Final Report Summary - VACCINATION TO CMV (Dissection of T cell cosignaling and development of vaccination strategies to cytomegalovirus)
Vaccination to cytomegalovirus (CMV):
Dissection of T cell co-signaling and development of vaccination strategies to CMV
Human cytomegalovirus (HCMV) is a beta-herpesvirus that establishes a lifelong infection in the vast majority of the world's population. HCMV is responsible for the morbidity of many immunocompromised patients and neonatal disabilities. In addition, HCMV significantly contributes to the immune senescence of the elderly, leading to an increased susceptibility to infections as one age. Efforts to generate a CMV vaccine have been unsuccessful thus far.
The current study examined viral immunomodulatory strategies of cytomegalovirus (CMV) that interfere with cosignaling and exploited this knowledge for the development of novel vaccination development strategies. The immune evasion mechanisms will be studied in mouse models using mouse-adapted CMV (MCMV). The genome of MCMV is colinear with that of HCMV, is roughly as large (approximately 230kB), encodes about 200 protein products and displays a similar course of infection to that of HCMV, making it an excellent animal model for studying the requirements for host immune control of CMV infection. The proposed studies are innovative in that we examine virus-host interactions from both the virus perspective and host perspective, which provide novel insights for rational design of vaccines against cytomegalovirus.
The primary objectives of this proposal are;
1) to examine the mechanisms of the virus that counteract the host costimulatory pathways
2) to identify the positive and negative costimulatory molecules of the host that contribute to control CMV infection
3) to target the relevant costimulatory pathways to develop a CMV vaccine
1. Determination of the mechanisms of the virus that counteract the host costimulatory pathways.
B7.1 and B7.2 are the costimulatory ligands that bind the main costimulatory receptor on T cells, CD28, which is counteracted (at later timepoints) through binding of the B7 molecules to the inhibitory receptor CTLA-4. Specific MCMV gene products down-regulate B7.1 (m138) and B7.2 (m147.5) strongly suggesting that B7.1/2 molecules play an important role in regulating immunity to this virus. HCMV also down-regulates B7.1 and B7.2 on the surface of infected dendritic cells (DCs), although the genes mediating the down-regulation have not yet been identified. The specific targeting of B7.1 and B7.2 by CMV strongly suggests that this cosignaling system may contribute to immune defence against this persistent virus. Moreover, it provides for the first time to investigate the importance of B7-CD28 signaling in establishing lifelong host-virus equilibrium, from both the virus and host perspective.
To test the importance of B7-down-modulation from the virus perspective, we generated MCMV mutants that lack m138 and m147.5 (MCMV-deltaB7) (Arens et al. Journal of Virology, 2011). Using these mutants we have addressed in detail the importance of this down-modulating effect on B7 expression by the virus with respect to the generation and maintenance of CD8 and CD4 T cell responses and viral replication. Our findings showed significant lower titers in the livers of MCMV-deltaB7 infected mice as compared to MCMV-WT, which was accompanied by increased CD4 T cell responses (Arens et al. Journal of Virology, 2011). Thus the MCMV-deltaB7, as compared to MCMV, induces stronger T cell responses due to increased availability of B7 on infected DCs and this will result in the observed decreased viral replication.
To determine the role of viral immunomodulatory genes on expression of costimulatory TNF family members we have examined the expression of CD70, OX40 ligand and 4-1BB ligand on infected DCs. For this dendritic cells (DCs) were infected with MCMV-expressing Green Fluorescent Protein (GFP) to distinguish between infected and non-infected DCs and expression of costimulatory molecules was kinetically monitored. Our data show that the TNF-like molecules CD70 (CD27L) and OX40L (CD134L) are downmodulated on CMV-infected dendritic cells but 4-1BBL was not downmodulated. Further experiments using deletion-mutants of MCMV were performed to identify which region(s) of MCMV are causing the downmodulation of CD70 and OX40L (manuscript in preparation).
2. Determination of the positive and negative costimulatory molecules of the host that contribute to control CMV infection
To determine the impact of host B7 expression on immunity to MCMV, we assessed the role of the B7.1/2 molecules in the induction of MCMV-specific T cell responses and the control of viral replication (Arens et al., Journal of Immunology 2011; Arens et al. Journal of Virology, 2011). Early after infection significant differences in the frequencies of MCMV-specific CD4 and CD8 T cells were observed between wild-type and B7.1/2 dKO mice in both the spleens and lungs. The quality of the T cell responses in WT and B7.1/2 dKO mice were significantly different with respect to autocrine IL-2 expression but not cytotoxicity. Together, these results reveal that host B7-CD28 costimulation regulates the magnitude and kinetics of the multifaceted T cell response that develops during CMV infection.
To evaluate if manipulation of the cosignaling molecules that dampen immune responses, PD-1 (receptor for PDL-1 and PDL-2) and CTLA-4 (inhibitory receptor for B7.1 and B7.2) had an effect on shaping the MCMV-specific CD4 and CD8 T cell response and MCMV replication we infected WT and B7.1/2 knockout mice with MCMV and treated them with PD-L1 (clone MIH-5),CTLA-4 (clone UC10) or control monoclonal antibody (mAb) during the first week after infection. In wild-type mice, blocking of PD-L1 resulted in a modest (but significant) increase in most of the MCMV-specific CD4 and CD8 T cell responses examined and as a consequence a reduction in viral titers. Remarkably, PDL-1 blockade in B7 deficient mice resulted in a marked increase in MCMV-specific T cell responses, indicating that the lack of positive costimulatory signals (i.e. CD28) sensitizes for usage of PD-1. Blocking of CTLA-4 resulted in increased expansion (percentage and absolute numbers) of both MCMV-specific CD4 and CD8 T cell responses in wild-type mice and as anticipated not in B7 deficient mice (data not shown).
To address the role of CD27-CD70 during MCMV infection in detail, we infected wild-type mice with and without blocking CD70 mAb and examined the viral replication and MCMV-specific CD4 and CD8 T cell responses in detail (kinetics, polyfunctional cytokine expression, cytotoxicity). The MCMV production in the salivary glands was significantly higher with CD70 blockade. Both during the acute and persistent infection the magnitude and quality of the T cell response was impaired by CD70 blockade. Adoptive transfer studies confirmed that CD27 expression on T cells is important (manuscript submitted).
3. Targeting of costimulatory pathways to develop a CMV vaccine
For this aim we first tested whether dominant MCMV-specific CD4 and CD8 epitopes were able to induce T cell expansion. Single and combinations of the peptides were injected subcutaneously along with CpG adjuvants (TLR 9 ligands) into wild-type mice and the T cell responses to these peptides were analysed. Both at day 8 and 40 polyfunctional T cell responses were detected. Although these responses were lower than natural CMV infection, they still protected against recurrent CMV infection (manuscript in preparation). Importantly, by using blocking antibodies to negative cosignaling molecules (PD-1, CTLA-4) and agonistic antibodies to CD27 and OX40 we could enhance the peptide vaccine-induced immunity to CMV.
The objectives of the proposal have been successfully achieved and have led to novel insights into the role of costimulatory molecules in combatting persistent viral infections such as CMV. This could lead to development of novel vaccines based on targeting costimulation.
Dissection of T cell co-signaling and development of vaccination strategies to CMV
Human cytomegalovirus (HCMV) is a beta-herpesvirus that establishes a lifelong infection in the vast majority of the world's population. HCMV is responsible for the morbidity of many immunocompromised patients and neonatal disabilities. In addition, HCMV significantly contributes to the immune senescence of the elderly, leading to an increased susceptibility to infections as one age. Efforts to generate a CMV vaccine have been unsuccessful thus far.
The current study examined viral immunomodulatory strategies of cytomegalovirus (CMV) that interfere with cosignaling and exploited this knowledge for the development of novel vaccination development strategies. The immune evasion mechanisms will be studied in mouse models using mouse-adapted CMV (MCMV). The genome of MCMV is colinear with that of HCMV, is roughly as large (approximately 230kB), encodes about 200 protein products and displays a similar course of infection to that of HCMV, making it an excellent animal model for studying the requirements for host immune control of CMV infection. The proposed studies are innovative in that we examine virus-host interactions from both the virus perspective and host perspective, which provide novel insights for rational design of vaccines against cytomegalovirus.
The primary objectives of this proposal are;
1) to examine the mechanisms of the virus that counteract the host costimulatory pathways
2) to identify the positive and negative costimulatory molecules of the host that contribute to control CMV infection
3) to target the relevant costimulatory pathways to develop a CMV vaccine
1. Determination of the mechanisms of the virus that counteract the host costimulatory pathways.
B7.1 and B7.2 are the costimulatory ligands that bind the main costimulatory receptor on T cells, CD28, which is counteracted (at later timepoints) through binding of the B7 molecules to the inhibitory receptor CTLA-4. Specific MCMV gene products down-regulate B7.1 (m138) and B7.2 (m147.5) strongly suggesting that B7.1/2 molecules play an important role in regulating immunity to this virus. HCMV also down-regulates B7.1 and B7.2 on the surface of infected dendritic cells (DCs), although the genes mediating the down-regulation have not yet been identified. The specific targeting of B7.1 and B7.2 by CMV strongly suggests that this cosignaling system may contribute to immune defence against this persistent virus. Moreover, it provides for the first time to investigate the importance of B7-CD28 signaling in establishing lifelong host-virus equilibrium, from both the virus and host perspective.
To test the importance of B7-down-modulation from the virus perspective, we generated MCMV mutants that lack m138 and m147.5 (MCMV-deltaB7) (Arens et al. Journal of Virology, 2011). Using these mutants we have addressed in detail the importance of this down-modulating effect on B7 expression by the virus with respect to the generation and maintenance of CD8 and CD4 T cell responses and viral replication. Our findings showed significant lower titers in the livers of MCMV-deltaB7 infected mice as compared to MCMV-WT, which was accompanied by increased CD4 T cell responses (Arens et al. Journal of Virology, 2011). Thus the MCMV-deltaB7, as compared to MCMV, induces stronger T cell responses due to increased availability of B7 on infected DCs and this will result in the observed decreased viral replication.
To determine the role of viral immunomodulatory genes on expression of costimulatory TNF family members we have examined the expression of CD70, OX40 ligand and 4-1BB ligand on infected DCs. For this dendritic cells (DCs) were infected with MCMV-expressing Green Fluorescent Protein (GFP) to distinguish between infected and non-infected DCs and expression of costimulatory molecules was kinetically monitored. Our data show that the TNF-like molecules CD70 (CD27L) and OX40L (CD134L) are downmodulated on CMV-infected dendritic cells but 4-1BBL was not downmodulated. Further experiments using deletion-mutants of MCMV were performed to identify which region(s) of MCMV are causing the downmodulation of CD70 and OX40L (manuscript in preparation).
2. Determination of the positive and negative costimulatory molecules of the host that contribute to control CMV infection
To determine the impact of host B7 expression on immunity to MCMV, we assessed the role of the B7.1/2 molecules in the induction of MCMV-specific T cell responses and the control of viral replication (Arens et al., Journal of Immunology 2011; Arens et al. Journal of Virology, 2011). Early after infection significant differences in the frequencies of MCMV-specific CD4 and CD8 T cells were observed between wild-type and B7.1/2 dKO mice in both the spleens and lungs. The quality of the T cell responses in WT and B7.1/2 dKO mice were significantly different with respect to autocrine IL-2 expression but not cytotoxicity. Together, these results reveal that host B7-CD28 costimulation regulates the magnitude and kinetics of the multifaceted T cell response that develops during CMV infection.
To evaluate if manipulation of the cosignaling molecules that dampen immune responses, PD-1 (receptor for PDL-1 and PDL-2) and CTLA-4 (inhibitory receptor for B7.1 and B7.2) had an effect on shaping the MCMV-specific CD4 and CD8 T cell response and MCMV replication we infected WT and B7.1/2 knockout mice with MCMV and treated them with PD-L1 (clone MIH-5),CTLA-4 (clone UC10) or control monoclonal antibody (mAb) during the first week after infection. In wild-type mice, blocking of PD-L1 resulted in a modest (but significant) increase in most of the MCMV-specific CD4 and CD8 T cell responses examined and as a consequence a reduction in viral titers. Remarkably, PDL-1 blockade in B7 deficient mice resulted in a marked increase in MCMV-specific T cell responses, indicating that the lack of positive costimulatory signals (i.e. CD28) sensitizes for usage of PD-1. Blocking of CTLA-4 resulted in increased expansion (percentage and absolute numbers) of both MCMV-specific CD4 and CD8 T cell responses in wild-type mice and as anticipated not in B7 deficient mice (data not shown).
To address the role of CD27-CD70 during MCMV infection in detail, we infected wild-type mice with and without blocking CD70 mAb and examined the viral replication and MCMV-specific CD4 and CD8 T cell responses in detail (kinetics, polyfunctional cytokine expression, cytotoxicity). The MCMV production in the salivary glands was significantly higher with CD70 blockade. Both during the acute and persistent infection the magnitude and quality of the T cell response was impaired by CD70 blockade. Adoptive transfer studies confirmed that CD27 expression on T cells is important (manuscript submitted).
3. Targeting of costimulatory pathways to develop a CMV vaccine
For this aim we first tested whether dominant MCMV-specific CD4 and CD8 epitopes were able to induce T cell expansion. Single and combinations of the peptides were injected subcutaneously along with CpG adjuvants (TLR 9 ligands) into wild-type mice and the T cell responses to these peptides were analysed. Both at day 8 and 40 polyfunctional T cell responses were detected. Although these responses were lower than natural CMV infection, they still protected against recurrent CMV infection (manuscript in preparation). Importantly, by using blocking antibodies to negative cosignaling molecules (PD-1, CTLA-4) and agonistic antibodies to CD27 and OX40 we could enhance the peptide vaccine-induced immunity to CMV.
The objectives of the proposal have been successfully achieved and have led to novel insights into the role of costimulatory molecules in combatting persistent viral infections such as CMV. This could lead to development of novel vaccines based on targeting costimulation.