JPH01500432A - Compositions and methods for immunizing against viral agents of AIDS and ARC - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Compositions and compositions for immunizing against viral agents of AIDS and ARC How to This invention is subject to the following NIH approvals: AI-22307-01, AI-23619 -01 and A1-23472-01 with government assistance. It was done. The government has certain rights to this invention.

This is “synthetic peptides and their use in diagnosis and vaccination of AIDS and ARC.” U.S. Patent Application No. 790,830, dated October 24, 1985, entitled and 198 entitled “Polyamide resin and method for preparing immunodiagnostic reagents.” This is a continuation-in-part of U.S. Patent Application No. 858,216, filed April 30, 2006.

Nine miles to 11 The present invention relates to acquired immunodeficiency syndrome (AIDS) and AIDS-related diseases (ARC). The present invention relates to a vaccination composition and a vaccination method for. In particular, the present invention Immunization methods against viral causative agents of DS and ARC, and AIDS and polyamide resin-synthetic polymers that can be used in ARC diagnostic assays. Concerning the putido complex.

AIDS is a severe immune system that has led to reports of opportunistic infections occurring among homosexual men. It was first discovered as a deficiency disorder [Gottlieb, M, S. ) et al., 305N, Engl, J., Med, 1425-1431 (1981 ); Masur, H., et al., 305N, Engl., J., Med. 1431-1438 (1981)]. “Acquired immunodeficiency syndrome ( The incidence of this new human disease, called AIDS, was rapidly increasing and -500432 (2) sexual transmission is thought to be the main method of transmission; It has been reported that the disease is transmitted through blood transfusions [see the above-mentioned article by Gottlieb et al. Light]. The causative agent of this disease is human T cell tropic virus type II (HTLV-1). Lymph node disease-associated virus (LAV) (Barre-S1 noussi.

F.) et al., 2205 science 868-871 (1983)), or Hitore 1-lowi, variously referred to as AIDS-related retroviruses (ARV) It turned out to be Luz.

Seroepidemiological studies have shown that HTLV was detected in the serum of most patients with AIDS or ARC. -I [Specific antibody identified. Serum obtained from AIDS patients and hemophiliacs The main antigen recognized by the antibodies in the protein is related to the coat glycoprotein. moreover, The most immunogenic of the human T cell-tropic viruses, HTLV-1 and HTLV-I Some proteins are glycoproteins expressed on the cell surface [Chen , I, S, ) et al., 305 Nature (London) 502 (1983 )].

The envelope (env) gene product of HTLV-1 [1 is used as a polyprotein precursor. It is synthesized and then glycosylated within infected cells.

Its glycosylated polyprotein (gp160) with an estimated molecular weight of 160 kd is an amino acid. gp12Q and carboxyl transmembrane subunit Processed by gp41. gp41 subunit purified virus preparation It is one of the main polypeptides contained in

Antibodies from AIDS and ARC patients exhibit virus-neutralizing activity, however , infection appears to occur in those patients before the development of neutralizing antibodies, r! ! 5 It is unknown whether induction of neutralizing antibodies prior to infection confers protective immunity. The general view is that the plateau determinants or epitopes involved in the induction of neutralizing antibodies are The tope is associated with the glycoprotein envelope [Holden et al. en, H, T,) and Taniyama (T, Taniyama), 150J, Exp, Med, 1367 (1979); Flyer (F 1yer, D, See C. et al., 305 Nature (London > 815 (1983)). sea bream〕. Until the present invention, no association has been established for the AIDSFI family of viruses. There wasn't. As explained below, now gp41, gp120 and gpis.

The coat glycoprotein is the most immunogenic epitope in individuals exposed to the virus. It proved to be taupe.

The present invention shows that there are two types of critical epitopes involved in the induction of protective virus-neutralizing antibodies. Associated with viral coat glycoprotein subunits, gp120 and gp41 It is based on the hypothesis that In addition, the present invention provides a solid-phase resin (on this resin, immunogenic When the polypeptide portion of the subunit is synthesized), AIDS and and their immunization to elicit an immune response against the full causative agent of ARC. It is based on the use of the polypeptide portion of the subunit. These polypeps Tide subunits are conveniently synthesized on solid phase resins. Solid phase peptide synthesis proteins and peptides (collectively referred to herein as “brotides”). This is a valuable method for elucidating the structure and mechanism of action. Most of this Such synthetic methods require that the brotide is immobilized (usually via a linker molecule) during its synthesis. A cross-linked polystyrene resin is used as the solid phase. Its synthesis protects Amino acids are added to a solid phase, and the protecting groups of the amino acids are selectively removed (deprotected). , and by an iterative cycle consisting of adding another suitably protected amino acid. [Merrifield, R.B. ), “Solid-phase peptide synthesis’°, 32 Adv, Enzymo1ogy22 1 (1969)].

Cross-linked polystyrene resins are the most commonly used supports in solid-phase brotide synthesis. used, but their resistance to the polar organic media necessary to solubilize the reactants The relatively hydrophobic nature poses problems in brotide chain synthesis. This kind of medium depends on The brotides that are being grown in the polystyrene matrices are free to solvate. Reduced diffusion and steric hindrance of reagents within the polymer lattice, resulting in insufficient swelling of the polymer This can cause loss of efficiency during the coupling cycle and reduce the final yield on an iterative basis. decrease.

In the early stages of synthesis, the resin to brotide mass ratio is high and the physical properties of the support are This drop in efficiency is particularly sharp when quality is dominant.

These disadvantages are that they are highly polar in nature and that the solvents required for peptide synthesis are free. This led to the development of a cross-linked polydimethylacrylamide-based support that penetrates into water. assert Atherton, E, Clive (D, L, J, C1) ive) and Sheppard (R, C, S heppard), “Polypeptide Synthesis. 97 J, A5ier, Chem.

S oc, 6584 (1975) A rshady , R, ), Atherton (E, Atherton), Gate (M, J , Galt), Lee (K, Lee) and Sheppard (R, C, S heppa) rd), °′ Easily prepared polar heftide/oligonucleotide synthesis for solid phase heftide/oligonucleotide synthesis "Support", 1979J, C,S.

See Chem, Co+am, 425 (1979). This polyamide tree Fats (e.g. aminomethyl derivatives) can be used with a suitable linker molecule (with the C-terminal residue as a support). Adapt to a synthetic strategy that incorporates alternative protection strategies by choosing be able to. However, peptides synthesized on this resin are It must be separated from the resin and purified, both of which are time-consuming processes. Reduces the final yield of brotides.

A significant advantage of the compositions and methods of the present invention is that the protide is It can be used to induce an immune response in laboratory animals without being separated or purified from fat. The point is that Resin-brotide complexes synthesized in this way are used for many assays. It can be used on the way. certain polyamide resin-peptide complexes, especially with immunogens. The gp120 and gp41 subunit portions of the Bp160 coat glycoprotein are of particular interest in the present invention.

Synthetic peptides with sequences similar to those contained in virus-encoded proteins are The following findings are useful for identifying natural antigenic determinants associated with such proteins. has been proven before. Several laboratories have tested various hepatitis B antigen (HBsAg) compounds. reported a study on the antigenic activity of the synthetic peptide. Dreesman (Drees…& n.

G, R, et al., 295 Nature 158 (1982); Lern er, R, A,) et al., 78Proc, Natl, Acad, Sci, U 5A3 403 (1981) Prince, A. M. et al., 79 Pro. c, Natl, Acad, Sci, U SA 579 (1982). stomach. Induction of antibody responses against HBsAg by using peptides of this type Although the peptide was found to be relatively weak, it It was possible to strengthen this by combining the two. The above literature by Lerner et al.; Sanna x (S anchez, Y,) et al., 18 I intervirolog y 209 (1982). In addition, tobacco mosaic virus [ Anderer (Anderer, F, A,) and Juranberger (Sch lumberger, H.

D,), 97B iochiw+, B1ophys, Aeta 503-50 9 (1965); Ampler and Juranberger, 115B 1ochis +, B 1ophys, Acta222-224 (1966) Near Iani (F Earnney, F, J,), Leung, C.

Y, ), Young (J, D, Young) and Benj, E. amini), 243B iochim, B1ophys, Acta509- 514 (1971)], foot-and-mouth disease virus [Bittle, J.L. ) et al., 298 Nature 30-33 (1982); Pfaff, E., et al., IEMBOJ, 869-874 (1982)], and polio Virus [Emini, E, A, >, Jamson (B, A, J Ameson) and Reimer (E, Wi++mer), 304 Natu RE 699-703 (1983)] Synthetic peptides corresponding to the acid sequences can be used to detect neutralizing epitopes associated with intact virus particles. was used to determine the

Immunogenic AIDS and ARC proteins based on primary sequence analysis of these immunogens Since inference of quality antigenic determinants is not accurate, it is difficult to identify putative epitopes by searching. The identification of AIDS and ARC viruses along with synthetic peptides is a painstaking task. including the description of antigenic sequences specific to virus-causing agents, and the purification and purification of synthetic peptides. Compositions and methods that do not require conjugation of the peptide to a carrier protein would be very advantageous.

Mitsuho Mga Therefore, the object of the present invention is to provide immunity against the viral causative agents of AIDS and ARC. To provide a composition comprising a polyamide resin and a synthetic peptide that can induce an epidemic response. The synthetic peptide is HTLV-[1, ARV or LAV) gp12 0 mari? j: has a sequence homologous to part of the amino acid sequence of gp41 coat glycoprotein It consists of an amino acid chain that contains a parent tree region in its sequence.

To achieve this goal, we first developed a number of synthetic compounds that could induce such a response. It was necessary to identify peptide candidates. Therefore, HTLV-III The amino acids of the most immunogenic proteins (i.e. 8p120 and gp41) determined the amino acid sequences and determined the amino acid sequences for these proteins and the complete virus particle. The amino acid sequences of these proteins where binding sites for antibodies are most likely to exist are determined. Identify the structural conformation and then identify the portion of the amino acid sequence that contains the binding site. Immunogenic synthetic peptides with identical or sufficiently homologous sequences and structures It is also an object of the present invention to synthesize

Another object of the present invention is to provide a polyamide resin and a method for producing the same. Their synthetic peptides were synthesized on the resin by solid-phase synthesis and injected into experimental animals. When this happens, the immune response to the viral causative agent of AIDS and ARC is teat. It is not necessary to separate the synthetic peptide from the resin before injecting it into experimental animals. 〉

Another object of the present invention is to use a polyamide resin-peptide complex as a ligand for specific binding pairs. (where the binding pair is a ligand and a parent specific for the ligand). HTLV-I [1 , a portion of the gp120 or 8p41 coat glycoprotein of ARV or LAV. consisting of amino acid chains with the same sequence); the complex is brought into contact with serum from an animal. and thereby eliminate the viral causative agent of AIDS or ARC present in the serum. bind all antibodies to the complex; then bind the bound antibodies to the specific binding partner. contact with an anti-ligand; Antibodies against the viral causative agent of AIDS or ARC in suspected individuals The object of the present invention is to provide an assay method for detecting.

Another object of the invention is to prepare HTLV-III, ARV or LA on polyamide resin. It has a sequence homologous to a part of gp120 or gp41 coat glycoprotein of V, and A peptide consisting of an amino acid chain containing the parent tree region in its sequence is synthesized, and Administering an effective amount of the polyamide resin-peptide complex to experimental animals. immunization of laboratory animals against the viral causative agent of AIDS or ARC, consisting of The goal is to provide a method to do so.

α11-deficient skin Figure 1 shows Chou-F asIIla's research on secondary structure. HTLV-Ill generated by a computer program that utilizes the estimation plan of n) , gp120 and gp41env glycoproteins 1p1 of LAV and ARV Average hydrophilicity value of each amino acid residue for the amino acid sequence of 60 glycoprotein precursors This is a depiction of the plot.

Figure 2 shows an actual computer program of the amino acid sequence segment of the plot in Figure 1. It is a cut.

Figure 3 HT L V - [[, LAV and A RV (7) Before gp160 The secondary structure of the amino acid sequence is shown in the plot of Figure 1 showing the difference from the secondary structure of the precursor. It is a schematic diagram.

Figure 4 shows the binding of peptide 6 by rabbit antibody using enzyme-linked immunosorbent assay. Horses immunized with the indicated gp120 peptide 503-532 (peptide 6 in Table H). FIG. 2 is a graph of optical density versus back dilution of antiserum from Heron. Figure 4A is pepti Figure 4B shows binding of control peptide (see Example 15).

Data from anti-peptide antiserum derived from rabbit 1 are shown in (·) and from rabbit 1. Data from anti-peptide antiserum derived from G. 2 are indicated by (○) and derived from each rabbit. Data from pre-immune sera are shown as (mu) and (△), respectively.

Figure 5 shows reverse transcriptase (RT) activity (co-H-TTP uptake; see Example 16g) A3. measured by testing. HTLV-I [1 u. Indicates virus proliferation. Figure 5A is a 10 dilution, Figure 5B is a 10-2 dilution, and Figure 5C is a 10-2 dilution. The figure shows virus replication at a 10-3 dilution and Figure 5D shows virus replication at a 10-4 dilution.

1 to 9 skins As mentioned above, the present invention is directed to the most immunogenic of the viral causative agents of AIDS and ARC. The gptzo and gp41 subunits of the coat glycoprotein are It is based in part on the hypothesis that As explained below, this hypothesis The validity is now explained by the amino acids of the gp120 and gp41 subunits. determine the amino acid sequences and identify those coated glycoproteins where antibody binding sites are most likely to exist. It was necessary to select parts of protein subunits. This selection is gp12 achieved using computer modeling of the structure of the 0 and gp41 subunits. It was.

Once the most likely antibody binding site has been identified, the binding sites on the polyamide resin are A amino acid chain was synthesized and the amino acid sequence at each site was duplicated. polystyrene A common method for coupling synthetic peptides to system resins is benzyl ester derivatives. Separation of the peptide from the resin is usually achieved by acid or base cleavage. be done. Although benzyl esters are susceptible to several such cleavage methods, Stable through the numerous deprotection, neutralization, and coupling reactions characteristic of phase synthesis methods . Hydrazine was also used to separate the brotide from the resin [Gessler (K Essler, W.) and Iselin (B, I5elin), 49 He 1v, Chim.

A [see cta 1330 (1966)], and various ammonia decomposition [manin Manning, M, >, 90 J, A m, c hem, S o 1348 (1968)] and other methods were used as well. Shigashi However, these methods avoid degradation of the peptide and subsequently produce synthetic by-products ( amino acids, short peptides, resin degradation products, and often incompletely protected groups. Appropriate steps must be taken to purify the peptides (including the removed peptides). There is a point. Purification is sometimes achieved by direct crystallization, but contaminating peptides are For syntheses of similar size and composition to the desired product, a more selective approach is recommended. must be adopted. Regardless of the separation and purification method, these requirements must be met. peptides add time-consuming steps to their synthesis and often reduce the total yield of synthetic peptides. Let it go down. The polyamide resin and method of the present invention do not require such separation and purification at all. This reduces the synthesis time and increases the yield of peptides. Melt.

The polyamide resin of the polyamide resin-peptide complex of the present invention is a diaminoalkaline N , N'-bis-argenoyl-diaminoalkane) in an aqueous solution. Produced by crosslinking methylacrylamide amniotes. current suitable fruit In embodiments, the crosslinking agent is Halpern and Spar N,N'-bisacryloyl-1,3-dia produced according to the method of Minopropane or N,N'-bisacryloyl-1,3-diaminobutane Either way (J.

A, Halpern and J, T, Sparrow, 'Improved N,N'-bisacrylic Loyldiaminoalkane synthesis method “’, 10SyntheticComm, 569 (1980); the entire text of which is hereby incorporated by reference].

The use of propanes a, the polymer it is obtained by the use of ethyl analogues is preferred because it results in polymers with larger pore sizes and improved swellability. suitable. However, a person skilled in the art who has the benefit of the present invention will appreciate the information published in said document. and other diaminoalkanes, N,N'-bisacryloyl-1,2-diaminoethyl Tan and N,N'-bisacryloyl-1,6-diamithexane are also included in the present invention. It will be appreciated that it can be used in the production of fat.

The crosslinked resin includes a functional monomer. The term "functional monomer" refers to synthetic peptides. alkenylamines used to fix the C-terminal amino acid of Tido to the resin. means. Methylsulfonylethyloxycarbonyl (MSC> group protected) [Tesser, G, I,) and Barba] -Togiers (1, C, Ba1vert-G eers), “Methylsulfony ethyloxycarbonyl group, a new amino protecting group that can be used in many ways”), 7 1 nt, J, Pepticle P rotein Res, 295 (197 5)], the functional monomers are called MSC alkenylamines. these The functional monomer is prepared by the reaction of a commercially available chloride derivative with an alkenylamine. and the MSC protecting group is then removed with base. However, M3C No base is required. Polyamide resin is simply added with excess allylamine and then filtered. manufactured by separating fine particles obtained by filtration or other methods Can be done. The amount of functional monomer added ranges from about 0.1 mmol to about 0 per gram of resin. ．． 5 mmol, preferably in the range of about 0.2 mmol to about 0.4 mmol. chosen to bring about change. Initiators can be selected from those skilled in the art such as persulfate or riboflavin. may be any initiator known in the art, preferably ammonium pervfL It is.

Since the above substances are mixed in an aqueous solution, they are collectively referred to as the “aqueous phase”. . In the polyamide resin production of the polyamide resin-synthetic peptide complex of the present invention The next step is to combine the aqueous and organic phases. The term “organic phase” refers to the aqueous phase. organic substances that, when stirred together, form a suspension (from which a resin is obtained) means solvent. In the presently preferred embodiment, the organic phase is hexane and a tetrasalt. It consists of a mixture of carbonized carbon.

An emulsifier is added during stirring to form beads of uniform size. emulsifier may be any surfactant known to those skilled in the art, and in the presently preferred embodiment Sorbitan sesquioleate, sorbitan monolaurate, or sorbitan It can be any of the following: The amount of surfactant added is approximately uniform. Adjustments are made to obtain fine spherical resin. As the amount of surfactant decreases, the An emulsion containing a large amount of amorphous material is formed, which is gradually increasing in size. Increasing the amount of surfactant will result in a reduction of the internal amino acid chains The amount of fine material is large and this fine material cannot be removed without loss of significant amounts of resin. It is difficult to These fine particles are present in the reaction vessel of the peptide synthesizer, as well as in the accompanying It can also clog pipes and valves.

Thereafter, the monomers in suspension are polymerized and promoted to form the polyamide resin beads of the present invention. Accelerators are added to promote growth.

Although many accelerators are known to those skilled in the art, in the production of polyamide resins N, When using N, N', N'-tetramethylethylenediamine (TEMED) achieved particular success. The resulting beads are then filtered and washed (if any). The M3C group (if present) is removed with base and the beads are dried. The beads are Can be screened through a mesh sieve to ensure relatively uniform size. Ru. The overall yield using the method of the invention ranges from about 87-94% from the starting monomer. Ta.

The resulting aminomethyl, cross-linked polydimethylacrylamide resin is a synthetic peptide gives maximum exposure of the peptide in aqueous solution and resin-polymer The backbone does not sterically restrict the peptide; exposure of the peptide is highly hydrated. This is a result of the polyamide resin's ability to swell to several times its dry bed volume when .

The synthetic peptide is coupled to a linker attached to the resin by an activator. Synthesized on beads by The term "linker" refers to the first linker of a synthetic peptide. A bonding group that bonds the carboxyl group of an amino acid to a resin. current preferred In embodiments, the linker is an oxyalkyl ammonium, acetate, aromatic acid (OBA). An amino acid residue is coupled to this to become the first amino acid of the peptide chain. . An OBA linker is used to attach the C-terminal amino acid to the polyamide resin. side chain protecting groups from the peptide without significant loss of synthetic peptide from the resin. Fluoride-free hydrogen fluoride can be used to remove. In the following examples , the selected amino acids were protected with t-butyloxycarbonyl (t-BOC) protecting group. However, those skilled in the art will understand whether the amino acid is any amino acid. It can often be the first amino acid of the peptide to be synthesized, and may also be the first amino acid of the peptide to be synthesized. It will be appreciated that protecting groups can be used as well, glycine residues are It serves as another spacer group between the lipid-polymer main body.

BOC-glycyl-4-(oxymethyl)benzoic acid, a presently preferred linker was prepared using a modification of the method of Mitchell et al. l, A, R,), Ghent (S, B, H.

Kent), Engelhard (M, EnBe1hard) and Maryfi Merrifield, R.B., “Improved Support for Solid-Phase Peptide Synthesis.” carrier, t-butyloxycarbonylaminoacyl-4-(oxymethyl)phenylene "A new synthetic route to ruacetamidomethyl-resin", 43 J, Org, Cb C, 2845 (1978); the entirety of which is hereby incorporated by reference]. Mi An important modification of the method of Chell et al. is the use of dimethylformamide as a solvent. I stopped doing that.

This solvent is difficult to evaporate and as a result even if evaporation raises the temperature Even if speeded up, this method still takes time. Manufactured as above The activator used to attach the linker to the prepared polyamide resin is diisopropylene. lopylcarbodiimide and 4-dimethylaminopyridine, but those skilled in the art Other active agents such as cyclohexylcarbodiimide and 4-methylpyrrolidinopyridine It will be appreciated that sexing agents can be used for this purpose as well.

After synthesizing the peptide on the polyamide resin, the polyamide resin-brotide of the present invention The complex can be used for many purposes, e.g. in experimental animals as A, IDS or To elicit an immunogenic response against the viral causative agent of ARC, HTLV- IIi, in vitro for the presence of antibodies against ARV or LA, V o to assay or antigens on the viral causative agent of AIDS or ARC. Used for mapping determinants. For example, in vitro assays Grind the polyamide resin-synthetic peptide complex with a motor and pestle, The ground complex was prepared in a microtiter test plate using a neutral pH buffer. This is done by adsorption onto a solid phase such as -1. A, I D S again serum or other body fluids suspected of containing antibodies to the viral causative agent of ARC. is then incubated with the adsorbed complex, washed to remove unbound antibody, and The bound antibodies can be analyzed using enzyme-linked immunosorbent assays, biotin-avidin amplification assays, or those skilled in the art. detection by other assays such as those known in the art.

Polyamide resin-synthetic peptide conjugates are also sometimes used during peptide synthesis. Take a portion of the amide resin, deprotect the peptide, and process it in a stepwise manner. Each removed portion was tested to determine if the peptide was a virus for AIDS and ARC. By determining that point during synthesis that binds to antibodies specific to the virus-causing agent. , to map antigenic determinants on the viral causative agent of AIDS or ARC. used for This method requires separation and purification steps that are required in other synthetic methods. This was made possible by eliminating the . The complex can also be ground and microtied. The antibody binding ability can be determined by adsorbing it onto a carrier such as a test plate. and certified as described above.

Separation of the peptide from the resin and purification of the peptide are essential for such assays. Not necessary.

The polyamide resin-brotide composite can also be used as a compound for A, IDS or ARC. useful as an immunogen against cancer-causing agents. This complex is prepared using an adjuvant. used directly to immunize laboratory animals with or without. “Experimental animal” As used herein, the term refers to any animal capable of an immune response. .

Although the experimental animals of primary interest are mammals, immunogenic responses can be determined using the method of the present invention. It can also be induced in other laboratory animals such as birds.

For example, the specific immune response to the viral agents of AIDS and ARC is , the envelope protein of HTLV-III virus as measured by radioimmunoprecipitation. Made of synthetic peptide with an amino acid sequence corresponding to protein and polyamide resin. was induced by immunizing rabbits using the conjugate.

These synthetic peptides then induce rabbit antibodies that bind to the AIDS virus. The antibodies were tested for their ability to bind human anti-HTLV-1 antibodies and Heron anti-HTLV-I [1 antibodies showed that they were able to neutralize the virus in tissue culture. It was tested to find out whether Once the most exempt Once epidemiogenic synthetic peptides have been identified, they can be used as vaccines or IDS and ARC patients and viral agents of AIDS and ARC. used in diagnostic assays to identify affected individuals.

The amino acid sequences of 811120 and gp41 subunit are HTLV-■ Inferences based on the nucleotide sequence of 3[H]leucine, 3JS)cysti NH2-terminus by Edman degradation of 3[H]valine- and 3[H]valine-labeled proteins These sequences were determined by verification of sequence analysis.

gp120 and gp41 coat glycoproteins (and their precursor gp16 The immunogenicity of 0) was demonstrated by screening serum samples from AIDS and ARC patients. ) Indirect cell membrane immunofluorescence using the 19/HTLV-III cell line (MIF> and 35[: Sl cystine-labeled H9/HTLV-I[1 cells were used. Radiation immunoprecipitation method/sodium dodecyl sulfate-polyacrylamide gel electrolysis Blood containing antibodies against HTLV-1 by electrophoresis (RI P/5DS-PAGE) It cannot be obtained by identifying the supernatant. A typical antibody-positive serum is also Hiramamelek. Glycoprotein preparation of H9/HTLV-III cells concentrated through a column of Chin Tested on objects.

These results show that antibody-positive serum contains antibodies that recognize gp120 and gp160. All samples containing antibodies against other epitopes had the highest proportion of gp12 It was found that it contains antibodies that recognize 0, pl, and 60.

gp120. The most immunogenic proteins on the gp41 and gp160 coat glycoproteins Certain site selections include hops (1-(opp, T, P, >) and wood (K.

R, W oods), 78Proc, Nat'l ＾ead, sci, U 5 A3824-3828 (1981) HTLV from HTLV-II, LAV and ARV -II [Estimating the location of hydrophilic regions associated with the coat gp160 glycoprotein The amino acid sequence of these glycoproteins was also achieved by Chu Fassman. Estimated proposal [Chou, P.

Y,) and Fasman (E, D, Fasman), f3B iochemis try2Z2 (1974)] was used to analyze the secondary structure. P The parent tree region is compared with the predicted secondary structure and most exposed on the surface of the glycoprotein. The parent tree area that is likely to be damaged was identified. These regions also contain viral coat proteins. Previous studies using the Since we reported that the hydrophilic region represents the surface region of the virus where the immunogen is located, We also researched the existence of the Dreesman (Dreesman).

G, R, et al., 295Nature (London) 158-160 (1982) (Hepatitis B surface antigen): Hopf and Wood's above-mentioned literature (hepatitis B surface antigen) ; Henderson, L., E., et al., 258J. , Biol, chem, 8400-8406 (1981) (Raucher rat leukemia virus); Gingeras, T., R., et al., 25 7 J, B iol, Che@, 13475-13491 (1,983) (Adenovirus spike protein): Watson.

R, J.) et al., 218 Science 381-384 (1982) (Simplicity (See Viral Coat Protein D)].

HTLV-[1, amino acid of gp160 glycoprotein derived from LAV and ARV Now that the amino acid sequence and likely immunogenic regions within the sequence have been identified, the next step is to The process involves identifying the same amino acid sequence (or binding to its epitope) as that region of the glycoprotein. Polymers with sufficiently similar sequences to be processed in the same way by matching antibodies The goal was to synthesize peptides. The synthesis was performed using the solid phase method described above. In all Ten types of synthetic peptides were synthesized, and each synthetic peptide was The selection was made based on the above-mentioned tests. The respective amino acids of these synthetic peptides The acid sequences are shown in Table H.

After that, 10 types of synthetic peptides were transferred to rabbits using polyamide resin-synthetic peptide complexes. It was used to induce an immune response in rabbits by injecting it into rabbits. to the rabbit may also be conjugated to a carrier after separation from the resin (on which the peptide is synthesized). The combined synthetic peptide was also injected. Antibody titers in rabbit serum are compared with bovine serum albumin. The antibody was tested for its ability to bind to a peptide conjugated to BSA (BSA). this Their results demonstrate that inhibition of rabbit anti-peptide binding to peptide-BSA is This was confirmed by conducting a harm test.

Rabbit anti-peptide antibodies were then used to detect natural proteins associated with HTLV-I. Their ability to recognize HT labeled with 35(S)-cystine was investigated. LV-I [[5 stained T cell line was used for immunoprecipitation and anti-peptide serum was released. It was investigated whether radioactive target 1 binds to HTLV-III natural protein. 5DS- Autoradiography by PAGE showed that the rabbit anti-peptide antibody was ■Specifically detects a single protein corresponding to the 6p160 precursor coat glycoprotein. It was revealed that it precipitates. When the precursor gp160 product is cleaved, the major Generates Ep120 coat glycoprotein and gp41 transmembrane glycoprotein Ru. The gp41 coat subunit is the amine terminal group of the precursor gp160 glycoprotein. To the same extent: not radiolabeled with 15(S)-cystine and detected by immunoprecipitation It wasn't done. However, “[S])-methionine was used as a label. If binding is detected by immunoprecipitation, the results are This was confirmed using the Western transfer method.

The anti-peptide antibodies generated in this way are then used to identify whether they are AIDS or ARC. The test was conducted to see if it could neutralize the virus causing the virus. This test This can be done in many ways. In one method, a polyamide resin-peptide conjugate The bodies are ground with a motor and pestle to create a suspension of resin in buffered saline. That page The emulsion was adsorbed onto the solid phase of a microtiter plate and then non-specifically Block the heterologous site with 10% normal goat serum. Binding of rabbit antibody to peptide is Biotin-goat antibody against rabbit IgG and avidin-conjugated horseradish periodine Detect using oxidase. Peroxidase activity is activated by 1.2'- Azinodi (3-ethyl-benzthiazoline-sulfonic acid) and H20□ Measure using A resin-bound peptide corresponding to the hepatitis B surface antigen sequence served as a control. Binding of the rabbit anti-peptide was detected using a plate reader at 410 nm. quantified by vector photometry.

In the second method, purified virus and rabbit anti-peptide antibodies were used in infected T helper cells. – incubate with the cell line and then transfer the lysed cells to Western Trans Anti-peptides were tested for the presence of viruses by immunoprecipitation and immunoprecipitation. The neutralizing ability of these antibodies was tested. A third method involves reducing reverse transcriptase (RT) activity. By measuring the rabbit anti-polyamide resin-synthetic peptide complex, The body's neutralizing ability was evaluated. These results were verified by radioimmunoprecipitation.

The most immunogenic synthetic peptides were then used in diagnostic assays for AIDS and ARC. used as a vaccine or as a vaccine.

When used in diagnostic assays, the preferred method is detection of antibodies against the disease-causing agent.

This assay can be performed using, for example, a column of polystyrene beads or a microwell test plate. the viral agent of AIDS or ARC in an inert matrix such as a synthetic peptide or carrier containing an amino acid sequence related to one epitope of – Applying a synthetic peptide conjugated to a protein (i.e. bovine serum albumin) It is done by doing. Alternatively, an inert matrix with several epitaxial A large number of different synthetic peptides containing topic amino acid sequences (°゛cocktail゛°) to clothe Another method is to use a polyamide resin-synthetic peptide complex as a motor. It can also be ground with a pestle and adsorbed onto a solid phase as described above.

A sample of biological fluid taken from a suspected patient is immunocaptured with predetermined antibodies. For this purpose, incubate with a matrix coated with synthetic peptides. Uncaptured The matrix separated from the harvested sample is then measurable, if present. If the body fluids of a given antibody are present in sufficient quantities to bind large numbers of human antibodies, anti-human antibodies would be the antibodies of interest). 1es) and then incubated with a biotinylated antibody directed against The product matrix separated from the uncaptured biotinylated antibody and matrix was If present, enough labeled avidity binds a measurable number of antibodies. (preferably avidin labeled with an enzyme such as alkaline phosphatase) Incubate with. The product matrix is separated from uncaptured avidin and The enzyme is preferably detected by adding a substrate specific for the enzyme and/or quantifies and thereby indirectly determines the presence of antibodies against the AIDS virus in a sample. Measure to. Antibodies can also be directly enzyme-labeled, in which case the matrix The substrate is incubated with an enzyme-reactive substrate, followed by a change in the substrate (e.g., color change). change or occurrence of fluorescence). If the label is an antibody, enzyme, or biotin-avi antigens and antibodies or enzymes, whether or not the enzymes are labeled with enzymes. The binding pair formed by the substrate is the “ligand” and “antiligand” of the specific binding pair. ” will be called.

Diagnostic assays are also performed for the detection of antigens rather than specific antibodies. antigen test In order to carry out this test, the solid phase matrix must be coated with anti-AIDS viral agents. antibodies, i.e. synthetic peptides such as the peptides of the present invention or polyamide resins. - produced by immunization with a synthetic peptide conjugate (preferably several peptides) Apply the applied antibody. Next, patients suspected of having been infected with the AIDS virus. A sample of biological fluid taken from a sample is added to the matrix, followed by biotinylated antibody. (This antibody is an antibody that binds to the AIDS virus produced as described above. ) is added. Then add an avidin-labeled enzyme and add a substrate specific to that enzyme. , detecting a change in color or the occurrence of fluorescence. These assays can also be used as inhibition assays. In that case, the bound antigen is labeled with biotin for A, IDS virus. Instead of adding antibodies, biotin-synthetic peptides or biotin-polyamide trees can be added. Add lipid-synthetic peptide complex.

The polyamide resin-synthetic peptide complex of the present invention is used as the virus-causing substance of AIDS According to the teachings of the present invention, polyamide resin- About 100 to 1 synthetic peptide or synthetic peptides made as a synthetic peptide complex 000 μg was administered to individuals together with adjuvant. Also used for synthesis Synthetic peptides that are conjugated to carriers after separation of the peptides from the resin are also administered. did. A suitable carrier contains the toxoid component and is compatible with the animal to be injected. Any one of several naturally occurring large protein-containing 'PfJ substances' has adjuvant activity. Any of several small peptide preparations that exhibit or liposomes. Toxoid component is tetanus toxoid or diphtheria It can be an atoxoid. “Large proteins foreign to the animal to be injected.” The expression ``keyhole limbet'' refers to (pet) hemocyanin (KLH) or substances such as BSA. Aju small peptide preparations with proven bunt activity and also act as carriers. are muramyl dipeptides, murabtidine, and polyamino acids (e.g. poly-L- glutamic acid or poly-L-lysine). Synthesis of about 10-100 molecules The peptide is chronic-maleimidobenzyl-N-hydroxysuccinimide ester ( MBS) [Liu, F, T, > et al., 18 B 1oche@1s try 690 (1979); Green (Green, N,) et al. , 28 Ce1l 477 (1982)], glutaraldehyde, carbo diimide, succinic anhydride or N-succinimidyl-3-[2-pyridyldith] Using a heterotrifunctional crosslinker such as Ocy-10 bionate, one molecule of carrier can be Connected to the rear.

Suitable adjuvants include alum (aluminum hydroxide) and those known to those skilled in the art. Contains other adjuvants. Polyamide resin-peptide complex and carrier Both rear-synthetic peptide complexes were prepared in distilled water, phosphate buffered saline, and citrate. Pharmaceutical solutions such as buffer solutions or neutral pHH solutions (i.e. buffers of about pH 6-8) Administered with an acceptable diluent.

The polyamide resin-synthetic peptide complex of the present invention also Used to screen putative vaccine candidates against RC Ru. Such screening involves the use of polyamides such as those described above in an inert matrix. This is best achieved by applying ground beads of resin-synthetic peptide complex. administered. After that, the vaccine candidate substance was 18G-rabbit anti-peptide-biotin anti- Antibodies against peptides, such as a 1:1000 dilution of the body, do not contain biotin. Incubate with When using biotinylated antibodies, Add biotin-enzyme complex (if biotin is not used, add biotin-labeled goat anti-wine). Add biotin-labeled anti-species antibody such as heron IgG, then add avidin-enzyme. ), then add the substrate and detect the reaction.

The polyamide resin-synthetic peptide complex of the present invention can also be used for AIDS or A.R.C. Used to serotype virus isolates from patients. By serotype Classification is performed in the same manner as described above for screening vaccine candidates. administered.

Because in both cases, the anti-peptide antibodies are completely isolated from the AIDS virus causative agent. This is because it must be combined with quality. However, virus single MfM blood In the case of type-based classification, a portion of the isolate is sequentially labeled with a large number of bound anti-peptide antibodies (such as Each antibody is specific for a different polyamide resin-synthetic peptide complex. (added to a separate inert matrix).

The invention will be better understood by reference to the following non-limiting examples. cormorant.

Example 1. HTLV-I[1! 2 types of HT with '43JII holding and shooting markings LV-]11 producing cell lines, H-9 and MOLT-3, 20% fetal bovine serum, Supplemented with 2mM glutamine, non-essential amino acids and 0.1% N a HCO3 It was grown in R　P　M　I-1,640 (maintenance medium). Remove cells from maintenance medium. After 1 hour, 35[S]-cystine (1 50μCi/*Il) and '(H)-glucosamine (20μCi/ml, 2 Cell cultures were labeled by adding >4 hours. Low speed centrifugation of cells (1,000xy, 10 minutes) from the tissue culture supernatant.

Example 2, 120 and 41 subunit exemption of HT L V-1 is one certificate. Friend In 1983-1984, clinics and clinics in high-risk areas for AIDS and ARC Serum samples collected from subjects visiting hospitals in the region were collected by Essex et al. 1-19 / HT L V' according to the description in Science 859 (1983) HTLV-III cells by indirect membrane immunofluorescence (MIF) Antibodies against I[1 were screened. In summary, this method involves the following steps: The cells were separated from the medium described in Example 1 above, with 1 x LO' to 2 x 10' cells. Wash cells twice with phosphate-buffered saline (PBS) and pre-centrifuge them. Place in 40 μk of a 1:4 dilution of the separated serum for 30 minutes at 37°C. Each preparation solution The samples were then washed twice with PBS and washed with a (e) against human immunoglobulin (IgA + I6G + IgM) 1:20 dilution of fluorescein-conjugated F(ab')2 fragment of antiserum 40/iR I reacted. Each sample was again incubated at 37°C for 30 minutes and then incubated with PBS for 2 to 30 minutes. Washed twice and examined by fluorescence microscopy. At least 50% (or 40%, as indicated) The serum sample was determined to be positive if the cells in . Each sample was coded and read in a double-blind manner for positive and negative human serum samples. included as standard. The results of this screening are shown in Table 1. Table ■ Positive for: AIDS 50 48 (96) 49 (98) A R, C5043 (86) 46 (92) Healthy male homosexual 73 34 (47) 36 (49) Healthy laboratory Staff 2700 1) Assay for ITLV-I [[Membrane Antigen (HTLV-III-MA).

According to the MIF described in Essex et al., 220 Science 859 (1983) implementation.

Assay of εp120 envelope glycoprotein of 2HT L V-1.

F IPSDS- as described in Essex et al., 220 Science 859 (1983) to PAGE For all samples obtained from the same 190 individuals, ff5(S)cystine-labeled H9/HT Radiolabeled immunoprecipitation using LV-I[r and unstained H9 cells. mIIIunoprecipitation) and sodium dodecyl sulfate- We also conducted tests using polyacrylamide gel electrophoresis (RIP/5DS-PAGE). It was. In summary, the method is as follows. The labeled cells were soaked in RIPAi buffer (0 , 15M -NaCIf', 0.05M) Lis-HCL pH 7, 2, 1% Tora Triton X - 100, 1% deoxycholic acid, and 0.1% After disruption with 5DS), the cells were centrifuged at 100,0 OOXy for 1 hour. cell Add protein to the upper layer of the lysate - Sepharose CL-4B (Protein iA beads) Standard negative control serum bound to 10μ! After clarification once, each part was tested on humans. It was reacted with 10 μl of serum. Use immunoreactive 11Th as sample buffer! c (0,1M Ku Lera 7 straw, 2% SDS, 0.08M 1. Jsu-H (J, pH 6, 8, 10 % glycerin, and 0.2% bromine (7x/- Le Blue) at 100°C. Elution was performed by boiling for 2 minutes. 12.5% solution containing 3.5% deposited gel Samples were analyzed in a resolving acrylamide gel (Laemmle, 227 Nature (London)). 68° (1970) discontinuous buffer system), surface labeling was done using Lactopel. It was carried out by an oxidase-catalyzed radioiodination method.

The results are shown in Table ■.

For typical antibody-positive sera, use a lentil lectin column. Glycoprotein preparation of H91HTL V-III cells concentrated using Tests were also conducted at HTLV-Iff glycoprotein as lentil lectin - Incubate with Sepharose 4B for 4 hours, then 0.2M Methylman eluted with Noside.

The resulting protein was then immunoprecipitated with HTLV-III standard serum to Quarantine - Sepharose-bound precipitate was dissolved in 0.1% SDS and 0.15M citric acid. Antibodies are deoxidized by boiling for 2 minutes in the presence of sodium hydroxide (pH 5.5). Equal amounts were incubated at 37°C in the presence of endoglycosidase H0, for 25 μs. or for 3 hours in the absence. Addition of 5 volumes of cold 95% ethanol Stop the reaction and precipitate the protein overnight at -20"C. Centrifuge at 2000Xy for 15 minutes and reconstitute proteins with electrophoresis sample buffer. The mixture was boiled for 3 minutes and subjected to electrophoresis. From samples obtained from four antibody-positive AIDS patients Proteins of approximately 120 kD, 160 kD and 41 kD were precipitated. similar results in two antibody-positive ARC patients and two antibody-positive healthy homosexual men. I got it though. Serum obtained from a healthy male homosexual who is antibody negative or who appears to be healthy. No proteins of relevant size were detected in serum obtained from laboratory personnel. . None of the human serum samples tested showed HTLV-[[other epitopes on the virus]. and contains no antibodies against at least gp120 and gp160. It also contained no easily detectable antibodies.

Table ■ Residue No., 341-304-728-735-846-503-733-55- 650-465-370 752752 Riverside ser thr leu asp ala val gly ala hisu glyarg arB pro argile ala pro thr se rvglyala pro ile pro arg"pro asp thr 　Ieu　asn×Iys”asn　pro　glukhis　thr　arc 　thr　ile”ser’trp　asn　arg　Hly　Ile　Iys pro leu glu asnasn asn gly ile pron ala argqphe glu asn2asn thr pro FIlu argIys gly ser ser glutenhr hrB asp g lu argargile ala gln 5erIeu Iys argg lu ile arg Hlu ser asn glu glu ile gl ukgly gln val glu ala gln phe array i1e a rg gly glu gly gln gly’ Iystglu arga spdile ile arc Ieu arg gly city 1ys pr.

seregln’ glu asp glu glu Hlu tt$f-as n glyleu gly gILI asp arg asp thr gl n5IIFargpro gly argala arg gtu glu aspg! u gly gly ser vat arg’val Ieu m etgln arg glu gly ser his leu argphe ila arg 1leOile asn glu aspgly phe asp gly arg val leu asnasn thr asp 1 eupval ala arglys ile arg phe thr 5e rthr ser leu his giuile gly ala 1eu i1efphe cys phe l eu a　gln　ARV中 h ala LAV c glu ARV medium d val in ARV alae lys in LAV f　vat　ARV g asn in ARV h tyr ARV in progress i None for ARV j His ARV in progress k asp ARV medium + HTLV-I [[There is a possibility of asp m leu ARV in n his ARV o In ARV, there is val between gly and ile pmet in ARV q HTLV-[[, there is a possibility of Hlu r HTLV-[[, ARV and The actual sequence of this peptide in LAV includes asp between arg and arg. s cys HTLV-I t arg ARV in progress u tyr in ARV v thr ARV in progress w leu ARV in progress xL^■ does not have the following three residues; ARV has three asn instead of asn During ARV z asp ARV medium aa Hly l-^■ Medium bb vat ARV Example 3. 12041 and 160 envelope proteins tentative gp16 from three virus isolates HTLV-I[[, LAV and ARV The deduced amino acid sequence of the 0 precursor glycoprotein was ・R Woods (Mol, Im5unol,) 483-489 (1983)) using parameters and hydrophilicity values. This was done using a computer program. The computer program is Apple -Written in RASIC. The program is based on Chow-Fasman's plan. Measurement method (P. Wai Chou and E. D. Fassman, 13 Biochemistry Amino acid sequences are recorded on the disk in a format consistent with It was created so that it could be recorded. The hydrophilicity program extends throughout the length of the hexapeptide. Calculate the average value of the hydrophilic groups, thereby increasing the accuracy of the estimation. Asx or Gl x, that is, the amide form of acidic amino acid residues, has no hydrophilic value, so these codes The three AIDS virus glycoproteins must be removed before doing the calculation. Regarding the quality, the average hydrophilic group per residue is plotted against the number of amino acid sequences. Figure 1 shows the AIDS/ARC virus whose characteristics have been clarified. Computer graphics of hydrophilicity plots obtained for three types of russe-causing substances This is a chart depicting the output. The highest peak (fi is also hydrophilic) has three types of sequences. The highest hydrophilic index is shown in a similar area for all and ARV at residues 739, 744 and 738, respectively. Second The highest hydrophilic region is amino acid residues 653-659 on the amino terminal side of beak 1. Centered nearby. The third highest hydrophilic region is close to beak 1 and contains three ring proteins. Each protein is centered around amino acid residues 733-739.

HTLV-1[[Actual computer program output of one segment of the sequence Shown in Figure 2. HTLV-1[[Because the entire sequence is long, only the - segment is shown. Ta. Proline residues are shown as "P" on the graph. Two consecutive items in this array or three aromatic amino acids are represented as "0". aromatic atom in an array The presence of amino acids indicates that there are regions with a high degree of hydrogen bonding potential. . Hydrogen bonding can affect the overall identity of the protein. These de The data indicate that these regions may be exposed on the surface of the ring protein.

HTLV-11 [Predicted secondary structure of ring protein (Chow-Fasman's predicted structure In this putative secondary structure (determined by ) shown in Figure 3) ITLV-111, The main differences between LAV and A-R,V are shown in the boxed area. these The regions are HTLV-I[1, LAV and ARV<, residues 127-1 with m, respectively. 150, 127-155, and 126-148. Here, the phase of the residue is Only about 40% are homogeneous, which changes the β-rotation potential. Sara There were significant differences in ARV regions 319-330 and 398-408. LA, V's 323-333 and 401-415, and 313 of HTLV-I[1! − 328 and 396-411. Contrasting hydrophilicity with secondary structure Mark 1 was shown to contain four potential β-turns within this region, starting at amino acid 73. The region centered around 9-744 is the first candidate as a potential antigenic determinant. Ru. Hydrophilic peak 2 also contains a putative β-rotation region, and this region is the ring protein of the envelope. Suggests that the quality is exposed on the surface.

Example 4,554 de 1 leakage zuL bottom.

A synthetic hebutide with the amino acid sequence shown in Table ■ under the heading “Peptide 4” (This is due to gp120 glycoprotein, the viral causative agent of AIDS and ARC. corresponding to residue numbers 735-752) by biosearch sum n (B1ose Synthesized using a solid phase method using a peptide synthesizer (Arch Samll) Bea Merrifield, 327 Doba, Enzymol, (Adv, Enzymol , >221 (1969)). dicyclohexylcarbodiimide in a catalytic amount of 4-N , butylene bound to polystyrene using N-dimethylaminopyridine. Oxycarbonyl-8-4-methylbenzyl-cystine was used as a solid support for the synthesis. It was used as The four amino groups are replaced by t-butyloxycarbonyl (t-BOC). The side chain protecting group was as follows. Regarding the hydroxyl group of serine, ben Dichlorobenzyl ether and dichlorobenzyl ether for the phenolic hydroxyl group of tyrosine. for the carboxyl groups of tel, glutamic acid and aspartic acid, respectively. γ- and β-benzyl esters were used.

The t-BOC was removed using trifluoroacetic acid (40% in CHZCjh) and the obtained The resulting salt was neutralized with N,N-diisopropylethylamine (10% in CH2C12). did. t-BOC amino acids were attached using diisopropylcarbodiimide . The individual steps of this synthesis were described by J.T. Sbarrow, 41 J. Olga, Ge. Mi, (J, Org.

Chem, ) 1350 (1976), the entirety of which is hereby incorporated by reference. and quote.

The protecting groups were removed and the solution containing 10% anisole and 1% ethanedithiol was prepared at 0°C. The peptide was dissociated from the resin using anhydrous hydrogen fluoride as a scavenger. . The hydrogen fluoride reagent was removed under vacuum at 0°C, then the peptide was precipitated and washed with anhydrous ether. Washed with water. After extracting the peptides from the resin using trifluoroacetic acid , the solvent was evaporated at 215° C. and the peptide was again precipitated with ether. centrifuge Afterwards, the ether was decanted and dissolved in 5% acetic acid containing 6M guanidine HC. The mixture was dissolved.

The above solution was applied to a Biogel (B1oGel) P2 column equilibrated in 5% acetic acid. The peptide-containing fractions were desalted and the peptide-containing fractions were pooled and lyophilized. Adding a cysteine residue to the boxyl terminus converts the peptide into a carrier protein. A functional -3H group was provided for attachment. Adding a glycine residue after cysteine In addition, between the above bonded cysteine residue and the amino acid sequence corresponding to gp160 Do not apply spacer amino acid to. Tyrosine residue for radiolabeling with +2SJ was added to the amino terminus to measure the peptide-carrier protein binding efficiency, The peptide being purified was also confirmed by absorbance at 278 nm.

After desalting in acetic acid on a Biogel P2 column and lyophilization, the peptide had the expected amino acid analysis results (see Table ■) and A linear concentration gradient of 0.05% trifluoroacetic acid and 2-propanol was used. It eluted as one peak (92%).

Example 5. Synthesis of Peptide 6 on Polyamide Resin Heading of Peptide 6 in Table ■ A synthetic peptide with the sequence shown as Corresponds to the sequence of residue numbers 503-532 of the causative substance gp120 glycoprotein ) was synthesized on polyamide as follows. Numbers used throughout this specification was adopted from the numbers assigned to the amino acid residues of HTLV-1 [[. This is Elle Ratner et al., “Complete nucleotide sequence of AIDS virus HTLV-■” 313 277-284 (1985), the entire text of which is hereby incorporated by reference. I quote it as a consideration. The sequence of the polyamide-peptide 6 conjugate is as follows. Ta.

H2N-VAL-ALA-PR○-THR-LYS-ALA-LYS-ARG- ARG-VAL-VAL-GLN-ARG-GLU-LYS-ARG-ALA- VAL-GLY-I LE-GLY-ALA-LEU-PHE-LEU-GLY -PHE-LEU-GLY-A, preparation of functional monomer 2-Methylsulfonylethyloxycarbonyl chloride (MSC chloride>(K + Laboz, ICN) 5g (26.8 mmol) to 15 acetonitrile companies Dissolve and redistilled allylamine (Kodak) 2.1zi! (28 mmol) and Redistilled diisopropylethylamine (DIEA)4. ! 3yl (28mm) 2.1ffi! in acetonitrile (in HL) for 20 minutes under stirring. It was added dropwise over a period of time. (DIEA, (Aldrich) refluxed over ninhydrin and redistilled. ) The solution was stirred for a further 2 hours and the solvent was evaporated. Residue in acetic acid The mixture was placed in ethyl 250y1 and left for 1 to 2 hours. Most of DIEA hydrochloride is acicular It precipitated as crystals. After filtration and evaporation, the @ material is treated with a minimum amount of chloroform. A silica gel G-60 column (60y> loaded into. Elution with chloroform gave pure MSC-allylamine (T RF-164 on LC (solvent = CHCl: l: CHz OH19: 1 　)).

The remaining DIEA salt was adsorbed onto the column under this condition. Occasionally TLC smell The substance that migrated to the vicinity of the front end of the solvent was mixed into the MSC-allylamine column fraction.

This material is MSC-allylamine crystallized from methylene chloride-hexane at -20°C. It was removed by changing the Yield: 4.8 g (86%, from MSC chloride) there were.

B. Preparation of crosslinking agent The crosslinking agent N,N'-bisacrylyl-1,3-diaminopropane is It was prepared according to the method described by John and Sparrow (supra). summarized below . Dissolve diaminopropane (Aldrich) in acetonitrile and add acrylic chloride. The mixture was added dropwise to the leu-aceI nitrile solution at 4°C, heated to room temperature, and stirred. Ji Separate the aminopropane dihydrochloride, wash with warm acetonitrile, and combine with P solution. Concentrated in vacuo. N,N'-bisacrylyl-1,3-diaminopropane is 4 C., overnight, the platelets obtained were filtered and dried in vacuo.

C. Preparation of polyamide resin Glass 21 volume cylindrical, trayed with nitrogen inlet tube and mechanically driven glass stirrer 490 ml of hexane and 290 zl of carbon tetrachloride were added to a polymerization vessel equipped with a reactor. melt The solution was purged with nitrogen for 15 minutes to remove oxygen. Example 5. According to the description of B. N,N'-bisacrylyl-1,3-diaminopropane (2,9, , 15,9 mmol) and N,N-dimethylacrylamide. (Polysciences) 18.2 zl (175 mmol). Example 5. A's MSC allylamine 10y (48 mmol) and water 12 prepared as described 0 ml was added and the solution was filtered, defoamed and added to the organic phase. 400-45 While stirring at 0 RPM, the density of the resulting mixture was adjusted to obtain a homogeneous suspension. . Ammonium persulfate (Bio-Rad) (H205d in o, sy>, and sesqui Added sorbitan oleate or sorbitan monolaurate (Sigma) 1111 added.

Next, N, N, N'', N'-tetramethylethylene diamide in H202xl TEMED (Bio-Rad) 3zf solution (pH 6.5-7.5, concentrated HCI ) was added to the suspension. The suspended emulsion was stirred for 2 hours under nitrogen atmosphere. . Next, the obtained bead-like material was filtered, and water (11), methanol (1, Dioxane:methanol=2N NaOH mixture Th<1.4:5:1.21, A iS for removal of C), water (21), IN-HCI<21>, water (2 t') and then washed with methanol (2f>).The resin was suspended in methanol and washed with Adjusted by canting (3 times). Methylene chloride (Baker, HPLC After swelling in dioxins, the resin was shrunk in hexane and dried in vacuo.

By sieving the resin with an 80 mesh (180 micron) sieve, it becomes a large amorphous material. Material removed.

Using diisopropylcarbodiimide as an activator, 4-dimethylaminopyridine ( BOC-alani was added to 100jy of resin using 100jy of resin (recrystallized from ethyl acetate) as a catalyst. The degree of functionalization was investigated by attaching ions. Amino acid analysis is lot-specific. The addition of allylamine Depending on the amount, about 0.1 to about 0.5 mmol/2 (resin) of resin was prepared. The treated resin showed no detectable staining with vicrylsulfonic acid, indicating that unreacted free This indicated the absence of amines. These beads swell in methylene chloride This occupied about 2.5 times their drying bed capacity. Dimethylformamide also When swollen in an aqueous solution, the beads have a dry bed capacity of about 4 and 4, respectively. It accounted for 6 times more.

D. Preparation of linker The linker, BOC-glycyl-4-(oxymethyl)benzoic acid, is a Mitchell It was prepared by a modification of the method of et al. (supra). In summary, 4-(bromomethyl) Cheap! , fragrant phenylacyl ester prepared as follows. redistilled diisopro pyramine 10.3zl and bromoacetophenone 12.059 (60.6ml) (remol) was dissolved in ethyl acetate 450. 4-(bromomethyl)benzikoic acid 13 ．． 89 g (60.6 mmol) was divided into 7 equal parts and heated at 40-50°C for 3 hours. Added to stirred solution. Stirring was continued for an additional 2 hours at room temperature. Precipitated Et Remove the iN HBr solution and add each 5011 10% citric acid, saturated salt to the ethyl acetate solution. 4 times with sodium chloride, saturated sodium bicarbonate and saturated sodium chloride aqueous solution Washed. The organic phase was dried over anhydrous magnesium sulfate and evaporated by rotary evaporation under reduced pressure. The solvent was removed. The residue was purified from CH2Cl2-petroleum ether (1:3 v/v). Crystallization gave 4-(bromomethyl)benzoic acid phenyl acyl ester.

4-(bromomethyl)benzoic acid phenylacyl ester was converted to BOC-glue as follows. It was converted to lysyl-4-(oxymethyl)benzoic acid. BOC-L-glycine (2 5 mmol, 4.38 g) in methanol 1511, tetramethyl hydroxide Ammonium (25% in methanol) was added dropwise until neutral. Solvent in vacuum and the salt was dissolved in 150M1 acetonitrile.

To this solution, under stirring, was added 4-(bromomethyl)benzoic acid phenyl prepared as described above. 5.8 g (17.5 mmol) of syl ester were added. - After mixing at night, The precipitated tetramethylammonium bromide was filtered and the solvent was evaporated. Residue in acetic acid Dissolved in 400 ml of ethyl and filtered the solution. The organic phase was then sequentially treated with 10% quenchant. Aqueous acid solution (3x75z1), 0.5M sodium bicarbonate: 0.5M potassium carbonate (2:1), pH 9.5 (8x 75.vf), then water (3x 75y1) ). The solution was dried (MgSOJ, the solvent was removed in vacuo. The residue was dissolved in 85% acetic acid 200z1 and acid washed with zinc powder 231? add Ta. Stir the mixture until no more phenacyl ester is visible by TLC. Stir (4-5 hours), filter the zinc, wash with 50 d' of acetic acid, and combine the solutions. Lyophilized. The residue was suspended in 100 ml of water: 30 ml of ethyl acetate, and the pH was adjusted to 1. ．． Adjusted to 5 (concentrated HC1) The extracts were combined and washed with water (100ii'), dried ti (MfISO4) and After evaporation and evaporation, BOC-glycyl-4-(oxymethyl)benzoic acid is converted into methyl chloride. Ren: Purified by recrystallization from hexane at -10°.

The yield is 4-51? (14.5 mmol, 83%, from phenacyl ester) It was.

E. Example 5 of linker binding to polyamide resin. BOC prepared as described in D. -Glycyl-4-(oxymethyl)benzoic acid and Example 5. Prepared as described in C. Amine methylpolyamide resin (1, h>), Biosearch Sam ■ Automatic peptide The synthesizer produces dicyclohexylcarbodiimide and dimethylaminopyridi in a 1:1 methylene chloride:dimethylformamide solution using Combined. Methylene chloride (Baker, HPLC grade) and dimethylformamide (for Baker and Photorex) without further purification. used for. After treatment with hydrogen fluoride, 50 ag of glycyl-resin is 0.15 It was found by amino acid analysis that it contained mmol/g. Amino acid analysis is below carried out by any of the following.

(1) Hydrolyze the resin-bound peptide for 2 hours (12N-HC1: propion acid, 1:1.135°C) or after 24 hours of hydrolysis (6N-HCl , 110°C) using a Peckman Model 119 amino acid analyzer. or (2) 2 hours After hydrolysis (12N-HCI: propion fl, 1:1.0.05% (containing phenol, 135°C) using a Beckman Model 7300 Amino Acid Analyzer.

The results of amino acid analysis are shown in Table ■. Peptides 1, 2.7 and 10 are the above resins The corresponding polyamide resin-peptide conjugate was synthesized in the same manner as above to obtain the corresponding polyamide resin-peptide conjugate. .

Example 6-10 Synthesize peptides 3, 5° 8 and 9 listed in Table ■ using the method described in Example 4 above. did. Each of these corresponds to the amino acid sequence in the table.

Example 11. Peptide 4 to Yaria Synthetic peptide 4 (see table ■) Through the -SH group of the tin residue, keyhole limpit (keyhole I) i+apet, limpet) hemocyanin (KLH) (for rabbit immunization) and On the amine group of bovine serum albumin (BSA) (for assaying anti-peptide activity), Heteronifunctional crosslinker M-maleimidobenzyl-N-hydroxysuccinimide Coupling was done using ester (MBS). Details of this method can be found in F.T. et al., 18 Biochemistry 690 (1979) and F. Green et al., 2 8 Cell 477 (1982). Both of these are cited in their entirety and here as a reference. use

Table ■ Residue Before HF treatment After HF treatment Thr, 75(1)”, 85(1)G lu/G In 2.30(2) 2.15 (2) Pro N, D, b (1) 1.07 (1) G ly 4. 85<5) 5.35 (5) A Ia 4.70 (5) 5.19 (5) Val 3.60 (4) 3.59 (4) 11e O, 94 (1) 1.04 (1) L eu 2.70 (3) 3.12 (3) P he 2.00 (2) 2.00 ( 2) L ys 2.73 (3) 4.10 (3) A rg 4.00 (4) 3 ．． 90 (4) μmol/g 71 50 a Values are not corrected for destruction during hydrolysis. Numbers in parentheses are individual represents the theoretical yield of each amino acid for the sequence.

b. Not measured.

It is summarized below. KLH or B in 10mM sodium phosphate (pH 7,2) M B S4xg and 800% SAlzy in dimethylformamide, respectively. ug for 30 minutes at 25°C. 50mM sodium phosphate A Sephadex PD-10 column equilibrated in a buffer solution (pH 6.0) was used to remove the unreacted The corresponding MBS and solvent were removed. 100 molar excess over KLH or BSA Approximately 500,000cp#l of peptide 4 was 125(:　engineering) labeled peptide 4 to the reaction mixture and incubated for an additional 3 hours at 25°C. Ta Peptides that did not bind to the protein carrier were removed by repeated dialysis.

The binding efficiency is determined by the amount of 125[■] peptide associated with KLl-1 and BSA. The rates were determined to be approximately 62% and 56% for KLH and BSA, respectively. Ta.

The sequence of the carrier-peptide 4 conjugate was as follows.

GLY-I LE-GLtJ-GLU-GLU-GLY-GLY-GLU-AR G-ASP-ARG-ASP-H the law of nature! lI OOH Peptides 3, 5.8 and 9 (see Table ■) were also attached to the carrier in a similar manner. there was.

Example 12.䆆兎圀Namimitamayitsu-Kite] U Peptide 4-KLH complex emulsified in incomplete adjuvant (as described above) Preparation) Two domestic rabbits were each immunized using 100μ2/time. 2 weeks for rabbit one intramuscular injection at each interval for a total of three injections), and serum was collected after each immunization. did.

A solid-phase radioimmunoassay method was adopted to titrate the rabbit anti-peptide antiserum. Below Summarized below. Peptide 4 conjugated to BSA (prepared as described in Example 11) 20 Adsorb 0ni+ into the wells of a polyvinyl microtiter plate and incubate at 4°C. Incubated at night. Add 10% normal goat serum (Nats) to remove non-specific portions. After blocking the site, add rabbit anti-peptide antiserum diluted in 10% NGtS. and incubated at 37°C for 2 hours. Antisera were obtained 14 days after each immunization. It was done. Fill microtiter wells with Tween 20 phosphate buffered saline (T-P). Wash with +25[) goat anti-rabbit comma globulin (50μ! 0.0OOcpJ was added. After incubating for 1 hour at 37°C, the wells The cells were washed with T-PBS to remove excess radioactivity and counted in a gamma counter. capacity are all 50μ! The antibebutide titers shown in Table ■ are based on the endpoint titer dilution (immunization expressed as the reciprocal of the maximum dilution of the antiserum that gave a higher ep- than the treated rabbit serum. be done. Endpoint titers are based on 5-fold dilutions and represent the average of triplicate values. .

Before immunization treatment 10 21 Tertiary 31.250 Before immunization treatment 10 Primary 1250 Secondary 6250 22 Tertiary 156.250 The results shown in Table ■ show that two rabbits were detectable after a single injection of peptide-KLH. Indicates that an anti-peptide response occurred (measured by peptide-BSA). Immunization The samples previously obtained from each rabbit did not bind the peptide significantly (titer below 10). . Antibebutide titers increased after each peptide injection and after the third injection. It ranged from 31,250 to 156,250.

The specificity of the antibody response is determined by the fact that the anti-peptide serum binds the control peptide bound to BSA. demonstrated by not getting it. Furthermore, HTLV-@peptide 728-745 ( Peptide 3in completely inhibited the binding of rabbit anti-peptide to peptide-BSA, (100%). Two domestic rabbits also developed high antibody titers against KL) ( Rabbit anti-KLH did not bind peptide-BSA.

M13. Company stop, Lead: Polyamide L person Bev de Δ Two rabbits were each treated with polyamide resin-Bev in complete Freund's adjuvant. Immunization was performed with Tidro conjugate (peptide 6.200 μ9/time). For rabbits every 2 weeks (1 intramuscular injection for a total of 3 injections). Subsequent injections should be administered at one or more blade intervals. and serum was obtained 30 days after the fourth injection. The control anti-peptide antiserum was KLH simian virus 40 tumor antigen peptide (in Example 11, peptide to KLH) (by the method described for the binding of hepatitis B surface antigen resin) or hepatitis B surface antigen resin-conjugated peptide. was produced in rabbits immunized with Tide (prepared according to the method of Example 5).

Example 14. House - HTLV by peptide body - J Tan Setsu Otsu” @Momo -: Bepudo carrier Rabbit antibodies against peptide 4 recognized the natural protein associated with HTLV-I. The ability to recognize was tested as follows. MOLT-3, i.e. HTLV-II[f fi-stained T cell lines were labeled with "[:S:]-cystia" and immunolabeled as described in Example 2 above. The anti-peptide serum was used in the epidemiological precipitation method to obtain radiolabeled HTLV-III. It was investigated whether or not it binds to natural protein H,6. Carrier-peptide 4 conjugate ( Rabbit anti-peptide antibodies obtained from rabbits immunized with (prepared as described in Example 11) , approximately 160,00 as shown by 5DS-PAGE autoradiograph. A single protein of 0 daltons was precipitated. This protein is located before HTLV-1. The precursor substance is envelope glycoprotein gp160. Rabbit blood before immunization When the supernatant was used for immunoprecipitation experiments, HTLV-1 [[for protein No reactivity was demonstrated. The above rabbit anti-peptide is a human anti-peptide obtained from AIDS patients. When serum is used for immunoprecipitation, HIS(s)-cystine labeled MOLT -Failure to recognize 8p120 envelope subunit detected by 3m cells It was. The gp41 envelope subunit is converted to gρ by “CS)-cystine. 120 is not radiolabeled to the same extent and is difficult to detect by immunoprecipitation. Ru.

The difficulty of preparing 8p41 with relatively high levels of specific activity is avoided by It was done. HTLV-II [anger-stained M OI-T-3 cells were stained with 1s(S)-methio Double labeling was achieved by addition of both nin and HIS(S)-cystine. Then this A collection of glycoproteins present in double cystine-methionine labeled cell lysates of The complex was concentrated by affinity chromatography as described in Example 2 above. Ta. As analyzed by the radioimmunoprecipitation experiment described in Example 2 above, When the putido serum reacted with these glycoprotein-enriched fractions, gp160 and Both gp41 and gp41 glycoproteins were observed.

Rabbit anti-peptides were produced using the Western transcription method for HTLV proteins. It was proven that the virus recognized 8p41. This method uses infected H9 cells (biolabeled Laboratories, Suchmond, California) or MOLT-3 Thin A stock solution of cell lysate is used as the HTLV-III protein source. With this assay method 5×lO’ cells were injected with 1% Zwittergen. t) Solubilize for 5 minutes in 3-14 (Calbiohemubering) solution lzf, Centrifuge at 000Xy for 10 minutes. The obtained upper layer solution was added with an equal volume of disruption buffer 1 kM) Lis-HCJ, pH 6,8, glycerin and 0.01% bromophenol Le Bleu) and boil for 3 minutes.

Destroyed cell lysate 8μ! 4-25% acrylamide linear concentration in adjacent rows. 24 hours in a gradient gel (1,5x 17x 14CJF) at 50v/ge. electrophoresis under constant voltage. The electrophoretically separated concentration gradient gel was then separated by 1 At 10"C for 90 minutes at a constant current of aIIIρ, Taubin et al. National, Akade, Sai, (Proc, Natl, A, cad.

Sci., ) 4350 (1979). )・Transfer to. This document is incorporated herein by reference in its entirety.

A pre-colored molecular weight marker (BRL) was also electrophoresed, and nitrocellulose as a standard for estimating the molecular weight of the transcribed HTLV-■ peptide. Using.

After completion of transcription, o, oot%III/v methiolate and 0.0001Xy/ Re-incubate in PBS containing Antifoam A (Sigma). Hydrated 5% w/v skim milk powder1. Place the nitrocellulose sheet together with OOw 1. Incubate for 30 minutes at warm temperature. Serum obtained from rabbits immunized with peptide 4 Serial dilutions were then incubated with nitrocellulose sheets for 1 hour at 37°C. The nitrocellulose sheet was then treated with Tween 20 phosphate buffered saline. Washed with 1.0 Oy1 of solution (T-PBS) To examine the binding of rabbit anti-peptide antibodies, rabbit IgG (5 μg/y/) was added to the It was incubated with the cellulose sheet for 1 hour at 37°C. nitrocellulose The sheets were washed again with T-PBS and then washed with avidin-labeled horseradish. Peroxidase (AC-HRP) 1. Add ug/y1 and incubate at room temperature for 20 minutes. After washing again with T-PBS, peroxidase chromogen bisubstrate solution (PBS) 0.2xg/rt1) O-cyanisdine + 30% H2021μm/w in BS Add l>100zf to the nitrocellulose membrane until a precipitate is observed on the membrane. (10-15 minutes), the peroxidase-catalyzed reaction was carried out on a nitrocellulose sheet. was stopped by washing in 2% SDS in water. This western transcription a Control assays included the use of normal human serum, as well as antiserum and infected and uninfected samples. A parallel comparison of reactivity with cell lysates is included. Binding with gp41 protein and binding to gp120 subunit was observed.

Example 15. Viral 7, Polyamide-mu pepti by house r development of human antibodies against the viral causative agent of AIDS and ARC Enzyme-linked immunosorbent assay (ELISA) was employed for detection.

The polyamide resin-bebutydro conjugate prepared as described in Example 5 was prepared in a mortar and pestle. Crushed and crushed conjugate in borate buffered saline (BS) (pH 8,0) A suspension was prepared. Approximately 10 μg of peptide 6 (weight calculated based on amino acid composition) 100 μp of this emulsion containing (Dynatech I mmunolon) II Microtiter Play The solid phase was absorbed in BBS (pH 8,0) at 4° C. for 8 hours. Non The specificity site was isolated to 10% normal in Tween 20 phosphate buffered saline (T-PBS). Blocked with goat serum (NGtS) and then washed with 'IPBs.

Rabbit serum diluted in 10% NGtS was then added to the peptide 6-coated plate. After incubating for 1 hour at 37°C, the cells were washed with T-PBS. Next, domestic rabbit Ig from Vector Laboratories (Burlingame, CA) Biotin-goat antibody against G was incubated with rabbit serum for 1 hour at 37°C. did. Each well was then washed and bound to horseradish peroxidase. Avidin (Av-HRP) was added and incubated at room temperature for 20 minutes. Then each well was washed with T-PBS to remove unbound Av-HRP, and 1,2′-azino Di(3-ethyl-benzothiazoline-sulfonic acid) (Sigma Chemical Company) 1 Peroxidase activity using mM solution and 0.03% H2O2 (as substrate) The reaction in which the properties were measured was stopped with 5% (w/v) sodium dodecyl sulfate. spectrophotometrically at 410 nm using a dynamic plate reader. was quantified. The optimal dilution of each reagent was selected by titration. Specific, except for the substrate All reagents for measuring binding were diluted in 10% NGtS as described in Example 13. Resin-bound hepatitis 8 surface antigen was used as a control. The results are shown in Figure 4. here The results for the polyamide resin-bebutydro conjugate are shown in graph A, and Results for the amide resin-Hepatitis B peptide conjugate are shown in Graph B. domestic rabbit Anti-peptide antiserum was obtained from rabbit no. 1 [·) and rabbit no. 2 (○). trial All experiments were performed using the triple test method, and brackets indicate numerical ranges.

Example 16. AIDS and A by antibodies against polyamide resin-bebutydro conjugate RC virus original Japanese A, HTLV-I [1 replication Rabbit anti-polyamide resin-bebutydro antiserum inhibits HTLV-II staining in vitro. Before examining the ability to neutralize sex, we investigated HTLV-1 replication in the human T cell system. I needed to check the speed of Various dilutions of HTLV-11 virus stock solution were tested successfully. In addition to human serum and immunized rabbit serum, anti-peptide reagents were used to remove viruses. The cells were incubated in the same manner as used to examine the sum. 1 hour with serum After incubation, T. Fawkes et al. (1985, Brosi, National , Akade, Sai, U.S.A. (Proe.

Nat'l Acad, Sci, USA) 4539-4543 (1985)) HTLV-I in susceptible A3.01 cell cultures maintained according to the method described in II virus dilution (the above document is hereby incorporated by reference in its entirety). . Cultures infected with HTLV-Itl were maintained for 15 days, and the supernatant fluid from the cultures was Harvested on 5.8.10.12 and 15th day 1.

HT L V-I [1 replication is due to the presence of reverse transcriptase (RT) activity in the culture supernatant It was evaluated by

Methods for measuring reverse transcriptase activity have been described in detail elsewhere (F. Valesinosi et al. 220 Science 868-871 (1983). It is summarized below. A3.01 15μp of each supernatant obtained from the 5-stained culture was added to virus dilution buffer (0.05M). Squirrel-HCJ, p) (7,8,0,1M-NaCl1,0,15wg/w1 di Add 50 μe of thiothreitol (DTT>, 0.1% Triton X-100) The cells were added to a microtiter plate with 96 wells. 50μ! reaction mixture ( 1M Tris-1-I C&, 3M-KCl, 0.1.5MHCj! 2.10% Ryton, PolyA, Oligo DT and 3H-thymidine-triphosphate ('H-T TP) was added and incubated at 37°C for 1 hour. of incubation Hey, the mixture is 50μ! was dropped onto nitrocellulose filter paper. These many papers are sequentially ( 1) 5% trichloracetic acid Fi (TCA) and 5% sodium birophosphate; (2) Each beaker was washed with 5% TCA, and (3) 50% ethanol.

The filter paper was counted with an automatic scintillation counter and the cp- of 3H-TTP was measured. .

To prepare various dilutions of HTLV-III virus stock solution, normal human serum 1 Seeds and four types of immunized rabbit serum were heat-inactivated at 56°C for 1 hour, and 0.2μ crane It was sterilized by filtration using a filter. 1:5 dilution 150JiNt! -HTI-V- I [1 isolated body (N Y -5) 10-', 10-2.

Ink for 1 hour at 37°C with equal volumes of 10-', 10-' and 10-S diluents. It was incubated. The NY-5 isolate was measured in the human T cell line A3.01 to It has an infectious titer of 0-5 units. After incubation, antibody-treated virus mixture The compound was added to 10' A3.01 cells.

This mixture was heated to 37°C using 1μ3/11 POLYBRENE (cal). biohemi) for 2 hours. This arbitrary dye A, 3.01 cells Wash and resuspend in RP M1 medium 1112 containing 10% heat-inactivated calf blood. and dispersed in a microtiter plate with 24 wells. Top layer of spent medium 500 μe of the solution was taken out on 5.8.10.12 and 15th day after 8 staining, and reverse transcriptase was added. The cells were frozen at -135°C until activity was determined. After removing the supernatant liquid, add R to each culture. Supplemented with PMI + fetal bovine serum 500p1. Each culture was performed in duplicate Ta. RT activity was measured by counts/min of 3H-T''P incorporated.

Human AIDS serum that tested positive by ELISA and Western blot methods The pool is available from Dr. Thomas Foulkes at the Immunomodulatory Research Institute. , NIAID, Bethesda, Maryland). Human serum and rabbit anti-peptide anti-blood The supernatant was processed as described above. In each case, the immunized serum of the rabbit was T L V-■ Infectivity (I' (T activity (c It was used as a negative control, showing no neutralization of the phthalate (determined by pm). Immunized tube blood 10-1.10 of the NY-5 isolate as described above. -2. Incubated with 10-co, 10-' and 10-5 dilutions.

Each culture was performed in duplicate. Infection A, 5.8.10 from 3.01 cells. On days 12 and 15, the supernatant was removed, frozen at -135°C, and assayed for RT activity. I said yes. The inhibition rate (%) of RT activity was determined by the following formula.

cpm, RT assay of anti-peptide antiserum cultures 1---X100 eplm, 200 to 200 before calculating the RT assay inhibition rate of immunotreated tube serum cultures. A background count of 750 cpm was subtracted from each measurement.

Four dilutions of HTLV-II [A, 10-'; BJO-2; C, 10-'; D.

1O-4) reaction rate (8 dye t & 5, 8.10.12 o and R' on the 15th day (based on F activity) is shown in FIG. Each point on the curve is 5 times performed using the double experimental method. represents the average cpm of 3H-TTP uptake based on different measurements of . A bar indicating the range - means standard error of the mean.

Significant replication of HTL V-III diluted to 10-I and 10″2 Dye f! It didn't happen on the 10th day. By the 12th day, these viruses are actively replicating. By day 15, RT activity decreased, and these HTLV-III dilutions The fluid was shown to have cytolytic activity against sensitive target cells. HTLV-I Replication of the 10-3 dilution was observed on days 12 and 15, whereas the 10-3 dilution of the virus 4 dilutions showed little or no replication even by day 15.200 5 and 8 days for all virus dilutions with 12 measurements showing below epm. Day 10 for 10-3 and 10-' dilutions, and 10-4 dilutions 11 of 12 and 15 days) are greater than the mean value ep1m or Based on the fact that we have shown the standard deviation and standard error of the mean equal to 3H-incorporation readings of 2000 cpm or more were measured using HTLV-I [1 as an indication for replication. I chose.

The standard deviation and standard error of the mean are equal to the mean cp transfer in those dilutions. The finding that individual eplm values are highly variable and that low RT activity is due to specific neutralization or simply random variation of the RT assay. This indicated that it was difficult to determine which of the following actions was attributable to the problem. overwhelmingly Antibodies cannot effectively neutralize the virus when large numbers of infectious pilions are present there is a possibility. Based on kinetic studies and properties of HTLV-1 [1 virus stock] Neutralization of infectivity of HTLV-I by human and rabbit anti-peptide antisera are to, tz and for 10-1 and 10-2 dilutions of HTLV-■. Day 15 was determined to be day 12 and day 15 for the 10-bow dilution.

B. Peptide blood neutralization Polyamide resin obtained from domestic rabbit n011, as shown by the results shown in Table V. - Antisera against peptidoloconjugates are comparable to pooled human sera from AIDS patients. In contrast, H on day 10 in both 10-1 and 10-2 dilutions of virus. Effectively reduced TLV-Ill replication. no, 2 for this peptide Rabbit serum failed to reduce HTLV-I[1 replication and RT assay It was used as a control antiserum throughout. This rabbit received a similar immunogen and was tested. Despite showing a possible anti-peptide reaction, this antiserum contained anti-HTL. No V-■ activity was detected by radiolabeled immunoprecipitation. HTLV-Ill The antiserum neutralized both gp120 and gp160 envelope glycoproteins. Don't detect either one. Rabbit no. 1 antiserum has 12 and 1 antisera compared with human AIDS serum. Lower efficacy in neutralizing HTLV-III was observed on day 15. R The rate of decline in T activity decreased by day 12 for 10-1 and 10-2 dilutions of virus. and decreased from over 90% (on day 10) to 23 and 45%, respectively. Ui J The weaker the response, the greater the decrease in RT activity on the 12th day, and the neutralization of antiserum. The efficacy was shown to be dependent on the amount of virus.

Both rabbit antisera against the polyamide resin-bebutydro conjugate showed 1 on day 10. 0-2 virus dilution was neutralized. However, both serum samples were obtained from human AIDS patients. or a higher concentration of rabbit anti-polyamide resin-bebutydro conjugate. It was not very effective in neutralizing the virus (10-'). Rabbit n03 3 effectively neutralized the 10-3 virus dilution on both days 12 and 15. (more than 95%). For antisera obtained from rabbit no. 4 to 15 days, RT No decrease in activity was observed. This is a comparison of rabbit n013 and rabbit no.4. This may be reflected in the fact that they showed higher antibody titers against HTLV-11. On day 15, all antiserum showed RT activity against high concentration of virus. No decrease was observed. This is due to the presence of 8-infectious virus in the culture and the presence of antiserum. In humans, this indicates that the virus was not effective at inhibiting viral replication at this time. Both antisera obtained from a patient with I.D. and rabbit no. RT activity was suppressed for the 10-3 dilution. Control rabbit antiserum (binds to KLH) Controls such as non-HTLV-■ envelope glycoprotein and hepatitis 8 surface antigen (produced against resin-bound peptide preparations) were tested for in vitro neutralization. At similar dilutions used, no significant inhibition of RT activity was observed (more than 25%). under).

Table ■ human rabbit RT activity reduction rate (10th day) 10-' 98% 96% 0% 0% 0%10-'' 97% 94% 0% 90% 97% 1O-3nd nd nd nd ndRT activity reduction rate (1 2nd day) 10-’ 91% 23% 0% 0% 0% 10-297% 45% 0% 67% 67% 10 bow 100% 70% 0% 100% nd'LRT activity Decrease rate (15th 7th) 10 inquiries 0% 0% 0% 0% 0% 10-20% 0% 0% 0% 0% 90% 24% 0% 95% 0% a If the cpIII number of 3H incorporation in the culture is below 2000 c- ゛...was 'I Example 17. Assay for AIDS or ARC collar: Body detection insoluble support Polyamide resin-synthetic peptide matrix prepared as described in Example 5 above. 5 μg of each conjugate was added to borate buffered saline (BBS) (pH 8.0> coat for 8 hours at 4°C (or coat the matrix for 1 hour at 37°C). coaching is also available). Conjugates were prepared in 10% normal goat serum (NGtS). Block for 20 min and 3 times with Tween 20 phosphate buffered saline (T-PBS). Wash. Contains antibodies against the viral causative agents of AIDS and/or ARC. Add the suspected serum sample and incubate for 1 hour at 37°C. support The body matrix was washed three times with T-PBS and biotin-labeled goat anti-Humanoid 8 ( 1 part 1000 of 5 stomach g/wl in 10% NGtS, Vector Labs, Califor Burlingame, Lunia). Wash the matrix 3 times with T-PBS ,'5H/yl avidin-horseradish oxidase 1:200 0 and incubate for 20 minutes at room temperature. Matrix eT-PBS Wash three times and remove the substrate, i.e. 2,2'-azinodi(3-ethylbenzothiazoli). Add the diammonium salt of sulfonic acid (ABTS) along with 820□. fermentation The elementary reaction was stopped with 10% SDS and the optical density was adjusted to 410 nm as described in Example 15. and read it.

Example 18. Assay for AIDS or ARC collar: 2. Presence of AIDS antigen A polyamide resin-peptide bond prepared as described in Example 5 for the detection of Antibiotics produced by immunizing experimental animals with one or more Coat the solid phase matrix with the body, block the antibody and wash as described above. Ru. Next, organisms suspected of having AIDS or ARC viral causative agents Add biological fluid sample and wash.

Assays can be performed as direct binding assays or as inhibition assays. can. When performing direct binding assays, AIDS and 7 produced as described above A biotin-labeled antibody against ARC or ARC is added and washed. Then Abhiji The labeled enzyme is added as described above, washed, and the substrate is added as described above. anti Stop the reaction and read the optical density.

When performed as an inhibition assay, biotin-labeled antibodies are added to the AIDS virus. Instead, add biotin-labeled peptide and insoluble support matrix. Wash. Next, an avidin-labeled enzyme is added and washed. Add substrate; Stop the reaction and read the optical density. From human or chimpanzee AIDS-containing serum The obtained IgG was subjected to ion exchange chromatography on a Whatman DE-52 anion exchange column. Purify by chromatography. IgG obtained from rabbit anti-peptide was used as protein A- Purify by Sepharose 4B column (Pharmacia). Biotin-N-hydro IgG using roxysuccinamide ester (Boehringer Mannheim) Biotinylate.

Example 19. AIDS and ARC care in experimental animals In order to vaccinate against the viral causative agent, synthetic antibiotics were prepared as described in Example 5 above. [Petide]-9 is synthesized on polyamide resin. Polyamide resin-synthetic peptide bond 10 in alum as an adjuvant, or a mixture of several conjugates. Animals are injected as a bolus of 0-1000 μg of synthetic peptide. intramuscular or subcutaneously on three separate occasions per week until a measurable response to the virus is detected. inject. Other intervals, e.g. 0, 1 and 6 months, for injection of synthetic peptides may be adopted.

Example 20. Screening for AIDS vaccines The synthetic peptides of the present invention have potential Screening for the efficacy of AIDS vaccine candidates in inducing immunogenic responses in test animals. It can also be used for leaning. One type of polyamide resin-synthetic peptide conjugate or coated on an insoluble matrix as described in Example 15 above. do. Next, the vaccine candidate is treated with an antibody (biotin-labeled) against the above peptide. , or without). If biotinylated , add avidin enzyme. If unlabeled, biotin anti-species antibody, For example, add biotin goat anti-rabbit IgG, read the optical density, and determine the vaccine candidate. determine the efficacy of blocking peptide binding.

The above examples are presented for purposes of illustration and not limitation. this Modifications to these methods will be obvious to those skilled in the art and may be incorporated into the invention as set forth in the claims below. We believe that any changes of this kind can be made without departing from the spirit and scope of the It will be done.

Engraving (no discrepancies in content) AA# AA Co-r HJ1 can H-F-ihr-7vp AA POS, + h 8 Hydrophilic Pro v1・ v-1(17,2 LAV 1-ITLV-Hl ”■-2 Jyo@(Contents are missing) A pit = a gully or a rumor-monger. Procedural amendment Date: June 1986 1.Display of the incident PCT/US 87101733 2. Name of the invention Compositions for immunizing against AIDS and A) virus causative agents and Method 3: Person making the correction Relationship to the incident: Patent applicant address Name: Southwest Foundation for Biomedical Research search Phone (270)-6641~6 5. Subject of correction Translation of the written specification and claims 6, contents of amendments Procedural amendment

Claims (14)

[Claims] 1. 8p120 or gp41 coat sugar of HTLV-III, ARV or LAV It has a sequence homologous to a part of the amino acid sequence of a protein and contains a hydrophilic region. AIDS and synthetic peptides consisting of amino acid chains and polyamide resins. and a composition capable of inducing an immune response against a viral causative agent of ARC. 2. 2. The composition of claim 1, wherein the amino acid chain includes a β turn. 3. the polyamide resin is a crosslinked polydimethylacrylamide resin; A composition according to claim 1. 4. Claim: the amino acid chain is attached to the polyamide resin via a linker. The composition according to item 1. 5. Claim 4, wherein the linker is an oxyalkylbenzoic acid derivative. composition. 6. gp120 or gp41 coat sugar of HTLV-III, ARV or LAV An amino acid chain that has a sequence homologous to a part of a protein and contains a hydrophilic region within it. A peptide consisting of AIDS and anti-inflammatory drug therapy, which consists of administering a lyamide resin-peptide complex to laboratory animals. and a method of immunizing laboratory animals against the viral causative agent of ARC. 7. The polyamide resin-peptide complex is combined with a pharmaceutically acceptable diluent. 7. The method of claim 6, wherein the method is administered to the animal. 8. 8. The method of claim 7, wherein the diluent further comprises an adjuvant. 9. Claim 7, wherein the diluent is distilled water or a pH buffer solution. Method. 10. A polyamide resin-synthetic peptide complex is attached to a specific binding pair of ligands. wherein said binding pair has a specific affinity for said ligand. and the synthetic peptide consists of an anti-ligand such as HTLV-III, ARV or A sequence homologous to part of the gp120 or gp41 coat glycoprotein of Tahashi AV Consists of an amino acid chain with; The complex is contacted with serum from an animal, thereby eliminating AIDS or AIDS in the serum. also binds to the complex an antibody against the viral causative agent of ARC; and then contacting the bound antibody with the anti-ligand of said specific binding pair; A method for detecting antibodies against viral causative agents of AIDS and ARC, comprising: . 11. The ligand is an enzyme, and the antiligand is a group specific to the enzyme. 11. The method of claim 10, wherein the method is: 12. The enzyme is horseradish peroxidase and the substrate is peroxidase. 12. The method of claim 11, wherein the hydrogen is hydrogen. 13. The ligand is an antibody, and the anti-ligand is an anti-ligand specific for the antibody. 11. The method according to claim 10, wherein the method is an original. 14. The ligand is an antigen, and the antiligand is an antibody specific for the antigen. 11. The method of claim 10.