[Show abstract][Hide abstract] ABSTRACT: Pandemic and seasonal influenza viruses cause considerable morbidity and mortality in the general human population. Protection from severe disease may result from vaccines that activate antigen-presenting DC for effective stimulation of influenza-specific memory T cells. Special attention is paid to vaccine-induced CD8+ T-cell responses, because they are mainly directed against conserved internal influenza proteins thereby presumably mediating cross-protection against circulating seasonal as well as emerging pandemic virus strains. Our study showed that influenza whole virus vaccines of major seasonal A and B strains activated DC more efficiently than those of pandemic swine-origin H1N1 and pandemic-like avian H5N1 strains. In contrast, influenza split virus vaccines had a low ability to activate DC, regardless which strain was investigated. We also observed that whole virus vaccines stimulated virus-specific CD8+ memory T cells much stronger compared to split virus counterparts, whereas both vaccine formats activated CD4+ Th cell responses similarly. Moreover, our data showed that whole virus vaccine material is delivered into the cytosolic pathway of DC for effective activation of virus-specific CD8+ T cells. We conclude that vaccines against seasonal and pandemic (-like) influenza strains that aim to stimulate cross-reacting CD8+ T cells should include whole virus rather than split virus formulations.
PLoS ONE 07/2014; 9(7):e103392. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The mechanisms that lead to the tegumentation of herpesviral particles are only poorly defined. The phosphoprotein 65 (pp65) is the most abundant constituent of the virion tegument of human cytomegalovirus (HCMV). It is, however, non-essential for virion formation. This seeming discrepancy has not met with a satisfactory explanation regarding the role of pp65 in HCMV particle morphogenesis. Here we addressed the question how the overall tegument composition of the HCMV virion depended on pp65 and how the lack of pp65 influenced the packaging of particular tegument proteins. To investigate this, we analyzed the proteome of pp65pos and pp65neg virions by label-free quantitative mass spectrometry and determined the relative abundance of tegument proteins. Surprisingly, only pUL35 was elevated in pp65neg virions. As the abundance of pUL35 in the HCMV tegument is low, it unlikely replaced pp65 as a structural component in pp65neg virions. A subset of proteins, including the third most abundant tegument protein pUL25 as well as pUL43, pUL45, pUL71 were reduced in pp65neg or pp65low virions, indicating that the packaging of these proteins was related to pp65. The levels of tegument components, like pp28 and the capsid associated tegument proteins pp150, pUL48 and pUL47, were unaffected by the lack of pp65. Our analyses demonstrate that deletion of pp65 is not compensated by other viral proteins in the process of virion tegumentation. The results are concordant with a model of pp65 serving as an optional scaffold protein that facilitates protein upload into the outer tegument of HCMV particles.
[Show abstract][Hide abstract] ABSTRACT: Immunoevasive proteins ("evasins") of human CMV (HCMV) modulate stability and localization of MHC class I (MHC I) molecules, and their supply of antigenic peptides. However, it is largely unknown to what extent these evasins interfere with recognition by virus-specific CD8 T cells. We analyzed the recognition of HCMV-infected cells by a panel of CD8 T cells restricted through one of nine different MHC I allotypes. We employed a set of HCMV mutants deleted for three or all four of the MHC I modulatory genes US2, US3, US6, and US11. We found that different HCMV evasins exhibited different allotype-specific patterns of interference with CD8 T cell recognition of infected cells. In contrast, recognition of different epitopes presented by the same given MHC I allotype was uniformly reduced. For some allotypes, single evasins largely abolished T cell recognition; for others, a concerted action of evasins was required to abrogate recognition. In infected cells whose Ag presentation efficiency had been enhanced by IFN-γ pretreatment, HCMV evasins cooperatively impared T cell recognition for several different MHC I allotypes. T cell recognition and MHC I surface expression under influence of evasins were only partially congruent, underscoring the necessity to probe HCMV immunomodulation using specific T cells. We conclude that the CD8 T cell evasins of HCMV display MHC I allotype specificity, complementarity, and cooperativity.
The Journal of Immunology 05/2014; · 5.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Human cytomegalovirus (HCMV) particle morphogenesis in infected cells is an orchestrated process that eventually results in the release of enveloped virions. Proteomic analysis has been employed to reveal the complexity in the protein composition of these extracellular particles. Only limited information is however available regarding the proteome of infected cells preceding the release of HCMV virions. We used quantitative mass spectrometry to address the pattern of viral and cellular proteins in cells, infected with derivatives of the AD169 laboratory strain. Our analyses revealed a remarkable conservation in the patterns of viral and of abundant cellular proteins in cells, infected for 2 hours, 2 days, or 4 days. Most viral proteins increased in abundance as the infection progressed over time. Of the proteins that were reliably detectable by mass spectrometry, only IE1 (pUL123), pTRS1, and pIRS1 were downregulated at 4 days after infection. In addition, little variation of viral proteins in the virions of the different viruses was detectable, independent of the expression of the major tegument protein pp65. Taken together these data suggest that there is little variation in the expression program of viral and cellular proteins in cells infected with related HCMVs, resulting in a conserved pattern of viral proteins ultimately associated with extracellular virions.
[Show abstract][Hide abstract] ABSTRACT: Dendritic cells play a central role in the immune control of human cytomegalovirus (HCMV) infection. This work aimed at investigating the impact of non-infectious, subviral dense bodies of HCMV on maturation and activation of dendritic cells (DC). Treatment of immature DC with dense bodies led to the maturation of these cells and significantly increased their capacity for cytokine release and antigen presentation. Dense bodies activated DC may thereby contribute to the development of antiviral immunity.
[Show abstract][Hide abstract] ABSTRACT: Control of human cytomegalovirus (HCMV) depends on CD8+ T cell responses that are shaped by an individual's repertoire of MHC molecules. MHC class I presentation is modulated by a set of HCMV-encoded proteins. Here we show that HCMV immunoevasins differentially impair T cell recognition of epitopes from the same viral antigen, immediate-early 1 (IE-1), that are presented by different MHC class I allotypes. In the presence of immunoevasins, HLA-A- and HLA-B-restricted T cell clones were ineffective, but HLA-C*0702-restricted T cell clones recognized and killed infected cells. Resistance of HLA-C*0702 to viral immunoevasins US2 and US11 was mediated by the alpha3 domain and C-terminal region of the HLA heavy chain. In healthy donors, HLA-C*0702-restricted T cells dominated the T cell response to IE-1. The same HLA-C allotype specifically protected infected cells from attack by NK cells that expressed a corresponding HLA-C-specific KIR. Thus, allotype-specific viral immunoevasion allows HCMV to escape control by NK cells and HLA-A- and HLA-B-restricted T cells, while the virus becomes selectively vulnerable to an immunodominant population of HLA-C-restricted T cells. Our work identifies a T cell population that may be of particular efficiency in HCMV-specific immunotherapy.
[Show abstract][Hide abstract] ABSTRACT: Suppression of major histocompatibility complex (MHC) class I-mediated presentation of human cytomegalovirus (HCMV) peptides is an important mechanism to avoid CD8 T lymphocyte recognition and killing of infected cells. Of particular interest is how MHC class I presentation of essential regulatory immediate-early (IE) proteins of HCMV can be effectively compromised at times when known viral immunoevasins are not abundantly expressed. The tegument protein pp71 had been suggested to be involved in MHC class I downregulation. Intriguingly, this polypeptide is also critically engaged in the initial de-repression of the major IE gene locus, leading to enhanced expression of IE proteins IE1-pp72 and IE2-pp86. Using a set of viral mutants, we addressed the role of pp71 in MHC class I presentation of IE1-pp72-derived peptides. We show that the amount of "incoming" pp71 positively correlates with IE1-pp72 protein levels and with the presentation of IE1-derived peptides. This indicates that the amount of the IE1-protein, induced by pp71, rather than a putative immunoevasive function of the tegument protein determines MHC class I antigen presentation of IE1-derived peptides. This process proved to be independent of the presence of pp65, which had been reported to interfere with IE1 presentation. It may thus be beneficial for the success of HCMV replication to limit the level of pp71 delivered from infecting particles in order to avoid critical levels of MHC class I presentation of IE protein-derived peptides.
[Show abstract][Hide abstract] ABSTRACT: Human cytomegalovirus (CMV) infections and relapse of disease remain major problems after allogeneic stem cell transplantation (allo-SCT), in particular in combination with CMV-negative donors or cordblood transplantations. Recent data suggest a paradoxical association between CMV-reactivation after allo-SCT and reduced leukemic relapse. Given the potential of Vä2-negative γδT-cells to recognize CMV-infected cells and tumor cells, the molecular biology of distinct γδT-cell-subsets expanding during CMV-reactivation after allo-SCT was investigated. Vä2(neg) γδT-cell expansions after CMV-reactivation were observed not only with conventional but also cordblood donors. Expanded γδT-cells were capable of recognizing both CMV-infected cells as well as primary leukemic blasts. CMV- and leukemia-reactivity were restricted to the same clonal population, whereas other Vä2(neg) T-cells interact with dendritic cells (DCs). Cloned Vä1-TCRs mediated leukemia-reactivity and DC-interactions, but surprisingly not CMV-reactivity. Interestingly, CD8áá expression appeared to be a signature of γδT-cells after CMV exposure. However, functionally CD8áá was primarily important in combination with selected leukemia-reactive Vä1-TCRs, demonstrating for the first time a co-stimulatory role of CD8áá for distinct γδTCRs. Based on these observations, we advocate the exploration of adoptive transfer of unmodified Vä2(neg) γδT-cells after allo-SCT to tackle CMV-reactivation and residual leukemic blasts, as well as application of leukemia-reactive Vä1-TCR-engineered T-cells as alternative therapeutic tools.Leukemia accepted article preview online, 1 January 2013; doi:10.1038/leu.2012.374.
Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K 01/2013; · 10.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Human cytomegalovirus (HCMV) interferes with MHC class I-restricted antigen presentation and thereby reduces recognition by CD8+ T cells. This interference is primarily mediated by ER-resident glycoproteins that are encoded in the US2-11 region of the viral genome. Such a suppression of recognition would be of particular importance immediately after infection, because several immunodominant viral antigens are already present in the cell in this phase. However, it is not known which of the evasion proteins gpUS2-11 interfere with antigen presentation to CD8+ T cells at this time of infection. Here we address this question, using recombinant viruses (RV) that express only one of the immunoevasins gpUS2, gpUS3, or gpUS11. Infection with RV-US3 had only a limited impact on the presentation of peptides from the CD8+ T cell antigens IE1 and pp65 under immediate-early conditions imposed by cycloheximide-actinomycin D blocking. Unexpectedly, both RV-US2 and RV-US11 considerably impaired the recognition of IE1 and pp65 by CD8+ T cells, and both US2 and, to lesser extent, US11 were transcribed under IE conditions. Thus, gpUS2 and gpUS11 are key effectors of MHC class I immune evasion immediately after HCMV infection.
Journal of General Virology 10/2012; · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The phosphoprotein 65 (pp65) of human cytomegalovirus is a prominent target of the antiviral CD8 T lymphocyte response. This study focused on investigating the properties of pp65 that render it a privileged antigen. It was found that pp65 was metabolically stable. The tegument protein was introduced into MHC class I presentation following its delivery via non-replicating dense bodies. No ubiquitination was found on particle-associated pp65. Proof was obtained that pp65 was a nucleocytoplasmic shuttle protein, using heterokaryon analyses. Based on this finding, inhibition experiments showed that presentation of particle-derived pp65 by HLA-A2 was sensitive to the impairment of the CRM1-mediated nuclear export pathway. The data support the idea that particle-derived pp65 can serve as a nuclear reservoir for proteasomal processing and MHC class I presentation, following its CRM1-dependent nuclear export. The presentation of pp65-derived peptides was also impaired by CRM1-inhibition following de novo synthesis of the tegument protein. However, pp65 protein levels were also reduced when blocking CRM1-mediated export after transient expression. This indicated that pp65 expression rather than direct interference with its own nuclear export was responsible for its reduced presentation in this case. The functionality of CRM1-mediated nuclear export is thus important for the presentation of pp65-derived peptides in the context of MHC class I on organ cells, both after exogenous uptake and after de novo synthesis of the tegument protein, but different mechanisms may account for either case.
Medical Microbiology and Immunology 09/2012; 201(4):567-79. · 3.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Current protocols used to select CMV-specific T cells for adoptive immunotherapy focus on virus-specific memory T cells from seropositive donors. However, this strategy is not feasible in patients undergoing allogeneic haematopoietic stem-cell transplantation (HSCT) from CMV-seronegative donors. Here, we redirected T cells of CMV-seronegative donors with a human genetically engineered TCR recognizing an HLA-A*0201-binding peptide epitope of CMVpp65. To facilitate clinical translation of this approach, we used a non-viral expression system based on in vitro transcribed RNA and electroporation. Although memory and naïve-derived T-cell subsets were both efficiently transfected by TCR-RNA, memory-derived T cells showed much stronger levels of HLA-A*0201-restricted cytolytic activity to CMV-infected fibroblasts and maintained acquired function for 5-10 days. In addition to redirection of CD8(+) cytotoxic T cells, TCR-RNA transfection was capable of redirecting CD4(+) T cells into potent Ag-specific Th cells that efficiently triggered maturation of DCs. Our data suggest that memory rather than naïve-derived T cells are the preferred subset for transient TCR expression by RNA electroporation, providing more efficient and sustained virus-specific CD4(+) and CD8(+) T-cell function. CMV TCR-RNA may represent a suitable therapeutic 'off-the-shelf' reagent to be used in severe CMV infections of HSCT patients when endogenous CMV-specific T-cell immunity is insufficient.
European Journal of Immunology 08/2012; · 4.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Human cytomegalovirus (HCMV), a member of the Herpesviridae family, is proficient at establishing lifelong persistence within the host in part due to immune modulating genes that limit immune recognition. HCMV encodes at least five glycoproteins within its unique short (US) genomic region that interfere with MHC class I antigen presentation, thus hindering viral clearance by cytotoxic T lymphocytes (CTL). Specifically, US3 retains class I within the endoplasmic reticulum (ER), while US2 and US11 induce class I heavy chain destruction. A cooperative effect on class I down-regulation during stable expression of HCMV US2 and US3 has been established. To address the impact of US3 on US11-mediated MHC class I down-regulation, the fate of class I molecules was examined in US3/US11-expressing cells and virus infection studies. Co-expression of US3 and US11 resulted in a decrease of surface expression of class I molecules. However, the class I molecules in US3/US11 cells were mostly retained in the ER with an attenuated rate of proteasome destruction. Analysis of class I levels from virus-infected cells using HCMV variants either expressing US3 or US11 revealed efficient surface class I down-regulation upon expression of both viral proteins. Cells infected with both US3 and US11 expressing viruses demonstrate enhanced retention of MHC class I complexes within the ER. Collectively, the data suggests a paradigm where HCMV-induced surface class I down-regulation occurs by diverse mechanisms dependent on the expression of specific US genes. These results validate the commitment of HCMV to limiting the surface expression of class I levels during infection.
[Show abstract][Hide abstract] ABSTRACT: After allogeneic hematopoietic stem-cell transplantation patients are at increased risk for herpes zoster as long as varicella-zoster virus specific T-cell reconstitution is impaired. This study aimed to identify immunodominant varicella-zoster virus antigens that drive recovery of virus-specific T cells after transplantation.
Antigens were purified from a varicella-zoster virus infected cell lysate by high-performance liquid chromatography and were identified by quantitative mass spectrometric analysis. To approximate in vivo immunogenicity for memory T cells, antigen preparations were consistently screened with ex vivo PBMC of varicella-zoster virus immune healthy individuals in sensitive interferon-γ ELISpot assays. Candidate virus antigens identified by the approach were genetically expressed in PBMC using electroporation of in vitro transcribed RNA encoding full-length proteins and were then analyzed for recognition by CD4(+) and CD8(+) memory T cells.
Varicella-zoster virus encoded glycoproteins B and E, and immediate early protein 62 were identified in immunoreactive lysate material. Predominant CD4(+) T-cell reactivity to these proteins was observed in healthy virus carriers. Furthermore, longitudinal screening in allogeneic stem-cell transplantation patients showed strong expansions of memory T cells recognizing glycoproteins B and E after onset of herpes zoster, while immediate early protein 62 reactivity remained moderate. Reactivity to viral glycoproteins boosted by acute zoster was mediated by both CD4(+) and CD8(+) T cells.
Our data demonstrate that glycoproteins B and E are major targets of varicella-zoster virus specific CD4(+) and CD8(+) T-cell reconstitution occurring during herpes zoster after allogeneic stem-cell transplantation. Varicella-zoster virus glycoproteins B and E might form the basis for novel non-hazardous zoster subunit vaccines suitable for immunocompromised transplant patients.
[Show abstract][Hide abstract] ABSTRACT: Polyethylenimines are cationic polymers with potential as delivery vectors in gene therapy and with proven antimicrobial activity. However, the antiviral activity of polyethylenimines has not been addressed in detail thus far. We have studied the inhibitory effects of a linear 25-kDa polyethylenimine on infections with human papillomaviruses and human cytomegaloviruses. Preincubation of cells with polyethylenimine blocked primary attachment of both viruses to cells, resulting in a significant reduction of infection. In addition, the dissemination of human cytomegalovirus in culture cells was efficiently reduced by recurrent administration of polyethylenimine. Polyethylenimine concentrations required for inhibition of human papillomavirus and cytomegalovirus did not cause any cytotoxic effects. Polyethylenimines and their derivatives may thus be attractive molecules for the development of antiviral microbicides.
Antimicrobial Agents and Chemotherapy 10/2011; 56(1):75-82. · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Immunodominance hierarchies operating in immune responses to viral Ags limit the diversity of the elicited CD8 T cell responses. We evaluated in I-A(b+)/A2-HHD-II and HLA-DR1(+)/A2-DR1 mice the HLA-A*0201-restricted, multispecific CD8 T cell responses to the human CMV tegument phosphoprotein pp65 (pp65) Ag. Vaccination of mice with pp65-encoding DNA elicited high IFN-γ(+) CD8 T cell frequencies to the pp65(495-503)/(e6) epitope and low responses to the pp65(320-328)/(e3) and pp65(522-530)/(e8) epitopes. Abrogation of the e6-specific immunity efficiently enhanced e3- and e8-specific T cell responses by a pp65(Δ501-503) DNA vaccine. The immunodominant e6-specific (but not the e3- and e8-specific) CD8 T cell response critically depends on CD4 T cell help. Injection of monospecific DNA- or peptide-based vaccines encoding the e3 or e8 (but not the e6) epitope into mice elicited CD8 T cells. Codelivering the antigenic peptides with different heterologous CD4 T cell helper epitopes enhanced e6-specific (but not e3- or e8-specific) CD8 T cell responses. Similarly, homologous CD4 T cell help, located within an overlapping (nested) pp65(487-503) domain, facilitated induction of e6-specific CD8 T cell responses by peptide-based vaccination. The position of the e6 epitope within this nested domain is not critical to induce the immunodominant, e6-specific CD8 T cell response to the pp65 Ag. Distant CD4 T cell epitope(s) can thus provide efficient help for establishing pp65-e6 immunodominance in vaccinated mice. These results have practical implications for the design of new T cell-stimulating vaccines.
The Journal of Immunology 09/2011; 187(5):2172-80. · 5.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The tegument protein pp65 of human cytomegalovirus (HCMV) is abundant in lytically infected human foreskin fibroblasts (HFF), as well as in virions and subviral dense bodies (DB). Despite this, we showed previously that pp65 is dispensable for growth in HFF. In the process of refining a DB-based vaccine candidate, different HCMV mutants were generated, expressing a dominant HLA-A2-presented peptide of the IE1 protein fused to pp65. One of the mutant viruses (RV-VM1) surprisingly showed marked impairment in virus release from HFF. We hypothesized that analysis of the phenotypic alterations of RV-VM1 would provide insight into the functions of pp65, poorly defined thus far. RV-VM1 infection resulted in nuclear retention of the fusion protein and reorganization of nuclear inclusion bodies. Coimmunoprecipitation experiments suggested that wild-type (wt) pp65 and pp65-VM1 were substrates of the viral pUL97 kinase in vitro and formed a complex with the viral RNA-export protein pUL69 and with pUL97 in lysates of infected cells. No evidence for an impairment of pUL97 within this complex was found. However, RV-VM1 replication in infected cells was resistant to a pUL97 inhibitor, and pUL97 inhibitors mimicked the mutant in terms of pp65 being retained in the nucleus. The results suggest that the life cycle of RV-VM1 was impeded at the stages of early-late transcription, RNA export or capsid maturation. wt-pp65 may play a role at these stages of infection, and complex formation with pUL69 and pUL97 may be important for that function.
Journal of General Virology 10/2010; 91(Pt 10):2531-41. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Control of human cytomegalovirus (HCMV) infection correlates with the reconstitution of antiviral T lymphocytes in haematopoietic stem cell transplant recipients. A vaccine to foster this reconstitution and to ameliorate the severe consequences of HCMV reactivation is yet unavailable. This work focused on providing a rationale for the amendment of the yields and the antigenic composition of a vaccine, based on subviral dense bodies (DB) of HCMV. Modified DB were generated that contained the HLA-A2 presented IE1 model peptide TMYGGISLL, integrated at different positions in the major DB protein pp65. Insertion at position W175 of pp65 allowed efficient formation of recDB in the cytoplasm of infected cells and resulted in considerable yields of these particles. Even in the absence of adjuvant, these particles proved to be highly immunogenic with respect to CD8 and CD4 T cell and neutralizing antibody responses.
[Show abstract][Hide abstract] ABSTRACT: Impairment of cellular immunity upon hematopoietic stem cell transplantation (HSCT) may lead to serious clinical manifestations induced by human cytomegalovirus (HCMV) and varicella-zoster virus (VZV) infections. Although the clinical presentations, preferential organ involvement and clinical courses are different, infections with both herpesviruses are similar with respect to many pathophysiological aspects and the therapeutic strategies that are employed to combat them. Antiviral drug prophylaxis and therapy are successfully used to limit the risk of reactivated HCMV and VZV infections, but are unable to absolutely prevent episodes of virus disease in long-term follow-up after HSCT. Control of infection requires the re-establishment of protective antiviral cellular immunity in the host. Here we review the most recent developments in the field of HCMV and VZV immunotherapy with specific emphasis on the question of how vaccines against HCMV and VZV may aid in enhancing the reconstitution of antiviral immunity after HSCT, and thereby support the control of these two agents by transplant recipients.
Expert Review of Vaccines 09/2009; 8(8):999-1021. · 4.22 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The capacity of human cytomegalovirus (HCMV) to establish and maintain a latent infection from which it can later reactivate ensures its widespread distribution in the population, but the mechanisms enabling maintenance of latency in the face of a robust immune system are poorly understood. We examined the role of the HCMV UL111A gene, which encodes homologs of the immunosuppressive cytokine interleukin-10 in the context of latent infection of myeloid progenitor cells. A UL111A deletion virus was able to establish, maintain, and reactivate from experimental latency in a manner comparable with parental virus, but major histocompatibility complex class II levels increased significantly on the surfaces of cells infected with the deletion virus. Importantly, there was an increase in both allogeneic and autologous peripheral blood mononuclear cells and CD4(+) T-cell responses to UL111A deletion virus-infected myeloid progenitors, indicating that loss of the capacity to express viral interleukin-10 during latency results in latently infected cells becoming more readily recognizable by a critical arm of the immune response. The detection of a viral gene that suppresses CD4(+) T-cell recognition of latently infected cells identifies an immune evasion strategy that probably enhances the capacity of HCMV to persist in a latent state within the human host.