Patent Grant
9566329
This disclosure relates to the field of viral vectors, specifically to a live, attenuated rubella viral vector platform capable of expressing a heterologous antigen, and the use of this platform to induce an immune response.
The worldwide spread of human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) has created an urgent need for HIV immunogens that can elicit broad protection against infection. Natural HIV infection in man and SIV infection in monkeys often elicit a strong antibody-mediated (B cell) and T cell-mediated immune response. A number of viral antigens targeted by broadly reactive neutralizing antibodies and antigen specific T cells have been identified. The challenge is to develop vaccines with sufficient potency to stimulate an effective immune response to these important viral antigens. Live viral vectors can enhance both T cell and B cell responses by presenting antigens in the most immunogenic way, in the context of an acute infection.
Weak immunogenicity of vaccine antigens is one of the main problems in vaccine development. Several approaches have been tried to improve presentation and enhance immunogenicity of these antigens, including DNA vaccines, non-replicating vectors and live viral vectors (
Some of the most successful vaccines, such as oral polio virus and measles, mumps, and rubella virus vaccine, consist of live attenuated viruses. These are given at very low doses, so the vaccine strain must grow in the host to produce sufficient viral antigens to elicit an immune response. By simulating a viral infection, they can elicit innate and adaptive immune responses, resulting in antigen-specific T cells and antibody-producing B cells. Through a process of attenuation, the vaccine strains have retained the growth and immunogenicity of wild type virus while losing its pathogenicity and virulence. However, for many pathogenic viruses, such as HIV, SW, RSV and/or hepatitis, it has not been possible to produce a live attenuated vaccine. Thus, additional approaches for creating vaccines for pathogenic viruses are needed.
Disclosed herein is a live attenuated rubella viral vector platform capable of stably expressing a heterologous antigen, such as a vaccine antigen (e.g., an HIV antigen (for example, an envelope glycoprotein antigen, such as, a gp41 or a gp120), a Gag antigen (such as an HIV or SIV Gag antigen), hepatitis B surface antigen (HBsAg), or an RSV antigen and the use of this platform to induce an immune response. The inventors utilized the rubella vaccine strain RA27/3 as a viral vector because of its safety and immunogenicity. Its safety has been demonstrated in millions of children around the world. If the rubella vector losses its insert, it reverts to the vaccine strain. At the same time, it is immunogenic and elicits both humoral and mucosal immunity. The inventors surprisingly found that rubella viral vector can accommodate foreign inserts at either of two sites, in the nonstructural region or between two structural genes. Each site was found to be controlled by a different viral promoter, resulting in expression of the insert as an early or late antigen of rubella. At the nonstructural site the insert was expressed as a fusion protein, while at the structural site it was expressed with the structural polyprotein and cleaved to a mature protein. The mature protein was incorporated into virions and co-sedimented with viral particles in sucrose density gradients. The vectors stably expressed the insert, while replicating for at least eight to ten passages in culture.
One advantage of rubella vectors for vaccine research is their ability to infect rhesus macaques. This animal model allows the testing of vector replication and immunogenicity in vivo. Rhesus macaques are also the animal of choice for SW and SHIV challenge studies; thus, the immune response can be evaluated for durability and protection against SIV or SHIV challenge. Production of rubella vectors is efficient, as they grow to high titer in cell culture, yet they immunize at a low dose. Their one limitation, eliciting antibodies to the vector, is overcome by combining a live rubella vector with another vaccine delivery platform in a prime and boost strategy.
The disclosed vectors are of interest to vaccine researchers as they reprogram the rubella vaccine strain to present new vaccine antigens. In the developed world, where virtually all rubella infections are caused by design (vaccination), the disclosed rubella vectors can be used to immunize against two or more infectious agents instead of one. In the third world, where rubella vaccine is not currently used, there is a window of opportunity for the vector to be given prior to the age of natural rubella infection, which ranges from two to six years old in most countries. It is contemplated that the disclosed vectors can be used against a variety of infectious diseases, including HIV, SIV, RSV and hepatitis.
In some embodiments, an isolated rubella viral vector includes a rubella non-structural protein open reading frame (ORF) without an in-frame deletion, a rubella structural protein ORF, and a heterologous antigenic insert. In some examples, the heterologous antigenic insert is positioned within the rubella structural protein ORF. In other examples, the heterologous antigenic insert is positioned in between the genes encoding structural proteins E2 and E1. In some examples, the heterologous antigenic insert is positioned between the genes encoding structural protein capsid and E2. In some examples, the antigenic insert comprises an amino acid sequence set forth as SEQ ID NO: 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 138, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 158, 159, 160, 161, or 162.
Exemplary antigenic inserts can include cytotoxic T lymphocyte (CTL) epitopes, HIV envelope protein epitopes, RSV antigen and HBsAg inserts. For example, an antigenic insert can include a CTL epitope of HIV or SIV Gag, an epitope of HIV gp41, including a membrane proximal region (MPER or MPR) or an epitope of HIV gp120. The antigenic insert can include repeats of any one of the disclosed antigenic epitopes, such as one to ten copies of one or more of the disclosed antigenic envelope or CTL epitopes.
In some examples, an antigenic insert is a wildtype or variant of a CTL epitope of a Gag polypeptide or a fragment thereof. In some examples, the antigenic peptide includes one or more major CTL epitopes of Gag, and can be from about 8 to about 350 amino acids in length, such from about 10 to about 280 amino acids in length, such as 20 to about 270 amino acids in length, such as from about 40 to about 250 amino acids in length, including 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 57, 60, 63, 65, 67, 70, 73, 75, 77, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349 or 350. In some examples, an antigenic insert includes one or more CTL epitopes, such as one or more CTL HIV or SIV epitopes, including those set forth as SEQ ID NOs: 77, 78, 79, 80, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, and 102. In some examples, an antigenic insert includes one or more antigenic polypeptide fragments of Gag, such as one or more antigenic polypeptides with an amino acid sequence provided by SEQ ID NOs: 82-88, 90, 91, 132, 133, 134, 135, 138, 139, 140, 141 or 142.
In some examples, an antigenic insert is a wildtype or variant gp41 polypeptide or a fragment thereof. In some examples, a gp41 antigenic insert can include (a) an antigenic polypeptide fragment of gp41 and (b) a transmembrane spanning region of gp41. For example, the gp41 antigenic insert includes (a) an antigenic polypeptide fragment, such as an antigenic polypeptide fragment with the amino acid sequence set forth in SEQ ID NO:1 (in which wherein X1, X2 and X3 are any amino acid) and the polypeptide is between 8 and 400 amino acids in length, such as between 10 and 300 amino acids in length, such as from about 12 to about 250, such as from about 16 to about 160 amino acids, such as from about 28 to about 150 amino acids in length, such as from about 28 to about 140 amino acids in length, including 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 120, 125, 130, 135, 140, or 145 amino acids; and (b) a transmembrane spanning gp41 region, such as a transmembrane spanning gp41 region with the amino acid sequence set forth in SEQ ID NO: 25 (in which X4, X5, and X6 are any hydrophobic amino acid) and is between 22 and 40 amino acids in length, such as about 23 and 38 amino acids in length, including 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 amino acids. In certain examples, an antigenic insert includes an antigenic polypeptide fragment of gp41 with an amino acid sequence provided by SEQ ID NOs: 1-22, 30, 81 or 89 and a transmembrane region of gp41 with an amino acid sequence provided by SEQ ID NOs: 25-28.
In some examples, an antigenic insert is a wildtype or variant gp120 polypeptide. In an example, a wildtype gp120 polypeptide has an amino acid provided by SEQ ID NO: 63 or a fragment thereof. In other examples, a variant gp120 polypeptide includes a gp120 polypeptide in which at least 8 consecutive residues of the fourth conserved loop (C4) between residues 419 and 434 of gp120 according HXB2 numbering of SEQ ID NO: 63 are deleted. This deletion within the β20-21 loop of the gp120 polypeptide exposes the CD4 binding site thereby providing improved antibody binding and antibody induction. In one example, a variant gp120 polypeptide is a gp120 polypeptide in which at least 8 consecutive residues, such as between 8-12, 8-11, 8-10, or 8-9 (for example, 9, 10, 11 or 12) consecutive residues of C4 between residues 419 and 434 of gp120 of SEQ ID NO: 63 have been deleted.
In a particular example, a variant gp120 polypeptide includes a gp120 polypeptide in which residues 424-432 are deleted. Additional variant gp120 polypeptides include deletions of INMWQKVGK (residues 424-432 of SEQ ID NO: 63), INMWQKVGKA (residues 424-433 of SEQ ID NO: 63), INMWQKVGKAM (residues 424-434 of SEQ ID NO: 63), RIKQIINMWQKVGK (residues 419-432 of SEQ ID NO: 63), IKQIINMWQKVGK (residues 420-432 of SEQ ID NO: 63), KQIINMWQKVGK (residues 421-432 of SEQ ID NO: 63), QIINMWQKVGK (residues 422-432 of SEQ ID NO: 63), or IINMWQKVGK (residues 423-432 of SEQ ID NO: 63). In other embodiments, variant gp120 polypeptides include combinations of the amino and carboxyl ends between residues 419 and 434.
In some embodiments, a variant gp120 polypeptide does not include a variant in which residues 419-428 of SEQ ID NO: 63 are deleted. In other embodiments, a variant gp120 polypeptide does not include a variant in which residues 437-452 of SEQ ID NO: 63 are deleted. In certain examples, an antigenic insert includes an amino acid sequence set forth by SEQ ID NOs: 63, 66, 67, 69, 71, 73 or 74.
In some embodiments, an isolated rubella viral vector comprises an antigenic insert comprising an amino acid sequence set forth as SEQ ID NO: 126, 127, 131-156 or 158-162.
In some embodiments, an isolated rubella viral vector comprises an antigenic insert consisting of an amino acid sequence set forth as SEQ ID NO: 126, 127, 131-156 or 158-162.
In some embodiments, a disclosed isolated rubella viral vector comprises an antigenic insert comprising a Gag antigenic insert, comprising the amino acid sequence as set forth by SEQ ID NO: 141, SEQ ID NO. 142, SEQ ID NO: 82 or SEQ ID NO: 83.
In some embodiments, a disclosed isolated rubella viral vector comprises an antigenic insert comprising the amino acid sequence set forth as one of SEQ ID NOs: 143-155; SEQ ID NO. 156; SEQ ID NOs: 158-162; or SEQ ID NOs: 84-91.
In some embodiments, a disclosed isolated rubella viral vector comprises an antigenic insert consisting of the amino acid sequence set forth as one of SEQ ID NOs: 143-155; SEQ ID NO. 156; SEQ ID NOs: 158-162; or SEQ ID NOs: 84-91.
Viral-like particles including any of the disclosed isolated viral vector constructs are provided herein. Compositions comprising the viral-like particles are also provided.
The disclosed isolated viral vectors can be used to induce an immune response, such as a protective immune response, when introduced into a subject. The provided rubella viral vector platforms can also be used in assays to diagnose a viral infection, such as an HIV infection, an SIV infection, RSV infection or hepatitis. Thus, methods are provided for inhibiting a viral infection, such as an HIV infection in a subject, for inducing an immune response to HIV in a subject, and for diagnosing HIV infection in a subject. The disclosed viral vector platforms can also be used for measuring host range, testing sensitivity to neutralizing antibodies, or screening antiviral drugs. In one example, methods of screening antiviral drugs including methods of identifying protease inhibitors are disclosed.
The foregoing and other features of the disclosure will become more apparent from the following detailed description, which proceeds with reference to the accompanying sequence listing and figures.
The nucleic and amino acid sequences listed herein are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. All sequence database accession numbers referenced herein are understood to refer to the version of the sequence identified by that accession number as it was available on the designated date.
SEQ ID NO: 1 is a consensus amino acid sequence for the membrane proximal region (MPR) of gp41 of HIV-1. An X represents specific amino acids where alterations can be tolerated.
SEQ ID NO: 2 is a consensus amino acid sequence based on each clade consensus sequence of the MPR region from HIV-1.
SEQ ID NO: 3 is the ancestral amino acid sequence of the MPER region from HIV-1 clade M. This sequence is also the consensus amino acid sequence of the MPR region from HIV-1 clade AG.
SEQ ID NO: 4 is the consensus amino acid sequence of the MPER region from HIV-1 clade A1. This sequence is also the ancestral amino acid sequence of the MPER region from HIV-1 clade A1.
SEQ ID NO: 5 is the consensus amino acid sequence of the MPER region from HIV-1 clade A2.
SEQ ID NO: 6 is the consensus amino acid sequence of the MPER region from HIV-1 clade B. This sequence is also the ancestral amino acid sequence of the MPER region from HIV-1 clade B.
SEQ ID NO: 7 is the consensus amino acid sequence of the MPER region from HIV-1 clade C.
SEQ ID NO: 8 is the ancestral amino acid sequence of the MPER region from HIV-1 clade C.
SEQ ID NO: 9 is the consensus amino acid sequence of the MPER region from HIV-1 clade D.
SEQ ID NO: 10 is the consensus amino acid sequence of the MPER region from HIV-1 clade F1.
SEQ ID NO: 11 is the consensus amino acid sequence of the MPER region from HIV-1 clade F2.
SEQ ID NO: 12 is the consensus amino acid sequence of the MPER region from HIV-1 clade G.
SEQ ID NO: 13 is the consensus amino acid sequence of the MPER region from HIV-1 clade H.
SEQ ID NO: 14 is the consensus amino acid sequence of the MPER region from HIV-1 clade AE.
SEQ ID NO: 15 is the consensus amino acid sequence of the MPER region from HIV-1 clade AB.
SEQ ID NO: 16 is the consensus amino acid sequence of the MPER region from HIV-1 clade 04CPX.
SEQ ID NO: 17 is the consensus amino acid sequence of the MPER region from HIV-1 clade 06CPX.
SEQ ID NO: 18 is the consensus amino acid sequence of the MPER region from HIV-1 clade 08BC.
SEQ ID NO: 19 is the consensus amino acid sequence of the MPER region from HIV-1 clade 10CD.
SEQ ID NO: 20 is the consensus amino acid sequence of the MPER region from HIV-1 clade 11CPX.
SEQ ID NO: 21 is the consensus amino acid sequence of the MPER region from HIV-1 clade 12BF.
SEQ ID NO: 22 is the consensus amino acid sequence of the MPER region from HIV-1 clade 14BG.
SEQ ID NOs: 23-24 are oligonucleotide primers used to amplify a rubella sequence flanking a zGFP insert.
SEQ ID NO: 25 is a consensus amino acid sequence for the transmembrane region of gp41. An X represents any hydrophobic amino acid.
SEQ ID NOs: 26-28 are amino acid sequences for a transmembrane spanning region of gp41.
SEQ ID NO: 29 is an amino acid sequence for a disclosed isolated immunogen in which the first transmembrane domain of hepatitis B surface antigen is replaced with the MPER and transmembrane domain of gp41.
SEQ ID NO: 30 is an exemplary MPER region from HIV-1 amino acid sequence.
SEQ ID NO: 31 is an exemplary wildtype amino acid sequence of HBsAg.
SEQ ID NO: 32 is an example of a nucleotide sequence for a T helper cell epitope.
SEQ ID NO: 33 is an example of an amino acid sequence for a T helper cell epitope.
SEQ ID NO: 34 is the CAAX amino acid sequence, where C is cystein, A is an aliphatic amino acid and X is any amino acid.
SEQ ID NO: 35 is the core amino acid sequence of the 2F5 epitope.
SEQ ID NO: 36 is the core amino acid sequence of the 4E10 epitope.
SEQ ID NO: 37 is the linker sequence GPGP.
SEQ ID NO: 38 (ggagctcgtcgacagcaa) is a forward primer for amplification of the HBsAg.
SEQ ID NO: 39 (gctctagacccgatgtacaccca) is a reverse primer for amplification of the HBsAg.
SEQ ID NO: 40 (gctctagaaacgagcaggagctgctg) is a forward primer for amplification of MPER.
SEQ ID NO: 41 (cgcggatcctcaccccttgatgtaccacagccactt) is a reverse primer for amplification of MPER.
SEQ ID NO: 42 (cgcggatcctcaatggtgatggtgatggtgggg) is a reverse primer for amplification of MPER-Foldon.
SEQ ID NO: 43 (gctctagagccgtggagcggtacctg) is a forward primer for amplification of C-heptad.
SEQ ID NO: 44 (ctcggatcctcaaatcatgatgaaaatcttgat) is a reverse primer for amplification of MPER-Tm5.
SEQ ID NO: 45 (ctcggatcctcacaccaggccaccaacaat) is a reverse primer for amplification of MPER-Tm10.
SEQ ID NO: 46 (ctcggatcctcacaccagcctcaggcccac) is a reverse primer for amplification of MPER-Tm15.
SEQ ID NO: 47 (ctcggatcctcaggcgggcgc) is a reverse primer for amplification of MPER-Tm23.
SEQ ID NO: 48 (ccctgcaagacctgcaccaccaccggtcagggcaactccaagttcccc) is a forward primer for amplification of the MPER region with AgeI.
SEQ ID NO: 49 (ggggaacttggagttgccctgaccggtggtggtgcaggtcttgcaggg) is a reverse primer for amplification of the MPER region with AgeI.
SEQ ID NO: 50 (ggcaccggtaacgagcaggagctgctg) is a forward primer for amplification of the MPER region with AgeI.
SEQ ID NO: 51 (ggcaccggtccccttgatgtaccacagccactt) is a reverse primer for amplification of the MPER region with AgeI.
SEQ ID NO: 52 (agcgaattcaacgagcaggagctgctg) is a forward primer for amplification of the MPER region with HBsAg (MPER SAG or MPER-N-term).
SEQ ID NO: 53 (cgcggatcctcacccgatgtacaccca) is a reverse primer for amplification of the MPER region with HBsAg (MPER SAG or MPER-N-term).
SEQ ID NO: 54 (caggaagccggaggtgatgaaccccttgatgtaccacagc cactt) is a forward primer for amplification of SAGMPER-R1 (HBsAg at the N-terminus of MPER).
SEQ ID NO: 55 (aagtggctgtggtacatcaaggggttcatcacctccggcttcctg) is a reverse primer for amplification of SAGMPER-R1 (HBsAg at the N-terminus of MPER).
SEQ ID NO: 56 is an nucleic acid sequence which encodes the Gag antigenic insert with an amino acid sequence set forth as SEQ ID NO: 103
SEQ ID NO: 57 (MKTIIALSYIFCLVFAQDLPGNDNNSEFNEKELLELDKWASL WNWFDITNWLWYIRLFIMIVGGLIGLRIVFAVLSIPQSLDSWWTSLNFLGGSPVCLGQNSQS PTSNHSPTSCPPICPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLIPGSTTTSTGP CKTCTTPAQGNSKFPSCCCTKPTDGNCTCIPINEKELLELDKWASLWNWFDITNWLWYIRL FIMIVGGLIGLRIVFAVLSIVVGLSPTVWLSAIWMMWYWGPSLYSIVSPFIPLLPIFFCLWVY IG) is an amino acid sequence for a disclosed isolated immunogen in which the first and third transmembrane domains of hepatitis B surface antigen are each replaced with the MPER and transmembrane domain of gp41.
SEQ ID NO: 58 is a nucleic acid sequence for a disclosed isolated immunogen in which the third transmembrane domains of HBsAg is replaced with the MPER and transmembrane domain of gp41.
SEQ ID NO: 59 is an amino acid sequence for a disclosed isolated immunogen in which the third transmembrane domain of hepatitis B surface antigen is replaced with the MPER and transmembrane domain of gp41.
SEQ ID NO: 60 (NEKELLELDKWASLW) is an amino acid sequence of the MPER region in the TM32 or TM32F constructs.
SEQ ID NO: 61 (NEKELLELDKWASLWNWFDITNWLW) is an amino acid sequence of the MPER region in the TM34 construct.
SEQ ID NO: 62 (MKTIIALSYIFCLVFAQDLPGNDNNSEFNEKELLELDKWASLW NWFDITNWLWYIRLFIMIVGGLIGLRIVFAVLSIPQSLDSWWTSLNFLGGSPVCLGQNSQSP TSNHSPTSCPPICPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLIPGSTTTSTGPC KTCTTPAQGNSKFPSCCCTKPTDGNCTCISINEKELLELDKWASLWNWFDITNWLWSSLW AIKYLWEWASVRFSWLSLLVPFVQWFVGLSPTVWLSAIWMMWYWGPSLYSIVSPFIPLLPI FFCLWVYIG) is an amino acid sequence of a disclosed variant HbsAg construct (TM16+34) in which the first domain is replaced with a MPER and transmembrane domain of gp41 and an additional MPER is inserted between a second and third domain in the variant HBsAg.
SEQ ID NO: 63 is an amino acid sequence of a variant gp120 with a V1V2 deleted gp120.
SEQ ID NO: 64 (MKTIIALSYIFCLVFAQDLPGNDNNSEFNEKELLELDKWASL WNWFDITNWLWYIRLFIMIVGGLIGLRIVFAVLSIPQSLDSWWTSLNFLGGSPVCLGQNSQS PTSNHSPTSCPPICPGYRWMCLRRFIIFLFILLLCIFLLVLLDYQGMLPVCPLIPGSTTTSTGPC KTCTTPAQGNSKFPSCCCTKPTDGNCTCISINEKELLELDKWASLWAINEKELLELDKWAS LWAINEKELLELDKWASLWAINEKELLELDKWASLWAIKYLWEWASVRFSWLSLLVPFV QWFVGLSPTVWLSAIWMMWYWGPSLYSIVSPFIPLLPIFFCLWVYIG) is an amino acid sequence of a disclosed variant HbsAg construct (TM16+32F) in which the first domain is replaced with a MPER and transmembrane domain of gp41 and four additional MPERs are inserted between a second and third domain in the variant HBsAg.
SEQ ID NO: 65 (MKTIIALSYIFCLVFAQDLPGNDNNSEFITSGFLGPLLVLQAGF FLLTRILTIPQSLDSWWTSLNFLGGSPVCLGQNSQSPTSNHSPTSCPPICPGYRWMCLRRFIIF LFILLLCLIFLLVLLDYQGMLPVCPLIPGSTTTSTGPCKTCTTPAQGNSKFPSCCCTKPTDGN CTCISINEKELLELDKWASLWAINEKELLELDKWASLWAINEKELLELDKWASLWAINEKE LLELDKWASLWAIKYLWEWASVRFSWLSLLVPFVQWFVGLSPTVWLSAIWMMWYWGPS LYSIVSPFIPLLPIFFCLWVYIG) is an amino acid sequence of a disclosed variant HbsAg construct (32F) in which four MPERs are inserted between a second and third domain in the variant HBsAg.
SEQ ID NO: 66 is an amino acid sequence for a variant gp120 with a V1V2 deletion with a beta 20-21 loop deletion.
SEQ ID NO: 67 is an amino acid sequence for a variant gp120 from HIV isolate JR-FL.
SEQ ID NO: 68 is a nucleic acid sequence for a variant gp120 from HW isolate JR-FL.
SEQ ID NO: 69 is an amino acid sequence for a variant gp120 from HW isolate AD8.
SEQ ID NO: 70 is a nucleic acid sequence for a variant gp120 from HIV isolate AD8.
SEQ ID NO: 71 is an amino acid sequence for a variant gp120 from HIV isolate BaL.
SEQ ID NO: 72 is a nucleic acid sequence for a variant gp120 from HIV isolate BaL.
SEQ ID NO: 73 is an amino acid sequence for a variant gp120 from HIV isolate IIIB
SEQ ID NO: 74 is a nucleic acid sequence for a variant gp120 from HIV isolate IIB.
SEQ ID NOS: 75-76 are oligonucleotide primers used to amplify Zoanthus sp. green fluorescent protein (zGFP).
SEQ ID NOS: 77-80 are amino acid sequences of disclosed variant rubella constructs in which one MPER is inserted into the structural open reading frame of the rubella construct.
SEQ ID NO: 81 is an amino acid sequence of MPERf which contains the epitope recognized by neutralizing monoclonal antibody 2F5.
SEQ ID NOS: 82-88 are amino acid sequences of Gag antigenic inserts.
SEQ ID NO: 89 is an amino acid sequence of MPERe which contains the epitope recognized by neutralizing monoclonal antibody 4E1.
SEQ ID NOs: 90-91 are amino acid sequences of Gag antigenic inserts.
SEQ ID NOs: 92-96 are amino acid sequences of CTL epitopes of SIV Gag.
SEQ ID NOs: 97-102 are amino acid sequences of CTL epitopes of HIV Gag.
SEQ ID NO: 103 is an amino acid sequence of a Gag antigenic insert.
SEQ ID NOs: 104-125 are oligonucleotide primer sequences.
SEQ ID NOs: 126-127 are amino acid sequences of MPERF inserts.
SEQ ID NO: 128 is an amino acid sequence of MPERF insert.
SEQ ID NO: 129 is an amino acid sequence of MPERE insert.
SEQ ID NO: 130 is an amino acid sequence of MPER insert.
SEQ ID NO: 131 is an amino acid sequence of MPER-HIVTM insert.
SEQ ID NO: 132 is an amino acid sequence of an SW Gag insert (sGag-E2TM amino acids 41-211) expressed at the structural site.
SEQ ID NO: 133 is an amino acid sequence of an SW Gag insert (SGag-E2TM, amino acids 135-271) expressed at the structural site.
SEQ ID NO: 134 is an amino acid sequence of an SW Gag insert (full p28-sGag-E2TM) expressed at the structural site.
SEQ ID NO: 135 is an amino acid sequence of an SW Gag insert (full p28 plus-sGag-E2TM) expressed at the structural site.
SEQ ID NOS: 136 is an amino acid sequence of a disclosed variant rubella constructs in which one MPER is inserted into the structural open reading frame of the rubella construct.
SEQ ID NO: 137 is an amino acid sequence of an MPER construct incorporating the gp41 trimerization domain (N and C domains) before MPER, such arrangement can increase MPER valency and size.
SEQ ID NOs: 138-140 are amino acid sequences of HIV Gag inserts expressed at the rubella structural site.
SEQ ID NOs: 141-162 are sequences of inserts expressed at the rubella structural site.
The Sequence Listing is submitted as an ASCII text file in the form of the file named Sequence.txt, which was created on Apr. 8, 2013, and is 196,048 bytes, which is incorporated by reference herein.
Many of the most successful vaccines are based on live attenuated vaccine strains of pathogens. As they replicate, they expose the host to exponentially increasing amounts of viral antigens, while presenting the antigens in the most immunogenic way, in the context of an acute infection. They combine innate and adaptive immunity to elicit antigen specific T cells and neutralizing antibodies. In the past, these strains were derived from virulent viruses by repeated passage under selective conditions, until virulence genes were mutated or lost, while retaining the ability to replicate and elicit an immune response in the host.
Human immunodeficiency virus (HIV) infection often elicits a strong immune response, including T cell immunity and neutralizing antibodies. Similarly, prior infection with nef deleted simian immunodeficiency virus (SIV) can protect animals against a subsequent challenge. However, it seems unlikely that HIV virus could be attenuated sufficiently for vaccine use. In part, this is due to the retroviral life cycle, with its obligatory integration into host DNA. It is also due to the high rate of mutation, with the risk of reversion to wild type. In addition, no virulence genes have been identified that could be mutated or deleted to produce a reliably attenuated vaccine strain.
Instead of classical attenuation, the inventors have developed live attenuated rubella viral vectors to combine the safety and immunogenicity of the vector with the antigenicity of the HIV and SIV protein inserts. Previous vectors have included DNA viruses, such as vaccinia virus and modified vaccinia Ankara, adenovirus and cytomegalovirus (CMV). They also have included RNA viruses, such as Venezuelan equine encephalomyelitis replicons, attenuated vesicular stomatitis virus, and yellow fever vaccine. Some vectors are defective and replicate poorly or not at all in vivo, while others grow freely. Rubella virus has a number of desirable properties for a live vector. The live attenuated rubella vaccine strain is immunogenic in humans at a dose of just 5,000 PFU. The RA27/3 vaccine strain is a licensed product, and its safety record has been established in millions of children around the world. It is immunogenic: one dose protects for life (against rubella infection), and it elicits mucosal as well as systemic immunity. It has no DNA intermediate, cannot integrate into host DNA, and does not persist after the acute infection. A full length, infectious cDNA clone is available, both for wild type rubella and for the RA27/3 vaccine strain, making it possible to manipulate rubella genetically. Rubella readily infects rhesus macaques. These are the animal model of choice for demonstrating protection against SIV or simian-human immunodeficiency virus (SHIV) challenge.
Despite these desirable properties, the use of rubella virus for a live vector has been unsuccessful because of the inability to maintain stable expression of foreign genes in a live rubella vector. Moreover, it has remained unclear which foreign genes could be inserted, where to insert them, and how large an insert could be accommodated in viral RNA and packaged into virions. For example, if the insert exceeded the size limit, selective pressure resulted in an unstable construct with loss of gene expression.
Disclosed herein is a rubella viral vector construct that is capable of expressing foreign genes at a high level without interfering with expression of essential rubella genes and packaging of live virus. This vector construct maintains stable expression of foreign genes for multiple passages. Thus, the inventors have created a new way to use rubella vaccine as a viral vector to express an additional protein antigen of a second virus. In this way, the safety and immunogenicity of a rubella vaccine can be combined with the antigenicity of another virus.
For example, previous vectors, with up to 1000 fold less potency, have been tested for immunization with HIV antigens, and they all failed to protect against HIV infection. The vector constructs disclosed herein are believed to be capable of actually immunizing against this pathogen. Further, unlike previous vectors, the safety and immunogenicity of a live attenuated rubella vaccine has been demonstrated in tens of millions of children throughout the world. Vaccine potency is based on the fact that this is a replicating vector that simulates infection. One dose protects for life against rubella. The vaccine induces mucosal as well as systemic immunity. Each of these properties would be desirable in a vaccine against HIV or other pathogens, such as SIV, RSV or hepatitis.
At the same time, a disclosed vector construct can also be the lowest cost vector for virtually any viral pathogen, since the immunizing dose is so low that one ml of culture fluid can make up to 1,000 doses of vaccine. The market could be more than 100 million doses per year. Moreover, in the United States, the disclosed vector construct can be used to generate a rubella vaccine that could be substituted for the current rubella vaccine, at almost zero cost, and used to immunize against rubella plus the inserted antigen. Without vaccination, the average age of becoming seropositive to rubella is about 9 years old in many parts of the world. Thus, it could be given to 1-2 year olds with a boost at 9 years old, with a high likelihood of success in immunizing against rubella as well as the foreign antigen (such as HBsAg or HIV, SIV or RSV antigen).
Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).
Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The term “comprises” means “includes.” All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
To facilitate review of the various embodiments of this disclosure, the following explanations of specific terms are provided:
Amplification: Of a nucleic acid molecule (e.g., a DNA or RNA molecule) refers to use of a technique that increases the number of copies of a nucleic acid molecule in a specimen. An example of amplification is the polymerase chain reaction (PCR), in which a biological sample collected from a subject is contacted with a pair of oligonucleotide primers, under conditions that allow for the hybridization of the primers to a nucleic acid template in the sample. The primers are extended under suitable conditions, dissociated from the template, and then re-annealed, extended, and dissociated to amplify the number of copies of the nucleic acid. The product of amplification may be characterized by electrophoresis, restriction endonuclease cleavage patterns, oligonucleotide hybridization or ligation, and/or nucleic acid sequencing using standard techniques. Other examples of amplification include strand displacement amplification, as disclosed in U.S. Pat. No. 5,744,311; transcription-free isothermal amplification, as disclosed in U.S. Pat. No. 6,033,881; repair chain reaction amplification, as disclosed in WO 90/01069; ligase chain reaction amplification, as disclosed in EP-A-320 308; gap filling ligase chain reaction amplification, as disclosed in U.S. Pat. No. 5,427,930; and NASBA™ RNA transcription-free amplification, as disclosed in U.S. Pat. No. 6,025,134.
Antibody: Immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, that is, molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen.
A naturally occurring antibody (e.g., IgG, IgM, IgD) includes four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. However, it has been shown that the antigen-binding function of an antibody can be performed by fragments of a naturally occurring antibody. Thus, these antigen-binding fragments are also intended to be designated by the term “antibody.” Specific, non-limiting examples of binding fragments encompassed within the term antibody include (i) a Fab fragment consisting of the VL, VH, CL and CH1 domains; (ii) an Fd fragment consisting of the VH and CH1 domains; (iii) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (iv) a dAb fragment (Ward et al., Nature 341:544-546, 1989) which consists of a VH domain; (v) an isolated complimentarity determining region (CDR); and (vi) a F(ab′)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region.
Methods of producing polyclonal and monoclonal antibodies are known to those of ordinary skill in the art, and many antibodies are available. See, e.g., Coligan, Current Protocols in Immunology Wiley/Greene, NY, 1991; and Harlow and Lane, Antibodies: A Laboratory Manual Cold Spring Harbor Press, NY, 1989; Stites et al., (eds.) Basic and Clinical Immunology (4th ed.) Lange Medical Publications, Los Altos, Calif., and references cited therein; Goding, Monoclonal Antibodies: Principles and Practice (2d ed.) Academic Press, New York, N.Y. 1986; and Kohler and Milstein, Nature 256: 495-497, 1975. Other suitable techniques for antibody preparation include selection of libraries of recombinant antibodies in phage or similar vectors. See, Huse et al., Science 246: 1275-1281, 1989; and Ward et al., Nature 341: 544-546, 1989. “Specific” monoclonal and polyclonal antibodies and antisera (or antiserum) will usually bind with a KD of at least about 0.1 μM, preferably at least about 0.01 μM or better, and most typically and preferably, 0.001 μM or better.
Immunoglobulins and certain variants thereof are known and many have been prepared in recombinant cell culture (e.g., see U.S. Pat. No. 4,745,055; U.S. Pat. No. 4,444,487; WO 88/03565; EP 256,654; EP 120,694; EP 125,023; Faoulkner et al., Nature 298:286, 1982; Morrison, J. Immunol. 123:793, 1979; Morrison et al., Ann Rev. Immunol 2:239, 1984). Detailed methods for preparation of chimeric (humanized) antibodies can be found in U.S. Pat. No. 5,482,856. Additional details on humanization and other antibody production and engineering techniques can be found in Borrebaeck (ed), Antibody Engineering, 2nd Edition Freeman and Company, NY, 1995; McCafferty et al., Antibody Engineering, A Practical Approach, IRL at Oxford Press, Oxford, England, 1996, and Paul Antibody Engineering Protocols Humana Press, Towata, N.J., 1995.
Antigen: A compound, composition, or substance that can stimulate the production of antibodies or a T cell response in an animal, including compositions that are injected or absorbed into an animal. An antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous immunogens. The term is used interchangeably with the term “immunogen.” The term “antigen” includes all related antigenic epitopes. An “antigenic polypeptide” is a polypeptide to which an immune response, such as a T cell response or an antibody response, can be stimulated. “Epitope” or “antigenic determinant” refers to a site on an antigen to which B and/or T cells respond. In one embodiment, T cells respond to the epitope when the epitope is presented in conjunction with an MHC molecule. Epitopes can be formed both from contiguous amino acids (linear) or noncontiguous amino acids juxtaposed by tertiary folding of an antigenic polypeptide (conformational). Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. Normally, a B-cell epitope will include at least about 5 amino acids but can be as small as 3-4 amino acids. A T-cell epitope, such as a CTL epitope, will include at least about 7-9 amino acids, and a helper T-cell epitope at least about 12-20 amino acids. Normally, an epitope will include between about 5 and 15 amino acids, such as, 9, 10, 12 or 15 amino acids. The amino acids are in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and multi-dimensional nuclear magnetic resonance spectroscopy. The term “antigen” denotes both subunit antigens, (for example, antigens which are separate and discrete from a whole organism with which the antigen is associated in nature), as well as killed, attenuated or inactivated bacteria, viruses, fungi, parasites or other microbes. Antibodies such as anti-idiotype antibodies, or fragments thereof, and synthetic peptide mimotopes, which can mimic an antigen or antigenic determinant, are also captured under the definition of antigen as used herein. Similarly, an oligonucleotide or polynucleotide which expresses an antigen or antigenic determinant in vivo, such as in gene therapy and DNA immunization applications, is also included in the definition of antigen herein.
An “antigen,” when referring to a protein, includes a protein with modifications, such as deletions, additions and substitutions (generally conservative in nature) to the native sequence, so long as the protein maintains the ability to elicit an immunological response, as defined herein. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the antigens.
Antigen Delivery Platform or Epitope Mounting Platform: In the context of the present disclosure, the terms “antigen delivery platform” and “epitope mounting platform” refer to a macromolecular complex including one or more antigenic epitopes. Delivery of an antigen (including one or more epitopes) in the context of an epitope mounting platform enhances, increases, ameliorates or otherwise improves a desired antigen-specific immune response to the antigenic epitope(s). The molecular constituents of the antigen delivery platform may be antigenically neutral or may be immunologically active, that is, capable of generating a specific immune response. Nonetheless, the term antigen delivery platform is utilized to indicate that a desired immune response is generated against a selected antigen that is a component of the macromolecular complex other than the platform polypeptide to which the antigen is attached. Accordingly, the epitope mounting platform is useful for delivering a wide variety of antigenic epitopes, including antigenic epitopes of pathogenic organisms such as bacteria and viruses. The antigen delivery platform of the present disclosure is particularly useful for the delivery of complex peptide or polypeptide antigens, which may include one or many distinct epitopes.
Antigenic polypeptide fragment: A polypeptide that is antigenic. In an example, an antigenic polypeptide fragment includes an HIV antigenic polypeptide fragment, such as a Gag, gp41 or gp120 antigenic polypeptide fragment or a HBsAg antigenic polypeptide fragment.
Conservative variants: “Conservative” amino acid substitutions are those substitutions that do not substantially affect or decrease a desired activity of a protein or polypeptide. For example, in the context of the present disclosure, a conservative amino acid substitution does not substantially alter or decrease the immunogenicity of an antigenic epitope. Similarly, a conservative amino acid substitution does not substantially affect the structure or, for example, the stability of a protein or polypeptide. Specific, non-limiting examples of a conservative substitution include the following examples:
The term conservative variation also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid, provided that antibodies raised to the substituted polypeptide also immunoreact with the unsubstituted polypeptide. Non-conservative substitutions are those that reduce an activity or antigenicity or substantially alter a structure, such as a secondary or tertiary structure, of a protein or polypeptide.
cDNA (complementary DNA): A piece of DNA lacking internal, non-coding segments (introns) and regulatory sequences that determine transcription. cDNA is typically synthesized in the laboratory by reverse transcription from messenger RNA extracted from cells.
Cytotoxic T lymphocyte (CTL): A type of lymphocyte (white blood cell) that is involved in the immune defenses of the body. Cytotoxic T cells are capable of inducing the death of inducing the death of infected somatic or tumor cells. They are also capable of killing cells infected with viruses (or other pathogens) or are otherwise damaged or dysfunctional. Most CTLs express T-cell receptors that can recognize a specific antigenic peptide bound to Class I MHC molecules.
Deletion: Removal or loss of a sequence of nucleic or amino acids. In one example, a deletion is an “in-frame deletion” (a deletion of a number of base pairs that is a multiple of three and thus constitutes a codon, and therefore does not disrupt the triplet reading frame.)
Diagnostic: Identifying the presence or nature of a pathologic condition, such as, but not limited to a condition induced by a viral or other pathogen. Diagnostic methods differ in their sensitivity and specificity. The “sensitivity” of a diagnostic assay is the percentage of diseased individuals who test positive (percent of true positives). The “specificity” of a diagnostic assay is 1 minus the false positive rate, where the false positive rate is defined as the proportion of those without the disease who test positive. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it suffices if the method provides a positive indication that aids in diagnosis. “Prognostic” is the probability of development (or for example, the probability of severity) of a pathologic condition, such as a symptom induced by a viral infection or other pathogenic organism, or resulting indirectly from such an infection.
Epitope: An antigenic determinant. These are particular chemical groups or peptide sequences on a molecule that are antigenic, that is, that elicit a specific immune response. An antibody specifically binds a particular antigenic epitope on a polypeptide. Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5, about 9, or 8-10 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and multi-dimensional nuclear magnetic resonance spectroscopy. See, e.g., “Epitope Mapping Protocols” in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed (1996). In some embodiments, an epitope binds an MHC molecule, e.g., an HLA molecule or a DR molecule. In some embodiments, an epitope is a cytotoxic T lymphocyte (CTL) epitope, such as a CTL epitope of Gag.
Expression Control Sequences: Nucleic acid sequences that regulate the expression of a heterologous nucleic acid sequence to which it is operatively linked. Expression control sequences are operatively linked to a nucleic acid sequence when the expression control sequences control and regulate the transcription and, as appropriate, translation of the nucleic acid sequence. Thus, expression control sequences can include appropriate promoters, enhancers, transcription terminators, a start codon (typically, ATG) in front of a protein-encoding gene, splicing signal for introns, maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons. The term “control sequences” is intended to include, at a minimum, components whose presence can influence expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.
Glycoprotein 41 (gp41): An HIV-1 envelope glycoprotein that mediates receptor binding and HIV entry into a cell. Gp41 includes a membrane proximal region (MPR or MPER) and a transmembrane spanning domain. Gp41 is immunogenic and induces a variety of neutralizing antibodies, such as neutralizing antibodies directed to 2F5, 4E10 and Z13. These three gp41 neutralizing antibodies recognize the MPER of the HW-1 gp41 glycoprotein.
Gp41 antigenic insert: A peptide fragment that includes a MPER of gp41 and a transmembrane spanning region of gp41. In an example, the MPER (also referred to as the antigenic polypeptide fragment) of gp41 includes the amino acid sequence of SEQ ID NO: 1 and a transmembrane spanning region of gp41 including the amino acid sequence set forth as SEQ ID NO: 25 (X4FIMIVGGLX5GLRIVFTX6LSIV, X1, X2 and X3 are any amino acid and X4, X5, and X6 are any hydrophobic amino acid). For example, the antigenic polypeptide fragment of gp41 is between 16 and 150 amino acids in length (such as 28 and 150 amino acids in length) and the transmembrane spanning region of gp41 is between 22 and 40 amino acids in length and wherein the transmembrane spanning region of gp41 is C-terminal to the antigenic polypeptide fragment of gp41.
Glycoprotein 120 (gp120): An envelope protein from Human Immunodeficiency Virus (HIV). The envelope protein is initially synthesized as a longer precursor protein of 845-870 amino acids in size, designated gp160. Gp160 forms a homotrimer and undergoes glycosylation within the Golgi apparatus. It is then cleaved by a cellular protease into gp120 and gp41. Gp41 contains a transmembrane domain and remains in a trimeric configuration; it interacts with gp120 in a non-covalent manner. Gp120 contains most of the external, surface-exposed, domains of the envelope glycoprotein complex, and it is gp120 which binds both to the cellular CD4 receptor and to the cellular chemokine receptors (such as CCR5).
Mature gp120 wildtype polypeptides have about 500 amino acids in the primary sequence. Gp120 is heavily N-glycosylated (approximately 20 to 25 sites) giving rise to an apparent molecular weight of 120 kD. Exemplary sequence of wt gp160 polypeptides are shown on GENBANK®, for example accession numbers AAB05604 and AAD12142 incorporated herein by reference in their entirety as available on Oct. 16, 2009.
The gp120 core has a unique molecular structure, which comprises two domains: an “inner” domain (which faces gp41) and an “outer” domain (which is mostly exposed on the surface of the oligomeric envelope glycoprotein complex). The two gp120 domains are separated by a “bridging sheet” that is not part of either domain. The gp120 core comprises 25 beta strands, 5 alpha helices, and 10 defined loop segments. The 10 defined loop segments include five conserved regions (C1-C5) and five regions of high variability (V1-V5).
Gp120 polypeptides also include “gp120-derived molecules” which encompasses analogs (non-protein organic molecules), derivatives (chemically functionalized protein molecules obtained starting with the disclosed protein sequences) or mimetics (three-dimensionally similar chemicals) of the native gp120 structure, as well as proteins sequence variants (such as mutants, for example deletions, such as loop deletions, insertions or point mutation in any combination), genetic alleles, fusions proteins of gp120, or combinations thereof.
The numbering used in gp120 polypeptides disclosed herein is relative to the HXB2 numbering scheme as set forth in Numbering Positions in HIV Relative to HXB2CG Bette Korber et al., Human Retroviruses and AIDS 1998: A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences. Korber B, Kuiken C L, Foley B, Hahn B, McCutchan F, Mellors J W, and Sodroski J, Eds. Theoretical Biology and Biophysics Group, Los Alamos National Laboratory, Los Alamos, N. Mex. which is incorporated by reference herein in its entirety.
As used herein, a variant gp120 polypeptide is a gp120 polypeptide in which one or more amino acids have been altered (e.g., deleted or substituted). In one example, a variant gp120 polypeptide is a gp120 polypeptide in which at least 8 consecutive residues, such as 9, 10, 11 or 12 consecutive residues, of the fourth conserved loop (C4) between residues 419 and 434 of gp120 of SEQ ID NO: 63 has been deleted. In a particular example, a variant gp120 polypeptide includes a gp120 polypeptide in which residues 424-432 are deleted. Additional variant gp120 polypeptides include deletions of INMWQKVGK (residues 424-432 of SEQ ID NO:63), INMWQKVGKA (residues 424-433 of SEQ ID NO: 63), INMWQKVGKAM (residues 424-434 of SEQ ID NO: 63), RIKQIINMWQKVGK (residues 419-432 of SEQ ID NO: 63), IKQIINMWQKVGK (residues 420-432 of SEQ ID NO: 63), KQIINMWQKVGK (residues 421-432 of SEQ ID NO: 63), QIINMWQKVGK (residues 422-432 of SEQ ID NO: 63), IINMWQKVGK (residues 423-432 of SEQ ID NO: 63). In other embodiments, variant gp120 polypeptides include combinations of the amino and carboxyl ends between residues 419 and 434.
Any of the disclosed variant gp120 polypeptide including deletions in C4 can also include a deletion in the V1V2 loop region (with an amino acid sequence set forth in SEQ ID NO: 63); see SR Pollard and DC Wiley, EMBO J. 11:585-91, 1992 which is hereby incorporated by reference in its entirety.
In some examples, an antigenic insert includes a gp120 polypeptide with an amino acid sequence set forth by SEQ ID NOs: 152, 153, 154, 155, 156, 158, 159 or 160.
Group-specific antigen (Gag): A gene which encodes core structural proteins of HIV or SIV. In particular, Gag contains approximately 1500 nucleotides and encodes four separate proteins (capsid protein (p24), matrix protein (p17), nucleocapsid (p9) and p6) which form the building blocks for the viral core. Gag forms a spherical shell underlying the membrane of an immature viral particle. After proteolytic maturation of Gag, the capsid (CA) domain of Gag reforms into a conical shell enclosing the RNA genome. This mature shell contains 1,000-1,500 CA proteins assembled into a hexameric lattice with a spacing of 10 nm. Exemplary nucleic acid and amino acid sequences are known to those of skill in the art including the amino acid sequences provided for Gag herein.
Hepatitis B Surface Antigen (HBsAg): HBsAg is composed of 3 polypeptides, preS1, preS2 and S that are produced from alternative translation start sites. The surface proteins have many functions, including attachment and penetration of the virus into hepatocytes at the beginning of the infection process. The surface antigen is a principal component of the hepatitis B envelope. HBsAg has four membrane spanning domains. Exemplary nucleic acid and amino acid sequences are known to those of skill in the art and are shown on GENBANK®, for example accession numbers NM_001166119, NM_001130714, NM_001130713, NM_016269, BAF48754, BAF48753, BAF48752, BAF48751, AAA35977, AAA35976, AAA35975, AAA35974 and AAA35973 all of which are incorporated herein by reference in their entirety as available on Oct. 16, 2009.
As used herein, a variant HBsAg can include natural variants or recombinant variants such as a HBsAg that includes a MPER from gp41. In a particular example, a variant HBsAg includes a MPER and a membrane spanning domain from gp41.
Heterologous antigenic insert: An insert with an antigenic sequence that is not normally (in the wild-type sequence) found adjacent to a second sequence. In one embodiment, the antigenic insert is from a different genetic source, such as a virus or organism, than the second sequence. In one particular example, the antigenic insert is an HIV envelope protein or an HIV Gag protein. For example, the heterologous antigenic insert is a MPER of the HIV-1 gp41 glycoprotein. In other examples, the heterologous antigenic insert is not a rubella structural protein, such as a capsid.
Host cells: Cells in which a polynucleotide, for example, a polynucleotide vector or a viral vector, can be propagated and its DNA expressed. The cell may be prokaryotic or eukaryotic. The term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. However, such progeny are included when the term “host cell” is used.
Human Immunodeficiency Virus (HIV): A virus, known to cause AIDS, which includes HIV-1 and HIV-2. HIV-1 is composed of two copies of single-stranded RNA enclosed by a conical capsid including the viral protein p24, typical of lentiviruses. The capsid is surrounded by a plasma membrane of host-cell origin.
The RNA genome has at least three genes, gag, pol, and env, which contain information needed to make the structural proteins for new virus particles. The envelope protein of HIV-1 is made up of a glycoprotein called gp160. The mature, virion associated envelope protein is a trimeric molecule composed of three gp120 and three gp41 subunits held together by weak noncovalent interactions. This structure is highly flexible and undergoes substantial conformational changes upon gp120 binding with CD4 and chemokine coreceptors, which leads to exposure of the fusion peptides of gp41 that insert into the target cell membrane and mediate viral entry. Following oligomerization in the endoplasmic reticulum, the gp160 precursor protein is cleaved by cellular proteases and is transported to the cell surface. During the course of HIV-1 infection, the gp120 and gp41 subunits are shed from virions and virus-infected cells due to the noncovalent interactions between gp120 and gp41 and between gp41 subunits.
The HIV-1 envelope glycoproteins (gp120-gp41), which mediate receptor binding and entry, are major targets for neutralizing antibodies. Although the envelope glycoproteins are immunogenic and induce a variety of antibodies, the neutralizing antibodies that are induced are strain-specific, and the majority of the immune response is diverted to non-neutralizing determinants. Broadly neutralizing monoclonal antibodies have been isolated only rarely from natural HIV infection. For example, only three gp41-directed neutralizing antibodies (2F5, 4E10 and Z13) and a few gp120-directed neutralizing antibodies have been identified to date.
Immune response: A response of a cell of the immune system, such as a B cell, T cell, or monocyte, to a stimulus. In some cases, the response is specific for a particular antigen (that is, an “antigen-specific response”). In some cases, an immune response is a T cell response, such as a CD4+ response or a CD8+ response. Alternatively, the response is a B cell response, and results in the production of specific antibodies. For purposes of the present invention, a “humoral immune response” refers to an immune response mediated by antibody molecules, while a “cellular immune response” is one mediated by T-lymphocytes and/or other white blood cells. A “protective immune response” is an immune response that inhibits a detrimental function or activity (such as a detrimental effect of a pathogenic organism such as a virus), reduces infection by a pathogenic organism (such as, a virus), or decreases symptoms that result from infection by the pathogenic organism. A protective immune response can be measured, for example, by the inhibition of viral replication or plaque formation in a plaque reduction assay or ELISA-neutralization assay (NELISA), or by measuring resistance to viral challenge in vivo.
An immunogenic composition can induce a B cell response. The ability of a particular antigen to stimulate a B cell response can be measured by determining if antibodies are present that bind the antigen. In one example, neurtralizing antibodies are produced.
One aspect of cellular immunity involves an antigen-specific response by cytolytic T-cells (“CTL”s). CTLs have specificity for peptide antigens that are presented in association with proteins encoded by the major histocompatibility complex (MHC) and expressed on the surface of cells. CTLs help induce and promote the destruction of intracellular microbes, or the lysis of cells infected with such microbes. Another aspect of cellular immunity involves an antigen-specific response by helper T-cells. Helper T-cells act to help stimulate the function, and focus the activity of, nonspecific effector cells against cells displaying peptide antigens in association with MHC molecules on their surface. A “cellular immune response” also refers to the production of cytokines, chemokines and other such molecules produced by activated T-cells and/or other white blood cells, including those derived from CD4+ and CD8+ T-cells.
The ability of a particular antigen to stimulate a cell-mediated immunological response may be determined by a number of assays, such as by lymphoproliferation (lymphocyte activation) assays, CTL cytotoxic cell assays, or by assaying for T-lymphocytes specific for the antigen in a sensitized subject. Such assays are well known in the art. See, for example, Erickson et al. (1993) J. Immunol. 151:4189-4199; Doe et al. (1994) Eur. J. Immunol. 24:2369-2376. Recent methods of measuring cell-mediated immune response include measurement of intracellular cytokines or cytokine secretion by T-cell populations, or by measurement of epitope specific T-cells (for example, by the tetramer technique) (reviewed by McMichael and O'Callaghan (1998) J. Exp. Med. 187(9)1367-1371; Mcheyzer-Williams et al. (1996) Immunol. Rev. 150:5-21; Lalvani et al. (1997) J. Exp. Med. 186:859-865).
Thus, an immunological response as used herein may be one which stimulates the production of CTLs, and/or the production or activation of helper T-cells. The antigen of interest may also elicit an antibody-mediated immune response. Hence, an immunological response may include one or more of the following effects: the production of antibodies by B-cells; and/or the activation of suppressor T-cells and/or gamma-delta T-cells directed specifically to an antigen or antigens present in the composition or vaccine of interest. These responses may serve to neutralize infectivity, and/or mediate antibody-complement, or antibody dependent cell cytotoxicity (ADCC) to provide protection to an immunized host. Such responses can be determined using standard immunoassays and neutralization assays, well known in the art.
Immunogenic composition: A composition comprising at least one epitope of a virus, or other pathogenic organism, that induces a measurable CTL response, or induces a measurable B cell response (for example, production of antibodies that specifically bind the epitope). It further refers to isolated nucleic acids encoding an immunogenic epitope of virus or other pathogen that can be used to express the epitope (and thus be used to elicit an immune response against this polypeptide or a related polypeptide expressed by the pathogen). For in vitro use, the immunogenic composition may consist of the isolated nucleic acid, protein or peptide. For in vivo use, the immunogenic composition will typically include the nucleic acid, protein or peptide in pharmaceutically acceptable carriers or excipients, and/or other agents, for example, stabilizers. An immunogenic polypeptide (such as an antigenic polypeptide), or nucleic acid encoding the polypeptide, can be readily tested for its ability to induce a CTL or antibody response by art-recognized assays.
Immunogenic peptide: A peptide which comprises an allele-specific motif or other sequence such that the peptide will bind an MHC molecule and induce a cytotoxic T lymphocyte (“CTL”) response, or a B cell response (e.g. antibody production) against the antigen from which the immunogenic peptide is derived.
Inhibiting an infection: Inhibiting infection by a pathogen such as a virus, such as a lentivirus, or other virus, refers to inhibiting the full development of a disease either by avoiding initial infection or inhibiting development of the disease process once it is initiated. For example, inhibiting a viral infection refers to lessening symptoms resulting from infection by the virus, such as preventing the development of symptoms in a person who is known to have been exposed to the virus, or to lessening virus number or infectivity of a virus in a subject exposed to the virus.
Isolated: An “isolated” biological component (such as a nucleic acid or protein or organelle) has been substantially separated or purified away from other biological components in the cell of the organism in which the component naturally occurs, for example, other chromosomal and extra-chromosomal DNA and RNA, proteins and organelles. Nucleic acids and proteins that have been “isolated” include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.
Label: A detectable compound or composition that is conjugated directly or indirectly to another molecule to facilitate detection of that molecule. Specific, non-limiting examples of labels include fluorescent tags, affinity tags, enzymatic linkages, and radioactive isotopes. An affinity tag is a peptide or polypeptide sequence capable of specifically binding to a specified substrate, for example, an organic, non-organic or enzymatic substrate or cofactor. A polypeptide including a peptide or polypeptide affinity tag can typically be recovered, for example, purified or isolated, by means of the specific interaction between the affinity tag and its substrate. An exemplary affinity tag is a poly-histidine (e.g., six-histidine) affinity tag which can specifically bind to non-organic metals such as nickel and/or cobalt. Additional affinity tags are well known in the art.
Linking peptide: A linking peptide (or linker sequence) is an amino acid sequence that covalently links two polypeptide domains. Linking peptides can be included between a polypeptide and an antigenic epitope to provide rotational freedom to the linked polypeptide domains and thereby to promote proper domain folding. Linking peptides, which are generally between 2 and 25 amino acids in length, are well known in the art and include, but are not limited to the amino acid sequences glycine-proline-glycine-proline (GPGP)(SEQ ID NO: 37) and glycine-glycine-serine (GGS), as well as the glycine(4)-serine spacer described by Chaudhary et al., Nature 339:394-397, 1989. In some cases multiple repeats of a linking peptide are present.
Lymphocytes: A type of white blood cell that is involved in the immune defenses of the body. There are two main types of lymphocytes: B cells and T cells. “T lymphocytes” or “T cells” are non-antibody producing lymphocytes that constitute a part of the cell-mediated arm of the immune system. T cells arise from immature lymphocytes that migrate from the bone marrow to the thymus, where they undergo a maturation process under the direction of thymic hormones. Here, the mature lymphocytes rapidly divide increasing to very large numbers. The maturing T cells become immunocompetent based on their ability to recognize and bind a specific antigen. Activation of immunocompetent T cells is triggered when an antigen binds to the lymphocyte's surface receptors. T cells include, but are not limited to, CD4+ T cells and CD8+ T cells. A CD4+ T lymphocyte is an immune cell that carries a marker on its surface known as “cluster of differentiation 4” (CD4). These cells, also known as helper T cells, help orchestrate the immune response, including antibody responses as well as killer T cell responses. CD8+ T cells carry the “cluster of differentiation 8” (CD8) marker. In one embodiment, a CD8 T cell is a cytotoxic T lymphocyte. In another embodiment, a CD8 cell is a suppressor T cell.
Membrane proximal region (MPER) of gp41: A region that is immediately N-terminal of the transmembrane region of gp41. The MPER is also known and referred to herein as the MPER (Membrane proximal external region). The MPER is highly hydrophobic (50% of residues are hydrophobic) and is highly conserved across many HIV clades (Zwick, M. B., et al., J Virol, 75 (22): p. 10892-905, 2001). The conserved MPER of HIV-1 gp41 is a target of two broadly neutralizing human monoclonal antibodies, 2F5 and 4E10. The core of the 2F5 epitope has been shown to be ELDKWAS (SEQ ID NO: 35). With this epitope, the residues D, K, and W were found to be involved in recognition by 2F5. The core of the 4E10 epitope, NWFDIT (SEQ ID NO: 36), maps just C-terminal to the 2F5 epitope on the gp41 ectodomain.
Oligonucleotide: A linear polynucleotide sequence of up to about 100 nucleotide bases in length.
Open reading frame (“ORF”): A series of nucleotide triplets (codons) coding for amino acids without any internal termination codons. These sequences are usually translatable into a polypeptide (peptide or protein). In one example, an open reading frame is a rubella non-structural protein open reading frame, such as one coding amino acids that include two NotI restriction enzyme sites. In other examples, an open reading frame is a rubella structural protein open reading frame.
Operatively linked: A first nucleic acid sequence is operatively linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operatively linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operatively linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame, for example, two polypeptide domains or components of a fusion protein.
Pharmaceutically acceptable carriers and/or pharmaceutically acceptable excipients: The pharmaceutically acceptable carriers or excipients of use are conventional. Remingtons: The Science and Practice of Pharmacy, University of the Sciences in Philadelphia, Lippincott Williams & Wilkins, Philadelphia, Pa., 21st Edition (2005), describes compositions and formulations suitable for pharmaceutical delivery of the constructs disclosed herein.
In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (e.g., powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
A “therapeutically effective amount” is a quantity of a composition used to achieve a desired effect in a subject. For instance, this can be the amount of the composition necessary to inhibit viral (or other pathogen) replication or to prevent or measurably alter outward symptoms of viral (or other pathogenic) infection. When administered to a subject, a dosage will generally be used that will achieve target tissue concentrations (for example, in lymphocytes) that has been shown to achieve an in vitro effect.
Polynucleotide: The term polynucleotide or nucleic acid sequence refers to a polymeric form of nucleotide at least 10 bases in length. A recombinant polynucleotide includes a polynucleotide that is not immediately contiguous with both of the coding sequences with which it is immediately contiguous (one on the 5′ end and one on the 3′ end) in the naturally occurring genome of the organism from which it is derived. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA) independent of other sequences. The nucleotides can be ribonucleotides, deoxyribonucleotides, or modified forms of either nucleotide. The term includes single- and double-stranded forms of DNA.
Polypeptide: Any chain of amino acids, regardless of length or post-translational modification (for example, glycosylation or phosphorylation), such as a protein or a fragment or subsequence of a protein. The term “peptide” is typically used to refer to a chain of amino acids of between 3 and 30 amino acids in length. For example an immunologically relevant peptide may be between about 7 and about 25 amino acids in length, e.g., between about 8 and about 10 amino acids.
In the context of the present disclosure, a polypeptide can be a fusion protein comprising a plurality of constituent polypeptide (or peptide) elements. Typically, the constituents of the fusion protein are genetically distinct, that is, they originate from distinct genetic elements, such as genetic elements of different organisms or from different genetic elements (genomic components) or from different locations on a single genetic element, or in a different relationship than found in their natural environment. Nonetheless, in the context of a fusion protein the distinct elements are translated as a single polypeptide. The term monomeric fusion protein (or monomeric fusion protein subunit) is used synonymously with such a single fusion protein polypeptide to clarify reference to a single constituent subunit where the translated fusion proteins assume a multimeric tertiary structure.
Probes and primers: A probe comprises an isolated nucleic acid attached to a detectable label or reporter molecule. Primers are short nucleic acids, preferably DNA oligonucleotides, for example, a nucleotide sequence of about 15 nucleotides or more in length. Primers may be annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, and then extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification of a nucleic acid sequence, for example, by the polymerase chain reaction (PCR) or other nucleic-acid amplification methods known in the art. One of skill in the art will appreciate that the specificity of a particular probe or primer increases with its length. Thus, for example, a primer comprising 20 consecutive nucleotides will anneal to a target with a higher specificity than a corresponding primer of only about 15 nucleotides. Thus, in order to obtain greater specificity, probes and primers may be selected that comprise 20, 25, 30, 35, 40, 50 or more consecutive nucleotides.
Promoter: A promoter is a minimal sequence sufficient to direct transcription. Also included are those promoter elements which are sufficient to render promoter-dependent gene expression controllable for cell-type specific, tissue-specific, or inducible by external signals or agents; such elements may be located in the 5′ or 3′ regions of the gene. Both constitutive and inducible promoters are included (see e.g., Bitter et al., Methods in Enzymology 153:516-544, 1987). For example, when cloning in bacterial systems, inducible promoters such as pL of bacteriophage lambda, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may be used. In one embodiment, when cloning in mammalian cell systems, promoters derived from the genome of mammalian cells (for example, metallothionein promoter) or from mammalian viruses (for example, the retrovirus long terminal repeat; the adenovirus late promoter; the vaccinia virus 7.5K promoter) can be used. Promoters produced by recombinant DNA or synthetic techniques may also be used to provide for transcription of the nucleic acid sequences.
Purified: The term “purified” does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified nucleic acid is one in which the nucleic acid is more enriched than the nucleic acid in its natural environment within a cell. Similarly, a purified peptide preparation is one in which the peptide or protein is more enriched than the peptide or protein is in its natural environment within a cell. In one embodiment, a preparation is purified such that the protein or peptide represents at least 50% (such as, but not limited to, 70%, 80%, 90%, 95%, 98% or 99%) of the total peptide or protein content of the preparation.
Recombinant: A recombinant nucleic acid is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence, for example, a polynucleotide encoding a fusion protein. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques.
Respiratory syncytial virus (RSV): A virus which is a leading cause of acute upper and lower respiratory tract infection in adults, young children and infants. Serological evidence indicates that in the western world approximately 95% of all children have been infected with RSV by the age of two and 100% of children have been exposed by the time they reach adulthood. In most cases the RSV infections will only cause minor upper respiratory illness with symptoms resembling that of the common cold. However, severe infection with the virus may result in bronchiolitis or pneumonia which may result in hospitalization or death. In a given year, around 91,000 infants are hospitalized with RSV infection in the United States. Infants who have been born prematurely or have a pre-existing lung disease are a high risk of severe infection and complications. These infections are responsible for 40 to 50% of hospitalizations for pediatric bronchiolitis and 25% of hospitalizations for pediatric pneumonia. Since the immune response to RSV infection is not protective, RSV infections reoccur throughout adulthood. In adults and older children, RSV infection has been associated with upper respiratory infection, tracheobronchitis, and otitis media. However, RSV in the institutionalized elderly can be more serious and is characterized by severe pneumonia and mortality rates of up to 20 and 78%, respectively. Adults with a previous history of heart or lung conditions are at a high risk for RSV infection. The infection has been linked to exacerbation of patients with chronic obstructive pulmonary disease. Significant mortality has been observed in immunocompromised patients, particularly those undergoing bone marrow transplantation.
RSV is a member of the order Mononegavira{umlaut over (ι)}es, which includes the non-segmented negative strand RNA viruses in the Families Paramyxoviridae, Rhandoviridae and Filoviridae. RSV of humans (often also termed RSV or HRSV) is a member of the Pneumovirus genus of the sub-family Pneumovirinae within the Family Paramyxoviridae. Based on genetic and antigenic variations in the structural proteins, RSV is classified into two subgroups, A and B (Mufson, M. et al., J. Gen. Virol. 66:2111-2124). Other members of the Pneumovirus genus include viruses such as bovine RSV (BRSV), ovine RSV (ORSV) and pneumonia virus of mice (PVM) amongst others. The sub-family Pneumovirinae also includes the genus Metapneumovirus which contains the human pathogen human metapneumovirus (hMPV).
hMPV causes respiratory illness ranging from mild upper respiratory symptoms to severe lower respiratory disease such as bronchiolitis and pneumonia. Depending on the patient population sampled, between 5 and 15% of respiratory infections in young children may be attributable to hMPV infection. hMPV is also associated with 12 to 50% of otitis media in children. In the Netherlands, 55% of tested individuals were seropositive for hMPV by age 2, and almost all individuals 5 years and older were seropositive.
In addition to the genome features described above, Family characteristics include a lipid envelope containing one or more glycoprotein species considered to be associated with attachment and entry of the host cell. Entry is considered to require a process by which the viral envelope fuses with the membrane of the host cell. Fusion of infected cells with, for example, their neighbors, can also result in the formation of fused multinucleate cells known as syncytia in some cases. The fusion process is believed to be glycoprotein mediated and is a feature shared with diverse enveloped viruses in other taxonomic groups. In the case of the Parctmyxoviridae viruses of all genera characteristically express a fusion glycoprotein (F) which mediates membrane fusion.
Rubella: A small, quasi-spherical, enveloped, nonsegmented, plus-strand RNA virus that is a member of the rubivirus genus of the togavirus family (Togaviridae). Molecular biology of rubella virus is summarized by Frey, T. K. in Adv. Virus Res. 44:69-160 (1994) which is hereby incorporated by reference in its entirety. The rubella virion (virus particle) includes a single-stranded RNA encapsidated in an icosahedral nucleocapsid surrounded by a lipid envelope. This virion has at least two RdRp nonstructural proteins (nsP150 and nsP90) and three structural proteins (Capsid (C), E2 and E1). Multiple copies of a viral protein, designated the C protein (molecular weight (MW)=32,000-38,000 daltons), make up the nucleocapsid. Two types of viral glycoprotein, designated E1 and E2 (MW=53,000-58,000 daltons and 42,000-48,000 daltons, respectively), are embedded in the envelope. The E2 glycoprotein has been further subdivided into two subgroups, designated E2a and E2b, by their ability to migrate differently when resolved by polyacrylamide gel electrophoresis. E1 is the viral hemagglutinin. Neutralizing epitopes have been found on both E1 and E2. In one example, MPER and other HIV antigenic determinants are linked to E2 and E1 to elicit similar neutralizing antibodies against HIV.
The rubella genome consists of RNA of positive polarity that is roughly 10,000 nucleotides long and is capped and polyadenylated. In infected cells, three viral RNA species are synthesized: the genomic RNA, which also is the mRNA for translation of the nonstructural proteins (whose function is in viral RNA synthesis) from a long open reading frame (ORF) at the 5′ end of the genome; a complementary genome-length RNA of minus polarity which is the template for synthesis of plus-strand RNA species; and a subgenomic (SG) RNA which is initiated internally and contains the sequences of the 3′-terminal one-third of the genome (3327 nucleotides) and serves as the mRNA for the translation of the structural proteins. The structural proteins are proteolytically processed from a polyprotein precursor during translation. The order of these proteins in the polyprotein is NH2-C-E2-E1-COOH.
Sequence identity: The similarity between amino acid (and polynucleotide) sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity); the higher the percentage, the more similar are the primary structures of the two sequences. In general, the more similar the primary structures of two amino acid sequences, the more similar are the higher order structures resulting from folding and assembly. However, the converse is not necessarily true, and polypeptides with low sequence identity at the amino acid level can nonetheless have highly similar tertiary and quaternary structures.
Methods of determining sequence identity are well known in the art. Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J. Mol. Biol. 48:443, 1970; Higgins and Sharp, Gene 73:237, 1988; Higgins and Sharp, CABIOS 5:151, 1989; Corpet et al., Nucleic Acids Research 16:10881, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988. Altschul et al., Nature Genet. 6:119, 1994, presents a detailed consideration of sequence alignment methods and homology calculations.
The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, Md.) and on the internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. A description of how to determine sequence identity using this program is available on the NCBI website on the internet.
Another indicia of sequence similarity between two nucleic acids is the ability to hybridize. The more similar are the sequences of the two nucleic acids, the more stringent the conditions at which they will hybridize. The stringency of hybridization conditions are sequence-dependent and are different under different environmental parameters. Thus, hybridization conditions resulting in particular degrees of stringency will vary depending upon the nature of the hybridization method of choice and the composition and length of the hybridizing nucleic acid sequences. Generally, the temperature of hybridization and the ionic strength (especially the Na+ and/or Mg+ concentration) of the hybridization buffer will determine the stringency of hybridization, though wash times also influence stringency. Generally, stringent conditions are selected to be about 5° C. to 20° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Conditions for nucleic acid hybridization and calculation of stringencies can be found, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001; Tijssen, Hybridization With Nucleic Acid Probes, Part I: Theory and Nucleic Acid Preparation, Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Ltd., NY, N.Y., 1993. and Ausubel et al. Short Protocols in Molecular Biology, 4th ed., John Wiley & Sons, Inc., 1999.
For purposes of the present disclosure, “stringent conditions” encompass conditions under which hybridization will only occur if there is less than 25% mismatch between the hybridization molecule and the target sequence. “Stringent conditions” may be broken down into particular levels of stringency for more precise definition. Thus, as used herein, “moderate stringency” conditions are those under which molecules with more than 25% sequence mismatch will not hybridize; conditions of “medium stringency” are those under which molecules with more than 15% mismatch will not hybridize, and conditions of “high stringency” are those under which sequences with more than 10% mismatch will not hybridize. Conditions of “very high stringency” are those under which sequences with more than 6% mismatch will not hybridize. In contrast nucleic acids that hybridize under “low stringency conditions include those with much less sequence identity, or with sequence identity over only short subsequences of the nucleic acid.
For example, a specific example of progressively higher stringency conditions is as follows: 2×SSC/0.1% SDS at about room temperature (hybridization conditions); 0.2×SSC/0.1% SDS at about room temperature (low stringency conditions); 0.2×SSC/0.1% SDS at about 42° C. (moderate stringency conditions); and 0.1×SSC at about 68° C. (high stringency conditions). One of skill in the art can readily determine variations on these conditions (e.g., Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989). Washing can be carried out using only one of these conditions, e.g., high stringency conditions, or each of the conditions can be used, e.g., for 10-15 minutes each, in the order listed above, repeating any or all of the steps listed. However, as mentioned above, optimal conditions will vary, depending on the particular hybridization reaction involved, and can be determined empirically.
Subject: Living multi-cellular vertebrate organisms, a category that includes both human and veterinary subjects, including human and non-human mammals.
Therapeutically active polypeptide: An agent, such as an epitope of a virus or other pathogen that causes induction of an immune response, as measured by clinical response (for example increase in a population of immune cells, increased cytolytic activity against the epitope). Therapeutically active molecules can also be made from nucleic acids. Examples of a nucleic acid based therapeutically active molecule is a nucleic acid sequence that encodes an epitope of a protein of a virus or other pathogen, wherein the nucleic acid sequence is operatively linked to a control element such as a promoter.
Therapeutically Effective Amount: An amount of a composition that alone, or together with an additional therapeutic agent(s) (for example nucleoside/nucleotide reverse transcriptase inhibitors, a non-nucleoside reverse transcriptase inhibitors, protease inhibitors, fusion/entry inhibitors or integrase inhibitors) induces the desired response (e.g., prevention, inhibition, or reduction in a virus, such as inhibition or reduction of viral infection or replication). In one example, a desired response is to inhibit or reduce HIV replication in a cell to which the therapy is administered. HIV replication does not need to be completely eliminated for the composition to be effective. For example, a composition can decrease HIV replication by a desired amount, for example by at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination of HIV), as compared to HIV replication in the absence of the composition.
In another example, a desired response is to inhibit HIV infection. The HIV infected cells do not need to be completely eliminated for the composition to be effective. For example, a composition can decrease the number of HIV infected cells by a desired amount, for example by at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination of detectable HIV infected cells), as compared to the number of HIV infected cells in the absence of the composition.
A therapeutically effective amount of a composition can be administered in a single dose, or in several doses, for example daily, during a course of treatment. However, the therapeutically effective amount can depend on the subject being treated, the severity and type of the condition being treated, and the manner of administration. For example, a therapeutically effective amount of such agent can vary from about 1 μg-10 mg per 70 kg body weight if administered intravenously.
Transformed or Transfected: A transformed cell is a cell into which a nucleic acid molecule has been introduced by molecular biology techniques. As used herein, the term introduction or transformation encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, including transfection with viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration.
Transmembrane spanning region or membrane spanning domain of gp41: A region or domain of gp41 that is immediately C-terminal to the membrane proximal region of gp41. An example of a transmembrane spanning region is provided in SEQ ID NO: 25.
Treating a disease: “Treatment” refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or other pathological condition, such as an infection, for example a sign or symptom of HIV. Treatment can also induce remission or cure of a condition, such as elimination of detectable HIV infected cells. In particular examples, treatment includes preventing a disease, for example by inhibiting the full development of a disease, such as HIV, by inhibiting HIV replication or infection or the development of AIDS. Prevention of a disease does not require a total absence of disease. For example, a decrease of at least 50% can be sufficient.
Vaccine: A vaccine is a pharmaceutical composition that elicits a prophylactic or therapeutic immune response in a subject. In some cases, the immune response is a protective immune response. Typically, a vaccine elicits an antigen-specific immune response to an antigen of a pathogen, for example, a bacterial or viral pathogen, or to a cellular constituent correlated with a pathological condition. A vaccine may include a polynucleotide, a peptide or polypeptide, a virus, a bacteria, a cell or one or more cellular constituents. In some cases, the virus, bacteria or cell may be inactivated or attenuated to prevent or reduce the likelihood of infection, while maintaining the immunogenicity of the vaccine constituent.
Vector: A nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell. A vector may include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector may also include one or more selectable marker gene and other genetic elements known in the art.
Virus: Microscopic infectious organism that reproduces inside living cells. A virus consists essentially of a core of a single nucleic acid surrounded by a protein coat, and has the ability to replicate only inside a living cell. “Viral replication” is the production of additional virus by the occurrence of at least one viral life cycle. A virus may subvert the host cells' normal functions, causing the cell to behave in a manner determined by the virus. For example, a viral infection may result in a cell producing a cytokine, or responding to a cytokine, when the uninfected cell does not normally do so.
“Retroviruses” are RNA viruses wherein the viral genome is RNA. When a host cell is infected with a retrovirus, the genomic RNA is reverse transcribed into a DNA intermediate which is integrated very efficiently into the chromosomal DNA of infected cells. The integrated DNA intermediate is referred to as a provirus. The term “lentivirus” is used in its conventional sense to describe a genus of viruses containing reverse transcriptase. The lentiviruses include the “immunodeficiency viruses” which include human immunodeficiency virus (HIV) type 1 and type 2 (HIV-1 and HIV-2), simian immunodeficiency virus (SIV), and feline immunodeficiency virus (FIV). HIV-1 is a retrovirus that causes immunosuppression in humans (HIV disease), and leads to a disease complex known as AIDS. “HIV infection” refers to the process in which HIV enters macrophages and CD4+ T cells by the adsorption of glycoproteins on its surface to receptors on the target cell followed by fusion of the viral envelope with the cell membrane and the release of the HIV capsid into the cell. “HIV disease” refers to a well-recognized constellation of signs and symptoms (including the development of opportunistic infections) in persons who are infected by an HIV virus, as determined by antibody or western blot studies. Laboratory findings associated with this disease are a progressive decline in T cells.
Virus-like particle or VLP: A nonreplicating, viral shell, derived from any of several viruses. VLPs are generally composed of one or more viral proteins, such as, but not limited to, those proteins referred to as capsid, coat, shell, surface and/or envelope proteins, or particle-forming polypeptides derived from these proteins. VLPs can form spontaneously upon recombinant expression of the protein in an appropriate expression system. Methods for producing particular VLPs are known in the art. The presence of VLPs following recombinant expression of viral proteins can be detected using conventional techniques known in the art, such as by electron microscopy, biophysical characterization, and the like. See, for example, Baker et al. (1991) Biophys. J. 60:1445-1456; Hagensee et al. (1994) J. Virol. 68:4503-4505. For example, VLPs can be isolated by density gradient centrifugation and/or identified by characteristic density banding. Alternatively, cryoelectron microscopy can be performed on vitrified aqueous samples of the VLP preparation in question, and images recorded under appropriate exposure conditions.
Rubella vectors expressing a variety of antigens, such as SIV, HIV, RSV or hepatitis antigens are demonstrated herein to be useful for evaluating new immunogens and for developing prime and boost strategies for eliciting protective immunity.
The inventors have discovered that rubella virus can be adapted for protein expression by making a permissive deletion in the nonstructural p150 gene (Not I deletion), to make room for the insert or by inserting positioning an insert in between two structural proteins within the rubella virus without making a deletion in the nonstructural p150 gene. The resulting rubella vectors replicate with kinetics similar to wild type virus, while stably expressing a reporter gene coding for Zoanthus sp. green fluorescent protein (zGFP) for at least 12 passages in cell culture. The inventors also report herein live rubella vectors expressing heterologous antigens, such as HIV and SIV vaccine antigens. These vectors express the HIV membrane-proximal external region (MPER) or SIV Gag antigens at either of two insertion sites: the Not I site or an insertion site in the structural gene region. The foreign antigens are controlled by early or late rubella promoters, depending on the insertion site. At the structural insertion site, HIV MPER was highly expressed as part of the structural polyprotein, processed to a free protein, and incorporated into virions. The vectors are based on rubella vaccine strain RA27/3 and grow to high titers in vitro. These vectors provide an ideal vaccine platform for testing vector replication and immunogenicity in vivo, as well as protection against SW or SHIV challenge.
In some embodiments, an isolated rubella viral vector construct includes a rubella non-structural protein ORF with an in-frame deletion, a rubella structural protein ORF, and a heterologous antigenic insert. In one example, the in-frame deletion within the rubella non-structural protein ORF is an in-frame deletion between two NotI restriction enzyme sites. In some examples, the heterologous antigenic insert is positioned within the rubella non-structural protein ORF. In other examples, the heterologous antigenic insert is positioned within the rubella structural protein ORF.
In some embodiments, an isolated rubella viral vector includes a rubella non-structural protein open reading frame (ORF) without an in-frame deletion, a rubella structural protein ORF, and a heterologous antigenic insert. In some examples, the heterologous antigenic insert is positioned within the rubella structural protein ORF. In other examples, the heterologous antigenic insert is positioned in between the genes encoding structural proteins E2 and E1.
In some examples, the antigenic insert comprises an amino acid sequence set forth as SEQ ID NO: 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 158, 159, 160, 161 or 162.
Exemplary antigenic inserts include an HIV antigenic insert (such as a Gag antigenic insert, a gp41 antigenic insert or a gp120 antigenic insert) or a hepatitis B antigenic insert. In some examples, a Gag antigenic insert includes an antigenic polypeptide fragment with an amino acid sequence provided by SEQ ID NOs: 82, 83, 84, 85, 86, 87, 88, 90, 91, 132, 133, 134, 135, 138, 139, 140, 141, or 142. In some examples, a gp41 antigenic insert includes an antigenic polypeptide fragment of gp41 with an amino acid sequence provided by SEQ ID NOs: 1-22, 30, 81 or 89 and a transmembrane region of gp41 with an amino acid sequence provided by SEQ ID NOs: 25-28. In certain examples, a gp120 antigenic insert includes an amino acid sequence set forth by SEQ ID NOs: 63, 66, 67, 69, 71, 73, 74, 152, 153, 154, 155, 156, 158, 159 or 160. For example, the gp120 antigenic insert includes a variant gp120 polypeptide comprising a deletion of at least 8 consecutive residues of the fourth conserved loop (C4) between residues 423 and 433 of SEQ ID NO: 63. In some examples, an antigenic insert includes one or more CTL epitopes, such as one or more CTL HIV or SIV epitopes, including those set forth as SEQ ID NOs: 77, 78, 79, 80, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, and 102.
Viral-like particles including the isolated viral vector construct are provided herein. Compositions comprising the viral-like particles are also provided.
Also disclosed are methods of using the disclosed isolated viral vectors to induce an immune response, such as a protective immune response, when introduced into a subject or to diagnose an HIV infection. For example, methods are provided for inhibiting HIV infection in a subject, for inducing an immune response to HIV in a subject, and for diagnosing HIV infection in a subject. Also disclosed are methods for measuring host range, testing sensitivity to neutralizing antibodies, or screening antiviral drugs, such as protease inhibitors.
A. Rubella Viral Vector Constructs
Disclosed herein are rubella viral vector constructs. In one example, an isolated rubella viral vector construct is disclosed that includes a rubella non-structural ORF with an in-frame deletion, a rubella structural protein ORF, and a heterologous antigenic insert. In some examples, an isolated rubella viral vector includes a sub genomic promoter. For example, the sub genomic promoter can control the expression of the structural proteins.
In one example, an in-frame deletion is within the rubella non-structural protein ORF. For example, the in-frame deletion is within nsP150. In one particular example, the in-frame deletion is an in-frame deletion between two NotI restriction enzyme sites located in nsP150, such as between base pairs (bp) 1685 and 2192. For example, the heterologous antigenic insert can be positioned within the rubella non-structural protein ORF. For example, the heterologous antigenic insert is positioned into nsP150, such as into the site of the Not I deletion (see
In other examples, the heterologous antigenic insert is positioned at either end of the three rubella structural proteins, capsid (C), E2 and E1 in a rubella viral construct that does not include a deletion, such as a Not I deletion, in the non-structural protein ORF.
In some embodiments of this example, the heterologous antigenic insert is positioned in between the genes encoding structural proteins C, E2 and E1. For example, the heterologous antigenic insert is positioned in between the genes encoding structural protein E2 and E1.
In some examples, the antigenic insert comprises an amino acid sequence set forth as SEQ ID NO: 77, 78, 79, 80, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 158, 159, 160, 161 or 162.
In one example, the heterologous antigenic insert sequence is expressed at the carboxyl end of rubella capsid protein without a deletion being made, such as an in-frame deletion within nsP150, in the non-structural protein ORF. For example, the heterologous antigenic insert sequence is expressed at the amino end of envelope protein E2 and no deletion is present, such as an in-frame deletion within nsP150, in the non-structural protein ORF. In another exemplary construct, the heterologous antigenic insert is expressed at the carboxyl end of E2 and no deletion is present, such as an in-frame deletion within nsP150, in the non-structural protein ORF. In another example, the heterologous antigenic insert sequence is expressed at the amino end of envelope protein E1.
In some examples, the MPER sequence is expressed at the carboxyl end of rubella capsid protein. In other examples, the MPER sequence is expressed at the amino end of envelope protein E2. In another exemplary construct, MPER is expressed at the carboxyl end of E2. In another example, the MPER sequence is expressed at the amino end of envelope protein E1.
In additional examples, a deletion is made within the P150 of the rubella construct, such as in the middle in a size comparable to the Not I deletion to allow for the insertion and expression of genes coding for heterologous antigens, such as one or more HIV envelope protein. In one particular example, a deletion comparable to the Not I deletion is present in the middle of the P150 of the rubella vaccine strain RA27/3. Various heterologous antigens, including any of those described herein are inserted into either the nonstructural ORF or structural ORF in this strain known to be safe and immunogenic in humans.
For use, the disclosed vector constructs are chemically introduced into susceptible culture cells, for example, E. coli, for amplification and production of large amounts of the cDNA clone by methods known to those of ordinary skill in the art, including chemical introduction. In one particular example, the purified infectious clone is digested with an restriction endonuclease such as EcoRI (New England Biolabs, Beverly, Mass.) for linearization at the termination of the rubella virus cDNA sequences. The linearized plasmid is then transcribed in vitro with an RNA polymerase such as SP6 RNA polymerase, which results in production of RNA transcripts. The resulting RNA transcripts are used to transfect the cells by transfection procedures known to those skilled in the art. The cells, in turn, will produce both the native structural proteins of the rubella virus and the protein encoded by the foreign gene (such as HIV antigens, SIV antigens or HBsAgs). The replication of the RNA sequences and the expression of the encoded protein by the cells may be monitored by various means known to the ones skilled in the art. In some examples, the cells will further produce recombinant virus particles which, in turn, may be used to infect cells or organisms.
The recombinant virus particles can be recovered in quantity using any purification process known to those of skill in the art, such as a nickel (NTA-agarose) affinity chromatography purification procedure. These particles can be combined with a pharmaceutically acceptable carrier to provide a safe, effective vaccine, such as a HIV, SIV, RSV or Hepatitis B vaccine. The carrier can be oil, water, saline, phosphate buffer, polyethylene glycol, glycerine, propylene glycol, and combinations thereof, or other vehicles routinely used by the pharmaceutical industry for these purposes (as described in detail below). The vaccine is usually provided in lyophilized form and therefore is free of preservatives.
The disclosed recombinant virus particles can also be used to identify antibodies, such as antibodies within a subject. The immunogenic compositions of this disclosure can be employed to generate antibodies that recognize the antigens disclosed herein and the antigen from which the disclosed antigen was derived. The methods include administering to a subject an immunogenic composition including a disclosed antigen or administering to the subject a polynucleotide encoding a disclosed antigen to generate antibodies that recognize the disclosed antigen. The subject employed in this embodiment is one typically employed for antibody production. Mammals, such as, rodents, rabbits, goats, sheep, etc., are preferred.
The antibodies generated can be either polyclonal or monoclonal antibodies. Polyclonal antibodies are raised by injecting (for example subcutaneous or intramuscular injection) antigenic polypeptides into a suitable animal (for example, a mouse or a rabbit). The antibodies are then obtained from blood samples taken from the animal. The techniques used to produce polyclonal antibodies are extensively described in the literature. Polyclonal antibodies produced by the subjects can be further purified, for example, by binding to and elution from a matrix that is bound with the polypeptide against which the antibodies were raised. Those of skill in the art will know of various standard techniques for purification and/or concentration of polyclonal, as well as monoclonal, antibodies. Monoclonal antibodies can also be generated using techniques known in the art.
i. Wildtype and Variant Gp41 Antigenic Inserts
In some examples, isolated rubella viral vectors disclosed herein include an antigenic insert that is a wildtype or variant gp41 polypeptide. In an example, an antigenic insert is a wildtype gp41 polypeptide or a fragment thereof. Exemplary sequence of wildtype gp41 polypeptides are shown on GENBANK®, for example accession number CAD23678 incorporated herein by reference in its entirety as available on Oct. 15, 2009. In other examples, a gp41 antigenic insert can include (a) an antigenic polypeptide fragment of gp41 and (b) a transmembrane spanning region of gp41.
In an example, the gp41 antigenic insert includes (a) an antigenic polypeptide fragment, such as an antigenic polypeptide fragment with the amino acid sequence set forth in SEQ ID NO:1 and is between 8 and 400 amino acids in length, including 10 and 300 amino acids in length, such as from about 10 to about 150, such as about 16 to about 160 amino acids, such as from about 28 to about 150 amino acids in length, such as from about 28 to about 140 amino acids in length, including 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 120, 125, 130, 135, 140, 141, 142, 143, 144 or 145 amino acids; and (b) a transmembrane spanning gp41 region, such as a transmembrane spanning gp41 region with the amino acid sequence set forth in SEQ ID NO: 25 (in which wherein X1, X2 and X3 are any amino acid; and X4, X5, and X6 are any hydrophobic amino acid) and is between 22 and 40 amino acids in length, such as about 23 and 38 amino acids in length, including 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 amino acids.
In one example, the antigenic polypeptide includes the amino acid sequence of NEX1X2LLX3LDKWASLWN (SEQ ID NO: 1). In this sequence, X1, X2 and X3 are any amino acid. The antigenic epitope can include repeats of this sequence, such as one to five copies of SEQ ID NO: 1. As noted above, the antigenic peptide includes one or more antigenic epitopes, such as one or more envelope proteins of HIV-1, and, including SEQ ID NO: 1, can be from about 10 to about 200 amino acids in length, such as from about 16 to about 160 amino acids, such as from about 28 to about 150 amino acids in length, such as from about 28 to about 140 amino acids in length, including 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 120, 125, 130, 135, 140, 141, 142, 143, 144 or 145 amino acids.
In several examples, the antigenic polypeptide includes one or more of the amino acid sequences set forth below:
In some examples, the antigenic polypeptide can include one of the amino acid sequences set forth as SEQ ID NOs: 2-22, 30, 81, 89, 126-131, 136 or 137. A single copy of one of SEQ ID NOs: 2-22, 30, 81, 89 or 137 can be included as the antigenic polypeptide. Alternatively, multiple copies of one of SEQ ID NOs: 2-22, 30, 81, 89, 126-131, 136 or 137 can be included as the antigenic polypeptide. Thus, one, two, three, four or five copies of one of the amino acid sequences set forth as SEQ ID NOs: 2-22, 30, 81, 89, 126-131, 136 or 137 can be included as the antigenic polypeptide.
In additional embodiments, more than one of these sequences can be included in the antigenic polypeptide. Thus, in several examples, two, three, four or five of the amino acid sequences set forth as SEQ ID NOs: 2-22, 30, 81, 89, 126-131, 136 or 137 can be included as the antigenic polypeptide in tandem. Each amino acid sequence included in the antigenic polypeptide can be present only a single time, or can be repeated.
In some embodiments, the transmembrane spanning gp41 region includes the amino acid sequence set forth in SEQ ID NO: 25. In this sequence, X1, X2 and X3 are any amino acid and X4, X5, and X6 are any hydrophobic amino acid and the transmembrane spanning gp41 region is between 22 and 40 amino acids in length, such as 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 amino acids. In several examples, the antigenic polypeptide includes one or more of the amino acid sequences set forth below:
A gp41 polypeptide can be covalently linked to a carrier, which is an immunogenic macromolecule to which an antigenic molecule can be bound. When bound to a carrier, the bound polypeptide becomes more immunogenic. Carriers are chosen to increase the immunogenicity of the bound molecule and/or to elicit higher titers of antibodies against the bound molecule which are diagnostically, analytically, and/or therapeutically beneficial. Covalent linking of a molecule to a carrier can confer enhanced immunogenicity and T cell dependence (see Pozsgay et al., PNAS 96:5194-97, 1999; Lee et al., J. Immunol. 116:1711-18, 1976; Dintzis et al., PNAS 73:3671-75, 1976). Useful carriers include polymeric carriers, which can be natural (for example, polysaccharides, polypeptides or proteins from bacteria or viruses), semi-synthetic or synthetic materials containing one or more functional groups to which a reactant moiety can be attached. Bacterial products and viral proteins (such as HBsAg and core antigen) can also be used as carriers, as well as proteins from higher organisms such as keyhole limpet hemocyanin, horseshoe crab hemocyanin, edestin, mammalian serum albumins, and mammalian immunoglobulins. Additional bacterial products for use as carriers include bacterial wall proteins and other products (for example, streptococcal or staphylococcal cell walls and lipopolysaccharide (LPS)).
ii. Wildtype and Variant Gp120 Antigenic Inserts
In some examples, isolated rubella viral vectors disclosed herein include an antigenic insert that is a wildtype or variant gp120 polypeptide. In an example, a wildtype gp120 polypeptide has an amino acid provided by SEQ ID NO: 63 or a fragment thereof. In other examples, a variant gp120 polypeptide includes a gp120 polypeptide in which at least 8 consecutive residues of the fourth conserved loop (C4) between residues 419 and 434 of gp120 according HXB2 numbering of SEQ ID NO: 63 are deleted. This deletion within the β20-21 loop of the gp120 polypeptide exposes the CD4 binding site thereby providing improved antibody binding and antibody induction. In one example, a variant gp120 polypeptide is a gp120 polypeptide in which at least 8 consecutive residues, such as between 8-12, 8-11, 8-10, or 8-9 (for example, 9, 10, 11 or 12) consecutive residues of C4 between residues 419 and 434 of gp120 of SEQ ID NO: 63 have been deleted.
In a particular example, a variant gp120 polypeptide includes a gp120 polypeptide in which residues 424-432 are deleted. Additional variant gp120 polypeptides include deletions of INMWQKVGK (residues 424-432 of SEQ ID NO: 63), INMWQKVGKA (residues 424-433 of SEQ ID NO: 63), INMWQKVGKAM (residues 424-434 of SEQ ID NO: 63), RIKQIINMWQKVGK (residues 419-432 of SEQ ID NO: 63), IKQIINMWQKVGK (residues 420-432 of SEQ ID NO: 63), KQIINMWQKVGK (residues 421-432 of SEQ ID NO: 63), QIINMWQKVGK (residues 422-432 of SEQ ID NO: 63), or IINMWQKVGK (residues 423-432 of SEQ ID NO: 63). In other embodiments, variant gp120 polypeptides include combinations of the amino and carboxyl ends between residues 419 and 434.
In some embodiments, a variant gp120 polypeptide does not include a variant in which residues 419-428 of SEQ ID NO: 63 are deleted. In other embodiments, a variant gp120 polypeptide does not include a variant in which residues 437-452 of SEQ ID NO: 63 are deleted.
Any of the disclosed variant gp120 polypeptide including deletions in C4 can also include a deletion in the V1V2 loop region (spanning from amino acids 125 to 205 of wild-type gp120, such as demonstrated in SEQ ID NO: 63); see SR Pollard and DC Wiley, EMBO J. 11:585-91, 1992 which is hereby incorporated by reference in its entirety.
The gp120 polypeptides disclosed herein can be chemically synthesized by standard methods, or can be produced recombinantly. An exemplary process for polypeptide production is described in Lu et al., Federation of European Biochemical Societies Letters. 429:31-35, 1998. They can also be isolated by methods including preparative chromatography and immunological separations.
Exemplary sequences for the amino acid sequence for full-length gp120 can be found on Genbank, EMBL and SwissProt websites. Exemplary non-limiting sequence information can be found for example, as SwissProt Accession No. P04578, (includes gp41 and gp120, initial entry Aug. 13, 1987, last modified on Jul. 15, 1999) and Genbank Accession No. AAF69493 (Oct. 2, 2000, gp120), all of which are incorporated herein by reference.
In other embodiments, the antigenic insert is a fusion protein. For example, fusion proteins are provided including a first and second polypeptide moiety in which one of the protein moieties includes a variant gp120 polypeptide such as a variant gp120 polypeptide with an amino acid sequence in which INMWQKVGK (residues 424-432 of SEQ ID NO:63), INMWQKVGKA (residues 424-433 of SEQ ID NO: 63), INMWQKVGKAM (residues 424-434 of SEQ ID NO: 63), RIKQIINMWQKVGK (residues 419-432 of SEQ ID NO: 63), IKQIINMWQKVGK (residues 420-432 of SEQ ID NO: 63), KQIINMWQKVGK (residues 421-432 of SEQ ID NO: 63), QIINMWQKVGK (residues 422-432 of SEQ ID NO: 63), or IINMWQKVGK (residues 423-432 of SEQ ID NO: 63) has been deleted. The other moiety is a heterologous protein such as a carrier protein and/or an immunogenic protein. Such fusions also are useful to evoke an immune response against gp120. In certain embodiments the gp120 polypeptides disclosed herein are covalent or non-covalent addition of toll like receptor (TLR) ligands or dendritic cell or B cell targeting moieties to produce self-adjuvanting proteins (e.g., IL-21).
In certain embodiments, a variant gp120 includes a V1V2 deletion without a beta 20-21 loop deletion with an amino acid sequence as set forth as:
In some embodiments, a variant gp120 includes a V1V2 deletion with a beta 20-21 loop deletion with an amino acid sequence as set forth as:
Sequences for deletion to generate gp120 variant with an amino acid sequence set forth in SEQ ID NO: 66 are shown in bold.
In other embodiments, a variant gp120 from a HIV isolate JRFL includes an amino acid sequence as set forth in SEQ ID NO: 67 and nucleic acid sequence set forth in SEQ ID NO: 68:
In other embodiments, a variant gp120 from a HIV isolate AD8 includes an amino acid sequence as set forth in SEQ ID NO: 69 or nucleic acid sequence set forth in SEQ ID NO: 70:
In other embodiments, a variant gp120 from a HIV isolate BaL includes an amino acid sequence as set forth in SEQ ID NO: 71 or a nucleic acid sequence as set forth in SEQ ID NO: 72:
In other embodiments, a variant gp120 from a HIV isolate IIIB includes an amino acid sequence as set forth in SEQ ID NO: 73 or a nucleic acid sequence as set forth in SEQ ID NO: 74:
A gp120 polypeptide can be covalently linked to a carrier, which is an immunogenic macromolecule to which an antigenic molecule can be bound. When bound to a carrier, the bound polypeptide becomes more immunogenic. Carriers are chosen to increase the immunogenicity of the bound molecule and/or to elicit higher titers of antibodies against the bound molecule which are diagnostically, analytically, and/or therapeutically beneficial. Covalent linking of a molecule to a carrier can confer enhanced immunogenicity and T cell dependence (see Pozsgay et al., PNAS 96:5194-97, 1999; Lee et al., J. Immunol. 116:1711-18, 1976; Dintzis et al., PNAS 73:3671-75, 1976). Useful carriers include polymeric carriers, which can be natural (for example, polysaccharides, polypeptides or proteins from bacteria or viruses), semi-synthetic or synthetic materials containing one or more functional groups to which a reactant moiety can be attached. Bacterial products and viral proteins (such as HBsAg and core antigen) can also be used as carriers, as well as proteins from higher organisms such as keyhole limpet hemocyanin, horseshoe crab hemocyanin, edestin, mammalian serum albumins, and mammalian immunoglobulins. Additional bacterial products for use as carriers include bacterial wall proteins and other products (for example, streptococcal or staphylococcal cell walls and lipopolysaccharide (LPS)).
iii. Wildtype and Variant Gag Antigenic Inserts
In some examples, isolated rubella viral vectors disclosed herein include an antigenic insert that is a wildtype or variant CTL epitope, such as a CTL epitope from HIV or SW. In some examples, a HIV or SIV CTL epitope is a CTL epitope of Gag or a fragment thereof. In some examples, the antigenic peptide includes one or more major CTL epitopes of Gag, and can be from about 5 to about 400 amino acids in length, including about 8 to about 350 amino acids in length, such from about 10 to about 280 amino acids in length, such as 20 to about 270 amino acids in length, such as from about 40 to about 250 amino acids in length, including 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 57, 60, 63, 65, 67, 70, 73, 75, 77, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349 or 350.
Exemplary sequences of wildtype CTL epitopes are publically available such as those provided on the World Wide Web addresshiv.lanl.gov/content/immunology/tables/ctl_summary which is incorporated herein by reference in its entirety as available on Oct. 15, 2010. In some examples, the antigenic peptide is more than 5 amino acids in length and is encoded by SIVagm Gag (1566 bp, GenBank Accession No. M30931), SIVmac239 gag (1533 bp, GenBank Accession No. M33262) or HIV-1 gag (1491 bp; CRF02_AG), each of which is hereby incorporated in reference in its entirety as of Apr. 6, 2012. In some examples, the antigenic peptide is more than 5 amino acids in length, such as from about 5 to about 400 amino acids in length, including about 8 to about 350 amino acids in length, such as from about 10 to about 280 amino acids in length, such as 20 to about 270 amino acids in length, such as from about 40 to about 250 amino acids in length, including 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 57, 60, 63, 65, 67, 70, 73, 75, 77, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349 or 350 amino acids and is encoded by SIVagm Gag (1566 bp, GenBank Accession No. M30931), SIVmac239 gag (1533 bp, GenBank Accession No. M33262), HIV-1 gag (1491 bp; CRF02_AG) or any of the SIV or HIV Gag sequences disclosed herein. In some examples, the antigenic peptide is between 135 and 180 amino acids and is encoded by SIVagm Gag (1566 bp, GenBank Accession No. M30931), SIVmac239 gag (1533 bp, GenBank Accession No. M33262) or HIV-1 gag (1491 bp; CRF02_AG). In some examples, the antigenic peptide has an amino acid sequence corresponding to amino acids 41-211 and/or amino acids 135-269 encoded by SIVmac239 gag (1533 bp, GenBank Accession No. M33262). In some examples, the antigenic peptide is approximately 250 amino acids and corresponds to p27 which is encoded by SIVmac239 gag (1533 bp, GenBank Accession No. M33262).
In some examples, a CTL epitope is a CTL epitope of Gag listed in Tables 1 and 2 below.
TRANSPTRR
NSPTRREL
PTRRELQVW
PTSRELQVW
AGAERQGTL
FSFPQITLW
In some examples, an antigenic polypeptide includes one or more of CTL epitopes of Gag, such as one or more of the epitopes listed in Table 1 or 2 or provided in present FIGS., including
In some examples, an antigenic polypeptide includes the amino acid sequence FQALSEGCTPYDINQMLNCVGDHQAAMQIIRDIINEEA (SEQ ID NO: 83) or REGSQKILSVLAPLVPTGSENLKSLYNTVSVIWSIHAED (SEQ ID NO: 82). For example, the isolated rubella viral vector includes a Gag antigenic insert including the amino acids FQALSEGCTPYDINQMLNCVGDHQAAMQIIRDIINEEA (SEQ ID NO: 83). In some examples, the Gag antigenic insert includes the amino acids LPLSPRTLNAWVKLIEEKKFGAEVVPG (residues 1 to 27 of SEQ ID NO: 84). In additional examples, the Gag antigenic insert includes the amino acids SQKILSVLAPL (residues 4-14 of SEQ ID NO: 82). In some examples, the antigenic insert includes a Gag antigenic insert, comprising the amino acids GSENLKSLYNT (residues 4-14 of SEQ ID NO: 88).
The antigenic epitope can include repeats of any one of these sequences, such as at least two repeats, such as between two to ten copies, such as three to five copies, such as one, two, three, four, five, six, seven, eight, nine or ten copies of SEQ ID NOs: 82-88 or combinations thereof (e.g., one or more copies of FQALSEGCTPYDINQMLNCVGDHQAAMQIIRDIINEEA (SEQ ID NO: 83); LPLSPRTLNAWVKLIEEKKFGAEVVPG (residues 1 to 27 of SEQ ID NO: 84). SQKILSVLAPL (residues 4-14 of SEQ ID NO: 82); or GSENLKSLYNT (residues 4-14 of SEQ ID NO: 88)).
In several examples, the antigenic polypeptide includes one or more of the amino acid sequences set forth below:
The antigenic polypeptide can include one of the amino acid sequences set forth as SEQ ID NOs: 82-88, 90 and 91. A single copy of one of SEQ ID NOs: 82-88, 90 and 91 can be included as the antigenic polypeptide. Alternatively, multiple copies of one of SEQ ID NOs: 82-88, 90 and 91 can be included as the antigenic polypeptide. Thus, one, two, three, four, five, six, seven, eight, nine or more copies of one of the amino acid sequences set forth as SEQ ID NOs: 82-88, 90 and 91 can be included as the antigenic polypeptide.
In additional embodiments, more than one of these sequences can be included in the antigenic polypeptide. Thus, in several examples, two, three, four or five of the amino acid sequences set forth as SEQ ID NOs: 82-88, 90 and 91, can be included as the antigenic polypeptide in tandem. Each amino acid sequence included in the antigenic polypeptide can be present only a single time, or can be repeated.
In some examples, an antigenic polypeptide includes an amino acid sequence set forth as
and is encoded by a nucleic acid sequence set forth as
An antigenic insert of a CTL epitope of a Gag polypeptide can be covalently linked to a carrier, which is an immunogenic macromolecule to which an antigenic molecule can be bound. When bound to a carrier, the bound polypeptide becomes more immunogenic. Carriers are chosen to increase the immunogenicity of the bound molecule and/or to elicit higher titers of antibodies against the bound molecule which are diagnostically, analytically, and/or therapeutically beneficial. Covalent linking of a molecule to a carrier can confer enhanced immunogenicity and T cell dependence (see Pozsgay et al., PNAS 96:5194-97, 1999; Lee et al., J. Immunol. 116:1711-18, 1976; Dintzis et al., PNAS 73:3671-75, 1976). Useful carriers include polymeric carriers, which can be natural (for example, polysaccharides, polypeptides or proteins from bacteria or viruses), semi-synthetic or synthetic materials containing one or more functional groups to which a reactant moiety can be attached. Bacterial products and viral proteins (such as HBsAg and core antigen) can also be used as carriers, as well as proteins from higher organisms such as keyhole limpet hemocyanin, horseshoe crab hemocyanin, edestin, mammalian serum albumins, and mammalian immunoglobulins. Additional bacterial products for use as carriers include bacterial wall proteins and other products (for example, streptococcal or staphylococcal cell walls and lipopolysaccharide (LPS)).
iv. Wildtype and Variant HBsAgs
In an example, a disclosed isolated rubella viral vector includes a wildtype or variant HBsAg. Suitable amino acid sequences for HBsAg are known in the art, and are disclosed, for example, in PCT Publication No. WO 2002/079217, which is incorporated herein by reference. Additional sequences for hepatitis B surface antigen can be found, for example, in PCT Publication No. 2004/113369 and PCT Publication No. WO 2004/09849. An exemplary HBsAg amino acid sequence, and the sequence of a nucleic acid encoding HBsAg, is shown in Berkower et al., Virology 321: 74-86, 2004, which is incorporated herein by reference in its entirety. An amino acid sequence of an exemplary HBsAg is set forth as follows:
Naturally occurring variants of HBsAg are found in other hepadnaviruses and also self assemble. These include: woodchuck hepatitis, ground squirrel hepatitis and duck hepatitis virus variants. Any of these naturally occurring HBsAg variants can be included within the disclosed constructs.
By itself, HBsAg assembles into approximately 22 nm virus-like particles. When expressed together with an HIV-1 antigenic epitope, the HBsAg fusion proteins assemble spontaneously and efficiently into virus-like particles (see Berkower et al., Virology 321: 75-86, 2004, which is incorporated herein by reference). Without being bound by theory, the multimeric form expresses the one or more antigenic epitopes at the lipid-water interface. These epitopes can be used to induce an immune response, such as to induce the production of neutralizing antibodies.
The preparation of HBsAg is well documented. See, for example, Harford et al. (1983) Develop. Biol. Standard 54:125; Greg et al. (1987) Biotechnology 5:479; EP-A-0 226 846; and EP-A-0 299 108.
Fragments and variants of HBsAgs as disclosed herein are fragments and variants that retain the ability to spontaneously assemble into virus-like particles. By “fragment” of an HBsAg is intended a portion of a nucleotide sequence encoding a HBsAg, or a portion of the amino acid sequence of the protein. By “homologue” or “variant” is intended a nucleotide or amino acid sequence sufficiently identical to the reference nucleotide or amino acid sequence, respectively.
It is recognized that the gene or cDNA encoding a polypeptide can be considerably mutated without materially altering one or more the polypeptide's functions. The genetic code is well known to be degenerate, and thus different codons encode the same amino acids. Even where an amino acid substitution is introduced, the mutation can be conservative and have no material impact on the essential functions of a protein (see Stryer, Biochemistry 4th Ed., W. Freeman & Co., New York, N.Y., 1995). Part of a polypeptide chain can be deleted without impairing or eliminating all of its functions. Sequence variants of a protein, such as a 5′ or 3′ variant, can retain the full function of an entire protein. Moreover, insertions or additions can be made in the polypeptide chain for example, adding epitope tags, without impairing or eliminating its functions (Ausubel et al., Current Protocols in Molecular Biology, Greene Publ. Assoc. and Wiley-Intersciences, 1998). Specific substitutions include replacing one or more transmembrane spanning domains of HBsAg with a gp41 transmembrane spanning domain, such as replacing the first domain and/or third domain of HBsAg with a gp41 transmembrane spanning domain. Other modifications that can be made without materially impairing one or more functions of a polypeptide include, for example, in vivo or in vitro chemical and biochemical modifications or the incorporation of unusual amino acids. Such modifications include, for example, acetylation, carboxylation, phosphorylation, glycosylation, ubiquitination, labeling, such as with radionucleides, and various enzymatic modifications, as will be readily appreciated by those well skilled in the art. A variety of methods for labeling polypeptides and labels useful for such purposes is well known in the art, and includes radioactive isotopes such as 125I or 3H, ligands that bind to or are bound by labeled specific binding partners (such as antibodies), fluorophores, chemiluminescent agents, enzymes, and antiligands or crosslinkers to produce dimers or multimers.
Functional fragments and variants of HBsAg include those fragments and variants that are encoded by nucleotide sequences that retain the ability to spontaneously assemble into virus-like particles. Functional fragments and variants can be of varying length. For example, a fragment may consist of 10 or more, 25 or more, 50 or more, 75 or more, 100 or more, or 200 or more amino acid residues of a HBsAg amino acid sequence.
A functional fragment or variant of HBsAg is defined herein as a polypeptide that is capable of spontaneously assembling into virus-like particles and/or self-aggregating into stable multimers. This includes, for example, any polypeptide six or more amino acid residues in length that is capable of spontaneously assembling into virus-like particles. Methods to assay for virus-like particle formation are well known in the art (see, for example, Berkower et al. (2004) Virology 321:75-86, herein incorporated by reference in its entirety).
“Homologues” or “variants” of a HBsAg are encoded by a nucleotide sequence sufficiently identical to a nucleotide sequence of hepatitis B surface antigen, examples of which are described above. By “sufficiently identical” is intended an amino acid or nucleotide sequence that has at least about 60% or 65% sequence identity, about 70% or 75% sequence identity, about 80% or 85% sequence identity, about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity over its full length as compared to a reference sequence, for example using the NCBI Blast 2.0 gapped BLAST set to default parameters. Alignment may also be performed manually by inspection. For comparisons of amino acid sequences of greater than about 30 amino acids, the Blast 2 sequences function is employed using the default BLOSUM62 matrix set to default parameters (gap existence cost of 11, and a per residue gap cost of 1). When aligning short peptides (fewer than around 30 amino acids), the alignment should be performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties). In one embodiment, the HBsAg protein is at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identical to the polypeptide set forth as SEQ ID NO: 31.
One or more conservative amino acid modifications can be made in the HBsAg amino acid sequence, whether an addition, deletion or modification, that does not substantially alter the 3-dimensional structure of the polypeptide. For example, a conservative amino acid substitution does not affect the ability of the HBsAg polypeptide to self-aggregate into stable multimers. HBsAg proteins having deletions of a small number of amino acids, for example, less than about 20% (such as less than about 18%, less than about 15%, less than about 10%, less than about 8%, less than about 5%, less than about 2%, or less than about 1%) of the total number of amino acids in the wild type HBsAg protein can also be included in the fusion proteins described herein. The deletion may be a terminal deletion, or an internal deletion, so long as the deletion does not substantially affect the structure or aggregation of the fusion protein.
In certain embodiments, a variant HBsAg can include a linker sequence. This peptide is a short amino acid sequence providing a flexible linker that permits attachment of an antigenic polypeptide, such as an HIV antigen (such as a gp41 or gp120 polypeptide), without disruption of the structure, aggregation (multimerization) or activity of the self-aggregating polypeptide component. Typically, a linear linking peptide consists of between two and 25 amino acids. Usually, the linear linking peptide is between two and 15 amino acids in length. In one example, the linker polypeptide is two to three amino acids in length, such as a serine and an arginine, or two serine residues and an arginine residue, or two arginine residues and a serine residue.
In other examples, the linear linking peptide can be a short sequence of alternating glycines and prolines, such as the amino acid sequence glycine-proline-glycine-proline. A linking peptide can also consist of one or more repeats of the sequence glycine-glycine-serine. Alternatively, the linear linking peptide can be somewhat longer, such as the glycine(4)-serine spacer described by Chaudhary et al., Nature 339:394-397, 1989.
Directly or indirectly adjacent to the remaining end of the linear linking peptide (that is, the end of the linear linking peptide not attached to the self-aggregating polypeptide component of the fusion protein) is a polypeptide sequence including at least one antigenic epitope of HIV-1, such as an epitope of gp41, such as at least one antigenic epitope of the membrane proximal region. The antigenic polypeptide can be a short peptide sequence including a single epitope. For example the antigenic polypeptide can be a sequence of amino acids as short as eight or nine amino acids, sufficient in length to provide an antigenic epitope in the context of presentation by a cellular antigen presenting complex, such as the major histocompatibility complex (MHC). The antigenic polypeptide can also be of sufficient in length to induce antibodies, such as neutralizing antibodies. Larger peptides, in excess of 10 amino acids, 20 amino acids or 30 amino acids are also suitable antigenic polypeptides, as are much larger polypeptides provided that the antigenic polypeptide does not disrupt the structure or aggregation of the HBsAg polypeptide component.
In some examples, the variant HBsAg includes one or more epitopes of the envelope protein of HIV-1 or major CTLs of HIV or SIV Gag, and is about 20 to about 200 amino acids in length, such as about 25 to about 150 amino acids in length, such as about 25 to about 100 amino acids in length. In several additional examples, the antigenic polypeptide includes one or more antigenic epitopes of HIV-1 gp41, such as the membrane proximal region (MPER) of gp41.
Exemplary sequences for HIV-1, as well as the amino acid sequence for full-length gp41 and gp120 and CTLs of Gag can be found on Genbank, EMBL and SwissProt websites. Exemplary non-limiting sequence information can be found for example, as SwissProt Accession No. P04578, (includes gp41 and gp120, initial entry Aug. 13, 1987, last modified on Jul. 15, 1999); Genbank Accession No. HIVHXB2CG (full length HIV-1, including RNA sequence and encoded proteins, Oct. 21, 2002); Genbank Accession No. CAD23678 (gp41, Apr. 15, 2005); Genbank Accession No. CAA65369 (Apr. 18, 2005); all of which are incorporated herein by reference. Similar information is available for HIV-2.
Suitable Env proteins are known in the art and include, for example, gp160, gp120, gp41, and gp140. Any clade of HIV is appropriate for antigen selection, including HIV clades A, B, C, and the like. HIV Gag, Pol, Nef and/or Env proteins from HIV clades A, B, C, as well as nucleic acid sequences encoding such proteins and methods for the manipulation and insertion of such nucleic acid sequences into vectors, are known (see, for example, HIV Sequence Compendium, Division of AIDS, National Institute of Allergy and Infectious Diseases, 2003, HIV Sequence Database (on the world wide web at hiv-web.lanl.gov/content/hiv-db/mainpage.html), Sambrook et al., Molecular Cloning, a Laboratory Manual, 2d edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1989, and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Association. Exemplary Env polypeptides, for example, corresponding to clades A, B and C are represented by the sequences of Genbank® Accession Nos. U08794, K03455 and AF286227, respectively.
Variant HBsAgs can form a self-aggregating multimeric spherical or rod-shaped structure upon expression in a host cell. Similarly, the variant HBsAgs can assemble spontaneously (self-aggregate) when placed in suspension in a solution of physiological pH (for example, a pH of about 7.0 to 7.6). Thus, in the present disclosure, wherever a single or monomeric variant HBsAg is disclosed, polymeric forms are also considered to be described.
In some embodiments, an isolated rubella viral vector includes a variant HBsAg with one or more transmembrane domains of the HBsAg replaced with a gp41 antigenic insert. The gp41 antigenic insert can include (a) an antigenic polypeptide fragment of gp41 and (b) a transmembrane spanning region of gp41. In an example, the gp41 antigenic insert includes (a) an antigenic polypeptide fragment, such as an antigenic polypeptide fragment with the amino acid sequence set forth in SEQ ID NO:1 and is between 28 and 350 amino acids in length and (b) a transmembrane spanning gp41 region, such as a transmembrane spanning gp41 region with the amino acid sequence set forth in SEQ ID NO: 25 (in which wherein X1, X2 and X3 are any amino acid; and X4, X5, and X6 are any hydrophobic amino acid) and is between 22 and 40 amino acids in length.
In one example, the antigenic polypeptide includes the amino acid sequence of NEX1X2LLX3LDKWASLWN (SEQ ID NO: 1). In this sequence, X1, X2 and X3 are any amino acid. The antigenic epitope can include repeats of this sequence, such as one to five copies of SEQ ID NO: 1. As noted above, the antigenic peptide includes one or more epitopes of the envelope protein of HIV-1, and, including SEQ ID NO: 1, about 10 to about 300 amino acids in length, such as from about 16 to about 160 amino acids, such as from about 28 to about 150 amino acids in length, such as from about 28 to about 140 amino acids in length.
In several examples, the antigenic polypeptide includes one or more of the amino acid sequences set forth in SEQ ID NOs: 2-22, 30, 81-102, 126-156 and 158-162. A single copy of one of SEQ ID NOs: 2-22, 30, 81-102, 126-156 and 158-162 can be included as the antigenic polypeptide. Alternatively, multiple copies of one of SEQ ID NOs: 2-22, 30, 81 or 89 can be included as the antigenic polypeptide. Thus, one, two, three, four or five copies of one of the amino acid sequences set forth as SEQ ID NOs: 2-22, 30, 81 or 89 can be included as the antigenic polypeptide.
In additional embodiments, more than one of these sequences can be included in the antigenic polypeptide. Thus, in several examples, two, three, four or five of the amino acid sequences set forth as SEQ ID NOs: 2-22, 30, 81 and 89 can be included as the antigenic polypeptide in tandem. Each amino acid sequence included in the antigenic polypeptide can be present only a single time, or can be repeated.
The HBsAg variants can include one or more transmembrane spanning domains that include one of the amino acid sequences set forth as SEQ ID NOs: 26-28. A single gp41 transmembrane can be included in the variant HBsAg. Alternatively, multiple gp41 transmembrane domains with amino acid sequences set forth as SEQ ID NOs: 26-28 can be included within the variant HBsAg. Thus, one, two, three, four or five gp41 transmembrane domains with one of the amino acid sequences set forth as SEQ ID NOs: 26-28 can be included in the variant HBsAg.
In one particular embodiment, an isolated rubella viral construct includes a variant HBsAg in which the first transmembrane spanning domain of the HBsAg is replaced by a gp41 antigenic insert. For example, the gp41 antigenic insert replaces at least the first 29 amino acid residues of SEQ ID NO:20, for example amino acid residues 1-35 of SEQ ID NO: 31. In another example, the gp41 antigenic insert replaces amino acid residues 1-32 of SEQ ID NO: 31. In yet another example, the gp41 antigenic insert replaces amino acid residues 1-29 of SEQ ID NO: 31. In a particular example, an isolated construct includes a variant HBsAg in which the first transmembrane spanning domain of the HBsAg is replaced by a gp41 antigenic insert that has the amino acid sequence set forth as SEQ ID NO: 29.
In another particular embodiment, an isolated construct includes a variant HBsAg in which the third transmembrane spanning domain of the HBsAg is replaced by a gp41 antigenic insert. For example, the gp41 antigenic insert replaces at least 29 amino acids residues of SEQ ID NO: 31, for example amino acid residues 150-190 of SEQ ID NO: 31. In another example, the gp41 antigenic insert replaces amino acid residues 153-187 of SEQ ID NO: 31. In yet another example, the gp41 antigenic insert replaces amino acid residues 156-185 of SEQ ID NO: 31. In a particular example, an isolated construct includes a variant HBsAg in which the third transmembrane spanning domain of the HBsAg is replaced by a gp41 antigenic insert has the amino acid sequence set forth as SEQ ID NO: 59.
In an even more particular embodiment, an isolated rubella viral construct includes a variant HBsAg in which more than one transmembrane spanning domains of HBsAg have been replaced with an antigenic insert. In one example, an isolated construct includes a variant HBsAg in which the first and the third transmembrane spanning domains of the HBsAg are replaced by a gp41 antigenic insert. For example, the gp41 antigenic insert replaces amino acid residues 1-35 and 150-190 of SEQ ID NO: 31. In another example, the gp41 antigenic insert replaces amino acid residues 1-32 and 153-187 of SEQ ID NO: 31. In yet another example, the gp41 antigenic insert replaces amino acid residues 1-29 and 156-185 of SEQ ID NO: 31. In a particular example, an isolated construct including a variant HBsAg in which the third transmembrane spanning domain of the HBsAg is replaced by a gp41 antigenic insert has the amino acid sequence set forth as:
In one example of an isolated construct, in which the first transmembrane domain of HBsAg is replaced with the MPER and transmembrane domain of gp41 has the amino acid sequence set forth as:
In one example of the isolated construct, the third transmembrane domain of HBsAg is replaced with the MPER and transmembrane domain of gp41 has the amino acid sequence set forth as:
In an example, an isolated construct is provided in which the first transmembrane domain and third domain of HBsAG is each replaced with the MPER and transmembrane domain of gp41 and has the amino acid sequence set forth as:
In one example, an isolated construct is provided in which the first transmembrane domain of HBsAg is replaced with the MPER and transmembrane domain of gp41 and an additional MPER is inserted just proximal to the third membrane spanning domain of HBsAg. In another example, an isolated construct is provided in which multiple MPERs are inserted within the HBsAg, such as two, three, four or more MPERs are inserted just proximal to the third membrane spanning domain of HBsAg. In yet another example, an isolated construct is provided in which a MPER and transmembrane domain of gp41 is inserted following the fourth HBsAg membrane spanning domain.
The variant HBsAg can optionally include additional elements, such as a leader sequence or a suitable T cell epitope. Generally, a T cell epitope is about eight to about ten amino acids in length, such as about nine amino acid in length, and binds major histocompatibility complex (MHC), such as HLA 2, for example, HLA 2.2. Examples of suitable T cell epitopes include, but are not limited to, ASLWNWFNITNWLWY (SEQ ID NO: 32) and IKLFIMIVGGLVGLR (SEQ ID NO: 33).
The variant HBsAg may also include a CAAX (SEQ ID NO: 34) sequence, for isoprenyl addition in vivo. In this sequence, C is cysteine, A is an aliphatic amino acid and X is any amino acid. The X residue determines which isoprenoid will be added to the cysteine. When X is a methionine or serine, the farnesyl-transferase transfers a farnesyl, and when X is a leucine or isoleucine, the geranygeranyl-transferase I transfers a geranylgeranyl group. In general, aliphatic amino acids have protein side chains containing only carbon or hydrogen atoms. Aliphatic amino acids include proline (P), glycine (G), alanine (A), valine (V), leucine (L), and isoleucine (I), presented in order from less hydrophobic to more hydrophobic. Although methionine has a sulphur atom in its side-chain, it is largely non-reactive, meaning that methionine effectively substitutes well with the true aliphatic amino acids.
B. Therapeutic Methods and Pharmaceutical Compositions
The disclosed isolated rubella viral vector constructs including antigenic inserts, such as HIV polypeptides (e.g., Gag, gp41 or gp120), RSV polypeptides or HBsAgs polypeptides (natural and recombinant) described herein can be used to produce pharmaceutical compositions, including compositions suitable for prophylactic and/or therapeutic administration. These compositions can be used to induce an immune response to HIV, SIV, RSV or Hepatitis B, such as a protective immune response. However, the compositions can also be used in various assays, such as in assays designed to detect an HIV-1 or Hepatitis B infection.
The disclosed isolated rubella viral constructs including can be administered to a subject in order to generate an immune response to HIV-1, SIV or Hepatitis B. In one example, the immune response is a protective immune response. Thus, the constructs disclosed herein can be used in a vaccine, such as a vaccine to inhibit subsequent infection with HIV, SIV or Hepatitis B. In some examples the disclosed constructs are administered as a virus like particle.
A therapeutically effective amount of a rubella viral construct, a virus-like particle including this construct, or a composition including the construct or virus-like particle can be administered to a subject to prevent, inhibit or to treat a condition, symptom or disease, such as AIDS. As such, the constructs can be administered as vaccines to prophylactically or therapeutically induce or enhance an immune response. For example, the pharmaceutical compositions described herein can be administered to stimulate a protective immune response against HIV, such as a HIV-1, SIV or Hepatitis B. In some examples, a disclosed composition is administered to a subject either alone or in combination with other HIV, SIV or Hepatitis B therapeutic agents. A single administration can be utilized to prevent or treat an HIV or Hepatitis B infection, or multiple sequential administrations can be performed.
In exemplary applications, compositions are administered to a subject infected with HIV or Hepatitis B, or likely to be exposed to an infection, in an amount sufficient to raise an immune response to HIV or Hepatitis B. Administration induces a sufficient immune response to reduce viral load, to prevent or lessen a later infection with the virus, or to reduce a sign or a symptom of HIV or Hepatitis B infection. Amounts effective for this use will depend upon various clinical parameters, including the general state of the subject's health, and the robustness of the subject's immune system, amongst other factors. A therapeutically effective amount of the compound is that which provides either subjective relief of one or more symptom(s) of HIV or Hepatitis B infection, an objectively identifiable improvement as noted by the clinician or other qualified observer, a decrease in viral load, an increase in lymphocyte count, such as an increase in CD4 cells, or inhibition of development of symptoms associated with infection. In one particular example, the administration of the composition will result in in vivo protein expression of the proteins encoded by the open reading frames contained in the expression vector construct. For example, the administration of the composition will result in the induction of immunity against the viruses whose proteins are encoded by the open reading frames.
The compositions can be administered by any means known to one of skill in the art (see Banga, A., “Parenteral Controlled Delivery of Therapeutic Peptides and Proteins,” in Therapeutic Peptides and Proteins, Technomic Publishing Co., Inc., Lancaster, Pa., 1995) such as by intramuscular, subcutaneous, or intravenous injection, but even oral, nasal, or anal administration is contemplated.
In some examples, the compositions are administered in a formulation including a carrier or excipient. A wide variety of suitable excipients are known in the art, including physiological phosphate buffered saline (PBS), and the like. Optionally, the formulation can include additional components, such as aluminum hydroxylphophosulfate, alum, diphtheria CRM197, or liposomes. To extend the time during which the peptide or protein is available to stimulate a response, the peptide or protein can be provided as an implant, an oily injection, or as a particulate system. The particulate system can be a microparticle, a microcapsule, a microsphere, a nanocapsule, or similar particle. A particulate carrier based on a synthetic polymer has been shown to act as an adjuvant to enhance the immune response, in addition to providing a controlled release. Aluminum salts may also be used to produce an immune response.
In a specific example, the composition is administered as a vaccine subcutaneously at a concentration range from 102 to 104 TCID50/person (TCID is an abbreviation for tissue culture infectious doses). For example, the vaccine is provided to the physician in a lyophilized form, reconstituted in an appropriate solvent such as deionized water or saline, and administered as a single injection.
In one embodiment, the construct is mixed with two or more of a stabilizing detergent, a micelle-forming agent, and an oil. Suitable stabilizing detergents, micelle-forming agents, and oils are detailed in U.S. Pat. No. 5,585,103; U.S. Pat. No. 5,709,860; U.S. Pat. No. 5,270,202; and U.S. Pat. No. 5,695,770, all of which are incorporated by reference. A stabilizing detergent is any detergent that allows the components of the emulsion to remain as a stable emulsion. Such detergents include polysorbate, 80 (TWEEN) (Sorbitan-mono-9-octadecenoate-poly(oxy-1,2-ethanediyl; manufactured by ICI Americas, Wilmington, Del.), TWEEN 40™, TWEEN 20™, TWEEN 60™, ZWITTERGENT™ 3-12, TEEPOL HB7™, and SPAN 85™. These detergents are usually provided in an amount of approximately 0.05 to 0.5%, such as at about 0.2%. A micelle forming agent is an agent which is able to stabilize the emulsion formed with the other components such that a micelle-like structure is formed. Such agents generally cause some irritation at the site of injection in order to recruit macrophages to enhance the cellular response. Examples of such agents include polymer surfactants described by BASF Wyandotte publications, for example, Schmolka, J. Am. Oil. Chem. Soc. 54:110, 1977; and Hunter et al., J. Immunol 129:1244, 1981, PLURONIC™ L62LF, L101, and L64, PEG1000, and TETRONIC™ 1501, 150R1, 701, 901, 1301, and 130R1. The chemical structures of such agents are well known in the art. In one embodiment, the agent is chosen to have a hydrophile-lipophile balance (HLB) of between 0 and 2, as defined by Hunter and Bennett, J. Immun. 133:3167, 1984. The agent can be provided in an effective amount, for example between 0.5 and 10%, or in an amount between 1.25 and 5%.
The oil included in the composition is chosen to promote the retention of the antigen in oil-in-water emulsion, such as to provide a vehicle for the desired antigen, and preferably has a melting temperature of less than 65° C. such that emulsion is formed either at room temperature (about 20° C. to 25° C.), or once the temperature of the emulsion is brought down to room temperature. Examples of such oils include squalene, Squalane, EICOSANE™, tetratetracontane, glycerol, and peanut oil or other vegetable oils. In one specific, non-limiting example, the oil is provided in an amount between 1 and 10%, or between 2.5 and 5%. The oil should be both biodegradable and biocompatible so that the body can break down the oil over time, and so that no adverse effects, such as granulomas, are evident upon use of the oil.
An adjuvant can be included in the composition. In one example, the adjuvant is a water-in-oil emulsion in which antigen solution is emulsified in mineral oil (such as Freund's incomplete adjuvant or montanide-ISA). In one embodiment, the adjuvant is a mixture of stabilizing detergents, micelle-forming agent, and oil available under the name PROVAX® (IDEC Pharmaceuticals, San Diego, Calif.). Other examples of suitable adjuvants are listed in the terms section of this specification.
Actual methods for preparing administrable compositions will be known or apparent to those skilled in the art and are described in more detail in such publications as Remington: The Science and Practice of Pharmacy, University of the Sciences in Philadelphia, Lippincott Williams & Wilkins, Philadelphia, Pa., 21st Edition (2005). The compositions can be administered, either systemically or locally, for therapeutic treatments, such as to treat an HIV infection. In therapeutic applications, a therapeutically effective amount of the composition is administered to a subject infected with HIV, such as, but not limited to, a subject exhibiting signs or symptoms of AIDS. Single or multiple administrations of the compositions can be administered depending on the dosage and frequency as required and tolerated by the subject. In one embodiment, the dosage is administered once as a bolus, but in another embodiment can be applied periodically until a therapeutic result is achieved. Generally, the dose is sufficient to treat or ameliorate symptoms or signs of the HIV infection without producing unacceptable toxicity to the subject.
Controlled release parenteral formulations can be made as implants, oily injections, or as particulate systems. For a broad overview of protein delivery systems, see Banga, Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, Technomic Publishing Company, Inc., Lancaster, Pa. (1995). Particulate systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles. Microcapsules contain the therapeutic protein as a central core. In microspheres, the therapeutic agent is dispersed throughout the particle. Particles, microspheres, and microcapsules smaller than about 1 μm are generally referred to as nanoparticles, nanospheres, and nanocapsules, respectively. Capillaries have a diameter of approximately 5 μm so that only nanoparticles are administered intravenously. Microparticles are typically around 100 μm in diameter and are administered subcutaneously or intramuscularly (see Kreuter, Colloidal Drug Delivery Systems, J. Kreuter, ed., Marcel Dekker, Inc., New York, N.Y., pp. 219-342 (1994); Tice & Tabibi, Treatise on Controlled Drug Delivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, N.Y., pp. 315-339 (1992)). In one example, virus like particles are in the range of 10-30 nm.
Polymers can be used for ion-controlled release. Various degradable and nondegradable polymeric matrices for use in controlled drug delivery are known in the art (Langer, Accounts Chem. Res. 26:537, 1993). For example, the block copolymer, polaxamer 407 exists as a viscous yet mobile liquid at low temperatures but forms a semisolid gel at body temperature. It has shown to be an effective vehicle for formulation and sustained delivery of recombinant interleukin-2 and urease (Johnston et al., Pharm. Res. 9:425, 1992; and Pec, J. Parent. Sci. Tech. 44(2):58, 1990). Alternatively, hydroxyapatite has been used as a microcarrier for controlled release of proteins (Ijntema et al., Int. J. Pharm. 112:215, 1994). In yet another aspect, liposomes are used for controlled release as well as drug targeting of the lipid-capsulated drug (Betageri et al., Liposome Drug Delivery Systems, Technomic Publishing Co., Inc., Lancaster, Pa., 1993). Numerous additional systems for controlled delivery of therapeutic proteins are known (e.g., U.S. Pat. No. 5,055,303; U.S. Pat. No. 5,188,837; U.S. Pat. No. 4,235,871; U.S. Pat. No. 4,501,728; U.S. Pat. No. 4,837,028; U.S. Pat. No. 4,957,735; and U.S. Pat. No. 5,019,369; U.S. Pat. No. 5,055,303; U.S. Pat. No. 5,514,670; U.S. Pat. No. 5,413,797; U.S. Pat. No. 5,268,164; U.S. Pat. No. 5,004,697; U.S. Pat. No. 4,902,505; U.S. Pat. No. 5,506,206; U.S. Pat. No. 5,271,961; U.S. Pat. No. 5,254,342; and U.S. Pat. No. 5,534,496).
C. Immunodiagnostic Reagents and Kits
In addition to the therapeutic methods provided above, any of the disclosed rubella viral constructs herein can be utilized to produce antigen specific immunodiagnostic reagents, for example, for serosurveillance. Immunodiagnostic reagents can be designed from any of the constructs including antigenic polypeptides described herein. For example, the presence of serum antibodies to HIV, SIV, RSV or Hepatitis B can be monitored using the isolated immunogens disclosed herein, such as to detect an HIV, SIV, RSV or Hepatitis B infection. Generally, the method includes contacting a sample from a subject, such as, but not limited to a blood, serum, plasma, urine or sputum sample from the subject with one or more of the variant molecules disclosed herein and detecting binding of antibodies in the sample to the variant molecule. For example, the method can include contacting a sample from a subject, such as, but not limited to a blood, serum, plasma, urine or sputum sample from the subject with one or more of the constructs disclosed herein and detecting binding of antibodies in the sample to the antigenic insert. The binding can be detected by any means known to one of skill in the art, including the use of labeled secondary antibodies that specifically bind the antibodies from the sample. Labels include radiolabels, enzymatic labels, and fluorescent labels.
Any such immunodiagnostic reagents can be provided as components of a kit. Optionally, such a kit includes additional components including packaging, instructions and various other reagents, such as buffers, substrates, antibodies or ligands, such as control antibodies or ligands, and detection reagents.
Methods are further provided for a diagnostic assay to monitor a particular disease and/or to monitor the response of the subject to immunization by a particular vaccine, such as HIV, SIV, RSV or Hepatitis B vaccine. In one example, a diagnostic assay is to monitor an HIV-1 induced disease in a subject and/or to monitor the response of the subject to immunization by an HIV vaccine. By “HIV-1 induced disease” is intended any disease caused, directly or indirectly, by HIV. An example of an HIV-1 induced disease is acquired autoimmunodeficiency syndrome (AIDS). The method includes contacting a disclosed construct with a sample of bodily fluid from the subject, and detecting binding of antibodies in the sample to the disclosed antigens. In addition, the detection of the HIV-1 binding antibody also allows the response of the subject to immunization by a HIV vaccine to be monitored. In still other embodiments, the titer of the HIV-1 binding antibodies is determined. The binding can be detected by any means known to one of skill in the art, including the use of labeled secondary antibodies that specifically bind the antibodies from the sample. Labels include radiolabels, enzymatic labels, and fluorescent labels. In other embodiments, a disclosed construct is used to isolate antibodies present in a subject or biological sample obtained from a subject.
The provided isolated viral vectors can also be used in screening antiviral drugs. In one example, methods of screening antiviral drugs including methods of identifying protease inhibitors are disclosed. Viral inhibitory peptides correspond to the sequence of a protease target site for rubella. Inhibitory peptides contain synthetic amino acids, such as dehydro-alanine, on either side of the expected cleavage site. When these groups are attacked by the active site cysteine of the protease, they react with it and form a covalent bond. This attaches irreversibly to the free SH-group of the active site of the enzyme and results in non-competitive inhibition of the protease.
In one particular example, a method of identifying protease inhibitors is provided that utilizes a disclosed rubella-GFP construct to detect the effect of protease inhibition. Typically, in the absence of a protease inhibitor, GFP is expressed well by days 3 to 7 of infection. However, when an inhibitory peptide is added, the virus fails to cleave its nonstructural protein precursor into two fragments: P150 and active RNA polymerase. Without this cleavage, no RNA transcription can occur, either of the negative strand template RNA, or of the positive strand RNA coding for the nonstructural and structural proteins. In particular, protease inhibition prevents expression of GFP linked to P150, as manifested by a loss of green fluorescence. Therefore, a protease inhibitor is identified by detecting a decrease or inhibition of GFP expression or a decrease or inhibition in expression of one or more of the rubella nonstructural and structural proteins, such as a 10%, 20%, 30% 40%, 50%, 60%, 70%, 80%, 90% or more reduction in expression. In one particular example, a method of identifying a protease inhibitor includes contacting a cell expressing one or more of the disclosed rubella-GFP viral constructs with one or more test agents and with an amount of an agent capable of inhibiting protease activity. Expression of GFP or one or more of the rubella nonstructural and/or structural proteins is subsequently measured, whereby a decrease in expression of one or more of this proteins indicates that the agent is a protease inhibitor.
Besides reducing GFP expression, protease inhibitors disrupt the localization of P150-GFP to viral replicating centers in the cytoplasm. P150-GFP preserves the normal functions of P150 and localizes correctly to sites of viral RNA synthesis as noted by P150-GFP localizing to these centers as intensely fluorescent dots in the cytoplasm. When protease is inhibited, even partially, this is detected as a reduction in replicating centers. It is believed that rubella will be just as sensitive to protease inhibitors as is HIV as both viruses work by similar mechanisms. Additionally, other alphaviruses, such as VEE, have a similar requirement for protease activation of RNA replication, and their inhibitory peptides can be designed similarly, based on the known targets for viral protease.
The disclosure is illustrated by the following non-limiting Examples.
This example describes the materials and methods used in Example 2.
Construction of Rubella Expression Plasmids and Mutant Viruses.
A full length, infectious cDNA clone coding for wild type rubella of the Therien strain (GenBank accession M15240), called Robo 302, was kindly provided by Dr. Teryl Frey of Georgia State University (Pugachev et. al., J. Virol. 71, 562-568, 1999). For the generation of pRubΔNot 1, Robo 302 was digested with Not I and re-ligated, resulting in deletion of a 507 bp fragment from the non-structural region (bp 3661-4168 of the plasmid), without disruption of the reading frame. For the generation of pRubΔNot 1-GFP, a 3807 bp Hind III-Sca 1 fragment from pRubΔNot 1 was cloned into pBR322, resulting in plasmid pBR322-3818ΔNot1.
This construct contained a single Not I site. Zoanthus sp. green fluorescent protein (zGFP) was PCR-amplified from pZsGreen1-C1 (Cat. No. 632447, Clontech Laboratories, Inc. Mountain View, Calif.) using upstream primer 5′ CGGCGGCCGCACCGGTCGCCACCATGGCCCAGTCCAAGCACGGCCTGACC-3′ (SEQ ID NO: 75) and downstream primer 5′-TGGCGGCCGCTCTAGATCCGGTGGATCCCGGGCCCGCGGTACCGTCG-3′ (SEQ ID NO: 76) and the resulting 804 bp product was digested with Not I and ligated into the Not 1 site of pBR322-3818 ΔNot 1 in frame with the non structural sequence, generating pBR322-3818ΔNot1-GFP. A 4096 bp Bsu36 I-Sca 1 restriction fragment from pBR322-3818ΔNot1-GFP, comprising GFP cloned into the Not I deletion in Rub non-structural coding sequences, was cloned into corresponding sites of Robo302 to generate pRub-ΔNot1-GFP.
Vero Cell Culture.
African Green monkey kidney (Vero) cells were maintained in DMEM containing L-Glutamine, penicillin/streptomycin (MediaTech, Inc. Herndon Va.) and 10% fetal bovine serum at 37° C. and 5% CO2 in a humidified incubator. All microscopy was carried out using an inverted Nikon Diaphot microscope equipped for fluorescence detection and digital image capture.
Generation of Capped, Infectious Rubella Virus RNA and Cell Transfection.
In each of the pRub plasmids, the viral genome is located downstream of an SP6 promoter. The plasmids were linearized by digestion with Spe I and transcribed using the RiboMAX Large scale RNA Production System (Promega Corp., Madison Wis.). The transcription reaction contained 1× SP6 transcription buffer, 0.8U RNAsin, 5 mM rATP, 5 mM rCTP, 5 mM rUTP, 1 mM rGTP, 2 mM Ribo m7G(5′)ppp(5′)G cap analog, 5-10 μg linear ds-DNA template and 1 μL of SP6 RNA polymerase. The reaction mixture was incubated at 37° C. for 3 hours, followed by another hour in the presence of 1 μl of RNAse-free DNAse. Capped infectious rubella RNA was transfected into Vero cells as follows. The RNA was combined with 1 mL of OptiMEM, while 14 μL of DMRIEC lipid reagent (Invitrogen Corporation Carlsbad, Calif.) was added to 1 mL of OptiMEM. The two mixtures were combined and mixed gently. Cells were seeded 12-18 hours before and were washed twice with OptiMEM before being overlaid with the transfection mixture and incubated for 6-12 hours at 37° C. and 5% CO2 in a humidified incubator.
Western Blot Analyses.
Rubella structural proteins were detected by western blot. Vero cell lysates in RIPA buffer were sonicated for 30 seconds and then electrophoresed on a denaturing Nupage 4-12% acrylamide Bis-Tris gel (Invitrogen Corporation, Carlsbad, Calif.). Proteins were transferred to 0.2 μm Nitrocellulose filters followed by blocking for 30 min in TBS with 3% bovine serum albumin at room temperature on a rocking platform. Primary antibody binding was performed with polyclonal goat anti-rubella antibodies at a 1:400 dilution (Fitzgerald Industries International, Inc. Concord, Mass. Cat#20-RG04) in TBS with 0.2% Tween, 0.3% BSA at 4° C. overnight on a rocking platform. Blots were washed 3 times with 10 ml TBS with 0.2% Tween for 10 minutes each on an orbital shaker. Horseradish peroxidase-conjugated rabbit anti-goat IgG at a dilution of 1:5000 (Santa Cruz Biotechnology, Santa Cruz, Calif.) was added in TBS with 0.2% Tween for 40 minutes at room temperature on a rocking platform, followed by the same washing procedure as above. Proteins were visualized by enhanced chemiluminescence (Amersham Biosciences, Buckinghamshire, England) and autoradiography.
Analysis of the zGFP Insert in Viral RNA.
Viral RNA was extracted by treating infected cells or pelleted virus with TRIzol (Invitrogen Corporation, Carlsbad, Calif.) and purified according to the manufacturer's instructions. Reverse transcription was performed using a SuperScript III kit (Invitrogen Corporation, Carlsbad, Calif.) and random hexamers. The cDNA was PCR amplified using an Advantage-GC PCR kit (Clonetech Laboratories, Mountain View, Calif.) and primers specific for rubella sequences flanking the insert. The 5′ primer Robo-55 was 5′-CCATTAAGCGGTTCCTCGGTAGC (SEQ ID NO: 23), and the 3′ primer was 5′-GAGTGCCGCGAGCGTCCGAGTGC (SEQ ID NO: 24), resulting in a product of 1.2 Kb. Amplified cDNA was analyzed by gel electrophoresis, purified using a Qiagen kit, and sequenced using the same PCR primers. To analyze zGFP function, the amplified cDNA was cloned into E. coli and each colony was examined for fluorescence. The cDNA was PCR amplified using a puReTaq Ready-To-Go PCR kit (GE Healthcare) and 5′ primers containing an NcoI restriction site and 3′ primers containing an EcoRI restriction site. The purified PCR products and a pZsGreen plasmid (Clontech Laboratories, Mountain View, Calif.) were cleaved using NcoI and EcoRI restriction endonucleases (NEB), gel purified, and ligated together to produce a functional zGFP plasmid. The products were transformed into competent DH5-α cells (Invitrogen Corporation, Carlsbad, Calif.), plated overnight in LB/Amp medium containing 100 uM IPTG, and visualized in an inverted fluorescent Nikon Diaphot microscope. Fifty five to 76 colonies per passage were analyzed for green fluorescence. Representative colonies of either type were grown in LB/Amp medium, and the plasmid was isolated (Qiagen Mini-prep kit) and sequenced (FDA core facility).
This example illustrates that a foreign gene, zGFP, can be inserted into the Not I site in the nonstructural gene nsP150 of a rubella vector, resulting in an infectious rubella hybrid that expresses the foreign protein for many generations. Although this example utilizes the foreign gene zGFP, it is contemplated that other foreign genes such as HIV antigens, SIV antigens, RSV antigens or HBsAgs can also be incorporated in a similar manner.
To determine if a deletion of 507 bp between two Not I restriction sites was permissive for subgenomic transcription and viral replication, each full length rubella cDNA was transcribed, capped, and transfected into Vero cells as described above. Expression of rubella structural proteins was detected by western blot of the P0 cell lysates on day 12. Wild type rubella expressed capsid, E2 and E1 proteins (
Normal expression of the structural proteins indicated that viral RNA polymerase made negative strand template RNA, followed by plus strand subgenomic RNA coding for the structural proteins. The deletion was located in a region of unknown function, and created a potential space for insertion of a foreign gene without interfering with essential viral functions. The reporter gene zGFP was inserted into the site of the Not I deletion. This added 792 bp of DNA, for a net increase of 285 bp, and preserved the open reading frame. Vero cells were transfected with capped viral RNA, and viral supernatants were transferred onto fresh cultures. The rubella-GFP hybrid virus expressed normal levels of rubella structural proteins, as shown by western blot (
Stability of GFP expression was tested during serial passage of culture supernatants on Vero cells. GFP expression was manifested by multiple bright fluorescent foci that appeared by day 4 and spread throughout the culture by day 7 to 10. Each intermediate passage showed GFP expression, and there was no fluorescence from an uninfected control run in parallel.
Genetic stability of zGFP was examined by recovering viral RNA from culture supernatants or infected cells after passages 5, 6, 10, and 11 and generating cDNA by RT-PCR of the insert. The overall GFP sequence was unchanged at passages 5 and 6, but showed a deletion of 27 bp at passages 10 and 11. This finding contrasted with fluorescence observed at later passages and suggested the possibility of a mixed population of wild type viruses giving fluorescence and mutants giving the observed sequence. To test this, the cDNAs were cloned into E. coli and individual colonies were examined for GFP expression. At passage 5, 76% of colonies (42 out of 55) were fluorescent, but by passages 10 and 11 only 5% of colonies (5 out of 100) expressed functional GFP.
Several clones of each phenotype were sequenced. At passage 5, four non-fluorescing clones constituted a swarm of different mutants. Two clones had point mutations Leu 46 to Pro or Phe 83 to Ser, while two others had deletions of 9 or 54 bp coding for amino acids Ile 79-Asp 81 or Lys 39-Phe 56. The deletion mutant that predominated at later passages was not detected at this passage. By passage 11, all five non-fluorescing clones had deletions ranging from 9 to 27 bp. Three had the predominant 27 bp deletion coding for amino acids Phe 83-Tyr 91 that was identical to the bulk culture. Two others had deletions coding for Asp 78-Val 80 or Thr 73-Asp 81. In contrast, two fluorescent clones at passage 11 showed the full wild type zGFP sequence. The loss of 9, 27, or 54 bp suggests persistent selective pressure to reduce the size of the zGFP insert. However, the emergence after 10 passages of a predominant clone with a 27 bp deletion that preserves the reading frame suggests that zGFP may achieve stability at a size of about 765 bp.
Rubella-GFP allowed the host range and sensitivity to interferon to be examined.
These studies support the notion that rubella can be a vector for delivering vaccine antigens, since it can express genes as large as most viral antigens, while growing to high enough titers for vaccine production and immunization. For example, the current insert size of 792 bp (or 765 bp after deletion) is larger than hepatitis B surface antigen (680 bp) and most HIV antigens, including p24 (660 bp) and the gp41 ectodomain (570 bp). In addition, rubella hybrids can achieve sufficiently high titers for efficient vaccine production. After four passages on Vero cells, the titer reached 4×106 fluorescent foci per ml. Live attenuated rubella is one of the most efficient vaccines, with a recommended human dose of 5,000 PFU (Plotkin et al., Rubella Vaccine. In: Plotkin, S. A., and Orenstein W. A. (Eds.), Vaccines, 4th ed. Saunders, Philadelphia, pp. 707-7432004), so this culture supernatant could provide 800 doses per ml.
This example describes a particular method that can be used to prevent or treat HIV in a human subject by administration of one or more compositions that includes an effective amount of any of the disclosed isolated immunogens. Although particular methods, dosages, and modes of administrations are provided, one skilled in the art will appreciate that variations can be made without substantially affecting the treatment.
Based upon the teaching disclosed herein, a virus, such as HIV, such as HIV type 1, can be prevented and/or treated by administering a therapeutically effective amount of a composition that includes a viral-like particle produced by an isolated rubella viral vector with an HIV antigenic insert to prevent, reduce or eliminate HIV infection, replication or a combination thereof. The method can include screening subjects to determine if they are HIV sero-positive, for example infected with HIV-1. Subjects having HIV infection are selected. In one example, subjects having increased levels of HIV antibodies in their blood (as detected with an enzyme-linked immunosorbent assay, Western blot, immunofluorescence assay, or nucleic acid testing, including viral RNA or proviral DNA amplification methods) are selected. In one example, a clinical trial would include half of the subjects following the established protocol for treatment of HIV (such as a highly active antiretroviral therapy). The other half would follow the established protocol for treatment of HIV (such as treatment with highly active antiretroviral compounds) in combination with administration of the compositions including an isolated rubella viral vector with an HIV antigenic insert (as described herein). In another example, a clinical trial would include half of the subjects following the established protocol for treatment of HIV (such as a highly active antiretroviral therapy). The other half would receive a composition including the isolated rubella viral vector with an HIV antigenic insert.
In another example, a population of subjects at high risk of HIV infection would be screened for rubella antibodies. The seronegative subjects would be randomized to receive conventional rubella vaccine without an insert or the rubella vectors with HIV inserts. The subjects would be observed over time for protection against rubella or HIV. If the vectors elicit protective immunity to HIV, the number of HIV infections in the rubella control group would greatly exceed the number in the rubella vector group. The cohort size and risk per year would be large enough to produce at least 30 infections in the control group. Under these conditions, a reduction of 50% or greater would be statistically significant protection.
Screening Subjects
In particular examples, the subject is first screened to determine if they are infected with HIV. Examples of methods that can be used to screen for HIV infection include a combination of measuring a subject's CD4+ T cell count and the level of HIV in serum blood levels or determine whether a subject is sero-positive for HIV antibodies.
In some examples, HIV testing consists of initial screening with an enzyme-linked immunosorbent assay (ELISA) to detect antibodies to HW, such as to HIV-1. Specimens with a nonreactive result from the initial ELISA are considered HIV-negative unless new exposure to an infected partner or partner of unknown HIV status has occurred. Specimens with a reactive ELISA result are retested in duplicate. If the result of either duplicate test is reactive, the specimen is reported as repeatedly reactive and undergoes confirmatory testing with a more specific supplemental test (e.g., Western blot or an immunofluorescence assay (IFA)). Specimens that are repeatedly reactive by ELISA and positive by IFA or reactive by Western blot are considered HIV-positive and indicative of HIV infection. Specimens that are repeatedly ELISA-reactive and occasionally provide an indeterminate Western blot result, which may be either an incomplete antibody response to HIV in an infected subject, or nonspecific reactions in an uninfected person. IFA can be used to confirm infection in these ambiguous cases. In some instances, a second specimen will be collected more than a month later and retested for subjects with indeterminate Western blot results. In additional examples, nucleic acid testing (e.g., viral RNA or proviral DNA amplification method) can also help diagnosis in certain situations.
The detection of HIV in a subject's blood is also indicative that the subject has HIV and is a candidate for receiving the therapeutic compositions disclosed herein. Moreover, detection of a CD4+ T cell count below 350 per microliter, such as 200 cells per microliter, suggests that the subject is likely to have HIV. However, lack of HIV antibodies could indicate that the subject is not infected and is a candidate for HIV prevention by the rubella vector treatment.
Pre-screening is not required prior to administration of the therapeutic compositions disclosed herein.
Pre-Treatment of Subjects
In particular examples, the subject is treated prior to administration of a therapeutic composition that includes one or more of the disclosed viral constructs. However, such pre-treatment is not always required, and can be determined by a skilled clinician. For example, the subject can be treated with an established protocol for treatment of HIV (such as a highly active antiretroviral therapy).
Administration of Therapeutic Compositions
Following subject selection, a therapeutic effective dose of the composition including one or more of the disclosed rubella viral constructs including an HIV antigen is administered to the subject (such as an adult human or a newborn infant either at risk for contracting HIV or known to be infected with HIV). Administration induces a sufficient immune response to reduce viral load, to prevent or lessen a later infection with the virus, or to reduce a sign or a symptom of HIV infection. Additional agents, such as anti-viral agents, can also be administered to the subject simultaneously or prior to or following administration of the disclosed compositions. Administration can be achieved by any method known in the art, such as oral administration, intranasal administration, inhalation, intravenous, intramuscular, intraperitoneal, or subcutaneous.
The amount of the composition administered to prevent, reduce, inhibit, and/or treat HIV or a condition associated with it depends on the subject being treated, the severity of the disorder, and the manner of administration of the therapeutic composition. Ideally, a therapeutically effective amount of an agent is the amount sufficient to prevent, reduce, and/or inhibit, and/or treat the condition (e.g., HIV) in a subject without causing a substantial cytotoxic effect in the subject. An effective amount can be readily determined by one skilled in the art, for example using routine trials establishing dose response curves. In addition, particular exemplary dosages are provided above. The therapeutic compositions can be administered in a single dose delivery, via continuous delivery over an extended time period, in a repeated administration protocol (for example, by a daily, weekly, or monthly repeated administration protocol). In one example, therapeutic compositions that include rubella viral vector constructs including an HIV antigenic insert, such as a Gag, gp41 or gp120 antigenic insert are administered intramuscularly to a human. As such, these compositions may be formulated with an inert diluent or with a pharmaceutically acceptable carrier.
In one specific example, a composition including an isolated rubella viral vector with an HIV antigenic insert is administered intravenously from 0.1 pg to about 100 mg per kg per day. In an example, the composition is administered continuously. Administration of the therapeutic compositions can be taken long term (for example over a period of months or years). In another example, the composition is administered at 50 μg per kg given twice a week for 2 to 3 weeks. In another example, the composition is administered at a dose of 1 pg to 10 ng and given at 0, 1, and 6 months to achieve a maximum immune response. It may also be given at escalating doses of 1 to 10 pg for the first dose, 100 pg to 1 ng for the second dose, and 10 ng to 100 ng for the third dose. This will allow immunity to the carrier virus to be overcome and to boost immunity to the heterologous antigen, including MPER.
Assessment
Following the administration of one or more therapies, subjects having HIV (for example, HIV-1 or HIV-2) can be monitored for reductions in HIV levels, increases in a subjects CD4+ T cell count, or reductions in one or more clinical symptoms associated with HIV. In particular examples, subjects are analyzed one or more times, starting 7 days following treatment. Subjects can be monitored using any method known in the art. For example, biological samples from the subject, including blood, can be obtained and alterations in HW or CD4+ T cell levels evaluated.
For uninfected subjects, HIV antibodies and neutralizing antibodies elicited by the vectors can have provide protection against subsequent HIV infection, particularly if the overall level of antibodies, or the level of neutralizing antibodies, exceeds some protective level.
Additional Treatments
In particular examples, if subjects are stable or have a minor, mixed or partial response to treatment, they can be re-treated after re-evaluation with the same schedule and preparation of agents that they previously received for the desired amount of time, including the duration of a subject's lifetime. A partial response is a reduction, such as at least a 10%, at least 20%, at least 30%, at least 40%, at least 50%, or at least 70% in HIV infection, HIV replication or combination thereof. A partial response may also be an increase in CD4+ T cell count such as at least 350 T cells per microliter.
This example illustrates the methods of monitoring serum antibodies to HIV or Hepatitis B.
Based upon the teachings disclosed herein, the presence of serum antibodies to HIV or Hepatitis B can be monitored using the isolated rubella viral vector construct platforms disclosed herein, such as to detect an HIV or Hepatitis B infection. Generally, the method includes contacting a sample from a subject, such as, but not limited to a blood, serum, plasma, urine or sputum sample from the subject with one or more of the disclosed compositions including one or more rubella viral vector constructs with an HIV or Hepatitis B antigenic insert and detecting binding of antibodies in the sample to the one or more constructs. The binding can be detected by any means known to one of skill in the art, including the use of labeled secondary antibodies that specifically bind the antibodies from the sample. Labels include radiolabels, enzymatic labels, and fluorescent labels.
Based upon the teaching herein, the utility of a rubella viral vector construct with an HIV antigenic insert to identify sera that contain neutralizing antibodies against the HIV antigenic insert included with the rubella viral vector construct can be determined by screening a set of weakly and broadly neutralizing human HIV-1 positive sera and HW-IgG for binding to one or more of the disclosed constructs or virus like particles that include one or more disclosed viral constructs. Human sera from HIV-1 positive patients and antibodies specific for HIV antigens can be serially diluted and analyzed for binding to HIV antigens and particles containing such polypeptides in ELISA format.
Based upon the teaching herein, subjects are immunized with a dose of 1 pg to 100 ng and given at 0, 1, and 6 months of the disclosed rubella viral vector including one or more HIV antigens or virus-like particles containing the disclosed vector including one or more of such antigens by intramuscular route. Sera from the subject is analyzed for binding to one or more of the HIV antibodies by ELISA.
In addition, the sera can be checked for their neutralizing ability in a viral neutralization assay using luciferase-based HIV entry assay. If the neutralizing titers are high enough, the subject is challenged with SHIV virus bearing the same envelope glycoproteins as HIV. Alternatively, SIV antigens can be incorporated into the disclosed viral vector, monkeys can be immunized with rubella-SIV, and challenged with virulent SIV strains. In one particular example, the subject is a rhesus monkey.
This example illustrates that SW and HIV antigens can be inserted into the Not I site of a rubella vector, resulting in an infectious rubella hybrid that expresses the foreign protein for multiple generations.
In a first set of studies, full length RNA coding for the rubella vector plus the insert was transcribed, capped, and the vector genes were transfected into Vero cells (passage 0) as described previously in Example 1. Growth of vector with the insert was then determined by measuring rubella proteins by Western blot analysis. Alternatively, expression of the insert gene product was determined by Western blot using antibodies specific for the insert. For example, this could be the Gag genes of SIV or the MPER antigen of HIV.
In a second set of studies, Vero cells were transfected as in the original method with rubella viral constructs containing SIV or HIV sequences inserted at the Not I site. These cells were called passage P0. After 7 days, virus and cells were transferred together as a cell suspension. The suspension was made by scraping a quarter of the cell monolayer, and then diluting the cell suspension to 2 ml, followed by transferring 0.1 to 0.2 ml directly onto a new monolayer of Vero cells to make passage P1. This procedure was repeated for multiple passages.
For example,
Further,
These studies show stable expression of SIV and HIV epitopes in a rubella vector resulting in an infectious rubella hybrid that expresses the foreign protein for sufficient generations to allow expansion in a fermentor, followed by propagation and expression as a vaccine antigen in the immunized host.
This example describes the materials and methods used in Example 9.
Analysis of Insert Stability.
To verify stability of the inserts in rubella, the rubella flanking regions, as well as the entire insert, was sequenced. Cells were infected with the vector for seven days and then treated with TRIzol (Invitrogen). RNA was purified from the aqueous phase with RNeasy Mini Kit, according to the manufacture's protocol (Qiagen GmbH, Germany). Reverse transcription was performed using a High Capacity RNA-to-cDNA Kit (Applied Biosystems), followed by PCR amplification using illustra puReTaq Ready-to-Go PCR beads (GE Healthcare). Oligonucleotide primers specific for rubella sequences flanking the Not I insertion site: Robo-seq67 (forward primer: 5′-gatgacgaggcgctcatcc, SEQ ID NO: 104) and Robo-seq6A (reverse primer: 5′-gagtgccgcgggcgtccgagtgc, SEQ ID NO: 105); or Robo-seq25 (forward primer: 5′-cgaactggtgagccccatgg, SEQ ID NO: 106) and Robo-seqSbfDn4 (reverse primer: 5′-gatctcgcaaatgcaggctccagtg, SEQ ID NO: 107) for inserts in the structural region. The PCR products were analyzed by gel electrophoresis, purified from the gel using MinElute Gel Extraction Kit (Qiagen) and sequenced using the same primers.
Antibodies and Antigens.
Antibodies were obtained from the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID. Monoclonals 2F5 and 4E10 were obtained from Dr. Hermann Katinger. Polyclonal goat antibodies to rubella structural proteins were purchased from Fitzgerald Industries International, Inc. (Concord, Mass.). Rabbit antibodies to rubella nonstructural protein P150 were provided by Dr. Tero Ahola (University of Helsinki, Finland). Aldrithiol-2 inactivated SHIV virions with 89.6 envelope and SIV Gag proteins were a gift of Drs. Larry Arthur and Jeffrey Lifson at the AIDS Vaccine Program, NCI.
Construction of cDNA Plasmids Coding for Infectious RNA.
Plasmid p10RA coding for full-length infectious cDNA of the RA27/3 vaccine strain of rubella and plasmid pRobo302, coding for cDNA of the Therien strain of wild type rubella, were provided by Dr. T. Frey (Georgia State University, Atlanta). Wild type rubella cDNA was cloned into pBR322 to produce plasmid pBRobo3226. Infectious rubella virus RNA was generated by transcribing from the SP6 promoter, followed by RNA capping.
For cloning purposes, a sub-clone of p10RA (from Hind III to Bgl II), pBR322-Hind-Bgl-RA was created in pBR322. To create space for potential inserts, a deletion was made between the two Not I sites at positions 3661 and 4168 in the non-structural protein region, as reported for wild type rubella. Hind III-Cla I fragment from the sub-clone carrying the Not I deletion was cloned back to p10RA, giving pBRA3226-dNotI plasmid. The constructs were verified by sequencing.
Construction of Vectors with Insertions in the Nonstructural Gene Region.
Recognition sequences for Avr II and Nsi I restriction enzymes were cloned into the Not I deletion site. These unique restriction sites were then used for directional cloning of inserts into the Not I-deletion site. In the case of MPERF, MPERE, SGag2L-A, SGag2L-B, SGag2L-C, and BC-SGag2, cloning was done using SpeI and Nsi I sites. Spe I and Avr II have compatible sticky ends. Each insert was PCR amplified using primers listed in Table 4 and HIV-1 89.6 gp160 or SIV mac239 gag DNA as templates, cut with restriction enzymes, and ligated into the matching restriction sites. The amino acid sequences of these inserts are listed in
Construction of Vectors with Insertions in the Structural Region.
For cloning purposes, a sub-clone of pBRobo3226 plasmid coding for wild type rubella was created. The new plasmid pBR322-NotI-BamHI contained rubella cDNA from the Not I site to BamH I in the structural protein region. DNA inserts coding for MPERF-E2TM and MPERF-E1TM, containing the 2F5 epitope, transmembrane domains of E2 and E1 rubella proteins, respectively, and E1 signal peptide sequence were synthesized by GeneArt (Regensburg, Germany). The inserts were flanked by Sbf I and Kpn I restriction sites, which were used to clone them into pBR322-NotI-BamHI. These plasmids were cut with Sbf I and BamH I restriction enzymes, and the products were cloned into plasmid pBRobo3226-dNotI, resulting in plasmids, which combined inserts in the structural genes with the deletion in the non-structural region at Not I. A similar construct on the RA27/3 background was made by inserting synthetic DNA coding for MPER, the transmembrane domain of HW-1 gp41 and the E1 signal peptide, flanked by Sbf I restriction sites, into the plasmid pBRA3226-dNotI coding for RA27/3 vaccine strain of rubella with the Not I deletion. All constructs were verified by sequencing. The amino acid sequences of these inserts are given in
Generation of Rubella Virus.
The protocol for generation of capped, infectious rubella virus RNA from plasmid DNA and subsequent transfection of Vero cells was described previously. Virus was propagated on Vero cells maintained in DMEM containing L-glutamine, penicillin/streptomycin (MediaTech, Inc., Herndon, Va.) and 10% heat-inactivated fetal bovine serum at 37° C. in a humidified incubator with 5% CO2.
After 7-10 days of infection, culture supernatant (P0) was centrifuged to produce cell-free supernatant and 0.1 mL of the supernatant was transferred onto fresh Vero cells to begin the next passage P1. Alternatively, in early passages, it was found that passing infected cells from the previous culture along with virus, helped in propagating new viral vectors. Infected cells were continued to be passed for the first four passages, after that cell-free virus was just as efficient. Infected cells were tested for expression of rubella structural proteins, as a measure of vector replication, and insert expression was evaluated by Western blot, using specific antibodies. To expand a viral stock, virus from passage 4 or 5 was grown for 7 to 10 days on a Vero cell monolayer in a T75 flask, and multiple aliquots were frozen.
Detection of Rubella Proteins and MPER Inserts by Western Blot.
Infected Vero cells were lysed in RIPA buffer (Thermo Scientific, Rockford, Ill.) with addition of BD BaculoGold Protease inhibitor cocktail (BD Biosciences, San Jose, Calif.). Cell lysates were sonicated, run on a NuPAGE 4-12% polyacrylamide Bis-Tris gel (Invitrogen, Carlsbad, Calif.), transferred to nitrocellulose, and blocked with 2% BSA in TBS. Primary antibody was incubated in TBS with 0.2% Tween20, 0.2% BSA for 1½ hours at room temperature. Expression of rubella structural proteins was detected with goat anti-rubella polyclonal antibodies at 1:700 dilution, while rubella non-structural protein P150 was detected using rabbit polyclonal antibodies at a dilution of 1:1000 (30). Expression of MPER-derived inserts was detected with human monoclonal antibodies 2F5 or 4E10 at 1 μg/ml. Blots were washed three times with TBS/0.2% Tween20, followed by incubation with secondary antibody diluted in the same buffer for 60 min at room temperature on a rocking platform. The second antibody was either horseradish peroxidase-conjugated rabbit anti-goat IgG, goat anti-rabbit IgG or goat anti-human IgG at 1:5000 dilutions (Santa Cruz Biotechnology, CA). After the same washing procedure as above, blots were visualized with enhanced chemiluminescence (GE Healthcare).
This example illustrates deletion/insertion strategies to construct rubella viral vectors carrying a foreign gene.
The sGag inserts were derived from SIV Gag amino acids 41-211 (
Each insert was ligated into the Not I site of the rubella vaccine strain RA27/3, as shown in
Replication of rubella vectors expressing MPERF, MPERE and sGag2L at passage Po was detected by Western blot (
Expression of the MPERF insert was detected by Western blot with monoclonal antibody 2F5 (
The seven vectors with sGag inserts included four of the five major epitopes from this region of Gag that are known targets of T cell immunity against SIV. Their replication was monitored by Western blot of rubella structural proteins (
Due to lack of a monoclonal antibody specific for the T cell epitopes of SIV Gag, expression of BC-sGag2 at the Not I site was detected indirectly, by measuring a shift in the P150 band due to the size of the p150-BCs-Gag2 fusion protein (
In some cases, vectors appeared to be replicating after the initial RNA transfection (P0) but failed to propagate further by passing clarified culture supernatants. In these early passages, propagation efficiency was increased by transferring virus via infected cells, rather than culture supernatants. After passage P4, cell-free passage was performed. This method helped to propagate vectors with inserts as large as BC-sGag2.
In addition, the insert was expanded to full length MPER, which included both the 2F5 and 4E10 epitopes, and anchored it to the membrane with the transmembrane domain of HIV gp41 linked to the E1 signal peptide (
A schematic of inserts at the structural site is shown in
Since MPER was expressed with the rubella structural proteins, it was examined whether it could be incorporated into virions. After banding MPER-HIVTM virus (P8) in sucrose density gradients, each fraction was analyzed for rubella viral proteins (
Rubella vectors would not be practical as a vaccine platform unless they could achieve high enough titers for vaccine production. Twenty ml stocks of rubella vectors were produced on the vaccine strain background (RA27/3). The vectors had MPERF or BC-sGag2 inserts at the Not I site, or an MPER-HIVTM insert in the structural site. Viral RNA content of the stocks were measured by real time RT-PCR, as described previously. As shown in Table 7, culture supernatants of MPERF contained 3.1×107 RNA copies/ml, BC-sGag2 grew to 2.5×106 RNA copies/ml, and MPER-HIVTM reached 1.3×107 RNA copies/ml. Comparing these vectors to a sample of rubella vaccine of known titer, titers were estimated of about 5.0×106 PFU/ml, 4.1×105 PFU/ml and 2.2×106 PFU/ml, respectively.
These studies identified two insertion sites where rubella can accommodate inserts into the viral genome: one is located between two Not I restriction sites in the nonstructural genes, while the other is located between the structural genes. When a protein antigen (zGFP, BC-sGag2 or MPERF) is expressed at the Not I site, it forms a fusion protein with rubella nonstructural protein P150. It is expressed as an early viral antigen, under control of the rubella genomic promoter, with maximal expression between days 2 to 5 of infection. Based on the results with P150-zGFP and prior studies of P150, the P150 fusion protein localizes to the cytoplasm, where it participates in viral replicative centers. This location is near the entry point for proteasomal processing and could deliver SIV Gag antigen to the main processing pathway leading to antigen presentation with MHC class I for induction of T cell immunity. However, expression as a fusion protein with P150 also places constraints on the insert, as P150 function must be preserved. Within viral replication centers, P150 acts as an essential cofactor in RNA synthesis, both for plus and minus strand RNA. Loss of P150 function would be a lethal event for the virus. This could occur if an insert prevented normal P150 folding, interfered with its interaction with RNA polymerase, or caused instability through proteolytic degradation or aggregation.
The second insertion site is located in the structural region of rubella, between genes coding for envelope glycoproteins E2 and E1. An MPER insert at this site was expressed as a late viral protein, under control of the strong subgenomic promoter. The ability to express the full MPER determinant at this site, but not at the Not I site, suggested greater flexibility in protein expression at the structural site. MPER expression was maximal between days 5 and 10 after infection. It was expressed as part of the structural polyprotein and then processed to free MPER antigen. Sedimentation in sucrose gradients showed that most of the MPER was incorporated into virions, and almost none remained as a free protein. As shown in the schematic (
The antigens expressed by recombinant rubella vectors include the MPER determinant of HW transmembrane protein gp41 and SIV Gag sequences, which are rich in T cell determinants. The MPER region is a target of broadly reactive neutralizing antibodies against HIV, such as monoclonal antibodies 2F5, 4E10, and Z13e. However, despite many attempts to elicit antibodies to MPER, there has been little success, presumably because MPER is a weak immunogen by itself, lacks the native conformation, or resembles self antigens. Its expression as part of a live, replicating viral vector may enhance the immune response to MPER by eliciting T cell help for MPER-specific B cells. Rubella vectors expressing MPER could also be combined with another vector in a prime and boost strategy or they could precede a boost with virus-like particles expressing MPER.
The SIV Gag determinants were derived from a region of Gag (aa 41-211) that is rich in targets of T cell immunity, including five T cell epitopes. Two epitopes, CM9 and GY9, are restricted by Mamu A01 and A02, respectively, and they have been identified as predominant targets of CTLs early after SIV infection. For these epitopes, virus escape occurs late, if at all. However, when it does occur, escape mutants at CM9 have been associated with loss of control of viremia and disease progression. These sites may be slow to mutate because they perform viral functions for SIV that require compensatory mutations before they can escape. By eliciting T cells specific for these epitopes prior to infection, the BC-sGag2 vector could potentially control a subsequent SIV infection.
Rubella's biological properties favor its use as a vaccine platform. Its safety has been demonstrated in millions of children around the world. The SIV Gag and HIV-1 MPER inserts at the Not I site were made with the RA27/3 vaccine strain, and our inserts at the structural site have been re-derived on this background. The vectors replicate to high titers during vaccine production, and the inserted genes are stably expressed for at least eight to ten passages in cell culture. The vaccine strain can be used safely in the host without further attenuation. Even if a vector loses its insert, it reverts to the vaccine strain. Rubella virus possesses little risk of viral persistence, has no DNA intermediates and does not integrate into host genomes as retroviral vectors do. Also, there is no interference with antigen processing and presentation, as with CMV and adenovirus vectors. While growing to high titers in cell culture, rubella vaccine immunizes at a low dose. Since the minimum recommended dose of the RA27/3 strain is 103 PFU for humans, the titers obtained with rubella vectors (Table 7) correspond to between 410 and 5,000 human doses per milliliter. At this level, without further optimization, the entire US birth cohort of four million children could be immunized with the product of a 10 L fermentor.
Live attenuated rubella vaccine is immunogenic, and it elicits mucosal as well as systemic immunity. A live vector can immunize with a much lower dose than a non-replicating vector; through exponential growth, it exposes the host to more antigen over time. Rubella has low cytotoxicity, so infected cells can present antigen for 5 days at the Not I site and up to 10 days at the structural site. Persistent expression may help the induction of antigen specific T cells and B cells. Antigen presented in the context of an acute infection may combine innate and adaptive immunity to elicit a stronger immune response.
This example characterizes rubella viral vector constructs carrying a foreign gene within the structural site without a deletion at the Not I site within the non-structural site.
As shown in
Not only were the vector constructs viable, but such rubella vectors grew to high titers in vitro as illustrated in
Subsequent studies were performed to determine if these rubella vector constructs were infectious in vivo and effective at immunizing subjects.
The difference between vectors that did or did not grow in vivo was apparently due to the Not I deletion. The missing gene is probably required to deal with host defenses, most likely interferon. It was found that Not I deleted rubella viruses with a structural site insertion can only grow in cell lines, such as Vero cells, that are unable to make interferon. They do not grow in MRC-5 or WI-38 cells that are classic hosts for rubella and produce interferon. In contrast, vectors with the same structural insert but without a Not I deletion grow well in these cells. Thus, a vector's ability to grow in MRC-5 or WI-38 cells correlates well with successful immunization of monkeys and man. Thus, these studies demonstrate that rubella vectors lacking a Not I deletion are infectious in vivo and immunize macaques efficiently.
Further studies were performed which also demonstrated rubella vectors lacking a Not I deletion replicate well in vivo, as detected by PCR of oral swabs.
Additional studies were performed to determine if the rubella vectors could elicit an immune response to the vaccine insert.
A set of studies was performed which determined that rubella constructs lacking a deletion at the Not I site were more robust and could accommodate much larger inserts at the structural site when compared to those including a deletion. As shown in
This example illustrates rubella vectors stably express SIV Gag and HIV MPER determinants at the structural site.
Vector replication in Vero cells was shown by Western blot with antibodies to the rubella structural proteins C and E1 (
Rubella vector stocks for the monkey studies were produced and characterized rubella vector stocks for monkey studies. For each vector type, we made one vector with an MPER insert and the other with a Gag insert. The MPER insert at the structural site consisted of the complete membrane proximal external region, followed by the HIV transmembrane domain and an E1 SP signal sequence. The Gag insert consisted of four known T cell epitopes linked in tandem (called BC-sGag2), followed by the E2TM domain of rubella and the E1 SP signal peptide. For each vector stock, we demonstrated insert expression by Western blot.
Viral titers were determined by quantitative RT-PCR. The titers were estimated by comparison to a rubella reference sample of known PFU titer. Viral titers were 7.7×106 PFU/ml, or greater, which is equivalent to about 1500 human doses per ml. Viral sequencing showed that the insert was stable and in reading frame after at least five passages. The vector doses given to macaques, based on viral titer, were between two and ten times the typical human dose of rubella vaccine (about 5,000 PFU/dose).
This example demonstrates that Rubella vectors bearing Gag and MPER determinants are highly immunogenic.
Monkeys were immunized according to a protocol. Group 1 macaques received three doses of DNA vaccine first, followed by a rubella vector boost at week 25. Group 3 macaques received three doses of non-replicating rubella vectors first, as we searched for a replication competent vector. They finally received replicating vectors at week 18, and this was followed by DNA vaccine given at weeks 25 and 31. A successful vaccine “take” was detected as the appearance of antibodies to the structural proteins of the rubella vector. All six macaques made antibodies to the replicating vector, as shown by ELISA assay on plates coated with rubella antigens. (These plates are used for diagnosis of human infections.)
Antibodies to the SIV Gag insert were shown. As shown in
The kinetics of the response to SIV Gag are shown for individual macaques in
This example demonstrates that antibodies elicited by rubella vectors are persistent and boostable.
The persistence of anti-Gag antibodies in Group 3 macaques for nine months after immunization was measured. This was compared to the persistence of anti-rubella antibodies in the same animals (
Anti-Gag antibodies were measured in the same macaques at the same time points (
The inventors waited up to a year for rubella antibodies to decline to a level where they could boost with rubella vectors bearing new antigens. For group 4 macaques, which were primed with rubella vaccine without an insert, these antibodies declined about 2.5-fold after 6 months, and then remained constant or rose slightly by 1 year. After one year (group 4) or 6 months (group 1), we boosted the macaques with rubella vectors expressing MPER and SIV Gag antigens (week 57 of the study). All five animals showed a prompt rise in antibodies to rubella.
The anti-SIV Gag antibody response was measured four weeks after the boost (week 61 of the study,
With other vaccines and vectors, the immune response to SIV and HIV antigens has been short-lived and lacked memory B cells. Transient antibody responses are considered one of the major obstacles to HIV vaccine development. Using live rubella vectors, it is shown herein that anti-Gag antibodies persist for over nine months and decline with nearly the same half-life as antibodies to rubella proteins. In general, persistent antibody titers are thought to depend on long-lived plasma cells, while boosting depends on memory B cells, and both of these are signs of germinal center function during immunization. The primary immune response to these vectors produced memory B cells, as shown by boosting 6 months later. The secondary response depended on successful priming, as control animals that were not primed to SIV Gag lacked memory B cells and could not respond to the boost. Potentially, two doses of live rubella vectors, given several years apart, could boost and update immunity to circulating strains of HIV. In addition, the ability to prime and boost memory B cells would allow us to combine rubella vectors with other viral vectors bearing similar HIV vaccine inserts.
This example demonstrates immunization with two rubella vectors at the same time elicits antibodies to both inserts.
Macaques of groups 1 and 3 were immunized with two rubella vectors concurrently. One vector expressed SIV Gag, and the other expressed HIV MPER with a transmembrane domain from HW. Antibodies to the MPER determinant were detected by ELISA assay on plates coated with gp140 trimers. As shown in
When two rubella vectors were given simultaneously, they both replicated side by side, as shown by RT-PCR, and they elicited antibodies to both inserts. This means that rubella vectors can be used to immunize against two or more antigens at the same time.
This example demonstrates that immunization with MPER determinants elicits broadly crossreactive neutralizing antibodies.
Additional MPER constructs are listed in the Table 8, part B. These are intended to improve signal peptidase cleavage, or to enhance immunogenicity by linkage to the SIV Gag sequence or by duplicating important epitopes recognized by neutralizing antibodies 2F5 and 10E8. The MPER determinants perform an essential viral function during membrane fusion and cell entry, and they are conserved sequences. They are also the target of broadly neutralizing antibodies. If MPER can be made more immunogenic, the resulting antibodies should have broadly crossreactive neutralizing activity.
LTAVVLQGYNPPAYG
AAFVLLVPWVLIFMVCRRTCRRRGAAAALTAVVLQGYNPPAYG
VLQGYNPPAYG
LTAVVLQGYNPPAYG
FLAGLLLAAVAVGTARAG
AAAALTAVVLQGYNPPAYG
AAAALTAVVLQGYNPPAYG
ataccctggccgcgttcgtgctcctcgtgccatgggtgctcatctttatggtctgtcggaggacctgcaga
AVVLQGYNPPAYG
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This example demonstrates expression of large Gag inserts for the induction of CTLs.
Rubella is a small RNA virus that replicates exclusively in the cytoplasm. This location is ideal for eliciting T cell immunity, since it delivers antigens directly into the proteasomal pathway leading to antigen presentation with MHC class I. The Gag proteins are important targets of CTLs during infection. The inventors have discovered that priming with DNA vaccine and boosting with the rubella-BCsGag2 vector gave high levels of Gag specific CD8+ T cells, ranging from 1 to 8% of all T cells. The inventors have also discovered that the structural insertion site can accommodate much larger Gag inserts than before. The large Gag inserts are shown schematically in
Expression of two large Gag inserts was demonstrated by western blot in
This example illustrates the expression of the outer domain of gp120 scaffolded on parts of the inner domain
HIV envelope gp120 is an important target of neutralizing antibodies. These depend on the native conformation of gp120. Gp120 has inner and outer domains. The main target of neutralizing antibodies binding is located in the outer domain, but its folding depends on the inner domain. The inventors have developed a variety of gp120 constructs for use as vaccine inserts. In each of these, the outer domain is preserved, but the inner domain is deleted at either end. Deletion at the amino end produces a truncated gp120. A deletion at the carboxyl end of the inner domain would produce an internal deletion in gp120.
Four examples of internally deleted gp120 are shown in Table 8, part C. These all have amino acids 79-213, followed by an SIGG linker, followed by 252-506. In the examples given, they are followed by different transmembrane domains, from rubella E2 protein, HIV gp41, sindbis virus, or VSV. Expression of the first three of these inserts by rubella vectors are shown in
Four examples of truncated gp120 are also given: these are amino acids 79-506, 88-506, 93-506, and 93-484. The amino acid sequences are given in Table 8, part C. Expression of these truncated forms of gp120 by rubella vectors are shown in
In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are only examples of the invention and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as my invention all that comes within the scope and spirit of these claims.
This is the U.S. National Stage of International Application No. PCT/US2013/035634, filed Apr. 8, 2013, which was published in English under PCT Article 21(2), which in turn claims the benefit of U.S. Provisional Application No. 61/621,394, filed Apr. 6, 2012, and 61/642,333, filed May 3, 2012, each of which is incorporated herein by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2013/035634 | 4/8/2013 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2013/152352 | 10/10/2013 | WO | A |
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| 6958237 | Frey | Oct 2005 | B2 |
| 9181306 | Berkower | Nov 2015 | B2 |
| 20030130498 | Frey et al. | Jul 2003 | A1 |
| 20120207784 | Berkower et al. | Aug 2012 | A1 |
| Number | Date | Country |
|---|---|---|
| WO2011047340 | Apr 2011 | AU |
| WO 2011047340 | Apr 2011 | WO |
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| Number | Date | Country | |
|---|---|---|---|
| 20150079123 A1 | Mar 2015 | US |
| Number | Date | Country | |
|---|---|---|---|
| 61621394 | Apr 2012 | US | |
| 61642333 | May 2012 | US |
