WO1991004493A1

WO1991004493A1 - Hiv-related epitopes for diagnosis and immunoprotection

Info

Publication number: WO1991004493A1
Application number: PCT/US1990/005331
Authority: WO
Inventors: Myron E. Essex; Tun-Hou Lee; Wan-Jr Su
Original assignee: President And Fellows Of Harvard College
Priority date: 1989-09-20
Filing date: 1990-09-19
Publication date: 1991-04-04
Also published as: AU6521690A

Abstract

Two epitopes of the envelope glycoprotein (gp120) of Human Immunodeficiency Viruses (HIV-I related viruses) provide immunoprotection and are useful serological markers for protective antibodies. One epitope (448C) is located between amino acid residue 448 and the C-terminus of gp120. The other epitope (KD) is located between amino acids 42 and 129. Polypeptides containing one or both of these epitopes, but substantially lacking certain other interfering epitopes, are useful for immunoassays to predict the course of HIV-1 related disease and to generate protective antibodies (by active or passive immunization).

Description

HIV-RELATED EPITOPES FOR DIAGNOSIS AND IMMUNOPROTECTION Background of the Invention This invention generally concerns immunodiagnosis and treatment of acquired immunodeficiency syndrome

(AIDS) and other mammalian diseases connected with human immunodeficiency virus type I (HIV-I) family of retroviruses.

Human immunodeficiency virus type I (HIV-1) , formerly called HTLV-III, LAV, or ARV belongs to the exogenous non-oncogenic retrovirus family. This virus is generally described in Barre-Sinoussi et al. (Science 220:868, 1983), Gallo et al. (Science 224:500, 1984), Popovic et al. (Science 224:497, 1984) and Levy et al. (Science 225:840, 1984), each of which is hereby incorporated by reference. Various isolates of this virus have been obtained from North America, Western Europe and Central Africa. These isolates differ somewhat in their nucleotide sequence, but the proteins encoded by these viruses are generally antigenically cross-reactive—i.e., antibodies specific for a protein of one isolate generally will react with corresponding proteins of other isolates—and certain regions are highly conserved at the amino acid level. We use the term "HIV-I-related viruses" to include all strains and variants of HIV-1, HTLV-III, LAV, ARV, and other exogenous non-oncogenic type C lymphotrophic retroviruses in that family causing AIDS, ARC, or related mammalian diseases. HIV-1 is a causative agent of AIDS in humans.

Infection by HIV-1 elicits a strong antibody response to the envelope glycoprotein gpl20, and antibodies to gp 120 are readily found in AIDS patients. There are conflicting opinions as to whether these antibodies play a significant role in preventing disease progression. Robert-Guroff et al. , J. Immuno. 13:83731 (1987) suggest that naturally produced gpl20 antibodies may neutralize HIV-1 virus in vivo, which may result in lesser disease manifestation. More specifically, it has been suggested that the binding of gpl20 to the CD4 receptor is the first step leading to HIV infection. Dalgleish et al., Nature 312:763-766 (1984); Klatzmann et al., Nature 312:767-768 (1984); Maddon et al., Cell 42:333-348 (1986). It has also been suggested that it would be desirable to block gpl20-CD4 receptor binding, which has a binding constant greater than 10⁹M. Lasky et al., Cell 50:975-985 (1987); Smith et al. Science 2 8.:1704-1707 (1987).

There are reports that map the gpl20 CD4 binding site to the C-terminal region of gpl20. Lasky et al., Cell .50:975-985 (1987); Kowalski et al., Science 237:1351-1355 (1987); Cordonnier et al., Nature 140:571-574 (1989); Lesley et al., J^. Virology 62.:3695-3702 (1988). There are also reports studying other gpl20 fragments including the N-terminal region of gpl20. Shaffer an et al., AIDS Research and Human Retroviruses 5:33-39 (1989).

There are also reports suggesting that there are two types of gpl20-binding antibodies. One, designated gpl20 (NR) , is directed against an epitope that requires the presence of disulfide bonds to maintain its conformation. The other (gpl20(R)) is directed to epitopes whose antigenicity is not affected by dithiothreitol reduction. It is suggested that the lack of gpl20(R) antibody is associated with a poor clinical outcome. That report also suggests that the portion of gpl20 proviral segment between a Dral site (amino acid number 343) and the C-terminal-encoding end encodes a polypeptide of 169 amino acids which serves as a marker for the presence of gpl20(R) binding antibodies. See Lee et al., "Association Between Antibody To Envelope Glycoprotein gpl20 And The Outcome Of Human Immunodeficiency Virus Infection" pp. 373-377 in Vaccines 88 Cold Spring Harbor Press 1988.

Repligen has isolated a 24-amino acid protein located in the vicinity of amino acids 301-324 of gpl20, See Boucher et al., AIDS 3(2. :71-76 (1989).

Summary of the Invention We have discovered that certain epitopes of gpl20, the envelope glycoprotein of HIV-1-related viruses, are particularly effective as serological markers for predicting the course of HIV-l-related viral pathology and for eliciting an immune response that has a beneficial effect in controlling the progress of

HIV-related viral pathology. We have also discovered that it may be useful to isolate these epitopes from other less useful but more dominant gpl20 epitopes. We use the term eptiope to mean an amino acid sequence which is a binding site for a monospecific antibody, and which contains sufficient information, when presented to the immune system as part of a larger molecule, to elicit antibodies that bind to it.

The invention particularly features two epitopes of gpl20 of HIV-related viruses which we will designate the 448C epitope and the KD epitope. The 448C epitope refers to the epitope defined generally by the C-terminal 67-70 amino acids of gpl20 (glycosylated or unglycosylated) . The KD epitope refers to the epitope extending along about 17% of the gpl20 amino acid sequence, beginning about 40-42 amino acids from the N-terminus. More specifically, the 448C epitope extends approximate from gpl20 amino acid 448 to the gpl20 C-terminus, and the KD epitope extends approximately from gpl20 amino acid residue 42 to amino acid residue 129, using the numbering system of clone HXB2 reported by Fisher et al., Nature 316:262-265 (1985), which is hereby incorporated by reference. Thus, a 448C epitope or an KD epitope is an amino acid sequence that binds to antibodies that are specific for the 448C or the KD region of gpl20, respectively.

One aspect of the invention generally features a method for detecting anti-448C specific antibodies in a sample, by incubating the sample with a polypeptide comprising a 448C epitope and substantially lacking the HIV-1 gpl20 epitope between gpl20 amino acid residues 343 and 447. We use the term polypeptide to describe short polypeptides as well as longer polypeptides and proteins. The presence of an immunocomplex between the polypeptide and sample antibody is detected and indicates that the sample contains anti-448C-specific antibodies. Preferably, the sample is incubated with a polypeptide fragment of gpl20 containing a sequence of at least 10 consecutive gpl20 amino acid residues between amino acid 448 and the C-terminus of gpl20 and it lacks any sequences longer than 5 amino acids from the gpl20 sequence 343-447. Most preferably the sample is incubated with a polypeptide containing the entire gpl20 amino acid sequence between amino acid 448 the gpl20 C-terminus. However, conservative alterations of that sequence that retain cross-reactivity with the 448C sequence are within the scope of the invention.

This aspect of the invention is useful for predicting the course of HIV-related viral pathology in that the presence of anti-448C specific antibodies correlates with retarded progress of that pathology. Another aspect of the invention features the polypeptides used in the above-described method for detecting: i.e. polypeptides comprising a 448C epitope and substantially lacking any gpl20 epitope between amino acid residues 343 and 447; preferably the polypeptide contains a sequence of at least 10 consecutive amino acid residues between amino acid 448 and the gpl20 C-terminus, and it lacks any sequences longer than five amino acids from the gpl20 sequence 343-447. Most preferably the polypeptide contains the entire gpl20 amino acid sequence between amino acid residue 448 and the gpl20 C-terminus.

A third aspect of the invention features injecting the above-described polypeptide into a mammal to elicit an immune response that is protective against an HIV-1-related virus. In active vaccination, an immunologically effective amount of the polypeptide in a suitable carrier is administered (e.g. by injection) to the individual at risk. In passive vaccination procedures, the globulin fraction of the mammal is recovered and an effective amount is administered to the individual at risk—e.g., an HIV patient. Alternatively, a cell producing anti-448C antibody is isolated from the host and immortalized by known techniques to provide a source of antibody that is then administered to an HIV patient.

A fourth aspect of the invention features a polypeptide comprising the KD epitope and substantially lacking at least one of the following gpl20 epitopes: gpl20 amino acid residues 130-288 (the "DrP epitope") ; gpl20 amino acid residues 343-447; or gpl20 amino acid residues 307-321. Preferably the polypeptide comprises a sequence of at least 10 (most preferably all) of the amino acid residues between gpl20 residue 42-129. The presence of KD-specific antibodies is also predictive of a less severe disease course.

A fifth aspect of the invention features injecting the above-described polypeptide into a mammal in order to elicit an immune response that is protective against an HIV-related virus.

Without being bound to any theory, it appears that certin gpl20 epitopes may be dominant yet ineffective, in that they elicit a high antibody titre which is not protective. Other epitopes elicit a more protective immune response, but the response is relatively weak. Also, there may be other reasons why it is convenient to generate a particular antigen free from some or all other gpl20 epitopes. The invention identifies two protective epitopes that can be presented free from epitopes that do not elicit protection. The invention also permits ready synthesis of important epitopes individually, without also synthesizing epitopes that, while useful, are conveniently synthesized separately and connected to each other.

It also appears that the CD4 receptor binding domain of gpl20 involves at least two discontinuous regions at opposite ends of gpl20. It may be desirable to employ vaccines with multiple gpl20 fragments to block multiple receptor binding sites, to thereby generate a cooperative binding affinity greater than that of virus-receptor interaction to more effectively compete against HIV binding that leads to HIV infection. Thus, preferred aspects of the fifth aspect of the invention feature the use of polypeptides that include the KD epitope and the 448C epitope, but lack the DrP epitope, the 343-447 epitope, the 307-321 epitope, or combinations thereof. Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.

Description of the Preferred Embodiments The Figures Fig. 1 is a diagrammatic representation of HIV proviral DNA encoding gpl20, showing restriction sites and showing gpl20 fragments including the KD fragment, the DRP fragment, and the 343C fragment, which includes the sub-fragments 343-448 and 448C.

Fig. 2 shows the DNA and inferred amino acid sequence of gpl20 of two HIV isolates BH8 and BH10, reported by Ratner et al. cited below.

Fig. 3 shows the DNA and inferred amino acid sequence of a third HIV isolate reported by Muesing et al. cited below.

Fig. 4 shows partial sequence of the KD epitope. The 448C Epitope

Referring to Fig. 1, the 343C segment of HIV-1 gpl20 is a region between amino acid position 343 and amino acid position 511 in gpl20. This defines a 169 amino acid polypeptide. Two subportions of this peptide are shown as 448C and 343-448, each of which represent approximately half of the 343C peptide separated at amino acid position 448.

The sequence of gpl20 is shown in Figs. 2 and 3 which includes portions of the HIV-1 sequence reported by Ratner et al., Nature 313:277-284 (1985) and Muesing et al., Nature 313:450-458 (1985), hereby incorporated by reference. The preferred 448C epitope is the 60-70 C-terminal amino acids of the gpl20 envelope glycoprotein of the HIV-related virus in question. One preferred 448C epitope consists of the 67 amino acids at the C-terminus of gpl20. Those skilled in the field will appreciate that conservatively substituted modifications of the 448C epitope generally will retain the immunological properties that are useful in the invention—viz. binding to 448C-specific antibodies and raising antibodies that bind specifically to the 448C region of gpl20. The KD Epitope

The KD sequence is shown in Figs. 2-4, and in Fisher et al., cited above. Preferably, the KD sequence spans from gpl20 residue 42 to 129. Fig. 4 locates the KD (Kpn 1 to Dra 1) segment of the HXB2 isolate reported by Fisher et al. and provides partial sequence data for that segment.

More specific methods for preparing 448C and KD polypeptide are as follows. Example 1

Vector pX-2 is modified from plasmids pUC-18 and pXVR (Feig et al., Proc. Nat. Acad. Sci. USA 83:4607. 1986) and contains a 3.1 kb Stul-Pstl fragment from plasmid pBK-28 (Kornfeld et al., cite). Plasmid pX-2 contains an EcoRl fragment having a tac promotor linked to the first 333 base pairs of the V-RAS^H gene. Downstream from the V-RAS^H coding sequence is the pUC-18 polylinker cloning site. A suitable DNA sequence encoding the 448C or KD polypeptide including a stop codon are inserted into the pUC-18 polylinker cloner site. Recombinant plasmids with the correct inserts are identified, and further verified by DNA sequencing.

Recombinant 448 or KD gene product is prepared by growing an overnight culture of Ε__ coli strain X-90 (Palaas et al., General Biology 60:1075, 1986) bearing pX-2. These cells are inoculated into 10 ml of standard LB media containing 50 micrograms per ml of ampicillin and grown to an optical density of 0.3 at 550 nm. 5 ml cultures are then induced with 5 nm isopropyl-beta- D-thiogalactopiranoside (IPTG) and the remaining culture used as an uninduced control. After three hours at 37°C the cells are collected by centrifugation, washed once with 15 mM NaCl, and 10 mM Tris-chloride, pH 7.5. The gene product is expressed as a fusion protein. Example 2 A 3.1 kilobase Sal 1 to Xho 1 fragment of the cloned HIV proviral DNA designated HXB2 (Fisher et al., Nature 316:262-265 (1985)) is generated and cloned into an appropriate cloning vector. A subclone covering the epitope of interest is cloned into an appropriate expression vector, such as pBaby disclosed in Rekosh et al., Proc. Nat'1. Acad. Sci. USA £5:334-338 (1988).

Variants of the epitope encoding DNA segment can be prepared by standard techniques such as site-directed mutagenesis techniques using synthetic oligonucleotides. Kunkel et al., Proc. Nat'1. Acad. Sci. USA 82:488-492 (1985) .

The resulting expression vactors are expressed by standard techniques (Rekosh et al.) and the protein is recovered by immunopurification techniques (Robey et al., Proc. Nat'1. Acad. Sci. USA 81:7023-7027 (1986). See also, Yu et al. Nature 335:262-265 (1985) and Matsuda et al., Proc. Nat'1. Acad. Sci. USA 85:6968-6972 (1988) . Diagnosis

This fusion protein is recognized by antibodies of HIV-1 sero-positive people who have specific antibodies for the epitope in question, and who therefore have a better prognosis than those who lack such antibodies.

Detection of serum antibodies to the 448C or KD gene product is performed using recombinant polypeptide as described above, partially purified as follows. The cells were lysed in 500μl of Laemmli sample buffer (Laemmli, Nature 227:680 (1970)) and boiled for two minutes before loading on 15% polyacrylamide gels using the Laemmli buffer system. The purification procedure of Pallas et al., ___ Virol. 6_0:1075 (1986) is generally used. Coomassie blue staining is used. The purified and isolated 448C or KD polypeptide can be employed as a standard antigen in any conventional assay procedure for detection in biological specimens of the presence of antibodies specific for 448C or KD, respectively.

The antigen can be labelled by conventional procedures with ¹²⁵I or ³⁵S or ³H for use in radioimmunoassay, with fluorescein for fluorescent immunoassay, with enzyme for enzyme immunoassay or with biotin, for biotin-avidin linked assays. It can be employed, labelled or unlabelled as desired, in competitive immunoassays, as well as in double antibody assays or other assays.

Alternatively, antigen could be immobilized on an insoluble phase, such as an insoluble resin, and detection of the anti-448C or KD antibodies is carried out by measuring their binding to the insoluble phase. Insoluble phases also include latex particles, which, when coated with the antigen subjected to reactive antibody, will agglutinate. Yet other insoluble phases include test tubes, vials, titration wells, and the like, to which antigen can be bound, and antibody thereto detected by double antibody techniques or Protein-A dependent techniques. The elements necessary for carrying out the diagnostic methodology described hereinbefore may be present in a kit. Such kit comprises a carrier being compartmentalized to receive therein one or more containers, each of said containers comprising one or more elements necessary to carry out the tests.

For example, the first container may contain the purified antigen (i.e., 448C or KD gene product or an immunologically cross-reactive polypeptide) in detectably labelled or in insolubilized form. A second container may comprise anti IgG antibody, polyclonal or monoclonal, useful in double antibody binding assay, or elements needed for detection of the label on the antigen (e.g. chromogenic substrates) .

Additional containers may comprise varying amounts of antigen which can be used to prepare a standard curve into which experimental results can be interpolated. The materials may be present in the kit by themselves, in solution, freeze-dried, or in admixture with other inert materials, such as inert proteins, and the like.

The biological specimens tested may include blood, serum, urine, tissues, saliva, feces, and the like.

Immunoprotection

Immunoprotection is provided by providing a substantially purified epitope-containing polypeptide as described above, and suspending the polypeptide in a physiologically acceptable solution such as saline solution. For example, the purified epitope containing polypeptide is suspended in saline solution and injected into a mammal at a dosage of 10-10,000 micrograms/kg of body weight. For active immunization (e.g. where the goal is prophylaxis or control of infection in an individual whose immune system is still functional) the above technique is used.

It is also possible to use passive immunization techniques described above. See generally, Kasper U.S. Re 31,672. Alternatively, chimeric monoclonal antibodies such as those described by Syu et al. , J. Virology (September 1989) pp. 3579-3585, can be produced and used to make antibodies used for passive immunization, i.e. by recovering an antibody-producing cell from a challenged mammal (e.g. a mouse or rat) and immortalizing the cell to produce antibody administered in an effective amount to the patient.

Other embodiments are within the following claims.

Claims

Claims 1. A method for detecting antibodies in a sample that are specific for a gpl20 448C epitope comprising incubating the sample with a polypeptide comprising a 448C epitope and substantially lacking any gpl20 epitope between amino acid residues 343 and 447 for a time and under conditions permitting formation of an immunocomplex, and detecting formation of an immunocomplex between the polypeptide and antibody in the sample.

2. The method of claim 1 in which the polypeptide is a gpl20 fragment containing a sequence of at least 10 consecutive gpl20 amino acid residues from the 448C epitope.

3. The method of claim 1 in which the polypeptide contains the complete gpl20 sequence from amino acid 448 to the gpl20 C-terminus.

4. The method of claim 1 in which the polypeptide lacks any sequence of five consecutive amino acids indentical to a five amino acid sequence of gpl20 between amino acid residues 343 and 447.

5. A substantially purified polypeptide comprising a 448C epitope and substantially lacking any gpl20 epitope between amino acid residues 343 and 447.

6. The polypeptide of claim 5 in which the polypeptide is a gpl20 fragment containing a sequence of at least 10 consecutive gpl20 amino acid residues from the 448C epitope.

7. The polypeptide of claim 5 in which the polypeptide contains the complete gpl20 sequence from amino acid 448 to the gpl20 C-terminus.

8. The polypeptide of claim 5 in which the polypeptide lacks any sequence of five consecutive amino acids indentical to a five amino acid sequence of gpl20 between amino acid residues 343 and 447.

9. A method of generating antibodies that are protective against HIV infection comprising injecting into a mammal an immunologically effective amount of a polypeptide according to any one of claims 5-8.

10. The method of claim 9 further comprising recovering an antibody-producing cell from said mammal, immortalizing said cell to produce said antibodies, and administering effective amount of said antibodies to an individual.

11. The method of claim 9 in which said mammal is a human.

12. A substantially purified polypeptide comprising the KD epitope of gpl20 and substantially lacking at least one of the following gpl20 epitopes: gpl20 residues 130-288 (the "Drp" epitope) , the 307-321 epitope, or amino acid residues 343-447.

13. The polypeptide of claim 12 in which said polypeptide comprises a sequence of at least 10 gpl20 amino acid residues between gpl20 residues 42-129.

14. The polypeptide of claim 13 in which said polypeptide comprises substantially the entire gpl20 amino acid sequence between residues 42-129.

15. The method of claim 10 in which said polypeptide comprises both the KD epitope and the 448C epitope.