WO1998059074A1 - Human immunodeficiency viruses causing aids in a nonhuman primate - Google Patents

Human immunodeficiency viruses causing aids in a nonhuman primate Download PDF

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WO1998059074A1
WO1998059074A1 PCT/US1998/012990 US9812990W WO9859074A1 WO 1998059074 A1 WO1998059074 A1 WO 1998059074A1 US 9812990 W US9812990 W US 9812990W WO 9859074 A1 WO9859074 A1 WO 9859074A1
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hiv
arg
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glu
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PCT/US1998/012990
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French (fr)
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Francis J. Novembre
Harold M. Mcclure
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Emory 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
    • 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
    • C12N2740/16211—Human Immunodeficiency Virus, HIV concerning HIV gagpol
    • C12N2740/16222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • This invention concerns unique isolates of human immunodeficiency virus type 1 (HIV-l jC ) which are highly infectious in vivo and produce acquired immune deficiency syndrome (AIDS) in a nonhuman primate.
  • HIV-l jC human immunodeficiency virus type 1
  • HIV-1 human immunodeficiency viruses
  • HIV-2 retroviruses which have been implicated as the causative agents of acquired immune deficiency syndrome (AIDS)
  • AIDS acquired immune deficiency syndrome
  • skepticism about the exact cause of AIDS has arisen (Duesberg [1991] Proc. Natl. Acad. Sci. 88:1575-1579)
  • a large amount of data has accumulated which supports HIV as the biologic agent of this disease.
  • the most convincing evidence includes: mother to child transmission; transmission via blood transfusion; and transmission via contaminated blood products (Curran et al. [1984] N. Engl. J. Med. 310:69-75).
  • HIV-1 human immunodeficiency virus type 1
  • gibbons Lusso et al. [1988] J. Immunol. 141:2467- 2473
  • pig-tailed macaques Barre-Sinoussi et al., supra
  • chimpanzees Alter et al. [1984] Science 226:549-552; Fultz et al. [1986] J. Virol. 58:116-124.
  • AIDS the development of disease
  • chimpanzees have been a major focus in the development of animal models for HIV-1 infection and therapy.
  • the present invention provides HIV-1 isolates that are infectious to nonhuman primates such as chimpanzees and induce AIDS in inoculated nonhuman primates including, but not limited to, chimpanzees. Further, this invention provides for the first time a proper animal model for HIV-1 infection and for the development of AIDS.
  • a first aspect of the present invention are the isolated human immunodeficiency virus type 1 (HIV-1) isolate having the identifying characteristics of HIV- 1 isolate JC (HIV-1 JC ) and assigned AIDS Reagent Program Catalog Number 3523 and the isolate having the identifying characteristics of HIV-1 isolate NC (HIV-1 NC ). These viruses are useful for the preparation of recombinant, attenuated and subunit vaccines, as well as for the preparation of challenge stocks. Each is also useful in screening for the presence of HIV in biological samples.
  • HIV-1 JC isolated human immunodeficiency virus type 1 isolate having the identifying characteristics of HIV- 1 isolate JC
  • HIV-1 NC HIV-1 NC
  • a second aspect of the invention is a biological sample, e.g., a biological fluid or tissue, containing an HIV-1 having the identifying characteristics of HIV-l Jc or HIV-1 NC .
  • a biological sample e.g., a biological fluid or tissue
  • an HIV-1 having the identifying characteristics of HIV-l Jc or HIV-1 NC e.g., primate blood specimens containing the HIV-1 JC or the HIV-1 NC of the present invention were used to cause HIV infection and to induce AIDS in a nonhuman primate, as specifically exemplified by the chimpanzee.
  • a third aspect of the invention are a biologically pure culture of host cells containing an HIV-1 having the identifying characteristics of HIV-l Jc and a biologically pure culture of host cells containing an HIV-1 having the identifying characteristics of HIV-1 NC .
  • a further aspect of the invention are isolated DNA molecules which produce infectious HIV-1 having the identifying characteristics of HIV-1 JC or which encode an antigenic fragment thereof or having the identifying characteristics of HIV-1 NC or encode an antigenic fragment thereof.
  • the substantially full length sequence for infectious molecular clone of HIV-l jC is given in SEQ ID NO:l 1
  • the corresponding sequence for the infectious molecular clone of HIV-1 NC is given in SEQ ID NO:12.
  • An additional aspect of the invention provides isolated DNA encoding an HIV-1 envelope protein having the amino acid sequence of SEQ ID NO:2.
  • the DNA sequence encoding an HIV-1 envelope protein contains the nucleotide sequence of SEQ ID NO: 1.
  • a further aspect of the invention are a composition comprising an antigenic preparation derived from the HIV-1 JC of the invention and a composition comprising an antigenic preparation derived from the HIV-1 NC of the present invention.
  • compositions comprising an immunogenic amount of an antigenic preparation derived from the HIV-1 JC of the invention in a pharmaceutically acceptable carrier or HIV-1 NC in a pharmaceutically acceptable carrier.
  • a further aspect of the present invention is a kit for detecting the presence of HIV- 1 antibodies, comprising an antigenic preparation derived from the foregoing HIV-1 JC or from HIV-1 NC as described herein.
  • Another aspect of the invention is a method of inducing in a subject antibodies to the HIV-1 JC or to HIV-1 NC of the invention, comprising the step of administering to the subject an immunogenic amount of an antigenic preparation derived from the HIV-l Jc or from the HIV- I NC .
  • the induced antibodies can be harvested and find use, for example, as hybridization probes in methods for HIV scanning.
  • a further aspect of the invention is a method of immunizing a subject against infection by HIV, comprising the step of administering to the subject an immunogenic amount of an antigenic preparation derived from the HIV-1 JC or derived from HIV-1 NC of the present invention.
  • HIV-1 JC induced AIDS in chimpanzees, e.g., in a chimpanzee referred to as C499.
  • HIV-1 JC contained in a biological sample from a chimpanzee having AIDS (C499) induces AIDS in a second chimpanzee and in a different primate species.
  • HIV-1 NC induced AIDS in chimpanzees e.g., in a chimpanzee referred to as C534.
  • HIV-1 NC contained in a biological sample from a chimpanzee having AIDS induces AIDS in a second chimpanzee and in a different primate species.
  • a further aspect of the invention provides a biological fluid or tissue sample, obtained from a chimpanzee having HIV-l JC -induced AIDS, containing an antigenic HIV fragment for inducing AIDS in a nonhuman primate.
  • a further aspect of the invention provides a biological fluid or tissue sample, obtained from a chimpanzee having HIV-l NC -induced AIDS, containing an antigenic HIV fragment for inducing AIDS in a nonhuman primate.
  • the present invention further provides various vaccine formulations containing active immunogenic agents derived from the foregoing HIV-1 JC , DNA encoding the HIV-1 JC , and DNA encoding antigenic fragments of the HIV- 1 JC from the foregoing HIV-1 NC.
  • An antigenic fragment contains one or more epitopes which bind antibodies directed to the HIV-1 JC and/or HT -I NC of the invention.
  • Fig. 1 shows the CD4+ -cell decline and plasma virus loads in chimpanzee C455 following transfusion with blood from C499.
  • blood was collected from C455 for in vitro analyses of CD4+ -cell levels and plasma virus loads.
  • Absolute peripheral CD4+ cells in C455 showed a dramatic decrease by 2 weeks post-transfusion. This rapid decline continued and, by 14 weeks after transfusion, the number of CD4+ cells decreased to 10/ ⁇ l. These low cell numbers have been maintained to date (42 weeks post-transfusion).
  • Plasma HIV-1 RNA loads in C455 showed high levels of virus present by two weeks after transfusion. Results obtained at the 4-week point suggest very high levels of virus replication in C455. However, HIV-1 levels appeared to be somewhat controlled by 5 to 9 weeks post-transfusion.
  • the cutoff level of 10 4 equivalents/ml ( — ) is the lower limit of the assay.
  • Fig. 2 illustrates CD4+ -cell decline in chimpanzee C534 following transfusion with blood from C455. Immediately before and at various times after transfusion, blood was collected from C534 for in vitro analyses of CD4+ -cell levels. Absolute peripheral CD4+ cells in C534 showed a dramatic decrease by 20 days post-transfusion.
  • Figure 3 illustrates the strategy used in PCR amplification of subgenomic fragments from FIIV-1 and the location and orientation of primers on the viral genome are shown. All the primers were designed from the HIV-1 LAI nucleotide sequence and their coordinates are described below. PCR amplified fragments were cloned in TA vectors and the corresponding name designations for the recombinant plasmids are shown in bracket.
  • the 5'-LTR- containing Apa I fragment amplified from HIV-1 JC PBMC genomic DNA was subcloned in pJC to generate plasmid pHIV-l JCI6 while the Apal-Ncol fragment containing the 5'-LTR region amplified from HIV-1 NC PBMC genomic DNA was subcloned in pHIV-l JC16 to generate pHIV-l NCJC chimeric plasmid.
  • Plasmid pHIV-l NC was constructed by subcloning the e «v-containing Ncol-Xhol fragment amplified from HIV-1 NC genomic DNA to pHIV-l N cj C chimeric plasmid.
  • FIG. 4A shows replication of cloned and uncloned HIV-1 isolates in con- A stimulated and unstimulated chimpanzee PBMC (cPBMC). Stimulated cPBMC infected with (Fig. 4A) HIV-a LAV- lb and SF2 parental strains and the highly cytopathic DH12 isolate, (Fig.
  • Chimpanzee PBMC (1.1 x 10 7 ) in T-25 cm 2 were infected with 20 ng of either HIV-1 NC (uncloned), HIV-1 SF2 , HIV-l LAV.lb , or HIV-1 DH12 virus and incubated for a total of 17 days at 37°C. Supernatant aliquots were made on 3, 7, 10, 14, and 17 days post infection. Reverse transcriptase (RT) assays were performed as outlined hereinbelow.
  • Figure 5 shows replication of HIV- 1 isolates in chimpanzee monocyte-derived macrophages (MDM).
  • Purified PBMC (6 x lOVwell) were used to obtain MDM.
  • Ten ng of each of virus HIV-1 JC16 (molecular clone), HIV-1 JC (uncloned), HIV-1 NC (molecular clone), HIV-I NC (uncloned), HIV-1 SF2 , HIV-l LAV . lb , and HIV-1 DH]2 was used for infection and on days 7 and 14 post infection supernatants were harvested.
  • the mount of virus in the supernatants was determined using the p24 HIV-1 antigen capture ELISA (Coulter). Supernatants from control uninfected cultures are represented.
  • HIV-1 isolate JC or HIV-1 JC or HIV JC or HIV-1 having the identifying characteristics of HIV-1 JC as used herein refers to the particular HIV-1 isolated from a chimpanzee (C499) that developed AIDS 10 years after infection with HIV-1 (Novieri et al. [1997] J. Virol. 71:4086-4091).
  • the DNA sequence of the cloned HIV-1 JC is given in SEQ ID NO:l l.
  • HIV-1 isolate NC or HIV-1 NC or HIV NC or HIV-1 having the identifying characteristics of HIV-1 NC as used herein refers to the particular HIV-1 isolated from a chimpanzee (C455) that developed AIDS after infection with HIV-1 JC (Novieri et al. [1997] J. Virol. 71:4086-4091).
  • the DNA sequence of the cloned HIV-1 NC is given in SEQ ID NO: 12.
  • antigenic preparation of HIV-1 JC or antigenic fragment of HIV-1 JC as used herein refers to the whole viral particle of the HIN-1 JC or to a fragment thereof, wherein such fragment encodes at least one epitope or antigenic determinant.
  • antigenic preparation of HIV-1 ⁇ C or antigenic fragment of HIV-1 NC as used herein refers to the whole viral particle of the HIV-1 JC or to a fragment thereof, wherein such fragment encodes at least one epitope or antigenic determinant.
  • animal or subject refers to a mammal and, more frequently, to a primate.
  • an effective amount refers to the quantity of active ingredient necessary to effect in an animal a change in a specific biochemical or immunological parameter.
  • an effective amount refers to the amount of HIV- 1 administered to a subject such that viral infection and AIDS developed.
  • antigenic amount refers to the quantity of active antigen necessary to effect an interaction with corresponding antibodies.
  • immunogenic amount refers to the quantity of active antigen necessary to stimulate the immune system in response to a specific antigen.
  • labeled is used herein to refer to the conjugating or covalent bonding of any suitable detectable group, including enzymes (e.g., horseradish peroxidase, ⁇ -glucuronidase, alkaline phosphatase, and ⁇ -D-galactosidase), fluorescent labels (e.g., fluorescein, luciferase), and radiolabels (e.g., 14 C, 131 1, 3 H, 32 P, and 35 S) to the compound being labeled.
  • enzymes e.g., horseradish peroxidase, ⁇ -glucuronidase, alkaline phosphatase, and ⁇ -D-galactosidase
  • fluorescent labels e.g., fluorescein, luciferase
  • radiolabels e.g., 14 C, 131 1, 3 H, 32 P, and 35 S
  • the viruses of the present invention resulted from the development of AIDS in a first chimpanzee infected with HIV-1 for over 10 years and by the rapid development of immunosuppression in a second chimpanzee transfused with blood from the first chimpanzee.
  • a cohort of 12 chimpanzees was inoculated with several strains of HIV- 1 at the Yerkes Center in the mid- 1980s.
  • a member of this cohort, C499 was described as part of a previously reported superinfection study (Fultz et al. [1987] J. Virol.
  • HIV-1 SF2 in 1985
  • HIV-I NDK m 1987 The first inoculation resulted in infection, as determined by positive virus isolation and persistent HIV- 1 -specific antibody response.
  • C499 developed progressive nonregenerative anemia (hematocrit levels of 37.5% in December 1995 and 27.2% in January 1996, with respective hemoglobin values of 12.1 and 8.4 g/dl; reticulocyte counts were 0.0% since November 1995). Due to progressive hematologic abnormalities, chronic diarrhea, and continued immunosuppression, the animal was euthanized in February 1996.
  • HIV-1 antibody endpoint titers HIV-1 whole- virus enzyme-linked immunosorbent assay, Genetic Systems, Redmond, WA
  • HIV-1 JC The virus strain derived from C499 (HIV-1 JC ) and other HIV-1 isolates (HIV-1 LAI and HIV-1 SF2 ) were tested for the ability to induce syncytium formation in cPBMC.
  • Virus stocks were prepared in cPBMC (HIV-1 JC ) or in human PBMC (HIV-1 LAI and HIV-1 SF2 ). Cells were incubated with virus overnight and were then washed. Cultures were examined daily for evidence of cytopathic effects.
  • cPBMC four days after infection with an HIV-1 isolate (HIV- l j c) from C499 showed beginnings of syncytium formation and separated syncytia.
  • the only virus to induce significant cytopathic effects in cPBMC was HIV-l j c- All cultures were examined for 14 days following infection. All cultures, regardless of the virus used, became positive for virus replication by seven days postinfection. Thus, it is concluded that genetic changes which confer the ability to induce syncytium formation occurred in the virus present in C499.
  • DNA prepared from HIV-l JC -infected cPBMC was used as a template in typical PCR assays with primers (forward, no. 384:
  • Tables 2A-2C illustrate amino acid alignment of V1-V2 clones obtained from HIV- l JC -infected cPBMC and prototypes HIV-1 LAI and HIV-1 SF2 , and HIV-I ND - env clones of HIV- l jC encompassing the VI -V2 region were sequenced by the dideoxy chain termination method (Sequenase; Amersham Life Science, Arlington Heights, IL). With the Intelligenetics Suite of programs (Intelligenetics, Beaverton, OR), sequences of HIV-1 JC env fragments were used to derive corresponding amino acid sequences.
  • HIV JC 10 Deduced amino acid sequences from five of these clones (HIV JC 10, HIV JC 17, HIV JC 45, HIV JC 48, and HIV JC 55) were then aligned with the corresponding region in HIV-1 LAI , HIV-1 SF2 , and HIV-I NDK isolates.
  • Amino acid sequences of HIV-1 LAI ,HIV-1 SF2 and HIV-I ⁇ K were obtained from the Human Retroviruses and AIDS Database.
  • tissue samples obtained by biopsy during the acute diarrheal stage (September 1995) and at necropsy were subjected to histopathological analyses.
  • lymphoid depletion revealed marked lymphoid depletion within the cortical area, with a few follicles remaining, as compared with a lymph node from an age-matched, uninfected chimpanzee, which lacked follicular development and had a very cellular cortex.
  • in situ hybridization experiments with digoxigenin-labeled riboprobes encompassing the entire HIV-1 genome (derived from the HIV-1 BH10 molecular clone (Hahn et al. [1984] Nature 312:166-169) were used to probe formalin-fixed lymph node sections.
  • Virus was isolated from all lymphoid organs including inguinal, axillary, and mesenteric lymph nodes as well as from the spleen and thymus. Virus was also isolated from the kidney and liver but not from the brain or cerebrospinal fluid.
  • results of titration analysis of PBMC and plasma from C499 show that in the 40 ml of blood, 1 x 10 4 50%) tissue culture infective doses (TCID 50 ) of virus was in PBMC and 2 x 10 4 TCID 50 of virus was in plasma. Thus, C455 received a total of 3 x 10 4 TCID 50 of virus.
  • Analysis of peripheral CD4 + cell levels in C455 revealed a precipitous decline beginning by 2 weeks after transfusion, when absolute numbers of CD4 + cells decreased from 1,240 to 320 cells/ ⁇ l (Fig. 1). This decline continued, reaching a minimum value of 10 cells/ ⁇ l (1% of total T cells) by 14 weeks post-transfusion.
  • the total percentage of T cells that the CD4 + population encompasses has remained constant at 1%, with only a slight rise in the absolute number of CD4 + cells (20 cells/ ⁇ l at the latest time point).
  • Levels of peripheral CD8 + cells showed an initial decline from 1,590 to 880 cells/ ⁇ l in the first two weeks after transfusion. However, these levels quickly rebounded to 2,010 CD8 + cells/ ⁇ l by 8 weeks after transfusion. Since this time, the number of CD8 + cells in circulation has been maintained between 550 and 4,320/ ⁇ l, with the level in most recent sample being 840 cells/ ⁇ l.
  • This invention presents an HIV-1 infected chimpanzee that developed AIDS as defined by the Centers for Disease Control and Prevention classification system (CDCP 1992, supra). Progression of clinical disease (anemia, thrombocytopenia, and chronic diarrhea) in this animal was associated with several key findings, including the following: (i) the presence of a virus which is cytopathic for cPBMC in vitro and in vivo and is genetically distinct from those used for inoculation, (ii) an increase in viral load; (iii) CD4 + -cell depletion, (iv) lymph node depletion, and (v) the presence of Cryptosporidium organisms in the intestine.
  • Table 3 shows the antibody response in chimpanzee C534 following transfusion with blood from C455. Immediately before and at various times after transfusion, blood was collected from C534 for in vitro analyses of antibody response. Plasma HIV-1 RNA loads in C534 showed high levels of virus present by two weeks after transfusion. Results obtained at the 3-week point indicate very high levels of virus replication in C455. The cutoff level of 10 4 equivalents/ml is the lower limit of the assay.
  • This invention presents HIV-1 isolates NC and JC which infected chimpanzees such that the HIV-1 infected chimpanzees develop AIDS as defined by the Centers for Disease Control and Prevention classification system (CDCP 1992, supra). Progression of clinical disease (anemia, thrombocytopenia, and chronic diarrhea) in this animal is associated with several key findings, including the following: (i) the presence of a virus which is cytopathic for cPBMC in vitro and in vivo and is genetically distinct from that used for inoculation, (ii) an increase in viral load; (iii) CD4 + -cell depletion, (iv) lymph node depletion, and (v) the presence of Cryptosporidium organisms in the intestine.
  • the adaptation of HIV- 1 from long-term chimpanzee infection to a pathogenic form provides a critical link for the adaptation of HIV- 1 to growth in more readily available nonhuman primate species.
  • the instant invention further contemplates the growth of HIV-1 JC isolated from C499 and/or HIV- I NC fr o C 55 in chimpanzees as well as in pig-tailed or rhesus macaques.
  • the present invention provides for continued biological and genetic characterization of HIV-1 JC and HIV-I NC with further key insights into the pathogenesis of HIV- 1 infection in humans and chimpanzees, for example, for the development of drugs and vaccines for the treatment and prevention of AIDS.
  • chimpanzee C499 was initially infected with HIV-1 SF2 in 1985 [Fultz et al. (1986) J. Virol. 58, 116-124] and later inoculated with HIV-l LAV.lb and HIV-I NDK in 1986 and 1987 respectively.
  • Superinfection with HIV-l LAV . ]b was demonstrated by restriction enzyme analysis of PBMC genomic DNA.
  • HIV-1 JC virus isolate
  • HIV-1 JC [JC16] and HIV-1 NC [NC7] Two clones, one from each group (HIV-1 JC [JC16] and HIV-1 NC [NC7]), were positive by RT and also showed massive syncytia formation (2 to 3 days post transfection), similar to that observed with uncloned virus.
  • 293 cells were transfected with molecularly cloned DNAs followed by amplification with cPBMC as outlined hereinbelow.
  • JC16 and NC7 The complete nucleotide sequences of JC16 and NC7 were determined as described in Example 1, and the sequences are given in SEQ ID NO:l 1 and SEQ ID NO: 12, respectively.
  • the genomes were determined to be 9193 nt (JC16) and 9196 nt (NC7) in length and contained open reading (ORFs) for all HIV- 1 -specific structural, regulatory and accessory genes. Alignment of JC16 and NC7 DNA sequences revealed that the two genomes were very similar, but contained a number of nucleotide changes spread throughout the genome.
  • the LTR sequences of JC16 and NC7 had percent nucleotide identities of 92.1% (LAV), 93.9% SF2), and 89.1% (NDK)-lower than that
  • the most divergent region between NC7 and JC16 was the V5 region of the env gene.
  • JC16 contained a 6 bp deletion in the gag gene relative to NC7 while NC7 had 3 bp deletion in the env gene region relative to JC16.
  • In the LTR region there was a 98.7% nucleotide identity between JC16 and NC7 with all the changes being localized to the U5 region (Table 4).
  • the deduced amino acid sequences for all proteins of HIV-1 JC and HIV-1 NC were generated using the Intelligenetics suite of programs. Using the Lasergene program (DNASTAR Inc., Madison, WI), multiple alignments of all proteins were constructed to examine similarities between HIV-1 NC , HIV-1 JC and the parental viruses (Table 4). Based upon percent homology calculations, Gag, Pol, Vif, Tat, Rev, Env, and Vpu of JC16 and NC7 were most closely related to LAV, with vpr and Nef being most closely related to SF2. In no case was it apparent that the NDK isolate was the origin of a protein sequence.
  • JC16 and NC7 While most changes involved amino acid point mutations, several proteins of JC16 and NC7 contained amino acid insertions or deletions relative to the parental inoculating strains. A closer analysis of amino acid alignments revealed that the percent homologies could be misleading with regards to the origin of the protein. For example, in Tat, JC16 and NC7 were more homologous to LAV than to SF2 (based upon percent homologies). However, the JC16 and NC7 Tat proteins contained 15 amino acid deletions with respect to LAV ⁇ similar to that present in the SF2 isolate. Similar findings were observed in Gag and Pol.
  • Table 5 shows amino acid alignments of Gag, Nef, and Env proteins from JC16, NC7 and the inoculating viruses, LAV, SF2, and NDK.
  • the deduced amino acid sequences for the Gag (a), Nef (b), and Env proteins of HIV-1 were determined using the Intelligenetics Suite of programs (Intelligenetics, Beaverton, OR) and the CLUSTAL method (DNASTAR Inc., Madison, WI) was used for the alignment of proteins.
  • the virus isolates are shown by the designations at the beginning of every line as LAI (HIV-1 LAV ), JC16 (HIV-1 JC16 ), NC7 (HIV- 1 NC7 ), and SF2 (HIV-1 SF2 ).
  • Dashes (-) denote amino acid deletion while dots (.) denote amino acid identity.
  • the numbers after the amino acid sequence on the right show the position of the right-most amino acid in the line.
  • the functional domains on Gag (a) and Nef (b) are indicated.
  • MBD (a) refers to membrane-binding domain while MTD (b) refers to membrane- targeting domain.
  • the hypervariable regions in the Env glycoprotein (c) are shown as VI to V5 and the CD4 binding domain is shown above the sequence.
  • Gpl20 (SU) is the outer surface membrane Env glycoprotein.
  • the NDK Env, LAI Env, and SFS Env protein sequences are given in SEQ ID Nos: 3, 4 and 5, respectively.
  • JC16 and NC7 Env protein sequences are given in SEQ ID Nos: 2 and 23, respectively. There were 7 (JC16 and LAI sequences) and 5 (NC7 sequence) amino acid deletions in the matrix protein (pi 7) relative to SF2 isolate sequence. At the C-terminal end of Gag polyprotein, the progeny viruses and SF2 virus had 12 amino acid deletions in p6 protein relative to LAV virus sequence (Table 5). However, the capsid (p24) and nucleocapsid (p7) proteins were generally well conserved including the cysteine residues within the zinc-finger domains. Point mutations unique to JC16 and NC7 were present in pi 7, p24, p7 and p6 peptides.
  • the Lck binding domain (proline-rich region) within Nef was well conserved with only one point mutation in LAI (Table 5). However, there were 4 amino acid insertions in the SF2 sequence relative to the other viruses at the N-terminal portion of Nef. Sequence analysis of eight other non- infectious clones (4 from JC and 4 from NC) confirmed observations made for the Gag and Nef deletions suggesting that these characteristics are a general property of the viruses obtained from C499 and C455.
  • the gp 120 (SU) glycoprotein contained the highest number of mutations.
  • the V1-V2, V3, V4, and V5 hypervariable regions contained 10, 8, 8, and 3 point mutations respectively specific to JC16 and NC7.
  • the VI -V2 region of JC16 and NC7 also contained insertions relative to the other viruses, resulting in amino acid lengths of 72 (progeny viruses), 69 (LAV), 70 (SF2), and 61 (NDK).
  • Both the CD4- binding domain and the proteolytic cleavage site (REKR) at the SU/TM junction were perfectly conserved.
  • the V3 region was the most interesting of the hypervariable regions. While the parental strains contained only 9 basic amino acids (Arg, Lys, and His positively charged residues) JC16 and NC7 HIV-1 isolates had 12 basic and 2 negatively charged residues (Asp and Glu). This gave the progeny viruses a net positive charge of 10 in the entire V3 region and an overall positive charge of +1 (LAV) or +2 (SF2) compared to the parental strains. Eight of the 10 resultant positive charges for JC and NC isolates are located between residues 10 and 27 of V3 (Fig. 3) compared with 5 of 9 (LAV) and 4 of 9 (SF2) in the same region.
  • JC and NC isolates seem to have a relatively high net positive charge of 4 (relative to SF2) and 3 (relative to LAV).
  • Other researchers have shown that changes in basic amino acids in the middle portion of V3 loop (residues 10 to 27 in Table 5) can alter the syncytium-inducing properties and phenotype of the virus [Bhattacharyya et al. ( 996) AIDS Res. Hum. Retroviruses 12, 83-90; De Wolf et al. (1994) AIDS Res. Hum. Retroviruses 10, 1387-1400; Okada et al. (1994) AIDS Res. Hum. Retroviruses 10, 803-811].
  • Figures 4A, 4B and 4C shows the results of replication studies in mitogen-stimulated cPBMC.
  • the SF2 isolate grew very slowly and to low titers in cPBMC.
  • the LAV- lb isolate grew very well and with rapid kinetics, with a high titer of virus already present by day 7 post infection.
  • the SF2 isolate was unable to induce detectable syncytium formation in cPBMC.
  • the LAV- lb isolate under these conditions, induced very few syncytia, in contrast to previously reported results [Watanabe et al. (1991) J. Virol. 65, 3344-3348]. Included in these analyses was the DH12 isolate of HIV-1 [Shibata et al. (1995) J. Virol. 69, 4453-4462].
  • This primary isolate from a human has been shown to be highly cytopathic for cPBMC. While this virus quickly established infection in the stimulated cell population, it did not grow to high titers.
  • the numerous syncytia formed infection with DH12 could account for the lack of growth observed.
  • the uncloned and cloned viruses of JC (Fig. 4B) and NC (Fig. 4C) replicated to levels comparable to those of LAV- lb for the same period of time. The rates of replication for cloned and uncloned viruses were indistinguishable .
  • DH12 isolate While the DH12 isolate was able to replicate in unstimulated cells, the levels of virus achieved were much less than that of the other viruses.
  • the SF2 isolate was unable to replicate in unstimulated PBMC.
  • Virus recovered from the molecular clones displayed the intrinsic replicative properties exhibited by the viruses they were derived from.
  • FIG. 5 shows virus production in macrophages at 7 and 14 days post infection. Both the HIV-1 JC and NC virus isolates (cloned and uncloned) replicated in MDM as determined by the levels of p24 antigen produced. However, the amount of virus produced by the HIV-1 JC16 molecular clone at 14 days post infection was twice that produced by the HIV-1 JC .
  • HIV-l LAV.]b HIV-1 DH12 also infected MDM and produced virus which is consistent with previous observations made by Gendelman et al. [Gendelman et al. (1991) J. Virol. 65, 3853-3863] and Shibata et al. [Shibata et al. (1995) J. Virol. 69, 4453-4462] respectively.
  • the other parental virus, HIV-1 SF2 did not replicate in MDM.
  • Genomic DNA was isolated and nested PCR was used to amplify the VI -V2 and V3-V5 regions of HIV- 1 envelope gene. These hypervariable regions of env gene were selected because most viral heterogeneity has been associated with them. Equal amounts of PCR products were mixed, heat denatured, and then reannealed with analogous fragments derived from standards (SF162 subtype B3 and ZM18 subtype C2) or from JC16 and NC7 molecular clones.
  • the distance between homoduplex and heteroduplex bands formed between JC16 and NC7 molecular clones was larger than that between bands formed from homoduplexes derived from genomic DNA.
  • the SF162 (subtype B3) heteroduplex bands migrated at almost the same rate as the ssDNA but the ZM18 (subtype C2) heteroduplex bands had the slowest migration.
  • Animal AIDS models include, but are not limited to, chimpanzees, the gibbons, pig-tailed macaques, and rhesus macaques infected with the HIV-1 JC or HIV-1 NC virus of the invention.
  • an immunodeficient pig-tailed macaque and also an immunodeficient rhesus macaque both infected with the HIN-1 JC virus of the invention isolated from the C499 chimpanzee which exhibited HIV-1-induced AIDS or the HIV-1 ⁇ C virus isolated from the C455 chimpanzee.
  • the macaques are infected with HIV-1 isolate JC or HIV-1 NC and are used as an animal model for human AIDS in essentially the same manner as chimpanzees as described above.
  • the transmission of HIV-1 JC or the HIV-1 NC virus into a macaque proceeds via injection of an isolated viral suspension or transfection of a biological fluid or tissue specimen from an HIV- 1 -infected and AIDS-bearing primate.
  • the HIV- 1 - infected donor specimen is introduced into a recipient by any suitable means, such as intraperitoneal injection, intravenous injection, surgical implantation and combinations thereof.
  • Donor tissue may be introduced as organized tissue (e.g., thymus, lymph node, etc.) or as discrete cells.
  • Antigenic fragments of the present invention are peptides which contain at least one epitope (antibody binding site) which binds antibodies which bind to at least one HIV-1 isolate of the present invention.
  • the antigenic fragments are preferably capable of inducing an immune response when administered to a nonhuman primate. DNA encoding such antigenic fragments may be used to transform host cells to thereby produce such antigenic fragments.
  • Antigenic fragments may be identified by a variety of means.
  • a protein from HIV-1 JC and/or from HIV-1 NC, such as an envelope protein, may be fragmented with a protease, and the fragments tested to determine whether or not various ones react with antiserum against the protein. See, e.g., J. Robinson et al, Mol. Cell Biochem. 21:23-32 (1978).
  • Another technique is to synthesize peptides which are fragments of the entire protein and determine whether the individual fragments are recognized by neutralizing antibodies against the protein. See, e.g., J.
  • Still another method useful for obtaining immunogenic fragments of a protein is by isolation and identification of monoclonal escape mutants. In this strategy, HIV-1 is produced in the presence of a monoclonal antibody to the virus. The only viruses which can grow under these conditions are those with a mutation in the nucleotide sequence which codes for an epitope to which the monoclonal antibody binds.
  • a mutant virus which grows under these conditions is referred to as the "monoclonal escape mutant.”
  • the monoclonal escape mutant is then sequenced and the mutant sequence compared with the nucleotide sequence of the HIV-1 JC isolate or the HIV-1 NC isolate to find the specific location of the mutation.
  • the mutation is located in a region which codes for a protective epitope, or an "immunogenic fragment.” See, e.g., J. Lopez et al., J. Virol. 64:927 (1990).
  • Antigenic preparations of the present invention are useful as reagents in immunoassay diagnostic studies of retroviruses.
  • Immunochemical methods for detecting retroviruses include, for example, immunofluorescence assays or immunoenzymatic assays.
  • Immunofluorescence assays typically involve incubating, for example, serum from the subject to be tested with preparations of the pathogenic virus or fragments thereof. Immune complexes formed are detected using either direct or indirect methods, for example, the use of antibodies to which fluorescent labels such as rhodamine or fluorescein have been coupled.
  • Immunoenzymatic assays typically involve viral extracts or other antigen-containing compositions bound to a surface.
  • Serum from a subject to be tested for the presence of antibodies directed against one or more antigens is contacted with the surface and, after a period of incubation, unbound substances are washed away.
  • the presence of immune complexes is detected using antibodies labeled with an enzyme such as horseradish peroxidase, alkaline phosphatase, or beta-galactosidase, which is capable of converting a colorless or nearly colorless substrate into a highly colored product, or an enzyme which emits light in the presence of the proper substrate.
  • the amount of product formed is detected visually, spectrophotometrically, or luminometrically and is compared to a similarly treated control.
  • the presence of antibodies in biological fluids may also be detected by agglutination.
  • Viral lysates or antigen compositions are used to coat, for example, latex particles.
  • Diagnostic tests utilizing the present invention may be carried out in accordance with known techniques.
  • Such techniques provide a method of detecting the presence of HIV- 1 by detecting the presence of HIV- 1 antibodies.
  • Such methods comprise collecting an antibody- containing biological sample (e.g., blood, blood sera, blood plasma, cerebrospinal fluid, tissue samples) from the subject, contacting the sample with an antigenic preparation of the viral particles of the present invention as given herein, and then detecting the formation of a reaction product between the antibodies in the sample and the antigenic preparation.
  • Any suitable assay format such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA) may be employed, in accordance with known techniques. See, e.g., Immunology: Basic Processes, 162-175 (J. Bellanti, [Ed.] 2d ed., W.B. Saunders Co. (1985).
  • kits for the detection of HTV infection comprise a container containing an antigenic preparation of the viral particles of the present invention, which may be lyophilized.
  • the antigenic preparation may comprise, e.g., the HIV-1 envelope protein (env) or the group antigen (gag) protein of the HIV-ljc and/or HrV-l NC of the invention.
  • a method of inducing antibodies to HIV-1 in a subject comprises administering to a subject an immunogenic amount of infectious viral particles of the present invention. This method may be used to make polyclonal or monoclonal antibodies, which may be used in diagnostic assays. Suitable subjects include mammals (such as, for example, rats, rabbits, mice, and horses) and primates.
  • primates is herein intended to encompass any members of the order Primata (for example, lemurs, mandrills, rhesus monkeys, macaques, and chimpanzees) and to include humans. Suitable subjects include those in which antibodies to HIV may be raised (e.g., rabbit, horse).
  • viral antigenic preparations of the present invention may be combined with any suitable pharmaceutically acceptable carrier (such as sterile, pyrogen-free physiological saline solution, or sterile, pyrogen-free phosphate- buffered saline solution).
  • suitable pharmaceutically acceptable carrier such as sterile, pyrogen-free physiological saline solution, or sterile, pyrogen-free phosphate- buffered saline solution.
  • the viral antigens are included in an effective immunogenic amount.
  • the precise amount to be administered to a given subject is determined by techniques known in the art and will vary depending on the route of administration, the subject and the desired response. Administration to the subject may occur by any suitable route (e.g., by intramuscular injection, subcutaneous injection, intraperitoneal injection, or intravenous injection).
  • the appropriate immunogenic dosage will depend upon the particular subject and the desired outcome.
  • the active agent may be given in an amount of from 0.05 to 50 ⁇ g per kg body weight (e.g., 0.5 or 1.0 ⁇ g per kg).
  • the invention also provides for a variety of different vaccines based on the structures of the HIV-l j c isolate and/or the HIV-1 NC of the invention and a method for vaccinating a population against HTV.
  • active agents used for the preparation of a vaccine of the invention include the live attenuated HTV-1 JC and or HIV-1 NC isolate, fixed whole virus, host cells expressing virus antigen, preparations of virus fragments, purified proteins, antigenic fragments of proteins and antigenic peptides which are derivatives of the antigenic fragments.
  • HIV-1 JC - and or HIV-l NC -derived compositions or vaccines are useful for preinfection immunization of primates as well as for postinfection (therapeutic) immunization of HIV-infected primates (see Fultz et al. [1989], Lerner et al., eds. Cold Spring Harbor, NY).
  • Live attenuated HIV isolate JC virus (or HIV-1 NC virus) is prepared by serial passage of the virus in tissue culture or genetically altered by recombinant techniques, in accordance with known procedures. Fixed virus is made by contacting live virus (attenuated or unattenuated) to a suitable fixative, such as formalin.
  • Preparations of viral fragments are made by lysing host cells, such as E coli cells, transformed with a vector encoding an HIV-1 isolate of the present invention (or both) or a portion thereof.
  • the lysate may be used in crude or partially purified form, or a particular viral protein (or antigenic fragment thereof) such as the envelope protein, can be purified to homogeneity and used as an active agent for a vaccine against HIV-1.
  • Host cells such as yeast cells may be transformed with vectors of the present invention capable of expressing HIV-1 proteins, or antigenic fragments thereof, on the surface of the host cells, and the transformed host cells used as an active vaccine agent as such or fixed (e.g., with formalin) and used as an active agent.
  • Antigenic peptides are selected from the group consisting of antigenic fragments of HIV isolate JC and/or NC proteins, such as the envelope protein, and the antigenic equivalents thereof (i.e., analogs or derivatives). Antigenic peptides may be chemically synthesized or produced by recombinant techniques.
  • Viral antigenic preparations and cells producing viral antigens and/or fragments thereof may be formulated into immunogenic compositions as neutral or salt forms.
  • cells are used they are of avirulent strains, or the cells are killed before use.
  • Pharmaceutically acceptable salts include but are not limited to the acid addition salts (formed with free amino groups of the peptide) which are formed with inorganic acids, e.g., hydrochloric acid or phosphoric acids; and organic acids, e.g., acetic, oxalic, tartaric, or maleic acid.
  • Salts formed with the free carboxyl groups may also be derived from inorganic bases, e.g., sodium, potassium, ammonium, calcium, or ferric hydroxides, and organic bases, e.g., isopropylamine, trimethylamine, 2-ethylamino-ethanol, histidine, and procaine.
  • inorganic bases e.g., sodium, potassium, ammonium, calcium, or ferric hydroxides
  • organic bases e.g., isopropylamine, trimethylamine, 2-ethylamino-ethanol, histidine, and procaine.
  • antigenic equivalents refers to proteins or peptides which bind to an antibody which binds to the protein or peptide with which equivalency is sought to be established. Antibodies which are used to select such antigenic equivalents are referred to as “selection antibodies” herein. Preferred selection antibodies are monoclonal antibodies which bind to HIV isolate JC and/or to HIV-1 NC , but not to prior isolates of HIV- 1 such as the HIV-1 isolates NDK, LAI and SF2, for example.
  • One or more amino acids of an antigenic peptide sequence may be replaced by one or more other amino acids which do not affect the antigenicity of that sequence.
  • Such changes can be guided by known similarities between amino acids in physical features such as charge density, hydrophobicity/hydrophilicity, size and configuration, For example, threonine and serine can be interchanged, or aspartic acid and glutamic acid, or leucine and isoleucine, and the like.
  • Antigenic equivalents may be formed by modifying reactive groups within a natural sequence or modifying the N-terminal amino and/or C-terminal carboxyl group. Such equivalents include salts formed with acids and/or bases, particularly physiologically acceptable inorganic and organic acids and bases. Other equivalents include modified carboxyl and/or amino groups on the synthetic peptide to produce esters or amides, or amino acid protecting groups such as N-t-butoxycarbonyl. Preferred modifications are those which provide a more stable, active peptide which will be less prone to enzymatic degradation in vivo.
  • polyclonal and/or monoclonal antibodies capable of specifically binding to a particular epitope of at least one HIV-1 isolate of the invention.
  • the term antibody is used to refer both to a homogenous molecular entity (monoclonal antibody) and a mixture (such as a serum product) made up of a plurality of different molecular entities (polyclonal antibody).
  • Monoclonal or polyclonal antibodies, and preferably monoclonal, specifically reacting with a particular epitope of interest can be made by methods known in the art.
  • immunogenic compositions may be formulated by any of the means known in the art.
  • Such vaccines are typically prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. The preparation may also, for example, be emulsified, or the protein encapsulated in liposomes.
  • Such vaccines may be administered to the subject by any suitable means, for example, by intramuscular injection, by subcutaneous injection, by intravenous injection, by intraperitoneal injection, by oral injection, and by nasal spray.
  • the vaccine or other immunogenic composition may be given in a single dose or multiple dose schedule.
  • a multiple dose schedule is one in which a primary course of vaccination may include 1 to 10 or more separate doses, followed by other doses administered at subsequent time intervals as required to maintain and or reinforce the immune response, e.g., at 1 to 4 months for a second dose and, if needed, a subsequent dose(s) after several months.
  • the immunogenic peptide antigen compositions are administered in a manner compatible with the dosage formulation and in such amount as will be prophylactically and/or therapeutically effective.
  • the quantity to be administered which is generally in the range of about 100 to 1,000 ⁇ g of protein per dose, more generally in the range of about 5 to 500 ⁇ g of protein per dose, depends on the subject to be treated, the capacity of the individual's immune system to synthesize antibodies, and the degree of protection desired. Precise amounts of the active ingredient required to be administered may depend on the judgment of the physician and may be peculiar to each individual, but such a determination is within the skill of such a practitioner.
  • Vaccine formulations of the present invention comprise the active agent mixed with excipients or carriers which are pharmaceutically acceptable and compatible with the active ingredient.
  • excipients include but are not limited to water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof.
  • concentration of the immunogenic polypeptide in injectable formulations is usually in the range of 0.2 to 5 mg/ml.
  • the vaccines may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants which enhance the effectiveness of the vaccine.
  • auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants which enhance the effectiveness of the vaccine.
  • adjuvants which may be effective include but are not limited to: aluminum hydroxide; aluminum phosphate; plant and animal oils; synthetic polymers; e.g., N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr- MDP); N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as nor- MDP); N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-( 1 '-2'-dipalmitoyl-sn-glycer
  • the vaccine formulations may also contain one or more stabilizer, for example, carbohydrates such as sorbitol, mannitol, starch, sucrose, dextrin, and glucose, proteins such as albumin or casein, and buffers such as alkaline metal phosphates and the like.
  • stabilizer for example, carbohydrates such as sorbitol, mannitol, starch, sucrose, dextrin, and glucose, proteins such as albumin or casein, and buffers such as alkaline metal phosphates and the like.
  • an antibody particularly a monoclonal antibody, which is specific for a particular epitope directed to a particular protein of the HIV-1 JC isolate and/or the HIV-1 NC isolate, can be used to screen for other HIV-1 isolates having similar epitopes recognized by that (monoclonal) antibody.
  • Antibodies generated against specific epitopes of the HIV-1 JC of the invention are useful, for example, as probes for screening DNA expression libraries or for detecting the presence of HIV- 1 strains in a test sample.
  • Antigens can be synthesized and conjugated to a suitable carrier protein (e.g., bovine serum albumin or keyhole limpet hemocyanin) for use in vaccines or in raising specific antibodies.
  • a suitable carrier protein e.g., bovine serum albumin or keyhole limpet hemocyanin
  • the polypeptides and antibodies will be labeled by joining, either covalently or noncovalently, a substance which provides a detectable signal.
  • Suitable labels include but are not limited to radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent agents, chemiluminescent agents, magnetic particles and the like. United States Patents describing the use of such labels include but are not limited to Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,355,241.
  • Immunogenic carriers may be used to enhance the immunogenicity of an active agent.
  • Such carriers include but are not limited to proteins and polysaccharides, liposomes, and bacterial cells and membranes. Protein carriers may be joined to the capsular polysaccharide molecules to form fusion proteins by recombinant or synthetic means or by chemical coupling. Useful carriers and means of coupling such carriers to polypeptide antigens are known in the art. The art knows how to administer immunogenic compositions so as to generate protective immunity where immunity is most helpful.
  • compositions and immunogenic preparations including vaccine compositions comprising substantially purified antigens derived from an HIV-1 isolate JC and or NC and a suitable carrier therefor are provided.
  • Immunogenic compositions are those which result in specific antibody production when injected into a human or an animal. Such immunogenic compositions are useful, for example, in immunizing primates against infection by HIV-1 strains.
  • the immunogenic preparations comprise an immunogenic amount of, as specifically exemplified, at least one antigenic determinant derived from the HIV-1 JC isolate of the invention and a suitable carrier.
  • the immunogenic composition can comprise host cells harboring an antigenic agent from the specifically exemplified HIV-1 JC strain and a suitable carrier.
  • a functionally equivalent, recombinant mutant of HIV-1 JC and or HIV-1 NC can be produced by the introduction of the cloned DNA containing the insertion mutations responsible for a desired characteristic. It is also within the scope of the present invention and readily within the grasp of the ordinary skilled artisan to generate other types of genetically stable mutations in the structural or enzyme genes of HIV- 1.
  • Such immunogenic compositions are useful, for example, in immunizing an animal, especially humans, against AIDS and related diseases resulting from infection by HIV-1 species.
  • Such immunogenic compositions can also elicit the production of antibodies which will cross react with proteins of other HIV-1 and HIV-2 strains expressing epitopes in common with those of the starting HIV-1 JC isolate.
  • immunogenic compositions may comprise one or more protein or the immunogenic cellular component.
  • immunogenic amount is meant an amount capable of eliciting the production of antibodies directed against an antigenic agent of HIV-1 JC in an animal or human to which the vaccine or immunogenic composition has been administered.
  • the nucleotide sequence of the HIV-1 JC isolate or the HIV-1 NC isolate can be used to generate hybridization probes which specifically bind to HIV-1 JC genetic material, or to DNA of HIV- 1 isolates having the identifying characteristics of the HIV-1 isolates JC or NC, to determine the presence of such HIV-1 in primates.
  • the hybridization probe may be selected so that it does not bind to other known HIV-1 isolates such as NDK, LAI, SF2, etc.
  • the hybridization probes can be cDNA fragments or polynucleotides and may be labeled with a detectable group, as is well-known in the art. Pairs of probes can serve as PCR primers for synthesis and amplification processes in accordance with the description, for example in U.S. Patent Nos. 4,683,202 and 4,683,195.
  • probes of the invention comprise DNA sequences of HIV- l jC or sequences encoding antigenic fragments thereof or sequences having identity thereto.
  • the production of DNA, vectors, transformed host cells, HIV-1 virus, proteins, and protein fragments of the present invention by genetic engineering techniques can be carried out in accordance with methods known in the art. See, e.g., U.S. Pat. No. 4,761,371, U.S. Pat. No. 4,877,729, U.S. Pat. No. 4,912,038, and U.S. Pat. No. 4,879,224, among others.
  • a nucleotide sequence (polynucleotide) or fragment thereof is substantially homologous (or substantially similar) to another polynucleotide if, when optimally aligned (with appropriate nucleotide insertions or deletions) with another polynucleotide, there is nucleotide sequence identity for approximately 80% of the nucleotide bases, usually approximately 90%, more preferably about 95% to 100%) of the nucleotide bases. Gaps introduced to optimize alignments are treated as mismatches.
  • substantial homology exists when a polynucleotide or fragment thereof will hybridize to another polynucleotide under selective or stringent hybridization conditions.
  • Selectivity of hybridization exists under stringent hybridization conditions which allow one to distinguish the target polynucleotide of interest from other polynucleotides.
  • selective hybridization will occur when there is approximately 75%) similarity over a stretch of about 14 nucleotides, preferably approximately 80% similarity, more preferably approximately 85%) similarity, and most preferably approximately 90%) similarity. See Kanehisa (1984) Nucl. Acids Res., 12:203-213.
  • the length of homology comparison, as described, may be over longer stretches, and in certain embodiments will often be over a stretch of about 17 to 20 nucleotides, preferably 21 to 25 nucleotides, more preferably 26 to 35 nucleotides, and more preferably about 36 or more nucleotides.
  • hybridization of polynucleotides is affected by such conditions as salt concentration, temperature, or organic solvents, in addition to the base composition, length of the complementary strands, and the number of nucleotide base mismatches between the hybridizing polynucleotides, as will be readily appreciated by those skilled in the art.
  • Stringent temperature conditions will generally include temperatures in excess of 30°C, typically in excess of 37°C, and preferably in excess of 45°C.
  • Stringent salt conditions will ordinarily be less than 1 M, typically less than 500 mM, and preferably less than 200 mM. However, the combination of parameters is much more important than the measure of any single parameter (Wetmur and Davidson [1968] J. Mol. Biol. 31:349-370).
  • An isolated or substantially pure polynucleotide is a polynucleotide which is substantially separated from other polynucleotide sequences which naturally accompany a native sequence.
  • the term embraces a polynucleotide sequence which has been removed from its naturally occurring environment, and includes recombinant or cloned DNA isolates, chemically synthesized analogues and analogues biologically synthesized by heterologous systems.
  • a polynucleotide is said to encode a polypeptide if, in its native state or when manipulated by methods known to those skilled in the art, it can be transcribed and/or translated to produce the polypeptide of a fragment thereof.
  • the anti-sense strand of such a polynucleotide is also said to encode the sequence.
  • Vectors are replicable DNA constructs used to either amplify or express DNA of the present invention.
  • An expression vector is a replicable DNA construct in which DNA of the present invention is operably linked to control sequences capable of expressing that DNA in a suitable host.
  • control sequences include a transcriptional promoter, an optional operator sequence to control transcription, a sequence encoding suitable mRNA ribosomal binding sites, and sequences which control the termination of transcription and translation.
  • Suitable vectors include plasmids, viruses (e.g., vaccinia virus, adenovirus, baculovirus, cytomegalo virus) phage, and integratable DNA fragments (i.e., fragments integratable into the host genome by recombination).
  • DNA regions are operably linked or operably associated when they are functionally related to each other.
  • a promoter is operably linked to a coding sequence if it controls the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to permit translation.
  • Transformed host cells are cells which have been transformed or transfected with vectors as described above. Transformed host cells ordinarily express the DNA of the present invention. Suitable host cells include prokaryote, yeast or higher eukaryotic cells such as mammalian cells and insect cells.
  • Prokaryote host cells include gram negative or gram positive organisms, for example Escherichia coli (E. coli) or Bacilli.
  • Exemplary host cells are E. coli W3110 (ATCC 27,325), E. coli B, E. coli XI 776 (ATCC 31,537), E. coli 294 (ATCC 31,446).
  • a broad variety of suitable prokaryotic and microbial vectors are available.
  • E. coli is typically transformed using pBR322.
  • Promoters most commonly used in recombinant microbial expression vectors include the ⁇ -lactamase (penicillinase) and lactose promoter systems (Change et al., Nature 275:615 [1978]; and Goeddel et al, Nature 281:544 [1979], a tryptophan (trp) promoter system (Goeddel et al. [1980] Nuc. Acids Res. 8:4057, and EPO App. Publ. No. 36J66) and the tac promoter (H. De Boer et al, Proc. Natl. Acad. Sci. USA 80:21 [1983]).
  • the promoter and Shine-Dalgarno sequence are operably linked to the DNA of the invention, i.e., they are positioned so as to promote transcription of messenger RNA from the DNA.
  • Eukaryotic microbes such as yeast cultures may also be transformed with vectors of the present invention. See, e.g., U.S. Pat. No. 4,745,057. Saccharomyces cerevisiae is the most commonly used yeast, although other yeast may also be used. Host cells such as insect cells (e.g., cultured Spodoptera frugiperda cells) and expression vectors such as the baculovirus expression vector may be employed in carrying out the present invention, as described in U.S. Pat. Nos. 4,745,051 and 4,879,236 to Smith et al.
  • Examples of useful mammalian host cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, and WI 138, BHK, COS-7, CV, and MDCK cell lines.
  • the transcriptional and translational control sequences in expression vectors to be used in transforming vertebrate cells are often provided by viral sources.
  • commonly used promoters are derived from polyoma, Adenovirus 2, and Simian Virus 40 (SV40). See, e.g., U.S. Pat. No. 4,599,308.
  • An origin of replication may be provided either by construction of the vector to include an exogenous origin, such as may be derived from SV40 or other viral (e.g., Polyoma, Adenovirus, VSV, or BPV) source, or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter is often sufficient. Rather than using vectors which contain viral origins of replication, one can transform mammalian cells by the method of cotransformation with a selectable marker and DNA of the present invention, as described in U.S. Pat. No. 4,399,216.
  • a cohort of 12 chimpanzees was inoculated with several strains of HIV- 1 at the Yerkes Center.
  • a member of this cohort, chimpanzee 499 was inoculated on three different occasions with three different HIV-1 isolates: HIV-1 SF2 in 1985, HIV-1 LAI in 1986 and HIV- I NDK in 1987 ( See Fultz et al - 1 1991 - J - Infect - Dis - 163:441-447 and Novembre et al. [1997] J. Virol. 71:4086-4091).
  • a chimpanzee be bred in captivity, be seropositive for Epstein-Barr virus and cytomegalovirus and be seronegative and PCR negative for HIV prior to being used as an animal model.
  • HIV-1 JC The original source of the HIV-1 JC isolate was the chimpanzee C499. HIV was easily isolated from the peripheral blood mononuclear cells (PBMC) of this animal. Cocultivation of PBMC derived from C499 with uninfected chimpanzee PBMC (cPBMC) resulted in the isolation of a virus (HIV-1 JC ) which induced syncytium formation in chimpanzee cells. The nucleotide sequence of this virus was distinguished from the isolates used for the initial inoculations and from other known HIV-1 isolates.
  • PBMC peripheral blood mononuclear cells
  • HIV-l j c isolate was deposited with the AIDS Reagent Program, McKesson Bioservices, 685 Lofstrand Lane, Rockville, MD 20850 USA, a division of the NIH AIDS Research and Reference Reagent Program, in May 1997 and has been assigned Catalog Number 3523.
  • a substantially full-length infectious molecular clone has a nucleotide sequence as given in SEQ ID NO: 11.
  • probes of the invention comprise DNA sequences as set forth in SEQ ID NO:l 1 and/or sequences of at least 15 contiguous nucleotides derived therefrom or sequences complementary thereto.
  • the original source of the HIV-1 JC isolate was the chimpanzee C455. HIV was easily isolated from the peripheral blood mononuclear cells (PBMC) of this animal. Cocultivation of PBMC derived from C455 with uninfected chimpanzee PBMC (cPBMC) resulted in the isolation of a virus (HIV-1 NC ) which induced syncytium formation in chimpanzee cells.
  • PBMC peripheral blood mononuclear cells
  • cPBMC uninfected chimpanzee PBMC
  • the nucleotide sequence of this virus is distinguished from other known HIV-1 isolates.
  • a substantially full-length infectious molecular clone has a nucleotide sequence as given in SEQ ID NO: 12.
  • probes of the invention comprise DNA sequences as set forth in SEQ ID NO: 12 and/or sequences, of at least 15 contiguous nucleotides derived therefrom or sequences complementary thereto.
  • the HIV-1 isolates used to inoculate C499 included LAV (lymphadenopathy- associated virus)-l BRU (Barre-Sinoussi [1983] Science 220:868-871), SF2 (previously designated ARV [AIDS-related virus]-2) (Levy et al. [1984] Science 225:840-842), and NDK, a highly cytopathic HIV-1 of African origin (Spire et al. [1989] Gene 81:275-284). Inoculations of virus were done intravenously with 1-ml aliquots of undiluted or diluted virus stocks.
  • C499 was part of a study to assess the effects of therapeutic vaccination on immunity and viral status in HIV- 1 -infected chimpanzees.
  • C499 was given two intramuscular injections, 4 weeks apart, of 500 ⁇ g of recombinant SF2 p53 gag, produced in yeast and formulated with 100 ⁇ g of muramyl tripeptide (Ciba-Geigy, Summit, NJ) in 4% squalene and 0.008% Tween 80 (Fultz et al. [1989] in Vaccines 89, supra).
  • the HIV-1 isolates used to inoculate C455 was JC, deposited with the AIDS Reagent Program, Catalog Number 3523. Inoculations of virus were done intravenously with 1-ml aliquots of undiluted or diluted virus stock.
  • C534 was given two intramuscular injections, 4 weeks apart, of 500 ⁇ g of recombinant SF2 p53 gag, produced in yeast and formulated with 100 ⁇ g of muramyl tripeptide (Ciba-Geigy, Summit, NJ) in 4%> squalene and 0.008% Tween 80 (Fultz et al. [1989] in Vaccines 89, supra).
  • Serum samples were tested by indirect ELISA for antibodies to specific HIV-1 proteins using a series of recombinant antigens. These antigens have been described (Fultz et al. [1989] in Vaccines 89, supra) and included p25 gag and p53 gag, produced in yeast; p31 pol; and e «v2-3(SF2) and e»v2-3 (IIIB), nonglycosylated polypeptides corresponding to full- length gpl20.
  • Antibody titers to whole- virus preparations were determined with an HIV enzyme immunoassay kit (EIA; Genetic Systems, Seattle, WA). Neutralization assays were performed as described (Fultz et al. [1986] Proc. Natl.
  • titers were based on >80% inhibition of reverse transcriptase (RT) activity after preincubation of virus with serum and infection of normal human PBMC.
  • RT reverse transcriptase
  • Antibodies cross-reactive with histone H2B were identified by immunoblot using purified calf thymus histones as described previously (Strickler et al. [1987] Nature 327:710-713).
  • CD8 + lymphocytes were washed once; 10 7 cells from C499 were placed in fresh RPMI-IL-2, cultured without indicator cells, and monitored for HIV-1 production.
  • CD4 + - and CD8 + -enriched populations were obtained by panning with monoclonal antibodies specific for the CD8 + antigen or with CD8 + -coated magnetic beads (Dynabeads; Robbins Scientific, Mountain View, CA).
  • CD8 + lymphocytes were recovered from the plates or magnetic beads by additional overnight incubation and repeated washes with medium. After stimulation with ConA, cultures were established either with CD4 + -enriched cells only or with CD4 + - and CD8 + -enriched cells in various ratios. Percentages of CD4 + and CD8 + cells in enriched populations were determined by analysis with FACScan (Becton-Dickinson, Mountain View, CA).
  • Replication kinetics in PBMC and macrophages were tested as follows. 1 x 10 7 freshly isolated or Con-A stimulated PBMC from HIV-1 negative chimpanzee were infected overnight (at 37°C) with 20 ng of the indicated virus (p24 antigen concentration). The cells were centrifuged at 1000 rpm for 10 min, resuspended in 10 ml complete RPMI medium containing 10%) FBS and IL-2 (IL-2 medium) and were incubated at 37 °C. Samples of supernatants (1 ml) were harvested on days 3, 7, 10, 14 and 17 post infection. IL-2 medium was added to the cultures following the sampling to maintain the original volume. Supernatants were used in RT assays to determine the relative amounts of virus produced.
  • cPBMC were resuspended in macrophage media (6 x lOVwell in RPMI 1640 containing 15% human serum [AB + ], 1% HEPES, 0.008 ng/ml GM- CSF, 0.03 ng/ml M-CSF, 1% antibiotic-antimycotic solution (Sigma, St. Louis, MO) and seeded in a 24-well plate and incubated at 37 °C for 4 hrs. The cells were mixed by pipetting up and down before the incubation was continued for 4 days. Non-adherent cells were removed by gently washing the wells. Fresh medium (2 ml/well) was added and the cells were cultured for an additional 3 days to allow full macrophage differentiation.
  • macrophage media 6 x lOVwell in RPMI 1640 containing 15% human serum [AB + ], 1% HEPES, 0.008 ng/ml GM- CSF, 0.03 ng/ml M-CSF, 1% antibiotic-antimycotic solution (Sigma
  • Infections were initiated by adding 10 ng of virus (p24) to the cells in 500 ⁇ l media and adsorbed overnight. The inoculum was removed and the cells were washed twice before fresh macrophage medium (2 ml) was added. On days 7 and 14 post infection, aliquots of 0.5 ml were taken for determination of p24 antigen levels using the HIV-1 p24 Antigen kit (Coulter Corp, Miami, FL) according to the manufacturer's instructions.
  • Lymphocyte proliferative responses to mitogens were tested by incorporation of [ 3 H]thymidine into the DNA of PBMC seeded in triplicate at 5 x 10 4 PBMC per well into 96- well plates.
  • PBMC were incubated for four days with different concentrations of phytohemagglutinin (PHA) or ConA, pulsed overnight with 1 ⁇ Ci of [ 3 H]thymidine per well, and harvested with a cell harvester (Skatron, Sterling, VA); counts per minute (cpm) incorporated were determined in a ⁇ counter.
  • PHA phytohemagglutinin
  • ConA cell harvester
  • Cytotoxic T lymphocyte (CTL) responses against HIV-1 env- and g ⁇ g-encoded antigens were assayed using fresh PBMC from C499 as effector cells and autologous Epstein Barr virus-transformed B cells as target cells.
  • Nonhuman primates infected with HIV-1 isolate JC or infected with HIV-1 NC are useful as a model system for the study of AIDS.
  • Chimpanzees and other monkey species used for this purpose are preferably specific pathogen-free animals, which are available from primate centers, e.g., the Yerkes Regional Primate Research Center, Emory University, Atlanta, GA.
  • Infected primates are preferably maintained as a single colony of two or more animals, all inoculated with HIV-1 isolate JC or a colony of two or more animals, all inoculated with HIV-1 NC .
  • a colony may be maintained in a single room with each primate housed in an appropriate cage, in accordance with standard practices for the maintenance of animals established by the Animal Welfare Act and the NIH guide for care and use of laboratory animals.
  • the primates are infected with the HIV-1 JC virus or the HIV-1 NC virus by any suitable means, such as intraperitoneal, intravenous or subcutaneous injection with a solution containing HIV-1 isolate JC or HIV-1 NC .
  • the solution may also be a body fluid or tissue (e.g., blood) from a previously infected primate, a blood fraction containing peripheral blood mononuclear cells from a previously infected primate, a pharmaceutically acceptable carrier such as saline solution containing HIV-1 isolate JC or HIV-1 isolate NC, etc.
  • Nonhuman primates infected with HIV- JC or HIV-1 NC are particularly useful as a model system for AIDS because of the concomitant decrease in CD4 + cells and increase in HTV-1 loads in plasma.
  • the development of AIDS has not been previously documented in any nonhuman primate species.
  • a primate(s) infected with HIV-l j c or HIV-I NC virus is subjected to a treatment useful in combating AIDS in humans and thereafter the progress of the infection and related diseases is monitored.
  • a control (placebo) group of HIV-1 JC or HIV-l NC -infected animals is left untreated for comparative purposes.
  • a slowing in the progression of the development of AIDS in infected animals indicates that the treatment may be useful for combating AIDS in humans and additional screening and toxicological testing is prescribed.
  • Such treatment includes but is not limited to a vaccine, a drug (e.g., an antiretroviral compound) or a drug combination (e.g., antiviral nucleosides such as AZT, DDI, etc.), a peptide, a protein, etc.) or a vaccine/drug combination, etc.
  • the progress of the disease is monitored by any suitable parameters including, but not limited to, (a) decline in CD4 + cell levels, (b) increase in viral loads in plasma, (c) presence of HIV-1 JC virus or HIV-1 NC virus, (d) weight loss, (e) general appearance, and other symptoms characteristic of AIDS.
  • Example 8 Virus Cloning and Sequence Analysis cPBMC infected with either HIV-1 JC or HIV-1 NC were used for isolation of DNA using the Puregene Kit (Gentra systems, Minneapolis, MN) as directed by the manufacturer.
  • the strategy used for PCR amplification and primer location on the HIV-1 viral genome is shown in Figure 3.
  • PCR primers MSF12 (5'-AAA TCT CTA GCA GTG GCG CCC GAA CAG-3', (SEQ ID NO:18); HIV-1 LAV nt 169 to 195) and MSR5 (5'-GCA CTC AAG GCA AGC TTT ATT GAG GCT-3', (SEQ ID NO:19); HIV-1 LAV nt 9225 to 9198) [Salminen et al.
  • Virology 213, 80-86 were used to amplify a 9056 bp product from PBMC genomic DNA prepared from HIV-l JC -infected cells.
  • Another PCR primer pair 527 (5'-CAC ACA CAA GGC TAC TTC CCT GAT TGG CAG A-3', SEQ ID NO:20, HIV-1 LAV nt 5302 to 5274) was used to amplify 5' LTR-containing fragments (5699 bp) from the same DNA source.
  • PCR primer pairs 527-528 and 529 (5'-ATG GAA CAA GCC CCA GAA GAC CAA GGG CCA CAG-3', SEQ ID NO:21, HIV-1 LAV nt 5141 to 5173) and 530 (5'-GGT CTG AGG GAT CTC TAG TTA CCA GAG TCA C-3', SEQ ID NO:22, HIV-1 LAV nt 151 to 121) were used to generate the 5'-half (5699 bp) and 3'-half (4142 bp) PCR products respectively from HIV-1 NC genomic PBMC DNA. Primers were synthesized on an Applied Biosystems 392 DNA synthesizer (Applied Biosystems, Foster City, CA).
  • PCR was performed using the reagents from the Expand Long Template kit (Boehringer Mannheim, Indianapolis, IN) and 200 ng of DNA template, according to the manufacturer's instructions. After an initial DNA denaturation of 94°C for 2 min, the PCR consisted of 10 cycles of 94°C for 15s, 61 °C for 30s, 68°C for 8 min followed by 20 cycles of 94 °C for 15s, 61 °C for 30s, and 68° C for 8 min with a 5 second addition to each extension. The samples were incubated at 72 °C for 30 min after the last cycle and then cooled to 4°C. Results of PCR reactions were evaluated on 0.9% agarose gels.
  • PCR products representing the correct sized fragments were isolated from agarose and were directly cloned into the pCR II plasmid and amplified in Escherichia coli bacteria (TA cloning kit, Invitrogen Corp., San Diego, CA) according to the manufacturer's protocol. Single bacterial colonies containing plasmids with inserts of the correct size were grown at 30°C overnight and plasmid DNA prepared by the alkaline lysis method.
  • a chimeric plasmid (pHIV-l NC jc) was generated by subcloning the PCR amplified 5' half of HIV-1 NC into pHIV- I j ci ⁇ - F° r HIV-I NC full-length clone, the Nco l-Xho I ettv-containing fragment from the 3' half PCR product was gel purified and subcloned into the Nco l-Xho I large fragment of plasmid pHTV-l NC jc containing the 5' half of HIV- 1 NC . Multiple restriction enzymes were used for analysis of both viral DNAs to confirm the full length clones.
  • the cells were washed twice in 10 ml complete medium before they were resuspended in 10 ml complete medium, transferred to a T-25 cm 2 flask and incubated at 37°C (5%> CO 2 ). The cells were checked daily for cytopathic effects (syncytia formation) and aliquots of cultures were tested for the presence of reverse transcriptase (RT) activity using standard assay methods.
  • RT reverse transcriptase
  • 2 x 10 5 cells in 6-well plates were transfected with 2 ⁇ g of viral DNA using lipofectin (Life Technologies, Gaithersburg, MD) or DOTAP (Boehringer Mannheim, Indianapolis, IN) according to the manufacturers' instructions.
  • the transfected cells were overlaid with 2 x 10 6 /well of uninfected cPBMC previously stimulated with concanavalin A (Con- A) for 4 days. After an additional 2 day incubation, the non-adherent cell population (cPBMC) were transferred to a T-25 flask and additional stimulated cPBMC added for virus amplification. Culture supernatants were assayed for RT activity and the cells were observed daily for development of syncytia. Cell-free stocks of molecularly cloned viruses were prepared at peak RT activity, aliquoted and stored under liquid nitrogen.
  • Con- A concanavalin A
  • Primers for sequencing were constructed from conserved regions of aligned sequences of HIV-1 LAI and HIV-1 SF2 and were synthesized on an applied Biosystems 392 DNA synthesizer.
  • the DNA sequence of each full-length clone virus was determined by the dideoxy-chain termination method using the sequenase system (Amersham Life Sciences, Arlington Heights, IL) and 35 S-dATP.
  • Nucleotide sequence alignments were performed with the Intelligenetics Suite of programs (Intelligenetics, Beaverton, OR) whild the phylogenetic analysis of amino acid sequence was done with CLUSTAL method (DNASTAR, Inc., Madison, WI).
  • nucleotide sequences for HIV-1 JC16 and HIV-1 NC have been assigned Genbank accession numbers AF049494 and AF049495 respectively.
  • the heteroduplex mobility assay kit (NIH AIDS Research and Reference Reagent Program) based on the method described by Delwart et al. (1993) Science 262, 1257-1261 was used. Briefly, equal amounts (5 ⁇ l each) of second-round PCR products (VI -V2 and V3- V5) from infected cPBMC genomic DNA were mixed with the reference PCR products to obtain heteroduplexes. After adding 1.1 ⁇ l of lOx annealing buffer (1 M NaCl, 100 mM Tris [pH 7.8], 20 mM EDTA), the mixed DNAs were denatured at 94 °C for 2 min and then reannealed by rapidly cooling in ice.
  • lOx annealing buffer (1 M NaCl, 100 mM Tris [pH 7.8], 20 mM EDTA
  • Virus levels were determined using the Chiron B-DNA assay as directed by the manufacturer. Plasma samples were stored at - 80°C until use. c ND, not done (no plasma sample was available for this date).
  • Table 2A Alignment of HIV-1 JC (SEQ ID NO:2) and H-V-l ⁇ env (SEQ ID NO:3) Protein Sequences. The consensus sequence corresponds to SEQ ID NO:6.
  • HIVJCENV 1 MRvkEncqhl rwgWK GIMLLGMLMiCSAtEkL VTVYYGVPvWKEtTTTLFCASDAKAY
  • HIVNDKENV 1 MRarE kemcqnlWKWGIMLLGMLMtCSAaEdLWVTVYYGVPiWKEaTTTLFCASDAKAY consensus MR- -En WK GIMLLGM___M-CSA-E- VTVYYGVP-WKE-TTTLFCASDAKAY
  • HIVJCENV 123 TPLCVTLNCTDlknEtktNSsdaNsnsgEimgnEeiKNCSFNVstgapgkvqkeYa fyal
  • HIVNDKENV 122 TPLCVTLNCTD E lrNS kgNgkveE eEkrKNCSFNVrdkreqvyalfYk div consensus TP CVT NCTDlknEt- -NSs--N Ei g-E- -KNCSFNV Y-Lf
  • HIVJCENV 184 dlvsikNenNSTshmLtsCnTSvsTQACPKvSFEPIPIHyCAPAGFAILKCnDKKFNGTGP
  • HIVJCENV 245 CnNVSTVQCTHGIRPWSTQLLLNGSvAEEEwlRSaNfsdNaKTIIVQLN SveltCTRP
  • HIVNDKENV 234 CsNVSTVQCTHGIRPWSTQLLLNGSlAEEEiiiRSeNltnNvKTIIVQLNaSivInCTRP consensus C-NVSTVQCTHGIRPWSTQLLLNGS-AEEE RS-N N-KTIIVQLN-S--I-CTRP
  • HIVJCENV 306 nynetkkirlhrgygrsfvT vrKlGdrkQAHCt nRtk dnALkQiAsKLreqfNKTal
  • HIVNDKENV 295 ykytrqrtsIglrqslytiTgkkkKtGyigQAHCkisRaeWnkALqQvAtKLgnllNKTtl consensus 1 Tgk--K-G QAHC R-- --AL-Q-A-KL NKT-I
  • HIVNDKENV 356 tFkpSSGGDpEItsHmlNCGGdfFYCNTsrLFNST NqTnstgfnngtvTLPCRIkQiVNl consensus -F- -SSGGD-EI- -H- -NCGG- -FYCNT- -LFNSTWN-T TLPCRI-Q-VN-
  • HIVJCENV 426 WQkVGKAMYAPPsdGqlrCtSNITGLLLTRDGGhndNNtnnETfRPGrGDMRDNWRSELYK
  • HIVJCENV 487 YKViKIEPlGVAPTKAkRRWqREKRAvGmvGAmFLGFLGAAGSTMGAASlTLTVQARQLl
  • HIVNDKENV 476 YKVvKIEPiGVAPTKArRRWeREKRAiG IGAvFLGFLGAAGSTMGAASvTLTVQARQLm consensus YKV-KIEP-GVAPTKA-RRW-REKRA-Gm-GA-FLGFLGAAGSTMGAAS-TLTVQARQL-
  • HIVJCENV 548 SGIVqQQNNLLRAIEAQQHLLQLT GIKQLQARVLAVERYLkDQQLLGIWGCSGklICTT HIVNDKENV 536 SGIVhQQNNLLRAI ⁇ AQQHLLQLTVWGIKQLQARVLAVERYLrDQQLLGI GCSGrhlCTT consensus SGIV-QQNNLLRAIEAQQHLLQLTVWGIKQLQARVLAVERYL-DQQLLGI GCSG- - ICTT
  • HIVJCENV 609 tVP NaS SNkSLDqI nNMT mEWdREIaNYTnLIhhLIEESQnQQEKNEqELLELDK A HIVNDKENV 597 nVPWNsSWSNrSLDelWqNMTWmEWeREIdNYTgLIysLIEESQiQQEKNEkELLELDKWA consensus -VP N-S SN-SLD-I -NMT -E -REI-NYT-LI--LIEESQ-QEKNE-ELLELDK A
  • HIVJCENV 670 SLWsWFdlsnWL YIKiFIMIVaGLvGLRIVFAVLSiVNRVRQGYSPLSFQThfPaPRGPD
  • HIVNDKENV 658 SL nWFsItk L YIKlFIMIVgGLiGLRIVFAVLSvVNRVRQGYSPLSFQTllPvPRGPD consensus SLW-WF-I--WLWYIK-FIMIV-GL-GLRIVFAVLS-VNRVRQGY ⁇ PLSFQT--P-PRGPD
  • HIVJCENV 731 RPdglEgEGGERdRDRSvRLVdGflALlWeDLRNLCLFSYHRLRDllLIvtRIVELLGRRG HIVNDKENV 719 RPeelEeEGGERgRDRSiRLVnGlfALfWdDLRNLCLFSYHRLRDsiLIaaRIVELLGRRG consensus RP- - IE-EGGER-RDRS-RLV-G- -AL- -DLRNLCLFSYHRLRD- -LI- -RIVELLGRRG
  • HIVJCENV 792 WEALKYL sLLQY SQ ⁇ LkNSAvnLfnTtAIvVAEgTDRilEWQRlCRAILhiPRRIRQG
  • HIVNDKENV 780 EALKYL nLLQY SQELrNSAssLldTiAIaVAErTDRvIEWQRaCRAILnvPRRIRQG consensus EALKYL -LLQYWSQEL-NSA--L--T-AI-VAE-TDR-IEWQR-CRAIL--PRRIRQG
  • HIVJCENV 853 LERiLL HIVNDKENV 841 LERILL consensus LER-LL
  • Alignment score 541.00 Table 2B. Alignment of HIV-1 JC and HIV-1 LAI env Protein Sequences (SEQ ID NO:2 and SEQ ID NO:4, respectively). The consensus sequence corresponds to SEQ ID NO:7.
  • HIVJCENV 1 MRVKEncQHL RWGWKWGiMLLGmlMICSATEKiWVTVYYGVPVWKEtTTTLFCASDAKAY
  • HIVlaienvt 1 MRVKEkyQHL RWGWK GtMLLGiLMICSATEKL VTVYYGVPVWKEaTTTLFCASDAKAY consensus MRVKE- -QHL R G KWG-MLLG-LMICSATEKL VTVYYGVPVWKE-TTTLFCASDAKAY
  • HIVJCENV 62 eeEVHNV ATHACVPTDPNPQEiVLaNVTEdFNM KNeMVEQMHtDIISL DeSLKPCVKL
  • HIVJCENV 123 TPLCVtLnCTDLkNeTkTNSSdaNSnSGEiM gneEIKNCSFNvSTgapGKVQKEYalFYa
  • HIVlaienvt 123 TPLCVsLkCTDLgNaTnTNSSntNSsSGEmMmekgEIKNCSFNiSTsirGKVQKEYafFYk consensus TPLCV-L-CTDL-N-T-TNSS--NS-SGE-Mm EIKNCSFN-ST GKVQKEY--FY-
  • HIVJCENV 183 LDIvsIkneNnsTShmLTSCNTSVsTQACPKVSFEPIPIHYCAPAGFAILKCNdKkFNGTG
  • HIVlaienvt 184 LDI IpidNdtTSytLTSCNTSViTQACPKVSFEPIPIHYCAPAGFAILKCNnKtFNGTG consensus LDIvsI N- -TS- -LTSCNTSV-TQACPKVSFEPIPIHYCAPAGFAILKCN-K-FNGTG
  • HIVJCENV 244 PCnNVSTVQCTHGIRPWSTQLLLNGSvAEEEWlRSANFsDNAKTIIVQLNhSVEItCTR
  • HIVJCENV 305 PNyNetKklRIhRGyGRsFVTvrKlGdrkQAHCtmnRtK dnaLKQIASKLREQFnNktal
  • HIVlaienvt 304 PNnNtrKsIRIqRGpGRaFVTigKiGnmrQAHCnisRaK natLKQIASKLREQFgNnktl consensus PN-N- -K- IRI-RG-GR-FVT- -K-G QAHC R-K LKQIASKLREQFgN 1
  • HIVlaienvt 365 IFkqSSGGDpEIvtHSFNCGGEfFYCNsTqLFNSTWfNsTwsTEgSNnteGsdtlTLPCRI consensus IF--SSGGD-EI--HSFNCGGE-FYCN-T-LFNSTWfN-T sTEgSNn--Gs--ITLPCRI
  • HIVJCENV 420 rQFvNMWQkVGKAMYAPPsdGQIRCtSNITGLLLTRDGGhNdNNtnnEtFRPGrGDMRDN
  • HIVJCENV 481 RSELYKYKViKIEPLGVAPTKAKRRWQREKRAVGmvGAmFLGFLGAAGSTMGAaSlTLTV
  • HIVlaienvt 485 RSELYKYKVvKI ⁇ PLGVAPTKAKRRWQREKRAVG iGAlFLGFLGAAGSTMGArSmTLTV consensus RSELYKYKV-KIEPLGVAPTKAKRRWQREKRAVGm-GA-FLGFLGAAGSTMGA-S-TLTV
  • HIVJCENV 542 QARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARvLAVERYLKDQQLLGIWGCSG
  • HIVlaienvt 545 QARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARiLAVERYLKDQQLLGIWGCSG consensus QARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQAR- AVERYLKDQQLLGIWGCSG
  • HIVJCENV 603 KLICTTtVPWNASWSNKSLdQIWNNMTWmEWDREIaNYTnLIHhLIEESQNQQEKNEQELL
  • HIVlaienvt 606 KLICTTaVPWNASWSNKSLeQIWNNMTWmEWDREInNYTsLIHsLIEESQNQQEKNEQELL consensus KLICTT-VPWNASWSNKSL-QIWNNMTW-EWDREI-NYT-LIH-LIEESQNQQEKNEQELL
  • HIVJCENV 664 ELDKWASLWsWFdlsNWLWYIKIFIMIVaGLVGLRIVFAVLSIVNRVRQGYSPLSFQTHfP
  • HIVJCENV 725 aPRGPDRPdGIEgEGGERDRDRSvRLVdGfLALlWeDLRnLCLFSYHRLRDLLLIVTRIVE
  • HIVJCENV 786 LLGRRGWEALKYlWsLLQYWSQELKNSAVnLfNtTAIvVAEGTDRilEWQrlCRAIlHIP
  • HIVlaienvt 789 LLGRRGWEALKYwWnLLQYWSQELKNSAVsLlNaTAIaVAEGTDRvIEWQgaCRAIrHIP consensus LLGRRGWEALKY-W-LLQYWSQELKNSAV-L-N-TAI-VAEGTDR- IEWQ- -CRAI-HIP
  • Alignment score 657.00
  • Table 2C Alignment of HIV- 1 JC and HIV- 1 SF2 env Protein Sequences (SEQ ID NO:2 and SEQ ID NO:5, respectively). The consensus sequence corresponds to SEQ ID NO:8.
  • HIVsf2envt 1 MkVK gtrrnyqhlWrWGtlLLGMLMICSATEKLWVTVYYGVP KEaTTTLFCASDArAY consensus M-VKe W-WG- -LLGMLMICSATEKLWVTVYYGVPVWKE-TTTLFCASDA-AY
  • HIVJCENV 62 eeEVHNVWATHACVPTDPNPQEiVLaNVTEdFNMWKNeMVEQMhtDIISLWDeSLKPCVKL
  • HIVsf2envt 61 dtEVHNVWATHACVPTDPNPQEvVLgNVTEnFNMWKNnMVEQMqeDIISLWDqSLKPCVKL consensus --EVHNVWATHACVPTDPNPQE-VL-NVTE-ETiIMWKN-MVEQM--DIISLWD-SLKPCVKL
  • HIVJCENV 123 TPLCVTLNCTDLkneTkTNSSdaNsnsgeimgneeikncsfnvstgapgKvqkeyalfyal
  • HIVsf2envt 122 TPLCVTLNCTDLgkaTnTNSS NwkeeikgeikncsfnittsirdkiqKenalfrnldw consensus TPLCVTLNCTDL T-TNSSdaN K
  • HIVJCENV 245 CnNVSTVQCTHGIRPvVSTQLLLNGSvAEEEWlRSaNFsdNAKTIIVQLNhSVeltCTRP
  • HIVsf2envt 242 CtNVSTVQCTHGIRPiVSTQLLLNGSlAEEEWiRSdNFtnNAKTIIVQLNeSValnCTRP consensus C-NVSTVQCTHGIRP-VSTQLLLNGS-AEEEW-RS-NF--NAKTIIVQLN-SV-I-CTRP
  • HIVsf2envt 303 NnNtrKsIylgpG rafhtTgRiiGDirkAHCnisRaqWnNtLeQIvkKLREQFgNnktlv consensus N-N--K-I-I--Gy T-R--GD AHC R--W-N-L-QI--KLREQFgN 1-
  • HIVJCENV 366 FNrSSGGDlEIeMHSFNCgGElFYCNTTkLFNsTW netTEsngkgenltLPCRIrQfvNM
  • HIVsf2envt 363 FNqSSGGDpEIvMHSFNCrGEfFYCNTTqLFNnTWrlnhTEgtkgndtliLPCRIkQiiNM consensus FN-SSGGD-EI-MHSFNC-GE-FYCNTT-LFN-TWr TE I-LPCRI-Q--NM
  • HIVJCENV 426 WQkVGKAMYAPPsdGQIrCtSNITGLLLTRDGGhNdnNtnnEtFRPGrGDMRDNWRSELYK
  • HIVsf2envt 424 WQeVGKAMYAPPigGQIsCsSNITGLLLTRDGGtNvtN dtEvFRPGgGDMRDNWRSELYK consensus WQ-VGKAMYAPP--GQI-C-SNITGLLLTRDGG-N--Nt--E-FRPG-GDMRDNWRSELYK Table 2C. (Continued)
  • HIVJCENV 487 YKVIKIEPLGvAPTKAKRRWQREKRAVGmVGAMFLGFLGAAGSTMGAaSLTLTVQARQLL
  • HIVsf2envt 484 YKVIKIEPLGiAPTKAKRRWQREKRAVGiVGAMFLGFLGAAGSTMGAvSLTLTVQARQLL consensus YKVIKIEPLG-APTKAKRRWQREKRAVG-VGAMFLGFLGAAGSTMGA-SLTLTVQARQLL
  • HIVJCENV 548 SGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARVLAVERYLkDQQLLGIWGCSGKLICTT
  • HIVsf2envt 545 SGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARVLAVERYLrDQQLLGIWGCSGKLICTT consensus SGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARVLAVERYL-DQQLLGIWGCSGKLICTT
  • HIVJCENV 609 tVPWNASWSNKSLdqlWnNMTWmeWdREIaNYTNlIhhLiEESQNQQEKNEQELLELDKWA
  • HIVsf2envt 606 aVPWNASWSNKSLedlWdNMTWmqWeREIdNYTNtlytLlEESQNQQEKNEQELL ⁇ LDKWA consensus -VPWNASWSNKSL- - IW-NMTW- -W-REI-NYTN- I- -L-EESQNQEKNEQELLELDKWA
  • HIVJCENV 670 SLWsWFdlsNWLWYIKIFIMIVaGLVGLRIVFAVLSIVNRVRQGYSPLSFQThf aPRGPD
  • HIVsf2envt 667 SLWnWFsItNWLWYIKIFIMIVgGLVGLRIVFAVLSIVNRVRQGYSPLSFQTrlPvPRGPD consensus SLW-WF-I-NWLWYIKIFIMIV-GLVGLRIVFAVLSIVNRVRQGYSPLSFQT--P-PRGPD
  • HIVJCENV 731 RPDGIEgEGGERDRDRSVRLVDGFLALlWEDLRnLCLFSY RLRDLLLIVtRiVElLGrRG
  • HIVsf2envt 728 RPDGIEeEGGERDRDRSVRLVDGFLALiWEDLRsLCLFSYrRLRDLLLIaaRtV ⁇ iLGhRG consensus RPDGIE-EGGERDRDRSVRLVDGFI_AL-WEDLR-LCLFSY-RLRDLLLI--R-VE-LG-RG
  • HIVJCENV 792 WEALKYlWSLLQYWsQELKNSAVnlfNtTAIvVaEGTDRilEWQRlcRAILHIpRRIRQG
  • HIVsf2envt 789 WEALKYwWSLLQYWiQELKNSAVs lNaTAIaVtEGTDRvIEVaQRayRAILHIhRRIRQG consensus WEALKY-WSLLQYW-QELKNSAV N-TAI-V-EGTDR-IEV-QR- -RAILHI-RRIRQG
  • HIVsf2envt 850 LERILL consensus LER-LL Alignment score 614.00
  • HIV-1LAI gag T HIV-1LAI gag T
  • HIV-1LAI gag L 51 HIV-1LAI gag L 51
  • HIV-1JC16 env Q I AS KLREQ FNNKT A I I FNRS S GGDLE I EMHS FNCGGE L F YCNTTKL FNS T W NE 403
  • Trp Gly lie Met Leu Leu Gly Met Leu Met lie Cys Ser Ala Thr Glu 20 25 30
  • AAA AAT GAA ATG GTA GAA CAG ATG CAT ACT GAT ATA ATC AGT TTA TGG 336 Lys Asn Glu Met Val Glu Gin Met His Thr Asp lie lie Ser Leu Trp 100 105 110
  • Ala Pro Ala Gly Phe Ala lie Leu Lys Cys Asn Asp Lys Lys Phe Asn
  • Thr lie lie Val Gin Leu Asn His Ser Val Glu lie Thr Cys Thr Arg 290 295 300
  • Trp Met Glu Trp Glu Arg Glu He Asp Asn Tyr Thr Gly Leu He Tyr 625 630 635 640
  • Trp Asn Ala Ser Trp Ser Asn Lys Ser Leu Glu Asp He Trp Asp Asn 610 615 620
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • Trp Gly He Met Leu Leu Gly Met Leu Met Xaa Ser Ala Xaa Glu Xaa 20 25 30
  • Trp Xaa Glu Trp Xaa Arg Glu He Xaa Asn Tyr Thr Xaa Leu He Xaa 625 630 635 640
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO

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Abstract

An isolated human immunodeficiency virus (HIV) type 1, having the identifying characteristics of HIV isolate JC and assigned AIDS Reagent Program Catalog Number 3523, was isolated from an HIV-infected chimpanzee that developed AIDS. This chimpanzee represents the first known animal model of HIV-1 induced AIDS. The substantially full-length (infectious) nucleotide sequences of the HIV-1JC and of HIV-1NC molecular clones are provided herein. This HIV-1JC and HIV-1NC isolates are useful for the preparation of recombinant, attenuated and subunit vaccines, as well as for the preparation of challenge stocks. It is also used as a diagnostic reagent in screening for the presence of HIV-1 in biological samples.

Description

HUMAN IMMUNODEFICIENCY VIRUSES CAUSING AIDS IN A NONHUMAN PRIMATE
CROSS REFERENCE TO RELATED APPLICATIONS This application claims priority from United States Provisional Patent Application No. 60/050,548, filed June 23, 1997 and from United States Provisional Patent Application No. 60/057,606, filed September 4, 1997.
ACKNOWLEDGEMENT OF FEDERAL RESEARCH SUPPORT This invention was made, at least in part, with funding from Public Health Service Grant RR-00165 from the National Center for Research Resources. The United States government may have certain rights in this invention.
BACKGROUND OF THE INVENTION
This invention concerns unique isolates of human immunodeficiency virus type 1 (HIV-ljC) which are highly infectious in vivo and produce acquired immune deficiency syndrome (AIDS) in a nonhuman primate.
The human immunodeficiency viruses types 1 and 2 (HIV-1, HIV-2) are retroviruses which have been implicated as the causative agents of acquired immune deficiency syndrome (AIDS) (Barre-Sinoussi et al. [1983] Science 220:868-871). While skepticism about the exact cause of AIDS has arisen (Duesberg [1991] Proc. Natl. Acad. Sci. 88:1575-1579), a large amount of data has accumulated which supports HIV as the biologic agent of this disease. The most convincing evidence includes: mother to child transmission; transmission via blood transfusion; and transmission via contaminated blood products (Curran et al. [1984] N. Engl. J. Med. 310:69-75). The development of disease in an animal inoculated with HIV- 1 would provide confirmatory evidence for the etiology of AIDS. While AIDS-like disease has been recently demonstrated in HIV-2-infected baboons (Barnett et al. [1994] Science 266:642-646), to date, no species, other than humans, has developed AIDS following infection with HIV-1. Thus, the most appropriate animal model for HIV-1 infection currently does not exist. However, an effective alternative animal model has been developed using simian immunodeficiency virus (SIV) infection of macaques. This model has been extremely useful for investigating the pathogenesis of immunodeficiency virus infection and for the testing of vaccines and therapies (Gardner et al. [1994] in 77ze Retroviruses, vol. 3 [Levy, ed.] Plenum Press, New York).
Experimental infection of nonhuman primates with human immunodeficiency virus type 1 (HIV-1) has been described for gibbons (Lusso et al. [1988] J. Immunol. 141:2467- 2473), pig-tailed macaques (Barre-Sinoussi et al., supra), and chimpanzees (Alter et al. [1984] Science 226:549-552; Fultz et al. [1986] J. Virol. 58:116-124). However, the development of disease (AIDS) has not been documented in these animals or in any animal species infected with HIV-1. For many years, chimpanzees have been a major focus in the development of animal models for HIV-1 infection and therapy. The ability to consistently infect chimpanzees with several HIV-1 subtypes and to reisolate the virus over extended periods (Fultz et al., supra; Johnson et al. [1993] AIDS Res. Hum. Retroviruses 9:375-378) has made chimpanzees useful for testing vaccine candidates (Berman et al. [1990] Nature 345:622-625; Fultz et al. [1992] Science 256:1687-1690; Girard et al. [1995] J. Virol. 69:6239-6248). However, the lack of disease in HIV- 1 -infected chimpanzees has raised concern over the relevance of these vaccine studies.
The present invention provides HIV-1 isolates that are infectious to nonhuman primates such as chimpanzees and induce AIDS in inoculated nonhuman primates including, but not limited to, chimpanzees. Further, this invention provides for the first time a proper animal model for HIV-1 infection and for the development of AIDS. SUMMARY OF THE INVENTION
A first aspect of the present invention are the isolated human immunodeficiency virus type 1 (HIV-1) isolate having the identifying characteristics of HIV- 1 isolate JC (HIV-1JC) and assigned AIDS Reagent Program Catalog Number 3523 and the isolate having the identifying characteristics of HIV-1 isolate NC (HIV-1NC). These viruses are useful for the preparation of recombinant, attenuated and subunit vaccines, as well as for the preparation of challenge stocks. Each is also useful in screening for the presence of HIV in biological samples.
A second aspect of the invention is a biological sample, e.g., a biological fluid or tissue, containing an HIV-1 having the identifying characteristics of HIV-lJcor HIV-1NC. In a particular embodiment of the invention, primate blood specimens containing the HIV-1JC or the HIV-1NC of the present invention were used to cause HIV infection and to induce AIDS in a nonhuman primate, as specifically exemplified by the chimpanzee.
A third aspect of the invention are a biologically pure culture of host cells containing an HIV-1 having the identifying characteristics of HIV-lJcand a biologically pure culture of host cells containing an HIV-1 having the identifying characteristics of HIV-1NC.
A further aspect of the invention are isolated DNA molecules which produce infectious HIV-1 having the identifying characteristics of HIV-1JC or which encode an antigenic fragment thereof or having the identifying characteristics of HIV-1NC or encode an antigenic fragment thereof. The substantially full length sequence for infectious molecular clone of HIV-ljC is given in SEQ ID NO:l 1, and the corresponding sequence for the infectious molecular clone of HIV-1NC is given in SEQ ID NO:12.
An additional aspect of the invention provides isolated DNA encoding an HIV-1 envelope protein having the amino acid sequence of SEQ ID NO:2. In a particular embodiment, the DNA sequence encoding an HIV-1 envelope protein contains the nucleotide sequence of SEQ ID NO: 1. A further aspect of the invention are a composition comprising an antigenic preparation derived from the HIV-1JC of the invention and a composition comprising an antigenic preparation derived from the HIV-1NC of the present invention.
An additional aspect of the invention are pharmaceutical compositions comprising an immunogenic amount of an antigenic preparation derived from the HIV-1JC of the invention in a pharmaceutically acceptable carrier or HIV-1NC in a pharmaceutically acceptable carrier.
A further aspect of the present invention is a kit for detecting the presence of HIV- 1 antibodies, comprising an antigenic preparation derived from the foregoing HIV-1JC or from HIV-1NC as described herein.
Another aspect of the invention is a method of inducing in a subject antibodies to the HIV-1JC or to HIV-1NC of the invention, comprising the step of administering to the subject an immunogenic amount of an antigenic preparation derived from the HIV-lJcor from the HIV- INC. The induced antibodies can be harvested and find use, for example, as hybridization probes in methods for HIV scanning.
A further aspect of the invention is a method of immunizing a subject against infection by HIV, comprising the step of administering to the subject an immunogenic amount of an antigenic preparation derived from the HIV-1JC or derived from HIV-1NC of the present invention.
It is another aspect of the invention to provide a method for inducing acquired immune deficiency syndrome (AIDS) in a nonhuman primate, comprising the step of administering to said primate an effective amount of an antigenic preparation derived from the HIV-ljC and/or HIV-1NC of the invention such that said primate develops AIDS. In a specific embodiment of the invention, HIV-1JC induced AIDS in chimpanzees, e.g., in a chimpanzee referred to as C499. In a different embodiment of the invention, HIV-1JC contained in a biological sample from a chimpanzee having AIDS (C499) induces AIDS in a second chimpanzee and in a different primate species. It is another aspect of the invention to provide a method for inducing acquired immune deficiency syndrome (AIDS) in a nonhuman primate, comprising the step of administering to said primate an effective amount of an antigenic preparation derived from the HIV-1NC of the invention such that said primate develops AIDS. In a specific embodiment of the invention, HIV-1NC induced AIDS in chimpanzees, e.g., in a chimpanzee referred to as C534. In a different embodiment of the invention, HIV-1NC contained in a biological sample from a chimpanzee having AIDS (C455) induces AIDS in a second chimpanzee and in a different primate species.
A further aspect of the invention provides a biological fluid or tissue sample, obtained from a chimpanzee having HIV-lJC-induced AIDS, containing an antigenic HIV fragment for inducing AIDS in a nonhuman primate.
A further aspect of the invention provides a biological fluid or tissue sample, obtained from a chimpanzee having HIV-lNC-induced AIDS, containing an antigenic HIV fragment for inducing AIDS in a nonhuman primate.
It is yet another aspect of the invention to provide a method for inducing AIDS in a nonhuman primate, comprising the step of administering to said primate an effective amount of a biological fluid or tissue sample, obtained from a primate having HIV-lJC-induced or HIV-lNC-induced AIDS, containing an antigenic HIV fragment for inducing AIDS in a nonhuman primate.
The present invention further provides various vaccine formulations containing active immunogenic agents derived from the foregoing HIV-1JC, DNA encoding the HIV-1JC, and DNA encoding antigenic fragments of the HIV- 1JC from the foregoing HIV-1NC. DNA encoding the HIV-1NC_ and DNA encoding antigenic fragments of the HIV-1NC- An antigenic fragment contains one or more epitopes which bind antibodies directed to the HIV-1JC and/or HT -INC of the invention. BRIEF DESCRIPTION OF THE FIGURES
Fig. 1 shows the CD4+ -cell decline and plasma virus loads in chimpanzee C455 following transfusion with blood from C499. Immediately before and at various times after transfusion, blood was collected from C455 for in vitro analyses of CD4+ -cell levels and plasma virus loads. Absolute peripheral CD4+ cells in C455 showed a dramatic decrease by 2 weeks post-transfusion. This rapid decline continued and, by 14 weeks after transfusion, the number of CD4+ cells decreased to 10/μl. These low cell numbers have been maintained to date (42 weeks post-transfusion). Plasma HIV-1 RNA loads in C455 showed high levels of virus present by two weeks after transfusion. Results obtained at the 4-week point suggest very high levels of virus replication in C455. However, HIV-1 levels appeared to be somewhat controlled by 5 to 9 weeks post-transfusion. The cutoff level of 104 equivalents/ml ( — ) is the lower limit of the assay.
Fig. 2 illustrates CD4+ -cell decline in chimpanzee C534 following transfusion with blood from C455. Immediately before and at various times after transfusion, blood was collected from C534 for in vitro analyses of CD4+ -cell levels. Absolute peripheral CD4+ cells in C534 showed a dramatic decrease by 20 days post-transfusion.
Figure 3 illustrates the strategy used in PCR amplification of subgenomic fragments from FIIV-1 and the location and orientation of primers on the viral genome are shown. All the primers were designed from the HIV-1LAI nucleotide sequence and their coordinates are described below. PCR amplified fragments were cloned in TA vectors and the corresponding name designations for the recombinant plasmids are shown in bracket. The 5'-LTR- containing Apa I fragment amplified from HIV-1JC PBMC genomic DNA was subcloned in pJC to generate plasmid pHIV-lJCI6 while the Apal-Ncol fragment containing the 5'-LTR region amplified from HIV-1NC PBMC genomic DNA was subcloned in pHIV-lJC16 to generate pHIV-lNCJC chimeric plasmid. Plasmid pHIV-lNC was constructed by subcloning the e«v-containing Ncol-Xhol fragment amplified from HIV-1NC genomic DNA to pHIV-lNcjC chimeric plasmid. All recombinant plasmids (pHIV-lJcl6, pHIV-lNCJC, and pHIV-lNC7) lacked 55 bp at the 5' end of the genome (U3 region) and all of U5 region in the 3' LTR region. Figures 4A-4F show replication of cloned and uncloned HIV-1 isolates in con- A stimulated and unstimulated chimpanzee PBMC (cPBMC). Stimulated cPBMC infected with (Fig. 4A) HIV-a LAV- lb and SF2 parental strains and the highly cytopathic DH12 isolate, (Fig. 4B) JC (uncloned) and JC16 (cloned) isolates of HIV- 1, and (Fig. 4C) NC (uncloned) and NC7 (cloned) isolates of HIV. Unstimulated cPBMC infected with: (Fig. 4D) LAV- lb, SF2, and DH12; (Fig. 4E) JC, JC16; and (Fig. 4F) NC, NC7. Chimpanzee PBMC (1.1 x 107) in T-25 cm2 were infected with 20 ng of either HIV-1NC (uncloned), HIV-1SF2, HIV-lLAV.lb, or HIV-1DH12 virus and incubated for a total of 17 days at 37°C. Supernatant aliquots were made on 3, 7, 10, 14, and 17 days post infection. Reverse transcriptase (RT) assays were performed as outlined hereinbelow.
Figure 5 shows replication of HIV- 1 isolates in chimpanzee monocyte-derived macrophages (MDM). Purified PBMC (6 x lOVwell) were used to obtain MDM. Ten ng of each of virus HIV-1JC16 (molecular clone), HIV-1JC (uncloned), HIV-1NC (molecular clone), HIV-INC (uncloned), HIV-1SF2, HIV-lLAV.lb, and HIV-1DH]2) was used for infection and on days 7 and 14 post infection supernatants were harvested. The mount of virus in the supernatants was determined using the p24 HIV-1 antigen capture ELISA (Coulter). Supernatants from control uninfected cultures are represented.
DETAILED DESCRIPTION OF THE INVENTION
The following definitions are given in order to provide clarity as to the intent or scope of their usage in the specification and claims.
The term HIV-1 isolate JC or HIV-1JC or HIVJC or HIV-1 having the identifying characteristics of HIV-1JC as used herein refers to the particular HIV-1 isolated from a chimpanzee (C499) that developed AIDS 10 years after infection with HIV-1 (Novembre et al. [1997] J. Virol. 71:4086-4091). The DNA sequence of the cloned HIV-1JC is given in SEQ ID NO:l l. The term HIV-1 isolate NC or HIV-1NC or HIVNC or HIV-1 having the identifying characteristics of HIV-1NC as used herein refers to the particular HIV-1 isolated from a chimpanzee (C455) that developed AIDS after infection with HIV-1JC (Novembre et al. [1997] J. Virol. 71:4086-4091). The DNA sequence of the cloned HIV-1NC is given in SEQ ID NO: 12.
The term antigenic preparation of HIV-1JC or antigenic fragment of HIV-1JC as used herein refers to the whole viral particle of the HIN-1JC or to a fragment thereof, wherein such fragment encodes at least one epitope or antigenic determinant. The term antigenic preparation of HIV-1ΝC or antigenic fragment of HIV-1NC as used herein refers to the whole viral particle of the HIV-1JC or to a fragment thereof, wherein such fragment encodes at least one epitope or antigenic determinant.
The term animal or subject as used herein refers to a mammal and, more frequently, to a primate.
The term effective amount as used herein refers to the quantity of active ingredient necessary to effect in an animal a change in a specific biochemical or immunological parameter. For example, in a particular embodiment of this disclosure, an effective amount refers to the amount of HIV- 1 administered to a subject such that viral infection and AIDS developed.
The term antigenic amount as used herein refers to the quantity of active antigen necessary to effect an interaction with corresponding antibodies.
The term immunogenic amount as used herein refers to the quantity of active antigen necessary to stimulate the immune system in response to a specific antigen.
The term labeled is used herein to refer to the conjugating or covalent bonding of any suitable detectable group, including enzymes (e.g., horseradish peroxidase, β-glucuronidase, alkaline phosphatase, and β-D-galactosidase), fluorescent labels (e.g., fluorescein, luciferase), and radiolabels (e.g., 14C, 1311, 3H, 32P, and 35S) to the compound being labeled. Techniques for labeling various compounds, including proteins, peptides and antibodies are well known. See, e.g., Morrison, Methods in Enzymology 32B, 103 (1974); Syvanen et al., J. Biol. Chem. 284, 3762 [1973]; Bolton and Hunter, Biochem. J. 133, 529 [1973].
The viruses of the present invention resulted from the development of AIDS in a first chimpanzee infected with HIV-1 for over 10 years and by the rapid development of immunosuppression in a second chimpanzee transfused with blood from the first chimpanzee. To investigate the ability of HIV- 1 to induce AIDS in non-human primates, a cohort of 12 chimpanzees was inoculated with several strains of HIV- 1 at the Yerkes Center in the mid- 1980s. A member of this cohort, C499, was described as part of a previously reported superinfection study (Fultz et al. [1987] J. Virol. 61:4026-4029) and was inoculated on three separate occasions with three different HIV-1 isolates: HIV-1SF2 in 1985, HIV-1LA[ in 1986, and HIV-INDK m 1987. The first inoculation resulted in infection, as determined by positive virus isolation and persistent HIV- 1 -specific antibody response. Clinically, the animal remained healthy except for the development of thrombocytopenia and lymphopenia in 1988, which resolved without treatment (Fultz et al. [1991] J. Infect. Dis. 163:441-447). (All HIV- infected chimpanzees are maintained in Biosafety level 3 isolation facilities.)
In 1993, after the resumption of yearly monitoring (monitoring of all HIV- 1 -infected chimpanzees at the Yerkes Center was suspended from March 1990 to May 1993), a decrease in the levels of platelets and CD4+ cells in C499 was observed (24,000 and 390/μl, respectively) (Table 1). Thrombocytopenia and CD4+-cell lymphopenia were persistent in this animal from this point onward. In addition, C499 displayed other significant clinical signs of disease. Beginning in March 1995, C499 developed chronic, intermittent diarrhea for which no enteric pathogens were identified and which was not resolved with antibiotic treatments. In September 1995, this animal developed acute fulminant diarrhea which was associated with large numbers of Blastocystis hominis and Balantidium coli. At this time, CD4+ cells decreased to extremely low levels (minimum of 10/μl:2% of total T cells) (Table 1), indicative of severe immunosuppression. Similarly, declines were observed in the levels of total lymphocytes and CD8+ cells (Table 1). Treatment with fluid replacement and antimicrobial and antiprotozoal therapy (doxycycline, ceftriaxone, enrofloxacin, gentamicin, and albendazole) resulted in the resolution of acute diarrhea within five days.
Because this was the first chimpanzee to develop AIDS, treatment with antiretroviral therapies was not administered in order to more fully characterize virological, immunological, and pathological parameters in this animal. As the acute diarrhea was resolved with treatment, CD4+-cell levels rose to a maximum of 180/μl (4% of total T cells) but declined again. Subsequently, chronic, intermittent diarrhea resumed and continued unresolved. During this period, C499 exhibited no lymphadenopathy or wasting. However, beginning in the latter part of 1995 and extending into 1996, C499 developed progressive nonregenerative anemia (hematocrit levels of 37.5% in December 1995 and 27.2% in January 1996, with respective hemoglobin values of 12.1 and 8.4 g/dl; reticulocyte counts were 0.0% since November 1995). Due to progressive hematologic abnormalities, chronic diarrhea, and continued immunosuppression, the animal was euthanized in February 1996.
Concomitant with the decrease in CD4+ cells was an increase in HIV-1 loads in plasma (Table 1). The increase in the level of the virus was detected in plasma samples dating from May 1993, when the CD4+-cell decline was first noted, but not before the suspension of monitoring in 1990. These levels are significantly higher than those for five other chimpanzees at the Yerkes Center which received cell-free or cell-associated HIV-1LAI or HIV-1SF2 inoculations (all have undetectable plasma viral RNA levels and are not immunosuppressed). These results suggest that pathogenic effects began to occur sometime between 1990, when CD4+-cell counts were at normal levels and viral loads were undetectable, and 1993, when alterations in the number of CD4+ cells and significant virus loads were present.
The ability to isolate virus from C499 varied since C499's first exposure to HIV-1. In general, early after inoculation, HIV was easily isolated from the peripheral blood mononuclear cells (PBMC) of this animal; however, after several months, virus could no longer be isolated. From August 1988 until the suspension of monitoring in 1990, the virus was consistently isolated from C499 on a monthly basis. Subsequently, after the resumption of monitoring in mid- 1993, HIV-1 continued to be easily isolated from this animal. Quantitative titration of PBMC viral load following the development of acute diarrhea in C499 in September 1995 until the time of euthanasia revealed that 104 to 105 PBMC was consistently required for virus isolation. The immune response of C499 to HIV-1 infection was very strong up to the time of euthanasia. HIV-1 antibody endpoint titers (HIV-1 whole- virus enzyme-linked immunosorbent assay, Genetic Systems, Redmond, WA) ranged from 51,200 to 204,800 since 1993. Because of the deteriorating condition of this animal and because of the severe decline in CD4+ cells, it was hypothesized that the HIV-1 present in this animal had evolved to become more cytopathic for chimpanzee CD4+ cells. Cocultivation of PBMC derived from C499 (obtained at the time of acute diarrhea) with uninfected chimpanzee PBMC (cPBMC) resulted in the isolation of a virus (HIV-1JC) which induced syncytium formation in chimpanzee cells (Fig. 1 A-D). This characteristic has been previously described for only three HIV-1 isolates (Ghosh et al. [1993] Virology 194:858- 864; Schuitemaker et al. [1993] J. Infect. Dis. 168:1140-1147; Shibata et al. [1995] J. Virol. 69:4453-4462), none of which were used for inoculation of C499.
The virus strain derived from C499 (HIV-1JC) and other HIV-1 isolates (HIV-1LAI and HIV-1SF2) were tested for the ability to induce syncytium formation in cPBMC. Virus stocks were prepared in cPBMC (HIV-1JC) or in human PBMC (HIV-1LAI and HIV-1SF2). Cells were incubated with virus overnight and were then washed. Cultures were examined daily for evidence of cytopathic effects. cPBMC four days after infection with an HIV-1 isolate (HIV- ljc) from C499 showed beginnings of syncytium formation and separated syncytia. cPBMC four days after infection with HIV-1LAI lacked of syncytium formation, and normal cell clusters were present. cPBMC four days after infection with HIV-1SF2 also showed no syncytium formation. The only virus to induce significant cytopathic effects in cPBMC was HIV-ljc- All cultures were examined for 14 days following infection. All cultures, regardless of the virus used, became positive for virus replication by seven days postinfection. Thus, it is concluded that genetic changes which confer the ability to induce syncytium formation occurred in the virus present in C499. To confirm that the virus present in C499 was different from the viruses used for inoculation, DNA prepared from HIV-lJC-infected cPBMC was used as a template in typical PCR assays with primers (forward, no. 384:
5'CCCTTCGAAGAGGATATAATCAGTTTATGGGATCAAAGC3* [SEQ ID NO:9]; reverse, no. 383: S'CCCTTCGAACTCTTCTTCTGCTAGACTGCCATπ' [SEQ ID NO: 10]) designed to amplify a 507-bp fragment of the env gene containing the VI and V2 regions. Genetic analysis of 16 HIV-1JC VI -V2 clones obtained by ligation of the amplification products with the vector pGEM7ZF (Promega, Madison, WI) showed amino acid homologies of 80 to 84% with FflV-lLAI, 73 to 80% with HIV-1SF2, and 63- to 68% with HIV-I^K. Thus, there appears to be considerable divergence between the virus present in C499 at the time of acute disease and the viruses used to inoculate this animal. This divergence is further illustrated in Fig. Tables 3A-3C, which show amino acid alignments of five HIV-1JC clones, HIV-INDK. HIV-1LAI, and HIV-1SF2. Comparative analyses between the 16 HIV-1JC clones showed that amino acid homologies ranged from 81 to 96%, with no clones being identical. These results suggest that the virus population in C499 consisted of a large quasispecies. Furthermore, the data, when combined with the in vitro analyses described above, indicate that the virus adapted after years of replication and mutation, becoming more pathogenic for the chimpanzee. While no evidence of recombination is evident from analyses performed in this small area, the possibility of recombination cannot be ruled out for other portions of the HIV-ljC genome.
Tables 2A-2C illustrate amino acid alignment of V1-V2 clones obtained from HIV- lJC-infected cPBMC and prototypes HIV-1LAI and HIV-1SF2, and HIV-IND - env clones of HIV- ljC encompassing the VI -V2 region were sequenced by the dideoxy chain termination method (Sequenase; Amersham Life Science, Arlington Heights, IL). With the Intelligenetics Suite of programs (Intelligenetics, Beaverton, OR), sequences of HIV-1JC env fragments were used to derive corresponding amino acid sequences. Deduced amino acid sequences from five of these clones (HIVJC10, HIVJC17, HIVJC45, HIVJC48, and HIVJC55) were then aligned with the corresponding region in HIV-1LAI, HIV-1SF2, and HIV-INDK isolates. Amino acid sequences of HIV-1LAI ,HIV-1SF2 and HIV-I^K were obtained from the Human Retroviruses and AIDS Database. To investigate additional pathogenic effects of HIV- 1 infection on C499, tissue samples obtained by biopsy during the acute diarrheal stage (September 1995) and at necropsy were subjected to histopathological analyses. First, examination of a peripheral lymph node (obtained from C499 in September 1995) revealed marked lymphoid depletion within the cortical area, with a few follicles remaining, as compared with a lymph node from an age-matched, uninfected chimpanzee, which lacked follicular development and had a very cellular cortex. To detect virus expression in tissues, in situ hybridization experiments with digoxigenin-labeled riboprobes encompassing the entire HIV-1 genome (derived from the HIV-1 BH10 molecular clone (Hahn et al. [1984] Nature 312:166-169) were used to probe formalin-fixed lymph node sections. Detection of bound probes was performed with a sheep anti-digoxigenin-alkaline phosphatase-labeled Fab monoclonal antibody and with Nitro Blue Tetrazolium-5-bromo-4-chloro-3-indolylphosphate toluidinium as the substrate chromogen, according to previously described methods (Hirsch et al. [1995] J. Virol. 69:955-967). In situ hybridization studies of C499's lymph node demonstrated the presence of HIV- 1 RNA in follicular trapping patterns, with few positively staining cells. These findings are similar to changes observed in HIV- 1 -infected persons developing AIDS (Pantaleo et al. [1995] N. Engl. J. Med. 332:209-216). Control samples, which included sense riboprobes and lymph nodes from uninfected chimpanzees, did not show any staining. Additional lymph nodes obtained from C499 at necropsy showed a similar depleted pattern. However, a few lymph nodes were not as depleted but did contain multinucleated giant cells which stained positive for the HIV-1 p24 antigen. These giant cells are often found in the lymph nodes of simian immunodeficiency virus (SΙV)-infected macaques and are occasionally found in the lymph nodes in HIV- 1 -infected persons.
Finally, histopathologic analysis of intestinal tissue from C499 revealed pathologic changes in the ileum, with significant blunting of the villi and intense infiltration of mononuclear cells and plasma cells. Examination of the intestinal mucosa at higher magnification revealed extensive infection with Cryptosporidium, which was present throughout the small intestine, lining the apical surfaces of intestinal epithelial cells. This organism is an AIDS-defϊning opportunistic pathogen (Centers for Disease Control and Prevention. 1992. 1993 revised classification system for HIV infection and expanded surveillance case definition for AIDS among adolescents and adults. Morbid. Mortal. Weekly Rep. 41:1-19) and probably accounted for the chronic intermittent diarrhea and intestinal pathology in C499.
Most tissues obtained from C499 following euthanasia appeared grossly normal, with no lymphadenopathy or splenomegaly. However, the spleen showed moderate congestion and lacked follicle development. Bone marrow specimens obtained at euthanasia showed some functional impairment in CFU granulocyte macrophage and CFU formation, although levels of CD4+ cells were not altered (Villinger et al. [1997] J. Med. Primatol 26:181-189. Virus was isolated from all lymphoid organs including inguinal, axillary, and mesenteric lymph nodes as well as from the spleen and thymus. Virus was also isolated from the kidney and liver but not from the brain or cerebrospinal fluid.
At the time of acute diarrhea in C499, a blood transfusion was performed to determine the effects of passage of virus from this animal to an uninfected chimpanzee. Forty milliliters of blood obtained by venipuncture from C499 was immediately transfused intravenously into C455. This chimpanzee, which was bred in captivity, was seropositive for Epstein-Barr virus and cytomegalovirus and was seronegative and PCR negative for HIV prior to the transfusion. Results of titration analysis of PBMC and plasma from C499 show that in the 40 ml of blood, 1 x 104 50%) tissue culture infective doses (TCID50) of virus was in PBMC and 2 x 104 TCID50 of virus was in plasma. Thus, C455 received a total of 3 x 104 TCID50 of virus. Analysis of peripheral CD4+ cell levels in C455 revealed a precipitous decline beginning by 2 weeks after transfusion, when absolute numbers of CD4+ cells decreased from 1,240 to 320 cells/μl (Fig. 1). This decline continued, reaching a minimum value of 10 cells/μl (1% of total T cells) by 14 weeks post-transfusion. Since this time, the total percentage of T cells that the CD4+ population encompasses has remained constant at 1%, with only a slight rise in the absolute number of CD4+ cells (20 cells/μl at the latest time point). Levels of peripheral CD8+ cells showed an initial decline from 1,590 to 880 cells/μl in the first two weeks after transfusion. However, these levels quickly rebounded to 2,010 CD8+ cells/μl by 8 weeks after transfusion. Since this time, the number of CD8+ cells in circulation has been maintained between 550 and 4,320/μl, with the level in most recent sample being 840 cells/μl. Because CD4+-cell levels declined so rapidly, the level of virus present in C455 was investigated by quantitation of plasma HIV-1 RNA with the Chiron B-DNA assay (Fig. 1). Two weeks after transf sion, plasma viral loads were 2 x 107 RNA equivalents/ml and reached maximum levels by 4 weeks post-transfusion (6.2 x 107 RNA equivalents/ml). Following a decline (which corresponded with the development of anti-HIV-1 specific antibody), plasma HIV-1 levels have been maintained at ~ 1.1 x 105 RNA equivalents/ml. Virus has been easily isolated from C455 at all times post-transfusion. Quantitative coculture of PBMC from C455 has shown that early after transfusion, virus could be isolated from as few as 102 PBMC, while at more recent time points, up to 105 to 106 cells was required for virus isolation. In vitro analysis of virus isolated from this animal at several time points has shown that the ability to induce syncytium formation in cPBMC has been retained, further implicating this virus in the pathogenesis of CD4+-cell decline. Antibody responses to HIV-1 have been moderate in C455. Enzyme-linked immunosorbent assay titers have been maintained between 1,600 and 6,400 since four weeks post-transfusion. Clinically, C455 has appeared normal, except for episodic incidences of a rash on the chest and in the scrotum area. In addition, the animal has experienced no weight loss, lymphadenopathy, or anemia.
This invention presents an HIV-1 infected chimpanzee that developed AIDS as defined by the Centers for Disease Control and Prevention classification system (CDCP 1992, supra). Progression of clinical disease (anemia, thrombocytopenia, and chronic diarrhea) in this animal was associated with several key findings, including the following: (i) the presence of a virus which is cytopathic for cPBMC in vitro and in vivo and is genetically distinct from those used for inoculation, (ii) an increase in viral load; (iii) CD4+-cell depletion, (iv) lymph node depletion, and (v) the presence of Cryptosporidium organisms in the intestine. It appears that the critical change(s) associated with clinical progression may have developed during the period in which C499 was not monitored (mid- 1990 through mid- 1993). The precipitous CD4+-cell decline concomitant with high viral loads displayed in C455, transfused with blood from C499, suggest that the HIV present in C499 has evolved to become more pathogenic for chimpanzees. The increased pathogenicity of a lentivirus after passage into a new host has been previously observed with the adaptation of SIV from sooty mangabeys to pig-tailed macaques, resulting in the development of the acutely lethal strain SIVsmmPBj l4 (Fultz et al. [1989] AIDS Res. Hum. Retroviruses 5:397-409).
Forty milliliters of blood obtained by venipuncture from C455 was immediately transfused intravenously into C534. This chimpanzee, which was bred in captivity, was seropositive for Epstein-Barr virus and cytomegalovirus and was seronegative and PCR negative for HIV prior to the transfusion. Results of titration analysis of PBMC and plasma from C455 show that in the 40 ml of blood, 1 x 104 50% tissue culture infective doses (TCID50) of virus was in PBMC and 2 x 104 TCID50 of virus was in plasma. Thus, C534 received a total of 3 x 104 TCID50 of virus. Analysis of peripheral CD4+ cell levels in C534 revealed a precipitous decline beginning by one week after transfusion, when absolute numbers of CD4+ cells decreased from approximately 1,450 to 700 cells/μl (Fig. 2). The total percentage of T cells that the CD4+ population encompasses has remained constant at approximately 1%, with only a slight rise in the absolute number of CD4+ cells (20 cells/μl at the latest time point). Levels of peripheral CD8+ cells showed an initial decline in the first two weeks after transfusion. However, these levels quickly rebounded after transfusion.
Table 3 shows the antibody response in chimpanzee C534 following transfusion with blood from C455. Immediately before and at various times after transfusion, blood was collected from C534 for in vitro analyses of antibody response. Plasma HIV-1 RNA loads in C534 showed high levels of virus present by two weeks after transfusion. Results obtained at the 3-week point indicate very high levels of virus replication in C455. The cutoff level of 104 equivalents/ml is the lower limit of the assay.
Because CD4+-cell levels declined so rapidly, the level of virus present in C534 was investigated by quantitation of plasma HIV-1 antibody response (Table 4). One week after transfusion, the plasma antibody titer was still 0. At two weeks after transfusion, the antibody titer rose to 400 and remained at 1600 at three and four weeks post-transfusion. Virus has been easily isolated from C455 at all times after transfusion. Quantitative coculture of PBMC from C534 has shown that early after transfusion, virus could be isolated from as few as 102 PBMC, while at more recent time points, up to 105 to 106 cells was required for virus isolation. In vitro analysis of virus isolated from this animal at several time points has shown that the ability to induce syncytium formation in cPBMC has been retained, further implicating this virus in the pathogenesis of CD4+-cell decline. Antibody responses to HIV-1 have been moderate in C534. Enzyme-linked immunosorbent assay titers have been maintained between 1,600 and 6,400 since four weeks post-transfusion. Clinically, C534 has appeared normal, except for episodic incidences of a rash on the chest and in the scrotum area. In addition, the animal has experienced no weight loss, lymphadenopathy, or anemia (normal hematocrit levels).
This invention presents HIV-1 isolates NC and JC which infected chimpanzees such that the HIV-1 infected chimpanzees develop AIDS as defined by the Centers for Disease Control and Prevention classification system (CDCP 1992, supra). Progression of clinical disease (anemia, thrombocytopenia, and chronic diarrhea) in this animal is associated with several key findings, including the following: (i) the presence of a virus which is cytopathic for cPBMC in vitro and in vivo and is genetically distinct from that used for inoculation, (ii) an increase in viral load; (iii) CD4+-cell depletion, (iv) lymph node depletion, and (v) the presence of Cryptosporidium organisms in the intestine. It is expected that the critical change(s) associated with clinical progression develop during the period spanning approximately 3 to 5 years after HIV-1 infection. The precipitous CD4+-cell decline concomitant with high viral loads displayed in C534, transfused with blood from C455, indicates that the HIV present in C455 remains pathogenic for chimpanzees.
In the past, relevance of the HIV- 1 -infected chimpanzee as a model for vaccine evaluation was questioned due to lack of disease development. The lack of an animal model which supports pathogenic HIV-1 infection has been a continuing problem for vaccine development. Although the time of progression to disease (>10 years), the currently limited numbers of animals available for use, and the overall high costs associated with working with chimpanzees are deterrents to their widespread use in AIDS research, the potential usefulness of this model cannot be disregarded. The development of AIDS in C499, the fact that additional HIV-infected chimpanzees have depressed CD4+-cell counts (<500/μl) and thrombocytopenia, and the rapid progression of the CD4+-cell decline in C455 support the role that this animal model provides in AIDS-related studies. The adaptation of HIV- 1 from long-term chimpanzee infection to a pathogenic form provides a critical link for the adaptation of HIV- 1 to growth in more readily available nonhuman primate species. The instant invention further contemplates the growth of HIV-1JC isolated from C499 and/or HIV- INC fro C 55 in chimpanzees as well as in pig-tailed or rhesus macaques. In addition, the present invention provides for continued biological and genetic characterization of HIV-1JC and HIV-INC with further key insights into the pathogenesis of HIV- 1 infection in humans and chimpanzees, for example, for the development of drugs and vaccines for the treatment and prevention of AIDS.
As described above, chimpanzee C499 was initially infected with HIV-1SF2 in 1985 [Fultz et al. (1986) J. Virol. 58, 116-124] and later inoculated with HIV-lLAV.lb and HIV-INDK in 1986 and 1987 respectively. Superinfection with HIV-lLAV.]b (but not HIV-I^ ) was demonstrated by restriction enzyme analysis of PBMC genomic DNA. At the time of disease development in C499, a virus isolate, termed HIV-1JC, was obtained by co-culture of C499 PBMC with normal cPBMC. At that time, sequence analysis of the VI -V2 region of env suggested that HIV-1JC was most closely related to HIV-1LAV. Also at the time of disease development, blood from C499 was transfused into an uninfected chimpanzee (C455) which resulted in a dramatic decline of CD4+ cells by 2 weeks post transfusion. The depressed CD4+ cell count is still maintained to date in this animal. One month post transfusion 50 μl of plasma from C455 was used for in vitro infection of normal chimpanzee PBMC and the resultant virus was designated HIV-1NC-
To perform a more thorough analysis of the genetic makeup of the HIV-1JC and HIV- INC viruses, we constructed substantially full-length, infectious molecular clones as described in the Examples and in Figure 3. Both clones lacked 55 nucleotides at the 5' end (5' LTR, U3 region) and all of the U5 region in the 3' LTR. Several clones (representing both HIV-1JC and HIV-1NC) which appeared to be the correct size were tested for biological activity by transfection of CEMxl74 cells. Supernatants from transfected cells were used in RT assays to monitor virus production. Two clones, one from each group (HIV-1JC [JC16] and HIV-1NC [NC7]), were positive by RT and also showed massive syncytia formation (2 to 3 days post transfection), similar to that observed with uncloned virus. To prepare stock viruses for use in in vitro assays, 293 cells were transfected with molecularly cloned DNAs followed by amplification with cPBMC as outlined hereinbelow.
The complete nucleotide sequences of JC16 and NC7 were determined as described in Example 1, and the sequences are given in SEQ ID NO:l 1 and SEQ ID NO: 12, respectively. The genomes were determined to be 9193 nt (JC16) and 9196 nt (NC7) in length and contained open reading (ORFs) for all HIV- 1 -specific structural, regulatory and accessory genes. Alignment of JC16 and NC7 DNA sequences revealed that the two genomes were very similar, but contained a number of nucleotide changes spread throughout the genome.
compared with the parental inoculating viruses, and with gaps introduced to optimize alignment and treated as mismatches, the LTR sequences of JC16 and NC7 had percent nucleotide identities of 92.1% (LAV), 93.9% SF2), and 89.1% (NDK)-lower than that The most divergent region between NC7 and JC16 was the V5 region of the env gene. JC16 contained a 6 bp deletion in the gag gene relative to NC7 while NC7 had 3 bp deletion in the env gene region relative to JC16. In the LTR region there was a 98.7% nucleotide identity between JC16 and NC7 with all the changes being localized to the U5 region (Table 4).
Comparative analyses were performed between JC16, NC7, and the parental inoculating viruses, SF2, LAV, and NDK (The sequence of LAV- lb has not yet been determined). When observed upon direct comparison of JC16 and NC7 (Table 3). Most of the host/virus transcription binding factor sequences (sites for AP-1, NF-AT, NF-kB and Sp- 1) and the TAR CORE and the Lys-tRNA sites were conserved (or had single point mutations) between the parental (SF2 and LAV strains) and progeny viruses. However, there were 3 point mutations unique to JC16 and NC7 at the NRF/NRE binding site.
The deduced amino acid sequences for all proteins of HIV-1JC and HIV-1NC were generated using the Intelligenetics suite of programs. Using the Lasergene program (DNASTAR Inc., Madison, WI), multiple alignments of all proteins were constructed to examine similarities between HIV-1NC, HIV-1JC and the parental viruses (Table 4). Based upon percent homology calculations, Gag, Pol, Vif, Tat, Rev, Env, and Vpu of JC16 and NC7 were most closely related to LAV, with vpr and Nef being most closely related to SF2. In no case was it apparent that the NDK isolate was the origin of a protein sequence. While most changes involved amino acid point mutations, several proteins of JC16 and NC7 contained amino acid insertions or deletions relative to the parental inoculating strains. A closer analysis of amino acid alignments revealed that the percent homologies could be misleading with regards to the origin of the protein. For example, in Tat, JC16 and NC7 were more homologous to LAV than to SF2 (based upon percent homologies). However, the JC16 and NC7 Tat proteins contained 15 amino acid deletions with respect to LAV~similar to that present in the SF2 isolate. Similar findings were observed in Gag and Pol.
Table 5 shows amino acid alignments of Gag, Nef, and Env proteins from JC16, NC7 and the inoculating viruses, LAV, SF2, and NDK. The deduced amino acid sequences for the Gag (a), Nef (b), and Env proteins of HIV-1 were determined using the Intelligenetics Suite of programs (Intelligenetics, Beaverton, OR) and the CLUSTAL method (DNASTAR Inc., Madison, WI) was used for the alignment of proteins. The virus isolates are shown by the designations at the beginning of every line as LAI (HIV-1LAV), JC16 (HIV-1JC16), NC7 (HIV- 1NC7), and SF2 (HIV-1SF2). Dashes (-) denote amino acid deletion while dots (.) denote amino acid identity. The numbers after the amino acid sequence on the right show the position of the right-most amino acid in the line. The functional domains on Gag (a) and Nef (b) are indicated. MBD (a) refers to membrane-binding domain while MTD (b) refers to membrane- targeting domain. The hypervariable regions in the Env glycoprotein (c) are shown as VI to V5 and the CD4 binding domain is shown above the sequence. Gpl20 (SU) is the outer surface membrane Env glycoprotein. The NDK Env, LAI Env, and SFS Env protein sequences are given in SEQ ID Nos: 3, 4 and 5, respectively. The JC16 and NC7 Env protein sequences are given in SEQ ID Nos: 2 and 23, respectively. There were 7 (JC16 and LAI sequences) and 5 (NC7 sequence) amino acid deletions in the matrix protein (pi 7) relative to SF2 isolate sequence. At the C-terminal end of Gag polyprotein, the progeny viruses and SF2 virus had 12 amino acid deletions in p6 protein relative to LAV virus sequence (Table 5). However, the capsid (p24) and nucleocapsid (p7) proteins were generally well conserved including the cysteine residues within the zinc-finger domains. Point mutations unique to JC16 and NC7 were present in pi 7, p24, p7 and p6 peptides. The Lck binding domain (proline-rich region) within Nef was well conserved with only one point mutation in LAI (Table 5). However, there were 4 amino acid insertions in the SF2 sequence relative to the other viruses at the N-terminal portion of Nef. Sequence analysis of eight other non- infectious clones (4 from JC and 4 from NC) confirmed observations made for the Gag and Nef deletions suggesting that these characteristics are a general property of the viruses obtained from C499 and C455.
The vast majority of point mutations, deletions, and insertions in these clones, relative to the parental viruses, were found in the env glycoprotein region (Tables 4 and 5). Alignment of the Env proteins revealed that all the cysteine residues resident in the protein were conserved between the viruses. There were a total of 30 predicted N-linked glycosylation sequences (Asn-X-Thr or Asn-X-Ser) for the parental strains compared to 27 (NC7) and 29 (JC16) for the progeny viruses. Most of the glycosylation sites reside in the SU portion of Env with HIV-1SF2 containing the highest number and HIV-1 isolate NC the least (25 for SF2, 24 for LAI and JC, and 23 for NC). Glycosylation of glycoproteins has been shown to influence the immune response toward virus infection. As expected, the gp 120 (SU) glycoprotein contained the highest number of mutations. The V1-V2, V3, V4, and V5 hypervariable regions contained 10, 8, 8, and 3 point mutations respectively specific to JC16 and NC7. The VI -V2 region of JC16 and NC7 also contained insertions relative to the other viruses, resulting in amino acid lengths of 72 (progeny viruses), 69 (LAV), 70 (SF2), and 61 (NDK). There were multiple amino acid deletions in the V4 regions and single amino acid insertions in V5 regions of JC16 and NC7 relative to the parental strains. Both the CD4- binding domain and the proteolytic cleavage site (REKR) at the SU/TM junction were perfectly conserved.
The V3 region was the most interesting of the hypervariable regions. While the parental strains contained only 9 basic amino acids (Arg, Lys, and His positively charged residues) JC16 and NC7 HIV-1 isolates had 12 basic and 2 negatively charged residues (Asp and Glu). This gave the progeny viruses a net positive charge of 10 in the entire V3 region and an overall positive charge of +1 (LAV) or +2 (SF2) compared to the parental strains. Eight of the 10 resultant positive charges for JC and NC isolates are located between residues 10 and 27 of V3 (Fig. 3) compared with 5 of 9 (LAV) and 4 of 9 (SF2) in the same region. At least within this region, JC and NC isolates seem to have a relatively high net positive charge of 4 (relative to SF2) and 3 (relative to LAV). Other researchers have shown that changes in basic amino acids in the middle portion of V3 loop (residues 10 to 27 in Table 5) can alter the syncytium-inducing properties and phenotype of the virus [Bhattacharyya et al. ( 996) AIDS Res. Hum. Retroviruses 12, 83-90; De Wolf et al. (1994) AIDS Res. Hum. Retroviruses 10, 1387-1400; Okada et al. (1994) AIDS Res. Hum. Retroviruses 10, 803-811].
To study the biological activities of the cloned and uncloned viruses derived from C499 and C455, we conducted in vitro replication studies. Figures 4A, 4B and 4C shows the results of replication studies in mitogen-stimulated cPBMC. Two of the three viruses used for inoculation of C499, SF2 and LAV- lb, were able to replicate in stimulated cPBMC, albeit with different kinetics (Fig. 4A). The SF2 isolate grew very slowly and to low titers in cPBMC. In contrast, the LAV- lb isolate grew very well and with rapid kinetics, with a high titer of virus already present by day 7 post infection. The SF2 isolate was unable to induce detectable syncytium formation in cPBMC. The LAV- lb isolate, under these conditions, induced very few syncytia, in contrast to previously reported results [Watanabe et al. (1991) J. Virol. 65, 3344-3348]. Included in these analyses was the DH12 isolate of HIV-1 [Shibata et al. (1995) J. Virol. 69, 4453-4462]. This primary isolate from a human has been shown to be highly cytopathic for cPBMC. While this virus quickly established infection in the stimulated cell population, it did not grow to high titers. The numerous syncytia formed infection with DH12 could account for the lack of growth observed. The uncloned and cloned viruses of JC (Fig. 4B) and NC (Fig. 4C) replicated to levels comparable to those of LAV- lb for the same period of time. The rates of replication for cloned and uncloned viruses were indistinguishable .
The ability of HIV- 1 virus isolates to replicate in unstimulated cPBMC was similarly evaluated. Results of this assay (Fig. 4D, 4E and 4F) showed that only the NC (cloned and uncloned) and the LAV- lb isolates of HIV- 1 were capable of significant replication in unstimulated cPBMC. Replication rates for these viruses were less than 10% those observed in stimulated cPBMC. Additionally, the kinetics of virus production in unstimulated cells was much slower than that observed in stimulated cPBMC. Interestingly, JC (cloned and uncloned) isolates of HIV- 1 failed to replicate in cPBMC, reflecting an inherent biological difference between the JC and NC viruses. While the DH12 isolate was able to replicate in unstimulated cells, the levels of virus achieved were much less than that of the other viruses. The SF2 isolate was unable to replicate in unstimulated PBMC. Virus recovered from the molecular clones displayed the intrinsic replicative properties exhibited by the viruses they were derived from.
Reports of HIV- 1 isolates able to replicate in chimpanzee macrophages has been controversial. To investigate the ability of these viruses and their respective clones to replicate in macrophages, we conducted in vitro assays using purified chimpanzee monocyte- derived macrophages (MDM). Figure 5 shows virus production in macrophages at 7 and 14 days post infection. Both the HIV-1 JC and NC virus isolates (cloned and uncloned) replicated in MDM as determined by the levels of p24 antigen produced. However, the amount of virus produced by the HIV-1JC16 molecular clone at 14 days post infection was twice that produced by the HIV-1JC. The titers for cloned and uncloned HIV-1NC viruses were comparable for the same period of time. Among the viruses that were tropic for MDM, HIV- IJC (uncloned) produced the least amount of virus. HIV-lLAV.]b and HIV-1DH12 also infected MDM and produced virus which is consistent with previous observations made by Gendelman et al. [Gendelman et al. (1991) J. Virol. 65, 3853-3863] and Shibata et al. [Shibata et al. (1995) J. Virol. 69, 4453-4462] respectively. Unlike HIV-lLAV.lb, the other parental virus, HIV-1SF2, did not replicate in MDM.
Because the cloned viruses displayed such similar in vitro biologic activities as the uncloned stocks, we sought to examine whether these viruses represented major species in the viral mix. Genomic DNA was isolated and nested PCR was used to amplify the VI -V2 and V3-V5 regions of HIV- 1 envelope gene. These hypervariable regions of env gene were selected because most viral heterogeneity has been associated with them. Equal amounts of PCR products were mixed, heat denatured, and then reannealed with analogous fragments derived from standards (SF162 subtype B3 and ZM18 subtype C2) or from JC16 and NC7 molecular clones. Electrophoresis of these fragments on polyacrylamide non-denaturing gels revealed differences in migration rates with homoduplexes moving faster in the gel than heteroduplexes. Homoduplexes based on genomic DNA (HIV-1JC or HIV-1NC) resulted in homoduplex (lower) and heteroduplex (upper) bands that had comparable intensities and were close together lane. The HIV-1JC/HIV-1JC16 (but not HIV-1NC/HIV-1NC7) heteroduplex band was between the homoduplex and heteroduplex bands obtained with genomic DNA homoduplexes. The distance between homoduplex and heteroduplex bands formed between JC16 and NC7 molecular clones was larger than that between bands formed from homoduplexes derived from genomic DNA. The SF162 (subtype B3) heteroduplex bands migrated at almost the same rate as the ssDNA but the ZM18 (subtype C2) heteroduplex bands had the slowest migration.
An advantage of establishing a model system for AIDS in smaller nonhuman primates is the decreased cost associated with the housing and upkeep of smaller animals. Animal AIDS models include, but are not limited to, chimpanzees, the gibbons, pig-tailed macaques, and rhesus macaques infected with the HIV-1JC or HIV-1NC virus of the invention.
Disclosed herein are an immunodeficient pig-tailed macaque and also an immunodeficient rhesus macaque; both infected with the HIN-1JC virus of the invention isolated from the C499 chimpanzee which exhibited HIV-1-induced AIDS or the HIV-1ΝC virus isolated from the C455 chimpanzee. The macaques are infected with HIV-1 isolate JC or HIV-1NC and are used as an animal model for human AIDS in essentially the same manner as chimpanzees as described above. The transmission of HIV-1JC or the HIV-1NC virus into a macaque proceeds via injection of an isolated viral suspension or transfection of a biological fluid or tissue specimen from an HIV- 1 -infected and AIDS-bearing primate. The HIV- 1 - infected donor specimen is introduced into a recipient by any suitable means, such as intraperitoneal injection, intravenous injection, surgical implantation and combinations thereof. Donor tissue may be introduced as organized tissue (e.g., thymus, lymph node, etc.) or as discrete cells. Antigenic fragments of the present invention are peptides which contain at least one epitope (antibody binding site) which binds antibodies which bind to at least one HIV-1 isolate of the present invention. The antigenic fragments are preferably capable of inducing an immune response when administered to a nonhuman primate. DNA encoding such antigenic fragments may be used to transform host cells to thereby produce such antigenic fragments.
Antigenic fragments may be identified by a variety of means. A protein from HIV-1JC and/or from HIV-1NC, such as an envelope protein, may be fragmented with a protease, and the fragments tested to determine whether or not various ones react with antiserum against the protein. See, e.g., J. Robinson et al, Mol. Cell Biochem. 21:23-32 (1978). Another technique is to synthesize peptides which are fragments of the entire protein and determine whether the individual fragments are recognized by neutralizing antibodies against the protein. See, e.g., J. Gerin et al., in Vaccines 85: Molecular and chemical Basis of Resistance to Parasitic, Bacterial and Viral Diseases, 235-239 (Lerner et al., eds. 1985). Still another method useful for obtaining immunogenic fragments of a protein is by isolation and identification of monoclonal escape mutants. In this strategy, HIV-1 is produced in the presence of a monoclonal antibody to the virus. The only viruses which can grow under these conditions are those with a mutation in the nucleotide sequence which codes for an epitope to which the monoclonal antibody binds. A mutant virus which grows under these conditions is referred to as the "monoclonal escape mutant." The monoclonal escape mutant is then sequenced and the mutant sequence compared with the nucleotide sequence of the HIV-1JC isolate or the HIV-1NC isolate to find the specific location of the mutation. The mutation is located in a region which codes for a protective epitope, or an "immunogenic fragment." See, e.g., J. Lopez et al., J. Virol. 64:927 (1990).
Antigenic preparations of the present invention are useful as reagents in immunoassay diagnostic studies of retroviruses. Immunochemical methods for detecting retroviruses include, for example, immunofluorescence assays or immunoenzymatic assays. Immunofluorescence assays typically involve incubating, for example, serum from the subject to be tested with preparations of the pathogenic virus or fragments thereof. Immune complexes formed are detected using either direct or indirect methods, for example, the use of antibodies to which fluorescent labels such as rhodamine or fluorescein have been coupled. Immunoenzymatic assays typically involve viral extracts or other antigen-containing compositions bound to a surface. Serum from a subject to be tested for the presence of antibodies directed against one or more antigens is contacted with the surface and, after a period of incubation, unbound substances are washed away. The presence of immune complexes is detected using antibodies labeled with an enzyme such as horseradish peroxidase, alkaline phosphatase, or beta-galactosidase, which is capable of converting a colorless or nearly colorless substrate into a highly colored product, or an enzyme which emits light in the presence of the proper substrate. The amount of product formed is detected visually, spectrophotometrically, or luminometrically and is compared to a similarly treated control. The presence of antibodies in biological fluids may also be detected by agglutination. Viral lysates or antigen compositions are used to coat, for example, latex particles.
Diagnostic tests utilizing the present invention may be carried out in accordance with known techniques. Such techniques provide a method of detecting the presence of HIV- 1 by detecting the presence of HIV- 1 antibodies. Such methods comprise collecting an antibody- containing biological sample (e.g., blood, blood sera, blood plasma, cerebrospinal fluid, tissue samples) from the subject, contacting the sample with an antigenic preparation of the viral particles of the present invention as given herein, and then detecting the formation of a reaction product between the antibodies in the sample and the antigenic preparation. Any suitable assay format, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA) may be employed, in accordance with known techniques. See, e.g., Immunology: Basic Processes, 162-175 (J. Bellanti, [Ed.] 2d ed., W.B. Saunders Co. (1985).
Also disclosed herein are kits for the detection of HTV infection. Such kits comprise a container containing an antigenic preparation of the viral particles of the present invention, which may be lyophilized. The antigenic preparation may comprise, e.g., the HIV-1 envelope protein (env) or the group antigen (gag) protein of the HIV-ljc and/or HrV-lNC of the invention. A method of inducing antibodies to HIV-1 in a subject, as disclosed herein, comprises administering to a subject an immunogenic amount of infectious viral particles of the present invention. This method may be used to make polyclonal or monoclonal antibodies, which may be used in diagnostic assays. Suitable subjects include mammals (such as, for example, rats, rabbits, mice, and horses) and primates. The term primates is herein intended to encompass any members of the order Primata (for example, lemurs, mandrills, rhesus monkeys, macaques, and chimpanzees) and to include humans. Suitable subjects include those in which antibodies to HIV may be raised (e.g., rabbit, horse).
In the above methods of inducing antibodies, viral antigenic preparations of the present invention may be combined with any suitable pharmaceutically acceptable carrier (such as sterile, pyrogen-free physiological saline solution, or sterile, pyrogen-free phosphate- buffered saline solution). The viral antigens are included in an effective immunogenic amount. The precise amount to be administered to a given subject is determined by techniques known in the art and will vary depending on the route of administration, the subject and the desired response. Administration to the subject may occur by any suitable route (e.g., by intramuscular injection, subcutaneous injection, intraperitoneal injection, or intravenous injection). The appropriate immunogenic dosage will depend upon the particular subject and the desired outcome. Techniques to determine a particular immunogenic amount of the viral particles of the present invention will be apparent to those of ordinary skill in the art. See, e.g., Johnson et al., Proc. Natl. Acad. Sci. USA 89:2175 (1992). For example, the active agent (viral particles or preparations thereof) may be given in an amount of from 0.05 to 50 μg per kg body weight (e.g., 0.5 or 1.0 μg per kg).
The invention also provides for a variety of different vaccines based on the structures of the HIV-ljc isolate and/or the HIV-1NC of the invention and a method for vaccinating a population against HTV. Examples of active agents used for the preparation of a vaccine of the invention include the live attenuated HTV-1JC and or HIV-1NC isolate, fixed whole virus, host cells expressing virus antigen, preparations of virus fragments, purified proteins, antigenic fragments of proteins and antigenic peptides which are derivatives of the antigenic fragments. According to the present invention, HIV-1JC- and or HIV-lNC-derived compositions or vaccines are useful for preinfection immunization of primates as well as for postinfection (therapeutic) immunization of HIV-infected primates (see Fultz et al. [1989], Lerner et al., eds. Cold Spring Harbor, NY).
Live attenuated HIV isolate JC virus (or HIV-1NC virus) is prepared by serial passage of the virus in tissue culture or genetically altered by recombinant techniques, in accordance with known procedures. Fixed virus is made by contacting live virus (attenuated or unattenuated) to a suitable fixative, such as formalin.
Preparations of viral fragments are made by lysing host cells, such as E coli cells, transformed with a vector encoding an HIV-1 isolate of the present invention (or both) or a portion thereof. The lysate may be used in crude or partially purified form, or a particular viral protein (or antigenic fragment thereof) such as the envelope protein, can be purified to homogeneity and used as an active agent for a vaccine against HIV-1. Host cells such as yeast cells may be transformed with vectors of the present invention capable of expressing HIV-1 proteins, or antigenic fragments thereof, on the surface of the host cells, and the transformed host cells used as an active vaccine agent as such or fixed (e.g., with formalin) and used as an active agent.
Antigenic peptides are selected from the group consisting of antigenic fragments of HIV isolate JC and/or NC proteins, such as the envelope protein, and the antigenic equivalents thereof (i.e., analogs or derivatives). Antigenic peptides may be chemically synthesized or produced by recombinant techniques.
Viral antigenic preparations and cells producing viral antigens and/or fragments thereof may be formulated into immunogenic compositions as neutral or salt forms. Preferably, when cells are used they are of avirulent strains, or the cells are killed before use. Pharmaceutically acceptable salts include but are not limited to the acid addition salts (formed with free amino groups of the peptide) which are formed with inorganic acids, e.g., hydrochloric acid or phosphoric acids; and organic acids, e.g., acetic, oxalic, tartaric, or maleic acid. Salts formed with the free carboxyl groups may also be derived from inorganic bases, e.g., sodium, potassium, ammonium, calcium, or ferric hydroxides, and organic bases, e.g., isopropylamine, trimethylamine, 2-ethylamino-ethanol, histidine, and procaine.
The term "antigenic equivalents," as used herein, refers to proteins or peptides which bind to an antibody which binds to the protein or peptide with which equivalency is sought to be established. Antibodies which are used to select such antigenic equivalents are referred to as "selection antibodies" herein. Preferred selection antibodies are monoclonal antibodies which bind to HIV isolate JC and/or to HIV-1NC, but not to prior isolates of HIV- 1 such as the HIV-1 isolates NDK, LAI and SF2, for example.
One or more amino acids of an antigenic peptide sequence may be replaced by one or more other amino acids which do not affect the antigenicity of that sequence. Such changes can be guided by known similarities between amino acids in physical features such as charge density, hydrophobicity/hydrophilicity, size and configuration, For example, threonine and serine can be interchanged, or aspartic acid and glutamic acid, or leucine and isoleucine, and the like.
Antigenic equivalents may be formed by modifying reactive groups within a natural sequence or modifying the N-terminal amino and/or C-terminal carboxyl group. Such equivalents include salts formed with acids and/or bases, particularly physiologically acceptable inorganic and organic acids and bases. Other equivalents include modified carboxyl and/or amino groups on the synthetic peptide to produce esters or amides, or amino acid protecting groups such as N-t-butoxycarbonyl. Preferred modifications are those which provide a more stable, active peptide which will be less prone to enzymatic degradation in vivo.
In a particular embodiment of the invention, polyclonal and/or monoclonal antibodies capable of specifically binding to a particular epitope of at least one HIV-1 isolate of the invention are provided. The term antibody is used to refer both to a homogenous molecular entity (monoclonal antibody) and a mixture (such as a serum product) made up of a plurality of different molecular entities (polyclonal antibody). Monoclonal or polyclonal antibodies, and preferably monoclonal, specifically reacting with a particular epitope of interest can be made by methods known in the art. See, e.g., Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratories; Goding (1986) Monoclonal Antibodies: Principles and Practice, 2d ed., Academic Press, New York. Also, recombinant immunoglobulins may be produced by methods known in the art, including but not limited to the methods described in U.S. Patent No. 4,816,567, incorporated by reference herein. Monoclonal antibodies with affinities of 108 M"1, preferably 109 to 1010 or more are preferred.
For use as a vaccine, immunogenic compositions may be formulated by any of the means known in the art. Such vaccines are typically prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. The preparation may also, for example, be emulsified, or the protein encapsulated in liposomes. Such vaccines may be administered to the subject by any suitable means, for example, by intramuscular injection, by subcutaneous injection, by intravenous injection, by intraperitoneal injection, by oral injection, and by nasal spray.
The vaccine or other immunogenic composition may be given in a single dose or multiple dose schedule. A multiple dose schedule is one in which a primary course of vaccination may include 1 to 10 or more separate doses, followed by other doses administered at subsequent time intervals as required to maintain and or reinforce the immune response, e.g., at 1 to 4 months for a second dose and, if needed, a subsequent dose(s) after several months.
The immunogenic peptide antigen compositions are administered in a manner compatible with the dosage formulation and in such amount as will be prophylactically and/or therapeutically effective. The quantity to be administered, which is generally in the range of about 100 to 1,000 μg of protein per dose, more generally in the range of about 5 to 500 μg of protein per dose, depends on the subject to be treated, the capacity of the individual's immune system to synthesize antibodies, and the degree of protection desired. Precise amounts of the active ingredient required to be administered may depend on the judgment of the physician and may be peculiar to each individual, but such a determination is within the skill of such a practitioner.
Vaccine formulations of the present invention comprise the active agent mixed with excipients or carriers which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients include but are not limited to water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. The concentration of the immunogenic polypeptide in injectable formulations is usually in the range of 0.2 to 5 mg/ml.
In addition, if desired, the vaccines may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants which enhance the effectiveness of the vaccine. Examples of adjuvants which may be effective include but are not limited to: aluminum hydroxide; aluminum phosphate; plant and animal oils; synthetic polymers; e.g., N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr- MDP); N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as nor- MDP); N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-( 1 '-2'-dipalmitoyl-sn-glycero- 3-hydroxyphosρhoryloxy)-ethylamine (CGP 19835A, referred to as MTP-PE); and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate; cell wall skeleton (MPL+TDM+CWS) in a 2% squaline/Tween 80 emulsion; etc.
In addition, the vaccine formulations may also contain one or more stabilizer, for example, carbohydrates such as sorbitol, mannitol, starch, sucrose, dextrin, and glucose, proteins such as albumin or casein, and buffers such as alkaline metal phosphates and the like.
In view of the similarities in protein molecules making up different isolates of HIV- 1, the skilled artisan understands that an antibody, particularly a monoclonal antibody, which is specific for a particular epitope directed to a particular protein of the HIV-1JC isolate and/or the HIV-1NC isolate, can be used to screen for other HIV-1 isolates having similar epitopes recognized by that (monoclonal) antibody. Antibodies generated against specific epitopes of the HIV-1JC of the invention are useful, for example, as probes for screening DNA expression libraries or for detecting the presence of HIV- 1 strains in a test sample. Antigens can be synthesized and conjugated to a suitable carrier protein (e.g., bovine serum albumin or keyhole limpet hemocyanin) for use in vaccines or in raising specific antibodies. Frequently, the polypeptides and antibodies will be labeled by joining, either covalently or noncovalently, a substance which provides a detectable signal. Suitable labels include but are not limited to radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent agents, chemiluminescent agents, magnetic particles and the like. United States Patents describing the use of such labels include but are not limited to Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,355,241.
Immunogenic carriers may be used to enhance the immunogenicity of an active agent. Such carriers include but are not limited to proteins and polysaccharides, liposomes, and bacterial cells and membranes. Protein carriers may be joined to the capsular polysaccharide molecules to form fusion proteins by recombinant or synthetic means or by chemical coupling. Useful carriers and means of coupling such carriers to polypeptide antigens are known in the art. The art knows how to administer immunogenic compositions so as to generate protective immunity where immunity is most helpful.
Compositions and immunogenic preparations including vaccine compositions comprising substantially purified antigens derived from an HIV-1 isolate JC and or NC and a suitable carrier therefor are provided. Immunogenic compositions are those which result in specific antibody production when injected into a human or an animal. Such immunogenic compositions are useful, for example, in immunizing primates against infection by HIV-1 strains. The immunogenic preparations comprise an immunogenic amount of, as specifically exemplified, at least one antigenic determinant derived from the HIV-1JC isolate of the invention and a suitable carrier. Alternatively, the immunogenic composition can comprise host cells harboring an antigenic agent from the specifically exemplified HIV-1JC strain and a suitable carrier. It is understood by one of ordinary skill in the art that a functionally equivalent, recombinant mutant of HIV-1JC and or HIV-1NC can be produced by the introduction of the cloned DNA containing the insertion mutations responsible for a desired characteristic. It is also within the scope of the present invention and readily within the grasp of the ordinary skilled artisan to generate other types of genetically stable mutations in the structural or enzyme genes of HIV- 1. Such immunogenic compositions (or vaccines) are useful, for example, in immunizing an animal, especially humans, against AIDS and related diseases resulting from infection by HIV-1 species. Such immunogenic compositions can also elicit the production of antibodies which will cross react with proteins of other HIV-1 and HIV-2 strains expressing epitopes in common with those of the starting HIV-1JC isolate. It is understood that where whole cells are formulated into the immunogenic composition, the cells are preferably inactivated, especially if the cells are of a virulent strain. Such immunogenic compositions may comprise one or more protein or the immunogenic cellular component. By "immunogenic amount" is meant an amount capable of eliciting the production of antibodies directed against an antigenic agent of HIV-1JC in an animal or human to which the vaccine or immunogenic composition has been administered.
The nucleotide sequence of the HIV-1JC isolate or the HIV-1NC isolate can be used to generate hybridization probes which specifically bind to HIV-1JC genetic material, or to DNA of HIV- 1 isolates having the identifying characteristics of the HIV-1 isolates JC or NC, to determine the presence of such HIV-1 in primates. The hybridization probe may be selected so that it does not bind to other known HIV-1 isolates such as NDK, LAI, SF2, etc. The hybridization probes can be cDNA fragments or polynucleotides and may be labeled with a detectable group, as is well-known in the art. Pairs of probes can serve as PCR primers for synthesis and amplification processes in accordance with the description, for example in U.S. Patent Nos. 4,683,202 and 4,683,195.
In specific embodiments, probes of the invention comprise DNA sequences of HIV- ljC or sequences encoding antigenic fragments thereof or sequences having identity thereto. In particular are provided probes having a DNA sequence as set forth in SEQ ID NO:l or a sequence having identity thereto. The production of DNA, vectors, transformed host cells, HIV-1 virus, proteins, and protein fragments of the present invention by genetic engineering techniques can be carried out in accordance with methods known in the art. See, e.g., U.S. Pat. No. 4,761,371, U.S. Pat. No. 4,877,729, U.S. Pat. No. 4,912,038, and U.S. Pat. No. 4,879,224, among others.
A nucleotide sequence (polynucleotide) or fragment thereof is substantially homologous (or substantially similar) to another polynucleotide if, when optimally aligned (with appropriate nucleotide insertions or deletions) with another polynucleotide, there is nucleotide sequence identity for approximately 80% of the nucleotide bases, usually approximately 90%, more preferably about 95% to 100%) of the nucleotide bases. Gaps introduced to optimize alignments are treated as mismatches.
Alternatively, substantial homology (or similarity) exists when a polynucleotide or fragment thereof will hybridize to another polynucleotide under selective or stringent hybridization conditions. Selectivity of hybridization exists under stringent hybridization conditions which allow one to distinguish the target polynucleotide of interest from other polynucleotides. Typically, selective hybridization will occur when there is approximately 75%) similarity over a stretch of about 14 nucleotides, preferably approximately 80% similarity, more preferably approximately 85%) similarity, and most preferably approximately 90%) similarity. See Kanehisa (1984) Nucl. Acids Res., 12:203-213. The length of homology comparison, as described, may be over longer stretches, and in certain embodiments will often be over a stretch of about 17 to 20 nucleotides, preferably 21 to 25 nucleotides, more preferably 26 to 35 nucleotides, and more preferably about 36 or more nucleotides.
The hybridization of polynucleotides is affected by such conditions as salt concentration, temperature, or organic solvents, in addition to the base composition, length of the complementary strands, and the number of nucleotide base mismatches between the hybridizing polynucleotides, as will be readily appreciated by those skilled in the art. Stringent temperature conditions will generally include temperatures in excess of 30°C, typically in excess of 37°C, and preferably in excess of 45°C. Stringent salt conditions will ordinarily be less than 1 M, typically less than 500 mM, and preferably less than 200 mM. However, the combination of parameters is much more important than the measure of any single parameter (Wetmur and Davidson [1968] J. Mol. Biol. 31:349-370).
An isolated or substantially pure polynucleotide is a polynucleotide which is substantially separated from other polynucleotide sequences which naturally accompany a native sequence. The term embraces a polynucleotide sequence which has been removed from its naturally occurring environment, and includes recombinant or cloned DNA isolates, chemically synthesized analogues and analogues biologically synthesized by heterologous systems.
A polynucleotide is said to encode a polypeptide if, in its native state or when manipulated by methods known to those skilled in the art, it can be transcribed and/or translated to produce the polypeptide of a fragment thereof. The anti-sense strand of such a polynucleotide is also said to encode the sequence.
Vectors are replicable DNA constructs used to either amplify or express DNA of the present invention. An expression vector is a replicable DNA construct in which DNA of the present invention is operably linked to control sequences capable of expressing that DNA in a suitable host. Generally, control sequences include a transcriptional promoter, an optional operator sequence to control transcription, a sequence encoding suitable mRNA ribosomal binding sites, and sequences which control the termination of transcription and translation. Suitable vectors include plasmids, viruses (e.g., vaccinia virus, adenovirus, baculovirus, cytomegalo virus) phage, and integratable DNA fragments (i.e., fragments integratable into the host genome by recombination).
DNA regions are operably linked or operably associated when they are functionally related to each other. For example, a promoter is operably linked to a coding sequence if it controls the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to permit translation. Transformed host cells are cells which have been transformed or transfected with vectors as described above. Transformed host cells ordinarily express the DNA of the present invention. Suitable host cells include prokaryote, yeast or higher eukaryotic cells such as mammalian cells and insect cells.
Prokaryote host cells include gram negative or gram positive organisms, for example Escherichia coli (E. coli) or Bacilli. Exemplary host cells are E. coli W3110 (ATCC 27,325), E. coli B, E. coli XI 776 (ATCC 31,537), E. coli 294 (ATCC 31,446). A broad variety of suitable prokaryotic and microbial vectors are available. E. coli is typically transformed using pBR322. Promoters most commonly used in recombinant microbial expression vectors include the β-lactamase (penicillinase) and lactose promoter systems (Change et al., Nature 275:615 [1978]; and Goeddel et al, Nature 281:544 [1979], a tryptophan (trp) promoter system (Goeddel et al. [1980] Nuc. Acids Res. 8:4057, and EPO App. Publ. No. 36J66) and the tac promoter (H. De Boer et al, Proc. Natl. Acad. Sci. USA 80:21 [1983]). The promoter and Shine-Dalgarno sequence are operably linked to the DNA of the invention, i.e., they are positioned so as to promote transcription of messenger RNA from the DNA.
Eukaryotic microbes such as yeast cultures may also be transformed with vectors of the present invention. See, e.g., U.S. Pat. No. 4,745,057. Saccharomyces cerevisiae is the most commonly used yeast, although other yeast may also be used. Host cells such as insect cells (e.g., cultured Spodoptera frugiperda cells) and expression vectors such as the baculovirus expression vector may be employed in carrying out the present invention, as described in U.S. Pat. Nos. 4,745,051 and 4,879,236 to Smith et al.
Examples of useful mammalian host cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, and WI 138, BHK, COS-7, CV, and MDCK cell lines. The transcriptional and translational control sequences in expression vectors to be used in transforming vertebrate cells are often provided by viral sources. For example, commonly used promoters are derived from polyoma, Adenovirus 2, and Simian Virus 40 (SV40). See, e.g., U.S. Pat. No. 4,599,308. An origin of replication may be provided either by construction of the vector to include an exogenous origin, such as may be derived from SV40 or other viral (e.g., Polyoma, Adenovirus, VSV, or BPV) source, or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter is often sufficient. Rather than using vectors which contain viral origins of replication, one can transform mammalian cells by the method of cotransformation with a selectable marker and DNA of the present invention, as described in U.S. Pat. No. 4,399,216.
Except as noted hereafter, standard techniques for cloning, DNA isolation, amplification and purification, for enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like, and various separation techniques are those known and commonly employed by those skilled in the art. A number of standard techniques are described in Sambrook et al. (1989) Molecular Cloning, Second Edition, Cold Spring Harbor Laboratory, Plainview New York; Maniatis et al. (1982) Molecular Cloning, Cold Spring Harbor Laboratory, Plainview, New York; Wu (ed.) (1993) Meth. Enzymol. 218:Part I; Wu (ed.) (1979) Meth. Enzymol. 68; Wu et al. (eds.) (1983) Meth. Enzymol. 100 and 101; Grossman and Moldave (eds.) Meth. Enzymol. 65; Miller (ed.) (1972) Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Plainview, New York; Old and Primrose (1981) Principles of Gene Manipulation, University of California Press, Berkely; Schleif and Wensink (1982) Practical Methods in Molecular Biology; Glover (ed.) (1985) DNA Cloning Vol. I and II, IRL Press, Oxford, UK; Hames and Higgins (eds.) (1985) Nucleic Acid Hybridization, IRL Press, Oxford, UK; and Setlow and Hollaender (1979) Genetic Engineering: Principles and Methods, Vols. 1-4, Plenum Press, New York. Abbreviations and nomenclature, where employed, are deemed standard in the field and commonly used in professional journals such as those cited herein.
All publications, patent applications and patents cited herein are incorporated by reference to the extent that they are not inconsistent with the present disclosure.
The foregoing discussion and the following examples are provided for illustrative purposes, and they are not intended to limit the scope of the invention as claimed herein. Modifications and variations which may occur to one of ordinary skill in the art are within the intended scope of this invention. The present invention is further described in the non- limiting examples set forth below.
EXAMPLES
Example 1. Animal Subjects
All animals (e.g., chimpanzees) were maintained in accordance with the guidelines established by the Animal Welfare Act and the NIH guide for care and use of laboratory animals. The Yerkes Center is fully accredited by the American Association for the Accreditation of Laboratory Animal Care (AAALAC).
A cohort of 12 chimpanzees was inoculated with several strains of HIV- 1 at the Yerkes Center. A member of this cohort, chimpanzee 499 was inoculated on three different occasions with three different HIV-1 isolates: HIV-1SF2 in 1985, HIV-1LAI in 1986 and HIV- INDK in 1987 (See Fultz et al- 11991- J- Infect- Dis- 163:441-447 and Novembre et al. [1997] J. Virol. 71:4086-4091).
It is preferred that a chimpanzee be bred in captivity, be seropositive for Epstein-Barr virus and cytomegalovirus and be seronegative and PCR negative for HIV prior to being used as an animal model.
Example 2. Isolation and Purification of HIV-1J Isolate
The original source of the HIV-1JC isolate was the chimpanzee C499. HIV was easily isolated from the peripheral blood mononuclear cells (PBMC) of this animal. Cocultivation of PBMC derived from C499 with uninfected chimpanzee PBMC (cPBMC) resulted in the isolation of a virus (HIV-1JC) which induced syncytium formation in chimpanzee cells. The nucleotide sequence of this virus was distinguished from the isolates used for the initial inoculations and from other known HIV-1 isolates.
The HIV-ljc isolate was deposited with the AIDS Reagent Program, McKesson Bioservices, 685 Lofstrand Lane, Rockville, MD 20850 USA, a division of the NIH AIDS Research and Reference Reagent Program, in May 1997 and has been assigned Catalog Number 3523. A substantially full-length infectious molecular clone has a nucleotide sequence as given in SEQ ID NO: 11.
In a specific embodiment of the invention, probes of the invention comprise DNA sequences as set forth in SEQ ID NO:l 1 and/or sequences of at least 15 contiguous nucleotides derived therefrom or sequences complementary thereto.
The original source of the HIV-1JC isolate was the chimpanzee C455. HIV was easily isolated from the peripheral blood mononuclear cells (PBMC) of this animal. Cocultivation of PBMC derived from C455 with uninfected chimpanzee PBMC (cPBMC) resulted in the isolation of a virus (HIV-1NC) which induced syncytium formation in chimpanzee cells. The nucleotide sequence of this virus is distinguished from other known HIV-1 isolates. A substantially full-length infectious molecular clone has a nucleotide sequence as given in SEQ ID NO: 12.
In a specific embodiment of the invention, probes of the invention comprise DNA sequences as set forth in SEQ ID NO: 12 and/or sequences, of at least 15 contiguous nucleotides derived therefrom or sequences complementary thereto.
Example 3. Virus and Viral Antigens
The HIV-1 isolates used to inoculate C499 included LAV (lymphadenopathy- associated virus)-lBRU (Barre-Sinoussi [1983] Science 220:868-871), SF2 (previously designated ARV [AIDS-related virus]-2) (Levy et al. [1984] Science 225:840-842), and NDK, a highly cytopathic HIV-1 of African origin (Spire et al. [1989] Gene 81:275-284). Inoculations of virus were done intravenously with 1-ml aliquots of undiluted or diluted virus stocks. In January 1988, 33 months after inoculation HIV-1SF2, C499 was part of a study to assess the effects of therapeutic vaccination on immunity and viral status in HIV- 1 -infected chimpanzees. C499 was given two intramuscular injections, 4 weeks apart, of 500 μg of recombinant SF2 p53 gag, produced in yeast and formulated with 100 μg of muramyl tripeptide (Ciba-Geigy, Summit, NJ) in 4% squalene and 0.008% Tween 80 (Fultz et al. [1989] in Vaccines 89, supra).
The HIV-1 isolates used to inoculate C455 was JC, deposited with the AIDS Reagent Program, Catalog Number 3523. Inoculations of virus were done intravenously with 1-ml aliquots of undiluted or diluted virus stock. C534 was given two intramuscular injections, 4 weeks apart, of 500 μg of recombinant SF2 p53 gag, produced in yeast and formulated with 100 μg of muramyl tripeptide (Ciba-Geigy, Summit, NJ) in 4%> squalene and 0.008% Tween 80 (Fultz et al. [1989] in Vaccines 89, supra).
Example 4. Serologic Assays
Serum samples were tested by indirect ELISA for antibodies to specific HIV-1 proteins using a series of recombinant antigens. These antigens have been described (Fultz et al. [1989] in Vaccines 89, supra) and included p25 gag and p53 gag, produced in yeast; p31 pol; and e«v2-3(SF2) and e»v2-3 (IIIB), nonglycosylated polypeptides corresponding to full- length gpl20. Antibody titers to whole- virus preparations were determined with an HIV enzyme immunoassay kit (EIA; Genetic Systems, Seattle, WA). Neutralization assays were performed as described (Fultz et al. [1986] Proc. Natl. Acad. Sci. 83:5286-5290); titers were based on >80% inhibition of reverse transcriptase (RT) activity after preincubation of virus with serum and infection of normal human PBMC. Antibodies cross-reactive with histone H2B were identified by immunoblot using purified calf thymus histones as described previously (Strickler et al. [1987] Nature 327:710-713).
Example 5. Virus Assays
Assays to detect virus in plasma samples or to recover virus from chimpanzee PBMC were performed with normal human PBMC as indicator cells, unless otherwise indicated, and have been described (Fultz et al. [1986] J. Virol. 58:116-124). Medium was RPM 1640 with 10%) fetal bovine serum, 8 units of recombinant interleukin-2 (IL-2)/ml, glutamine, and antibiotics (RPM-IL-2). Infectious PBMC were quantified by serial 1:5 limiting dilution and cocultivation with human PBMC (Fultz et al. [1986], supra). HIV-1 was detected in all assays by the presence of RT activity in cell-free culture supernatants. To detect HIV antigen in serum or plasma samples, a commercially available HIV-1 antigen capture kit (Coulter, Hialeah, FL) was used.
To assess the influence of CD8+ lymphocytes on viral replication and recovery, after 3 days of stimulation with concanavalin A (ConA 10 μg/ml) in RPMI 1640, PBMC were washed once; 107 cells from C499 were placed in fresh RPMI-IL-2, cultured without indicator cells, and monitored for HIV-1 production. In other experiments, the overnight incubation of C499's PBMC to remove adherent cells, CD4+- and CD8+-enriched populations were obtained by panning with monoclonal antibodies specific for the CD8+ antigen or with CD8+-coated magnetic beads (Dynabeads; Robbins Scientific, Mountain View, CA). After removal of the CD8"{??1 cells, CD8+ lymphocytes were recovered from the plates or magnetic beads by additional overnight incubation and repeated washes with medium. After stimulation with ConA, cultures were established either with CD4+-enriched cells only or with CD4+- and CD8+-enriched cells in various ratios. Percentages of CD4+ and CD8+ cells in enriched populations were determined by analysis with FACScan (Becton-Dickinson, Mountain View, CA).
Replication kinetics in PBMC and macrophages were tested as follows. 1 x 107 freshly isolated or Con-A stimulated PBMC from HIV-1 negative chimpanzee were infected overnight (at 37°C) with 20 ng of the indicated virus (p24 antigen concentration). The cells were centrifuged at 1000 rpm for 10 min, resuspended in 10 ml complete RPMI medium containing 10%) FBS and IL-2 (IL-2 medium) and were incubated at 37 °C. Samples of supernatants (1 ml) were harvested on days 3, 7, 10, 14 and 17 post infection. IL-2 medium was added to the cultures following the sampling to maintain the original volume. Supernatants were used in RT assays to determine the relative amounts of virus produced.
For replication in macrophages, cPBMC were resuspended in macrophage media (6 x lOVwell in RPMI 1640 containing 15% human serum [AB+], 1% HEPES, 0.008 ng/ml GM- CSF, 0.03 ng/ml M-CSF, 1% antibiotic-antimycotic solution (Sigma, St. Louis, MO) and seeded in a 24-well plate and incubated at 37 °C for 4 hrs. The cells were mixed by pipetting up and down before the incubation was continued for 4 days. Non-adherent cells were removed by gently washing the wells. Fresh medium (2 ml/well) was added and the cells were cultured for an additional 3 days to allow full macrophage differentiation. Infections were initiated by adding 10 ng of virus (p24) to the cells in 500 μl media and adsorbed overnight. The inoculum was removed and the cells were washed twice before fresh macrophage medium (2 ml) was added. On days 7 and 14 post infection, aliquots of 0.5 ml were taken for determination of p24 antigen levels using the HIV-1 p24 Antigen kit (Coulter Corp, Miami, FL) according to the manufacturer's instructions.
Example 6. Cell-mediated Immune Responses
Lymphocyte proliferative responses to mitogens were tested by incorporation of [3H]thymidine into the DNA of PBMC seeded in triplicate at 5 x 104 PBMC per well into 96- well plates. PBMC were incubated for four days with different concentrations of phytohemagglutinin (PHA) or ConA, pulsed overnight with 1 μCi of [3H]thymidine per well, and harvested with a cell harvester (Skatron, Sterling, VA); counts per minute (cpm) incorporated were determined in a β counter.
Cytotoxic T lymphocyte (CTL) responses against HIV-1 env- and gαg-encoded antigens were assayed using fresh PBMC from C499 as effector cells and autologous Epstein Barr virus-transformed B cells as target cells. Target cells were infected at MOI=10 with recombinant vaccinia viruses expressing HIV-1 gpl 20 env of p24 gag or with wild-type vaccinia as control. Infected targets were labeled with 200 μCi of [51Cr] sodium chromate (Du Pont, Boston) for 2 hours and washed, and 105 target cells (T) were mixed with effector cells (E) at various ratios in 96-well plates. The plates ere incubated for 6 hours at 37°C in 5% CO2, then 0.1 ml of medium was removed from each well and counted in a γ counter. Maximum release (max) was determined by adding % Triton X to labeled target cells, and spontaneous release (spon), by adding only medium. Percentage of specific lysis was determined: [cpm(experimental) - cpm (spon)]/[cpm(max) - cpm(spon)] x 100.
Example 7. Nonhuman Primate Model Systems for AIDS
Nonhuman primates infected with HIV-1 isolate JC or infected with HIV-1NC are useful as a model system for the study of AIDS. Chimpanzees and other monkey species used for this purpose are preferably specific pathogen-free animals, which are available from primate centers, e.g., the Yerkes Regional Primate Research Center, Emory University, Atlanta, GA.
Infected primates are preferably maintained as a single colony of two or more animals, all inoculated with HIV-1 isolate JC or a colony of two or more animals, all inoculated with HIV-1NC. A colony may be maintained in a single room with each primate housed in an appropriate cage, in accordance with standard practices for the maintenance of animals established by the Animal Welfare Act and the NIH guide for care and use of laboratory animals.
The primates are infected with the HIV-1JC virus or the HIV-1NC virus by any suitable means, such as intraperitoneal, intravenous or subcutaneous injection with a solution containing HIV-1 isolate JC or HIV-1NC. The solution may also be a body fluid or tissue (e.g., blood) from a previously infected primate, a blood fraction containing peripheral blood mononuclear cells from a previously infected primate, a pharmaceutically acceptable carrier such as saline solution containing HIV-1 isolate JC or HIV-1 isolate NC, etc.
Nonhuman primates infected with HIV-JC or HIV-1NC are particularly useful as a model system for AIDS because of the concomitant decrease in CD4+ cells and increase in HTV-1 loads in plasma. The development of AIDS has not been previously documented in any nonhuman primate species. When used as a model system, a primate(s) infected with HIV-ljc or HIV-INC virus is subjected to a treatment useful in combating AIDS in humans and thereafter the progress of the infection and related diseases is monitored. A control (placebo) group of HIV-1JC or HIV-lNC-infected animals is left untreated for comparative purposes. A slowing in the progression of the development of AIDS in infected animals indicates that the treatment may be useful for combating AIDS in humans and additional screening and toxicological testing is prescribed. Such treatment includes but is not limited to a vaccine, a drug (e.g., an antiretroviral compound) or a drug combination (e.g., antiviral nucleosides such as AZT, DDI, etc.), a peptide, a protein, etc.) or a vaccine/drug combination, etc. After treatment initiation, the progress of the disease is monitored by any suitable parameters including, but not limited to, (a) decline in CD4+ cell levels, (b) increase in viral loads in plasma, (c) presence of HIV-1JC virus or HIV-1NC virus, (d) weight loss, (e) general appearance, and other symptoms characteristic of AIDS.
Example 8. Virus Cloning and Sequence Analysis cPBMC infected with either HIV-1JC or HIV-1NC were used for isolation of DNA using the Puregene Kit (Gentra systems, Minneapolis, MN) as directed by the manufacturer. The strategy used for PCR amplification and primer location on the HIV-1 viral genome is shown in Figure 3. PCR primers MSF12 (5'-AAA TCT CTA GCA GTG GCG CCC GAA CAG-3', (SEQ ID NO:18); HIV-1LAV nt 169 to 195) and MSR5 (5'-GCA CTC AAG GCA AGC TTT ATT GAG GCT-3', (SEQ ID NO:19); HIV-1LAV nt 9225 to 9198) [Salminen et al. (1995) Virology 213, 80-86] were used to amplify a 9056 bp product from PBMC genomic DNA prepared from HIV-lJC-infected cells. Another PCR primer pair 527 (5'-CAC ACA CAA GGC TAC TTC CCT GAT TGG CAG A-3', SEQ ID NO:20, HIV-1LAV nt 5302 to 5274) was used to amplify 5' LTR-containing fragments (5699 bp) from the same DNA source. For the HIV-1NC viral genome, PCR primer pairs 527-528 and 529 (5'-ATG GAA CAA GCC CCA GAA GAC CAA GGG CCA CAG-3', SEQ ID NO:21, HIV-1LAV nt 5141 to 5173) and 530 (5'-GGT CTG AGG GAT CTC TAG TTA CCA GAG TCA C-3', SEQ ID NO:22, HIV-1LAV nt 151 to 121) were used to generate the 5'-half (5699 bp) and 3'-half (4142 bp) PCR products respectively from HIV-1NC genomic PBMC DNA. Primers were synthesized on an Applied Biosystems 392 DNA synthesizer (Applied Biosystems, Foster City, CA). Briefly, PCR was performed using the reagents from the Expand Long Template kit (Boehringer Mannheim, Indianapolis, IN) and 200 ng of DNA template, according to the manufacturer's instructions. After an initial DNA denaturation of 94°C for 2 min, the PCR consisted of 10 cycles of 94°C for 15s, 61 °C for 30s, 68°C for 8 min followed by 20 cycles of 94 °C for 15s, 61 °C for 30s, and 68° C for 8 min with a 5 second addition to each extension. The samples were incubated at 72 °C for 30 min after the last cycle and then cooled to 4°C. Results of PCR reactions were evaluated on 0.9% agarose gels. PCR products representing the correct sized fragments were isolated from agarose and were directly cloned into the pCR II plasmid and amplified in Escherichia coli bacteria (TA cloning kit, Invitrogen Corp., San Diego, CA) according to the manufacturer's protocol. Single bacterial colonies containing plasmids with inserts of the correct size were grown at 30°C overnight and plasmid DNA prepared by the alkaline lysis method.
The strategy for preparing full-length molecular clones is illustrated in Figure 3. Several restriction enzymes were used to generate restriction maps for the positive clones. The Apa I fragment (1947 bp) from the 5' half PCR product was gel-purified and subcloned into the large fragment (7675 bp) of pJC using standard cloning procedures [Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY] to generate plasmid pHIV-lJC]6 (Fig. 1). A chimeric plasmid (pHIV-lNCjc) was generated by subcloning the PCR amplified 5' half of HIV-1NC into pHIV- Ijciό- F°r HIV-INC full-length clone, the Nco l-Xho I ettv-containing fragment from the 3' half PCR product was gel purified and subcloned into the Nco l-Xho I large fragment of plasmid pHTV-lNCjc containing the 5' half of HIV- 1NC. Multiple restriction enzymes were used for analysis of both viral DNAs to confirm the full length clones.
5 x 106 CEMxl74 cells in T-25 flasks were transfected with 2 μg of either pHIV-lJC]6 orNC7 DNA in transfection buffer (25 mM Tris-HCl)(pH 7.5, 140 mM NaCl, 5 mM Kcl, 0.7 mM K2HPO43H2O) containing 4 μl of DEAE-dextran (60 mg/ml) for 20 minutes at room temperature. Five miUiliters of complete medium (RPMI 1640 supplemented with 10%) fetal bovine serum (FBS) and 2 mM L-glutamine) was added to stop the reaction followed by centrifugation of cells at 1000 rpm for 10 minutes. The cells were washed twice in 10 ml complete medium before they were resuspended in 10 ml complete medium, transferred to a T-25 cm2 flask and incubated at 37°C (5%> CO2). The cells were checked daily for cytopathic effects (syncytia formation) and aliquots of cultures were tested for the presence of reverse transcriptase (RT) activity using standard assay methods. For 293 cell lines, 2 x 105 cells in 6-well plates were transfected with 2 μg of viral DNA using lipofectin (Life Technologies, Gaithersburg, MD) or DOTAP (Boehringer Mannheim, Indianapolis, IN) according to the manufacturers' instructions. After 24 hours, the transfected cells were overlaid with 2 x 106/well of uninfected cPBMC previously stimulated with concanavalin A (Con- A) for 4 days. After an additional 2 day incubation, the non-adherent cell population (cPBMC) were transferred to a T-25 flask and additional stimulated cPBMC added for virus amplification. Culture supernatants were assayed for RT activity and the cells were observed daily for development of syncytia. Cell-free stocks of molecularly cloned viruses were prepared at peak RT activity, aliquoted and stored under liquid nitrogen.
Primers for sequencing were constructed from conserved regions of aligned sequences of HIV-1LAI and HIV-1SF2 and were synthesized on an applied Biosystems 392 DNA synthesizer. The DNA sequence of each full-length clone virus was determined by the dideoxy-chain termination method using the sequenase system (Amersham Life Sciences, Arlington Heights, IL) and 35S-dATP. Nucleotide sequence alignments were performed with the Intelligenetics Suite of programs (Intelligenetics, Beaverton, OR) whild the phylogenetic analysis of amino acid sequence was done with CLUSTAL method (DNASTAR, Inc., Madison, WI).
Example 9. Heteroduplex Mobility Assays.
The nucleotide sequences for HIV-1JC16 and HIV-1NC have been assigned Genbank accession numbers AF049494 and AF049495 respectively.
The heteroduplex mobility assay kit (NIH AIDS Research and Reference Reagent Program) based on the method described by Delwart et al. (1993) Science 262, 1257-1261 was used. Briefly, equal amounts (5 μl each) of second-round PCR products (VI -V2 and V3- V5) from infected cPBMC genomic DNA were mixed with the reference PCR products to obtain heteroduplexes. After adding 1.1 μl of lOx annealing buffer (1 M NaCl, 100 mM Tris [pH 7.8], 20 mM EDTA), the mixed DNAs were denatured at 94 °C for 2 min and then reannealed by rapidly cooling in ice. Three μl of loading dye (25% Ficoll, 1% Orange G) was added to the cooled DNA mixture and the samples loaded onto 5% polyacrylamide gel in lx TBE (88 mM Tris-borate, 89 mM boric acid, 2 mM EDTA) buffer and electrophoresed at a constant voltage of 250 V for 2.5 hrs. The gels were stained with ethidium bromide and visualized under ultra-violet (UV) light. TABLE 1 Platelet counts, l m hoc te subset levels and lasma HIV-1 loads in C499
Figure imgf000049_0001
* Lymphocyte subset counts in peripheral blood were determined by FACScan analysis as described previously (Ahmed-
Ansari et al. [1989] Am. J. Primatol. 17:107-131). Virus levels were determined using the Chiron B-DNA assay as directed by the manufacturer. Plasma samples were stored at - 80°C until use. c ND, not done (no plasma sample was available for this date).
Table 2A. Alignment of HIV-1JC (SEQ ID NO:2) and H-V-l^ env (SEQ ID NO:3) Protein Sequences. The consensus sequence corresponds to SEQ ID NO:6.
HIVJCENV 1 MRvkEncqhl rwgWK GIMLLGMLMiCSAtEkL VTVYYGVPvWKEtTTTLFCASDAKAY
I I I 0 0 0 I I I I I M M I M I I I I I E i 1 1 f 1 1 1 1 1 i 1 1
HIVNDKENV 1 MRarE kemcqnlWKWGIMLLGMLMtCSAaEdLWVTVYYGVPiWKEaTTTLFCASDAKAY consensus MR- -En WK GIMLLGM___M-CSA-E- VTVYYGVP-WKE-TTTLFCASDAKAY
HIVJCENV 62 eeE HN ATHACrVPTDPNPQEI LaNVTEdFNM KNeW^:QMHtDIIS WDeSLKPCVKL
HIVNDKENV 61 kkEaHNi ATHACVPTDPNPQEIeLeNVTEnE^M KNnMVEQMHeDIISLVTOqS KPCVKL consensus --E-HN- TH CVPTDPNPQEI- -NV E-FN_WKN-WEQMH-DIIS ro-SLKPCV
HIVJCENV 123 TPLCVTLNCTDlknEtktNSsdaNsnsgEimgnEeiKNCSFNVstgapgkvqkeYa fyal
HIVNDKENV 122 TPLCVTLNCTD E lrNS kgNgkveE eEkrKNCSFNVrdkreqvyalfYk div consensus TP CVT NCTDlknEt- -NSs--N Ei g-E- -KNCSFNV Y-Lf
HIVJCENV 184 dlvsikNenNSTshmLtsCnTSvsTQACPKvSFEPIPIHyCAPAGFAILKCnDKKFNGTGP
HIVNDKENV 174 pi dnnNrtNSTnyrLinCdTStiTQACPKiSFEPIPIHfCAPAGFAILKCrDKKFNGTGP consensus -Iv N--NST L--C-TS--TQACPK-SFEPIPIH-CAPAGFAILKC-DKKFNGTGP
HIVJCENV 245 CnNVSTVQCTHGIRPWSTQLLLNGSvAEEEwlRSaNfsdNaKTIIVQLN SveltCTRP
HIVNDKENV 234 CsNVSTVQCTHGIRPWSTQLLLNGSlAEEEiiiRSeNltnNvKTIIVQLNaSivInCTRP consensus C-NVSTVQCTHGIRPWSTQLLLNGS-AEEE RS-N N-KTIIVQLN-S--I-CTRP
HIVJCENV 306 nynetkkirlhrgygrsfvT vrKlGdrkQAHCt nRtk dnALkQiAsKLreqfNKTal
HIVNDKENV 295 ykytrqrtsIglrqslytiTgkkkKtGyigQAHCkisRaeWnkALqQvAtKLgnllNKTtl consensus 1 Tgk--K-G QAHC R-- --AL-Q-A-KL NKT-I
HIVJCENV 365 iFnrSSGGDlEIemHsfNCGGelFYCNTtkLFNST NeTtesngkgeniTLPCRIrQfVNm
HIVNDKENV 356 tFkpSSGGDpEItsHmlNCGGdfFYCNTsrLFNST NqTnstgfnngtvTLPCRIkQiVNl consensus -F- -SSGGD-EI- -H- -NCGG- -FYCNT- -LFNSTWN-T TLPCRI-Q-VN-
HIVJCENV 426 WQkVGKAMYAPPsdGqlrCtSNITGLLLTRDGGhndNNtnnETfRPGrGDMRDNWRSELYK
HIVNDKENV 417 WQrVGKAMYAPPieGlIkCsSNITGLLLTRDGG aNNsshETiRPGgGDMRDNWRSELYK consensus WQ-VGKAMYAPP--G-I-C-SNITGLLLTRDGGhn-NN ET-RPG-GDMRDN RSELYK Table 2A. (Continued)
HIVJCENV 487 YKViKIEPlGVAPTKAkRRWqREKRAvGmvGAmFLGFLGAAGSTMGAASlTLTVQARQLl HIVNDKENV 476 YKVvKIEPiGVAPTKArRRWeREKRAiG IGAvFLGFLGAAGSTMGAASvTLTVQARQLm consensus YKV-KIEP-GVAPTKA-RRW-REKRA-Gm-GA-FLGFLGAAGSTMGAAS-TLTVQARQL-
HIVJCENV 548 SGIVqQQNNLLRAIEAQQHLLQLT GIKQLQARVLAVERYLkDQQLLGIWGCSGklICTT HIVNDKENV 536 SGIVhQQNNLLRAIΞAQQHLLQLTVWGIKQLQARVLAVERYLrDQQLLGI GCSGrhlCTT consensus SGIV-QQNNLLRAIEAQQHLLQLTVWGIKQLQARVLAVERYL-DQQLLGI GCSG- - ICTT
HIVJCENV 609 tVP NaS SNkSLDqI nNMT mEWdREIaNYTnLIhhLIEESQnQQEKNEqELLELDK A HIVNDKENV 597 nVPWNsSWSNrSLDelWqNMTWmEWeREIdNYTgLIysLIEESQiQQEKNEkELLELDKWA consensus -VP N-S SN-SLD-I -NMT -E -REI-NYT-LI--LIEESQ-QQEKNE-ELLELDK A
HIVJCENV 670 SLWsWFdlsnWL YIKiFIMIVaGLvGLRIVFAVLSiVNRVRQGYSPLSFQThfPaPRGPD
III II I MINI Mill II MINIUM MIMIMMIMM I Mill
HIVNDKENV 658 SL nWFsItk L YIKlFIMIVgGLiGLRIVFAVLSvVNRVRQGYSPLSFQTllPvPRGPD consensus SLW-WF-I--WLWYIK-FIMIV-GL-GLRIVFAVLS-VNRVRQGYΞPLSFQT--P-PRGPD
HIVJCENV 731 RPdglEgEGGERdRDRSvRLVdGflALlWeDLRNLCLFSYHRLRDllLIvtRIVELLGRRG HIVNDKENV 719 RPeelEeEGGERgRDRSiRLVnGlfALfWdDLRNLCLFSYHRLRDsiLIaaRIVELLGRRG consensus RP- - IE-EGGER-RDRS-RLV-G- -AL- -DLRNLCLFSYHRLRD- -LI- -RIVELLGRRG
HIVJCENV 792 WEALKYL sLLQY SQΞLkNSAvnLfnTtAIvVAEgTDRilEWQRlCRAILhiPRRIRQG
IMIIIM IMMIIII III I I II III III 01 Mill 010
HIVNDKENV 780 EALKYL nLLQY SQELrNSAssLldTiAIaVAErTDRvIEWQRaCRAILnvPRRIRQG consensus EALKYL -LLQYWSQEL-NSA--L--T-AI-VAE-TDR-IEWQR-CRAIL--PRRIRQG
HIVJCENV 853 LERiLL HIVNDKENV 841 LERILL consensus LER-LL
Alignment score = 541.00 Table 2B. Alignment of HIV-1JC and HIV-1LAI env Protein Sequences (SEQ ID NO:2 and SEQ ID NO:4, respectively). The consensus sequence corresponds to SEQ ID NO:7.
HIVJCENV 1 MRVKEncQHL RWGWKWGiMLLGmlMICSATEKiWVTVYYGVPVWKEtTTTLFCASDAKAY
HIVlaienvt 1 MRVKEkyQHL RWGWK GtMLLGiLMICSATEKL VTVYYGVPVWKEaTTTLFCASDAKAY consensus MRVKE- -QHL R G KWG-MLLG-LMICSATEKL VTVYYGVPVWKE-TTTLFCASDAKAY
HIVJCENV 62 eeEVHNV ATHACVPTDPNPQEiVLaNVTEdFNM KNeMVEQMHtDIISL DeSLKPCVKL
HIVlaienvt 62 dtEvπNVWATH Cr P DPNPQEvV vrVTEnFNM KNdMVEQI^eDIISL DqSLKPCVKL consensus --EVHNV ATHACVPTDPNPQE-VL-NVTE-FNMWKN-MVEQMH-DIISL D-SLKPCVKL
HIVJCENV 123 TPLCVtLnCTDLkNeTkTNSSdaNSnSGEiM gneEIKNCSFNvSTgapGKVQKEYalFYa
HIVlaienvt 123 TPLCVsLkCTDLgNaTnTNSSntNSsSGEmMmekgEIKNCSFNiSTsirGKVQKEYafFYk consensus TPLCV-L-CTDL-N-T-TNSS--NS-SGE-Mm EIKNCSFN-ST GKVQKEY--FY-
HIVJCENV 183 LDIvsIkneNnsTShmLTSCNTSVsTQACPKVSFEPIPIHYCAPAGFAILKCNdKkFNGTG
HIVlaienvt 184 LDI IpidNdtTSytLTSCNTSViTQACPKVSFEPIPIHYCAPAGFAILKCNnKtFNGTG consensus LDIvsI N- -TS- -LTSCNTSV-TQACPKVSFEPIPIHYCAPAGFAILKCN-K-FNGTG
HIVJCENV 244 PCnNVSTVQCTHGIRPWSTQLLLNGSvAEEEWlRSANFsDNAKTIIVQLNhSVEItCTR
HIVlaienvt 243 PCtNVSTVQCTHGIRPWSTQLLLNGSlAEEEWiRSANFtDNAKTIIVQLNqSVEInCTR consensus PC-NVSTVQCTHGIRPWSTQLLLNGS-AEEΞW-RSANF-DNAKTIIVQLN-SVEI-CTR
HIVJCENV 305 PNyNetKklRIhRGyGRsFVTvrKlGdrkQAHCtmnRtK dnaLKQIASKLREQFnNktal
HIVlaienvt 304 PNnNtrKsIRIqRGpGRaFVTigKiGnmrQAHCnisRaK natLKQIASKLREQFgNnktl consensus PN-N- -K- IRI-RG-GR-FVT- -K-G QAHC R-K LKQIASKLREQFgN 1
HIVJCENV 365 IFnrSSGGDlEIemHSFNCGGElFYCNtTkLFNST NeT TE SN gkG enlTLPCRI
HIVlaienvt 365 IFkqSSGGDpEIvtHSFNCGGEfFYCNsTqLFNSTWfNsTwsTEgSNnteGsdtlTLPCRI consensus IF--SSGGD-EI--HSFNCGGE-FYCN-T-LFNSTWfN-T sTEgSNn--Gs--ITLPCRI
HIVJCENV 420 rQFvNMWQkVGKAMYAPPsdGQIRCtSNITGLLLTRDGGhNdNNtnnEtFRPGrGDMRDN
HIVlaienvt 426 kQFiNMWQeVGKAMYAPPisGQIRCsSNITGLLLTRDGG N NNngsEiFRPGgGDMRDNW consensus -QF-NMWQ-VGKAMYAPP--GQIRC-SNITGLLLTRDGGhNdNN E-FRPG-GDMRDNW Table 2B. (Continued)
HIVJCENV 481 RSELYKYKViKIEPLGVAPTKAKRRWQREKRAVGmvGAmFLGFLGAAGSTMGAaSlTLTV
HIVlaienvt 485 RSELYKYKVvKIΞPLGVAPTKAKRRWQREKRAVG iGAlFLGFLGAAGSTMGArSmTLTV consensus RSELYKYKV-KIEPLGVAPTKAKRRWQREKRAVGm-GA-FLGFLGAAGSTMGA-S-TLTV
HIVJCENV 542 QARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARvLAVERYLKDQQLLGIWGCSG
HIVlaienvt 545 QARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARiLAVERYLKDQQLLGIWGCSG consensus QARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQAR- AVERYLKDQQLLGIWGCSG
HIVJCENV 603 KLICTTtVPWNASWSNKSLdQIWNNMTWmEWDREIaNYTnLIHhLIEESQNQQEKNEQELL
HIVlaienvt 606 KLICTTaVPWNASWSNKSLeQIWNNMTWmEWDREInNYTsLIHsLIEESQNQQEKNEQELL consensus KLICTT-VPWNASWSNKSL-QIWNNMTW-EWDREI-NYT-LIH-LIEESQNQQEKNEQELL
HIVJCENV 664 ELDKWASLWsWFdlsNWLWYIKIFIMIVaGLVGLRIVFAVLSIVNRVRQGYSPLSFQTHfP
HIVlaienvt 667 ELDKWASLWnWFnItNWLWYIKIFIMIVgGLVGLRIVFAVLSIVNRVRQGYSPLSFQTHIP consensus ELDKWASLW-WF-I-NWLWYIKIFIMIV-GLVGLRIVFAVLSIVNRVRQGYSPLSFQTH-P
HIVJCENV 725 aPRGPDRPdGIEgEGGERDRDRSvRLVdGfLALlWeDLRnLCLFSYHRLRDLLLIVTRIVE
HIVlaienvt 728 tPRGPDRPeGIEeEGGERDRDRSiRLVnGsLALiWdDLRsLCLFSYHRLRDLLLIVTRIVE consensus -PRGPDRP-GIE-EGGERDRDRS-RLV-G-LAL-W-DLR-LCLFSYHRLRDLLLIVTRIVE
HIVJCENV 786 LLGRRGWEALKYlWsLLQYWSQELKNSAVnLfNtTAIvVAEGTDRilEWQrlCRAIlHIP
HIVlaienvt 789 LLGRRGWEALKYwWnLLQYWSQELKNSAVsLlNaTAIaVAEGTDRvIEWQgaCRAIrHIP consensus LLGRRGWEALKY-W-LLQYWSQELKNSAV-L-N-TAI-VAEGTDR- IEWQ- -CRAI-HIP
HIVJCENV 847 RRIRQGLERiLL
HIVlaienvt 850 RRIRQGLERiLL consensus RRIRQGLER-LL
Alignment score = 657.00 Table 2C. Alignment of HIV- 1 JC and HIV- 1 SF2 env Protein Sequences (SEQ ID NO:2 and SEQ ID NO:5, respectively). The consensus sequence corresponds to SEQ ID NO:8.
HIVJCENV 1 MrVKencqhl rwgWkWGimLLGMI_MICSATEKLWVTVYYGVPVWKEtTTTLFCASDAkAY
HIVsf2envt 1 MkVK gtrrnyqhlWrWGtlLLGMLMICSATEKLWVTVYYGVP KEaTTTLFCASDArAY consensus M-VKe W-WG- -LLGMLMICSATEKLWVTVYYGVPVWKE-TTTLFCASDA-AY
HIVJCENV 62 eeEVHNVWATHACVPTDPNPQEiVLaNVTEdFNMWKNeMVEQMhtDIISLWDeSLKPCVKL
IMMMMMMMIMM II MM Mill Mlllll MIMMI
HIVsf2envt 61 dtEVHNVWATHACVPTDPNPQEvVLgNVTEnFNMWKNnMVEQMqeDIISLWDqSLKPCVKL consensus --EVHNVWATHACVPTDPNPQE-VL-NVTE-ETiIMWKN-MVEQM--DIISLWD-SLKPCVKL
HIVJCENV 123 TPLCVTLNCTDLkneTkTNSSdaNsnsgeimgneeikncsfnvstgapgKvqkeyalfyal
HIVsf2envt 122 TPLCVTLNCTDLgkaTnTNSS NwkeeikgeikncsfnittsirdkiqKenalfrnldw consensus TPLCVTLNCTDL T-TNSSdaN K
HIVJCENV 184 dlvsiknenNsTshmLtsCNtSVsTQACPKVSFEPIPIHYCaPAGFAILKCNdKkFNGtGP
HIVsf2envt 181 pldnastttNyTnyrLihCNrSViTQACPKVSFEPIPIHYCtPAGFAILKCNnKtFNGkGP consensus -I N-T L- -CN-SV-TQACPKVSFEPIPIHYC-PAGFAILKCN-K-FNG-GP
HIVJCENV 245 CnNVSTVQCTHGIRPvVSTQLLLNGSvAEEEWlRSaNFsdNAKTIIVQLNhSVeltCTRP
HIVsf2envt 242 CtNVSTVQCTHGIRPiVSTQLLLNGSlAEEEWiRSdNFtnNAKTIIVQLNeSValnCTRP consensus C-NVSTVQCTHGIRP-VSTQLLLNGS-AEEEW-RS-NF--NAKTIIVQLN-SV-I-CTRP
HIVJCENV 306 NyNetKklrl rGygrsf TvRklGDrkqAHCtmnRtkWdNaLkQIasKLREQFnNktali
HIVsf2envt 303 NnNtrKsIylgpG rafhtTgRiiGDirkAHCnisRaqWnNtLeQIvkKLREQFgNnktlv consensus N-N--K-I-I--Gy T-R--GD AHC R--W-N-L-QI--KLREQFgN 1-
HIVJCENV 366 FNrSSGGDlEIeMHSFNCgGElFYCNTTkLFNsTW netTEsngkgenltLPCRIrQfvNM
HIVsf2envt 363 FNqSSGGDpEIvMHSFNCrGEfFYCNTTqLFNnTWrlnhTEgtkgndtliLPCRIkQiiNM consensus FN-SSGGD-EI-MHSFNC-GE-FYCNTT-LFN-TWr TE I-LPCRI-Q--NM
HIVJCENV 426 WQkVGKAMYAPPsdGQIrCtSNITGLLLTRDGGhNdnNtnnEtFRPGrGDMRDNWRSELYK
II 00010 III I MMMMIMM I I I MM IMMMMMM
HIVsf2envt 424 WQeVGKAMYAPPigGQIsCsSNITGLLLTRDGGtNvtN dtEvFRPGgGDMRDNWRSELYK consensus WQ-VGKAMYAPP--GQI-C-SNITGLLLTRDGG-N--Nt--E-FRPG-GDMRDNWRSELYK Table 2C. (Continued)
HIVJCENV 487 YKVIKIEPLGvAPTKAKRRWQREKRAVGmVGAMFLGFLGAAGSTMGAaSLTLTVQARQLL
0 0 i M M I M M I M M M M M M M M I M M I M M 1 1 1 1 1 1 1 I I i 1 1
HIVsf2envt 484 YKVIKIEPLGiAPTKAKRRWQREKRAVGiVGAMFLGFLGAAGSTMGAvSLTLTVQARQLL consensus YKVIKIEPLG-APTKAKRRWQREKRAVG-VGAMFLGFLGAAGSTMGA-SLTLTVQARQLL
HIVJCENV 548 SGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARVLAVERYLkDQQLLGIWGCSGKLICTT
HIVsf2envt 545 SGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARVLAVERYLrDQQLLGIWGCSGKLICTT consensus SGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARVLAVERYL-DQQLLGIWGCSGKLICTT
HIVJCENV 609 tVPWNASWSNKSLdqlWnNMTWmeWdREIaNYTNlIhhLiEESQNQQEKNEQELLELDKWA
1 1 1 1 1 1 1 1 1 1 1 1 I I M M I I I I M M I I I I I I I I I I I I I 1 1 I I I I I I I I
HIVsf2envt 606 aVPWNASWSNKSLedlWdNMTWmqWeREIdNYTNtlytLlEESQNQQEKNEQELLΞLDKWA consensus -VPWNASWSNKSL- - IW-NMTW- -W-REI-NYTN- I- -L-EESQNQQEKNEQELLELDKWA
HIVJCENV 670 SLWsWFdlsNWLWYIKIFIMIVaGLVGLRIVFAVLSIVNRVRQGYSPLSFQThf aPRGPD
HIVsf2envt 667 SLWnWFsItNWLWYIKIFIMIVgGLVGLRIVFAVLSIVNRVRQGYSPLSFQTrlPvPRGPD consensus SLW-WF-I-NWLWYIKIFIMIV-GLVGLRIVFAVLSIVNRVRQGYSPLSFQT--P-PRGPD
HIVJCENV 731 RPDGIEgEGGERDRDRSVRLVDGFLALlWEDLRnLCLFSY RLRDLLLIVtRiVElLGrRG
HIVsf2envt 728 RPDGIEeEGGERDRDRSVRLVDGFLALiWEDLRsLCLFSYrRLRDLLLIaaRtVΞiLGhRG consensus RPDGIE-EGGERDRDRSVRLVDGFI_AL-WEDLR-LCLFSY-RLRDLLLI--R-VE-LG-RG
HIVJCENV 792 WEALKYlWSLLQYWsQELKNSAVnlfNtTAIvVaEGTDRilEWQRlcRAILHIpRRIRQG
HIVsf2envt 789 WEALKYwWSLLQYWiQELKNSAVs lNaTAIaVtEGTDRvIEVaQRayRAILHIhRRIRQG consensus WEALKY-WSLLQYW-QELKNSAV N-TAI-V-EGTDR-IEV-QR- -RAILHI-RRIRQG
HIVJCENV 853 LERiLL
HIVsf2envt 850 LERILL consensus LER-LL Alignment score = 614.00 Table 3. HIV-1 Antibody Response of C534 After Transfusion From C455
Figure imgf000056_0001
Table 4. Comparative Analyses of Chimpanzee HIV-1 Isolates With Inoculating Viruses
Figure imgf000057_0001
'Amino acid homology
2 Amino acids or nucleotides not observed in parental viruses
'Nucleotide identity Table 5
-MBD l-> matrix protein (pl7)
HIV-1JC16 gag MGARAS VLSGGDLDRWEKIRLRPGGKKKYMLKHI VWASRELERFAVNPG
HIV-1NC7 gag
HIV-1LAI gag E K
HIV-1SF2 gag E. . K K
HIV-INDK gag K. . T . . R A. . . LI TL o HIV-1JC16 gag GS EELRSLYNTI ATLYGVHQKIEVKDTKEALDKI EEEQNKSKKKAQQAA HIV-1NC7 gag C E V HIV-1LAI gag V. . . . C . . . R. . I HIV-1SF2 gag V. . . . C . . .R.D E HIV-INDK gag . . . . I V. . . . C . . ER VE .M T capsid protein (p24) j HIV-1JC16 gag I VQNLQGQMVHQAI S PRTLNAWVKVIEEKAFS PEVI PMFS ALS EGATPQ
HIV-1NC7 gag V SJX HIV-1LAI g 3ag 3 . .. . I V < HIV-1SF2 gag V j HIV-INDK gag J HIV-1JC16 gag T INEEAAEWDRVHP VQ GL I PGQMRE PRGS D I AGTTSTLQEQ I GWMTS
HIV-1NC7 gag
HIV-1LAI gag H. . P N
HIV-1SF2 gag H . . P N
HIV-INDK gag . . . D L . . . H . . PV A
HIV-1JC16 gag VRM YS P I S I LD I RQG P KE P FRD Y VDR F YKTLR AEQ S QE VKNWMTE TLL
HIV-1NC7 gag
HIV-1LAI gag T
HIV-1SF2 gag T D
HIV-INDK gag V D p24 <-l I p2 1-> Nucleocapsid [
HIV-1JC16 gag EEMMTACQGVGGPGHKARVLAEAMSQVT - NS AA I MMQRGNF KNQRKMVK
HIV-1NC7 gag -
HIV-1LAI gag - . . . T R. . . . I
HIV-1SF2 gag - . P .N R . . . .T
HIV-INDK gag GS T .V GP . . SI
Figure imgf000058_0001
Table 5 (continued)
[ zinc finger ] p7 <-l pi l-> p6
HIV-1JC16 gag C WKCGKEGHQMKDC I E S Q ANFLGK I W P SHKGR PGNFLQ S R P E PTAP P EESLRSGIETT 47
HIV-1NC7 gag R 47
HIV-1LAI gag T.R Y FLQSRPEPTAPP . . . F . . . V . . . 48
HIV-1SF2 gag . .R. .R T.R Y . . . F. F.E.K. 47
HIV-INDK gag R . . . . 42 0 p6 <-\
HIV-1JC16 gag TP S QKQE P I DKE VYPLT S LRS L FGND P S S Q 50 o
HIV-1NC7 gag 50
HIV-1LAI gag L 51
HIV-1SF2 gag L 50
HIV-INDK gag 42 0
)
Table 5 (continued)
-MTD-
HIV-1JC16 nef MGGKW S KS S I VG WPT I RERMKRAG P AADG VG AAS RDLE KHG A I S S T AATNADC A 56
HIV-1NC7 nef R 56
HIV-1LAI nef V V. . . . R . . E A. . 56
HIV-1SF2 nef R . MG . . S A R . . EPRAE V 60 HIV-INDK nef L . . . . A . . . . IRKTD V S . . DT . . 56 LCK binding domain J I I ' ' HIV-1JC16 nef WLEAQEE - EE VG F P VRPQ VP LR PM YKAG I DL S H FLKE KGGLEGL VWS QRRQD I LD W I Y 115 j HIV-1NC7 nef - 115 HIV-1LAI nef - T AV IH 115 ! HIV-1SF2 nef - AL . I I E 119 HIV-INDK nef S EAV I . . KK . . E V . 116 HIV-1JC16 nef HTQGYFPDWQNYTPGPGIRYPLTFGWCFKLVPVEPDKVEEANEGEDNILLHPMCLHGMED 175 tπ HIV-1NC7 nef 175 "^ HIV-1LAI nef V Y K. . NTS . . . . VS . . . . D . 175 HIV-1SF2 nef E N. S S 179 } HIV-INDK nef N. . . I Q. . . .D.QE . . . .T.R. . .C QQ- . . . 176
HIV-1JC16 nef AE KEVL VWR FD S KL AFHH V ARELH P E YYKDC 207
HIV-1NC7 nef 207
HIV-1LAI nef P . R . . . E R F .N. 207
HIV-1SF2 nef M 211
HIV-INDK nef P . RQ . . M . . . N . R . . LE . K F . . . . 208
Figure imgf000060_0001
Table 5 (continued) signal peptide <-l->gpl20 (SU)
HIV-1JC16 env MRVKE NCQHLWR WG WKWG I MLLGMLM I C S ATE KL WVT VY YG VP VWKETTTTL FC AS D 57
HIV-1NC7 env .Y A 57
HIV-1LAI env KY T . . . . I A 57
HIV-1SF2 env . K. .GTRR.Y TL A 56
HIV-INDK env . . AR . KER . . .N. . T . . .A.D I . . . A 56 117 117 117 116 116 176 176 177 170 163 233
Figure imgf000061_0001
233
HIV-1LAI env . . F. . K. . . IP .D .DTT . YT I 232
HIV-1SF2 env N . . . RN . . V . P . D . ASTT . NYTNYR . IH . . R . . I T 230
HIV-INDK env K. . . . P . D .N. RTN . - TNYR . IN. D . . TI I F 222
HIV-1JC16 env CNDKKFNGTG P CNNVS T VQCTHG I R VVS TQLLLNG S VAE E EV VLRS ANFS DNAKT I I VQ 293
HIV-1NC7 env L 293
HIV-1LAI env . .N. T T L I T 292
HIV-1SF2 env . .N. T . . . K. . . T I L I . . D . . TN 290
HIV-INDK env . R S L. . . . III . . E . LTN.V 282
I V3
HIV-1JC16 env LNHSVEITCTRPNYNETKKIRIHRjGYGRS ! FVTV RKLGDRKQAHCTMNRTKWDNALK 349
HIV-1NC7 env R . . . . . . . 349
HIV-1LAI env . .Q. . . .N N.TR. S . . .Q. . P . .A . . . IG KI- . NMR . . . . NIS . A. . NAT . . 348
HIV-1SF2 env . . E . .A.N N.TR. S .Y. - - . P. .A -H. TG . II . . IRK . . . NIS . AQ . N . T . E 345
HIV-INDK env . .A. IV.N. . . . YKYTRQRTS . ■ LRQ LY . ITGKKK. T. YIG . . . . KIS .AE .NK. .Q 340
Table 5 (continued)
HIV-1JC16 env Q I AS KLREQ FNNKT A I I FNRS S GGDLE I EMHS FNCGGE L F YCNTTKL FNS T W NE 403
HIV-1NC7 env - . . 402
HIV-1LAI env G . NKT . . . KQ P . . VT F . . . . S . Q FNSTWST . 408
HIV-1SF2 env . . VK G . NKT . V . . Q P . .V R. . F Q . . .N. WRLN 400
HIV-INDK env .V. T. . -GNLL . . . T. T . KP P . - TS .ML. . . . DF SR . Q 393 j CD4 binding V4 1 I I I V5-- | HIV-1JC16 env TTESNGKGENI TLPCR IRQFVNMWQKVGKAMYAPPSDGQ I RCTSNITGLLLTRDGGHNDN 463 HIV-1NC7 env GPSD 462 HIV-1LAI env GSNNTEGSDT K. . I . . . . E IS S N. - . 467 | HIV-1SF2 env H . . GTKGNDT .1 K. II . . . . E IG. . . S . S T.VT 460 HIV-INDK env .NSTGFNNGTV K. I . . L . . R IE . L . K. S A. - - 451 V5 I gpl20 (SU) <-l->gp41 (TM) l HIV-1JC16 env NTNNE T FR PGRGDMRDNWR S E L YKYKV I K I E P LG VAP TKAKRR VVQRE KRAVGMVG AMFL 523 HIV-1NC7 env . KTDK G 522 <g HIV-1LAI env . -GS . I . . . .G V I- . . L. . 525 HIV-1SF2 env . -DT.V. . . . G I I 519
HIV-INDK env . SSH. . I . . . G V. . . . I R . . . . E I . L- . .V. . 510
HIV-1JC16 env G FLG AAG S TMG AAS LTLT VQ ARQLLS G I VQQQNNLLR A I E AQQHLLQLTVWG I KQLQ ARV 583
HIV-1NC7 env 582
HIV-1LAI env R .M I 585
HIV-1SF2 env V 579
HIV-INDK env V M. . . . H 570
HIV-1JC16 env LAVER YLKDQQLLG I WGC S GKL I CTTT VP NAS WSNKS DQ I WNNMT ME DRE I ANYTN 643
HIV-1NC7 env L 642
HIV-1LAI env A E N. . . S 645
HIV-1SF2 env R A ED . . D Q . E . . . D . . . . 639
HIV-INDK env R RH . . . .N. . . . S . . . . R. . . E . . Q E . . . D . . . G 630
HIV-1JC16 env L I HHL I EE S QNQQEKNEQE LLE LDKWAS L S WFD I SNWL Y I KI F I M I VAGL VGLR I VF A 703
HIV-1NC7 env 702
HIV-1LAI env .. .S N. .N.T G 705
HIV-1SF2 env T.YT.L N..S.T G 699
HIV-INDK env . .YS I K N. .S.TK L G. .I 690
Table 5 (continued)
HIV-1JC16 env VLS I VNR VRQG YS P S FQTH F P APRG PDR PDG I EGEGGERDRDRS VRLVDG FL ALL WEDL 763
HIV-1NC7 env 762
HIV-1LAI env L.T E. . . E I . . .N. S . . . I .D. . 765
HIV-1SF2 env RL . V E I . . . . 759
HIV-INDK env . . .V LL .V EE . . E G . . . . I . . .N. LF . . F .D . . 750 HIV-1JC16 env RNL C L FS YHRLRDLLL I VTR I VE LLGRRG E ALKY WS LLQ YWS QELKNS AVNL FNTT A I 823
HIV-1NC7 env W R . . 822 HIV-1LAI env . S W.N S . L .A. . . 825 J HIV-1SF2 env . S R AA. T . . I . .H W I SWL .A. . . 819 HIV-INDK env SI . .AA N R. . . SS . LD . I . . 810 HIV-1JC16 env VVAE GTDR I I E VVQRL CR A I LH I PRR I RQGLER I LL 860
HIV-1NC7 env F . . 859 * HIV-1LAI env A V GA . . . . R 862 SJX HIV-1SF2 env A. T V. . .A. .AY H L. . 856 S t? HIV-INDK env A . . . R . . . V A NV L . . 847 >
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: EMORY UNIVERSITY
(ii) TITLE OF INVENTION: Human Immunodeficiency Viruses Causing AIDS in a Nonhuman Primate
(iii) NUMBER OF SEQUENCES: 23
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Greenlee, Winner and Sullivan, P.C.
(B) STREET: 5370 Manhattan Circle, Suite 201
(C) CITY: Boulder
(D) STATE: Colorado
(E) COUNTRY: US
(F) ZIP: 80303
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentin Release #1.0, Version #1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: WO
(B) FILING DATE: 23-JUN-1998
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 60/050,548
(B) FILING DATE: 23-JUN-1997
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 60/057,606
(B) FILING DATE: 04-SEP-1997
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Ferber M. , Donna
(B) REGISTRATION NUMBER: 33,878
(C) REFERENCE/DOCKET NUMBER: 66-97 WO
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (303) 499-8080
(B) TELEFAX: (303) 499-8089
(2) INFORMATION FOR SEQ ID NO : 1 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2577 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS : double
(D) TOPOLOGY: not relevant
60 ( ii ) MOLECULE TYPE : cDNA
( iii ) HYPOTHETICAL : NO ( iv) ANTI - SENSE : NO
( ix) FEATURE :
(A) NAME/KEY: CDS
(B) LOCATION: 1..2577
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1 :
ATG AGA GTG AAG GAG AAC TGT CAG CAC TTG TGG AGA TGG GGG TGG AAA 48 Met Arg Val Lys Glu Asn Cys Gin His Leu Trp Arg Trp Gly Trp Lys 1 5 10 15
TGG GGC ATC ATG CTC CTT GGG ATG TTA ATG ATC TGT AGT GCT ACA GAA 96 Trp Gly lie Met Leu Leu Gly Met Leu Met lie Cys Ser Ala Thr Glu 20 25 30
AAA TTG TGG GTC ACA GTC TAT TAT GGG GTA CCT GTG TGG AAG GAA ACA 144 Lys Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp Lys Glu Thr 35 40 45
ACT ACC ACT CTA TTT TGT GCA TCA GAT GCT AAA GCA TAT GAA GAA GAG 192 Thr Thr Thr Leu Phe Cys Ala Ser Asp Ala Lys Ala Tyr Glu Glu Glu 50 55 60
GTA CAT AAT GTT TGG GCC ACA CAT GCC TGT GTA CCC ACA GAC CCC AAC 240 Val His Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp Pro Asn 65 70 75 80
CCA CAA GAA ATA GTA TTG GCA AAT GTG ACA GAA GAT TTT AAC ATG TGG 288 Pro Gin Glu lie Val Leu Ala Asn Val Thr Glu Asp Phe Asn Met Trp 85 90 95
AAA AAT GAA ATG GTA GAA CAG ATG CAT ACT GAT ATA ATC AGT TTA TGG 336 Lys Asn Glu Met Val Glu Gin Met His Thr Asp lie lie Ser Leu Trp 100 105 110
GAT GAA AGC CTA AAA CCA TGT GTA AAA TTA ACC CCA CTC TGT GTT ACT 384 Asp Glu Ser Leu Lys Pro Cys Val Lys Leu Thr Pro Leu Cys Val Thr 115 120 125
TTA AAT TGC ACT GAT TTG AAG AAT GAA ACT AAG ACC AAT AGT AGT GAT 432 Leu Asn Cys Thr Asp Leu Lys Asn Glu Thr Lys Thr Asn Ser Ser Asp 130 135 140
GCC AAT AGT AAT AGC GGG GAA ATA ATG GGG AAC GAA GAG ATA AAA AAT 480 Ala Asn Ser Asn Ser Gly Glu lie Met Gly Asn Glu Glu lie Lys Asn 145 150 155 160
TGC TCT TTC AAT GTC AGC ACA GGC GCA CCA GGT AAG GTG CAG AAA GAA 528 Cys Ser Phe Asn Val Ser Thr Gly Ala Pro Gly Lys Val Gin Lys Glu 165 170 175
61 TAT TCA CTT TTT TAT GCA CTT GAT ATA GTA TCA ATA AAG AAT GAA AAT 576 Tyr Ser Leu Phe Tyr Ala Leu Asp lie Val Ser lie Lys Asn Glu Asn 180 185 190
AAT AGT ACC AGC CAT ATG TTG ACA AGT TGT AAC ACC TCA GTC AGT ACA 624
Asn Ser Thr Ser His Met Leu Thr Ser Cys Asn Thr Ser Val Ser Thr 195 200 205
CAG GCC TGT CCA AAG GTA TCC TTT GAG CCA ATT CCC ATA CAT TAT TGT 672
Gin Ala Cys Pro Lys Val Ser Phe Glu Pro lie Pro lie His Tyr Cys 210 215 220
GCC CCG GCT GGT TTT GCA ATT CTA AAA TGT AAT GAT AAG AAG TTC AAT 720
Ala Pro Ala Gly Phe Ala lie Leu Lys Cys Asn Asp Lys Lys Phe Asn
225 230 235 240
GGA ACA GGA CCA TGT AAC AAT GTC AGC ACA GTA CAA TGT ACA CAT GGA 768
Gly Thr Gly Pro Cys Asn Asn Val Ser Thr Val Gin Cys Thr His Gly 245 250 255
ATT AGA CCA GTA GTG TCA ACT CAA CTG CTG TTA AAT GGC AGT GTA GCA 816 lie Arg Pro Val Val Ser Thr Gin Leu Leu Leu Asn Gly Ser Val Ala 260 265 270
GAA GAA GAG GTA GTA CTT AGA TCT GCC AAT TTC TCA GAC AAT GCT AAA 864
Glu Glu Glu Val Val Leu Arg Ser Ala Asn Phe Ser Asp Asn Ala Lys 275 280 285
ACC ATA ATA GTA CAG CTG AAC CAC TCT GTA GAA ATT ACT TGT ACA AGA 912
Thr lie lie Val Gin Leu Asn His Ser Val Glu lie Thr Cys Thr Arg 290 295 300
CCC AAC TAC AAT GAA ACA AAG AAA ATC CGT ATC CAC AGA GGA TAT GGA 960
Pro Asn Tyr Asn Glu Thr Lys Lys lie Arg lie His Arg Gly Tyr Gly
305 310 315 320
AGA TCA TTT GTT ACA GTA AGA AAA TTG GGA GAT AGG AAA CAA GCA CAT 1008
Arg Ser Phe Val Thr Val Arg Lys Leu Gly Asp Arg Lys Gin Ala His 325 330 335
TGT ACC ATG AAT AGA ACG AAA TGG GAC AAC GCT TTA AAA CAG ATA GCT 1056
Cys Thr Met Asn Arg Thr Lys Trp Asp Asn Ala Leu Lys Gin lie Ala 340 345 350
AGC AAA TTA AGA GAA CAA TTT AAT AAA ACA GCA ATA ATC TTT AAC CGG 1104
Ser Lys Leu Arg Glu Gin Phe Asn Lys Thr Ala lie lie Phe Asn Arg 355 360 365
TCC TCA GGA GGG GAC CTA GAA ATT GAA ATG CAC AGT TTT AAT TGC GGA 1152
Ser Ser Gly Gly Asp Leu Glu lie Glu Met His Ser Phe Asn Cys Gly 370 375 380
GGG GAA TTG TTC TAC TGT AAT ACA ACA AAA CTG TTT AAT AGT ACT TGG 1200
Gly Glu Leu Phe Tyr Cys Asn Thr Thr Lys Leu Phe Asn Ser Thr Trp
385 390 395 400
62 AAT GAG ACT ACA GAG TCA AAT GGC AAG GGA GAA AAT ATC ACA CTC CCA 1248 Asn Glu Thr Thr Glu Ser Asn Gly Lys Gly Glu Asn lie Thr Leu Pro 405 410 415
TGC AGA ATA AGA CAA TTT GTA AAC ATG TGG CAG AAA GTA GGA AAA GCA 1296 Cys Arg lie Arg Gin Phe Val Asn Met Trp Gin Lys Val Gly Lys Ala 420 425 430
ATG TAT GCC CCT CCC AGC GAT GGA CAA ATT AGG TGT ACA TCA AAT ATT 1344 Met Tyr Ala Pro Pro Ser Asp Gly Gin lie Arg Cys Thr Ser Asn lie 435 440 445
ACT GGG CTA CTA TTA ACA AGA GAT GGG GGT CAT AAT GAT AAC AAC ACT 1392 Thr Gly Leu Leu Leu Thr Arg Asp Gly Gly His Asn Asp Asn Asn Thr 450 455 460
AAC AAC GAG ACC TTC AGA CCG GGA AGA GGA GAT ATG AGG GAC AAT TGG 1440 Asn Asn Glu Thr Phe Arg Pro Gly Arg Gly Asp Met Arg Asp Asn Trp 465 470 475 480
AGA AGT GAA TTA TAT AAA TAT AAA GTA ATA AAA ATT GAA CCA TTA GGA 1488 Arg Ser Glu Leu Tyr Lys Tyr Lys Val lie Lys lie Glu Pro Leu Gly 485 490 495
GTA GCA CCC ACC AAG GCA AAG AGA AGA GTG GTG CAG AGA GAA AAA AGA 1536 Val Ala Pro Thr Lys Ala Lys Arg Arg Val Val Gin Arg Glu Lys Arg 500 505 510
GCA GTG GGA ATG GTA GGA GCT ATG TTC CTT GGG TTC TTG GGA GCA GCA 1584 Ala Val Gly Met Val Gly Ala Met Phe Leu Gly Phe Leu Gly Ala Ala 515 520 525
GGA AGC ACT ATG GGC GCA GCG TCA TTG ACG CTG ACG GTA CAG GCC AGA 1632 Gly Ser Thr Met Gly Ala Ala Ser Leu Thr Leu Thr Val Gin Ala Arg 530 535 540
CAA TTA TTG TCT GGT ATA GTG CAG CAG CAG AAC AAT CTG CTG AGA GCT 1680 Gin Leu Leu Ser Gly lie Val Gin Gin Gin Asn Asn Leu Leu Arg Ala 545 550 555 560
ATT GAG GCG CAA CAA CAT CTG TTG CAA CTC ACA GTC TGG GGC ATC AAG 1728 lie Glu Ala Gin Gin His Leu Leu Gin Leu Thr Val Trp Gly lie Lys 565 570 575
CAG CTC CAG GCA AGA GTC CTG GCT GTA GAA AGA TAC CTA AAG GAT CAA 1776 Gin Leu Gin Ala Arg Val Leu Ala Val Glu Arg Tyr Leu Lys Asp Gin 580 585 590
CAG CTC CTG GGG ATC TGG GGT TGC TCT GGA AAA CTC ATT TGC ACC ACT 1824 Gin Leu Leu Gly lie Trp Gly Cys Ser Gly Lys Leu lie Cys Thr Thr 595 600 605
ACT GTG CCT TGG AAT GCT AGT TGG AGT AAT AAA TCT TTG GAT CAG ATT 1872 Thr Val Pro Trp Asn Ala Ser Trp Ser Asn Lys Ser Leu Asp Gin lie 610 615 620
63 TGG AAT AAC ATG ACC TGG TTG GAG TGG GAC AGA GAA ATT GCC AAT TAC 1920 Trp Asn Asn Met Thr Trp Leu Glu Trp Asp Arg Glu lie Ala Asn Tyr 625 630 635 640
ACA AAC TTA ATA CAT CAC TTA ATT GAA GAA TCG CAA AAC CAG CAA GAA 1968 Thr Asn Leu lie His His Leu lie Glu Glu Ser Gin Asn Gin Gin Glu 645 650 655
AAG AAT GAA CAA GAA TTA TTG GAA TTA GAT AAA TGG GCA AGT TTG TGG 2016 Lys Asn Glu Gin Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu Trp 660 665 670
AGT TGG TTT GAC ATA TCA AAC TGG CTG TGG TAT ATA AAA ATA TTC ATA 2064 Ser Trp Phe Asp lie Ser Asn Trp Leu Trp Tyr lie Lys lie Phe lie 675 680 685
ATG ATA GTA GCA GGC TTA GTA GGT TTA AGA ATA GTT TTT GCT GTG CTT 2112 Met lie Val Ala Gly Leu Val Gly Leu Arg lie Val Phe Ala Val Leu 690 695 700
TCT ATA GTA AAT AGA GTT AGG CAG GGA TAC TCA CCA TTG TCA TTC CAG 2160 Ser lie Val Asn Arg Val Arg Gin Gly Tyr Ser Pro Leu Ser Phe Gin 705 710 715 720
ACC CAC TTC CCA GCT CCG AGG GGA CCC GAC AGG CCA GAC GGA ATC GAA 2208 Thr His Phe Pro Ala Pro Arg Gly Pro Asp Arg Pro Asp Gly lie Glu 725 730 735
GGA GAA GGT GGA GAG AGA GAC AGA GAC AGA TCC GTG CGA TTA GTG GAT 2256 Gly Glu Gly Gly Glu Arg Asp Arg Asp Arg Ser Val Arg Leu Val Asp 740 745 750
GGA TTC TTA GCA CTT CTC TGG GAA GAC CTG CGC AAC CTG TGC CTC TTC 2304 Gly Phe Leu Ala Leu Leu Trp Glu Asp Leu Arg Asn Leu Cys Leu Phe 755 760 765
AGC TAC CAC CGC TTG AGA GAC TTA CTC TTG ATT GTA ACG AGG ATT GTG 2352 Ser Tyr His Arg Leu Arg Asp Leu Leu Leu lie Val Thr Arg lie Val 770 775 780
GAA CTT CTC GGA CGC AGG GGG TGG GAA GCC CTC AAA TAT TTG TGG AGT 2400 Glu Leu Leu Gly Arg Arg Gly Trp Glu Ala Leu Lys Tyr Leu Trp Ser 785 790 795 800
CTC CTA CAG TAT TGG AGT CAG GAG CTA AAG AAT AGT GCT GTC AAC TTG 2448 Leu Leu Gin Tyr Trp Ser Gin Glu Leu Lys Asn Ser Ala Val Asn Leu 805 810 815
TTC AAT ACC ACA GCT ATA GTA GTA GCT GAG GGG ACA GAT AGG ATC ATA 2496 Phe Asn Thr Thr Ala lie Val Val Ala Glu Gly Thr Asp Arg lie lie 820 825 830
GAA GTA GTA CAA AGA CTT TGT AGA GCT ATT CTC CAC ATA CCT AGA AGA 2544 Glu Val Val Gin Arg Leu Cys Arg Ala lie Leu His lie Pro Arg Arg 835 840 845
64 ATT AGA CAG GGC TTG GAA AGA TTT TTG CTA TAA 2577
He Arg Gin Gly Leu Glu Arg Phe Leu Leu * 850 855
(2) INFORMATION FOR SEQ ID NO : 2 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 859 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Met Arg Val Lys Glu Asn Cys Gin His Leu Trp Arg Trp Gly Trp Lys 1 5 10 15
Trp Gly He Met Leu Leu Gly Met Leu Met He Cys Ser Ala Thr Glu 20 25 30
Lys Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp Lys Glu Thr 35 40 45
Thr Thr Thr Leu Phe Cys Ala Ser Asp Ala Lys Ala Tyr Glu Glu Glu 50 55 60
Val His Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp Pro Asn 65 70 75 80
Pro Gin Glu He Val Leu Ala Asn Val Thr Glu Asp Phe Asn Met Trp 85 90 95
Lys Asn Glu Met Val Glu Gin Met His Thr Asp He He Ser Leu Trp 100 105 110
Asp Glu Ser Leu Lys Pro Cys Val Lys Leu Thr Pro Leu Cys Val Thr 115 120 125
Leu Asn Cys Thr Asp Leu Lys Asn Glu Thr Lys Thr Asn Ser Ser Asp 130 135 140
Ala Asn Ser Asn Ser Gly Glu He Met Gly Asn Glu Glu He Lys Asn 145 150 155 160
Cys Ser Phe Asn Val Ser Thr Gly Ala Pro Gly Lys Val Gin Lys Glu 165 170 175
Tyr Ser Leu Phe Tyr Ala Leu Asp He Val Ser He Lys Asn Glu Asn 180 185 190
Asn Ser Thr Ser His Met Leu Thr Ser Cys Asn Thr Ser Val Ser Thr 195 200 205
Gin Ala Cys Pro Lys Val Ser Phe Glu Pro He Pro He His Tyr Cys 210 215 220
65 Ala Pro Ala Gly Phe Ala He Leu Lys Cys Asn Asp Lys Lys Phe Asn 225" 230 235 240
Gly Thr Gly Pro Cys Asn Asn Val Ser Thr Val Gin Cys Thr His Gly 245 250 255
He Arg Pro Val Val Ser Thr Gin Leu Leu Leu Asn Gly Ser Val Ala 260 265 270
Glu Glu Glu Val Val Leu Arg Ser Ala Asn Phe Ser Asp Asn Ala Lys 275 280 285
Thr He He Val Gin Leu Asn His Ser Val Glu He Thr Cys Thr Arg 290 295 300
Pro Asn Tyr Asn Glu Thr Lys Lys He Arg He His Arg Gly Tyr Gly 305 310 315 320
Arg Ser Phe Val Thr Val Arg Lys Leu Gly Asp Arg Lys Gin Ala His 325 330 335
Cys Thr Met Asn Arg Thr Lys Trp Asp Asn Ala Leu Lys Gin He Ala 340 345 350
Ser Lys Leu Arg Glu Gin Phe Asn Lys Thr Ala He He Phe Asn Arg 355 360 365
Ser Ser Gly Gly Asp Leu Glu He Glu Met His Ser Phe Asn Cys Gly 370 375 380
Gly Glu Leu Phe Tyr Cys Asn Thr Thr Lys Leu Phe Asn Ser Thr Trp 385 390 395 400
Asn Glu Thr Thr Glu Ser Asn Gly Lys Gly Glu Asn He Thr Leu Pro 405 410 415
Cys Arg He Arg Gin Phe Val Asn Met Trp Gin Lys Val Gly Lys Ala 420 425 430
Met Tyr Ala Pro Pro Ser Asp Gly Gin He Arg Cys Thr Ser Asn He 435 440 445
Thr Gly Leu Leu Leu Thr Arg Asp Gly Gly His Asn Asp Asn Asn Thr 450 455 460
Asn Asn Glu Thr Phe Arg Pro Gly Arg Gly Asp Met Arg Asp Asn Trp 465 470 475 480
Arg Ser Glu Leu Tyr Lys Tyr Lys Val He Lys He Glu Pro Leu Gly
485 490 495
Val Ala Pro Thr Lys Ala Lys Arg Arg Val Val Gin Arg Glu Lys Arg
500 505 510
Ala Val Gly Met Val Gly Ala Met Phe Leu Gly Phe Leu Gly Ala Ala 515 520 525
66 Gly Ser Thr Met Gly Ala Ala Ser Leu Thr Leu Thr Val Gin Ala Arg " 530 535 540
Gin Leu Leu Ser Gly He Val Gin Gin Gin Asn Asn Leu Leu Arg Ala 545 550 555 560
He Glu Ala Gin Gin His Leu Leu Gin Leu Thr Val Trp Gly He Lys 565 570 575
Gin Leu Gin Ala Arg Val Leu Ala Val Glu Arg Tyr Leu Lys Asp Gin 580 585 590
Gin Leu Leu Gly He Trp Gly Cys Ser Gly Lys Leu He Cys Thr Thr 595 600 605
Thr Val Pro Trp Asn Ala Ser Trp Ser Asn Lys Ser Leu Asp Gin He 610 615 620
Trp Asn Asn Met Thr Trp Leu Glu Trp Asp Arg Glu He Ala Asn Tyr 625 630 635 640
Thr Asn Leu He His His Leu He Glu Glu Ser Gin Asn Gin Gin Glu 645 650 655
Lys Asn Glu Gin Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu Trp 660 665 670
Ser Trp Phe Asp He Ser Asn Trp Leu Trp Tyr He Lys He Phe He 675 680 685
Met He Val Ala Gly Leu Val Gly Leu Arg He Val Phe Ala Val Leu 690 695 700
Ser He Val Asn Arg Val Arg Gin Gly Tyr Ser Pro Leu Ser Phe Gin 705 710 715 720
Thr His Phe Pro Ala Pro Arg Gly Pro Asp Arg Pro Asp Gly He Glu 725 730 735
Gly Glu Gly Gly Glu Arg Asp Arg Asp Arg Ser Val Arg Leu Val Asp 740 745 750
Gly Phe Leu Ala Leu Leu Trp Glu Asp Leu Arg Asn Leu Cys Leu Phe 755 760 765
Ser Tyr His Arg Leu Arg Asp Leu Leu Leu He Val Thr Arg He Val 770 775 780
Glu Leu Leu Gly Arg Arg Gly Trp Glu Ala Leu Lys Tyr Leu Trp Ser 785 790 795 800
Leu Leu Gin Tyr Trp Ser Gin Glu Leu Lys Asn Ser Ala Val Asn Leu 805 810 815
Phe Asn Thr Thr Ala He Val Val Ala Glu Gly Thr Asp Arg He He 820 825 830
67 Glu Val Val Gin Arg Leu Cys Arg Ala He Leu His He Pro Arg Arg 835 840 845
He Arg Gin Gly Leu Glu Arg Phe Leu Leu * 850 855
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 854 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS : single
(D) TOPOLOGY: not relevant
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 3 :
Met Arg Ala Arg Glu Lys Glu Arg Asn Cys Gin Asn Leu Trp Lys Trp 1 5 10 15
Gly He Met Leu Leu Gly Met Leu Met Thr Cys Ser Ala Ala Glu Asp 20 25 30
Leu Trp Val Thr Val Tyr Tyr Gly Val Pro He Trp Lys Glu Ala Thr 35 40 45
Thr Thr Leu Phe Cys Ala Ser Asp Ala Lys Ala Tyr Lys Lys Glu Ala 50 55 60
His Asn He Trp Ala Thr His Ala Cys Val Pro Thr Asp Pro Asn Pro 65 70 75 80
Gin Glu He Glu Leu Glu Asn Val Thr Glu Asn Phe Asn Met Trp Lys 85 90 95
Asn Asn Met Val Glu Gin Met His Glu Asp He He Ser Leu Trp Asp 100 105 110
Gin Ser Leu Lys Pro Cys Val Lys Leu Thr Pro Leu Cys Val Thr Leu 115 120 125
Asn Cys Thr Asp Leu Lys Asn Glu Thr Lys Thr Asn Ser Ser Asp Ala 130 135 140
Asn Ser Asn Ser Gly Glu He Met Gly Asn Glu Glu He Lys Asn Cys 145 150 155 160
Ser Phe Asn Val Ser Thr Gly Ala Pro Gly Lys Val Gin Lys Glu Tyr 165 170 175
Ser Leu Phe Tyr Ala Leu Asp Asn Asn Asn Arg Thr Asn Ser Thr Asn 180 185 190
68 - Tyr Arg Leu He Asn Cys Asp Thr Ser Thr He Thr Gin Ala Cys Pro 195 200 205
Lys He Ser Phe Glu Pro He Pro He His Phe Cys Ala Pro Ala Gly 210 215 220
Phe Ala He Leu Lys Cys Arg Asp Lys Lys Phe Asn Gly Thr Gly Pro 225 230 235 240
Cys Ser Asn Val Ser Thr Val Gin Cys Thr His Gly He Arg Pro Val 245 250 255
Val Ser Thr Gin Leu Leu Leu Asn Gly Ser Leu Ala Glu Glu Glu He 260 265 270
He He Arg Ser Glu Asn Leu Thr Asn Asn Val Lys Thr He He Val 275 280 285
Gin Leu Asn Ala Ser He Val He Asn Cys Thr Arg Pro Tyr Lys Tyr 290 295 300
Thr Arg Gin Arg Thr Ser He Gly Leu Arg Gin Ser Leu Tyr Thr He 305 310 315 320
Thr Gly Lys Lys Lys Lys Thr Gly Tyr He Gly Gin Ala His Cys Lys 325 330 335
He Ser Arg Ala Glu Trp Asn Lys Ala Leu Gin Gin Val Ala Thr Lys 340 345 350
Leu Gly Asn Leu Leu Asn Lys Thr Thr He Thr Phe Lys Pro Ser Ser 355 360 365
Gly Gly Asp Pro Glu He Thr Ser His Met Leu Asn Cys Gly Gly Asp 370 375 380
Phe Phe Tyr Cys Asn Thr Ser Arg Leu Phe Asn Ser Thr Trp Asn Gin 385 390 395 400
Thr Asn Ser Thr Gly Phe Asn Asn Gly Thr Val Thr Leu Pro Cys Arg 405 410 415
He Lys Gin He Val Asn Leu Trp Gin Arg Val Gly Lys Ala Met Tyr 420 425 430
Ala Pro Pro He Glu Gly Leu He Lys Cys Ser Ser Asn He Thr Gly 435 440 445
Leu Leu Leu Thr Arg Asp Gly Gly Ala Asn Asn Ser Ser His Glu Thr 450 455 460
He Arg Pro Gly Gly Gly Asp Met Arg Asp Asn Trp Arg Ser Glu Leu 465 470 475 480
Tyr Lys Tyr Lys Val Val Lys He Glu Pro He Gly Val Ala Pro Thr 485 490 495
69 Lys Ala Arg Arg Arg Val Val Glu Arg Glu Lys Arg Ala He Gly Leu 500 505 510
Gly Ala Val Phe Leu Gly Phe Leu Gly Ala Ala Gly Ser Thr Met Gly 515 520 525
Ala Ala Ser Val Thr Leu Thr Val Gin Ala Arg Gin Leu Met Ser Gly 530 535 540
He Val His Gin Gin Asn Asn Leu Leu Arg Ala He Glu Ala Gin Gin 545 550 555 560
His Leu Leu Gin Leu Thr Val Trp Gly He Lys Gin Leu Gin Ala Arg 565 570 575
Val Leu Ala Val Glu Arg Tyr Leu Arg Asp Gin Gin Leu Leu Gly He 580 585 590
Trp Gly Cys Ser Gly Arg His He Cys Thr Thr Asn Val Pro Trp Asn 595 600 605
Ser Ser Trp Ser Asn Arg Ser Leu Asp Glu He Trp Gin Asn Met Thr 610 615 620
Trp Met Glu Trp Glu Arg Glu He Asp Asn Tyr Thr Gly Leu He Tyr 625 630 635 640
Ser Leu He Glu Glu Ser Gin He Gin Gin Glu Lys Asn Glu Lys Glu 645 650 655
Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu Trp Asn Trp Phe Ser He 660 665 670
Thr Lys Trp Leu Trp Tyr He Lys Leu Phe He Met He Val Gly Gly 675 680 685
Leu He Gly Leu Arg He Val Phe Ala Val Leu Ser Val Val Asn Arg 690 695 700
Val Arg Gin Gly Tyr Ser Pro Leu Ser Phe Gin Thr Leu Leu Pro Val
705 710 715 720
Pro Arg Gly Pro Asp Arg Pro Glu Glu He Glu Glu Glu Gly Gly Glu
725 730 735
Arg Gly Arg Asp Arg Ser He Arg Leu Val Asn Gly Leu Phe Ala Leu
740 745 750
Phe Trp Asp Asp Leu Arg Asn Leu Cys Leu Phe Ser Tyr His Arg Leu
755 760 765
Arg Asp Ser He Leu He Ala Ala Arg He Val Glu Leu Leu Gly Arg
770 775 780
Arg Gly Trp Glu Ala Leu Lys Tyr Leu Trp Asn Leu Leu Gin Tyr Trp
785 790 795 800
70 Ser Gin Glu Leu Arg Asn Ser Ala Ser Ser Leu Leu Asp Thr He Ala 805 810 815
He Ala Val Ala Glu Arg Thr Asp Arg Val He Glu Val Val Gin Arg 820 825 830
Ala Cys Arg Ala He Leu Asn Val Pro Arg Arg He Arg Gin Gly Leu 835 840 845
Glu Arg Leu Leu Leu Xaa 850
(2) INFORMATION FOR SEQ ID NO : 4 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 862 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS : single
(D) TOPOLOGY: not relevant
(ii) MOLECULE TYPE: protein
(i i) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 4 :
Met Arg Val Lys Glu Lys Tyr Gin His Leu Trp Arg Trp Gly Trp Lys 1 5 10 15
Trp Gly Thr Met Leu Leu Gly He Leu Met He Cys Ser Ala Thr Glu 20 25 30
Lys Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp Lys Glu Ala 35 40 45
Thr Thr Thr Leu Phe Cys Ala Ser Asp Ala Lys Ala Tyr Asp Thr Glu 50 55 60
Val His Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp Pro Asn 65 70 75 80
Pro Gin Glu Val Val Leu Val Asn Val Thr Glu Asn Phe Asn Met Trp 85 90 95
Lys Asn Asp Met Val Glu Gin Met His Glu Asp He He Ser Leu Trp 100 105 110
Asp Gin Ser Leu Lys Pro Cys Val Lys Leu Thr Pro Leu Cys Val Ser 115 120 125
Leu Lys Cys Thr Asp Leu Gly Asn Ala Thr Asn Thr Asn Ser Ser Asn 130 135 140
Thr Asn Ser Ser Ser Gly Glu Met Met Met Glu Lys Gly Glu He Lys 145 150 155 160
71 Asn Cys Ser Phe Asn He Ser Thr Ser He Arg Gly Lys Val Gin Lys 165 170 175
Glu Tyr Ala Phe Phe Tyr Lys Leu Asp He He Pro He Asp Asn Asp 180 185 190
Thr Thr Ser Tyr Thr Leu Thr Ser Cys Asn Thr Ser Val He Thr Gin 195 200 205
Ala Cys Pro Lys Val Ser Phe Glu Pro He Pro He His Tyr Cys Ala 210 215 220
Pro Ala Gly Phe Ala He Leu Lys Cys Asn Asn Lys Thr Phe Asn Gly 225 230 235 240
Thr Gly Pro Cys Thr Asn Val Ser Thr Val Gin Cys Thr His Gly He 245 250 255
Arg Pro Val Val Ser Thr Gin Leu Leu Leu Asn Gly Ser Leu Ala Glu 260 265 270
Glu Glu Val Val He Arg Ser Ala Asn Phe Thr Asp Asn Ala Lys Thr 275 280 285
He He Val Gin Leu Asn Gin Ser Val Glu He Asn Cys Thr Arg Pro 290 295 300
Asn Asn Asn Thr Arg Lys Ser He Arg He Gin Arg Gly Pro Gly Arg 305 310 315 320
Ala Phe Val Thr He Gly Lys He Gly Asn Met Arg Gin Ala His Cys 325 330 335
Asn He Ser Arg Ala Lys Trp Asn Ala Thr Leu Lys Gin He Ala Ser 340 345 350
Lys Leu Arg Glu Gin Phe Gly Asn Asn Lys Thr He He Phe Lys Gin 355 360 365
Ser Ser Gly Gly Asp Pro Glu He Val Thr His Ser Phe Asn Cys Gly 370 375 380
Gly Glu Phe Phe Tyr Cys Asn Ser Thr Gin Leu Phe Asn Ser Thr Trp 385 390 395 400
Phe Asn Ser Thr Trp Ser Thr Glu Gly Ser Asn Asn Thr Glu Gly Ser 405 410 415
Asp Thr He Thr Leu Pro Cys Arg He Lys Gin Phe He Asn Met Trp 420 425 430
Gin Glu Val Gly Lys Ala Met Tyr Ala Pro Pro He Ser Gly Gin He 435 440 445
Arg Cys Ser Ser Asn He Thr Gly Leu Leu Leu Thr Arg Asp Gly Gly 450 455 460
72 Asn Asn Asn Asn Gly Ser Glu He Phe Arg Pro Gly Gly Gly Asp Met 465 470 475 480
Arg Asp Asn Trp Arg Ser Glu Leu Tyr Lys Tyr Lys Val Val Lys He 485 490 495
Glu Pro Leu Gly Val Ala Pro Thr Lys Ala Lys Arg Arg Val Val Gin 500 505 510
Arg Glu Lys Arg Ala Val Gly He Gly Ala Leu Phe Leu Gly Phe Leu 515 520 525
Gly Ala Ala Gly Ser Thr Met Gly Ala Arg Ser Met Thr Leu Thr Val 530 535 540
Gin Ala Arg Gin Leu Leu Ser Gly He Val Gin Gin Gin Asn Asn Leu 545 550 555 560
Leu Arg Ala He Glu Ala Gin Gin His Leu Leu Gin Leu Thr Val Trp 565 570 575
Gly He Lys Gin Leu Gin Ala Arg He Leu Ala Val Glu Arg Tyr Leu 580 585 590
Lys Asp Gin Gin Leu Leu Gly He Trp Gly Cys Ser Gly Lys Leu He 595 600 605
Cys Thr Thr Ala Val Pro Trp Asn Ala Ser Trp Ser Asn Lys Ser Leu 610 615 620
Glu Gin He Trp Asn Asn Met Thr Trp Met Glu Trp Asp Arg Glu He 625 630 635 640
Asn Asn Tyr Thr Ser Leu He His Ser Leu He Glu Glu Ser Gin Asn 645 650 655
Gin Gin Glu Lys Asn Glu Gin Glu Leu Leu Glu Leu Asp Lys Trp Ala 660 665 670
Ser Leu Trp Asn Trp Phe Asn He Thr Asn Trp Leu Trp Tyr He Lys 675 680 685
He Phe He Met He Val Gly Gly Leu Val Gly Leu Arg He Val Phe 690 695 700
Ala Val Leu Ser He Val Asn Arg Val Arg Gin Gly Tyr Ser Pro Leu 705 710 715 720
Ser Phe Gin Thr His Leu Pro Thr Pro Arg Gly Pro Asp Arg Pro Glu 725 730 735
Gly He Glu Glu Glu Gly Gly Glu Arg Asp Arg Asp Arg Ser He Arg 740 745 750
Leu Val Asn Gly Ser Leu Ala Leu He Trp Asp Asp Leu Arg Ser Leu 755 760 765
73 Cys Leu Phe Ser Tyr His Arg Leu Arg Asp Leu Leu Leu He Val Thr 770 775 780
Arg He Val Glu Leu Leu Gly Arg Arg Gly Trp Glu Ala Leu Lys Tyr 785 790 795 800
Trp Trp Asn Leu Leu Gin Tyr Trp Ser Gin Glu Leu Lys Asn Ser Ala 805 810 815
Val Ser Leu Leu Asn Ala Thr Ala He Ala Val Ala Glu Gly Thr Asp 820 825 830
Arg Val He Glu Val Val Gin Gly Ala Cys Arg Ala He Arg His He 835 840 845
Pro Arg Arg He Arg Gin Gly Leu Glu Arg He Leu Leu Xaa 850 855 860
(2) INFORMATION FOR SEQ ID NO : 5 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 856 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: not relevant
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 5 :
Met Lys Val Lys Gly Thr Arg Arg Asn Tyr Gin His Leu Trp Arg Trp 1 5 10 15
Gly Thr Leu Leu Leu Gly Met Leu Met He Cys Ser Ala Thr Glu Lys 20 25 30
Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp Lys Glu Ala Thr 35 40 45
Thr Thr Leu Phe Cys Ala Ser Asp Ala Arg Ala Tyr Asp Thr Glu Val 50 55 60
His Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp Pro Asn Pro 65 70 75 80
Gin Glu Val Val Leu Gly Asn Val Thr Glu Asn Phe Asn Met Trp Lys 85 90 95
Asn Asn Met Val Glu Gin Met Gin Glu Asp He He Ser Leu Trp Asp 100 105 110
Gin Ser Leu Lys Pro Cys Val Lys Leu Thr Pro Leu Cys Val Thr Leu 115 120 125
74 Asn Cys Thr Asp Leu Gly Lys Ala Thr Asn Thr Asn Ser Ser Asn Trp 130 135 140
Lys Glu Glu He Lys Gly Glu He Lys Asn Cys Ser Phe Asn He Thr 145 150 155 160
Thr Ser He Arg Asp Lys He Gin Lys Glu Asn Ala Leu Phe Arg Asn 165 170 175
Leu Asp Val Val Pro He Asp Asn Ala Ser Thr Thr Thr Asn Tyr Thr 180 185 190
Asn Tyr Arg Leu He His Cys Asn Arg Ser Val He Thr Gin Ala Cys 195 200 205
Pro Lys Val Ser Phe Glu Pro He Pro He His Tyr Cys Thr Pro Ala 210 215 220
Gly Phe Ala He Leu Lys Cys Asn Asn Lys Thr Phe Asn Gly Lys Gly 225 230 235 240
Pro Cys Thr Asn Val Ser Thr Val Gin Cys Thr His Gly He Arg Pro 245 250 255
He Val Ser Thr Gin Leu Leu Leu Asn Gly Ser Leu Ala Glu Glu Glu 260 265 270
Val Val He Arg Ser Asp Asn Phe Thr Asn Asn Ala Lys Thr He He 275 280 285
Val Gin Leu Asn Glu Ser Val Ala He Asn Cys Thr Arg Pro Asn Asn 290 295 300
Asn Thr Arg Lys Ser He Tyr He Gly Pro Gly Arg Ala Phe His Thr 305 310 315 320
Thr Gly Arg He He Gly Asp He Arg Lys Ala His Cys Asn He Ser 325 330 335
Arg Ala Gin Trp Asn Asn Thr Leu Glu Gin He Val Lys Lys Leu Arg 340 345 350
Glu Gin Phe Gly Asn Asn Lys Thr He Val Phe Asn Gin Ser Ser Gly 355 360 365
Gly Asp Pro Glu He Val Met His Ser Phe Asn Cys Arg Gly Glu Phe 370 375 380
Phe Tyr Cys Asn Thr Thr Gin Leu Phe Asn Asn Thr Trp Arg Leu Asn 385 390 395 400
His Thr Glu Gly Thr Lys Gly Asn Asp Thr He He Leu Pro Cys Arg 405 410 415
He Lys Gin He He Asn Met Trp Gin Glu Val Gly Lys Ala Met Tyr 420 425 430
75 Ala Pro Pro He Gly Gly Gin He Ser Cys Ser Ser Asn He Thr Gly 435 440 445
Leu Leu Leu Thr Arg Asp Gly Gly Thr Asn Val Thr Asn Asp Thr Glu 450 455 460
Val Phe Arg Pro Gly Gly Gly Asp Met Arg Asp Asn Trp Arg Ser Glu 465 470 475 480
Leu Tyr Lys Tyr Lys Val He Lys He Glu Pro Leu Gly He Ala Pro 485 490 495
Thr Lys Ala Lys Arg Arg Val Val Gin Arg Glu Lys Arg Ala Val Gly 500 505 510
He Val Gly Ala Met Phe Leu Gly Phe Leu Gly Ala Ala Gly Ser Thr 515 520 525
Met Gly Ala Val Ser Leu Thr Leu Thr Val Gin Ala Arg Gin Leu Leu 530 535 540
Ser Gly He Val Gin Gin Gin Asn Asn Leu Leu Arg Ala He Glu Ala 545 550 555 560
Gin Gin His Leu Leu Gin Leu Thr Val Trp Gly He Lys Gin Leu Gin 565 570 575
Ala Arg Val Leu Ala Val Glu Arg Tyr Leu Arg Asp Gin Gin Leu Leu 580 585 590
Gly He Trp Gly Cys Ser Gly Lys Leu He Cys Thr Thr Ala Val Pro 595 600 605
Trp Asn Ala Ser Trp Ser Asn Lys Ser Leu Glu Asp He Trp Asp Asn 610 615 620
Met Thr Trp Met Gin Trp Glu Arg Glu He Asp Asn Tyr Thr Asn Thr 625 630 635 640
He Tyr Thr Leu Leu Glu Glu Ser Gin Asn Gin Gin Glu Lys Asn Glu 645 650 655
Gin Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu Trp Asn Trp Phe 660 665 670
Ser He Thr Asn Trp Leu Trp Tyr He Lys He Phe He Met He Val 675 680 685
Gly Gly Leu Val Gly Leu Arg He Val Phe Ala Val Leu Ser He Val 690 695 700
Asn Arg Val Arg Gin Gly Tyr Ser Pro Leu Ser Phe Gin Thr Arg Leu 705 710 715 720
Pro Val Pro Arg Gly Pro Asp Arg Pro Asp Gly He Glu Glu Glu Gly 725 730 735
76 Gly Glu Arg Asp Arg Asp Arg Ser Val Arg Leu Val Asp Gly Phe Leu 740 745 750
Ala Leu He Trp Glu Asp Leu Arg Ser Leu Cys Leu Phe Ser Tyr Arg 755 760 765
Arg Leu Arg Asp Leu Leu Leu He Ala Ala Arg Thr Val Glu He Leu 770 775 780
Gly His Arg Gly Trp Glu Ala Leu Lys Tyr Trp Trp Ser Leu Leu Gin 785 790 795 800
Tyr Trp He Gin Glu Leu Lys Asn Ser Ala Val Ser Trp Leu Asn Ala 805 810 815
Thr Ala He Ala Val Thr Glu Gly Thr Asp Arg Val He Glu Val Ala 820 825 830
Gin Arg Ala Tyr Arg Ala He Leu His He His Arg Arg He Arg Gin 835 840 845
Gly Leu Glu Arg Leu Leu Leu Xaa 850 855
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 854 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: not relevant
(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO
(ix) FEATURE:
(A) NAME/KEY: Region
(B) LOCATION: 1..854
(D) OTHER INFORMATION: /note= "Xaa indicates residues which are not specified in the consensus sequence . "
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 6 :
Met Arg Xaa Xaa Glu Asn Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Trp Lys
1 5 10 15
Trp Gly He Met Leu Leu Gly Met Leu Met Xaa Ser Ala Xaa Glu Xaa 20 25 30
Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Xaa Lys Glu Thr Thr Thr 35 40 45
Leu Phe Cys Ala Ser Asp Ala Lys Ala Tyr Xaa Xaa Glu Xaa His Asn 50 55 60
77 Xaa Trp Ala Thr His Ala Cys Val Pro Thr Asp Pro Asn Pro Gin Glu 65 70 75 80
He Xaa Leu Xaa Asn Val Thr Glu Xaa Phe Asn Met Trp Lys Asn Xaa 85 90 95
Met Val Glu Gin Met His Xaa Asp He He Ser Leu Trp Asp Xaa Ser 100 105 110
Leu Lys Pro Cys Val Lys Leu Thr Pro Leu Cys Val Thr Leu Asn Cys 115 120 125
Thr Asp Leu Lys Asn Glu Thr Xaa Xaa Asn Ser Ser Xaa Xaa Asn Xaa 130 135 140
Xaa Xaa Xaa Glu He Met Gly Xaa Glu Xaa Xaa Lys Asn Cys Ser Phe 145 150 155 160
Asn Val Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Tyr Xaa Leu 165 170 175
Phe Xaa Xaa Xaa Xaa He Val Xaa Xaa Xaa Asn Xaa Xaa Asn Ser Thr 180 185 190
Xaa Xaa Xaa Leu Xaa Xaa Cys Xaa Thr Ser Xaa Xaa Thr Gin Ala Cys 195 200 205
Pro Lys Xaa Ser Phe Glu Pro He Pro He His Xaa Cys Ala Pro Ala 210 215 220
Gly Phe Ala He Leu Lys Cys Xaa Asp Lys Lys Phe Asn Gly Thr Gly 225 230 235 240
Pro Cys Xaa Asn Val Ser Thr Val Gin Cys Thr His Gly He Arg Pro 245 250 255
Val Val Ser Thr Gin Leu Leu Leu Asn Gly Ser Xaa Ala Glu Glu Glu 260 265 270
Xaa Xaa Xaa Arg Ser Xaa Asn Xaa Xaa Xaa Asn Xaa Lys Thr He He 275 280 285
Val Gin Leu Asn Xaa Ser Xaa Xaa He Xaa Cys Thr Arg Pro Xaa Xaa 290 295 300
Xaa Xaa Xaa Xaa Xaa Xaa Xaa He Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 305 310 315 320
Xaa Thr Gly Lys Xaa Xaa Lys Xaa Gly Xaa Xaa Xaa Gin Ala His Cys 325 330 335
Xaa Xaa Xaa Arg Xaa Xaa Trp Xaa Xaa Ala Leu Xaa Gin Xaa Ala Xaa 340 345 350
Lys Leu Xaa Xaa Xaa Xaa Asn Lys Thr Xaa He Xaa Phe Xaa Xaa Ser 355 360 365
78 Ser Gly Gly Asp Xaa Glu He Xaa Xaa His Xaa Xaa Asn Cys Gly Gly 370 375 380
Xaa Xaa Phe Tyr Cys Asn Thr Xaa Xaa Leu Phe Asn Ser Thr Trp Asn 385 390 395 400
Xaa Thr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Thr Leu Pro Cys 405 410 415
Arg He Xaa Gin Xaa Val Asn Xaa Trp Gin Xaa Val Gly Lys Ala Met 420 425 430
Tyr Ala Pro Pro Xaa Xaa Gly Xaa He Xaa Cys Ser Asn He Thr Gly 435 440 445
Leu Leu Leu Thr Arg Asp Gly Gly His Asn Xaa Asn Asn Xaa Xaa Xaa 450 455 460
Glu Thr Xaa Arg Pro Gly Xaa Gly Asp Met Arg Asp Asn Trp Arg Ser 465 470 475 480
Glu Leu Tyr Lys Tyr Lys Val Xaa Lys He Glu Pro Xaa Gly Val Ala 485 490 495
Pro Thr Lys Ala Xaa Arg Arg Val Val Xaa Arg Glu Lys Arg Ala Xaa 500 505 510
Met Gly Ala Xaa Phe Leu Gly Phe Leu Gly Ala Ala Gly Ser Thr Met 515 520 525
Gly Ala Ala Ser Xaa Leu Thr Val Gin Ala Arg Gin Leu Xaa Ser Gly 530 535 540
He Val Xaa Gin Gin Asn Asn Leu Leu Arg Ala He Glu Ala Gin Gin 545 550 555 560
His Leu Leu Gin Leu Thr Val Trp Gly He Lys Gin Leu Gin Ala Arg 565 570 575
Val Leu Ala Val Glu Arg Tyr Leu Xaa Asp Gin Gin Leu Leu Gly He 580 585 590
Trp Gly Cys Ser Gly Xaa Xaa He Cys Thr Thr Xaa Val Pro Trp Asn 595 600 605
Xaa Ser Trp Ser Asn Xaa Ser Leu Asp Xaa He Trp Xaa Asn Met Thr 610 615 620
Trp Xaa Glu Trp Xaa Arg Glu He Xaa Asn Tyr Thr Xaa Leu He Xaa 625 630 635 640
Xaa Leu He Glu Glu Ser Gin Xaa Gin Gin Glu Lys Asn Glu Xaa Glu 645 650 655
Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu Trp Xaa Trp Phe Xaa He 660 665 670
79 Xaa Xaa Trp Leu Trp Tyr He Lys Xaa Phe He Met He Val Xaa Gly 675 680 685
Leu Xaa Gly Leu Arg He Val Phe Ala Val Leu Ser Xaa Val Asn Arg 690 695 700
Val Arg Gin Gly Tyr Ser Pro Leu Ser Phe Gin Thr Xaa Xaa Pro Xaa 705 710 715 720
Pro Arg Gly Pro Asp Arg Pro Xaa Xaa He Glu Xaa Glu Gly Gly Glu 725 730 735
Arg Xaa Arg Asp Arg Ser Xaa Arg Leu Val Xaa Gly Xaa Xaa Ala Leu 740 745 750
Xaa Trp Xaa Asp Leu Arg Asn Leu Cys Leu Phe Ser Tyr His Arg Leu 755 760 765
Arg Asp Xaa Xaa Leu He Xaa Xaa Arg He Val Glu Leu Leu Gly Arg 770 775 780
Arg Gly Trp Glu Ala Leu Lys Tyr Leu Trp Xaa Leu Leu Gin Tyr Trp 785 790 795 800
Ser Gin Glu Leu Xaa Asn Ser Ala Xaa Xaa Leu Xaa Xaa Thr Xaa Ala 805 810 815
He Xaa Val Ala Glu Xaa Thr Asp Arg Xaa He Glu Val Val Gin Arg 820 825 830
Xaa Cys Arg Ala He Leu Xaa Xaa Pro Arg Arg He Arg Gin Gly Leu 835 840 845
Glu Arg Xaa Leu Leu Xaa 850
(2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 866 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: not relevant
(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO
(ix) FEATURE:
(A) NAME/KEY: Region
(B) LOCATION: 1..866
(D) OTHER INFORMATION: /note= "Xaa residues are not specified in this consensus sequence . "
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 7 :
80 Met Arg Val Lys Glu Xaa Xaa Gin His Leu Trp Arg Trp Gly Trp Lys
1 5 10 15
Trp Gly Xaa Met Leu Leu Gly Xaa Leu Met He Cys Ser Ala Thr Glu 20 25 30
Lys Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp Lys Glu Xaa 35 40 45
Thr Thr Thr Leu Phe Cys Ala Ser Asp Ala Lys Ala Tyr Xaa Xaa Glu 50 55 60
Val His Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp Pro Asn 65 70 75 80
Pro Gin Glu Xaa Val Leu Xaa Asn Val Thr Glu Xaa Phe Asn Met Trp 85 90 95
Lys Asn Xaa Met Val Glu Gin Met His Xaa Asp He He Ser Leu Trp 100 105 110
Asp Xaa Ser Leu Lys Pro Cys Val Lys Leu Thr Pro Leu Cys Val Xaa 115 120 125
Leu Xaa Cys Thr Asp Leu Xaa Asn Xaa Thr Xaa Thr Asn Ser Ser Xaa 130 135 140
Xaa Asn Ser Xaa Ser Gly Glu Xaa Met Met Xaa Xaa Xaa Glu He Lys 145 150 155 160
Asn Cys Ser Phe Asn Xaa Ser Thr Xaa Xaa Xaa Gly Lys Val Gin Lys 165 170 175
Glu Tyr Xaa Xaa Phe Tyr Xaa Leu Asp He Val Ser He Xaa Xaa Xaa 180 185 190
Asn Xaa Xaa Thr Ser Xaa Xaa Leu Thr Ser Cys Asn Thr Ser Val Xaa 195 200 205
Thr Gin Ala Cys Pro Lys Val Ser Phe Glu Pro He Pro He His Tyr 210 215 220
Cys Ala Pro Ala Gly Phe Ala He Leu Lys Cys Asn Xaa Lys Xaa Phe 225 230 235 240
Asn Gly Thr Gly Pro Cys Xaa Asn Val Ser Thr Val Gin Cys Thr His 245 250 255
Gly He Arg Pro Val Val Ser Thr Gin Leu Leu Leu Asn Gly Ser Xaa 260 265 270
Ala Glu Glu Glu Val Val Xaa Arg Ser Ala Asn Phe Xaa Asp Asn Ala 275 280 285
Lys Thr He He Val Gin Leu Asn Xaa Ser Val Glu He Xaa Cys Thr 290 295 300
81 Arg Pro Asn Xaa Asn Xaa Xaa Lys Xaa He Arg He Xaa Arg Gly Xaa 305 310 315 320
Gly Arg Xaa Phe Val Thr Xaa Xaa Lys Xaa Gly Xaa Xaa Xaa Gin Ala 325 330 335
His Cys Xaa Xaa Xaa Arg Xaa Lys Trp Xaa Xaa Xaa Leu Lys Gin He 340 345 350
Ala Ser Lys Leu Arg Glu Gin Phe Gly Asn Xaa Xaa Xaa He He Phe 355 360 365
Xaa Xaa Ser Ser Gly Gly Asp Xaa Glu He Xaa Xaa His Ser Phe Asn 370 375 380
Cys Gly Gly Glu Xaa Phe Tyr Cys Asn Xaa Thr Xaa Leu Phe Asn Ser 385 390 395 400
Thr Trp Phe Asn Xaa Thr Trp Ser Thr Glu Gly Ser Asn Asn Xaa Xaa 405 410 415
Gly Ser Xaa Xaa He Thr Leu Pro Cys Arg He Xaa Gin Phe Xaa Asn 420 425 430
Met Trp Gin Xaa Val Gly Lys Ala Met Tyr Ala Pro Pro Xaa Xaa Gly 435 440 445
Gin He Arg Cys Xaa Ser Asn He Thr Gly Leu Leu Leu Thr Arg Asp 450 455 460
Gly Gly His Asn Asp Asn Asn Xaa Xaa Xaa Glu Xaa Phe Arg Pro Gly 465 470 475 480
Xaa Gly Asp Met Arg Asp Asn Trp Arg Ser Glu Leu Tyr Lys Tyr Lys 485 490 495
Val Xaa Lys He Glu Pro Leu Gly Val Ala Pro Thr Lys Ala Lys Arg 500 505 510
Arg Val Val Gin Arg Glu Lys Arg Ala Val Gly Met Xaa Gly Ala Xaa 515 520 525
Phe Leu Gly Phe Leu Gly Ala Ala Gly Ser Thr Met Gly Ala Xaa Ser 530 535 540
Xaa Thr Leu Thr Val Gin Ala Arg Gin Leu Leu Ser Gly He Val Gin 545 550 555 560
Gin Gin Asn Asn Leu Leu Arg Ala He Glu Ala Gin Gin His Leu Leu 565 570 575
Gin Leu Thr Val Trp Gly He Lys Gin Leu Gin Ala Arg Xaa Leu Ala 580 585 590
Val Glu Arg Tyr Leu Lys Asp Gin Gin Leu Leu Gly He Trp Gly Cys 595 600 605
82 Ser Gly Lys Leu He Cys Thr Thr Xaa Val Pro Trp Asn Ala Ser Trp 610 615 620
Ser Asn Lys Ser Leu Xaa Gin He Trp Asn Asn Met Thr Trp Xaa Glu 625 630 635 640
Trp Asp Arg Glu He Xaa Asn Thr Xaa Leu He His Xaa Leu He Glu 645 650 655
Glu Ser Gin Asn Gin Gin Glu Lys Asn Glu Gin Glu Leu Leu Glu Leu 660 665 670
Asp Lys Trp Ala Ser Leu Trp Xaa Trp Phe Xaa He Xaa Asn Trp Leu 675 680 685
Trp Tyr He Lys He Phe He Met He Val Xaa Gly Leu Val Gly Leu 690 695 700
Arg He Val Phe Ala Val Leu Ser He Val Asn Arg Val Arg Gin Gly 705 710 715 720
Tyr Ser Pro Leu Ser Phe Gin Thr His Xaa Pro Xaa Pro Arg Gly Pro 725 730 735
Asp Arg Pro Xaa Gly He Glu Xaa Glu Gly Gly Glu Arg Asp Arg Asp 740 745 750
Arg Ser Xaa Arg Leu Val Xaa Gly Xaa Leu Ala Leu Xaa Trp Xaa Asp 755 760 765
Leu Arg Xaa Leu Cys Leu Phe Ser Tyr His Arg Leu Arg Asp Leu Leu 770 775 780
Leu He Val Thr Arg He Val Glu Leu Leu Gly Arg Arg Gly Trp Glu 785 790 795 800
Ala Leu Lys Tyr Xaa Trp Xaa Leu Leu Gin Tyr Trp Ser Gin Glu Leu 805 810 815
Lys Asn Ser Ala Val Xaa Leu Xaa Asn Xaa Thr Ala He Xaa Val Ala 820 825 830
Glu Gly Thr Asp Arg Xaa He Glu Val Val Gin Xaa Xaa Cys Arg Ala 835 840 845
He Xaa His He Pro Arg Arg He Arg Gin Gly Leu Glu Arg Xaa Leu 850 855 860
Leu Xaa 865
(2) INFORMATION FOR SEQ ID NO : 8 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 836 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
83 (D) TOPOLOGY: not relevant
(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO
(ix) FEATURE:
(A) NAME/KEY: Region
(B) LOCATION: 1..836
(D) OTHER INFORMATION: /note= "Xaa residues are not specified in this consensus sequence . "
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 8 :
Met Val Lys Glu Trp Trp Gly Leu Leu Gly Met Leu Met He Cys Ser 1 5 10 15
Ala Thr Glu Lys Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp 20 25 30
Lys Glu Thr Thr Thr Leu Phe Cys Ala Ser Asp Ala Ala Tyr Glu Val 35 40 45
His Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp Pro Asn Pro 50 55 60
Gin Glu Val Leu Asn Val Thr Glu Phe Asn Met Trp Lys Asn Met Val 65 70 75 80
Glu Gin Met Asp He He Ser Leu Trp Asp Ser Leu Lys Pro Cys Val 85 90 95
Lys Leu Thr Pro Leu Cys Val Thr Leu Asn Cys Thr Asp Leu Xaa Xaa 100 105 110
Xaa Thr Xaa Thr Asn Ser Ser Asp Ala Asn Xaa Xaa Xaa Xaa Xaa Xaa 115 120 125
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 130 135 140
Xaa Xaa Xaa Lys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 145 150 155 160
He Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn Xaa Thr Xaa Xaa Xaa Leu Xaa 165 170 175
Xaa Cys Asn Xaa Ser Val Xaa Thr Gin Ala Cys Pro Lys Val Ser Phe 180 185 190
Glu Pro He Pro He His Tyr Cys Xaa Pro Ala Gly Phe Ala He Leu 195 200 205
Lys Cys Asn Xaa Lys Xaa Phe Asn Gly Xaa Gly Pro Cys Xaa Asn Val 210 215 220
84 Ser Thr Val Gin Cys Thr His Gly He Arg Pro Xaa Val Ser Thr Gin 225 230 235 240
Leu Leu Leu Asn Gly Ser Xaa Ala Glu Glu Glu Val Val Xaa Arg Ser 245 250 255
Xaa Asn Phe Xaa Xaa Asn Ala Lys Thr He He Val Gin Leu Asn Xaa 260 265 270
Ser Val Xaa He Xaa Cys Thr Arg Pro Asn Xaa Asn Xaa Xaa Lys Xaa 275 280 285
He Xaa He Xaa Xaa Gly Tyr Xaa Xaa Xaa Xaa Xaa Thr Xaa Arg Xaa 290 295 300
Xaa Gly Asp Xaa Xaa Xaa Ala His Cys Xaa Xaa Xaa Arg Xaa Xaa Trp 305 310 315 320
Xaa Asn Xaa Leu Xaa Gin He Xaa Xaa Lys Leu Arg Glu Gin Phe Gly 325 330 335
Asn Xaa Xaa Xaa He Xaa Phe Asn Xaa Ser Ser Gly Gly Asp Xaa Glu 340 345 350
He Xaa Met His Ser Phe Asn Cys Xaa Gly Glu Xaa Phe Tyr Cys Asn 355 360 365
Thr Thr Xaa Leu Phe Asn Xaa Thr Trp Arg Xaa Xaa Xaa Thr Glu Xaa 370 375 380
Xaa Xaa Xaa Xaa Xaa Xaa He Xaa Leu Pro Cys Arg He Xaa Gin Xaa 385 390 395 400
Xaa Asn Met Trp Gin Xaa Val Gly Lys Ala Met Tyr Ala Pro Pro Xaa 405 410 415
Xaa Gly Gin He Xaa Cys Xaa Ser Asn He Thr Gly Leu Leu Leu Thr 420 425 430
Arg Asp Gly Gly Xaa Asn Xaa Xaa Asn Thr Xaa Xaa Glu Xaa Phe Arg 435 440 445
Pro Gly Xaa Gly Asp Met Arg Asp Asn Trp Arg Ser Glu Leu Tyr Lys 450 455 460
Tyr Lys Val He Lys He Glu Pro Leu Gly Xaa Ala Pro Thr Lys Ala 465 470 475 480
Lys Arg Arg Val Val Gin Arg Glu Lys Arg Ala Val Gly Xaa Val Gly 485 490 495
Ala Met Phe Leu Gly Phe Leu Gly Ala Ala Gly Ser Thr Met Gly Ala 500 505 510
Xaa Leu Thr Leu Thr Val Gin Ala Arg Gin Leu Leu Ser Gly He Val 515 520 525
85 Gin Gin Gin Asn Asn Leu Leu Arg Ala He Glu Ala Gin Gin His Leu 530 535 540
Leu Gin Leu Thr Val Trp Gly He Lys Gin Leu Gin Ala Arg Val Leu 545 550 555 560
Ala Val Glu Arg Tyr Leu Xaa Asp Gin Gin Leu Leu Gly He Trp Gly 565 570 575
Cys Ser Gly Lys Leu He Cys Thr Thr Xaa Val Pro Trp Asn Ala Ser 580 585 590
Trp Ser Asn Lys Ser Leu Xaa Xaa He Trp Xaa Asn Met Thr Trp Xaa 595 600 605
Xaa Trp Xaa Arg Glu He Xaa Asn Tyr Thr Asn Xaa He Xaa Xaa Leu 610 615 620
Xaa Glu Glu Ser Gin Asn Gin Gin Glu Lys Asn Glu Gin Glu Leu Leu 625 630 635 640
Glu Leu Asp Lys Trp Ala Ser Leu Trp Xaa Trp Phe Xaa He Xaa Asn 645 650 655
Trp Leu Trp Tyr He Lys He Phe He Met He Val Xaa Gly Leu Val 660 665 670
Gly Leu Arg He Val Phe Ala Val Leu Ser He Val Asn Arg Val Arg 675 680 685
Gin Gly Tyr Ser Pro Leu Ser Phe Gin Thr Xaa Xaa Pro Xaa Pro Arg 690 695 700
Gly Pro Asp Arg Pro Asp Gly He Glu Xaa Glu Gly Gly Glu Arg Asp 705 710 715 720
Arg Asp Arg Ser Val Arg Leu Val Asp Gly Phe Leu Ala Leu Xaa Trp 725 730 735
Glu Asp Leu Arg Xaa Leu Cys Leu Phe Ser Tyr Xaa Arg Leu Arg Asp 740 745 750
Leu Leu Leu He Xaa Xaa Arg Xaa Val Glu Xaa Leu Gly Xaa Arg Gly 755 760 765
Trp Glu Ala Leu Lys Tyr Xaa Trp Ser Leu Leu Gin Tyr Trp Xaa Gin 770 775 780
Glu Leu Lys Asn Ser Ala Val Xaa Xaa Xaa Asn Xaa Thr Ala He Xaa 785 790 795 800
Val Xaa Glu Gly Thr Asp Arg Xaa He Glu Val Xaa Gin Arg Xaa Xaa 805 810 815
Arg Ala He Leu His He Xaa Arg Arg He Arg Gin Gly Leu Glu Arg 820 825 830
86 Xaa Leu Leu Xaa 835
(2) INFORMATION FOR SEQ ID NO : 9 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "Oligonucleotide."
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 9 :
CCCTTCGAAG AGGATATAAT CAGTTTATGG GATCAAAGC 39
(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "Oligonucleotide."
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
CCCTTCGAAC TCTTCTTCTG CTAGACTGCC ATT 33
(2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9193 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: not relevant
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI -SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: GGTCTCTCTG GTTAGACCAG ATCTGAGCCT GGGAGCTCTC TGGCTAGCTA GGGAACCCAC 60 TGCTTAAGCC TCAATAAAGC TTGCCTTGAG TGCTTCAAGT AGTGTGTGCC CGTCTGTTGT 120
87 GTGACTCTGG TAACTAGAGA TCCCTCAGAC CCTTTTAGTC AGTGTGGAAA AATCTCTAGC 180
AGTGGCGCCC GAACAGGGAC CGGAAAGCAA AAGGGAAACC AGAGAAGCTC TCTCGACGCA 240
GGACTCGGCT TGCTGAAGCG CGCACAGCAA GAGGCGAGGG GCGGCGACTG GTGAGTACGC 300
CGATATTTTT GACTAGCGGA GGCTAGAAGG AGAGAGATGG GTGCGAGAGC GTCGGTATTA 360
AGCGGGGGAG ATTTAGATCG ATGGGAAAAA ATTCGGTTAA GGCCAGGGGG AAAGAAAAAA 420
TATATGTTAA AACATATAGT ATGGGCAAGC AGGGAGCTAG AACGATTCGC AGTCAATCCT 480
GGCCTGTTAG AAACATCAGA AGGCTGTAGA CAAGTACTGG AACAGCTACA GCCATCCCTT 540
CAGACAGGAT CAGAAGAACT TAGATCCTTA TATAATACAA TAGCAACCCT CTATGGTGTG 600
CATCAAAAAA TAGAGGTAAA AGACACCAAG GAAGCTTTAG ACAAAATAGA GGAAGAGCAA 660
AACAAAAGTA AGAAAAAAGC ACAGCAAGCA GCAGCTGACA CAGGACACAG CAGCCAGGTC 720
AGCCAAAATT ACCCTATAGT GCAGAACCTA CAGGGGCAAA TGGTACATCA GGCCATATCA 780
CCTAGAACTT TAAATGCATG GGTAAAAGTA ATAGAAGAGA AGGCTTTCAG CCCAGAAGTG 840
ATACCCATGT TTTCAGCATT ATCAGAAGGA GCCACCCCAC AAGATTTAAA CACCATGTTA 900
AACACAGTGG GGGGACATCA AGCAGCCATG CAAATGTTAA AAGAGACCAT CAATGAGGAA 960
GCTGCAGAAT GGGATAGAGT GCATCCAGTG CAGGCAGGGC TTATTGCACC AGGCCAGATG 1020
AGAGAACCAA GGGGAAGTGA CATAGCAGGA ACTACTAGTA CCCTTCAGGA ACAAATAGGA 1080
TGGATGACAA GTAATCCACC TATCCCAGTA GGAGAAATTT ATAAAAGGTG GATAATCCTG 1140
GGCTTAAATA AAATAGTAAG AATGTATAGC CCTATCAGCA TTCTAGACAT AAGACAAGGA 1200
CCAAAAGAAC CCTTTAGAGA CTATGTAGAC CGGTTCTATA AAACTCTAAG AGCCGAGCAA 1260
GCTTCACAGG AAGTAAAAAA TTGGATGACA GAAACCTTGC TGGTCCAAAA TGCGAACCCA 1320
GATTGTAAGA CTATCTTAAA AGCATTAGGA CCAGGAGCTA CACTAGAAGA AATGATGACA 1380
GCATGTCAGG GAGTGGGAGG ACCCGGCCAT AAGGCAAGAG TTTTAGCTGA AGCAATGAGC 1440
CAAGTAACAA ATTCAGCTGC CATAATGATG CAGAGAGGCA ATTTTAAGAA CCAAAGAAAG 1500
ATGGTTAAGT GTTTCAATTG TGGCAAAGAG GGGCACGTAG CCAGAAATTG CAGGGCCCCT 1560
AGAAAAAAGG GCTGTTGGAA ATGTGGAAAG GAAGGACACC AAATGAAAGA TTGTATTGAG 1620
AGCCAGGCTA ATTTTTTAGG GAAAATCTGG CCTTCCCACA AGGGAAGGCC AGGGAATTTC 1680
CTTCAGAGCA GACCAGAGCC AACAGCCCCA CCAGAAGAGA GCCTCAGGTC TGGGATAGAG 1740
ACAACAACTC CCTCTCAGAA GCAGGAGCCA ATAGACAAGG AAGTGTATCC TTTAACTTCC 1800
CTCAGATCAC TCTTTGGCAA CGACCCCTCG TCACAATAAA GATAGGGGGG CAACTAAAGG 1860
88 AAGCTCTATT AGATACAGGA GCAGATGATA CAGTATTAGA AGAAATGAAT TTGCCAGGAA 1920
GATGGAAGCC AAAAATGATA GGGGGCATTG GAGGTTTTAT CAAAGTAAGA CAGTATGATC 1980
AGATACCCAT AGAAATCTGT GGACATAAAG CTATAGGTAC AGTATTAGTA GGACCTACAC 2040
CTGTCAACAT AATTGGAAGA AATCTGTTGA CTCAGATTGG TTGCACTTTA AATTTTCCCA 2100
TTAGTCCTAT TGAAACTGTA CCAGTGAGAT TAAAGCCAGG AATGGATGGC CCAAAAGTTA 2160
AACAATGGCC ATTGACAGAA GAAAAAATAA AAGCATTAGT AGAAATTTGT ACAGAAATGG 2220
AAAAGGAAGG GAAAATTTCA AAAATTGGGC CTGAAAATCC ATACAATACT CCAGTATTTG 2280
CCATAAAGAA AAAAGACAGT ACTAAATGGA GAAAATTAGT AGATTTCAGA GAACTTAATA 2340
AGAGAACTCA AGACTTCTGG GAAGTTCAAT TAGGAATACC ACATCCCTCA GGTTTAAAAA 2400
AGAAAAAATC AGTAACAGTA CTAGATGTGG GTGATGCATA TTTTTCAGTT CCCTTAGATG 2460
AAGATTTCAG GAAGTATACT ACATTTACCA TACCTAGTAT AAACAATCAG ACACCAGGGA 2520
TTAGATATCA GTACAATGTG CTTCCACAGG GATGGAAAGG ATCACCAGCA ATATTCCAAA 2580
GTAGCATGAC AAAAATCTTA GAGCCTTTTA GAAAACAGAA TCCAGACATA GTTATCTATC 2640
AATACATGGA TGATTTGTAT GTAGGATCTG ACTTAGAAAT AGAGCAGCAT AGAACAAAAA 2700
TAGAGGAACT GAGACAGCAT CTGTTGAGGT GGGGATTTAC CACACCAGAC AAAAAACATC 2760
AGAAAGAACC TCCATTCCTC TGGATGGGGT ATGAACTCCA TCCTGATAAA TGGACAGTAC 2820
AGCCTATAGT GCTGCCAGAA AAAGACAGCT GGACTGTCAA TGACATACAG AAGTTAGTGG 2880
GAAAATTGAA TTGGGCAAGT CAGATTTACC CAGGGATTAA AGTAAGGCAA TTATGTAAAC 2940
TCCTTAGAGG AGCCAAAGCA CTAACAGAAG TAATACCACT AACAGAAGGA GCAGAGCTAG 3000
AACTGGCAGA AAACAGAGAG ATTCTAAAAG AACCAGTACA TGGAGTGTAT TATGACCCAT 3060
CAAAAGACTT AATAGCAGAA ATACAGAAGC AGGGGCAAGG CCAATGGACA TATCAAATTT 3120
ATCAAGAGCC ATTTAAAAAT CTGAAAACAG GAAAATATGC AAGAACGAGG GGTGCCCACA 3180
CTAATGATGT AAAACAATTA ACAGAGGCAG TGCAAAAAAT AGCCACAGAA AGCATAGTAA 3240
TATGGGGGAA GACTCCTAAA TTTAAACTGC CCATACAAAA GGAAACATGG GAAACATGGT 3300
GGACAGAGTA TTGGCAAGCC ACTTGGATTC CTGAATGGGA GTTTGTCAAT ACCCCTCCTT 3360
TAGTAAAATT ATGGTACCAG TTAGAGAAAG AACCCATAGT AGGAGCAGAA ACTTTCTATG 3420
TAGATGGGGC AGCTAACAGG GAGACTAAAT TAGGAAAAGC AGGATATGTT ACTAACAGAG 3480
GAAGACAAAA GGTTGTCACC CTGACTAACA CAACAAATCA GAAGACTGAG TTACAAGCAA 3540
TTCATCTAGC TTTACAGGAT TCAGGATCAG AAGTAAACAT AGTAACAGAC TCACAATATG 3600
89 CATTAGGAAT CATTCAAGCA CAACCAGATC AAAGTGAATC AGAGTTAGTC AATCAAATAA 3660
TAGAGCAGTT AATAAAAAAG GAAAAGGTCT ATCTGGCATG GGTACCAGCA CACAAAGGAA 3720
TTGGAGGAAA TGAACAAGTA GATAAATTAG TCAGTGCTGG AATCAGAAAA GTACTATTTT 3780
TAGATGGAAT AGATAAGGCC CAAGAAGAAC ATGAGAAATA TCACAATAAT TGGAGAGCAA 3840
TGGCTAGTGA TTTTAACCTG CCACCTGTAG TAGCAAAAGA AATAGTAGCC AGCTGTGATA 3900
AATGTCAGCT AAAAGGAGAA GCTATGCATG GACAAGTAGA CTGTAGTCCA GGAATATGGC 3960
AACTAGATTG TACACATTTA GAAGGAAAAG TTATCCTGGT AGCAGTTCAT GTAGCCAGTG 4020
GATATATAGA AGCAGAAGTT ATTCCAGCAG AAACAGGGCA GGAAACAGCA TACTTTCTCT 4080
TAAAATTAGC AGGAAGATGG CCAGTAAAAA CAATACATAC AGACAATGGC AGCAATTTCA 4140
CCAGTGCTGC GGTTAAGGCC GCCTGTTGGT GGGCAGGAGT CAAACAAGAA TTTGGAATTC 4200
CCTACAATCC CCAAAGTCAA GGAGTAGTAG AATCTATGAA TAAAGAATTA AAGAAAATTA 4260
TAGGACAGGT AAGAGATCAA GCTGAACATC TTAAGACAGC AGTACAAATG GCAGTATTTG 4320
TCCACAATTT TAAAAGAAAA GGGGGGATTG GGGGGTACAG TGCAGGGGAA AGAATAATAG 4380
ACATAATAGC AACAGACATA CAAACTAGAG AACTACAAAA ACAAATTACA AAAATTCAAA 4440
ATTTTCGGGT TTATTACAGG GACAGCAGAG ATCCACTTTG GAAAGGACCA GCAAAGCTCC 4500
TCTGGAAAGG TGAAGGGGCA GTAGTAATAC AAGATAATAG TGACATAAAA GTAGTGCCAA 4560
GAAGAAAAGC AAAGATCATT AGGGATTATG GAAAACAGAT GGCAGGTGAT GATTGTGTGG 4620
CAAGTAGACA GGATGAGGAT TAGAACATGG AAAAGTTTAG TAAAACATCA TGTATATGTT 4680
TCAAAGAAAG CTAGGGGATG GTTTTATAGA CATCACTATG AAAGCACTCA TCCAAAAATA 4740
AGTTCAGAAA TACACATCCC ACTAGGGGAT GCTAGATTGG TAGTAACAAC ATATTGGGGT 4800
CTGCATACAG GAGAAAGAGA ATGGCATTTG GGTCATGGAG TCTCCATAGA ATGGAGGAAA 4860
AGGAGCTATA GCACACAAGT AGACCCTGAA CTAGCAGACC AACTAATTCA TCTGTATTAC 4920
TTTGATTGTT TTTCAGACTC TGCTATAAGA AAGGCCTTAT TAGAACACAT AGTTAGCCCT 4980
AGGTGTGAAT ATCGAGCAGG ACATTCCAAG GTAGGATCTC TACAATACTT GGCACTATCA 5040
GCCTTAATAA CACCAAAGAA GATAAAGCCA CCTTTGCCTA GTGTTACGAA ACTGACAGAG 5100
GATAGATGGA ACAAGCCCCA GAAGACCAAG GGCCACAGAG GGAGCCATAC AATGAATGGA 5160
CACTAGAGCT TTTAGAGGAG CTTAAGGGAG AAGCTGTTAG ACATTTTCCT AGGCCATGGC 5220
TCCATAGCTT AGGACAACAT ATCTATGAAA CTTATGGAGA TACTTGGGCA GGAGTGGAAG 5280
CCATAATAAG AATTCTGCAA CAATTGCTGT TTATTCATTT CAGAATTGGG TGTCAACATA 5340
90 GCAGAATAGG CATTATTCAA CAGAGGAGAG CAAGAAGAAA TGGAGCCAGT AGATCCTAAT 5400
CTAGAGCCCT GGAAGCATCC AGGAAGTCAG CCTAAAACTG CTTGTACCAA ATGCTATTGT 5460
AAAAGGTGTT GCTTTCATTG CCAAGTTTGT TTCACAACAA AAGCCTTAGG CATCTCCTAT 5520
GGCAGGAAGA AGCGGAGACA GCGACGAAGA CCTCGTCAGG GCAGCCAGGC TCATCAAGCT 5580
TCTCTATCAG AGCAGTAAGT AGTATATGTA ATGCAACTTA TATTAATTGT AACAATAGTA 5640
GCTTTAGTAG TAACATTAAT AATAGCAATA GTTGTGTGGT CCATAGTACT CATAGAATAT 5700
AGGAAAATAT TAAGACAAAG GAAAATAGAC AAGTTAATTA ATAGACTAGT AGAAAGAGCA 5760
GAAGACAGTG GCAATGAGAG TGAAGGAGAA CTGTCAGCAC TTGTGGAGAT GGGGGTGGAA 5820
ATGGGGCATC ATGCTCCTTG GGATGTTAAT GATCTGTAGT GCTACAGAAA AATTGTGGGT 5880
CACAGTCTAT TATGGGGTAC CTGTGTGGAA GGAAACAACT ACCACTCTAT TTTGTGCATC 5940
AGATGCTAAA GCATATGAAG AAGAGGTACA TAATGTTTGG GCCACACATG CCTGTGTACC 6000
CACAGACCCC AACCCACAAG AAATAGTATT GGCAAATGTG ACAGAAGATT TTAACATGTG 6060
GAAAAATGAA ATGGTAGAAC AGATGCATAC TGATATAATC AGTTTATGGG ATGAAAGCCT 6120
AAAACCATGT GTAAAATTAA CCCCACTCTG TGTTACTTTA AATTGCACTG ATTTGAAGAA 6180
TGAAACTAAG ACCAATAGTA GTGATGCCAA TAGTAATAGC GGGGAAATAA TGGGGAACGA 6240
AGAGATAAAA AATTGCTCTT TCAATGTCAG CACAGGCGCA CCAGGTAAGG TGCAGAAAGA 6300
ATATGCACTT TTTTATGCAC TTGATATAGT ATCAATAAAG AATGAAAATA ATAGTACCAG 6360
CCATATGTTG ACAAGTTGTA ACACCTCAGT CAGTACACAG GCCTGTCCAA AGGTATCCTT 6420
TGAGCCAATT CCCATACATT ATTGTGCCCC GGCTGGTTTT GCAATTCTAA AATGTAATGA 6480
TAAGAAGTTC AATGGAACAG GACCATGTAA CAATGTCAGC ACAGTACAAT GTACACATGG 6540
AATTAGACCA GTAGTGTCAA CTCAACTGCT GTTAAATGGC AGTGTAGCAG AAGAAGAGGT 6600
AGTACTTAGA TCTGCCAATT TCTCAGACAA TGCTAAAACC ATAATAGTAC AGCTGAACCA 6660
CTCTGTAGAA ATTACTTGTA CAAGACCCAA CTACAATGAA ACAAAGAAAA TCCGTATCCA 6720
CAGAGGATAT GGAAGATCAT TTGTTACAGT AAGAAAATTG GGAGATAGGA AACAAGCACA 6780
TTGTACCATG AATAGAACGA AATGGGACAA CGCTTTAAAA CAGATAGCTA GCAAATTAAG 6840
AGAACAATTT AATAATAAAA CAGCAATAAT CTTTAACCGG TCCTCAGGAG GGGACCTAGA 6900
AATTGAAATG CACAGTTTTA ATTGCGGAGG GGAATTGTTC TACTGTAATA CAACAAAACT 6960
GTTTAATAGT ACTTGGAATG AGACTACAGA GTCAAATGGC AAGGGAGAAA ATATCACACT 7020
CCCATGCAGA ATAAGACAAT TTGTAAACAT GTGGCAGAAA GTAGGAAAAG CAATGTATGC 7080
91 CCCTCCCAGC GATGGACAAA TTAGGTGTAC ATCAAATATT ACTGGGCTAC TATTAACAAG 7140
AGATGGGGGT CATAATGATA ACAACACTAA CAACGAGACC TTCAGACCGG GAAGAGGAGA 7200
TATGAGGGAC AATTGGAGAA GTGAATTATA TAAATATAAA GTAATAAAAA TTGAACCATT 7260
AGGAGTAGCA CCCACCAAGG CAAAGAGAAG AGTGGTGCAG AGAGAAAAAA GAGCAGTGGG 7320
AATGGTAGGA GCTATGTTCC TTGGGTTCTT GGGAGCAGCA GGAAGCACTA TGGGCGCAGC 7380
GTCATTGACG CTGACGGTAC AGGCCAGACA ATTATTGTCT GGTATAGTGC AGCAGCAGAA 7440
CAATCTGCTG AGAGCTATTG AGGCGCAACA ACATCTGTTG CAACTCACAG TCTGGGGCAT 7500
CAAGCAGCTC CAGGCAAGAG TCCTGGCTGT AGAAAGATAC CTAAAGGATC AACAGCTCCT 7560
GGGGATCTGG GGTTGCTCTG GAAAACTCAT TTGCACCACT ACTGTGCCTT GGAATGCTAG 7620
TTGGAGTAAT AAATCTTTGG ATCAGATTTG GAATAACATG ACCTGGATGG AGTGGGACAG 7680
AGAAATTGCC AATTACACAA ACTTAATACA TCACTTAATT GAAGAATCGC AAAACCAGCA 7740
AGAAAAGAAT GAACAAGAAT TATTGGAATT AGATAAATGG GCAAGTTTGT GGAGTTGGTT 7800
TGACATATCA AACTGGCTGT GGTATATAAA AATATTCATA ATGATAGTAG CAGGCTTAGT 7860
AGGTTTAAGA ATAGTTTTTG CTGTGCTTTC TATAGTAAAT AGAGTTAGGC AGGGATACTC 7920
ACCATTGTCA TTCCAGACCC ACTTCCCAGC TCCGAGGGGA CCCGACAGGC CAGACGGAAT 7980
CGAAGGAGAA GGTGGAGAGA GAGACAGAGA CAGATCCGTG CGATTAGTGG ATGGATTCTT 8040
AGCACTTCTC TGGGAAGACC TGCGCAACCT GTGCCTCTTC AGCTACCACC GCTTGAGAGA 8100
CTTACTCTTG ATTGTAACGA GGATTGTGGA ACTTCTCGGA CGCAGGGGGT GGGAAGCCCT 8160
CAAATATTTG TGGAGTCTCC TACAGTATTG GAGTCAGGAG CTAAAGAATA GTGCTGTCAA 8220
CTTGTTCAAT ACCACAGCTA TAGTAGTAGC TGAGGGGACA GATAGGATCA TAGAAGTAGT 8280
ACAAAGACTT TGTAGAGCTA TTCTCCACAT ACCTAGAAGA ATTAGACAGG GCTTGGAAAG 8340
GATTTTGCTA TAAGATGGGT GGCAAGTGGT CAAAAAGTAG TATAGTTGGA TGGCCTACTA 8400
TAAGGGAAAG AATGAAACGA GCTGGACCAG CAGCAGATGG GGTGGGAGCA GCATCTCGAG 8460
ACCTAGAAAA ACATGGAGCA ATCACAAGTA GCAATACAGC AGCTACCAAT GCTGATTGTG 8520
CCTGGCTAGA AGCACAAGAG GAGGAAGAGG TGGGTTTTCC AGTCAGACCT CAGGTACCTT 8580
TAAGACCAAT GACTTACAAG GCAGGTATAG ATCTTAGCCA CTTTTTAAAA GAAAAGGGGG 8640
GACTGGAAGG GCTAGTTTGG TCCCAAAGAA GACAAGATAT CCTTGATCTG TGGATCTACC 8700
ACACACAAGG CTACTTCCCT GATTGGCAGA ACTACACACC AGGGCCAGGG ATCAGATATC 8760
CACTGACCTT TGGATGGTGC TTCAAGCTAG TACCAGTTGA GCCAGATAAG GTAGAAGAGG 8820
92 CCAATGAAGG AGAGGACAAC ATCTTGTTAC ACCCTATGTG TCTACATGGA ATGGAGGACG 8880
CGGAGAAAGA AGTGTTAGTG TGGAGATTTG ACAGTAAACT AGCCTTCCAT CACGTAGCCC 8940
GAGAGCTGCA TCCGGAGTAC TACAAAGACT GCTGACATCG AGCTTTCTAC AAGGGACTTT 9000
CCGCTGGGGA CTTTCCAGGG AGGCGTGATC TGGGTGGGAC TGGGGAGTGG CGTGCCCTCA 9060
GATGCTGCAT ATAAGCAGCT GCTTTTGCCT GTACTGGGTC TCTCTGGTTA GACCAGATCT 9120
GAGCCTGGGA GCTCTCTGGC TAGCTAGGGA ACCCACTGCT TAAGCCTCAA TAAAGCTTGC 9180
CTTGAGTGCT TCA 9193
93
Figure imgf000098_0001
HIVNCFULL7. SEQ ggtctctctggttagaccagatctgagcctgggagctctctggctagctagggaacccactgcttaagcc tcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagaga tccctcagacccttaaagtcagtgtggaaaaatctctagcagtggcgcccgaacagggacgcgaaagcga aagtagaaccagagaagctctctcgacgcaggactcggcttgctgaagcgcgcacagcaagaggcgaggg gaggcgactggtgagtacgccaatatttttgactagcggaggctagaaggagagagatgggtgcgagagc gtcggtattaagcgggggagatttagatcgatgggaaaaaattcggttaaggccagggggaaagaaaaaa tatatgttaaaacatatagtatgggcaagcagggagctagaacgattcgcagttaatcctggcctgttag aaacagctgaaggctgtagacaaatactagaacagctacagccatcccttcagacaggatcagaagaact taga cc tatataatacaatagcaaccctctattgtgtgcatcaaaaaatagaggtaaaagacaccaag gaagctttagaaaaaatagaggaagagcaaaacaaaagtaagaaaaaagtacagcaagcagcagctgcaa ctggcacaggaaacagcagccaggtcagccaaaattaccctatagtgcagaacctacaggggcaaatggt acatcaggccatatcacctagaactttaaatgcatgggtaaaagtaatagaagagaaggctttcagccca gaagtgatacccatgttttcagcattatcagaaggagccaccccacaagatttaaacaccatgctaaaca cagtggggggacatcaagcagccatgcaaatgttaaaagagaccatcaatgaggaagctgcagaatggga tagagtgcatccagtgcaggcagggc ta gcaccaggccagatgagagaaccaaggggaagtgacata gcaggaactactagtacccttcaggaacaaataggatggatgacaagtaatccacctatcccagtaggag aaatttataaaagatggataattttgggcttaaataaaatagtaagaatgtatagccctatcagcattct ggatataagacaaggaccaaaagaaccctttagagactatgtagaccggttctataaaactctaagagcc gagcaagcttcacaggaagtaaaaaattggatgacagaaaccttgttggtccaaaatgcgaacccagatt gtaagac a ttaaaagcattaggaccaggagctacattagaagaaatgatgacagcatgtcagggagt gggaggacccggccataaggcaagggttttagctgaagcaatgagccaagtaacaaattcagctgccata tgatgcagagaggcaattttaaaaaccaaagaaagatggttaagtgtttcaattgtggcaaagaagggc atgtagccagaaattgcagggcccctagaaaaaagggctgttggaaatgtggaaaggaaggacaccaaat gaaagattgtattgaaagacaggctaattttttagggaaaatctggccttcccacaagggaaggccaggg aat ttcttcagagcagaccagagccaacagccccaccagaagagagcctcaggtctgggatagagacaa caactccctctcagaagcaggagccaatagacaaggaagtgtatcctttaacttccctcagatcactctt tggcaacgacccctcgtcacaataaagataggggggcaactaaaggaagctc a tagatacaggagcag a gatac gtattagaagaaatgaatttgccaggaagatggaaaccaaaaatgatagggggaattggagg ttt atcaaagtaagacagtatgatcagatacccatagaaatctgtggacataaagctataggtacagta ttagtaggacctacacctgtcaacataattggaagaaatctgttgactcagattggttgcactttaaatt ttcccattagtcctattgaaactgtaccagtaaaattaaagccaggaatggatggcccaaaagttaaaca atggccattgacagaagaaaaaataaaagcattaatagaaatttgtacagaaatggaaaaggaagggaaa a tcaaaaattgggcctgaaaatccatacaatactccagtatttgccataaagaaaaaagacagtacta aatggagaaaattagtagatttcagagaacttaataagagaactcaagacttctgggaagttcaattagg aataccacatcccgcagggttaaaaaagaaaaaatcagtaacagtactagatgtgggtgatgcatatttt tcagttcccttagatgaagatttcaggaagtatactgcatttaccatacctagtataaacaatgagacac cagggattagatatcagtacaatgtgctgccacagggatggaaaggatcaccagcaatattccaaagtag catgacaaaaatcttagagccttttagaaaacagaatccagacatagttatctatcaatatatggatgat ttgtatgtaggatctgacttagaaatagagcagcatagaacaaaaatagaggaactgagacagcatctgt tgaagtggggatttaccacaccagacaaaaaacatcagaaggaacctccattcctctggatgggttatga actccatcctgataaatggacagtacagcctatagtgctgccagagaaagacagctggactgtcaatgac atacagaagttagtgggaaaattaaattgggcaagtcagatttacccagggattaaagtaaggcaattat gtaaac ccttaggggagccaaagcactaacagatgtaataccactaacagaaggagcagagctagaact ggcagaaaacagagagattctaaaagaaccagtacatggagtgtattatgacccatcaaaagacttaata gcagaaatacagaagcagggacaaggtcaatggacatatcagatttatcaagagccatttaaaaatctga gaacaggaaaatatgcaagaacgaggggtgcccacactaatgatataaaacaattaacagaggcagtgca aaaaatagccacagaaagcatagtaatatggggaaagac cc aaatttaaactgcccatacaaaaggaa acatgggaaacatggtggacagagtattggcaagctacctggattcctgagtgggagtttgtcaataccc ctcc ttagtaaaattatggtaccagttagagaaagaacccatagtaggagcagaaactttctatgtaga tggggcagctaacagggagactaaattaggaaaagcaggatatgttactaacagaggaagacaaaaggta gtccccctgactaacacaacaaatcagaagactgagttacaagcaattcatctagctttacaggattcag gatcagaagtaaacatagtaacagactcacaatatgcattaggaatcattcaagcacaaccagatcaaag tgaatcagagttagtcaatcaaataatagaacagttaataaaaaaggaaaaggtctatctggcatgggta ccagcacacaaaggaattggaggaaatgaacaagtagataaattagtcagtgctggaatcaggaaagtac ta tctagatggaatagataaggcccaagaagaacatgagaaatatcataataattggagagcaatggc tagtgattttaacctgccacctgtagtagcaaaggaaatagtagccagctgtgataaatgtcagctaaaa
94 ggagaagccatgcatggacaagtagactgtagtccaggaatatggcaactagattgtacacatttagaag gaaaagttatcctagtagcagttcatgtagccagtggatatatagaagcagaagttattccagcagaaac agggcaggaaacagcatactttctcttaaaattagcaggaagatggccagtaaaaacaatacacacagat aatggcagcaatttcaccagtgctgcggttaaggccgcctgttggtgggcgggagtcaagcaggaatttg gaattccctacaatccccaaagtcaaggagtagtagaatctatgaataaagaattaaagaaaattatagg acaggtaagagatcaagctgaacatcttaagacagcagtacaaatggcagtatttgtccacaattttaaa agaaaaggggggattggggggtacagtgcaggggaaagaataatagacataatagcaacagacatacaaa ctagagaactacaaaaacaaattacaaaaattcaaaattttcgggt ta acagggacagcagagaccc actttggaaaggaccagcaaagc cc ctggaaaggtgaaggggcagtagtaatacaagataatagtgac ataaaagtagtgccaagaagaaaagcaaagatcattagggattatggaaaacagatggcaggtgatgatt gtgtggcaagtagacaggatgaggattagaacatggacaagtttagtaaaacatcatgtatatgtttcaa agaaagctaggggatggttttatagacatcactatgaaagcactcatccaaaaataagttcagaaataca cattccactaggggatgctagattggtagtaacaacatattggggtctgcatacaggagaaagagaatgg ca tgggtcatggagtttccatagaatggaggaaaaggagctatagcacacaagtagaccctgaactag cagaccaactaattcatctgtattactttgattgttttgcagactctgctataagaaaggccttattagg acacatagttagccctaggtgtgcatatcaagcaggacattccaaggtaggatctctacaatacttggca cta cagccttaataacaccaaagaagataaagccacctttgcctagtgttacaaaactgacagaggata gatggaacaagccccagaagaccaagggccacagagggagccacacaatgaatggacactagagctttta gaggagcttaagacagaagctgttagacattttcctaggccatggctccatagcttaggacaacatatct atgaaacttatggagatacttgggcaggagtggaagccataataagaattctgcaacaattgctgtt at tc tttcagaattgggtgtcaacatagcagaataggcattattcaacagaggagagcaagaagaaatgga gccagtagatcctaatctagagccctggaagcatccaggaagtcagcctaaaactgcttgtaccaaatgc tattgtaaaaagtgttgctttcattgccaagtttgtttcacaacaaaagccttaggcatctcc atggca ggaagaagcggagacagcgacgaagagc cc cagggcagccaggc ca caagcttctctatcagagca gtaagtagtatatgtaatgcaacttatattaatagtaacaatagtagctttagtagtaacattaataata gcaatagttgtgtggtccatagtactcatagaatataggaaa at aagacaaaggaaaatagacaagt taattaatagactagtagaaagagcagaagacagtggcaatgagagtgaaggagaactatcagcacttgt ggagatgggggtggaaatggggca ca gc ccttgggatgttaatgatctgtagtgctacagaaaaatt gtgggtcacagtctattatggggtacctgtgtggaaggaagcaaccaccactctattttgtgcatcagat gctaaagcatatgaagaagaggtacataatgtttgggccacacatgcctgtgtacccacagaccccaacc cacaagaaatagtattggcaaatgtgacagaagattttaacatgtggaaaaatgaaatggtagaacagat gcatactgatataatcagtttatgggatgaaagcctaaaaccatgtgtaaaattaaccccactctgtgtt ac taaattgcactgatttgaagaatgaaactaagaccaatagtagtgatgccaatagtaatagcgggg aaa aatggggaacgaagagataaaaaattgctctttcaatgtcagcacaggcgcaccaggtaaggtgca gaaagaatatgcacttttttatgcacttgatatagtatcaataaagaatgaaaataatagtaccagccat atgttgacaagttgtaacacctcagtcagtacacaggcctgtccaaaggta cc ttgagccaattccca taca a gtgccccggctggttttgcaattctaaaatgtaatgataagaagttcaatggaacaggacc atgtaacaatgtcagcacagtacaatgtacacatggaattaggccagtagtatcaactcaactgctgtta aatggcagtctagcagaagaagaggtagtacttagatctgccaatttctcagacaatgctaaaaccataa tagtacagctgaaccactctgtagaaattacttgtacaagacccaactacaatgaaacaaaaagaatccg tatccacagaggatatggaagatcat tgttacagtaagaaaattgggagataggaaacaagcacattgt accatgaatagaacgaaatgggacaacgctttaaaacagatagctagcaaattaagagaacaatttaata aaacagcaataatctttaaccggtcctcaggaggggacctagaaattgaaatgcacagttttaattgcgg aggggaattgttctactgtaatacaacaaaactgtttaatagtacttggaatgagactacagagtcaaat ggcaagggagaaaatatcacactcccatgcagaataagacaatttgtaaacatgtggcagaaagtaggaa aagcaatgtatgcccctcccagcgatggacaaattaggtgtacatcaaatattactgggctactattaac aagagatgggggtggtcctagtgataacaaaaccgacaagaccttcagaccaggaggaggagatatgagg gacaattggagaagtgaattatataaatataaagtaataaaaattgaaccattaggagtagcacccacca aggcaaagagaagagtggtgcaaagagaaaaaagagcagtgggaatggtaggagctatg cc tgggtt cttgggagcagcaggaagcactatgggcgcagcgtcattgacgctgacggtacaggccagacaattattg tctggtatagtgcagcagcagaacaatctgctgagagctattgaggcgcaacaacatctgttgcaactca cagtctggggcatcaagcagctccaggcaagagtcctggctgtagaaagatacctaaaggatcaacagct cctggggatctggggttgctctggaaaactca tgcaccactactgtgccttggaatgctagttggagt aataaatctctggatcagatttggaataacatgacctggttggagtgggacagagaaattgccaattaca caaacttaatacatcacttaattgaagaatcgcaaaaccagcaagaaaagaatgaacaagaattattgga attagataaatgggcaagtttgtggagttggtttgacatatcaaactggctgtggtatataaaaatattc ataatgatagtagcaggcttagtaggtttaagaatagtttttgctgtgctttctatagtaaatagagtta ggcagggatactcaccattgtcattccagacccacttcccagctccgaggggacccgacaggccagacgg
95 aatcgaaggagaaggtggagagagagacagagacagatccgtgcgattagtggatggattcttagcactt ctctgggaagacctgcgcaacctgtgcctcttcagctaccaccgcttgagagacttactcttgattgtaa cgaggattgtggaacttctgggacgcagggggtgggaagccctcaaatattggtggagtctcctacagta ttggagtcaggagctaaagaatagtgctgtcaacttgttcaataccagagctatagtagtagctgagggg acagataggatcatagaagtagtacaaagactttgtagagctattctccacatacctagaagaattagac agggcttggaaagatttttgctataagatgggtggcaagtggtcaaaaagtagtatagttggatggccta ctataagggaaagaatgaaacgagctggaccagcagcagatggggtgggagcagcatctcgagacctaga aaaacatcgagcaatcacaagtagcaatacagcagctaccaatgctgattgtgcctggctagaagcacaa gaggaggaagaggtgggttttccagtcagacctcaggtacctttaagaccaatgacttacaaggcaggta tagatcttagccactttttaaaagaaaaggggggactggaagggctagtttggtcccaaagaagacaaga tatccttgatctgtggatctaccacacacaaggctacttccctgattggcagaactacacaccagggcca gggatcagatatccactgacctttggatggtgcttcaagctagtaccagttgagccagataaggtagaag aggccaatgaaggagaggacaacatcttgttacaccctatgtgtctacatggaatggaggacgcggagaa agaagtgttagtgtggagatttgacagtaaactagccttccatcacgtagcccgagagctgcatccggag tactacaaagactgctgacatcgagctttctacaagggactttccgctggggactttccagggaggcgtg atc gggtgggactggggagtggcgtgccctcagatgctgcatataagcagctgcttttgcctgtactgg gtctctctggttagaccagatctgagcctgggagctctctggctagctagggaacccactgcttaagcct caataaagcttgccttgagtgcttca
96 FOR SEQ ID NO: 13 THROUGH SEQ ID NO: 17, PLEASE SEE TABLE 5.
(2) INFORMATION FOR SEQ ID NO: 18
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "Oligonucleotide."
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18
AAATCTCTAG CAGTGGCGCC CGAACAG 27
(2) INFORMATION FOR SEQ ID NO: 19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "Oligonucleotide."
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:
GCACTCAAGG CAAGCTTTAT TGAGGCT 27
(2) INFORMATION FOR SEQ ID NO: 20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "Oligonucleotide."
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:
CACACACAAG GCTACTTCCC TGATTGGCAG A 31
(2) INFORMATION FOR SEQ ID NO: 21:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 base pairs
97 (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "Oligonucleotide."
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:
ATGGAACAAG CCCCAGAAGA CCAAGGGCCA CAG 33
(2) INFORMATION FOR SEQ ID NO: 22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "Oligonucleotide."
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:
GGTCTGAGGG ATCTCTAGTT ACCAGAGTCA C 31
(2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2404 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: not relevant
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
Met Glu Thr Ala Arg Gly Val Ala Leu Leu Tyr Ser Gly Leu Ala Ser 1 5 10 15
Asn Thr Tyr Arg Gly Leu Asn His He Ser Leu Glu Thr Arg Pro Ala 20 25 30
Arg Gly Thr Arg Pro Gly Leu Tyr Thr Arg Pro Leu Tyr Ser Thr Arg 35 40 45
Pro Gly Leu Tyr He Leu Glu Met Glu Thr Leu Glu Leu Glu Gly Leu 50 55 60
98 Tyr Met Glu Thr Leu Glu Met Glu Thr He Leu Glu Cys Tyr Ser Ser 65 70 75 80
Glu Arg Ala Leu Ala Thr His Arg Gly Leu Leu Tyr Ser Leu Glu Thr 85 90 95
Arg Pro Val Ala Leu Thr His Arg Val Ala Leu Thr Tyr Arg Thr Tyr 100 105 110
Arg Gly Leu Tyr Val Ala Leu Pro Arg Val Ala Leu Thr Arg Pro Leu 115 120 125
Tyr Ser Gly Leu Ala Leu Ala Thr His Arg Thr His Arg Thr His Arg 130 135 140
Leu Glu Pro His Glu Cys Tyr Ser Ala Leu Ala Ser Glu Arg Ala Ser 145 150 155 160
Pro Ala Leu Ala Leu Tyr Ser Ala Leu Ala Thr Tyr Arg Gly Leu Gly 165 170 175
Leu Gly Leu Val Ala Leu His He Ser Ala Ser Asn Val Ala Leu Thr 180 185 190
Arg Pro Ala Leu Ala Thr His Arg His He Ser Ala Leu Ala Cys Tyr 195 200 205
Ser Val Ala Leu Pro Arg Thr His Arg Ala Ser Pro Pro Arg Ala Ser 210 215 220
Asn Pro Arg Gly Leu Asn Gly Leu He Leu Glu Val Ala Leu Leu Glu 225 230 235 240
Ala Leu Ala Ala Ser Asn Val Ala Leu Thr His Arg Gly Leu Ala Ser 245 250 255
Pro Pro His Glu Ala Ser Asn Met Glu Thr Thr Arg Pro Leu Tyr Ser 260 265 270
Ala Ser Asn Gly Leu Met Glu Thr Val Ala Leu Gly Leu Gly Leu Asn 275 280 285
Met Glu Thr His He Ser Thr His Arg Ala Ser Pro He Leu Glu He 290 295 300
Leu Glu Ser Glu Arg Leu Glu Thr Arg Pro Ala Ser Pro Gly Leu Ser 305 310 315 320
Glu Arg Leu Glu Leu Tyr Ser Pro Arg Cys Tyr Ser Val Ala Leu Leu 325 330 335
Tyr Ser Leu Glu Thr His Arg Pro Arg Leu Glu Cys Tyr Ser Val Ala 340 345 350
Leu Thr His Arg Leu Glu Ala Ser Asn Cys Tyr Ser Thr His Arg Ala 355 360 365
99 Ser Pro Leu Glu Leu Tyr Ser Ala Ser Asn Gly Leu Thr His Arg Leu 370 375 380
Tyr Ser Thr His Arg Ala Ser Asn Ser Glu Arg Ser Glu Arg Ala Ser 385 390 395 400
Pro Ala Leu Ala Ala Ser Asn Ser Glu Arg Ala Ser Asn Ser Glu Arg 405 410 415
Gly Leu Tyr Gly Leu He Leu Glu Met Glu Thr Gly Leu Tyr Ala Ser 420 425 430
Asn Gly Leu Gly Leu He Leu Glu Leu Tyr Ser Ala Ser Asn Cys Tyr 435 440 445
Ser Ser Glu Arg Pro His Glu Ala Ser Asn Val Ala Leu Ser Glu Arg 450 455 460
Thr His Arg Gly Leu Tyr Ala Leu Ala Pro Arg Gly Leu Tyr Leu Tyr 465 470 475 480
Ser Val Ala Leu Gly Leu Asn Leu Tyr Ser Gly Leu Thr Tyr Arg Ser 485 490 495
Glu Arg Leu Glu Pro His Glu Thr Tyr Arg Ala Leu Ala Leu Glu Ala 500 505 510
Ser Pro He Leu Glu Val Ala Leu Ser Glu Arg He Leu Glu Leu Tyr 515 520 525
Ser Ala Ser Asn Gly Leu Ala Ser Asn Ala Ser Asn Ser Glu Arg Thr 530 535 540
His Arg Ser Glu Arg His He Ser Met Glu Thr Leu Glu Thr His Arg 545 550 555 560
Ser Glu Arg Cys Tyr Ser Ala Ser Asn Thr His Arg Ser Glu Arg Val 565 570 575
Ala Leu Ser Glu Arg Thr His Arg Gly Leu Asn Ala Leu Ala Cys Tyr 580 585 590
Ser Pro Arg Leu Tyr Ser Val Ala Leu Ser Glu Arg Pro His Glu Gly 595 600 605
Leu Pro Arg He Leu Glu Pro Arg He Leu Glu His He Ser Thr Tyr 610 615 620
Arg Cys Tyr Ser Ala Leu Ala Pro Arg Ala Leu Ala Gly Leu Tyr Pro 625 630 635 640
His Glu Ala Leu Ala He Leu Glu Leu Glu Leu Tyr Ser Cys Tyr Ser 645 650 655
Ala Ser Asn Ala Ser Pro Leu Tyr Ser Leu Tyr Ser Pro His Glu Ala 660 665 670
100 Ser Asn Gly Leu Tyr Thr His Arg Gly Leu Tyr Pro Arg Cys Tyr Ser 675 680 685
Ala Ser Asn Ala Ser Asn Val Ala Leu Ser Glu Arg Thr His Arg Val 690 695 700
Ala Leu Gly Leu Asn Cys Tyr Ser Thr His Arg His He Ser Gly Leu 705 710 715 720
Tyr He Leu Glu Ala Arg Gly Pro Arg Val Ala Leu Val Ala Leu Ser 725 730 735
Glu Arg Thr His Arg Gly Leu Asn Leu Glu Leu Glu Leu Glu Ala Ser 740 745 750
Asn Gly Leu Tyr Ser Glu Arg Leu Glu Ala Leu Ala Gly Leu Gly Leu 755 760 765
Gly Leu Val Ala Leu Val Ala Leu Leu Glu Ala Arg Gly Ser Glu Arg 770 775 780
Ala Leu Ala Ala Ser Asn Pro His Glu Ser Glu Arg Ala Ser Pro Ala 785 790 795 800
Ser Asn Ala Leu Ala Leu Tyr Ser Thr His Arg He Leu Glu He Leu 805 810 815
Glu Val Ala Leu Gly Leu Asn Leu Glu Ala Ser Asn His He Ser Ser 820 825 830
Glu Arg Val Ala Leu Gly Leu He Leu Glu Thr His Arg Cys Tyr Ser 835 840 845
Thr His Arg Ala Arg Gly Pro Arg Ala Ser Asn Thr Tyr Arg Ala Ser 850 855 860
Asn Gly Leu Thr His Arg Leu Tyr Ser Leu Tyr Ser He Leu Glu Ala 865 870 875 880
Arg Gly He Leu Glu His He Ser Ala Arg Gly Gly Leu Tyr Thr Tyr 885 890 895
Arg Gly Leu Tyr Ala Arg Gly Ser Glu Arg Pro His Glu Val Ala Leu 900 905 910
Thr His Arg Val Ala Leu Ala Arg Gly Leu Tyr Ser Leu Glu Gly Leu 915 920 925
Tyr Ala Ser Pro Ala Arg Gly Leu Tyr Ser Gly Leu Asn Ala Leu Ala 930 935 940
His He Ser Cys Tyr Ser Thr His Arg Met Glu Thr Ala Ser Asn Ala 945 950 955 960
Arg Gly Thr His Arg Leu Tyr Ser Thr Arg Pro Ala Ser Pro Ala Ser 965 970 975
101 Asn Ala Leu Ala Leu Glu Leu Tyr Ser Gly Leu Asn He Leu Glu Ala 980 985 990
Leu Ala Ser Glu Arg Leu Tyr Ser Leu Glu Ala Arg Gly Gly Leu Gly 995 1000 1005
Leu Asn Pro His Glu Ala Ser Asn Leu Tyr Ser Thr His Arg Ala Leu 1010 1015 1020
Ala He Leu Glu He Leu Glu Pro His Glu Ala Ser Asn Ala Arg Gly 1025 1030 1035 1040
Ser Glu Arg Ser Glu Arg Gly Leu Tyr Gly Leu Tyr Ala Ser Pro Leu 1045 1050 1055
Glu Gly Leu He Leu Glu Gly Leu Met Glu Thr His He Ser Ser Glu 1060 1065 1070
Arg Pro His Glu Ala Ser Asn Cys Tyr Ser Gly Leu Tyr Gly Leu Tyr 1075 1080 1085
Gly Leu Leu Glu Pro His Glu Thr Tyr Arg Cys Tyr Ser Ala Ser Asn 1090 1095 1100
Thr His Arg Thr His Arg Leu Tyr Ser Leu Glu Pro His Glu Ala Ser 1105 1110 1115 1120
Asn Ser Glu Arg Thr His Arg Thr Arg Pro Ala Ser Asn Gly Leu Thr 1125 1130 1135
His Arg Thr His Arg Gly Leu Ser Glu Arg Ala Ser Asn Gly Leu Tyr 1140 1145 1150
Leu Tyr Ser Gly Leu Tyr Gly Leu Ala Ser Asn He Leu Glu Thr His 1155 1160 1165
Arg Leu Glu Pro Arg Cys Tyr Ser Ala Arg Gly He Leu Glu Ala Arg 1170 1175 1180
Gly Gly Leu Asn Pro His Glu Val Ala Leu Ala Ser Asn Met Glu Thr 1185 1190 1195 1200
Thr Arg Pro Gly Leu Asn Leu Tyr Ser Val Ala Leu Gly Leu Tyr Leu 1205 1210 1215
Tyr Ser Ala Leu Ala Met Glu Thr Thr Tyr Arg Ala Leu Ala Pro Arg 1220 1225 1230
Pro Arg Ser Glu Arg Ala Ser Pro Gly Leu Tyr Gly Leu Asn He Leu 1235 1240 1245
Glu Ala Arg Gly Cys Tyr Ser Thr His Arg Ser Glu Arg Ala Ser Asn 1250 1255 1260
He Leu Glu Thr His Arg Gly Leu Tyr Leu Glu Leu Glu Leu Glu Thr 1265 1270 1275 1280
102 His Arg Ala Arg Gly Ala Ser Pro Gly Leu Tyr Gly Leu Tyr Gly Leu 1285 1290 1295
Tyr Pro Arg Ser Glu Arg Ala Ser Pro Ala Ser Asn Leu Tyr Ser Ala 1300 1305 1310
Ser Pro Leu Tyr Ser Gly Leu Thr His Arg Pro His Glu Ala Arg Gly 1315 1320 1325
Pro Arg Gly Leu Tyr Gly Leu Tyr Gly Leu Tyr Ala Ser Pro Met Glu 1330 1335 1340
Thr Ala Arg Gly Ala Ser Pro Ala Ser Asn Thr Arg Pro Ala Arg Gly
1345 1350 1355 1360
Ser Glu Arg Gly Leu Leu Glu Thr Tyr Arg Leu Tyr Ser Thr Tyr Arg 1365 1370 1375
Leu Tyr Ser Val Ala Leu He Leu Glu Leu Tyr Ser He Leu Glu Gly 1380 1385 1390
Leu Pro Arg Leu Glu Gly Leu Tyr Val Ala Leu Ala Leu Ala Pro Arg 1395 1400 1405
Thr His Arg Leu Tyr Ser Ala Leu Ala Leu Tyr Ser Ala Arg Gly Ala 1410 1415 1420
Arg Gly Val Ala Leu Val Ala Leu Gly Leu Asn Ala Arg Gly Gly Leu
1425 1430 1435 1440
Leu Tyr Ser Ala Arg Gly Ala Leu Ala Val Ala Leu Gly Leu Tyr Met 1445 1450 1455
Glu Thr Val Ala Leu Gly Leu Tyr Ala Leu Ala Met Glu Thr Pro His 1460 1465 1470
Glu Leu Glu Gly Leu Tyr Pro His Glu Leu Glu Gly Leu Tyr Ala Leu 1475 1480 1485
Ala Ala Leu Ala Gly Leu Tyr Ser Glu Arg Thr His Arg Met Glu Thr 1490 1495 1500
Gly Leu Tyr Ala Leu Ala Ala Leu Ala Ser Glu Arg Leu Glu Thr His
1505 1510 1515 1520
Arg Leu Glu Thr His Arg Val Ala Leu Gly Leu Asn Ala Leu Ala Ala 1525 1530 1535
Arg Gly Gly Leu Asn Leu Glu Leu Glu Ser Glu Arg Gly Leu Tyr He 1540 1545 1550
Leu Glu Val Ala Leu Gly Leu Asn Gly Leu Asn Gly Leu Asn Ala Ser 1555 1560 1565
Asn Ala Ser Asn Leu Glu Leu Glu Ala Arg Gly Ala Leu Ala He Leu 1570 1575 1580
103 Glu Gly Leu Ala Leu Ala Gly Leu Asn Gly Leu Asn His He Ser Leu 1585 1590 1595 1600
Glu Leu Glu Gly Leu Asn Leu Glu Thr His Arg Val Ala Leu Thr Arg 1605 1610 1615
Pro Gly Leu Tyr He Leu Glu Leu Tyr Ser Gly Leu Asn Leu Glu Gly 1620 1625 1630
Leu Asn Ala Leu Ala Ala Arg Gly Val Ala Leu Leu Glu Ala Leu Ala 1635 1640 1645
Val Ala Leu Gly Leu Ala Arg Gly Thr Tyr Arg Leu Glu Leu Tyr Ser 1650 1655 1660
Ala Ser Pro Gly Leu Asn Gly Leu Asn Leu Glu Leu Glu Gly Leu Tyr 1665 1670 1675 1680
He Leu Glu Thr Arg Pro Gly Leu Tyr Cys Tyr Ser Ser Glu Arg Gly 1685 1690 1695
Leu Tyr Leu Tyr Ser Leu Glu He Leu Glu Cys Tyr Ser Thr His Arg 1700 1705 1710
Thr His Arg Thr His Arg Val Ala Leu Pro Arg Thr Arg Pro Ala Ser 1715 1720 1725
Asn Ala Leu Ala Ser Glu Arg Thr Arg Pro Ser Glu Arg Ala Ser Asn 1730 1735 1740
Leu Tyr Ser Ser Glu Arg Leu Glu Ala Ser Pro Gly Leu Asn He Leu 1745 1750 1755 1760
Glu Thr Arg Pro Ala Ser Asn Ala Ser Asn Met Glu Thr Thr His Arg 1765 1770 1775
Thr Arg Pro Leu Glu Gly Leu Thr Arg Pro Ala Ser Pro Ala Arg Gly 1780 1785 1790
Gly Leu He Leu Glu Ala Leu Ala Ala Ser Asn Thr Tyr Arg Thr His 1795 1800 1805
Arg Ala Ser Asn Leu Glu He Leu Glu His He Ser His He Ser Leu 1810 1815 1820
Glu He Leu Glu Gly Leu Gly Leu Ser Glu Arg Gly Leu Asn Ala Ser 1825 1830 1835 1840
Asn Gly Leu Asn Gly Leu Asn Gly Leu Leu Tyr Ser Ala Ser Asn Gly 1845 1850 1855
Leu Gly Leu Asn Gly Leu Leu Glu Leu Glu Gly Leu Leu Glu Ala Ser 1860 1865 1870
Pro Leu Tyr Ser Thr Arg Pro Ala Leu Ala Ser Glu Arg Leu Glu Thr 1875 1880 1885
104 Arg Pro Ser Glu Arg Thr Arg Pro Pro His Glu Ala Ser Pro He Leu 1890 1895 1900
Glu Ser Glu Arg Ala Ser Asn Thr Arg Pro Leu Glu Thr Arg Pro Thr 1905 1910 1915 1920
Tyr Arg He Leu Glu Leu Tyr Ser He Leu Glu Pro His Glu He Leu 1925 1930 1935
Glu Met Glu Thr He Leu Glu Val Ala Leu Ala Leu Ala Gly Leu Tyr 1940 1945 1950
Leu Glu Val Ala Leu Gly Leu Tyr Leu Glu Ala Arg Gly He Leu Glu 1955 1960 1965
Val Ala Leu Pro His Glu Ala Leu Ala Val Ala Leu Leu Glu Ser Glu 1970 1975 1980
Arg He Leu Glu Val Ala Leu Ala Ser Asn Ala Arg Gly Val Ala Leu 1985 1990 1995 2000
Ala Arg Gly Gly Leu Asn Gly Leu Tyr Thr Tyr Arg Ser Glu Arg Pro 2005 2010 2015
Arg Leu Glu Ser Glu Arg Pro His Glu Gly Leu Asn Thr His Arg His 2020 2025 2030
He Ser Pro His Glu Pro Arg Ala Leu Ala Pro Arg Ala Arg Gly Gly 2035 2040 2045
Leu Tyr Pro Arg Ala Ser Pro Ala Arg Gly Pro Arg Ala Ser Pro Gly 2050 2055 2060
Leu Tyr He Leu Glu Gly Leu Gly Leu Tyr Gly Leu Gly Leu Tyr Gly 2065 2070 2075 2080
Leu Tyr Gly Leu Ala Arg Gly Ala Ser Pro Ala Arg Gly Ala Ser Pro 2085 2090 2095
Ala Arg Gly Ser Glu Arg Val Ala Leu Ala Arg Gly Leu Glu Val Ala 2100 2105 2110
Leu Ala Ser Pro Gly Leu Tyr Pro His Glu Leu Glu Ala Leu Ala Leu 2115 2120 2125
Glu Leu Glu Thr Arg Pro Gly Leu Ala Ser Pro Leu Glu Ala Arg Gly 2130 2135 2140
Ala Ser Asn Leu Glu Cys Tyr Ser Leu Glu Pro His Glu Ser Glu Arg 2145 2150 2155 2160
Thr Tyr Arg His He Ser Ala Arg Gly Leu Glu Ala Arg Gly Ala Ser 2165 2170 2175
Pro Leu Glu Leu Glu Leu Glu He Leu Glu Val Ala Leu Thr His Arg 2180 2185 2190
105 Ala Arg Gly He Leu Glu Val Ala Leu Gly Leu Leu Glu Leu Glu Gly 2195 2200 2205
Leu Tyr Ala Arg Gly Ala Arg Gly Gly Leu Tyr Thr Arg Pro Gly Leu 2210 2215 2220
Ala Leu Ala Leu Glu Leu Tyr Ser Thr Tyr Arg Thr Arg Pro Thr Arg 2225 2230 2235 2240
Pro Ser Glu Arg Leu Glu Leu Glu Gly Leu Asn Thr Tyr Arg Thr Arg 2245 2250 2255
Pro Ser Glu Arg Gly Leu Asn Gly Leu Leu Glu Leu Tyr Ser Ala Ser 2260 2265 2270
Asn Ser Glu Arg Ala Leu Ala Val Ala Leu Ala Ser Asn Leu Glu Pro 2275 2280 2285
His Glu Ala Ser Asn Thr His Arg Ala Arg Gly Ala Leu Ala He Leu 2290 2295 2300
Glu Val Ala Leu Val Ala Leu Ala Leu Ala Gly Leu Gly Leu Tyr Thr 2305 2310 2315 2320
His Arg Ala Ser Pro Ala Arg Gly He Leu Glu He Leu Glu Gly Leu 2325 2330 2335
Val Ala Leu Val Ala Leu Gly Leu Asn Ala Arg Gly Leu Glu Cys Tyr 2340 2345 2350
Ser Ala Arg Gly Ala Leu Ala He Leu Glu Leu Glu His He Ser He 2355 2360 2365
Leu Glu Pro Arg Ala Arg Gly Ala Arg Gly He Leu Glu Ala Arg Gly 2370 2375 2380
Gly Leu Asn Gly Leu Tyr Leu Glu Gly Leu Ala Arg Gly Pro His Glu 2385 2390 2395 2400
Leu Glu Leu Glu
106

Claims

We claim:
1. An isolated human immunodeficiency virus type 1 (HIV-1), which is HIV-1 JC, identified by a nucleotide sequence as given in SEQ ID NO:l 1, or which is HIV-1NC, identified by a nucleotide sequence as given in SEQ ID NO: 12.
2. A biological sample comprising the HIV- 1 JC of claim 1.
3. A biological sample comprising the HIV-1NC of claim 1.
4. The biological sample of claim 1 wherein said sample is a sample of blood.
5. A biologically pure culture of host cells comprising the HIV-1JC or HIV-1NC of claim
1.
6. The culture of claim 5 wherein the host cells are peripheral blood mononuclear cells.
7. A composition comprising an antigenic preparation of the HIV- 1 JC of claim 1.
8. A composition comprising an antigenic preparation of the HIV-1NC of claim 1.
9. A kit for detecting the presence of HIV- 1 antibodies comprising an antigenic preparation of the HIV-1JC or HIV-1NC of claim 1.
10. An immunogenic composition comprising an antigenic preparation of the HIV-1JC or HIV-1NC of claim 1 and a pharmaceutically acceptable carrier.
11. A vaccine comprising an antigenic preparation of the HIV- 1 JC or HIV- 1 NC of claim 1.
12. An isolated DNA molecule comprising a nucleotide sequence encoding an infectious molecular clone for HIV-1JC or an antigenic fragment thereof or for HIV-1NC or an antigenic fragment thereof.
107
13. The isolated DNA molecule of claim 12, wherein the DNA comprises a nucleotide sequence encoding an HIV-1JC envelope protein as given in SEQ ID NO:2. an HIV-1JC gag, nef or p24 protein, or an antigenic fragment of any of the foregoing..
14. The isolated DNA molecule of claim 13, wherein the DNA comprises the nucleotide sequence of SEQ ID NO: 1 or a portion thereof specifying an antigenic fragment of said HIV-1JC envelope protein.
15. The isolated DNA molecule of claim 12, wherein the DNA comprises a nucleotide sequence encoding an HIV-1NC envelope protein as given in SEQ ID NO:23, an HIV- 1NC gag, nef or p24 protein, or an antigenic fragment of any of the foregoing.
16. A method of inducing antibodies to HIV-1JC in a nonhuman mammalian subject comprising the steps of (a) administering to the subject an immunogenic amount of an antigenic preparation of the HIV-1JC and optionally (b) harvesting said antibodies to HIV-1JC.
17. The method of claim 16 wherein said subject is a primate.
18. A method of inducing antibodies to HIV- 1 NC in a nonhuman mammalian subj ect comprising the steps of (a) administering to the subject an immunogenic amount of an antigenic preparation of the HIV-1NC and optionally (b) harvesting said antibodies to HIV-1NC.
19. The method of claim 18 wherein said subject is a primate.
20. A method of immunizing a subject against the development of acquired immune deficiency syndrome (AIDS) comprising the step of administering to said subject an immunogenic amount of an antigenic preparation of HIV-1JC or HIV-1NC such that antibodies directed to HIV-1JC or HIV-1NC are produced in said subject and thereby symptoms of AIDS are diminished or prevented.
21. The method of claim 20 wherein said subject is a primate.
108
22. A method for inducing acquired immune deficiency syndrome (AIDS) in a nonhuman primate, comprising the step of administering to said primate an effective amount of an infections preparation of the HIV-1 of claim 1 such that said primate develops AIDS.
23. The method of claim 22 wherein said infections preparation of HIV- 1 is a biological specimen obtained from a nonhuman primate infected with HIV-1 JC and exhibiting AIDS.
24. The method of claim 22 wherein said infections preparation of HIV- 1 is a biological specimen obtained from a nonhuman primate infected with HIV-1NC and exhibiting AIDS.
25. A method of screening for HIV-1 in a biological sample comprising the step of introducing into said biological sample a hybridization probe comprising a nucleotide sequence comprising at least about 15 contiguous nucleotides from SEQ ID NO:l 1 or a sequence complementary thereto or from SEQ ID NO: 12 or a nucleotide sequence complementary thereto under stringent conditions such that said hybridization probe binds to an HIV-1JC gene or a nucleotide sequence having at least about 95% nucleotide sequence identity thereto or an HIV-1NC gene or a nucleotide sequence having at least about 95% nucleotide sequence identity thereto.
26. A nonhuman primate model infected with at least one HIV-1 of claim 1 and exhibiting symptoms of AIDS, useful for the development of a drug or vaccine for the treatment or prevention of AIDS.
27. The primate model of claim 26 wherein said primate is a chimpanzee or a macaque.
28. The primate model of claim 27 wherein said HIV-1 is HIV-1JC.
29. The primate model of claim 27 wherein said HIV-1 is HIV-1NC.
109
PCT/US1998/012990 1997-06-23 1998-06-23 Human immunodeficiency viruses causing aids in a nonhuman primate WO1998059074A1 (en)

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US7217554B2 (en) 1999-08-31 2007-05-15 Novozymes A/S Proteases and variants thereof
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