Vaccine Vectors Harnessing the Power of Cytomegaloviruses.

Cytomegalovirus (CMV) species have been gaining attention as experimental vaccine vectors inducing cellular immune responses of unparalleled strength and protection. This review outline the strengths and the restrictions of CMV-based vectors, in light of the known aspects of CMV infection, pathogenicity and immunity. We discuss aspects to be considered when optimizing CMV based vaccines, including the innate immune response, the adaptive humoral immunity and the T-cell responses. We also discuss the antigenic epitopes presented by unconventional major histocompatibility complex (MHC) molecules in some CMV delivery systems and considerations about routes for delivery for the induction of systemic or mucosal immune responses. With the first clinical trials initiating, CMV-based vaccine vectors are entering a mature phase of development. This impetus needs to be maintained by scientific advances that feed the progress of this technological platform

Immune Protection against Virus Challenge in Aging Mice Is Not Affected by Latent Herpesviral Infections.

Latent herpesvirus infections alter immune homeostasis. To understand if this results in aging-related loss of immune protection against emerging infections, we challenged old mice carrying latent mouse cytomegalovirus (CMV), herpes simplex virus 1 (HSV-1), and/or murine gammaherpesvirus 68 (MHV-68) with influenza virus, West Nile virus (WNV), or vesicular stomatitis virus (VSV). We observed no increase in mortality or weight loss compared to results seen with herpesvirus-negative counterparts and a relative but not absolute reduction in CD8 responses to acute infections. Therefore, the presence of herpesviruses does not appear to increase susceptibility to emerging infections in aging patients

Murine cytomegalovirus infection via the intranasal route offers a robust model of immunity upon mucosal CMV infection.

Cytomegalovirus (CMV) is a ubiquitous virus, causing the most common congenital infection in humans, yet a vaccine against this virus is not available. The experimental study of immunity against CMV in animal models of infection, such as the infection of mice with the mouse CMV (MCMV), has relied on systemic intraperitoneal infection protocols, although the infection naturally transmits by mucosal routes via body fluids containing CMV. To characterize the biology of infections by mucosal routes, we have compared the kinetics of virus replication, the latent viral load, and CD8 T cell responses in lymphoid organs upon experimental intranasal and intragastric infection to intraperitoneal infection of two unrelated mouse strains. We have observed that intranasal infection induces robust and persistent virus replication in lungs and salivary glands, but a poor one in the spleen. CD8 T cell responses were somewhat weaker than upon intraperitoneal infection, but showed similar kinetic profiles and phenotypes of antigen-specific cells. On the other hand, intragastric infection resulted in abortive or poor virus replication in all tested organs, and poor T cell responses to the virus, especially at late times after infection. Consistent with the T cell kinetics, the MCMV latent load was high in the lungs, but low in the spleen of intranasally infected mice and lowest in all tested organs upon intragastric infection. In conclusion, we show here that intranasal, but not intragastric infection of mice with MCMV represents a robust model to study short and long-term biology of CMV infection by a mucosal route

Peptide Processing Is Critical for T-Cell Memory Inflation and May Be Optimized to Improve Immune Protection by CMV-Based Vaccine Vectors.

Cytomegalovirus (CMV) elicits long-term T-cell immunity of unparalleled strength, which has allowed the development of highly protective CMV-based vaccine vectors. Counterintuitively, experimental vaccines encoding a single MHC-I restricted epitope offered better immune protection than those expressing entire proteins, including the same epitope. To clarify this conundrum, we generated recombinant murine CMVs (MCMVs) encoding well-characterized MHC-I epitopes at different positions within viral genes and observed strong immune responses and protection against viruses and tumor growth when the epitopes were expressed at the protein C-terminus. We used the M45-encoded conventional epitope HGIRNASFI to dissect this phenomenon at the molecular level. A recombinant MCMV expressing HGIRNASFI on the C-terminus of M45, in contrast to wild-type MCMV, enabled peptide processing by the constitutive proteasome, direct antigen presentation, and an inflation of antigen-specific effector memory cells. Consequently, our results indicate that constitutive proteasome processing of antigenic epitopes in latently infected cells is required for robust inflationary responses. This insight allows utilizing the epitope positioning in the design of CMV-based vectors as a novel strategy for enhancing their efficacy

Immune protection by C-terminal epitope localization in a CMV vaccine vector.

<p>(<b>A-C</b>) Mice were prime/boosted at 4 weeks intervals with recombinant MCMVs or control virus and challenged with 2.5x10⁴ TC-1 cells/mouse at least 10 weeks after priming. Tumor size measured by caliper (mean +/- SEM is shown). <b>(A)</b> Immunization was performed with 10⁶ PFU of MCMV^E6+E7 (n = 9) and tumor growth compared to unvaccinated (naïve) controls (n = 10) (<b>B</b>) Mice were immunized with 10⁵ PFU of MCMV^ie2E7, and compared to MCMV^ie2SL or PBS control (n = 10 in each group) (<b>C</b>) Mice were prime/boosted with 10⁵ PFU of MCMV^ieE6-7Full (n = 9) and compared to unvaccinated controls (n = 12) (<b>D</b>) Representative flow cytometry plots of dextramer-stained blood lymphocytes from mice infected with 10⁵ PFU of MCMV^E6+E7, MCMV^ie2E7, MCMV^ie2E6-7Full or MCMV^ie2SL and analyzed by D(b) E7_49-57 dextramer staining for the presence of E7-specific CD8 T cells at 21 weeks post-priming. (<b>E</b>) Group values from dextramer staining as in panel D are shown (each symbol is a mouse; horizontal line shows the median). Significance was assessed by Kruskal—Wallis test followed by Dunn’s post hoc analysis for indicated columns. **<i>p</i> < 0.01, ns—not significant.</p

Peptide C-terminal localization results in better protection and induction of effector memory CD8 T-cell response.

<p><b>(A</b>) 129Sv female mice were i.p. infected with 2x10⁵ PFU MCMV^WT, MCMV^ie2SL or MCMV^M45SL (n = 10 in each group) and 8 months later challenged with 10⁶ PFU of VACV^SL. Seven days post challenge, ovaries were titrated for infectious vaccinia by plaque assay. Histograms show group means, error bars are standard deviations. Significance was assessed by Kruskal—Wallis test followed by Dunn’s post hoc analysis for indicated columns. **<i>p</i> < 0.01, ns—not significant. <b>(B-C)</b> 129/Sv mice were infected intraperitoneally (i.p.) with 2x10⁵ PFU of MCMV^ie2SL, MCMV^M45SL or 10⁶ PFU of VACV^SL. Blood leukocytes were stimulated with the SSIEFARL peptide at 7, 14, 28, 60, 90, 120, 180 dpi. Cells were surface-stained for CD3, CD4, CD8, CD11a, CD44, KLRG1, CD127 and intracellularly for IFNγ expression and analyzed by flow cytometry. (<b>B</b>) Representative dot plots of KLRG1 and CD127 expression in IFNγ producing cells upon 6h SSIEFARL in vitro re-stimulation on days 7 and 180 p.i‥ (<b>C</b>) Left graph—epitope specific cells with the CM phenotype (CD127⁺KLRG1^-). Right graph—epitope specific cells with the EM phenotype (CD127^-KLRG1⁺). The experiment was performed three times independently, at 5 mice per group in each experiment, and grouped averages +/- SEM from all three experiments are shown. Significance on day 180 p.i. was assessed by Kruskal—Wallis test followed by Dunn’s post hoc analysis. *<i>p</i><0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001, ****<i>p</i> < 0.0001, ns—not significant.</p

C-terminal localization of the HGIRNASFI peptide allows its presentation on the surface of infected cells.

<p>(<b>A</b>) Cell surface expression of MHC class I molecule (D^b). LSECs (C57BL/6) were infected with the indicated viruses at MOI of 5 with centrifugal enhancement as described. D^b expression was measured by flow cytometry at 16h p.i‥ Fluorescence histograms for a representative experiment are shown. (<b>B</b>) LSECs were infected with the indicated viruses or incubated with a corresponding dose of UV inactivated virus (UV dose– 150J) at an MOI of 0.2 with centrifugal enhancement and co-cultured with HGIRNASFI-specific CTLs at an E:T ratio 3:1. Co-culture was performed for 15h, upon which the T cells were collected and stained for intracellular IFNγ. Where indicated, virus was inactivated by UV light. Two independent experiments were performed, with 4 or 5 wells per experimental condition. Representative dot plots are shown. (<b>C</b>) Relative intensity of signals measured by targeted nanoLC-MS³ from D^b-immunoprecipitates of cells infected for 24 hours with indicated viruses at an MOI of 2 with centrifugal enhancement. Two high abundant endogenous control peptides (KALINADEL and AALENTHLL) and a low abundant (FGPVNHEEL) endogenous control peptide were present in all IP samples, indicating valid sample processing in all cases. The MCMV target peptide HGIRNASFI was detected only in the cells infected with the MCMV^M45Cterm recombinant. (<b>D</b>) Grouped means +/- SEM of blood CD8 T-cells stained by HGIRNASFI-D^b tetramers in bone-marrow chimeric mice, where C57BL/6 mice received TAP^-/- or C57BL/6 bone marrow at 3 months before infection with 10⁶ PFU/mouse of MCMV^WT (top panel) or MCMV^M45Cterm (bottom panel). The experiment was performed three times at 5 mice per group and pooled results are shown. Significance on day 7 p.i. was assessed by a Mann—Whitney <i>U</i> test. ****<i>p</i> < 0.0001, ns—not significant.</p