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WO2011028963A2 - Compositions immunogènes du vih - Google Patents

Compositions immunogènes du vih Download PDF

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Publication number
WO2011028963A2
WO2011028963A2 PCT/US2010/047754 US2010047754W WO2011028963A2 WO 2011028963 A2 WO2011028963 A2 WO 2011028963A2 US 2010047754 W US2010047754 W US 2010047754W WO 2011028963 A2 WO2011028963 A2 WO 2011028963A2
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Prior art keywords
epitope
hiv
composition
immunodominant
immune
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WO2011028963A3 (fr
Inventor
Peter L. Nara
Gregory J. Tobin
George Lin
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Biological Mimetics Inc
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Biological Mimetics Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/21—Retroviridae, e.g. equine infectious anemia virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/24011—Poxviridae
    • C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
    • C12N2710/24141—Use of virus, viral particle or viral elements as a vector
    • C12N2710/24143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • Class One pathogens such as measles, mumps and rubella viruses
  • Class One pathogens are those pathogens, which, in general: (1) infect or cause the most serious disease in children/young adults; (2) carry a relatively stable microbial genome; (3) have a natural history of disease which results in spontaneous recovery; and (4) induce durable memory, associated with polyclonal and
  • Class Two pathogens such as, viral influenza, HIV-1, malaria, tuberculosis, trypanosomes, schistosomes, leishmania, anaplasma, enterovirus, astrovirus, rhinovirus, Norwalk viruses, toxigenic/pathogenic E. coli, Neisseria, Streptomyces, nontypeable Haemophilus influenza, hepatitis C, streptococcus, staphylococcus, cancer cells etc. are characterized by quite opposite features.
  • Class Two pathogens (1) tend to infect and are transmitted in a significantly extended host age range, with infections occurring and reoccurring from childhood through the geriatric period; (2) exhibit genetic instability in defined
  • 02a31b28 1 regions of their genome (a hallmark of the successful evolution of such pathogens); (3) in some cases, spontaneous recovery of disease frequently leaves the host vulnerable to multiple repeated annual infections and/or the establishment of either a chronic/active or chronic/latent infectious state; (4) induce oligoclonal, early immune responses that are directed to a very limited set of immunodominant epitopes which provide either narrow strain-specific protection, no protection and/or enhanced infection, in many cases, induced immune responses that only provide strain-specific immunity and thus do not provide broader protective immunity that would be desirable for many pathogens that undergo mutation; and (5) cause immune dysregulation following infection or vaccination, e.g.
  • an immunodominant decoy, dysregulating or immune steering/biasing epitope or its equivalent ligand-receptor interaction of any of the innate host defense system molecules that someway alters the hosts acquired/adaptive immune response to the pathogen is considered to be the main focus and target of technologies designed to modify, alter or eliminate them from the molecule so as to result in the host immune system responding in an alternate manner resulting in an either/and or earlier and more broadly protective immune response following active immunization of the host.
  • HIV-1 immunodeficiency virus type 1
  • 02a31b28 2 are selected and maintained across host and microbial taxa.
  • the operational failures of the vertebrate immune system for example, by Deceptive Imprinting, are fundamentally the same whether infected with HIV-1 or with a common cold virus for an average of 2-6 times a year for 60 years.
  • Antigenic variation is an evolved mechanism to ensure rapid sequence variation of specific pathogen gene(s) encoding homologues of an individual protein antigen, usually involving multiple, related gene copies, resulting in a change in the structure of an antigen on the surface of the pathogen.
  • the host immune system during infection or re-infection is less capable of recognizing the pathogen and must make new antibodies to recognize the changed antigens before the host can continue to combat the disease.
  • the host cannot stay completely immune to the viral disease. That phenomenon stands as one of the more, if not, most daunting problem challenging modern vaccine development today.
  • Original antigenic sin was not considered pathogenic and a means to avoid the immune system until 1990 (Nara et al., J. Virol. 62:2622-2628, 1988) when it was uncovered that original antigenic sin was a means, for example, for NA viruses such as HIV to avoid the host immune system.
  • the immune avoidance strategy was observed to be implemented by a number of microbiological and possibly eukaryotic organisms.
  • pathogens often express epitopes which are preferentially recognized by a host immune system, so called immunodominant epitopes or determinants.
  • those epitopes may not serve an important function for pathogen viability or function, for example.
  • immunodominant epitopes are essentially decoys. That observation indicates that immunodominant epitopes can be modified practicing methods known in the art which then will enable a host to raise an immune response to other epitopes displayed by the pathogen.
  • the immunologic mechanisms for immunodominance are not fully understood, and no one mechanism yet fully explains how or why certain epitopes have evolved to be immunoregulatory and immunodominant.
  • the range of immune responses observed in the phenomena include the induction of highly strain-specific or isolate-specific neutralizing antibody capable of inducing passive protection in experimental animal model-viral challenge systems all the way to the induction of a binding non-protective/non-neutralizing, blocking and even pathogen-enhancing antibody that in some cases prevents the host immune system from recognizing nearby adjacent epitopes, to interfering with CD 4 T-cell help.
  • the immune response to HIV is composed of an initial cell-mediated immune response followed by the subsequent development of neutralizing antibodies.
  • virus titers in the blood fall coincident with the induction of anti-HIV cellular and humoral immune responses.
  • the fall in viremia correlates with the appearance of anti-HIV major histocompatibility complex (MHC) class I-restricted CD 8 + cytotoxic T cells.
  • MHC major histocompatibility complex
  • Recent evidence has shown a strong correlation of anti-HIV CD 4 + T cell responses and reduced viral loads. Therefore, the presentation of HIV antigens in the context of MHC class II molecules to CD 4 + T cells may be a key aspect of the control of HIV infection.
  • Vaccines based on subunit immunogens although extremely safe, are limited in the breadth of antigens that are presented to the immune system because only one or a few of the viral proteins are utilized as immunogens and choice of subunit antigens may limit the likelihood of cross protection, for example, among clades of HIV. Also, the production of vaccines based on subunit immunogens requires the molecular manipulation of the viral proteins into cloning or expression vectors, perhaps leading to increased production time and cost.
  • the strains of HIV-1 can be classified into four groups: the "major” group M, the “outlier” group O, and two new groups, N and P (Hemelaar et al., AIDS 20(16)W 13-23, 2006). Those four groups may represent four separate introductions of simian immunodeficiency virus into humans. Group O appears to be restricted to west-central Africa and group N, a strain discovered in 1998 in Cameroon, is extremely rare. In 2009, a new strain closely relating to gorilla simian
  • HIV-1 group P More than 90% of HIV-1 infections belong to HIV-1 group M.
  • group M there are known to be at least nine genetically distinct subtypes (or clades), A, B, C, D, F, G, H, J and K.
  • Subtype B is prevalent in the Western Hemisphere, while subtypes A, C and D commonly occur in Africa. In Asia, the most frequently found subtypes are
  • the most effective vaccines for invoking a strong and complete immune response may also carry the most risk of harming the individual while the safer alternatives (subunit) may induce as complete a set of immunity as a whole virus vaccine mentioned above but require a properly chosen antigen from the virus that is immune refocused and delivered in a licensed and safe adjuvant.
  • the invention relates, in part, to novel HIV antigens with enhanced or novel immunogenicity.
  • An HIV composition of interest can serve as an improved vaccine, resulting from modifications providing the virus or viral antigen with a different array of and/or newly recognizable epitopes.
  • Figure 1 provides a portion of Table 1 listing certain mutations.
  • Figure 2 is a continuation of Table 1.
  • Figure 3 provides a portion of Table 2 providing primers.
  • Figure 4 is a continuation of Table 2.
  • Figure 5 is a continuation of Table 2.
  • Wild type refers to a naturally occurring organism.
  • the term also relates to forms of nucleic acids and of proteins found in a natural occurring organism of a naturally occurring population arising from natural processes, such as seen in polymorphisms arising from natural mutation and maintained by genetic drift, natural selection and so on, and does not include a nucleic acid or protein with a sequence obtained by, for example, purposeful modifications of the sequences through either a biologic or chemical selective process, or through molecular manipulations, such as a mutagenesis method.
  • a wild type allele or antigen usually is but need not be the most prevalent form in a population. Also, herein, wild type is used as an equivalent of the parental form. Thus, if allele A is mutated to form allele A*, then A is the parental or wildtype form in that relations. Allele A itself can be a rare mutation in a population.
  • Immunogen and "antigen” are used interchangeably herein as a molecule that elicits a specific immune response, for example, containing an antibody that binds to that molecule or eliciting T cells, such as CD 4 + or CD 8 + cells, capable of
  • That molecule can contain one or more sites to which a specific antibody binds. As known in the art, such sites are known as epitopes or determinants.
  • An antigen can be polypeptide, polynucleotide, polysaccharide, a lipid and so on, as well as a combination thereof, such as a glycoprotein or a lipoprotein.
  • An immunogenic compound or product, or an antigenic compound or product is one which elicits a specific immune response, which can be a humoral, cellular or both.
  • a vaccine is an immunogen or antigen used to generate an immunogen or antigen used to generate an immunogen or antigen used to generate an immunogen or antigen used to generate an immunogen or antigen used to generate an immunogen or antigen used to generate an immunogen or antigen used to generate an immunogen or antigen used to generate an immunogen or antigen used to generate an immunogen or antigen used to generate an immunogen or antigen used to generate an immunogen or antigen used to generate an
  • the dosage is derived, extrapolated and/or determined from preclinical and clinical studies, as known in the art. Multiple doses can be administered as known in the art, and as needed to ensure a prolonged prophylactic or anamnestic (memory) state.
  • the successful endpoint of the utility of a vaccine for the purpose of this invention is the resulting presence of an induced immune response (e.g.
  • the induced antibody in some way combines with a compound, molecule and the like carrying the cognate antigen or immunogen or directs the host to neutralize, reduce or prevent and/or eliminate a viral pathogen from infecting and causing serious clinical disease.
  • Successful measurement of vaccine outcome may include, but is not exclusively confined to, immunity that either protects against infection or which reduces disease or inactivity
  • Immunoprotection for the purposes of the instant invention is another endpoint and is a marker of inducing presence of such circulating anti-pathogen antibody that binds the immunogen or the pathogen, or cells that express an antigen of the pathogen. That can be determined using, for example, any known immunoassay, such as an ELISA. Alternatively, one can use a viral neutralization assay to ascertain presence of circulating anti-viral antibody. For the purposes of the instant invention, observing immunoprotection, that is, presence of circulating antibody or presence of immune cells reactive with virus or virus-infected cells, is evidence of efficacy of a vaccine of interest.
  • the period of immunoprotection in a host can be at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 8 days, at least 10 days, at least 14 days, at least 15 days, at least 20 days, at least 21 days, at least 25 days, at least 28 days, at least 30 days, at least 35 days, at least 40 days, at least 42 days, at least 45 days, at least 49 days, at least 56 days, at least 60 days, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 1 year, at least 2 years or longer.
  • the immunoprotection is observed in outbred populations, different geographic populations, clades and so on.
  • an antigen or epitope can be a toleragen. It is known that tolerance can be obtained, for example, at low dose or at high does. In other circumstances, situations known as passive immunity or passive immunization when antibody is administered to a host not to act as an antigen but as an antibody, sometimes is called a passive vaccine or passive vaccination.
  • Immunodominant epitope is an epitope that selectively provokes an immune response in a host organism to the effective or functional exclusion, which may be partial or complete, of other epitopes on the same or another antigen of the pathogen.
  • An immunodominant epitope may be specific for a strain, a clade, a group, a family, a species, a genus and so on.
  • an immunodominant epitope that is modified in one strain, clade and so on may reveal an epitope that is generic to a much wider range of pathogen.
  • an immunodominant epitope may reside in a particular site of a structural protein, for example, the envelope protein, such as the V3 loop, and modifying a V3 loop immunodominant epitope may reveal epitopes in regions aside from V3, such as those associated with the VI loop, V2 loop, V4 loop, V5 loop, A loop, B loop, E loop, an a helix region and so on (Kwong et al., Nature 393:648-659, 1998).
  • immunodampen and equivalent terms, do not necessarily have to result in the complete removal of said epitope. Also, immunodampening can exert an influence on epitopes distal from the modified site. It may be necessary or beneficial to modify plural sites of an epitope or plural epitopes of a pathogen. Hence, an
  • 02a31b28 12 epitope that is immunomodified is one which is not as or is no longer
  • Immunodampening of an immunodominant epitope of an antigen can result in the production in a host organism of high titer antibodies or T cell responses against non-dominant epitopes on that antigen and/or new titers of antibodies or T cell responses to otherwise relatively immune silent epitopes or those which previously were poorly immunogenic.
  • Such immunodampened antigens can serve as effective vaccines against organisms that have an antigen with a moderately or highly variable and/or conserved immunodominant epitope.
  • the instant invention also relates to an antibody or a cell that is reactive to an epitope previously not recognized or poorly recognized by a host because of one or more immunodominant epitopes.
  • the antibody or cell is one which binds a non-immunodominant epitope or binds to an antigen which does not comprises or is devoid of an immunodominant epitope.
  • Said antibody, or binding portion thereof, or cell can be derived, for example, as a monoclonal antibody, and used for diagnostic or therapeutic purposes, such as passive immunization using a human or humanized antibody as known in the art, see for example, W02009032661.
  • the invention also relates to complexes of an antibody and an HIV antigen.
  • the HIV antigen may comprise an immunodominant epitope, an immunodampened
  • immunodominant epitope or does not comprise an immunodominant epitope.
  • An immunodominant epitope can be identified by any of a variety of means and methods, for example, computationally, serologically, cellularly, using a
  • T cell-based peptide assay structural analyses and so on. Multiple approaches can be used. Often, patient samples prior to infection are used as a control. Thus, serum antibodies or T cell reactivity of a host organism before and once infected with the pathogenic organism can be tested. The serum is evaluated for content of antibodies that bind to the identified antigens, usually either as pre-existing antibodies (naive human or animal) or occurring within a short amount of time after exposure or immunization that are likely to cause an immune response in a host organism.
  • an immunodominant epitope substantially many antibodies in the serum will bind to and/or T cells will recognize the immunodominant epitope(s), with reduced to no binding/recognition to other epitopes present in the antigen. Regions other than immunodominant B and T cell epitopes may be targets for immune refocusing. Other domains that engage various host cell receptors (receptotopes) involved in host defense of the pathogens protein(s) may have evolved to stimulate a dysregulatory response (engaging host defense pathways that are inappropriate, e.g. those early signals and pathways that determine which antibody or cell-mediated response should be activated, thus sending the early immune response in the wrong direction).
  • Dysregulating epitopes may cause, for example, stimulation of deleterious or suboptimal expression and release of various cytokines and chemokines which turn on and off various immune cells needed for activation of a pathway.
  • immunodominant epitope is modified as taught herein using the materials and methods taught herein and as known in the art as a design choice.
  • 02a31b28 14 immunodampening can be performed through any of a variety of methods, including, but not limited to, site-specific mutagenesis, antibody-induced evolution,
  • sequences that leads to post-translational glycosylation can be introduced or eliminated to dampen an immunodominant epitope.
  • sequences that encode, for example, N-linked glycosylation sequons (NXT/S) can be altered or added in or near immunodominant epitopes. Methods for altering nucleic acids of sequons are taught herein.
  • N-linked carbohydrate can be determined by the primary amino acid sequence of the polypeptide.
  • a triplet amino acid sequence consisting of asparagine, followed by any amino acid, and ending with a serine or threonine (N-X-S/T), where X is any amino acid other than proline or aspartic acid is a target for N-linked CHO addition for dampening an epitope or antigen from immune system recognition.
  • N-X-S/T serine or threonine
  • X is any amino acid other than proline or aspartic acid
  • a particular amino acid of the immunodominant epitope can be replaced, substituted or deleted to dampen immunogenicity, for example, by substituting, removing or adding amino acids, modifying amino acids, by adding or removing a lipidation or glycosylation site, such as an O-glycosylation site or an N-glycosylation site, and so on.
  • lipid groups can be added to polypeptides, sometimes referred to as prenylation (Young, J Lipid Res 46:2529-30, 2005) although other hydrophobic groups can be added to a polypeptide, such as, myristoyl and palmitoyl groups (Magee, J Cell Sci 97:581-584, 1990).
  • 02a31b28 15 polypeptide can be altered by adding, deleting or substituting one or more molecules, groups, compounds and the like at a target site or at an epitope, or in or at an adjacent or distal site which alters the target site or epitope.
  • a particular amino acid can be derivatized or functional groups thereon can be removed, modified or added, for example, chemically, to effect such a change, such as, adding a saccharide group thereto, such as a glycol, such as a polyglycol, such as polyethylene glycol.
  • Immunodampening can occur by replacing, substituting or eliminating one amino acid, two amino acids, three amino acids or more of the immunodominant epitope, for example, by site-directed mutagenesis of the nucleic acid encoding the antigen to express another amino acid which is less immunogenic or which changes the pattern or hierarchy of immunogenicity.
  • Methods for altering nucleic acids and/or polypeptides are provided herein, and are known in the art.
  • a recombinant gpl20 of HIV B clade that displays a molecularly introduced N-linked sequon (NXT/S), which resulted in the addition of a supernumerary N-linked glycan in the immunodominant V3 domain, exhibited novel antigenic properties, such as the inability to bind antibodies that recognize wildtype V3 epitopes while inducing antibody responses to other previously silent or less immunogenic epitopes. Presence of the supernumerary carbohydrate moiety did not compromise the infectious viability of the HIV-1 recombinant virus.
  • Test animals immunized with the recombinant glycoprotein showed moderate to high titers of antibodies that neutralize infection to both homologous and heterologous wildtype HIV-1 in vitro.
  • immunodampening of the immunodominant epitope within the V3 domain of gp 120/ 160 caused the immune response to refocus on other neutralizing
  • Suitable sites of HIV for immune modification include, but are not limited to the VI loop, V2 loop, V3 loop, V4 loop, V5 loop, CI region, C2 region, C3 region, superantigen regions and so on of gpl20; and the a helix, N terminus, C terminus and so on of gp 41.
  • Suitable other sites of HIV for epitope modification include non-env proteins, gag, protease, pol, vif, tat, T, nef, rev, and other viral proteins.
  • Suitable sites of immune modification also include RNA domains such as TAR and RRE.
  • a plurality of modifications can occur in more than one of the above regions.
  • the one or more modifications to the above-noted regions are coupled to a modification in the V3 loop.
  • phrases and terms, as well as combinations thereof, "functional fragment, portion, variant, derivative or analog” and the like, as well as forms thereof, of a pathogen, antigen, component, subunit and the like thereof, such as, gpl20, gpl60, gag, pol and the like relate to an element having qualitative biological activity in common with the wild-type or parental element from which the variant, derivative, analog and the like was derived.
  • a functional portion, fragment or analog of HIV is one which stimulates an immune response as does native HIV, although the response may be to different epitopes on the HIV or a derived molecule.
  • “functional equivalents” includes the virus and portions thereof with the ability to stimulate an immune response to HIV, although modified.
  • a pathogen such as, HIV of interest, such as a membrane preparation carrying, for example, gpl20, gpl40 or gpl60, as well as preparations of any other HIV antigens, such as gag or pol, can be obtained practicing methods known in the art.
  • a pathogen such as, HIV of interest
  • intramolecular modifications e.g. deletions, charge changes, adding one or more N-linked sequons and so on
  • the altered epitope can induce a new hierarchy of immune responses at either or both the B and T cell levels (Garrity et al, J Immunol. (1997) 159( 0:279-89) against subdominant or previously silent epitopes. That technology as described herein is known as "Immune efocusing," which is synonymous with other equivalent terms used herein
  • gpl20 gp41 and or gpl60 subunit protein vaccine that is immunomodified as taught herein can be sufficient to protect against challenge from plural strains of HIV.
  • immunodampening can be implemented by any of a variety of techniques such as, altering or deleting specific amino acids of the epitope, or adding, for example, a glycosylation site at or near the epitope.
  • the changes can occur at the level of the polypeptide or at the level of the polynucleotide, practicing methods known in the art.
  • 02a31b28 18 antigen with defined antisera known to react with the immunodominant epitope such as, by an ELISA or Western blot, for example.
  • Candidates demonstrating reduced reactivity with those defined antisera are chosen for testing in vivo to determine whether those dampened antigens are immunogenic and the host generates an immune response thereto.
  • a mouse or other animal is immunized with the dampened or disabled antigen as known in the art, serum is obtained and is tested in an in vitro assay for reactivity therewith. That antiserum then can be tested on wildtype virus to determine if the antibody still recognizes the wild type epitope or the wild type antigen.
  • Those candidate immunodampened or immunodisabled antigens less or no longer reactive with known antisera that bind to or exhibit cell-mediated immunoreactivity to the parent immunodominant antigen, yet remain immunogenic in hosts, are selected as candidate vaccines for further testing.
  • either the immune antiserum or immune cells can be tested for reactivity with a number of pathogen strains or clades in standardized assays to determine how generic that antibody or reactive cell is, that is, whether the newly recognized epitopes on the dampened antigen are generic to a wide range of pathogen strains, clades, variants, varieties and so on, and if the antibody or cell has antipathogen activity.
  • immunodominant epitope can be modified to express an immunodampened epitope.
  • the nucleic acid sequence can be modified by, for example, site-directed mutagenesis to express amino acid substitutions, insertions, deletions and the like, some of which may introduce further modification at or near the immunodominant epitope, such as, introducing a glycosylation site, such as, mutations which cause N-glycosylation or O-glycosylation at or near the immunodominant epitope and so on.
  • One procedure for obtaining epitope mutants or muteins is "alanine scanning mutagenesis" (Cunningham & Wells, Science 244: 1081-1085 (1989); and Cunningham & Wells, Proc Nat. Acad Sci USA 84:6434-6437 (1991)).
  • One or more residues are replaced by alanine or polyalanine residue(s).
  • Those residues demonstrating functional sensitivity to the substitutions then can be refined by introducing further or other mutations at or for the sites of substitution.
  • the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined. Similar substitutions can be attempted with other amino acids, depending on the desired property of the scanned residues.
  • a more systematic method for identifying amino acid residues to modify comprises identifying residues involved in immune system stimulation or immunodominant antibody recognition and those residues with little or no
  • Modification can involve deletion (removal) or substitution of a residue or an amino acid, or insertion (addition) of one or more residues or amino acids adjacent to a residue or amino acid of interest.
  • a conservative substitution can be a first substitution. If such a substitution results in reduction of immune system stimulation or reduced reactivity with known immunodominant antibody, then another conservative substitution can be made to determine if more substantial changes are obtained.
  • the naturally occurring amino acids can be divided into groups based on common side chain properties:
  • hydrophobic methionine (M or met), alanine (A or ala), valine (V or val), leucine (L or leu) and isoleucine (I or ile);
  • cysteine C or cys
  • serine S or ser
  • threonine T or thr
  • asparagine N or asn
  • glutamine Q or gin
  • amino acids can fit more than one of the above categories, which is not intended to be the only means to classify amino acids.
  • Non-conservative substitutions can entail exchanging an amino acid with an amino acid from another group.
  • Conservative substitutions can entail exchange of one amino acid for another from within a group.
  • Preferred amino acid substitutions are those which dampen or disable an immunodominant epitope, but can also include those which, for example:
  • Analogs can include various muteins of a sequence other than the naturally occurring peptide sequence.
  • a conservative amino acid substitution generally should not substantially change the structural characteristics of the parent sequence (e.g., a replacement amino acid should not tend to break a helix that occurs in the parent sequence, or to disrupt other types of secondary structure that characterizes the parent sequence) unless of a change in the bulk or conformation of the R group or side chain (Proteins, Structures and Molecular Principles (Creighton, ed., W. H. Freeman and Company, New York (1984);
  • the epitope mutant with altered biological and/or structural properties will have an amino acid sequence having at least 75% amino acid sequence identity or similarity with the amino acid sequence of the parent molecule, at least 80%, at least 85%, at least 90% and often at least 95% identity.
  • Identity or similarity with respect to parent amino acid sequence is defined herein as the percentage of amino acid residues in the candidate sequence that are identical (i.e., same residue) or similar (i.e., amino acid residue from the same group based on common side chain properties, supra) with the parent molecule residues, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity.
  • sequence identity can be less than 75%o, less than 70%, less than 65%, less than 60%>, less than 55%, less than 50%, less
  • Covalent modifications of the molecules of interest are included within the scope of the invention. Such may be made by chemical synthesis or by enzymatic or chemical cleavage of the molecule, if applicable. Other types of covalent modifications of the molecule can be introduced into the molecule by reacting targeted amino acid residues of the molecule with an organic derivatizing agent that is capable of reacting with selected side chains or with the N-terminal or C-terminal residue.
  • the use of an antibody that is naturally derived from infection or by immunization to the immunodominant epitopes as either a polyclonal or monoclonal antibody can be used.
  • an Ab or an Ig molecule can be one from any of the Ig family of molecules, IgG, IgA, IgM, IgD or IgE, or equivalents from other animals including birds, camels or invertebrates (any living organism capable of making an antibody, an antibody-like molecule or an antigen-binding molecule), or minimal fragments (F a b 2 , F a b, or mini body light or heavy chain pieces etc.) of the epitope-paratope antibody binding site can be used.
  • the antibody or epitope-binding fragments thereof can be derived naturally or experimentally from the whole Ig, enzymatic cleavage or synthetically using either peptide chemistry or recombinant protein expression technology.
  • the antibody is made as known in the art, such as by immunizing
  • 02a31b28 24 an animal, recombinant expression, phage display and so on, and modified, such as, truncated, as known in the art.
  • WO05/35726 teaches various methods for introducing, modifying, changing, replacing and so on substituents found on biomolecules.
  • Cysteinyl residues can be reacted with a-haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to yield carboxylmethyl or carboxyamidomethyl derivatives. Cysteinyl residues also can be derivatized by reaction with bromotrifiuoroacetone, a-bromo-P-(5- imidozoyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2- pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate,
  • Histidyl residues can be derivatized by reaction with
  • Lysinyl and a terminal residues can be reacted with succinic or other carboxylic acid anhydrides to reverse the charge of the residues.
  • suitable reagents for derivatizing a amino-containing residues include imidoesters, such as, methyl picolinimidate, pyridoxal phosphate, pyridoxal chloroborohydride, trmitrobenzenesulfonic acid, O-methylisourea and 2,4-pentanedione, and the amino acid can be transaminase-catalyzed with glyoxylate.
  • Arginyl residues can be modified by reaction with one or several conventional reagents, such as, phenylglyoxal, 2,3-butanedione,
  • tyrosyl residues can be made with aromatic diazonium compounds or tetranitromethane.
  • aromatic diazonium compounds or tetranitromethane.
  • N-acetylimidizole and tetranitromethane can be used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively.
  • Tyrosyl residues can be iodinated using methods known in the art.
  • carbodiimide or l-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide Furthermore, aspartyl and glutamyl residues can be converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.
  • Glutaminyl and asparaginyl residues are frequently deamidated to the corresponding glutamyl and aspartyl residues, respectively, under neutral or basic conditions.
  • the deamidated form of those residues falls within the scope of the instant invention.
  • Another type of covalent modification involves chemically or enzymatically coupling glycosides to the molecules of interest.
  • the sugar(s) may be attached to: (a) arginine and histidine; (b) free carboxyl groups; (c) free sulfhydryl groups, such as those of cysteine; (d) free hydroxyl groups, such as those of serine, threonine or hydroxyproline; (e) aromatic residues such as those of phenylalanine, tyrosine or tryptophan; or (f) the amide group of glutamine.
  • a polyglycol can be appended to an amino acid.
  • deglycosylation for example, can require exposure of the molecule to the compound, trifluoromethanesulfonic acid, or an equivalent compound, resulting in cleavage of most or all sugars except the linking sugar (N-acetylglucosamine or
  • RNA or DNA encoding the g l20, gp 160 and the like of HIV is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to the relevant genes, Innis et al. in PCR Protocols. A Guide to Methods and Applications, Academic (1990), and Sanger et al, Proc Natl Acad Sci 74:5463 (1977)). Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells, such as, E.
  • RNA or DNA also may be modified, for example, by substituting bases to optimize for codon usage in a particular host or by covalently joining to the coding sequence of a heterologous polypeptide. Such an approach could be the basis for developing a subunit vaccine.
  • the gpl20 of HIV was selected as a target for refocusing the host immune response to other nondominant sites on gpl20 as novel targets for an immunoprotective response, preferably one of broad scope and spectrum active on a wide variety of strains, clades and so on.
  • RNA, proteins, structures and domains can be altered by site-specific mutation to nucleotides that participate in the formation of the structure or domain. In some cases, mutations,
  • 02a31b28 28 such as nucleotide base substitutions, can be made without changing the sequence of the encoded protein, (e.g silent mutation-synonymous), essentially recoding.
  • TSE transmissible spongiform encephalopathies
  • plasmids harboring the vaccine-encoding sequence carry a non-antibiotic selection marker, since it is not always ideal to use antibiotic resistance markers for selection and maintenance of plasmids in hosts.
  • the present invention provides a selection strategy in which, for example, a catabolic enzyme is utilized as a selection marker by enabling the growth of bacteria in medium containing a substrate of said catabolic enzyme as a carbon source.
  • a catabolic enzyme includes, but is not restricted to, lacYZ encoding lactose uptake and ⁇ -galactosidase (Genbank Nos. J01636, J01637, K01483 or K01793).
  • selection markers that provide a metabolic advantage in a defined medium include, but are not restricted to, galTK (GenBank No. X02306) for galactose utilization, sacPA (GenBank No. J03006) for sucrose utilization, trePA (GenBank No. Z54245) for trehalose utilization, xylAB (GenBank No. CAB 13644 and
  • the administered product is a nucleic acid encoding a modified epitope of interest.
  • the administered product is a nucleic acid vector carrying the coding sequence of a modified epitope of interest.
  • the nucleic acid vector then expresses the modified epitope.
  • a suitable vector can be an adenoviral vector, an influenza vector, another viral vector, a bacterial vector, a yeast vector, a mammalian cell vector and so on.
  • a modified epitope of interest is produced in an edible product for ingestion by a target host to be immunized, such as a plant, a food and so on, such as a vegetable, a fruit, a milk, a milk product, a grain, a grain product and so on.
  • the modified antigen or epitope of interest then is manipulated using materials and methods known in the art. Often, the materials and methods relate to recombinant methods.
  • the antigen of interest can be secreted or excreted in a culture medium, can be cell bound or can be contained within a cell. Standard procedures are practiced to isolate the product of interest.
  • the immunogen of the present invention may be used to treat a mammal.
  • the immunogen of interest can be administered to a nonhuman mammal for the purpose of obtaining preclinical data or for treatment, for example.
  • exemplary nonhuman mammals include nonhuman primates, dogs, cats, livestock, such as, cattle, goat, sheep, rabbits and so on, poultry, such as, domesticated fowl, turkey and duck, rodents and other mammals.
  • Such mammals may be established animal models for a
  • 02a31b28 30 disease may be used to study toxicity of the immunogen of interest.
  • dose escalation studies may be performed in the mammal.
  • a product of the invention of interest can be used to treat same.
  • a formulation of interest can include a physiological buffer (U.S. Pat. No. 5,589,466) or other suitable carrier.
  • an adjuvant Such molecules suitably are present in combination in amounts that are effective for the purpose intended.
  • the adjuvant can be administered sequentially, before or after antigen administration or simultaneously therewith. Examples include aluminum phosphate or aluminum hydroxyphosphate (e.g. Ulmer et al., Vaccine, 18: 18 (2000)), monophosphoryl-lipid A (also referred to as MPL or MPLA; Schneerson et al., J. Immunol, 147:2136-2140 (1991); e.g. see Sasaki et al, Inf. Immunol, 65:3520-3528
  • the formulation herein also may contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely impact each other. Hence, a
  • 02a31b28 31 compound of interest can be administered with another antiviral compound.
  • a compound of interest is administered with an antibody or
  • a compound of interest and antibody or antigen-binding portion thereof are administered as a complex wherein said antibody is bound to said compound of interest.
  • the immunogen of interest can be used with a second
  • the therapeutic moiety can be any drug, vaccine and the like used for an intended purpose.
  • the therapeutic moiety can be a biological, a small molecule and so on.
  • the immunogen of interest can be administered concurrently or sequentially with a second HIV immunogenic composition, immunodampened or not, for example.
  • an immunodampened antigen of interest can be combined with an existing vaccine, although that approach would minimize the use thereof if the existing vaccine, for example, is made in eggs, or an administered formulation can comprise two or more immunodampened epitopes.
  • small molecule and analogous terms include, but are not limited to, peptides, peptidomimetics, amino acids, amino acid analogues,
  • polynucleotides polynucleotide analogues, carbohydrates, lipids, nucleotides, nucleotide analogues, organic or inorganic compounds (i.e., including heterorganic
  • 02a31b28 32 and/organometallic compounds which can have a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds, which can have a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds, which can have a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds, which can have a molecular weight less than about 500 grams per mole, and salts, esters, combinations thereof and other pharmaceutically acceptable forms of such compounds which stimulate an immune response or are immunogenic, or have a desired pharmacologic activity.
  • the immunogen of the invention may be administered alone or in combination with other types of treatments, including a second immunogen or a treatment for the disease being treated.
  • the second component can be an
  • the immunogen of the instant invention may be conjugated to various effector molecules such as heterologous polypeptides, drugs, and the like.
  • An immunogen may be conjugated to a therapeutic moiety such as an antibiotic (e.g., a therapeutic agent or a radioactive metal ion (e.g., a emitters such as, for example, 213 Bi)), an adjuvant or a targeting molecule.
  • a therapeutic moiety such as an antibiotic (e.g., a therapeutic agent or a radioactive metal ion (e.g., a emitters such as, for example, 213 Bi)), an adjuvant or a targeting molecule.
  • Therapeutic compounds of the invention alleviate at least one symptom associated with a disease, disorder, or condition associated with HIV amenable for treatment with an immunogen of interest.
  • the products of the invention may be provided in pharmaceutically acceptable compositions as known in the art or as
  • Pharmacopeia for use in animals and more particularly in humans.
  • the products of interest can be administered in any acceptable manner.
  • Methods of introduction include, but are not limited to, parenteral, subcutaneous, intraperitoneal, intrapulmonary, intranasal, epidural, inhalation and oral routes, and if desired for immunosuppressive treatment, intralesional administration.
  • Parenteral infusions include intramuscular, intradermal, intravenous, intraarterial or
  • the products or compositions may be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal, genital and intestinal mucosa etc.) and may be administered together with other biologically active agents. Administration can be systemic or local.
  • intraventricular catheter for example, attached to a reservoir, such as an Ommaya reservoir.
  • the product can be suitably administered by pulse infusion, particularly with declining doses of the products of interest.
  • the dosing can be given by injection, such as, intravenous or subcutaneous injections, depending, in part, on whether the administration is brief or chronic.
  • 02a31b28 34 Various other delivery systems are known and can be used to administer a product of the present invention, including, e.g., encapsulation in liposomes, microparticles, microcapsules (see Langer, Science 249: 1527 (1990); Liposomes in the Therapy of Infectious Disease and Cancer, Lopez -Berestein et al., eds., (1989)), cochleates or other synthetic carrier structure or compound.
  • the active ingredients may be entrapped in a microcapsule prepared, for example, by coascervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin microcapsule and
  • poly-(methylmethacylate) microcapsule respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules
  • macroemulsions for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules.
  • Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.
  • the composition of interest may also be administered into the lungs of a patient in the form of a dry powder composition, see e.g., U.S. Pat. No. 6,514,496.
  • 02a31b28 35 Preferably, when administering a product of the invention, care is taken to use materials to which the protein does not absorb or adsorb.
  • compositions can take the form of solutions, suspensions, an emulsion, tablets, pills, capsules, powders, sustained release formulations, depots and the like.
  • the composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
  • Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate etc. Examples of suitable carriers are described in "Remington's Pharmaceutical Sciences," Martin.
  • Such compositions will contain an effective amount of the immunogen preferably in purified form, together with a suitable amount of carrier so as to provide the proper and desired form for administration to the patient.
  • the formulation will be constructed to suit the mode of administration.
  • the product can be delivered in a controlled release system.
  • a pump may be used (see Langer, Science 249: 1527 (1990); Sefton, CRC Crit Ref Biomed Eng 14:201 (1987);
  • polymeric materials can be used (see Medical Applications of Controlled Release, Langer et al., eds., CRC Press (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen et al., eds., Wiley (1984); Ranger et al, J Macromol Sci Rev Macromol Chem 23:61 (1983); see also Levy et al, Science 228: 190 (1985); During et al, Ann Neurol 25:351 (1989);
  • a controlled release system can be placed in proximity of the therapeutic target.
  • Sustained release preparations may be prepared for use with the products of interest.
  • Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing the immunogen, which matrices are in the form of shaped articles, e.g., films or matrices.
  • Suitable examples of such sustained release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethylmethacrylate), poly(vinyl alcohol)), polylactides (U.S. Pat. No.
  • copolymers of L-glutamic acid and ethyl-L-glutamate non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers (such as injectable microspheres composed of lactic acid-glycolic acid copolymer) and poly-D-(-)-3-hydroxybutyric acid.
  • polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release cells, proteins and products for and during shorter time periods. Rational strategies can be devised for stabilization depending on the mechanism involved.
  • an amino acid substitution or other change can stabilize an epitope, particularly if there is another change therein which might disrupt the normal secondary, tertiary or quaternary structure of the protein carrying said epitope, said epitope or regions adjacent to said epitope.
  • Therapeutic formulations of the product may be prepared for storage as lyophilized formulations or aqueous solutions by mixing the product having the desired degree of purity with optional pharmaceutically acceptable carriers, diluents, excipients or stabilizers typically employed in the art, i.e., buffering
  • An immune refocused polypeptide (which includes an antigen, a portion thereof, an epitope, a determinant and so on) which can be produced as a subunit substantially free of contaminating proteins, including other pathogen proteins, in combination with other viral or non-viral polypeptides; as an
  • immunodisabled polypeptide of interest which can be expressed or produced in recombinant viruses, VLP's or in combination with one or more proteins of virus or cell origin; as an immunodisabled polypeptide which can be expressed or produced as an isolated molecule and then combined with one or more proteins of virus or cell origin; and so on); can be obtained or made in substantially pure form.
  • the epitope of interest can be expressed on a structure and used as is, for example, on a virion, a virus-like particle, a liposome, other carrier molecule, for example, constructed of saccharides or polypeptides, or combinations thereof and so on.
  • an epitope of interest can be incorporated into any known delivery means.
  • An “isolated” or “purified” immunogen is substantially free of contaminating proteins from the medium from which the immunogen is obtained, or substantially free of chemical precursors or other chemicals in the medium used which contains components that are chemically synthesized.
  • substantially free of subcellular material includes preparations of a cell in which the
  • 02a31b28 38 cell is disrupted to form a component mixture which can be separated from
  • an immunogen that is substantially free of subcellular material includes preparations of the immunogen having less than about 30%, 20%, 25%o, 20%o, 10%), 5%o, 2.5%. or 1%>, (by dry weight) of subcellular contaminants or any other element that differs from the product of interest.
  • the terms "stability" and “stable” in the context of a liquid formulation comprising an immunogen refer to the resistance of the immunogen in a formulation to, for example, thermal and chemical aggregation, degradation, fragmentation or other change which degrades an intended function or functions under given manufacture, preparation, transportation and storage conditions, such as, for one week, for two weeks, for three weeks, for one month, for two months, for three months, for four months, for five months, for six months or more.
  • the “stable” formulations of the invention retain biological activity equal to or more than 80%, 85%, 90%, 95%, 98%, 99% or 99.5% of the original measured property under given manufacture, preparation, transportation and storage conditions over the above-noted time periods.
  • the stability of said immunogen preparation can be assessed by degrees of aggregation, degradation or fragmentation by methods known to those skilled in the art, including, but not limited to, physical observation, such as, with a microscope, particle size and count determination and so on, compared to a reference.
  • the metric can be a biologic property or activity.
  • the instant invention encompasses formulations, such as, liquid formulations that are maintained or stored at temperatures found in a commercial refrigerator and freezer found in the office of a physician or laboratory, such as from about -20° C to about 5° C, said stability assessed, for example, by microscopic analysis, for storage purposes, such as for about 7 days, 10 days, 14 days, 20 days, 30 days, 45 days, 60 days, for about 120 days, for about 180 days, for about a year, for about 2 years or more.
  • the liquid formulation of the present invention also exhibit stability, as assessed, for example, by particle analysis, at room temperatures, for at least a few hours, such as one hour, two hours or about three hours prior to use.
  • Stability can also be measured using a biologic or functional property.
  • carrier refers to a diluent, adjuvant, excipient or vehicle with which the therapeutic is administered.
  • physiological carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a suitable carrier when the pharmaceutical composition is administered intravenously.
  • Saline solutions and aqueous dextrose and glycerol solutions also can be employed as liquid carriers, particularly for injectable solutions.
  • Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like.
  • the composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • Buffers are preferably present at a concentration ranging from about 0.1 mM to about 50 mM.
  • Suitable buffering agents for use with the instant invention include both organic and inorganic acids, and salts thereof, such as citrate buffers (e.g., monosodium citrate-disodium citrate mixture, citric acid-trisodium citrate mixture, citric acid-monosodium citrate mixture etc.), succinate buffers (e.g., succinic acid-monosodium succinate mixture, succinic acid-sodium hydroxide mixture, succinic acid-disodium succinate mixture etc.), tartrate buffers (e.g., tartaric acid-sodium tartrate mixture, tartaric acid-potassium tartrate mixture, tartaric acid- sodium hydroxide mixture etc.), fumarate buffers (e.g., fumaric acid-monosodium fumarate mixture, fumaric acid-disodium fumarate mixture, monosodium fuma
  • the present invention can provide liquid formulations of an immunogen having a pH ranging from about 5.0 to about 7.0; about 5.5 to about 6.5;
  • 02a31b28 41 about 5.8 to about 6.2; about 6.0; about 6.0 to about 7.5; about 6.5 to about 7.0; and so on.
  • Preservatives may be added to retard microbial growth, and may be added in amounts ranging from 0.1%-1% (w/v). Suitable preservatives for use with the present invention include phenol, benzyl alcohol, m-cresol,
  • octadecyldimethylbenzyl ammonium chloride e.g., benzylconium halides (e.g., chloride, bromide and iodide), hexamethonium chloride, alkyl parabens, such as, methyl or propyl paraben, catechol, resorcinol, cyclohexanol and 3-pentanol.
  • Isotonicifiers are present to ensure physiological isotonicity of liquid compositions of the instant invention and include polyhydric sugar alcohols, such as, trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol.
  • Polyhydric alcohols can be present in an amount of between about 0.1% to about 25%, by weight, about 1% to about 5%>, and so on, taking into account the relative amounts of the other ingredients.
  • Stabilizers refer to a broad category of excipients which can range in function from a bulking agent to an additive which solubilizes the therapeutic agent or helps to prevent denaturation or adherence to the container wall.
  • Typical stabilizers can be polyhydric sugar alcohols; amino acids, such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine,
  • 2-phenylalanine, glutamic acid, threonine etc. organic sugars or sugar alcohols, such as lactose, trehalose, stachyose, arabitol, erythritol, mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol and the like, including cyclitols such as inositol; polyethylene glycol; amino acid polymers; sulfur-containing reducing agents, such as
  • a-monothioglycerol and sodium thiosulfate low molecular weight polypeptides (i.e., ⁇ 10 residues); proteins, such as human serum albumin, bovine serum albumin, gelatin or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone, saccharides, monosaccharides, such as xylose, mannose, fructose or glucose;
  • Stabilizers can be present in the range from 0.1 to 10,000 w/w per part of immunogen.
  • Additional miscellaneous excipients include bulking agents,
  • chelating agents e.g., EDTA
  • antioxidants e.g., ascorbic acid, methionine or vitamin E
  • cosolvents e.g., ascorbic acid, methionine or vitamin E
  • surfactant refers to organic substances having amphipathic structures, namely, are composed of groups of opposing solubility tendencies, typically an oil-soluble hydrocarbon chain and a water-soluble ionic group. Surfactants can be classified, depending on the charge of the surface-active moiety, into anionic, cationic and nonionic surfactants. Surfactants often are used as wetting, emulsifying, solubilizing and dispersing agents in pharmaceutical compositions and preparations of biological materials.
  • Nonionic surfactants or detergents may be added to help solubilize the therapeutic agent, as well as to protect the therapeutic protein against agitation-induced aggregation, which also permits the formulation to be exposed to shear surface stresses without causing denaturation of the protein.
  • Suitable nonionic surfactants include polysorbates (20, 80 etc.),
  • Nonionic surfactants may be present in a range of about 0.05 mg/ml to about 1.0 mg/ml, about 0.07 mg/ml to about 0.2 mg/ml, and so on.
  • inorganic salt refers to any compound, containing no carbon, that results from replacement of part or all of the acid hydrogen or an acid by a metal or a group acting like a metal, and often is used as a tonicity adjusting compound in pharmaceutical compositions and preparations of biological materials.
  • Common inorganic salts are NaCl, KC1, NaH 2 P0 4 etc.
  • diluents include a phosphate-buffered saline, buffer for buffering against gastric acid in the bladder, such as citrate buffer (pH 7.4) containing sucrose, bicarbonate buffer (pH 7.4) alone, or bicarbonate buffer (pH 7.4) containing ascorbic acid, lactose or aspartame.
  • examples of carriers include proteins, e.g., as found in skim milk, sugars, e.g., sucrose, or polyvinylpyrrolidone. Typically these carriers would be used at a concentration of about 0.1-90% (w/v) but also at a range of 1-10% (w/v).
  • the formulations to be used for in vivo administration must be sterile. That can be accomplished, for example, by filtration through sterilization or filtration membranes.
  • the subcellular formulations of the present invention may be sterilized by filtration.
  • the immunogen composition will be formulated, dosed and administered in a manner consistent with good medical practice. Factors for consideration include the particular disorder being treated, the particular mammal
  • 02a31b28 44 being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to practitioners.
  • the amount of antigen is not critical to the present invention but is typically an amount sufficient to induce the desired humoral and/or
  • the amount of immunogen of the present invention to be administered will vary depending on the species and the physical properties and characteristics of the subject, as well as the disease or condition that is being treated. Generally, the dosage employed can be about
  • the term "effective amount” refers to the amount of a therapy (e.g., a prophylactic or therapeutic agent), which is sufficient to reduce the severity and/or duration of a targeted disease, ameliorate one or more symptoms thereof, prevent the advancement of a targeted disease or cause regression of a targeted disease, or which is sufficient to result in the prevention of the development, recurrence, onset, or progression of a targeted disease or one or more symptoms thereof.
  • a therapy e.g., a prophylactic or therapeutic agent
  • a treatment of interest can increase survivability of the host, based on baseline or a normal level, by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%.
  • an effective amount of a therapeutic or a prophylactic agent reduces the symptoms of a targeted disease, such as a symptom of HIV by at least 5%, preferably at least 10%,
  • 02a31b28 45 at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%.
  • an effective amount is one which generates in an in vitro system a specific immune response, whether humoral or cellular.
  • an immunodisabled peptide reacts with a specific HIV antibody or a cell reacts to or with HIV-infected cells (for example, as evidenced by DNA synthesis)
  • an effective amount is that which provides a response above background.
  • an effective amount is that which generates in vivo, in a host of design choice, a specific immune response to the disabled peptide, be that response humoral or cellular.
  • the composition may also include a solubilizing agent and a local anesthetic such as lidocaine or other "caine” anesthetic to ease pain at the site of the injection.
  • a solubilizing agent such as lidocaine or other "caine” anesthetic to ease pain at the site of the injection.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or concentrate in a sealed container, such as an ampule or sachet indicating the quantity of active agent.
  • a dry lyophilized powder or concentrate in a sealed container, such as an ampule or sachet indicating the quantity of active agent.
  • the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampule of sterile water for injection or saline can be provided, for example, in a kit, so that the ingredients may be mixed prior to administration.
  • the article of manufacture can comprise a container and a label.
  • Suitable containers include, for example, bottles, vials, syringes and test tubes.
  • the containers may be formed from a variety of materials such as glass or plastic.
  • the container holds a composition which is effective for preventing or treating a targeted condition or disease and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • the label on or associated with the container indicates that the composition is used for treating the condition of choice.
  • the article of manufacture may further comprise a second container comprising a pharmaceutically acceptable buffer, such as, water, phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes and package inserts with instructions for use.
  • a pharmaceutically acceptable buffer such as, water, phosphate-buffered saline, Ringer's solution and dextrose solution.
  • buffers, diluents, filters, needles, syringes and package inserts with instructions for use including buffers, diluents, filters, needles, syringes and package inserts with instructions for use.
  • kits e.g., comprising an immunogenic composition of interest, homolog, derivative thereof and so on, for use, for example, as a vaccine, and instructions for the use of same and so on.
  • the instructions may include directions for using the composition, derivative and so on.
  • the composition can be in liquid form or presented as a solid form, for example, desiccated or lyophilized.
  • the kit can contain suitable other reagents, such as a buffer, a reconstituting solution and other necessary ingredients for the intended use. A packaged combination of reagents in predetermined amounts with instructions for
  • 02a31b28 47 use thereof such as for a therapeutic use is contemplated.
  • other additives may be included, such as, stabilizers, buffers and the like.
  • the relative amounts of the various reagents may be varied to provide for concentrates of a solution of a reagent, which provides user flexibility, economy of space, economy of reagents and so on.
  • Serologic data from CM235 isotype (Genbank accession number L03698) immunized baboons are used to identify immunodominant epitopes within gpl20, especially those within linear domains. Analysis using 118 12mer CM235 envelope peptides overlapping by 8 amino acids show 4 immunodominant epitopes including the V2 and V3 domains, V2 being the most immunodominant. In this example, both V2 and V3 domains are targeted for immunodampening. In other examples, additional domains of the gpl60 glycoprotein are targeted for immunodampening.
  • Immune dampening strategies are developed to completely dampen the VI /V2 immunodominant domain. Examples of immune dampened mutations of this region are provided in Table 1 in Figures 1 and 2.
  • V3 domain of CM235 is:
  • Immunodominant V3 peptides identified by the CM235 immune sera from baboons include: peptide 306 SNNTRTSIPIGP (residues 5-16 of SEQ ID NO: 2) peptide 310 RTSIPIGPGQAF (residues 9-20 of SEQ ID NO: 2) peptide 314 PIGPGQAFYRTG (residues 13-24 of SEQ ID NO:
  • vaccinia virus transfer vector After immune refocusing mutations are introduced into the gpl20, gpl40, and gpl60 genes, they are subcloned into the pSC65 vaccinia virus transfer vector. Vaccinia virus expression is used as a convenient method for the production of preclinical antigens.
  • the pSC65 vector permits identification of recombinant virus via coexpression of the LacZ gene product and subsequent staining
  • the pSC65 vector recombines into the TK gene of vaccinia leading to dual selection (TK " and blue staining plaques) in cell lines lacking thymidine kinase activity.
  • HeLa cells are transfected with the pSC65 transfer vector containing the gpl20, gpl40 and gpl60 genes, and then infected with the WR strain of vaccinia virus. After three days, the crude lysates of the cultures are prepared, diluted and used to infect TK " cell monolayers in the presence of BrdU. Once the infection proceeded, the culture fluids are replaced with agarose overlays containing X-gal and blue recombinant plaques are picked. Plaques are screened for expression of Env proteins. Positive plaques are purified to homogeneity through multiple rounds of plaque purification.
  • a series of deletion and multiple point mutations are designed to dampen the immunodominant variable domains of gpl20E. Mutations are designed to introduce codons to direct the addition of glycosylation residues into immunodominant epitopes, replace charged amino acids with uncharged residues, and delete peptides thought to dysregulate immune responses. The deletions are designed to retain some of the proposed structural elements of the glycoproteins. Examples of immune refocused mutations are presented in Table 1. Oligonucleotides used to engineer the mutations are shown in Table 2.
  • the mutated gp 120/ 140/ 160 fragments are cloned into the vaccinia transfer vector pSC65 such that a series of gpl20, gpl40, and gpl60 genes are derived.
  • Recombinant vaccinia viruses are engineered by transfecting the pSC65 vectors into HeLa cells as described above.
  • membrane-bound proteins are recovered from HeLa cells infected with recombinant vaccinia virus by extraction using buffers containing nonionic detergents such as 20 mM Tris-HCl, pH 8.0 with 0.1% Tween-20.
  • Recombinant gpl20 and gpl40 glycoproteins are expressed as secreted proteins and are recovered from the conditioned culture supernatant.
  • Recombinant gpl20, gpl40, and gpl60 glycoproteins are purified using lectin affinity chromatography with lentil lectin sepharose and galanthus nivalis lectin agarose. The glycoproteins are analyzed by immunoblotting, SDS-PAGE and total protein composition.
  • sites of the gpl60 molecule are targets of immune re focusing. These sites may include amino acids in or near contact points for antibodies that have strain-specific or nonprotective activities, cell receptors, or coreceptors and sites that may contribute to deceptive imprinting.
  • a domain of the gp41 region may be a target of immune refocusing by the two mutations, M14 (CSGKIICT (SEQ ID NO: 3) -» CSNGTICT) (SEQ ID NO: 48) and M15 (GLWGCSGKIIC (SEQ ID NO: 4) -> GLAGCSGAIIC) (SEQ ID NO: 49).
  • M14 introduces an N-linked glycosylation site into the C-C loop and Ml 5 introduces nonconservative amino acid substitutions into the same loop.
  • Additional domains of the gp41 region may also be targeted by immune refocusing including an immune refocusing including an immunodominant epitope in the following section of the Clade B sequence: VQQQSNLLRAIEAQQHLLQLTVWGIKQLQARVLAVE RYLKDQKFLGLWGCSGKIICTT (SEQ ID NO:5).
  • an alpha helix structure of gp41 may also be targeted by immune refocusing.
  • the Clade B sequence, SNLLRAIEAQQHLLQLTVWGIKQLQARVLAVERYLK can be immune refocused by changing charged or other residues in either the faces of the helix or the sequences N-terminal or C-terminal to the helix.
  • M22 QARVLAV ERYLK (SEQ ID NO: 7)-> QAGVLAVQGYLK) (SEQ ID NO: 50) and M23 (LRAIEA (SEQ ID NO: 8) -> LGAIOA) (SEQ ID NO: 51) introduce charge changes to the alpha helix while M24 (LLQLTVWGIKQLQARVL (SEQ ID NO: 9) -> LGQLTVWGIKQLQARGL) (SEQ ID NO: 52) and M25 (LLRAIEAQQ
  • HLLQLTVWGIKQLQ (SEQ ID NO: 10) -> LGRAIEAQGHLLQLTGWGIKQGQ) (SEQ ID NO: 53) introduce changes to the face of the helix.
  • a superantigen site in gpl20 of Clade B can be immune refocused with mutations such as Ml 8 (PCKIKQIINM (SEQ ID NO: 11) PCAIVQIINM) (SEQ ID NO: 54).
  • the intervening peptide sequence between epitopes thought to convey broadened immunity may be immune dampened.
  • the gpl20 sequence between epitopes of monoclonal antibodies 3b and 2F5 may be immune dampened with modifications such as those of Ml 9
  • immune refocused immunogens may be composed of combinations of mutations at different sites.
  • Ml 3 may consist of a V1/V2 mutation in the presence of a V3 mutation while Ml 6 may consist of the M13 mutation in the presence of M14 or M15.
  • the immune refocused Env antigens may be used to raise antibodies that have novel and broadened therapeutic, diagnostic or investigatory properties.
  • Animals such as, but not restricted to mice, may be immunized with purified gpl20, gpl40, and/or gpl60 glycoproteins by using either single immune refocused antigens or a combination of immune-refocused antigens.
  • Polyclonal or monoclonal antibodies may be derived using methods known to the art.
  • novel and broadly reactive antibodies, antibody fragments, or their derivatives (such as, minibodies) may be derived from animal or humans that have been exposed, infected or naive to a pathogen, immune refocused or antibody immune refocused or other altered antigens.
  • TNA or DNA from the immune cells e.g. B cells, B cell precursors or pluripotential stem cells, for example.
  • relevantt genes or gene fragments encoding an immunoglobulin or portions thereof are cloned directly from the nucleic acid or cloned into an expression library for screening with the same or wild-type antigens or other structural mimetic probes.
  • the antibodies may be tested for antiviral activities against a panel of HIV- 1 isolates. Antibodies that crossneutralize or otherwise inactivate strains of virus different from the strain from which the antigen had been derived are considered to contain broadened crossreactive activities. The antibodies may be further characterized by antiviral testing against multiple strains with increasing evolutionary distance from the parental strain. Broadly reactive antibodies may be manufactured for use as therapeutics, preventatives, diagnostics, or experimental reagents.
  • Antibodies may be used to immune refocus antigens by binding to decoy epitopes, receptotopes, immunodominant epitopes or other domains of the antigen.
  • Antibodies raised from either unmodified or immune refocused antigens, or parts thereof, may be used to form antibody-antigen complexes for the purpose of producing a vaccine or deriving an antibody.
  • immunization with gpl20, gpl40, or gpl60 antigens or infection with virus can stimulate the induction of strain-specific antibodies to the immunodominant epitopes.
  • antibodies prepared in an immunization or infection process can be purified and bound to the antigen to form an immune stimulatory complex. Immunization with the complex may result in the stimulation of broadened crossprotective immunity.
  • the antibody in the complex function to reduce the accessibility or to coat the immunodominant, strain-specific
  • 02a31b28 55 epitopes so as to refocus the immune system to sites not covered by the bound antibody.
  • the sites not covered may be more highly conserved and elicit broadened immunity.
  • Antibody fractions may be used to immune refocus antigens by binding to decoy epitopes.
  • Antibody molecules e.g., IgG
  • Antibody molecules may be digested with papain or trypsin to yield F a b or F( a b) 2 fragments which, due to their smaller size relative to whole IgG, penetrate and bind the strain-specific decoy epitope more efficiently than whole IgG.
  • F a b or F( a b) 2 fragments can be complexed with gpl20, gpl40, or gpl60 antigens to form an immune stimulatory complex which may stimulate broadened crossprotective immunity or stimulate the production of antibodies with broadened therapeutic activities.
  • Recombinant and single chain antibodies and antibody fragments may be used to immune refocus antigens by binding to decoy epitopes using a similar technique.
  • the safety, toxicity and potency of recombinant immunogens are evaluated according to the guidelines in 21 CFR 610, which include: (i) general safety test; (ii) stringent safety test in immunocompetent mice; (iii) guinea pig safety test; and (iv) acute and chronic toxicity tests, as described below.
  • mice Groups of eight BALB/c mice are inoculated intraperitoneally with 100 ⁇ of immunogen containing 300 ⁇ g of the immunogen of interest. Suitable negative and positive controls are used.
  • 02a31b28 56 [00158] The animals are monitored for general health and body weight for 14 days post infection. Similar to animals that receive placebo, animals that receive the immunogen remain healthy, and do not lose weight or display overt signs of disease during the observation period.
  • mice in each group are sacrificed and the spleen, lung and liver homogenates are analyzed for immunogen.
  • week 4 8, 12, and 16 post infection, 3 mice in each group are sacrificed and spleen, live and lung homogenates are obtained and analyzed to assess presence of the immunogen.
  • mice are given a total of 3 doses of vaccine at 0, 14 and 60 days and the immune response to HIV is measured by ELISA using sera collected from the tail vein of individual mice at 10 day intervals, as described. The neutralization of HIV is measured in the collected 80 days after the first vaccination.
  • the results of the study show that the vaccine of interest has the capacity to substantially increase the magnitude and potency of the humoral response to HIV and therefore possesses useful adjuvant properties.
  • HIV-1 the confounding variables of virus

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Abstract

L'invention concerne de nouvelles compositions qui s'utilisent en tant qu'immunogènes du VIH. Les compositions permettent une réponse de l'hôte envers des sites normalement non reconnus par un hôte. L'invention porte également sur des anticorps élevés contre de tels immunogènes. L'invention concerne également des complexes d'anticorps ou de parties de liaison à l'antigène de ceux-ci et d'antigènes du VIH.
PCT/US2010/047754 2009-09-03 2010-09-02 Compositions immunogènes du vih Ceased WO2011028963A2 (fr)

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US10092637B2 (en) 2011-10-20 2018-10-09 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Dengue virus E-glycoprotein polypeptides containing mutations that eliminate immunodominant cross-reactive epitopes

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US5585250A (en) * 1993-08-20 1996-12-17 The United States Of America As Represented By The Department Of Health & Human Services Dampening of an immunodominant epitope of an antigen for use in plant, animal and human compositions and immunotherapies
WO2004053100A2 (fr) * 2002-12-11 2004-06-24 The Scripps Research Institute Polypeptides gp120 du virus de l'immunodeficience humaine mutants immunogenes, et leurs procedes d'utilisation
EP1667631A4 (fr) * 2003-09-15 2010-04-14 Novartis Vaccines & Diagnostic Approches combinees pour produire des reponses immunitaires

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10092637B2 (en) 2011-10-20 2018-10-09 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Dengue virus E-glycoprotein polypeptides containing mutations that eliminate immunodominant cross-reactive epitopes
US10568956B2 (en) 2011-10-20 2020-02-25 The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Dengue virus e-glycoprotein polypeptides containing mutations that eliminate immunodominant cross-reactive epitopes
US11154608B2 (en) 2011-10-20 2021-10-26 The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Dengue virus e-glycoprotein polypeptides containing mutations that eliminate immunodominant cross-reactive epitopes

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