AU2007203565B2

AU2007203565B2 - Genetic vaccine that mimics natural viral infection and induces long-lasting immunity to pathogens

Info

Publication number: AU2007203565B2
Application number: AU2007203565A
Authority: AU
Inventors: Danher Wang
Original assignee: GenPhar Inc
Current assignee: GenPhar Inc
Priority date: 2000-06-02
Filing date: 2007-07-31
Publication date: 2010-08-12
Anticipated expiration: 2021-06-04
Also published as: AU2007203565A1

Description

AUSTRALIA Patents Act 1990 ORIGINAL COMPLETE SPECIFICATION INVENTION TITLE: GENETIC VACCINE THAT MIMICS NATURAL VIRAL INFECTION AND INDUCES LONG-LASTING IMMUNITY TO PATHOGENS The following statement is a full description of this invention, including the best method of performing it known to us: 11i/hJIY7 .t I 67R frnn n-I GENETIC VACCINE THAT MIMICS NATURAL VIRAL INFECTION AND INDUCES LONG-LASTING IMMUNITY TO PATHOGENS Field of the Invention 5 This invention relates to vaccines for stimulating immune responses in human and other hosts, and, in particular, relates to recombinant viruses that express heterologous antigens of pathogenic viruses, such as Ebola virus, HIV, hepatitis viruses, and influenza viruses. 10 Background of the Invention Current techniques for developing vaccines are largely based on the concept of using denatured virus or purified viral proteins made from bacteria. These types of vaccines may be effective for only a limited 15 number of infectious agents, and the protection rates are limited. For viruses that contain membrane (envelope) glycoproteins (GPs), including the Ebola virus and the HIV virus, use of denatured virus or purified viral proteins often does not work satisfactorily. There may be several reasons for this. First, the GPs of these viruses are 20 sensitive to the denaturing procedures so that the epitopes of the proteins are altered by the denaturing process. Second, the sugar moieties of the GPs are important antigenic determinants for neutralizing antibodies. In comparison, proteins made in bacteria are not properly glycosylated and can fold into somewhat different structures that can 25 have antigenecities different from those of the natural viral proteins. Further, many vaccines that are based on attenuated or denatured virus provide a weak immune response to poorly immunogenic antigens. In addition, the vaccine preparations frequently offer only limited protection, not life-long immunity as desired. 30 Other vaccine approaches express antigens by plasmids directly injected into the body, the so-called naked DNA or DNA vaccine 1 a technology. These methods involve the deliberate introduction of a DNA plasmid carrying an antigen-coding gene by transfecting cells with the plasmid in vivo. The plasmid expresses the antigen that causes an immune response. The immune response stimulated by DNA vaccine 5 can be very inefficient, presumably due to low levels of uptake of the plasmid and low levels of antigen expression in the cells. DNA vaccines are also characterized by an extremely short antigen expression period due to vector degradation. In addition, DNA vaccines are difficult and costly to produce in large amounts. 10 Replication-competent, live vaccinia viruses have also been modified for expression of the genes for hepatitis B (HBV), human immunodeficiency virus (HIV), influenza and malaria antigens. In some instances, though, the immune response of recombinant vaccines is often of limited nature and magnitude. Thus, for example, while 15 peripheral immunization with vaccinia influenza recombinants provides good protection against lower respiratory tract infections, it fails to induce immunity in the upper respiratory tract. On the other hand, peripheral immunization with recombinant vaccines may prove ineffective when local rather than systemic immunity is required, as in, 20 for example, the gastro-intestinal tract. Vaccination with recombinant vaccinia virus expressing Ebola virus GP has been attempted to confer partial protection in guinea pigs. Gilligan, K.J., et al., Vaccines, 97:87-92 (1997). Vaccination with DNA constructs expressing either GP or nucleocapsid protein (NP) protects 25 mice from lethal challenge with Ebola virus. Vanderzanden, L., et al., Virology, 246(1):134-44 (1998). However, each of these approaches has its own set of limitations that make them less then ideal choices for Ebola virus vaccines in humans. For example, vaccinia virus rapidly kills vector-infected cells. Consequently, the vaccine antigen is expressed 30 for only a short time. However, the major limitation for this type of approaches is that the replication of vaccina virus causes the immune 2 system to react mainly to the vaccinia proteins, only small portion of the immune responses is targeted to the antigen of the pathogenic virus. This phenomenon has been termed "antigen dilution". Previous attempts to remedy these deficiencies, including expression of 5 vaccine antigens through viruses having stronger promoters, such as poxvirus, have not met with significant success. As yet, no vaccine has been effective in conferring protection against HIV infection. Attempts to develop vaccines have thus far failed. Certain antibodies reactive with HIV, notably anti-GP160/120 are present at high levels throughout both 10 the asymptomatic and symptomatic phases of the HIV infection, suggesting that rather than playing a protective role, such antibodies may in fact promote the attachment and penetration of the virus into the host cell. More significantly, current vaccines do not induce efficient cellular responses against the infected cells, the source of newly released virions. 15 The present application is a divisional application of Australian Patent Application No. 2001271288, the disclosures of which are herein incorporated by reference. SUMMARY OF THE INVENTION 20 The present invention provides a replication incompetent recombinant adenovirus, comprising: one or more antigen sequences heterologous to a native progenitor of the recombinant adenovirus inserted in an El, E3 or E4 region of the adenovirus, each 25 antigen sequence expressing a viral antigen from a pathogenic virus under control of a first promoter located in the same region as the antigen sequences; and one or more immunostimulator sequences heterologous to the native progenitor of the recombinant adenovirus inserted in an E4, E3 or El region of the adenovirus that does not comprise the antigen sequence, each immunostimulator 30 sequence expressing an immunostimulator under the control of a second promoter located in the same region as the immunostimulator sequences; and 24/06/10,dc 16748 amended specification pages 2406 10.doc,3 3 characterised in that the first promoter either bicistronically or polycistronically controls the expression of one or more antigen sequences and that the second promoter either bicistronically or polycistronically controls the expression of one or more immunostimulator sequences, wherein the bicistronic control is via an 5 internal ribosomal entry site or a splicing donor-acceptor mechanism and the polycistronic control is via a splicing donor-acceptor mechanism. It is preferred that the second promoter bicistronically controls expression of two immunostimulator sequences via an internal ribosomal entry site or via a splicing donor-acceptor mechanism when the adenovirus comprises two 10 heterologous immunostimulator sequences. It is preferred that the first promoter polycistronically controls expression of at least two of the antigen sequences via a splicing donor-acceptor mechanism when the adenovirus comprises a plurality of antigen sequences. It is further preferred that the first promoter controls expression of at least 15 two of the antigen sequences as a fusion protein. Genetic viral vaccines are provided. These vaccines are designed to mimic natural infection of pathogenic viruses without causing diseases that are naturally associated with the pathogenic viruses in a host to be immunised, such as human, domestic animals, and other mammals. 20 The vaccines are recombinant benign viruses that are replication deficient or incompetent. The benign viruses may be designed to express antigens from a wide variety of pathogens such as viruses, bacteria and parasites, and thus may be used to treat this wide variety of viruses, bacteria, and parasites that natively express these antigens. Infection of the benign virus causes host cells to express the antigens of the 25 pathogenic virus and presents the antigen in its natural conformation and pathway as if the cell were infected by the pathogenic virus, and induces a strong and long lasting immune response in the host. A recombinant benign virus is provided for eliciting an immune response in a host infected by the virus. The recombinant virus comprises: an antigen sequence 30 heterologous to the benign virus that encodes a viral antigen from a pathogenic virus, expression of the viral antigen eliciting an immune response directed against the pathogenic virus and cells expressing the viral antigen in the host upon infection of 18/03/ 1 O,dc 16748 amended specification pages.doc,4 4 the host by the recombinant virus; and an immuno-stimulator sequence heterologous to the benign virus that encodes an immuno-stimulator whose expression in the host enhances the immunogenicity of the viral antigen. The recombinant virus is replication-incompetent and does not cause disease that is associated with the 5 pathogenic virus in the host The recombinant benign virus may be a replication-incompetent virus such as adenovirus, adeno-associated virus, SV40 virus, retrovirus, herpes simplex virus or vaccinia virus. Preferably, the benign virus does not have the pathologic regions of the native progenitor of the benign virus but retains its infectivity. 10 It is preferred that the benign virus is a replication-incompetent adenovirus, more preferably adenovirus type 5. The heterologous antigen sequence may be positioned in the El, E3 or E4 region of the adenovirus. The immuno-stimulator sequence may be positioned in the E4, E3 or El region of the adenovirus. It is preferred that the heterologous antigen sequence and the immuno 15 stimulator sequence are positioned in the El, E3 or E4 region of the adenovirus, where the heterologous antigen sequence and the immuno-stimulator sequence are expressed from a promoter bicistronically via an internal ribosomal entry site or via a splicing donor-acceptor mechanism. Expression of the viral antigen or the immuno-stimulator may be controlled 20 by a promoter homologous to the native progenitor of the recombinant virus. Alternatively, expression of the viral antigen may be controlled by a promoter heterologous to the native progenitor of the recombinant virus. For example, the promoter heterologous to the native progenitor of the recombinant virus may be a eukaryotic promoter such as insulin promoter, human cytomegalovirus (CMV) 25 promoter and its early promoter, simian virus SV40 promoter, Rous sarcoma virus LTR promoter/enhancer, the chicken cytoplasmic P-actin promoter, and inducible promoters such as the tetracycline-inducible promoter. The pathogenic virus may be any pathogenic virus that causes pathogenic effects or disease in human or other animals. Thus, the recombinant benign virus can 30 be used as a vaccine for protecting the host from infection of the pathogenic virus. Alternatively, it is preferred that the pathogenic virus may be various strains of human immunodeficiency virus (HIV), such as HIV-1 and HIV-2. The viral 18/03/ 10,dc 16748 amended specification pages.doc.5 5 antigen may be an HIV glycoprotein (or surface antigen) such as HIV GP120 and GP41, or a capsid protein (or structural protein) such as HIV P24 protein. Alternatively it is preferred that the pathogenic virus may be Ebola virus. The viral antigen may be an Ebola glycoprotein or surface antigen such as Ebola 5 GPl or GP2 protein. Alternatively, it is preferred that the pathogenic virus may be hepatitis virus such as hepatitis A, B, C, D or E virus. For example, the viral antigen may be a surface antigen or core protein of hepatitis B virus such as the small hepatitis B surface antigen (SHBsAg) (also referred to as the Australia antigen), the middle 10 hepatitis B surface antigen (MHBsAg) and the large hepatitis B surface antigen (LHBsAg). The viral antigen may be a surface antigen or core protein of hepatitis C virus such as NS3, NS4 and NS5 antigens. In an alternative preferred embodiment, the pathogenic virus may be a respiratory syncytial virus (RSV). For example, the RSV viral antigen may be the 15 glycoprotein (G-protein) or the fusion protein (F-protein) of RSV, for which the sequences are available from GenBank. Alternatively, it is preferred that the pathogenic virus may be a herpes simplex virus (HSV) such as HSV-l and HSV-2. For example, the HSV viral antigen may be the glycoprotein D from HSV-2. 20 Alternatively, it is preferred that the viral antigen may be a tumor antigen, such as Her 2 of breast cancer cells and CD20 on lymphoma cells, a viral oncogene such as E6 and E7 of human papilloma virus, or a cellular oncogene such as mutated ras. It is noted that, other virus-associated proteins or antigens are readily 25 available to those of skill in the art. Selection of the pathogenic virus and the viral antigen associated with the pathogenic virus is not a limiting factor. The recombinant virus also expresses an immuno-stimulator to mimic cytokine-releasing response of a host cell upon viral infection and further augments the immune response to the viral antigen co-expressed from the recombinant virus. 30 The immuno-stimulator may preferably be a cytokine. Examples of cytokine include, but are not limited to, interleukin-2, interleukin-8, interleukin-12, p 18/03/10,dc 16748 amended specification pages.doc.6 6 interferon, X-interferon, 7-interferon, granulocyte colony stimulating factor (G-CSF), and granulocyte-macrophage colony stimulating factor (GM-CSF). The viral antigen may be a full-length antigenic viral protein or a portion of the antigenic viral protein that contains the predominant antigen, neutralizing 5 antigen, or epitope of the pathogenic virus. Alternatively, the viral antigen contains the constant region of glycoproteins of at least two strains of the pathogenic virus. It is preferred that the viral antigen may be a modified antigen that is mutated from a glycoprotein of the pathogenic virus such that the viral antigen is rendered non-functional as a viral component but retains its antigenicity. Such 10 modification of the viral antigen includes deletions in the proteolytic cleavage site of the glycoprotein, and duplications and rearrangement of immunosuppressive peptide regions of the glycoprotein. It is preferred that a recombinant virus is provided for eliciting an immune response in a host infected by the virus. The recombinant virus comprises: an 15 antigen sequence heterologous to the recombinant virus that encodes a viral antigen from a pathogenic virus, expression of the viral antigen eliciting an immune response directed against the viral antigen and cells expressing the viral antigen in the host upon infection of the host by the recombinant virus. The recombinant virus is preferably a replication-incompetent adeno 20 associated virus, SV40 virus, retrovirus, herpes simplex virus or vaccinia virus. The recombinant virus is capable of infecting the the host and preferably does not comprise pathologic regions native to the native progenitor of the recombinant virus. Preferably, the recombinant virus includes an immuno-stimulator sequence heterologous to the recombinant virus that encodes an immuno-stimulator whose 25 expression in the host enhances the immunogenicity of the viral antigen. Preferably, there are viral vaccines that present multiple antigens to the host to further mimic natural infection of a native pathogenic virus and induce strong and long-lasting immune response to various strains or types of the pathogenic virus in the host. 30 Preferably, a recombinant virus is provided as a viral vaccine for eliciting an immune response in a host infected by the virus. The recombinant virus comprises: a plurality of antigen sequences heterologous to the recombinant virus, each encoding 18/03/10,dc 16748 amended specification pages.doc.7 7 a viral antigen from a same pathogenic virus, different strains of a pathogenic virus, or different kinds of pathogenic viruses, expression of the plurality of the antigen sequences eliciting an immune response directed against the viral antigen and cells expressing the viral antigen in the host upon infection of the host by the recombinant 5 virus. The recombinant virus may preferably be replication-incompetent and not cause malignancy in the host naturally associated with pathogenic virus. Preferably, the recombinant virus may be any virus, preferably replication incompetent adenovirus, adeno-associated virus, SV40 virus, retrovirus, herpes simplex virus or vaccinia virus. The benign virus may also preferably have the 10 pathologic regions of the native progenitor of the benign virus deleted but retain its infectivity. Preferably, the plurality of the antigen sequences may be multiple copies of the same antigen sequence or multiple antigen sequences that differ from each another. 15 Preferably, at least two of the plurality of the antigen sequences are expressed from a promoter bicistronically via an internal ribosomal entry site or via a splicing donor-acceptor mechanism. Preferably, at least two of the plurality of the antigen sequences are expressed from a promoter to produce a fusion protein. 20 Preferably, the viral genome further comprises at least one promoter heterologous to the native progenitor of the recombinant virus that controls the expression of at least two of the plurality of the antigen sequences. Examples of the promoter heterologous to the native progenitor of the recombinant virus include, but are not limited to, insulin promoter, CMV promoter and its early promoter, SV40 25 promoter, retrovirus LTR promoter/enhancer, the chicken cytoplasmic p-actin promoter, and inducible promoters such as tetracycline-inducible promoter. Preferably, the plurality of antigen sequences may be a combination of antigens from at least two strains of the pathogenic virus. Preferably, the plurality of antigen sequences may be a combination of 30 antigens from at least two different pathogenic viruses. For example, the plurality of antigen sequences may be a combination of antigens from HIV-1, HIV-2, herpes 18/03/10,dc 16748 amended specification pages.doc,8 8 simplex virus type 1, herpes simplex virus type 2, Ebola virus, Marburg virus, and hepatitis A, B, C, D, and E viruses. Preferably, the recombinant virus may further comprise one or more immuno-stimulator sequences that are heterologous to the benign virus and encodes 5 an immuno-stimulator whose expression in the host enhances the immunogenicity of the viral antigen. For example, the immuno-stimulator may be a cytokine. Examples of the cytokine include, but are not limited to, interleukin-2, interleukin-4, interleukin-12, 0- interferon, X-interferon, y-interferon, G-CSF, and GM-CSF. Preferably one or more immuno-stimulator sequences may be multiple 10 copies of the same immuno-stimulator sequence or multiple immuno-stimulator sequences that differ from each other. Preferably, at least two of the immuno-stimulator sequences may be expressed from a promoter multicistronically via an internal ribosomal entry site or via a splicing donor-acceptor mechanism. Alternatively, it is preferred that at least 15 two of the immuno-stimulator sequences may be expressed from a promoter to form a fusion protein. There are genetic vaccines that elicit strong and long-lasting immune response to pathogenic bacteria. A recombinant virus is provided as a genetic bacteria vaccine for eliciting an immune response in a host infected by the 20 recombinant virus. The recombinant virus comprises: a plurality of antigen sequences heterologous to the recombinant virus, each encoding a bacterial antigen from a pathogenic bacteria, expression of the plurality of the bacterial antigen sequences eliciting an immune response directed against the bacterial antigen and cells expressing the bacterial antigen in the host upon infection of the host by the 25 recombinant virus. The recombinant virus may preferably be replication incompetent and not cause malignancy naturally associated with the pathogenic bacteria in the host. The pathogenic bacteria may be any pathogenic bacteria that causes pathogenic effects or diseases in a host, such as bacillus tuberculoses, bacillus 30 anthracis, and spirochete Borrelia burgdorferi that causes the Lyme disease in animals. The plurality of antigen sequences may encode lethal factors, protective antigen, edema factors of the pathogenic bacteria, or combinations thereof. 18/03/10,dc 16748 amended specification pages.doc,9 9 The vaccines against parasites elicit strong and long-lasting immune response to pathogenic parasites. A recombinant virus is provided as a parasite vaccine for eliciting an immune response in a host infected by the recombinant virus. The recombinant virus comprises: a plurality of antigen sequences heterologous to 5 the benign virus, each encoding a parasitic antigen from a pathogenic parasite, expression of the plurality of the parasitic antigen sequences eliciting an immune response directed against the parasitic antigen and cells expressing the parasitic antigen in the host upon infection of the host by the recombinant virus. The recombinant virus may preferably be replication-incompetent and not cause a 10 malignancy naturally associated with the pathogenic parasite in the host. The pathogenic parasite may be any pathogenic parasites that cause pathogenic effects or diseases in a host, such as malaria and protozoa such as Cryptosporidium, Eimeria, Histomonas, Leucocytozoon, Plasmodium, Toxoplasma, Trichomonas, Leishmania, Trypanosoma, Giardia, Babesia, and Theileria. The 15 plurality of antigen sequences may encode coat proteins, attachment proteins of the pathogenic parasites, or combinations thereof. Preferably pharmaceutical compositions include the viral vaccines. The pharmaceutical composition may include any of the recombinant viruses described above and a pharmaceutically acceptable carrier or diluent. 20 The pharmaceutical composition may also include an adjuvant for augmenting the immune response to the viral antigen expressed from the recombinant virus. Examples of the adjuvant include, but are not limited to, bacillus Calmette-Guerin, endotoxin lipopolysaccharide, keyhole limpet hemocyanin, interleukin-2, GM-CSF, and cytoxan. 25 Preferably, the kits may include any one or more vaccines in combination with a composition for delivering the vaccine to a host and/or a device, such as a syringe, for delivering the vaccine to a host. Preferably, there are methods for enhancing the immunity of a host with the recombinant viruses described above. 30 The method comprises: administering to the host a recombinant virus in an amount effective to induce an immune response. The in the recombinant virus comprises: an antigen sequence heterologous to the recombinant virus that encodes a 18/03/i0,dc 16748 amended specification pages.doc,10 10 viral antigen from a pathogenic virus, expression of the viral antigen eliciting an immune response directed against the viral antigen and cells expressing the viral antigen in the host upon infection of the host by the recombinant virus; and an immuno-stimulator sequence heterologous to the benign virus that encodes an 5 immuno-stimulator whose expression in the host enhances the immunogenicity of the viral antigen. The recombinant virus may preferably be replication-incompetent and not cause malignancy naturally associated with the pathogenic virus in the host. The recombinant virus may be administered to the host via any pharmaceutically acceptable route of administration. The recombinant virus may be 10 administered to the host via a route of intramuscular, intratracheal, subcutaneous, intranasal, intradermal, rectal, oral and parental administration. Preferably, a method is provided for enhancing the immunity of a host to a pathogenic virus with multiple antigens. The method comprises: administering to the host a recombinant virus in an amount effective to induce an immune response. The 15 recombinant virus comprises: a plurality of antigen sequences heterologousto the benign virus, each encoding a viral antigen from a pathogenic virus, expression of the plurality of the antigen sequences eliciting an immune response directed against the viral antigen and cells expressing the viral antigen in the host upon infection of the host by the recombinant virus. The recombinant virus may preferably be 20 replication-incompetent and not cause malignancy naturally associated with the pathogenic virus in the host. Preferably, the recombinant virus may further comprise one or more immuno-stimulator sequences heterologous to the recombinant virus that encodes an immuno-stimulator whose expression in the host enhances the immunogenicity of the 25 viral antigen. Preferably, a method is provided for enhancing the immunity of a host to a pathogenic virus by using multiple recombinant viral vaccines (or viruses). Multiple recombinant viruses may carry different antigens in each recombinant virus. The multiple recombinant viruses may be administered simultaneously or step-wise to the 30 host. The method comprises: administering to a host a first and second recombinant viruses in an amount effective to induce an immune response. The first 18103/1O,dc 16748 amended specification pages.doc,I I 11 recombinant virus comprises: an antigen sequence heterologous to the first recombinant virus that encodes a viral antigen from a pathogenic virus, expression of the viral antigen eliciting an immune response directed against the viral antigen and cells expressing the viral antigen in the host upon infection of the host by the 5 recombinant virus. The second recombinant virus comprises: an immuno-stimulator sequence heterologous to the recombinant virus that encodes an immuno-stimulator whose expression in the host enhances the immunogenicity of the viral antigen. The first and second recombinant viruses may preferably be replication-incompetent and not cause a malignancy naturally associated with the pathogenic virus in the host. 10 Preferably, the first and second recombinant virus may be any benign virus, such as replication-incompetent adenovirus, adeno-associated virus, SV40 virus, retrovirus, herpes simplex virus and vaccinia virus. Preferably, both the first and second recombinant viruses may be replication-incompetent adenovirus. Preferably, one of the first and second recombinant viruses may be recombinant adenovirus and 15 the other may be recombinant vaccinia virus. Preferably, a method is provided for enhancing the immunity of a host to a pathogen. The method comprises: administering to the host a recombinant virus and one or more immuno-stimulators. The recombinant virus may be any of the recombinant viruses described above. In particular, the recombinant virus comprises 20 one or more antigen sequences heterologous to the recombinant virus that encode one or more antigens from the pathogen. Expression of the antigen elicits an immune response directed against the antigen and cells expressing the antigen in the host upon infection of the host by the recombinant virus. The recombinant virus is preferably replication-incompetent and does not cause a malignancy naturally 25 associated with the pathogen in the host. The pathogen may be a pathogenic virus such as HIV, hepatitis virus and Ebola virus, a pathogenic bacteria or parasite. Preferably, the immuno-stimulator may be any molecule that enhances the immunogenicity of the antigen expressed by the cell infected by the recombinant virus. Preferably, the immuno-stimulator is a cytokine, including, but not limited to 30 interleukin-2, interleukin-8, interleukin-12, p- interferon, X-interferon, y-interferon, granulocyte colony stimulating factor, granulocyte-macrophage colony stimulating factor, and combinations thereof. The cytokine may be administered into the host in 18/03/1 0,dc 16748 amended specification pagcs.doc, 12 12 a form of purified protein alone or formulated with one or more pharmaceutically acceptable excipients. It is preferred that the cytokine may be administered in a form of expression vector that expresses the coding sequence of the cytokine upon transfecting or transducing the cells of the host. 5 BRIEF DESCRIPTION OF THE FIGURES Figures 1A-1C illustrate an example of how to construct a genetic vaccine of the present invention. 10 Figure 1A illustrates an example of a shuttle vector pLAd.Antigen carrying multiple antigen genes such as Antigen 1 and Antigen 2 which can be expressed from a CMVie promoter bicistronically via a splicing donor-acceptor mechanism at the SD and SA sites. Figure lB illustrates an example of a shuttle vector pRAd.Cytokines 15 carrying multiple cytokine genes such as IL-2, INF, and IL-8 genes which can be expressed from a CMVie promoter bicistronically via an internal ribosomal entry site IRES and a splicing donor-acceptor mechanism at the SD and SA sites. Figure 1C illustrates an example of constructing a genetic vaccine by ligating with an adenoviral backbon with a fragment that is derived from the shuttle 20 vector pLAd.Antigen and contains multiple antigen genes and a fragment that is derived from the shuttle vector pRAd.Cytokines and contains multiple cytokine genes. Figure 2 illustrates the wild-type GP gene, which encodes the two forms of glycoproteins (sGP and GP), contains a RNA editing signal 25 30 18/03/ 10,dc 16748 amended specification pages.doc, 13 13 THIS PAGE LEFT INTENTIONALLY BLANK 18/03/10,dc 16748 amended specification pages.doc, 14 14 that results in un-edited and edited mRNAs. The sGP is synthesized from an un-edited mRNA and the GP is synthesized from an edited mRNA (having an insertion in one of the seven uridines). Figure 2 also depicts the modifications made to the RNA to prevent the synthesis of 5 sGP. The RNA editing site is modified from UUU UUU U to UUC UUC UU. This modification removes the editing signal and results in the mRNA coding only for the GP. Figure 3 illustrates the modification of the immunosuppressive peptide (IS) located in GP2. Figure 3A shows the wild type GP. Figure 10 3B shows GP with the 10 amino acid deletion of the IS peptide. Figure 3C shows the IS peptide, which is split, reversed and duplicated. Abbreviations: FP, Fusion peptide; IS, Immunosuppressive peptide; TM, Transmembrane domain. Figures 4A and 4B illustrate a procedure used to create a 15 recombinant adenoviral vector as a genetic vaccine against Ebola virus. Figures 4A illustrates a shuttle vector pLAd/EBO-GP carrying the GP gene of Ebola virus an antigen, and a shuttle vector pRAdlL2,4 carrying the IL-2 and IL-4 gene. 20 Figure 4B illustrates the construction of a recombinant adenoviral vector by ligating an adenoviral backbone with a fragment that is derived from the shuttle vector pLAd/EBO-GP and contains the GP gene and a fragment that is derived from the shuttle vector pRAdIL2,4 and contains IL-2 and IL-4 genes. 25 Figure 5 illustrates a complex adenoviral vector as an example of the genetic vaccine of the present invention. The Ebola viral GP gene is expressed by a CMVie promoter in the El region. The GP gene is followed by INF-y and GM-CSF which are expressed by two IRES sequences. This configuration allows for the expression of three 30 proteins from a single mRNA. Expression of IL-2 and IL-4 is controlled by a second CMVie promoter as a bi-cistronic cassette, and followed by 15 a second bi-cistronic cassette that expressed the two subunits of IL12 in the E4 region by a SV40 early promoter. Figure 6 shows relative titers of antibody against HIV antigens in a group of mice. 5 Figure 7 shows relative titers of antibody against HIV antigens in another group of mice. Figure 8A-C show INF-y secretion from activated splenocytes harvested from mice inoculated with adenoviral vectors in response to target cell stimulation. 10 Figure 9 show granzyme A secretion from activated splenocytes harvested from mice inoculated with adenoviral vectors in response to target cell stimulation. Figure 1 OA shows relative titers of antibody against HBV surface antigen in a group of mice. 15 Figure 1OB shows relative titers of antibody against HBV surface antigen in another group of mice. Figure 11A shows relative titers of antibody against HBV core antigen in a group of mice. Figure 11 B shows relative titers of antibody against HBV core 20 antigen in another group of mice. Figure 12 shows relative titers of antibody against HiV antigen gp120 in mice. Figure 13 shows relative titers of antibody against HiV antigen Gag in mice. 25 Figure 14 shows relative titers of antibody against HiV antigen Rev in mice. Figure 15 A shows INF-y secretion from activated splenocytes harvested (week 4 post-inoculation) from mice inoculated with adenoviral vectors in response to target cell stimulation. 16 Figure 15 B shows INF-y secretion from activated splenocytes harvested (week 8 post-inoculation) from mice inoculated with adenoviral vectors in response to target cell stimulation. Figure 15 C shows INF-y secretion from activated splenocytes 5 harvested (week 12 post-primary inoculation and week 1 post secondary inoculation) from mice inoculated with adenoviral vectors in response to target cell stimulation. DETAILED DESCRIPTION OF THE INVENTION 10 The present invention provides genetic vaccines, pharmaceutical compositions including the vaccines and methods of immunizing a host against infection of a wide range of pathogenic viruses, bacteria and parasites. The genetic vaccines are recombinant benign viruses that 15 are replication deficient and do not cause malignancy in the host to be immunized. Vaccination using the genetic vaccines of the present invention mimics natural viral infection in that the antigen(s) expressed by the cell infected by the genetic vaccine is presented to the host immune system in its natural conformation and by a "inside-out" 20 mechanism, as compared with the conventional "outside-in" approach of vaccination using denatured protein or virus as a vaccine. In addition, the cell infected by the genetic vaccine also releases high levels of cytokine, thereby mimicking the natural response of the cell under stress induced by viral infection and yet not causing pathogenic effects on the 25 cells. Mistaken by such a "signal of pathogenic viral infection", the host immune system mounts a strong immune defense against the antigen presented by the infected cell. Therefore, in a sense, the genetic vaccine of the present invention behaves like a "sheep in wolf's clothing", presenting the viral antigen to induce a strong immune 30 response and yet not causing the detrimental effects that the pathogens would cause on the host. 17 In one embodiment, a recombinant virus is provided for eliciting an immune response in a host infected by the virus. The recombinant virus comprises: an antigen sequence heterologous to the recombinant virus that encodes a viral antigen from a pathogenic virus, expression of 5 the viral antigen eliciting an immune response directed against the viral antigen and cells expressing the viral antigen in the host upon infection of the host by the recombinant virus; and an immuno-stimulator sequence heterologous to the recombinant virus that encodes an immuno-stimulator whose expression in the host enhances the 10 immunogenicity of the viral antigen. The recombinant virus is replication-incompetent and does not cause the malignancy naturally associated with the pathogenic virus in the host. In another embodiment, a recombinant virus is provided as a viral vaccine for eliciting an immune response against multiple antigens in a 15 host infected by the virus. The recombinant virus comprises: a plurality of antigen sequences heterologous to the benign virus, each encoding a different viral antigen from one or more pathogenic viruses, expression of the plurality of the antigen sequences eliciting an immune response directed against the viral antigens and cells expressing the viral antigen 20 in the host upon infection of the host by the recombinant virus. The recombinant virus may preferably be replication-incompetent and not cause the malignancy that is naturally associated with the pathogenic virus(es) in the host. The vaccines of the present invention can be used to immunize 25 the host against a wide variety and different strains of pathogenic viruses such as HIV-1, HIV-2, herpes simplex virus type 1, herpessimplex virus type 2, Ebola virus, Ebola virus, and hepatitis A, B, C, D, and E viruses, or pathogenic bacteria such as bacillus tumerculoses and bacillus anthracis. 30 The recombinant vaccine of the present invention is a recombinant virus that contains nucleic acid sequences encoding one or 18 more viral antigens in the viral genome. When a host is immunized by the recombinant vaccine, i.e., infected by the recombinant virus, the infection of the virus in a host cell results in expression of the viral antigen which is present on the surface of the infected cell. Since 5 expression of the viral antigen is driven by a strong promoter, expession can be maintained at a high level. Upon recognizing the large amount viral antigen on the cell surface, the host immune system mounts a strong defense against the viral antigen, thereby achieving long-lasting immunity against the pathogenic virus from which the viral antigen is 10 derived. Compared with immunization with vaccines that are isolated proteins expressed by bacteria, yeast or insect cells, the viral antigen expressed from the recombinant virus of the present invention better mimics the natural viral antigen in its structure and function. Isolated 15 protein vaccine may not adopt the native conformation of the natural viral antigen and may not be properly glycosylated in the bacteria, yeast or insect cells. When such an isolated protein vaccine is injected into the host, this antigen is presented from the outside of the host cell. This conventional "outside-in" approach often does not generate strong, 20 long-lasting immune response, presumably due to the altered antigenicity of the vaccine and quick clearance of the protein vaccine by the immune scavenging cells. In contrast, the genetic vaccine of the present invention, i.e., the recombinant virus, presents the viral antigen by an "inside-out" 25 mechanism. The viral antigen is expressed after infection of the recombinant virus in the host cells. This better mimics the natural production and presentation of the viral antigen by the pathogenic virus. By using a replication incompetent virus that is incapable of spreading beyond initially infected cells, the present invention 30 dramatically reduces the risk of side effects that may potentially be generated by using replication-competent, live virus. For example, 19 vaccines based on live vaccinia virus can replicate in the host cells, which can impose a high level of stress on the host cell and eventually lead to cell death. Moreover, compared to the approach of using attenuated or 5 inactive virus as a vaccine, the process of making the genetic vaccine of the present invention is much safer. Vaccination of a large population of people or animals demand large amounts of vaccines. For virulent viruses such as Ebola virus and HIV, large-scale production of attenuated or inactive virus from the live virus can pose a great danger 10 to the environment and people who handle the live virus. The recombinant virus of the present invention can be used to express multiple antigen sequences simultaneously from the same viral vector. Thus, the recombinant virus may encode multiple antigens from the same strain of pathogenic virus, from different strains of the same 15 pathogenic viruses, or from different antigens from different kind of viruses, bacteria or parasites. This enables the vaccines of the present invention to be utilized to immunize against a broad-spectrum of viruses and other infectious agents. Since these multiple antigen sequences are rearranged in the recombinant viral genome, the risk of potential 20 recombination of these viral sequences to generate a pathogenic virus is virtually eliminated. The genetic vaccine of the present invention also preferably express large amount of immunuo-stimulator, such as cytokine. In a natural process of viral infection, virus-infected cells display viral 25 antigens on their surface in the context of the MHC-I receptor, while viral particles are digested by the professional antigen-presenting cells which display antigens in association with MHC-l1 receptors. In response to viral infection, a full range of cytokines and interferons are produced, resulting in a strong humoral and cellular response to the viral antigens. 30 At the same, large numbers of memory cells remain to defeat any new infection. In vaccinations using isolated protein vaccines, the protein is 20 quickly cleared by the immune scavenging cells. During this process, only MHC-1l antigen presentation occurs and the cytokine-releasing response is absent or greatly diminished. As a result, little cellular response is generated and few "memory" cells are produced. 5 In comparison, co-expression of viral antigen and cytokine from the recombinant virus of the present invention effectively mimics the natural response of the host cell to viral infection by presenting the antigen on the surface of the infected and producing large amount of immuno-modulating cytokines. With the high levels of cytokine 10 expressed from the host cells infected by the genetic vaccine, the host immune system would be "tricked" to mount a strong response to vaccine, thereby resulting in a longer-lasting immunity. Additionally, although vaccination with the genetic vaccine mimics the natural viral infection of a pathogenic virus, the vaccine itself is a 15 benign virus that does not have the detrimental effects of the pathogenic virus. For example, infection of a pathogenic virus such as HIV, influenza virus and Ebola virus has profound immuno-suppressing effects on the host, presumably due to the immuno-suppressing functions of the glycoproteins of the virus. According to the present 20 invention, the viral antigen sequence carried by the genetic vaccine is preferred to have its pathogenic or immuno-suppressing regions deleted. In a sense, the genetic vaccine of the present invention behaves like a "sheep in wolf's clothing", presenting the viral antigen to induce strong immune response and yet not causing detrimental effects 25 on the host. 1. The Genetic Vaccines of the Present Invention The present invention is directed to vaccines that mimic the features of a native pathogenic virus, but without eliciting immuno 30 suppression and pathogenicity, thus causing the host to mount an effective defense, while not being in any actual danger of infection. The 21 genetic vaccines are replication incompetent or defective viruses into which one or more DNA sequences encoding one or more viral antigens are inserted into the regions of the viral genome non-essential to its infectivity. The recombinant virus expresses the viral antigens and 5 elicits a cell-mediated immune response in vivo directed against the antigens and cells expressing the antigens. In one embodiment, a recombinant virus is provided for eliciting an immune response in a host infected by the virus. The recombinant virus comprises: an antigen sequence heterologous to the recombinant 10 virus that encodes a viral antigen from a pathogenic virus, expression of the viral antigen eliciting an immune response directed against the viral antigen and cells expressing the viralantigen in the host upon infection of the host by the recombinant virus; and an immuno-stimulator sequence heterologous to the recombinant virus that encodes an 15 immuno-stimulator whose expression in the host enhances the immunogenicity of the viral antigen. The recombinant virus is replication-incompetent and does not cause a malignancy naturally associated with the pathogenic virus in the host. The recombinant virus may be constructed from any virus as long 20 as the native progenitor is rendered replication incompetent. For example, replication-incompetent adenovirus, adeno-associated virus, SV40 virus, retrovirus, herpes simplex virus or vaccinia virus may be used to generate the recombinant virus by inserting the viral antigen into the region non-essential to the infectivity of the recombinant virus. 25 Therefore, it is preferred that the recombinant virus does not have the pathologic regions of the native progenitor of the benign virus but retains its infectivity to the host. In a preferred embodiment, the recombinant virus is a replication incompetent adenovirus. 30 The recombinant adenovirus of the present invention can direct high levels of antigen expression that provide strong stimulation of the 22 immune system. The antigen expressed by cells infected by adenovirus is processed and displayed in the infected cells in a way that mimics pathogen-infected cells. This phase is believed to be very important in inducing cellular immunity against infected cells, and is completely 5 lacking when conventional vaccination approaches are used. Further, the recombinant adenovirus may infect dendritic cells which are very potent antigen-presenting cells. Further, the recombinant adenovirus may also carry genes encoding immuno-enhancing cytokines to further boost immunity. Moreover, the recombinant adenovirus may naturally 10 infect airway and gut epithelial cells in humans, and therefore the vaccine may be delivered through nasal spray or oral ingestion. In addition, the recombinant adenovirus of the present invention should be safe because it is replication-incompetent. The heterologous antigen sequence may be positioned in the 15 El, E3 or E4 region of the adenovirus. The immuno-stimulator sequence may be positioned in the El, E3 or E4 region of the adenovirus. In a variation of the preferred embodiment, the heterologous antigen sequence and the immuno-stimulator sequence are positioned 20 in the El, E3 or E4 region of the adenovirus, where the heterologous antigen sequence and the immuno-stimulator sequence are expressed from a promoter bicistronically via an internal ribosomal entry site or via a splicing donor-acceptor mechanism. The expression of the viral antigen or the immuno-stimulator may 25 be controlled by a promoter homologous to the native progenitor of the recombinant virus. Alternatively, the expression of the viral antigen may be controlled by a promoter heterologous to the native progenitor of the recombinant virus. For example, the promoter heterologous to the native progenitor of the recombinant virus may be a eukaryotic promoter 30 such as insulin promoter, human cytomegalovirus (CMV) promoter and its early promoter, simian virus SV40 promoter, Rous sarcoma virus LTR 23 promoter/enhancer, the chicken cytoplasmic p-actin promoter, and inducible promoters such as the tetracycline-inducible promoter. The pathogenic virus may be any pathogenic virus that causes pathogenic effects or disease in a host such as human, domestic 5 animals or other mammals. Thus, the recombinant virus can be used as a vaccine for protecting the host from infection of the pathogenic virus. In a variation, the pathogenic virus may be various strains of human immunodeficiency virus (HIV), such as HIV-1 and HIV-2. The viral antigen may be a HIV glycoprotein (or surface antigen) such as HIV 10 GP120 and GP41, a capsid protein (or structural protein) such as HIV P24 protein, or other HIV regulatory proteins such as Tat, Vif and Rev proteins. In another variation, the pathogenic virus may be influenza virus. The viral antigen may be an influenza glycoprotein such as influenza 15 HAI, HA2 and NA. In another variation, the pathogenic virus may be Ebola virus. The viral antigen may be an Ebola glycoprotein or surface antigen such as Ebola GP1 and GP2 protein. In yet another variation, the pathogenic virus may be hepatitis 20 virus such as hepatitis A, B, C, D or E virus. The viral antigen may be a surface antigen or core protein of hepatitis A, B, C, D or E virus. For example, the viral antigen may be a surface antigen or core protein of hepatitis B virus such as the small hepatitis B surface antigen (SHBsAg) (also referred to as the Australia antigen), the middle hepatitis B surface 25 antigen (MHBsAg) and the large hepatitis B surface antigen (LHBsAg). The viral antigen may also be a surface antigen or core protein of hepatitis C virus such as NS3, NS4 and NS5 antigens. In yet another variation, the pathogenic virus may be a respiratory syncytial virus (RSV). For example, the RSV viral antigen 30 may be the glycoprotein (G-protein) or the fusion protein (F-protein) of RSV, for which the sequences are available from GenBank. 24 In yet another variation, the pathogenic virus may be a herpes simplex virus (HSV) such as HSV-1 and HSV-2. For example, the HSV viral antigen may be the glycoprotein D from HSV-2. In yet another variation, the viral antigen may be a tumor antigen 5 or viral oncogene such as E6 and E7 of human papilloma virus, or cellular oncogenes such as mutated ras or p53. It is noted that, other virus-associated proteins or antigens are readily available to those of skill in the art. Selection of the pathogenic virus and the viral antigen is not a limiting factor in this invention. 10 The viral antigen may be a full-length antigenic viral protein or a portion of the antigenic viral protein that contains the predominant antigen, neutralizing antigen, or epitope of the pathogenic virus. Alternatively, the viral antigen contains the conserved region of glycoproteins between at least two strains of the same pathogenic virus. 15 In a variation, the viral antigen may be a modified antigen that is mutated from a glycoprotein of the pathogenic virus such that the viral antigen is rendered non-functional as a viral component but retains its antigenicity. Such modification of the viral antigen includes deletions in the proteolytic cleavage site of the glycoprotein, and duplications and 20 rearrangement of immunosuppressive peptide regions of the glycoprotein. The recombinant virus also expresses an immuno-stimulator to mimic cytokine-releasing response of a host cell upon viral infection and further augments immune response to the viral antigen co-expressed 25 from the recombinant virus. The immuno-stimulator may preferably be a cytokine. Examples of cytokine include, but are not limited to, interleukin-2, interleukin-4, interleukin-12, P- interferon, %-interferon, =y interferon, granulocyte colony stimulating factor (G-CSF), and granulocyte-macrophage colony stimulating factor (GM-CSF). 30 In another embodiment, a recombinant virus is provided for eliciting an immune response in a host infected by the virus. The 25 recombinant virus comprises: an antigen sequence heterologous to the recombinant virus that encodes a viral antigen from a pathogenic virus, expression of the viral antigen eliciting an immune response directed against the viral antigen and cells expressing the viral antigen in the 5 host upon infection of the host by the recombinant virus. According to this embodiment, the recombinant virus is preferably be replication-incompetent adeno-associated virus, SV40 virus, retrovirus, herpes simplex virus or vaccinia virus. The benign virus may preferably have the pathologic regions of the native progenitor of the 10 benign virus deleted but retains its infectivity to the host. Optionally, the recombinant virus includes an immuno-stimulator sequence heterologous to the recombinant virus that encodes an immuno-stimulator whose expression in the host enhances the immunogenicity of the viral antigen. 15 The present invention also provides genetic vaccines that elicit strong and long-lasting immune response to pathogenic bacteria. In one embodiment, a recombinant virus is provided as a genetic bacteria vaccine for eliciting an immune response in a host infected by the recombinant virus. The viral genome of the recombinant virus 20 comprises: a plurality of antigen sequences heterologous to the recombinant virus, each encoding a bacterial antigen from a pathogenic bacteria, expression of the plurality of the bacterial antigen sequences eliciting an immune response directed against the bacterial antigen and cells expressing the bacterial antigen in the host upon infection of the 25 host by the recombinant virus. The recombinant virus may preferably be replication-incompetent and not cause a malignancy naturally associated with the pathogenic bacteria in the host. The pathogenic bacteria may be any pathogenic bacteria that causes pathogenic effects or diseases in a host, such as bacillus 30 tuberculoses, bacillus anthracis, and spirochete Borrelia burgdorfen' that causes the Lyme disease in animals. The plurality of antigen sequences 26 may encode lethal factors, protective antigen, edema factors of the pathogenic bacteria, or combination thereof. The present invention also provides parasites vaccines that elicit strong and long-lasting immune response to pathogenic parasites. In 5 one embodiment, a recombinant virus is provided as a parasite vaccine for eliciting an immune response in a host infected by the benign virus. The recombinant virus comprises: a plurality of antigen sequences heterologous to the recombinant virus, each encoding a parasitic antigen from a pathogenic parasite, expression of the plurality of the 10 parasitic antigen sequences eliciting an immune response directed against the parasitic antigen and cells expressing the parasitic antigen in the host upon infection of the host by the recombinant virus. The recombinant virus may preferably be replication-incompetent and not a cause malignancy naturally associated with the pathogenic parasite in 15 the host. The pathogenic parasite may be any pathogenic parasite that causes pathogenic effects or diseases in a host, such as malaria and protozoa such as Cryptosporidium, Eimeria, Histomonas, Leucocytozoon, Plasmodium, Toxoplasma, Trichomonas, Leishmania, 20 Trypanosoma, Giardia, Babesia, and Theileria. The plurality of antigen sequences may encode coat proteins, attachment proteins of the pathogenic parasites, or combinations thereof. The present invention also provides viral vaccines that present multiple antigens to the host to further mimic natural infection of a native 25 pathogenic virus and induce strong and long-lasting immune response to various strains or types of the pathogenic virus in the host. In one embodiment, a recombinant virus is provided as a viral vaccine for eliciting an immune response in a host infected by the virus. The recombinant virus comprises: a plurality of antigen sequences 30 heterologous to the recombinant virus, each encoding a viral antigen from a pathogenic virus, expression of the plurality of the antigen 27 sequences eliciting an immune response directed against the viral antigen and cells expressing the viral antigen in the host upon infection of the host by the recombinant virus. The recombinant virus may preferably be replication-incompetent and not cause a malignancy 5 naturally associated with the pathogenic virus in the host. According to the embodiment, the recombinant virus may be any virus, preferably replication-incompetent adenovirus, adeno-associated virus, SV40 virus, retrovirus, herpes simplex virus or vaccinia virus. The recombinant virus may also preferably have the pathologic regions of 10 the native progenitor of the benign virus deleted but retain its infectivity to the host. Also according to the embodiment, the plurality of the antigen sequences may be multiple copies of the same antigen sequence or multiple antigen sequences that differ from each another. 15 In a variation of the embodiment, at least two of the plurality of the antigen sequences are expressed from a promoter bicistronically via an internal ribosomal entry site or via a splicing donor-acceptor mechanism. Alternatively, at least two of the plurality of the antigen sequences 20 are expressed from a promoter to form a fusion protein. Also according to the embodiment, the recombinant virus further comprises at least one promoter heterologous to the native progenitor of the recombinant virus that controls the expression of at least two of the plurality of the antigen sequences. Examples of the promoter 25 heterologous to the native progenitor of the recombinant virus include, but are not limited to, insulin promoter, CMV promoter and its early promoter, SV40 promoter, Rous sarcoma virus LTR promoter/enhancer, the chicken cytoplasmic p-actin promoter, and inducible promoters such as tetracycline-inducible promoter. 28 Also according to the embodiment, the plurality of antigen sequences may be a combination of antigens from at least two strains of the pathogenic virus. Optionally, the plurality of antigen sequences may be a 5 combination of antigens from at least two different pathogenic viruses. For example, the plurality of antigen sequences may be a combination of antigens from HIV-1, HIV-2, herpes simplex virus type 1, herpes simplexvirus type 2, influenza virus, Marburg virus, Ebola virus, and hepatitis A, B, C, D, and E viruses. 10 In a variation of the embodiment, the viral genome of the recombinant virus may further comprise one or more immuno-stimulator sequences that is heterologous to the recombinant virus and encodes an immuno-stimulator whose expression in the host enhances the immunogenicity of the viral antigen. For example, the immuno 15 stimulator may be a cytokine. Examples of the cytokine include, but are not limited to, interleukin-2, interleukin-4, interleukin-12, P- interferon, X interferon, y-interferon, G-CSF, and GM-CSF. According to the variation, the one or more immuno-stimulator sequences may be multiple copies of the same immuno-stimulator 20 sequence or multiple immuno-stimulator sequences that differ from each other. Optionally, at least two of the immuno-stimulator sequences may be expressed from a promoter bicistronically via an internal ribosomal entry site or via a splicing donor-acceptor mechanism. Alternatively, at 25 least two of the immuno-stimulator sequences may be expressed from a promoter to form a fusion protein. The DNA sequence encoding viral antigen(s) is inserted into any non-essential region of the replication defective virus. In the case of adenovirus, for example, the nucleic acid is preferably inserted into the 30 El, E3 and/or E4 region of the adenovirus and most preferably into the E4 region. Because the El, E3 and E4 regions are available as 29 insertion sites, the present invention also contemplates separate insertion of more than one encoding sequence. In the recombinant viral vector vaccines of the present invention, the selected nucleotide sequences of the viral antigens are operably 5 linked to control elements that direct transcription or expression thereof in the subject in vivo. Either homologous or heterologous viral control sequences can be employed. Useful heterologous control sequences generally include those derived from sequences encoding hostian or viral genes. Examples include, but are not limited to a cytomegalovirus 10 (CMV) promoter such as the CMV immediate early promoter region (CMVI), SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter (AdMLP), a herpes simplex virus promoter,and a retrovirus LTR promoter. Preferably, any strong constitutive promoter may be operatively linked to viral antigens or 15 cytokines. More preferably the viral promoter is CMV immediate early promoter (CMV). Figures 1A-1C illustrate a method for constructing a recombinant adenoviral vector as a genetic vaccine of the present invention. The recombinant adenoviral vector of the present invention is constructed by 20 using shuttle plasmids or vectors carrying multiple antigen genes and multiple cytokine genes. Figure 1A illustrates a shuttle plasmid (pLAd.Antigen) containing two antigen genes, Antigen I and Antigen 2.. The shuttle plasmid pLAd.Antigen contains the left end of the adenoviral genome including 25 the left long terminal repeats L-TR, and an adenoviral packaging signal (y). The El region of the adenovirus is replaced by a multiple gene expression cassette and CMV. promoter. Genes encoding Antigen 1 and Antigen 2 are placed under the transcriptional control of the CMV, promoter by a splicing mechanism at 30 the SD and SA sites. The plasmid pLAd.Antigen also contains a SV40 30 polyadenylation site, as well as prokaryotic replication origin and ampicillin-resistance gene for DNA propagation in bacteria. Figure 1B illustrates another shuttle plasmid (pRAd.Cytokines) containing multiple cytokine genes such as IL-2, INF, and IL-8. The 5 shuttle plasmid pRAd.Cytokines contains the right end of the adenoviral genome including the right long terminal repeats R-TR. Most of the E4 region (except orf6) is replaced by the cytokine genes. Expression of cytokine genes is under the transcriptional control of the CMV, promoter via an internal ribosomal entry site (IRES) and by a splicing mechanism 10 at the SD and SA sites. The plasmid pRAd.Cytokines also contains a bovine growth hormone (BGH) polyadenylation site, as well as a prokaryotic replication origin and ampicillin-resistance gene for DNA propagation in bacteria. The recombinant adenoviral genome is assembled from the two 15 shuttle plasmids, pLAd.Antigen and pRAd.Cytokines, which carries the left and right end of the adenoviral genome, respectively. The shuttle plasmids pLAd.Antigen and pRAd.Cytokines are digested with restriction enzymes such as Xbal and EcoRI, respectively. As illustrated in Figure IC, the fragments corresponding to the 20 left end and right end of adenovirus from these two shuttle plasmids, pLAd.Antigen and pRAd.Cytokines, are isolated and ligated to the middle section of the adenoviral genome (the adenovirus backbone). The ligated vector genome DNA is then transfected into 293HK cells that express the El proteins of adenovirus. In the presence of El 25 proteins, the vector genome in which the El has been deleted can replicate and be packaged into viral particle, i.e. producing the recombinant adenoviral vector that can be used as a genetic vaccine of the present invention. The El region which is preserved in a native adenoviral genome but deleted from the recombinant viral genome is an 30 example of the pathologic region native to the native progenitor of the recombinant virus: the wild type adenovirus. 31 Figure 5 illustrates an example of a genetic vaccine constructed by using the method described above. The replication defective adenovirus, type 5, is the vector backbone into which viral antigen and cytokines are inserted in the El region. The viral antigens are 5 expressed using the CMVie promoter. The gene for the viral antigen is followed by the gene encoding INF-y and GM-CSF, utilizing 2 IRES sequences to achieve expression of the three proteins from a single mRNA. 1L2 and 1L4 are controlled by a second CMV, promoter as a bi cistronic cassette, followed by a second bi-cistronic cassette that 10 express the two subunits of IL12 in the E4 region. Those skilled in the art will appreciate that the present invention is not limited to the structure discussed above, but that alternative cytokines may be used alone or in combination with these and/or other cytokines. The detailed information about of these cytokines are described in the following section. 15 2. Cytokines Co-Expressed With Viral Antigens The recombinant virus of the present invention may also express an immuno-stimulator to mimic cytokine-releasing response of a host cell upon viral infection and further augment immune response to the 20 viral antigen co-expressed from the recombinant virus. The immuno stimulator may be an immunoenhancing cytokine to further stimulate the immune system. The recombinant virus may encode one or multiple cytokines in any combination. Alternatively, multiple cytokines may be expressed by more than one recombinant virus or delivered to the host 25 by using other techniques such as delivery via naked DNA plasmids or injection of cytokine proteins. Examples of cytokine include, but are not limited to, interleukin-2, interleukin-4, interleukin-8, interleukin-12, P- interferon, X-interferon, y interferon, granulocyte colony stimulating factor (G-CSF), and 30 granulocyte-macrophage colony stimulating factor (GM-CSF). 32 Cytokines are immunodmodulatory molecules particularly useful in the vaccines of the invention as they are pleitropic mediators that modulate and shape the quality and intensity of the immune response. Cytokines are occasionally autocrines or endocrines, but are largely 5 paracrine hormones produced in nature by lymphocytes and monocytes. As used herein, the term "cytokine" refers to a member of the class of proteins or peptides that are produced by cells of the immune system and that regulate or modulate an immune response. Such regulation can occur within the humoral or the cell mediated immune 10 response and includes modulation of the effector function of T cells, B cells, NK cells, macrophages, antigen presenting cells or other immune system cells. Cytokines are typically small proteins or glycoproteins having a molecular mass of less than about 30 kDa. As used herein the term 15 cytokine encompasses those cytokines secreted by lyphocytes andother cell types (often designated as lymphokines) as well as cytokines secreted by monocytes and macrophages and other cell types (often designated as monokines). As used herein, the term cytokine encompasses those cytokines secreted by lymphocytes andother cell 20 types as well as cyotkines secreted by monocytes and macrophages and other cell types. The term cytokine includes the interleukins, such as IL-2, IL-4, IL-8 and IL-12, which are molecules secreted by leukocytes that primarily affect the growth and differentiation of hematopoietic and immune system cells. The term cytokine also 25 includes hematopoietic growth factors and, in particular, colony stimulating factors such as colony stimulating factor-1, granulocyte colony stimulating factor and granulocyte macrophage colony stimulating factor. The cytokines can have the sequence of a naturally occurring 30 cytokine or can have an amino acid sequence with substantial amino acid sequence similarity, e.g., 60-95% amino acid sequence similarity, 33 preferably 70-98% amino acid sequence, and most preferably 75-95% amino acid sequence similarity to the sequence of a naturally occurring cytokine. Thus, it is understood that limited modifications to a naturally 5 occurring sequence can be made without destroying the biological function of the cytokine. For example, minor modifications of gamma interferon that do not destroy its function fall within the definition of gamma interferon. These modifications can be deliberate, as through site-directed mutagenesis, or can be accidental such as through 10 mutation. The preferred cytokines are IL-2, IL-8, IL-12, or y- interferon, p-interferon, X-interferon, GM-CSF, or G-CSF or a combination thereof. Interleukin -2 is a lymphokine produced by helper T cells and is active in controlling the magnitude and type of the immune response. Smith, K. A., Ann. Rev. Immunol. 2, 319-333 (1984). Other functions 15 have also been ascribed to IL-2 including the activation of NK cells (Minato, N. et al., J. Exp. Med. 154, 750 (1983)) and the stimulation of cell division in large granular lymphocytes and B cells. Tsudo, M. et al.. J. Exp. Med. 160, 612-616 (1984). Studies in mice and humans have demonstrated that deficient immune responsiveness both in vivo and in 20 vitro can be augmented by IL-2. For example, exogenous IL-2 can restore the immune response in cyclophosphamide-induced immunosuppressed mice (Merluzzi, V. J. et al. Cancer Res. 41, 850-853 (1981)) and athymic (nude) mice. Wagner, H. et al. Nature 284, 278-80 (1982). Furthermore, IL-2 canrestore responsiveness of lymphocytes 25 from patients with various immunodeficiency states such as leprosy and cancer. Vose, B. M. et al. Cancer Immuno. 13,105-111 (1984). The genes for murine (Yokota, T. et al. Proc. Natl. Acad. Sci. USA 82, 68-72 (1985)) and human (Taniguchi, T. et al. Nature, 302, 305-307 (1983)) IL 2 have been cloned and sequenced. 30 Interleukin-4 is a T cell derived factor that acts as an induction factor on resting B cells, as a B cell differentiation factor and as a B cell 34 growth factors. Sevenusar, E. Eur. J. Immunol. 17, 67-72 (1987). The gene for human IL-4 has been isolated and sequenced. Lee, F. et al. Proc. Nati. Acad. Sci. USA 83, 2061-2065 (1986). IL-12 is a recently characterized heterodimeric cytokine that has 5 a molecular weight of 75 kDa and is composed of disulfide-bonded 40 kDa and 35 kDa subunits. It is produced by antigen presenting cells such as macrophages, and binds to receptors on activated T, B and NK cells (Desai, B. B., et al., J. Immunol., 148:3125-3132 (1992); Vogel, L. A., et al., nt. Immunol., 8:1955-1962 (1996)). It has several effects 10 including 1) enhanced proliferation of T cells and NK cells, 2) increased cytolytic activities of T cells, NK cells, and macrophages, 3) induction of IFN- production and to a lesser extent, TNF-a and GM-CSF, and 4) activation of TH1 cells. (Trinchieri, G., et al., Blood, 84:4008-4027 (1994). IL-12 has been shown to be an important costimulator of 15 proliferation in Thl clones (Kennedy et al., Eur. J. Immunol. 24:2271 2278 (1994)) and leads to increased production of IgG2a antibodies in serum (Morris, S. C., et al., J. Immunol. 152:1047-1056 (1994); Germann, T. M., et al., Eur. J. Immunol., 25:823-829 (1995); Sher, A., et al., Ann. N. Y. Acad. Sci., 795:202-207 (1996); Buchanan, J. M., et al., 20 Int. Immunol., 7:1519-1528 (1995); Metzger, D. W. et al., Eur. J. Imunol., 27:1958-1965 (1997)). Administration of IL-12 can also temporarily decrease production of IgG1 antibodies (Morris, S. C., et al., J. Immunol. 152:1047-1056 (1994); McKnight, A. J., J. Immunol. 152:2172-2179 (1994); Buchanan, J. M., et al., nt. immunol., 7:1519-1528 (1995)), 25 indicating suppression of the Th2 response. The purification and cloning of IL-12 are disclosed in WO 92/05256and WO 90/05147, and in EP 322,827 (identified as "CLMF"). All of the above effects were observed in adult animals. Interferons (IFNs) are relatively small, species-specific, single 30 chain polypeptides, produced by hostian cells in response to exposure to a variety of inducers such as viruses, polypeptides, mitogens and the 35 like. They exhibit antiviral, antiproliferative and immunoregulatory properties and are, therefore, of great interest as therapeutic agents in the control of cancer and various other antiviral diseases (J. Desmyter et al., Lancet 11, 645-647 (1976); R. Derynck et al., Nature 287, 193 5 (1980)). Human interferons are grouped into three classes based on their cellular origin and antigenicity: a-interferon (leukocytes), p interferon (fibroblasts) and y-interferon (B cells). Recombinant forms of each group have been developed and are commercially available. y-interferon is also a T cell derived molecule which has profound 10 effects on the immune response. The molecule promotes the production of immunoglobulin by activated B cells stimulated with interleukin-2. y-interferon also increases the expression of histocompatability antigens on cells which associated with viral antigens to stimulate cytotoxic T cells. The gene for human y-interferon has been 15 isolated and sequenced. Gray, P. W. et al., Nature 295, 503-508 (1982). Human alpha interferons (also known as Leukocyte interferons) comprise a family of about 30 protein species, encoded by at least 14 different genes and about 16 alleles. Some of these alpha interferon 20 protein species have been shown to have antiviral, antigrowth and immunoregulatory activities. See, e.g., Pestka et al., Ann. Rev. Biochem., 56:727 (1987). The therapeutic efficacy of human alpha interferons has been established for human cancers and viral diseases. For example, recombinant interferons (IFN alpha-2a, IFN alpha-2b, IFN 25 alpha-2c), cell-line derived interferon (IFN alpha-ni) and interferon derived from leukocytes (IFN alpha-n3) are currently used for the treatment of Condyloma acuminata, hepatitis (Weck et al., Am. J. Med., 85(Suppl 2A):159 (1988); Korenman et al., Annal. Intern. Med., 114.:629 (1991); Friedman-Kien et al., JAMA, 259:533 (1988)), for the regression 30 of some malignancies (Baron et al., JAMA, 266:1375 (1991)), for the treatment of AIDS related Kaposi's sarcoma (Physicians Desk 36 Reference, 47th edit., eds. Medical Economics Data, Montvale, N.J., p. 2194 and 2006 (1993)) and are currently being considered for the treatment of human acquired immunodeficiency syndrome (AIDS) either alone or in combination with other antiviral agents (Hirsch, Am. J. Med., 5 85(Suppl 2A):182 (1988)). p-interferon has been shown to be a glycoprotein by chemical measurement of its carbohydrate content. It has one N-glycosidyl attachment site (E. Knight, Jr., Proc. Natl. Acad. Sci., 73, 520 (1976); E. Knight, Jr., and D. Fahey, J. Interferon Res., 2 (3), 421 (1982)). Even 10 though not much is known about the kinds of sugars which make up the carbohydrate moiety of p-interferon, it has been shown that the carbohydrate moiety is not essential for its antigenicity, biological activity or hydrophobicity (T. Taniguchi et al., supra; E. Knight, Jr., supra; and E. Knight, Jr. and D. Fahey, supra). Beta-interferon can be induced in 15 fibroblasts by viral challenge and contains about 165 amino acids. The sequence of -interferon is known. Fiers et al. Philos. Tmas. R. Soc. Lond., B, Biol. Sci. 299:29-38 (1982). GM-CSF is a cytokine important in the maturation and function of dendritic cells. It binds receptors on dendritic cells and stimulates these 20 cells to mature, present antigen, and prime naive T cells. Dendritic cells form a system of highly efficient antigen-presenting cells. After capturing antigen in the periphery, dendritic cells migrate to lymphoid organs and present antigens to T cells. These potent antigen presenting cells are unique in their ability to interact with active naive T 25 cells. The potent antigen-presenting capacity of dendritic cells may be due in part to their unique life cycle and high level expression of major histocompatibility complex class I and Il molecules and co-stimulatory molecules. The sequence of human GM-CSF is known. Wong et al., Science 228:810-815 (1985). 30 Granulocyte colony stimulating factor (G-CSF) is one of the hematopoietic growth factors, also called colony stimulating factors, that 37 stimulate committed progenitor cells to proliferate and to form colonies of differentiating blood cells. G-CSF preferentially stimulates the growth and development of neutrophils, and is useful for treating in neutropenic states. Welte et at., PNA S-USA 82: 1526-1530 (1985); Souza et at., 5 Science 232: 61-65 (1986) and Gabrilove, J. Seminars in Hematology 26: (2) 1-14 (1989). G-CSF increases the number of circulating granulocytes and has been reported to ameliorate infection in sepsis models. G-CSF administration also inhibits the release of tumor necrosis factor (TNF), a cytokine important to tissue injury during sepsis 10 and rejection. See, e.g., Wendel et al., J. Immunol., 149:918-924 (1992). The cDNAs for human (Nagata et al., Nature 319;415, 1986) and mouse G-CSF (Tsuchiya et al., PNAS 83, 7633, 1986) have been isolated, permitting further structural and biological characterization of G-CSF. 15 In humans, endogenous G-CSF is detectable in blood plasma. Jones et al., Bailliere's Clinical Hematology 2 (1): 83-111 (1989). G CSF is produced by fibroblasts, macrophages, T cells trophoblasts, endothelial cells and epithelial cells and is the expression product of a single copy gene comprised of four exons and five introns located on 20 chromosome seventeen. Transcription of this locus produces a mRNA species which is differentially processed, resulting in two forms of G CSF mRNA, one version coding for a protein of 177 amino acids, the other coding for a protein of 174 amino acids. Nagata et at., EMBO J 5: 575-581 (1986). The form comprised of 174 amino acids has been 25 found to have the greatest specific in vivo biological activity. G-CSF is species cross-reactive, such that when human G-CSF is administered to another host such as a mouse, Canine or monkey, sustained neutrophil leukocytosis is elicited. Moore et at. PNAS-USA 84: 7134-7138 (1987). The present invention provides an effective means for enhancing 30 the immune response to the specific foreign antigenic polypeptides of recombinant viruses. Although any foreign antigenic polypeptide can be 38 used in the vaccine of the present invention, the vaccine is particularly useful in vaccines against the HIV virus and the Ebola virus, since these viruses have a negative effect on the host's immune system. The vaccine is also very useful for immunization against hepatitis B and C 5 virus. 3. Genetic Vaccines Against HIV Infection The genetic vaccine of the present invention also addresses the need for an efficient vaccine against the HIV virus. According to the 10 present invention the genetic vaccine may be a recombinant benign virus in which the viral genome carries one or more antigens from HIV, such as HIV glycoproteins (e.g. GP120 and GP41) or capsid proteins (e.g. P24). Sequences of these HIV antigens may be modified such as deletion of the immunosuppressive regions of the HIV glycoproteins. 15 The HIV virus causes the disease known as Acquired Immune Deficiency Syndrome (AIDS). AIDS has been described as a modem plague since its first description in 1981, it has claimed over 60,000 victims, and accounted for over 32,000 deaths in the United States alone. The disease is characterized by a long aysmptomatic period 20 followed by a progressive degeneration of the immune systemand the central nervous system. The virus may remain latent in infected individuals for five or more years before symptoms appear, and thus, the true impact of the disease has yet to be felt. Many Americans may unknowingly be infected and capable of infecting others who might 25 come into contact with their body fluids. Thus, if unchecked, the personal, social and economic impact of AIDS will be enormous. The HIV virus is a retrovirus. Thus, its genetic matierial is RNA, which encodes the information for viral replication. Upon infection of a host cell, the RNA acts as a template for the transcription to DNA, which 30 is catalyzed by an enzyme called reverse transcriptase. The DNA so produced enters the cell nucleus where it is integrated into the host DNA 39 as a provirus. When properly activated, the retroviral-derived DNA is transcribed and translated to produce RNA containing virions, which are then released from the cell by a budding process. When an individual becomes infected with HIV, the virus 5 preferentially attaches to and enters a particular class of white blood cells, called T4 lymphocytes, which are characterized by the presence of a cell surface marker termed CD4. These white blood cells play an integral role in the immune system, functioning as critical components of both the humoral and cellular immune response. Much of the 10 deleterious effect of HIV can be attributed to the functional depression or destruction of T4 lymphocytes. The intact HIV virion is roughly spherical and is approximately 10 nm in diameter. The virion has an outer membrane covered with spike like structures made up of glycoprotein, gp160/120. In addition, there 15 exists a transmembrane protein termed gp4l. Inside the virion are two structural proteins: an outer shell composed of the phosphoprotein, p17, and an inner nucleoid or central core made up of the phosphoprotein, p24. The viral RNA is present inside the core along with two copies of the reverse transcriptase enzyme, p66/51, which is necessary for the 20 synthesis of viral DNA from the RNA template. The HIV RNA genome encodes three major structural genes: gag, pol and env, which are flanked at either end by long terminal repeat (LTR) sequences. The gag gene codes for the group-specific core proteins, p55, p39, p24, p17 and p15. The pol genes code for the reverse transcriptase, p66/p51, and 25 the protease, p31. The env genes encode the outer envelope glycoprotein, gp120, and its precursor, gp160, and the transmembrane glycoprotein, gp4l. Some of the genes tend to be highly variable, particularly the env genes. In addition, there are five other genes, not shared by other retroviruses, which are either involved in transcriptional 30 or translational regulation or encode other structural proteins. The entire 40 HIV genome has now been sequenced. See Ratner et al. Nature 313:277 (1985), which is incorporated herein by reference. The HIV envelope protein has been extensively described, and the amino acid and RNA sequences encoding HIV envelope from a 5 number of HIV strains are known. See Myers, G. et al., Human Retroviruses and AIDS: A compilation and analysis of nucleic acid and amino acid sequences, Los Alamos National Laboratory, Los Alamos, N.M. (1992). The env genes of various strains of HIV are predicted to encode proteins of 850 to 880 amino acids. Extensive glycosylation of 10 the Env precursor polyprotein during synthesis produces gp160 (about 160 kilodaltons) which is also the major form of the env gene product detected in infected cells. Gp160 forms a homotrimers and undergoes glycosylation with the Golgi apparatus. The functional domains of gp160 includes, starting from N 15 terminus, Signal peptide, Variable regions 1 through 5 which encompass CD4 binding sites (e.g., Thr27, Trp 4 27 , Aspa"/Glu" 7 ", and Asp 4 57), Proteolytic processing site (also called the cleavage site between gp120 and gp4l), Fusion domain, Leucine zipper motif, transmembrane domain, and Lentivirus lytic peptides (LLP) 1 and 2. 20 Although the nucleotide and amino acid sequences of gp120 and the numbering thereof from various isolates and strains of HIV may differ, the region encoding the functional domains can be readily identified by the teaching in Luciw (1996) in "Fundamental Virology", 3 'd ed., eds., Fields et al., Lippincott-Raven Publishers, Philadelphia, Chapter 27, pp. 25 845-916. The signal peptide at the N-terminus of the Env precursor gp160 directs ribosomes translating the nascent protein to the endoplasmic reticulum; an intracellular proteinase removes this signal peptide during Env gp biogenesis. The Env precursor gp160 is cleaved at the 30 processing site by a cellular protease to produce gp120 (designated SU subunit) and gp4l (designated TM subunit). Gp120 contains most of 41 the external, surface-exposed, domains of the envelope glycoprotein complex. Gp4l contains a transmembrane domain and remains in a trimeric configuration, and it interacts with gp120 in a non-covalent manner. The subunits of gp4l include: Fusion peptide, Leucine zipper 5 like region, transmembrane domain (TM), LLP1 and LLP2. The gp120 subunit contains five variable regions and six conserved regions. The variable (V) domains and conserved (C) domains of gp120 are specified according to the nomenclature of Modrow et al. (1987) "Computer-assisted analysis of envelope protein 10 sequences of seven human immunodeficiency virus isolates: predictions of antigenic epitopes in conserved and variable regions", J. Virol. 61:570-578. The gp120 molecule consists of a polypeptide core of 60,000 daltons, which is extensively modified by N-linked glycosylation to 15 increase the apparent molecular weight of the molecule to 120,000 daltons. The positions of the 18 cysteine residues in the gp120 primary sequence, and the positions of 13 of the approximately 24 N-linked glycosylation sites in the gp120 sequence are common to all gp120 sequences. The hypervariable domains contain extensive amino acid 20 substitutions, insertions and deletions. Sequence variations in these domains result in up to 30% overall sequence variability between gp120 molecules from the various viral isolates. Despite this variation, all gp120 sequences preserve the virus's ability to bind to the viral receptor CD4 and to interact with gp4l to induce fusion of the viral and host cell 25 membranes. The HIV virus attaches to host cells by an interaction of the envelope glycoproteins with a cell surface receptor. It appears that when HIV makes contact with a T4 cell, gp120 interacts with the CD4 receptor. Recently, the crystal structure of the core domain of HIV-1 30 gp120 (strain HXB-2, a clade B virus) has been solved by complexing the protein with a fragment of human CD and an antigen-binding 42 fragment from a virus-neutralizing antibody that blocks chemokine receptor binding. Kwong et al. (1998) "Structure of an HIV gp12O envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody", Nature 393:648-659. These studies 5 revealed that the gp120 core has a unique molecular structure that comprises two domains-an "inner domain" (which faces gp4l) and an "outer" domain (which is mostly exposed on the surface of the oligomeric envelope glycoprotein complex). The two gp120 domains are separated by a "bridging sheet" that is not part of either domain. 10 Binding to CD4 causes a conformational change in gp120 which exposes the bridging sheet and may move the inner and outer domains relative to each other. It was also found that most of the carbohydrate molecules which are added to gp120 are added to the outer domain. This is consistent with the idea that that virus uses carbohydrate 15 molecules to mask external antigenic epitopes on gp120. Gpl 20 not only binds to the cellular CD4 receptor but also to HIV coreceptors such as the cellular chemokine receptors (e.g. CCR5). Upon binding to the receptor and/or coreceptor, the viral envelope is then fused with the cell membrane and the inner core of the virus enters 20 the infected cell where the transcription of RNA into a DNA provirus is catalyzed by reverse transcriptase. The provirus may remain in the cell in a latent form for some months or years, during which time the infected individual is asymptomatic. However, if the virus is later activated causing viral replication and immuno- suppression the individual will 25 than be susceptible to the opportunistic infections associated with AIDS. In one embodiment of the HIV vaccine of jhe present invention, a recombinant virus is provided for eliciting strong immune response against infection of HIV. The recombinant virus comprises: an antigen sequence heterologous to the recombinant virus that encodes an 30 antigen from human immunodeficiency virus (HIV), expression of the HIV antigen eliciting an immune response directed against the HIV 43 antigen and cells expressing the HIV antigen in a host upon infection of the host by the recombinant virus; and an immuno-stimulator sequence heterologous to the recombinant virus that encodes an immuno stimulator whose expression in the host enhances the immunogenicity 5 of the HIV antigen. In a preferred embodiment, the recombinant virus is replication-incompetent and does not cause a malignancy naturally associated with HIV in the host. The recombinant virus is used as a genetic vaccine to be administered to a host to induce or elicit strong and long-lasting immunity against HIV infection. 10 In comparison with other approaches for developing HIV vaccine using denatured or attenuated HIV virion, the approach of the present invention should be safer and more efficient in eliciting strong immune response but not creating risks of reactivation of HIV, probably through recombination with the wild type HIV infecting the host. 15 According to the present invention, the HIV antigen expressed by the genetic vaccine may be any antigen derived from a HIV virus, such as HIV surface, core/capsid, regulatory, enzyme and accessory proteins. Examples of HIV surface protein include, but are limited to the products of the env gene such as gp120 and gp4l. Examples of HIV 20 capsid protein include, but are limited to the products of the gag gene such as the cleavage products of the Pr559" by the viral encoded protease PR: the mature capsid proteins MA (p17), CA (p24), p2, NC (p7), p1 and p6. Herderson et al. (1992) J. Virol. 66:1856-1865. Examples of viral regulatory proteins include, but are not limited to the 25 products of the tat and rev genes: Tat and Rev. Examples of viral enzyme proteins include, but are not limited to the products of the pol gene: p11 (protease or PR), p51 (reverse transcriptase or RT), and p32 (integrase or IN). Examples of viral accessory proteins include, but are not limited to the products of the vif, vpr, vpx, vpu and nef genes: Vif, 30 Vpr, Vpx, Vpu and Nef. 44 In one embodiment, HIV Nef protein may serve as the HIV antigen expressed by the recombinant virus of the present invention. For example, sequence encoding Nef (e.g., the nef sequence at position 8152-8523 for BH10 strain of HIV and at position 8787-9407 for pNL4-3 5 strain of HIV) may be inserted into the vector. In another embodiment, HIV Rev protein may serve as the HIV antigen expressed by the recombinant virus of the present invention. For example, sequence encoding Rev (e.g., the rev1 sequence at position 5969-6044 and the rev2 sequence at position 8369-8643 for 10 pNL4-3 strain of HIV) may be inserted into the vector. In yet another embodiment, full length HIV Gag protein may serve as the HIV antigen expressed by the recombinant virus of the present invention. For example, sequence encoding full leng Gag (e.g., the gag sequence at position 112-1650 for BH10 strain of HIV and at position 15 790-2292 for pNL4-3 strain of HIV) may be inserted into the vector. Alternatively, capsid protein from HIV Gag protein (e.g. p24 CA) may serve as the HIV antigen expressed by the recombinant virus of the present invention. For example, sequence encoding p24CA (e.g., the sequence at position 1186-1878 for BHIO strain of HIV and at position 20 508-1200 for pNL4-3 strain of HIV) may be inserted into the vector. In yet another embodiment, the HIV antigen expressed by the recombinant virus is derived from the env gene products. For example, the antigen is derived from the Env protein. According to the embodiment, modifications or mutagenesis may 25 be used to delete or mutate in certain region(s) of Env to render it non functional and yet still contains neutralizing epitopes for its natural genicity. For example, the proteolytic processing site of Env may be deleted or mutated to render it resistant to cleavage by cellular protease to produce gpl20 and gp4l fragments. Deletion or mutation may also 30 be carried out on the transmembrane and cytoplasmic domains of gp41 such as the TM, LLP-1 and LLP-2 domains. Compared to the wild type 45 Env, the mutated Env protein should have a reduced risk of being incorporated into a wild type HIV that infects the host and being exploited by HIV in its furtherance of the goal: destruction the host's immune system. 5 For example, wildtype HIV Env can be modified in the following ways. Wildtype gp120 sequence from BH10 strain of HIV and containing Env, Tat, and Rev coding sequences can be digested with restriction enzymes EcoR I and Xho I to produce a fragment starting from nucleotide 5101 and ending at nucleotide 8252. The cytosolic domain 10 of Env can be removed by deleting nucleotides from the coding sequence at position 7848-8150 for BH10 strain, and 8610-8785 for pNL4-3 strain of HIV. The cleavage site of Env can be removed by deleting 12 nucleotides encoding amino acid sequence REKR at position 7101-7112 for BH10 strain, and 7736-7747 for pNL4-3 strain of 15 HIV. Also according to this embodiment, the modified Env protein may contain deletions in the regions that do not contain neutralizing epitopes. For example, the V1 and V5 domains of gp120 may be deleted without sacrificing the natural antigenicity of gp120. Portions of the V2 and V3 20 domains of gp120 that do not contain neutralizing epitopes may also be deleted. Although the principle neutralizing domain (PND) has been found in the V3 domain, V2 and C4 domains of gp120 have also been found to contain neutralizing epitopes. Among various strains or clades of HIV, the amino acid sequences of the neutralizing epitopes may be 25 variable. However, it has been found that the amount of variation is highly constrained. Thus, the sequences not containing the neutralizing epitopes should be readily determined. For example, sequence encoding V1 region of Env can be deleted at position 5961-6032 for BH10 strain, and 6602-6673 for pNL4 30 3 strain of HIV. Sequence encoding V2 region of Env can be deleted at position 6060-6161 for BH10 strain, and 6700-6796 for pNL4-3 strain of 46 HIV. Optionally, sequence encoding both V1 and V2 regions of Env can be deleted at position 5961-6161 for BH10 strain, and 6602-6796 for pNL4-3 strain of HIV. Alternatively, the HIV antigen expressed by the recombinant virus 5 may be a subunit of gp120 which contains one or more selected variable (V) and/or conserved (C) domains. For example, the HIV antigen may be a gpl20 subunit containing V2, V3 and C4 domains, or V3 and C4 domains. The location of neutralizing epitopes in the V3 domain is well known. It has been found that neutralizing epitopes in the V2 and C4 10 domains are located between residues 163 and 200 and between about 420 and 440, respectively. In addition, residues for antibody binding also include residues 171, 174, 177, 181, 183, 187, 188 in the V2 domain and residues 429 and 432 in the C4 domains. Berman et al. (1999) Virology 265:1-9; and Berman (1998) AIDS Res. Human 15 Retroviruses 15:115-132. In another embodiment, the HIV antigen expressed by the recombinant virus of the present invention may be a modified Env protein that contains deletions and/or mutations in the glycosylation sites. The gp120 of HIV-1 contains 24 potential sites for N-linked 20 glycosylation (Asn-X-Ser/Thr); about 13 of the 24 glycosylation motifs are conserved in the different viral isolates. Analysis of HIV-1 Env gp proteins has demonstrated that 17 of 24 potential glycosylation sites are modified with carbohydrate side chains. Mizuochi et al. (1990) J. Biol. Chem. 265:8519-8524; and Leonard et al. (1990) J. Biol. Chem. 25 265:10373-10382. Because of the extensive glycosylation of Env gp proteins, very few regions of the peptide backbone of gp120 protrude from the carbohydrate mass. Some of the glycosylation sites have been found in non-neutralizing epitopes that dilute the immunity against true neutralizing epitopes or serve as decoy epitopes. Thus, deletion or 30 mutation of these glycosylation sites may enhance immunity of the antigen by unmasking the true neutralizing epitopes. 47 In another embodiment, the different HIV antigens may be expressed by the same recombinant virus of the present invention. For example, both Env, Tat and Rev proteins may be expressed from the same promoter such as a CMV early promoter via a retroviral splicing 5 donor-acceptor mechanism. Optionally, HIV Gag protein, either in full length or a truncated or modified form (e.g., capsid protein p24), may also be expressed together with other HIV antigens such as Env, Tat and Rev. Further, these HIV antigens may be expressed together with the immuno-stimulator(s) (e.g., IL-2, IL-12, INF-y, and GMCSF) in single 10 or multiple copies by the same recombinant viral vector. For example, the sequences encoding the HIV antigens may be inserted into El region of an adenoviral vector and expressed from a CMV early promoter via a retroviral splicing donor-acceptor mechanism or an IRES mechanism. The sequences encoding the immuno 15 stimulators may be inserted into E4 region of the same adenoviral vector and expressed from another CMV early promoter via a retroviral splicing donor-acceptor mechanism or an IRES mechanism. In yet another embodiment, the sequence encoding the HIV antigen in the recombinant virus of the present invention is a mosaic 20 antigen that contains sequences from different strains, isolates and/or clades of HIV viruses. A strain of HIV is the HIV isolated from an individual (an isolate), characterized and given a strain name (e.g., MN, LAl). Because of the heterogenecity of HIV, not two isolates are exactly the same. A group of related HIV isolates are classified according to 25 their degree of genetic similarity such as of their envelop proteins. There are currently two groups of HIV-1 isolates, M and 0. The M group consists of at least 9 clades (also called subtypes), A through I. The 0 group may consist of a similar number of clades. Clades are genetically distinct but are all infectious. It is believed that by using a 30 mosaic HIV antigen in the design of the genetic vaccine of the present invention the vaccine produced should have an enhanced ability to 48 stimulate the production of anti-HIV antibodies and HIV-specific cytotoxic T lymphocytes (CTLs) against a wider spectrum of "wild type" HIV strains. In one embodiment, the mosaic HIV antigen in the recombinant 5 virus contains antigens from multiple clades of HIV-1, including clade A (ATCC No: HIV-1 92UG037WHO.0108HED), B (ATCC No: pNL4-3), C (ATCC No: HIV-1 92BR025WHO.109HED), D (ATCC No: HIV-1 92UG024.2), E (ATCC No: HIV-1 93TH976.17), F (ATCC No: HIV-1 93BR020.17), and G (ATCC No: HIV-1 92RU131.9). Optionally, 10 multiple repeats of restriction fragments of HIV antigen (e.g., Ava I fragments) from different clades may be linked head-to-tail to generate an even more complex mosaic HIV antigen. For example, an adenoviral vector may be constructed to the V3 loops of multiple clades as the mosaic HIV antigen. Optionally, HIV 15 antigens with gp41 deletion from multiple clades may serve as the mosaic HIV antigen. Alternatively, HIV antigens from multiple clades with V1 and V2 loops deleted from clade B (pNL4-3) may serve as the mosaic HIV antigen. Yet optionally, an.human gene Thy-1 GPA anchor sequence 20 encoding amino acid sequence SWLLLLLLSLSLLQATDFMSL [SEQ ID NO: 9] may be added to the recombinant viral construct. In another embodiment, the mosaic HIV antigen contains an Env protein which comprises variable and constant domains of gp120 derived from different strains, isolates and/or clades of HIV viruses. For 25 example, V2 domain from clade B of the M group may be mixed with V3 and C4 domains from clade C of the 0 group to generate a mosaic HIV antigen. Vaccination of individuals with such a mosaic antigen may stimulate CTLs with cross-clade activity. In another word, these CTLs can recognize and kill target cells infected HIV from different clades. 30 Alternatively, the recombinant virus may express a plurality of HIV antigens, each of which is an antigen from a different strain, isolate or 49 clade of HIV. For example, env genes from different clades of HIV can be cloned into the recombinant virus and expressed in tandem to produces various Env proteins from these clades in the host cells. It is believed that expressing various Env proteins from different strains, 5 isolates or clades of HIV in the host cells should enhance the ability of the genetic vaccine of the present invention to stimulate the production of anti-HIV antibodies and HIV-specific cytotoxic T lymphocytes (CTLs) against a wider spectrum of "wild type" HIV strains. The host vaccinated with such a vaccine would be able to be immunized from infection of 10 various strains of HIV. By using the genetic vaccine of the present invention, individuals not infected by HIV may be immunized against HIV. For HIV-infected individuals the vaccine may also be used boost their immune response and help fight against this virulent virus. Since the genetic vaccine can 15 express high level of antigens and/or a variety of HIV glycoproteins and capsid proteins simultaneously, the vaccinated individuals should be immunized against various strains of HIV, such as HIV-1 and HIV-2. Additionally, since the genetic vaccine can express high levels of cytokines to mimic the body's response to natural viral infection, the 20 body's immune response to such a genetic vaccine against HIV should be strong and long-lasting, thereby achieving a life-long immunity against this deadly virus. 4. Genetic Vaccines Against Hepatitis Viruses 25 The genetic vaccine of the present invention also addresses the need for an efficient vaccine against hepatitis viruses such as hepatitis A, B, C, D, and E viruses. According to the present invention the genetic vaccine may be a recombinant benign virus in which the viral genome carries one or more antigens from a hepatitis virus, such as 30 glycoproteins and core proteins of the hepatitis virus. Sequences of 50 these HIV antigens may be modified such as deletion of the pathogenic regions of the hepatitis glycoproteins or coreproteins. In particular, the recombinant virus of the present invention can be used as a vaccine to immunize individuals against Hepatitis B 5 infections. Viral hepatitis B is caused by the Hepatitis B virus (HBV). HBV is estimated to have infected 400 million people throughout the world, making HBV one of the most common humanpathogens. Hepatocellular carcinomas (HCC), one of the most common cancers afflicting humans, is primarily caused by chronic HBV infection. 10 HBV is a mostly double-stranded DNA virus in the Hepadnaviridae family. The HBV genome is unique in the world of viruses due to its compact form, use of overlapping reading frames, and dependence on a reverse-transcriptase step, though the virion contains primarily DNA. The HBV genome has four genes: pol, env, pre-core 15 and X that respectively encode the viral DNA polymerase, envelope protein, pre-core protein (which is processed to viral capsid) and protein X. The function of protein X is not clear but it may be involved in the activation of host cell genes and the development of cancer. The diagnosis of HBV infection is generally made on the basis of 20 serology. Virtually all individuals infected with HBV will have detectable serum hepatitis surface antigens (HBsAg). Despite notable successes of vaccines against HBV infection, it is still an on-going task. A review on modern hepatitis vaccines, including a number of key references, may be found in the Eddleston, The Lancet, p. 1142, May 12, 1990. See 25 also Viral Hepatitis and Liver Disease, Vyas, B. N., Dienstag, J. L., and Hoofnagle, J. H., eds., Grune and Stratton, Inc. (1984) and Viral Hepatitis and Liver Disease, Proceedings of the 1990 International Symposium, eds F. B. Hollinger, S. M. Lemon and H. Margolis, published by Williams and Wilkins. According to the present 30 invention, the viral antigen may be a surface antigen or core protein of hepatitis B virus such as the small hepatitis B surface antigen (SHBsAg) 51 (also referred to as the Australia antigen), the middle hepatitis B surface antigen (MHBsAg) and the large hepatitis B surface antigen (LHBsAg). Antigens of different types of HBV, such as Asian type C and America type A, may be expressed by the recombinant virus to elicit 5 immune response to these types of HBV. The HBV surface antigen (HBsAg) or the core antigen (HBcAg) may be expressed by the recombinant virus of the present invention, separately or in combination (HBsAg + HBcAg). For example, the sequences encoding multiple HBV antigens 10 may be inserted into El or E4 region of an adenoviral vector and expressed from a CMV early promoter via a retroviral splicing donor acceptor mechanism or an IRES mechanism. Further, these HBV antigens may be expressed in combination with one or more immuno stimulators such as IL-2, IFN-y and GMCSF in single or multiple copies. 15 Sequences encoding these cytokines may be inserted into E4 or El region that is not occupied by the antigen sequences and expressed from another CMV early promoter via a retroviral splicing donor-acceptor mechanism or an IRES mechanism. Specific combinations of inserts include, but are not limited to, 20 HBsAg + HBcAg; HBsAg + HBcAg + IL-2; HBsAg + HBcAg + IFN-y + GMCSF; and HBsAg + IFN-y + IFN-y + GMCSF. The sequences encoding the immuno-stimulators may be inserted into E4 region of the same adenoviral vector and expressed from another CMV early promoter via a retroviral splicing donor-acceptor 25 mechanism or an IRES mechanism. Also according to the present invention, the viral antigen may be a surface antigen or core protein of hepatitis C virus such as NS3, NS4 and NS5 antigens. For example, sequence(s) encoding the HCV antigen(s) may be 30 inserted into El or E4 region of an adenoviral vector and expressed 52 separately or in combination with one or more immuno-stimulators such as IL-2, IL-12, IFN-y and GMCSF in single or multiple copies. Specific combinations include, but are not limited to, (1) HCV wildtype E2 + wildtype El; 5 (2) core of HCV; (3) HCV E2 + E1 + core; (4) HCV E2 + E1 + core + IL-2; (5) HCV E2 + El + core + IL-2 + IFN-y + GMCSF; and (6) HCV E2 + El + core + IL-2 + IFN-y + IL-12. 10 In another embodiment, multi copies of hypervariable regions (HVR) of HCV El and E2, e.g., five copies of HVR (5xHVR), may serve as the viral antigen in the recombinant virus, and may be expressed alone or in combination with one or more immuno-stimulators such as 15 IL-2, IL-12, IFN-y and GMCSF in single or multiple copies. Specific combinations include, but are not limited to, (1) E2-5xHVR + El; (2) E2-5xHVR + El + IL-2; (3) E2-5xHVR + El + core + IL-2; 20 (4) E2-5xHVR + El + core + IL-2 + IFN-y + GMCSF; and (5) E2-5xHVR + El + core + IL-2 +IL-12. By using the genetic vaccine of the present invention, non hepatitis-infected individuals may be immunized against hepatitis virus. 25 For hepatitis virus-infected individuals the vaccine may also be used boost their immune response and help fight against the hepatitis virus. Since the genetic vaccine can express high level of antigens and/or a variety of hepatitis glycoproteins and coreproteins simultaneously, the vaccinated individuals should be immunized against various strains 30 and/or types of hepatitis virus, such as hepatitis A, B, C, D, and E virus. Additionally, since the genetic vaccine can express high levels of 53 cytokines to mimic the body's response to natural viral infection, the body's immune response to such a genetic vaccine against hepatitis should be strong and long-lasting, thereby achieving a life-long immunity against the hepatitis virus. 5 5. Genetic Vaccines Against Ebola Virus The genetic vaccine of the present invention also addresses the need for an efficient vaccine against the deadly virus, Ebola virus. According to the present invention the genetic vaccine may be a 10 recombinant benign virus in which the viral genome carries one or more antigens from Ebola hepatitis, such as glycoproteins (e.g. GP1 and GP2) of Ebola virus. Sequences of these Ebola antigens may be modified such as deletion of the immunosuppressive regions and/or other pathogenic regionsof the Ebola virus. 15 Ebola virus is one of the most lethal viruses known to mankind with a mortality rate of up to 90%. Johnson, K.M., Ann Intern Med 91(1):1 17-9 (1979). Victims of Ebola virus infection are subjected to a horrible hemorrhagic diseases which kills in a matter of days. The natural reservoir of the virus remains unknown, as do the specifics of 20 pathogenesis of the infection. The virus has a very specific tropism for liver cells and cells of the reticuloendothelial system, such as macrophages. Massive destruction of the liver is hallmark feature of the disease. Although Ebola virus infection is rare, there is concern by public 25 health officials about the potential for the disease to become an international epidemic as the Ebola virus is easily transmitted through human contact and is extremely contagious. Outbreaks like those that have recently occurred in Africa could happen in industrialized countries due to the rapid and extensive nature of modern travel. Recent cases of 30 Ebola virus infection in Africa send strong warnings to be prepared for the outbreaks of this extremely dangerous infectious disease. In 54 addition, Ebola virus has a terrifying potential if used as a biological weapon byterrorist nations or organizations. As in most cases of viral infection, the best approach to prevent an outbreak of Ebola virus is through vaccination. However, there currently is no effective vaccine 5 nor treatment available against Ebola virus infection. Ebola viruses are enveloped, negative strand RNA viruses, which belong to the family Filoviridae. There are three strains of filoviruses: Ebola, Marburg and Reston. The Ebola virus can enter the body a number of different ways such as an opening through which air is taken 10 in because the virus can travel on airborne particles and it can also enter the body through any opening in the skin, such as cuts. The Ebola virus has a non-segmented RNA genome that encodes all the viral structural proteins (nucleoprotein, matrix proteins VP24 and VP40), non-structural proteins (VP30, VP35) and viral 15 polymerase. Peters, C.J., West J Medl64(1):36-8 (1996). Among the viral proteins, the envelope glycoproteins (GP) exist in two forms, a secreted glycoprotein (50-70 kDa) and a transmembrane glycoprotein (130-170 kDa) generated by transcriptional editing. Sanchez, A. et al., Proc Natl Acad Sci U.S.A., 93(8):3602-7 (1996). Although the two forms 20 of GP share 295 amino acid homology, they have distinct binding specificities, suggesting that they play different roles in the course of viral infection. The secreted glycoprotein (sGP) is the predominant form synthesized and secreted by the infected cells. It may play a role in suppressing the host immune system (Yang,Z., et al., Science 25 279(5353):1034-7 (1998)) and may serve as a decoy to allow the virus particle to escape from neutralizing antibodies, since the two forms of GPs partly share their antigenicity. Analysis of monoclonal antibodies from the human survivors of Ebola virus Zaire infection has revealed that the vast majority of them were specific to the sGP, and only a few 30 bound weakly to GP. Maruyama, T., et al., J Infect Dis, 179 Suppl 1:S235-9 (1999), Maruyama, T., et al., J Virol, 73(7):6024-30 (1999). 55 Although the exact mechanism by which the sGP may suppress the immune system is not clearly understood, the large amounts of sGP synthesized in the early phase of the infection are probably responsible for the inhibition of neutrophil infiltration of the infected sites (Yang, Z., et 5 al., Science 279(5353):1034-7 (1998)) and the absence of humoral immune response in Ebola virus infected patients. Baize, S., et al., Nat Med, 5(4):423-6 (1999). This protein may also act to over-activate many types of immune cells which can lead to massive intravascular apoptosis - essentially a shut-down of the immune system. Baize, S., 10 et al., Nat Med, 5(4):423-6 (1999). The importance of the sGP to the Ebola virus life-cycle is also suggested by the fact it is present in all Ebola virus strains examined to date. Feldmann, H., et al., Arch Virol Suppl, 15:159-69 (1999). The membrane glycoproteins are responsible for the attachment 15 and penetration of the virions into target cells by mediating receptor binding and viral-cellular membrane fusion. Wool-Lewis, et al., J. Virol, 72(4):3155-60 (1998), Ito H., et al., J. Virol, 73(10):8907-12 (1999). They are synthesized as a single peptide precursor and cleaved by cellular enzymes (furin or cathepsin B) into the two mature forms, GP1 20 and GP2. The two GPs remain associated through a disulfide bond linkage and remain anchored in the viral membrane by a transmembrane (TM) domain. Ito H., et al., J. Virol, 73(10):8907-12 (1999); Malashkevich, V.N., et al., Proc Natl Acad Sci U.S.A., 96(6):2662-7 (1999). The proteolytic cleavage site is composed of 4-5 25 basic amino acid residues that are similar to those found in the GPs of retrovirus, influenza, and paramyxoviruses. Garten, W., et al., Biochimie, 76(3-4):217-25 (1994). The cleavage event is essential for viral infectivity and is likely carried out by the same enzymes that cleave GPs of retrovirus or influenza viruses. Garten, W., et al., Biochimie, 30 76(3-4):217-25 (1994); Volchkov, V.E., et al., Virology, 245(1):110-9 (1994). In addition, Ebola virus GP may share a common mechanism of 56 mediating viral infection with retroviral and influenza glycoproteins. Weissenhom, W., et al., Mol MembrBiol, 16(1):3-9 (1999). Because membrane-bound GPs play critical roles in initiating virus infection and are also the predominant proteins exposed on the surface of the virions, 5 they are the primary targets for neutralizing antibodies against the virus. One of the properties of Ebola viruses that make them lethal to the host is their ability to suppress the host immune system. Serologic analysis of patients who died of the Ebola virus infection showed no signs of humoral or cellular immune responses. Baize, S., et al., Nat 10 Med, 5(4):423-6 (1999). In contrast, antibodies against viral proteins and virus-specific T-cell activities were detected in a few survivors. Baize, S., et al., Nat Med, 5(4):423-6 (1999). Although the immunosuppressive mechanisms are yet to be understood, it is probable that the high levels of sGP and the immunosuppressive 15 peptide in the GP are to blame for the absence of humoral and cellular immune responses in Ebola virus-infected patients. The proteins that are responsible for the initial inflection of Ebola virus are the viral glycoproteins. Therefore, they are the target for neutralizing antibodies. However, Ebola virus has evolved "tricks" to 20 prevent or delay the host immune response until it is too late to recover from the infection. Conventional approaches in producing vaccines against Ebola virus are likely to be ineffective for the following reasons: (1) viral glycoproteins produced in bacteria, yeast or insect cells are not properly glycosylated and therefore do not have the true antigenicity of 25 the viral proteins; (2) Ebola virus is too dangerous to be produced in large amounts as an inactivated-virus vaccine; and (3) procedures of inactivating the virus often destroy the conformation of the proteins, and therefore alter their antigenicity. A preferred embodiment of the present invention is a recombinant 30 viral vaccine having nucleic acids encoding one or more antigens of 57 Ebola virus. Restriction maps and full sequence information of the Ebola virus, including the Zaire strain, is available through GenBank. The genetic vaccine is a recombinant benign virus which is replication defective or incompetent and therefore is incapable of 5 spreading beyond initially infected cells. For example, a recombinant adenoviral vaccine of the present invention mediates high levels of Ebola viral antigen expression for a period of two or more weeks, even though Ebola viral proteins have no functional relevance to recombinant virus function. 10 In another embodiment of the invention, the recombinant virus expresses one or more modified Ebola virus antigens. The modified Ebola virus antigens are preferably Ebola virus envelope glycoproteins and/or immunogically active parts thereof. Preferably the glycoproteins are modified GP and sGP glycoproteins. The Ebola virus GP and sGP 15 glycoproteins are modified to destroy their pathogenic and immunosuppressive functions, but retain most of their natural antigenicity, since they are expressed, folded, glycosylated, and targeted to the cellular membrane inside the cells that can be productively infected by the Ebola virus. The modifications are carried 20 out using standard molecular genetic manipulation techniques such as restriction digests and polymerase chain reaction. A preferred modification of the Ebola virus envelope glycoprotein destroys the infective function of the Ebola virus GP. Any modification that destroys the infective function of Ebola virus can be used, but 25 preferably the modification is a five amino acid deletion in the cleavage site of the GP. See Example 1. This cleavage site is composed of five basic amino acid residues, RRTRR, at position 501 from the start of the open reading frame. This deletion may be introduced into the Ebola virus GP cDNA using PCR amplification, which is performed by methods 30 well known in the art. 58 Another preferred modification of the Ebola virus viral genome prevents synthesis of the sGP. Any modification that prevents synthesis may be employed. Preferably the modification is directed to altering the RNA editing site from UUUUUUU to UUCUUCUU. See example 1. 5 Another preferred modification to Ebola virus antigen used in the present vaccines is immunosuppressive (IS) peptide located in GP2. The IS peptide motif is located at amino acids 585-609. A ten amino acid deletion between amino acide 590-600 removes its function. Second, each half of the IS peptide motif is reversed and duplicated. 10 See Figure 2. This further ensures that its function has been destroyed and also increases its antigenicity. Further it is readily apparent to those skilled in the art that variations or derivatives of the nucleotide sequences encoding Ebola virus antigen(s) of the present invention can be produced, which alter 15 the amino acid sequence of the encoded protein. The altered expressed antigen(s) may have an altered amino acid sequence, yet still elicit immuneresponses that react with Ebola virus antigen(s), and are considered functional equivalents. In addition, fragments of the full length genes that encode portions of the full-length protein may also be 20 constructed. These fragments may encode a protein or peptide which elicits antibodies which react with Ebola virus antigen(s), and are considered functional equivalents. Vaccination of an individual with the vaccines of the present invention results in entrance of adenoviral particles into cells and 25 expression of Ebola virus antigen(s), such as the envelope glycoproteins, and the immune-stimulating cytokines. The expression of Ebola virus antigen(s) in cells induces strong and persistent immune responses as if an infection has occurred. The genetic vaccine has all of the immunogenicity of a natural infection, including expression of the 30 natural viral proteins and long-lasting antigen stimulation, but does not have the pathogenicity of a true viral infection. In the vaccines of the 59 present invention, the immunosuppressive mechanisms of Ebola virus are disabled, the antigens occur in their natural forms and are associated with the cell membrane, and immune stimulation lasts for weeks. The effects of this novel vaccine are long lasting and provide 5 high rates of protection against Ebola virus infection. The present invention is also directed to a method of immunizing a human against Ebola virus infection comprising administering the vaccines described above. The techniques for administering these vaccines to humans are known to those skilled in the health fields. 10 By using the genetic vaccine of the present invention, individuals may be immunized against Ebola virus. Since the genetic vaccine can express high levels of antigens and/or a variety of glycoproteins simultaneously, the vaccinated individuals should be immunized against various strains Ebola virus. Additionally, since the genetic vaccine can 15 express high levels of cytokines to mimic the body's response to natural viral infection, the body's immune response to such a genetic vaccine against Ebola virus should be strong and long-lasting, thereby achieving a life-long immunity against the Ebola virus. 20 5. Formulation and Routes of Administration The present invention also relates to a pharmaceutical composition comprising the vaccine(s) described above, and a pharmaceutically acceptable diluent, carrier, or excipient carrier. Additionally the vaccine may also contain an aqueous medium or a 25 water containing suspension, often mixed with other constituents in order to increase the activity and/or the shelf life. These constituents may be salt, pH buffers, stabilizers (such as skimmed milk or casein hydrolysate), emulsifiers, and preservatives. An adjuvant may be included in the pharmaceutical composition 30 to augment the immune response to the viral antigen expressed from the recombinant virus. Examples of the adjuvant include, but are not 60 limited to, muramyl dipeptide, aluminum hydroxide, saponin, polyanions, anamphipatic substances, bacillus Calmette-Guerin (BCG), endotoxin lipopolysaccharides, keyhole limpet hemocyanin (GKLH), interleukin-2 (IL-2), granulocyte-macrophage colony-stimulating factor (GM-CSF) and 5 cytoxan, a chemotherapeutic agent which is believed to reduce tumor induced suppression when given in low doses. The present invention also provides kits for enhancing the immunity of a host to a pathogen. These kits may include any one ore more vaccines according to the present invention in combination with a 10 composition for delivering the vaccine to a host and/or a device, such as a syringe, for delivering the vaccine to a host. The vaccine according to the invention can be administered in a conventional active immunization scheme: single or repeated administration in a manner compatible with the dosage formulation, and 15 in such amount as will be prophylactively effective, i.e. the amount of immunizing antigen or recombinant microorganism capable of expressing the antigen that will induce immunity in humans against challenge by the pathogenic virus or bacteria, such virulent Ebola virus, HIV, hepatitis A, B, C, D, and E virus, and bacillus tuberculous. 20 Immunity is defined as the induction of a significant level of protection after vaccination compared to an unvaccinated human. The vaccine of the present invention, i.e. the recombinant virus, may be administered to a host, preferably a human subject, via any pharmaceutically acceptable routes of administration. The routes of 25 administration include, but are not limited to, intramuscular, intratracheal, subcutaneous, intranasal, intradermal, rectal, oral and parental route of administration. Routes of administration may be combined, if desired, or adjusted depending upon the type of the pathogenic virus to be immunized against and the desired body site of 30 protection. 61 Doses or effective amounts of the recombinant virus may depend on factors such as the condition, the selected viral or bacterial antigen, the age, weight and health of the host, and may vary among hosts. The appropriate titer of the recombinant virus of the present invention to be 5 administered to an individual is the titer that can modulate an immune response against the viral or bacterial antigen and elicits antibodies against the pathogenic virus or bacteria from which the antigen is derived. An effective titer can be determined using an assay for determining the activity of immunoeffector cells following administration 10 of the vaccine to the individual or by monitoring the effectiveness of the therapy using well known in vivo diagnostic assays. For example, a prophylactically effective amount or dose of a recombinant adenovirus of the present invention may be in the range of from about 100 pfl to about 10 ml of saline solution containing concentrations of from about 1x10 4 to 15 1x10" plaque forming units (pfu) virus/mI. One skilled in the art understands that the amount of virus particles to be administered depends, for example, on the number of times the vaccine is administered and the level of response desired. 20 6. Methods of Enhancing the Immunity of a Host to Pathogens The present invention also provides methods for enhancing the immunity of a host host to pathogens with the recombinant viruses described above. In one embodiment, the method is provided for enhancing the 25 immunity of a host host to a pathogenic virus. The method comprises: administering to the host a recombinant virus in an amount effective to induce an immune response. The recombinant virus comprises: an antigen sequence heterologous to the benign virus that encodes a viral antigen from a pathogenic virus, expression of the viral antigen eliciting 30 an immune response directed against the viral antigen and cells expressing the viral antigen in the host upon infection of the host by the 62 recombinant virus; and an immuno-stimulator sequence heterologous to the benign virus that encodes an immuno-stimulator whose expression in the host enhances the immunogenicity of the viral antigen. The recombinant virus may preferably be replication-incompetent and not 5 cause a malignancy naturally associated with the pathogenic virus in the host. The recombinant virus may be administered to the host via any pharmaceutically acceptable route of administration. The recombinant virus may be administered to the host via a route of intramuscular, 10 intratracheal, subcutaneous, intranasal, intradermal, rectal, oral and parental administration. In another embodiment, a method is provided for immunizing a host against a pathogenic virus with multiple antigens that elicit strong and long-lasting immune response to the multiple antigens. The method 15 comprises: administering to the host a recombinant virus in an amount effective to induce an immune response. The recombinant virus comprises: a plurality of antigen sequences heterologous to the recombinant virus, each encoding a different viral antigen from one or more pathogenic viruses, expression of the plurality of the antigen 20 sequences eliciting an immune response directed against the viral antigen and cells expressing the viral antigen in the host upon infection of the host by the recombinant virus. The recombinant virus may preferably be replication-incompetent and not cause malignancy that is naturally associated with the pathogenic virus(es) in the host. 25 Optionally, the recombinant virus may also comprise one or more immuno-stimulator sequences heterologous to the recombinant virus that encodes an immuno-stimulator whose expression in the host enhances the immunogenicity of the viral antigen. In yet another embodiment, a method is provided for immunizing 30 a host against a pathogenic virus by using multiple genetic vaccines or viruses. Multiple recombinant viruses may carry different antigens in 63 each recombinant virus. The multiple recombinant viruses may be administered simultaneously or step-wise to the host. The method comprises: administering to a host a first and second recombinant viruses in an amount effective to induce an immune 5 response, wherein antibodies are produced. The first recombinant benign virus comprises: an antigen sequence heterologous to the first recombinant virus that encodes a viral antigen from a pathogenic virus, expression of the viral antigen eliciting an immune response directed against the viral antigen and cells expressing the viral antigen in the 10 host upon infection of the host by the recombinant virus. The second recombinant virus comprises: an immuno-stimulator sequence heterologous to the second recombinant virus that encodes an immuno stimulator whose expression in the host enhances the immunogenicity of the viral antigen. The first and second recombinant viruses may 15 preferably be replication-incompetent and not cause malignancy naturally associated with the pathogenic virus in the host. According to the embodiment, the first and second recombinant virus may be any of a benign virus, such as replication-incompetent adenovirus, adeno-associated virus, SV40 virus, retrovirus, herpes 20 simplex virus and vaccinia virus. Optionally, both the first and second recombinant viruses may be replication-incompetent adenovirus. Also optionally, one of the first and second recombinant viruses may be recombinant adenovirus and the other may be recombinant vaccinia virus. 25 In yet another embodiment, a method is provided for enhancing the immunity of a host to a pathogen. The method comprises: administering to the host a recombinant virus and one or more immuno stimulators. The recombinant virus may be any of the recombinant viruses described above. In particular, the recombinant virus comprises 30 one or more antigen sequences heterologous to the recombinant virus that encode one or more antigens from the pathogen. Expression of the 64 antigen elicits an immune response directed against the antigen and cells expressing the antigen in the host upon infection of the host by the recombinant virus. The recombinant virus is preferably replication incompetent and does not cause a malignancy naturally associated with 5 the pathogen in the host. The pathogen may be a pathogenic virus such as HIV, hepatitis virus and Ebola virus, a pathogenic bacteria or parasite. According to this embodiment, the immuno-stimulator may be any molecule that enhances the immunogenicity of the antigen expressed by 10 the cell infected by the recombinant virus. Preferably, the immuno stimulator is a cytokine, including, but not limited to interleukin-2, interleukin-8, interleukin-12, P- interferon, X-interferon, y-interferon, granulocyte colony stimulating factor, granulocyte-macrophage colony stimulating factor, and combinations thereof. The cytokine may be 15 administered into the host in a form of purified protein. Alternatively, the cytokine may be administered in a form of expression vector that expresses the coding sequence of the cytokine upon transfecting or transducing the cells of the host. Standard procedures for endonuclease digestion, ligation and 20 electrophoresis are carried out in accordance with the manufacturer's or supplier's instructions. Standard techniques are not described in detail and will be well understood by persons skilled in the art. Practicing the present invention employs, unless otherwise indicated, conventional methods of virology, microbiology, molecular 25 biology and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See e.g. Sambrook, et al. Molecular Cloning: A laboratory Manual; DNA Cloning: A Practical Approach, vol I & II (D. Glover ed.); Oligonucleotide Synthesis (N. Giat, ed.); Nucleic Acid Hybridization (B. Hames & S. 30 Higgins, eds., Current Edition); Transcription and Translation (B. Hames 65 & S. Higgins, eds., Current Edition); Fundamental Virology, 2nd Edition, vol. I & 11 (B. N. Fields and D. M. Knipe, eds.) The following examples are provided to illustrate the present invention without, however limiting the same thereto. 5 EXAMPLES The following procedures are described to illustrate how to make a genetic vaccine of the present invention against various pathogenic 10 viruses. The genetic vaccine is based on an adenoviral vector with modified antigens derived from the pathogenic virus (e.g., Ebola virus, Hepatitis B virus and HIV) inserted into the adenoviral backbone. Additionally, the recombinant adenovirus also carries multiple genes encoding various cytokines. The recombinant adenovirus is replication 15 incompetent but still retains adenoviral infectivity. It is noted that genetic vaccine against other pathogenic viruses, bacteria and parasites may be constructed by one with ordinary skill in the art following similar procedures described in details below. 1. Genetic Vaccine Against Ebola Virus 20 Embodiments of the genetic vaccine against Ebola virus and methods of their construction are described in detail as follows. 1) Genetic modification of the Ebola virus membrane lvcoproteins 25 The modifications are carried out using standard molecular genetic manipulation techniques, such as restriction enzyme digests and polymerase chain reaction (PCR). The glycoproteins of Ebola virus are modified to produce the optimal antigen for Ebola virus vaccine. Two modified forms of the GP 30 proteins are constructed to have inactivated immunosuppressive and infectious mechanisms, but retain full natural antigenicity of the wild-type 66 glycoproteins. The mRNA editing signal is deleted to prevent the production of the secreted glycoprotein (sGP), which is immunosuppressive; and (2) the proteolytic cleavage site of the glycoprotein precursor is deleted to prevent the formation of the 5 functional glycoproteins (GP1 and GP2). Sanchez, A., et al., Proc Nat/ Acad Sci U.S.A. 93(8):3602-7 (1996). In one form the immunosuppressive peptide region is deleted to prevent its function, and in the other form, the immunosuppressive peptide motif is split in order to destroy its function, but retain its immunogenicity. These steps 10 produce effective and safe antigens for the vaccine. The envelope glycoproteins (GP) of the Ebola virus are synthesized as a single precursor protein and cleaved into the two subunits (GP1 and GP2) by a cellular enzyme (furin) during transport. Volchkov, V.E., et al., Proc Natl Acad Sci U.S.A., 95(10):5762-7 (1998). 15 This proteolytic cleavage is essential for the formation of the mature glycoproteins and the release of the fusion peptide located at the C Terminus of the cleavage site. The mature glycoproteins are incorporated into virions as trimers (each monomer is a heterodimer of GPI and GP2 linked by a disulfide bond). Sanchez, A., et al., J. Virol 20 72(8):6442-7 (1998). The glycoproteins of Ebola virus are the major proteins exposed on the viral membrane surface, and are responsible for initiating virus entry into host cells. Therefore, they are a primary target for neutralizing antibodies. The glycoprotein cleavage site is composed of five basic amino 25 acid residues (RRTRR [SEQ ID NO: 10]) at position 501 from the start site of the open reading frame. The Ebola virus glycoprotein cleavage site is similar to the conserved sequences found in glycoproteins of other viruses, such as in the envelope protein of RSV or MuLV. We have previously shown that deletions or point mutations at these basic 30 amino acid residues can block cleavage and render the glycoproteins non-functional in RSV. Dong, J.Y, et al., J. Viro/ 66(2):865-74 (1992). 67 To destroy the infective functions of the Ebola virus glycoprotein, the five basic amino acid residues in the cleavage site are deleted. This deletion is introduced into the Ebola virus GP cDNA using PCR amplification. Alternatively, the cleavage site can be altered, such as by 5 site specific mutation resulting in elimination of cleavage. Another important feature of the Ebola virus is that two forms of glycoproteins are synthesized from a single gene, a secreted from (sGP) and a membrane-bound form (GP). The two forms are generated as a result of an alternative RNA editing event at a sequence of seven 10 uridines (at location 1020-1028 from the start site), which is highly conserved among all four Ebola virus subtypes. Sanchez, A., et al., Proc Natl Acad Sci U.S.A. 93(8):3602-7 (1996). The sGP is synthesized from un-edited mRNA and likely has immunosuppressive functions. The GP is synthesized from an edited mRNA and likely has 15 immunosuppressive functions. The GP is synthesized from an edited mRNA with insertion in one of the seven uridines. This RNA editing causes a frame-shift and results in a translation of the second reading frame that encodes the complete transmembrane glycoprotein (GP2). To prevent the synthesis of sGP, the RNA editing site is modified 20 from UUUUUUU [SEQ ID NO: 2] to UUCUUCUU [SEQ ID NO: 3]. In the cDNA, the equivalent sequence is AAAAAAA [SEQ ID NO: 4] and AAGAAGAA [SEQ ID NO: 5], respectively. This modification accomplishes two things: (1) all mRNAs encode only the GP (equivalent to the edited form with -1 frame shift); and (2) UUUUUU [SEQ ID NO: 6] 25 encodes the same animo acid residues as UUCUUC [SEQ ID NO: 7], but prevents the possibility of further polymerase slipping at the stretch of the six uridines. The additional editing would cause deletion of one more uridine and further (-2) frame shifting. The mechanism of this modification is diagramed in Figure 2. 30 A third modification may be introduced into the Ebola virus glycoprotein relating to a deletion of the immunosuppressive (IS) peptide 68 located in GP2. The IS peptide motif (amino acid 585-609, form the start site) is highly conserved in filoviruses and has a high degree of homologywith a motif in the glycoproteins of oncogenic retroviruses that has been shown to be immunosuppressive. Volchkov, V.E., et al., 5 FEBS Lett 305(3):181-4 (1992); Will, C., et al., J. ViroI67(3):1203-10 (1993); Mitani, M., et al., Proc Natl Acad Sci U.S.A. 84(1):237-40 (1987); Gatot, J.S., et al., J. Biol Chem 273(21):12870-80 (1998); Denner, J., et al., J Acquir Immune Defic Syndr Hum Retrovirol 12(5):442-50 (1996). First, a ten amino acid deletion is introduced in the core region of the 10 motif (between amino acid 590-600) to remove its function. Second, each half of the motif is reversed and duplicated to destroy function and increase antigenicity. It is believed that antibodies against the IS peptide may inhibit the immunosuppressive function of the Ebola viruses during an infection. The basic strategy of this modification is 15 diagrammed in Figures 3A-3C. As illustrated in Figure 3A-3C, modification of the immunosuppressive peptide (IS) is made on the GP2 gene. Figure 3A illustrates the wild type GP. Figure 3B illustrates GP with the 10 amino acid deletion of the IS peptide. Figure 3C illustrates the IS peptide, 20 which is split, reversed and duplicated. With these modifications, Ebola virus glycoproteins are generated that are non-functional, not immunosuppressive, yet they retain the natural antigenicity of GP. These modified GP sequences are used to generate antigens in the vaccines of the present invention against Ebola 25 virus. DNA sequences of the resulting altered GP genes are confirmed by sequence analysis. The modified GP sequences are then cloned intoplasmid vectors containing DNA elements necessary for efficient expression of these GPs in hostian cells. Expression and correct 30 localization to the cellular membrane is determined by transient transfections of HeLa or 293 cells and analyzed by Western blot and 69 FACS, using polyclonal antibodies from hyperimmunized equine serum and anti-horse secondary antibodies labeled with horse radish peroxidase (HRP) or fluorescent tags, respectively. 5 2) Construction of a series of replication-defective adenoviral vaccines that mediate high levels of expression of the modified Ebola virus GPs The vaccines of the present invention utilize a recombinant 10 benign virus to carry modified antigens of Ebola virus to trick the host into mounting a robust immune defense against the Ebola virus. The preferred benign virus is a replication-defective adenovirus. These vectors are an excellent choice for vaccine expression, for several reasons. First, adenoviral vectors direct high levels of antigen 15 expression that provide strong stimulation of the immune system. Second, the antigen that they express is processed and displayed in the transduced cells in a way that mimics pathogen-infected cells. This phase is believed to be very important in inducing cellular immunity against infected cells, and is completely lacking when conventional 20 vaccination approaches are used. Third, adenoviral vectors infect dendritic cells which are very potent antigen-presenting cells. Diao, J. et al., Gene Ther 6(5):845-53 (1999); Zhong, L., et al., Eur J Immuno/ 29(3):964-72 (1999); Wan, Y., et al., /nt J Oncol 14(4):771-6 (1999); Wan, Y., et al., Hum Gene Ther 8(11):1355-63 (1997). Fourth, these 25 vectors can be engineered to carry immunoenhancing cytokine genes to further boost immunity. Fifth, adenoviruses naturally infect airway and gut epithelial cells in humans, and therefore the vaccine may be delivered through nasal spray or oral ingestion. And finally, the adenoviral vectors of this invention are safe because they are 30 replication-defective andhave been used in high doses (10' to 1012 i.p./dose) in clinical trials for gene therapy studies. Gahery-Segard, H., 70 et al., J. C/in Invest 100(9):2218-26 (1997); Bellon, G.,et al., Hum Gene Ther 8(l):15-25 (1997); Boucher, R.C., et al., Hum Gene Ther 5(5):615 39 (1994). Indeed, even live viruses have been safely used in military recruits to prevent common colds. 5 This vector-construction system is also used to establish complex vectors that express multiple genes or regulatory mechanisms. For example, the vector construct is used to express multiple cytokines along with Ebola GP antigens in a single complex vector to further enhance the immune induction. Alternatively, antigens and cytokines 10 are placed in separate vectors. This enables the manipulation of different combinations of cytokines and antigens by co-transduction (infection) with two or three vectors. Construction of the adenoviral vectors is diagramed in Figure 4. The cDNA encoding a modified GP(s) is cloned into the left-end (El 15 region) of the adenovirus genome using a shuttle vector pLAd (Fig. 4A left side), resulting in a shuttle vector pLAd/EBO-GP. The pLAd/EBO GP vector contains the left end of the adenoviral genome including the left long terminal repeats L-TR and the adenoviral packaging signal Xy. Genes encoding cytokines such as IL-2 and IL-4 are inserted into E4 20 region of the adenovirus vector using the shuffle vector pRAd (Figure 4A, right side), resulting in a shuffle vector pRAdIL2,4. The pRAdlL2,4 contains the right end of the adenoviral genome including the right long terminal repeats R-TR. To construct an adenoviral vector carrying the GP gene only, the 25 shuttle vector pLAd/EBO-GP is digested with appropriate restriction enzymes such as Xba 1. The fragment containing the GP gene is ligated to an adenoviral backbone and pRAd vector. To construct an adenoviral vector carrying both the GP gene in the El region and cytokine genes in the E4 region, both pLAd/EBO-GP 30 and pRAdIL2, 4 are linearized and ligated to the backbone of the adenovirus (Fig. 4B). 71 To generate recombinant adenoviral vectors, the ligated vector genome is transfected into 293 cells, in which only the correctly ligated genome with the two adenovirus terminal repeats can replicate and generate infectious viral particles. Human 293 cells (Graham et al., J. 5 Gen. Virol., 36: 59-72 (1977)), available from the ATCC under Accession No.: CRL1573), has adenovirus Ela and Elb genes stably integrated in its genome. The 293 cells supplement the essential El gene of adenovirus that has been deleted from the vector backbone. The final vector has El, E3 and partial E4 deleted and can only replicate 10 in 293 cells, but not in target cells. The adenoviral vectors are amplified in 293 cells and purified by ultracentrifugation in cesium chloride gradients. Titers of vectors are determined by serial dilutions and counting of the infectious particle (ip) after infection of 293 cells. 15 3) Determination of immune responses to the genetic vaccine An in vitro assay is used to quantitate the amount of neutralizing antibodies developed in response to the vaccine. The assay is based on a retroviral vector system which is based on a Moloney Murine 20 Leukemia virus system. Vectors and packaging cells expressing GAG and POL proteins have been extensively characterized and are commercially available. A packaging vector construct that carries a p galactosidase gene as a reporter is used. A novel vector construct expressing the membrane form of the Ebola virus GP is co-transfected 25 with the P-Gal reporter vector resulting in a GAG-POL packaging cell line, which generates retroviral vector particles with the Ebola virus GP instead of its original envelope protein. 4) modified GP antien provides better 30 production of neu tibodies in animal models 72 The adenoviral vaccine vectors carrying the two GP variants are tested for their ability to induce an immune response to the Ebola virus GP in CD-1 mice (Charles River Laboratories; outbred stock of Swiss mice from Rockefeller Institute). Specifically, the neutralizing antibody 5 titers and cytolytic T-lymphocyte (CTL) activities to the Ebola virus GP antigens induced by the GP variants with and without the IS motif are compared. Three groups of 30 8-week old mice are injected subcutaneously with 105 ip of adenoviral vectors expressing GP variant I (with IS peptide deleted), GP variant 2 (with IS peptide split and 10 inverted) and -Galactosidase (control vector), respectively. Six mice from each group are sacrificed (by CO 2 asphyxiation and cervical dislocation) at 1, 2, 4, 8 and 16 weeks post-vaccination, and their blood and spleens are harvested. In addition, 6 mice are mock-vaccinated with saline and sacrificed 2 days later to provide preimmunization 15 controls. From mice injected with the control p-Gal vector, tissue sections from the sites of the vector injection are taken, fixed, and stained with the X-gal solution to determine the number and type of vector transduced cells at various time-points post-infection. In addition, 20 hemolysin staining isperformed to determine the degree of infiltration of various immune cells (neutrophils, macrophages, monocytes, etc.) at the site of the vector delivery. Sera from vaccinated animals is assayed for total GP-binding antibodies using a standard 96-well plate ELISA protocol, as has been 25 described. Van Ginkel, F.W., et al., Hum Gene Ther6(7):895-903 (1995); Van Ginkel, F.W., et al., J Immunol 159(2):685-93 (1997). Neutralizing activity of the sera is analyzed by monitoring the infectious activity of the Ebola virus GP-pseudotyped retroviral vector (Wool Lewis, et al., J. Virol, 72(4):3155-60 (1998)) on HeLa cells after the 30 vector has been incubated with various serum concentrations. Expression of P-galactosidase in infected cell lysates serves as an 73 indicator of the neutralizing activity of the serum (the lower the P-gal activity, the more EBO-p-Gal vectors have been neutralized) and is measured using a very sensitive fluorogenic substrate (Galacto-Light kit J) and a fluorescence plate reader. Anti-GP serum-neutralized infection 5 rates are compared to infection rates in the absence of serum and in the presence of non-GP activated serum. Cytotoxic lymphocytes (CTLs) are extracted from mouse spleens as has been previously described. Van Ginkel, F.W., et al., Hum Gene Ther 1995;6(7):895-903; Dong, J.Y., et al., Hum Gene Ther 10 1996;7(3):319-31. They are mixed with a constant number of detached LnCaP cells (prostate carcinoma cells of epithelial origin) transduced with an adenoviral vector carrying an unmodified Ebola virus GP protein. Ratios of effector: target cells of 10:1, 3:1, and 1:1 are used. The cells are seeded into 96-well plates, and 24 hour later all unattached cells 15 (which include all of the effector CTLs and dead or dying LnCaP cells) are removed, and the remaining viable (adherent) cells are quantitated by the MTT (3-(4,5-dimethylthiazol-20-yl) 2,5-diphenyl tetrazolium bromide) cleavage assay. This assay has been employed in detecting the lymphocyte cytotoxic activity (Ni, J., et al., J Clin Lab Anal 20 1996;10(1):42-52) and compares favorably with the radioactive assays in terms of sensitivity, reliability and speed. 5) Immuno-enhancinga functions of multiple cytokines and their effects on the efficacy of the genetic vaccines 25 To augment the effects of the vaccine, a vector-mediated gene transfer to express the immunoenhancing cytokines, such as IL2, IL4, IL12, INF-y, and GM-CSF is used. Initially, each cytokine is separately cloned or the cytokines are cloned in various combinations into 30 adenoviral vectors separate from the vectors encoding viral antigens. The immunoenhancing effects of individual cytokines or their 74 combinations are studied by co-infecting with a vector encoding the cytokine and the vector carrying the antigens. The titers of serum antibodies are compared, as well as the time it takes to reach effective titers in animals inoculated with vaccines in combination with different 5 cytokine-expressing vectors. These experiments allow the determination of whether immunoenhancing cytokines induce higher levels of antibodies, shorten the induction time, and prolong the immunity against the Ebola virus. After determining the best-performing modified GP variant, the 10 extent that the immune response elicited by it is enhanced by co delivery to the immunization site of vectors carrying various cytokines is analyzed. Interleukin-2, either by itself or in combination with IL-4 or IL 12, has been demonstrated to strongly enhance the activation and proliferation of cytotoxic T-cells, natural killer (NK) cells and B-cells. 15 Michael, B.N., et al., Cell Immunol 1994;158(1);105-15; Bruserud, 0., et al.,EurJ. Haematol 1992;48(4);221-7; Jacobsen, S.E., et al., Res Immunol 1995;146(7-8):506-14; Wolf, S.F., et al., Res Immunol 1995; 146(7-8);486-9; Tepper, R.I., Res Immunol 1993; 144(8):633-7; O'Garra A., et al., Res Immunol 1993;144(8):620-5; Ohe, Y., et al., /nt J 20 Cancer 1993;53(3):432-7; Delespesse, G., et al., Res Immunol 1995;146(7-8):461-6. [36-43]. INF-y stimulates the humoral immune response and increases the permeability of the blood vessel walls at the site of its secretion (Chensue, S.W., et al., J /mmunol 154(11):5969-76 (1995); Szente, B.E., et al., Biochem Biophys Res Commun 25 203(3):1645-54 (1994); Adams, R.B., et al., J. Immunol 150(6):2356 63(1993)), while Gm-CSF activates and attracts macrophages and other professional APCs to the site of the infection. Bober, L.A., et al., /mmunopharmacology29(2):111-9 (1995); Dale, D.C., et al., Am J. Hematol 57(1):7-15 (1998); Zhao, Y., et al., Chung Hua / Hsueh Tsa 30 Chih 77(10): 32-6 (1997). 75 Six groups of 30 8-week old mice are injected subcutaneously with a mixture of 5x1 04 ip of the selected GP variant vector and 5x 104 ip of one of the following vectors: Ad-P-Gal, Ad-IL2, Ad-1L2/1L4, Ad IL2/IL12, Ad-IFN-y and Ad-GM-CSF. 5 Six mice from each group are sacrificed and analyzed at 1, 2, 4, 8 and 16 weeks as described in Example 4. Analysis of total IgG is peformed using ELISA, neutralizing activity is assayed as interference with the ability of EBO-GP pseudotyped retroviral vector to infect HeLa cells, and anti-GP CTL activity is performed by mixing spleen-extracted 10 CTLs with target LnCaP cells transduced with Ad-EBO-GP construct as described in Example 4. Levels of various cytokines in the serum are also quantitated by ELISA using available commercial assays. In some cases, these assays can distinguish between human and murine versions of the same cytokine, providing direct information on the 15 expression levels of cytokines delivered using Adenovirus vectors and how they correlate with the development of the immune response. After the individual cytokines are analyzed, those that performed best are tested in combinations. Four groups of 30 8-week old mice are injected subcutaneously with a mixture of 5x104 i.p. of the selected GP 20 variant vector and 5x10 4 i.p. of up to 3 selected cytokine-expressing vectors (if fewer than 3 cytokine vectors are used, i.p. counts are made up with Ad-p-Gal vector). Six mice from each group are sacrificed at weeks 1, 2, 4, 8 and 16, and analyzed as described above. To verify the robust and reproducible nature of the immune 25 response to the GP vector and multiple cytokines in different species, the experiment as described above is reproduced in rabbits. Five groups of six white New Zealand rabbits are injected into the thigh muscle with one of the following vector combinations: the Ad-p-Gal vector (10' ip), the selected GP vector (2.5 x 10' ip plus 7.5x10' ip of Ad 30 p-Gal), and three cytokine vector combinations (2.5x1 0. of each cytokine vector) plus the GP vector (2.5x1 0' ip), as described above. 76 The animals are bled 2 days prior to vaccination (pre-immune bleed) and then according to the schedule described above. 5 to 10 ml of blood will be extracted per session. Analysis is performed in a similar fashion to that of mice (see above). 5 Because genes coding for human cytokines are used in mouse and rabbit models, it is possible that their immune systems will have a non-identical (to human) response to those proteins. However, a high degree of homology exists between human and mouse cytokines and their receptors, and published reports on experiments using human or 10 other hostian cytokines in mice indicate a high level of equivalency. If necessary, species-specific versions of these cytokines can be obtained and cloned into the adenoviral vectors of the present invention for species-targeted cytokine activity studies. 15 6) Optimizing the efficiency and rates of administration of the vaccine and conducting safety and pathogen challenge studies in non primate and primate animal models After determining the best combinations of the cytokines and 20 antigens, the final version of the vaccine vectors are constructed. These complex recombinant adenoviral vectors deliver combinations of cytokines and antigens into target cells using a single vector. Dose-titer analysis in mice and rabbits are conducted to identify the lowest dose required to generate maximallevels of immune responses. Different 25 routes of vaccine administration, such as intramuscular and intravenous injection, oral ingestion and nasal sprays are compared. For safety studies, dose escalation experiments in mice and rabbits are conducted until toxicity is observed or until levels ten times the effective dose have been reached. Finally, additional safety and pathogen challenge 30 experiments are conducted in primates. 77 2. Genetic Vaccine Against HIV Embodiments of the genetic vaccine against HIV and methods of their construction are described in detail as follows. 5 1) Construction of replication-defective adenoviral vaccines agains HLV a) Ad.tat.env.L2 An adenoviral vector, Ad.tat.env.L2, was constructed to carry 10 coding sequences for multiple HIV antigens including Env, Tat, and Rev proteins, and interleukin-2 (IL-2) in the same vector. Expression of the HIV antigens and IL-2 is separately controlled by promoters located in different regions of the adenoviral vector. This design is believed to be able to ensure high level expression of both the viral antigens and the 15 immuno-stimulator IL-2 and to enhance immunogenicity of the adenoviral vaccine. As shown by experimental data presented in the next section, this adenoviral vector is capable of eliciting strong humoral immune response in animals against HIV antigens. The adenoviral vector, Ad.tat.env.lL2, was constructed using 20 strategies similar to those for constructing the adenoviral vaccines against Ebola virus as described in detail above. Briefly, EcoRl/Xhol restriction fragment from HIV-1 strain BH10 that encodes full length gp160, full length Tat and full length Rev was inserted into the left end (El region) of the adenoviral genome using a shuttle vector pLAd 25 (Figure 4A, left side), resulting in a shuttle vector pLAd-CMV-Env-Tat Rev. The sequence encoding IL-2 (with a silent mutation caused by deletion of Xba I site) was inserted into E4 region of the adenoviral genome using the shuttle vector pRAd (Figure 4A, right side), resulting 30 in a shuttle vector pRAd-CMV-IL2. 78 Both pLAd-CMV-Env-Tat-Rev and pRAd-CMV-IL2 were linearized using appropriate restriction enzymes such as Xba I and EcoRI and ligated to the backbone of the adenovirus (Figure 4B), resulting in the recombinant adenoviral vector Ad.tat.env.L2. 5 b) Ad.3C.env.gag Another adenoviral vector, Ad.3C.env.gag, was constructed to carry coding sequences for multiple HIV antigens including a modified Env (gp160 with deletion of the cleavage site (REKR [SEQ ID NO: 11]) 10 and the cytoplasmic domain of 33 amino acids in length) and Gag proteins, and three different cytokines (IL-2 with silent mutation to destroy Xbal site, INF-y, and GMCSF) in the same vector. Expression of the HIV antigens and the cytokines is separately controlled by promoters located in different regions of the adenoviral vector. This 15 design is believed to be able to ensure high level expression of both the viral antigens and the immuno-stimulators and to enhance immunogenicity of the adenoviral vaccine. As shown by experimental data presented in the next section, this adenoviral vector is capable of eliciting strong humoral immune response in animals against HIV 20 antigens. The adenoviral vector, Ad.env.gag.3C, was constructed using strategies similar to those for constructing the adenoviral vaccines against Ebola virus as described in detail above. Briefly, the sequence from HIV-1 strain BH10 that encodes Env/gp160 was modified to delete 25 the sequences encoding the cleavage site (the REKR site) and the cytoplasmic domain of 99 nucleotides in length, and then, along with the sequence encoding a full length Gag, inserted into the left end (El region) of the adenoviral genome using a shuttle vector pLAd (Figure 4A, left side). 30 These two HIV antigens are expressed separately from a CMV promoter via a retroviral splicing donor (SD) and acceptor (SA) mechanism at 79 three splicing acceptor sites, SA 1 , and SA 2 . The shuttle vector produced is designated pLAd-CMV-SD/SA-Env-SA 2 Gag. Sequences encoding multiple immuno-stimulators, including IL-2 (with a silent mutation caused by deletion of Xba I site), INF-y, and 5 GMCSF, were inserted into E4 region of the adenoviral genome using the shuttle vector pRAd (Figure 4A, right side). These three immuno stimulators are expressed separately from another CMV promoter via a retroviral splicing donor (SD) and acceptor (SA) mechanism at three splicing acceptor sites, SA,, SA 2 , and SA 3 . The shuttle vector produced 10 is designated pRAd-CMV-SD/SA-IL2-SA. INFy-SA 3 -GMCSF. Both pLAd-CMV-SD/SA-Env-SArGag and pRAd-CMV-SD/SA, IL2-SA 2 -lNFy-SA 3 -GMCSF were linearized using appropriate restriction enzymes such as Xba I and EcoRi and ligated to the backbone of the adenovirus (Figure 4B), resulting in the recombinant adenoviral vector 15 Ad.3C.env.gag. c) Ad.3C.env.rev.gag Another adenoviral vector, Ad.env.rev.gag.3C, was constructed to carry coding sequences for multiple HIV antigens including a modified 20 Env (gp160 with deletion of the cleavage site (REKR [SEQ ID NO: 11]) and the cytoplasmic domain of 33 amino acids in length), full length Rev and Gag proteins, and three different cytokines (IL-2 with silent mutation to destroy Xbal site, INF-y, and GMCSF) in the same vector. Expression of the HIV antigens and the cytokines is separately 25 controlled by promoters located in different regions of the adenoviral vector. This design is believed to be able to ensure high level expression of both the viral antigens and the immuno-stimulators and to enhance immunogenicity of the adenoviral vaccine. As shown by experimental data presented in the next section, this adenoviral vector is 30 capable of eliciting strong humoral immune response in animals against HIV antigens. 80 The adenoviral vector, Ad.3C.env.rev.gag, was constructed using strategies similar to those for constructing the adenoviral vaccines against Ebola virus as described in detail above. Briefly, the sequence from HIV-1 strain BH10 that encodes Env/gpl60 was modified to delete 5 the sequences encoding the cleavage site (the REKR site) and the cytoplasmic domain of 99 nucleotides in length, and then, along with sequences encoding full length Gag and full length Rev, inserted into the left end (El region) of the adenoviral genome using a shuttle vector pLAd (Figure 4A, left 10 side). These three HIV antigens are expressed separately from a CMV promoter via a retroviral splicing donor (SD) and acceptor (SA) mechanism at three splicing acceptor sites, SA 1 , SA 2 , and SA.. The shuttle vector produced is designated pLAd-CMV-SD/SA-Env-SA 2 -Gag 15 SA 3 -Rev. Sequences encoding multiple immuno-stimulators, including IL-2 (with a silent mutation caused by deletion of Xba I site), INF-y, and GMCSF, were inserted into E4 region of the adenoviral genome using the shuttle vector pRAd (Figure 4A, right side). These three immuno 20 stimulators are expressed separately from another CMV promoter via a retroviral splicing donor (SD) and acceptor (SA) mechanism at three splicing acceptor sites, SA,, SA 2 , and SA 3 . The shuttle vector produced is designated pRAd-CMV-SD/SArl L2-SA 2 - INFy-SA 3 -GMCSF. Both pLAd-CMV-SD/SA-Env-SA 2 rGag-SA 3 Rev and pRAd-CMV 25 SD/SA-lL2-SA 2 -lNFy-SA 3 -GMCSF were linearized using appropriate restriction enzymes such as Xba I and EcoRi and ligated to the backbone of the adenovirus (Figure 4B), resulting in the recombinant adenoviral vector Ad.3C.env.rev.gag. 30 2) Immune responses of animals to the adenoviral vaccine against HIV antiaens 81 Experimental mice were inoculated with the adoviral vaccine constructed above, Ad.tat.env.lL2, to elicit immune response to the HIV antigens expressed by this vector. Immunogenicity of the adenoviral vector was determined by measuring titers of antibody against HIV tat 5 and env. Figures 6 and 7 show the immunogenicity of Ad.tat.env.L2 against the HIV Env protein in two groups of mice, respectively. These groups of C57BL/6 mice (supplied by Charles River Laboratories. Wilmington, MA) were injected intramuscularly with 107 pfu 10 Ad.tat.env.L2 on different dates as indicated in the figures. Blood (about 150-500 pl for each animal) was collected from four animals every two weeks following inoculation and serum was prepared. At 77 days post-inoculation, these mice were re-challenged with an additional 107 pfu of Ad.tat.env.L2. Blood was collected from three animals every 15 day following secondary challenge. Titers of antibody elicited against HIV tat and env were determined by ELISA against Ad.tat.env.L2 infected HeLa cell lysates. Briefly, lysates of the HeLa cells infected with Ad.tat.env.L2 were prepared as follows. HeLa cells were infected with Ad.tat.env.lL2 at a 20 multiplicity of infection (MOI) of 20. Fourty-eight hours post infection, HeLa cells were harvested and resuspended in a buffer that contained 1% TritonX-100. A post-nuclear supernatant was obtained by centrifuging the lysates at 15,000 x g for 5 min. The lysates were diluted to 10 pg/ml for coating wells of ELISA plates. Standard ELISA assays 25 were performed to measure OD450 of the sera and relative titers of antibody against HIV tat and env proteins were calculated by normalizing against the mean of the CD450 of mouse pre-immunization sera. As shown in Figure 6, the three mice in this group had strong 30 immune responses to the HIV antigens expressed by the adenoviral vector Ad.tat.env. 1L2, with the highest titer of antibody against HIV 82 antigens reached in about 42 days post inoculation. The second inoculation with Ad.tat.env.IL2 boosted the immune reponse again and very high titers were achieved within about 5 days of the second inoculation. 5 As shown in Figure 7, the three mice in this group also had strong immune responses to the HIV antigens expressed by the adenoviral vector Ad.tat.env.L2, with the highest titer of antibody against HIV antigens reached in about 70 days post inoculation. The second inoculation with Ad.tat.env.L2 boosted the immune reponse 10 again and very high titers were achieved within about 5 days of the second inoculation. Figures 12, 13 and 14 show the immunogenicity of the recombinant adenoviral vectors Ad.3C.env.gag and Ad.3C.env.gag.rev against HIV antigens gp120, Gag and Rev, respectively. C57BL/6 mice 15 were injected intramuscularly with 10' pfu Ad.3C.env.gag or Ad.3C.env.gag.rev. Six weeks post-inoculation, relative antibody titers of these mice were determined by ELISA against recombinant purified gp120, Gag, or Rev proteins (obtained from the NIH AIDS Research and Reference Reagent Program, Bethesda, MD). Sera were diluted 1:250. 20 Each value represents the mean from 4 individual mice. Background absorbance has been subtracted. As shown in Figures 12 and 13, the mice inoculated with Ad.3C.env.gag or Ad.3C.env.gag.rev had strong immune responses to the HIV antigens gp120 and Gag, as compared with those of the non 25 immunized mice. 3) Activation of cytotoxic T lymphocytes (CTL) by immunization with the adenoviral vaccines against HIV antigens Activation of cytotoxic T lymphocytes (CTL) by immunization with 30 the adenoviral vaccine against HIV antigens was measured by using two independent assays: an IFNy assay and a granzyme A assay. The IFNy 83 and granzyme assays were designed to detect antigen-specific activation of T-cells. IFNy is secreted by activated CTL and T.1 helper T cells which function specifically in the cellular immune pathway. Granzyme A is also secreted by activated CTL. The basic approach is 5 to incubate splenocytes with target cells that express antigens of interest and look for secretion of IFNy or granzyme A into the medium. a) IFNy assay This assay is a modification of the standard ''Cr-release lytic 10 assay (Current Protocols in Immunology, Coligan et al., eds.) except that the target cells are not radiolabeled prior to incubation with the splenocytes. Detailed procedures for this assay are described in Di Fabio et al. (1994) "Quantitation of human influenza virus-specific cytotoxic T lymphocytes: correlation of cytotoxicity and increased 15 numbers of IFN-gamma-(or IFNy-) producing CD8+ T cells" Int. Immunol. 6:11-9. Briefly, about 1x10 5 splenocytes were incubated with 10' target cells (e.g., infected with appropriate viruses carrying the target antigens) in a total volume of 100 pl. Cells were incubated for 4h at 37 0 C. IFNy was measured by ELISA from 25 pl medium. 20 Activation of CTL in mice inoculated with the adenoviral vaccine against HIV antigens was determined by using the IFNy assay described above. Briefly, twelve C57BU/6 mice were injected intramuscularly with 10i pfu Ad.tat.env.lL2. Spleens were harvested from 4 inoculated mice at the time points indicated in Figures 8A-C. Splenocytes were 25 activated by incubation with B16-Fl cells (a melanoma cell line from C57BU6, ATCC No: CRL-6323) that had been infected with Ad.tat.env.lL2. At day seven after stimulation, activated splenocytes were mixed with B16-F1 cells infected with the indicated viruses. IFNy secretion into the medium was determined by ELISA (R&D Systems, 30 Minneapolis, MN). 84 Figures 8A-C show percent increases in the amount of IFNy secreted into the medium over the period of time ranging from 4-8 weeks post inoculation. As shown in Figure 8A, secretion of IFNy increased significantly in splenocytes of the four mice harvested 4 5 weeks post inoculation with Ad.tat.env.lL2. In contrast, little increase in IFNy secretion occurred when the splenocytes were incubated with B16 F1 cells infected with an adenoviral vector expressing non-specifc protein p-Gal (Ad.lacZ) or uninfected B16-F1 cells. Secrection of IFNy increased more in the splenocytes of mice 10 harvested 6 weeks post inoculation as shown in Figure 8B. Noticeably, there was near 100% increase in secrection of IFNy in splenocytes of mouse 5 (Figure 8B). Secrection of IFNy increased more dramatically in the splenocytes of mice harvested 8 weeks post inoculation as shown in 15 Figure 8C. There was more than 300% increase in secrection of IFNy in splenocytes of mouse 11 (Figure 8C). These results demonstrate that strong humoral immune responses against HIV, such as induction of high titer antibody and activation of CTL specifically targeting HIV antigens, have been 20 achieved by inoculating animals with the adenoviral vaccine expressing both HIV viral antigens and an immuno-stimulator such as IL-2. The immune responses resemble those during a recovering of viral infection diseases. These results tend to show that the genetic vaccines of the present invention that mimic natural viral infection hold great promises 25 as efficacious vaccines for humans against HIV. b) Granzyme A assay Granzyme A assay was performed using a protocol modified from the one described in Deitz et al. (2000) "MHC I-dependent antigen 30 presentation is inhibited by poliovirus protein 3A" Proc. NatI. Acad. Sci. 97:13790-13795. The granzyme A assay described in Deitz et al. was 85 a modification of a protocol described in: Kane et al. (1989) "Cytolytic

T

lymphocyte response to isolated class I H-2 proteins and influenza peptides" Nature (London) 340:157-159. Granzyme A Assays were performed following similar procedures 5 as for IFNy assays with the following exceptions. Granzyme A secretion into the medium was determined by an enzymatic assay. Units of granzyme A were determined by calculating the slope of activity during the linear phase of the reaction. One unit of granzyme A was defined as the amount of enzyme required to convert the substrate to 1 OD 4 , in 10 one hour. Briefly, about 1x1 0' activated splenocytes and about lx105 target cells were incubated together as for the IFNy assays. Granzyme A activity was determined by combining 20 pl medium with 180 pl reaction mixture (0.2 mM BLT (N--a-benzyloxycarbonyl-L-lysinethiobenzy ester, 15 Sigma, St. Louis, MO), 0.22 mM DTNB (5,5'-dithio-bis(2 nitrobenzoicacid, Sigma, St. Louis, MO)) in 96-well plates and incubating at room temperature. Absorbance at 405 nm was monitored over a period of several hours. Slopes of enzyme activity were determined for the linear phase of the reaction and converted to units of 20 enzyme. Figure 9 shows increases in the amount of granzyme A secreted into the medium for splenocytes of mice harvested 8 weeks post inoculation. As shown in Figure 9, secretion of granzyme A increased significantly in splenocytes of the four mice harvested 8 weeks post 25 inoculation with Ad.tat.env.lL2. In contrast, much less increase in granzyme A secretion occurred when the splenocytes were incubated with B16-Fl cells infected with an adenoviral vector expressing non specifc protein P-Gal (Ad.lacZ), an adenoviral vector expressing both hepatitis B surface antigen and IL-2 (Ad.HBsAg/lL2) or uninfected B16 30 F1 cells. Similarly, there is little spontaneous granzyme A secrection in these splenocytes not incubated with the target cells. 86 These results, obtained by using the granzyme A assay independent from the IFNy assay, again demonstrate that strong activation of CTL specifically targeting HIV antigens was induced by inoculating mice with the adenoviral vaccine expressing both HIV viral 5 antigens and an immuno-stimulator such as IL-2. These results also support the belief that the genetic vaccines provided by the present invention hold great promises as efficacious vaccines for humans against HIV. 10 c) ELISPOT Assay ELISPOT assays were performed to determine CTL activation in mice inoculated with the recombinant adenoviral vectors, Ad.3C.env.gag and Ad.3C.env.gag.rev. C57BL16 mice were inoculated with 107 pfu Ad.3C.env.gag or Ad.3C.env.gag.rev. Mice were sacrificed at two week 15 intervals and splenocytes were prepared (se Current Protocols in Immunology, Coligan et al. eds.). At week 11, mice were inoculated with a second dose of 107 pfu of Ad.3C.env.gag or Ad.3C.env.gag.rev. 2 x 10' splenocytes were incubated with 4 x 104 MC57G cells (ATCC #CRL-2295) that had been infected with vaccinia viruses expressing 20 either Env, Gag, or Rev, in 96-well, mouse IFNy, ELISPOT plates (R&D Systems, Minneapolis, MN) for 30h. Non-specific activation was monitored following the addition of 4 pg/ml PHA (Sigma, St. Louis, MO) instead of antigen-expressing cells. IFNy spots were visualized as per the kit instructions and counted. Wild type and recombinant vaccinia 25 viruses were obtained from the NIH AIDS Research and Reference Reagent Program, Bethesda, MD. Figures 15 A-C show the results of the ELISPOT assays above in week 4 post-inoculation, week 8 post-inoculation, and week 12 post primary inoculation and week 1 post-secondary inoculation, respectively. 30 As shown in these figures, immunization of mice with the genetic 87 vaccines of the present invention induced strong activation of CTL against specific HIV antigens. 3. Genetic Vaccine Against Hepatitis B Virus 5 Embodiments of the genetic vaccine against hepatitis B virus and methods of their construction are described in detail as follows. 1) Construction of replication-defective adenoviral vaccines against hepatitis B virus 10 Two adenoviral vectors, Ad.HBsAg.lL2 and Ad.HBcAg.lL2, were constructed to carry the coding sequences for a hepatitis B surface antigen (HBsAg) and a HBV core antigen (HBcAg), respectively. In the same vector, DNA sequence encoding interleukin-2 (IL-2) was also 15 included and expressed by a promoter different from that for expressing the viral antigen. This design is believed to be able to ensure high level expression of both the viral antigens and the immuno-stimulator IL-2 and to enhance immunogenicity of the adenoviral vaccine. As shown by experimental data presented in the next section, both of these two 20 adenoviral vectors are capable of eliciting strong and long-lasting immune responses in animals against hepatitis B antigens. These two adenoviral vectors, Ad.HBsAg.lL2 and Ad.HBcAg.lL2, were constructed using strategies similar to those for constructing the adenoviral vaccines against Ebola virus as described in detail above. 25 a) Ad.HBsAg.1L2 Briefly, full length HBsAg (with a silent mutation caused by deletion of Xba I site) was inserted into the left end (El region) of the adenoviral genome using a shuttle vector pLAd (Figure 4A, left side), 30 resulting in a shuttle vector pLAd-CMV-HBsAg. 88 The sequence encoding IL-2 (with a silent mutation caused by deletion of Xba I site) was inserted into E4 region of the adenoviral genome using the shuttle vector pRAd (Figure 4A, right side), resulting in a shuttle vector pRAd-CMV-IL2. 5 Both pLAd-CMV-HBsAg and pRAd-CMV-IL2 were linearized using appropriate restriction enzymes such as Xba I and EcoRI and ligated to the backbone of the adenovirus (Figure 4B), resulting in the recombinant adenoviral vector designated Ad.HBsAg.1L2. 10 a) Ad.HBcAg.1L2 Briefly, sequences encoding full length HBsAg (with a silent mutation caused by deletion of Xba I site) and full length HBcAg were inserted into the left end (El region) of the adenoviral genome using a shuttle vector pLAd (Figure 4A, left side). HBsAg and HBcAg are 15 expressed separately from another CMV promoter via a retroviral splicing donor (SD) and acceptor (SA) mechanism at two splicing acceptor sites, SA, and SA 2 . The shuttle vector produced is designated pLAd-CMV-SD/SA-HBsAg-SA-HbcAg. Sequences encoding multiple immuno-stimulators, including IL-2 20 (with a silent mutation caused by deletion of Xba I site), INF-y, and GMCSF, were inserted into E4 region of the adenoviral genome using the shuttle vector pRAd (Figure 4A, right side). These three immuno stimulators are expressed separately from another CMV promoter via a retroviral splicing donor (SD) and acceptor (SA) mechanism at three 25 splicing acceptor sites, SA 1 , SA 2 , and SA 3 . The shuttle vector produced is designated pRAd-CMV-SD/SArl L2-SA 2 - INFy-SA 3 -GMCSF. Both pLAd-CMV-SD/SA-HBsAg-SA 2 -HbcAg and pRAd-CMV SD/SA-1L2-SA 2 - INFy-SA 3 GMCSF were linearized using appropriate restriction enzymes such as Xba I and EcoRI and ligated to the 30 backbone of the adenovirus (Figure 4B), resulting in the recombinant adenoviral vector designated Ad.HBcAg.IL2. 89 2) Immune responses of animals to the adenoviral vaccines against HBV antigens 5 Experimental mice were inoculated with the adoviral vaccine constructed above, Ad.HBsAg.1L2 and Ad.HBcAg.lL2, to elicit immune response to the hepatitis B surface antigen and core antigen expressed by these two vectors, respectively. Immunogenicity of these adenoviral vectors was determined by measuring titers of antibodies against 10 HBsAg and HbcAg, respectively. a) HBV surface antigen (HBsAg) antibody titers CD-1 mice (Charles River Laboratories, Wilmington, MA) were injected intramuscularly with several different concentrations of 15 Ad.HBsAg.lL2: 10", 1x10", 1x10 7 , 1x108, 5 x 105, 5 x 106, and 5 x 107 pfu virus. Figure IOA shows the relative Anti-HBsAg antibody titers measured for sera harvested from mice inoculated with 1 x 105 and 5 x 105 pfu. Serum in each measurement was diluted 1:500. Figure 1OB shows the relative Anti-HbsAg antibody titers measured for sera 20 harvested from mice inoculated with 1x10 7 and 1xi0' pfu. Serum in each measurement was diluted 1:1500. To measure the relative titers of the Anti-HbsAg antibody elicited by Ad.HBsAg.IL2, blood (about 150-500 pl) from each animal was collected from immunized mice every two weeks and serum was 25 prepared. Blood was incubated at room temperature for 2-3 h to allow for clotting. The blood was then chilled overnight at 4 0 C to shrink the clot. Unclotted liquid was transferred to a clean tube and centrifuged at 2000 x g for 5 min. The supernatant was transferred to another clean tube. Sodium azide (NaNa) was added to 0.05% as a preservative 30 Small aliquots were kept at 4 0 C for short-term storage. Long-term storage was at -80 0 C. 90 Relative anti-HBsAg titers were determined by ELISA against recombinant HBsAg purified from yeast (from Aldevron, LLC, Fargo, ND). As shown in Figure 10A, the mice in group 1 had increasingly strong immune responses to HBsAg expressed by the adenoviral vector, 5 Ad.HBsAg.1L2, within 8 weeks post inoculation. This vector with a titer as low as 5 x 105 pfu was sufficient to elicit high levels of antibody specifically against HBsAg. Figure 10B shows the immunogenicity of Ad.HBsAg.L2 with higher titers. As shown in Figure 10B, immunogenicity of Ad.HBsAg.L2 10 increased dramatically as the titer of the adenoviral vector was increased from 1 x 107 pfu to 1x 108 pfu. These results demonstrate that the adenoviral vector expressing both hepatitis B surface antigen and IL-2 can induce strong immune response specifically targeting the viral antigen in mice inoculated with 15 this vector. These results also support the belief that the genetic vaccines provided by the present invention hold great promises as efficacious vaccines for humans against hepatitis B virus. b) HBV core antigen (HBcAg) antibody titers 20 Groups of C57BL/6 mice (Charles River Laboratories, Wilmington, MA) were injected intramuscularly with 1x107 pfu Ad.HBcAg.1L2 on different dates. Blood was collected from four animals every two weeks following inoculation and serum was prepared. At 91 days (Group 3, Figure I1A) or 84 days (Group 4, Figure 11B) post 25 inoculation, mice were re-challenged with an additional 1x107 pfu virus. Blood was collected from three animals every day following secondary challenge. Antibody titer was determined by ELISA against recombinant HBcAg purified from E. coli (from Chemicon International, Inc., Temecula, CA). 30 As shown in Figure 11A, mice in group 3 had strong immune response to the hepatitis core antigen HBcAg expressed by the 91 adenoviral vector Ad.HBcAg.IL2, with the highest titer of antibody against HBcAg reached in about 28 days post inoculation. The second inoculation with Ad.HBcAg.IL2 boosted the immune response again and very high titers were achieved within about 3 days of the second inoculation. 5 As shown in Figure 11B, mice in group 4 also had strong immune response to the hepatitis core antigen HBcAg expressed by the adenoviral vector Ad.HBcAg.IL2, with the high titer of antibody against HBcAg reached in about 34 days post inoculation. The second inoculation with Ad.HBcAg.lL2 boosted the immune response again and very high titers were achieved within about 3 days of the 10 second inoculation. These results demonstrate that the adenoviral vector expressing both hepatitis B core antigen and IL-2 can also induce strong immune response specifically targeting the viral antigen in mice inoculated with this vector. These results once again support the belief that the genetic vaccines provided by the present 15 invention hold great promises as efficacious vaccines for humans against hepatitis B virus. Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or 20 group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form or suggestion that the prior art forms part of the common general knowledge in Australia. 18/03/10.de 16748 amended specification pages.doc.92 92

Claims

1. A replication incompetent recombinant adenovirus, comprising: one or more antigen sequences heterologous to a native progenitor of the 5 recombinant adenovirus inserted in an El, E3 or E4 region of the adenovirus, each antigen sequence expressing a viral antigen from a pathogenic virus under control of a first promoter located in the same region as the antigen sequences; and one or more immunostimulator sequences heterologous to the native progenitor of the recombinant adenovirus inserted in an E4, E3 or El region of the 10 adenovirus that does not comprise the antigen sequence, each immunostimulator sequence expressing an immunostimulator under the control of a second promoter located in the same region as the immunostimulator sequences; and characterised in that the first promoter either bicistronically or polycistronically controls the expression of one or more antigen sequences and that 15 the second promoter either bicistronically or polycistronically controls the expression of one or more immunostimulator sequences, wherein the bicistronic control is via an internal ribosomal entry site or a splicing donor-acceptor mechanism and the polycistronic control is via a splicing donor-acceptor mechanism.

2. The replication incompetent recombinant adenovirus according to 20 claim 1, wherein the second promoter bicistronically controls expression of two immunostimulator sequences via an internal ribosomal entry site or via a splicing donor-acceptor mechanism when the adenovirus comprises two heterologous immunostimulator sequences.

3. The replication incompetent recombinant adenovirus according to 25 claim 1, wherein the first promoter polycistronically controls expression of at least two of the antigen sequences via a splicing donor-acceptor mechanism when the adenovirus comprises a plurality of antigen sequences.

4. The replication incompetent recombinant adenovirus according to claim 1, wherein the first promoter controls expression of at least two of the antigen 30 sequences as a fusion protein.

5. The replication incompetent recombinant adenovirus according to any one of claim 1 to claim 4, wherein the plurality of antigen sequences are 18/03/10,dc 16748 amended specification pages.doc.93 93 different antigens from the same pathogenic virus, a combination of antigens from at least two strains of the pathogenic virus or a combination of antigens from at least two different pathogenic virus.

6. The replication incompetent recombinant adenovirus according to 5 any one of claim I to claim 5, wherein the plurality of antigen sequences are a combination of antigens selected from at least two of the following group: HIV type 1; HIV type 2; herpes simplex virus type 1; herpes simplex virus type 2; Ebola virus; Hepatitis A; Hepatitis B; Hepatitis C; Hepatitis D; and Hepatitis E virus.

7. The replication incompetent recombinant adenovirus according to 10 any one claim I to claim 6, wherein the second promoter controls expression of at least two of the immunostimulant sequences as a fusion protein.

8. The replication incompetent recombinant adenovirus according to any one claim 1 to claim 7, wherein the adenovirus is adenovirus type 5..

9. The replication incompetent recombinant adenovirus according to 15 any one of claim I to claim 8, wherein the first promoter or the second promoter or a combination thereof is a promoter homologous or heterologous to the native progenitor of the recombinant virus, wherein the heterologous promoter is selected from any one of the following group: a CMV promoter; a SV40 promoter; a retrovirus LTR promoter; and a chicken cytoplasmic b-actin promoter. 20 10. The replication incompetent recombinant adenovirus according to any one of claim I to claim 9, wherein the viral antigen is a full-length antigenic viral protein or a portion of the antigenic viral protein that contains the predominant antigen, neutralising antigen, or epitope of the pathogenic virus or a region conserved between glycoproteins of at least two strains of the pathogenic virus or is a modified 25 antigen comprising mutated glycoprotein of the pathogenic virus, wherein said mutation includes deletions in the proteolytic cleavage site of the glycoprotein and duplications and rearrangement of immunosuppressive peptide regions of the glycoprotein such that the viral antigen is rendered non-functional as a viral component but retains its antigenicity. 30 ]1. The replication incompetent recombinant adenovirus according to any one of claim 1 to claim 10, wherein the pathogenic virus is human immunodeficiency virus, wherein an antigen of said HIV is a HIV surface protein 18/03/10,dc 16748 amended specification pages.doc,94 94 comprising a HIV surface protein which is encoded by env gene of HIV, a HIV Gag protein, a HIV capsid protein comprising a p17, p24, p2, p7 or p6 capsid protein, a HIV regulatory protein, a HIV enzyme, a HIV accessory protein, a HIV glycoprotein comprising one or more of V2, V3, C4 domains of HIV, a modified HIV protein 5 comprising a modified HIV gp160 containing mutation(s) in the proteolytic processing site between gp120 and gp41, a modified HIV gpl60 with deletion of the cytoplasmic domain, a modified HIV gp120 containing mutations in one or more glycosylation sites, or combination thereof or a HIV mosaic antigen that includes variable and conserved domains of HIV from different strains, isolates or clades of 10 HIV, includes one or more domains from at least two clades of the group M HIV, includes one or more domains from at least two clades of the group 0 HIV or a combination thereof.

12. The replication incompetent recombinant adenovirus according to any one of claim I to claim 10, wherein the pathogenic virus is influenza virus, 15 wherein an antigen of the influenza virus is an influenza glycoprotein, wherein said glycoprotein is anyone of: HAl; HA2; and NA of the influenza virus.

13. The replication incompetent recombinant adenovirus according to any one of claim I to claim 10, wherein the pathogenic virus is Ebola virus, wherein an antigen of the Ebola virus comprises an Ebola glycoprotein, wherein said 20 glycoprotein is Ebola GPI or Ebola GP2 protein.

14. The replication incompetent recombinant adenovirus according to any one of claim I to claim 10, wherein the pathogenic virus is any one of: hepatitis A; hepatitis B; hepatitis C; hepatitis D and hepatitis E virus, wherein an antigen of the hepatitis virus is a surface antigen or a core protein of hepatitis B virus 25 comprising any one of: SHBsAg; MHBsAg; and LHBsAg of hepatitis B virus or a surface antigen or core protein of hepatitis C virus comprising any one of: NS3; NS4; and NS5 antigen of hepatitis C virus.

15. The replication incompetent recombinant adenovirus according to any one of claim I to claim 10, wherein the pathogenic virus is respiratory syncytial 30 virus, wherein an antigen of the respiratory syncytial virus comprises a glycoprotein or a fusion protein of the respiratory syncytial virus. 18/03/1O,dc 16748 amended specification pages.doc,95 95

16. The replication incompetent recombinant adenovirus according to any one of claim 1 to claim 10, wherein the pathogenic virus is herpes simplex virus type 1 or type 2, wherein an antigen of the herpes simplex virus comprises a glycoprotein D from herpes simplex virus type 2. 5 17. The replication incompetent recombinant adenovirus according to any one of claim 1 to claim 10, wherein the pathogenic virus is an E6 or an E7 human papilloma virus.

18. The replication incompetent recombinant adenovirus according to any one of claim 1 to claim 17, wherein the immunostimulator is a cytokine, wherein 10 said cytokine is selected from any one of the following group: interleukin-2; interleukin-4; interleukin-12; b-interferon; 1-interferon; g-interferon; granulocyte colony stimulating factor; and granulocyte-macrophage colony stimulating factor.

19. The use of the replication incompetent recombinant adenovirus according to any one of claim 1 to claim 18 in the preparation of a vaccine to 15 enhance the immunity of a host to one or more pathogenic viruses.

20. The use of the replication incompetent recombinant adenovirus according to claim 19, wherein the replication incompetent recombinant adenovirus is administered to the host by any of the following methods: intramuscularly; intratracheally; subcutaneously; intranasally; intradermally; rectally; orally; and 20 parentally.

21. A replication incompetent recombinant adenovirus, substantially as hereinbefore described with reference to any one of the Examples.

22. A replication incompetent recombinant adenovirus, substantially as hereinbefore described with reference to any one of the accompanying drawings. 25 18/03/10,dc 16748 amended specification pages.doc,96 96