US20020106798A1 - DNA expression vectors and methods of use - Google Patents

DNA expression vectors and methods of use Download PDF

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Publication number
US20020106798A1
US20020106798A1 US09/798,675 US79867501A US2002106798A1 US 20020106798 A1 US20020106798 A1 US 20020106798A1 US 79867501 A US79867501 A US 79867501A US 2002106798 A1 US2002106798 A1 US 2002106798A1
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Prior art keywords
hiv
vector
dna
env
measles
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Harriet Robinson
James Smith
Ted Ross
Rick Bright
Jian Hua
Dennis Ellenberger
Donald Hildebrand
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Emory University
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Emory University
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Priority to US09/798,675 priority Critical patent/US20020106798A1/en
Assigned to EMORY UNIVERSITY reassignment EMORY UNIVERSITY ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: HUA, JIAN, ROBINSON, HARRIET L., BRIGHT, RICK ARTHUR, ELLENBERGER, DENNIS, HILDEBRAND, DONALD G., SMITH, JAMES M., ROSS, TED M.
Priority to US10/093,953 priority patent/US20040105871A1/en
Publication of US20020106798A1 publication Critical patent/US20020106798A1/en
Priority to US10/336,566 priority patent/US8623379B2/en
Priority to US11/009,063 priority patent/US7795017B2/en
Priority to US11/333,770 priority patent/US20070048861A1/en
Assigned to NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF HEALTH AND HUMAN SERVICES (DHHS), U.S. GOVERNMENT reassignment NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF HEALTH AND HUMAN SERVICES (DHHS), U.S. GOVERNMENT CONFIRMATORY LICENSE (SEE DOCUMENT FOR DETAILS). Assignors: EMORY UNIVERSITY
Priority to US12/749,164 priority patent/US20150231227A1/en
Priority to US14/137,095 priority patent/US9254319B2/en
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Definitions

  • the present invention is directed generally to the fields of molecular genetics and immunology. More particularly, the present invention describes novel DNA expression vectors, novel vectors comprising DNA encoding an immunogenic protein, and novel methods of immunizing animals including humans by administering the novel vectors comprising DNA encoding an immunogenic protein.
  • Vaccines have had profound and long lasting effects on world health. SmaIl pox has been eradicated, polio is near elimination, and diseases such as diphtheria, measles, mumps, pertussis, and tetanus are contained. Nonetheless, microbes remain major killers with current vaccines addressing only a handful of the infections of man and his domesticated animals. Common infectious diseases for which there are no vaccines cost the United States $120 billion dollars per year (Robinson et al., 1997). In first world countries, emerging infections such as immunodeficiency viruses, as well as reemerging diseases like drug resistant forms of tuberculosis, pose new threats and challenges for vaccine development. The need for both new and improved vaccines is even more pronounced in third world countries where effective vaccines are often unavailable or cost-prohibitive. Recently, direct injections of antigen-expressing DNAs have been shown to initiate protective immune responses.
  • DNA-based vaccines use bacterial plasmids to express protein immunogens in vaccinated hosts.
  • Recombinant DNA technology is used to clone cDNAs encoding immunogens of interest into eukaryotic expression plasmids.
  • Vaccine plasmids are then amplified in bacteria, purified, and directly inoculated into the hosts being vaccinated.
  • DNA typically is inoculated by a needle injection of DNA in saline, or by a gene gun device that delivers DNA-coated gold beads into skin.
  • the plasmid DNA is taken up by host cells, the vaccine protein is expressed, processed and presented in the context of self-major histocompatibility (MHC) class I and class II molecules, and an immune response against the DNA-encoded immunogen is generated.
  • MHC self-major histocompatibility
  • DNA vaccines also known as “genetic immunization”
  • Gene therapy studies on DNA delivery into muscle revealed that pure DNA was as effective as liposome-encapsulated DNA at mediating transfection of skeletal muscle cells (Wolff et al., 1990).
  • This unencapsulated DNA was termed “naked DNA,” a fanciful term that has become popular for the description of the pure DNA used for nucleic acid vaccinations.
  • Gene guns which had been developed to deliver DNA into plant cells, were also used in gene therapy studies to deliver DNA into skin.
  • HIV-1 is projected to infect 1% of the world's population by the year 2000, making vaccine development for this recently emergent agent a high priority for world health.
  • Preclinical trials on DNA vaccines have demonstrated that DNA alone can protect against highly attenuated HIV-1 challenges in chimpanzees (Boyer et al., 1997), but not against more virulent SIV challenges in macaques (Lu et al., 1997).
  • a combination of DNA priming plus an envelope glycoprotein boost has raised a neutralizing antibody-associated protection against a homologous challenge with a non-pathogenic chimera between SIV and HIV (SHIV-IIIb) (Letvin et al., 1997).
  • Protocols which proved less effective at containing challenge infections included immunization by both priming and boosting by intradermal or gene gun DNA inoculations, immunization by priming with intradermal or gene gun DNA inoculations and then boosting with a protein subunit; immunization by priming with gene gun DNA inoculations and boosting with recombinant fowl pox virus, immunization with protein only, and immunization with recombinant fowl pox virus only (Robinson et al,1999).
  • Early clinical trials of DNA vaccines in humans have revealed no adverse effects (MacGregor et al., 1996) and the raising of cytolytic T-cells (Calarota et al., 1998).
  • a number of studies have screened for the ability of co-transfected lymphokines and co-stimulatory molecules to increase the efficiency of immunization (Robinson and Pertmer, in press).
  • DNA vaccine approaches include the limitation of immunizations to products encoded by DNA (e.g., proteins) and the potential for atypical processing of bacterial and parasitic proteins by eukaryotic cells.
  • DNA e.g., proteins
  • Another significant problem with existing approaches to DNA vaccines is the instability of some vaccine insert sequences during the growth and amplification of DNA vaccine plasmids in bacteria.
  • One possible cause of instability is exposure during plasmid growth of secondary structures in vaccine inserts or the plasmid backbone that can be recognized by bacterial endonucleases.
  • the present invention provides novel pGA constructs.
  • the novel pGA constructs are useful as vectors for the delivery of DNA vaccines.
  • the present invention also provides novel pGA constructs having vaccine inserts.
  • the pathogen vaccine inserts can include the DNA transcription unit of any virus, bacteria, parasite and/or fungi.
  • the present invention describes novel methods of immunizing patients by administering therapeutically effective amounts of the novel pGA constructs comprising pathogen vaccine inserts.
  • the present invention describes novel methods of immunizing patients by administering therapeutically effective amounts of the novel pGA constructs comprising pathogen vaccine inserts followed by booster immunizations with live vectored vaccines such as recombinant modified vaccinia Ankara (MVA) vectors comprising the same vaccine inserts.
  • live vectored vaccines such as recombinant modified vaccinia Ankara (MVA) vectors comprising the same vaccine inserts.
  • the present invention also describes novel methods of raising mult-epitope CD8 T-cell responses by administering therapeutically effective amounts of the novel pGA constructs comprising pathogen vaccine inserts followed by booster immunizations with a live vectored vaccine such as recombinant modified vaccinia Ankara (MVA) vectors comprising the same vaccine inserts.
  • a live vectored vaccine such as recombinant modified vaccinia Ankara (MVA) vectors comprising the same vaccine inserts.
  • FIG. 1 illustrates a novel pGA1 construct of the present invention. Designations are identities and positions of elements in the vector. Designations in italic print are unique restriction endonuclease sites useful for cloning vaccine inserts into the vector.
  • FIG. 2 illustrates the DNA sequence SEQ ID NO: 1 of the novel pGA1 construct shown in FIG. 1. The positions of elements in the plasmid are indicated below the nucleotide sequence.
  • FIG. 3 illustrates a novel pGA2 construct of the present invention. Designations are identities and positions of elements in the vector. Designations in italic print are unique restriction endonuclease sites useful for cloning vaccine inserts into the vector.
  • FIG. 4 illustrates the DNA sequence SEQ ID NO: 2 of the novel pGA2 construct shown in FIG. 3. The positions of elements in the plasmid are indicated below the nucleotide sequence.
  • FIG. 5 illustrates a novel pGA3 construct of the present invention. Designations are identities and positions of elements in the vector. Designations in italic print are unique restriction endonuclease sites useful for cloning vaccine inserts into the vector.
  • FIG. 6 illustrates the DNA sequence SEQ ID NO: 3 of the novel pGA3 construct shown in FIG. 5. The positions of elements in the plasmid are indicated below the nucleotide sequence.
  • FIG. 7 compares the levels of anti-HA IgG raised by the influenza HI hemagglutinin expressed in a pGA vector (pGA3/H1) and in the pJW4303 research vector (pJW4303/H1).
  • BALB/c mice were immunized and boosted with a low dose (0.1 ⁇ g) or a high dose (1 ⁇ g), of the indicated plasmids using gene gun inoculations. A priming immunization was followed by a booster immunization at 4 weeks.
  • FIG. 8A presents a schematic of the parent wt BH10 provirus from which constructs producing non-infectious virus like particles (VLPs) were produced. Dotted regions indicate sequences that were deleted in the VLP constructs. Positions and designations of the various regions of the BH10 provirus are indicated in the rectangular boxes. The U3RU5 regions which encode the long terminal repeats contain transcriptional control elements. All other indicated regions encode proteins. For clarity, products expressed by pol (Prt, RT, Int) and env (SU and TM) are indicated.
  • FIG. 8B depicts the JS2 vaccine insert.
  • This 6.7 kb vaccine insert expresses the Gag, Prt, and RT sequences of the BH10 strain of HIV-1-IIIb, Tat and Vpu proteins that are from ADA, and Rev and Env proteins that are chimeras of ADA and BH10 sequences.
  • the Gag sequences include mutations of the zinc fingers to limit packaging of viral RNA.
  • the RT sequences encompass three point mutations to eliminate reverse transcriptase activity. Designations are the same as in FIG. 8A.
  • the bracketed area indicates the region of BH10 in which sequences from ADA have been substituted for the BH10 sequences to introduce a CCR-5 using Env.
  • the x's indicate safety mutations.
  • FIG. 8C depicts the JS5 insert.
  • JS5 is a 6 kb vaccine insert that expresses Gag, Prt, RT, Vpu Tat, and Rev.
  • JS5 is comprised of the same sequences as JS2 except that sequences in Env have been deleted. The deleted sequences are indicated in FIG. 8B as a filled rectangle. Designations are the same as in FIGS. 8A and 8B.
  • the Rev responsive element (RRE) which is in the 3′ region of Env is retained in the construct.
  • FIGS. 9A and 9B show Gag and Env expression, respectively, for intermediates in the construction of the JS2 vaccine insert. Data are from transient transfections in 293T cells.
  • pGA1/JS1 ADA VLP
  • pGA1/JS1 produced higher levels of both Gag (FIG. 9A) and Env (FIG. 9B) than wild type HIV-1 ADA or HIV-1 IIIb proviruses, and a VLP-producing DNA (dPol) used in previous studies.
  • FIG. 10 shows the expression of p24 capsid in transiently transfected cells by vaccine vectors expressing inserts without safety mutations (JS1 and JS4), inserts with point mutations in the zinc fingers and in RT (JS2 and JS5), and point mutations in the zinc fingers, RT, and protease (JS3 and JS6). Note that the safety mutations in the zinc fingers and RT supported active VLP expression whereas the safety mutation in Prt did not. JS2 and JS5 were chosen for continued vector development based on their high levels of expression in the presence of safety mutations.
  • FIGS. 11A and 11B show Gag and Env expression, respectively, of novel candidate vaccine constructs expressed by pGA vectors with and without intron A.
  • PGA1 but not pGA2 contains intron A.
  • pGA2/JS2 and pGA1/JS5 were chosen for use in vaccines based on their favorable levels of expression.
  • FIGS. 12 A- 12 D shows Western blots of cell lysates and tissue culture supernatants from 293T cells transfected with (1) mock, (2) pGA2/JS2, and (3) pGA1/JS5, where the primary antibody was pooled from anti-HIV Ig from infected patients (FIG. 12A), anti-p24 (FIG. 12B), anti-gp120 (FIG. 12C), and anti-RT (FIG. 12D) respectively.
  • FIG. 13 illustrates pGA
  • FIG. 14 compares Gag expression levels between pGA2/89.6, pGA1/Gag-Pol and pGA2/JS2. Comparative studies for expression were performed on transiently transfected 293T cells.
  • FIGS. 15 A- 15 C show the temporal frequencies of Gag-specific T cells.
  • FIG. 15A Gag-specific CD8 T Cell responses raised by DNA priming and rMVA booster immunization.
  • the schematic presents Gag-CM9-tetramer data generated in the high-dose i.d. DNA-immunized animals.
  • FIG. 15B Gag-CM9-Mamu-A*0l tetramer-specific T cells in Mamu-A*01 vaccinated and control macaques at various times before challenge and at two weeks after challenge.
  • the number at the upper right corner of each plot represents the frequency of tetramer-specific CD8 T cells as a % of total CD8 T cells.
  • the numbers above each column of plots designate individual animals.
  • FIG. 15C Gag-specific IFN-y ELISPOTs in A *01 and non-A *01 (hatched bars) vaccinated and non-vaccinated macaques at various times before challenge and at two weeks after challenge. Three pools of approximately 10-13 Gag peptides (22-mers overlapping by 12) were used for the analyses. The numbers above data bars represent the arithmetic mean ⁇ the standard deviation for the ELISPOTs within each group. The numbers at the top of the graphs designate individual animals. *, data not available; #, ⁇ 20 ELISPOTs per 1 ⁇ 10 6 PBMC.
  • FIGS. 16 A- 16 B shows the height and breadth of IFN-y-producing ELISPOTs against Gag and Env in the DNA/MVA memory response.
  • FIG. 16A Responses against individual Gag and Env peptide pools. Data for animals within a group are designated by the same symbol.
  • FIG. 16B Averages of the height and breadth of ELISPOT responses for the different groups.
  • the heights are the mean ⁇ the standard deviation for the sums of the Gag and Env ELISPOTs for animals in each group.
  • the breadths are the mean ⁇ the standard deviation for the number of Gag and Env pools recognized by animals in each group.
  • ELISPOT responses were determined in PBMC, during the memory phase, at 25 weeks after the rMVA booster (four weeks prior to challenge) using 7 pools of Gag peptides (approximately seven 22-mers overlapping by 12) representing about 70 amino acids of Gag sequence, and 21 pools of Env peptides (approximately ten 15-mers overlapping by 11) representing about 40 amino acids of Env sequence.
  • FIG. 17 shows the DNA sequence SEQ ID NO: 4 of a pGA2 construct comprising the vaccine insert, where the pathogen vaccine insert.
  • JS2 expresses clade B HIV-1 VLP. Both the nucleotide sequence SEQID NO: 4 and encoded proteins are indicated.
  • FIG. 18 shows the DNA sequence of a pGA1 construct comprising a pathogen vaccine insert, where the pathogen vaccine insert.
  • JS5 expresses lade B HIV-1 Gag-pol insert. Both the sequence and the encoded proteins are shown.
  • FIGS. 19 A- 19 E show temporal viral loads, CD4 counts and survival after challenge of vaccinated and control animals.
  • FIG. 19A Geometric mean viral loads
  • FIG. 19B geometric mean CD4 counts for vaccine and control groups at various weeks post-challenge. The key for the groups is in panel B.
  • FIG. 19C Survival curve for vaccinated and control animals. The dotted line represents all 24 vaccinated animals.
  • FIG. 19D viral loads
  • FIG. 19E CD4 counts for individual animals in the vaccine and control groups. The key to animal numbers is presented in FIG. 19E. Assays for the first 12 weeks post challenge had a background of 1000 copies of RNA per ml of plasma. Animals with loads below 1000 were scored with a load of 500. For weeks 16 and 20, the background for detection was 300 copies of RNA/ml. Animals with levels of virus below 300 were scored at 300.
  • FIGS. 20 A- 20 C show Post-challenge T-cell responses in vaccine and control groups.
  • FIG. 20A temporal tetramer+ cells and viral loads.
  • FIG. 20B Intracellular cytokine assays for IFN- ⁇ production in response to stimulation with the Gag-CM9 peptide at two weeks post-challenge. This ex vivo assay allows evaluation of the functional status of the peak post-challenge tetramer+ cells displayed in FIG. 15A.
  • FIG. 20C Proliferation assay at 12 weeks post-challenge. Gag-Pol-Env (open bars) and Gag-Pol (hatched bars) produced by transient transfections were used for stimulation. Supernatants from mock-transfected cultures served as control antigen. Proteins were used at approximately 1 ⁇ g per ml of p27 Gag for stimulations. Stimulation indices are the growth of cultures in the presence of viral antigens divided by the growth of cultures in the presence of mock antigen.
  • FIGS. 21 A- 21 E show lymph node histomorphology and viral loads at 12 weeks post-challenge.
  • FIG. 21A Typical lymph node from a vaccinated macaque showing evidence of follicular hyperplasia characterized by the presence of numerous secondary follicles with expanded germinal centers and discrete dark and light zones.
  • FIG. 21B Typical lymph node from an infected control animal showing follicular depletion and paracortical lymphocellular atrophy.
  • FIG. 21C A representative lymph node from an age-matched, uninfected macaque displaying non-reactive germinal centers.
  • FIG. 21D The percent of the total lymph node area occupied by germinal centers was measured to give a non-specific indicator of follicular hyperplasia. Data for uninfected controls are for four age-matched rhesus macaques.
  • FIG. 21E Lymph node virus burden was determined by in situ hybridization using an antisense riboprobe cocktail that was complementary to SHIV-89.6 gag and pol. All of the examined nodes were inguinal lymph nodes.
  • FIGS. 22 A- 22 D show temporal antibody responses following challenge.
  • Micrograms of total Gag (FIG. 22A) or Env (FIG. 22B) antibody were determined using enzyme linked immunosorbent assays (ELISAs).
  • the titers of neutralizing antibody for 89.6 (FIG. 22C) and 89.6P (FIG. 22D) were determined using MT-2 cell killing and neutral red staining. Titers are the reciprocal of the serum dilution giving 50% neutralization of the indicated viruses grown in human PBMC. Symbols for animals are the same as in FIG. 19.
  • FIGS. 23 A- 23 E show correlations and dose response curves for the vaccine trial (FIGS. 23A and B). Inverse correlations between peak vaccine raised IFN- ⁇ ELISPOTs and viral loads at 2 (FIG. 23A) and 3 (FIG. 23B) weeks post-challenge. Only twenty-three of the 24 vaccinated animals are included in the correlations because of the loss of the peak DNA/MVA ELISPOT sample for animal 3 (see FIG. 15C). (FIG. 23C) Dose response curves for the average height of Gag ELISPOTS at the peak DNA-MVA response (data from FIG. 15C). (FIG.
  • FIG. 24 shows anti-HA IgG raised by gene gun inoculation of DNAs expressing HA proteins.
  • FIG. 25 Shows avidity of the anti HA IgG raised by the three different HA DNA vaccines.
  • FIG. 26 shows protection from weight loss after virus challenge.
  • FIG. 27 illustrates the importance of including Env in the vaccine.
  • FIGS. 28 A- 28 D illustrates the importance of including Env in vaccines administered to animals challenged interectally with SHIV-89.6P.
  • FIG. 29 is a schematic representation of vector DNA vaccine constructs.
  • FIG. 30 shows Western blot results showing expression of vaccine constructs in vitro.
  • FIG. 31 is a temporal curve of measles virus neutralizing antibody.
  • This invention relates to novel vectors, novel vectors comprising pathogen vaccine inserts, and novel methods of immunizing patients against a pathogen.
  • the novel immunization methods elicit both cell-mediated and humoral immune responses that may limit the infection, spread or growth of the pathogen and result in protection against subsequent challenge by the pathogen.
  • nucleic acid refers to any natural and synthetic linear and sequential arrays of nucleotides and nucleosides, for example cDNA, genomic DNA, mRNA, tRNA, oligonucleotides, oligonucleosides and derivatives thereof.
  • nucleic acids may be collectively referred to herein as “constructs,” “plasmids,” or “vectors.”
  • Representative examples of the nucleic acids of the present invention include bacterial plasmid vectors including expression, cloning, cosmid and transformation vectors such as, but not limited to, pBR322, animal viral vectors such as, but not limited to, modified adenovirus, influenza virus, polio virus, pox virus, retrovirus, and the like, vectors derived from bacteriophage nucleic acid, and synthetic oligonucleotides like chemically synthesized DNA or RNA.
  • nucleic acid further includes modified or derivatised nucleotides and nucleosides such as, but not limited to, halogenated nucleotides such as, but not only, 5-bromouracil, and derivatised nucleotides such as biotin-labeled nucleotides.
  • isolated nucleic acid refers to a nucleic acid with a structure (a) not identical to that of any naturally occurring nucleic acid or (b) not identical to that of any fragment of a naturally occurring genomic nucleic acid spanning more than three separate genes, and includes DNA, RNA, or derivatives or variants thereof.
  • the term includes, but is not limited to, the following: (a) a DNA which has the sequence of part of a naturally occurring genomic molecule but is not flanked by at least one of the coding sequences that flank that part of the molecule in the genome of the species in which it naturally occurs; (b) a nucleic acid incorporated into a vector or into the genomic nucleic acid of a prokaryote or eukaryote in a manner such that the resulting molecule is not identical to any vector or naturally occurring genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), ligase chain reaction (LCR) or chemical synthesis, or a restriction fragment; (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein, and (e) a recombinant nucleotide sequence that is part of a hybrid
  • nucleotide sequence is in purified form.
  • purified in reference to nucleic acid represents that the sequence has increased purity relative to the natural environment.
  • polypeptide and “protein” refer to a polymer of amino acids of three or more amino acids in a serial array, linked through peptide bonds.
  • polypeptide includes proteins, protein fragments, protein analogues, oligopeptides and the like.
  • polypeptides contemplates polypeptides as defined above that are encoded by nucleic acids, produced through recombinant technology, isolated from an appropriate source, or are synthesized.
  • polypeptides further contemplates polypeptides as defined above that include chemically modified amino acids or amino acids covalently or noncovalently linked to labeling ligands.
  • fragment refers to an isolated portion of the subject nucleic acid constructed artificially (e.g., by chemical synthesis) or by cleaving a natural product into multiple pieces, using restriction endonucleases or mechanical shearing, or a portion of a nucleic acid synthesized by PCR, DNA polymerase or any other polymerizing technique well known in the art, or expressed in a host cell by recombinant nucleic acid technology well known to one of skill in the art.
  • fragment as used herein may also refer to an isolated portion of a polypeptide, wherein the portion of the polypeptide is cleaved from a naturally occurring polypeptide by proteolytic cleavage by at least one protease, or is a portion of the naturally occurring polypeptide synthesized by chemical methods well known to one of skill in the art.
  • gene refers to nucleic acid sequences (including both RNA or DNA) that encode genetic information for the synthesis of a whole RNA, a whole protein, or any portion of such whole RNA or whole protein.
  • foreign genes genes that are not naturally part of a particular organism's genome
  • heterologous genes or “exogenous genes”
  • endogenous genes genes that are naturally a part of a particular organism's genome are referred to as “endogenous genes”.
  • RNA nucleic acid molecule at least complementary in part to a region of one of the two nucleic acid strands of the gene.
  • expression also refers to the translation from said RNA nucleic acid molecule to give a protein or polypeptide or a portion thereof.
  • locus refers to the site of a gene on a chromosome. Pairs of genes control hereditary traits, each in the same position on a pair of chromosomes. These gene pairs, or alleles, may both be dominant or both be recessive in expression of that trait. In either case, the individual is said to be homozygous for the trait controlled by that gene pair. If the gene pair (alleles) consists of one dominant and one recessive trait, the individual is heterozygous for the trait controlled by the gene pair.
  • allelic variants Natural variation in genes or nucleic acid molecules caused by, for example, recombination events or resulting from mutation, gives rise to allelic variants with similar, but not identical, nucleotide sequences. Such allelic variants typically encode proteins with similar activity to that of the protein encoded by the gene to which they are compared, because natural selection typically selects against variations that alter function. Allelic variants can also comprise alterations in the untranslated regions of the gene as, for example, in the 3′ or 5′ untranslated regions or can involve alternate splicing of a nascent transcript, resulting in alternative exons being positioned adjacently.
  • transcription regulatory sequences refers to nucleotide sequences that are associated with a gene nucleic acid sequence and which regulate the transcriptional expression of the gene.
  • the “transcription regulatory sequences” may be isolated and incorporated into a vector nucleic acid to enable regulated transcription in appropriate cells of portions of the vector DNA.
  • the “transcription regulatory sequence” may precede, but are not limited to, the region of a nucleic acid sequence that is in the region 5′ of the end of a protein coding sequence that may be transcribed into mRNA.
  • Transcriptional regulatory sequences may also be located within a protein coding region, in regions of a gene that are identified as “intron” regions, or may be in regions of nucleic acid sequence that are in the region of nucleic acid.
  • coding region refers to a continuous linear arrangement of nucleotides that may be translated into a protein.
  • a full length coding region is translated into a full length protein; that is, a complete protein as would be translated in its natural state absent any post-translational modifications.
  • a full length coding region may also include any leader protein sequence or any other region of the protein that may be excised naturally from the translated protein.
  • probe when referring to a nucleic acid, refers to a nucleotide sequence that can be used to hybridize with and thereby identify the presence of a complementary sequence, or a complementary sequence differing from the probe sequence but not to a degree that prevents hybridization under the hybridization stringency conditions used.
  • the probe may be modified with labels such as, but not only, radioactive groups, biotin, or any other label that is well known in the art.
  • nucleic acid vector refers to a natural or synthetic single or double stranded plasmid or viral nucleic acid molecule that can be transfected or transformed into cells and replicate independently of, or within, the host cell genome.
  • a circular double stranded plasmid can be linearized by treatment with an appropriate restriction enzyme based on the nucleotide sequence of the plasmid vector.
  • a nucleic acid can be inserted into a vector by cutting the vector with restriction enzymes and ligating the pieces together.
  • the nucleic acid molecule can be RNA or DNA.
  • expression vector refers to a nucleic acid vector that may further include at least one regulatory sequence operably linked to a nucleotide sequence coding for the Mago protein. Regulatory sequences are well recognized in the art and may be selected to ensure good expression of the linked nucleotide sequence without undue experimentation by those skilled in the art.
  • regulatory sequences includes promoters, enhancers, and other elements that may control expression. Standard molecular biology textbooks such as Sambrook et al. eds “Molecular Cloning: A Laboratory Manual” 2nd ed. Cold Spring Harbor Press (1989) may be consulted to design suitable expression vectors, promoters, and other expression control elements. It should be recognized, however, that the choice of a suitable expression vector depends upon multiple factors including the choice of the host cell to be transformed and/or the type of protein to be expressed.
  • transformation and “transfection” as used herein refer to the process of inserting a nucleic acid into a host.
  • Many techniques are well known to those skilled in the art to facilitate transformation or transfection of a nucleic acid into a prokaryotic or eukaryotic organism. These methods involve a variety of techniques, such as treating the cells with high concentrations of salt such as, but not only a calcium or magnesium salt, an electric field, detergent, or liposome mediated transfection, to render the host cell competent for the uptake of the nucleic acid molecules.
  • salt such as, but not only a calcium or magnesium salt, an electric field, detergent, or liposome mediated transfection
  • the term “recombinant cell” refers to a cell that has a new combination of nucleic acid segments that are not covalently linked to each other in nature.
  • a new combination of nucleic acid segments can be introduced into an organism using a wide array of nucleic acid manipulation techniques available to those skilled in the art.
  • a recombinant cell can be a single eukaryotic cell, or a single prokaryotic cell, or a mammalian cell.
  • the recombinant cell can harbor a vector that is extragenomic. An extragenomic nucleic acid vector does not insert into the cell's genome.
  • a recombinant cell can further harbor a vector or a portion thereof that is intragenomic.
  • the term intragenomic defines a nucleic acid construct incorporated within the recombinant cell's genome.
  • nucleic acid refers to combinations of at least two nucleic acid sequences that are not naturally found in a eukaryotic or prokaryotic cell.
  • the nucleic acid sequences may include, but are not limited to nucleic acid vectors, gene expression regulatory elements, origins of replication, sequences that when expressed confer antibiotic resistance, and protein-encoding sequences.
  • recombinant polypeptide is meant to include a polypeptide produced by recombinant DNA techniques such that it is distinct from a naturally occurring polypeptide either in its location, purity or structure. Generally, such a recombinant polypeptide will be present in a cell in an amount different from that normally observed in nature.
  • patients refers to animals, preferably mammals, and more preferably humans.
  • immunizing refers to the production of an immune response in a patient that protects (partially or totally) from the manifestations of infection (i.e., disease) caused by a pathogen.
  • a patient immunized by the present invention will not be infected by the pathogen or will be infected to a lesser extent than would occur without immunization.
  • Immunizations may be either prophylactic or therapeutic in nature. That is, both previously uninfected and infected patients may be immunized with the present invention.
  • DNA transcription unit refers to a polynucleotide sequence that includes at least two components: antigen-encoding DNA and transcriptional promoter elements.
  • a DNA transcription unit may optionally include additional sequences, such as enhancer elements, splicing signals, termination and polyadenylation signals, viral replicons, and/or bacterial plasmid sequences.
  • the DNA transcription unit can be produced by a number of known methods. For example, DNA encoding the desired antigen can be inserted into an expression vector to construct the DNA transcription unit, as described in Maniatis et al, Molecular Cloning: A Laboratory Manual, 2d, Cold Spring Harbor Laboratory Press (1989), the disclosure of which is incorporated by reference in its entirety.
  • the term “vaccine insert” as used herein refers to the DNA transcription unit of a pathogen.
  • the vaccine insert is a DNA transcription unit that can generate an immune responses in a patient.
  • th evaccine insert is a pathogen vaccine insert encoding antigens derived from any virus, bacteria, parasite and/or fungi.
  • Exemplary viruses include herpesvirus, orthomyxoviruses, rhinoviruses, picornaviruses, adenoviruses, paramyxoviruses, coronaviruses, rhabdoviruses, togaviruses, flaviviruses, bunyaviruses, rubella virus, reovirus, measles, hepadna viruses, Ebola, retroviruses (including human immunodeficiency virus), and the like.
  • Exemplary bacteria include tuberculosis, mycobateria, spirochetes, rickettsias, chlamydia, mycoplasma and the like.
  • Exemplary parasites include malaria and the like.
  • Exemplary fungi include yeasts, molds, and the like.
  • antigen refers to any protein, carbohydrate, or other moiety expressed by a pathogen that is capable of eliciting a protective response against a pathogen.
  • the antigen may or may not be a structural component of the pathogen.
  • encoded antigens that can be translation products or polypeptides of various lengths. Antigens undergo normal host cell modifications such as glycosylation, myristoylation or phosphorylation. In addition, they can be designed to undergo intracellular, extracellular or cell-surface expression. Furthermore, they can be designed to undergo assembly and release from cells.
  • adjuvant means a substance added to a vaccine to increase a vaccine's immunogenicity.
  • the mechanism of how an adjuvant operates is not entirely known. Some adjuvants are believed to enhance the immune response by slowly releasing the antigen, while other adjuvants are strongly immunogenic in their own right and are believed to function synergistically.
  • Known vaccine adjuvants include, but are not limited to, oil and water emulsions (for example, complete Freund's adjuvant and incomplete Freund's adjuvant), Corynebacterium parvum , Bacillus Calmette Guerin, aluminum hydroxide, glucan, dextran sulfate, iron oxide, sodium alginate, Bacto-Adjuvant, certain synthetic polymers such as poly amino acids and co-polymers of amino acids, saponin, “REGRESSIN” (Vetrepharm, Athens, Ga.), “AVRIDINE” (N, N-dioctadecyl-N′,N′-bis(2-hydroxyethyl)-propanediamine), paraffin oil, and muramyl dipeptide.
  • Adjuvants also encompass genetic adjuvants such as immunomodulatory molecules encoded in a co-inoculated DNA.
  • the co-inoculated DNA can be in the same vaccine construct as the vaccine immunogen or in a
  • the term “pharmaceutically acceptable carrier” means a vehicle for containing the vaccine that can be injected into a bovine host without adverse effects.
  • Suitable pharmaceutically acceptable carriers known in the art include, but are not limited to, sterile water, saline, glucose, dextrose, or buffered solutions.
  • Carriers may include auxiliary agents including, but not limited to, diluents, stabilizers (i.e., sugars and amino acids), preservatives, wetting agents, emulsifying agents, pH buffering agents, viscosity enhancing additives, colors and the like.
  • selectable marker gene refers to an expressed gene that allows for the selection of a population of cells containing the selectable marker gene from a population of cells not having the expressed selectable marker gene.
  • the “selectable marker gene” may be an “antibiotic resistance gene” that can confer tolerance to a specific antibiotic by a microorganism that was previously adversely affected by the drug. Such resistance may result from a mutation or the acquisition of resistance due to plasmids containing the resistance gene transforming the microorganism.
  • terminal sequence refers to nucleotide sequences that function to stop transcription.
  • transcription or “transcribe” as used herein refers to the process by which RNA molecules are formed upon DNA templates by complementary base pairing. This process is mediated by RNA polymerase.
  • VLP refers to virus-like particles and, as used, also refers to aggregates of viral proteins.
  • the major immunological advantage of DNA-based immunizations is the ability of the immunogen to be presented by both MHC class I and class II molecules. Endogenously synthesized proteins readily enter processing pathways for the loading of peptide epitopes onto MHC I as well as MHC II molecules. MHC I-presented epitopes raise cytotoxic T-cells (Tc) responses whereas MHC II-presented epitopes raise helper T-cells (Th). By contrast, immunogens that are not synthesized in cells are largely restricted to the loading of MHC II epitopes and the raising of Th but not Tc.
  • DNA vaccines When compared with live attenuated vaccines or recombinant viral vectors that produce immunogens in cells and raise both Th and Tc, DNA vaccines have the advantages of not being infectious and of focusing the immune response on only those antigens desired for immunization. DNA vaccines also are advantageous because they can be manipulated relatively easily to raise type 1 or type 2 T-cell help. This allows a vaccine to be tailored for the type of immune response that will be mobilized to combat an infection. DNA vaccines are also cost effective because of the ease with which plasmids can be constructed using recombinant DNA technology, the ability to use a generic method for vaccine production (growth and purification of plasmid DNA), and the stability of DNA over a wide range of temperatures.
  • CMV cytomegalovirus
  • transcriptional control elements useful in the present invention include a strong polyadenylation signal such as, for example, that derived from a bovine growth hormone encoding gene, or a rabbit ⁇ globin polyadenylation signal (Bohm et al., 1996; Chapman et al., 1991; Hartikka et al., 1996; Manthorpe et al., 1993; Montgomery et al., 1993).
  • the CMV immediate early promoter may be used with or without intron A (Chapman et al., 1991).
  • intron A increases the expression of many antigens from RNA viruses, bacteria, and parasites, presumably by providing the expressed RNA with sequences which support processing and function as an eukaryotic mRNA. It will be appreciated that expression also may be enhanced by other methods known in the art including, but not limited to, optimizing the codon usage of prokaryotic mRNAs for eukaryotic cells (Andre et al., 1998; Uchijima et al., 1998). Multi-cistronic vectors may be used to express more than one immunogen or an immunogen and a immunostimulatory protein (Iwasaki et al., 1997a; Wild et al., 1998).
  • Immunogens can also be engineered to be more or less effective for raising antibody or Tc by targeting the expressed antigen to specific cellular compartments. For example, antibody responses are raised more effectively by s antigens that are displayed on the plasma membrane of cells, or secreted therefrom, than by antigens that are localized to the interior of cells (Boyle, Koniaras, and Lew, 1997; Inchauspe et al., 1997).
  • Tc responses may be enhanced by using N-terminal ubiquitination signals which target the DNA-encoded protein to the proteosome causing rapid cytoplasmic degradation and more efficient peptide loading into the MHC I pathway (Rodriguez, Zhang, and Whitton, 1997; Tobery and Siliciano, 1997; Wu and Kipps, 1997).
  • N-terminal ubiquitination signals which target the DNA-encoded protein to the proteosome causing rapid cytoplasmic degradation and more efficient peptide loading into the MHC I pathway.
  • Recombinant DNA molecules encoding a string of MHC epitopes from different pathogens can elicit Tc responses to a number of pathogens (Hanke et al., 1998b). These strings of Tc epitopes are most effective if they also include a Th epitope (Maecker et al., 1998; Thomson et al., 1998).
  • Vaccination by saline injections can be intramuscular (i.m.) or intradermal (i.d.) (Fynan et al., 1993).
  • the dose of DNA needed to raise a response depends upon the method of delivery, the host, the vector, and the encoded antigen. The most profound effect is seen for the method of delivery. From 10 ⁇ g to 1 mg of DNA is generally used for saline injections of DNA, whereas from 0.2 ⁇ g to 20 ⁇ g of DNA is used for gene gun deliveries of DNA. In general, lower doses of DNA are used in mice (10-100 ⁇ g for saline injections and 0.2 ⁇ g to 2 ⁇ g for gene gun deliveries), and higher doses in primates (100 ⁇ g to 1 mg for saline injections and 2 ⁇ g to 20 ⁇ g for gene gun deliveries). The much lower amount of DNA required for gene gun deliveries reflect the gold beads directly delivering DNA into cells.
  • Protein boosts have been used to increase neutralizing antibody responses to the HIV-1 Env.
  • Recombinant pox virus boosts have been used to increase both humoral and cellular immune responses.
  • a number of different pox viruses can be used for the pox boost.
  • a vaccinia virus termed modified vaccinia Ankara (MVA) has been particularly effective in mouse models (Schneider et al., 1998). This may reflect MVA, which is replication defective in mammalian models, being attenuated for the ability to evade host immune responses.
  • MVA modified vaccinia Ankara
  • DNA vaccines for immunodeficiency viruses such as HIV-1 encounter the challenge of sufficiently limiting an incoming infection such that the inexorable long-term infections that lead to AIDS are prevented.
  • Complicating this is that neutralizing antibodies is both difficult to raise and specific against particular viral strains (Burton and Montefiori, 1997; Moore and Ho, 1995).
  • Much effort has focused on raising cell-mediated responses of sufficient strength to severely curtail infections.
  • the best success at raising high titers of Tc have come from immunization protocols using DNA primes followed by recombinant pox virus boosters.
  • the novel pGA vectors of the present invention have a prokaryotic origin of replication, a selective marker gene for plasmid selection, and a transcription cassette for eukaryotic cells.
  • Unique to the pGA vectors of the present invention is the inclusion of the lambda terminator in the same transcriptional orientation, and following, the selective marker gene. This terminator sequence prevents read-through from the kanamycin cassette into vaccine sequences while the plasmid is being produced in bacteria. Prevention of transcriptional read-through stabilizes vaccine insert sequences by limiting the exposure of secondary structures that can be recognized by bacterial endonucleases.
  • a transcription cassette as incorporated in the pGA vectors of the present invention uses sequences from the cytomegalovirus immediate early promoter (CMVIE) and from the bovine growth hormone polyadenylation sequences (BGHpA) to control transcription.
  • CMVIE cytomegalovirus immediate early promoter
  • BGHpA bovine growth hormone polyadenylation sequences
  • a leader sequence that is a synthetic homolog of the tissue plasminogen activator gene leader sequence (tPA) is optional in the transcription cassette.
  • the vectors of the present invention differ in the sites that can be used for accepting vaccine inserts and in whether the transcription cassette includes intron A sequences in the CMVIE promoter. Both intron A and the tPA leader sequence have been shown in certain instances to supply a strong expression advantage to vaccine inserts (Chapman et al., 1991).
  • pGAl is a 3894 bp plasmid.
  • pGA1 comprises a promoter (bp 1-690), the CMV-intron A (bp 691-1638), a synthetic mimic of the tPA leader sequence (bp 1659-1721), the bovine growth hormone polyadenylation sequence (bp1761-1983), the lambda T0 terminator (bp 1984-2018), the kanamycin resistance gene (bp 2037-2830) and the Co1EI replicator (bp 2831-3890).
  • the DNA sequence of the pGA1 construct (SEQ ID NO: 1) is shown in FIG. 2.
  • the indicated restriction sites are single cutters useful for the cloning of vaccine inserts.
  • the ClaI or BspD1 sites are used when the 5′ end of a vaccine insert is cloned upstream of the tPA leader.
  • the NheI site is used for cloning a sequence in frame with the tPA leader sequence.
  • the sites listed between SmaI and B1nI are used for cloning the 3′ terminus of a vaccine insert.
  • pGA2 is a 2947 bp plasmid lacking the 947 bp of intron A sequences found in pGA1.
  • pGA2 is the same as pGA1, except for the deletion of intron A sequences.
  • pGA2 is valuable for cloning sequences which do not require an upstream intron for efficient expression, or for cloning sequences in which an upstream intron might interfere with the pattern of splicing needed for good expression.
  • FIG. 3 presents a map of pGA2 with useful restriction sites for cloning vaccine inserts
  • FIG. 4 shows the DNA sequence SEQ ID NO: 2.
  • the use of restriction sites for cloning vaccine inserts into pGA2 is the same as that used for cloning fragments into pGA1.
  • pGA3 is a 3893 bp plasmid that contains intron A.
  • pGA3 is the same as pGA1 except for the cloning sites that can be used for the introduction of vaccine inserts.
  • inserts cloned upstream of the tPA leader sequence use a Hind III site.
  • Sequences cloned downstream from the tPA leader sequence use sites between the SmaI and the B1nI site. In pGA3, these sites include a BamHI site.
  • FIG. 5 shows the map for pGA3
  • FIG. 6 shows the DNA sequence SEQ ID NO: 3.
  • any vaccine insert known in the art can be used in the novel pGA constructs described herein, including but not limited to viral pathogens like HIV, influenza, measles, herpes, Ebola, and the like.
  • the present invention contemplates inserts from immunodeficiency virus, more preferably HIV, including all clades of HIV-1 and HIV-2 and modifications thereof; influenza virus genes including all subtypes and modifications thereof; and vaccine inserts derived from measles genes.
  • immunodeficiency virus more preferably HIV, including all clades of HIV-1 and HIV-2 and modifications thereof; influenza virus genes including all subtypes and modifications thereof; and vaccine inserts derived from measles genes.
  • the immunodeficiency virus vaccine inserts of the present invention were designed to express non-infectious virus like particles (VLPs) from a single DNA. This was achieved using the subgenomic splicing elements normally used by immunodeficiency viruses to express multiple gene products from a single viral RNA. Important to the subgenomic splicing patterns are (i) splice sites and acceptors present in full length viral RNA, (ii) the Rev responsive element (RRE) and (iii) the Rev protein. The splice sites in retroviral RNAs use the canonical sequences for splice sites in eukaryotic RNAs.
  • VLPs virus like particles
  • the RRE is an ⁇ 200 bp RNA structure that interacts with the Rev protein to allow transport of viral RNAs from the nucleus to the cytoplasm.
  • the 10 kb RNA of immunodeficiency virus undergoes splicing to the mRNAs for the regulatory genes Tat, Rev, and Nef. These genes are encoded by exons present between RT and Env and at the 3′ end of the genome.
  • the singly spliced mRNA for Env and the unspliced mRNA for Gag and Pol are expressed in addition to the multiply spliced mRNAs for Tat, Rev, and Nef.
  • the expression of non-infectious VLPs from a single DNA affords a number of advantageous features to an immunodeficiency virus vaccine.
  • the expression of a number of proteins from a single DNA affords the vaccinated host the opportunity to respond to the breadth of T- and B-cell epitopes encompassed in these proteins.
  • the expression of proteins containing multiple epitopes affords the opportunity for the presentation of epitopes by diverse histocompatibility types. By using whole proteins, one offers hosts of different histocompatibility types the opportunity to raise broad-based T-cell responses.
  • Antibody responses are often best primed by multi-valent vaccines that present an ordered array of an epitope to responding B-cells (Bachmann, Zinkemagel, 1997).
  • Virus-like particles by virtue of the multivalency of Env in the virion membrane, will facilitate the raising of anti-Env antibody responses. These particles will also present non-denatured and normal forms of Env to the immune system.
  • novel vectors of the present invention can be administered to a patient in the presence of adjuvants or other substances that have the capability of promoting DNA uptake or recruiting immune system cells to the site of the inoculation.
  • Embodiments include combining the DNA vaccine with conventional adjuvants or genetic adjuvants.
  • Conventional adjuvants including reagents that favor the stability and uptake of the DNA, recruit immune system cells to the site of inoculation, or facilitate the immune activation of responding lymphoid cells, include but are not limited to oil and water emulsions (for example, complete Freund's adjuvant and incomplete Freund's adjuvant), Corynebacterium parvum , Bacillus Calmette Guerin, aluminum hydroxide, glucan, dextran sulfate, iron oxide, sodium alginate, Bacto-Adjuvant, certain synthetic polymers such as poly amino acids and co-polymers of amino acids, saponin, “REGRESSIN” (Vetrepharm, Athens, Ga.), “AVRIDINE” (N,N-dioctadecyl-N′,N′-bis(2-hydroxyethyl)-propanediamine), paraffin oil, and muramyl dipeptide.
  • the present invention also contemplates the use of genetic adjuvants such
  • a vaccine according to the present invention can be administered in a variety of ways including through any parenteral or topical route.
  • an individual can be inoculated by intravenous, intraperitoneal, intradermal, subcutaneous or intramuscular methods.
  • Inoculation can be, for example, with a hypodermic needle, needleless delivery devices such as those that propel a stream of liquid into the target site, or with the use of a gene gun that bombards DNA on gold beads into the target site.
  • the vector comprising the pathogen vaccine insert can be administered to a mucosal surface by a variety of methods including intranasal administration, i.e., nose drops or inhalants, or intrarectal or intravaginal administration by solutions, gels, foams, or suppositories.
  • the vector comprising the vaccine insert can be orally administered in the form of a tablet, capsule, chewable tablet, syrup, emulsion, or the like.
  • vectors can be administered transdermally, by passive skin patches, iontophoretic means, and the like.
  • any appropriate physiologically acceptable medium is suitable for introducing the vector comprising the pathogen vaccine insert into the patient.
  • suitable pharmaceutically acceptable carriers known in the art include, but are not limited to, sterile water, saline, glucose, dextrose, or buffered solutions.
  • Carriers may include auxiliary agents including, but not limited to, diluents, stabilizers (i.e., sugars and amino acids), preservatives, wetting agents, emulsifying agents, pH buffering agents, viscosity enhancing additives, colors and the like.
  • pGA1 as illustrated in FIG. 1 and FIG. 2 contains the Co1E1 origin of replication, the kanamycin resistance gene for antibiotic selection, the lambda T0 terminator, and a eukaryotic expression cassette including an upstream intron.
  • the Co1E1 origin of replication is a 600 nucleotide DNA fragment that contains the origin of replication (ori), encodes an RNA primer, and encodes two negative regulators of replication initiation. All enzymatic functions for replication of the plasmid are provided by the bacterial host.
  • the original constructed plasmid that contained the Co1E1 replicator was pBR322 (Bolivar, et al. 1977; Sutcliffe, et al. 1978).
  • the kanamycin resistance gene is an antibiotic resistance gene for plasmid selection in bacteria.
  • the lambda T0 terminator prevents read through from the kanamycin resistance gene into the vaccine transcription cassette during prokaryotic growth of the plasmid (Scholtissek and Grosse, 1987). By preventing read through into the vaccine expression cassette, the terminator helps stabilize plasmid inserts during growth in bacteria.
  • the eukaryotic expression cassette is comprised of the CMV immediate early promoter including intron A (CMVIE-IA) and termination sequences from the bovine growth hormone polyadenylation sequence (BGHpA).
  • CMVIE-IA CMV immediate early promoter including intron A
  • BGHpA bovine growth hormone polyadenylation sequence
  • tPA tissue plasminogen activator
  • Cloning sites within the transcription cassette include a Clal site upstream of the tPA leader, a NheI site for cloning in frame with the tPA leader, and XmnI, SmaI, RsrII, AvrII, and B1nI sites for cloning prior to the BGHpA.
  • the Co1E1 replicator, the Kanamycin resistance gene and transcriptional control elements for eukaryotic cells were combined in one plasmid using polymerase chain reaction (PCR) fragments from a commercial vector, pZErO-2 (Invitrogen, Carlsbad, Calif.) and a eukaryotic expression vector , pJW4303 (Lu et al., 1997).
  • PCR polymerase chain reaction
  • a 2040 bp fragment from pJW4303 from nt 376 to nt 2416 included the CMVIE promoter with intron A, a synthetic homolog of the tissue plaminogen activator leader (tPA), and the bovine growth hormone polyadenylation site (BGHpA). Fragments were amplified by polymerase chain reaction (PCR) with oligonucleotide primers containing SalI sites.
  • PCR polymerase chain reaction
  • a ligation product with the transcription cassettes for Kanamycin resistance from pZeRO2 and the eukaryotic transcription cassette form pJW4303 in opposite transcriptional orientations was identified for further development. Nucleotide numbering for this parent for the pGA vectors was started from the first bp of the 5′ end of the CMV promoter.
  • the T0 terminator was introduced into this parent for the pGA vectors by PCR amplification of a 391 bp fragment with a BamH1 restriction endonuclease site at its 5′end and an XbaI restriction endonuclease site at its 3′end.
  • the initial 355 bp of the fragment were sequences in the BGHpA sequence derived from the pJW4303 transcription cassette, the next 36 bases in a synthetic oligonuclotide introduced the T0 sequence and the XbaI site.
  • the introduced T0 teminator sequences comprised the nucleotide sequence as follows: 5′-ATAAAAAACGCCCGGCGGCAACCGAGCGTTCTGAA-3′ (SEQ ID NO:)
  • T0 terminator containing BamH1-XbaI fragment was substituted for the homologous fragment without the T0 terminator in the plasmid created from pXeRO 2 and pJW4303.
  • the product was sequenced to verify the TO orientation.
  • a region in the eukaryotic transcription cassette between nucleotides 1755-1845 contained the last 30bp of the reading frame for SIV nef. This region was removed from pGA by mutating the sequence at nt1858 and generating an Avr II restriction endonuclease site. A naturally occurring Avr II site is located at nt1755. Digestion with Avr II enzyme and then religation with T4 DNA ligase allowed for removal of the SIV segment of DNA between nucleotides 1755-1845. To facilitate cloning of HIV-1 sequences, into pGA vectors a ClaI site was introduced at bp1645 and an RsrII site at bp 1743 using site directed mutagenesis. Constructions were verified by sequence analyses.
  • pGA2 is identical to pGA1 except for deletion of the intron A sequences from the CMVIE promoter.
  • pGA2 was created from pGA1 by introducing a ClaI site 8 bp downstream from the mRNA cap site in the CMVIE promoter.
  • the ClaI site was introduced using oligonucleotide-directed mutagensis using the complimentary primers 5′-CCGTCAGATCGCATCGATACGCCATCCACG-3′ (SEQ ID NO:) and 5′-CGTGGATGGCGTATCGATGCGATCTGACGG-3′ (SEQ ID NO:).
  • pGA1 was digested with ClaI to remove the 946 bp ClaI fragment from pGA1, and then religated to yield pGA2.
  • pGA3 as shown in FIG. 5 and FIG. 6 is identical to pGA1 except for the introduction of a HindIII site in stead of the ClaI site at nt 1645 and a BamHI site instead of the RsrII site at nucleotide 1743.
  • pGW4303 plasmid has been used for DNA vaccinations in mice, rabbits, and rhesus macaques (Robinson et al. 1999; Robinson et al., 1997; Pertmer, et al., 1995; Feltquate, et al. 1997; Torres, et al. 1999).
  • the pGA3/H1 and pJW4303/H1 vaccine plasmids expressed similar levels of H1 in eukaryotic cells, as summarized below: TABLE 5 In Vitro Expression Levels of HA plasmids.
  • Relative HA Units Plasmids Supernatant Cell Lysate PGA3/H1 0.1 ⁇ 0.1 5.7 ⁇ 0.6 pGA vector 0.0 ⁇ 0.0 0.2 ⁇ 0.1 PJW4303/H1 0.3 ⁇ 0.05 4.8 ⁇ 0.5 pJW4303 0.0 ⁇ 0.0 0.1 ⁇ 0.1
  • mice were vaccinated with DNA coated gold particles via gene gun. Mice were primed and boosted with a low dose (0.1 ⁇ g) or a high dose (1 ⁇ g) of the plasmid DNAs. The booster immunization was given at 4 weeks after the priming immunization.
  • the amount of anti-H1 IgG raised in response to immunizations was as high or higher following immunization with pGA3/H1 than following immunization with pJW4303/H1 (FIG. 7).
  • the pGA vector proved to be as effective, or more effective, than the pJW4303 vector at raising immune responses.
  • Immunodeficiency virus vaccine inserts expressing virus like particles have been developed in pGA1 and pGA2.
  • the VLP insert was designed with dade B HIV-1 sequences so that it would match HIV-1 sequences that are endemic in the United States. Within clade B, different isolates exhibit clustal diversity, with each isolate having overall similar diversity from the consensus sequence for the lade (Subbarao, Schochetman, 1996). Thus, any lade B isolate can be used as a representative sequence for other lade B isolates. HIV-1 isolates use different chemokine receptors as co-receptors. The vast majority of viruses that are undergoing transmission use the CCR-5 co-receptor (Berger, E. A., 1997). Therefore the vaccine insert was designed to have a CCR-5 using Env.
  • VLPs with an R5-Env by a HIV-1 DNA vaccine also has the advantage of supporting Env-mediated entry of particles into professional antigen presenting cells (APCs) such as dendritic cells and macrophages. Both dendritic cells and macrophages express the CD4 receptor and the CCR-5 co-receptor used by CCR-5-tropic (R5) HIV-1 Envs.
  • APCs professional antigen presenting cells
  • dendritic cells and macrophages express the CD4 receptor and the CCR-5 co-receptor used by CCR-5-tropic (R5) HIV-1 Envs.
  • R5 Env in the vaccine, the VLP expressed in a transfected non-professional APC (for example keratinocyte or muscle cells) can gain entry into the cytoplasm of an APC by Env-mediated entry.
  • DNA-based immunizations rely on professional APCs for antigen presentation (Corr et al., 1996; Fu, et al., 1997; Iwasaki A, et al., 1997). Much of DNA-based immunization is accomplished by direct transfection of professional APC (Condon et al., 1996; Porgador et al., 1998). Transfected muscle cells or keratinocytes serve as factories of antigen but do not directly raise immune response (Torres et al., 1997). By using an expressed antigen that is assembled and released from transfected keratinocytes or muscle cells and then actively enters professional APC, the efficiency of the immunization may be increased.
  • candidate vaccines were constructed from 7 different HIV-1 sequences, as shown in the following table: TABLE I Comparison of candidate vaccine inserts Ability Plasmid Sequences to grow Expression Expression designation tested plasmid of Gag of Env Comment BH10-VLP BH10 good good good good X4 Env 6A-VLP 6A env in poor not tested not tested BH10-VLP BAL-VLP BAL env in good poor poor BH10-VLP ADA-VLP ADA env in good good good good chosen for vaccine, BH10-VLP renamed pGA1/JS1 CDC-A-VLP CDC-A env in good good poor BH10-VLP CDC-B-VLP CDC-B-env in good good good good good not as favorable BH10-VLP expression as ADA CDC-C-VLP CDC -C env good good good good good not as favorable in BH10-VLP expression as ADA
  • pBH10-VLP An initial construct, pBH10-VLP, was prepared from IIIb sequences that are stable in bacteria and have high expression in eukaryotic cells.
  • the BH10 sequences were obtained from the NIH-sponsored AIDS Repository (catalog #90).
  • the parental pBH10 was used as the template for PCR reactions to construct pBH 10-VLP.
  • Primers were designed to yield a Gag-Rt PCR product (5′ PCR product) encompassing from 5′ to 3′ 105 bp of the 5′ untranslated leader sequence and gag and pol sequences from the start codon for Gag to the end of the RT coding sequence.
  • the oligonucleotide primers introduced a ClaI site at the 5′ end of the PCR product and EcoRI and NheI sites at the 3′ end of the PCR product.
  • Sense primer 1 (5′-GAGCTCTATCGATGCAGGACTCGGCTTGC-3′ (SEQ ID NO:)) and antisense primer 2 (5′-GGCAGGTTTTAATCGCTAGCCTATGCTCTCC-3′ (SEQ ID NO: )) were used to amplify the 5′PCR product.
  • the PCR product for the env region of HIV-1 encompassed the vpu, tat, rev, and env sequences and the splice acceptor sites necessary for proper processing and expression of their respective mRNAs.
  • An EcoRI site was introduced at the 5′ end of this product and NheI and RsrII sites were introduced into the 3′ end.
  • Sense primer 3 (5′-GGGCAGGAGTGCTAGCC-3′ (SEQ ID NO:)
  • antisense primer 4 (5′-CCACACTACTTTCGGACCGCTAGCCACCC-3′ (SEQ ID NO:) were used to amplify the 3′PCR product).
  • the 5′ PCR product was cloned into pGA1 at the ClaI and NheI sites and the identity of the construct confirmed by sequencing.
  • the 3′ PCR product was then inserted into the 5′ clone at the EcoRI and NheI sites to yield pBH10-VLP.
  • the construction of this VLP resulted in proviral sequences that lacked LTRs, integrase, vif, and vpr sequences (see FIG. 8A).
  • the ADA-VLP with the zinc finger and RT mutations was found to express Gag and Env more effectively than the VLP plasmid without the mutations (FIG. 10).
  • the mutation that inactivated the protease gene markedly reduced VLP expression (FIG. 10) and was not included in the further development of the vaccine plasmid.
  • the ADA-VLP without mutations was designated JS1 and the ADA-VLP with mutations, JS2.
  • the JS5 insert, a plasmid expressing Gag, RT, Tat, and Rev was constructed from JS2 by deleting a BglII fragment in the ADA Env (FIG. 8). This deletion removed sequences from nt 4906-5486 of the pGA2/JS2 sequence and results in a premature stop codon in the env gene leading to 269 out of the 854 amino acids of Env being expressed while leaving the tat, rev, and vpu coding regions the RRE and splice acceptor sites intact.
  • the DNA sequence of pGA1/JS5 is shown in FIG. 18.
  • JS2 and JS5 vaccine inserts were originally constructed in pGA1, a vector that contains the ⁇ 1 kb intron A of the CMVIE promoter upstream of the vaccine insert.
  • pGA2 vectors lacking intron A were constructed expressing the JS2 and JS5 vaccine inserts.
  • pGA2 proved to have as good an expression pattern as pGA1 for JS2 (FIG. 11).
  • JS5 was expressed much more effectively by pGA1 than pGA2 (FIG. 11).
  • the absence of intron A resulted in 2-3-fold lower levels of expression than in the presence of intron A.
  • the deletions and zinc finger mutations in the JS2 and JS5 vaccine inserts (Table 2) reduced the levels of viral RNA in particles by at least 1000-fold. Particles pelleted from the supernatants of transiently transfected cells were tested for the efficiency of the packaging of viral RNA.
  • the VLPs were treated with DNase, RNA was extracted and the amount of RNA standardized by p24 levels before RT PCR. The RT PCR reaction was followed by nested PCR using primers specific for viral sequences. End point dilution of the VLP RNA was compared to the signal obtained from RNA packaged in wt HIV-1 Ba1 virus.
  • Packaging for both JS2 and JS5 was restricted by the deletions in the plasmid by 500-1000-fold, as summarized below: TABLE 3 Packaging of viral RNA is reduced in pGA2/JS2 and pGA1/JS5 VLPs Vaccine Copies vRNA relative Construct Deletions/Mutations to wt HIV-1 b HIV-1 bal Wt 1 pGA1/JS1 VLP Deleted: LTRs, int, vif, .002 vpr, nef pGA1/JS2 VLP Deleted: LTRs, int, vif, .0001 vpr, nef, Mutations in Zn fingers and RT pGA1/JS4 VLP Deleted: LTRs, int, vif, .001 vpr, nef pGA1/JS5 VLP Deleted: LTRs, int, vif, .001 vpr, nef, env; Mutations
  • the zinc finger mutations decreased the efficiency of packaging for the JS2 particles a further 20-fold but did not further affect the efficiency of packaging for the JS5 particles. This pattern of packaging was reproducible for particles produced in independent transfections.
  • FIGS. 12 A-D Western blot analyses, shown in FIGS. 12 A-D, revealed the expected patterns of expression of pGA2/JS2 and pGA1/JS5. Both immature and mature proteins were observed in cell lysates, whereas only the mature forms of Gag and Env were found in the VLP-containing lysates (FIGS. 12B and 12C). Reverse transcriptase was readily detected in cell lysates (FIG. 12D).
  • SHIVs are hybrids of simian and human immunodeficiency virus sequences that grow well in macaques (Li et al., 1992). By using a SHIV, vaccines that are at least partially of HIV-1 origin can be tested for efficacy in macaque models.
  • pGA2/89.6 expresses sequences from SHIV-89.6 (Reimann, Li, Voss, et al., 1996; Reimann, Li, Veazey, et al., 1996).
  • the 89.6 Env represents a patient isolate (Collman et al., 1992).
  • the SHIV-89.6 virus is available as a highly pathogenic challenge stock, designated SHIV-89.6P (Reimann, Li, Voss, et al., 1996; Reimann, Li, Veazey, et al., 1996), which allows a rapid determination of vaccine efficacy.
  • the SHIV-89.6P challenge can be administered via both intrarectal and intravenous routes.
  • SHIV-89.6 and SHIV-89.6P do not generate cross-neutralizing antibody.
  • pGA2/89.6 (FIG. 13) has many of the design features of pGA2/JS2. Both express immunodeficiency virus VLPs: HIV-1 VLP in the case of pGA2/JS2, while the VLP expressed by pGA2/89.6 is a SHIV VLP.
  • the gag-pol sequences in pGA2/89.6 are from SIV239, while the tat, rev, and env sequences are from HIV-1-89.6.
  • pGA2/89.6 also differs from pGA2/JS2 in that the integrase, vif and vpr sequences have not been deleted, nor has the reverse transcriptase gene been inactivated by point mutations.
  • the zinc fingers in NC have been inactivated by a deletion and not by point mutations.
  • pGA1/Gag-Pol was also constructed to allow evaluation of the protective efficacy of a Gag-Pol expressing vector with the Gag-Pol-Env expresssing pGA2/89.6.
  • This vector was constructed from pGA1/JS5 and pGA2/89.6 (FIG. 13).
  • rhesus macaque model was used to investigate the ability of systemic DNA priming followed by a recombinant MVA (rMVA) booster to protect against a mucosal challenge with the SHIV-89.6P challenge strain (Amara et al, 2001).
  • rMVA recombinant MVA
  • the DNA component of the vaccine (pGA2/89.6) was made as described in Example 11 and and expressed eight immunodeficiency virus proteins (SIV Gag, Pol, Vif, Vpx, and Vpr and HIV Env, Tat, and Rev) from a single transcript using the subgenomic splicing mechanisms of immunodeficiency viruses.
  • the rMVA booster (89.6-MVA) was provided by Dr. Bernard Moss (NIH) and expresses both the HIV 89.6 Env and the SIV 239 Gag-Pol, inserted into deletion II and deletion III of MVA respectively, under the control of vaccinia virus early/late promoters (Wyatt and Moss, unpublished results).
  • the 89.6 Env protein was truncated for the C-terminal 115 amino acids of gp41.
  • the modified H5 promoter controlled the expression of both foreign genes.
  • the vaccination trial compared i.d. and i.m. administration of the DNA vaccine and the ability of a genetic adjuvant, a plasmid expressing macaque GM-CSF, to enhance the immune response raised by the vaccine inserts.
  • Vaccination was accomplished by priming with DNA at 0 and 8 weeks and boosting with rMVA at 24 weeks.
  • 1-100 ⁇ l i.d. inoculation was given with a solution containing 2.5 mg of pGA2/89.6 and 2.5 mg per ml of pGM-CSF.
  • the vaccination protocol is summarized as follows: TABLE 4 Vaccination Trial Group, Prime at Boost at (# macaque) 0 and 8 weeks Immunogen 24 weeks Immunogen 1 (6) i.d. bioject 2.5 mg VLP DNA i.d. + i.m. MVA gag-pol-env 2 (6) i.m. bioject 2.5 mg VLP DNA i.d. + i.m. MVA gag-pol-env 3 (6) i.d bioject 250 ug VLP DNA i.d. + i.m MVA gag-pol-env 4 (6) i.m. bioject 250 ug VLP DNA i.d. + i.m. MVA gag-pol-env 5 (6) i.d.
  • DNA priming followed by rMVA boosting generated high frequencies of virus-specific T cells that peaked at one week following the rMVA booster, as shown in FIG. 15.
  • the frequencies of T cells recognizing the Gag-CM9 epitope were assessed using Mamu-A*01-tetramers; and the frequencies of T cells recognizing epitopes throughout Gag and Env, using pools of overlapping Gag and Env peptides and an enzyme linked immunospot (ELISPOT) assay.
  • ELISPOT enzyme linked immunospot
  • PBMC peripheral blood mononuclear cells
  • CD3 FN-18, Biosource International, Camarillo, Calif.
  • CD8 SKi, Becton Dickinson, San Jose, Calif.
  • Gag-CM9 CTPYDINQM-Mamu-A*01 tetramer conjugated to FITC, PerCP and APC respectively, in a volume of 100 ⁇ l at 8-10° C. for 30 min.
  • Cells were washed twice with cold PBS containing 2% FBS, fixed with 1% paraformaldehyde in PBS and analyses acquired within 24 hrs. on a FACScaliber (Becton Dickinson, San Jose, Calif.).
  • Cells were initially gated on lymphocyte populations using forward scatter and side scatter and then on CD3 cells. The CD3 cells were then analyzed for CD8 and tetramer-binding cells. Approximately 150,000 lymphocytes were acquired for each sample. Data were analyzed using FloJo software (Tree Star, Inc. San Carlos, Calif.).
  • IFN- ⁇ ELISPOTs For IFN- ⁇ ELISPOTs, MULTISCREEN 96 well filtration plates (Millipore Inc. Bedford, Mass.) were coated overnight with anti-human IFN- ⁇ antibody (Clone B27, Pharmingen, San Diego, Calif.) at a concentration of 2 ⁇ g/ml in sodium bicarbonate buffer (pH 9.6) at 8-10° C. Plates were washed two times with RPMI medium then blocked for one hour with complete medium (RPMI containing 10% FBS) at 37° C. Plates were washed five more times with plain RPMI medium and cells were seeded in duplicate in 100 ⁇ l complete medium at numbers ranging from 2 ⁇ 10 4 to 5 ⁇ 10 5 cells per well.
  • Peptide pools were added to each well to a final concentration of 2 ⁇ g/ml of each peptide in a volume of 100 ⁇ l in complete medium.
  • Cells were cultured at 37° C. for about 36 hrs under 5% CO 2 . Plates were washed six times with wash buffer (PBS with 0.05% Tween-20) and then incubated with 1 ⁇ g of biotinylated anti-human IFN-y antibody per ml (clone 7-86-1, Diapharma Group Inc., West Chester, Ohio) diluted in wash buffer containing 2% FBS. Plates were incubated for 2 hrs at 37° C. and washed six times with wash buffer.
  • wash buffer PBS with 0.05% Tween-20
  • ELISPOTs The frequencies of ELISPOTs are approximate because different dilutions of cells have different efficiencies of spot formation in the absence of feeder cells (34). The same dilution of cells was used for all animals at a given time point, but different dilutions were used to detect memory and peak effector responses.
  • Gag-CM9 tetramer analyses were restricted to macaques that expressed the Mamu-A*01 histocompatibility type, whereas ELISPOT responses did not depend on a specific histocompatibility type.
  • Temporal T cell assays were designed to score both the acute (peak of effector cells) and long-term (memory) phases of the T cell response (FIG. 15A). As expected, the DNA immunizations raised low levels of memory cells that expanded to high frequencies within one week of the rMVA booster (FIG. 15). In Mamu-A*01 macaques, cells specific to the Gag-CM9 epitope expanded to frequencies as high as 19% of total CD8 T cells (see animal 2 FIG. 15B).
  • Viral RNA from 150 ⁇ l of ACD anticoagulated plasma was directly extracted with the QIAamp Viral RNA kit (Qiagen), eluted in 60 ⁇ l AVE buffer, and frozen at ⁇ 80° C. until SHIV RNA quantitation was performed. 5 ⁇ l of purified plasma RNA was reverse transcribed in a final 20 ⁇ l volume containing 50 mM KCI, 10 mM Tris-HCl, pH 8.3, 4 mM MgCl 2 , 1 mM each dNTP, 2.5 ⁇ M random hexamers, 20 units MultiScribe RT, and 8 units RNase inhibitor. Reactions were incubated at 25° C.
  • a Perkin Elmer Applied Biosystems 7700 Sequence Detection System was used with the PCR profile: 95° C. for 10 min., followed by 40 cycles at 93° C. for 30 sec., 59.5° C. for 1 min.
  • PCR product accumulation was monitored using the 7700 sequence detector and a probe to an internal conserved gag gene sequence, where FAM and Tamra denote the reporter and quencher dyes.
  • SHIV RNA copy number was determined by comparison to an external standard curve consisting of virion-derived SIVmac239 RNA quantified by the SIV bDNA method (Bayer Diagnostics, Emeryville, Calif.). All specimens were extracted and amplified in duplicate, with the mean result reported.
  • the intra-assay coefficient of variation is ⁇ 20% for samples containing >10 4 SHIV RNA copies/ml, and ⁇ 25% for samples containing 10 3 -10 4 SHIV RNA copies/ml.
  • the following modifications to increase the sensitivity of the SHIV RNA assay were made: 1) Virions from ⁇ 1 ml of plasma were concentrated by centrifugation at 23,000 g, 10° C.
  • DNA-primed group had a geometric mean of 6 ⁇ 10 3 copies of viral RNA and the non-vaccinated controls, a geometric mean of 2 ⁇ 10 6 .
  • the unvaccinated controls were succumbing to AIDS.
  • the 24 vaccinated animals only one animal, in the low dose i.m. group, had intermittent viral loads above 1 ⁇ 10 4 copies per ml (FIG. 19D).
  • PBMC peripheral blood mononuclear cells
  • Cells were surface stained with antibodies to CD8 conjugated to PerCP (clone SK1, Becton Dickinson) at 8°-10° C. for 30 min., washed twice with cold PBS containing 2% FBS, fixed and permeabilized with Cytofix/Cytopern solution (Phanningen, Inc.). Cells were then incubated with antibodies to human CD3 (clone FN-18, Biosource International, Camarillo, Calif.) and IFN- ⁇ (Clone B27, Pharmingen) conjugated to FITC and PE, respectively, in Perm wash solution (Pharmingen) for 30 min at 4° C. Cells were washed twice with Perm wash once with plain PBS, resuspended in 1% para-formaldehyde in PBS. Approximately 150,000 lymphocytes were acquired on the FACScaliber and analyzed using FloJo software.
  • PBMC peripheral blood mononuclear cells
  • PBMC peripheral blood mononuclear cells
  • Supernatants from 293T cells transfected with the DNA expressing either SHIV-89.6 Gag and Pol or SHIV-89.6 Gag, Pol and Env were used directly as antigens.
  • Supernatants from mock DNA (vector alone) transfected cells served as negative controls. On day six cells were pulsed with 1 ⁇ Ci of tritiated-thymidine per well for 16-20 hrs.
  • Stimulation indices are the counts of tritiated-thymidine incorporated in PBMC stimulated with 89.6 antigens divided by the counts of tritiated-thymidine incorporated by the same PBMC stimulated with mock antigen.
  • Tetramer+ cells peaked at less than 1% of total CD8 cells (FIG. 20A), and IFN-y-producing T cells were present at a mean frequency of about 300 as opposed to the much higher frequencies of 1000 to 6000 in the vaccine groups (FIG. 15C) (P ⁇ 0.05).
  • the tetramer+ cells in the control group like those in the vaccine group, were largely IFN- ⁇ producing following stimulation with the Gag-CM9 peptide (FIG. 20B).
  • 3 of the 4 controls had undetectable levels of IFN- ⁇ -producing T cells (data not shown). This rapid loss of anti-viral CD8 cells in the presence of high viral loads may reflect the lack of CD4 help.
  • T cell proliferative responses demonstrated that virus-specific CD4 cells had survived the challenge and were available to support the antiviral immune response (FIG. 20C).
  • mean stimulation indices for Gag-Pol-Env or Gag-Pol proteins ranged from 35 to 14 in the vaccine groups but were undetectable in the control group.
  • intracellular cytokine assays demonstrated the presence of virus-specific CD4 cells in vaccinated but not control animals (data not shown).
  • the overall rank order of the vaccine groups for the magnitude of the proliferative response was 2.5 mg i.d. >2.5 mg i.m. >250 ⁇ g i.d. >250 ⁇ g i.m.
  • lymph nodes from the vaccinated animals were morphologically intact and responding to the infection whereas those from the infected controls had been functionally destroyed (FIG. 5).
  • Nodes from vaccinated animals contained large numbers of reactive secondary follicles with expanded germinal centers and discrete dark and light zones (FIG. 5A).
  • lymph nodes from the non-vaccinated control animals showed follicular and paracortical depletion (FIG. 5B), while those from unvaccinated and unchallenged animals displayed normal numbers of minimally reactive germinal centers (FIG. 5C).
  • Germinal centers occupied ⁇ 0.05% of total lymph node area in the infected controls, 2% of the lymph node area in the uninfected controls, and up to 18% of the lymph node area in the vaccinated groups (FIG. 5D).
  • the lymph node area occupied by germinal centers was about two times greater for animals receiving low-dose DNA priming than for those receiving high-dose DNA priming, suggesting more vigorous immune reactivity in the low-dose animals (FIG. 5D).
  • in situ hybridization for viral RNA revealed rare virus-expressing cells in lymph nodes from 3 of the 24 vaccinated macaques, whereas virus-expressing cells were readily detected in lymph nodes from each of the infected control animals (FIG. 5E).
  • the cytomorphology of infected lymph node cells was consistent with a macrophage phenotype (data not shown).
  • ELISAs for total anti-Gag antibody used bacterial produced SIV gag p27 to coat wells (2 ⁇ g per ml in bicarbonate buffer).
  • ELISAs for anti-Env antibody used 89.6 Env produced in transiently transfected 293T cells captured with sheep antibody against Env (catalog number 6205; International Enzymes, Fairbrook Calif.). Standard curves for Gag and Env ELISAs were produced using serum from a SHIV-89.6-infected macaque with known amounts of anti-Gag or anti-Env IgG.
  • Bound antibody was detected using goat anti-macaque IgG-PO (catalog # YNGMOIGGFCP, Accurate Chemical, Westbury, N.Y.) and TMB substrate (Catalog # T3405, Sigma, St. Louis, Mo.). Sera were assayed at 3-fold dilutions in duplicate wells. Dilutions of test sera were performed in whey buffer (4% whey and 0.1% tween 20 in 1 ⁇ PBS). Blocking buffer consisted of whey buffer plus 0.5% non-fat dry milk. Reactions were stopped with 2M H 2 SO 4 and the optical density read at 450 nm. Standard curves were fitted and sample concentrations were interpolated as ⁇ g of antibody per ml of serum using SOFTmax 2.3 software (Molecular Devices, Sunnyvale, Calif.).
  • the dose of DNA had significant effects on both cellular and humoral responses (P ⁇ 0.05) while the route of DNA administration had a significant effect only on humoral responses (FIGS. 23 C-E).
  • the route and dose of DNA had no significant effect on the level of protection.
  • the high-dose DNA-primed animals had slightly lower geometric mean levels of viral RNA (7 ⁇ 10 2 and 5 ⁇ 10 2 ) than the low-dose DNA-primed animals (9 ⁇ 10 2 and 1 ⁇ 10 3 ).
  • the animal with the highest intermittent viral loads (macaque 22) was in the low dose i.m.-primed group (FIG. 19D).
  • the low dose i.m.-primed group which was slow to control viremia (FIG. 19A) may have poorer long term protection.
  • the breadth of the response did not have an immediate effect on the containment of viral loads, but with time may affect the frequency of viral escape.
  • the DNA/MVA vaccine did not prevent infection. Rather, the vaccine controlled the infection, rapidly reducing viral loads to near or below 1000 copies of viral RNA per ml of blood. Containment, rather than prevention of infection, affords the virus the opportunity to establish a chronic infection (Chun et al., 1998). Nevertheless, by rapidly reducing viral loads, a multiprotein DNA/MVA vaccine will extend the prospect for long-term non-progression and limit HIV transmission.
  • a DNA vaccine expressing a fusion of measles H and the C3d component of complement was used to determine if vaccination could achieve earlier and more efficient anti-H antibody responses.
  • the fusion of two or three copies of C3d to a model antigen, hen egg lysozyme increased the efficiency of immunizations by more than 1000-fold (Dempsey et al, 1996). This resulted in more rapid appearance of hemagglutination inhibition (HI) activity and protective immunity (Ross et al, 2000 and Ross et al., 2001).
  • HI hemagglutination inhibition
  • C3d In the human immune system, one consequence of complement activation is the covalent attachment of the C3d fragment of the third complement protein to the activating protein.
  • C3d in turn binds to CD21 on B lymphocytes, a molecule with B cell stimulatory functions that amplify B lymphocyte activation.
  • the H moiety of the fusion In a measles H-C3d fusion protein, the H moiety of the fusion would bind to anti-H Ig receptors on B cells and signal through the B cell receptor, while the C3d moiety of the fusion would bind to CD21 and signal through CD19.
  • a B cell responding to an H-C3d fusion protein would undergo more effective signaling than a B cell responding to H alone.
  • Mice vaccinated with DNA expressing a secreted H-fused to three copies of C3d (sH-3C3d) generated a more rapid appearance and higher levels of neutralizing antibody activity than DNA expressing sH only.
  • Plasmid DNA pTR600, a eukaryotic expression vector, was constructed to contain two copies of the cytomegalovirus immediate-early promoter (CMV-IE) plus intron A (IA) for initiating transcription of eukaryotic inserts and the bovine growth hormone polyadenylation signal (BGH poly A) for termination of transcription.
  • CMV-IE cytomegalovirus immediate-early promoter
  • IA intron A
  • BGH poly A bovine growth hormone polyadenylation signal
  • the vector contains a multi-cloning site for the easy insertion of gene segments and the Col El origin of replication for prokaryotic replication and the Kanamycin resistance gene (Kanr) for selection in antibiotic media (FIG. 29A).
  • Hemagglutinin (H) cDNA sequences from the Edmonton strain and C3d sequences were cloned as previously described and transferred into the pTR600 vaccine vector using unique restriction endonuclease sites (FIG. 29B).
  • the secreted version was generated by deleting the transmembrane and cytoplasmic domains of H. This was accomplished using PCR to clone a fragment of the H gene in frame with an N-terminal synthetic mimic of the tissue plasminogen activator (tpA) leader sequence (Torres, et al, 2000).
  • the vectors expressing sH-C3d fusion proteins were generated by cloning three tandem repeats of the mouse homologue of C3d in frame at the 3′ end of the sH gene as previously described (Dempsey, 1996; Ross et al, 2000; and Ross et al, 2001).
  • the construct design was based upon Dempsey et al. and used sequences from pSLG-C3d.
  • Linkers composed of two repeats of 4 glycines and a serine ⁇ (G 4 S) 2 ⁇ were fused at the junctures of H and C3d and between each C3d repeat.
  • Potential proteolytic cleavage sites between the junctions of C3d and the junction of sH and C3d were mutated by using Bam HI and Bgl II fusion to mutate an Arg codon to a Gly codon.
  • the plasmids were amplified in Escherichia coli strain, DH5a, purified using anion-exchange resin columns (Qiagen, Valencia, CA) and stored at ⁇ 20° C. in dH 2 O. Plasmids were verified by appropriate restriction enzyme digestion and gel electrophoresis. Purity of DNA preparations was determined by optical density reading at 260 nm and 280 nm.
  • mice and DNA immunizations Six to 8 week old BALB/c mice (Harlan Sprague Dawley, Indianapolis, Ind.) were used for inoculations. Briefly, mice were anesthetized with 0.03-0.04 ml of a mixture of 5 ml ketamine HCl (100 mg/ml) and 1 ml xylazine (20 mg/ml). Mice were immunized with two gene gun doses containing 0.5 ⁇ g of DNA per 0.5 mg of approximately 1- ⁇ m gold beads (DeGussa-Huls Corp., Ridgefield Park, N.J.) at a helium pressure setting of 400 psi.
  • the human embryonic kidney cell line 293T (5 ⁇ 10 5 cells/transfection) was transfected with 2 ⁇ g of DNA using 12% lipofectamine according to the manufacture's guidelines (Life Technologies, Grand Island, N.Y.). Supernatants were collected and stored at ⁇ 20° C. Quantitative antigen capture ELISAs for H were conducted as previously described (Cardoso et al, 1998).
  • a quantitative ELISA was performed to assess anti-H specific IgG levels. Briefly, Ltk-cells constitutively expressing the H protein of MV (24) were grown in 96-well plates. Antisera dilutions were incubated with the intact cells expressing H antigen. The anti-H antibodies were allowed to bind to the cells for 30 min following which the cells were fixed in acetone (80%). The specific antibody responses were detected with biotinylated anti-mouse IgG antibodies and the streptavidine-phosphatase alkaline system (Sigma). Antibody binding to Ltk-cells not expressing H antigen was used to standardize the system. The results were expressed as the endpoint dilution titer.
  • Neutralization assays were conducted on Vero cells grown in six well plates (25). Briefly, 100-200 p.f.u. of the Edmonton strain of measles virus were mixed with serial dilution of sera, incubated for 1 h at 37° C. and then inoculated onto plates. Plates were incubated at 37° C. for 48 h and containing either the sH or sH-3C3d compared to transmembrane-associated forms of the antigen. Human 293T cells were transiently transfected with 2 ⁇ g of plasmid and both supernatants and cell lysates were assayed for H using an antigen capture ELISA.
  • H protein Approximately 75% of the H protein was secreted into the supernatant for both sH-DNA and sH-3C3d-DNA transfected cells. As expected, ⁇ 99% of the H antigen was detected in the cell lysate of cells transfected with plasmids expressing transmembrane form of H.
  • mice were vaccinated by DNA coated gold particles via gene gun with either a 0.1 ⁇ g or a 1 ⁇ g inoculum. At 4 and 26 weeks post vaccination, mice were boosted with the same dose of DNA given in the first immunization. The temporal pattern for the appearance of anti-H antibody showed a faster onset in mice vaccinated with the C3d fusion expressing DNA compared to mice vaccinated with sH DNA. Good titers of antibody were raised by the first immunization . These were boosted by the 2 nd and 3 rd immunizations. following the third immunization, titers were 5-6 times higher in the sH-3C3D vaccinated mice than in those vaccinated with sH DNA.
  • sH secreted form of H
  • sH-3C3d C3d-fusion of the secreted form of H
  • FIG. 29 The sH represented the entire ectodomain of H, but excluded the transmembrane and cytoplasmic region.
  • the cloning placed the N-terminal synthetic mimic of the tissue plasminongen activator (tPA) leader sequence in frame with the H sequence.
  • tPA tissue plasminongen activator
  • the sH-3C3d fusion protein was generated by cloning three tandem repeats of the mouse homologue of C3d in frame with the secreted H gene (FIG. 29B).
  • the proteolytic cleavage sites found at the junction between each C3d molecule as well as the junction between the H protein and the first C3d coding region, were destroyed by mutagenesis.
  • Measles virus H was expressed at slightly lower levels by plasmids sH expressing DNA. Mice vaccinated with sH-3C3d expressing plasmids had a sharp rise in neutralizing antibody levels that reached a plateau by week 14. In contrast, it took a third vaccination with sH expressing DNA to elicit detectable levels of neutralizing antibodies. After 28 weeks post-vaccination, sera from mice vaccinated with sH-3C3d-DNA had neutralizing titers (>250) that could reduce plaque formation of MV infection by 90%.
  • mice vaccinated with DNA expressing sH-3C3d had low levels of neutralizing antibody even after the third vaccination (180 for 50% plaque reduction) (FIG. 31).
  • Plasmid vector construction and purification procedures have been previously described for JW4303 (Torres, et al. 1999; Pertmer et al. 1995; Feltquate et al. 1997).
  • influenza hemagglutinin (HA) sequences from A/PR/8/34 (H1N1) were cloned into either the pJW4303 or pGA eukaryotic expression vector using unique restriction sites.
  • HA a secreted(s) and a transmembrane (tm) associated
  • vectors expressing sHA or tmHA in pJW4303 were designated pJW/sHA and pJW/tmHA respectively and the vectors expressing sHA, tmHA, or sHA-3C3d in pGA were designated pGA/sHA, pGA/tmHA, and pGA/sHA-3C3d respectively.
  • Vectors expressing HA-C3d fusion proteins were generated by cloning three tandem repeats of the mouse homolog of C3d and placing the three tandem repeats in-frame with the secreted HA gene.
  • the construct designed was based upon Dempsey et al (1996).
  • Linkers composed of two repeats of 4 glycines and a serine [(G 4 S) 2 ] were fused at the joints of each C3d repeat.
  • the pGA/sHA-3C3d plasmid expressed approximately 50% of the protein expressed by the pGA/sHA vector. However, the ratio of sHA-3C3d found in the supernatant vs.
  • the cell lysate was similar to the ratio of antigen expressed by pGA/sHA. More than 80% of the protein was secreted into the supernatant. In western analysis, a higher molecular weight band was detected at 120 kDa and represented the sHA-3C3d fusion protein. Therefore, the sHA-3C3d fusion protein is secreted into the supernatant as efficiently as the sHA antigen.
  • mice Six to 8 week old BALB/c mice (Harlan Sprague Dawley, Indianapolis, Ind.) were used for inoculations. Mice, housed in microisolator units and allowed free access to food and water, were cared for under USDA guidelines for laboratory animals. Mice were anesthetized with 0.03-0.04 ml of a mixture of 5 ml ketamine HCl (100 mg/ml) and 1 ml xylazine (20 mg/ml).
  • Gene gun immunizations were performed on shaved abdominal skin using the hand held Accell gene delivery system and immunized with two gene gun doses containing 0.5 ⁇ g of DNA per 0.5 mg of approximately 1- ⁇ m gold beads (DeGussa-Huls Corp., Ridgefield Park, N.J.) at a helium pressure setting of 400 psi.
  • mice were vaccinated by DNA coated gold particles via gene gun with either a 0.1 ⁇ g or 1 ⁇ g dose inoculum. At 4 weeks post vaccination, half of the mice in each group were boosted with the same dose of DNA given in the first immunization.
  • Total anti-HA IgG induced by the sHA-3C3d- and tmHA-expressing plasmids were similar in the different experimental mouse groups and 3-5 times higher then the amount raised by the sHA expressing plasmids (FIG. 24).
  • the amount of anti-HA antibody elicited increased relative to the amount of DNA used for vaccination in a dose dependent manner (FIGS. 24 E- 24 F).
  • the dose response curves and temporal pattern for the appearance of anti-HA antibody were similar in the mice vaccinated with tmHA-DNA or sHA-3C3d-DNA, but lower and slower, in the mice vaccinated with sHA-DNA.
  • the booster immunization both accelerated and increased the titers of antibodies to HA.
  • Sodium thiocyanate (NaSCN) displacement ELISAs demonstrated that the avidity of the HA-specific antibody generated with sHA-3C3d expressing DNA was consistently higher than antibodies from sHA-DNA or tmHA-DNA vaccinated mice (FIG. 25).
  • the avidity of specific antibodies to HA was compared by using graded concentrations NaSCN, a chaotropic agent, to disrupt antigen-antibody interactions. The binding of antibodies with less avidity to the antigen is disrupted at lower concentrations of NaSCN than that of antibodies with greater avidity to the antigen.
  • the ED 50 had increased to 1.8 M for antibodies from both sHA-DNA and tmHA-DNA vaccinated mice (FIG. 25C).
  • HI Hemagglutination-inhibition assays
  • mice vaccinated with sHA-3C3d-DNA had titers greater than 1:640.
  • the only vaccinated mice that had a measurable HI titer (1:160) at week 8 were boosted mice vaccinated with 1 ⁇ g dose sHA-3C3d-DNA.
  • mice were challenged with a lethal dose of A/PR/8/34 influenza virus (HlNl) and monitored daily for morbidity (as measured by weight loss) and mortality. Weight loss for each animal was plotted as a percentage of the average pre-challenge weight versus days after challenge (FIG. 26).
  • HlNl A/PR/8/34 influenza virus
  • Virus-challenged naive mice and pGA vector only vaccinated mice showed rapid weight loss with all the mice losing >20% of their body weight by 8 days post-challenge (FIG. 26).
  • PBS mock-challenged mice showed no weight loss over the 14 days of observation. All boosted mice survived challenge, 14 weeks after vaccination, regardless of the dose of DNA plasmid administered. However, boosted mice vaccinated with a 0.1 ⁇ g dose of sHA-DNA did drop to 92% of their initial body weight at 8 days post-challenge before recovering (FIG. 26).
  • mice to survive challenge were sHA-3C3d- and tmHA-DNA vaccinated mice, albeit with greater weight loss than was observed from mice challenged at 14 weeks after vaccination.
  • the only 0.1 ⁇ g dose, boosted mice to survive challenge at 8 weeks after vaccination were the sHA-3C3d vaccinated mice (FIG. 26).
  • pGA was constructed as described in Example 1 above to contain the cytomegalovirus immediate-early promoter (CMV-IE) plus intron A (IA) for initiating transcription of eukaryotic inserts and the bovine growth hormone polyadenylation signal (BGH poly A) for termination of transcription.
  • CMV-IE cytomegalovirus immediate-early promoter
  • IA intron A
  • BGH poly A bovine growth hormone polyadenylation signal
  • the first 32 amino acids were deleted from the N-terminus of each sgp 120 and replaced with a leader sequenced from the tissue plasminogen activator (tpA).
  • the vectors expressing sgp120-C3d fusion proteins were generated by cloning three tandem repeats of the mouse homologue of C3d in frame with the sgp120 expressing DNA.
  • the construct design was based upon Dempsey et al (1996).
  • Linkers composed of two repeats of 4 glycines and a serine ⁇ (G 4 S) 2 ⁇ were fused at the junctures of HA and C3d and between each C3d repeat.
  • proteolytic cleavage sites between the junctions of C3d and the junction of 3C3d were mutated by ligating Bam HI and Bgl II restriction endonuclease sites to mutate an Arg codon to a Gly codon.
  • the plasmids were amplified in Escherichia coli strain-DH5 ⁇ , purified using anion-exchange resin columns (Qiagen, Valencia, Calif.) and stored at ⁇ 20° C. in dH 2 O. Plasmids were verified by appropriate restriction enzyme digestion and gel electrophoresis. Purity of DNA preparations was determined by optical density reading at 260 nm and 280 nm.
  • mice Six to 8 week old BALB/c mice (Harlan Sprague Dawley, Indianapolis, Ind.) were vaccinated as described in Example 17 above. Briefly, mice were immunized with two gene gun doses containing 0.5 ⁇ g of DNA per 0.5 mg of approximately 1- ⁇ m gold beads (DeGussa-Huls Corp., Ridgefield Park, N.J.) at a helium pressure setting of 400 psi.
  • An endpoint ELISA was performed to assess the titers of anti-Env IgG in immune serum using purified HIV-1-IIIB gp120 CHO-expressed protein (Intracell) to coat plates as described (Richmond et al., 1998). Alternatively, plates were coated with sheep anti-Env antibody (International Enzymes Inc., Fallbrook, Calif.) and used to capture sgp120 produced in 293T cells that were transiently transfected with sgp120 expression vectors. Mouse sera from vaccinated mice was allowed to bind and subsequently detected by anti-mouse IgG conjugated to horseradish peroxidase. Endpoint titers were considered positive that were two fold higher than background.
  • Avidity ELISAs were performed similarly to serum antibody determination ELISAs up to the addition of samples and standards. Samples were diluted to give similar concentrations of specific IgG by O.D. Plates were washed three times with 0.05% PBS-Tween 20. Different concentrations of the chaotropic agent, sodium thiocyanate (NaSCN) in PBS, were then added (0M, 1 M, 1.5 M, 2 M, 2.5 M, and 3 M NaSCN). Plates were allowed to stand at room temperature for 15 minutes and then washed six times with PBS-Tween 20. Subsequent steps were performed similarly to the serum antibody determination ELISA and percent of initial IgG calculated as a percent of the initial O.D. All assays were done in triplicate.
  • NaSCN sodium thiocyanate
  • Antibody-mediated neutralization of HIV-1 IIIB and 89.6 was measured in an MT-2 cell-killing assay as described previously (Montefiori et al., 1988). Briefly, cell-free virus (50 ⁇ l containing 10 8 TCID 50 of virus) was added to multiple dilutions of serum samples in 100 ⁇ l of growth medium in triplicate wells of 96-well microtiter plates coated with poly-L-lysine and incubated at 37° C. for 1 h before MT-2 cells were added (10 5 cells in 100 ⁇ l added per well). Cell densities were reduced and the medium was replaced after 3 days of incubation when necessary.
  • Neutralization was measured by staining viable cells with Finter's neutral red when cytopathic effects in control wells were >70% but less than 100%. Percentage protection was determined by calculating the difference in absorption (A 540 ) between test wells (cells+virus) and dividing this result by the difference in absorption between cell control wells (cells only) and virus control wells (virus only). Neutralizing titers are expressed as the reciprocal of the plasma dilution required to protect at least 50% of cells from virus-induced killing.
  • Env was expressed at overall similar levels by plasmids containing either the secreted form of the antigen, but at a two-four-fold lower level by the sgp120-C3d expressing plasmids.
  • Human 293T cells were transiently transfected with 2 ⁇ g of plasmid and both supernatants and cell lysates were assayed for gp120 using an antigen capture ELISA.
  • the sgp120 constructs expressed from 450 to 800 ng per ml, whereas the 3C3d fusions expressed from 140 to 250 ng per ml.
  • mice were vaccinated by DNA coated gold particles via gene gun with a 1 ⁇ g dose inoculum. Mice were vaccinated at day 1 and then boosted at 4, 14, and 26 weeks with the same DNA given in the first immunization. When sera were assayed on gp120-IIB-coated plates, mice vaccinated with the DNAs expressing the C3d fusion proteins had anti-Env antibodies 3-7 times higher then the amount of antibody raised by the counterpart sgp120 expressing plasmids.
  • mice vaccinated with sgp120-(IIIB)-3C3d had the highest levels of antibody and mice vaccinated with sgp120-(ADA)-3C3d expressing DNA had the lowest levels of anti-Env antibodies.
  • the temporal pattern for the appearance of anti-Env antibody revealed titers being boosted at each of the inoculations for all constructs tested.
  • Sodium thiocyanate (NaSCN) displacement ELISAs demonstrated that the avidity of the antibody generated with sgp120-3C3d expressing DNA was consistently higher than that from sgp120-DNA vaccinated mice.
  • Avidity assays were conducted on sera raised by sgp120-(IIIB) and sgp120-(IIIB)-3C3d because of the type specificity of the raised antisera and the commercial availability of the IIIB protein (but not the other proteins) for use as capture antigen.
  • the avidity of specific antibodies to Env was compared by using graded concentrations NaSCN, a chaotropic agent, to disrupt antigen-antibody interaction. Results indicatedthat the antibody from sgp120-3C3d-DNA vaccinated mice underwent more rapid affinity maturation than antibody from sgp120-DNA vaccinated mice.
  • mice vaccinated with DNA expressing the fusion of sgp120 and 3C3d proteins elicited a faster onset of antibody (3 vaccinations), as well as higher levels of antibodies.
  • Plasmid DNA expressing a secreted or a nonsecreted form of hepatitis C virus nucleocapsid comparative studies of antibody and T-helper responses following genetic immunization. DNA Cell Biol 16(2), 185-95.
  • HIV-1 neutralization the consequences of viral adaptation to growth on transformed T cells. Aids 9(Suppl A), S 117-36.
  • Torres C A Iwasaki A, Barber B H, Robinson H L. Differential dependence on target site tissue for gene gun and intramuscular DNA immunizations. J Immunol 1997;158(10):4529-32.

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WO2012036993A1 (fr) 2010-09-14 2012-03-22 University Of Pittsburgh-Of The Commonwealth System Of Higher Education Antigènes contre la grippe à réactivité large optimisés par ordinateur
WO2012177760A1 (fr) 2011-06-20 2012-12-27 University Of Pittsburgh-Of The Commonwealth System Of Higher Education Antigènes réactifs à large spectre optimisés par le calcul pour le virus de la grippe h1n1
WO2013119683A1 (fr) 2012-02-07 2013-08-15 University Of Pittsburgh - Of The Commonwealth System Of Higher Education Antigènes à large spectre optimisés in silico pour les virus grippaux de type h3n2, h2n2 et b
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EP3431100A1 (fr) 2010-09-14 2019-01-23 University of Pittsburgh- Of the Commonwealth System of Higher Education Antigènes contre la grippe à réactivité large optimisés par ordinateur
WO2012177760A1 (fr) 2011-06-20 2012-12-27 University Of Pittsburgh-Of The Commonwealth System Of Higher Education Antigènes réactifs à large spectre optimisés par le calcul pour le virus de la grippe h1n1
EP3199545A2 (fr) 2011-06-20 2017-08-02 University of Pittsburgh - Of the Commonwealth System of Higher Education Antigènes réactifs à large spectre optimisés par le calcul pour le virus de la grippe h1n1
WO2013119683A1 (fr) 2012-02-07 2013-08-15 University Of Pittsburgh - Of The Commonwealth System Of Higher Education Antigènes à large spectre optimisés in silico pour les virus grippaux de type h3n2, h2n2 et b
EP3305806A1 (fr) 2012-02-13 2018-04-11 University of Pittsburgh - Of the Commonwealth System of Higher Education Antigènes largement réactifs optimisés par ordinateur pour la grippe h5n1 humaine et aviaire

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CN1523999A (zh) 2004-08-25
CA2401974A1 (fr) 2001-12-06
EP1418940A4 (fr) 2006-09-20
EP1418940A2 (fr) 2004-05-19
AU2002211948B2 (en) 2007-09-13
US20060051839A1 (en) 2006-03-09
EP2388015A1 (fr) 2011-11-23
US7795017B2 (en) 2010-09-14
CN1311871C (zh) 2007-04-25
CA2401974C (fr) 2013-07-02

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