KR20010085326A - Prevention and treatment of viral disease - Google Patents

Abstract

The present invention is directed to the production of fusion-related molecular structures (FRMSs) consisting of one or a plurality of transitional fusion-related crystal regions. High-efficiency vaccines can be constructed that provide a fusion-related determinant in the immune system that produces unique antibodies capable of strongly neutralizing a wide range of primary isolates from worldwide and different phylogenetic viral tissues. The present invention relates to the use of FRMS in a variety of compositions and methods, including methods for preparing, isolating, and purifying FRMS and for use as vaccine immunogens, diagnostics, and therapeutics. The present invention relates to FRMS capable of inducing neutralizing antibodies against viral immunogens and viral primary isolates. Antibodies generated against FRMS can be used to study molecular pathways in fusion of viruses and host cells and to identify antibody-mediated blocking points of these pathways. The invention also relates to the use of the antibodies of the invention for antiviral drugs, blood product additives, contraceptive addition passive immunity or fetal immunity in post-exposure treatment.

Description

Prevention and treatment of viral diseases {PREVENTION AND TREATMENT OF VIRAL DISEASE}

2. Background Technology

The references mentioned in this article are not sources of the invention.

Viruses are infectious agents that drive a variety of diseases in humans, animals, bacteria, plants, and are therefore medically and commercially important. Broadly speaking, free viral particles, collectively referred to as virions, consist of the genome (RNA or DNA), related proteins or polyamides, and coated proteins, collectively referred to as capsids. Some virions may be surrounded by a membrane envelope. Capsids and envelope structures serve to protect the genome from nucleases present in the environment and to facilitate viral attachment and entry into cells that the virus infects and replicates.

2.1. Virus entry

In order to replicate, the virus must use the biosynthetic process of the host cell. Viruses use biosynthetic pathways and substrates available in animals to maintain and propagate their genetic information. To access the cell's biopath, the virus must enter or infect the cell. Viral proteins bind to these receptors, which initiate a complex series of tasks, through which a viral nucleic acid or nucleoprotein is released intracellularly.

In the case of enveloped viruses, the fusion of the viral envelope with the cell membrane (eg, cell surface membrane or inner membrane) facilitates infection. Various enveloped viruses are involved in human and animal diseases.

2.2. HIV

A clinically important example of enveloped virus is HIV. Human immunodeficiency virus (HIV) is thought to be a major cause of degenerative immune system disease, collectively referred to as AIDS (Barre-Sinoussi, F., et al., 1983, Science 220: 868-870; Gallo, R., et al., 1984, Science 224: 500-503). In the human body, HIV replication occurs mainly in the CD4 ⁺ T lymphocyte population, which leads to depletion of these cell types and thus to immunodeficiency, opportunistic infections, neurological dysfunction, neoplastic growth, and ultimately death. There are two or more HIV types: HIV-1 (Barre-Sinoussi, F., et al., 1983, Science 220: 868-870; Gallo, R., et al., 1984, Science 224: 500-503 ) And HIV-2 (Clavel, F., et al., 1986, Science 223: 343-346; Guyader, M., et al., 1987, Nature 326: 662-669). In addition, important genetic advection exists within each population.

HIV is a member of the retroviral rental family of viruses (Teich, N., et al., 1984, RNA Tummor Viruses, Weiss, R., et al., Eds., CSH-Press, pp. 949-956). Retroviruses are small enveloped viruses that carry a single stranded RNA genome and replicate through DNA intermediates produced by virus-encoded reverse transcriptase (RNA-dependent DNA polymerase) (Varmus, H., 1988, Science). 240: 1427-1439).

HIV viral particles consist of a viral core (a portion of the capsid protein and the viral RNA genome) and enzymes required for initial replication. Myristylized gag protein constitutes the outer shell of the core of the virus core, which is surrounded by a lipid membrane envelope derived from the infected cell membrane. HIV envelope surface glycoproteins are synthesized into a single precursor of 160 kilodaltons that are split into two glycoproteins gp41 and gp120 by cellular proteases during viral emergence. gp41 is a transmembrane glycoprotein and gp120 is an extracellular glycoprotein that is non-covalently bound to gp41 in trimer or multimeric form (Hammarskjold, M., et al., 1989, Biochem.Bjophys. Acta 989: 269-280 ).

HIV targets CD4 ⁺ cells because CD4 cell membrane protein (CD4) acts as a cellular receptor for the HIV-1 virus (Dalgleish, A., et al., 1984, Nature 312: 763-767; Klatzmann et al., 1984, Nature 312: 767-768; Maddon et al., 1986, Cell 47: 333-348). Cell entry into the virus depends on gp120 binding to intracellular CD4 receptor molecules (McDougal, JS, et al., 1986, Science 231: 382-385; Maddon, PJ, et al., 1986, Cell 47: 333- 348), which explains the HIV preference for CD4 ⁺ cells.

Immediately after the interaction of the viral envelope protein with the cell receptor, the envelope protein-CD4 complex is subsequently morphed to facilitate interaction with the host cell co-receptor and constitute a trimolecular complex. Additional morphological changes in the trimolecular complex allow the fusion of the viral envelope and the additional cell membrane to release the genetic material into the cell.

2.3. Virus treatment strategies

2.3.1. Drug therapy

While considerable efforts are being made to devise effective therapies, there are no antiviral therapeutic drugs for AIDS yet. Attempts to develop such drugs have focused on several stages of the HIV life cycle (Mitsuyam H., et al., 1991, FASEB J. 5: 2369-2381). Various viral targets have been proposed to interfere with the HIV life cycle, because the view that harmful side effects occur when interfering with host cell proteins is analogous. For example, viral encoded reverse transcriptase has become a focus of drug development. A number of reverse-transcriptase-targeted drugs have been developed, including 2,3, -dideoxynucleoside analogs that show antagonistic activity against HIV, such as AZT, ddI, ddC, d4T (Mitsuya, H. , et al., 1991, Science 249: 1533-1544).

Reverse transcriptase (RT) such as azidomidine (AZT), lamivudine (3TC), dideoxyinosine (ddI), dideoxycytosine (ddC) in combination with HIV protease inhibitors in a novel treatment regimen for HIV-1 It has been found that the anti-HIV complex compounds targeting to show a much improved effect (2 or 3 log reduction) compared to AZT alone (approximately 1 log reduction). For example, using a mixture of AZT, ddI, 3TC, ritonavir, improved results were obtained (Perelson, AS, et al., 1996, Science 15: 1582-1586). However, long-term use of mixtures of these drugs is particularly likely to cause toxicity to the bone marrow. In addition, long-term cytotoxic therapies include killer cell activity (Blazevic, V., et al., 1995, AIDS Res. Hum.Retroviruses 11: 1335-1342) and inhibitory factors, mainly Rantes, MIP-1α, MIP-1β ( Cocchi, F., et al., 1995, Science 270: 1811-1815) releases CD8 ⁺ T cells essential for the regulation of HIV. Another concern with long-term anti-retroviral chemotherapy is the development of HIV mutations with partial or complete resistance (Lange, JM, 1995, AIDS Res. Hum. Retroviruses 10: S77-82). Such mutations are considered inevitable results of anti-viral therapy. The emergence of variant viruses due to the disappearance and treatment of wild-type viruses with a decrease in the number of CD4 ⁺ T cells strongly suggests that the emergence of viral variants due to at least some compounds is a major cause of AIDS therapy failure.

In addition, failure of conventional drugs can also be attributed to HIV reservoirs that lie in a slowly growing cell population and do not actively produce viruses, thus avoiding many conventional treatments.

The development of drugs that can inhibit the cell entry of the virus has been attempted. In these studies, the focus is on CD4, the cell surface receptor for HIV. For example, recombinant soluble CD4 appears to inhibit CD4 ⁺ T cell infection of some HIV-1 strains (Smith, DH, et al., 1987, Science 238: 1704-1707). However, certain primary HIV-1 isolates are significantly insensitive to inhibition by recombinant CD4 (Daar, E., et al., 1990, Proc. Natl. Acad. Sci. USA 87: 6574-6579). In addition, in clinical trials of recombinant soluble CD4, inconclusive results were obtained (Schooley, R., et al., 1990, Ann. Int. Med. 112: 247-253; Kahn, JO, et al., 1990, Ann. Int. Med. 112: 254-261; Yarchoan, R., et al., 1989, Proc. Vth Int. Conf. On AIDS, p. 564, MCP 137).

The later stages of HIV replication, in which the major virus-specific process of certain viral encoded proteins are made, are also proposed as anti-HIV drug targets. Since later processes depend on the activity of viral proteases, drugs that inhibit these proteases are being developed (Erickson, J., 1990, Science 249: 527-533).

Recently, chemokines produced by CD8 ⁺ T cells have been known to be involved in the inhibition of HIV infection (Paul, WE, 1994, Cell 82: 177; Bolognesi, DP, 1993, Semin. Immunol. 5: 203). Chemokines RANTES, MIP-1α, MIP-1β secreted by CD8 ⁺ T cells have been shown to inhibit HIV-1 p24 antigen production in vitro, in cells infected with HIV-1 or HIV-2 isolates (Cocchi , F, et al., 1995, Science 270: 1811-1815). Thus, these chemokines may be useful in therapy for HIV infection. However, the clinical results of these candidate drugs are still questionable.

2.3.2. vaccine

Another major strategy in antiviral treatment and prevention is the development of vaccines. Due to the potential spread of viral infections such as HIV, effective vaccines are needed.

In general, conventional methods of making vaccines include the use of inactivated or attenuated pathogens. Moderately inactivated pathogenic microorganisms are harmless to use as biological drugs but do not destroy immunogenicity. Injection of these "killed" particles into the host induces an immune response that can prevent future infection by living microorganisms. However, a disadvantage of using killed vaccines (inactivated pathogens) is that they cannot inactivate all microbial particles. Even if this is achieved, multiple vaccinations are required because the killed pathogens do not proliferate in the host or because of their incomplete immunogenicity due to other unknown reasons, the survival is short. Finally, microorganism antigens may be altered during inactivation and these antigens may not be effective as immunogens.

Attenuation refers to the production of pathogenic microbial strains that have lost disease-producing capacity. One way to achieve this is by treating the microorganisms to specific growth conditions or by frequent passage in cell culture. Subsequently, variants are selected that have lost virulence but can still induce an immune response. Attenuated pathogens are excellent immunogens in that they replicate in host cells and induce immunity for long periods of time. However, using live vaccines presents several problems, the biggest of which is the risk of insufficient attenuation and virulence.

Another alternative is to use subunit vaccines. This includes immunization with ingredients that have relevant immune substances.

For HIV, for example, envelope proteins (gp160, gp120, gp41) appear to be the major antigens against anti-HIV antibodies present in AIDS patients (Barin et al., 1985, Science 228: 1094-1096). Thus, these proteins are the most common after that serve as immunogens for anti-HIV vaccine development. Some speakers have begun using various proteins of gp160, gp120 or gp41 as immunotargets for the host immune system (Ivanoff, L., et al., US Pat. No. 5,141,867; Saith, G., et al., WO92 / 22,654; Shafferman, A., WO 91 / 09,872; Formoso, C., et al., WO 90 / 07,119).

Because HIV envelope proteins mediate early binding and entry stages in infection, many vaccine strategies have been developed that focus on blocking the function of viral envelope proteins. In 1993, two recombinant forms of the surface gp120 subunit of HIV envelope protein (rgp120) emerged as candidates for candidate vaccines in large-scale efficacy studies supported by the National Institutes of Health (NIH). In previous clinical trials, the rgp120 vaccine was found to be able to induce antibodies that are safe and strongly neutralize the relevant laboratory-adaptive isolates of HIV (Belshe, RB, et al., 1994, Journal of the American Medical Association 272: 475; Kahn, JO, et al., 1994, Journal of Infectious Diseases 170: 1288). However, since it was discovered that the primary isolate ("PI") virus resists neutralization by the rgp120 vaccine serum, no further progress has been made (Cohen, J., 1993, Science 262: 980; Cohen, J., 1994, Science 264: 1839).

2.4. Necessity of Neutralization of Primary Separator

With the spread of HIV infection, more than 40 million people are threatened with infection worldwide by 2000 (UNAIDS Report (www.unaids.org/unaids/report)). Effective HIV vaccines are urgently needed, but progress is not made for this purpose because vaccine candidates fail to induce antibodies that can neutralize the infection of various primary isolates (PIs) in HIV infected individuals (Wrin, T). , et al., 1995, Journal of Virology 69:39; Mooore, JP, et al., 1995, AIDS 9 (suppl A), S117; Mascola, JR, et al., 1996. Journal of Infectious Diseases 173: 340).

Unfortunately, until now, speakers have failed to produce vaccines that produce antibodies that block various primary isolates of HIV. Primary isolates are taken directly from infected individuals and grow limited in primary laboratory peripheral blood lymphocytes (PB 1s). Thus, primary HIV isolates are much more clinically desirable than laboratory-adapted strains. Laboratory adaptation strains are those strains that continue to grow in established T cells in the laboratory (Wrin et al., 1995, J. of Virology 69:39). Previous vaccine candidates were to induce neutralizing antibodies against laboratory-adapted strains. For example, Wrin et al. (1995, Journal of Virology 69:39) tested HIV envelope proteins derived from laboratory adapted strains using recombinant DNA technology. Antibodies generated by these recombinant protein immunizations neutralized only laboratory-adapted strains of virus. Recombinant viral vectors and DNA used to produce native oligomeric envelope proteins in situ also failed to induce neutralizing antibodies against primary isolates. In addition, immunogens produced using recombinant technology and composed of gp120 and soluble CD4 receptors induced antibodies to gp120 (Gershoni, 1993, FASEB Journal 7: 1185), but these antibodies did not neutralize primary isolates.

Neutralizing antibodies are important in preventing the binding and fusion of viruses and host cells. The action of the antibody to neutralize the laboratory-adapted isolate was analyzed. Neutralizing antibodies that specifically migrate to the CD4 binding domain on gp120 interfere with CD4 binding. However, most neutralizing antibodies interfere with later stages of CD4 binding. For example, some neutralizing antibodies inhibit the interaction with co-receptors (Trkola et al., 1998, Journal of Virology 72: 1876; Wu, et al., 1996, Nature 384: 179-83).

Identification of immunogens that induce neutralizing antibodies in the host is essential for the development of effective vaccines. Neutralizing antibodies should act not only on laboratory-adapted isolates, but also on clinically relevant primary isolates. As mentioned above, there is still a need for novel immunogen molecules to produce antibodies that effectively neutralize various primary isolates.

3. Summary of the Invention

The present invention provides a surprising discovery that high-efficiency vaccines can be constructed that provide a transient fusion-related determinant to the immune system that produces unique antibodies capable of strongly neutralizing a wide range of primary isolates from worldwide and different phylogenetic viral tissues. To The vaccine of the present invention comprises a fusion-related molecular structure (FRMS) consisting of one or a plurality of fusion-related crystal regions as immunogens. The present invention is also based on the finding that the extensive and unique neutralization of different primary isolates suggests that the critical determinants presented by FRMS are highly conserved and are directly linked to fundamental functions in binding and fusion of envelope proteins. The present invention relates to the use of FRMS in a variety of compositions and methods, including use as a vaccine immunogen, diagnostic drug, therapeutic drug.

The present invention also relates to FRMS capable of inducing neutralizing antibodies against enveloped viral pathogens. Epitopes that induce these neutralizing antibodies are caused by morphological changes during the binding and fusion of the viral envelope and host cell membrane.

In one embodiment, the complex is due to the interaction of the viral protein with one or more host cell receptors or co-receptors, wherein the complex is co-cultured with cells transfected with nucleic acid expressing the viral protein and cells expressing the host cell receptor. Make. Preferably, the cells recombinantly express host cell receptors. In another embodiment, the culture is immobilized at the start of cell-cell interaction to preserve the fusion-competent immunogen. In a suitable embodiment, the fusion-related immunogen is generated as a result of the interaction of major human immunodeficiency virus type 1 (HIV-1) envelope proteins, host cell receptor CD4, host cell coreceptor CCR5.

Vaccination of the FRMS of the present invention with an immunogen results in an immune response against viral pathogens and induces the production of neutralizing antibodies in the immunized host. In addition to using FRMS as a vaccine, antibodies generated against FRMS can be used to study molecular pathways in the fusion of viruses and host cells and to identify antibody-mediated blocking points for these pathways. In addition, specific high-affinity "labels" are expressed as part of the envelope protein or host cell receptor, thereby facilitating the identification and purification of the FRMS of the present invention.

The present invention relates to the development of monoclonal antibodies (mAbs) induced by these FRMS immunogens. Monoclonal antibodies are provided that can neutralize primary isolates. In addition, neutralizing mAbs with functionally preserved fusion-dependent epitopes is useful for passive immunity in fetal or post-exposure treatment.

The present invention is produced due to the binding of the envelope protein and one or more cell membrane proteins of the envelope virus, wherein the envelope protein and the membrane protein are necessary and sufficient under conditions suitable for the fusion of the cell membrane with the cell membrane protein and the viral envelope.

The present invention provides a combination of (a) an HIV envelope protein or variant thereof combined into a viral envelope; (b) provide an isolated molecular structure comprising epitopes produced by the binding of human CD4 and co-receptors for HIV fusion. In one embodiment, the co-receptor is chemokine receptor CCR5 or CXCR4. In another embodiment, the molecular structure is produced by the binding of HIV gp41 variants that are fusion-defective. In other embodiments, the variant has one or more mutations selected from V2E, G10V, V570R, Y586E. In another embodiment, the molecular structure is produced by the binding of wild type HIV envelope protein.

The present invention relates to (a) a variant envelope protein of the envelope virus, wherein the envelope protein acts on the fusion of the host cell membrane and the viral envelope in the wild type; (b) provides an isolated molecular structure comprising an epitope produced by the binding of one or a plurality of host cell membrane proteins acting as receptors for the envelope protein.

In one embodiment, the virus is a retroviridae, Rhabdoviridae, Caronaviridae, Filoviridae, Poxviridae, Bunyaviridae, Flaviviraceae It is selected from Flaviviridae, Togaviridae, Orthomyxoviridae, Paramyxoviridae and Herpesviridae. In another embodiment, the envelope protein is El and E2 of HCV and the cell membrane protein is CD81. In another embodiment, the molecular structure is a cross-linked cell membrane structure. In another embodiment, the molecular structure is separated from cell lysate. In one embodiment, the cellular molecular structure consists of cells that recombinantly express the envelope protein. In another embodiment, the cell lysate is obtained from a plurality of cells consisting of cells that recombinantly express the envelope protein. In another embodiment, the cell molecular structure further comprises a cell that recombinantly expresses one or a plurality of host cell membrane proteins.

The present invention provides a recombinant enveloped virus, wherein the virus recombinantly expresses cellular receptors for the native enveloped protein of the virus on the envelope. In one embodiment, the cell receptor is a co-receptor for human CD4 or HIV, or the virus recombinantly expresses human CD4 and co-receptor at the same time. In other embodiments, suitable conditions include low pH. In another embodiment, the cross-linked molecular structure further comprises cross-linked viral particles of said virus carrying envelope proteins.

The present invention provides a vaccine composition consisting of an immunological amount of the molecular structure and a pharmaceutically acceptable carrier.

The present invention provides a monoclonal antibody against the molecular structure. In one embodiment, the antibody is labeled.

The present invention provides purified polyclonal antisera specific for molecular structure.

The present invention provides a contraceptive jelly, foam, cream or ointment containing an effective amount of an antibody that inhibits or reduces viral infection.

The present invention provides a contraceptive jelly, foam, cream or ointment containing an effective amount of an antibody that inhibits or reduces HIV infection.

The present invention provides a contraceptive jelly, foam, cream or ointment containing an effective amount of antisera that inhibits or reduces HIV infection.

The present invention provides a sample of mammalian blood, wherein the antibody in the blood sample adds an effective amount that can inhibit or reduce viral infection.

The present invention provides a sample of human blood, wherein the antibody in the blood sample adds an effective amount that can inhibit or reduce viral infection. In one embodiment, the envelope protein or host cell membrane protein further comprises an affinity label. In another embodiment, the envelope protein, CD4 or co-receptor further comprises an affinity label. In another embodiment, the antibody is labeled.

The present invention provides a kit for holding labeled monoclonal antibodies directed against said molecular structure in one or a plurality of containers.

The present invention provides a kit for holding labeled monoclonal antibodies directed against said molecular structure in one or a plurality of containers. In one embodiment, the kit retains the molecular structure in a separate container.

The present invention provides a cell line that recombinantly expresses a coat protein of a coat virus, or a fusion-defective coat protein, which acts upon fusion of a viral coat with a host cell membrane, wherein the cell line acts as a receptor for the coat protein. Expresses one or more cell membrane proteins. In one embodiment, one or a plurality of proteins that act as receptors are recombinantly expressed.

The present invention provides a cell line that recombinantly expresses HIV gp160, which expresses CD4 and co-receptors for HIV, while lacking a functional protease that divides gp160 into gp120 and gp41.

The present invention provides a method for treating or preventing a viral infection in a subject, said method comprising administering to said subject an immunologically effective amount of said molecular structure capable of treating or inhibiting a viral infection. In one embodiment, the subject is a human. In other embodiments, the subject is a livestock.

The present invention provides a method of treating or preventing HIV infection in a human, the method comprising administering to a human an immunologically effective amount of the molecular structure capable of treating or inhibiting an HIV infection.

The present invention provides a method of treating or preventing a viral infection in a subject, which method comprises administering to the subject an effective amount of a monoclonal antibody that can treat or inhibit the viral infection.

The present invention provides a method of treating or preventing an HIV infection in a human, which method comprises administering to the human an effective amount of a monoclonal antibody capable of treating or inhibiting an HIV infection. In one embodiment, the person is at high risk of HIV infection. In one embodiment, the method is for treating human HIV.

The present invention provides a method for treating or preventing HIV infection in a human fetus, which method comprises administering to the pregnant subject an effective amount of a monoclonal antibody capable of treating or inhibiting an HIV infection.

The present invention provides a method of treating or preventing a viral infection in a blood sample, which method comprises administering to the blood sample an effective amount of a monoclonal antibody capable of treating or inhibiting a viral infection.

The present invention provides a method of treating or preventing HIV infection in a blood sample of a human, the method comprising administering to the blood sample an effective amount of a monoclonal antibody capable of treating or inhibiting an HIV infection.

The present invention provides a method for removing contaminants of a surgical or dental tool, which method comprises contacting the tool with an effective amount of a monoclonal antibody that can inhibit viral infection.

The present invention provides a method of removing contaminants of a surgical or dental tool, which method comprises contacting the tool with an effective amount of monoclonal antibody that can inhibit HIV infection.

The present invention provides a method for monitoring antibody production of the molecular structure in a subject previously administered an amount of molecular structure, the method comprising separating a sample containing serum from the subject; Detecting the presence of any antibody to the molecular structure in the serum, the detection is carried out by a competitive immunoassay method using a labeled antibody against the molecular structure.

The present invention provides a method for producing an immunogen that is used as a vaccine for treating or preventing a viral infection, wherein the method comprises (a) an envelope protein of the envelope virus or a chimeric form thereof, wherein the envelope protein is a cell membrane and a viral envelope. Contacting a mutant or chimeric form of said envelope protein that is fusion-defective and one or more cellular proteins or chimeric forms thereof that act as receptors for said envelope protein; (b) exposing the envelope protein or chimeric type or mutant thereof or chimeric type and one or a plurality of host cell proteins or chimeric types thereof to a cross-linking drug, and (c) the envelope protein or chimeric type or mutation thereof It consists of separating the cross-linked structure that bears the form or chimeric form thereof.

The present invention provides a method of producing an immunogen for use as a vaccine for treating or preventing HIV infection, which method comprises: (a) a HIV envelope protein or a chimeric type thereof, or a fusion-defective mutant or chimeric type thereof; Co-culturing the first cell recombinantly expressing and (i) human CD4 or a chimeric type thereof against HIV and (ii) a second cell expressing a co-receptor or chimeric type thereof; (b) exposing the envelope protein or its chimeric or mutant or chimeric and CD4 or chimeric and co-receptor or chimeric forms thereof to a cross-linking drug, and (c) the envelope protein or chimeric type or Consists of isolating cross-linked structures bearing the mutant or chimeric form thereof. In one embodiment, the virus is HIV and the host cell protein is CD4 and co-receptor for HIV. In one embodiment, the second cell recombinantly expresses CD4 or a co-receptor, both CD4 and a co-receptor, or any of the chimeric types described above.

In other embodiments, the first and second cells are the same cell type. In another embodiment, the envelope protein or chimeric type or mutant or chimeric type thereof is present in a virus particle or virus-like particle. In another embodiment, the cross-linked structure is a cross-linked cell complex.

In another embodiment, the virus is a family of retroviridae, Rhabdoviridae, Caronaviridae, Filoviridae, Poxviridae, Bunyaviridae, Flavianvirus family Flaviviridae, Togaviridae, Orthomyxoviridae, Paramyxoviridae, Herpesviridae.

In another embodiment, the contacting step consists of contacting the cells expressing the host cell protein with the envelope protein or its chimeric or mutant or virus expressing its chimeric type. In another embodiment, the chimeric type of the envelope protein protein or the chimeric type of the host cell protein is contacted, said chimeric type comprising an affinity label. In another embodiment, the chimeric type of the envelope protein or the chimeric type of CD4 or co-receptor is contacted, said chimeric type comprising an affinity label.

The present invention provides a method of producing an immunogen for use as a vaccine for treating or preventing HIV infection, which method comprises: (a) a HIV envelope protein or a chimeric type thereof, or a fusion-defective mutant or chimeric type thereof; Co-cultivating a recombinantly expressing first cell and (i) a human CD4 or chimeric form thereof against HIV and (ii) a second cell expressing a co-receptor or chimeric type thereof, wherein the affinity label comprises an affinity label At least one of said chimeric forms is expressed; (b) lysing co-cultured cells under non-denaturing conditions to produce cell lysates; (c) separating the envelope protein or its chimeric or mutant or chimeric form from the cell lysate in a specific manner, wherein the specific method comprises contacting the cell lysate bearing the binding partner with an affinity label and The molecular structure bound to the affinity label is recovered.

The present invention provides a cross-linked structure that is the product of the process.

The present invention provides monoclonal antibodies to structures which neutralize the following HIV primary isolates: 92US657, 92US660, 92RW023, 93IN101, 92UG035, 92TH023.

The present invention provides a pill, jelly, foam, cream or ointment containing an effective amount of an antibody that inhibits or reduces HIV infection.

The present invention provides a method for treating or preventing a viral infection in a subject, said method comprising administering to said subject an immunologically effective amount of said molecular structure capable of treating or inhibiting a viral infection.

The present invention provides a method of treating or preventing HIV infection in a human, the method comprising administering to a human an immunologically effective amount of the molecular structure capable of treating or inhibiting an HIV infection.

The present invention provides a method of removing contaminants from a surgical or dental tool, which method comprises contacting the tool with an effective amount of a monoclonal antibody that can inhibit HIV infection.

The present invention provides a method for removing contaminants from a surgical or dental tool, which method comprises contacting the tool with an effective amount of a monoclonal antibody that can inhibit viral infection.

The present invention provides a method for screening molecular structure in terms of vaccine effect, wherein the method immunizes a transgenic non-human mammal with the molecular structure, wherein the transgenic non-human mammal is one or more plural. Simultaneously express CD4 and co-receptors for HIV from the exogenous gene; Detecting any neutralizing antibody against HIV produced by the mammal.

The present invention provides a method for screening molecular structure in terms of vaccine effect, which method comprises: (a) immunizing a transgenic non-human mammal with the molecular structure, wherein the transgenic non-human mammal is one Or expresses one or a plurality of host cell membrane proteins from a plurality of exogenous genes; (b) detecting any neutralizing antibody against the virus produced by the mammal. In one embodiment, the mammal is a mouse. In another embodiment, the first cell recombinantly expresses an HIV envelope protein or chimeric type thereof.

The invention, consisting of monoclonal antibodies and affinity labeled viral envelope proteins, respectively, against FRMS, was made with government support provided by the National Institutes of Health, under license numbers R01 AI44669-01 and R21 AI44312-02.

This application claims priority to US split application 60 / 014,806 (August 3, 1998) and US split application 60 / 095,105 (June 29, 1999).

1. Introduction

The present invention provides a surprising discovery that high-efficiency vaccines can be constructed that provide transient fusion-related determinants in the immune system that produce unique antibodies capable of strongly neutralizing a wide range of primary isolates from worldwide and different phylogenetic viral tissues. To The present invention is directed to the production of fusion-related molecular structures (FRMS) consisting of one or a plurality of fusion-related crystal regions. The present invention also relates to the discovery that the extensive and unique neutralization of different primary isolates suggests that the critical determinants presented by FRMS are highly conserved and are directly linked to fundamental functions in binding and fusion of envelope proteins. The present invention relates to the use of FRMS in a variety of compositions and methods, including methods for preparing, isolating, and purifying FRMS and for use as vaccine immunogens, diagnostics, and therapeutics.

The present invention relates to FRMS capable of inducing neutralizing antibodies against viral immunogens and viral primary isolates. Antibodies generated against FRMS can be used to study molecular pathways in fusion of viruses and host cells and to identify antibody-mediated blocking points of these pathways. The invention also relates to the use of the antibodies of the invention for antiviral drugs, blood product additives, contraceptive addition passive immunity or fetal immunity in post-exposure treatment.

1 shows neutralization of homologous 168P PI virus by FC and FI vaccine sera. The transgenic mice (hu CD4 +, hu CCR5 +, mouse CD4 +) were FC immunogens (COS-env with U87-CD4-CCR5; square; n = 3 mice) or cell control (U87-CD4-CCR5 alone or pseudo- Co-cultured with transfected COS; original; n = 3 mice) Non-immunized mice (triangles; n = 2 mice) were also used, serum was either CXCR4 (black symbol) or CCR5. Neutralization of 168P was tested using U87-CD4 expressing (white symbol) The data is the average of three to six neutralization assays analyzed for serum obtained two weeks after the second and third immunizations.

(A) in FIGS. 2A-B shows neutralization of P168 using only FC vaccine serum except FI. The transgenic mice had FC immunogen (black square; n = 4), FI immunogen (COS-env with U87 cells; gray circle; n = 4; COS-env with U87-CD4 cells, n = 3; gray diamond , n = 3; COS-env with sCD4, white diamond, n = 2; COS-env with U87-CD4-CCR5 cells, each fixed individually before mixing for immunization, gray square, n = 2) . Unimmunized mice (white triangles; n = 2 mice) were also used. Neutralization is independent of specific coreceptor use and data is the average of three to six neutralization assays in U87-CD4-CXCR4 or -CCR5 cells. In some cases, serum from all animals in each experimental group was pooled. (B) shows neutralization of P168 using only FI vaccine serum, excluding FC. Neutralization of homologous 168P PI virus in human PBL (lymphocyte) cultures. PBLs were isolated, stimulated with hemagglutinin and grown in the presence of interleukin-2; Neutralization was measured. HIV p24 antigen was measured after 5 days of culture by ELISA (Coulter Coporation) and the values were normalized to the virus control (36 ng / ml). Asterisks indicate p24 antigen levels below the limit of detection at dilution used in ELISA.

3A-B show that FC vaccine serum does not neutralize the milk-type HIV virion bearing amphiphilic MLV envelope protein (A) or primary SIVmac251 (B). For HIV with amphiphilic MLV envelope protein (ampho MLV pseudotype), neutralization susceptibility of the collected FC to FI antiserum was measured in U87-CD4-CXCR4 cells. For primary isolate SIV mac251, neutralization was measured at U87-CD4-CCR5. Indications are as follows: FC immunogen (black square) and FI immunogen (Cos-env + U87 cells, gray circle).

4A-B show neutralization of TCLA 168C virus by FI vaccine serum. Neutralizing sensitivity (A) of 168P PI virus and its neutralizing sensitivity (B) of TCLA derivative 168C were collected in serum: FC immunogen (black square), FI immunogen (COS-env + U87 cells, gray source; COS-env + U87 -CD4 cells, gray diamonds; COS-env + sCD4, white diamonds), were tested in U87-CD4-CXCR4 cells with a pseudo-transfected cell control (white circle).

5 shows neutralization of various PI viruses from tissues A-E. Primary isolates expanded in human PBLs and neutralized serum collected: FC immunogen (black square), FI immunogen (COS-env + U87 cells, gray circles; COS-env + U87-CD4 cells, gray diamond; COS- env + sCD4, white diamond), pseudo-transfected cell control (white circles) and measured in permeable U87-CD4-CCR5 (or -CXCR4). Virus biotypes are indicated in the lower right corner with available SI or NSI. Virus isotypes are displayed at the bottom corner of each graph.

6 shows the uptake of PI virus neutralization by formaldehyde-fixed FC COS-env cells. FC vaccine serum is repeatedly cultured with formaldehyde-fixed COS-env cells (grey squares) or control COS cells (white squares). Serum obtained just prior to FC immunization was similarly absorbed (gray and white circles, respectively). Starting FC and pre-immune sera are indicated by black squares or black circles, respectively. Serum was tested for neutralization of 168P using U87-CD4-CXCR4 cells.

Figure 7 shows that PI virus neutralizing activity is not taken up by circular U87-CD4-CCR5 cells. Collected FC vaccine serum was incubated with U87-CD4 CCR5 cells (white squares) or in empty microculture wells (fake, black squares). Collected FI serum (COS-env + U98-CD4) was treated similarly (white and black circles, respectively). Serum was tested for remaining neutralization of 168P with U87-CD4-CCR5 cells.

8 shows the neutralizing activity of two HIV tissue B primary isolates using hybridoma supernatants.

9 shows serum titers of mice immunized with the fusion-competent vaccine immunogen. Open squares represent fusion-compound (FRMS) immunogens (env + CD4 / CCR5); Black circles represent immunogens obtained from cells expressing envelope protein immunogens and cells not expressing both CD4 and CCR5 (env); White indicates cells expressing envelope protein and cells expressing CD4 immunogen (env + CD4); White triangles represent cell controls expressing CD4 and CCR5 immunogens (CD4 / CCR5).

10 shows cross-neutralization of 320SI primary virus isolates by antibodies obtained from mice immunized with the fusion-complex immunogen complexes of the present invention. Black circles represent fusion-compound (FRMS) immunogens (env + CD4 / CCR5); White squares represent immunogens obtained from cells expressing envelope proteins; White circles represent immunogens obtained from cells expressing the envelope protein and CD4 (env + CD4).

Figure 11 shows detection of envelope proteins co-purified with CD4-Spep and CCR5-Spep by Western blot analysis. a) 168P envelope protein isolated after co-culture with cells expressing CD4-Spep, b) 168P envelope protein isolated after co-culture with cells expressing CD4 and CCR5-Spep, c) 168P envelope protein After co-culture with temporary-transfected cells, they were not detected.

12 shows co-culture of BSC40 cells infected with rV-168Penv and rV-CD4 / CCR5 24 hours after infection.

5. Detailed Description of the Invention

The present invention relates to a fusion-related molecular structure (FRMS) capable of inducing neutralizing antibodies against enveloped viral pathogens. The FRMS of the present invention consists of epitopes produced due to the binding of one or more cell molecules and one or more viral envelope molecules. The process of fusion of the cell membrane with the viral envelope is initiated after binding of the viral envelope protein and specific cell receptors or co-receptors on the host cell, including the fusion of the surface or internal cell membrane with the viral envelope. Fusion is driven by morphological changes that occur in the envelope-receptor / coreceptor complex. Without being limited to specific mechanisms, it is believed that new and unique immunogenic epitopes for neutralizing antibodies are exposed by morphological changes that occur during fusion. The fusion-related molecular structure of the present invention is similar to the intermediate form material produced during fusion. The FRMS of the present invention can be used for a variety of purposes, including the purposes set forth in paragraphs 5.5 and 5.7. For example, in a suitable embodiment of the present invention, when FRMS is used for immunization of a host animal, it induces neutralizing antibodies against the infecting virus, and thus, the vaccine composition of the present invention for the treatment or prevention of viral infections and undesired consequences. Provide new and effective ingredients.

Accordingly, the present invention provides a separate molecular structure consisting of (a) an envelope protein of the envelope virus and (b) an epitope produced by the binding of one or a plurality of membrane proteins, wherein the envelope protein and the membrane protein are cell membrane proteins. It is necessary under conditions suitable for the fusion of the cell membrane with the viral envelope. In this article, cell membrane proteins include proteins of cell surface membranes and inner membranes (including vesicle membranes). Such cell membrane proteins are integrated membrane proteins (eg, transmembrane proteins), attached to the membrane by adhesion lipids (fatty acid chains or prenyl groups) or polysaccharides (eg, phospholipids, phosphatidylinositols), or non-covalent interactions (eg, Binding to the inner membrane protein).

The present invention also relates to (a) the envelope protein of the envelope virus, which envelope protein acts on the fusion of the host cell membrane with the viral envelope in the wild type; (b) provides an isolated molecular structure comprising an epitope produced by the binding of one or a plurality of host cell membrane proteins acting as receptors for the envelope protein.

In certain embodiments, the present invention provides a kit comprising: (a) an HIV envelope protein or variant thereof combined into a viral envelope; (b) provide an isolated molecular structure comprising epitopes produced by the binding of human CD4 and co-receptors for HIV fusion. In one embodiment, the co-receptor is chemokine receptor CCR5 or CXCR4. In another embodiment, the molecular structure is produced by the binding of HIV gp41 variants that are fusion-defective. In another specific embodiment, the variant has one or more mutations selected from V2E, G10V, V570R, Y586E. In another embodiment, the molecular structure is produced by the binding of wild type HIV envelope protein. In another specific embodiment, the envelope protein is El and E2 of the flavivirus HCV and the cell membrane protein is CD81.

The present invention also provides a method of producing an immunogen for use as a vaccine for treating or preventing a viral infection, the method comprising: (a) an envelope protein of the envelope virus or a chimeric form thereof, wherein the envelope protein comprises a cell membrane; Contacting a mutant or chimeric form of said envelope protein that is fusion-defective and one or more cellular proteins or chimeric forms thereof that act as receptors for said envelope protein; (b) exposing the envelope protein or chimeric type or mutant thereof or chimeric type and one or a plurality of host cell proteins or chimeric types thereof to a cross-linking drug, and (c) the envelope protein or chimeric type or mutation thereof It consists of separating the cross-linked structure that bears the form or chimeric form thereof. In one embodiment, the virus is HIV and the host cell protein is CD4 and co-receptor for HIV.

In certain embodiments, the present invention provides a method of producing an immunogen for use as a vaccine for treating or preventing an HIV infection, wherein the method comprises (a) an HIV envelope protein or a chimeric form thereof, or a mutant that is fusion-defective Or co-culturing a first cell recombinantly expressing its chimeric type and (i) human CD4 or its chimeric type against HIV and (ii) a second cell expressing a co-receptor or chimeric type thereof; (b) exposing the envelope protein or its chimeric or mutant or chimeric and CD4 or chimeric and co-receptor or chimeric forms thereof to a cross-linking drug, and (c) the envelope protein or chimeric type or Consists of isolating cross-linked structures bearing the mutant or chimeric form thereof.

In one embodiment, the second cell recombinantly expresses CD4 or a co-receptor, both CD4 and a co-receptor, or any of the chimeric types described above.

In another specific embodiment, the present invention provides a method of producing an immunogen for use as a vaccine for treating or preventing an HIV infection, wherein the method comprises (a) an HIV envelope protein or chimeric type thereof, or a fusion-defective mutation. Co-culturing the first cell recombinantly expressing the type or chimeric type thereof and (i) human CD4 or chimeric type thereof against HIV and (ii) a second cell expressing a co-receptor or chimeric type thereof, wherein At least one of said chimeric forms comprising an affinity label is expressed; (b) lysing co-cultured cells under non-denaturing conditions to produce cell lysates; (c) separating the envelope protein or its chimeric or mutant or chimeric form from the cell lysate in a specific manner, wherein the specific method comprises contacting the cell lysate bearing the binding partner with an affinity label and The molecular structure bound to the affinity label is recovered.

5.1. Generation of FRMS

The present invention is directed to a FRMS composed of epitopes resulting from the binding of one or a plurality of viral envelope molecules with one or a plurality of host cell molecules. The FRMS of the present invention includes a structure produced by the combination of one or more viral coat proteins and one or more host cell proteins. For example, in the FRMS of the present invention, a fusion-complex complex (e.g., a complex generated during fusion of a viral envelope and a cell membrane), a fusion-defective complex (e.g., a complex generated during fusion of a fusion-defective viral envelope and a cell membrane) ), Complexes generated by the binding of one or more viral envelope molecules with one or more cell receptors or co-receptors to the virus. In the case of HIV, FRMS is produced by binding of the HIV envelope protein and CD4 to chemokine receptors.

Therefore, the FRMS of the present invention is composed of epitopes capable of inducing neutralizing antibodies against viruses.

The FRMS of the present invention is made by the various methods presented in this article. In a suitable embodiment, FRMS is made by culturing cells expressing one or more components that bind to produce FRMS. All components can be expressed on a cell, or the first component can be expressed on a cell, the second component can be expressed in a different cell and the third component can be expressed in one of the cells described above or in a different cell. Alternatively, one of the cells can be replaced with a virus-like particle containing viral particles, pseudoviral, recombinant virus or surface components thereof. The component (viral envelope protein, host cell receptor or any necessary host cell co-receptor) may be endogenous or recombinantly expressed by the cell. Thus, FRMS can be made by the interaction of exogenous molecules (eg, recombinantly expressed), endogenous molecules or any mixture thereof.

In another embodiment of the present invention, the soluble form of the viral envelope protein of the envelope virus is used to prepare the FRMS of the present invention. For example, in one embodiment soluble coat proteins are added to host cells in vitro, which host cells express receptors for viruses. Thus, binding of soluble viral coat protein and host cell receptor results in FRMS.

In another embodiment of the invention, one or a plurality of soluble cell receptors are used for the preparation of FRMS. In one embodiment, soluble cell receptors that bind to viral envelope proteins to produce FRMS can be used for FRMS preparation by contacting viral envelope proteins with said soluble cell receptors. In another specific embodiment, all components that bind to form FRMS are in soluble form and the FRMS is reconstituted in vitro. In this embodiment, a mAb can be added to the FRMS to facilitate reconstitution of the FRMS.

In another embodiment of the invention, FRMS is made upon pH reduction. For example, such pH-mediated cell fusion enables the production of FRMS for enveloped viruses, where the membrane proteins are located in the inner cell membrane.

5.1.1. Exogenous Expression of Complex Components

Cellular components involved in the production of FRMS for specific viruses include cell receptors or co-receptors for specific viruses. In certain embodiments, at least some of these components are endogenously expressed by the cell. Thus, FRMS can be prepared using any cell known in the art expressing the cellular components of FRMS.

Cells expressing native cell receptors or co-receptors for viral proteins can be used. For example, the human CD4 presented here is known in the art as an intracellular receptor for HIV. In one embodiment of the present invention, FRMS is prepared using cells endogenously expressing CD4.

Cells endogenously expressing the components of the complex of the present invention may comprise one or a plurality of cellular components used for the production of FRMS. For example, cells which express all the necessary components in the cells of the FRMS or cells which do not express all the necessary components can be used. For example, in the case of HIV FRMS, any cell that expresses CD4 and HIV co-receptors at the same time, for example CCR5, can be used to prepare HIV FRMS. Alternatively, in any cell that endogenously expresses CD4 but does not endogenously express an HIV co-receptor, the non-endogenously expressed complex component can be introduced into the cell and expressed recombinantly as described below. In certain embodiments of the invention, the coreceptor CXCR4 is located primarily in the T cell line. In another specific embodiment of the invention, the coreceptor CCR5 is located primarily in macrophages. In certain embodiments of the invention, the coreceptor CXCR4 is located primarily in the T cell line. In certain embodiments of the invention, the coreceptor CCR5 is located primarily in macrophages. The exogenous component can be introduced into the cell by any method known in the art, including the method described in paragraph 5.1.2.

In another embodiment of the invention, cells transformed with viral genes or genomic integration to express viral components of FRMS can be used for the preparation of fusion related complexes. In this way, the cells are believed to endogenously express viral components important for FRMS construction. Such virus-transformed cells may also endogenously express one or more cellular components of FRMS. Alternatively, viral particles can be used as a source of endogenous expression of FRMS viral components.

5.1.2. Recombinant Expression of Components Important for FRMS Production

All components important for the construction of FRMS, fragments thereof or derivatives thereof can be introduced into cells and expressed as endogenous components within the cells.

The present invention includes expression systems (eukaryotic and prokaryotic expression vectors), which can be used to express components important for the preparation of the FRMS, fragments thereof or derivatives thereof of the present invention.

In another embodiment of the invention, the viral envelope protein may be expressed in a host cell. In this embodiment, the exogenous viral envelope protein can be introduced into the cell by transfection with a viral vector as disclosed herein or by infection with a live or attenuated virus bearing the viral envelope protein. Thus, viral infection may induce the production of viral envelope protein in a suitable embodiment, wherein expression of the viral envelope protein on the cell membrane is enhanced by modifying (eg, by recombinant means) the viral envelope protein and at the cell surface or other cell membrane. Increase expression. For example, targeting signals are known in the art to enhance the delivery of intracellular proteins to specific intracellular vesicles or locations. For example, by manipulating the endoplasmic reticulum signal KDEL (a Chinese character amino acid code) in a viral envelope protein, it is possible to promote the movement of the protein to the ER when the cell is infected with the virus.

The present invention also provides cells expressing viral and intracellular components important for FRMS production. In one embodiment, the cell is constructed to express a cellular receptor or co-receptor for the virus or to recombinantly express the viral envelope protein. Such cells may endogenously express host cell receptors or co-receptors for viruses, or recombinantly express one or both of these proteins. When viral and intracellular proteins are recombinantly expressed, such proteins can be expressed in different vectors or in a single vector, wherein the vector consists of multiple genes linked to and operated by a single promoter. Thus, all components necessary for the production of FRMS are expressed in individual cells. In certain embodiments, the cells are constructed to recombinantly express CD4, coreceptor CCR5 or CXCR4, HIV gp160. Alternatively, gp120 and gp41 can be used in place of gp160. When individual cells are constructed to express co-receptors for gp160, CD4, HIV-1, the viral envelope protein is also constructed to lack the unique protease position that governs the cleavage of gp160 into gp120 and gp41. In such embodiments, an alternative protease site is constructed in the viral envelope protein, which is not native to the host cell (eg, bacterial protease). FRMS production in cells expressing three proteins is caused by contacting the cell with a non-native protease (eg, by adding an effective amount to the cell medium sufficient to induce cleavage). Various proteases and protease positions are known in the art and can be used in accordance with the present invention.

Nucleotide sequences encoding viral envelope proteins, intracellular receptors, functionally active derivatives or fragments or other derivatives thereof of the present invention may be used for the transcription and translation of an appropriate expression vector, e. You can insert it into a vector containing the elements you need to execute.

The present invention relates to the use of DNA expression vectors or viral vectors carrying coding sequences of FRMS or analogues, derivatives or components of these fragments that operate in conjunction with regulatory elements that direct expression of the coding sequence and expression of the coding sequence in a host cell. Included are the use of a engineered host cell having any of the above-described coding sequences operating in conjunction with the oversight regulatory element.

DNA expression vectors and viral vectors carrying nucleic acids encoding components important for the production of FRMS of the present invention can be made by recombinant DNA techniques using techniques known in the art. Thus, this article provides a method for preparing the expression vector and the viral vector of the present invention by expressing a retained nucleic acid having a sequence encoding a FRMS or an analog, a derivative or a component important for the preparation of these fragments. Methods known to those of skill in the art can be used to construct expression vectors carrying gene product coding sequences and appropriate transcriptional and translational control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques, and genetic recombination (Sambrook, et al., 1989, Molecular Cloning, A Laboratory Manyal, Second Edition, Cold Spring Harbor Press, NY, and Ausubel, et al., 1989-1999, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, NY). Alternatively, gene product sequences can be chemically synthesized using oligonucleotide synthesizers ("Oligonucleotide Synthesis", 1984, Gait, MJ, IRL Press, Oxford). Gene expression can be regulated in a variety of ways known in the art (Mizuno, T. et al., 1984, Proc. Natl. Acad Sci USA. 81 (7): 1966-70).

Nucleic acids or DNAs encoding one or a plurality of components that produce FRMS can be achieved by various methods, such as liposomes, electroporation, transfection, viral vectors, bacteriophages, and the like.

In one embodiment, the nucleic acid or DNA encoding one or a plurality of components to produce a FRMS packs the plasmid DNA consisting of a nucleic acid encoding said component into liposomes or lipids plasmid DNA consisting of a nucleic acid encoding said component. Or by mixing with liposomes to construct DNA-lipids or DNA-liposomal complexes. Liposomes consist of cationic lipids and neutral lipids commonly used to transfect cells in vitro. Cationic lipids complex with plasmid DNA to form liposomes.

Cations and neutral liposomes are contemplated in the present invention. Cationic liposomes can form complexes by mixing with negatively-charged bioactive molecules (eg, DNA) and allowing them to charge-bond. Cationic liposomes can be usefully used for nucleic acids by the negative charge of the nucleic acids. Examples of cationic liposomes include lipofectin, lipofectamine, lipofectase, DOTAP (Hawley-Nelson et al., 1992, Focus 15 (3): 73-83; Felgner et al., 1992, Proc. Natl.Acad. Sci. USA 84: 7413; Stewart et al., 1992, Human Gene Therapy 3: 267-275). Commercially available cationic lipids such as dimethyldioctadecammonium (DDAB); Biodegradable lipid 1,2-bis (oleoloxy) -3- (trimethylammonio) propane (DOTAP) can be used; These liposomes can be mixed with neutral lipids such as L-α dioleyl phosphatidylethanolamine (DOPE) or cholesterol (Chol), two neutral lipids commonly used for systemic delivery. DNA: liposomal ratios can be optimized using methods known in the art (Nicolau et al., 1987, Methods Enzymol. 149: 157) and the complexes of the invention can be placed in a container using conventional techniques in the art. .

In another embodiment of the invention, plasmid DNA encoding a gene or nucleic acid encoding one or a plurality of components that produce the FRMS of the invention can be delivered via a polycation, wherein the polycation carries multiple positive charges. And in vitro, in vivo, and in delivery of the gene (Boletta et al., 1996, J. Am. Soc. Nephrol. 7: 1728).

Recombinant viruses can also be used to deliver components that bind and produce FRMS. Cells infected with these viruses or transformed by the viral genome will therefore express the desired components. In other embodiments of the invention, live recombinant vectors or inactivated recombinant viral vectors expressing one or a plurality of components disclosed herein can be engineered. In this respect, various viruses can be genetically engineered to express components important for FRMS production. In certain cases, the recombinant virus needs to adapt to, or be sensitive to, or at low temperatures.

In accordance with the present invention, various viruses and viral vectors are used to deliver nucleotide sequences encoding one or a plurality of components important for the production of FRMS of the present invention, examples of which are described below.

Retroviruses are commonly used to deliver genes to host cells. Retroviral vectors are extremely effective gene transfer vehicles that do not cause detectable harm when entering cells. Retroviral nucleic acids are integrated into the host chromosomal DNA, which lasts for a long time and is stably delivered later, which vectors are useful for the delivery of one or more components that produce FRMS. An example of a suitable retroviral vector is a lentiviral that has the advantage of infecting and transducing non-differentiated cells. In this embodiment, a lentiviral vector that encodes a packable RNA vector genome and operates in conjunction with a promoter that lacks all functional retroviral cogenes delivers DNA encoding an important component for the production of the FRMS of the present invention. use. Examples of such vectors are given in WO 98/17815, WO 98/17816, WO 98/17817.

In another embodiment, non-integrated viral vectors, such as adenoviruses, which infect and transduce non-differentiated cells are used to deliver components important for FRMS production. Adenovirus vectors are beneficial in that they avoid the risks associated with the facilitation of research modifications or insertion mutagenesis of the host cell genome. Adenoviruses are one of the optimal non-integrated viral vectors developed so far and can be used to deliver expression cassettes up to 75 kb. Recombinant adenoviruses can be produced at high titers, which efficiently deliver genes to a wide range of non-replicating and replicating cells, making them ideal for mammalian gene delivery in vivo.

Adenovirus-based vectors are relatively safe and can be engineered to be inactivated in terms of their ability to encode the desired components while replicating into the normal lytic virus life cycle. Adenoviruses have a natural flavor for airway epithelial cells. Thus, adenovirus-based vectors are particularly preferred for epithelial delivery. In a suitable embodiment, the adenovirus-based gene therapy vector consists of the adenovirus 2 antigenic genome, wherein the Ela and Elb regions on the genome involved in the early stages of viral replication are deleted and replaced with the nucleotide sequence of interest. In other embodiments, the adenovirus-based gene therapy vector only contains the necessary open reading frame (ORF3 or ORF6 of adenovirus early region 4 (E4)) and all other E4 open reading frames are missing, or additional in the E3 region. It may also have a deletion (US Patent No. 5,670,488). In another embodiment, the adenovirus-based gene vector may be pseudo-adenovirus (PAV), which holds a non-hazardous viral gene and may theoretically accept approximately 36 kb of foreign material.

In another embodiment, adeno-associated virus (AAV) systems can be used to deliver components useful for the preparation of the FRMS of the present invention. AAV is widely available, and recently AAV vectors have been devised that do not require helper viruses. Examples of such AAV vectors are given in WO97 / 17458.

Vaccinia virus vectors can be used according to the invention because attenuated vaccinia variants have been described in which most fragments of DNA are easily cloned into the genome and recombined (Meyer, et al., 1991, J. Gen. Virol. 72: 1031-1038). In one embodiment of the present invention, vaccinia virus vectors are used to deliver components important for FRMS production and to express these components in recombinant vaccinia infected cells. In certain embodiments, the component is human CD4, co-receptor (eg, CCR5 or CXCR4), HIV envelope protein. In this particular embodiment, infecting the cells with recombinant vaccinia results in the expression of components and the generation of HIV FRMS.

In another specific embodiment, two different recombinant vaccinia viruses are constructed, wherein the first virus encodes an HIV env protein and the second virus encodes human CD4 and chemokine receptors. In certain embodiments, the first virus is used to infect a first population of cells in which the HIV env protein is expressed. The second virus is used to infect a second population of cells, which, when infected, expresses CD4 and chemokine receptors. Co-culture of the first infected cell population and the second infected cell population results in HIV FRMS. In other embodiments, the co-cultured cells can be cross-linked.

Orthomyxoviruses, including influenza; Paramyxoviruses including respiratory syncytial virus and Sendai virus; Robdoviruses can be engineered to express mutations that produce an attenuated phenotype (U.S.Patent Serial No. 5,578,473, issued November 26, 1996). These viral genomes can be engineered to express foreign nucleotide sequences, such as components important for the production of FRMS of the present invention (U.S. Patent Serial No. 5,166,057, November 24, 1992).

Reverse genetic techniques can be used to engineer negative and positive strand RNA viral genes to introduce mutations that produce an attenuated phenotype as shown in influenza viruses, measles viruses, sindbis viruses, and polioviruses (Palese et al. , 1996, Proc. Natl. Acad. Sci. USA 93: 11354-11358). These techniques can also be used to introduce foreign DNA, ie, DNA encoding components or proteins important for FRMS production, to make recombinant viral vectors according to the present invention. In addition, attenuated adenovirus viruses and retroviruses can be engineered to express components important for FRMS production. Thus, various viruses can be engineered to deliver components that are important for the production of the FRMS of the invention.

Bacteriophage can be used to specifically infect bacterial cells (Soothill, J.S., 1992, J. Med. Microbilo. 37: 358-261).

In the viral vectors of the invention, non-viral DNA (eg, DNA encoding cellular receptors) or non-native viral DNA (eg, DNA encoding viral envelope proteins) may identify any component that is important for FRMS production. You can code

Thus, those skilled in the art will appreciate that the viral envelope protein, cell receptor or co-receptor used to construct the FRMS of the present invention may be virions, virus-infected cells or chemically / genetically inactivated virions, viral vectors, viral replicons (ie It can be appreciated that non-replicating viral constructs (eg, VEE replicons), virus-like particles produced by recombinant DNA, can be delivered to cells or naked cells to DNA.

As mentioned above, the recombinant expression system may include regulatory elements, including but not limited to inducible promoters, non-inducible promoters, enhancers, operators. Any of the methods described above for inserting DNA or nucleic acid fragments into a vector can be used to construct expression vectors carrying genes or chimeric genes composed of appropriate transcriptional / translational regulatory signals and protein coding sequences. These methods include in vitro recombinant DNA and synthetic techniques. In general, expression of a nucleic acid sequence encoding a protein or peptide fragment is controlled by a second nucleic acid sequence such that the protein or peptide is expressed in a host cell transformed by the recombinant DNA molecule. For example, the expression of viral envelope proteins is controlled by any promoter / enhancer element known in the art. A promoter / inserter is homologous (ie unique) or heterozygous (ie non-unique). Promoters used to regulate the expression of proteins include the SV40 early promoter region (Benoist et al., 1981, Nature 290: 304-310), promoters contained in the 3 'terminal repeat of the Raus sarcoma virus (Yanamoto et al., 1980, Cell 22: 787-797), the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. USA 78: 1441-1445), the regulatory sequence of the metallothionein gene (Brinster et al. , 1982, Nature 296: 39-42), plant expression vector (Herrera-Estrella et al., Nature 303: 209-213), cauliflower mosaic virus 35S RNA promoter (Gardner et al., 1981, Nucl.Acids Res. 9: 2871), promoters of the photosynthetic enzyme ribulose diphosphate carboxylase (Herrera-Estrella et al., 1984, Nature 310: 115-120), promoter elements from yeast or other fungi ( Eg, Gal4-reactive promoter), ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol Kinase) promoter, alkaline phosphatase promoter, the following animal transcriptional regulatory regions (used for transgenic animals with tissue specificity): elastase I gene regulatory regions that are active in pancreatic acinar cells (swift et al., 1984, Cell 38: 639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biil. 50: 399-409; MacDonald, 1987, Hepatology 7: 425-515); Gene regulatory regions that are active in pancreatic beta cells (Hanahan, 1985, Nature 315: 115-122); Immunoglobulin gene regulatory regions that are active in lymphocytes (Grosschedl et al., 1984, Cell 38: 647-658; Adames et al., 1985, Nature 318: 533-538; Alexander et al., 1987, Mol. Cell. Biol. 7: 1436-1444); Mouse breast tumor virus regulatory regions that are active in testes, breasts, lymphocytes, mast cells (Leder et al., 1986, Cell 45: 485-495); Aluminin gene regulatory regions that are active in the liver (Pinkert et al., 1987, Genes and Devel. 1: 268-276); Alpha-fetoprotein gene regulatory region (Krumlauf et al., 1985, Mol. Cell. Biol. 5: 1639-1648; Hammer et al., 1987, Science 235: 53-58); Alpha 1-antitrypsin gene regulatory region showing activity in the liver (Kelsey et al., 1987, Genes and Devel. 1: 161-171); Beta-globin gene regulatory regions that are active in erythrocyte cells (Mogram et al., 1985, Nature 315-338-340; Kollias et al., 1986, Cell 46: 89-94); Myelin based protein gene regulatory region showing activity in oligodendrocytes in the brain (Readhead et al., 1987, Cell 48: 703-712); Myosin light chain-2 gene regulatory region showing activity in skeletal muscle (Sani, 1985, Nature 314: 283-286); Progesterone releasing hormone gene regulatory regions that are active in the hypothalamus (Mason et al., 1986, Science 234: 1372-1378) are included, but are not limited to these.

In certain embodiments, with a promoter that operates in conjunction with a nucleic acid encoding a viral protein, an intracellular protein or a chimeric protein and one or more origins of replication, optionally one or more selectable marks (eg, antibiotic resistance genes) Use a constructed vector. In other embodiments, two or more promoters can be used to direct expression of two or more genes in a single plasmid.

In certain embodiments, CMV (IE) contiguous early (IE) promoters are used to direct expression of nucleic acids encoding viral proteins, intracellular proteins or chimeric proteins.

The promoter may be inducible or structural. Expression from certain promoters can be enhanced in the presence of certain inducers; Thus, expression of the engineered protein chimera can be regulated. Inducible promoters can be used to regulate the expression of the proteins of the invention to produce proteins in the presence of inducers.

The present invention also provides a method for stably expressing a component producing the FRMS of the present invention. For long term high-efficiency production of recombinant proteins, stable expression is possible. For example, cell lines that stably express one or a plurality of cell receptors and co-receptors for viruses can be engineered. Instead of using expression vectors with viral replication origin, host cells are transformed with DNA regulated by appropriate expression elements (e.g., promoter sequences, enhancer sequences, transcription terminators, polyadenylation sites, etc.) and selectable marks. Can be converted. For example, after introducing foreign DNA, the engineered cells are grown for 1-2 days in culture medium and then transferred to selective medium. Selectable marks in recombinant plasmids confer resistance to selection lesions (eg, integrating the plasmid into chromosomes), allowing cells to grow and form, which can be cloned and expanded into cell lines. This method is used primarily for manipulating cell lines. Such cell lines are particularly useful for screening and evaluating compounds that affect the selected gene product.

Multiple selection systems can be used to create stably-expressing cell lines, including the herpes simplex virus thymidine kinase (Wigler, et al., 1977, Cell), which can be used for tk-, hgprt- or aprt- cells, respectively. 11: 223) (Szybalska et al., 1962, Proc. Natl. Acad. Sci. USA 48: 2026), hyposankin-guanine phosphoribosyltransferase (Szybalska et al., 1962, Proc. Natl. Acad. Sci USA 48: 2026), but not limited to adenine phosphoribosyltransferase (Lowy, et al., 1980, Cell 22: 817). In addition, anti-metabolite resistance can be used as the basis for selection of the following genes: DHFR (Wigler, et al., 1980, Natl. Acad. Sci. USA 77: 3567; O'Hare, et al, which confers resistance to methotrexate). , 1981, Proc. Natl. Acad. Sci. USA 78: 1527); Gpt conferring resistance to mycophenolic acid (Mulligan et al., 1981, Proc. Natl. Acad. Sci. USA 78: 2072); NeoG-418 (Colberre-Garapin, et al., 1981, J. Mol. Biol. 150: 1) that confers resistance to aminoglycoside G-418; Hygro confers resistance to hygromycin (Santerre, et al., 1984, Gene 30: 147).

5.1.3. Cells that express components that, when combined, produce FRMS

The present invention includes expression of components that, when bound, produce FRMS. In some embodiments of the invention, the expression is in primary cells, animal cells, insect cell lines. In accordance with the present invention, various primary or secondary cells or cell lines can be used, including, but not limited to, cells isolated from skin, bone marrow, liver, spleen, pancreas, kidney, adrenal gland, and neural tissue. Other cell types that can be used in the present invention are immune cells (e.g., T-cells, B-cells, natural killer cells, etc.), macrophages / monocytes, adipocytes, heart cells, fibroblasts, neurons, reticular cells, etc. . In other embodiments, secondary cell lines, including but not limited to hepatocytes (CWSV, NR, Chang hepatocytes) or other cell lines (eg, CHO, VERO, BHK, Hela, COS, MDCK, 293,373, CaSki, W138 cell lines) Can be used. In a suitable embodiment, the host cell comprises one or a plurality of receptors to facilitate binding or viral infection.

In a suitable embodiment of the invention, the cells used in the invention are eukaryotic cell lines, preferably mammals, most preferably humans. Cells can be obtained from humans (eg HeLa cells), ledgers, mice, rabbits, chickens, etc., but can also be obtained from non-human transgenic animals. Many eukaryotic cell lines are available from ATCC (American Type CultureCollection, Rockville, MD). In the most suitable embodiment, the eukaryotic cells are obtained from humans. In other embodiments, the cells are obtained from a mouse, monkey or rat. The production of FRMS of the present invention preferably uses non-tumor cell lines and autologous cells.

Accordingly, the present invention provides a cell comprising one or a plurality of components for producing the FRMS of the present invention. The invention also provides a cell comprising all of the components necessary for the preparation of the FRMS of the invention. In one embodiment, the invention provides a cell that recombinantly expresses a coat protein of a coat virus that acts on the fusion of a viral coat with a host cell membrane, or a mutant type of said coat protein that is fusion-defective. In other embodiments, the cell expresses one or a plurality of cell membrane proteins that act as receptors for envelope proteins. In certain embodiments, the present invention provides cells recombinantly expressing HIV gp160, human CD4, co-receptors for HIV.

The present invention also provides a cell line which recombinantly expresses the envelope protein of the envelope virus that acts on the fusion of the viral envelope and the host cell membrane, or a mutant form of said envelope protein that is fusion-defective. In other embodiments, the cell line expresses one or a plurality of cell membrane proteins that act as receptors for envelope proteins. In certain embodiments, the present invention provides a cell line that recombinantly expresses HIV gp160, wherein the cell line expresses CD4 and co-receptors for HIV; The cell line lacks a functional protease that cleaves gp160 to produce gp120 and gp41.

In another embodiment of the invention, cells containing the FRMS of the invention are administered to an individual as an immunogen (see paragraph 5.5). In a suitable embodiment, the cell is a human primary cell when the subject is a human. In a more suitable embodiment, the cell is autologous.

5.1.4. Use of Fusion Defect Variants in Complex Formation

In one embodiment, any viral envelope protein that possesses wild type fusion activity is used to make FRMS. Alternatively, fusion-defective envelope proteins are used as set forth in this paragraph.

In the present invention, the viral envelope protein or host cell receptor or co-receptor protein is mutated, and the mutation inhibits the termination of the process of viral envelope and cell membrane fusion. The present invention provides a fusion-defective mutant that does not allow termination of the process of viral envelope-cell membrane fusion but can produce the fusion-related molecular structure of the invention. Fusion-defective mutants are used to construct epitopes consisting of FRMS. In one embodiment of the invention, FRMS accumulates when the fusion defect envelope protein and cellular receptor bind. Thus, the fusion-defective mutants of the present invention can be used to prepare FRMS.

Any mutagenesis technique known in the art can be used to modify individual nucleotides on a DNA sequence, the purpose of which is to make amino acid substitutions in the expressed peptide sequence, to create / remove restriction sites, or It is to attach affinity label. Such techniques include chemical mutagenesis, in vitro site-specific mutations (Hutchinson, C., et al., 1978, J. Biol. Chem 253: 6551), and specific oligonucleotide mutagenesis (Smith, 1985, Ann. Rev. Genet. 19: 423-463; Hill et al., 1987, Methods Enzymol. 155: 558-568), PCR-based redundant expansion (Ho et al., 1989, Gene 77: 51-59), PCT-based megaprimers Mutagenesis (Sarkar et al., 1990, Biotechniques, 8: 404-407), but not limited to these. Modifications can be confirmed by DNA sequencing.

Fusion-defective mutants can be constructed against any envelope virus. In certain embodiments, mutants can be constructed that affect the termination of the fusion. The selection of mutants is based on existing literature or on molecular modeling according to known or hypothetical expected structures. Preferred are mutants that do not adversely affect envelope protein expression and migration to the cell surface.

In another embodiment, the fusion-defective variant is identified by expressing a mutated gene in a cell such as COS 293T (eg, the method of the invention) and analyzing the fusion ability. The cells are then analyzed for cell surface expression of the mutant (by methods known in the art such as cytometry or by live cell indirect immunofluorescence). In addition, proteolytic treatment of variant proteins, such as the HIV gp160 variant, can be analyzed by Western blot analysis or other methods known in the art. The fusion ability of the variant components was analyzed as shown in paragraph 5.7 (eg, by the follicle formation assay for HIV, U87-CD4-coceptor cell fusion assay).

In another embodiment of the invention, the binding-defective, fusion-defective or fusion-stopped variant envelope protein tests for the ability to induce primary isolated virus neutralizing antibodies in transgenic mouse immunoassays. In this embodiment, the immunogen consists of cells expressing an experimental variant envelope protein co-cultured with, for example, U87-CD4-CCR5 cells (when examining HIV-associated mutants).

In suitable embodiments of the invention, mutants that induce extensive PI virus neutralization develop into subunits and recombinant virus-based immunogens. Table I provides a list of variants for HIV. As one of ordinary skill in the art will recognize, similar mutants can be constructed for other enveloped viruses.

Table I

Protein / Location Mutant gp120-gp41 cutting position gp120 C terminus REKR to REKT Fusion Peptide Mutants gp41 V2E, G10V gp41 twisted-helix mutant Sheath V570R, Y586E, L568A, W571R, Q577R, N656L Enhanced gp120 cutting Modification at furin protease cleavage site of gp120: C-REKR to RSKR

For example, additional mutants in the crosslinked sheet of the gp120 core structure include those of Kwong et al. (Kwong PD, et al., 1998, Nature 393: 648-59; Kwong PD, ET AL., 1999, J. Biol Chem). 274: 4115-23) and the putative gp41 / gp120 interaction region.

In another embodiment, the FRMS of the present invention is made using a viral envelope protein having binding and fusion-related variants. In one embodiment, the viral protein binds to the host cell receptor, but the mutation stops the fusion process before or during the fusion of the viral envelope and the host cell membrane. Thus, where the variant protein used in the present invention produces an epitope that induces PI neutralizing antibodies, the FRMS of the present invention may comprise one or more viruses, including proteins that are fusion-defective, fusion-interrupted, or binding-defective. Epitopes produced due to the binding of envelope proteins and one or a plurality of cell membrane proteins. Protein complexes suspended during the fusion process can be isolated and used in methods and compositions including, but not limited to, immunogens, diagnostics, kits, vaccines, compositions.

For example, in certain embodiments the FRMS consists of the major viral envelope protein of HIV-1. There are a variety of fusion-related mutants that can be used to construct the complexes of the invention. Regardless of the mechanism, the oligo envelope protein complex gp160, consisting of the gp120 surface and the gp41 transmembrane region, first binds to CD4 on the host cell during HIV-1 and host cell fusion. The conformational change between the envelope protein and the CD4 receptor facilitates interaction with co-receptor molecules on the surface of the host cell. Due to the additional morphology on the viral protein-CD4-co-receptor trimomeric complex, the viral gp41 pre-hairpin intermediate is exposed and the N-terminal gp41 hydrophobic fusion domain is inserted into the host cell membrane. The helix heptad-repeat region in the all-hairpin intermediate then collapses to form the trimer twisted-helix core of the fusion activity gp41. The twisted-helix core drives the ultimate fusion of the added cell and viral membrane.

Viral envelope protein

Viral envelope proteins with mutants that stop proteolytic cleavage of the gp160 precursor protein to prevent the release of the gp41 hydrophobic fusion domain can be used to construct the complex of the invention. In particular, mutants that modified the highly conserved lys / arg-X-lys / arg-arg position at the gp120 C-terminus appear to block fusion induction (Lee CN, et al., 1994, AIDS Res. Hum. Retroviruses. 10: 1065-9).

In addition, mutants that affect the N-terminal gp41 fusion peptide can be used to make the complex of the present invention. Since the hydrophobic N-terminal region of gp41 takes on a helical morphology, it is inserted into the cell membrane to destabilize the lipid bilayer, thereby mediating fusion. If a change occurs in a particular amino acid in this region, the coat protein will not fuse. Synthetic peptides with these changes cannot bind to the lipid bilayer and thus do not induce fusion. The properties of two fusion-defective mutants: V2E and G10V (Kliger Y, et al., 1997, J Biol Chem. 272: 13496-505) at gp41 are already known. The former has polar substitution on the hydrophobic peptide, while the latter affects the helix structure of the lipid bilayer. These mutants a specific site - 168P envelope gene by any method known in the art or mutagenesis (gp41: ala- val -gly-ile -gly-val-leu-phe-leu- gly -phe-leu-gly ... can be introduced, the envelope protein is tested for expression and fusion.

In addition, envelope proteins with mutants that have modified the twisted-helix core region of gp41 can be used to construct the structures of the present invention. Highly-conserved twisted-helix motifs are found in proteins involved in fusion with many viruses, and similar structures have been identified in intracellular proteins that mediate vesicle fusion. Synthetic peptides that mimic N- or C-helix regions (e.g., DP107 and DP178) (Furuta et al., 1998, Nature Structural Biology 5: 276-279) bind to homologous helix regions to prevent disruption of all-hairpin intermediates. Interference significantly neutralizes viral infectivity. The N- and C-regions of gp41 saturate mutagenesis in addition to detailed structural information from crystallography. Several mutants that possess and combine normal envelope protein expression have been shown to be non-fused due to disruption of the interface between the N- and C-helix coils (eg, V570R and Y586E) (Weng et al., 1998, Journal of Virology 72: 9676-9682).

Compound or mutated viral envelope proteins and cell receptors or co-receptors can be incorporated into viral vectors or diploid cells by methods known in the art, including those described herein. Viral genes encoding viral proteins are routinely cloned for expression in bacterial cells, eukaryotic or prokaryotic cells, or in viruses or DNA vectors. Similarly, human intracellular receptors can be cloned and expressed to create models for studying dangerous pathogenic diseases. Viral genes or host receptors can be integrated into foreign DNA using transfection or viral vectors.

5.1.5. Alternative way to generate FRMS

In addition, another method of initiating the preparation of the structure of the present invention is to manipulate pH conditions, temperatures, salt concentrations. In addition, monoclonal antibodies that bind strongly enough to coat proteins may induce the conformational changes necessary to produce the structures of the present invention. Thus, the FRMS of the present invention can be produced by the interaction of viral proteins and various animal cell components including those described above.

5.2. FRMS capture

Once the FRMS is made, a variety of methods are used to preserve or "capture" the FRMS in an immunogenic form that provides vaccine efficacy against the primary virus isolate.

In one embodiment, the FRMS of the invention is made by the interaction of one or a plurality of viral coat proteins and one or a plurality of host cell receptors or co-receptors. In one embodiment, the complex “captures” by immobilizing or cross-linking the FRMS. For example, FRMS generated from co-expressed cells expressing viral and cellular components, respectively, can be captured by immobilizing the cells with a cross-linking drug.

In certain embodiments, cells transformed with nucleic acids expressing viral envelope proteins and cells transformed with nucleic acids expressing host cell receptors and co-receptors are cultured together. Viral proteins expressed on the surface of the first cell bind to receptors expressed on the surface of the second cell. The cells are fixed during initiation of cell-cell interaction. Regardless of the mechanism, crosslinking “captures” the intermediate fusion complex and provides a fusion-related determinant that can be isolated and used as a vaccine component. Any technique known in the art can be used to express the viral component or cellular component of the fusion complex in cells (see paragraph 5.1.1).

In capturing FRMS, fusion cells or fusion-defective mutant cells can be fixed or cross-linked by any technique known in the art. Cross-linking drugs include formaldehyde glutaraldehyde, formalin, p-angidobenzoyl hydrazide, N- (4- [p-azidosalcyclamido] -butyl) -3 ') 2'-pyridyldithio ) -Propionamide, bis (beta- [4-azidosalcyclamido] -ethyl) disulfide, 1,4-bismaleimidyl-2,3-dihydroxybutane, 1,6-bismaleimidohexane, 1,5-difluoro-2,4-dinitrobenzene, dimethyl adipimidate-2HCl, dimethyl suverimidite-2HCl, dimethyladipodidate-2HCl, dimethyl pimelimidate-2HCl, disuccinimi Dill glutarate, disuccinimidyl tartrate, 1-ethyl-3 [3-dimethylanonopropyl] carbodimide hydrochloride, (N-hydroxy succinimidyl) -4-azidosalicylic acid, sul Posuccinimidyl 2- [7-azido-4-methyl-coumarin-3-acetamidomethyl-1,3-aminopropionate, N-succinimidyl-4-iodoacetylamidine Benzoate, N-succinimidyl-3- [2-pyridylthio] propionate, succinimidyl 6- [3- (2-pyridylathio) -propionamide] hexanoate (Pierce Chemical Co., Rockford, IL), but is not limited to these.

In a suitable embodiment of the invention, the cross-linking drug is formaldehyde. In certain embodiments, low grade formaldehyde (0.2%) is used to preserve antigenicity. In other embodiments, high concentrations may be used to optimize stability. In making the complex of the present invention, formaldehyde at a concentration of 0.01% to 8% can be used. Other drugs that can be used to cross-link fusion cells include, but are not limited to, glutaraldehyde (0.05-05%). Those skilled in the art will appreciate that fixed or cross-linking conditions (ie, drug used, drug concentration, time, temperature) can be varied to determine optimal conditions for immunogenicity of FRMS.

In another embodiment, the complexes of the present invention can be made by infecting human diploid cells on a cell culture with recombinant vaccinia virus, eg, expressing viral envelope protein and CD4 / coceptor (if not endogenously present). During fusion, cells cross-link to capture fusion-related structures and crystal sites.

In another embodiment of the present invention, when a cell expressing a cell receptor or co-receptor for a virus is directly infected by a live or attenuated virus, the infected cell is cross-linked to capture FRMS.

In another embodiment of the invention, a single cell can be engineered to express all components important for FRMS production. In this embodiment, these cells are fixed or cross-linked to capture FRMS. In this embodiment, cell-cell interaction does not need to occur at a fixed time because all the components are expressed in individual cells.

The present invention provides viral particles engineered to express cellular components or proteins that are important for FRMS production in the viral envelope. In this embodiment, the recombined viral particles cross-link to capture the FRMS produced in the viral particles.

In another embodiment of the invention, the complex does not need to be fixed or cross-linked. The complexes of the present invention were found to form in solution cell lysate. Thus, for example, simply lysing cells transfected or infected with HIV-1 envelope protein and CD4 / coreceptor to form the complex of the present invention, wherein FRMS is produced in solution. Cytolysis protocols are known in the art (Sambrook et al., Supra ). Cytolysis protocols include detergent (eg , 1% NP40) lysis, freeze thaw lysis, ultrasonic lysis, or any method known in the art. In certain embodiments, the cell cultures are fixed upon occurrence of cell-cell interactions by immobilization in ice cold 0.2% formaldehyde dissolved in phosphate buffered saline. In another embodiment of the invention, detergent dissolution of the cells comprises the use of detergent Brij 97.

Thus, the present invention provides molecular structures that are cross-linked and separated from cell lysates. In another embodiment, the present invention provides fusion-defective mutants capable of FRMS accumulation and FRMS capture.

5.3. FRMS Separation

In other embodiments, cells expressing components that bind to produce FRMS (eg, cross-linked cells) can be used as immunogens in the vaccines of the invention. Alternatively, the molecular structure consisting of FRMS can be separated into immunogens.

Once FRMS is generated, it can be isolated and purified by standard methods, including chromatography (eg, ion exchange, affinity, size column chromatography), centrifugation, fractional solubility, or any other standard technique for protein purification. In addition, ingredients important for the production of FRMS can be synthesized by including affinity labels that facilitate recovery and purification. Peptide labels may bind to any region of the protein, but such binding should not alter the epitope morphology produced by the binding of the modified component to other complex components. In various embodiments, such chimeric proteins comprising an affinity label can be made by ligating the gene sequence encoding the component with the sequence encoding the peptide label in an appropriate translation frame and recombinantly expressing the protein. Care should be taken to ensure that the modified gene is in the same translation frame and is not interrupted by the translation termination signal.

Peptide modifications employ various peptide labels known in the art, and examples of such peptide labels include polyhistidine sequences (Petty, 1996, Metal-chelate affinitychromatography, in Current Protocols in Molecular Biology, Vol. 2, Ed.Ausubel et al., Greene Publish.Assoc. & Wiley Interscience), glutathione S-transferase (GST; Smith, 1993, Methods Mol. Coll Bio. 4: 220-229), E. coli maltose binding protein (Guan et. al., 1987, Gene 67: 21-30), various cellulose binding domains (US Patent Nos. 5,496,934; 5,202,247; 5,137,819; Tomme et al., 1994, Protein Eng. 7: 117-123), STAG ^TM System (Novagen, Inc.), but it is not limited to these. Other usable peptide labels include, but are not limited to, FLAG epitopes, myc epitopes at amino acids 408-439, influenza virus hemagglutinin (HA) epitopes available to monoclonal antibodies. Other peptide labels are recognized by a specific binding partner, thus facilitating separation by affinity binding with the binding partner, which is preferably anchored to the solid phase surface. As will be appreciated by those skilled in the art, a variety of methods can be used to obtain the coding regions of the peptides described above, including but not limited to DNA cloning, DNA amplification, and synthetic methods. Some peptide labels and reagents for their deletion and isolation are commercially available.

DNA sequences that are readily available from libraries or suppliers or that encode known known peptide labels are suitable for the practice of the present invention. Such peptide labels are recognized by specific binding partners and facilitate separation by affinity binding with binding pairs that can be immobilized on a solid phase surface. Chimeric protein gene products can be made using recombinant DNA technology. For example, a gene sequence encoding a component important for FRMS production can be introduced into a vector having a peptide labeled sequence, which is then expressed as a peptide-labeled chimeric protein. Peptide labels recognized by a specific binding partner are used for affinity binding with a binding partner immobilized on a solid phase surface.

In a suitable embodiment, the poly-histidine labeled fusion-related protein is constructed by inserting a fusion-related protein or gene fragment into an expression vector, eg, the pET15 vector series (Novagen), wherein the vector is an N-terminal poly- Express a sequence inserted into a chimeric protein carrying a histidine label. Proteins that have six or more histidine residues in succession at the amino or carboxy termini exhibit a strong binding affinity for nickel. The poly-histidine-labeled chimeric protein will specifically bind to the surface of the solid phase coated with chelated nickel. In suitable embodiments, microtiter plates coated with metal chelates are used to separate poly-histidine-labeled chimeric proteins (Pierce).

Alternatively, the system presented by Janknecht et al. Allows for easy purification of non-denatured chimeric proteins in human cell lines (Jenknecht, et al., 1991, Proc. Natl. Acad. Sci. USA 88,8972-8976 ). In such a system, the gene of interest is subcloned into a vaccinia recombinant plasmid, the open reading frame of which is fused and translated into an amino-terminal label consisting of six histidine residues. Extracts of cells infected with recombinant vaccinia virus are loaded onto a Ni ²⁺ nitriloacetic acid-agarose column and histidine-labeled proteins are eluted selectively with imidazole-containing buffer.

In another specific embodiment, the expression construct subclones the fusion-related protein into an EcoRI restriction site of each of the three pGEX vectors (glutathione S-transferase expression vector; Smith et al., 1988, Gene 7: 31-40). I can make it. This enables the expression of chimeric proteins consisting of fusion-related proteins, each of which is bound to the GST binding domain on each translation frame, in which the GST binding activity is also maintained in the resulting chimeric protein. GST chimeric peptides exhibit a strong binding affinity for glutathione substrates. In certain embodiments, FRMS comprising a GST label is separated from the mixture by contacting a binding mixture consisting of cell lysates or immobilized cells with a glutathione-bound solid phase surface (eg, glutathione sepharose beads). The mixture is then added to the column or beads in a manner that allows for interaction and binding. At the end of the reaction, the unbound material can be washed off. The interaction between the chimeric protein and glutathione-agarose beads allows the separation of the complex.

In other embodiments, the chimeric protein can be readily purified using antibodies specific for the chimeric protein expressed.

In another embodiment, an affinity-labeled FRMS can be constructed by conjugating an affinity compound to a fusion-related protein. Affinity compounds can be used, including but not limited to biotin, photobiotin or other compounds known in the art. In one embodiment, the affinity compound or affinity label can be conjugated to a fusion-related protein via a multifunctional cross-linker, preferably a bifunctional molecule. Multifunctional crosslinkers in this article include molecules with one or more functional groups that act on functional groups on fusion-related proteins. In general, such crosslinkers form covalent bonds with amino or silpyl drill groups on the polypeptide. For example, biotin N-hydroxysuccinimide esters are used.

In a suitable embodiment of the invention, the highly specific method of isolating FRMS through binding to CD4, chemok co-receptor, envelope protein is collectively referred to as the 15 amino acid S-peptides of RNase A and RNase S. S-peptide affinity labeled molecules using specific, high affinity interactions (K _d = 10 ^-9 M) of larger subtilisin-cut fragments (S-proteins; amino acids 21-124 of RNase A) (Potts et al., 1963; Dorai et al., 1994). This S-TAG ^™ system was developed commercially by Novagen, Inc. for the detection and isolation of recombinant chimeric proteins expressed in bacteria and baculovirus systems, and has recently been extended to mammalian chimeric proteins. Fusion-related proteins of FRMS are labeled with S-peptide. The present invention provides examples of complex components in which the C-terminus of a CD4, CCR5 or envelope protein molecule is labeled with an S-peptide. Labeled complexes were easily isolated and purified by S-protein agarose affinity chromatography.

In one embodiment, the membrane-impermeable cross-linking drug can be used to immobilize cells bearing a labeled complex. In certain embodiments, immobilization of cells bearing a labeled complex with an impermeable cross-linking drug may avoid inactivation of the cytoplasmic S-peptide label. Alternatively, the S-peptide sequence (ala-glu-thr-ala-ala-ala-ala-phe-glu-arg-gln-his-met-asp-ser) no longer contains vulnerable lysine residues. Modifications can be made to formaldehyde without inactivating the cytoplasmic label. The fusion cells can be immobilized with a wide range of impermeable cross-linking drugs, such as BS ³ and DTSSP (respectively bifunctional N-hydroxy that react with amines to form non-cleavable or reversible bonds). Succinimidyl (NHS) ester); Sulfo-SMPB (heterobifunctional NHS esters and maleimides that irreversibly cross-link amine groups and sulfidyl groups); Sulfo-SANPAH and SASD (heterologous bifunctional NHS esters and photoreactive phenylazide cross-linkers, respectively, which form non-cleavable or reversible bonds) are included, but are not limited to these. Don't let that happen.

The labeled complex is purified to separately prepare a fusion-related component for use in the vaccine composition. Purified or isolated labeling complexes can also be used as a diagnostic standard for in vitro analysis. In addition, functional assays of envelope-mediated fusions in labeled complexes can be performed.

Accordingly, the present invention provides a method for purifying a protein complex consisting of an envelope protein of human immunodeficiency virus type 1 that functionally interacts with human CD4 and human CCR5, the method comprising: a) labeling the complex with a peptide sequence; b) separating the labeled complex.

In another specific embodiment, the present invention provides a protein complex consisting of an envelope protein of human immunodeficiency virus type 1 that functionally interacts with human CD4 and human CCR5.

5.4. FRMS type

As will be appreciated by those skilled in the art, any envelope virus can be used in the methods of the present invention for constructing or using FRMS. For example, it is used as FRMS vaccine immunogen of the present invention. One advantage of vaccines consisting of FRMS is that these immunogens provide significantly higher primary isolate neutralization rates. Enveloping viruses that can be used include, but are not limited to, retroviruses, coronaviruses, filoviruses, arenaviruses, Fallsviruses, Bunyaviruses, Flaviviruses, Togaviruses, Orthomyxoviruses, Paramyxoviruses, Herpes Viruses, and Iridoviruses. It is not limited to these.

In one embodiment of the invention, retroviral FRMS makes the method of the invention. For example, in a suitable embodiment the FRMS of the present invention consists of a major viral envelope immunogen of human immunodeficiency virus type 1 (HIV-1) and is made by interacting with a host cell receptor for HIV-1 and a host cell co-receptor. The host cell co-receptor can be CCR5, CXCR4, CCR3, CCR2b or others known in the art. FRMS exemplified in the present invention is made by the interaction of HIV-1 isolate 168P virus envelope protein and host cell receptor CD4 with host cell co-receptor CCR5. As will be appreciated by those skilled in the art, other recombinant HIV envelope proteins, including those that function independently of CD4, can be used to create FRMS that fall within the scope of the present invention.

In certain embodiments for HIV vaccines, the immunogens of the invention are envelope-proteins expressing COS-7 cells (LaCasse et al. 1998, Science, 283: 357-360) and human CD4 and CCR5 co-receptors (U87-CD4). Cells are co-cultured expressing -CCR5). In this embodiment, the envelope-expressing cells are harvested using 0.5 mM EDTA after 24 hours of transfection (30-60% transfection efficiency) and co-cultured with an equal number of U87-CD4-CCR5 target cells. The fusion progress is assessed by staining the envelope-expressing cells and monitoring their integration into the multinuclear complexes under a microscope. Fusion is terminated within 12-24 hours. The complex is formed immediately. After 1-5 hours of co-culture fixed cells are estimated to be 10-30% of the optimal coform formation. The initial time point of fusion was chosen early to capture the transition intermediates to be fused. At other time points (eg, 10 hours, 24 hours or more), the fusion-related complexes of the present invention can be obtained. Complexes obtained later in the fusion process may still expose neutralizing epitopes, which can be used to tabulate the shape and pathway of important immunogens over time.

In another embodiment, the Flavivirus FRMS is made by the method of the present invention. Viruses belonging to the Flaviviridae family include hepatitis C virus (HCV), dengue virus, yellow fever virus, tick-borne encephalitis virus, and bovine viral diarrhea virus. The Flaviviridae family is similar to the Togavirus family, including alphaviruses (eg, Venezuelan encephalomyelitis virus, Sindbis virus, Semiki forest virus) and Rubella virus. In contrast to HIV, flavivirus enters the cell by receptor-mediated endocytosis, and then pH-induced fusion occurs in the endosome.

Flavivirus Intraviral viruses encode envelope (E) proteins, which are proteolytically digested in a number of intraviral viruses and complete with E1 and E2 proteins. Especially in the case of HCV (hepatitis C virus), the E2 protein is the receptor binding moiety (binding with the cell membrane protein CD81) and E1 is the putative viral fusion protein.

In another embodiment of the invention, flavivirus or togavirus FRMS is made by the method of the invention. For example, the HCV FRMS of the present invention is made of a combination of E1, E2, CD81. In certain embodiments, HCV E1 and E2 proteins can co-express on acceptable cell substrates, which are located locally in cell vesicles (ER) and are not very present at the cell surface. In one embodiment, these cell cultures are cultured in media buffered at pH 5 to 6.8 to initiate E1E2 fusion of the cell membrane. Cultures are treated with cross-linked drugs to capture intermediate fusion-complex structures, which are used directly as vaccine vaccines (or just after separation of the intracellular ER membrane).

In another specific embodiment, the disrupted intracellular ER membrane bearing the E1E2 protein is isolated from the inverted micelles and then incubated with appropriate infiltrating cells expressing the E2 receptor CD81. E1E2-bearing micelles are absorbed internally, resulting in E1-mediated fusion within the endosome. The cells are then cross-linked and used as vaccine immunogens. Alternatively, the E1E2 protein is engineered to modify the ER retention signal, thus eliminating the need to isolate the ER membrane by expressing the E1E2 protein on the cell surface.

In another specific embodiment, a recombinant DNA-derived HCV virus-like particle or a pseudotyped virion (eg, alphavirus or VSV) bearing HCV E1E2 is made and cultured with receptor-expressing cells in a medium adjusted to pH 5-6.8 Fuse. Cultures are treated with cross-linking drugs such as formaldehyde and FRMS is used as vaccine immunogen. Alternatively, virions can be cultured with CD81-expressing cells; The internally absorbed virions are allowed to fuse in the endosome, which can be cross-linked to capture the vaccine immunogen of the present invention.

In another embodiment of the invention, the orthomyxovirus FRMS is made by the method of the invention. The orthomyxovirus family includes all influenza viruses, including influenza A, influenza B, and influenza C. Thus, in a suitable embodiment, influenza FRMS induces neutralizing antibodies against a wide range of influenza, enabling drift-free influenza vaccines.

Similar to flaviviruses and togaviruses, orthomyxoviruses enter target cells by receptor-mediated endocytosis and are fused in endosomes. In this case, the viral nuuraminidase (NA) protein acts as a sialic acid receptor binding domain (uniquely expressed cell surface carbohydrate) and the hemagglutinin (HA) protein acts on pH-induced fusion. In contrast to flaviviruses, influenza viruses emerge from the cell surface, so that expression of recombinant NA and HA proteins occurs at the cell surface.

In another embodiment, influenza FRMS is made by the method of the present invention. Influenza FRMS is made in a manner similar to that described above for flaviviruses and induces pH-triggered HA-mediated fusion between appropriate cells, between cells and isolated cell membranes, or between virion particles, which are NA-mediated. The complex is captured with a cross-linking drug that allows endocytosis and subsequent endosomal fusion.

In another embodiment of the invention, paramyxovirus FRMS can be made by the methods of the invention. Paramyxoviridae can be made by the method of the present invention. The Paramyxovirida family includes several subgroups of medically and veterinary importance, including morbiliviruses (measles virus, dog distemper virus, and virus virus), rubula virus (mumphs virus, Newcastle disease), and paramyxoviruses. (Human and bovine parainfluenza) and pneumovirus (respiratory syncope virus), but are not limited to these. These viruses are similar to orthomyxoviruses, with the exception that paramyxovirus fusion occurs at the cell surface rather than the endosomes, which is independent of pH. For paramyxoviruses, the nuuraminidase-hemagglutinin (HN) (or simply hemagglutinin (H)) protein mediates cell adhesion, while the fusion (F) protein mediates virus-cell or cell-cell fusion. do. For example, for measles virus, H protein binds to cellular CD46 receptor and F protein mediates membrane fusion. In certain embodiments, cells that express H and F proteins and cells expressing CD46 from MV are co-cultured to create cell-cell fusions, which can be captured by cross-linking or by any of the other methods described above. Alternatively, in other embodiments the co-receptor may be expressed in fusion-allowing cells expressing exogenous CD46. Certain embodiments (as described for HIV and other viruses described above) can also be used.

In one embodiment of the invention, the herpes virus FRMS is made by the method of the invention. Herpes virus consists of a large family of medically and veterinary members. Human herpesviruses include, but are not limited to, HSV1 and HSV2, bicellovirus, Epstein-Barrvirus, cytomegalovirus, Kaposi herpes virus HHV 8.

For example, in the case of HSV1, binding and entry involves several viral glycoproteins (gD, gC, gH / gL) and several cellular receptors and adhesion molecules (glycosamine glycans and herpes virus entry mediator (Hve) protein AC). It's a complicated process. Binding and fusion occur at the cell surface in a pH-independent manner.

In certain embodiments, HSV FRMS is constructed using cells that express a cell receptor and adhesion molecules (eg, human MRC5 cells) and co-culture with cells expressing HSV glycoproteins gD, gC, gH, gL. Cell-cell fusion is preferably inhibited by fixation with cross-linked drugs and captures FRMS.

As noted in this article, viral proteins bind to host cell receptors, such as herpesviruses, poxviruses, paramyxoviruses, measles, mumps, rubella, respiratory syncytial viruses, influenza, hepatitis C, ebola, and flavi. You can create FRMS for viruses, including viruses (eg dengue and yellow fever). In addition, the FRMS of the present invention may include viral proteins of veterinary critical viruses, including rabbits, feline leukemia, feline immunodeficiency virus, and virus.

Table II presents a list of cell receptors for specific enveloped viruses, which are expressed with the viral envelope protein and bound to the envelope protein to produce FRMS.

Other viral diseases that can be prevented or treated by the methods of the present invention include hepatitis C, influenza, chickenpox, herpes simplex virus type I (HSV-I), herpes simplex virus type II (HSV-II), basin, respiratory syncytial virus , Cytomegalovirus, epidermal virus, arbovirus, hantavirus, mumps virus, measles virus, rubella virus, human immunodeficiency virus type I (HIV-1), human immunodeficiency virus type II (HIV-2), arbitrary Togaviruses (eg dengue fever virus), alphaviruses, flaviviruses, coronaviruses, rabies virus, Marburg disease virus, ebolavirus, rapinefluenzavirus, orthomyxovirus, bunyavirus, arenavirus, human T-cell leukemia Virus type I, human T-cell leukemia virus type II, monkey immunodeficiency virus, lentivirus, Epstein-Barr virus, Human herpes viruses, long-tailed monkey herpes virus 1 (B virus), poxviruses are included, but are not limited to these.

Table III presents a list of envelope proteins for specific envelope viral families, which are expressed together with the cytoreceptor protein to produce FRMS when bound to the cytoreceptor protein.

In another embodiment of the invention, the three-strand twisted-helix structure of the orthomyxovirus family (eg influenza), phyllovirus family (eg Ebola) or retroviral family (eg HIV) facilitates FRMS production.

In other embodiments, synthetic peptides can be used to inhibit viral infection. For example, synthetic peptides consisting of one of the gp41 helix coils can be combined with homologous helix regions to broadly inhibit viral infection (Wild, C., et al., 1992, Proceedings of the National Academy of Sciences USA 89: 10537). Furutaa, RA, et a., 1998, Nature Structural Biology 5: 276).

In another embodiment, the neutralizing antibody against the FRMS immunogen targets the structure involved in fusion activation.

In other embodiments of the invention, viral strains that do not require host cell receptors for viral binding but require host cell co-receptors may be used. For HIV, for example, HIV strains that do not require CD4 for viral binding can be used in the methods of the present invention (Hoxie, et al., 1998, J. Reprod. Immunol. 41: 197-211.). Thus, expression of CD4 is not necessary when using such strains.

5.5. Vaccine Compositions and Administration

The present invention relates to a method of vaccinating an individual or animal to prevent infection by the virus and to increase the immune response to the virus. The FRMS of the present invention can be provided as a vaccine using several means. The FRMS described above can be administered to the vaccine as fixed cells complexed with an adjuvant. Separately purified FRMS can also be administered as a vaccine.

FRMS can be made in situ by vaccine vaccination simultaneously with transfected cells expressing viral proteins and expressing host cell receptors and co-receptors. Similarly, such immunization reaction strategies can employ vectors comprising DNA encoding viral proteins and vectors comprising DNA encoding receptor proteins. Vectors include viral vectors such as vaccinia and vectors used in DNA immunization reactions. At the same time after immunization, the interaction between the viral protein and the host cell receptor protein takes place in vivo , resulting in the formation of the FRMS of the invention, thus exposing the vaccine to a unique epitope capable of inducing neutralizing antibodies.

Another way to make FRMS in situ involves using receptors and co-receptors on host cells. For example, in preparing an HIV envelope resulting from interaction with CD4 and CCR5, a vector containing DNA encoding transfected cells or proteins expressing HIV glycoproteins is injected or targeted into lymphocytes of the host. (Using VEE or another vector). Viral proteins bind to host cells, including viral receptors and pupil receptors, in vivo to initiate fusion to form FRMS, and thus the vaccine is exposed to newly produced neutralizing epitopes.

In certain embodiments, the vaccine composition consists of an isolated protein complex and a pharmaceutically acceptable carrier composed of envelope proteins of human immunodeficiency virus type I capable of functionally interacting with human CD4 and human CCR5. In another embodiment, the present invention provides a method of immunizing an animal against a virus, which is comprised of one or more viral proteins capable of functionally interacting with one or more host cell receptors or co-receptors that mediate viral binding. A vaccine composition consisting of a protein complex is administered to an animal to produce a neutralizing antibody against the virus.

In one embodiment, the vaccine composition of the present invention comprises: (a) a first nucleic acid encoding the envelope protein of the envelope virus; (b) providing a vaccine composition consisting of a second nucleic acid encoding one or more cell membrane proteins, wherein the envelope protein and the cell membrane protein are necessary to fuse the envelope of the virus to the cell membrane comprising the cell membrane protein under appropriate conditions. Conditions, envelope proteins and cell membrane proteins are expressed in the individual to produce neutralizing antibodies against the virus; (c) a pharmaceutically acceptable carrier.

In one embodiment, the present invention provides a method of treating a host exposed to a virus or preventing infection of the host with a virus, wherein the method is a human immunodeficiency capable of functionally interacting with human CD4 and human CCR5. An isolated protein complex consisting of a viral type 1 envelope protein consists of injecting the host with an antibody produced by immunizing the animal in an amount effective to treat or prevent infection of the host.

In another embodiment, the invention relates to a method for providing a protein complex consisting of one or more viral proteins capable of functionally interacting with one or more host cell receptors or co-receptors that mediate virus binding, invasion and infection. It comprises (a) culturing a first cell expressing one or more viral proteins; (b) culturing a second cell expressing a co-receptor or one or more host cell receptors for one or more viral proteins; c) co-culture of the first and second cells; (d) immobilize the co-culture during cell-cell fusion; (e) isolating the fixed cells.

Using various methods, the vaccine composition of the present invention can be introduced, for example, oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal or oocyte method (using biped needles). To scrape off the upper layers of the skin), but not limited thereto.

The patient or individual receiving the vaccine is suitable for mammals and most appropriately for humans, but for humans such as cattle, horses, sheep, pigs, poultry (chicken), goats, cats, dogs, hamsters, mice and rats. Animals may also be used.

Patients or individuals administered with the vaccine include, but are not limited to, non-human wildlife such as lions, cheetahs, elephants, giraffes, African antelopes, leopards, black panthers, and the like. Accordingly, in this embodiment, the present invention provides a wildlife maintenance means that provides a method for preventing or treating a wild animal or a high population of animals with a high risk of being infected with a viral disease.

In some embodiments herein, the subject is a human. In another embodiment, the person is at high risk for HIV infection. In another embodiment, the individual is a livestock product.

The present invention provides a method of treating or preventing a viral disease or immunizing an animal in an animal, by administering the vaccine of the present invention to an animal in an amount having an immune effect.

The vaccine composition of the present invention can also be used to make an antibody for use in passive immunotherapy, whereby a pre-formed antibody is administered to a heterologous organism to protect the host for a short time.

Vaccination of mice with an immunogen consisting of the FRMS of the present invention can induce neutralizing antibodies in immunized animals. In an embodiment, the complex is administered with a vaccine consisting of immobilized cells comprising an adjuvant. Those skilled in the art will appreciate that other vaccine strategies may be used in practicing the present invention. For example, isolated purified FRMS or an epitope thereof may be administered as a subunit vaccine. Alternatively, as already mentioned, a gene encoding a complex having a function is constructed and placed in a vector or plasmid for use in a live vector or DNA plasmid vaccine.

The viral vaccine composition of the present invention consists of at least one FRMS and an pharmaceutically acceptable carrier or excipient in an effective amount of immunization as the vaccine immunogen of the present invention. Boosting can also be considered. In one embodiment, the vaccine composition of the present invention consists of a "pharmaceutically acceptable carrier". As described herein, a pharmaceutically acceptable carrier refers to a substance that does not interfere with the action of the immunogen and is not toxic to animals. Pharmaceutically acceptable carriers are well known in the art and include, for example, salts, buffer salts, dextrose, water, glycerol, isotonic isotonic water soluble buffers, oil-in-water emulsions, or oil-in-water emulsions, Water-soluble compositions, liposomes, microbeads, microsomes, adjuvant compounds, composites thereof, and the like, but are not limited thereto. One example of such a pharmaceutically acceptable carrier may be a physiologically balanced culture medium comprising one or more stabilizers such as stabilized, hydrolyzed proteins, lactose. The carrier must be sterile. The composition should be suitable for the mode of administration.

If desired, the composition may include small amounts of wet or emulsion materials, pH buffers. The composition may be a liquid solution, suspension, emulsion, tablet, pill, capsule, delayed release composition or powder. Oral compositions include standard carriers such as pharmaceutically acceptable grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate and the like.

Generally, the ingredients are supplied separately or mixed together in unit dosage form, e.g. dry lyophilized powder or moisture free concentrates in a sterile sealed container such as an ampoule or bag in which the amount of active substance is known. Can be. When the composition is administered by injection, a sterile dilution ampoule can be added and the ingredients mixed before injection.

In certain embodiments, a lyophilized fusion complex of the invention is provided in a first container, the second container consisting of 50% glycine, 0.25% phenol and an aqueous solution of a preservative (eg , 0.005% brilliant green) Diluents are included.

The exact dosage of immunogen or FRMS used in the composition depends on the route of administration and on the characteristics of the patient, which the physician should judge according to the circumstances of each patient based on standard clinical techniques. The effective immunization amount should be an amount sufficient to create an immune response to the complex in the host to which the fusion-dependent complex or vaccine immunogen is administered.

In certain embodiments, the effective amount of vaccine immunogen to a human subject of the present invention is 10 to 100 mg / kg body weight, more suitably 0.1 to 10 mg / kg body weight, and may be boosted but is not preferred.

How accurate the amount of vaccine immunogen that can be used in a given formulation is not a major problem if a minimum sufficient amount is used to elicit an immune response. A dosage range of at least 10 μg up to several mg is also contemplated. In one example, in certain embodiments, the individual dose is 50-650 μg per immunization process. In another embodiment, the dosage depends on the composition of the vaccine immunogen (eg, purified mimitope and purified FRMS, whole cell preparation).

In a suitable embodiment, the vaccine composition consists of an immunizing effective amount of an FRMS immunogen, an immune stimulant, a pharmaceutically acceptable carrier. As used herein, an immunostimulant includes any compound or composition that has the ability to enhance the activity of the immune system, in combination with a particular immunogen to exhibit specific potentiating effects, or one or more elements of an immune response. When activated, it can simply produce an effect independently. Some of the immunostimulants commonly used in vaccine compositions are adjuvant alum or muramyl dipeptides (MDP) and analogs thereof. Methods of using such materials are known in the art, and those skilled in the art will readily be able to determine the appropriate amount of stimulant for a given viral vaccine. One or more immunostimulants may be used in a given composition.

Using standard methods, purified immunogens or complex vaccines can be used. For example, purified proteins should be adjusted to appropriate concentrations, formulated with appropriate vaccine adjuvant and packaged for use. Suitable adjuvants include mineral gels such as aluminum hydroxide; Surface active materials such as lysolecithin, pluronic polyols; Polyanions; Peptides; Oil emulsions; Alum; Including but not limited to MDP. The immunogen may be bound to liposomes or may be bound to polysaccharides or other polymers used in vaccine compositions. If the complex is a hapten, for example, if the antigenic molecule is capable of selectively reacting with antibodies of the same species but is not a immunogen capable of eliciting an immune response, the hapten is covalently bound to the carrier or immunogen molecule. Can be; For example, large proteins such as serum albumin may confer immunogenicity to the binding of the hapten. Hapten-carriers can be used as vaccines.

The effect dose (immunization amount) of the vaccine of the invention can be extrapolated in a dose-response curve derived from an animal model test system.

Accordingly, the present invention provides a method of treating or preventing a viral infection in a subject, wherein the subject is administered an effective immunization amount of FRMS effective for treating or preventing the viral infection. In certain embodiments, the invention is to administer to a human an effective amount of FRMS immunogenic effect effective to treat or prevent infection by HIV, in order to treat or prevent infection by HIV in humans.

The present invention also provides a method of treating or preventing a viral infection in a subject, wherein the subject is administered an effective amount of FRMS effective for treating or preventing the viral infection. In certain embodiments, the invention is to administer to a human an effective amount of a monoclonal antibody effective to treat or prevent infection by HIV, in order to treat or prevent infection by HIV in humans.

5.6. Antibody production

The present invention relates to making polyclonal and monoclonal antibodies that are reactive with the FRMS of the present invention. According to the present invention, an antibody capable of immunospecifically binding to such an immunogen can be made using FRMS, fragments thereof and derivatives thereof, and analogs thereof as immunogens. Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab fragments, Fab expression libraries, and the like.

The invention also relates to a method of making an immune response, such as antibody production in an animal. In one embodiment, the animal is administered FRMS of the present invention. Preferably the FRMS consists of HIV envelope protein, which is caused by interaction with CD4 and CCR5. FRMS is administered such that an immune response can occur in the animal against the immunogen. Preferably, antibodies against epitopes on FRMS are made. Most suitably the antibodies produced include neutralizing antibodies. In more suitable embodiments, the resulting antibodies can neutralize various primary isolates of the virus.

Various procedures known in the art can be used to produce polyclonal antibodies against FRMS or derivatives or analogs thereof. In certain embodiments, rabbit polyclonal antibodies against an epitope of FRMS, a substring thereof, can be obtained. To produce antibodies, a variety of host animals can be immunized by injecting native FRMS or synthetic FRMS or derivatives thereof (fragments or chimeras), including but not limited to rabbits, mice, rats, and the like. . Various adjuvants are used to increase the immunological response, which depends on the host species, for example Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lyso lecithin, phonics Polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenols, human adjuvants such as bacille Calmette-Guerin (BCG), corynebacterium parvum It is not limited to this.

In order to prepare monoclonal antibodies against FRMS, derivatives or analogs thereof, any technique capable of producing antibody molecules by continuous cell lines in culture can be used. For example, hybridoma technology and trioma technology, human B-cell hybridoma technology (Kozbor et al., 1983) developed by Kohler and Milstein, (Kohler et al., 1975, Nature 256: 495-497). , Immunology Today 4:72) and EBV-Hybridoma technology can be used to make human monoclonal antibodies (Cole et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). In a further embodiment of the invention, monoclonal antibodies can also be made in sterile animals ( see eg, PCT / US90 / 022548). According to the present invention, it is also possible to use a human antibody, a hybridoma of a human: in the (Cole et al, 1983, Proc Natl Acad Sci USA 80..... 2026-2030) or the human B cells with EBV virus It can also be obtained by transformation in vitro (Cole et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, pp. 77-96).

In certain embodiments, spleens of mice immunized with FRMS are obtained and splenocytes are isolated from the culture medium to separate splenocytes. Splenocytes are fused with HPRT-negative mouse myeloma cells using polyethylene glycol (PEG). Cell pellets with a 4: 1 ratio of myeloma cells to splenocytes were resuspended and treated with serum-free medium containing 50% pretested PEG-4000 (2.5 min contact). The cell suspension is then slowly diluted with serum free medium and the cells are HAT selection medium on 96 well culture dishes; 20% pretested fetal calf serum, 10% NCTC-109 (Gibco), 1% non-essential amino acids, 1X glutamine and 1X Pen-Strep, 1X OPI (15 mg / ml oxalacetate, 5 mg / ml pyruvate sodium, 20 units / ml insulin), 100 μM hypoxanthine, 0.4 μM aminopterin, DMEM medium containing 16 μM thymidine]. Wells containing hybridomas were monitored under a microscope and supernatants were obtained as needed for antibody production testing.

In fact, according to the present invention, chimeric antibodies (Morrison et al., 1984, Proc. Natl. Acad. Sci. USA 81: 6851-6855; Neuberger et al., 1984, Natjure 312: 604-608; Takeda et al., 1985, Nature 314: 452-454), a technique developed for production, ie, the joining of genes derived from proximal antibody molecules specific for FRMS with genes derived from human antibody molecules with appropriate biological activity. Can be used and such antibodies are within the scope of the present invention. In another embodiment, the "fluidized" antibody also comprises U.S. Patent No. 5,225,539.

According to the present invention, the techniques used to produce single chain antibodies (U.S. Patent No. 4,946,778) can be used to make FRMS-specific single chain antibodies. A further embodiment of the invention utilizes techniques for the construction of Fab 'expression libraries (Huse et al., 1989, Science 246: 1275-1281) to produce monoclonal Fabs with the desired specificity for tumor suppressor proteins, derivatives or analogs. Fragments can be identified quickly and easily.

Antibody fragments comprising the molecule's idiotype can be obtained using known techniques. For example, such fragments include F (ab ') ₂ fragments obtained by pepsin treatment of antibody molecules, Fab' fragments obtained by reducing disulfide bonds of F (ab ') ₂ fragments, and antibody molecules with papain and a reducing agent. Treatment includes, but is not limited to, Fab fragments, Fav fragments, and the like.

Fab fragments obtained from complex libraries can also be considered in the present invention (see e.g., Chanock et al., 1993, Infect Agents Dis. 2: 118-31).

In producing antibodies, methods for screening for desired antibodies can be made using techniques known in the art (eg, enzyme-linked immunosorbent assays or ELISAs). For example, to screen for antibodies that specifically recognize FRMS, the resulting hybridomas can be examined for products that bind to FRMS epitopes but not to epitopes on other proteins. First, to select antibodies that specifically bind to one FRMS but not specifically to the other FRMS verb, the selection is based on a positive binding to the first FRMS verb but no binding to the second verb. do. Screening of antibodies can be performed by virus neutralization assays (known in the art).

Provide antibodies specific for the domain of FRMS. Antibodies specific for epitopes of FRMS proteins are also provided.

The generated antibodies can be separated by techniques known in the art (eg, immunoaffinity chromatography, centrifugation, precipitation, etc.) and can also be used for diagnostic immunoassays. Antibodies can be used to monitor disease progression or treatment. Any immunoassay system known in the art (such as those mentioned above) can be used for this purpose, for example, radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), "sandwich" immunoassay, Using techniques including recipe response, gel acuity precipitin response, immunodiffusion test, eglutinin test, complement-fixed test, immunoradioactivity test, fluorescence immunoassay, protein A immunoassay, immunoelectrophoresis test, etc. Competitive and non-competitive inspections include, but are not limited to.

When using the FRMS of the present invention as an immunogen, it has the ability to induce neutralizing antibodies against viral pathogens. In a suitable embodiment, FRMS induces neutralizing antibodies against various categories of viral primary isolates. In certain embodiments, FRMS induces neutralizing antibodies against primary isolates of HIV-1. Neutralizing antibodies that are not induced by static HIV envelope proteins may at least weakly bind to the static envelope while the antibody completes binding with high affinity to critical intermediate structures, and may have envelope proteins through binding and fusion processes. . Such neutralizing antibodies are, in fact, moved to an important site that is available at critical moments irrespective of the site of action of the cell, in particular independent of the cell site of membrane fusion (eg, the cell surface or an inner membrane such as an endosomal membrane). . Thus, viral envelope proteins serve as vectors to bring antibodies into the cell, cytoplasmic, or histocompatibility-providing process (eg, linked to toxins, other proteins, vaccine immunogens).

The hybridoma cell supernatants of the present invention are examined for their ability to neutralize homologous viruses and for the ability to neutralize genetically diverse viruses. The ability to neutralize genetically diverse viruses means recognition by mAbs in conserved crystal regions. In addition, mAb is tested for inhibition of envelope-injected cell-cell fusion. MAbs are also tested for their ability to bind or immunologically precipitate to receptor-associated complexes including envelope proteins and envelope proteins. Of particular interest is that these mAbs specific for fusion-dependent epitopes bind weakly to static envelope proteins.

In one embodiment, hybridomas can be identified using a significant amount of screening test to detect PI virus neutralization and the mAb can be characterized by determining the molecular target and width of the PI virus neutralization.

In various embodiments of the invention, antibodies made by the methods of the invention can be used individually (eg a single mAb) or in combination (one or more mAbs related to the same or different epitopes). Combination applications of antibodies include multiple mAbs related to the same virus or to other viruses. In some embodiments of the present invention, there is an advantage in that multiple mAbs for the same virus or multiple mAbs for the same FRMS of the virus can be used.

5.7. Antibody binding and virus infection inhibition test

The present invention can be tested in a variety of ways for the ability of antibodies or derivatives or analogs to bind to FRMS and for the ability to interfere with viral infection.

Binding can be tested with Susan known in the art. For example, biopsies are performed by exposing mAb derivatives or analogs to cells known to express chemokine receptors to test or test known effects. Alternatively, mAbs, derivatives or analogs are tested for their ability to bind chemokine receptors, host cell receptors or viral envelope proteins using a variety of procedures, including, for example, protein affinity chromatography, affinity. Library-based methods such as blotting, immunoprecipitation, cross-linking, protein brobbing, phage display, dual hybrid systems ( see, generally, Phizicky et al., 1995, Microbilo. Rev. 59: 94-123) It is not limited to this.

Large amounts of screening for mAb, derivative or analog binding can be performed using methods known in the art, such as flow cytometry methods. In certain embodiments, cells expressing human CD4 and one of the HIV officials (CC CKR-5, CxC CKR4, CCR5, CXCR4 etc) are treated with biotinylated mAb, derivatives, analogs thereof, and bound to FRMS The cell surface can be detected by avidin FITC conjugate. Alternatively, the flow cytometry system can be used for competition binding assays using the monoclonal antibodies of the present invention in the following manner or using any method known in the art. In certain embodiments, mAbs of the invention are labeled and tested for their ability to bind to the FRMS of the invention as compared to the ability of a test antibody to bind to the same FRMS. Test antibodies of the invention can be derived from a sample such as serum from a subject or from a production sample such as a hybridoma supernatant.

In another embodiment, the anti-viral activity exhibited by the mAb, mAb derivative or analog of the present invention can be measured using readily viable in vitro assays, which inhibit the infection by acellular viruses. Or the ability of a compound to inhibit coalescence formation can be tested and the effect of the compound on cell proliferation and viability can be assessed. Applying such tests, one can determine the strain or strain specific inhibitory activity of the mAb, its derivatives, the related anti-viral activity exerted by the analog or the immunodeficiency virus composition best suited for the virus.

In one embodiment, cell fusion assays are used to test for the ability of mAbs, derivatives and analogs to inhibit virus-induced coalescence formation in vitro . In certain embodiments, cell fusion assays are used to test the ability of a mAb derivative or analog of the present invention to inhibit HIV-induced coalescence formation in vitro . In such embodiments, the test is culturing uninfected CD4 ⁺ cells in the presence of a composition comprising chronic HIV-infected cells and a mAb, derivative or analog to be tested. In each case, the concentration range can be tested. This range should also include reference cultures without addition of mAb, derivatives, or analogs. Standard conditions for culturing known in the art can be used. After incubation for 24 hours at an appropriate time, such as at 37 ° C., the presence of multinuclear giant cells in the culture is observed under a microscope, which indicates cell fusion and coalescence formation.

In another embodiment, the in vitro infection test can be performed using the major macrophage, macrophage-property isolate HIV-1 _BaL9 , wherein the macrophage attribute HIV-1 isolate is Gartner et al., 1986, Science 233 First explained in: 215. According to this test, the major macrophages isolated according to methods known in the _art are infected with HIV-1 _BaL , which proliferate and maintain only in the major macrophages. Influx immunodeficiency virus is incubated with major macrophages in the presence of the mAb, derivative or analog to be tested. After a period of infection, unbound virus is washed away and cells are left in culture. Viral replication levels in such assays may include, but are not limited to, techniques known in the art, such as measuring reverse transcriptase levels (RT) or releasing extracellular p24 core antigens on days after infection. It can be evaluated by the method. The level of inhibition of viral infection or replication is determined by measuring HIV p24 levels (or another measure of viral infection or replication, such as RT), generated against a baseline test run without mAb, derivative or analog. Suitably the mAb derivative or analog reduces viral levels, as measured by p24, by about ≧ 50% relative to the baseline test run without the test compound. The presence of p24 can be determined using methods known in the art, such as commercially available enzyme-linked immunosorbent assays (Coulter, Hialeah, Florida; Avvott Laboratories, Hvalstad, Norway). Alternatively, RT activity was determined by Goff et al., (Goff et al., 1981, J. Virol. 38: 239-248) and Willey et al., (Willey et al., 1988, J. Virol. 62: 139- Testing can be done by monitoring cell free supernatants using standard techniques such as described in 147).

In another embodiment of the invention, the mAb of the invention may be tested in a major isolate neutralization assay. Various neutralization tests are known in the art and are within the scope of the present invention. In one embodiment, mAbs of the invention were examined in a focus test. In one embodiment of the focus test, the test antibody is incubated with the measured amount of virus before adding the virus to the host cell. Infection of host cells can be measured by staining the cells using immunohistochemical methods known in the art using antibodies specific for viral proteins. In general, baseline tests are performed side by side without the use of test antibodies. Test antibodies that inhibit (eg, prevent or reduce) infection when compared to a reference infection can be considered neutralizing antibodies. In another embodiment of the invention, PBL assays can be used to test for neutralization reactions by test antibodies. In one embodiment of the PBL test, the test antibody is incubated with the measured amount of virus prior to adding the virus to the host cell. Even if the host cell culture is incubated for several days, the infection can be examined by measuring virions in the host cell supernatant sample. In general, baseline tests are performed side by side without the use of test antibodies. Test antibodies that inhibit (eg, prevent or reduce) infection when compared to a reference infection can be referred to as neutralizing antibodies.

5.7.1. Transformed Mouse Model

In the present invention, using a transformed animal other than a person expressing one or more components, the expressed components are combined to make the FRMS of the present invention. Specific embodiments in which these components are combined to form FRMS are listed in Tables 2 and 3. The transformed animal model of the present invention can be used as a resistance model or an infection model. In the resistance model, all cellular components important for FRMS formation are expressed in the transformed animal. In the case of infection models, one or more of the components important for FRMS formation are expressed so that the transformed animal can be infected by a virus that induces FRMS.

Expression of components capable of forming FRMS using animals including, but not limited to, mice, rats, rabbits, guinea pigs, pigs, micro pigs, goats, sheep and non-human primates such as baboons, monkeys, and chimpanzees. To make a transformed animal. As used herein, an "transformed" animal is one that is capable of expressing one or more components and forming FRMS gene sequences that can be obtained from other species (e.g., a human's sequence of humans encoding one or more components). Animals that are genetically engineered to overexpress an endogenous (same species) sequence that is capable of being expressed, to form FRMS) and one or more components that are associated to form a FRMS, or to form a FRMS. Refers to animals genetically engineered (ie, "knocked out") and their descendants so that they no longer express endogenous genes encoding more than one component.

Animals can also be introduced into genes encoding one or more components that can be combined to form FRMS transgenes using any technique in the art to form transformed animal strains. Such techniques include pronuclear injection (Hoppe and Wagner, 1989, US Pat. No. 4,873,191); Gene transfer transfected with embryos (Van der Putten, et al., 1985, Proc. Natl. Acad. Sci., USA 82, 6148-6152); Gene targeting to embryonic cells (Thompson, et al., 1989, Cell 56, 313-321); Electroporation of the embryo (Lo, 1983, Mol. Cell. Biol. 3, 1803-1814); Sperm-injected gene delivery (Lavitrano et al., 1989, Cell 57,717-723) (Gordon 1989, Transgenic Animals, Intl. Rev. Cytol. 115,171-229), and the like.

Any technique known in the art can be used to make a transformed animal clone comprising one or more components that will be associated to form a FRMS transgene, for example, oocytes extracted from embryos cultured with a nucleus. Deliver to the nucleus and induce fetal or adult cells to stop (Campbell, et al., 1996, Nature 380, 64-66; Wilmut et al., Nature 385, 810-813).

The present invention provides transgenic animals having transgenes in all cells of one or more components that form FRMS in association and animals, such as mosaic animals, which have transgenes in some but not all cells. The transgenes can be combined into a single transgene or in concatamers (eg, head-head arrangement or head-tail arrangement). Transgenes can be selectively introduced and activated in specific cell types using the following method (Lasko et al., (Lasko et al., 1992, Proc. Natl. Acad. Sci. USA 89,6232-6236). The regulatory sequences required for such cell-type specific activation may vary depending on the particular cell type desired, which is well understood by those of skill in the art If gene transgene is desired to bind to the chromosomal region of an endogenous gene, gene targeting Briefly, when using such a technique, a vector comprising some nucleotide sequences homologous to the endogenous gene is designed for binding, and homologous recombination with the chromosomal sequence is used to bind the nucleotide sequence of the endogenous gene. Bind to destroy the function of the nucleotide sequence. The transgene can be selectively introduced into a specific cell type, It can be united to disable the endogenous gene encoding the one or more components to form a FRMS (Gu, et al., (Gu, et al., 1994, Science 265,103-106)). Such cell-type specific The regulatory sequence required for the inactivation depends on the particular cell type desired, but this is the part recognized by those skilled in the art.

Once the transformed animal is made, the expression of the recombinant components that are associated to form the FRMS gene can be tested using standard techniques. Initial screening can be performed using Southern blot analysis or PCR techniques to analyze animal tissue to determine if transgene binding has occurred. MRNA levels of transgenes in the tissues of transformed animals can be assessed using techniques including but not limited to Northern blot analysis, in situ hybridization, reverse transcriptase PCR (RT-PCR), etc. of tissue samples obtained from animals. have. Tissue samples expressing component genes can be assessed immunohistochemically using antibodies specific for the component transgene product.

In certain embodiments, the present invention can use transformed mice to create a model system for screening test vaccines intended for use as vaccines in species other than mice. In a suitable embodiment, the test vaccine is a vaccine intended for use in humans.

Transformed mouse models of the present invention include mice constructed to express co-receptors and host cell receptors of viral species that are not native to mice. Such mice are beneficial for analyzing virus neutralization because the effect of neutralizing antibodies on host cell receptors is excluded. Such mice can be provided as a model system for viral resistance. For example, transgenic mice can be constructed or utilized to express human cell receptors of HIV, such as HIV, that can infect humans.

In one embodiment of the present invention, vaccine studies can utilize transformed mice expressing human CD4 and CCR5 co-receptors under the control of CD4 regulatory elements that restrict co-expression in thymic cells and T-helper lymphocytes. (Killeen et al., 1993, EMBO J., 12: 1547-53). In this regard, the immune response mimics that in humans; Immunogenic epitopes are limited to the envelope, the HIV-dependent appearance of CD4, and the official. Although late entry restrictions for HIV replication limit the usefulness of such mice in infection studies to date, the present invention provides new uses that can be used as a system for analyzing HIV vaccines. In this model, the transgene functions to provide resistance to the human CD4 and CCR5 components of the vaccine.

To perform this test, transformed animals are immunized one or more times with the FRMS of the present invention. Alternatively, reference immunogens can be used to vaccinate other mice that serve as the reference. The FRMS immunogen can be in any form described herein, for example in cell lysate, in solution, in cross-linked structure, isolated, purified form. FRMS immunogens can be introduced into mice as vaccine preparations.

Serum or antibodies made from mice immunized with FRMS or reference immunogens were then collected and tested for their ability to neutralize the PI virus. Serum is collected after an appropriate time after the immunization so that an immune response can occur in mice. Such times are known in the art. In one embodiment, serum is collected two weeks after each immunization injection.

Suitably the serum or antibody is tested for the ability to neutralize two, three or four petrol isolates of the virus. More suitably, serum or antibodies are tested for their ability to neutralize four, six, eight major isolates of the virus. Most suitably, serum or antibodies are tested for their ability to neutralize 10-15, 15-25 or major isolates of the virus.

The neutralizing capacity of the main isolates can be examined by known methods such as those presented here. In one embodiment, neutralization can be determined to the extent of infectious inhibition with respect to culturing the viral sample used for infection with Sets antibodies. Antibodies that inhibit (reduce or block) viral infection refer to neutralizing antibodies. In another embodiment, the serum or antibody is tested for the ability to inhibit coalescence or the ability to inhibit infection by acellular viruses and assess the effect of the compound on cell proliferation and viability. In a suitable embodiment of the invention, the test antibody is a monoclonal antibody. In a suitable embodiment of the invention, the test antibody is a human monoclonal antibody produced in response to the FRMS immunogen. As will be appreciated by those skilled in the art, it is expected that no activity will be detected in the serum or antibody obtained from mice immunized with the reference immunogen to inhibit or neutralize the infection.

In one embodiment of the invention, the virus neutralization test is performed on at least one major isolate. In suitable embodiments, virus neutralization assays are performed on 2-5, 5-10, 10-15 major isolates. In suitable embodiments, the neutralization reaction test is performed on at least 15-20, 20-30 or at least 30 major isolates. In another suitable embodiment, the major isolates tested are from one or more viruses.

Neutralization can be characterized by a ratio that neutralizes the number of major isolates. In general, in one embodiment, a single assay can be used to assess antibody neutralization. In one embodiment, the antibodies of the invention neutralize 30-40%, 40-50%, 50-60% of the major isolates of the virus. In a suitable embodiment, the antibodies of the invention neutralize 60-75%, 70-85%, 80-90% of virus major isolates. In the most suitable embodiment, the antibodies of the invention neutralize 95%, 95-98%, 99-100% of the major isolates of the virus. In a suitable embodiment, when testing for neutralization reaction of the major isolates against mAbs, the neutralization reaction will be expressed as the number of lesions in the presence of mAb (or hybridoma supernatant) compared to the number in the presence of only embryos. Can be.

In certain embodiments, transformed mice (hu CD4 +, hu CCR5 +, mouse CD4 ⁺ ) are immunized with an HIV FRMS immunogen (co-culture with cross-linked COS-env and U87-CD4-CCR5 cells) or by cell reference ( U87-CD4-CCR5 cells alone or co-cultured with simulated transfected COS cells). The sensitivity of homologous 168P virus to neutralization by vaccine sera can be determined by U87-CD4CCR5 cells expressing CCR5 or CXCR4 co-receptors ( see, LaCasse, et al., 1998, Science 72: 2491; Follis, KE, et al., 1998, Journal of Virology 72: 7603).

In another specific embodiment of the invention, where the HIV primary isolate is used for neutralization reaction testing, such primary isolates include 92US657, 92US660, 93TH014, 89.6, 320NSI, 320OSI, SHIV89.6P, 168P, 92RW023, 92UG031. , 92UG037, 92RW008, 93IN101, 93IN999, 93IN905, 93IN904, 92UG035, 92UG021,92UG024, 92UG046, 92TH023, 92TH024, 93TH051, 93TH053, and the like.

In one embodiment of the invention, antibodies made against HIV FRMS by the method of the present invention (paragraph 5.6) can neutralize 60-70%, 70-80%, 80-85% of the major isolates of HIV. . In suitable embodiments, it is possible to neutralize 80-90%, 90-95%, 95-99% of the major isolates of HIV FRMS. In the most suitable embodiment, antibodies made against HIV FRMS can neutralize 100% of such major isolates of HIV.

In another measurement embodiment, the major isolates of HIV include one or more isolates of A, B, C, D, E, F, G, H, I. In another embodiment, the major isolates of HIV include one or more isolates such as M-, O-, N-groups.

In another embodiment, neutralization activity may be described by antibody-mediation by methods known in the art. For example, serum may be adsorbed to a solid support comprising Protein-A and Protein-G. When the antibody is responsible for the viral neutralization activity of the serum, serum depleted of protein-A and protein-G binding proteins contains little virus neutralizing activity and is eluted from protein-A and protein-G solid substrates. It is expected to contain virus neutralization activity.

Accordingly, the present invention provides a method for screening a molecular structure having a vaccine effect consisting of mammals other than a transformed human having a molecular structure, wherein the non-transformed mammal is human CD4 and one or more transgenes from one or more transgenes. It expresses a co-receptor of HIV, which can detect any neutralizing antibody against HIV produced by a mammal.

The present invention provides a method for screening a molecular structure having a vaccine effect consisting of mammals, not transformed humans having a molecular structure, wherein the mammalian non-transformed mammalian host cell membrane protein from one or more transgenes. When expressed, it can detect any neutralizing antibody against HIV produced by the mammal.

5.7.2. Determine vaccine effect

The immunity of one or more immunogens of the invention can be determined by monitoring the immune response of the test animal after immunization with FRMS, using any immunoassay known in the art. Humoral (antibody) responses and cell mediated immunity occur means that there is an immune response. Test animals include mice, hamsters, dogs, cats, monkeys, rabbits, and chimpanzees, but ultimately become human.

As described in 5.10, methods for introducing a vaccine include oral, through the brain, standard routes for transdermal, intramuscular, subcutaneous, venous, peritoneal, nasal or any immunization reactions. The immune response of the test subject can be analyzed by various methods, for example, by the reactivity of the generated immune serum to the immunogen of the present invention using a known technique, the method used is enzyme-linked immunity Adsorption tests (ELISA), immunoblot, radioimmunoassay (RIA), radioimmunoprecipitation. Alternatively, protection of the immunized host from infection by the pathogen or attenuation of symptoms from infection by the pathogen in the immunized host indicates a vaccine effect.

As an example of an animal suitable for testing viral vaccines, the vaccines of the present invention can be tested in rabbits for their ability to induce antibody responses against FRMS immunogens. Young New Zealand white rabbits (SPF) free of male specific pathogens were used. Each test group received a fixed concentration of vaccine. Reference group was injected with 1 mM Tris-HCl pH9.0 without FRMS immunogen. The presence of antibodies specific for the immunogen was examined using ELISA.

In a suitable embodiment of the invention, the animal testing the virus vaccine effect can be used in a transformed animal model as described in 5.7.1.

The invention also provides a method for optimizing and quantifying a vaccine consisting of FRMS. Suitably, the complex can be made using autogenous cells that deny responsiveness to host cell components. However, vaccine compositions consisting of complexes made using non-autogenous cells can be evaluated using transgenic animals engineered genetically engineered to be resistant to the host cell components of the complex. Using such transformed animals, one can determine whether the success achieved by vaccination with the fusion-complex of the present invention is due to the novel fusion-capable epitope provided by the complex. For example, in one embodiment, FRMS is formed by the interaction of HIV (168P) viral coat protein and human proteins CD4 and CCR5. As described in paragraph 6, the vaccine composition was tested in transformed mice genetically engineered to be resistant to the human component of the vaccine. Mice expressing both human CD4 receptors and human CCR5 co-receptors are used to assess the complex, indicating that the neutralizing antibodies issued are related to envelope proteins or immunological epitopes defined for HIV-dependent CD4 and co-receptor shapes. Explain that. The use of transformed animals to screen vaccine immunogens is also a new use of such mice. Transformed mice that are resistant to the host portion of the vaccine can be used to optimize vaccine components, test variants, and to evaluate the adjuvant.

It will also be appreciated by those skilled in the art that this principle can be applied to any virus model to provide a fast and safe method of screening antivirus vaccines. In addition, transgenic animals other than the transgenic mice may be used to screen for the recommended vaccine.

In one embodiment, the present invention provides a method for screening a molecular structure having a vaccine effect, which comprises immunizing a mammal, not a human, transformed with a molecular structure having the following structure, wherein the molecular structure is an enveloped virus Coat protein; (b) an epitope formed by the association of one or more cell membrane proteins, wherein the envelope protein and cell membrane protein are sufficient conditions for the viral envelope to fuse to the cell membrane comprising the cell membrane protein under appropriate conditions, wherein Mammalian non-transformed mammals consist of expressing one or more host cell membrane proteins from one or more transgenes to detect the presence of any neutralizing antibody against the virus produced by the mammal.

In certain embodiments, the present invention provides a method for screening a molecular structure having a vaccine effect, consisting of immunizing a mammal other than a human transformed with a molecular structure having the structure: ) HIV envelope protein; (b) consisting of an epitope formed by the association of co-receptors for human CD4 and HIV fusion, wherein the non-transformed mammal expresses co-receptors of human CD4 and HIV from one or more transgenes, It consists of detecting the presence of any neutralizing antibody against the virus produced by the virus. In a suitable embodiment, the mammal is a mouse. In another embodiment the molecular structure is isolated. The molecular structure can be separated.

5.8. Use of Antibodies

Antibodies produced by the vaccine composition of the antibodies of the invention can be used in methods known in the art. For example, vaccine immunogens or antibodies generated against FRMS immunized with FRMS of the present invention may be used for diagnostic immunoassays, passive immunotherapy, viral infection removal, anti-viral treatment and prevention, generation of anti-idiotype antibodies. It has a useful use such as.

The present invention includes antibodies directed by novel epitopes of the FRMS of the present invention, including antibodies that can be used as a reference or component in diagnostic tests and kits. In addition, the mAb for FRMS of the present invention can be used as a marker for major determinants in reference standards, diagnostic agents, binding and fusion for in vitro and in vivo biopsies. For example, during large scale production of the FRMS of the invention, the mAb of the invention is useful for determining the amount of FRMS produced in each batch. In such embodiments, the mAb is useful for determining the amount of change between lot-lot (ELISA, or RIA mAb). Alternatively, labeled FRMS can be used to test competition using test FRMS to bind mAbs specific for FRMS.

Similar tests can be used to determine the antibody titer of a sample from an individual. In such a test, a serum sample of an individual is taken at some time after administration of the vaccine of the present invention. Sample serum is tested for its ability to neutralize the virus. Serum is tested for the ability to bind to immunogen FRMS or to compete with neutralizing mAbs that bind to immunogen FRMS. If the serum sample is weak or lacking the ability to neutralize the virus or bind to FRMS, it means that the subject should be booster vaccinated with the FRMS immunogen. Thus, the present invention provides a method for monitoring antibody production against molecular structure in a subject previously administered molecular structure, which comprises separating a sample of serum from the subject; It consists in detecting the presence of antibodies to the molecular structure in the serum. In one embodiment, the detection is done by performing a competitive immunoassay with labeled antibodies against molecular structure.

In addition, polyclonal serum or monoclonal antibodies can be administered to a subject for the purpose of treating or preventing a viral infection. In certain embodiments, such antibodies may be administered for the purpose of preventing maternal virus from spreading to the fetus or preventing later exposure through a passive immunization reaction. The humoralized mAbs of the invention can be used for prophylaxis or treatment in the event of maternal to fetal viral infection or later exposure. MAbs of the present invention are useful for passive immunological response studies in hu-PBL-scid mouse models of HIV-infected and SHIV studies in Rhesus monkeys. Accordingly, the present invention provides a method of treating or preventing an infection by HIV in a human fetus, which comprises administering to a pregnant woman a monoclonal antibody that is effective for treating or preventing infection by HIV in a fetus.

MAb production for FRMS of the present invention can be used to describe biochemical immunochemical pathways for infection and to define important epitopes for PI virus neutralization. Monoclonal antibodies (mAb) generated against the FRMS of the present invention can be used to analyze biochemical basis for binding and invasion, and immunochemical basis for major isolate virus neutralization. In such embodiments, the immunogen selected for superior MAb development in immunogenicity studies in the vaccination process model induces a potent, broad range of major isolate virus neutralizing antibodies.

Antibodies of the invention can also be used to produce anti-idiotype antibodies. Anti-idiotype antibodies can then be used in the immunization reaction to produce a subset of antibodies capable of binding to the initial antigen of the pathogenic organism (Jerne, 1974, Ann. Immunol. (Paris) 125c: 373; Jerne). , et al., 1982, EMBO J. 1: 234).

In addition, mAb can be used as an anti-viral substance in blood and blood products. The present invention provides neutralizing antibodies that can be used as an additive to remove contamination from blood or blood products contaminated with or suspected of being virus contaminated. to provide. For example, the mAb of the present invention can be used as an additive in donor blood (eg, blood in a blood bank) that can be used to treat an individual. The mAb of the present invention can be used as an additive in somatic bags, gloves, and blood sample collection containers. In certain embodiments of the invention, one or more mAb (s) of the invention is used to clean the birth canal prior to birth of the child to prevent infection from the mother to the fetus. The effective amount of the mAb of the present invention as blood and blood additive is 1 µg / ml to 10 mg / ml. As will be appreciated by those skilled in the art, the effective amount depends on the antibody affinity, additives, and composition. Use an amount that is effective enough to inhibit (eg, reduce or prevent) viral infection. In suitable embodiments, neutralizing mAb mixtures for various viruses are used for decontamination. In certain embodiments, an effective neutralizing mAb of the invention immunospecific for HIV is added to a human blood sample prior to injecting blood into a subject to be transfused. In such embodiments, HIV is neutralized by mAb. Accordingly, the present invention provides a mammalian blood sample to which an antibody of the present invention is added which is effective for inhibiting or reducing infection by a virus. In certain embodiments, the invention provides a human blood sample to which an antibody of the invention is added that is effective to inhibit or reduce infection by a virus. The present invention also provides a method of inhibiting viral infection in a blood sample, wherein the effective amount of monoclonal antibody effective in inhibiting viral infection is contacted with the blood sample. In certain embodiments, the invention is contacting a blood sample with a monoclonal antibody effective to inhibit infection by HIV in a method that inhibits infection with HIV in a human blood sample.

The neutralizing antibody of the present invention is useful for decontamination of any object contaminated with a virus having the antibody. Objects decontaminated with the mAb of the present invention include, but are not limited to, surgical and dental instruments (eg, drills, picks, surgical scalpels). In suitable embodiments, neutralizing mAb mixtures for various viruses are used to remove contamination. In suitable embodiments, an individual that is not available for single use (eg, a computer-assisted tool, a robotic tool, a specific tool) may be decontaminated with one or more mAbs of the present invention. The effective amount of mAb of the present invention used to remove contamination is 1 µg / ml to 10 mg / ml. As will be appreciated by those skilled in the art, the effective amount may be affected by antibody affinity, additives and composition. After decontamination, the effective amount is sufficient to inhibit viral infection. In certain embodiments, HIV is neutralized during decontamination. Accordingly, the present invention provides a method for decontamination of a surgical or dental tool, which consists in contacting the tool with a monoclonal antibody effective to inhibit infection by a virus. In certain embodiments, the present invention provides a method for decontamination of a surgical or dental tool, consisting of contacting the tool with a monoclonal antibody that is effective to inhibit infection by HIV.

In another embodiment of the invention, antibodies to FRMS can be used to screen for molecules composed of epitopes recognized by the antibody. For example, the mAb of the invention can be used to screen small molecules for the presence of epitopes recognized by the antibody. Such small molecules are tested for their potential as immunogens or therapeutics. In another embodiment, the mAb of the invention can be used to identify peptides that bind to antibodies (phage display library). For example, such peptides are tested for immunogenicity (eg mimetopes).

The mAbs of the invention can be used as anti-viral materials in contraceptive or bactericidal products. Preference is given to viruses known to be sexually transmitted when the addition of one or more mAbs of the invention to a contraceptive or bactericidal product. Accordingly, the present invention provides a contraceptive product consisting of an effective amount of one or more neutralizing mAbs of the present invention. Contraceptive or bactericidal products to which the mAb of the present invention is added include, but are not limited to, creams, foams, jelly, ointments, condoms, pessaries and the like. Contraceptive products may include substances that kill sperm. In suitable embodiments, neutralizing mAb mixtures for various viruses can be used in the contraceptive product. The effective amount of the mAb of the invention in the contraceptive or sterile product is from 1 μg / ml to 10 mg / ml. As will be appreciated by those skilled in the art, the effective amount may vary depending on antibody affinity, additives and composition. Any amount that can inhibit viral infection is a randomly effective amount. For example, when used in anti-viral contraceptives, the amount that can inhibit infection when the virus is transmitted by sexual intercourse is effective. In certain embodiments, the contraceptive or bactericidal product consists of a mAb of the invention that is immunospecific for HIV or other sexually transmitted viruses. Accordingly, the present invention provides a jelly, foam, cream, ointment contraceptive or sterilization product composed of an effective amount of the antibody of the present invention effective to inhibit or reduce infection by a virus. In certain embodiments, the invention provides a jelly, foam, cream, ointment contraceptive or bactericidal product composed of an effective amount of an antibody of the invention effective to inhibit or reduce infection by HIV.

5.9. Therapeutic Composition

The present invention provides a method of inducing anti-FRMS antibody production in a subject by administering FRMS. FRMS, functionally active fragments, analogs, derivatives of the present invention; Nucleic acids encoding FRMS of the invention; Antibodies that can immunospecifically bind to FRMS can be used as therapeutic agents.

The present invention also provides pharmaceutical compositions. Such compositions consist of a therapeutically effective amount and a pharmaceutically acceptable carrier. In certain embodiments, "pharmaceutically acceptable" means U.S. A for use in an animal, especially a human. It means that it has been approved by a federal or state official in a pharmacopoeia or other generally recognized pharmacopeia. "Carrier" refers to a diluent, adjuvant, excipient, vehicle that can be administered with a therapeutic agent. Such pharmaceutical carriers may be sterile liquids such as water and oils, including, for example, petroleum, animal and plant or synthetic peanut oil, soybean oil, mineral oil, sesame oil and the like. When the pharmaceutical composition is administered intravenously, water is a suitable carrier. Salt solutions, water soluble dextrose and glycerol solutions can be used as liquid carriers, which are particularly suitable for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, solution, rice, flour, whistle, silica gel, sodium stearate, monostearate glycerol, talc, sodium chloride, dried skim milk, glycerol, propylene glycol , Water and ethanol are included. If desired, the composition comprises a small amount of a wetting or emulsifying agent, pH buffer. Such compositions may be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, delayed release compositions and the like. The compositions may also be formulated as suppositories using conventional binders and carriers such as triglycerides. Oral compositions may include standard carriers such as pharmaceutically acceptable grades of mannitol, lactose, starch, magnesium stearate sodium sarakin, cellulose, magnesium carbonate, and the like. Suitable pharmaceutically usable carriers are described in "Remington's Pharmaceutical Sciences" by E.W.Martin. Such compositions are in a form suitable for administration to a patient, including a therapeutically effective amount of a purified form and an appropriate amount of carrier. The composition may be suitable for the mode of administration.

In suitable embodiments, the composition may be formulated to the extent that it can be used as a pharmaceutical composition suitable for human intravenous administration. In general, compositions for intravenous administration are sterile isotonic aqueous buffers. If necessary, the composition includes a local anesthetic such as lingocaine and a soluble agent to relieve pain at the injection site. In general, the components may be supplied in unit dosage form separately or in mixed form, for example in the form of lyophilized or moisture-free concentrates in sterile sealed containers, such as ampoules or bags, in which the amount of active ingredient is known. Can be When the composition is injected and administered, it is dispensed into an infusion bottle containing sterile pharmaceutically available grade of water or salt. When the composition is administered by injection, an ampoule containing sterile water or salt for injection may be provided, and the ingredients may be mixed before administration.

The therapeutic agents of the invention may be formulated in the form of neutrals or salts. Pharmaceutically acceptable salts include those made from hydrogen chloride, phosphoric acid, acetic acid, oxalic acid, tartaric acid, and the like free, and sodium, potassium, ammonium, calcium ferric hydroxide, isopropylamine, triethylamine, 2-ethylamino And those formed from free carboxyl groups derived from ethanol, histidine, procaine and the like.

5.10. Gene therapy

Gene therapy refers to a therapy performed by administering a nucleic acid molecule to an individual. In an embodiment of the invention, the nucleic acid molecule produces a protein encoded therein, thereby making FRMS in vivo to mediate the therapeutic effect.

In certain embodiments, a nucleic acid consisting of sequences encoding one or more components that associate with each other to form the FRMS of the present invention is administered to create an immunological response to the FRMS through gene therapy. In a more particular embodiment, nucleic acids encoding viral envelope proteins, host cell receptors, host cell co-receptors or functional derivatives thereof of the envelope virus are administered via gene therapy. In another embodiment, the viral envelope protein of the enveloped virus is administered via gene therapy.

Any method for gene therapy that is available in the art is used in accordance with the present invention. An exemplary method is described below.

General methods for gene therapy are described in Goldspiel et al., 1993, Clinical Pharmacy 12: 488-505; Wu and Wu, 1991, Biotherapy 3: 87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32: 573-596; Mulligan, 1993, Science 260: 926-932; Morgan and Anderson, 1993, Ann. REV. Biochem. 62: 191-217; and May, 1993, TIBTECH 11: 155-215. Methods commonly known in the art for recombinant DNA techniques that can be used are Ausubel et al. (Eds.), 1993, Current Protocols in Molecular Biology, John Wiley & Sons, NY) and Kriegler, 1990, Gene Transfer and See Expression, A Laboratory Manual , Stockton Press, NY.

In certain suitable embodiments, the therapeutic agent consists of an HIV envelope protein, a human CD4 receptor, a co-receptor (eg, chemokine receptor CCR5 or CXCR4) in a suitable host. In another suitable embodiment, nucleic acid molecules can be used, wherein the HIV envelope protein coding sequence is carried to an individual to express the native CD4 and native co-receptor in the individual.

The delivery of nucleic acid to a patient is a direct method, i.e. exposing the nucleic acid or the vector with the nucleic acid to the patient, and indirectly, transfecting the cell with the nucleic acid in vitro and implanting it into the patient. do. These two methods are known as in vivo and ex vivo gene therapy.

In certain embodiments, the nucleic acid is administered in vivo to produce an encoded product. This can be accomplished by a variety of methods known in the art, for example, by constructing and administering an appropriate nucleic acid expression vector and infecting it with a defective or attenuated retrovirus or other viral vector to be intracellular ( US Pat. No. 4,980,286), direct injection of DNA only or microparticle release (e.g., gene gun; Biolistic, Dupont) or coated with lipid or cell-surface receptors or infectious agents or encapsulated in liposomes, microparticles, microcapsules Or in conjunction with a peptide known to be able to enter the nucleus or nucleus, or by prolonged administration to a ligand for receptor-mediated endocytosis (Wu and Wu, 1987, J. Biol. Chem. 262: 4429-4432). In this case, a specific cell type capable of expressing a receptor can be used as a target. In another embodiment, the nucleic acid-ligand complex is made so that the ligand consists of a fusionable viral product that destroys the endosome so that the nucleic acid is not destroyed by the lysosome. In another embodiment, the nucleic acid is targeted in vivo to allow the cell to be specifically incorporated, thereby targeting and expressing specific receptors (see, eg ., International Patent Publications WO 92/06180 by Wy et al. , WO 92/22635 by Wilson et al., WO 92/20316 by Findeis et al., WO 93/14188 by Clarke et al., And WO 93/20221 by Young. Alternatively, nucleic acids can be introduced into host cells for expression and bound using homologous recombination (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86: 8932-8935, Zijlstra et al., 1989, Nature). 342: 435-438).

In certain embodiments, viral vectors can be used that include viral envelope proteins, nucleic acids encoding host cell membrane proteins. Any viral vector described in paragraph 5.1.2 can be used for gene therapy purposes. For example, retroviral vectors may be used (see Miller et al., 1993, Meth. Enzymol. 217: 581-599). Such retroviral vectors are modified to cut out retroviral sequences that are not necessary to bind to the package of the viral genome and host cell DNA. Other documents describing the use of retroviral vectors in gene therapy include Clowes et al., 1994, j. clin. Invest. 93: 644-651, Kiem et al., 1994, Blood 83: 1467-1473, Salmons and Gunzberg, 1993, Human Gene Therapy 4: 129-141, and Grossman and Wilson, 1993, Curr. Opin. in Genetics and Devel. 3: 110-114.

As described in 5.1.2, adenoviruses are other viral vectors that can be used for gene therapy. Adenoviruses are particularly suitable vehicles for delivering genes to the respiratory epithelium. Other targets that may be used in adeno-based delivery systems include the liver, central nervous system, epithelial cells, and muscle. Adenoviruses have the advantage of infecting non-dividing cells. Kozarsky and Wilson, 1993, Current Opinion in Genetics and Development 3: 499-503 present a review of adenovirus-based gene therapy. Bout et al., 1994, Human Gene Therapy 5: 3-10 describe the use of adenovirus vectors to transfer genes to the respiratory epithelium of rhesus macaques. Other uses of adenoviruses in such gene therapy include Rosenfeld et al., 1991, Science 252: 431-434, Rosenfeld et al., 1992, Cell 68: 143-155, and Mastrangeli et al., 1993, J. Clin. Invest. 91: 225-234.

Adeno-associated virus (AAV) is recommended for gene therapy (Walsh et al., 1993, Proc. Soc. Exp. Biol. Med. 204: 289-300).

Other methods of gene therapy transfer genes to cells in tissue culture using methods such as electroporation, lipofection, calcium phosphate mediated transfection, viral infection. Any method known in the art for delivering genes to cells can be used in conjunction with the gene therapy of the invention, including the methods described in 5.1.2. Usually, the method of delivery involves delivering a selectable label to the cell. The cells then enter the screening process to isolate the expressed cells by taking the transferred genes. Such cells are provided to the patient.

In such embodiments, the nucleic acid is introduced into the cells prior to injection of the resulting recombinant cells in vivo . This includes, but is not limited to, transfection, electroporation, microinjection, infection with viral or bacteriophage vectors with nucleic acid sequences, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, and the like. Any method that is not limited can be used. Techniques for introducing foreign genes into cells are variously known in the art (see, eg ., Loeffler and Behr, 1993, Meth. Enzymol. 217: 599-618, Cohen et al., 1993, Meth. Enzymol. 217: 618-644, Cline, 1985, Pharmac. Ther. 29: 69-92), in accordance with the invention, can be used within the scope of not destroying the development and physiologically essential functions of the recipient cells. This technique must safely transport the nucleic acid to the cell, and the nucleic acid is expressed by the cell to be delivered to the posterior cell for expression.

The resulting recombinant cells can be delivered to the patient in a variety of ways known in the art. In suitable embodiments, it may be provided to epithelial cells. In another embodiment, the recombinant skin cells may be provided through skin transplantation to a patient. Recombinant blood cells (eg, hematopoietic liver or descendant cells) are preferably administered intravenously. Depending on the desired effect of the amount of cells used, the condition of the patient, this can be determined by one skilled in the art.

Cells into which nucleic acids are to be introduced for gene therapy include available cell types, including epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes, blood cells, T lymphocytes, B lymphocytes, and single cells. , Macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; Various liver or progenitor cells, particularly hematopoietic liver or progenitor cells, including but not limited to cells taken from bone marrow, cord blood, peripheral blood, fetal liver, and the like.

In suitable embodiments, the cells used in gene therapy are autografted in the patient.

In embodiments that use recombinant cells for gene therapy, nucleic acid molecules that encode components that associate to form FRMS are used. In certain embodiments, for example, the nucleic acid encodes an HIV viral envelope protein, human CD4, and a co-receptor (such a chemokine receptor CCR5 or CXCR4). The nucleic acid molecule encoding is introduced into the cell so that the gene is expressed in the cell or their descendants, and the recombinant cell is administered in vivo for the therapeutic effect. In certain embodiments, liver or progenitor cells may be used. Any liver or progenitor cells isolated and maintained in vitro can be used in accordance with embodiments of the present invention. Such hepatocytes include hematopoietic stem cells (HSC), epithelial tissue such as skin, lining of the intestine, embryonic heart muscle cells, liver hepatocytes (WO 94/0859), and neural stem cells (Stemple and Anderson, 1992, Cell 71). (973-985), but is not limited to such.

In certain embodiments, a nucleic acid introduced for gene therapy consists of an inducible promoter linked to a coding moiety (paragraph 5.1.2) so that expression of the nucleic acid can be controlled with or without an inducer that appropriately induces transcription.

Additional methods can be used to deliver the nucleic acid molecules encoding the components to make the FRMS of the present invention, which one of ordinary skill in the art would recognize, also belongs to the present invention.

Accordingly, the present invention provides a method for treating or preventing infection by a virus in a subject, comprising: (a) a first nucleic acid encoding the envelope protein of the envelope virus; And (b) a second nucleic acid encoding one or more cell membrane proteins, wherein the envelope of the virus and the cell membrane protein are necessary and sufficient conditions to fuse the envelope of the virus to the cell membrane comprising the cell membrane protein under appropriate conditions. The same envelope proteins and cell membrane proteins are expressed in the individual, producing neutralizing antibodies against the virus. In one embodiment, the first and second nucleic acids are identical. In other embodiments, the first and second nucleic acids are different nucleic acid vectors. In certain embodiments, the envelope protein is an envelope protein of HIV and the cell membrane protein is a CD4 and HIV co-receptor.

5.11. Dosing method

The present invention provides a method for treating and preventing viral infections and diseases by administering to a subject in need of treatment with a therapeutically effective amount of the therapeutic agent of the present invention. The subject includes, but is not limited to, animals such as monkeys, cows, pigs, horses, chickens, cats, dogs, preferably mammals most suitably humans. In certain embodiments, the subject is a person who is not infected with HIV.

Various delivery systems are known and are used to administer the therapeutic agents of the invention, including, for example, liposomes, microparticles, microcapsules, recombinant cells capable of expressing therapeutic agents, receptor-mediated endocytosis (Wu et al., 1987, J. Biol. Chem. 262: 4429-4432), construction of therapeutic nucleic acid as part of a retrovirus or other vector. Methods of introduction include, but are not limited to, transdermal, muscle, peritoneal, venous, subcutaneous, nasal, dural and oral routes. The compound may be administered via any conventional other route, for example by infusion or buccal injection or absorption through epithelial or mucosal linings (eg, oral mucosa, rectal and visceral mucosa), which are biologically It can also be administered with an active substance. Administration can be systemic or local. It is also possible to introduce a pharmaceutical composition of the invention comprising the antibody into the central nervous system by any suitable route, such as intraventricular or intrathecal injection, and injection into the ventricular catheter, ie a reservoir such as an Ommaya reservoir. It is carried out using the attached catheter. It can also be administered to the lung, in which case it can be carried out by using an inhaler or a nebulizer, prepared by aerosol material.

In certain embodiments, pharmaceutical compositions composed of antibodies of the invention may be administered topically to the treatment site. For example, injection, catheter use, suppositories, or transplantation may be used, but in the case of implantation, it may be a porous, non-porous, gelatinous material. In an embodiment, the pharmaceutical composition of the invention is administered to a wound of a human, the wound being a suspected part of HIV exposure.

In other embodiments, the therapeutic agent may be delivered via a vehicle, such as liposomes ( see , Langer, 1990, Science 249: 1527-1533; Treat et al., 1989 in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-). Berestein and Fidler (eds.), Less, New York, pp. 353-365; Lopez-Berestein, Science ., Pp. 317-327; see generally Science .

In another embodiment, the therapeutic agent can be delivered via a system in which release is controlled. In one embodiment, pumps may be used ( see Langer, supra ; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14: 201; Buchwald et al., 1980, Surgery 88: 507; Saudek et al., 1989, N. Engl. J. Med. 321: 574). In another embodiment, polymeric materials may be used ( see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance). , Somlen and Ball (eds.), Wiley, New York (1984); Ranger et al., 1983, J. Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al., 1989, J Neurosurg. 71: 105). In another embodiment, the release control system is placed in close proximity to the therapeutic target and only provides about one aliquot of the eczema dose ( see , eg ., Goodson, in Medical Applications of Controlled Release, 1984, supra , vol. 2). , pp. 115-138).

Langer mentions other emission control systems (Langer, 1990, Science 249: 1527-1533).

If desired, the composition is provided in a pack or dispensing device in one or more unit dosage forms containing the active ingredient. The pack consists of a metal or plastic foil such as a foam pack. The pack or dispensing device may be accompanied by instructions for administration. The compositions of the present invention formed in comparable carriers are prepared, placed in appropriate containers and labeled for the treatment of the disease.

5.1.2. Explain the usefulness of HIV

The present invention provides a test for testing the anti-HIV effect on HIV-FRMS. The present invention also monitors the effect of FRMS vaccines in patients or individuals by examining the antibodies in serum samples derived from the patients or individuals after vaccination. The therapeutic agents of the invention are tested in vitro and tested in vivo for the desired therapeutic or prophylactic activity before use in humans. In vitro or in vivo assays known herein to measure viral infection or production can be used to test the effectiveness of the therapeutic agents of the invention.

In an embodiment of the invention, a method is provided for screening a formulation consisting of mAbs for anti-HIV, wherein the assay consists of examining the formulation for its ability to inhibit HIV infection.

For example, to test a therapeutic in vitro , the effect of the therapeutic agent on HIV infection in cultured cells can be examined. Briefly, cultured hematopoietic cells (eg, primary PBMCs, isolated macrophages, isolated CD4 ⁺ T cells, or cultured H9 human T cells) can acutely infect cells in vitro and in vitro. Infect with HIV-1 using titers known to be present, such as 10 ⁵ TCID ₅₀ / ml. The appropriate amount of therapeutic agent is then added to the cell culture medium. Cultures are tested for HIV-1 production by measuring HIV antigen using ELISA assays at 3 and 10 days after HIV-1 infection. If the HIV antigen level is reduced by more than the level observed in the untreated criteria, then the treatment is effective in treating HIV infection. Alternatively, comparative testing can be performed before and after treatment with any commercially available HIV ELISA.

In addition, HIV-1 LTR induced in transcription can be tested to test the effectiveness of the therapeutic agents of the invention. In particular, a reporter gene such as a gene having a protein or RNA product that can be detected immediately, for example, a gene of chloramphenicol acetyl transferase (CAT), is cloned into the DNA plasmid structure, and transcription of the reporter gene is linked to the HIV-1 LTR promoter. Drive by. The resulting structure is introduced into cultured cell lines such as human CD4 ⁺ T cell line HUT 78 using transfection or any other method known in the art.

After exposing the transformed cells to the therapeutic agent, transcription in HIV-1 LTR is determined by measuring CAT activity using techniques known in the art. The reduced transcription driven by HIV-1 LTR indicates that the therapeutic agent is useful for the treatment or prevention of HIV infection.

Tests in animal models are described in 5.7.1.

The effectiveness of the therapeutic agents of the invention can be determined in SIV infected rhesus monkeys ( see , Letrin, NL, et al., 1990, J. AIDS 3: 1023-1040), particularly in rhesus monkeys infected with SIV _mac251 . , SIV strains can induce symptoms very similar to human AIDS in experimentally infected monkeys (Kestler, H., et al., 1990, Science 248: 1109-1112). In particular, monkeys are infected with acellular SIV _mac251 with a titer of 10 ^4.5 TCID ₅₀ / ml. Infection is monitored for the presence of SIV p27 antigen in PBMCs. The utility of therapeutic agents is characterized by gaining normal weight, decreasing SIV titers in PBMCs, and increasing in CD4 ⁺ T cells. Alternatively, the SHIV model can be used, in which HIV env proteins are constructed in the basic structure of SIV. In one embodiment, the monkey is immunized with a FRMS vaccine, ie a vaccine consisting of cells expressing HIV env and a cell expressing the CD4 and CCR5 receptors of the rhesus monkey. Once a suitable in vitro neutralization reaction is obtained, the animal is infected with an infectious SHIV virus containing neutralization-sensitive HIV env. In particular, monkeys are vaccinated with an immunogen consisting of one or more FRMS and challenged with SHIV 89.6P primary isolates ( see e.g., Montefiori et al., 1998, J Virol. 72: 3427-31).

Once the drug has been tested in vitro , and appropriately in animal models other than humans, the utility of the drug should be confirmed in human subjects. Therapeutic effects with therapeutic agents can be determined using various variables of HIV infection and HIV related diseases. In particular, changes in viral load can be determined using quantitative tests for serum HIV-1 RNA using quantitative RT-PCR (Van Gemen, B., et al., 1994, J. Virol. Methods 49: 157-168; Chen, YH, et al., 1992, AIDS 6: 533-539) or as a test for virus production from isolated PBMCs. Virus production in PBMCs can be determined by measuring HIV-1 titers using coculture of PBMCs and infected PBLs in an individual and ELISA on p24 antigen levels (see eg, Wrin, T., et al., 1995, Science 69:39; Popovic, M., et al., 1984 Science 204: 497-500). Therapeutic administration can be assessed by assessing changes in CD4 ⁺ T cell levels, body weight, HIV infection, or any other physical condition associated with AIDS or AIDS related complexes (ARCs). Reduction of HIV viral load or production, increase in CD4 ⁺ T cells, or elimination of HIV-related symptoms mean that it is useful to administer the therapeutic agent in the treatment or prevention of HIV infection.

5.13. Kit

The present invention relates to kits composed of fusion-capable complexes and antibodies issued by such complexes. Kits containing such complexes or antibodies can be used to analyze vaccines, run stability studies or display vaccine release. Antibodies and complexes can be used in diagnostic kits to detect the presence of antibodies or viruses in a host system. For example, a standard diagnostic enzyme-linked immunosorbent assay (ELISA) or competitive ELISA is performed using an antibody or complex as a substrate or reagent in a kit.

The present invention also provides a pharmaceutical pack or kit, which consists of one or more containers containing one or more components of the vaccine composition of the present invention. Such containers shall be labeled with the government's use of pharmaceuticals, pharmaceuticals or biological substances to regulate the manufacturer and the sale of such substances, and such labels shall be approved by the manufacturer or available for human use.

In one embodiment, the invention provides a kit comprising a labeled monoclonal antibody against a molecular structure in one or more containers, the molecular structure comprising (a) the envelope protein of the envelope virus, (b) one or more cell membrane proteins And the envelope protein and cell membrane protein are sufficient conditions for the viral envelope to be fused under appropriate conditions to the cell membrane including the cell membrane protein. In another embodiment, the present invention provides a kit comprising a molecular structure in a separate container, wherein the molecular structure is (a) an envelope protein of the envelope virus, (b) an epitope formed by associating one or more cell membrane proteins Wherein the envelope protein and cell membrane protein are sufficient conditions for the viral envelope to fuse under appropriate conditions to the cell membrane comprising the cell membrane protein.

In certain embodiments, the present invention provides a kit consisting of labeled monoclonal antibodies directed against a molecular structure in one or more containers, the molecular structure comprising (a) a mutation thereof combined with an HIV envelope protein or viral envelope; (b) co-receptors for human CD4 and HIV fusion are composed of epitopes formed in association. The present invention provides a kit consisting of labeled monoclonal antibodies directed against molecular structure in separate containers, wherein the molecular structure comprises (a) its mutations that are sampled into an HIV envelope protein or viral envelope; (b) co-receptors for human CD4 and HIV fusion are composed of epitopes formed in association.

In one embodiment, the present invention provides a kit comprising a molecular structure in one or more containers, wherein the molecular structure comprises (a) an envelope protein of the envelope virus, (b) an epitope formed by associating one or more cell membrane proteins. Wherein the envelope protein and cell membrane protein are sufficient conditions for the viral envelope to fuse under appropriate conditions to the cell membrane comprising the cell membrane protein. In suitable embodiments, the molecular structure is isolated. In another embodiment, the present invention provides a kit further comprising a pharmaceutically acceptable carrier, wherein the molecular structure is present in an immunological amount.

In another embodiment, the invention comprises (a) a first nucleic acid encoding the envelope protein of the envelope virus, (b) a second nucleic acid encoding the one or more cell membrane proteins in one or more containers, In this case, the envelope protein and the cell membrane protein are sufficient conditions for the viral envelope to be fused under the appropriate conditions to the cell membrane including the cell membrane protein, and the envelope protein and the cell membrane protein are expressed in the individual to produce neutralizing antibodies against the virus. .

6. Examples

The inventors of the present invention have made a surprising finding that the ability of the PI virus to neutralize is related to the provision of envelope proteins that function in active infections as compared to the lack of static proteins of the rgp 120 vaccine.

As described herein, HIV envelope proteins are assembled into a series of complexes by protein-protein interactions to make structural changes, resulting in the fusion of the virus and cell membranes, and infecting the cells. When bound to CD4, the envelope protein changes shape and interacts in succession with one of several co-receptor molecules, mainly CC chemokine receptor 5 (CCR5) or CXC chemokine receptor 4 (CXCR4) (Berger, EA, 1997, AIDS 11 (suppl A), S3; Moore, JP, et al., 1997, Current Opinions in Immunology 9: 551; Doranz, BJ, et al., 1997, Immunology Research 16:15). Without being limited to the mechanism, the interaction with the co-receptor induces a shape change in the envelope protein and exposes the transmembrane and hydrophobic fusion domains of the gp41 subunit, which mediates the fusion of side-by-side cell and viral membranes. Based on this model of HIV binding and invasion, HIV vaccine immunogens that bind to apparently functioning intermediate structures have been developed.

The examples described below illustrate the methods and compositions of the present invention.

Example 1: Neutralization of a wide range of primary isolates from HIV-FRMS immunogens

In one embodiment of the invention, the HIV vaccine immunogen is made to have a transient structure that occurs during HIV binding and fusion. In the transformed mouse immunization model, formaldehyde-fixed whole cell vaccines induced antibodies capable of neutralizing 23 infectivity of 24 primary HIV isolates isolated from various geopolitical locations and genetic clade A-E. The development of such fusion-dependent immunogens provides an HIV vaccine with clinically significant and broad effects.

As described herein, HIV envelope proteins aggregate complexes that have undergone protein-protein interactions and structural changes, causing virus and cell membranes to fuse, infecting cells. When bound to CD4, the envelope protein changes shape and interacts in succession with one of several co-receptors, mainly CC chemokine receptor 5 (CCR5) or CXC chemokine receptor 4 (CXCR4). in Berher, EA, 1997, AIDS 11 (suppl A), S3; Moore, JP, et al., 1997, Current Opinions in Immunology 9: 551; Doranz, BJ, et al., 1997, Immunology Research 16:15) . Interaction with the co-receptor induces a shape change in the envelope protein and in the exposure of the transmembrane and the hydrophobic fusion domain of the gp41 subunit, mediating the polymerization of side-by-side cell and viral membranes. This example describes a method of developing an HIV vaccine immunogen that explicitly binds to such a mediated structure.

One measure of envelope protein function is the ability to mediate cell-cell fusion. When cells expressing the envelope protein are cocultured with cells expressing CD4 and co-receptors, multinuclear coalescence is formed within 6-24 hours. For example, binding and fusion processes can be captured in the process by formaldehyde-crosslinking prior to significant coalescence-forming.

FRMS Preparation

In this study, functioning envelope proteins were derived from T-lymphocyte PI virus obtained from Amsterdam Cohort (ACH168.10; 168P). The molecular cloned envelope gene of ACH168.10 was identified in pCR3.1-Uni plasmid (Invitrogen) (Tersmette, M., et al., 1989, Journal of Virology 63: 2118; Wrin, T., et al., 1995) in PCR. , Science 69:39; LaCasse, RA, et al., 1998, Journal of Virology 72: 2491). Molecular cloned envelope proteins and parent coalescence-induced (SI) viruses utilize CCR5 and CXCR4 co-receptors.

Cos-7 cells are transfected to express the envelope protein (COS-env) and subsequently co-cultured with human U87 glioma cells expressing CD4 and CCR5 co-receptors (U87-CD4-CCR5). 168P (168P23) envelope gene is precipitated calcium phosphate, which functions as a well-known (20 ㎍ DNA / 10 ⁶ cells / culture dish 10㎝) COS-7 cells (Anerican Type Culture Collection, Manassus, VA) using the method (see eg ., Jordan, M., et al., 1996, Nucleic Acids Research 24: 596). Transiently expressed COS-7 cells were obtained after 2 days using 0.5 mM EDTA / phosphate buffer (PBS) and U87-CD4-CCR5 fusion partner (Hill, CM, et al., 1997, Journal of Virology 71 (6296) is prepared using 0.1 mM EDTA / PBS. Co-culture is started by mixing the two cell types (each 1.5 × 10 ⁶ cells) in a 10 cm culture dish. The time course of cell-cell fusion can be monitored under a microscope and by immunochemical staining (HIVIG) in side-by-side co-cultures (LaCasse, RA, et al., 1998, Science 72: 2491). Formaldehyde fixation for 4-5 hours generally yields co-cultures, with no apparent coalescence formation.

Capture of FRMS

To capture the detoxification intermediates during the binding and fusion process, the coculture is fixed in 0.2% formaldehyde after 5 hours when multinucleated cells appear. In particular, the cultures are fixed overnight at 4 ° C. with 0.2% formaldehyde / PBS, using techniques known in the art ( see eg ., Yamamoto, JK, et al., 1991, AIDS Research and Hunan Retroviruses). 7: 911; Verschoor, EJ, et al., 1995, Veterinary Immunology and Immunologypathology 46: 139). Cells were scraped off, washed twice with PBS, resuspended to a normal density of 3 × 10 ⁶ cells / 0.1 ml in PBS containing 10% DMSO, frozen at −80 ° C. for storage, and described below. It can also be used directly as an immunogen.

Immunization with FRMS Immunogen

Formaldehyde treated whole cell preparations are used as fusion-capable (FC) or FRMS immunogens. In order to test whether such immunogens induce neutralizing antibodies, it is necessary to limit the immune response against viruses and virus-derived epitopes. Otherwise, antibodies to CD4 and CCR5 can be produced that block self infectivity. Therefore, an animal model immunologically resistant to human CD4 and CCR5 components of the vaccine was used. Thus, immunogenicity studies are conducted with transformed mice expressing hu CD4 and hu CCR5 receptors.

CD4 targeted deletion and hu CD4 transformed mouse constructs were performed as described in Killeen, N., et al., 1993 EMBO Journal 12: 1547. The hu CCR5 transformed mouse design can be as follows; Molecular cloning of the 1.15 kb hu CCR5 cDNA into the assembled SalI site at exon 2 of the murine CD4 expression cassette (construct c in (Sawada, et al., 1994, Cell 77: 917). Enhancer, CD4 promoter, first (non-coding) exon, internally deficient intron 1 with removal of CD4 silence, and methods known in the art can be used to identify transformation basis with flow cytometry with mAb. Such animals are grown in hu CD4 expressing mice to give rise to descendants expressing hu CD4, hu CCR5, mouse CD4 Hu CD4, hu CCR5, mouse CD4 by flow cytometry using a Coulter EPICS ELITE flow cytometer. The following antibody reagents were screened: mice α-human CD4 / CyChrome (Pharmingen), α-human CCR5 MAB 180 (R & D Systems) + goat α-mouse Ig / FITC (Caltag), Rat α-mouse CD4 L3T4 / PE.

Transformed mice expressing hu CD4, hu CCR5, mice are immunized with FRMS immunogen or cell reference (co-culture with U87-CD4-CCR5 cells, or sham-transfected COS cells). The vaccine consists of formaldehyde-fixed whole cells (3x10 ⁶ cells / 0.1 ml) and the same amount of Ribi adjuvant (R-700; reconstituted with PBS, half the recommended amount), and in some experiments the initial immunization reaction was An adjuvant containing cell wall material (R-730). Mice were given a 0.05 ml vaccine at four subcutaneous sites. Booster immune response is performed every three weeks, and mice are allowed to grow for 10-28 days after immunization on the tail. Serum antibodies against gp120 can be quantified using gp120 ELISA by known methods ( see, Moore, J., et al., 1989, AIDS 3: 155).

Virus neutralization

Sensitivity to neutralization of homologous 168P virus by vaccine sera is determined using U87-CD4 cells expressing CCR5 or CXCR4 co-receptors. Such PI virus neutralization assays have been identified for standard neutralization assays in PBL cultures (LaCasse, RA, et al., 1998, Science 72: 2491; Follis, KE, et al., 1998, Journal of Virology 72 : 7603), can be determined by running in wells in the presence of mouse serum. All serums are inactivated by heat prior to use in the neutralization test.

The neutralization reaction test of homologous 168P virus using fusion-competence (FC) and fusion-incompetence (FI) vaccine sera was performed as follows. Transformed mice (hu CD4 +, hu CCR5 +, mouse CD4 +) were either FC immunogens (COS-env and U87-CD4-CCR5; □, n = 3 mice) or cells (U87-CD4-CCR5 cells alone or sham-transfect) Immunized with sensitized COS cells, co-cultured with ○, n = 3 mice). Only unimmunized mice were performed (Δ; n = 2 mice). Serum was tested for 168 P neutralization using U87-CD4 cells expressing CXCR4 (black) or CCR5 (white). The data represent the average of three to six extraneous neutralization assays using serum obtained two weeks after the second and third immune responses.

As can be seen in Figure 1, the results of the neutralization test showed that the infectivity in the serum of mice immunized on a cellular basis was not inhibited, which is transformed mice are resistant to hu CD4 and CCR5, other foreign cells Reactivity indicates that it does not interfere with the viral infectivity test (FIG. 1). Serum from mice immunized with FRMS immunogen neutralizes homologous 168PI virus. Neutralizing activity can be described as antibody-mediated, and such activity can be eluted from a solid support including Protein-A and Protein-G. In particular, serum is adsorbed to Protein-A Sepharose (sigma) and Protein-G agarose (Sigma) in turn at 4 ° C. Adsorption of antibodies can be confirmed using gp120 ELISA. Combined with the solid support, the antibody is eluted using 100 mM glycine pH 2.5. The eluate was neutralized and dialyzed using ultrafiltration using centrifugation (Microcon-100; Amicon). Neutralization in FC serum was> 99% in the PBL assay; In the U87-CD4-coR cell test, neutralization by FC serum was found to be 80-90%.

In sensitive PBL neutralization assays, the "incompetent-capable" vaccine sera are less timely than the "fusion-capable" vaccine sera, but to some extent inhibit the PI virus replication (see, LaCasse, 2000). RA, et al., 1999, Science 283: 357-62). This minimal effect is intermittently claimed in the literature on PI virus-neutralization with rgp120 vaccine sera (Devico, A, A Silver, et al., 1996 Virology 218: 258-63; Berman, PW, et al., 1998 AIDS Res & Hum Retrovirus 14 (suppl 3) S277-S89). The molecular basis based on the difference in neutralization sensitivity among PI viruses is not yet fully understood at this point and may or may not be correlated with the phylogenetic clade. Depending on the serotype, apparent crystal sites occur regardless of multiple clades. Thus, "fusion-capable" envs derived from clades other than B can be used to determine the prototype defining the "fusion-capable" neutralization serotype. Regardless of the total number of HIV serotypes, 80% of the tested PI viruses can be significantly neutralized (> 70%) using a single but properly provided Claude B env.

CXCR4 co-receptors can replace CCR5.

Neutralization of 168P virus by FC sera was observed independent of the co-receptors used in the U87-CD4 cell infection test (FIG. 1). Some reports generally explain that neutralization tests are independent of the use of specific co-receptors (LaCasse, R.A., et al., 1998, Science 72: 2491); Trkola, A. et al., 1998 Journal of Virology 72: 1876; Montefiori, D.C., et al., 1998, Journal of Virology 72: 3427; Follis, K.E., et al., 1998, Science 72: 7603).

The fact that neutralization with co-receptor CXCR4 not exposed to animals was observed here indicates that the neutralization did not directly target the CCR5 component of the vaccine. In addition, the CXCR4 co-receptor can be replaced with CCR5.

Immunogenic Immunity

The role of fusion-dependent crystals in inducing PI virus neutralization was examined. A fusion-competent (FI) immunogen expresses env but does not have cell-cell fusion. This includes COS-env co-cultured with U87 cells (CD4 or CCR5 co-receptor), COS-env co-cultured with U87-CD4 cells, and COS-env cells with soluble CD4 (sCD4). In particular, the cultures expressing the envelope are incubated with sCD4 (Berger, EA, et al., 1988, Proceedings of the National Academy of Sciences USA 85: 2357) (5 μg / ml; 1 hr at 37 ° C.) and continuous By washing to remove unbound sCD4. All FI immunogens are fixed with formaldehyde, as described above. Additional FI immunogens consist of COS-env and U87-CD4-CCR5 cells, which are separately fixed in formaldehyde prior to mixing during vaccine preparation.

In stark contrast to FC or FRMS immunogens, all FI immunogens did not induce neutralization of homologous PI virus (FIG. 2A). In particular, P168 is neutralized by FC but not by FI immunogen. As can be seen in FIG. 2A, the transformed mice contained FC immunogen (■; n = 4), FI immunogen (COS-env + U87 cells; gray circles; n = 4); COS-env + U87-CD4 cells; Gray diamonds; n = 3; COS-env + sCD4; White diamonds; n = 2; COS env + U87-CD4-CCR5 cells, each immobilized separately before mixing for immunization; Gray square, n = 2) or sham-transfected cos cell immunogen (co-cultured with U87-CD4-CCR5 cells, white circle, n = 2). Unimmunized mice (white triangle, n = 2) can be used. Neutralizing reactions are independent of specific co-receptor use, where data represent the mean of three to six neutralization assays in U87-CD4-CXCR4 or -CCR5 cells. In some cases, serum is collected from all animals within each experimental population. These results are in agreement with literature that has failed for the fgp120 vaccine to induce PI virus neutralization. Differences in neutralization with FC and FI vaccines can be observed in tests using human primary blood lymphocytes (PBLs) (FIG. 2B).

The neutralization of homologous 168PI viruses can be explained by FC immunogens rather than FI immunogens in human PBL cultures. As can be seen in Figure 2B, lymphocytes are isolated, stimulated with phytohemagglutinin and grown in the presence of interleukin-2. Neutralization reactions can be observed as described herein. HIV p24 antigen is determined after 5 days of incubation according to ELISA (Coulter Corporation) and the values are normalized to viral standards (36 ng / ml). * Indicates sub-limiting p24 antigen levels at dilution used in ELISA. The vaccine group was as defined in FIG. 2A and serum was collected for this test.

Specificity of FRMS Immunogen

To rule out the possibility that FC vaccine serum inhibits viral infectivity in a nonspecific manner, FC vaccine serum has not been shown to inhibit the infectivity of mock HIV virions with amphoteric murine leukemia virus (MLV) envelope protein (envelope). Defective HIV NL4-3-Luc-R ^- E ^- proviruses are simulated using amphoteric MLV envelope proteins (Deng, H., et al., 1996, Nature 381: 661). In addition, FC vaccine sera have not been shown to neutralize major isolates of monkey immunodeficiency virus SIVmac251. A major isolate of SIVmac251 produced from Rhesus monkey PBLs was used (Langlois, AL, et al., 1998, Journal of Virology 72: 6950).

As can be seen in FIG. 3, the FC vaccine did not neutralize the mock HIV virion with the amphoteric MLV envelope protein or the major SIVmac251. HIV with amphoteric MLV envelope protein (ampho MLV mockup) is used for neutralization response with FC and FI antiserum determined in U87-CD4-CXCR4 cells. Neutralization of the major isolate SIVmac251 was determined in U87-CD4-CCR5 cells. The symbols defined in FIG. 2 are as follows; FC immunogen (black squares); FI immunogen (Cos env + U87 cells, black circle).

Laboratory-Adapted Separator Neutralization

Like conventional rgp120 vaccines, FI vaccines were able to induce neutralization of associated laboratory-adapted HIV isolates, derivatives of T-cell line adapted 168P, and 168C (Wrin, T., et al., 1995, Science 69 La39, RA, et al., 1998, Science 72: 2491). Neutralization sensitivity of 168P PI virus and its TCLA derivative (168C) was examined in U87-CD4-CXCR4 cells. Vaccine groups and symbols are defined in Figure 2; Serum was collected for this analysis. The results presented in FIG. 4 suggest neutralization of TCLA 168C virus by FI vaccine serum. Neutralizing titers of the TCLA 168C virus were compared with FC or FI vaccine serum, like anti-gp120 antibody titers, with similar levels of inherent immunogenicity between vaccines.

In the transgenic mouse model, the FI vaccine does not induce PI virus neutralization, and thus the specificity of neutralization induced by the FC vaccine is more remarkable. Statistical comparisons were made based on data generated from all experimental animals and all virus neutralization assays. Using a simple model for virus-antibody binding, the 'coupling constant' K for each assay was calculated, and the average K value for each mouse was measured. Further experimentation revealed significant differences in mean neutralization between FC and FI immunogens (p <0.01). In all experiments, the response in the experimental animals was constantly constant.

In addition, failure to inhibit PI virus infectivity of FI vaccine sera strongly suggests that it does not target indeterminate human cell targets that have made initial studies of SIV vaccines inactivated in immune response (Cranage, MP, et al. , 1993, AIDS Research and Human Retroviruses 9:13; Putkonen, P., et al., 1993, Journal of Medical Primatology 22: 100; Arthur, LO, et al., 1992, Science 258: 1935). In contrast, the present invention recognizes that FC or FRMS immunogens present unique determinants that mediate neutralization of PI viruses.

Primary isolate neutralization by FRMS vaccine serum

An important issue in HIV vaccine development focuses on the ability of vaccine sera to neutralize a wide range of PI viruses. To determine the breadth of PI virus neutralization induced by FRMS immunogens, eg, FC immunogens, the susceptibility of representative PI viruses obtained from five widely spread and geographically diverse phylogenetic tissues was examined. Infectious Proviruses ACH320.2A.1.2 (320SI) and ACH320.2A.2.1 (320NSI) (Groenink, M., et al., 1991, Journal of Virology 65: 1968; Guillon, C., et al., 1995, AIDS Research and Human Retroviruses 11: 1537) were obtained from NIBSC (UK) (AIDS Reagent Program). HIV89.6 (Collman, R., et al., 1992, Journal of Virology 66: 7517), SHIV89.6, SHIV89.6P (Reimann, KA, et al., 1996, Journal of Virology 70: 3198) Was used. All other isolates were obtained through the NIH AIDS Research and Reference Reagent Program and the UNAIDS Network for HIV-1 Isolation and Characterization. PI virus has been limited to expanded in PHA-activated PBLs (Wrin, T., et al., 1995, Science 69:39).

As described in FIG. 5, FC serum induced with functional tissue B envelope protein was obtained from PI experimental virus-North America / Europe (tissue B), Africa (tissue A and D), Thailand (tissue B and E), India (tissue). 23 of 24 mononuclear / NSI and lymphocyte-affinity / SI viruses obtained in C) were neutralized. Vaccine groups and symbols are defined in Figure 2; Serum was collected for this analysis.

Despite the sequence diversity between these isolates, most showed similar susceptibility to neutralization by FC vaccine serum. One isolate (92RW008) did not achieve> 50% neutralization, and the other two isolates (93IN904 and 92UG024) had limited neutralization above 50%; This exception to a wide range of neutralization patterns strongly suggests that the FC immunogen of the present invention targets primary viral determinants.

FI serum did not neutralize these heterologous PI viruses. Extensive and uniform neutralization of various PI viruses by FRMS immunogens suggests that the critical sites presented by FRMS immunogens (eg FC immunogens) are highly conserved and closely related to the basic functions of the envelope in binding and fusion. do.

Neutralizing antibody

To more strictly define the molecular target in PI virus neutralization by FC vaccine, neutralizing antibodies were transferred from FC vaccine serum immediately after incubation with the envelope protein expressed on the surface of infected COS cells.

Therefore, formaldehyde-fixed COS cells expressing 168P envelope protein were incubated with FC serum and the collected serum was tested for PI virus neutralization. FC vaccine serum was taken up sequentially four times with approximately 10 ⁶ formaldehyde-fixed COS cells expressing the 168P envelope. Incubate at 4 ° C. for 1 hour with stirring. Controls include whole blood serum and formaldehyde-fixed, trans-transfected COS cells. The absorbance of the mixed anti-gp120 antibodies was monitored by gp120 ELISA. Final sera were tested for HIV 168P neutralization using U87-CD4-CXCR4 cells. Neutralizing activity in the FC vaccine was removed by incubation with the envelope-expressing cells, but only a small fraction when incubated with the COS cell control (FIGS. 6 and 7). In particular, FC vaccine serum was repeatedly cultured with formaldehyde-fixed COS-env or control COS cells, tested for residual neutralization of 168P using U87-CD4-CXCR4 cells. Serum obtained before FC immunization was similarly absorbed.

Production of neutralizing antibodies

In order to demonstrate that the method of the present invention is useful for the production of neutralizing antibodies, the method of the present invention made a hybridoma supernatant capable of neutralizing primary isolates (COS-env + U87-CD4-CCR5 as an immunogen). So). In brief, hybridomas were analyzed for virus neutralization in the U87-CD4-CXCR4 cell assay described above. Hybridoma supernatants were tested for their ability to neutralize two representative HIV tissue B isolates (ACH168.10 (168P) and 92US657). Neutralization is indicated by comparing the number of lesions in the presence of media alone with the number of lesions in the presence of hybridoma supernatant. As shown in Figure 8, most hybridomas neutralized both PI viruses by 60-80% (0.4-0.2 aliquot residual infectivity, respectively).

Thus, the FRMS immunogens of the present invention can induce neutralizing monoclonal antibodies against primary isolated viruses. Taken together these examples show that moderately-existing tissue B envelope protein can strongly neutralize most of the PI viruses obtained in multiple HIV tissues, which requires unlimited HIV antigen types for many vaccine defenses. Imply that you do not.

In addition, although the identity of the envelope proteins does not serve as an effective immunogen in the present invention, it is recognized that important fusion-related epitopes are sufficiently present in the identity proteins to allow binding. The FRMS immunogens of the invention used as vaccine compositions provide a wide range of protection against various viral antigenic types and tissues.

Example 2: Fusion-defective Mutants

Various mutants were tested in the method of the invention.

For HIV, three classes of envelope protein mutants include (1) mutants that have stopped proteolysis of the gp160 proprotein; (2) mutants affecting the N-terminal gp41 fusion peptide domain; (3) mutants that affect the twisted-helix domain are included, but are not limited to these.

Mutations that stop proteolysis of the gp160 proprotein include certain mutants with modifications of the highly conserved K / RXK / RR site at the gp120 C-terminus, in which case the gp160 glycoprotein is not proteolytic (Freed, EO, et al., 1989, J. Virol. 63: 4670-5; Guo HG, et al., 1990, Virology, 174: 217-24; Bosch V., et al., 1990, J. Virol. 1990: 2337-44; Dubay, JW, et al., 1995, J. Virol., 69: 4675-82).

Site-mutation induction (QuikChange, Stratagene) was used to introduce cleavage mutations in the 168P envelope proteins (REKR to REKT). The cleavage-defective 168P envelope protein gp160 was expressed on the cell surface but was unable to mediate cell-cell fusion or infection by the pseudotyped HIV virion.

Mutations that affect the N-terminal gp41 fusion peptide domain include mutations in the hydrophobic N-terminal region of gp41 that are thought to mediate fusion by inserting into the cell membrane and destabilizing the lipid bilayer. If a specific amino acid in the region is changed, the coat protein becomes a non-fusion-producing peptide.

The nature of the mutant in the N-terminal fusion peptide of gp41 is well known: V2E (Freed, EO, et al., 1992, Proc. Natl. Acad. Sci. USA 1992: 70-4; Pereira, FB, et al , 1997, AIDS Res. & Hum Retrovirus 13: 1203-11), G10V (Delahunty, MD, et al., 1996, Virology 218-94-102). The former has polar substitution in the hydrophobic peptide, while the latter affects the helix structure of the lipid bilayer. These mutants were introduced into the 168P envelope gene (gp4: A V GIGVLFL G FLG ..) by site-mutation and the envelope proteins tested for expression are fusion. These mutations can be integrated into pAbT4587 to easily produce the homologous vaccinia virus of the present invention.

Mutations that change the twisted-helix region include fusion proteins with many viruses, such as mutations in the highly conserved twisted-helix motif found in V570R and Y586E of HIV (Weissenhorn, W., et al., 1998 Proc. Natl. Acad. Sci. USA 95: 6032-6). In one embodiment, a change is introduced into the 168P envelope gene, the expression and fusionality is examined in a cell assay, and then transferred to pAbT4587-168Penv for production of homologous recombinant vaccinia virus.

Example 3: FRMS Preparation

Fusion-competent immunogens express molecularly cloned HIV 168P envelope proteins (from ACH168.10a complex-derived primary isolate using CCR5 and CXCR4) (LaCasse, RA, et al., 1998, Science 72: 2491). Calcium phosphate-transfected COS-7 (American Type Culture Collection, Rockville, MD) cells and the same number of cell fusion pairs were prepared by co-culture. Cell fusion pairs are U87-CD4 cells expressing CCR5 (U87-CD4-CCR5) (Dan Littman, Skirball Institute of Biomolecular Medicine, NYU Medical Center). The culture was terminated by fixing ice-cold 0.2% formaldehyde in phosphate-buffered saline at the beginning of cell-cell interaction (3-5 hours). Collection is performed at the time of capture of the transitional intermediate that induces cell-cell fusion. In preliminary and simultaneous immunostaining investigations, the maximum complex production at time of harvest was found to be 10-30%. Cells were fixed overnight and scraped off.

Example 4- Mice Immunization with FRMS

Transgenic mice expressing human CD4 and CCR5 co-receptors (Dan Littman, Skirball Institute of Biomolecular Medicine, NYU Medical Center) were immunized three times with cells prepared and harvested in Example 1 at 21-day intervals. Cells were prepared with the vaccine using Ribi adjuvant (Ribi ImmunoChem Research, Inc.) (2 × 10 ⁶ cells / 0.2 ml / mouse). Blood was collected from the tail vein. The obtained serum was used for neutralization assay of U87-CD4 cell line expressing CCR5 or CXCR4. This rapid PI virus neutralization assay was confirmed in comparison with standard neutralization assays in peripheral blood lymphocyte (PBL) cultures (LaCasse, RA, et al., 1998, Science 72: 2491), which can be effectively performed in the presence of mouse serum. have. In three separate experiments, the fusion-competent vaccine was administered to two mice. Virus polymerization assay using homologous PI virus 168P was performed using U87-CD4-CCR5 and -CXCR4 cells, and some sera were analyzed after the second and third immunizations. In all cases, the results are consistent, the mean data of which is shown in FIG. 9. Strong neutralization of homologous 168P PI virus was observed continuously; ≧ 80% neutralization levels were generally obtained in 1: 100 diluted mouse serum. Neutralization was observed using CXCR4 co-receptors where the animals were not exposed.

Several important control immunizations were performed. One fundamental control is to confirm that non-functional envelope protein immunogens do not induce PI virus neutralizing antibodies in the transgenic mouse vaccination model. In a particular study, a mouse was administered a vaccine consisting of 168P envelope / COS cells co-cultured with U87 cells that did not express CD4 and co-receptors. A total of three analyzes showed some neutralization of the 168P PI virus in two blood, but the titer and content differed significantly from that observed using the fusion-competent vaccine.

The morphological changes induced upon CD4 binding are thought to be important for later interaction with co-receptors. Thus, antibodies to these novel envelope proteins are useful for neutralizing PI viruses. In parallel with the envelope protein control vaccine, other mice received a control vaccine consisting of COS cells expressing 168P envelope co-cultured with U87-CD4 cells. When using a fusion-composite system, no composite formation was observed. CD4 addition does not seem to affect the minimal neutralization obtained with the envelope protein alone.

Residual neutralization generated from antibodies to CD4 and CCR5 may be resistant to incomplete transgenes. Thus, a control vaccine consisting of U87-CD4-CCR5 cells incubated alone or in combination with temporary-transfected COS cells was tested. In three studies, no inhibition of viral infection was observed. These data confirmed resistance in transgenic mice and the validity of the vaccination model. Specific immune responses appear to be limited to viruses and virus-derived epitopes. Although these mice are resistant to human CD4 and CCR5 as in human vaccines, antibodies to other cellular proteins do not appear to interfere with viral infection in these assays.

The possibility that the antibody response is partially mediated by DNA immunity was investigated. During transfection, the envelope-expressing cells were exposed to plasmid DNA in μg. To examine whether the animal responds to DNA immunity, 20 μg 168P23 plasmid (pCR3.1-Uni expression vector) was injected subcutaneously in two mice. Although the route of administration is not suitable for DNA immunity, it is similar to DNA immunization with transfected cell vaccines. DNA was not formalin-inactivated in this experiment. In all eight neutralization assays, no PI virus neutralization was observed at all immediately following DNA immunization.

Example 5: Neutralization of Additional PI with Antibody to FRMS

ACH320.2A.1.2 (320SI) (Amsterdam Cohort) is a molecular cloned T-lymphocyte-affinity PI virus, which is particularly resistant to neutralization by HIV-IG, CD4-Ig, IgG _1- b12. The residual serum remaining in Example 3 was used to examine the susceptibility of the PI virus to the fusion-complex immunogen and the fusion-non-complex immunogen. Although limited to a fusion-competent immunogen diluted 1: 100, this heterologous Tissue B isolate showed a clear sensitivity to neutralization. In contrast to the partial neutralization seen for homologous 168P virus, it was also interesting that the fusion-non-compound serum (env and env + CD4) did not neutralize the heterologous 320SI virus (FIG. 10).

An important issue in the development of HIV vaccines is the ability of vaccine antisera to neutralize a wide range of PI viruses. To determine the extent of PI virus neutralization induced by fusion-competent immunogens, the susceptibility of representative PI viruses obtained from five widely distributed and geographically diverse phylogenetic tissues was examined. Neutralization assay of U87-CD4-coceptor cells was the same as described in Example 1 above. Fusion-Compound Serum Induced by Functional Tissue B Envelope Proteins from PI Experimental Virus-North America / Europe (Tissue B), Africa (Tissues A and D), Thailand (Tissues B and E), India (Tissue C) 23 of 24 mononuclear / NSI and lymphocyte-affinity / SI viruses could be neutralized. Despite the sequence diversity between these isolates, most showed similar sensitivity to neutralization by fusion-competent vaccine sera. One isolate (92RW008) did not achieve> 50% neutralization and the other two isolates (93IN904 and 92UG024) had limited neutralization above 50%. Control serum did not evenly neutralize these heterologous PI viruses, which coincides with the failure of the rgp120 immunogen. Extensive and uniform neutralization of different PI viruses induced by a representative single tissue B envelope protein is highly conserved in the critical sites presented by the fusion-competent immunogen and is closely associated with the basic function of the envelope protein in binding and fusion. Imply that it is. These findings suggest that the number of different HIV neutralizing antigens required to protect the world from HIV infection should be limited.

Example 6: Preparation and Separation of Labeled FRMS

The FRMS of the present invention was labeled using a commercially available system to facilitate separation and purification. Since the molecular manipulation methods used to construct CD4, CCR5, CXCR4, HIV envelope labeled S-peptides are similar to each other, only CD4-Spep will be described in detail. The molecule labeled the S-peptide at the C-terminus of the CD4 molecule. The S peptide-encoded sequence (lys-glu-thr-ala-ala-ala-lys-phe-glu-arg-gln-his-met-asp-ser) is a synthetic oligonucleotide and high performance XL PCR (PE Applied Biosystems). Was inserted between the cytoplasmic C-terminal isoleucine and the stop codon (TGA) of the CD4 cDNA expression plasmid (CD4-Spep). Functional expression of the modified CD4 was confirmed by infection of COS-7 cells expressing CD4-Spep and CXCR4 co-receptors. C-terminally labeled CD4 could support infection with 168P virus compared to native CD4. S-peptide labeled CD4 was also easily isolated using S-protein affinity chromatography (Novagen, Inc.). Using a similar method, homologous S-peptide labeled CCR5 and CXCR4 co-receptors and HIV envelope molecules were constructed. All constructs appeared to act on binding and fusion. COS-7 cells were transfected with CXCR4 and CD4-Spep or native CD4 (in pcDNA3.1 expression vector), and after 2 days biotin (EZ-Link Sulfo-NHS-LC-Biotin; Pierce Chemical Co.) Was labeled. Lysates were made with protease inhibitors in 0.5% Triton X-100. CD4 molecules were purified by 1) immunoinfiltration with MAb T4 (Coulter Corp., Hialeah, FL) and rabbit-α-mouse Ig antibody-loaded Protein A agarose or 2) affinity purification with S-protein agarose. It was. The protein was boiled and released in sodium dodecyl sulfate (SDS) sample buffer, dissociated by 10% polyacrylamide-SDS gel electrophoresis and transferred to nitrocellulose. Biotin-treated proteins were detected with avidin-horseradish peroxidase (Pierce) and nickel-fortified DAB substrates. The 62 kD CD4-Spep was specifically defined with S-protein agarose to yield amounts and purity exceeding that of α-CD4-antibody mediated immunoinfiltration.

The time course of S-peptide separation after formalin crosslinking was examined. COS-7 cells were transfected with CD4-Spep (± CXCR4) and then harvested in 0.5% Triton X-100 lysis buffer (control) or subjected to ice-cooled formaldehyde fixation before lysis. CD4-Spep protein was purified by S-protein agarose and analyzed by Western blot using S-protein HRP (Novagen, Inc.). Minimal loss of S-peptide bonds (or recovery) was observed at 0.2% formaldehyde fixation for 1 hour; There was a slight loss of 2% formaldehyde fixation for more than 1 hour and 3 hours. Under intermediate reaction conditions (eg 0.2% formaldehyde, 1-2 hours), effective cross-linked complexes can be effectively separated.

In order to identify specific CD4-Spep complexes, experiments were conducted using formaldehyde fixation and the specific cross-linking drug Pierce Chemical Company (DTSSP). DTSSP is a homobifunctional N-hydroxysuccinimidyl ester that reacts with primary amines and aldehydes. DTSSPs are expected to capture cytoplasmic S-peptide labels as opposed to formaldehyde because they do not penetrate or pass through the cell membrane due to negative polarity. Unlike formaldehyde, DTSSP cross-linking is reversible; Internal disulfide bonds can cleave to release cross-linked components.

COS-7 cells were transfected with 168P envelope or CD4-Spep plasmid; Enveloped cells and cells transfected were metabolized in 10 cm dishes for 8 hours using 1 mCi total ³⁵ S-methionine and cysteine. Afterwards, the envelope (or sham) and CD4-Spep expressing cells were co-cultured for 4 hours for cell-cell interaction and cross-formaldehyde (0.2%, 1 hour on ice) or DTSSP (2 mM, 2 × 10 minutes on ice) Combined. Lysates were made and S-peptide bearing complexes were isolated by S-protein agarose affinity chromatography.

Formaldehyde cross-linked complexes were analyzed by 6% polyacrylamide SDS gel electrophoresis. High molecular weight polymers not found in the sham + CD4-Spep control were identified. In addition, viral proteins gp120 and gp160 can be seen, possibly separated via non-covalent association with CD4-Spep. Detection of S-protein affinity-purified (ie, CD4-Spep) and metabolic-labeled (eg, enveloped) high molecular weight complexes suggests that cross-linked complexes can be separated for further analysis.

To continue analyzing these high molecular weight complexes, the DTSSP cross-linked complexes were examined. S-protein affinity purified complexes were analyzed by 6% polyacrylamide / SCS gel electrophoresis with or without 50 mM DTT pretreatment. Envelope-specific complexes that were intact with DTSSP cross-linking were not clearly dissociated beyond complexes isolated from sham + CD4-Spep controls. gp120 / gp160 was rarely detected. However, when the DTSSP cross-linking is reversed, the gp120 and gp160 proteins are specifically released. These data demonstrate the envelope-CD4 complex formation observed using formaldehyde fixation, empowering the use of reversible cross-linked drugs.

These data support the use of CD4-Spep affinity labeling and chemical cross-linking methods in the isolation of fusion-active CD4-related complexes. S-peptide labeled co-receptors CCR5 (CCR5-Spep), CXCR4 (CXCR4-Spep), viral envelope (Env-Spep) can be produced and used to isolate fusion-active complexes. Separated complexes can be used to define the molecular structure involved in the progress of skin-mediated membrane fusion.

An investigation was conducted to determine whether the envelope protein could be co-purified with CD4-Spep or CCR5-Spep. Human 293T cells were transfected individually with 168 envelope protein and a) 168P CD4-Spep, b) CCR5-Spep and CD4 or c) sham. Envelope-expressing cells were co-cultured with the fusion pair (ac) for 5 hours, and the complexes were lysed using 1% Brij-97 cleaner (Lapham et al., 1996) and separated by S-protein affinity chromatography. . Co-purified coat proteins isolated using Brij-97 were detected by Western blot analysis using HIV V3-induced mAb 50.1 (FIG. 11). As expected, after co-culture with cells expressing CD4-Spep, large amounts of 168P coat protein were isolated. In addition, after co-culture with cells expressing CD4 and CCR5-Spep, a smaller but significant amount of envelope protein was isolated. Contaminated enveloped protein was not detected in co-culture with temporary transfected cells. In a similar experiment, the complexes were separated from formaldehyde cross-linked fusion-composite co-cultures.

These results strongly suggest that complexes of the envelope proteins, CD4, CCR5, are produced early in the fusion, which can be separated by S-protein affinity chromatography. These S-protein purified molecules and complexes can be used as "cleavage virus" or subunit vaccine immunogens.

Example 7: Recombinant vaccinia virus producing fusion-competent FRMS in cell culture or vaccination sites

Other acceptable vaccine methods for diverting the present invention to specific uses include the use of viral vectors. For example, recombinant vaccinia virus expressing envd and CD4 / CCR5, respectively, can be co-administered to induce in situ cell-cell fusion. In one embodiment, two recombinant vaccinia viruses (the first virus expresses HIV 168P envelope protein (rV-168Penv) and the second virus expresses CD4 and CCR5 co-receptors (rV-CD4 / CCR5)). Co-cultures of cells infected with rV-168env and cells infected with rV-CD4 / CCR5 are fusion-competent and produce multinuclear complexes. rV-168Penv cells, rV-CD4 / CCR5 cells, or cross-linked co-cultured rV-168Penv and rV-CD4 / CCR5 cells were used as FRMS for vaccination into transgenic mice as described above.

Serum obtained from mice vaccinated with cross-linked and co-cultured rV-168Penv and rV-CD4 / CCR5 cells can neutralize the virus in a variety of HIV tissues, which viral vectors have been successfully incorporated into the method for preparing FRMS of the present invention. Suggests that it can be used.

As those skilled in the art will recognize, all examples and embodiments disclosed herein are intended to illustrate the invention, and various modifications and variations thereof are intended to be included within the spirit of the invention and the appended claims.

Embodiments disclosed in this article do not limit the invention. Indeed, those skilled in the art will readily recognize various modifications of the invention from the foregoing specification and the accompanying drawings. Such modifications are included in the scope of the appended claims.

Various materials disclosed in this article, including patent applications, patent publications, and publications, are incorporated herein by reference.

Claims (106)

In a separate molecular structure (a) envelope proteins of envelope viruses; (b) consists of an epitope formed by the association of one or more cell membrane proteins, wherein the envelope protein and cell membrane protein are essential and capable of fusion under appropriate conditions with the cell membrane comprising the viral envelope and the cell membrane protein. Isolated molecular structure, characterized in that consisting of. In an isolated molecular structure, (a) mutant forms thereof that can be assembled into HIV envelope proteins or viral envelopes; (b) An isolated molecular structure, characterized in that it consists of epitopes formed in association with co-receptors for human CD4 and HIV fusion. The isolated molecular structure of claim 2, wherein the co-receptor is CCR5 or CXCR4. 3. The isolated molecular structure according to claim 2, wherein the HIV gp41 mutants lacking fusion ability are formed in association. The isolated molecular structure of claim 4, wherein the mutant comprises one or more mutants selected from V2E, G10V, V570R, Y576E. 3. The isolated molecular structure of claim 2, wherein the wild type HIV envelope protein is formed in association. In an isolated molecular structure, (a) the mutated coat protein of the enveloped virus, the coat protein in the wild type, functions in the fusion of the viral coat and the host cell membrane, and the mutated coat protein lacks the fusion ability; (b) An isolated molecular structure, consisting of epitopes formed by associating one or more host cell membrane proteins that function as receptors for envelope proteins. 8. The virus according to claim 1 or 7, wherein the virus is retroviridae, Rhabdoviridae, Caronaviridae, Filoviridae, Poxviridae, Bunyaviridae. , Flaviviridae (Flaviviridae), Togaviridae (Togaviridae), Ortomyxoviridae (Orthomyxoviridae), Paramyxoviridae (Paramyxoviridae), herpesviridae (Herpesviridae), characterized in that the molecular structure. 8. The isolated molecular structure according to claim 1 or 7, wherein the envelope protein is E1 and E2 of HCV and the cell membrane protein is CD81. 8. The isolated molecular structure according to claim 1 or 7, wherein the molecular structure is a cross-linked cell membrane molecular structure. The isolated molecular structure according to claim 2, wherein the molecular structure is a cross-linked cell membrane molecular structure. 8. The isolated molecular structure according to claim 1 or 7, which is separated from cell lysate. The isolated molecular structure of claim 2, wherein the molecular structure is separated from the cell lysate. 11. The molecular structure according to claim 10, wherein the cell molecular structure consists of cells expressing the coat protein by synthesizing. 12. The molecular structure according to claim 11, wherein the cell molecular structure consists of cells expressing the coat protein by synthetase. 13. The molecular structure according to claim 12, wherein the cell lysate is obtained from a plurality of cells expressing the envelope protein by synthesis. 15. The molecular structure of claim 14, wherein the cellular molecular structure includes cells that recombinantly express one or more host cell membrane proteins. In a recombinant enveloped virus, the recombinant enveloped virus is characterized in that the virus recombinantly expresses cellular receptors for the native envelope protein of the virus on the envelope. 19. The virus of claim 18, wherein the virus is HIV, the cell receptor is human CD4 or a co-receptor of HIV, and the virus recombinantly expresses both the human CD4 and the co-receptor of HIV. The molecular structure according to claim 1, wherein suitable conditions refer to lowering the pH. 12. The molecular structure of claim 11 wherein the crosslinked cellular molecular structure consists of crosslinked viral particles of a virus comprising envelope proteins. A vaccine composition comprising an immunogenic effect of the molecular structure according to claims 1 to 4, 6 and 8 and a pharmaceutically acceptable carrier. Monoclonal antibody against the molecular structure according to claim 1. Monoclonal antibody against the molecular structure according to claim 2. Purified polyclonal antiserum specific for the molecular structure according to claim 2. A contraceptive jelly, foam, cream or ointment comprising an effective amount of the antibody according to claim 23 effective to inhibit or reduce infection by a virus. A contraceptive jelly, foam, cream or ointment comprising an effective amount of the antibody according to claim 24 effective to inhibit or reduce infection by a virus. A contraceptive jelly, foam, cream or ointment comprising an effective amount of the antibody according to claim 25 effective to inhibit or reduce infection by a virus. A mammalian blood sample comprising an effective amount of an antibody according to claim 23 effective to inhibit or reduce infection by a virus. A mammalian blood sample comprising an effective amount of an antibody according to claim 24 effective to inhibit or reduce infection by a virus. 8. The molecular structure of claim 1 or 7, wherein the envelope protein or host cell membrane protein comprises an affinity tag. 3. The molecular structure of claim 2 wherein the envelope protein, CD4, co-receptor comprises an affinity tag. The antibody of claim 23, wherein the antibody is labeled. A kit comprising at least one container with a monoclonal antibody labeled with a molecular structure according to claim 1. A kit comprising at least one monoclonal antibody labeled with a molecular structure according to claim 2 in at least one container. 35. The kit according to claim 34, comprising the molecular structure according to claim 1 in a separate container. 36. The kit according to claim 35, comprising the molecular structure according to claim 2 in a separate container. In a cell line that recombinantly expresses a coat protein of a coat virus or a mutant form of a fusion-defected coat protein that functions upon fusion of a host cell membrane with a viral coat, the cell line functions as a receptor for the coat protein. A cell line characterized by expressing at least one cell membrane. The cell line of claim 38, wherein at least one protein that functions as a receptor is recombinantly expressed. A cell line that recombinantly expresses HIV gp160, wherein the cell line expresses the co-receptors of CD4 and HIV, and the cell line lacks a protease that functions to cleave gp160 to make gp120 and gp41. A method of treating and preventing infection by a virus in a subject, said method comprising administering to the subject an immunogenic effective amount of the molecular structure according to claim 1 effective in treating or preventing the infection by a virus. How to feature. A method for treating and preventing infection by a virus in a subject, the method comprising administering to the subject an immunogenic effective amount of the molecular structure according to claim 10 effective to treat or prevent infection by the virus. . A method for treating and preventing infection by a virus in a subject, the method comprising administering to the subject an immunogenic effective amount of the molecular structure according to claim 12 effective to treat or prevent infection by the virus. . A method of treating and preventing infection by HIV in humans, the method comprising administering to a human an immunogenic effective amount of the molecular structure according to claim 2 effective to treat or prevent infection by HIV. . A method of treating and preventing infection by HIV in humans, comprising: administering to a human an immunogenic effective amount of the molecular structure according to any one of claims 3 to 6 that is effective for treating or preventing infection by HIV. How to feature. The method of claim 41, wherein the subject is a human. The method of claim 41, wherein the subject is a livestock. A method of treating and preventing infection by a virus in a subject, the method comprising administering to the subject an effective amount of a monoclonal antibody according to claim 23 effective to treat or prevent infection by the virus. A method of treating and preventing infection with HIV in humans, the method comprising administering to the subject an effective amount of a monoclonal antibody according to claim 24 effective to treat or prevent infection with HIV. The method of claim 49, wherein the human is at high risk for HIV infection. A method of treating and preventing HIV infection in a human fetus, the method comprising administering to a pregnant person an effective amount of a monoclonal antibody according to claim 24 effective to treat or prevent infection with HIV in a fetus. How to. The method of claim 49, wherein the method is for treating AIDS in a human. A method of inhibiting infection by a virus in a blood sample, the method comprising contacting the blood sample with an effective amount of a monoclonal antibody according to claim 23 effective to inhibit infection by a virus. A method of inhibiting infection by a virus in a blood sample, the method comprising contacting the blood sample with an effective amount of a monoclonal antibody according to claim 24 effective to inhibit infection by a virus. A method for preventing contamination of a surgical or dental tool, the method comprising contacting the tool with an effective amount of the monoclonal antibody according to claim 23 effective to inhibit infection by a virus. A method for the prevention of contamination of a surgical or dental tool, the method comprising contacting the tool with an effective amount of the monoclonal antibody according to claim 24 effective to inhibit infection by HIV. A method for monitoring whether an antibody against the molecular structure according to claim 1 or 7 is produced in a subject previously administered an effective amount of the molecular structure according to claim 1, wherein the sample consisting of serum is collected. And detecting whether an antibody against the molecular structure of claim 1 is present in the serum. 58. The method of claim 57, wherein the detection is performed using a competitive immunoassay with a labeled antibody according to claim 1 or 7. A method of producing an immunogen that can be used as a vaccine to treat or prevent infection by a virus; (a) contacting the envelope protein of the envelope virus or its chimeric type with the cell membrane; Wherein the envelope protein is brought into contact with one or more proteins or chimeric forms thereof which function to fuse the viral envelope, or which function as mutants of the envelope protein lacking the fusion function or as chimeric and receptors of the envelope protein; And (b) exposing the envelope protein or chimeric type thereof or mutant thereof or chimeric type thereof, one or more host cell types or chimeric types thereof to cross-binding agents; And (c) isolating a cross-linked structure composed of the envelope protein or chimeric type thereof or mutant thereof or chimeric type thereof. 60. The method of claim 59, wherein the virus is HIV and the host cell protein is a co-receptor for CD4 and HIV. In the method for producing an immunogen that can be used as a vaccine for treating or preventing infection by HIV, (a) a mutant or chimeric type thereof, or a chimeric type thereof lacking a fusion ability of a first cell or fusion ability capable of synthetically expressing an HIV envelope protein or chimeric type thereof, and (i) a human CD4 or chimeric type thereof (ii) Co-culture with second cells capable of expressing a co-receptor for HIV or a chimeric type thereof; And (b) exposing the coat protein or chimeric type thereof or mutant thereof or mykeratype thereof, CD4 or chimeric type thereof and co-receptor for HIV or chimeric type thereof to a crosslinker; And (c) isolating a cross-linked structure consisting of an envelope protein or a chimeric type thereof or a mutant or chimeric type thereof. 62. The method of claim 61, wherein the second cell is capable of recombinantly expressing CD4 or a co-receptor, both CD4 and a co-receptor or chimeric forms thereof. 62. The method of claim 61, wherein the first and second cells are the same cell type. 62. The method of claim 61, wherein the first and second cells are different cell types. 60. The method of claim 59, wherein in step (a), the envelope protein or chimeric type thereof or mutant thereof or chimeric type thereof is present in the virus particle or particle similar to the virus. 62. The method of claim 61, wherein the crosslinked structure is a crosslinked cell complex. 60. The virus of claim 59, wherein the virus is Retroviridae, Rhabdoviridae, Caronaviridae, Filoviridae, Poxviridae, Bunyaviridae, Flaviviridae (Flaviviridae), Togaviridae, Orthomyxoviridae, Paramyxoviridae, Herpesviridae. 66. The method of claim 65, wherein the contacting step is performed by infecting a cell expressing the host cell protein with a virus expressing the envelope protein or chimeric type thereof or mutant thereof or chimeric type thereof. 60. The method of claim 59, wherein one of the chimeric or host cell proteins of the envelope protein is contacted and the chimeric type consists of an affinity tag. 62. The method of claim 61, wherein the chimeric type or CD4 or co-receptor of the envelope protein is contacted and the chimeric type consists of an affinity tag. In the method for producing an immunogen that can be used in a vaccine for treating or preventing infection by HIV, (a) a mutant or chimeric type thereof, or a chimeric type thereof lacking a fusion ability of a first cell or fusion ability capable of synthetically expressing an HIV envelope protein or chimeric type thereof, and (i) a human CD4 or chimeric type thereof (ii) Co-culture with second cells capable of expressing a co-receptor for HIV or a chimeric type thereof; Wherein at least one of the chimeric types consisting of affinity tags is expressed; And (b) hemolytically co-cultured cells to obtain cell lysates under undenatured conditions; And (c) by using a method of contacting the cell lysate with the binding partner for the affinity tag to recover the molecular structure bound to the affinity tag, from the cell lysate the envelope protein or its chimeric type or a mutant or its mutant. Separating the molecular structure consisting of chimeric forms. A cross-linked structure produced by the method according to claim 59. A cross-linked structure produced by the method according to claim 60. A cross-linked structure produced by the method according to claim 61. A cross-linked structure produced by the method according to claim 62. A monoclonal antibody against the structure according to claim 60, wherein the primary isolate of HIV, ie, 92US657, 92US660, 92RW023, 92IN101, 92UG035, 92TH023, can be neutralized in vitro. A contraceptive jelly, foam, cream or ointment, comprising an effective amount of the antibody according to claim 76 to inhibit or reduce infection by HIV. A method of treating or preventing a viral infection in a subject, the method comprising administering to the subject an immunogenic effective amount of the structure of claim 72 that is effective for treating or preventing the viral infection. A method of treating or preventing infection with HIV in a human, the method comprising administering to the human an immunogenic effective amount of the structure of claim 73 effective to treat or prevent infection with HIV. A method of treating or preventing infection with HIV in a human, the method comprising administering to a human an immunogenic effective amount of the structure of claim 74 effective to treat or prevent infection with HIV. 76. A method of preventing contamination of a surgical or dental tool, the method comprising contacting the tool with an effective amount of the monoclonal antibody of claim 76 effective to inhibit infection by a virus. A method of preventing contamination of a surgical or dental tool, the method comprising contacting the tool with an effective amount of the monoclonal antibody of claim 23 effective to inhibit infection by a virus. A method for screening molecular structures with vaccine potency, wherein the molecular structure according to claim 2 is immunized with a non-human transgenic mammal, wherein the non-transgenic mammal is human CD4 and HIV from one or more transgenes. Expressing co-receptors of; And And detecting any neutralizing antibody against HIV produced by the mammal. A method for screening molecular structures with vaccine efficacy, comprising immunizing a non-human transgenic mammal with the molecular structure according to claim 1 wherein the non-transgenic mammal is one or more hosts from one or more transgenes. Express cell membrane proteins; And And detecting any neutralizing antibody against HIV produced by the mammal. 84. The method of claim 83, wherein the non-human mammal is a mouse. 62. The method of claim 61, wherein the first cell is capable of recombinantly expressing an HIV envelope protein or chimeric type thereof. Kit comprising the molecular structure according to claim 1 in at least one container. 88. The kit of claim 87, wherein the container comprises a pharmaceutically acceptable carrier and the molecular structure is present in an immunological amount. An isolated protein complex comprising a coat protein of human immunodeficiency virus type I capable of functionally interacting with human CD4 and human CCR5. 90. A vaccine composition comprising a protein complex according to claim 89 and a pharmaceutically acceptable carrier. A method of immunizing an animal against a virus, comprising a protein complex consisting of one or more host cell receptors or one or more viral proteins capable of functional interaction with a co-receptor that mediates viral binding, viral invasion and infection 90. A vaccine composition according to claim 90 is administered to an animal; Wherein the neutralizing antibody to the virus is generated. A method of preparing a protein complex consisting of one or more host cell receptors or one or more viral proteins capable of functional interaction with a co-receptor that mediates viral binding, chipping or infection, a) culturing a first cell expressing one or more viral proteins; b) culturing a second cell capable of expressing a co-receptor or one or more host cell receptors for one or more viral proteins; c) co-culture of the first and second cells; d) immobilizing the co-culture during cell-cell fusion; And e) separating the immobilized cells. 93. A fixed cell made according to the method of claim 92. A method of purifying a protein complex composed of an envelope protein of human immunodeficiency virus type I capable of functional interaction with human CD4 and human CCR5, a) attaching the peptide sequence to the complex for purification; b) separating the sequenced complexes. A method of treating or preventing a viral infection in a subject, comprising: (a) a first nucleic acid encoding the envelope protein of the enveloped virus and (b) a second nucleic acid encoding one or more cell membrane proteins To administer; At this time, the envelope protein and the cell membrane protein are sufficient conditions for fusing the envelope of the virus and the cell membrane including the cell membrane protein under appropriate conditions. Characterized in that the method. 97. The method of claim 95, wherein the first and second nucleic acids are identical. 97. The method of claim 95, wherein the first and second nucleic acids are different nucleic acid vectors. 97. The method of claim 95, wherein the envelope protein is an envelope protein of HIV and the cell membrane protein is a CD4 and HIV co-receptor. In the vaccine composition, (a) a first nucleic acid encoding the envelope protein of the envelope virus; (b) a second nucleic acid encoding one or more cell membrane proteins, wherein the envelope protein and cell membrane protein are sufficient conditions for the viral envelope to fuse under appropriate conditions to the cell membrane comprising the cell membrane protein, Proteins and cell membrane proteins are expressed in individuals to produce neutralizing antibodies to viruses; (c) a vaccine composition comprising a pharmaceutically acceptable carrier. A method for treating a host exposed to a virus or preventing a host from being infected by a virus, the method comprising: an antibody produced by immunizing an animal with an effective amount of the protein complex according to claim 89 to prevent or treat infection of the host And administering to the host. In an isolated protein complex, it consists of a human immunodeficiency virus type I envelope protein capable of functional interaction with human CD4 and human CCR5, wherein at least one of the envelope proteins CD4 or CCR5 is tagged with a peptide. Isolated protein complexes. (a) a first nucleic acid encoding the envelope protein of the envelope virus; (b) a second nucleic acid encoding one or more cell membrane proteins is contained in one or more containers, wherein the envelope protein and cell membrane protein are fused to the cell membrane where the envelope of the virus comprises the cell membrane protein under appropriate conditions. The kit is characterized in that sufficient conditions are required, and the envelope protein and cell membrane protein are expressed in a host to produce a neutralizing antibody against the virus. (a) the monoclonal antibody according to claim 23; (b) envelope proteins of envelope viruses; And (c) a composition comprising a pharmaceutically acceptable carrier. A method of treating or preventing infection by a virus in a subject, the method comprising administering to the subject an effective amount of the composition of claim 103 effective to treat or prevent infection by the virus. 93. The method of claim 92, wherein the vaccinia virus vector is used to express one or more host cell receptors or co-receptors in the second cell. 93. The method of claim 92, wherein the vaccinia virus vector is used to express one or more host cell receptors or co-receptors in the first cell.