DK168892B1 - DNA sequence which encodes proteins which are immunologically reactive with antibodies against LAV/HTLV- III, a recombinant plasmid which contains the DNA, a bacterial cell which is transformed with a plasmid, a process for preparing the protein, and the use of the protein in analyses - Google Patents

Description

in DK 168892 B1

Technical area

The invention relates to a DNA sequence encoding proteins that are immunologically reactive with antibodies against LAV / HTLV-III, recombinant plasmid containing the DNA, bacterial cell transformed with 5 µm of asthma, method for producing the protein and using the protein by assays.

Background

Acquired immunodeficiency syndrome (AIDS) is a defect in the transmissible cellular immunity that is characterized by opportunistic infections and certain rare diseases. The dominant risk groups for AIDS include gay men, intravenous drug users, transfusion recipients and blood products, and heterosexual partners and children of high-risk individuals, which suggests that an infectious agent transmitted through intimate contact or blood products is involved.

Recent evidence indicates that the infectious agent responsible for the transmission of disease is a new lymphotropic retrovirus known as lymphadenopathy-associated virus (LAV) (Barré-Sinoussi et al., Science 220: 868 (1983)). Similar viruses have been reported by other science groups (Popovic et al., Science 224: 497 (1984); Levy et al., Science 225: 840 (1984); Vilmer et al., Lancet 1: 753 (1983)) and have have been designated human T-cell Lymphotropic virus type III (HTLV-III), AIDS-associated retrovirus (ARV) or immunodeficiency-associated virus (IDAV). Even more recent results indicate that LAV, HTLV-III, ARV and IDAV share several important characteristics including essential nucleotide homology (Wain-Hobson et al., Cell 40: 9 (1985); Muesing et al., Nature 313: 450 (1985). ; Sanchez-Pescador et al., Science 227: 484 (1985)), and that they should be considered in the Sun 30 of the same virus, although there is a likelihood of strain-to-strain variations among the viral isolates. In addition to exhibiting substantial nucleotide homology, the isolates are similar in morphology, cytopathology, requirements for optimal reverse transcriptase activity, and at least share some antigenic properties (Levy, see above; Schupbach et al., Science 224: 503 (1984)).

As stated above, it is known that the virus can be transmitted through blood products (blood, blood serum, blood plasma and fractions thereof), making it important to screen blood products to determine if the donor has been exposed to the virus. This can be done in any way by several methods, including enzyme-linked immunosorbent assay (ELISA) for the detection of LAV antibodies and related viruses. People whose blood contains antibodies 5 against LAV are said to be "seropositive". Blood from seropositive donors can by detection be eliminated from the blood supply, thereby helping to prevent the spread of the disease.

Individuals' immune response to LAV varies. Antibodies can be produced against several viral proteins, including p13, 10p18, p25, p36, gp43, p55, gp110, etc. (Schupbach et al., N.Enol. J. Med. 312: 265 (1985)). Not all individuals will produce antibodies against the same proteins or against the same epitope on a particular protein.

Detection of Seropositive Persons As Currently Performed, 15 have several inherent problems. Most prominent among these problems is the need to isolate antigen from whole viruses for immunological assays. This isolation requires the handling of large volumes of live, potentially infectious virus and as such poses a significant security risk. Furthermore, there are problems in relation to the yield, purity and reproducibility of viruses from one preparation to the next. This can result in an unacceptable number of false positives and / or negatives. Accordingly, in the art, alternative methods are needed to produce viral antigens which are useful in blood screening assays and further provide 25 similar advantages.

EXPLANATION OF THE INVENTION Briefly, the present invention relates to DNA sequences comprising a portion of the group-specific antigenic region (gag) of the LAV genome, the portion which is essentially pGAG-1, pGAG-2 or pGAG-3. and encoding a protein that is immunologically reactive with antibodies against LAV / HTLV-III.

The invention also relates to a recombined plasmid capable of replication in bacterial host cells, which includes 35 prokaryotic transcription and translation signals for expression, followed by the read phase of the DNA sequence described above.

In a preferred embodiment, signals are selected from an operon such as the trp operon. which can be induced and / or suppressed.

The invention further relates to bacterial cells, such as E. coli, which have been transformed with the recombined plasmid described above.

The invention further relates to a method for producing proteins that are immunologically reactive with antibodies against LAV / HTLV-III, which comprises introducing into the bacterial host cell the recombinant plasmid described above. After introduction of the p ismid, the bacterial host is grown in a suitable medium. Expression of the protein is then induced and the protein product with the sequence is isolated from the bacterial host. The protein product 10 can then be purified after isolation, e.g. by gel chromatography.

A further aspect of the invention is a method for determining the presence of LAV / HTLV-III antibodies in a biological fluid. The method comprises incubating the biological fluid with a protein produced by bacterial cells transformed with a recombinant plasmid described above to produce a reaction mixture and then analyzing the reaction mixture to determine the presence of the antibodies. In a preferred embodiment, the analysis step of the reaction mixture comprises contacting the reaction mixture with a labeled specific binding partner to the antibody.

Yet another aspect of the invention relates to a method for determining the presence of LAV / HTLV-III antigen in a biological fluid, which method comprises incubating the biological fluid with a labeled protein produced by bacterial cells transformed with the above described recombined plasmid, and either sequentially or simultaneously with an antibody for the protein so that specific binding occurs. Then, the reaction mixture formed during the incubation is analyzed to determine the amount of labeling associated with the antibody.

In a further aspect of the invention, there is disclosed a method for determining the presence of antibodies against LAV / HTLV-III in a biological fluid, which comprises latex spheres conjugated with a protein produced by bacterial cells transformed with a recombined plasmid. capable of replicating in 35 bacterial host cells, which plasmid includes prokaryotic transcriptional and translational signals for expression. The signals are followed by a DNA sequence comprising a portion of the gag region of the LAV genome, the portion encoding a protein that is immunologically reactive with antibodies against LAV / HTLV-III. The biological fluid is then incubated with the latex sphere / protein conjugate to form a reaction mixture. The reaction mixture is then analyzed to determine the presence of the antibodies.

Other aspects of the invention will become apparent from the following detailed description and accompanying drawings.

Brief description of the drawing

Fig. 1 illustrates the construction of pSS-5 and pBS-5 from AJ19.

Figure 2 illustrates the trp-E exorcation vectors pJH12 and pJH14 including the polylinker sequences.

Fig. 3 illustrates the origin of the LAV insertions in pGAG-2 and pGAG-3.

Fig. 4 illustrates the construction of pGAG-2 from pJH12 and pSM002. Fig. 5 illustrates the construction of pGAG-3 from pJH14 and pBPB14.

Fig. 6 illustrates the trp-E expression vector pATH10 including the reading frames for the cleavage sites in the polylinker region.

Fig. 7 illustrates the origin of the LAV insertions in pGAG1.

Fig. 8 illustrates the construction of pGAG1 from pBS-5, pUC18 and pATH10.

Fig. 9 is a histogram of optical density values obtained from serum samples.

Best Embodiment of the Invention

Prior to carrying out the invention, in order to understand it, it may be helpful to present definitions of certain terms used herein.

Lymphadenopathy-associated virus (LAVI: A human T-lymphotropic retrovirus.) For the purposes of the present invention, a virus is believed to be the same or equivalent to LAV if it meets substantially the following criteria: (a) the virus is tropical for T lymphocytes, especially T helper cells (CD4 +, according to the international nomenclature defined in

Bernard et al., Leucocyte Typing. New York: Springer Verlag (1984)).

(B) the virus is cytopathic for infected CD4 + cells (rather than transforming, as for HTLV-I and II); (c) the virus encodes an RNA-dependent DNA polymerase (reverse 2+ transcriptase) which is Mg-dependent (optimal concentration 5 mM, optimal pH 7.8, cannot be inhibited by actinomycin D) and can use 5 DK 168892 B1 oligo 12-18 as a primer for reverse transcription from its 3'-LTR; (d) the virus forms in a sucrose gradient band having a density of ca. 1.16; o (e) the virus may be labeled with [H] -uridine; (f) the virus can be distinguished by members of the HTLV-I / II family of viruses by immunological and nucleotide sequence criteria (with this criterion HTLV-III should not be considered a member of the HTLV-I / II family); ; (G) the virus cross-reacts substantially immunologically with the proteins encoded by the gag and env regions of LAV; and (h) the virus shares substantial nucleotide homology (75-100%) and amino acid sequence homology (75-100%) with LAV.

Immunologically reactive; An antigen and an antibody are said to be

"immunologically reactive" if they are able to bind specifically to each other, typically having an affinity of at least 106 µm, more frequently at least 10 µm

Transformed or transformed: the process of stable and hereditary modification of the genotype of a recipient cell or organism by introduction of purified DNA.

Lymphadenopathy-associated virus (LAV) can be isolated from patients with AIDS or lymphadenopathy syndrome. Lymph nodes from such patients are typically subjected to biopsy and placed in culture medium supplemented as needed to produce growth. A mitogen such as interleukin-2 (IL-2) or phytohemagglutinin (PHA) may be included; antiserum against human interferon may also be included. Reverse transcriptase activity typically appears after ca. 15 days of culture, indicating the presence of virus. The virus can be concentrated from the culture supernatant using a nonionic detergent followed by banding in a sucrose gradient. These and other methods of purification are well known in the art and are described e.g. in Montelaro et al., J. Virology 42: 1029 (1982).

LAV can be propagated in any of a number of ways.

It can be grown in T lymphocytes derived from the umbilical cord or from peripheral blood or from bone marrow. Alternatively, it can be propagated in dead T cells or B cells; see e.g. Popovic et al., Science 224: 6 DK 168892 B1 497 (1984) and Montagnier et al., Science 225: 63 (1984). The growth of the virus is usually measured in the presence of reverse transcriptase activity.

A LAV genomic clone can be prepared by any of several well known methods in the art, including but not limited to those described by Hahn et al., Nature 312: 166 (1984); Alizon et al., Nature 312: 757 (1984); Luciw et al., Nature 312: 760 (1984); and Muesing et al., Nature 313: 450 (1985).

Briefly, one of these methods is the following (Alizon et al.): DNA

isolated from LAV-infected T cells in a healthy donor, partially digested with a restriction endonuclease such as HindIII, and the resulting digestion mixture is electrophoretically fractionated. Fragments corresponding in size to the size of the entire LAV genome (about 9.2 15 kb) are eluted from the gel, precipitated, resuspended and ligated into the ends of a vector treated with a suitable restriction enzyme. The ligation mixture is packed in bacteriophage particles. Bacteria are transformed with the bacteriophage and the clones are screened for LAV insert! see using a suitable probe (such as cDNA prepared from LAV RNA). From a positive clone, the desired region of LAV may be subcloned into a bacterial plasmid vector such as pUC 18. Additional subcloning may be desirable to remove unwanted sequences and add additional attack sites (in the form of a polylinker) at both ends for the purpose of facilitate cloning in an expression vector.

The LAV sequences can be subcloned into an inducible expression vector. A number of expression vectors are known in the art, and they include Eight 11: Tn5 (Hall et al., Nature 311: 379 (1984); trp E (Paul et al., Eur. J. Cell. Biol 31: 171 (1983) pINIII (Masui et al., Biotechnology Jan.1984 p.81).

The proteins obtained can be partially purified and used as antigens in immunoassays. For use as antigens in immunoassays, the proteins can be used in labeled or unlabeled form. Once labeled, the labels may include radioisotopes, fluorophores, enzymes, luminescent substances or particles. These and other labels are well known in the art and are described in e.g. the following US patents: 3,766,162, 3,791,932, 3,817,837, 3,996,345 and 4,233,402.

Assays utilizing the recombined proteins of the present invention may be heterogeneous (i.e., require a separation step) or homogeneous. If the analysis is heterogeneous can. a number of separation methods, including centrifugation, filtration, chromatography or magnetism, are used.

A preferred assay for screening blood products or other physiological fluids for the presence of antibodies is an ELISA. Typically, antigen (in this case, a recombined protein or a combination of recombined proteins) is adsorbed to the surface of a microtiter well. Residual protein binding sites on the surface are then blocked with a suitable agent such as bovine serum albumin (BSA), heat-inactivated normal goat serum (NGS) or BLOTTO (a buffered solution of fat-free dry milk which also contains a preservative, salts and anti-foaming agent). The well is then incubated with a sample suspected of containing specific antibodies. The sample can be added unmixed or, more often, it can be diluted, usually in a buffered solution containing a small amount (0.1-5.0% by weight) of protein such as BSA, NGS or BLOTTO. After incubation for a sufficient time to allow specific binding to take place, the well is washed to remove unbound protein and incubated with labeled anti-human immunoglobulin (Q-Hulg). The label can be selected from a variety of enzymes, including horseradish peroxidase (HRP), β-galactosidase, alkaline phosphatase and glucose oxidase. Sufficient time is allowed for specific binding to take place, after which the well is washed again to remove unbound conjugate and the substrate for the enzyme is added. The color is allowed to develop and the optical density of the contents of the well 25 is determined visually or by means of an instrument.

For convenience, reagents for ELISA can be provided in the form of kits. These kits may include microtiterpads to which viral proteins produced by recombinant methods may be preabsorbed, several diluents and buffers, labeled 30 conjugates for the detection of specifically bound antibodies, and other signal-derived reagents such as enzyme substrates, cofactors and chromogens.

Sera of LAV-infected individuals contain antibodies to many LAV proteins, including p13, p18, p25, p36, gp43, p55, gp110, and others. Although not all individuals produce antibodies against the same proteins, individual sera are most consistently reactive with antibodies against the gag proteins, p25 and p18, and against the env proteins, gp43 and gp110. Differences between people can be due to several factors, including the progression of the disease. There are e.g. Some evidence that antibodies to nuclear proteins are dominant in the early stages of the disease, but decreases with the progress of immune suppression. In contrast, antibody titer against the envelope glycoproteins is thought to persist during the later stages of the disease.

Further variation in response may be due to polymorphism in the genes encoding viral proteins. Various isolates of LAV exhibit significant changes in the env protein. Interestingly, the gag protein sequences are highly conserved.

In any seropositive sample tested, anti-substances have been seen against at least one of the gag proteins (p18 or p25) or one of the env proteins (gp43 or gp10). However, none of these proteins is universally recognized by seropositive individuals. Therefore, it appears essential that blood be screened for antibodies to at least one 15 gag protein and one env protein. In a previous patent application, U.S. Patent No. 721,237, entitled "Expression of Immunologically Reactive Viral Proteins," the present invention utilizes portions of the envelope region (env) of the LAV genome encoding a protein that is immunologically reactive with LAV antibodies. . In the present invention, portions of the gag region of the LAV genome that encode a protein that is also immunologically reactive with LAV antibodies are used. In a combination, proteins encoded by the gag and env regions can be used to detect high-sensitivity seropositive individuals.

In the following example, two overlapping regions of LAV gag (Figure 3) and another part of the gag region, including most of p18 and parts of p25 (Figure 7), were selected for expression. The choice was influenced by the finding that p18 and p25 were the gag proteins that were most reproducibly reactive with sera from LAV-infected individuals. Our selection within these sequences was dictated by the localization of hydrophilic regions and protease attack sites (both of which may be exposed to the surface of the protein and immunogenic) as well as the size limitation of effective expression in the selected vectors (trg_E).

The LAV genomic clone, designated AJ19, was subcloned into the bacterial plasmid vector, pUC18. The resulting subclone, designated pBT-1, was further subcloned to generate pSS-5 and pBS-5, which mainly contained gag and null sequences.

In one procedure (Example I), the gag sequences were further subcloned into pIC19R (forming pi azimides pSM002 and pBPB14) and regions of the gag sequence were transferred into the trp E-inducible expression vector. The gag DNA was inserted into the reading frame downstream of the trp E gene, resulting in the expression of a trp E-gag fusion protein when E. coli was transformed with this construct. The resulting proteins were partially purified and characterized by their reactivity in ELISA with sera from known seropositive and known seronegative subjects. Two useful constructs, designated pGAG-2 and pGAG-3, were identified.

In another procedure (Example II), part of the gag sequence (bp 375-547) was further subcloned into pUC18amp (forming plasmid 0674-14-10). Another region of the gag sequence (bp 505-961) was transferred into plasmid 0674-14-10), forming plasmid 0674-27-38. The gag sequences (gp 347-961) were transferred into the tro-E-inducible expression vector pATH10. The gag DNA was inserted into the reading frame of the downstream faith E-a. resulting in expression of a true E-gag fusion protein when E. coli was transformed with this construct. The resulting proteins were partially purified and characterized by their reactivity in ELISA with sera from known seropositive 20 and known seronegative subjects. A useful construct, designated pGAG-1, was identified.

The following examples are provided for illustration only, but are not limiting in any way.

Example I

A. Construction of the trp-gag expression vectors

Any of several bacterial expression systems can be used to express foreign proteins, the trp E system was chosen for the expression of LAV gag sequences because it contains a strong inducible promoter, but its expression can also be suppressed so that foreign (and potentially toxic) protein does not accumulate in the bacteria for long periods of time.

Expression vectors are restricted by type and reading frame in their restriction sites. Eg. believe E-excitation vectors require that the DNA insert have termini which are compatible with the recognition sites of Barn HI, HindIII or EcoRI. Greater diversity can be introduced by first subcloning the region of interest into an intermediate vector which has a wider region and an altered structure of restriction sites. The region of interest can then be introduced into an expression vector using restriction sites provided by the intermediate vector.

Our strategy was therefore to first subclone most of the case domain of the LAV genome into a transfer vector, pIC19R. Then, 5 different fragments of these subclones were ligated into one of two trp expression vectors (pJH12, pJH14), which differed only in reading frame at the restriction sites in the polylinker region (see Figure 2).

1. Subcloning of the LAV Aenome 10 a. Preparation of Phage DNA.

The entire LAV genome was obtained from the Pasteur Institute in the form of g-phage particles containing a 9.2 kb genomic DNA insert at the HindiII recognition site in phage AL47.1. This clone is referred to as AJ19 and is described in Wain-Hobson et al., Cel! 40: 9 (1985). AJ19 phage particles were transfected into the Q359 strain of E. coli K-12 (genotype of Q359 is hsdRk ", hsdMk +, supF, $ 80, P2) according to the procedure of Maniatis et al., Molecular Cloning: A Laboratory Manual. New York : Cold Spring Harbor Laboratory, 1982, p.64.

A single plaque was collected and the phage was multiplied by the 20 plate lysate method (Maniatis, see above, p.65). After 9 hours of incubation at 37 ° C, the plates (100 mm diameter) containing confluent plaques were overlaid with 5 ml of 100 mM NaCl / 20 mM MgSO 4/50 mM Tris, pH 7.5. After incubation for 12 hours at 4 ° C, the liquid was collected and extracted twice with an equal volume of chloroform.

To 10 ml of the resulting aqueous phase containing phage particles was added 2 ml of 0.25 M EDTA / 2.5% SDS / 0.5M Tris, pH 9, and the suspension was incubated at 70 ° C for 15 minutes to open the phage. 2.5 ml of 8M potassium acetate was added and the solution was incubated on ice for 15 minutes, then centrifuged for 10 minutes at 12,000 x g at 4 ° C to pellet protein. The supernatant was transferred to a 50 ml polypropylene centrifuge gel and extracted with an equal volume of phenol (pH 8, equilibrated with 1 M Tris, pH

8) at 20 ° C. The aqueous phase was then extracted with an equal 35 s volume of chloroform: isoamylal carbon (24: 1) at 20 ° C. To the aqueous phase, 2.5 volumes of 95% ethanol were added to precipitate the DNA. After centrifugation, the DNA pellet was dried and resuspended in 10 mM Tris HCl, pH 7.4 / lMM EDTA.

b. Subcloning of the gag region.

11 DK 168892 B1

About 12 mg of AJ19 DNA prepared in A.l.a. above was digested completely with the restriction enzyme Sst I (Bethesda Research Labs, Bethesda, MD), which intersects only in the LTR regions of this in the sol of LAV. The digestion mixture was subjected to electrophoresis 5 at 1 V / cm through 0.9% agarose in 0.089 M Tris-borate / 0.089 M boric acid / µM EDTA. The position of the 9.1 kb fragment was determined relative to molecular weight standards after staining with ethidium bromide. This band was electroeluted in NA45 paper (Schleicher and Schuell, Keene, NH). The DNA was recovered from the paper according to the instructions given by the supplier.

The 9.1 kb Sstl fragment was ligated into the Sstl pre-digested vector pUC18 in a ratio of 10 inserted molecules: 1 vector molecule. E. coli strain HB101 was transformed with the ligation mixture by the CaClg procedure of Maniatis et al. (See above 15) and plated on LB plus ampicillin (200 mg / ml) agar plates.

Single colonies were collected and diluted in 3 ml of LB plus ampicillium medium and grown overnight at 37 ° C with constant shaking. Plasmid DNA was prepared by the alkaline lysis method (Maniatis et al., Supra, p.368). 1 colony was selected which contained the 9.1 kb Sstl insert in such orientation that the EcoRI recognition site in the polylinker was closest to the 5 'end of the LAV genome, as determined by restriction analysis of the plasmid DNA . This subclone was designated pBT1 (ATCC deposit number 53069) (Figure 1).

25 Two subclones from plasmid pBT-1 were prepared (see Figure 1). First, (pSS-5), pBT-1 was digested with Sal-I and then ligated. Second, pBS-5, pBT-1 was digested with Barn HI and BglII and then ligated. In both cases, the 5 'end of the LAV genome was retained in the vector. HB101 cells were transformed with 3 ° the ligated DNA and colonies containing the pSS-5 and pBS-5 insertions were identified by restriction analysis of the purified plasmid DNA.

Areas of pSS-5 and pBS-5 were then subcloned into the intermediate vector, pIC19R. As discussed above, this provided the necessary restriction sites in the correct reading frame for transfer to the expression vectors.

Specifically, the HindiII fragment of pBS-5 (bp 631 to bp 1258 of the LAV genome; numbered according to Wain-Hobson et al., Cell 40: 9 (1985)) was ligated into HindIII and pIC19R digested with 12 DK 168892 B1. calf intestinal phosphatase (Figure 4). The ligated DNA was taken up by CaClg shock-treated E.coli TB-1. Using the chromogenic substrate 5-bromo-4-chloro-3-indolyl - /} - galactoside (Sigma), ampicillin-resistant colonies were screened for inactivation of 5 /} -gal actosidase due to insertion of the gag sequence.

The orientation of the insert was determined by digesting plasmid DNA with Pvu II. The resulting plasmid is referred to as pSM002.

The pvuII-BglII fragment (bp 691 to bp 1642 of the LAV genome) was ligated into SmaI and BglII digested pIC19R (Figure 5). The ligated DNA was taken up by E. coli HB101, and the resulting ampicillin-resistant colonies were screened with the chromogen described above.

Suitable colonies were further screened by restriction analysis of plasmid DNA. The resulting plasmid is referred to as pBPB14.

2. Inserting the gag sequence into trp vectors.

The expression vectors contained the E. coli trp operon promoter, the operator and the trp E gene inserted into pBR322 (Figure 2). The trp E gene was truncated at its Bgl-II recognition site closest to the 5 'end by insertion of a polylinker sequence (Konopka et al., J. Virol. 51: 223 20 (1984)). The different trp vectors (pJH12 and pJH14) differed with respect to reading frame at the restriction sites in the polylinker region.

Insertion of an open reading frame into the appropriate vector resulted in the generation of a fusion protein with trp_E sequences at the amino terminal end (Spindler et al., J. Virol. 49: 132 (1984)).

pGAG-2 (ATCC deposit number 53111) was constructed by digesting the HindIII gag subclone (pSM002, Figure 4) with BglII and BamHI.

The BglII and BamHI attack sites were located within the surrounding polylinker region of pIC19R. The gag fragment was purified and ligated into BamHI digested pJH12 (Figure 4). Ligated DNA was taken up by CaCl2 shock-treated E. coli HB101, and colonies were grown in the presence of ampicillin (100 µg / ml) and tryptophan (40 mg / ml). Tryptophan was used to suppress expression of the foreign protein, where accumulation of this would have been destructive to the bacteria. Suitable ampicillin-resistant colonies were identified by hybridization with a radiolabeled gag DNA probe. Restriction analysis of minilysates was used to confirm the presence and orientation of the insert.

pGAG-3 (ATCC accession number 53112) was constructed by EcoRI digestion of pBPB14, which contains the gag region between PVU-II

13 DK 168892 B1 (bp 691) and Bgl-II (bp 1642), as shown in Figure 5. The EcoRI attack sites are in the polylinker sequences surrounding the gag sequences. The gag fragment was gel purified and ligated into pJH14 treated with Eco RI and calf phosphatase. E. coli HB101 cells were transformed with the ligated DNA, plated as described above, and screened by restriction analysis to confirm the presence and orientation of the gag sequence in pJH14.

B. Protein Expression 10 1. Transformation of E. coli with the trp-gag constructs

Each of the recombinant trp-gag expression plasmids was transferred from E. coli HB101 to E. coli C600, because the latter is a better host for protein production. Transfer involved transformation of CaCl2 - shock treated C600 with supercoil DNA from mini-15 2 lysates of HB101. Bacteria were plated in the presence of ampicillin and tryptophan as described (Konopka et al., J. Virol 51: 223 (1984)). Ampici11 in nresi stent colonies were screened by minilysates to confirm the presence of the appropriate plasmid.

2. Expression of trp-gag proteins Growth and induction of E. coli C600 transformed with the trp expression vectors was performed as described (Spindler et al., J.Virol. 49: 132 (1984); Konopka et al. , J. Virol. 51: 223 (1984)). In brief, minimal medium containing tryptophan (40 mg / ml) and ampicillin (100 / zg / ml) were inoculated with transformed bacteria from glycerol strain cultures. The cultures were grown with aeration at 37 ° C overnight. The overnight cultures were then inoculated at 1: 100 in fresh minimal medium containing ampicillin (10 ° -g / ml) but not 3Q tryptophan. These cultures were grown with aeration for 2-3 hours (up to early log phase) at 37 ° C. The inducer, 3β-indolacrylic acid (Sigma), was added to a final concentration of 20 µg / ml from freshly prepared stock solutions at 20 mg / ml in 95¾ ethanol.

Induced cultures were grown at 37 ° C with aeration for from 35 to 4-5 hours and then pelleted and optionally frozen. Protein exchange from pGAG-2 and pGAG-3 was typically between 10-30 mg / liter.

14 Isolation and purification of trp-gag proteins

Fusion proteins were partially purified from cell pellets as described. (Konopka et al., J. Virol. 51: 223 (1984)). Briefly, bacteria were resuspended in 100 ml of 50 mM Tris, pH 7.5 / 0.5 mM EDTA / 150 mM NaCl (TNE) per ml. liter of induced culture. Lysozyme (Sigma) was added to a final concentration of 1 mg / ml. After 15 minutes at 0 ° C, NP40 was added to the mixture to a final concentration of between 0.05% and 0.2% for 10 minutes at 0 ° C. 10 1-2 mg of DNase (Sigma) was then added with 150 ml of DNase buffer (1.5 M NaCl / 12 mM

MgClg). Reaction mixtures were incubated until they were no longer viscous, usually from several hours to overnight. Insoluble proteins were then pelleted by centrifugation for 15 minutes at 8000 x g at 0 ° C. Pellets were washed 2 times in TNE and then analyzed for the presence of insoluble proteins by denaturing polyacrylamide gel electrophoresis. Proteins were visualized by staining with Coomassie brilliant blue.

Alternatively, the insoluble pellet was denatured and reduced by resuspension of the pellet from ca. 200 ml cells in 180 µl 0.14 20 M Tris HCl, pH 7.8 / 6% SDS and 20 ml b-mercaptoethanol and heating at 95 ° C-100 ° C for 20 minutes. The sample was then dried in a Speed

Vac concentrator (Savant Instruments, Hicksville, NY). To remove the SDS, the sample was extracted as follows: The pellet was resuspended in 1 ml of acetone / triethylenamine / acetic acid / water 25 (17/1/1/1) and vigorously treated on vortex, the suspension was cooled on ice for 1 hour. hour, centrifuged and the supernatant was discarded. This extraction was repeated twice with 0.4 ml of the above acetone mixture and then twice with 0.4 ml of acetone alone. The pellet was then dried in the Speed Vac concentrator.

30

The protein pellet was then dissolved in 1.4 ml of 6 M guanidine hydrochloride / 0.1 M Tris HCl, pH 8.5 / 10 mM DTT and incubated at 37 ° C for 4 hours. Any particulate material was then removed by centrifugation. The sample contained in the supernatant was placed on a 1.6 x 96 cm Sephacryl S-300 column (PHarmacia, Piscataway, NJ) equilibrated in 6 M guanidine hydrochloride / 10 mM EDTA / 1 mM DTT. The column was eluted with the same buffer and the absorbance of the eluted material was measured at 280 nm.

The fractions were analyzed by ELISA with LAV seropositive sera and with E. coli seropositive sera to determine the specific activity and the contaminating activity, respectively. The fractions that showed reactivity with the former but not with the latter serum were used in subsequent ELISA on patient sera.

5 D. Immunological reactivity of trp-gag proteins 1. Western blot analysis

Sub-samples of the insoluble protein preparations expressed by pGAG-2 and pGAG-3 were dissolved in 2% sodium dodecyl sulfate / 100 mM Tris, pH 6.8 / 20% glycerol / 1.5 M / M mercaptoethanol and electrophoresed on denaturing polyacrylamide gel. . Proteins were electro-transferred on nitrocellulose (BA85, Schleicher and Schuell, Keene, NH), and the filters were blocked with 5% bovine serum albumin (Sigma). The filters were then probed with E. coli adsorbed human sera pool one from AIDS patients. The filters were then developed with HRP-conjugated goat-a-Hulg. The pool was reactive with both trp-gag fusion proteins but not with faith E protein alone.

2. Analysis by ELISA

Column-purified GAG-2 and GAG-3 proteins were diluted in 0.05M carbonate / bicarbonate buffer (pH 9.6) to a final concentration of 0.3 µg / ml and 3.4 µg / m 2, respectively. Sub-samples of 50 µl per ml were used. well and they were incubated at 4 ° C overnight. Plates were then blocked with BLOTTO (5% + w / v µm (dry milk without fat / - 0.01% thimerosol / 0.01% antifoam A in 0.01 M sodium phosphate, pH 2.5 7.2 / 0.15 M Sera were diluted 1: 100 with a 1: 1 mixture of BLOTTO and PBS (0.01 M sodium phosphate, pH 7.3 / 0.15 M NaCl), and 50 µl diluted sera was added per well for 1 hour at 37 ° C. The sera was removed, and the plates were washed 3 times in wash buffer (0.15 M NaCl / 0.05% [w / v] Tween 20) before addition of 100 / t of goat anti-human IgG / horseradish peroxide seconjugate (50% stock diluted 1: 10,000 in 50mM NaCitrate / -0.05% Tween 20/1% heat-inactivated normal goat serum; obtained from Antibodies, Inc., Davis , CA) for 1 hour at 37 ° C. The conjugate was removed and the plates washed 3 times with 0.15 M NaCl / 0.05% (w / v) Tween 20. The ELISA plate was developed by adding 100 μΐ per well of substrate solution (10 mg 3,3 ', 5,5'-tetramethyl-benzidine in 50 ml 0.05 M sodium mcitrate, pH 7.0) for 30 minutes at room temperature. The reactions were stopped by adding 100 µl / well of 3N H2SO4 and the optical density at 450 nm was determined on an automated ELISA reader. Proteins produced by pGAG-2 and pGAG-3 were both found to be reactive with a panel of known seropositive sera.

The panel included sera from two healthy heterosexuals, 5 5 people diagnosed as LAS (lymphadenopathy syndrome) and / or homosexual, 1 person with AIDS and a pool of sera from AIDS patients. Sera from 2 heterosexuals were scored as negative in a helivirus ELISA. All AIDS, LAS, and / or homosexual sera were confirmed to be seropositive in a whole-virus ELISA and by 10 radiolabelled immunoprecipitation of LAV antigens. The results from these sera are shown in Table I. These findings demonstrate that sera reactive with LAV antigens are also reactive with bacterially expressed gag proteins.

3. Fluorescence Sensor Glass Test for Detection of Serum Antibody

against LOW

Soluble protein prepared as described above was conjugated to latex beads and the protein / bead preparation was ethanol fixed to slides. A sub-sample of patient serum is incubated with the protein / beads on a slide. The slides were washed and FITC-labeled anti-human immunoglobulin was added in Evans blue counterstain. The slides were washed and mounting medium was added to each as well as a cover glass.

Alternatively, the protein / sphere preparation was placed in test tube 25 for incubation with patient serum. The tubes were centrifuged and washed and the FITC-labeled anti-human immunoglobulin was added in Evans blue counterstain. The glasses were centrifuged and the supernatant aspirated. A sub-sample of the spheres was placed on a microscopy slide and ethanol fixed and coverslips were mounted.

All slides were examined by fluorescence microscopy. If test serum is antibody positive, the beads appear as fluorescent green beads; if test serum is antibody negative, the beads appear as red beads.

35 5

Table 1 17 DK 168892 B1

Comparison of GAG-2 and GAG-3 with a whole virus lysate in an ELISA for detection of LAV antibodies.

Whole - Confirmed virus- like sero-

Serumnr. Diagnosis 1 GAT-2 GAG-3 positive

Yl / CDC positive 2,000 1,292 1,580 yes 10 control pool 124 LAS and / or 1,189 0.762 0.396 yes gay 127 LAS and / or 1,046 0.291 1.093 yes gay 15 130 LAS and / or 0.912 0.420 1.200 yes gay 153 LAS and / or 2,000 1.137 1.073 yes gay 501 AIDS 1,109 1,049 1,556 yes 20 666 Unknown 2,000 nd 1,747 yes 637 Rapid 0.097 0.113 0.116 Non Sero-heterosexual Positive 641 Fast 0.199 0.116 0.069 Non Sero-heterosexual Positive 25 4. Reactivity of the combination of trp-gag and trp-env proteins

A trp-gafl protein was combined with a trp-env protein in one ml of crotiter well. ELISA was performed as described above for GAG-2 or GAG-3 alone. Table II shows that the combination of GAG-3 and ENV-3 has a greater sensitivity for detection of seropositive individuals than for each protein alone. Of the seropositive samples, 7/7 was detected when the proteins were combined, while 6/7 was detected with GAG-3 or ENV-3 alone.

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19 DK 168892 B1

Example II

A. Construction of tr ^ -gag expression vectors

Any of several bacterial expression systems can be used to express foreign proteins, the trp E system was chosen for the expression of LAV gag sequences because it contains a highly inducible promoter, but its expression can also be suppressed, thus that foreign (and potentially toxic) protein does not accumulate in the bacteria for long periods of time.

Expression vectors are limited by the type and reading frame in their restriction sites. Eg. the trp E expression vector pATH10 requires the DNA insert to have termini compatible with the recognition sites of BamHI, Cla I, Hind III, Pst I, Sac I,

Sal I, Sma I, Xba I, Xma I or EcoRI. Larger diversity can be introduced by first subcloning the region of interest into an intermediate vector which has a wider region and an altered structure at restriction sites. The region of interest can then be introduced into an expression vector by using restriction sites provided by the intermediate vector.

Therefore, the strategy was first to subclone the desired gag region 20 of the LAV genome into a transfer vector, pUC18amp. Then, the gag sequences of this subclone were ligated into the faith exorcation vector pATH10, which contained a polylinker region with appropriate restriction sites in the reading frame followed by expression of the gag insert (Figure 6).

25 1. The subclonin of the LAV genome

a. Phage DNA Preparation The entire LAV genome was obtained from the Pasteur Institute in the form of λ phage particles containing a 9.2 kb genomic DNA insert into the HindIII site of attack in phage AL47.1. This clone is referred to as XJ19 and is described in Wain-Hobson et al., Cel! 40: 9 (1985). AJ19 phage particles were transfected into the Q359 strain of E. coli K-12 (the genotype of Q359 is hsdRK ', hsdMk +, supF, $ 80, P2) according to the procedure of Maniatis et al., Molecular Cloning: A Laboratory Manual. New York: Cold Spring Harbor Laboratory, 1982, p.64. A single plaque was collected and the plaque multiplied by the plate lysate method (Maniatis, see above, p.65). After 9 hours of incubation at 37 ° C, the plates (100 mm diameter) containing confluent plaques were poured with 5 ml of 100 mM NaCl / 20 mM MgSO 2/50 mM Tris, pH

DK 168892 B1 7.5. After incubation for 12 hours at 4 ° C, the liquid was collected and extracted twice with an equal volume of chloroform.

To 10 ml of the resulting aqueous phase containing phage particles was added 2 ml of 0.25 M EDTA / 2.5% SDS / 0.5M Tris, pH 9, and the suspension was incubated at 70 ° C for 15 minutes to disrupt the phage. 2.5 ml of 8 M potassium acetate was added and the solution was incubated on ice for 15 min, then centrifuged for 10 min at 12,000 x g at 4 ° C to pellet protein. The supernatant was transferred to a 50 ml polypropylene centrifuge gel and extracted with an equal volume of phenol (pH 8, equilibrated with 1 M Tris, pH 8) at 20 ° C. The aqueous phase was then extracted with an equal volume of chloroform: isoamylal carbon (24: 1) at 20 ° C. To the aqueous phase, then, 2.5 volumes of 95% ethanol were added to precipitate the DNA. After centrifugation, the DNA pellet was dried and resuspended in 10 mM Tris HCl, pH 7.4 / 1 mM EDTA.

b. Subcloning of the aag region.

About 12 increase AJ19 DNA produced in A.l.a. above was completely digested with the restriction enzyme Sst I (Bethésda 20 Research Labs, Bethesda, MD), which intersects only in the LTR regions of this isolate of LAV. The digestion mixture was electrophoresed at 1 V / cm through 0.9% agarose in 0.089 M Tris-borate / 0.089 M boric acid / 1 mM EDTA. The position of the 9.1 kb fragment was determined relative to molecular weight standards after staining with ethidium bromide. This band was electroeluted in NA45 paper (Schleicher and Schuell, Keene, NH). The DNA was recovered from the paper according to the instructions of the supplier.

The 9.1 kb SstI fragment was ligated into the SstI digested vector pUC18 at a ratio of 10 inserted molecules: 1 vector-30 molecule. E. coli strain HB101 was transformed with the ligation mixture by the CaClg procedure of Maniatis et al. (see above) and plated on LB plus ampicillin (200 µg / ml) agar plates.

Single colonies were collected and diluted in 3 ml of LB plus ampicillin medium and grown overnight at 37 ° C under constant shaking. Plasmid DNA was prepared by the alkaline lysis method (Maniatis et al., Supra, p.368). 1 colony was selected which contained the 9.1 kb Sstl insert in such an orientation that the EcoRI site of attack in the polylinker was near the 5 'end of the LAV genome, as determined by plasmid restriction analysis. DNA. This subclone was designated pBT-1 (ATCC deposit number 53069) (Figure 1).

pBT-1 was digested with BamHI and BglII and then ligated. The 5 'portion of the LAV genome was conserved within the vector.

5 HB101 cells were transformed with the ligated DNA and colonies containing the pBS-5 insert (see Figure I) were identified by restriction analysis of the purified plasmid DNA.

The qaa coding sequence for pGAG1 was further subcloned into pUC18amp, which provided restriction sites necessary for proper insertion into the ΐτβ expression vector. Two subcloning steps were necessary to concatenate the required gag sequence. In the first step, pBS-5 was digested with Sau3A and the 172 bp fragment ranging from bp 375 to bp 547 (numbered according to Wain-Hobson et al., Cell 40: 9 (1985)) became gel-15 purified. This fragment was ligated into BamHI digested pUC18amp. The ligated DNA was taken up in CaCl 2 shock treated E. coli JM83 and the resulting ampicillas in resistant colonies were screened for the presence of the insert by the chromagen 5-bromo-4-chloro-3-indolyl-β-galactoside. Suitable colonies were screened by restriction analysis of plasmid DNA. The resulting plasmid was referred to as 0674-14-10.

In the second step, 0674-14-10 was treated with Accl and Pst! and ligated into a 456 bp fragment from pBS-5 extending from the AccI site of attack at bp 505 to the PstI site of attack at bp 961. The 25 ligated DNA was taken up by CaClg shock-treated JM83, and ampicillin-resistant colonies were screened by restriction analysis of plasmid DNA. The resulting plasmid, referred to as "0674-27-38", contained the gag coding sequence from bp 375 to bp 961 (see Figure 7).

2. Insertion of the gag sequence into trp vectors pGAG1 (ATCC accession number 53379) was constructed by digesting 0674-27-38 at the PstI and EcoRI polylinker restriction sites surrounding the gag sequence. This fragment was gel purified and ligated to EcoRI and PstI digested pATH10 (see Figure 8). The ligated DNA was taken up by CaCl2 shock-treated E. coli C600, and the ampi-ci11 intrinsic colonies were screened by restriction analysis of plasmid DNA to confirm the presence of the gag sequence.

22 DK 168892 B1 B. Protein Expression 1. Expression of trp-gag Proteins Growth and induction of E. coli C600 transformed with the trp expression vectors was as described (Spindler et al., J. Virol. 49 · * 132 (1984) ), Konopka et al., J. Virol. 51: 223 (1984)). Briefly, minimal medium containing tryptophan (40 µg / ml) and ampicillin (100 µg / ml) were inoculated with transformed bacteria from glycerol parent cultures. The cultures were grown with aeration at 37 ° C overnight. The night cultures were then inoculated at 1: 100 in fresh minimal medium containing ampicillin (100 µg / ml) but not tryptophan. These cultures were grown with aeration for from 2 to 3 hours (up to early log phase) at 37 ° C. The inducer, 3- / Mndolacrylic acid (Sigma), was added to a final concentration of 20g / ml from freshly prepared stock solutions at 20mg / ml in 95% ethanol.

Induced cultures were grown at 37 ° C with aeration for from 4 to 5 hours and then pelleted and optionally frozen. Protein yields from pGAG-1 were typically from 20 to 40 mg / liter.

C. Isolation and purification of trp-qag proteins

Fusion proteins were partially purified from cell pellets as described (Konopka et al., J. Virol. 51: 223 (1984)). Briefly, bacteria were resuspended in 100 ml of 50 mM Tris, pH 7.5 / 0.5 mM EDTA / 150 mM NaCl (TNE) per ml. liter of induced culture. Lysozyme 25 (Sigma) was added to a final concentration of 1 mg / ml. After 15 minutes at 0 ° C, NP40 was added to the mixture to a final concentration of between 0.05% and 0.2% for 10 minutes at 0 ° C. Then 1-2 mg of DNase (Sigma) with 150 ml of DNase buffer (1.5 M NaCl / 12 mM MgClg) was added. Reaction mixtures were incubated until they were no longer viscous, usually from several hours to overnight. Insoluble proteins were then pelleted by centrifugation for 15 minutes at 8000 x g at 0 ° C. Pellets were washed 2 times in TNE and then analyzed for the presence of insoluble proteins by denaturing polyacrylamide gel electrophoresis. Proteins were visualized by staining with Coomassie brilliant blue.

Alternatively, fusion proteins were purified by SDS-polyacrylamide gel electrophoresis. About 0.5 ml of insoluble pellet, representing fusion protein from ca. 200 ml of cells, washed 3 times with 2 ml of 2% deoxycholate / 1 M KCl, and then washed 2 times with TNE. The pellet was then resuspended in 0.4 ml of 5% SDS / 100 mM Tris, pH

6.8 / 20% glycerol / 1.4 M β-mercaptoethanol by vortexing and heating at 100 ° C for 10 minutes. Traces of insoluble material were centrifuged and the supernatant was placed on an 8% polyacrylamide gel. Protein bands were visualized by staining marker 5 lanes at the edges of the gel with Coomassie brilliant blue. The region of the gel containing the fusion proteins was excised and placed in a dialysis bag filled with a buffer of 0.1% SDS / 25 mM Tris, pH 8.0. The fusion protein was then electrophoresed out of the gel and collected in the buffer.

10 D. Immunological reactivity of trp-gag proteins 1. Western blot analysis.

Sub-samples of the insoluble protein preparations expressed by pGAG-1 were dissolved in 2% sodium dodecyl sulfate / 100 mM Tris, pH 6.8 / 20% glycerol / 1.5 M / 3-mercaptoethanol and subjected to electrophoresis on denaturing polyacrylamide gels. Proteins were electro-transferred on nitrocellulose (BA85, Schleicher and Schuell, Keene, NH), and the filters were blocked with 5% bovine serum albumin (Sigma). The filters were then analyzed with E. coli adsorbed human sera collected from 20 AIDS patients. The filter was then developed with HRP-conjugated goat aHulg. The pool was reactive with the pGAG-1 fusion proteins, but not with true E protein alone.

2. Analysis by ELISA 25

Electrophoretically purified GAG-1 protein was diluted in 0.05 M carbonate / bicarbonate buffer (pH 9.6) to a final concentration of 2 µg / ml. Sub-samples of 50 μ / pr were used. microtiter wells and they were incubated at 4 ° C overnight. The plates were then blocked with BLOTTO (5% [w / v] dry milk without fat / 0.01% thimerosol / 0.01% antifoam A in 0.01 M sodium phosphate, pH 7.2 / 0.15 M sodium chloride). 1 hour at room temperature. Sera diluted 1: 100 with a 1: 1 mixture of BLOTTO and PBS (0.01 M sodium phosphate, pH 7.3 / 0.15 M NaCl), and 50 µl diluted sera was added per day. well for 1 hour at 37 ° C. The sera was removed and the plates washed 3 times in wash buffer (0.15 M NaCl / 0.05% [w / v] Tween 20) before adding 100 beer goat anti-human IgG / horseradish peroxidase conjugate (50% stock solution diluted 1: 10,000 in 50 mM NaCitrate / 0.05% Tween 20/1% heat-inactivated normal goat serum; obtained from Antibodies, Inc., Davis, CA) for 1 hour at 37 ° C. The conjugate was removed, the plates were washed 3 times with 0.15 M NaCl / 0.05% (w / v) Tween 20. The ELISA was developed by adding 100 ml of substrate solution to each well (10 mg 3). 3 ', 5,5'-tetramethylbenzidine in 50 ml of 0.05 M sodium citrate, pH 7.0) for 30 minutes at room temperature.

The reactions were quenched at 100 ml / well of 3N HgSO4 and the optical density at 450 nm determined in an automated ELISA reader. Protein produced by pGAG-1 was found to be reactive with a panel of known seropositive sera.

The panel included 241 sera positive against LAV and 270 seranega-10 ten against LAV as defined in a virion-based ELISA. Status of

these sera had been confirmed by immunoprecipitation of radiolabeled LAV antigens. Table III shows that both positive and negative serum samples were drawn from individuals in both the high-risk and low-risk groups. Antibodies reacting with GAG1 were found in all diagnostic groups, although to varying degrees. In particular, the reactivity to GAG1 was found in fewer individuals who were on a developmental stage with ARC and AIDS, compared with individuals who were healthy or in earlier stages of the progression to AIDS (eg PGL

or persistent generalized lymphadenopathy). This is in line with reports showing reactivity to nuclear proteins early after exposure to LAV, but a loss of reactivity to nuclear proteins as the disease progresses. Figure 9 is a histogram of optical density values obtained with all serum samples.

The utility of pGAG1 as an antigen was further tested by ELISA in a minor assay in which sera that was weakly reactive or non-reactive with ENV3, an env recombined protein, was tested for reactivity with pGAG1. Table IV shows ELISA values for 8 positive and 4 negative sera tested against pENV3 and 30 pGAG1 individually and in combination. Three of these sera (14-0085, 07-3915 and 14-0100) were either non-reactive or weakly reactive to pENV3, but were highly reactive to the virus and pGAG1. Two sera (08-0030 and 10-0056) that were weakly reactive to the virus were very weakly reactive to pENV3 alone or pGAG1 alone. However, reactions with both 35 recombined proteins present yielded positive ELISA values that were more easily distinguishable from seronegative controls. These results indicate that pGAG1 is useful for detecting positive sera that have low reactivity to pENV3 and that the combination of pGAG1 and pENV3 is more effective than both alone in distinguishing between seropositive and seronegative individuals.

26 DK 168892 B1

Table III

* GAG1 reactivity in diaanose rupture

Number Percent Number Percent negative non-reactive positive reactive Group samples with GAG1 samples with GAG!

Low risk

Donor 105 100.0% 11 90.9%

Autoimmune disease 20 100.0% 0

Other 11 100.0% 5 80.0% High risk Healthy gay men 53 96.2% 38 86.6% IV drug users 31 90.3% 22 - 77.3%

Among product recipients 10 100.0% 11 81.8%

Sexual partners 10 100.0% 13 84.6% PGL 28 85.7% 62 "" 88.7% ARC 1 100.0% 31 74.2% AIDS 1 100.0% 48 56.3%

Total 270 96.7% 241 77.6% * cut-off value = 0.288 27 DK 168892 B1

3. Fluorescence fluorescence test for detection of serum antibody against LAV

Soluble protein prepared as described above was conjugated to latex beads, and the protein / bead preparation ethanol-5 was fixed on microscopy objects. A sub-sample of patient serum was incubated with the protein / beads on a slide. The slides were washed and FITC-labeled anti-human immunoglobulin was added in Evans blue counter-color. The slides were washed and each mounting medium and a cover glass were added to each.

Alternatively, the protein / sphere preparation was placed in reagent glass for incubation with patient serum. The tubes were centrifuged and washed, and FITC-labeled anti-human immunoglobulin was added in Evans blue counterstain. The tubes were centrifuged and the supernatant aspirated. A sub-sample of the spheres was placed on a mica cup in slides and ethanol fixed and coverslips were placed.

All the slides were examined by fluorescence microscopy. If test serum is antibody positive, the beads appear as fluorescent green beads; if test serum is antibody negative, the 20 spheres appear as red spheres.

4. Reactivity of the combination of trp-gag and trp-env proteins

A trp-gag protein was combined with a trp-env protein in a microtiter well. ELISA was performed as described above for GAG-1 or ENV-3 alone. Table IV shows that the combination of GAG-1 and ENV-3 has a greater sensitivity for detection of seropositive individuals than both proteins alone. Of the seropositive samples, 7/7 was detected when the proteins were combined, while 6/7 was detected with GAG-1 or ENV-3 alone.

5 DK 168892 B1 28

Table IV

Comparison of ELISA values using virus lysate or pGAG1 and pENV3 recombinant proteins.

Designation

Serum Whole pENV3 + by virion

number virus pENV3 pGAGl pGAGI RIP & EIA

Yl / CDC 2,000 1,414 2,039 2,213 Seropositive 10 501 1,109 0,882 1,754 2,008 Seropositive 127 1,046 1,458 1,718 1,974 Seropositive 08-0030 0,443 0,143 0,276 0,473 Seropositive 14-0085 0,951 0,075 0,793 1,136 Seropositive 07-3915 2,286 0.372 1.290 1.628 Seropositive 10-0056 0.378 0.188 0.144 0.435 Seropositive 08.0083 0.036 0.089 0.074 0.139 Seronegative 08-0090 0.043 0.062 0.062 0.103 Seronegative 08-0091 0.027 0.06 0.063 0.085 Seronegative 08-0096 0.025 0.041 0.058 0.085 Seronegative

Claims (9)

29 DK 168892 B1 DNA sequence, characterized in that it comprises a portion of the gag region of the LAV genome, which portion is substantially pGAG-1, pGAG-2 or pGAG-3 and encodes a protein which is immunologically reactive with antibodies against LAV / HTLV-III. Recombinant plasmid capable of replicating in bacterial host cells, characterized in that in asthma it includes prokaryotic transcriptional and translational signals for expression, followed by reading frame of a DNA sequence according to claim 1, which sequence encodes for a protein that is immunologically reactive with antibodies against LAV / HTLV-III. Recombined plasmid according to claim 2, characterized in that the signals originate from the belief operon. Bacterial cell, characterized in that it is transformed with a recombined plasmid according to any one of the preceding claims 2-3. Transformed cell according to claim 4, characterized in that the bacterial cell is E. coli. Process for producing proteins that are immunologically reactive with antibodies to LAV / HTLV-III, characterized in that a bacterial host cell is introduced into a bacterial host plasmid according to claims 2-3, that the bacterial host is grown in a suitable medium and that the protein product of this sequence is isolated from the bacterial host. Process according to claim 6, characterized in that after isolation the protein product is purified by gel penetration chromatography. Protein for use in an assay for determining the presence of LAV / HTLV-III antibodies in a biological fluid, characterized in that it is prepared according to any one of claims 6-7. Protein for use in an assay to determine the presence of LAV / HTLV-1 II antigen in a biological fluid, characterized in that it is prepared according to any one of claims 6-7. DK 168892 B1 SslI (S5) Sstl (9129) / \ XJ19 ^ -U (* B?) X. / Sstl Digestion with / Xv Ssti / Ligation 0, SolI '' Bom HI all (5331) Digestion with / Water Digest BglE. BamHI / \ with Sal1 / Ligation Λ Ligation @ {pSS-5 i (533 * .Bgll / BomH1 I ^ r 1 FIG.l DK 168892 B1 • c 'Polylinker region (ompr ij) Pvun FIG.2 rBamHI-] P JH12: 5 'GAG ATC CCC GGG GAT CCT CTA GAG TCG ACC f — HindHI— | TGC AGC CCA AGC TT 3' pBomHI-, pJH14: 5 'GAG ATC CCC CCG AAT TCG GGGGGATCC TCT r— HindlH—, AGA GTC GAC CTG C AG CCC A AG CTT 3 'DK 168892 B1 pl8 p25 pl3 Amino Acids ...... I "1"' I '' '' I * * * (hundreds) 0 1 2 3 4 5 Hind Π Hind I In pGAGj2 In FIG. 3 PvuH BglE I_pGAG 3_I _pi 8 _ p25__p | 3 Amino Acids I 1 1 '' I 'I' '' i '' '__ (hundreds) O l 2 3 4 5 Sau3a! (Bp375) Pstl (bp96l) FIG.7 I_pGAG -Ι I DK 168892 B1 / γ HindID 4, ampr J / pICI9R j HindIII '\ ^^ and ^^ / ^ omp ^ y II ·, τΐτ ^ Digestion with / KS ?, ® \ T Hindm J Ligation / ^ X / Bgl11 Bom HI f V ~ HindΙΠΜ224Ι (PSM002 1 / n \ y ompr J (, ιμλ \\ --- 'J ^ -HindlE (63i) I pJH12 J # ^ BamHI \ ΟΓΠΡ JJ j Digestion with BamHI.BglH Ligation BgH / BomHI - ^^ BomHI / ^ rp-Qag \ \\ (pGAG2)) \ ampr JJ FIG.4 DK 168892 B1 / Smol BglH Pvui-lv ampr I f) ici9R \ ^ Ayoog å V ompr J BglH \ j Digestion (, 642} Digestion N. / with Bglll.Smol with PvuI, BglII \ ^ / Ligerirg ^ EcoRI EcoRI / (PBPBI41 1 © Wy T ^ BglUne «) / EcoRI Digestion / V see with / \ IEcoRI 1 Week's EcoRI EcoRI \ j / ^^ * t \ / ^ Up * gogNN \ f pGAG3 jl ompr JJ FIG.5 DK 168892 Bl ^^ Polylinker region f omp '<j) \ j ^ / PvuH pATHIO Cleavage site relative to Restriction endonuclease__ reading frame _ Child HI 3 Cla I 1 EcoRI 3 Hind III 3 Pst i '1 Sac I * Sal I · 3 .. Sma I 3 Xba I 3 Xma I 1 FIG.6 DK 168892 B1 BamHI (pBS'r5] / ^ ucieN Sau3al -jU amp J lampr J Sau3al (547) X Digestion with / BamHI / -Ligation - Pst! S * 'yi £ \ ^ Sau3al / BamHI / nRR ς \ i 0674-14 \ (547) ip ° ~ r \ -10 1-Accl I amPr] l ampr I (505) (505) Digestion with N - ^^ αυ3άΙ / BamHI Accl and Pst I y (375) PstI \ PstI EcoRI (961) \ igering f 1 / V ~ Se I OS7fe27 1 / PATHIO \\. ai $ I 1 0ΠΦΓ) j \ Λacci \ IV (505) V // 0 ^ Sau3al / BamHI .EcoRI (375) / n. / Digestion with Digestion \ / ps | I and with PstI and EcoRI / Ligation Sau3al / BamHI PstI (375) (961) .N ^ W ^ coRI / trp-gag FIG.8 \ ampr JJ DK 168892 B1 io :: oT®; ;; ci®: ci® O - I pT '*' C'j £ £!: / -u 4-I fl m «&«: «*" rioo) $ m PH i ^ s,; (Ν '- ID * ; cj "· —v © Γ -“ r · 4J 1 '<”1. 4J I .H _ * CO«: s 1. ® ω J · ~ »ω" u Cl Cl ^ PJ O j - I. - -: - m oro µ! o ^ r o- wm. © w © '«-. ....... .......... o- cn ^ - ------ ------- - '(U o> I-1 t - ♦ - I - II —..... iuQOOOOQ © <5 µ Ϊ CN O OO O v P <Pn * ““ "