| PATENT ASSIGNEE'S COUNTRY | USA |
| UPDATE | 06.00 |
| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | 13.06.00 |
| PATENT TITLE |
Immuno-reactive peptide CTL epitopes of human cytomegalovirus |
| PATENT ABSTRACT |
The invention provides a plurality of peptides (and immunologically functional variants thereof) which are immunogenic epitopes recognized by CD8.sup.+ class I MHC restricted cytotoxic T-lymphocytes of patients harboring latent cytomegalovirus (HCMV) infection. The peptides are capable of activating CTLs and CTLp's in the absence of active viral replication, and thus are useful for eliciting a cellular immune response against HCMV by normal and immunodeficient subjects. Polypeptide and lipopeptide vaccines, with and without adjuvants, also are disclosed. |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | 11.05.98 |
| PATENT REFERENCES CITED |
Rammensee et al. , "Immunogenetics", 41(4): 178-228 (1995). Livingston et al., J. Immunol. 159: 1383-1392 (1997). Quinnan et al., "HLA-Restricted T-Lymphocyte and Non-T-Lymphocyte Cytotoxic Responses Correlate with Recovery from Cytomegalovirus Infection in Bone-Marrow-Transplant Recipients", The New England Journal of Medicine, Cytomegalovirus Infection 307(1): 7-13 (1982). Borysiewicz et al., "Human Cytomegalovirus-Specific Cytotoxic T Lymphocytes: Requirements for in vitro Generation and Specificity", Eur. J. Immunol. 13:804-809 (1983). Clark et al., "Isolation and Partial Chemical Characterization of a 64,000-Dalton Glycoprotein of Human Cytomegalovirus", Journal of Virology, Notes 49(1):279-282 (1984). Forman et al., "A 64,000 Dalton Matrix Protein of Human Cytomegalovirus Induces In Vitro Immune Responses Similar to Those of Whole Viral Antigen", The Journal of Immunology, 134(5):3391-3395 (1985). Meyers et al., "Risk Factors for Cytomegalovirus Infection After Human Marrow Transplantation", The Journal of Infectious Diseases 153(3):478-488 (1986). Miller et al., "Retrovirus-Mediated Gene Transfer into Human Skin Fibroblasts", Mar. 1988 Meeting at Cold Spring Harbor. Borysiewicz et al., "Relative Frequency of Stage-Specific CTL Recognizing the 72-kD Immediate Early Protein and Glycoprotein B Expressed by Recombinant Vaccinia Viruses", J. Exp. Med. 168:919-931 (1988). Miller et al., "Design of Retrovirus Vectors for Transfer and Expression of the Human .beta.-Globin Gene", Jounal of Virology 62(11):4337-4345 (1988). Townsend et al., "Recognition of Influenza Virus Proteins by Cytotoxic T Lymphocytes", Phil. Trans. R. Soc. Lond. B 323:527-533 (1989). Gelinas et al., "Regulated Expression of the Human .beta.-Globin Gene After Retroviral Transfer into Murine and Human Hematopoietic Cells", Hemoglobin Switching, Part B: Cellular and Molecular Mechanisms, pp. 235-249 (1989). Pande et al., "Structural Analysis of a 64-kDa Major Structural Protein of Human Cytomegalovirus (Towne): Identification of a Phosphorylation Site and Comparison to pp65 of HCMV (AD169)", Virology 178:6-14 (1990). Falk et al., "Allele-Specific Motifs Revealed by Sequencing of Self-Peptides Eluted from MHC Molecules", Nature 351:290-296 (1991). Schmidt et al., "A Randomized, Controlled Trial of Prophylactic Ganciclovir for Cytomegalovirus Pulmonary Infection in Recipients of Allogeneic Bone Marrow Transplants", The New England Journal of Medicine, 324(15):1005-1011 (1991). Del Val et al., "Efficient Processing of an Antigenic Sequence for Presentation by MHC Class I Molecules Depends on Its Neighboring Residues in the Protein", Cell 68:1145-1153 (1991). Penna et al., "Cytotoxic T Lymphocytes Recognize an HLA-A2-Restricted Epitope Within the Hepatitis B Virus Nucleocapsid Antigen", J. Exp. Med. 174:1565-1570 (1991). Goodrich et al., "Early Treatment with Ganciclovir to Prevent Cytomegalovirus Disease After Allogenei Bone Marrow Transplantation", The New England Journal of Medicine 325(23):1601-1607 (1991). Reusser et al., "Cytotoxic T-Lymphocyte Response to Cytomegalovirus After Human Allogeneic Bone Marrow Transplantation: Pattern of Recovery and Correlation With Cytomegalovirus Infection and Disease", Blood 78(5):1373-1380 (1981). Riddell et al., "Restoration of Viral Immunity in Immunodeficient Humans by the Adoptive Transfer of T Cell Clones", Science 257:238-241 (1992). Missale et al., "HLA-A31 and HLA-Aw68-Restricted Cytotoxic T Cell Responses to a Single Hepatitis B Virus Nucleocapsid Epitope During Acute Viral Hepatitis", J. Exp. Med. 177:751-762 (1993). Gilbert et al., "Selective Interference with Class I Major Histocompatibility Complex Presentation of the Major Immediate-Early Protein Following Infection with Human Cytomegalovirus", Journal of Virology 67(6):3461-3469 (1993). Bertoletti et al., "Definition of a Minimal Optimal Cytotoxic T-Cell Epitope within the Hepatitis B Virus Nucleocapside Protein", Journal of Virology 67(4):2376-2380 (1993). Kast et al., "Human Leukocyte Antigen-A2.1 Restricted Candidate Cytotoxic T Lymphocyte Epitopes of Human Papillomavirus Type 16 E6 and E7 Proteins Identified by Using the Processing-Defective Human Cell Line T2", Journal of Immunotherapy 14:115-120 (1993). Ralston et al., "Characterization of Hepatitis C Virus Envelope Glycoprotein Complexes Expressed by Recombinant Vaccinia Viruses", Journal of Virology 67(11):6753-6761 (1993). Goodrich et al., "Ganciclovir Prophylaxis To Prevent Cytomegalovirus Disease after Allogeneic Marrow Transplant", Annals of Internal Medicine 118:173-178 (1993). Winston et al., "Ganciclovir Prophylaxis of Cytomegalovirus Infection and Disease in Allogeneic Bone Marrow Transplant Recipients", Annals of Internal Medicine 118:179-184 (1993). Riddell et al., "Therapeutic Reconstitution of Human Viral Immunity by Adoptive Transfer of Cytotoxic T Lymphocyte Clones", Current Topics in Microbiology and Immunology 189:9-34 (1994). Li et al., "Recovery of HLA-Restricted Cytomegalovirus (CMV)-Specific T-Cell Responses After Allogeneic Bone Marrow Transplant: Correlation with CMV Diseas and Effect of Ganciclovir Proophylaxis", Blood 83(7):1971-1979 (1994). Johnson et al., "Induction of a Major Histocompatibility Complex Class I-Restricted Cytotoxic T-Lymphocyte Response to a Highly Conserved Region of Human Immunodeficiency Virus Type 1 (HIV-1) gp 120 in Seronegative Humans Immunized with a Candidate HIV-1 Vaccine", Journal of Virology 68(5):3145-3153 (1994). Speir et al., "Potential Role of Human Cytomegalovirus and p53 Interaction in Coronary Restenosis", Science 265:391-394 (1994). McLaughlin-Taylor et al., "Identification of the Major Late Human Cytomegalovirus Matrix Protein pp65 as a Target Antigen for CD8+ Virus-Specific Cytotoxic T Lymphocytes", Journal of Medical Virology 43:103-110 (1994). Vitiello et al., "Development of a Lipopeptide-based Therapeutic Vaccine to Treat Chronic HBV Infection", J. Clin. Invest. 95:341-349 (1995). Walter et al., "Reconstitution of Cellular Immunity Against Cytomegalovirus in Recipients of Allogeneic Bone Marrow by Transfer of T-Cell Clones from the Donor", The New England Journal of Medicine 333(16):1038-1044 (1995). Drijfhout et al., "Detailed Motifs for Peptide Binding to HLA-A* 0201 Derived from Large Random Sets of Peptides Using a Cellular Binding Assay", Human Immunology 43:1-12 (1995). D'Amaro et al., "A Computer Program for Predicting Possible Cytotoxic T Lymphocyte Epitopes Based on HLA Class I Peptide-Binding Motifs", Human Immunology 43:13-18 (1995). Rassmussen, "Immune Response to Human Cytomegalovirus Infection", Current Topics in Microbiology and Immunology 154:222-254 (1990). Zhou et al., "Association Between Prior Cytomegalovirus Infection and the Risk of Restenosis After Coronary Atherectomy", The New England Journal of Medicine 335(9):624-630 (1996). Moss, "Vaccinia Virus Vectors", Construction of Recombinant Viruses, Chapter 15, pp. 345-362. Pande et al., "Direct DNA Immunization of Mice with Plasmid DNA Encoding the Tegument Protein pp65 (ppUL83) of Human Cytomegalovirus Induces High Levels of Circulating Antibody to the Encoded Protein", Scand J Infect Dis Suppl 99, (Sweden) 1995 p 117-120. Khattab, B. A., et al.: "Three T-Cell Epitopes Within the C-Terminal 265 Amino Acids of the Matrix Protein pp65 of Human Cytomegalovirus Recognized by Human Lymphocytes," Journal of Medical Virology, 52:68-76 (1997). Ogg, G. H., et al. "HLA-peptide tetrameric complexes," Immunology, 10:393-396 (1998). Pande, H., et al.: "Human Cytomegalovirus Strain Towne pp65 Gene: Nucleotide Sequence and Expression in Escherichia coli," Virology, 182: 220-228 (1991). Parker, Kenneth C., et al: "Scheme for Ranking Potential HLA-A2 Binding Peptides Based on Independent Binding of Individual Peptide Side-Chains," Journal of Immunology, 152: 163-175 (1994). Wills, MR et al: "The human CTL response to Cytomegalovirus is dominated by structural protein," J. Virology, 70(11): 7569-7579, ( 1996). Tsunoda, T. et al: "Seriologically identically HLA B35 alleles which do not cross-present minimal cytotoxic epitopes to CD8+ CTL," J. Cell. Biochem., vol. Suppl.0(19A): 298 (1995) Abstr. No. J2-218. Diamond, DJ, et al: "Development of a candidate HLA A* 0201 restricted peptide based vaccine against HCMV infection," Blood, 90(5): 1751-1767 (1997). Deres, K. et al: "In vivo priming of virus-specific cytotoxic T lymphocytes with synthetic lipopeptide vaccine," Nature, 342: 561-564 (1989). Gonczol E. et al.: "Preclinical evaluation of an ALVAC (canarypox)-human cytomegalovirus glycoprotein B vaccine candidate," Vaccine, 13(12): 1080-1085 (1995). Primary Examiner: Mosher; Mary E. Assistant Examiner: Salimi; Ali R. Attorney, Agent or Firm: Rothwell, Figg, Ernst & Kurz -------------------------------------------------------------------------------- |
| PATENT GOVERNMENT INTERESTS |
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH This invention was made with government support in the form of grant no. CA30206 from the United States Department of Health and Human Services, National Cancer Institute. The government may have certain rights in the invention. |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
We claim: 1. A vaccine against human cytomegalovirus which elicits an MHC Class I cellular immune response to human cytomeaglovirus comprising a peptide selected from the group consisting of NX.sub.1 VPMVATX.sub.2, wherein X.sub.1 is L, I, M, T, or V and X.sub.2 is V, A, C, I, L or T (SEQ ID NO: 2) with the proviso that the peptide is not NLVPMVATV (SEQ ID NO:1); YXEHPTFSQY, wherein X is S, T or L (SEQ ID NO: 4); FX.sub.1 FPKDVALX.sub.2, wherein X.sub.1 is V or T and X.sub.2 is L, R, or K (SEQ ID NO: 6); and TPRVTGGGAX, wherein X is L, M, or F (SEQ ID NO: 8); and (SEQ ID NO: 9). 2. A cellular vaccine against human cytomegalovirus which elicits an MHC Class I cellular immune response to human cytomegalovirus comprising antigen presenting cells that have been treated in vitro so as to present on their surface a peptide selected from the group consisting of NX.sub.1 VPMVATX.sub.2, wherein X.sub.1 is L, I, M, T, or V and X.sub.2 is V, A, C, I, L or T (SEQ ID NO: 2) with the proviso that the peptide is not NLVPMVATV (SEQ ID NO:1); YXEHPTFSQY, wherein X is S, T or L (SEQ ID NO: 4); FX.sub.1 FPTKDVALX.sub.2, wherein X.sub.1 is V or T and X.sub.2 is L, R, or K (SEQ ID NO: 6); TPRVTGGGAX, wherein X is L, M, or F (SEQ ID NO: 8); and FPTKDVAL (SEQ ID NO: 9). 3. A cellular vaccine against human cytomegalovirus of claim 2 wherein the antigen presenting cells are autologous. 4. A cellular vaccine against human cytomegalovirus of claim 2 wherein the antigen presenting cells are allogeneic. |
| PATENT DESCRIPTION |
BACKGROUND OF THE INVENTION 1. Technical Field This invention relates to human cytomegalovirus (HCMV), and in particular to peptide fragments from a single subunit protein that function as T-cell epitopes of HCMV in human beings. The peptide fragments are capable of directing human cytotoxic T lymphocytes (CTL) to recognize and lyse human cells infected with HCMV. The peptide fragments can independently direct HCMV-specific CTL to lyse cells incubated with the peptide and which express HLA A, B or C genes. 2. Description of the Background Art The HCMV genome is relatively large (about 235 k base pairs) and has the capacity to encode more than two hundred proteins. HCMV is composed of a nuclear complex of nucleic acid (double-stranded DNA) surrounded by capsid proteins having structural or enzymatic functions, and an external glycopeptide- and glycolipid-containing membrane envelope. HCMV is a member of the herpes virus family and has been associated with a number of clinical syndromes. HCMV infection is relatively common and is usually self-limiting in the healthy, immunocompetent child or adult (L. Rasmussen, Curr. Top. Microbiol. Immunol. 154:221-254, 1990). Approximately ten percent (10%) of all newborn infants carry HCMV and the virus can cause severe congenital disease in the fetus or infant. Some of these newborn infants suffer congenital birth defects. Other newborn infants carry cytomegalovirus for some time before they actually show symptoms of the disease. For example, HCMV is a common cause of mental retardation in children who acquire the infection in utero from mothers carrying an active infection. Several studies have begun to question whether persistent and apparently asymptomatic HCMV infection in an otherwise healthy adult poses health risks in certain individuals. For example, individuals who have undergone coronary angioplasty sometimes subsequently develop restenosis as a result of arterial remodeling. In one study, about one third of such patients with restenosis had detectable HCMV DNA in their arterial lesions (E. Speir et al., Science 265:391-394 (1994)), whereas in another study CMV seropositive patients were five times more likely to develop restenosis than their seronegative counterparts (Y. F. Zhou et al., New Enaland J. Med. 335:624-630 (1996)). These studies suggest that decreasing the number of HCMV infected host cells can benefit certain individuals. HCMV also has been associated with morbidity and mortality in immuno-compromised patients. HCMV is an important consideration in the treatment of patients suffering from Acquired Immunodeficiency Syndrome (AIDS). The defining complication is retinitis, which, if left untreated, can lead to blindness. Historically, CMV disease has been one of the more devastating of the opportunistic infections (OI) that beset HIV-1-infected individuals whose CD4.sup.+ T cell level diminishes below 100/mm.sup.3. Other disease manifestations of CMV viremia also appear as the CD4.sup.+ T cell counts drops below 100/mm.sup.3, including encephalitis, enteritis and pneumonia. At autopsy there is multi-organ involvement of CMV disease in the preponderance of AIDS patients who had severe CMV retinitis. Patients infected with HCMV often suffer impairment of some of their vital organs, including the salivary glands, brain, kidney, liver and lungs, as a result of the effects of the disease. Furthermore, HCMV is associated with a wide spectrum of classical syndromes including mononucleosis and interstitial pneumonia. HCMV also has an oncogenic potential and a possible association with certain types of malignancies including Kaposi's sarcoma. HCMV can cause opportunistic infections resulting in a variety of complications in, for example, immunosuppressed organ transplant patients. Prior to the use of antiviral chemotherapy, HCMV infection had been responsible for a substantial proportion of post-bone marrow transplantation (BMT) complications (J. Meyers et al., J. Infect Dis. 153:478-488 (1986)). The advent of drugs such as ganciclovir with substantial anti-CMV activity dramatically reduced complications associated with post-BMT CMV infections (G. Schmidt et al. New Enaland J. Med. 324:1005-1011 (1991) and J. M. Goodrich et al., New Enaland J. Med. 325:1601-1607 (1991)). Ganciclovir is most effective when administered prophylactically before diagnosis of HCMV infection. This approach has several negative aspects including a higher proportion of recipients becoming neutropenic (one third) and increased numbers of concomitant fatal bacterial and fungal diseases (J. M. Goodrich et al., Ann. Intern. Med. 118:173-178 (1993)). An alternative approach in which ganciclovir was given when HCMV antigens or DNA are first detected by culture methods provided no survival advantage compared to prophylaxis or treatment post-disease for all patients (D. J. Winston et al., Ann. Intern. Med. 118:179-184 (1993)). Finally, because of the acute nature of the side-effects, there is a need for increased hospitalization and growth factor administration to treated patients which, coupled with the cost of ganciclovir prophylaxis, increases the cost of BMT after-care. Because human cytomegalovirus is relatively common, yet is associated with extremely serious health conditions, a considerable effort has been made to study the biology of the virus with the aims of improving diagnosis of the disease as well as developing preventative and therapeutic strategies. The mounting of a CD8.sup.+ CTL response is believed to be an important mammalian host response to certain acute viral infections. The observations that HCMV infection is widespread and persistent, and may be reactivated and become clinically evident in the immunosuppressed patient, have suggested that virus-specific T-cells, including HCMV-specific CTL, play an important role in the control of persistent infection and the recovery from CMV disease. In humans, protection from the development of CMV disease in immunosuppressed BMT recipients correlates with the recovery of measurable CD8.sup.+ CMV-specific class I MHC-restricted T cell responses (Quinnan et al., N. Eng. J. Med. 307:7-13 (1982); Reusser et al., Blood 78:1373-1380 (1991)). These observations led investigators to carry out clinical trials in which donor-derived CMV-specific CD8.sup.+ CTL were infused into BMT recipients as an alternative to ganciclovir prophylaxis and therapy (S. R. Riddell et al., Science 257:238-241 (1992)). The transfer of CD8.sup.+ CTL clones to allogeneic bone marrow transplant recipients results in detectable CTL-based CMV immunity, and statistically significant diminution of CMV disease after BMT (E. A. Walter et al., N. Eng. J. Med. 333:1038-1044 (1995)). Although successful in application, this approach has the disadvantage that it requires a sophisticated laboratory setup (which is also highly labor-intensive and costly) to derive the HCMV-specific CTL in vitro to be reinfused into a patient. A desirable alternative would be to deliver a vaccine derived from HCMV that would impart immunity to a BMT recipient, a solid organ recipient, a heart patient, an AIDS patient or a woman of child-bearing years, without the need for ex vivo expansion of HCMV-specific CTL. To develop such a vaccine, the viral proteins which cause the host to recognize HCMV in a protective manner must be identified, so that their amino acid sequence information can be determined. No such vaccine presently is available, however. The viral life cycle provides insight as to the most effective time frame for targeting a vaccine to maximally disrupt virus production and spread. Following HCMV entry into the host cell and uncoating, the viral genome is expressed sequentially via immediate early (0-2 hour), early (2-24 hour) and late (>24 hour) viral proteins. However, certain viral structural proteins such as pp65 are chaperoned into the cell because of their existence in large quantity in the viral particle. Much attention has focused upon structural virion proteins as potential immunodominant target antigens for HCMV-specific CTL responses. One viral structural protein, pp65, has been identified as a target antigen for CMV-specific class I MHC restricted CTL derived from the peripheral blood of most asymptomatic CMV seropositive individuals (E. Mclaughlin-Taylor et al., J. Med. Virol. 43:103-110 (1994)). Importantly, CD8.sup.+ class I MHC restricted CTL specific for pp65 will recognize autologous HCMV-infected cells without the requirement for viral gene expression, presumably as a result of processing of the internal depot of pp65 that is transferred into the cell during infection (M. J. Gilbert et al., J. Virology 67:3461-3469 (1993)). CTL against pp65 or pp150 (another matrix protein that is recognized frequently) are able to recognize and lyse HCMV-infected cells in vitro within an hour of infection in the absence of viral gene expression (S. R. Riddell and P. D. Greenberg, Curr. Top. Microbiol. Immunol. 189:9-34 (1994)). Thus, these CTL may represent an important effector cell for limiting HCMV reactivation and progression to CMV disease, and such a cellular immune response in both immunocompromised and normal individuals would be extremely important (C. R. Li et al., Blood 83:1971-1979 (1994)). Alternatively, CTL recognizing envelope proteins are not a substitute for pp65 and pp150 CTL because they are rarely found, arising late in infection and they are poor lytic effectors because of the down-regulation of the required Class I MHC molecules (M. J. Gilbert et al., J. Virology 67:3461-3469 (1993)). Finally, the HCMV major protein IE, produced abundantly early after infection, is specifically inhibited from being a stimulator of CD8.sup.+ CTL by a CMV-dependent blockade of its presentation (M. J. Gilbert et al., Nature [London] 383:720-722, 1996). Therefore, vaccines stimulating immunity against pp65 or pp150 would be the preferred mechanism for eliciting protective immunity against CMV infection. It has been established that individual MHC Class I molecules preferentially bind peptides of a given motif and that the amino acid sequence of specific positions of the motif are invariant, allowing a given peptide to bind to MHC Class I molecules with high affinity. These are referred to as "anchor positions" (K. Falk et al., Nature 351:290-296 (1991)). Later studies have suggested that amino acid positions other than the anchor positions also contribute to the specificity of peptide binding to MHC Class I molecules. Additionally, residues at positions within the CTL epitope which do not interact with MHC may interact with T cells, presumably by binding the T Cell receptor (TCR). The binding of peptide amino acid residues to MHC or TCR structures is independently governed, so that substitution of TCR binding amino acid residues in many cases will not interfere with binding to the MHC molecule on the surface of an antigen presenting cell. Edman degradation followed by N-terminal sequence analysis has been used to sequence the peptide mixture which is bound to the MHC class I peptide binding groove. In most cases the length of these peptides is between 9 and 11 amino acids. Mass spectrometry of HPLC separated peptide mixtures can elucidate the primary sequence of individual peptides. Peptide fragments which bind to MHC identified in this manner are referred to as "naturally processed epitopes." Alternatively, one can predict which peptides of a given length, between 9-11 amino acids, will optimally bind to individual HLA Class I alleles based on their conformity to a motif (K. Falk et al., Nature 351:290-296 (1991)). One such motif has been established for HLA A*0201. Positions 2 and 9 of a nonapeptide are anchor residues for HLA A*0201, with minor contributions to binding from positions 1, 4, 3, 5, 6, 7, 8 in decreasing order of importance to binding strength (J. W. Drijfhout et al., Human Immunology 43:1-12 (1995)). Similar motifs have been established for decamers and undecamers for HLA A*0201. Correspondingly, unique amino acid motifs have been established for a subset of other HLA A and B alleles to predict binding peptides between 8-11 amino acids (H. G. Rammensee et al., Immungaenetics 41 (4):178-228 (1995)). It is recognized that CTL are an important mechanism by which a mammalian organism defends itself against infection by viruses and possibly cancer. A processed form of, e.g., a viral protein minimal cytotoxic epitope (MCE) in combination with MHC Class I molecules is recognized by T cells, such as CD8.sup.+ CTL. Functional studies of viral and tumor-specific T cells have confirmed that an MCE of 8-12 amino acids can prime an antigen presenting cell (APC) to be lysed by CD8.sup.+ CTL, as long as the APC expresses on the cell surface the correct MHC molecule that will bind the peptide. It has been shown that the route of entry of a protein into the cell determines whether it will be processed as an antigen bound to either MHC Class I or II molecules. The endogenous or Class I pathway of protein degradation is often used by infectious viruses when they are present within cells. Viral nucleoproteins which may never reach the cell surface as full length molecules are still processed within the cell, and degraded portions are transported to the surface via MHC Class I molecules. Viral envelope glycoproteins, merely because they are cell surface molecules, do not obligatorily induce CTL recognition. Rather, it has been found that viral nucleoproteins, predominantly in the form of processed epitopes are the target antigens recognized by CD8.sup.+ CTL (A. Townsend et al., Philos. Trans. R. Soc. Lond.(Biol). 323:527-533 (1989)). As it has become apparent that antigens entering the cell through exogenous pathways (pinocytosis, etc.) are not typically processed and presented by Class I MHC molecules, methods to introduce proteins directly into the cytoplasm have become the focus of vaccine developers. An approach that had gained favor was to use recombinant vaccinia viruses to infect cells, delivering a large amount of intracellular antigen. The enthusiasm for using vaccinia viruses as vaccines has diminished, however, because these viruses have the potential to cause disease in immunosuppressed people, such as BMT recipients. Another approach to vaccination is to mix an antigenic protein with an adjuvant and introduce the mixture under the skin by subcutaneous injection. Yet another potential approach to immunization to elicit CTL is to use the MCE defined for a viral antigen in the context of a particular MHC restriction element to boost a CTL memory response to a virus. The ability of an MCE to provide protective immunity to challenge by a lethal dose of an infectious virus has been discussed in the literature. Vaccine developers have developed increasing interest in utilizing the MCE as the vaccine because it is capable of binding to MHC Class I molecules through external binding of the cell surface molecules without the need for internalization or processing. The MCE has been most effective as an immunogen when synthesized as a lipidated peptide together with a helper CD4 epitope (A. Vitiello et al., J. Clin. Invest. 95:341-349 (1995) and B. Livingston et al., J. Immunol. 159:1383-1392, 1997). Other modifications of the bivalent vaccine include inclusion of a signal sequence (KDEL) for endoplasmic reticulum retention and targeting to attain maximum activity. There is also evidence in the literature that an MCE presented by particular types of APC (e.g. dendritic cells) may cause a primary immune response to occur in the absence of viral infection or prior contact with the virus or tumor cell. Accordingly, in spite of significant efforts towards identifying the HCMV proteins that are recognized by CTLs, as well as the specific identification of the HCMV late structural protein pp65, improved methods of preventing and treating HCMV infection are needed. Introduction of CMV-specific CTL into a recipient is not a universally applicable and practical strategy to confer immunity to all those at-risk individuals who may need to be immunized against HCMV infection. SUMMARY OF THE INVENTION Accordingly, one aspect of the present invention relates to immunologically active peptides, and functional variants thereof, capable of eliciting a cellular immune response to HCMV in humans. The peptides are capable of directing human CTL to recognize and lyse human cells infected with HCMV. Such immunologically active peptides, in association with an MHC Class I molecule, are recognized by CTLs of individuals having a latent (inactive) HCMV infection. Another aspect of the present invention provides a method of augmenting the immune system of a patient in need thereof (i.e., a patient harboring a latent or active CMV infection) by administering at least one immunologically active peptide according to the present invention that will be recognized by CTLs and/or CTLps (CTL precursors) of the patient. In yet another aspect of the invention, at least one immunologically active peptide is administered to uninfected individuals to provide immunity against future infections by HCMV. Such a peptide may be administered in the form of a peptide or lipopeptide vaccine, optionally with an adjuvant. Alternatively, the peptide(s) may be administered in the form of a cellular vaccine via the administration of autologous or allogeneic antigen presenting cells or dendritic cells that have been treated in vitro so as to present the peptide on their surface. Yet another aspect of the invention is a method to augment the immune response of an individual who is latently infected with CMV and is at risk for reactivation of CMV infection, wherein T cells are removed from an individual and treated in vitro with a peptide of the present invention. The resulting CMV-reactive CTL are reinfused autologously to the patient or allogeneically to, for example, a BMT recipient. In yet another aspect, a method to confer immunity against an HCMV infection to a previously uninfected individual includes the steps of removing T cells from the individual, exposing the T cells in vitro to a peptide of the present invention and then reinfusing the resulting HCMV-reactive CTL to the individual. The peptides of the present invention also may be administered to previously infected or uninfected patients, or in vitro to T cells, in the form of a polynucleotide (DNA-based) vaccine, wherein a suitable gene transfer vector, such as a plasmid or an engineered viral vector that contains DNA encoding the peptide fragment under the control of appropriate expression regulatory sequences, is administered to the patient or to T cells in culture. In yet another of its aspects, the present invention provides a vaccinia, canarypox or other eukaryotic virus vector containing a DNA sequence encoding the immunologically active peptide fragment. The vector infects an antigen presenting cell which in turn presents antigen that will be recognized by CTLs of patients having a latent (inactive) HCMV infection. An additional aspect of the invention relates to diagnostic reagents for detection of the presence of active versus quiescent HCMV infections. The peptides according to the present invention can directly stimulate CTLp in vitro and therefore can be used in an assay to determine the degree of immunostimulation being caused by HCMV. The peptides can also be used to distinguish individuals who are seropositive from those who have not been exposed to HCMV (seronegative individuals). T cells from a patient can be contacted in vitro with APC that have been primed with a peptide according to the present invention. BRIEF DESCRIPTION OF THE DRAWING FIG. 1 shows the cytotoxic response elicited by peptides in the absence of lipidation. FIG. 2 shows the cytotoxic response elicited by monolipidated peptides. FIG. 3 shows the cytotoxic response elicited by dilipidated peptides. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS According to one aspect of the present invention, a nonapeptide (9 amino acid peptide) of the sequence NLVPMVATV (pp65.sub.495-503) (SEQ ID NO:1) is an immunogenic epitope of pp65 from CMV laboratory strains AD169 and Towne and all wild type isolates examined to date which is recognized by CD8.sup.+ Class I MHC restricted cytotoxic T-lymphocytes of patients harboring latent CMV infection. The peptide is capable of activating CTLs in the absence of active viral replication, and thus is useful for augmenting the immune system of normal and immunodeficient patients, as well as in the study of the Class I antigen processing pathway for HCMV proteins. The nonapeptide has amino acid residues in positions 2 and 9 which are the preferred residues at those positions for interaction with the HLA A*0201 and certain subtypes of HLA A*02XX, where XX=subtypes 02-22 (J. W. Drijfhout et al., Human Immunology 43:1-12 (1995)). Nonetheless, other less preferred amino acid residues may replace the preferred anchors, whereupon the peptide can continue to exhibit the capacity to bind HLA A*0201 and certain subtypes of HLA A*02XX with the ability to stimulate HCMV-specific CD8.sup.+ CTL. Thus, in one aspect, the present invention provides an immunologically active peptide, capable of eliciting a cellular immune response to human cytomegalovirus infection, of the preferred sequence: NLVPMVATV (SEQ ID NO:1). Sequence variants of the preferred peptide include peptides of the sequence NX.sub.1 VPMVATX.sub.2 wherein X.sub.1 is L,I,M,T or V, and X.sub.2 is V,A,C,I,L or T (SEQ ID NO:2). The invention includes the construction and selection of other functional sequence variants, which can be carried out by those skilled in the art based upon the present disclosure. The peptide or the structural variants disclosed herein also can be a functional part of a longer peptide which produces the immunological effects disclosed herein. Other immunologically active peptides according to the present invention include the peptides: YSEHPTFTSQY (SEQ ID NO:3) which binds to HLA A*01XX including A*0101 and subtypes thereof. Sequence variants of this peptide include peptides of the sequence YXEHPTFTSQY wherein X is S, T or L (SEQ ID NO:4). The invention includes the construction and selection of other functional sequence variants, which can be carried out by those skilled in the art based upon the present disclosure. The peptide or the structural variants disclosed herein also can be a functional part of a longer peptide which produces the immunological effects disclosed herein. FVFPTKDVALR (SEQ ID NO:5) which binds to HLA A*68XX including A*6801 and subtypes thereof. Sequence variants of this peptide include peptides of the sequence FX.sub.1 FPTKDVALX.sub.2 wherein X.sub.1 is V or T and X.sub.2 is L, R or K (SEQ ID NO:6). The invention includes the construction and selection of other functional sequence variants, which can be carried out by those skilled in the art based upon the present disclosure. The peptide or the structural variants disclosed herein also can be a functional part of a longer peptide which produces the immunological effects disclosed herein. TPRVTGGGAM (SEQ ID NO:7) which binds to HLA B*07XX including B*0702 and subtypes thereof. Sequence variants of this peptide include peptides of the sequence TPRVTGGGAX wherein X is L, F, or M (SEQ ID NO:8). The invention includes the construction and selection of other functional sequence variants, which can be carried out by those skilled in the art based upon the present disclosure. The peptide or the structural variants disclosed herein also can be a functional part of a longer peptide which produces the immunological effects disclosed herein. FPTKDVAL (SEQ ID NO:9) which binds to HLA B*35XX including B*3502, B*3504, B*3506 and other subtypes thereof with compatible peptide binding sites. The invention includes the construction and selection of other functional sequence variants, which can be carried out by those skilled in the art based upon the present disclosure. The peptide or the structural variants disclosed herein also can be a functional part of a longer peptide which produces the immunological effects disclosed herein. RPHERNGFTVL (SEQ ID NO:19) which binds to HLA B*07XX including B*0702 and other subtypes thereof with compatible peptide binding sites. The invention includes the construction and selection of other functional sequence variants, which can be carried out by those skilled in the art based upon the present disclosure. The peptide or the structural variants disclosed herein also can be a functional part of a longer peptide which produces the immunological effects disclosed herein. SVLGPISGHVLK (SEQ ID NO:20) which binds to HLA A*11XX including A*1101 and other subtypes thereof with compatible peptide binding sites. The invention includes the construction and selection of other functional sequence variants, which can be carried out by those skilled in the art based upon the present disclosure. The peptide or the structural variants disclosed herein also can be a functional part of a longer peptide which produces the immunological effects disclosed herein. |
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