Ignacio Mena

Coxsackievirus B3-Induced Myocarditis : Perforin Exacerbates Disease, But Plays No Detectable Role in Virus Clearance

Viral myocarditis is remarkably common, being detected in approximately 1% of unselected asymptomatic individuals. Many cases are attributable to enteroviral infection, and in particular to coxsackievirus B3. The underlying pathogenesis is controversial, but most studies admit the important immunopathological role of infiltrating CD8+ (cytotoxic) T lymphocytes (CTLs). We have previously shown that CTLs play conflicting roles in coxsackievirus B (CVB) myocarditis; they assist in controlling virus replication, but also are instrumental in causing the extensive inflammatory disease, which often results in severe myocardial scarring. A role for perforin, the major CTL cytolytic protein, in CVB myocarditis has been suggested, but never proven. In the present study we use perforin knockout (PKO) mice to show that perforin plays a major role in CVB infection; in broad terms, perforin is important in immunopathology, but not in CVB clearance. For example, PKO mice are better able to withstand a normally lethal dose of CVB (100% survival of PKO mice compared with 90% death in +/+ littermates). In addition, PKO mice given a nonlethal dose of CVB develop only a mild myocarditis, whereas their perforin+ littermates have extensive myocardial lesions. The myocarditis in PKO mice resolves more quickly, and these mice show minimal histological sequelae; in contrast, late in disease the perforin+ mice develop severe myocardial fibrosis. PKO mice, despite lacking this major CTL effector function, can control the infection and eradicate the virus; growth kinetics and peak CVB titers are indistinguishable in PKO and perforin+ mice. Therefore, the immunopathological and antiviral effects of CTLs can be uncoupled by ablation of perforin; this offers a promising target for therapy of myocarditis. Furthermore, we evaluate the possible roles of apoptosis, and of chemokine expression, in CVB infection. In perforin+ mice, apoptotic cells are detected within the inflammatory infiltrate, whereas in their PKO counterparts, apoptotic myocyte nuclei are seen. Chemokine expression in both PKO and perforin+ mice precedes and parallels the course of myocarditis. Several chemokines are detectable earlier in PKO mice than in perforin+ mice, but PKO mice show reduced peak levels, and chemokine expression decays sooner. In particular, MIP-1alpha expression is barely detectable at any time point in PKO mice, but it is readily identified in perforin+ animals, peaking just before the time of maximal myocarditis; this is particularly interesting, given that MIP-1alpha knockout mice are resistant to CVB myocarditis, but remain able to control viral infection. Thus, the chemokine pathway offers a second route of intervention to diminish myocarditis and its sequelae, while permitting the host to eradicate the virus.

Cell Cycle Status Affects Coxsackievirus Replication, Persistence, and Reactivation In Vitro

ABSTRACT Enteroviral persistence has been implicated in the pathogenesis of several chronic human diseases, including dilated cardiomyopathy, insulin-dependent diabetes mellitus, and chronic inflammatory myopathy. However, these viruses are considered highly cytolytic, and it is unclear what mechanisms might permit their long-term survival. Here, we describe the generation of a recombinant coxsackievirus B3 (CVB3) expressing the enhanced green fluorescent protein (eGFP), which we used to mark and track infected cells in vitro. Following exposure of quiescent tissue culture cells to either wild-type CVB3 or eGFP-CVB3, virus production was very limited but increased dramatically after cells were permitted to divide. Studies with cell cycle inhibitors revealed that cells arrested at the G1 or G1/S phase could express high levels of viral polyprotein and produced abundant infectious virus. In contrast, both protein expression and virus yield were markedly reduced in quiescent cells (i.e., cells in G0) and in cells blocked at the G2/M phase. Following infection with eGFP-CVB3, quiescent cells retained viral RNA for several days in the absence of infectious virus production. Furthermore, RNA extracted from nonproductive quiescent cells was infectious when transfected into dividing cells, indicating that CVB3 appears to be capable of establishing a latent infection in G0 cells, at least in tissue culture. Finally, wounding of infected quiescent cells resulted in viral protein expression limited to cells in and adjacent to the lesion. We suggest that (i) cell cycle status determines the distribution of CVB3 during acute infection and (ii) the persistence of CVB3 in vivo may rely on infection of quiescent (G0) cells incapable of supporting viral replication; a subsequent change in the cell cycle status may lead to virus reactivation, triggering chronic viral and/or immune-mediated pathology in the host.

Using Recombinant Coxsackievirus B3 To Evaluate the Induction and Protective Efficacy of CD8+ T Cells during Picornavirus Infection

ABSTRACT Coxsackievirus B3 (CVB3) is a common human pathogen that has been associated with serious diseases including myocarditis and pancreatitis. To better understand the effect of cytotoxic T-lymphocyte (CTL) responses in controlling CVB3 infection, we have inserted well-characterized CTL epitopes into the CVB3 genome. Constructs were made by placing the epitope of interest upstream of the open reading frame encoding the CVB3 polyprotein, separated by a poly-glycine linker and an artificial 3Cpro/3CDpro cleavage site. This strategy results in the foreign protein being translated at the amino- terminus of the viral polyprotein, from which it is cleaved prior to viral assembly. In this study, we cloned major histocompatibility complex class I-restricted CTL epitopes from lymphocytic choriomeningitis virus (LCMV) into recombinant CVB3 (rCVB3). In vitro, rCVB3 growth kinetics showed a 1- to 2-h lag period before exponential growth was initiated, and peak titers were ∼1 log unit lower than for wild-type virus. rCVB3 replicated to high titers in vivo and caused severe pancreatitis but minimal myocarditis. Despite the high virus titers, rCVB3 infection of naive mice failed to induce a strong CD8+ T-cell response to the encoded epitope; this has implications for the proposed role of “cross-priming” during virus infection and for the utility of recombinant picornaviruses as vaccine vectors. In contrast, rCVB3 infection of LCMV-immune mice resulted in direct ex vivo cytotoxic activity against target cells coated with the epitope peptide, demonstrating that the rCVB3-encoded LCMV-specific epitope was expressed and presented in vivo. The preexisting CD8+memory T cells could limit rCVB replication; compared to naive mice, infection of LCMV-immune mice with rCVB3 resulted in ∼50-fold-lower virus titers in the heart and ∼6-fold-lower virus titers in the pancreas. Although the inserted CTL epitope was retained by rCVB3 through several passages in tissue culture, it was lost in an organ-specific manner in vivo; a substantial proportion of viruses from the pancreas retained the insert, compared to only 0 to 1.8% of myocardial viruses. Together, these results show that expression of heterologous viral proteins by recombinant CVB3 provides a useful model for determining the mechanisms underlying the immune response to this viral pathogen.

Coxsackievirus replication and the cell cycle: a potential regulatory mechanism for viral persistence/latency

Coxsackieviruses (CV) are characterized by their ability to cause cytopathic effects in tissue culture and by their capacity to initiate acute disease by inducing apoptosis within targeted organs in vivo. These viruses are considered highly cytolytic, but can establish persistence/latency in susceptible cells, indicating that a regulatory mechanism may exist to shut off viral protein synthesis and replication under certain situations. The persistence of coxsackieviral RNA is of particular medical interest due to its association with chronic human diseases such as dilated cardiomyopathy and chronic inflammatory myopathy. Here, we discuss the potential mechanisms regulating coxsackievirus replication, and the ability of viral RNA to remain in an apparent latent state within quiescent cells.

Chimeric calicivirus-like particles elicit protective anti-viral cytotoxic responses without adjuvant

We have analyzed the potential of virus-like particles (VLPs) from rabbit hemorrhagic disease virus (RHDV) as a delivery system for foreign T cell epitopes. To accomplish this goal, we generated chimeric RHDV-VLPs incorporating a CD8(+) T cell epitope (SIINFEKL) derived from chicken ovalbumin (OVA). The OVA epitope was inserted in the capsid protein (VP60) of RHDV at two different locations: 1) the N-terminus, predicted to be facing to the inner core of the VLPs, and 2) a novel insertion site predicted to be located within an exposed loop. Both constructions correctly assembled into VLPs. In vitro, the chimeric VLPs activated dendritic cells for TNF-alpha secretion and they were processed and presented to specific T cells. In vivo, mice immunized with the chimeric VLPs without adjuvant were able to induce specific cellular responses mediated by cytotoxic and memory T cells. More importantly, immunization with chimeric VLPs was able to resolve an infection by a recombinant vaccinia virus expressing OVA protein.

Self-Assembly of the Recombinant Capsid Protein of a Swine Norovirus into Virus-Like Particles and Evaluation of Monoclonal Antibodies Cross-Reactive with a Human Strain from Genogroup II

ABSTRACT Noroviruses (NoVs) are responsible for the majority of gastroenteritis outbreaks in humans. Recently, NoV strains which are genetically closely related to human genogroup II (GII) NoVs have been detected in fecal specimens from swine. These findings have raised concern about the possible role of pigs as reservoirs for NoVs that could infect humans. To better understand the epidemiology of swine NoVs in both the swine and the human populations, rapid immunoassays are needed. In this study, baculovirus recombinants were generated to express the capsid gene of a swine NoV GII genotype 11 (GII.11) strain which self-assembled into virus-like particles (VLPs). Subsequently, the purified VLPs were used to evoke monoclonal antibodies (MAbs) in mice. A panel of eight promising MAbs was obtained and evaluated for their ability to bind to heterologous VLPs, denaturated antigens, and truncated capsid proteins. The MAbs could be classified into two groups: two MAbs that recognized linear epitopes located at the amino-terminal half (shell domain) of the swine NoV GII.11 VLPs and that cross-reacted with human GII.4 NoV VLPs. The other six MAbs bound to conformational epitopes and did not cross-react with the human GII.4 VLPs. To our knowledge, this is the first report on the characterization of MAbs against swine NoVs. The swine NoV VLPs and the MAbs described here may be further used for the design of diagnostic reagents that could help increase our knowledge of the prevalence of NoV infections in pigs and the possible role of pigs as reservoirs for NoVs.

Theiler's Virus Infection of Primary Cultures of Bone Marrow-Derived Monocytes/Macrophages

ABSTRACT Theilers virus, a murine picornavirus, causes a persistent infection of macrophage/microglial cells in the central nervous systems of SJL/J mice. Viral replication is restricted in the majority of infected cells, whereas a minority of them contain large amounts of viral RNA and antigens. For the present work, we infected primary cultures of bone marrow monocytes/macrophages from SJL/J mice with Theilers virus. During the first 10 h postinfection (p.i.), infected monocytes/macrophages were round and covered with filopodia and contained large amounts of viral antigens throughout their cytoplasm. Later on, they were large, flat, and devoid of filopodia and they contained only small amounts of viral antigens distributed in discrete inclusions. These two types of infected cells were very reminiscent of the two types of infected macrophages found in the spinal cords of SJL/J mice. At the peak of virus production, the viral yield per cell was approximately 200 times lower than that for BHK-21 cells. Cell death occurred in the culture during the first 24 h p.i. but not thereafter. No infected cells could be detected after 4 days p.i., and the infection never spread to 100% of the cells. This restriction was unchanged by treating the medium at pH 2 but was abolished by treating it with a neutralizing alpha/beta interferon antiserum, indicating a role for this cytokine in limiting virus expression in monocyte/macrophage cultures. The role of alpha/beta interferon was confirmed by the observation that monocytes/macrophages from IFNA/BR−/− mice were fully permissive.

Epitope Insertion at the N-Terminal Molecular Switch of the Rabbit Hemorrhagic Disease Virus T=3 Capsid Protein Leads to Larger T=4 Capsids.

ABSTRACT Viruses need only one or a few structural capsid proteins to build an infectious particle. This is possible through the extensive use of symmetry and the conformational polymorphism of the structural proteins. Using virus-like particles (VLP) from rabbit hemorrhagic disease virus (RHDV) as a model, we addressed the basis of calicivirus capsid assembly and their application in vaccine design. The RHDV capsid is based on a T=3 lattice containing 180 identical subunits (VP1). We determined the structure of RHDV VLP to 8.0-Å resolution by three-dimensional cryoelectron microscopy; in addition, we used San Miguel sea lion virus (SMSV) and feline calicivirus (FCV) capsid subunit structures to establish the backbone structure of VP1 by homology modeling and flexible docking analysis. Based on the three-domain VP1 model, several insertion mutants were designed to validate the VP1 pseudoatomic model, and foreign epitopes were placed at the N- or C-terminal end, as well as in an exposed loop on the capsid surface. We selected a set of T and B cell epitopes of various lengths derived from viral and eukaryotic origins. Structural analysis of these chimeric capsids further validates the VP1 model to design new chimeras. Whereas most insertions are well tolerated, VP1 with an FCV capsid protein-neutralizing epitope at the N terminus assembled into mixtures of T=3 and larger T=4 capsids. The calicivirus capsid protein, and perhaps that of many other viruses, thus can encode polymorphism modulators that are not anticipated from the plane sequence, with important implications for understanding virus assembly and evolution.

Infection of Macrophage Primary Cultures by Persistent and Nonpersistent Strains of Theiler's Virus: Role of Capsid and Noncapsid Viral Determinants

ABSTRACT We compared the infection of bone marrow macrophages by the DA and GDVII strains of Theilers virus and by two viruses constructed by exchanging the DA and GDVII capsids. The replication of the GDVII strain and of both chimeric viruses was restricted in macrophages. Therefore, the infection of macrophages requires both capsid and noncapsid viral determinants.

Rabbit hemorrhagic disease virus capsid, a versatile platform for foreign B-cell epitope display inducing protective humoral immune responses

Virus-like particles (VLPs), comprised of viral structural proteins devoid of genetic material, are tunable nanoparticles that can be chemically or genetically engineered, to be used as platforms for multimeric display of foreign antigens. Here, we report the engineering of chimeric VLPs, derived from rabbit hemorrhagic disease virus (RHDV) for presentation of foreign B-cell antigens to the immune system. The RHDV capsid comprises 180 copies of a single capsid subunit (VP60). To evaluate the ability of chimeric RHDV VLPs to elicit protective humoral responses against foreign antigens, we tested two B-cell epitopes: a novel neutralizing B-cell epitope, derived from feline calicivirus capsid protein, and a well characterized B-cell epitope from the extracellular domain of influenza A virus M2 protein (M2e). We generated sets of chimeric RHDV VLPs by insertion of the foreign B-cell epitopes at three different locations within VP60 protein (which involved different levels of surface accessibility) and in different copy numbers per site. The immunogenic potential of the chimeric VLPs was analyzed in the mouse model. The results presented here indicated that chimeric RHDV VLPs elicit potent protective humoral responses against displayed foreign B-cell epitopes, demonstrated by both, in vitro neutralization and in vivo protection against a lethal challenge.