Rat Cytomegalovirus Vaccine Prevents Accelerated Chronic Rejection in CMV-Naïve Recipients of Infected Donor Allograft Hearts.

Cytomegalovirus accelerates transplant vascular sclerosis (TVS) and chronic rejection (CR) in solid organ transplants; however, the mechanisms involved are unclear. We determined the efficacy of a CMV vaccine in preventing CMV-accelerated rat cardiac allograft rejection in naïve recipients of CMV+ donor hearts. F344 donor rats were infected with RCMV 5 days prior to heterotopic cardiac transplantation into CMV-naïve or H2 O2 -inactivated RCMV-vaccinated Lewis recipients. Recipients of RCMV-infected donor hearts rejected at POD59, whereas vaccinated recipients exhibited a significantly prolonged time to rejection-POD97, similar to recipients of uninfected donor hearts (POD108). Although all of the donor hearts were preinfected, the vaccinated recipients had lower graft and PBMC viral loads at POD 7 compared to unvaccinated controls. Adoptive T cell and passive antibody transfers from vaccinated Lewis rats into naïve recipients demonstrate that both T-cell and B-cell arms of the adaptive immune response provide protection against CMV-accelerated rejection. Similar findings were obtained when testing three different adjuvants in passive transfer experiments. We have determined that the timing of the vaccine prior to transplantation and the specific adjuvant play critical roles in mediating anti-viral responses and promoting graft survival. CMV vaccination prior to transplantation may effectively increase graft survival.

Cytomegalovirus latency promotes cardiac lymphoid neogenesis and accelerated allograft rejection in CMV naïve recipients.

Human cytomegalovirus (HCMV) infection is associated with the acceleration of transplant vascular sclerosis (TVS) and chronic allograft rejection (CR). HCMV-negative recipients of latently HCMV infected donor grafts are at highest risk for developing CMV disease. Using a rat heart transplant CR model, we have previously shown that acute rat CMV (RCMV) infection following transplantation significantly accelerates both TVS and CR. Here, we report that RCMV-naïve recipients of heart allografts from latently RCMV-infected donors undergo acceleration of CR with similar kinetics as acutely infected recipients. In contrast to acutely infected recipients, treatment of recipients of latently infected donor hearts with ganciclovir did not prevent CR or TVS. We observed the formation of tertiary lymphoid structures (TLOs) containing macrophages and T cells in latently infected hearts prior to transplantation but not in uninfected rats. Moreover, pathway analysis of gene expression data from allografts from latently infected donors indicated an early and sustained production of TLO-associated genes compared to allografts from uninfected donors. We conclude that RCMV-induced TLO formation and alteration of donor tissue T cell profiles prior to transplantation in part mediate the ganciclovir-insensitive rejection of latently infected donor allografts transplanted into naïve recipients by providing a scaffold for immune activation.

Mechanisms of cytomegalovirus-accelerated vascular disease: induction of paracrine factors that promote angiogenesis and wound healing.

Human cytomegalovirus (HCMV) is associated with the acceleration of a number of vascular diseases such as atherosclerosis, restenosis, and transplant vascular sclerosis (TVS). All of these diseases are the result of either mechanical or immune-mediated injury followed by inflammation and subsequent smooth muscle cell (SMC) migration from the vessel media to the intima and proliferation that culminates in vessel narrowing. A number of epidemiological and animal studies have demonstrated that CMV significantly accelerates TVS and chronic rejection (CR) in solid organ allografts. In addition, treatment of human recipients and animals alike with the antiviral drug ganciclovir results in prolonged survival of the allograft, indicating that CMV replication is a requirement for acceleration of disease. However, although virus persists in the allograft throughout the course of disease, the number of directly infected cells does not account for the global effects that the virus has on the acceleration of TVS and CR. Recent investigations of up- and downregulated cellular genes in infected allografts in comparison to native heart has demonstrated that rat CMV (RCMV) upregulates genes involved in wound healing (WH) and angiogenesis (AG). Consistent with this result, we have found that supernatants from HCMV-infected cells (HCMV secretome) induce WH and AG using in vitro models. Taken together, these findings suggest that one mechanism for HCMV acceleration of TVS is mediated through induction of secreted cytokines and growth factors from virus-infected cells that promote WH and AG in the allograft, resulting in the acceleration of TVS. We review here the ability of CMV infection to alter the local environment by producing cellular factors that act in a paracrine fashion to enhance WH and AG processes associated with the development of vascular disease, which accelerates chronic allograft rejection.

The role of angiogenic and wound repair factors during CMV-accelerated transplant vascular sclerosis in rat cardiac transplants.

Human cytomegalovirus (HCMV) accelerates transplant vascular sclerosis (TVS), a consequence of angiogenesis (AG) and wound repair (WR). While HCMV can be localized to TVS lesions, the low number of infected cells suggests a global effect on target tissues. We used microarray analysis followed by real-time-polymerase chain reaction (RT-PCR) in an RCMV-accelerated TVS rat cardiac transplant model to determine whether CMV activates host WR and AG factors. Dysregulated cellular genes in allografts from RCMV-infected recipients were compared to those from uninfected recipients and native hearts. We demonstrated that RCMV upregulates the genes involved in WR and AG, which was highest during the critical time of TVS acceleration (21-28 days). Using a standard in vitro AG assay, virus and serum-free supernatants collected at 48 h postinfection significantly induced endothelial cell (EC) migration, branching and tubule formation compared to supernatants from mock-infected cells. Supernatants from ultraviolet (UV)-inactivated RCMV-infected cells failed to induce AG, indicating that virus replication is required. Upregulation of WR and AG genes occurs during the critical period of CMV-accelerated TVS. Targeting these genes may prevent this process and improve allograft survival.

Cytomegalovirus accelerates chronic allograft nephropathy in a rat renal transplant model with associated provocative chemokine profiles.

BACKGROUND: Studies have shown that rat cytomegalovirus (RCMV) infection accelerates transplant vascular sclerosis (TVS) in rat heart and small bowel allotransplants. In these models, RCMV-accelerated TVS results from increased graft infiltration of inflammatory cells through up-regulation of chemokine expression. The aim of this study was to determine if RCMV infection accelerates renal transplant chronic allograft nephropathy (CAN), and the role of chemokines in this process. METHODS: F344 kidneys were transplanted into Lewis recipients with and without RCMV infection. To monitor CAN, serum creatinine (Cr) levels were measured starting at 4 weeks posttransplantation. At 7 and 21 days, and at terminal rejection, grafts were examined for histologic changes, inflammatory cell infiltrates, viral load, and chemokine expression profiles. RESULTS: By week 8, serum Cr showed significant elevation (P < .01) in the RCMV-infected group vs uninfected group, and remained significantly elevated through the end of the study. RCMV+ renal allografts had significant inflammatory cell infiltration and increased CAN at postoperative day (POD) 28. The CC chemokines RANTES, MCP-1, and MIP-1alpha, and the CXC chemokine IP-10 were up-regulated in RCMV-infected vs uninfected allografts. IP-10 was significantly up-regulated early in the process, whereas RANTES and MCP-1 were induced at a later time. CONCLUSIONS: RCMV infection accelerates CAN, with associated graft inflammatory infiltrates, which is paralleled by an increase in expression of CC and CXC chemokines. Our findings suggest that the early induction of IP-10 in the infected allografts promotes alterations in T-cell and monocyte migration to the graft, which initiates accelerated inflammatory and fibrotic changes associated with CAN.

Do pathogens accelerate atherosclerosis?

Infection with the pathogens human cytomegalovirus (HCMV) or Chlamydia pneumonia (CP) is linked to the development of vascular disease, including atherosclerosis. The role of pathogens in vasculopathies has been controversial. However, animal models have demonstrated a direct link between infection with CP and herpesviruses and the development of vascular disease. Clinical studies have shown a direct association of HCMV and CP with the acceleration of vascular disease. This article will review the evidence supporting the role for CP and HCMV in the development of vascular disease and will suggest a potential mechanism for HCMV acceleration of the disease process. Vascular diseases are the result of either mechanical or immune-related injury followed by inflammation and subsequent smooth muscle cell (SMC) proliferation and/or migration from the vessel media to the intima, which culminates in vessel narrowing. A number of in vitro and in vivo models have provided potential mechanisms involved in pathogen-mediated vascular disease. Recently, we have demonstrated that HCMV infection of arterial but not venous SMC results in significant cellular migration in vitro. Migration was dependent on expression of the HCMV-encoded chemokine receptors, US28, and the presence of the chemokines, RANTES or MCP-1. Migration involved chemotaxis and provided the first evidence that viruses may induce migration of SMC toward sites of chemokine production through the expression of a virally encoded chemokine receptor in infected SMC. Because SMC migration into the neointimal space is the hallmark of vascular disease, these observations provide a molecular link between HCMV and the development of vascular disease.

Reactivation of latent human cytomegalovirus in CD14(+) monocytes is differentiation dependent.

We have previously demonstrated reactivation of latent human cytomegalovirus (HCMV) in myeloid lineage cells obtained from healthy donors. Virus was obtained from allogenically stimulated monocyte-derived macrophages (Allo-MDM), but not from macrophages differentiated by mitogenic stimulation (ConA-MDM). In the present study, the cellular and cytokine components essential for HCMV replication and reactivation were examined in Allo-MDM. The importance of both CD4(+) and CD8(+) T cells in the generation of HCMV-permissive Allo-MDM was demonstrated by negative selection or blocking experiments using antibodies directed against both HLA class I and HLA class II molecules. Interestingly, contact of monocytes with CD4 or CD8 T cells was not essential for reactivation of HCMV, since virus was observed in macrophages derived from CD14(+) monocytes stimulated by supernatants produced by allogeneic stimulation of peripheral blood mononuclear cells. Examination of the cytokines produced in Allo-MDM and ConA-MDM cultures indicated a significant difference in the kinetics of production and quantity of these factors. Further examination of the cytokines essential for the generation of HCMV-permissive Allo-MDM identified gamma interferon (IFN-gamma) but not interleukin-1 or -2, tumor necrosis factor alpha, or granulocyte-macrophage colony-stimulating factor as critical components in the generation of these macrophages. In addition, although IFN-gamma was crucial for reactivation of latent HCMV, addition of IFN-gamma to unstimulated macrophage cultures was insufficient to reactivate virus. Thus, this study characterizes two distinct monocyte-derived cell types which can be distinguished by their ability to reactivate and support HCMV replication and identifies the critical importance of IFN-gamma in the reactivation of HCMV.

The HCMV chemokine receptor US28 is a potential target in vascular disease.

The human cytomegalovirus (HCMV) has been implicated in the acceleration of vascular disease for some time. The development of vascular disease involves a chronic inflammatory process with many contributing factors, and of these, chemokines and their receptors have recently been identified as key mediators. Interestingly, HCMV encodes four potential chemokine receptors (US27, US28, UL33 and UL78). Of these virally-encoded chemokine receptors, US28 has been the most widely characterized. US28 binds many of the CC-chemokines, and this class of chemokines contributes to the development of vascular disease. Importantly, HCMV infection mediates in vitro SMC migration, which is dependent upon expression of US28 and CC-chemokine binding. US28 and the US28 functional homologues that are capable of inducing the migration of SMC represent potential targets in the treatment of CMV-accelerated vascular disease such as atherosclerosis, restenosis, and transplant vascular sclerosis.

The human cytomegalovirus IE86 protein can block cell cycle progression after inducing transition into the S phase of permissive cells.

Human cytomegalovirus (HCMV) infection of permissive cells has been reported to induce a cell cycle halt. One or more viral proteins may be involved in halting progression at different stages of the cell cycle. We investigated how HCMV infection, and specifically IE86 protein expression, affects the cell cycles of permissive and nonpermissive cells. We used a recombinant virus that expresses the green fluorescent protein (GFP) to determine the effects of HCMV on the cell cycle of permissive cells. Fluorescence by GFP allowed us to select for only productively infected cells. Replication-defective adenovirus vectors expressing the IE72 or IE86 protein were also used to efficiently transduce 95% or more of the cells. The adenovirus-expressed IE86 protein was determined to be functional by demonstrating negative autoregulation of the major immediate-early promoter and activation of an early viral promoter in the context of the viral genome. To eliminate adenovirus protein effects, plasmids expressing GFP for fluorescent selection of only transfected cells and wild-type IE86 protein or a mutant IE86 protein were tested in permissive and nonpermissive cells. HCMV infection induced the entry of U373 cells into the S phase. All permissive cells infected with HCMV were blocked in cell cycle progression and could not divide. After either transduction or transfection and IE86 protein expression, the number of all permissive or nonpermissive cell types in the S phase increased significantly, but the cells could no longer divide. The IE72 protein did not have a significant effect on the S phase. Since IE86 protein inhibits cell cycle progression, the IE2 gene in a human fibroblast IE86 protein-expressing cell line was sequenced. The IE86 protein in these retrovirus-transduced cells has mutations in a critical region of the viral protein. The locations of the mutations and the function of the IE86 protein in controlling cell cycle progression are discussed.

The human cytomegalovirus chemokine receptor US28 mediates vascular smooth muscle cell migration.

Human cytomegalovirus (HCMV) infection of smooth muscle cells (SMCs) in vivo has been linked to a viral etiology of vascular disease. In this report, we demonstrate that HCMV infection of primary arterial SMCs results in significant cellular migration. Ablation of the chemokine receptor, US28, abrogates SMC migration, which is rescued only by expression of the viral homolog and not a cellular G protein-coupled receptor (GPCR). Expression of US28 in the presence of CC chemokines including RANTES or MCP-1 was sufficient to promote SMC migration by both chemokinesis and chemotaxis, which was inhibited by protein tyrosine kinase inhibitors. US28-mediated SMC migration provides a molecular basis for the correlative evidence that links HCMV to the acceleration of vascular disease.

Cyclophilin a modulates processing of human immunodeficiency virus type 1 p55Gag: mechanism for antiviral effects of cyclosporin A.

The molecular chaperone cyclophilin A (Cyp A) modulates human immunodeficiency virus type 1 (HIV-1) infectivity through its interactions with Gag structural proteins. The molecular mechanism for CypA in HIV-1 replication is not known. We studied chaperone effects on Gag precursor processing using cyclosporin A (CsA) to bind CypA and prevent its interaction with p55Gag. CsA treatment inhibited p55Gag processing in extracellular virus-like particles produced from COS cells. We confirmed the effect of CsA on Gag processing by examining virions produced from CEMx174 cells infected with HIV-1LAI. Particles accumulated in the presence of CsA displayed mostly immature virion morphology and lacked condensed capsids. CsA has a direct effect on HIV-1 Gag processing that implicates CypA as having an important role in the maturation of HIV-1 particles.

Gag protein from human immunodeficiency virus type 1 assembles in the absence of cyclophilin A.

Human immunodeficiency virus type 1 (HIV-1) replication requires coordinated activities of host and viral factors. We reported previously that interactions of the host factor cyclophilin A with HIV-1 Gag polyproteins affected Gag processing and maturation of virus particles (Streblow et al., 1998. Virology 245, 197-202). We now use in vitro translation and physical analysis of Gag structures to refine our understanding of how cyclophilin A affects HIV-1 replication. Gag assembled into oligomeric structures in vitro in the presence or absence of cyclophilin A, and proteins synthesized under the two conditions were equally susceptible to cleavage by exogenous HIV-1 protease. These and previous data show that Cyclophilin A is required at a step between Gag assembly and Gag processing/virion morphogenesis. Cyclophilin A may be required for Gag conformational changes subsequent to assembly, that are required for efficient dimerization and activation of the viral protease.

Therapeutic approaches to HIV infection based on virus structure and the host pathogen interaction.

The HIV-1 infection of central nervous system, with attendant neuropathy and dementia, poses a unique challenge for antiviral therapy. For practical considerations, it is important to define carefully the precise therapeutic objectives. (1) Is it necessary to inhibit spreading HIV-1 infection in the central nervous system? (2) What is the role of inflammatory responses in central nervous system disease during HIV-1 infection? (3) Is there a correlation between pathology and dementia? (4) Are virions or virus gene products toxic in the central nervous system? (5) Is there a role for immune suppression and opportunistic pathogens in AIDS dementia? The development of therapeutic agents for HIV-1 infection is guided by our knowledge of virus structure, the function of viral proteins, the interactions with host components, and detailed features of the virus life cycle. In each case, unique features of the virus can be identified and established as targets for unique antiviral compounds. Drugs acting as inhibitors of virus enzymatic functions are plagued by the rapid development in vivo of drug-resistant virus variants, although combination or alternating chemotherapeutic regimens may obviate some of these concerns. Novel approaches to inhibiting virus are flourishing. In vitro studies show the value of agents as diverse as molecular decoys for tat activity to efforts to mutagenize integrated proviruses by modified oligonucleotides that form triple helices with chromosomal genes. As each particular clinical situation is better defined, the design and application of these agents can be refined to inhibit HIV-1 replication and reduce the associated morbidity.

Selective amplification of simian immunodeficiency virus genotypes after intrarectal inoculation of rhesus monkeys.

Animal models for sexual transmission of human immunodeficiency virus can define the influences of virus type, dose, and route of inoculation on infection and clinical outcome. We used an uncloned simian immunodeficiency virus stock (SIVmac) to inoculate cells in vitro and to inoculate rhesus monkeys by intravenous and intrarectal routes. The distribution of virus genotypes present in each of these infection examples was characterized by DNA sequence analysis of viral long terminal repeats (LTRs). Our analysis of LTR sequences from in vitro and in vivo infections revealed three main genotypes: one genotype was observed only for in vitro infection, and two other genotypes were recovered only from infected animals. By comparing animals inoculated with high intrarectal doses of SIVmac and those inoculated with low doses, we demonstrated that unique subsets of the stock were selected after intrarectal infection. Our findings indicate that minor genotypes present in the stock cross the rectal mucosa and are amplified selectively to become prominent in peripheral blood mononuclear cells from acutely infected animals. Studies with a molecular recombinant of SIV and human immunodeficiency virus type 1 sequences, SHIV, showed that viral LTR sequences do not undergo especially rapid sequence variation or rearrangement after intrarectal inoculation. The mucosal barrier exerts a significant influence on infection and disease progression by reducing the efficiency of SIVmac infection and by permitting distinct, pathogenic genotypes to become established in the host.