WO1996009066A2 - Procede de traitement d'infections dues au virus de l'immunodeficience humaine (vih) - Google Patents

Procede de traitement d'infections dues au virus de l'immunodeficience humaine (vih) Download PDF

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Publication number
WO1996009066A2
WO1996009066A2 PCT/US1995/011943 US9511943W WO9609066A2 WO 1996009066 A2 WO1996009066 A2 WO 1996009066A2 US 9511943 W US9511943 W US 9511943W WO 9609066 A2 WO9609066 A2 WO 9609066A2
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virus
siv
hiv
tropic
envelope
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PCT/US1995/011943
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English (en)
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WO1996009066A3 (fr
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Janice E. Clements
Michael Murphey-Corb
M. Christine Zink
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The Johns Hopkins University School Of Medicine
The Administrators Of The Tulane Educational Fund
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Priority to AU35930/95A priority Critical patent/AU3593095A/en
Priority to EP95933167A priority patent/EP0784484A2/fr
Publication of WO1996009066A2 publication Critical patent/WO1996009066A2/fr
Publication of WO1996009066A3 publication Critical patent/WO1996009066A3/fr

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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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1036—Retroviridae, e.g. leukemia viruses
    • C07K16/1045—Lentiviridae, e.g. HIV, FIV, SIV
    • C07K16/1063—Lentiviridae, e.g. HIV, FIV, SIV env, e.g. gp41, gp110/120, gp160, V3, PND, CD4 binding site
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • This invention relates generally to the field of immunology and specifically to improved enhanced immunological response against human immunodeficien ⁇ cy virus (HIV) infection.
  • HIV human immunodeficien ⁇ cy virus
  • AIDS Acquired immune deficiency syndrome
  • HAV human immunodeficiency virus
  • the World Health Organization estimates that there are currently between eight and ten million people presently infected with HIV and that this number will rise to 15-20 million by the end of this decade. By this time, the cumulative total of AIDS patients will be in the region of 16 million people which will pose an impossible burden for health care systems. Governments worldwide have allocated vast sums of money to publicize the risks of HIV infection and to educate people about the ways in which infection can be avoided, but despite all these efforts, the AIDS epidemic continues unabated. At present, even the best anti-AIDS drugs have a limited efficacy and are associated with detrimental side effects.
  • the primary cellular targets for human immunodeficiency virus type 1 are CD4+ lymphocytes and monocyte-derived macrophages.
  • the lymphocyte is the major infected cell type in the blood while the macrophage is the predominant infected cell type in the brain and spinal cord.
  • the decline in CD4+ lymphocyte concentrations in HIV-infected individuals clearly contrib ⁇ utes to the eventual development of AIDS. Less appreciated and understood is the role of monocytes and macrophages.
  • a vaccine contains immunodominant antigens which elicit an effective immune response.
  • HIV infection can cause profound lymphopenia, primarily of the CD4 subset of T lymphocytes.
  • Affected individuals have decreased or absent delayed-type hypersensitivity, extreme susceptibility to opportunistic infections and may acquire certain unusual malignancies such as Kaposi's sarcoma or Burkitt's lymphoma.
  • HIV also causes polyclonal expansion of B lymphocytes, leading to hypergamma- globulinemia.
  • affected individuals are incapable of mounting a primary immune response to newly encountered antigens.
  • the syndrome has been recog ⁇ nized primarily in "at risk” groups, including homosexually active males, intravenous drug users, recipients of blood or blood products, and certain populations from Central Africa and the Caribbean.
  • the syndrome has also been recognized in heterosexual partners of individuals in all "at risk” groups and in infants of affected mothers.
  • a vaccine should artificially stimulate the immune system in such a way that a subsequent entry of the live pathogenic virus into the body results in the inhibition and, preferably, the elimination of the virus and its progeny before disease occurs.
  • One of the major problems associated with HIV is that the mechanisms of disease induction are largely unknown. Seropositive individuals can remain healthy for many years carrying very low levels of HIV within the body. During this period, the anti-HIV immune response remains very strong and yet, at a certain point in time, the virus begins to replicate rapidly and AIDS develops. Vaccines in general allow the pathogen some degree of replication, but prevent the onset of disease. However, given the failure of the vigorous immune response to prevent the low levels of virus during the latent phase from suddenly expanding to cause disease, it has long been assumed that an AIDS vaccine must also prevent the initial establish ⁇ ment of infection.
  • SIV exhibits extensive similarity to HIV in genomic organization, gene sequences, and biological properties (Desro- siers, R., Annu. Rev. Immunol., 8:557, 1990; Gardner, ef al., AIDS 2 (Suppl.1):S 3, 1988). Molecularly cloned SIV mac 239 causes AIDS and death in the common rhesus monkey (Macaca mulatta) (Kestler, et al., Science
  • HIV came using what is perhaps the simplest form of a vaccine, i.e., whole inactivated virus.
  • groups throughout the world predominantly the group of Murphey-Corb in the United States, successfully protected rhesus macaques against subsequent challenge with a lethal dose of homologous virus using whole virus particles inactivated by formalin (Science, 246:1293,
  • Simian Immunodeficiency Virus (SIV)-infected macques have now proven to be a valuable animal model for the study of human AIDS.
  • SIV Simian Immunodeficiency Virus
  • SIV/Delta was originally isolated from rhesus monkeys with an experimentally transmissible immunodeficiency disease characterized by wasting, chronic diarrhea, lymphadenopathy or lymphoid depletion, opportunistic infections and increased incidence of B-cell lympho- mas.
  • This virus is tropic for CD4 positive T-lymphocy es from rhesus monkeys and humans, but is less cytotoxic for human T-lymphocytes than HIV.
  • Numerous inoculations of SIV/Delta have been performed in juvenile rhesus monkeys over the past several years at the Tulane Regional Primate Research Center (TRPRC) in an attempt to understand the pathogenesis of this virus. To date over 400 rhesus have been inoculated with pathogenic isolates of SIV/Delta; the current mortality in these experimental infections is greater than 90%, with 50% of the deaths occurring within 6 months post- inoculation.
  • the present invention provides a method for stimulating in a subject, a cross-protective immune responses induced by an attenuated macrophage-tropic clone of HIV, and demonstrates that rapid, protective responses appear concomitantly with broad neutralizing antibodies.
  • the present invention is based on the unexpected discovery that administra ⁇ tion to a host of an attenuated immunodeficiency lentivirus, which is macro ⁇ phage-tropic, as opposed to the often studied immunodeficiency lentivirus, which is only lymphocyte-tropic, induces a rapid, immune response in the host that is associated with production of broad range neutralizing antibodies.
  • the invention shows that a live attenuated virus containing macrophage-tropic specific nucleotide sequences induces a protective immune response by 1 ) proper presentation of antigenic sequences to the immune system by replication in macrophages/monocytes and 2) by presentation of the correct conformational structure of specific polypeptide sequences which may be unique to macrophage-tropic proteins for induction of the appropriate immune response.
  • monocyte/macrophages reside in high concentra ⁇ tions in mucosal surfaces
  • selective antigen presentation by these cells may confer a selective advantage in the induction of mucosal immunity which is necessary to block the spread of lentiviruses.
  • Presentation of the polypeptide antigen that provides macrophage specific tropism may include a cellular determinant, therefore alloantigens may also play a role in proper induction of a protective immune response.
  • the invention also provides pharmaceutical compositions comprising an attenuated virus comprising a retroviral nucleotide sequence encoding a macrophage-tropic polypeptide which stimulates an immune response, in a pharmaceutically acceptable carrier.
  • the retrovirus nucleotide se ⁇ quence encodes a macrophage-tropic polypeptide such as an HIV virus enve- lope (env) polypeptide.
  • FIGURES 1 A-C show a schematic illustration of the strategy for amplifying SIV envelope sequences.
  • FIGURES 2A-C show the detection of mutations by sequencing of PCR products from SIV infected monkey brain DNA.
  • FIGURE 3 shows the amino acid differences in the env genes of wild type and recombinant SIV.
  • FIGURE 4 shows the amino acid sequence of the env genes of wild type and recombinant SIV.
  • FIGURE 5 shows a flow cytometric analysis of T lymphocyte populations in the peripheral blood of monkey L238 after inoculation with SIV/17E-CI (•, CD4+; °, CD8+; *, CD4+CD29+).
  • FIGURE 6 shows neutralizing antibody titers (10 log 10 ) over time in sera from monkeys infected with SIV/17E-CI (°, L235; ⁇ , L238; ⁇ , M118; •, L652; and ⁇ , L471 ).
  • FIGURE 7 shows the avidity of serum antibodies for SIV envelope glycopro- teins (o, L235; ⁇ , L238; ⁇ , M118; •, L652; and D, L471 ).
  • FIGURE 8 shows the conformational dependence of serum antibodies to SIV envelope glycoproteins as measured by (A) native and (B) denatured viral envelope glycoprotein substrates (°, L235; ⁇ , L238; ⁇ , M118; •, L652; and D, L471 ).
  • FIGURE 9 is a table showing antigen-specific CTL and antibody responses in monkeys immunized with /.e -deleted monocyte and lymphocyte-tropic SIV clones.
  • the present invention provides a method and compositions for immunological protection against the human immunodeficiency virus (HIV).
  • HBV human immunodeficiency virus
  • SIV simian immunodeficiency virus
  • SIV-infected monkeys were reactive not only with the infecting virus, but several heterologous isolates of SIV as well.
  • This model serves as the basis for a comparable method and compositions useful for inducing a protective immune response against HIV in humans.
  • the invention provides an immunotherapeutic method of treating a host having or at risk of having a lentivirus infection, comprising administering to the host a therapeutical ly effective amount of an attenuated virus comprising a retrovirus nucleotide sequence encoding a macrophage-tropic polypeptide which stimulates an immune response.
  • An immunotherapeutic method in accordance with this invention entails the administration of the attenuated virus comprising a retrovirus nucleotide sequence encoding a macrophage- tropic polypeptide.
  • the attenuated virus can be administered by injection or infusion, for example, prior to (prophylaxis) or following (therapy) the onset of infection with the lentivirus.
  • the amount of attenuated virus required to induce an immune response to the lentivirus depends on such factors as the type and severity of the infection, the size and weight of the infected subject, and the effectiveness of other concomitantly employed modes of prophylaxis or therapy. This amount should be sufficient to induce an immune response in an immunized individual which ameliorates the particular lentiviral disease as compared to the immune response in a non-immunized individual.
  • the retrovirus nucleotide sequence encoding the macrophage-tropic polypep ⁇ tide can be derived from any retrovirus and preferably is derived from a lentivirus sequence.
  • the lentivirus family includes such viruses as human immunodeficiency virus (HIV) (including HIV type-1 and type-2), simian immunodeficiency virus (SIV), visna virus of sheep, caprine arthritis-encephali ⁇ tis virus and equine infectious anemia virus.
  • HIV human immunodeficiency virus
  • SIV simian immunodeficiency virus
  • visna virus of sheep equine infectious anemia virus.
  • the macrophage- tropic nucleotide sequence used in the immunotherapeutic method of the invention is derived from SIV, when the host is a non-human primate, and from HIV, when the host is a human.
  • the immunotherapeutic method of the invention includes a prophylactic method directed to those hosts at risk for the lentivirus infection.
  • the method is useful for humans at risk for HIV infection.
  • a "prophylactically effective" amount of the attenuated virus comprising a retrovirus nucleotide sequence encoding a macrophage-tropic polypeptide which stimulates an immune response refers to that amount which is capable of inducing an immune response to HIV which produces some degree of protection as compared to non-immunized individuals.
  • Transmission of HIV occurs by at least three known routes: sexual contact, blood (or blood product) transfusion and via the placenta. Infection via blood includes transmission among intravenous drug users. Since contact with HIV does not necessarily result in symptomatic infection, as determined by seroconversion, all humans may be potentially at risk and, therefore, should be considered for prophylactic treatment by the immunotherapeutic method of the invention.
  • the term "therapeutically effective" means that the amount of attenuated virus administered is of sufficient quantity to increase the subject's immune response to the virus, for example, to HIV.
  • the dosage ranges for the administration of the virus composition are those large enough to produce the desired effect in which the HIV epitopes are focused on the surface of the APCs, thereby allowing a more efficient antigen presentation and therefore a more effective vaccination.
  • antigen presentation may include cellular determinants as well as HIV determinants.
  • the dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions and the like.
  • the dosage will vary with the age, condition, sex, and extent of the disease in the patient and can be determined by one skilled in the art.
  • the dosage can be adjusted by the individual physician in the event of any contraindications and can be readily ascertained without resort to undue experimentation.
  • the effectiveness of treatment can be determined by monitoring the level of CD4+ T-cells in a patient. An increase or stabilization in the relative number of CD4+ cells should correlate with recovery of the patient's immune system.
  • the attenuated virus used in the method of the invention can be administered to a patient prior to infection with HIV (i.e., prophylactically) or at any of the stages described below, after initial inaction.
  • the HIV infection may run any of the following courses: 1) approximately 15% of infected individuals have an acute illness, characterized by fever, rash, and enlarged lymph nodes and meningitis within six weeks of contact with HIV. Following this acute infection, these individuals become asymptomatic.2) The remaining individuals with HIV infection are not symptomatic for years. 3) Some individuals develop persistent generalized lymphadenopathy (PGL), characterized by swollen lymph nodes in the neck, groin and axilla. Five to ten percent of individuals with PGL revert to an asymptomatic state.
  • PGL generalized lymphadenopathy
  • ARC AIDS-related complex
  • the retroviral macrophage-tropic nucleotide sequence preferably encodes the envelope polypeptide including gp120 and the amino terminal 189 amino acids of gp41 (gp120/gp41(189)) (Anderson, et al., Virology, 195:616, 1993, for nucleotide sequence).
  • the sequence may include a fewer or greater number of nucleotide sequences, as long as the sequence still retains the macrophage-tropic activity of gp120/gp41(189). While not wishing to be bound by a particular theory, it is believed that the 3-D conformation or quaternary structure and folding of the gp120/gp41 (189) amino acid sequence confers the macrophage-tropism ability to the virus.
  • the attenuated virus comprising a retrovirus nucleotide sequence encoding a macrophage-tropic polypeptide, such as an attenuated virus containing an HIV envelope nucleotide sequence
  • a retrovirus nucleotide sequence encoding a macrophage-tropic polypeptide such as an attenuated virus containing an HIV envelope nucleotide sequence
  • the composition can be administered intravenously, intraperitoneally, intramuscularly, subcutane- ously, intracavity, orally, mucosally, or transdermally.
  • Preparations for parenteral administration are contained in a "pharma ⁇ ceutically acceptable carrier".
  • Such carriers include sterile aqueous or non- aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents include propylene glycol, polyethylene glycol, metabolizable oils such as, olive oil, squalene or squalane, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
  • Attenuated viruses comprising a retrovirus nucleotide sequence encoding a macrophage-tropic polypeptide are contemplated in the method of the invention including, but not limited to, live attenuated virus
  • Attenuation can be achieved by various methods well known to those of skill in the art, including deleting the retrovirus nef gene or other non-structural gene, or constructing a recombi- nant, infectious but non-pathogenic virus.
  • the method of the invention further envisions administration of nucleotide sequences encoding a macrophage-tropic polypeptide or the polypeptide itself, as synthetic peptides, DNA vaccines, natural viral products, and recombinant DNA products via various delivery vehicles.
  • Naked DNA molecules can be directly administered in vivo, either by injection into muscle (Fynan, E.F., et al., Proc. Natl. Acad. Sci. USA, 90:11478-11482; 1993; Robinson, H.L., et al., Vaccine, 11:957-960, 1993; Ulmer, J.B., et al. commonly Science, 259:1745-1749, 1993; Wang, B., Proc. Natl. Acad. Sci.
  • Vaccination with live attenuated wild-type or recombinant virus is contem- plated, either alone or in combination with adjuvant, such as aluminum hydroxide or Freund's adjuvant in a non-toxic, prophylactic or therapeutic amount.
  • adjuvant such as aluminum hydroxide or Freund's adjuvant in a non-toxic, prophylactic or therapeutic amount.
  • no adjuvant is utilized, however, when administered in the form of a polypeptide, an adjuvant as described above is preferably used.
  • An advantage of using attenuated live viral vaccine is the small amount of material necessary to generate a strong immune response.
  • the virus can be attenuated using methods well known in the art.
  • HIV-1jrfl, HIV-1bal, HIV- 1ada, HIV-1-89.6 and HIV-1sf162 are non-limiting examples of known strains of macrophage-tropic HIV isolates that are publicly available.
  • any HIV isolate that is macrophage-tropic and can stimulate production of antibodies in a patient, which cross-react with other infectious HIV can be used as a vaccine in the practice of this invention.
  • Methods for determining whether an HIV isolate is macrophage-tropic and methods for culturing macrophage-tropic isolates are known to those of skill in the art (see for example, Coligan, etal., Current Protocols in Immunology, Greene Publishing Associates, Inc.
  • Delivery of macrophage-tropic specific polynucleotide can be achieved using vehicles such as a recombinant expression vector, e.g., a chimeric virus, or a colloidal dispersion system.
  • a recombinant expression vector e.g., a chimeric virus
  • a colloidal dispersion system for therapeutic delivery of nucleotide sequences is the use of liposomes. Production of such vehicles are well known in the art.
  • viral vectors which can be utilized for gene therapy as taught herein include adenovirus, adeno-associated virus, herpes virus, vaccinia, or an RNA virus such as a retrovirus.
  • adenovirus adeno-associated virus
  • herpes virus vaccinia
  • RNA virus such as a retrovirus.
  • Known techniques of molecular biology can be used to insert genes for antigenic epitopes for HIV virus into vectors as vehicles.
  • Vaccinia virus has been used as one such vector. (See for example, Current Protocols in Molecular Biology, Ed. by F.M. Ausubel, Current Protocols, Vol. 2, ⁇ 16.17, 1993).
  • the genes for the gp 120 and amino terminus of gp41 HIV-macrophage-tropic virus are available and can be inserted into a suitable vector using techniques well known in the art.
  • retroviral vectors in which a single foreign gene can be inserted include, but are not limited to: Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus
  • retroviral vectors can incorporate multiple genes. All of these vectors can transfer or incorporate a gene for a selectable marker so that transduced cells can be identified and generated.
  • a macrophage-tropic specific nucleotide sequence into the viral vector, along with another gene which encodes the ligand for a receptor on a specific target cell, for example, the vector is now target specific.
  • specific polynucleotide sequences which can be inserted into the retroviral genome to allow target specific delivery of the retroviral vector containing the macrophage-tropic specific polynucleotide.
  • helper cell lines that contain plasmids encoding all of the structural genes of the retrovirus under the control of regulatory sequences within the LTR. These plasmids are missing a nucleotide sequence which enables the packaging mechanism to recognize an RNA transcript for encapsidation.
  • Helper cell lines which have deletions of the packaging signal include but are not limited to ⁇ 2, PA317 and PA12, for example. These cell lines produce empty virions, since no genome is packaged.
  • a retroviral vector is introduced into such cells in which the packaging signal is intact, but the structural genes are replaced by other genes of interest, the vector can be packaged and vector virion produced.
  • the vector virions produced by this method can then be used to infect a tissue cell line, such as NIH 3T3 cells, to produce large quantities of chimeric retroviral virions.
  • Another delivery system for attenuated viruses comprising macrophage- specific polypolynucleotides or polypeptides is a colloidal dispersion system. Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in- water emulsions, micelles, mixed micelles, and liposomes.
  • the preferred colloidal system of this invention is a liposome.
  • Liposomes are artificial membrane vesicles which are useful as delivery vehicles in vitro and in vivo. It has been shown that large unilamellar vesicles (LUV), which range in size from 0.2-4.0 um can encapsulate a substantial percentage of an aqueous buffer containing large macromolecules. RNA, DNA and intact virions can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (Fraley, et al., Trends Biochem. Sci., 6:77, 1981). In addition to mammalian cells, liposomes have been used for delivery of polynucleotides in plant, yeast and bacterial cells.
  • a liposome In order for a liposome to be an efficient gene transfer vehicle, the following characteristics should be present: (1 ) encapsulation of the genes of interest at high efficiency while not compromising their biological activity; (2) preferential and substantial binding to a target cell in comparison to non-target cells; (3) delivery of the aqueous contents of the vesicle to the target cell cytoplasm at high efficiency; and (4) accurate and effective expression of genetic information (Mannino, et al.,
  • liposomes including unilamellar bodies comprising a single lipid bilayer, can be used as vectors to deliver viral proteins, such as polypeptides specific for determining macrophage-tropism, to vaccinate against HIV virus.
  • viral proteins such as polypeptides specific for determining macrophage-tropism
  • the targeting of liposomes has been classified based on anatomical and mechanistic factors.
  • Anatomical classification is based on the level of selectivity, for example, organ-specific, cell-specific, and organelle-specific.
  • Mechanistic targeting can be distinguished based upon whether it is passive or active. Passive targeting utilizes the natural tendency of liposomes to distribute to cells of the reticulo-endothelial system (RES) in organs which contain sinusoidal capillaries.
  • RES reticulo-endothelial system
  • Active targeting involves alteration of the liposome by coupling the liposome to a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein, or by changing the composition or size of the liposome in order to achieve targeting to organs and cell types other than the naturally occurring sites of localization.
  • a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein
  • the viral proteins and portions thereof, prepared as described above, may also be used in the preparation of subunit vaccines prepared by known techniques. Polypeptides displaying antigenic regions capable of eliciting protective immune response are selected and incorporated in an appropriate carrier. Alternatively, an antigenic portion of a viral protein or proteins may be incorporated into a larger protein by expression of fused proteins.
  • the preparation of subunit vaccines for other viruses is described in various references, including Lemer, etal., Proc. Natl. Acad. Sci. USA, 78:3403, 1981 and Bhatanagar, etal., Proc. Natl. Acad. Sci. USA, 79:4400, 1982. See also, U.S. Patent Nos.
  • 4,565,697 where a naturally-derived viral protein is incorporated into a vaccine composition
  • 4,528,217 and 4,575,495 where synthetic peptides forming a portion of a viral protein are incorporated into a vaccine composition.
  • Other methods for forming vaccines employing only a portion of the viral proteins are described in U.S. Patent Nos. 4,552,757; 4,552,758; and 4,593,002. The relevant portions of each of these patents are incorporated herein by reference.
  • Such vaccines are useful for raising an immune response against HIV, for example a protective antibody titer, in humans susceptible to the virus.
  • the attenuated viruses or vehicles containing macrophage-tropic specific sequences, prepared as described above, may be administered in any conventional manner, including nasally, subcutaneously, or intramuscularly.
  • Adjuvants will also find use with subcutaneous and intramuscular injection of completely inactivated vaccines to enhance the immune response.
  • Live attenuated viruses can also be incorporated into immunostimulating complexes (ISCOM) for use as a vaccine using methods well known in the art.
  • ISCOM immunostimulating complexes
  • SIV recombinant vaccine containing macrophage-tropic specific sequences, gp120 and the amino terminus of gp41 are shown in the present invention to raise high neutralization antibody titers after about 10 TCIDgo (tissue culture infectious dose; grown in primary rhesus peripheral blood mononuclear cells and injected into rhesus macaque monkeys (EXAMPLE 5).
  • the recombinant SIV or HIV can be incorporated into ISCOM particles which are useful for prophylactic or therapeutic vaccination against SIV or HIV infection, respectively.
  • the presentation of viral protein antigens in ISCOM particles has three main advantages: 1) no replicating viral nucleic acid is introduced into the host, 2) high levels of neutralizing antibodies are achieved, and 3) a cellular immunity is evoked, including cytotoxic T-cells induced under restriction of MHC class II.
  • the methodology for making ISCOM vaccines is well known in the art (B. Morein, et al., Nature, 308:457-60, 1984).
  • the invention provides a novel pharmaceutical composition which may be useful in the immunotherapeutic method of the invention, for example.
  • the pharmaceutical composition comprises an attenuated virus comprising a retrovirus nucleotide sequence encoding a macrophage-tropic polypeptide which stimulates an immune response, in a pharmaceutically acceptable carrier, as described above.
  • the retroviral nucleotide sequence encoding a macrophage-tropic polypeptide preferably encodes a virus envelope (env) polypeptide, most preferably, the HIV envelope.
  • the nucleotide sequence encoding the HIV envelope protein for example, is in operable linkage in a lentivirus genome, thereby allowing efficient transcription and translation of the envelope.
  • the pharmaceutical composition may comprise a recombinant chimeric lentivirus which - is macrophage-tropic, in a pharmaceutically acceptable carrier.
  • the composition comprises a recombinant chimera containing at least a gene encoding the lentivirus envelope protein.
  • the chimera includes a gene that encodes an HIV macrophage-tropic envelope protein which replaces an SIV envelope encoding gene, in an SIV genome.
  • the HIV envelope gene is inserted in operable linkage in the SIV genome so that it is efficiently transcribed and translated.
  • the SIV background genome may be macrophage-tropic or it may be lymphocyte-tropic.
  • the pharmaceutical composition may include a recombinant virus which includes a macrophage-tropic HIV envelope gene inserted in a lymphocyte-tropic or other macrophage-tropic HIV genome in operable linkage.
  • a pharmaceutical composition of the invention includes vehicles for delivery of nucleotide sequences encoding a macrophage-tropic polypep- tide or the polypeptide itself, such as synthetic peptides, DNA vaccines, natural viral products, and recombinant DNA products, in a pharmaceutically acceptable carrier.
  • vehicles may include, but are not limited to, RNA and DNA virus vectors and liposomes.
  • SIV ⁇ .239 was serially passaged in monkeys to obtain SIV mac 239/R71-BR from the brain of macaque R71 (Sharma, etal., J. Virol., 66:3550-3556, 1992a).
  • This virus hereafter referred to as R71 virus, was inoculated intracerebrally into the brain of macaque 17E and gave rise to SIV mac 239/17E-BR, referred to as 17E virus.
  • the HUT-78 and C8166 (Salahuddin, etal., Blood, 68:281-284, 1983) T-cell lines, and the CEMx174 T-cell/B-cell fusion cell line (Salter, etal., Immunogenetics, 21:235- 246, 1985) and the U937 monocyte/macrophage cell line were maintained according to standard culture technique, using RPMI medium with 10% fetal bovine serum (GIBCO) and 2 mM glutamine (GIBCO). Primary rhesus macaque peripheral blood mononuclear cells PBMCs were isolated and maintained using standard techniques, as previously described (Sharma, et al., supra,).
  • PCR was used to directly amplify the SIV DNA from frozen brain tissue of the R71 and 17E monkeys. Tissues were homogenized individually with a frozen mortar and pestle into a suspension using a solution of 1% sodium dodecyl sulfate (SDS), 100 mM NaCI, 50 mM Tris-HCI (pH 7.5), 1 mM EDTA, and 0.1 mg/ml proteinase K (Boehringer-Mannheim). Subsequent DNA isolation was carried out as previously described (Sharma, et al., supra). To control for contamination, parallel samples of uninfected HUT-78 cells were lysed similarly and carried through subsequent procedures.
  • SDS sodium dodecyl sulfate
  • PCR was done using standard conditions as previously described (Sharma, et al., supra) with several modifications. Primers were synthesized complementary to SIV mac 239 sequences homologous to conserved regions of HIV-1 (Starcich, et al., Cell, 45:637-648, 1986; Hahn, et al., Nature (London), 232:1548-1553, 1986; Alizon, et al., Cell, 46:63-74, 1986; Benn, et al., Science, 230:949-951 , 1985) on an Applied Biosystems DNA synthesizer. Overlapping fragments of 1.0 to 1.3 kilobases (kb) were amplified.
  • R71 DNA An initial amplification for R71 DNA was first carried out using the following primer set ⁇ '-AAGCTTGGATCCGCATGC- TATAACACATGCTATTGT-3'(5.1) (SEQ ID NO: 1) and 5'-AAGCTTGAATTC- GGAGGTTCTTTGTTCCCCAGACGG-3'(3.1) (SEQ ID NO:2), which are complementary to bases 6446 to 6469 and 8379 to 8402 of SIV mac 239, respectively (Regier and Desrosiers, supra). Both primers have 12 non- homologous bases at the 5' end.
  • Primer 5.2 also has 12 non-homologous bases on the 5' end.
  • the 5.1 to 3.1 reaction was used as starting material for reactions with primers 5.2 and 388 as well as reactions with primers 235 and 3.2
  • the 235 to 443 reaction was used as starting material for reactions with primers 279 to 3.1 , as well as primers 239 and 443.
  • the initial reactions were done with primer set 5.1/388 and also with primer set 235/443.
  • the 5.1/388 reaction was used as starting material for reactions with primer set 5.2/388.
  • the 235/443 reaction was used for starting material for reactions with primer set 279/3.1, as well as primer set 239/443.
  • the primers and amplified products for R71 are shown schematically in FIGURE 1A.
  • the 17E envelope (along with tat and rev 5' ORF) was amplified by PCR from DNA isolated from brain homogenates that were co-cultivated with primary monkey macrophages. Sequences from the Eco47lll restriction site (bp 6351 ) to the Nhe ⁇ site (bp8742) were amplified by a single PCR reaction (described above).
  • the 5* primer was made complementary to bases 6343 to 6362 (481 ) ⁇ '-AAGCTTGGATCCCTCCAACGAGCGCTCTTAAT 3' (SEQ ID NO: 9) and the 3' primer was made complementary to bases 8743 to 8763 (407) 5'- AAGCTTGGATCCCCCCTGCCTTAACTTAGCTAG 3' (SEQ ID NO: 10). Both primers have Hind W sites at their 5' ends to facilitate intermediate molecular cloning and nucleotide sequence analyses. The Eco74lll to Nhe ⁇ fragment from the PCR amplification was cloned into the SIV mac 239 molecular clone (p239).
  • PCR products were sequenced directly using previously described methods (Kusukawa, et al., BioTechniques, 9:66-72, 1990). Briefly, products were precipitated by adding 0.6 volume of 20% polyethylene glycol and 2.5 M NaCI, which left the majority of the PCR primers in solution. Primers used for sequence analysis were end-labeled with 2 P, annealed to the PCR products by boiling of the labeled primer and template followed by rapid cooling. Klenow (Pharmacia) was added, and this reaction was added to mixtures of cold deoxy and dideoxy nucleotides.
  • Primers used for this test were complementary to regions of the LTR of SIV mac 239 and SIV mac 251 which were identical. They were 5'-AAGCTTCTCGAGCATTTGGC- TGGCTATGGAAATTAG-3' (SEQ ID NO: 11) and 5'-AAGTTGGATCCCTCTA- CCTGCTAGTGCTG-3' (SEQ ID NO: 12), which are complementary to bases 124 to 147 and 567 to 584, respectively, of SIV ⁇ S ⁇ .
  • PCR was done using standard reaction conditions, 25 cycles of 1 minute of denaturation at 94°, 1 minute of annealing at 55°, and 1 minute of extension at 72° with either 10 ng of SIV. ⁇ 251 DNA, 5 ng of SIV. ⁇ 39 DNA plus 5 ng of SIV mac 251 DNA or 1 ng of SIV mac 239 DNA plus 9ng of SIV mac 251 DNA as plates.
  • the PCR product was prepared and sequenced as above.
  • Envelope sequences from R71 were subcloned into pBS- (Stratagene) cloning vector.
  • the nucleotide sequence of these clones was determined to identify the nucleotide changes in individual env genes.
  • the PCR products were generated using an overlapping mutagenesis technique (Higuchi, e* al., Academic Press, pp.177-183, 1990) which incorporates a single bast . ⁇ r silent mutation that abrogated recognition of the T/V.I H I site at nucleotide 8315 by that restriction enzyme.
  • the product of this mutagenesis was confirmed both by the loss of recognition by Tth , 111 as well as by nucleotide sequence analysis.
  • the mutagenesis of the Tth 1111 site was done to facilitate the construction of recombinant viruses (see below).
  • Two initial reactions were done using the following primer sets 5'-AAGCTTGAATTCGCA- TCAGCAAAAGTAGACATGG-3; (406) (SEQ ID NO: 13) and 5'-CTCTTGAC- CACATCCAACAGCTG-3' (408) (SEQ ID NO: 14), which were complementary to bases 7015 to 7036 and 8302 to 8324, respectively; and ⁇ '-CAGCTGTT- GGATGTGGTCAAGAG-3' (409) (SEQ ID NO: 15) and 5'-AAGCTTGGATCC-
  • CCCCTGCCTTAACTTAGCTAG-3' (407) (SEQ ID NO: 16), which are complementary to bases 8302 to 8324 and 8743 to 8763, respectively.
  • the bases in bold type in 408 and 409 are the mismatches between these primers and SIV mac 239, creating silent mutation.
  • Primers 406 and 407 have 12 nonhomologous bases that contain the recognition sequences for EcoRI and
  • Primers 408 and 409 are complementary to each other and therefore the PCR products were expected to hybridize and subsequently be filled in by Taq polymerase as shown in FIGURE 2. PCR products were digested with EcoRI and BamHI and subcloned into the pBS-vector. Subclones were sequenced using the dideoxy method (Sanger, et al., Proc. Natl. Acad. Sci. USA, 74:5463-5467, 1977).
  • the SIV mac 239 clone was digested with EcoRI, and the complete infectious provirus and flanking cellular DNA were subcloned into pUC19.
  • the plasmid, p239 which is infectious, was digested with Nhe ⁇ and Tth 111 and purified by agarose gel electrophoresis.
  • Envelope subclones in pBS were digested with Nhe ⁇ and TtM 111, purified by agarose gel electrophoresis, and ligated into the p239 provirus to create p239-R71-1-1 , p239-R71-2, p239-R71-10, p239-R71- 13, and p239-R71 -14.
  • Two control recombinants were made placing either the env region from SIV. ⁇ 39 or SlV ⁇ l into the T./.1111 and Nhe ⁇ sites of p239, using the same procedures as with the R71 recombinants.
  • CEMx174 cells were added to PBMC cultures on Day 7 to amplify virus production. Additional experiments were done by electroporation, using the
  • Bio-Rad Gene Pulser Conditions used for PBMCs were according to manufacturer's specifications, with a pulse of 300v and 500 ⁇ F.
  • CEMx174, HUT-78, and U937 cells were electroporated according to manufacturer's specif cations also, with 200v and 960 ⁇ F.
  • Two electroporation experiments were one using PBMCs and CEMx174 cells and one each in HUT-78 and U937 cells. Cells were assayed for viral replication, a subset of cultures from different experiments was assayed for SIV p27 levels in the supernatant using a commercial immunoassay (Abbott) for detection of p24 of HIV.
  • Abbott commercial immunoassay
  • FIGURE 1 shows the strategy for amplifying SIV envelope sequences. Primers are shown as arrows, primer number is next to arrow, and direction of arrow indicates 5' to 3' orientation.
  • the "X" in primers 408 and 409 indicates a point mutation which alters the second T./71111. This base pair change does not alter the amino acid sequence.
  • Primers 408 and 409 are complementary, so the two initial products shown, containing the mutation, will hybridize to each other in a second reaction. This second reaction gives the product shown, from nucleotide 7015 to 8763.
  • the jagged lines indicate nonhomologous sequences which contain restriction enzyme sites to facilitate cloning.
  • PCR amplification products were obtained from uninfected HUT-78 DNA samples.
  • the primers used for PCR were made complementary to env regions that would be expected to be conserved by analogy to the HIV envelope. Overlapping fragments were generated to determine if in fact the regions to which the primers bound were conserved. Nucleotide sequence analysis showed that the internal primers were in fact complementary to sequences that had remained identical to SIV mac 239 (see below).
  • the population of envelope genes present in the brain was amplified by PCR, and the nucleotide sequence of the amplified DNA was determined to identify nucleotides that were distinct from those of SIV mac 239.
  • a ⁇ P end-labeled primer complementary to nucleotides (nt) 6917 to 6938 was used for reactions in FIGURE 2A and a similarly labeled oligonucleotide complementary to bases 7917 to 7938 was used for reactions in FIGURE 2B.
  • Unique PCR products from R71 DNA (reactions 1 and 2), unique products from 17E DNA (reactions 3 and 4), and unique products from control cloned SW/ ⁇ DNA (reaction 5) are shown in FIGURE 2A and FIGURE 2B.
  • Sequenc ⁇ ing lanes are in the following order: A.C.G, and T, and the sequence is shown on the left of each reaction.
  • the oligonucleotide in (B) is complementary to the plus-strand and gives minus-strand sequences.
  • the autoradiograph that is shown was turned over to yield the sequences of the plus-strand.
  • the complete A-G transition at nt 7025 is shown in (A), which is predicted to change lysine 141 to arginine.
  • a mixture of both a T and a C residue at no 7864 is shown in (B), which encodes either serine or proline at the position of proline 421.
  • FIGURE 2C is a control experiment in which direct sequencing was carried out on PCR products from the LTR of cloned SIV mac 251 DNA (reaction 1 ), cloned SIV ⁇ .239 DNA and cloned 81 ⁇ ,- 251 DNA present in equal amounts in the initial PCR reaction (reaction 2), and cloned SIV mac 239 DNA and cloned SIV mac 251 DNA (reaction 3) present in a 1.9 molar ratio as starting material (reaction 3).
  • the appearance of the C residue in reaction 2 at the position indicated by the arrow is due to the SIV mac 239 sequence, and is barely detectable in reaction 3.
  • FIGURES 3 and 4 A diagram of the envelope gene of SIV mac 239 is shown to scale with the position of HIV-1 variable regions and the CD4 binding domain.
  • the T./.1111 and the Nhe ⁇ sites used for subcloning envelope regions as well as the gp 120-gp32 cleavage site are shown with the amino acid position indicated beneath (FIGURE 3).
  • the wild-type SIV mac 239 amino acids which are altered in R71 are given for reference, with their positions alternating between above and below the one letter amino acid code.
  • Mutated amino acids determined by direct sequencing of the PCR products from R71 brain DNA are as shown, with boxed amino acids indicating a complete loss of the nucleotide found in SIV mac 239 that was predicted to give the "wild-type" amino acid. Mutations not boxed indicate a mixture of the mutant and wild-type amino acids at that site. The asterisks indicate silent mutations.
  • Results from sequencing individual clones from a different PCR reaction are shown (R71 to R71-14).
  • the (-) indicates predicted amino acids identical to SIV ⁇ S ⁇ , yet different from the R71 population. Changes here are also indicated by the one letter abbreviation.
  • the ($) symbol in R71-2 indicates mutations to stop codons. Data from direct sequencing of 17E brain DNA are shown in the same manner as the R71 direct sequencing.
  • FIGURE 4 The complete amino acid sequence of the env gene of SIV mac from amino acids 1-738 is listed in FIGURE 4, with the changes preser ⁇ the brain isolates and molecular clones. An asterisk indicates a silent m_..ation, while a dash indicates no change from the SIV mac 239 sequence.
  • the amino acid changes found to contribute to macrophage-tropism in SIV mac 239/316 are shown in the last lines marked 316 (Mori, et al., J. Virol., 66:2067, 1992). Regions analogous to the HIV-1 variable regions are bracketed (V1-V5) and the surface membrane protein-transmembrane protein cleavage site is indicated by an arrow.
  • nucleotide changes were found in the R71 envelope sequence when compared to SIV mac 239. These nucleotide changes were located throughout the envelope gene.
  • the envelope region from nucleotides (nt) 7015 to 8763 was amplified in a separate reaction, as shown in FIGURE 1 B. This approach was also employed to facilitate the construction of recombinant clones of SIV. ⁇ 39 with R71 envelope sequences.
  • the R71 envelope sequences were inserted between the unique Nhe ⁇ site at nt 8746 and the TtM 111 site at nt 7034, as discussed below.
  • Recombinant viruses were subsequently generated using the infectious molecular clone of SIV. ⁇ ,239 and the PCR derived envelope sequences of R71 from the T.r.1111 site to the Nhe site.
  • the recombinant DNAs were transfected into primary rhesus PBMCs as well as four cell lines. However, none of these recombinants produced infectious virus.
  • the env gene from the DNA obtained from macaque 17E was amplified from the Eco47lll (bp 6351 ) site within the 5'ORF of the tat gene to the Nhe ⁇ site (bp8742). This DNA fragment was inserted in the SIV mac 239 infectious clone (p239) and transfected into CEMx174 cells (lymphocyte cell line). Five infectious recombinant viruses were obtained cloned 17E-2, 17E-3, 17E-5, 17E-6, and 17E-8. Two of these clones have been characterized further. The complete nucleotide sequence of the Eco47lll to Nhe ⁇ site of clones 17E-2 and 17E-3 was determined. The clones had identical nucleotide sequences.
  • amino acid changes found in clones 17E-2 and 17E-3 are shown in FIGURE 3 and 4. Most of the amino acid changes are in common with those identified in R71 and 17E described above.
  • One base pair change was found in the tat and rev 5'ORFs. This change does not alter the amino acid sequence of rev but it causes a conservative change in the tat protein located outside any functional domains that has been identified.
  • SIV/17E-CI contains the gp120 and a portion of the gp41 sequences (to amino acid 730) from the macrophage-trop- ic/neurotropic virus strain SIV/17E-Br in the background of the infectious molecular clone SIV ⁇ S ⁇ , which replicates poorly in monocyte/macrophages (H. Kestler, et al., Science, 248:1190, 1990).
  • TCID 5 o produced in the particular cell type and titrated in CEMX174 cells.
  • the Tulane Regional Primate Research Center takes responsibility for humane care and use of laboratory animals used in projects awarded by the Public Health Service.
  • the present invention complied with the Principles for Use of Animals, The Guide for the Care and Use of Laboratory Animals, the Provisions of the Animal Welfare Act, and other applicable laws and regula- tions.
  • the Center's statement of assurance is on file with the PHS Office for Protection from Research Risks (Assurance number A3701 -01 ).
  • This facility is accredited by the American Association for Accreditation of Laboratory Animal Care. Animals are anesthetized with ketamine prior to all procedures that require the removal of animals from their cages. No restraining devices are necessary during these procedures. When necessary, moribund animals are euthanized by intravenous inoculation of a lethal dose of sodium pentabar- bitol).
  • PBMC Peripheral blood mononuclear cells
  • Each PCR reaction mixture contained 10 mM Tris-HCL, pH 9.0 at 25°, 50 mM KCI, 1.75 mM MgCI 2 , 0.01% (w.v) gelatin, 2 mM dNTP, 20 pM 5' and 3' oligonucleotide primers, and 2.5 U Taq polymerase (Promega).
  • One microgram of DNA was then amplified by 30 cycles in a DNA Thermocycler (Perkin-Elmer Corp., Norwalk, CT). The first cycle consisted of denaturation at 94° for 1 minute, annealing at 55° for 1 minute, and extension at 72° for 1 minute plus 10 seconds for each 30 cycles.
  • a second nested round was denatured for 1 minute at 94°, annealed at 45° for 1 minute, and extended at 60° for 1 minute plus 10 seconds for each of 30 cycles. Fifteen percent of the amplified product was then electrophoresed through a 2% agarose gel and visualized by ethidium bromide staining.
  • the sequences of the LTR-specific primer pairs used in the first round were (5") 5'-ATAGTTGCAGTACATGTGGCTAGTG-3' (SEQ ID NO: 17) and (3') 5'-TCTCTGCCTCTTTCTCTGTAATAGAC-3' (SEQ ID NO: 17) and (3') 5'-TCTCTGCCTCTTTCTCTGTAATAGAC-3' (SEQ ID NO: 17) and (3') 5'-TCTCTGCCTCTTTCTCTGTAATAGAC-3' (SEQ ID NO: 17) and (3') 5'-TCTCTGCCTCTTTCTCTGTAATAGAC-3' (SEQ ID NO: 17) and (3
  • fragments were Southern blotted after electrophoresis to a Magna NT Nylon Transfer Membrane (MSI, Westboro, MA) and hybridized to a 32 P-labeled oligonucleotide complementary to sequences within the primers used in the amplification reaction.
  • the sequence of the oligonucleotide probe was (5') 5'- AGCAGGTAGAGCCTGGGTGTTC-3' (SEQ ID NO:21).
  • a Macrophages were cultured from the peripheral blood and virus was assayed both by titration of supernatants and by the co-cultivation with CEMx174 cells at 14 days after isolation.
  • b Lymphocytes were cultured from the peripheral blood and virus was assayed both by titration of supernatants and by the development of virus induced CPE (cytopathic effect) at 7 and 14 days after isolation.
  • AIDS-like disease was monitored in all 8 SIV/17E-CI- infected monkeys by detection of viral p26 serum and by flow cytometric measurement of changes in T lymphocyte populations in the peripheral blood.
  • Of particular interest was either a reciprocal decline in CD4+ (helper) and an increase in CD8+ (suppressor) T lymphocytes, or a selective decline in the CD4+CD29+ (helper-inducer) T lymphocyte population.
  • a selective decline in CD4+CD29+ T lymphocytes has been shown to be a reliable early indicator of disease progression in monkeys infected with the pathogenic isolate SIV/DeltaB670 (M. Murphey-Corb, et al., Science, 246:1293, 1989).
  • FIGURE 6 shows neutralizing antibody titers (10log 10 ) over time in sera from monkeys infected with SIV/17E-CI.
  • Neutralization of SIV/17E-CI was performed in 96-well tissue culture plates containing RPMI supplemented with 10% fetal bovine serum. Five-fold serial dilutions of plasma (heat inactivated at 56°C and clarified by centrifugation) were added to each, each with 10-100TCID 50 of virus and incubated 1 hour at 37°C. 1x10 6 CEMx174 cells were added to each well and the development of CPE was recorded at 7 days.
  • the 50% neutralization endpoint was calculated using the method of Karber (G. Karber, Arch Exp. Path. Pharmakoi, 162:480, 1931 ). L235 (o), L238 ( ⁇ ), M118 ( ⁇ ), L652 (•), and L471 (D). These titers rose rapidly and peaked at 5 months postinfection and remained constant throughout the following year. Neutralization assays were routinely done in a T-cell line (CEMx174), however, when the assays were done in primary rhesus macrophages, an equivalent level of neutralizing antibody was measured.
  • Neutralization assays were done in primary macaque macrophages as described in FIGURE 2 except that primary macaque macrophages were cultured in 96 well plates for 5 days prior to the addition of virus or virus incubated with serial dilutions of plasma. The endpoint was determined at fourteen days by the addition of 2 x 10 6 CEMxl 74 cells/well, the CPE assessed, and the 50% neutralization endpoint determined.
  • the production of high levels of neutralizing antibodies in monkeys infected with SIV/17E-CI is in direct contrast to the absence or low levels of neutralizing antibodies made in response to infection with the parental strain SIV ⁇ .
  • envelope sequences in SIV/17E-CI that confer macrophage-tropism also appear to be responsible for eliciting a strong neutralizing antibody response in vivo.
  • neutralizations were done at monthly intervals using SIV mac 239, the uncloned parental strain SIV/17E-Br, another recombinant clone that contains the entire env gene from SIV mac /17E-CI called SIV(, ac 17E-Fr, and a heterologous primary isolate of sooty mangabey monkey origin, SIV/DeltaB670 (Table 3).
  • SIV/17E-CI which consists of a single genotype of SlV ⁇ l lineage
  • SIV/DeltaB670 is a primary isolate consisting of a swarm of genetic variants cultured from the lymph node of a rhesus monkey infected with SIV from a sooty mangabey monkey.
  • SIV/17E-CI induces, over time, neutralizing antibodies against a genetically diverse strain of SIV.
  • SIV envelope glycoprotein-specific antibody responses were examined.
  • native viral glycoproteins from purified virus preparations were anchored onto concanavalin A in microtiter plates for the respective immunoassays.
  • Serum antibody avidities were determined by measuring the stability of antibody- antigen complexes to a urea wash as described previously (K. Hedman and S.A. Rousseau, J. Med. Virol., 27:288, 1989; K. Hedman, ef al., J. Med.
  • FIGURE 7 shows avidity of serum antibodies for SIV envelope glycoproteins.
  • the antibody avidity index (Al) K. Hedman, et al., supra
  • Con A-ELISA J.E. Robinson, et al., J. Immunol. Meth., 132:63. 1990
  • Con A-anchored native envelope glycoprotein substrate was prepared from Triton X-100-disrupted SIVB7 in microtiter plates as described in FIGURE 4.
  • SIVB7 is a noninfectious virus derived from CEMx174 cells chronically infected with
  • the avidity index (Al) was calculated from the ratio (A/B x 100%) of the absorbency values obtained with the urea treatment FIGURE 7(A) compared to the absorbency observed with the PBS treatment, FIGURE 7(B). Al values (30% are designated as low avidity antibodies, 30%-50% as intermediate avidity antibodies, and )50% as high avidity antibodies (K. Hedman, et al., supra).
  • the data in FIGURE 7 demonstrate that the envelope glycoprotein-specific antibody responses in the 5 SIV/17E-CI infected monkeys evaluated longitudi ⁇ nally increase in avidity over the first 7 months post infection and apparently level off at an intermediate avidity of about 40% thereafter.
  • the relatively slow evolution of antibody avidity in the glycoprotein-specific antibody response indicates an ongoing maturation of humoral immune responses to this chronic infection for at least 7 months postinfection.
  • the avidity appears to reach a maximum level that is maintained even after subsequent virus challenge 8 months postinfection.
  • This slow increase in the avidity of antibodies to the SIV envelope glycoprotein is in distinct contrast to the relatively rapid increase in avidities observed during other viral infections (A. Salmi, Curr. Opin. Immunol., 3:503, 1992), perhaps indicating an important escape mechanism by which SIV eludes immune responses soon after infection.
  • the conformation-dependence of envelope-specific antibody responses was also measured in the longitudinal panel of serum samples taken from 5 of the 8 SIV/17E-CI-infected monkeys.
  • the relative reactivity of serum antibodies at a standard dilution was measured in parallel against con A-anchored native viral glycoprotein and against "denatured" viral glycoprotein produced by reductive carboxymethylation of protein cysteine sulfhydryl groups.
  • this assay compares the reactivity of serum antibodies with a native viral glycoprotein complex to that with envelope glycoproteins in which all disulfide bonds have been irreversibly reduced to alter protein tertiary structure, without deliberate denaturation of the envelope protein secondary structure.
  • FIGURE 8 shows conformational dependence of serum antibodies to SIV envelope glycoproteins.
  • the conformational dependence of envelope-specific antibodies elicited by infection with SIV/17D-CI was determined by measuring in Con A-ELISA (J.E. Robinson, et al., J. Immunol. Meth., 132:63, 1990) the antibody reactivities against a native (panel A) and denatured (panel B) viral envelope glycoprotein substrates prepared by reduction and carboxymethylation of protein sulfhydryl groups.
  • Con A-ELISA J.E. Robinson, et al., J. Immunol. Meth., 132:63, 1990
  • gradient- purified M. Murphey-Corb, etal., supra
  • SIVB7 particles disrupted with 1% Triton X-100 was used as the "native" glycoprotein substrate.
  • SIVB7 was treated with dithiothreotol to reduce disulfide bonds and then with iodoacetic acid to achieve an irreversible carboxymethylation of the reduced sulfhydryl groups (A.M. Crestfield, etal., J. Biol. Chem., 38:622, 1963).
  • These reaction conditions were chosen because they should quantitatively disrupt envelope glycoprotein disulfide bonds and affect tertiary protein structure without extensive alterations in protein secondary structural properties, as would be expected from treatments with chaotropic salts or ionic detergents.
  • test serum 100 ⁇ l per well of 5% nonfat dry mild in PBS (blotto) for 1 hour at room temperature. After removing the blocking solution, 50 ⁇ l of appropriately diluted test serum were added to each well and incubated for 1 hour at room temperature. All test sera were diluted in blotto to produce an absorbency at 450 nm of about 1.0 in the standard Con A-ELISA procedure. After serum incubation, the wells were washed with PBS-TX, and 50 ⁇ l per well of a 1 :1000 dilution of peroxidase-conjugated anti-human IgG in blotto was added for 1 hour at room temperature.
  • TMBlue (TSI) substrate was added for approximately 15 minutes before color development was terminated by the addition of 50 ⁇ l per well of 1 N sulfuric acid.
  • Antibody reactivity to the Con A-anchored native or denatured envelope glycoprotein substrates was then determined by measuring the absorbency at 450 nm. (Monkeys L235 (o), L238 ( ⁇ ), M118 ( ⁇ ), L652 (•), and L471 ( ⁇ )).
  • Serum antibodies at all time points tested displayed a 2-3 fold greater reactivity with the native viral glycoprotein substrate compared to the denatured viral glycoprotein antigen (FIGURE 8).
  • the predominance of conformation dependent antibodies consistently identified in sera of SIV/17E- Cl-infected monkeys is reminiscent of similar antibodies produced in chronic HIV-1 (J.P. Moore and D.D. Ho, Virol., 67:863, 1993) and SIV (B.W. McBride, et al., Gen. Virol., 74:1033, 1993) infections.
  • This is the first report of the in kinetics of induction of this conformational dependent response over the course of an experimental infection, thereby revealing the early dominance of this response.
  • the conformational dependence properties of the antibody response to SIV/17E-C1 infection differ from the avidity properties which clearly evolve more slowly over the first 7 months postinfection.
  • the genetic diversity of gp120 sequences in virus present in two of the animals that were challenged was examined nine days after inoculation in the peripheral blood mononuclear cells and at 244 days after inoculation in mononuclear cells from both lymph nodes and the peripheral blood.
  • the entire gp120 sequence 3 (nucleotides 6342-8222; (D.A. Regier and R.C. Desrosiers, AIDS Res. Hum. Retroviruses, 6:1221 1990).
  • sequence analysis amplification of the entire gp120 envelope sequence was performed by nested PCR using the conditions described in EXAMPLE 5 and primers
  • Sequences specific for the external domain of gp41 were similarly amplified using primers (5') 5'-GAACATACATTT- ATTGGCATCCTAG-3' (SEQ ID NO:26) and (3') 5'- AAGCAGAAAGGGTCCTAACAGACCAGGGT-3' (SEQ. ID NO:27) for the first round and (5') 5'-CCATTGGTCAAACATCCCACATATACTGGA-3' (SEQ ID NO:28) and (3') 5'-CCAGGCGGCGACTAGGAGAGATGGGAACAG-3' (SEQ ID NO:29) for the second round.
  • Sequences specific for the external domain of gp41 were similarly amplified using primers (5') 5'- GAACATACATTTATTGGCATCCTAG-3' (SEQ ID NO:30) and (3') 5'- AAGCAGAAAGGGTCCTAACAGACCAGGGT-3' (SEQ ID NO:31 ) for the first round and (5') 5'-CCATTGGTCAAACATCCCACATATACTGGA-3' (SEQ ID NO:32) and (3') 5'-CCAGGCGGCGACTAGGAGAGATGGGAACAG-3' (SEQ ID NO:33) for the second round. Second round products were cloned into the TA cloning vector (Invitrogen, San Diego, CA).
  • infected macaques were inoculated intravenously with 50 animal infectious doses of rhesus-grown SIV/DeltaB670 either prior to 7 months (3 macaques) or after 8 months (2 macaques) postinfection (TABLE 4). Lymph node biopsies were performed immediately prior to and 14 days after challenge and, together with PBMC-derived cells, were evaluated for SIV/DeltaB670- specific sequences by PCR.
  • SIV-specific sequences when present, were further analyzed by sequence analysis of cloned PCR products containing the V1 hypervariable domain of gp120 (Sequences specific for the V1 hypervariable region of gp120 were determined on PCR-amplified products as described above using primers (5') 5'-CCTCCAACGAGCGCTCTTCAT-3' (SEQ ID NO:34) and (3') 5'-CCTGCTGTTGCGAGAAAACCCAAG-
  • AACCCTAGC-3' (SEQ ID NO:35) for the first round and (5" 5'- CAGTCACAGAACAGGCAATAGA-3' (SEQ ID NO:36) and (3') 5'- CCTGCTGTTGCGAGAAAACCCAAGAACCCTAGC-3' (SEQ ID NO:37) for the second round.
  • monkey M462 The major variant found within the challenge inoculum was identified in monkey M462, whereas only a minor variant (representing less than 10% of the variants) could be identified in monkey M697.
  • Similar analysis of naive monkeys inoculated with SIV/De- ltaB670 has shown that the major variant present in the initial inoculum consistently emerges as the dominant form following in vivo infection.
  • these data suggest that the clonal emergence of a minor form of the inoculum observed in monkey M697 may have been influenced by the low level of neutralizing antibody detected in the serum of this animal at challenge.
  • Both M462 and M697 have lived beyond 280 days postchallenge, but are showing persistent declines in CD4+ and increases in CD8+ T lymphocytes in the peripheral blood.
  • monocytotropic SIV/17E-CI serves as an effective vaccine by inducing protective responses that are, at least in part, humoral.
  • SIVmac17E ⁇ nef and SIVmac239 ⁇ nef were constructed using the same deletion strategy originally described by Dr. Ron Desrosiers for SIVmac239 ⁇ nef (Desrosiers, et al., supra.) Six monkeys were inoculated with each strain and monitored for infection, disease status, and immune responses to viral infection. All monkeys became persistently infected and have shown no signs of disease at 8 months postinfection.
  • SIV/17E-CI infected monkeys The early (within 3 weeks) induction of vigorous type-specific neutralizing antibody responses in SIV/17E-CI infected monkeys is in dramatic contrast to the absence of detectable neutralizing antibody in monkeys infected with the parent clone SIV mac 239 where weak responses only appear after 6 months postinfection.
  • the genetic difference between SIV. ⁇ 39 and SIV/17E-CI is restricted to 7 amino acid changes in gp160; these conservative changes not only confer the ability to replicate in monocyte/macrophages but also enable the early induction of vigorous neutralizing activity in vivo. These changes may promote conformational changes in the envelope which expose neutralizing epitopes, or neutralizing antibody may be more readily induced by presentation to the immune system by infected monocyte/macrophages, or both.
  • Macrophage-tropic strains of HIV-1 appear to be transmitted most efficiently and to establish initial infections in humans (T.F.W. Wolfs, et al., Virol., 189:103, 1992; S.M. Wolinsky, et al., Science,
  • NAME Haile, Ph.D., Lisa A.
  • MOLECULE TYPE cDNA
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  • FRAGMENT TYPE (vi) ORIGINAL SOURCE:
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  • FRAGMENT TYPE (vi) ORIGINAL SOURCE:
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  • MOLECULE TYPE cDNA (ill) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE
  • MOLECULE TYPE cDNA (in) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE:
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE (vi) ORIGINAL SOURCE:
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE
  • MOLECULE TYPE cDNA (ill) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE:
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • MOLECULE TYPE cDNA (ill) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE:
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • MOLECULE TYPE cDNA (ill) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE:
  • Val Tyr Gly lie Tyr Cys Thr Leu Tyr Val Thr Val Phe Tyr Gly Val 20 25 30 Pro Ala Trp Arg Asn Ala Thr lie Pro Leu Phe Cys Ala Thr Asn Lys 35 40 45
  • Val Asn Glu Thr Ser Ser Cys lie Ala Gin Asp Asn Cys Thr Gly Leu 145 150 155 160
  • Glu Gin Glu Gin Met lie Ser Cys Lys Phe Asn Met Thr Gly Leu Lys
  • Trp Asp Ala lie Arg Phe Arg Tyr Cys Ala Pro Pro Gly Tyr Ala Leu 225 230 235 240 Leu Arg Cys Asn Asp Thr Asn Tyr Ser Gly Phe Met Pro Lys Cys Ser

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Virology (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • AIDS & HIV (AREA)
  • Hematology (AREA)
  • Oncology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne un traitement prophylactique et curatif de l'infection causée chez un humain par le virus de l'immunodéficience humaine (VIH). Un modèle virus de l'immunodéficience de singe (VIS)-macaque montre que l'infection avec un VIS de recombinaison non pathogène mais visant les macrophages, assure une immunité systémique de l'hôte en stimulant la production d'anticorps neutralisants. Les essais montrent que les anticorps neutralisants produits ont une large réactivité vis-à-vis de plusieurs souches hétérologues de VIS. L'invention concerne également des compositions pharmaceutiques utiles pour le traitement d'un sujet avant ou après l'infection avec le VIH.
PCT/US1995/011943 1994-09-23 1995-09-19 Procede de traitement d'infections dues au virus de l'immunodeficience humaine (vih) WO1996009066A2 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU35930/95A AU3593095A (en) 1994-09-23 1995-09-19 Method of treatment of human immunodeficiency virus (hiv) infection
EP95933167A EP0784484A2 (fr) 1994-09-23 1995-09-19 Procede de traitement d'infections dues au virus de l'immunodeficience humaine (vih)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US31142494A 1994-09-23 1994-09-23
US08/311,424 1994-09-23

Publications (2)

Publication Number Publication Date
WO1996009066A2 true WO1996009066A2 (fr) 1996-03-28
WO1996009066A3 WO1996009066A3 (fr) 1996-05-23

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PCT/US1995/011943 WO1996009066A2 (fr) 1994-09-23 1995-09-19 Procede de traitement d'infections dues au virus de l'immunodeficience humaine (vih)

Country Status (3)

Country Link
EP (1) EP0784484A2 (fr)
AU (1) AU3593095A (fr)
WO (1) WO1996009066A2 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7943375B2 (en) 1998-12-31 2011-05-17 Novartis Vaccines & Diagnostics, Inc Polynucleotides encoding antigenic HIV type C polypeptides, polypeptides and uses thereof
US8133494B2 (en) 2001-07-05 2012-03-13 Novartis Vaccine & Diagnostics Inc Expression cassettes endcoding HIV-1 south african subtype C modified ENV proteins with deletions in V1 and V2

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994021806A1 (fr) * 1993-03-19 1994-09-29 Medical Research Council Systeme d'administration commande par des facteurs associes au vih et a la cellule

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994021806A1 (fr) * 1993-03-19 1994-09-29 Medical Research Council Systeme d'administration commande par des facteurs associes au vih et a la cellule

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
JOURNAL OF VIROLOGY, vol. 69, no. 5, - May 1995 pages 2737 -2744, JANICE E. CLEMENTS ET AL. 'Cross-Protective Immune Responses Induced in Rhesus Macaques by Immunization with Attenuated Macrophage-Tropic Simian Immunodeficiency Virus' *
NATURE, vol. 328, 1987 pages 348-351, FERNANDO PLATA ET AL. 'AIDS virus-specific cytotoxic T lymphocytes in lung disorders' *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7943375B2 (en) 1998-12-31 2011-05-17 Novartis Vaccines & Diagnostics, Inc Polynucleotides encoding antigenic HIV type C polypeptides, polypeptides and uses thereof
US8133494B2 (en) 2001-07-05 2012-03-13 Novartis Vaccine & Diagnostics Inc Expression cassettes endcoding HIV-1 south african subtype C modified ENV proteins with deletions in V1 and V2

Also Published As

Publication number Publication date
WO1996009066A3 (fr) 1996-05-23
AU3593095A (en) 1996-04-09
EP0784484A2 (fr) 1997-07-23

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