KR100242596B1 - Human thymic cell leukemia virus-I and II peptide antigens and preparation method thereof - Google Patents

Abstract

The present invention relates to novel HTLV-I and HTLV-II peptide antigens for use in diagnostic assays for screening and identifying HTLV-I and HTLV-II antiserum. These peptides are derived from the same region as the HTLV-I and HTLV-II gp46 membrane proteins and are characterized by immunoreactivity with HTLV-II specific monoclonal antibodies and antigen reactivity with HTLV-I and HTLV-II specific antibodies. Can be distinguished.

Description

[Name of invention]

Human thymic cell leukemia virus-I and II peptide antigens and preparation method thereof

Detailed description of the invention

This application claims the benefit of U.S. Patent No. 653091, entitled "HTLV-I Peptide Antigens and Methods," which is U.S. Patent No. 653,091, entitled "HTLV-I Peptide Antigens and Methods." US Patent Application No. 366,313, filed June 13, 1989, filed December 31, 1986, under the name “HTLV-I peptide antigen and method” of the invention. US Patent Application No. 948,270, which is now abandoned.

[Field of Invention]

The present invention relates to HTLV-I specific antigens and methods of making and using the antigens.

[references]

Cwirla, S.E., et al., Proc Nat Acad. Sci, USA, 87: 6378 (1990).

Huynh, T. V., et al., In “DNA Cloning, Volume 1,” ed. D.M.Glover, Washington, D.C. IRL Press, 1985 (Chapter 2).

Laemmli, U.K., Nature, 227: 680 (1970).

Lipka, J. J., et al., J Infect Disk, 162: 353 (1990).

Lipka, J. J., et al., Proceedings of the 43 Meeting (1990).

Maniatis, T., et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (1982).

Matsushita, S., et al., Proc Natl Acad Sci (USA), 83: 2672 (1986).

Miyoshi, I. et al., Nature, 294: 770 (1981).

Poiesz, B.J., et al., Proc Natl Acad Sci (USA), 77: 7415 (1980).

Popovic, M., et al., Science, 29: 856 (1983).

Samuel, K. P., et al., Science, 226: 1094 (1984).

Samuel, K.P., Gene Anal Tech, 2: 60 (1985)

Scott, J. T., et al., Science, 249: 386 (1990).

Seiki, M., et al., Proc Natl Acad Sci (USA), 80: 3618 (1983).

Shimotokno, K., et al., Proc Natl Acad Sci, USA, 82: 3101 (1985).

[Background of invention]

Human T-cell leukemia virus (HTLV) represents the T-cell retroviral family, which consists of three known members. HTLV type I (HTLV-I) exhibits transgenic activity in vitro and is etiologically related to adult T-cell leukemia, which is known to be endemic worldwide. HTLV-II is another retrovirus that has the ability to transform in vitro and is isolated from patients with T-cell strains of chorionic leukemia.

HTLV-III, also known as lymphadenopathy-associated virus and now known as human immunodeficiency virus (HIV), is soluble in certain types of T-cells and is involved in the pathogenesis of acquired immunodeficiency syndrome (AIDS). Unlike HTLV-I or HTLV-II viruses, HTLV-III is not known to have in vitro transformation activity.

Diagnosis of HTLV-I infection is usually based on serum antibody responses to HTLV-I peptide antigens. Such diagnostic methods usually include an initial screening assay to identify HTLV-I antibodies based on enzymatic immunoassay methods (EIA) using HTLV-I virion peptides. This assay used for blood screening detects HTLV-I and HTLV-II positive of 0.5-0.05%, of which about 4/5 are false positive. Therefore, positive sera should be further tested by Western blot methods or detection assays using radioimmunoprecipitation assays to detect antibody responses to specific HTLV-I peptide antigens.

Current blood test methods require an identity test based on an immune response between HTLV-I p24 gag protein and one or more of envelope proteins gp46, gp21 or gp68. When test antigens are prepared from virion proteins, only gp46 alone provides a high rate of antibody response to true HTLV-I seropositives. The reaction with gp46 can then be detected by further testing the antigen using a more sensitive radioimmunoprecipitation assay. The above screening and confirmatory test methods confirm positive HTLV-I and HTLV-II, but cannot distinguish between the two HTLV viruses.

Therefore, it is desirable to provide an improved method for detecting HTLV-I positive serum. Specifically, the improved test method should be able to detect all HTLV-I and HTLV-II positive sera with a minimum number of false positives and be able to distinguish HTLV-I positive sera from HTLV-II positive sera. do.

[Summary of invention]

It is therefore an object of the present invention to provide an improved method and kit for detecting HTLV-I and HTLV-II positive human serum.

Another object of the present invention is to provide a method and kit that can distinguish between HTLV-I and HTLV-II positive sera.

The patent application for “HTLV-I Peptide Antigen and Assay” discloses an HTLV-I gp 46 envelope protein region that is immunoreactive to .5α monoclonal antibody (Mab) prepared by ATCC cell line HB 8755 (Matsushita). An HTLV-I peptide consisting of is disclosed. This region is contained in the 42 amino acid overlapping region of three gp46 peptide antigens represented by MTA-1, MTA-4 and MTA-5. The 42 amino acid sequence overlapping region includes the sequence Ser-Leu-Leu-Val-Asp-Ala-Pro-Gly-Tyr-Asp-Pro-Ile-Trp-Phe-Leu-Asn-Thr-Glu-Pro-Ser-Gln-Leu -Pro-Pro-Thr-Ala-Pro-Pro-Leu-Leu-Pro-His-Ser-Asn-Leu-Asp-His-Ile-Leu-Glu-Pro-Ser containing 42 amino acid sequence C The terminal Ser residue contains the additional residue Ile-Pro-Trp-Lys-Ser-Lys. The consensus amino acid sequence in recombinant and synthetic peptides immunoreactive with .5α Mab is Thr-Ala-Pro-Pro-Leu-Leu-Pro-His-Ser-Asn-Leu-Asp-His-Ile-Leu-Glu-Pro -Ser.

In another aspect, the present invention includes a kit for detecting whether human serum is infected with HTLV-I. The kit includes a solid support on which the gp46 peptide antigen is supported, and a reporter system for detecting the presence of human antibodies bound to the peptide antigen.

In one embodiment, the kits of the invention are provided in the form of EIA, which screens human serum for HTLV-I antibodies. In another embodiment, the peptide antigen is immobilized on a strip in the form of a western blot identifying HTLV-I serum antibodies, along with one or more identifying HTLV-I antigens.

In another embodiment of the present invention, the kit of the present invention includes an HTLV-II specific antigen capable of specifically reacting with an antibody from HTLV-II positive serum, as defined below. This kit enables the specific detection of HTLV-I and HTLV-II positive sera.

The present invention also includes methods for detecting HTLV-I positive human serum. In this method, test serum, expressed as .5α Mab, is reacted with a peptide antigen that is immunoreactive with an anti-HTLV-I monoclonal antibody (Mab) derived from ATCC cell line HB 8755. The presence of anti-HTLV-I antibodies bound to the antigen is detected by a suitable reporter-labeled anti-human antibody.

.5α Mab-reactive peptides may be prepared by any-sequence selection method, preferably by introducing a mixture of any sequence polynucleotides encoding 5-10 amino acid residues into a suitable expression vector to form a library of any sequence vector. Can be. Expression products of the library vector can be screened for the presence of amino acid sequences that are immunoreactive with respect to .5α Mab. Then, library clones expressing these immunoreactive amino acid sequences can be isolated and used to prepare polypeptides encoded by the inserted sequences.

Also in the present invention, the amino acid sequence Met-Thr-Leu-Leu-Val-Asp-Ala-Pro-Gly-Tyr-Asp-Pro-Leu consisting of less than about 50 amino acids derived from the HTLV-II envelope protein gp46 -Trp-Phe-Ile-Thr-Ser-Glu-Pro-Thr-Gln-Pro-Pro-Pro-Thr-Ser-Pro-Pro-Leu-Val-His-Asp-Ser-Asp-Leu-Glu-His HTLV-II peptide antigens comprising immunogenic regions formed from -Val-Leu-Thr-Pro-Ser-Thr-Ser-Trp-Thr-Thr-Lys are disclosed. The consensus amino acid sequence in recombinant and synthetic peptides immunoreactive with HTLV-II antiserum is Ser-Pro-Pro-Leu-Val-His-Asp-Ser-Asp-Leu-Glu-His-Val-Leu-Thr-Pro- Ser, or at the Ser C-terminus of this sequence, has the sequence extended by the amino acid sequence Thr-Ser-Trp-Thr-Thr-Lys.

Peptide antigens are used in test kits to detect whether human serum is infected with HTLV-II. The kit includes a solid support having a peptide antigen and a reporter system capable of detecting whether a human antibody is bound to the peptide antigen.

Objects and other objects of the present invention as described above will become more apparent by the detailed description of the present invention with reference to the drawings.

[Brief Description of Drawings]

Figure 1 (a) shows a portion of the HTLV-I genome containing the gp46 envelope protein coding sequence in the upper line, and the lower line contains the sequence encoding the overlapping HTLV-I peptide antigen formed according to the present invention. A portion of the gp46 coding region is shown and Figure 1 (b) shows MTA-4, MTA-1 and MTA-5,

2 shows the HTLV-I code sequence and amino acid sequence corresponding to a portion of the HTLV-I coat protein,

3 shows the amino acid sequences of the homologous regions of HTLV-I and HTLV-II gp46 in the peptide antigen regions of the invention, and shows the peptide sequences (upper part of the figure) of several HTLV-I gp46 peptide antigens according to the invention. Depicts the peptide sequence of the HTLV-II peptide antigen (bottom of figure),

4 (a) and 4 (b) show the antigenicity to the MTA 1 peptide and the corresponding HTLV-II gp46 peptide,

5 shows a recombinant method for preparing and selecting an arbitrary sequence peptide according to the present invention,

6 shows the HTLV-II coding sequence and the corresponding amino acid sequence within the region of the gp46 envelope protein from which the HTLV-II peptide of the invention is derived,

FIG. 7 shows a modified western blot containing HTLV-I virus lysate and recombinant proteins p21E and MTA-4, wherein lanes A-F and G-R are HTLV-I and HTLV-II antiserum, respectively.

Detailed description of the invention

[I. Preparation of HTLV-I Peptide Antigen]

This section describes the preparation of HTLV-I peptide antigens that are immunoreactive against anti-HTLV-I antibodies found in individuals with HTLV-I related T-cell leukemia. Antigens were prepared using any HTLV-I gene sequence, cloned in a suitable expression vector, 100-300 base pairs in length, and then selected with antibodies for expression of immunoreactive peptides.

[A. HTLV-I Genome Library]

From cellular DNA containing the HTLV-I proviral genome, a genomic library of HTLV-I was prepared by conventional methods. Double helix DNA has been found to produce T-cells, or HTLV-I virus, isolated in patients found to be infected with the HTLV-I virus, such as HUT 102-B2 (Poiesz), MT-2 (Miyoshi), and MJ-. It can be prepared from HTLV-I infected cells, including known cell lines, such as tumor cells. The viral genome is integrated into the host DNA in these cells. Methods of making cell lines containing the HTLV-I genome are described in detail in the references above.

All host genomic DNA from the above cell lines are partially cleaved using common cleavage enzymes such as HaeIII or A1ul, for example, under conditions that result in partial cleavage in the range of 15-20 kb, and the cleaved material is for example sucrose. 15-20 kb fragments are isolated by fractionation by gradient centrifugation. The fragments were then cloned into a phage cloning vector that can efficiently incorporate a suitable cloning vector, preferably a 15-20 kb insert. In a preferred method, the isolated fragments are treated with EcoRI methylase, the EcoRI linker is linked to the end of the fragment under standard conditions (Maniatis), and then phage such as λ Charon 4a with a unique EcoRI insertion site. Cloned into vector.

Cloned genomic fragments were screened with probes complementary to the desired sequences of complete copies of the HTLV-I genome. HTLV-I sequences are known as known in the methods for preparing radiolabeled synthetic oligonucleotide probes for a given sequence (Seiki). In addition, synthetic oligonucleotides of a particular sequence are prepared by a salesman such as Synthetic Genetics, Inc .; San Diego, CA. Using this oligonucleotide probe, molecular clones containing the HTLV-I sequence were isolated from the library by standard hybridization methods (Maniatis, p. 322). The clones can be analyzed by restriction site analysis to confirm the presence of the entire viral genome sequence, which is manifested by the presence of a direct long term repeat unit that contacts the integrated viral genome. The identified molecular clones can be cleaved with a suitable endonuclease to generate a complete copy of the viral genome. Preferred endonuclease for this purpose is Sacl, which cleaves one end of the viral genome in either LTR of the viral coding sequence but does not cut inside. If the clone's HTLV-I genome is a variant with a third Sacl site, the appropriate restriction enzyme can be selected to isolate the full length genome. The purified total copy sequence is about 9.5 kb fragment. Alternatively, only fragments of the genome representing the env gene sequence can be purified for expression library preparation.

Alternatively, cloning vectors containing HTLV-I double helix DNA complete copies have been reported (Seiki) and can be obtained directly from the researchers as described in Example 1.

To prepare the desired HTLV-I genomic library, complete copies of the HTLV-I inserts can be cleaved from the cloning vector, eg by complete cleavage using Sacl, and separated into 9.5 kb fragments as described in Example 1. have. The complete copy fragments of the isolated fragments are cut to produce DNA fragments, preferably any fragments of mainly about 100-300 base pairs in size. Example 1 discloses a method for preparing such fragments by DNAase cleavage. Since it is desirable to obtain peptide antigens with between about 30-100 amino acids, it is preferred that the cleavage fragments are fractionated in size, for example by gel electrophoresis, to select fractions with a size range of about 100-300 base pairs. .

Genomic cleavage fragments are preferably inserted into an appropriate cloning vector, preferably an expression vector that expresses the code for the peptide in a suitable host. One of the preferred expression vectors is λ gt11 containing a unique EcoRI insertion site 53 base pairs upstream of the transcription termination code of the β-galactosidase gene. Thus, the inserted sequence can be expressed as β-galactosidase. Thus, the insert gene is a β-galactosidase fusion protein containing the N-terminal portion of most of the β-galactosidase gene, a heterologous peptide, and at least some C-terminal regions of the β-galactosidase gene. Can be expressed as. This vector also produces a temperature-sensitive inhibitor (cl857) that causes virus lysogeny at an acceptable temperature, such as 32 ° C., to induce lysis of the virus at elevated temperatures such as 42 ° C. The advantage of this vector is that (1) it is very efficient for recombinant formation and (2) has the ability to select lysogenic host cells based on the growth of the host separator at acceptable temperatures, but at non-acceptable temperatures (3) the level of production of recombinant fusion proteins is high. In addition, since phages containing heterozygous inserts produce inactive β-galactosidase enzymes, phages with inserts can be readily identified by β-galactosidase colored substrate reactions.

Cleavage fragments inserted into the expression vector may be modified according to conventional methods, if desired, to contain the desired restriction site linker, such as, for example, the EcoRI linker. Example 1 describes methods for cloning cleavage fragments into λgt11, which blunt-ending the end of the fragment, adding an EcoRI linker, and cutting the fragments into an EcoRI cleaved λgt11. Ligation is included. The resulting viral genome library is checked to confirm that a relatively large (typical) library has been produced. This step, in the case of λgt11 vectors, can be performed by infecting a suitable bacterial host, plating the bacteria, and then examining the plaque for loss of β-galactosidase activity. Using the method described in Example 1, about 60% of the plaques showed a loss of enzyme activity when compared to the background level of the phage exhibiting a loss of enzyme activity as shown in FIG.

[B. Peptide Antigen Expression]

The previously formed genomic library is screened for the preparation of peptide antigens (expressed as fusion proteins) that are immunoreactive with human anti-HTLV-I of interest. One particularly preferred antibody for diagnosing HTLV-I infection is a .5α monoclonal antibody (Mab) with the same specificity as the antibody present in patients with T-cell leukemia associated with HTLV-I infection, as mentioned above. to be. This antibody is prepared by an EBV-transformed B-lymph cell line with ATCC accession number HC8755 (see Example 2).

In a preferred screening method, the host cells infected with the phage library vector are plated as above and the plate is blotted with a nitrocellulose filter to transfer the recombinant antigens produced in the cells to the filter. The filter is then reacted with an anti-HTLV-I antibody, washed to remove unbound antibody, and the reporter labeled anti-human antibody bound to the filter is reacted in the form of a sandwich through the anti-HTLV-I antibody. .

Typically, phage plaques identified by the preparation of the desired recombinant antigen are tested again for the production of antibody reactive fusion proteins at relatively low concentrations. The screening method described in Example 2 is for illustration. In the process, several recombinant phage clones were identified that produce immunoreactive recombinant antigens.

It is preferred to analyze one or more library vectors identified as above by nucleic acid sequencing to determine the location of the peptide coding region in the HTLV-I genome. The method of cleaving the heterologous insert (including the contiguous code sequence of the fusion protein, if necessary) from the selected library vector and purifying and sequencing the cut fragments is carried out by the known methods summarized in Example It is common to be. Coding sequences of three peptides found to be immunoreactive with .5α Mab are shown in the figure. Three heterologous sequences are associated with known HTLV-I sequences (Seiki). As discussed more fully in Example 3, these sequences all fall within the 5565 to 5895 base pairs of the HTLV-I genome in the gene encoding the HTLV-I envelope protein gp46 (part A in the drawing), and the 5664 to 5790 base pairs. In between (part B) are overlapping coding sequences (indicated by two arrows in the drawing). As shown in the figure, the C portion, which is an overlapping sequence, encodes a 42 amino acid peptide antigen having the following amino acid sequence: Ser-Leu-Leu-Val-Asp-Ala-Pro-Gly-Tyr-Asp -Pro-Ile-Trp-Phe-Leu-Asn-Thr-Glu-Pro-Ser-Gln-Leu-Pro-Pro-Thr-Ala-Pro-Pro-Leu-Leu-Pro-His-Ser-Asn-Leu -Asp-His-Ile-Leu-Glu-Pro-Ser. Screening studies conducted under the present invention showed that the MTA-1 peptide yielded the best percentage with respect to HTLV-I positive serum, especially in Japanese subjects. As shown in FIG. 3, the MTA-1 peptide includes additional ILe-Pro-Trp-Lys-Ser-Lys residues at the Ser C-terminus of the sequence. In a preferred embodiment of the present invention, the HTLV-I specific peptide contains an immunogenic region of a C-terminal 48 amino acid MTA-1 sequence that is immunoreactive with 0.55α Mab.

More generally, the HTLV-I peptides of the present invention include immunogenic regions of the amino acid sequence that are reactive with 0.5 α Mab. As defined herein, certain sequences include some neutral amino substituents that do not at least reduce the immunoreactivity of the peptide antigen against .5α Mab. Such amino substituents can be selected based on hydrophobicity, size, charge, hydrophobic capacity, and similarity in secondary structural effects according to known amino acid substitution principles.

Selected clones can be used for scale-up preparation for the purpose of identity of recombinant proteins. The quantitative preparation method comprises: (a) solubilizing a suitable host, such as, for example, E. coli with a desired λgt11 recombinant, (b) culturing the transduced cells under conditions where a high level of heterologous peptide is obtained, and (c) Purifying the recombinant antigen from lysed cells.

In a preferred method comprising the λgt11 cloning vector, high-producer E. coli host BNN 103 was infected with selected library phage and the replicates replicated on two plates. One of the plates that the viral toleration develops is grown at 32 ° C. and the other in the lysis stage is grown at 42 ° C. and then cell growth is inhibited. As a result, cells grown at lower temperatures but not at higher temperatures were successfully tolerated.

Next, in a liquid culture condition in which the virus insert-containing fusion protein was prepared at high levels, the solubilized host cell was grown and lysed by quenching to release the desired fusion protein. These methods are described in detail in Example 4.

HTLV-I coding sequences from λgt11 clones expressing the peptide antigen MTA-1 are prepared by PCR amplification as described in paragraph II below and cloned into pGEX-1 expression vectors (Pharmacia, Piscataway, NJ). Inserts cloned into pGEX-1 are expressed as fusion proteins with protein Sj26, a 26 Kdal glutathione S-transferase derived from the parasite Schistosoma japonicum. Limited paneling of pGEX-MTA-1 against serum free from individuals infected with HTLV-I or HTLV-II shows no significant difference between the reactivity of pGEX-MTA-1 to β-gal-MTA-1. Did.

[C. Peptide Purification]

Recombinant proteins were purified by standard protein purification methods including differential precipitation, molecular sieve chromatography, ion exchange chromatography, isoelectric focusing, gel electrophoresis and affinity chromatography. For example, in the case of fusion proteins such as the β-galactosidase-fusion protein prepared as above, the protein isolation technique used may be combined with the technique originally used for isolation of the protein. Therefore, in the isolation of galactosidase fusion proteins, the protein can be easily shorted by simple affinity chromatography that allows cell lysate to pass through a solid support having a surface-bound anti-galactosidase antibody. . This method was tried in Example 4.

[II. Peptide Immune Reactivity with .5α Mab]

In another aspect, the present invention also relates to HTLV-I positive human serum by reacting HTLV-I positive human serum with a peptide antigen that is immunoreactive with HTLV-I Mab, ie .5α Mab, produced by ATCC cell line HB 8755. A method of detecting is disclosed. The presence of HTLV-I specific antibodies in the serum is detected by the reporter-labeled anti-human antibody described in Example 7.

[A. HTLV-I Inducing Peptide]

In one embodiment, the peptide contains immunogenic regions derived from the 42 amino acid overlap regions from the MTA-1, MTA-4 and MTA-5 HTLV-I peptides. These peptide antigens are further specified to allow for identification of the immunoreactive region in the 42 amino acid sequence overlap region. The location of the immunoreactive region in the C-terminal portion of the overlap region is shown by two lines of evidence. Initially, .5α Mab was reported to react specifically with HTLV-I envelope protein, ie no reaction with HTLV-II or HTLV-III (HIV-1) envelope protein was observed. Applicants and co-workers have learned from this fact that the gp46 peptide antigens MTA-1 and MTA-4 described above are reactive with HTLV-I but not with HTLV-II or HTLV-III antiserum (Lipka).

Secondly, in the comparison of the amino acid sequence of the MTA-1 peptide with the corresponding region in the corresponding HTLV-II gp46 protein (Figure 3), the homogeneity in the N-terminal half of the peptide is C-terminal half It was shown to be substantially larger than in (the central region of the HTLV-I and HTLV-II sequences shown in FIG. 3). These results indicate that the maximum difference in antigenicity is found in the C-terminal half of the peptide region.

This can be more clearly confirmed in the antigenicity diagrams for the two corresponding peptide regions shown in Figures 4 (a) and 4 (b) for the HTLV-I and HTLV-II peptides, respectively. A diagram of antigenicity is provided by the standard hydrophobic program “Antigen” of PC Gene from Intelligenetics (Palo Alto, Calif.). As shown, the two figures overlap substantially at residues 3-28, but are clearly inconsistent at residues 28-40. Nonoverlapping residues include the following HTLV-I sequences:

Leu-Pro-His-Ser-Asn-Leu-Asp-His-Ile-Leu-Glu-Pro-Ser

Multiple peptide antigens containing the C-terminal region shown immediately above were prepared and tested for binding to .5α Mab and HTLV-I and HTLV-II antiserum. The sequence of some of these peptides is shown at the top of FIG. 3 along with the sequences of the MTA-1, MTA-4 and MTA-5 peptide antigens. Peptides were prepared by solid phase synthesis according to standard methods. In short, N-α-protected amino acid anhydrides are prepared in crystallized form and used to carry out a continuous amino acid addition reaction at the N-terminus. In each residue addition reaction, the growing peptide is treated with an acid (on a solid support) to remove the N-α-protecting group and washed several times to remove residual acid and at the same time accessibility of the peptide end to the reaction medium. Accelerated. The peptide is then reacted with activated N-protected amino acid symmetry anhydride and the solid support is washed off.

In each residue-addition step, the amino acid addition reaction may be repeated in a total of two or three separate addition reactions to increase the percentage of growth peptide molecules that react. Typically, 1-2 reaction cycles were used for the addition of the first 12 residues and 2-3 reaction cycles were used for the remaining residues. After completion of the growing peptide chain, the protected peptide resin was treated with liquid hydrofluoric acid to release and release the peptide from the support.

Peptides were tested for specific immunoreactivity with .5α Mab by substantially binding competition studies as described in Example 6. K163 peptides containing 18 C-terminal residues of MTA-4 or MTA-1 strongly inhibit the binding of .5α Mab to MTA-4. However, no binding interference was observed for peptide K162 containing only 11 C-terminal residues of MTA-4. Peptide K164, containing six C-terminal residues and an additional C-terminal 13 residues of MTA-4, weakly inhibits the binding between .5α Mab and MTA-4 or MTA-1.

The results indicate that the most potent immunoreactive region for .5α Mab in the gp46 peptide is in the region comprising peptide K163, which is the sequence identity and antigen between the HTLV-I and HTLV-II sequences in that region. Inconsistent with the gender diagram. Weak binding of .5α Mab to K164 peptide indicates that the desired epitope is in the overlapping region His-Ile-Leu-Glu-Pro-Ser-His-Ile-Leu between K163 and K164, wherein the adjacent N-terminus or C It may indicate that the terminal sequences are required for antigen expression, or that the K164 peptide contains additional epitope regions with weak immune reactivity with .5α Mab.

The peptides were also tested for their ability to inhibit the antiserum of HTLV-I infected patients from binding to MTA-4. In general, the ability of any particular peptide to inhibit .5α Mab binding to MTA-4 can bind to HTLV-I antiserum in the ELISA binding protocol (Example 6B) or by Western blot assay method (Example 8C). It was found that in human HTLV-I antiserum similar to the ability to inhibit binding to MTA-4 or MTA-1. Therefore, peptide K162 does not react with any HTLV-I serum of the ELISA protocol and does not inhibit J-254 serum binding to MTA-1 or MTA-4.

[B. Any sequence peptide]

In another embodiment, .5α Mab-reactive peptides for use in the methods of the invention are prepared by the selection of any sequence peptides. Recently, it has been demonstrated that antibodies corresponding to specifically short (5-10 residues) peptides can be used to screen libraries of randomly occurring peptides for immunoreactive species (Scott; Cwirla et al.). This method was used to identify novel sequences that were immunoreactive with respect to .5α monoclonal antibodies in the present invention.

In a preferred method, about 10 ⁸ new hepta peptides are prepared by constructing an epitope library using a filamentous paja fUSE5 as a vector. It is thought that other filamentous phage vectors are equally useful for expressing these libraries.

5 schematically illustrates the sequence of steps required to generate and screen fUSE5 filamentous phage epitope libraries. In short, fUSE5 RF DNA is cut with restriction endonuclease Sfil to create an insertion site for insertion of foreign DNA. Synthetic (15 + 3m) containing a modified sequence of the form (NNK) m, where N represents A, G, C or T, K represents G or T, and m varies from 2 to 15 ) Base pair (bp) BgII DNA fragments are prepared. In a preferred embodiment of the present invention, m is in the range of 5 to 10 and the base is optionally added only once to the primer of the template. Another way to achieve the optional addition of codons encoding 20 amino acids is to optionally attach trinucleotide codons representing each amino acid to the template primer.

The insert can be ligated to the cloning vector and then amplification of the filamentous phage vector by transfection of E. coli cells. The presence of the marker accompanying the vector determines the successful transfection. In a preferred embodiment of the present invention, such markers are tetracycline resistant. Next, the recombinant phage is isolated from the bacterial cell. Phages having the desired sequence are isolated by an antibody panning method in which the phages are incubated with .5α Mab or Fab fragment thereof. Next, a biotinylated secondary antibody (anti-human IgG of chlorine) is added, and the complex containing biotinylated secondary antibody, .5α Mab, and an immunoreactive peptide containing phage is added to the streptavidin coated plate. By attachment it is isolated from unreacted antibody and phage. Next, the phage containing the immunoreactive sequence was eluted to determine its DNA sequence.

The foreign DNA sequence present in the filamentous phage fusion protein pIII determines the sequence of the immunoreactive peptide. Peptides found to be immunoreactive through this procedure can be prepared as immunogens by synthesis by standard peptide synthesis and conjugation to a suitable peptide carrier.

[III. HTLV-II Peptide Antigen]

This paragraph relates to methods for the identification and cloning of HTLV-II antisera and particularly immunoreactive HTLV-II peptides. These peptides are derived from the HTLV-II gp46 envelope protein region, which is homologous to the MTA-1 peptide from the previously described HTLV-I gp46 region.

By cloning the HTLV-II coding sequence corresponding to the desired peptide sequence, the HTLV-II peptide (FIG. 3) designated GH2-K15 corresponding to the HTLV-I peptide MTA-1 was prepared. 147 base pair (bp) HTLV-II DNA fragments corresponding to 5648 to 5794 nucleotides of the HTLV-II genome using the polymerase chain reaction (PCR) method (Perkin-Elmer / Cetus GeneAmp kit) were subjected to HTLV-II clone pM04 (plasmid amplified from most of the HTLV-II genome cloned into the BamH I site of pBR 322).

Forward and reverse primers are shown in FIG. The amplified DNA was ligated into the EcoRI site of the λgt11 phage vector to form a clone such as 3K15 containing the 147 HTLV-II DNA insert to be inserted into the -galactosidase gene of λgt11. Recombinant phage was used to transfect E. coli strain BNN103. Details regarding this are described in Example 5.

In preliminary experiments, serum from about 150 subjects not infected, as well as serum from about 200 subjects infected with HTLV-I or HTLV-II as determined by PCR method, were paneled against GH2-K15 antigen. . 98% of serum collected from individuals infected with HTLV-II reacted with GH2-K15. Neither HTLV-I infected or uninfected serum reacted with GH2-K15. Screening results demonstrated that the GH2-K15 peptide had specific immunoreactivity with HTLV-II positive serum.

Several smaller peptides containing the GH2-K15 amino acid sequence were prepared by the recombinant method described in paragraph I. In summary, peptides were prepared by PCR amplification of HTLV-II genomic DNA using PCR primers designed to facilitate the amplification of the indicated sequences as detailed in Example 5. Of these peptides, five designated (GH2-) K14, K16, K24, K35, and K34 have the sequence shown in FIG.

The recombinant HTLV-II peptide described above was immunoscreened against several HTLV-II and HTLV-I by ELISA method, as described in Example 8. The results are shown in Table 1. All λgt11 HTLV-II clones except GH2-K16 were recognized by at least one of the six HTLV-II sera tested. GH2-K16, the only non-reactive clone, deletes the 22 amino acids at the carboxy terminus contained in GH2-K15. All other clones tested contained 17 or more amino acids present in peptide K125: Ser-Pro-Pro-Leu-Val-His-Asp-Ser-Asp-Leu-Glu-His-Val-Leu -Hr-pro-ser

In addition, as shown in Table 1, none of the peptides tested reacted with HTLV-I serum or .5α Mab at all.

Three of the original HTLV-II clones, GH2-K15, GH2-K35 and GH2-K16, were cloned into pGEX-1 expression vectors. Recombinant proteins expressed by the three pGEX-1 HTLV-II clones GH2-K15, GH2-K25 and GH2-K35 were all recognized by J-317 HTLV-II serum.

TABLE 1

Many peptide antigens containing amino acid sequences in the K15 sequence were prepared by the solid phase method described in paragraph III above. Of these peptides, the sequences of the five peptides designated (GH2-) K169, K170, K125, K126 and K128 are shown in FIG. These peptides were tested for immune reactivity with several HTLV-I and HTLV-II positive sera by ELISA methods, and some of these peptides were also tested for their ability to inhibit HTLV-II antibodies from binding to the K15 antigen. Tested.

K125 peptide was recognized by multiple HTLV-II sera when analyzed by ELISA method. In one experiment, six of the 12 HTLV-II sera could bind K125 effectively. In the same experiment, none of the 7 HTLV-I sera bound peptide K125. K125 peptide also inhibited the binding of J-317, a highly reactive HTLV-II serum, to GH2-K15 subjected to western blotting. The ability of serum J-317 to bind GH2-K15 is not affected by incubation with HTLV-I peptide K163 or HTLV-II peptide K128.

HTLV-II peptide K170 is recognized by multiple HTLV-II sera in an ELISA-based assay and in the same manner not by HTLV-I sera. K169 peptides are not recognized by HTLV-I serum in assay methods based on ELISA.

Data from both HTLV-II recombinant antigen analysis and analysis of synthetic HTLV-II peptides show that HTLV-II specific epitopes are included in the following 17 amino acid sequences in the GH2-K15 peptide: Ser-Pro-Pro-Leu Val-His-Asp-Ser-Asp-Leu-Glu-His-Val-Leu-Thr-Pro-Ser.

The data obtained by extensive paneling of the HTLV-I antigens MTA-1 and MTA-4 described above show that the six final amino acids Thr-Ser-Trp-Thr-Thr-Lys of GH2-K15 were recognized by HTLV-II antiserum. It may also contribute to epitopes.

[IV. HTLV-I and HTLV-II diagnosis method]

In the following, three basic types of diagnostic applications of the HTLV-I and HTLV-II peptide antigens of the present invention will be described. The first application is based on inhibiting supplement-mediated, antibody-dependent cytolysis by the peptide. In this method, serum from a test subject is reacted with a clone to HTLV-I or HTLV-II infected T-cells in the presence of a supplement. The presence of anti-HTLV-I or anti-HTLV-II antibodies is evidenced by cell lysis determined by, for example, exclusion of trypan blue dye.

If cytolysis is observed, the specificity of the anti-HTLV-I antibody against the HTLV-I peptide by reacting the serum with excess HTLV-I or HTLV-II peptide and then mixing the serum with the cells in the presence of the supplement. This is proven. The presence of HTLV-I or HTLV-II antibodies would result in a substantial reduction in cell lysis. This method is described in Example 6A.

Using this method, antibody titers in assay serum can also be quantified by titrating serum while increasing the amount of peptide and measuring peptide concentrations where significant effects are observed on the degree of cell lysis.

A second general assay type is the method of enzyme-immunoassay of human serum against HTLV-I or HTLV-II infection. In this assay, a solid reagent having a surface-bound HTLV-I or HTLV-II gp46 peptide antigen is reacted with analyte serum under conditions that allow the antibody to bind to the peptide on the reagent. After washing the reagent to remove unbound serum components, the reagent is reacted with an enzyme-labeled anti-human antibody to bind the enzyme to the reagent in proportion to the amount of bound anti-HTLV-I antibody on the solid support. The reagent is washed again to remove unbound antibody and the amount of enzyme bound to the reagent is measured. One exemplary method of using an anti-human antibody labeled with alkaline phosphatase is described in detail in Example 7 for a direct HTLV-I screening assay. In the present invention, the enzyme-labeled antibody and the reagents required for the enzyme detection method are also referred to as reporter means for detecting the presence of a human antibody bound to a peptide antigen on a solid support.

Solid surface reagents in the assay method are prepared by known techniques for attaching protein materials to solid support materials such as polymer beads, dip sticks or filter materials. Such attachment methods generally include a method of non-specific adsorption of the protein to a support (filter support disclosed in Example 8), or a protein via a free amino group such as an activated carboxyl, hydroxyl or aldehyde group on a solid support. A method of covalently attaching to the same chemical reactor is included.

A third type of general assay is the Western Block Assay for use in identifying HTLV-I or HTLV-II antiserum. This assay method includes, in addition to the gp46 peptide antigen of the present invention, one or more identifying HTLV-I or HTLV-II antigens effective for detection of HTLV-I or HTLV-II antiserum. In one preferred method, identification peptides include p24 gag protein from HTLV-I virus lysate and p21E recombinant envelope protein containing most of the HTLV-I gp21 envelope protein (Samuel, 1984, 1985). p24 lysate protein takes most of HTLV-I and HTLV-II positive serum but not all of it. The p21E recombinant protein not only acquires virtually all HTLV-I and HTLV-II, but also provides some false positives. This modified western blot assay method has been reported by Applicants and co-workers (Lipka). Detailed blotting assay methods are described in Example 8.

As described above, and as detailed in Example 8, the modified Western blot method was tested for all HTLV-Is, as evidenced by immune responses with viral lysate protein p24 and recombinant protein p21E. And HTLV-II positive serum (panel of 95) is detected. In addition, the MTA-4 peptide is immunoreactive only against identified HTLV-I sera. Using this modified blotting assay, HTLV-I or HTLV-II antiserum can be identified and the two types of HTLV virus can be distinguished by selective immunoreactivity with the peptides of the invention.

In another embodiment of the Western Blot Assay, the HTLV-I peptide antigen is replaced with the HTLV-II gp peptide antigen described in paragraph III. In this method, the HTLV-I virus lysate protein and the p21E recombinant protein allow for identification of HTLV-I or HTLV-II antiserum as described above. HTLV-II specific peptides take HTLV-II antiserum but do not take HTLV-I antiserum, thus providing a positive confirmation for HTLV-II antiserum.

The two methods can be combined to provide both HTLV-I and HTLV-II specific peptide antigens to provide a positive confirmation of any of the HTLV antiserum.

[V. Vaccine composition]

The present invention also includes vaccine compositions containing HTLV-I gp 46 peptides and antigen carriers such as immunogenic proteins to which antigen peptides bind. The peptide contains an immunogenic region formed by the 42- or 47-amino acid overlap of the MTA-1, MTA-4 and MTA-5 peptides described in paragraph I above, which region is anti-HTLV-I .5α. It is immunoreactive with Mab, an antibody derived from ATCC cell line HB8755. More specifically, the peptide contains immunogenic regions of the following peptide sequences: Thr-Ala-Pro-Pro-Leu-Leu-Pro-His-Ser-Asn-Leu-Asp-His-Ile-Leu -Glu-Pro-Ser.

Since .5a Mab is a neutralizing antibody, the antibody generated by the peptide is expected to be a neutralizing antibody.

In another method, the vaccine composition comprises an amino acid sequence: Met-Thr-Leu-Leu-Val-Asp-Ala-Pro-Gly-Tyr-Asp-Pro-Leu-Trp-Phe-Ile-Thr-Ser-Glu-Pro -Thr-Gln-Pro-Pro-Pro-Thr-Ser-Pro-Pro-Leu-Val-His-Asp-Ser-Asp-Leu-Glu-His-Val-Leu-Thr-Pro-Ser-Thr-Ser -Trp-Thr-Thr-Lys, preferably amino acid sequence: Ser-Pro-Pro-Leu-Val-His-Asp-Ser-Asp-Leu-Glu-His-Val-Leu-Thr-Pro-Ser-Thr HTLV- formed by Ser-Trp-Thr-Thr-Lys, or Ser-Pro-Pro-Leu-Val-His-Asp-Ser-Asp-Leu-Glu-His-Val-Leu-Thr-Pro-Ser HTLV-II gp46 peptide containing II specific immunogenic regions.

Particularly useful protein carriers for the peptide (s) include hemocyanin (KLH) from keyhole parasols, tetanus toxins, poly-1- (Lys: Glu), coagulation of peanuts, poly-D Lysine, diphtheria toxin, ovalbumin, soybeen agglutinate, bovine serum albumin (BSA), human serum albumin, and the like.

The immunogenic peptide (s) can be bound to the carrier using many known methods and genetic engineering techniques, including chemical induction methods. Such genetic engineering techniques are described in more detail in Gerald Quinnan, “Proceedings of a Workshop”, November 13-14, 1984. Vaccines and inoculum of the present invention may be administered by intramuscular or subcutaneous injection method, orally by enteric capsule or tablet means, by suppository or intranasal spray, or by other suitable route of administration. For patients, the appropriate polypeptide dosage will depend in part on the route of administration chosen and on a number of other factors. These factors include the weight of the mammal to be immunized, the carrier material used, the adjuvant used, and the number of vaccinations to be used.

Each inoculum for human patients may contain from about 10 μg to about 100 mg unit dose of polypeptide in addition to any carrier capable of binding to the polypeptide. If desired, a series of doses may be administered over a period of time for optimal immunity. If desired, a unit dosage form of a vaccine containing the aforementioned amount of polypeptide may be provided.

In any case, the immunogen contained in the vaccine or inoculum is present in an “effective amount”, which amount is well known in the art of immunology, for example the weight of the mammal to be immunized, the adjuvant, the desired duration of protection, and It depends on several factors, such as the immunization protocol.

In the following Examples, the present invention will be described in detail without limiting the scope of the present invention even if at all.

[matter]

The materials used in the following examples are as follows:

Enzyme: DNAase I and alkaline phosphatase were purchased from Boehringer Mannheim Biochemicals (BMB, Indianapolis, IN); EcoRI, EcoRI methylase, DNA ligase, and polymerase I were purchased from New England Biolabs (NEB, Beverly, Mass.); RNase was purchased from Sigma; St. Louis, Mo.

Other reagents: EcoRI linker from NEB; Nitroblue tetrazolium (NBT), 5-bromo-4-chloro-3-indolyl phosphate (BCIP), 5-bromo-4-chloro-3-indolyl-D-galactopyranoside from Sigma (X-gal) and isopropyl-D-thiogalactopyranoside (IPTG) were purchased.

Example 1

[Preparation of HTLV-I Genome Library]

[Source of genomic material]

R. C. of the Laboratory of Tumor cell Biology (Bethesda, MD), National Institutes of Health. Dr. R.C. Gallo and F. Dr. F. Wong-Staal received a bacteriophage containing a complete copy DNA insert derived from the HTLV-I genome. The bacteriophage was completely cleaved with Sacl to release the viral genome insert. The cleaved material was electrophoresed on a standard 1% agarose gel and the 9.5 kb simple obtained by electroeluting was extracted with phenol / chloroform and ethanol precipitated.

Purified genomic DNA was dispersed in standard cleavage buffer (0.5M Tris HCl, pH 7.5; 1 mg / ml BSA; 10 mM MnCl ₂ ) at a concentration of about 1 mg / ml and digested with DNAase I for about 5 minutes at room temperature. The above reaction conditions were determined from previous measurement studies, in which the incubation time required to prepare mainly 100-300 base pair fragments was determined. The materials were extracted with phenol / chloroform and then precipitated with ethanol.

Under standard conditions (Huynh), the ends of the genome fragments were blunt-terminated with DNA Pol I, extracted with phenol / chloroform and precipitated with ethanol. Smooth terminated material was linked to EcoRI linker under standard conditions (Maniatis, pp. 396-397) and then cleaved with EcoRI to remove excess linker ends. This material was then agarose gel-fractionated to remove linkers not yet ligated and selected by size (see below).

The fragments prepared in the previous step were analyzed by electrophoresis (5-10V / cm) on 1.2% agarose gel using X174 / HaeIII and X174 / HindIII size markers. Fractions from 100 to 300 bp were eluted onto NA45 strips (Schleicher and Schuell), which were then placed in a 1.5 mL micro test tube containing eluent (1 M NaCl, 50 mM arginine, pH 9.0) at 67 ° C. for 30-60 minutes. Incubate. DNA in the current solution was extracted with phenol / chloroform and precipitated with ethanol. The pellet formed was redispersed in 20 μl of TE (0.01 M Tris HCl, pH 7.5, 0.001 M EDTA).

gt11 phage vector (Huynh) was purchased from Promega Biotec (Madison, Wis.). This cloning vector has a unique EcoRI site 53 base pairs upstream of the β-galactosidase translation end codon. 0.5-1.0 μg EcoRI-cleaved gt11, 0.5-3 μl of the HTLV-I genomic fragment, 0.5 μl 10 × ligation buffer (above), 0.5 μl ligase (200 units), and distilled water up to 5 μl By mixing, the genome fragment was introduced into the EcoRI site. The mixture was incubated overnight at 14 ° C. and then packaged in vitro according to standard methods (Maniatis, pp. 256-268). (Maniatis, pp. 256-268j).

The packaged phage was used to infect E. coli strain KM392 obtained from Kevin Moore of Dr. DNAX (Palo Alto, Calif.). Alternatively, you can use E. coli Y1090 (ATCC # 37197), available from the American Type Culture Collection. Infected bacteria were plated and the resulting colonies were checked for loss of β-galactosidase activity (clear plaques) in the presence of X-gal using standard X-gal substrate plaque assay (Maniafis). Table 2 below shows the number of recombinant (transparent) plaques (line 1) obtained by inserting EcoRI-terminated HTLV-I fragments. The same was done for the EcoRI linker control (line 2) and vector alone (line 3). As shown, about 50% of phage plaques showed a loss of enzyme (recombination). The level of background in the presence or absence of EcoRI linker is 15% or less, indicating successful development of the HTLV-I epitope library. The phage library contained about 106 plaque-forming units (pfu) / ml.

TABLE 2

Example 2

[Screening of gp 46 code inserts]

Purified .5 antibody from human cell line (ATCC # C8755) was provided by Dr. Samuel Broder of National Cancer Institute (Bethesda, MD) of the National Institute of Health. Mouse anti-human IgG antibodies induced by covalent binding with alkaline phosphatase were obtained from Promega Biotech (Madison, WI).

KM392 cells infected with about 10 ⁴ pfu of phage stock from Example 1 were prepared on 150 mm plates, incubated at 37 ° C. for 5-8 hours and inverted. The infected cells were spread on nitrocellulose sheets and allowed to transfer HTLV-I recombinant protein from plaque to paper. Plates and filters are marked so as to match the positions of the corresponding plates and filters.

The filter was washed twice in TBST buffer (10 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Tween 20), blocked with AIB (1% gelatin in TBST buffer) and washed again in TBST, .5 Monoclonal antibodies (diluted to 1-2 μg / ml in AIB, 12-15 mL / plate) were added and then incubated overnight. The sheets were washed twice in TBST and then contacted with enzyme-labeled anti-human antibodies to attach labeled-antibodies to filter sites containing antigens recognized by .5 antibodies. After the final wash, filter was removed from substrate medium containing 33 μl of NBT (50 mg / ml stock maintained at 5 ° C.) in 5 ml alkaline phosphatase buffer (100 mM Tris, 9.5, 100 mM NaCl, 5 mM MgCl ₂ ). Developed. The substrate that reacted appeared only at the point where the antigen was made, as recognized by 0.5α Mab.

The preparation region of the antigen determined in the previous step was replated at about 100-200 pfu on an 82 mm plate. Repeating the previous step, starting with incubation for 5-8 hours and undergoing NBT / BCIP development, identified plaques that secreted antigen capable of reacting with .5Mab. Identified plaques were taken and eluted in phage buffer (Maniatis, p. 443). Three recombinant phage plaques secreting antibody-reactive peptides were selected for sequencing analysis according to the method of Example 3. Corresponding infection phages are designated MTA4-, MTA-1 and MTA5.

Example 3

[Purification and DNA Extraction of Phage]

Phage MTA-4, MTA-1 and MTA-5 were isolated from plate cultures of infected E. coli Y1088 bacteria. These cells were obtained from ATCC (ATCC # 31195). Phage dilution buffer was added to collect phage (maniatis), the material was purified from bacterial fragments by low speed centrifugation, and the supernatant was poured into SW27 tubes. RNase and DNase were added until the concentration was from 1 mg / ml of the stock solution to 1 µg / ml, respectively. Samples were incubated at 37 ° C. for 30 minutes and an equal volume of polyethylene glycol (PEG), 5.8 g of NaCl, 2.0 g of MgSO ₄ 7H ₂ O, pH 7.5, 1M Tris Cl, and 2% gelatin were added. The sample was placed in an ice bath for 1 hour to allow phage particles to form a precipitate, which was isolated by centrifugation at 10k for 10 minutes at 4 ° C.

The supernatant was decanted and the pellet was resuspended in 0.6 mL PDB buffer (5.8 g NaCl, 2.0 g MaSO ₄ .7H ₂ O, 50 mL 1M Tris Cl, pH 7.5 and 5 mL 2% gelatin). Transfer to ml polypropylene microtubes. 5 μl of 10% SDS, 5 μl 0.5M EDTA and 2.5 μl protease K (20 mg / ml) were added and the samples were incubated at 50 ° C. for 15 minutes.

Detergent and enzyme treated materials were extracted with the same volume of phenol / chloroform and centrifuged to ensure phase separation. The aqueous phase was transferred to a new test tube and the extraction / centrifugation method was repeated with a mixture of chloroform and isoamyl alcohol. The same volume of isopropanol was added and mixed upside down several times and then cooled to -70 ° C for 20 minutes. Samples were centrifuged for 5 minutes and the supernatant decanted. The pellet was washed in 70% ethanol and slightly dried in a thermal block at 37 ° C. and resuspended in 100 μl TE buffer at pH 7.5.

Isolated phage DNA was digested with Kpnl and Sacl and then combined with the Kpnl / Sacl cleaved plasmid vector pGEM-3 (Promega Biotec) to isolate plasmid recombinants with the desired insert. The HTLV-I insert was then sequenced using standard dideoxy sequencing methods and forward and reverse primers for the λgt1 sequence flanked by the EcoRI insertion site.

In the figure, for each of the three fusion proteins tested, the coding sequence and corresponding amino acid sequence of a portion of the fusion protein formed by the above method is shown. The proximal CC base of the env gene contributed by the terminal G base of the β-gal gene and each of the three insertion sequences produces a GCC (Ala) codon, resulting in a Ser codon occurring at the codon position of all three env inserts. Replace. As shown, the insert in MTA-4 contains 225 base pair sequences spanning 5564 to 5790 bases of the HTLV-I coding region. The insert of MTA 5 phage extends from base 5664 to base 5895. The 231 base pair sequence spans amino acids 162 to 240 of the gp46 protein.

The insert overlap regions of 5664-5790 comprise the 42 amino acid sequences of amino acids 162-203 of the original gp46 protein.

Example 4

[Isolation of HLV-I Peptide Antigens]

[A. Composition of Lysogen]

At Stanford University (Stanford, CA). Eco RI Ltd. C600 was obtained from Dr. R. Davis. Alternatively, Eco RI Y 1089 (ATCC # 37196) may be used. 10 ml of eluted plaques were adsorbed to 50 µl of bacterial culture overnight, thereby infecting 1 ml of overnight saturated cultured cells with one of the three phages from Example 3. Infected bacteria were spread out only on LB agar plates (Maniatis, p. 440) and incubated at 32 ° C. Individual colonies were picked with sterile tongs and placed in corresponding grids on two separate plates. One of the plates was incubated at 32 ° C and the other at 42 ° C. Cells grown at low temperatures (due to cell lysis) that are not high temperature (representing a lytic state produced by the presence of phage inhibitory proteins) are presumed to be soluble. Many lysogenic colonies from each of the three phage types have been found.

[B. Recombinant Antigen Induction from a Source]

In this example, the induction of recombinant proteins containing HTLV-I epitopes from the λgt11 source prepared in Example 4 with MTA-4 phage will be described. As previously described, the antigen is prepared in the form of a -galactosidase fusion protein containing the N-terminal portion of the phage -gal protein.

A superbroth was prepared containing 35 g of bactotriptone, 2 g of bacto-yeast extract, 5 g of NaCl and 5 ml of 1N NaOH in one 1dHO. 500 ml of super broth was inoculated at 1: 100 with a saturated culture in which the EcoRI λgt11 source prepared in the previous example was incubated overnight. Cultures were incubated with A ₆₀₀ = 0.4-0.5 with strong ventilation.

To maximize the production of the protein, the temperature sensitive-galactosidase inhibitor gene was inactivated by raising the temperature of the culture to 43-44 ° C. The temperature was maintained at 43 ° C. for 15 minutes in a 65 ° C. water bath with ventilation. IPTG, which induces galactosidase expression by competitive binding to galactosidase inhibitors, was added to the baths to further increase protein production. The culture was transferred back to a vibrator at 38 ° C. and vibrated for about 1 hour. Cells were then pelleted at 37 ° C. at 6000 × g for 15 minutes and resuspended in lysis buffer (10 mM Tris, pH 7.4, 2% Triton × -100, 1% aprotinin and 50 μg PMSF) immediately to liquid N _2. I put it in. When the frozen sample is thawed, dissolution is complete.

[C. Purification of Fusion Protein]

The cell lysate obtained in the previous example was thawed and warmed to 37 ° C. 10 μl of DNAse (1 μg / ml) was added and the mixture was incubated until the viscosity decreased. The dissolved material is rapidly cooled on ice and cleared for 5 minutes at 4 ° C. in a microfuge, which is loaded onto a 6 ml column of anti-galactosidase bound to Sepharoce 4B (Pharmacia). It was. Equilibrate the column for 1-2 hours, wash with 7 volumes (column volume) of TX buffer (10 mM Tris, pH 8.0, 2% Triton X-100, 50 μg / ml PMSF) and then 5 mM 3,5 in TX buffer. -Washed with 2 volumes of diiodosalicylic acid. The fusion protein was then eluted from the column with 35 mM 3,5-dioiosalicylic acid in TX buffer. Most of the protein was eluted to the first 3-4 volumes and after 7 volumes substantially complete removal.

Eluted samples were desalted and concentrated using an Amicon filter (Danvers, MA).

Example 5

[Preparation of HTLV-II Antigen]

[A. Synthesis and Cloning of HTLV-II DNA Sequence]

Cloning HTLV-II DNA sequences were generated using the polymerase chain reaction (PCR) method. Six 30 bp DNA primers were synthesized. All six primers had an gt11 sequence of 3 bp and had an EcoRI site at its 5 ′ end. This is followed by the 21 bp HTLV-II sequence. Three forward primers contain HTLV-II sequences corresponding to nucleotides 5648-5668, 5687-5707 and 5726-5745. Three reverse primers contain HTLV-II sequences corresponding to nucleotides 5794-5774, 5776-5756, and 5728-5708.

PCR was performed according to the manufacturer's instructions (Perkin Elmer / Cetus), and all PCR reactions contained 2 ng of the HTLV clone as template and 1 μm of the appropriate PCR primer. PCR amplifications were performed 25 times. Each cycle includes performing a 1 minute transformation at 94 ° C., annealing the primer for the template at 50 ° C. for 2 minutes, and extending the primer for 2 minutes to 72 ° C. The amplified DNA was then purified and cleaved with EcoRI. The cleaved DNA was ligated to the Ecol site of lambda gt11. The frequency of non-recombinant phage is then determined by packaging the recombinant phage DNA and plating the non-recombinant phage in the presence of 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside. It was.

The ratio of recombinant phage to non-recombinant phage was about 50/1. Multiple isolated plaques from each of the six recombinant phage clones were taken and screened using PCR using λgt11 flanking primers 11F and 11R and / or HTLV-II plaques described above. Next, clones containing exactly sized and oriented inserts were amplified and used for subsequent immunoscreening assays. Next, EcoRI fragments from three clones GH2-K15, GH2-K16 and GH2-K35 were subcloned into pGEX-1 plasmid and sequenced DNA. The obtained sequences are precisely bound to the reported sequence (Shimotokno) for the desired HTLV-II region.

[B. Immunological Analysis of HTLV-II Clones]

Recombinant phage was mixed with wild type gt11 to 1/1 and this infected E. coli strain KM-392. After allowing the phage to grow for about 5 hours, the expressed proteins were bound to the nitrocellulose filter overnight. The filter was then washed three times with TBS (0.5M NaCl, 20 mM Tris pH 8.0), cut into portions and blocked with TBS and 1% gelatin. The filter portion was then incubated overnight with one step antibody, serum from HTLV-I or HTLV-II infected individuals diluted 1/100 in TBS + gelatin. After washing with TBS, the filter was incubated for at least 1 hour with goat anti-human serum conjugated with alkaline phosphatase. The bound antibody was detected by washing the filter with TBS and then incubating the filter in a solution of nibroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolylphosphate. If plaques derived from recombinant phage can be clearly distinguished from plaques of wild type gt11, certain sera were evaluated as positive.

[C. Expression and Purification of Recombinant Antigens]

First, β-galactosidase fusion protein was expressed by generating a lysate. E. coli strain BNN103 was infected using recombinant gt11 phage and two plates were grown at 32 ° C and 42 ° C to confirm the identity. Preparation of the fusion protein can be induced by raising the temperature of the culture to 42 ° C. for 15 minutes on the log of the source. Isopropyl thiogalactoside was then added to a final concentration of 1.6 Mm and the cultures were grown at 37 ° C. for an additional hour. Cells are then pelleted by centrifugation at 5000 × g for 15 minutes and resuspended in lysis buffer (2% Triton × -100, 1% aprotinin, 10 mM Tris, pH 7.4) at 1/50 of the original culture volume. I was. The solution was then frozen by dipping in a dry ice / ethanol bath and then thawed. DNasel was added to a final concentration of 1 μg / ml, and the lysates were incubated at room temperature for 5 minutes. Insoluble debris were then pelleted by centrifugation at 10,000 × g for 10 minutes. The supernatant was then centrifuged as before. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, Lammeli) analysis of a sample of pellet and supernatant fractions showed that GH2-K15 was found in the supernatant fractions by default.

The supernatant fractions were then combined with 2 ml of Protosorb Lacz adsorbent (Promega) and incubated at 25 ° C. for 2 hours with shaking. The column resin was then poured into a disposable column and washed twice with 10 mL of TX buffer (1% Aprotinin, 10 mM Tris pH 7.4). The bound protein was eluted with 14 mL of carbonate buffer pH 10.8 and 2 mL fractions were collected in a test tube which already contained 1 mL of 2M Tris buffer (pH 7.5). The fractions were then concentrated using Centricon 30s (Amicon) according to the manufacturer's instructions. Fractions were washed with 2 ml of MTBS buffer (150 mM NaCl, 4 mM NaH ₂ PO ₄ , 16 mM Na ₂ HPO ₄ , pH 7.3) and then concentrated again.

SDS-PAGE was used to determine the location, yield and purity of the purified fusion protein. After one pass through the immunoaffinity column, the purity of the GH2-K15 recombinant antigen was ˜70%. Fractions containing the fusion protein were pooled and the specimen was used for the next step western blot test. Western blot analysis was basically performed as previously described (Lipka). In the titration experiments, the optimal loading of purified GH2-K15 antigen was determined to be 3 μg of protein per cm of nitrocellulose. Peptide competition experiments are described in Example 6C. Next, nitrocellulose strips containing the blotted antigen were added to the serum samples and incubated to develop normal.

[D. HTLV antiserum]

Serum samples were obtained from HTLV-I, HTLV-II, or ELISA reactive-HTLV negative individuals. These samples were obtained from a number of different sources and regions. In addition, many seropositive serum samples were typed for HTLV-I or HTLV-II infection using PCR with species specific DNA primers. HTLV-I serum samples include 58 PCR proven samples, consisting of 45 samples from Jamaican food cookers, 2 IVDUs from New Orleans, and 11 northern California blood providers. Included. In addition, a total of 238 HTLV-I serum samples were obtained in Japan. PCR data could not be obtained from samples in Japan, and infections were categorized by Western blotting analysis of serum samples for HTLV-I antigen using the characteristics set forth above (Lipka et al., JID). HTLV-II serum samples included 57 PCR identified samples consisting of 6 IVDUs from New Orleans, 24 IVDUs from Northern California, and 27 blood suppliers from Northern California. HTLV negative sera included samples from one food handler in Jamaica, one California blood provider, and 29 blood providers in Japan. PCR analysis was performed on serum samples as above (Lipka).

Example 6

[Immunoreactive Detection of Peptide Antigens]

[A. Inhibition of Cell Lysis]

HUT 102-B2 cells were obtained from LTCB, NIH. Obtained from Dr. Gallo. This is a long term cultured T-cell line known to produce HTLV-I.

Human IgG as a .5α antibody (˜5 μg / ml IgG) or homozygous control group was preincubated for 30 minutes at room temperature with MTA-4 recombinant peptide or unrelated recombinants. 50 μl of this mixture was then added to 5 × 10 5 HUT 102B2 cells in 96-well microtiter plates and incubated for 30 minutes at room temperature. 30 μl of rabbit supplements were added per well and incubated at 37 ° C. for 1 hour. The viability of the cells was tested by microscopy. Lysis of cells can be visually inhibited by the addition of the MTA-4 peptide antigen, but not by preincubation with irrelevant recombinant peptide antigen. After preliminary incubation with the recombinant antigen or irrelevant recombinant peptide antigen, human IgG paired with the same type had no effect on HUT 102-B2 viability.

[B. ELISA assay method]

HTLV-I and HTLV-II peptides were tested by ELISA assay to determine the ability of serum from HTLV infected individuals to bind the synthetic peptides. In brief, ELISA assays include binding a fixed amount of synthetic peptide to microtiter plates and then adding serum from HTLV infected individuals. The unbound serum was then washed away and the antibody bound to the peptide was detected by the secondary antibody. The secondary antibody was conjugated to an enzyme that converts a colorless substrate to a colored product. The amount of colored product prepared represents the amount of serum antibody bound to the peptide. Signals obtained from specific serum for bound peptide were subtracted from signals obtained from serum from wells that contained no peptide at all. The value obtained after subtracting the negative peptide background value should be 2.5 times the background value considered to be positive.

[C. Antibody binding inhibition]

Inhibition assays include incubating excess synthetic peptides with serum from an HTLV infected individual, and then placing a nitrocellulose strip containing the HTLV-I or HTLV-II recombinant antigen blotted thereon. . If the serum can be bound to the peptide, the vast excess of peptide in the solution containing the antibody prevents the antibody from significantly binding to the relatively small amount of antigen present on the nitrocellulose strip. The amount of antibody bound to the recombinant antigen on the nitro cellulose strip is measured using an enzyme conjugated secondary antibody, in a similar manner as described above for the ELISA assay. Experiments on the control include incubating HTLV-I serum with HTLV-II peptide K125 or HTLV-II serum with HTLV-I peptide and then determining the ability of the serum to recognize suitable recombinant antigens. Steps are included.

Example 7

[EIA analysis]

As described in Example 4, a purified MTA-4 peptide antigen was prepared, blotted onto a nitro cellulose filter, and then used in a solid phase assay method to determine the patient with T-cell leukemia (HTLV). Serum antigens of 6 patients infected with -I) were measured. In each case, 0.1 ml of various serum dilutions diluted 1: 100 to 1: 50,000 from each individual were added to the filter and allowed to stand for 30 minutes at room temperature. The filter was then washed twice with TBST buffer (Example 2) and incubated with anti-human antibody conjugated with alkaline phosphatase as in Example 2. In addition, as in Example 2, the presence of the antibody was measured by color expression in NBT and BCIP.

Example 8

[Modified Western Blot Method to Identify HTLV-I Positive Serum]

Recombinant MTA-I was prepared as in Example 4. Recombinant p21E was prepared as previously described (Samuel, 1984, 1985). HTLV-II virus lysates were prepared from chronically infected cell line MT-2 (Hillcrest Biologicals, Cypress, CA). This HTLV-I antigen was mixed and then separated under reduced pressure on a 11.5% acrylamide SDS / PAGE gel (Laemmli). The redissolved protein was electrically blotted on nitrocellulose (nitrocellulose membrane) blocked by blotting (100 mM Tris-HCl, 5% skim milk powder in pH 7.4, serum of 2.5% normal goats) and then air dried. And cut into 3 mm wide strips.

In the assay method, the test strips above were first hydrated in TBS buffer and these strips were incubated overnight with human test serum diluted 1:50 in blotting solution. The strips were washed several times with wash buffer and then incubated for 1 hour with goat anti-human IgG conjugated with alkaline phosphatase (Bio-Rad, Richmond, CA). After washing, color expression was induced by incubating the strip in a substrate solution containing 5-bromo-4-chloro-3-indolyl phosphate, and nitroblue tetrazolium in 100 mM Tris-HCl buffer, pH 9.5, 50 mM MgCl ₂ . Achieved. Color development was continued until a uniform background color developed on the strip, and the strip was rinsed twice with deionized water to stop expression.

Panels of HTLV-I or HTLV-II positive sera were tested. These panels were previously identified as HTLV-I or HTLV-II positive by PCR assay (Lipka). The results are shown in Figure 6, where panel A-G is HTLV-I antiserum and panel H-S is HTLV-II antiserum. Virus lysate protein gp24 is immunoreactive with all serum samples such as recombinant peptide gp21E. MTA-4 is immunoreactive only against HTLV-I serum samples.

While specific embodiments of the present invention, methods of construction, and uses have been described, it will be apparent to those skilled in the art that various other uses, formulations, and methods of implementation are within the scope of the present invention.

Claims (7)

An immunoreactive peptide antigen consisting of about 50 amino acids derived from HTLV-II envelope protein gp46 and used to detect the presence of HTLV-I infection in human serum, wherein the antigen is the amino acid sequence Met-Thr-Leu-Leu- Val-Asp-Ala-Pro-Gly-Tyr-Asp-Pro-Leu-Trp-Phe-Ile-Thr-Ser-Glu-Pro-Thr-Gln-Pro-Pro-Pro-Thr-Ser-Pro-Pro- Immune Reactivity Including Immunogenic Regions Formed by Leu-Val-His-Asp-Ser-Asp-Leu-Glu-His-Val-Leu-Thr-Pro-Ser-Thr-Ser-Trp-Thr-Thr-Lys Peptide antigen. An immunoreactive peptide antigen consisting of less than about 50 amino acids derived from HTLV-II envelope protein gp46 and used to detect the presence of HTLV-I infection in human serum, wherein the antigen is the amino acid sequence Ser-Pro-Pro- Immune reactive peptide antigens composed of immunogenic regions formed by Leu-Val-His-Asp-Ser-Asp-Leu-Glu-His-Val-Leu-Thr-Pro-Ser-Thr-Ser-Trp-Thr-Thr . An immunoreactive peptide antigen consisting of less than about 50 amino acids derived from HTLV-II envelope protein gp46, wherein the antigen is an amino acid sequence Ser-Pro-Pro-Leu-Val-His-Asp-Ser-Asp-Leu-Glu- An immunoreactive peptide antigen consisting of an immunogenic region formed by His-Val-Leu-Thr-Pro-Ser. A kit for detecting and distinguishing infections of HTLV-I and HTLV-II, wherein the amino acid sequence Thr-Ala-Pro-Pro-Leu-Leu- is immunoreactive with an anti-HTLV-I monoclonal antibody derived from ATCC HB8755. A first stationary peptide antigen containing a peptide region consisting of Pro-His-Ser-Asn-Leu-Asp-His-Ile-Leu-Glu-Pro-Ser derived from HTLV-I envelope gp46, and an amino acid sequence Ser-Pro About 50 amino acids derived from HTLV-II envelope protein gp46 containing a peptide region consisting of -Pro-Leu-Val-His-Asp-Ser-Asp-Leu-Glu-His-Val-Leu-Thr-Pro-Ser A solid support supported by a second peptide antigen, wherein the second antigen immobilized at one or more positions on the solid support is spaced from the position of the HTLV-I derived gp46 peptide antigen, and the serum is Serum antibodies when attached and the reporter means for detecting the presence of human antibodies is bound to the support Kit, which is configured such that the support is in contact with the fixed antigen peptide. The method of claim 4, wherein the HTLV-II specific peptide antigen has an amino acid sequence Ser-Pro-Pro-Leu-Val-His-Asp-Ser-Asp-Leu-Glu-His-Val-Leu-Thr-Pro-Ser-Thr A kit characterized by containing a peptide region consisting of -Ser-Trp-Thr-Thr. The method of claim 4, wherein the HTLV-II specific peptide antigen has an amino acid sequence Met-Thr-Leu-Leu-Val-Asp-Ala-Pro-Gly-Tyr-Asp-Pro-Leu-Trp-Phe-Ile-Thr-Ser -Glu-Pro-Thr-Gln-Pro-Pro-Pro-Thr-Ser-Pro-Pro-Leu-Val-His-Asp-Ser-Asp-Leu-Glu-His-Val-Leu-Thr-Pro-Ser Kit containing a peptide region consisting of -Thr-Ser-Trp-Thr-Thr-Lys. The method of claim 4, wherein the HTLV-I specific peptide antigen has an amino acid sequence Ser-Leu-Leu-Val-Asp-Ala-Pro-Gly-Tyr-Asp-Pro-Ile-Trp-Phe-Leu-Asn-Thr-Glu -Pro-Ser-Gln-Leu-Pro-Pro-Thr-Ala-Pro-Pro-Leu-Leu-Pro-His-Ser-Asn-Leu-Asp-His-Ile-Leu-Glu-Pro-Ser-Ile Kit comprising a peptide region consisting of -Pro-Trp-Lys-Ser-Lys.