Kinase‐Deficient CMVpp65 Triggers a CMVpp65 Specific T‐Cell Immune Response in HLA‐A*0201.K<sup>b</sup> Transgenic Mice after DNA Immunization

Gallez-Hawkins, Ghislaine; Lomeli, Norma A.; Li, X. L.; Zheng, Yao; Rosa, Corinna La; Diamond, Don J.; Zaia, John A.

doi:10.1046/j.1365-3083.2002.01099.x

Cited by 10 publications

(14 citation statements)

References 57 publications

(59 reference statements)

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“…The murine model HHD II is also a transgenic HLA A‫1020ء‬ mouse that contains a disrupted murine major histocompatibility complex (MHC) molecule, forcing the mouse to present all its immunological epitopes through the HLA pathway (10). Both have become useful models to investigate peptide recognition of specific proteins that can be extrapolated to human subjects.The immunodominant HLA A‫-1020ء‬restricted peptide (pp65 [495][496][497][498][499][500][501][502][503] ) of CMV pp65 has been well studied (3,6,12,30), whereas there is no immunodominant peptide recognized for the CMV IE1 protein, despite the prevalence of an IE1-specific CTL response (11,14,16,17). Three reports have described the stimulatory effect of peptides IE1 p315-323 , IE1 p316-324 , and IE1 p354-362 from CMV IE1 in the context of HLA A‫1020ء‬ (11, 17, 24) in a cytokine flow cytometry or CTL assay.…”

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“…The immunodominant HLA A‫-1020ء‬restricted peptide (pp65 [495][496][497][498][499][500][501][502][503] ) of CMV pp65 has been well studied (3,6,12,30), whereas there is no immunodominant peptide recognized for the CMV IE1 protein, despite the prevalence of an IE1-specific CTL response (11,14,16,17). Three reports have described the stimulatory effect of peptides IE1 p315-323 , IE1 p316-324 , and IE1 p354-362 from CMV IE1 in the context of HLA A‫1020ء‬ (11,17,24) in a cytokine flow cytometry or CTL assay.…”

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confidence: 99%

“…Six-to eight-week-old A2Kb mice (1) were immunized three times at 4-week intervals with various combinations of DNA expressing CMV pp65mII, CMV IE1, and granulocyte-macrophage colony-stimulating factor (GM-CSF). The genes for pp65mII, IE1, and murine GM-CSF were inserted into the mammalian expression vector pcDNA3.1 ϩ (Invitrogen, San Diego, Calif.) containing the intron A as described previously (12). Mice M1 to M4 were inoculated intramuscularly with 100 g of pcDNA-IE1 and 100 g of pcDNAGM-CSF, M5 to M8 were inoculated with 100 g of pcDNA-IE1 and 100 g of pcDNApp65mII, and M9 to M12 were inoculated with 50 g of pcDNA-IE1, 50 g of pcDNApp65mII, and 50 g of pcDNAGM-CSF.…”

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confidence: 99%

“…The control target T2 cells (no peptides) or nontransfected A293 cells were not lysed by the effector cells (data not shown). The pp65mII DNA was considered a positive control that resulted in an immune response in 50% of immunized mice (12). The GM-CSF DNA was added as a cytokine adjuvant to increase the immune response to DNA immunization in mice (25,27).…”

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Use of Transgenic HLA A*0201/Kb and HHD II Mice To Evaluate Frequency of Cytomegalovirus IE1-Derived Peptide Usage in Eliciting Human CD8 Cytokine Response

Gallez-Hawkins

Villacres

et al. 2003

J Virol

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Unlike the pp65 protein of human cytomegalovirus (CMV), which has an immunodominant peptide, pp65 [495][496][497][498][499][500][501][502][503] , recognized by human CD8؉ cells in the context of HLA A‫,1020ء‬ the fine peptide specificity for CMV IE1 has shown no such immunodominance. With the use of transgenic HLA A‫/1020ء‬Kb and HHD II mice, a selected pool of IE1 peptides, including IE1 p256-264 , IE1 p297-304 , and IE1 p316-324 , were shown to stimulate cytolytic T-lymphocyte lysis in the context of HLA A‫.1020ء‬ Based on an intracellular gamma interferon response, IE1 p297-304 , a previously unrecognized CD8 epitope, triggered a prominent response to CMV IE1 in HLA A‫1020ء‬ subjects.Murine models to predict human cytomegalovirus (CMV) vaccine immunogenicity are available to detect the presence of a specific immune response, such as cytolytic T-lymphocyte (CTL) function (2, 5, 28), CD4ϩ gamma interferon (IFN-␥) helper response (15, 22), or antibody response to specific CMV proteins (18,20,21). Of interest is the use of the transgenic A2Kb mouse containing the human HLA A‫1020ء‬with the murine ␣3 chain (Kb) (9,19,29). The murine model HHD II is also a transgenic HLA A‫1020ء‬ mouse that contains a disrupted murine major histocompatibility complex (MHC) molecule, forcing the mouse to present all its immunological epitopes through the HLA pathway (10). Both have become useful models to investigate peptide recognition of specific proteins that can be extrapolated to human subjects.The immunodominant HLA A‫-1020ء‬restricted peptide (pp65 [495][496][497][498][499][500][501][502][503] ) of CMV pp65 has been well studied (3,6,12,30), whereas there is no immunodominant peptide recognized for the CMV IE1 protein, despite the prevalence of an IE1-specific CTL response (11,14,16,17). Three reports have described the stimulatory effect of peptides IE1 p315-323 , IE1 p316-324 , and IE1 p354-362 from CMV IE1 in the context of HLA A‫1020ء‬ (11, 17, 24) in a cytokine flow cytometry or CTL assay. However, not all CMV-seropositive subjects respond to these peptides (17). This report describes the use of HLA A2 transgenic mice to identify a novel IE1 peptide, IE1-297, that is recognized by murine CTLs as well as human CD8 cells by cytokine flow cytometry.T2 stabilization. T2 cells are defective for endogenous class I presentation, but the presence of peptide binding to the MHC molecule will stabilize its expression on the cell surface. The stabilized MHC molecule can be detected by flow cytometry with a monoclonal antibody to the HLA A‫1020ء‬ molecule (13). The peptide sequences used in this study were collected from two algorithms for HLA peptide predicted motifs: SYFPEITHI (23)

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confidence: 99%

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confidence: 99%

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Use of Transgenic HLA A*0201/Kb and HHD II Mice To Evaluate Frequency of Cytomegalovirus IE1-Derived Peptide Usage in Eliciting Human CD8 Cytokine Response

Gallez-Hawkins

Villacres

et al. 2003

J Virol

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“…Despite this potential for cell toxicity, CMV pp65 has been proposed to be a critical antigen in any anti-CMV vaccine (17,32). CMV pp65 has been shown to have protein kinase activity (8,39), and a mutation at a critical phosphate binding site (CMV pp65mII) removed the kinase activity without altering the antigenicity (39). UL83 is considered an earlylate gene, with synthesis beginning between 12 and 24 h after infection, during which time the protein product accumulates in the nucleus.…”

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Biologic and Immunologic Effects of Knockout of Human Cytomegalovirus pp65 Nuclear Localization Signal

Zaia

Li²,

Franck³

et al. 2009

Clin Vaccine Immunol

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The human cytomegalovirus (CMV) pp65 protein contains two bipartite nuclear localization signals (NLSs) at amino acids (aa) 415 to 438 and aa 537 to 561 near the carboxy terminus of CMV pp65 and a phosphate binding site related to kinase activity at lysine-436. A mutation of pp65 with K436N (CMV pp65mII) and further deletion of aa 537 to 561 resulted in a novel protein (pp65mIINLSKO, where NLSKO indicate NLS knockout) that is kinaseless and that has markedly reduced nuclear localization. The purpose of this study was to biologically characterize this protein and its immunogenicity compared to that of native pp65. Unlike the native CMV pp65, following either DNA-or recombinant adeno-associated virus-based transduction of CMV pp65mIINLSKO into cells in vitro, the first observation of pp65mIINLSKO expression was in the cytoplasm and pp65mIINLSKO was expressed at higher levels than the native protein. The CMV pp65mIINLSKO mRNA was more abundant earlier than CMV pp65 mRNA (at 4 h and 8 h, respectively), but the half-lives of the proteins were the same. This modification altered the antigenic processing of CMV pp65 in vitro, as measured by the improved efficiency of cytotoxic killing in a pp65mIINLSKO-transduced human HLA A*0201 target cell line. In HHDII mice expressing HLA A*0201, pp65mIINLSKO was as immunogenic as CMV pp65. By RNA microarray analysis, expression of the CMV pp65mIINLSKO had less of an effect on cell cycle pathways than the native CMV pp65 did and a greater effect on cell surface signaling pathways involving immune activity. It is concluded that the removal of the primary NLS motif from pp65 does not impair its immunogenicity and should be considered in the design of a vaccine.A major immunodominant protein of human cytomegalovirus (CMV) is the tegument protein CMV pp65 (UL83) (15,16,23,36). The biologic function of CMV pp65 is unclear, but as a nucleotropic protein which enters the nucleus immediately after infection (10,28,31,33), CMV pp65 binds to polo-like kinase 1 (PLK-1), an enzyme important in mitosis (11), and it is likely that the protein has specific effects on cell cycle events (14,22,30). Despite this potential for cell toxicity, CMV pp65 has been proposed to be a critical antigen in any anti-CMV vaccine (17, 32). CMV pp65 has been shown to have protein kinase activity (8, 39), and a mutation at a critical phosphate binding site (CMV pp65mII) removed the kinase activity without altering the antigenicity (39). UL83 is considered an earlylate gene, with synthesis beginning between 12 and 24 h after infection, during which time the protein product accumulates in the nucleus. However, at late times after infection, this CMV pp65 is exported back to the cytoplasm by means of the exportin system (31). CMV pp65 contains elements of the prototypic nuclear localization signal (NLS) in which arginine and lysine predominate within a bipartite motif in which short regions of basic amino acids are separated by 10 or more nonconserved amino acids (13,24,35). The nuclear localization signals of CMV pp65...

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Cytomegalovirus and Varicella-Zoster Virus Vaccines

Hunt

2006

Encyclopedia of Molecular Cell Biology and Molecular Medicine

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Kinase‐Deficient CMVpp65 Triggers a CMVpp65 Specific T‐Cell Immune Response in HLA‐A*0201.K^b Transgenic Mice after DNA Immunization

Cited by 10 publications

References 57 publications

Use of Transgenic HLA A*0201/Kb and HHD II Mice To Evaluate Frequency of Cytomegalovirus IE1-Derived Peptide Usage in Eliciting Human CD8 Cytokine Response

Use of Transgenic HLA A*0201/Kb and HHD II Mice To Evaluate Frequency of Cytomegalovirus IE1-Derived Peptide Usage in Eliciting Human CD8 Cytokine Response

Biologic and Immunologic Effects of Knockout of Human Cytomegalovirus pp65 Nuclear Localization Signal

Cytomegalovirus and Varicella-Zoster Virus Vaccines

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Kinase‐Deficient CMVpp65 Triggers a CMVpp65 Specific T‐Cell Immune Response in HLA‐A*0201.Kb Transgenic Mice after DNA Immunization

Cited by 10 publications

References 57 publications

Use of Transgenic HLA A*0201/Kb and HHD II Mice To Evaluate Frequency of Cytomegalovirus IE1-Derived Peptide Usage in Eliciting Human CD8 Cytokine Response

Use of Transgenic HLA A*0201/Kb and HHD II Mice To Evaluate Frequency of Cytomegalovirus IE1-Derived Peptide Usage in Eliciting Human CD8 Cytokine Response

Biologic and Immunologic Effects of Knockout of Human Cytomegalovirus pp65 Nuclear Localization Signal

Cytomegalovirus and Varicella-Zoster Virus Vaccines

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Kinase‐Deficient CMVpp65 Triggers a CMVpp65 Specific T‐Cell Immune Response in HLA‐A*0201.K^b Transgenic Mice after DNA Immunization