Note: Descriptions are shown in the official language in which they were submitted.
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Novel compositions
FIELD OF THE INVENTION
The present invention relates to particular immunogenic compositions
comprising a gp120
related polypeptide and an adjuvant, wherein the adjuvant comprises a saponin
and a
lipopolysaccharide. Methods for the preparation of such immunogenic
compositions and
related kits are also provided.
BACKGROUND OF THE INVENTION
HIV is the primary cause of acquired immune deficiency syndrome (AIDS) which
is regarded
as one of the world's major health problems. There were approximately 34
million people
living with HIV in 2011 (WHO HIV/AIDS Fact sheet number 360, June 2013) and
over 4 million
new infections are occurring every year. HIV has claimed more than 25 million
lives over the
past three decades. Although antiretroviral therapy is prolonging the lives of
many people
infected with HIV, to be effective antiretroviral therapy requires strict
adherence to often
complex multidrug administration regimes and does not cure the infection. New
infections are
greatly exceeding the number of people able to be treated through current
global financial
efforts. There is a need for a vaccine to prevent new infections but the
development of a safe
and effective HIV vaccine poses a significant challenge.
Two types of HIV have been characterized: HIV-1 and HIV-2. HIV-1 is highly
virulent and
infective and is the cause of the majority of HIV infections globally, whilst
HIV-2 has a lower
virulence and infectivity and is largely confined to West Africa (Gilbert et
al., Stat In Med
22(4):573-593 (2003) and Reeves and Doms J Gen Vir 83:1253-1265 (2002)). There
are
many genetically distinct subtypes (also known as 'clades') of HIV-1 and the
amino acid
sequences of just the envelope glycoproteins (gp120 and gp41) can vary from 25-
30%
between subtypes (Kalish et al., AIDS 9:851-857 (1995)). The genetic diversity
of HIV-1 along
with the high mutation rate are major obstacles for HIV-1 vaccine development.
Although extensive research throughout the world has been conducted to produce
a vaccine,
much work is still required.
CA 02851852 2014-05-08
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The HIV envelope glycoprotein gp120 and other HIV-1 proteins
gp120 is the viral protein that is used for attachment to a host cell. This
attachment is
mediated by gp120 binding surface molecules of helper T cells and macrophages
including
one of the two chemokine receptors CCR-5 or CXCR-4. The gp120 protein is first
expressed
as a larger precursor molecule (gp160), which is then cleaved post-
translationally to yield
gp120 and gp41. The gp120 protein is retained on the surface of the virion,
non-covalently
associated with the gp41 molecule, which is inserted into the viral membrane.
Three non-
covalently associated envelope glycoprotein gp120 and gp41 heterodimers form
the trimeric
Env spike found on the surface of HIV-1.
Non-envelope proteins of HIV-1 have been described and include for example
internal
structural proteins such as other products of the Env gene (such as gp160 and
gp41) the
products of the gag and pot genes (such as MA, CA, SP1, NC, SP2 and P6; and
RT, RNase H,
IN and PR, respectively) and other non-structural proteins such as Rev, Nef,
Vif, Vpr, Vpu and
Tat (Greene et al., New Eng J Med, 324, 5, 308 et seq (1991) and Bryant et al.
(Ed. Pizzo),
Pediatr Infect Dis J, 11, 5, 390 et seq (1992)).
Previous work in the field
gp120 was among the first targets of HIV vaccine research and was considered
to be useful as
an antigenic component in vaccines intended to elicit cell-mediated immune
responses. The
gp120 protein contains epitopes that are recognized by cytotoxic T lymphocytes
(CTL). These
effector cells are able to eliminate virus-infected cells, and therefore
constitute an antiviral
immune mechanism. Some CTL epitopes appear to be relatively conserved among
different
HIV strains. However, current CTL-based vaccines do not protect against
infection in animal
models of HIV (Amara et at., Science 292(5514):69-74 (2001)). Furthermore, CTL-
based
vaccines studied to date in humans have not induced these intended responses
in all
recipients (Goepfert et at., J Infect Dis 192(7):1249-1259 (2005)). Such CTL-
based vaccines
were not able to protect against infection, nor impact viral load post
infection (the "HVTN-505
study").
The gp120 protein is a major target of neutralising antibodies (Pantophlet et
at., Annu Rev
Immunol 24:739-769 (2006)) and neutralising antibodies were previously thought
to be a
potential correlate of protection (Bruck et al., Vaccine 12(12):1141-1148
(1994) and Plotkin,
Pediatr Infect Dis J 20:63-75 (2001)).
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One region of the gp120 protein in particular, the 'V3 loop', is targeted by
neutralising
antibodies. Antibodies present in immune sera of infected individuals bind to
V3 loop peptides
(Spenlehauer et al., J Vir, 72(12):98559864 (1998)). Accordingly, much
research was
focussed on the V3 loop of the gp120 protein. However, the V3 loop is
unfortunately highly
variable and highly strain specific (Vaine et al., PLoS One, 5(11):e13916,
(2010), Jones et al.,
J Infect Dis, 179:558-566 (1999) and McCormack et al., Vaccine 18(13):1166-
1177 (2000)).
It has been shown that vaccination with gp120 will not always induce
neutralising antibodies
19 against HIV and SHIV, and when neutralising antibodies are induced, these
antibodies rarely
confer protection against divergent viruses (Voss et al., J Vir 77(2):1049-
1058 (2003), Plotkin,
Pediatr Infect Dis J 20:63-75 (2001), Wren and Kent, Hum Vacc 7(4):466-473
(2011)).
Use of recombinant HIV-1 NefTat, gp120W61D and SIV Nef proteins formulated
with the ASO2A
adjuvant system (5Oug of QS-21 and 5Oug of 3D-MPL in an oil-in-water emulsion)
was shown
to protect against AIDS in a rhesus macaque SHIV animal model system when
animals were
challenged with SIV/HIV strain SH1V896p. However, HIV-1 gp1200,61D formulated
alone with
ASO2A did not provide protection. The lack of protection after immunisation
with gp12106
-..61D
formulated alone with ASO2A was suggested to be related to the heterologous
nature of the
challenge virus (20.2% sequence difference for gp120), since HIV-1 gp120w6m
formulated in
ASO2A had previously been shown to induce sterile immunity against homologous
SHIVw61D
challenge (Voss et al., J Virol 77(2):1049-1058 (2003) and Mooij et al., AIDS
12:F15-F22
(1998)).
NefTat and gp120w61D formulated in ASO2A has been administered to HIV
seronegative
individuals in a safety and immunogenicity study. However, the neutralising
antibodies elicited
by this vaccine had poor cross-subtype reactivity (Leroux-Roels et al.,
Vaccine 28:7016-7024
(2010) - the "PRO HIV-002 trial"). Furthermore, gp120 had a negative impact on
antibody and
T cell responses (geometric mean antibody titre and lymphocyte proliferation)
when comparing
administration of NefTat and gp120 versus NefTat alone (Goepfert et al.,
Vaccine 25:510-518
(2007) - the "HVTN-041" study). Accordingly, it was found that (i) a vaccine
comprising gp120
with an adjuvant comprising QS-21 and 3D-MPL had poor cross-subtype reactivity
and (ii) it
appears that gp120 may have a detrimental impact on previously accepted
potential markers
of efficacy for HIV vaccines containing other antigens.
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In light of the findings illustrated above, the use of gp120 as a vaccine
antigen to elicit humoral
responses (particularly when administered with an adjuvant comprising QS-21
and 3D-MPL)
was thought to be of limited use for a broadly protective vaccine and,
consequently, interest in
this protein has reduced.
The RV144 HIV-1 vaccine trial was the first to demonstrate evidence of
protection against HIV-
1 infection, with an estimated vaccine efficacy of 31.2% (Rerks-Ngarm et al, N
Engl J Med
361:2209-2220 (2009)). The protocol consisted of four priming injections of
ALVAC-HIV
(vCP1521), and two booster injections of AIDSVAX B/E. ALVAC-HIV was
administered at
baseline (day 0), 4 weeks, 12 weeks and 24 weeks. AIDSVAX B/E was administered
at weeks
12 and 24. ALVAC-HIV (vCP1521) is a recombinant canary pox viral vector
containing gp120
from HIV-1 subtype E (CRF01_AE) strain 92TH023, linked to the transmembrane-
anchoring
portion of gp41 (carrying a deletion in the immunodominant region) from HIV-1
subtype B
strain LAI. The vector also contained HIVLA1 gag and pol genes. AIDSVAX B/E is
a
preparation of recombinant HIV-1 subtype B MN gp120, CM244 subtype E A244
gp120 and an
alum adjuvant.
In order to identify correlates of risk of HIV-1 infection in RV144, plasma
specimens from
RV144 study participants were analysed (Haynes et al., N Engl J Med 366:1275-
1286 (2012)).
Assays were performed on samples, obtained two weeks after final immunisation,
from 41
vaccinees who became infected and 205 uninfected vaccinees. These assays
examined the
roles of T-cell, IgG antibody and IgA antibody responses in the modulation of
infection risk. It
was found that levels of antibodies specific for gp7O-V1V2 (a scaffolded
protein carrying the
first and second variable regions of HIV-1 gp120 fused to murine leukemia
virus gp70 ¨ see
Pinter et al., Vaccine 16(19):1803-1811 (1998)) were correlated with a lower
risk of infection
and the binding of plasma IgA antibodies to envelope proteins correlated a
higher risk of
infection. Furthermore, since this analysis was performed, it has been found
that V2
antibodies induced in the RV144 trial cross-react with multiple HIV-1 subtypes
(Zolla-Pazner et
al., PlosOne, 8(1):e53629 (2013)). Accordingly, for the above reasons, it was
concluded that
vaccines which induce higher levels of V1V2 antibodies may have improved
efficacy against
HIV-1 infection.
To produce an HIV vaccine, it is therefore highly desirable to identify a
composition capable of
eliciting a high level of antibodies specific for the V1V2 region of gp120.
To summarise, previous work in the field has demonstrated that:
CA 02851852 2014-05-08
= gp120 in A9D2A aciuvant (C2S-21 and 31D-IVFL cil-in-vketer ernision) cid
nct proAcie
prctection against challenge Wth heterolcgous virus in a primate rrrclel,
= gp120 may have a detrimental irrpact on markers ct vaccine efficecy and
5 = antibodies against the V1V2 region of gp120 are a correlate of
HIV-1 protection.
Immunogenic compositions of the present invention may have one or more of the
following
advantages compared to compositions of the prior art:
(i) achieve a stronger humoral immune response, for example a higher serum
titre
of antibodies binding the V1V2 region of gp120,
(ii) achieve a stronger cellular immune response, for example proliferation
of and
cytokine release by polyfunctional T cells,
(iii) achieve a broader humoral immune response, for example a achieving a
titre of
antibodies binding the V1V2 region of gp120 in a larger proportion of
vacinees,
(iv) achieve a broader cellular immune response, for example proliferation
of and
cytokine release by polyfunctional T cells in a larger proportion of vacinees,
(v) achieve a stronger humoral immune response against a particular subtype
of
HIV-1,
(vi) achieve a stronger cellular immune response against a particular
subtype of
HIV-1,
(vii) require fewer components,
(viii) require non-live components,
(ix) involve a more simple dosage regime,
(x) be simpler to produce,
(xi) be more easily stored,
(xii) be of utility in the treatment or prevention of HIV-1 infection,
(xiii) be of utility in the treatment or prevention of HIV-1 infection by a
first HIV-1
subtype when the gp120 related polypeptide of the composition is derived from
a second HIV-1 subtype,
(xiv) achieve a more durable immune response, for example based on magnitude
of
response and/or responder rates,
(xv) achieve a greater reduction in viral load,
(xvi) induce a higher level of protection against infection.
SUMMARY OF THE INVENTION
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The present inventors administered compositions containing either (a)
gp120W61D and ASO1B
or (b) gp120w6lo, ASO1B and NefTat to mice and analysed the serological
response of the
mice.
The present inventors found that, surprisingly, mice administered with
gp120W610 and ASO1B
(without NefTat) had a higher mean level of anti-V1V2 antibodies in their
serum compared to
mice administered with gp120w61D, ASO1B and NefTat.
It may be expected that the claimed immunogenic compositions elicit a high
level of anti-V1V2
antibodies, and have utility the in the prophylaxis of HIV infection.
The present invention provides an immunogenic composition which is in the form
of a human
dose comprising a gp120 related polypeptide and an adjuvant, wherein the
adjuvant comprises
between 10-4Oug of a lipopolysaccharide and between 10-4Oug of an
immunologically active
saponin fraction derived from the bark of Quillaja Saponaria Molina presented
in the form of a
liposome.
Also provided is an immunogenic composition comprising a gp120 related
polypeptide and an
adjuvant, wherein the adjuvant comprises a lipopolysaccharide and an
immunologically active
saponin fraction derived from the bark of Quillaja Saponaria Molina presented
in the form of a
liposome wherein:
(i) the conductivity of the composition is 13 mS/cm or lower; and/or
(ii) the concentration of salts in said composition is 130 mM or lower; and/or
(iii) the concentration of sodium chloride in said composition is 130 mM or
lower.
Further provided is an immunogenic composition comprising a gp120 related
polypeptide and
an adjuvant, wherein the adjuvant comprises a lipopolysaccharide and an
immunologically
active saponin fraction derived from the bark of Quillaja Saponaria Molina
presented in the
form of a liposome and wherein the composition is substantially free of a
NefTat related
polypeptide wherein the NefTat related polypeptide is a polypeptide consisting
of SEQ ID NO:
4.
DESCRIPTION OF THE FIGURES
Figure 1: Mouse anti-V1V2 serology comparison
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DESCRIPTION OF THE SEQUENCES
SEQ ID NO: 1 gp120w6ip
SEQ ID NO: 2 Nef
SEQ ID NO: 3 Tat
SEQ ID NO: 4 NefTat
SEQ ID NO: 5 gp120zmiti
SEQ ID NO: 6 polynucleotide sequence encoding gp120zmi8
SEQ ID NO: 7 native gp120zmia
SEQ ID NO: 8 polynucleotide sequence encoding native gp120zmi8
DETAILED DESCRIPTION OF THE INVENTION
Polypeptides
As used herein, the term 'a gp120 related polypeptide' refers to a polypeptide
comprising the
V1V2 region of SEQ ID NO: 1 or to an immunogenic derivative or fragment of the
V1V2 region
of SEQ ID NO: 1, or to a polypeptide comprising the V1V2 region of SEQ ID NO:
5 or to an
immunogenic derivative or fragment of the V1V2 region of SEQ ID NO: 5.
Suitably, the gp120
related polypeptide refers to a polypeptide consisting of the V1V2 region of
SEQ ID NO: 1 or to
an immunogenic derivative or fragment of the V1V2 region of SEQ ID NO: 1, or
to a
polypeptide consisting of the V1V2 region of SEQ ID NO: 5 or to an immunogenic
derivative or
fragment of the V1V2 region of SEQ ID NO: 5.
The V1V2 region of a gp120 related polypeptide is the stretch of residues
defined by two
particular cysteine residues which form a disulphide bridge in the folded
state of the
polypeptide. The V1V2 region itself excludes these cysteine residues. An
example of the
V1V2 region in the case of the gp1200,61D polypeptide (SEQ ID NO: 1) is the
stretch of residues
from residue 90 to 184. The skilled person will appreciate that the position
of these cysteine
residues and therefore the position of the V1V2 region within a given gp120
related
polypeptide can vary. This is illustrated by another gp120 related
polypeptide, gp120D.418 (SEQ
ID NO: 5) wherein the V1V2 region is the stretch of residues from residue 90
to 172.
Suitably, the term 'a gp120 related polypeptide' refers to a polypeptide
comprising SEQ ID NO:
1 or to an immunogenic derivative or fragment of SEQ ID NO: 1, or to a
polypeptide comprising
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SEQ ID NO: 5 or to an immunogenic derivative or fragment of SEQ ID NO: 5.
Suitably, the
gp120 related polypeptide refers to a polypeptide consisting of SEQ ID NO: 1
or to an
immunogenic derivative or fragment of SEQ ID NO: 1, or to a polypeptide
consisting of SEQ ID
NO: 5 or to an immunogenic derivative or fragment of SEQ ID NO: 5.
As used herein, the term "derivative" refers to a polypeptide that is modified
relative to the
reference sequence. Immunogenic derivatives are sufficiently similar to the
reference
sequence to remain capable of eliciting an immune response against the V1V2
region of the
reference sequence. Suitably, immunogenic derivatives are sufficiently similar
to the reference
sequence to retain other key immunogenic properties of the reference sequence
such as
avoiding the introduction of immunodominant epitopes. A derivative may, for
example,
comprise a modified version of the reference sequence or alternatively may
consist of a
modified version of the reference sequence.
Suitably, the gp120 related polypeptide comprises or consists of a gp120
polypeptide derived
from HIV-1 or HIV-2, suitably from groups M, N, 0 or P of HIV-1, suitably from
subtype A, B, C,
D, E, F, G, H, I, J or K of group M. Suitably, the gp120 related polypeptide
comprises or
consists of a gp120 polypeptide derived from HIV-1, suitably from group M,
more suitably from
subtype B. For example, the gp120 related polypeptide comprises or consists of
the gp120
polypeptide from a HIV-1 or HIV-2, suitably from an M, N, 0 or P group HIV-1,
suitably from a
subtype A, B, C, D, E, F, G, H, I, J or K of group M HIV-1. Suitably, the
gp120 related
polypeptide comprises or consists of the gp120 polypeptide from an HIV-1,
suitably from an
HIV-1 group M, more suitably from an HIV-1 group M subtype B.
The skilled person will recognise that individual substitutions, deletions or
additions to the
gp120 related polypeptide which alter, add or delete a single amino acid or a
small percentage
of amino acids is an "immunogenic derivative" where the alteration(s) results
in the
substitution/deletion/addition of residues which do not substantially impact
the immunogenic
function. By "not substantially impact the immunogenic function" is meant at
least 50%,
suitably at least 75% and suitably at least 90% activity of the reference
sequence in an assay
of the level of antibodies binding the gp120 V1V2 region (e.g. ELISA) produced
at a given time
point after immunisation with the gp120 related polypeptide.
Conservative substitution tables providing functionally similar amino acids
are well known in
the art. In general, such conservative substitutions will fall within one of
the amino-acid
groupings specified below, though in some circumstances other substitutions
may be possible
CA 02851852 2014-05-08
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without substantially affecting the immunogenic properties of the antigen. The
following eight
groups each contain amino acids that are typically conservative substitutions
for one another:
1) Alanine (A), Glycine (G);
2) Aspartic acid (D), Glutamic acid (E);
3) Asparagine (N), Glutamine (Q);
4) Arginine (R), Lysine (K);
5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);
6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);
7) Serine (S), Threonine (T); and
8) Cysteine (C), Methionine (M)
(see, e.g., Creighton, Proteins 1984).
Suitably such substitutions do not occur in the region of an epitope, and do
not therefore have
a significant impact on the immunogenic properties of the antigen. Suitably an
immunogenic
derivative will contain substitutions of up to 20 residues (for example up to
5 residues) relative
to the reference sequence.
Immunogenic derivatives may also include those wherein additional amino acids
are inserted
compared to the reference sequence. Suitably such insertions do not occur in
the region of an
epitope, and do not therefore have a significant impact on the immunogenic
properties of the
antigen. Suitably an immunogenic derivative will contain additions of up to 5
residues (for
example 1 or 2 residues) at 0-5 locations (for example 0-2 locations) relative
to the reference
sequence. One example of insertions includes a short stretch of histidine
residues (e.g. 6
residues) to aid purification of the antigen in question.
Immunogenic derivatives include those wherein amino acids have been deleted
compared to
the reference sequence. Suitably such deletions do not occur in the region of
an epitope, and
do not therefore have a significant impact on the immunogenic properties of
the antigen.
Suitably an immunogenic derivative will contain deletions of up to 5 residues
(for example 1 or
2 residues) at 0-5 locations (for example 0-2 locations) relative to the
reference sequence.
Suitably the gp120 related polypeptide will comprise, such as consist of, an
immunogenic
derivative of the V1V2 region of SEQ ID NO: 1 having a small number of
deletions, insertions
and/or substitutions, such as a derivative of the V1V2 region of SEQ ID NO: 1
having deletions
of up to 5 residues (for example 1 or 2 residues) at 0-5 locations (for
example 0-2 locations),
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insertions of up to 5 residues (for example 1 or 2 residues) at 0-5 five
locations (for example 0-
2 locations) and substitutions of up to 20 residues (for example up to 5
residues).
Alternatively, the gp120 related polypeptide will comprise, such as consist
of, an immunogenic
5 derivative of the V1V2 region of SEQ ID NO: 5 having a small number of
deletions, insertions
and/or substitutions, such as a derivative of the V1V2 region of SEQ ID NO: 5
having deletions
of up to 5 residues (for example 1 or 2 residues) at 0-5 locations (for
example 0-2 locations),
insertions of up to 5 residues (for example 1 or 2 residues) at 0-5 five
locations (for example 0-
2 locations) and substitutions of up to 20 residues (for example up to 5
residues).
Suitably the gp120 related polypeptide will comprise, such as consist of, an
immunogenic
derivative of SEQ ID NO: 1 having a small number of deletions, insertions
and/or substitutions,
such as a derivative of SEQ ID NO: 1 having deletions of up to 5 residues at 0-
5 locations,
insertions of up to 5 residues at 0-5 five locations and substitutions of up
to 20 residues.
Alternatively, the gp120 related polypeptide will comprise, such as consist
of, an immunogenic
derivative of SEQ ID NO: 5 having a small number of deletions, insertions
and/or substitutions,
such as a derivative of SEQ ID NO: 5 having deletions of up to 5 residues at 0-
5 locations,
insertions of up to 5 residues at 0-5 five locations and substitutions of up
to 20 residues.
Suitably, the gp120 related polypeptide comprises a polypeptide with at least
70% identity,
more suitably at least 80% identity, more suitably at least 85% identity, more
suitably at least
90% identity, more suitably at least 95% identity, more suitably at least 98%
identity, more
suitably at least 99% identity with the V1V2 region of SEQ ID NO: 1. Suitably
the gp120
related polypeptide comprises the V1V2 region of SEQ ID NO: 1.
Suitably, the gp120 related polypeptide consists of a polypeptide with at
least 70% identity,
suitably at least 80% identity, more suitably at least 85% identity, more
suitably at least 90%
identity, more suitably at least 95% identity, more suitably at least 98%
identity, more suitably
at least 99% identity with the V1V2 region of SEQ ID NO: 1. Suitably, the
gp120 related
polypeptide consists of the V1V2 region of SEQ ID NO: 1.
Alternatively, the gp120 related polypeptide comprises a polypeptide with at
least 70% identity,
more suitably at least 80% identity, more suitably at least 85% identity, more
suitably at least
90% identity, more suitably at least 95% identity, more suitably at least 98%
identity, more
CA 02851852 2014-05-08
11
suitably at least 99% identity with the V1V2 region of SEQ ID NO: 5. Suitably
the gp120 related
polypeptide comprises the V1V2 region of SEQ ID NO: 5.
Suitably, the gp120 related polypeptide consists of a polypeptide with at
least 70% identity,
suitably at least 80% identity, more suitably at least 85% identity, more
suitably at least 90%
identity, more suitably at least 95% identity, more suitably at least 98%
identity, more suitably
at least 99% identity with the V1V2 region of SEQ ID NO: 5. Suitably the gp120
related
polypeptide consists of the V1V2 region of SEQ ID NO: 5.
Suitably, the gp120 related polypeptide comprises a polypeptide with at least
70% identity,
more suitably at least 80% identity, more suitably at least 85% identity, more
suitably at least
90% identity, more suitably at least 95% identity, more suitably at least 98%
identity, more
suitably at least 99% identity with SEQ ID NO: 1. Suitably, the gp120 related
polypeptide
comprises SEQ ID NO: 1.
Suitably, the gp120 related polypeptide consists of a polypeptide with at
least 70% identity,
suitably at least 80% identity, more suitably at least 85% identity, more
suitably at least 90%
identity, more suitably at least 95% identity, more suitably at least 98%
identity, more suitably
at least 99% identity with SEQ ID NO: 1. Suitably, the gp120 related
polypeptide consists of
SEQ ID NO: 1.
Alternatively, the gp120 related polypeptide comprises a polypeptide with at
least 70% identity,
more suitably at least 80% identity, more suitably at least 85% identity, more
suitably at least
90% identity, more suitably at least 95% identity, more suitably at least 98%
identity, more
suitably at least 99% identity with SEQ ID NO: 5. Suitably, the gp120 related
polypeptide
comprises SEQ ID NO: 5.
Suitably, the gp120 related polypeptide consists of a polypeptide with at
least 70% identity,
suitably at least 80% identity, more suitably at least 85% identity, more
suitably at least 90%
identity, more suitably at least 95% identity, more suitably at least 98%
identity, more suitably
at least 99% identity with SEQ ID NO: 5. Suitably, the gp120 related
polypeptide consists of
SEQ ID NO: 5.
The terms "identical" or "% identity," in the context of two or more
polypeptide sequences, refer
to two or more sequences or sub-sequences that are the same or have a
specified percentage
of amino acid residues that are the same over a specified region, when
compared and aligned
CA 02851852 2014-05-08
12
for maximum correspondence over a comparison window, or designated region as
measured
using one of the following sequence comparison algorithms or by manual
alignment and visual
inspection. This definition also refers to the compliment of a test sequence.
Optionally, the
identity exists over a region that is at least 300 amino acids in length, such
as at least 400
amino acids or at least 500 amino acids. Most suitably, the comparison is
performed over a
window corresponding to the entire length of the reference sequence (as
opposed to the
derivative sequence).
For sequence comparison, one sequence acts as the reference sequence, to which
the test
sequences are compared. When using a sequence comparison algorithm, test and
reference
sequences are entered into a computer, subsequence coordinates are designated,
if
necessary, and sequence algorithm program parameters are designated. Default
program
parameters can be used, or alternative parameters can be designated. The
sequence
comparison algorithm then calculates the percentage sequence identities for
the test
sequences relative to the reference sequence, based on the program parameters.
A "comparison window", as used herein, refers to a segment in which a sequence
may be
compared to a reference sequence of the same number of contiguous positions
after the two
sequences are optimally aligned. Methods of alignment of sequences for
comparison are well-
known in the art. Optimal alignment of sequences for comparison can be
conducted, e.g., by
the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482
(1981), by the
homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443
(1970), by the
search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA
85:2444
(1988), by computerised implementations of these algorithms (GAP, BESTFIT,
FASTA, and
TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group,
575
Science Dr., Madison, WI), or by manual alignment and visual inspection (see,
e.g., Curr Pro
Mol Biol (Ausubel et al., eds. 1995 supplement)).
An example of an algorithm that is suitable for determining percent sequence
identity and
sequence similarity are the BLAST and BLAST 2.0 algorithms, which are
described in Altschul
et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol.
215:403-410
(1990), respectively. Software for performing BLAST analyses is publicly
available through the
National Center for Biotechnology Information (website at
www.ncbi.nlm.nih.gov/). This
algorithm involves first identifying high scoring sequence pairs (HSPs) by
identifying short
words of length W in the query sequence, which either match or satisfy some
positive-valued
threshold score T when aligned with a word of the same length in a database
sequence. T is
CA 02851852 2014-05-08
13
referred to as the neighbourhood word score threshold (Altschul et al.,
supra). These initial
neighbourhood word hits act as seeds for initiating searches to find longer
HSPs containing
them. The word hits are extended in both directions along each sequence for as
far as the
cumulative alignment score can be increased. Cumulative scores are calculated
using, for
nucleotide sequences, the parameters M (reward score for a pair of matching
residues; always
> 0) and N (penalty score for mismatching residues; always <0). For amino acid
sequences, a
scoring matrix is used to calculate the cumulative score. Extension of the
word hits in each
direction are halted when: the cumulative alignment score falls off by the
quantity X from its
maximum achieved value; the cumulative score goes to zero or below, due to the
accumulation of one or more negative-scoring residue alignments; or the end of
either
sequence is reached. The BLAST algorithm parameters W, T, and X determine the
sensitivity
and speed of the alignment. The BLASTN program (for nucleotide sequences) uses
as
defaults a wordlength (W) of 11, an expectation (E) or 10, M=5, N=-4 and a
comparison of both
strands. For amino acid sequences, the BLASTP program uses as defaults a
wordlength of 3,
and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff &
Henikoff, Proc.
Natl. Acad. Sci. USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of
10, M=5, N=-4,
and a comparison of both strands.
The BLAST algorithm also performs a statistical analysis of the similarity
between two
sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-
5787 (1993)).
One measure of similarity provided by the BLAST algorithm is the smallest sum
probability
(P(N)), which provides an indication of the probability by which a match
between two
nucleotide or amino acid sequences would occur by chance. For example, a
nucleic acid is
considered similar to a reference sequence if the smallest sum probability in
a comparison of
the test nucleic acid to the reference nucleic acid is less than about 0.2,
more suitably less
than about 0.01, and most suitably less than about 0.001.
An "immunogenic fragment" will contain a contiguous sequence of amino acids
from the gp120
related polypeptide of which it is a fragment. Suitably, the fragment contains
at least 15 to 50
amino acids, at least 51 to 150, at least 300 amino acids, at least 350, at
least 400, or at least
450 contiguous amino acids from SEQ ID NO: 1.
Alternatively the fragment contains at least 15 to 50 amino acids, at least 51
to 150 amino
acids, at least 300, at least 350, at least 400, or at least 450 contiguous
amino acids from SEQ
ID NO: 5.
CA 02851852 2014-05-08
14
The immunogenic fragment will remain capable of eliciting an immune response
against the
V1V2 region of the reference sequence. By "remain capable of eliciting an
immune response
against the V1V2 region of the reference sequence" is meant at least 50%,
suitably at least
75% and suitably at least 90% activity of the reference sequence in an assay
of the level of
antibodies binding the gp120 V1V2 region (e.g. EL1SA) produced at a given time
point after
immunisation with the gp120 related polypeptide.
The gp120 related polypeptide may for example contain 2000 amino acid residues
or fewer,
such as 1500 amino acid residues or fewer, in particular 1000 amino acid
residues or fewer,
especially 800 amino acid residues or fewer.
Fusion proteins (also known as chimeric proteins) are proteins created through
the covalent
linkage of two or more polypeptide sequences which are not joined in nature,
such as through
a peptide linkage. For example, the gp120 related polypeptide may be provided
in the form of
a fusion protein also comprising a second HIV antigen.
The dose of a gp120 related polypeptide is suitably able to produce an
adequate immune
response in a human while having an acceptable reactogenicity profile.
Suitably the immunogenic composition comprises around 1 to 200 ug of gp120
related
polypeptide, suitably between 1 to 100 ug, suitably between 2 to 50 ug, such
as between 3 to
ug, in particular between 5 to 15 ug or between 16 to 25 ug, between 9 to 11
ug or between
19 to 21 ug, most suitably 10 ug or 20 ug.
25 Suitably, the immunogenic composition is provided in a volume which is
suitable for
administration to a human as a single dose. The volume which may be
administered to a
human is dependent on the method, route and/or location of administration. In
one
embodiment the human dose is between 0.1 and 1m1, more suitably between 0.3
and 0.75m1,
such as between 0.45 and 0.55m1, in particular 0.5m1. Volumes of between 0.1
and 1m1 are
30 particularly suitable for administration through routes such as
subcutaneous and in particular
intramuscular delivery. In another embodiment the human dose is between 0.05
and 0.2m1,
suitably between 0.075 and 0.15m1, in particular 0.1m1. Volumes of between
0.05 and 0.2ml
are particularly suitable for administration through routes wherein a limited
volume can be
administered, such as intradermal delivery.
CA 02851852 2014-05-08
A gp120 polypeptide may be prepared by methods described in the art or methods
analogous
thereto.
As used herein, the term 'a Nef related polypeptide' refers to the polypeptide
provided in SEQ
5 ID NO: 2, or to an immunogenic derivative or fragment thereof.
As used herein, the term 'a Tat related polypeptide' refers to the polypeptide
provided in SEQ
ID NO: 3, or to an immunogenic derivative or fragment thereof.
10 As used herein, the term 'a NefTat related polypeptide' refers to the
polypeptide provided in
SEQ ID NO: 4, or to an immunogenic derivative or fragment thereof.
Suitably the Nef related polypeptide will comprise, such as consist of, an
immunogenic
derivative of SEQ ID NO: 2, optionally having a small number of deletions,
insertions and/or
15 substitutions. An example is a derivative of SEQ ID NO: 2 having
deletions of up to 5 residues
at 0-5 locations, insertions of up to 5 residues at 0-5 five locations and
substitutions of up to 20
residues.
Suitably the Tat related polypeptide will comprise, such as consist of, an
immunogenic
derivative of SEQ ID NO: 3, optionally having a small number of deletions,
insertions and/or
substitutions. An example is a derivative of SEQ ID NO: 3 having deletions of
up to 5 residues
at 0-5 locations, insertions of up to 5 residues at 0-5 five locations and
substitutions of up to 20
residues.
Suitably the NefTat related polypeptide will comprise, such as consist of, an
immunogenic
derivative of SEQ ID NO: 4, optionally having a small number of deletions,
insertions and/or
substitutions. An example is a derivative of SEQ ID NO: 4 having deletions of
up to 5 residues
at 0-5 locations, insertions of up to 5 residues at 0-5 five locations and
substitutions of up to 20
residues.
Suitably, the Nef related polypeptide comprises SEQ ID NO: 2. Suitably the Nef
related
polypeptide comprises a polypeptide with at least 99% identity, suitably at
least 98% identity,
more suitably at least 95% identity, more suitably at least 90% identity, more
suitably at least
85% identity, more suitably at least 80% identity, more suitably at least 75%
identity, more
suitably at least 70% identity with SEQ ID NO: 2.
CA 02851852 2014-05-08
16
Suitably, the Tat related polypeptide comprises SEQ ID NO: 3. Suitably the Tat
related
polypeptide comprises a polypeptide with at least 99% identity, suitably at
least 98% identity,
more suitably at least 95% identity, more suitably at least 90% identity, more
suitably at least
85% identity, more suitably at least 80% identity, more suitably at least 75%
identity, more
suitably at least 70% identity with SEQ ID NO: 3.
Suitably, the NefTat related polypeptide comprises SEQ ID NO: 4. Suitably the
NefTat related
polypeptide comprises a polypeptide with at least 99% identity, suitably at
least 98% identity,
more suitably at least 95% identity, more suitably at least 90% identity, more
suitably at least
85% identity, more suitably at least 80% identity, more suitably at least 75%
identity, more
suitably at least 70% identity with SEQ ID NO: 4.
Suitably, the Nef related polypeptide consists of SEQ ID NO: 2. Suitably the
Nef related
polypeptide consists of a polypeptide with at least 99% identity, suitably at
least 98% identity,
more suitably at least 95% identity, more suitably at least 90% identity, more
suitably at least
85% identity, more suitably at least 80% identity, more suitably at least 75%
identity, more
suitably at least 70% identity with SEQ ID NO: 2.
Suitably, the Tat related polypeptide consists of SEQ ID NO: 3. Suitably the
Tat related
polypeptide consists of a polypeptide with at least 99% identity, suitably at
least 98% identity,
more suitably at least 95% identity, more suitably at least 90% identity, more
suitably at least
85% identity, more suitably at least 80% identity, more suitably at least 75%
identity, more
suitably at least 70% identity with SEQ ID NO: 3.
Suitably, NefTat related polypeptide consists of SEQ ID NO: 4. Suitably the
NefTat related
polypeptide consists of a polypeptide with at least 99% identity, suitably at
least 98% identity,
more suitably at least 95% identity, more suitably at least 90% identity, more
suitably at least
85% identity, more suitably at least 80% identity, more suitably at least 75%
identity, more
suitably at least 70% identity with SEQ ID NO: 4.
In one embodiment a Nef related polypeptide is a NefTat related polypeptide.
In another
embodiment a Tat related polypeptide is a NefTat related polypeptide.
Particular derivatives of a Nef related polypeptide, Tat related polypeptide
or NefTat related
polypeptide include those with additional His residues at the N-terminus (e.g.
a polyhistidine
tag of six His residues, which may be used for nickel affinity purification).
CA 02851852 2014-05-08
17
Suitably the compositions of the present invention are substantially free of a
Nef related
polypeptide. Suitably, the compositions of the present invention contain a
ratio of a Nef related
polypeptide:gp120 related polypeptide of less than 1:20, suitably less than
1:25, suitably less
than 1:50, suitably less than 1:100, suitably less than 1:200, suitably less
than 1:500, more
suitably less than 1:1000 by weight.
Suitably the compositions of the present invention contain a Nef related
polypeptide at a level
of less than 5ug, suitably less than 4ug, suitably less than 3ug, suitably
less than 2ug, suitably
less than lug, suitably less than 0.5ug, suitably less than 0.2ug, suitably
less than 0.1ug,
suitably less than 0.05ug, more suitably less than 0.0lug per human dose.
Most suitably the compositions of the present invention are free of a Nef
related polypeptide.
Suitably the compositions of the present invention are substantially free of a
Tat related
polypeptide. Suitably, the compositions of the present invention contain a
ratio of a Tat related
polypeptide:gpl 20 related polypeptide of less than 1:20, suitably less than
1:25, suitably less
than 1:50, suitably less than 1:100, suitably less than 1:200, suitably less
than 1:500, more
suitably less than 1:1000 by weight.
Suitably the compositions of the present invention contain a Tat related
polypeptide at a level
of less than 5ug, suitably less than 4ug, suitably less than 3ug, suitably
less than 2ug, suitably
less than lug, suitably less than 0.5ug, suitably less than 0.2ug, suitably
less than 0.1ug,
suitably less than 0.05ug, more suitably less than 0.0lug per human dose.
Most suitably the compositions of the present invention are free of a Tat
related polypeptide.
Suitably the compositions of the present invention are substantially free of a
NefTat related
polypeptide. Suitably, the compositions of the present invention contain a
ratio of a NefTat
related polypeptide:gp120 related polypeptide of less than 1:20, suitably less
than 1:25,
suitably less than 1:50, suitably less than 1:100, suitably less than 1:200,
suitably less than
1:500, more suitably less than 1:1000 by weight.
Suitably the compositions of the present invention contain a NefTat related
polypeptide at a
level of less than 5ug, suitably less than 4ug, suitably less than 3ug,
suitably less than 2ug,
CA 02851852 2014-05-08
18
suitably less than lug, suitably less than 0.5ug, suitably less than 0.2ug,
suitably less than
0.1ug, suitably less than 0.05ug, more suitably less than 0.0lug per human
dose.
Most suitably the compositions of the present invention are free of a NefTat
related
polypeptide.
The presence of further HIV proteins may increase the efficacy of the
composition of the
invention. Suitably the composition of the invention may comprise additional
proteins which
may be further products of the Env gene (such as gp160 and gp41 or a further
gp120 related
polypeptide) the products of the gag and pol genes (such as MA, CA, SP1, NC,
SP2 and P6;
and RT, RNase H, IN and PR, respectively) and, other non-structural proteins
such as Rev,
Vif, Vpr and Vpu (or immunogenic derivatives or fragments thereof).
The HIV gag gene encodes a precursor protein p55, which can assemble
spontaneously into
immature virus-like particles (VLPs). The precursor is proteolytically cleaved
into the major
structural proteins CA (capsid) and MA (matrix), and into several smaller
proteins. Both the
precursor protein p55 and its major derivatives CA and MA may be considered as
appropriate
vaccine antigens which may increase the efficacy of the composition of the
invention. The
precursor p55 and the capsid protein CA may be used as VLPs or as monomeric
proteins.
Suitably the composition of the present invention may comprise one or more of
these proteins.
Suitably, the composition of the present invention may comprise a
polynucleotide encoding a
polypeptide described above including for example a gp120 related polypeptide.
The
polynucleotide may be in the form of plasmid DNA or in the form of a
recombinant live vector.
In one embodiment, the composition of the present invention does not comprise
additional HIV
antigens. In a second embodiment 'of the invention the composition comprises 1-
5 additional
HIV antigens, such as 1 or 2 additional HIV antigens. Additional HIV antigens
may be
provided in the form of proteins or polynucleotides encoding proteins.
Suitably, the immunogenic composition comprises a total of around 1 to 500 ug
of antigenic
material, suitably between 1 to 200 ug, such as between 5 to 100 ug, most
suitably between 5
to 50 ug.
Suitably, the polypeptides and polynucleotides used in the present invention
are isolated. An
"isolated" polypeptide or polynucleotide is one that is removed from its
original environment.
CA 02851852 2014-05-08
19
For example, a naturally-occurring protein is isolated if it is separated from
some or all of the
coexisting materials in the natural system. Preferably, such polypeptides are
at least about
90% pure, more suitably at least about 95% pure and most suitably at least
about 99% pure.
A polynucleotide is considered to be isolated if, for example, it is cloned
into a vector that is not
a part of its natural environment.
Adjuvant
Sapon ins
The immunogenic composition of the invention comprises an immunologically
active saponin
fraction ("a saponin") as an adjuvant or as a component of an adjuvant. A
particularly suitable
saponin for use in the present invention is Quil A and its derivatives. Quil A
is a saponin
preparation isolated from the South American tree Quillaja Saponaria Molina
and was first
described by Dalsgaard et al. in 1974 ("Saponin adjuvants", Archiv. fur die
gesamte
Virusforschung, Vol. 44, Springer Verlag, Berlin, p243-254) to have adjuvant
activity. Purified
fragments of Quil A have been isolated by HPLC which retain adjuvant activity
without the
toxicity associated with Quil A (US5604106), for example QS-7 and QS-21 (also
known as
QA7 and QA21). QS-21 is a natural saponin derived from the bark of Quillaja
saponaria
Molina, which induces CD8+ cytotoxic T cells (CTLs), Thl cells and a
predominant IgG2a
antibody response and is a preferred saponin in the context of the present
invention.
In a suitable form of the present invention, the saponin adjuvant within the
immunogenic
composition is a derivative of saponaria molina quil A, suitably an
immunologically active
fraction of Quil A, such as QS-7 or QS-21, suitably QS-21. In one embodiment
the
compositions of the invention contain the immunologically active saponin
fraction in
substantially pure form. Suitably the compositions of the invention contain QS-
21 in
substantially pure form, that is to say, the QS-21 is at least 90% pure, for
example at least 95%
pure, or at least 98% pure.
In a specific embodiment, QS-21 is provided in a less reactogenic composition
where it is
quenched with an exogenous sterol, such as cholesterol for example. Several
particular forms
of less reactogenic compositions wherein the lytic activity of QS-21 is
quenched with an
exogenous cholesterol exist. In a specific embodiment, the saponin/sterol is
in the form of a
liposome structure (US6846489, Example 1). In this embodiment the liposomes
suitably
contain a neutral lipid, for example phosphatidylcholine, which is suitably
non-crystalline at
CA 02851852 2014-05-08
room temperature, for example eggyolk phosphatidylcholine, dioleoyl
phosphatidylcholine
(DOPC) or dilauryl phosphatidylcholine. The liposomes may also contain a
charged lipid which
increases the stability of the lipsome-QS-21 structure for liposomes composed
of saturated
lipids. In these cases the amount of charged lipid is suitably 1-20% w/w,
suitably 5-10%. The
5 ratio of sterol to phospholipid is 1-50% (mol/mol), suitably 20-25%.
Suitable sterols include beta-sitosterol, stigmasterol, ergosterol,
ergocalciferol and cholesterol.
In one particular embodiment, the adjuvant composition comprises cholesterol
as sterol.
These sterols are well known in the art, for example cholesterol is disclosed
in the Merck
10 Index, 11th Edn., page 341, as a naturally occurring sterol found in
animal fat.
The sterol according to the invention is taken to mean an exogenous sterol,
i.e. a sterol which
is not endogenous to the organism from which the antigenic preparation is
taken but is added
to the antigen preparation or subsequently at the moment of formulation.
Typically, the sterol
15 may be added during subsequent formulation of the antigen preparation with
the saponin
adjuvant, by using, for example, the saponin in its form wherein its lytic
activity is quenched
with the sterol. Suitably the exogenous sterol is associated to the saponin
adjuvant as
described in US6846489.
20 Where the active saponin fraction is QS-21, the ratio of QS-21 : sterol
will typically be in the
order of 1:100 to 1:1 (w/w), suitably between 1:10 to 1:1 (w/w), and suitably
1:5 to 1:1 (w/w).
Suitably excess sterol is present, the ratio of QS-21 : sterol being at least
1:2 (w/w). In one
embodiment, the ratio of QS-21 : sterol is 1:5 (w/w).
Other saponins which have been described in the literature include Escin,
which has been
described in the Merck index (12th ed: entry 3737) as a mixture of saponins
occurring in the
seed of the horse chestnut tree, Lat: Aesculus hippocastanum. Its isolation is
described by
chromatography and purification (Fiedler, Arzneimittel-Forsch. 4, 213 (1953)),
and by ion-
exchange resins (Erbring et al., US 3,238,190). Fractions of escin have been
purified and
shown to be biologically active (Yoshikawa M, et al. (Chem Pharm Bull (Tokyo)
1996
44(8):1454-1464)). Sapoalbin from Gypsophilla struthium (R. Vochten et al.,
1968, J Pharm
BeIg, 42, 213-226) has also been described in relation to ISCOM production for
example.
Another useful saponin is those derived from the plant Gyophilla struthium.
CA 02851852 2014-05-08
21
Suitably, the total amount of saponin in the immunogenic composition of the
present invention,
particularly in a human dose of the immunogenic composition of the present
invention. is
between 1-10Oug.
In one embodiment, there is provided an immunogenic composition comprising QS-
21 at a
level of around 50 ug, for example between 38-100 ug, suitably between 40-75
ug or between
45-60 ug, more suitably 49-51, most suitably 50 ug.
In a further embodiment, there is provided an immunogenic composition
comprising QS-21 at
a level of around 25 ug, for example between 10-37 ug, suitably between 15-30
ug or between
20-27 ug, more suitably 24-26, more suitably 25 ug.
In another embodiment, there is provided an immunogenic composition in a
volume which is
suitable for a human dose which human dose of the immunogenic composition
comprises QS-
21 at a level of around 50 ug, for example between 38-100 ug, suitably between
40-75 ug or
between 45-60 ug, more suitably 49-51, most suitably 50 ug.
In another embodiment, there is provided an immunogenic composition in a
volume which is
suitable for a human dose which human dose of the immunogenic composition
comprises QS-
21 at a level of around 25 ug, for example between 10-37 ug, suitably between
15-30 ug or
between 20-27 ug, more suitably 24-26, more suitably 25 ug.
The dose of QS-21 is suitably able to enhance an immune response to an antigen
in a human.
In particular a suitable QS-21 amount is that which improves the immunological
potential of the
composition compared to the unadjuvanted composition, or compared to the
composition
adjuvanted with another QS-21 amount, whilst being acceptable from a
reactogenicity profile.
Lipopolysaccharide adjuvants
Lipopolysaccharides (LPS) are the major surface molecule of, and occur
exclusively in, the
external leaflet of the outer membrane of gram-negative bacteria. LPS impede
destruction of
bacteria by serum complements and phagocytic cells, and are involved in
adherence for
colonisation. LPS are a group of structurally related complex molecules of
approximately
10,000 Da!tons in size and consist of three covalently linked regions:
(i) an 0-specific polysaccharide chain (0-antigen) at the outer region
CA 02851852 2014-05-08
22
(ii) a core oligosaccharide central region
(iii) lipid A - the innermost region which serves as the hydrophobic
anchor, it comprises
glucosamine disaccharide units which carry long chain fatty acids.
The biological activities of LPS, such as lethal toxicity, pyrogenicity and
adjuvanticity, have
been shown to be related to the lipid A moiety. In contrast, immunogenicity is
associated with
the 0-specific polysaccharide component (0-antigen). Both LPS and lipid A have
long been
known for their strong adjuvant effects, but the high toxicity of these
molecules has precluded
their use in vaccine formulations. Significant effort has therefore been made
towards reducing
the toxicity of LPS or lipid A while maintaining their adjuvanticity.
The Salmonella minnesota mutant R595 was isolated in 1966 from a culture of
the parent
(smooth) strain (Luderitz et al. 1966 Ann N Y Acad Sci 133:349-374). The
colonies selected
were screened for their susceptibility to lysis by a panel of phages, and only
those colonies
that displayed a narrow range of sensitivity (susceptible to one or two phages
only) were
selected for further study. This effort led to the isolation of a deep rough
mutant strain which is
defective in LPS biosynthesis and referred to as S. minnesota R595.
In comparison to other LPS, those produced by the mutant S. minnesota R595
have a
relatively simple structure.
(i) they contain no 0-specific region - a characteristic which is
responsible for the shift
from the wild type smooth phenotype to the mutant rough phenotype and results
in a
loss of virulence
(ii) the core region is very short - this characteristic increases the
strain susceptibility to a
variety of chemicals
(iii) the lipid A moiety is highly acylated with up to 7 fatty acids.
4'-monophosporyl lipid A (MPL), which may be obtained by the acid hydrolysis
of LPS
extracted from a deep rough mutant strain of gram-negative bacteria, retains
the adjuvant
properties of LPS while demonstrating a toxicity which is reduced by a factor
of more than
1000 (as measured by lethal dose in chick embryo eggs) (Johnson et al. 1987
Rev Infect Dis 9
Suppl:S512-S516). LPS is typically refluxed in mineral acid solutions of
moderate strength
(e.g. 0.1 M HCI) for a period of approximately 30 minutes.
This process results in
dephosphorylation at the 1 position, and decarbohydration at the 6' position,
yielding MPL.
CA 02851852 2014-05-08
23
3-0-deacylated monophosphoryl lipid A (3D-MPL), which may be obtained by mild
alkaline
hydrolysis of MPL, has a further reduced toxicity while again maintaining
adjuvanticity, see
US4912094 (Ribi lmmunochemicals). Alkaline hydrolysis is typically performed
in organic
solvent, such as a mixture of chloroform/methanol, by saturation with an
aqueous solution of
weak base, such as 0.5 M sodium carbonate at pH 10.5. Further information on
the
preparation of 3D-MPL is available in, for example, US4912094 (Corixa
Corporation).
The composition further comprises an additional adjuvant which is a
lipopolysaccharide,
suitably a non-toxic derivative of lipid A, particularly monophosphoryl lipid
A or more
particularly 3-deacylated monophoshoryl lipid A (3D-MPL).
3D-MPL is sold under the name MPL by GlaxoSmithKline Biologicals N.A. and is
referred
throughout the document as MPL or 3D-MPL. see, for example, US4436727;
US4877611;
US4866034 and US4912094. 3D-MPL can be produced according to the methods
disclosed
in US4912094. Chemically it is a mixture of 3-deacylated monophosphoryl lipid
A with 3, 4, 5
or 6 acylated chains. Suitably in the compositions of the present invention
small particle 3D-
MPL is used. Small particle 3D-MPL has a particle size such that it may be
sterile-filtered
through a 0.22um filter. Such preparations are described in US5776468.
Suitably, the total amount of lipopolysaccharide in the immunogenic
composition of the present
invention, particularly in a human dose of the immunogenic composition of the
present
invention, is between 1-10Oug.
In one embodiment, there is provided an immunogenic composition comprising 3D-
MPL at a
level of around 50 ug, for example between 38-100 ug, suitably between 40-75
ug or between
45-60 ug, more suitably 49-51, most suitably 50 ug.
In a further embodiment, there is provided an immunogenic composition
comprising 3D-MPL at
a level of around 25 ug, for example between 10-37 ug, suitably between 15-30
ug or between
20-27 ug, more suitably 24-26, more suitably 25 ug.
In another embodiment, there is provided an immunogenic composition in a
volume which is
suitable for a human dose which human dose of the immunogenic composition
comprises 3D-
MPL at a level of around 50 ug, for example between 38-100 ug, suitably
between 40-75 ug or
between 45-60 ug, more suitably 49-51, most suitably 50 ug.
CA 02851852 2014-05-08
24
In another embodiment, there is provided an immunogenic composition in a
volume which is
suitable for a human dose which human dose of the immunogenic composition
comprises 3D-
MPL at a level of around 25 ug, for example between 10-37 ug, suitably between
15-30 ug or
between 20-27 ug, more suitably 24-26, more suitably 25 ug.
Suitable compositions of the invention are those wherein liposomes are
initially prepared
without MPL (as described in US6846489), and MPL is then added, suitably as
small particles
of below 100 nm particles or particles that are susceptible to sterile
filtration through a 0.22 urn
membrane. The MPL is therefore not contained within the vesicle membrane
(known as MPL
out). Compositions where the MPL is contained within the vesicle membrane
(known as MPL
in) also form an aspect of the invention. The antigen can be contained within
the vesicle
membrane or contained outside the vesicle membrane. Suitably soluble antigens
are outside
and hydrophobic or lipidated antigens are either contained inside or outside
the membrane.
The invention comprises both lipopolysaccharide and immunologically active
saponin. In a
specific embodiment of the invention, the lipopolysaccharide is 3D-MPL and the
immunologically active saponin is QS-21. In an embodiment of the invention,
the composition
comprises a lipopolysaccharide and immunologically active saponin in a
liposomal formulation.
Suitably in one form of these embodiments, the composition comprises 3D-MPL
and QS-21,
with optionally a sterol which is suitably cholesterol.
In a further embodiment of the invention, the adjuvant composition comprises
in a liposomal
formulation lipopolysaccharide and immunologically active saponin in
combination with one or
more further immunostimulants or adjuvants. Suitably in one form of this
embodiment the
lipopolysaccharide is 3D-MPL and the immunologically active saponin is QS-21.
In a specific embodiment, QS-21 and 3D-MPL are present in a weight ratio of
between 1:2 to
2:1. Suitably QS-21 and 3D-MPL are present in same final amount per human dose
of the
immunogenic composition. In one aspect of this embodiment, a human dose of
immunogenic
composition comprises a final level of 50 ug of 3D-MPL and 50 ug of QS-21. In
another
aspect, a human dose of immunogenic composition comprises a final level of 25
ug of 3D-MPL
and 25 ug of QS-21. In a further embodiment, a human dose of immunogenic
composition
comprises a final level of bug each of MPL and QS-21.
Vaccine preparation is generally described in New Trends and Developments in
Vaccines,
edited by Voller et al., University Park Press, Baltimore, Maryland, U.S.A.
1978. Encapsulation
CA 02851852 2014-05-08
within liposomes is described, for example, in US4235877. Conjugation of
proteins to
macromolecules is disclosed, for example, in US4372945 and in US4474757.
Salt concentration
5
Some antigens are sensitive to the presence of salts. Without being limited by
theory, it is
believed that these antigens are detrimentally impacted by a phenomenon known
as "salting
out" which may be defined as the precipitation of a protein from its solution
by interaction with
salts, such as sodium chloride. These antigens aggregate and precipitate at a
concentration of
10 sodium chloride as low as 150 mM. Consequently, the stability of
immunogenic compositions
comprising a gp120 related polypeptides may be improved by a reduction in the
concentration
of sodium chloride.
The immunogenic compositions of the invention will suitably be aqueous
preparations.
Accordingly, the present invention provides an immunogenic composition
comprising a gp120
related polypeptide, wherein the conductivity of the composition is 13 mS/cm
or lower. In
particular, the present invention provides immunogenic compositions wherein
the conductivity
of the immunogenic composition is 12 mS/cm or lower, for example 10 mS/cm or
lower, 8
mS/cm or lower, 6 mS/cm or lower, 5 mS/cm or lower, 4 mS/cm or lower, or 3
mS/cm or lower.
In a particular embodiment the conductivity of the immunogenic composition is
2.5 mS/cm or
lower, such as 2.25 mS/cm or lower, or 2.0 mS/cm or lower. In a further
specific embodiment
the conductivity of the immunogenic composition is 1.5 to 2.5 mS/cm.
Additionally provided is an immunogenic composition comprising a gp120 related
polypeptide,
wherein the concentration of salts in said composition is 130 mM or lower. In
particular, the
present invention provides immunogenic compositions wherein the concentration
of salts in
said composition is 100 mM or lower, for example 90 mM or lower, 80 mM or
lower, 70 mM or
lower, 60 mM or lower, 50 mM or lower, or 40 mM or lower. In a particular
embodiment the
concentration of salts in said composition is 35 mM or lower, such as 30 mM or
lower, or 25
mM or lower. In a further specific embodiment the concentration of salts in
said composition is
20 to 40 mM, such as 25 to 35 mM.
The present invention also provides an immunogenic composition comprising a
gp120 related
polypeptide, wherein the concentration of sodium chloride in said composition
is 130 mM or
lower. In particular, the present invention provides immunogenic compositions
wherein the
CA 02851852 2014-05-08
26
concentration of sodium chloride is 100 mM or lower, for example 90 mM or
lower, 80 mM or
lower, 70 mM or lower, 60 mM or lower, 50 mM or lower, 40 mM or lower, 30 mM
or lower, 20
mM or lower or 15 mM or lower. In a particular embodiment the concentration of
sodium
chloride in the immunogenic composition is 10 mM or lower, such as 7.5 mM or
lower.
Suitably the concentration of sodium chloride in the immunogenic composition
is at or below 5
mM. In a further specific embodiment, the immunogenic composition is
essentially free of
sodium chloride. By essentially free is meant that the concentration of sodium
chloride is at or
very near to zero mM (such as 3 mM or less, 2 mM or less or 1 mM or less).
Suitably, the concentration of CaCl2 in the immunogenic compositions will be
40 mM or lower,
30 mM or lower, 20 mM or lower, 15 mM or lower or 10 mM or lower.
Suitably, the concentration of MgSO4 in the immunogenic compositions will be
80 mM or lower,
60 mM or lower, 40 mM or lower, 30 mM or lower, 20 mM or lower or 10 mM or
lower
Suitably, the total concentration of NH, Mg2+ and Ca2+ ions in the immunogenic
compositions
will be 80 mM or lower, 60 mM or lower, 40 mM or lower, 30 mM or lower, 20 mM
or lower or
10 mM or lower.
It is well known that for parenteral administration solutions should have a
pharmaceutically
acceptable osmolality to avoid cell distortion or lysis. A pharmaceutically
acceptable osmolality
will generally mean that solutions will have an osmolality which is
approximately isotonic or
mildly hypertonic. Suitably the immunogenic compositions of the present
invention will have
an osmolality in the range of 250 to 750 mOsm/kg, for example, the osmolality
may be in the
range of 250 to 550 mOsm/kg, such as in the range of 280 to 500 mOsm/kg.
Osmolality may be measured according to techniques known in the art, such as
by the use of a
commercially available osmometer, for example the Advanced Model 2020
available from
Advanced Instruments Inc. (USA).
An "isotonicity agent" is a compound that is physiologically tolerated and
imparts a suitable
tonicity to a formulation to prevent the net flow of water across cell
membranes that are in
contact with the formulation.
In a particular embodiment there are provided immunogenic compositions further
comprising a
non-ionic tonicity agent. A non-ionic tonicity agent for use in an immunogenic
composition will
CA 02851852 2014-05-08
27
itself need to be pharmaceutically acceptable, e.g. suitable for use in
humans, as well as being
compatible with the gp120 related antigen and further compatible with other
components such
as the immunostimulant(s).
In one embodiment of the present invention, suitable non-ionic tonicity agents
are polyols,
sugars (in particular sucrose, fructose, dextrose or glucose) or amino acids
such as glycine. In
one embodiment the polyol is a sugar alcohol, especially a C3-6 sugar alcohol.
Exemplary
sugar alcohols include glycerol, erythritol, threitol, arabitol, xylitol,
ribitol, sorbitol, mannitol,
dulcitol and iditol. In a specific example of this embodiment, a suitable non-
ionic tonicity agent
is sorbitol. The skilled person will recognise that an appropriate osnnolality
may be attained
through the use of a mixture of different tonicity agents. In a particular
embodiment of the
invention the non-ionic tonicity agent in the compositions of the invention
incorporates sucrose
and/or sorbitol.
In one embodiment, a suitable concentration of polyol within the immunogenic
composition is
between about 2.5 and about 15% (w/v), in particular between about 2.5 and
about 10% (w/v)
for example between about 3 and about 7% (w/v), such as between about 4 and
about 6%
(w/v). In a specific example of this embodiment, the polyol is sorbitol.
In another embodiment, the immunogenic composition comprises sucrose and
sorbitol. In
such circumstances the immunogenic composition may suitably contain between
about 2.5
and about 15% (w/v) of sucrose and between about 2.5 and about 15% (w/v) of
sorbitol, in
particular between about 2.5 and about 10% (w/v) of sucrose and between about
2.5 and
about 10% (w/v) of sorbitol, for example, between about 3 and about 7% (w/v)
of sucrose and
between about 3 and about 7% (w/v) of sorbitol, such as between about 4 and
about 6% (w/v)
of sucrose and between about 4 and about 6% (w/v) of sorbitol.
The pH of the immunogenic compositions should be suitable for parenteral
administration.
Typically the pH will be in the range of 6.0 to 9Ø Suitably the pH will be
in the range 7.0 to
9.0, especially 7.25 to 8.75, such as 7.5 to 8.5, in particular pH 7.75 to
8.25. A pH of about
8.0 is of particular interest.
The pH may be controlled by the use of buffers, including for example Iris or
phosphate
buffers.
CA 02851852 2014-05-08
28
The conductivity of an immunogenic composition of the invention can be
measured using
techniques known in the art, for example using a dedicated conductivity meter
or other
instrument with the capability to measure conductivity. One suitable
instrument is the
Zetasizer Nano ZS from Malvern Instruments (UK).
The skilled person can readily test for the concentration of both sodium (Na)
and chloride (Cr)
ions using known techniques and kits. For example, sodium can be determined
using a kit
such as the Sodium Enzymatic Assay Kit (Catalogue Number: BQ011EAEL) from
Biosupply.
Chloride can be determined using a kit such as Chloride Enzymatic Assay Kit
(Catalogue
Number: BQ006EAEL) from Biosupply.
Immunogenic properties of the immunogenic composition of the present invention
In the present invention the immunogenic composition is suitably capable of
inducing humoral
response in a mammal, such as a human, administered with the immunogenic
composition.
Humoral responses can be detected using an appropriate antibody-based assay.
For
example, the presence or absence in serum of an immunoglobulin G (IgG)
antibody response
to a gp120 related polypeptide can be analyzed using ELISA. The induction of
humoral
responses such as IgG antibodies, suitably IgG antibodies binding the V1V2
region of gp120,
indicates the immunogenicity of the immunogenic compositions of the invention.
In a further embodiment, the immunogenic composition is capable of inducing an
improved
CD4 T-cell immune response.
By "improved CD4 1-cell immune response" is meant that a higher CD4 response
is obtained
in a mammal, such as a human, after administration of the adjuvanted
immunogenic
composition.
In particular but not exclusively, said 'improved CD4 1-cell immune response'
is obtained in an
immunologically unprimed patient, i.e. a patient who is seronegative to HIV.
The improved CD4 1-cell immune response (which may be provided by
`polyfunctionar T cells)
may be assessed by measuring the number of cells producing any of the
following immune
markers:
= CD4 T cells that express at least one immune marker
CA 02851852 2014-05-08
29
= cells producing at least two different immune markers (e.g. CD4OL, IL-2
and/or IFN-
gamma, TNF-alpha)
= cells producing at least CD4OL and another immune marker (e.g. IL-2, TNF-
gamma,
and/or IFN-gamma)
= cells producing at least IL-2 and another immune marker (e.g. CD4OL, TNF-
alpha
and/or IFN-gamma)
= cells producing at least IFN-gamma and another immune marker (e.g. IL-2,
TNF-alpha
and/or CD4OL)
= cells producing at least TNF-alpha and another immune marker (e.g. IL-2,
CD4OL
and/or IFN-gamma)
There will be an improved CD4 T-cell immune response when cells producing any
of the above
immune markers are in a higher amount following administration. Typically at
least one,
suitably two of the six conditions mentioned herein above will be fulfilled.
In a particular
embodiment, the cells producing all four immune markers will be present at a
higher amount.
The improved CD4 1-cell immune response conferred by the gp120 composition of
the present
invention may be ideally obtained after one single administration.
In another embodiment, the administration of said immunogenic composition
induces an
improved B-memory cell response in a mammal, such as a human, administered
with the
immunogenic composition. An improved B-memory cell response is intended to
mean an
increased frequency of peripheral blood B lymphocytes capable of
differentiation into antibody-
secreting plasma cells upon antigen encounter as measured by stimulation of in-
vitro
differentiation.
In a specific embodiment, the administration of said immunogenic composition
induces at least
two of the following responses: (i) an improved CD4 1-cell immune response,
(ii) an improved
B-memory cell response, (iii) an improved humoral response, against at least
one of the
component antigen(s) or antigenic composition compared to either immune
response obtained
with other compositions.
The magnitude of an immune response can also be expressed as the titre (or
concentration) of
antigen-specific antibodies induced by the immunogenic composition as
determined by an
appropriate serological test. The magnitude of a T cell response can be
expressed as the
CA 02851852 2014-05-08
frequency (or number) of antigen-specific cells induced by the immunogenic
composition
among the total population of T cells, which can be monitored by cytokine
production.
Suitably the composition of the present invention elicits an immune response
capable of cross-
5 reactivity. Cross-reactivity is herein taken to mean the ability of
immune responses induced by
an immunogenic composition of the invention to recognize strains of HIV-1 from
subtypes that
are not represented in the immunogenic composition. For example, an
immunogenic
composition of the invention comprising a gp120 related polypeptide comprising
from a strain
of HIV-1 from subtype B is considered cross-reactive if the HIV-specific
immune response,
10 such as HIV-specific antibody or CD4+ T cell response (in particular an
antibody response to
the V1V2 loop of gp120), induced by the composition reacted with one or more
different
strains of HIV-1 not in the composition, for example, with a strain of HIV-1
from a subtype other
than subtype B. Suitably, cross-reactivity will be in respect of an HIV-1
strain from a different
sub-type, in particular in respect of an HIV-1 strain from a different group.
Suitably, the level of cross-reactivity observed is up to 10%, up to 15%, up
to 20%, up to 25%,
up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 55%, up to 60%,
up to 65% up
to 70%, up to 80%, up to 90% or up to 100% of antigen-specific cells induced
by the
immunogenic composition among the total population of T cells or titre (or
concentration) of
antigen-specific antibodies induced by the immunogenic composition.
When measuring cross-reactivity in terms of the percentage of responders to
the strains of
HIV-1 from different subtypes, the number or percentage of vaccinated
individuals that show a
positive response in an immunological assay after subsequent challenge can be
measured. A
responder can respond to one or more epitopes on an antigen. A responder can
also respond
to one or more polypeptides in an immunogenic composition of the invention
and/or to one or
more antigens in an immunogenic composition of the invention.
Immunological assays such as serological tests that can be used to analyse the
percentage of
responders or the magnitude of an immune response are known in the art.
Examples of such
assays are known to a person skilled in the art.
Suitably, the level of cross-reactivity observed is up to 10%, up to 15%, up
to 20%, up to 25%,
up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 55%, up to 60%,
up to 65% up
to 70%, up to 80%, up to 90% or up to 100% of subjects in a sample are
responders.
CA 02851852 2014-05-08
31
In an embodiment, the immunogenic composition of the invention is for use in
eliciting high and
long-lasting numbers of HIV-1-specific antibodies in an individual not
infected with HIV.
In a further embodiment, the immunogenic composition of the invention is for
use in eliciting
high and long-lasting numbers of HIV-1-specific antibodies in an individual at
risk of infection
with an HIV-1 strain from one or more clades different from the one or more
HIV-1 clades from
which the gp120 related polypeptide in the immunogenic composition is derived.
In an embodiment, the immunogenic composition of the invention is for use in
controlling or
reducing viremia in an individual infected with HIV.
Suitably, after administration of the composition, the viral load of the
subject remains below
100,000 copies/m1 for at least four months after administration. In a further
embodiment, the
viral load of the subject remains below 100,000 copies/ml of serum for at
least six months, at
least twelve months, at least eighteen months, at least two years, at least
three years, at least
four years, at least five years, at least six years, at least seven years, at
least eight years, at
least nine years, or at least ten years. In another embodiment, the subject
maintains a viral
load below 50,000 copies/ml, below 10,000 copies/ml, below 5000 copies/ml,
below 1000
copies/ml, or below 500 copies/ml. Suitably, viral load is maintained or
reduced for at least six
months, at least twelve months, at least eighteen months, at least two years,
at least three
years, at least four years, at least five years, at least six years, at least
seven years, at least
eight years, at least nine years, or at least ten years after administration
of the composition.
Suitably, administration of the inventive composition results in a durable
response. A durable
response is for example the ability to detect, in the serum of an individual,
IgG antibody
capable of binding to the V1V2 region of the gp120 related polypeptide of the
composition at
least 24 weeks, at least 48 weeks, at least 72 weeks, at least 96 weeks, at
least two years, at
least three years, at least four years, at least five years, at least six
years, at least seven years,
at least eight years, at least nine years, or at least ten years after the
only administration of the
composition, or the first administration of the composition in a course of
repeat administrations,
to the individual. Suitably, antibody levels will be detected at a level of at
least 5%, more
suitably at least 10% and in particular at least 20% of the serum titre two
weeks following the
first administration. Suitably the antibody will be detectable in at least 50%
of individuals, more
suitably at least 60% of individuals and in particular at least 75%.
CA 02851852 2014-05-08
32
Suitably, a durable response is for example the ability to detect, in the
serum of an individual,
IgG antibody binding the V1V2 region of the gp120 related polypeptide of the
composition at
least 2 weeks, at least 6 months, at least 12 months, at least 18 months, at
least two years, at
least three years, at least four years, at least five years, at least six
years, at least seven years,
at least eight years, at least nine years, or at least ten years after the
final administration of the
composition in a course of repeat administrations to the individual. Suitably,
antibody levels
will be detected at a level of at least 5%, more suitably at least 10% and in
particular at least
20% of the serum titre two weeks following the final administration. Suitably
the antibody will
be detectable in at least 50% of individuals, more suitably at least 60% of
individuals and in
particular at least 75%.
Suitably, the present invention is capable of achieving a more durable immune
response
based on responder rates. Suitably, up to 10%, up to 15%, up to 20%, up to
25%, up to 30%,
up to 35%, up to 40%, up to 45%, up to 50%, up to 55%, up to 60%, up to 65% up
to 70%, up
to 80%, up to 90% or up to 100% of vaccinated individuals mount an increased
humoral
response such as an increased serum level of IgG antibody binding the V1V2
region of the
gp120 related polypeptide of the composition.
Vaccination means
The immunogenic compositions of the invention may be administered by any
suitable delivery
route, such as intradermal, mucosal e.g. intranasal, oral, intramuscular or
subcutaneous.
Other delivery routes are well known in the art. The intramuscular delivery
route is preferred
for the immunogenic composition.
Intradermal delivery is another suitable route. Any suitable device may be
used for intradermal
delivery, for example short needle devices such as those described in
US4886499. Intradermal
vaccines may also be administered by devices which limit the effective
penetration length of a
needle into the skin. Also suitable are jet injection devices which deliver
liquid vaccines to the
dermis via a liquid jet injector or via a needle which pierces the stratum
corneum and produces
a jet which reaches the dermis. Jet injection devices are described for
example in
US5480381. Also suitable are ballistic powder/particle delivery devices which
use compressed
gas to accelerate vaccine in powder form through the outer layers of the skin
to the dermis.
Additionally, conventional syringes may be used in the classical mantoux
method of
intradermal administration.
CA 02851852 2014-05-08
33
Another suitable administration route is the subcutaneous route. Any suitable
device may be
used for subcutaneous delivery, for example classical needle. Suitably, a
needle-free jet
injector service is used, such as that published in US6623446. More suitably
said device is
pre-filled with the liquid vaccine formulation.
Alternatively the vaccine is administered intranasally. Typically, the vaccine
is administered
locally to the nasopharyngeal area, suitably without being inhaled into the
lungs. It is desirable
to use an intranasal delivery device which delivers the vaccine formulation to
the
nasopharyngeal area, without or substantially without it entering the lungs.
In a specific aspect of the present invention, the immunogenic composition may
be given
intramuscularly for the first administration, and a boosting composition may
be administered
through a different route, for example intradermal, subcutaneous or
intranasal.
In one aspect of the invention a schedule for vaccination with the immunogenic
composition
may comprise the sequential ("prime-boost"), concomitant or simultaneous
administration of
the immunogenic composition and DNA encoding any of the above-mentioned
proteins.
In one embodiment the schedule for vaccination with the immunogenic
composition consists of
three to five (for example four) administrations of the immunogenic
composition to an individual
over a period of four to eight (for example five to seven) months. Suitably,
the schedule for
vaccination with the immunogenic composition consists of four administrations
of the
immunogenic composition to an individual over a period of five to seven
months.
Suitably, the gp120 related polypeptide of the invention may be substituted by
a polynucleotide
encoding the gp120 related polypeptide of the invention. Suitably, the
polynucleotide is codon-
optimised.
The DNA may be delivered as plasmid DNA or in the form of a recombinant live
vector, e.g. a
poxvirus vector or any other suitable live vector such as retrovirus,
lentivirus, adenovirus,
adeno-associated virus and modified vaccinia ankara (MVA) virus. Suitably, an
adenovirus is
used. Alternatively a canary pox virus may be used.
The immunogenic composition may be injected once or several times followed by
one or more
DNA administrations. DNA may be used first for one or more administrations
followed by one
CA 02851852 2014-05-08
34
or more immunisations with the immunogenic composition. Alternatively, the
immunogenic
composition may be injected once or several times in conjunction with DNA
administrations.
Antiretrovirals
HIV is a retrovirus. The conversion of its RNA to DNA is accomplished through
the action of
the enzyme reverse transcriptase. Compounds that inhibit the function of
reverse
transcriptase inhibit replication of HIV in infected cells. Drugs
incorporating such compounds
are useful in the prevention or treatment of HIV infection in humans and may
be utilised in
conjunction with a composition of the present invention.
The composition of the present invention may be administered in conjunction
with (i.e. before,
during or after administration of) antiretroviral therapy (ART) such as
nucleoside or non-
nucleoside reverse transcriptase inhibitors, protease inhibitors, fusion
inhibitors, entry
inhibitors, maturation inhibitors, cellular inhibitors and integrase strand
transfer inhibitors.
Antiretroviral drugs include lamivudine and zidovudine, emtricitabine (FTC),
zidovudine (ZDV),
azidothymidine (AZT), lamivudine (310), zalcitabine, dideoxycytidine (ddC),
tenofovir
disoproxil fumarate (TDF), didanosine (ddl), stavudine (d4T), abacavir sulfate
(ABC),
etravirine, delavirdine (DLV), efavirenz (EFV), nevirapine (NVP), amprenavir
(APV), tipranavir
(TPV), indinavir (IDV), saquinavir, saquinavir mesylate (SQV), lopinavir
(LPV), ritonavir (RTV),
fosamprenavir calcium (FOS-APV), ritonavir, RTV, darunavir, atazanavir sulfate
(ATV),
nelfinavir mesylate (NFV), enfuvirtide, T-20, maraviroc, dolutegravir and
raltegravir. ART drugs
can also include antibodies, such as ibalizumab, targeting HIV proteins or
cellular proteins
associated with disease progression. Also included are immune-based therapies,
such as IL-2,
IL-12 and alpha-epibromide. Each of these drugs can be administered alone or
in combination
with any other ART drug. Information about ART drugs and their administration
can be found
many pharmacopeia, such as the United States Pharmacopeia (USP) or accessed
online,
such as at www.aidsmeds.com (accessed 5 September 2013) Commercial names of
these
drugs and combinations of these drugs include Atripla (efavirenz,
emtricitabine and tenofovir
disoproxil fumarate), Complera (emtricitabine, rilpivirine, and tenofovir
disoproxil fumarate),
Stribild (elvitegravir, cobicistat, emtricitabine, tenofovir disoproxil
fumarate), Combivir
(lamivudine and zidovudine), Emtriva (emtricitabine, FTC), Epivir (lamivudine,
3TC), Epzicom
(abacavir and lamivudine), Hivid (zalcitabine, dideoxycytidine, ddC), Retrovir
(zidovudine,
azidothymidine, AZT, ZDV), Trizivir (abacavir, zidovudine, and lamivudine),
Truvada (tenofovir
disoproxil fumarate and emtricitabine), Videx EC (enteric coated didanosine,
ddl EC), Videx
CA 02851852 2014-05-08
(didanosine, dideoxyinosine, ddl), Viread (tenofovir disoproxil fumarate,
TDF), Zerit (stavudine,
d4T), Ziagen (abacavir sulfate, ABC), rilpivirine, etravirine, Rescriptor
(delavirdine, DLV),
Sustiva (efavirenz, EFV), Viramune (nevirapine, NVP), Agenerase (amprenavir,
APV), Aptivus
(tipranavir, TPV), saquinavir, Invirase (saquinavir mesylate, SQV), Kaletra
(lopinavir and
5 ritonavir, LPV/RTV), Lexiva (Fosamprenavir Calcium, FOS-APV), Norvir
(ritonavir, RTV),
darunavir, Reyataz (atazanavir sulfate, ATV), Viracept (nelfinavir mesylate,
NFV), Fuzeon
(enfuvirtide, 1-20), maraviroc, raltegravir, dolutegravir.
In one embodiment, the immunogenic composition is administered to a patient
who is also
10 taking antiretrovirals. In a further embodiment, the immunogenic
composition is administered
to a patient who has previously taken antiretrovirals. In a further
embodiment, the
immunogenic composition is administered to a patient who is not taking and has
not previously
taken antiretrovirals.
15 In one embodiment, the immunogenic composition is administered to a patient
having a CD4
count of 200 or more, suitably 500 or more, most suitably between 500-1700
cells per cubic
millimetre of blood. In a further embodiment, the immunogenic composition is
administered to
a patient having a nadir C04 count of 200 or more, more suitably 500 or more
cells per cubic
millimetre of blood.
In one embodiment the immunogenic composition is administered to a subject,
such as a
human, who is not infected with HIV. In one embodiment the immunogenic
composition is
administered to a subject, such as a human, who is infected with HIV.
In one aspect of the invention, there is provided a gp120 related protein for
use in the
manufacture of an immunogenic composition as described herein. Such as for the
prophylaxis
of HIV infection. Alternatively, the composition may be intended for the
treatment of HIV
infection.
In a further aspect there is provided a method for the prophylaxis of HIV
infection by
administration of an immunogenic composition as described herein to a mammal,
such as a
human. Such administration may reduce the risk of HIV infection and/or the
severity of HIV
infection.
CA 02851852 2014-05-08
36
In a further aspect there is provided a method for the treatment of HIV
infection by
administration of an immunogenic composition as described herein to a mammal
infected with
HIV, such as a human. Such administration may reduce the severity of HIV
infection.
In a further aspect there is provided a method for reducing the risk of HIV
transmission from an
HIV-infected individual to a partner of said HIV-infected individual, by
administration of an
immunogenic composition as described herein to the HIV-infected individual.
There is also provided an immunogenic composition as described herein for use
in the
prophylaxis of HIV infection.
There is also provided an immunogenic composition as described herein for use
in the
treatment of HIV infection.
The invention is illustrated by way of the following clauses:
Clause 1. An immunogenic composition comprising a gp120 related polypeptide
and an
adjuvant, wherein the adjuvant comprises a lipopolysaccharide and an
immunologically active saponin fraction derived from the bark of Quillaja
Saponaria Molina presented in the form of a liposome and wherein the
composition is substantially free of a NefTat related polypeptide wherein the
NefTat related polypeptide is a polypeptide consisting of SEQ ID NO: 4.
Clause 2. An immunogenic composition which is in the form of a human
dose comprising
a gp120 related polypeptide and an adjuvant, wherein the adjuvant comprises
between 10-40ug of a lipopolysaccharide and between 10-4Oug of an
immunologically active saponin fraction derived from the bark of Quillaja
Saponaria Molina presented in the form of a liposome.
Clause 3. An immunogenic composition comprising a gp120 related polypeptide
and an
adjuvant, wherein the adjuvant comprises a lipopolysaccharide and an
immunologically active saponin fraction derived from the bark of Quillaja
Saponaria Molina presented in the form of a liposome wherein:
(i) the conductivity of the composition is 13 mS/cm or lower; and/or
(ii) the concentration of salts in said composition is 130 mM or lower; and/or
(iii) the concentration of sodium chloride in said composition is 130 mM or
lower.
CA 02851852 2014-05-08
37
Clause 4. The immunogenic composition according to either clause 2 or 3, which
is
substantially free of a NefTat related polypeptide wherein the NefTat related
polypeptide is a polypeptide consisting of SEQ ID NO: 4.
Clause 5. The immunogenic composition according to either clause 1 or 4,
which contains
a ratio of NefTat related polypeptide:gpl 20 related polypeptide of less than
1:20.
Clause 6. The immunogenic composition according to either clause 1 or 4, which
contains
less than lug of NefTat related polypeptide.
Clause 7. The immunogenic composition according to either clause 5 or 6
wherein the
composition is free of NefTat related polypeptide.
Clause 8. The immunogenic composition according to any one of clause 1 or 4 to
7
wherein the NefTat related polypeptide comprises a polypeptide with at least
99% identity with SEQ ID NO: 4.
Clause 9. The immunogenic composition according to clause 8 wherein the
NefTat related
polypeptide comprises a polypeptide with at least 90% identity with SEQ ID NO:
4.
Clause 10. The immunogenic composition according to clause 9 wherein the
NefTat related
polypeptide comprises a polypeptide with at least 80% identity with SEQ ID NO:
4.
Clause 11. The immunogenic composition according to clause 10 wherein the
NefTat
related polypeptide comprises a polypeptide with at least 70% identity with
SEQ
ID NO: 4.
Clause 12. The immunogenic composition according to either clausel or 4,
which contains
a ratio of Net related polypeptide:gpl 20 related polypeptide of less than
1:20.
Clause 13. The immunogenic composition according to either clause 1 or 4,
which contains
less than lug of Net related polypeptide.
Clause 14. The immunogenic composition according to either clause 12 or 13
wherein the
composition is free of Net related polypeptide.
Clause 15. The immunogenic composition according to any one of clauses 1, 4 or
12 to 14
wherein the Nef related polypeptide comprises a polypeptide with at least 99%
identity with SEQ ID NO: 2.
Clause 16. The immunogenic composition according to clause 15 wherein the
Nef related
polypeptide comprises a polypeptide with at least 90% identity with SEQ ID NO:
2.
CA 02851852 2014-05-08
38
Clause 17. The immunogenic composition according to clause 16 wherein the Nef
related
polypeptide comprises a polypeptide with at least 80% identity with SEQ ID NO:
2.
Clause 18. The immunogenic composition according to clause 17 wherein the
Nef related
polypeptide comprises a polypeptide with at least 70% identity with SEQ ID NO:
2.
Clause 19. The immunogenic composition according to either clause 1 or 4,
which contains
a ratio of Tat related polypeptide:gp120 related polypeptide of less than
1:20.
Clause 20. The immunogenic composition according to either clause 1 or 4,
which contains
less than lug of Tat related polypeptide.
Clause 21. The immunogenic composition according to either clause 19 or 20
wherein the
composition is free of Tat related polypeptide.
Clause 22. The immunogenic composition according to any one of clauses 1, 4 or
19 to 21
wherein the Tat related polypeptide comprises a polypeptide with at least 99%
identity with SEQ ID NO: 3.
Clause 23. The immunogenic composition according to clause 22 wherein the Tat
related
polypeptide comprises a polypeptide with at least 90% identity with SEQ ID NO:
3.
Clause 24. The immunogenic composition according to clause 23 wherein the
Tat related
polypeptide comprises a polypeptide with at least 80% identity with SEQ ID NO:
3.
Clause 25. The immunogenic composition according to clause 24 wherein the Tat
related
polypeptide comprises a polypeptide with at least 70% identity with SEQ ID NO:
3.
Clause 26. The immunogenic composition according to any one of clauses 1 or
3 to 25
wherein the lipopolysaccharide is present at a level of between 10-10Oug.
Clause 27. The immunogenic composition according to clause 26 wherein the
lipopolysaccharide is present at a level of between 15-8Oug.
Clause 28. The immunogenic composition according to clause 27 wherein the
lipopolysaccharide is present at a level of between 20-65ug.
Clause 29. The immunogenic composition according to clause 28 wherein the
lipopolysaccharide is present at a level of between 30-6Oug.
Clause 30. The immunogenic composition according to clause 29 wherein the
lipopolysaccharide is present at a level of between 45-55ug.
CA 02851852 2014-05-08
39
Clause 31. The immunogenic composition according to clause 30 wherein the
lipopolysaccharide is present at a level of 5Oug.
Clause 32. The immunogenic composition according to any one of clauses 1
or 3 to 31
wherein the saponin is present at a level of between 10-10Oug.
Clause 33. The immunogenic composition according to clause 32 wherein the
saponin is
present at a level of between 15-8Oug.
Clause 34. The immunogenic composition according to clause 33 wherein the
saponin is
present at a level of between 20-65ug.
Clause 35. The immunogenic composition according to clause 34 wherein the
saponin is
present at a level of between 30-6Oug.
Clause 36. The immunogenic composition according to clause 35 wherein the
saponin is
present at a level of between 45-55ug.
Clause 37. The immunogenic composition according to clause 36 wherein the
saponin is
present at a level of 5Oug.
Clause 38. The immunogenic composition according to any one of clauses 1 to
25 wherein
the lipopolysaccharide is present at a level of between 15-35ug.
Clause 39. The immunogenic composition according to clause 38 wherein the
lipopolysaccharide is present at a level of between 20-3Oug.
Clause 40. The immunogenic composition according to clause 39 wherein the
lipopolysaccharide is present at a level of 25ug.
Clause 41. The immunogenic composition according to any one of clauses 1
to 31 wherein
the saponin is present at a level of between 15-35ug.
Clause 42. The immunogenic composition according to clause 41 wherein the
saponin is
present at a level of between 20-3Oug.
Clause 43. The immunogenic composition according to clause 42 wherein the
saponin is
present at a level of 25ug.
Clause 44. The immunogenic composition according to any one of clauses 1
to 43 wherein
the lipopolysaccharide is 3D-MPL.
Clause 45. The immunogenic composition according to any one of clauses 1 to
44 wherein
the saponin is QS-21.
Clause 46. The immunogenic composition according to any one of clauses 1,
2 or 4 to 45
wherein:
(i) the conductivity of the composition is 13 mS/cm or lower; and/or
(ii) the concentration of salts in said composition is 130 mM or lower; and/or
(iii) the concentration of sodium chloride in said composition is 130 mM or
lower.
CA 02851852 2014-05-08
Clause 47. The immunogenic composition according to clause 46 wherein the
conductivity
of the composition is 13 mS/cm or lower.
Clause 48. The immunogenic composition according to clause 47 wherein the
conductivity
of the composition is 6 mS/cm or lower.
5 Clause 49. The immunogenic composition according to clause 48
wherein the conductivity
of the composition is 1.5 to 2.5 mS/cm.
Clause 50. The immunogenic composition according to clause 46 wherein the
concentration of salts in said composition is 130 mM or lower.
Clause 51. The immunogenic composition according to clause 50 wherein the
10 concentration of salts in said composition is 60 mM or lower.
Clause 52. The immunogenic composition according to clause 51 wherein the
concentration of salts in said composition is 20 to 40 mM.
Clause 53. The immunogenic composition according to clause 46 wherein the
concentration of sodium chloride in said composition is 130 mM or lower.
15 Clause 54. The immunogenic composition according to clause 53
wherein the
concentration of sodium chloride in said composition is 60 mM or lower.
Clause 55. The immunogenic composition according to clause 54 wherein the
concentration of sodium chloride in said composition is 10 mM or lower.
Clause 56. The immunogenic composition according any one of clauses 1 to
55, wherein
20 the concentration of CaC12 in the immunogenic composition is 30 mM
or lower.
Clause 57. The immunogenic composition according any one of clauses 1 to
56, wherein
the concentration of MgSO4 in the immunogenic composition is 60 mM or lower.
Clause 58. The immunogenic composition according any one of clauses 1 to
57, total
concentration of NH:' Mg2+ and Ca2+ ions is 40 mM or lower.
25 Clause 59. The immunogenic composition according to any one of
clauses 1 to 58 which is
an aqueous solution.
Clause 60. The immunogenic composition according to any one of clauses 1 to
59 which is
a single human dose.
Clause 61. The immunogenic composition according to any one of clauses 1 to
60 wherein
30 the human dose is between 0.1 and 1m1.
Clause 62. The immunogenic composition according to clause 61 wherein the
human dose
is between 0.3 and 0.75m1.
Clause 63. The immunogenic composition according to clause 62 wherein the
human dose
is 0.5m1.
35 Clause 64. The immunogenic composition according to any one of
clauses 1 to 60 wherein
the human dose is between 0.05 and 0.2ml.
CA 02851852 2014-05-08
41
Clause 65. The immunogenic composition according to any one of clauses 1 to 64
wherein
the osmolality is in the range of 250 to 750 mOsm/kg.
Clause 66. The immunogenic composition according to clause 65 wherein the
osmolality is
in the range of 250 to 550 mOsm/kg.
Clause 67. The immunogenic composition according to any one of clauses 1 to
66,
comprising a non-ionic tonicity agent.
Clause 68. The immunogenic composition according to clause 67, wherein the non-
ionic
tonicity agent is a polyol.
Clause 69. The immunogenic composition according to clause 68, wherein
the polyol is
sorbitol.
Clause 70. The immunogenic composition according to clause 69, wherein
the
concentration of sorbitol is between about 4 and about 6% (w/v).
Clause 71. The immunogenic composition according to any one of clauses 1 to
70, wherein
the concentration of sucrose is between about 4 and about 6% (w/v).
Clause 72. The immunogenic composition according to any one of clauses 1 to
71 wherein
the pH is in the range of 6.0 to 9Ø
Clause 73. The immunogenic composition according to any one of clauses 1 to 72
wherein
the composition comprises the gp120 related polypeptide at a level of around 1
to 100 ug.
Clause 74. The immunogenic composition according to clause 73 wherein the
composition
comprises the gp120 related polypeptide at a level of 3 to 30 ug.
Clause 75. The immunogenic composition according to clause 74 wherein the
composition
comprises the gp120 related polypeptide at a level of 5 to 15 ug.
Clause 76. The immunogenic composition according to clause 74 wherein the
composition
comprises the gp120 related polypeptide at a level of 16 to 25 ug.
Clause 77. The immunogenic composition according to any one of clauses 1 to 76
wherein
the composition comprises 1-5 additional HIV antigens.
Clause 78. The immunogenic composition according to clause 77 wherein the
composition
comprises additional HIV antigens selected from the list consisting of gp160,
gp41, MA, CA, SP1, NC, SP2, P6, RT, RNase H, IN, PR, Rev, Vif, Vpr and Vpu.
Clause 79. The immunogenic composition according to any one of clauses 1 to 78
wherein
the composition comprises a total of around 1 to 500 ug of antigenic material.
Clause 80. The composition according to any one of clauses 1 to 79 wherein the
gp120
related polypeptide comprises a gp120 polypeptide derived from HIV-1.
Clause 81. The composition according to clause 80 wherein the gp120 related
polypeptide
comprises a gp120 polypeptide derived from HIV-1 group M.
CA 02851852 2014-05-08
42
Clause 82. The composition according to clause 81 wherein the gp120 related
polypeptide
comprises a gp120 polypeptide derived from HIV-1 group M subtype C.
Clause 83. The composition according to clause 82 wherein the gp120 related
polypeptide
comprises a gp120 polypeptide derived from HIV-1 group M subtype B.
Clause 84. The composition according to any one of clauses 1 to 81 and 83
wherein the
gp120 related polypeptide comprises a polypeptide with at least 70% identity
with the V1V2 region of SEQ ID NO: 1.
Clause 85. The composition according to clause 84 wherein the gp120 related
polypeptide
comprises a polypeptide with at least 80% identity with the V1V2 region of SEQ
ID NO: 1.
Clause 86. The composition according to clause 85 wherein the gp120 related
polypeptide
comprises a polypeptide with at least 90% identity with the V1V2 region of SEQ
ID NO: 1.
Clause 87. The composition according to clause 86 wherein the gp120
related polypeptide
comprises a polypeptide with at least 95% identity with the V1V2 region of SEQ
ID NO: 1.
Clause 88. The composition according to clause 87 wherein the gp120 related
polypeptide
comprises a polypeptide with at least 98% identity with the V1V2 region of SEQ
ID NO: 1.
Clause 89. The composition according to clause 88 wherein the gp120 related
polypeptide
comprises a polypeptide with at least 99% identity with the V1V2 region of SEQ
ID NO: 1.
Clause 90. The composition according to clause 89 wherein the gp120 related
polypeptide
comprises the V1V2 region of SEQ ID NO: 1.
Clause 91. The composition according to any one of clauses 1 to 81 and 83 to
90 wherein
the gp120 related polypeptide consists of a polypeptide with at least 70%
identity with the V1V2 region of SEQ ID NO: 1.
Clause 92. The composition according to clause 91 wherein the gp120
related polypeptide
consists of a polypeptide with at least 80% identity with the V1V2 region of
SEQ
ID NO: 1.
Clause 93. The composition according to clause 92 wherein the gp120 related
polypeptide
consists of a polypeptide with at least 90% identity with the V1V2 region of
SEQ
ID NO: 1.
CA 02851852 2014-05-08
43
Clause 94. The composition according to clause 93 wherein the gp120 related
polypeptide
consists of a polypeptide with at least 95% identity with the V1V2 region of
SEQ
ID NO: 1.
Clause 95. The composition according to clause 94 wherein the gp120
related polypeptide
consists of a polypeptide with at least 98% identity with the V1V2 region of
SEQ
ID NO: 1.
Clause 96. The composition according to clause 95 wherein the gp120 related
polypeptide
consists of a polypeptide with at least 99% identity with the V1V2 region of
SEQ
ID NO: 1.
Clause 97. The composition according to clause 96 wherein the gp120 related
polypeptide
consists of the V1V2 region of SEQ ID NO: 1.
Clause 98. The composition according to any one of clauses 1 to 81 and 83 to
97 wherein
the gp120 related polypeptide comprises a polypeptide with at least 70%
identity with SEQ ID NO: 1.
Clause 99. The composition according to clause 98 wherein the gp120 related
polypeptide
comprises a polypeptide with at least 80% identity with SEQ ID NO: 1.
Clause 100. The composition according to clause 99 wherein the gp120 related
polypeptide
comprises a polypeptide with at least 90% identity with SEQ ID NO: 1.
Clause 101. The composition according to clause 100 wherein the gp120 related
polypeptide
comprises a polypeptide with at least 95% identity with SEQ ID NO: 1.
Clause 102. The composition according to clause 101 wherein the gp120 related
polypeptide
comprises a polypeptide with at least 98% identity with SEQ ID NO: 1.
Clause 103. The composition according to clause 102 wherein the gp120 related
polypeptide
comprises a polypeptide with at least 99% identity with SEQ ID NO: 1.
Clause 104. The composition according to clause 103 wherein the gp120 related
polypeptide
comprises SEQ ID NO: 1.
Clause 105. The composition according to any one of clauses 1 to 81 and 83 to
104 wherein
the gp120 related polypeptide consists of a polypeptide with at least 70%
identity with SEQ ID NO: 1.
Clause 106. The composition according to clause 105 wherein the gp120 related
polypeptide
consists of a polypeptide with at least 80% identity with SEQ ID NO: 1.
Clause 107. The composition according to clause 106 wherein the gp120 related
polypeptide
consists of a polypeptide with at least 90% identity with SEQ ID NO: 1.
Clause 108. The composition according to clause 107 wherein the gp120 related
polypeptide
consists of a polypeptide with at least 95% identity with SEQ ID NO: 1.
CA 02851852 2014-05-08
44
Clause 109. The composition according to clause 108 wherein the gp120 related
polypeptide
consists of a polypeptide with at least 98% identity with SEQ ID NO: 1.
Clause 110. The composition according to clause 109 wherein the gp120 related
polypeptide
consists of a polypeptide with at least 99% identity with SEQ ID NO: 1.
Clause 111. The composition according to clause 110 wherein the gp120 related
polypeptide
consists of SEQ ID NO: 1.
Clause 112. The composition according to any one of clauses 1 to 82 wherein
the gp120
related polypeptide comprises a polypeptide with at least 70% identity with
the
V1V2 region of SEQ ID NO: 5.
Clause 113. The composition according to clause 112 wherein the gp120 related
polypeptide
comprises a polypeptide with at least 80% identity with the V1V2 region of SEQ
ID NO: 5.
Clause 114. The composition according to clause 113 wherein the gp120 related
polypeptide
comprises a polypeptide with at least 90% identity with the V1V2 region of SEQ
ID NO: 5.
Clause 115. The composition according to clause 114 wherein the gp120 related
polypeptide
comprises a polypeptide with at least 95% identity with the V1V2 region of SEQ
ID NO: 5.
Clause 116. The composition according to clause 115 wherein the gp120 related
polypeptide
comprises a polypeptide with at least 98% identity with the V1V2 region of SEQ
ID NO: 5.
Clause 117. The composition according to clause 116 wherein the gp120 related
polypeptide
comprises a polypeptide with at least 99% identity with the V1V2 region of SEQ
ID NO: 5.
Clause 118. The composition according to clause 117 wherein the gp120 related
polypeptide
comprises the V1V2 region of SEQ ID NO: 5.
Clause 119. The composition according to any one of clauses 1 to 82 and 112 to
118
wherein the gp120 related polypeptide consists of a polypeptide with at least
70% identity with the V1V2 region of SEQ ID NO: 5.
Clause 120. The composition according to clause 119 wherein the gp120 related
polypeptide
consists of a polypeptide with at least 80% identity with the V1V2 region of
SEQ
ID NO: 5.
Clause 121. The composition according to clause 120 wherein the gp120 related
polypeptide
consists of a polypeptide with at least 90% identity with the V1V2 region of
SEQ
ID NO: 5.
CA 02851852 2014-05-08
Clause 122. The composition according to clause 121 wherein the gp120 related
polypeptide
consists of a polypeptide with at least 95% identity with the V1V2 region of
SEQ
ID NO: 5.
Clause 123. The composition according to clause 122 wherein the gp120 related
polypeptide
5
consists of a polypeptide with at least 98% identity with the V1V2 region of
SEQ
ID NO: 5.
Clause 124. The composition according to clause 123 wherein the gp120 related
polypeptide
consists of a polypeptide with at least 99% identity with the V1V2 region of
SEQ
ID NO: 5.
10 Clause
125. The composition according to clause 124 wherein the gp120 related
polypeptide
consists of the V1V2 region of SEQ ID NO: 5.
Clause 126. The composition according to any one of clauses 1 to 82 and 112 to
125
wherein the gp120 related polypeptide comprises a polypeptide with at least
70% identity with SEQ ID NO: 5.
15 Clause
127. The composition according to clause 126 wherein the gp120 related
polypeptide
comprises a polypeptide with at least 80% identity with SEQ ID NO: 5.
Clause 128. The composition according to clause 127 wherein the gp120 related
polypeptide
comprises a polypeptide with at least 90% identity with SEQ ID NO: 5.
Clause 129. The composition according to clause 128 wherein the gp120 related
polypeptide
20 comprises a polypeptide with at least 95% identity with SEQ ID NO:
5.
Clause 130. The composition according to clause 129 wherein the gp120 related
polypeptide
comprises a polypeptide with at least 98% identity with SEQ ID NO: 5.
Clause 131. The composition according to clause 130 wherein the gp120 related
polypeptide
comprises a polypeptide with at least 99% identity with SEQ ID NO: 5.
25 Clause
132. The composition according to clause 131 wherein the gp120 related
polypeptide
comprises SEQ ID NO: 5.
Clause 133. The composition according to any one of clauses 1 to 82 and 112 to
132
wherein the gp120 related polypeptide consists of a polypeptide with at least
70% identity with SEQ ID NO: 5.
30 Clause
134. The composition according to clause 133 wherein the gp120 related
polypeptide
consists of a polypeptide with at least 80% identity with SEQ ID NO: 5.
Clause 135. The composition according to clause 134 wherein the gp120 related
polypeptide
consists of a polypeptide with at least 90% identity with SEQ ID NO: 5.
Clause 136. The composition according to clause 135 wherein the gpl 20 related
polypeptide
35 consists of a polypeptide with at least 95% identity with SEQ ID
NO: 5.
CA 02851852 2014-05-08
46
Clause 137. The composition according to clause 136 wherein the gp120 related
polypeptide
consists of a polypeptide with at least 98% identity with SEQ ID NO: 5.
Clause 138. The composition according to clause 137 wherein the gp120 related
polypeptide
consists of a polypeptide with at least 99% identity with SEQ ID NO: 5.
Clause 139. The composition according to clause 138 wherein the gp120 related
polypeptide
consists of SEQ ID NO: 5.
Clause 140. The immunogenic composition according to any one of clauses 1 to
139
wherein the composition further comprises a sterol, wherein the ratio of
saponin:sterol is from 1:1 to 1:100 w/w.
Clause 141. The immunogenic composition according to clause 140 wherein the
ratio of
saponin:sterol is from 1:1 to 1:5 w/w.
Clause 142. The immunogenic composition according to either clause 140 or 141
wherein
said sterol is cholesterol.
Clause 143. The immunogenic composition according to any one of clauses 1 to
142, which
is provided in a multi-dose presentation.
Clause 144. The immunogenic composition according to any one of clauses 1 to
143, which
is provided in presentation containing an overage of 1-50% to allow for
wastage
during administration.
Clause 145. The immunogenic composition according to any one of clauses 1 to
144, for use
as a medicament.
Clause 146. The immunogenic composition according to clause 145, for use in
the treatment
or prevention of HIV-1 from group M, N, 0 or P.
Clause 147. The immunogenic composition according to clause 146, for use in
the treatment
or prevention of HIV-1 group M subtype A, B, C, D, E, F, G, H, I, J or K.
Clause 148. The immunogenic composition according to any one of clauses 1 to
147,
wherein the gp120 related polypeptide is derived from a first HIV-1 subtype,
for
use in the treatment or prevention of HIV-1 infection by a second HIV-1
subtype.
Clause 149. The immunogenic composition according to any one of clauses 1 to
148,
wherein the gp120 related polypeptide is derived from a first HIV-1 subtype,
for
use in the treatment or prevention of HIV-1 infection by a second HIV-1
subtype, wherein the first and second HIV-1 subtypes have different native
gp120 polypeptide sequences.
Clause 150. The immunogenic composition according to clause 149, wherein the
first HIV-1
subtype is selected from the list consisting of A, B, C, D, E, F, G, H, I, J
or K; of
HIV-1 group M.
CA 02851852 2014-05-08
47
Clause 151. The immunogenic composition of any one of clauses 1 to 150 for use
in
inducing a humoral immune response against HIV-1 strains from one or more
subtypes different from the HIV-1 subtype from which the gp120 related
polypeptide of the composition is derived.
Clause 152. The immunogenic composition according to any one of clauses 1 to
151 for use
in eliciting antibodies against the V1V2 loop of HIV-1 gp120.
Clause 153. A method of treatment or prophylaxis of HIV-1 infection comprising
the step of
administering a composition according to any one of clauses 1 to 152 to an
individual.
Clause 154. The method of treatment or prophylaxis of HIV-1 infection
according to clause
153 further comprising concomitant administration of an antiretroviral drug.
Clause 155. The method of treatment or prophylaxis of HIV-1 infection
according to either
clause 153 or 154 wherein IgG antibody capable of binding to the V1V2 region
of the gp120 related polypeptide of the composition is detectable in the serum
of an individual at least 24 weeks after the only administration of the
composition or the first administration of the composition in a course of
repeat
administrations to the individual.
Clause 156. The method of treatment or prophylaxis of HIV-1 infection
according to clause
155 wherein the IgG antibody is detectable at least 48 weeks after the only
administration of the composition or the first administration of the
composition in
a course of repeat administrations to the individual.
Clause 157. The method of treatment or prophylaxis of HIV-1 infection
according to clause
156 wherein the IgG antibody is detectable at least 72 weeks after the only
administration of the composition or the first administration of the
composition in
a course of repeat administrations to the individual.
Clause 158. The method of treatment or prophylaxis of HIV-1 infection
according to clause
157 wherein the IgG antibody is detectable at least 96 weeks after the only
administration of the composition or the first administration of the
composition in
a course of repeat administrations to the individual.
Clause 159. The method of treatment or prophylaxis of HIV-1 infection
according to either
clause 153 or 154 wherein IgG antibody capable of binding to the V1V2 region
of the gp120 related polypeptide of the composition is detectable in the serum
of an individual at least 2 weeks after the final administration of the
composition
in a course of repeat administrations to the individual.
Clause 160. The method of treatment or prophylaxis of HIV-1 infection
according to clause
159 wherein the IgG antibody is detectable at least 6 months after the final
CA 02851852 2014-05-08
48
administration of the composition in a course of repeat administrations to the
individual.
Clause 161. The method of treatment or prophylaxis of HIV-1 infection
according to clause
160 wherein the IgG antibody is detectable at least 12 months after the final
administration of the composition in a course of repeat administrations to the
individual.
Clause 162. The method of treatment or prophylaxis of HIV-1 infection
according to clause
161 wherein the IgG antibody is detectable at least 18 months after the final
administration of the composition in a course of repeat administrations to the
individual.
Clause 163. The method of treatment or prophylaxis of HIV-1 infection
according to any one
of clauses 155 to 158 wherein antibody levels are detectable at at least 5% of
the serum titre two weeks following the only administration.
Clause 164. The method of treatment or prophylaxis of HIV-1 infection
according to any one
of clauses 155 to 162 wherein antibody levels are detectable at at least 5% of
the serum titre two weeks following the first administration.
Clause 165. The method of treatment or prophylaxis of HIV-1 infection
according to any one
of clauses 159 to 162 wherein antibody levels are detectable at at least 5% of
the serum titre two weeks following the final administration.
Clause 166. The method of treatment or prophylaxis according to any one of
clauses 155 to
165 wherein the antibody will be detectable in at least 50% of individuals
administered with the composition.
Clause 167. The method of treatment or prophylaxis according to clause 166
wherein the
antibody will be detectable in at least 80% of individuals administered with
the
composition.
Clause 168. The method of treatment or prophylaxis according to any one of
clauses 153 to
167 wherein a polynucleotide encoding a gp120 related polypeptide is
administered to the individual and subsequently the composition according to
any one of clauses 1 to 152 is administered to the individual.
Clause 169. The method of treatment or prophylaxis according to any one of
clauses 153 to
167 wherein the composition according to any one of clauses 1 to 152 is
administered to the individual and subsequently a polynucleotide encoding a
gp120 related polypeptide is administered to the individual.
Clause 170. A method of reducing the risk of HIV transmission from an HIV-
infected
individual to a partner of said HIV-infected individual comprising the step of
CA 02851852 2014-05-08
49
administering the immunogenic composition of any one of clauses 1 to 152 to
the HIV-infected individual.
Clause 171. A method of making the composition according to any one of clauses
1 to 152,
comprising adding a gp120 derived polypeptide to a lipopolysaccharide and an
immunologically active saponin fraction derived from the bark of Quillaja
Saponaria Molina presented in the form of a liposome.
Clause 172. A kit for making an immunogenic composition according to any one
of clauses 1
to 152, comprising a first container and a second container, wherein the first
container comprises a gp120 derived polypeptide and the second container
comprises a lipopolysaccharide and an immunologically active saponin fraction
derived from the bark of Quillaja Saponaria Molina presented in the form of a
liposome.
Clause 173. The kit according to clause 172, wherein the second container
comprises an
aqueous solution.
Clause 174. A viral vector comprising a polynucleotide encoding a polypeptide
comprising
the gp120w6lo polypeptide of SEQ ID No: 1.
Clause 175. The viral vector of clause 174 comprising a polynucleotide
encoding a
polypeptide consisting of the gp120w6lo polypeptide of SEQ ID No: 1.
Clause 176. A viral vector comprising a polynucleotide encoding a polypeptide
comprising
the gp120zmi8 polypeptide of SEQ ID NO: 5.
Clause 177. The viral vector of clause 176 comprising a polynucleotide
encoding a
polypeptide consisting of the gp120D,418 polypeptide of SEQ ID NO: 5.
Clause 178. The viral vector of any one of clauses 174 to 177 which is an
adenovirus,
canary pox virus or MVA virus.
The present invention will now be further described by means of the following
non-limiting
examples.
EXAMPLES
Example 1: Seroloov comparison
Method
CA 02851852 2014-05-08
A first composition containing 2ug gp12Ch.
-.y61D and 50u1 ASO1B and a second composition
containing 2ug gp120w
-610, 50u1 ASO1B and Zug NefTat were prepared. At three timepoints
(days 0, 14 and 28), the first composition was administered intramuscularly
(IM) to a first group
of 25 CB6F1 mice and at the same three timepoints the second composition was
administered
5 IM to a second group of 25 CB6F1 mice.
The titre of anti-V1V2 antibodies in the sera of the mice in each group was
compared at 14
days after the second administration and at 14 days after the third
administration. To evaluate
the quantity of anti-V1V2 IgG antibodies present in each serum sample, the HIV-
1 subtype B
10 case A2 gp120 V1V2 region scaffolded onto a murine gp70 protein was used in
an lgtot ELISA
binding antibody assay. During the development of the assay, the CH58 (the
anti-V2
monoclonal antibody used in the RV144 trial) was used as a positive control to
detect
successful binding of serum lgtot antibodies to scaffolded V1V2. In addition,
gp70 protein (not
bound to a V1V2 region) was used as a negative control to assess specificity
during
15 development of the assay.
Results
On both timepoints tested, a trend for a higher anti-V1V2 antibody response
was observed in
20 serum samples from mice which had been administered with the composition
which did not
contain NefTat as compared to serum samples from mice which had been
administered with
the composition containing NefTat. This was particularly notable at 14 days
after the third
administration (see Figure 1).
All references referred to in this application, including patent and patent
applications, are
incorporated herein by reference to the fullest extent possible.
Throughout the specification and the claims which follow, unless the context
requires
otherwise, the word 'comprise', and variations such as 'comprises' and
'comprising', will be
understood to imply the inclusion of a stated integer, step, group of integers
or group of steps
but not to the exclusion of any other integer, step, group of integers or
group of steps.
The application of which this description and claims forms part may be used as
a basis for
priority in respect of any subsequent application. The claims of such
subsequent application
CA 02851852 2014-05-08
51
may be directed to any feature or combination of features described herein.
They may take
the form of product, composition, process, or use claims and may include, by
way of example
and without limitation, the following claims.
