EP0996730A1

EP0996730A1 - Nucleic acid vaccines encoding g protein of respiratory syncytial virus

Info

Publication number: EP0996730A1
Application number: EP98934710A
Authority: EP
Inventors: Xiaomao Li; Suryaprakesh Sambhara; Michel H. Klein
Original assignee: Connaught Laboratories Ltd
Current assignee: Sanofi Pasteur Ltd
Priority date: 1997-07-18
Filing date: 1998-07-16
Publication date: 2000-05-03
Also published as: WO1999004010A1; AU756222B2; JP2001512662A; CA2296089A1; NZ502626A; CN1174099C; AU8427898A; CN1271389A; BR9815496A

Abstract

Non-replicating vectors, such as plasmid vectors, containing a nucleotide sequence coding for a G protein of respiratory syncytial virus (RSV) and a promoter for such sequence, preferably a cytomegalovirus promoter, are described. Such vectors also may contain a further nucleotide sequence located adjacent to the RSV G protein encoding sequence to enhance the immunoprotective ability of the RSV G protein when expressed in vivo. Such non-replicating vectors may be used to immunize a host, including a human host, against RSV infection by administration thereto. Such non-replicating vectors also may be used to produce antibodies for detection of RSV infection in a sample.

Description

TITLE OF INVENTION NUCLEIC ACID VACCINES ENCODING G PROTEIN OF RESPIRATORY SYNCYTIAL VIRUS

FIELD OF INVENTION The present invention is related to the field of respiratory syncytial virus (RSV) vaccines and is particularly concerned with vaccines comprising nucleic acid sequences encoding the attachment (G) protein of RSV.

BACKGROUND OF INVENTION Respiratory syncytial virus (RSV) , a negative- strand RNA virus belonging to the Paramyxoviridae family of viruses, is the major viral pathogen responsible for bronchiolitis and pneumonia in infants and young children (ref. 1 - Throughout this application, various references are referred to in parenthesis to more fully describe the state of the art to which this invention pertains. Full bibliographic information for each citation is found at the end of the specification, immediately preceding the claims. The disclosures of these references are hereby incorporated by reference into the present disclosure) . Acute respiratory tract infections caused by RSV result in approximately 90,000 hospitalizations and 4,500 deaths per year in the United States (ref. 2). Medical care costs due to RSV infection are greater than $340 M annually in the United States alone (ref. 3) . There is currently no licensed vaccine against RSV. The main approaches for developing an RSV vaccine have included inactivated virus, live- attenuated viruses and subunit vaccines.

A protective immune response against RSV is thought to require the induction of neutralizing antibodies against the surface fusion (F) and attachment (G) glycoproteins (ref. 4) . In addition, cytotoxic T lymphocytes (CTL) responses are involved in viral clearance. The F protein is conserved amongst the RSV A and B subgroups .

The G protein (33 kDa) of RSV is heavily O- glycosylated giving rise to a glycoprotein of apparent molecular weight of 90 kDa (ref. 5) . Two broad subtypes of RS virus have been defined: A and B (ref. 6) . The major antigenic differences between these subtypes are found in the G glycoprotein (refs. 3, 7).

The use of RSV proteins as vaccines may have obstacles. Parenterally administered vaccine candidates have so far proven to be poorly immunogenic with regard to the induction of neutralizing antibodies in seronegative chimpanzees. The serum antibody response induced by these antigens may be further diminished in the presence of passively acquired antibodies, such as the transplacentally acquired maternal antibodies which most young infants possess . A subunit vaccine candidate for RSV consisting of purified fusion (F) glycoprotein from RSV infected cell cultures and purified by immunoaffinity or ion-exchange chromatography has been described (ref. 8). Parenteral immunization of seronegative or seropositive chimpanzees with this preparation was performed and three doses of 50 μg were required in seronegative animals to induce an RSV serum neutralizing titre of approximately 1:50. Upon subsequent challenge of these animals with wild-type RSV, no effect of immunization on virus shedding or clinical disease could be detected in the upper respiratory tract. The effect of immunization with this vaccine on virus shedding in the lower respiratory tract was not investigated, although this is the site where the serum antibody induced by parenteral immunization may be expected to have its greatest effect . Safety and immunogenicity studies have been performed in a small number of seropositive individuals. The vaccine was found to be safe in seropositive children and in three seronegative children (all > 2.4 years of age). The effects of immunization on lower respiratory tract disease could not be determined because of the small number of children immunized. One immunizing dose in seropositive children induced a 4 -fold increase in virus neutralizing antibody titres in 40 to 60% of the vaccinees. Thus, insufficient information is available from these small studies to evaluate the efficacy of this vaccine against RSV-induced disease. A further problem facing subunit RSV vaccines is the possibility that inoculation of seronegative subjects with immunogenic preparations might result in disease enhancement. In the 1960 's, vaccination of infants with a formalin-inactivated RSV preparation (FI-RSV) resulted in enhanced lung disease upon subsequent exposure to live virus, also referred to as immunopotentiation (refs. 9, 10). These vaccinees developed strong serological responses, but were not protected against infection and some developed severe, occasionally fatal respiratory tract disease upon natural infection.

Although precise mechanisms remain unknown, it has been suggested that this form of immune enhancement might reflect either structural alterations of RSV antigens (ref. 11), residual serum and/or cellular contaminants (ref. 12), a specific property of the viral attachment (G) protein (refs. 13,14) or an imbalanced cell-mediated immune response (refs. 13,15). It has been demonstrated that the FI-RSV vaccine induced a TH2-type immune response in mice whereas immunization with live RSV, which does not cause immunopotentiation, elicits a TH1 response (ref.15).

In some studies, the immune response to immunization with a synthetic RSV FG fusion protein resulted in disease enhancement in rodents resembling that induced by a formalin-inactivated RSV vaccine. Immunization of mice with a recombinant vaccinia virus expressing the RSV G protein resulted in G-specific T cell responses in the lungs which are exclusively recruited from the CD4+T cell sublineage and are strongly Th2-biased. G-specific T cells induce lung haemmorrage, pulmonary neutrophil recruitment (shock lung) , intense pulmonary eosinophilia, and sometimes death in the adoptively transferred murine recipients (ref. 14) . The association of immunization with disease enhancement using certain vaccine preparations including non-replicating antigens suggests caution in their use as vaccines in seronegative humans .

Live attenuated vaccines against disease caused by RSV may be promising for two main reasons. Firstly, infection by a live vaccine virus induces a balanced immune response comprising mucosal and serum antibodies and cytotoxic T-lymphocytes . Secondly, infection of infants with live attenuated vaccine candidates or naturally acquired wild-type virus is not associated with enhanced disease upon subsequent natural reinfection. It will be challenging to produce live attenuated vaccines that are immunogenic for younger infants who possess maternal virus-neutralizing antibodies and yet are attenuated for seronegative infants greater than or equal to 6 months of age. Attenuated live virus vaccines also have the risks of residual virulence and genetic instability.

Injection of plasmid DNA containing sequences encoding a foreign protein has been shown to result in expression of the foreign protein and the induction of antibody and cytotoxic T-lymphocyte (CTL) responses to the antigen in a number of studies (see, for example, refs. 16, 17, 18) . The use of plasmid DNA inoculation to express viral proteins for the purpose of immunization may offer several advantages over the strategies summarized above. Firstly, DNA encoding a viral antigen can be introduced in the presence of antibody to the virus itself, without loss of potency due to neutralization of virus by the antibodies. Secondly, the antigen expressed in vivo should exhibit a native conformation and the appropriate glycosylation. Therefore, the antigen should induce an antibody response similar to that induced by the antigen present in the wild-type virus infection. In contrast, some processes used in purification of proteins can induce conformational changes which may result in the loss of immunogenicity of protective epitopes and possibly immunopotentiation. Thirdly, the expression of proteins from injected plasmid DNAs can be detected in vivo for a considerably longer period of time than that in virus- infected cells, and this has the theoretical advantage of prolonged cytotoxic T-cell induction and enhanced antibody responses. Fourthly, in vivo expression of antigen may provide protection without the need for an extrinsic adjuvant.

The ability to immunize against disease caused by RSV by administration of a DNA molecule encoding an RSV G protein was unknown before the present invention. In particular, the efficacy of immunization against RSV induced disease using a gene encoding a secreted form of the RSV G protein was unknown. Infection with RSV leads to serious disease. It would be useful and desirable to provide isolated genes encoding RSV G protein and non- replicating vectors, including plasmid vectors, for in vivo administration and for use in immunogenic preparations, including vaccines, for protection against disease caused by RSV and for the generation of diagnostic reagents and kits. In particular, it would be desirable to provide vaccines that are immunogenic and protective in humans, including seronegative infants, that do not cause disease enhancement (immunopotentiation) .

SUMMARY OF INVENTION The present invention relates to a method of immunizing a host against disease caused by respiratory syncytial virus, to non-replicating vectors containing nucleic acid molecules used in immunogenic compositions for such purpose, and to diagnostic procedures utilizing the vectors and nucleic acid molecules. In particular, the present invention is directed towards the provision of nucleic acid vaccines encoding the G protein of respiratory syncytial virus.

In accordance with one aspect of the invention, there is provided an immunogenic composition for in vivo administration to a host for the generation in the host of protective antibodies to respiratory syncytial virus (RSV) G protein, comprising a non-replicating vector comprising: a first nucleotide sequence encoding a RSV G protein or a RSV G protein fragment that generates antibodies that specifically react with RSV G protein, a promoter sequence operatively coupled to said first nucleotide sequence for expression of said RSV G protein in the host, and a second nucleotide sequence located between said first nucleotide sequence and said promoter sequence to increase expression of said RSV G protein in vivo from said vector in the host, and a pharmaceutically-acceptable carrier therefor. The first nucleotide sequence may be that which encodes a full-length RSV G protein. The first nucleotide sequence may comprise the nucleotide sequence shown in Figure 2 (SEQ. ID No: 1) or encode a full length RSV G protein having the amino acid sequence shown in Figure 2 (SEQ. ID no: 2) . Alternatively, the first nucleotide sequence may be that which encodes an RSV G protein from which the transmembrane coding sequence and sequences upstream thereof are absent. The first nucleotide sequence encoding the truncated RSV G protein may comprise the nucleotide sequence shown in Figure 3 (SEQ. ID no: 3) or may comprise a nucleotide sequence encoding the truncated RSV G protein having the amino acid sequence shown in Figure 3 (SEQ ID no: 4) . The lack of expression of the transmembrane region results in a secreted form of the RSV G protein.

The non-replicating vector may further comprise a heterologous signal peptide encoding nucleotide sequence immediately upstream of the 5' -terminus of the first nucleotide sequence. The signal peptide encoding sequence may encode the signal peptide of human tissue plasminogen activator.

The promoter sequence may be an immediate early cytomegalovirus (CMV) promoter. The second nucleotide sequence may comprise the human cytomegalovirus Intron

A.

The non-replicating vector generally is a plasmid vector. Plasmid vectors encoding the G protein and included in the immunogenic composition provided by this aspect of the invention may specifically be pXL5 or pXL6, constructed and having their characterizing elements, as seen in Figures 4 or 5, respectively.

In accordance with a further aspect of the present invention, there is provided a method of immunizing a host against disease caused by infection with respiratory syncytial virus (RSV) , which comprises administering to the host an effective amount of a non- replicating vector comprising: a first nucleotide sequence encoding an RSV G protein or a RSV G protein fragment that generates antibodies that specifically react with RSV G protein, a promoter sequence operatively coupled to said first nucleotide sequence for expression of said RSV G protein in the host, and a second nucleotide sequence located between said first nucleotide sequence and said promoter sequence to increase expression of said RSV G protein in vivo from said vector in the host .

The immunization method may be effected to induce a balanced Thl/Th2 immune response.

The present invention also includes a novel method of using a gene encoding respiratory syncytial virus (RSV) G protein or a RSV G protein fragment that generates antibodies that specifically react with RSV G protein, to protect a host against disease caused by infection with respiratory syncytial virus, which comprises : isolating the gene; operatively linking the gene to at least one control sequence to produce a non-replicating vector, said control sequence directing expression of the RSV G protein when said vector is introduced into a host to produce an immune response to the RSV G protein, and introducing the vector into the host . The procedure provided in accordance with this aspect of the invention may further include the step of: operatively linking the gene to an immunoprotection enhancing sequence to produce an enhanced immunoprotection by the RSV G protein in the host, preferably by introducing the immunoprotection enhancing sequence between the control sequence and the gene, including introducing immunostimulatory CpG sequences in the vector.

In addition, the present invention includes a method of producing a vaccine for protection of a host against disease caused by infection with respiratory syncytial virus (RSV), which comprises: isolating a first nucleotide sequence encoding an RSV G protein or a RSV G protein fragment that generates antibodies that specifically react with RSV G protein, operatively linking the first nucleotide sequence to at least one control sequence to produce a non- replicating vector, the control sequence directing expression of the RSV G protein when introduced into a host to produce an immune response to the RSV G protein when expressed in vivo from the vector in a host, operatively linking the first nucleotide sequence to a second nucleotide sequence to increase expression of the RSV G protein in vivo from the vector in a host, and formulating the vector as a vaccine for in vivo administration.

The vector may be a plasmid vector selected from pXL5 and pXL6. The invention further includes a vaccine for administration to a host, including a human host, produced by this method.

As noted previously, the vectors provided herein are useful in diagnostic applications. In a further aspect of the invention, therefore, there is provided a method of determining the presence of a respiratory syncytial virus (RSV) G protein in a sample, comprising the steps of:

(a) immunizing a host with a non-replicating vector to produce antibodies specific for the RSV G protein, the non-replicating vector comprising a first nucleotide sequence encoding an RSV G protein or an RSV G protein fragment that generates antibodies that specifically react with RSV G protein, a promoter sequence operatively coupled to the first nucleotide sequence for expression of the RSV G protein in the host and a second nucleotide sequence located between the first nucleotide sequence and the promoter sequence to increase expression of the RSV G protein in vivo from the vector in the host;

(b) isolating the RSV G protein-specific antibodies;

(c) contacting the sample with the isolated antibodies to produce complexes comprising any RSV G protein present in the sample and the RSV G protein-specific antibodies; and

(d) determining production of the complexes.

The non-replicating vector employed to elicit the antibodies may be a plasmid vector pXL5 or pXL6. The invention also includes a diagnostic kit for detecting the presence of a respiratory syncytial virus (RSV) G protein in a sample, comprising:

(a) a non-replicating vector capable of generating antibodies specific for the RSV G protein when administered to a host, said non- replicating vector comprises a first nucleotide sequence encoding an RSV G protein or an RSV G protein fragment that generates antibodies that specifically react with RSV G protein, a promoter sequence operatively coupled to the first nucleotide sequence for expression of the RSV G protein in a host, and a second nucleotide sequence located between the first nucleotide sequence and the promoter sequence to increase expression of the RSV G protein in vivo from the vector in the host;

(b) isolation means to isolate the RSV G protein specific antibodies;

(c) contacting means to contact the isolated RSV G protein-specific antibodies with the sample to produce a complex comprising any RSV G protein present in the sample and RSV G protein specific antibodies; and

(d) identifying means to determine production of the complex. The present invention further is directed to a method for producing antibodies specific for a G protein of a respiratory syncytial virus (RSV) comprising:

(a) immunizing a host with an effective amount of a non-replicating vector to produce RSV G-specific antibodies, said non-replicating vector comprising: a first nucleotide sequence encoding a RSV G protein or a RSV G protein fragment that generates antibodies that specifically react with RSV G protein, a promoter sequence operatively coupled to said first nucleotide sequence for expression of said RSV G protein in the host, and a second nucleotide sequence located between said first nucleotide sequence and said promoter sequence to increase expression of said RSV G protein in vivo from said vector in the host; and

(b) isolating the RSV G specific antibodies from the host . The present invention is also directed to a method for producing monoclonal antibodies specific for a G protein of respiratory syncytial virus (RSV) , comprising the steps of:

(a) constructing a vector comprising a first nucleotide sequence encoding a RSV G protein or a

RSV G protein fragment that generates antibodies that specifically react with RSV G protein, a promoter sequence operatively coupled to the first nucleotide sequence for expression of the RSV G protein in the host and a second nucleotide sequence located between the first nucleotide sequence and the promoter sequence to increase expression of the RSV G protein when in vivo from the vector in a host; (b) administering the vector to at least one mouse to produce at least one immunized mouse;

(c) removing B-lymphocytes from the at least one immunized mouse;

(d) fusing the B-lymphocytes from the at least one immunized mouse with myeloma cells, thereby producing hybridomas;

(e) cloning the hybridomas;

(f) selecting clones which produce anti-RSV G protein antibody; (g) culturing the anti-RSV G protein antibody- producing clones; and

(h) isolating anti-RSV G protein monoclonal antibodies . Such monoclonal antibodies may be used to purify RSV G protein from virus.

In this application, the term "RSV G protein" is used to define a full-length RSV G protein, such proteins having variations in their amino acid sequences including those naturally occurring in various strains of RSV, a secreted form of RSV G protein lacking a transmembrane region, as well as functional analogs of the RSV G protein. In this application, a first protein is a "functional analog" of a second protein if the first protein is immunologically related to and/or has the same function as the second protein. The functional analog may be, for example, an immunologically-active fragment of the protein or an immunologically-active substitution, addition or deletion mutant thereof. BRIEF DESCRIPTION OF THE FIGURES The present invention will be further understood from the following General Description and Examples with reference to the Figures of the accompanying drawings, in which:

Figure 1 illustrates a restriction map of the gene encoding a G protein of respiratory syncytial virus (RSV) ;

Figure 2 illustrates the nucleotide sequence of a gene encoding a membrane bound form of the G protein of respiratory syncytial virus (SEQ ID No: 1) as well as the amino acid sequence of the RSV G protein encoded thereby (SEQ ID No: 2) ;

Figure 3 illustrates the nucleotide sequence of a gene encoding the secreted form of the RSV G protein lacking the transmembrane domain (SEQ ID No: 3) as well as the amino acid sequence of a truncated RSV G protein lacking the transmembrane domain encoded thereby (SEQ ID NO : 4 ) ; Figure 4 shows the construction of plasmid pXL5 containing a gene encoding a full-length membrane attached form of the RSV G protein and containing the CMV Intron A sequence;

Figure 5 shows the construction of plasmid pXL6 containing a gene encoding a secreted form of the RSV G protein lacking the transmembrane domain and containing the CMV Intron A sequence as well as a nucleotide sequence encoding a signal peptide of the human tissue plasminogen activator (TPA) ; Figure 6 shows the nucleotide sequence for the plasmid VR-1012 (SEQ ID No. 5) ;

Figure 7 shows the nucleotide sequence for the 5 ' untranslated region and the signal peptide of the human tissue plasminogen activator (TPA) (SEQ. ID no: 6) and Figure 8 shows the lung cytokine expression profile in DNA immunized mice after RSV challenge.

GENERAL DESCRIPTION OF INVENTION

As described above, the present invention relates generally to polynucleotide, including DNA, immunization to obtain protection against infection by respiratory syncytial virus (RSV) and to diagnostic procedures using particular non-replicating vectors. In the present invention, several recombinant plasmid vectors were constructed to contain a nucleotide sequence encoding an RSV G protein.

The nucleotide sequence of the full length RSV G gene is shown in Figure 2 (SEQ ID No: 1) . Certain constructs provided herein include the nucleotide sequence encoding the full-length RSV G (SEQ ID No : 2) protein while others include an RSV G gene modified by deletion of the transmembrane coding sequence and nucleotides upstream thereof (see Figure 3, SEQ ID No: 3) , to produce a secreted or truncated RSV G protein lacking the transmembrane domain (SEQ ID No. 4) . The nucleotide sequence encoding the RSV G protein is operatively coupled to a promoter sequence for expression of the encoded RSV G protein in vivo . The promoter sequence may be the human immediately early cytomegalovirus (CMV) promoter. This promoter is described in ref. 19. Any other convenient promoter may be used, including constitutive promoters, such as, the Rous Sarcoma Virus LTRs, and inducible promoters, such as the metallothionin promoter, and tissue specific promoters . The non-replicating vectors provided herein, when administered to an animal in the form of an immunogenic composition with a pharmaceutically-acceptable carrier, effect in vivo RSV G protein expression, as demonstrated by an antibody response in the animal to which it is administered. Such antibodies may be used herein in the detection of RSV protein in a sample, as described in more detail below. The administration of the non- replicating vectors, specifically plasmids pXL5 and pXL6, produced anti-G antibodies, virus neutralizing antibodies, a balanced Thl/Th2 response in the lungs post viral challenge and conferred protection in mice against live RSV infection, as seen from the Examples below.

The recombinant vector also may include a second nucleotide sequence located adjacent the RSV G protein encoding nucleotide sequence to enhance the immunoprotective ability of the RSV G protein when expressed in vivo in a host . Such enhancement may be provided by increased in vivo expression, for example, by increased mRNA stability, enhanced transcription and/or translation. This additional sequence generally is located between the promoter sequence and the RSV G protein-encoding sequence. This enhancement sequence may comprise the immediate early cytomegalovirus Intron A sequence.

The non-replicating vector provided herein may also comprise an additional nucleotide sequence encoding a further antigen from RSV, an antigen from at least one other pathogen or at least one immunomodulating agent, such as a cytokine . Such vector may contain the additional nucleotide sequence in a chimeric or a bicistronic structure. Alternatively, vectors containing the additional nucleotide sequence may be separately constructed and coadministered to a host, along with the non-replicating vectors provided herein.

The non-replicating vector may further comprise a nucleotide sequence encoding a heterologous viral or eukaryotic signal peptide, such as the human tissue plasminogen activator (TPA) signal peptide, in place of the endogenous signal peptide for the truncated RSV G protein. Such nucleotide sequence may be located immediately upstream of the RSV G encoding sequence in the vector.

The immunogenicity of the non-replicating DNA vectors may be enhanced by inserting immunostimulatory CpG sequences in the vector.

It is clearly apparent to one skilled in the art, that the various embodiments of the present invention have many applications in the fields of vaccination, diagnosis and treatment of RSV infections. A further non-limiting discussion of such uses is further presented below. 1. Vaccine Preparation and Use

Immunogenic compositions, suitable to be used as vaccines, may be prepared from the RSV G genes and vectors as disclosed herein. The vaccine elicits an immune response in an animal which includes the production of anti-RSV G antibodies. Immunogenic compositions, including vaccines, containing the nucleic acid may be prepared as injectables, in physiologically- acceptable liquid solutions or emulsions for polynucleotide administration. The nucleic acid may be associated with liposomes, such as lecithin liposomes or other liposomes known in the art, as a nucleic acid liposome (for example, as described in WO 9324640, ref. 20) or the nucleic acid may be associated with an adjuvant, as described in more detail below. Liposomes comprising cationic lipids interact spontaneously and rapidly with polyanions, such as DNA and RNA, resulting in liposome/nucleic acid complexes that capture up to

100% of the polynucleotide. In addition, the polycationic complexes fuse with cell membranes, resulting in an intracellular delivery of polynucleotide that bypasses the degradative enzymes of the lysosomal compartment. Published PCT application WO 94/27435 describes compositions for genetic immunization comprising cationic lipids and polynucleotides . Agents which assist in the cellular uptake of nucleic acid, such as calcium ions, viral proteins and other transfection facilitating agents, may advantageously be used.

Polynucleotide immunogenic preparations may also be formulated as microcapsules, including biodegradable time-release particles. Thus, U.S. Patent 5,151,264 describes a particulate carrier of a phospholipid/glycolipid/polysaccharide nature that has been termed Bio Vecteurs Supra Moleculaires (BVSM) . The particulate carriers are intended to transport a variety of molecules having biological activity in one of the layers thereof.

U.S. Patent 5,075,109 describes encapsulation of the antigens trinitrophenylated keyhole limpet hemocyanin and staphylococcal enterotoxin B in 50:50 poly (DL-lactideco-glycolide) . Other polymers for encapsulation are suggested, such as poly (glycolide) , poly (DL-lactide-co- glycolide), copolyoxalates, polycaprolactone, poly (lactide-co-caprolactone) , poly (esteramides) , polyorthoesters and poly (8- hydroxybutyric acid), and polyahhydrides . Published PCT application WO 91/06282 describes a delivery vehicle comprising a plurality of bioadhesive microspheres and antigens. The microspheres being of starch, gelatin, dextran, collagen or albumin. This delivery vehicle is particularly intended for the uptake of vaccine across the nasal mucosae . The delivery vehicle may additionally contain an absorption enhancer.

The RSV G gene containing non-replicating vectors may be mixed with pharmaceutically acceptable excipients which are compatible therewith. Such excipients may include, water, saline, dextrose, glycerol, ethanol, and combinations thereof. The immunogenic compositions and vaccines may further contain auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, or adjuvants to enhance the effectiveness thereof. Immunogenic compositions and vaccines may be administered parenterally, by injection subcutaneously, intravenously, intradermally or intramuscularly, possibly following pretreatment of the injection site with a local anesthetic. Alternatively, the immunogenic compositions formed according to the present invention, may be formulated and delivered in a manner to evoke an immune response at mucosal surfaces. Thus, the immunogenic composition may be administered to mucosal surfaces by, for example, the nasal or oral (intragastric) routes. Alternatively, other modes of administration including suppositories and oral formulations may be desirable. For suppositories, binders and carriers may include, for example, polyalkylene glycols or triglycerides. Oral formulations may include normally employed incipients, such as, for example, pharmaceutical grades of saccharine, cellulose and magnesium carbonate.

The immunogenic preparations and vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective, protective and immunogenic. The quantity to be administered depends on the subject to be treated, including, for example, the capacity of the individual's immune system to synthesize the RSV G protein and antibodies thereto, and if needed, to produce a cell-mediated immune response. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner. However, suitable dosage ranges are readily determinable by one skilled in the art and may be of the order of about 1 μg to about 2 mg of the RSV G gene-containing vectors. Suitable regimes for initial administration and booster doses are also variable, but may include an initial administration followed by subsequent administrations. The dosage may also depend on the route of administration and will vary according to the size of the host . A vaccine which protects against only one pathogen is a monovalent vaccine. Vaccines which contain antigenic material of several pathogens are combined vaccines and also belong to the present invention. Such combined vaccines contain, for example, material from various pathogens or from various strains of the same pathogen, or from combinations of various pathogens . Immunogenicity can be significantly improved if the vectors are co-administered with adjuvants, commonly used as 0.05 to 0.1 percent solution in phosphate- buffered saline. Adjuvants enhance the immunogenicity of an antigen but are not necessarily immunogenic themselves. Adjuvants may act by retaining the antigen locally near the site of administration to produce a depot effect facilitating a slow, sustained release of antigen to cells of the immune system. Adjuvants can also attract cells of the immune system to an antigen depot and stimulate such cells to elicit immune responses .

Immunostimulatory agents or adjuvants have been used for many years to improve the host immune responses to, for example, vaccines. Thus, adjuvants have been identified that enhance the immune response to antigens.

Some of these adjuvants are toxic, however, and can cause undesirable side-effects, making them unsuitable for use in humans and many animals. Indeed, only aluminum hydroxide and aluminum phosphate (collectively commonly referred to as alum) are routinely used as adjuvants in human and veterinary vaccines.

A wide range of extrinsic adjuvants and other immunomodulating material can provoke potent immune responses to antigens. These include saponins complexed to membrane protein antigens to produce immune stimulating complexes (ISCOMS) , pluronic polymers with mineral oil, killed mycobacteria in mineral oil, Freund's complete adjuvant, bacterial products, such as muramyl dipeptide (MDP) and lipopolysaccharide (LPS) , as well as monophoryl lipid A, QS 21 and polyphosphazene.

In particular embodiments of the present invention, the non-replicating vector comprising a first nucleotide sequence encoding an G protein of RSV may be delivered in conjunction with a targeting molecule to target the vector to selected cells including cells of the immune system.

The immunogenicity of the non-replicating vector may be enhanced by coadministering plasmid DNA vectors expressing cytokines or chemokines or by coexpressing such molecules in a bis-cistronic or fusion construct.

The non-replicating vector may be delivered to the host by a variety of procedures, for example, Tang et al . (ref. 21) disclosed that introduction of gold microprojectiles coated with DNA encoding bovine growth hormone (BGH) into the skin of mice resulted in production of an i-BGH antibodies in the mice, while Furth et al . (ref. 22) showed that a jet injector could be used to transfect skin, muscle, fat and mammary tissues of living animals. 2. Iππnunoassays

The RSV G genes and vectors of the present invention are useful as immunogens for the generation of anti-G antibodies for use in immunoassays, including enzyme-linked immunosorbent assays (ELISA) , RIAs and other non-enzyme linked antibody binding assays or procedures known in the art. In ELISA assays, the non- replicating vector first is administered to a host to generate antibodies specific to the RSV G protein. These RSV G-specific antibodies are immobilized onto a selected surface, for example, a surface capable of binding the antibodies, such as the wells of a polystyrene microtiter plate. After washing to remove unadsorbed antibodies, a non-specific protein, such as a solution of bovine serum albumin (BSA) that is known to be antigenically neutral with regard to the test sample, may be bound to the selected surface. This allows for blocking of non-specific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific bindings of antisera onto the surface.

The immobilizing surface is then contacted with a sample, such as clinical or biological materials, to be tested in a manner conducive to immune complex (antigen/antibody) formation. This procedure may include diluting the sample with diluents, such as solutions of BSA, bovine gamma globulin (BGG) and/or phosphate buffered saline (PBS)/Tween. The sample is then allowed to incubate for from about 2 to 4 hours, at temperatures such as of the order of about 20° to 37°C. Following incubation, the sample-contacted surface is washed to remove non-immunocomplexed material. The washing procedure may include washing with a solution, such as PBS/Tween or a borate buffer. Following formation of specific immunocomplexes between the test sample and the bound RSV G specific antibodies, and subsequent washing, the occurrence, and even amount, of immunocomplex formation may be determined.

BIOLOGICAL MATERIALS Certain plasmids that contain the gene encoding the RSV G protein and referred to herein have been deposited with the American Type Culture Collection (ATCC) located at 12301 Parklawn Drive, Rockville, Maryland, 20852, U.S.A., pursuant to the Budapest Treaty and prior to the filing of this application. Samples of the deposited plasmids will become available to the public upon grant of a patent based upon this United States patent application and all restrictions on access to the deposits will be removed at that time. Samples of the deposited plasmids will be replaced if the depository is unable to dispense viable samples. The invention described and claimed herein is not to be limited in scope by plasmids deposited, since the deposited embodiment is intended only as an illustration of the invention. Any equivalent or similar plasmids that encode similar or equivalent antigens as described in this application are within the scope of the invention.

Plasmid ATCC Designation Date Deposited pXL5 209143 July 16, 1997 pXL6 209144 July 16, 1997

EXAMPLES The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific Examples. These Examples are described solely for purposes of illustration and are not intended to limit the scope of the invention. Changes in form and substitution of equivalents are contemplated as circumstances may suggest or render expedient . Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitations.

Methods of molecular genetics, protein biochemistry, and immunology used but not explicitly described in this disclosure and these Examples are amply reported in the scientific literature and are well within the ability of those skilled in the art. Example 1

This Example describes the construction of vectors containing the RSV G gene.

Figure 1 shows a restriction map of the gene encoding the G protein of respiratory syncytial virus and Figure 2 shows the nucleotide sequence of the gene encoding the full-length RSV G protein (SEQ ID No: 1) and the deduced amino acid sequence (SEQ ID No: 2) . Figure 3 shows the gene encoding the secreted RSV G protein (SEQ ID No: 3) and the deduced amino acid sequence (SEQ ID No: 4) .

Plasmid pXL5 (Figure 4) was prepared for the expression of the full-length RSV G protein as follows:

A recombinant Bluescript plasmid (RSV G12) containing the cDNA encoding the full-length G protein of a clinical RSV isolate (subgroup A) was used to construct vectors for RSV DNA-G immunization. RSV G12 was digested with Afllll and EcoRI and filled-in with the Klenow subunit of DNA polymerase. The resulting

1.23 kb fragment containing the coding sequence for the full-length G protein was gel-purified and ligated to

VR-1012 (Vical) (Figure 6) previously linearized with EcoRV. This procedure placed the RSV G cDNA downstream of the immediate-early cytomegalovirus (CMV) promoter and Intron A sequences of human cytomegalovirus (CMV) and upstream of the bovine growth hormone (BGH) poly-A site. The junctions of the cDNA fragments in the plasmid construct were confirmed by sequencing analysis. The resulting plasmid was designated pXL5.

Plasmid pXL6 (Figure 5) was prepared for the expression of a secretory RSV G protein as follows:

RSV G12 was digested with EcoRI, filled-in with Klenow and digested again with BamHI . The BamHI cleavage resulted in the generation of a cDNA fragment encoding a RSV G protein with N-terminal truncation. This DNA segment was gel-purified and ligated in the presence of a pair of 11 mer oligodeoxynucleotides (5 ' GATCCACTCAG 3') (SEQ ID no : 7)

3* GTGAGTCCTAG 5' (SEQ ID no : 8) to VR-1020 (Vical) previously digested with Bglll, filled in with Klenow, digested again with BamHI and gel-purified. This procedure placed the truncated RSV G cDNA (lacking the coding region for the N-terminal 91 amino acid residues including the transmembrane domain) downstream of the immediate-early CMV promoter and Intron A sequences of human CMV and upstream of the BGH poly-A site. In addition, there was the introduction of approximately 100 bp of 5' untranslated region and the coding sequence for the signal peptide of human plasminogen activator protein (Figure 7) fused in frame to the N-terminus of the RSV G protein coding sequence downstream of the CMV promoter/Intron A sequences. The junctions of the cDNA fragments in the plasmid construct were confirmed by sequencing analysis. The resulting plasmid was designated pXL6. Example 2

This Example describes the immunization of mice. Mice are susceptible to infection by RSV as described in ref. 24.

Plasmid DNA was purified through double CsCl centrifugations . For intramuscular (i.m.) immunization, tibialis anterior muscles of BALB/c mice (male, 6 to 8 week old) (Jackson Lab., Bar Harbor, ME, USA) were bilaterally injected with 2 x 50μg (lμg/μL in PBS) of either pXL5, pXL6 or V-1012. Five days prior to DNA injection, the muscles were treated with 2 x 50μL (lOμM in PBS) of cardiotoxin (Latoxan, France) to increase DNA uptake and enhance immune responses, as reported by Davis et al (ref. 23) . The animals were boosted with the same dose of plasmid DNA 6 weeks and 13 weeks later, respectively. For intradermal (i.d.) immunization, lOOμg of the plasmid DNA (2μg/μL in PBS) of were injected at the base of the tail and boosted 6 weeks and 13 weeks later, respectively. Mice in the positive control group were immunized intranasally (i.n.) with 10⁶ plaque forming units (pfu) of a clinical RSV strain of the A2 subtype grown in Hep2 cells kindly provided by Dr. B. Graham (ref. 24).

Four weeks after the third immunization, mice were challenged intranasally with 10⁶ pfu of the RSV A2 strain. Lungs were asceptically removed 4 days later, weighed and homogenized in 2 mL of complete culture medium (ref. 25) . The number of pfu in lung homogenates was determined in duplicate as previously described (ref. 26) using vaccine-quality Vero cells. Example 3

This Example describes the immunogenicity and protection by polynucleotide immunization.

Antisera obtained from immunized mice were analyzed for anti-RSV G IgG antibody titres using specific enzyme-linked immunosorbent assay (ELISA) and for RSV- specific plaque-reduction titres. ELISAs were performed using 96-well plates coated with immunoaffinity-purified RSV G protein (50 ng/mL) and 2-fold serial dilutions of immune sera. A goat anti -mouse IgG antibody conjugated to alkaline phosphatase (Jackson ImmunoRes., Mississauga, Ontario, Canada) was used as secondary antibody. Plaque reduction titres were determined according to Prince et al (ref. 26) using vaccine- quality Vero cells. Four-fold serial dilutions of immune sera were incubated with 50 pfu of the RSV Long strain (ATCC) in culture medium at 37°C for 1 hr in the presence of 5% C0₂ and the mixtures were used to infect Vero cells. Plaques were fixed with 80% methanol and developed 5 days later using a mouse anti-RSV F monoclonal IgGl antibody and donkey anti-mouse IgG antibody conjugated to peroxidase (Jackson ImmunoRes . , Mississauga, Ontario, Canada) . The RSV-specific plaque reduction titre was defined as the dilution of serum sample yielding 60% reduction in plaque number. Both ELISA and plaque reduction assays were performed in duplicate and data are expressed as the means of two determinations.

The results obtained are reproduced in Tables I and II below:

Table I. Immunogenicity of DNA-G in BALB/c Mice

Immunogen Anti-RSV G IgG Titre RSV-Specffic (Los 2(titre/100) Plaque Reduction

Titre

6 weeks 10 weeks 17 weeks (Log 2 titre) 17 weeks

VR-1012(i.m.) 0.00 + 0.00 0.00 + 0.00 0.00 + 0.00 0.00 + 0.00

pXL5 (i.m.) 3.10 ±2.77 9.70+1.06 8.60+1.17 5.40+1.65 pXL6 (i.m.) 5.78 + 1.20 9.30 + 0.82 8.89+1.54 7.26 + 0.82 pXL5 (i.d.) 1.50+1.27 8.60+1.43 8.30+1.25 7.92 + 0.59 pXL6 (i.d.) 3.70+1.25 10.30+1.06 9.44+1.24 6.92 + 0.94

RSV (i.n.) 6.83+0.41 9.67 + 0.52 9.83 + 0.41 11.80 + 0.08

Table π. Immunoprotective Ability of DNA-G in BALB/c Mice

Mean Virus Lung

Immunogen No. Mice Ηtre* (pfu/g lung) No. Fully (Log lO±SD) Protected MiceW

VR-1012 (i.m.) 6 4.81 + 0.01 0

ρXL5 (i.m.) 6 0.29 + 0.90 5 ρXL6 (i.m.) 6 0.40 + 1.20 5 pXL5 (i.d.) 6 0.30 + 1.10 5 pXL6 (i.d.) 6 0.29 + 0.90 5

RSV (i.n.). 6 0.00 + 0.00 6

* Sensitivity of the assay: 10¹"⁹⁶ pfu/g lung.

# The term, fully protected mice, refers to animals with no detectable RSV in the lungs 4 days post viral challenge.

As seen in Table I, plasmids pXL5 and pXL6 were found to be immunogenic following either i.m. or i.d. immunization producing anti-G antibodies and virus neutralizing antibodies. In addition, as seen in Table II, the plasmids pXL5 and pXL6 protected immunized mice against primary RSV infection of the lower respiratory tract . The control vector produced no immune response and did not confer protection. Example 4 This Example describes the determination of the local lung cytokine expression profile in mice immunized with pXL5 and pXL6 after RSV challenge.

BALB/c mice were immunized at 0 and 6 weeks with lOOμg of pXL5 and 6, prepared as described in Example 1, and challenged with RSV i.n. at 10 weeks. Control animals were immunized with placebo PI -RSV and live RSV and challenged with RSV according to the same protocol. In addition, animals were immunized with pXL2 , as described in copending United States Patent Application no. 08/476,397 filed June 7, 1995 (WO 96/40945) and challenged with RSV, also following the same protocol. Four days post viral challenge, lungs were removed from immunized mice and immediately frozen in liquid nitrogen. Total RNA was prepared from lungs homogenized in TRIzol/β-mercaptoethanol by chloroform extraction and isopropanol precipitation. Reverse transcriptase- polymerase chain reaction (RT-PCR) was then carried out on the RNA samples using either IL-4, IL-5 or IFN-γ specific primers from CloneTech. The amplified products were then liquid-hybridized to cytokine-specific ³²P- labeled probes from CloneTech, resolved on 5% polyacrylamide gels and quantitated by scanning of the radioactive signals in the gels. Three mouse lungs were removed from each treatment group and analyzed for lung cytokine expression for a minimum of two times. The data is presented in Figure 8 and represents the means and standard deviations of these determinations.

As may be seen from the data presented in Figure 8: 1. Immunization with live RSV intranasally (i.n.) resulted in a balanced cytokine profile (IFN-γ, IL- 4 and IL-5) , whereas that with FI-RSV intramuscularly (i.m.) resulted in a Th2 predominance (elevated IL-4 and IL-5) . These results are similar to those reported in the literature.

2. Immunization with pXL5 or pXL6 via either the i.m. or intradermal (i.d.) route gave rise to a balanced cytokine profile similar to that with live RSV immunization.

3. The magnitude of the cytokine responses with i.m. pXL6 (RSV G) and pXL2 (RSV F) immunization using the construct expressing a secretory form of the protein (SEC) is significantly higher than that with live RSV immunization.

4. The magnitude of the cytokine response with pXL5 immunization using constructs expressing a full-length membrane-associated RSV G protein (MA) and i.d. pXL6 was somewhat higher than that with live RSV immunization.

5. The balanced local cytokine response observed with DNA-G immunization contrasts with that reported by Openshaw et al (ref. 13). Using a recombinant vaccinia virus expressing the G protein, these investigators reported a local Th2 response by analysis of bronchoalveolar lavage. The results herein, which were obtained through a monogenic approach, indicate that the Th2 response is not necessarily an intrinsic property of the G protein. SUMMARY OF THE DISCLOSURE In summary of this disclosure, the present invention provides certain novel non-replicating vectors containing genes encoding RSV G proteins, methods of immunization using such vectors and methods of diagnosis using such vectors. Modifications are possible within the scope of this invention.

REFERENCES

1. Mclntosh K. , Canock, R.M. In: Fields B.N, Knipe, DM, editors. Virology. New York: Raven Press: 1990: 1045-1072

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3. Wertz GW, Sullender WM. , Biotechnology 1992; 20: 151-176

4. Murphy, B. R. et al , 1994, Virus Res. 32: 13-36.

5. Levine, S., Kleiber-France, R., and Paradiso, P.R. (1987) J. Gen. Virol. 69, 2521-2524.

6. Anderson, L.J., Hierholzer, J.C., Tsou, C, Hendry, R.M. , Fernie, B.F., Stone, Y. and Mclntosh, K.

(1985) J. Infect. Dis. 151, 626-633.

7. Johnson et al . , J. Virol 1987, 61: 3163-3166

8. Crowe, J.E., Vaccine 1995, 13: 415-421

9. Kapikian, A.Z. et al 1969, Am. J. Epidemiol. 89: 405-421.

10. Kim, H.W., et al 1969 Am. J. Epidemiol. 89: 422- 434.

11. Murphy, B.R. et al 1986 J. Clin. Microbiol . 24: 197-202.

12. Vaux-Peretz, F. et al 1992 Vaccine 10: 113-118.

13. Openshaw, P.J. 1995 Springer-Semin Immunopathol . 17: 187-201.

14. Alwan et al 1994 J. Exp. Med. 179:81-89.

15. Graham, B.S. 1995 Am. J. Respir. Crit. Care Med. 152:563-6

16. WO 90/11092

17. WO 94/21797

18. Ulmer, Current Opinion, Invest Drugs, 1993, 2: 983- 989

19. Chapman, B.S.; Thayer, R.M. ; Vincent, K.A. and Haigwood, N.L., Nucl. Acids. Res. 1991, 19: 3979- 3986. 20. Nabel, G.J. 1993, Proc . Natl . Acad. Sci . USA 90: 11307-11311.

21. Tang et al . , Nature 1992, 356: 152-154

22. Furth et al . Analytical Biochemistry, 1992, 205:

365-368

23. Davis et al . , Vaccine 1994, 12: 1503-1509

24. Graham, B.S. ; Perkins M.D.; Wright, P.F. and

Karzon, D.T. J. Mod. Virol. 1988 26: 153-162.

25. Du, R.P et al . 1994., Bio Technology 12: 813-818.

26. Prince, G.A. ϊt al, 1978. Ame. J. Pathol . 93: 771- 790.

Claims

CLAIMS What we claim is:

1. An immunogenic composition for in vivo administration to a host for the generation in the host of protective antibodies to respiratory syncytial virus (RSV) G protein, comprising a non-replicating vector comprising: a first nucleotide sequence encoding a RSV G protein or a RSV G protein fragment that generates antibodies that specifically react with RSV G protein, a promoter sequence operatively coupled to said first nucleotide sequence for expression of said RSV G protein in the host, and a second nucleotide sequence located between said first nucleotide sequence and said promoter sequence to increase expression of said RSV G protein in vivo from said vector in the host, and a pharmaceutically-acceptable carrier therefor.

2. The composition of claim 1 wherein said first nucleotide sequence encodes a full-length RSV G protein.

3. The composition of claim 2 wherein said nucleotide sequence comprises the nucleotide sequence shown in Figure 2 (SEQ ID NO:l).

4. The composition of claim 2 wherein said first nucleotide sequence comprises the nucleotide sequence encoding a full length RSV G protein having the amino acid sequence shown in Figure 2 (SEQ ID NO: 2) .

5. The composition of claim 1 wherein said first nucleotide sequence encodes a RSV G protein from which the transmembrane coding sequence and sequences upstream thereto are absent .

6. The composition of claim 5 wherein said non- replicating vector further comprises a heterologous signal peptide encoding nucleotide sequence immediately upstream of the 5' -terminus of said first nucleotide sequence .

7. The composition of claim 6 wherein said signal peptide encoding sequence encodes the signal peptide for human tissue plasminogen activator.

8. The composition of claim 5 wherein said first nucleotide sequence comprises the nucleotide sequence shown in Figure 3 (SEQ ID NO: 3) .

9. The composition of claim 5 wherein said first nucleotide sequence comprises a nucleotide sequence encoding a truncated RSV G protein having the amino acid sequence shown in Figure 3 (SEQ ID NO: 4) .

10. The composition of claim 1 wherein said promoter sequence is a immediate early cytomegalovirus promoter.

11. The composition of claim 1 wherein said second nucleotide sequence is the human cytomegalovirus Intron A.

12. The composition of claim 1 wherein the non- replicating vector is a plasmid vector.

13. The composition of claim 12 wherein the plasmid vector is pXL5 as shown in Figure 4.

14. The composition of claim 12 wherein the plasmid vector is pXL6 as shown in Figure 5.

15. A method of immunizing a host against disease caused by infection with respiratory syncytial virus (RSV) , which comprises administering to said host an effective amount of a non-replicating vector comprising: a first nucleotide sequence encoding an RSV G protein or a RSV G protein fragment that generates antibodies that specifically react with RSV G protein, a promoter sequence operatively coupled to said first nucleotide sequence for expression of said RSV G protein in the host, and a second nucleotide sequence located between said first nucleotide sequence and said promoter sequence to increase expression of said RSV G protein in vivo from said vector in the host .

16. The method of claim 15 wherein said first nucleotide sequence encodes a full-length RSV G protein.

17. The method of claim 16 wherein said nucleotide sequence comprises the nucleotide sequence shown in Figure 2 (SEQ ID NO:l) .

18. The method of claim 16 wherein said first nucleotide sequence comprises the nucleotide sequence encoding a full length RSV G protein shown in Figure 2 (SEQ ID N0:2) .

19. The method of claim 14 wherein said first nucleotide sequence encodes an RSV G protein from which the transmembrane coding sequence and sequences upstream thereto are absent .

20. The method of claim 19 wherein said non-replicating vector further comprises a heterologous signal peptide encoding nucleotide sequences immediately upstream of the 5' -terminus of said first nucleotide sequence.

21. The method of claim 20 wherein said signal peptide encoding sequence encodes the signal peptide for human tissue plasminogen activator.

22. The method of claim 19 wherein said first nucleotide sequence comprises the nucleotide sequence shown in Figure 3 (SEQ ID NO: 3) .

23. The method of claim 19 wherein said first nucleotide sequence comprises a nucleotide sequence encoding a transverse RSV G protein shown in Figure 3 (SEQ ID NO: 4) .

24. The method of claim 15 wherein said promoter sequence is an immediate early cytomegalovirus promoter.

25. The method of claim 15 wherein said second nucleotide sequence is the human cytomegalovirus Intron A.

26. The method of claim 1 wherein the non-replicating vector is a plasmid vector.

27. The method of claim 26 wherein said plasmid vector is pXL5 as shown in Figure 4.

28. The method of claim 26 wherein said vector is pXL6 as shown in Figure 5.

29. The method of claim 15 wherein a balanced Thl/Th2 immune response is induced.

30. A method of using a gene encoding a respiratory syncytial virus (RSV) G protein or a RSV G protein fragment that generates antibodies that specifically react with RSV G protein, to produce an immune response in a host , which comprises : isolating said gene, operatively linking said gene to at least one control sequence to produce a non-replicating vector, said control sequence directing expression of said RSV G protein when introduced into a host to produce an immune response to said RSV G protein, and introducing said vector into a host.

31. The method of claim 30 wherein said gene encoding an RSV G protein encodes a full length RSV G protein.

32. The method of claim 30 wherein said gene encoding an RSV G protein encodes an RSV G protein lacking the transmembrane domain and sequences upstream thereto .

33. The method of claim 32 wherein said vectgor further comprises a signal peptide encoding nucleotide sequences immediately upstream of the 5 '-terminus of said first nucleotide sequence.

34. The method of claim 33 wherein said signal peptide encoding sequence encodes the signal peptide for human tissue plasminogen activator.

35. The method of claim 30 wherein said at least one control sequence comprises the immediate early cytomegalovirus promoter.

36. The method of claim 35 including the step of: operatively linking said gene to an immunoprotection enhancing sequence to produce an enhanced immunoprotection to said RSV G protein in said host.

37. The method of claim 36 wherein said immunoprotection enhancing sequence is introduced into said vector between said control sequence and said gene.

38. The method of claim 37 wherein said immunoprotection enhancing sequence is the human cytomegalovirus Intron A.

39. The method of claim 30 wherein said gene is contained within a plasmid selected from the group consisting of pXL5 and pXL6.

40. A method of producing a vaccine for protection of a host against disease caused by infection with respiratory syncytial virus (RSV), which comprises: isolating a first nucleotide sequence encoding an RSV G protein or a RSV G protein fragment that generates antibodies that specifically react with RSV G protein, operatively linking said first nucleotide sequence to at least one control sequence to produce a non- replicating vector, the control sequence directing expression of said RSV G protein when introduced to a host to produce an immune response to said RSV G protein, operatively linking said first nucleotide sequence to a second nucleotide sequence to increase expression of said RSV G protein in vivo from the vector in the host , and formulating said vector as a vaccine for in vivo administration to a host.

41. The method of claim 40 wherein said vector is selected from group consisting of pXL5 and pXL6.

42. A vaccine produced by the method of claim 40.

43. A method of determining the presence of a respiratory syncytial virus (RSV) G protein in a sample, comprising the steps of:

(a) immunizing a host with a non-replicating vector to produce antibodies specific for the RSV G protein, said non-replicating vector comprising: a first nucleotide sequence encoding a RSV G protein or a RSV G protein fragment that generates antibodies that specifically react with RSV G protein, a promoter sequence operatively coupled to said first nucleotide sequence for expression of said RSV G protein in the host, and a second nucleotide sequence located between said first nucleotide sequence and said promoter sequence to increase expression of said RSV G protein in vivo from said vector in the host,

(b) isolating the RSV G protein specific antibodies;

(c) contacting the sample with the isolated antibodies to produce complexes comprising any RSV G protein present in a sample and said isolated RSV G protein-specific antibodies; and

(d) determining the production of the complexes.

44. The method of claim 43 wherein said vector is selected from the group consisting of pXL5 and pXL6.

45. A diagnostic kit for detecting the presence of a respiratory syncytial virus (RSV) G protein in a sample, comprising:

(a) a non-replicating vector capable of generating antibodies specific for the RSV G protein when administered to a host, the non- replicating vector comprising: a first nucleotide sequence encoding a RSV G protein or a RSV G protein fragment that generates antibodies that specifically react with RSV G protein, a promoter sequence operatively coupled to said first nucleotide sequence for expression of said RSV G protein in the host, and a second nucleotide sequence located between said first nucleotide sequence and said promoter sequence to increase expression of said RSV G protein in vivo from said vector in the host;

(b) isolation means to isolate said RSV G protein- protein-specific antibodies;

(c) contacting means to contact the isolated RSV G specific antibodies with the sample to produce a complex comprising any RSV G protein in the sample and RSV G protein specific antibodies, and

(d) identifying to determine production of the complex.

46. The diagnostic kit of claim 45 wherein said vector is selected from the group consisting of pXL5 and pX 6.

47. A method for producing antibodies specific for a G protein of respiratory syncytial virus (RSV) comprising:

(b) isolating the RSV G-specific antibodies from the host .

48. A method of producing monoclonal antibodies specific for a G protein of respiratory syncytial virus (RSV) comprising the steps of:

(a) constructing a vector comprising: a first nucleotide sequence encoding a RSV G protein or a RSV G protein fragment that generates antibodies that specifically react with RSV G protein, a promoter sequence operatively coupled to said first nucleotide sequence for expression of said RSV G protein in the host, and a second nucleotide sequence located between said first nucleotide sequence and said promoter sequence to increase expression of said RSV G protein in vivo from said vector in the host;

(b) administering the vector to at least one mouse to produce at least one immunized mouse;

(c) removing B-lymphocytes from the at least one immunized mouse;

(e) cloning the hybridomas;

(f) selecting clones which produce anti-RSV G protein antibody;

(g) culturing the anti-RSV G protein antibody- producing clones; and then

(h) isolating anti-RSV G protein antibodies from the cultures .