WO1992014489A1 - Poliovirus-based vaccines - Google Patents

Poliovirus-based vaccines Download PDF

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Publication number
WO1992014489A1
WO1992014489A1 PCT/US1992/001303 US9201303W WO9214489A1 WO 1992014489 A1 WO1992014489 A1 WO 1992014489A1 US 9201303 W US9201303 W US 9201303W WO 9214489 A1 WO9214489 A1 WO 9214489A1
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poliovirus
hybrid
loop
hiv
epitope
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PCT/US1992/001303
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French (fr)
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Michael G. Murray
Scott D. Putney
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Repligen Corporation
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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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • 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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/32011—Picornaviridae
    • C12N2770/32611—Poliovirus
    • C12N2770/32622—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This invention relates to a poliovirus-based vaccine.
  • Poliovirus is a picornavirus which exists in nature as three stable ser ⁇ types, PV-1, PV-2 and PV-3. These viruses each display four distinct neutralizing antigenic ("N-Ag") regions designated N-AgI, N-Agll, N- AglllA, and N-AglllB (Murdin and Wimmer, 1989, J. Virol . 63:5251). The three-dimensional structure of the poliovirion has been determined, and reveals that the N- Ags occur as specific surface projections or "loops" on the viral particle (Hogle et al. , 1985, Science 229:1358).
  • N-AgI includes a continuous linear sequence of amino acids, designated N-AgIA, located on a well- exposed surface loop linking ⁇ sheets B and C, designated the BC loop of VP1.
  • N-AgI also includes residues from the DE loop of VP1 and, in PV-3 (Sabin vaccine strain) , residues from the EF and GH loops, designated N-AglB.
  • N- Agll, N-AglllA and N-AglllB are also discontinuous in structure and are made up of two or more polypeptide chains coming together in three dimensions to form points of attachment for neutralizing antibodies. Poliovirus has been used as a vehicle for carrying heterologous immunogenic epitopes (Murray et al.
  • Girard et al. discloses the construction of hybrid picornaviruses in which one of the immunogenic epitopes on the virus is replaced with an epitope from a heterologous virus, for example, a hepatitis A or HIV epitope.
  • Evans et al. (1989, Nature 339:385) discloses the construction of a poliovirus antigen chimera containing an epitope from the transmembrane glycoprotein of HIV.
  • HIV vaccines have been developed which are based on a subunit of the AIDS virus and thus are aimed at raising an immune response towards one or several immunogenic components of the HIV virus; one immunogenic HIV component is the envelope protein, located on the surface of the virus.
  • the HIV envelope protein is translated as an 88 kDa precursor and subsequently glycosylated to give the 160 kDa glycoprotein, gpl60.
  • gpl60 is cleaved by a cellular protease to yield the external envelope, gpl20, and the transmembrane glycoprotein, gp41.
  • the gpl20 molecule contains an approximately 40 amino acid region called the principal neutralizing domain (PND) .
  • the PND is a linear determinant that has been localized to the third variable domain (V3) of the external envelope protein, between amino acids in the region of 303 and 338, inclusive, and is flanked by two cysteine residues which are joined to each other by a disulfide cross- bridge and thus create a looped structure in native gpl20 (Putney et al., 1986, Science 234:1392; Rusche et al., 1988 Proc. Nat . Aca. Sci . 85:3198; Javaherian et al, 1989, Proc. Nat . Aca . Sci . 86:6768).
  • HIV-neutralizing antibodies which are present in the majority of HIV-infected individuals, bind primarily, if not exclusively, to the envelope protein. The importance of the
  • HIV-1 PND in viral infection is evident in the observations that protective immunity to HIV-1 correlates with the presence of antibodies reactive with PND (Emini et al, 1990, J. Virol. 64:3674; Berman et al., 1990, Nature, 345:622; Girard et al., 1990, J. Cellular
  • the invention features a hybrid poliovirus which contains an epitope from a heterologous protein (i.e., other than a native poliovirus protein) inserted into the BC loop of poliovirus, wherein between zero and ten amino acids, inclusive, of the BC loop are retained in the hybrid poliovirus, wherein the hybrid poliovirus is capable of eliciting antibodies directed against the heterologous epitope and the hybrid virus further contains a mutation at a site outside of the BC loop which results in a viral titer that is at least 5-fold higher than the viral titer of the virus that lacks the mutation.
  • a heterologous protein i.e., other than a native poliovirus protein
  • the "BC loop” refers to the neutralizing antigenic region whose sequence extends from amino acid residue 95 to amino acid residue 104 of VPl of poliovirus, and is flanked on each side by a ⁇ sheet; ⁇ sheet B ( ⁇ B) extends through and ends at residue 94 and ⁇ sheet C ( ⁇ C) extends from and begins at residue 105.
  • ⁇ sheet B ⁇ B
  • ⁇ sheet C ⁇ C
  • the portion of the amino acid sequence of the VPl capsid protein spanning the N-AgI epitope of various types and strains of poliovirus is shown in Fig. 1.
  • Epipe can refer to any region of two or more amino acids of a protein that is immunogenic, i.e., capable of eliciting an immune response.
  • "Titer” refers to the number of individual virions capable of infection.
  • the invention also features a hybrid poliovirus which retains between two and six, inclusive, amino acid residues from the poliovirus BC loop region, and contains an epitope of a heterologous protein inserted into the BC loop region of poliovirus.
  • the hybrid poliovirus thus contains a recombinant BC loop region, retaining at least two amino acids in the BC loop region adjacent the ⁇ B sheet. Replacement of poliovirus sequences with heterologous sequences in this fashion is compatible with viral proliferation.
  • the heterologous sequence is an active determinant participating in the induction of antibodies capable of reacting with the heterologous protein.
  • the two retained BC loop amino acid residues which lie adjacent the ⁇ B sheet are the proline and alanine residues at positions 95-96 of the N-AgIA epitope of type 1 poliovirus [PV-1 (Mahoney strain) ], or corresponding residues of another strain of poliovirus (e.g.
  • the Sabin strain contains serine and alanine residues at these positions)
  • the replaced residues include the threonine, asparagine, lysine, and aspartate residues at positions 99-102, respectively; preferably, the replaced residues may further include one or more of the serine, threonine, lysine, and leucine residues at positions 97, 98, 103 and 104, respectively, or the corresponding residues of another strain of poliovirus; alternatively, the BC loop residues retained in the hybrid poliovirus may further include one or more of the amino acid residues at positions 97, 98, 103 and 104 of strain PV-1, or the corresponding residues of another strain of poliovirus.
  • any of the hybrid polioviruses described above may further include a mutation at a site outside of the BC loop, the mutation resulting in a viral titer that is at least 5-fold higher than the titer of the virus that lacks the mutation.
  • the viral titer of the hybrid poliovirus is lxlO 6 TCID 50 /ml
  • the viral titer of the hybrid virus containing a mutation outside of the BC loop must be at least 5xl0 6 TCID 5(J /ml.
  • the epitope from the HIV-1 envelope protein is capable of eliciting neutralizing antibodies; preferably, the neutralizing epitope includes all or a portion of the PND of HIV-1; most preferably, the PND epitope is the HIV-1-IIIB, of the sequence isoleucine-glutamine-arginine-glycine- proline-glycine-arginine-alanine-phenylalanine-valine.
  • the hybrid poliovirus of the invention may also retain between one and seven, inclusive, amino acid residues from the poliovirus BC loop region, and contain the HIV-l-MN PND, the MN PND region including the sequence isoleucine-histidine- isoleucine-glycine- proline-glycine-arginine-alanine-phenylalanine-tyrosine inserted into the BC loop region of poliovirus, or a PND of a viral variant of the HIV-MN prototype.
  • the HIV-1 PND epitope may also be one that elicits broadly neutralizing antibodies; and the length of the PND sequence is between approximately 5 and 40, inclusive, amino acids in length; more preferably, between approximately 7 and 10 amino acids; most preferably, 10 amino acids in length.
  • PND epitope refers to a portion of HIV-1 which is recognized by an antibody that is capable of inhibiting HIV infection, e.g., by either the IIIB or MN strains or an MN variant strain, of cells by cell-free virions, or fusion of infected and uninfected cells, or both.
  • the HIV-IIIB strain is described in Ratner et al., 1985, Nature 313:277.
  • the HIV-MN strain is described in USSN 537,441, by Charles F. Scott et al., assigned to the same assignee and hereby incorporated by reference.
  • the MN prototype virus is defined by a particular amino acid sub-sequence within the principal neutralizing domain (i.e., the loop region of the gpl20 envelope protein) having positions A 1 -A 17 : K-R-K-R-I-H-I- G-P-G-R-A-F-Y-T-T-K (i.e., lysine-arginine-lysine- arginine-isoleucine-histidine-isoleucine-glycine-proline- glycine-arginine-alanine-phenylalanine-tyrosine- threonine-threonine-lysine) .
  • K-R-K-R-I-H-I- G-P-G-R-A-F-Y-T-T-K i.e., lysine-arginine-lysine- arginine-isoleucine-histidine-isoleucine-glycine-proline-
  • MN viral variants are herein defined as variants which exhibit complete amino acid sequence homology at residues I-G-P-G-R (isoleucine- glycine-proline-glycine-arginine) , i.e., at positions A 7 - A 11 , and at least 36% homology with the remaining 12 amino aids of the MN sequence given above.
  • a “broadly neutralizing epitope” refers to a portion of the hybrid polio/HIV virus which is recognized by an antibody that is capable of inhibiting HIV infection by two or more HIV strains, of cells by cell- free virions, or fusion of infected and uninfected cells, or both.
  • a broadly neutralizing epitope will be most useful if an antibody to that epitope is capable of neutralizing one HIV strain which has either the IIIB or MN sequences or MN variant sequence within the PND, and a second HIV strain having a different PND sequence.
  • a broadly neutralizing epitope will include an amino acid sequence within the PND that is present on at least two HIV strains.
  • the invention also features a method of vaccinating a human patient against HIV-1 or treating a human patient infected with HIV-1, which includes administering any one of the hybrid polioviruses described above according to standard poliovirus vaccination methods; and an HIV-1 vaccine which contains a hybrid poliovirus of the invention; preferably, the vaccine is admixed with a pharmaceutically acceptable carrier.
  • a hybrid poliovirus of the invention may be prepared as an attenuated viral vaccine or may be chemically inactivated prior to administration.
  • the invention also features a method of constructing a hybrid poliovirus of the invention, including the steps of providing DNA encoding an epitope, providing poliovirus DNA or viral variants thereof and encoding the BC loop of poliovirus, wherein the BC loop includes ten amino acids, performing separate replacement steps, each step including replacing the BC loop-encoding DNA with epitope-encoding DNA by selecting a site within the BC loop-encoding DNA for each replacement, wherein successive sites are selected for each replacement step, beginning at the amino-terminal encoding end within the ⁇ sheet B, e.g., within the BC loop region itself or at the naturally-occurring SphI restriction site within the ⁇ B sheet, and testing the hybrid poliovirus for its ability to elicit an immune response that is capable of preventing or reducing HIV-1 infection.
  • the testing step can be performed in vitro or in vivo, by assaying for reduction of viral infectivity of cells by cell-free virions, or fusion of infected and uninfected cells, or fusion of cells infected with vaccinia virus that express the HIV envelope protein, or by assaying for the presence of neutralizing antibodies in the serum.
  • replacement indicates that DNA encoding one or more antigen amino acids may be substituted for DNA encoding one or more BC loop amino acids or one to amino acids within the ⁇ sheet B that flank the BC loop.
  • the hybrid poliovirus of the invention provides a method of vaccinating an individual against HIV-1 by introducing the HIV-1 immunogenic epitope in a conformation that mimics the native structure of HIV-1 in order to induce an effective immune response.
  • the exposed linear epitope of N-AgIA can be used as a target for in vitro mutagenesis, allowing for the selective expression of defined regions of heterologous proteins in a position that is exposed to the immune surveillance of the vaccinate, thereby enabling a specific immune response to be induced.
  • administration of the vaccine of the invention to a patient will not result in integration of the HIV genome into host cell DNA and its attendant side-effects, e.g., HIV infection.
  • a poliovirus-based vaccine offers the advantages of both replicating and non-replicating viral vaccines, as it may induce a secretory as well as a systemic humoral immune response.
  • the presence of secretory antibodies in mucous fluids may reduce the efficiency of transmission of HIV.
  • the hybrid polio-HIV vaccines of the invention may also be used therapeutically to treat HIV seropositive patients.
  • Figure 2 is a schematic representation of a strategy for constructing poliovirus/HIV hybrid viruses.
  • Figure 3 is a schematic representation of the BC loop in native PV-l(M) and the comparable region within the hybrid virus M1/HIV1IIIB-1D-PND+5.
  • Figures 4a and 4b show the amino acid sequence of the BC loop region in a series of PV-l/HIV hybrids.
  • the invention provides a hybrid poliovirus containing a neutralizing epitope from another virus, e.g., the HIV-1 virus, for use as a vaccine.
  • a portion of the N- AglA loop is replaced with a sequence from an immunogenic region of the other virus, e.g., the PND region of the HIV envelope protein, so as to maintain the viability of the hybrid virus; i.e., to produce a vaccine.
  • a viable poliovirus/HIV PND hybrid Described in detail below by way of example is the construction of a viable poliovirus/HIV PND hybrid; however, an immunogenic region from any heterologous protein, or any one of a number of other viruses may be inserted into the BC region of poliovirus, using conventional genetic engineering techniques.
  • poliovirus/HIV-III D hybrid virus A number of hybrid polioviruses containing the HIV PND sequence were constructed using the mutagenesis cartridge strategy of Murray et al (1988, Proc. Natl . Acad. Sci . , 85:3203-3207 and 1988, In Vaccines 89 f Cold Spring Harbor Laboratories, CSH, NY) . Briefly, a synthetic oligonucleotide "cartridge" encoding an amino acid sequence from the PND region of HIV gpl20 was inserted into the poliovirus cDNA sequence replacing the sequence encoding all or a portion of the N-AgI loop.
  • Sequences from the central portion, or "tip" of the PND loop were chosen for insertion in poliovirus.
  • This region is highly conserved among HIV variants and comprises the G-P-G sequence occurring at or about positions 311 to 313 of the HIV envelope and flanking amino acid residues. This region has been shown to be capable of binding and eliciting HIV neutralizing antibodies.
  • the PND sequence from any strain of HIV-1 can be substituted, but preferably is a sequence that is common to a large proportion of HIV isolates and therefore will be capable of eliciting broadly neutralizing antibodies.
  • Nrul and SnaBI restriction sites at PV nucleotide numbers 1174 and 2954, respectively, a 1782 base pair fragment was excised from a full length infectious cDNA of PV-1 (Mahoney) called pEV104 (Semler et al. Nucleic Acids Research 12:5123). This NruI-SnaBI fragment was transferred into a modified pBR322 vector.
  • the modified vector (Hindlll position 29 to PvuII 2064 was excised, the ends blunted and religated) contains an EcoRI-NruI-SnaBI-EcoRI polylinker inserted at the unique pBR322 EcoRI site and lacks restriction sites to be used in forming the cartridge.
  • the EcoRI fragment containing the PV sequences was then transferred into the EcoRI site of M13mpl0 and standard mismatch oligonucleotide mutagenesis procedures were used to create the mutagenesis cartridge. Specifically, a Hindlll site was created by changing A2791T (i.e., A changed to T) and the SphI site at 2926 was changed to a Nsil site by changing G2923C and C2929A. These changes leave the naturally occurring SphI (2737) and the newly-created Hindlll (2787) sites unique in this fragment. The EcoRI - 11 - cartridge containing DNA fragment was then placed back into the modified pBR322 vector described above and used for various synthetic cartridge exchanges.
  • the 484 base pair Nhel (PV 2470) to SnaBI (PV 2954) cartridge containing fragment was then transferred into plasmid PT7PV1XL.
  • the mismatch oligonucleotide used to create the Hindlll site coincidentally corrected a mutation (G2798A) in the pT7PVl-5 (van der Werf et al. Proc. Nat . Acad. Sci . 83:2330) sequences outside the mutagenesis cartridge, resulting in a M1107V correction to the wild type PV-l (Mahoney) sequence (Kitamura et al. Nature 291:547).
  • This corrected plasmid is designated pT7PVlXL.
  • pP3sub The insertion of synthetic oligonucleotides which encode PV-l specific amino acids from the SphI site to the Sail site within the cartridge and PV-3 (Leon) sequences in the BC loop was named pP3sub.
  • pP3sub was then used as the cartridge containing intermediate plasmid into which all PND encoding synthetic oligonucleotides were inserted.
  • the PV-3 (Leon) sequences from the Sail site to the Hindlll site were exchanged for the various PND encoding oligonucleotides.
  • the 484 (+/- depending upon the particular construction) base pair Nhel-SnaBI PND containing fragment was then transferred into pT7PVlXL.
  • Fig. 1 shows the amino acid sequence spanning the NAg-I region of the polio/HIV hybrid virus envelope protein.
  • the amino acid sequence is numbered according to the sequence of VPl of PV-l(M).
  • PV-l(M), PV-2(L), and PV-3(L) refer to polioviruses type 1(Mahoney) , type 2(Lansing) and type 3(Leon) , respectively.
  • “Sabin” refers to the attenuated vaccine strain.
  • Fig. 2 The construction of one hybrid, which contains the PND sequence from the HIV-1 IIIB strain, and the first five N-AgIA amino acids (proline-alanine- serine-threonine-threonine, i.e., P-A-S-T-T) on the ⁇ B proximal side of the loop, is exemplified in Fig. 2.
  • the nucleotide sequence of the oligonucleotide cartridge and the amino acid sequence encoded by the cartridge are shown.
  • the boxed amino acid residues constitute the tip of the PND loop sequence from the HIV-1 IIIB strain.
  • the resulting hybrid is denoted M1/HIV-IIIB-1D-PND+5.
  • FIG 3 is a schematic representation of the N- AglA loop in native PV-l(M) and the comparable region within the hybrid virus M1/HIV1IIIB-1D-PND+5.
  • Figs. 4a and b show the amino acid sequence of the N-AgIA loop region in a series of PV-l/HIV hybrids. Shaded areas denote the PV sequences, while the exchanged sequences are unshaded. The "+” indicates the number of poliovirus BC loop amino acids retained.
  • Synthetic cartridges were designed to encode 0, 1, 2, 3, 4, or 5 amino acids from the ⁇ B proximal side of the N-AgI loop, and PND sequences from HIV strains IIIB (Fig. 4a) or MN (Fig. 4b) .
  • the poliovirus sequence spanning nucleotides 2470 to 2956 was excised by cleavage with Nhel and SnaBI, and transferred back into the full- length poliovirus sequence in plasmid pT7PVlXL.
  • RNA transcripts were prepared using a commercially available T7 RNA polymerase transcription kit according to the manufacturer's instructions (Stratagene, LaJolla, CA) . Transfection of HeLa cells with RNA transcripts synthesized in vitro was essentially as described by van der Werf et al. (1986, supra) . The hybrid transcripts yielded a hybrid virus in which amino acids 1097, 1098, 1099, 1100, and 1102 of PV-l(M) were replaced by ten amino acids of HIV-1 ( Figure 4) .
  • This hybrid virus was designated M1/HIV1IIIB-1D-PND+2, M1/HIV1IIIB-1D-PND+3, M1/HIV1IIIB-1D-PND+4, M1/HIV1IIIB-1D-PND+5, according to the nomenclature of Bernstein et al. (1986, J. Virol . 60:1040). All four hybrid viruses were titered by standard TCID 50 and pfu methods as described by Reed et al., 1938, Amer. J " . Hygiene 27:49; Golding et al., 1976, Res . Vet . Sci .
  • hybrid viruses described above were inoculated into rabbits and guinea pigs using standard inoculation protocols and adjuvants. Approximately 15 to 75 days later, the animals were bled and their serum tested for the ability to neutralize wild type HIV-l in a standard virus neutralization assay and for the ability to inhibit fusion induced by infection of CD4+ cells with a recombinant vaccinia virus which expresses the HIV-l envelope protein.
  • Hybrid virus M1/HIV1IIIB-1D-PND+3 was found to elicit antibodies in rabbits which neutralized the HIV-l IIIB virus at a titer of 1:80.
  • this virus also was capable of eliciting antibodies which inhibited the vac-env induced fusion of CD4+ cells induced by vaccinia virus expressing the HIV-l MN envelope at a titer of 1:20.
  • a hybrid poliovirus of the invention may be serial passaged in culture and mutants with improved phenotype characteristics (e.g., improved growth kinetics, improved viability, improved temperature stability, and improved receptor affinity) may be obtained.
  • a variant exhibiting improved growth kinetics and/or viability and/or temperature stability and/or receptor affinity will grow to at least a 5-fold higher titer than the hybrid virus prior to serial passaging.
  • the titer of a virus is measured according to standard in vitro viral titration assays, e.g., the TCID 50 assay described by Reed et al., supra: Golding et al., 1976, supra. or the PFU assay described by Emini et al., 1983, supra.
  • the hybrid viruses of the invention may be administered as an attenuated virus or as a chemically inactivated virus, in a pharmaceutically acceptable carrier. Attenuation may be achieved by serial passage of the virus in tissue culture until a virus is obtained that is incapable of attacking the spinal cord of the patient (non-neurovirulent) . Chemical inactivation may be achieved using an acceptable agent, e.g., formalin, as used in preparation of the Salk inactivated polio vaccine.
  • the virus of the invention may be administered in a dosage depending upon its viability and ability to elicit neutralizing antibodies.
  • Mechanism of Action The poliovirus capsid is a highly structured icosahedral particle, the three dimensional structure of which is described in detail in Hogle et al.
  • Each core is composed of an eight-stranded antiparallel beta barrel with two flanking helices.
  • Four strands (referred to as ⁇ sheets B,I,D, and G) make up a large twisted beta sheet which forms the front and bottom surfaces of the barrel.
  • the strands which form the front and back surfaces are joined at one end by four short "loops", giving the barrel the shape of a triangular wedge.
  • VPl is located near the five-fold axis on the icosahedral surface of the virion. Due to the positioning of VPl, three of the loops connecting the beta strands are exposed at the summit.
  • the top loop, or BC (residues 95 to 104) , connects ⁇ sheets B and C, and twists out from the surface of the subunit so that it is particularly well exposed, giving the five-fold peak a ribbed appearance.
  • poliovirus N-AgI region e.g., other HIV neutralizing regions, HRV-Nims, HAV-N-Ags, poliovirus N-Ags, as long as the appropriate PV amino acids, e.g., the proline and alanine residues for polio/HIV-IIIB hybrids, of the N-AgI region are retained.
  • PV amino acids e.g., the proline and alanine residues for polio/HIV-IIIB hybrids
  • Lys Arg Lys Arg lie His lie Gly Pro Gly Arg Ala Phe Tyr Thr Lys
  • Cys Val Ala lie lie Thr Val Asp Asn Ser Ala Ser Thr Lys Asn Lys
  • Cys Val Ala lie lie Glu Val Asp Asn Asp Ala Pro Thr Lys Arg Ala
  • Cys Val Ala lie lie Glu Val Asp Asn Asp Ala Pro Thr Lys Arg Ala
  • Cys Val Ala lie lie Glu Val Asp Asn Glu Gin Pro Thr Arg Ala Gin
  • Cys Val Ala lie lie Glu Val Asp Asn Glu Gin Pro Thr Arg Ala Gin

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Abstract

A hybrid poliovirus which contains an epitope from a heterologous protein inserted into the BC loop of poliovirus, wherein between zero and ten amino acids, inclusive, of the BC loop are retained in the hybrid poliovirus, wherein the BC loop of the hybrid poliovirus is capable of eliciting antibodies directed against the heterologous protein and the hybrid virus further conains a mutation at a site outside of the BC loop which results in a viral titer that is at least 5-fold higher than the viral titer of the virus that lacks the mutation.

Description

"OLIOVIRUS-BASED VACCINES
Background of the Invention This invention relates to a poliovirus-based vaccine.
Poliovirus (PV) is a picornavirus which exists in nature as three stable serόtypes, PV-1, PV-2 and PV-3. These viruses each display four distinct neutralizing antigenic ("N-Ag") regions designated N-AgI, N-Agll, N- AglllA, and N-AglllB (Murdin and Wimmer, 1989, J. Virol . 63:5251). The three-dimensional structure of the poliovirion has been determined, and reveals that the N- Ags occur as specific surface projections or "loops" on the viral particle (Hogle et al. , 1985, Science 229:1358). N-AgI includes a continuous linear sequence of amino acids, designated N-AgIA, located on a well- exposed surface loop linking β sheets B and C, designated the BC loop of VP1. N-AgI also includes residues from the DE loop of VP1 and, in PV-3 (Sabin vaccine strain) , residues from the EF and GH loops, designated N-AglB. N- Agll, N-AglllA and N-AglllB are also discontinuous in structure and are made up of two or more polypeptide chains coming together in three dimensions to form points of attachment for neutralizing antibodies. Poliovirus has been used as a vehicle for carrying heterologous immunogenic epitopes (Murray et al. , 1987, Abstract, Seventh International Congress of Virology, Edmonton, Alberta, Canada; Murray et al., 1988, Proc. ΛTat. Aca . Sci . 85:3203; Murray et al., 1988, Science 241:213; Burke et al, 1988, Nature 339:81; Murray et al., in Vaccines 1989 , Cold Spring Harbor, NY; Murdin and Wimmer, 1989, J.Virol . 63:5251; Evans et al., 1989, Nature 339:385) .
Bradley et al. (1989, in Molecular Aspects of Picornavirus Infections and Detection, Amer.Soc.Microbiol. Review) discloses that the BC loop can be replaced by heterologous sequences representing antigenic determinants of other poliovirus serotypes or other pathogens. Burke et al. (1988, Nature 332:81) and Murray et al. (1988, Proc. Nat . Aca . Sci . 85:3203) have reported that a hybrid virus carrying neutralization antigenic sites of two different serotypes of poliovirus can be neutralized by corresponding type-specific neutralizing antibodies and can elicit the corresponding type-specific antibodies.
Girard et al. (EPO 302 801) discloses the construction of hybrid picornaviruses in which one of the immunogenic epitopes on the virus is replaced with an epitope from a heterologous virus, for example, a hepatitis A or HIV epitope. Evans et al. (1989, Nature 339:385) discloses the construction of a poliovirus antigen chimera containing an epitope from the transmembrane glycoprotein of HIV.
Popovic et al. (1984, Science 224:497) first identified HIV as the causative agent of AIDS. AIDS vaccines have been developed which are based on a subunit of the AIDS virus and thus are aimed at raising an immune response towards one or several immunogenic components of the HIV virus; one immunogenic HIV component is the envelope protein, located on the surface of the virus. The HIV envelope protein is translated as an 88 kDa precursor and subsequently glycosylated to give the 160 kDa glycoprotein, gpl60. gpl60 is cleaved by a cellular protease to yield the external envelope, gpl20, and the transmembrane glycoprotein, gp41. The gpl20 molecule contains an approximately 40 amino acid region called the principal neutralizing domain (PND) . The PND is a linear determinant that has been localized to the third variable domain (V3) of the external envelope protein, between amino acids in the region of 303 and 338, inclusive, and is flanked by two cysteine residues which are joined to each other by a disulfide cross- bridge and thus create a looped structure in native gpl20 (Putney et al., 1986, Science 234:1392; Rusche et al., 1988 Proc. Nat . Aca. Sci . 85:3198; Javaherian et al, 1989, Proc. Nat . Aca . Sci . 86:6768).
HIV-neutralizing antibodies, which are present in the majority of HIV-infected individuals, bind primarily, if not exclusively, to the envelope protein. The importance of the
HIV-1 PND in viral infection is evident in the observations that protective immunity to HIV-1 correlates with the presence of antibodies reactive with PND (Emini et al, 1990, J. Virol. 64:3674; Berman et al., 1990, Nature, 345:622; Girard et al., 1990, J. Cellular
Biochem. , Supplement 14D) , and, where there is either a low or no measurable titer of PND-directed neutralizing antibodies, there was no measurable protection against HIV (Kennedy et al., 1987, Vaccines 87, Cold Spring Harbor Laboratory, p. 250-255; Arthur et al., 1989, J.Virol . 63:5046; Berman et al., 1988, Proc. Nat . Aca . Sci . 85:5200; Hu et al., 1987, Nature, 328:721; and Prince et al., 1988, Proc. Nat . Aca . Sci . 85:6944).
Rιιτnτnaτy nf the Invention The invention features a hybrid poliovirus which contains an epitope from a heterologous protein (i.e., other than a native poliovirus protein) inserted into the BC loop of poliovirus, wherein between zero and ten amino acids, inclusive, of the BC loop are retained in the hybrid poliovirus, wherein the hybrid poliovirus is capable of eliciting antibodies directed against the heterologous epitope and the hybrid virus further contains a mutation at a site outside of the BC loop which results in a viral titer that is at least 5-fold higher than the viral titer of the virus that lacks the mutation.
As used herein, the "BC loop" refers to the neutralizing antigenic region whose sequence extends from amino acid residue 95 to amino acid residue 104 of VPl of poliovirus, and is flanked on each side by a β sheet; β sheet B (βB) extends through and ends at residue 94 and β sheet C (βC) extends from and begins at residue 105. The portion of the amino acid sequence of the VPl capsid protein spanning the N-AgI epitope of various types and strains of poliovirus is shown in Fig. 1. "Epitope" can refer to any region of two or more amino acids of a protein that is immunogenic, i.e., capable of eliciting an immune response. "Titer" refers to the number of individual virions capable of infection.
The invention also features a hybrid poliovirus which retains between two and six, inclusive, amino acid residues from the poliovirus BC loop region, and contains an epitope of a heterologous protein inserted into the BC loop region of poliovirus. The hybrid poliovirus thus contains a recombinant BC loop region, retaining at least two amino acids in the BC loop region adjacent the βB sheet. Replacement of poliovirus sequences with heterologous sequences in this fashion is compatible with viral proliferation. Moreover, the heterologous sequence is an active determinant participating in the induction of antibodies capable of reacting with the heterologous protein.
In preferred embodiments, the two retained BC loop amino acid residues which lie adjacent the βB sheet are the proline and alanine residues at positions 95-96 of the N-AgIA epitope of type 1 poliovirus [PV-1 (Mahoney strain) ], or corresponding residues of another strain of poliovirus (e.g. , the Sabin strain contains serine and alanine residues at these positions) , and the replaced residues include the threonine, asparagine, lysine, and aspartate residues at positions 99-102, respectively; preferably, the replaced residues may further include one or more of the serine, threonine, lysine, and leucine residues at positions 97, 98, 103 and 104, respectively, or the corresponding residues of another strain of poliovirus; alternatively, the BC loop residues retained in the hybrid poliovirus may further include one or more of the amino acid residues at positions 97, 98, 103 and 104 of strain PV-1, or the corresponding residues of another strain of poliovirus.
Any of the hybrid polioviruses described above may further include a mutation at a site outside of the BC loop, the mutation resulting in a viral titer that is at least 5-fold higher than the titer of the virus that lacks the mutation. For example, if the viral titer of the hybrid poliovirus is lxlO6 TCID50/ml, then the viral titer of the hybrid virus containing a mutation outside of the BC loop must be at least 5xl06 TCID5(J/ml. In other preferred embodiments, the epitope from the HIV-1 envelope protein is capable of eliciting neutralizing antibodies; preferably, the neutralizing epitope includes all or a portion of the PND of HIV-1; most preferably, the PND epitope is the HIV-1-IIIB, of the sequence isoleucine-glutamine-arginine-glycine- proline-glycine-arginine-alanine-phenylalanine-valine. The hybrid poliovirus of the invention may also retain between one and seven, inclusive, amino acid residues from the poliovirus BC loop region, and contain the HIV-l-MN PND, the MN PND region including the sequence isoleucine-histidine- isoleucine-glycine- proline-glycine-arginine-alanine-phenylalanine-tyrosine inserted into the BC loop region of poliovirus, or a PND of a viral variant of the HIV-MN prototype. Preferably, the HIV-1 PND epitope may also be one that elicits broadly neutralizing antibodies; and the length of the PND sequence is between approximately 5 and 40, inclusive, amino acids in length; more preferably, between approximately 7 and 10 amino acids; most preferably, 10 amino acids in length.
As used herein, "PND epitope" refers to a portion of HIV-1 which is recognized by an antibody that is capable of inhibiting HIV infection, e.g., by either the IIIB or MN strains or an MN variant strain, of cells by cell-free virions, or fusion of infected and uninfected cells, or both. The HIV-IIIB strain is described in Ratner et al., 1985, Nature 313:277. The HIV-MN strain is described in USSN 537,441, by Charles F. Scott et al., assigned to the same assignee and hereby incorporated by reference. The MN prototype virus is defined by a particular amino acid sub-sequence within the principal neutralizing domain (i.e., the loop region of the gpl20 envelope protein) having positions A1-A17: K-R-K-R-I-H-I- G-P-G-R-A-F-Y-T-T-K (i.e., lysine-arginine-lysine- arginine-isoleucine-histidine-isoleucine-glycine-proline- glycine-arginine-alanine-phenylalanine-tyrosine- threonine-threonine-lysine) . MN viral variants are herein defined as variants which exhibit complete amino acid sequence homology at residues I-G-P-G-R (isoleucine- glycine-proline-glycine-arginine) , i.e., at positions A7- A11, and at least 36% homology with the remaining 12 amino aids of the MN sequence given above.
A "broadly neutralizing epitope" refers to a portion of the hybrid polio/HIV virus which is recognized by an antibody that is capable of inhibiting HIV infection by two or more HIV strains, of cells by cell- free virions, or fusion of infected and uninfected cells, or both. A broadly neutralizing epitope will be most useful if an antibody to that epitope is capable of neutralizing one HIV strain which has either the IIIB or MN sequences or MN variant sequence within the PND, and a second HIV strain having a different PND sequence. A broadly neutralizing epitope will include an amino acid sequence within the PND that is present on at least two HIV strains.
The invention also features a method of vaccinating a human patient against HIV-1 or treating a human patient infected with HIV-1, which includes administering any one of the hybrid polioviruses described above according to standard poliovirus vaccination methods; and an HIV-1 vaccine which contains a hybrid poliovirus of the invention; preferably, the vaccine is admixed with a pharmaceutically acceptable carrier. A hybrid poliovirus of the invention may be prepared as an attenuated viral vaccine or may be chemically inactivated prior to administration. The invention also features a method of constructing a hybrid poliovirus of the invention, including the steps of providing DNA encoding an epitope, providing poliovirus DNA or viral variants thereof and encoding the BC loop of poliovirus, wherein the BC loop includes ten amino acids, performing separate replacement steps, each step including replacing the BC loop-encoding DNA with epitope-encoding DNA by selecting a site within the BC loop-encoding DNA for each replacement, wherein successive sites are selected for each replacement step, beginning at the amino-terminal encoding end within the β sheet B, e.g., within the BC loop region itself or at the naturally-occurring SphI restriction site within the βB sheet, and testing the hybrid poliovirus for its ability to elicit an immune response that is capable of preventing or reducing HIV-1 infection. The testing step can be performed in vitro or in vivo, by assaying for reduction of viral infectivity of cells by cell-free virions, or fusion of infected and uninfected cells, or fusion of cells infected with vaccinia virus that express the HIV envelope protein, or by assaying for the presence of neutralizing antibodies in the serum. As used herein, "replacement" indicates that DNA encoding one or more antigen amino acids may be substituted for DNA encoding one or more BC loop amino acids or one to amino acids within the β sheet B that flank the BC loop.
The hybrid poliovirus of the invention provides a method of vaccinating an individual against HIV-1 by introducing the HIV-1 immunogenic epitope in a conformation that mimics the native structure of HIV-1 in order to induce an effective immune response. The exposed linear epitope of N-AgIA can be used as a target for in vitro mutagenesis, allowing for the selective expression of defined regions of heterologous proteins in a position that is exposed to the immune surveillance of the vaccinate, thereby enabling a specific immune response to be induced. In addition, administration of the vaccine of the invention to a patient will not result in integration of the HIV genome into host cell DNA and its attendant side-effects, e.g., HIV infection. A poliovirus-based vaccine offers the advantages of both replicating and non-replicating viral vaccines, as it may induce a secretory as well as a systemic humoral immune response. The presence of secretory antibodies in mucous fluids may reduce the efficiency of transmission of HIV. The hybrid polio-HIV vaccines of the invention may also be used therapeutically to treat HIV seropositive patients.
Other features and advantages of the invention will be apparent from the description of embodiments of the invention, and from the claims. Detailed Description of Emhod-j-ments of the Invention Before describing the invention in detail, the drawings will be described. Drawings Figure 1 shows the amino acid sequence of the region spanning N-AgIA of various poliovirus types and strains.
Figure 2 is a schematic representation of a strategy for constructing poliovirus/HIV hybrid viruses. Figure 3 is a schematic representation of the BC loop in native PV-l(M) and the comparable region within the hybrid virus M1/HIV1IIIB-1D-PND+5.
Figures 4a and 4b show the amino acid sequence of the BC loop region in a series of PV-l/HIV hybrids. In one embodiment, the invention provides a hybrid poliovirus containing a neutralizing epitope from another virus, e.g., the HIV-1 virus, for use as a vaccine. In the hybrid viruses of the invention, a portion of the N- AglA loop is replaced with a sequence from an immunogenic region of the other virus, e.g., the PND region of the HIV envelope protein, so as to maintain the viability of the hybrid virus; i.e., to produce a vaccine. Described in detail below by way of example is the construction of a viable poliovirus/HIV PND hybrid; however, an immunogenic region from any heterologous protein, or any one of a number of other viruses may be inserted into the BC region of poliovirus, using conventional genetic engineering techniques.
Construction of poliovirus/HIV-IIID hybrid virus A number of hybrid polioviruses containing the HIV PND sequence were constructed using the mutagenesis cartridge strategy of Murray et al (1988, Proc. Natl . Acad. Sci . , 85:3203-3207 and 1988, In Vaccines 89f Cold Spring Harbor Laboratories, CSH, NY) . Briefly, a synthetic oligonucleotide "cartridge" encoding an amino acid sequence from the PND region of HIV gpl20 was inserted into the poliovirus cDNA sequence replacing the sequence encoding all or a portion of the N-AgI loop. Sequences from the central portion, or "tip" of the PND loop were chosen for insertion in poliovirus. This region is highly conserved among HIV variants and comprises the G-P-G sequence occurring at or about positions 311 to 313 of the HIV envelope and flanking amino acid residues. This region has been shown to be capable of binding and eliciting HIV neutralizing antibodies. The PND sequence from any strain of HIV-1 can be substituted, but preferably is a sequence that is common to a large proportion of HIV isolates and therefore will be capable of eliciting broadly neutralizing antibodies.
Using unique Nrul and SnaBI restriction sites at PV nucleotide numbers 1174 and 2954, respectively, a 1782 base pair fragment was excised from a full length infectious cDNA of PV-1 (Mahoney) called pEV104 (Semler et al. Nucleic Acids Research 12:5123). This NruI-SnaBI fragment was transferred into a modified pBR322 vector. The modified vector (Hindlll position 29 to PvuII 2064 was excised, the ends blunted and religated) contains an EcoRI-NruI-SnaBI-EcoRI polylinker inserted at the unique pBR322 EcoRI site and lacks restriction sites to be used in forming the cartridge. The EcoRI fragment containing the PV sequences was then transferred into the EcoRI site of M13mpl0 and standard mismatch oligonucleotide mutagenesis procedures were used to create the mutagenesis cartridge. Specifically, a Hindlll site was created by changing A2791T (i.e., A changed to T) and the SphI site at 2926 was changed to a Nsil site by changing G2923C and C2929A. These changes leave the naturally occurring SphI (2737) and the newly-created Hindlll (2787) sites unique in this fragment. The EcoRI - 11 - cartridge containing DNA fragment was then placed back into the modified pBR322 vector described above and used for various synthetic cartridge exchanges. The 484 base pair Nhel (PV 2470) to SnaBI (PV 2954) cartridge containing fragment was then transferred into plasmid PT7PV1XL. The mismatch oligonucleotide used to create the Hindlll site coincidentally corrected a mutation (G2798A) in the pT7PVl-5 (van der Werf et al. Proc. Nat . Acad. Sci . 83:2330) sequences outside the mutagenesis cartridge, resulting in a M1107V correction to the wild type PV-l (Mahoney) sequence (Kitamura et al. Nature 291:547). This corrected plasmid is designated pT7PVlXL. The insertion of synthetic oligonucleotides which encode PV-l specific amino acids from the SphI site to the Sail site within the cartridge and PV-3 (Leon) sequences in the BC loop was named pP3sub. pP3sub was then used as the cartridge containing intermediate plasmid into which all PND encoding synthetic oligonucleotides were inserted. To construct the hybrid PV/PND plasmids, the PV-3 (Leon) sequences from the Sail site to the Hindlll site were exchanged for the various PND encoding oligonucleotides. The 484 (+/- depending upon the particular construction) base pair Nhel-SnaBI PND containing fragment was then transferred into pT7PVlXL. A series of hybrids were constructed which retained an increasing number of poliovirus amino acids on the βB proximal side of the BC loop (Fig. 1) . Fig. 1 shows the amino acid sequence spanning the NAg-I region of the polio/HIV hybrid virus envelope protein. The amino acid sequence is numbered according to the sequence of VPl of PV-l(M). PV-l(M), PV-2(L), and PV-3(L) refer to polioviruses type 1(Mahoney) , type 2(Lansing) and type 3(Leon) , respectively. "Sabin" refers to the attenuated vaccine strain. The construction of one hybrid, which contains the PND sequence from the HIV-1 IIIB strain, and the first five N-AgIA amino acids (proline-alanine- serine-threonine-threonine, i.e., P-A-S-T-T) on the βB proximal side of the loop, is exemplified in Fig. 2. The nucleotide sequence of the oligonucleotide cartridge and the amino acid sequence encoded by the cartridge are shown. The boxed amino acid residues constitute the tip of the PND loop sequence from the HIV-1 IIIB strain. The resulting hybrid is denoted M1/HIV-IIIB-1D-PND+5. Figure 3 is a schematic representation of the N- AglA loop in native PV-l(M) and the comparable region within the hybrid virus M1/HIV1IIIB-1D-PND+5. Figs. 4a and b show the amino acid sequence of the N-AgIA loop region in a series of PV-l/HIV hybrids. Shaded areas denote the PV sequences, while the exchanged sequences are unshaded. The "+" indicates the number of poliovirus BC loop amino acids retained. Synthetic cartridges were designed to encode 0, 1, 2, 3, 4, or 5 amino acids from the βB proximal side of the N-AgI loop, and PND sequences from HIV strains IIIB (Fig. 4a) or MN (Fig. 4b) .
After the hybrid N-AgI region was constructed in the plasmid subclone (pP3sub) , the poliovirus sequence spanning nucleotides 2470 to 2956 was excised by cleavage with Nhel and SnaBI, and transferred back into the full- length poliovirus sequence in plasmid pT7PVlXL.
Infectious RNA transcripts were prepared using a commercially available T7 RNA polymerase transcription kit according to the manufacturer's instructions (Stratagene, LaJolla, CA) . Transfection of HeLa cells with RNA transcripts synthesized in vitro was essentially as described by van der Werf et al. (1986, supra) . The hybrid transcripts yielded a hybrid virus in which amino acids 1097, 1098, 1099, 1100, and 1102 of PV-l(M) were replaced by ten amino acids of HIV-1 (Figure 4) . This hybrid virus was designated M1/HIV1IIIB-1D-PND+2, M1/HIV1IIIB-1D-PND+3, M1/HIV1IIIB-1D-PND+4, M1/HIV1IIIB-1D-PND+5, according to the nomenclature of Bernstein et al. (1986, J. Virol . 60:1040). All four hybrid viruses were titered by standard TCID50 and pfu methods as described by Reed et al., 1938, Amer. J". Hygiene 27:49; Golding et al., 1976, Res . Vet . Sci . 20:142, or the PFU assay described by Emini et al., 1983, Nature 304:699, and were found to grow to titers of approximately 1x10s TCID50/ml. Their identity was confirmed by sequencing a PCR generated fragment of the viral cDNA which included the inserted sequences.
All of the hybrid viruses described above were inoculated into rabbits and guinea pigs using standard inoculation protocols and adjuvants. Approximately 15 to 75 days later, the animals were bled and their serum tested for the ability to neutralize wild type HIV-l in a standard virus neutralization assay and for the ability to inhibit fusion induced by infection of CD4+ cells with a recombinant vaccinia virus which expresses the HIV-l envelope protein. Hybrid virus M1/HIV1IIIB-1D-PND+3 was found to elicit antibodies in rabbits which neutralized the HIV-l IIIB virus at a titer of 1:80. Moreover, this virus also was capable of eliciting antibodies which inhibited the vac-env induced fusion of CD4+ cells induced by vaccinia virus expressing the HIV-l MN envelope at a titer of 1:20.
These results demonstrate that M1/HIV1IIIB-1D- PND+3 is capable of eliciting antibodies which will neutralize the HIV-l virus and may be useful as a novel vaccine for AIDS. Second Site Mutation
A hybrid poliovirus of the invention may be serial passaged in culture and mutants with improved phenotype characteristics (e.g., improved growth kinetics, improved viability, improved temperature stability, and improved receptor affinity) may be obtained. A variant exhibiting improved growth kinetics and/or viability and/or temperature stability and/or receptor affinity will grow to at least a 5-fold higher titer than the hybrid virus prior to serial passaging. The titer of a virus is measured according to standard in vitro viral titration assays, e.g., the TCID50 assay described by Reed et al., supra: Golding et al., 1976, supra. or the PFU assay described by Emini et al., 1983, supra. Use
The hybrid viruses of the invention may be administered as an attenuated virus or as a chemically inactivated virus, in a pharmaceutically acceptable carrier. Attenuation may be achieved by serial passage of the virus in tissue culture until a virus is obtained that is incapable of attacking the spinal cord of the patient (non-neurovirulent) . Chemical inactivation may be achieved using an acceptable agent, e.g., formalin, as used in preparation of the Salk inactivated polio vaccine. The virus of the invention may be administered in a dosage depending upon its viability and ability to elicit neutralizing antibodies. Mechanism of Action The poliovirus capsid is a highly structured icosahedral particle, the three dimensional structure of which is described in detail in Hogle et al. (supra) . The three major capsid proteins, VPl, VP2 and VP3, are remarkably similar to each other, each consisting of a structurally identical "core". Each core is composed of an eight-stranded antiparallel beta barrel with two flanking helices. Four strands (referred to as β sheets B,I,D, and G) make up a large twisted beta sheet which forms the front and bottom surfaces of the barrel. The strands which form the front and back surfaces are joined at one end by four short "loops", giving the barrel the shape of a triangular wedge.
VPl is located near the five-fold axis on the icosahedral surface of the virion. Due to the positioning of VPl, three of the loops connecting the beta strands are exposed at the summit. The top loop, or BC (residues 95 to 104) , connects β sheets B and C, and twists out from the surface of the subunit so that it is particularly well exposed, giving the five-fold peak a ribbed appearance.
Without being bound to any theory, it is possible that the similarity in the conformation of the N-AgI loop and the conformation of the PND loop of HIV-l suggested that the N-AgI loop could be replaced with all or a portion of the PND loop and HIV-l neutralizing antibodies would be elicited by the hybrid virus. Poliovirus would thus serve as a vehicle for delivery of an HIV vaccine. We discovered, however, that when the N-AgI loop sequence was completely replaced with an HIV PND sequence, viable virus was not produced. This could be due to inefficient proteolytic processing of the poliovirus polyprotein, interference with poliovirus assembly pathway or inefficient receptor binding of the hybrid virus. In order to produce a viable virus, we discovered that certain elements of the natural PV sequence must be retained in order to maintain the viability of the virus. In particular, we found that, for HIV-IIIB and HIV-MN hybrid polioviruses, certain amino acids within the loop that are on the internal edge of the loop, i.e. adjacent to βB (Fig. 1) , must be retained in order to maintain viability of the virus. When an acceptable sequence of amino acids is maintained, high titers of the hybrid virus were observed. Moreover, hybrid virus Ml/HIV-IIIB- 1D-PND+3 elicited HIV-l neutralizing antibodies. Other Embodiments Other embodiments are within the following claims. For example, other immunogenic regions of viruses may be inserted into the poliovirus N-AgI region, e.g. , other HIV neutralizing regions, HRV-Nims, HAV-N-Ags, poliovirus N-Ags, as long as the appropriate PV amino acids, e.g., the proline and alanine residues for polio/HIV-IIIB hybrids, of the N-AgI region are retained.
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(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
TCGACAACCC AGCTTCCACC ACGATACAGA GAGGACCCGG GAGAGCTTTC GTGA 54
(2) INFORMATION FOR SEQ ID NO: 14: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 56
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
GTTGGGTCGG AAGGTGGTGC TATGTCTCTC CCTGGGCCCT CTCGAAAGCA CTTCGA 56
(2) INFORMATION FOR SEQ ID NO: 15: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19
(B) TYPE: amino acid (D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
Cys Val Thr lie Met Thr Val Asp Asn Pro Ala Ser Thr Thr Asn Lys
5 10 15 sp Lys Leu
(2) INFORMATION FOR SEQ ID NO: 16: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10
(B) TYPE: amino acid (D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: Ile Gin Arg Gly Pro Gly Arg Ala Phe Val
5 10
(2) INFORMATION FOR SEQ ID NO: 17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10
(B) TYPE: amino acid (D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:
He His He Gly Pro Gly Arg Ala Phe Tyr
5 10
ΛZ

Claims

Claims
1. A hybrid poliovirus comprising an epitope of a heterologous protein inserted into the BC loop of poliovirus, wherein between zero and ten amino acids, inclusive, of said BC loop are retained in said hybrid poliovirus, wherein said hybrid poliovirus is capable of eliciting in a mammal antibodies said heterologous protein, wherein said hybrid virus further comprises a mutation at a site outside of said BC loop, which results in a viral titer that is at least 5-fold higher than the viral titer of the virus that lacks said mutation.
2. A hybrid poliovirus comprising an epitope of a heterologous protein inserted into the BC loop of poliovirus, wherein between two and six amino acids, inclusive, of said BC loop are retained in said hybrid poliovirus, two of said retained amino acids being those located closest to the poliovirus internal βB sheet, wherein said BC loop of said hybrid poliovirus is capable of eliciting in a mammal antibodies specifically reactive with said heterologous protein.
3. The hybrid poliovirus of claim 2 wherein said retained poliovirus BC loop residues which lie closest to the βB sheet comprise the proline and alanine residues at positions 95-96 of said BC loop of strain PV1, or corresponding residues of another strain of poliovirus.
4. The hybrid poliovirus of claim 3 wherein said retained residues further comprise one or both of the lysine or leucine residues at positions 103 and 104, respectively, of the BC loop of strain PV1, or corresponding residues of another strain of poliovirus.
5. The hybrid poliovirus of claim 3 or 4 wherein said retained residues further comprise one or both of the serine or threonine residues at positions 97 and 98, respectively, of the BC loop of strain PV1, or corresponding residues of another strain of poliovirus.
6. The hybrid poliovirus of claim 1, 2 , 3, or 4 wherein said heterologors epitope is an epitope of the HIV-l envelope protein which is capable of eliciting HIV- 1 in a mammal neutralizing antibodies.
7. The hybrid poliovirus of claim 6 wherein said HIV neutralizing epitope comprises all or a portion of the PND of HIV.
8. The hybrid poliovirus of claim 7 wherein said PND contains at least 10 amino acids.
9. The hybrid poliovirus of claim 7 wherein said PND comprises the HIV-IIIB PND.
10. The hybrid poliovirus of claim 9 wherein said HIV-IIIB sequence comprises isoleucine-glutamine- arginine-glycine-proline-glycine-arginine-alanine- phenylalanine-valine.
11. A hybrid poliovirus comprising the PND of HIV-l-MN inserted into the BC loop of poliovirus, wherein between one and seven amino acids, inclusive, of said BC loop are retained in said hybrid poliovirus and one of said retained amino acids lies adjacent the poliovirus internal βB sheet, wherein said hybrid poliovirus is capable of eliciting in a mammal HIV-l neutralizing antibodies.
12. The hybrid poliovirus of claim 11 wherein said HIV-MN PND sequence comprises isoleucine-histidine- isoleucine-glycine-proline-glycine-arginine-alanine- phenylalanine-tyrosine.
13. The hybrid poliovirus of claim 6 wherein said HIV epitope raises broadly neutralizing antibodies.
14. The hybrid poliovirus of any of claims 2-5 and 7-13 further comprising a mutation at a site outside of said BC loop, said mutation resulting in a viral titer that is at least 5-fold higher than the viral titer of the virus that lacks said mutation.
15. The hybrid poliovirus of claim 6 further comprising a mutation at a site outside of said BC loop, said mutation resulting in a viral titer that is at least 5-fold higher than the viral titer of the virus that lacks said mutation.
16. A method of vaccinating a human patient against HIV-l or treating a human patient infected with HIV-l, said method comprising administering the hybrid poliovirus of claim 1, 2, or 11.
17. An HIV-l vaccine comprising the hybrid poliovirus of claim 1, 2 or 11 admixed with a pharmaceutically acceptable carrier.
18. A method of constructing the hybrid poliovirus of claim 2 or 11, said method comprising providing DNA encoding an eptiope, providing DNA comprising poliovirus or viral variants thereof and encoding a portion of the β sheet B of poliovirus. performing separate replacement steps, each said step comprising replacing said epitope-encoding DNA into said β sheet B-encoding DNA by selecting a site within said β sheet B-encoding DNA for each replacement, wherein successive sites are selected for each said replacement step, beginning at the amino-terminal encoding end of said β sheet B, and testing said hybrid poliovirus for its ability to prevent HIV-l infection.
19. A method of constructing the hybrid poliovirus of claim 18, said method comprising providing DNA encoding an epitope, providing DNA comprising poliovirus or viral variants thereof and encoding a portion of the BC loop of poliovirus, performing separate replacement steps, each said step comprising replacing said epitope-encoding DNA into said BC loop-encoding DNA by selecting a site within said BC loop-encoding DNA for each replacement, wherein successive sites are selected for each said replacement step, beginning at the amino-terminal encoding end of said BC loop, and testing said hybrid poliovirus for its ability to prevent HIV-l infection.
PCT/US1992/001303 1991-02-14 1992-02-14 Poliovirus-based vaccines WO1992014489A1 (en)

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Title
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NATURE, Volume 339, issued 01 June 1989 (London, GB), EVANS et al., "An engineered poliovirus chimaera elicits broadly reactive HIV-1 neutralizing antibodies", see pages 385-388. *
SEMLER et al., "Molecular Aspects of Picornavirus Infection and Detection", published 1989, by AMERICAN SOCIETY FOR MICROBIOLOGY (WASHINGTON, DC), see page 3-25. *
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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5651972A (en) * 1989-04-21 1997-07-29 University Of Florida Research Foundation, Inc. Use of recombinant swine poxvirus as a live vaccine vector
US6217882B1 (en) 1989-04-21 2001-04-17 University Of Florida Research Foundation, Inc. Use of recombinant swine poxvirus as a live vaccine vector
WO1994026900A2 (en) * 1993-05-13 1994-11-24 Connaught Laboratories Limited Hybrid picornaviruses expressing chlamydial epitopes
WO1994026900A3 (en) * 1993-05-13 1995-02-16 Connaught Lab Hybrid picornaviruses expressing chlamydial epitopes
US5622933A (en) * 1993-09-13 1997-04-22 Armel S.A. Multiple branch peptide constructions for use against HIV
WO2012138774A2 (en) 2011-04-04 2012-10-11 University Of Iowa Research Foundation Methods of improving vaccine immunogenicity
JP2014512182A (en) * 2011-04-04 2014-05-22 ユニバーシティー オブ アイオワ リサーチ ファウンデーション Methods to improve vaccine immunogenicity
EP2694102A4 (en) * 2011-04-04 2015-03-04 Univ Iowa Res Found Methods of improving vaccine immunogenicity
AU2012240231B2 (en) * 2011-04-04 2017-05-25 University Of Iowa Research Foundation Methods of improving vaccine immunogenicity
US10059746B2 (en) 2011-04-04 2018-08-28 University Of Iowa Research Foundation Methods of improving vaccine immunogenicity

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