CMVpp65, a candidate component of human cytomegalovirus (CMV) vaccines, has phosphokinase (PK) ac... more CMVpp65, a candidate component of human cytomegalovirus (CMV) vaccines, has phosphokinase (PK) activity that could affect vaccine safety. A mutated form of CMVpp65 substituting asparagine for lysine at the adenosine triphosphate (ATP)-binding site (CMVpp65mII) is kinase-deficient. Using DNA immunizations in a transgenic human leucocyte antigen (HLA)A*0201.Kb mouse model, the mutated CMVpp65 induced cytotoxic T lymphocytes (CTL) immunity similarly to native CMVpp65. Murine CTL lines generated from these immunizations killed human cells either after sensitization with CMVpp65-specific peptides or after infection with either CMV-Towne strain or rvac-pp65. It is proposed that CMVpp65mII be evaluated in candidate vaccines for CMV.
Supplemental Figures. S1: Chemokine/Cytokine Quantification in Serum and in Tumor Microenvironmen... more Supplemental Figures. S1: Chemokine/Cytokine Quantification in Serum and in Tumor Microenvironment. S2: Blocking of CXCR3 suppresses the infiltration of CD8 T cell. S3: Administration of 7DW8-5 results in activation of NKT cells, NK cells, and DC in mouse spleen.
Human CMV continues to be a major risk factor in patients undergoing allogeneic hematopoietic ste... more Human CMV continues to be a major risk factor in patients undergoing allogeneic hematopoietic stem cell (HSCT) and solid organ transplant (SOT), despite advancement of antiviral therapy. CMV tegument protein pp65 and CMV immediate early gene product IE1 are both considered immunodominant targets of cell-mediated immunity (CMI) and potentially capable of controlling CMV infection. Similar to healthy adults, CMV tegument protein pp65 and IE1 are frequently recognized by human CD4 and CD8 T cells in HCT and SOT recipients. To better assess their role in host defense, we have constructed a novel attenuated poxvirus (MVA) transfer vector named pZWIIA and generated a recombinant MVA (rMVA) expressing both full-length pp65 and exon4 of IE1 (pp65-IE1-MVA) at high levels, followed by the genetic removal of the bacterial marker gene used to distinguish recombinant forms during the screening process. Immunogenicity evaluation indicates that pp65-IE1-MVA not only can induce robust primary CMI to both antigens in HLA A2.1, B7 and A11 transgenic mice, but also can stimulate vigorous expansion of memory T lymphocyte responses to pp65 and IE1 in PBMC of CMV-positive donors. The recent discovery that additional early antigens of CMV are also well-recognized by healthy adults prompted us to include the IE2 antigen in the vaccine. Evaluation of in vivo immunogenicity of the vaccine expressing IE2 in both transgenic mice and in vitro antigenicity in human peripheral blood lymphocytes of HSCT and SOT recipients will be presented. A novel overlapping peptide library spanning full-length IE2 has been constructed in our laboratory, and used in conjunction with the vaccine expressing IE2, along with IE1 and pp65. Since MVA has a foreign gene capacity of over 30 kilobases (Kb), we have developed a strategy to maximize coverage of haplotypes through insertion of 6 additional antigens into novel insertion sites of the virus, for a total of just 12.0 Kb of the CMV genome. The selected antigens cover 83% of the population, while representing over 22% of the combined CD4 and CD8 T cell repertoire in seropositives. Our studies of the recognition of these antigens expressed in MVA and monitored with our own synthesized over-lapping peptide libraries in both HSCT and SOT recipients will be presented. These properties make the MVA-based vaccine ideal for the dual role of priming and boosting CMV-specific T cell immunity as a means to control CMV disease in recipients of HCT or SOT. pZWIIA alone or in combination with other MVA transfer vectors can be used to generate MVA based multiple-antigen vaccine which have application in vaccine development for a wide spectrum of infectious diseases and cancer.
Recent studies using direct stimulation of PBMC from CMV-positive individuals with optimal CTL ep... more Recent studies using direct stimulation of PBMC from CMV-positive individuals with optimal CTL epitope peptides have identified new antigens that are targets of the host immune response. The growing number of targets of the cellular immune response has prompted an evaluation of which antigens are potentially associated with protective immunity. A panel of seven human cytomegalovirus (CMV) epitope peptides and the corresponding MHC-I tetramers was used to evaluate cellular immunity in six healthy seropositive donors and in six hematopoietic stem cell transplant recipients (HSCT). Broad CMV-specific responses were found to epitopes within several CMV polypeptides that were restricted by multiple HLA alleles in several individuals. The results document the simultaneous expansion of several of these CD8+ T-lymphocyte populations following allogeneic HSCT. The combined levels of CMV epitope-specific T-cells exceeded 20% of CD8+ T-lymphocytes in some individuals. The cytotoxic functionality of 26 different populations of CMV-specific T-lymphocytes detected within this group of 12 individuals was addressed by utilizing a recently described assay that measures transient surface levels of the lysosomal membrane proteins LAMP-1 (CD107a) and LAMP 2 (CD107b) after peptide stimulation. This degranulation/mobilization assay can be combined with tetramer staining of antigen-specific CD8+ T-lymphocytes, and has potential as a surrogate marker for cytotoxic function. We found that a significant proportion of CD8+ T-lymphocytes specific for epitopes within the CMV pp65 and pp50 gene products had functional potential as measured by this assay (median percentage of cells within 14 T-cell populations staining with pp65 or pp50 tetramers that degranulated on stimulation with cognate peptide = 26.0% and 19.8%). By contrast, CD8+ T-lymphocytes specific for epitopes within the CMV IE-1 gene product had markedly reduced functionality (median percentage of cells within 12 T-cell populations staining with IE-1 tetramers that degranulated on stimulation with cognate peptide = 5.6%). This difference was significant (p= 0.003 by an F-test after adjusting for HLA and using interaction of subjects and epitopes as the error). This reduced degranulation efficiency of IE-1-specific T cells is consistent with their inefficient cytotoxic recognition of CMV-infected autologous fibroblasts. Further characterization of this functional dichotomy includes comparison of cell surface marker phenotype and cytokine release in response to antigenic presentation. These functional differences between T-lymphocyte populations within the same individual have implications for choosing antigens that are both protective and necessary to include in a CMV vaccine for transplant patients at risk for infectious complications after viral reactivation.
Background: Vaccines for SARS-CoV-2 have been considerably effective in reducing rates of infecti... more Background: Vaccines for SARS-CoV-2 have been considerably effective in reducing rates of infection and severe COVID-19. However, many patients, especially those who are immunocompromised due to cancer or other factors, as well as individuals who are unable to receive vaccines or are in resource-poor countries, will continue to be at risk for COVID-19. We describe clinical, therapeutic, and immunologic correlatives in two patients with cancer and severe COVID-19 who were treated with leflunomide after failing to respond to standard-of-care comprising remdesivir and dexamethasone. Both patients had breast cancer and were on therapy for the malignancy. Methods: The protocol is designed with the primary objective to assess the safety and tolerability of leflunomide in treating severe COVID-19 in patients with cancer. Leflunomide dosing consisted of a loading dose of 100 mg daily for the first three days, followed by daily dosing, at the assigned dose level (Dose Level 1: 40 mg, Dose Le...
Adoptive transfer of ex vivo expanded CMV-specific T cells is an effective approach, and an attra... more Adoptive transfer of ex vivo expanded CMV-specific T cells is an effective approach, and an attractive alternative to using anti-virals to manage CMV infection for HSCT recipients. We recently published a robust approach to expanding CMV-specific CTL based on infection of autologous EBV-LCL with the attenuated poxvirus, Modified Vaccinia Ankara (MVA), expressing CMV pp65, pp150, and IE1 proteins. This approach causes vigorous, up to 500fold expansions in as little as 12–14 days of memory CD8+ T cells specific for these immunodominant antigens. In order to improve the specificity of the expanded T cells, a method was sought to derive effective antigen presenting cells (APC) that avoided the use of EBV-LCL. Of equal importance is to develop an expansion approach that avoids the need to involve virally infected APC in developing a clinical product. Our preliminary observation is that rMVA can infect PBMC in vitro, causing high levels of expression of recombinant CMV antigens. To be per...
Haploidentical hematopoietic cell transplantation (HaploHCT) using high-dose post-transplant cycl... more Haploidentical hematopoietic cell transplantation (HaploHCT) using high-dose post-transplant cyclophosphamide (PTCy) has been increasingly used in patients with hematologic disorders with promising results. However, limited data are available on the incidence, pattern, and risk factors including donor/recipient KIR genotypes for cytomegalovirus (CMV) reactivation after HaploHCT with PTCy. Furthermore, the impact of CMV reactivation on HaploHCT outcomes is not yet well-described. In this retrospective study, we evaluated a series of 119 consecutive patients who underwent HaploHCT with PTCy at City of Hope for hematological diseases, between January 2009 and December 2016. CMV reactivation was monitored by our institutional PCR assay (quantitative detection limit: 500 gc/ml, qualitative limit: 250 gc/ml) at least once a week for 100 days post-transplant, with preemptive anti-CMV therapy for positive PCR according to our institutional guidelines. The median age of the cohort was 43 yea...
Natural killer (NK) cells can control malignant as well as virus-infected cells. We and others ha... more Natural killer (NK) cells can control malignant as well as virus-infected cells. We and others have recently described clonal expansion of NK cells expressing the activating receptor NKG2C, the maturation marker CD57, and self-educating inhibitory killer immunoglobulin-like receptors (KIRs) during the course of CMV infection. These NK cells persist long after the resolution of infection and are potent producers of interferon-γ, as such displaying adaptive or memory properties. While it is known that CMV drives a unique program of NK cell maturation, it is unclear how latent CMV may affect maturation of NK cells and other lymphocyte subsets following hematopoietic cell transplantation (HCT). 292 sibling and umbilical cord blood (UCB) HCT recipients were assessed for expression of NKG2C and CD57 on NK (total CD56+CD3- cells) or T cells (CD56+CD3+ and CD56-CD3+) during immune reconstitution. Data was analyzed based on pre-transplant CMV serostatus and post-HCT CMV-reactivation dete...
As human cytomegalovirus (HCMV) is a common cause of disease in newborns and transplant recipient... more As human cytomegalovirus (HCMV) is a common cause of disease in newborns and transplant recipients, developing an HCMV vaccine is considered a major public health priority. Yet an HCMV vaccine candidate remains elusive. Although the precise HCMV immune correlates of protection are unclear, both humoral and cellular immune responses have been implicated in protection against HCMV infection and disease. Here we describe a vaccine approach based on the well-characterized modified vaccinia virus Ankara (MVA) vector to stimulate robust HCMV humoral and cellular immune responses by an antigen combination composed of the envelope pentamer complex (PC), glycoprotein B (gB), and phosphoprotein 65 (pp65). We show that in mice, multiantigenic MVA vaccine vectors simultaneously expressing all five PC subunits, gB, and pp65 elicit potent complement-independent and complement-dependent HCMV neutralizing antibodies as well as mouse and human MHC-restricted, polyfunctional T cell responses by the i...
Proinflammatory signals promote prostate tumorigenesis and progression, but their origins and dow... more Proinflammatory signals promote prostate tumorigenesis and progression, but their origins and downstream effects remain unclear. We recently demonstrated that the expression of an innate immune receptor, TLR9, by prostate cancer cells is critical for their tumor-propagating potential. We investigated whether cancer cell-intrinsic TLR9 signaling alters composition of the prostate tumor microenvironment. We generated Ras/Myc (RM9) and Myc-driven (Myc-CaP) prostate cancer cells expressing the tetracycline-inducible gene Tlr9 (Tlr9(ON) ) or the control LacZ (LacZ(ON) ). When engrafted into mice and treated with tetracycline, Tlr9(ON) , but not LacZ(ON) , tumors showed accelerated growth kinetics compared with tumors in PBS-treated mice. Tlr9 upregulation in cancer cells triggered the selective accumulation of CD11b(+)Ly6G(HI)Ly6C(LO) myeloid cells, phenotypically similar to PMN-MDSCs. The PMN-MDSCs from tetracycline-treated RM9-Tlr9(ON) tumors increased the immunosuppressive activity of...
CMVpp65, a candidate component of human cytomegalovirus (CMV) vaccines, has phosphokinase (PK) ac... more CMVpp65, a candidate component of human cytomegalovirus (CMV) vaccines, has phosphokinase (PK) activity that could affect vaccine safety. A mutated form of CMVpp65 substituting asparagine for lysine at the adenosine triphosphate (ATP)-binding site (CMVpp65mII) is kinase-deficient. Using DNA immunizations in a transgenic human leucocyte antigen (HLA)A*0201.Kb mouse model, the mutated CMVpp65 induced cytotoxic T lymphocytes (CTL) immunity similarly to native CMVpp65. Murine CTL lines generated from these immunizations killed human cells either after sensitization with CMVpp65-specific peptides or after infection with either CMV-Towne strain or rvac-pp65. It is proposed that CMVpp65mII be evaluated in candidate vaccines for CMV.
Supplemental Figures. S1: Chemokine/Cytokine Quantification in Serum and in Tumor Microenvironmen... more Supplemental Figures. S1: Chemokine/Cytokine Quantification in Serum and in Tumor Microenvironment. S2: Blocking of CXCR3 suppresses the infiltration of CD8 T cell. S3: Administration of 7DW8-5 results in activation of NKT cells, NK cells, and DC in mouse spleen.
Human CMV continues to be a major risk factor in patients undergoing allogeneic hematopoietic ste... more Human CMV continues to be a major risk factor in patients undergoing allogeneic hematopoietic stem cell (HSCT) and solid organ transplant (SOT), despite advancement of antiviral therapy. CMV tegument protein pp65 and CMV immediate early gene product IE1 are both considered immunodominant targets of cell-mediated immunity (CMI) and potentially capable of controlling CMV infection. Similar to healthy adults, CMV tegument protein pp65 and IE1 are frequently recognized by human CD4 and CD8 T cells in HCT and SOT recipients. To better assess their role in host defense, we have constructed a novel attenuated poxvirus (MVA) transfer vector named pZWIIA and generated a recombinant MVA (rMVA) expressing both full-length pp65 and exon4 of IE1 (pp65-IE1-MVA) at high levels, followed by the genetic removal of the bacterial marker gene used to distinguish recombinant forms during the screening process. Immunogenicity evaluation indicates that pp65-IE1-MVA not only can induce robust primary CMI to both antigens in HLA A2.1, B7 and A11 transgenic mice, but also can stimulate vigorous expansion of memory T lymphocyte responses to pp65 and IE1 in PBMC of CMV-positive donors. The recent discovery that additional early antigens of CMV are also well-recognized by healthy adults prompted us to include the IE2 antigen in the vaccine. Evaluation of in vivo immunogenicity of the vaccine expressing IE2 in both transgenic mice and in vitro antigenicity in human peripheral blood lymphocytes of HSCT and SOT recipients will be presented. A novel overlapping peptide library spanning full-length IE2 has been constructed in our laboratory, and used in conjunction with the vaccine expressing IE2, along with IE1 and pp65. Since MVA has a foreign gene capacity of over 30 kilobases (Kb), we have developed a strategy to maximize coverage of haplotypes through insertion of 6 additional antigens into novel insertion sites of the virus, for a total of just 12.0 Kb of the CMV genome. The selected antigens cover 83% of the population, while representing over 22% of the combined CD4 and CD8 T cell repertoire in seropositives. Our studies of the recognition of these antigens expressed in MVA and monitored with our own synthesized over-lapping peptide libraries in both HSCT and SOT recipients will be presented. These properties make the MVA-based vaccine ideal for the dual role of priming and boosting CMV-specific T cell immunity as a means to control CMV disease in recipients of HCT or SOT. pZWIIA alone or in combination with other MVA transfer vectors can be used to generate MVA based multiple-antigen vaccine which have application in vaccine development for a wide spectrum of infectious diseases and cancer.
Recent studies using direct stimulation of PBMC from CMV-positive individuals with optimal CTL ep... more Recent studies using direct stimulation of PBMC from CMV-positive individuals with optimal CTL epitope peptides have identified new antigens that are targets of the host immune response. The growing number of targets of the cellular immune response has prompted an evaluation of which antigens are potentially associated with protective immunity. A panel of seven human cytomegalovirus (CMV) epitope peptides and the corresponding MHC-I tetramers was used to evaluate cellular immunity in six healthy seropositive donors and in six hematopoietic stem cell transplant recipients (HSCT). Broad CMV-specific responses were found to epitopes within several CMV polypeptides that were restricted by multiple HLA alleles in several individuals. The results document the simultaneous expansion of several of these CD8+ T-lymphocyte populations following allogeneic HSCT. The combined levels of CMV epitope-specific T-cells exceeded 20% of CD8+ T-lymphocytes in some individuals. The cytotoxic functionality of 26 different populations of CMV-specific T-lymphocytes detected within this group of 12 individuals was addressed by utilizing a recently described assay that measures transient surface levels of the lysosomal membrane proteins LAMP-1 (CD107a) and LAMP 2 (CD107b) after peptide stimulation. This degranulation/mobilization assay can be combined with tetramer staining of antigen-specific CD8+ T-lymphocytes, and has potential as a surrogate marker for cytotoxic function. We found that a significant proportion of CD8+ T-lymphocytes specific for epitopes within the CMV pp65 and pp50 gene products had functional potential as measured by this assay (median percentage of cells within 14 T-cell populations staining with pp65 or pp50 tetramers that degranulated on stimulation with cognate peptide = 26.0% and 19.8%). By contrast, CD8+ T-lymphocytes specific for epitopes within the CMV IE-1 gene product had markedly reduced functionality (median percentage of cells within 12 T-cell populations staining with IE-1 tetramers that degranulated on stimulation with cognate peptide = 5.6%). This difference was significant (p= 0.003 by an F-test after adjusting for HLA and using interaction of subjects and epitopes as the error). This reduced degranulation efficiency of IE-1-specific T cells is consistent with their inefficient cytotoxic recognition of CMV-infected autologous fibroblasts. Further characterization of this functional dichotomy includes comparison of cell surface marker phenotype and cytokine release in response to antigenic presentation. These functional differences between T-lymphocyte populations within the same individual have implications for choosing antigens that are both protective and necessary to include in a CMV vaccine for transplant patients at risk for infectious complications after viral reactivation.
Background: Vaccines for SARS-CoV-2 have been considerably effective in reducing rates of infecti... more Background: Vaccines for SARS-CoV-2 have been considerably effective in reducing rates of infection and severe COVID-19. However, many patients, especially those who are immunocompromised due to cancer or other factors, as well as individuals who are unable to receive vaccines or are in resource-poor countries, will continue to be at risk for COVID-19. We describe clinical, therapeutic, and immunologic correlatives in two patients with cancer and severe COVID-19 who were treated with leflunomide after failing to respond to standard-of-care comprising remdesivir and dexamethasone. Both patients had breast cancer and were on therapy for the malignancy. Methods: The protocol is designed with the primary objective to assess the safety and tolerability of leflunomide in treating severe COVID-19 in patients with cancer. Leflunomide dosing consisted of a loading dose of 100 mg daily for the first three days, followed by daily dosing, at the assigned dose level (Dose Level 1: 40 mg, Dose Le...
Adoptive transfer of ex vivo expanded CMV-specific T cells is an effective approach, and an attra... more Adoptive transfer of ex vivo expanded CMV-specific T cells is an effective approach, and an attractive alternative to using anti-virals to manage CMV infection for HSCT recipients. We recently published a robust approach to expanding CMV-specific CTL based on infection of autologous EBV-LCL with the attenuated poxvirus, Modified Vaccinia Ankara (MVA), expressing CMV pp65, pp150, and IE1 proteins. This approach causes vigorous, up to 500fold expansions in as little as 12–14 days of memory CD8+ T cells specific for these immunodominant antigens. In order to improve the specificity of the expanded T cells, a method was sought to derive effective antigen presenting cells (APC) that avoided the use of EBV-LCL. Of equal importance is to develop an expansion approach that avoids the need to involve virally infected APC in developing a clinical product. Our preliminary observation is that rMVA can infect PBMC in vitro, causing high levels of expression of recombinant CMV antigens. To be per...
Haploidentical hematopoietic cell transplantation (HaploHCT) using high-dose post-transplant cycl... more Haploidentical hematopoietic cell transplantation (HaploHCT) using high-dose post-transplant cyclophosphamide (PTCy) has been increasingly used in patients with hematologic disorders with promising results. However, limited data are available on the incidence, pattern, and risk factors including donor/recipient KIR genotypes for cytomegalovirus (CMV) reactivation after HaploHCT with PTCy. Furthermore, the impact of CMV reactivation on HaploHCT outcomes is not yet well-described. In this retrospective study, we evaluated a series of 119 consecutive patients who underwent HaploHCT with PTCy at City of Hope for hematological diseases, between January 2009 and December 2016. CMV reactivation was monitored by our institutional PCR assay (quantitative detection limit: 500 gc/ml, qualitative limit: 250 gc/ml) at least once a week for 100 days post-transplant, with preemptive anti-CMV therapy for positive PCR according to our institutional guidelines. The median age of the cohort was 43 yea...
Natural killer (NK) cells can control malignant as well as virus-infected cells. We and others ha... more Natural killer (NK) cells can control malignant as well as virus-infected cells. We and others have recently described clonal expansion of NK cells expressing the activating receptor NKG2C, the maturation marker CD57, and self-educating inhibitory killer immunoglobulin-like receptors (KIRs) during the course of CMV infection. These NK cells persist long after the resolution of infection and are potent producers of interferon-γ, as such displaying adaptive or memory properties. While it is known that CMV drives a unique program of NK cell maturation, it is unclear how latent CMV may affect maturation of NK cells and other lymphocyte subsets following hematopoietic cell transplantation (HCT). 292 sibling and umbilical cord blood (UCB) HCT recipients were assessed for expression of NKG2C and CD57 on NK (total CD56+CD3- cells) or T cells (CD56+CD3+ and CD56-CD3+) during immune reconstitution. Data was analyzed based on pre-transplant CMV serostatus and post-HCT CMV-reactivation dete...
As human cytomegalovirus (HCMV) is a common cause of disease in newborns and transplant recipient... more As human cytomegalovirus (HCMV) is a common cause of disease in newborns and transplant recipients, developing an HCMV vaccine is considered a major public health priority. Yet an HCMV vaccine candidate remains elusive. Although the precise HCMV immune correlates of protection are unclear, both humoral and cellular immune responses have been implicated in protection against HCMV infection and disease. Here we describe a vaccine approach based on the well-characterized modified vaccinia virus Ankara (MVA) vector to stimulate robust HCMV humoral and cellular immune responses by an antigen combination composed of the envelope pentamer complex (PC), glycoprotein B (gB), and phosphoprotein 65 (pp65). We show that in mice, multiantigenic MVA vaccine vectors simultaneously expressing all five PC subunits, gB, and pp65 elicit potent complement-independent and complement-dependent HCMV neutralizing antibodies as well as mouse and human MHC-restricted, polyfunctional T cell responses by the i...
Proinflammatory signals promote prostate tumorigenesis and progression, but their origins and dow... more Proinflammatory signals promote prostate tumorigenesis and progression, but their origins and downstream effects remain unclear. We recently demonstrated that the expression of an innate immune receptor, TLR9, by prostate cancer cells is critical for their tumor-propagating potential. We investigated whether cancer cell-intrinsic TLR9 signaling alters composition of the prostate tumor microenvironment. We generated Ras/Myc (RM9) and Myc-driven (Myc-CaP) prostate cancer cells expressing the tetracycline-inducible gene Tlr9 (Tlr9(ON) ) or the control LacZ (LacZ(ON) ). When engrafted into mice and treated with tetracycline, Tlr9(ON) , but not LacZ(ON) , tumors showed accelerated growth kinetics compared with tumors in PBS-treated mice. Tlr9 upregulation in cancer cells triggered the selective accumulation of CD11b(+)Ly6G(HI)Ly6C(LO) myeloid cells, phenotypically similar to PMN-MDSCs. The PMN-MDSCs from tetracycline-treated RM9-Tlr9(ON) tumors increased the immunosuppressive activity of...
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