Suppression of costimulation by human cytomegalovirus promotes evasion of cellular immune defenses.

CD58 is an adhesion molecule that is known to play a critical role in costimulation of effector cells and is intrinsic to immune synapse structure. Herein, we describe a virally encoded gene that inhibits CD58 surface expression. Human cytomegalovirus (HCMV) UL148 was necessary and sufficient to promote intracellular retention of CD58 during HCMV infection. Blocking studies with antagonistic anti-CD58 mAb and an HCMV UL148 deletion mutant (HCMV∆UL148) with restored CD58 expression demonstrated that the CD2/CD58 axis was essential for the recognition of HCMV-infected targets by CD8+ HCMV-specific cytotoxic T lymphocytes (CTLs). Further, challenge of peripheral blood mononuclear cells ex vivo with HCMV∆UL148 increased both CTL and natural killer (NK) cell degranulation against HCMV-infected cells, including NK-driven antibody-dependent cellular cytotoxicity, showing that UL148 is a modulator of the function of multiple effector cell subsets. Our data stress the effect of HCMV immune evasion functions on shaping the immune response, highlighting the capacity for their potential use in modulating immunity during the development of anti-HCMV vaccines and HCMV-based vaccine vectors

ADAM17 targeting by human cytomegalovirus remodels the cell surface proteome to simultaneously regulate multiple immune pathways

Human cytomegalovirus (HCMV) is a major human pathogen whose life-long persistence is enabled by its remarkable capacity to systematically subvert host immune defenses. In exploring the finding that HCMV infection up-regulates tumor necrosis factor receptor 2 (TNFR2), a ligand for the pro-inflammatory antiviral cytokine TNFα, we found that the underlying mechanism was due to targeting of the protease, A Disintegrin And Metalloproteinase 17 (ADAM17). ADAM17 is the prototype ‘sheddase’, a family of proteases that cleaves other membrane-bound proteins to release biologically active ectodomains into the supernatant. HCMV impaired ADAM17 surface expression through the action of two virally-encoded proteins in its UL/b’ region, UL148 and UL148D. Proteomic plasma membrane profiling of cells infected with an HCMV double-deletion mutant for UL148 and UL148D with restored ADAM17 expression, combined with ADAM17 functional blockade, showed that HCMV stabilized the surface expression of 114 proteins (P < 0.05) in an ADAM17-dependent fashion. These included reported substrates of ADAM17 with established immunological functions such as TNFR2 and jagged1, but also numerous unreported host and viral targets, such as nectin1, UL8, and UL144. Regulation of TNFα-induced cytokine responses and NK inhibition during HCMV infection were dependent on this impairment of ADAM17. We therefore identify a viral immunoregulatory mechanism in which targeting a single sheddase enables broad regulation of multiple critical surface receptors, revealing a paradigm for viral-encoded immunomodulation

Μελέτη των κυτταρικών, σηματοδοτικών μονοπατιών κατά τη διάρκεια λανθάνουσας μόλυνσης και επανενεργοποίησης από τον ιό του απλού έρπητα τύπου 1 (Herpes Simpex Virus type 1, HSV-1)

HSV-1 is a DNA virus of the herpesviruses family (Herpesviridae) and specifically belongs to the Alpha subfamily (Alphaherpesvirinae). It is a neurotropic virus that alternates from a lytic cycle in epithelial cells to a latent cycle in neurons. Its lytic cycle takes place in three phases, the immediate early (IE), the early (E) and the late (L). In the present thesis, we studied the mechanisms that govern infection by Herpes Simplex virus type-1 investigating two directions; the effect of CD40L on the outcome of infection and the methylation profile of host genes during the course of infection.In the first part, the effect of CD40L on HSV-1 infection was studied and it was found that CD40L directly inhibits infection by HSV-1 following entry of the virus in the host cell. Different stages of viral infection were analyzed as well as antiviral mechanisms with a particular emphasis on autophagy. Collectively, it was demonstrated that HSV-1 is directly inhibited by the activation of the CD40L pathway by a mechanism that is PI3K-dependent and autophagy-independent.At the second part of the study, the methylation profile of the host cell genome was analyzed during lytic and latent infection by HSV-1 with particular interest on the enzymes associated to epigenetic phenomena. PCR-array analysis showed that there is a great variation in the methylation profile during the immediate early (IE) and early (E) phase of infection while in the late (L) phase of infection and in latently infected cells the methylation profile remains stable, however, different from the steady state methylation of the host. Several histone deacetylase genes were identified as targets for alterations in DNA methylation and an effort was made to correlate the changes in DNA methylation to gene expression and their impact on the progeny virus.Ο HSV-1 είναι ένας DNA ιός που ανήκει στην οικογένεια των ερπητοϊών (Herpesviridae) και συγκεκριμένα στην οικογένεια των α-ερπητοϊών (Alphaherpesvirinae). Είναι νευροτρόπος ιός ο οποίος πραγματοποιεί λυτικό κύκλο στα επιθηλιακά κύτταρα ενώ στα νευρικά κύτταρα βρίσκεται σε λανθάνουσα φάση. Ο λυτικός κύκλος του HSV-1 λαμβάνει χώρα σε τρεις φάσεις, την άμεσα πρώιμη (α), την πρώιμη (β) και την όψιμη(γ) και αποτελεί χαρακτηριστικό τρόπο έκφρασης των γονιδίων των ερπητοϊών. Στα πλαίσια της παρούσας διδακτορικής διατριβής διερευνήθηκαν μηχανισμοί που διέπουν την μόλυνση από τον ιό του απλού έρπητα τύπου Ι (HSV-1). Συγκεκριμένα, διερευνήθηκε η δράση του CD40L και ο ρόλος του στην έκβαση της μόλυνσης από τον HSV-1. Επιπλέον, διερευνήθηκε το πρότυπο μεθυλίωσης κυτταρικών γονιδίων σε διάφορα στάδια της μόλυνσης από τον ιό.Ως προς το πρώτο σκέλος της εργασίας, κατά την οποία διερευνήθηκε η δράση του CD40L στην μόλυνση από τον HSV-1, βρέθηκε ότι ο CD40L παρεμποδίζει την εξέλιξη της μόλυνσης άμεσα, μετά την είσοδο του ιού στο κύτταρο. Μελετήθηκαν διάφορα στάδια της μόλυνσης καθώς κ αντιϊκοί μηχανισμοί ενώ έμφαση δόθηκε στον μηχανισμό της αυτοφαγίας. Συνολικά, ο ιός παρεμποδίζεται από την ενεργοποίηση του μονοπατιού του CD40L μέσω ενός μηχανισμού που σχετίζεται με την κινάση PI3K και είναι ανεξάρτητος της αυτοφαγίας.Ως προς το δεύτερο σκέλος της διατριβής, μελετήθηκε το πρότυπο μεθυλίωσης του κυτταρικού γονιδιώματος σε λυτική κ λανθάνουσα μόλυνση με τον ιό HSV-1 δίνοντας ιδιαίτερη έμφαση στο πρότυπο μεθυλίωσης γονιδίων που κωδικοποιούν για ένζυμα που σχετίζονται με επιγενετικά φαινόμενα. Βρέθηκε ότι στην άμεσα πρώιμη κ πρώιμη φάση του λυτικού κύκλου υπάρχει έντονη διακύμανση των επιπέδων μεθυλίωσης των κυτταρικών γονιδίων ενώ στην όψιμη φάση της μόλυνσης και κατά τη λανθάνουσα κατάσταση το πρότυπο μεθυλίωσης είναι διαφορετικό από την κατάσταση ηρεμίας αλλά σταθερό

Suppression of costimulation by human cytomegalovirus promotes evasion of cellular immune defenses.

CD58 is an adhesion molecule that is known to play a critical role in costimulation of effector cells and is intrinsic to immune synapse structure. Herein, we describe a virally encoded gene that inhibits CD58 surface expression. Human cytomegalovirus (HCMV) UL148 was necessary and sufficient to promote intracellular retention of CD58 during HCMV infection. Blocking studies with antagonistic anti-CD58 mAb and an HCMV UL148 deletion mutant (HCMV∆UL148) with restored CD58 expression demonstrated that the CD2/CD58 axis was essential for the recognition of HCMV-infected targets by CD8+ HCMV-specific cytotoxic T lymphocytes (CTLs). Further, challenge of peripheral blood mononuclear cells ex vivo with HCMV∆UL148 increased both CTL and natural killer (NK) cell degranulation against HCMV-infected cells, including NK-driven antibody-dependent cellular cytotoxicity, showing that UL148 is a modulator of the function of multiple effector cell subsets. Our data stress the effect of HCMV immune evasion functions on shaping the immune response, highlighting the capacity for their potential use in modulating immunity during the development of anti-HCMV vaccines and HCMV-based vaccine vectors

Monoclonal antibodies targeting nonstructural viral antigens can activate ADCC against human cytomegalovirus.

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that causes severe disease following congenital infection and in immunocompromised individuals. No vaccines are licensed, and there are limited treatment options. We now show that the addition of anti-HCMV antibodies (Abs) can activate NK cells prior to the production of new virions, through Ab-dependent cellular cytotoxicity (ADCC), overcoming viral immune evasins. Quantitative proteomics defined the most abundant HCMV proteins on the cell surface, and we screened these targets to identify the viral antigens responsible for activating ADCC. Surprisingly, these were not structural glycoproteins; instead, the immune evasins US28, RL11, UL5, UL141, and UL16 each individually primed ADCC. We isolated human monoclonal Abs (mAbs) specific for UL16 or UL141 from a seropositive donor and optimized them for ADCC. Cloned Abs targeting a single antigen (UL141) were sufficient to mediate ADCC against HCMV-infected cells, even at low concentrations. Collectively, these findings validated an unbiased methodological approach to the identification of immunodominant viral antigens, providing a pathway toward an immunotherapeutic strategy against HCMV and potentially other pathogens

Monoclonal antibodies targeting nonstructural viral antigens can activate ADCC against human cytomegalovirus.

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that causes severe disease following congenital infection and in immunocompromised individuals. No vaccines are licensed, and there are limited treatment options. We now show that the addition of anti-HCMV antibodies (Abs) can activate NK cells prior to the production of new virions, through Ab-dependent cellular cytotoxicity (ADCC), overcoming viral immune evasins. Quantitative proteomics defined the most abundant HCMV proteins on the cell surface, and we screened these targets to identify the viral antigens responsible for activating ADCC. Surprisingly, these were not structural glycoproteins; instead, the immune evasins US28, RL11, UL5, UL141, and UL16 each individually primed ADCC. We isolated human monoclonal Abs (mAbs) specific for UL16 or UL141 from a seropositive donor and optimized them for ADCC. Cloned Abs targeting a single antigen (UL141) were sufficient to mediate ADCC against HCMV-infected cells, even at low concentrations. Collectively, these findings validated an unbiased methodological approach to the identification of immunodominant viral antigens, providing a pathway toward an immunotherapeutic strategy against HCMV and potentially other pathogens