Stress granules regulate double-stranded RNA-dependent protein kinase activation through a complex containing G3BP1 and Caprin1.

UNLABELLED: Stress granules (SGs) are dynamic cytoplasmic repositories containing translationally silenced mRNAs that assemble upon cellular stress. We recently reported that the SG nucleating protein G3BP1 promotes antiviral activity and is essential in double-stranded RNA-dependent protein kinase (PKR) recruitment to stress granules, thereby driving phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α). Here, we delineate the mechanism for SG-dependent PKR activation. We show that G3BP1 and inactive PKR directly interact with each other, dependent on both the NTF2-like and PXXP domains of G3BP1. The G3BP1-interacting protein Caprin1 also directly interacts with PKR, regulates efficient PKR activation at the stress granule, and is also integral for the release of active PKR into the cytoplasm to engage in substrate recognition. The G3BP1-Caprin1-PKR complex represents a new mode of PKR activation and is important for antiviral activity of G3BP1 and PKR during infection with mengovirus. Our data links stress responses and their resultant SGs with innate immune activation through PKR without a requirement for foreign double-stranded RNA (dsRNA) pattern recognition. IMPORTANCE: Our previous work indicates that stress granules have antiviral activity and mediate innate immunity through functions of G3BP1; however, the mechanistic details of these functions were not resolved. We show that much of the antiviral activity of stress granules is contingent on the function of PKR in a complex with G3BP1 and Caprin1. The PKR-G3BP1-Caprin1 complex undergoes dynamic transitioning within and outside stress granules to accomplish PKR activation and translational repression. This mechanism appears to function distinctly from canonical pattern recognition of double-stranded RNA by PKR. Therefore, this mechanism bridges the stress response with innate immunity, allowing the cell to respond to many cellular stressors and amplify the pathogen pattern recognition systems of innate immunity.

Suppression of injuries caused by a lytic RNA virus (mengovirus) and their uncoupling from viral reproduction by mutual cell/virus disarmament.

Viruses often elicit cell injury (cytopathic effect [CPE]), a major cause of viral diseases. CPE is usually considered to be a prerequisite for and/or consequence of efficient viral growth. Recently, we proposed that viral CPE may largely be due to host defensive and viral antidefensive activities. This study aimed to check the validity of this proposal by using as a model HeLa cells infected with mengovirus (MV). As we showed previously, infection of these cells with wild-type MV resulted in necrosis, whereas a mutant with incapacitated antidefensive ("security") viral leader (L) protein induced apoptosis. Here, we showed that several major morphological and biochemical signs of CPE (e.g., alterations in cellular and nuclear shape, plasma membrane, cytoskeleton, chromatin, and metabolic activity) in cells infected with L(-) mutants in the presence of an apoptosis inhibitor were strongly suppressed or delayed for long after completion of viral reproduction. These facts demonstrate that the efficient reproduction of a lytic virus may not directly require development of at least some pathological alterations normally accompanying infection. They also imply that L protein is involved in the control of many apparently unrelated functions. The results also suggest that the virus-activated program with competing necrotic and apoptotic branches is host encoded, with the choice between apoptosis and necrosis depending on a variety of intrinsic and extrinsic conditions. Implementation of this defensive suicidal program could be uncoupled from the viral reproduction. The possibility of such uncoupling has significant implications for the pathogenesis and treatment of viral diseases.

Lower respiratory tract infection induced by a genetically modified picornavirus in its natural murine host.

Infections with the picornavirus, human rhinovirus (HRV), are a major cause of wheezing illnesses and asthma exacerbations. In developing a murine model of picornaviral airway infection, we noted the absence of murine rhinoviruses and that mice are not natural hosts for HRV. The picornavirus, mengovirus, induces lethal systemic infections in its natural murine hosts, but small genetic differences can profoundly affect picornaviral tropism and virulence. We demonstrate that inhalation of a genetically attenuated mengovirus, vMC(0), induces lower respiratory tract infections in mice. After intranasal vMC(0) inoculation, lung viral titers increased, peaking at 24 h postinoculation with viral shedding persisting for 5 days, whereas HRV-A01a lung viral titers decreased and were undetectable 24 h after intranasal inoculation. Inhalation of vMC(0), but not vehicle or UV-inactivated vMC(0), induced an acute respiratory illness, with body weight loss and lower airway inflammation, characterized by increased numbers of airway neutrophils and lymphocytes and elevated pulmonary expression of neutrophil chemoattractant CXCR2 ligands (CXCL1, CXCL2, CXCL5) and interleukin-17A. Mice inoculated with vMC(0), compared with those inoculated with vehicle or UV-inactivated vMC(0), exhibited increased pulmonary expression of interferon (IFN-α, IFN-ß, IFN-λ), viral RNA sensors [toll-like receptor (TLR)3, TLR7, nucleotide-binding oligomerization domain containing 2 (NOD2)], and chemokines associated with HRV infection in humans (CXCL10, CCL2). Inhalation of vMC(0), but not vehicle or UV-inactivated vMC(0), was accompanied by increased airway fluid myeloperoxidase levels, an indicator of neutrophil activation, increased MUC5B gene expression, and lung edema, a sign of infection-related lung injury. Consistent with experimental HRV inoculations of nonallergic, nonasthmatic human subjects, there were no effects on airway hyperresponsiveness after inhalation of vMC(0) by healthy mice. This novel murine model of picornaviral airway infection and inflammation should be useful for defining mechanisms of HRV pathogenesis in humans.

Differential IFN-alpha/beta production suppressing capacities of the leader proteins of mengovirus and foot-and-mouth disease virus.

Picornaviruses encompass a large family of RNA viruses. Some picornaviruses possess a leader (L) protein at the N-terminus of their polyprotein. The L proteins of encephalomyocarditis virus, a cardiovirus, and foot-and-mouth disease virus (FMDV), an aphthovirus, are both dispensable for replication and their major function seems to be the suppression of antiviral host cell responses. Previously, we showed that the L protein of mengovirus, a strain of encephalomyocarditis virus, inhibits antiviral responses by inhibiting type I interferon (IFN-alpha/beta) gene transcription. The L protein of the FMDV is a protease (L(pro)) that cleaves cellular factors to reduce cytokine and chemokine mRNA production and to inhibit cap-dependent cellular host mRNA translation, thereby limiting the production of proteins with antiviral activity. In this study, we constructed a viable chimeric mengovirus that expresses FMDV L(pro) in place of the authentic L protein in order to compare the efficiency of the immune evasion mechanisms mediated by L and L(pro) respectively. We show that in this mengovirus background the L protein is more potent than FMDV L(pro) in suppressing IFN-alpha/beta responses. Yet, FMDV L(pro) is important to antagonize infection-limiting responses both in vitro and in vivo.

A rat model of picornavirus-induced airway infection and inflammation.

BACKGROUND: Infection of the lower airways by rhinovirus, a member of the picornavirus family, is an important cause of wheezing illnesses in infants, and plays an important role in the pathogenesis of rhinovirus-induced asthma exacerbations. Given the absence of natural rhinovirus infections in rodents, we investigated whether an attenuated form of mengovirus, a picornavirus whose wild-type form causes systemic rather than respiratory infections in its natural rodent hosts, could induce airway infections in rats with inflammatory responses similar to those in human rhinovirus infections. RESULTS: After inoculation with 10(7) plaque-forming units of attenuated mengovirus through an inhalation route, infectious mengovirus was consistently recovered on days 1 and 3 postinoculation from left lung homogenates (median Log10 plaque-forming units = 6.0 and 4.8, respectively) and right lung bronchoalveolar lavage fluid (median Log10 plaque-forming units = 5.8 and 4.0, respectively). Insufflation of attenuated mengovirus, but not vehicle or UV-inactivated virus, into the lungs of BN rats caused significant increases (P < 0.05) in lower airway neutrophils and lymphocytes in the bronchoalveolar lavage fluid and patchy peribronchiolar, perivascular, and alveolar cellular infiltrates in lung tissue sections. In addition, infection with attenuated mengovirus significantly increased (P < 0.05) lower airway levels of neutrophil chemoattractant CXCR2 ligands [cytokine-induced neutrophil chemoattractant-1 (CINC-1; CXCL1) and macrophage inflammatory protein-2 (MIP-2; CXCL2)] and monocyte chemoattractant protein-1 (MCP-1; CCL2) in comparison to inoculation with vehicle or UV-inactivated virus. CONCLUSION: Attenuated mengovirus caused a respiratory infection in rats with several days of viral shedding accompanied by a lower airway inflammatory response consisting of neutrophils and lymphocytes. These features suggest that mengovirus-induced airway infection in rodents could be a useful model to define mechanisms of rhinovirus-induced airway inflammation in humans.

Delivery of mengovirus-derived RNA replicons into tumoural liver enhances the anti-tumour efficacy of a peripheral peptide-based vaccine.

Hepatocellular carcinoma is a deadly cancer with growing incidence for which immunotherapy is one of the most promising therapeutic approach. Peptide-based vaccines designed to induce strong, sustained CD8+ T cell responses are effective in animal models and cancer patients. We demonstrated the efficacy of curative peptide-based immunisation against a unique epitope of SV40 tumour antigen, through the induction of a strong CD8+ T cell-specific response, in our liver tumour model. However, as in human clinical trials, most tumour antigen epitopes did not induce a therapeutic effect, despite inducing strong CD8+ T cell responses. We therefore modified the tumour environment to enhance peptide-based vaccine efficacy by delivering mengovirus (MV)-derived RNA autoreplicating sequences (MV-RNA replicons) into the liver. The injection of replication-competent RNA replicons into the liver converted partial tumour regression into tumour eradication, whereas non-replicating RNA had no such effect. Replicating RNA replicon injection induced local recruitment of innate immunity effectors (NK and NKT) to the tumour and did not affect specific CD8+ T cell populations or other myelolymphoid subsets. The local delivery of such RNA replicons into tumour stroma is therefore a promising strategy complementary to the use of peripheral peptide-based vaccines for treating liver tumours.

The mengovirus leader protein blocks interferon-alpha/beta gene transcription and inhibits activation of interferon regulatory factor 3.

Viral infection of mammalian cells triggers the synthesis and secretion of type I interferons (i.e. IFN-alpha/beta), which induce the transcription of genes that cause cells to adopt an antiviral state. Many viruses have adapted mechanisms to evade IFN-alpha/beta-mediated responses. The leader protein of mengovirus, a picornavirus, has been implicated as an IFN-alpha/beta antagonist. Here, we show that the leader inhibits the transcription of IFN-alpha/beta and that both the presence of a zinc finger motif in its N-terminus and phosphorylation of threonine-47 are required for this function. Transcription of IFN-alpha/beta genes relies on the activity of a number of transcription factors, including interferon regulatory factor 3 (IRF-3). We show that the leader interferes with the transactivation activity of IRF-3 by interfering with its dimerization. Accordingly, mutant viruses with a disturbed leader function were impaired in their ability to suppress IFN-alpha/beta transcription in vivo. By consequence, the leader mutant viruses had an impaired ability to replicate and spread in normal mice but not in IFNAR-KO mice, which are incapable of mounting an IFN-alpha/beta-dependent antiviral response. These results suggest that the leader, by suppressing IRF3-mediated IFN-alpha/beta production, plays an important role in replication and dissemination of mengovirus in its host.

Increased antiviral activity of microscale-purified HuIFN alpha 8 (human interferon alpha 8) over HuIFN alpha 2b in Hep-2 cells challenged with Mengo virus.

Human proteins are not routinely expressed at high levels in Escherichia coli for, among other reasons, different codon usage. Several purification procedures have been applied to recover recombinant proteins for further biological characterization. However, the vast majority involve costly chromatography procedures. In the present study, both (Hu)IFN(alpha 2b) (human interferon alpha 2b) and (Hu)IFN(alpha 8) were expressed efficiently in E. coli BL21-codonplus-RIL. Subsequently, both recombinant proteins were purified to homogeneity by passive elution from reverse-stained SDS/PAGE gels, a cost-effective purification procedure. After purification, both recovered proteins were biologically active. The (Hu)IFN(alpha 8) subtype induced 1.46-fold more antiviral activity than (Hu)IFN(alpha 2b) using Hep-2 human laryngeal carcinoma cell challenged with Mengo virus.

Transcutaneous immunization of domestic animals: opportunities and challenges.

Transcutaneous immunization (TCI), the topical application of antigen and adjuvant directly onto intact skin, can safely and effectively elicit systemic immune responses in mice and humans against a variety of antigens. This novel method of vaccine delivery has the potential to provide a safe and convenient method by which vaccines may be delivered to elicit protective immunity in domestic animals. To date, however, immune responses induced by TCI in companion and production animals has not been reported. In this report, we demonstrate that TCI may be widely applicable to many animals. Immune responses elicited by TCI require further optimization for each antigen and species, and success may depend upon the structure and composition of the skin of the target species. The prospect of TCI as a practical and broadly applicable approach to vaccination in veterinary medicine is discussed in the context of these challenges.

Quantitative and qualitative analyses of the immune responses induced by a multivalent minigene DNA vaccine.

Vaccines containing minigenes - isolated antigenic epitopes encoded by short open reading frames - can, under certain circumstances, confer protective immunity upon the vaccinee. Here we evaluate the efficacy of the minigene vaccine approach using DNA immunization and find that, to be immunogenic, a minigene-encoded epitope requires a perfect "Kozak" translational initiation region. In addition, using intracellular cytokine staining, we show that immunization with a plasmid encoding a full-length protein induces epitope-specific CD8(+) T cells which are detectable directly ex vivo, and constitute approximately 2% of the vaccinee's splenic CD8(+) T cells. In contrast, such cells are undetectable directly ex vivo in recipients of a minigene vaccine. Nevertheless, the minigene plasmid does induce a low number of epitope-specific CD8(+) T cells, which can be amplified to detectable levels by in vivo stimulation. Indeed, 4 days after in vivo stimulation (by virus infection), all vaccinated mice - regardless of whether they had been vaccinated with the minigene or with the full-length gene - had similar numbers of epitope-specific CD8(+) T cells. However, despite these strong responses at 4 days post-infection, recipients of the minigene vaccine showed no enhanced ability to limit virus replication and dissemination. We therefore observe a dichotomy; minigene vaccinees are not protected, despite the presence of strong virus-specific immune responses at 4 days post-challenge. We suggest that the protective benefits of vaccination exert themselves very soon - perhaps within minutes or hours - after virus challenge. If the vaccine-induced immune response is too low to achieve this early protective effect, virus-specific T cells will expand rapidly, but ineffectually, leading to the strong but non-protective response measured at 4 days post-infection. Thus, vaccine-induced immunity should be monitored very early in infection, since the extent to which these responses may later be amplified is largely irrelevant to the protection observed.

Genetically engineered Mengo virus vaccination of multiple captive wildlife species.

Encephalomyocarditis virus (EMCV), has caused the deaths of many species of animals in zoological parks and research institutions. The Audubon Park Zoo, (New Orleans, Louisiana, USA) attempted vaccination of several species with a killed EMCV vaccine with mixed results. This paper reports an attempt at vaccination against EMCV using a genetically engineered, live attenuated Mengo virus (vMC0) at the Audubon Park Zoo and Miami Metro Zoo, (Miami, Florida, USA) from December 1996 to June 1997. Several species of animals were vaccinated with vMC0, which is serologically indistinguishable from the field strain of EMCV. Serum samples were taken at the time of vaccination and again 21 days later, then submitted for serum neutralization titers against EMCV. The vaccinate species included red capped mangebey (Cercocebus torquatus), colobus (Colobus guereza), angolan colobus (Colobus angolensis), ruffed lemur (Lemur variegatus ruber and Lemur variegatus variegatus), back lemur (Lemur macaco), ring-tailed lemur (Lemur catta), siamang (Hylobates syndactylus), diana guenon (Cercopithicus diana), spider monkey (Ateles geoffroyi), common marmoset (Callithrix jacchus), talapoin monkey (Cercopithecus talapoin), Brazilian tapir (Tapirus terrestris), Baird's tapir (Tapirus bairdii), Malayan tapir (Tapirus indicus), dromedary camel (Camelus dromedarius), bactrian camel (Camelus bactrianus), gerenuk (Litocranius walleri), guanaco (Lama glama guanicoe), black duiker (Cephalophus niger), Vietnamese potbellied pig (Sus scrofa), babirusa (Babyrousa babyrussa), collard peccary (Tayass tajacu), and African crested porcupine (Hystrix africaeaustralis). The vaccine response was variable, with high virus neutralizing antibody titer responses in some primate species and mixed to poor responses for other species. No ill effects were seen with vaccination.

Protection against lethal encephalomyocarditis virus infection in the absence of serum-neutralizing antibodies.

Although the ability of serum-neutralizing antibodies to protect against picornavirus infection is well established, the contribution of cell-mediated immunity to protection is uncertain. Using major histocompatibility complex class II-deficient (RHAbeta-/-) mice, which are unable to mediate CD4(+) T-lymphocyte-dependent humoral responses, we demonstrated antibody-independent protection against lethal encephalomyocarditis virus (EMCV) infection in the natural host. The majority of RHAbeta-/- mice inoculated with 10(4) PFU of attenuated Mengo virus (vMC24) resolved infection and were resistant to lethal challenge with the highly virulent, serotypically identical cardiovirus, EMCV. Protection in these mice was in the absence of detectable serum-neutralizing antibodies. Depletion of CD8(+) T lymphocytes prior to lethal EMCV challenge ablated protection in vMC24-immunized RHAbeta-/- mice. The CD8(+) T-lymphocyte-dependent protection observed in vivo may, in part, be the result of cytotoxic T-lymphocyte (CTL) activity, as CD8(+) T splenocytes exhibited in vitro cytolysis of EMCV-infected targets. The existence of virus-specific CD8(+) T-lymphocyte memory in these mice was demonstrated by increased expression of cell surface activation markers CD25, CD69, CD71, and CTLA-4 following antigen-specific reactivation in vitro. Although recall response in vMC24-immunized RHAbeta-/- mice was intact and effectual shortly after immunization, protection abated over time, as only 3 of 10 vMC24-immunized RHAbeta-/- mice survived when rechallenged 90 days later. The present study demonstrating CD8(+) T-lymphocyte-dependent protection in the absence of serum-neutralizing antibodies, coupled with our previous results indicating that vMC24-specific CD4(+) T lymphocytes confer protection against lethal EMCV in the absence of prophylactic antibodies, suggests the existence of nonhumoral protective mechanisms against picornavirus infections.

Study of the immunogenicity of different recombinant Mengo viruses expressing HIV1 and SIV epitopes.

Recombinant Mengo viruses expressing heterologous genes have proven to be safe and immunogenic in both mice and primates, and to be able to induce both humoral and cellular immune responses (Altmeyer et al., 1995, 1996). Several recombinant Mengo viruses expressing either a large region (aa 65-206) of the HIV1 nef gene product, or cytotoxic T lymphocyte (CTL) epitopic regions from the SIV Gag (aa 182-190), Nef (aa 155-178) and Pol (aa 587-601) gene products were engineered. The heterologous antigens were expressed either as fusion proteins with the Mengo virus leader (L) protein, or in cleaved form through autocatalytic cleavage by the foot-and-mouth disease virus 2A protein. Rhesus macaques and BALB/c mice inoculated with the Mengo virus SIV recombinants failed to develop CTL responses against the SIV gene products, while one of the HIV-Nef recombinants induced a weak CTL response in mice directed to an HIV1 Nef peptide spanning positions 182-198. In contrast, BALB/c mice immunized with vaccinia virus recombinants expressing HIV1 Nef developed a strong CTL response to the 182-198 peptide and also responded to a second peptide spanning positions 73-81. These results indicate that Mengo virus recombinants expressing HIV1 Nef and SIV CTL epitopes are weak immunogens. One of the fusion recombinants expressing SIV CTL epitopes failed to infect macaques even when used at high doses, while the recombinant expressing HIV1 Nef as a fusion protein failed to infect BALB/c mice. These results demonstrate that the expression of certain heterologous sequences as fusion proteins with L can result in the loss of the ability of the recombinant to infect normally susceptible animals.

Theiler's virus and Mengo virus induce cross-reactive cytotoxic T lymphocytes restricted to the same immunodominant VP2 epitope in C57BL/6 mice.

C57BL/6 mice develop a virus-specific cytotoxic T-lymphocyte (CTL) response after intraperitoneal inoculation with either the DA strain of Theiler's virus or Mengo virus, two members of the Cardiovirus genus. These CTLs contribute to viral clearance in the case of Theiler's virus but do not protect the mice from the fatal encephalomyelitis caused by Mengo virus. In this study we show that DA and Mengo virus-induced CTLs are cross-reactive. The cross-reactivity is due to a conserved, H-2Db-restricted epitope located between amino acid residues 122 and 130 of the VP2 capsid protein (VP2(122-130)). This epitope is immunodominant in C57BL/6 mice infected with Theiler's virus. The VP2(122-130) epitope, initially identified for Mengo virus, is the first CTL epitope described for Theiler's virus.

A multivalent minigene vaccine, containing B-cell, cytotoxic T-lymphocyte, and Th epitopes from several microbes, induces appropriate responses in vivo and confers protection against more than one pathogen.

The development of safe and effective vaccines remains a major goal in the prevention, and perhaps treatment, of infectious diseases. Ideally, a single vaccine would confer protection against several pathogens and would induce both cellular and humoral arms of the immune response. We originally demonstrated that two virus-specific cytotoxic T-lymphocyte (CTL) epitopes, from the same virus but presented by different major histocompatibility complex alleles, when linked in tandem as minigenes in a recombinant vaccinia virus, could confer complete protection against subsequent viral challenge. In the study, we extended this approach, which we termed string of beads, expanding the immunogenic scope in two ways: first, by introduction of T helper (Th) and B-cell (antibody) epitopes alongside CTL epitopes and second, by including immunogenic sequences from a variety of infectious agents, five viruses and one bacterium. The vaccine (VV-sv) comprises CTL epitopes from Sendai virus, respiratory syncytial virus, and lymphocytic choriomeningitis virus (LCMV); Th epitopes from vesicular stomatitis virus and Mycobacterium tuberculosis; and an antibody epitope from mengovirus. The construct contains a single start codon, and the epitopes are linked directly, without intervening spacer amino acids. There was some concern that the combination of several normally immunodominant epitopes might result in a new hierarchy of dominance, in which certain epitopes predominated and others exhibited reduced immunogenicity. However we show that when analyzed in tissue culture and in vivo, all six epitopes are expressed. CTL and Th cells are induced in vivo, along with neutralizing antibody. The induced immunity is biologically relevant: after VV-sv immunization, the antimengovirus antibody confers protection against mengovirus challenge. Similarly, CTL induced by the LCMV epitope protected mice against challenge with this agent. Thus, a polyvalent, minigene-based vaccine can simultaneously induce several classes of immune response and thereby can confer protection against diverse pathogens.

The immunogenic and pathogenic potential of short poly(C) tract Mengo viruses.

We have shown previously that genetically engineered Mengo viruses with artificial deletions in their 5' noncoding polyribocytidylic acid (poly(C)) tracts are highly attenuated for the natural murine host and also for other animals such as baboons, macaques, and domestic pigs. The present report further characterizes select short poly(C) tract Mengo viruses in the natural murine host. A positive correlation was found between the length of the poly(C) tract and murine virulence, as measured by virus brain titers and brain lesion scores after infection. Histological examination of brain tissue collected from infected animals clearly showed that the short poly(C) tract viruses did not induce the devastating pathological effects characteristic of animals inoculated with wild-type virus. Instead, the short-tract Mengo viruses proved excellent immunological agents. A dose of only 100 plaque-forming units of vMC24 (poly(C) tract: C13UC10), injected subcutaneously, protected 80% of recipient animals against a normally lethal dose of encephalomyocarditis virus. The protection was long-lived, and animals similarly immunized with vMCo virus (poly(C) tract: Co) still had protective neutralizing antibody titers up to 16 months after inoculation. In addition, the short-tract viruses proved genetically stable, in that the vMC24 virus did not yield detectable pathogenic revertants even after multiple, forced passages in 4-week-old mice. These studies suggest that Mengo viruses containing deletions in their poly(C) tracts are biologically safe and potent immunogens and imply that they may have uses as cardiovirus vaccines.

Protection of non-murine mammals against encephalomyocarditis virus using a genetically engineered Mengo virus.

Genetically engineered Mengo viruses with artificial deletions in the 5' noncoding poly(C) tracts are highly attenuated for pathogenicity when introduced as live vaccines into the natural murine host. Inoculation produces lifelong protective immunity without disease or viral persistence. This report extends the vaccination studies to non-murine hosts, including baboons, macaques and domestic pigs, all of which are susceptible to severe cardiovirus epizootics. All animals of these species that were inoculated with vMC24, an engineered strain of Mengo, seroconverted. When the immunized animals were challenged, they were protected against lethal doses of encephalomyocarditis virus (EMCV) derived from currently circulating epizootic strains. In baboons, the neutralizing antibody titers induced by vMC24 were significantly higher than from an inactivated EMCV vaccine. Moreover, terminal histopathology on baboons (inoculated intramuscularly), macaques (inoculated intracerebrally), and pigs (inoculated intramuscularly) showed few, if any, gross lesions characteristic of EMCV-like disease, in the vMC24 vaccinates. We suggest that genetically engineered, short poly(C) Mengo viruses may be universally potent attenuated vaccines for many types of animals and can possibly provide safe, efficacious protection against all cardioviruses of the EMCV serotype.

Characterization of Mengo virus neutralization epitopes. II. Infection of mice with an attenuated virus.

A panel of five neutralizing monoclonal antibodies was generated from mice immunized with an attenuated strain of Mengo virus. Four of the antibodies were used to select mutants of Mengo virus which were able to escape neutralization by the selecting antibody, but it was not possible to select mutants which could escape neutralization by the fifth antibody. The capsid coding region of the RNA genome of each mutant was directly sequenced to identify the mutation(s) responsible for the neutralization escape phenotype. These results are compared to those of a previous study in which immunogenic determinants recognized by neutralizing antibodies generated against pentameric capsid subunits were located on the external surface of the Mengo virion. We have confirmed the existence of the previously identified immunogenic determinant in VP3 (site 2) as well as an immunodominant determinant in VP2 (site 1). Two previously uncharacterized determinants, located in surface loops of VP1 (sites 3 and 4A), were also identified. None of the mutations conferring the neutralization escape phenotype was found near the surface depressions on the virion which are believed to be the receptor binding sites.

Cytotoxic T cell response to Mengo virus in mice: effector cell phenotype and target proteins.

The Mengo virus specific cytotoxic T lymphocyte (CTL) response was investigated after intraperitoneal infection of mice with the attenuated Mengo virus strain vMC24. A high level of CTL activity was detected in spleen cell cultures obtained from infected C3H/HeJ (H-2k) or C57BL/6 (H-2b) mice after a secondary in vitro stimulation with Mengo virus-infected cells. The CTL activity, which was MHC class I-restricted, was shown to be mediated by CD8+ T cells. Recombinant vaccinia viruses that expressed capsid proteins VP0, VP1 or VP3 were produced and used to identify the protein(s) recognized by the Mengo virus-specific CTLs. In both C3H/HeJ and C57BL/6 mice, analysis of CTL activity against target cells expressing each capsid protein showed that VP0 was the only capsid protein recognized by the CD8+ CTLs. The CTL epitope(s) could be further located in the C-terminal half of VP0, i.e. in capsid protein VP2. Moreover, using unlabelled target cells expressing VP0 as cold competitors, we were able to almost completely inhibit recognition and lysis of Mengo virus-infected cells by specific CD8+ CTLs. Thus, the CTL response directed against VP2 was immunodominant in both C3H/HeJ- and C57BL/6-infected mice.

Attenuated Mengo virus: a new vector for live recombinant vaccines.

Several features make Mengo virus an excellent candidate for use as a vaccine vector. The virus has a wide host range, including rodents, pigs, monkeys, and most likely humans, and expresses its genome exclusively in the cytoplasm of the infected cell. Stable attenuated strains exist which are deleted for part of the 5' noncoding region of the genome. Here we report an attenuated Mengo virus recombinant, vLCMG4, that encodes an immunodominant cytotoxic T-lymphocyte epitope of the lymphocytic choriomeningitis virus (LCMV) nucleo-protein. vLCMG4 induced protective immunity against lethal LCMV infection after a single, low-dose immunization in BALB/c mice and elicited an LCMV-specific CD8+ cytotoxic T lymphocyte response. This demonstrates the potential of recombinant Mengo virus vaccines to confer protection against infectious diseases by the induction of cellular immune responses.