The orchestrated functions of innate leukocytes and t cell subsets contribute to humoral immunity, virus control, and recovery from secondary poxvirus challenge

A pivotal role for antigen-specific recall responses to secondary virus infection is well established, but the contribution of innate immune cells to this process is unknown. Recovery of mice from a primary orthopoxvirus (ectromelia virus [ECTV]) infection requires the function of natural killer (NK) cells, granulocytes, plasmacytoid dendritic cells (pDC), T cells, and B cells. However, during a secondary challenge, resolution of infection is thought to be dependent on antibody but not T cell function. We investigated the contribution of NK cells, granulocytes, and pDC to virus control during a secondary virus challenge in mice that had been primed with an avirulent, mutant strain of ECTV. Mice depleted of NK cells, granulocytes, or pDC effectively controlled virus, as did mice depleted of both CD4 and CD8 T cell subsets. However, mice concurrently depleted of all three innate cell subsets had elevated virus load, but this was significantly exacerbated in mice also depleted of CD4 and/or CD8 T cells. Increased viral replication in mice lacking innate cells plus CD4 T cells was associated with a significant reduction in neutralizing antibody. Importantly, in addition to T-dependent neutralizing antibody responses, the function of CD8 T cells was also clearly important for virus control. The data indicate that in the absence of innate cell subsets, a critical role for both CD4 and CD8 T cells becomes apparent and, conversely, in the absence of T cell subsets, innate immune cells help contain infectio

Evidence for Persistence of Ectromelia Virus in Inbred Mice, Recrudescence Following Immunosuppression and Transmission to Naive Mice

Orthopoxviruses (OPV), including variola, vaccinia, monkeypox, cowpox and ectromelia viruses cause acute infections in their hosts. With the exception of variola virus (VARV), the etiological agent of smallpox, other OPV have been reported to persist in a variety of animal species following natural or experimental infection. Despite the implications and significance for the ecology and epidemiology of diseases these viruses cause, those reports have never been thoroughly investigated. We used the mouse pathogen ectromelia virus (ECTV), the agent of mousepox and a close relative of VARV to investigate virus persistence in inbred mice. We provide evidence that ECTV causes a persistent infection in some susceptible strains of mice in which low levels of virus genomes were detected in various tissues late in infection. The bone marrow (BM) and blood appeared to be key sites of persistence. Contemporaneous with virus persistence, antiviral CD8 T cell responses were demonstrable over the entire 25-week study period, with a change in the immunodominance hierarchy evident during the first 3 weeks. Some virus-encoded host response modifiers were found to modulate virus persistence whereas host genes encoded by the NKC and MHC class I reduced the potential for persistence. When susceptible strains of mice that had apparently recovered from infection were subjected to sustained immunosuppression with cyclophosphamide (CTX), animals succumbed to mousepox with high titers of infectious virus in various organs. CTX treated index mice transmitted virus to, and caused disease in, co-housed naïve mice. The most surprising but significant finding was that immunosuppression of disease-resistant C57BL/6 mice several weeks after recovery from primary infection generated high titers of virus in multiple tissues. Resistant mice showed no evidence of a persistent infection. This is the strongest evidence that ECTV can persist in inbred mice, regardless of their resistance status

Poxvirus-Encoded Gamma Interferon Binding Protein Dampens the Host Immune Response to Infection

Ectromelia virus (ECTV), a natural mouse pathogen and the causative agent of mousepox, is closely related to variola virus (VARV), which causes smallpox in humans. Mousepox is an excellent surrogate small-animal model for smallpox. Both ECTV and VARV encode a multitude of host response modifiers that target components of the immune system and that are thought to contribute to the high mortality rates associated with infection. Like VARV, ECTV encodes a protein homologous to the ectodomain of the host gamma interferon (IFN-γ) receptor 1. We generated an IFN-γ binding protein (IFN-γbp) deletion mutant of ECTV to study the role of viral IFN-γbp (vIFN-γbp) in host-virus interaction and also to elucidate the contribution of this molecule to the outcome of infection. Our data show that the absence of vIFN-γbp does not affect virus replication per se but does have a profound effect on virus replication and pathogenesis in mice. BALB/c mice, which are normally susceptible to infection with ECTV, were able to control replication of the mutant virus and survive infection. Absence of vIFN-γbp from ECTV allowed the generation of an elective host immune response that was otherwise diminished by this viral protein. Mice infected with a vIFN-γbp deletion mutant virus, designated ECTV-IFN-γbpΔ, produced increased levels of IFN-γ and generated robust cell-mediated and antibody responses. Using several strains of mice that exhibit differential degrees of resistance to mousepox, we show that recovery or death from ECTV infection is determined by a balance between the host's ability to produce IFN-γ and the virus' ability to dampen its effects

Loss of Actin-Based Motility Impairs Ectromelia Virus Release In Vitro but Is Not Critical to Spread In Vivo

Ectromelia virus (ECTV) is an orthopoxvirus and the causative agent of mousepox. Like other poxviruses such as variola virus (agent of smallpox), monkeypox virus and vaccinia virus (the live vaccine for smallpox), ECTV promotes actin-nucleation at the surface of infected cells during virus release. Homologs of the viral protein A36 mediate this function through phosphorylation of one or two tyrosine residues that ultimately recruit the cellular Arp2/3 actin-nucleating complex. A36 also functions in the intracellular trafficking of virus mediated by kinesin-1. Here, we describe the generation of a recombinant ECTV that is specifically disrupted in actin-based motility allowing us to examine the role of this transport step in vivo for the first time. We show that actin-based motility has a critical role in promoting the release of virus from infected cells in vitro but plays a minor role in virus spread in vivo. It is likely that loss of microtubule-dependent transport is a major factor for the attenuation observed when A36R is deleted.This work was funded by the National Health and Medical Research Council through the grants APP100790 (G.K) and GNT0632785 (T.N)

Identification of poxvirus CD8+ T cell determinants to enable rational design and characterization of smallpox vaccines

The large size of poxvirus genomes has stymied attempts to identify determinants recognized by CD8+ T cells and greatly impeded development of mouse smallpox vaccination models. Here, we use a vaccinia virus (VACV) expression library containing each of the predicted 258 open reading frames to identify five peptide determinants that account for approximately half of the VACV-specific CD8+ T cell response in C57BL/6 mice. We show that the primary immunodominance hierarchy is greatly affected by the route of VACV infection and the poxvirus strain used. Modified vaccinia virus ankara (MVA), a candidate replacement smallpox vaccine, failed to induce responses to two of the defined determinants. This could not be predicted by genomic comparison of viruses and is not due strictly to limited MVA replication in mice. Several determinants are immunogenic in cowpox and ectromelia (mousepox) virus infections, and immunization with the immunodominant determinant provided significant protection against lethal mousepox. These findings have important implications for understanding poxvirus immunity in animal models and bench-marking immune responses to poxvirus vaccines in humans

A Natural Genetic Variant of Granzyme B Confers Lethality to a Common Viral Infection

Many immune response genes are highly polymorphic, consistent with the selective pressure imposed by pathogens over evolutionary time, and the need to balance infection control with the risk of auto-immunity. Epidemiological and genomic studies have identified many genetic variants that confer susceptibility or resistance to pathogenic micro-organisms. While extensive polymorphism has been reported for the granzyme B (GzmB) gene, its relevance to pathogen immunity is unexplored. Here, we describe the biochemical and cytotoxic functions of a common allele of GzmB (GzmBW) common in wild mouse. While retaining ‘Asp-ase ’ activity, GzmBW has substrate preferences that differ considerably from GzmBP, which is common to all inbred strains. In vitro, GzmBW preferentially cleaves recombinant Bid, whereas GzmBP activates pro-caspases directly. Recombinant GzmBW and GzmBP induced equivalent apoptosis of uninfected targets cells when delivered with perforin in vitro. Nonetheless, mice homozygous for GzmBW were unable to control murine cytomegalovirus (MCMV) infection, and succumbed as a result of excessive liver damage. Although similar numbers of anti-viral CD8 T cells were generated in both mouse strains, GzmBW-expressing CD8 T cells isolated from infected mice were unable to kill MCMV

Expression of the interferon-inducible chemokines MuMig and Crg-2 following vaccina virus infection in viro

MuMig (monokine induced by gamma interferon) and Crg-2 (cytokine responsive gene) are chemokines of the CXC subfamily. They share activity as T and NK cell chemoattractants. Crg-2 has been shown to be inducible by IFN, TNF, IL-1 and LPS, whereas the expression of MuMig is thought to be strictly dependent on IFN-γ. In the present study, the kinetics of expression of the genes for MuMig and Crg-2 were analysed by northern blot analysis in organs of normal mice and in groups of gene knockout mice deficient in IFN-γ (IFN- γ-/-) or receptors for IFN-α/β and IFN-γ (double R(-/-); DR+) after vaccinia virus infection. MuMig mRNA was not expressed in IFN-γ(-/-) mice in all organs examined, whereas Crg-2 mRNA levels were marginally reduced. In contrast, MuMig and Crg-2 mRNA transcripts were completely abolished in the DR(-/-) mice