This application is a § 371 national stage of PCT International Application No. PCT/IB2017/001101, filed Jul. 26, 2017, claiming priority of European Patent Application No. 16382364.4, filed Jul. 27, 2016, the contents of each of which are hereby incorporated by reference into the application.
This application incorporates-by-reference nucleotide and/or amino acid sequences which are present in the file named “200724 90822_Substitute_Sequence_Listing_AWG.txt”, which is 78 kilobytes in size, and which was created Jul. 23, 2020 in the IBM-PC machine format, having an operating system compatibility with MS-Windows, which is contained in the text file filed Jul. 24, 2020 as part of this application.
The present invention relates to fusion proteins and virus-like particles (VLPs) useful in human and veterinary medicine. The invention refers also to a fusion protein comprising a polypeptide of interest, a transmembrane domain and an HIV gag polypeptide and their functionally equivalent variants. The invention relates additionally to the polynucleotides, vectors, host cells and VLPs particles expressing or presenting said fusion proteins, and to the pharmaceutical compositions, immunogenic or vaccines composition containing said fusion proteins, polynucleotides, vectors, host cells and VLPs and their use in treatment and prevention on infectious diseases.
Virus-like particles (VLP) represent an excellent vaccine platform that has been proven effective against non-enveloped viruses such as the hepatitis B virus (HBV), human papilloma virus (HPV) or hepatitis E virus (HEV). See Lua L, et al., Biotechnol. Bioeng. 2014; 111:425-440. In these cases, VLPs contain a single capsid protein that is produced in prokaryotic or eukaryotic cell systems to spontaneously form VLP with excellent immunogenic results. The success and versatility of VLP production platforms has prompted the exploration of more complex designs. Some examples could be the generation of VLPs with heterologous antigen presentation by engrafting exogenous sequences in viral capsid proteins and the generation of enveloped VLPs, which contain a lipid bilayer derived from the VLP-producer cell and show higher heterogeneity than non-enveloped VLPs. See Bryant M, et al., Proc. Natl. Acad. Sci. USA 1990; 87:523-527 and Visciano M, et al., Vaccine 2011; 29(31):4903-4912.
Human immunodeficiency Virus (HIV) vaccine research has also approached the VLP technology. As for other retroviruses, HIV particles are enveloped structures formed by the multimerization of Gag structural proteins on the inner part of plasma membrane of infected cells through the myrystoylation of its N-term end. See Bryant, 1990, supra. During the budding process, Gag polymers incorporate genomic viral RNA, different viral enzymes and accessory proteins and recruit cell surface expressed envelope glycoprotein complexes to ensure infectivity. However, it is well known that expression of a retroviral gag gene is sufficient to produce VLP structures lacking infectivity. See Visciano, 2011, supra and Cervera L, et al., J. Biotechnol. 2013; 166:152-165. These nude Gag VLPs can be modified either to incorporate proteins or RNAs and act as delivery vectors or to incorporate proteins on their surface to act as vaccine preparations. See Visciano, 2011, supra.
Over the last years, several groups have developed the production process of enveloped VLP focusing on HIV vaccine research. See Visciano, 2011 and Cervera, 2013, supra. However, the optimal generation of enveloped VLPs is not the only obstacle in these approaches. A second major difficulty is the complexity of HIV envelope glycoprotein (Env). See Mao Y, et al., Nat. Struct. Mol. Biol. 2012; 19:893-899. This large glycoprotein contains two subunits gp120 and gp41 capable of generating neutralizing and non-neutralizing antibodies. Despite multiple approaches to redesign protein mimics that act as immunogens to elicit neutralizing antibodies against Env, few positive results have been achieved See Mao, 2012, supra. Nevertheless, despite the efforts made to date, there still exists a continuing need in the art for novel ways of introducing antigen epitopes in VLPs compounds, as well as developing VLPs useful in eliciting an immune response against particular antigen epitopes.
In a first aspect, the present invention relates to a fusion protein which comprises from the N- to the C-terminus:
(a) a polypeptide of interest, or a functionally equivalent variant thereof,
(b) a transmembrane domain, or a functionally equivalent variant thereof, and
(c) an HIV gag polypeptide, or a functionally equivalent variant thereof.
In an additional aspect, the invention relates to a polynucleotide encoding a fusion protein according to the invention.
In a further aspect, the invention refers to a vector comprising a polynucleotide according to the invention.
In another aspect, the invention is directed to a host cell comprising a fusion protein or a vector according to the invention.
In a still further aspect, the invention relates to a method for preparing a VLP loaded with an immunogenic polypeptide comprising:
(a) expressing in a cell a fusion protein according to the invention, and
(b) recovering the VLP from the extracellular medium.
Additionally, the present invention relates to a virus like-particle comprising the fusion protein according to the invention or obtained through using a method of the invention.
In a further aspect, the invention refers to a pharmaceutical composition comprising a fusion protein, a polynucleotide, a vector, a host cell or a virus-like particle according to the invention, or a combination thereof.
In another aspect, the invention is directed to a fusion protein, a polynucleotide, a vector, a host cell, a virus-like particle or a pharmaceutical composition according to the invention, or combination thereof, for use in medicine.
In a still further aspect, the invention relates to a fusion protein, a polynucleotide, a vector, a host cell, a virus-like particle or a pharmaceutical composition according to the invention, or combination thereof, for use in the treatment or prevention of a disease caused by an infection or tumor in a subject. In an alternative form of this aspect, the invention relates to a method of treating or preventing disease caused by an infection or tumor in a subject which comprises the administration of a therapeutically effective amount of a fusion protein, a polynucleotide, a vector, a host cell, a virus-like particle or a pharmaceutical composition according to the invention, or combination thereof, to a subject. In a further alternative form of this aspect, the invention refers to the use of a fusion protein, a polynucleotide, a vector, a host cell, a virus-like particle or a pharmaceutical composition according to the invention, or combination thereof, in the manufacture of a medicament for the treatment or prevention of a disease caused by an infection or tumor in a subject.
Additionally, the present invention relates to a kit comprising a fusion protein, a polynucleotide, a vector, a host cell, a virus-like particle or a pharmaceutical composition according to the invention, or a combination thereof.
The plasmid pcDNA3.1/MINGAG-3.7 was deposited on Jul. 25, 2012, under accession number DSM 26214 at the DSMZ—Deutsche Sammlung von Mikroorganismen and Zellkulturen, InhoffenstraBe 7 B, D-38124 Braunschweig, Federal Republic of Germany.
The nucleic and amino acid sequences depicted in the accompanying sequence listing are shown using the standard letter abbreviations and codes applied conventionally in the art. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. The accompanying sequence listing an integral part of the present invention.
One of the main limitations of the generation of antibodies is the poor incorporation of antigens of interest into VLPs in vitro. The present invention describes novel VLPs using gp41-based and other proteins, which include a transmembrane domain fused to gag for overcoming this limitation. This design disclosed in the present invention: (i) allows for the membrane localization of these proteins, (ii) maintains the VLP self-packing ability of Gag, (iii) shows increased gp41 surface density and (iv) improves the correct spatial presentation of antigens, thus (v) enhances the immunogenic responses to said antigens. The results clearly demonstrate the increased immunogenicity of the VLPs of the invention.
The term “AIDS”, as used herein, refers to the symptomatic phase of HIV infection, and includes both Acquired Immune Deficiency Syndrome (commonly known as AIDS) and “ARC,” or AIDS-Related Complex. See Adler M, et al., Brit. Med. J. 1987; 294: 1145-1147. The immunological and clinical manifestations of AIDS are well known in the art and include, for example, opportunistic infections and cancers resulting from immune deficiency.
The term “allergen”, as used herein, refers to a peptide, polypeptide or protein to which a subject is sensitive and that may cause an immune reaction (e.g. allergen extracts of pollens, insects, food or food products; components present in saliva, insect claws or stings; plant components).
The term “amino acid”, as used herein, refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Amino acids may be referred to herein by either their commonly known three-letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
The term “codon optimized”, as used herein, refers to the alteration of codons in nucleic acids to reflect the typical codon usage of the host organism to improve the expression of a reference polypeptide without altering its amino acid sequence. There are several methods and software tools known in the art for codon optimization. See Narum D, et al., Infect. Immun. 2001; 69(12):7250-7253), Outchkourov N, et al., Protein Expr. Purif 2002; 24(1):18-24, Feng L, et al., Biochemistry 2000; 39(50):15399-15409 and Humphreys D, et al., Protein Expr. Purif 2000; 20(2):252-264.
The term “comprising” or “comprises”, as used herein, discloses also “consisting of” according to the generally accepted patent practice.
The terms “Env” or “gp160”, as used herein, refer to a glycoprotein having either the antigenic specificity or the biological function of the outer envelope protein (Env) of HIV and encompassing two subunits, the gp120 and the gp41 glycoproteins. Exemplary sequences of wild-type (wt) gp160 polypeptides are available. See GenBank accession nos. AAB05604 and AAD12142.
The term “epitope”, as used herein, refers to that portion of a given immunogenic substance that is the target of (i.e. is bound by) an antibody or cell-surface receptor of a subject's immune system that has mounted an immune response against said immunogenic substance, as determined by any methods known in the art. Further, an epitope may be defined as a portion of an immunogenic substance that is recognized by a humoral or cellular response, and in particular, an antibody response or a T-cell response in a subject, as determined by any method available in the art. An epitope can be a portion of any immunogenic substance, such as a protein, polynucleotide, polysaccharide, an organic or inorganic chemical, or any combination thereof. The term “epitope” may also be used interchangeably with “antigenic determinant” or “antigenic determinant site”.
The terms “express”, “expresses” or “expression”, as used herein, refers to the transcription of a nucleic acid molecule, and optionally, its translation. Typically, transcription and translation of a coding sequence will result in the production of polypeptide such as the fusion proteins of the invention.
The expression “functionally equivalent variant”, as used herein, refers to: (i) a polypeptide resulting from the modification, deletion or insertion or one or more amino acids and which substantially preserves the activity of its reference polypeptide and (ii) a polynucleotide resulting from the modification, deletion or insertion of one or more bases and which substantially preserves the activity of the polypeptide expressed by the reference nucleic acid. Functionally equivalent variants contemplated in the context of the present invention, include polypeptides which show at least 60%, 70%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, 99% of similarity or identity with sequences SEQ ID NOs: 1-2, 5, 15-28, 30, 32, 34, 36 and 38 and which retain substantially equivalent ability to induce VLP formation in the case of a gag polypeptide variant or retains the ability to be embedded into the plasma membrane in the case of a functionally equivalent variant of the HIV gp41 polypeptide. Functionally equivalent variants contemplated in the context of the present invention include also polynucleotides which show at least 60%, 70%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, 99% of similarity or identity with sequences SEQ ID NOs: 3-4, 6-14, 29, 31, 33, 35 and 37 and that, when expressed, retain substantially equivalent ability to induce VLP formation in the case of a gag polypeptide variant or retains the ability to be embedded into the plasma membrane in the case of a functionally equivalent variant of the HIV gp41 polypeptide. The degree of identity or similarity between two polypeptides or two polynucleotides is determined by using computer-implemented algorithms and methods that are widely known in the art. The identity and similarity between two sequences of amino acids is preferably determined using the BLASTP algorithm. See Altschul S, et al., “BLAST Manual” (NCBI NLM NIH, Bethesda, Md., USA, 2001).
The term “fusion protein”, as used herein, relates to proteins generated by gene technology which consist of two or more functional domains derived from different proteins. A fusion protein may be obtained by conventional means (e.g. by means of gene expression of the nucleotide sequence encoding for said fusion protein in a suitable cell).
The term “gag polypeptide”, as used herein relates to a primary protein product of the gag gene of HIV, which provides the basic physical infrastructure of the virus, and which is processed by viral protease during maturation to MA (matrix protein, p17), CA (capsid protein, p24), SP1 (spacer peptide 1, p2), NC (nucleocapsid protein, p7), SP2 (spacer peptide 2, p1) and P6 protein.
The term “gp41”, as used herein, refers to human immunodeficiency virus-I envelope glycoprotein gp4 1. Gp4 1 is a subunit which forms the Env glycoprotein of HIV-1 together with gp120. Env is a trimer composed of three external subunits (gp120) and three transmembrane subunits (gp41). The extracellular moiety of gp41 protein contains three essential functional regions: a fusion peptide (FP), a N-terminal heptad repeat (HR1) and a C-terminal heptad repeat (HR2). The HR1 and HR2 regions contain several leucine zipper-like motifs which have tendency to form coiled structures. See Peisajovich S, Shai Y, Biochem. Biophys. Acta 2003; 1614:122-129; Suarez T, et al., FEBS Lett. 2000; 477:145-149; Chan D, et al., Cell 1997; 89:263-273. The nucleic acid and amino acid sequences of a large number of HIV gp-41 are readily available to the public. See HIV Sequence Database, hiv.1an1.gov, July 2017.
The term “gp120”, as used herein, refers to a glycoprotein having either the antigenic specificity or the biological function of the outer envelope protein (env) of HIV. A “gp120 protein” is a molecule derived from a gp120 region of an Env polypeptide. The amino acid sequence of gp120 is approximately 511 amino acids. Gp120 is a heavily N-glycosylated protein with an apparent molecular weight of 120 kDa. Gp120 contains five relatively conserved domains (C1-C5) interspersed with five variable domains (V1-V5). The variable domains contain extensive amino acid substitutions, insertions and deletions. A “gp120 polypeptide” includes both single subunits and multimers. The gp41 portion is anchored in (and spans) the membrane bilayer of the virion, while the gp120 segment protrudes into the surrounding environment. The receptor binding domain of gp120 is localized to N-terminal half of the protein. This is followed by a proline rich region (PRR), which behaves either as a hinge or trigger to communicate receptor binding to the fusion machinery. The C-terminus of gp120 is highly conserved and interacts with gp41. See GenBank accession nos. AAB05604 and AAD12142.
The terms “heptad repeat 2” or “HR2”, as used herein, refer, but is not limited to, a heptad repeat region located at the carboxy terminus of the extracellular portion of the wild-type gp41. See Eggink D, et al., J. Virol. 2008; 82(13):6678-6688. A heptad repeat is a motif in which a hydrophobic amino acid is repeated every seven residues. Such motifs are designated a through g. See Lupas A, Trends Biochem. Sci. 1996; 21:375-382. Heptad repeats which contain hydrophobic or neutral residues at the a and d positions can form alpha helices and are able to interact with other heptad repeats by forming coiled coils. See Chambers P, et al., J. Gen. Virol. 1990; 71:3075-3080 and Lupas, 1996, supra.
The term “HIV”, as used herein, include HIV-1 and HIV-2, SHIV and SIV. “HIV-1” means the human immunodeficiency virus type-1. HIV-1 includes, but is not limited to, extracellular virus particles and the forms of HIV-1 associated with HIV-1 infected cells. The HIV-1 virus may represent any of the known major subtypes (Classes A, B, C, D E, F, G and H) or outlying subtype (Group 0) including laboratory strains and primary isolates. “HIV-2” means the human immunodeficiency virus type-2. The HIV-1 strains include, but are not limited to, the HIV-1JR-FL, HIV-1DH123, HIV-1Gun-1, HIV-189.6, and HIV-1HXB2 strains. HIV-2 includes, but is not limited to, extracellular virus particles and the forms of HIV-2 associated with HIV-2 infected cells. The term “SIV” refers to simian immunodeficiency virus which is an HIV-like virus that infects monkeys, chimpanzees, and other nonhuman primates. SIV includes, but is not limited to, extracellular virus particles and the forms of SIV associated with SIV infected cells.
The term “HIV exposure”, as used herein, refers to the contact of an uninfected subject with a subject having an HIV infection or AIDS, or the contact with body fluids from such HIV-infected subject, in which such fluids from the infected subject contact a mucous membrane, a cut or abrasion in the tissue (e.g. needle stick, unprotected sexual intercourse), or other surface of the uninfected subject in such a way that the virus could be transmitted from the infected subject or infected subject's body fluids to the uninfected subject.
The term “HIV infection”, as used herein, refers to indications of the presence of the HIV virus in an individual including asymptomatic seropositivity, AIDS-related complex (ARC), and acquired immunodeficiency syndrome (AIDS).
The term “HIV-1JR-FL”, as used herein, refers to an HIV-1 strain originally isolated from the brain tissue of an AIDS patient taken at autopsy and co-cultured with lectin-activated normal human PBMCs.
The terms “host cell” and “recombinant host cell”, as used interchangeably herein, refer to a cell into which a nucleic acid or vector according to the invention has been introduced. “Host cell” and “recombinant host cell” includes also the progeny, or potential progeny, of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but can be still included within the scope of the term as used herein.
The terms “identical” or percent “identity” in the context of two or more nucleic acids or polypeptides refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity. The percent of identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software are known in the art which can be used to obtain alignments of amino acid or nucleotide sequences. Examples of algorithms suitable for determining sequence similarity include, but are not limited to, the BLAST, Gapped BLAST, and BLAST 2.0, WU-BLAST-2, ALIGN, and ALIGN-2 algorithms. See Altschul S, et al., Nuc. Acids Res. 1977; 25:3389-3402, Altschul S, et al., J. Mol. Biol. 1990; 215:403-410, Altschul S, et al., Meth. Enzymol. 1996; 266:460-480, Karlin S, et al., Proc. Natl. Acad. Sci. USA 1990; 87:2264-2268, Karlin S, et al., Proc. Natl. Acad. Sci. USA 1993; 90:5873-5877, Genentech Corp, South San Francisco, Calif., US, blast.ncbi.nlm.nih.gov, July 2017. Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, for instance, by the Smith-Waterman local homology algorithm, by the Needleman-Wunsch homology alignment algorithm, by the Pearson-Lipman similarity search method, by computerized implementations of these algorithms or by manual alignment and visual inspection. See Smith T, et al., Adv. Appl. Math. 1981; 2:482-489, Needleman S, et al., J. Mol. Biol. 1970; 48:443-453, Pearson W, et al., Lipman D, Proc. Natl. Acad. Sci. USA 1988; 85:2444-2448, the GAP, BESTFIT, FASTA and TFASTA programs, Wisconsin Genetics Software Package, Genetics Computer Group, Madison, Wis., USA; Ausubel F, et al., Eds., “Short Protocols in Molecular Biology”, 5th Ed. (John Wiley and Sons, Inc., New York, N.Y., USA, 2002).
The term “immunogenic composition”, as used herein, refers to a pharmaceutical composition capable of eliciting a humoral (e.g. antibodies) or cellular (e.g. cytotoxic T-cell) immune response in a subject against a specific immunogen.
The term “immunogenic polypeptide”, as used herein, relates to a polypeptide from a pathogenic organism, tumor marker or allergen that, when administered to a subject, can induce a protective immune response against the organism, tumor marker or allergen.
The term “immunologically functional equivalent”, as used herein, refers to a variant of a polypeptide that retains substantially equivalent ability to induce an immune response in a subject as the reference polypeptide. The term “immunologically functional equivalent” is well known in the art and is further defined in detail herein. Immunologically functional equivalents may increase the antigenicity of a polypeptide, maintain the same level of antigenicity of the reference polypeptide, or decrease the antigenicity of a polypeptide only slightly so that it maintains its usefulness as an antigen in an immunogenic composition.
The term “infectious disease”, as used herein, relates to diseases caused by pathogens such as viruses, bacteria, fungi, protozoa and parasites.
The term “kit”, as used herein, refers to a product containing the different reagents necessary for carrying out the uses and methods according to the invention which is packed as to allow their transport and storage. Materials suitable for packing the components of the kit include crystal, plastic (e.g. polyethylene, polypropylene, polycarbonate), bottles, vials, paper or envelopes.
The term “KLH”, as used herein, relates to a large, multisubunit, oxygen-carrying, metalloprotein that is found in the hemolymph of the giant keyhole limpet, Megathura crenulata, a species of keyhole limpet that lives off the coast of California, from Monterey Bay to Isla Asuncion off Baja California.
The terms “linker region” or “linker”, as used herein, refer to any heterologous polypeptide of at least 1, 2, 3, 4 or 5 or more amino acids in length, which when inserted between a first and second region yields a functional linkage joining both regions and wherein each region can preserve its functional and immunological properties.
The terms “membrane-proximal external region” or “MPER”, as used herein, refer, but are not limited to, a highly conserved region of the gp41 ectodomain adjacent to the viral membrane.
The terms “neutralizing antibody” or “nAb”, as used herein, is any antibody or antigen-binding fragment that binds to an extracellular molecule (e.g. a protein or a protein domain in the surface of a pathogenic virus or bacteria) and interferes with the ability of the pathogen to infect a cell or modulates its activity. In the context of the invention, the pathogen is preferably HIV, and more specifically, the gp120 protein of the HIV viral envelope. In particular, the term “HIV neutralizing antibody” refers to a neutralizing antibody with affinity to the CD4 binding site of gp120 such as IgGb12. The term “neutralizing antibodies” includes the subclass of bnAbs. As used herein, “broadly neutralizing antibody” or “bnAb” is understood as an antibody obtained by any method that, when delivered at an effective dose, can be used as a therapeutic agent for the prevention or treatment of HIV infection or AIDS against more than 7 strains of HIV, preferably more than 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more strains of HIV.
The terms “NL4-3” and “BaL”, as used herein, refer to two different HIV isolates commonly used in the laboratory. The NL4-3 isolate was cloned from NY5 and LAV proviruses. See Adachi A, et al., J. Virol. 1986; 59:284-291. The BaL isolate was obtained from a primary culture of adherent cells grown from explanted lung tissue. See Gartner S, et al., Science 1986; 233:215-219.
The terms “nucleic acid”, “polynucleotide” and “nucleotide sequence”, as used interchangeably herein, relate to any polymeric form of nucleotides of any length and composed of ribonucleotides or deoxyribonucleotides. The terms include both single-stranded and double-stranded polynucleotides, as well as modified polynucleotides (e.g. methylated, protected). Typically, the nucleic acid is a “coding sequence” which, as used herein, refers to a DNA sequence that is transcribed and translated into a polypeptide in a host cell when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5′ (amino) terminus and a translation stop codon at the 3′ (carboxy) terminus. A coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g. mammalian) DNA, and even synthetic DNA sequences. A transcription termination sequence will usually be located 3′ to the coding sequence.
The term “operably linked”, as used herein, means that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence (e.g. in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). See Auer H, Nature Biotechnol. 2006; 24: 41-43.
The expression “parenteral administration” and “administered parenterally”, as used herein, means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and, intrasternal injection and infusion.
The expression “peripheral blood mononuclear cell” or its abbreviation “PBMC”, as used herein, refer to any peripheral blood cell having a round nucleus. These cells consist of lymphocytes (i.e. T cells, B cells, NK cells) and monocytes.
The expression “pharmaceutically acceptable carrier”, as used herein, includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents that are physiologically compatible with the fusion proteins, polynucleotides, vectors, host cells or VLP according to the invention.
The term “pharmaceutical composition”, as used herein, refers to a combination of at least one fusion protein, polynucleotide, vector, host cell or VLP according to the invention and at least one other ingredient, including but not limited to a pharmaceutically acceptable carrier, that render the combination suitable for administration to a subject. The term “pharmaceutical composition” includes, but is not limited to, immunogenic and vaccine compositions.
The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues, which may contain more than one amino acid residue (e.g. dipeptide, tripeptide, oligopeptide). The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.
The terms “prevent,” “preventing” and “prevention”, as used herein, refer to inhibiting the inception or decreasing the occurrence of a disease in a subject. The prevention may be complete (e.g. the total absence of pathological cells in a subject). The prevention may also be partial, such as, for example, lowering the occurrence of pathological cells in a subject. Prevention also refers to a reduced susceptibility to a clinical condition. Within the context of the present invention, the terms “prevent,” “preventing” and “prevention”, refer specifically to averting or reducing the probability of HIV infection in a subject sustaining HIV exposure.
The expression term “product result from the processing of the Env protein”, as used herein, refers to any of the proteins produced by the processing of the gp160 protein.
The term “recombinant bacterium”, as used herein, refers to any bacterium modified by the introduction of heterologous DNA. “Wild-type” or “control” bacterium include bacterium as found in its natural state without genetic manipulation or that are substantially identical to the recombinant bacterium, but do not express one or more of the proteins encoded by the heterologous DNA (e.g. contains a plasmid without the coding sequence of the heterologous polypeptide of interest). The term is intended to include progeny of the bacterium originally modified by the introduction of heterologous DNA.
The expression “stringent hybridization conditions”, as used herein, refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acid, but to no other sequences.
The term “subject”, as used herein, refers to an individual, plant or animal, such as a human, a nonhuman primate (e.g. chimpanzees and other apes and monkey species); farm animals, such as birds, fish, rabbits, goats, sheep, pigs, cattle and horses; companion animals, such as dogs and cats; laboratory animals including rodents, such as mice, rats and guinea pigs. The term does not denote a particular age or sex. The term “subject” encompasses an embryo and a fetus. In a preferred embodiment, the subject is a human.
The term “substantially equivalent”, as used herein, refers to variants which are able to generate an immune response which is differs from the immune response generated with the native region by no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%.
The term “therapeutic agent” as used herein, refers to an atom, molecule or compound useful in the treatment or prevention of a disease. Examples of therapeutic agents include, but are not limited to, epitopes, antigens, immunogens, antibodies, antibody fragments, drugs, cytotoxic agents, pro-apopoptotic agents, toxins, nucleases (e.g. DNAses and RNAses), hormones, immunomodulators, chelators, photoactive agents or dyes, anti-angiogenic agents, chemotherapeutic agents, cytokines, chemokines, prodrugs, enzymes, binding proteins, peptides or combinations thereof.
The term “therapeutically effective amount”, as used herein, refers to the dose or amount of a fusion protein, polynucleotide, vector, host cell, VLP, pharmaceutical composition or immunogenic or vaccine composition according to the invention, or a combination thereof, that produces a therapeutic response or desired effect in a subject.
The terms “transmembrane domain” and “TM domain”, as used herein, refer to an amino acid sequence of approximately hydrophobic residues with an occasional polar residue of integral proteins that pass across membrane.
The expression “transmembrane domain of the HIV gp41 polypeptide”, as used herein, refers to the region of the gp41 polypeptide that is embedded into the plasma membrane once gp41 has acquired its native topology.
The terms “treat” and “treatment”, as used herein, refer to the administration of a fusion protein, polynucleotide, vector, host cell, VLP, pharmaceutical composition, immunogenic or vaccine composition according to the invention, or a combination thereof, for controlling the progression of a disease after its clinical signs have appeared. Control of the disease progression is understood to mean the beneficial or desired clinical results that include, but are not limited to, reduction of the symptoms, reduction of the duration of the disease, stabilization of pathological states (specifically to avoid additional deterioration), delaying the progression of the disease, improving the pathological state and remission (both partial and total). The control of progression of the disease also involves an extension of survival compared with the expected survival if treatment was not applied. Within the context of the present invention, the terms “treat” and “treatment” refer specifically to stopping or slowing the infection and destruction of healthy CD4+ T cells in an HIV infected subject. It also refers to the stopping and slowing of the onset of symptoms of the acquired immunodeficiency disease such as extreme low CD4+ T cell count and repeated infections by opportunistic pathogens. Beneficial or desired clinical results include, but are not limited to, an increase in absolute naïve CD4+ T cell count (range 10-3520), an increase in the percentage of CD4+ T cell over total circulating immune cells (range 1-50%), or an increase in CD4+ T cell count as a percentage of normal CD4+ T cell count in an uninfected subject (range 1-161%). “Treatment” can also mean prolonging survival of the infected subject as compared to expected survival if the subject does not receive any HIV targeted treatment.
The terms “vaccine” and “vaccine composition”, as used herein, refers to an immunogenic composition for in vivo administration to a subject, to confer protection against disease, and particularly, a bacterial or viral disease.
The term “vector”, as used herein, refers to a nucleic acid molecule, linear or circular, that comprises a nucleic acid according to the invention operably linked to additional segments that provide for its autonomous replication in a host cell or according to the expression cassette of the nucleic acid molecule.
The terms “virus-like particle vector” and “VLP”, as used herein, refer to non-infectious particles resembling viruses that do not contain any viral genetic material. VLPs are the result of the expression of viral structural proteins, such as capsid proteins, and their self-assembly.
In a first aspect, the present invention relates to a fusion protein which comprises from the N- to the C-terminus:
(a) a polypeptide of interest, or a functionally equivalent variant thereof,
(b) a transmembrane domain, or a functionally equivalent variant thereof, and
(c) an HIV gag polypeptide, or a functionally equivalent variant thereof.
In a preferred embodiment, the polypeptide of interest comprises a therapeutic agent, a region of a therapeutic agent, or a functionally equivalent variant thereof. Examples of therapeutic agents include, but are not limited to, epitopes, antigens, immunogens, antibodies, antibody fragments, drugs, cytotoxic agents, pro-apopoptotic agents, toxins, nucleases (e.g. DNAses and RNAses), hormones, immunomodulators, chelators, photoactive agents or dyes, anti-angiogenic agents, chemotherapeutic agents, cytokines, chemokines, prodrugs, enzymes, binding proteins, peptides, or a region thereof. Preferably, the therapeutic agent is an epitope, antigen, immunogen, or a functionally equivalent variant thereof. In another embodiment, the polypeptide of interest includes a combination of one or more therapeutic agents, their regions or functionally equivalent variants. Examples of these combinations include, but are not limited to, a polypeptide of interest comprising two or more therapeutic agents of different origin (e.g. viral antigen/bacterial antigen, tumor marker/bacterial antigen) or of the same origin (e.g. two or more antigens of the same pathogen).
Preferably, the HIV gag polypeptide, or the functionally equivalent variant thereof, is from HIV-1HXB2. The full sequence of the gag polypeptide from HXB2 has accession number P04585-1 in the UniProt database (6 Jul. 2016) and comprises sequence SEQ ID NO: 1. Preferably, the functional equivalent variants of the HIV gag polypeptide show at least by 60%, 70%, 80%, 90%, 95%, 97%, 98% or 99% of the formation activity of its reference polypeptide. There are several techniques known in the art for evaluating the formation activity of the functionally equivalent variants of the VLPs according to the invention.
Accordingly, a functionally equivalent variant of the HIV gag polypeptide shows a degree of similarity of at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% with respect to the amino acid sequence of the corresponding native HIV gag polypeptide, provided the VLP formation ability of the protein is maintained. Preferably, the similarity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50-100 amino acids or nucleotides in length. An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically deemed substantially identical to a second polypeptide, for example, when the two peptides differ only by conservative substitutions. The degree of similarity between two peptides can be determined using computer algorithms and methods, which are widely known by the persons skilled in the art. The identity between two amino acid sequences of two peptides is preferably determined using the BLASTP algorithm. See Altschul, S, et al., “BLAST Manual”, (NCBI NLM NIH Bethesda, Md. 20894, Altschul S, et al., J., 1990, Mol. Biol. 215:403-410). In a preferred embodiment, the degree of identity of the functionally equivalent variant with respect to gag is determined along the complete sequence of the variant, of gag or both.
In a preferred embodiment, the HIV gag polypeptide, or the functionally equivalent variant thereof, lacks the myristoylation sequence. Myristoylation is a lipidation modification where a myristoyl group derived from myristic acid, is covalently attached by an amide bond to the alpha-amino group of an N-terminal glycine residue. In the HIV gag polypeptide, the glycine susceptible to be myristoylated is the glycine in position 2 of SEQ ID NO: 1. The first methionine is removed by the host cell machinery. Preferably, the functionally equivalent variant of HIV gag lacking the myristoylation sequence comprises sequence SEQ ID NO: 2.
Any single-pass transmembrane protein may be used in the present invention. Examples include, but are not limited, to the transmembrane domain of CD22 (SEQ ID NO: 17), CD36 (SEQ ID NO: 18), CD44 (SEQ ID NO: 19) and the R696A mutant of HIV-1 Env (SEQ ID NO: 30). The capability of a sequence for forming a transmembrane domain can be analyzed by various assays known in the art.
In a preferred embodiment, the transmembrane domain is the transmembrane domain of the HIV gp41 polypeptide or a functionally equivalent variant thereof.
In a particular embodiment, the transmembrane region of the HIV gp41 polypeptide comprises sequence SEQ ID NO: 16
The fusion protein according to the invention comprises the immunogenic polypeptide located N-terminal to the HIV gag polypeptide, or the functionally equivalent variant.
The immunodominant polypeptide may comprise one or more epitopes. Generally, but not always, such immunodominant polypeptide or epitopes are highly immunogenic when tested according to methods that are known to those of skill in the art.
The proteins or peptides capable of inducing an immune response can be recombinant proteins or peptides, identical or similar to the natural antigens of a specific microorganism.
In a preferred embodiment, the polypeptide of interest comprises an immunogenic polypeptide. Preferably, the immunogenic polypeptide is a polypeptide capable of (suitable or designed for) inducing an immune response against an infectious disease in a subject, such as a disease caused by pathogenic microorganisms (e.g. virus, bacteria, fungi, mycoplasma, endo and ectoparasites). Preferably, the subject is a human being, a companion or farm animal. More preferably, the subject is human.
Viral pathogens include, but are not limited to, RNA viruses; DNA viruses; adenovirdiae (e.g. mastadenovirus, aviadeno virus); herpesviridae (e.g. herpes simplex virus 1, herpes simplex virus 2, herpes simplex virus 5, herpes simplex virus 6); leviviridae (e.g. levivirus, enterobacteria phase MS2, allolevirus); poxyiridae (e.g. chordopoxyirinae, parapoxvirus, avipoxvirus, capripoxvirus, leporipoxvirus, suipoxvirus, molluscipox virus, entomopoxyirinae); papovaviridae (e.g. polyomavirus, papillomavirus); paramyxoviridae (e.g. paramyxovirus, parainfluenza virus 1, mobillivirus (e.g. measles virus), rubulavirus (e.g. mumps virus); pneumonoviridae (e.g. pneumovirus, human RSV); metapneumovirus (e.g. avian pneumovirus, human metapneumo virus); picornaviridae (e.g. enterovirus, rhinovirus, hepatovirus (e.g. human hepatitis A virus), cardiovirus, apthovirus); reoviridae (e.g. orthoreo virus, orbivirus, rotavirus, cypo virus, fijivirus, phytoreo virus, oryzavirus); retroviridae (e.g. mammalian type B retroviruses, mammalian type C retroviruses, avian type C retroviruses, type D retrovirus group, BLV-HTLV retroviruses, lentivirus (e.g. HIV-1, HIV-2, spuma virus)); flaviviridae (e.g. hepatitis C virus); hepadnaviridae (e.g. hepatitis B virus); togaviridae (e.g. alphavirus (such as sindbis virus and rubivirus such as rubella virus); rhabdoviridae (e.g. vesiculovirus, lyssavirus, ephemera virus, cytorhabdovirus, necleorhabdovirus); arenaviridae (e.g. arenavirus, lymphocytic choriomeningitis virus, Ippy virus, lassa virus); and coronaviridae (e.g. coronavirus, torovirus); Cytomegalovirus (mononucleosis); Dengue virus (dengue fever, shock syndrome); Epstein-Barr virus (mononucleosis, Burkitt's lymphoma); Human T-cell lymphotropic virus type 1 (T-cell leukemia); Influenza A, B, and C (respiratory disease); Japanese encephalitis virus (pneumonia, encephalopathy); Poliovirus (paralysis); Rhinovirus (common cold); Rubella virus (fetal malformations); Vaccinia virus (generalized infection); Yellow fever virus (jaundice, renal and hepatic failure); and Varicella zoster virus (chickenpox). Examples of antigens of these viral pathogens include, hepatitis B surface antigen, rotavirus antigens such as VP4 and VP7, influenza virus antigens such as hemagglutinin or nucleoprotein and the thymidine kinase herpes simplex antigen.
The viral pathogens also include HIV antigens capable of generating an HIV-specific immune response. HIV antigens include, but are not limited to, peptides derived from HIV early regulatory proteins including HIV Tat, Rev and Nef (e.g. Nef-V3) proteins, or other HIV proteins such as Gag, Pol, Env, Vif, Vpr and Vpu. In particular, peptide epitopes of these proteins are those that are capable of generating neutralizing antibodies. Numerous HIV CTL/CD8+ and T-helper/CD4+ epitopes are known in the art and are contemplated for use in compositions effective for generating an HIV-specific immune response and are contemplated for use in the fusion proteins, VLPs and pharmaceutical compositions of the invention. See Korber B, et al., Eds., “HIV Molecular Immunology”, (Los Alamos National Laboratory, Los Alamos, N. Mex., USA, 2006/2007).
Examples of specific viral pathogens which are important in veterinary medicine include, but are not limited to, Classical swine fever virus, PCV2 (postweaning multisystemic wasting syndrome) and Pseudorabies virus (Aujeszky's disease) (pigs); BHV-1 (bovine herpesvirus type 1) (cattle); Equine influenza virus and WNV (West Nile virus) (horses); Avian influenza virus (e.g. H5N1, HKN3), HTV (Turkey herpesvirus), MDV (Marek's disease virus) and IBDV (infectious bursal disease virus) and NVD (Newcastle disease virus) (poultry); Feline leukemia virus and Rabies virus (cats); and Canine coronavirus, Canine distemper virus, Canine parvovirus and IHN virus (dogs).
Bacterial pathogens include, but are not limited to, Actinornyces israelli, Bacillus anthraces, Bacteroides spp., Bordetella pertussis, Borrelia burgdorferi, Brucella spp., Campylobacter spp., Campylobacter jejuni, Chlamydia spp., Chlamydia trachomatis, Clostridium botulinum, Clostridium perfringens, Clostridium tetani, Corynebacterium dipththeriae, Enterococcus spp., Erysipelothrix rhusiopathiae, Escherichia coli, enterohemorrhagic E. coli, enterotoxigenic E. coli, Fusobacterium nucleatum, Haemophilus influenzae type B and non-typable, Helicobacter pylori, Klebsiella pneumoniae, Legionella pneumophila, Leptospira spp. Listeria monocytogenes, Mycobacterium spp. (e.g. M. avium, M. gordonae, M. intracellulare, M. kansaii, M. leprae, M. tuberculosis), Mycobacterium tuberculosis, Neisseria gonorrhoeae, Neisseria meningitidis, Pasteurella spp., Pasteurella multocida, Pneumococcus spp., Pseudomonas aeruginosa, Rickettsia spp., Salmonella spp., Shigella spp., Staphylococcus aureus, Streptococcus spp., Streptococcus (anaerobic species), Streptococcus (viridans group), Streptococcus agalactiae (Streptococcus Group B), Streptococcus B, Streptococcus bovis, Streptococcus faecalis, Streptobacillus moniliformis, Streptococcus mutans, Streptococcus pneumoniae, Streptococcus pyogenes, Group A beta hemolytic Streptococcus, Treponema pallidum, Treponema pertenue, Vibrio cholera and Yersinia pestis.
Examples of specific bacterial pathogens which are important in veterinary medicine include, but are not limited to, Actinobacillus pleuropneumoniae and Lawsonia intracellularis (pigs); Anaplasma centrale, Anaplasma marginate, Brucella abortus, Brucella melitensis and Ehrlichia ruminantium (cattle); Streptococcus equi (horses); Chlamydophila abortus (sheep); Bordetella avium, Mycoplasma gallisepticum, Mycoplasma synoviae and Salmonella spp. (poultry); Aeromonas salmonicida, Vibrio anguillarum and Yersinia ruckeri (fish); and Porphyromonas gulae, Porphyromonas denticanis and Porphyromonas salivosa (dogs).
Pathogenic fungi include, but are not limited to, the genera Aspergillus, Blastomyces, Candida, Coccidiodes, Cryptococcus, Histoplasma, Phycomyces, Tinea corporis, Tinea unguis, Sporothrix schenckii and Pneumocystis carinii. Specific examples of pathogenic fungi include, but are not limited to, Blastomyces dermatitides, Candida albicans, Chlamydia trachomatis, Coccidioides immitis, Cryptococcus neoformans and Histoplasma capsulatum.
Pathogenic parasites include, but are not limited to, the genera Dirofilaria, Leishmania, Plasmodium, Schistosoma, Toxoplasma and Tripanosoma. Specific examples of pathogenic parasites include, but are not limited to, Dirofilaria immitis, Leishmania major, Leishmania donovani, Leishmania infantum, Leishmania braziliensis, Leishmania. panamensis, Leishmania mexicana, Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale Plasmodium vivax, Toxoplasma gondii, Tripanosoma brucei and Tripanosoma cruzi. Examples of specific antigens for these pathogenic parasites include Plasmodium spp. circumsporozoite antigen, Plasmodium spp. merozoite surface antigen and Leishmania spp. gp63.
Examples of specific pathogenic fungi and parasites which are important in veterinary medicine include, but are not limited to, Babesia bigemina, Babesia bovis, Dictyocaulus viviparous, Fasciola gigantica, Fasciola hepatica, Neospora caninum, Theileria annulata, Theileria hirci and Theileria parva (cattle); Sarcocystis neurona (horses); Toxoplasma gondii (sheep); Eimeria spp. (coccidiosis), Eimeria maxima and Eimeria tenella (poultry); and Babesia canis, Giardia duodenalis and Leishmania donovani (dogs).
In another preferred embodiment, the immunogenic polypeptide is a polypeptide associated with tumors or cancers (“tumor markers”) capable of (suitable or designed for) inducing or modulating an immune response against a tumor or cancer cell. Therefore, the fusion proteins according to the invention could be used for the treatment or prevention of cancers by means of the stimulation of an antigen-specific immune response against a tumor antigen.
Over a thousand proteins are differentially expressed in human cancers and thus may serve as tumo rmarkers. Such proteins play a role in cancer-related processes including, but not limited to, angiogenesis, apoptosis, cell differentiation, cell signaling, hematopoiesis, hormonal control and immune reactions. Exemplary tumor markers include, but are not limited to, carcinoembryonic antigen (CEA) for both malignant pleural effusion and peritoneal cancer dissemination; human epidermal growth factor receptor 2 (HER-2/neu) for stage IV breast cancer; bladder tumor antigen for urothelial cell carcinoma; thyroglobulin for thyroid cancer metastasis; α-fetoprotein for hepatocellular carcinoma; prostate specific antigen (PSA) for prostate cancer; cancer antigen 125 (CA 125) for non-small cell lung cancer; cancer antigen 19.9 (CA 19.9) for pancreatic cancer; cancer antigen 15.3 (CA 15.3) for breast cancer; the combination of leptin, prolactin, osteopontin, and insulin-like growth factor II (IGF-II) for ovarian cancer; the combination of CD98, fascin, secreted chain of the polymeric immunoglobulin receptor (sPIgR), and 14-3-3 eta proteins for lung cancer; troponin I for myocardial infarction, and B-type natriuretic peptide for congestive heart failure.
Other common tumor markers include the estrogen receptor/progesterone receptor (ER/PR), HER-2/neu, and epidermal growth factor receptor (EGFR) for breast cancer, and tissue inhibitor of metalloproteinases (TIMP-1)-associated with serum HER2-positive breast cancer; Kirsten Ras oncogene (KRAS) and UDP glucuronosyltransferase family 1 member A (UGT1A1) for colorectal cancer; HER-2/neu for gastric cancer, c-KIT, CD20 antigen, CD30, and factoril interacting with PAPOLA and CPSF1-platelet-derived growth factor receptor alpha fusion protein (FIP1L1-PDGRF alpha), and platelet-derived growth factor receptor (PDGFR) for gastrointestinal stromal tumor (GIST); Philadelphia Chromosome (BCR/ABL)/PML/RAR alpha and anaplastic lymphoma kinase (TPMT/UGT1A1/ALK EGFR) for leukemia/lymphoma; KRAS/EGFR for lung cancer, and BRAF and S100 for melanoma.
Other examples of tumor markers include tumor suppressors that are lost in cancers, such as Breast Cancer Gene 1 (BRCA1), Breast Cancer Gene 2 (BRCA2); RNA such as mRNA, microRNA; proteins found in body fluids or tissue such as prostate specific antigen and CA-125; gene and protein based biomarkers; and nonspecific biomarkers such as glycosaminoglycans in body fluids; alkaline phosphatase and urinary hydroxyproline in skeletal involvement; hyaluronic acid excretion and urinary hydroxyproline in bone disease, and combinations thereof.
Tumor markers associated with important veterinary malignancies suchs as Canine malignant melanoma (CMM), the most common oral tumor in dogs and bovine ocular squamous cell carcinoma, can also be presented using the fusion proteins and VLPs of the invention.
In another preferred embodiment, the immunogenic polypeptide is a polypeptide capable of (suitable or designed for) inducing an immune response against an allergen.
Non-limiting illustrative examples of allergens include, but are not limited to, protein extracts of pollens from trees and flowers (e.g. Lolium perenne, Poa pratense, Phleum pratense, Cynodon dactylon, Festuca pratensis, Dactylis glomerata, Secale cereale, Hordeum vulgare, Avena sativa, Triticum sativa, Artemisia vulgaris, Chenopodium album, Plantago lanceolata, Taraxacum vulgare, Parietaria judaica, Salsola kali, Urtica dioica, Olea europea, Platanus spp., Cupressus spp), protein extracts of insects (e.g. Dermatophagoides pteronyssinus, Dermatophagoides farinae, Acari spp. (e.g. Acarus siro), Blomia tropicalis, Euroglyphus maynei, Glyciphagus domesticus, Lepidoglyphus destructor, Tyrophagus putrescentiae), protein extracts of fungi or of animal dander (e.g. Penicillium spp., Alternaria alternata, Cladosporium herbarum, dog dander, cat dander, horse dander) and protein extracts of food or food products.
In a preferred embodiment, the immunogenic polypeptide is from a protein of a virus. Preferably, the virus is HIV.
In a preferred embodiment, the immunogenic polypeptide does not contain an epitope from the HIV gag polypeptide.
In another preferred embodiment, the immunogenic polypeptide is from an Env protein of the HIV virus or a product results from the processing of the Env protein.
In a preferred embodiment, the sequence of the Env protein of HIV comprises SEQ ID NO: 20, or a functionally equivalent variant thereof.
In a preferred embodiment, the product resulting from the processing of the Env protein, is gp41, or a functionally equivalent variant.
The functional equivalent variants of the gp41 polypeptide have preferably at least 60%, 70%, 80%, 90%, 95%, 97%, 98% or 99% the formation activity of their reference polypeptide. The skilled person knows several techniques to detect VLP formation of a functional variant of an HIV gag polypeptide.
In a preferred embodiment, the gp41 variant sequence comprises SEQ ID NO: 5 and comprises the gp41 HR2 region and the MPER region.
In another preferred embodiment, the size of the immunogenic polypeptide has less than 90 amino acids, less than 80 amino acids, less than 70 amino acids or less than 60 amino acids.
In another preferred embodiment, the immunogenic polypeptide comprises from the N to C terminus:
The gp41 HR1 and HR2 sequences are well known in the art. In a preferred embodiment, the HR2 region comprises SEQ ID NO: 21. Preferably, the MPER region comprises SEQ ID NO: 21, in the particular case of the HXB2 Env protein.
As modifications and changes may be made in the structure of the regions forming the gp41 variants according to the invention and still obtain molecules having like or otherwise desirable characteristics, such functional equivalents are also encompassed within the scope of the present invention.
For the purposes of the present invention, a polypeptide that is useful as an antigen in an immunogenic composition (i.e. has sufficient “immunogenic activity”) can be identified by any method commonly known in the art for measuring the ability of a given polypeptide to trigger the generation of antibodies specific for said polypeptide when administered to a host organism. The ability of a given variant of the polypeptide according to the invention to be an immunologically equivalent variant may be determined using standard neutralization assays, wherein the suspected variant is inoculated into a test animal and the resulting antibodies are tested for their ability to neutralize the infection of susceptible cells by HIV strains.
Immunologically functional equivalents of the immunogenic polypeptide according to the invention can be obtained by modifying the polypeptide. Said modifications can be, without limitation, amino acid changes, deletions, truncations, polypeptide fragments, fusions to other polypeptides, insertions or any combination thereof. Accordingly, immunologically functional equivalents of the immunogenic polypeptide show a degree of identity with respect to the amino acid sequence shown of the corresponding region in the native immunogenic polypeptide of at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% provided that the immunogenic activity of the reference polypeptide is maintained. Preferably, the identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is at least 50-100 amino acids or nucleotides in length. An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. The degree of identity between two peptides can be determined using computer algorithms and methods, which are widely known by the persons skilled in the art. The identity between two amino acid sequences of two peptides is preferably determined using the BLASTP algorithm. See Altschul, S. et al., “BLAST Manual”, (NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al., J., 1990, Mol. Biol. 215:403-410).
As an example of modifications contemplated to be within the scope of the present invention, certain amino acids may be substituted for other amino acids in a polypeptide structure without appreciable loss of interactive binding capacity of the structure such as, for example, the epitope of an antigen that is recognized and bound by an antibody. Since it is the interactive capacity and nature of a polypeptide that defines its biological (e.g. immunological) functional activity, certain amino acid sequence substitutions can be made in an amino acid sequence (or its underlying DNA coding sequence) and nevertheless obtain a polypeptide with comparable properties. Various changes may be made to the amino acid sequences of the antigens of the present invention without appreciable loss of immunogenic activity.
It is understood in the art that for making functionally equivalent amino acid substitutions, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biological function on a polypeptide is generally understood in the art. See Kyte J, et al., J. Mol. Biol. 1982; 15(1):105-132. It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. In making changes based upon the hydropathic index, the substitution of amino acids whose hydropathic indices are within plus or minus 2 is preferred, those which are within plus or minus 1 are particularly preferred, and those within plus or minus 0.5 are even more particularly preferred. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics; these are: isoleucine (+4.5), valine (+4.2), leucine (+3.8), phenylalanine (+2.8), cysteine/cystine (+2.5), methionine (+1.9), alanine (+1.8), glycine (−0.4), threonine (−0.7), serine (−0.8), tryptophan (−0.9), tyrosine (−1.3), proline (−1.6), histidine (−3.2), glutamate (−3.5), glutamine (−3.5), aspartate (−3.5), asparagine (−3.5), lysine (−3.9) and arginine (−4.5).
It is also understood in the art that the substitution of similar amino acids can be made effectively based on their hydrophilicity; particularly where the immunologically functional equivalent polypeptide thereby created is intended for use in immunological embodiments, as in certain embodiments of the present invention. The greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity. See Hopp T, U.S. Pat. No. 4,554,101. In making changes based upon similar hydrophilicity values, the substitution of amino acids whose hydrophilicity values are within plus or minus 2 is preferred, those which are within plus or minus 1 are particularly preferred, and those within plus or minus 0.5 are even more particularly preferred. The following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0), lysine (+3.0), aspartate (+3.0 plus or minus 1), glutamate (+3.0 plus or minus 1), serine (+0.3), asparagine (+0.2), glutamine (+0.2), glycine (0), threonine (−0.4), proline (−0.5 plus or minus 1), alanine (−0.5), histidine (−0.5), cysteine (−1.0), methionine (−1.3), valine (−1.5), leucine (−1.8), isoleucine (−1.8), tyrosine (−2.3), phenylalanine (−2.5) and tryptophan (−3.4).
It is well known in the art that where certain residues are shown to be particularly important to the immunological or structural properties of a protein or peptide, like for example, residues in binding regions or epitopes, such residues may not generally be exchanged. In this manner, functional equivalents are defined herein as those polypeptides, which maintain a substantial amount of their native immunological activity. In general, the shorter the length of the molecule, the fewer changes can be made to the molecule without affecting its function. Longer domains may have an intermediate number of changes. The full-length protein will have the most tolerance for a larger number of changes. However, it must be appreciated that certain molecules or domains that are highly dependent upon their structure may tolerate little or no modification.
Immunologically equivalent variants can also be obtained by the expression from nucleic acid sequences which are substantially identical to the molecule encoding the regions in the native immunogenic polypeptide or their complements hybridize to each other under stringent conditions, as described below, provided that the immunogenic activity of the polypeptide encoded by said nucleic acids is preserved.
Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. See Tijssen S, “Overview of principles of hybridization and the strategy of nucleic acid assays”, Laboratory Techniques in Biochemistry and Molecular Biology (Elsevier Science Publishers B.V., Amsterdam, N L 1993). Generally, stringent conditions are selected to be about 5-10° C. degrees lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH. The Tm is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50 percent of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm 50 percent of the probes are occupied at equilibrium). Stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g. 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g. greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For high stringency hybridization, a positive signal is at least two times background, preferably 10 times background hybridization. Exemplary high stringency or stringent hybridization conditions include: 50 percent formamide, 5×SSC and 1 percent SDS incubated at 42° C. or 5×SSC and 1 percent SDS incubated at 65° C., with a wash in 0.2×SSC and 0.1 percent SDS at 65° C.
A person skilled in the art will appreciate that the different components which form part of the fusion protein according to the present invention may be functionally linked directly or indirectly through a linker region.
In a preferred embodiment, the fusion protein according to the invention further comprises at least one linker. Preferably, the linker comprises glycine residues and, more preferably glycine and serine residues. Even more preferably, the linker comprises three glycine residues and one serine residue. Preferably, the linker comprises sequences SEQ ID NO: 23 or SEQ ID NO: 24. More preferably, the linker sequence is selected from the group consisting of sequences SEQ ID NO: 23 and SEQ ID NO: 24.
In another preferred embodiment, the fusion protein according to the invention further comprises a tag which may be used for the detection or for the purification of the gp41 variant using reagents showing specific affinity towards said tags. Adequate detection/purification tags includes hexa-his (metal chelate moiety), hexa-hat GST (glutathione 5-tranferase) glutathione affinity, calmodulin-binding peptide (CBP), strep-tag, cellulose binding domain, maltose binding protein, S-peptide tag, chitin binding tag, immuno-reactive epitopes, epitope tags, E2tag, HA epitope tag, Myc epitope, FLAG epitope, AU1 and AU5 epitopes, Glu-Glu epitope, KT3 epitope, IRS epitope, Btag epitope, protein kinase-C epitope, VSV epitope or any other tag as long as the tag does not affect the stability of the protein or the immunogenicity of the antigen attached thereto.
Several types of amino acid sequences may be added to the fusion proteins according to the invention to attain different objectives such as, for example, facilitating their handling or monitoring their expression. The addition of these peptide sequences to the fusion protein according to the invention does not affect their functional or immunogenic capabilities.
In a preferred embodiment, the linker sequence comprises sequences SEQ ID NO: 25, SEQ ID NO: 26 or SEQ ID NO: 27. Preferably, the linker sequence is selected from the group consisting of sequences SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27.
In another preferred embodiment, the fusion protein according to the invention comprises sequence SEQ ID NO: 28.
a) Nucleic Acids
In an additional aspect, the invention relates to a polynucleotide encoding a fusion protein according to the invention.
In a preferred embodiment, the polynucleotide according to the invention comprises sequence SEQ ID NO: 9.
In a preferred embodiment, the sequence of the polynucleotide according to the invention is codon-optimized according to the host selected for its expression. In another preferred embodiment, the codon-optimized sequence coding for the immunogenic polypeptide according to the invention comprises sequence SEQ ID NO: 11. In an additional preferred embodiment, the codon-optimized sequence coding for the HIV gag polypeptide according to the invention comprises sequence SEQ ID NO:13
Many methods and software tools for codon optimization are known to the skilled person. Codon-optimized nucleic acids for use according to the present invention can be prepared by replacing the codons of the nucleic acid encoding the immunogen by codons with appear with a high frequency in the selected host genome (e.g. “humanized” codons, codons that appear frequently in highly expressed human genes).
In a preferred embodiment, the codon-optimized polynucleotide according to the invention (i.e. human) comprises sequence SEQ ID NO: 14.
The polynucleotide of this invention can be operably linked to any promoter or enhancer capable of driving expression of the nucleic acid following introduction into a host cell. A promoter is an array of nucleic acid control sequences that directs transcription of a nucleic acid. A promoter includes necessary nucleic acid sequences (which can be) near the start site of transcription, such as in the case of a polymerase II type promoter (a TATA element). A promoter also can include distal enhancer or repressor elements which can be located as much as several thousand base pairs from the start site of transcription. Both constitutive and inducible promoters are included.
The polynucleotides according to the invention can be incorporated into a vector, for the purposes of cloning, performing other laboratory manipulations, manufacturing recombinant peptides or gene delivery.
b) Vectors
In a further aspect, the invention refers to a vector comprising a polynucleotide according to the invention.
A person skilled in the art will understand that there is no limitation regarding the type of vector that can be used with the present invention. The vector can be a cloning vector, suitable for propagation and for obtaining polynucleotides, gene constructs or expression vectors incorporated to several heterologous organisms. Thus, suitable vectors according to the present invention include prokaryotic expression vectors (e.g. pUC18, pUC19, Bluescript and their derivatives), mp18, mp19, pBR322, pMB9, CoIEl, pCR1, RP4, phages and shuttle vectors (e.g. pSA3 and pAT28), yeast expression vectors (e.g. vectors of the type of 2 micron plasmids), integration plasmids, YEP vectors, centromeric plasmids and the like, insect cell expression vectors (e.g. the pAC series and pVL series vectors), plant expression vectors, such as vectors of expression in plants (e.g. pIBI, pEarleyGate, pAVA, pCAMBIA, pGSA, pGWB, pMDC, pMY, pORE series vectors), and eukaryotic expression vectors based on viral vectors (e.g. adenoviruses, viruses associated to adenoviruses as well as retroviruses and lentiviruses), as well as non-viral vectors such as pSilencer 4.1-CMV (Ambion, Applied Biosystems, Foster City, Calif., USA), pcDNA3, pcDNA3.1/hyg pHCMV/Zeo, pCR3.1, pEFl/His, pIND/GS, pRc/HCMV2, pSV40/Zeo2, pTRACER-HCMV, pUB6/V5-His, pVAX1, pZeoSV2, pCI, pSVL and pKSV-10, pBPV-1, pML2d and pTDT1. In a preferred embodiment, the vector is pcDNA3.1.
In a particular embodiments, the vector is an expression vector that can include a regulatory element, such as the cytomegalovirus hCMV immediate early gene, the early promoter of SV40 adenovirus, the late promoter of SV40 adenovirus, the lac system, the TAC system, the TRC system, the major operator and promoter regions of phage, the control regions of fd coat protein, the promoter for 3-phosphoglycerate kinase, the promoters of acid phosphatase or the promoters of the yeast mating factors. Exemplary vectors include, but are not limited to, bacterial artificial chromosomes, cosmids, yeast artificial chromosomes, phage, plasmids, lipid vectors and viral vectors (e.g. retrovirus). Suitable expression vectors for fusion protein have been previously described. See Kayman, U.S. Pat. No. 5,643,756.
c) Host Cells
In another aspect, the invention is directed to a host cell comprising the fusion proteins according to the invention, the polynucleotides encoding them or the vectors comprising said polynucleotides.
Cells suitable in the present invention include, without limitation, mammal, plant, insect, fungal and bacterial cells. Bacterial cells include, but are not limited to, Gram-positive bacterial cells such as species of the Bacillus, Streptomyces and Staphylococcus genus and Gram-negative bacterial cells such as cells of the Escherichia and Pseudomonas genus. Fungal cells preferably include yeast cells such as Saccharomyces, Pichia pastoris and Hansenula polymorpha. Insect cells include, but are not limited to, Drosophila cells and Sf9 cells. Plant cells include, among others, cells of crop plants such as cereals, medicinal, ornamental or bulbous plants. Mammalian cells include, but are not limited to, Chinese hamster ovary (CHO) cells such as CHO-K1 (ATCC accession number CCL-61), DG44 (Chasin L, et al., Som. Cell Mol. Genet. 1986; 12:555-556; and Kolkekar A, et al., Biochemistry 1997; 36:10901-10909), CHO-K1 Tet-On (Clontech, Mountain View, Calif., USA), CHO designated ECACC 85050302 (CAMR, Salisbury, Wiltshire, UK), CHO clone 13 (GEIMG, Genova, IT), CHO clone B (GEIMG, Genova, IT), CHO-K1/SF designated ECACC 93061607 (CAMR, Salisbury, Wiltshire, UK), RR-CHOK1 designated ECACC 92052129 (CAMR, Salisbury, Wiltshire, UK), CHO cells negative for dihydrofolate reductase (CHO/−DHFR, Urlaub G, et al., Proc. Natl. Acad. Sci. USA 1980; 77:4216-4220), SV40-transformed monkey kidney CV1 cells (COS, COS-7, ATCC accession number CRL-1651); human embryonic kidney cells (HEK-293, HEK-293T cells); baby hamster kidney cells (BHK, ATCC accession number CCL-IO); monkey kidney cells (CV1, ATCC accession number CCL-70); African Green Monkey kidney cells (VERO-76, ATCC accession number CRL-1587; VERO, ATCC accession number CCL-81); mouse Sertoli cells (TM4, Mather J, Biol. Reprod. 1980; 23:243-251); human cervical carcinoma cells (HELA, ATCC accession number CCL-2); dog kidney cells (MDCK, ATCC accession number CCL-34); human lung cells (W138, ATCC accession number CCL-75); human hepatoma cells (HEP-G2, HB 8065); mouse mammary tumor cells (MMT 060562, ATCC accession number CCL-51); buffalo rat liver cells (BRL 3A, ATCC accession number CRL-1442); TRI cells (Mather J, Ann. NY Acad. Sci. 1982, 383:44-68); MCR 5 cells and FS4 cells. Preferably, the host cells utilized are HEK-293T cells.
In a preferred embodiment, the host cell according to the invention is a recombinant bacterium.
In a preferred embodiment, the bacterium is a Gram-negative bacterial cell, and this term is intended to include all facultative Gram-negative cells of the family Enterobacteriaceace such as Escherichia spp., Shigella spp., Citrobacter spp., Salmonella spp., Klebsiella spp., Enterobacter spp., Erwinia spp., Kluyvera spp., Serratia spp., Cedecea spp., Morganella spp., Hafhia spp., Edwardsiella spp., Providencia spp., Proteus and Yersinia spp.
All the terms and embodiments previously described in relation to the fusion proteins the invention are equally applicable to polynucleotides, vectors or host cells according to the invention.
In a still further aspect, the invention relates to a method for preparing a VLP loaded with a polypeptide of interest comprising:
(a) expressing in a cell a fusion protein according to the invention, and
(b) recovering the VLP from the extracellular medium.
The first step of the method according to the invention comprises the transformation of a host cell with a vector comprising the polynucleotide according to the invention. Said transformation can be carried out by conventional techniques that are well known to those of ordinary skill in the art. Where the host is prokaryotic, such as E. coli, competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaCl2) method using procedures well known in the art. Alternatively, MgCl2 or RbCl can be used. Transformation can also be performed after forming a protoplast of the host cell if desired, or by electroporation.
When the host is a eukaryote, such methods of transfection of DNA as calcium phosphate coprecipitates, conventional mechanical procedures such as microinjection, electroporation, insertion of a plasmid encased in liposomes or virus vectors can be used. Eukaryotic cells can also be co-transformed with polynucleotide sequences encoding an immunogenic gp120 polypeptide, and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene. Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express the protein
The cell culture should be maintained under suitable conditions to allow the polynucleotide according to the invention to be expressed.
The second step of the method according to the invention comprises recovering the VLP from the extracellular medium, for example by centrifugation and filtration through 0.45 μm filters.
In the case of the VLPs obtained using the fusion proteins according to the invention, the VLPs are characterized for having a viral capsid enclosed by a lipidic envelope, which lipid envelope is derived from the cell that formed the VLP.
In another aspect, the invention relates to a virus like-particle comprising the fusion protein according to the invention or obtained using the method according to the invention.
All the terms and embodiments previously described are equally applicable to the method for preparing a VLP according to the invention and to the VLP according to the invention.
In a further aspect, the invention refers to a pharmaceutical composition comprising a fusion protein, a polynucleotide, a vector, a host cell or a virus-like particle according to the invention, or a combination thereof.
The pharmaceutical composition according to the invention may further comprise a pharmaceutically acceptable carrier.
A pharmaceutically acceptable carrier is essentially non-toxic to recipients at the dosages and concentrations employed, and is compatible with other ingredients of the formulation. For example, the carrier for a formulation containing polypeptides would not normally include oxidizing agents and other compounds that are known to be deleterious to polypeptides. Suitable carriers include, but are not limited to water, dextrose, glycerol, saline, ethanol, and combinations thereof. The carrier can contain additional agents such as wetting or emulsifying agents, pH buffering agents, or adjuvants which enhance the effectiveness of the formulation.
The pharmaceutical composition according to the invention can be administered by any means known to one skilled in the art, such as by intramuscular, subcutaneous or intravenous injection, and oral, nasal, or anal administration. See Banga A, “Parenteral Controlled Delivery of Therapeutic Peptides and Proteins,” in Therapeutic Peptides and Proteins (Technomic Publishing Co., Inc., Lancaster, Pa., USA, 1995). The polypeptide of interest according to the invention can be provided as an implant, an oily injection or as a particulate system to prolong it therapeutic benefits (e.g. increase immunogenic response). The particulate system can be a microparticle, a microcapsule, a microsphere, a nanocapsule or similar particle. See Banga, 1995, supra. Preferably, the pharmaceutical compositions according to the invention are administered parenterally.
The pharmaceutical compositions according to the invention can be formulated in unit dosage form, suitable for individual administration of precise dosages. A therapeutically effective amount of pharmaceutical composition can be administered in a single dose, or in multiple doses, for example daily, during a course of treatment or as part of a prevention protocol. In specific, non-limiting examples, pulse doses of a pharmaceutical composition according to the invention include at least daily, at least weekly and at least monthly administration regimens. The dosage, frequency and route of administration of the pharmaceutical composition may be varied by the skilled person according to the needs of the subject and following the applicable protocols in human and veterinary medicine.
The therapeutically effective amount of the pharmaceutical composition according to the invention can depend on the severity of the disease and the age, weight, general state of the subject, and other clinical factors, such as the subject's sensitivity to the polypeptide of interest. Thus, the final determination of the appropriate treatment regimen will be made by the attending clinician. Typically, dosages used in vitro can provide useful guidance in the amounts useful for in situ administration of the pharmaceutical composition, and animal models may be used to determine effective dosages for treatment of particular disorders. See Gilman R, et al., Eds., Goodman and Gilman's: The Pharmacological Basis of Therapeutics, 8th Ed. (Pergamon Press, New York, N.Y., US, 1990), and Gennaro A, Ed., Remington's Pharmaceutical Sciences, 18th Ed. (Mack Publishing Co., Easton, Pa., US, 1990). Typically, the dose range is from about 0.1 μg/kg body weight to about 100 mg/kg body weight. Other suitable ranges include doses of from about 1 μg/kg to 10 mg/kg body weight. In one example, the dose is about 1.0 μg to about 50 mg, for example, 1 μg to 1 mg, such as 1 mg peptide per subject.
a) Immunogenic or Vaccine Compositions
In a preferred embodiment, the pharmaceutical composition according to the invention is an immunogenic or vaccine composition. Said immunogenic or vaccine composition comprises a therapeutically effective amount a fusion protein, a polynucleotide, a vector, a host cell or a virus-like particle according to the invention, or a combination thereof.
The immunogenic or vaccine composition according to the invention may further comprise a pharmaceutically acceptable carrier and other excipients such as adjuvant. Examples of adjuvant that can be used with the immunogenic or vaccine compositions according to the invention include, but are not limited to, AlK(SO4)2, AlNa(SO4)2, AlNH4(SO4), silica, alum, Al(OH)3, Ca3(PO4)2, kaolin, carbon, aluminum hydroxide, muramyl dipeptides, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-DMP), N-acetyl-nornuramyl-L-alanyl-D-isoglutamine (CGP 11687, also referred to as nor-MDP), N-acetylmuramyul-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′2′-dipalmitoyl-sn-glycero-3-hydroxphosphoryloxy)-ethylamine (CGP 19835A, also referred to as MTP-PE), RIBI (MPL+TDM+CWS) in a 2 percent squalene/Tween-80® emulsion, lipopolysaccharides and its various derivatives, including lipid A, Freund's Complete Adjuvant (FCA), Freund's Incomplete Adjuvants, Merck Adjuvant 65, polynucleotides (for example, poly IC and poly AU acids), wax D from Mycobacterium tuberculosis, substances found in Corynebacterium parvum, Bordetella pertussis, and members of the genus Brucella spp., Titermax, ISCOMS, Quil A, ALUN, Lipid A derivatives, choleratoxin derivatives, HSP derivatives, LPS derivatives, synthetic peptide matrixes or GMDP, interleukin 1, interleukin 2, Montanide ISA-51 and QS-21, CpG oligonucleotide, poly I:C and GM-CSF. See Hunter, U.S. Pat. No. 5,554,372, and Jager, WO1997028816. A particulate carrier based on a synthetic polymer has been shown to act as an adjuvant to enhance the immune response, in addition to providing a controlled release. Aluminum salts can also be used as adjuvants to produce an immune response.
In a preferred embodiment, the immunogenic or vaccine composition according to the invention comprises a therapeutically effective amount of the virus-like particles according to the invention and is devoid of adjuvant. In a more preferred embodiment, the immunogenic or vaccine composition according to the invention is essentially free from aluminum phosphate.
The immunogenic or vaccine compositions according to the invention can be administered whenever its therapeutic effect is desired (e.g. decreased signs, symptoms, or laboratory results of HIV-1 infection). Generally, the dose is sufficient to treat or ameliorate symptoms or signs of disease without producing unacceptable toxicity to the subject. Systemic or local administration can be utilized. The immunogenic or vaccine compositions may be administered to a subject according to a prime/boost protocol, whereby a first administration of the composition is followed by at least one second administration at a different date using the same or a different dosage to reinforce or maintain the subject's immune response against the polypeptide of interest (i.e. immunogen).
All the terms and embodiments previously described are equally applicable to the immunogenic composition or the vaccine composition according to the invention.
In another aspect, the invention is directed to a fusion protein, a polynucleotide, a vector, a host cell, a virus-like particle or a pharmaceutical composition according to the invention, or combination thereof, for use in medicine. In a preferred embodiment, the fusion protein, polynucleotide, vector, host cell, virus-like particle or pharmaceutical composition according to the invention, or combination thereof, is for use in human medicine. In another preferred embodiment, the fusion protein, polynucleotide, vector, host cell, virus-like particle or pharmaceutical composition according to the invention, or combination thereof, is for use in veterinary medicine.
In a still further aspect, the invention relates to a fusion protein, a polynucleotide, a vector, a host cell, a virus-like particle or a pharmaceutical composition according to the invention, or combination thereof, for use in the treatment or prevention of a disease caused by an infection or tumor in a subject. In a preferred embodiment, the subject is a human being. In another preferred embodiment, the subject is a farm or companion animal. In an alternative form of this aspect, the invention relates to a method of treating or preventing a disease caused by an infection or tumor in a subject which comprises the administration of a therapeutically effective amount of a fusion protein, a polynucleotide, a vector, a host cell, a virus-like particle or a pharmaceutical composition according to the invention, or combination thereof, to a subject. In a further alternative form of this aspect, the invention refers to the use of a fusion protein, a polynucleotide, a vector, a host cell, a virus-like particle or a pharmaceutical composition according to the invention, or combination thereof, in the manufacture of a medicament for the treatment or prevention of a disease caused by an infection or tumor in a subject.
In an additional aspect, the invention relates to a fusion protein, a polynucleotide, a vector, a host cell, a virus-like particle or a pharmaceutical composition according to the invention, or combination thereof, for use in the delivery of therapeutic agents to cells or in diagnosis.
In a preferred embodiment, the polypeptide of interest is an HIV polypeptide. In a more preferred embodiment, the HIV polypeptide comprises the MPER region of gp41 or a functionally equivalent variant thereof. Preferably, the subject is a human, chimpanzee, macaque or baboon. More preferably, the subject is a human.
Other diseases that may be treated or prevented using the fusion proteins, polynucleotides, vectors, host cells, virus-like particles or pharmaceutical compositions according to the invention, or their combinations, include infectious diseases caused by viruses, bacteria, fungi, protozoa and parasites. Examples of virus or viral infections that may be treated or prevented by using the fusion proteins, polynucleotides, vectors, host cells, virus-like particles or pharmaceutical compositions according to the invention, or their combinations, include, but are not limited to, adenovirus, cytomegalovirus, Epstein-Barr virus, hanta virus, dengue, hepatitis A, hepatitis B, hepatitis C, herpes simplex type I, herpes simplex type II, human immunodeficiency virus (HIV), human papilloma virus (HPV), influenza, measles, mumps, papova virus, polio, respiratory syncytial virus, rinderpest, rhinovirus, rotavirus, rubella, SARS virus, smallpox and viral meningitis. Examples of suitable bacteria or bacterial infection include, but are not limited to, Bacillus antracis, Borrelia burgdorferi, Campylobacter jejuni, Chlamydia trachomatis, Clostridium botulinum, Clostridium tetani, Diptheria spp., E. coli, Legionella spp., Helicobacter pylori, Mycobacterium rickettsia, Mycoplasma nesisseria, Pertussis spp., Pseudomonas aeruginosa, S. pneumonia, Streptococcus spp., Staphylococcus spp., Vibria cholerae and Yersinia pestis. Examples of suitable fungi or fungal infections include, but are not limited to, Aspergillus fumigatus, Blastomyces dermatitides, Candida albicans, Coccidioides immitis, Cryptococcus neoformans, Histoplasma capsulatum and Penicillium marneffei. Examples of suitable protozoa and parasites or fungi or protozoal and parasitic infections include, but are not limited, Chlamydia spp., Leishmania spp., Plasmodium spp. (e.g. malaria), Rickettsia spp. and Trypanosoma spp.
The fusion proteins, polynucleotides, vectors, host cells, virus-like particles or pharmaceutical compositions according to the invention, or their combinations, could also be useful for counteracting pathogens involved in bioterrorism such as, for example, Botulinum toxin, Bacillus anthraces, Clostridium perfringens, Bacillus subtilus, Bacillus thuringiensis, hemorrhagic conjunctivitis virus (Enterovirus 70) and rotavirus.
The fusion proteins, polynucleotides, vectors, host cells, virus-like particles or pharmaceutical compositions according to the invention, or their combinations, could be additionally useful for the treatment or prevention of chronic conditions, such as Alzheimer's disease, Parkinson's disease and rheumatoid arthritis or other conditions, such as allergies and tumors. An example of the latter could consist of a chimeric protein simultaneously including antibodies or fragment of antibodies against tumoral markers or radioactive elements for radiotherapy. The fusion proteins according to the present invention, and the antibodies derived elicited by from them, could also be used for the diagnosis of fertility and pregnancy, or of diseases such as colon, lung, breast and prostate cancer. These fusion proteins could also be used to monitor and control drug abuse or the progress of therapeutic treatments.
The present invention further relates to preventing or reducing symptoms associated with HIV infection. These include symptoms associated with the minor symptomatic phase of HIV infection, including, for instance, shingles, skin rash and nail infection, mouth sores, recurrent nose and throat infection and weight loss. In addition, further symptoms associated with the major symptomatic phase of HIV infection, include, for example, oral and vaginal thrush (Candida), persistent diarrhea, weight loss, persistent cough, reactivated tuberculosis, and recurrent herpes infections, such as cold sores (herpes simplex). Symptoms of full-blown AIDS which can be treated in accordance with the present invention, include, for instance, diarrhea, nausea and vomiting, thrush and mouth sores, persistent, recurrent vaginal infections and cervical cancer, persistent generalized lymphadenopathy (PGL), severe skin infections, warts and ringworm, respiratory infections, pneumonia, especially Pneumocystis carinii pneumonia (PCP), herpes zoster (or shingles), nervous system problems, such as pains, numbness or “pins and needles” in the hands and feet, neurological abnormalities, Kaposi's sarcoma, lymphoma, tuberculosis, and other opportunistic infections.
The fusion proteins, polynucleotides, vectors, host cells, virus-like particles, and pharmaceutical compositions according to the invention, or their combination, can induce the expression of antibodies in a subject. In one embodiment, the antibodies are neutralizing antibodies (i.e. nAbs). In a preferred embodiment, the fusion proteins, polynucleotides, vectors, host cells, virus-like particles, and pharmaceutical compositions according to the invention, or their combination, are used for preventing HIV infection or AIDS in a non-infected subject under HIV exposure.
The fusion proteins according to the invention may also have multiple uses in the breeding of companion and farm animals and fish. These fusion proteins may be used to prepare immunogenic or vaccine compositions against animal retroviruses, such as Feline Leukemia Virus (FeLV), but also against non-viral pathogens such as Brucella spp., a bacterial agent that causes many problems in cattle, or against Piscirickettsia salmonis, which affects mainly the commercial breeding of salmon. Other possible uses of the fusion proteins according to the invention are the preparation of vaccines against Mycoplasma hyopneumoniae, a pneumonic agent that produces great losses in pigs. It would also be possible to use the fusion proteins according to the invention for modulating the expression of certain hormones to accelerate the growth rate and increase the production of milk, in farming animals, or make their meat leaner. An example of these non-conventional indications would be the use of these entities for the immunocastration of farm animals, such like pigs.
Typically, the neutralizing antibodies used in the method of the present invention bind to the surface of the pathogen and inhibit or reduce infection by the pathogen by at least 99%, 95%, 90%, 85%, 80%, 75%, 70%, 60%, 50%, 45%, 40%, 35%, 30%, 25%, 20% or 10% relative to the infection by the pathogen in the absence of said antibody(ies) or in the presence of a negative control. The nAbs can then be tested to determine if they are broadly neutralizing (i.e. bnAbs) using any of the methods provided herein. If the nAbs or bnAbs were raised in a non-human animal, the CDRs or the complete sequence can be transferred from the non-human framework to a human framework to generate an antibody suitable for administration to a human. Methods for determining whether an antibody is a nAb have been described in the art. See Li M, et al., J. Virol. 2005; 79:10108-10125, Wei X, et al., Nature 2003; 422:307-312, and Montefiori D, Curr. Protoc. Immunol. 2005; January, Chapter 12:Unit 12.11. These methods are based on the determination of the reduction in expression of a reporter gene after a single round of viral infection using a receptive cell line using a virus which encodes the reporter gene.
In another preferred embodiment the fusion protein, the polynucleotide, the vector, the host cell or the virus like-particle is for use according to the invention, wherein the subject is previously treated with a conjugate comprising the immunogenic polypeptide forming part of the fusion protein coupled to a carrier.
In a preferred embodiment, the immunogenic polypeptide corresponds to any region of HIV gp41 polypeptide or a functionally equivalent variant thereof. Preferably, the region is the MPER region. More preferably, the MPER region comprises SEQ ID NO: 22.
In another preferred embodiment, the carrier is KLH (Keyhole Limpet hemocyanin)
In a further aspect, the present invention refers to kits comprising at least one of the fusion proteins, polynucleotides, vectors, host cells, virus-like particles or pharmaceutical compositions according to the invention, or a combination thereof. The components of the kits according to the invention may be optionally packed in suitable containers and be labeled for their intended use, including the treatment or prevention of infectious diseases. In one embodiment, the infectious disease is HIV, AIDS or their related diseases or conditions. The components of the kits may be stored in unit or multi-dose containers as an aqueous, preferably sterile, solution or as a lyophilized, preferably sterile, formulation for reconstitution. The containers may be formed from a variety of materials such as glass or plastic and may have a sterile access port (e.g. the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The kits may further comprise more containers comprising a pharmaceutically acceptable carrier. They may further include other materials desirable from a commercial and user standpoint, including, but not limited to, buffers, diluents, filters, needles, syringes, culture medium for one or more of the suitable host cells or other active agents. The kits can contain instructions customarily included in commercial packages for therapeutic products that contain information, for example, about the indications, usage, dosage, manufacture, administration, contraindications or warnings concerning the use of such therapeutic products.
All publications mentioned herein are incorporated in their entirety by reference. Having now generally described the invention, the same will be more readily understood through reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the present invention, unless specified.
The pMIN mammalian expression plasmid coding for a small protein containing the C-terminal HR2 region, the MPER and TM domain has been described previously. See Molinos-Albert L, et al., Retrovirology 2014, 11:44. A similar plasmid pMINFULL coding for a gp41-derived protein that contains the MIN sequence and the cytoplasmic tail of gp41 was also generated. See
Gag sequences were amplified from the pGag-EGFP plasmid that codes for a Rev-independent HIV-1 Gag protein fused in frame to the enhanced GFP. The plasmid was obtained from the NIH AIDS Reagent Program (Cat No. 11468). See Dhillon A, et al., J. Virol. 2007; 81:6548-6562. The gag amplicon was subcloned in frame into a pcDNA3.1MIN plasmid to generate the pcDNA3.1/MINGAG-3.7 plasmid that codes for the fusion protein MINGAG. A pGAG plasmid coding for the full Gag sequence was generated by excision of the gag coding sequence from the pGag-EGFP plasmid and subcloning in a pcDNA3.1 plasmid (Invitrogen, Carlsbad, Calif., USA; Cat. Nos. K4900-01, K4900-40) See
The C34, T-20 and MPER peptides (HXB2 sequence) and Keyhole Limpet hemocyanin (KLH) and MPER coupled to KLH (KLH-MPER) were employed (Thermo Fisher Scientific, Waltham, Mass., USA). 15-mer overlapping peptides covering the gp41 HR2 and MPER regions were kindly provided by C. Brander (IrsiCaixa, Badalona, ES). The MIN protein containing a C-terminal 6×His tag was produced in E. coli BL21 DE3 strain (Invitrogen, Thermo Fisher Scientific, Waltham, Mass., USA). See
VLP were obtained by transfection of different derivatives of HEK-293T cells according to the production scale. For stable transfection, we transfected HEK-293T cells with pMINFULL and pGAG, pGAG or pcDNA3.1/MINGAG-3.7 using calphos mammalian transfection kit (Clontech®, Takara Bio Inc., Otsu, JP), and selected the transfected cells by using geneticine plus hygromicine, hygromicine or geneticine, respectively.
Given the poor yield of MINGAG VLPs, large scale production of GAG and MINGAG VLPs was carried out in a different setting. Large amounts of the pcDNA3.1/MINGAG-3.7 expression plasmid were produced using maxiprep kits (Qiagen NV, Venlo, NL) and were used for transient transfection of HEK-293T cells. Gag VLP were produced in 1 L culture flask, while MINGAG VLP were produced in a 5 L bag. 48 hours after transfection, supernatants were collected, ultrafiltered, concentrated (to 1 L in the case of MINGAG) and ultracentrifuged. Both preparations contained large amounts of gag protein as assessed by Nanoparticle Tracking Analysis (NTA, NanoSight Ltd., Malvern, GB) and electron microscopy.
To evaluate the antigen exposure on the surface of VLPs, HEK-293T cells were transfected with fusion proteins including the extracellular epitope of interest and the full HIV gag sequence. 24 hours after transfection cells were harvested and stained with specific antibodies against cell surface antigens (anti gp41 antibodies 2F5, 4E10 or 10E8 or specific antibodies against other proteins of interest), after washing the excess of antibody, cells were fixed and permeabilized using FIX and PERM® Cell Fixation and Cell Permeabilization Kit (Thermo Fisher Scientific, Waltham, Mass., USA) and intracellularly stained with the anti-gag antibody Kc57 (Beckman Coulter, Brea Calif., USA). After a final wash, cells were analyzed in a LSRII flow cytometer (BD Biosciences, San Jose, Calif., USA). The frequency of cell-surface antigen positive cells among GAG positive cells was used as a measure of antigen exposure.
Female C57BL/6 mice (Harlan Sprague Dawley Inc., Indianapolis, Ind., USA) aged 6-8 weeks were used for murine immunization protocols. Groups of mice (5 mice per group) were immunized subdermally with 50 μg of recombinant proteins or VLPs at 3-week intervals. Blood samples were collected by maxillary artery puncture before immunization and at 3 weeks. Mice were observed daily to record body weight changes.
For rabbit immunization, 3- to 4-month-old New Zealand White rabbits were inoculated subcutaneously with different immunogens. A control rabbit group (n=4) was primed with full gag DNA and nude VLP, and the immunized twice at weeks 0 and 3 with nude VLP. A second group of rabbits (n=4) was primed with DNA coding for the HTI immunogen, full HIV gag and full HIV env genes and then immunized at week 0 and 3 with MINGAG VLP. See Mothe B, et al., J. Transl. Med. 2011; 9:208. Sera were collected at weeks 1, 3, 4 and at sacrifice.
All animal and husbandry experiments were conducted according to the applicable rules and regulations.
Peptides C34, T20, MPER, overlapping 15mer gp41 peptides or recombinant GAG or MIN protein were used to coat 96-well Maxisorp Nunc-immuno plates (Thermo Fisher Scientific, Waltham, Mass., USA). After blocking, plates were incubated with 100 μL of previously diluted plasma samples overnight at 4° C. Plates were then washed and 100 μL of a horseradish peroxidase (HRP)-conjugated F(ab)2 goat anti-mouse IgG (Fc specific, Jackson ImmunoResearch Labs, Inc., West Grove, Pa., USA) were dispensed for one hour at room temperature. Plates were developed with 100 μl of 0-phenylenediamine dihydrochloride (OPD) substrate (Sigma-Aldrich, Saint Louis, Mo., USA) and stopped with 100 μL of 4N H2504. Optical density was measured at 492 nm for specific signal and at 620 nm for background.
HIV-2 chimeras were made in the context of the full-length p7312A HIV-2 molecular clone (GenBank accession number L36874). Expression vectors for the wild type HIV-2 (p7312A) and HIV-2 chimeras containing the HIV-1 gp41 MPER (p7312A-C1), the 2F5 (p7312A-C3) or 4E10 epitopes (p7312A-C4), were kindly provided by G. M. Shawn (University of Pennsylvania). See Dhillon, 2007, supra. Pseudoviruses were generated by transfection of plasmids in HEK-293T cells. After 24 hours post-transfection, supernatants were harvested, filtered at 0.45 micron and viral stocks frozen at −80° C.
HIV-1 isolates NL4.3, BaL, AC10 and SVPB16 were generated as pseudoviruses using Env expression plasmids and the pSG3 vector as previously described. See Sanchez-Palomino S, et al., Vaccine 2011, 29:5250-5259. Cell-free virus neutralization by plasma samples was tested by a standard TZM-b1 based assay. See Sanchez-Palomino S, 2011, supra. Vaccine 2011, 29:5250-5259. Briefly, in a 96-well culture plate, 100 μL of previously diluted plasma samples were preincubated with 50 μL of pseudovirus stock, using 200 TCID50, at 37° C., one hour. Then, 100 μL containing 10,000 TZM-b1 luciferase-reporter target cells per well were added. Plates were cultured at 37° C. and 5% CO2 for 48 hours. 2F5, 4E10 and IgGb12 (Polymun Scientific GmbH, Klosterneuburg, AT), and anti-CD4 clone SK3 (BD Biosciences Corp., Franklin Lakes, N.J., USA) were used as controls. Plasma samples were inactivated (56° C., 30 minutes) prior to the assay. TZM-b1 reporter cells were treated with dextran (Sigma-Aldrich, Saint Louis, Mo., USA) to enhance infectivity. Luciferase substrate, Britelite Plus (PerkinElmer, Inc., Waltham, Mass., USA) was used for the read out of HIV infectivity.
The MIN protein was selected due to its high expression and proper MPER exposure on the cell-surface of transfected cells. See Molinos-Albert, 2014, supra. As a first approach to test its potential immunogenicity, both the recombinant MIN protein and the VLP displaying the selected MIN protein were produced. See
For VLPs, the cytoplasmic domain of gp41 was added to the MIN sequence (MINFULL construction) in order to increase the interaction with Gag during VLP budding. A HEK-293T cell clone stably expressing both MINFULL and GAG HIV-1 proteins was selected according to protein expression and VLP production. The selected clone (4G2A) showed cell surface MPER exposure and an intracellular GAG content comparable to a GAG expressing HEK-293T cell clone selected in parallel and lacking gp41 expression. See
Considering the low amounts of MIN protein incorporated in VLP and the low number of Env spikes reported in HIV virions, it was hypothesized that the poor incorporation of MIN protein into the VLPs could be a limiting factor in the above described approach. Then, it was considered whether increasing the density of MPER epitopes on the surface of VLP could enhance its immunogenic potential. See
The release of VLP by transfected cells was assessed by ELISA in cleared supernatant collected 48 hours after transfection. Although VLP production was 8-fold lower for the MINGAG construct than for nude VLPs, both proteins showed similar levels of particulate material, suggesting proper particle generation. See
The immunogenicity of the MINGAG VLPs in C57BL/6 mice was tested. Five animals per group were injected with a 200 μL preparation of nude or MINGAG VLPs with or without aluminum phosphate as adjuvant. Immunogens were prepared maintaining the same Gag antigen concentration between groups, wherein the only difference was the presence or not of the MIN fragment in MINGAG VLPs. Accordingly, all animals were injected with four doses (at 3 weeks interval) of 32 ng of gag antigen.
The time course of anti-gp41 IgG responses (measured using the MIN antigen) showed high and specific immunogenicity in both MINGAG immunized groups, with a faster elicitation in adjuvanted animals but higher final levels in animals immunized only with VLP. See
These results indicate that the preparation of MINGAG VLPs is immunogenic, as some of the animals immunized with this preparation could mount a strong IgG response. However, no significant neutralizing activity was observed in both sera and purified IgG obtained from control or immunized animals against two laboratory adapted viruses (i.e. NL43 and BaL). See
To increase the response to the MPER of our MINGAG protein, a second immunization procedure was designed in which animals were immunized with MPER peptides coupled to KLH (four doses every three weeks) before VLP immunization (as the immunization protocol described above) in the absence of adjuvant. Again, all animals showed similar anti-Gag responses, while only MINGAG immunized animals showed strong anti-gp41 IgG responses. See
A similar approach to Example 3 was used to test the immunogenicity of the MINGAG VLP in rabbits. VLP were injected (two doses of MINGAG VLPs) in the absence of adjuvants and animals were primed with gag and env DNA. All animals generated significant anti-gp41 IgG responses (as measured by the T-20 peptide and the C34 peptides) that mapped to the HR2 and the MPER sequences of the immunogen protein, with some overlap with the 2F5 neutralizing epitope See
A codon optimized (cop) version of the nucleotide sequences coding for the GAG protein and MINGAG fusion protein were cloned in pcDNA3.1 vectors to yield the plasmids pcDNA3.1/GAGcop and pcDNA3.1/MINGAGcop. Transient transfection of HEK-293T cells with original pcDNA3.1/MINGAG-3.7 and the new codon optimized pcDNA3.1/MINGAGcop resulted in a significant higher expression of the codon optimized version. See
The higher expression of codon optimized MINGAGcop fusion proteins led to exploring the immunogenicity of naked DNA in murine models. pVax′ expression vectors (Thermo Fisher Scientific, Waltham, Mass., USA) coding for MINGAG fusion protein, GAG protein and an empty pVax vector were constructed, purified using EndoFree Plasmid Giga Kit (Qiagen NV, Venlo, NL) and injected to C57BL/6 mice. Five animals per group were injected with a 100 μg of each plasmid, four times at 3-weeks intervals.
The time course of anti-gp41 IgG responses (measured using the MIN antigen) showed specific immunogenicity exclusively in the pVaxMINGAGcop immunized group. See
The general structure of fusion proteins according to the invention includes a small linker region between the TM and the GAG sequences. Whether changes in this small linker sequence affect the expression and production of VLPs was evaluated. For addressing this issue, different versions of the codon optimized pcDNA3.1/MINGAGcop were generated containing a GGGS (SEQ ID: 23), no linker, an alanine/glutamic acid and lysine linker (SEQ ID NO: 26) or an alanine/proline linker (SEQ ID NO: 27) sequences. See
Similarly, the effect of the TM sequence was analyzed, by replacing the TM region of HIV by:
(a) the TM region of the human CD22 molecule (SEQ ID NO: 17),
(b) the TM region of the human CD36 molecule (SEQ ID NO: 18),
(c) the TM region of the human CD44 molecule (SEQ ID NO: 19) or
(d) the R696A mutant of HIV-1 Env (SEQ ID NO: 30).
The expression of epitopes 2F5, 4E10 and 10E8 as well as the production of VLP was assessed.
The different TM sequences strongly affected the exposure of relevant MPER epitopes. Compared to the wild type gp41 TM, the CD22 and CD36 sequences notably decreased the expression of antigenic sites (mainly the 4E10 and 10E8 epitopes), while both the CD44 TM and the mutated gp41 TM sequences greatly enhanced epitope exposure. This effect was specially observed for the 4E10 antigenic site, indicating that the latter constructs offer a better and more balanced exposure of the MPER than the wild type MINGAGcop construct. See
Furthermore, when the production of VLP was assessed and compared to the wild type MINGAGcop construct, the CD22, CD36 and CD44 constructs had no effect on VLP recovery, while a significant higher yield was observed for the mutated gp41 TM sequence, whose production was 1.5-fold higher than the production observed for the MINGAGcop reference. See
To demonstrate the ability of MIN/GAG fusion proteins to expose different epitopes, a neutralizing epitope of the HCV E2 protein was engrafted into the extracellular moiety of MIN. The 2F5 epitope of MIN/GAGcop (aa 655-667 of gp41) was replaced by the peptide KQNIQLINTNGSWH corresponding to aa 408-421 of HCV E2 sequence to generate a MINHCVGAG chimeric protein (SEQ ID NO: 32). The nucleotide sequence (SEQ ID NO: 31) was cloned into pcDNA3.1 vector and expressed in HEK-293T cells.
The capacity of the external immunogenic moiety of the GAG based fusion protein for accommodating antigens different from HIV was demonstrated. Several antigens relating to widely different pathologies were selected:
A diagram of these constructs is shown in
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WO2018/020324 | 2/1/2018 | WO | A |
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4554101 | Hopp | Nov 1985 | A |
5554372 | Hunter | Sep 1996 | A |
5643756 | Kayman et al. | Jul 1997 | A |
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20190315807 A1 | Oct 2019 | US |