WO1997028265A9

WO1997028265A9 - Measles immunization by dna transcription unit inoculation

Info

Publication number: WO1997028265A9
Application number: PCT/US1997/001982
Authority: WO
Filing date: 1997-02-05
Publication date: 1997-10-30

Abstract

This invention relates to methods and compositions for immunizing a mammal against measles virus, comprising introducing into the mammal at least one DNA transcription unit comprising DNA encoding a measles virus antigen operatively linked to DNA which is a promoter region, resulting in the expression of the antigen, thereby eliciting an immune response. The elicited immune response can provide protection against disease caused by the measles virus. The host can be any mammal, including a human.

Description

MEASLES IMMUNIZATION BY DNA TRANSCRIPTION UNIT INOCULATION

Background of the Invention

Measles, or Rubeola, is a highly contagious acute viral disease characterized by fever, severe cough, acute rhinitis, conjunctivitis and mucous membrane lesions (Koplik's spots), followed by a generalized maculopapular rash. Despite the development of vaccines, it remains a worldwide health problem. In less developed countries, more than one million children a year die as a result of measles disease and its complications. It is the third largest killer of children under age five.

The measles virus is a member of the genus Morbillivirus in the family Paramyxoviridae. Other members of the Morbillivirus genus include canine distemper virus, peste des petits ruminants virus, and rinderpest virus. Unlike other members of the paramyxoviridae family, Morbilliviruses lack neuraminidase. The measles virion is a spherical enveloped particle with a nucleocapsid, composed of a central core of ribonucleic acid (RNA) with a helically arranged protein coat.

The entire genome of the original measles viral isolate (the Edmonston strain) has been sequenced. The genome is a nonsegmented, linear, single strand of negative sense RNA. It has an estimated molecular weight of 4.5 x IO⁶ daltons. It is approximately 15,900 nucleotides in length, but the exact number may vary by several nucleotides between virus strains and even between viruses of the same strain with different passage histories. The genome encodes six structural proteins. The two envelope proteins are the hemagglutinin (H) and fusion (F) transmembrane glycoproteins. Together, the F and H proteins mediate fusion of the viral and host cell membranes and viral entry into the host cell. The matrix (M) protein is thought to play a key role in virus maturation. The other three proteins complex with the viral RNA. The major internal protein is the nucleocapsid (N) protein. There are minor variations in the molecular weight of the N protein of different measles virus strains. The other two internal virion proteins are the large protein or polymerase (L) and phosphoprotein (P) proteins, which are present in small quantities. They are assumed to represent parts of the transcription complex.

Both humoral and cell-mediated immunity appear to be capable of preventing infection in normal individuals exposed to the virus, although cell- mediated immunity is necessary to clear measles virus infection. Virology. Fields, et al., 2d Ed., Vol. 1, 1035 (1990) . Antibodies to F and H proteins appear to protect against infection in vivo, although antibody to H protein alone can neutralize viral invasion. Measles Control — Resetting the Agenda. A Report of the Children's Vaccine Initiative's Ad Hoc Committee on an Investment Strategy for Measles Control, page 9, Bellagio, Italy, March 15, 1993. Although the measles virus has antigenically stable H and F antigens, minor antigenic differences have been observed in neutralization tests comparing laboratory strains and fresh isolates from cases of acute measles and subacute sclerosing panencephalitis (SSPE) . Measles antibody titers and serum are slightly increased in patients with multiple sclerosis and are often increased in patients with active chronic hepatitis and connective tissue diseases such as disseminating lupus erythematosus.

Cell-mediated immunity to measles virus can be measured with antigen specific T cell proliferation or cytotoxic T lymphocyte assays. The cytotoxic T cells have specificity not only for measles virus surface antigens, such as H and F, but also for internal antigens, especially the N protein. No effective inactivated vaccine is yet available, but live attenuated vaccines are widely used. Attenuation of the wild-type human measles virus, the Edmonston strain, is achieved by its adaptation to and serial passage in various cell lines, leading to a mutant whose activity is partially restricted in humans. One problem with the use of this vaccine is that immunized infants often retain maternally derived antibodies which restrict replication of the live measles virus, decreasing its immunogenicity, and, therefore, decreasing the efficacy of the vaccine. This is particularly problematic in developing countries where measles infection and mortality rates are high in infants under one year of age, an age where they are likely to retain maternal measles neutralizing antibodies. The live attenuated vaccines are also limited in that they do not raise as high, or as long- lived, neutralizing antibody responses as wild-type infections. It would be advantageous to have additional vaccines against measles available.

Summary of the Invention

This invention relates to methods and compositions for immunization against the measles virus using subunit vaccination. Specifically, this invention relates to a method of immunizing a mammal against the measles virus, comprising introducing into the mammal a DNA transcription unit (or units) . DNA transcription units are taken up and expressed in the host cells of the mammal, resulting in production of the measles antigen or antigens. An immune response, such as a humoral immune response, a cell-mediated immune response or both is/are elicited in the mammal, providing protection against measles infection. The host can be any mammal, including a human. The invention also relates to compositions comprising at least one DNA transcription unit and a pharmaceutically acceptable (physiologically acceptable) carrier. The invention also relates to DNA transcription units for use in the claimed methods and compositions.

Each DNA transcription unit used in the claimed methods and compositions comprises DNA encoding at least one measles virus antigen operatively linked to DNA which is a transcriptional promoter element or elements (the promoter region) . The promoter region can be of retroviral or nonretroviral origin. Measles virus antigen(s) encoded by the DNA in the transcription unit are one or more of the following: hemagglutinin (H) , matrix (M) , fusion protein (F) , nucleocapsid protein (N) , large polymerase (L) , phosphoprotein (P) , and nonstructural protein (C) of the measles virus. Each DNA transcription unit can comprise multiple copies of the same antigen, and/or it can comprise different antigens. The antigens can be from the same or different strains of measles virus. A DNA transcription unit can be used to express any measles virus antigen, such as hemagglutinin or fusion protein, as well as one or more antigenic fragments and/or peptides that have been experimentally determined to be effective in immunizing a mammal against infection by measles virus. Furthermore, a DNA transcription unit can be designed to produce internal, surface, secreted, or assembled forms of the antigens being used as immunogens. For example, the antigen can be in secreted form or a precursor form. Each antigen can be selected from a subset of T cell-recognized determinants or epitopes in a measles virus protein. Alternatively, or in addition, each antigen can be selected from a subset of B cell-recognized determinants or epitopes in a measles virus protein. In one embodiment of the present invention, the antigen is a measles virus hemagglutinin protein. In another embodiment, the antigen is a measles virus fusion protein. The antigen can be a measles virus hemagglutinin protein in a secreted form, for example, sHA4, or in a transmembrane form, such as HA7.

In the claimed methods, a single DNA transcription unit or multiple DNA transcription units can be administered to a mammal to achieve immunization against one antigen or multiple antigens. Likewise, a composition can contain a single DNA transcription unit or multiple DNA transcription units, in addition to a physiologically acceptable carrier. Furthermore, the compositions and DNA transcription units can comprise different measles antigens, for example, measles antigens of different strains, and/or they can comprise antigens from pathogens or infectious agents other than those of the measles virus. In a preferred method embodiment, the mammal is inoculated with both F DNA and H DNA. In a preferred composition embodiment, the composition comprises both H DNA and F DNA.

The DNA transcription units and compositions can be administered through various routes, including the parental route or the mucosal route. The mucosal route can be oral or respiratory (including nasal and tracheal mucosal surfaces) . Alternatively, the DNA transcription units and compositions can be administered through a route of administration selected from the group consisting of: intravenous, intramuscular, intraperitoneal, intradermal, and subcutaneous routes. The DNA transcription units can be administered in a pharmaceutically acceptable carrier. The DNA transcription unit or composition can be microsphere-encapsulated. In one embodiment, the DNA transcription unit or composition is coated onto gold beads for administration by particle bombardment delivery, for example, the gene gun. In one embodiment of the present invention, the individual is immunized through one or more parenteral routes of inoculation. DNA transcription units administered to the skin can be delivered with a gene gun. In a second embodiment, the individual is immunized by contacting a mucosal surface, such as a respiratory mucosal surface or tracheal mucosal surface, with DNA transcription units in such a manner that the transcription units are taken up by (i.e., enter the cells of) the mucosal surface. DNA transcription units for mucosal administration can be microsphere-encapsulated.

There are numerous advantages to the current invention. For example, immunization can be accomplished for any antigen encoded by measles DNA.

Furthermore, the encoded measles antigens are expressed as "pure" antigens which can undergo host cell modifications in a manner similar to the modifications undergone by antigens expressed by the wild type strain. In addition, the DNA is easily and inexpensively manipulated and is stable as a dry product or in solution over a wide range of temperatures. This is particularly useful in less- developed countries where refrigeration is unavailable. Moreover, the efficacy of subunit DNA vaccines is not reduced by the presence of persistent maternal antibodies in infants. Thus, this technology is valuable for the development of highly effective subunit vaccines against the measles virus.

Brief Description of the Drawings

Figure 1 is a schematic representation of the pJW4303 vector comprising SV40 origin (SV40 Ori) , bovine growth hormone polyadenylation sequences (BGHpA) , the Edmonston Hemagglutinin H and sH sequence inserts, and a CMV immediate promoter sequence (CMV Pro) which includes the sequence encoding the CMV intron A. The tridents on the inserts represent codons for glycosylation sites. The striped region labeled TM on the H insert indicates the transmembrane region. The numbers are nucleotide positions in the cDNAs used for preparation of inserts.

Figure 2 is a schematic representation of the pJW4303 vector comprising SV40 origin (SV40 Ori) , bovine growth hormone polyadenylation sequences (BGHpA) , the Edmonston Fusion sequence insert F₀, and a CMV immediate promoter sequence (CMV Pro) which includes the sequence encoding the CMV intron A. FI and F2 represent the subunits of fusion protein following the posttranslational cleavage process. The tridents on the inserts represent codons for glycosylation sites. The striped region labeled TM on the insert indicates the transmembrane region. The numbers are nucleotide positions in the cDNAs used for preparation of inserts.

Figure 3A is a graph depicting temporal curves of neutralization titers of antibodies raised in mice by gene gun inoculation of H DNA. DNA deliveries are indicated on dotted lines. Each curve represents data for an individual animal.

Figure 3B is a graph depicting temporal curves of neutralization titers of antibodies raised in mice by gene gun inoculation of sH DNA. DNA deliveries are indicated on dotted lines. Each curve represents data for an individual animal.

Figure 4 iε a bar graph depicting the neutralization titers of antibodies raised in mice by gene gun inoculations of H DNA, F_o DNA, and a combination of H DNA and F_Q DNA. Solid bars represent results from mice receiving one inoculation; striped bars, mice receiving two inoculations, 4 weeks apart. Figures 5A, 5B and 5C are graphs depicting temporal curves of neutralization titers of antibodies raised in mice by gene gun inoculations of H DNA (Figure 5A) , F DNA (Figure 5B) and both H DNA and F DNA (Figure 5C) . DNA deliveries are indicated on dotted lines. Each curve represents data for sera pooled from six mice. Data for groups receiving a single inoculation are presented as open circles, whereas data for groups receiving the boost are presented as closed circles.

Figure 6 depicts temporal curves of enzyme linked immunoabsorbent assay (ELISA) titers of antibodies raised in two rabbits by gene gun H DNA inoculations.

Figure 7A depicts temporal curves of titers of neutralizing antibodies raised in two rabbits by gene gun H DNA inoculations. DNA deliveries are indicated on dotted lines. Each curve represents data for an individual animal.

Figure 7B depicts temporal curves of titers of neutralizing antibodies raised in two rabbits by gene gun F DNA inoculations. DNA deliveries are indicated on dotted lines. Each curve represents data for an individual animal.

Figure 8 depicts temporal curves of titers of neutralizing antibodies raised in Rhesus macaques by inoculations of H DNA, F DNA, and a combination of H DNA and F DNA. The black and white squares depict the curves of animals inoculated with H DNA intradermally and by gene gun, respectively. The black circles depict the curve of animals inoculated with F DNA intradermally. The black and white diamonds depict the curves of animals inoculated with a combination of H DNA and F DNA intradermally and by gene gun.

Detailed Description of the Invention

This invention relates to a method of immunizing mammals, including humans, against measles virus, thereby eliciting humoral and/or cell-mediated immune responses which interfere with the activity of a measles antigen or antigens, or which limit the spread or growth of the virus and result in protection against subsequent disease caused by the virus. In the method of the present invention, a DNA transcription unit or units is/are administered to an individual in whom immunization is desired. The present invention also relates to DNA transcription units which comprise DNA encoding a measles virus antigen or antigens, and DNA which is a transcriptional promoter element or elements. The invention also relates to products comprising at least one DNA transcription unit comprising DNA encoding an antigen of measles virus operatively linked to DNA which is a promoter region. Alternatively, they can comprise more than one DNA transcription unit, each transcription unit comprising DNA encoding an antigen of measles virus operatively linked to DNA which is a promoter region, wherein the antigen of measles virus for one transcription unit is different from the antigen of measles virus of the other transcription unit, or each of the other transcription units. The present invention further relates to compositions comprising one or more DNA transcription units and a physiologically acceptable carrier.

The term "immunizing" refers herein to producing an immune response in a mammal which protects the mammal (partially or totally) from the pathological effects of the virus. That is, a mammal immunized by the present invention is protected, either partially or totally, against infection by the measles virus. Thus, an immunized mammal will not be infected with measles virus; will be infected to a lesser extent than would occur without immunization; or, if infected, will not exhibit disease associated with the measles virus or will exhibit disease of less severity than would occur without immunization.

A "DNA transcription unit" is a polynucleotide sequence which includes at least two components: DNA encoding at least one measles virus antigen and a transcriptional promoter element or elements (region) . DNA encoding measles virus antigen can encode one antigen, or multiple antigens, such as antigens from two or more different measles proteins from the same strain of measles virus or antigens from two or more different strains of measles virus. The DNA can also encode antigenic fragments, peptides or derivatives that have been experimentally determined to be effective in immunizing a mammal. In one embodiment, the DNA transcription unit can be of retroviral or nonretroviral origin. The DNA transcription unit can be administered without additional DNA or it can be inserted into a vector which includes sequences for replication of the DNA transcription unit. A DNA transcription unit can optionally include additional sequences, such as: enhancer elements, splicing signals, termination and polyadenylation signals, viral replicons, antigens from pathogens other than measles virus, and bacterial plasmid sequences. The DNA transcription unit(s) can additionally comprise an antigen or antigens from an infectious agent or pathogen other than measles virus. The infectious agent or pathogen can be selected from the group including, but not limited to: influenza, rotavirus, tetanus, respiratory syncytial virus, diphtheria, pertussis, mumps and rubella.

The DNA encoding the antigen(s) are operatively linked to the DNA which is the transcriptional promoter element(s) . The term "operatively linked" indicates that these components are linked so aε to facilitate expression of the antigen(s) .

The "measles virus antigen" can be any antigen or combination of antigens expressed by measles virus, or any antigen or combination of antigens that has been determined to be capable of eliciting a protective response against measles. The antigen or antigens can be naturally occurring (wild type antigens) , or can be mutated or specially modified. That is, the antigen or antigens can have the sequence of all or a portion of a naturally-occurring antigen or a modified sequence (a sequence which differs from a naturally-occurring antigen by the addition, deletion, substitution or modification of at least one amino acid) . The antigen or antigens can represent different forms, such as secreted or soluble, or different strains of measles virus. These antigens may or may not be structural components of the measles virus. The encoded antigens can be translation products or polypeptides. The polypeptides can be of various lengths. The antigens can be selected from a subset of T cell-recognized determinants in a measles protein. The antigen(s) can be selected from a subset of B cell-recognized epitopes in a measles protein. They can undergo normal host cell modifications such as glycosylation, myristolation and 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.

In a preferred embodiment, the DNA transcription unit is noninfectious and non-replicative. In one method, a single DNA transcription unit

(i.e., one type of DNA transcription unit) can be administered, or a combination of two or more types of DNA transcription units can be administered. For example, the units can comprise antigens from different measles virus proteins, or from different strains of measles virus. In a preferred embodiment, both H DNA and F DNA are administered. A composition may contain one or more DNA transcription units, and a physiologically acceptable carrier. The DNA transcription units within a composition can be the same type or different types. In a preferred embodiment, the composition comprises both H DNA and F DNA.

The DNA transcription unit or units can be produced by a number of known methods. For example, using known methods, DNA encoding the desired antigen can be inserted into an expression vector. See Maniatis et al.. Molecular Cloning. A Laboratory Manual. 2d. Cold Spring Harbor Laboratory Press (1989) . DNA encoding the desired antigen can be obtained from sources in which it occurs in nature or can be produced using known methods, such as amplification techniques, recombinant methodologies, and synthetic methods.

The DNA transcription unit can be administered to an individual with adjuvants or other substances that have the capability of promoting DNA uptake or recruiting immune system cells to the site of the inoculation. In one embodiment, the DNA transcription unit can be coated onto gold beads for administration by particle bombardment delivery. It should be understood that the DNA transcription unit itself is directly or indirectly expressed in the host cell by transcription factors provided by the host cell, or provided by a DNA transcriptional unit.

The DNA transcription unit can be administered to an individual by any parenteral route. For example, the DNA transcription unit can be introduced by intravenous, intraperitoneal, intradermal, subcutaneous or intramuscular methods, or by a particle bombardment method, such as a gene gun. The DNA transcription unit can also be introduced through any mucosal route. The DNA transcription unit can be administered by contact with a mucosal surface by a variety of methods, including DNA-containing nose-drops, inhalants, suppositories or by microsphere encapsulated DNA. For example, the DNA transcription unit can be administered to a respiratory mucosal surface, such as the nares or the trachea. Alternatively, the DNA transcription unit can be administered to an oral mucosal surface.

Any appropriate physiologically compatible medium, such as saline, is suitable for introducing the DNA transcription unit into an individual.

Immunization as described herein was accomplished with various DNA transcription units (e.g., vectors) that express different measles proteins. The DNA transcription units described herein are representative of the types of DNA transcription units that can be used in the current invention.

In one embodiment of the current invention, immunization was accomplished using a DNA transcription unit encoding measles virus hemagglutinin glycoprotein. The hemagglutinin glycoprotein mediates attachment of the virus to host cells and is a major target for neutralizing antibodies. Measles virus hemagglutinin protein can be expressed in both full-length membrane- associated (H) and truncated secreted (sH) structural forms. In one embodiment, the measles virus hemagglutinin is HA7. In another embodiment, the measles virus hemagglutinin is sHA4.

In another embodiment, immunization was accomplished using a DNA transcription unit expressing the measles virus fusion (F) glycoprotein. The fusion glycoprotein mediates the delivery of the virion nucleocapsid into the cytoplasm of the host cell after the virion becomes attached to the host cell. The fusion protein is a major target for both neutralizing and fusion inhibiting antibodies. Fusion proteins are synthesized by the natural virus in the cell host as inactive precursors (F₀) , comprising 540-580 amino acids, which gain fusion function by a posttranslation cleavage process that is mediated by cellular trypsin- like enzymes. In another embodiment, the F₀ precursor antigen was used. A mixture of DNA transcription units, comprising DNA encoding antigens from both forms of hemagglutinin and DNA encoding fusion protein, can be used in the current invention.

The products of this invention can be used in therapy, prophylaxis, and diagnosis. As described in the following Examples, inoculation trials using measles virus DNA were designed to raise immune responses against measles virus antigens. The trials used murine, rabbit, and Rhesus macaque models to test the ability of DNA expression vectors comprising DNA transcription units to raise neutralizing antibodies. The vectors comprised DNA encoding H, sH, or F measles proteins. Standard curves for ELISA assays used a rabbit anti-mouse F(ab')2 fragment (Jackson Laboratories, West Grove, PA) or a chicken anti-rabbit F(ab')2 fragment (ICN Inc., Costa Mesa, CA) to capture known amounts of purified mouse or rabbit IgG. Bound anti-H or anti-F antibody was detected with biotinylated anti-mouse or anti- rabbit IgG (heavy and light chain specific) and streptavidin horseradish peroxidase (Vector

Laboratories, Burlingame, CA) . Neutralization titers were assayed at 50% and 90% plaque reduction.

In the murine models, neutralizing antibody titers were raised in mice receiving DNA transcription units comprising DNA encoding H or sH forms of hemagglutinin, DNA transcription units comprising DNA encoding F fusion protein, or both DNA transcription units comprising H DNA and DNA transcription units comprising F DNA, whereas mice receiving control DNA did not exhibit raised antibody titers. The H and sH DNA appeared to raise antibody with similar effectiveness (ratio of neutralizing activity to ELISA activity) . In the rabbit models, high neutralizing antibody titers were raised in rabbits receiving DNA transcriptional units comprising H DNA and in rabbits receiving DNA transcriptional units comprising F DNA. In the Rhesus macaque models, neutralizing antibody titers were raised in animals receiving DNA transcription units comprising H DNA, comprising F DNA, and comprising both H DNA and F DNA, whether the route of administration was intradermal or by gene gun. The current invention is illustrated by the following examples, which are not to be construed as limiting in any way.

Example 1 DNA Constructs for Immunization Against

Measles Virus A series of DNA transcription units were prepared for immunizations against measles virus. The series uses pJW4303 expression vectors developed at James I. Mullins laboratory (Stanford University, Palo Alto, CA) . The JW4303 vectors and accompanying oligonucleotides are designed to facilitate the cloning of polymerase chain reaction - amplified fragments of an antigen, including an antigen of any isolate of measles virus.

The JW4303 plasmid uses approximately 2000 bp from the cytomegalovirus (CMV) immediate early promoter and sequences from the bovine growth hormone (BGH) for insert expression (Figs. 1-2) . Sequences from the CMV immediate early promoter include sequences encoding the CMV intron A. This intron can enhance the expression of inserted genes (Chapman, et al. , Nucleic Acids

Research 14:3979-3986 (1991))- The tissue plasminogen activator (tPA) leader facilitates synthesis and secretion of glycosylated proteins (Haigwood, et al.. Prot. Eng. 2:611-620 (1989)) . This synthetic leader provides the start site for antigen expression.

Polymerase chain reaction amplification from designer oligonucleotides is used to create antigen fragments that are inserted in-frame with the tPA leader.

Polymerase chain reactions using fifteen cycles of amplification by the Taq polymerase (Promega, Madison WI) and standard reaction conditions were used to produce fragments for subcloning into the pJW4303 expression vector. Hemagglutinin-expressing vaccine plasmids were prepared from a cDNA for the hemagglutinin from the Edmonston strain of measles virus (Genbank accession number, M14877) (Fig. 1) . For the full length H, a 5' sense primer (GATCaagcttATGTCACCACAACGAGAC) and a 3' antisense primer (GATCggatccCTATCTGCGATTGGTTCC) were used to prepare a Hindlll to BamHl fragment for subcloning into Hindlll plus BamHl digested pJW4303. The Hindlll to BamHl sites in the primers are in lower case. The natural start and stop codons for H are underlined. For the transmembrane truncated form of H (sH) , a 5' sense plasmid (GATCgctagcGGCATTAGACTTCATCGG) and the same 3' antisense primer were used to prepare a Nhel to BamHl fragment for subcloning into Nhel plus BamHl digested pJW4303. This cloning placed an N-terminal synthetic mimic of the tissue plasminogen activator leader sequence in frame with H sequences. Tissue plasminogen activator and H sequences were fused immediately 3' to the transmembrane domain of H (Figure 1) . The Nhel site used for this cloning is presented in lower case and the junction codon with H sequences (amino acid 56) is underlined. The F-expressing plasmid was prepared from a cDNA for the Edmonston fusion protein (Genbank accession number M14915) (Figure 2) . The complete F protein cDNA was subcloned into Hindlll plus Nhel digested pJW4303. This was accomplished using flanking sites in the original cloning vector (Hindlll, created by mutation of a Xbol site, and Xbal in Bluescript SK; Stratagene, La Jolla, CA) . Expression of pJW4303/H and pJW4303/sH was verified using transiently transfected COS cells and indirect immunofluorescence. In these tests for H and secreted H expression, the anti-hemagglutinin monoclonal antibodies HA15 and HA85 were used as the first antibody. Expression of a secreted form of H by pJW4303/sH was verified using an antigen capture ELISA to test cell lysates and culture medium. This assay used pJW4303/H raised polyclonal mouse sera for capture and detection. Expression of pJW4303/F was verified using a western blot analysis and the F-ost3 monoclonal antibody as the first antibody.

Example 2 Antibody Responses to Inoculation of

Mice Using H DNA. sH DNA and F DNA The ability of hemagglutinin (both membrane- associated and secreted) and fusion glycoproteins to raise neutralizing antibody was examined. Four groups of 6-8 week old BALB/c mice (Taconic Farms, Tarrytown, NY) (six per group) were inoculated with JW4303 plasmids: one group receiving H DNA, one group receiving sH DNA, one group receiving F DNA, and a control group receiving the plasmids with no antigen DNA insert.

To deliver the DNA, an Accell® particle bombardment device, the gene gun (Agracetus, Inc. , Middleton, WI) , was employed to deliver DNA-coated gold beads to the freshly shaved abdominal epidermis of the mice. These gene-gun delivery experiments were done in collaboration with Dr. Joel R. Haynes of Agracetus, Inc. For gene-gun delivery, plasmid DNA was affixed to 0.95 micron particles by adding gold powder (Degussa, South Plainfield, NJ) , and plasmid DNA to a centrifuge tube containing spermidine and calcium. Plasmid DNA and gold were coprecipitated during vortex mixing, after which the precipitate was allowed to settle and was washed with absolute ethanol and resuspended in ethanol. The gold/DNA suspension was transferred to a capped vial and immersed in a sonicating water bath to resolve clumps. Then the DNA/gold suspension was layered onto Mylar sheets and allowed to settle for several minutes, after which the meniscus was broken and excess ethanol was removed by aspiration. DNA/gold-coated mylar sheets were dried and stored under vacuum. The total amount of DNA per sheet was a function of the DNA/gold ratio. The DNA/gold ratio was 2.5 μg DNA/1.0 mg gold, resulting in 1 μg DNA per sheet and 0.4 mg. gold per sheet.

The BALB/c mice were anesthetized with Ketaset/Rompun (ketamine) . Abdominal target areas were prepared using known techniques. Target areas were thoroughly rinsed with water prior to gene delivery. DNA-coated gold particles were delivered into abdominal skin with an electric discharge gene gun, which employs an electric spark discharge as the motive force (Yang, M.S. et al.. Proc. Natl. Acad. Sci. USA 87: 9568-9572 (1990)). Each mouse received four deliveries per inoculation, at a discharge voltage of 17kV. The beads deliver DNA into cells, where the DNA dissolves and can be expressed. (Yang, M.S., supra; Williams, R.S. et al.. Proc. Natl. Acad. Sci. USA 88: 2726-2730 (1991)). One group of six mice was inoculated with H DNA, another group of six mice was inoculated with sH DNA, another group of six mice was inoculated with F DNA, and the last group of six mice was inoculated with beads that contained control plasmids. The mice in each group were inoculated twice, with the second inoculation (the boost) occurring approximately four weeks after the first. For each initial inoculation, the gun delivered four shots, each with 1 μg of DNA per 0.4 mg of gold. For each boost, the gun delivered four shots, each consisting of 0.4 μg of DNA per 0.8 mg of gold.

The quality of the raised antibody was assessed by comparing neutralizing activity and ELISA activity. To perform antibody assays, sera were collected immediately prior to each DNA inoculation, and at eleven times after the second inoculation. For sera collection, anesthetized mice were bled from the eye vein into microhematocrit tubes. The sera were stored in aliquots at -80°.

At weeks 16 and 50, pooled sera from each test group of mice were tested for the presence of neutralizing antibodies which inhibit replication of the Chicago-1 strain (a 1989 US wild type isolate) of measles virus. Vero cells were obtained from the American Type Culture Collection (Rockville, MD) , and the Chicago-1 strain was obtained from W. Bellini (Centers for Disease Control, Atlanta, GA) . Preimmune sera served as negative controls. These tests were conducted by mixing 100-200 pfu of the Chicago-1 strain of measles virus with serial dilutions of mouse sera for one hour at 37° and then inoculated onto 6-well tissue culture plates (Corning Inc, Corning, NY) in which Vero cells were grown. The plates were inoculated at 37° for 48-60 hours. Plaques were counted using an inverted microscope following staining with 0.1% crystal violet or neutral red. The results of the assays are summarized in Table 1, below. The neutralizing titers were defined as the reciprocals of the last dilution of sera giving >90% inhibition of viral plaque formation.

Levels of antibodies were scored using an ELISA using known methods. See Ausubel et a_l. , Current

Protocols in Molecular Biology. 2nd. John Wiley & Sons, Inc. (1995) . Corning Easy Wash microtiter plates (Corning Inc., Corning, NY) were coated with extracts of Ltk cells expression H or F proteins: Extracts were prepared by typsinizing cells from one confluent T-75 flask, washing the cells twice with PBS, and then resuspending the cells in 2 ml of PBS supplemented with 0.1% Na-deoxycholate. Extracts were frozen in aliquots at -80°. A 1:20 dilution of the frozen extract was used for coating plates. Assays were read at 450 nm and analyzed using the SOFTmax® ver 2.31 software (Molecular Devices Corp., Sunnyvale, CA) . Data were given as relative ELISA units. Optical densities for sera of the control group (mice receiving no antigen DNA) were subtracted from values obtained for the experimental groups (mice receiving H DNA_f sH DNA or F DNA) .

To determine the quality of the neutralizing antibodies raised by the antigen DNA, the neutralization titer was divided by the number of ELISA units, IgG. The results are summarized in Table 1.

10 Both DNA-expressed H and sH raised long-lived neutralizing responses of similar quality, with significant levels of antibodies that persisted at fifty weeks (See Table 1) .

Table l: Quality of Neutralizing Antibody Raised by 15 Full-Length and Secreted Forms of Hemagglutinin

♦Edmonston strain

0 To demonstrate the temporal immunological effects of the inoculations, the sera of each of the H DNA- immunized mice and the sH DNA-immunized mice were tested over a course of twenty-eight weeks for presence of neutralizing antibodies. The titrations of 5 antibodies were done as described in Current Protocols in Molecular Biology, supra. Titers of neutralizing activity are the reciprocals of the highest dilution of sera giving complete neutralization of 200 50% tissue culture infectious doses of the Chicago-1 strain of 0 virus. The sera of mice from both groups demonstrated peak levels of titer four to eight weeks after the second inoculation. Results are shown in Figure 3A and Figure 3B. The sera of mice inoculated with H DNA demonstrated a high level of antibodies over the course of the twenty-eight week assay. Sera from the H- inoculated mice developed neutralizing titers of 1,000 to 10,000, whereas sera from sH inoculated mice developed lower titers of 100 to 3,000. These titers have been stable for the first year of the experiment.

Example 3 Antibody Responses to Inoculation of Mice Using F DNA and Membrane-Associated

H DNA To test a vaccine which included both H and F DNA, ten test groups of BALB/c mice (six mice per group) received gene deliveries of either H DNA, F DNA, or both H and F DNA. For each type of DNA delivered

(i.e., H, F, or both), one group of mice was inoculated once, and one group of mice was inoculated twice, with the second inoculation occurring four weeks after the first inoculation. Each mouse was bled prior to each inoculation and seven weeks after the first inoculation.

A helium pulse Accell® gene gun (Agracetus, Inc., Middleton, WI) was used to deliver DNA coated beads to the abdominal epidermis of the mice. The DNA beads were prepared as described in the previous example, but with a DNA/gold ratio of 1.0 μg DNA to 1.0 mg gold. The mice were anesthetized with Ketaset/Rompun (ketamine) and the abdominal target areas were prepared using known techniques. The DNA beads were delivered to the target areas in four deliveries per inoculation, at a pressure of 450 p.s.i., each delivery consisting of 0.5 μg DNA and 0.5 mg gold.

The first two experimental groups of mice received beads coated with H DNA, group 1 receiving one inoculation, and group 2 receiving two inoculations. The next two groups received beads coated with F DNA, group 3 receiving one inoculation and group 4 receiving two inoculations. Each of the next two groups received beads coated with H DNA and beads coated with F DNA, group 5 receiving one inoculation of the separate H- coated beads and F-coated beads and group 6 receiving two inoculations of the separate H-coated beads and F- coated beads. The next two groups received inoculations where each bead was coated with a mixture of both H and F DNA, group 7 receiving one inoculation of the mixture beads and group 8 receiving two inoculations. The final two groups were controls, wherein the mice were inoculated with beads coated with pJW4303 plasmids containing no antigen inserts, group 9 receiving one inoculation of control beads and group 10 receiving two inoculations of control beads.

To determine the ability of these inoculations to raise neutralizing antibodies, the mice were bled at six weeks and the sera from the mice in each test group was pooled and assayed for neutralizing antibodies using the methods described in Example 2, using the Edmonston strain of measles virus obtained from the American Type Culture Collection (Rockville, MD) . (See Figure 4 and Figures 5A-5C) . The neutralization titers are the reciprocals of the highest dilution of sera giving complete neutralization of 200 50% tissue culture infectious doses of virus. The sera of mice from the control group did not demonstrate appreciable neutralization titers.

Single inoculations of the H, the F and a mixture of the H and F expressing DNAs also proved capable of raising good titers of neutralizing antibody (Figures 5A-5C) . In this experiment, half of the twelve animals receiving each plasmid (or combination of plasmids) were boosted at 4 weeks (data represented as closed circles in Figures 5A-5C) . Neutralization assays tested sera for 50% plaque reduction of the Edmonston strain of measles virus. In the singly inoculated animals, antibody responses rose over the first eight weeks, with the titers of antibody at 8 weeks being 4 to 10 times higher than the titers of antibody at 4 weeks. Interestingly, the boost increased the plateau titers of the pooled sera by only 2 to 4 fold over the titers that were achieved by the single inoculations. Lower neutralizing responses were raised by the F-DNA inoculations. Inoculations with both H plus F DNA (which used one half the amount of H or F DNA used for the immunizations with a single plasmid) achieved an intermediate titer of neutralizing antibody. This intermediate titer would be consistent with the DNA doses for raising anti-H antibody being on the linear range of a dose/response curve. The neutralizing antibody to F showed poorer persistence than the neutralizing antibody to H. The H DNA-inoculated mice had neutralization titers at 90% plaque reduction of 320-420, and the F DNA-inoculated mice had neutralization titers at 90% plaque reduction of less than or equal to 50.

Example 4 Antibody Responses to Inoculation of

Rabbits Using H DNA and F DNA A further experiment was undertaken in young, female 4-5 lbs. NZW- rabbits (Millbrook Farms, Amherst, MA) using the Accell® helium pulse gene gun, to deliver DNA-coated gold beads into their abdominal epidermis.

Four rabbits were anesthetized with Ketaset/Rompun (ketamine) , and their abdominal target areas were prepared. Two rabbits were inoculated with pJW4303/H DNA and two with pJW4303/F DNA, using the gene gun method described in Example 3 above, with each rabbit receiving 36 deliveries per inoculation, each delivery consisting of 0.25 μg DNA per 0.5 mg gold at a pressure of 450 p.s.i. The DNA/gold ratio for bead preparation was 0.5 μg DNA/1.0 mg gold. Each rabbit was inoculated three times, at intervals of four weeks. The rabbits were bled prior to each inoculation and at varying intervals after the inoculations.

The rabbits were then analyzed for antibody responses to the H DNA administrations. Levels of antibodies were scored using ELISA using the methods described in Example 2, above. The results are depicted in Figure 6. Antibodies were raised after the first inoculation and were boosted by the second inoculation. Neutralizing activity in the H DNA-inoculated and F DNA-inoculated rabbits was also tested. The titrations of antibodies were done as described in Examples 2 and 3, above, using the Edmonston strain. Titers of neutralizing activity are the reciprocals of the highest dilution of sera giving complete neutralization of 200 50% tissue culture infectious doses of virus. Data are presented in Figure 7A (H DNA) and Figure 7B (F DNA) . The results of both the ELISA and the neutralization titer assays demonstrated that both of the rabbits immunized with H DNA presented demonstrable rises in antibodies after each inoculation, peaking within four weeks of the third inoculation. The F-immunized rabbits also demonstrated raised neutralizing antibody titers, although the titers did not rise significantly until the ninth week. A series of inoculations at one month intervals were used to raise polyclonal and anti-H and anti-F sera in NZW rabbits. Sera were tested for 50% reduction of Edmonston plaques. Antibody responses raised by the H- expressing plasmid rose more rapidly than those raised by the F-expressing DNA. After 3 immunizations, slightly higher titers of neutralizing antibody had been raised by the H-expressing DNA (5000 to 10,000) than the F-expressing DNA (~5000) . Unlike the mice, the titers of neutralizing antibody for H fell with time. The rabbits inoculated with H DNA exhibited neutralization titers of 950-1300, 90% plaque reduction. The rabbits inoculated with F DNA exhibited neutralization titers of 1,500-2,000, 90% plaque reduction.

Example 5 Antibody Responses to Inoculation of Rhesus Macaques Using H DNA and F DNA

A Rhesus macaque model was used to test immune responses to inoculation with H DNA, F DNA, and both H DNA and F DNA, both by intradermal administration and by gene gun administration by using an Accell® helium pulse gene gun (Agracetus, Inc., Middleton, WI) . Five groups of one- and two-year-old Rhesus macaques (Johns Hopkins University, Baltimore, MD) were inoculated with JW4303 plasmids: One group of 3 macaques was inoculated with 125 μg of H DNA intradermally (id) , one group of 3 macaques was inoculated with 8 μg of H DNA (eight deliveries of 1 μg DNA per inoculation) on gold beads by gene gun, one group of 2 macaques was inoculated with 125 μg of F DNA intradermally, one group of 3 macaques was inoculated with both 125 μg H DNA and 125 μg F DNA (for a total of 250 μg DNA/macaque) intradermally, and one group of 3 macaques received both 8 μg H DNA and 8 μg F DNA (for a total of 16 μg DNA/macaque) by gene gun. The macaques were anaesthetized with Ketaset/Rompun (ketamine) and their target areas were prepared. Each macaque was either inoculated intradermally in the back or inoculated with a gene gun at the abdominal epidermis. The gene gun method used is described in Example 3 above, with each macaque receiving 1 μg DNA per delivery at a pressure of 400 p.s.i.

To determine the ability of these inoculations to raise neutralizing antibodies in a 50% plaque reduction neutralization test (PRNT50) , plasma from each macaque was drawn before immunization and at 4 weeks and 12 weeks after immunization. The plasma was tested after each bleeding. The titrations of antibodies were done as described as in Example 2 above. Essentially all macaques produced measles virus neutralizing antibody (See Figure 8) . The highest levels of antibody were produced by macaques that received H DNA intradermally.

Equivalents Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. These and all other such equivalents are intended to be encompassed by the following claims.

Claims

1. A product for use in therapy in a mammal, e.g., immunization, and comprising a DNA transcription unit comprising DNA encoding an antigen of measles virus operatively linked to DNA which is a promoter region.

2. A product comprising a DNA transcription unit, wherein the transcription unit(s) comprise(s) DNA encoding an antigen of measles virus operatively linked to DNA which is a promoter region, for use in therapy, e.g. for use in immunization of a mammal.

3. Use of a DNA transcription unit comprising DNA encoding an antigen of measles virus operatively linked to DNA which is a promoter region, for the immunization of a mammal.

4. A method of immunizing a mammal, said method comprising administering to the mammal a DNA transcription unit comprising DNA encoding an antigen of measles virus operatively linked to DNA which is a promoter region.

5. A product comprising more than one DNA transcription unit, each transcription unit comprising DNA encoding an antigen of measles virus operatively linked to DNA which is a promoter region, wherein the antigen of measles virus for one transcription unit is different from the antigen of measles virus of the other transcription unit, or each of the other transcription units, for use in therapy, e.g. for use in immunization of a mammal.

6. A product for use in therapy comprising more than one DNA transcription unit, each transcription unit comprising DNA encoding an antigen of measles virus operatively linked to DNA which is a promoter region, wherein the antigen of measles virus for one transcription unit is different from the antigen of measles virus of the other transcription unit, or each of the other transcription units, e.g. for use in a method of immunizing a mammal.

7. Use of a product comprising more than one DNA transcription unit for immunizing a mammal by administering to said mammal the product, wherein each transcription unit comprises DNA encoding an antigen of measles virus operatively linked to DNA which is a promoter region, wherein the antigen of measles virus for one transcription unit is different from the antigen of measles virus of the other transcription unit, or each of the other transcription units.

8. A method of immunizing a mammal, said method comprising administering to a mammal more than one DNA transcription unit, each transcription unit comprising DNA encoding an antigen of measles virus operatively linked to DNA which is a promoter region, wherein the antigen for one transcription unit is different from the antigen of the other transcription unit, or each of the other transcription units.

9. A product, use or method according to any one of the preceding claims, wherein the promoter region, or each of the promoter regions, is/are of nonretroviral origin.

10. A product, use, or method according to any one of the preceding claims, wherein the promoter region, or each of the promoter regions, is/are of retroviral origin.

11. A product, use, or method according to any one of the preceding claims, wherein the antigen, or at least one antigen, is a measles virus hemagglutinin.

12. The product, use or method according to any one of the preceding claims, wherein the antigen, or at least one antigen, is a measles virus fusion protein.

13. The product, use or method of claim 12, wherein the measles virus fusion protein is F₀.

14. A product, use or method according to any one of the preceding claims wherein the antigen, or each of the antigens, is capable of eliciting a protective response against a disease caused by the measles virus.

15. A product, use or method according to claims 5-8 or 14 wherein each antigen is from a different strain of the measles virus.

16. A product, use or method according to claims 5-8 or 14 wherein each antigen is from a different measles virus protein.

17. A product, use or method according to any one of the preceding claims wherein the antigen, or each of the antigens, is a measles virus protein selected from the group consisting of: hemagglutinin, fusion protein, matrix protein, nucleocapsid protein, phosphoprotein, large polymerase protein, and nonstructural protein.

18. A product, use or method according to claims 5-8 or 14, wherein each antigen is a measles virus hemagglutinin from a different strain of the measles virus.

19. A product, use or method according to claims 5-8 or 14 wherein each antigen is a measles virus fusion protein from a different strain of the measles virus.

20. A product, use or method according to claims 5-8 or 14 wherein at least one of the antigens is a measles virus hemagglutinin and at least one of the antigens is a measles virus fusion protein.

21. A product, use or method according to any one of the preceding claims, wherein the antigen or each of the antigens is a secreted form of a protein selected from the group consisting of: hemagglutinin and fusion protein.

22. A product, use or method according to any one of the preceding claims wherein the antigen, or each of the antigens, is selected from subset of T cell-recognized determinants in a measles virus protein.

23. A product, use or method according to any one of the preceding claims wherein the antigen, or each of the antigens, is selected from subset of the B- cell recognized epitopes in a measles virus protein.

24. The product, use or method according to any one of the preceding claims, wherein the transcription unit, or each of the transcription units, is microsphere encapsulated.

25. The product, use or method according to any one of the preceding claims, wherein the transcription unit, or each of the transcription units, is coated onto gold beads for administration to the mammal by particle bombardment delivery.

26. The product, use or method according to any one of the preceding claims wherein the transcription unit, or each of the transcription units, in a physiologically acceptable carrier, is administered to the mammal through a route of administration selected from the group consiεting of intravenous, intramuscular, intraperitoneal, intradermal and subcutaneous.

27. The product, use or method according to any one of the preceding claims wherein the transcription unit, or each of the transcription units, in a physiologically acceptable carrier, is administered to the mammal parenterally.

28. The product, use or method according to any one of claims 1 to 25 wherein the transcription unit, or each of the transcription units, is administered to the mammal by contact to a mucosal surface.

29. The product, use or method according to claim 28, wherein the mucosal surface is a respiratory mucosal surface, such as a nasal mucosal surface or a tracheal mucosal surface.

30. The product, use or method of any of the preceding claims, wherein at least one transcription unit further comprises at least one antigen which is not from the measles virus.

31. The product, use or method of Claim 30, wherein at least one antigen is from a pathogen selected from the group consisting of: influenza, rotavirus tetanus, respiratory syncytial virus, diphtheria, pertussis, mumps and rubella.

32. The product, use or method of any one of the preceding claims, wherein the transcription unit is directly expressed by host cell factors.

33. The product, use or method of any one of the preceding claims, wherein the mammal is a human.

34. Use of a DNA transcription unit comprising DNA encoding an antigen of measles virus operatively linked to DNA which is a promoter region, for the manufacture of a medicament for use in immunization of a mammal.

35. Use of a product comprising more than one DNA transcription unit for the manufacture of a medicament for immunizing a mammal by administering to said mammal the product, wherein each transcription unit comprises DNA encoding an antigen of measles virus operatively linked to DNA which is a promoter region, wherein the antigen of measles virus for one transcription unit is different from the antigen of measles virus of the other transcription unit, or each of the other transcription units. -

36. A product, use or method of any one of the preceding claims whereby a humoral immune response and/or cell-mediated immune response is/are elicited against the antigen or each of the antigens.

37. A method of immunizing a mammal against measles virus, said method comprising administering to the mammal a DNA transcription unit comprising DNA encoding a measles virus antigen of the virus operatively linked to DNA which is a promoter region, wherein the DNA transcription unit is expressed in cells of the mammal, whereby the mammal is protected from the measles disease.

38. The method of Claim 37, wherein the measles virus antigen is a measles virus hemagglutinin.

39. The method of Claim 38, wherein the measles virus hemagglutinin is HA7.

40. The method of Claim 38, wherein the measles virus hemagglutinin is sHA4.

41. The method of Claim 37, wherein the measles virus antigen is a measles virus fusion protein.

42. The method of Claim 41, wherein the measles virus fusion protein is F₀.

43. A method of immunizing a mammal against measles virus, said method comprising administering to the mammal one or more DNA transcription units, each comprising DNA encoding an antigen or antigens of the measles virus operatively linked to DNA which is a promoter region, wherein the DNA transcription unit or units are expressed in cells of the mammal, thereby eliciting a humoral immune response, a cell-mediated immune response or both, against the antigen or antigens, whereby the mammal is protected from the measles disease.

44. The method of Claim 33, wherein the DNA transcription unit is administered in combination with one or more additional DNA transcription units, each comprising DNA encoding a different antigen of the measles virus operatively linked to a promoter region.

45. A composition comprising at least one DNA transcription unit comprising DNA encoding an antigen of measles virus operatively linked to DN_A which is a promoter region, and a physiologically acceptable carrier.