Note: Descriptions are shown in the official language in which they were submitted.
CA 02697370 2010-02-22
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HIV ENV PROTEINS WITH MODIFICATIONS IN THE V3 LOOP
This application claims priority from US provisional application 60/966,048,
the full contents of
which are incorporated herein by reference.
GOVERNMENT SUPPORT
The application was made with support from the United States NIAID-NIH HIVRAD
under
Grant No. 5P01 A148225-03. Thus, the U.S. Government may have certain rights
in this
invention.
TECHNICAL FIELD
This invention is in the field of human immunodeficiency virus (HIV) and, in
particular, the viral
envelope glycoproteins and their manipulation.
BACKGROUND OF THE INVENTION
The various proteins encoded within the HIV genome include the envelope
glycoprotein (Env)
and the trans-activating transcriptional factor (Tat).
In both HIV-1 and HIV-2 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. For convenience, these proteins are hereafter referred to by the
standard HIV-1
nomenclature i.e. the precursor is `gp160', the membrane glycoprotein is
`gp120' and the
transmembrane glycoprotein is 'gp4l'. These names are based on approximate
molecular weights
of the HIV-1 glycoproteins.
Situated on the surface of HIV virions, gp120 can interact with the host cell
CD4 receptor. This
interaction induces a conformational transition in gp120, leading to the
exposure of its `V3' loop.
Deletion studies have suggested that accessibility of the V3 region is
influenced by the 'Vl' and
`V2' loops, see, Sourial et al. (2006) Curr HIV Res. 4(2):229-37. For example,
Srivastava et al.
(2003) J Virol 77(4):2310-20 reports that deletion of the V2 loop can alter
the immunogenicity of
the V 1 and V3 loops.
Because of its surface exposure, gp120 has been the main focus of HIV vaccine
research over the
last 20 years. While anti-Env antibodies that arise during natural infection
have been found to
neutralize primary HIV isolates, however, the same has not been true of
antibodies elicited by
Env-based subunit vaccines. Improvements to Env-based vaccines are therefore
required.
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.
W02005/090391 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
CA 02697370 2010-02-22
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combination of Env and Tat polypeptides is also disclosed in e.g., Ensoli et
al. (2005) Microbes
Infect 7:1392-9.
It is an object to provide Env-derived polypeptides with altered Tat-binding
properties.
SUMMARY OF THE INVENTION
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. In one
embodiment, the HIV Tat polypeptide and the HIV Env polypeptide form a
complex. In certain
embodiments, 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. In one embodiment, 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.
In certain embodiments, 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. In certain embodiments, the polypeptide of (ii) is <30
amino acids long. In
other embodiments, the polypeptide of (ii) is cyclic. In further embodiments,
the fragment of (ii)
has an amino acid sequence selected from group consisting of SEQ ID NOs: 30 to
37.
In certain embodiments, the Tat polypeptide of the mixtures, processes or
polypeptides of the
invention has amino acid sequence SEQ ID NO: 12.
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The present invention is also directed to a pharmaceutical composition
comprising a mixture of
the invention. In certain embodiments, 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.
BRIEF DESCRIPTION OF THE DRAWING
Figure 1 shows the results of a Far-Western assay. Lanes are: (1) markers at
35, 50, 75, 105, 160
and 250 kDa; (2) gpl40AV2; (3) gpl400V2 + CD4; (4) gpl40dV3-22; (5) gpl40dV3-
22 + CD4;
(6) gp120; (7) gp120 + CD4; and (8) CD4.
DETAILED DESCRIPTION OF THE INVENTION
In general terms, 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
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WO 2009/029569 PCT/US2008/074179
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.
The Env polypeptide
In some embodiments, mixtures of the invention include a HIV Env polypeptide
that has one or
more mutations in its V3 loop.
Various forms of Env polypeptide can be used, from HIV-1 or HIV-2. For
example, the mixture
may include a full-length gp160 Env polypeptide with V3 mutation(s), a gp120
Env polypeptide
with V3 mutation(s), a truncated gp120 or gp160 Env polypeptide with V3
mutation(s), gp160 or
gp120 polypeptide with V3 mutation(s) and one or more deletions outside V3, a
fusion protein
including a gp120 or gp160 polypeptide with V3 mutation(s), etc. Rather than
being a full-length
Env precursor with V3 mutation(s), however, the invention will typically use a
shortened protein.
The amino acid sequence of the full-length HIV-1 Env precursor from the REFSEQ
database
(GI:9629363) is a 856mer shown below (SEQ ID NO: 1 herein; V3 loop
underlined):
MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWA
THACVPTDPNPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPCVKLTPLCVSLKCTDLKNDTNT
NSSSGRMIMEKGEIKNCSFNISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYKLTSCNTSVITQACPKVSF
EPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCTHGIRPWSTQLLLNGSLAEEEWIRSVNFTDN
AKTIIVQLNTSVEINCTRPNNNTRKRIRIQRGPGRAFVTIGKIGNMRQAHCNISRAKWNNTLKQIASKLR
EQFGNNKTIIFKQSSGGDPEIVTHSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTITLPCRI
KQIINMWQKVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSNNESEIFRPGGGDMRDNWRSELYKYKVVK
IEPLGVAPTKAKRRWQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQLLSGIVQQQNNLLRAIE
AQQHLLQLTVWGIKQLQARILAVERYLKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWNHTTWME
WDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWFNITNWLWYIKLFIMIVGGLVGLRIVFA
VLSIVNRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGERDRDRSIRLVNGSLALIWDDLRSLCLFSYHR
LRDLLLIVTRIVELLGRRGWEALKYWWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIEWQGACRAI
RHIPRRIRQGLERILL
This wild-type HIV-1 precursor protein is cleaved to give the surface
glycoprotein gp120 (e.g.
amino acids 29-511 of SEQ ID NO: 1; SEQ ID NO: 2 herein) and the transmembrane
domain
gp4l (e.g. amino acids 512-856 of SEQ ID NO: 1; SEQ ID NO: 3 herein):
MRVKEKYQHLWRWGWRWGTMLLGMLMIC/SATEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVW
ATHACVPTDPNPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWDQSLKPCVKLTPLCVSLKCTDLKNDTN
TNSSSGRMIMEKGEIKNCSFNISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYKLTSCNTSVITQACPKVS
FEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCTHGIRPWSTQLLLNGSLAEEEWIRSVNFTD
NAKTIIVQLNTSVEINCTRPNNNTRKRIRIQRGPGRAFVTIGKIGNMRQAHCNISRAKWNNTLKQIASKL
REQFGNNKTIIFKQSSGGDPEIVTHSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTITLPCR
IKQIINMWQKVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSNNESEIFRPGGGDMRDNWRSELYKYKVV
KIEPLGVAPTKAKRRWQREKR/AVGIGALFLGFLGAAGSTMGAASMTLTVQARQLLSGIVQQQNNLLRA
IEAQQHLLQLTVWGIKQLQARILAVERYLKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWNHTTW
MEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWFNITNWLWYIKLFIMIVGGLVGLRIV
FAVLSIVNRVRQGYSPLSFQTHLPTPRGPDRPEGIEEEGGERDRDRSIRLVNGSLALIWDDLRSLCLFSY
HRLRDLLLIVTRIVELLGRRGWEALKYWWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIEWQGACR
AIRHIPRRIRQGLERILL
The hypervariable regions within the gp120 region are located as follows,
numbered according to
SEQ ID NO: 1: V1 = 131-157; V2 = 157-196; V3 = 296-331; V4 = 385-418; and V5 =
461-471.
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Within the overall CI-V1-V2-C2-V3-C3-V4-C4-V5-C5 arrangement of gp120,
therefore, 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-1 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
For instance, the Env sequence (SEQ ID NO: 38) after removal of the leader is
shown below for
the SF162 strain (SEQ ID NO: 7; V3 loop underlined). It is cleaved after Arg-
475 to give the
mature proteins, including the gp120 (SEQ ID NO: 50 herein, namely amino acids
1-475 of SEQ
ID NO: 7):
SAVEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEIVLENVTENFNMWKN
NMVEQMHEDIISLWDQSLKPCVKLTPLCVTLHCTNLKNATNTKSSNWKEMDRGEIKNCSFKVTTSIRNKM
QKEYALFYKLDVVPIDNDNTSYKLINCNTSVITQACPKVSFEPIPIHYCAPAGFAILKCNDKKFNGSGPC
TNVSTVQCTHGIRPVVSTQLLLNGSLAEEGWIRSENFTDNAKTIIVQLKESVEINCTRPNNNTRKSITI
GPGRAFYATGDIIGDIRQAHCNISGEKWNNTLKQIVTKLQAQFGNKTIVFKQSSGGDPEIVMHSFNCGGE
FFYCNSTQLFNSTWNNTIGPNNTNGTITLPCRIKQIINRWQEVGKAMYAPPIRGQIRCSSNITGLLLTRD
GGKEISNTTEIFRPGGGDMRDNWRSELYKYKWKIEPLGVAPTKAKR.RWQREKRAVTLGAMFLGFLGAA
GSTMGARSLTLTVQARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARVLAVERYLKDQQLLGIW
GCSGKLICTTAVPWNASWSNKSLDQIWNNMTWMEWEREIDNYTNLIYTLIEESQNQQEKNEQELLELDKW
ASLWNWFDISKWLWYIKIFIMIVGGLVGLRIVFTVLSIVNRVRQGYSPLSFQTRFPAPRGPDRPEGIEEE
GGERDRDRSSPLVHGLLALIWDDLRSLCLFSYHRLRDLILIAARIVELLGRRGWEALKYWGNLLQYWIQE
LKNSAVSLFDAIAIAVAEGTDRIIEVAQRIGRAFLHIPRRIRQGFERALL
The hypervariable regions in the SF162 strain, numbered according to SEQ ID
NO: 7, are:
V1 = 98-128; V2 = 128-167; V3 = 267-301; V4 = 354-381; and V5 = 423-435.
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):
MCGKSLLCVASLLASAYLVYCTQYVTVFYGVPVWRNASIPLFCATKNRDTWGTIQCKPDNDDYQEITLNV
TEAFDAWDNTVTEQAVEDVWSLFETSIKPCVKLTPLCVAMSCNSTTNNTTTTGSTTGMSEINETSPSYSD
NCTGLGKEEIVNCQFYMTGLERDKKKQYNETWYSKDVVCESNNTKDGKNRCYMNHCNTSVITESCDKHYW
DAIKFRYCAPPGYALLRCNDTNYSGFEPKCSKWASTCTRMMETQTSTWFGFNGTRAENRTYIYWHGRDN
RTIISLNKYYNLSIHCKRPGNKTVVPITLMSGLVFHSQPINTRPRQAWCWFKGKWREAMQEVKQTLIKHP
RYKGTNDTKNINFTKPGRGSDPEVAYMWTNCRGEFLYCNMTWFLNWVENRPNQTQHNYAPCHIRQIINTW
HKVGKNVYLPPREGQLTCNSTVTSIIANIDVNSNQTNITFSAEVAELYRLELGDYKLIEVTPIGFAPTRE
KRYSSAPVRNKRGVFVLGFLGFLATAGSAMGAASLTLSAQSRTLLAGIVQQQQQLLDVVKRQQEMLRLTV
WGTKNLQARVTAIEKYLKDQAQLNSWGCAFRQVCHTTVPWVNDSLSPDWNNMTWQEWEKQVRYLEANISQ
SLEQAQIQQEKNMYELQKLNSWDVFGNWFDLTSWIKYIQYGVYIWGVIVLRIAIYIVQLLSRLRKGYRP
VFSSPPGYLQQIHIHTDRGQPANEGTEEDDRDDDGYDLXPWPINYIHFLIHLLTRLLTGLYKICRDLLST
NSPTHRLISQNLTAIRDWLRLKAAYLQYGGEWIQEAFQAFAKTTRETLASAWGGLCAAVQRVGRGILAVP
RRIRQGAEIALL
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
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NRTIISLNKYYNLSIHCKRPGNKTWPITLMSGLVFHSQPINTRPRQAWCWFKGKWREAMQEVKQTLIKH
PRYKGTNDTKNINFTKPGRGSDPEVAYMWTNCRGEFLYCNMTWFLNWVENRPNQTQHNYAPCHIRQIINT
WHKVGKNVYLPPREGQLTCNSTVTSIIANIDVNSNQTNITFSAEVAELYRLELGDYKLIEVTPIGFAPTR
EKRYSSAPVRNKR/GVFVLGFLGFLATAGSAMGAASLTLSAQSRTLLAGIVQQQQQLLDVVKRQQEMLRL
TVWGTKNLQARVTAIEKYLKDQAQLNSWGCAFRQVCHTTVPWVNDSLSPDWNNMTWQEWEKQVRYLEANI
SQSLEQAQIQQEKNMYELQKLNSWDVFGNWFDLTSWIKYIQYGVYIVVGVIVLRIAIYIVQLLSRLRKGY
RPVFSSPPGYLQQIHIHTDRGQPANEGTEEDDRDDDGYDLXPWPINYIHFLIHLLTRLLTGLYKICRDLL
STNSPTHRLISQNLTAIRDWLRLKAAYLQYGGEWIQEAFQAFAKTTRETLASAWGGLCAAVQRVGRGILA
VPRRIRQGAEIALL
The hypervariable regions etc. can, again, be identified by sequence alignment
and by reference
to the alignments in the Los Alamos HIV Sequence Compendia
Other specific Env sequences that can be used include those disclosed in
W000/39302,
W003/020876, WO2005/007808, W003/004620, W000/39304
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.
An Env polypeptide used with the invention may further include mutations
outside the V3 loop.
For example, 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.
For instance, it is known to make a soluble form of the Env precursor by
removing its
transmembrane domain and cytoplasmic tail. This polypeptide, which includes
the gp120
sequence and the ectodomain of gp4l, is known as `gp140' (Zhang et al. (2001)
J. Biol. Chem.
276:39577-85), and has been reported to be a better immunogen than gp120 (Earl
et al. (2001) J
Virol 75:645-53). Thus the precursor is truncated at its C-terminus e.g. after
Ile-682 of SEQ ID
NO:1 (giving a gp140 sequence having amino acids Ser-29 to Ile-682 of SEQ ID
NO: 1; SEQ ID
NO: 49 herein), or after Ile-673 of SEQ ID NO: 38 (giving a gp140 sequence
having amino acids
Ser-28 to Ile-673 of SEQ ID NO: 38; SEQ ID NO: 39 herein,). Thus an Env
polypeptide used
with the invention may include a portion of gp4l but not include its
transmembrane domain.
It is also known to make deletions within the V2 loop of the Env precursor, to
give 'AV2'
mutants. For instance, 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. In particular, Env/Tat complexes may not
be seen with
monomeric gp120 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
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with a Gly-Ala-Gly tripeptide. For example, a AV2 mutant may have the
following sequence
(SEQ ID NO: 8):
SATEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEVVLVNVTENFNMWKN
DMVEQMHEDIISLWDQSLKPCVKLTPLCVSLKCTDLKNDTNTNSSSGRMIMEKGEIKNCXCNTSVITQAC
PKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPCTNVSTVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSV
NFTDNAKTIIVQLNTSVEINXNISRAKWNNTLKQIASKLREQFGNNKTIIFKQSSGGDPEIVTHSFNCGG
EFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTITLPCRIKQIINMWQKVGKAMYAPPISGQIRCSSNIT
GLLLTRDGGNSNNESEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKRRVVQREKR
where: (i) the `X' at position 130 represents a mutant V2 loop e.g. with
between 4 and 15 amino
acids; and (ii) the `X' at position 231 represents a mutant V3 loop.
Another useful AV2 mutant, based on SF162, may have the following sequence
(SEQ ID NO:
45):
SAVEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEIVLENVTENFNMWKN
NMVEQMHEDIISLWDQSLKPCVKLTPLCVTLHCTNLKNATNTKSSNWKEMDRGEIKNCXCNTSVITQACP
KVSFEPIPIHYCAPAGFAILKCNDKKFNGSGPCTNVSTVQCTHGIRPVVSTQLLLNGSLAEEGVVIRSEN
FTDNAKTIIVQLKESVEINCXCNISGEKWNNTLKQIVTKLQAQFGNKTIVFKQSSGGDPEIVMHSFNCGG
EFFYCNSTQLFNSTWNNTIGPNNTNGTITLPCRIKQIINRWQEVGKAMYAPPIRGQIRCSSNITGLLLTR
DGGKEISNTTEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPTKAKRRVVQREKR
where: (i) the `X' at position 129 represents a mutant V2 loop e.g. with
between 4 and 15 amino
acids; and (ii) the `X' at position 231 represents a mutant V3 loop.
A particularly preferred Env polypeptide for use with the invention is a gp140
protein with a AV2
mutation from HIV-1 strain SF162. In its mature form, after cleavage of a
signal sequence and
secretion (see, e.g., Figure 24 of W000/39302), this polypeptide has the
following amino acid
sequence (SEQ ID NO: 9):
SAVEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEIVLENVTENFNMWKN
NMVEQMHEDIISLWDQSLKPCVKLTPLCVTLHCTNLKNATNTKSSNWKEMDRGEIKNCSFKVGAGKLINC
NTSVITQACPKVSFEPIPIHYCAPAGFAILKCNDKKFNGSGPCTNVSTVQCTHGIRPWSTQLLLNGSLA
EEGVVIRSENFTDNAKTIIVQLKESVEINCTRPNNNTRKSITIGPGRAFYATGDIIGDIRQAHCNISGEK
WNNTLKQIVTKLQAQFGNKTIVFKQSSGGDPEIVMHSFNCGGEFFYCNSTQLFNSTWNNTIGPNNTNGTI
TLPCRIKQIINRWQEVGKAMYAPPIRGQIRCSSNITGLLLTRDGGKEISNTTEIFRPGGGDMRDNWRSEL
YKYKWKIEPLGVAPTKAISSWQSEKSAVTLGAMFLGFLGAAGSTMGARSLTLTVQARQLLSGIVQQQN
NLLRAIEAQQHLLQLTVWGIKQLQARVLAVERYLKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLDQIW
NNMTWMEWEREIDNYTNLIYTLIEESQNQQEKNEQELLELDKWASLWNWFDISKWLWYI
In the present invention, the wild-type SF162 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.
As the HIV genome is in a state of constant flux, and contains several domains
that exhibit
relatively high degrees of variability between isolates, the invention is not
limited to the use of
Env polypeptides having the exact sequence of a known HIV polypeptide. Thus
the Env
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polypeptide used according to the invention may be selected from one of the
following, provided
that it includes a mutant V3 loop:
(i) a 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;
(ii) a 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;
(iii) 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);
(iv) a 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
(v) 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 x=y identical aligned monomers in
each window
of x amino acids moving from N-terminus to C-terminus, where: p>x; there are p-
x+l
windows; x is selected from 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100,
150, 200, 250,
300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800 or 850; 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, ifx=y
is not an integer, it is rounded up to the nearest integer.
These 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.
Within category (ii), 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).
Identity
between polypeptides is preferably determined by the Smith-Watennan homology
search
algorithm as implemented in the MPSRCH program (Oxford Molecular), using an
affine gap
search with parameters gap open penalty=12 and gap extension penalty=l.
Within category (iii), 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
8
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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. 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, etc.) single amino acid deletions 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. 1, 2, 3,
4, 5, 6, 7, 8, 9, 10,
etc.) single amino acid insertion relative to SEQ ID NO: 1, 2, 4, 5, 7, 8, 9,
38, 39, 43, 45, 49 or
50. The 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. aspartate, glutamate; (2) basic i.e. lysine,
arginine, histidine; (3) non-polar
i.e. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine,
tryptophan; and
(4) uncharged polar i.e. glycine, asparagine, glutamine, cysteine, serine,
threonine, tyrosine.
Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as
aromatic amino acids.
In general, substitution of single amino acids within these families does not
have a major effect
on the biological activity. Various substitutions have been described for use
with Env
polypeptides e.g. it is known to inactivate the cleavage site between gp120
and gp4l (e.g. by a
Lys--*Ser substitution) in order to provide a polypeptide that remains in full-
length form, or to
remove the `clipping' site in the V3 loop (W091/15238), or to delete or
substitute glycosylation
sites, particularly N-glycosylation sites (i.e. asparagine residues).
Within category (iv), the value of 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. 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 Mol Biol
36:207-223) or
similar methods), or they can be predicted (e.g. using the Jameson-Wolf
antigenic index
(Jameson, BA et al. 1988, CABIOS 4(1):181-186), matrix-based approaches
(Raddrizzani &
Hammer (2000) Brief Bioinform 1(2):179-189), TEPITOPE (De Lalla et al. (1999)
J. Immunol.
163:1725-1729), neural networks (Brusic et al. (1998) Bioinformatics 14(2):121-
130), OptiMer &
EpiMer (Meister et al. (1995) Vaccine 13(6):581-591; Roberts et al. (1996)
AIDS Res Hum
Retroviruses 12(7):593-610), ADEPT (Maksyutov & Zagrebelnaya (1993) Comput
Appl Biosci
9(3):291-297), Tsites (Feller & de la Cruz (1991) Nature 349(6311):720-1),
hydrophilicity (Hopp
(1993) Peptide Research 6:183-190), antigenic index (Welling et al. (1985)
FEBS Lett. 188:215-
218) or the methods disclosed in Davenport et al. (1995) Immunogenetics 42:392-
297).
Within category (v), the preferred pairwise alignment algorithm is the
Needleman-Wunsch global
alignment algorithm (Needleman& Wunsch (1970) J. Mol. Biol. 48:443-453), using
default
parameters (e.g. with Gap opening penalty = 10.0, and with Gap extension
penalty = 0.5, using
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the EBLOSUM62 scoring matrix). 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, AV2
mutants of gp140 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 gp120, unless its
V2 loop is
mutated, but are formed from trimeric gp 140 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. As mentioned above, Env polypeptides in mixtures
of the invention
will have one or more mutations in the V3 loop relative to a wild-type Env
sequence.
In wild-type HIV-1 Env sequence SEQ ID NO: 2, the V3 loop consists of amino
acids Cys-268 to
Cys-303 (SEQ ID NO: 13):
CTRPNNNTRKRIRIQRGPGRAFVTIGKIGNMRQAHC
In wild-type HIV-1 Env sequence from strain SF162 (SEQ ID NO: 7), the V3 loop
consists of
amino acids Cys-267 to Cys-301 (SEQ ID NO: 46):
CTRPNNNTRKSITIGPGRAFYATGDIIGDIRQAHC
In wild-type HIV-2 Env sequence SEQ ID NO: 4, the V3 loop consists of amino
acids Cys-296 to
Cys-329 (SEQ ID NO: 14):
CKRPGNKTVVPITLMSGLVFHSQPINTRPRQAWC
The location of the 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:
SF162 CTRPNNNTRKSITIGPGRAFYATGDIIGDIRQAHC (SEQ ID NO: 44)
TV1 CTRPNNNTRKSVRIGPGQAFYATNDVIGNIRQAHC (SEQ ID NO: 25)
MJ4 CTRPGNNTRRSVRIGPGQAFYATGDIIGDIRAAHC (SEQ ID NO: 26)
CM235 CTRPSNNTRTSITIGPGQVFYRTGDIIGDIRKAYC (SEQ ID NO: 27)
Q461 CIRPGNNTRKSVRIGPGQAFYATGDITGDIRNAHC (SEQ ID NO: 28)
Consensus CTRPNNNTRKSVRIGPGQAFYATGDIIGDIRQAHC (SEQ ID NO: 29)
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.
CA 02697370 2010-02-22
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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).
Mutations in the V3 loop of Env have previously been reported, and any of
these can be used
with invention. For instance, Su et al. (2000) AIDS Res Hum Retroviruses.
16(1):37-48 reports
viruses with deletion of the conserved Gly-Pro-Gly motif from the centre of
the V3 loop.
Kmieciak et al. (1998) J Immunol. 160(11):5676-83 analyzed the effect of
deletion of the V3
loop, and the AV3 mutants showed increased CTL activities in vitro against
conserved epitopes
of the env protein. The authors of Jagodzinski et al. (1996) Virology
226(2):217-27 deleted the
V3 loop and also transplanted V3 loops between different HIV strains. Hansen
et al. (1996) Arch
Virol 141(2):291-300 mutated Thr and Ser residues in the V3 loop to prevent 0-
glycosylation,
substituting Ala residues instead. Similarly, Kang et al. (2005) Virology
331(1):20-32 mutated
glycosylation sites in the V3 loop. Chiou et al. (1992) AIDS Res Hum
Retroviruses 8(9):1611-8
prepared Env proteins with a series of deletions in the V3 loop. Pollard et
al. (1992) EMBO J.
11(2):585-91 used deletions to show that 62 N- and 20 C-terminal residues
along with the V1, V2
and V3 variable regions of gp 120 were unnecessary for CD4 binding. Sanders et
al. (2000) J
Virol 74(11):5091-100 characterized gp140 variants with deletions in the Vl,
V2 and/or V3
loops. Yang et al. (2004) J Virol. 78(8):4029-36 shortened the stem of the V3
loop by selective
deletions, and progressive shortening of the stem abolished immunogenicity and
functional
activity of HIV Env, suggesting that highly conserved subregions within V3 may
be relevant
targets for eliciting neutralizing antibody responses, for affecting HIV
tropism, and for increasing
the immunogenicity of vaccines. Gzyl et al. (2004) Virology 318(2):493-506
reports that partial
deletions in the VI, V2 and/or V3 loops may facilitate approaches for boosting
cross-reactive
cellular and antibody responses to the Env glycoprotein.
Examples of mutant V3 loop sequences for use in Env polypeptides of the
invention, each
including several consecutive amino acid deletions, are shown below as SEQ ID
NOS: 15 to 22:
CTRPNNNGAGDIRQAHC (SEQ ID NO: 15)
CTITIGPGRAFYATGDIIGDIRQAHC (SEQ ID NO: 16)
CTRPNNNTRKSITIGPGRAFYATQAHC (SEQ ID NO: 17)
CTRPNNNTRFYATGDIIGDIRQAHC (SEQ ID NO: 18)
CTRPNNNTRGDIIGDIRQAHC (SEQ ID NO: 19)
CTFYATGDIIGDIRQAHC (SEQ ID NO: 20)
CTRPNNNTRQAHC (SEQ ID NO: 21)
CTGAGHC (SEQ ID NO: 22)
CTRPNNNTRGAGQAHC (SEQ ID NO: 23)
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A further example of a V3 loop sequence for use in Env polypeptides of the
invention, with three
substitutions relative to the wild-type SF162 V3 sequence, is shown below (SEQ
ID NO: 24):
CTRPNNNTRKSITIGPGRAFYATGDIIGNMRQAHC (SEQ ID NO: 24)
The consensus sequence (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
V3fragments
In some embodiments, 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.mil/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.
Eight specific V3-derived polypeptides for use with the invention are shown
below:
TRKSITIGPGRAFYATGD (SEQ ID NO: 30)
RPNNNTRKS (SEQ ID NO: 31)
GDIIGDIR (SEQ ID NO: 32)
KSITIGPGRA (SEQ ID NO: 33)
KSITIGPGRAFYAT (SEQ ID NO: 34)
RPNNNTRKSITIGPGRA (SEQ ID NO: 35)
KSITIGPGRAFYATGDIIGDIR (SEQ ID NO: 36)
RPNNNTRKSITIGPGRAFYATGDIIGDIRQA (SEQ ID NO: 37)
The Tat polypeptide
Mixtures of the invention include a HIV Tat polypeptide, and various forms of
Tat polypeptide
can be used from HIV-1 or HIV-2. The length of the Tat polypeptide varies
depending on virus
strain. The amino acid sequence of the full-length HIV-1 Tat polypeptide from
the REFSEQ
database (GI:9629358) is a 86mer shown below (SEQ ID NO: 10 herein):
MEPVDPRLEPWKHPGSQPKTACTNCYCKKCCFHCQVCFITKALGISYGRKKRRQRRRAHQNSQTHQASLS
KQPTSQPRGDPTGPKE
Within the various HIV-1 Tat polypeptide sequences, Cys-22 and Cys-37 are
conserved and form
an intramolecular disulfide bond. The RKKRRQRRR 9-mer (SEQ ID NO: 51) is a
nuclear
localization signal. These features can be identified in other HIV-1 Env
sequences by performing
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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.
The amino acid sequence of a full-length HIV-2 Tat polypeptide (GI:41056781)
is a 130mer
shown below (SEQ ID NO: 11 herein):
METPLKAPESSLMSYNEPSSCTSERDVGSQELAKQGEELLSQLHRPLEPCNNKCYCKGCCFHCQLCFLNK
GLGICYDRKGRRRRTPKKTKAHSSSASDKSISTRTGNSQPEKKQKKTLETTLETARGLGR
An alignment of this and other 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 W000/39302,
W003/020876, W02005/007808, W003/004620 and W099/27958.
A particularly useful Tat polypeptide for use with the invention is from HIV-1
strain BH10. This
polypeptide has the following amino acid sequence (SEQ ID NO: 12;
GI:62291022):
MEPVDPRLEPWKHPGSQPKTACTNCYCKKCCFHCQVCFITKALGISYGRKKRRQRRRPPQGSQTHQVSLS
KQPTSQSRGDPTGPKE
As the HIV genome is in a state of constant flux, and contains several domains
that exhibit
relatively high degrees of variability between isolates, the invention is not
limited to the use of
Tat polypeptides having the exact sequence of a known HIV polypeptide. Thus
the Tat
polypeptide used according to the invention may be selected from:
(i) a polypeptide comprising an amino acid sequence selected from SEQ ID NOs:
10, 11 and
12;
(ii) a polypeptide comprising an amino acid sequence that has sequence
identity to an amino
acid sequence selected from SEQ ID NOs: 10, 11 and 12;
(iii) a 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;
(iv) a 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; or
(v) 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 x=y 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
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, if x=y is not an integer, it is rounded up to the nearest integer.
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These 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.
Within category (ii), 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).
Identity
between polypeptides is preferably determined by the Smith-Watennan homology
search
algorithm as implemented in the MPSRCH program (Oxford Molecular), using an
affine gap
search with parameters gap open penalty=12 and gap extension penalty=l.
Within category (iii), 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.
Within category (iv), the value of 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.
Within category (v), the preferred pairwise alignment algorithm is the
Needleman-Wunsch global
alignment algorithm as described above.
Env/Tat mixtures
Mixtures of the invention include Env and Tat polypeptides. In the mixture,
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-1 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-1, 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
14
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WO 2009/029569 PCT/US2008/074179
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
CRF (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-1 nomenclature is discussed in more detail in Robertson et al.
(2000) Science
288:55-6.
The use of 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. For instance, the Env and Tat polypeptides may both be from the SF162
strain (subtype
B), or the invention may use Env from one strain (e.g. SF162) and Tat from
another strain (e.g.
BH 10).
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. Thus 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. As described in US provisional patent application
60/786,947 filed March 28
2006, 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.
Where a complex binds specifically to CD4, this binding activity can be
assessed using known
assays e.g. as described in W091/13906. 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 W091/13906). 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.
Where the complex binds specifically to an anti-Tat monoclonal antibody, a
preferred
monoclonal antibody is 8D1.8, 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.
CA 02697370 2010-02-22
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77:5415-27, Avraham et al. (2004) J Immunol 173:6228-33, Liu et al. (2002) J
Virol 76:6689-
700.
EnvlTat covalent cross-linking
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)
Chemistry ofprotein conjugation and cross-linking. ISBN 0-8493-5886-8 and
Hermanson (1996)
Bioconjugate techniques. ISBN 0-12-342336-8. For example, 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, a-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 `BS3'), 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.
Methods for cross-linking proteins using these reagents are known in the art.
Generally, the
invention will involve mixing Env polypeptide, Tat polypeptide and a linking
reagent under
conditions that permit the covalent linking reaction to proceed. In some two-
step procedures,
however, such as those using a heterobifunctional reagent, 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. If a
linker 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. As an alternative, the photoreactive group can be used first.
As mentioned above, 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. Thus
the concentration of
the Env and Tat proteins, the concentration of the cross-linking reagent(s),
the pH, the reaction
16
CA 02697370 2010-02-22
WO 2009/029569 PCT/US2008/074179
temperature and the reaction time can be controlled to give the desired degree
of cross-linking.
When testing a particular combination of Env, Tat and cross-linking reagent
then an initial series
of reactions can be performed to evaluate suitable reaction conditions.
Pharmaceutical compositions
Mixtures 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. In a
patient already infected
with HIV, the immune response may reduce the severity of the infection (e.g.
reduce viral load)
and may even result in clearance of HIV infection. In a patient who is not
infected with HIV, the
immune response may reduce the risk of future HIV infection and may even be
protective against
future HIV infection. These effects arising from administration of the
immunogenic composition
of may be augmented by, or also require, the use of other anti-HIV strategies
e.g. the
administration of antivirals, including but not limited to nucleoside reverse
transcriptase
inhibitors, non-nucleoside reverse transcriptase inhibitors, protease
inhibitors, entry inhibitors,
fusion inhibitors, etc.
Immunogenic compositions will include an immunologically effective amount of a
polypeptide.
By `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 1 g and 10mg 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.
To control tonicity, it is preferred to include a physiological salt, such as
a sodium salt. Sodium
chloride (NaCI) is preferred, which may be present at between 1 and 20 mg/ml.
Other salts that
may be present include potassium chloride, potassium dihydrogen phosphate,
disodium
phosphate dehydrate, magnesium chloride, calcium chloride, etc.
17
CA 02697370 2010-02-22
WO 2009/029569 PCT/US2008/074179
Compositions will generally have an osmolality of between 200 mOsm/kg and 400
mOsm/kg,
preferably between 240-3 60 mOsm/kg, and will more preferably fall within the
range of 290-310
mOsm/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 5-20mM 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-octylphenoxypolyethoxy-ethanol), etc.
Vaccines may be administered in a dosage volume of about 0.5m1.
Vaccine adjuvants
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 W000/23105. Aluminum salt
adjuvants are
described in more detail below.
= An oil-in-water emulsion, as 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. (2003) Nucleic
Acids
Research 31:2393-2400, W002/26757 and W099/62923 disclose possible analog
18
CA 02697370 2010-02-22
WO 2009/029569 PCT/US2008/074179
substitutions e.g. replacement of guanosine with 2'-deoxy-7-deazaguanosine.
The
adjuvant effect of CpG oligonucleotides is further discussed in Krieg (2003)
Nature
Medicine 9:831-835, McCluskie et al. (2002) FEMS Immunology and Medical
Microhiology 32:179-185, W098/40100, US patents 6,207,646, 6,239,116 and
6,429,199.
A CpG sequence may be directed to TLR9, such as the motif GTCGTT or TTCGTT
(Kandimalla et al. (2003) Biochemical Society Transactions 31 (part 3):654-
658). The
CpG sequence may be specific for inducing a Thl 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.
Preferably, the CpG is a CpG-A ODN. Preferably, the CpG oligonucleotide is
constructed
so that the 5' end is accessible for receptor recognition. Optionally, 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-
861 and W003/035836. A useful CpG adjuvant is 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.
= 3-0-deacylated monophosphoryl lipid A(`3dMPL', also known as `MPLTM') (Myers
et
al. (1990) pages 145-156 of Cellular and molecular aspects of endotoxin
reaction,
Vaccine Adjuvants: Preparation Methods and Research Protocols (Volume 42 of
Methods in Molecular Medicine series). ISBN: 1-59259-083-7. Ed. O'Hagan pages
273-
282, Johnson et al. (1999) JMed Chem 42:4640-9 and Baldrick et al. (2002)
Regulatory
Toxicol Pharmacol 35:398-413). 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
(i.e. at positions 2 and 2'), and there is also 0-acylation at the 3'
position.
= An imidazoquinoline compound, such as Imiquimod ("R-837") (US patents
4,680,338;
4,988,815) Resiquimod ("R-848") (W092/15582), 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
Res. 64(2):79-83, Vasilakos et al. (2000) Cell Immunol. 204(1):64-74, US
patents
4689338, 4929624, 5238944, 5266575, 5268376, 5346905, 5352784, 5389640,
5395937,
19
CA 02697370 2010-02-22
WO 2009/029569 PCT/US2008/074179
5482936, 5494916, 5525612, 6083505, 6440992, 6627640, 6656938, 6660735,
6660747,
6664260, 6664264, 6664265, 6667312, 6670372, 6677347, 6677348, 6677349,
6683088,
6703402, 6743920, 6800624, 6809203, 6888000 and 6924293 and Jones (2003) Curr
Opin Investig Drugs 4:214-218.
= A thiosemicarbazone compound, such as those disclosed in W02004/060308.
Methods of
formulating, manufacturing, and screening for active compounds are also
described in
W02004/060308. The thiosemicarbazones are particularly effective in the
stimulation of
human peripheral blood mononuclear cells for the production of cytokines, such
as TNF-
a.
= A tryptanthrin compound, such as those disclosed in W02004/064759. Methods
of
formulating, manufacturing, and screening for active compounds are also
described in
reference W02004/064759. The thiosemicarbazones are particularly effective in
the
stimulation of human peripheral blood mononuclear cells for the production of
cytokines,
such as TNF-a.
= A nucleoside analog, such as: (a) Isatorabine (ANA-245; 7-thia-8-
oxoguanosine):
O
S
J~ ~
N N N
O
0 H
OO
and prodrugs thereof; (b) ANA975; (c) ANA-025-1; (d) ANA380; (e) the compounds
disclosed in US patents 6,924,271, 5,658,731 and US2005/0070556; (f) a
compound
having the formula:
R,
N R5
9
R~N R4
R3
wherein:
Rl and R2 are each independently H, halo, -NRaR, -OH, C1_6 alkoxy, substituted
C1_6 alkoxy, heterocyclyl, substituted heterocyclyl, C6_10 aryl, substituted
C6_1o
aryl, C1_6 alkyl, or substituted C1_6 alkyl;
R3 is absent, H, C1_6 alkyl, substituted C1_6 alkyl, C6_10 aryl, substituted
C6_10 aryl,
heterocyclyl, or substituted heterocyclyl;
CA 02697370 2010-02-22
WO 2009/029569 PCT/US2008/074179
R4 and R5 are each independently H, halo, heterocyclyl, substituted
heterocyclyl,
-C(O)-Rd, C1-6 alkyl, substituted C1_6 alkyl, or bound together to form a 5
membered ring as in R4-5:
,MXj
DrRa
X2 R4-5
R9
the binding being achieved at the bonds indicated by a
X1 and X2 are each independently N, C, 0, or S;
R8 is H, halo, -OH, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -OH, -NRaRb, -
(CH2)n-0-
Rc, -O-(C1-6 alkyl), -S(O)pRe, or -C(O)-Rd;
R9 is H, C1-6 alkyl, substituted C1-6 alkyl, heterocyclyl, substituted
heterocyclyl or
R9a, wherein R9a is:
0
RfO R9a
Rlo R11
the binding being achieved at the bond indicated by a
Rlo and Ril are each independently H, halo, C1_6 alkoxy, substituted C1-6
alkoxy, -
NRaRb, or -OH;
each Ra and Rb is independently H, C1-6 alkyl, substituted CI-6 alkyl, -
C(O)Rd, C6-10
aryl;
each R, is independently H, phosphate, diphosphate, triphosphate, C1-6 alkyl,
or
substituted C1-6 alkyl;
each Rd is independently H, halo, C1-6 alkyl, substituted C1_6 alkyl, C1-6
alkoxy,
substituted C1-6 alkoxy, -NH2, -NH(C1_6 alkyl), -NH(substituted CI-6 alkyl), -
N(CI-
6 alkyl)2, -N(substituted C1-6 alkyl)2, C6-1o aryl, or heterocyclyl;
each Re is independently H, C1-6 alkyl, substituted C1-6 alkyl, C6_10 aryl,
substituted C6-10 aryl, heterocyclyl, or substituted heterocyclyl;
each Rf is independently H, C1-6 alkyl, substituted C1_6 alkyl, -C(O)Rd,
phosphate,
diphosphate, or triphosphate;
each n is independently 0, 1, 2, or 3;
each p is independently 0, 1, or 2; or
or (g) a pharmaceutically acceptable salt of any of (a) to (f), a tautomer of
any of (a) to
(f), or a pharmaceutically acceptable salt of the tautomer.
= Loxoribine (7-allyl-8-oxoguanosine) (US patent 5,011,828).
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WO 2009/029569 PCT/US2008/074179
= Compounds disclosed in W02004/87153, including: Acylpiperazine compounds,
Indoledione compounds, Tetrahydraisoquinoline (THIQ) compounds,
Benzocyclodione
compounds, Aminoazavinyl compounds, Aminobenzimidazole quinolinone (ABIQ)
compounds (US 6,605,617, W002/18383), Hydrapthalamide compounds, Benzophenone
compounds, Isoxazole compounds, Sterol compounds, Quinazilinone compounds,
Pyrrole
compounds (W02004/018455), Anthraquinone compounds, Quinoxaline compounds,
Triazine compounds, Pyrazalopyrimidine compounds, and Benzazole compounds
(W003/082272).
= Compounds disclosed in W02006/00242, including 3,4-di(1H-indol-3-yl)-1H-
pyrrole-
2,5-diones, staurosporine analogs, derivatized pyridazines, chromen-4-ones,
indolinones,
quinazolines, and nucleoside analogs.
= 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.
= Small molecule immunopotentiators (SMIPs) such as:
N2-methyl-l-(2-methylpropyl)-1 H-imidazo[4,5-c]quinoline-2,4-diamine
N2,N2-dimethyl-l-(2-methylpropyl)-1 H-imidazo [4,5-c]quinoline-2,4-diamine
N2-ethyl-N2-methyl- l -(2-methylpropyl)-1 H-imidazo[4,5-c]quinoline-2,4-
diamine
N2-methyl-l-(2-methylpropyl)-N2-propyl-1 H-imidazo[4,5-c]quinoline-2,4-diamine
1-(2-methylpropyl)-N2-propyl-1 H-imidazo [4,5-c]quinoline-2,4-diamine
N2-butyl-l-(2-methylpropyl)-1 H-imidazo[4,5-c]quinoline-2,4-diamine
N2-butyl-N2-methyl-l-(2-methylpropyl)-1 H-imidazo[4,5-c]quinoline-2,4-diamine
N2-methyl-l-(2-methylpropyl)-N2-pentyl-1 H-imidazo [4,5-c]quinoline-2,4-
diamine
N2-methyl- l -(2-methylpropyl)-N2-prop-2-enyl-1 H-imidazo [4,5-c]quinoline-2,4-
diamine
1-(2-methylpropyl)-2-[(phenylmethyl)thio]-1 H-imidazo[4,5-c]quinolin-4-amine
1-(2-methylpropyl)-2-(propylthio)-1 H-imidazo[4,5-c]quinolin-4-amine
2-[[4-amino-l-(2-methylpropyl)-1 H-imidazo[4,5-c]quinolin-2-
yl] (methyl)amino]ethano l
2-[[4-amino-l-(2-methylpropyl)-1 H-imidazo[4,5-c]quinolin-2-
yl](methyl)amino]ethyl
acetate
4-amino-l-(2-methylpropyl)-1,3-dihydro-2H-imidazo [4, 5-c]quinolin-2-one
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WO 2009/029569 PCT/US2008/074179
N2-butyl-l-(2-methylpropyl)-N4,N4-bis(phenylmethyl)-1 H-imidazo[4,5-
c]quinoline-2,4-
diamine
N2-butyl-N2-methyl-l-(2-methylpropyl)-N4,N4-bis(phenylmethyl)-1 H-imidazo [4,5-
c]quinoline-2,4-diamine
N2-methyl-l-(2-methylpropyl)-N4,N4-bis(phenylmethyl)-1 H-imidazo[4,5-
c] quinol ine-2,4-diamine
N2,N2-dimethyl-l-(2-methylpropyl)-N4,N4-bis(phenylmethyl)-1 H-imidazo [4,5-
c]quinoline-2,4-diamine
1-{4-amino-2-[methyl(propyl)amino]-1 H-imidazo[4,5-c]quinolin-l-yl} -2-
methylpropan-2-
ol
1-[4-amino-2-(propylamino)-1 H-imidazo[4,5-c]quinolin-l-yl]-2-methylpropan-2-
ol
N4,N4-dibenzyl-l-(2-methoxy-2-methylpropyl)-N2-propyl-1 H-imidazo [4, 5-
c]quinoline-2,4-diamine.
= 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, QS17, QS18, QS21, QH-A, QH-B and QH-C. Preferably,
the
saponin is QS21. A method of production of QS21 is disclosed in US 5,057,540.
Saponin
formulations may also comprise a sterol, such as cholesterol (W096/33739).
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. Preferably, the ISCOM includes one or more of QuilA, QHA & QHC. ISCOMs
are further described in W096/33739, EP-A-0 109942 and W096/1 1 71 1.
Optionally, the
ISCOMS may be devoid of additional detergent (W000/07621). A review of the
development of saponin based adjuvants can be found in Barr et al. (1998)
Advanced
Drug Delivery Reviews 32:247-271 and Sjolanderet et al. (1998) Advanced Drug
Delivery
Reviews 32:321-338.
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WO 2009/029569 PCT/US2008/074179
= Bacterial ADP-ribosylating toxins (e.g. the E.coli heat labile enterotoxin
"LT", cholera
toxin "CT", or pertussis toxin "PT") and detoxified derivatives thereof, such
as the
mutant toxins known as LT-K63 and LT-R72 (Sjolanderet et al. (1998) Advanced
Drug
Delivery Reviews 32:321-338). The use of detoxified ADP-ribosylating toxins as
mucosal
adjuvants is described in W095/1721 and as parenteral adjuvants in W098/42375.
= Bioadhesives and mucoadhesives, such as esterified hyaluronic acid
microspheres (Singh
et al] (2001) JCont Release 70:267-276) or chitosan and its derivatives
(W099/2796).
= Microparticles (i.e. a particle of -100nm to -150gm in diameter, more
preferably
-200nm to -30 m in diameter, or -500nm to -10 m in diameter) formed from
materials
that are biodegradable and non-toxic (e.g. a poly(a-hydroxy acid), a
polyhydroxybutyric
acid, a polyorthoester, a polyanhydride, a polycaprolactone, etc.), with
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 (Chapters 13 & 14 of Vaccine Design: The Subunit and Adjuvant
Approach
(eds. Powell & Newman) Plenum Press 1995 (ISBN 0-306-44867-X)). Examples of
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 (W099/52549). Such
formulations
further include polyoxyethylene sorbitan ester surfactants in combination with
an
octoxynol (WO01/21207) 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-(1'-
2'dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine ("MTP-PE").
= 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. Such complexes
include
"IVX- 908", a complex comprised of Neisseria meningitidis outer membrane and
LPS.
24
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WO 2009/029569 PCT/US2008/074179
= Methyl inosine 5'-monophosphate ("MIMP") (Signorelli & Hadden (2003) Int
Immunopharmacol 3(8):1177-86).
= A polyhydroxlated pyrrolizidine compound (W02004/064715), such as one having
formula:
HO ~ ~Jw
Ra....._.~ OH
cHZcaH
where 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-a-D-
glucopyranose,
3-epi-casuarine, 7-epi-casuarine, 3,7-diepi-casuarine, etc.
= A gamma inulin (Cooper (1995) Pharm Biotechnol 6:559-80) or derivative
thereof, such
as algammulin.
= A compound of formula I, II or III, or a salt thereof:
I II 111
xt~~,--Y~ /X~~"Y~
( HE)a ~CHZIo
0-P-OH X.~ -0 O=P-O' 1! Z2 A: \N
WO ~-0 mm
R= ~ ~ b=
0 0
1 Ctl~jq tw. a X ~
NT' ~ Fi2)C ~~ 2)a` F+~' p2 fif ~ Rz W\ rowy~. t 5~ ~
R2 Ff ~~ [ a3c \~~~ a ~~(~Hz1a (GW2}e' ir da' ~
~, 4
pa G2 !_ C'`pa ~ pa He y
as defined in W003/011223, such as `ER 803058', `ER 803732', `ER 804053', ER
804058', `ER 804059', `ER 804442', `ER 804680', `ER .804764', ER 803022 or `ER
804057' e.g.:
CA 02697370 2010-02-22
WO 2009/029569 PCT/US2008/074179
0
o" 'Cõ[1zs
a =
p- I1-C)~(T
/_j () Na [Iti ,,[IJ,3
~ (:
HN
HN ER804057
O-P-QO' v `G,H~s
[23
() Va [iN Y-Y C;i.F
( ) 0
N
A
J _O(O 0 0
O 1~~ ER-803022:
p, A
0 0 0
0
= Derivatives of lipid A from Escherichia coli such as OM-174 (described in
Meraldi et al.
(2003) Vaccine 21:2485-2491, Pajak et al. (2003) Vaccine 21:836-84.
= A formulation of a cationic lipid and a (usually neutral) co-lipid, such as
aminopropyl-
dimethyl-myri stoleyloxy-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)-1-
propanaminium
salts are preferred (US patent 6586409)
= Compounds containing lipids linked to a phosphate-containing acyclic
backbone, such as
the TLR4 antagonist E5564 (Wong et al. (2003) J Clin Pharmacol 43(7):735-42,
US2005/0215517):
26
CA 02697370 2010-02-22
WO 2009/029569 PCT/US2008/074179
0 0 0 ,oPO(orn,
C1Ia(> U U
(CIIiIgCII.j
- If
These and other adjuvant-active substances are discussed in more detail in
references Vaccine
Design: The Subunit and Adjuvant Approach (eds. Powell & Newman) Plenum Press
1995 (ISBN
0-306-44867-X) and Vaccine Adjuvants: Preparation Methods and Research
Protocols (Volume
42 of Methods in Molecular Medicine series). ISBN: 1-59259-083-7. Ed. O'Hagan.
Compositions may include two or more of said adjuvants.
Antigens and adjuvants in a composition will typically be in admixture.
Oil-in-water emulsion adjuvants
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. Peanut oil,
soybean oil, coconut
oil, and olive oil, the most commonly available, exemplify the nut oils.
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. For example, 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.
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WO 2009/029569 PCT/US2008/074179
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, is also a preferred oil. 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-1,2-
ethanediyl) groups, with
octoxynol-9 (Triton X-100, or t-octylphenoxypolyethoxyethanol) being of
particular interest;
(octylphenoxy)polyethoxyethanol (IGEPAL CA-630/NP-40); phospholipids such as
phosphatidylcholine (lecithin); polyoxyethylene fatty ethers derived from
lauryl, cetyl, stearyl
and oleyl alcohols (known as Brij surfactants), such as triethyleneglycol
monolauryl ether (Brij
30); and sorbitan esters (commonly known as the SPANs), such as sorbitan
trioleate (Span 85)
and sorbitan monolaurate. Preferred surfactants for including in the emulsion
are Tween 80
(polyoxyethylene sorbitan monooleate), Span 85 (sorbitan trioleate), lecithin
and Triton X-100.
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 (% by weight) 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. 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' (W090/14837, 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
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WO 2009/029569 PCT/US2008/074179
Approach (eds. Powell & Newman) Plenum Press 1995 of Vaccine Adjuvants:
Preparation
Methods and Research Protocols (Volume 42 of Methods in Molecular Medicine
series).
The MF59 emulsion advantageously includes citrate ions e.g. 10mM sodium
citrate buffer.
= An emulsion of squalene, a tocopherol, and Tween 80. The emulsion 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 90m1 of this solution with a mixture of (5g of
DL-a-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.
= An emulsion of squalene, a tocopherol, and a Triton detergent (e.g. Triton X-
100). 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 a-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,
ll0 g/ml Triton X-100 and 100 g/ml a-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-1" 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 L121 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. As described in W095/11700, 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
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WO 2009/029569 PCT/US2008/074179
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.
= An emulsion in which a saponin (e.g. QuilA or QS21) and a sterol (e.g. a
cholesterol) are
associated as helical micelles (W02005/09718).
= 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) (W02006/113373).
= 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) (W02006/11337).
The emulsions may be mixed with antigen extemporaneously, at the time of
delivery. Thus 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.
Aluminum salt adjuvants
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 andAdjuvant 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.
The adjuvants known as "aluminium hydroxide" are typically aluminium
oxyhydroxide salts,
which are usually at least partially crystalline. Aluminium oxyhydroxide,
which can be
represented by the formula AlO(OH), can be distinguished from other aluminium
compounds,
such as aluminium hydroxide Al(OH)3, by infrared (IR) spectroscopy, in
particular by the
presence of an adsorption band at 1070cm I and a strong shoulder at 3090-
3100cm 1[chapter 9
of Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell & Newman)
Plenum Press
1995]. 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. 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) is typical
for aluminium hydroxide adjuvants. The pI of aluminium hydroxide adjuvants is
typically about
CA 02697370 2010-02-22
WO 2009/029569 PCT/US2008/074179
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.
The adjuvants known as "aluminium phosphate" are 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 P04/Al molar ratio between 0.3 and 1.2.
Hydroxyphosphates can be distinguished from strict A1PO4 by the presence of
hydroxyl groups.
For example, an IR spectrum band at 3164cm'1 (e.g. when heated to 200 C)
indicates the presence
of structural hydroxyls [ch.9 of Vaccine Design: The Subunit and Adjuvant
Approach (eds.
Powell & Newman) Plenum Press 1995.
The PO4/A13+ 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 PO4/Al molar ratio between 0.84 and
0.92,
included at 0.6mg A13+/ml. 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-l04m) after any antigen adsorption.
Adsorptive capacities of
between 0.7-1.5 mg protein per mg Al at pH 7.4 have been reported for
aluminium phosphate
adjuvants.
The point of zero charge (PZC) of aluminium phosphate is inversely related to
the degree of
substitution of phosphate for hydroxyl, and this degree of substitution can
vary depending on
reaction conditions and concentration of reactants used for preparing the salt
by precipitation.
PZC is also altered by changing the concentration of free phosphate ions in
solution (more
phosphate = more acidic PZC) or by adding a buffer such as a histidine buffer
(makes PZC more
basic). Aluminium phosphates used according to the invention will generally
have a PZC of
between 4.0 and 7.0, more preferably between 5.0 and 6.5 e.g. about 5.7.
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. In
this case 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.
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The concentration of AI~ in a composition for administration to a patient is
preferably less than
10mg/ml e.g. <5 mg/ml, <4 mg/ml, <3 mg/mi, <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. Thus 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. For instance, 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.
In a preferred arrangement, 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.
In another preferred arrangement, the two kit components are held together but
separately in the
same syringe e.g. a dual-chamber syringe, such as those disclosed in
W02005/089837,
W000/07647, W099/17820, EP-A-0520618, W098/01174, US patents 6,692,468,
5,971,953,
4,060,082. When the syringe is actuated (e.g. during administration to a
patient) then the contents
of the two chambers are mixed. This arrangement avoids the need for a separate
mixing step at
the time of use.
The kit components will generally be in aqueous form. In some arrangements, a
component
(typically the antigen component rather than the adjuvant component) is in dry
form (e.g. in a
lyophilised form), with the other component being in aqueous form. 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-filled
syringe and a
lyophilised antigen component in a vial.
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Methods of treatment, and administration of vaccines
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.).
General
The term "comprising" encompasses "including" as well as "consisting" e.g. a
composition
"comprising" X may consist exclusively of X or may include something
additional e.g. X + Y.
The word "substantially" does not exclude "completely" e.g. a composition
which is
"substantially free" from Y may be completely free from Y. Where necessary,
the word
"substantially" may be omitted from the definition of the invention.
The term "about" in relation to a numerical value x means, for example, x+10
/a.
Unless specifically stated, a process comprising a step of mixing two or more
components does
not require any specific order of mixing. Thus 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.
Where 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.
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Where 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.
The term "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. Also included within the definition are, for example, polypeptides
containing one or
more analogs of an amino acid (including, for example, unnatural amino acids,
etc.), as well as
other modifications known in the art. 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. As
an alternative to chemical synthesis, 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. of 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,
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CA 02697370 2010-02-22
WO 2009/029569 PCT/US2008/074179
non-myristoylated, monomeric, multimeric, particulate, denatured, etc.). For
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.
EXAMPLES
The full-length SF162 strain Env sequence has the following amino acid
sequence (SEQ ID NO:
38):
MRVKGIRKNYQHLWRGGTLLLGMLMICSAVEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWAT
HACVPTDPNPQEIVLENVTENFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLHCTNLKNATNTK
SSNWKEMDRGEIKNCSFKVTTSIRNKMQKEYALFYKLDWPIDNDNTSYKLINCNTSVITQACPKVSFEP
IPIHYCAPAGFAILKCNDKKFNGSGPCTNVSTVQCTHGIRPVVSTQLLLNGSLAEEGWIRSENFTDNAK
TIIVQLKESVEINCTRPNNNTRKSITIGPGRAFYATGDIIGDIRQAHCNISGEKWNNTLKQIVTKLQAQF
GNKTIVFKQSSGGDPEIVMHSFNCGGEFFYCNSTQLFNSTWNNTIGPNNTNGTITLPCRIKQIINRWQEV
GKAMYAPPIRGQIRCSSNITGLLLTRDGGKEISNTTEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPT
KAKRRVVQREKRAVTLGAMFLGFLGAAGSTMGARSLTLTVQARQLLSGIVQQQNNLLRAIEAQQHLLQLT
VWGIKQLQARVLAVERYLKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLDQIWNNMTWMEWEREIDNYT
NLIYTLIEESQNQQEKNEQELLELDKWASLWNWFDISKWLWYIKIFIMIVGGLVGLRIVFTVLSIVNRVR
QGYSPLSFQTRFPAPRGPDRPEGIEEEGGERDRDRSSPLVHGLLALIWDDLRSLCLFSYHRLRDLILIAA
RIVELLGRRGWEALKYWGNLLQYWIQELKNSAVSLFDAIAIAVAEGTDRIIEVAQRIGRAFLHIPRRIRQ
GFERALL
For expression purposes, 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 gp160 sequence can be modified to a gp140 form (SEQ ID NO: 39):
SAVEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWAT
HACVPTDPNPQEIVLENVTENFNMWKNNMVEQMHEDIISLWDQSLKPCVKLTPLCVTLHCTNLKNATNTK
SSNWKEMDRGEIKNCSFKVTTSIRNKMQKEYALFYKLDVVPIDNDNTSYKLINCNTSVITQACPKVSFEP
IPIHYCAPAGFAILKCNDKKFNGSGPCTNVSTVQCTHGIRPWSTQLLLNGSLAEEGVVIRSENFTDNAK
TIIVQLKESVEINCTRPNNNTRKSITIGPGRAFYATGDIIGDIRQAHCNISGEKWNNTLKQIVTKLQAQF
GNKTIVFKQSSGGDPEIVMHSFNCGGEFFYCNSTQLFNSTWNNTIGPNNTNGTITLPCRIKQIINRWQEV
GKAMYAPPIRGQIRCSSNITGLLLTRDGGKEISNTTEIFRPGGGDMRDNWRSELYKYKVVKIEPLGVAPT
KAKRRVVQREKRAVTLGAMFLGFLGAAGSTMGARSLTLTVQARQLLSGIVQQQNNLLRAIEAQQHLLQLT
VWGIKQLQARVLAVERYLKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLDQIWNNMTWMEWEREIDNYT
NLIYTLIEESQNQQEKNEQELLELDKWASLWNWFDISKWLWYIT"'T''T""TV^^T "^T DTT' '"VT
nrWrnsro
GFEP.A.Lb
It can be further modified to include five amino acid mutations at the
cleavage site, to produce
oligomeric gp140. This sequence (SEQ ID NO: 43) is known as `gp140mut7':
SAVEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEIVLENVTENFNMWKN
NMVEQMHEDIISLWDQSLKPCVKLTPLCVTLHCTNLKNATNTKSSNWKEMDRGEIKNCSFKVTTSIRNKM
QKEYALFYKLDWPIDNDNTSYKLINCNTSVITQACPKVSFEPIPIHYCAPAGFAILKCNDKKFNGSGPC
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TNVSTVQCTHGIRPWSTQLLLNGSLAEEGVVIRSENFTDNAKTIIVQLKESVEINCTRPNNNTRKSITI
GPGRAFYATGDIIGDIRQAHCNISGEKWNNTLKQIVTKLQAQFGNKTIVFKQSSGGDPEIVMHSFNCGGE
FFYCNSTQLFNSTWNNTIGPNNTNGTITLPCRIKQIINRWQEVGKAMYAPPIRGQIRCSSNITGLLLTRD
GGKEISNTTEIFRPGGGDMRDNWRSELYKYKWKIEPLGVAPTKAISSVVQSEKSAVTLGAMFLGFLGAA
GSTMGARSLTLTVQARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARVLAVERYLKDQQLLGIW
GCSGKLICTTAVPWNASWSNKSLDQIWNNMTWMEWEREIDNYTNLIYTLIEESQNQQEKNEQELLELDKW
ASLWNWFDISKWLWYI
The 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):
SAVEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEIVLENVTENFNMWKN
NMVEQMHEDIISLWDQSLKPCVKLTPLCVTLHCTNLKNATNTKSSNWKEMDRGEIKNCSFKVTTSIRNKM
QKEYALFYKLDVVPIDNDNTSYKLINCNTSVITQACPKVSFEPIPIHYCAPAGFAILKCNDKKFNGSGPC
TNVSTVQCTHGIRPVVSTQLLLNGSLAEEGVVIRSENFTDNAKTIIVQLKESVEINCTRPNNNTRGAGQA
HCNISGEKWNNTLKQIVTKLQAQFGNKTIVFKQSSGGDPEIVMHSFNCGGEFFYCNSTQLFNSTWNNTIG
PNNTNGTITLPCRIKQIINRWQEVGKAMYAPPIRGQIRCSSNITGLLLTRDGGKEISNTTEIFRPGGGDM
RDNWRSELYKYKVVKIEPLGVAPTKAKRRVVQREKRAVTLGAMFLGFLGAAGSTMGARSLTLTVQARQLL
SGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARVLAVERYLKDQQLLGIWGCSGKLICTTAVPWNASWS
NKSLDQIWNNMTWMEWEREIDNYTNLIYTLIEESQNQQEKNEQELLELDKWASLWNWFDISKWLWYI
A construct encoding SEQ ID NO: 40 (`gp140dV3-22') 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.
A modified form of SEQ ID NO: 40 (`gp1400V2dv3-22') in which the V2 loop is
replaced by
SEQ ID NO: 42 (CSFKVGAGKLINC) is prepared in the same way. Its sequence is SEQ
ID NO:
41:
SAVEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEIVLENVTENFNMWKN
NMVEQMHEDIISLWDQSLKPCVKLTPLCVTLHCTNLKNATNTKSSNWKEMDRGEIKNCSFKVGAGKLINC
NTSVITQACPKVSFEPIPIHYCAPAGFAILKCNDKKFNGSGPCTNVSTVQCTHGIRPVVSTQLLLNGSLA
EEGVVIRSENFTDNAKTIIVQLKESVEINCTRPNNNTRGAGQAHCNISGEKWNNTLKQIVTKLQAQFGNK
TIVFKQSSGGDPEIVMHSFNCGGEFFYCNSTQLFNSTWNNTIGPNNTNGTITLPCRIKQIINRWQEVGKA
MYAPPIRGQIRCSSNITGLLLTRDGGKEISNTTEIFRPGGGDMRDNWRSELYKYKWKIEPLGVAPTKAI
SSWQSEKSAVTLGAMFLGFLGAAGSTMGARSLTLTVQARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWG
IKQLQARVLAVERYLKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLDQIWNNMTWMEWEREIDNYTNLI
YTLIEESQNQQEKNEQELLELDKWASLWNWFDISKWLWYI
This protein is purified in the same manner as described above.
Equivalent Env-derivatives re also made for strain TV 1.
Example 1
After confirming purity, oligomerization and CD4-binding activity for both
gp140dV3-22 and
gp1400V2dv3-22 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:
Parameter Off rate (s-1 x 10=5) Half life (hours)
Ligand CD4 Tat Tat-cys CD4 Tat Tat-cys
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9p140sF162 5.30 + 0.76% 3.88 + 3.13% 0.13 + 96.4% 3.64 4.96 150.1
gp140AV2sF162 7.60 + 0.67% 5.50 + 1.37% 4.02 + 1.80% 2.53 3.50 4.79
gp140dV3-22sF162 5.48 + 0.63% 6.24 + 2.03% 4.61 + 1.82% 3.51 3.08 4.17
gp140Tv, 6.48 + 0.82% 10.0 + 1.22% 7.38 + 1.69% 2.97 1.92 2.61
gp140dV3-22Tvj 3.47 + 0.67% 6.98 + 2.82% 3.84 + 4.35% 5.55 2.76 5.01
Thus all the trimeric variants of Env bound to both CD4 and the tat
polypeptide. Monomeric
gp120 binds to CD4 as expected, but binding to tat is not observed.
BIACORETM can also be used to test binding of the SF162-derived proteins to
(i) CD4, (ii)
neutralizing antibody b12, which binds to gp120'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
gp120. Dissociation constants (M-1 x 10-10) and Rmax values (using 4.8d & CD4)
are as follows:
CD4 Kd b1 Kd 4.8d Kd x Up-reg 4.8d* Rmax
gp120 770 970 40 1.7 416
gp140AV2 160 34 3.8 2.6 281
gp140dV3-22 180 200 75 1.4 660
gp140 AV2 dV3-22 650 1000 79 1.8 602
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 BlAcore signal observed for env mixed with CD4 as a function of the signal
observed for env
without CD4, on the 4.8d antibody.
Thus the modifications that are introduced into Env do not substantially alter
its CD4-binding
properties. Moreover, monoclonal antibody binding remained at reasonable
levels for all variants.
Example 2
Tat-binding for the SF162-derived proteins (with or without CD4) is also
tested by a Far-Western
assay. Results are shown in Figure 1. Thus both in a kinetic experimental
environment (i.e.
BlAcore) and under conditions of equilibrium (i.e. 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 gpI20AV2.
Overall, the data show no significant difference between the magnitude of
binding of Tat to the
AV2 and dV3-22 forms of Env. However, the off rate for the dV3-22 trimeric Env
was faster (i.e.
shorter half-life) when compared to the AV2 trimeric protein.
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Example 3
Alternative 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 SF162.
SF162dV3-20 CTRPNNN------------------GAGDIRQAHC (SEQ ID NO: 15)
SF162dV3-9 CT--------- ITIGPGRAFYATGDIIGDIRQAHC (SEQ ID NO: 16)
SF162dV3-6 CTRPNNNTRKSITIGPGRAFYAT--------QAHC (SEQ ID NO: 17)
SF162dV3-10 CTRPNNNTR----------FYATGDIIGDIRQAHC (SEQ ID NO: 18)
SF162dV3-12 CTRPNNNTR--------------GDIIGDIRQAHC (SEQ ID NO: 19)
SF162dv3-17 CT----------------- FYATGDIIGDIRQAHC (SEQ ID NO: 20)
SF162dV3-22 CTRPNNNTR----------------------QAHC (SEQ ID NO: 21)
SF162dV3-28sub CTRPNNNTR------------------- GAGQAHC (SEQ ID NO: 23)
SF162dV3-28 CT----------------------------GAGHC (SEQ ID NO: 22)
SF162V3sub CTRPNNNTRKSITIGPGRAFYATGDIIGNMRQAHC (SEQ ID NO: 24)
The deleted peptide sequences (SEQ ID NOS: 30 to 37) are synthesized and
evaluated for their
ability to bind to Tat.
Example 4
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 Vl-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 V1-
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.
It will be understood that the invention has been described by way of example
only and
modifications may be made whilst remaining within the scope and spirit of the
invention.
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