EP2192918A1 - Hiv env proteins with modifications in the v3 loop - Google Patents
Hiv env proteins with modifications in the v3 loopInfo
- Publication number
- EP2192918A1 EP2192918A1 EP08798605A EP08798605A EP2192918A1 EP 2192918 A1 EP2192918 A1 EP 2192918A1 EP 08798605 A EP08798605 A EP 08798605A EP 08798605 A EP08798605 A EP 08798605A EP 2192918 A1 EP2192918 A1 EP 2192918A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- polypeptide
- env
- loop
- hiv
- seq
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/16011—Human Immunodeficiency Virus, HIV
- C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
- C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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- C—CHEMISTRY; METALLURGY
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- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/16011—Human Immunodeficiency Virus, HIV
- C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
- C12N2740/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
Definitions
- This invention is in the field of human immunodeficiency virus (HIV) and, in particular, the viral envelope glycoproteins and their manipulation.
- HIV human immunodeficiency virus
- the various proteins encoded within the HIV genome include the envelope glycoprotein (Env) and the trans-activating transcriptional factor (Tat).
- the Env protein is initially expressed as a long precursor protein that is subsequently cleaved to give an exterior membrane glycoprotein and a transmembrane glycoprotein.
- these proteins are hereafter referred to by the standard HIV-I nomenclature i.e. the precursor is 'gpl60', the membrane glycoprotein is 'gpl20' and the transmembrane glycoprotein is 'gp41 '.
- These names are based on approximate molecular weights of the HIV-I glycoproteins.
- gpl20 can interact with the host cell CD4 receptor. This interaction induces a conformational transition in gpl20, leading to the exposure of its 'V3' loop.
- Tat protein is important in regulating HIV gene expression. Although it is a transcription factor, it has also been found to be released by infected cells and has been proposed as a vaccine antigen.
- WO2005/090391 discloses that Env and Tat proteins can interact to form a complex. The interaction is said to require the presence of the V3 loop in the Env protein.
- a vaccine based on a combination of Env and Tat polypeptides is also disclosed in e.g., Ensoli et al. (2005) Microbes Infect 7:1392-9.
- the present invention is directed to a mixture of (i) a HIV Tat polypeptide and (ii) a HIV Env polypeptide, wherein the Env polypeptide has one or more mutations in its V3 loop.
- the HIV Tat polypeptide and the HIV Env polypeptide form a complex.
- the HIV Tat polypeptide and the HIV Env polypeptide are covalently linked to each other.
- the present invention is also directed to a process for preparing a mixture of (i) a HIV Tat polypeptide and (ii) a HIV Env polypeptide, wherein the Env polypeptide has one or more mutations in its V3 loop.
- the process comprises mixing (i) a HIV Tat polypeptide with (ii) a HIV Env polypeptide, wherein the Env polypeptide has one or more mutations in its V3 loop. In other embodiments, the process comprises combining a HIV Env polypeptide with a HIV Tat polypeptide, wherein the Env polypeptide has one or more mutations in its V3 loop. In certain embodiments, the process comprises a further step of: determining if the Env and Tat polypeptides have formed a complex.
- the present invention is also directed to a HIV Env polypeptide having a mutant V3 loop sequence, wherein the mutant sequence is selected from the group consisting of SEQ IDs 15, 16, 17, 18, 19, 20, 21, 22, 23 and 24.
- the Env polypeptide of the mixtures, processes or polypeptides of the invention includes one or more mutation(s) outside the V3 loop. In other embodiments, the Env polypeptide lacks the wild-type transmembrane domain and cytoplasmic tail. In still other embodiments, the Env polypeptide includes one or more deletion(s) within the V2 loop. In certain embodiments, the Env polypeptide has a V3 loop with an amino acid sequence selected from the group consisting of SEQ ID NOs: 15 to 24. In other embodiments, the Env polypeptide has a V3 loop with amino acid sequence SEQ ID NO: 29.
- the invention is also directed to a mixture of (i) a HIV Tat polypeptide and (ii) a polypeptide comprising a fragment of a V3 loop of a HIV Env polypeptide, wherein the polypeptide of (ii) is no longer than 100 amino acids and includes at least 5 consecutive amino acids from a HIV Env polypeptide V3 loop.
- the polypeptide of (ii) is ⁇ 30 amino acids long.
- the polypeptide of (ii) is cyclic.
- the fragment of (ii) has an amino acid sequence selected from group consisting of SEQ ID NOs: 30 to 37.
- the Tat polypeptide of the mixtures, processes or polypeptides of the invention has amino acid sequence SEQ ID NO: 12.
- the present invention is also directed to a pharmaceutical composition comprising a mixture of the invention.
- the pharmaceutical composition of the invention includes a vaccine adjuvant.
- the present invention is also directed to a method of raising an immune response in a patient, comprising the step of administering a composition of the invention the patient.
- Figure 1 shows the results of a Far- Western assay. Lanes are: (1) markers at 35, 50, 75, 105, 160 and 250 kDa; (2) gpl40 ⁇ V2; (3) gpl40 ⁇ V2 + CD4; (4) gpl40dV3-22; (5) gpl40dV3-22 + CD4; (6) gpl20; (7) gpl20 + CD4; and (8) CD4.
- the invention concerns modification of the V3 loop of the HIV envelope glycoprotein in order to alter its Tat-binding properties.
- the invention provides a mixture of (i) a HIV Tat polypeptide and (ii) a HIV Env polypeptide, wherein the Env polypeptide has one or more mutations in its V3 loop.
- the Tat and Env polypeptides may form a complex and may, as described in more detail below, be covalently linked.
- the invention also provides a process for preparing a polypeptide mixture, comprising a step of mixing (i) a HIV Tat polypeptide with (ii) a HIV Env polypeptide, wherein the Env polypeptide has one or more mutations in its V3 loop.
- the invention also provides a method comprising a step of: combining a HIV Env polypeptide with a HIV Tat polypeptide, wherein the Env polypeptide has one or more mutations in its V3 loop.
- the method may comprise a further step of: determining if the Env and Tat polypeptides have formed a complex. This further step allows the effect of different V3 mutations on Tat- binding activity to be determined.
- any complexes formed by the V3 mutants can be compared to complexes formed by wild-type Env.
- the mutants may form weaker or stronger complexes than wild-type Env (e.g. tighter association constant), or may form complexes more quickly or slowly, etc.
- the invention also provides an Env polypeptide with one or more mutations in its V3 loop, identified by such methods as having a higher affinity for Tat than wild-type Env.
- the invention also provides a HIV Env polypeptide having a V3 loop sequence selected from the group consisting of SEQ IDs 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 and 29.
- the invention also provides a mixture of (i) a HIV Tat polypeptide and (ii) a polypeptide comprising a fragment of a V3 loop of a HIV Env polypeptide.
- the polypeptide of (ii) will usually be no longer than 100 amino acids, but the fragment will usually have at least 5 amino acids from a V3 loop (usually 5 consecutive amino acids from a V3 loop).
- the two polypeptides may form a complex, and may be covalently linked.
- mixtures of the invention include a HIV Env polypeptide that has one or more mutations in its V3 loop.
- Env polypeptide can be used, from HIV-I or HIV-2.
- the mixture may include a full-length gpl60 Env polypeptide with V3 mutation(s), a gpl20 Env polypeptide with V3 mutation(s), a truncated gpl20 or gpl60 Env polypeptide with V3 mutation(s), g ⁇ l60 or gpl20 polypeptide with V3 mutation(s) and one or more deletions outside V3, a fusion protein including a gpl20 or gpl60 polypeptide with V3 mutation(s), etc.
- the invention will typically use a shortened protein.
- amino acid sequence of the full-length HIV-I Env precursor from the REFSEQ database (GI:9629363) is a 856mer shown below (SEQ ID NO: 1 herein; V3 loop underlined):
- This wild-type HIV-I precursor protein is cleaved to give the surface glycoprotein gpl20 ⁇ e.g. amino acids 29-511 of SEQ ID NO: 1; SEQ ID NO: 2 herein) and the transmembrane domain gp41 ⁇ e.g. amino acids 512-856 of SEQ ID NO: 1 ; SEQ ID NO: 3 herein):
- the subdomains are as follows (numbered according to SEQ ID NO: 2): 1-102; 103-129; 129-168; 169-267; 268-303; 304-356; 357-390; 391-432; 433-443; and 444-483.
- the coordinates of these subdomains can be identified in other HIV-I Env sequences by performing a suitable sequence alignment. Pre-aligned sequences from numerous strains, annotated with these features, can also be found in the Los Alamos HIV Sequence Compendia http://hiv-web.lanl.gov
- the Env sequence (SEQ ID NO: 38) after removal of the leader is shown below for the SF 162 strain (SEQ ID NO: 7; V3 loop underlined). It is cleaved after Arg-475 to give the mature proteins, including the gpl20 (SEQ ID NO: 50 herein, namely amino acids 1-475 of SEQ IDNO: 7):
- the amino acid sequence of a full-length HIV-2 Env precursor (GI:2144996) is a 852mer shown below (SEQ ID NO: 4 herein; V3 loop underlined):
- the HIV-2 Env precursor protein is cleaved to give the surface glycoprotein ⁇ e.g. amino acids 20-502 of SEQ ID NO: 4; SEQ ID NO: 5 herein) and the transmembrane domain ⁇ e.g. amino acids 503-852 of SEQ ID NO: 4; SEQ ID NO: 6 herein): MCGKSLLCVASLLASAYLV/YCTQYVTVFYGVPVWRNASIPLFCATKNRDTWGTIQCKPDNDDYQEITLN VTEAFDAWDNTVTEQAVEDVWSLFETSIKPCVKLTPLCVAMSCNSTTNNTTTTGSTTGMSEINETSPSYS DNCTGLGKEEIVNCQFYMTGLERDKKKQYNETWYSKDWCESNNTKDGKNRCYMNHCNTSVITESCDKHY WDAIKFRYCAPPGYALLRCNDTNYSGFEPKCSKWASTCTRMMETQTSTWFGFNGTRAENRTYIYWHGRD NRTIISLNKYYNLSIHC
- the hypervariable regions etc. can, again, be identified by sequence alignment and by reference to the alignments in the Los Alamos HIV Sequence Compendia
- Env polypeptides used with the invention will have, relative to a wild-type sequence, a V3 loop having one or more mutations. Such V3 mutations are described in more detail below.
- Env polypeptide used with the invention may further include mutations outside the V3 loop.
- the invention will typically use a shortened Env polypeptide.
- the shortening will involve the removal of one of more amino acids from the full-length sequence e.g. truncation at the C-terminus and/or N-terminus, deletion of internal residues, removal of subdomains other than V3 US patent 5,792,459, and combinations of these approaches.
- an Env polypeptide used with the invention may include a portion of gp41 but not include its transmembrane domain.
- V2 loop of the Env precursor it is also known to make deletions within the V2 loop of the Env precursor, to give ' ⁇ V2' mutants.
- one or more amino acids within the 40-mer V2 loop can be deleted (e.g. at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 or more amino acids).
- Deletions within the V2 loop have been reported to improve immunogenicity of Env polypeptides (Barnett et al. (2001) J Virol 75:5526-40; Srivastava et al. (2003) J Virol 77:2310- 20).
- Env polypeptides with deletions and/or substitutions in the V2 loop have been found to be useful in forming Env/Tat complexes.
- Env/Tat complexes may not be seen with monomeric gpl20 unless its V2 loop is mutated.
- Amino acids deleted from the V2 loop may be substituted with other amino acids e.g. it is known to replace the central portion of the V2 loop with a Gly-Ala-Gly tripeptide.
- a ⁇ V2 mutant may have the following sequence (SEQ ID NO: 8):
- Another useful ⁇ V2 mutant based on SF 162, may have the following sequence (SEQ ID NO:
- Env polypeptide for use with the invention is a gpl40 protein with a ⁇ V2 mutation from HIV-I strain SF 162.
- this polypeptide In its mature form, after cleavage of a signal sequence and secretion (see, e.g., Figure 24 of WO00/39302), this polypeptide has the following amino acid sequence (SEQ ID NO: 9):
- the wild-type SF 162 V3 loop (underlined; SEQ ID NO: 44) will be replaced by a mutant V3 loop as described elsewhere herein.
- Env polypeptides used with the invention may retain the ability of natural Env to bind to CD4.
- Residues that have been identified as important for CD4 binding include (numbered according to SEQ ID NO: 1) Asp-368, Glu-370, Trp-427, Val-430 and Pro-438.
- the invention is not limited to the use of Env polypeptides having the exact sequence of a known HIV polypeptide.
- the Env polypeptide used according to the invention may be selected from one of the following, provided that it includes a mutant V3 loop:
- polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 1, 2, 4, 5, 7, 8, 9, 38, 39, 43, 45, 49 and 50;
- polypeptide comprising an amino acid sequence that has sequence identity to an amino acid sequence selected from SEQ ID NOs: 1, 2, 4, 5, 7, 8, 9, 38, 39, 43, 45, 49 and 50;
- a polypeptide comprising an amino acid sequence that, compared to an amino acid sequence selected from SEQ ID NOs: 1, 2, 4, 5, 7, 8, 9, 38, 39, 43, 45, 49 and 50, has one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) substitutions and/or deletions and/or insertions (inside and, optionally, outside the V3 loop);
- polypeptide comprising an amino acid sequence comprising a fragment of at least n consecutive amino acids from an amino acid sequence selected from SEQ ID NOs: 1, 2, 4, 5, 7, 8, 9, 38, 39, 43, 45, 49 and 50, where n is 7 or more; or
- a polypeptide comprising a sequence of p amino acids that, when aligned with an amino acid sequence selected from SEQ ID NOs: 1, 2, 4, 5, 7, 8, 9, 38, 39, 43, 45, 49 and 50 using a pairwise alignment algorithm, has at least xy identical aligned monomers in each window of x amino acids moving from N-terminus to C-terminus, where: p>x; there are p-x+1 windows; x is selected from 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250,
- y is selected from 0.50, 0.60, 0.70, 0.75, 0.80, 0.85, 0.90, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, or 0.99; and, ifxy is not an integer, it is rounded up to the nearest integer.
- polypeptides include homologs, orthologs, allelic variants and mutants of SEQ ID NOs 1 , 2, 4, 5, 7, 8, 9, 38, 39, 43, 45, 49 and 50. For instance, it is known to mutate natural Env sequences to improve resistance to proteases.
- the polypeptides also include fusion polypeptides, in which the Env sequence is fused to non-Env sequence. For instance, it is known to fuse Env sequences without the native leader peptide to leader peptides from non-Env proteins e.g. from tissue plasminogen activator.
- the degree of sequence identity may be greater than 50% (e.g. 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more).
- each substitution involves a single amino acid
- each deletion preferably involves a single amino acid
- each insertion preferably involves a single amino acid.
- These changes may arise deliberately (e.g. by site-directed mutagenesis) or naturally (e.g. through virus evolution or through spontaneous mutation).
- the polypeptides in category (iii) may have one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) single amino acid substitutions relative to SEQ ID NO: 1, 2, 4, 5, 7, 8, 9, 38, 39, 43, 45, 49 or 50.
- These polypeptides may have one or more (e.g.
- substitutions, insertions and/or deletions may be at separate locations or may be contiguous. Substitutions may be conservative i.e. replacements of one amino acid with another which has a related side chain. Genetically-encoded amino acids are generally divided into four families: (1) acidic i.e.
- n may be greater than 7 e.g.
- the fragment may comprise at least one T-cell and/or B-cell epitope of the sequence.
- T- and B-cell epitopes can be identified empirically (e.g. using PEPSCAN Geysen et al. (1984) PNAS USA 81:3998-4002; Carter (1994) Methods MoI Biol 36:207-223) or similar methods), or they can be predicted (e.g. using the Jameson- Wolf antigenic index (Jameson, BA et al.
- This algorithm is conveniently implemented in the needle tool in the EMBOSS package (Rice et al. (2000) Trends Genet 16:276-277).
- Env polypeptide is found in oligomeric form on the HIV virion, and preferred Env polypeptides used with the invention can also form oligomers, and in particular trimers. For instance, ⁇ V2 mutants of gpl40 have been shown to form trimers (Barnett et al. (2001) J Virol 75:5526-40). Env/Tat complexes generally do not form when using monomeric gpl20, unless its V2 loop is mutated, but are formed from trimeric gpl40 without requiring any V2 mutation.
- Preferred Env polypeptides used with the invention have, in addition to a mutant V3 loop, a mutant V2 loop (e.g. a mutant V2 as in SEQ ID NO: 9). Mutant V3 loops
- the wild-type V3 loop in Env has cysteine residues at both termini, which may be covalently linked in a disulfide bridge.
- Env polypeptides in mixtures of the invention will have one or more mutations in the V3 loop relative to a wild-type Env sequence.
- V3 loop consists of amino acids Cys-268 to Cys-303 (SEQ ID NO: 13):
- V3 loop consists of amino acids Cys-267 to Cys-301 (SEQ ID NO: 46): CTRPNNNTRKSITIGPGRAFYATGDI IGDIRQAHC
- V3 loop consists of amino acids Cys-296 to Cys-329 (SEQ ID NO: 14):
- V3 loop in other Env sequences can readily be identified by performing a suitable sequence alignment and, as mentioned above, pre-aligned sequences from numerous strains annotated to show the V3 loop can also be found in the Los Alamos HIV Sequence Compendia. For instance, the wild-type V3 loops of five specific strains from different subtypes are aligned below, together with a consensus sequence:
- a mutation in the V3 loop may independently be a deletion of a single amino acid, the substitution of a single amino acid with a single amino acid, or the insertion of one or more amino acids.
- An Env polypeptide used with the invention may have one or more of such mutations e.g. one or more deletions and/or one or more substitutions and/or one or more insertions.
- Mutant V3 loops with at least one deletion are typical e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or more deletion(s). Where there is more than one deletion, it is possible to group them (i.e. two or more neighboring wild-type amino acids may both be deleted) and/or separate them (i.e. two deleted amino acids are separated by at least one wild-type amino acid).
- mutant V3 loop sequences for use in Env polypeptides of the invention are shown below as SEQ ID NOS: 15 to 22: CTRPNNNGAGDIRQAHC (SEQ ID NO: 15)
- CTITIGPGRAFYATGDIIGDIRQAHC SEQ ID NO: 16
- CTRPNNNTRFYATGDIIGDIRQAHC SEQ ID NO: 18
- CTRPNNNTRGDIIGDIRQAHC SEQ ID NO: 19
- CTFYATGDIIGDIRQAHC SEC ID NO: 20
- CTRPNNNTRQAHC (SEQ ID NO: 21)
- CTRPNNNTRGAGQAHC (SEQ ID NO: 23) A further example of a V3 loop sequence for use in Env polypeptides of the invention, with three substitutions relative to the wild-type SF 162 V3 sequence, is shown below (SEQ ID NO: 24):
- CTRPNNNTRKSITIGPGRAFYATGDI IGNMRQAHC SEQ ID NO : 24
- the consensus sequence SEQ ID NO: 29
- SEQ ID NO: 29 may also be used in place of a wild-type V3 loop.
- Preferred mutant V3 loops retain the ability to interact with a Tat polypeptide.
- the interaction may be weaker/stronger, quicker/slower, etc. than seen with the wild-type V3 sequence
- a mixture of the invention includes a polypeptide comprising a fragment of a V3 loop of a HIV Env polypeptide.
- This polypeptide will usually be no longer than 100 amino acids ⁇ e.g. ⁇ 90, ⁇ 80, ⁇ 70, ⁇ 60, ⁇ 50, ⁇ 40, ⁇ 30, ⁇ 20 amino acids).
- the V3 loop fragment will include at least 5 amino acids from a wild-type V3 loop ⁇ e.g. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acids from a V3 loop).
- the polypeptide may include amino acids to the C-terminus and/or N-terminus of a V3 loop fragment.
- the polypeptide may be linear and/or branched. Cyclic polypeptides may be used ⁇ e.g. DTIC report ADA322181 (1994) http://handle.dtic.mii/100.2/ADA322181 discloses a cyclic 35mer peptide formed from a V3 loop). If a polypeptide includes more than one cysteine residue, these may be linked to form a disulfide bridge.
- TRKSITIGPGRAFYATGD SEQ ID NO: 30
- KSITIGPGRAFYAT SEQ ID NO: 34
- RPNNNTRKSITIGPGRA SEQ ID NO: 35
- the Tat polypeptide Mixtures of the invention include a HIV Tat polypeptide, and various forms of Tat polypeptide can be used from HIV-I or HIV-2.
- the length of the Tat polypeptide varies depending on virus strain.
- the amino acid sequence of the full-length HIV-I Tat polypeptide from the REFSEQ database (GI:9629358) is a 86mer shown below (SEQ ID NO: 10 herein):
- the amino acid sequence of a full-length HIV-2 Tat polypeptide is a 130mer shown below (SEQ ID NO: 11 herein): METPLKAPESSLMSYNEPSSCTSERDVGSQELAKQGEELLSQLHRPLEPCNNKCYCKGCCFHCQLCFLNK GLGICYDRKGRRRRTPKKTKAHSSSASDKSISTRTGNSQPEKKQKKTLETTLETARGLGR
- HIV-2 Tat sequences can be found in the Los Alamos HIV Sequence Compendia.
- Other specific tat sequences that can be used include those disclosed in references WO00/39302, WO03/020876, WO2005/007808, WO03/004620 and WO99/27958.
- a particularly useful Tat polypeptide for use with the invention is from HIV-I strain BHlO.
- This polypeptide has the following amino acid sequence (SEQ ID NO: 12; GL62291022):
- the invention is not limited to the use of Tat polypeptides having the exact sequence of a known HIV polypeptide.
- Tat polypeptide used according to the invention may be selected from:
- polypeptide comprising an amino acid sequence that has sequence identity to an amino acid sequence selected from SEQ ID NOs: 10, 11 and 12;
- polypeptide comprising an amino acid sequence that, compared to an amino acid sequence selected from SEQ ID NOs: 10, 11 and 12, has one or more ⁇ e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) substitutions and/or deletions and/or insertions;
- polypeptide comprising an amino acid sequence comprising a fragment of at least n consecutive amino acids from an amino acid sequence selected from SEQ ID NOs: 10, 11 and 12, where n is 7 or more;
- a polypeptide comprising a sequence of p amino acids that, when aligned with an amino acid sequence selected from SEQ ID NOs: 10, 11 and 12 using a pairwise alignment algorithm, has at least xy identical aligned monomers in each window of x amino acids moving from N-terminus to C-terminus, where: p>x; there are p-x+1 windows; x is selected from 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 or 85; y is selected from
- polypeptides include homologs, orthologs, allelic variants and mutants of SEQ ID NOs 10, 11 and 12. They also include fusion polypeptides, in which the Tat sequence is fused to non-Tat sequence.
- the degree of sequence identity may be greater than 50% (e.g. 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more).
- each substitution involves a single amino acid, each deletion preferably involves a single amino acid, and each insertion preferably involves a single amino acid. These changes may arise deliberately (e.g. by site-directed mutagenesis) or naturally (e.g. through virus evolution or through spontaneous mutation).
- the polypeptides in category (iii) may have one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) single amino acid substitutions relative to SEQ ID NO: 10, 11 or 12. These polypeptides may have one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) single amino acid deletions relative to SEQ ID NO: 10, 11 or 12. These polypeptide s may have one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) single amino acid insertion relative to SEQ ID NO: 10, 11 or 12.
- the substitutions, insertions and/or deletions may be at separate locations or may be contiguous. As mentioned above, substitutions may be conservative.
- n may be greater than 7 e.g. 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850 or more.
- the fragment may comprise at least one T-cell and/or B-cell epitope of the sequence. As described above, such epitopes can be identified empirically or can be predicted.
- the preferred pairwise alignment algorithm is the Needleman-Wunsch global alignment algorithm as described above.
- Mixtures of the invention include Env and Tat polypeptides.
- these may form complexes, in which the Env and Tat polypeptides may be associated non-covalently and/or covalently.
- Particularly useful complexes have essentially a 1:1 molar ratio of Env and Tat. Where the Env is in the form of a trimer, therefore, the preferred complex includes three Tat monomers.
- the Env and Tat polypeptides in a mixture may be from the same type if HIV e.g. both are from
- HIV-I or both are from HIV-2. Where the same HIV types are used, it is also useful to mix Env and Tat polypeptides from the same group e.g. within HIV-I, both are from group M, group N or group O. Within group M, it is useful to mix Env and Tat polypeptides from the same subtype (or clade) e.g. from subtype A, B, C, D, F, G, H, J or K. It is also possible to use Env or Tat from a CRP (circulating recombinant form) subtype, such as an A/B or A/E CRF. Where a subtype includes sub-subtypes then the Env and Tat polypeptides may be from the same sub-subtype. Using Env and Tat from different groups, subtypes, sub-subtypes and/or clades is not, however, excluded. HIV-I nomenclature is discussed in more detail in Robertson et al. (2000) Science 288:55-6.
- Env and Tat from subtype B or C is preferred. Within a single subtype (or, where applicable, sub-subtype) it is possible to use Env and Tat from the same strain or from different strains.
- the Env and Tat polypeptides may both be from the SF 162 strain (subtype B), or the invention may use Env from one strain (e.g. SF162) and Tat from another strain (e.g. BHlO).
- Env/Tat complexes of the invention may bind specifically to (a) CD4 and/or (b) a monoclonal antibody that specifically binds to HIV Tat polypeptide and/or (c) a monoclonal antibody that specifically binds to HIV Env polypeptide.
- the complexes may retain the CD4-binding activity of the uncomplexed Env polypeptide and/or the mAb-binding activity of the uncomplexed Tat or Env polypeptide.
- Complexes with both of binding activities (a) and (b) are particularly useful.
- retaining these two activities in a covalent complex requires an appropriate degree of cross-linking between Env and Tat. Although this degree of cross-linking can vary within a fairly broad range, and thus does not need to be controlled with absolute precision, too little cross-linking leads to unstable complexes and too much cross-linking leads to a loss of binding activity.
- a complex binds specifically to CD4, this binding activity can be assessed using known assays e.g. as described in WO91/13906.
- the assay does not need to use native CD4, however, and it is more typical to use a purified soluble form of CD4 based on its external domain (e.g. see example 5 of WO91/13906).
- the CD4 may also be labeled to facilitate the assay.
- the CD4 is preferably human CD4. At least 250 SNPs have so far been described for CD4, and any of these polypeptides can be used, such as the REFSEQ CD4 (GI:10835167).
- the uncomplexed Env will specifically bind to CD4, and this specific binding activity can be retained in the Env/Tat complex. Although the binding activity is not removed, however, the actual binding affinity may change.
- a preferred monoclonal antibody is 8Dl.8, which is available through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH (http://www.aidsreagent.org/UploadDocs/ds4672_003.pdf ).
- the use of this antibody in Tat- binding assays has previously been disclosed e.g. in references Rohr et al. (2003) J Virol. 77:5415-27, Avraham et al (2004) J Immunol 173:6228-33, Liu et al (2002) J Virol 76:6689- 700.
- Env and Tat proteins may be covalently linked together in complexes of the invention.
- Various methods for covalently linking proteins are known in the art e.g. see references Wong (1991)
- covalent linking may involve the use of homobifunctional cross-linkers, heterobifunctional cross-linkers or zero-length cross-linkers. It may involve reagents directed to sulfhydryl groups in proteins, reagents directed to amino groups in proteins, reagents directed to carboxyl groups in proteins, tyrosine-selective reagents, arginine-specific reagents, histidine-specific reagents, methionine-alkylating reagents, tryptophan-specific reagents, serine-modifying reagents, etc.
- a preferred group of cross-linking reagents for use with the invention includes aldehydes, and in particular includes formaldehyde and the dialdehydes.
- Suitable dialdehydes include glyoxal, malondialdehyde, succinialdehyde, adipaldehyde, ⁇ -hydroxyadipaldehyde, glutaraldehyde and phthalaldehyde.
- Glutaraldehyde and its derivatives are particularly preferred, including 2-methoxy-2,4-dimethylglutaraldehyde, 3-methoxy-2,4-dimethylglutaraldehyde and
- 3-methylglutar-aldehyde Pyridoxal phosphates can also be used.
- Other amino group-directed cross-linkers include bis-imidoesters, bis-succinimidyl derivatives (e.g. bis(sulfosuccinimidyl)suberate, or 'BS 3 '), bifunctional aryl halides, bifunctional acylating agents (including di-isocyanates, di-isothiocyanates, bifunctional sulfonyl halides, bis-nitrophenyl esters and bifunctional acylazides), diketones, p-benzoquinone, 2-iminothiolane, erythritolbiscarbonate, mucobromic acid, mucochloric acid, ethylchloroformate and multidiazonium compounds.
- the invention will involve mixing Env polypeptide, Tat polypeptide and a linking reagent under conditions that permit the covalent linking reaction to proceed.
- a linking reagent under conditions that permit the covalent linking reaction to proceed.
- one of the two polypeptides will be reacted with the linking reagent first, to form an activated polypeptide, and then the activated polypeptide will be reacted with the second polypeptide.
- Heterobifunctional linkers with a photoreactive group are also useful.
- thermoreactive group has one thermoreactive group and one photoreactive group then a first step can involve attachment via the thermoreactive group, and then conjugation to make the complex can be initiated by the use of e.g. UV light.
- the photoreactive group can be used first.
- the cross-linking reaction may be performed to an extent that is not so great as to eliminate critical binding activities of the Env and Tat proteins.
- concentration of the Env and Tat proteins, the concentration of the cross-linking reagent(s), the pH, the reaction temperature and the reaction time can be controlled to give the desired degree of cross-linking.
- an initial series of reactions can be performed to evaluate suitable reaction conditions.
- compositions of the invention can be used as active ingredients in immunogenic compositions. These compositions can be administered to animals in order to elicit an immune response.
- the immune response preferably includes a humoral (e.g. an antibody response, such as a neutralizing antibody response) and/or a cellular response against Env and/or Tat.
- the immune response may reduce the severity of the infection (e.g. reduce viral load) and may even result in clearance of HIV infection.
- the immune response may reduce the risk of future HIV infection and may even be protective against future HIV infection.
- Immunogenic compositions will include an immunologically effective amount of a polypeptide.
- 'immunologically effective amount it is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for the desired treatment or prevention.
- This amount can vary depending upon the health and physical condition of the individual to be treated, age, the taxonomic group of individual to be treated (e.g. non-human primate, primate, etc.), the capacity of the individual's immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials, and a typical quantity of complex per dose is between l ⁇ g and lOmg per antigen.
- Immunogenic compositions of the invention are pharmaceutically acceptable. They usually include components in addition to the complexes e.g. they typically include one or more pharmaceutical carrier(s) and/or excipient(s). A thorough discussion of such components is available in Gennaro (2000) Remington: The Science and Practice of Pharmacy. 20th edition, ISBN: 0683306472.
- compositions will generally be in aqueous form.
- compositions will generally have an osmolality of between 200 m ⁇ sm/kg and 400 m ⁇ sm/kg, preferably between 240-360 m ⁇ sm/kg, and will more preferably fall within the range of 290-310 m ⁇ sm/kg.
- Compositions may include one or more buffers.
- Typical buffers include: a phosphate buffer; a Tris buffer; a borate buffer; a succinate buffer; a histidine buffer; or a citrate buffer. Buffers will typically be included in the 5-2OmM range.
- the pH of a composition will generally be between 5 and 8, and more typically between 6 and 7.
- the composition is preferably sterile.
- the composition is preferably non-pyrogenic e.g. containing ⁇ 1 EU (endotoxin unit, a standard measure) per dose, and preferably ⁇ 0.1 EU per dose.
- the composition is preferably gluten free.
- compositions of the invention may include detergent e.g. a polyoxyethylene sorbitan ester surfactant (known as 'Tweens'), an octoxynol (such as octoxynol-9 (Triton X-100) or t-octy lphenoxypolyethoxy-ethanol), etc .
- a polyoxyethylene sorbitan ester surfactant known as 'Tweens'
- an octoxynol such as octoxynol-9 (Triton X-100) or t-octy lphenoxypolyethoxy-ethanol
- Vaccines may be administered in a dosage volume of about 0.5ml.
- Vaccine adjuvants may be administered in a dosage volume of about 0.5ml.
- compositions of the invention may advantageously include an adjuvant, which can function to enhance the immune responses (humoral and/or cellular) elicited in a patient who receives the composition.
- adjuvants that can be used with the invention include, but are not limited to:
- a mineral-containing composition including calcium salts and aluminum salts (or mixtures thereof).
- Calcium salts include calcium phosphate (e.g. the "CAP" particles disclosed in US patent 6355271).
- Aluminum salts include hydroxides, phosphates, sulfates, etc., with the salts taking any suitable form (e.g. gel, crystalline, amorphous, etc.). Adsorption to these salts is preferred.
- the mineral containing compositions may also be formulated as a particle of metal salt WO00/23105. Aluminum salt adjuvants are described in more detail below.
- An immunostimulatory oligonucleotide such as one containing a CpG motif (a dinucleotide sequence containing an unmethylated cytosine linked by a phosphate bond to a guanosine), a TpG motif WO01/22972,a double-stranded RNA, an oligonucleotide containing a palindromic sequence, or an oligonucleotide containing a poly(dG) sequence.
- Immunostimulatory oligonucleotides can include nucleotide modifications/analogs such as phosphorothioate modifications and can be double- stranded or (except for RNA) single-stranded. Kandimalla et al.
- a CpG sequence may be directed to TLR9, such as the motif GTCGTT or TTCGTT
- the CpG sequence may be specific for inducing a ThI immune response, such as a CpG-A ODN (oligodeoxynucleotide), or it may be more specific for inducing a B cell response, such a CpG-B ODN.
- CpG-A and CpG-B ODNs are discussed in Blackwell et al. (2003) J Immunol 170:4061-4068, Krieg (2002) Trends Immunol 23:64-65, WO01/95935.
- the CpG is a CpG-A ODN.
- the CpG oligonucleotide is constructed so that the 5' end is accessible for receptor recognition.
- two CpG oligonucleotide sequences may be attached at their 3' ends to form "immunomers". See, for example, Kandimalla et al. (2003) Biochemical Society Transactions 31 (part 3):654- 658, Kandimalla et al. (2003) BBRC 306:948-953, Bhagat et al (2003) BBRC 300:853-
- CpG7909 also known as ProMuneTM (Coley Pharmaceutical Group, Inc.).
- Immunostimulatory oligonucleotides will typically comprise at least 20 nucleotides. They may comprise fewer than 100 nucleotides.
- 3dMPL has been prepared from a heptoseless mutant of Salmonella minnesota, and is chemically similar to lipid A but lacks an acid-labile phosphoryl group and a base-labile acyl group. Preparation of 3dMPL was originally described in UK patent application GB-A-2220211.
- 3dMPL can take the form of a mixture of related molecules, varying by their acylation (e.g. having 3, 4, 5 or 6 acyl chains, which may be of different lengths).
- the two glucosamine (also known as 2-deoxy-2-amino-glucose) monosaccharides are N-acylated at their 2-position carbons
- An imidazoquinoline compound such as Imiquimod (“R-837”) (US patents 4,680,338; 4,988,815) Resiquimod (“R-848”) (WO92/15582), and their analogs; and salts thereof (e.g. the hydrochloride salts). Further details about immunostimulatory imidazoquinolines can be found in Stanley (2002) Clin Exp Dermatol 27:571-577, Wu et al (2004) Antiviral
- a thiosemicarbazone compound such as those disclosed in WO2004/060308. Methods of formulating, manufacturing, and screening for active compounds are also described in WO2004/060308. The thiosemicarbazones are particularly effective in the stimulation of human peripheral blood mononuclear cells for the production of cytokines, such as TNF- ⁇ . • A tryptanthrin compound, such as those disclosed in WO2004/064759. Methods of formulating, manufacturing, and screening for active compounds are also described in reference WO2004/064759. The thiosemicarbazones are particularly effective in the stimulation of human peripheral blood mononuclear cells for the production of cytokines, such as TNF- ⁇ . • A nucleoside analog, such as: (a) Isatorabine (ANA-245; 7-thia-8-oxoguanosine):
- Ri and R 2 are each independently H, halo, -NR 3 R b , -OH, C ]-6 alkoxy, substituted Ci.6 alkoxy, heterocyclyl, substituted heterocyclyl, C 6-I o aryl, substituted C ⁇ -io aryl, C] -6 alkyl, or substituted Ci -6 alkyl;
- R 3 is absent, H, Ci -6 alkyl, substituted Ci -6 alkyl, C 6- io aryl, substituted C 6-I0 aryl, heterocyclyl, or substituted heterocyclyl;
- R 4 and R 5 are each independently H, halo, heterocyclyl, substituted heterocyclyl, -C(O)-R d , Q -6 alkyl, substituted Q -6 alkyl, or bound together to form a 5 membered ring as in R 4 ⁇ :
- Xi and X 2 are each independently N, C, O, or S;
- R 8 is H, halo, -OH, C ]-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, -OH, -NR a R b , -(CH 2 ) n -0- R c , -0-(C 1-6 alkyl), -S(O) p R e , or -C(O)-Ra;
- R 9 is H, Ci -6 alkyl, substituted Cj -6 alkyl, heterocyclyl, substituted heterocyclyl or R 9a , wherein Rg a is:
- Rio and Rn are each independently H, halo, Ci -6 alkoxy, substituted Ci -6 alkoxy, - NR 3 R b , or -OH; each R a and R b is independently H, C 1-6 alkyl, substituted Cj -6 alkyl, -C(O)Rj, C 6 .io aryl; each R 0 is independently H, phosphate, diphosphate, triphosphate, C] -6 alkyl, or substituted Ci -6 alkyl; each R d is independently H, halo, Ci -6 alkyl, substituted Ci -6 alkyl, Ci -6 alkoxy, substituted Ci -6 alkoxy, -NH 2 , -NH(Ci -6 alkyl), -NH(substituted C ]-6 alkyl), -N(Ci- 6 alkyl) 2 , -N(substituted Ci -6 alkyl) 2 , C 6-J0 aryl, or heterocycl
- Loxoribine (7-allyl-8-oxoguanosine) (US patent 5,011,828). • Compounds disclosed in WO2004/87153, including: Acylpiperazine compounds, Indoledione compounds, Tetrahydraisoquinoline (THIQ) compounds, Benzocyclodione compounds, Aminoazavinyl compounds, Aminobenzimidazole quinolinone (ABIQ) compounds (US 6,605,617, WO02/18383), Hydrapthalamide compounds, Benzophenone compounds, Isoxazole compounds, Sterol compounds, Quinazilinone compounds, Pyrrole compounds (WO2004/018455), Anthraquinone compounds, Quinoxaline compounds, Triazine compounds, Pyrazalopyrimidine compounds, and Benzazole compounds (WO03/082272).
- An aminoalkyl glucosaminide phosphate derivative such as RC-529 (Johnson et al. (1999) Bioorg Med Chem Lett 9:2273-227, Evans et al. (2003) Expert Rev Vaccines 2:219-22).
- a phosphazene such as poly[di(carboxylatophenoxy)phosphazene] ("PCPP") as described, for example, in Andrianov et al. (1998) Biomaterials 19:109-115 and Payne et al. ( 1998) Adv Drug Delivery Review 31 : 185- 196.
- SIPs Small molecule immunopotentiators
- N2-methyl-l-(2-methylpropyl)-N2-pentyl-lH-imidazo[4,5-c]quinoline-2,4-diamine N2-methyl-l-(2-methylpropyl)-N2-prop-2-enyl-lH-imidazo[4,5-c]quinoline-2,4- diamine l-(2-methylpropyl)-2-[(phenylmethyl)thio]-lH-imidazo[4,5-c]quinolin-4-amine l-(2-methylpropyl)-2-(propylthio)-lH-imidazo[4,5-c]quinolin-4-amine
- Saponins [chapter 22 of Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell & Newman) Plenum Press 1995 ISBN 0-306-44867-X], which are a heterologous group of sterol glycosides and triterpenoid glycosides that are found in the bark, leaves, stems, roots and even flowers of a wide range of plant species. Saponin from the bark of the Quillaia saponaria Molina tree have been widely studied as adjuvants.
- Saponin can also be commercially obtained from Smilax ornata (sarsaprilla), Gypsophilla paniculata (brides veil), and Saponaria officianalis (soap root).
- Saponin adjuvant formulations include purified formulations, such as QS21, as well as lipid formulations, such as ISCOMs.
- QS21 is marketed as StimulonTM.
- Saponin compositions have been purified using HPLC and RP-HPLC. Specific purified fractions using these techniques have been identified, including QS7, QS 17, QSl 8, QS21, QH-A, QH-B and QH-C.
- the saponin is QS21.
- Saponin formulations may also comprise a sterol, such as cholesterol (WO96/33739).
- a sterol such as cholesterol
- Combinations of saponins and cholesterols can be used to form unique particles called immunostimulating complexes (ISCOMs) [chapter 23 of Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell & Newman) Plenum Press 1995 (ISBN 0-306- 44867-X)].
- ISCOMs typically also include a phospholipid such as phosphatidylethanolamine or phosphatidylcholine. Any known saponin can be used in ISCOMs.
- the ISCOM includes one or more of QuilA, QHA & QHC.
- ISCOMS may be devoid of additional detergent (WO00/07621).
- additional detergent WO00/07621.
- Bacterial ADP-ribosylating toxins e.g. the E.coli heat labile enterotoxin "LT”, cholera toxin "CT”, or pertussis toxin "PT”
- LT heat labile enterotoxin
- CT cholera toxin
- PT pertussis toxin
- the use of detoxified ADP-ribosylating toxins as mucosal adjuvants is described in WO95/1721 and as parenteral adjuvants in WO98/42375.
- Bioadhesives and mucoadhesives such as esterified hyaluronic acid microspheres (Singh et al] (2001) J Cont Release 70:267-276) or chitosan and its derivatives (WO99/2796).
- Microparticles i.e. a particle of -lOOnm to ⁇ 150 ⁇ m in diameter, more preferably ⁇ 200nm to ⁇ 30 ⁇ m in diameter, or ⁇ 500nm to ⁇ 10 ⁇ m in diameter
- materials that are biodegradable and non-toxic e.g. a poly( ⁇ -hydroxy acid), a polyhydroxybutyric acid, a polyorthoester, a polyanhydride, a polycaprolactone, etc.
- poly(lactide-co-glycolide) being preferred, optionally treated to have a negatively- charged surface (e.g. with SDS) or a positively-charged surface (e.g. with a cationic detergent, such as CTAB).
- Liposomes Choapters 13 & 14 of Vaccine Design: The Subunit and Adjuvant Approach
- liposome formulations suitable for use as adjuvants are described in US 5,916,588, 6,090,406 and EP-A-0626169.
- Polyoxyethylene ethers and polyoxyethylene esters (WO99/52549). Such formulations further include polyoxyethylene sorbitan ester surfactants in combination with an octoxynol (WOO 1/21207) as well as polyoxyethylene alkyl ethers or ester surfactants in combination with at least one additional non-ionic surfactant such as an octoxynol (WO01/2115).
- Preferred polyoxyethylene ethers are selected from the following group: polyoxyethylene-9-lauryl ether (laureth 9), polyoxyethylene-9-steoryl ether, polyoxytheylene-8-steoryl ether, polyoxyethylene-4-lauryl ether, polyoxyethylene-35- lauryl ether, and polyoxyethylene-23-lauryl ether.
- Muramyl peptides such as N-acetylmuramyl-L-threonyl-D-isoglutamine (“thr-MDP"), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylglucsaminyl-N- acetylmuramyl-L-Al-D-isoglu-L-Ala-dipalmitoxy propylamide ("DTP-DPP", or "TheramideTM), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(l'-
- thr-MDP N-acetylmuramyl-L-threonyl-D-isoglutamine
- nor-MDP N-acetyl-normuramyl-L-alanyl-D-isoglutamine
- MTP-PE 2'dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine
- An outer membrane protein proteosome preparation prepared from a first Gram-negative bacterium in combination with a liposaccharide (LPS) preparation derived from a second Gram-negative bacterium, wherein the outer membrane protein proteosome and LPS preparations form a stable non-covalent adjuvant complex.
- LPS liposaccharide
- IVX- 908 a complex comprised of Neisseria meningitidis outer membrane and LPS. Methyl inosine 5'-monophosphate (“MIMP”) (Signorelli & Hadden (2003) Int Immunopharmacol 3(8): 1177-86).
- MIMP Methyl inosine 5'-monophosphate
- a polyhydroxlated pyrrolidine compound (WO2004/064715), such as one having formula:
- R is selected from the group comprising hydrogen, straight or branched, unsubstituted or substituted, saturated or unsaturated acyl, alkyl ⁇ e.g. cycloalkyl), alkenyl, alkynyl and aryl groups, or a pharmaceutically acceptable salt or derivative thereof.
- examples include, but are not limited to: casuarine, casuarine-6- ⁇ -D-glucopyranose, 3-e/?/-casuarine, 7-ep/-casuarine, 3,7-diepz-casuarine, etc.
- a gamma inulin (Cooper (1995) Pharm Biotechnol 6:559-80) or derivative thereof, such as algammulin.
- ⁇ R 803058', 'ER 803732', 'ER 804053', ER 804058', ⁇ R 804059', ⁇ R 804442', ⁇ R 804680', 'ER 804764', ER 803022 or ⁇ R 804057' e.g.:
- a formulation of a cationic lipid and a (usually neutral) co-lipid such as aminopropyl- dimethyl-myristoleyloxy-propanaminiumbromide-diphytanoylphosphatidyl-ethanolamine ("VaxfectinTM”) or aminopropyl-dimethyl-bis-dodecyloxy-propanaminiumbromide- dioleoylphosphatidyl-ethanolamine ("GAP-DLRIE:DOPE”).
- Formulations containing (+)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(syn-9-tetradeceneyloxy)-l-propanaminium salts are preferred (US patent 6586409)
- Compositions may include two or more of said adjuvants.
- Antigens and adjuvants in a composition will typically be in admixture.
- Oil-in-water emulsions are particularly useful as adjuvants.
- Various such emulsions are known, and they typically include at least one oil and at least one surfactant, with the oil(s) and surfactant(s) being biodegradable (metabolizable) and biocompatible.
- the oil droplets in the emulsion are generally less than 5 ⁇ m in diameter, and may even have a sub-micron diameter, with these small sizes being achieved with a microfluidizer to provide stable emulsions. Droplets with a size less than 220nm are preferred as they can be subjected to filter sterilization.
- the invention can be used with oils such as those from an animal (such as fish) or vegetable source. Sources for vegetable oils include nuts, seeds and grains.
- Jojoba oil can be used e.g. obtained from the jojoba bean.
- Seed oils include safflower oil, cottonseed oil, sunflower seed oil, sesame seed oil and the like. In the grain group, corn oil is the most readily available, but the oil of other cereal grains such as wheat, oats, rye, rice, teff, triticale and the like may also be used.
- 6- 10 carbon fatty acid esters of glycerol and 1,2-propanediol while not occurring naturally in seed oils, may be prepared by hydrolysis, separation and esterification of the appropriate materials starting from the nut and seed oils.
- Fats and oils from mammalian milk are metabolizable and may therefore be used in the practice of this invention.
- the procedures for separation, purification, saponification and other means necessary for obtaining pure oils from animal sources are well known in the art.
- Most fish contain metabolizable oils which may be readily recovered.
- cod liver oil, shark liver oils, and whale oil such as spermaceti exemplify several of the fish oils which may be used herein.
- a number of branched chain oils are synthesized biochemically in 5-carbon isoprene units and are generally referred to as terpenoids.
- Shark liver oil contains a branched, unsaturated terpenoids known as squalene, 2,6,10,15,19,23- hexamethyl-2,6,10,14,18,22-tetracosahexaene, which is particularly preferred herein.
- Squalane the saturated analog to squalene
- Fish oils, including squalene and squalane, are readily available from commercial sources or may be obtained by methods known in the art. Other preferred oils are the tocopherols (see below). Mixtures of oils can be used.
- Surfactants can be classified by their 'HLB' (hydrophile/lipophile balance). Preferred surfactants of the invention have a HLB of at least 10, preferably at least 15, and more preferably at least 16.
- the invention can be used with surfactants including, but not limited to: the polyoxyethylene sorbitan esters surfactants (commonly referred to as the Tweens), especially polysorbate 20 and polysorbate 80; copolymers of ethylene oxide (EO), propylene oxide (PO), and/or butylene oxide (BO), sold under the DOWFAXTM tradename, such as linear EO/PO block copolymers; octoxynols, which can vary in the number of repeating ethoxy (oxy-l,2-ethanediyl) groups, with octoxynol-9 (Triton X-IOO, or t-octylphenoxypolyethoxyethanol) being of particular interest; (octylphenoxy)polye
- Preferred surfactants for including in the emulsion are Tween 80 (polyoxyethylene sorbitan monooleate), Span 85 (sorbitan trioleate), lecithin and Triton X-IOO.
- Mixtures of surfactants can be used e.g. Tween 80/Span 85 mixtures, or Tween80/Triton-X100 mixtures.
- a combination of a polyoxyethylene sorbitan ester such as polyoxyethylene sorbitan monooleate (Tween 80) and an octoxynol such as t-octylphenoxypolyethoxyethanol (Triton X- 100) is also suitable.
- Another useful combination comprises laureth 9 plus a polyoxyethylene sorbitan ester and/or an octoxynol.
- Preferred amounts of surfactants are: polyoxyethylene sorbitan esters (such as Tween 80) 0.01 to 1%, in particular about 0.1 %; octyl- or nonylphenoxy polyoxyethanols (such as Triton X-100, or other detergents in the Triton series) 0.001 to 0.1 %, in particular 0.005 to 0.02%; polyoxyethylene ethers (such as laureth 9) 0.1 to 20 %, preferably 0.1 to 10 % and in particular 0.1 to 1 % or about 0.5%.
- Specific oil-in-water emulsion adjuvants useful with the invention include, but are not limited to:
- a submicron emulsion of squalene, Tween 80, and Span 85 A submicron emulsion of squalene, Tween 80, and Span 85.
- the composition of the emulsion by volume can be about 5% squalene, about 0.5% polysorbate 80 and about 0.5% Span 85. In weight terms, these ratios become 4.3% squalene, 0.5% polysorbate 80 and 0.48% Span 85.
- This adjuvant is known as 'MF59' (WO90/14837, Podda & Del Giudice (2003) Expert Rev Vaccines 2:197-203, and Podda (2001) Vaccine 19: 2673-2680), as described in more detail in Chapter 10 of Vaccine Design: The Subunit and Adjuvant Approach (eds.
- the MF59 emulsion advantageously includes citrate ions e.g. 1OmM sodium citrate buffer.
- An emulsion of squalene, a tocopherol, and Tween 80 may include phosphate buffered saline. It may also include Span 85 ⁇ e.g. at 1%) and/or lecithin. These emulsions may have from 2 to 10% squalene, from 2 to 10% tocopherol and from 0.3 to 3% Tween 80, and the weight ratio of squalene tocopherol is preferably ⁇ 1 as this provides a more stable emulsion.
- One such emulsion can be made by dissolving Tween 80 in PBS to give a 2% solution, then mixing 90ml of this solution with a mixture of (5g of DL- ⁇ -tocopherol and 5ml squalene), then microfluidising the mixture.
- the resulting emulsion may have submicron oil droplets e.g. with an average diameter of between 100 and 250nm, preferably about 180nm.
- the emulsion may also include a 3d-MPL (see below).
- the emulsion may contain a phosphate buffer.
- An emulsion comprising a polysorbate ⁇ e.g. polysorbate 80), a Triton detergent ⁇ e.g. Triton X-100) and a tocopherol ⁇ e.g. an ⁇ -tocopherol succinate).
- the emulsion may include these three components at a mass ratio of about 75:11:10 ⁇ e.g. 750 ⁇ g/ml polysorbate 80, HO ⁇ g/ml Triton X-100 and lOO ⁇ g/ml ⁇ -tocopherol succinate), and these concentrations should include any contribution of these components from antigens.
- the emulsion may also include squalene.
- the emulsion may also include a 3d-MPL (see below).
- the aqueous phase may contain a phosphate buffer.
- An emulsion of squalane, polysorbate 80 and poloxamer 401 (“PluronicTM L121").
- the emulsion can be formulated in phosphate buffered saline, pH 7.4. This emulsion is a useful delivery vehicle for muramyl dipeptides, and has been used with threonyl-MDP in the
- SAF-I adjuvant (Allison & Byars (1992) Res Immunol 143:519-25), (0.05-1% Thr-MDP, 5% squalane, 2.5% Pluronic L121 and 0.2% polysorbate 80). It can also be used without the Thr-MDP, as in the "AF" adjuvant (Hariharan et al. (1995) Cancer Res 55:3486-9) (5% squalane, 1.25% Pluronic L 121 and 0.2% polysorbate 80). Microfluidization is preferred. • An emulsion having from 0.5-50% of an oil, 0.1-10% of a phospholipid, and 0.05-5% of a non-ionic surfactant.
- preferred phospholipid components are phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidic acid, sphingomyelin and cardiolipin.
- Submicron droplet sizes are advantageous. • A submicron oil-in-water emulsion of a non-metabolizable oil (such as light mineral oil) and at least one surfactant (such as lecithin, Tween 80 or Span 80).
- Additives may be included, such as QuilA saponin, cholesterol, a saponin-lipophile conjugate (such as GPI- 0100, described in US patent 6,080,725, produced by addition of aliphatic amine to desacylsaponin via the carboxyl group of glucuronic acid), dimethyidioctadecylammonium bromide and/or N,N-dioctadecyl-N,N-bis (2-hydroxyethyl)propanediamine.
- a saponin e.g. QuilA or QS21
- a sterol e.g. a cholesterol
- An emulsion comprising a mineral oil, a non-ionic lipophilic ethoxylated fatty alcohol, and a non-ionic hydrophilic surfactant (e.g. an ethoxylated fatty alcohol and/or polyoxyethylene-polyoxypropylene block copolymer) (WO2006/113373).
- An emulsion comprising a mineral oil, a non-ionic hydrophilic ethoxylated fatty alcohol, and a non-ionic lipophilic surfactant (e.g. an ethoxylated fatty alcohol and/or polyoxyethylene-polyoxypropylene block copolymer) (WO2006/11337).
- the emulsions may be mixed with antigen extemporaneously, at the time of delivery.
- the adjuvant and antigen may be kept separately in a packaged or distributed vaccine, ready for final formulation at the time of use.
- the antigen will generally be in an aqueous form, such that the vaccine is finally prepared by mixing two liquids.
- the volume ratio of the two liquids for mixing can vary (e.g. between 5:1 and 1:5) but is generally about 1:1.
- the adjuvants known as aluminum hydroxide and aluminum phosphate may be used. These names are conventional, but are used for convenience only, as neither is a precise description of the actual chemical compound which is present (e.g. see chapter 9 of Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell & Newman) Plenum Press 1995).
- the invention can use any of the "hydroxide” or "phosphate” adjuvants that are in general use as adjuvants.
- aluminium hydroxide typically aluminium oxyhydroxide salts, which are usually at least partially crystalline.
- Aluminium oxyhydroxide which can be represented by the formula AlO(OH)
- IR infrared
- the degree of crystallinity of an aluminium hydroxide adjuvant is reflected by the width of the diffraction band at half height (WHH), with poorly-crystalline particles showing greater line broadening due to smaller crystallite sizes.
- WHH diffraction band at half height
- the surface area increases as WHH increases, and adjuvants with higher WHH values have been seen to have greater capacity for antigen adsorption.
- a fibrous morphology e.g. as seen in transmission electron micrographs
- the pi of aluminium hydroxide adjuvants is typically about 11 i.e. the adjuvant itself has a positive surface charge at physiological pH. Adsorptive capacities of between 1.8-2.6 mg protein per mg Al +++ at pH 7.4 have been reported for aluminium hydroxide adjuvants.
- aluminium hydroxyphosphate typically aluminium hydroxyphosphates, often also containing a small amount of sulfate (i.e. aluminium hydroxyphosphate sulfate). They may be obtained by precipitation, and the reaction conditions and concentrations during precipitation influence the degree of substitution of phosphate for hydroxyl in the salt.
- Hydroxyphosphates generally have a PO 4 /A1 molar ratio between 0.3 and 1.2.
- Hydroxyphosphates can be distinguished from strict AIPO 4 by the presence of hydroxyl groups. For example, an IR spectrum band at 3164cm "1 (e.g. when heated to 200 0 C) indicates the presence of structural hydroxyls [ch.9 of Vaccine Design: The Subunit and Adjuvant Approach (eds.
- the P(VAl 3+ molar ratio of an aluminium phosphate adjuvant will generally be between 0.3 and 1.2, preferably between 0.8 and 1.2, and more preferably 0.95+0.1.
- the aluminium phosphate will generally be amorphous, particularly for hydroxyphosphate salts.
- a typical adjuvant is amorphous aluminium hydroxyphosphate with PO 4 /A1 molar ratio between 0.84 and 0.92, included at 0.6mg Al 3+ AnI.
- the aluminium phosphate will generally be particulate (e.g. plate-like morphology as seen in transmission electron micrographs). Typical diameters of the particles are in the range 0.5-20 ⁇ m (e.g. about 5-10 ⁇ m) after any antigen adsorption.
- Adsorptive capacities of between 0.7-1.5 mg protein per mg Al +4"1" at pH 7.4 have been reported for aluminium phosphate adjuvants.
- Suspensions of aluminium salts used to prepare compositions of the invention may contain a buffer (e.g. a phosphate or a histidine or a Tris buffer), but this is not always necessary.
- the suspensions are preferably sterile and pyrogen-free.
- a suspension may include free aqueous phosphate ions e.g. present at a concentration between 1.0 and 20 mM, preferably between 5 and 15 mM, and more preferably about 10 mM.
- the suspensions may also comprise sodium chloride.
- the invention can use a mixture of both an aluminium hydroxide and an aluminium phosphate.
- there may be more aluminium phosphate than hydroxide e.g. a weight ratio of at least 2:1 e.g. >5:1, >6:1, >7:1, >8:1, >9:1, etc.
- concentration of Al "1"1"1" in a composition for administration to a patient is preferably less than 10mg/ml e.g. ⁇ 5 mg/ml, ⁇ 4 mg/ml, ⁇ 3 mg/ml, ⁇ 2 mg/ml, ⁇ 1 mg/ml, etc.
- a preferred range is between 0.3 and 1 mg/ml.
- Kits of the invention Where a composition includes two components for delivery to a patient, such as an Env/Tat mixture and an adjuvant, these may be mixed during manufacture, or they may be mixed extemporaneously, at the time of delivery.
- the invention provides kits including the various components ready for mixing.
- the kit allows the adjuvant and the complex to be kept separately until the time of use. This arrangement is particularly useful when using an oil-in-water emulsion adjuvant.
- the components are physically separate from each other within the kit, and this separation can be achieved in various ways.
- the two components may be in two separate containers, such as vials.
- the contents of the two vials can then be mixed e.g. by removing the contents of one vial and adding them to the other vial, or by separately removing the contents of both vials and mixing them in a third container.
- one of the kit components is in a syringe and the other is in a container such as a vial.
- the syringe can be used ⁇ e.g. with a needle) to insert its contents into the second container for mixing, and the mixture can then be withdrawn into the syringe.
- the mixed contents of the syringe can then be administered to a patient, typically through a new sterile needle. Packing one component in a syringe eliminates the need for using a separate syringe for patient administration.
- the two kit components are held together but separately in the same syringe e.g. a dual-chamber syringe, such as those disclosed in WO2005/089837, WO00/07647, WO99/17820, EP-A-0520618, WO98/01174, US patents 6,692,468, 5,971,953, 4,060,082.
- a dual-chamber syringe such as those disclosed in WO2005/089837, WO00/07647, WO99/17820, EP-A-0520618, WO98/01174, US patents 6,692,468, 5,971,953, 4,060,082.
- the kit components will generally be in aqueous form.
- a component typically the antigen component rather than the adjuvant component
- the two components can be mixed in order to reactivate the dry component and give an aqueous composition for administration to a patient.
- a lyophilised component will typically be located within a vial rather than a syringe.
- Dried components may include stabilizers such as lactose, sucrose or mannitol, as well as mixtures thereof e.g. lactose/sucrose mixtures, sucrose/mannitol mixtures, etc.
- One possible arrangement uses an aqueous adjuvant component in a pre-f ⁇ lled syringe and a lyophilised antigen component in a vial.
- the invention provides a method of raising an immune response in a patient, comprising the step of administering a composition of the invention to the patient.
- the compositions of the invention are particularly suitable for administration to human patients, but can also be administered to other mammals for investigational purposes, for raising antisera, etc.
- the invention also provides a kit or composition of the invention for use as a medicament.
- the invention also provides the use of an Env/Tat mixture of the invention in the manufacture of a medicament for raising an immune response in a patient.
- compositions of the invention can be administered in various ways.
- the most preferred immunization route is by injection (e.g. intramuscular, subcutaneous, intravenous), but other available routes include, but are not limited to, intranasal, oral, intradermal, transcutaneous, transdermal, pulmonary, etc.
- Treatment can be by a single dose schedule or a multiple dose schedule. Multiple doses may be used in a primary immunization schedule and/or in a booster immunization schedule. In a multiple dose schedule the various doses may be given by the same or different routes e.g. a parenteral prime and mucosal boost, a mucosal prime and parenteral boost, etc. Administration of more than one dose (typically two doses) is typical. Multiple doses will typically be administered at least 1 week apart (e.g. about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 8 weeks, etc.).
- composition comprising X may consist exclusively of X or may include something additional e.g. X + Y.
- a process comprising a step of mixing two or more components does not require any specific order of mixing.
- components can be mixed in any order. Where there are three components then two components can be combined with each other, and then the combination may be combined with the third component, etc.
- animal (and particularly bovine) materials are used in the culture of cells, they should be obtained from sources that are free from transmissible spongiform encaphalopathies (TSEs), and in particular free from bovine spongiform encephalopathy (BSE). Overall, it is preferred to culture cells in the total absence of animal-derived materials.
- TSEs transmissible spongiform encaphalopathies
- BSE bovine spongiform encephalopathy
- a protein or a complex "binds specifically" to a particular target (e.g. to CD4 or to a monoclonal antibody), it will typically bind to that target with at least 10-fold greater affinity than to a control protein e.g. than to CD3 or than to an anti-Rev antibody. Specific binding and non-specific binding can be distinguished by standard techniques e.g. by checking the effect of control proteins on the interaction, by checking dose-responsiveness, etc.
- polypeptide refers to amino acid polymers of any length.
- the polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acid components.
- the terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component.
- polypeptides containing one or more analogs of an amino acid including, for example, unnatural amino acids, etc.
- Polypeptides can occur as single chains or associated chains.
- Polypeptides of the invention can be naturally or non-naturally glycosylated (i.e. the polypeptide has a glycosylation pattern that differs from the glycosylation pattern found in the corresponding naturally occurring polypeptide).
- Env and Tat polypeptides for use with the invention can be prepared in many ways e.g. by chemical synthesis (in whole or in part), by digesting longer polypeptides using proteases, by translation from RNA, by purification from cell culture (e.g. from recombinant expression), from the organism itself (e.g. after bacterial culture, or direct from patients), etc.
- a preferred method for production of peptides ⁇ 40 amino acids long involves in vitro chemical synthesis (Bodanszky (1993) Principles of Peptide Synthesis (ISBN: 0387564314), Fields et al. (1997) Meth Enzymol 289: Solid-Phase Peptide Synthesis. ISBN: 0121821900).
- Solid-phase peptide synthesis is particularly preferred, such as methods based on tBoc or Fmoc (Chan & White (2000) Fmoc Solid Phase Peptide Synthesis. ISBN: 0199637245) chemistry. Enzymatic synthesis (Kullmann (1987) Enzymatic Peptide Synthesis. ISBN: 0849368413) may also be used in part or in full.
- biological synthesis may be used e.g. the polypeptides may be produced by translation. This may be carried out in vitro or in vivo.
- Biological methods are in general restricted to the production of polypeptides based on L-amino acids, but manipulation of translation machinery (e.g.
- aminoacyl tRNA molecules can be used to allow the introduction of D-amino acids (or of other non natural amino acids, such as iodotyrosine or methylphenylalanine, azidohomoalanine, etc.) (Ibba (1996) Biotechnol Genet Eng Rev 13:197- 216). Where D-amino acids are included, however, it is preferred to use chemical synthesis. Polypeptides of the invention may have covalent modifications at the C-terminus and/or N- terminus.
- Env and Tat polypeptides can take various forms (e.g. native, fusions, glycosylated, non-glycosylated, lipidated, non-lipidated, phosphorylated, non-phosphorylated, myristoylated, non-myristoylated, monomeric, multimeric, particulate, denatured, etc.).
- Env oligomeric glycosylated polypeptides are preferred.
- Env and Tat polypeptides are preferably provided in purified or substantially purified form i.e. substantially free from other polypeptides (e.g. free from naturally-occurring polypeptides), particularly from other HIV or host cell polypeptides, and are generally at least about 50% pure (by weight), and usually at least about 90% pure i.e. less than about 50%, and more preferably less than about 10% (e.g. 5% or less) of a composition is made up of other expressed polypeptides.
- the leader (amino acids 1-27; SEQ ID NO: 47) can be replaced by a leader sequence from tpa (SEQ ID NO: 48, MDAMKRGLCCVLLLCGAVFVSP).
- the gpl60 sequence can be modified to a gpl40 form (SEQ ID NO: 39):
- c gpl40mut7' SAVEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEIVLENVTENFNMWKN
- V3 loop of SEQ ID NO: 39 can be replaced with SEQ ID NO: 23, in which a central 22mer has been deleted and a flexible sequence inserted (SEQ ID NO: 40; 627mer):
- a construct encoding SEQ ID NO: 40 ('gpl40dV3-22') can be expressed in 293T cells and purified. Proteins are initially purified using GNA lectin, and are then re-purified using a ceramic hydroxyapatite column (CHAP). Small-scale and large-scale purifications are performed.
- GNA lectin GNA lectin
- CHAP ceramic hydroxyapatite column
- SEQ ID NO: 40 A modified form of SEQ ID NO: 40 ('gpl40 ⁇ V2dv3-22') in which the V2 loop is replaced by SEQ ID NO: 42 (CSF 1 KVGAGKLINC) is prepared in the same way. Its sequence is SEQ ID NO: 41 :
- Example 1 After confirming purity, oligomerization and CD4-binding activity for both gpl40dV3-22 and gpl40 ⁇ V2dv3-22 proteins using HPLC, are assayed for both CD4- and tat-binding activity using the BIACORETM system. Results using immobilized CD4, immobilized tat or immobilized tat-cys (a mutant of tat that still binds to env (Ensoli et al. (2005) Microbes Infect 7:1392-9) are summarized below:
- gp140 S F162 5.30 + 0.76% 3.88 + 3.13% 0.13 + 96.4% 3.64 4.96 150.1 gp140 ⁇ V2 S Fi62 7.60 + 0.67% 5.50 + 1.37% 4.02 + 1.80% 2.53 3.50 4.79 gp140dV3-22sFi62 5.48 + 0.63% 6.24 + 2.03% 4.61 + 1.82% 3.51 3.08 4.17 gp140 ⁇ vi 6.48 + 0.82% 10.0 + 1.22% 7.38 + 1.69% 2.97 1.92 2.61 gp140dV3-22 ⁇ vi 3.47 + 0.67% 6.98 + 2.82% 3.84 + 4.35% 5.55 2.76 5.01
- BIACORETM can also be used to test binding of the SF162-derived proteins to (i) CD4, (ii) neutralizing antibody bl2, which binds to gpl20's CD4-binding site (Zwick et al. (2003) J Virol 77:5863-76), and (iii) non-neutralizing antibody 4.8d, which binds to a conformational epitope on gpl20.
- Dissociation constants M "1 x 10 "10
- Rmax values using 4.8d & CD4
- 4.8d is a CD4 inducible epitope antibody. It recognizes env alone at a low level, once env has undergone its conformational change due to CD4 binding.
- the 'x 4.8d up-reg' figure indicates the BIAcore signal observed for env mixed with CD4 as a function of the signal observed for env without CD4, on the 4.8d antibody.
- Tat-binding for the SF162-derived proteins is also tested by a Far- Western assay. Results are shown in Figure 1.
- a kinetic experimental environment i.e. BIAcore
- a Far Western analysis trimeric envelope and its variants are able to bind to tat.
- Monomeric gpl20 does not generally show any evidence of binding to the tat polyprotein in either experiment, unless the V2 loop is deleted as in gpl20 ⁇ V2.
- V3 loop substitutions may be designed. Alignment of SEQ ID NO: 44 with SEQ ID NOS: 25 to 29 suggests that the middle of the V3 loop is more variable than the outer flanking regions. In mutant loop SEQ ID NO: 15, the middle portion is deleted and the flanking regions remain intact. In SEQ ID NOs: 16 and 17 the N- or C- terminus flanking region is deleted. In SEQ ID NOs: 18 to 21 contact sites for monoclonal antibody 447D are removed. In SEQ ID NO: 22, the loop is replaced with a flexible Gly-Ala-Gly sequence. In SEQ ID NO: 23 this flexible sequence is also inserted into the SEQ ID NO: 21 loop. In SEQ ID NO: 24, a different V3 loop is substituted into SF 162. SF162dV3-20 CTRPNMM GAGDIRQAHC (SEQ ID NO: 15)
- the deleted peptide sequences (SEQ ID NOS: 30 to 37) are synthesized and evaluated for their ability to bind to Tat.
- Biologically active Tat binds the HIV Env through high affinity interactions with the V3 loop. This requires exposure and/or conformational transitions of the V3 loop that are induced upon Env oligomerization or V2 loop deletion. Shortening of the V1-V2 loop is a key feature of virus isolates emerging during early infection, which, however, are much more sensitive to neutralization. These data point to a key role of Tat in shielding these isolates from the mounting humoral immune response. Further, binding of the V3 loop of Env to Tat resembles the interaction of Env with the CCR5 co-receptor, a process that is also potentially enhanced by Vl- V2 deletion or shortening. This indicates that the Tat-Env complex may impact virus entry and transmission to T cells, in particular the low CCR5 expressing CD4+ T cells that appear to be early targets of infection in mucosal tissues.
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