Rapid Determination of HLA B*07 Ligands
Rapid Determination of HLA B*07 Ligands
Defined T cell epitopes for West Nile (WN) virus may be useful for developing subunit vaccines against WN virus infection and diagnostic reagents to detect WN virus-specific immune response. We applied a bioinformatics (EpiMatrix) approach to search the WN virus NY99 genome for HLA B*07 restricted cytotoxic T cell (CTL) epitopes. Ninety-five of 3,433 WN virus peptides scored above a predetermined cutoff, suggesting that these would be likely to bind to HLA B*07 and would also be likely candidate CTL epitopes. Compared with other methods for genome mapping, derivation of these ligands was rapid and inexpensive. Major histocompatibility complex ligands identified by this method may be used to screen T cells from WN virus-exposed persons for cell-mediated response to WN virus or to develop diagnostic reagents for immunopathogenesis studies and epidemiologic surveillance.
West Nile (WN) virus is the cause of a potentially fatal form of viral encephalitis that suddenly emerged in the New York City area during 1999. The virus is a member of the Flavivirus family, which includes St. Louis encephalitis (SLE), Japanese encephalitis (JE), hepatitis C, and dengue viruses. WN virus is common in West Asia, Africa, and the Middle East but was not reported in the Americas until the New York outbreak in 1999. The source of virus introduction to New York City is unknown; potential sources include an infected host (human or bird), an infected vector (mosquito), or bioterrorism. The WN-NY99 virus associated with the New York 1999 outbreak appears to have been circulating in Israel since 1997. Other close relatives to the WN-NY99 virus were isolated in Italy (1998), Morocco (1996), Romania (1996), and Africa (1989, 1993, and 1998).
Surveillance data indicate intensified transmission and geographic expansion of the WN virus-NY99 outbreak in the northeastern United States during 1999 and 2000. Twelve states and the District of Columbia reported WN virus activity in 2000, a substantial increase over the four states reporting activity in 1999. WN-NY99 is expected to continue to spread along the east coast of the United States in 2001 and thereafter, as a result of overwintering of mosquitoes and avian migratory patterns.
WN virus's genome is 11,000 nucleotides long. The following structural proteins have been identified: envelope glycoprotein (env gp E), capsid (C), and premembrane protein (prM). The following nonstructural proteins have also been identified: NS-1, NS-2A/NS-2B, NS3, NS-4A/NS-4B; and NS-5 (RNA-directed polymerase). Isolates that have been completely sequenced include the WN-NY99 virus originally obtained from a Chilean flamingo, a WN-NY99 equine isolate, the Italy 1998 virus, the Romania 1996 virus, and the prototype Eg101 virus. Although the latter viruses are closely related to WN-NY99, they are not identical to each other or to WN-NY99. A virus isolated in Israel, Israel 1998, appears to be identical to WN-NY99; completion of its genome sequence is under way at the Institute Pasteur, France.
An extensive body of research is available on the immunology of flaviviruses in the murine model; however, relatively little research has been done on human immune response to WN virus. Some information on human T cell responses to related viruses (e.g., JE virus, dengue) has been obtained. Both CD4 T helper cells and cytotoxic T cells that respond to JE virus and dengue proteins have been identified, and their epitopes have been mapped. Some of the JE virus CD4 T epitopes are identical or nearly identical to sequences in WN virus. Langerhans cells in the epidermis may play a role in the upregulation of immune response to the virus, processing antigen and presenting it to T cells. Mobilization of dendritic cells and antigen presentation by these cells to T cells in the lymphoid follicles may be involved in the development of immune responses to WN virus.
Cytotoxic T cell responses (restricted by class I major histocompatibility complex [MHC] and MHC class II) and T helper responses (restricted by class II MHC) appear to be critical components of human immune response to members of the flavivirus family. Cell-mediated immunity to WN virus may prove to be an important barrier to infection of the central nervous system, and vaccines that promote the development of T effector cells may provide protection from WN virus encephalitis or may be used to treat patients who have WN virus-related illnesses. Further research to test these hypotheses will require the development of reagents such as the T cell epitopes defined in this study.
New bioinformatics tools developed by the TB/HIV Research Lab and EpiVax (Providence, RI) enable researchers to move rapidly from genome sequence to epitope selection. EpiMatrix is a computer-driven pattern-matching algorithm that identifies T cell epitopes. BlastiMer permits the analysis of protein sequences for homology with other known proteins.
The goal of this project was to demonstrate the utility of a bioinformatics and computational immunology approach for the rapid selection of T cell epitope reagents. Defining these reagents will permit the evaluation of cell-mediated responses in the immunopathogenesis of WN virus, promote the development of diagnostic reagents such as tetramers, and provide components for epitope-based preventive or therapeutic vaccines. A secondary goal was to determine the time required to select and screen epitope candidates in vitro, since time may be a critical factor in the development of vaccines and diagnostic reagents in response to emerging infectious pathogens.
On the basis of experience with the EpiMatrix HLA B*07 prediction tool, we selected peptides for this pilot study that were expected to be restricted by HLA B*07. In studies of HIV-1 peptides, 60% of peptides selected by EpiMatrix HLA B*07 stimulated T cell responses in vitro. We therefore expected that approximately 60% of WN virus peptides selected by the same criteria would bind to HLA B*07 and stimulate T cell responses.
We screened 16 WN virus peptides and identified 12 epitope candidates, 5 of which exhibited strong binding to HLA B*07 at a range of peptide concentrations in vitro. The largest source of delay in the screening process was peptide synthesis (4 weeks from placement of order to receipt of the first set of peptides and 8 weeks until delivery of the final set of peptides). This process could be accelerated if more rapid access to MHC ligands were necessary.
The binding studies we describe are a first step to confirming immunogenicity. In cases such as WN virus, in which access to T cells from infected persons is limited, both the bioinformatics step and the binding assays can be carried out without clinical specimens. Once the epitope candidates selected by this method are confirmed in cytotoxic T cell (CTL) assays, they may be useful for 1) screening exposed persons for T cell responses, 2) investigating the immunopathogenesis of WN virus disease in humans, 3) as components of diagnostic kits developed for WN virus surveillance, 4) as reagents for measuring WN virus vaccine-related immune responses, and possibly 5) as components of a subunit vaccine for WN virus. Confirmation of T cell response to the peptides will depend on availability of peripheral blood cells from WN virus-infected patients during the 2001 transmission season. Additional peptides also need to be identified and screened for binding to other HLA alleles, to broaden the MHC specificity of the diagnostic reagent or immunopathogenesis tools developed by this approach.
Defined T cell epitopes for West Nile (WN) virus may be useful for developing subunit vaccines against WN virus infection and diagnostic reagents to detect WN virus-specific immune response. We applied a bioinformatics (EpiMatrix) approach to search the WN virus NY99 genome for HLA B*07 restricted cytotoxic T cell (CTL) epitopes. Ninety-five of 3,433 WN virus peptides scored above a predetermined cutoff, suggesting that these would be likely to bind to HLA B*07 and would also be likely candidate CTL epitopes. Compared with other methods for genome mapping, derivation of these ligands was rapid and inexpensive. Major histocompatibility complex ligands identified by this method may be used to screen T cells from WN virus-exposed persons for cell-mediated response to WN virus or to develop diagnostic reagents for immunopathogenesis studies and epidemiologic surveillance.
West Nile (WN) virus is the cause of a potentially fatal form of viral encephalitis that suddenly emerged in the New York City area during 1999. The virus is a member of the Flavivirus family, which includes St. Louis encephalitis (SLE), Japanese encephalitis (JE), hepatitis C, and dengue viruses. WN virus is common in West Asia, Africa, and the Middle East but was not reported in the Americas until the New York outbreak in 1999. The source of virus introduction to New York City is unknown; potential sources include an infected host (human or bird), an infected vector (mosquito), or bioterrorism. The WN-NY99 virus associated with the New York 1999 outbreak appears to have been circulating in Israel since 1997. Other close relatives to the WN-NY99 virus were isolated in Italy (1998), Morocco (1996), Romania (1996), and Africa (1989, 1993, and 1998).
Surveillance data indicate intensified transmission and geographic expansion of the WN virus-NY99 outbreak in the northeastern United States during 1999 and 2000. Twelve states and the District of Columbia reported WN virus activity in 2000, a substantial increase over the four states reporting activity in 1999. WN-NY99 is expected to continue to spread along the east coast of the United States in 2001 and thereafter, as a result of overwintering of mosquitoes and avian migratory patterns.
WN virus's genome is 11,000 nucleotides long. The following structural proteins have been identified: envelope glycoprotein (env gp E), capsid (C), and premembrane protein (prM). The following nonstructural proteins have also been identified: NS-1, NS-2A/NS-2B, NS3, NS-4A/NS-4B; and NS-5 (RNA-directed polymerase). Isolates that have been completely sequenced include the WN-NY99 virus originally obtained from a Chilean flamingo, a WN-NY99 equine isolate, the Italy 1998 virus, the Romania 1996 virus, and the prototype Eg101 virus. Although the latter viruses are closely related to WN-NY99, they are not identical to each other or to WN-NY99. A virus isolated in Israel, Israel 1998, appears to be identical to WN-NY99; completion of its genome sequence is under way at the Institute Pasteur, France.
An extensive body of research is available on the immunology of flaviviruses in the murine model; however, relatively little research has been done on human immune response to WN virus. Some information on human T cell responses to related viruses (e.g., JE virus, dengue) has been obtained. Both CD4 T helper cells and cytotoxic T cells that respond to JE virus and dengue proteins have been identified, and their epitopes have been mapped. Some of the JE virus CD4 T epitopes are identical or nearly identical to sequences in WN virus. Langerhans cells in the epidermis may play a role in the upregulation of immune response to the virus, processing antigen and presenting it to T cells. Mobilization of dendritic cells and antigen presentation by these cells to T cells in the lymphoid follicles may be involved in the development of immune responses to WN virus.
Cytotoxic T cell responses (restricted by class I major histocompatibility complex [MHC] and MHC class II) and T helper responses (restricted by class II MHC) appear to be critical components of human immune response to members of the flavivirus family. Cell-mediated immunity to WN virus may prove to be an important barrier to infection of the central nervous system, and vaccines that promote the development of T effector cells may provide protection from WN virus encephalitis or may be used to treat patients who have WN virus-related illnesses. Further research to test these hypotheses will require the development of reagents such as the T cell epitopes defined in this study.
New bioinformatics tools developed by the TB/HIV Research Lab and EpiVax (Providence, RI) enable researchers to move rapidly from genome sequence to epitope selection. EpiMatrix is a computer-driven pattern-matching algorithm that identifies T cell epitopes. BlastiMer permits the analysis of protein sequences for homology with other known proteins.
The goal of this project was to demonstrate the utility of a bioinformatics and computational immunology approach for the rapid selection of T cell epitope reagents. Defining these reagents will permit the evaluation of cell-mediated responses in the immunopathogenesis of WN virus, promote the development of diagnostic reagents such as tetramers, and provide components for epitope-based preventive or therapeutic vaccines. A secondary goal was to determine the time required to select and screen epitope candidates in vitro, since time may be a critical factor in the development of vaccines and diagnostic reagents in response to emerging infectious pathogens.
On the basis of experience with the EpiMatrix HLA B*07 prediction tool, we selected peptides for this pilot study that were expected to be restricted by HLA B*07. In studies of HIV-1 peptides, 60% of peptides selected by EpiMatrix HLA B*07 stimulated T cell responses in vitro. We therefore expected that approximately 60% of WN virus peptides selected by the same criteria would bind to HLA B*07 and stimulate T cell responses.
We screened 16 WN virus peptides and identified 12 epitope candidates, 5 of which exhibited strong binding to HLA B*07 at a range of peptide concentrations in vitro. The largest source of delay in the screening process was peptide synthesis (4 weeks from placement of order to receipt of the first set of peptides and 8 weeks until delivery of the final set of peptides). This process could be accelerated if more rapid access to MHC ligands were necessary.
The binding studies we describe are a first step to confirming immunogenicity. In cases such as WN virus, in which access to T cells from infected persons is limited, both the bioinformatics step and the binding assays can be carried out without clinical specimens. Once the epitope candidates selected by this method are confirmed in cytotoxic T cell (CTL) assays, they may be useful for 1) screening exposed persons for T cell responses, 2) investigating the immunopathogenesis of WN virus disease in humans, 3) as components of diagnostic kits developed for WN virus surveillance, 4) as reagents for measuring WN virus vaccine-related immune responses, and possibly 5) as components of a subunit vaccine for WN virus. Confirmation of T cell response to the peptides will depend on availability of peripheral blood cells from WN virus-infected patients during the 2001 transmission season. Additional peptides also need to be identified and screened for binding to other HLA alleles, to broaden the MHC specificity of the diagnostic reagent or immunopathogenesis tools developed by this approach.
