (syncytium formation 20%); ++ = = complete suppression of syncytium. Monoclonal antibodies that stably blocked all tested viruses with the exception of MRMU were: Leu 3a, OKT4a, F101-69, F101-5, EDU-2, NUTH-1, T4, 19thy 5D7, 94b1, 91d6, OKT4, OKT4b, OKT4d , YIT4, MT151, MT321, MT310, T4 / 18T3A9, 13B8.2 (group 1).

 Antibodies that do not block any of the tested strains were: OKT4 and OKT4C (group 2).

 The results shown in Table 8 show the same VSV (HIV) inhibition pattern for typical mAbS groups 1 and 2 as for syncytium inhibition.

 Suppression of the infectivity of the VSV pseudotype (HIV) under the influence of CD4 mAbS. VSV pseudotypes (HIV) were obtained by VSV superinfection of HIV-producing H9 subline cells, they were titrated on fused CD4-bearing CDR-CEM cells pretreated with mAb. VSV platelet counts were calculated 48 hours after infection with the pseudotype infection (++ means a decrease in platelets> 90%; + means a decrease in platelets 40-90%; - means no decrease in platelets).

The expression of the surface of CD4 antigen cells on CD4-bearing cell lines uninfected or infected with HIV-1, HIV-2 or SIV was measured by direct binding of ¹²⁵ I labeled CD4 mAbS. Table 9 shows that CD4 mAbS decreases to the same extent after infection with any of these viruses. This has been demonstrated with mAbS for up to two different epitopes of CD4 antigen. In contrast to these results, infection with D-type virus without using CD4 as its receptor does not affect the expression of CD4.

The binding of CD4 mAbS to uninfected and infected HIV and IV T cell lines was determined as follows. CD4 mAbS, labeled with ¹²⁵ I (17) were incubated for 30 min at ⁴ ° C with 2x10 ⁴ cells in the cells 96-μ mesh plate -titratora in the presence of 2% BSA carrier protein (concentration of labeled mAbS previously standardized, so as to produce roughly 1000-2000 bound mAbs per minute with CEM cells). The plates were washed three times, and bound mAbs were measured on a gamma counter. Results are expressed as the average of three samples. The specificity of binding of labeled mAbS was determined by pre-incubation of uninfected cells with an excess (20 μg / ml) of the same unlabeled mAbS.

 PRI me R 7. The use of mimotypes for the extraction of antibodies.

 A. Synthetic peptides and antigens HIV.

 The synthesis of the sequences of peptide 735-752 dr160 from HTLV-IIIB isolates was carried out by the method of solid-phase synthesis of peptides. This peptide corresponds to the second major predicted hydrophilic zone in dr41. The amino acid sequence of this peptide is known.

 A control peptide containing the recognition point of the nuclear transport signal of the large tumor antigen (T-ag) of the monkey virus (SV40) was synthesized. The purified peptide recombinant p160 peptide produced in the baculovirus expression vector system was used.

 B. Antibodies and their purification.

 The chimpanzees used in this study were part of a study of a synthetic peptide vaccine for HIV. Two chimpanzees (X-99) and (X-183) received four injections of 200 μg of peptide 735-752 conjugated to KLH before infection with HIV. The control chimpanzee (X-189) received the same injections of the SV40 nuclear transfer peptide conjugated to KLH. Serum from individual chimpanzees was collected after the third and fourth immunizations and before infection with the HIV virus. The first immunization was carried out in a complete adjuvant synergist of Friend (FCA), and subsequent injections were carried out in an incomplete adjuvant synergist of Friend.

 Three New Zealand white rabbits received three fortnightly injections of 100 μg of peptide 735-752 conjugated to KLH in FCA. The specificity of rabbit antiserum has been known. Characteristics of chimpanzee antiserum are summarized in Table 10.

Prior to purification by affinity chromatography in columns with an immunosorbent containing a peptide, chimpanzee and rabbit immunoglobulins were precipitated with 16% sodium sulfate (w / v). Each globulin preparation is resuspended in 20 l of saline containing borate buffer solution (BBS) and stirred overnight at ⁴ ° C with the immunoadsorbent (Sepharose 4B or Affi-Gel 501) containing peptide. After washing with BBS to remove unbound serum proteins, the antipeptide antibodies were eluted with 5 M potassium iodide. Protein-containing fractions were merged with IgG fractions completely dialyzed on BBS, purified from the resulting antipeptide preparation by filtration through Sephadex G-200 gel. The concentration of immunoglobulin was calculated using an extinction coefficient of 13.5 and 15% for 1% of the preparation of chimpanzee antibodies and rabbits, respectively.

 C. Preparation and purification of anti-Id antibodies.

 The preparation of xenogenic anti-Id antibodies of New Zealand white rabbits was carried out in a known manner. Rabbits were immunized two weeks with the IgG peptide of anti-HIV chimpanzee X-99, the resulting antisera were re-adsorbed on a 4B Sepharose column containing IgG of the pre-immunized chimpanzee X-99 until all detectable reactivity with isotypic and allotypic determinants disappeared. A control anti-Id was obtained by immunizing rabbits with the IgG peptide of anti-SV40 chimpanzee X-189 and subsequent adsorption to remove anti-isotype and anti-allotype specificities using immunosorbent columns containing IgG of pre-immunized chimpanzees. These methods of preparing these columns with an IgG-containing immunoadsorbent by activation with cyanogen bromide have been described in other publications. Rabbit IgG was purified from antiserum containing adsorbed anti-Id by using staphylococcal protein A agarose column.

 Detection of anti-immunoglobulin antibodies.

The presence of the anti-isotype and anti-allotype along with other anti-Id antibodies was determined by direct binding analysis using IgG obtained from the pre-immune and antipeptide-containing chimpanzee serum in the ELISA of the immunosorbent bound by the biotin-avidin-enhanced enzyme. 200 ng in 50 μl of chimpanzee IgG were adsorbed in the cells of the microtiter plates. Nonspecific points were blocked, the plates were washed as described above. Rabbit antisera containing anti-Id were introduced into the chimpanzee IgG coating of a microtiter cell. Specific binding was determined using biotinylated goat anti-rabbit gamma depth and horse radish peroxidase conjugated with streptavidin. The substrates were 2,2-azino-di- (3-ethyl-benzthiaxoline sulphonic acid) and H ₂ O ₂ , the colorimetric reaction was stopped by adding sodium dodecyl sulphate. The optical density of each cell was shown on an ELISA plate reader at 410 nm, as previously described.

 Coupling with KLH and precipitation with alum.

 Methods for coupling anti-Id with KLH along with alum precipitation were carried out as described in example 2.

 F. Insertion of anti-Id and peptide 735 into 752 KLH conjugate.

 Adult female BALB / s ByJ mice. (Jaclson Laboratory Bar Harbor) was injected with bi-weekly injections of 20 μg IgG as alum precipitate or conjugated with KLH precipitated with alum. Mice were bled 14 and 28 days after the last rabbit anti-Ig injection. Serum obtained from mice prior to immunization served as a negative control. Mice receiving peptide 735-752 conjugated to one KLH received 14 days apart three intraperitoneal injections of 20 μg peptin-KLH as an alum precipitate. These antisera were tested 14 days after the final injection.

 G. Determination of antipeptide and anti-HIV activity.

The antiseptide 735-752 response in murine antisera was determined on a biotin-avidin-enhanced ELISA using biotin-labeled goat anti-mouse IgG (b-gamg; Vecter Laboratories Eg., Burlingame, CA). The serum content of the antipeptide was quantitatively determined as the final titer value, it reflects the last dilution of the serum containing the antipeptide, which gives a different OD ₄₁₀ -3 value compared to the OD ₄₁₀ value obtained by diluting the pre-immune serum 1:10.

Anti-HIV activity was determined in a similar manner using the baculovirus gp160 recombinant containing the amino acid sequence associated with the HTLV-IIIB isolate. Ten recombinants panogrammov gp160 (Mikrogenezis) deposited on the surface of individual cells in BBS ELISA plates overnight at 4 ^C. The analysis was performed by methods previously described. Anti-HIV titers are likewise expressed as final titers of the last dilution.

 H. The specificity of the anti-Id antibody.

To determine the specificity of rabbit anti-Id, the following binding assay was performed. For coating microtiter plates triple cells using different concentrations of rabbit anti-Id IgG preparation in 50 l BBS, which was adsorbed to cells for 1 hour at 37 ° ^C. Points nonspecific binding blocked with 10% normal goat serum (NGS), then insert washed in Tween 20, including saline containing phosphate buffered saline (T-PBS). Biotinylated preparations of anti-peptide IgG of chimpanzees injected dose of about 10 ng per well, in binding was carried out for about 1 hour at 37 ^° C IgG, which do not provide specific binding of anti-Ig, was removed by washing with T-PBS. The determination of the binding of Id-anti-Id was carried out as described for anti-immunoglobulin by ELISA.

 I. Suppression analysis.

The suppression of the Id-anti-Id reaction was carried out in order to study the ability to suppress various antibodies of chimpanzees and rabbits or mouse antiserum. A constant amount of rabbit anti-Id IgG (156 ng in 50 ul) was adsorbed on the solid phase for 1 hour at 37 ^C. This concentration of anti-Id correspond to the linear portion of the binding curve Id. Points unspecific binding were blocked by adding 10% TGS for 30 min at ³⁷ ° C and then the plates were washed in PBS-T. Biotinylated Id chimpanzee X-99 IgG (b-Id, 10 ng / well) were mixed with various concentrations of different inhibitors were administered three times and then the cells where incubated for 1 hour at 37 ^° C Octalnaya part of the analysis was carried out as described for anti-immunoglobulin (ELISA). Maximum binding (no inhibition) was observed in the case of cells in which b-Id was administered without an inhibitor.

1-

J. Биотинилирование антител.In order to determine whether anti-Id can suppress I binding of Id to peptide 735-752, or with gp160, a competitive binding inhibition assay was performed. The inhibition of Id binding to either the peptide or gp160 was studied in the same way as the Id-anti-Id reaction with the following modifications: 200 ng of peptide 735-752 conjugated to BSA, or 10 ng of gp160 recombinant adsorbed to the solid phase during overnight at 4 ^C. After blocking with 10% NGS ELISA plates were washed three times in PBS-T. Biotinylated Id (10 ng / well) was mixed with an equal volume of anti-Id of various concentrations, then applied three times to microtiter cells. Horse radish avidin peroxidase and substrate were administered as described in other assays. The suppression of the reactions Id-anti-Id or Id-peptide was calculated by the above formula. The percentage of suppression is
100 ×

1-

J. Biotinylation of antibodies.

5 mg of purified IgG in 0.5 ml of BBS was mixed with 80 μl of 0.5 M NaHCO ₃ , pH 9, 40 μl of dH ₂ O and 10 μl of a 50 mg / ml solution of biotin-N-oxysuccinimide ester (Plerce Chemical Co., Rockford IL.) in anhydrous dimethylformamide (JT Baker Chemical Co., Phillipsturg, NJ.) and incubated for 1 hour at 25 ^C. The reaction was stopped with 25 .mu.l of input I M MH ₄ Cl and the mixture is then dialyzed overnight in BBS at 4 ° ^C. The biotinylated IgG was mixed with glycerin and kept at ^-20 ° C in the dark.

 K. Preparation of immunoadsorbents.

Peptide 735-752 was conjugated to cyanogen bromide activated Sepharose 4B. Approximately 3 mg of peptide 735-752 was conjugated to 1 ml of sepharose. The amount of non-conjugated peptide in this stream was determined by light absorption at 275 nm. The efficiency of conjugated binding was 92%. In addition, the peptide 735-752 and the transfer peptide SV 40 T-ag were also conjugated to affinity 501 (Bio-Rad Laboratories, Richmand, CA). Affi-gel 501 is an organomercury derivative that readily forms a mercaptide bond with free SH groups. Since both peptides contain terminal cysteine groups, this base was used to prepare the peptide agent column. The presence of the free reduced sulfohydryl group on the peptide before conjugation was determined by the reaction of the free H group with Ellman's reagent. When present less than the theoretical amount of free sulfhydryl groups, the peptides are recovered either 5 M equivalent of sodium borohydride at ⁴ ° C or dithiothreitol.

Affi-gel 501 was balanced in 10 mM NaHPO ₄ , pH 6, about 2 mg of the peptide was introduced into 10 mM NaHPO ₄ per 1 ml of gel, and the mixture was stirred overnight at room temperature. The base was washed with 50 mM sodium acetate, pH 5, the amount of peptide was determined, which was not conjugated by measuring the optical absorption of the stream at 275 nm. The free functional points on the gel were saturated with 0.05 M cysteine. This gel was washed and aged in 50 mM NaPO ₄ , pH 7, before its practical use. The conjugation efficiency of the peptide 735-752 and the SV 40 transfer peptide was 62 and 77%, respectively. To prepare the chimpanzee X-99 IgG antipeptide for immunization of rabbits, cyanogen bromide activated sepharose 4B was used. According to the binding to the peptide 735-752, gp160 and anti-Id, no difference was found with the chimpanzee antibody using two different chromatographic bases.

 L. Spot analysis of Western.

Western spot analysis was carried out using the Bio-Rad immuno-point system (Bio-Rad Labortories, Ricbimond CA). Nitrocellulose bands on which HIV antigens were spotted precipitated were blocked with 20 mM Tris-HCl, 150 mM NaCl buffer solution (9) 7.4 containing 1% BSA and 02% Tween 20. After blocking nonspecific strip points interacted with AIDS collected human serum (1: 100) or mouse serum (1:10) overnight at 4 ° ^C. The strips were washed with buffered Tris-Hcl solution to remove unbound anti ate. The reactivity of human and mouse antibodies with goat anti-human and anti-mouse Ig conjugated to alkaline phosphatase (Sigma, St donis, MO), respectively, was determined. The substrate used was obtained from Bio-Rad laboratories. Characteristics of the serum containing the chimpanzee antipeptide in relation to the binding of HIV coat antigens are given in Table 10.

 Both chimpanzee antisera interacted with gp41 according to Western point analysis and gp160 according to radioimmunoprecipitation. Antiserum bound peptide 735-752 and solid phase recombinant gp160 (ELISA). These antisera were not effective in neutralizing HIV infectivity under in vitro conditions; only a partial decrease in the activity of reversible transcriptase HIV was observed.

 When infected with an infectious HIV virus, a reaction of developing chimpanzee antibodies (antibodies to gag proteins) is observed, which is an indicator of viral replication. Prior to HIV infection, an antiserum was selected from X-99 chimpanzee as an Id preparation that should be used to generate anti-Id.

 M. Rabbit antibody to chimpanzee X-99 IgG antipeptide recognizes Id determinants.

In order to demonstrate the specificity of the anti-Id rabbit antibody preparation, a binding assay was performed using Id along with other chimpanzee IgG preparations. Two hundred nanograms of IgG of various chimpanzees bound to the solid phase and the rabbit antibody preparation was administered in various dilutions. When diluted 1: 100, the rabbit antibody and anti-Id bind to the idiotype (OD ₄₀₁ 0.47) of the IgG chimpanzee antipeptide, while no reactivity with IgG from chimpanzee X-99 to immunization (OD ₄₁₀ 0.02) or with pre-immune IgG and IgG peptide anti-SV40 Ta from chimpanzee X-189 (OD ₄₁₀ 0.008). In addition, anti-Id bound the IgG of the anti-HIV chimpanzee X-183 peptide (DM 0.19) with its pre-immune IgG preparation (OD ₄₁₀ 0.006). These data show that rabbit anti-Id recognizes the determinants of Id associated with the anti-HIV peptide immunogen from two different chimpanzees. This determinant was not present in their respective IgG preparations prior to immunization with the peptide. In contrast, the anti-Id preparation of the anti-SV40T peptide only binds the Id immunogen (chimpanzee X-189), but not the pre-immune IgG or the anti-HIV IgG peptide preparation.

 N. Rabbit anti-Id recognizes the connecting point of the Id antigen.

Four types of Ab-2 were obtained by immunization with Ab-1. Ab-2a recognizes an Id different from the connecting point of the antigen. The reaction of this anti-Id is not inhibited by binding to the Ab-1 antigen used to induce Ab-1. Ab-2 β or anti-Id internal display takes the form of a three-dimensional structure of the antigen recognized by Ab-1. The Id-anti-Id reaction is suppressed by the antigen. These anti-Id bind Ab-I produced in various species immunized with a nominal antigen. Ab-2 anti-Id has been demonstrated to include potential vaccine activities due to the mimicking ability of the antigen. Ab-2 γ also recognizes Id suppressed by the antigen, but does not have any internal imaging ability. These anti-Ids are usually limited to certain types of inbred strains within the given species, and antigen suppression may be due to spatial difficulties. The final category of anti-Id is Ab-2 ^c or epithelium. Such anti-Id recognize Id and epitope on Ab-1 and antigen, respectively.

 Further, a study was conducted to determine whether anti-Id can inhibit Ag-1 chimpanzees by binding to its antigen peptide 735-752 along with gp160. As shown in table 11, 10 μg of anti-Id can suppress peptide binding by 47%. The control anti-Id is unable to inhibit the binding of Ab-1 chimpanzee. In addition, anti-Id also partially inhibits the binding of Ab-1 to gp160 (24%).

 Higher concentrations of anti-Id do not inhibit the binding of Id to a higher degree of either peptide or gp160. Along with this, these data confirm that anti-Id recognizes the determinant associated in part with the binding point of the chimpanzee Ab-1 antigen. The fact that only partial suppression is observed confirms that anti-Id recognizes other determinants associated with Ab-1 that cannot participate in antigen binding. This is not surprising, since anti-Id is polyclonal in nature and may contain a mixture of various classes of Ab-2, including Ab-2 β or Ab-2 γ, which are responsible for binding to two preparations of Ab-1 anti-HIV chimpanzee along with suppression binding of Id to antigen. The determinants of the nonantigen binding point, together with the fact that anti-Id binds Ab-1 homologous to a greater extent than Ab-1 heterologous chimpanzee X-183, confirms that anti-Id can also contain Ab-2 α population.

 Since the Ab-1 preparation was purified by affinity chromatography and some peptides may be contaminated with the Ab-1 immunogen, the ability of rabbit anti-Id to directly bind peptide 735-752 and gp160 was studied. Neither antipeptide activity nor anti-HIV was detected in rabbit anti-Id preparations. Thus, the suppression by means of anti-Id binding of the Ab-1 antigen and the detection of total Id in two preparations of the chimpanzee antipeptide was not the result of contamination of the Ab-1 preparations with the peptide and the subsequent antipeptide reaction in the rabbit antibody preparation.

 A. Rabbit anti-Id primarily recognizes the chimpanzee idiotype.

 In order to determine whether anti-Id has internal imaging ability, the ability of rabbit and mouse chimpanzee antipeptides antibodies to inhibit the Id-anti-Id reaction is studied. These experiments were also carried out in order to confirm that isotopic and allotype determinants are not recognized in this analysis. Only one of the three preparations of the rabbit antipeptide suppressed and none of the three preparations of the BALB / c mouse monoclonal antibody inhibited the Id-anti-Id reaction (Table 12).

 At a concentration of 2.5 μg, the chimpanzee peptide inhibitor X-99 and X-183 inhibited the Id-anti-Id reaction by 85% and 28%, respectively. Thus, homologous Id was a significantly better inhibitor of the reaction. In support of direct binding studies, there was no significant inhibition of the reaction by pre-immune chimpanzee IgG and anti-SV40 T-A peptide. Only one single rabbit anti-HIV peptide preparation showed some inhibition of the Id-anti-Id response (40%). These data confirm that the anti-Id preparation does not consist mainly of anti-Id internal display due to the inability to recognize a common interspecific idiotype. In addition, homologous Id was the best inhibitor of the Id-anti-Id reaction. This fact, along with the inability of some monoclonal antipeptide preparations of rabbits and mice to serve as inhibitors, further confirms that the Ab-1 component may also be present in anti-Id.

 P. Induction of the anti-HIV reaction in mice receiving anti-Id.

 Previous studies have shown that rabbit anti-Id, consisting mainly of Ab-2 β or Ab-2 α, can induce a surface antigen hepatitis B antigen in mice without subsequent antigenic stimulation. In the following example, a study was conducted to determine if this anti-Id could induce an anti-HIV reaction specific for peptide 735-752.

 Four groups of mice, five in each group, received five intraperitoneal injections of rabbit anti-Id in the form of alum precipitate or in the state of conjugated binding to KLH. Two groups received the control anti-Id and the other two groups received anti-Id specific for the Ab-I preparation of chimpanzee X-99. A fifth group of mice was immunized with three injections of peptide 735-752 conjugated to KLH. Sera from all groups were tested for antipeptide and anti-HIV activity between 14 and 28 days after the final immunization. Table 13 shows the anti-Id titers. The group that received the anti-Id for chimpanzee X-99 as an alum precipitate gave both an antipeptide and an anti-HIV reaction. Compared to mice that received the peptide conjugated to KLH, the average reciprocal titer of the final antipeptide in the treated anti-Id mice was approximately 3 times lower (600 compared to 2000). However, anti-HIV titers between the two groups were comparable (average 400 compared to 600). None of the two immunized anti-Id control groups showed any antipeptide or anti-HIV activity at a 1:20 serum dilution. Thus, the antipeptide or anti-HIV reaction is not the result of an anti-rabbit IGG or anti-KLH reaction in these mice. Immunization with anti-Id conjugated to KLH also induces both an antipeptide response and an anti-KLH reaction in these mice. Immunization with anti-Id conjugated to KLH also induces both an antipeptide reaction and an anti-HIV reaction.

 The titer of antibody reactions in this group of mice is 2-3 times less than in the group of mice receiving precipitated alum anti-Id without conjugate binding. Thus, the conjugated binding of the anti-Id used in this study to KLH is not required to either induce or to increase the antibody response in vivo.

 Induction of the anti-tobacco mosaic virus and surface antigen hepatitis B antigen reactions have been previously demonstrated with both Ab-2 α and Ab-2 β with internal imaging.

 Q. Anti-Id indicates anti-HIV, which is part of the chimpanzee X99 antideptide Id.

 Previous studies of the response of a mouse antibody to hepatitis B surface antigen (HBsAg) have demonstrated that anti-Id with internal imaging induces an antibody response to HBsAg that expresses a similar Id when compared with mice immunized with HBsAg. In addition, anti-Id Ab-2 α induces an antibody response to HBsAg that expresses an Id that is not present on antibodies from mice immunized with HBsAg.

 Ab-2α induces normally inactive clones, causing secretion of antibodies in HBsAg, which expresses a different Id when compared with stimulation of the nominal antigen. A strong serological difference between the anti-Id and the peptide immunized groups of mice was that the treated anti-Id mice suppressed the Id-anti-Id response, while no inhibition was detected in the peptide immunized mice (see table 13) . These data show that the treated anti-Id mice express the same Id also present in chimpanzees immunized with a peptide conjugated to KLH. This Id is usually not expressed in the serum of mice immunized with a peptide conjugated to KLH. Thus, it is clear that anti-Id can be used to induce a serological reaction that usually does not dominate the immune response when an antigen is used as an immunogen. The anti-Id reaction has serological characteristics similar to Ab-1 used to create anti-Id. Serum from mice immunized with the control anti-Id (SV40) did not show the ability to inhibit the reaction of the antigen peptide 735-752 of chimpanzee X-99 anti-Id (Table 13). These results suggest that pre-injection of rabbit anti-Id leads to the expression of positive Id antibodies that bind gp160 and are specific for peptide 735-752. Clone activation, usually not induced during an immune response, i.e. inactive clones was detected during a mouse immune response to a bacterial levan. Immunization with anti-KLH-conjugated anti-Id induced an anti-levana or anti-HBsAg population that comprised Id, along with an Ab-1 population that was not normally produced during the immune response to bacterial levan and HBsAg.

 In the following example, the specificity of the anti-Id-induced anti-HIV reaction was determined and the ability of both peptide 735-752 and gp160 to inhibit binding to this peptide was studied (Table 14). The gp41 peptide at a concentration of 10 μg inhibited the binding of anti-HIV mouse anti-Id to the peptide by 89%. The control peptide SV40 inhibited this reaction by only 6%. At similar inhibitor concentrations, gp160 was a less effective inhibitor of the anti-Id anti-HIV peptide compared to this peptide (52, unlike 89%). These data confirm that anti-Id induces an antipeptide reaction that is not fully associated with the antinative gp160 reaction. The specificity of the induced anti-Id antibody reaction comprises the antipeptide and anti-gp160 component.

 The reaction of the natural component of the anti-HIV reaction induced by anti-Id antibodies was confirmed by Western point analysis. The immunized anti-Id mouse serum interacted with a 41 kDaltons molecular weight region. No reactivity was detected with a control group of anti-mouse Id serum. Control human serum from AIDS patients showed reactivity with sheath antigens (160, 120, and 41 kD) along with gag proteins (55, 24, and 18 kD). These data indicate that the anti-HIV response in mouse antiserum is associated with gp41. The reason that the mouse antiserum does not recognize gp160 along with gp41 is not known in the Western point analysis. However, the predominant antibody response in mice and chimpanzees immunized with gp41 peptide conjugated to KLH, as determined by Western point analysis, is gp41.

 The inability of the murine anti-Id-induced antiserum to bind to gp160 may reflect a relatively specific antibody response directed towards the region associated with gp4AI. In addition, the concentration in gp160 antigen in this particular Western point assay is optimal because only a mild anti-gp160 reaction was detected in human AIDS serum. This may be another possible reason explaining why the anti-Id-induced anti-HIV reaction recognizes only gp41, but not gp160.

Claims (1)

 A METHOD FOR OBTAINING AN AIDS VACCINE, including administering an antigen to an animal, isolating antibodies, then administering them to another animal, isolating antibody-producing spleen cells, producing hybrids, culturing them to produce anti-idiotype antibodies, characterized in that, in order to increase immunogenicity and expansion spectrum of activity, fragment 735 - 752 of the envelope protein of dr 41 is used as antigen, and the anti-idiotypic antibody is capable of binding to the recognition epitope of an SDC cell marker to inhibition of binding between the SDC cell marker and any of the Leu 3A and OCTCH marker antibodies.