Abstract:
Protective and therapeutic vaccines are disclosed. Vaccines for preventing or treating herpes simplex virus infection are disclosed. Methods for preventing herpes simplex virus infection and treating individuals who have been infected with herpes simplex virus and to compositions for and methods of making prophylactic and therapeutic vaccines for herpes simplex virus are disclosed.

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     This application claims the benefit of U.S. Provisional Application Ser. No. 60/028,754, filed Oct. 23, 1996 (now abandoned), and U.S. Provisional Application Ser. No. 60/053,206, filed Jul. 21, 1997 (now abandoned), which are both incorporated herein by reference. 
    
    
     FIELD OF THE INVENTION 
     The present invention relates to protective and therapeutic vaccines including vaccines for herpes simplex virus, to methods for preventing herpes simplex virus infection and treating individuals who have been infected with herpes simplex virus and to compositions for and methods of making prophylactic and therapeutic vaccines for herpes simplex virus. 
     BACKGROUND OF THE INVENTION 
     Herpes simplex virus (HSV), which includes both herpes simplex virus 1 (HSV1) and herpes simplex virus 2 (HSV2), presents a serious health concern to those infected by the virus as well as to uninfected members of the population. A great deal of effort has been expended to identify effective therapeutic compositions and methods to alleviate symptoms and reduce or eliminate viral flair-ups in which the dormant virus becomes active and presents itself as sores on the genital or oral tissues. In addition, vaccines are under development to prevent infection in uninfected individuals. One type of vaccine under development is a subunit vaccine containing purified glycoprotein D (gD). The gD protein may be derived from HSV-1 (gD-1) or HSV-2 (gD-2). 
     While such therapeutic compositions and vaccines may offer some benefits, there remains a need for effective compositions and methods of immunizing individuals prophylactically against HSV infection and methods of treating HSV infected individuals. There is a need for compositions and methods for making such prophylactics and therapeutics. 
     SUMMARY OF THE INVENTION 
     The present invention relates to isolated herpes simplex virus genes including HSV2 gD2 and to modified forms of that gene. The modified forms of HSV2 gD2 include those which lack a functional transmembrane region and/or a functional signal peptide. 
     The present invention relates to plasmids which comprise nucleotide sequences that encode either HSV2 gD2 or a modified version of HSV gD2 operably linked to regulatory elements necessary for expression in eukaryotic cells. 
     The present invention relates to a method of inducing, in an individual, an immune response against HSV2 gD2 comprising the step of administering to an individual, a plasmid which comprises a nucleotide sequence that encodes HSV2 gD2 or a modified form of HSV gD2 operably linked to regulatory elements necessary for expression in eukaryotic cells. 
     The present invention relates to a method of treating an individual who is infected with HSV comprising the step of administering to the individual, a plasmid which comprises a nucleotide sequence that encodes HSV2 gD2 or a modified form of HSV gD2 operably linked to regulatory elements necessary for expression in eukaryotic cells. 
     The present invention relates to a method of preventing an individual from becoming infected by HSV which comprises administering a nucleotide sequence that encodes HSV2 gD2 or a modified form of HSV gD2 operably linked to regulatory elements necessary for expression in eukaryotic cells. 
     The present invention relates to DNA vaccines which comprise nucleotide sequences that encode HSV2 gD2 or a modified form of HSV gD2 operably linked to regulatory elements necessary for expression in eukaryotic cells. In some embodiments, the vaccine comprises plasmids which include the nucleotide sequences that encode HSV2 gD2 or a modified from thereof. In some embodiments, the vaccines are facilitated DNA vaccines which further comprise a polynucleotide function enhancer or other facilitating composition. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIGS. 1A-1D show gD constructs. 
     FIGS. 2A-2E shows the sequence for HSV gD from Genbank (SEQ ID NO.1). 
     FIGS. 3A-3E show various inserts and plasmids having gD2 deletions in the HSV gD2 coding sequences. 
     FIGS. 4A-4E show humoral responses in immunized mice. AntigD2 specific serum antibodies in mice immunized with the indicated gD2 expression vectors were measured at day 79 by standard ELISA. Serum samples were diluted 1:100. 
     FIG. 5 shows antibody isotyping. The relative levels of IgG1 and IgG2A gD2-specific antibodies at day 79 were determined by standard ELISA. 
     FIGS. 6A-6E show lymphoproliferative responses of mouse spleen cells following immunization. 10 5  whole spleen cells from mice immunized with 40 μg of DNA were cultured with 20 ng gD2/well. After 4 days of incubation, cells were incubated with 1 μCi  3  H-thymidine for 18 hours. Lymphoproliferation was then measured as a function of  3  H-thymidine uptake. 
     FIGS. 7A-7C shows constructs according to the invention. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The present invention relates to improved DNA vaccines and vaccine protocols, and compositions and methods for making the same. The present invention relates to improved DNA anti-HSV vaccines and vaccine protocols, and compositions and methods for making the same. The present invention provides HSV2 gD2 cDNA which can be incorporated into vaccines. Additionally, the present invention provides coding sequences for modified HSV2 gD2 which can be incorporated into vaccines. In some embodiments, the modified HSV gD2 lacks a functional signal peptide. In some embodiments, the modified HSV gD2 lacks a functional transmembrane region (TMR). In some embodiments, the modified HSV gD2 lacks a functional signal peptide and a functional transmembrane region. 
     A wild type--or full length construct contains a signal peptide that directs the gD polypeptide into the endoplasmic reticulum where it is glycosylated. It travels to the cell surface through the secretory pathway and remains associated with the surface membrane via the transmembrane region (TMR), a hydrophobic region located near the C-terminus. 
     A TMR deletion construct was made in order to be secreted from cells and be available to be picked up by antigen presenting cells and boost the humoral response to gD. A cellular response may also be boosted. We have demonstrated that cells transferred with this construct secrete it into the media and that there is no detectable gD associated with the cell membrane. 
     Signal peptide deletion constructs were made to be localized to the cytoplasm and perhaps be misfolded. This provides a means by which more gD is transported to the proteosome, resulting in more gD derived peptide being able to complex with MHC Class 1 molecules. This boosts the cellular response to gD. There are two signal peptide deletion type constructs. One has a TMR and the other does not. Both types of proteins are expected to be localized to the cytoplasm but may have differences in distribution within the cytoplasm based on differences in their hydrophobicities. 
     We have demonstrated that cells transfected with these constructs express gD that is localized to the cytoplasm. There is no detectable gD associated with the cell membrane and no gD detected in the media of transfected cells. Based on the molecular weight of the expressed gD, it appears to be unglycosylated. 
     FIG. 1A shows a diagram of the HSV2 gD2 protein. In some embodiments, a nucleotide sequence that encodes this protein under the control of regulatory sequences is included in a vaccine. In some preferred embodiments, the vaccine is a DNA vaccine. 
     FIG. 1B shows a diagram of a HSV2 gD2 protein with a TMR deletion. In some embodiments, the entire TMR is deleted. In some embodiments, the TMR function is inhibited by deleting most of the TMR coding sequence. In some embodiments, a nucleotide sequence that encodes this protein under the control of regulatory sequences is included in a vaccine. In some preferred embodiments, the vaccine is a DNA vaccine. 
     FIG. 1C shows a diagram of a HSV2 gD2 protein with a signal peptide deletion. In some embodiments, the entire signal peptide is deleted. In some embodiments, the signal peptide function is inhibited by deleting most of the signal peptide coding sequence. In some embodiments, a nucleotide sequence that encodes this protein under the control of regulatory sequences is included in a vaccine. In some preferred embodiments, the vaccine is a DNA vaccine. 
     FIG. 1D shows a diagram of a HSV2 gD2 protein with a TMR deletion and a signal peptide deletion. In some embodiments, the entire TMR is deleted. In some embodiments, the TMR function is inhibited by deleting most of the TMR coding sequence. In some embodiments, the entire signal peptide is deleted. In some embodiments, the signal peptide function is inhibited by deleting most of the signal peptide coding sequence. In some embodiments, a nucleotide sequence that encodes this protein under the control of regulatory sequences is included in a vaccine. In some preferred embodiments, the vaccine is a DNA vaccine. 
     The data in FIGS. 4A-4E, 5 and 6A-6E demonstrate that by removing the TMR of the HSV gD protein, a shift in IGg antibody isotype from IgG2a to IgG1 was obtained. This shift is considered a surrogate marker indicating a shift from a predominantly TH1 to a predominantly TH2 response, or from a cellular to an antibody response. Cytokines released by the cells would also be expected to differ accordingly. 
     A similar effect may be achieved by deleting or mutating the sequence coding for the TMR or membrane binding region of any protein normally anchored in the membrane. e.g., other herpes virus envelope proteins, HSV1, HSV2, EBV, CMV, HZV. This list of viruses is only a partial list and those having ordinary skill in the art can readily select other viruses which can be used to practice the invention. Further, other proteins which can be used include cell envelope associated protein. Likewise, proteins that enter the secretory pathway but contain other membrane retention signals such as endoplasmic retention signals from other viruses as well as host cellular proteins that are cell envelope associated could be made to secrete by removing or otherwise deleting the TMR or other membrane retention signal. In addition, proteins encoded by cells or viruses that are not envelope associated can be designed to be secreted by adding a signal peptide and removing membrane or cell compartment localization signals. 
     Secretion, with a resulting shift to a predominantly TH2 response, can be achieved where this is desired by the following changes to the construct: 
     1) removing or mutating the TMR or membrane binding region; 
     2) adding a signal or leader sequence; 
     3) co-expressing with a protease that clips the membrane binding region at an added protease site, or, 
     4) adding a sequence coding for an intein, which will self-cleave. The intein coding sequence would be inserted into the gene in a way that would result in a cleavage that would separate the TMR from the rest of the gene. In this way you would maintain the TMR protein expression which may contain an immunological epitope and yet render the TMR incapable anchoring the protein to the cell envelope. 
     If retention of the protein in the cell is desired, a signal or leader sequence can be removed, or if targeting specifically to the ER (endoplasmic reticulum) is desired, an ER retention signal and a sequence for a secretory peptide may be added. 
     The ability to shift the immune response from primarily Th1 to primarily Th2 allows for the design of improved vaccine protocols. In some embodiments, the primary and possibly first boost is designed to yield a Th1 response. The first boost or subsequent boosts may be designed to drive a Th2 response, thus affording the vaccinee improved protection. 
     In some preferred embodiments, the constructs described in FIGS. 1A-1D are incorporated into DNA vaccines. DNA vaccines are described in U.S. Pat. No. 5,589,466 and U.S. Pat. No. 5,593,971, which are incorporated herein by reference, PCT/US90/01515, PCT/US93/02338, PCT/US93/048131, PCT/US94/00899, and the priority applications cited therein, which are each incorporated herein by reference, and U.S. Ser. No. 08/642,045 filed May 6, 1996 which is incorporated herein by reference. In addition to the delivery protocols described in those applications, alternative methods of delivering DNA are described in U.S. Pat. Nos. 4,945,050 and 5,036,006, which are both incorporated herein by reference. 
     Using DNA vaccine technology, plasmid DNA that includes the coding sequences described in FIG. 1A-1D operably linked to regulatory elements required for gene expression is administered to individuals. The cells of the individual take up the plasmid DNA and the coding sequence is expressed. The antigen so produced becomes a target against which an immune response is directed. The immune response directed against the antigen provides the prophylactic or therapeutic benefit to the individual against HSV. 
     DNA vaccines include naked and facilitated vaccines. Further, they may be administered by a variety of techniques including several different devices for administering substances to tissue. The published literature includes several review articles that describe aspects of DNA vaccine technology and cite some of the many reports of results obtained using the technology. The following review articles which are each incorporated herein by reference as are each of the references cited in each review article discuss DNA vaccine technology: McDonnel W. M and F. K. Askari 1996 New Engl. J. Med. 334(1)42-45; Robinson, A. 1995 Can. Med. Assoc. J. 152(10):1629-1632; Fynan, E. F. et al. 1995 Int. J. Immunopharmac. 17(2)79-83; Pardoll, D. M. and A. M. Beckerleg 1995 Immunity 3:165-169; and Spooner et al. 1995 Gene Therapy 2:173-180. 
     According to the present invention, the coding sequence of the inserts described in FIG. 1A-1D are inserted into the plasmid which is then used in a vaccine composition. 
     As used herein, the term insert is meant to refer to a nucleotide sequence that encodes a gD2 protein described in FIG. 1A-1D including nucleotide sequences that encode a gD2 protein which comprises a non-functional TMR and/or a non-functional signal peptide. 
     As used herein, the term genetic construct is meant to refer to plasmids which comprise an insert operably linked to regulatory elements required for expression of the insert in eukaryotic cells. Regulatory elements for DNA expression include a promoter and a polyadenylation signal. In addition, other elements, such as a Kozak region, may also be included in the genetic construct. Initiation and termination signals are required regulatory elements which are often considered part of the coding sequence. The coding sequences of genetic constructs of the invention include functional initiation and termination signals. 
     The present invention relates to methods of introducing genetic material into the cells of an individual in order to induce immune responses against HSV. The methods comprise the steps of administering to the tissue of said individual, DNA that includes a coding sequence for an insert such as those shown in FIGS. 1A-1D operably linked to regulatory elements required for expression. 
     The present invention provides genetic constructs useful as DNA vaccines that include coding sequences for inserts such as those shown in FIGS. 1A-1D. 
     In some embodiments, the cDNA reported by Watson et al. 1983 Gene 26:307-312, which is incorporated herein by reference is used to construct inserts. The sequence is published in Genbank accession number K01408, which is incorporated herein by reference, and shown in FIG. 2. The coding sequence of the Watson clone spans nucleotides 268-1449. The sequence encoding the signal peptide includes nucleotides 268-342. The TMR is encoded by nucleotides 1249-1446. 
     In some embodiments, the insert comprises the entire coding sequence. In some embodiments, the insert consists of the entire coding sequence. 
     In some embodiments, the insert comprises the entire coding sequence which includes a frame shift or deletion or insertion that renders the signal peptide inoperable without effecting the remaining portions of the protein. In some embodiments, the sequence encoding the signal peptide is deleted and the insert comprises the remaining coding sequence. In some embodiments, less than the complete sequence that encodes the signal peptide is included, such as for example, inserts that include only nucleotides 287-1449, 297-1449, 307-1449, 317-1449, 327-1449 and 337-1449. 
     In some embodiments, the insert comprises the entire coding sequence which includes a frame shift or deletion or insertion that renders the TMR inoperable without effecting the remaining portions of the protein. In some embodiments, the sequence encoding the TMR is deleted and the insert comprises the remaining coding sequence. In some embodiments, less than the complete sequence that encodes the TMR is included, such as for example, inserts that include only nucleotides 268-1426, 268-1406, 268-1386, 268-1366, 268-1346, 268-1326, 268-1306, 268-1286, 268-1266 and 268-1246. 
     In some embodiments, the insert comprises the entire coding sequence which includes a frame shift or deletion or insertion that renders the signal peptide inoperable and a frame shift or deletion or insertion which renders the TMR inoperable without effecting the remaining portions of the protein. In some embodiments, the sequence encoding the signal peptide is deleted and the insert comprises the remaining coding sequence with a deleted or inoperable TMR. In some embodiments, the sequence encoding the TMR is deleted and the insert comprises the remaining coding sequence with a deleted or inoperable signal peptide. In some embodiments, the sequences encoding the signal peptide and TMR are deleted. In some embodiments, the insert consists of nucleotides 278-1426, 288-1386, 298-1346, 308-1306, 318-1266, 328-1246, and 342-1248. 
     In some embodiments, the insert is inserted into plasmid described in PCT/US94/00899 filed Jan. 26, 1994 and published as WO 94/16737 on Aug. 4, 1994, which is incorporated herein by reference. In some embodiments, the insert is inserted into plasmid described in U.S. Ser. No. 08/642,045 filed May 6, 1996, which is incorporated herein by reference. 
     According to the present invention, compositions and methods are provided which prophylactically and/or therapeutically immunize an individual against HSV, which includes HSV1 and HSV2, particularly HSV2. The genetic material, i.e. an insert, encodes a target protein, i.e. gD2 with or without a functioning signal peptide and/or TMR. The genetic material is expressed by the individual&#39;s cells and serves as an immunogenic target against which an immune response is elicited. 
     The present invention is useful to elicit immune responses against HSV2 gD2 protein. The immune response elicited may cross react with HSV1 gD1 protein. The present invention is useful to immunize individuals against HSV, particularly HSV2, such that an immune response against HSV2 gD2 provides protective immunity against HSV. The present invention is useful to combat HSV in infected individuals by eliciting an immune response against HSV gD2 which can be directed at infected cells that are expressing viral proteins. 
     According to the present invention, DNA encodes the unmodified or modified HSV2 gD2 protein operably linked to regulatory elements. Regulatory elements for DNA expression include a promoter and a polyadenylation signal. In addition, other elements, such as a Kozak region, may also be included in the genetic construct. 
     As used herein, the term &#34;expressible form&#34; refers to gene constructs which contain the necessary regulatory elements operably linked to the insert such that when present in the cell of the individual, the insert will be expressed. 
     When taken up by a cell, the genetic construct(s) may remain present in the cell as a functioning extrachromosomal molecule and/or integrate into the cell&#39;s chromosomal DNA. DNA may be introduced into cells where it remains as separate genetic material in the form of a plasmid or plasmids. Alternatively, linear DNA which can integrate into the chromosome may be introduced into the cell. When introducing DNA into the cell, reagents which promote DNA integration into chromosomes may be added. DNA sequences which are useful to promote integration may also be included in the DNA molecule. Alternatively, RNA may be administered to the cell. It is also contemplated to provide the genetic construct as a linear minichromosome including a centromere, telomeres and an origin of replication. Gene constructs may remain part of the genetic material in attenuated live microorganisms or recombinant microbial vectors which live in cells. Gene constructs may be part of genomes of recombinant viral vaccines where the genetic material either integrates into the chromosome of the cell or remains extrachromosomal. 
     Genetic constructs include regulatory elements necessary for gene expression of a nucleic acid molecule. The elements include: a promoter, an initiation codon, a stop codon, and a polyadenylation signal. In addition, enhancers are often required for gene expression of the sequence that encodes the immunogenic target protein. It is necessary that these elements be operably linked to the sequence that encodes the desired proteins and that the regulatory elements are operable in the individual to whom they are administered. 
     Initiation codons and stop codon are generally considered to be part of a nucleotide sequence that encodes the immunogenic target protein. However, it is necessary that these elements are functional in the individual to whom the gene construct is administered. The initiation and termination codons must be in frame with the coding sequence. 
     Promoters and polyadenylation signals used must be functional within the cells of the individual. 
     Examples of promoters useful to practice the present invention, especially in the production of a genetic vaccine for humans, include but are not limited to promoters from Simian Virus 40 (SV40), Mouse Mammary Tumor Virus (MMTV) promoter, Human Immunodeficiency Virus (HIV) such as the HIV Long Terminal Repeat (LTR) promoter, Moloney virus, ALV, Cytomegalovirus (CMV) such as the CMV immediate early promoter, Epstein Barr Virus (EBV), Rous Sarcoma Virus (RSV) as well as promoters from human genes such as human Actin, human Myosin, human Hemoglobin, human muscle creatine and human metalothionein. 
     Examples of polyadenylation signals useful to practice the present invention, especially in the production of a genetic vaccine for humans, include but are not limited to SV40 polyadenylation signals and LTR polyadenylation signals. In particular, the SV40 polyadenylation signal which is in pCEP4 plasmid (Invitrogen, San Diego, Calif.), referred to as the SV40 polyadenylation signal, is used. 
     In addition to the regulatory elements required for DNA expression, other elements may also be included in the DNA molecule. Such additional elements include enhancers. The enhancer may be selected from the group including but not limited to: human Actin, human Myosin, human Hemoglobin, human muscle creatine and viral enhancers such as those from CMV, RSV and EBV. 
     Genetic constructs can be provided with a mammalian origin of replication in order to maintain the construct extrachromosomally and produce multiple copies of the construct in the cell. Plasmids pCEP4 and pREP4 from Invitrogen (San Diego, Calif.) contain the Epstein Barr virus origin of replication and nuclear antigen EBNA-1 coding region which produces high copy episomal replication without integration. 
     An additional element may be added which serves as a target for cell destruction if it is desirable to eliminate cells receiving the genetic construct for any reason. A herpes thymidine kinase (tk) gene in an expressible form can be included in the genetic construct. The drug gangcyclovir can be administered to the individual and that drug will cause the selective killing of any cell producing tk, thus, providing the means for the selective destruction of cells with the genetic construct. 
     In order to maximize protein production, regulatory sequences may be selected which are well suited for gene expression in the cells into which the construct is administered. Moreover, codons may be selected which are most efficiently transcribed in the cell. One having ordinary skill in the art can produce DNA constructs which are functional in the cells. 
     The method of the present invention comprises the steps of administering nucleic acid molecules to tissue of the individual. In some preferred embodiments, the nucleic acid molecules are administered intramuscularly, intranasally, intraperatoneally, subcutaneously, intradermally, or topically or by lavage to mucosal tissue selected from the group consisting of vaginal, rectal, urethral, buccal and sublingual. 
     In some embodiments, the nucleic acid molecule is delivered to the cells in conjunction with administration of a facilitating agent. Facilitating agents are also referred to as polynucleotide function enhancers or genetic vaccine facilitator agents. Facilitating agents are described in U.S. Ser. No. 08/008,342 filed Jan. 26, 1993, U.S. Ser. No. 08/029,336 filed Mar. 11, 1993, U.S. Ser. No. 08/125,012 filed Sep. 21, 1993 now issued as U.S. Pat. No. 5,593,972, and International Application Ser. No. PCT/US94/00899 filed Jan. 26, 1994 published on Aug. 4, 1994 as International Publication No. WO94/16737, which are each incorporated herein by reference. In addition, facilitating agents are described in PCT application Ser. No. PCT/US95/04071 filed Mar. 30, 1995, published as International Publication No. WO95/26718 on Oct. 12, 1995, which is incorporated herein by reference. Facilitating agents which are administered in conjunction with nucleic acid molecules may be administered as a mixture with the nucleic acid molecule or administered separately simultaneously, before or after administration of nucleic acid molecules. In addition, other agents which may function as transfecting agents and/or replicating agents and/or inflammatory agents and which may be co-administered with or without a facilitating agent include growth factors, cytokines and lymphokines such as -interferon, gamma-interferon, platelet derived growth factor (PDGF), GC-SF, GM-CSF, TNF, epidermal growth factor (EGF), IL-1, IL-2, IL-4, IL-6, IL-8, IL-10 and B7.2 as well as fibroblast growth factor, surface active agents such as immune-stimulating complexes (ISCOMS), Freund&#39;s incomplete adjuvant, LPS analog including monophosphoryl Lipid A (MPL), muramyl peptides, quinone analogs and vesicles such as squalene, and squalene, and hyaluronic acid may also be administered in conjunction with the genetic construct. 
     In some preferred embodiments, the genetic constructs of the invention are formulated with or administered in conjunction with a facilitator selected from the group consisting of benzoic acid esters, anilides, amidines, urethans and the hydrochloride salts thereof such as those of the family of local anesthetics. 
     The facilitator in some preferred embodiments may be a compound having one of the following formulae: 
     
         Ar--R.sup.1 --O--R.sup.2 R.sup.3 
    
     or 
     
         Ar--N--R.sup.1 --R.sup.2 --R.sup.3 
    
     or 
     
         R.sup.4 --N--R.sup.5 --R.sup.6 
    
     or 
     
         R.sup.4 --O--R.sup.1 --N--R.sup.7 
    
     wherein: 
     Ar is benzene, p-aminobenzene, m-aminobenzene, o-aminobenzene, substituted benzene, substituted p-aminobenzene, substituted m-aminobenzene, substituted o-aminobenzene, wherein the amino group in the aminobenzene compounds can be amino, C 1  -C 5  alkylamine, C 1  -C 5 , C 1  -C 5  dialkylamine and substitutions in substituted compounds are halogen, C 1  -C 5  alkyl and C 1  -C 5  alkoxy; 
     R 1  is C═O; 
     R 2  is C 1  -C 10  alkyl including branched alkyls; 
     R 3  is hydrogen, amine, C 1  -C 5  alkylamine, C 1  -C 5 , C 1  -C 5  dialkylamine; 
     R 2  +R 3  can form a cyclic alkyl, a C 1  -C 10  alkyl substituted cyclic alkyl, a cyclic aliphatic amine, a C 1  -C 10  alkyl substituted cyclic aliphatic amine, a heterocycle, a C 1  -C 10  alkyl substituted heterocycle including a C 1  -C 10  alkyl N-substituted heterocycle; 
     R 4  is Ar, R 2  or C 1  -C 5  alkoxy, a cyclic alkyl, a C 1  -C 10  alkyl substituted cyclic alkyl, a cyclic aliphatic amine, a C 1  -C 10  alkyl substituted cyclic aliphatic amine, a heterocycle, a C 1  -C 10  alkyl substituted heterocycle and a C 1  -C 10  alkoxy substituted heterocycle including a C 1  -C 10  alkyl N-substituted heterocycle; 
     R 5  is C═NH; 
     R 6  is Ar, R 2  or C 1  -C 5  alkoxy, a cyclic alkyl, a C 1  -C 10  alkyl substituted cyclic alkyl, a cyclic aliphatic amine, a C 1  -C 10  alkyl substituted cyclic aliphatic amine, a heterocycle, a C 1  -C 10  alkyl substituted heterocycle and a C 1  -C 10  alkoxy substituted heterocycle including a C 1  -C 10  alkyl N-substituted heterocycle; and. 
     R 7  is Ar, R 2  or C 1  -C 5  alkoxy, a cyclic alkyl, a C 1  -C 10  alkyl substituted cyclic alkyl, a cyclic aliphatic amine, a C 1  -C 10  alkyl substituted cyclic aliphatic amine, a heterocycle, a C 1  -C 10  alkyl substituted heterocycle and a C 1  -C 10  alkoxy substituted heterocycle including a C 1  -C 10  alkyl N-substituted heterocycle. 
     Examples of esters include: benzoic acid esters such as piperocaine, meprylcaine and isobucaine; para-aminobenzoic acid esters such as procaine, tetracaine, butethamine, propoxycaine and chloroprocaine; meta-aminobenzoic acid esters including metabuthamine and primacaine; and para-ethoxybenzoic acid esters such as parethoxycaine. Examples of anilides include lidocaine, etidocaine, mepivacaine, bupivacaine, pyrrocaine and prilocaine. Other examples of such compounds include dibucaine, benzocaine, dyclonine, pramoxine, proparacaine, butacaine, benoxinate, carbocaine, methyl bupivacaine, butasin picrate, phenacaine, diothan, luccaine, intracaine, nupercaine, metabutoxycaine, piridocaine, biphenamine and the botanically-derived bicyclics such as cocaine, cinnamoylcocaine, truxilline and cocaethylene and all such compounds complexed with hydrochloride. 
     In preferred embodiments, the facilitator is bupivacaine. The difference between bupivacaine and mepivacaine is that bupivacaine has a N-butyl group in place of an N-methyl group of mepivacaine. Compounds may have at that N, C 1  -C 10 . Compounds may be substituted by halogen such as procaine and chloroprocaine. The anilides are preferred. 
     The facilitating agent is administered prior to, simultaneously with or subsequent to the genetic construct. The facilitating agent and the genetic construct may be formulated in the same composition. 
     Bupivacaine-HCl is chemically designated as 2-piperidinecarboxamide, 1-butyl-N-(2,6-dimethylphenyl)-monohydrochloride, monohydrate and is widely available commercially for pharmaceutical uses from many sources including from Astra Pharmaceutical Products Inc. (Westboro, Mass.) and Sanofi Winthrop Pharmaceuticals (New York, N.Y.), Eastman Kodak (Rochester, N.Y.). Bupivacaine is commercially formulated with and without methylparaben and with or without epinephrine. Any such formulation may be used. It is commercially available for pharmaceutical use in concentrations of 0.25%, 0.5% and 0.75% which may be used on the invention. Alternative concentrations, particularly those between 0.05%-1.0% which elicit desirable effects may be prepared if desired. According to the present invention, about 250 μg to about 10 mg of bupivacaine is administered. In some embodiments, about 250 μg to about 7.5 mg is administered. In some embodiments, about 0.05 mg to about 5.0 mg is administered. In some embodiments, about 0.5 mg to about 3.0 mg is administered. In some embodiments about 5 to 50 μg is administered. For example, in some embodiments about 50 μl to about 2 ml, preferably 50 μl to about 1500 μl and more preferably about 1 ml of 0.25-0.50% bupivacaine-HCl and 0.1% methylparaben in an isotonic pharmaceutical carrier are administered at the same site as the vaccine before, simultaneously with or after the vaccine is administered. Similarly, in some embodiments, about 50 μl to about 2 ml, preferably 50 μl to about 1500 μl and more preferably about 1 ml of 0.25-0.50% bupivacaine-HCl in an isotonic pharmaceutical carrier is administered at the same site as the vaccine before, simultaneously with or after the vaccine is administered. Bupivacaine and any other similarly acting compounds, particularly those of the related family of local anesthetics may be administered at concentrations which provide the desired facilitation of uptake of genetic constructs by cells. 
     In some embodiments of the invention, the individual is first subject to injection of the facilitator prior to administration of the genetic construct. For example, up to a about a week to ten days prior to administration of the genetic construct, the individual is first injected with the facilitator. In some embodiments, the individual is injected with facilitator about 1 to 5 days, in some embodiments 24 hours, before or after administration of the genetic construct. Alternatively, if used at all, the facilitator is administered simultaneously, minutes before or after administration of the genetic construct. Accordingly, the facilitator and the genetic construct may be combined to form a single pharmaceutical composition. 
     In some embodiments, the genetic constructs are administered free of facilitating agents, that is in formulations free from facilitating agents using administration protocols in which the genetic constructs are not administered in conjunction with the administration of facilitating agents. 
     The Herpes simplex 2 glycoprotein D (gD) gene encodes a glycoprotein that is associated with the viral envelope and the infected cell plasma membrane. A gD encoded signal peptide directs the translocation of the nascent polypeptide into the lumen of the endoplasmic reticulum where it enters the secretory pathway, is glycosylated and folded. The protein remains associated with the plasma membrane via a hydrophobic C-terminal domain referred to as the transmembrane region or TMR. 
     DNA immunization with a plasmid expressing Herpes simplex 2 glycoprotein D gene has been shown to induce humoral and cellular immune responses in several animal models. In mice, the immune response generated by the initial gD-expressing plasmid was found to be predominantly a TH1 or cellular response. We have demonstrated that a TH2 or humoral response may be induced during immunization if the gene is modified so that a predominantly secreted version of gD is expressed by the plasmid component of the vaccine. The encoded protein differs from the native gD 2 protein only in the deletion of the last 66 amino acids which encode the TMR. We have demonstrated that a construct engineered to encode a TMR-deleted protein expresses a protein which is predominantly secreted into the media of transfected cells. Only a small amount of protein remains cell-associated. High levels of soluble antigen have been shown to stimulate Th2 responses, while low doses of soluble antigen stimulate the production of IL-12, leading to Th1 responses (Abbas, A. K., Murphy, K. M., Sher, A. (1996). Functional diversity of helper T lymphocytes. Nature 381:787-793). A construct designed to favor secretion of antigen would promote Th2 immune responses. 
     Accordingly, the invention relates to engineered polynucleotide constructs capable of expressing an antigenic protein which will induce a desired TH1 or TH2 immune response, to plasmid or other vector constructs containing and capable of expressing such engineered polynucleotide constructs, and to methods of immunizing a mammal with such constructs in order to achieve a desired TH1 or TH2 immune response. In one desired embodiment, the invention relates to a method of engineering a gene or a group of genes so that the encoded protein(s) is secreted from the cell, thus enabling a TH2 response. In another desired embodiment, the invention relates to a method of immunizing wherein a mammal is first immunized at least once with a plasmid that encodes a protein that induces a TH1 response and subsequent immunizations are with a plasmid system that allows for the efficient secretion of that protein such that the response may be subsequently pushed towards a TH2 response. In some applications, the invention relates to a method wherein the mammal is first immunized with a polynucleotide vaccine that induces a TH2 response and then boosted with a vaccine that pushes a TH1 response. In another embodiment of the invention, both TH1 and Th2 responses may be achieved via simultaneous or contemporaneous immunization with one or more vaccine composition(s) that push(es) both a TH1 response and a TH2 response. 
     The constructs of the invention may engineered as follows: 
     In one preferred embodiment, a modified construct contains a TMR deletion which results in enhanced secretion of the expressed antigenic protein into the extracellular compartment, thereby producing an enhanced TH2 or humoral immune response. In one preferred embodiment, a modified construct contains a signal or leader peptide deletion which results in intracellular localization to the cytosol compartment, thereby resulting in an enhanced TH1 or cellular immune response. In another preferred embodiment, both signal and TMR deletions are made, resulting in the expression of an immunogenic protein which localizes to the cytosolic compartment, thereby resulting in an enhanced TH1 or cellular immune response. 
     The following describes a list of ways to enable secretion of cell associated proteins. It is first necessary to engineer a signal peptide at the amino terminus of a protein for cell associated proteins that do not enter the secretory pathway. This may be all that is needed for the efficient secretion of some of these proteins but other of these proteins will need further modification(s). For example, some proteins that are normally not secreted may contain domains that interact with membranes and this interaction may inhibit secretion of those proteins. Alternatively, these proteins may contain motifs that localize the protein to certain sub-cellular compartments such as the nucleus and it is possible that these sequences may also prevent efficient secretion of the protein. It would therefore be necessary to destroy these domains by deletion or mutation. In many instances these sequences will already be known, in other cases, homology to known domains and localization motifs can be identified by scanning the sequence. In other cases, unidentified inhibitory sequences can be mapped and destroyed via a selection based mutagenesis approach. 
     For proteins that normally enter the secretory pathway, but remain associated with the cell via membrane retention domains or domains that localize the protein to a sub-compartment within the secretory pathway, it will first be necessary to remove those domains. These can be removed via deletion or mutation. In some instances, the natural signal peptides encoded by these proteins may be inefficient at translocating the protein into the ER. In these cases, a heterologous signal peptide can be used in place of the native signal peptide. An example of a heterologous signal peptide would be the one encoded by the HSV2 gD gene. 
     Deletion of sequences can be through deletion or mutation of the sequences in the construct itself (FIG. 7). Alternatively, in some cases where the inhibitory sequences exist as distinct domains (i.e, they are not marbled throughout the protein) and they are localized to the C-terminal end of the protein, and it is desirable to retain these sequences in the construct for immunogenicity purposes, the construct can be engineered in such a way that these sequences are not covalently joined to the portion of the protein destined for secretion. This can be done by encoding a protease site between the part of the protein to be secreted and the part of the protein with sequences inhibitory for secretion. This protease site would be a cleavage site for a protease that is endogenous to the cells expressing the vaccine protein. Alternatively, the protease could be provided in trans on either the same construct as the encoded vaccine protein or on a separate plasmid that would be co-injected with the vaccine plasmid. In this instance, cleavage would not be dependent on a protease that naturally occurs within cells that express the vaccine plasmid. It is also feasible to include a self-cleaving protease such as the polio 3C protease or an intein between the domains of the protein to be separated. There are some instances where it would not be feasible to remove domains by a protease approach. For example, a sub-cellular localization domain is first expressed as a part of a precursor polypeptide (prior to proteolysis) and could in effect interfere with nascent polypeptide translocation into the ER. In this instance, the domain would have to be removed through deletion or site-directed mutation. Also, one would have to ascertain if the desired proteolysis reaction will occur in the ER. 
     Another consideration for targeted secretion is the stability of the protein in the extracellular compartment. If the protein is unstable, it may be possible to increase its stability and maintain its antigenicity by fusing it to another peptide or protein such as a fusion protein. In some instances, the fusion proteins may be able to assemble into serum stable particles. 
     The invention also relates to polynucleotide constructs engineered to express fusion proteins which assemble to form particles, as well as methods of immunization with such polynucleotide constructs. Not only are such proteins stable, but they are of the size which is preferentially taken up by APCs (antigen presenting cells) and are processed in such a way that they are presented by both MHC class 1 and class 2 molecules. 
     Constructs of the invention modified to encode proteins that are predominantly secreted would be appropriate for a number of antigens for which a TH2 or humoral response would be required, as in a prophylactic vaccine against viral, parasitic and bacterial infections. The proteins that are chosen to be expressed would be those antigenic proteins that make up the viral particles, parasite, bacteria or spore. However, proteins that are not a part of the infecting organism per se, but are associated particularly with the infected cell membrane might also be targets for expression since a humoral response against these antigens could result in cell death via the complement pathway or ADCC pathway. 
     EXAMPLES 
     Example 1 
     Insert TMR consists of 37 nucleotides of HSV2 gD2 5&#39; flanking sequence and sequences encoding the HSV gD2 leader peptide and the first 302 amino acids of the mature protein. 66 amino acids are deleted from the carboxy terminus. The construct has 1 nucleotide of the 3&#39; HSV gD2 flanking sequence. 
     The insert is cloned into vector APL-400-004 to produce APL-400-004 TMR shown in FIG. 3A. 
     In some embodiments, a second construct, APL-400-024 TMR is prepared. That plasmid is identical to APL-400-004 TMR except the chimeric kanamycin resistance construct of U.S. Ser. No. 08/642,045 filed May 6, 1996 is inserted in place of the kanamycin resistance gene in the vector APL-400-004. 
     Example 2 
     Insert L -1  consists of 9 bp of authentic 5&#39; sequence flanking the ATG of HSV2 gD2 followed by the ATG and then the coding sequence for the mature protein coding region starting with amino acid 26. The coding sequence for the first 25 amino acids which comprise the leader peptide has been deleted. The insert also includes approximately 550 bp of 3&#39; sequences flanking the stop codon. 
     The insert is cloned into vector APL-400-004 to produce APL-400-004 L -1  shown in FIG. 3B and further comprises 39 bp 5&#39; of the authentic flanking (5&#39;) sequences is from the TA vector (PCR II, Invitrogen). 
     In some embodiments, a second construct, APL-400-024 L -1  is prepared. That plasmid is identical to APL-400-004 L -1  except it contains the chimeric kanamycin resistance construct of U.S. Ser. No. 08/642,045 filed May 6, 1996 in place of the kanamycin resistance gene in the vector APL-400-004. 
     Example 3 
     Insert L -II , consists of 41 bp of authentic 5&#39; sequences flanking the ATG of HSV2 gD2 followed by ATG and then the coding sequences for the mature protein coding region starting with amino acid 26. The coding sequence for the first 25 amino acids which comprise the leader peptide have been deleted. The insert also includes approximately 550 bp of 3&#39; sequences following the stop codon. 
     The insert is cloned into vector APL-400-004 to produce APL-400-004 L -II  shown in FIG. 3C. 
     In some embodiments, a second construct, APL-400-024 L -II  is prepared. That plasmid is identical to APL-400-004 L -II  except having the chimeric kanamycin resistance construct of U.S. Ser. No. 08/642,045 filed May 6, 1996 in place of the kanamycin resistance gene in the vector APL-400-004. 
     Example 4 
     Insert L -3  consists of 41 bp of authentic 5&#39; sequences flanking the ATG of HSV2 gD2 followed by ATG and 6 bp after the ATG in order to preserve the Kozak site, and then the coding sequences for the mature protein coding region starting with amino acid 26. The coding sequence for the first 25 amino acids which comprise the leader peptide has been deleted. The insert also includes approximately 550 bp of 3&#39; sequences following the stop codon. 
     The insert is cloned into vector APL-400-004 to produce APL-400-004 L -3  shown in FIG. 3D. 
     In some embodiments, a second construct, APL-400-024 L -3  is prepared. That plasmid is identical to APL-400-004 L0 -3  except it the chimeric kanamycin resistance construct of U.S. Ser. No. 08/642,045 filed May 6, 1996 in place of the kanamycin resistance gene in the vector APL-400-004. 
     Example 5 
     Insert L -3  TMR consists of 41 bp of authentic 5&#39; sequences flanking the ATG of HSV2 gD2 followed by ATG and 6 bp after the ATG in order to preserve the Kozak site, and then the coding sequences for the mature protein coding region starting with amino acid 26. The coding sequence for the first 25 amino acids which comprise the leader peptide and the coding sequence for the 66 amino acids at the carboxy terminus of the mature protein which comprise the transmembrane region have been deleted. The insert also includes 1 bp of 3&#39; sequences following the stop codon. 
     The insert is cloned into vector APL-400-004 to produce APL-400-004 L -3  TMR shown in FIG. 3E. 
     In some embodiments, a second construct, APL-400-024 L -3  TMR is prepared. That plasmid is identical to APL-400-004 L -3  TMR except has the chimeric kanamycin resistance construct of U.S. Ser. No. 08/642,045 filed May 6, 1996 in place of the kanamycin resistance gene in the vector APL-400-004. 
     Example 6 
     Mice were immunized I.M. at day 0 and day 14 with 20 μg DNA/0.4% bupivacaine. Mice were bled at day 14 and day 42 and sera assayed for the presence of anti-gD antibodies. Mice immunized with the TMR deletion appeared to have mounted a higher humoral response than did mice immunized with the full length HSVgD construct. No sero-conversion was detected in mice immunized with either of the signal peptide deletions. 
     
         __________________________________________________________________________#             SEQUENCE LISTING- &lt;160&gt; NUMBER OF SEQ ID NOS: 2- &lt;210&gt; SEQ ID NO 1&lt;211&gt; LENGTH: 1635&lt;212&gt; TYPE: DNA&lt;213&gt; ORGANISM: Herpes simplex virus type 2&lt;220&gt; FEATURE:&lt;221&gt; NAME/KEY: CDS&lt;222&gt; LOCATION: (268)..(1446)- &lt;400&gt; SEQUENCE: 1- cttggggggg ggggggaaga aactaaaaac acatcaagcc cacaacccat cc - #cacaaggg  60- gggttatggc ggacccaccg caccaccata ctccgattcg accacatatg ca - #accaaatc 120- acccccagag gggaggttcc atttttacga ggaggaggag tataatagag tc - #tttgtgtt 180- taaaacccgg ggtcggtgtg gtgttcggtc ataagctgca ttgcgaacca ct - #agtcgccg 240#tcc ggc gtc ggg    294 tcacggc atg ggg cgt ttg acc#            Met Gly Arg Le - #u Thr Ser Gly Val Gly#           5  1- acg gcg gcc ctg cta gtt gtc gcg gtg gga ct - #c cgc gtc gta tgc gcc 342Thr Ala Ala Leu Leu Val Val Ala Val Gly Le - #u Arg Val Val Cys Ala# 25- aaa tac gcc tta gca gac ccc tcg ctt aag at - #g gcc gat ccc aat cga 390Lys Tyr Ala Leu Ala Asp Pro Ser Leu Lys Me - #t Ala Asp Pro Asn Arg#                 40- ttt cgc ggg aag aac ctt ccg gtt ttg gac ca - #g ctg acc gac ccc ccc 438Phe Arg Gly Lys Asn Leu Pro Val Leu Asp Gl - #n Leu Thr Asp Pro Pro#             55- ggg gtg aag cgt gtt tac cac att cag ccg ag - #c ctg gag gac ccg ttc 486Gly Val Lys Arg Val Tyr His Ile Gln Pro Se - #r Leu Glu Asp Pro Phe#         70- cag ccc ccc agc atc ccg atc act gtg tac ta - #c gca gtg ctg gaa cgt 534Gln Pro Pro Ser Ile Pro Ile Thr Val Tyr Ty - #r Ala Val Leu Glu Arg#     85- gcc tgc cgc agc gtg ctc cta cat gcc cca tc - #g gag gcc ccc cag atc 582Ala Cys Arg Ser Val Leu Leu His Ala Pro Se - #r Glu Ala Pro Gln Ile#105- gtg cgc ggg gct tcg gac gag gcc cga aag ca - #c acg tac aac ctg acc 630Val Arg Gly Ala Ser Asp Glu Ala Arg Lys Hi - #s Thr Tyr Asn Leu Thr#               120- atc gcc tgg tat cgc atg gga gac aat tgc gc - #t atc ccc atc acg gtt 678Ile Ala Trp Tyr Arg Met Gly Asp Asn Cys Al - #a Ile Pro Ile Thr Val#           135- atg gaa tac acc gag tgc ccc tac aac aag tc - #g ttg ggg gtc tgc ccc 726Met Glu Tyr Thr Glu Cys Pro Tyr Asn Lys Se - #r Leu Gly Val Cys Pro#       150- atc cga acg cag ccc cgc tgg agc tac tat ga - #c agc ttt agc gcc gtc 774Ile Arg Thr Gln Pro Arg Trp Ser Tyr Tyr As - #p Ser Phe Ser Ala Val#   165- agc gag gat aac ctg gga ttc ctg atg cac gc - #c ccc gcc ttc gag acc 822Ser Glu Asp Asn Leu Gly Phe Leu Met His Al - #a Pro Ala Phe Glu Thr170                 1 - #75                 1 - #80                 1 -#85- gcg ggt acg tac ctg cgg cta gtg aag ata aa - #c gac tgg acg gag atc 870Ala Gly Thr Tyr Leu Arg Leu Val Lys Ile As - #n Asp Trp Thr Glu Ile#               200- aca caa ttt atc ctg gag cac cgg gcc cgc gc - #c tcc tgc aag tac gct 918Thr Gln Phe Ile Leu Glu His Arg Ala Arg Al - #a Ser Cys Lys Tyr Ala#           215- ctc ccc ctg cgc atc ccc ccg gca gcg tgc ct - #c acc tcg aag gcc tac 966Leu Pro Leu Arg Ile Pro Pro Ala Ala Cys Le - #u Thr Ser Lys Ala Tyr#       230- caa cag ggc gtg acg gtc gac agc atc ggg at - #g tta ccc cgc ttt atc1014Gln Gln Gly Val Thr Val Asp Ser Ile Gly Me - #t Leu Pro Arg Phe Ile#   245- ccc gaa aac cag cgc acc gtc gcc cta tac ag - #c tta aaa atc gcc ggg1062Pro Glu Asn Gln Arg Thr Val Ala Leu Tyr Se - #r Leu Lys Ile Ala Gly250                 2 - #55                 2 - #60                 2 -#65- tgg cac ggc ccc aag ccc ccg tac acc agc ac - #c ctg ctg ccg ccg gag1110Trp His Gly Pro Lys Pro Pro Tyr Thr Ser Th - #r Leu Leu Pro Pro Glu#               280- ctg tcc gac acc acc aac gcc acg caa ccc ga - #a ctc gtt ccg gaa gac1158Leu Ser Asp Thr Thr Asn Ala Thr Gln Pro Gl - #u Leu Val Pro Glu Asp#           295- ccc gag gac tcg gcc ctc tta gag gat ccc gc - #c ggg acg gtg tct tcg1206Pro Glu Asp Ser Ala Leu Leu Glu Asp Pro Al - #a Gly Thr Val Ser Ser#       310- cag atc ccc cca aac tgg cac atc ccg tcg at - #c cag gac gtc gcg ccg1254Gln Ile Pro Pro Asn Trp His Ile Pro Ser Il - #e Gln Asp Val Ala Pro#   325- cac cac gcc ccc gcc gcc ccc agc aac ccg gg - #c ctg atc atc ggc gcg1302His His Ala Pro Ala Ala Pro Ser Asn Pro Gl - #y Leu Ile Ile Gly Ala330                 3 - #35                 3 - #40                 3 -#45- ctg gcc ggc agt acc ctg gcg gcg ctg gtc at - #c ggc ggt att gcg ttt1350Leu Ala Gly Ser Thr Leu Ala Ala Leu Val Il - #e Gly Gly Ile Ala Phe#               360- tgg gta cgc cgc cgc gct cag atg gcc ccc aa - #g cgc cta cgt ctc ccc1398Trp Val Arg Arg Arg Ala Gln Met Ala Pro Ly - #s Arg Leu Arg Leu Pro#           375- cac atc cgg gat gac gac gcg ccc ccc tcg ca - #c cag cca ttg ttt tac1446His Ile Arg Asp Asp Asp Ala Pro Pro Ser Hi - #s Gln Pro Leu Phe Tyr#       390- tagaggagtt tccccgttcc cgtgtacctc tgggcccgtg tgggagggtg gc - #cggggtat1506- ttgggtggga cttggactcc gcataaaggg agtctcgaag gagggaaact ag - #gacagttc1566- ataggccggg agcgtggggc gcgcaccgcg tcccgacgat tagccaccgc gc - #ccacagcc1626#       1635- &lt;210&gt; SEQ ID NO 2&lt;211&gt; LENGTH: 393&lt;212&gt; TYPE: PRT&lt;213&gt; ORGANISM: Herpes simplex virus type 2- &lt;400&gt; SEQUENCE: 2- Met Gly Arg Leu Thr Ser Gly Val Gly Thr Al - #a Ala Leu Leu Val Val#                 15- Ala Val Gly Leu Arg Val Val Cys Ala Lys Ty - #r Ala Leu Ala Asp Pro#             30- Ser Leu Lys Met Ala Asp Pro Asn Arg Phe Ar - #g Gly Lys Asn Leu Pro#         45- Val Leu Asp Gln Leu Thr Asp Pro Pro Gly Va - #l Lys Arg Val Tyr His#     60- Ile Gln Pro Ser Leu Glu Asp Pro Phe Gln Pr - #o Pro Ser Ile Pro Ile# 80- Thr Val Tyr Tyr Ala Val Leu Glu Arg Ala Cy - #s Arg Ser Val Leu Leu#                 95- His Ala Pro Ser Glu Ala Pro Gln Ile Val Ar - #g Gly Ala Ser Asp Glu#           110- Ala Arg Lys His Thr Tyr Asn Leu Thr Ile Al - #a Trp Tyr Arg Met Gly#       125- Asp Asn Cys Ala Ile Pro Ile Thr Val Met Gl - #u Tyr Thr Glu Cys Pro#   140- Tyr Asn Lys Ser Leu Gly Val Cys Pro Ile Ar - #g Thr Gln Pro Arg Trp145                 1 - #50                 1 - #55                 1 -#60- Ser Tyr Tyr Asp Ser Phe Ser Ala Val Ser Gl - #u Asp Asn Leu Gly Phe#               175- Leu Met His Ala Pro Ala Phe Glu Thr Ala Gl - #y Thr Tyr Leu Arg Leu#           190- Val Lys Ile Asn Asp Trp Thr Glu Ile Thr Gl - #n Phe Ile Leu Glu His#       205- Arg Ala Arg Ala Ser Cys Lys Tyr Ala Leu Pr - #o Leu Arg Ile Pro Pro#   220- Ala Ala Cys Leu Thr Ser Lys Ala Tyr Gln Gl - #n Gly Val Thr Val Asp225                 2 - #30                 2 - #35                 2 -#40- Ser Ile Gly Met Leu Pro Arg Phe Ile Pro Gl - #u Asn Gln Arg Thr Val#               255- Ala Leu Tyr Ser Leu Lys Ile Ala Gly Trp Hi - #s Gly Pro Lys Pro Pro#           270- Tyr Thr Ser Thr Leu Leu Pro Pro Glu Leu Se - #r Asp Thr Thr Asn Ala#       285- Thr Gln Pro Glu Leu Val Pro Glu Asp Pro Gl - #u Asp Ser Ala Leu Leu#   300- Glu Asp Pro Ala Gly Thr Val Ser Ser Gln Il - #e Pro Pro Asn Trp His305                 3 - #10                 3 - #15                 3 -#20- Ile Pro Ser Ile Gln Asp Val Ala Pro His Hi - #s Ala Pro Ala Ala Pro#               335- Ser Asn Pro Gly Leu Ile Ile Gly Ala Leu Al - #a Gly Ser Thr Leu Ala#           350- Ala Leu Val Ile Gly Gly Ile Ala Phe Trp Va - #l Arg Arg Arg Ala Gln#       365- Met Ala Pro Lys Arg Leu Arg Leu Pro His Il - #e Arg Asp Asp Asp Ala#   380- Pro Pro Ser His Gln Pro Leu Phe Tyr385                 3 - #90__________________________________________________________________________