Title:  Methods for vaccination and vaccines therefor

United States Patent:  6,689,757

Issued:  February 10, 2004

Inventors:  Craig; Roger K. (Cheshire, GB)

Assignee:  M.L. Laboratories PLC (London, GB)

Appl. No.:  221050

Filed:  December 28, 1998

Abstract

The invention relates to methods of and compositions for vaccinating a mammal against a disease, wherein a mixture is administered which includes (i) a nucleic acid which encodes a first epitope and (ii) a peptide containing a second epitope such that both of the nucleic acid and the second epitope are taken up by and the nucleic acid is expressed in a professional antigen presenting cell of the mammal, and the first and second epitopes are processed in the cell such that an immune response is elicited in the mammal to the epitopes.

DETAILED DESCRIPTION OF THE INVENTION

The invention is based on the observation that an immune response to an antigen may be rendered highly specific and effective by delivery to antigen presenting cells of a mixture comprising an antigen in its peptide or polypeptide form and a nucleic acid encoding an antigen.

The invention also is based on the observation that an immune response to an antigen may be rendered highly effective by delivery to antigen presenting cells of a complex comprising an antigen in its peptide or polypeptide form and a nucleic acid encoding an antigen.

The invention also is based on the observation that an immune response to an antigen may be rendered highly effective by delivery to antigen presenting cells of a complex comprising an antigen in its peptide or polypeptide form and a nucleic acid. In this aspect of the invention, the nucleic acid need not encode an antigen, but is useful to promote uptake of the antigen by the antigen presenting cell.

In the simplest form, the peptide antigen and the nucleic acid encoded antigen described herein are the same. However, it is believed that a more effective immune response may be obtained using a first peptide antigen in combination with a second different nucleic acid-encoded antigen, or wherein several different peptide antigens are administered in combination with one or several different nucleic acid-encoded antigens. A "more effective" immune response will be evident, as it relates to prior art vaccination procedures and compositions, as a two-fold and preferably a five-fold to ten-fold higher immune response, or by the finding that both a cellular and a humoral immune response is elicited by complexes or mixtures of the invention. Methods and compositions for making and carrying out the invention are described in detail below.

VACCINATION VIA ANTIGEN PRESENTING CELL-RESTRICTED GENE

Expression According to the Invention

The invention provides for vaccination against a disease or pathogen via administration to a mammal of a mixture or complex described according to the invention and, in some cases, of antigen presenting cell--(i.e., tissue--) restricted expression of a nucleic acid encoding an antigen associated with the disease or pathogen. Tissue-restricted expression of a gene encoding an antigen for which an immune response is desired, wherein the tissue to which expression is restricted comprises antigen presenting cells, as defined herein or subsets thereof is obtained as follows.

Two modes of effective antigen presenting cell-restricted gene expression are contemplated according to the invention: 1) via targeted delivery of a mixture or complex comprising an antigenic peptide and a gene (preferably encoding an antigen for which an immune response is desired) to antigen presenting cells, and 2) via either targeted or untargeted gene delivery (and thus possible delivery of the gene to different cell types) wherein control of expression of the gene is effected using genetic control elements which limit gene expression to the desired antigen presenting cells or subset of cells.

Delivery of Nucleic Acid to Host Cell

It is contemplated according to the invention that a mixture or complex according to the invention may be delivered to an antigen presenting host cell non-specifically or specifically (i.e., to a designated subset of host cells). Three modes of delivery are contemplated. First, wherein no targeting ligand is used. Second and third, where a targeting ligand is employed; in the case of non-specific delivery to cells, a non-specific ligand is used that targets a cell surface receptor that is present on the target cell population as well as on other cells; in the case of specific delivery, a ligand is used that targets a specific subset of cells. Therefore, the complex or mixture may be delivered generally to any cell, or may be delivered to antigen presenting cells, or may be delivered to a subset of antigen presenting cells, these subsets and targeting ligands and cognate receptors being described herein.

1. Non-viral Delivery to APCs

Non-viral delivery to APCs may or may not employ a targeting ligand. Targeted delivery to APCs, their stem cells, or other precursor cell types can be achieved by receptor-mediated gene transfer using a complex containing a ligand which is targeted to a cognate receptor on a cell surface. Targeting ligands useful according to the invention include but are not limited to the following: (a) for hemopoietic stem cells: anti-CD34 monoclonal antibody, or the Stem cell factor (c-Kit or CD117), or flk-2 ligand (human homolog STK-1); (b) for monocyte/macrophage/dendritic cell precursors: anti-CD33 monoclonal antibody; (c) for differentiated macrophage/dendritic cells: glycosylated DNA binding peptides carrying mannose groups may be used to target to specific receptors, for example the mannose receptor; and (d) for MHC class II bearing cells: an antibody that is specific for the constant region of MHC class II proteins or a ligand that binds MHC class II, for example soluble CD4; for example, one subset of MHC class II-bearing cells, B lymphocytes, may be targeted using soluble CD4 or using antibodies to or ligands for CD80, CD19, or CD22; for endothelial cells, y-interferon; and (e) for APCs or T cells, co-stimulatory molecules such as B7-1, B7-2 or CD28, CTLA-4, respectively.

Targeted delivery vehicles for delivery of DNA constructs to cells are known in the art and include DNA/polycation complexes which are specific for a cell surface receptor, as described in, for example, Wu and Wu (1988) J. Biol. Chem 263:14621; Wilson et al. (1992) T. Biol. Chem. 267:963-967, and U.S. Pat. No. 5,166,320, and, for example, U.S. Ser. No. 60/011,531, assigned to the same assignee and hereby incorporated by reference. In this co-pending application, a self-assembling virus-like particle is described and includes the DNA of interest and condensing peptides which are heteropeptides with respect to their amino acid composition (i.e., containing at, least two different amino acids which are preferably basic and thus good DNA binding and DNA condensing peptides) and which have low polydispersion (i.e., a given preparation of a heteropeptide which has low polydispersion contains peptides of very similar, if not identical lengths, such that the preparation is essentially monodispersed).

The invention thus also relates to a nucleic acid construct which is delivered to a cell using a synthetic virus like particle for transfecting nucleic acid into a mammalian cell. The synthetic virus like particle includes a recombinant nucleic acid, a plurality of nucleic acid condensing peptides, the peptides being non-covalently associated with the recombinant nucleic acid such that the nucleic acid is in condensed form, wherein each nucleic acid condensing peptide is a heteropeptide, and plurality of nucleic acid condensing peptides has low polydispersion.

The plural nucleic acid condensing peptides may include a first nucleic acid condensing peptide and a second nucleic acid condensing peptide, wherein the first nucleic acid condensing peptide comprises a first functional group covalently bound thereto. The first nucleic acid condensing peptide may further include a second functional group which may be directly bound to the peptide or may be covalently bound to the first functional group, where the first functional group is bound to the peptide.

Alternatively, a second nucleic acid condensing peptide also may include a second functional group covalently bound thereto, the second functional group being different from the first functional group. The first and second nucleic acid condensing peptides may have identical or different amino acid sequences.

The functional groups which are bound to peptides useful according to the invention include antigenic peptides or proteins, such as influenza nucleoprotein (NP), or a ligand that targets a specific cell-type such as a monoclonal antibody, insulin, transferrin, asialoglycoprotein, or a sugar. The ligand thus may target cells in a non-specific manner or in a specific manner that is restricted with respect to cell type.

The first nucleic acid condensing peptide may include 8-24 positively charged amino acid side groups; for example, the number of positively charged amino acid side groups may be in the range of 12-18.

The ratio of positive/negative charges in a synthetic virus like particle that is capable of targeting a specific mammalian cell type is within the range 0.5-3 per phosphate residue in the nucleic acid; this ratio thus also may be within the range 0.8-1.2.

The ratio of positive/negative charges in a synthetic virus like particle that is unrestricted with respect to the type of cell it targets is in within the range of 0.5-5 per phosphate residue in the nucleic acid, and thus also may be within the range of 1.2-2.

A nucleic acid condensing peptide which is particularly useful for condensing the nucleic acid construct and therefore for delivering nucleic acid to a cell includes a peptide of the generic formula

NH2--A--(X₁ X₂ Y₁ Y₂)_n X₃ X₄ --(Z₁ Z₂ Z₃ Z₄)--(X₅ X₆ Y₃ Y₄)_m X₇ X₈ BCOOH

wherein each of X_1-8 is, independently, an amino acid having a positively charged group on the side chain; wherein each of Y_1-4 is, independently, a naturally occurring amino acid which promotes alpha helix formation; wherein each of Z_1-4 is, independently, a naturally occurring amino acid with at least 3 amino acids having a high propensity to form a stabilized turn structure; wherein A is an amino-terminal serine or threonine residue; wherein B is any amino acid and wherein n=2-4 and m=2.

Other peptides are those wherein each of X, ₈ is, independently, lysine, arginine, 2,4-diamino-butyric acid or omithine; wherein each of Y_1-4 is, independently, glutamic acid, alanine, leucine, methionine, glutamine, tryptophan or histidine; wherein each of Z_1-4 is, independently, asparagine, glycine, proline, serine, or aspartic acid; wherein B is any one of alanine, glutamic acid or cysteine.

It is also contemplated according to the invention that peptides useful in this embodiment of the invention which involves delivery of a complex according to the invention to a cell either ex vivo or in vivo may contain one or more internal Serine, Threonine, or Cysteine residues, preferably at a position in the sequence which will be exposed for conjugation to a selected ligand, and thus not on the positively charged (nucleic acid oriented) face of the a-helix. This positioning of selected reactive amino acid residues within the peptide are oriented such that they do not contact the face of the peptide that contacts nucleic acid permits conjugation of the peptide with other functional peptides by bonds of selected and defined stability. Cysteine allows specific conjugation via the thiol side chain to compounds containing other reactive thiol groups (via disulfide), alkylating functions (to form thioether bonds), or other thiol reactive groups such as maleimide derivatives.

Peptides which fall within this generic sequence include: NBC7 TRRAWRRAKRRAARRCGVSARRAARRAWRRE-OH; (SEQ ID NO:1) and, NBC11 H-TKKAWKKAEKKAAKKCGVSAKKAAKKAWKKA-NH₂. (SEQ ID NO:2) Thus, a nucleic acid condensing peptide useful for delivery of a nucleic acid may contain: 1) helix-forming amino acids, 2) a repeating three-dimensional structure that contacts the major groove of the nucleic acid, 3) suitable chromophores for quantitation, and 4) a number of "handles" (i.e., reactive sites) for regio-specific conjugation of ligands which form accessory functional domains.

Nucleic acid condensing peptides also may include portions of HI (sequence I, II or III below) which are identified herein as sequences which possess the ability to condense nucleic acid. Therefore, a nucleic acid condensing peptide can include a linear combination of the following three consensus sequences where the total sequence length is>17 residues:

Sequence I: -K-K-X-P-K-K-Y-Z-B-P-A-J (SEQ ID NO:3) where: K is Lysine, P is Proline; A is Alanine; X is Serine, Threonine or Proline; Y is Alanine or Valine; Z is Alanine, Threonine or Proline; B is Lysine, Alanine, Threonine or Valine; and J is Alanine or Valine.

Sequence II---X-K-S-P-A-K-A-K-A- (SEQ ID NO:4) where: X is Alanine or Valine; K is Lysine; S is Serine; P is Proline; and A is Alanine.

Sequence III: -X-Y-V-K-P-K-A-A-K-Z-K-B (SEQ ID NO:5) where: X is tysine or Arginine; Y is Alanine or Threonine; Z is Proline, Alanine or Serine; B is Lysine, Threonine or Valine; K is Lysine; P is Proline; A is Alanine.

One such peptide is NBC1, which has the following structure:

NH2-[SV40 NLS]-[Seq I]-[Seq II]-[Seq III]-[SV40 NLS]-[Seq I]-C--COOH, where --C-- is Cysteine; where the SV40 NLS has the sequence Pro-Lys-Lys-Lys-Arg-Lys-Val-Gln (SEQ ID NO:6); and the sequence H-PKKKRKVEKKSPKKAKKPAAKSPAKAKAKAVKPKAAKPKKPKKKRKVEKKSP KKAKKPAAC (Acm)-OH. (SEQ ID NO:7)

Another such nucleic acid condensing peptide of the invention will have a peptide that falls within the following generic sequence: NH2-X-(Y)_n --C--COOH, where X is either absent or Serine or Threonine; Y is sequence I, II or III as defined above; n is 2-6; and C is Cysteine.

Other such peptides have the following structures and sequences:

NBC2 has the structure: NH₂ -[Seq III][SV40 NLS1]-[Seq I]-C--COOH, where --C-- is Cysteine.

NBC8 has the structure: NH₂ -[Seq I]-[Seq I]-C--COOH, where --C-- is Cysteine.

NBC9 has the structure: NH₂ -[Seq I]-[Seq I]-[Seq I]-C--COOH, where --C-- is Cysteine.

NBC10 has the structure: NH₂ -[Seq I]-[Seq I]-[Seq I]-[Seq I]-C--COOH

where --C-- is Cysteine; the amino acid sequences of which are as follows:

NBC2 H-KAVKPKAAKPKKPKKKRKVEKKSPKKAKKPAAC(Acm)-OH (SEQ ID NO:8);

(NBC8 H-KKSPKKAKKPAAKKSPKKAKKPAAC(Acm)-OH (SEQ ID NO:9);

NBC9 H-KKSPKKAKKPAAKKSPKKAKKPAAKKSPKKAKKPAAC(Acm)-OH (SEQ ID NO:10);

NBC10 -KKSPKKAKKPAAKKSPKKAKKPAAKKSPKKAKKPAAKKSPKKAKKP (Acm)-OH (SEQ ID NO:11);

As described above, nucleic acid condensing peptides having a low polydispersion index (PDI) are useful for delivery to a cell of a nucleic acid encoding an antigen according to the invention. The PDI for such peptides may be calculated from analysis of the peptides by electro-spray mass spectrometry. This method gives the exact mass of each component to within 0.001%. The PDI values of the peptide preparations useful in the present invention are in the range of 1.0-1.100. Peptide preparations which are especially useful in the invention possess a PDI<1.01, and even <1.001.

Preferred delivery vehicles are prepared as follows. A synthetic virus like particle is formulated such that the nucleic acid encoding an antigen and the peptide preparation are prepared in equal volumes of the same buffer. The nucleic acid is shaken or vortexed while the condensing peptide preparation is added at the rate of 0.1 volume per minute. The complex is left at room temperature for at least 30 minutes prior to addition to the target cells or prior to administration to a subject, and can be stored at 4^oC. The particle is centrifuged to remove any aggregated material.

In addition to the above-described DNA/polycation complexes for cell targeting, methods are known in the prior art for preparing cell-targeting liposomes containing nucleic acid. An example of targeting liposomes is immunoliposomes, which are prepared, for example, by adsorption of proteins (e.g., immunoglobulin) on the liposomal surface; incorporation of native protein into the liposome membrane during its formation (e.g., by ultrasonication, detergent dialysis or reverse phase evaporation); covalent binding (direct or via a spacer group) of a protein to reactive compounds incorporated into the liposomes membrane; noncovalent hydrophobic binding of modified proteins during liposome formation or by the incubation with preformed liposomes); and indirect binding, including covalent binding of immunoglobulin protein via a polymer to the liposome (see Torchilin, V. P. CRC Critical reviews in Therapeutic Drug Carrier Systems, vol. 2(1)). Binding of the nucleic acid-ligand complex to the receptor facilitates uptake of the nucleic acid by receptor-mediated endocytosis.

A nucleic acid-ligand complex linked to adenovirus capsids, which naturally disrupt endosomes, thereby releasing material into the cytoplasm, can be used to avoid degradation of the complex by intracellular lysosomes (see for example Curiel et al. (1991) Proc. Natl. Acad. Sci. USA 88:8850; Cristiano et al. (1993) Proc. Natl. Acad. Sci. USA 90:2122-2126).

Receptor-mediated nucleic acid uptake can be used to introduce nucleic acid into cells either in vitro or in vivo and, additionally, has the added feature that nucleic acid can be selectively targeted to a particular cell type by use of a ligand which binds to a receptor selectively expressed on a target cell of interest, or can be non-selective with respect to the target cell type.

The precise stoichiometric ratio of the various components of the complex or mixture according to the invention can be varied in order to control the magnitude of the initial immune response, the efficiency of delivery and the degree of specific targeting to APCs or related cells. Generally, the ratio of the nucleic acid encoding a first epitope to the amino acid sequence encoding the second epitope will be in the range of 1:10,000 to 1,000:1, with a preferred range of 1:1,000 to 100:1, and a most preferred range of 1:200 to 10:1.

A) Nucleic Acid Vectors Useful for Non-viral Delivery

The invention contemplates the use of a vector containing the gene of interest (i.e., the gene encoding an antigen for which an immune response is desired). The vector may be carried in a delivery vehicle which is targeted or untargeted for cell delivery, as described above. Vectors useful according to the invention will include vectors that integrate into host cell nuclear DNA or stable episomal vectors.

Episomal Vectors

Extrachromosomal replicators, generally, in addition to their origin function, encode functions that assure equal distribution of replicated molecules between daughter cells at cell division. In higher organisms, different mechanisms exist for partitioning of extrachromosomal replicators. For example, artificial (ARS-containing) plasmids in yeast utilize chromosomal centromeres as extrachromosomal replicators (Struhl et al., 1979, Proc. Natl. Acad. Sci. USA, 76:1035-1039). In metazoan cells, one well studied example of a stable extrachromosomal replicator exists--the latent origin oriP from Epstein-Barr Virus (EBV). The maintenance function of EBV requires the viral replication factor EBNA-1 and a series of binding sites for EBNA-1 termed the family of repeats (FR). Replication from oriP requires cis-acting elements (the Family of Repeats--FR and the dyad symmetry element) and the viral origin-binding protein, EBNA-1 (Yates et al., Proc. Natl. Acad Sci. USA, 81, 3806-3810 (1984); Yates et al., Nature 313:812-815 (1985)). FR has an effect on the stable extrachromosomal replication of the oriP by nuclear retention of the FR containing plasmids in mitosis. This activity directs plasmids into the newly forming nucleus in the telophase stage of the cell division (Krysan et al., Mol. Cell. Biol. 9:1026-1033 (1989)).

Particularly preferred vectors useful according to the invention are maintained at a high copy number in dividing and non-dividing cells of a patient. This may be achieved by employing an episomal vector such as the BPV-1 vector system described in WO 94/12629 and in Piirsoo et al., 1996, EMBO Jour. 15:1, comprising a plasmid harboring the BPV-1 origin of replication (minimal origin plus minichromosomal maintenance element) and optionally the E1 and E2 genes. The BPV-1 E1 and E2 genes are required for stable maintenance of a BPV episomal vector. These factors ensure that the plasmid is replicated to a stable copy number of up to thirty copies per cell independent of cell cycle status. The gene construct therefore persists stably in both dividing and non-dividing cells. This allows the maintenance of the gene construct in cells such as hemopoietic stem cells and more committed precursor cells.

"Minimal origin of replication" (MO) refers to a minimal cis-sequence within a papilloma virus that is necessary for initiation of DNA synthesis. The MO of BPV-1 is located at the 3' end of the upstream regulatory region within a 60 base pair DNA fragment (7914-7927) including an AT-rich region, a consensus sequence to which all papilloma viral E2 proteins bind, and an E1 protein binding site spanning nucleotide 1. The MO of HPV is located in the URR fragment (nt 7072-7933/1-99) (Chiang et al. Px,oc. Natl. Acad. Sci. USA 1992).

"E1" refers to the protein encoded by nt 849-2663 of BPV subtype 1; or to nt 832-2779 of HPV of subtype 11, or to equivalent E1 proteins of other papillomaviruses, or to functional fragments or mutants of a papillomavirus E1 protein, i.e., fragments or mutants of E1 which possess the replicating properties of E1.

"E2" refers to the protein encoded by nt 2594-3837 of BPV subtype 1; or to nt 2723-3823 of HPV subtype 11, or to equivalent E2 proteins of other papillomaviruses, or to functional fragments or mutants of a papillomavirus E2 protein, i.e., fragments or mutants of E2 which possess the replicating properties of E2.

"Minichromosomal maintenance element, (MME) refers to a region of the papilloma viral genome to which viral or human proteins essential for papilloma viral replication bind, which region is essential for stable episomal maintenance of the papilloma viral MO in a host cell, as described in Piirsoo et al. Preferably, the MME is a sequence containing multiple binding sites for the transcriptional activator E2. The MME in BPV is herein defined as the region of BPV located within the upstream regulatory region which includes a minimum of about six sequential E2 binding sites, and which gives optimum stable maintenance with about ten sequential E2 binding sites. E2 binding site 9 is a preferred sequence for this site, as described hereinbelow, wherein the sequential sites are separated by a spacer of about 4-10 nucleotides, and optimally 6 nucleotides. E1 and E2 can be provided to the plasmid either in cis or in trans, also as described in WO 94/12629 and in Piirsoo et al. "E2 binding site" refers to the minimum sequence of papillomavirus double-stranded DNA to which the E2 protein binds. An E2 binding site may include the sequence 5' ACCGTTGCCGGT 3', (SEQ NO:12) which is high affinity E2 binding site 9 of the BPV-1 URR; alternatively, an E2 binding site may include permutations of binding site 9, which permutations are found within the URR, and which consist essentially of the consensus sequence 5'ACCN6GGT3', (SEQ NO:13) where N is, independent of its position, any nucleotide, and 6 refers to six independent nucleotides (N). One or more transcriptional activator E2 binding sites are, in most papillomaviruses, located in the upstream regulatory region, as in BPV and HPV.

A vector useful according to the invention may include a region of BPV between 6959-7945/1-470 on the BPV genetic map (see WO 94/12629), which region includes an origin of replication, a first promoter operatively associated with a gene encoding an antigen or epitope thereof, the BPV E1 gene operatively associated with a second promoter to drive transcription of the E1 gene; and the BPV E2 gene operatively associated with a third promoter to drive transcription of the E2 gene.

The promoters which drive expression of the E1 and E2 genes may be identical or different, and may be a tissue-specific promoter, such as the immunoglobulin, heavy chain promoter/enhancer for B-cell and the heavy or light chain promoters for blood cell expression, or from ubiquitously expressed genes, for example from the phosphoglycerolkinase, IE-CMV, RSV-LTR or DHFR genes. The arrangement of E1 and E2 genes relative to the BPV origin of replication may mimic the natural orientations of the sequences in the BPV genome, or it may assume a variety of other orientations, the choices of which will be apparent to one of skill in the art One skilled in the art will recognize that a variety of vectors will work according to the invention.

2. Delivery to APCs Mediated by Viral Vectors

In another preferred approach for introducing nucleic acid and an antigen into an antigen presenting cell, a viral vector containing the nucleic acid is used for transfer. Infection of cells with a viral vector has the advantage that a large proportion of cells receive the nucleic acid. Additionally, molecules encoded within the viral vector are expressed efficiently in cells which have taken up the vector nucleic acid.

1. Retroviruses: Defective retroviruses are well characterized for use in gene transfer for gene therapy purposes (for a review see Miller, A. D. (1990 Blood 76:271). A recombinant retrovirus can be constructed having a nucleic acid encoding an antigen of interest inserted into the retroviral genome. Additionally, portions of the retroviral genome can be removed to render the retrovirus replication defective. The replication defective retrovirus is then packaged into virions which can be used to infect a target cell through the use of a helper virus by standard techniques. Protocols for producing recombinant retroviruses and for infecting cells in vitro or in vivo with such viruses can be found in Current Protocols in Molecular Biology, Ausubel, F. M. et al. (eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14, and other standard laboratory manuals. Examples of suitable retroviruses include pLJ, pZIP, pWE and pEM which are well known to those skilled in the art. Examples of suitable packaging virus lines include .psi.Crip, .psi.Cre, .psi.2, and .psi.Am. Retroviruses have been used to introduce a variety of genes into many different cell types, including epithelial cells, endothelial cells, lymphocytes, myoblasts, hepatocytes, bone marrow cells, in vitro and/or in vivo (see for example Eglitis, et al. (1985) Science 230:1395-1398; Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464; Wilson et al. (1988) Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et al. (1990) Proc. Natl. Acad. Sci. USA 87:6141-6145; Huber et al. (1991) Proc. Natl. Acad. Sci. USA 88:8039-8043; Ferry et al. (1991) Proc. Natl. Acad Sci. USA 88:8377-8381; Chowdhury et al. (1991, Science 254:1802-1805; van Beusechem et al. (1992) Proc. Natl. Acad Sci. USA 89:7640-7644; Kay et al. (1992) Human Gene Therapy 3:641-647; Dai et al. (1992) Proc. Natl. Acad Sci. USA 89:10892-10895; Hwu et al. (1993) J. Immunol. 150:4:104-115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCT Application WO 89/07136; PCT Application WO 89/02468; PCT Application WO 89/05345; and PCT Application WO 92/07573).

2. Adenoviruses: The genome of an adenovirus can be manipulated such that it encodes and expresses a gene product of interest but is inactivated in terms of its ability to replicate in a normal lytic viral life cycle. See for example Berkner et al. (1988) BioTechniques 6:616; Rosenfeld et al. (1991) Science 252:431-434; and Rosenfeld et al. (1992) Cell 68:143-155. Suitable adenoviral vectors derived from the adenovirus strain Ad type 5 d1324 or other strains of adenovirus (e.g., Adz, Ad3, Ad7 etc.) are well known to those skilled in the art. Recombinant adenoviruses are advantageous in that they do not require dividing cells to be effective gene delivery vehicles and can be used to infect a wide variety of cell types, including airway epithelium (Rosenfeld et al. (1992) cited supra), endothelial cells (Lemarchand et al. (1992) Proc. Natl. Acad. Sci. USA 89:6482-6486), hepatocytes (Herz and Gerard (1993) Proc. Natl. Acad. Sci. USA 90:2812-2816) and muscle cells (Quantin et al. (1992) Proc. Natl. Acad. Sci. USA 89:2581-2584). Many replication-defective adenoviral vectors are deleted for all or parts of the viral E1 and E3 genes but retain as much as 80% of the adenoviral genetic material.

3. Adeno-Associated Viruses: Adeno-associated virus (AAV) is a naturally occurring defective virus that requires another virus, such as an adenovirus or a herpes virus, as a helper virus for efficient replication and a productive life cycle. (For a review see Muzyczka et al. Curr. Topics in Micro. and Immunol. (1 992) 158:97-129). It exhibits a high frequency of stable integration (see for example Flotte et al. (1992) Am. J. Respir. Cell. Mol. Biol. 7:349-356; Samulski et al. (1989) J. Virol. 63:3822-3828; and McLaughlin et al. (1989) J. Virol 62:1963-1973). Vectors containing as little as 300 base pairs of AAV can be packaged and can integrate. Space for exogenous nucleic acid is limited to about 4.5 kb. An AAV vector such as that described in Tratschin et al. (1985) Mol. Cell. Biol. 5:3251-3260 can be used to introduce nucleic acid into cells. A variety of nucleic acids have been introduced into different cell types using AAV vectors (see for example Hermonat et al. (1984) Proc. Natl. Acad. Sci. USA 81 :6466-6,470; Tratschin et al. (1985) Mol. Cell. Biol. 4:2072-2081; Wondisford et al. (1988) Mol. Endocrinol. 2:32-39; Tratschin et al. (1984) J. Virol.51 :611 619; and Flotte et al. (1993) J. Biol. Chem. 268:3781-3790).

The delivery vehicle also may comprise a viral antigen such as any of those described above, wherein one or more of the viral proteins has been modified to include an epitope(s) to which an immune response is desired.

3. Tissue-restricted Gene Expression

Alternatively or in addition to the use of targeted delivery vehicles, DNA regulatory elements which lead to expression in APCs, their stem cells or other precursor cell types can be used according to the invention.

Tissue specific expression is provided according to the invention using genetic control elements which restrict expression of the gene with which the element is associated to a tissue for which the element is specific. Examples of such genetic control elements include locus control regions and tissue-specific promoters and enhancers.

Locus Control Regions (LCRS) (Grosveld et al., Cell 51:975-985, 1987), also known as Dominant Activator Sequences, Locus Activating Regions or Dominant Control Regions, are responsible for conferring tissue specific, integration-site independent, copy number dependent expression on transgenes integrated into chromatin in host cells. First discovered in the human globin gene system, which was prone to strong position effects when integrated into the chromatin of transgenic mice or mouse erythro-leukaemia (MEL) cells (Magram et al., Nature 315:338-340, 1985; Townes et al., EMBO J. 4:1715-1723, 1985; Kollias et al., Cell 46:89-94, 1986; Antoniou et al., EMBO J. 7:377-384, 1988), LCRs have the ability to overcome such position effects when linked directly to transgenes (Grosveld et al., supra). Numerous LCRs have been defined in the art, including but not limited to the 0-globin and CD2 LCRs (Greaves et al., 1989), the macrophagespecific lysozyme LCR. (Bonifer et al., 1985, 1990), and a class II MHC LCR (Carson et al., Nucleic Acids Res. 21, 9:2065-2072, 1993).

Preferred regulatory elements include locus control regions (LCRS) such as the MHC class II LCR. Other control regions, for example, the Ig LCR for B cells, may also be used provided they lead to expression in APCs, their stem cells or other precursor cell types.

Vectors encoding elements permitting immune system evasion. Vectors useful according to the invention also may encode a sequence which serves to protect certain antigenic determinants encoded by the vector from viability to the host immune system. Thus, if the host mounts an immune response to determinants encoded by the vector, or carried on a vehicle used to deliver the nucleic acid to the antigen presenting cell, then this antigenicity may be eliminated or reduced by introducing into the open reading frame of the antigenic protein sequences from the Epstein bar virus EBNA-1 protein encoding a glycine-containing repeat, that suppresses antigen presentation of amino acid sequences linked in cis. See for example, Levitskaya et al., 1995, Nature 375:685.

A DNA sequence encoding a Glycine-containing sequence (for example, residues 93-325 of the Epstein-Barr virus nuclear antigen-1, carried on an Ncol-Apal fragment of EBNA1 (strain B95.8)) is inserted into a selected site within a gene encoding a protein which is believed or known to be immunogenic, but for which no immune response is desired upon administration of a nucleic acid according to the invention. The Glycine-encoding DNA sequence may, for example, be inserted downstream of the protein which is desired to be non-immunogenic, or downstream of an epitope of the protein which is known to be immunogenic; for example, it may be joined to the protein via an in-frame fusion at the carboxy or amino terminus of the protein, or inserted anywhere within the protein to render that protein nonimmunogenic. It is preferred that the Glycine-containing sequence is inserted within the protein at a distance wherein either terminal residue of the glycine-containing sequence is at a distance of between about 1 and 300 residues from an immunogenic epitope of the protein. In the case of a protein which contains multiple epitopes, it is believed that all epitopes of the antigenic protein will be rendered non-antigenic according to this embodiment of the invention. Preferably, the distance between the site of insertion in the protein. of the glycine-containing sequence and one amino acid of the protein epitope is between about 1 and 200 residues, more preferably 10-100 residues, and most preferably between about 20 and 50 residues. As used herein, "epitope" refers to an antigenic region of about 6-20 amino acids within a protein that is immunologically recognized as foreign.

For example, where the vector used to deliver a nucleic acid encoding an antigen to antigen presenting cells according to the invention contains the BPV E1 and E2 proteins, it is possible that the E1 and E2 proteins may cause an undesired immune response. Therefore, insertion of a glycine-containing sequence into each reading frame of these proteins may be accomplished in order to reduce or eliminate an immune response to the E1 and E2 proteins, without appreciably affecting the desired immune response to the antigen encoded by the vaccinating nucleic acid.

Glycine-containing sequence useful for obtaining masking of selected foreign proteins or immunogenic epitopes, such as those carried in vectors useful according to the invention, comprise a recombinant glycine-containing amino acid sequence having the formula: [(Gly_{a)X(Glyb)Y(Glyc)Z]n, whe}rein each Gly_a, Gly_b, Gly_c, independently, may be one, two, or three sequential glycine residues; each of X, Y and Z is, independently, a hydrophobic or polar amino acid without a ring structure and having a side-chain of less than 3 atoms, wherein each of X, Y and Z, respectively, need not be identical from n repeat to n repeat; and n may be from 1-66.

It is preferred that each of X, Y and Z is, independently, selected from the group consisting of Ala, Ser, Val, Ile, Leu and Thr; more preferably, each of X, Y and Z is, independently, one of Ala and Ser; most preferably, each of X, Y and Z is, independently, Ala; more preferably, each of X and Y is not Met or Cys.

It is also preferred that the glycine-containing sequence comprises (GlyGlyXGlyYGlyZ) and n=7 repeats; or (GlyGlyXGlyYGlyGlyZ) in n=9 repeats where the remaining n repeats comprise (Gly_{a)X(Glyb)Y(Glyc)Z up} to a total repeat number of 66, and preferably 28 total repeats; or the glycine-containing sequence is (GlyGlyXGlyYGlyGlyGlyZ) in n=7 repeats where the remaining n repeats comprise (Gly_{a)X(Glyb)Y(Glyc)Z up} to a total repeat number of 66, and preferably 28 total repeats; or the glycine-containing sequence comprises (GlyGlyAlaGlyAlaGlyGlyla (SEQ ID NO:14) in n=9 repeats where the remaining n repeats comprise (Gly_{s,)X(Glyb)Y(Glyc)Z up} to a total repeat number of 66, and preferably 28 total repeats.

It is also preferred that the glycine-containing sequence is (GlyGlyAlaGlyAlaGlyGlyGlyAla) (SEQ ID NO:15) in n=7 repeats where the remaining n repeats comprise (Gly_{a)X(Glyb)Y(Glyc)Z up} to a total repeat number of 66, and preferably 28 total repeats; or the glycine-containing sequence is (GlyGlyXGlyYGlyGlyZ) in n=9 repeats and (GlyGlyXGlyYGlyGlyGlyZ) in n=7 repeats where the remaining n repeats comprise (Gly.) X(Gly_b)Y(Gly,)Z up to a total repeat number of 66.

The glycine-containing sequence (GlyGlyAlaGlyAlaGlyGlyAla) in n=9 repeats and (GlyGlyAlaGlyAlaGlyGlyGlyAla) in n=7 repeats is also preferred, where the remaining n repeats comprise (Gly_{a)X(Glyb)Y(Glyc)Z up} to a total repeat number of 66.

Antigens and Genes Encoding Antigens Useful According to the Invention

It is contemplated according to the invention that an immune response may be elicited via presentation of any protein or peptide capable of eliciting such a response. It is preferred that the antigen is a key epitope which gives rise to a strong immune response to a particular agent of infectious disease. If desired, more than one antigen or epitope may be encoded by the nucleic acid in order to increase the likelihood of an immune response. Thus, the vector may include a multi-gene construct in which the antigens encoded by the genes are coordinately expressed. If desired, all of the open reading frames of a pathogen's genome may be arranged together (as an expression library) in a vector in order to form a region of coding sequences which are expressed and presented by the antigen presenting cell. The use of expression libraries is described in Barry et al., 1995, Nature 377:632.

Alternatively, DNA sequences encoding antigens specific for different diseases or infectious agents-may be arranged together in a vector for delivery to and expression in an antigen presenting cell. Therefore, immune protection may be elicited against a number of diseases specified by the antigens encoded within the construct.

In a further embodiment of the present invention the antigen encoding nucleic acid sequence may contain a signal sequence for secretion of the antigen outside the cell. Signal sequences useful in the present invention are well known to those skilled in the art and are, for example, described in Blobel and Dobberstein (1975), J. Cell Biol., 6-1, 852-862. The secreted antigen will be taken up by the secreting cell and other neighbouring cells, processed as an exogenous antigen, and presented in association with Class II MHC. The delivery vehicle of the present invention may contain a mixture of nucleic acids encoding an antigen, some with, and some without, the secretion signal sequence. Thus secreted and non-secreted antigen can be produced in the same cell and both Class I and Class II MHC presentation can be produced after a single transfection event.

Antigens Useful According to the Invention are as Follows

1. Viral Antigens

Examples of viral antigens include, but are not limited to, retroviral antigens such as retroviral antigens from the human immunodeficiency virus (HIV) antigens such as gene products of the gag, pol, and env genes, the Nef protein, reverse transcriptase, and other HIV components; hepatitis viral antigens such as the S, M, and L proteins of hepatitis B virus, the pre-S antigen of hepatitis B virus, and other hepatitis, e.g., hepatitis A, B, and C, viral components such as hepatitis C viral RNA; influenza viral antigens such as hemagglutinin and neuraminidase and other influenza viral components; measles viral antigens such as the measles virus fusion protein and other measles virus components; rubella viral antigens such as proteins E1 and E2 and other rubella virus components; rotaviral antigens such as VP7sc and other rotaviral components; cytomegaloviral antigens such as envelope glycoprotein B and other cytomegaloviral antigen components; respiratory syncytial viral antigens such as the RSV fusion protein, the M2 protein and other respiratory syncytial viral antigen components; herpes simplex viral antigens such as immediate early proteins, glycoprotein D, and other herpes simplex viral antigen components; varicella zoster viral antigens such as gpI, gpII, and other varicella zoster viral antigen components; Japanese encephalitis viral antigens such as proteins E, M-E, M-E-NS 1, NS 1, NS 1-NS2A, 80%E, and other Japanese encephalitis viral antigen components; rabies viral antigens such as rabies glycoprotein, rabies nucleoprotein and other rabies viral antigen components. See Fundamental Virology, Second Edition, eds. Fields, B. N. and Knipe, D. M. (Raven Press, New York, 1991) for additional examples of viral antigens.

2. Bacterial Antigens

Bacterial antigens which can be used in the compositions and methods of the invention include, but are not limited to, pertussis bacterial antigens such as pertussis toxin, filamentous hemagglutinin, pertactin, FIM2, FIM3, adenylate cyclase and other pertussis bacterial antigen components; diptheria bacterial antigens such as diptheria toxin or toxoid and other diptheria bacterial antigen components; tetanus bacterial antigens such as tetanus toxin or toxoid and other tetanus bacterial antigen components; streptococcal bacterial antigens such as M proteins and other streptococcal bacterial antigen components; gram-negative bacilli bacterial antigens such as lipopolysaccharides and other gram-negative bacterial antigen components; Mycobacterium tuberculosis bacterial antigens such as mycolic acid, heat shock protein 65 (HSP65), the 30 kDa major secreted protein, antigen 85A and other mycobacterial antigen components; Helicobacter pylori bacterial antigen components; pneumococcal bacterial antigens such as pneumolysin, pneumococcal capsular polysaccharides and other pneumococcal bacterial antigen components; haemophilus influenza bacterial antigens such as capsular polysaccharides and other haemophilus influenza bacterial antigen components; anthrax bacterial antigens such as anthrax protective antigen and other anthrax bacterial antigen components; rickettsiae bacterial antigens such as romps and other rickettsiae bacterial antigen components. Also included with the bacterial antigens described herein are any other bacterial, mycobacterial, mycoplasmal, rickettsial, or chlamydial antigens.

3. Fungal Antigens

Fungal antigens which can be used in the compositions and methods of the invention include, but are not limited to, candida fungal antigen components; histoplasma fungal antigens such as heat shock protein 60 (HSP60) and other histoplasma fungal antigen components; cryptococcal fungal antigens such as capsular polysaccharides and other cryptococcal fungal antigen components; coccidiodes fungal antigens such as spherule antigens and other coccidiodes fungal antigen components, and tinea fungal antigens such as trichophytin and other coccidiodes fungal antigen components.

4. Parasite Antigens

Examples of protozoa and other parasitic antigens include, but are not limited to, plasmodium falciparum antigens such as merozoite surface antigens, sporozoite surface antigens, circumsporozoite antigens, gametocyte/gamete surface antigens, blood-stage antigen pf 1 55/RESA and other plasmodial antigen components; toxoplasma antigens such as SAG-1, p30 and other toxoplasma antigen components; schistosomae antigens such as glutathione-S-transferase, paramyosin, and other schistosomal antigen components; leishmania major and other leishmaniae antigens such as gp63, lipophosphoglycan and its associated protein and other leishmanial antigen components; and trypanosoma cruzi antigens such as the 75-77 kDa antigen, the 56 kDa antigen and other trypanosomal antigen components

5. Tumor Antigens

Tumor antigens which can be used in the compositions and methods of the invention include, but are not limited to, prostate specific antigen (PSA), telomerase; multidrug resistance proteins such as P-glycoprotein; MAGE-1, alpha fetoprotein, carcinoembryonic antigen, mutant p53, papillomavirus antigens, gangliosides or other carbohydrate-containing components of melanoma or other tumor cells. It is contemplated by the invention that antigens from any type of tumor cell can be used in the compositions and methods described herein.

6. Antigens Involved in Autoimmunity

Antigens which have been shown to be involved in autoimmunity and could be used in the delivery vehicles of the present invention to induce tolerance include, but are not limited to, myelin basic protein, myelin oligodendrocyte glycoprotein and proteolipid protein of multiple sclerosis and CII collagen protein of rheumatoid arthritis.

It is known that bacterial DNA can have immunostimulatory properties (Tokunaga et al., J. Nat. Cancer Insti., 1984, 72:955; Togunaga et al., Microbiol. Immunol. 1992, 36:55). Thus, for a nucleic acid encoding any one or more of the above-described antigens, it is preferred that the nucleic acid has immunostimulatory properties and may contain specifically identified CpG motifs that have been demonstrated to confer immunostimulation (Krieg et al., Nature, 1995, 374:546; Yi et al., Jour. Immunol., 1996, 156:558)

DOSAGE, ROUTE OF ADMINISTRATION AND PHARMACEUTICAL FORMULATIONS

According to the present invention, nucleic acid encoding one or more antigens or epitopes thereof may be used in gene therapy in order to provide immune protection against various infectious agents and diseases. The nucleic acid may be delivered to antigen presenting cells as part of a vehicle for delivering a nucleic acid to a cell which may specifically target a cell type or which may be untargeted with respect to the recipient cell. Delivery of a nucleic acid to APCs according to the present invention may be accomplished via ex vivo gene therapy or in in vivo gene therapy. For prophylactic and therapeutic vaccines, in vivo gene therapy is preferred, and for therapeutic vaccines, ex vivo gene therapy also is appropriate. A gene may be delivered to cells cultured ex vivo prior to reinfusion of the transfected cells into the patient or the gene may be delivered in a gene delivery vehicle complex by direct in vivo injection into the patient's vascular system or in a body area rich in the target cells. The in vivo injection may be made subcutaneously, intravenously, intramuscularly or intraperitoneally. The delivery vehicle may also be delivered by oral, nasal, vaginal or urethral routes, and may be delivered in combination with other delivery vehicles such as microspheres, gene gun, or Bioject.TM.. Techniques for ex vivo and in vivo gene therapy are known to those skilled in the art. Generally, the compositions are administered in a manner compatible with the dosage formulation, and in such amount as will be prophylactically and/or therapeutically effective. The quantity to be administered depends on the subject to be treated, including, e.g., capacity of the subject's immune system to synthesize antibodies, and the degree of protection desired. Suitable dosage ranges are on the order of, where ex vivo transfected cells are administered to a patient, 10⁵ -10⁸, and optionally 10⁶ -10⁷ cells are administered in a single dose; where a gene/delivery vehicle complex is administered 100 ng-10 mg, or 1 .mu.g-1 mg, or optionally 1 .mu.g-10 .mu.g of complex or mixture is administered in a single dose. Suitable regimens for initial administration and booster shots are also variable but are typified by an initial administration followed by subsequent inoculations or other administrations. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and may be peculiar to each subject. It will be apparent to those of skill in the art that the therapeutically effective amount of a composition of this invention will depend, inter alia, upon the administration schedule, the unit dose of antigen administered or expressed by an encoding nucleic acid that is administered, whether the compositions are administered in combination with other therapeutic agents, the immune status and health of the recipient, and the therapeutic activity of the particular nucleic acid molecule, delivery complex, or ex vivo transfected cell.

Complexes and mixtures according to the invention also may be mixed in a physiologically acceptable diluent such as water, phosphate buffered saline, or saline, and further may include an adjuvant. Adjuvants such as incomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide, or alum are materials well known in the art. Upon administration with a composition as described herein, via injection, oral, transdermal, or other routes, the immune system of the host will respond to the polypeptide antigen or the nucleic acid encoded antigen and the polypeptide antigen by producing either an effective cellular or humoral immune response to the antigen(s) or both effective cellular and humoral immune responses, as described herein.

Compositions of the invention can be given in a single dose schedule, or in a multiple dose schedule. A multiple dose schedule is one in which a primary course of vaccination can include 1-10 separate doses, followed by other doses given at subsequent time intervals required to maintain and or reinforce the immune response, for example, at 1-4 months for a second dose, and if needed, a subsequent dose(s) after several months. Periodic boosters at intervals of 1-5 years, usually 3 years, may be desirable to maintain the desired levels of protective immunity. The course of the immunization can be followed by in vitro proliferation assays of peripheral blood lymphocytes (PBLS) co-cultured with ESAT6 or ST-CF, and by measuring the levels of IFN-.gamma. released from the primed lymphocytes. The assays can be performed using conventional labels, such as radionucleotides, enzymes, fluorescent labels and the like. These techniques are known to one skilled in the art and can be found in U.S. Pat. Nos. 3,791,932; 4,174,384; and 3,949,064, which are hereby incorporated by reference.

In addition to allowing maximization of the immune response, the ability to co-deliver DNA and antigen to APCs has the advantage of being able to modulate the type of immune response induced. It is known that DNA immunization tends to produce a T helper-type 1 (Th1) response, whereas protein immunization induces a T helper-type 2 (Th2) response (Raz et al., 1996, PNAS 93:5141). Thus, by manipulation of the DNA and protein/peptide components of the complex or mixture of the invention, the type of immune response may be manipulated by varying the relative amounts of each component.

Complexes and mixtures of the invention also may find use as diagnostic reagents. For example, they may be used to determine the susceptibility of a particular individual to a treatment regimen which employs the complexes or mixtures, and thus may be helpful in modifying an existing treatment protocol or in determining a prognosis for an affected individual. In addition, they may be used to predict which individuals will be at substantial risk for developing an infection by an organism, pathogen or agent for which vaccination is desired.

The invention may be used to generate both prophylactic and therapeutic immune responses. Optionally, the immune system can be primed and boosted either using the same complex or mixture or using combinations of different complexes or mixtures described herein, or combinations of complexes or mixtures described herein with conventional vaccines known in the art, e.g., recombinant protein vaccines or killed virus vaccines.

EXEMPLIFICATION

1. The Following Examples Demonstrate Cell Type-specific Expression in Antigen Presenting Cells in Animal Models

Cell Type-specific Expression of the Human Glucocerebrosidase Gene in Cells of the Monocyte-macrophage Lineage in several Lines of Transgenic Mice

A construct was generated in which the mouse MHC class II Ea gene Locus Control Region (LCR, Carson and Wiles, Nucl. Acids Res., 21, (2065-2072 (1993)) was linked to the human gene encoding glucocerebrosidase (GC). Several transgenic lines were made with linearized DNA of this construct. Expression of human GC and functional activity was detectable in the spleen cells of most transgenic lines. Non-transgenic mice (ntg) had no detectable human GC activity. The spleen contains both B lymphocytes and macrophages/dendritic cells , and so to definitively demonstrate expression in macrophages/dendritic cells, enriched populations of activated, transgenic macrophages w ere produced by thioglycollate injection into the peritoneum of transgenic mice. Analysis of the resulting macrophage populations for expression of human GC showed that F4/80-positive macrophages (Gordon et al., Curr. Topics. Microbiol. immunol., (1992) 181, 1-37) abundantly expressed human GC (as detected by monoclonal antibody 8E4). This demonstrates APC-specific expression of the heterologous gene rising the MHC LCR.

In Vivo Vaccination: Generation of Transgenic Mice Expressing Influenza NP Under MHC LCR Control, and Generation of Anti-NP Immunity in Syngeneic Non-transgenic Littermates by Transplantation of Transgenic Spleen and Bone Marrow Cells

To demonstrate that expression of foreign proteins in the APC can generate protective cellular and/or-humoral immune responses, transgenic lines are made in which the Influenza NP gene is expressed under the control of the MHC class II Ea LCR as described above.

From these mice, spleen cells or bone marrow cells are isolated and infused into non-transgenic syngeneic mice. Mice are then analyzed for expression of the NP gene, which is expected to be present in the donor cells and their differentiated descendants. Analysis of the transplant recipients is expected to reveal that some have successfully generated NP-specific cytotoxic T cells and anti-NP antibody responses.

Anti-influenza Vaccines to Generate Immunity Directed Against the Hemagglutinin Component of the Viral Capsid

Transgenic lines are made in which the Influenza Hemagglutinin (HA) gene is expressed under the control of the MHC class II Ea LCR described above. From these mice, spleen cells or bone marrow cells are isolated and infused into non-transgenic syngeneic mice. Mice are then analyzed for expression of the HA gene, which is shown to be present in the donor cells and their differentiated descendants. Analysis of the transplant recipients is expected to reveal that some have successfully generated HA-specific cytotoxic T cells and anti-HA antibody responses.

Anti-HIV Vaccines

Transgenic mice are made expressing HIV-1 tat, rev, nef or gag genes, and combinations thereof, under the control of the MHC class II Ea LCR described above. Transplantation of transgenic spleen cells to syngeneic non-transgenic mice is expected to elicit a cytotoxic T cell response which can be measure in vitro using Chromium⁵¹ release assays for T cell-mediated cytotoxicity. To measure the T cell response, T cells from transplantation recipients are challenged with syngeneic, non-transgenic, irradiated, Cr⁵¹ -labeled target Antigen-Presenting Cells in the presence of immunodominant peptides from the HIV-1 nef, rev, gag and tat proteins. Effective T cell responses are detected by measuring Cr⁵¹ release as a result of target cell lysis. Serum antibody responses against HIV-1 nef, rev, gag and tat are also measured in the transplant recipients, to confirm the generation of a humoral response.

Anti-hepatitis B Virus (HBV) Vaccines

Transgenic mice are made expressing the HBc, HBe, S, pre-S and pX genes, and combinations thereof, under the control of the MHC class II Ea LCR. Transplantation of transgenic spleen cells to syngeneic non-transgenic mice is expected to elicit a cytotoxic T cell response which can be measured in vitro using Chromium⁵¹ release assays for T cell-mediated cytotoxicity. To measure the T cell response, T cells from transplantation recipients are challenged with syngeneic, non-transgenic, irradiated, Cr⁵¹ -labeled target Antigen-Presenting Cells in the presence of immunodominant peptides from the HBc, HBe, S, pre-S, and pX proteins. Effective T cell responses are detected by measuring Cr⁵¹ release as a result of target cell lysis. Serum antibody responses against the HBc, HBe, S, pre-S, and pX proteins are also measured in the transplant recipients, to confirm the generation of a humoral response.

Anti-hepatitis C Virus (HC7V) Vaccines

Transgenic mice are made expressing nucleocapsid protein C22-3, the NS3 and NS4-region derived C200 and C33c proteins, and combinations thereof, under the control of the MHC class II Ea LCR. Transplantation of transgenic spleen cells to syngeneic non-transgenic mice is expected to elicit a cytotoxic T cell response which can be measured in vitro using Chromium⁵ release assays for T cell-mediated Cytotoxicity. To measure the T cell response, T cells from transplantation recipients are challenged with syngeneic, non-transgenic, irradiated, Cr⁵¹ -labeled target Antigen-Presenting Cells in the presence of immunodominant peptides from nucleocapsid protein C22-3, the NS3 and NS4- region derived C200 and C33c proteins. Effective T cell responses are detected by measuring Cr⁵¹ release as a result of the target cell lysis. Serum antibody responses against the nucleocapsid protein C22-3, the NS3 and NS4- region derived C200 and C22c proteins are also measured in the transplant recipients, to confirm the generation of a humoral response.

Anti-human Papilloma Virus (HPV) Vaccines

Transgenic mice are made expressing the HPV 16 proteins E1, E2, E7, E5 and E6, and combinations thereof, under the control of the MHC class II Ea LCR. Transplantation of transgenic spleen cells to syngeneic non-transgenic mice is expected to elicit a cytotoxic T cell response which can be measured in vitro using Chromium⁵¹ release assays for T cell-mediated cytotoxicity. To measure the T cell response, T cells from transplantation recipients are challenged with syngeneic, non-transgenic, irradiated, Cr⁵¹ -labeled target Antigen-Presenting cells in the presence of immunodominant peptides from the E1, E2, E7, E5 and E6 proteins. Effective T cell responses are detected by measuring Cr⁵¹ release as a result of target cell lysis. Serum antibody responses against the E1, E2, E7 E5 and E6 proteins are also measured in the transplant recipients, to confirm the generation of a humoral response.

Anti-tumor Vaccines

Lines of transgenic mice are made expressing the genes encoding the melanoma-specific antigen MAGE-1, tyrosinase, the murine homologue of the HER2/neu proto-oncogene which is mutated in ovarian and breast tumors, the HPV 3.6 E7 protein, and the connexin 37 protein which is mutated in the 3LL lung carcinoma. Bone marrow and spleen cells of these transgenic mice are transplanted to syngeneic non-transgenic mice to elicit cytotoxic T-cell mediated and humoral immune response to the proteins encoded by the transgenes. MAGE-1/tyrosinase, HER2/neu, HPV-16 E7, and connexin 37 transgenic-transplant recipients and untransplanted controls are subsequently injected subcutaneously with tumor cells from the B16 mouse melanoma, murine mammary carcinoma T5O/80, the 3LL mouse lung carcinoma, or the C3 mouse tumor transfected with HPV 16 genomic DNA, respectively, and tumor growth is monitored. Transplant recipients are expected to show much reduced tumor growth than non-transplanted controls, confirming that protective anti-tumorimmune responses-have been generated.

Moreover, non-transplanted recipients already bearing tumors of the types described above are expected to be cured of their cancers by an infusion of bone marrow or spleen cells from syngeneic mice transgenic for the corresponding tumor-specific antigen-coding gene also described above.

2. Examples of Gene Therapy Using the Delivery Vehicle of the Present Invention

Anti-influenza Vaccines to Generate Immunity Directed Against Hemagglutinin Component of the Viral Capsid

A delivery vehicle comprising a plasmid vector in which the Influenza Hemagglutinin (HA) gene is expressed under the control of the MHC class II Ea LCR described above, is administered to subjects by direct intravascular injection or by transfection of cultured bone marrow-derived cells in vitro which are then reinfused into patients intravenously. Subsequent analysis of the patients is expected to reveal that some patients have successfully generated HA-specific cytotoxic T cells and anti-HA antibody responses.

A delivery vehicle comprising a plasmid vector in which nucleotide sequences from the influenza matrix protein gene encoding a peptide including the HLA A2 restricted epitope located at residues 57-68 (Goton et al, 1987, Nature 326:331-332. Moss et al 1991, PNAS 88:8987-8990) are expressed under the control of the MHC class II Ea LCR described above is administered to patients by direct intravascular injection or by transfection of cultured bone marrow-derived cells in vitro which are then reinfused into patients intravenously. Subsequent analysis of the patients is expected to reveal that some patients have successfully generated HA-specific, cytotoxic T cells.

Anti-HIV Vaccines

A delivery vehicle comprising a plasmid vector in which the HIV-1 tat, rev, nef or gag genes, and combinations thereof, are expressed under the control of the MHC class II Ea LCR is administered to patients by direct intravascular injection or by transfection of cultured bone marrow-derived cells in vitro which are then reinfused into patients intravenously. Subsequent analysis of the patients is expected to reveal that some patients have successfully generated CTL responses against the HIV-1 nef, rev, tat and gag proteins. Effective T cell responses are detected by measuring Cr⁵¹ release as a result of target cell lysis. Serum antibody responses against HIV-1 nef, rev, tat and gag are also measured in the transplant recipients, confirming the generation of humoral response.

A delivery vehicle comprising a plasmid vector containing nucleotide sequences from the HIV-1 gag gene which encode peptides including the HLAB27 and B8 restricted epitopes in HIV p17 Gag and HIV p24 Gag (Nixon et al. 1988, Nature 336:484-487, Nixon and McMichael, 1991 AIDS 5:1 eO49-1059) and combinations thereof, expressed under the control of the MHC class II Ea LCR, is administered to patients by direct intravascular injection or by transfection of cultured bone marrow-derived cells in vitro which are then reinfused into patients intravenously. Subsequent analysis of the patients is expected to reveal that some patients have successfully generated CTL responses against Gag protein sequences. Effective T cell responses are detected by measuring Cr⁵¹ release as a result of target cell lysis.

Anti-hepatitis B Virus (HBV) Vaccines

A delivery vehicle comprising a plasmid vector in which HBV HBc, HBe, S, pre-S, and pX genes, and combinations thereof, are expressed under the control of the MHC class II Ea LCR is administered to patients by direct intravascular injection or by transfection of cultured bone marrow-derived cells in vitro which are then reinfused into patients intravenously. Subsequent analysis of the patients is expected to reveal that some patients have successfully generated CTL responses against the HBV HBc, HBe, S, pre-S, and pX proteins. Effective T cell responses are detected by measuring Cr⁵¹ release as a result of target cell lysis. Serum antibody responses against HBV HBc, HBe, S, pre-S, and pX proteins are also measured in the transplant recipients, confirming the generation of humoral response.

Anti-hepatitis C Virus (HCV) Vaccines

A delivery vehicle comprising a plasmid vector in which the genes encoding the HCB nucleocapsid protein C22-3, the NS3 and NS4-region-derived C200 and C33c proteins, and combinations thereof, are expressed under the control of the MHC class II Ea LCR is administered to patients by direct intravascular injection or by transfection of cultured bone marrow-derived cells in vitro which are then reinfused into patients intravenously. Subsequent analysis of the patients is expected to reveal that some patients have successfully generated CTL responses against nucleocapsid protein C22-3, the NS3 and NS4-region-derived C200 and C22c proteins. Effective T cell responses are detected by measuring Cr⁵¹ release as a result of target cell lysis. Serum antibody responses against nucleocapsid protein C22-3, the NS3 and NS4 region-derived C200 and C33c proteins are also measured in the transplant recipients, confirming the generation of a humoral response.

Anti-human Papilloma Virus (HPV) Vaccines

A delivery vehicle comprising a plasmid vector in which genes encoding the HPV 16 proteins E1, E2, E7 E5 and E6, and combinations thereof, are expressed under the control of the MHC class II Ea LCR, is administered to patients by direct intravascular injection or by transfection of cultured bone marrow-derived cells in vitro which are then reinfused into patients intravenously. Subsequent analysis of the patients is expected to reveal that some patients have successfully generated CTL responses against E1, E2, E7 E5 and E6 proteins. Effective T cell responses are detected by measuring Cr⁵¹ release as a result of target cell lysis. Serum antibody responses are also measured in the transplant recipients, confirming the generation of a humoral response.

Anti-tumor Vaccines

A delivery vehicle comprising a plasmid vector in which genes encoding the melanoma-specific antigen MAGE-1, tyrosinase, the murine homologue of the HER2/neu proto-oncogene which is mutated in ovarian and breast tumors, and the HPV 16 E7 protein are expressed under the control of the MHC class II Ea LCR, is administered to patients by direct intravascular injection or by transfection of cultured bone marrow-derived cells in vitro which are then reinfused into patients intravenously. Subsequent analysis of the patients is expected to reveal that some patients have successfully generated CTL responses against MAGE-1, tyrosinase, HER2/neu or HPV E6 or E7 proteins. Effective T cell responses are detected by measuring Cr⁵¹ release as a result of target cell lysis. Serum antibody responses against MAGE-1, tyrosinase, HER2/neu, or HPV E6 or E7 proteins are also measured in the transplant recipients, confirming the generation of humoral response. In addition tumor regression may be observed.

3. Examples of Delivery Systems That Allow Presentation of Antigen in Association With Class I and Class II MHC (In Vivo)

The following examples teach one of skill in the art how to differentiate MHC Class I and Class II responses in vivo, and how to obtain an increased MHC Class II-mediated immune response using a construct prepared and administered according to the invention.

Addition of Pentide/Protein to the Delivery Vehicle

To demonstrate that administration of a delivery system containing peptide/protein antigens gives rise to a greater helper T cell response (i.e. predominantly a class II restricted response) than administration of a delivery vehicle in which the antigen is solely encoded on the nucleic acid, mice are vaccinated with a delivery vehicle with or without antigenic peptides. The complex intended for delivery will have previously been tested for delivery of both antigen and DNA in vivo. Testing is performed by screening complex formulations for the ability to generate an immune response within the animals. Possible complex formulations include, but are not limited to, those described in International Patent Application No. PCT/GB96/01396. The DNA contained within the delivery vehicle either encodes no antigenic gene or the gene for influenza nucleoprotein. The antigenic peptides are appropriate, Class II presented, epitopes of NP or the whole NP protein. T cells from the immunized mice are then assayed for class I- and class II-restricted responses. When the delivery vehicle contained antigenic peptide/protein but no antigen gene a predominantly class II-restricted response is observed, whereas in the absence of both antigenic peptides/protein, and antigen gene, no class II-restricted response is seen. In the presence of the antigen gene, class I and class II responses are observed but the class II response is increased when antigenic peptides/protein are included on the delivery vehicle. Thus, addition of antigenic peptide/protein to the delivery vehicle produces an increased class II-restricted response. Class I-restricted CTL responses are measured by Cr⁵¹ release assay and Class II-restricted helper T cell responses are measured by T cell proliferation assay or cytokine release assay.

Addition of Secretion Signal Sequence to Antigen Gene

To demonstrate that administration of a delivery vehicle containing DNA encoding an antigen gene with a secretion signal sequence that is functional in mammalian cells gives rise to a greater Class II response than administration of a delivery vehicle in which an non-secreted version of the antigen is produced, mice are vaccinated with a delivery vehicle containing a DNA molecule encoding the influenza nucleoprotein gene with or without a secretary signal sequence T cells from the immunized mice are then assayed for CTL and helper T cell responses in order to determine the predominantly class I- and class II-restricted response, respectively. The mice which had been immunized with the secreted form of NP show increased helper T cell (i.e. predominantly class II-restricted) responses when compared to immunization with the gene encoding the non-secreted antigen. Thus, addition of secretory signal sequence to the antigenic gene produces an increased helper T cell response (i.e. predominantly a class II-restricted response).

Claim 1 of 53 Claims

What is claimed is:

1. A method of eliciting an immune response in a mammal comprising intramuscularly or subcutaneously administering to said mammal a mixture comprising (i) a nucleic acid encoding at least a first immunogenic epitope operably linked to transcriptional regulatory elements for expression of said immunogenic epitope and (ii) a peptide comprising a second immunogenic epitope such that the nucleic acid and the peptide are taken up by and the nucleic acid is expressed in a professional antigen presenting cell of the mammal as said first immunogenic epitope, wherein an immune response is elicited in the mammal to said first immunogenic epitope and said second immunogenic epitope, provided that said mixture is not a virus.

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