Targeted Deletion of Regions Rich in Immune-Evasive Genes from the Cytomegalovirus Genome as a Novel Vaccine Strategy

Abstract

Human cytomegalovirus (CMV), a ubiquitous human pathogen, is a leading cause of congenital infections and represents a serious health risk for the immunosuppressed patient. A vaccine against CMV is currently not available. CMV is characterized by its large genome and by multiple genes modulating the immunity of the host, which cluster predominantly at genome termini. Here, we tested whether the deletion of gene blocks rich in immunomodulatory genes could be used as a novel concept in the generation of immunogenic but avirulent, herpesvirus vaccines. To generate an experimental CMV vaccine, we selectively deleted 32 genes from the mouse cytomegalovirus (MCMV) genome. The resulting mutant grew to titers similar to that of wild-type MCMV in vitro. In vivo, the mutant was 10,000-fold attenuated and well tolerated, even by highly susceptible mice deficient for B, T, and NK cells or for the interferon type I receptor. Equally relevant for safety concerns, immune suppression did not lead to the mutant's reactivation from latency. Immunization with the replicationcompetent mutant, but not with inactivated virus, resulted in protective immunity, which increased over time. Vaccination induced MCMV-specific antibodies and a strong T-cell response. We propose that a targeted and rational approach can improve future herpesvirus vaccines and vaccine vectors.The human cytomegalovirus (HCMV), a betaherpesvirus subfamily member, is a ubiquitous human pathogen that causes congenital infections and also represents a major morbidity risk for immune-suppressed or immunodeficient patients (56).HCMV carries approximately 200 genes, which represent a large antigenic potential. However, despite previous efforts, (59, 60), no effective vaccine has been generated so far (67). Although the Towne strain was shown in numerous studies to be a safe and immunogenic vaccine (1, 29, 30), its immunogenicity was lower than that of the wild-type (WT) virus, and it failed to confer immune protection against infection by natural contact (2). Several features of HCMV infection make vaccine development uniquely difficult. A large number of HCMV genes modulate the innate and adaptive host immune responses to the advantage of the pathogen (43,50,74). Natural CMV infection provides only partial protection, and reinfection can cause congenital CMV disease even in infants of mothers with preconceptional immunity (5, 22). Moreover, persistence of the virus in state of latency, with the possibility of reactivation over the course of the patient's life, represents a safety concern when a live attenuated herpesvirus vaccine is used. Finally, cytomegaloviruses are characterized by strict species specificity, and there is no animal model for direct studies of HCMV infection and immunity in vivo.The infection of mice with mouse CMV (MCMV) represents a widely used in vivo model of CMV infection (40).Studies with the MCMV model revealed that protective immunity against CMV infection requires both B and T cells (31)(32)(33)64). Therefore, it is not surprising that sub...

Recombinant mouse cytomegalovirus expressing a ligand for the NKG2D receptor is attenuated and has improved vaccine properties

“…The vaccination potential of CMV mutants lacking nonessential viral genes has already been proven (33,34). Also, spread-deficient MCMV lacking the essential gene M94 induced a virus-specific immune response and proved to be safe in an immunodeficient host (31).…”

“…Still, a live, attenuated vaccine approach has several characteristics that render it attractive. Unlike subunit vaccines, which induce cellular or humoral immune response to selected antigens only, live vaccines induce a much broader immunity that may mimic protection acquired following natural infection (31,33,(66)(67)(68)(69). Cellular immunity against CMV follows unique kinetics characterized by maintenance or even expansion of the virus-specific CD8 + T cell response over time (70,71).…”

“…Various fluorescently conjugated Abs were used: CD8α (clone 53-6.7), CD27 (LG.7F9), CD62L (MEL-14), CD122 (5H4), CD127 (A7R34), KLRG-1 (2F1), NKG2A/C/E (20D5), NKG2D (M1-6), CTLA-4 (UC10-4B9), PD-1 (J43), IL-2 (JES6-SH4), IFN-γ (XMG1.2), TNF-α (MP6-XT22), CD3β (H57-597), CD11c (N418), NKp46 (29A1.4), PDCA-1 (eBio927), RAE-1γ (CX1), RAE-1αβδε (clone 199205), MULT-1 (clone 237104), H60 (clone 205326). An in vitro assay to detect cytokine production and degranulation was preformed as previously described (33 323 ) for 6 hours at 37°C, with brefeldin A (eBioscience) added for the last 4 hours of stimulation. For degranulation assays, CD107a mAb (D4B) and monensin (eBioscience) were added to the cultured cells during peptide stimulation.…”

“…Their use, however, carries the risk of CMV disease caused by the vaccine strain or reactivation in the immunocompromised state, unless the vaccine virus is efficiently controlled by residual immunity. One approach to generating such an immunogenic, yet safe live vaccine is deletion of viral genes that subvert the host immune response (33,34) or essential genes resulting in spread-deficient virus (31). The other approach is the insertion of a ligand recognized by the activating receptor on immune cells into the CMV genome.…”

The Context of Gene Expression Defines the Immunodominance Hierarchy of Cytomegalovirus Antigens

“…Taken together, these results indicate that gene context is critical in defining the immunodominance of Ags encoded by MCMV. CD8 T cells specific for the SSIEFARL peptide encoded by either of the recombinant MCMVs are functional MCMV has been shown to suppress immunity by modulation of PD-L1 expression on dendritic cells, which is associated with diminished CD8 and CD4 T cell responses (24), and by induction of suboptimal CD8 IFN-g responses (25). It was therefore necessary to determine whether expression context affected SSIEFARL-specific CD8 T cell effector functionality.…”