Recombinant protein bodies as immunogen-specific adjuvants

Abstract

An immunogen-specific is adjuvant for a vaccine or inoculum is disclosed. The adjuvant is comprised of particulate recombinant protein body-like assemblies (RPBLAs) that contain a recombinant fusion protein that contains two portions peptide-linked together. A first portion is a protein body-inducing sequence (PBIS) and a second portion is a T-cell stimulating immunogenic polypeptide whose sequence is that of a pathogenic polypeptide sequence present in or induced by a vaccine or inoculum. The adjuvant, when used as an inoculum in a host animal without a prior priming vaccination or inoculation, does not induce production of antibodies or T cell activation to the pathogenic sequence.

Claims

1. A method for inducing a T-cell mediated immune response against an immunogenic peptide in a subject in need thereof, the method comprising administering to a subject in need thereof a vaccine comprising a nucleic acid molecule that encodes a recombinant fusion protein, said recombinant fusion protein comprising a protein body-inducing sequence (PBIS) and an immunogenic polypeptide, wherein the PBIS comprises a signal peptide sequence that directs the fusion protein towards the endoplasmic reticulum of the expressing cell and wherein the PBIS retains the fusion protein in the endoplasmic reticulum of the expressing cell.

2. The method as defined in claim 1 wherein the PBIS comprises a prolamin.

3. The method as defined in claim 2 wherein the prolamin is selected from the group consisting of gamma-zein, alpha-zein, delta-zein, beta-zein, rice prolamin and gamma-gliadin.

4. The method as defined in claim 1 wherein the immunogenic polypeptide is selected from the group consisting of (i) a polypeptide encoded by the HPV E7 gene, (ii) a polypeptide encoded by the HIV-1 gag gene and (iii) a polypeptide encoded by the HIV-1 pol gene.

5. The method as defined in claim 1 wherein the administration is preceded by administration of a priming vaccination or inoculation using a composition comprising the immunogenic polypeptide or a nucleic acid encoding the immunogenic polypeptide.

6. The method as defined in claim 5 wherein the composition used in the priming vaccination or inoculation comprises: (i) a particulate recombinant protein body-like assembly (RPBLA) comprising a recombinant fusion protein, said recombinant fusion protein comprising a protein body-inducing sequence (PBIS) and the immunogenic polypeptide, (ii) a nucleic acid molecule that encodes the immunogenic polypeptide, or (iii) a nucleic acid molecule that encodes a recombinant fusion protein, said recombinant fusion protein comprising a protein body-inducing sequence (PBIS) and the immunogenic polypeptide, wherein the PBIS includes a signal peptide that directs the fusion protein towards the endoplasmic reticulum of the expressing cell and wherein the PBIS retains the fusion protein in the endoplasmic reticulum of the expressing cell.

7. The method as defined in claim 1 wherein the vaccine is administered intramuscularly.

8. The method of claim 2, wherein the PBIS consists essentially of RX3.

9. The method of claim 1, wherein immunogenic polypeptide is encoded by HIV-1.

10. A method for inducing a T-cell mediated immune response against an immunogenic peptide in a subject in need thereof, the method comprising: (i) administering a priming vaccination or inoculation comprising a nucleic acid molecule that encodes a recombinant fusion protein, said recombinant fusion protein comprising a protein body-inducing sequence (PBIS) and an immunogenic polypeptide, wherein the PBIS includes a signal peptide that directs the fusion protein towards the endoplasmic reticulum of the expressing cell, and (ii) administering a vaccine composition comprising a particulate recombinant protein body-like assembly (RPBLA) comprising a recombinant fusion protein, said recombinant fusion protein comprising a protein body-inducing sequence (PBIS) an immunogenic polypeptide and wherein the PBIS retains the fusion protein in the endoplasmic reticulum of the expressing cell.

11. The method as defined in claim 1, wherein the PBIS comprises the sequence of SEQ ID NO: 6.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) In the drawings forming a part of this disclosure,

(2) FIG. 1 in three panels (FIG. 1A, FIG. 1B and FIG. 1C) shows the analysis by western blot of RPBLA fractions isolated from tobacco plants agroinfiltrated with RX3-p24, RX3-p41 and RX3-RT. The presence of full length RX3 fusion proteins in the corresponding RPBLA fraction preparation was checked by using the following antibodies: (i) R8 which recognizes RX3, (ii) p24 which recognizes p41 and p24 antigens and (iii) RT which recognizes RT antigen.

(3) FIG. 2 contains two graphs that show an IFN- (FIG. 2A) and IL-2 (FIG. 2B) ELISPOT analysis of p24 cell responses after vaccination of BALB/c mice. Inoculations with the indicated immunogens were given as specified in the methods. Reactions in the corresponding ELISPOT assay were done in triplicate with the indicated Gag peptides, an irrelevant peptide (Irrel pept) or absence of peptide (Med), and bars are the average number of spot forming units (sfu)SD/106 splenocytes. Data are from a representative study with splenocytes pooled from 5 mice per group.

(4) FIG. 3 shows western blot detection of anti-Gag antibodies in mouse serum. The content of anti-Gag antibody in mouse serum was detected using commercial western blot strips as described in the methods. Pos, positive control serum; Neg, negative control serum; d40, mouse serum taken at day 40 after inoculation as indicated and described in methods; d0, pre-inoculation mouse serum. The inoculation regimen for each set of strips is indicated on the right of the strips: these were (i) single gag DNA inoculation (pTHGagx1), (ii) gag DNA prime-gag DNA boost (pTHGagx2), (iii) gag DNA primeRX3-p24 boost (pTHGagC+RX3-p24), (iv) single RX3-p24 inoculation (RX3-p24).

(5) FIG. 4 contains two graphs that show an IFN- (FIG. 4A) and IL-2 (FIG. 4B) ELISPOT analysis of p41 cell responses after vaccination of BALB/c mice. Inoculations with the indicated immunogens were given as specified in the methods. Reactions in the corresponding ELISPOT assay were done in triplicate with the indicated Gag peptides, an irrelevant peptide TYSTVASSL (SEQ ID NO:1; irrel pept) or absence of peptide (Med) and bars are the average number of spot forming units (sfu)SD/106 splenocytes. Data are from a representative study with splenocytes pooled from 5 mice per group.

(6) FIG. 5 contains two graphs that show an IL-2 (FIG. 5A) and IFN- (FIG. 5B) ELISPOT analysis of RT cell responses after vaccination of BALB/c mice. Inoculations with the indicated immunogens were given as specified in the methods. Reactions in the corresponding ELISPOT assay were done in triplicate with the indicated Gag peptides, an irrelevant peptide TYSTVASSL (SEQ ID NO:1; irrel pept) or absence of peptide (Med) and bars are the average number of spot forming units (sfu)SD/106 splenocytes. Data are from a representative study with splenocytes pooled from 5 mice per group.

(7) FIG. 6 is a map of the artificial HPV-16 E7SH gene. The HPV-16 E7 wild-type gene (E7WT, above) was dissected at the positions corresponding to the pRB binding site (nt 72/73) and between the two C-X-X-C motifs (nt 177/178 and nt 276/277). The resulting four fragments a, b, c and d were rearranged (shuffled) forming the core element with the sequence a, d, c, b. To avoid loss of putative CTL epitopes at the junctions a-b, b-c and c-d, these sequences (327 nt=39 amino acids) were added as an appendix forming the complete HPV-16 E7SH gene. To minimize the potential risk of back-to-wild-type recombination the codons of the core element were optimized for expression in humans according to the Kazusa codon usage database that can be found at kazusa.or.jp/codon/. A Kozak sequence was added in front of the gene to enhance translation.

(8) FIG. 7 shows the analysis by western blot of RPBLA fractions isolated from tobacco plants agroinfiltrated with RX3-E7SH. The presence of full length RX3 fusion proteins in the corresponding RPBLA fraction preparation was checked by using E7SH antibody.

(9) FIG. 8 contains two graphs (FIG. 8A and FIG. 8B) that illustrate CTL responses in C57BL/6 mice after DNA and RPBLAs immunization. Four mice per group were immunized once intra-muscularly in each musculus tibialis anterior with: (i) 50 g empty plasmid (pTHamp), (ii) 50 g plasmid expressing E7SH (pTHamp-E7SH), (iii) or subcutaneously into the flank with 5 g of RPBLAs containing RX3-Gfp fusion protein (RX3-Gfp), (iv) 5 g of RPBLAs containing RX3-E7SH fusion protein (RX3-E7SH) or (v) 5 g of RPBLAs containing RX3-E7SH fusion protein and 100 l of IFA (5 g RX3-E7SH in 100 l buffer+100 l IFA). Ex vivo IFN- and Granzyme B Elispot assays were performed and each bar represents the number of activated T cells from an individual animal.

(10) FIG. 9 is a graph of CTL responses in C57BL/6 mice after RPBLAs immunization. Four mice per group were immunized once intramuscularly or sc (as above) with: (i) 5 g of RPBLAs containing RX3-Gfp fusion protein (RX3-Gfp), (ii) 5 g of RPBLAs containing RX3-E7SH fusion protein (RX3-E7SH), (iii) 5 g of RPBLAs containing RX3-E7SH fusion protein and 100 l of IFA (RX3-E7SH/IFA), (iv) 5 g of ovalbumin (OVA) or (v) 5 g of ovalbumin and 100 l of IFA (OVA/IFA) in each musculus tibialis anterior. Ex vivo Granzyme B Elispot assays were performed and each bar represents the number of activated T cells from an individual animal.

(11) FIG. 10 in two parts as FIG. 10A and FIG. 10B illustrate growth of C3 tumors in C57BL/6 mice after immunization with: (i) 100 g empty plasmid (pTHamp), (ii) 100 g plasmid expressing E7SH (pTHamp-E7SH), (iii) 5 g of RPBLAs containing RX3-Gfp fusion protein (RX3-Gfp), (iv) 5 g of RPBLAs containing RX3-E7SH fusion protein (RX3-E7SH) or (v) 5 g of RPBLAs containing RX3-E7SH fusion protein and 100 l of IFA (RX3-E7SH/IFA). Data shown provide the surface area tumor size from days 0 to 14. FIG. 10A illustrates a comparison of DNA vs RPBLAs immunization effect on tumor regression, whereas FIG. 10B illustrates that there is no unspecific tumor regression effect in DNA and RPBLAs immunizations lacking the E7SH antigen.

(12) FIG. 11 is a graph showing the results of tumor growth on rechallenge studies after immunization with: (i) 100 g plasmid expressing E7SH (pTHamp-E7SH), (ii) 5 g of RPBLAs containing RX3-E7SH fusion protein (RX3-E7SH) or (iii) 5 g of RPBLAs containing RX3-E7SH fusion protein and 100 l of IFA (RX3-E7SH/IFA). Those mice that showed complete regression after the tumor regression study of FIG. 10 were injected again with 0.510.sup.6 C3 cells s.c. in 100 l PBS into the flank 3 weeks after completion of the tumor regression experiment. As a control, the same number of non-immunized mice received the same treatment. Twenty days after this injection, all control mice showed tumor growing, whereas none of the immunized mice developed tumors.

DEFINITIONS

(13) The word antigen has been used historically to designate an entity that is bound by an antibody or receptor, and also to designate the entity that induces the production of the antibody or cellular response such as that of a CD4+ T cell. More current usage limits the meaning of antigen to that entity bound by an antibody or receptor, whereas the word immunogen is used for the entity that induces antibody production or cellular response. Where an entity discussed herein is both immunogenic and antigenic, reference to it as either an immunogen or antigen is typically made according to its intended utility.

(14) Antigenic determinant refers to the actual structural portion of the antigen that is immunologically bound by an antibody combining site or T-cell receptor. The term is also used interchangeably with epitope.

(15) As used herein, the term fusion protein designates a polypeptide that contains at least two amino acid residue sequences not normally found linked together in nature that are operatively linked together end-to-end (head-to-tail) by a peptide bond between their respective carboxy- and amino-terminal amino acid residues. A fusion protein of the present invention is a chimer of a protein body-inducing sequence (PBIS) linked to a second sequence that is a T-cell stimulating polypeptide (e.g., peptide or protein) that is present in the pathogen (target) at which the vaccine or inoculum is directed.

(16) The term immunogen-specific is used herein to distinguish the adjuvanticity of a contemplated recombinant adjuvant and a more general adjuvant. More particularly, a contemplated immunogen-specific adjuvant enhances the cellular (T-cell) immune response toward an immunogen that includes an amino acid residue sequence of the adjuvant and does not generally activate the immune system. Thus, the vaccine or inoculum shares an amino acid residue sequence or encodes a shared sequence with the adjuvant.

(17) An inoculum is a composition that comprises an immunogenically effective amount of immunogenic chimer particles dissolved or dispersed in a pharmaceutically acceptable diluent composition that typically also contains water. When administered to a host animal in need of immunization or in which antibodies or activated T cells are desired to be induced such as a mammal (e.g., a mouse, dog, goat, sheep, horse, bovine, monkey, ape, or human) or bird (e.g., a chicken, turkey, duck or goose), an inoculum induces a B cell and/or T cell response (stimulation) in an inoculated host animal such as production of antibodies that immunoreact with the immunogen of the chimer and/or induces T cells that respond to the immunogen. A vaccine is a type of inoculum in which the vaccine-induced antibodies not only immunoreact with the immunogen or activated T cells respond to that immunogen, but also immunoreact with the pathogen from which the immunogen is derived in vivo, and provide protection from that disease state.

(18) The expression T-Cell-mediated immunity refers to an immune response that does not involve antibodies or complement but rather involves the activation of macrophages, natural killer cells (NK), antigen-specific cytotoxic T-lymphocytes, and the release of various cytokines in response to an antigen. Historically, the immune system was separated into two branches: humoral immunity, for which the protective function of immunization could be found in the humor (cell-free bodily fluid or serum) and cellular immunity, for which the protective function of immunization was associated with cells. CD4 cells or helper T cells provide protection against different pathogens. T-Cell-mediated immunity is an immune response produced when T cells, especially cytotoxic T cells, that are sensitized to foreign antigens attack and lyse target cells. In addition to direct cytotoxicity, T cells can stimulate the production of lymphokines that activate macrophages. Cell-mediated immune responses are important in defense against pathogens, autoimmune diseases, some acquired allergies, viral infection, some tumors and other immune reactions.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

(19) The present invention contemplates an immunogen-specific adjuvant for a vaccine or inoculum. The adjuvant is comprised of particulate recombinant protein body-like assemblies (RPBLAs) that contain a recombinant fusion protein. The recombinant fusion protein contains two sequences peptide-linked together in which one sequence is a protein body-inducing sequence (PBIS) such as a preferred prolamin sequence and the other is a T-cell stimulating immunogenic polypeptide whose sequence (a) is present in a pathogenic polypeptide sequence present in a polypeptide-containing vaccine or inoculum or (b) is encoded by a nucleic acid vaccine or inoculum. The adjuvant, at the concentration used in an inoculum in a host animal without a prior priming by vaccination or inoculation of a host animal or additional immunogen, does not induce production of antibodies in that host animal that immunoreact with or induce T cell activation to the pathogenic sequence.

(20) The invention also contemplates a method for inducing an T-cell mediated immune response in a subject in need thereof against an immunogenic peptide which comprises the administration to a subject in need thereof of a vaccine selected from the group of (i) a particulate recombinant protein body-like assemblies (RPBLAs) that contain a recombinant fusion protein, said recombinant fusion protein containing two portions peptide-linked together in which a first portion is a protein body-inducing sequence (PBIS) and a second portion is a immunogenic polypeptide and (ii) a nucleic acid molecule that encodes a fusion protein, said recombinant fusion protein containing two portions peptide-linked together in which a first portion is a protein body-inducing sequence (PBIS) and a second portion is a immunogenic polypeptide.

(21) In a preferred embodiment, the method of the invention is carried out using a RPBLA wherein the PBIS forming part of the first portion includes a prolamin sequence. In a still more preferred embodiment, the prolamin sequence is present in a prolamin selected from the group consisting of gamma-zein, alpha-zein, delta-zein, beta-zein, rice prolamin and gamma-gliadin.

(22) In a preferred embodiment, the PBIS sequence further includes a signal peptide sequence that directs a protein towards the endoplasmic reticulum (ER) of the RPBLA-expressing cell.

(23) In a preferred embodiment, the immunogenic peptide used in fusion protein forming the RPBLA is a peptide capable of stimulating the T-cell immune response.

(24) In another preferred embodiment, the method of the invention is carried out using a RPBLA comprising a second portion wherein the immunogenic polypeptide sequence is selected from the group of (i) a polypeptide encoded by the HPV E7 gene, (ii) a polypeptide encoded by the HIV-1 gag gene and (iii) a polypeptide encoded by the HIV-1 pol gene

(25) In a preferred embodiment, the method of the invention is carried out using a particulate recombinant protein body-like assemblies (RPBLAs) are assembled in vitro from the purified recombinant fusion protein.

(26) In a more preferred embodiment, the administration step of the method of the invention is preceded by a priming vaccination or inoculation step using a composition comprising immunogenic polypeptide or a nucleic acid encoding said immunogenic polypeptide.

(27) In a still more preferred embodiment, the composition comprising the immunogenic polypeptide used in the priming vaccination or stimulation step is selected from the group of (i) a particulate recombinant protein body-like assemblies (RPBLAs) that contain a recombinant fusion protein, said recombinant fusion protein containing two portions peptide-linked together in which a first portion is a protein body-inducing sequence (PBIS) and a second portion is the T-cell stimulating immunogenic polypeptide, (ii) a nucleic acid molecule that encodes the immunogenic polypeptide and (iii) a nucleic acid molecule that encodes a fusion protein, said recombinant fusion protein containing two portions peptide-linked together in which a first portion is a protein body-inducing sequence (PBIS) and a second portion is the immunogenic polypeptide.

(28) In a preferred embodiment, the immunogenic peptide used in fusion protein forming the RPBLA is a peptide capable of stimulating the T-cell immune response.

(29) In a more preferred embodiment, the vaccine is administered intramuscularly.

(30) In another aspect, the invention relates to vaccine for use in a method for inducing an T-cell mediated immune response in a subject in need thereof against an immunogenic peptide wherein the vaccine is selected from the group of (i) a particulate recombinant protein body-like assemblies (RPBLAs) that contain a recombinant fusion protein, said recombinant fusion protein containing two portions peptide-linked together in which a first portion is a protein body-inducing sequence (PBIS) and a second portion is a immunogenic polypeptide and (ii) a nucleic acid molecule that encodes a fusion protein, said recombinant fusion protein containing two portions peptide-linked together in which a first portion is a protein body-inducing sequence (PBIS) and a second portion is a immunogenic polypeptide.

(31) A contemplated adjuvant can be administered along with or separately as a boost to an anti-pathogen vaccine or inoculum. Such a vaccine or inoculum can contain an attenuated live or killed pathogen such as a bacterium or virus, a subunit vaccine or inoculum that contains only a protein portion of a pathogen, or a vaccine or inoculum that contains an immunogen that is comprised of a polypeptide linked to a carrier, wherein the immunogenic portion of the vaccine or inoculum contains a polypeptide sequence that is also present in the adjuvant. Where the vaccine or inoculum is a nucleic acid preparation such as a DNA or RNA vaccine, the nucleic acid encodes an immunogenic amino acid sequence that is also present in the adjuvant. Nucleic acid vaccines and inocula are themselves well known.

(32) A contemplated adjuvant can be administered as a preparation of expressed RPBLAs, or as a nucleic acid preparation, such as a single or double stranded DNA sequence, that encodes the RPBLAs. In the latter circumstance, the RPBLAs are expressed in vivo in the host animal. In either situation, media in which the expressed RPBLAs or nucleic acids are dissolved or dispersed to form adjuvant compositions are also well known.

(33) Illustrative nucleic acid sequences are provided hereinafter that encode specific portions of the RPBLAs. As is well known in the art, particular codons are preferred for encoding amino acid residues in different animals, and as a consequence the skilled worker can revise specific nucleic acid sequences to provide desired degrees of expression. In addition, several vectors are well known for expressing foreign nucleic acids and their encoded proteins in animal hosts, including humans. On expression, the polypeptides encoded self-assemble in vivo to form RPBLAs.

(34) T-cell stimulating immunogenic polypeptide portions of a number of illustrative adjuvants are discussed hereinafter that relate to the HIV-1 virus. In those adjuvants, a DNA vaccine that comprises all of parts of the gag gene is utilized. The HIV-1 gag gene encodes four proteins: the P24 capsid (CA), P17 matrix (MA), and two nucleocapsid proteins (NC) P6 and P9. Illustrative adjuvants' fusion proteins contain the gag-encoded P24 sequence or the P41 sequence that results from fusion of the P24 and P17 sequences, and the reverse transcriptase (RT) that is encoded by the HIV-1 pol gene.

(35) Analogously, a vaccine or inoculum against hepatitis B virus (HBV) the utilizes one or more of the surface (HBsAg) proteins as immunogen can utilize an adjuvant whose T-cell stimulating immunogenic polypeptide portion includes a sequence illustrated of a surface protein that includes the PreS1 and/or PreS2 portions of the surface protein in the table of T Cell Epitopes that follows. One such vaccine is that sold under the name RECOMBIVAX HB hepatitis B vaccine that is a non-infectious subunit viral vaccine derived from the hepatitis B surface antigen (HBsAg) produced in yeast cells and developed in the Merck Research Laboratories. Similarly, the HBV core-based vaccine of U.S. Pat. No. 7,351,413, can be provided an adjuvant by utilization of one or more core sequences set out in the table of T Cell Epitopes that follows. Additional adjuvants can be prepared as discussed herein using the sequences in that following table or other T cell epitopes obtained from the literature.

(36) A contemplated adjuvant is typically used in an adjuvant-effective amount dissolved or dispersed in a pharmaceutically acceptable diluent as an adjuvant composition. The amount utilized can vary widely in different host animals, with the T-cell stimulating immunogenic polypeptide portion used, and the construct used. Typical amounts are about 1 microgram (g) of RPBLAs per kilogram (kg) of host body weight (g/kg) to about 1 milligram (mg) of RPBLAs per kilogram of host body weight (mg/kg). More usual amounts are about 5 g/kg of host body weight to about 0.5 mg/kg host body weight.

(37) The diluent is typically aqueous-based and can include one or more additional adjuvants, buffers, salts and viscosity enhancing agents. The ingredients of the diluent are those materials that are often present in a vaccine or inoculum as are discussed hereinafter.

(38) Protein Bodies and Protein Body-Inducing Sequences

(39) Inasmuch as protein bodies (PBs) are appropriately so-named only in seeds, similar structures produced in other plant organs and in non-higher plants are referred to generally as synthetic PBs or recombinant protein body-like assemblies (RPBLAs). Such RPBLAs are membrane-enclosed fusion proteins that are found associated with the endoplasmic reticulum (ER) of a cell.

(40) Purified RPBLAs are membrane free preparations of RPBLAs in which the membrane has been removed, usually by chemical reduction as with a mercaptan-containing reagent, and the fusion protein purified as by chromatographic means to free the fusion protein from the membrane and other expression-associated impurities. The resulting purified protein is then reassembled in vitro to form purified RPBLA particles. That reformation of particles typically takes place in an aqueous composition in the presence of salts and an oxidizing environment. The formation of such purified RPBLAs is illustrated hereinafter.

(41) A contemplated RPBLA is a recombinantly prepared fusion protein (polypeptide) that is expressed in a cell foreign to the nucleic acids used to transform the cell. The cell(s) in which the polypeptide is expressed is a host cell(s), and can be a cell preparation or cells of an intact organism. The intact organism can itself be a group of single celled organisms such as bacteria or fungi, or multi-celled plants or animals, including humans. When a human is the host, the person is the recipient of a nucleic acid-encoded form of the adjuvant and the adjuvant is administered as part of a treatment regimen.

(42) In living organisms, the amino acid residue sequence of a protein or polypeptide is directly related via the genetic code to the deoxyribonucleic acid (DNA) sequence of the gene that codes for the protein. Thus, through the well-known degeneracy of the genetic code additional DNAs and corresponding RNA sequences (nucleic acids) can be prepared as desired that encode the same fusion protein amino acid residue sequences, but are sufficiently different from a before-discussed gene sequence that the two sequences do not hybridize at high stringency, but do hybridize at moderate stringency.

(43) High stringency conditions can be defined as comprising hybridization at a temperature of about 50-55 C. in 6SSC and a final wash at a temperature of 68 C. in 1-3SSC. Moderate stringency conditions comprise hybridization at a temperature of about 50 C. to about 65 C. in 0.2 to 0.3 M NaCl, followed by washing at about 50 C. to about 55 C. in 0.2SSC, 0.1% SDS (sodium dodecyl sulfate).

(44) A nucleic sequence (DNA sequence or an RNA sequence) that (1) itself encodes, or its complement encodes, a fusion protein containing a protein body-inducing sequence (PBIS) and a polypeptide of interest is also contemplated herein. As is well-known, a nucleic acid sequence such as a contemplated nucleic acid sequence is expressed when operatively linked to an appropriate promoter in an appropriate expression system as discussed elsewhere herein.

(45) Different hosts often have preferences for a particular codon to be used for encoding a particular amino acid residue. Such codon preferences are well known and a DNA sequence encoding a desired fusion protein sequence can be altered, using in vitro mutagenesis for example, so that host-preferred codons are utilized for a particular host in which the fusion protein is to be expressed.

(46) The RPBLAs are usually present in a generally spherical form having a diameter of about 0.5 to about 3 microns () and usually about 1. In some instances, RPBLAs are amorphous in shape and can vary widely in dimensions, but are still found associated with the ER.

(47) The density of RPBLAs is typically greater than that of substantially all of the endogenous host cell proteins, and is typically about 1.1 to about 1.35 g/ml. The high density of contemplated RPBLAs is due to the general ability of the recombinant fusion proteins to assemble as multimers and accumulate.

(48) A contemplated RPBLA used as an adjuvant need not be expressed in a plant. Rather, as disclosed in published US application 20060121573, RPBLAs can be expressed in other transformed eukaryotes, particularly in transformed mammalian cells.

(49) A fusion protein of the adjuvant RPBLAs contains two proteinaceous sequences linked together by a peptide bond as is found in a naturally occurring protein or in a polypeptide expressed by a genetically engineered nucleic acid. In a contemplated fusion protein, one sequence is a protein body-inducing sequence (PBIS) such as that of a prolamin, and the other is a biologically active immunogenic polypeptide. Either of the two portions can be at the N-terminus of the fusion protein. However, it is preferred to have the PBIS at the N-terminus.

(50) A contemplated protein body-inducing sequence (PBIS) is preferably in whole or part from a higher plant. Illustrative, non-limiting examples of PBIS include storage proteins or modified storage proteins, as for instance, prolamins or modified prolamins, prolamin domains or modified prolamin domains. Prolamins are reviewed in Shewry et al., 2002 J. Exp. Bot. 53(370):947-958. A preferred PBIS sequence is present in a prolamin compound sequence such as gamma-zein, alpha-zein, delta-zein, beta-zein, rice prolamin and gamma-gliadin that are discussed hereinafter.

(51) A PBIS includes a sequence that directs a protein towards the endoplasmic reticulum (ER) of the RPBLA-expressing cell. That sequence often referred to as a leader sequence or signal peptide can be from the same plant as the remainder of the PBIS or from a different plant or an animal or fungus. Illustrative signal peptides are the 19 residue gamma-zein signal peptide sequence shown in WO 2004003207 (US 20040005660), the 19 residue signal peptide sequence of alpha-gliadin or 21 residue gamma-gliadin signal peptide sequence (see, Altschuler et al., 1993 Plant Cell 5:443-450; Sugiyama et al., 1986 Plant Sci. 44:205-209; and Rafalski et al., 1984 EMBO J. 3(6):1409-11415 and the citations therein). The pathogenesis-related protein of PR10 class includes a 25 residue signal peptide sequence that is also useful herein. Similarly functioning signal peptides from other plants and animals are also reported in the literature.

(52) The characteristics of the signal peptides responsible for directing the protein to the ER have been extensively studied (von Heijne et al., 2001 Biochim. Biophys. Acta December 12 1541(1-2):114-119). The signal peptides do not share homology at a primary structure, but have a common tripartite structure: a central hydrophobic h-region and hydrophilic N- and C-terminal flanking regions. These similarities, and the fact that proteins are translocated through the ER membrane using apparently common pathways, permits interchange of the signal peptides between different proteins or even from different organisms belonging to different phyla (See, Martoglio et al., 1998 Trends Cell Biol. October; 8(10):410-415). Thus, a PBIS can include a signal peptide of a protein from a phylum different from higher plants.

(53) It is to be understood that an entire prolamin sequence is not required to be used. Rather, as is discussed hereinafter, only portions are needed although an entire prolamin sequence can be used.

(54) Gamma-Zein, a maize storage protein whose DNA and amino acid residue sequences are shown hereinafter, is one of the four maize prolamins and represents 10-15 percent of the total protein in the maize endosperm. As other cereal prolamins, alpha- and gamma-zeins are biosynthesized in membrane-bound polysomes at the cytoplasmic side of the rough ER, assembled within the lumen and then sequestered into ER-derived protein bodies (Herman et al., 1999 Plant Cell 11:601-613; Ludevid et al., 1984 Plant Mol. Biol. 3:277-234; Torrent et al., 1986 Plant Mol. Biol. 7:93-403).

(55) Gamma-Zein is composed of four characteristic domains: i) a peptide signal of 19 amino acids, ii) the repeat domain containing eight units of the hexapeptide PPPVHL (SEQ ID NO:2) [(53 amino acid residues (aa)], iii) the ProX domain where proline residues alternate with other amino acids (29 aa) and iv) the hydrophobic cysteine rich C-terminal domain (lll aa).

(56) The ability of gamma-zein to assemble in ER-derived RPBLAs is not restricted to seeds. In fact, when gamma-zein-gene was constitutively expressed in transgenic Arabidopsis plants, the storage protein accumulated within ER-derived PBLS in leaf mesophyl cells (Geli et al., 1994 Plant Cell 6:1911-1922). Looking for a signal responsible for the gamma-zein deposition into the ER-derived protein bodies (prolamins do not have KDEL signal for ER-retention), it has been demonstrated that the proline-rich N-terminal domain including the tandem repeat domain was necessary for ER retention. In this work, it was also suggested that the C-terminal domain could be involved in protein body formation, however, recent data (WO2004003207A1) demonstrate that the proline-rich N-terminal domain is necessary and sufficient to retain in the ER and to induce the protein body formation. However, the mechanisms by which these domains promote the protein body assembly are still unknown, but evidence from in vitro studies suggests that the N-terminal portion of gamma-zein is able to self-assemble into ordered structures.

(57) It is preferred that a gamma-zein-based PBIS include at least one repeat and the amino-terminal nine residues of the ProX domain, and more preferably the entire Pro-X domain. The C-terminal portion of gamma-zein is not needed, but can be present. Those sequences are shown in US 20040005660 and designated as RX3 and P4, respectively, and are noted hereinafter.

(58) Zeins are of four distinct types: alpha, beta, delta, and gamma. They accumulate in a sequential manner in the ER-derived protein bodies during endosperm development. Beta-zein and delta-zein do not accumulate in large amount in maize PBs, but they were stable in the vegetative tissues and were deposited in ER-derived protein body-like structures when expressed in tobacco plants (Bagga et al., 1997 Plant Cell September 9(9):1683-1696). This result indicates that beta-zein, as well as delta-zein, can induce ER retention and PB formation.

(59) The wheat prolamin storage proteins, gliadins, are a group of K/HDEL-less proteins whose transport via the ER appears to be complex. These proteins sequester in to the ER where they are either retained and packaged into dense protein bodies, or are transported from the ER via the Golgi into vacuoles. (Altschuler et al., 1993 Plant Cell 5:443-450.)

(60) The gliadins appear to be natural chimeras, containing two separately folded autonomous regions. The N-terminus is composed of about 7 to about 16 tandem repeats rich in glutamine and proline. The sequence of the tandem repeats varies among the different gliadins, but are based on one or the other consensus sequences PQQPFPQ (SEQ ID NO:3), PQQQPPFS (SEQ ID NO:4) and PQQPQ (SEQ ID NO:5). The C-terminal region of the protein contains six to eight cysteines that form intramolecular disulfide bonds. The work of the Altschuler et al. indicates that the N-terminal region and consensus sequences are responsible for PB formation in the ER from gamma-gliadin. (Altschuler et al., 1993 Plant Cell 5:443-450.)

(61) Illustrative useful prolamin-type sequences are shown in the Table below along with their GenBank identifiers.

(62) TABLE-US-00001 PROTEIN NAME GENBANK ID -Zein (22 kD) M86591 Albumin (32 kD) X70153 -Zein (27 kD) X53514 -Zein (50 kD) AF371263 -Zein (18 kD) AF371265 7S Globulin or Vicilin type NM_113163 11S Globulin or Legumin type DQ256294 Prolamin 13 kD AB016504 Prolamin 16 kD AY427574 Prolamin 10 kD AF294580 -Gliadin M36999 -Gliadin precursor AAA34272

(63) Further useful sequences are obtained by carrying out a BLAST search in the all non-redundant GenBank CDS translations+PDB+SwissProt+PIR+PRF (excluding environmental samples) data base as described in Altschul et al., 1997 Nucleic Acids Res. 25:3389-3402 using a query such as those shown below:

(64) RX3 Query

(65) TABLE-US-00002 SEQIDNO:6 PPPPVHLPPPVHLPPPVHLPPPVHLPPPVHLPPPVHLPPPVHVPPPVHL PPPP
Alpha-Zein

(66) TABLE-US-00003 SEQIDNO:7 QQQQQFLPALSQLDVVNPVAYLQQQLLASNPLALANVAAYQQQQQLQQF LPALSQLAMVNPAAYL
Rice Prolamin Query

(67) TABLE-US-00004 SEQIDNO:8 QQVLSPYNEFVRQQYGIAASPFLQSATFQLRNNQVWQQLALVAQQSHCQ DINIVQAIAQQLQLQQFGDLY

(68) An illustrative modified prolamin includes (a) a signal peptide sequence, (b) a sequence of one or more copies of the repeat domain hexapeptide PPPVHL (SEQ ID NO: 2) of the protein gamma-zein, the entire domain containing eight hexapeptide units; and (c) a sequence of all or part of the ProX domain of gamma-zein. Illustrative specific modified prolamins include the polypeptides identified below as R3, RX3 and P4 whose DNA and amino acid residue sequences are also shown below.

(69) Particularly preferred prolamins include gamma-zein and its component portions as disclosed in published application WO2004003207, the rice rP13 protein and the 22 kDa maize alpha-zein and its N-terminal fragment. The DNA and amino acid residue sequences of the gamma-zein, rice and alpha-zein proteins are shown below.

(70) Gamma-Zein of 27 kD

(71) DNA Sequence:

(72) TABLE-US-00005 SEQIDNO:9 atgagggtgttgctcgttgccctcgctctcctggctctcg 40 ctgcgagcgccacctccacgcatacaagcggcggctgcgg 80 ctgccagccaccgccgccggttcatctaccgccgccggtg 120 catctgccacctccggttcacctgccacctccggtgcatc 160 tcccaccgccggtccacctgccgccgccggtccacctgcc 200 accgccggtccatgtgccgccgccggttcatctgccgccg 240 ccaccatgccactaccctactcaaccgccccggcctcagc 280 ctcatccccagccacacccatgcccgtgccaacagccgca 320 tccaagcccgtgccagctgcagggaacctgcggcgttggc 360 agcaccccgatcctgggccagtgcgtcgagtttctgaggc 400 atcagtgcagcccgacggcgacgccctactgctcgcctca 440 gtgccagtcgttgcggcagcagtgttgccagcagctcagg 480 caggtggagccgcagcaccggtaccaggcgatcttcggct 520 tggtcctccagtccatcctgcagcagcagccgcaaagcgg 560 ccaggtcgcggggctgttggcggcgcagatagcgcagcaa 600 ctgacggcgatgtgcggcctgcagcagccgactccatgcc 640 cctacgctgctgccggcggtgtcccccacgcc 672
Protein Sequence:

(73) TABLE-US-00006 SEQIDNO:10 MetArgValLeuLeuValAlaLeuAlaLeuLeuAlaLeuAlaAlaSer 151015 AlaThrSerThrHisThrSerGlyGlyCysGlyCysGlnProProPro 202530 ProValHisLeuProProProValHisLeuProProProValHisLeu 354045 ProProProValHisLeuProProProValHisLeuProProProVal 505560 HisLeuProProProValHisValProProProValHisLeuProPro 65707580 ProProCysHisTyrProThrGlnProProArgProGlnProHisPro 859095 GlnProHisProCysProCysGlnGlnProHisProSerProCysGln 100105110 LeuGlnGlyThrCysGlyValGlySerThrProIleLeuGlyGlnCys 115120125 ValGluPheLeuArgHisGlnCysSerProThrAlaThrProTyrCys 130135140 SerProGlnCysGlnSerLeuArgGlnGlnCysCysGlnGlnLeuArg 145150155160 GlnValGluProGlnHisArgTyrGlnAlaIlePheGlyLeuValLeu 165170175 GlnSerIleLeuGlnGlnGlnProGlnSerGlyGlnValAlaGlyLeu 180185190 LeuAlaAlaGlnIleAlaGlnGlnLeuThrAlaMetCysGlyLeuGln 195200205 GlnProThrProCysProTyrAlaAlaAlaGlyGlyValProHisAla 210215220
RX3
DNA Sequence:

(74) TABLE-US-00007 SEQIDNO:11 atgagggtgttgctcgttgccctcgctctcctggctctcg 40 ctgcgagcgccacctccacgcatacaagcggcggctgcgg 80 ctgccagccaccgccgccggttcatctaccgccgccggtg 120 catctgccacctccggttcacctgccacctccggtgcatc 160 tcccaccgccggtccacctgccgccgccggtccacctgcc 200 accgccggtccatgtgccgccgccggttcatctgccgccg 240 ccaccatgccactaccctactcaaccgccccggcctcagc 280 ctcatccccagccacacccatgcccgtgccaacagccgca 320 tccaagcccgtgccagacc 339
Protein Sequence:

(75) TABLE-US-00008 SEQIDNO:12 MetArgValLeuLeuValAlaLeuAlaLeuLeuAlaLeuAlaAlaSer 151015 AlaThrSerThrHisThrSerGlyGlyCysGlyCysGlnProProPro 202530 ProValHisLeuProProProValHisLeuProProProValHisLeu 354045 ProProProValHisLeuProProProValHisLeuProProProVal 505560 HisLeuProProProValHisValProProProValHisLeuProPro 65707580 ProProCysHisTyrProThrGlnProProArgProGlnProHisPro 859095 GlnProHisProCysProCysGlnGlnProHisProSerProCysGln 100105110 Tyr
R3
DNA Sequence:

(76) TABLE-US-00009 SEQIDNO:13 atgagggtgttgctcgttgccctcgctctcctggctctcg 40 ctgcgagcgccacctccacgcatacaagcggcggctgcgg 80 ctgccagccaccgccgccggttcatctaccgccgccggtg 120 catctgccacctccggttcacctgccacctccggtgcatc 160 tcccaccgccggtccacctgccgccgccggtccacctgcc 200 accgccggtccatgtgccgccgccggttcatctgccgccg 240
Protein Sequence:

(77) TABLE-US-00010 SEQIDNO:14 MetArgValLeuLeuValAlaLeuAlaLeuLeuAlaLeuAlaAlaSer 151015 AlaThrSerThrHisThrSerGlyGlyCysGlyCysGlnProProPro 202530 ProValHisLeuProProProValHisLeuProProProValHisLeu 354045 ProProProValHisLeuProProProValHisLeuProProProVal 505560 HisLeuProProProValHisValProProProValHisLeuProPro 65707580 ProProCysHisTyrProThrGlnProProArgTyr 8590
P4
DNA Sequence:

(78) TABLE-US-00011 SEQIDNO:15 atgagggtgttgctcgttgccctcgctctcctggctctcg 40 ctgcgagcgccacctccacgcatacaagcggcggctgcgg 80 ctgccagccaccgccgccggttcatctgccgccgccacca 120 tgccactaccctacacaaccgccccggcctcagcctcatc 160 cccagccacacccatgcccgtgccaacagccgcatccaag 200 cccgtgccagacc 213
Protein Sequence:

(79) TABLE-US-00012 SEQIDNO:16 MetArgValLeuLeuValAlaLeuAlaLeuLeuAlaLeuAlaAlaSer 151015 AlaThrSerThrHisThrSerGlyGlyCysGlyCysGlnProProPro 202530 ProValHisLeuProProProProCysHisTyrProThrGlnProPro 354045 ArgProGlnProHisProGlnProHisProCysProCysGlnGlnPro 505560 HisProSerProCysGlnTyr 6570
X10
DNA Sequence:

(80) TABLE-US-00013 SEQIDNO:17 atgagggtgttgctcgttgccctcgctctcctggctctcg 40 ctgcgagcgccacctccacgcatacaagcggcggctgcgg 80 ctgccaatgccactaccctactcaaccgccccggcctcag 120 cctcatccccagccacacccatgcccgtgccaacagccgc 160 atccaagcccgtgccagacc 180
Protein Sequence:

(81) TABLE-US-00014 SEQIDNO:18 MetArgValLeuLeuValAlaLeuAlaLeuLeuAlaLeuAlaAlaSer 151015 AlaThrSerThrHisThrSerGlyGlyCysGlyCysGlnCysHisTyr 202530 ProThrGlnProProArgProGlnProHisProGlnProHisProCys 354045 ProCysGlnGlnProHisProSerProCysGlnTyr 505560
rP13rice prolamin of 13 kD homologous to the cloneAB016504 Sha et al., 1996 Biosci. Biotechnol. Biochem. 60(2):335-337; Wen et al., 1993 Plant Physiol. 101(3):1115-1116; Kawagoe et al., 2005 Plant Cell 17(4):1141-1153; Mullins et al., 2004 J. Agric. Food Chem. 52(8):2242-2246; Mitsukawa et al., 1999 Biosci. Biotechnol. Biochem. 63(11):1851-1858
Protein Sequence:

(82) TABLE-US-00015 SEQIDNO:19 MKIIFVFALLAIAACSASAQFDVLGQSYRQYQLQSPVLLQQQVLSPYNEF VRQQYGIAASPFLQSATFQLRNNQVWQQLALVAQQSHCQDINIVQAIAQQ LQLQQFGDLYFDRNLAQAQALLAFNVPSRYGIYPRYYGAPSTITTLGGVL
DNA Sequence:

(83) TABLE-US-00016 SEQIDNO:20 atgaagatcattttcgtctttgctctccttgctattgctgcatgcagcg cctctgcgcagtttgatgttttaggtcaaagttataggcaatatcagct gcagtcgcctgtcctgctacagcaacaggtgcttagcccatataatgag ttcgtaaggcagcagtatggcatagcggcaagccccttcttgcaatcag ctacgtttcaactgagaaacaaccaagtctggcaacagctcgcgctggt ggcgcaacaatctcactgtcaggacattaacattgttcaggccatagcg cagcagctacaactccagcagtttggtgatctctactttgatcggaatc tggctcaagctcaagctctgttggcttttaacgtgccatctagatatgg tatctaccctaggtactatggtgcacccagtaccattaccacccttggc ggtgtcttg
22aZt N-terminal fragment of the maize alpha-zein of 22 kDV01475 Kim et al., 2002 Plant Cell 14(3):655-672; Woo et al., 2001 Plant Cell 13(10):2297-2317; Matsushima et al., 1997 Biochim. Biophys. Acta 1339(1):14-22; Thompson et al., 1992 Plant Mol. Biol. 18(4):827-833.
Protein Sequence (Full Length):

(84) TABLE-US-00017 SEQIDNO:21 MATKILALLALLALFVSATNAFIIPQCSLAPSAIIPQFLPPVTSMGFEHL AVQAYRLQQALAASVLQQPINQLQQQSLAHLTIQTIATQQQQQFLPALSQ LDVVNPVAYLQQQLLASNPLALANVAAYQQQQQLQQFLPALSQL
DNA Sequence (Full Length):

(85) TABLE-US-00018 SEQIDNO:22 atggctaccaagatattagccctccttgcgcttcttgccctttttgtgag cgcaacaaatgcgttcattattccacaatgctcacttgctcctagtgcca ttataccacagttcctcccaccagttacttcaatgggcttcgaacaccta gctgtgcaagcctacaggctacaacaagcgcttgcggcaagcgtcttaca acaaccaattaaccaattgcaacaacaatccttggcacatctaaccatac aaaccatcgcaacgcaacagcaacaacagttcctaccagcactgagccaa ctagatgtggtgaaccctgtcgcctacttgcaacagcagctgcttgcatc caacccacttgctctggcaaacgtagctgcataccaacaacaacaacaat tgcagcagtttctgccagcgctcagtcaacta
Gamma-Gliadin precursorAAA34272Scheets et al., 1988 Plant Sci. 57:141-150.
Protein Sequence:

(86) TABLE-US-00019 SEQIDNO:23 NMQVDPSGQVQWPQQQPFPQPQQPFCQQPQRTIPQPHQTF HHQPQQTFPQPQQTYPHQPQQQFPQPQQPQQPFPQPQQTF PQQPQLPFPQQPQQPFPQPQQPQQPFPQSQQPQQPFPQPQ QQFPQPQQPQQSFPQQQQPAIQSFLQQQMNPCKNFLLQQC NHVSLVSSLVSIILPRSDCQVMQQQCCQQLAQIPQQLQCA AIHSVAHSIIMQQEQQQGVPILRPLFQLAQGLGIIQPQQP AQLEGIRSLVLKTLPTMCNVYVPPDCSTINVPYANIDAGIGGQ
DNA Sequence (M36999)

(87) TABLE-US-00020 SEQIDNO:24 gcatgcattgtcaaagtttgtgaagtagaattaataacct tttggttattgatcactgtatgtatcttagatgtcccgta gcaacggtaagggcattcacctagtactagtccaatatta attaataacttgcacagaattacaaccattgacataaaaa ggaaatatgatgagtcatgtattgattcatgttcaacatt actacccttgacataaaagaagaatttgacgagtcgtatt agcttgttcatcttaccatcatactatactgcaagctagt ttaaaaaagaatyaaagtccagaatgaacagtagaatagc ctgatctatctttaacaacatgcacaagaatacaaattta gtcccttgcaagctatgaagatttggtttatgcctaacaa catgataaacttagatccaaaaggaatgcaatctagataa ttgtttgacttgtaaagtcgataagatgagtcagtgccaa ttataaagttttcgccactcttagatcatatgtacaataa aaaggcaactttgctgaccactccaaaagtacgtttgtat gtagtgccaccaaacacaacacaccaaataatcagtttga taagcatcgaatcactttaaaaagtgaaagaaataatgaa aagaaacctaaccatggtagctataaaaagcctgtaatat gtacactccataccatcatccatccttcacacaactagag cacaagcatcaaatccaagtaagtattagttaacgcaaat ccaccatgaagaccttactcatcctaacaatccttgcgat ggcaacaaccatcgccaccgccaatatgcaagtcgacccc agcggccaagtacaatggccacaacaacaaccattccccc agccccaacaaccattctgccagcaaccacaacgaactat tccccaaccccatcaaacattccaccatcaaccacaacaa acatttccccaaccccaacaaacatacccccatcaaccac aacaacaatttccccagacccaacaaccacaacaaccatt tccccagccccaacaaacattcccccaacaaccccaacta ccatttccccaacaaccccaacaaccattcccccagcctc agcaaccccaacaaccatttccccagtcacaacaaccaca acaaccttttccccagccccaacaacaatttccgcagccc caacaaccacaacaatcattcccccaacaacaacaaccgg cgattcagtcatttctacaacaacagatgaacccctgcaa gaatttcctcttgcagcaatgcaaccatgtgtcattggtg tcatctctcgtgtcaataattttgccacgaagtgattgcc aggtgatgcagcaacaatgttgccaacaactagcacaaat tcctcaacagctccagtgcgcagccatccacagcgtcgcg cattccatcatcatgcaacaagaacaacaacaaggcgtgc cgatcctgcggccactatttcagctcgcccagggtctggg tatcatccaacctcaacaaccagctcaattggaggggatc aggtcattggtattgaaaactcttccaaccatgtgcaacg tgtatgtgccacctgactgctccaccatcaacgtaccata tgccaacatagacgctggcattggtggccaatgaaaaatg caagatcatcattgcttagctgatgcaccaatcgttgtag cgatgacaaataaagtggtgtgcaccatcatgtgtgaccc cgaccagtgctagttcaagcttgggaataaaagacaaaca aagttcttgtttgctagcattgcttgtcactgttacattc actttttatttcgatgttcatccctaaccgcaatcctagc cttacacgtcaatagctagctgcttgtgctggcaggttac tatataatctatcaattaatggtcgacctattaatccaag taataggctattgatagactgctcccaagccgaccgagca cctatcagttacggatttcttgaacattgcacactataat aattcaacgtatttcaacctctagaagtaaagggcattttagtagc
Beta zeinAF371264Woo et al., (2001) Plant Cell 13 (10), 2297-2317.
DNA

(88) TABLE-US-00021 SEQIDNO:25 atgaagatggtcatcgttctcgtcgtgtgcctggctctgtcagctgccag cgcctctgcaatgcagatgccctgcccctgcgcggggctgcagggcttgt acggcgctggcgccggcctgacgacgatgatgggcgccggcgggctgtac ccctacgcggagtacctgaggcagccgcagtgcagcccgctggcggcggc gccctactacgccgggtgtgggcagccgagcgccatgttccagccgctcc ggcaacagtgctgccagcagcagatgaggatgatggacgtgcagtccgtc gcgcagcagctgcagatgatgatgcagcttgagcgtgccgctgccgccag cagcagcctgtacgagccagctctgatgcagcagcagcagcagctgctgg cagcccagggtctcaaccccatggccatgatgatggcgcagaacatgccg gccatgggtggactctaccagtaccagctgcccagctaccgcaccaaccc ctgtggcgtctccgctgccattccgccctactactga
Protein

(89) TABLE-US-00022 SEQIDNO:26 MKMVIVLVVCLALSAASASAMQMPCPCAGLQGLYGAGAGLTTMMGAGGLY PYAEYLRQPQCSPLAAAPYYAGCGQPSAMFQPLRQQCCQQQMRMMDVQSV AQQLQMMMQLERAAAASSSLYEPALMQQQQQLLAAQGLNPMAMMMAQNMP AMGGLYQYQLPSYRTNPCGVSAAIPPYY
Delta zein 10 kDAF371266Woo et al., (2001) Plant Cell 13 (10), 2297-2317, and Kirihara et al., (1988) Gene. November 30; 71(2):359-70.
DNA

(90) TABLE-US-00023 SEQIDNO:27 atggcagccaagatgcttgcattgttcgctctcctagctctttgtgcaag cgccactagtgcgacgcatattccagggcacttgccaccagtcatgccat tgggtaccatgaacccatgcatgcagtactgcatgatgcaacaggggctt gccagcttgatggcgtgtccgtccctgatgctgcagcaactgttggcctt accgcttcagacgatgccagtgatgatgccacagatgatgacgcctaaca tgatgtcaccattgatgatgccgagcatgatgtcaccaatggtcttgccg agcatgatgtcgcaaatgatgatgccacaatgtcactgcgacgccgtctc gcagattatgctgcaacagcagttaccattcatgttcaacccaatggcca tgacgattccacccatgttcttacagcaaccctttgttggtgctgcattc tag
Protein

(91) TABLE-US-00024 SEQIDNO:28 MAAKMLALFALLALCASATSATHIPGHLPPVMPLGTMNPCMQYCMMQQGL ASLMACPSLMLQQLLALPLQTMPVMMPQMMTPNMMSPLMMPSMMSPMVLP SMMSQMMMPQCHCDAVSQIMLQQQLPFMFNPMAMTIPPMFLQQPFVGAAF
Signal Peptides
Gamma-Zein

(92) TABLE-US-00025 SEQIDNO:29 MetArgValLeuLeuValAlaLeuAlaLeuLeuAla LeuAlaAlaSerAlaThrSer
Alpha-Gliadin

(93) TABLE-US-00026 SEQIDNO:30 MetLysThrPheLeuIleLeuValLeuLeuAlaIle ValAlaThrThrAlaThrThrAla
Gamma-Gliadin

(94) TABLE-US-00027 SEQIDNO:31 MetLysThrLeuLeuIleLeuThrIleLeuAlaMet AlaIleThrIleGlyThrAlaAsnMet
PR10

(95) TABLE-US-00028 SEQIDNO:32 MetAsnPheLeuLysSerPheProPheTyrAlaPhe LeuCysPheGlyGlnTyrPheValAlaValThrHis Ala
T-Cell Stimulating Immunogenic Polypeptides

(96) A large number of T-cell-stimulating immunogenic polypeptide sequences have been identified in the literature. A partial list is provided below in the table below using the single letter code.

(97) TABLE-US-00029 TCellEpitopes SEQ Organism Protein Sequence* Citation IDNO HIV P24 GPKEPFRDY- 1 33 VDRFYKC Corynebacterium toxin FQVVHNSYN- 2 34 diptheriae RPAYSPGC Borrelia ospA VEIKEGTVTLKRE- 3 35 burgdorferi IDKNGKVTVSLC TLSKNISKSG- 4 36 EVSVELNDC InfluenzaVirus HA SSVSSFERFEC 5 37 A8/PR8 LIDALLGDPC 6 38 TLIDALLGC 6 39 NP FWRGENGRKTRS- 7 40 AYERMCNILKGK LRVLSFIRGTKV- 7 41 SPRGKLSTRG SLVGIDPFKLLQ- 7 42 NSQVYSLIRP AVKGVGTMVMEL- 7 43 IRMIKRGINDRN Trypanosoma SHNFTLVASVII- 8 44 cruzi EEAPSGNTC Plasmodium MSP1 SVQIPKVPYPNGIVYC 9 45 falciparum DFNHYYTLKTGLEADC 46 PSDKHIEQYKKI- 10 47 KNSISC EYLNKIQNSLST- 11 48 EWSPCSVT P.vivax YLDKVRATVGTE- 22 49 WTPCSVT P.yoelii EFVKQISSQLTE- 22 50 EWSQCSVT Streptococcus AgI/II KPRPIYEAKL- 12 51 sobrinus AQNQKC AKADYEAKLA- 52 QYEKDLC LCMV(lymphocytic NP RPQASGVYM- 13 53 choriomeningitisvirus) GNLTAQC Clostridium tox QYIKANSKFIG- 14 54 tetani ITELC Neisseria PorB AIWQVEQKASIAGTDSGWC 21 55 meningitidis NYKNGGFFVQYGGAYKRHC 21 56 HNSQTEVAATLAYRFGNVC 21 57 PorB TPRVSYAHGFKGLVDDADC 21 58 RFGNAVPRISYAHGFDFIC 21 59 AFKYARHANVGRNAFELFC 21 60 SGAWLKRNTGIGNYTQINAC 21 61 AGEFGTLRAGRVANQC 21 62 IGNYTQINAASVGLRC 21 63 GRNYQLQLTEQPSRTC 21 64 SGSVQFVPAQNSKSAC 21 65 HANVGRDAFNLFLLGC 21 66 LGRIGDDDEAKGTDPC 21 67 SVQFVPAQNSKSAYKC 21 68 NYAFKYAKHANVGRDC 21 69 AHGFDFIERGKKGENC 21 70 GVDYDFSKRTSAIVSC 21 71 HDDMPVSVRYDSPDFC 21 72 RFGNAVPRISYAHGFD3 FIERGKKGENC 21 73 NYAFKYAKHANVGRDA- 21 74 FNLFLLGC SGAWLKRNTGIGNYTQ- 21 75 INAASVGLRC SGSVQFVPAQNSKSAYTPAC 21 76 OpaB TGANNTSTVSDYFRNRITC 21 77 IYDFKLNDKFDKFKPYIGC 21 78 Opa-5d LSAIYDFKLNDKFKPYIGC 21 79 Opac NGWYINPWSEVKFDLNSRC 21 80 HepatitisB Surface MGTNLSVPN- 15,16 81 PreS1 PLGFFPDHQLDP PLGFFPDH 82 PLGFFPDHQL 83 PreS2 MQWNSTAFHQ- 15 84 TLQDPRVRG- LYLPAGG MQWSTAFHQ- 85 TLQDP MQWNSTALHQ- 86 ALQDP QDPRVR 17 87 Core MDIDPYKEFGAT- 18 88 VELLSFLP RDLLDTASALYR- 18 89 EALESPEHCSPHH TWVGVNLEDPAS- 18 90 RDLVVSYVNTNMG VVSYVNTNMGL- 18 91 KFRQL LLWFHISCLTF- 18 92 GRETVIEYLV LLWFHISCLTF- 18 93 VSFGVWIRTPP- 18 94 AYRPPNAPIL VSFGVWIRTPPA 18 95 PPAYRPPNAPIL 18 96 WIRTPPAYRPPN 18 97 PHHTALRQAIL- 19 98 CWGELMTLA M.tuberculosis 65KDProtein AVLEDPYILLVSSKV 20 99 LLVSSKVSTVKDLLP 20 100 LLPLLEKVIGAGKPL 20 101 AILTGGQVISEEVGL 20 102 IAFNSGLEPGVVAEK 20 103 ARRGLERGLNAL- 20 104 ADAVKV EKIGAELVKEVAKK 20 105 GLKRGIEKAVEKVETL 20 106 IEDAVRNAKAAVEEG 20 107 HPV-16 E6Protein TIHDIILEC 23 121 FAFRDLCIVY 23 122 E7Protein YMLDLQPETT 23 123 LEDLLMGTL 23 124 DLYCYEQLN 24 125 *Underlined C (C) is not from the native sequence.
Citations: 1. U.S. Pat. No. 5,639,854. 2. EPO 399001 B1. 3. Bockenstedt et al. (1996) J. Immunol., 157(12):5496-5502. 4. Zhong et al. (1996) Eur. J. Immunol., 26(11):2749. 5. Brumeanu et al. (1996) Immunotechnology, 2(2):85-95. 6. Brown et al. (1993) J. Virol., 67(5):2887-2893. 7. Brett et al., (1991) J. Immunol., 147(3):984-991. 8. Kahn et al. (1997) J. Immunol., 159(9):4444. 9. Ohta et al. (1997) Int. Arch. Allergy Immunol., 114(1):15. 10. U.S. Pat. No. 4,886,782. 11. Calvo-Calle et al. (1997) J. Immunol. 159(3):1362-1373. 12. Staffileno et al. (1990) Arch. Oral Biol., 35: Suppl. 47S. 13. Saron et al. (1997) Proc. Natl. Acad. Sci. USA 94(7):3314-3319. 14. Yang et al. (1997) Vaccine, 15(4):377-386. 15. Neurath et al., (1986) F. Brown et al. eds., Vaccines 85, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., pp. 185-189. 16. Milich et al., (1987) F. Brown et al. eds., Vaccines 87, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., pp. 50-55. 17. Kent et al., (1987) F. Brown et al. eds., Vaccines 86, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., pp. 365-369. 18. U.S. Pat. No. 4,882,145. 19. Alexander et al., (1994) Immunity 1:751-761. 20. U.S. Pat. No. 5,478,726 21. WO 03/072731 22. U.S. Pat. No. 6,942,866 23. U.S. Patent Publication 20060182763 24. U.S. Pat. No. 7,329,498.

(98) A group of preferred T-cell stimulating immunogenic sequences are present in HPV-16, in the E7 gene. In order to translate the previously discussed therapeutic DNA vaccine candidate [Osen et al., 2001 Vaccine 19(30):4276-4286] into a vaccine for use in a clinical trial, the safety features were further enhanced. For this reason, no heterologous genes were fused. Rather, immunogenicity was enhanced by placing a Kozak-sequence [Kozak et al., 1987 Nucleic Acids Res 15(20):8125-8148] in front of the gene [Steinberg et al., 2005 Vaccine 23(9):1149-1157]. A plasmid-vector pTHamp [Hanke et al., (1998) Vaccine 16(4):426-435] applicable to humans [Hanke et al., 2000 Nat Med 6(9):951-955] was selected. Many expression vectors are known and available for use for DNA vaccines. See for example, U.S. Pat. No. 7,351,813 B2 and EP 1 026 253 B1 and the citations therein.

(99) More importantly E7 itself was redesigned. The sequence was taken apart exactly at the positions that are critical for transforming properties of the protein (pRB-binding site, C-X-X-C motifs) and reassembled in a shuffled order as core gene. This sequence was codon optimized to humans (almost identical to mice). The original junctions destroyed by the dissection were added as an appendix with a non-codon optimized sequence to minimize recombination events reconstituting the wild-type sequences (see also FIG. 6).

(100) Tumor protection and regression studies provide a first impression on immunogenicity and effectivity of tumor vaccines. Those studies do not fully reflect, however, the responses induced in humans. In vitro immunization of human lymphocytes by antigen-loaded dendritic cells (DCs) can be used as a model of human responses [Norm et al., 2003 J Cancer Res Clin Oncol 129(9):511-520]. Loading of DCs by DNA transfection is a suitable technique [Lohmann et al., 2000 Cancer Gene Ther 7(4):605-614] and specific T cell priming verifies the potential immunogenicity of the DNA vaccine candidate.

(101) The results shown hereinafter illustrate that the HPV-16 E7SH DNA vaccine candidate of the second generation induces specific immunity in vivo in mice and after in vitro immunization of human lymphocytes and, therefore, can provide for a safe therapeutic HPV-vaccine.

(102) The sequence of the gene that expresses the HPV-16 E7SH protein is as follows from 5 to 3:

(103) TABLE-US-00030 SEQIDNO:126 CCCGCCGCCACCATGCACGGCGACACCCCCACCCTG CACGAGTACATGCTGGACCTGCAGCCCGAGACCACC GACCTGTACTGCATCTGCAGCCAGAAACCCAAGTGC GACAGCACCCTGCGGCTGTGCGTGCAGAGCACCCAC GTGGACATCCGGACCCTGGAGGACCTGCTGATGGGC ACCCTGGGCATCGTGTGCCCCTACGAGCAGCTGAAC GACAGCAGCGAGGAGGAGGATGAGATCGACGGCCCC GCCGGCCAGGCTGAGCCCGACCGGGCCCACTACAAC ATCGTGACCTTCTGCTGCCAACCAGAGACAACTGAT CTCTACTGTTATGAGCAATTAAATGACAGCTCAGAG CATTACAATATTGTAACCTTTTGTTGCAAGTGTGAC TCTACGCTTCGGTTGTGCATGGGCACACTAGGAATT GTGTGCCCCATCTGTTCTCAGAAACCATAA

(104) Another group of preferred T-cell stimulating immunogenic sequences are present in HIV-1. In particularly preferred practice, a polypeptide sequence present in HIV-1 is encoded by the HIV-1 gag gene. These sequences are thus present in the P24, P17, P6 or P9 proteins encoded by the gag gene, or a polypeptide such as the P41 polypeptide.

(105) A particular T-cell stimulating immunogenic sequence need not itself be present as a distinct polypeptide in HIV-1 or any other pathogen. Rather, such a sequence is present as a portion of a distinct polypeptide or proteinaceous material encoded by an open reading frame of a pathogenic genome.

(106) Specific T-cell stimulating immunogenic sequences useful herein are provided below.

(107) p41 DNA Sequence 5 to 3

(108) TABLE-US-00031 SEQIDNO:108 1 ATGGGTGCTAGAGCTTCTATTCTTAGAGGTGAAAAGCTTGATAAGTGGGAAAAGATTAGA 61 CTTAGACCAGGTGGTAAGAAGCATTATATGCTTAAGCATATTGTTTGGGCTTCTAGAGAA 121 CTTGAAAGATTTGCTCTTAATCCAGGTTTGCTTGAAACTTCTGAAGGTTGTAAGCAAATT 181 ATGAAGCAACTTCAACCAGCTCTTCAAACTGGTACTGAAGAACTTAAGTCTCTTTATAAT 241 ACTGTTGCTACTCTTTATTGTGTTCATGAAAAGATTGAAGTTAGAGATACTAAGGAAGCT 301 CTTGATAAGATTGAAGAAGAACAAAATAAGTGTCAACAAAAGACTCAACAAGCTAAGGCT 361 GCTGATGGTAAGGTTTCTCAAAATTATCCAATTGTTCAAAATCTTCAAGGTCAAATGGTT 421 CATCAAGCTATTTCTCCAAGAACTCTTAATGCTTGGGTTAAGGTTATTGAAGAAAAGGCT 481 TTTTCTCCAGAAGTTATTCCAATGTTTACTGCTCTTTCTGAAGGTGCTACTCCACAAGAT 541 CTTAATACTATGCTTAATACTGTTGGTGGTCATCAAGCTGCTATGCAAATGCTTAAGGAT 601 ACTATTAATGAAGAAGCTGCTGAATGGGATAGACTTCATCCAGTTCATGCTGGTCCAATT 661 GCTCCAGGTCAAATGAGAGAACCAAGAGGTTCTGATATTGCTGGTACTACTTCTACTCTT 721 CAAGAACAAATTGCTTGGATGACTTCTAATCCACCAATTCCAGTTGGTGATATTTATAAG 781 AGATGGATTATTCTTGGTCTTAATAAGATTGTTAGAATGTATTCTCCAGTTTCTATTCTT 841 GATATTAGACAAGGTCCAAAGGAACCATTTAGAGATTATGTTGATAGATTTTTTAAGACT 901 CTTAGAGCTGAACAAGCTACTCAAGAAGTTAAGAATTGGATGACTGATACTCTTCTTGTT 961 CAAAATGCTAATCCAGATTGTAAGACTATTCTTAGGGCTCTTGGTCCAGGTGCTACTCTT 1021 GAAGAAATGATGACTGCTTGTCAAGGTGTTGGTGGTCCAGGTCATAAGGCTAGAGTTCTT 1081 TAA
p41 Amino Acid Sequence
Translation of P41 (1-1083)
Universal code
Total amino acid number: 360, MW=40309
Max ORF starts at AA pos 1 (may be DNA pos 1) for 360 AA (1080 bases),
MW=40309
Origin

(109) TABLE-US-00032 SEQIDNO:109 1 MGARASILRGEKLDKWEKIRLRPGGKKHYMLKHIVWASRELERFALNPGLLETSEGCKQI 61 MKQLQPALQTGTEELKSLYNTVATLYCVHEKIEVRDTKEALDKIEEEQNKCQQKTQQAKA 121 ADGKVSQNYPIVQNLQGQMVHQAISPRTLNAWVKVIEEKAFSPEVIPMFTALSEGATPQD 181 LNTMLNTVGGHQAAMQMLKDTINEEAAEWDRLHPVHAGPIAPGQMREPRGSDIAGTTSTL 241 QEQIAWMTSNPPIPVGDIYKRWIILGLNKIVRMYSPVSILDIRQGPKEPFRDYVDRFFKT 301 LRAEQATQEVKNWMTDTLLVQNANPDCKTILRALGPGATLEEMMTACQGVGGPGHKARVL 361 *
p24 DNA Sequence 5 to 3

(110) TABLE-US-00033 SEQIDNO:110 1ATGCCAATTGTTCAAAATCTTCAAGGTCAAATGGTTCATCAAGCTATTTCTCCAAGAACT 61CTTAATGCTTGGGTTAAGGTTATTGAAGAAAAGGCTTTTTCTCCAGAAGTTATTCCAATG 121TTTACTGCTCTTTCTGAAGGTGCTACTCCACAAGATCTTAATACTATGCTTAATACTGTT 181GGTGGTCATCAAGCTGCTATGCAAATGCTTAAGGATACTATTAATGAAGAAGCTGCTGAA 241TGGGATAGACTTCATCCAGTTCATGCTGGTCCAATTGCTCCAGGTCAAATGAGAGAACCA 301AGAGGTTCTGATATTGCTGGTACTACTTCTACTCTTCAAGAACAAATTGCTTGGATGACT 361TCTAATCCACCAATTCCAGTTGGTGATATTTATAAGAGATGGATTATTCTTGGTCTTAAT 421AAGATTGTTAGAATGTATTCTCCAGTTTCTATTCTTGATATTAGACAAGGTCCAAAGGAA 481CCATTTAGAGATTATGTTGATAGATTTTTTAAGACTCTTAGAGCTGAACAAGCTACTCAA 541GAAGTTAAGAATTGGATGACTGATACTCTTCTTGTTCAAAATGCTAATCCAGATTGTAAG 601ACTATTCTTAGGGCTCTTGGTCCAGGTGCTACTCTTGAAGAAATGATGACTGCTTGTCAA 661GGTGTTGGTGGTCCAGGTCATAAGGCTAGAGTTCTTTAA
p24 Amino Acid Sequence
Translation of p24 (1-699)
Universal code
Total amino acid number: 232, MW=25660
Max ORF starts at AA pos 1 (may be DNA pos 1) for 232 AA (696 bases),
MW=25660
Origin

(111) TABLE-US-00034 SEQIDNO:111 1MPIVQNLQGQMVHQAISPRTLNAWVKVIEEKAFSPEVIPMFTALSEGATPQDLNTMLNTV 61GGHQAAMQMLKDTINEEAAEWDRLHPVHAGPIAPGQMREPRGSDIAGTTSTLQEQIAWMT 121SNPPIPVGDIYKRWIILGLNKIVRMYSPVSILDIRQGPKEPFRDYVDRFFKTLRAEQATQ 181EVKNWMTDTLLVQNANPDCKTILRALGPGATLEEMMTACQGVGGPGHKARVL
RT DNA Sequence 5 to 3

(112) TABLE-US-00035 SEQIDNO:112 1ATGAGGGTGTTGCTCGTTGCCCTCGCTCTCCTGGCTCTCGCTGCGAGCGCCACCTCCACG 61CATACAAGCGGCGGCTGCGGCTGCCAGCCACCGCCGCCGGTTCATCTACCGCCGCCGGTG 121CATCTGCCACCTCCGGTTCACCTGCCACCTCCGGTGCATCTCCCACCGCCGGTCCACCTG 181CCGCCGCCGGTCCACCTGCCACCGCCGGTCCATGTGCCGCCGCCGGTTCATCTGCCGCCG 241CCACCATGCCACTACCCTACTCAACCGCCCCGGCCTCAGCCTCATCCCCAGCCACACCCA 301TGCCCGTGCCAACAGCCGCATCCAAGCCCGTGCCAGACCATGGACGACGATGATAAGTGC 361GGCAAGAAGGCCATCGGCACCGTGCTGGTGGGCCCCACCCCCGTGAACATCATCGGCCGG 421AACATGCTGACCCAGCTGGGCTGCACCCTGAACTTCCCCATCAGCCCCATCGAGACCGTG 481CCCGTGAAGCTGAAGCCCGGCATGGACGGCCCCAAGGTGAAGCAGTGGCCCCTGACCGAG 541GTGAAGATCAAGGCCCTGACCGCCATCTGCGAGGAGATGGAGAAGGAGGGCAAGATCACC 601AAGATCGGCCCCGAGAACCCCTACAACACCCCCATCTTCGCCATCAAGAAGGAGGACAGC 661ACCAAGTGGCGGAAGCTGGTGGACTTCCGGGAGCTGAACAAGCGGACCCAGGACTTCTGG 721GAGGTGCAGCTGGGCATCCCCCACCCCGCCGGCCTGAAGAAGAAGAAGAGCGTGACCGTG 781CTGGACGTGGGCGACGCCTACTTCAGCGTGCCCCTGGACGAGGGCTTCCGGAAGTACACC 841GCCTTCACCATCCCCAGCATCAACAACGAGACCCCCGGCATCCGGTACCAGTACAACGTG 901CTGCCCCAGGGCTGGAAGGGCAGCCCCGCCATCTTCCAGGCCAGCATGACCAAGATCCTG 961GAGCCCTTCCGGGCCAAGAACCCCGAGATCGTGATCTACCAGTACATGGCCGCCCTGTAC 1021GTGGGCAGCGACCTGGAGATCGGCCAGCACCGGGCCAAGATCGAGGAGCTGCGGGAGCAC 1081CTGCTGAAGTGGGGCTTCACCACCCCCGACAAGAAGCACCAGAAGGAGCCCCCCTTCCTG 1141TGGATGGGCTACGAGCTGCACCCCGACAAGTGGACCGTGCAGCCCATCCAGCTGCCCGAG 1201AAGGACAGCTGGACCGTGAACGACATCCAGAAGCTGGTGGGCAAGCTGAACTGGACCAGC 1261CAGATCTACCCCGGCATCAAGGTGCGGCAGCTGTGCAAGCTGCTGCGGGGCACCAAGGCC 1321CTGACCGACATCGTGCCCCTGACCGAGGAGGCCGAGCTGGAGCTGGCCGAGAACCGGGAG 1381ATCCTGAAGGAGCCCGTGCACGGCGTGTACTACGACCCCAGCAAGGACCTGATCGCCGAG 1441ATCCAGAAGCAGGGCGACGACCAGTGGACCTACCAGATCTACCAGGAGCCCTTCAAGAAC 1501CTGAAAACCGGCAAGTACGCCAAGCGGCGGACCACCCACACCAACGACGTGAAGCAGCTG 1561ACCGAGGCCGTGCAGAAGATCAGCCTGGAGAGCATCGTGACCTGGGGCAAGACCCCCAAG 1621TTCCGGCTGCCCATCCAGAAGGAGACCTGGGAGATCTGGTGGACCGACTACTGGCAGGCC 1681ACCTGGATCCCCGAGTGGGAGTTCGTGAACAGCGGCCGCTTTCGAATCTAG
RT Amino Acid Sequence
Translation of RT (1-1731)
Universal code
Total amino acid number: 576, MW=65360
Max ORF starts at AA pos 1 (may be DNA pos 1) for 576 AA (1728 bases),
MW=65360
Origin

(113) TABLE-US-00036 SEQIDNO:113 1MRVLLVALALLALAASATSTHTSGGCGCQPPPPVHLPPPVHLPPPVHLPPPVHLPPPVHL 61PPPVHLPPPVHVPPPVHLPPPPCHYPTQPPRPQPHPQPHPCPCQQPHPSPCQTMDDDDKC 121GKKAIGTVLVGPTPVNIIGRNMLTQLGCTLNFPISPIETVPVKLKPGMDGPKVKQWPLTE 181VKIKALTAICEEMEKEGKITKIGPENPYNTPIFAIKKEDSTKWRKLVDFRELNKRTQDFW 241EVQLGIPHPAGLKKKKSVTVLDVGDAYFSVPLDEGFRKYTAFTIPSINNETPGIRYQYNV 301LPQGWKGSPAIFQASMTKILEPFRAKNPEIVIYQYMAALYVGSDLEIGQHRAKIEELREH 361LLKWGFTTPDKKHQKEPPFLWMGYELHPDKWTVQPIQLPEKDSWTVNDIQKLVGKLNWTS 421QIYPGIKVRQLCKLLRGTKALTDIVPLTEEAELELAENREILKEPVHGVYYDPSKDLIAE 481IQKQGDDQWTYQIYQEPFKNLKTGKYAKRRTTHTNDVKQLTEAVQKISLESIVIWGKTPK 541FRLPIQKETWEIWWTDYWQATWIPEWEFVNSGRFRI*
NSs DNA Sequence 5 to 3
NSs is a silencing suppressor used in the agroinfiltration of tobacco plants.

(114) TABLE-US-00037 SEQIDNO:114 1ATGTCTTCAAGTGTTTATGAGTCGATCATTCAGACAAAAGCTTCAGTCTGGGGATCAACT 61GCATCTGGTAAAGCTGTTGTAGATTCTTACTGGATTCATGAACTTGGTACTGGTTCTCCA 121CTAGTTCAAACCCAGCTGTATTCTGATTCAAGAAGCAAAAGTAGCTTTGGCTATACTGCA 181AAGGTAGGGAATCTTCCCTGTGAGGAAGAAGAAATTCTTTCTCAGCATGTGTATATCCCT 241ATTTTTGATGATGTTGATTTTAGCATCAATATTGATGACTCTGTTCTGGCACTGTCTGTT 301TGCTCCAACACAGTCAATACTAACGGAGTGAAACATCAAGGTCATTTGAAAGTTTTGTCT 361CCTGCTCAGCTCCACTCTATTGGATCTACCATGAACGGATCTGATATTACAGACCGATTC 421CAGCTCCAAGAAAAAGATATAATTCCCAATGACAGGTACATTGAAGCTGTAAACAAAGGC 481TCTTTGTCTTGTGTTAAAGAGCATACCTATAAGGTCGAGATGTGCTACAATCAAGCTTTA 541GGCAAAGTGAATGTTCTATCCCCTAACAGAAATGTCCATGAATGGCTGTACAGTTTCAAG 601CCAAATTTCAATCAAGTTGAAAGCAACAACAGAACTGTAAATTCTCTTGCAGTGAAATCT 661CTGCTCATGTCAGCAGGAAATAACATCATGCCTAACTCTCAGGCTTTTGTCAAAGCTTCC 721ACTGATTCTCATTTCAAGCTGAGCCTCTGGCTAAGAGTTCCAAAGGTTTTGAAGCAGATT 781TCCATTCAGAAATTGTTCAAAGTTGCAGGAGATGAAACTAACAAAACATTTTATTTATCT 841ATTGCTTGCATTCCAAACCATAACAGTGTTGAGACAGCTTTAAACATTTCTGTTATTTGC 901AAGCATCAGCTCCCAATCCGTAAATTTAAAGCTCCTTTTGAATTATCAATGATGTTTTCT 961GATTTAAAGGAGCCTTACAACATTGTTCATGATCCTTCATATCCTCAGAGGATTGTTCAT 1021GCTCTGCTTGAAACTCACACGTCTTTTGCACAAGTTCTTTGCAACAACTTGCAAGAAGAC 1081GTGATCATCTACACTTTGAACAACTATGAGCTAACTCCTGGAAAGTTAGATCTAGGTGAA 1141AGAACCTTAAATTACAGTGAAGATGTCTGCAAAAGGAAATATTTCCTCTCAAAAACACTT 1201GAATGTCTTCCATCTAACACACAAACTATGTCTTACTTAGACAGCATCCAAATCCCTTCC 1261TGGAAGATAGACTTTGCTAGGGGAGAAATTAAAATTTCTCCACAATCTGTTTCAGTTGCA 1321AAATCTTTGTTAAAGCTTGATTTAAGTGGGATCAAAAAGAAAGAATCTAAGATTTCGGAA 1381GCATGTGCTTCAGGATCAAAATAA
Translation of NSs (1-1404)
Universal code
Total amino acid number: 467, MW=52121
Max ORF starts at AA pos 1 (may be DNA pos 1) for 467 AA (1401 bases),
MW=52121
Origin

(115) TABLE-US-00038 SEQIDNO:115 1MSSSVYESIIQTKASVWGSTASGKAVVDSYWIHELGTGSPLVQTQLYSDSRSKSSFGYTA 61KVGNLPCEEEEILSQHVYIPIFDDVDFSINIDDSVLALSVCSNTVNTNGVKHQGHLKVLS 121PAQLHSIGSTMNGSDITDRFQLQEKDIIPNDRYIEAVNKGSLSCVKEHTYKVEMCYNQAL 181GKVNVLSPNRNVHEWLYSFKPNFNQVESNNRTVNSLAVKSLLMSAGNNIMPNSQAFVKAS 241TDSHFKLSLWLRVPKVLKQISIQKLFKVAGDETNKTFYLSIACIPNHNSVETALNISVIC 301KHQLPIRKFKAPFELSMMFSDLKEPYNIVHDPSYPQRIVHALLETHTSFAQVLCNNLQED 361VIIYTLNNYELTPGKLDLGERTLNYSEDVCKRKYFLSKTLECLPSNTQTMSYLDSIQIPS 421WKIDFARGEIKISPQSVSVAKSLLKLDLSGIKKKESKISEACASGSK*
Gag CD8 Peptide Amino Acid Sequence

(116) TABLE-US-00039 AMQMLKDTI SEQIDNO:116
Gag CD4 (13) Peptide Amino Acid Sequence

(117) TABLE-US-00040 NPPIPVGDIYKRWIIGLNK SEQIDNO:117
Gag CD4 (17) Peptide Amino Acid Sequence

(118) TABLE-US-00041 FRDYVDRFFKTLRAEQATQE SEQIDNO:118
RT CD4 Peptide Amino Acid Sequence

(119) TABLE-US-00042 PKVKQWPLTEVKIKALTAI SEQIDNO:119
RT CD8 Peptide Amino Acid Sequence

(120) TABLE-US-00043 VYYDPSKDLIA SEQIDNO:120

(121) T-cell stimulating immunogenicity of a contemplated adjuvant can be measured by a variety of well known techniques. In usual practice, a host animal is inoculated with a contemplated RPBLA vaccine or inoculum, and peripheral mononuclear blood cells (PMBC) are thereafter collected. Those PMBC are then cultured in vitro in the presence of the biologically active polypeptide (T cell immunogen) for a period of about three to five days. The cultured PMBC are then assayed for proliferation or secretion of a cytokine such as IL-2, GM-CSF of IFN-. Assays for T cell activation are well known in the art. See, for example, U.S. Pat. No. 5,478,726 and the art cited therein.

(122) A contemplated adjuvant is typically prepared from a recovered RPBLA particles by dispersing the RPBLAs in a physiologically tolerable (acceptable) diluent vehicle such as water, saline, phosphate-buffered saline (PBS), acetate-buffered saline (ABS), Ringer's solution, or the like to form an aqueous composition. The diluent vehicle can also include oleaginous materials such as peanut oil, squalane, squalene and the like as are well known.

(123) The preparation of adjuvants that contain proteinaceous materials as active ingredients is also well understood in the art. Typically, such adjuvants are prepared as parenterals, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared. The preparation can also be emulsified, which is particularly preferred.

(124) The immunogenically active RPBLAs are often mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, an adjuvant can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents that enhance the immunogenic effectiveness of the composition.

(125) Without further elaboration, it is believed that one skilled in the art can, using the preceding description and the detailed examples below, utilize the present invention to its fullest extent. The following preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limiting of the remainder of the disclosure in any way whatsoever.

Example 1

Plasmid Construction and Plant Transformation

(126) DNA encoding HIV-1 p24, p41 and RT from a cloned South African HIV isolate Du422 (GenBank accession no. AF544010) was fused to Zera using PCR and subsequently cloned into an A. tumefaciens binary expression vector pTRAc (Meyers, BMC Biotechnology 2008 8:53) in E. coli to yield the recombinant clone pTRAcRX3p24, pTRAcRX3p41 and pTRAcRX3RT.

(127) The HPV-16 E7SH gene was engineered by three consecutive PCR reactions as described in [Steinberg et al., 2995 Vaccine 23(9):1149-1157]. The resulting HPV16 E7SH gene was amplified by PCR and an enterokinase site was added to the 5 end of the protein. This construct was then fused to RX3 by replacing GFP in an A. tumefaciens binary vector pTRA C [Mclean et al., 2007 J Gen Virol 88:1460-1469] that contained a RX3 GFP fusion gene.

(128) The recombinant clone was purified from E. coli, and electroporated into competent host A. tumefaciens GV3101::pMP90RK cells. Recombinant A. tumefaciens cultures containing pTRAcRX3p24, pTRAcRX3p41, pTRAcRX3RT and pRX3E7SH were injection-infiltrated into the leaves of 6-week old N. benthamiana plants using a needle and syringe. The leaves were co-infiltrated with A. tumefaciens LBA4404 containing a silencing suppressor pBIN-NS to enhance transient protein expression. The infiltrated plants were grown at 22 C. under a 16 hour: 8 hour light:dark cycle.

(129) Harvesting and Purification of Transiently-Expressed RX3 Fusion Proteins

(130) Approximately 10 g of infiltrated leaf tissue was ground up in liquid nitrogen and resuspended in 20 ml of buffer PBP3 (100 mM Tris pH8, 50 mM KCl, 6 mM MgCl.sub.2, 10 mM EDTA and 0.4M NaCl). This suspension was homogenized for 3 minutes and then filtered through miracloth (a quick filtration material for gelatinous homogenates and for protoplast isolation that is composed of rayon polyester with a pore size of 22-25 mm and an acrylic binder that is available from Calbiochem, San Diego, Calif.). The filtrate was loaded on top of a density step gradient. The gradient comprised of 7 ml volumes of 15, 25, 35 and 45% concentrations of Optiprep density gradient medium made up in buffer PBP3. The gradient was centrifuged for 2 hours at 80 000Xg in a Beckman SW28 rotor at 4 C. The pellet was resuspended in 500 l of buffer PBP3 and an aliquot stored for analysis. The remainder was stored at 70 C.

Example 2

Immunization of Mice

(131) HIV Group Study

(132) Female BALB/c mice (8 to 10 weeks old) were divided into the appropriate number of groups (5 mice per group):

(133) Group 1vDNA prime

(134) Group 2vDNA prime+vDNA boost

(135) Group 3vDNA prime+RPBLAs boost

(136) Group 4RPBLAs prime

(137) The DNA vaccines (vDNA) used in the prime and boost inoculations correspond to: (i) pTHGagC that expresses the Du422 HIV-1 subtype C Gag (van Harmelen, 2003), was manufactured by Aldevron, Fargo, N. Dak., USA and resuspended at 1 mg DNA/ml saline and (ii) pVRCgrttn, that express five HIV-1 subtype C genes gag, reverse transcriptase (RT), tat, and nef (Burgers et al., AIDS Research and Human Retroviruses 2008 24(2):195-206). The vDNA (100 g DNA/100 l saline) was administered by injecting 50 l into each tibialis anterior muscle.

(138) For intramuscular inoculation with the RPBLAs containing the corresponding RX3 fusion proteins (RX3-p24, RX3-p41 and RX3-RT), 100 l of the corresponding RPBLAs fraction isolated as described above and resuspended in saline buffer The following amounts of RPBLAs were injected into the tibialis anterior muscle: 3.6 g for RX3-p24; 3.1 g for RX3-p41; and 4 g for RX3-RT.

(139) HPV Group Study

(140) DNA Vaccination

(141) Six- to eight-week-old female C57BL/6 mice were injected with 50 l of 10 M cardiotoxin into each tibialis anterior muscle 5-6 days prior to DNA injection. For vaccination, 50 l of plasmid DNA (1 g/l in PBS) was injected into each pretreated muscle. Ten days later, mice were sacrificed and splenocytes were isolated from the spleen.

(142) Tumor Protection and Regression Studies

(143) Tumor protection and regression studies were performed essentially as described in (hlschlager et al., Vaccine 2006 24(2):2880-2893). The specific modifications of the protocol are indicated in the corresponding experiments.

Example 3

IFN- and IL-2 ELISPOT Assay

(144) HIV Antigens

(145) A single cell suspension of splenocytes was prepared from spleens harvested on day 12 or 40 and pooled from 5 mice per group. IFN- ELISPOT responses were measured using a mouse IL-2 or IFN- ELISPOT set (BD Pharmingen). Splenocytes were plated in triplicate at 510.sup.5/well in a final volume of 200 l R10 culture medium (RPMI with 10% heat inactivated FCS, Gibco, containing 15 mM -mercaptoethanol, 100 U penicillin per ml, and 100 g streptomycin).

(146) The peptides (>95% pure, Bachem, Switzerland) GagCD8 AMQMLKDTI (SEQ ID NO:116) gag CD4(13) NPPIPVGDIYKRWIIGLNK (SEQ ID NO:117) gag CD4(17) FRDYVDRFFKTLRAEQATQE (SEQ ID NO:118), RT(CD8) VYYDPSKDLIA (SEQ ID NO:120), or RT(CD4) PKVKQWPLTEVKIKALTAI (SEQ ID NO:119), were used as stimuli in the assay at a final concentration of 4 g/ml. Reactions containing an irrelevant H-2K.sup.d binding peptide TYSTVASSL (SEQ ID NO:1), (obtained from Elizabeth Reap, AlphaVax) or without peptide served as background controls. Spots were detected with the detection antibody at 22 hours, developed with Nova Red then counted using a CTL Analyzer (Cellular Technology, Ohio, USA) with Immunospot Version 3.0 software. The average number of spots in triplicate wells was calculated and results are expressed as the average number of spot-forming units (SFU) per 10.sup.6 splenocytesthe standard deviation (SD). For each group of mice, the average background spots obtained in the absence of peptide and in the presence of the irrelevant peptide plus one standard deviation of this average was considered as the cut-off for a positive response.

(147) HPV Antigens

(148) In all studies, ELISPOTs were performed ex vivo essentially as described in (Steinberg et al., 2005 Vaccine 23:1149-1157). Murine IFN-gamma Elispot assays were performed ex vivo and 5 or 6 days after each in vitro restimulation as described earlier (Ohlschlager et al., 2006). The granzyme B Elispot assay was performed similarly to the IFN-gamma Elispot Assay. For this assay, the anti-mouse granzyme capture antibody (100 ng/well, clone R4-6A2; PharMingen, San Diego, USA) and the biotinylated anti-mouse granzyme detection antibody (50 ng/well, clone XMG1.2; PharMingen, San Diego, USA) were used.

Example 4

Western Blot of Antiserum

(149) Western blots were carried out using a LAV Blot I commercial kit (Biorad). Mouse serum from inoculated mice was used to detect antibodies with goat anti-mouse IgG conjugated to alkaline phosphatase.

Example 5

Isolation (Purification) of RPBLAs Containing RX3-p24, RX3-p41 or RX3-RT by Density Gradient from Agroinfiltrated Tobacco Leaves

(150) Approximately 10 g of leaf tissue agroinfiltrated with the corresponding construct (pRX3-p24, pRX3-p41 or pRX3-RT) was ground up in liquid nitrogen and resuspended in 20 ml of buffer PBP3 (100 mM Tris pH8, 50 mM KCl, 6 mM MgCl.sub.2, 10 mM EDTA and 0.4M NaCl). This was homogenized for 3 minutes on ice using a Polytron homogenizer and then filtered through miracloth. The corresponding filtrate was loaded on top of a density step gradient, comprising of 7 ml volumes of 15, 25, 35 and 45% concentrations of Optiprep density gradient medium made up in buffer PBP3. The gradient was centrifuged for 2 hours at 80,000g in a Beckman SW28 rotor at 4 C. The pellet was resuspended in 500 l buffer PBP3 to check for the presence of RPBLAs by optic microscopy, and an aliquot stored for analysis. The remainder was stored at 70 C.

(151) To verify that the RPBLAs fraction contained the corresponding RX3 fusion protein, an aliquot of it was analyzed by western blot using anti-RX3 and anti-p24 antibodies to verify the integrity of the fusion protein (FIG. 1). The amount of immunogen was quantified by densitometric analysis of a western blot dilutions of HIV-1 p17/p24 (also referred as p41) and HIV-1 RT as standards. The concentration of the corresponding immunogen was estimated to 36 ng/l for RX3-p24, 31 ng/l for RX3-p41 and approximately 40 ng/l for RX-RT.

Example 6

Determination of the Cellular Response Triggered by the Intramuscular Inoculation of RX3-p24

(152) To determine the cellular immune response induced by the administration of RX3-p24 containing RPBLAs, four groups of mice were inoculated as follows: (i) mice inoculated with the DNA vaccine (pTHGagx1), (ii) mice inoculated with the DNA vaccine and boosted with another dose of the same DNA vaccine (pTHGagx2), (iii) mice inoculated with the DNA vaccine and boosted with RPBLAs containing RX3-p24 and no further DNA (pTHGag+RX3-p24), and (iv) mice inoculated exclusively with RPBLAs containing RX3-p24 (RX3-p24).

(153) IFN- and IL-2 ELISPOT assays indicated that mice inoculated with the DNA vaccine alone (pTHGagx1) induced a cellular response. As shown in FIG. 2, CD4 as well as CD8 T-cells secreted a larger amount of IFN- and IL-2 when they were incubated with the stimulating peptides gag CD8, gag CD4(13) or gag CD4(17), compared to T-cells incubated with unrelated peptide. As expected and has been shown previously, the mouse group boosted with a second inoculation of the DNA vaccine (pTHGagx2) showed an even larger cellular response (4-fold compared to the pTHGag group).

(154) When the same assays were performed with the mouse group inoculated exclusively with the RPBLAs containing RX3-p24 (RX3-p24), no significant response was observed. This result suggested that the immunogen aggregated inside RPBLAs is not able to trigger the cellular response. Nevertheless, when IFN- and IL-2 ELISPOT assays were performed on T-cells from mice inoculated with the DNA vaccine and boosted with a second inoculation consisting of RPBLAs containing RX3-p24 and no further DNA (pTHGag+RX3-p24), a surprising 3-fold higher cellular response was observed compared to the pTHGagx1 group. The lack of cellular response observed in the p24 mouse group probably indicates that a higher amount of RPBLAs should be inoculated.

(155) These data indicate that RPBLAs are a suitable immunogen presentation vehicle able to induce a cellular response.

Example 7

Determination of the Humoral Response Triggered by the Intramuscular Inoculation of RX3-p24

(156) It has been shown that the risk of AIDS is greatly increased in individuals with falling titres of p24 antibodies, suggesting that high anti-p24 antibody titres might be necessary to maintain a disease-free state.

(157) To determine the presence of antibodies against the p24 antigen, strips containing a representation of the HIV virus proteins [LAV Blot I commercial kit (Biorad)] were incubated with mouse serum from the four inoculation groups (pTHGagx1, pTHGagx2, pTHGag+RX3-p24 and RX3-p24). Antibodies against the p24 protein were detected only in mice inoculated with the DNA vaccine and boosted with a second round of the DNA inoculation or with RPBLAs containing the RX3-p24 and no further DNA (FIG. 3; pTHGagx2 and pTHGag+RX3-p24 mouse groups). Interestingly, the antibodies generated from this second group recognized the full length Gag protein (p55) in addition to the p24 protein indicating that a higher titer of antibodies is produced in pTHGag+RX3-p24 mouse group compared to the pTHGagx1 one.

Example 8

Determination of the Cellular Response Triggered by the Intramuscular Inoculation of RX3-p41

(158) As indicated previously, p41 which results from the fusion of p17 and p24 fragments (p17/24) of the HIV Gag protein, contains the highest density of CTL epitopes in the HIV-1 genome (Novitsky et al., J. Virol. 2002 76(20):10155-10168). In this context the efficiency of RPBLAs containing the RX3-p41 fusion protein to trigger the cellular response of the immune system was examined.

(159) As occurred previously in Example 6, IFN- and IL-2 ELISPOT assays indicated that mice inoculated with a single dose of the DNA vaccine induced a small cellular response, which was significantly increased when those mice were boosted with a second inoculation with the DNA vaccine (compare pTHGagx1 versus pTHGagx2 in FIG. 4). It is interesting to point out that splenocytes from the mouse group boosted with the RX3-p41 (pTHGag+RX3-p41) secreted an even larger amount of IFN and IL-2 than the pTHGagx2 group when they were incubated with the gagCD4(13) and gagCD4(17) stimulating peptides (FIG. 4). Although the secretion of IFN and IL-2 was not increased by the incubation of splenocytes from the pTHGag+RX3-p41 mouse group with gagCD8-stimulating peptides, it can be concluded that the cellular response of the immune system is efficiently boosted by the inoculation of RPBLAs containing RX3-p41 fusion protein.

Example 9

Determination of the Cellular Response Triggered by Intramuscular Inoculation of RX3-RT

(160) As an effective multivalent vaccine against HIV includes several antigens, similar studies were performed with HIV viral protein RT.

(161) IFN ELISPOT assays indicated that mice inoculated with a single dose of the DNA vaccine (pVRCgrttnx1) induced a very poor cellular response. Exclusively CD8 T-cells incubated with the stimulating peptides RT(CD8) secreted a larger amount of IFN- than the same cells incubated with un irrelevant peptide TYSTVASSL (SEQ ID NO:1; FIG. 5). A boost with a second inoculation of the DNA vaccine (pVRCgrttnx2) or the RPBLAs containing RX3RT (pVRCgrttn+RX3-RT) was needed to observe a general induction of the cellular response. FIG. 5 shows that CD4 and CD8 T-cells incubated with the corresponding stimulating peptides secreted a larger amount of IFN- and IL-2 compared to the control treatments (absence or presence of an irrelevant peptide).

Example 10

Determination of the Immune Response Triggered by the Intramuscular Inoculation of a DNA Vaccine Expressing RX3-p24, RX3-41 or RX3-RT

(162) DNA vaccines encoding HIV antigens have been studied extensively and shown to induce both humoral and cellular immune responses in animal models as well as in humans (Estcourt et al., Immunol. Rev. 2004 199:144-155). However, although DNA vaccines have been shown to be safe, immunizations have generated low and transient levels of immune responses.

(163) pTHGag was shown in the mouse model to induce a potent cytotoxic lymphocyte response. Pr55Gag expressed in a variety of cell systems can assemble and bud through the plasma membrane to form highly immunogenic virus-like particles (VLPs). RPBLAs can not been considered as classical VLPs, because their assembly is induced by the aggregation capacity of RX3, which is not a viral protein involved in the formation of the virus particles. However, the suitability of a DNA vaccine expressing RPBLAs containing the RX3-24, RX3-41, RX3-RT and RX3E7SH was examined. Interestingly, once the corresponding pTH-derived vectors (pTHRX3-p24, pTHRX3-p41, pTHRX3-RT and pTHRX3-E7SH) were administrated as the pTHGag in previous studies, significant humoral and cellular immune responses were observed. This unexpected result indicates that RPBLAs can be administered by DNA vaccination; in spite of this organelles are stored in the ER and are not supposed to bud through the plasma membrane to form highly immunogenic virus-like particles (VLPs).

Example 11

Determination of the Immune Response Triggered by the Inoculation of RPBLAs Assembled In Vitro

(164) The isolation of RPBLAs by density gradient permits the recovery of a highly enriched fraction of RPBLAs, but a certain degree of contaminants are co-purified. To remove as much contaminants as possible, the RX3 fusion proteins (RX3-p24, RX3-p41, RX3-RT and RX3-E7SH) were solubilized from the corresponding RPBLA fraction in 20 mM Tris pH8, 2% DOC, 10 mM DTT incubated 1 hour at room temperature in soft agitation, and purified in RP-FPLC. The elution fractions containing the RX3 fusion proteins with more than 95% purity were pooled and lyophilized. The corresponding pellet was recovered in distilled water in the presence of 200 mM of NaCl and 50 mM of CaCl. In these conditions, the fusion proteins containing the RX3 peptide reassemble spontaneously to reform RPBLAs in vitro, outside of the plant ER.

(165) In vitro-assembled RPBLAs containing the corresponding RX3 fusion protein were inoculated into mice and the IFN, IL-2 and Granzyme B ELISPOT assays showed that RPBLAs boost significantly the cellular response in equivalent studies as the those performed using in vivo-formed RPBLAs. This surprising result indicates that in vitro-assembled RPBLAs maintain the capacity of inducing the cellular response.

(166) RX3 fusion proteins can be induced to assemble in vitro and form RPBLAs in the following conditions: (i) reducing the pH value of the solution, (ii) increasing salt content, (iii) reducing or removing the concentration of reducing agents, (iv) adding oxidizing agents, (v) decreasing the temperature, or a combination of this factors. Obviously, in vitro RPBLAs are not surrounded by a membrane. Preferred salts to induce the assembly in vitro are NaCl, CaCl and KCl and preferred pH values are below 7.

(167) As indicated before, a double immune response (cellular plus humoral) produces a more protective effect against AIDS. The presence of antibodies against the p24, p41 and RT antigens was shown by using the HIV strips (LAV Blot I commercial kit (Biorad)) in mice primed with the DNA vaccine and boosted with the corresponding in vitro assembled RPBLAs.

Example 12

Isolation (Purification) of RPBLAs Containing RX3-E7SH by Density Gradient from Agroinfiltrated Tobacco Leaves

(168) Approximately 10 g of leaf tissue agroinfiltrated with the HPV-16 antigen E7SH fused to RX3 (pRX3-E7SH) was ground up in liquid nitrogen and resuspended in 20 ml of buffer PBP3 (100 mM Tris pH8, 50 mM KCl, 6 mM MgCl.sub.2, 10 mM EDTA and 0.4M NaCl). This was homogenized for 3 minutes on ice using a Polytron homogenizer and then filtered through miracloth. The corresponding filtrate was loaded on top of a density step gradient, comprising of 7 ml volumes of 15, 25, 35 and 45% concentrations of Optiprep density gradient medium made up in buffer PBP3. The gradient was centrifuged for 2 hours at 80,000g in a Beckman SW28 rotor at 4 C. The pellet was resuspended in 500 l buffer PBP3 to check for the presence of RPBLAs by optic microscopy, and an aliquot stored for analysis. The remainder was stored at 70 C.

(169) The gene sequence for the RX2-E7SH fusion protein is shown below from 5 to 3:

(170) TABLE-US-00044 SEQIDNO:129 ATGAGGGTGTTGCTCGTTGCCCTCGCTCTCCTGGCTCTCGCTGCGAGC GCCACCTCCACGCATACAAGCGGCGGCTGCGGCTGCCAGCCACCGCCG CCGGTTCATCTACCGCCGCCGGTGCATCTGCCACCTCCGGTTCACCTG CCACCTCCGGTGCATCTCCCACCGCCGGTCCACCTGCCGCCGCCGGTC CACCTGCCACCGCCGGTCCATGTGCCGCCGCCGGTTCATCTGCCGCCG CCACCATGCCACTACCCTACTCAACCGCCCCGGCCTCAGCCTCATCCC CAGCCACACCCATGCCCGTGCCAACAGCCGCATCCAAGCCCGTGCCAG ACCATGGACGACGATGATAAGATGCACGGCGACACCCCCACCCTGCAC GAGTACATGCTGGACCTGCAGCCCGAGACCACCGACCTGTACTGCATC TGCAGCCAGAAACCCAAGTGCGACAGCACCCTGCGGCTGTGCGTGCAG AGCACCCACGTGGACATCCGGACCCTGGAGGACCTGCTGATGGGCACC CTGGGCATCGTGTGCCCCTACGAGCAGCTGAACGACAGCAGCGAGGAG GAGGATGAGATCGACGGCCCCGCCGGCCAGGCTGAGCCCGACCGGGCC CACTACAACATCGTGACCTTCTGCTGCCAACCAGAGACAACTGATCTC TACTGTTATGAGCAATTAAATGACAGCTCAGAGCATTACAATATTGTA ACCTTTTGTTGCAAGTGTGACTCTACGCTTCGGTTGTGCATGGGCACA CTAGGAATTGTGTGCCCCATCTGTTCTCAGAAACCATAA

(171) To verify that the RPBLAs fraction contained the corresponding RX3 fusion protein, an aliquot of it was analyzed by western blot using anti-RX3 to verify the integrity of the fusion protein (FIG. 7).

Example 13

Determination of the Cellular Response Triggered by the Inoculation of RX3-E7SH

(172) To determine the cellular immune response induced by the administration of RX3-E7SH containing RPBLAs, five groups of mice were inoculated as follows: (i) mice inoculated with the DNA vaccine expressing E7SH antigen (pTHamp-E7SH), (ii) mice inoculated with the corresponding DNA vaccine negative control (pTHamp) with the sequence:

(173) TABLE-US-00045 SEQIDNO:127 5GACGGATCGGGAGATCTCCCGATCCCCTATGGTCGACTCTCAGTAC AATCTGCTCTGATGCCGCATAGTTAAGCCAGTATCTGCTCCCTGCTTG TGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACAAC AAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAG GCGTTTTGCGCTGCTTCGCGATGTACGGGCCAGATATACGCGTTTTGA GATTTCTGTCGCCGACTAAATTCATGTCGCGCGATAGTGGTGTTTATC GCCGATAGAGATGGCGATATTGGAAAAATCGATATTTGAAAATATGGC ATATTGAAAATGTCGCCGATGTGAGTTTCTGTGTAACTGATATCGCCA TTTTTCCAAAAGTGATTTTTGGGCATACGCGATATCTGGCGATAGCGC TTATATCGTTTACGGGGGATGGCGATAGACGACTTTGGTGACTTGGGC GATTCTGTGTGTCGCAAATATCGCAGTTTCGATATAGGTGACAGACGA TATGAGGCTATATCGCCGATAGAGGCGACATCAAGCTGGCACATGGCC AATGCATATCGATCTATACATTGAATCAATATTGGCCATTAGCCATAT TATTCATTGGTTATATAGCATAAATCAATATTGGCTATTGGCCATTGC ATACGTTGTATCCATATCATAATATGTACATTTATATTGGCTCATGTC CAACATTACCGCCATGTTGACATTGATTATTGACTAGTTATTAATAGT AATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCG TTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACC CCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAA TAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTG CCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTA TTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACA TGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCA TCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTG GATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACG TCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAAT GTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTAC GGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCG CCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGG ACCGATCCAGCCTCCGCGGCCGGGAACGGTGCATTGGAACGCGGATTC CCCGTGCCAAGAGTGACGTAAGTACCGCCTATAGAGTCTATAGGCCCA CCCCCTTGGCTTCTTATGCATGCTATACTGTTTTTGGCTTGGGGTCTA TACACCCCCGCTTCCTCATGTTATAGGTGATGGTATAGCTTAGCCTAT AGGTGTGGGTTATTGACCATTATTGACCACTCCCCTATTGGTGACGAT ACTTTCCATTACTAATCCATAACATGGCTCTTTGCCACAACTCTCTTT ATTGGCTATATGCCAATACACTGTCCTTCAGAGACTGACACGGACTCT GTATTTTTACAGGATGGGGTCTCATTTATTATTTACAAATTCACATAT ACAACACCACCGTCCCCAGTGCCCGCAGTTTTTATTAAACATAACGTG GGATCTCCACGCGAATCTCGGGTACGTGTTCCGGACATGGGCTCTTCT CCGGTAGCGGCGGAGCTTCTACATCCGAGCCCTGCTCCCATGCCTCCA GCGACTCATGGTCGCTCGGCAGCTCCTTGCTCCTAACAGTGGAGGCCA GACTTAGGCACAGCACGATGCCCACCACCACCAGTGTGCCGCACAAGG CCGTGGCGGTAGGGTATGTGTCTGAAAATGAGCTCGGGGAGCGGGCTT GCACCGCTGACGCATTTGGAAGACTTAAGGCAGCGGCAGAAGAAGATG CAGGCAGCTGAGTTGTTGTGTTCTGATAAGAGTCAGAGGTAACTCCCG TTGCGGTGCTGTTAACGGTGGAGGGCAGTGTAGTCTGAGCAGTACTCG TTGCTGCCGCGCGCGCCACCAGACATAATAGCTGACAGACTAACAGAC TGTTCCTTTCCATGGGTCTTTTCTGCAGTCACCGTCCTTGACACGAAG CTTGGTACCGAGCTCGGATCCACTAGTAACGGCCGCCAGTGTGCTGGA ATTCTGCAGATATCCATCACACTGGCGGCCGCTCGAGCATGCATCTAG AGGGCCCTATTCTATAGTGTCACCTAAATGCTAGAGCTCGCTGATCAG CCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCC CCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCT AATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTA TTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAG ACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGG CGGAAAGAACCAGCTGGGGCTCGAGGGGGGATCGATCCCGTCGACCTC GAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGT TATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGT AAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTG CGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCAT TAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGC TCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTG CGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACA GAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAA AAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGG CTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGG TGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGA AGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATAC CTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCA CGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGC TGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGT AACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTG GCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGT GCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGG ACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAA AGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGT GGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCT CAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAAC GAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATC TTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAA AGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGT GAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCC TGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCT GGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCA GATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGT GGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGG GAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTT GCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCT TCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCC ATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTC AGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTG CATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACT GGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCG AGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGC AGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAA CTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACT CGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCT GGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGG GCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTAT TGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAA TGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGA AAAGTGCCACCTGACGTC3
(iii) mice inoculated with RPBLAs containing RX3-E7SH(RX3-E7SH), (iv) mice co-inoculated with RPBLAs containing RX3-E7SH and incomplete Freund's adjuvant (RX3-E7SH/IFA), and finally (v) mice inoculated with RPBLAs containing RX3 fused to Gfp (RX3-Gfp) as a negative control of RPBLAs.

(174) As expected, IFN- and Granzyme B ELISPOT assays indicated clearly that mice inoculated with the DNA vaccine (pTHamp-E7SH) induced a cellular response. As shown in FIG. 8, splenocyte cells coming from mice inoculated with the pTHamp-E7SH DNA vaccine secreted a significant larger amount of IFN- and Granzyme B, than the ones coming from mice inoculated with the DNA vaccine in the absence of E7SH antigen (pTHamp).

(175) Surprisingly, it was also observed that splenocytes isolated from mice inoculated with RPBLAs containing RX3-E7SH(RX3-E7SH) also released a large amount of IFN- and Granzyme B (equivalent to pTHamp-E7SH mice group) in the presence or absence of IFA co-administration FIG. 8. As a control, the negative results observed in RX3-Gfp group indicate that no unspecific cellular response against E7SH is triggered by the administration of RPBLAs.

(176) These results demonstrate clearly that E7SH antigen administered in fusion with RX3 in a RPBLAs particle is able to trigger efficiently a cellular response. The fact that no adjuvant was needed to achieve the maximum effect indicates that RX3-E7SH in RPBLAs is an efficient antigen presentation vehicle able to induce a cellular response. This conclusion was supported by the observation that it is necessary to co-administer IFA to ovalbumin (OVA) in order to induce an efficient cellular response FIG. 9.

(177) The amino acid sequence for ovalbumin in single letter code is shown below:

(178) TABLE-US-00046 MGSIGAASMEFCFDVFKELKVHHANENIFYCPIAIMSALA MVYLGAKDSTRTQINKVVRFDKLPGFGDSIEAQCGTSVNV HSSLRDILNQITKPNDVYSFSLASRLYAEERYPILPEYLQ CVKELYRGGLEPINFQTAADQARELINSWVESQTNGIIRN VLQPSSVDSQTAMVLVNAIVFKGLWEKTFKDEDTQAMPFR VTEQESKPVQMMYQIGLFRVASMASEKMKILELPFASGTM SMLVLLPDEVSGLEQLESIINFEKLTEWTSSNVMEERKIK VYLPRMKMEEKYNLTSVLMAMGITDVFSSSANLSGISSAE SLKISQAVHAAHAEINEAGREVVGSAEAGVDAASVSEEFR ADHPFLFCIKHIATNAVLFFGRCVSP

Example 14

Determination of the Cytolytic Activity of the Splenocytes Induced by the Inoculation of with RX3-E7SH

(179) To determine, if the specifically activated splenocytes had cytolytic activity, .sup.51Cr-release assays were performed. .sup.51Cr release assays were performed 5-6 days after an in vitro restimulation of murine spleen cells as described elsewhere [Steinberg et al., (2005) Vaccine 23(9):1149-1157.] An animal was scored positive when the specific lysis of the specific target (RX3-E7 or pTHamp-E7SH cells) was at least 10% above the lysis of the control target (RX3-Gfp or pTHamp cells) for the protein and DNA based vaccines. After a first round of in vitro restimulation strong specific cytolytic activity against the E7WT-expressing RMA-E7 transfectants was shown (see table below.)

(180) TABLE-US-00047 TABLE .sup.51C-release assay (after 1.sup.st in vitro Specific restimulation) Lysis (%) pTHamp 8 3 pTHamp-E7SH 12 4 RX3-Gfp 26 6 RX3-E7SH 33 5 RX3-E7SH/IFA 29 6

(181) Surprisingly, the mean of specific lysis of the RMA-E7 cells was comparable in the RX3-E7SH-group (33%) and the pTHamp-E7SH immunized animals (26%), and significantly higher than the corresponding control groups RX3-Gfp (12%) and the pTHamp (8%). This result indicates that RX3-E7SH RPBLAs was able to induce a specific cytolytic activity against E7 expressing cells as efficiently as has already been shown by using the DNA vaccine pTHamp-E7SH [hlschlager et al., (2006) Vaccine 24:2880-2893]. Moreover, the fact that the cytolytic activity was not increased when the RPBLAs containing RX3-E7SH fusion protein was co-administered with IFA (see RX3-E7SH/IFA-group in table) suggests that even a lower dose would be effective to trigger the cytolytic effect, supporting the idea that RPBLAs provide an efficient administration vehicle to trigger a specific cytolytic effect.

(182) It is important to add that it has been widely demonstrated that a cytolytic response is a crucial element for controlling tumor growth [Akazawa, 2004 Cancer Res 64:757-764] and viral infection (Vine et al., 2004 J Immunol 173:5121-5129].

Example 15

Determination of Tumor Growth in Mice Inoculated with RX3-E7SH

(183) The aim of a therapeutic tumor vaccine is the induction of an effective immune response eradicating established tumors. Therefore, vaccination with the E7SH gene was examined to determine whether a cellular immune response could be induced that was able to control established E7-expressing tumor cells in vivo. In four tumor regression studies, a total of 80 animals were transplanted with a tumorigenic dose of syngeneic C3-tumor cells (day 0). When the tumors had reached a mean size of 4-9 mm.sup.2 at days 5-18, the animals were inoculated with: (i) 100 g of the E7SH-encoding plasmid (pTHamp-E7SH), (ii) 100 g of empty pTHamp vector (pTHamp), (iii) RPBLAs containing 5 g of RX3-E7SH(RX3-E7SH), and (iv) the same amount of RX3-E7SH RPBLAs co-administered with IFA (RX3-E7SH/IFA) (day zero). Tumor size was determined every two days by measuring with a ruler until the end of the study (day 14).

(184) As shown in FIG. 10A, tumor size increased progressively in mice inoculated with the control DNA vector (pTHamp), reaching a maximum average size of 110 mm.sup.2 14 days after inoculation. Tumor growth was significantly reduced in those mice inoculated with RPBLAs containing RX3-E7SH. Through out the study, the RX3-E7SH mice group showed tumors with lower size compared to the control group, reaching a mean value of 40 mm.sup.2 at day 14. It is interesting to point out that the protective effect of RX3-E7SH inoculation is comparable to the DNA vaccine pTHamp-E7SH, which has been reported to be a good therapeutic vaccine against E7-expressing tumors [Ohlschlager et al., (2006) Vaccine 24:2880-2893]. Moreover, as indicated before, the fact that the co-administration of RPBLAs containing RX3-E7SH with IFA (RX3-E7SH/IFA) did not increase the protective effect of the same amount of RPBLAs containing RX3-E7SH in the absence of an adjuvant (RX3-E7SH) suggests that a lower amount of RX3-E7SH will be protective against E7-expressing tumor growth.

(185) To exclude an unspecific tumor growth reduction due to some contaminants present in the RPBLAs preparation, or by the RX3 polypeptide by itself, equivalent amounts of RPBLAs containing RX3-Gfp were inoculated in an independent study, and no effect on tumor growth was observed when compared to pTHamp DNA control group (see FIG. 10B).

(186) It must be pointed out that mice were inoculated only once with RPBLAs with RX3-E7SH. In a prime boost study it is expected to have an enhanced therapeutic effect.

Example 16

Protective Effect Against Tumor Growth in Mice Inoculated with RX3-E7SH

(187) Taking into consideration a goal of the application of a protective (prophylactic) vaccine based on RPBLAs in addition to a therapeutic vaccine, rechallenge studies were undertaken to determine whether the RPBLAs containing RX3-E7SH were able to protect animals from an outgrowth of E7-expressing syngeneic tumors.

(188) Those mice that showed complete regression after the tumor regression experiment were injected again with 0.510.sup.6 C3 cells s.c. in 100 l PBS into the left flank 3 weeks after completion of the tumor regression study. The first C3 inoculation was given into the right flank. As a control, the same number of non-immunised mice received the same treatment. Twenty days after this injection, all control mice showed tumors with a size range of 100-400 mm.sup.2, whereas the immunized mice developed no tumors, and therefore showed clear protection from tumor growth (see FIG. 11).

(189) Each of the patents, patent applications and articles cited herein is incorporated by reference. The use of the article a or an is intended to include one or more.

(190) The foregoing description and the examples are intended as illustrative and are not to be taken as limiting. Still other variations within the spirit and scope of this invention are possible and will readily present themselves to those skilled in the art.