Described herein are nucleic acid molecules which encode multiple highly conserved epitopes from HIV-1 proteins, and optionally also epitopes from CCR5; usually also included sequences that encode spacers between two or more of the epitopes. Some of the provided nucleic acid molecules further include sequences that encode targeting domains, useful for targeting the encoded protein into a pathway for enhancing epitope presentation in a vertebrate immune system. Also described are multivalent proteins encoded for by these nucleic acid molecules. The disclosure also encompasses immunogenic compositions that comprise one or more of the nucleic acid molecules, and/or one or more of the proteins encoded thereby, as well as methods of inducing an immune response against HIV-1 in a subject by administering to the subject an effective amount of a composition containing one or more of these molecules. Also provided are cultured host cells containing within them one or more of the described nucleic acid molecules.

Description of the Invention

SUMMARY OF THE DISCLOSURE

Multi-clade multivalent (MCMV) (polyepitope; multi-epitope) polypeptides and mixtures of polypeptides have been developed, which can be used to stimulate immune responses to HIV-1 in vertebrates. In various embodiments, these polypeptides and polypeptide mixtures include immunogenic CTL, T- and/or B-cell determinants that are capable of eliciting broad and effective immune responses against diverse subtypes of HIV-1. Immunogens described herein are designed to be subtype-independent and will provide both prime and boost reagents for worldwide use.

Also described herein are recombinant MCMV constructs that can be used directly or indirectly to protect subjects against infection by multiple HIV-1 subtypes. These constructs are designed to elicit T-cell, B-cell, or both T-cell and B-cell responses against highly conserved epitopes within multiple HIV-1 subtypes. The constructs, when integrated into a vector, can be used as immunogens, can be used as DNA vaccines, and can be used as sources of recombinant protein for stimulation of immune responses in subjects, as well as for protein boosts to subjects who have received a nucleic acid construct previously.

Without being bound by theory, it is believed that the MCMV HIV-1 constructs and polypeptides provide universal immune stimulants and vaccines, capable of effective use in any part of the world affected by the HIV-1 epidemic.

The construction and design of specific provided constructs are particularly useful in that they allow convenient addition/deletion of epitopes, and contain specific cellular targeting domains that optimize antigen processing and recognition.

The provided constructs and proteins encoded thereby also can be combined with other epitope-based constructs to generate, for instance, multi-pathogen vaccines.

The foregoing and other features and advantages will become more apparent from the following detailed description of several embodiments, which proceeds with reference to the accompanying figures.

Overview of Several Embodiments

Provided herein in various embodiments are multi-clade, multivalent recombinant polyepitope polypeptides, which are useful to induce immunogenic responses in vertebrate animals to HIV-1. These polypeptides include CTL-stimulatory epitopes, T-helper cell stimulatory epitopes, B-cell stimulatory epitopes, or combinations of two or more such types of epitopes. Epitopes in the polyepitope polypeptides are selected to provide multi-clade coverage. In particular, epitopes are selected to be at least 50% conserved across a plurality of HIV-1 subtypes, for instance, at least 2, 3, 4, 5, 6, or more HIV-1 subtypes. In particular embodiments, at least 30% of the epitopes included in a single polyepitope polypeptide are at least 60% conserved, at least 70% conserved, at least 80% conserved, or even more highly conserved across a plurality of HIV-1 subtypes.

In specific embodiments there are provided isolated polyepitope polypeptides, wherein adjacent polypeptide segments are linked by a spacer peptide. In some examples, the spacer peptide links multiple groups of polypeptide segments. Specific, non-limiting examples of the spacer peptide include the tri-amino acid lysine-alanine-alanine, or proline-glycine-proline.

In other examples, the isolated polyepitope polypeptides also include a targeting signal that targets the polyepitope polypeptides to a lysosome or to a proteosome. Specific, non-limiting, examples of the targeting signal include a targeting-competent fragment of lysosomal integral membrane protein-II or ubiquitin.

In still other examples, the isolated polyepitope polypeptides also include a plurality of amino acid segments from one or more HIV-1 coreceptors. A specific, non-limiting, example of a HIV-1 coreceptor is CCR5.

In further examples, the isolated polyepitope polypeptides include human cytotoxic T-lymphocyte stimulatory epitopes, human T-helper cell stimulatory epitopes, human B-cell stimulatory epitopes, or combinations of two or more epitopes thereof.

In additional embodiments there are provided isolated polyepitope polypeptides, which polypeptides comprise an amino acid sequence selected from the group consisting of sequences provided in SEQ ID NOs: 2, 4, 5, 6, 8 and 10. Also provided are mixtures of two or more isolated polyepitope polypeptides, including (but not limited to) mixtures of the polypeptides having sequences as shown in SEQ ID NOs: 2 and 8, 2 and 10, 4 and 8, 4 and 10, 5 and 8, 5 and 10, 6 and 8, and 6 and 10.

Other embodiments are isolated polynucleotides (nucleic acid molecules) which encode one of the polyepitope polypeptides described herein. Specific examples of such nucleic acid molecules comprise a sequence selected from the group consisting of sequences recited in SEQ ID NOs: 1, 3, 7, 9 and complements thereof. Other specific examples of nucleic acid molecules are the portions of each of SEQ ID NOs: 1, 3, 7, and 9 which correspond to and encode the polyepitope polypeptides shown in SEQ ID NOs: 2, 4, 8, and 10, respectively.

Also provided herein are genetic constructs that comprise at least one nucleic acid molecule encoding a polyepitope polypeptide, and host cells transformed with such a genetic construct.

Yet another embodiment is a composition comprising at least one polyepitope polypeptide or at least one nucleic acid molecule encoding a polyepitope polypeptide, and at least one component selected from the group consisting of pharmaceutically acceptable carriers and adjuvants. This disclosure further provides methods for eliciting and/or enhancing an immune response in a subject, which methods involve administering to the subject such a composition. In one specific, non-limiting example, the subject is infected with HIV-1

IV. Multi-Clade, Multivalent HIV-1 Constructs

The current disclosure provides multi-clade multivalent HIV-1 constructs useful for inducing immune responses in HIV-1-infected populations with diverse HLA alleles and HIV subtypes.

HIV-1 MCMV constructs comprise synthetic nucleic acid sequences to be used as HIV-1 immune-stimulatory and/or vaccine constructs to protect against multiple HIV-1 subtypes. These synthetic nucleic acid molecules or mixtures of nucleic acid molecules are designed to elicit both T-cell and B-cell responses against highly conserved epitopes within multiple HIV-1 subtypes. In specific embodiments, the synthetic genes are contained in plasmid constructs, which can be used as a DNA vaccine, as well as a source of recombinant protein for subsequent protein boosts.

Provided herein in various embodiments are multi-clade, multivalent polyepitope polypeptides, which are useful to induce immunogenic responses in vertebrate animals to HIV-1. These polypeptides include CTL-stimulatory epitopes, T-helper cell stimulatory epitopes, B-cell stimulatory epitopes, or combinations of two or more such types of epitopes. Epitopes in the polyepitope polypeptides are selected to provide multi-clade coverage. In particular, epitopes are selected to be at least 50% conserved across a plurality of HIV-1 subtypes, for instance, at least 2, 3, 4, 5, 6, or more HIV-1 subtypes. In particular embodiments, at least 30% of the epitopes included in a single polyepitope polypeptide are at least 60% conserved, at least 70% conserved, at least 80% conserved, or even more highly conserved across a plurality of HIV-1 subtypes.

One aspect of embodiments provided herein is that peptide sequences, each of which contains one or more antibody-binding or class I or class II MHC-binding epitopes, can be linked in tandem to form polyepitope polypeptides. These polyepitope polypeptides are proteolytically processed in cells to release the individual epitopes, and are useful for stimulating an immune response in a vertebrate animal.

When a MCMV polyepitope polypeptide is introduced into a cell, it is proteolytically processed into at least some of its constituent epitopes. At least some of the epitopes generated from the polypeptide can bind to MHC class I or MHC class II molecules present in the cell, though some of the epitopes may be specific for MHC class I or class II molecules present only on other cells. Included epitopes also may be B-cell epitopes, which elicit antibody-mediated immune responses upon binding to antibody receptors on the surface of a B-cell.

In one aspect, the disclosure features a nucleic acid encoding a polyepitope polypeptide that can include, in any order, a first, second, and third segment, each of which is at least nine amino acids in length. As used herein, a "segment" is an amino acid sequence which (a) corresponds to the sequence of a portion (that is, a fragment, less than all) of a naturally occurring protein, and (b) contains one or more epitopes. By "epitope" is meant a peptide which binds to the binding groove of an MHC class I or class II molecule, or to the antigen-binding region of an antibody. In addition, the polyepitope polypeptide can encode a targeting signal, for instance a peptide sequence that targets the protein to which it is fused to the lysosome or to the proteosome, as described in more detail herein.

In some embodiments, a segment of the polyepitope polypeptide has the amino acid sequence of a portion of (1) a naturally occurring HIV-1 protein or (2) a naturally occurring coreceptor (collectively referred to as "naturally occurring proteins"), that is at least nine amino acids in length. A second segment has the amino acid sequence of a second portion of the same or a different naturally occurring protein, is at least nine amino acids in length, and includes at least one epitope which is different from the epitope(s) of the first segment. Optionally, a third segment is included in the polyepitope polypeptide, and has the amino acid sequence of a third portion of the same or a different naturally occurring protein, is at least nine amino acids in length, and includes at least one epitope which is different from the epitope(s) of the first and second segments. Alternatively, the first, second and third portions may be portions of two or three different naturally occurring proteins (for example, two or three different HIV-1 proteins). The polyepitope polypeptide may optionally include additional segments, for example, it can include at least 4, 5, 10, 15, 20, 25, 30, 40, 50, 60, 75, 90 or even 100 or more segments, each being a portion of a naturally-occurring protein of a pathogenic agent and/or of a naturally occurring coreceptor which can be the same or different from the protein(s) from which the other segments are derived.

Each of these segments is at least nine amino acids in length, and each contains at least one epitope different from the epitope(s) of other segments in the polyepitope polypeptide. At least one (and in some embodiments, more) of the segments in the polyepitope polypeptide may contain class I or class II MHC-binding epitopes. Two, three, or more of the segments can be contiguous in the polyepitope polypeptide: that is, they are joined end-to-end, with no spacer between them. Alternatively, any two adjacent segments can be linked by a spacer amino acid or spacer peptide. In particular embodiments, the spacer comprises three amino acids. Specific non-limiting examples of spacers are the tri-amino acid KAA and the tri-amino acid PGP. Additionally, a spacer amino acid or spacer peptide can be used to link multiple groups of two, three, or more contiguous segments in the polyepitope polypeptide: that is, a spacer amino acid or spacer peptide is inserted between every two, three, or more segments.

A given segment of protein within the polyepitope polypeptide need not be any specified length, so long as it is sufficiently long to generate at least one epitope, for example, 2, 3, 4, 5, or more epitopes, and is at least 9 amino acids in length. For example, a given segment can have a length of at least 10 amino acids, for example, at least 11, 12, 13, 14, 15, 20, 25, 30, 40, or 50 amino acids. A given segment corresponds to a particular naturally occurring protein if any 9 (or more) consecutive amino acids of the segment are found in exactly the same order in a portion of the naturally occurring protein. In exemplary embodiments, the segments included in a polyepitope polypeptide are obtained from one or more HIV-1 proteins and/or coreceptors (for example, CCR5).

It is understood that the term "naturally occurring proteins" used above does not preclude modification of the sequence used in the polyepitope polypeptide, for instance by changing one or a few amino acids. In addition, it is understood that the nucleic acid molecule encoding the segment need not be identical to the "naturally occurring" sequence, as found in (for instance) the HV-1 genome. In particular, it is contemplated that the codon usage in the nucleic acid molecule can be modified, for instance to convert the encoding sequence to a codon optimized sequence. The codon optimization can be tailored for the host cell in which the construct will eventually be expressed. Thus, some constructs are engineered to be codon biased for expression in a prokaryotic cell, others to be expressed in a unicellular eukaryotic cell, and still others to be expressed in a cell of a multicellular eukaryote (for example, a vertebrate). Codon selection to take advantage of species biases is well known to those of ordinary skill.

The discovery of the HIV-1 coreceptors, together with a greater understanding of the Envelope-receptor mediated conformational changes resulting in the membrane fusion process, has identified several promising vaccine targets. These epitopes as well as others in the transmembrane envelope glycoprotein (gp41) have been identified as HIV-1 neutralizing epitopes. Likewise, epitopes in the CCR5 coreceptor have been identified as potential targets for interfering with receptor-env interactions. Any of these epitopes can be included in the polyepitope polypeptides described herein.

Construction of HIV-MCMV Immunogens

HIV-1-MCMV immunogen constructs comprised of a string of codon-optimized epitopes have been produced. The antigenic fragments/epitopes in examples of such constructs were selected using published studies including broad MHC allele recognition and were compiled from the Los Alamos sequence database. A representative pair of immunogen constructs (polyepitope polypeptides) contains multiple B-cell epitopes, CTL epitopes, and T-helper epitopes representing immunodominant regions for all subtypes of HIV-1 (see tables included in the examples, and FIGS. 1, 2, and 3, see Original Patent). The epitopes chosen are >80% homologous across diverse HIV-1 subtypes. B-cell epitopes in the virus binding domain of the human HIV coreceptor CCR5 are also included.

Without intending to be limited to a single interpretation, it is believed that antibodies to CCR5 together with neutralizing antibodies directed against the HIV-1 envelope glycoprotein and strong T-cell immunity will interfere with the viral entry process and is expected to induce sterilizing immunity.

Example immunogen constructs are shown in SEQ ID NOs: 2, 4, 5, 6, 8, and 10. The constructs shown in SEQ ID NOs: 2, 4, 5, and 6 include CTL epitopes (and therefore can be referred to generally as MCMV-CTL constructs); those in SEQ ID NOs: 8 and 10 include B-cell and T-helper epitopes (and therefore can be referred to generally as MCMV-AB/Th constructs).

Unique restriction enzyme digestion sites have been included in the nucleic acid constructs encoding the provided polyepitope polypeptides. These facilitate addition/deletion of epitopes, as well as the shuttling of the polyepitope cassette between a number of DNA vectors, including DNA vaccine constructs (for example, pVax-1, Invitrogen, Carlsbad, Calif.), eukaryotic yeast expression vectors (for example, pYes, Invitrogen, Carlsbad, Calif.), and multi-cell type expression vectors (for example, pTriEX-4, Novagen, Madison, Wis.). This enables the production of both a DNA based immunogen and vaccine, and ready production recombinant polyepitope polypeptide, which can be used directly as an immunogen or as a boost. The synthetic genes (which encode one or more polyepitope polypeptides) also can be incorporated into attenuated viral vectors such as Modified Vaccinia or Adenovirus to serve as a boosting agent.

Delivery and Immunogenicity by Inclusion of Targeting Sequences

Recent studies suggest that peptide spacers between epitopes and/or targeting sequences may increase the immunogenicity of certain epitopes. Targeting sequences such as the LIMP-II targeting sequence (which directs proteins to lysosomes and enhances class-II recognition), or targeting-competent fragments thereof, are used in certain provided embodiments to help enhance T-helper response. Likewise, proteosome targeting sequences (for example, ubiquitin or targeting-competent fragments thereof) that help induce class I recognition are included in specific embodiments, to provide improved CTL production. The chosen epitopes were back translated and human codon optimized for increased expression from the DNA construct.

In any of the described nucleic acids encoding polyepitope polypeptides, a spacer amino acid or spacer peptide can be included between any two adjacent segments of the construct. Optionally, in some embodiments the spacer is included between each epitope; in other embodiments, a spacer is included between every two, every three, every four, every five epitopes, or even less often. In particular embodiments, the spacer comprises three amino acids. Specific non-limiting examples of spacers are the tri-amino acid KAA and the tri-amino acid PGP.

Recognition of Epitopes Contained in the Constructs

Most vaccine constructs under development are subtype-specific. This has led to development of a number of country-specific subtype-specific HIV-1 vaccines, however, such vaccines will be difficult to implement due to emerging diversity and changing epidemic of HIV-1.

In contrast, the constructs provided herein comprise highly conserved immunogenic regions of HIV-1 that result in cross-protective immune responses across HIV-1 subtypes. The immune responses to the immunogenic epitopes can be tested, for instance, in recently-infected HIV-1 infected persons (Primary HIV-1 infection; PHI) or individuals that have a slow progression to disease.

V. Uses of MCMV Immunogens

In order to function effectively in vivo as a DNA-based immunogen, it is advantageous to include within the MCMV nucleic acid construct a control sequence that has the effect of enhancing or promoting the translation of the sequences encoding the antigens. Use of such promoters is well known to those of skill in the fields of molecular biology, cell biology, and viral immunology (See, "Molecular Cloning: A Laboratory Manual", 2nd Ed., Sambrook, Fritsch and Maniatis, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989; and "Current Protocols in Molecular Biology", Ausubel et al., John Wiley and Sons, New York 1987 (updated quarterly)).

In certain embodiments, the nucleic acid construct is intended for use as a vaccine in a mammalian host. Therefore it is advantageous to employ a promoter which operates effectively in mammalian cells. Particular embodiments relate to both prokaryotic and eukaryotic host cells. Many promoter sequences are known that are useful in either prokaryotic or eukaryotic host cells. A promoter is operably disposed with respect to the sequence(s) whose translation is to be promoted, so that it is capable of promoting translation. In certain embodiments, the promoter is the cytomegalovirus early promoter. In addition, in some embodiments, the sequences to be expressed are followed by a terminator sequence.

Preparation of the nucleic acids is readily accomplished by methods well known to workers of skill in the field of molecular biology. Procedures involved are set forth, for example, in Sambrook, Fritsch and Maniatis, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989, and "Current Protocols in Molecular Biology", Ausubel et al., John Wiley and Sons, New York 1987 (updated quarterly). Incorporation of promoters, such as the cytomegalovirus promoter, and of the polyadenylation signal, is likewise well known to skilled practitioners in molecular biology and recombinant DNA engineering.

When a nucleic acid molecule harboring a MCMV epitope chain is prepared, it may be obtained in larger quantities by methods that amplify a nucleic acid fragment. Such methods are widely known to workers skilled in molecular biology and recombinant DNA engineering. Examples of these methods include incorporation of the nucleic acid fragment into a plasmid for replication by culturing in a cell (for example, a prokaryotic cell) and harvesting the plasmid after growth of the culture, as well as amplification of the nucleic acid fragment by nucleic acid amplification methods, such as the PCR. These methods are exemplary only, and not intended to limit the ways in which the nucleic acid construct may be obtained.

The MCMV nucleic acid constructs may be introduced into appropriate host cells in many ways well known to those of ordinary skill in the fields of molecular biology and viral immunology. By way of example, these include, but are not limited to, incorporation into a plasmid or similar nucleic acid vector which is taken up by the host cells, or encapsulation within vesicular lipid structures such as liposomes, especially liposomes comprising cationic lipids, or adsorption to particles that are incorporated into the host cell by endocytosis.

In general, a host cell is a prokaryotic or eukaryotic cell harboring a MCMV nucleic acid, or into which such a MCMV molecule has been introduced. The constructs described herein induce the intracellular biosynthesis of the encoded multivalent HIV-1 antigens. A suitable host cell is one which has the capability for the biosynthesis of the gene products as a consequence of the introduction of the nucleic acid. In particular embodiments, a suitable host cell is one which responds to a control sequence and to a terminator sequence, if any, which may be included within the construct. In order to respond in this fashion, such a host cell contains within it components which interact with a control sequence and with a terminator, and act to carry out the respective promoting and terminating functions. When the host cell is cultured in vitro, it may be a prokaryote, a single-celled eukaryote or a vertebrate cell. In particular embodiments, the host cell is a mammalian cell

VI. Stimulation of Immunological Responses to HIV-1

With the provision herein of polyepitope polypeptide antigens specific to HIV-1, methods are now enabled for the stimulation of immune responses to such antigens in subjects. In certain embodiments, such immune responses will be protective against HIV-1 infection in the subject. MCMV polyepitope polypeptides (singly or in combination) can be used, for instance, as immunogenic agents in the inhibition, treatment, or amelioration of HIV-1. Subjects selected for this type of treatment are those who are known to have, or are suspected of having or are at risk of suffering, a HIV-1 infection.

The provided immunostimulatory MCMV polyepitope polypeptides, or constructs or vectors encoding such polypeptides, are combined with a pharmaceutically acceptable carrier or vehicle for administration as an immunostimulatory composition or a vaccine to human or animal subjects. In some embodiments, more than one polyepitope polypeptide may be combined to form a single preparation.

The immunogenic formulations may be conveniently presented in unit dosage form and prepared using conventional pharmaceutical techniques. Such techniques include the step of bringing into association the active ingredient and the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers. Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid carrier, for example, water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets commonly used by one of ordinary skill in the art.

In certain embodiments, unit dosage formulations are those containing a dose or unit, or an appropriate fraction thereof, of the administered ingredient. It should be understood that in addition to the ingredients particularly mentioned above, formulations encompassed herein may include other agents commonly used by one of ordinary skill in the art.

The compositions provided herein, including those for use as immunostimulatory agents or vaccines, may be administered through different routes, such as oral, including buccal and sublingual, rectal, parenteral, aerosol, nasal, intramuscular, subcutaneous, intradermal, and topical. They may be administered in different forms, including but not limited to solutions, emulsions and suspensions, microspheres, particles, microparticles, nanoparticles, and liposomes.

The volume of administration will vary depending on the route of administration. By way of example, intramuscular injections may range from about 0.1 ml to about 1.0 ml. Those of ordinary skill in the art will know appropriate volumes for different routes of administration.

The amount of protein in each vaccine dose is selected as an amount that induces an immunostimulatory or immunoprotective response without significant, adverse side effects. Such amount will vary depending upon which specific immunogen is employed and how it is presented. Initial injections may range from about 1 .mu.g to about 1 mg, with some embodiments having a range of about 10 .mu.g to about 800 .mu.g, and still other embodiments a range of from about 25 .mu.g to about 500 .mu.g. Following an initial vaccination, subjects may receive one or several booster immunizations, adequately spaced. Booster injections may range from about 1 .mu.g to about 1 mg, with other embodiments having a range of about 10 .mu.g to about 750 .mu.g, and still others a range of about 50 .mu.g to about 500 .mu.g. Periodic boosters at intervals of 1-5 years, for instance three years, may be desirable to maintain the desired levels of protective immunity.

As described in WO 95/01441, the course of the immunization may be followed by in vitro proliferation assays of PBL (peripheral blood lymphocytes) co-cultured with ESAT6 or ST-CF, and especially by measuring the levels of IFN-released from the primed lymphocytes. The assays are well known and are widely described in the literature, including in U.S. Pat. Nos. 3,791,932; 4,174,384 and 3,949,064.

A relatively recent development in the field of immune stimulatory compounds (for example, vaccines) is the direct injection of nucleic acid molecules encoding peptide antigens (broadly described in Janeway & Travers, Immunobiology: The Immune System In Health and Disease, page 13.25, Garland Publishing, Inc., New York, 1997; and McDonnell & Askari, N. Engl. J. Med. 334:42-45, 1996). Plasmids (vectors) that include nucleic acid molecules described herein, or that include a nucleic acid sequence encoding an immunogenic MCMV polyepitope polypeptide may be utilized in such DNA vaccination methods.

Thus, the terms "immunostimulatory preparation" and "vaccine" as used herein also include nucleic acid vaccines in which a nucleic acid molecule encoding a MCMV polyepitope polypeptide is administered to a subject in a pharmaceutical composition. For genetic immunization, suitable delivery methods known to those skilled in the art include direct injection of plasmid DNA into muscles (Wolff et al., Hum. Mol. Genet. 1:363, 1992), delivery of DNA complexed with specific protein carriers (Wu et al., J. Biol. Chem. 264:16985, 1989), co-precipitation of DNA with calcium phosphate (Benvenisty and Reshef, Proc. Natl. Acad. Sci. 83:9551, 1986), encapsulation of DNA in liposomes (Kaneda et al., Science 243:375, 1989), particle bombardment (Tang et al., Nature 356:152, 1992; Eisenbraun et al., DNA Cell Biol. 12:791, 1993), and in vivo infection using cloned retroviral vectors (Seeger et al., Proc. Natl. Acad. Sci. 81:5849, 1984).

Similarly, nucleic acid vaccine preparations can be administered via viral carrier.

It is also contemplated that the provided immunostimulatory molecules and preparations can be administered to a subject indirectly, by first stimulating a cell in vitro, which stimulated cell is thereafter administered to the subject to elicit an immune response.

VII. Immunological and Pharmaceutical Compositions

Immunological compositions, including immunological elicitor compositions and vaccines, and other pharmaceutical compositions containing latency-specific polypeptides or antigenic fragments thereof are useful for reducing, ameliorating, treating, or possibly preventing HIV infection, particularly HIV-1 infection. One or more of the polypeptides are formulated and packaged, alone or in combination with adjuvants or other antigens, using methods and materials known to those skilled in the vaccine art. An immunological response of a subject to such an immunological composition may be used therapeutically or prophylactically, and in certain embodiments provides antibody immunity and/or cellular immunity such as that produced by T-lymphocytes, such as cytotoxic T-lymphocytes or CD4.sup.+ T-lymphocytes.

The MCMV polyepitope polypeptides may be administered with an adjuvant in an amount effective to enhance the immunogenic response against the conjugate. At this time, the only adjuvant widely used in humans has been alum (aluminum phosphate or aluminum hydroxide). Saponin and its purified component Quil A, Freund's complete adjuvant and other adjuvants used in research and veterinary applications have toxicities which limit their potential use in human vaccines. However, chemically defined preparations such as muramyl dipeptide, monophosphoryl lipid A, phospholipid conjugates such as those described by Goodman-Snitkoff et al. (J. Immunol 147:410415, 1991), encapsulation of the conjugate within a proteoliposome as described by Miller et al. (J. Exp. Med 176:1739-1744, 1992), and encapsulation of the protein in lipid vesicles may also be useful.

The compositions provided herein, including those formulated to serve as vaccines, may be stored at temperatures of from about -100.degree. C. to about 4.degree. C. They may also be stored in a lyophilized state at different temperatures, including higher temperatures such as room temperature. The preparation may be sterilized through conventional means known to one of ordinary skill in the art. Such means include, but are not limited to, filtration, radiation and heat. The preparations also may be combined with bacteriostatic agents, such as thimerosal (ethyl(2-mercaptobenzoate-S)mercury sodium salt) (Sigma Chemical Co., St. Louis, Mo.), to inhibit bacterial growth.

A variety of adjuvants known to one of ordinary skill in the art may be administered in conjunction with the protein(s) in the provided vaccine composition. Such adjuvants include but are not limited to the following: polymers, co-polymers such as polyoxyethylene-polyoxypropylene copolymers, including block co-polymers; polymer P1005; Freund's complete adjuvant (for animals); Freund's incomplete adjuvant; sorbitan monooleate; squalene; CRL-8300 adjuvant; alum; QS 21, muramyl dipeptide; CpG oligonucleotide motifs and combinations of CpG oligonucleotide motifs; trehalose; bacterial extracts, including mycobacterial extracts; detoxified endotoxins; membrane lipids; or combinations thereof.

In a particular embodiment, a vaccine is packaged in a single dosage for immunization by parenteral (that is, intramuscular, intradermal or subcutaneous) administration or nasopharyngeal (that is, intranasal) administration. In certain embodiments, the vaccine is injected intramuscularly into the deltoid muscle. The vaccine may be combined with a pharmaceutically acceptable carrier to facilitate administration. The carrier is, for instance, water, or a buffered saline, with or without a preservative. The vaccine may be lyophilized for resuspension at the time of administration or in solution.

The carrier to which the polypeptide may be conjugated may also be a polymeric delayed release system. Synthetic polymers are particularly useful in the formulation of a vaccine to affect the controlled release of antigens.

Microencapsulation of the polypeptide will also give a controlled release. A number of factors contribute to the selection of a particular polymer for microencapsulation. The reproducibility of polymer synthesis and the microencapsulation process, the cost of the microencapsulation materials and process, the toxicological profile, the requirements for variable release kinetics and the physicochemical compatibility of the polymer and the antigens are all factors that must be considered. Examples of useful polymers are polycarbonates, polyesters, polyurethanes, polyorthoesters, polyamides, poly-(d,1-lactide-co-glycolide) (PLGA), and other biodegradable polymers.

Doses for human administration of a pharmaceutical composition or a vaccine may be from about 0.01 mg/kg to about 10 mg/kg, for instance about 1 mg/kg. Based on this range, equivalent dosages for heavier (or lighter) body weights can be determined. The dose may be adjusted to suit the individual to whom the composition is administered, and may vary with age, weight, and metabolism of the individual, as well as the health of the subject. Such determinations are left to the attending physician or another familiar with the subject and/or the specific situation. The vaccine may additionally contain stabilizers or physiologically acceptable preservatives, such as thimerosal.
 

Claim 1 of 10 Claims

1. An isolated recombinant polyepitope polypeptide comprising an amino acid sequence selected from the group consisting of sequences recited in SEQ ID NOs: 2, 4, 5, 6, 8, 10, and combinations of two or more thereof.

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