BE1021315B1 - NEW COMPOSITIONS - Google Patents

Abstract

The invention relates to a particular immunogenic composition comprising a gp120-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 10 and 40 µg of a lipopolysaccharide and between 10 and 40 µg of an immunologically active saponin derived from the bark Quillaja saponaria Molina presented in the form of a liposome or have a sodium chloride concentration of 130 mµ or less.

Description

NEW COMPOSITIONS

The present invention relates to particular immunogenic compositions comprising a gp120-related polypeptide and adjuvant, wherein the adjuvant comprises a saponin and a lipopolysaccharide. Methods for preparing such immunogenic compositions and related kits are also provided.

HIV is the leading cause of Acquired Immunodeficiency Syndrome (AIDS), which is considered 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 more than 4 million new infections occur each year. HIV has claimed more than 25 million lives in the past three decades. Although antiretroviral therapy prolongs the lives of many HIV-infected people, effective antiretroviral therapy requires strict compliance with often complex drug regimens and does not cure the infection. New infections far outnumber the number of people who can be treated with current global financial efforts. There is a need for a vaccine to prevent new infections, but the development of a healthy and effective HIV vaccine is a major challenge.

Two types of HIV have been characterized: HIV-1 and HIV-2. HIV-1 is highly virulent and infectious and is globally the cause of the majority of HIV infections, while HIV-2 has lower virulence and infectivity and is largely confined to West Africa (Gilbert et al., Stat In Med 22 (4): 573-593 (2003) and Reeves and Doms J Gen Vir 83: 1253-1265 (2002)). There are many genetically distinct subtypes (also known as "clades") of HIV-1, and the fair amino acid sequences of envelope glycoproteins (gp120 and gp41) can vary from 25 to 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 to the development of an HIV-1 vaccine.

Although extensive research has been conducted around the world to produce a vaccine, there is still much work to be done.

The gp! 20 envelope protein of HIV and other HIV-1 proteins

Gp120 is the viral protein that is used for attachment to a host cell. This attachment is mediated by gp120 binding surface molecules of helper T cells and macrophages including one of the two CCR-5 or CXCR-4 chemokine receptors. The gp120 protein is first expressed as a larger precursor molecule (gp160), which is then post-translationally cleaved to give gp120 and gp41. The gp120 protein is retained on the surface of the virion, non-covalently associated with the gp41 molecule, which is inserted into the viral membrane. . Three non-covalently associated glycoprotein envelope glycoproteins gp120 and gp41 form the trimeric trimellate on the surface of HIV-1.

Non-enveloped HIV-1 proteins have been described and include, for example, internal structural proteins such as other Env gene products (such as gp160 and gp41), gag and pol gene products (such as MA, CA). , SP1, NC, SP2 and P6, and RT, RNase H, IN and PR, respectively) and other non-structural proteins such as Rev, Nef, Vif, Vpr, Vpu and Tat (Greene et al., New Eng J Med, 324, 5, 308 and seq (1991) and Bryant et al (Ed Pizzo), Pediatr Infect Dis J, 11, 5, 390 and seq (1992)).

Previous work in the field

Gp120 was among the first targets of HIV vaccine research and has been considered 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 (CTLs). These effector cells are capable of eliminating virus-infected cells, and thus constitute an antiviral immune mechanism. Some CTL epitopes appear to be relatively conserved among the different strains of HIV. Nevertheless, current CTL vaccines do not protect against infection in animal models of HIV (Amara et al., Science 292 (5514): 69-74 (2001)). In addition, the CTL-based vaccines studied to date in humans have not induced these sought-after responses in all recipients (Goepfert et al., J Infect Dis 192 (7): 1249-1259 (2005) ). Such CTL vaccines could not protect against infection or impact post-infection viral load ("HVTN-505 study").

The gp120 protein is a major target of the neutralizing antibodies (Pantophlet et al., Annu Rev Imiaunol 24: 739-769 (2006)) and it has been previously thought that the neutralizing antibodies constitute a potential protective correlate Bruck et al. Vaccine 12 (12): 1141-1148 (1994) and Plotkin, Pediatr Infect Dis J 20: 63-75 (2001)).

One region of the gp120 protein in particular, the "V3 loop," is targeted by the neutralizing antibodies. Antibodies present in immune sera from infected individuals bind to peptides of the V3 loop (Spenlehauer et al., J. Vir., 72 (12): 98559864 (1998)). As a result, much of the research focused on the V3 loop of the gp120 protein. Nevertheless, the V3 loop is unfortunately highly variable and highly specific to the strain (Vaine et al., PLoS One, 5 (11): el3916, (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 does not always induce neutralizing antibodies against HIV and HIV, and when neutralizing 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)).

The use of NefTat recombinant HIV-1, gp120W6iD and Nef-Vis proteins formulated with the AS02A adjuvant system (50 μg of QS-21 and 50 μg of 3D-MPL in an oil-in-oil emulsion) has been shown to be effective. water) protected against AIDS in an animal model system of rhesus macaque HIVS when animals were challenged with HIV / SIV. However, gpl20W61D of HIV-1 formulated alone with AS02A did not confer protection. It was suggested that the lack of protection after immunization with gp120W6iD formulated alone with AS02A was related to the heterologous nature of the challenge virus (20.2% sequence difference for gp120), since it had previously been shown that gp120WeiD formulated in AS02A induces sterile immunity against a challenge to homologous HIVSW61D (Voss et al., J Virol 77 (2): 1049-1058 (2003) and Mooij et al., AIDS 12: F15-F22 (1998)).

NefTat and gpl2 0w6iD formulated in AS02A were administered to HIV-negative individuals in a safety and immunogenicity study. However, the neutralizing antibodies elicited by this vaccine had a poor inter-subtype activity (Leroux-Roels et al., Vaccine 28: 7016-7024 (2010) - "PRO HIV-002"). In addition, gp120 had a negative impact on antibody and T cell responses (geometric mean antibody titre and lymphocyte proliferation) compared with NefTat and gp120 against NefTat alone (Goepfert et al. , Vaccine 25: 510-518 (2007) -the study "HVTN-041"). Therefore, it was found that (i) a vaccine comprising gp120 with adjuvant comprising QS-21 and 3D-MPL had poor inter-subtype reactivity and (ii) it appears that gp120 could have an impact. detrimental to previously accepted potential markers of the efficacy of HIV vaccines containing other antigens. In light of the results illustrated above, it has been thought that 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 of limited use for a wide-ranging protection vaccine and, as a result, interest in this protein has been eroded. The HIV-1 RV144 vaccine trial was the first to demonstrate evidence of protection against HIV-1 infection, with an estimated vaccine efficacy of 31.2% (Rerks-Ngarm et al., N Engl J Med 361: 2209-220 (2009). The protocol consisted of four initial injections of ALVAC-HIV (vCP1521), and two booster injections of AIDSVAX B / E. ALVAC-HIV were 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 vector of canine pox virus containing gp120 from HIV-1 subtype E1 (CRF01_AE) strain 92TH023. , linked to the transmembrane anchor portion of gp41 (deleted in the immunodominant region) of the HIV-1 strain LAI subtype B. The vector also contained the gag and pol genes of HIV-1. AIDSVAX B / E is a recombinant HIV-1 subtype B gp120 MN preparation, CM244 gcl20 A244 subtype E, and alum adjuvant.

In order to identify correlates of risk of HIV-1 infection in RV144, plasma specimens from RV144 study participants were analyzed (Haynes et al., N Engl J Med 366: 1275-12266 (2012)). )). Assays were performed on samples obtained two weeks after final immunization from 41 vaccinates who became infected and 205 vaccinated uninfected. These assays examined the roles of T cell responses, IgG antibodies, and IgA antibodies in modulating the risk of infection. Specific antibody levels to gp70-VlV2 (a scaffolded protein carrying the first and second variable regions of gp120 of HIV-1 fused to murine leukemia virus gp70 - see Pinter et al., Vaccine 16 (19)) have been found. : 1803-1811 (1998)) were associated with a lower risk of infection and that plasma IgA binding to envelope proteins correlated with a higher risk of infection. Moreover, since this analysis was performed, it was found that V2 antibodies induced in the RV144 assay cross-reacted with multiple HIV-1 subtypes (Zolla-Pazner et al., PlosOne, 8 ( 1): e53629 (2013)). Therefore, for the above reasons, it has been concluded that vaccines that induce higher levels of V1V2 'antibodies may have improved efficacy against HIV-1 infection.

To produce an HIV vaccine, it is therefore highly desirable to identify a composition capable of eliciting a high level of antibodies specific for the V1V2 region of gp120.

In summary, previous work in the field has demonstrated that: • gp120 in adjuvant AS02A (oil-in-water emulsion of QS-21 and 3D-MPL) did not confer protection against a challenge to a heterologous virus in a primate model, • gp120 could have a detrimental impact on markers of vaccine efficacy, and • antibodies against the V1V2 region of gp120 were a protective correlate against HIV-1.

Immunogenic compositions of the present invention may have one or more of the following advantages over prior art compositions: (i) achieve a stronger humoral immune response, e.g., higher serum antibody titer binding to the V1V2 region of gp120, (ii) achieve a stronger cellular immune response, e.g., proliferation and release of cytokines by polyfunctional T cells, (iii) achieve a broader humoral immune response, eg, achieve an antibody titre of binding to the V1V2 region of gp120 in a greater proportion of vaccinated, (iv) achieve a broader cellular immune response, eg, proliferation and release of cytokines by polyfunctional T cells in a greater proportion of vaccinated, (v) achieve a stronger humoral immune response against a particular subtype of HIV-1, (vi) achieve a higher cellular immune response against a particular subtype of HIV-1, (vii) require a smaller number of components, (viii) require non-living components, (vii) involve a simpler dosing regimen, (x) be simpler to be produced, (xi) to be more readily stored, (xii) to have utility in the treatment or prevention of HIV-1 infection, (xiii) to have utility in the treatment or prevention of infection with HIV-1, HIV-1 by a first subtype of HIV-1 when the gp120-related polypeptide of the composition is derived from a second subtype of HIV-1, (xiv) achieve a more durable immune response, for example based on the magnitude of response rate and / or responder, (xv) achieve greater reduction in viral load, (xvi) induce a higher level of protection against infection.

The present inventors administered compositions containing either (a) gp120W6iD and AS01B or (b) gp120W6iD # AS01B and NefTat to mice and analyzed the serological response of the mice.

The present inventors have surprisingly found that mice given gp120w6iD and AS01B (without NefTat) had a higher average level of anti-VlV2 antibodies in their serum than mice administered gp120W6iD / AS01B and NefTat.

The claimed immunogenic compositions could be expected to elicit a high level of anti-VlV2 antibodies, and have utility in the prophylaxis of HIV infection.

The present invention provides an immunogenic composition which is in the form of a human dose comprising a gp120-related polypeptide and adjuvant, wherein the adjuvant comprises between 10 and 40 μg of a lipopolysaccharide and between 10 and 40 μg. μg of an immunologically active saponin fraction derived from the bark of Quillaja saponaria Molina presented as a liposome.

There is also provided an immunogenic composition comprising a gp120-related polypeptide and adjuvant, wherein the adjuvant comprises a lipopolysaccharide and an immunologically active saponin fraction derived from Quillaja saponaria Molina bark presented as a liposome wherein: (i) the conductivity of the composition is 13 mS / cm or less; and / or (ii) the salt concentration in said composition is 130 mM or less, and / or (iii) the sodium chloride concentration in said composition is 130 mM or less.

There is further provided an immunogenic composition comprising a gp120-related polypeptide and adjuvant, wherein the adjuvant comprises a lipopolysaccharide and an immunologically active saponin fraction derived from the bark of

Quillaja saponaria Molina presented as 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.

Figure 1: Comparison of mouse anti-VlV2 serology.

Description of the sequences

SEQ ID NO: 1 gpl2 0w6iD SEQ ID NO: 2 Nef SEQ ID NO: 3 Tat SEQ ID NO: 4 NefTat SEQ ID NO: 5 gpl2 0ZMi8 SEQ ID NO: 6 polynucleotide sequence encoding gpl2 0zMi8 SEQ ID NO: 7 gpl2 0ZMi8 native SEQ ID NO: 8 polynucleotide sequence encoding native gp120 zMi8.

polypeptides

As used herein, the term "a gp120-related polypeptide" refers to a polypeptide comprising the V1V2 region of SEQ ID NO: 1 or an immunogenic derivative or fragment of the V1V2 region of SEQ ID NO: 1, or a polypeptide comprising the V1V2 region of SEQ ID NO: 5 or an immunogenic derivative or fragment of the V1V2 region of SEQ ID NO: 5. Adequately, the gp120 related polypeptide refers to a V1V2 region polypeptide of SEQ ID NO: 1 or an immunogenic derivative or fragment of the V1V2 region of SEQ ID NO: 1, or a polypeptide consisting of the V1V2 region of SEQ ID NO: 5 or an immunogenic derivative or fragment of the V1V2 region of SEQ ID NO: 5 .

The VIV2 region of a gp120-related polypeptide is the stretching of residues defined by two particular cysteine residues that form a disulfide 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 gp120w6iD polypeptide (SEQ ID NO: 1) is the stretching of residues from residue 90 to residue 184. Those skilled in the art will appreciate that the position of these residues cysteine therefore the position of the V1V2 region in a polypeptide related to the given gp120 may vary. This is illustrated by another gp120-related polypeptide, gp120ZMi8 (SEQ ID NO: 5) in which the V1V2 region is the stretching of residues from residues 90 to 172.

Suitably, the term "a gp120-related polypeptide" refers to a polypeptide comprising SEQ ID NO: 1 or an immunogenic derivative or fragment of SEQ ID NO: 1, or a polypeptide comprising SEQ ID NO: 5 or a derivative or immunogenic fragment of SEQ ID NO: 5. Adequately, the gp120-related polypeptide refers to a polypeptide consisting of SEQ ID NO: 1 or an immunogenic derivative or fragment of SEQ ID NO: 1, or a polypeptide consisting of SEQ ID NO Or an immunogenic derivative or fragment of SEQ ID NO: 5.

As used herein, the term "derivative" refers to a polypeptide that is modified with respect to the reference sequence. The immunogenic derivatives are sufficiently similar to the reference sequence to remain able to elicit an immune response against the V1V2 region of the reference sequence. Adequately, the immunogenic derivatives are sufficiently similar to the reference sequence to retain other key immunogenic properties of the reference sequence such as avoiding the introduction of immunodominant epitopes. A derivative may, for example, comprise a modified version of the reference sequence or, alternatively, may consist of a modified version of the reference sequence.

Suitably, the gp120-related polypeptide comprises or consists of a gp120 polypeptide derived from HIV-1 or HIV-2, suitably M, N, O or P groups of HIV-1, appropriately from subtype A, B, M, D, E, F, G, H, I, J or K of the M group. Suitably, the gp120 related polypeptide comprises or consists of a polypeptide of gp120 derived from HIV-1, suitably from the M group, more suitably For example, the gp120 related polypeptide comprises or consists of the gp120 polypeptide from HIV-1 or HIV-2, suitably HIV-1 from the M, N, O, or P group. , suitably of a group M HIV-1 subtype A, B, C, D, E, F, G, H, I, J, or K. Adequately, the polypeptide related to. gp120 comprises or consists of the gp120 polypeptide from HIV-1, conveniently from an HIV-1 M group, more appropriately from a M subtype B group of HIV-1. Those skilled in the art will recognize that individual substitutions, deletions, or additions to the gp120-related polypeptide that modify, add, or delete a single amino acid or a small percentage of amino acids is an "immunogenic derivative" where the modification (s) lead to the substitution / deletion / addition of residues which do not substantially affect the immunogenic function. "Not substantially affecting immunogenic function" means at least 50%, suitably at least 75% and suitably at least 90% of the activity of the reference sequence in an antibody level assay. binding to the V1V2 region of gp120 (e.g., ELISA) produced at a given time after immunization with the gp120-related polypeptide.

Conservative substitution tables providing functionally similar amino acids are well known in the art. In general, such conservative substitutions fall into one of the amino acid groups specified below, although under certain circumstances other substitutions may be possible without substantially affecting the immunogenic properties of the antigen. The following eight groups each contain amino acids which are typically conservative substitutions for each other: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine (C), methionine (M) (see, e.g., Creighton, Proteins 1984).

Adequately, such substitutions do not occur in the region of an epitope, and therefore do not have a significant impact on the immunogenic properties of the antigen. Suitably, an immunogenic derivative will contain substitutions of up to 20 residues (e.g., up to 5 residues) relative to the reference sequence.

The immunogenic derivatives may also include those in which the additional amino acids are inserted relative to the reference sequence. Adequately, such insertions do not occur in the region of an epitope, and therefore do not have a significant impact on the immunogenic properties of the antigen. Suitably, an immunogenic derivative will contain additions of up to 5 residues (e.g. 1 or 2 residues) at 0 to 5 locations (e.g., 0 to 2 locations) relative to the reference sequence. An example of insertion includes a short stretch of histidine residues (e.g., 6 residues) to assist in the purification of the antigen in question.

Immunogenic derivatives include those in which the amino acids have been deleted with respect to the reference sequence. Adequately, such deletions do not occur in the region of an epitope, and therefore do not have a significant impact on the immunogenic properties of the antigen. Suitably, an immunogenic derivative will contain deletions of up to 5 residues (e.g. 1 or 2 residues) at 0 to 5 locations (e.g., 0 to 2 locations) relative to the reference sequence.

Suitably, the gp120-related polypeptide will comprise, as will be, 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 (eg 1 or 2 residues) in 0 to 5 locations (eg 0 to 2 locations), insertions up to 5 residues (eg 1 or 2 residues) at 0 to 5 locations (eg 0 to 2 locations) and substitutions of up to 20 residues (eg up to 5 residues).

Alternatively, the gp120-related polypeptide will comprise, as will be, 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 region. V1V2 of SEQ ID NO: 5 having deletions of up to 5 residues (e.g. 1 or 2 residues) at 0 to 5 locations (e.g. 0 to 2 locations), insertions of up to 5 residues (e.g. or 2 residues) at 0 to 5 locations (e.g., 0 to 2 locations) and substitutions of up to 20 residues (e.g., up to 5 residues).

Suitably, the gp120-related polypeptide will comprise, as will be, 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 in 0 to 5 locations, insertions of up to 5 residues in 0 to 5 locations and substitutions of up to 20 residues.

Alternatively, the gp120-related polypeptide will comprise, as will be, 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 in 0 to 5 locations, insertions of up to 5 residues in 0 to 5 locations and substitutions of up to 20 residues.

Suitably, the gp120-related polypeptide comprises a polypeptide with at least 70% identity, more suitably at least 80% identity, more suitably at least 85% identity, more suitably at least 90% identity, more suitably at least 95% identity, more suitably at least 98% identity, more suitably at least 99% identity with the V1V2 region of SEQ ID NO: 1. Adequately, the gp120-related polypeptide comprises the V1V2 region of SEQ ID NO: 1.

Suitably, the gp120-related polypeptide is a polypeptide with at least 70% identity, suitably at least 80% identity, more suitably at least 85% identity, more suitably at least 90% identity. , more suitably at least 95% identity, more suitably at least 98% identity, more suitably at least 99% identity with the V1V2 region of SEQ ID NO: 1. Adequately, the gp120-related polypeptide consists of in the V1V2 region of SEQ ID NO: 1.

Suitably, the gp120-related polypeptide comprises a polypeptide with at least 70% identity, more suitably at least 80% identity, more suitably at least 85% identity, more suitably at least 90% identity, more suitably at least 95% identity, more suitably at least 98% identity, more suitably at least 99% identity with the V1V2 region of SEQ ID NO: 5. Adequately, the gp120-related polypeptide comprises the VIV2 region of SEQ ID NO: 5.

Suitably, the gp120-related polypeptide is 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. Adequately, the gp120-related polypeptide consists of the V1V2 region of SEQ ID NO: 5.

Suitably, the gp120-related polypeptide comprises a polypeptide with at least 70% identity, more suitably at least 80% identity, more suitably at least 85% identity, more suitably at least 90% identity, more suitably at least 95% identity, more suitably at least 98% identity, more suitably at least 99% identity with SEQ ID NO: 1. Adequately, the gp120 related polypeptide comprises SEQ ID NO: 1 .

Suitably, the gp120-related polypeptide is a polypeptide with at least 70% identity, suitably at least 80% identity, more suitably at least 85% identity, more suitably at least 90% identity, more suitably at least 95% identity, more suitably at least 98% identity, more suitably at least 99% identity with SEQ ID NO: 1. Suitably, the gp120 related polypeptide consists of SEQ ID NO: 1.

Alternatively, the gp120-related polypeptide comprises a polypeptide with at least 70% identity, more suitably at least 80% identity, more suitably at least 85% identity, more suitably at least 90% identity. , more suitably at least 95% identity, more suitably at least 98% identity, more suitably at least 99% identity with SEQ ID NO: 5. Adequately, the gp120-related polypeptide comprises SEQ ID NO: 5.

Suitably, the gp120-related polypeptide is a polypeptide with at least 70% identity, suitably at least 80% identity, more suitably at least 85% identity, more suitably at least 90% identity. , more suitably at least 95% identity, more suitably at least 98% identity, more suitably at least 99% identity with SEQ ID NO: 5. Adequately, the gp120-related polypeptide is SEQ ID NO : 5.

The terms "identical" or "% identity", in the context of two or more polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues which are the same on a specified region, when compared and aligned for maximum match on a comparison window, or a 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 complement 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 made on a window corresponding to the entire length of the reference sequence (as opposed to the derived sequence).

For sequence comparison, a 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 may be used, or alternate parameters may be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequences, based on the program parameters.

A "comparison window" as used herein refers to a segment in which a sequence can be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Sequence alignment methods for comparison are well known in the art. An optimal sequence alignment for comparison can be conducted, for example, by the Smith & local homology algorithm. Waterman, Adv. Appl. Math. 2: 482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48: 443 (1970), by the similarity search method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA and TFASTA in the software package Wisconsin Genetics, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment or visual inspection (see, for example, Curr Pro Mol Biol (Ausubel et al., 1995 supplement)).

An example of an appropriate algorithm for determining sequence identity in percent and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nue. Acids Res. 25: 3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215: 403-410 (1990), respectively. BLAST analysis software is publicly available from the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov/). This algorithm first involves the identification of pairs of high-score sequences (PSH) by identifying short words of length W in the interrogation sequence, which are consistent with, or satisfy a threshold value T of positive value. when they are aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word side threshold (Altschul et al., Supra). These occurrences of early neighborhood words act as seeds for initiating research to find longer HHPs containing them. The word occurrences are extended in both directions along each sequence as long 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 concordant residues, always> 0) and N (penalty score for mismatched residues, always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. An extension of the occurrences of words in each direction is stopped when: the cumulative alignment score falls by the amount X relative to its maximum value reached; the cumulative rating goes to zero or less due to the accumulation of one or more negative residue alignment alignments; or the end of one or the other of the sequences is reached. The parameters W, T and X of the BLAST algorithm determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defects a word length (W) of 11, a wait (E) of 10, M = 5, N = -4 and a comparison of the two strands. For amino acid sequences, the BLASTP program uses as defects a word length of 3, a wait (E) of 10, and the scoring matrix BLOSUM62 (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, for example, Karlin & Altschul, Proc Nat'l, Acad Sci USA 90: 5873-5787 (1993)). A 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 to be similar to a reference sequence if the lowest probability of sum in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more suitably lower. at 0.01, and most suitably less than about 0.001.

An "immunogenic fragment" will contain a contiguous sequence of amino acids from the polypeptide related to gp120, 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 "remains capable of eliciting an immune response against the V1V2 region of the reference sequence" means at least 50%, suitably at least 75% and suitably at least 90% of the sequence activity in the assay the level of antibodies binding to the V1V2 region of gp120 (e.g., ELISA) produced at a given time after immunization with the gp120-related polypeptide.

The gp120-related polypeptide may, for example, contain 2,000 or fewer amino acid residues, such as 1,500 amino acid residues or less, particularly 1,000 amino acid residues or less, especially 800 amino acid residues or less. amino acids or less.

Fusion proteins (also known as chimeric proteins) are proteins created by the covalent binding of two or more polypeptide sequences that are not joined in nature, such as by a peptide bond. For example, the gp120-related polypeptide may be provided as a fusion protein also comprising a second HIV antigen.

The dose of a gp120-related polypeptide is adequately capable of producing an adequate immune response in a human while having an acceptable reactogenicity profile.

Suitably, the immunogenic composition comprises about 1 to 200 μg of gp120-related polypeptide, suitably between 1 and 100 μg, suitably between 2 and 50 μg, such as between 3 and 30 μg, particularly between 5 and 15 μg. or between 16 and 25 μg, between 9 and 11 μg or between 19 and 21 μg, most suitably 10 μg or 20 μg.

Suitably, the immunogenic composition is provided in a volume suitable for administration to a human as a single dose. The volume that can be administered to a human depends on the method, route and / or location of the administration. In one embodiment, the human dose is between 0.1 and 1 mL, more suitably between 0.3 and 0.75 mL, such as between 0.45 and 0.55 mL, particularly 0.5 mL. Volumes between 0.1 and 1 mL are more particularly suitable for administration by routes such as subcutaneous injection and in particular intramuscular injection. In another embodiment, the human dose is between 0.05 and 0.2 mL, suitably between 0.075 and 0.15 mL, particularly 0.1 mL. Volumes between 0.05 and 0.2 mL are particularly suitable for administration via routes in which a limited volume may be administered, such as intradermal delivery.

A gp120 polypeptide can 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 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 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 an immunogenic derivative or fragment thereof.

Suitably, the Nef-related polypeptide will comprise, as will be, an immunogenic 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 to 5 locations, insertions of up to 5 residues at 0 to 5 locations and substitutions of up to 20 residues.

Suitably, the Tat-related polypeptide will comprise, as will be, 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 to 5 locations, insertions of up to 5 residues at 0 to 5 locations and substitutions of up to 20 residues.

Suitably, the NefTat-related polypeptide will comprise, as will be, 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 to 5 locations, insertions of up to 5 residues at 0 to 5 locations, and substitutions of up to 20 residues.

Suitably, the Nef-related polypeptide comprises SEQ ID NO: 2. Adequately, the Nef-related polypeptide comprises a polypeptide with at least 99% identity, suitably at least 98% identity, more suitably at least 95% identity. identity, more than 90% identity, more than 85% identity, more than 80% identity, more than 75% identity, more than 70% identity 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. identity, more than 90% identity, more than 85% identity, more than 80% identity, more than 75% identity, more than 70% identity identity with SEQ ID NO: 3.

Suitably, the NefTat-related polypeptide comprises SEQ ID NO: 4. Adequately, the NefTat-related polypeptide comprises a polypeptide with at least 99% identity, suitably at least 98% identity, more suitably at least 95% identity. identity, more than 90% identity, more than 85% identity, more than 80% identity, more than 75% identity, more than 70% identity identity with SEQ ID NO: 4.

Suitably, the Nef-related polypeptide consists of SEQ ID NO: 2. Adequately, the Nef-related polypeptide consists of a polypeptide with at least 99% identity, suitably at least 98% identity, more suitably at least 95% identity. identity, more than 90% identity, more than 85% identity, more than 80% identity, more than 75% identity, more than 70% identity ID with SEQ ID NO: 2.

Suitably, the Tat-related polypeptide is SEQ ID NO: 3. Suitably, the Tat-related polypeptide consists of a polypeptide with at least 99% identity, adequately at least 98% identity, more suitably at least 95% identity. identity, more than 90% identity, more than 85% identity, more than 80% identity, more than 75% identity, more than 70% identity ID with SEQ ID NO: 3.

Suitably, the NefTat-related polypeptide consists of SEQ ID NO: 4. Adequately, the NefTat-related polypeptide consists of a polypeptide with at least 99% identity, adequately at least 98% identity, more suitably at least 95% identity. identity, more than 90% identity, more than 85% identity, more than 80% identity, more than 75% identity, more than 70% identity ID 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 N-terminal His residues (eg, a six-residue polyhistinine tag, which may be used for purification). by affinity to nickel).

Adequately, the compositions of the present invention are substantially free of a Nef-related polypeptide. Adequately, the compositions of the present invention contain a polypeptide ratio related to Nef: 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: 20, suitably less than 1: 500, more suitably less than 1: 1000 by weight.

Adequately, the compositions of the present invention contain a polypeptide related to Nef at a level of less than 5 μg, suitably less than 4 μg, suitably less than 3 μg, suitably less than 2 μg, suitably less than 1 μg. ig, adequately less than 0.5 ug, adequately less than 0.2 ug, adequately less than 0.1 ug, adequately less than 0.05 ug, more adequately less than 0.01 ug per dose human.

Most suitably, the compositions of the present invention are free of a Nef-related polypeptide.

Adequately, the compositions of the present invention are substantially free of a Tat-related polypeptide. Adequately, the compositions of the present invention contain a Tat-related polypeptide: gp120-related polypeptide ratio of less than 1:20, suitably less than 1:25, suitably less than 1:50, suitably less than 1: 10 0, suitably less than 1: 200, suitably less than 1: 50 0, more suitably less than 1: 1000 by weight.

Adequately, the compositions of the present invention contain a Tat-related polypeptide at a level of less than 5 μg, suitably less than 4 μg, suitably less than 3 μg, suitably less than 2 μg, suitably less than 1 μg. / ig, suitably less than 0.5 μg, adequately less than 0.2 μg, suitably less than 0.1 μg, adequately less than 0.05 μg, more appropriately less than 0, 01 / xg per human dose.

Most suitably, the compositions of the present invention lack a Tat-related polypeptide.

Adequately, the compositions of the present invention are substantially free of a NefTat-related polypeptide. Suitably, the compositions of the present invention contain a polypeptide related to NefTat: 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.

Adequately, the compositions of the present invention contain a NefTat-related polypeptide at a level of less than 5 μg, suitably less than 4 μg, suitably less than 3 μg, suitably less than 2 μg, suitably less than 1 mg, suitably less than 0.5 μg, suitably less than 0.2 μg, suitably less than 0.1 μg, suitably less than 0.05 μg, more suitably less than 0.01 μg per human dose.

Most suitably, the compositions of the present invention are free of a NefTat-related polypeptide.

The presence of additional HIV proteins may increase the effectiveness of the composition of the invention. Suitably, the composition of the invention may comprise additional proteins which may be additional products of the Env gene (such as gp160 and gp41 or an additional gp120 related polypeptide), gag and pol gene products (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 derivatives or immunogenic fragments thereof) .

The HIV gag gene encodes a p55 precursor protein, which can spontaneously assemble into immature virus-like particles (PSVs). The precursor is proteolytically cleaved into the major structural proteins CA (capsid) and MA (matrix), and into several smaller proteins. Both the p55 precursor protein and its major derivatives CA and MA can be considered as suitable vaccine antigens that can increase the effectiveness of the composition of the invention. The p55 precursor and the CA capsid protein can be used as PSVs or as monomeric proteins. Adequately, the composition of the present invention may comprise one or more of these proteins.

Suitably, the composition of the present invention may comprise a polynucleotide encoding a polypeptide described above including, for example, a gp120-related polypeptide. The polynucleotide may be in the form of plasmid DNA or in the form of a recombinant living vector.

In one embodiment, the composition of the present invention does not include additional HIV antigens. In a second embodiment of the invention, the composition comprises 1 to 5 additional HIV antigens, such as 1 or 2 additional HIV antigens. Additional HIV antigens can be provided in the form of proteins or polynucleotides encoding proteins.

Suitably, the immunogenic composition comprises a total of about 1 to 500 μg of antigenic material, suitably between 1 to 200 μg, such as between 5 to 100 μg, most suitably between 5 and 50 μg.

Adequately, the polypeptides and polynucleotides used in the present invention are isolated. An "isolated" polypeptide or polynucleotide is removed from its original environment. For example, a naturally occurring protein is isolated if it is separated from all or part of coexisting materials in the natural system. Preferably, such polypeptides are at least about pure. 9 0%, more suitably pure at about 99% and most suitably at least about 99% pure. A polynucleotide is considered isolated if, for example, it is cloned into a vector that is not part of its natural environment.

Saponines Adjuvant

The immunogenic composition of the invention comprises an immunologically active saponin moiety ("a saponin") as an adjuvant or component of an adjuvant. A saponin particularly suitable 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 has been described for the first time by Dalsgaard et al. in 1974 ("Saponin adjuvants", Archiv, für die gesamte Virusforschung, 44, Springer Verlag, Berlin, pp. 243-254) to have adjuvant activity. Purified fragments of Quil A were isolated by HPLC, which retain adjuvant activity without Quil A-associated toxicity (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 (CTL), Th1 cells and a predominant IgG2a antibody response and is a preferred saponin in the context of this invention. invention.

In a suitable form of the present invention, the saponin adjuvant within the immunogenic composition is a saponaria molina derivative which is suitably an immunologically active moiety of Quil A, such as QS-7 or QS-21, suitably QS- 21.

In one embodiment, the compositions of the invention contain the immunologically active saponin fraction in substantially pure form. Adequately, the compositions of the invention contain QS-21 in substantially pure form, i.e., QS-21 is at least 90% pure, for example at least 95% pure or at least 100% pure. 98%.

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 particulate forms of less reactogenic compositions in which the lytic activity of QS-21 is quenched with exogenous cholesterol exist. In a specific embodiment, the saponin / sterol is in the form of a liposome structure (US 6,846,489, Example 1). In this embodiment, the liposome suitably contains a neutral lipid, for example phosphatidylcholine, which is suitably non-crystalline at room temperature, for example egg yolk phosphatidylcholine, dioleoyl phosphatidylcholine (DOPC) or dilauryl phosphatidylcholine. The liposomes may also contain a charged lipid which increases the stability of the liposome-QS-21 structure for liposomes composed of saturated lipids. In these cases, the amount of lipid loaded is suitably from 1 to 20% w / w, suitably from 5 to 10%. The ratio sterol phospholipid is 1 to 50% (mol / mol), suitably 20 to 25%.

Suitable sterols include beta-sitosterol, stigmasterol, ergosterol, ergocalciferol and cholesterol. In a particular embodiment, the adjuvant composition comprises cholesterol as sterol. These sterols are well known in the art, for example the cholesterol disclosed in the Merck Index, 11th ed., Page 341, as a naturally occurring sterol found in animal fats.

The sterol according to the invention is taken to designate an exogenous sterol, that is to say a sterol which is not endogenous for the organism from which the antigenic preparation is taken but is added to the antigen preparation or subsequently at the time of formulation. Typically, the sterol may be added during further formulation of the antigen preparation with the saponin adjuvant, using, for example, saponin in its form in which its lytic activity is quenched with the sterol. Adequately, the exogenous sterol is associated with saponin adjuvant as described in US 6,846,489.

When the active saponin fraction is QS-21, the QS-21: sterol ratio will typically be in the range of 1: 100 to 1: 1 (w / w), suitably 1: 10 to 1: 1 (w / w). p), and suitably 1: 5 to 1: 1 (w / w) · Adequately, the excess sterol is present, the ratio QS-21: sterol being at least 1: 2 (w / w). In one embodiment, the QS-21: sterol ratio is 1: 5 (w / w). Other saponins that have been described in the literature include escin, which has been described in Merck Index (12th ed: entry 3737) as a mixture of saponins occurring in horse chestnut seed, Lat: Aesculus hippocastanum . Its isolation is described by chromatography and purification (Fiedler, Arzneimittel-Forsch, 4, 213 (1953)), and by ion exchange resins (Erbring et al., US 3,238,190). Escin fractions were purified and shown to be biologically active (Yoshikawa M. et al., Chem Pharm Bull (Tokyo) 1996 44 (8): 1454-1464)). Sapoalbine from Gypsophila struthium (R. Vochten et al., 1968, J Pharm Belg, 42, 213-226) has also been described in connection with the production of ISCOM, 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 and 100 μg.

In one embodiment, there is provided an immunogenic composition comprising QS-21 at a level of about 50 μg, for example between 38 and 100 μg, suitably between 40 and 75 μg or between 45 and 60 μg, more suitably 49 to 51, most suitably 50 ^ g.

In a further embodiment, there is provided an immunogenic composition comprising QS-21 at a level of about 25 μg, for example between 10 and 37 μg, suitably between 15 and 30 μg, or between 20 and 27 μg. ^ g, more suitably 24 to 26, more suitably 25 ^ g.

In another embodiment, there is provided an immunogenic composition in a volume that is suitable for a human dose, which human dose of the immunogenic composition comprises QS-21 at a level of about 50 μg, for example between 38 and 100 μg. suitably between 40 and 75 μg or between 45 and 60 μg, more suitably 49 to 51, most suitably 50 μg.

In another embodiment, there is provided an immunogenic composition in a volume that is suitable for a human dose, which human dose of the immunogenic composition comprises QS-21 at a level of about 25 μg, for example between 10 and 37 μg , suitably between 15 and 30 μg or between 20 and 27 μg, more suitably 24 to 26, more suitably 25 μg.

The dose of QS-21 is apt to enhance an immune response to an antigen in a human. In particular, a suitable amount of QS-21 is that which improves the immunological potential of the composition over the non-adjuvanted composition, or compared to the adjuvanted composition with another amount of QS-21, while being acceptable to a reactogenicity point of view.

Lipopolysaccharide adjuvants

Lipopolysaccharides (LPS) are the major surface molecule of, and occur exclusively in, the outer leaflet of the outer membrane of Gram-negative bacteria. LPS inhibit the killing of bacteria by serum supplements and phagocytic cells, and are involved in adhesion for colonization. LPS is a group of structurally related complex molecules of approximately 10,000 Daltons in size and consists of three covalently linked regions: (i) a 0-specific polysaccharide (O-antigen) chain at the outer region ( ii) a central region the core oligosaccharide (iii) lipid A - the innermost region which serves as a hydrophobic anchor, it comprises glucosamine disaccharide units which carry the long chain fatty acids.

It has been shown that the biological activities of LPS, such as lethal toxicity, pyrogenicity and the possibility of adjuvant use, are related to lipid fraction A. In contrast, immunogenicity is associated with the O-specific polysaccharide component (antigen). to 0). Both LPS and Lipid A have long been known for their strong adjuvant effects, but the high toxicity of these compounds has precluded their use in vaccine formulations. Significant efforts have therefore been made to reduce the toxicity of LPS or lipid A while maintaining their ability to serve as adjuvants.

The mutant R595 of Salmonella minnesota was isolated in 1966 from a culture of the parent (smooth) strain (Luderitz et al., 1966 Ann N Y Acad Sci 133: 349-374). The susceptibility to lysis of the selected colonies was screened by a panel of phages, and only those colonies which displayed a broad range of sensitivity (predisposition to one or two phages only) were selected for further study. This effort led to the isolation of a deep rough mutant strain that is deficient in LPS biosynthesis and designated S. minnesota R595.

In comparison to other LPS, those produced by the S. minnesota mutant R595 have a relatively simple structure. (i) they do not contain any O-specific region - a characteristic that is responsible for the passage of the wild-type smooth phenotype to the mutant rough phenotype and leads to a loss of virulence (ii) the core region is very short - this characteristic increases the sensitivity of the strain to a variety of chemicals (iii) the lipid A moiety is highly acylated with up to 7 fatty acids.

The 4'-monophosphoryl lipid A (MPL), which can be obtained by acid hydrolysis of LPS extracted from a deep-rooted mutant strain of Gram-negative bacteria, retains the adjuvant properties of LPS while demonstrating a toxicity that is reduced by a factor of more than 1,000 (as measured by the lethal dose in chicken embryo eggs) (Johnson et al., 1987 Rev Infect Dis 9 Suppl: S512 to S516). LPS is typically refluxed in mild strength mineral acid solutions (eg, 0.1M HCl) for a period of approximately 30 minutes. This process leads to dephosphorylation at position 1, and decarbohydration at the 6 'position, giving MPL.

3-O-deacylated monophosphoryl lipid A (3D-MPL), which can be obtained by mild alkaline hydrolysis of MPL, has a further reduced toxicity while still maintaining the possibility of being used as an adjunct, see US 4,912,094 ( Ribi Immunochemicals). Alkaline hydrolysis is typically carried out in an organic solvent, such as a chloroform / methanol mixture, by saturation with a weak aqueous base solution, such as 0.5 M sodium carbonate at pH 10.5. Additional information on the preparation of 3D-MPL is available, for example, in US 4,912,094 (Corixa Corporation).

The composition further comprises an additional adjuvant which is a lipopolysaccharide, suitably a nontoxic derivative of lipid A, in particular monophosphoryl lipid A or more particularly 3-deacylated monophosphoryl lipid A (3D-MPL).

3D-MPL is sold under the name MPL by GlaxoSmithKline Biologicals N.A. and is designated throughout the document by MPL or 3D-MPL. See, for example, US 4,436,727; U.S. 4,877,611; US 4,866,034 and US 4,912,094. 3D-MPL can be produced according to the methods disclosed in US 4,912,094. Chemically, it is a mixture of monophosphoryl lipid A 3-deacylated with 3,4 , 5 or 6 acylated chains. Adequately, in the compositions of the present invention, small particle 3D-MPL is used. The small particle 3D-MPL has a particle size such that it can be sterile filtered on a 0.25 μτη filter. Such preparations are described in US 5,776,468.

Adequately, 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 and 100 μg.

In one embodiment, there is provided an immunogenic composition comprising 3D-MPL at a level of about 50 μg, for example between 38 and 100 μg, suitably between 40 and 75 μg or between 45 and 60 μg, more suitably 49 to 51, most suitably 50 μg.

In a further embodiment, there is provided an immunogenic composition comprising 3D-MPL at a level of about 25 μg, for example between 10 and 37 μg, suitably between 15 and 30 μg or between 20 and 27 μg, more suitably 24. to 26, more suitably 25 μg.

In another embodiment, there is provided an immunogenic composition in a volume that is suitable for a human dose, which human dose of the immunogenic composition comprises 3D-MPL at a level of about 50 μg, for example between 38 and 100 μg, suitably between 40 and 75 μg or between 45 and 60 μg, more suitably 49 to 51, most suitably 50 μg.

In another embodiment, there is provided an immunogenic composition in a volume that is suitable for a human dose, which human dose of the immunogenic composition comprises 3D-MPL at a level of about 25 μg, for example between 10 and 37. μg, suitably between 15 and 30 μg or between 20 and 27 μg, more suitably 24 to 26, more suitably 25 μg.

Suitable compositions of the invention are those in which liposomes are initially prepared without MPL (as described in US 6,846,489), and MPL is then added, suitably as small particles of particles below 100 nm. or particles that are sensitive to sterile filtration on a 0.22 μιη membrane. MPL is therefore not contained in the vesicular membrane (known as MPL out). Compositions where MPL is contained within the vesicular membrane (known as MPL in) also form an aspect of the invention. The antigen may be contained in the vesicular membrane or contained outside the vesicular membrane. Adequately, soluble antigens are outside and hydrophobic or lipid antigens are contained either inside or outside the membrane. The invention comprises both a lipopolysaccharide and an immunologically active saponin. In a specific embodiment of the invention, the lipopolysaccharide is 3D-MPL and the immunologically active saponin is QS-21. In one embodiment of the invention, the composition comprises a lipopolysaccharide and an 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 that is properly cholesterol.

In a further embodiment of the invention, the adjuvant composition comprises in a liposomal formulation an immunologically active lipopolysaccharide and saponin in combination with one or more additional immunostimulants or adjuvants. Adequately, in one form of this embodiment, the lipopolysaccharide is 3D-MPL and the immunologically active saponin is QS-21.

In a specific embodiment, QS-21 and 3D-MPL are present in a weight ratio between 1: 2 and 2: 1. Adequately QS-21 and 3D-MPL are present in the 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 μg of 3D-MPL and 50 μg of QS-21. In another aspect, a human dose of immunogenic composition comprises a final level of 25 μg of 3D-MPL and 25 μg of QS-21. In a further embodiment, a human dose of immunogenic composition comprises a final level of 10 μg each of 3D-MPL and QS-21.

The vaccine preparation is generally described in New Trends and Developments in Vaccines, edited by Voiler et al., University Park Press, Baltimore, Maryland, USA 1978. Encapsulation within liposomes is described, for example, in US 4,235,877. The conjugation of proteins to macromolecules is disclosed, for example in US 4,372,945 and US 4,474,757.

Salt concentration

Some antigens are sensitive to the presence of salts. Without being a theory, it is believed that these antigens are adversely 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 sodium chloride concentration as low as 150 mM. Accordingly, the stability of immunogenic compositions comprising a gp120-related polypeptide can be improved by reducing the sodium chloride concentration.

The immunogenic compositions of the invention will suitably be aqueous preparations.

Accordingly, the present invention provides an immunogenic composition comprising a gp120-related polypeptide, wherein the conductivity of the composition is 13 mS / cm or less. In particular, the present invention provides immunogenic compositions in which the conductivity of the immunogenic composition is 12 mS / cm or less, for example 10 mS / cm or less, 8 mS / cm or less, 6 mS / cm or less, 5 mS / cm or less, 4 mS / cm or less or 3 mS / cm or less. In a particular embodiment, the conductivity of the immunogenic composition is 2.5 mS / cm or less, such as 2.25 mS / cm or less or 2.0 mS / cm or less. In a further specific embodiment, the conductivity of the immunogenic composition is 1.5 to 2.5 mS / cm.

In addition, there is provided an immunogenic composition comprising a gp120-related polypeptide, wherein the salt concentration in said composition is 130 mM or less. In particular, the present invention provides immunogenic compositions wherein the salt concentration in said composition is 100 mM or less, e.g. 90 mM or less, 80 mM or less, 70 mM or less, 60 mM or less, 50 mM or less, or 40 mM or less. In a particular embodiment, the salt concentration in said composition is 35 mM or less, such as 30 mM or less or 25 mM or less. In a further specific embodiment, the salt concentration 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 less. In particular, the present invention provides immunogenic compositions in which the concentration of sodium chloride is 100 mM or less, e.g. 90 mM or less, 80 mM or less, 70 mM or less, 60 mM or less, 50 mM or less, 40 mM or less, 30 mM or less, 20 mM or less, or 15 mM or less. In a particular embodiment, the concentration of sodium chloride in the immunogenic composition is 10 mM or less, such as 7.5 mM or less. Suitably, the concentration of sodium chloride in the immunogenic composition is 5 mM or below. In a further specific embodiment, the immunogenic composition is substantially free of sodium chloride. By essentially free, it means that the sodium chloride concentration is at or very near zero (such as 3 mM or less, 2 mM or less, or 1 mM or less).

Suitably, the concentration of CaCl 2 in the immunogenic compositions will be 40 mM or less, 30 mM or less, 20 mM or less, 15 mM or less or 10 mM or less.

Suitably, the concentration of MgSO4 in the immunogenic compositions will be 80 mM or less, 60 mM or less, 40 mM or less, 30 mM or less, 20 mM or less, or 10 mM or less.

Suitably, the total concentration of NH4 +, Mg2 + and Ca2 + ions in the immunogenic compositions will be 80mM or less, 60mM or less, 40mM or less, 30mM or less, 20mM or less, or 10mM or less. less.

It is well known that for parenteral administration the solutions must have a pharmaceutically acceptable osmolality to avoid cell distortion or lysis. Pharmacologically acceptable osmolality will generally mean that the solutions will have an osmolality that is approximately isotonic or slightly hypertonic. Suitably, the immunogenic compositions of the present invention will have an osmolality in the range of 250 to 750 mOsm / kg, for example, the osmolality may be in the range of 250 to 550 mOsm / kg, such as in the range of 280 to at 500 mOsm / kg. The osmolality can 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 proper tonicity to a formulation to prevent the net flow of water through cell membranes that are in contact with the formulation.

In a particular embodiment, there is provided immunogenic compositions further comprising a nonionic tonicity agent. A nonionic tonicity agent for use in an immunogenic composition will itself need to be pharmaceutically acceptable, for example suitable for use in humans, as well as compatible with the gp120-related antigen and further compatible with other components such as immunostimulant (s).

In one embodiment of the present invention, suitable nonionic tonicity agents are polyols, sugars (especially 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. Examples of sugar alcohols include glycerol, erythritol, threitol, arabitol, xylitol, ribitol, sorbitol, mannitol, dulcitol and iditol. In a specific example of this embodiment, a suitable nonionic tonicity agent is sorbitol. Those skilled in the art will recognize that appropriate osmolality can be achieved by the use of a mixture of different tonicity agents. In a particular embodiment of the invention, the nonionic tonicity agent in the compositions of the invention incorporates sucrose and / or sorbitol.

In one embodiment, a suitable polyol concentration in the immunogenic composition is between about 2.5 and about 15% (w / v), especially 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) sucrose and between about 2.5 and about 15% (w / v) of sorbitol, particularly between about 2 , 5 and about 10% (w / v) sucrose and between about 2.5 and about 10% (w / v) sorbitol, for example, between about 3 and about 7% (w / v) sucrose and between about 3 and about 7% (w / v) sorbitol, such as between about 4 and about 6% (w / v) sucrose and between about 4 and about 6% (w / v) sorbitol.

The pH of the immunogenic compositions should be suitable for parenteral administration. Typically, the pH will be in the range of 6.0 to 9.0. Suitably, the pH will be in the range of 7.0 to 9.0, especially 7.25 to 8.75, such as 7.5 to 8.5, especially pH 7.75 to 8.25. A pH of about 8.0 is particularly interesting.

The pH can 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 having the ability to measure conductivity. A suitable instrument is Zetasizer Nano ZS from Malvern Instruments (UK). One of ordinary skill in the art can readily test both sodium (Na +) and chloride (Cl -) ion concentration using known techniques and kits, for example, sodium can be determined using a kit. such as the Sodium Enzymatic Assay kit (catalog number: BQ011EAEL) from Biosupply Chloride can be determined using a kit such as the Chloride Enzymatic Assay kit (catalog number: BQ006EAEL) from Biosupply.

Immunogenic Properties of the Immunogenic Composition of the Present Invention

In the present invention, the immunogenic composition is suitably capable of inducing a humoral response in a mammal, such as a human, to which the immunogenic composition has been administered.

Humoral responses can be detected using an appropriate antibody-based assay. For example, the presence or absence in the serum of an immunoglobulin G (IgG) antibody response to a gp120-related polypeptide can be assayed by ELISA. The induction of humoral responses such as IgG antibodies, suitably IgG antibodies binding to 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 enhanced CD4 T cell immune response.

By "enhanced 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 "enhanced CD4 T cell immune response" is obtained in an immunologically un-triggered patient, i.e., a patient who is seronegative to HIV.

The enhanced CD4 T cell immune response (which can be provided by "polyfunctional" T cells) can be estimated by measuring the number of cells producing any of the following immunogenic markers: CD4 T cells that express at least one immunogenic marker • cells producing at least two different immunogenic markers (e.g. CD40L, IL-2 and / or IFN-gamma, TNF-alpha) • cells producing at least CD40L and another immunogenic marker (e.g. 2, TNF-gamma and / or IFN-gamma) • cells producing at least IL-2 and another immunogenic marker (e.g., CD40L, TNF-alpha and / or IFN-gamma) • cells producing at least IFN- gamma and another immunogenic marker (e.g., IL-2, TNF-alpha and / or CD40L) • cells producing at least TNF-alpha and another immunogenic marker (e.g., IL-2, CD40L and / or IFN- gamma).

There will be an enhanced CD4 T cell immune response when cells producing any of the foregoing immunogenic markers will be in a higher amount after administration. Typically at least one, suitably two of the six conditions mentioned above will be satisfied. In a particular embodiment, the cells producing each of the four immunogenic markers will be present in a larger amount.

The enhanced CD4 T cell immune response conferred by the gp120 composition of the present invention may be conveniently obtained after a single administration.

In another embodiment, the administration of said immunogenic composition induces an improved memory cell B response in a mammal, such as a human, to which the immunogenic composition is administered. An improved memory cell B response is intended to refer to an increased frequency of peripheral blood B cells capable of differentiation to antibody-secreting plasma cells upon encountering an antigen as measured by in vitro differentiation stimulation.

In a specific embodiment, administration of said immunogenic composition induces at least two of the following responses: (i) an enhanced CD4 T cell immune response, (ii) an improved memory cell B response, (iii) a response improved humoral, against at least one of the constitutive antigen (s) or the antigenic composition in comparison with one or other of the immune responses obtained with other compositions.

The magnitude of an immune response may also be expressed as the titer (or concentration) of antigen-specific antibodies induced by the immunogenic composition as determined by an appropriate serological test. The size 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.

Adequately, the composition of the present invention elicits an immune response capable of cross-reactivity. Here, cross-reactivity is to be understood as meaning the ability of immune responses induced by an immunogenic composition of the invention to recognize HIV-1 strains derived from subtypes that are not represented in the immunogenic composition. For example, an immunogenic composition of the invention comprising a gp120-related polypeptide comprising an HIV-1 subtype B strain is considered cross-reactive if the HIV-specific immune response, such as the specific antibody. to HIV or the CD4 + T cell response (particularly an antibody response to the gpl20 V1V2 loop), induced by the composition reacted with one or more different HIV-1 strains that are not in the composition for example, with a strain of HIV-1 derived from a subtype other than subtype B. Adequately, the cross-reactivity will be with respect to a strain of HIV-1 derived from a different subtype, particular with respect to a strain of HIV-1 from a different group.

Adequately, 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 the titer (or concentration) of antigen-specific antibodies induced by the immunogenic composition.

When measuring cross-reactivity in terms of the percentage of responders to HIV-1 strains from different subtypes, the number or percentage of vaccinated individuals who show a positive response in an immunoassay after subsequent challenge may be measured. An answering machine can respond to one or more epitopes of an antigen. An answering machine may also respond to one or more polypeptides in an immunogenic composition of the invention and / or to one or more antigens in an immunogenic composition of the invention.

Immunological assays such as serologic assays that can be used to analyze the percentage of responders or the size of an immune response are known in the art. Examples of such assays are known to those skilled in the art.

Adequately, 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 if they are responders.

In one embodiment, the immunogenic composition of the invention is for use in eliciting high and long-lasting numbers of HIV-1 specific antibodies in an uninfected individual.

In a further embodiment, the immunogenic composition of the invention is for use in eliciting high and long-lasting numbers of HIV-1-specific antibodies in an individual at risk of infection with an infection. an HIV-1 strain derived from one or more clades different from one or more HIV-1 clades from which the gp120-related polypeptide in the immunogenic composition is derived.

In one embodiment, the immunogenic composition of the invention is for use in controlling or reducing viremia in an HIV-infected individual.

Adequately, 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 subject's viral load 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 5,000 copies / mL, below 1,000 copies / mL or below. 500 copies / mL. Adequately, the viral load is maintained or reduced for at least six months, at least twelve months, at least eighteen months, at least two years, at least three years, at least four years, at least five years, at least six years. years, at least seven years, at least eight years, at least nine years or at least ten years after the administration of the composition.

Adequately, the administration of inventive composition leads to a sustainable response. A durable response is, for example, the ability to detect, in the serum of an individual, an IgG antibody capable of binding to the V1V2 region of the gp120-related polypeptide of the composition at least 24 weeks, at least 48 weeks, at 72 weeks, at least 96 weeks, at least two years, at least three years, at least four years, at least five years, at least six years, at least seven years, at least eight years, at least nine years, or at least at least ten years after the single administration of the composition, or the first administration of the composition during repeated administrations, to the individual. Adequately, the levels of antibodies 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 after the first administration. Adequately, the antibody will be detectable in at least 50% of individuals, more adequately 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, an IgG antibody binding to the V1V2 region of the gp120-related polypeptide of the composition at least 2 weeks, at least 6 months, at 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 at least ten years after the final administration of the composition during repeated administrations to the individual. Adequately, the 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 after the final administration. Adequately, the antibody will be detectable in at least 50% of individuals, more adequately at least 60% of individuals and in particular at least 75%.

Adequately, the present invention is capable of achieving a more durable immune response based on responder rates. Adequately, 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 an increased humoral response such as increased serum IgG antibody binding to the V1V2 region of the gp120-related polypeptide of the composition.

Means of vaccination

The immunogenic compositions of the invention may be administered by any suitable delivery route, such as intradermally, mucosally, for example intranasally, orally, intramuscularly or subcutaneously. Other delivery routes are well known in the art. The intramuscular delivery route is preferred for the immunogenic composition. Intradermal delivery is another suitable pathway. Any suitable device can be used for intradermal delivery, for example short needle devices such as those described in US 4,886,499. Intradermal vaccines can also be administered by devices which limit the effective penetration length of a needle in the skin. Also suitable are jet injection devices that deliver liquid vaccines to the dermis via a liquid jet injector or a needle that pierces the stratum corneum and produces a jet that reaches the dermis. Jet injection devices are described, for example, in US 5,480,381. Powder / ballistic particle delivery devices that use a compressed gas to accelerate the powdered vaccine through the outer layers of the powder are also suitable. the skin towards the dermis. Additionally, conventional syringes can be used in the conventional Mantoux intradermal method.

Another suitable route of administration is the subcutaneous route. Any suitable device can be used for subcutaneous delivery, for example a conventional needle. Suitably, a needleless jet injection device is used, as published in US 6,623,446. 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, adequately without being inhaled into the lungs. It is desirable to use an intranasal delivery device that delivers the formulation of. vaccine 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 by a different route, for example intradermal, subcutaneous or intranasal.

In one aspect of the invention, a vaccination program with the immunogenic composition may comprise concomitant or simultaneous sequential administration ("initial-boost injection") of the immunogenic composition and DNA encoding any of the aforementioned proteins.

In one embodiment, the vaccination program with the immunogenic composition consists of three to five (e.g. four) administrations of the immunogenic composition to an individual over a period of four to eight (e.g., five to seven) months. Suitably, the vaccination program with the immunogenic composition consists of four administrations of the immunogenic composition to an individual over a period of five to seven months.

Suitably, the gp120-related polypeptide of the invention may be replaced by a polynucleotide encoding the gp120-related polypeptide of the invention. Adequately, the polynucleotide is optimized in codon. The DNA may be provided in the form of plasmid DNA or in the form of a recombinant living vector, for example a poxvirus vector or any other suitable living vector such as a retrovirus, a lentivirus, an adenovirus or an adeno virus. -associated and modified Ankara vaccinia virus (VAM). Adequately, an adenovirus is used. Alternatively, a canary pox virus can be used.

The immunogenic composition may be injected one or more times after one or more administrations of DNA. The DNA can be used first for one or more administrations followed by one or more immunizations with the immunogenic composition. Alternatively, the immunogenic composition may be injected one or more times together with DNA administrations.

antiretrovirals

HIV is a retrovirus. The conversion of its RNA into DNA is accomplished by the action of the reverse transcriptase enzyme. Compounds that inhibit the function of reverse transcriptase inhibit HIV replication in infected cells. Drugs incorporating such compounds are useful in the prevention or treatment of HIV infection in humans and may be used in conjunction with a composition of the present invention.

The composition of the present invention may be administered in conjunction with (i.e., before, during, or after administration of) antiretroviral therapy (ART) such as nucleoside or non-nucleoside reverse transcriptase inhibitors, protease inhibitors, fusion inhibitors, input inhibitors, processing inhibitors, cell 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 (dof), stavudine (d4T), abacavir sulphate (ABC), andtravirine, 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), enfufirtide, T-20, maraviroc, dolutegravir and raltegravir. TAR drugs may also include antibodies, such as ibalizumab, targeting HIV proteins or cellular proteins associated with progression of the disease. Also included are immunotherapies, such as IL-2, IL-12 and alpha-epibromide. Each of these drugs can be administered alone or in combination with any other ART drug. Information about ART and its administration can be found in many pharmacopoeias, such as the United States Pharmacopoeia (USP) or by accessing online, such as www.aidsmeds.com (accessed September 5, 2013) . Trade names for 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, dof), Viread (tenofovir disoproxil fumarate, TDF), Zerit (stavudine, d4T), Ziagen ( abacavir sulfate, ABC), rilpivirine, etravirine, Rescriptor (delavirdine, DLV), Sustiva (efavirenz, EFV), Viramune (nevirapine, NVP), Agenerase (amprenavir, APV), Aptivus (tipranavir, TPV), saquinavir, Invirase (saquinavir mesylate, SQV), Kaletra (lopinavir and 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 does not take 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 and 1700 cells per cubic millimeter of blood. In a further embodiment, the immunogenic composition is administered to a patient having a CD4 nadir count of 200 or more, more suitably 500 or more cells per cubic millimeter of blood.

In one embodiment, the immunogenic composition is administered to a subject, such as a human, who is not infected with HIV. In one embodiment, the immunogenic composition is administered to a subject, such as a human, who is infected with HIV.

In one aspect of the invention, there is provided a gp120 related protein for use in the manufacture of an immunogenic composition as described herein, such as for prophylaxis or HIV infection. Alternatively, the composition may be for treating an HIV infection.

In a further aspect, there is provided a method of prophylaxis of HIV infection by administering 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 of treating an HIV infection by administering an immunogenic composition as described herein to an HIV-infected mammal, such as a human. Such an administration can reduce the severity of an HIV infection.

In a further aspect, there is provided a method of reducing the risk of HIV transmission from an HIV-infected individual to a partner of said HIV-infected individual by administering an immunogenic composition as described herein to individual infected with HIV.

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 with the aid of the following clauses.

Clause 1. Immunogenic composition comprising a polypeptide related to gpl20. and an adjuvant, wherein the adjuvant comprises a lipopolysaccharide and an immunologically active saponin fraction derived from Quillaja saponaria Molina bark presented as a liposome and wherein the composition is substantially free of NefTat-related polypeptide, in which the polypeptide related to

NefTat is a polypeptide consisting of SEQ ID NO: 4.

Clause 2. An immunogenic composition which is in the form of a human dose comprising a gp120-related polypeptide and an adjuvant, wherein the adjuvant comprises between 10 and 40 μg of a lipopolysaccharide and between 10 and 40 μg of an immunologically active saponin fraction derived from Quillaja saponaria Molina bark presented as a liposome.

Clause 3. An immunogenic composition comprising a gp120-related polypeptide and adjuvant, wherein the adjuvant comprises a lipopolysaccharide and an immunologically active saponin fraction derived from Quillaja saponaria Molina bark presented as a liposome in which (i) the conductivity of the composition is 13 mS / cm or less; and / or (ii) the salt concentration in said composition is 130 mM or less; and / or (iii) the concentration of sodium chloride in said composition is 130 mM or less.

Clause 4. An immunogenic composition according to any of Clauses 2 or 3, which is substantially free of a NefTat-related polypeptide, wherein the NefTat-related polypeptide is a polypeptide consisting of SEQ ID NO: 4.

Clause 5. An immunogenic composition according to any one of Clauses 1 or 4, which contains a NefTat-related polypeptide-related polypeptide ratio of less than 1: 20.

Clause 6. Immunogenic composition according to any of Clauses 1 or 4, which contains less than 1 of NefTat-related polypeptide.

Clause 7. An immunogenic composition according to any one of clauses 5 or 6 wherein the composition is free of NefTat-related polypeptide.

Clause 8. An immunogenic composition according to any one of Clauses 1 or 4 to 7, wherein the NefTat-related polypeptide comprises a polypeptide with at least 99% identity with SEQ ID NO: 4.

Clause 9. Immunogenic composition according to clause 8, in which the polypeptide related to

NefTat comprises a polypeptide with at least 90% identity with SEQ ID NO: 4.

Clause 10. An immunogenic composition according to clause 9, wherein the NefTat-related polypeptide comprises a polypeptide with at least 80% identity with SEQ ID NO: 4.

Clause 11. An immunogenic composition according to clause 10, wherein the NefTat-related polypeptide comprises a polypeptide with at least 70% identity with SEQ ID NO: 4.

Clause 12. An immunogenic composition according to any one of Clauses 1 or 4, which contains a polypeptide ratio related to Nef: gp120-related polypeptide of less than 1:20.

Clause 13. An immunogenic composition according to any of Clauses 1 or 4, which contains less than 1 of Nef-related polypeptide.

Clause 14. An immunogenic composition according to any of Clauses 12 or 13, wherein the composition is free of Nef-related polypeptide.

Clause 15. An immunogenic composition according to any one of Clauses 1, 4 or 12 to 14, wherein the Nef-related polypeptide comprises a polypeptide with at least 99% identity with SEQ ID NO: 2.

Clause 16. An immunogenic composition according to clause 15, wherein the Nef-related polypeptide comprises a polypeptide with at least 90% identity with SEQ ID NO: 2.

Clause 17. An immunogenic composition according to clause 16, wherein the Nef-related polypeptide comprises a polypeptide with at least 80% identity with SEQ ID NO: 2.

Clause 18. An immunogenic composition according to clause 17, wherein the Nef-related polypeptide comprises a polypeptide with at least 70% identity with SEQ ID NO: 2.

Clause 19. An immunogenic composition according to any one of Clauses 1 or 4, which contains a Tat-related polypeptide ratio: gp120-related polypeptide of less than 1: 20.

Clause 20. Immunogenic composition according to any one of Clauses 1 or 4, which contains less than 1 μg of Tat-related polypeptide.

Clause 21. An immunogenic composition according to any of Clauses 19 or 20, wherein the composition is free of Tat-related polypeptide.

Clause 22. An immunogenic composition according to any of Clauses 1, 4 or 19 to 21, wherein the Tat-related polypeptide comprises a polypeptide with at least 99% identity with SEQ ID NO: 3.

Clause 23. Immunogenic composition according to clause 22, wherein the Tat-related polypeptide comprises a polypeptide with at least 90% identity with SEQ ID'NO: 3.

Clause 24. An immunogenic composition according to clause 23, wherein the Tat-related polypeptide comprises a polypeptide with at least 80% identity with SEQ ID NO: 3.

Clause 25. An immunogenic composition according to clause 24, wherein the Tat-related polypeptide comprises a polypeptide with at least 70% identity with SEQ ID NO: 3.

Clause 26. Immunogenic composition according to any one of Clauses 1 or 3 to 25, wherein the lipopolysaccharide is present at a level between 10 and 100 μg.

Clause 27. An immunogenic composition according to clause 26, in which the lipopolysaccharide is present at a level between 15 and 80 μg.

Clause 28. An immunogenic composition according to clause 27, in which the lipopolysaccharide is present at a level between 20 and 65 μg.

Clause 29. Immunogenic composition according to Clause 28, in which the lipopolysaccharide is present at a level between 30 and 60 μg.

Clause 30. Immunogenic composition according to clause 29, in which the lipopolysaccharide is present at a level between 45 and 55 μg.

Clause 31. Immunogenic composition according to clause 30, in which the lipopolysaccharide is present at a level of 50 μg.

Clause 32. Immunogenic composition according to any one of the clauses 1 or 3 to 31, wherein the saponin is present at a level between 10 and 100 μg.

Clause 33. An immunogenic composition according to clause 32, wherein the saponin is present at a level between 15 and 80 μg.

Clause 34. An immunogenic composition according to clause 33, in which the saponin is present at a level between 20 and 65 μg.

Clause 35. Immunogenic composition according to clause 34, in which the saponin is present at a level between 30 and 60 μg.

Clause 36. An immunogenic composition according to clause 35, in which the saponin is present at a level between 45 and 55 μg.

Clause 37. Immunogenic composition according to clause 36, in which the saponin is present at a level of 50 μg.

Clause 38. An immunogenic composition according to any one of Clauses 1 to 25, wherein the lipopolysaccharide is present at a level of between 15 to 35 μg.

Clause 39. Immunogenic composition according to clause 38, in which the lipopolysaccharide is present at a level between 20 to 30 μg.

Clause 40. Immunogenic composition according to clause 39, in which the lipopolysaccharide is present at a level of 25 μg.

Clause 41. An immunogenic composition according to any one of Clauses 1 to 31, wherein the saponin is present at a level between 15 to 35 μg.

Clause 42. Immunogenic composition according to clause 41, wherein the saponin is present at a level between 20 to 30 μg.

Clause 43. Immunogenic composition according to clause 42, wherein the saponin is present at a level of 25 μg.

Clause 44. An immunogenic composition according to any one of Clauses 1 to 43, wherein the lipopolysaccharide is 3D-MPL.

Clause 45. An immunogenic composition according to any of Clauses 1 to 44, wherein the saponin is QS-21.

Clause 46. An 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 less; and / or (ii) the salt concentration in said composition is 130 mM or less, and / or (iii) the sodium chloride concentration in said composition is 130 mM or less.

Clause 47. Immunogenic composition according to clause 46, wherein the conductivity of the composition is 13 mS / cm or less.

Clause 48. An immunogenic composition according to clause 47, wherein the conductivity of the composition is 6 mS / cm or less.

Clause 49. Immunogenic composition according to clause 48, wherein the conductivity of the composition is 1.5 to 2.5 mS / cm.

Clause 50. Immunogenic composition according to clause 46, wherein the salt concentration in said composition is 130 mM or less.

Clause 51. An immunogenic composition according to clause 50, wherein the salt concentration in said composition is 60 mM or less.

Clause 52. An immunogenic composition according to Clause 51, wherein the salt concentration in said composition is 20 to 40 mM.

Clause 53. An immunogenic composition according to clause 46, wherein the concentration of sodium chloride in said composition is 130 mM or less.

Clause 54. An immunogenic composition according to clause 53, wherein the concentration of sodium chloride in said composition is 60 mM or less.

Clause 55. An immunogenic composition according to clause 54, wherein the concentration of sodium chloride in said composition is 10 mM or less.

Clause 56. An immunogenic composition according to any one of Clauses 1 to 55, wherein the concentration of CaCl 2 in the immunogenic composition is 30 mM or less.

Clause 57. An immunogenic composition according to any one of Clauses 1 to 56, wherein the concentration of MgSO4 in the immunogenic composition is 60 mM or less.

Clause 58. Immunogenic composition according to any one of clauses 1 to 57, the total concentration of NH4 +, Mg2 + and Ca2 + ions being 40 mM or less.

Clause 59. Immunogenic composition according to any one of Clauses 1 to 58, which is an aqueous solution.

Clause 60. An immunogenic composition according to any one of Clauses 1 to 59, which is a single human dose.

Clause 61. An immunogenic composition according to any one of Clauses 1 to 60, wherein the human dose is between 0.1 and 1 mL.

Clause 62. Immunogenic composition according to Clause 61, in which the human dose is between 0.3 and 0.75 mL.

Clause 63. Immunogenic composition according to Clause 62, in which the human dose is 0.5 mL.

Clause 64. An immunogenic composition according to any one of Clauses 1 to 60, wherein the human dose is between 0.05 and 0.2 mL. Clause 65. An immunogenic composition according to any one of Clauses 1 to 64, wherein the osmolality is in the range of 250 to 750 mOsm / kg.

Clause 66. An immunogenic composition according to clause 65, wherein the osmolality is in the range of 250 to 550 mOsm / kg.

Clause 67. An immunogenic composition according to any one of Clauses 1 to 66 comprising a nonionic tonicity agent.

Clause 68. An immunogenic composition according to clause 67, wherein the nonionic tonicity agent is a polyol.

Clause 69. Immunogenic composition according to Clause 68, in which the polyol is sorbitol.

Clause 70. An immunogenic composition according to clause 69, wherein the concentration of sorbitol is between about 4 and about 6% (w / v).

Clause 71. An immunogenic composition according to any one of Clauses 1 to 70, wherein the sucrose concentration is between about 4 and about 6% (w / v).

Clause 72. An immunogenic composition according to any of Clauses 1 to 71, wherein the pH is in the range of 6.0 to 9.0.

Clause 73. An immunogenic composition according to any one of Clauses 1 to 72, wherein the composition comprises the gp120 related polypeptide at a level of about 1 to 100 μg.

Clause 74. An immunogenic composition according to clause 73, wherein the composition comprises the gp120-related polypeptide at a level of 3 to 30 μg.

Clause 75. The immunogenic composition according to clause 74, wherein the composition comprises the gp120-related polypeptide at a level of 5 to 15 μg.

Clause 76. An immunogenic composition according to clause 74, wherein the composition comprises the gp120-related polypeptide at a level of from 16 to 25 μg.

Clause 77. An immunogenic composition according to any one of Clauses 1 to 76, wherein the composition comprises 1 to 5 additional HIV antigens.

Clause 78. An immunogenic composition according to Clause 77, wherein the composition comprises additional HIV antigens selected from the list consisting of gp160, gp41, MA, CA, SP1, NC, SP2, P6, RT, RNase H, IN, PR. , Rev, Vif, Vpr and Vpu.

Clause 79. An immunogenic composition according to any of Clauses 1 to 78, wherein the composition comprises a total of about 1 to 500 antigenic material.

Clause 80. A composition according to any one of Clauses 1 to 79, wherein the gp120-related polypeptide comprises a gp120 polypeptide derived from HIV-1.

Clause 81. A composition according to clause 80, wherein the gp120 related polypeptide comprises a group M-derived HIV-1 gp120 polypeptide.

Clause 82. A composition according to clause 81, wherein the gp120 related polypeptide comprises a gp120 polypeptide derived from HIV-1 group M subtype C.

Clause 83. Composition according to clause 82, wherein the gp120-related polypeptide comprises a gp120 polypeptide derived from HIV-1 group M subtype B.

Clause 84. A composition according to any of Clauses 1 to 81 and 83, wherein the gp120-related polypeptide comprises a polypeptide with at least 70% identity with the V1V2 region of SEQ ID NO: 1.

Clause 85. A composition according to clause 84, wherein the gp120-related polypeptide comprises a polypeptide with at least 80% identity with the V1V2 region of SEQ ID NO: 1.

Clause 86. A composition according to clause 85, wherein the gp120 related polypeptide comprises a polypeptide with at least 90% identity with the V1V2 region of SEQ ID NO: 1.

Clause 87. A composition according to clause 86, wherein the gp120 related polypeptide comprises a polypeptide with at least 95% identity to the V1V2 region of SEQ ID NO: 1.

Clause 88. A composition according to clause 87, wherein the gp120-related polypeptide comprises a polypeptide with at least 98% identity with the V1V2 region of SEQ ID NO: 1.

Clause 89. A composition according to clause 88, wherein the gp120-related polypeptide comprises a polypeptide with at least 99% identity to the V1V2 region of SEQ ID NO: 1.

Clause 90. A composition according to clause 89, wherein the gp120-related polypeptide comprises the V1V2 region of SEQ ID NO: 1.

Clause 91. A composition according to any one of Clauses 1 to 81 and 83 to 90, wherein the gp120-related polypeptide is a polypeptide with at least 70% identity to the V1V2 region of SEQ ID NO: 1.

Clause 92. Composition according to clause 91, wherein the gp120-related polypeptide is a polypeptide with at least 80% identity to the V1V2 region of SEQ ID NO: 1.

Clause 93. A composition according to clause 92, wherein the gp120-related polypeptide is a polypeptide with at least 90% identity to the V1V2 region of SEQ ID NO: 1.

Clause 94. A composition according to clause 93, wherein the gp120-related polypeptide is a polypeptide with at least 95% identity to the V1V2 region of SEQ ID NO: 1.

Clause 95. A composition according to clause 94, wherein the gp120-related polypeptide is a polypeptide with at least 98% identity to the V1V2 region of SEQ ID NO: 1.

Clause 96. Composition according to clause 95, wherein the gp120-related polypeptide is a polypeptide with at least 99% identity to the V1V2 region of SEQ ID NO: 1.

Clause 97. A composition according to clause 96, wherein the gp120-related polypeptide is the V1V2 region of SEQ ID NO: 1.

Clause 98. A composition according to any of Clauses 1 to 81 and 83 to 97, wherein the gp120-related polypeptide comprises a polypeptide with at least 70% identity with SEQ ID NO: 1.

Clause 99. A composition according to clause 98, wherein the gp120-related polypeptide comprises a polypeptide with at least 80% identity with SEQ ID NO: 1.

Clause 100. A composition according to clause 99, wherein the gp120-related polypeptide comprises a polypeptide with at least 90% identity with SEQ ID NO: 1.

Clause 101. A composition according to clause 100, wherein the gp120-related polypeptide comprises a polypeptide with at least 95% identity with SEQ ID NO: 1.

Clause 102. A composition according to clause 101, wherein the gp120-related polypeptide comprises a polypeptide with at least 98% identity with SEQ ID NO: 1.

Clause 103. A composition according to clause 102, wherein the gp120-related polypeptide comprises a polypeptide with at least 99% identity with SEQ ID NO: 1.

Clause 104. A composition according to clause 103, wherein the gp120 related polypeptide comprises SEQ ID NO: 1.

Clause 105. A composition according to any of Clauses 1 to 81 and 83 to 104, wherein the gp120-related polypeptide is a polypeptide with at least 70% identity with SEQ ID NO: 1.

Clause 106. A composition according to clause 105, wherein the gp120-related polypeptide is a polypeptide with at least 80% identity with SEQ ID NO: 1.

Clause 107. A composition according to clause 106, wherein the gp120-related polypeptide is a polypeptide with at least 90% identity with SEQ ID NO: 1.

Clause 108. Composition according to clause 107, wherein the gp120-related polypeptide is a polypeptide with at least 95% identity with SEQ ID NO: 1.

Clause 109. A composition according to clause 108, wherein the gp120-related polypeptide is a polypeptide with at least 98% identity with SEQ ID NO: 1.

Clause 110. A composition according to clause 109, wherein the gp120-related polypeptide is a polypeptide with at least 99% identity with SEQ ID NO: 1.

Clause 111. A composition according to clause 110, wherein the gp120-related polypeptide is SEQ ID NO: 1.

Clause 112. A composition according to any one of Clauses 1 to 82, wherein the gp120-related polypeptide comprises a polypeptide with at least 70% identity to the V1V2 region of SEQ ID NO: 5.

Clause 113. A composition according to clause 112, wherein the gp120-related polypeptide comprises a polypeptide with at least 80% identity with the V1V2 region of SEQ ID NO: 5.

Clause 114. A composition according to clause 113, wherein the gp120-related polypeptide comprises a polypeptide with at least 90% identity with the V1V2 region of SEQ ID NO: 5.

Clause 115. A composition according to clause 114, wherein the gp120 related polypeptide comprises a polypeptide with at least 95% identity to the V1V2 region of SEQ ID NO: 5.

Clause 116. A composition according to clause 115, wherein the gp120-related polypeptide comprises a polypeptide with at least 98% identity with the V1V2 region of SEQ ID NO: 5.

Clause 117. A composition according to clause 116, wherein the gp120-related polypeptide comprises a polypeptide with at least 99% identity to the V1V2 region of SEQ ID NO: 5.

Clause 118. A composition according to clause 117, wherein the gp120-related polypeptide comprises the V1V2 region of SEQ ID NO: 5.

Clause 119. A composition according to any of Clauses 1 to 82 and 112 to 118, wherein the gp120-related polypeptide is a polypeptide with at least 70% identity to the V1V2 region of SEQ ID NO: 5.

Clause 120. A composition according to clause 119, wherein the gp120-related polypeptide is a polypeptide with at least 80% identity to the V1V2 region of SEQ ID NO: 5.

Clause 121. A composition according to clause 120, wherein the gp120-related polypeptide is a polypeptide with at least 90% identity to the V1V2 region of SEQ ID NO: 5.

Clause 122. A composition according to clause 121, wherein the gp120 related polypeptide is a polypeptide with at least 95% identity to the V1V2 region of SEQ ID NO: 5.

Clause 123. A composition according to Clause 122, wherein the gp120-related polypeptide is a polypeptide with at least 98% identity to the V1V2 region of SEQ ID NO: 5.

Clause 124. A composition according to Clause 123, wherein the gp120-related polypeptide is a polypeptide with at least 99% identity to the VIV2 region of SEQ ID NO: 5.

Clause 125. A composition according to Clause 124, wherein the gp120-related polypeptide is the V1V2 region of SEQ ID NO: 5.

Clause 126. A composition according to any one of Clauses 1 to 82 and 112 to 125, wherein the gp120-related polypeptide comprises a polypeptide with at least 70% identity with SEQ ID NO: 5.

Clause 127. A composition according to clause 126, wherein the gp120-related polypeptide comprises a polypeptide with at least 80% identity with SEQ ID NO: 5.

Clause 128. A composition according to clause 127, wherein the gp120-related polypeptide comprises a polypeptide with at least 90% identity with SEQ ID NO: 5.

Clause 129. A composition according to clause 128, wherein the gp120-related polypeptide comprises a polypeptide with at least 95% identity with SEQ ID NO: 5.

Clause 130. A composition according to clause 129, wherein the gp120-related polypeptide comprises a polypeptide with at least 98% identity with SEQ ID NO: 5.

Clause 131. A composition according to Clause 130, wherein the gp120-related polypeptide comprises a polypeptide with at least 99% identity with SEQ ID NO: 5.

Clause 132. A composition according to Clause 131, wherein the gp120-related polypeptide comprises SEQ ID NO: 5.

Clause 133. A composition according to any one of Clauses 1 to 82 and 112 to 132, wherein the gp120-related polypeptide is a polypeptide with at least 70% identity with SEQ ID NO: 5.

Clause 134. A composition according to Clause 133, wherein the gp120-related polypeptide is a polypeptide with at least 80% identity with SEQ ID NO: 5.

Clause 135. A composition according to clause 134, wherein the gp120-related polypeptide is a polypeptide with at least 90% identity with SEQ ID NO: 5.

Clause 136. A composition according to clause 135, wherein the gp120-related polypeptide is a polypeptide with at least 95% identity with SEQ ID NO: 5.

Clause 137. A composition according to clause 136, wherein the gp120-related polypeptide is a polypeptide with at least 98% identity with SEQ ID NO: 5.

Clause 138. A composition according to clause 137, wherein the gp120-related polypeptide is a polypeptide with at least 99% identity with SEQ ID NO: 5.

Clause 139. A composition according to clause 138, wherein the gp120-related polypeptide is SEQ ID NO: 5.

Clause 140. An immunogenic composition according to any of Clauses 1 to 139, wherein the composition further comprises a sterol, wherein the saponin: sterol ratio is from 1: 1 to 1: 100 w / w.

Clause 141. Immunogenic composition according to clause 140, wherein the saponin: sterol ratio is 1: 1 to 1: 5 w / w.

Clause 142. Immunogenic composition according to any one of clauses 140 or 141, wherein said sterol is cholesterol.

Clause 143. Immunogenic composition according to any one of Clauses 1 to 142, which is provided in a multi-dose presentation.

Clause 144. Immunogenic composition according to any one of clauses 1 to 143, which is provided in a presentation containing a surplus of 1 to 50% to account for losses during administration.

Clause 145. Immunogenic composition according to any one of Clauses 1 to 144 for use as a medicament.

Clause 146. Immunogenic composition according to clause 145, for use in the treatment or prevention of HIV-1 from group M, N, O or P.

Clause 147. Immunogenic composition according to clause 146, for use in the treatment or prevention of HIV-1 group M subtype A, B, C, D, E, F, G, H, I, J or K.

Clause 148. An 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 a HIV-1 infection with a second subtype of HIV-1.

Clause 149. An immunogenic composition according to any one of Clauses 1 to 148, wherein the gp120-related polypeptide is derived from a first HIV-1 subtype, for use in the treatment or prevention of HIV-1 infection by a second subtype of HIV-1, wherein the first and second subtypes of HIV-1 have different native gp120 polypeptide sequences.

Clause 150. An immunogenic composition according to clause 149, wherein the first subtype of HIV-1 is selected from the list consisting of A, B, C, D, E, F, G, H, I, J or K; HIV-1 group M.

Clause 151. Immunogenic composition according to any one of clauses 1 to 150, for use in the induction of a humoral immune response against strains of HIV-1 derived from one or more subtypes different from the subtype of HIV-1 from which the polypeptide related to the gp120 of the composition is derived.

Clause 152. Immunogenic composition according to any of Clauses 1 to 151, for use in eliciting antibodies against the HIV-1 gp120 loop V1V2.

Clause 153. A method of treating or prophylaxis of an infection, comprising the step of administering a composition according to any one of clauses 1 to 152 to an individual.

Clause 154. A method of treating or prophylaxis of an HIV-1 infection according to clause 153, further comprising the concomitant administration of an antiretroviral drug.

Clause 155. A method of treating or prophylaxis of HIV-1 infection according to either of clauses 153 or 154, wherein an IgG antibody capable of binding to the V1V2 region of the polypeptide related to the gp120 of the composition is detectable in the serum of an individual at least 24 weeks after the single administration of the composition or the first administration of the composition during repeated administration to the individual.

Clause 156. A method of treating or prophylaxis of an HIV-1 infection according to clause 155, wherein the IgG antibody is detectable at least 48 weeks after the single administration of the composition or the first administration of the composition during repeated administrations to the individual.

Clause 157. A method of treating or prophylaxis of an HIV-1 infection according to clause 156, wherein the IgG antibody is detectable at least 72 weeks after the single administration of the composition or the first administration of the composition during repeated administrations to the individual.

Clause 158. A method of treating or prophylaxis of an HIV-1 infection according to clause 157, wherein the IgG antibody is detectable at least 96 weeks after the single administration of the composition or the first administration of the composition during repeated administrations to the individual.

Clause 159. A method of treatment or prophylaxis of HIV-1 infection according to either of clauses 153 or 154, wherein an IgG antibody capable of binding to the V1V2 region of the polypeptide related to the gp120 of the composition is detectable in the serum of an individual at least 2 weeks after the final administration of the composition during repeated administrations to the individual.

Clause 160. A method of treating or prophylaxis of an 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 administrations. repeated to the individual.

Clause 161. A method of treating or prophylaxis of an HIV-1 infection according to Clause 160, wherein the IgG antibody is detectable at least 12 months after the final administration of the composition during repeated dosing. the individual.

Clause 162. A method for the treatment or prophylaxis of an HIV-1 infection according to Clause 161, wherein the IgG antibody is detectable at least 18 months after the final administration of the composition during repeated dosing. the individual.

Clause 163. A 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 serum titre two weeks after only administration.

Clause 164. A method of treating or prophylaxing an HIV-1 infection according to any one of clauses 155 to 162, wherein the antibody levels are detectable at at least 5% serum titre two weeks after the first administration.

Clause 165. A method of treating or prophylaxis of an HIV-1 infection according to any one of clauses 159 to 162, wherein antibody levels are detectable at at least 5% of serum titer two weeks after infection. final administration.

Clause 166. A 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 the individuals to which the composition is administered.

Clause 167. A method of treatment or prophylaxis according to clause 166, wherein the antibody will be detectable in at least 80% of the individuals to which the composition is administered.

Clause 168. A 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 of the clauses 1 at 152 is administered to the individual.

Clause 169. A 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 polypeptide related to gp120 is administered to the individual.

Clause 170. A method of reducing the risk of HIV transmission from an HIV-infected individual to a partner of said HIV-infected individual comprising the step of administering the immunogenic composition according to any one of clauses 1 to 152 to the individual infected with HIV.

Clause 171. Process for the preparation of the composition according to any one of Clauses 1 to 152, comprising the addition of a polypeptide derived from gpl20 to a lipopolysaccharide and an immunologically active saponin fraction derived from Quillaja bark Molina saponaria presented in the form of a liposome.

Clause 172. A kit for preparing 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 polypeptide derived from gp120 and the second container comprises a lipopolysaccharide and an immunologically active saponin fraction derived from Quillaja saponaria Molina bark presented as a liposome.

Clause 173. Kit according to clause 172, wherein the second container comprises an aqueous solution.

Clause 174. Viral vector comprising a polynucleotide encoding a polypeptide comprising gp120SSiD of SEQ ID NO: 1.

Clause 175. Viral vector according to clause 174, comprising a polynucleotide encoding a polypeptide consisting of the polypeptide gpl2 0w6iD of SEQ XD NO: 1.

Clause 176. Viral vector comprising a polynucleotide encoding. a polypeptide comprising the gp120M18 polypeptide of SEQ ID NO: 5.

Clause 177. A viral vector according to clause 176, comprising a polynucleotide encoding a polypeptide consisting of the gp120M18 polypeptide of SEQ ID NO: 5.

Clause 178. A viral vector according to any one of clauses 174 to 177, which is an adenovirus, a canary pox virus or an MVA virus.

The present invention will now be further described by way of the following non-limiting examples.

Examples

Example 1: Comparison of Serology Method

A first composition containing 2 μg of gp120W6iD and 50 μg of AS01B was prepared and a second composition containing 2 μg of gp120w6D, 50 μg of AS01B and 2 μg of NefTat. At three instants (days 0, 14 and 28), the first intramuscular (IM) composition was administered to a first group of 25 CB6F1 mice and at the same three instants the second composition was administered to a second group of 25 CB6F1 mice.

The titre of anti-VlV2 antibody was compared in the sera of the mice in each group at 14 days after the second administration and at 14 days after the third administration. To evaluate the amount of anti-VlV2 IgG antibodies present in each serum sample, the V1V2 region of gp120 of HIV-1 subtype B case A2 scaffolded on murine gp70 protein was used in an ELISA antibody binding assay. Igtot. During the development of the assay, CH58 (the anti-V2 monoclonal antibody used in the RV144 assay) was used as a positive control to detect a successful binding of serum Igtot antibodies to scaffolded V1V2. In addition, the gp70 protein (not bound to a V1V2 region) was used as a negative control to estimate specificity during assay development. Results

At the two instants tested, one was observed. a tendency toward a higher anti-VlV2 antibody response in mouse serum samples administered the composition which did not contain NefTat as compared to serum samples from mice administered the composition containing NefTat. This is particularly noticeable at 14 days after the third administration (see Figure 1).

All references made in this application, including patents and patent applications, are hereby incorporated by reference to the greatest extent possible.

Throughout the Memorial and the claims that follow, unless the context otherwise requires, the word "understand", and variations such as "understands" and "including" will be understood to imply the inclusion of an integer, d a step, a group of integers or a group of steps, but not the exclusion of any other integer, step, group of integers or group of steps.

The application of which this description and these claims form part may be used as the basis of priority for any subsequent application. The claims of such a subsequent application may be directed to any particularity or combination of features described herein. These may take the form of product-type claims, composition, process or use and may include, by way of example and without limitation, the following claims.

Sequence Listing SEQ XD NO 1 - gpl2 0cj6iD

AEQLWVTVYYGVPVWKEATTTLFCASDAKAYDTEVHNVWATHACVPTDPNPQEW LGNVTEYFNMWKNNMVDQMHEDIISLWDQSLKPCVKLTPLCVTLDCDDVNTTNST TTTSNGWTGEIRKGEIKNCSFNITTSIRDKVQKEYALFYNLDWPIDDDNATTKN KTTRNFRLIHCNSSVMTQACPKVSFEPIPIHYCAPAGFAILKCNNKTFDGKGLCT NVSTVQCTHGIRPWSTQLLLNGSLAEEEWIRSDNFMDNTKTIIVQLNESVAIN CTRPNNNTRKGIHIGPGRAFYAARKIIGDIRQAHCNLSRAQWNNTLKQIVIKLRE HFGNKTIKFNQSSGGDPEIVRHSFNCGGEFFYCDTTQLFNSTWNGTEGNNTEGNS TITLPCRIKQIINMWQEVGKAMYAPPIGGQIRCSSNITGLLLTRDGGTEGNGTEN BEEN IFRPGGGDMRDNWRSELYKYKWKVE PLGVAPTRAKRRWQR VIV2 underlined region. SEQ ID NO: 2 - Nave

MGGKWSKSSWGWPTVRERMRRAEPAADGVGAASRDLEKHGAITSSNTAATNAAC

AWLEAQEEEEVGFPVTPQVPLRPMTYKAAVDLSHFLKEKGGLEGLIHSQRRQDIL

DLWIYHTQGYFPDWQNYTPGPGVRYPLTFGWCYKLVPVEPDKVEEANKGENTSLL

HPVSLHGMDDPEREVLEWRFDSRLAFHHVARELHPEYFKNC SEQ ID NO: 3 - Tat

EPVDPRLEPWKHPGSQPKTACTNCYCKKCCFHCQVCFITKALGISYGRKKRRQRR

RPPQGSQTHQVSLSKQPTSQSRGDPTGPKE SEQ ID NO: 4 - NefTat

MGGKWSKS SWGWPTVRERMRRAE PAADGVGAASRDLEKHGAITS SNTAATNAAC AWLEAQEEEEVGFPVTPQVPLRPMTYKAAVDLSHFLKEKGGLEGLIHSQRRQDIL DLWIYHTQGYFPDWQNYTPGPGVRYPLTFGWCYKLVPVEPDKVEEANKGENTSLL hpvslhgmddperevlewrfdsrlafhhvarelhpeyfknc ^ s | epvdprlepwk

HPGSQPKTACTNCYCKKCCFHCQVCFITKALGISYGRKKRRQRRRPPQGSQTHQV

SLSKQPTSQSRGDPTGPKE

Portion Nef: amino acids 1 to 206 Tat portion: amino acids 209 to 293 (underlined) Box: additional binding amino acids introduced into the fusion protein SEQ ID NO: 5 ~~ gpl20zMi8

GDNLWTVYYGVPVWKEAKTTLFCASDAKAYEREVHNVWATHACVPTDPNPQEIV

LGNVTENFNMWKNDMVDQMHEDIIRLWDQSLKPCVKLTPLCVTLECGNVNVTHEN

STKGEMKNCSFNATTELKDKKQRVYALFYKLDIVPLNENNNSSEDSSEYRLINCN

TSAITQACPKVTLDPIPIHYCAPAGYAILKCNNKTFNGTGPCHNVSTVQCTHGIK

PWSTQLLLNGSLAEEEIIIRSENLTNNAKTIIVHLNESVEIVCTRPSNNTRKSI

RIGPG

QAFYATGGIIGNIRQAHCNISKENWNKTLQKVGKKLAEHFPNKTIKFDQHSGGDL

EITTHSFNCRGEFFY

CNTSNLFNSTYKPNDTNSTYNPNDTITLPCRIKQIINMWQGVGQAMYAPPIAGNI

TCKSNITGLLLTRDG GSNDTTNTETFRPGGGDMRDNWRSELYKYKWEIKTLGIAPTAAKRRWETR underlined region V1V2. SEQ ID NO: 6 - polynucleotide sequence encoding gpl202Mi8 ggagacaacttgtgggtcacagtctattatggggtacctgtgtggaaagaagcaa aaactactttattctgtgcatcagatgctaaagcatatgagagagaagtgcataa tgtctgggctacacatgcctgtgtacccacagaccccaacccacaagaaatagtt ttgggaaatgtaacagaaaattttaacatgtggaaaaatgacatggtggatcaga tgcatgaggatataatcaggttatgggatcaaagcttaaagccatgtgtaaagtt gaccccactctgtgtcactttagaatgtggaaatgttaatgttacccatgagaat agcacgaagggggaaatgaaaaattgctctttcaatgcaaccacagaactaaaag ataaaaaacagagagtgtatgcacttttttataaa cttgatatagtaccacttaatgagaataacaactctagtgaggactctagtgagt atagattaataaattgtaatacctcagccataacacaagcctgtccaaaggtcac tttggacccaattcctatacattattgtgctccagctggatatgcgattctaaag tgtaataataagacattcaatgggacaggaccatgccataatgtcagcacagtac aatgtacacacggaatcaagccagtggtatcaactcaactactgttaaatggtag cctagcagaagaagagataataattaggtctgaaaatctaacaaacaatgccaaa acaataatagtacatcttaatgaatctgtagaaattgtgtgtacaagacccagca ataatacaagaaaaagtataaggataggaccagga caagcattctatgcaacaggtggcataataggaaacataagacaagcacattgta acattagtaaagagaactggaataaa actttacaaaaggtaggaaaaaaattagc agagcacttccctaataaaacaataaaatttgaccaacactcaggaggggaccta gaaattacaacacatagctttaattgtagaggagaatttttctattgcaatacat caaacctgtttaatagtacatataagcctaatgatacaaatagtacatataatcc taatgatacaatcacactcccatgcagaataaaacaaattataaacatgtggcag ggggtaggacaagcaatgtatgcccctcccattgcaggaaacataacatgtaaat caaatatcacaggactactattgacacgggatggagggtcaaatgataccacaaa cacagagacattcagacctggaggaggagatatgagggacaattggagaagtgaa ctatataaatataaagtggtagaaattaaaacattgggcatagcacccactgcgg caaaaaggagagtggtggagacgagataa SEQ ID NO: 7 - GPL2 native 0ZMi8

GDNLWVTVYYGVPVWKEAKTTLFCASDAKAYEREVHNVWATHACVPTDPNPQEIV

LGNVTENFNMWKNDMVDQMHEDIIRLWDQSLKPCVKLTPLCVTLECGNVNVTHEN

STKGEMKNCSFNATTELKDKKQRVYALFYKLDIVPLNENNNSSEDSSEYRLINCN

TSAITQACPKVTLDPIPIHYCAPAGYAILKCNNKTFNGTGPCHNVSTVQCTHGIK

PWSTQLLLNGSLAEEEIIIRSENLTNNAKTIIVHLNESVEIVCTRPSNNTRKSI

RIGPGQAFYATGGIIGNIRQAHCNISKENWNKTLQKVGKKLAEHFPNKTIKFDQH

SGGDLEITTHSFNCRGEFFYCNTSNLFNSTYKPNDTNSTYNPNDTITLPCRIKQI

INMWQGVGQAMYAPPIAGNITCKSNITGLLLTRDGGSNDTTNTETFRPGGGDMRD

NWRSELYKYKWEIKPLGIAPTAAKRRWETR

The native gp120G17 polypeptide sequence contains the underlined proline instead of threonine. SEQ ID NO: 8 - polynucleotide sequence encoding native GPL2 0ZMi8 ggagacaacttgtgggtcacagtctattatggggtacctgtgtggaaagaagcaa aaactactttattctgtgcatcagatgctaaagcatatgagagagaagtgcataa tgtctgggctacacatgcctgtgtacccacagaccccaacccacaagaaatagtt ttgggaaatgtaacagaaaattttaacatgtggaaaaatgacatggtggatcaga tgcatgaggatataatcaggttatgggatcaaagcttaaagccatgtgtaaagtt gaccccactctgtgtcactttagaatgtggaaatgttaatgttacccatgagaat agcacgaagggggaaatgaaaaattgctctttcaatgcaaccacagaactaaaag ataaaaaacagagagtgtatgcacttttttataaa cttgatatagtaccacttaatgagaataacaactctagtgaggactctagtgagt atagattaataaattgtaatacctcagccataacacaagcctgtccaaaggtcac tttggatccaattcctatacattattgtgctccagctggatatgcgattctaaag tgtaataataagacattcaatgggacaggaccatgccataatgtcagcacagtac aatgtacacacggaatcaagccagtggtatcaactcaactactgttaaatggtag cctagcagaagaagagataataattaggtctgaaaatctaacaaacaatgccaaa acaataatagtacatcttaatgaatctgtagaaattgtgtgtacaagacccagca ataatacaagaaaaagtataaggataggaccagga caagcattctatgcaacaggtggcataataggaaacataagacaagcacattgta acattagtaaagagaact ggaataaaactttacaaaaggtaggaaaaaaattagc agagcacttccctaataaaacaataaaatttgaccaacactcaggaggggaccta gaaattacaacacatagctttaattgtagaggagaatttttctattgcaatacat caaacctgtttaatagtacatataagcctaatgatacaaatagtacatataatcc taatgatacaatcacactcccatgcagaataaaacaaattataaacatgtggcag ggggtaggacaagcaatgtatgcccctcccattgcaggaaacataacatgtaaat caaatatcacaggactactattgacacgggatgga gggtcaaatgataccacaaacacagagacattcagacctggaggaggagatatga gggacaattggagaagtgaactatataaatataaagtggtagaaattaaaccatt gggcatagcacccactgcggcaaaaaggagagtggtggagacgagataa

To produce the gp120ZM1 polynucleotide sequence from the native gp120ZM18 polynucleotide sequence: a) a silent point mutation is introduced (C instead of T at position 597 (underlined)) b) a non-silent point mutation A instead of C is introduced, in position 1426 (underlined), modifying the native CCA codon (proline) for the ACA codon (threonine).

Sequence list <110> GlaxoSmithKline Biologicals s.a. <120> New compositions <130> VB 65567 <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 Tire Tyr Gly Val Pro Val Trp Lys 1 5 10 15

Glu Ala Thr Thr Thr Phe Cys Ala Ser Asp Ala Lys Ala Tyr Asp 20 25 30

Thr Glu Val His Asn Val Trp Ala Thr His Ala Cys Pro Val Thr Asp 35 40 45

Pro Asn Pro Gin Val Glu Val Leu Gly Asn Thr Thru Glu Tyr Phe Asn 50 55 60

Met Trp Lys Asn Asn Met Val Asp Gin Met His Glu Asp Ile Ile Ser 65 70 75 80

Leu Trp Asp Gin Ser Lys Pro Leuk Lys Pro Leu Leuk Pro Leu Cys 85 90 95

Val Thr Leu Asp Asp Cys Asp Asp Asn Thr Thrn Asn Thr Ser Thr Thr 100 105 110

Thr Ser Asn Gly Trp Thr Gly Glu Island Arg Lys Gly Glu Island Lys Asn 115 120 125

Cys ser phe Asn Thr Thr Ser Isle Arg Asp Asp Lys Val Gin Lys Glu 130 135 140

Tyr Ala Leu Phe Asn Tyr Leu Val Asp Val Val Asp Asp As Asp Asn Asp 145 150 155 160

Ala Thr Thr Lys Asn Lys Thr Thr Arg Asn Phe Arg Leu Ile His Cys 165 170 175

Asn Ser Ser Val Met Thr Gin Ala Cys Pro Lys Val Ser Phe Glu Pro 180 185 190

Ile Pro Ile His Tyr cys Ala Pro Ala Gly Phe Ala Ile Leu Lys Cys 195 200 205

Asn Asn Lys Thr Phe Asp Gly Lys Gly Leu Cys Thr Asn Val Ser Thr 210 215 220

Val Gin Cys Thr His Gly Arg Arg Val Val Ser Ser Gin Leu Leu 225 230 235 240

Leu Asn Gly Ser Leu Ala Glu Glu Glu Val Val Island Arg Ser Asp Asn 245 250 255

Phe Met Asp Asn Thr Lys Thr island island Val Gin Leu Asn Glu ser val 260 265 270

Ala Island Asn Cys Thr Arg Pro Asn Asn Asn Thr Arg Lys Gly Island His 275 280 285

Gly Pro Gly Arg Island Ala Phe Tyr Ala Ala Arg Lys Island Gly Asp Island 290 295 300

Arg Gin Island Ala His Cys Asn Leu Ser Arg Ala Gin Trp Asn Asn Thr 305 310 315 320

Leu Lys Gin Ile Val Ile Lys Leu Arg Glu His Phe Gly Asn Lys Thr 325 330 335

Ile Lys Phe Asn Gin Ser Ser Gly Gly Asp Pro Glu Island Val Arg His 340 345 350

Ser Phe Asn Cys Gly Gly Glu Phe Phe Tyr cys Asp Thr Thr Gin Leu 355 360 365

Phe Asn ser Thr Trp Asn Gly Thr Glu Gly Asn Asn Thr Glu Gly Asn 370 375 380

Ser Thr Island Thr Leu Pro cys Arg Island Lys Gin Island Asn Island Met Trp 385 390 395 400

Gin Glu Val Gly Lys Ala Met Tyr Ala Pro Pro Island Gly Gly Gin Island 405 410 415

Arg Cys Ser Ser Asn Island Thr Gly Leu Leu Leu Thr Arg Asp Gly Gly 420 425 430

Thr Glu Gly Asn Gly Thr Asu Glu Glu Thr Glu Phe Island 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 val val Gly Trp Pro Thr val 15 10 15

Arg Glu Arg Arg Arg Ala Glu Arg Ala Ala Asp Gly Val Gly Ala 20 25 30

Ala Ser Asp Asp Glu Glu Lys His Gly Ala Thr Island ser ser Asn Thr 35 40 45

Ala Ala Ala Asa Ala Ala Cly Ala Trp Leu Ala Glin Glu Glu Glu Glu 50 55 60

Glu Val Pro Gly Phe Pro Thr Pro Gin Val Pro Pro 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 Ile His Ser Gin Arg Arg Gin Asp Ile Leu Asp Leu 100 105 110

Trp Tire Island 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 Asp Asp Lys Val Glu Glu Ala Asn Lys Gly Glu 145 150 155 160

Asn Thr Ser Leu Leu His Pro Ser Ser His His Gly Asp Asp Pro 165 170 175

Glu Arg Glu Val Leu Glu Trp Arg Phe Asp 5er 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

Pro Glu Pro Pro Asp Pro Arg Glu Pro Pro Lys Lys Pro Gly Ser Gin 1 5 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 Island Thr Lys Ala Leu Gly Island Ser Tyr Gly Arg 35 40 45

Lys Arg Arg Lys Arg Gin Arg Arg Pro Pro Gin Gly Ser Gin Thr His 50 55 60

Gin Val Ser Ser Ser Lys Pro Gin Ser 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 <22 0> <223> Fusion NefTat <400> 4

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 Ala Ala Asp Aspa Gly Val Gly Ala 20 25 30

Ala Ser Asp Arg Asp Glu Gly Lys His Gly Ala Thread Ser Ser Ser Asn Thr 35 40 45

Ala Ala Ala Asa Ala Ala Cly Ala Trp Leu Ala Glin Glu Glu Glu Glu 50 55 60

Glu Val Pro Gly Phe Pro Thr Val Pro Gin Pro Arg Arg Pro Met Thr 65 70 75 80

Tyr Lys Ala Ala val Asp Leu ser Her Phe Leu Lys Glu Lys Gly Gly 85 90 95

Leu Glu Gly Leu Ile His Ser Gin Arg Arg Gin Asp Ile Leu Asp Leu 100 105 110

Trp Tire Island His Thr Gin Gly Tire Pro Asp Trp Gin Asn Tire Thr 115 120 125

Pro Pro Va ^ ΑΓ9 Tyr Pro Leu Thr Phe Gly Trp Cys Tyr Lys HO 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

Pro Glu Pro Pro Asp Pro Arg Glu Pro Gl Pro Pro Lys His 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

Cys Gin val cys Phe Island Thr Lys Ala Leu Gly Island Ser Tyr Gly Arg 245 250 255

Lys Arg Arg Lys Gin Arg Arg Pro Arg Pro Gin Gly Gin ser Thr His 260 265 270

Gin Val Ser Leu Ser Lys Pro Gin Ser Ser Gin Ser Arq Glv Pro Asd

275 280 285 P

Thr Gly Pro Glu Lys 290 <210> 5 <211> 472

<212> PRT <213> Artificial <220> <223> ZM18 polypeptide <4 O O> 5

Gly Asp Asn Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp Lys 15 10 15

Glu Ala Lys Thr Thr 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 Island Val Leu Gly Asn Thr Thru Glu Asn Phe Asn 50 55 60

Met Asp Trp Lys Asn Met Asp Asp Asp Met Gin Glu Asp Ile Ile 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 Isle val 130 135 140

Pro Leu Asn Asn Gln Asn Asn Ser Ser Glu Asp Ser Ser Glu Tyr Arg 145 150 155 160

Leu Ile Asn Cys Asn Thr Ser Ala Island Thr Gin Ala Cys Pro Lys Val 165 170 175

Thr Leu Asp Pro Island Pro Island His Tyr cys Ala pro Ala Gly Tyr Ala 180 185 190

Ile 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 Ile Lys Pro Val Val ser 210 215 220

Thr Gin Leu Leu Leu Asn Gly ser Leu Ala Glu Glu Glu Ile Ile Ile 225 230 235 240

Arg Ser Glu Asn Leu Thr Asn Asn Ala Lys Thr Ile Ile val His Leu 245 250 255

Asn Glu Ser Val Glu Island Val Cys Thr Arg Pro Ser Asn Asn Thr Arg 260 265 270

Lys Ser Arg Island Gly Pro Gly Gin Ala Phe Tyr Ala Thr Gly Gly 275 280 285

Island Gly Asn Island Arg Gin Island Ala His cys Asn Island ser Lys Glu Asn 290 295 300

Trp Asn Lys Thr Leu Gin Lys Val Gly Lys Leu Lys Ala Glu His Phe 305 310 315 320

Pro Asn Lys Thr Island Lys Phe Asp Gin His Ser Gly Gly Asp Leu Glu 325 330 335

Thr Thr Island His Ser Phe Asn Cys Arg Gly Glu Phe Phe Tire Cys Asn 340 345 350

Thr Ser Asn Leu Phe Asn Ser Thr Tyr Lys Pro Asn Asp Thr Asn ser 355 360 365

Thr Asn Asn Pro Asn Asp Thr Thr Island Pro Leu Pro cys Arg Island Lys Gin 370 375 380

Ile Ile Asn Met Trp Gin Gly Val Gly Gin Ala Met Tyr Ala Pro Pro 385 390 395 400

Ala Gly Asn Island Thr Island Cys Lys Ser Asn Island Thr Gly Leu Leu Leu 405 410 415

Thr Arg Asp Gly Gly ser Asn Asp Thr Thrn 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 Island Lys Thr Leu Gly Island Ala Pro Thr Ala Ala 450 455 460

Arg Arg Lys Arg Val Glu Thr Arg 465 470 <210> 6 <211> 1419

<212> DNA <213> Artificial <22 0> <223> polynucleotide ZM18 <4 00> 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 gtacaagac c 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 ttaaaacatt gggcatagca 1380 cccactgcgg caaaaaggag agtggtggag acgagataa 1419 <210> 7 <211> 472 <212> PRT <213> Artificial <220> <223> native polypeptide gpl20 ZM18 <400> 7

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 Island Val Leu Gly Asn Thr Thru Glu Asn Phe Asn 50 55 60

Met Trp Lys Asp Aspn Met Val Asp Gin Met His Glu Asp.Ile Arg Island 65 70 75 80

Leu Trp Asp Gin Ser Lys Pro Leuk Lys Pro Leu Leuk 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 -L-L3 12U 125 ASP Lily Lys Gin Arg Val Tyr Ala Leu Phe Tyr Lily Leu Asp Island Val uu us 140

Pro Leu Asn Glu Asn Asn Asn Ser Ser Glu Asp ser Ser Glu Tyr Arg ± 4i> 1SU 155 160

Leu Ile Asn cys Asn Thr Ser Ala Island Thr Gin Ala Cys Pro Lys Val 165 170 175

Thr Leu Asp Pro Island Pro Island His Tyr cys Ala Pro Ala Gly Tyr Ala 180 185 Igo Island 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 Island Lvs Val Val Pro 210 215 220

Thr Gin Leu Leu Leu Asn Gly Ser Ala Leu Glu Glu Glu Ile Ile Ile 225 230 235 240

Arg Ser Glu Asn Leu Thr Asn Asn Ala Lys Thr Island Ile Val His Leu 245 250 255

Asn Glu Ser val Glu Island Val Cys Thr Arg Pro ser Asn Asn Thr Arg 260 265 270

Lys Ser Arg Island Gly Gly Gin Ala Phe Tyr Ala Thr Gly Gly 275 280 285

Island Gly Asn Island Arg Gin Island Ala His Cys Asn Island Ser Lys Glu Asn 290 295 300

Trp Asn Lys Thr Leu Gin Lys Val Gly Lys Leu Lys Ala Glu His Phe 305 310 315 320

Pro Asn Lys Thr Island Lys Phe Asp Gin His Ser Gly Gly Asp Leu Glu 325 330 335

Thr Thr Island 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

Thr Tyr Asn Pro Asn Thr Asp Island Thr Leu Pro Cys Arg Island Lys Gin 370 375 380

Ile Ile Asn Met Trp Gin Gly Val Gly Gin Ala Met Tyr Ala Pro Pro 385 390 395 400

Ile Ala Gly Asn Island Thr cys Lys Ser Asn Island Thr Gly Leu Leu Leu 405 410 415

Thr Arg Asp Gly Gly Ser Asn As Thr Thrn Asn Thr Glu Thr Arg 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 Island Lys Pro Leu Gly Island 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> native polynucleotide gp120 ZM18 <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 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

Claims (29)

An immunogenic composition comprising a gp120-related polypeptide and adjuvant, wherein the adjuvant comprises a lipopolysaccharide and an immunologically active saponin fraction derived from Quillaja saponaria Molina bark 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, wherein said composition is for use in eliciting the production of antibodies against the V1V2 loop of gp120 of HIV-1. An immunogenic composition which is in the form of a human dose comprising a gp120-related polypeptide and adjuvant, wherein the adjuvant comprises between 10 and 40 μg of a lipopolysaccharide and between 10 and 40 μg of a An immunologically active saponin fraction derived from Quillaja saponaria Molina bark presented as a liposome, wherein this composition is for use in eliciting the production of antibodies against the HIV-1 gp120 loop V1V2. An immunogenic composition comprising a gp120-related polypeptide and adjuvant, wherein the adjuvant comprises a lipopolysaccharide and an immunologically active saponin fraction derived from Quillaja saponaria Molina bark presented as a liposome wherein: (i) the conductivity of the composition is 13 mS / cm or less; and / or (ii) the salt concentration in said composition is 130 mM or less, and / or (iii) the sodium chloride concentration in said composition is 130 mM or less; wherein said composition is for use in eliciting the production of antibodies against the HIV-1 gp120 loop V1V2. An immunogenic composition for use according to any one of claims 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. An immunogenic composition for use according to any one of claims 1, 3 or 4, wherein the lipopolysaccharide is present at a level between 10 and 100 μg. An immunogenic composition for use according to any one of claims 1 or 3 to 5, wherein the saponin is present at a level between 10 and 100 μg. An immunogenic composition for use according to any one of claims 1 to 6, wherein the lipopolysaccharide is 3D-MPL. An immunogenic composition for use according to any one of claims 1 to 7, wherein the saponin is QS-21. An immunogenic composition for use according to any one of claims 1, 2 or 4 to 8, wherein: (i) the conductivity of the composition is 13 mS / cm or less; and / or (ii) the salt concentration in said composition is 130 mM or less, and / or (iii) the sodium chloride concentration in said composition is 130 mM or less. An immunogenic composition for use according to any one of claims 1 to 9, which is an aqueous solution. Immunogenic composition for use according to any one of claims 1 to 10, which is a single human dose. An immunogenic composition for use according to any one of claims 1 to 11, wherein the human dose is between 0.1 and 1 mL. An immunogenic composition for use according to any one of claims 1 to 12 comprising a nonionic tonicity agent. The immunogenic composition for use according to any one of claims 1 to 13, wherein the composition comprises the gp120 related polypeptide at a level of about 1 to 100 μg. An immunogenic composition for use according to any one of claims 1 to 14, wherein the composition comprises 1 to 5 additional HIV antigens. The composition for use according to any one of claims 1 to 15, wherein the gp120-related polypeptide comprises a polypeptide with at least 70% identity with the V1V2 region of SEQ ID NO: 1. The composition for use 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. The composition for use according to any one of claims 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. 19. The composition for use 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. The immunogenic composition for use according to any one of claims 1 to 19, wherein the composition further comprises a sterol, wherein the saponin: sterol ratio is from 1: 1 to 1: 100 w / w. 21. An immunogenic composition for use according to any one of claims 1 to 20 for use in the treatment or prevention of HIV-1 from the group M, N, O or P. 22. Immunogenic composition for use 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, H, I, J or K. 23. An immunogenic composition for use 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 an HIV-1 infection by a second subtype of HIV-1, wherein the first and second subtypes of HIV-1 have different native gp120 polypeptide sequences. An immunogenic composition for use according to any one of claims 1 to 23 for use in the treatment or prophylaxis of an HIV infection in which a polynucleotide encoding a gp120-related polypeptide is administered to the individual and subsequently the composition of any one of claims 1 to 23 is administered to the individual. Immunogenic composition for use 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. The process for preparing the composition for use according to any one of claims 1 to 25, comprising adding a gp120 derived polypeptide to a lipopolysaccharide and an immunologically active saponin fraction derived from Quillaja bark. Molina saponaria presented in the form of a liposome. A kit for preparing an immunogenic composition for use according to any one of claims 1 to 25, comprising a first container and a second container, wherein the first container comprises a polypeptide derived from gp120 and the second container comprises a lipopolysaccharide and an immunologically active saponin fraction derived from the bark of Quillaja saponaria Molina presented as a liposome. A viral vector comprising a polynucleotide encoding a polypeptide comprising the gp120W6iD polypeptide of SEQ ID NO: 1. A viral vector comprising a polynucleotide encoding a polypeptide comprising the gp120ZMi8 polypeptide of SEQ ID NO: 5.