IE20130343A1

IE20130343A1 - Immunogenic compositions comprising a gp120 related polypeptide

Info

Publication number: IE20130343A1
Application number: IE20130343A
Authority: IE
Inventors: Patricia B Bourguignon; Marguerite Christine Koutsoukos; Clarisse Marie-Madeleine Lorin
Original assignee: Glaxosmithkline Biolog Sa
Priority date: 2013-09-16
Filing date: 2013-11-08
Publication date: 2015-03-25
Also published as: BE1021327B1; CA2851871A1; GB201316464D0; IE86613B1; WO2015036062A1

Abstract

The present invention relates to particular immunogenic compositions comprising a gpl2O related polypeptide and an adjuvant, wherein the adjuvant comprises a saponin and a lipopolysaccharide. Such compositions are substantially free of a NefTat related polypeptide, comprise between 1O-4Oug of a lipopolysaccharide and between 1O-4Oug of an immunologically active saponin fraction derived from the bark of Quillaja Saponaria Molina presented in the form of a liposome or have a concentration of sodium chloride of 130 mM or lower.

Description

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 Dorns 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.

VBS5568PCT ,E 1 3 Ο 3 4 3 The HIV envelope glycoprotein gp120 and other HIV-1 proteins gp120 is the viral protein that is used for attachment to a host ceil. 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 noncovalently 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 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, Nef, Vif, Vpr, Vpu and Tat (Greene et ai., 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 al., 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 al., 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 al., Αηηϋ 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)). / I· . · '•’•4 ' 'τ .·· : i s. ΐ ι / · VB65568PCT ,E 1 Τ 0 3 4 3 One region of the gp120 protein in particular, the !V3 loop1, 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 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, gp120W6iD and SIV Nef proteins formulated with the AS02A adjuvant system (50ug of QS-21 and 50ug of 3D-MPL in an oii-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 SHIV8s.6p· However, HIV-1 gp120W61D formulated alone with AS02A did not provide protection. The tack of protection after immunisation with gp120W6w formulated alone with AS02A was suggested to be reiated to the heterologous nature of the challenge virus (20.2% sequence difference for gp120), since HIV-1 gp120weiD formulated in AS02A had previously been shown to induce sterile immunity against homologous SHIV^id challenge (Voss et al., J Virol 77(2):1049-1058 (2003) and Mooij et al., AIDS 12:F15-F22 (1998)).

NefTat and gp120weiD formulated in AS02A 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.

VB65568PCT 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 HIV1 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 LAL The vector also contained HIVla, gag and pol genes. AtDSVAX B/E is a preparation of recombinant HIV-1 subtype B MN gp120, CM244 subtype Ε 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 gp70-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., PiosOne, 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 levei of antibodies specific for the V1V2 region of gp120.

To summarise, previous work in the field has demonstrated that: IEl 3 0 3 4 3 VB65568PCT • gp120 in AS02A adjuvant (QS-21 and 3D-MPL oii-in-water emulsion) did not provide protection against challenge with heterologous virus in a primate model, • gp120 may have a detrimental impact on markers of vaccine efficacy and • 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 HlV-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 The present inventors administered compositions containing either (a) gp120W6iD and AS01B or (b) gp120w6iD, AS01B and NefTat to mice and analysed the serological response of the mice.

VB65568PCT IEl 3 0 3 4 3 The present inventors found that, surprisingly, mice administered with gp120W6iD and AS01B (without NefTat) had a higher mean level of anti-V1V2 antibodies in their serum compared to mice administered with gp120weiD. AS01B and NefTat.

The present inventors analysed serum from 30 subjects who had previously taken part in the "PRO HIV-002" trial and had been vaccinated with gp120, NefTat and AS01B adjuvant (QS-21 and 3D-MPL in liposomal form).

The present inventors found that, surprisingly, subjects from the PRO HIV-002 trial had a high level of anti-V1V2 antibodies in their serum, which showed high persistence and exceeded the level of anti-V1V2 antibodies in serum from subjects ofthe RV144 trial.

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 poiypeptide and an adjuvant, wherein the adjuvant comprises between 10-40ug of a lipopolysaccharide and between 10-40ug 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.

I Further provided is an immunogenic composition comprising a gp12O related polypeptide and an adjuvant, wherein the adjuvant comprises a lipopolysaccharide and an immunologically V865568PCT Κ1 3 Ο 3 4 3 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 Figure 2: PRO HIV-002 and RV144 trial analysis: anti-V1V2 IgG comparison 10 Figure 3: PRO HIV-002 anti-V1V2 IgG responder rates Figure 4: PRO HIV-002 anti-V1V2 IgG cross-subtype reactivity as compared to RV144 Figure 5: PRO HIV-002 anti-V1V2 IgG durability DESCRIPTION OF THE SEQUENCES SEQ ID NO: 1 9p120weiD SEQ ID NO: 2 Nef SEQ ID NO: 3 Tat SEQ ID NO: 4 NefTat SEQ ID NO: 5 9p120zMie SEQ ID NO: 6 polynucleotide sequence encoding gp120a«8 SEQ ID NO: 7 native gp12Qan8 SEQ ID NO: 8 polynucleotide sequence encoding native gp120ZM18 DETAILED DESCRIPTION OF THE INVENTION Polypeptides As used herein, the term ‘a gp120 related polypeptide’ refers to a polypeptide comprising the 30 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.

VB65568PCT 8 JEH 0 3 4 j 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 gp120W61D 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, gp120ZM18 (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 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 simitar 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 Μ, N, O or P of HIV-1, suitably from subtype A, B, C, D, E, F, G, Η, I, J or K of group M. Suitably, the gp120 reiated 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 Μ, N, O or P group HIV-1, suitably from a subtype A, B, C, D, E, F, G, Η, 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. ' VB65568PCT IE; 3 0 3 4 3 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) resuits 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 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); ) 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 VB65568PCT IE J 3 0 3 4 3 sequence. One example of insertions includes a short stretch of histidine residues (e.g. 6 residues) to aid purification ofthe antigen in question. immunogenic derivatives include those wherein amino acids have been deleted compared to 5 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 wifi 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), insertions of up to 5 residues (for example 1 or 2 residues) at 0-5 five locations (for example ΟΙ 5 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 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 02 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 suitahly at least 90% identity, more suitably at least 95% identity, more suitably at least 98% identity, more j 7 w VB65568PCT ΙΕ ί 3 Ο 3 43 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, 5 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 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 (D 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 VB65568PCT IE 1 3 Ο 3 4 3 suitably at feast 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, 5 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 8% 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 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 wellknown 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 Needieman & Wunsch, J. Mol. Biot, 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proe. Nat’l. Acad. Sci. USA £5:2444 (1988), by computerised implementations of these algorithms (GAP, BESTFIT, FASTA, and VB65568PCT ,E’ 30 3 TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wl), 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 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 VB65568PCT IEl 3 0 3 43 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 5 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.

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. ELISA) 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 (aiso 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 30 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.

IE 1 3 Ο 3 43 VB65568PCT 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 1ml, more suitably between 0.3 and 0.75ml, such as between 0.45 and 0.55mi, in particular 0.5ml. Volumes of between 0.1 and 1ml are 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 0.2ml, suitably between 0.075 and 0.15mt, in particular 0.1ml. 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.

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 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.

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 25 derivative of SEQ ID NO: 2, optionally having a small number of deletions, insertions and/or 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.

VB65568PCT IEl 3 Ο 3 4 3 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.

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. fcl 3 ο VB65568PCT 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).

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 1ug, suitably less than 0.5ug, suitably less than 0.2ug, suitably less than 0.1 ug, suitably less than 0.05ug, more suitably less than 0.01 ug 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: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 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 1ug, suitably less than 0.5ug, suitably less than 0.2ug, suitably less than 0.1ug, suitably less than 0.05ug, more suitably less than 0.01 ug per human dose.

VB65568PCT IE 1 3 Ο 3 4 3 Most suitably the compositions ofthe present invention are free of a Tat related polypeptide.

Suitably the compositions of the present invention are substantiaily free of a NefTat related 5 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, suitably less than 1ug, suitably less than 0.5ug, suitably less than 0.2ug, suitably less than 0.1 ug, suitably less than 0.05ug, more suitably less than 0.01ug 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 ofthe 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.

VB65568PCT 1E1 3 0 3 4 3 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. 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 Saponins 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), Th1 cells and a predominant lgG2a 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 VB6S568PCT IE 1 3 Ο 3 43 compositions of the invention contain the immunologicaily 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 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 ratio of sterol to phospholipid is 1-50% (mol/mol), suitably 20-25%.

Suitable sterols include beta-sitosterol, stigmasterol, ergosteroi, 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 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 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.

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).

VB65568PCT JEf 3 0 3 4 3 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: Aescutus hippocastanum. Its isolation is described by chromatography and purification (Fiedler, Arzneimittel-Forsch. 4, 213 (1953)), and by ion5 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 Belg, 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.

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 1-100ug.

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 QS25 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 QS30 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.

VB65568PCT JE 1 3 Ο 3 43 Lipopolysaccharide adjuvants Lipopolysaccharides (LPS) are the major surface molecule of, and occur exclusively in, the 5 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 ,000 Daltons in size and consist of three covalently linked regions: (i) an O-specific polysaccharide chain (O-antigen) at the outer region (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 O-specific polysaccharide component (O-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 Ν 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 (susceptibfe 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 O-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 W’:· * VB65568PCT IEl 3 Ο 3 43 (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. 3-O-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 Immunochemicals). 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 3DMPL is used. Smail 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 1-100ug.

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.

VB65568PCT IE1 3 0 3 4 3 in a further embodiment, there is provided an immunogenic composition comprising 3D-MPL at a ievel of around 25 ug, for example between 10-37 ug, suitably between 15-30 ug or between -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 3DMPL 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 3DMPL 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 um 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.

VB65568PCT JE 1 3 ο 3 43 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 levei 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 10ug 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 within liposomes is described, for example, in US4235877. Conjugation of proteins to macromolecules is disclosed, for example, in US4372945 and in US4474757.

Salt concentration 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 sodium chloride as low as 150 mM. Consequently, the stability of immunogenic compositions comprising a gp120 reiated polypeptides may be improved by a reduction in the concentration of sodium chloride.

The immunogenic compositions ofthe invention wifi 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 IE 1 3 0 3 4 3 VB65568PCT 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 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 CaCI2 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 NH4+, Mg2+ and Ca2+ ions in the immunogenic compositions wili 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 wiil have an osmoiality 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 bein the range of 250 to 550 mOsm/kg, such as in the range of 280 to 500 mOsm/kg. a es VB65568PCT IEl 3 0 3 4 3 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 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 pofyols, 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 osmolality 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.

VB65568PCT IEl 30 3 3 The pH of the immunogenic compositions should be suitable for parenteral administration. Typically the pH will be in the range of 6.0 to 9.0. 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 Tris or phosphate buffers.

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 (Cl') 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 ofthe immunogenic composition ofthe 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. ί ' VB65568PCT IEl 3 Ο 3 4 3 By "improved CD4 T-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 T-cell immune response’ is obtained in an immunologically unprimed patient, i.e. a patient who is seronegative to HIV, The improved CD4 T-cell immune response (which may be provided by ‘polyfunctional’ 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 • cells producing at least two different immune markers (e.g. CD40L, IL-2 and/or IFNgamma, TNF-alpha) • cells producing at least CD40L 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. CD40L, TNF-alpha and/or IFN-gamma) • cells producing at least IFN-gamma and another immune marker (e.g. IL-2, TNF-alpha and/or CD40L) · cells producing at least TNF-alpha and another immune marker (e.g. IL-2, CD40L 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 T-cell immune response conferred by the gp120 composition ofthe present invention may be ideally obtained after one singie 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 antibody35 secreting plasma cells upon antigen encounter as measured by stimulation of in-vitro differentiation. 1E1 3 0 3 4 3 VB65568PCT In a specific embodiment, the administration of said immunogenic composition induces at least two of the following responses: (i) an improved CD4 T-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 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 crossreactivity. 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 ofthe 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, 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 HJV-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 ti C.

Vi 1E1 3 0 3 43 VB65568PCT 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.

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 ΗIV-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 ciades 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/ml 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 beiow 500 copies/ml. Suitably, viraf load is maintained or reduced for at least six months, at (east 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 VB65568PCT IEl 3 0 3 4 3 capable of binding to the V1V2 region of the gp120 related polypeptide of the composition at least 24 weeks, at ieast 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 ieast 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%.

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 ofthe 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 ieast 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 ofthe 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.

VB65568PCT IE1 3 0 3 4 3 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.

Another suitable administration route is the subcutaneous route. Any suitable device may be used for subcutaneous delivery, for exampie 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.

VB65568PCT IEl 3 Ο 3 43 Suitably, the gp120 related polypeptide of the invention may be substituted by a polynucleotide encoding the gp120 related polypeptide of the invention. Suitably, the polypeptide is codonoptimised.

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 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 non25 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 (3TC), 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 cellqlar proteins r. associated with disease progression. Also included are immune-based therapies, such as IL-2, ¢1 VB65568PCT IE 1 3 0 3 43 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 (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, fnvirase (saquinavir mesylate, SQV), Kaletra (lopinavir and ritonavir, LPV/RTV), Lexiva (Fosamprenavir Calcium, FOS-APV), Norvir (ritonavir, RTV), darunavir, Reyataz (atazanavir sulfate, ATV), Viracept (nelfinavir mesylate, NFV), Fuzeon (enfuvirtide, T-20), maraviroc, raltegravir, dolutegravir.

In one embodiment, the immunogenic composition is administered to a patient who is also 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.

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 CD4 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.

VB65568PCT ΙΕί 3 Ο 3 43 In one aspect of the invention, there is provided a gp120 reiated protein for use in the manufacture of an immunogenic composition as described herein. Such as for the prophyiaxis 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 prophyiaxis 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.

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.

Clause 2.

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 reiated polypeptide wherein the NefTat related polypeptide is a polypeptide consisting of SEQ ID NO: 4, 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 10-40ug of an VB65568PCT IE 1 3 0 3 4 3 Clause 3.

Clause 4.

Clause 5.

Clause 6.

Clause 7.

Clause 8.

Clause 9.

Clause 10.

Clause 11.

Clause 12.

Clause 13. immunologically active saponin fraction derived from the bark of Quillaja Saponaria Molina presented in the form of a liposome.

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. 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.

The immunogenic composition according to either clause 1 or 4, which contains a ratio of NefTat related polypeptide:gp120 related polypeptide of less than 1:20.

The immunogenic composition according to either clause 1 or 4, which contains less than 1ug of NefTat related polypeptide.

The immunogenic composition according to either clause 5 or 6 wherein the composition is free of NefTat related polypeptide.

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.

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.

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.

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.

The immunogenic composition according to either clausel or 4, which contains a ratio of Nef related polypeptide:gp120 related polypeptide of iess than 1:20. The immunogenic composition according to either clause 1 or 4, which contains less than 1ug of Nef related polypeptide.

VB65568PCT JEl 3 ο 3 4 3 Clause 14. The immunogenic composition according to either clause 12 or 13 wherein the composition is free of Nef related polypeptide. Clause 15. The immunogenic composition according to any one of clauses 1, 4 or 12 to 14 5 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: o 10 Clause 17. 4-- 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. The immunogenic composition according to clause 17 wherein the Nef related polypeptide comprises a poiypeptide with at least 70% identity with SEQ ID NO: Clause 18. 15 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 1ug of Tat related polypeptide. 20 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. 25 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. The immunogenic composition according to clause 23 wherein the Tat related polypeptide comprises a polypeptide with at least 80% identity with SEQ ID NO: Clause 24. 30 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. The immunogenic composition according to any one of clauses 1 or 3 to 25 Clause 26. 35 wherein the lipopolysaccharide is present at a level of between 10-100ug.

VB65568PCT •Ε 1 3 Ο 3 4 3 3S Clause 27. The immunogenic composition according to clause lipopolysaccharide is present at a level of between 15-80ug. 26 wherein the Clause 28. The immunogenic composition according to clause lipopolysaccharide is present at a levei of between 20-65ug. 27 wherein the Clause 29. The immunogenic composition according to clause lipopolysaccharide is present at a level of between 30-60ug. 28 wherein the Clause 30. The immunogenic composition according to clause lipopolysaccharide is present at a level of between 45-55ug. 29 wherein the Clause 31. The immunogenic composition according to clause lipopolysaccharide is present at a level of 50ug. 30 wherein the Clause 32.

Clause 33.

Clause 34.

Clause 35.

Clause 36.

Clause 37.

Clause 38.

Clause 39.

Clause 40.

Clause 41. 30 Clause 42.

Clause 43.

Clause 44.

The immunogenic composition according to any one of clauses 1 or 3 to 31 wherein the saponin is present at a level of between 10-100ug.

The immunogenic composition according to clause 32 wherein the saponin is present at a level of between 15-80ug.

The immunogenic composition according to ciause 33 wherein the saponin is present at a level of between 20-65ug.

The immunogenic composition according to clause 34 wherein the saponin is present at a level of between 30-60ug.

The immunogenic composition according to clause 35 wherein the saponin is present at a levei of between 45-55ug.

The immunogenic composition according to clause 36 wherein the saponin is present at a level of 50ug.

The immunogenic composition according to any one of clauses 1 to 25 wherein the lipopolysaccharide is present at a level of between 15-35ug.

The immunogenic composition according to clause 38 wherein the lipopolysaccharide is present at a level of between 20-30ug.

The immunogenic composition according to clause 39 wherein the lipopolysaccharide is present at a level of 25ug.

The immunogenic composition according to any one of clauses 1 to 31 wherein the saponin is present at a level of between 15-35ug.

The immunogenic composition according to clause 41 wherein the saponin is present at a level of between 20-30ug.

The immunogenic composition according to clause 42 wherein the saponin is present at a level of 25ug.

The immunogenic composition according to any one of clauses 1 to 43 wherein the lipopolysaccharide is 3D-MPL.

VB65568PCT IEl 3 0 3 4 3 Clause 45.

Clause 46. 5 Clause 47.

Clause 48.

Clause 49.

Clause 50.

Clause 51.

Clause 52.

Clause 53.

Clause 54.

Clause 55.

Clause 56.

Clause 57. 30 Clause 58.

Clause 59.

Clause 60.

The immunogenic composition according to any one of clauses 1 to 44 wherein the saponin is QS-21.

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. The immunogenic composition according to clause 46 wherein the conductivity of the composition is 13 mS/cm or lower.

The immunogenic composition according to clause 47 wherein the conductivity of the composition is 6 mS/cm or lower.

The immunogenic composition according to clause 48 wherein the conductivity of the composition is 1.5 to 2.5 mS/cm.

The immunogenic composition according to clause 46 wherein the concentration of salts in said composition is 130 mM or lower.

The immunogenic composition according to clause 50 wherein the concentration of saits in said composition is 60 mM or lower.

The immunogenic composition according to clause 51 wherein the concentration of salts in said composition is 20 to 40 mM.

The immunogenic composition according to clause 46 wherein the concentration of sodium chloride in said composition is 130 mM or lower.

The immunogenic composition according to clause 53 wherein the concentration of sodium chloride in said composition is 60 mM or lower.

The immunogenic composition according to clause 54 wherein the concentration of sodium chloride in said composition is 10 mM or lower.

The immunogenic composition according any one of clauses 1 to 55, wherein the concentration of CaCl2 in the immunogenic composition is 30 mM or lower. 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. The immunogenic composition according any one of clauses 1 to 57, total concentration of NH4+, Mg2+ and Ca2+ ions is 40 mM or lower.

The immunogenic composition according to any one of clauses 1 to 58 which is an aqueous solution.

The immunogenic composition according to any one of clauses 1 to 59 vyhich is a single human dose. U IE 1 3 Ο 3 4 3 VB65568PCT Clause 61.

Clause 62.

Clause 63.

Clause 64.

Clause 65.

Clause 66.

Clause 67.

Clause 68.

Clause 69.

Clause 70.

Clause 71.

Clause 72.

Clause 73.

Clause 74.

Clause 75.

Clause 76.

Clause 77. 35 The immunogenic composition according to any one of clauses 1 to 60 wherein the human dose is between 0.1 and 1mf.

The immunogenic composition according to clause 61 wherein the human dose is between 0.3 and 0.75ml.

The immunogenic composition according to clause 62 wherein the human dose is 0.5ml.

The immunogenic composition according to any one of clauses 1 to 60 wherein the human dose is between 0.05 and 0.2ml.

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.

The immunogenic composition according to clause 65 wherein the osmolality is in the range of 250 to 550 mOsm/kg.

The immunogenic composition according to any one of clauses 1 to 66, comprising a non-ionic tonicity agent.

The immunogenic composition according to clause 67, wherein the non-ionic tonicity agent is a polyol.

The immunogenic composition according to clause 68, wherein the polyol is sorbitol.

The immunogenic composition according to clause 69, wherein the concentration of sorbitol is between about 4 and about 6% (w/v).

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).

The immunogenic composition according to any one of clauses 1 to 71 wherein the pH is in the range of 6.0 to 9.0.

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.

The immunogenic composition according to clause 73 wherein the composition comprises the gp120 related polypeptide at a level of 3 to 30 ug.

The immunogenic composition according to clause 74 wherein the composition comprises the gp120 related polypeptide at a level of 5 to 15 ug.

The immunogenic composition according to clause 74 wherein the composition comprises the gp120 related polypeptide at a level of 16 to 25 ug.

The immunogenic composition according to any one of clauses 1 to 76 wherein the composition comprises 1-5 additional HIV antigens.

VB65568PCT JE 1 3 0 3 4 3 Clause 78.

Clause 79.

Clause 80.

Clause 81.

Clause 82.

Clause 83.

Clause 84.

Clause 85.

Clause 86.

Clause 87.

Clause 88.

Clause 89.

Clause 90.

Clause 91.

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. 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. The composition according to any one of clauses 1 to 79 wherein the gp120 related polypeptide comprises a gp120 polypeptide derived from HIV-1.

The composition according to clause 80 wherein the gp120 related polypeptide comprises a gp120 polypeptide derived from HIV-1 group M.

The composition according to clause 81 wherein the gp120 related polypeptide comprises a gp120 polypeptide derived from HIV-1 group M subtype C.

The composition according to clause 82 wherein the gp120 related polypeptide comprises a gp120 polypeptide derived from HIV-1 group M subtype B.

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.

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.

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.

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.

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.

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.

The composition according to clause 89 wherein the gp120 related polypeptide comprises the V1V2 region of SEQ ID NO: 1.

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 70ΰ/ό identity with the V1V2 region of SEQ ID NO: 1. ς VB65568PCT IEl 3 0 3 43 Clause 92.

Clause 93.

Clause 94.

Clause 95.

Clause 96.

Clause 97.

Clause 98.

Clause 99.

Clause 100.

Clause 101.

Clause 102.

Clause 103.

Clause 104.

Clause 105.

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.

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.

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.

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.

The composition according to clause 95 wherein the gp12O related polypeptide consists of a polypeptide with at ieast 99% identity with the V1V2 region of SEQ ID NO: 1.

The composition according to clause 96 wherein the gp120 related polypeptide consists of the V1V2 region of SEQ ID NO: 1.

The composition according to any one of clauses 1 to 81 and 83 to 97 wherein the gp12O related polypeptide comprises a polypeptide with at least 70% identity with SEQ ID NO: 1.

The composition according to clause 98 wherein the gp120 related polypeptide comprises a polypeptide with at least 80% identity with SEQ ID NO: 1.

The composition according to clause 99 wherein the gp120 related polypeptide comprises a polypeptide with at least 90% identity with SEQ ID NO: 1.

The composition according to clause 100 wherein the gp120 related polypeptide comprises a polypeptide with at least 95% identity with SEQ ID NO: 1.

The composition according to clause 101 wherein the gp120 related polypeptide comprises a polypeptide with at least 98% identity with SEQ ID NO: 1.

The composition according to clause 102 wherein the gp120 related polypeptide comprises a polypeptide with at least 99% identity with SEQ ID NO: 1.

The composition according to clause 103 wherein the gp120 related polypeptide comprises SEQ ID NO: 1.

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.

IE 1 3 0 3 4 3 VB65568PCT Clause 106.

Clause 107.

Clause 108.

Clause 109.

Clause 110.

Clause 111.

Clause 112.

Clause 113.

Clause 114.

Clause 115.

Clause 116.

Clause 117.

Clause 118.

Clause 119.

The composition according to ciause 105 wherein the gp120 related polypeptide consists of a polypeptide with at least 80% identity with SEQ ID NO: 1.

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.

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.

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.

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.

The composition according to clause 110 wherein the gp120 related polypeptide consists of SEQ ID NO: 1.

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.

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.

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.

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.

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.

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.

The composition according to clause 117 wherein the gp120 related polypeptide comprises the V1V2 region of SEQ ID NO: 5.

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 atjealt 70% identity with the V1V2 region of SEQ ID NO: 5. a IE 1 3 Ο 3 4 3 VB65568PCT Clause 120.

Clause 121.

Clause 122.

Clause 123.

Clause 124.

Clause 125.

Clause 126.

Clause 127.

Clause 126.

Clause 129.

Clause 130.

Clause 131.

Clause 132.

Clause 133.

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.

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.

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.

The composition according to clause 122 wherein the gp120 related polypeptide consists of a polypeptide with at least 98% identity with the V1V2 region of SEQ ID NO: 5.

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.

The composition according to clause 124 wherein the gp120 related polypeptide consists of the V1V2 region of SEQ ID NO: 5.

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 ieast 70% identity with SEQ ID NO. 5.

The composition according to clause 126 wherein the gp120 related polypeptide comprises a polypeptide with at least 80% identity with SEQ ID NO: 5.

The composition according to clause 127 wherein the gp120 related polypeptide comprises a polypeptide with at least 90% identity with SEQ ID NO: 5.

The composition according to clause 128 wherein the gp120 related polypeptide comprises a polypeptide with at least 95% identity with SEQ ID NO: 5.

The composition according to clause 129 wherein the gp120 related polypeptide comprises a polypeptide with at least 98% Identity with SEQ ID NO: 5.

The composition according to clause 130 wherein the gp120 related polypeptide comprises a polypeptide with at least 99% identity with SEQ ID NO: 5.

The composition according to clause 131 wherein the gp120 related polypeptide comprises SEQ ID NO: 5.

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.

VB65568PCT IEl 3 0 3 4 3 Clause 134.

Clause 135.

Clause 136.

Clause 137.

Clause 138.

Clause 139.

Clause 140.

Clause 141.

Clause 142.

Clause 143.

Clause 144.

Clause 145.

Clause 146.

Clause 147.

Clause 148.

Clause 149.

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.

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.

The composition according to clause 135 wherein the gp120 related polypeptide consists of a polypeptide with at least 95% identity with SEQ ID NO: 5.

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.

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.

The composition according to clause 138 wherein the gp120 related polypeptide consists of SEQ ID NO: 5.

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.

The immunogenic composition according to clause 140 wherein the ratio of saponin:sterol is from 1:1 to 1:5 w/w.

The immunogenic composition according to either clause 140 or 141 wherein said sterol is cholesterol.

The immunogenic composition according to any one of clauses 1 to 142, which is provided in a multi-dose presentation.

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.

The immunogenic composition according to any one of clauses 1 to 144, for use as a medicament.

The immunogenic composition according to clause 145, for use in the treatment or prevention of HIV-1 from group Μ, N, O or P.

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, Η, I, J or K.

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.

The immunogenic composition according to any one of clauses 1 tor14jB; wherein the gp120 related polypeptide is derived from a first HIV-1 subtype, for VB65S68PCT Clause 150.

Clause 151.

Clause 152.

Clause 153.

Clause 154.

Clause 155.

Clause 156.

Clause 157.

Clause 158.

IE 1 3 Ο 3 4 3 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.

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, Η, I, J or K; of HIV-1 group M.

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.

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.

A method of treatment or prophyiaxis of HIV-1 infection comprising the step of administering a composition according to any one of clauses 1 to 152 to an individual.

The method of treatment or prophylaxis of HIV-1 infection according to clause 153 further comprising concomitant administration of an antiretroviral drug.

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.

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.

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.

The method of treatment or prophylaxis of HIV-1 infection according to clause 157 wherein the IgG antibody is detectable at ieast 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.

VB65568PCT IE 1 3 0 3 4 3 Clause 159.

Clause 160.

Clause 161, Clause 162.

Clause 163.

Clause 164.

Clause 165.

Clause 166.

Clause 167.

Clause 168.

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.

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 administration of the composition in a course of repeat administrations to the individual.

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.

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.

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.

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.

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.

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.

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.

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. >-T VB65568PCT ΙΕ ΐ 3 Ο 3 4 3 Clause 169.

Clause 170.

Clause 171.

Clause 172.

Clause 173.

Clause 174.

Clause 175.

Clause 176.

Clause 177.

Clause 178.

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.

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 administering the immunogenic composition of any one of clauses 1 to 152 to the HIV-infected individual.

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.

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.

The kit according to clause 172, wherein the second container comprises an aqueous solution.

A viral vector comprising a polynucleotide encoding a polypeptide comprising the gp120W6iD polypeptide of SEQ ID No: 1.

The viral vector of clause 174 comprising a polynucleotide encoding a polypeptide consisting of the gp120W61D polypeptide of SEQ ID No: 1.

A viral vector comprising a polynucleotide encoding a polypeptide comprising the gp120zMis poiypeptide of SEQ ID NO: 5.

The viral vector of clause 176 comprising a polynucleotide encoding a polypeptide consisting of the gp120ZW8 polypeptide of SEQ ID NO: 5.

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.

VB65568PCT IE 1 3 0 3 4 3 EXAMPLES Example 1: Serology comparison Method A first composition containing 2ug gp120w6iD and 50ul AS01B and a second composition containing 2ug gp120w6iD, 50ul AS01B and 2ug 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 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 case A2 gp120 V1V2 region scaffolded onto a murine gp70 protein was used in an Igiot 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 Igtot antibodies to scaffolded V1V2. In addition, gp70 protein (not bound to a V1V2 region) was used as a negative control to assess specificity during development of the assay.

Results On both timepoints tested, a trend for a higher anti-V1V2 antibody response was observed in 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), Example 2: PRO HIV-002 and RV144 trial analysis - anti-V1V2 IgG comparison .

Background: The PRQ-HIV-002 trial // r/ This was a study aimed to determine the safety and immunogenicity of agp120/NefTat candidate HIV-1 vaccine formulated with one of three different adjuvant systems (AS02A, ΙΕ ι 3 0 3 43 VB65568PCT AS02V and AS01B) in healthy HIV-seronegative adults. See Leroux-Roels et al., Vaccine 28:7016-7024 (2010) for further details.

Background: The RV144 trial This was a study aimed to evaluate the efficacy of four priming injections of a recombinant canarypox vector vaccine (ALVAC-HIV [vCP1521]) plus two booster injections of a recombinant gp120 subunit vaccine (AIDSVAX B/E) at preventing HIV-1 infection in healthy adults. See Rerks-Ngarm et al, N Engl J Med 361:2209-2220 (2009) for further details.

Anti-V1V2 IgG comparison Method Serum samples were obtained from 30 subjects from the PRO-HIV-002 trial who had been vaccinated with 20ug gp120W6iD, 20ug NefTat and AS01B (liposome-based, 50ug MPL and 50ug QS-21) at 0, 1,3 and 6 months, according to the trial protocol. The serum had been collected at 9 different timepoints. These timepoints were baseline (the first of the four vaccinations, day 0), day 42 (2 weeks after the second vaccination), day 84, day 98 (2 weeks after the third vaccination), day 168, day 182 (2 weeks after the fourth vaccination), day 336, day 504 and day 672 (18 months after the fourth vaccination).

To evaluate the quantity of anti-V1V2 IgG antibodies present in each serum sample, the HIV-1 subtype B case A2 gp120 V1V2 region scaffolded onto a murine gp70 protein was used in an IgG multiplex binding antibody assay, alongside a gp70 control and ConSgp140 (group M consensus gp140). CH58 (the anti-V2 monoclonal antibody used in the RV144 trial) was used to detect successful binding of serum IgG antibodies to scaffolded V1V2. Samples were run at a dilution of 1:100.

The levels of anti-V1V2 ciade B antibodies in the serum samples from the 30 subjects of the PRO-HIV-002 trial were compared to those of 205 vaccinees in the case-control group who remained uninfected in the RV144 trial.

Results VB65568PCT IE 1 3 0 3 4 3 The serum samples from the PRO-HIV-002 trial subjects contained a higher level of anti-V1V2 clade B antibodies than the serum samples from the RV144 trial subjects at 2 weeks after the last vaccination in both trials (see Figure 2), The serum samples from the PRO-HIV-002 trial subjects showed a 100% response rate (average: 80.7ug/mi, median 62.2 ug/ml) compared to serum samples from the RV144 trial subjects showing a 64% response rate (average: 4.2 ug/ml, median 1.6 ug/ml).

It was found that anti-V1V2 clade B antibodies in the serum samples from the PRO-HIV-002 trial subjects persisted (87% responders) at 18 months after the fourth priming injection of gp120/NefTat/AS01 B (see Figure 2 and Figure 3). In the RV144 trial, responses to the gp120 V2 loop was reduced 6 months after the final vaccination (Excler et al., Poster TUP231 - IAS 2013 - Kuala Lumpur, Malaysia).

The serum samples from the PRO-HIV-002 trial subjects all contained anti-V1V2 antibodies after the third injection of gp120/NefTat/AS01 B (see Figure 3).

The serum samples from the PRO-HIV-002 trial subjects had a different reactivity profile against varying subtypes of HIV-1 compared to serum samples from the RV144 trial subjects 2 weeks after the last vaccination in both trials (see Figure 4).

All references referred to in this application, including patent and patent applications, are 25 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’, wili 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 subsequentapplication may be directed to any feature or combination of features described herein. They may take VB65568PCT JEl 3 Ο 3 43 the form of product, composition, process, or use ciaims and may include, by way of example and without limitation, the following claims.

VBS5568PCT ΙΕί 3 Ο 3 43 SEQUENCE LISTING SEQ ID NO: l-gp^Owem AEQLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEWLGNVTEYFNMWKNNM VDQMHEDIISLWDQSLKPCVKLTPLCVTLDCDDVNTTNSTTTTSNGWTGEIRKGEIKNCSFNITTSIRDK VQKEYAL FYHLDVVPIDDDMATTKNKT TRM FRLIHCNS S VMTQACPKVS FE PIPIHYCAPAG FAILKCNN KTFDGKGLCTNVSTVQCTHGIRPWSTQLLLNGSLAEEEWIRSDNFMDNTKTIIVQLNESVAINCTRPN NNTRKGIHIGPGRAFYAARKIIGDIRQAHCNLSRAQWNNTLKQIVIKLREHFGNKTIKFNQSSGGDPEIV RHSFNCGGEFFYCDTTQLFNSTWNGTEGNNTEGNSTITLPCRIKQIINMWQEVGKAMYAPPIGGQIRCSS NITGLLLTRDGGTEGNGTENETEIFRPGGGDMRDNWRSELYKYKWKVEPLGVAPTRAKRRWQR V1V2 region underlined.

SEQ ID NO: 2 - Nef MGGKWSKSSWGWPTVRERMRRAEPAADGVGAASRDLEKHGAITSSNTAATNAACAWLEAQEEEEVGFPV TPQVPLRPMTYKAAVDLSHFLKEKGGLEGLIHSQRRQDILDLWIYHTQGYFPDWQNYTPGPGVRYPLTFG WCYKLVPVEPDKVEEANKGENTSLLHPVSLHGMDDPEREVLEWRFDSRLAFHHVARELHPEYFKNC SEQ ID NO: 3-Tat EPVDPRLEPWKHPGSQPKTACTNCYCKKCCFHCQVCFITKALGISYGRKKRRQRRRPPQGSQTHQVSLSK QPTSQSRGDPTGPKE SEQ ID NO: 4 - NefTat MGGKWSKSSWGWPTVRERMRRAEPAADGVGAASRDLEKHGAITSSNTAATNAACAWLEAQEEEEVGFPV TPQVPLRPMTYKAAVDLSHFLKEKGGLEGLIHSQRRQDILDLWIYHTQGYFPDWQNYTPGPGVRYPLTFG wcyklvpvepdkveeankgentsllhpvslhgmddperevlewrfdsrlafhhvarelhpeyfknc|ts]e PVDPRLEPWKHPGSQPKTACTNCYCKKCCFHCQVCFITKALGI5YGRKKRRQRRRPPQGSQTHQVSLSKQ PTSQSRGDPTGPKE Nef portion: amino acids 1-206 Tat portion: amino acids 209-293 (underlined) Box: additional linking amino acids introduced in fusion protein SEQ ID NO: 5-gp1207M18 GDNLWVTVYYGVPVWKEAKTTLFCASDAKAYEREVHNVWATHACVPTDPNPQEIVLGNVTENFNMWKNDM VDQMHEDIIRLWDQS LKPCyKLTPLCVTLECGNyNyTHENSTKGEMKNCSFNATTELKDKKQRVYALFYK,, LDIVPLNENNNSSEDSSEYRLINCNTSAITQACPKVTLDPIPIHYCAPAGYAILKCNNKTFNGTGPCHNV ·' \ STVQCTHGIKPWSTQLLLNGSLAEEEIIIRSENLTNNAKTIIVHLNESVEIVCTRPSNiJXRKSIRIGPG > QAFYATGGIIGNIRQAHCNISKENWNKTLQKVGKKLAEHFPNKTIKFDQHSGGDLEiTTHSFNCRGEFFY ' > CNTSNLFNSTYKPNDTNSTYNPNDTITLPCRIKQIINMWQGVGQAMYAPPIAGNITCKSNITGI.LtTRDG GSNDTTNTETFRPGGGDMRDNWRSELYKYKWEIKTLGIAPTAAKRRVVETR _ X V1V2 region underlined.

VB65568PCT IEi 3 0 3 4 3 SEQ ID NO: 6 - polynucleotide sequence encoding gp120zMis ggagacaacttgtgggtcacagtctattatggggtacctgtgtggaaagaagcaaaaactactttattct gtgcatcagatgctaaagcatatgagagagaagtgcataatgtctgggctacacatgcctgtgtacccac agaccccaacccacaagaaatagttttgggaaatgtaacagaaaattttaacatgtggaaaaatgacatg gtggatcagatgcatgaggatataatcaggttatgggatcaaagcttaaagccatgtgtaaagttgaccc cactctgtgtcactttagaatgtggaaatgttaatgttacccatgagaatagcacgaagggggaaatgaa aaattgctctttcaatgcaaccacagaactaaaagataaaaaacagagagtgtatgcacttttttataaa cttgatatagtaccacttaatgagaataacaactctagtgaggactctagtgagtatagattaataaatt gtaatacctcagccataacacaagcctgtccaaaggtcactttggacccaattcctatacattattgtgc tccagctggatatgcgattctaaagtgtaataataagacattcaatgggacaggaccatgccataatgtc agcacagtacaatgtacacacggaatcaagccagtggtatcaactcaactactgttaaatggtagcctag cagaagaagagataataattaggtctgaaaatctaacaaacaatgccaaaacaataatagtacatcttaa tgaatctgtagaaattgtgtgtacaagacccagcaataatacaagaaaaagtataaggataggaccagga caagcattctatgcaacaggtggcataataggaaacataagacaagcacattgtaacattagtaaagaga actggaataaaactttacaaaaggtaggaaaaaaattagcagagcacttccctaataaaacaataaaatt tgaccaacactcaggaggggacctagaaattacaacacatagctttaattgtagaggagaatttttctat tgcaatacatcaaacctgtttaatagtacatataagcctaatgatacaaatagtacatataatcctaatg atacaatcacactcccatgcagaataaaacaaattataaacatgtggcagggggtaggacaagcaatgta tgcccctcccattgcaggaaacataacatgtaaatcaaatatcacaggactactattgacacgggatgga gggtcaaatgataccacaaacacagagacattcagacctggaggaggagatatgagggacaattggagaa gtgaactatataaatataaagtggtagaaattaaaacattgggcatagcacccactgcggcaaaaaggag agtggtggagacgagataa SEQ ID NO: 7 - native gp120zMi8 GDNLWVTVYYGVPVWKEAKTTLFCASDAKAYEREVHNVWATHACVPTDPNPQEIVLGNVTENFNMWKNDM VDQMHE DIIRLWDQS LKPCVKLT PLCVT LECGNVNVTHENS TKGEMKNCS FNAT T ELKDKKQRVYAL FYK LDIVPLNENNNSSEDSSEYRLINCNTSAITQACPKVTLDPIPIHYCAPAGYAILKCNNKTFNGTGPCHNV STVQCTHGIKPVVSTQLLLNGSLAEEEIIIRSENLTNNAKTIIVHLNESVEIVCTRPSNNTRKSIRIGPG QAFYATGGIIGNIRQAHCNISKENWNKTLQKVGKKLAEHFPNKTIKFDQHSGGDLEITTHSFNCRGEFFY CNTSNLFNSTYKPNDTNSTYNPNDTITLPCRIKQIINMWQGVGQAMYAPPIAGNITCKSNITGLLLTRDG GSNDTTNTETFRPGGGDMRDNWRSELYKYKWEIKPLGIAPTAAKRRWETR Native gp120ZM18 polypeptide sequence contains underlined proline instead of threonine.

SEQ ID NO: 8 - polynucleotide sequence encoding native gp120zMie ggagacaacttgtgggtcacagtctattatggggtacctgtgtggaaagaagcaaaaactactttattct gtgcatcagatgctaaagcatatgagagagaagtgcataatgtctgggctacacatgcctgtgtacccac agaccccaacccacaagaaatagttttgggaaatgtaacagaaaattttaacatgtggaaaaatgacatg gtggatcagatgcatgaggatataatcaggttatgggatcaaagcttaaagccatgtgtaaagttgaccc cactctgtgtcactttagaatgtggaaatgttaatgttacccatgagaatagcacgaagggggaaatgaa aaattgctctttcaatgcaaccacagaactaaaagataaaaaacagagagtgtatgcacttttttataaa cttgatatagtaccacttaatgagaataacaactctagtgaggactctagtgagtatagattaataaatt gtaatacctcagccataacacaagcctgtccaaaggtcactttggatccaattcctatacattattgtgc tccagctggatatgcgattctaaagtgtaataataagacattcaatgggacaggaccatgccataatgtc agcacagtacaatgtacacacggaatcaagccagtggtatcaactcaactactgttaaatggtagcctag cagaagaagagataataattaggtctgaaaatctaacaaacaatgccaaaacaataatagtacatcttaa tgaatctgtagaaattgtgtgtacaagacccagcaataatacaagaaaaagtataaggataggaccagga caagcattctatgcaacaggtggcataataggaaacataagacaagcacattgtaacattagtaaagaga actggaataaaactttacaaaaggtaggaaaaaaattagcagagcacttccctaataaaacaataaaatt tgaccaacactcaggaggggacctagaaattacaacacatagctttaattgtagaggagaatttttctat tgcaatacatcaaacctgtttaatagtacatataagcctaatgatacaaatagtacatataatcctaatg atacaatcacactcccatgcagaataaaacaaattataaacatgtggcagggggtaggacaagcaatgta tgcccctcccattgcaggaaacataacatgtaaatcaaatatcacaggactactattgacacgggatgga gggtcaaatgataccacaaacacagagacattcagacctggaggaggagatatgagggacaattggagaa gtgaactatataaatataaagtggtagaaattaaaccattgggcatagcacccactgcggcaaaaaggag agtggtggagacgagataa VB65568PCT IEl 3 Ο 3 4 3 To produce the gp120zMie polynucleotide sequence from the native gp120zM18 polynucleotide sequence: a) one silent point mutation is introduced (C instead of T at position 597 (underlined). b) one non-silent point mutation A instead of C is introduced, at position 1426 (underlined) modifying the CCA native codon (proline) for the ACA codon (threonine).

IEl 3 0 3 4 3 SEQUENCE LISTING File Ref. PT362 <110> GlaxoSmithKline Biologicals s.a. <120> Novel Compositions <130> VB <160> 8 <170> Patentln version 3.5 <210> 1 <211> 485 <212> PRT <213> Human immunodeficiency virus <400> 1 Ala Glu Gin Leu Trp Val Thr Val 1 5 Tyr Tyr Gly Val Pro Val Trp Lys 10 15 Glu Ala Thr Thr Thr Leu Phe Cys 20 Ala Ser Asp Ala Lys Ala Tyr Asp 25 30 Thr Glu Val His Asn Val Trp Ala 35 40 Thr His Ala Cys Val Pro Thr Asp 45 Pro Asn Pro Gin Glu Val Val Leu 50 55 Gly Asn Val Thr Glu Tyr Phe Asn 60 Met Trp Lys Asn Asn Met Val Asp 65 70 Gin Met His Glu Asp lie He Ser 75 80 Leu Trp Asp Gin Ser Leu Lys Pro 85 Cys Val Lys Leu Thr Pro Leu Cys 90 95 Val Thr Leu Asp Cys Asp Asp Val 100 Asn Thr Thr Asn Ser Thr Thr Thr 105 110 Thr Ser Asn Gly Trp Thr Gly Glu 115 120 He Arg Lys Gly Glu He Lys Asn 125 Cys Ser Phe Asn lie Thr Thr Ser 130 135 Tyr Ala Leu Phe Tyr Asn Leu Asp Val Val Pro lie Asp Asp Asp Asn 145 150 155 160 He Arg Asp Lys Val Gin Lys Glu 140 Phe Arg Leu lie His Cys 175 Ala Thr Thr Lys Asn Lys 165 Thr Thr Arg Asn 170 Asn Ser Ser Val Met 180 Thr Gin Ala Cys Pro 185 Lys Val Ser Phe Glu Pro 190 He Pro He His Tyr 195 Cys Ala Pro Ala Gly 200 Phe Ala He Leu Lys Cys 205 Asn Asn Lys Thr Phe 210 Asp Gly Lys Gly Leu 215 Cys Thr Asn Val Ser Thr 220 Val Gin Cys Thr His 225 Gly He Arg Pro Val 230 Val Ser Thr Gin Leu Leu 235 240 Leu Asn Gly Ser Leu 245 Ala Glu Glu Glu Val 250 Val lie Arg Ser Asp Asn 255 Phe Met Asp Asn Thr Lys Thr lie He 260 265 Val Gin Leu Asn Glu Ser Val 270 Ala He Asn Cys Thr Arg Pro Asn Asn 275 280 Asn Thr Arg Lys Gly lie His 285 He Gly Pro Gly Arg Ala Phe Tyr Ala 290 295 Ala Arg Lys lie lie Gly Asp 300 He Arg Gin Ala His Cys Asn Leu Ser 305 310 Arg Ala Gin Trp Asn Asn Thr 315 320 Leu Lys Gin lie VaL He Lys Leu Arg 325 Glu His Phe Gly Asn Lys Thr 330 335 He Lys Phe Asn Gin Ser Ser Gly Gly 340 345 Asp Pro Glu He Val Arg His 350 Ser Phe Asn Cys Gly Gly Glu Phe Phe 355 360 Tyr Cys Asp Thr Thr Gin Leu 365 Phe Asn Ser Thr Trp Asn Gly Thr Glu 370 375 Gly Asn Asn Thr Glu Gly Asn 380 Ser Thr He Thr Leu Pro Cys Arg lie 385 390 Lys Gin lie He. Asn Met Trp 395 400 Gin Glu Val Gly Lys Ala Met Tyr Ala Pro Pro lie Gly Gly Gin lie IE? 3 Ο 3 4 J 405 410 415 Arg Cys Ser Ser Asn lie Thr Gly Leu Leu Leu Thr Arg Asp Gly Gly 420 425 430 Thr Glu Gly Asn Gly Thr Glu Asn Glu Thr Glu lie Phe Arg Pro Gly 435 440 445 Gly Gly Asp Met Arg Asp Asn Trp Arg Ser Glu Leu Tyr Lys Tyr Lys 450 455 460 Val Val Lys Val Glu Pro Leu Gly Val Ala Pro Thr Arg Ala Lys Arg 465 470 475 480 Arg Val Val Gin Arg 485 <210> 2 <211> 206 <212> PRT <213> Human immunodeficiency virus <400> 2 Met Gly Gly Lys Trp Ser Lys Ser Ser Val Val Gly Trp Pro Thr Val 15 10 15 Arg Glu Arg Met Arg Arg Ala Glu Pro Ala Ala Asp Gly Val Gly Ala 20 25 30 Ala Ser Arg Asp Leu Glu Lys His Gly Ala lie Thr Ser Ser Asn Thr 35 40 45 Ala Ala Thr Asn Ala Ala Cys Ala Trp Leu Glu Ala Gin Glu Glu Glu 50 55 60 Glu Val Gly Phe Pro Val Thr Pro Gin Val Pro Leu Arg Pro Met Thr 65 70 75 80 Tyr Lys Ala Ala Val Asp Leu Ser His Phe Leu Lys Glu Lys Gly Gly 85 90 95 Leu Glu Gly Leu He His Ser Gin Arg Arg Gin Asp He Leu Asp Leu 100 105 110 Trp He Tyr His Thr Gin Gly Tyr Phe Pro Asp Trp Gin Asn. Tyr Thr 115 120 125 IEl 3 0 3 4 3 Pro Gly Pro Gly Val Arg Tyr Pro Leu Thr Phe Gly Trp Cys Tyr Lys 130 135 140 Leu Val Pro Val Glu Pro Asp Lys Val Glu Glu Ala Asn Lys Gly Glu 145 150 155 160 Asn Thr Ser Leu Leu His Pro Val Ser Leu His Gly Met Asp Asp Pro 165 170 175 Glu Arg Glu Val Leu Glu Trp Arg Phe Asp Ser Arg Leu Ala Phe His 180 185 190 His Val Ala Arg Glu Leu His Pro Glu Tyr Phe Lys Asn Cys 195 200 205 <210> 3 <211> 85 <212> PRT <213> Human immunodeficiency virus <400> 3 Glu Pro Val Asp Pro Arg Leu Glu Pro Trp Lys His Pro Gly Ser Gin 15 10 15 Pro Lys Thr Ala Cys Thr Asn Cys Tyr Cys Lys Lys Cys Cys Phe His 20 25 30 Cys Gin Val Cys Phe lie Thr Lys Ala Leu Gly lie Ser Tyr Gly Arg 35 40 45 Lys Lys Arg Arg Gin Arg Arg Arg Pro Pro Gin Gly Ser Gin Thr His 50 55 60 Gin Val Ser Leu Ser Lys Gin Pro Thr Ser Gin Ser Arg Gly Asp Pro 65 70 75 80 Thr Gly Pro Lys Glu 85 <210> 4 <211> 293 <212> PRT <213> Artificial <220> <223> NefTat Fusion <400> 4 Met Gly Gly Lys Trp Ser Lys Ser Ser Val Val Gly Trp Pro Thr Val 15 IEl 30 343 Arg Glu Arg Met Arg Arg Ala Glu Pro Ala Ala Asp Gly Val Gly Ala 20 25 30 Ala Ser Arg Asp Leu Glu Lys His Gly Ala lie Thr Ser Ser Asn Thr 35 40 45 Ala Ala Thr Asn Ala Ala Cys Ala Trp Leu Glu Ala Gin Glu Glu Glu 50 55 60 Glu Val Gly Phe Pro Val Thr Pro Gin Val Pro Leu Arg Pro Met Thr 65 70 75 80 Tyr Lys Ala Ala Val Asp Leu Ser His Phe Leu Lys Glu Lys Gly Gly 85 90 95 Leu Glu Gly Leu He His Ser Gin Arg Arg Gin Asp He Leu Asp Leu 100 105 110 Trp He Tyr His Thr Gin Gly Tyr Phe Pro Asp Trp Gin Asn Tyr Thr 115 120 125 Pro Gly Pro Gly Val Arg Tyr Pro Leu Thr Phe Gly Trp Cys Tyr Lys 130 135 140 Leu Val Pro Val Glu Pro Asp Lys Val Glu Glu Ala Asn Lys Gly Glu 145 150 155 160 Asn Thr Ser Leu Leu His Pro Val Ser Leu His Gly Met Asp Asp Pro 165 170 175 Glu Arg Glu Val Leu Glu Trp Arg Phe Asp Ser Arg Leu Ala Phe His 180 185 190 His Val Ala Arg Glu Leu His Pro Glu Tyr Phe Lys Asn Cys Thr Ser 195 200 205 Glu Pro Val Asp Pro Arg Leu Glu Pro Trp Lys His Pro Gly Ser Gin 210 215 220 Pro Lys Thr Ala Cys Thr Asn Cys Tyr Cys Lys Lys Cys Cys Phe His 225 230 235 240 IE 1 3 ο 3 4 3 Cys Gin Val Cys Phe lie Thr Lys Ala Leu Gly He Ser Tyr Gly Arg 245 250 255 Lys Lys Arg Arg Gin Arg Arg Arg Pro Pro Gin Gly Ser Gin Thr His 260 265 270 Gin Val Ser Leu Ser Lys Gin Pro Thr Ser Gin Ser Arg Gly Asp Pro 275 280 285 Thr Gly Pro Lys Glu 290 <210> 5 <2H> 472 <212> PRT <213> Artificial <220> <223> 2M18 polypeptide <400> 5 Gly Asp > Asn Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp Lys 1 5 10 15 Glu Ala Lys Thr Thr Leu Phe Cys Ala Ser Asp Ala Lys Ala Tyr Glu 20 25 30 Arg Glu Val His Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp 35 40 45 Pro Asn Pro Gin Glu He Val Leu Gly Asn Val Thr Glu Asn Phe Asn 50 55 60 Met Trp Lys Asn Asp Met Val Asp Gin Met His Glu Asp He lie Arg 65 70 75 80 Leu Trp Asp Gin Ser Leu Lys Pro Cys Val Lys Leu Thr Pro Leu Cys 85 90 95 Val Thr Leu Glu Cys Gly Asn Val Asn Val Thr His Glu Asn Ser Thr 100 105 110 Lys Gly Glu Met Lys Asn Cys Ser Phe Asn Ala Thr Thr Glu Leu Lys 115 120 125 Asp Lys Lys Gin Arg Val Tyr Ala Leu Phe Tyr Lys Leu Asp lie Val 130 135 140 rt'-' Pro Leu Asn Glu Asn Asn Asn Ser Ser Glu 145 150 Leu lie Asn Cys Asn Thr Ser Ala He Thr 165 170 Thr Leu Asp Pro lie Pro He His Tyr Cys 180 185 He Leu Lys Cys Asn Asn Lys Thr Phe Asn 195 200 Asn Val Ser Thr Val Gin Cys Thr His Gly 210 215 Thr Gin Leu Leu Leu Asn Gly Ser Leu Ala 225 230 Arg Ser Glu Asn Leu Thr Asn Asn Ala Lys 245 250 Asn Glu Ser Val Glu He Val Cys Thr Arg 260 265 Lys Ser He Arg He Gly Pro Gly Gin Ala 275 280 He lie Gly Asn He Arg Gin Ala His Cys 290 295 Trp Asn Lys Thr Leu Gin Lys Val Gly Lys 305 310 Pro Asn Lys Thr He Lys Phe Asp Gin His 325 330 He Thr Thr His Ser Phe Asn Cys Arg Gly 340 345 Thr Ser Asn Leu Phe Asn Ser Thr Tyr Lys 355 360 Ser Ser Glu Tyr Arg 160 Ala Cys Pro Lys 175 Val Pro Ala Gly 190 Tyr Ala Thr Gly 205 Pro Cys His Lys 220 Pro Val Val Ser Glu Glu He lie He 240 lie He Val His 255 Leu Ser Asn Asn 270 Thr Arg Tyr Ala 285 Thr Gly Gly He 300 Ser Lys Glu Asn Leu Ala Glu His Phe 320 Gly Gly Asp Leu 335 Glu Phe Phe Tyr 350 Cys Asn Asn Asp Thr Asn Ser 365 Thr Tyr Asn Pro Asn Asp Thr He Thr Leu Pro Cys Arg He Lys Gin 370 375 380 IEl 3 0 3 4 3 IE 1 3 0 3 4 3 He 385 He Asn Met Trp Gin Gly Val Gly Gin Ala Met Tyr Ala Pro Pro 400 390 395 lie Ala Gly Asn He Thr Cys Lys Ser Asn He Thr Gly Leu Leu Leu 405 410 415 Thr Arg Asp Gly Gly Ser Asn Asp Thr Thr Asn Thr Glu Thr Phe Arg 420 425 430 Pro Gly Gly Gly Asp Met Arg Asp Asn Trp Arg Ser Glu Leu Tyr Lys 435 440 445 Tyr Lys Val· Val Glu lie Lys Thr Leu Gly lie Ala Pro Thr Ala Ala 450 455 460 Lys Arg Arg Val Val Glu Thr Arg 465 470 <210> 6 <211> 1419 <212> DNA <213> Artificial <220> <223> ZM18 polynucleotide <400> 6 ggagacaact tgtgggtcac agtctattat ggggtacctg tgtggaaaga agcaaaaact 60 actttattct gtgcatcaga tgctaaagca tatgagagag aagtgcataa tgtctgggct 120 acacatgcct gtgtacccac agaccccaac ccacaagaaa tagttttggg aaatgtaaca 180 gaaaatttta acatgtggaa aaatgacatg gtggatcaga tgcatgagga tataatcagg 240 ttatgggatc aaagcttaaa gccatgtgta aagttgaccc cactctgtgt cactttagaa 300 tgtggaaatg ttaatgttac ccatgagaat agcacgaagg gggaaatgaa aaattgctct 360 ttcaatgcaa ccacagaact aaaagataaa aaacagagag tgtatgcact tttttataaa 420 cttgatatag taccacttaa tgagaataac aactctagtg aggactctag tgagtataga 480 ttaataaatt gtaatacctc agccataaca caagcctgtc caaaggtcac tttggaccca 540 attcctatac attattgtgc tccagctgga tatgcgattc taaagtgtaa taataagaca 600 ttcaatggga caggaccatg ccataatgtc agcacagtac aatgtacaca cggaatcaag 660 ccagtggtat caactcaact actgttaaat ggtagcctag cagaagaaga gataataatt 720- aggtctgaaa atctaacaaa caatgccaaa acaataatag tacatcttaa tgaatctgta 780 gaaattgtgt gtacaagacc cagcaataat acaagaaaaa gtataaggat aggaccagga 840- r\ o.. caagcattct atgcaacagg tggcataata ggaaacataa gacaagcaca ttgtaacatt 900 agtaaagaga actggaataa aactttacaa aaggtaggaa aaaaattagc agagcacttc 960 cctaataaaa caataaaatt tgaccaacac tcaggagggg acctagaaat tacaacacat 1020 agctttaatt gtagaggaga atttttctat tgcaatacat caaacctgtt taatagtaca 1080 tataagccta atgatacaaa tagtacatat aatcctaatg atacaatcac actcccatgc 1140 agaataaaac aaattataaa catgtggcag ggggtaggac aagcaatgta tgcccctccc 1200 attgcaggaa acataacatg taaatcaaat atcacaggac tactattgac acgggatgga 1260 gggtcaaatg ataccacaaa cacagagaca ttcagacctg gaggaggaga tatgagggac 1320 aattggagaa gtgaactata taaatataaa gtggtagaaa ttaaaacatt gggcatagca 1380 cccactgcgg caaaaaggag agtggtggag acgagataa 1419 <210> Ί <211> 472 <212> PRT <213> Artificial <220> <223> gpl20 ΖΜ18 native polypeptide <400> Ί Gly Asp Asn Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp Lys 15 10 15 Glu Ala Lys Thr Thr Leu Phe Cys Ala Ser Asp Ala Lys Ala Tyr Glu 20 25 30 Arg Glu Val His Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp 35 40 45 Pro Asn Pro Gin Glu lie Val Leu Gly Asn Val Thr Glu Asn Phe Asn 50 55 60 Met Trp Lys Asn Asp Met Val Asp Gin Met His Glu Asp lie He Arg 65 70 75 80 Leu Trp Asp Gin Ser Leu Lys Pro Cys Val Lys Leu Thr Pro Leu Cys 85 90 95 Val Thr Leu Glu Cys Gly Asn Val Asn Val Thr His Glu Asn Ser Thr 100 105 110 Lys Gly Glu Met Lys Asn Cys Ser Phe Asn Ala Thr Thr Glu Leu Lys 115 120 125 Asp Lys Lys Gin Arg Val Tyr Ala Leu Phe Tyr Lys Leu Asp lie Val 130 135 140 IE 1 3 Ο 3 4 3 Pro Leu Asn Glu Asn Asn Asn Ser Ser Glu Asp Ser Ser Glu Tyr Arg 145 150 155 160 Leu lie Asn Cys Asn Thr Ser Ala lie Thr Gin Ala Cys Pro Lys Val 165 170 175 Thr Leu Asp Pro lie Pro lie His Tyr Cys Ala Pro Ala Gly Tyr Ala 180 185 190 lie Leu Lys Cys Asn Asn Lys Thr Phe Asn Gly Thr Gly Pro Cys His 195 200 205 Asn Val Ser Thr Val Gin Cys Thr His Gly He Lys Pro Val Val Ser 210 215 220 Thr Gin Leu Leu Leu Asn Gly Ser Leu Ala Glu Glu Glu lie He He 225 230 235 240 Arg Ser Glu Asn Leu Thr Asn Asn Ala Lys Thr He He Val His Leu 245 250 255 Asn Glu Ser Val Glu He Val Cys Thr Arg Pro Ser Asn Asn Thr Arg 260 265 270 Lys Ser He Arg He Gly Pro Gly Gin Ala Phe Tyr Ala Thr Gly Gly 275 280 285 He He Gly Asn He Arg Gin Ala His Cys Asn He Ser Lys Glu Asn 290 295 300 Trp Asn Lys Thr Leu Gin Lys Val Gly Lys Lys Leu Ala Glu His Phe 305 310 315 320 Pro Asn Lys Thr He Lys Phe Asp Gin His Ser Gly Gly Asp Leu Glu 325 330 335 He Thr Thr His Ser Phe Asn Cys Arg Gly Glu Phe Phe Tyr Cys Asn 340 345 350 Thr Ser Asn Leu Phe Asn Ser Thr Tyr Lys Pro Asn Asp Thr Asn Ser 355 360 365 IE 1 3 0 3 4 3 Thr Tyr Asn Pro Asn Asp Thr lie Thr Leu Pro Cys Arg He Lys Gin 370 375 380 He He Asn Met Trp Gin Gly Val Gly Gin Ala Met Tyr Ala Pro Pro 385 390 395 400 He Ala Gly Asn lie Thr Cys Lys Ser Asn lie Thr Gly Leu Leu Leu 405 410 415 Thr Arq Asp Gly Gly Ser Asn Asp Thr Thr Asn Thr Glu Thr Phe Arg 420 425 430 Pro Gly Gly Gly Asp Met Arg Asp Asn Trp Arg Ser Glu Leu Tyr Lys 435 440 445 Tyr Lys Val Val Glu He Lys Pro Leu Gly He Ala Pro Thr Ala Ala 450 455 460 Lys Arg Arg Val Val Glu Thr Arg 465 470 <210> 8 <211> 1419 <212> DNA <213> Artificial <220> <223> gpl20 ZM18 native polynucleotide <400> 8 ggagacaact tgtgggtcac agtctattat ggggtacctg tgtggaaaga agcaaaaact 60 actttattct gtgcatcaga tgctaaagca tatgagagag aagtgcataa tgtctgggct 120 acacatgcct gtgtacccac agaccccaac ccacaagaaa tagttttggg aaatgtaaca 180 gaaaatttta acatgtggaa aaatgacatg gtggatcaga tgcatgagga tataatcagg 240 ttatgggatc aaagcttaaa gccatgtgta aagttgaccc cactctgtgt cactttagaa 300 tgtggaaatg ttaatgttac ccatgagaat agcacgaagg gggaaatgaa aaattgctct 360 ttcaatgcaa ccacagaact aaaagataaa aaacagagag tgtatgcact tttttataaa 420 cttgatatag taccacttaa tgagaataac aactctagtg aggactctag tgagtataga 480 ttaataaatt gtaatacctc agccataaca caagcctgtc caaaggtcac tttggatcca 540 attcctatac attattgtgc tccagctgga tatgcgattc taaagtgtaa taataagaca 600 ttcaatggga caggaccatg ccataatgtc agcacagtac aatgtacaca cggaatcaag 660 ccagtggtat caactcaact actgttaaat ggtagcctag cagaagaaga gataataatt 720 IE: 3 0 3 4 3 aggtctgaaa atctaacaaa caatgccaaa acaataatag tacatcttaa tgaatctgta 780 gaaattgtgt gtacaagacc cagcaataat acaagaaaaa gtataaggat aggaccagga 840 caagcattct atgcaacagg tggcataata ggaaacataa gacaagcaca ttgtaacatt 900 agtaaagaga actggaataa aactttacaa aaggtaggaa aaaaattagc agagcacttc 960 cctaataaaa caataaaatt tgaccaacac tcaggagggg acctagaaat tacaacacat 1020 agctttaatt gtagaggaga atttttctat tgcaatacat caaacctgtt taatagtaca 1080 tataagccta atgatacaaa tagtacatat aatcctaatg atacaatcac actcccatgc 1140 agaataaaac aaattataaa catgtggcag ggggtaggac aagcaatgta tgcccctccc 1200 attgcaggaa acataacatg taaatcaaat atcacaggac tactattgac acgggatgga 1260 gggtcaaatg ataccacaaa cacagagaca ttcagacctg gaggaggaga tatgagggac 1320 aattggagaa gtgaactata taaatataaa gtggtagaaa ttaaaccatt gggcatagca 1380 cccactgcgg caaaaaggag agtggtggag acgagataa 1419 r<

Claims

1. An immunogenic composition comprising a gp120 related polypeptide and an adjuvant, wherein the adjuvant comprises a lipopolysaccharide and an immunologically active 5 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. 10

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 10-40ug of an immunologically active saponin fraction derived from the bark of Quillaja Saponaria Molina presented in the form of a liposome.

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: 20 (i) the conductivity ofthe 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.

4. The immunogenic composition according to either claim 2 or 3, which is substantially 25 free of a NefTat related polypeptide wherein the NefTat related polypeptide is a polypeptide consisting of SEQ ID NO: 4.

5. The immunogenic composition according to any one of claims 1, 3 or 4 wherein the lipopolysaccharide is present at a level of between 10-100ug.

6. The immunogenic composition according to any one of claims 1 or 3 to 5 wherein the saponin is present at a level of between 10-100ug.

7. The immunogenic composition according to any one of claims 1 to 6 wherein the 35 lipopolysaccharide is 3D-MPL. Έί 3 0 3 4 3 VB65568PCT

8. The immunogenic composition according to any one of claims 1 to 7 wherein the saponin is QS-21.

9. The immunogenic composition according to any one of claims 1, 2 or 4 to 8 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 iower.

10. The immunogenic composition according to any one of claims 1 to 9 which is an aqueous solution.

11. The immunogenic composition according to any one of claims 1 to 10 which is a single human dose.

12. The immunogenic composition according to any one of claims 1 to 11 wherein the human dose is between 0.1 and 1ml.

13. The immunogenic composition according to any one of ciaims 1 to 12, comprising a non-ionic tonicity agent.

14. The immunogenic composition according to any one of claims 1 to 13 wherein the composition comprises the gp120 related polypeptide at a level of around 1 to 100 ug.

15. The immunogenic composition according to any one of claims 1 to 14 wherein the composition comprises 1-5 additional HIV antigens.

16. The composition according to any one of claims 1 to 15 wherein the gp12O related polypeptide comprises a polypeptide with at least 70% identity with the V1V2 region of SEQ ID NO: 1.

17. The composition according to any one of claims 1 to 16 wherein the gp120 related polypeptide comprises a polypeptide with at least 70% identity with SEQ ID NO: 1... \

18. The composition according to any one of ciaims 1 to 15 wherein the gp120 related polypeptide comprises a polypeptide with at least 70% identity with the V1V2 region of SEQ ID NO: 5. VB655S8PCT IEl 3 0 3 4 3

19. The composition according to any one of claims 1 to 16 wherein the gp120 related polypeptide comprises a polypeptide with at least 70% identity with SEQ ID NO: 5.

20. The immunogenic composition according to any one of claims 1 to 19 wherein the composition further comprises a sterol, wherein the ratio of saponin:sterol is from 1:1 to 1:100 w/w.

21. The immunogenic composition according to any one of claims 1 to 20, for use in the treatment or prevention of HIV-1 from group Μ, N, O or P.

22. The immunogenic composition according to any one of claims 1 to 20, for use in the treatment or prevention of HIV-1 group M subtype A, B, C, D, E, F, G, Η, I, J or K.

23. The immunogenic composition according to any one of claims 1 to 22, 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.

24. The immunogenic composition according to any one of claims 1 to 23 for use in eliciting antibodies against the V1V2 loop of HIV-1 gp120.

25. The immunogenic composition according to any one of claims 1 to 23 for use in the treatment or prophylaxis of HIV infection wherein a polynucleotide encoding a gp120 related polypeptide is administered to the individual and subsequently the composition according to any one of claims 1 to 24 is administered to the individual.

26. The immunogenic composition according to any one of claims 1 to 23 for use in the treatment or prophylaxis of HIV infection wherein the composition according to any one of claims 1 to 23 is administered to the individual and subsequently a polynucleotide encoding a gp120 related polypeptide is administered to the individual.

27. A method of treatment or prophylaxis of HIV-1 infection comprising the step of administering a composition according to any one of claims 1 to 26 to an individual. VB65568PCT IEH 0 3 4 3

28. A method of reducing the risk of H(V transmission from an HIV-infected individual to a partner of said HiV-infected individual comprising the step of administering the immunogenic composition of any one of claims 1 to 26 to the HIV-infected individual. 5

29. A method of making the composition according to any one of claims 1 to 26, 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. 10

30. A kit for making an immunogenic composition according to any one of claims 1 to 26, 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.

31. A viral vector comprising a polynucleotide encoding a polypeptide comprising the gp120weiD polypeptide of SEQ ID No: 1.

32. A viral vector comprising a polynucleotide encoding a polypeptide comprising the 20 gp120zMi8 polypeptide of SEQ ID NO: 5.