Patent 6,541,459

The invention is directed to a herpes simplex virus vaccine comprising a herpes simplex virus glycoprotein H-glycoprotein L complex. The invention is also directed to a vaccine comprising a DNA encoding a herpes simplex virus glycoprotein H-glycoprotein L complex. Also included is an antibody which specifically binds to a herpes simplex virus glycoprotein H-glycoprotein L complex and DNA encoding the same.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to the discovery that two HSV-1 specific glycoproteins, gH and gL, when complexed together and administered to animal, serve to protect the animal against infection by HSV. Thus, there has been discovered a subunit vaccine comprising a soluble HSV-1 gHt-gL complex, which vaccine is useful not only as a prophylactic therapeutic agent for protection of an animal against a herpesvirus infection, but is also useful as a therapeutic agent for treatment of an ongoing herpesvirus infection in an animal, particularly in an animal having a high propensity to reactivate a herpesvirus infection.

It is known that HSV-1 gH and gL form a molecular complex which is present on the virion envelope. This complex is essential for viral infectivity in that it is required for entry of virus into cells and for cell to cell spread of virus which is believed to occur via membrane fusion. In the experiments described herein, gH and gL have been stably expressed in and secreted from mammalian cells in culture as a soluble complex, named gHt-gL. This complex, when inoculated into an animal, elicits antibody which serves to neutralize virus in a virus neutralization cell culture assay. Further, when the gHt-gL complex is inoculated into an animal, it elicits an immune response which serves to protect the inoculated animal against disease when the animal is challenged with infectious virus. Thus, it has been discovered according to the present invention that a soluble herpes simplex virus type 1 gHt-gL complex functions to vaccinate an animal against herpes simplex virus disease.

By the term "soluble gHt-gL complex" as used herein, is meant a complex comprising a truncated HSV gH and a substantially full length HSV gL which are bound together in the complex and which are soluble in an aqueous solution.

The soluble gHt-gL complex of the invention may be obtained in large quantities for use as a vaccine for protection of humans against HSV infection, or for eliminating or diminishing the frequency of reactivation of the virus from the latent state thus, reducing the severity of recurrent HSV infection in humans. The complex is also useful as a diagnostic reagent for assessing the presence or absence of a herpesvirus infection in a human. Such an assessment is made by obtaining serum from the individual and reacting it with the complex in a standard immunoassay such as radioimmunoassay or enzyme linked immunoadsorbent assay (ELISA).

By the term "vaccine" as used herein, is meant a composition, a protein complex or a DNA encoding a protein complex which serves to protect an animal against a herpesvirus disease.

By the term "immunizing a human against herpes simplex virus infection" is meant administering to the human a composition, a protein complex, a DNA encoding a protein complex, an antibody or a DNA encoding an antibody, which elicits an immune response in the human which immune response provides protection to the human against a herpes simplex virus disease.

Homologs of the genes encoding HSV-1 gH and gL have been identified in most other herpesviruses including human CMV (Cranage et al., 1988, J. Virol. 62:1416), VZV (Davison and Scott, 1986, J. Gen. Virol. 67:1759) and EBV (McGeoch and Davison, 1986, Nucl. Acids Res. 4:4281). The CMV UL115 gene, a positional homolog of the HSV-1 gL gene, encodes a secreted protein which forms a complex with CMV gH and is therefore a positional and likely functional (although not a sequence) homolog to HSV-1 gL (Kaye et al., 1992, J. Gen. Virol. 73:2693; Spaete et al., 1993, Virology 193:853). HHV-6 (Josephs et al., 1991, J. Virol. 65:5597), pseudorabies virus (Klupp et al., 1991, Virology 182:732) and herpesvirus saimiri (Gompels et al., 1988, J. Gen. Virol. 69:2819) also each encode homologs of HSV-1 gH and gL.

The invention should not be construed to be limited to a soluble HSV-1 gHt-gL complex. Rather, the invention should be construed to encompass soluble gHt-gL complexes which are derived from both HSV-1 and HSV-2, which soluble complexes may be used as vaccines to protect humans from disease caused by either of these two types of viruses. As the data presented herein establish, antibody directed against soluble HSV-1 gHt-gL complex serves to neutralize infection of cells in culture by HSV-2. Thus, since antibodies raised against HSV-1 gHt-gL complex neutralize HSV-2, the invention should be construed to include gHt-gL complexes from either virus type which serve to protect cells and humans against infection by both HSV-1 and HSV-2.

The gHt-gL complex of the invention may therefore comprise one subunit derived from HSV-1 and another subunit derived from HSV-2, yielding at least four general classes of complexes which are encompassed by the invention. One complex comprises HSV-1 gH bound to HSV-1 gL. Another complex comprises HSV-1 gH bound to HSV-2 gL. A third complex comprises HSV-2 gH bound to HSV-1 gL and a fourth complex comprises HSV-2 gH bound to HSV-2 gL.

The gHt-gL complex of the invention comprises a truncated gH molecule which is complexed to a substantially full length gL molecule. It has been discovered in the present invention that it is necessary that the gH portion of the gHt-gL complex be truncated in order that the complex is secreted from the cell in soluble form. Truncated forms of gH (referred to herein as "gHt") include those containing amino acid residues selected from the group consisting of 1-792, 1-648, 1-475, 1-324 and 1-275.

As is customary in the field of herpes simplex virology, amino acids in proteins encoded by herpes simplex viruses are numbered from the first methionine in the protein.

By the term "truncated" as used herein as it refers to gH, is meant a molecule of gH which contains less than the complete number of amino acids found in a wild type protein. Particularly, the term truncated is used to mean a gH molecule which is not membrane anchored, i.e., which comprises a deletion or other mutation which facilitates secretion of gH from the cell. Mutations in the gH molecule which give rise to different lengths of gH may comprise insertion, deletion or point mutations. An insertion mutation is one where additional base pairs are inserted into a DNA molecule. A deletion mutation is one where base pairs have been removed from a DNA molecule. A point mutation is one where a single base pair alteration has been made in a DNA molecule. Each of these mutations is designed such that creation of any one of them in a DNA molecule effects an alteration in the nature of any polypeptide expressed by that DNA, which alteration results in a gH molecule capable of binding to gL to form a complex having biological activity as defined herein, and which gH-gL complex is secreted from a cell in which it is expressed.

The complex also includes a substantially full length gL molecule which may comprise all of the amino acids of gL, or may also be mutated comprise less than all of the amino acids of gL.

By the term "substantially full length herpesvirus gL" as used herein, is meant a herpesvirus gL molecule which comprises a sufficient number of amino acids so that the substantially full length gL is capable of binding to gHt, forming a complex therewith, which complex has biological activity as defined herein. Thus, a substantially full length gL molecule does not necessarily contain all of the amino acids which comprise herpesvirus gL, (although according to the invention, it may) but rather, the molecule comprises a substantial portion of the molecule sufficient for binding to gHt and forming a biologically active complex therewith.

Referring to gH and gL molecules encoded by HSV-1, it has been discovered in the present invention that a stable gHt-gL complex can be formed wherein the gHt component comprises amino acids 1-324 and the gL component is substantially fill length. A stable gHt-gL complex can also be formed wherein the gHt component comprises amino acids amino acids 1-792 and the gL component comprises amino acids 1-168. Thus, the invention should not be construed to be limited to any particular specific length of either gH or gL. Rather, the invention should be construed to encompass any length of a truncated gH which binds to any length of gL to form a complex which has gHt-gL biological activity as defined herein. The procedures which are used to generate plasmids expressing proteins of different lengths are well known in the art and the means for expressing gH and gL in a cell such that they form a biologically active complex are described in detail herein. Thus, it is well within the skill of those in the art to generate biologically active gHt-gL complexes, wherein the gHt-and gL each comprise an animo acid length which is different from the gHt and gL molecules disclosed in the experimental examples section herein.

The invention should also be construed to include any form of a gHt-gL complex having substantial homology to the HSV-1 gHt-gL complex disclosed herein. Preferably, a gHt-gL complex which is substantially homologous is about 50% homologous, more preferably about 70% homologous, even more preferably about 80% homologous and most preferably about 90% homologous to the gHt-gL complex secreted from HL-7 cells.

"Homologous" as used herein, refers to the subunit sequence similarity between two polymeric molecules, e.g., between two nucleic acid molecules, e.g., two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit position in both of the two molecules is occupied by the same monomeric subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous at that position. The homology between two sequences is a direct function of the number of matching or homologous positions, e.g., if half (e.g., five positions in a polymer ten subunits in length) of the positions in two compound sequences are homologous then the two sequences are 50% homologous, if 90% of the positions, e.g., 9 of 10, are matched or homologous, the two sequences share 90% homology. By way of example, the DNA sequences 3'ATTGCC 5' and 3'TAAGCC 5' share 50% homology. Also by way of example, the amino acid sequences CTAGYR and CTACRY (SEQ ID Nos: 1 and 2, respectively) share 50% homology.

To generate an HSV gHt-gL complex, following the teaching provided herein it is well within the skill of those in the art to take a plasmid encoding truncated gH and full length gL from other strains of HSV-1 and introduce it into a population of mammalian cells such that the cells become stably transfected with the plasmid and are caused to express and secrete a soluble form of gHt-gL complex as described herein. It is also well within the skill of those in the art to take yet another plasr nid encoding gH and gL (i.e., DNA obtained from various strains of HSV-2, which DNA encodes gH and gL) and generate cell lines which secrete soluble gHt-gL complex following the teaching contained herein.

The invention should not be construed to be limited to the particular method of introduction of herpesvirus DNA into mammalian cells described herein. Rather, other methods may be used to generate cells which express a soluble form of gH-gL complex. Such methods include, but are not limited to, the use of retroviral and other viral vectors for delivery of herpesvirus-specific gH-gL encoding DNA into cells and the use of other chemical means of transfection. In addition as described herein, the complex to be formed by cells encoding gH-gL may include a mixture of gH derived from one virus strain and gL derived from yet another virus strain. Generation of such mixed complexes is accomplished using the protocols described above and other protocols available to virologists, described for example in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y.).

The invention should also be construed to include gHt-gL complexes which are generated in mammalian cells as described herein, or which are generated by other means, such as by expression in a baculovirus system, or a yeast expression system. gHt-gL complexes which are generated by synthetic methods are also included in the invention.

Also contemplated by the invention is a subunit vaccine comprising an isolated nucleic acid, preferably, an isolated DNA, encoding a gHt-gL complex. Such a nucleic acid, preferably, a DNA molecule, may be used directly as a vaccine as described herein, or it may be used to transfect cells in order to produce large quantities of gHt-gL for use as a subunit vaccine.

To generate a DNA encoding gHt-gL, the desired gHt and gL coding sequences are ligated together in either of two configurations. In the first configuration, a plasmid is generated having the following elements: a promoter for expression of gHt which is positioned upstream of a desired gHt encoding sequence and a promoter for expression of gL which is positioned upstream of a desired gL coding sequence. The plasmid therefore encodes gHt and gL on the same molecule wherein expression of each of gHt and gL is under the control of an individual promoter sequence, preferably the same promoter sequence. Both gHt and gL are expressed individually from:this plasmid in a cell and form complex therein which is secreted from the cells as described herein.

Alternatively, a plasmid may be generated which has the following elements: a single a promoter which is positioned upstream of a desired gHt encoding and a desired gL encoding sequence, the gHt and gL encoding sequences being separated by a DNA sequence encoding a cleavage site for a protease. In this plasmid, the gHt and gL encoding sequences may be positioned in the plasmid in either orientation which respect to each other, such that either one of them is juxtaposed to the promoter sequence. DNA encoding the protease cleavage site which is positioned between the gHt and gL coding sequences may be any DNA known to encode a length of amino acids which are cleaved by any protease which is present in a majority of cells and which is particularly present in cells into which the DNA of the invention is introduced. gHt-gL which is expressed by this plasmid is initially expressed in a cell as a single length of protein comprising gHt and gL fused together via a protease cleavage site. Subsequent cleavage of the fused protein by a protease generates individual molecules of gHt and gL which form a complex which is secreted from the cell as desccribed herein.

The isolated DNA of the invention is not limited to a plasmid based DNA, but rather may include any form of DNA which encodes gHt-gL as described herein in the case of a plasmid DNA. Thus, the isolated DNA of the invention may include a viral vector, a non-viral vector, or a plasmid DNA.

The promoter sequence which is used to drive expression of gHt-gL in either type of configuration may be any constitutive promoter which drives expression of these proteins in cells. Such promoters therefore include, but are not limited to, the cytomegalovirus immediate early promoter/regulatory sequence, the SV40 early promoter/enhancer sequence, the Rous sarcoma virus promoter/enhancer and any other suitable promoter which is available in the art for constitutive expression of high levels of proteins in cells.

When the isolated DNA of the invention is used to generate large quantities of gHt-gL complex, cells are transfected with the DNA using the methodology disclosed herein or any other available transfection methodology, gHt-gL is expressed and is recovered from the cells as described herein.

When the isolated DNA is to be used as a vaccine, a DNA based vaccine is prepared following the disclosure described in Wang et al. (1993, Proc. Natl. Acad. Sci. USA 90:4156-4160). The vaccine comprises DNA encoding a gHt and a substantially fill length gL expressed under the control of any of the promoters disclosed herein. Antibodies are raised against the expressed protein by intramuscular injection of DNA into the hind limb of six to eight week old mice. The anesthetic bupivacaine (50 .mu.l of a 0.5% solution) is used to improve immunogenicity of the vaccine. The animals are immunized first with bupivacaine and then are immunized the following day with 50 .mu.g of plasmid DNA encoding gHt-gL. At about four weeks, animals are test bled to measure the level of anti-gHt-gL antibody and are re-injected with bupivacaine and DNA on successive days. On day 45, or thereabouts, serum is collected from the animals and is tested to determine whether antibodies contained therein neutralize virus in the virus neutralization assays described herein. DNA encoding other HSV glycoproteins such as, but not limited to gD, gC and gB may included for immunization of the animal using the same protocol.

To adapt this DNA based vaccine to human subjects, the amounts of DNA, the route of injection and the adjuvants to be used may vary from that just described. However, these variations will be readily apparent to the skilled artisan working in the field of DNA based vaccines.

The invention should be construed to include any and all isolated DNAs which are homologous to the gHt-gL DNA described and referenced herein, provided these homologous DNAs have the biological activity of gHt-gL complex as defined herein.

An "isolated DNA", as used herein, refers to a DNA sequence, segment, or fragment which has been purified from the sequences which flank it in a naturally occurring state, e.g., a DNA fragment which has been removed from the sequences which are normally adjacent to the fragment, e.g., the sequences adjacent to the fragment in a genome in which it naturally occurs. The term also applies to DNA which has been substantially purified from other components which naturally accompany the DNA, e.g., RNA or DNA or proteins which naturally accompany it in the cell.

The invention should also be construed to include DNAs which are substantially homologous to the DNA isolated according to the method of the invention. Preferably, DNA which is substantially homologous is about 50% homologous, more preferably about 70% ho mnologous, even more preferably about 80% homologous and most preferably about 90% homologous to DNA obtained using the method of the invention.

The invention should therefore be construed to include any form of a gHt-gL complex or DNA encoding a gHt-gL complex, which is homologous to the HSV-1 gHt-gL complex or it's DNA disclosed herein and which has or encodes gHt-gL complex biological activity as defined herein.

To purify a gHt-gL complex for use as a vaccine or other therapeutic, the examples given in the experimental details section may be followed. Essentially, a substantially pure preparation of a gHt-gL complex is obtained by immunoaffinity chromatography of supernatants obtained from cells which express and secrete gHt-gL complex using the monoclonal antibody (MAb), 53S or any other antibody which specifically binds gH, gL or the combination of the two. To purify the gH-gL complex, the supernatant is passed over an affinity column comprising anti-gHt-gL complex antibody, the column is washed with buffer and adsorbed proteins are eluted from the column in fractions using an elution buffer, such as 50 mM glycine buffer (pH 2.5) containing 0.5 M NaCl and 0.1% Triton X-100. Fractions so elute a are neutralized with a high pH buffer, for example, Tris-HCl, pH 9.0 and are then analyzed for the presence of gHt and gL by gel electrophoresis or other protein detection technology. Fractions containing the proteins are pooled and are concentrated using a commercially available concentrator, for example, a Centricon-10 concentrator.

By the term "substantially pure" as it refers to a gH-gL complex, is meant a complex which has been separated from the components which naturally accompany it in the cell or medium in which it resides. Typically, a compound is substantially pure when at least 10%, more preferably at least 20%, more preferably at least 50%, more preferably at least 60%, more preferably at least 75%, more preferably at least 90%, and most preferably at least 99% of the total material (by volume, by wet or dry weight, or by mole percent or mole fraction) in a sample is the compound of interest. Purity can be measured by any appropriate method, e.g, in the case of polypeptides by column chromatography, gel electrophoresis or HPLC analysis. A compound, e.g., a protein, is also substantially purified when it is essentially free of naturally associated components or when it is separated from the native contaminants which accompany it in its natural state.

The invention should be construed to include modifications of gHt or gL which in their modified form are capable of forming a complex having the biological activity of the gHt-gL complex disclosed herein. For example, conservative amino acid substitutions may be made in either or both of gHt or gL which alter the primary sequence of the proteins without significantly affecting the ability of these proteins to bind together and retain the biological activity of the gHt-gL complex. Conservative amino acid substitutions typically include substitutions within the following groups, but are not limited to these groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; phenylalanine, tyrosine. Also included are proteins and peptides which have been modified using ordinary molecular biological techniques so as to improve their resistance to proteolytic degradation, to optimize solubility properties, or to alter post-translational modification of the protein or peptide. The proteins and peptides of the invention are not limited to products of any of the specific exemplary processes listed herein.

By "biological activity" of gHt-gL as used herein, is meant a gHt-gL complex which when inoculated into an animal elicits an antibody, a virus neutralizing antibody, which neutralizes the infectivity of a herpesvirus in a virus neutralization assay and which protects an animal against disease when wild type virus is subsequently administered to the animal.

Typically, a virus neutralization assay involves incubation with a known titer of infectious virus of serial dilutions of serum obtained from an animal administered the vaccine for a period of time. Following the incubation period, the amount of infectious virus remaining is quantitated, usually by plaque assay.

The term "virus neutralizing effective amount" as used herein, means an amount of antigen which elicits an immune response when administered to an animal, which response is capable of neutralizing virus infectivity to a level which is less than 50% of normal infectivity in a standard virus neutralization assay.

A virus neutralizing immune response is also one which affords protection to the animal from lethal challenge with wild type virus. Protection against lethal challenge with wild type virus is typically assessed by first immunizing a series of animals with the subject antigen to generate serum capable of neutralizing virus infectivity in a standard virus neutralization assay. The animals are then inoculated with a serial dilutions of wild type virus, which dilutions contain sufficient virus to kill non-immunized animals. The death rate of the animals is quantitated and is compared to the level of the virus neutralizing immune response in each of the animals. Protection from lethal challenge has been effected when non-immunized animals die and immunized animals do not die as a result of infection with virus.

Also included in the term "virus neutralizing immune response" is a response which affords protection against HSV disease, such as zosteriform disease, the establishment of latency in ganglia that innervate the site of infection and the production of subclinical (asymptomatic) virus shedding. Protection against HSV disease is typically assessed by counting HSV lesions that develop at the site of virus challenge, or by scoring animals for erythema (redness) and swelling. In the zosteriform model, disease is scored as primary, i.e., at the site of infection, or secondary, ie., at a site remote from the initial site of infection but along the axis of the same dermatome.

Protection against the establishment of latency is typically assessed by removing the ganglia and determining the presence (and amount) or absence of HSV in the ganglia. Protection against subclinical virus shedding is typically determined by culturing virus from the animal at selected intervals post-challenge or by using standard PCR to measure levels of viral DNA.

By the term "virus neutralizing antibody" as used herein, is meant a reduction in the infectivity of a virus in the presence of the antibody compared with the infectivity of the virus in the absence of the antibody. Typically, an antibody is a virus neutralizing antibody when the infectivity of the virus is reduced by about 50% in the presence of the antibody at a dilution of the serum containing the antibody which is greater than 1:20. The higher the dilution of serum which neutralizes a constant amount of virus by 50%, the greater the estimate of the activity of the antibody contained within the serum.

The term "protect an animal against disease" is used herein to mean a reduction in the level of disease caused by a wild type virus in an animal inoculated with a gHt-gL complex compared with the level of disease caused by a wild type virus in an animal which as not been inoculated with a gHt-gL complex. As the data presented herein establish, the gHt-gL complex of the invention protected an animal against infection by HSV to the same extent as did gD when animals were similarly administered this glycoprotein. As noted herein, HSV gD as a subunit vaccine is currently in clinical trials and at present represents the "gold standard" of HSV vaccines. Thus, the gHt-gL subunit vaccine of the invention is capable of protecting an animal against HSV disease to a level at least as good as that observed when gD is used to immunize an animal.

To determine whether a gHt-gL complex generated using the methods described herein has biological activity, the following general protocols are followed. To assess biological activity of a gHt-gL complex, an animal is first immunized with the complex. Although the examples provided herein are directed to rabbits and mice, any other animal may be used. .

Using mice as an example, a mouse is immunized at about biweekly intervals with about four doses of approximately 10 .mu.g of gHt-gL per dose. Serum obtained from the mouse post-immunization is tested for the presence of an anti-gHt-gL complex antibody in any immunological assay, for example, an ELISA. A virus neutralization assay is performed wherein dilutions of serum obtained from the immunized animal are mixed with infectious virus. The mixture is added to cells and neutralization of virus by the antibody is measured as described herein.

To determine the efficacy of the gH-gL complex as a vaccine, the gH-gL complex is administered intraperitoneally to mice using an adjuvant system suitable for administration of proteins to mice, for example, the Ribi adjuvant system (RAS; Ribi Immunochemical Research, Hamnilton, Mont.), or other suitable adjuvant. Both pre- and post-immune serum is obtained from the mice and the presence or absence of antibodies is determined in the standard assays described herein. The ability of anti-gHt-gL antibodies to neutralize HSV is determined in a standard viral neutralization assay, such as but not limited to, a plaque reduction neutralization assay. Mice are administered a range of concentrations of gH-gL complex from about 0.1 to about 20 .mu.g per dose, using several different immunization schedules, i.e., weekly, biweekly, in order to determine the optimum conditions for effective immunization of the mice against HSV. Sera obtained from mice so immunized are tested for the ability to neutralize HSV-1 strain NS (or other strain of HSV depending on the virus from which the gHt-gL complex is derived) and other strains of both HSV-1 and HSV-2. Since the ability of an antibody to neutralize virus in culture is predictive of the protective activity of that antibody, neutralization of any one of the viruses listed above by antibody raised against the gHt-gL complex is predictive of the ability of gHt-gL complex to serve as a subunit vaccine candidate against that virus.

To assess whether antibody raised against gH-gL protects mice against in vivo challenge with virus, immunized and non-immunized mice are administered various concentrations of virus intraperitoneally at a time post-immunization when peak antibody levels are apparent following the experiments described above. The number of immunized animals which survive challenge by virus is indicative of the efficacy of the gHt-gL complex as a vaccine candidate. Although these studies may be conducted using an intraperitoneal route, studies on the vaccine capabilities of a gHt-gL complex may involve all possible routes of administration including, but not limited to, intramuscular, subcutaneous and even oral routes of administration. In addition, as described in the experimental details section herein, other animal models for herpesvirus infections, such as guinea pigs are used.

Furthermore, studies may be conducted to examine viral latency in gH-gL immunized animals surviving virus challenge and in animals which are administered the complex and are then tested in any of the available latency models of HSV infection. Such studies will be performed according to published protocols, such as that described by Stanberry (Pathogenesis of herpes simplex virus infection and animal models for its study. In: Current Topics in Microbiology and Immunology, 179: Herpes simplex virus: Pathogenesis and Control, Springer Verlag (Berlin), 1992, pp 15-30). Thus, the establishment of latency in ganglia that innervate the site of infection and the production of subclinical (asymptomatic) virus shedding may be examined as described herein.

The subunit vaccine of the invention may be formulated to be suspended in a pharmaceutically acceptable composition suitable for use in animals and in particular, in humans. Such formulations include the use of adjuvants: such as muramyl dipeptide derivatives (MDP) or analogs which are described in U.S. Pat. Nos. 4,082,735; 4,082,736; 4,101,536; 4,185,089; 4,235,771; and, 4,406,890. Other adjuvants which are useful include alum (Pierce Chemical Co.), lipid A, trehalose dimycolate and dimethyldioctadecylammonium bromide (DDA), Freund's adjuvant, and IL-12. Other components may include a polyoxypropylene-polyoxyethylene block polymer (Pluronic.RTM.), a non-ionic surfactant, and a metabolizable oil such as squalene (U.S. Pat. No. 4,606,918).

The subunit vaccine of the invention may be encapsulated into liposomes for administration to the animal. See for example, U.S. Pat. Nos. 4,053,585, 4,261,975 and 4,406,890.

The subunit vaccine of the invention is administered to a human by any suitable route of administration, for example, subcutaneously, intramuscularly, orally, intravenously, intradermally, intranasally or intravaginally. The complex is first suspended in a pharmaceutically acceptable carrier which is suitable for the chosen route of administration and which will be readily apparent to those skilled in the art of vaccine preparation and administration. The dose of vaccine to be used may vary dependent upon any number of factors including the age of the individual and the route of administration. Typically, the subunit vaccine is administered in a range of 1 .mu.g to 50 mg of protein per dose. Approximately 1-10 doses are administered to the individual at intervals ranging from once per day to once per week to once every few years.

The vaccine of the invention is useful for prevention of herpesvirus disease in an animal, preferably a human. However, the vaccine is also useful as a therapeutic agent for treatment of acute episodes of herpesvirus infection in order to boost the immune response in the animal. Thus the invention contemplates both prophylactic and therapeutic uses for the vaccine of the invention.

It should be appreciated that the subunit vaccine of the invention may be combined with other subunit vaccines, such as subunit vaccines comprising gD, gB or combinations thereof, gC, and the like each of which may be generated and used according to published protocols and the procedures described herein.

The antibodies which are produced in animals may themselves serve as therapeutic compounds for treatment of HSV infection, particularly in severely immunocompromised individuals, such as those infected with human immunodeficiency virus or those receiving transplants. The antibody may also be useful for administration to newborn infants infected with HSV and to adults at risk for developing HSV encephalitis. The invention should therefore be construed to include anti-gHt-gL antibodies as described herein and anti-gHt-gL antibodies which may be modified such that they are phage displayed and/or humanized using technology available in the art. It will be appreciated that the antibodies which are useful include those which specifically bind to a gHt-gL complex derived from either HSV-1 or HSV-2 and a gHt-gL complex wherein one component id derived from HSV-1 and the other component is derived from HSV-2. Similarly, DNA encoding antibodies which are now described may comprises DNAs encoding gHt and gL subunits derived from either of HSV-1 or HSV-2.

The generation of polyclonal and monoclonal antibodies is well known in the art and is described and referenced herein. Phage displayed and humanized antibodies are also well known in the art and are also described herein.

Given the advances in technology in cloning DNA encoding proteins comprising antibodies, the invention should also be construed to include an isolated DNA which encodes a gHt-gL antibody, or a portion or fragment of such antibody.

When the antibody of the invention is a monoclonal antibody, the nucleic acid encoding the antibody may be cloned and sequenced using technology which is available in the art, and is described, for example, in Wright et al. (1992, Critical Rev. in Immunol. 12(3,4):125-168) and the references cited therein. Further, the antibody of the invention may be "humanized" using the technology described in Wright et al., (supra) and in the references cited therein.

For example, to generate a phage antibody library, a cDNA library is first obtained from mRNA which is isolated from cells, e.g., the hybridoma, which express the desired protein to be expressed on the phage surface, e.g., the desired antibody. cDNA copies of the mRNA are produced using reverse transcriptase. cDNA which specifies immunoglobulin fragments are obtained by PCR and the resulting DNA is cloned into a suitable bacteriophage vector to generate a bacteriophage DNA library comprising DNA specifying immunoglobulin genes. The procedures for making a bacteriophage library comprising heterologous DNA are well known in the art and are described, for example, in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y.).

Bacteriophage which encode the desired antibody, may be engineered such that the protein is displayed on the surface thereof in such a manner that it is available for binding to its corresponding binding protein, e.g., the antigen against which the antibody is directed. Thus, when bacteriophage which express a specific antibody are incubated in the presence of a cell which expresses the corresponding antigen, the bacteriophage will bind to the cell. Bacteriophage which do not express the antibody will not bind to the cell. Such panning techniques are well known in the art and are described for example, in Wright et al., (supra).

Processes such as those described above, have been developed for the production of human antibodies using M13 bacteriophage display (Burton et al., 1994, Adv. Immunol. 57:191-280). Essentially, a cDNA library is generated from mRNA obtained from a population of antibody-producing cells. The mRNA encodes rearranged immunoglobulin genes and thus, the cDNA encodes the same. Amplified cDNA is cloned into M13 expression vectors creating a library of phage which express human Fab fragments on their surface. Phage which display the antibody of interest are selected by antigen binding and are propagated in bacteria to produce soluble human Fab immunoglobulin. Thus, in contrast to conventional monoclonal antibody synthesis, this procedure immortalizes DNA encoding human immunoglobulin rather than cells which express human immunoglobulin.

By the term "synthetic antibody" as used herein, is meant an antibody which is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage as described herein. The term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence technology which is available and well known in the art.

By the term "specifically binds," as used herein, is meant an antibody which recognizes and binds an HSV gHt-gL complex, but does not substantially recognize or bind other molecules in a sample.

The invention thus includes an isolated DNA encoding a gHt-gL antibody or a portion of the antibody of the invention. To isolate DNA encoding an antibody, for example, DNA is extracted from antibody expressing phage obtained according to the methods of the invention. Such extraction techniques are well known in the art and are described, for example, in Sambrook et al. (supra).

The anti-gHt-gL complex antibody of the invention may be conventionally administered to a mammal, preferably a human, parenterally, by injection, for example, subcutaneously, intravenously, intramuscularly, and the like. Additional formulations which are suitable for other modes of administration include suppositories, intranasal aerosols and, in some cases, oral formulations. The antibody may be administered in any of the described formulations either daily, several times daily, weekly, bi-weekly or monthly or several times a year in a dosage which will be apparent to the skilled artisan and will depend on the type of disease being treated. Preferably, the dosage will range from about 1 nanogram of antibody to several milligrams of antibody to even up to about 100 milligrams of antibody per dose.

It will be appreciated that the subunit vaccine of the invention, the DNA vaccine of the invention and the antibody of the invention may be used to prevent or treat HSV infections in a human in cases where the human is not yet infected, in cases where the human is infected and treatment is initiated in order to prevent more severe infection, such as, for example, HSV encephalitis, and in cases where the human is latently infected with the virus and has a high propensity to reactivate. In addition, the compositions of the invention are useful for treatment of neonates at risk for developing severe herpesvirus infection and immunosuppressed individuals at risk for developing severe herpes virus infection, such as is the case in patients having acquired immunodeficiency syndrome and in transplant patients and those requiring chemotherapy.

Claim 1 of 9 Claims

What is claimed is:

1. A vaccine comprising an isolated DNA encoding a soluble herpes simplex virus gHt-gL complex suspended in a pharmaceutically acceptable carrier.

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