KR0168447B1 - T-lymphotropic retrovirus monoclonal antibodies - Google Patents

Abstract

The present invention provides monoclonal antibodies for the detection of HIV-1 and HIV-2 gene products, cell lines producing these antibodies, peptides containing epitopes of the antibodies and the antibodies and peptides for detection of the gene products. It relates to the method used. In particular, the antibody reacts with p24 / p26 pepsid proteins, disclosed nonapeptides containing HIV-1 and HIV-2 conserved epitopes, and three monoclonals capable of detecting HIV-1 and HIV-2 simultaneously. Capture ELISA using a combination consisting of a knonal antibody is disclosed.

Description

[Name of invention]

T-lymphophilic Retrovirus Monoclonal Antibody

FIELD OF THE INVENTION

The present invention provides a monoclonal antibody, a peptide consisting of an epitope of the monoclonal antibody and a T-lymphotropic retrovirus, in particular HIV-1, HIV-2, using the monoclonal antibody and the peptide. And assays for detecting SIV.

Background of the Invention

T-lymphophilic retroviruses include monkey retrovirus SIV and human retrovirus HIV-1 (a presumed vehicle of AIDS) and HIV-2 among lentiviruses. Although HIV-1 and HIV-2 are evolutionarily related, nucleic acid sequencing results indicate that HIV-2 is more closely related to SIV than to HIV-1. Guyader et al. (1987) recognized only 42% of the total genomic sequence identity between the HIV-1 and HIV-2 isolates they compared. Despite HIV-1 related superstructural and biological characteristics, such as in vitro cytopathogenicity and CD4 affinity (Clavel et al., 1986), patients infected with HIV-2 are infected with AIDS, pure neuropathy or asymptomatic infections. (Kuhnel et al., 1988).

The HIV-1 genome and the HIV-2 genome encode LTR's and viral structural proteins. And It has a typical retrovirus structure consisting of sequences and regulatory elements encoding one or more enzymes, including regions, reverse transcriptases and other ORF's. HIV-1 The region encodes a precursor peptide known as p55. p55 is processed to produce a major core protein or capsid protein, also known as p24, among other proteins. For HIV-2, a similarity known as p57 The precursor is larger and the main core protein is known as p26. Of HIV-1 and HIV-2 The amino acid identity between the proteins was found to be only 58%. Even among dissimilar isolates among HIV-1 Protein identity is greater than 90%. These and other data support the hypothesis that, although HIV-1 and HIV-2 are somewhat related, they are remarkably distinguished retrovirus species.

HIV-1 and, in some cases, HIV-2 have a significant impact on the human immune system, which may lead to population selection, quality control in blood banks, diagnostics, and facilitation of understanding of these viruses to identify vaccines and treatments. For this reason, sensitive and rapid diagnostic assays that can be used to detect the presence of HIV are desirable and are in urgent need. For ease and convenience, the assay is preferably based on immunological methods such as ELISA, and because of the reproducibility, specificity and uniformity, the reagents are preferably monoclonal antibodies and defined antigenic peptides. p24 antigenemia is an early sign of HIV infection (Kessler et al., 1987; Wall et al., 1987), and clinical progression of AIDS sequelae is associated with a decrease in anti-p24, and AIDS-infected patients High titer levels have been shown to cause death, such as p24 / p26. Assays capable of detecting the product directly would be very useful. Weiss et al. (1988) identified human serum samples containing antibodies specific for the gp130 of HIV-2 by radioimmunoprecipitation assay and ELISA. These antibodies showed low HIV-1 cross reactivity in the neutralization reactions of the VSV pseudotype neutralization assay and the C8166 conjugate formation assay.

Minasian et al. Have described monoclonal antibodies generated against HIV-2 and identified as R1C7. R1C7, an anti-capsid antibody (p26), reacted with five HIV-1 isolates and seven SIV isolates tested as well as three HIV-2 isolates tested. In immunoprecipitation, R1C7 was bound to 55KD and 26KD HIV-2 protein, 24KD and 55KD HIV-1 protein and 28KD SIV protein.

Nidrig et al. Developed a panel of 29 monoclonal antibodies against HIV-1. One antibody reacted directly with p17 and its precursor p32, while the other reacted with p24, some of which also reacted with p55. The p17 antibody was found to be HIV-1 specific. Twenty of the 28 anti-p24 antibodies reacted with the corresponding capsid protein of HIV-2 (p26) in the immunoprecipitation reaction, and five of them also with the corresponding SIV protein, p28. Nidrig et al. Did not describe antibody titers, the efficacy of antibodies in antigen capture assays or which of the antibodies binds to p26, p55 or both. Some antibodies also appear in HIV-2 samples. Its function was reacted with an unknown 22KD protein.

Many diagnostic kits and assays have been developed for detecting HIV-1 in samples of serum, blood, blood products or other body tissues. These assays use a variety of methods, such as western-blot, enzyme-assisted immunosorbent assay (ELISA) or indirect immunofluorescence assays, and utilize antibodies against fully virus or purified virus antigens. See, for example, US Pat. No. 4,520,113 to Gallo et al .; See Sargadaran et al. (1984) and Robert Gerrough et al. (1982).

Summary of the Invention

The present invention provides monoclonal antibodies for the detection of HIV-1 and HIV-2 gene products as well as SIV gene products, cell lines for producing these antibodies, peptides containing epitopes of the antibodies and the gene products. It relates to an assay using the antibody and peptide for the detection of. In particular, the antibodies react with p24 / p26 capsid proteins. Nodapeptides comprising HIV-1 / HIV-2 covalent epitopes are described, and a capture ELISA method using a combination of three monoclonal antibodies capable of detecting HIV-1 and HIV-2 simultaneously is described. .

[Brief Description of Drawings]

1 is a graph showing the reactivity of the culture supernatant in the capture ELISA method.

The detailed description is given in Table 1.

2 is a photograph of immunoblot nitro cellulose strips measuring the specificity of anti-HIV antibodies.

Figure 3 used protein A-purified antibodies as probes for isolated HIV-2 proteins in immunoprecipitation. Lanes 1 and 2 are for positive control and lane 3 is for negative control.

4 is a picture of several recombinant p24 peptides used to map epitopes.

5 is a diagram of four regions of p24 to which various monoclonal antibodies bind.

6 is bloated And Western photograph showing the reaction of fragments with several monoclonals. Lane 1 in each photo contains the entire virus lysate. Lane 5 in each picture is the p24-plasmid for negative control and lane 6 in each picture is for the other negative control containing non-HIV infectious MOLT lysate.

FIG. 7 is a graph showing the results of ELISA using sequential overlapping nonapeptides as antigens to measure epitopes of 7-D4.

8 is a diagram showing epitopes mapped with sequential overlapping nonapeptides as antigens in ELISA.

9 is the composition of regions containing 7-D4 epitopes.

FIG. 10 is a graph showing the sensitivity of capture ELISA using two anti-P24 antibodies, 6-C10 and 5-B, solids, and HIV-1 strong salted MOLT3 lysate as antigen. The anti-HIV of HRP fused human was the reporter.

Figure 11 is a graph showing the sensitivity of capture ELISA with 6-C10 and 5-B4 in the solid phase with and without 7-D4 to detect p26 of HIV-2.

12 shows dose response curves for HIV-1 and HIV-2 in capture ELISA using 6-C10, 5-B4 and 7-D4.

Figure 13 compares HIV-1 administration response curves between three antibody capture ELISAs and reverse transcriptase assay.

Detailed description of the invention

The present invention relates to monoclonal antibodies and methods for their preparation, immunoassays and oligopeptides. The methods used are known in the art and are briefly described throughout this specification. Appropriate methods for practicing the invention are described in Meth Enzymology 121, (1986) and other references.

[Production of Monoclonal Antibody]

Monoclonal antibodies were produced by conventional methods (Kohler Milstein, 1975). Lysates (50%) of HIV-1 infected MOLT3 cells emulsified in complete Freund's adjuvant were immunized by repeated intraperitoneal injection into BALB / c mouse females. Sensitized splenocytes were fused with P3X63-Ag 8.653 myeloma cells using PEG 1500. Heterologous polynuclear cells were selected and cloned in HAT medium and tested for reactivity to HIV antigens by capture ELISA. IgG fractions of polyclonal human anti-HIV were coated onto wells of microtiter plates. HIV-1 (produced in MOLT3 cells) and culture supernatant were added to the wells simultaneously. Bound mouse antibodies were detected using enzyme-labeled anti-mouse Ig antibodies. Representative data of the screening process are shown in FIG. Sample numbers are as shown in Table 1 below.

[Western blots]

Further Western blot (Towbin et al., 1979) tests were performed on anti-HIV clone candidates. Lysates of HIV-infected MOLT3 cells were separated through 12% acrylamide gels under denaturing conditions. The protein was transferred to nitro cellulose and the individual strips were blocked and reacted with the culture supernatant. Bound antibodies were detected using enzyme-labeled goat anti-mouse Ig antibodies. Antibodies that specifically reacted with p24 were selected (Figure 2). The names of the individual strips are described in Table 2.

Anti-p24 antibodies were tested for cross-reactivity to p26 of HIV-2 by immunoblot method. HIV-2 lysates were isolated, blotted and reacted with anti-p24 antibody. Two antibodies, 7-D4 and 5-D9, reacted strongly with p26 (Figure 3). The names of the strips are described in Table 3.

In a related experiment, 7-D4 recognized a protein of about 27,000 molecular weight in the lysate of SIV.

[Epitope Mapping]

Amino acids containing the p24 epitope of 7-D4 were mapped in the following manner. Area and A portion of was subcloned into the transfection vector. Plasmids containing EcoRI / SstI gag / pol ORF by cleaving viral DNA [cDNA library HIV-1 _RF , clone HAT3 (Starcich et al., 1986)] of λ _HAT bacteriophage with EcoRI and ligation into pBR 322-derived plasmid pMLB1113. (Clone 29) was prepared. Clone 29 was cleaved with SstI to remove exogenous vector sequence and religated to make plasmid gag / pol 1.2. The latter plasmid was sonicated to make blunt ends and ligated with EcoRI linker. The mixture was then cleaved with EcoRI, ligated into λORF8 (Meissner et al., 1987) and packaged. λORF8 expression libraries were generated in E. coli and selected using human anti-HIV polyclonal antibodies and mouse anti-p24 (HIV-1) monoclonal antibodies. Positive reactants were selected, amplified and the expressed peptides were analyzed by western blotting, immunoassay and nucleotide sequencing. Recombinant p24 peptide 8, 126, 107 lessons 141 were expressed in E. coli. Separately, clone 29 is used as a template and oligonucleotides corresponding to the 5 'and 3' ends of the known sequence are used in the polymerase chain reaction. The complete sequence of protein p24 was prepared. The 5 'end contains the EcoRI site and the 3' end contains the BamHI site. The reaction product was digested with EcoRI and BamHI and ligated into PMLB1113. Recombinant p24 protein 24.5 were expressed in E. coli. The characteristics of the recombinant p24 peptide are shown in FIG.

Several recombinant p24 peptides were used as antigens in ELISA and Western blot to determine whether the provided monoclonal antibodies bound the provided peptides. The panel and reactivity pattern of the p24 peptide of either monoclonal antibody indicates the position of the recognized epitope in one of the four regions as shown in Tables 4 and 5 and 6.

7-D4 24.5 and Since only 126 is bound, it can be inferred that the 7-D4 epitope is mapped to region B, which ranges from amino residues 142 to 209.

To further locate the epitope of 7-D4, synthetic sequential overlapping nonapeptides were made for the B region of p24. Each nonapeptide served as a solid phase antigen in a series of ELISAs to determine the maximum binding affinity of the monoclonal antibody. A single peak of reactivity was found for linear domains comprising regions containing amino acids 142-158 (Figure 8) (Figure 7). Comparing the amino acid sequences of p24 of the HIV-1 isolate, p26 of the HIV-2 isolate and p27 of the SIV _MAC , Ser-Pro-Arg-Thr-Leu-Asn-Ala-Trp-Val-Lys within the epitope of p24 Decapeptides consisting of appeared to be common (FIG. 9). The region surrounding the decapeptide can be assumed to be the 7-D4 epitope of p26 in HIV-2 and p27 in SIV _MAC . The importance of constantly defined epitopes is known to those skilled in the art. One of the advantages is that it can produce novel antibodies that can react with such epitopes and similar epitopes. Synthetic peptides are formed along epitope sequences and used as antigens, either unmodified or conjugated to a carrier. For example, the peptide can be conjugated to PPD, tetanus toxoid, KLH or BSA using glutaraldehyde, carbodiimide or N-maleimido benzoylhydrosuccinimide ester. For information on using synthetic peptides as antigens, see Shiva Foundation Symposium 119 (1986) John Wiley and Sons (NY). Antibodies are generated in vivo, such as in mice, goats, or other laboratory animals, or in vitro using methods and material systems (Alameda, Calif.) Similar to Hannah Biologics' IVIS. Another advantage is that large amounts of epitope sequences can be prepared synthetically or using standard recombinant DNA techniques as described above, wherein the peptides are screened for circulating antibodies or circulating antigens in immunology-based assays and inhibitory assays. It can act as an antigen in a kit for. Another advantage relates to improving the assays as described herein. Without further investigation, whether epitopes identified in the HIV-1 isolates described herein are specific for the isolates and are further specific for the HIV-2 and SIV isolates described herein. It is unclear whether or not. As a reference, the sequences can be used to manipulate epitopes, i.e. to identify relevant sequences with high conservability among a wide variety of HIV isolates, or to obtain relevant sequences with higher reactivity. It can be manipulated by replacing more amino acids or by derivatizing epitopes. Although the nonapeptide assay is shared in HIV-2 and SIV, HIV-1 Individual linear epitopes of amino acids 142 to 158 have been disclosed, but the present invention is directed to monoclonal antibodies, epitopes of said monoclonal antibodies and HIV-1, HIV-2, and The present invention relates to an assay using the antibody and peptide capable of detecting an encoded protein.

[Capture ELISA] method

Each was used as a capture or HRP-conjugate antibody in a sandwich assay to select monoclonals that are useful for ELISA. In summary, the monoclonal antibody was coated onto the wells and 10 μl of rupture buffer was added. 100 μl of the antigen sample or control suspended in surfactant buffer was added and incubated at 37 ° C. for 90 minutes. After washing, enzyme conjugated anti-HIV reagent (horseradish peroxydase-conjugated-human anti-HIV IgG, affinity purified, 100 μl) was added to the wells and incubated at 37 ° C. for 30 minutes to detect bound antigen. did. After washing several times, 100 μl of substrate solution was added to the wells and incubated at room temperature for 30 minutes. 100 μl of the terminating reagent was added and the absorbance was measured at 450 nm using an air blank. Representative data are shown in Table 5.

Antibodies 5-B4, 6-C10 and 7-E10 performed well as both captured and conjugated antibodies. Maximum signal was obtained using HRP-human anti-HIV as conjugate. Various combinations of monoclonals were used as capture antibodies in ELISA. The combination of 5-B4 and 6-C10 showed the highest sensitivity for detecting p24 (Figure 10). 7-D4 was used as a capture antibody to detect p26 of HIV-2 (FIG. 11). Maximum sensitivity and intensity were found to occur when three antibodies 5-B4, 6-C10 and 7-D4 were combined as capture antibodies. Under these conditions, not only p24 but also p26 can be detected from some boundary clinical samples that were difficult to interpret when only 5-B4 and 6-C10 were used as capture antibodies. The sensitivity of the capture ELISA using these three antibodies is 10 pg / ml or less (1 pg / well or less) for HIV-1 p24 antigen and 0.5 ng / ml or less (HIV) for HIV-2 p26 antigen. Sensitivity is found despite the presence of antibodies in the clinical sample. Capture ELISA with three antibodies 5-B4, 6-C10 and 7-D4 was also compared to reverse transcriptase assay for detection of complete viruses. ELISA was 25000 times more sensitive than reverse transcriptase assay (Figure 13). The present invention has been described in the present patent application with reference to the detailed description of the preferred embodiments of the present invention, and the above description is intended to explain the present invention rather than limit the present invention, Modifications are easy without departing from the object of the invention and the appended claims.

references

Claims (33)

And a epitope of p24 of HIV-1 and an epitope of p26 of HIV-2, wherein said epitope is located within amino acid residues 140-160 of p24. The monoclonal antibody of claim 1, wherein the epitope is located within amino acid residues 142 to 158 of p24. The monoclonal antibody of claim 1, wherein the epitope is located within amino acid residues 144-158 of p24. The monoclonal antibody of claim 1, wherein the epitope comprises the amino acid sequence Ser-Pro-Arg-Thr-Leu-Asn-Ala-Trp-Val-Lys. The epitope of claim 1, wherein the epitope comprises the amino acid sequence Hi-XXX-Ser-Pro-Arg-Thr-Leu-Asn-Ala-Trp-Val-Lys-X, wherein X is an amino acid having a certain biological function Monoclonal antibodies. Reacts with an antigen comprising the amino acid sequence of His-XXX-Ser-Pro-Arg-Thr-Leu-Asn-Ala-Trp-Val-Lys-X, where X is a monoclonal, amino acid with constant biological function Antibodies. A monoclonal antibody that reacts with an antigen comprising the amino acid sequence Ser-Pro-Arg-Thr-Leu-Asn-Ala-Trp-Val-Lys. The monoclonal of claim 1, comprising the amino acid sequence His-XXX-Ser-Pro-Arg-Thr-Leu-Asn-Ala-Trp-Val-Lys-X, wherein X is an amino acid having a certain biological function. Epitopes that react with antibodies, An epitope comprising the amino acid sequence Ser-Pra-Arg-Thr-Leu-Asn-Ala-Trp-Val-Lys that reacts with the monoclonal antibody of claim 1. An amino acid sequence wherein X is His-X-X-X-Ser-Pro-Arg-Thr-Leu-Asn-Ala-Trp-Val-Lys-X with constant biological function. The amino acid sequence is Ser-Pro-Arg-Thr-Leu-Asn-Ala-Trp-Val-Lys. A diagnostic kit for the detection of HIV-1 and HIV-2 comprising at least one antibody reacting with an antigen of HIV-1 and the monoclonal antibody of claim 1. The diagnostic kit of claim 12, wherein the antibody is a motoclonal antibody. The diagnostic kit of claim 13, wherein the epitope of the antibody comprises the amino acid sequence Ser-Pro-Arg-Thr-Leu-Asn-Ala-Trp-Val-Lys. The diagnostic kit of claim 14, wherein the diagnostic kit contains two monoclonal antibodies that react with the antigen of HIV-1. The monoclonal antibody of claim 15, wherein one of said monoclonal antibodies that react with an antigen of HIV-1 binds to an epitope located within amino acid residues 142 to 209 of p24 and wherein said monoclonal antibody reacts with an antigen of HIV-1. A diagnostic kit, wherein the second antibody binds an epitope located within amino acid residues 263 to 344 of p24. A diagnostic kit for the detection of HIV-1 and HIV-2, comprising at least one antibody that reacts with an antigen of HIV-1 and the monoclonal antibody of claim 6, A diagnostic kit for detection of HIV-1 and HIV-2 comprising at least one antibody reacting with an antigen of HIV-1 and the monoclonal antibody of claim 7. HIV-1 and HIV-HIV-2 antigens in a sample, comprising contacting the sample with at least one antibody and the monoclonal antibody of claim 1 and measuring the formation of the antigen-antibody complexes with the antigen of HIV-1. Detection method. The method of claim 19, wherein the antibody is a monoclonal antibody. The method of claim 20, wherein the epitope of the antibody comprises the amino acid sequence of Ser-Pro-Arg-Thr-Leu-Asn-Ala-Trp-Val-Lys. The method of claim 21, which contains two monoclonal antibodies that react with the antigen of HIV-1. The monoclonal antibody of claim 22, wherein one of said monoclonal antibodies that react with an antigen of HIV-1 reacts with an epitope located within amino acid residues 142-209 of p24 and wherein said monoclonal antibody reacts with an antigen of HIV-1. Of which the second antibody reacts with an epitope located within amino acid residues 263 to 344 of p24. Detection of HIV-1 and HIV-2 antigens in a sample, comprising contacting the sample with at least one antibody and the monoclonal antibody of claim 6 and measuring the formation of an antigen-antibody complex Way. Detection of HIV-1 and HIV-2 antigens in a sample, comprising contacting the sample with at least one antibody and the monoclonal antibody of claim 7 and measuring the formation of an antigen-antibody complex Way. A method for detecting HIV-1 and HIV-2 antibodies in a sample comprising contacting the epitope of claim 8 with a sample and measuring the formation of an antigen-antibody complex. A method for detecting HIV-1 and HIV-2 antibodies in a sample comprising contacting the epitope of claim 9 with a sample and measuring the formation of an antigen-antibody complex. A method for detecting HIV-1 and HIV-2 antibodies in a sample comprising contacting the sample with the amino acid sequence of claim 10 and measuring the formation of an antigen-antibody complex. A method for detecting HIV-1 and HIV-2 antibodies in a sample, comprising contacting the sample with the amino acid sequence of claim 11 and measuring the formation of the senate-antibody complex. A diagnostic kit for the detection of HIV-1 and HIV-2 antibodies in a sample comprising the epitope of claim 8. A diagnostic kit for detecting HIV-1 and HIV-2 antibodies in a sample comprising the epitope of claim 9. A diagnostic kit for detecting HIV-1 and HIV-2 antibodies in a sample comprising the amino acid sequence of claim 10. A diagnostic kit for the detection of HIV-1 and HIV-2 antibodies in a sample comprising the amino acid sequence of claim 11.