WO1989009618A1 - Method for controlling hiv infectivity and vaccines for use therein - Google Patents

Method for controlling hiv infectivity and vaccines for use therein Download PDF

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Publication number
WO1989009618A1
WO1989009618A1 PCT/US1989/001205 US8901205W WO8909618A1 WO 1989009618 A1 WO1989009618 A1 WO 1989009618A1 US 8901205 W US8901205 W US 8901205W WO 8909618 A1 WO8909618 A1 WO 8909618A1
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hiv
gpl20
vaccine
polypeptide
serum
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PCT/US1989/001205
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French (fr)
Inventor
William M. Mitchell
W. Edward Robinson, Jr.
David C. Montefiori
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Vanderbilt University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the field of this invention is vaccines for controlling infection by the human immunodefi ⁇ ciency virus type-1 (HIV-1), and more particularly to subunit vaccines based on the HIV-1 viral envelope glycoproteins gpl20 and gp41 and their precursor glycoprotein gpl ⁇ O. Additionally, this invention relates to the detection and quantitation of HIV-1 antibody-dependent enhancement (ADE).
  • HIV-1 human immunodefi ⁇ ciency virus type-1
  • ADE HIV-1 antibody-dependent enhancement
  • HIV Human immunodeficiency virus
  • HIV-2 HIV-2
  • This application is primarily concerned with the control of HIV-1 infectivity. Be ⁇ cause of both the morbidity and the mortality associ ⁇ ated with HIV-1 infection and widespread concern over its prevalence, massive effort has been aimed at pro ⁇ ducing a safe, effective vaccine.
  • HIV-1 vaccines are currently being discussed as po ⁇ tential candidates: a reco binant, eu aryotic gpl20 (2), a recombinant, eukaryotic gpl ⁇ O (3), and a Vac ⁇ cinia virus recombinant (4).
  • This invention is based on the discovery that a high percent (greater than 60%) of HIV-1 seropositive individuals have an antibody which enhances the infectivity of HIV-1. This enhancement apparently involves action in concert with the alter ⁇ nate pathway of complement.
  • antibody-dependent enhancement (ADE) of certain viral infec ⁇ tions has been previously known to occur, this phe ⁇ nomenon had not been observed for AIDS infection.
  • the first report of a serum factor which enhances HIV-1 infection was made in December 1987 by appli ⁇ cants (17) . The initial investigation suggested that the factor was not a virus or an IgG-type antibody. The factor could not be identified and the role of complement was unrecognized.
  • the enhancement factor is an immunoglobulin which, with the alternate pathway of complement, can mask neutralizing antibodies and enhance HIV infec ⁇ tion.
  • the antibody has been shown to be directed toward the product of the HIV-1 env virogene (gpl60).
  • a specific region of gpl60 contains one or more antigenic domains which bind to the antibody.
  • An antigenic domain which produces infection enhancing antibodies has been localized by binding to a recom ⁇ binant gpl60 fragment, PENV9, corresponding to the gpl ⁇ O amino acids 467 to 752.
  • That region includes the i munodominant epitope of gp!20, amino acid sequence 497-511, and the immunodominant epitope of gp41, amino acid sequence 593-604. (The numbers refer to the Beck- man "Microgenie" listing. )
  • Polypeptide fragments for use in HIV-1 vaccines nominally must include one or more epitopes generating neutralizing antibodies to HIV-1, and Bcell memory, while being devoid of epitopes that elicit ADE of HIV-1 infection.
  • the vaccines of this invention are useful for preventing HIV-1 infection.
  • the vaccines can be administered to re ⁇ tard progression to the Acquired Immunodeficiency Syn ⁇ drome (AIDS), or the pre-AIDS conditions, AIDS Related Complex (ARC) and Lymphadenopathy Syndrome (LAS).
  • AIDS Acquired Immunodeficiency Syn ⁇ drome
  • ARC AIDS Related Complex
  • LAS Lymphadenopathy Syndrome
  • the present invention therefore provides a method of producing a safe and effective vaccine for controlling the infectivity of human immunodeficiency virus type 1 (HIV-1).
  • a polypeptide composed of a fragment of HIV-1 gpl60, which fragment has at least one epitope gener- ating HIV-1 neutralizing antibodies It is determined by in vitro laboratory tests that the polypeptide is incapable of generating HIV-1 infectivity enhancing antibodies (IEA). The polypeptide is then prepared in vaccine dose form. The in vitro tests for determining that polypeptide fragments will not generate IEA are subsequently described in detail.
  • HIV-1 infection-enhancing factor in three of eleven HIV-1 seropositive patients' sera was discovered by the applicants (17) during the routine titration of HIV-1 neutralizing antibody acr • civity, using sera that had not been previously heat treated but which had been stored at 4°C up to 1 month following collection for HIV-1 serology testing.
  • the three identified sera when preincubated for one hour with HIV-1, then added to target MT-2 cells in the ap ⁇ plicants' microtiter infection assay, caused pronounced HIV-1-induced cytopathology as evidenced by many large, nultinucleate giant synctia at dilutions from 1:4 to 1:64.
  • the virus control wells demonstrated none to few synctia when observed under phase contrast microscopy.
  • the increase in synctia formation was accompanied by a decrease in cell viability as determined by vital dye uptake.
  • the infection-enhancing activity was not present in HIV-1 seronegative serum.
  • filtration of sera con ⁇ taining enhancing activity through 50 nm filters failed to remove the observed activity.
  • a paucity of synctia was observed when greater than one multiplicity of infection (m.o.i.) of the HTLV- III » isolate of HIV-1 was added to MT-2 cells but a profound number of giant synctia were present when filtered sera were added to the virus one hour before the cells were added.
  • the factor is not a virus likely to be found in the sera of patients infected with HIV (i.e., including HIV-1 itself, CMV, EBV, Herpes simplex. Varicella zoster, HTLV1/II, etc.) and is suggestive of a bio ⁇ logical entity whose hydrodynamic volume radius of gyration is ⁇ 50 nm.
  • Enhancing Activity is a Multicomponent System consisting of a Ubiquitous Heat-Labile Component
  • the Heat Labile Component After several weeks at 4% our sera identified as having enhancing activity had lost the capacity to enhance HIV-1 infec ⁇ tion of target cells in vitro. When sera that had lost enhancing activity were mixed 1:1 with normal fresh serum, the activity was restored. The activity was not present in normal serum alone. Further, if normal human serum was heated at 60°C for one hour, it lost its ability to enhance HIV-1 infection when mixed with heat-inactivated serum with known enhancing ac ⁇ tivity. Table I demonstrates the neutralizing titer of either age-inactivated or heat-inactivated sera as well as the neutralizing or enhancing titer of that same serum following 1:1 mixing with normal human serum.
  • complement component Clq deficient serum a serum that contains less than 5% of classic complement pathway activity as determined by CH so titer, was able to enhance virus cytopathicity as well as normal, fresh human serum when combined with HIV-1 seropositive sera.
  • Factor B a factor utilized in the alternate complement pathway deficient serum was unable to decrease time to cytolysis when added to heat- inactivated patient serum known to contain enhancing activity.
  • complement component C3 defi ⁇ cient serum a complement component shared by both the alternate and classic pathways, was unable to decrease time to HIV-1 induced cytolysis when mixed with com ⁇ plement-inactivated enhancing serum.
  • guinea pig complement serum which can substitute for human serum in the classical but not the alternate complement pathways, was unable to substitute for normal human serum in the enhancing assay as it could not enhance cytopathicity of HIV-1 when combined with seropositive sera.
  • a substantial body of evidence demon ⁇ strates that the ubiquitous heat-labile component necessary for HIV-1 infection-enhancement found in all human serum is the alternate complement pathway.
  • the Heat-Stable Component The original data indicated that enhancing activity was not reduced when batch mixed with Protein-A Sepharose although neutralizing antibody activity was removed (i.e., staphylococcal protein-A binds IgG and some IgA and IgM from human sera) , and when covalently bound to an insoluble matrix such as Sepharose, antibodies with an affinity to Protein-A can be removed from solution. Further studies using Protein-A Sepharose columns, however, did result in removal of the heat-stable com ⁇ ponent. As Table II shows, the stable factor was al ⁇ most entirely removed by protein A column chromatog- raphy while the labile factor was not retained by the column.
  • ***P.A. is the flow-through of serum 763 subjected to protei ⁇ -A column chrom tograph .
  • ⁇ NT denotes not tested.
  • serum 281 demonstrated good neutralizing antibody titers against HIV-1 in the presence of fresh human serum, it was combined with three enhancing sera. Table III demonstrates that although serum 281 could still neutralize virus in the presence of enhancing serum, cytolysis of 50% or more MT-2 cells occured only at serum dilutions greater than 1:128 in the presence of sera 969 and 154 (data for serum 154 not shown) . When combined with serum 763, the most potent enhancer of HIV-1 infection, MT-2 cells were protected from HIV-1-induced pathology only at dilutions of neutralizing serum 281 less than 1:16. Serum 281 was capable of protecting MT-2 cells from cytolysis at a dilution of 1:128 when tested alone.
  • enhancing activity can be monitored in the chimpanzee, the only human surrogate animal model for vaccine testing.
  • the preferred em ⁇ bodiment for complement restoration in order to establish enhancing titer in the chimpanzee is fresh frozen human serum since chimpanzee complement yields a non-antibody dependent enhancement resulting in a high normal background not observed when fresh frozen human serum is used as a complement source.
  • PENV9 a cloned fragment of gpl60 referred to as PENV9 (13) and separate BSA column as a control for non-specific ad ⁇ sorption.
  • the PENV9 polypeptide fragment contains the amino acids of gpl ⁇ O from 467 to 756, which bridge the immunodominant regions of gpl20 (15) and gp41 (16).
  • Table V demonstrates the results of the af ⁇ finity chromatography.
  • serum 281 was neutralizing even in the presence of comple ⁇ ment, while sera 763, 772, and 154 were enhancing.
  • the PENV9 eluent fraction contained only enhancing ac ⁇ tivity for all four sera.
  • enhancing epitopes can be separated from neutralizing epitopes.
  • the BSA column data demonstrates that non-specific column- protein "stickiness" was not a factor since no activi ⁇ ty was present in the BSA column eluent.
  • Residue 1 methionine to residue 856 leucine follows the total sequence of Ratner (21) .
  • Vaccines designed in accordance with the principles of the present invention will be more ef ⁇ fective because they will lack capacity to induce antibody-dependent enhancement of HIV infectivity. Such vaccines differ in this important respect from the present vaccines utilizing gpl60 or gpl20 as the immunizing antigens.
  • the present invention comprises an improved method of controlling HIV-1 infectivity in human pa ⁇ tients.
  • the size of the fragments will be in excess of 170 amino acids from gpl60/gpl20, and preferably about 190 to 450 amino acids. Additional non-gpl60/gpl20 amino acids may be present at 5' or 3' ends of the functional fragment.
  • the polypeptide fragment can contain about 175, 193, or 448 amino acids, corresponding respectively to sequences 273 to 447, 273 to 465, and 1 to 447, all of which contain a neutralizing epitope and are free of infection- enhancing epitope. All of these can be readily pre ⁇ pared from restriction fragments of the gp!60 gene.
  • Another potentially desirable polypeptide can contain the gpl20 amino acid sequence from 105 through 456, a sequence of 352 amino acids including the neutraliz ⁇ ing epitope 291-307 and the virus attachment/T-cell antipathic regions 421-456/413-458.
  • polypeptide fragments of gpl60/gpl20 contain no more than 496 amino acids, such as the sequence 1 to 496, which includes both of the neutralizing regions so far identified in gpl20, while omitting the immuno ⁇ dominant region 497-511.
  • polypeptides can be utilized in nongly- cosylated form, but for optimum effectiveness it is preferred to utilize fully glycosylated polypeptides, i.e., corresponding in glycosylation to gpl60/120.
  • Other segments of gpl ⁇ O can be added or incorporated to the sequences described above, viz. by genetic engineering techniques and manipulations.
  • the entire sequence of gpl60 from 478 to 605 is omitted from the vaccine polypeptide.
  • the polypeptide vaccines of this invention can be prepared and administered by procedures and protocols similar to those being used for the gpl20 and gpl60 vaccines.
  • the carrier of the peptide may be a sterile aqueous solution, such as normal saline, and the polypeptide may be absorbed on alum (26, 27). Concentrations may range from 10 to 100 micrograms per illiliter. Concentration will be selected so that a single injected dose of 0.5 to 1.0 ml followed by booster doses will provide a sufficient amount of the polypeptide to consistently induce an immune protec ⁇ tive response.
  • the preferred route of injection is intramuscular, except in cases where the patient is at risk of hemorrhage (i.e, hemophiliacs).
  • subcutaneous injection can be used as an alternate to intramuscular injection.
  • a booster injection may be given at intervals of one month, six months, or yearly, as re ⁇ quired.
  • Titers of neutralizing antibodies can be monitored to determine the need for a booster injec ⁇ tion.
  • Truncated species of the HIV-1 gpl60 can be obtained by restriction endonuclease digestion of HIV-1 proviral DNA followed by insertion of the frag ⁇ ment into an expression vector plasmid which can be cloned in eukaryotic cells for the synthesis of the modified protein.
  • This genetic engineering of the HIV envelope gene utilizes materials which are available to the research community through biotechnology com ⁇ panies. Similar protocols have been developed and utilized for the cloning and expression of whole HIV-1 gpl60 and gpl20 products in eukaryotic cells.
  • Re ⁇ striction endonucleases are used to engineer a trun ⁇ cated HIV-1 gpl ⁇ O or gpl20 by eliminating epitopes responsible for eliciting antibody-dependent enhance ⁇ ment of HIV infection while retaining epitopes responsible for eliciting protective immunity. Since carbohydrate moities are known to be important for known to be important for antigenic stimulus, emphasis should be placed on expressing the genetically engi ⁇ neered protein in eukaryotic cells, which are most likely to synthesize a properly glycosylated product.
  • restriction fragment which encodes only desired epitopes is the Sspl fragment en ⁇ coding the first 448 amino acids of gp!20.
  • This frag ⁇ ment can be obtained from the HIV DNA on pHxB-2D available from Biotech Research Laboratories, Incor ⁇ porated. The fragment can be inserted in the proper vector AC373 and used to construct a recombinant baculovirus by cotransfection of the Spodoptera frugiperda cell line Sf9, as described in (14).
  • the recombinant baculovirus has the property of synthe ⁇ sizing large amounts of the cloned, tailored HIV gpl60 protein in infected Sf9 cells owing to the strong polyhedron gene promoter used to drive expression of this gene.
  • This mammalian system has the added advantage of producing a fully glycosylated product.
  • an E ⁇ coli expression system can be used, as described in (13).
  • a Bgl-II DNA fragment of the gpl60 gene can be used to prepare the 193 amino sequence from 273 to 465; or a Bgl-II/Sspl DNA fragment to prepare a 176 amino acid sequence from 273 to 448.
  • Synthesis and purification of HIV recombinant polypeptides can be accomplished by infecting mono- layers of Sf9 cells with genetically engineered bacu ⁇ lovirus at a multiplicity of infection of 3. Cells are harvested approximately 4 days later and lysed in a solution containing 20mM Tris-HCl, pH 7.5, lOmM Mg(OAc) 2 , 1% Triton X-100. The lysed cell suspen ⁇ sion is centrifuged at 15,000 xg and the pellet, which 22
  • glycoprotein contains recombinant glycoprotein, is solubilized in isotonic buffer and further purified by lentil lectin sepharose Cl-4b affinity chromatography. The presence of recombinant HIV glycoprotein can be confirmed by immunoblotting. The purified glycoprotein is then prepared for vaccine use as previously described.
  • Evaluation of gpl60 fragments for the absence of epitope which can induce formation of infection- enhancing antibodies can be achieved by three methods: (1) the decrease in time required for productive in ⁇ fection of ADE-sensitive target cells; (2) the differ ⁇ ential titer of neutralizing antibody activity as a function of fresh complement in ADE-sensitive target cells; and (3) the detection of antibodies which bind specifically to (i) PENV9 or (ii) a fraction of PENV9, or (iii) a synthetic peptide homologous to the anti ⁇ body dependent enhancement domains of PENV9. Methods 1 and 2
  • Methods 1 and 2 require infection of target cells by HIV-1.
  • the prototype target cell is a clone of the T-lymphoblastoid cell line MT-2 and is culti ⁇ vated as previously described (19).
  • MT-2 cells yield 100% cytosis in 4 days or less when challenged with HIV-1 at a multiplicity of infection (MOID) ⁇ l.
  • This cell line is a sensitive indicator for HIV-1 ADE due to its high content of complement receptors (CR2) and conventional HIV receptors (i.e., CR4) on its plasma membrane and serves as a useful indicator cell for HIV-1 ADE in both human and chimpanzee sera.
  • Alter ⁇ nate target cell should be used in the basic assay provided that they grow readily in 96 cell culture plates, are highly permissive for HIV-1 infection, and 23
  • Method 1 defines the titer of infection-enhancement as that highest titer of serum which yields a statistically significant reduction in time to cell cytolysis and/or HIV-1 antigen release into the supernatant as compared to control HIV-1 infection.
  • Method 2 depends on the differential neutralizing titer obtained on heat- inactivated serum (56C, 1/2 hr.) when titered in the presence of a constant amount of fresh serum. Serum with significant amounts of infection-enhancing antibody will demonstrate reduction or elimination of neutralizing antibody titers in the presence of fresh human or chimpanzee complement.
  • Method 3 defines the titer of infection-enhancement as that highest titer of serum which yields a statistically significant reduction in time to cell cytolysis and/or HIV-1 antigen release into the supernatant as compared to control HIV-1 infection.
  • Method 2 depends on the differential neutralizing titer obtained on heat- inactivated serum (56C, 1/2 hr.) when titered in the presence
  • This method is a noninfectious method which depends on the binding of infection-enhancing anti ⁇ bodies or enhancing/antibodies immunodominant domains of PENV9, a fraction of PENV9 containing antibody enhancing immunodominant domains, or synthetic pep- tides homologous with said enhancing/antibodies im ⁇ munodominant domains.
  • enhancing/antibody im ⁇ munodominant domains may be either in solution or attached to a solid matrix, the latter being the pre ⁇ ferred physical state.
  • the latter is washed x 3 with physiological saline to remove non- specifically bound antibody and the remaining anti ⁇ body quantitated by standard methods of analysis which include commercially available antibodies containing covalently bonded enzymes, fluorescent groups, or radioactive tracers and which are specific for human or chimpanzee antibody. Quantitation is made by the absolute amount of antibody bound from a given amount of serum or by qualitative detection as a function of serum titer.
  • the foregoing also represent new method of detection and quantitation of HIV-1 infection enhanc ⁇ ing antibodies (IEA) which can be used in humans and chimpanzees.
  • One method is based on the decrease in the time of HIV-1 induced target cell cytopathicity.
  • detection and quanti ⁇ tation of the infection enhancing antibodies can be based on a decrease in time of HIV antigen expres ⁇ sion.
  • Complement test procedure can also be used. Specifically, .IEA in humans and chimpanzees can be detected and quantitated based on differential neu ⁇ tralizing antibody titers as a function of the pres ⁇ ence or absence of fresh human or chimpanzee comple ⁇ ment. This may be determined by target cell cyto ⁇ pathicity and/or by HIV antigen expression.
  • Infection enhancing antibodies can also be detected and quantitated by binding to PENV9 or a fragment of PENV9 in solution or bound to a solid phase matrix. The amount of bound antibody can be measured by ELISA, fluorescence, or radioactive methods.
  • PENV9 synthetic peptides homolo ⁇ gous to the infection-enhancing epitopes of PENV9 can be used.

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Abstract

A method and vaccine are provided for controlling infectivity of human immunodeficiency virus type 1 (HIV-1). The method and vaccine are predicated on the discovery that HIV-1 gp160 and gp120 contain one or more epitopes generating HIV-1 infection-enhancing antibodies (IEA). The method and vaccine of this invention use a polypeptide fragment or fragments of the total amino acid sequence of gp160 which includes at least one epitope of gp120 generating neutralizing antibodies to HIV-1 while being devoid of any epitope of gp160 generating IEA.

Description

METHOD FOR CONTROLLING HIV INFECTIVITY AND VACCINES FOR USE THEREIN
FIELD OF INVENTION
The field of this invention is vaccines for controlling infection by the human immunodefi¬ ciency virus type-1 (HIV-1), and more particularly to subunit vaccines based on the HIV-1 viral envelope glycoproteins gpl20 and gp41 and their precursor glycoprotein gplβO. Additionally, this invention relates to the detection and quantitation of HIV-1 antibody-dependent enhancement (ADE).
BACKGROUND OF INVENTION
Human immunodeficiency virus (HIV), the causative agent of AIDS, is a highly variable retro- virus comprising two serological subgroups designated HIV-1 and HIV-2 (1). This application is primarily concerned with the control of HIV-1 infectivity. Be¬ cause of both the morbidity and the mortality associ¬ ated with HIV-1 infection and widespread concern over its prevalence, massive effort has been aimed at pro¬ ducing a safe, effective vaccine. At least three HIV-1 vaccines are currently being discussed as po¬ tential candidates: a reco binant, eu aryotic gpl20 (2), a recombinant, eukaryotic gplβO (3), and a Vac¬ cinia virus recombinant (4). The latter two are al¬ ready in clinical trials in the U.S. and abroad (3, 4). These vaccines are produced from the HIV-1 env gene product, either using the gpl50 precursor pro¬ tein, or the mature envelope glycoprotein gp 120 (5).
These vaccines are being tested because numerous studies suggest that gpl20 is responsible These vaccines are being tested because numerous studies suggest that gpl20 is responsible for the cellular tropis of the virus by binding to the CD-4 target on T-helper lymphocytes (6), as well as being sufficient for synctium formation and cytopath- ic effect (7). Thus, antibodies to this protein should be capable of neutralizing the HIV. Indeed, neutralizing antibodies to HIV have been described by a number of investigators (8-10). Further, anti¬ bodies to the gpl20 have been shown to prevent virus infection in vitro (11). Unfortunately, although patients may have significant neutralizing antibody titers, they can still progress to AIDS. Moreover, vaccine trials in chimpanzee models and with the re¬ lated Simian Immunodeficiency Virus (SIV) have been disappointing. Specifically, immunized animals are not protected from HIV challenge. Experience to date has led vaccine developers to view the prospects of an effective anti-HIV vaccine with pessimism (12).
Putney, et al. (13) in 1986 reported on studies of neutralizing epitopes of gpl20, using the deglycosylated polypeptide, and fragments produced without glycosylation. The fragments of gpl20 studied included PB1, corresponding to the COOH-terminal half, PB3, corresponding to the NH2-terminal half, and PENV9, corresponding to the COOH-end of gpl20 with the NH2-end of gp41 (encoded by the BgL II to BamHI fragment of the gplβO gene). BP1 was found to contain a neutralizing epitope, while PB3 and PENV9 elicited binding but not neutralizing antibodies. PB1 was suggested as a candidate for vaccine testing.
Ho, et al. (10) in 1987 reported on studies with a series of synthesized peptides corresponding to short segments of gplβO. Two neutralizing domains were indicated as present on gpl20 and two on gp41. It was suggested that a candidate vaccine might in¬ clude all of the neutralizing epitopes while excluding non-neutralizing epitopes that might elicit inter¬ fering antibodies. No data, however, was presented to support that hypothesis. Immunodominant domains pro¬ ducing non-neutralizing antibodies have been reported for gpl20/41 (15, 16). One of these is near the COOH- terminus of gpl20 (15), and the other is in the first third of gp41 (16).
SUMMARY OF INVENTION
This invention is based on the discovery that a high percent (greater than 60%) of HIV-1 seropositive individuals have an antibody which enhances the infectivity of HIV-1. This enhancement apparently involves action in concert with the alter¬ nate pathway of complement. Although antibody- dependent enhancement (ADE) of certain viral infec¬ tions has been previously known to occur, this phe¬ nomenon had not been observed for AIDS infection. The first report of a serum factor which enhances HIV-1 infection was made in December 1987 by appli¬ cants (17) . The initial investigation suggested that the factor was not a virus or an IgG-type antibody. The factor could not be identified and the role of complement was unrecognized.
Subsequently, applicants have determined that the enhancement factor is an immunoglobulin which, with the alternate pathway of complement, can mask neutralizing antibodies and enhance HIV infec¬ tion. Further, the antibody has been shown to be directed toward the product of the HIV-1 env virogene (gpl60). A specific region of gpl60 contains one or more antigenic domains which bind to the antibody. An antigenic domain which produces infection enhancing antibodies has been localized by binding to a recom¬ binant gpl60 fragment, PENV9, corresponding to the gplβO amino acids 467 to 752. That region includes the i munodominant epitope of gp!20, amino acid sequence 497-511, and the immunodominant epitope of gp41, amino acid sequence 593-604. (The numbers refer to the Beck- man "Microgenie" listing. ) For production of a safe and effective HIV-1 vaccine, it is therefore important to eliminate those epitopes which produce enhancing antibodies. Polypeptide fragments for use in HIV-1 vaccines nominally must include one or more epitopes generating neutralizing antibodies to HIV-1, and Bcell memory, while being devoid of epitopes that elicit ADE of HIV-1 infection.
The vaccines of this invention are useful for preventing HIV-1 infection. For persons found to be carrying H V-1, the vaccines can be administered to re¬ tard progression to the Acquired Immunodeficiency Syn¬ drome (AIDS), or the pre-AIDS conditions, AIDS Related Complex (ARC) and Lymphadenopathy Syndrome (LAS). Methods are described which allow the identification and quantitation of specific antibodies which mediate antibody-dependent enhancement (ADE) of HIV-1 infection. These methods are necessairy for the comprehensive eval¬ uation of any HIV-1 vaccine and the determination of whether said vaccine elicits ADE responses in the immunization recipient.
The present invention therefore provides a method of producing a safe and effective vaccine for controlling the infectivity of human immunodeficiency virus type 1 (HIV-1). In one preferred embodiment, there is obtained, such as by expression from a DNA vector, a polypeptide composed of a fragment of HIV-1 gpl60, which fragment has at least one epitope gener- ating HIV-1 neutralizing antibodies. It is determined by in vitro laboratory tests that the polypeptide is incapable of generating HIV-1 infectivity enhancing antibodies (IEA). The polypeptide is then prepared in vaccine dose form. The in vitro tests for determining that polypeptide fragments will not generate IEA are subsequently described in detail.
EXPERIMENTAL BASIS OF INVENTION
The existence of an HIV-1 infection-enhancing factor in three of eleven HIV-1 seropositive patients' sera was discovered by the applicants (17) during the routine titration of HIV-1 neutralizing antibody acr •civity, using sera that had not been previously heat treated but which had been stored at 4°C up to 1 month following collection for HIV-1 serology testing. The three identified sera, when preincubated for one hour with HIV-1, then added to target MT-2 cells in the ap¬ plicants' microtiter infection assay, caused pronounced HIV-1-induced cytopathology as evidenced by many large, nultinucleate giant synctia at dilutions from 1:4 to 1:64. The virus control wells (no serum) at the same time (48 hours) demonstrated none to few synctia when observed under phase contrast microscopy. The increase in synctia formation was accompanied by a decrease in cell viability as determined by vital dye uptake. The infection-enhancing activity was not present in HIV-1 seronegative serum. Moreover, filtration of sera con¬ taining enhancing activity through 50 nm filters failed to remove the observed activity. A paucity of synctia was observed when greater than one multiplicity of infection (m.o.i.) of the HTLV- III» isolate of HIV-1 was added to MT-2 cells but a profound number of giant synctia were present when filtered sera were added to the virus one hour before the cells were added. This result demonstrated that the factor is not a virus likely to be found in the sera of patients infected with HIV (i.e., including HIV-1 itself, CMV, EBV, Herpes simplex. Varicella zoster, HTLV1/II, etc.) and is suggestive of a bio¬ logical entity whose hydrodynamic volume radius of gyration is <50 nm.
Demonstration that Enhancing Activity is a Multicomponent System consisting of a Ubiquitous Heat-Labile Component
(Complement) and a Limiting Heat stable Component Immunoglobulin
1. The Heat Labile Component: After several weeks at 4% our sera identified as having enhancing activity had lost the capacity to enhance HIV-1 infec¬ tion of target cells in vitro. When sera that had lost enhancing activity were mixed 1:1 with normal fresh serum, the activity was restored. The activity was not present in normal serum alone. Further, if normal human serum was heated at 60°C for one hour, it lost its ability to enhance HIV-1 infection when mixed with heat-inactivated serum with known enhancing ac¬ tivity. Table I demonstrates the neutralizing titer of either age-inactivated or heat-inactivated sera as well as the neutralizing or enhancing titer of that same serum following 1:1 mixing with normal human serum. Some of the sera (137, 154, 890 and 969) were converted from high neutralizing titers (>1:64) to high enhancing titers (>1:64). Another group (4/16) had little neu¬ tralizing activity but could enhance infectivity to high titer. These results indicated that the enhancing activity observed was a multi-component system and sug¬ gested that the ubiquitous heat-labile activity uti¬ lized either the classical or alternative complement pathway.
Table I. Comparison of Reciprocal Neutralizing and Enhancing Titers of Complement inactivated HIV-Antibody Positive Sera as a function of Complement Restoration with Normal Serum
Complement
Depleted* Complement Restored Neutralizing Neutralizing Enhancing
Serum # Titer Titer Titer
763 16 0 256
154 128 0 128
969 128 0 128
479 0 0 64
283 4 0 32
193 0 0 128
772 0 0 128
885 128 0 0
137 64 0 128
890 128 0 64
281 128 96 0
600 32 64 0
612 128 16 0
Normal 0 0 0
♦Complement depleted by aging (>4 weeks at 4β) or by heat-inactivation (60°C) for one hour). **Sera mixed 1:1 with normal, fresh human serum. Studies by the applicants have shown that the observed enhancing activity of HIV-1 seropositive in¬ dividuals utilizes the alternate complement pathway. Cobra venom factor has been demonstrated to inactivate the alternate pathway of complement (18). It was found that serum 763 could cause a decrease in time to cy- tolysis at a dilution of 1:64 or less in the absence of cobra venom or in the presence of cobra venom added at the same time that the virus was added (0 hours), but could enhance cytolysis only to a dilution less than 1:8 in the presence of a one hour pre-incubation with cobra venom (1 hour). Cobra venom factor had the same effect on serum 969. Cobra venom factor alone had no effect either on the ability of normal serum to en¬ hance infection (there was no enhancement) or prevent infection of MT-2 cells by HIV-1 (there was no neu¬ tralization) .
In parallel studies we have demonstrated that complement component Clq deficient serum, a serum that contains less than 5% of classic complement pathway activity as determined by CHso titer, was able to enhance virus cytopathicity as well as normal, fresh human serum when combined with HIV-1 seropositive sera. Factor B (a factor utilized in the alternate complement pathway) deficient serum was unable to decrease time to cytolysis when added to heat- inactivated patient serum known to contain enhancing activity. Likewise, complement component C3 defi¬ cient serum, a complement component shared by both the alternate and classic pathways, was unable to decrease time to HIV-1 induced cytolysis when mixed with com¬ plement-inactivated enhancing serum.Finally, guinea pig complement serum, which can substitute for human serum in the classical but not the alternate complement pathways, was unable to substitute for normal human serum in the enhancing assay as it could not enhance cytopathicity of HIV-1 when combined with seropositive sera. Thus, a substantial body of evidence demon¬ strates that the ubiquitous heat-labile component necessary for HIV-1 infection-enhancement found in all human serum is the alternate complement pathway.
2. The Heat-Stable Component: The original data indicated that enhancing activity was not reduced when batch mixed with Protein-A Sepharose although neutralizing antibody activity was removed (i.e., staphylococcal protein-A binds IgG and some IgA and IgM from human sera) , and when covalently bound to an insoluble matrix such as Sepharose, antibodies with an affinity to Protein-A can be removed from solution. Further studies using Protein-A Sepharose columns, however, did result in removal of the heat-stable com¬ ponent. As Table II shows, the stable factor was al¬ most entirely removed by protein A column chromatog- raphy while the labile factor was not retained by the column. The data demonstrate the protein-A treated serum 763 had little activity even in the presence of 1:20 normal serum (titer <1:8). Protein-A did not, however, remove the labile factor because a column flow-through could decrease time to cytolysis in the presence of 1:20 heat-inactivated serum 763 to a dilution of 1:64. Further, the stable component was capable, when titered in the presence of a constant amount of labile component from normal serum (1:20) of enhancing cytolysis at a dilution of 1:5120. Thus, the heat stable component was not the limiting factor in serum and was removed by protein-A column chroma- tography. The labile factor was the limiting factor and was not removed by protein-A chromatography. 11
TABLE IX. Enhancing Factor Reciprocal Titer as a Function of Heat Lability and Protein-A Column Retention of Fresh Serum 763
Added Factor*
Heated
Normal Heated Normal
Serum 763 None Serum Serum 763 Serum
No treatment 12S NT§ NT 64
Heated** 0 5120 NT 0
P.A.*** 8 NT 64 8
P. . Heated NT 8 NT NT
*Serum as indicated was added so that the dilution of serum in growth medium was 1:20 in each well. Serum 763 treated various ways was then twofold diluted in this growth media containing the indicated 1:20 serum.
**Serum was heated at 60βC for one hour.
***P.A. is the flow-through of serum 763 subjected to proteiπ-A column chrom tograph .
§NT denotes not tested.
B, Demonstration that HIV-1-Enhancing Sera Can Inhibit the Biological Effects of Neutralizing Antibody
Since serum 281 demonstrated good neutralizing antibody titers against HIV-1 in the presence of fresh human serum, it was combined with three enhancing sera. Table III demonstrates that although serum 281 could still neutralize virus in the presence of enhancing serum, cytolysis of 50% or more MT-2 cells occured only at serum dilutions greater than 1:128 in the presence of sera 969 and 154 (data for serum 154 not shown) . When combined with serum 763, the most potent enhancer of HIV-1 infection, MT-2 cells were protected from HIV-1-induced pathology only at dilutions of neutralizing serum 281 less than 1:16. Serum 281 was capable of protecting MT-2 cells from cytolysis at a dilution of 1:128 when tested alone.
Table III. Reciprocal Neutralization Titer* of Serum 281, A Neutralizing Serum, Mixed with Several Enhancing Sera
Fresh, Normal
No Serum Serum 969 763
Serum 281 64 128 96** 6**
No Serum 0 0 64 16***
Fresh Serum - 0 0 § §
♦Highest dilution giving 50% protection from HIV-induced cytopathology relative to a virus control containing no serum. **Fresh serum was mixed 1:1:1 with enhancing serum and neutralizing serum. ***Serum 763 was heat-inactivated to give neutralizing titer value. §Denotes enhancing activity. C. Demonstration that Antibody-Dependent Enhancement of HIV-1 Infection is Induced in the Chimpanzee Challenged with Live HIV-1
Evidence that HIV antibody-dependent enhance¬ ment (ADE) occurs in species other than man has been obtained in the chimpanzee. Fresh frozen sera from two normal chimpanzees (X35 and X95) significantly reduced the time required for HIV induced cytopathicity in MT-2 cells; this non-antibody enhancement was not further stimulated by human anti-HIV sera. Conversely, as illustrated in Table IV, heat inactivated chimpanzee serum XI15 obtained from an animal chronically infected with HIV-1 (20) was able to utilize both chimpanzee and human complement to enhance HIV infection of MT-2 cells and to mask its constitutive neutralizing antibody ac¬ tivity towards HIV-1. Thus, enhancing activity can be monitored in the chimpanzee, the only human surrogate animal model for vaccine testing. The preferred em¬ bodiment for complement restoration in order to establish enhancing titer in the chimpanzee is fresh frozen human serum since chimpanzee complement yields a non-antibody dependent enhancement resulting in a high normal background not observed when fresh frozen human serum is used as a complement source.
Figure imgf000016_0001
TABLE IV. Comparison of Reciprocal Neutralizing and Enhancing Titers of Chimpanzee Complement- Inactivated, HIV-Antibody Positive and Negative Sera as a Function of Complement Restoration with Chimpanzee or Human Normal Serum
Complement£ Complement Restored Depleted Chimpanzee Complement Human Complement
Serum I Neutralizing Titer Neutralizing Tjter Enhancing Titer Neutralizing Titer Enhancing Titer
X115 128 16 2048 0 2048
Figure imgf000016_0002
X95v 0 0 0
a Heat-inactivated (56βC, one-half hour) . b Titered with constant 1:50 fresh-frozen, normal chimpanzee serum or 1:20 fresh-frozen, normal human serum as indicated, c HIV-negative chimpanzee. d A high background due to non-antibody complement fixation was observed with normal chimpanzee serum.
D. Demonstration that Antibody-Dependent Enhancement of HIV-1 Infection is Directed Towards a Region of the gp-160 Polyprotein which Contains a Portion of the C-terminal of gpl20 and the N-Terminus of gp41
Four enhancing sera each containing neutral¬ izing antibodies of varying titer were subjected to affinity chromatography against the protein of a cloned fragment of gpl60 referred to as PENV9 (13) and separate BSA column as a control for non-specific ad¬ sorption. The PENV9 polypeptide fragment contains the amino acids of gplβO from 467 to 756, which bridge the immunodominant regions of gpl20 (15) and gp41 (16).
Table V demonstrates the results of the af¬ finity chromatography. As demonstrated earlier, serum 281 was neutralizing even in the presence of comple¬ ment, while sera 763, 772, and 154 were enhancing. The PENV9 eluent fraction contained only enhancing ac¬ tivity for all four sera. Thus, enhancing epitopes can be separated from neutralizing epitopes. The BSA column data demonstrates that non-specific column- protein "stickiness" was not a factor since no activi¬ ty was present in the BSA column eluent.
TABLE V. Reciprocal HIV-1 Enhancing and Neutralizing Titers from BSA and PENV9 Column Fractions
Column Fraction
Serum* BSA effluent BSA eluent PENV9 effluent PENV9 eluent
763 (0)/>2048** (01/0 (0)/1024 (0)/1024
772 (16)/>2048 C0)/0 C1024)/0 (0)/512
154 C16)/>2048 (0)/0 (8)/1024 (0)/64
281 (32)/0 (0)/0 C>1024)/0 (0)/64
*Serum 281 was demonstrated to be a neutralizing serum in the presence of complement and had shown no ADE in assays. Sera 763, 772, and 154 have previously demonstrated ADE in in vitro assays.
**The highest dilution giving neutralization of HIV-1 is given in parentheses, the highest dilution demonstrating enhancement of HIV-1 infection is given without parentheses. In cases where both enhancement and neutralization occurred, enhancement was observed at the first dilution greater than the greatest dilution demonstrating neutralization.
VACCINE DESIGN In applying the discovery underlying the present inven¬ tion to vaccine design, consideration should be given to the func¬ tional regions of gplβO as summarized in the following Table A.
TABLE A
Locations of Proposed Functional Regions of gpl60 Precursor of gpl20 and gp41 from Amino Acid Residues 1 to 856
Microgenie ,,,
Residue Nos. Proposed Function References
1 (Met) Start gpl20 21
105-117 T-Cell Antipathic 22
262-264 Noncovalent gpl20/gp41 23 interaction
291-307 HIV-1 Neutralizing 10
413-456 Virus Attachment 24
421-436 T-Cell Antipathic 22
451-577 HIV-1 Neutralizing 10
497-511 Immunodominant 15
512 (Ala) Start gp41 25
593-604 Immunodominant 16
609-625 HIV-1 Neutralizing 10
721-744 HIV-1 Neutralizing 10
856 (Leu) End gp41 21
(1) Based on Bec man "Microgenie" printout, taking the ATG codon cf nucleotides 5802-5804 as encoding first amino acid ( ethioπine) , and following the sequence of Ratner et al. (21)
(2) In the cited references residue numbers for these sequences are shown with higher numbers: viz. T-cell antipathic (112-124) , neutralizing (298-314), T-cell antipathic (428-443), neutraliz¬ ing (458-484), immunodominant (504-518), immunodominant (598-604), neutralizing (616-632), neutralizing (728-751). 18
In describing polypeptide fragments for use in the present invention, the same residue numbering will be used as in Table A. Residue 1 methionine to residue 856 leucine follows the total sequence of Ratner (21) .
Vaccines designed in accordance with the principles of the present invention will be more ef¬ fective because they will lack capacity to induce antibody-dependent enhancement of HIV infectivity. Such vaccines differ in this important respect from the present vaccines utilizing gpl60 or gpl20 as the immunizing antigens.
The present invention comprises an improved method of controlling HIV-1 infectivity in human pa¬ tients. There is administered one or more polypeptide fragments of the amino acid sequences of HIV-1 or gplδO; the fragments including one or more epitopes generating neutralizing antibodies to HIV-1 while be¬ ing devoid of any epitope generating HIV infection enhancing antibodies. The size of the fragments will be in excess of 170 amino acids from gpl60/gpl20, and preferably about 190 to 450 amino acids. Additional non-gpl60/gpl20 amino acids may be present at 5' or 3' ends of the functional fragment.
In preferred embodiments, the polypeptide fragment can contain about 175, 193, or 448 amino acids, corresponding respectively to sequences 273 to 447, 273 to 465, and 1 to 447, all of which contain a neutralizing epitope and are free of infection- enhancing epitope. All of these can be readily pre¬ pared from restriction fragments of the gp!60 gene. Another potentially desirable polypeptide can contain the gpl20 amino acid sequence from 105 through 456, a sequence of 352 amino acids including the neutraliz¬ ing epitope 291-307 and the virus attachment/T-cell antipathic regions 421-456/413-458. Preferably the polypeptide fragments of gpl60/gpl20 contain no more than 496 amino acids, such as the sequence 1 to 496, which includes both of the neutralizing regions so far identified in gpl20, while omitting the immuno¬ dominant region 497-511.
The polypeptides can be utilized in nongly- cosylated form, but for optimum effectiveness it is preferred to utilize fully glycosylated polypeptides, i.e., corresponding in glycosylation to gpl60/120. Other segments of gplβO can be added or incorporated to the sequences described above, viz. by genetic engineering techniques and manipulations. In par¬ ticular, it may be desirable to add to the COOH- ter inal portion of the polypeptide one or more addi¬ tional HIV-1 neutralizing epitopes, such as those of gp41, the 609-625 or the 721-744 sequences. When such sequences are added, they should not include the im¬ munodominant 593-604. Preferably the entire sequence of gpl60 from 478 to 605 is omitted from the vaccine polypeptide.
The polypeptide vaccines of this invention can be prepared and administered by procedures and protocols similar to those being used for the gpl20 and gpl60 vaccines. The carrier of the peptide may be a sterile aqueous solution, such as normal saline, and the polypeptide may be absorbed on alum (26, 27). Concentrations may range from 10 to 100 micrograms per illiliter. Concentration will be selected so that a single injected dose of 0.5 to 1.0 ml followed by booster doses will provide a sufficient amount of the polypeptide to consistently induce an immune protec¬ tive response. The preferred route of injection is intramuscular, except in cases where the patient is at risk of hemorrhage (i.e, hemophiliacs). Where re¬ quired, subcutaneous injection can be used as an alternate to intramuscular injection. Following the initial injection, a booster injection may be given at intervals of one month, six months, or yearly, as re¬ quired. Titers of neutralizing antibodies can be monitored to determine the need for a booster injec¬ tion.
PREPARATION OF VACCINES
Truncated species of the HIV-1 gpl60 can be obtained by restriction endonuclease digestion of HIV-1 proviral DNA followed by insertion of the frag¬ ment into an expression vector plasmid which can be cloned in eukaryotic cells for the synthesis of the modified protein. This genetic engineering of the HIV envelope gene utilizes materials which are available to the research community through biotechnology com¬ panies. Similar protocols have been developed and utilized for the cloning and expression of whole HIV-1 gpl60 and gpl20 products in eukaryotic cells. Re¬ striction endonucleases are used to engineer a trun¬ cated HIV-1 gplδO or gpl20 by eliminating epitopes responsible for eliciting antibody-dependent enhance¬ ment of HIV infection while retaining epitopes responsible for eliciting protective immunity. Since carbohydrate moities are known to be important for known to be important for antigenic stimulus, emphasis should be placed on expressing the genetically engi¬ neered protein in eukaryotic cells, which are most likely to synthesize a properly glycosylated product.
An example of a restriction fragment which encodes only desired epitopes is the Sspl fragment en¬ coding the first 448 amino acids of gp!20. This frag¬ ment can be obtained from the HIV DNA on pHxB-2D available from Biotech Research Laboratories, Incor¬ porated. The fragment can be inserted in the proper vector AC373 and used to construct a recombinant baculovirus by cotransfection of the Spodoptera frugiperda cell line Sf9, as described in (14). The recombinant baculovirus has the property of synthe¬ sizing large amounts of the cloned, tailored HIV gpl60 protein in infected Sf9 cells owing to the strong polyhedron gene promoter used to drive expression of this gene. This mammalian system has the added advantage of producing a fully glycosylated product. For a non-glycosylated product, an E^ coli expression system can be used, as described in (13). Similarly, a Bgl-II DNA fragment of the gpl60 gene can be used to prepare the 193 amino sequence from 273 to 465; or a Bgl-II/Sspl DNA fragment to prepare a 176 amino acid sequence from 273 to 448.
Synthesis and purification of HIV recombinant polypeptides can be accomplished by infecting mono- layers of Sf9 cells with genetically engineered bacu¬ lovirus at a multiplicity of infection of 3. Cells are harvested approximately 4 days later and lysed in a solution containing 20mM Tris-HCl, pH 7.5, lOmM Mg(OAc)2, 1% Triton X-100. The lysed cell suspen¬ sion is centrifuged at 15,000 xg and the pellet, which 22
contains recombinant glycoprotein, is solubilized in isotonic buffer and further purified by lentil lectin sepharose Cl-4b affinity chromatography. The presence of recombinant HIV glycoprotein can be confirmed by immunoblotting. The purified glycoprotein is then prepared for vaccine use as previously described.
Methods for Vaccine Evaluation
Evaluation of gpl60 fragments for the absence of epitope which can induce formation of infection- enhancing antibodies can be achieved by three methods: (1) the decrease in time required for productive in¬ fection of ADE-sensitive target cells; (2) the differ¬ ential titer of neutralizing antibody activity as a function of fresh complement in ADE-sensitive target cells; and (3) the detection of antibodies which bind specifically to (i) PENV9 or (ii) a fraction of PENV9, or (iii) a synthetic peptide homologous to the anti¬ body dependent enhancement domains of PENV9. Methods 1 and 2
Methods 1 and 2 require infection of target cells by HIV-1. The prototype target cell is a clone of the T-lymphoblastoid cell line MT-2 and is culti¬ vated as previously described (19). MT-2 cells yield 100% cytosis in 4 days or less when challenged with HIV-1 at a multiplicity of infection (MOID) ≥l. This cell line is a sensitive indicator for HIV-1 ADE due to its high content of complement receptors (CR2) and conventional HIV receptors (i.e., CR4) on its plasma membrane and serves as a useful indicator cell for HIV-1 ADE in both human and chimpanzee sera. Alter¬ nate target cell should be used in the basic assay provided that they grow readily in 96 cell culture plates, are highly permissive for HIV-1 infection, and 23
are sensitive to ADE of HIV-1 infection. Method 1 defines the titer of infection-enhancement as that highest titer of serum which yields a statistically significant reduction in time to cell cytolysis and/or HIV-1 antigen release into the supernatant as compared to control HIV-1 infection. Method 2 depends on the differential neutralizing titer obtained on heat- inactivated serum (56C, 1/2 hr.) when titered in the presence of a constant amount of fresh serum. Serum with significant amounts of infection-enhancing antibody will demonstrate reduction or elimination of neutralizing antibody titers in the presence of fresh human or chimpanzee complement. Method 3
This method is a noninfectious method which depends on the binding of infection-enhancing anti¬ bodies or enhancing/antibodies immunodominant domains of PENV9, a fraction of PENV9 containing antibody enhancing immunodominant domains, or synthetic pep- tides homologous with said enhancing/antibodies im¬ munodominant domains. These enhancing/antibody im¬ munodominant domains may be either in solution or attached to a solid matrix, the latter being the pre¬ ferred physical state. Following binding of anti¬ bodies to the solid state peptide, the latter is washed x 3 with physiological saline to remove non- specifically bound antibody and the remaining anti¬ body quantitated by standard methods of analysis which include commercially available antibodies containing covalently bonded enzymes, fluorescent groups, or radioactive tracers and which are specific for human or chimpanzee antibody. Quantitation is made by the absolute amount of antibody bound from a given amount of serum or by qualitative detection as a function of serum titer. The foregoing also represent new method of detection and quantitation of HIV-1 infection enhanc¬ ing antibodies (IEA) which can be used in humans and chimpanzees. One method is based on the decrease in the time of HIV-1 induced target cell cytopathicity. Alternatively or additionally, detection and quanti¬ tation of the infection enhancing antibodies (IEA) can be based on a decrease in time of HIV antigen expres¬ sion.
Complement test procedure can also be used. Specifically, .IEA in humans and chimpanzees can be detected and quantitated based on differential neu¬ tralizing antibody titers as a function of the pres¬ ence or absence of fresh human or chimpanzee comple¬ ment. This may be determined by target cell cyto¬ pathicity and/or by HIV antigen expression.
Infection enhancing antibodies (IEA) can also be detected and quantitated by binding to PENV9 or a fragment of PENV9 in solution or bound to a solid phase matrix. The amount of bound antibody can be measured by ELISA, fluorescence, or radioactive methods. Instead of PENV9, synthetic peptides homolo¬ gous to the infection-enhancing epitopes of PENV9 can be used. REFERENCES
(1) Guyader, M. , et al.. Genome organization and transactivation of the human immunodeficiency virus type 2. Nature 1987; 326:662-669.
(2) Lasky, L.A., et al.. Neutralization of the AIDS retrovirus antibodies to a recombinant envelope glycoprotein. Science 1986; 233:209-212.
(3) Barnes, AIDS vaccine trial okayed. Science 1987; 237:973.
(4) Zagury, D.L., et al.. Immunization against AIDS in humans. Nature 1987; 326:249-250.
(5) Allan, J.S., et al.. Major glycoprotein antigens that induce antibodies in AIDS patients are en¬ coded by HTLV-III, Science 1985; 228:1091-1094.
(6) McDougal, J.S., et al.. Binding of HTLV-III/LAV to T4 T-Cells by a complex of the 110K viral protein and the T4 molecule. Science 1986; 231:382-385.
(7) Sodroski, J., et al.. Role of the HTLV-III/LAV envelope in syncytiu formation and cytopathic¬ ity. Nature 1986; 324:572-574.
(8) Weiss, R.A., et al.. Variable and conserved neutralization antigens of human immunode¬ ficiency virus. Nature 1986; 324:572-574. (9) Carter, W.A., et al.. Clinical, immunological and virological effects of Ampligen, a mis¬ matched double-stranded RNA, in patients with AIDS or AIDS-related complex. Lancet 1987; i:1286-1292.
(10) Ho, D.C., et al.. Human immunodeficiency virus neutralizing antibodies recognize several coserved domains on the envelope glycoproteins, J. Virol. 1987; 61:2024-2028.
(11) Robey, W.G., et al. , Prospect for prevention of human immunodeficiency virus infection: purified gpl20 kDa envelope glycoprotein induces neutral¬ izing antibody. Proc. Natl. Acad. Sci. (USA), 1986; 83:7023-7027.
(12) Kolata, G., The New York Times, Feb. 12, 1988.
(13) Putney, S.D., et al., HTLV-III/LAV-neutralizing antibodies to an E^ coli-produced fragment of the virus envelope. Science 1986; 234:1392-1395.
(14) Summer, M.D., et al. (1987), in "A Manual of Baculovirus Vector and Insect Culture Pro¬ cedures", Texas Agric. Exp. Sta. Bull. No. 1555.
(15) Palker, et al., A conserved region at the COOH terminus of human immunodeficiency virus gpl20 envelope protein contains an immunodominant epitope. Proc. Nat. Acad. Sci. USA 1987; 84:2479-2483. 27
(16) Gnann, J.W., Jr., et al.. Fine mapping of an immunodominant domain in the transmembrane glycoprotein of human immunodeficiency virus. J. Virol. 1987; 61:2639-2641.
(17) Robinson, W.E., et al., A human immunode¬ ficiency virus type 1 (HIV-1) infection- enhancing factor in human sera. Biochem. Biophys. Res. Comm. 1987; 149L693.699.
(18) Klein, P.G., et al. Multiple nature of the 3rd component of guinea pig complement
1) Separation and characterization of 3 factors a, b, and c, essential for haemol¬ ysis. Immunol. 1965; 8:590-603.
(19) Montefiore, D.C., et al.. Evaluation of anti¬ viral drugs and neutralizing antibodies against human immunodeficiency virus by a rapid and sensitive microtiter infection assay. J. Clin. Microbiol. 1988: 26:231-235.
(20) Eichberg, J.W., et al., T-cell responses to Human Immunodeficiency Virus (HIV) and its recombinant antigens in HIV-infected chimpan¬ zees. J. Virology, 1987; 61:3804-3808.
(21) Ratner, L., et al.. Complete nucleotide se¬ quence of the AIDS virus, HTLV-III. Nature 1985; 313:277-284. (22) Cease, K.B., et al.. Helper T-cell antigenic site identification in the acquired immunode¬ ficiency syndrome virus gpl20 envelope protein and induction of immunity in mice to the native protein using a 16-residue synthetic peptide. Proc. Natl. Acad. Sci. (USA) 1987; 84:4249-4253.
(23) Kowalski, M. , et al.. Functional regions of the envelope glycoprotein of Human Immunodeficiency Virus Type 1. Science 1987; 237:1351-1355.
(24) Lasky, L.A., et al.. Delineation of a region of the human immunodeficiency virus type 1 gpl2 glycoprotein critical for interaction with the CD4 receptor. Cell 1987; 50:975-985.
(25) Veronese, F.D., et al.. Characterization of gp41 as the transmembrane protein coded by the HTLV- III/LAV envelope gene. Science 1985; 229:1402- 1405.
(26) Bittle, J.L., et al.. Protection against foot-and- mouth disease by immunization with a chemically synthesized peptide predicted from the viral nucleotide sequence. Nature 1982; 298:30-33.
(27) Shinnick, T.M., et al.. Synthetic peptide immuno- gens as vaccines. Ann. Rev. Microbiol., 1983; 37:425-446.

Claims

CLAIMSWe claim:
1. A vaccine for preventing infection by the human immunodeficiency virus type 1 (HIV-1) and/or for inhibiting disease progression in HIV-1 seropositive patients, comprising a sterile carrier containing a polypeptide composed of a fragment or fragments of the total amino acid sequence of HIV-1 gpl60, said poly¬ peptide containing at least 170 amino acids and in¬ cluding at least one epitope of gpl20 generating neu¬ tralizing antibodies to HIV-1 while being devoid of any epitope of gpl60 generating HIV-1 infection- enhancing antibodies.
2. The vaccine of claim 1 in which said polypeptide is glycosylated and contains at least 170 amino acids of gpl20.
3. The vaccine of claims 1 or 2 in which said polypeptide contains from 190 to 450 amino acids of gpl20.
4. The vaccine of claims 1 or 2 in which said polypeptide is selected from the group of gpl20 amino acids sequences corresponding essentially to the sequences 1 to 447, 273 to 447, and 273 to 465.
5. The vaccine of claims 1 or 2 in which said polypeptide consists of at least amino acids 105 to 456 of gpl20, and includes at least two epitopes of gpl20 generating neutralizing antibodies to HIV while being devoid of any epitope of gpl60 generating HIV infection-enhancing antibodies.
6. The vaccine of claim 5 in which said polypeptide contains the sequence 1 to 447 of gpl20 but does not include the immunodominant sequence 497 to 511 of gpl20.
7. The method of producing a vaccine for controlling the infectivity of human immunodefi¬ ciency of virus type 1 (HIV-1), comprising obtaining a polypeptide fratment of HIV-1 gpl60 which has at least one epitope generating HIV-1 neutralizing antibodies, determining by in vitro testing that said polypeptide will not generate HIV-1 infectivity enhancing anti¬ bodies (IEA), and preparing said polypeptide fragment in vaccine dose form.
8. The method of claim 7 in which said poly¬ peptide fragment is a fragment of gpl20 and contains at least 170 amino acids but not more than 496 amino acids.
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Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMM., Vol. 149, No. 2, 16 December 1987, ROBINSON et al., see pages 693-699. *
JOURNAL OF IMMUNOLOGY, Vol. 135, No. 6, December 1985, ECKELS et al., see pages 4201-4203. *
JOURNAL OF VIROLOGY, Vol. 61, No. 6, June 1987, HO et al., see pages 2024-2028. *
JOURNAL OF VIROLOGY, Vol. 62, No. 11, November 1988, SKINNER et al., see pages 4195-4200. *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, U.S.A., Vol. 84, June 1987, NARA et al., see pages 3797-3801. *
SCIENCE, Vol. 234, 12 December 1986, PUTNEY et al., see pages 1392-1395. *
SCIENCE, Vol. 237, 11 September 1987, KOWALSKI et al., see pages 1351-1355. *

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