Interferon partly dictates a divergent transcriptional response in poxvirus-infected and bystander inflammatory monocytes.

Inflammatory monocytes (iMOs) and B cells are the main targets of the poxvirus ectromelia virus (ECTV) in the lymph nodes of mice and play distinct roles in surviving the infection. Infected and bystander iMOs control ECTV's systemic spread, preventing early death, while B cells make antibodies that eliminate ECTV. Our work demonstrates that within an infected animal that survives ECTV infection, intrinsic and bystander infection of iMOs and B cells differentially control the transcription of genes important for immune cell function and, perhaps, cell identity. Bystander cells upregulate metabolism, antigen presentation, and interferon-stimulated genes. Infected cells downregulate many cell-type-specific genes and upregulate transcripts typical of non-immune cells. Bystander (Bys) and infected (Inf) iMOs non-redundantly contribute to the cytokine milieu and the interferon response. Furthermore, we uncover how type I interferon (IFN-I) or IFN-γ signaling differentially regulates immune pathways in Inf and Bys iMOs and that, at steady state, IFN-I primes iMOs for rapid IFN-I production and antigen presentation.

Langerhans Cells Orchestrate the Protective Antiviral Innate Immune Response in the Lymph Node.

During disseminating viral infections, a swift innate immune response (IIR) in the draining lymph node (dLN) that restricts systemic viral spread is critical for optimal resistance to disease. However, it is unclear how this IIR is orchestrated. We show that after footpad infection of mice with ectromelia virus, dendritic cells (DCs) highly expressing major histocompatibility complex class II (MHC class IIhi DCs), including CD207+ epidermal Langerhans cells (LCs), CD103+CD207+ double-positive dermal DCs (DP-DCs), and CD103-CD207- double-negative dermal DCs (DN-DCs) migrate to the dLN from the skin carrying virus. MHC class IIhi DCs, predominantly LCs and DP-DCs, are the first cells upregulating IIR cytokines in the dLN. Preventing MHC class IIhi DC migration or depletion of LCs, but not DP-DC deficiency, suppresses the IIR in the dLN and results in high viral lethality. Therefore, LCs are the architects of an early IIR in the dLN that is critical for optimal resistance to a disseminating viral infection.

Migratory Dendritic Cells, Group 1 Innate Lymphoid Cells, and Inflammatory Monocytes Collaborate to Recruit NK Cells to the Virus-Infected Lymph Node.

Circulating natural killer (NK) cells help protect the host from lympho-hematogenous acute viral diseases by rapidly entering draining lymph nodes (dLNs) to curb virus dissemination. Here, we identify a highly choreographed mechanism underlying this process. Using footpad infection with ectromelia virus, a pathogenic DNA virus of mice, we show that TLR9/MyD88 sensing induces NKG2D ligands in virus-infected, skin-derived migratory dendritic cells (mDCs) to induce production of IFN-γ by classical NK cells and other types of group 1 innate lymphoid cells (ILCs) already in dLNs, via NKG2D. Uninfected inflammatory monocytes, also recruited to dLNs by mDCs in a TLR9/MyD88-dependent manner, respond to IFN-γ by secreting CXCL9 for optimal CXCR3-dependent recruitment of circulating NK cells. This work unveils a TLR9/MyD88-dependent mechanism whereby in dLNs, three cell types-mDCs, group 1 ILCs (mostly NK cells), and inflammatory monocytes-coordinate the recruitment of protective circulating NK cells to dLNs.

Cutting Edge: Protection by Antiviral Memory CD8 T Cells Requires Rapidly Produced Antigen in Large Amounts.

Numerous attempts to produce antiviral vaccines by harnessing memory CD8 T cells have failed. A barrier to progress is that we do not know what makes an Ag a viable target of protective CD8 T cell memory. We found that in mice susceptible to lethal mousepox (the mouse homolog of human smallpox), a dendritic cell vaccine that induced memory CD8 T cells fully protected mice when the infecting virus produced Ag in large quantities and with rapid kinetics. Protection did not occur when the Ag was produced in low amounts, even with rapid kinetics, and protection was only partial when the Ag was produced in large quantities but with slow kinetics. Hence, the amount and timing of Ag expression appear to be key determinants of memory CD8 T cell antiviral protective immunity. These findings may have important implications for vaccine design.

Matrix-M adjuvant enhances antibody, cellular and protective immune responses of a Zaire Ebola/Makona virus glycoprotein (GP) nanoparticle vaccine in mice.

Ebola virus (EBOV) causes severe hemorrhagic fever for which there is no approved treatment or preventive vaccine. Immunological correlates of protective immunity against EBOV disease are not well understood. However, non-human primate studies have associated protection of experimental vaccines with binding and neutralizing antibodies to the EBOV glycoprotein (GP) as well as EBOV GP-specific CD4(+) and CD8(+) T cells. In this report a full length, unmodified Zaire EBOV GP gene from the 2014 EBOV Makona strain (EBOV/Mak) was cloned into a baculovirus vector. Recombinant EBOV/Mak GP was produced in Sf9 insect cells as glycosylated trimers and, when purified, formed spherical 30-40 nm particles. In mice, EBOV/Mak GP co-administered with the saponin adjuvant Matrix-M was significantly more immunogenic, as measured by virus neutralization titers and anti-EBOV/Mak GP IgG as compared to immunization with AlPO4 adjuvanted or non-adjuvanted EBOV/Mak GP. Similarly, antigen specific T cells secreting IFN-γ were induced most prominently by EBOV/Mak GP with Matrix-M. Matrix-M also enhanced the frequency of antigen-specific germinal center B cells and follicular helper T (TFH) cells in the spleen in a dose-dependent manner. Immunization with EBOV/Mak GP with Matrix-M was 100% protective in a lethal viral challenge murine model; whereas no protection was observed with the AlPO4 adjuvant and only 10% (1/10) mice were protected in the EBOV/Mak GP antigen alone group. Matrix-M adjuvanted vaccine induced a rapid onset of specific IgG and neutralizing antibodies, increased frequency of multifunctional CD4+ and CD8(+) T cells, specific TFH cells, germinal center B cells, and persistence of EBOV GP-specific plasma B cells in the bone marrow. Taken together, the addition of Matrix-M adjuvant to the EBOV/Mak GP nanoparticles enhanced both B and T-cell immune stimulation which may be critical for an Ebola subunit vaccine with broad and long lasting protective immunity.

Sequential Activation of Two Pathogen-Sensing Pathways Required for Type I Interferon Expression and Resistance to an Acute DNA Virus Infection.

Toll-like receptor 9 (TLR9), its adaptor MyD88, the downstream transcription factor interferon regulatory factor 7 (IRF7), and type I interferons (IFN-I) are all required for resistance to infection with ectromelia virus (ECTV). However, it is not known how or in which cells these effectors function to promote survival. Here, we showed that after infection with ECTV, the TLR9-MyD88-IRF7 pathway was necessary in CD11c(+) cells for the expression of proinflammatory cytokines and the recruitment of inflammatory monocytes (iMos) to the draining lymph node (dLN). In the dLN, the major producers of IFN-I were infected iMos, which used the DNA sensor-adaptor STING to activate IRF7 and nuclear factor κB (NF-κB) signaling to induce the expression of IFN-α and IFN-ß, respectively. Thus, in vivo, two pathways of DNA pathogen sensing act sequentially in two distinct cell types to orchestrate resistance to a viral disease.

Targeted Blockade of JAK/STAT3 Signaling Inhibits Ovarian Carcinoma Growth.

Ovarian carcinoma is the fifth leading cause of death among women in the United States. Persistent activation of STAT3 is frequently detected in ovarian carcinoma. STAT3 is activated by Janus family kinases (JAK) via cytokine receptors, growth factor receptor, and non-growth factor receptor tyrosine kinases. Activation of STAT3 mediates tumor cell proliferation, survival, motility, invasion, and angiogenesis, and recent work demonstrates that STAT3 activation suppresses antitumor immune responses and supports tumor-promoting inflammation. We hypothesized that therapeutic targeting of the JAK/STAT3 pathway would inhibit tumor growth by direct effects on ovarian carcinoma cells and by inhibition of cells in the tumor microenvironment (TME). To test this, we evaluated the effects of a small-molecule JAK inhibitor, AZD1480, on cell viability, apoptosis, proliferation, migration, and adhesion of ovarian carcinoma cells in vitro. We then evaluated the effects of AZD1480 on in vivo tumor growth and progression, gene expression, tumor-associated matrix metalloproteinase (MMP) activity, and immune cell populations in a transgenic mouse model of ovarian carcinoma. AZD1480 treatment inhibited STAT3 phosphorylation and DNA binding, and migration and adhesion of cultured ovarian carcinoma cells and ovarian tumor growth rate, volume, and ascites production in mice. In addition, drug treatment led to altered gene expression, decreased tumor-associated MMP activity, and fewer suppressor T cells in the peritoneal TME of tumor-bearing mice than control mice. Taken together, our results show pharmacologic inhibition of the JAK2/STAT3 pathway leads to disruption of functions essential for ovarian tumor growth and progression and represents a promising therapeutic strategy.

Impact of distinct poxvirus infections on the specificities and functionalities of CD4+ T cell responses.

UNLABELLED: The factors that determine CD4+ T cell (TCD4+) specificities, functional capacity, and memory persistence in response to complex pathogens remain unclear. We explored these parameters in the C57BL/6 mouse through comparison of two highly related (>92% homology) poxviruses: ectromelia virus (ECTV), a natural mouse pathogen, and vaccinia virus (VACV), a heterologous virus that nevertheless elicits potent immune responses. In addition to elucidating several previously unidentified major histocompatibility complex class II (MHC-II)-restricted epitopes, we observed many qualitative and quantitative differences between the TCD4+ repertoires, including responses not elicited by VACV despite complete sequence conservation. In addition, we observed functional heterogeneity between ECTV- and VACV-specific TCD4+ at both a global and individual epitope level, particularly greater expression of the cytolytic marker CD107a from TCD4+ following ECTV infection. Most striking were differences during the late memory phase where, in contrast to ECTV, VACV infection failed to elicit measurable epitope-specific TCD4+ as determined by intracellular cytokine staining. These findings illustrate the strong influence of epitope-extrinsic factors on TCD4+ responses and memory. IMPORTANCE: Much of our understanding concerning host-pathogen relationships in the context of poxvirus infections stems from studies of VACV in mice. However, VACV is not a natural mouse pathogen, and therefore, the relevance of results obtained using this model may be limited. Here, we explored the MHC class II-restricted TCD4+ repertoire induced by mousepox (ECTV) infection and the functional profile of the responding epitope-specific TCD4+, comparing these results to those induced by VACV infection under matched conditions. Despite a high degree of homology between the two viruses, we observed distinct specificity and functional profiles of TCD4+ responses at both acute and memory time points, with VACV-specific TCD4+ memory being notably compromised. These data offer insight into the impact of epitope-extrinsic factors on the resulting TCD4+ responses.

MyD88-dependent immunity to a natural model of vaccinia virus infection does not involve Toll-like receptor 2.

UNLABELLED: Although the pattern recognition receptor Toll-like receptor 2 (TLR2) is typically thought to recognize bacterial components, it has been described to alter the induction of both innate and adaptive immunity to a number of viruses, including vaccinia virus (VACV). However, many pathogens that reportedly encode TLR2 agonists may actually be artifactually contaminated during preparation, possibly with cellular debris or merely with molecules that sensitize cells to be activated by authentic TLR2 agonists. In both humans and mice, the most relevant natural route of infection with VACV is through intradermal infection of the skin. Therefore, we examined the requirement for TLR2 and its signaling adaptor MyD88 in protective immunity to VACV after intradermal infection. We find that although TLR2 may recognize virus preparations in vitro and have a minor role in preventing dissemination of VACV following systemic infection with large doses of virus, it is wholly disposable in both control of virus replication and induction of adaptive immunity following intradermal infection. In contrast, MyD88 is required for efficient induction of CD4 T cell and B cell responses and for local control of virus replication following intradermal infection. However, even MyD88 is not required to induce local inflammation, inflammatory cytokine production, or recruitment of cells that restrict virus from spreading systemically after peripheral infection. Thus, an effective antiviral response does require MyD88, but TLR2 is not required for control of a peripheral VACV infection. These findings emphasize the importance of studying relevant routes of infection when examining innate sensing mechanisms. IMPORTANCE: Vaccinia virus (VACV) provides the backbone for some of the most widely used and successful viral vaccine vectors and is also related to the human pathogens Cantagalo virus and molluscum contagiosum virus that infect the skin of patients. Therefore, it is vital to understand the mechanisms that induce a strong innate immune response to the virus following dermal infection. Here, we compare the ability of the innate sensing molecule Toll-like receptor 2 (TLR2) and the signaling molecule MyD88 to influence the innate and adaptive immune response to VACV following systemic or dermal infection.

Crosstalk between the type 1 interferon and nuclear factor kappa B pathways confers resistance to a lethal virus infection.

Nuclear factor kappa B (NF-κB) and type 1 interferon (T1-IFN) signaling are innate immune mechanisms activated upon viral infection. However, the role of NF-κB and its interplay with T1-IFN in antiviral immunity is poorly understood. We show that NF-κB is essential for resistance to ectromelia virus (ECTV), a mouse orthopoxvirus related to the virus causing human smallpox. Additionally, an ECTV mutant lacking an NF-κB inhibitor activates NF-κB more effectively in vivo, resulting in increased proinflammatory molecule transcription in uninfected cells and organs and decreased viral replication. Unexpectedly, NF-κB activation compensates for genetic defects in the T1-IFN pathway, such as a deficiency in the IRF7 transcription factor, resulting in virus control. Thus, overlap between the T1-IFN and NF-κB pathways allows the host to overcome genetic or pathogen-induced deficiencies in T1-IFN and survive an otherwise lethal poxvirus infection. These findings may also explain why some pathogens target both pathways to cause disease.

Memory CD8⁺ T cells can outsource IFN-γ production but not cytolytic killing for antiviral protection.

Immunization with vaccinia virus (VACV), the virus comprising the smallpox vaccine, induces memory CD8(+) T cells that protect from subsequent infections with smallpox in humans or the related ectromelia virus (ECTV) in mice. Memory CD8(+) T cells largely mediate these effects by expanding into secondary effectors that secrete the antiviral cytokine interferon-γ (IFN-γ) and induce cytolysis via releasing factors such as perforin, which permeabilizes target cells. We show that protection from ECTV infection after VACV immunization depends on the initial memory cell frequency and ability of expanded secondary effectors to kill infected targets in a perforin-dependent manner. Although IFN-γ is essential for antiviral protection, it can be produced by either secondary effectors or concomitant primary effector CD8(+) T cells recruited to the response. Thus, during lethal virus challenge, memory CD8(+) T cells are required for cytolytic killing of infected cells, but primary effectors can play important roles by producing IFN-γ.

The mature virion of ectromelia virus, a pathogenic poxvirus, is capable of intrahepatic spread and can serve as a target for delayed therapy.

Orthopoxviruses (OPVs), which include the agent of smallpox (variola virus), the zoonotic monkeypox virus, the vaccine and zoonotic species vaccinia virus, and the mouse pathogen ectromelia virus (ECTV), form two types of infectious viral particles: the mature virus (MV), which is cytosolic, and the enveloped virus (EV), which is extracellular. It is believed that MVs are required for viral entry into the host, while EVs are responsible for spread within the host. Following footpad infection of susceptible mice, ECTV spreads lymphohematogenously, entering the liver at 3 to 4 days postinfection (dpi). Afterwards, ECTV spreads intrahepatically, killing the host. We found that antibodies to an MV protein were highly effective at curing mice from ECTV infection when administered after the virus reached the liver. Moreover, a mutant ECTV that does not make EV was able to spread intrahepatically and kill immunodeficient mice. Together, these findings indicate that MVs are sufficient for the spread of ECTV within the liver and could have implications regarding the pathogenesis of other OPVs, the treatment of emerging OPV infections, as well as strategies for preparedness in case of accidental or intentional release of pathogenic OPVs.

Characterization of ectromelia virus deficient in EVM036, the homolog of vaccinia virus F13L, and its application for rapid generation of recombinant viruses.

The orthopoxvirus (OPV) vaccinia virus (VACV) requires an intact F13L gene to produce enveloped virions (EV) and to form plaques in cell monolayers. Simultaneous introduction of an exogenous gene and F13L into F13L-deficient VACV results in expression of the foreign gene and restoration of plaque size. This is used as a method to rapidly generate VACV recombinants without the need for drug selection. However, whether other OPVs require the orthologs of F13L to generate EV and form plaques, whether F13L orthologs and EV are important for OPV pathogenesis in natural hosts, and whether a system based on F13L ortholog deficiency can be used to generate recombinant OPVs other than VACV have not been reported. The F13L ortholog in ectromelia virus (ECTV), the agent of mousepox, is EVM036. We show that ECTV lacking EVM036 formed small plaques and was highly attenuated in vivo but still induced strong antibody responses. Reintroduction of EVM036 in tandem with the DsRed gene resulted in a virus that expressed DsRed in infected cells but was indistinguishable from wild-type ECTV in terms of plaque size and in vivo virulence. Thus, our data show that, like F13L in VACV, EVM036 is required for ECTV plaque formation and that EVM036 and EV are important for ECTV virulence. Our experiments also suggest that OPVs deficient in F13L orthologs could serve as safer anti-OPV vaccines. Further, our results demonstrate that ECTV deficient in EVM036 can be exploited for the rapid generation of fully virulent ECTV expressing foreign genes of interest.

Antibody inhibition of a viral type 1 interferon decoy receptor cures a viral disease by restoring interferon signaling in the liver.

Type 1 interferons (T1-IFNs) play a major role in antiviral defense, but when or how they protect during infections that spread through the lympho-hematogenous route is not known. Orthopoxviruses, including those that produce smallpox and mousepox, spread lympho-hematogenously. They also encode a decoy receptor for T1-IFN, the T1-IFN binding protein (T1-IFNbp), which is essential for virulence. We demonstrate that during mousepox, T1-IFNs protect the liver locally rather than systemically, and that the T1-IFNbp attaches to uninfected cells surrounding infected foci in the liver and the spleen to impair their ability to receive T1-IFN signaling, thus facilitating virus spread. Remarkably, this process can be reversed and mousepox cured late in infection by treating with antibodies that block the biological function of the T1-IFNbp. Thus, our findings provide insights on how T1-IFNs function and are evaded during a viral infection in vivo, and unveil a novel mechanism for antibody-mediated antiviral therapy.

Direct presentation is sufficient for an efficient anti-viral CD8+ T cell response.

The extent to which direct- and cross-presentation (DP and CP) contribute to the priming of CD8(+) T cell (T(CD8+)) responses to viruses is unclear mainly because of the difficulty in separating the two processes. Hence, while CP in the absence of DP has been clearly demonstrated, induction of an anti-viral T(CD8+) response that excludes CP has never been purposely shown. Using vaccinia virus (VACV), which has been used as the vaccine to rid the world of smallpox and is proposed as a vector for many other vaccines, we show that DP is the main mechanism for the priming of an anti-viral T(CD8+) response. These findings provide important insights to our understanding of how one of the most effective anti-viral vaccines induces immunity and should contribute to the development of novel vaccines.

The amino acid sequences flanking an antigenic determinant can strongly affect MHC class I cross-presentation without altering direct presentation.

Direct presentation (DP) and cross presentation (CP) on MHC I by professional APCs are defined by the internal or external source of the Ag, respectively. Although some Ags are substrates for both DP and CP, others are only substrates for DP. The reasons for this difference remain largely unknown. In this study, we studied in tissue culture and also in vivo, the effects of altering the length and sequence of the amino acid chains flanking an MHC class I restricted determinant (the chicken OVA OVA(258-265), SIINFEKL) that is normally a good substrate for both DP and CP. We demonstrate that CP but not DP strictly requires flanking N and C-terminal extensions of minimal length. Furthermore, we show that removal but not replacement of just one amino acid 22 residues downstream from the determinant is sufficient to strongly affect CP without affecting either protein stability or DP. Thus, our work shows that the flanking residues of an antigenic determinant can differentially affect CP and DP, and that features of the Ag other than half-life can have a major impact in CP. Our studies may have implications for understanding CP in viral infections and possibly for the design of new vaccines.

The orthopoxvirus type I IFN binding protein is essential for virulence and an effective target for vaccination.

Nonliving antiviral vaccines traditionally target proteins expressed at the surface of the virion with the hope of inducing neutralizing antibodies. Orthopoxviruses (OPVs), such as the human smallpox virus and the mouse-equivalent ectromelia virus (ECTV; an agent of mousepox), encode immune response modifiers (IRMs) that can increase virulence by decreasing the host immune response. We show that one of these IRMs, the type I interferon (IFN) binding protein (bp) of ECTV, is essential for ECTV virulence and is a natural target of the antibody response. More strikingly, we demonstrate that immunization with recombinant type I IFN bp protects mice from lethal mousepox. Collectively, our experiments have important implications for our understanding of the role of IRMs in OPV virulence and of type I IFNs in OPV infections. Furthermore, our work provides proof of concept that effective antiviral vaccines can be made to prevent disease by targeting virulence factors as an alternative to the traditional approach that attempts to prevent infection by virus neutralization.

Memory CD8+ T cells are gatekeepers of the lymph node draining the site of viral infection.

It is uncertain how immunity protects against systemic viral diseases. Here, we demonstrate that in the absence of persistent virus, not only antibodies but also recall responses by long-lived memory CD8(+) T cells prevent mousepox, a disease caused by ectromelia virus, a close relative of the virus of human smallpox. Moreover, we show that to protect, recall CD8(+) T cells directly kill targets in the lymph node draining the primary site of infection thus curbing systemic viral spread. Therefore, our work provides the basis for a model where lymph nodes are not just organs where lymphocytes become activated and proliferate but also the sites where a major fight against virus spread takes place.