EA039968B1 - COMPOSITION OF THE HIV VACCINE - Google Patents

Description

Background of the invention

The Human Immunodeficiency Virus (HIV) affects millions of people worldwide, and preventing HIV with an effective vaccine remains paramount even in the era of widespread antiretroviral therapy. HIV-1 is the most common and pathogenic strain of the virus, with more than 90% of HIV/AIDS cases due to infection with HIV-1 group M. Group M is further subdivided into clades or subtypes. Ideally, an effective vaccine would be capable of eliciting both strong cellular responses and the production of broad-spectrum neutralizing antibodies capable of neutralizing HIV-1 strains of various clades.

The high genetic variability of HIV-1 makes the development of an HIV-1 vaccine an unprecedented challenge. In order to improve coverage of potential T-cell epitopes and improve cellular responses, HIV-1 Gag, Pol, and Env mosaic antigens derived from HIV group antigen (Gag), polymerase proteins (Pol), and envelope proteins (Env) of HIV have been described by other authors. and designed in an attempt to provide maximum coverage of potential T cell epitopes (eg, Barouch et al., Nat Med 2010, 16: 319-323). Mosaic antigens are similar in length and domain structure to naturally occurring wild-type HIV-1 antigens.

Sequences encoding mosaic antigens have been cloned into vectors such as those based on recombinant adenovirus serotype 26 (rAd26) and these recombinant vectors have been used in vaccines to generate immune responses to HIV (see, for example, Barouch et al. supra; and WO 2010/059732). Viral vectors expressing such HIV mosaic antigens have been shown to be effective in activating the immune response to HIV infection.

Another therapeutic strategy that has been investigated in inducing immune responses to HIV is the use of HIV trimeric envelope proteins such as gp140 as immunogens in vaccines. The native shell spike on the surface of HIV is trimeric. Examples of trimeric envelope proteins include the gp140 protein from clade C and the mosaic trimeric envelope protein such as those disclosed in WO 2014/042942 and WO 2014/107744.

The gp140 protein from clade C has been previously described, for example, in WO 2010/042942 and in Nkolola et al., 2010, but these disclosures did not focus on the development of any pharmaceutical formulation. In these disclosures, in some of the experiments, the protein was in phosphate buffered saline (PBS). Mosaic gp140 has been described previously, for example, in WO 2014/107744 and in Nkolola et al., 2014, but again no attention has been paid to the development of any pharmaceutical formulation in these disclosures. In these disclosures, in some of the experiments the protein was in 25 mM Tris at pH 7.5 and 150 mM NaCl.

Trimeric HIV envelope proteins such as gp140 are capable of inducing strong immune responses. To provide enhanced immunity against HIV, such envelope proteins can also be administered in combination with other HIV antigens, such as mosaic antigens. However, the stability of HIV envelope proteins in the form of trimers is not optimal under the conditions commonly used for clinical and commercial production. The trimeric envelope proteins of HIV are susceptible to both chemical and physical degradation. Moreover, many different factors, such as buffer composition, can affect the stability of proteins, and these effects are often unpredictable. For example, the use of HEPES buffer in protein formulations has been shown to generate hydrogen peroxide when exposed to natural light during the manufacturing process, which can affect the stability of both the protein and other formulation components such as surfactants. See, for example, Baicu et al., Cryobiology (2002) 45(1) 33-48; Lepe-Zuniga et al., J. Immunol. Methods (1987) 103(1), 145; and Zigler et al., In Vitro Cell. dev. Biol. (1985) 21(5), 282-287. It is desirable to have a gp140-based HIV vaccine formulation that is suitable for the stability of different variants of the gp140 protein, such as gp140 from clade C or mosaic gp140, and preferably formulated with an aluminum phosphate adjuvant in a single vial formulation (instead of being completely dependent on mixing in a pharmacy just prior to delivery of the vaccine) and furthermore would enable the manufacture of a drug product that satisfies the broad late phase and industrial scale requirements. As a rule, it is impossible to predict which combination of ingredients will result in a composition that will satisfy all of these requirements.

Therefore, there is a need in the art for improved formulations based on HIV gp140 proteins with better stability under clinical and commercial manufacturing conditions in order to realize the full therapeutic potential of such trimeric envelope proteins. These formulations must also be compatible for use with additional HIV antigen(s), including vectors expressing HIV antigen(s), and/or adjuvants.

Brief description of the invention

The invention relates to immunogenic compositions based on HIV gp140 proteins, which are characterized by improved stability. The immunogenic compositions of the present invention can be stored under refrigerated conditions for extended periods of time and are

- 1 039968 more optimal for use in production for clinical and commercial purposes. The immunogenic compositions of the present invention may also contain an adjuvant. The present invention also relates to methods for preparing immunogenic compositions and methods for using immunogenic compositions to induce an immune response to HIV.

In one general aspect, the present invention relates to an immunogenic composition containing, relative to the total volume of the composition

a) 0.05 to 5 mg/ml HIV gp140 protein;

b) 2 to 15% (w/v) sorbitol;

c) 0.01 to 0.05% (w/v) polysorbate 20;

d) 5 to 20 mM histidine buffer pH 5.5 to 7.0.

Preferably, the immunogenic composition according to an embodiment of the present invention comprises a stabilized trimeric HIV gp140 protein, such as the HIV gp140 protein, comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

In certain embodiments, the implementation of the present invention, the immunogenic composition contains from 0.2 to 1 mg/ml of HIV gp140 protein.

In certain embodiments, the implementation of the immunogenic composition according to the embodiment of the present invention contains from 5 to 12% (w/v) sorbitol.

In certain embodiments, the immunogenic composition contains 0.02% (w/v) polysorbate 20.

In certain embodiments, the implementation of the immunogenic composition contains 10 mm histidine buffer with a pH of 6.5.

According to embodiments of the present invention, the immunogenic composition may further comprise an adjuvant, such as an aluminum phosphate adjuvant. In some of these embodiments, aluminum phosphate may be present in the composition at a concentration of 0.7-5.0 mg/ml, such as 0.8-4.0 mg/ml, such as 0.85, 1, 1.5, 2, 0, 2.5, 3.0, 3.5, 3.84, 4.0 mg/mL. In certain embodiments, aluminum phosphate is present in the immunogenic composition at a concentration of 0.85 mg/ml. In certain embodiments, aluminum phosphate is present in the immunogenic composition at a concentration of 3.84 mg/mL.

In a preferred embodiment of the present invention, the immunogenic composition contains, relative to the total volume of the composition

a) 0.2 to 1 mg/ml HIV gp140 protein containing the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2;

b) 5 to 12% (w/v) sorbitol;

c) 0.02% (w/v) polysorbate 20;

d) 10 mM histidine buffer pH 6.5;

e) an aluminum phosphate adjuvant, preferably at a concentration of 0.7-4.0 mg/ml.

In another general aspect, the present invention relates to a method for preparing an immunogenic composition, comprising mixing

a) 0.05 to 5 mg/ml HIV gp140 protein;

b) 2 to 15% (w/v) sorbitol;

c) 0.01 to 0.05% polysorbate 20;

d) 5 to 20 mM histidine buffer pH 5.5 to 7.0, thus obtaining an immunogenic composition.

In certain embodiments, the immunogenic composition of the present invention contains an aluminum phosphate adjuvant, preferably at a concentration of 0.7-4.0 mg/ml, and is stable when stored at 2-8°C for at least one month , preferably at least 2, 3, 4, 5, 6 months, more preferably at least 7, 8, 9, 10, 11, 12 months, even more preferably at least 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 months, most preferably at least 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 months or longer, e.g. 1- 72 months e.g. 6-48 months e.g. 12-36 months. In certain embodiments, the immunogenic composition of the present invention contains an aluminum phosphate adjuvant, preferably at a concentration of 0.7-4.0 mg/ml, and is stable at 25°C for at least 6 months, for example 6- 12 months or 6-24 months. In certain embodiments, the immunogenic composition of the present invention contains an aluminum phosphate adjuvant, preferably at a concentration of 0.7-4.0 mg/ml, and is stable at 40°C for at least 1 week, for example 1- 12 weeks, such as at least 2 weeks, 3 weeks, 4 weeks, 1 month, such as 1-2 months, 1-3 months or 3-6 months.

And in another general aspect, the present invention relates to a method of inducing an immune response to human immunodeficiency virus (HIV) in a subject in need thereof, which comprises administering to the subject an effective amount of an immunogenic composition of the present invention.

In a specific embodiment, a method for inducing an immune response to HIV comprises

- 2 039968 introduction to a subject in need of it, an immunogenic composition containing, relative to the total volume of the composition

a) 0.2 to 1 mg/ml HIV gp140 protein containing the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2;

b) 5 to 12% (w/v) sorbitol;

c) 0.02% polysorbate 20;

d) 10 mM histidine buffer pH 6.5;

e) an aluminum phosphate adjuvant, preferably at a concentration of 0.7-4.0 mg/ml.

In certain embodiments, the method of inducing an immune response to HIV further comprises administering to the subject an effective amount of a second immunogenic composition comprising or encoding one or more additional HIV antigens, such as those comprising or encoding the amino acid sequence of SEQ ID NOs: 3-12 . Preferably, the method comprises administering one or more vectors, preferably adenovirus serotype 26 vectors, encoding one or more HIV antigens, such as those containing the amino acid sequence of SEQ ID NOs: 3-12. Methods for inducing an immune response to HIV may also include the introduction of one or more MVA-based vectors encoding one or more HIV antigens, such as those containing the amino acid sequence of SEQ ID NO: 3-12. One or more additional HIV antigens may also contain SEQ ID NO: 11, which has one or more mutations selected from the group consisting of (i) I529P (change Ile to Pro at position 529), (ii) K480E (change Lys to Glu at position 480) and (iii) combinations of EK479-4 0RRRR (i.e., replacement of GluLys at positions 479 and 480 by four Arg residues in succession), I529P (replacement of Ile by Pro at position 529), A471C (replacement of Ala with Cys at position 471) and T575C (replacement of Thr with Cys at position 575). In one embodiment, the antigen containing SEQ ID NO: 11 contains SEQ ID NO: 12.

In another general aspect, the present invention relates to a method for preparing an immunogenic composition that is stable under long-term storage conditions and contains the HIV gp140 protein, the method comprising (i) mixing the following components to obtain an immunogenic composition containing these components in amounts relative to the total volume of the composition :

(a) 0.05 to 5 mg/ml HIV gp140 protein;

(b) 2 to 15% (w/v) sorbitol;

(c) 0.01 to 0.05% (w/v) polysorbate 20;

(d) 5 to 20 mM histidine buffer pH 5.5 to 7.0;

(e) water;

(f) an aluminum phosphate adjuvant, preferably at a concentration of 0.7-4.0 mg/ml;

(ii) storing the composition at 2-8° C. for at least one month, such as 1-72 months, such as 6-48 months, such as 12-36 months.

Brief description of the figures

The foregoing summary, as well as the following detailed description of the present invention, will be better understood when read in conjunction with the accompanying drawings. It should be understood that the present invention is not limited to the specific embodiments shown in the drawings.

The figures show the following.

In FIG. 1A and 1B show results of analysis of HIV gp140 protein formulations prepared with acetate buffer, phosphate buffer and histidine buffer using SolvoVPE and results of analysis of HIV gp140 protein formulations prepared with sorbitol and sucrose by dynamic light scattering. (DLS), in particular in FIG. 1A shows the results of analysis using SolvoVPE; plotted the dependence of the change in absorbance at 350 nm (turbidity) on the change in absorbance at 280 nm (concentration); data points represent the change between samples analyzed at time 0 (T ₀ ) and after stressing the samples at 40° C. for 24 hours (T ₂₄ ); analysis using SolvoVPE was carried out as described in example 1;

in fig. 1B shows the results of the DLS analysis; the dependence of the change in radius (R _h ) from time 0 (T ₀ ) at 20°C to time 7 days (T _{7 days} ) at 70°C for sugar was plotted (comparison of sucrose and sorbitol); DLS analysis was carried out as described in example 1;

in fig. 2A-F show the results of high performance size exclusion chromatography (HP-SEC) analysis of the HIV gp140 formulations described in Example 2; the relative abundance of hexameric, trimeric, and high/low molecular weight structures based on the HIV gp140 protein from clade C was determined using HP-SEC of samples analyzed at time 0 (T0) and after stress exposure of the samples at 40°C for 24 h ( T ₂₄ ); Tri+Hex refers to the relative number of trimeric and hexameric structures based on the HIV gp140 protein from clade C; LMW refers to low molecular weight structures, such as cleavage or degradation products, in particular

- 3 039968 in FIG. 2A shows the relative abundance (%) of hexameric and trimeric structures of clade C HIV gp140 protein in _T0 samples at different protein concentrations (0.2 and 1.0 mg/mL);

in fig. 2B shows the relative abundance (%) of hexameric and trimeric structures of clade C HIV gp140 protein in _T0 samples at various surfactant concentrations (0.02% and 0.1%); the data shown is a combination of data collected from the analysis of samples containing PS20 and samples containing PS80;

in fig. 2C shows the relative abundance (%) of hexameric and trimeric structures of clade C HIV gp140 protein in _T24 samples at different protein concentrations (0.2 and 1.0 mg/mL);

in fig. 2D shows the relative abundance (%) of hexameric and trimeric structures of clade C HIV gp140 protein in T24 samples at various surfactant concentrations (0.02% and 0.1%);

the data shown is a combination of data collected from the analysis of samples containing PS20 and samples containing PS80;

in fig. 2E shows the relative abundance (%) of small molecule structures of the HIV gp140 protein from clade C, depending on the type of surfactant (comparison of PS20 and PS80) in samples T ₀ ;

in fig. 2F shows the relative abundance (%) of clade C HIV gp140 small molecule structures in T ₂₄ samples at different surfactant concentrations;

in fig. 3A and B show the results of stability studies of HIV gp140 protein formulations in accordance with embodiments of the present invention; HIV gp140 mosaic protein formulations were subjected to multiple freeze-thaw cycles as described in Example 3;

in fig. 3A shows protein concentration data after one, three and five freeze-thaw cycles determined by measuring absorbance at 280 nm;

in fig. 3B shows haze data for tested formulations after one, three and five freeze-thaw cycles, determined by measuring absorbance at 350 nm;

in fig. 4A-C show the results of stability studies of formulations based on HIV gp140 protein from clade C; clade C HIV gp140 protein formulations were stored at 25°C and 60% and 40°C relative humidity (RH) and 75% RH both with and without aluminum phosphate adjuvant and then tested using an analytical a method based on the use of SDS under reducing conditions, as described in example 4;

in fig. 4A shows relative protein concentration (%) in clade C HIV gp140 formulations stored at 25°C and 60% RH without aluminum phosphate adjuvant;

in fig. 4B shows relative protein concentration (%) in clade C HIV gp140 formulations stored at 40°C and 75% RH without aluminum phosphate adjuvant;

in fig. 4C shows relative protein concentration (%) in clade C HIV gp140 formulations stored at 25°C and 60% RH with an aluminum phosphate adjuvant;

in fig. 4D shows the relative protein concentration (%) in clade C HIV gp140 formulations stored at 40°C and 75% RH with an aluminum phosphate adjuvant.

Detailed description of the invention

Various publications, articles, and patents are cited or described in the Background section and throughout the specification; and each of these references is incorporated herein by reference in its entirety. The discussion of documents, acts, materials, devices, products, etc., which has been included in the present description, is intended to provide the context of the present invention. Such discussion is not an admission that any or all of these materials are part of the prior art with respect to any disclosed or claimed inventions.

Unless otherwise defined, all technical and scientific terms used in this document have the same meaning as is commonly understood by one of ordinary skill in the art to which the present invention pertains. Otherwise, certain terms used in this document have the meanings set forth in the description. All patents, published patent applications and publications cited in this document are incorporated by reference as if they were fully set forth in this document. It should be noted that the singular forms used herein and in the appended claims include reference to the plural unless the context clearly indicates otherwise.

Unless otherwise indicated, any numerical values such as concentration or range of concentrations described herein should in all cases be understood as modified by the term approximately. Thus, the numerical value usually includes values of ±10% of the specified value. For example, a concentration of 1 mg/mL includes all values from 0.9 to 1.1 mg/mL. In the same way, the concentration range from 1 to 10% (w/v) includes the range from 0.9% (w/v) to 11% (w/v). As used herein, the use of a numeric range unambiguously includes all possible subranges, all numeric values in that range, including integers in such ranges, and fractional values, unless the context explicitly states otherwise.

As used herein, subject means any animal, preferably a mammal, most preferably a human, to which an immunogenic

- 4 039968 composition according to embodiments of the present invention. As used herein, the term mammal encompasses any mammal. Examples of mammals include, but are not limited to, mice, rats, rabbits, guinea pigs, monkeys, humans, etc., more preferably humans.

The present invention relates generally to immunogenic compositions comprising the HIV gp140 protein and optionally an adjuvant, methods for preparing and storing such compositions, and methods for inducing an immune response to HIV in a subject using the immunogenic compositions alone or in combination with one or more additional HIV antigens, which are preferably expressed by one or more vectors.

HIV antigens

The human immunodeficiency virus (HIV) is a member of the genus Lentivirinae, which is part of the Retroviridae family. Two varieties of HIV infect humans: HIV-1 and HIV-2. HIV-1 is the most common strain of the HIV virus and is known to be more pathogenic than HIV-2. As used herein, the terms human immunodeficiency virus and HIV include, without limitation, HIV-1 and HIV-2.

HIV is classified into several clades with a high degree of genetic divergence. As used herein, the terms HIV clade or HIV subtype refer to related human immunodeficiency viruses classified according to their degree of genetic similarity. There are currently three groups of HIV-1 isolates: M, N, and O. Group M (major strains) consists of at least ten clades from A to J. Group O (distant strains) may consist of a similar number of clades. The N group is a new HIV-1 isolate that has not been assigned to any of the M or O groups.

As used herein, the terms HIV antigen, HIV antigenic protein, HIV antigenic polypeptide, and HIV immunogen refer to a polypeptide capable of inducing an immune response, such as a humoral and/or cell-mediated response, to HIV in a subject. An HIV antigen can be an HIV protein, a fragment or epitope thereof, or a combination of several HIV proteins or parts thereof, which can induce an immune response or confer immunity, such as protective immunity, to HIV in a subject.

The antigen is preferably capable of eliciting a protective immune response in the host, such as inducing an immune response to a viral disease or infection and/or conferring immunity to (i.e., vaccinating) the subject to the viral disease or infection that protects the subject from the viral disease or infection. . For example, the antigen may comprise the HIV protein or fragments thereof, such as the products of the HIV gag, pol, and env genes.

The HIV antigen may be any HIV-1 or HIV-2 antigen or fragment thereof. Examples of HIV antigens include, without limitation, gag, pol, and env gene products that encode structural proteins and essential enzymes. The gag, pol, and env gene products are synthesized as polyproteins, which are further processed into several other protein products. The main protein product of the gag gene is a polyprotein of the viral structural protein Gag, which is further processed to the protein products MA, CA, SP1, NC, SP2 and P6. The pol gene encodes viral enzymes (Pol, polymerase), and the main protein product is further processed into the protein products RT, RNase H, IN, and PR. The env gene encodes structural proteins, in particular the virion envelope glycoproteins. The main protein product of the env gene is gp160, which is further processed to gp120 and gp41.

In certain embodiments, the HIV antigen comprises an HIV Gag, Env, or Pol antigen, or any antigenic portion or epitope, or combination thereof, preferably an HIV-1 Gag, Env, or Pol antigen, or any antigenic portion, or epitope, or combination thereof.

HIV antigens can also be HIV mosaic antigens. As used herein, a mosaic antigen refers to a recombinant protein assembled from natural sequence fragments. Mosaic antigens are similar to natural antigens, but are optimized to maximize the coverage of potential T-cell epitopes found in natural sequences, which improves the spectrum and coverage of the immune response. The HIV mosaic antigens for use in the present invention are preferably Gag, Pol and/or Env HIV-1 mosaic antigens. HIV Gag, Pol and/or Env mosaic antigens are mosaic antigens containing multiple epitopes derived from one or more HIV Gag, Pol and/or Env polyprotein sequences. For example, a GagPol mosaic antigen contains a Gag mosaic sequence and a Pol mosaic sequence.

Examples of Gag, Pol and/or Env HIV mosaic antigens that can be used in the present invention include those described, for example, in US 20120076812; Barouch et al., Nat Med 2010, 16:319323; and Barouch et al., Cell 155:1-9, 2013, all of which are incorporated herein by reference in their entirety. Gag, Pol and/or Env HIV mosaic antigens for use in the present invention preferably include, without limitation, moslEnv (SEQ ID NO: 3), mos2Env (SEQ ID NO: 4), mos1Pol (SEQ ID NO: 5), mos2Pol ( SEQ ID NO: 6), mos1Gag (SEQ ID NO: 7), mos2Gag (SEQ ID

- 5 039968

NO: 8) and combinations thereof, such as moslGagPol (SEQ ID NO: 9) and mos2GagPol (SEQ ID NO: 10). Other examples of HIV mosaic antigens include synthetic HIV Env proteins, which are non-naturally occurring HIV envelope proteins optimized to induce an immune response or confer immunity to one or more naturally occurring strains of HIV, such as the one containing SEQ ID NO: 11 or SEQ ID NO: 11 which has one or more mutations selected from the group consisting of (i) I529P (change Ile to Pro at position 529), (ii) K480E (change Lys to Glu at position 480) and ( iii) combinations of EK479-480RRRR (i.e., replacement of GluLys at positions 479 and 480 with four Arg residues in succession), I529P (replacement of Ile with Pro at position 529), A471C (replacement of Ala with Cys at position 471) and T575C (replacing Thr with Cys at position 575) or, in preferred embodiments, the antigen comprising SEQ ID NO: 11 contains the amino acid sequence of SEQ ID NO: 12 as described in PCT/EP 2016/081159 (filed December 15, 2016 of the year on behalf of Janssen V accines & Prevention B.V.), which is incorporated herein by reference in its entirety.

HIV gp140 protein

As used herein, the term HIV gp140 protein refers to the uncleaved ectodomain of the gp160 trimeric coat protein, ie. (gp160) ₃ . Embodiments of the present invention relate to improved HIV gp140 formulations, preferably trimeric and/or hexameric gp140 subunits associated with the ectodomain of gp41 subunits lacking the gp41 transmembrane and cytoplasmic segments. The HIV env gene encodes the precursor protein gp160, which undergoes proteolytic cleavage to form two mature envelope glycoproteins gp120 and gp41. gp160 is first trimerized to (gp160) ₃ and then cleaved into two non-covalently linked proteins gp120 and gp41 by a cleavage reaction mediated by the host cell protease, furin, in a sequence that is highly conserved among retroviral envelope glycoprotein precursors.

Virus entry into the cell is subsequently mediated by the trimer of the gp120/gp41 heterodimers. Gp120 is a receptor binding fragment and binds to the CD4 receptor on a target cell that has such a receptor, such as, for example, a T helper cell. Gp41, which is non-covalently bound to gp120, is a fusion fragment and provides a second step through which HIV enters the cell. Gp41 is initially enveloped by the virus, but when gp120 binds to the CD4 receptor, gp120 changes its conformation, which causes gp41 to become accessible and in this case can facilitate fusion with the host cell. The HIV gp140 protein was used as an identifier for the native state of the split spike of the virus.

The expression of gp140 proteins has been described in several publications (e.g., Zhang et al., 2001; Sanders et al., 2002; Harris et al., 2011) and variants of this protein are now available from service providers, e.g. based on different strains of HIV. The gp140 protein of the present invention may have a cleavage site mutation such that the gp120 domain and gp41 ectodomain are covalently linked, or alternatively, the gp120 domain and gp41 ectodomain may be non-covalently linked (e.g., via a disulfide bridge, such as in SOSIP variants). gp140 proteins have been used in various vaccination experiments (e.g., Nkolola et al., 2010, 2014; Kovacs et al., 2012; Barouch et al., 2015; Sanders et al., 2015).

In accordance with embodiments of the present invention, the HIV gp140 protein may be a homotrimer (eg, trimers containing three identical polypeptide units) or a heterotrimer (eg, trimers containing three polypeptides, not all of which are identical). The HIV gp140 protein can also be a hexamer. Both the trimeric structures and the hexameric structures of the HIV gp140 protein are immunogenic against HIV or can induce immune responses to HIV in vivo. Preferably, the HIV gp140 protein is a trimer, and more preferably a homotrimer. The HIV gp140 protein may be a naturally occurring sequence, for example, a sequence isolated from any HIV clade such as clade A, clade B, clade C, etc., or a gp140 mosaic protein. The gp140 mosaic protein contains multiple epitopes derived from one or more gp140 sequences from one or more HIV clades.

Preferably, the HIV gp140 protein is a stabilized trimeric gp140 protein. A stabilized gp140 trimer protein is a gp140 protein that may have or be modified to include a polypeptide sequence, such as a trimerization domain that enhances the stability of the trimeric structure, or it may be a gp140 protein that is modified such that to contain mutations (when compared to native gp140 sequences) that stabilize the trimeric structure, such as SOSIP and/or other mutations. Examples of trimerization domains include, but are not limited to, a T4-fibrin foldon trimerization domain, for example, having the amino acid sequence GSGGYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 13); a supercoiled trimerization domain derived from GCN4, for example having the amino acid sequence MKQIEDKIEEILSKIYHIENEIARIKKLIGEV (SEQ ID NO: 14);

and the E. coli aspartate transcarbamoylase catalytic subunit as a trimerization marker. Such trimerization domains can be used to ensure the formation of a stable trimer (for stabilized trimers of gp140 proteins, see for example WO 2010/042942, Nkolola et al., 2010, WO 2014/107744 and Nkolola et al. 2014). The stabilized trimeric gp140 protein for use in the present invention may also contain cleavage site mutations that increase stability, such as furin cleavage sites and/or so-called SOSIP mutations (see, for example, Sanders et al., 2002, 2015). The stabilized trimeric gp140 protein can be obtained from the gp140 protein isolated from any HIV clade, eg clade A, clade B, clade C, etc. The stabilized gp140 trimer protein may also be a stabilized gp140 mosaic protein.

Exemplary HIV gp140 proteins that can be used in the present invention include clade C HIV gp140 protein (SEQ ID NO: 1), HIV gp140 mosaic protein (SEQ ID NO: 2), and combinations thereof. Both the HIV gp140 protein from clade C (SEQ ID NO: 1) and the HIV gp140 mosaic protein (SEQ ID NO: 2) are stabilized gp140 trimer proteins containing the T4-fibrin foldon trimerization domain.

Immunogenic compositions

In a first aspect, the present invention relates to an immunogenic composition comprising the HIV gp140 protein.

An immunogenic composition, as used herein, means a composition capable of inducing an immune response in a subject to which the present composition has been or will be administered. The immunogenic composition may be a vaccine. Vaccine means a composition that can provide protective immunity or a protective immune response in a subject or that can be used to vaccinate a subject. According to embodiments of the present invention, any HIV gp140 protein known in the art in light of the present invention may be used in the immunogenic composition of the present invention. The immunogenic composition may contain one or more HIV gp140 proteins, for example one, two or three HIV gp140 proteins.

Preferably, the immunogenic composition of the present invention contains a stabilized gp140 trimeric protein. In one preferred embodiment, the HIV gp140 protein is an HIV gp140 protein from clade C, such as that containing the amino acid sequence of SEQ ID NO: 1. In another preferred embodiment, the HIV gp140 protein is a mosaic HIV gp140 protein, such as that , which contains the amino acid sequence under SEQ ID NO: 2.

In other embodiments, the immunogenic composition comprises both the HIV gp140 protein from clade C and the HIV gp140 mosaic protein, such as those of SEQ ID NO: 1 and SEQ ID NO: 2. For example, the immunogenic composition may comprise a mixture of the HIV gp140 protein from clade C containing the amino acid sequence of SEQ ID NO: 1 and the HIV gp140 mosaic protein containing the amino acid sequence of SEQ ID NO: 2, for example in a ratio of 1:1.

The immunogenic composition of the present invention also contains water, preferably water for injection. It is added to the composition in sufficient quantity (q.s.), depending on the volume of the composition to be prepared.

In accordance with embodiments of the present invention, the immunogenic composition contains from 0.05 to 5 mg/ml HIV gp140 protein, for example 0.05, 0.1, 0.2, 0.5, 1, 2, 3, 4 or 5 mg /ml In specific embodiments of the present invention, the immunogenic composition comprises 0.2 or 1 mg/ml of the HIV gp140 protein, such as that containing SEQ ID NO: 1 or SEQ ID NO: 2. In certain other embodiments of the present invention, the immunogenic composition comprises from 0.05 to 5 mg/ml of a mixture of HIV gp140 proteins, such as those containing SEQ ID NO: 1 and SEQ ID NO: 2, for example in a ratio of 1:1.

The immunogenic compositions of the present invention further comprise sorbitol (sugar), polysorbate 20 (surfactant), and a histidine buffer. The inventors unexpectedly found that the stability of the HIV gp140 protein in the composition containing histidine was improved compared to that in compositions containing other amino acids. The inventors also unexpectedly found that the inclusion of Polysorbate 20 in the composition further improved the stability of the HIV gp140 protein in the composition. Typically, buffers used for protein formulations contain a combination of acetate and sorbitol, or a combination of histidine and sucrose (see, for example, Uchiyama, Biochimica Biophysics Acta (2014) 1844, 2041-2052). The combination of histidine and sorbitol is not normally used in a buffer for protein formulations. To the inventors' knowledge, the combination of histidine and sorbitol with polysorbate 20 (surfactant) has not been used in any commercial protein formulations. Thus, it was surprising to see that the combination of histidine and sorbitol improved the stability of the HIV gp140 protein.

In accordance with embodiments of the present invention, the concentration of sorbitol may

- 7 039968 be in the range from 2 to 15% (w/v), for example, be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15% (w/v). In one preferred embodiment, the concentration of sorbitol is 5% (w/v). In another preferred embodiment, the concentration of sorbitol is 12% (w/v).

In accordance with embodiments of the present invention, the concentration of polysorbate 20 may range from 0.01 to 0.05% (w/v), for example, be 0.01, 0.02, 0.03, 0.04 or 0 .05% (w/v). In one preferred embodiment, the concentration of polysorbate 20 is 0.02% (w/v).

According to embodiments of the present invention, the concentration of the histidine buffer is in the range of 5 to 20 mM, eg 5, 10, 15 or 20 mM, and preferably 10 mM. The pH value of the histidine buffer is in the range of 5.5 to 7.0, for example 5.5, 6.0, 6.5 or 7.0, and preferably 6.5. In one preferred embodiment, the concentration of the histidine buffer is 10 mM and the pH of the histidine buffer is 6.5. Any pH value described herein should in all cases be understood as modified by the term approximately, which, when used in relation to a pH value, includes ±0.5 of the indicated pH value. Unless otherwise indicated, all pH values refer to the pH of the histidine buffer itself, which is included in the immunogenic composition of the present invention.

In certain embodiments of the present invention, the immunogenic composition further comprises an adjuvant. The terms adjuvant and immunostimulant are used interchangeably herein and are defined as one or more substances that stimulate the immune system or enhance an immune response. For example, an adjuvant can be used to enhance the immune response to the HIV gp140 protein and/or immunogenic composition of the present invention when administered alone or additionally in combination with one or more adenoviral vectors encoding one or more HIV antigens. Adjuvants suitable for use in the present invention should be those that are potentially safe, well tolerated and effective in humans, such as, for example, QS-21, Iscomatrix, Detox-PC, MPL-SE, MoGM-CSF, TiterMax- G, CRL-1005, GERBU, TERamide, PSC97B, Adjumer, PG-026, GSKI, GcMAF, B-alethine, MPC-026, Adjuvax, CpG ODN, Betafectin, aluminum salts (e.g. aluminum hydroxide and/or aluminum phosphate; an example of an aluminum phosphate adjuvant is AdjuPhos, sterilized aluminum phosphate wet gel suspension), Adjuplex and MF59.

The preferred adjuvant is an aluminum phosphate adjuvant.

In accordance with embodiments of the present invention, an aluminum phosphate adjuvant can be included in the immunogenic composition at concentrations of from about 0.7 to 5.0 mg/ml, for example 0.7, 0.75, 0.8, 0.85 , 0.9, 0.95 or 1 mg/ml, for example at a fixed concentration of 0.85 mg/ml or, for example, 1.5, 2.0, 2.5, 3.0, 3.5, 3, 6, 3.7, 3.8, 3.85, 3.9, 3.95, 4.0, 4.5 or 5.0 mg/ml, for example at a fixed concentration of 3.84 mg/ml. One of the objectives of the present invention was to provide formulations of long term stability with an aluminum phosphate adjuvant present together with the gp140 protein immunogen. Surprisingly, the liquid formulations of the present invention, which contain HIV gp140, histidine buffer, sorbitol, and polysorbate 20, were found to be stable for at least 6 months at 2-8°C. It was also found that these compositions are stable for at least 6 months at an elevated temperature of 25°C. In addition, these formulations have even been found to be stable for at least 2 weeks, 1 month or even up to 3 months at 40° C. as measured by SDS PAGE under reducing conditions.

In an exemplary embodiment of the present invention, the immunogenic composition comprises, relative to the total volume of the composition

a) 0.2 to 1 mg/ml HIV gp140 protein containing the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2;

b) 5 to 12% (w/v) sorbitol;

c) 0.02% (w/v) polysorbate 20;

d) 10 mM histidine buffer pH 6.5;

e) 0.7 to 4.0 mg/ml (eg 0.85 mg/ml or 3.84 mg/ml) of an aluminum phosphate adjuvant.

The immunogenic compositions of the present invention may be formulated in any form suitable for administration to a subject to facilitate administration and enhance efficacy, including, but not limited to, oral (enteral) administration and parenteral injections. Parenteral injections, for example, may include subcutaneous injection, intramuscular injection, or intradermal injection. It is also possible to formulate the immunogenic compositions of the present invention for other routes of administration, for example transmucosal, rectal, sublingual, oral or intranasal. Preferably, the immunogenic composition is formulated for intramuscular injection.

The immunogenic compositions of the present invention have the advantage that the protein

- 8 039968 gp140 HIV can be stored in a stable state in liquid form at a refrigerated temperature, for example from 2°C to 8°C, for long periods of time, such as approximately 2 years. In certain embodiments, the immunogenic compositions contain an adjuvant, such as an aluminum phosphate adjuvant. Immunogenic compositions containing an adjuvant are also compatible with storage in liquid form under refrigerated conditions for extended periods of time. The immunogenic compositions of the present invention are also compatible with storage in lyophilized form. Thus, the compositions of the present invention are preferably liquid formulations, meaning that they are in liquid form at the preferred storage temperature, i.e. at 2-8°C. Typically, liquid formulations or compositions in accordance with the present invention are aqueous suspensions, which means that not all of the protein and/or particulate material, such as aluminum phosphate, may be completely dissolved. In such cases, it is recommended to stir the composition before use. It is preferable to store the compositions of the present invention, which contain an aluminum phosphate adjuvant, at a temperature above the freezing point of water, most preferably at 2-8°C, for example 2°C, 3°C, 4°C, 5°C, 6°C, 7°C or 8°C. The advantages are that no resource-intensive and costly lyophilization and associated re-dissolution is required prior to use, there is no need for bedside mixing and separate storage of the aluminum phosphate adjuvant, and the formulations can be stored ready for use. In addition, storage under refrigerated conditions, but not freezing, makes possible the use of compositions easier in case of limited resources, for example, if it is not possible to freeze. Moreover, the observed stability retention of the compositions of the present invention at elevated temperatures (e.g. 25°C and even 40°C) indicates that random temperature fluctuations, for example when the composition is temporarily exposed to room temperature, even in the case of a warm climate, do not will have an immediate adverse effect on the vaccine composition of the present invention.

The compositions of the present invention, including unexpectedly those containing aluminum phosphate, are stable when stored at a temperature of 2-8°C for at least one day, one week, two weeks, one month, preferably at least 2, 3, 4, 5, 6 months. More preferably, these compositions are stable under these conditions for at least 7, 8, 9, 10, 11, 12 months, even more preferably at least 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24 months, most preferably at least 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 months or longer, such as 1-72 months, such as 6-48 months, for example 12-36 months. Thus, the present invention, in certain embodiments, provides compositions according to the present invention that are storage stable at 2-8°C for at least one month, at least three months, at least 6 months. Preferably, such compositions are stable for at least 12 months, at least 18 months, at least 24 months, at least 30 months, at least 36 months. In certain embodiments, the present invention provides for the use of the immunogenic compositions of the present invention for vaccinating a subject, preferably a human subject, after storing the immunogenic compositions at 2-8°C for at least one day, at least one week, at least two weeks, at least three weeks, at least one month, at least two months, at least three months, at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 , 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 months.

For the purposes of the present invention, an immunogenic composition that contains the HIV gp140 protein and which, after storage for at least one week, preferably at least one month, at a given temperature, when analyzed on an SDS PAGE gel under reducing conditions, shows a level of degradation of said gp140 protein, constituting no more than 25%, preferably no more than 10%, is considered to be an immunogenic composition stable under long-term storage conditions at the specified temperature. An immunogenic composition that contains the HIV gp140 protein in accordance with the present invention is considered stable under certain conditions (for example, 2-8°C) for a specified time (for example, 6 months), if, under these conditions, after the specified time, the specified gp140 protein when analyzed on an SDS PAGE gel under reducing conditions, shows a degradation rate of no more than 25%, preferably less than 20%, preferably less than 15%, preferably less than 10%, most preferably less than 5% (compared to the initial measurement at t =0). Degradation is visually observed as additional bands under the band of gp140 with the desired molecular weight. Alternative assays, such as ELISA, can also be used to measure stability, and in certain embodiments, a composition that is stable as defined above also exhibits a degradation rate of no more than 50%, preferably less than 25% in the ELISA assay ( decrease compared to the original signal at t=0).

- 9 039968

Methods for preparing an immunogenic composition

The present invention also relates to a method for preparing the immunogenic composition of the present invention. In accordance with embodiments of the present invention, a method for preparing an immunogenic composition includes mixing HIV gp140 protein, sorbitol, polysorbate 20, and a histidine buffer in appropriate concentration ranges. One of ordinary skill in the art will be well aware of the conventional techniques used to prepare such compositions.

For example, immunogenic compositions can be prepared by mixing histidine buffer, sorbitol, and polysorbate 20 at desired concentrations. The HIV gp140 protein can then be added. The pH of the composition can be adjusted before or after the addition of the HIV gp140 protein. As another illustrative example, a buffer solution containing histidine and sorbitol can first be prepared. The HIV gp140 protein is prepared in buffer at the required concentration and then added to a buffer solution containing histidine and sorbitol, followed by the addition of polysorbate at the target concentration. The adjuvant may be added last to form the final composition. The present invention also provides methods for preparing a long-term storage stable immunogenic composition that contains the HIV gp140 protein. In certain embodiments, such methods comprise (i) providing an immunogenic composition according to the present invention (i.e., containing 0.05-5 mg/mL HIV gp140 protein, 2-15% (w/v) sorbitol, 0 01-0.05% polysorbate 20, 5-20 mM histidine buffer pH 5.5-7.0, water and preferably 0.7-4.0 mg/ml aluminum phosphate) and (ii) storing said composition at 2-8°C for at least one month, such as 1-72 months, such as 6-48 months, such as 12-36 months, such as 18-30 months. In certain embodiments, such methods include (i) mixing the following components to form an immunogenic composition containing these components in amounts relative to the total volume of the composition:

(a) 0.05 to 5 mg/ml HIV gp140 protein;

(b) 2 to 15% (w/v) sorbitol;

(c) 0.01 to 0.05% (w/v) polysorbate 20;

(d) 5 to 20 mM histidine buffer pH 5.5 to 7.0;

(e) water (preferably water for injection);

(f) an aluminum phosphate adjuvant, preferably at a concentration of 0.7-4 mg/ml;

(ii) storing the composition at 2-8° C. for at least one month, such as 1-72 months, such as 6-48 months, such as 12-36 months, such as 18-30 months.

Methods for inducing an immune response

The present invention also relates to methods of inducing an immune response to human immunodeficiency virus (HIV) in a subject in need thereof using the immunogenic composition of the present invention. An immune response may occur against one or more HIV clades. The methods described herein also include administering an immunogenic composition of the present invention in combination with one or more additional HIV antigens that are preferably expressed from one or more vectors such as adenoviral or MVA vectors, including priming and enhancing the immune response.

In one general aspect, a method of inducing an immune response to human immunodeficiency virus (HIV) in a subject in need thereof comprises administering to the subject an effective amount of an immunogenic composition comprising an HIV gp140 protein according to an embodiment of the present invention. Any of the immunogenic compositions described herein can be used in a method for inducing an immune response to HIV in a subject. Preferably, the composition comprises a stabilized trimeric HIV gp140 protein, such as the HIV gp140 protein from clade C containing the amino acid sequence of SEQ ID NO: 1, or the HIV gp140 mosaic protein containing the amino acid sequence of SEQ ID NO: 2, or a mixture thereof. The composition may further contain an adjuvant, such as an aluminum phosphate adjuvant. An exemplary embodiment of an immunogenic composition for use in the methods of the present invention comprises, relative to the total volume of the composition

a) 0.2 to 1 mg/ml HIV gp140 protein containing the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2;

b) 5 to 12% (w/v) sorbitol;

c) 0.02% (w/v) polysorbate 20;

d) 10 mM histidine buffer pH 6.5;

e) 0.7 to 4.0 mg/ml (eg 0.85 or 3.84 mg/ml) of an aluminum phosphate adjuvant.

According to embodiments of the present invention, the induction of an immune response, when used in relation to the methods and compositions described herein, encompasses providing protective immunity and/or vaccinating a subject against an infection, such as an infection caused by HIV, for prophylactic purposes, as well as calling a person in need of this subject required immune

- 10 039968 response or effect against an infection, such as an infection caused by HIV, for therapeutic purposes. The methods of the present invention are preferably for prophylactic purposes, such as for providing protective immunity. The immune response may be a cellular immune response and/or a humoral immune response.

As used herein, the term protective immunity or protective immune response means that the vaccinated subject is able to control the infection caused by the pathogen against which the vaccination was performed. Typically, a subject who has developed a protective immune response develops only mild to moderate clinical symptoms or no symptoms at all. Typically, a subject having a protective immune response to or protective immunity to a particular pathogen will not die as a result of infection with said pathogen.

Typically, the administration of immunogenic compositions according to embodiments of the present invention will have a prophylactic goal of generating an immune response to the HIV antigen prior to infection or development of symptoms. In other embodiments, the immunogenic compositions may be administered for post-exposure prophylaxis. The immunogenic compositions of the present invention may also be administered to a non-human mammal, for example for experimental purposes.

As used herein, an effective amount or an immunologically effective amount means an amount of a composition sufficient to induce the desired immune effect or immune response in a subject in need thereof. In one embodiment, an effective amount means an amount sufficient to induce an immune response in a subject in need thereof. In certain embodiments, an effective amount means an amount sufficient to induce immunity in a subject in need thereof, for example, to provide a protective effect against a disease, such as a viral infection. In certain embodiments, an effective amount means an amount sufficient to enhance an immune response in a subject in need thereof. For example, when used in combination with one or more other components or immunogenic compositions capable of influencing an immune response, such as in a primer-booster regimen, the effective amount may be an amount sufficient to enhance the immune response induced by one or more other components or immunogenic compositions.

The effective amount may vary depending on a variety of factors such as the subject's physical condition, age, weight, health status, etc.; a specific application, whether it be inducing an immune response or providing protective immunity; and the specific disease, such as a viral infection, against which immunity is required. One of ordinary skill in the art can easily determine the effective amount in light of the present invention. As a general guide, when used against the HIV gp140 protein, an effective amount can be in the range, e.g., about 0.3 to about 3000 μg, e.g. 1-1000 μg, e.g. 10-500 μg, e.g. , 200, 250, 300, 350, 400, 450 or 500 mcg.

An effective amount of the immunogenic composition may be administered in one composition or in multiple compositions, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 compositions (e.g. tablets, capsules and/or injectables). ), wherein the administration of multiple compositions (eg, tablets, capsules, and/or injectables) collectively provides an immunogenically effective amount to the subject. In a so-called primer-booster scheme, it is also possible to administer to a subject an effective amount of the immunogenic composition, followed by administering to the same subject another dose of an effective amount of the immunogenic composition, as described in more detail below.

According to embodiments of the present invention, the immunogenic composition may be administered to a subject by any means known in the art, including, without limitation, oral (enteral) administration and parenteral injections. Parenteral injections may include, for example, subcutaneous injection, intramuscular injection, or intradermal injection. Preferably, the immunogenic composition is administered by intramuscular injection.

It is also possible to administer the immunogenic compositions of the present invention together with one or more additional HIV antigens or one or more vectors, such as adenoviral vectors, encoding one or more additional HIV antigens. As used herein, co-delivery, co-administration, administration together, or administration in combination with refers to the simultaneous administration of two or more components, such as an immunogenic composition containing the HIV gp140 protein, or multiple viral expression vectors, such as adenoviral vectors. . Simultaneous administration may be the administration of two or more components on at least the same day. If the two components are administered together with each other, they can be administered in separate compositions sequentially within a short period of time, such as 24, 20, 16, 12, 8 or 4 hours, or within 1 hour or

- 11 039968 less, or they can be entered in one composition at the same time. Non-limiting examples of administering immunogenic compositions based on the gp140 protein with one or more additional HIV antigens encoded by vectors, such as adenoviral vectors, are provided in WO 2016/049287, the disclosure of which is incorporated herein by reference in its entirety.

Thus, in certain embodiments of the present invention, the method of inducing an immune response further comprises administering to the subject an effective amount of a second immunogenic composition comprising one or more HIV antigens or one or more vectors encoding HIV antigens. Any HIV antigen known to those skilled in the art may be used in light of the present invention, such as Nef, Gag, Env or Pol HIV antigens, or any antigenic portion or epitope or combination thereof. HIV mosaic antigens can also be used. Exemplary HIV antigens include, without limitation, Env, Gag, and/or Pol mosaic antigens and combinations thereof, such as those containing the amino acid sequences of SEQ ID NO: 3-12 or SEQ ID NO: 11, which has one or more mutations, selected from the group consisting of (i) I529P (change of Ile to Pro at position 529), (ii) K480E (change of Lys to Glu at position 480) and (iii) combination EK479-480RRRR (i.e. change of GluLys at positions 479 and 480 into four consecutive Arg residues), I529P (He replaced by Pro at position 529), A471C (Ala replaced by Cys at position 471), and T575C (Thr replaced by Cys at position 575). Additional HIV antigens can be administered to the subject, for example, as isolated proteins or polypeptides, or as vectors encoding these proteins or polypeptides.

Preferably, in a method comprising administering to a subject an effective amount of a second immunogenic composition, the second immunogenic composition comprises one or more vectors encoding one or more HIV antigens.

Any vector known to those skilled in the art may be used in light of the present invention.

Preferably the vector is an adenoviral vector, more preferably an adenovirus serotype 26 vector. The preparation of recombinant adenoviral vectors is well known in the art. The production of recombinant vectors based on adenovirus serotype 26 (rAd26) is described, for example, in WO 2007/104792 and Abbink et al. (2007) Virol 81(9): 4654-63. Exemplary sequences of the Ad26 genome are found in GenBank under accession number EF 153474 and under SEQ ID NO: 1 in WO 2007/104792. Examples of vectors useful in the present invention include, for example, the vectors described in WO 2012/082918, the disclosure of which is incorporated herein by reference in its entirety. Other adenoviral vectors that can be used in combination with the immunogenic compositions of the present invention include those described in WO 2016/049287 and PCT Application No. PCT/EP 2016/081159, the disclosure of which is incorporated herein by reference in its entirety.

In accordance with embodiments of the present invention, adenoviral vectors may contain one HIV antigen or more than one HIV antigen, for example two, three or four or more HIV antigens. The immunogenic compositions may contain one or more adenoviral vectors, for example two, three or four or more HIV antigens encoding one or more different HIV antigens. Also in accordance with embodiments of the present invention, the second composition may contain one adenoviral vector or more than one adenoviral vector, for example two, three or four or more adenoviral vectors. If the second composition contains more than one adenoviral vector, the adenoviral vectors may encode the same or different HIV antigens.

Adenovirus vectors for use in the methods of the present invention encoding one or more HIV antigens contain a nucleic acid region encoding an HIV antigen that is operably linked to a promoter, meaning that the nucleic acid region is under the control of the promoter. The promoter may be a homologous promoter (ie derived from the same genetic source as the vector) or a heterologous promoter (ie derived from a different vector or genetic source). Examples of suitable promoters include the cytomegalovirus (CMV) promoter and the Rous sarcoma virus (RSV) promoter. The promoter is preferably located 5' from the nucleic acid site in the expression cassette.

As a general guide, when used with an adenoviral vector, an effective amount can range from about 108 viral particles to about 10 ¹² viral particles, such as 108, 10 ⁹ , 10 ¹⁰ , 10 ¹¹ , or 10 ¹² viral particles. The preparation and use of immunogenic compositions containing adenovirus vectors, such as vectors based on adenovirus serotype 26, is well known to those of ordinary skill in the art. Liquid pharmaceutical compositions typically contain a liquid carrier such as water, petrolatum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may also be included.

- 12 039968

For example, a recombinant adenoviral vector can be stored in a buffer that is also used for the international standard for adenovirus (Hoganson et al., 2002, Bioprocessing J 1: 43-8): 20 mM Tris pH 8, 25 mM NaCl and 2.5% glycerol . Another useful adenovirus formulation buffer suitable for administration to humans is 20 mM Tris, 2 mM MgCl ₂ , 25 mM NaCl, 10% (w/v) sucrose, and 0.2% (w/v) polysorbate -80. Another formulation buffer that is suitable for recombinant adenovirus contains 10-25 mM citrate buffer, pH 5.9-6.2, 4-6% (w/w) hydroxypropyl-beta-cyclodextrin (HBCD), 70- 100 mM NaCl, 0.018-0.035% (w/w) polysorbate-80 and optionally 0.3-0.45% (w/w) ethanol. Other buffers may be used, and some examples of suitable formulations for storage and for pharmaceutical administration of purified vectors are known.

The introduction of immunogenic compositions containing one or more additional HIV antigens or one or more adenoviral vectors encoding one or more additional HIV antigens, is usually intramuscular, intradermal or subcutaneous. However, other routes of administration may also be contemplated, such as intravenous, rectal, transdermal, oral, nasal, etc. Intramuscular administration of immunogenic compositions can be carried out using a needle for injection of a suspension of expression vectors, for example adenoviral vectors, and/or antigenic polypeptides. An alternative is to use a needleless injection device to administer the composition (using, for example, Biojector™) or a lyophilized powder containing the vaccine.

In one embodiment, an immunogenic composition comprising a gp140 protein according to an embodiment of the present invention is used in combination with an adenoviral vector, preferably an adenovirus serotype 26 vector, encoding an HIV antigen comprising the amino acid sequence of SEQ ID NO: 3.

In one embodiment, an immunogenic composition comprising the HIV gp140 protein according to an embodiment of the present invention is used in combination with an adenoviral vector, preferably an adenovirus serotype 26 vector, encoding an HIV antigen comprising the amino acid sequence of SEQ ID NO: 4.

In one embodiment, an immunogenic composition comprising the HIV gp140 protein according to an embodiment of the present invention is used in combination with an adenoviral vector, preferably an adenovirus serotype 26 vector, encoding an HIV antigen comprising the amino acid sequence of SEQ ID NO: 5.

In one embodiment, an immunogenic composition comprising the HIV gp140 protein according to an embodiment of the present invention is used in combination with an adenovirus vector, preferably an adenovirus serotype 26 vector, encoding an HIV antigen comprising the amino acid sequence of SEQ ID NO: 6.

In one embodiment, an immunogenic composition comprising the HIV gp140 protein according to an embodiment of the present invention is used in combination with an adenoviral vector, preferably an adenovirus serotype 26 vector, encoding an HIV antigen comprising the amino acid sequence of SEQ ID NO: 7.

In one embodiment, an immunogenic composition comprising the HIV gp140 protein according to an embodiment of the present invention is used in combination with an adenoviral vector, preferably an adenovirus serotype 26 vector, encoding an HIV antigen comprising the amino acid sequence of SEQ ID NO: 8.

In one embodiment, an immunogenic composition comprising the HIV gp140 protein according to an embodiment of the present invention is used in combination with an adenoviral vector, preferably an adenovirus serotype 26 vector, encoding an HIV antigen comprising the amino acid sequence of SEQ ID NO: 9.

In one embodiment, an immunogenic composition comprising the HIV gp140 protein according to an embodiment of the present invention is used in combination with an adenoviral vector, preferably an adenovirus serotype 26 vector, encoding an HIV antigen comprising the amino acid sequence of SEQ ID NO: 10.

In one embodiment, an immunogenic composition comprising an HIV gp140 protein according to an embodiment of the present invention is used in combination with an adenovirus vector, preferably an adenovirus serotype 26 vector, encoding an HIV antigen comprising the amino acid sequence of SEQ ID NO: 11, wherein SEQ ID NO: 11 has one or more mutations selected from the group consisting of (i) I529P (change of Ile to Pro at position 529), (ii) K480E (change of Lys to Glu at position 480), and (iii) a combination of EK479-480RRRR (i.e. replacement of GluLys at positions 479 and 480 with four Arg residues in succession), I529P (replacement of Ile with Pro at position 529), A471C (replacement of Ala with Cys at position 471), and T575C (replacement of Thr with Cys at position 575) or SEQ ID NO: 12.

Upon administration of adenoviral vectors encoding one or more HIV antigens, the adenoviral vector expresses the encoded HIV antigens in such a way that the antigens are presented to the subject's immune system, thereby inducing the desired immune or other immune response.

- 13 039968 induction of an immune response. An immunogenic composition comprising the HIV gp140 protein according to an embodiment of the present invention may be administered together with one or more adenoviral vectors to prime the immune response and/or, if administered after one or more adenoviral vectors, to enhance the immune response.

Thus, in other embodiments of the present invention, a method of inducing an immune response to HIV in a subject in need thereof comprises (i) administering to the subject an effective amount of an immunogenic composition according to an embodiment of the present invention comprising the HIV gp140 protein and preferably further comprising an adjuvant, and (ii) administering to the subject an effective amount of a second immunogenic composition comprising one or more adenoviral vectors encoding one or more HIV antigens. Steps (i) and (ii) are performed in any order, with one of the steps being for primer immunization and the other for booster immunization. Optionally, the method may further include introducing one or more modified vaccinia ankara (MVA) vectors encoding one or more HIV antigens. MVA-based vectors can encode any HIV antigen described herein. Preferably, the MVA-based vectors encode one or more HIV antigens selected from the group consisting of SEQ ID NO: 3-12 or SEQ ID NO: 11 that has one or more mutations selected from the group consisting of (i) I529P ( substitution of Ile for Pro at position 529), (ii) K480E (replacement of Lys for Glu at position 480), and (iii) combinations of EK479-480RRRR (i.e. replacement of GluLys at positions 479 and 480 with four Arg residues following each other another), I529P (replacement of Ile with Pro at position 529), A471C (replacement of Ala with Cys at position 471), and T575C (replacement of Thr with Cys at position 575).

Examples of adenoviral vectors, MVA-based vectors, and primer-booster schedules that can be used in combination with an immunogenic composition containing the HIV gp140 protein according to an embodiment of the present invention include those described in WO 2016/049287, the disclosure of which is incorporated herein by reference. in its entirety.

For example, in one embodiment of the disclosed methods, one or more adenoviral vectors encoding one or more HIV antigens are used to prime an immune response. An immunogenic composition comprising the HIV gp140 protein of the present invention may be used in conjunction with one or more adenoviral vectors for priming immunization. The primer may be administered only once, but may also optionally be administered multiple times, such as with an initial primer at time 0 followed by another primer approximately 4-14 weeks, such as 10-14 weeks after the initial primer. An immunogenic composition comprising the HIV gp140 protein, optionally together with one or more additional adenoviral or MVA vectors encoding one or more additional HIV antigens, can be used to enhance the immune response. Booster immunization can also be performed once or several times, for example, first about 22-26 weeks after the initial primer, followed by another booster about 46-50 weeks after the initial primer. The immune response induced by immunization is monitored.

In other general aspects, the present invention relates to vaccine combinations for inducing an immune response to human immunodeficiency virus (HIV) in a subject in need thereof. According to embodiments of the present invention, the vaccine combination comprises an immunogenic composition comprising an HIV gp140 protein, such as that comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2; one or more adenovirus vectors, preferably vectors based on adenovirus serotype 26, encoding one or more HIV antigens, such as an HIV antigen containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-12, and wherein SEQ ID NO : 11 has one or more mutations selected from the group consisting of (i) I529P (change of Ile to Pro at position 529), (ii) K480E (change of Lys to Glu at position 480), and (iii) a combination of EK479-480RRRR ( i.e. the replacement of GluLys at positions 479 and 480 with four Arg residues in succession), I529P (replacement of Ile with Pro at position 529), A471C (replacement of Ala with Cys at position 471) and T575C (replacement of Thr with Cys at position 575), and optionally one or more MVA-based vectors encoding one or more HIV antigens, such as an HIV antigen containing an amino acid sequence selected from the group consisting of SEQ ID NOs 3-12, wherein SEQ ID NOs: 11 has one or more mutations, selected x from the group consisting of (i) I529P (change of He to Pro at position 529), (ii) K480E (change of Lys to Glu at position 480), and (iii) a combination of EK479-480RRRR (i.e. replacement of GluLys at positions 479 and 480 with four Arg residues following one another), I529P (replacement of Ile with Pro at position 529), A471C (replacement of Ala with Cys at position 471) and T575C (replacement of Thr with Cys at position 575) .

In accordance with embodiments of the present invention, vaccine combinations can be used in any of the methods described herein to induce an immune response to HIV in a subject in need thereof, including priming and enhancing immune

- 14 039968 response.

Embodiments

Embodiment 1 is an immunogenic composition containing, relative to the total volume of the composition

a) 0.05 to 5 mg/ml HIV gp140 protein;

b) 2 to 15% (w/v) sorbitol;

c) 0.01 to 0.05% (w/v) polysorbate 20;

d) 5 to 20 mM histidine buffer pH 5.5 to 7.0.

Embodiment 2 is the immunogenic composition of Embodiment 1 wherein the HIV gp140 concentration is 0.2 mg/mL.

Embodiment 3 is the immunogenic composition of Embodiment 1 wherein the HIV gp140 concentration is 1 mg/mL.

Embodiment 4 is an immunogenic composition according to any one of Embodiments 1-3, wherein the concentration of sorbitol is 5% (w/v).

Embodiment 5 is an immunogenic composition according to any one of Embodiments 1-3, wherein the concentration of sorbitol is 12% (w/v).

Embodiment 6 is an immunogenic composition according to any one of Embodiments 1-5, wherein the concentration of Polysorbate 20 is 0.02% (w/v).

Embodiment 7 is an immunogenic composition according to any one of Embodiments 1-6, wherein the concentration of the histidine buffer is 10 mM and the pH of the histidine buffer is 6.5.

Embodiment 8 is an immunogenic composition according to any one of Embodiments 1-7 further comprising an aluminum phosphate adjuvant, for example at a concentration of 0.7-1.0 mg/ml, preferably at a concentration of 0.85 mg/ml.

Embodiment 9 is an immunogenic composition according to any one of Embodiments 1-8, wherein the HIV gp140 protein comprises the amino acid sequence of SEQ ID NO: 1.

Embodiment 10 is an immunogenic composition according to any one of Embodiments 1-8, wherein the HIV gp140 protein comprises the amino acid sequence of SEQ ID NO: 2.

Embodiment 11 is an immunogenic composition containing, relative to the total volume of the composition

a) 0.2 to 1 mg/ml HIV gp140 protein containing the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2;

b) 5 to 12% (w/v) sorbitol;

c) 0.02% (w/v) polysorbate 20;

d) 10 mM histidine buffer pH 6.5;

e) an aluminum phosphate adjuvant, for example at a concentration of 0.7-4.0 mg/ml, for example 0.85 or 3.84 mg/ml.

Embodiment 12 is an immunogenic composition according to Embodiment 11 containing 0.2 mg/ml HIV gp140 protein containing the amino acid sequence of SEQ ID NO: 1 and 5% (w/v) sorbitol.

Embodiment 13 is an immunogenic composition according to Embodiment 11 containing 0.2 mg/ml HIV gp140 protein containing the amino acid sequence of SEQ ID NO: 2 and 5% (w/v) sorbitol.

Embodiment 14 is the immunogenic composition of Embodiment 11 containing 0.2 mg/ml HIV gp140 protein containing the amino acid sequence of SEQ ID NO: 1 and 12% (w/v) sorbitol.

Embodiment 15 is an immunogenic composition according to Embodiment 11 containing 0.2 mg/ml HIV gp140 protein containing the amino acid sequence of SEQ ID NO: 2 and 12% (w/v) sorbitol.

Embodiment 16 is an immunogenic composition according to Embodiment 11 containing 1.0 mg/ml HIV gp140 protein containing the amino acid sequence of SEQ ID NO: 1 and 5% (w/v) sorbitol.

Embodiment 17 is an immunogenic composition according to Embodiment 11 containing 1.0 mg/ml HIV gp140 protein containing the amino acid sequence of SEQ ID NO: 2 and 5% (w/v) sorbitol.

Embodiment 18 is an immunogenic composition according to Embodiment 11 containing 1.0 mg/ml HIV gp140 protein containing the amino acid sequence of SEQ ID NO: 1 and 12% (w/v) sorbitol.

Embodiment 19 is an immunogenic composition according to Embodiment 11 containing 1.0 mg/ml HIV gp140 protein containing the amino acid sequence of SEQ ID NO: 2 and 12% (w/v) sorbitol.

Embodiment 20 is an immunogenic composition according to any

- 15 039968 of embodiments 1-19, formulated for intramuscular injection.

Embodiment 21 is a method for preparing an immunogenic composition comprising mixing

a) 0.05 to 5 mg/ml HIV gp140 protein;

b) 2 to 15% (w/v) sorbitol;

c) 0.01 to 0.05% polysorbate 20;

d) 5 to 20 mM histidine buffer pH 5.5 to 7.0, thus obtaining an immunogenic composition.

Embodiment 22 is a method of inducing an immune response to HIV in a subject in need thereof, comprising administering to the subject an effective amount of an immunogenic composition containing, relative to the total volume of the composition

a) 0.05 to 5 mg/ml HIV gp140 protein;

b) 2 to 15% (w/v) sorbitol;

c) 0.01 to 0.05% polysorbate 20;

d) 5 to 20 mM histidine buffer pH 5.5 to 7.0.

Embodiment 23 is the method of Embodiment 22 wherein the immunogenic composition comprises, relative to the total volume of the composition

a) 0.2 to 1 mg/ml HIV gp140 protein containing the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2;

b) 5 to 12% (w/v) sorbitol;

c) 0.02% polysorbate 20;

d) 10 mM histidine buffer pH 6.5;

e) an aluminum phosphate adjuvant, for example at a concentration of 0.7-4.0 mg/ml, for example 0.85 or 3.84 mg/ml.

Embodiment 24 is a method of inducing an immune response to HIV in a subject in need thereof, comprising administering to the subject an effective amount of an immunogenic composition according to any of Embodiments 1-20 or any of Embodiments 36-37.

Embodiment 25 is the method of any one of embodiments 2224, further comprising administering to the subject an effective amount of one or more adenovirus vectors, preferably adenovirus serotype 26 vectors encoding one or more HIV antigens.

Embodiment 26 is the method of any one of embodiments 2225, further comprising administering to the subject an effective amount of one or more MVA-based vectors encoding one or more HIV antigens.

Embodiment 27 is the method of Embodiment 25 or Embodiment 26, wherein the one or more HIV antigens comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 3-12, and wherein SEQ ID NO: 11 has one or several mutations selected from the group consisting of (i) I529P (change of Ile to Pro at position 529), (ii) K480E (change of Lys to Glu at position 480), and (iii) a combination of EK479-480RRRR (i.e. change GluLys at positions 479 and 480 into four Arg residues following one another), I529P (replacement of Ile with Pro at position 529), A471C (replacement of Ala with Cys at position 471), and T575C (replacement of Thr with Cys at position 575).

Embodiment 28 is an immunogenic composition according to any of Embodiments 1-20 or any of Embodiments 36-37 which is stable at 2-8°C for at least six months.

Embodiment 29 is an immunogenic composition according to any of Embodiments 1-20 or any of Embodiments 36-37 which is stable at 2-8°C for at least 12 months, at least 18 months, at least at least 24 months, at least 30 months, at least 36 months.

Embodiment 30 is an immunogenic composition according to any of Embodiments 1-20 or any of Embodiments 36-37 which is stable at 2-8°C for 6-72 months.

Embodiment 31 is an immunogenic composition according to any of Embodiments 1-20 or any of Embodiments 36-37 which is stable at 25°C for at least six months.

Embodiment 32 is an immunogenic composition according to any of Embodiments 1-20 or any of Embodiments 36-37 which is stable at 40° C. for at least one week, preferably at least two weeks.

Embodiment 33 is a method for storing an immunogenic composition comprising the HIV gp140 protein, the method comprising obtaining an immunogenic composition according to any of Embodiments 1-20, or any of Embodiments 28-32, or any of Embodiments 36-37 and storage of said composition at 2-8°C for

at least one day, such as at least one week, such as at least one month, such as 1 day to 72 months, such as 6-48 months, such as 12-36 months, such as 1830 months.

Embodiment 34 is a method for preparing a long-term storage stable immunogenic composition that contains the HIV gp140 protein, the method comprising (i) providing an immunogenic composition that contains 0.05-5 mg/mL of the HIV gp140 protein, 215% (w/w ./vol.) sorbitol, 0.01-0.05% polysorbate 20, 5-20 mM histidine buffer pH 5.5-7.0, water and preferably 0.7-4.0 mg/ml aluminum phosphate, and (ii) storing said composition at 2-8°C for at least one week, such as at least one month, such as 1-72 months, such as 6-48 months, such as 12-36 months, such as 18-30 months.

Embodiment 35 is the use of an immunogenic composition according to any of Embodiments 1-20, or any of Embodiments 28-32, or any of Embodiments 36-37, for administration to a subject, preferably a human subject, to induce an immune response on HIV, wherein the immunogenic composition is stored at 2-8°C for at least one day, such as at least one week, such as at least two weeks, such as at least three weeks, such as at least one month, such as at least two months, such as at least three months, such as at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 months.

Embodiment 36 is an immunogenic composition according to any one of embodiments 1-9, wherein the HIV gp140 protein comprises a mixture of the HIV gp140 protein comprising the amino acid sequence of SEQ ID NO:1 and the HIV gp140 protein comprising the amino acid sequence of SEQ NO: 2.

Embodiment 37 is the immunogenic composition of Embodiment 36 wherein the HIV gp140 protein comprising the amino acid sequence of SEQ ID NO:1 and the HIV gp140 protein comprising the amino acid sequence of SEQ NO: 2 are present in a 1:1 mixture.

Examples

Example 1 Composition development study for HIV gp140 protein from clade C.

There are many possible variations of each of the components that can be included in a protein formulation, such as buffers, sugars, pH, surfactant, and so on. For example, various buffer options may include phosphate, acetate, HEPES, Tris, MOPS, etc.; different amino acid options may include histidine, arginine, lysine, alanine, etc.; various sugar options may include sucrose, sorbitol, glycerol, mannitol, trehalose, etc.; and various surfactant options may include Polysorbate 20, Polysorbate 80, Tween-20, Tween-80, etc. There are also many other types of excipients and possible variants of each that can be further included in the protein formulation, such as osmolytes (e.g. glycine, proline, glycerol, urea, etc.), salts (e.g. sodium chloride, potassium, sodium sulfate, etc.), carbohydrates (e.g. lactose), proteins and polymers (e.g. HSA, gelatin, PVP, PLGA, PEG, etc.), chelators and antioxidants (e.g. EDTA, DTPA , ethanol, etc.), preservatives (for example, benzyl alcohol, m-cresol, phenol, etc.), etc. See, for example, Kamerzell et al., Advanced Drug Delivery Reviews (2011) 63, 1118-1159. Therefore, there are many different theoretically possible combinations of components that can be used to identify the composition. However, which formulation is most suitable depends on the particular protein in the formulation.

For this reason, the inventors set out to find improved formulations that could meet the complex requirements in the unpredictable field of protein formulations, in particular an improved formulation based on HIV gp140, which allows the production of a drug formulation that satisfies the broad requirements specific to the late phase. and industrial scale, and contains an aluminum phosphate adjuvant and gp140 protein, which must be stored as a drug in one vial at refrigeration temperature and which prevents instability associated with some of the components present in currently used formulations. The formulations are intended to be stored in liquid form, but may be stored in lyophilized form and re-dissolved in liquid prior to injection.

Composition screening studies are designed to evaluate different buffers (histidine, phosphate and acetate), buffer concentrations (at a concentration of 10 to 50 mM), sugars (sucrose and sorbitol at a concentration of 2 to 12% w/v), surfactants (polysorbate 20 and polysorbate 80 at a concentration of 0.02 to 1% w/v) and pH values from 4.5 to 7.5 in relation to the stability of formulations based on HIV gp140 protein from clade C. Parameters were changed as shown in table. 1.

- 17 039968

Table 1. Scheme for studying the composition based on the HIV gp!40 protein from clade C

Buffer Histidine (His) Phosphate (Pho) acetate (ace) pH 5.5-6.5 6.5-7.5 4.5-5.5 Buffer concentration 10 mM, 20 mM or 50 mM Sugar Sucrose or sorbitol at a concentration of 2% or 12% (weight/rev.) Surface-active substance Polysorbate 20 (PS20) or Polysorbate 80 (PS80) at 0.02%, 0.05% or 0.10% (w/v)

Formulations were prepared by adding the buffer components at the tested pH, followed by the addition of sugar and surfactant. If necessary, the pH was adjusted. The gpl40 protein from clade C (SEQ ID NO: 1) was then added to a concentration of 1 or 0.2 mg/mL.

The stability of the composition was analyzed using the SolvoVPE system to evaluate the concentration and turbidity. Samples of each formulation were analyzed at time 0 (T _o ) and then after stress at 40° C. for 24 h (T24). The stability of the composition was also analyzed using dynamic light scattering (DLS).

SolvoVPE Analysis SolvoVPE (C Technologies, Inc.; Bridgeport, NJ, USA) was used to determine protein concentration by measuring the UV absorbance of samples of each formulation at 280 and 350 nm. The turbidity change was determined from the difference in absorbance at 350 nm between the _To and T ₂ 4 measurements. For the desired formulations, any change in turbidity should preferably be minimal. An increase in turbidity indicates that the protein is precipitating out of solution and that the composition is correspondingly less stable. The results of analysis using SolvoVPE are shown in FIG. 1.

From the SolvoVPE analysis, it can be seen that the acetate buffer formulations had the largest increase in turbidity (mean change +0.339), while the phosphate buffer formulations showed a moderate increase (mean change +0.177) and the histidine buffer show almost no change in turbidity (mean change was -0.010). Since a significant change in turbidity is undesirable, the turbidity data indicate that the histidine buffer is the most optimal buffer tested in terms of improving the stability of the HIV gpl40 protein.

Dynamic Light Scattering (DLS) Analysis DLS was used to assess colloid stability and specifically to assess whether protein aggregation occurs in protein formulations. Measured the change in radius (R _h ) from the moment T _about at 20°C to the moment T _{7 days} at 70°C. More specifically, measured Rh for the original sample at 20°C. Then the sample was heated to 70°C and maintained at 70°C for 7 days before measuring the final Rh. The difference between the initial R _h value and the final R _h values is plotted in FIG. 1B. A large change in the R _h value indicates that protein aggregation is occurring and that the composition is correspondingly less desirable.

It can be seen from the results of the DLS analysis that the formulations containing sorbitol had a lower change in R _h compared to formulations containing sucrose. Since a larger change in the R _h value is undesirable, the DLS data indicate that sorbitol is the most optimal sugar tested to improve the stability of the HIV gpl40 protein.

The results of the above studies also indicated that sugar, sugar concentration, surfactant, surfactant concentration and protein concentration had an effect on stability, so these parameters were further investigated as described in Example 2 below.

Example 2 Effect of sugar, surfactant and protein concentration on the stability of HIV gpl40 formulations from clade C.

Immunogenic formulations based on the HIV gpl40 protein were prepared in 10 mM histidine buffer pH 6.0+0.5, while changing the following parameters: sugar (sorbitol and sucrose), sugar concentration (2 and 12% w/v), polysorbate (PS20 and PS80), polysorbate concentration (0.02 and 0.1% w/v) and protein concentration (0.2 and 1.0 mg/ml). Formulations were prepared by adding 10 mM histidine buffer pH 6.0+0.5 followed by the addition of sugar and surfactant. If necessary, the pH was adjusted. HIV gpl40 protein from clade C (SEQ ID NO: 1) was added to the desired concentration. The formulations were then analyzed by high performance size exclusion chromatography (HP-SEC) as described below.

Received and tested compositions are presented below in table. 2.

- 18039968

Table 2. HIV gp140 protein formulations

Compound Sugar Sugar concentration (% w/v) superficially -active substance Surfactant Concentration (% w/v) Protein concentration (mg/ml) 1 Sorbitol 12 PS20 0.02 0.2 2 Sorbitol 12 PS80 0, 1 0.2 3 Sorbitol 2 PS20 0, 1 0.2 4 Sorbitol 2 PS20 0, 1 0.2 5 Sorbitol 2 PS80 0.02 1 6 Sorbitol 12 PS80 0.02 1 7 Sorbitol 12 PS20 0.02 1 8 Sorbitol 2 PS80 0, 1 1 9 Sorbitol 12 PS80 0, 1 1 10 sucrose 12 PS80 0.02 0.2 eleven sucrose 2 PS20 0.02 0.2 12 sucrose 12 PS80 0, 1 0.2 13 sucrose 2 PS80 0.02 0.2 14 sucrose 2 PS80 0, 1 1 15 sucrose 2 PS20 0.02 1 16 sucrose 2 PS20 0, 1 1 17 sucrose 12 PS20 0, 1 1 18 sucrose 12 PS20 0, 1 1

High performance size exclusion chromatography (HP-SEC) analysis: HP-SEC was used to analyze the amount of hexameric, trimeric, and high/low molecular weight structures in formulation samples by monitoring absorbance at 280 nm. The HIV gp140 protein exists predominantly as a trimer. Some hexameric structures of the HIV gp140 protein are also observed. Although the desired structure is a trimeric structure, HIV immunogenicity is observed for both trimeric structures and hexameric structures. The presence of high molecular weight (HMW) and low molecular weight (LMW) structures in the composition is undesirable. In particular, the presence of LMW structures indicates cleavage and/or degradation of the gp140 protein. The presence of HMW structures can be caused by a variety of factors such as gp140 protein aggregation. Samples of each formulation were analyzed at time 0 (T ₀ ) and then stressed at 40° C. for 24 h (T ₂₄ ). Formulations containing 1 mg/ml of clade C gp140 protein were diluted two-fold prior to injection into the HP-SEC system, while formulations containing 0.2 mg/ml of clade C gp140 protein were not diluted prior to administration.

The results of HP-SEC analysis are presented below in table. 3 and in FIG. 2A-F. The results indicate that the number of trimers and hexamers for both samples T ₀ and samples T ₂₄ depends on the concentration of surfactant and HIV gp140 protein. Formulations with a lower concentration of surfactant (0.02% w/v) had a higher observed value of trimeric+hexameric structures of the HIV gp140 protein, while a lower content of high/low molecular weight structures was observed (see Fig. 2B , D and F) both before and after the stress treatment of the sample. Formulations with a higher concentration of HIV gp140 protein (1.0 mg/mL) also had a higher observed value of HIV gp140 protein trimer+hexamer structures, with a lower content of high/low molecular weight structures observed (see Fig. 2B and D) both before and after stressing the sample (see FIGS. 2A and C). Formulations containing polysorbate 20, in contrast to those containing polysorbate 80, also had a lower amount of low molecular weight structures present (see FIG. 2E).

- 19 039968

Table 3. Results of HP-SEC analysis

Compound High molecular structures (%) Hexamer (%) Trimer (%) low mol cular structures (%) That T ₂ 4 That T ₂ 4 That T ₂₄ That T ₂₄ 1 0.57 0 10.69 10, 13 88.05 89.87 0 0 2 0.22 0 8, 48 9.55 80, 08 85.39 11.22 5.06 3 0.2 0 9, 15 9.51 84, 47 85.77 0 4.72 4 0 0 9.26 9.56 84, 63 86.71 0 3.73 5 0 0 10.72 12.27 88.41 87.73 0.86 0 6 0 0 10.78 11, 6 88, 44 88.4 0.78 0 7 0 0 11.08 11.7 88.6 88.3 0.32 0 8 0 0 10.46 11, 67 87.33 88.33 2.21 0 9 0 0 10.6 11.57 86, 91 88.43 2.48 0 10 0 0 9.79 10, 02 88.17 89, 98 2.05 0 eleven 0 0 10.52 10.54 88.75 89.46 0 0 12 0 0 8.9 9.23 79.79 83.51 11.31 7.26 13 0 0 9, 74 10.58 87.58 89.42 2, 68 0 14 0 0 10.37 11, 82 87.38 87.98 2.25 0.2 15 0 0 10.91 12.15 88.77 87.85 0.32 0 16 0 0 10.57 11, 42 87.71 87.72 1.72 0.86 17 0 0 10.63 11, 45 88.51 88.43 0 0.12 18 0 0 10.74 11.54 88.28 88.46 0 0

The data presented in table. 3 indicate that the combination of histidine buffer and sorbitol is preferred over the combination of histidine buffer and sucrose. For example, formulations containing histidine buffer and sorbitol, such as formulations 1, 4, and 7, show the smallest change in the number of hexameric and trimeric structures between T ₀ and T ₂₄ compared to that observed for formulations containing histidine buffer and sucrose. This result was surprising because, as a rule, for protein composition, histidine buffer and sorbitol are not used in combination with each other, while histidine buffer and sucrose are often used in combination with each other. Thus, it was unexpected that the most optimal composition contained histidine buffer (pH 6.0±0.5) and sorbitol, and not histidine buffer and sucrose. The above study also indicates that the inclusion of polysorbate 20 as a surfactant (see, for example, Fig. 2E) at a concentration of 0.2% (w/v) (see, for example, Fig. 2B, D and F) provides a formulation in which the stability of the HIV gp140 protein is further improved.

Advantages of the formulations identified herein over those currently used in clinical trials for a gp140-based drug product include that they use existing pharmacopoeia-grade qualified excipients that are readily available for large-scale production and do not suffer from known problems that may adversely affect stability in the long term, and they make it possible to store the bulk drug substance and the antigen-based drug product in a stable state. Moreover, these formulations allow storage and stability of adjuvant drugs in single vial drug form at refrigerated temperatures. These formulations are suitable for storage in liquid form, but can also potentially be stored in lyophilized form and then re-dissolved in liquid prior to injection, which is another advantage.

Example 3 Stability studies of HIV gp140 mosaic protein formulations.

The stability of two formulations containing the HIV gp140 mosaic protein (SEQ ID NO: 2) was studied in a freeze-thaw and temperature cycling study. The tested compositions are presented in table. 4.

- 20 039968

Table 4. HIV gp140 mosaic protein formulations

Compound Protein Buffer Sugar Surface-active substance fl 1.0mg/ml 10 mM histidine pH 6.5 12% sorbitol 0.02% PS20 F2 1.0 mg/ml 10 mM histidine, pH 6.5 2% sorbitol 0.02% PS20

The compositions were subjected to several freeze-thaw cycles. One freeze-thaw cycle was performed by freezing at -80°C or -40°C for 24 hours followed by thawing at -2°C to 8°C for 24 hours. Samples were analyzed at the end of 1, 3 and 5 cycles. freeze-thaw by measuring absorbance at 280 nm (concentration) and 350 nm (turbidity). The results are shown in FIG. 3A and B.

It can be seen from the results that the absorbance at 350 nm and at 280 nm did not change substantially for both formulations F1 and F2 after several freeze-thaw cycles, indicating that the concentration and turbidity of the formulation remained relatively unchanged. This demonstrates that the HIV gp140 protein in the formulations is freeze-thaw stable.

Example 4 Long term stability study of HIV gp140 protein formulations.

The stability of HIV gp140 protein formulations in histidine buffer, both with and without adjuvant, according to embodiments of the present invention, was compared with the stability of HIV gp140 protein formulation prepared in HEPES buffer with and without adjuvant. The tested compositions are presented in table. 5. All formulations contained 0.2 mg/ml HIV gp140 protein from clade C (SEQ ID NO: 1).

- 21 039968

Table 5. HIV gp140 formulations for long-term stability studies

Compound Buffer Composition Adjuvant? 1 20 mM HEPES, pH 6.5 Without adjuvant 90 mM NaCl 4% (w/v) sucrose 0.02% polysorbate 80 2 10 mM histidine buffer, pH 6.5 12% (w/v) sorbitol 0.02% (w/v) polysorbate 20 Without adjuvant 3 10 mM histidine buffer, pH 6.5 2% (w/v) sorbitol 0.02% (w/v) polysorbate 20 Without adjuvant 4 10 mM histidine buffer, pH 6.5 5% (w/v) sorbitol 0.02% (w/v) polysorbate 20 Without adjuvant 5 20 mM HEPES, pH 6.5 90 mM NaCl 4% (w/v) sucrose 0.02% polysorbate 80 + aluminum phosphate adjuvant (0.85 mg/ml) 6 10 mM histidine buffer, pH 6.5 12% (w/v) sorbitol 0.02% (w/v) polysorbate 20 + aluminum phosphate adjuvant (0.85 mg/ml) 7 10 mM histidine buffer, pH 6.5 2% (w/v) sorbitol 0.02% (w/v) polysorbate 20 + aluminum phosphate adjuvant (0.85 mg/ml) 8 10 mM histidine buffer, pH 6.5 5% (w/v) sorbitol 0.02% (w/v) polysorbate 20 + aluminum phosphate adjuvant (0.85 mg/ml)

The compositions were stored at 2 to 8°C; at 25°C and 60% relative humidity (RH); and at 40°C and 75% RH.

The study is ongoing and samples are being tested after storage for 2 weeks, 1, 2, 3, 6, 9, 12, 18, 24, 30 and 36 months.

Samples were tested using SDS under reducing conditions after storage for up to three months. When SDS-PAGE was carried out under reducing conditions, the trimeric and hexameric forms were reduced to the monomeric form, which was found on the gel as a single band. Any degradation of the protein to low molecular weight structures led to a change (decrease) in the level of the monomeric form of the protein observed on the gel. Data for samples stored at 25°C and 60% RH and at 40°C and 75% RH are shown in FIG. 4A-D. The data show that all formulations, both with and without aluminum phosphate adjuvant, when stored at 25° C. and 60% RH were stable for up to three months (FIGS. 4A and C). However, formulations containing histidine buffer and sorbitol were more stable than those containing

- 22 039968

HEPES and sucrose (FIGS. 4B and D), when stored at 40°C and 75% RH, both with and without aluminum phosphate adjuvant. In addition, after six months of storage at 25°C, the material formulated using histidine with 12% sorbitol without aluminum phosphate adjuvant showed better stability compared to the formulation prepared using HEPES buffer, as measured by SDS-PAGE (smaller % change from baseline measurement; data not shown). Moreover, it was noted that gp140, which was formulated using histidine buffer, sorbitol and aluminum phosphate, was stable at 25°C for at least six months (data not shown). Also, when formulated with histidine buffer and sorbitol, it was noted that at higher concentrations of aluminum phosphate adjuvant (3.84 mg/mL), the HIV gp140 protein was stable at 25°C for at least six months, and at 40° C. for at least three months as measured by SDS PAGE under reducing conditions (less than 25% recovery, data not shown); while ELISA measurements showed stability (less than 50% recovery) at 40° C. for at least two weeks, data not shown). These data further indicate that HIV gp140 protein-based formulations containing histidine buffer and sorbitol are more stable and thus can be stored as an adjuvant drug in a single vial at elevated temperatures, which is an advantage as often it is difficult to formulate an adjuvant protein that is storage stable at elevated temperatures. This means that the drug and adjuvant can be stored in the same vial at the manufacturing site or at the filling and packaging area, and separate storage with mixing at the pharmacy or at the patient's bedside immediately before administration is not required. In addition to the economic benefit, this is a significant advantage, especially for an HIV vaccine product that is also intended for use in a variety of settings where capacity and equipment may be limited. It also has the advantage of limiting the number of operations that need to be carried out on the medicinal product in the field, where a large number of subjects need to be vaccinated in one campaign. Thus, the compositions of the present invention unexpectedly improve the practical properties for the use of the finished vaccine, especially in the case of limited resources.

A combination of two HIV gp140 proteins, one of SEQ ID NO: 1 and one of SEQ ID NO: 2, is mixed (0.2-1.0 mg/ml total protein concentration, e.g. 0.1 mg/ml of each protein, resulting in to a total gp140 protein concentration of 0.2 mg/mL) and tested in the preferred formulation, i.e. 2 to 15% (w/v) sorbitol; from 0.01 to 0.05% (w/v), for example 0.02%, polysorbate 20; and 5 to 20 mM, e.g. 10 mM, histidine buffer pH 5.5 to 7.0, e.g. pH 6.5, and preferably aluminum phosphate, e.g. 0.7-4 mg/ml, e.g. 0.85 or 3.84 mg/ml, using the methods described above. This composition is also expected to be stable for at least six months at 2-8°C, as are the individual proteins in this composition, as indicated above.

It should be understood that the examples and embodiments described herein are for informational purposes only and that the above described embodiments may be modified without departing from their general inventive intent. Thus, it is to be understood that this invention is not limited to the specific embodiments disclosed, but is intended to cover all modifications within the spirit and scope of the present invention as defined in the appended claims.

Claims (17)

CLAIM 1. An immunogenic composition containing, relative to the total volume of the composition: a) 0.05 to 5 mg/ml HIV gp140 protein or a mixture of at least two HIV gp140 proteins; b) 2 to 15% (w/v) sorbitol; c) 0.01 to 0.05% (w/v) polysorbate 20; d) 5 to 20 mM histidine buffer pH 5.5 to 7.0. 2. The immunogenic composition of claim 1, wherein the concentration of HIV gp140 protein or mixture of HIV gp140 proteins is 0.2 mg/mL. 3. The immunogenic composition of claim 1, wherein the concentration of HIV gp140 protein or mixture of HIV gp140 proteins is 1 mg/mL. 4. An immunogenic composition according to any one of claims 1 to 3, wherein the concentration of sorbitol is 5% (w/v). 5. An immunogenic composition according to any one of claims 1 to 3, wherein the concentration of sorbitol is 12% (w/v). - 24 039968 6. An immunogenic composition according to any one of claims 1 to 5, wherein the concentration of polysorbate 20 is 0.02% (w/v). 7. An immunogenic composition according to any one of claims 1 to 6, wherein the concentration of the histidine buffer is 10 mM and the pH of the histidine buffer is 6.5. 8. An immunogenic composition according to any one of claims 1 to 7, further comprising an aluminum phosphate adjuvant. 9. The immunogenic composition according to claim 8, wherein the concentration of the aluminum phosphate adjuvant is 0.7-4.0 mg/ml. 10. An immunogenic composition according to any one of claims 1 to 9, wherein the HIV gp140 protein contains the amino acid sequence of SEQ ID NO: 1. 11. An immunogenic composition according to any one of claims 1-9, wherein the HIV gp140 protein comprises the amino acid sequence of SEQ ID NO: 2. 12. An immunogenic composition according to any one of claims 1 to 9, comprising a mixture of the HIV gp140 protein containing the amino acid sequence of SEQ ID NO: 1 and the HIV gp140 protein containing the amino acid sequence of SEQ ID NO: 2. 13. An immunogenic composition containing, relative to the total volume of the composition: a) 0.2 or 1 mg/ml of HIV gp140 protein containing the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or a mixture of HIV gp140 protein containing the amino acid sequence of SEQ ID NO: 1 and HIV gp140 protein containing amino acid sequence SEQ ID NO: 2; b) 5 or 12% (w/v) sorbitol; c) 0.02% (w/v) polysorbate 20; d) 10 mM histidine buffer pH 6.5; e) an aluminum phosphate adjuvant at a concentration of 0.85 or 3.84 mg/ml. 14. A method for producing an immunogenic composition according to claim 1, including mixing the HIV gp140 protein, sorbitol, polysorbate 20 and a histidine buffer. 15. The method of claim 14 further comprising admixing an aluminum phosphate adjuvant. 16. A method for producing an immunogenic composition according to claim 1, which is stable under storage conditions at 2-8°C for at least one month and contains the HIV gp140 protein, comprising mixing the following components to obtain an immunogenic composition containing these components in amounts relative to the total volume of the composition: a) 0.05 to 5 mg/ml HIV gp140 protein; b) 2 to 15% (w/v) sorbitol; c) 0.01 to 0.05% (w/v) polysorbate 20; d) 5 to 20 mM histidine buffer pH 5.5 to 7.0; e) water; f) an aluminum phosphate adjuvant, preferably at a concentration of 0.7-4.0 mg/ml. 17. The use of an immunogenic composition according to any one of claims 1 to 13 for administration to a human patient to induce an immune response to HIV.