Smad3 promotes adverse cardiovascular remodeling and dysfunction in doxorubicin-treated hearts.

Many anticancer therapies cause serious cardiovascular complications that degrade quality of life and cause early mortality in treated patients. Specifically, doxorubicin is known as an effective anticancer agent that causes cardiomyopathy in treated patients. There has been growing interest in defining the role of endothelial cells in cardiac damage by doxorubicin. We have shown in the present study that endothelial nuclei accumulate more intravenously administered doxorubicin than other cardiac cell types. Doxorubicin enhanced cardiac production of the transforming growth factor-ß (TGF-ß) ligands and nuclear translocation of phospho-Smad3 in both cultured and in vivo cardiac endothelial cells. To examine the role of the TGF-ß/mothers against decapentaplegic homolog 3 (Smad3) pathway in cardiac damage by doxorubicin, we used both Smad3 shRNA stable endothelial cell lines and Smad3-knockout mice. We demonstrated using endothelial transcriptome analysis that upregulation of the TGF-ß and inflammatory cytokine/cytokine receptor pathways, as well as suppression of cell cycle and angiogenesis by doxorubicin, were alleviated in Smad3-deficient endothelial cells. The results of transcriptomic analysis were validated using qPCR, immunoblotting, and ex vivo aortic ring sprouting assays. Similarly, increased cardiac expression of cytokines and chemokines observed in treated wild-type mice was diminished in treated Smad3-knockout animals. We also detected increased end-diastolic diameter and depressed systolic function in doxorubicin-treated wild-type but not Smad3-knockout mice. This work provides evidence for the critical role of the canonical TGF-ß/Smad3 pathway in cardiac damage by doxorubicin.NEW & NOTEWORTHY Microvascular endothelial cells in the heart accumulate more intravenously administered doxorubicin than nonendothelial cardiac cell types. The treatment enhanced the TGF-ß/Smad3 pathway and elicited endothelial cell senescence and inflammatory responses followed by adverse cardiac remodeling and dysfunction in wild-type but not Smad3-deficient animals. Our study suggests that the TGF-ß/Smad3 pathway contributes to the development of doxorubicin cardiomyopathy and the potential value of novel approaches to ameliorate cardiotoxicity by targeting the Smad3 transcription factor.

Highly conserved influenza T cell epitopes induce broadly protective immunity.

Influenza world-wide causes significant morbidity and mortality annually, and more severe pandemics when novel strains evolve to which humans are immunologically naïve. Because of the high viral mutation rate, new vaccines must be generated based on the prevalence of circulating strains every year. New approaches to induce more broadly protective immunity are urgently needed. Previous research has demonstrated that influenza-specific T cells can provide broadly heterotypic protective immunity in both mice and humans, supporting the rationale for developing a T cell-targeted universal influenza vaccine. We used state-of-the art immunoinformatic tools to identify putative pan-HLA-DR and HLA-A2 supertype-restricted T cell epitopes highly conserved among > 50 widely diverse influenza A strains (representing hemagglutinin types 1, 2, 3, 5, 7 and 9). We found influenza peptides that are highly conserved across influenza subtypes that were also predicted to be class I epitopes restricted by HLA-A2. These peptides were found to be immunoreactive in HLA-A2 positive but not HLA-A2 negative individuals. Class II-restricted T cell epitopes that were highly conserved across influenza subtypes were identified. Human CD4+ T cells were reactive with these conserved CD4 epitopes, and epitope expanded T cells were responsive to both H1N1 and H3N2 viruses. Dendritic cell vaccines pulsed with conserved epitopes and DNA vaccines encoding these epitopes were developed and tested in HLA transgenic mice. These vaccines were highly immunogenic, and more importantly, vaccine-induced immunity was protective against both H1N1 and H3N2 influenza challenges. These results demonstrate proof-of-principle that conserved T cell epitopes expressed by widely diverse influenza strains can induce broadly protective, heterotypic influenza immunity, providing strong support for further development of universally relevant multi-epitope T cell-targeting influenza vaccines.

Cytokine Activation by Antibody Fragments Targeted to Cytokine-Receptor Signaling Complexes.

Exogenous cytokine therapy can induce systemic toxicity, which might be prevented by activating endogenously produced cytokines in local cell niches. Here we developed antibody-based activators of cytokine signaling (AcCS), which recognize cytokines only when they are bound to their cell surface receptors. AcCS were developed for type I interferons (IFNs), which induce cellular activities by binding to cell surface receptors IFNAR1 and IFNAR2. As a potential alternative to exogenous IFN therapy, AcCS were shown to potentiate the biological activities of natural IFNs by ∼100-fold. Biochemical and structural characterization demonstrates that the AcCS stabilize the IFN-IFNAR2 binary complex by recognizing an IFN-induced conformational change in IFNAR2. Using IFN mutants that disrupt IFNAR1 binding, AcCS were able to enhance IFN antiviral potency without activating antiproliferative responses. This suggests AcCS can be used to manipulate cytokine signaling for basic science and possibly for therapeutic applications.

The TGF-ß pathway mediates doxorubicin effects on cardiac endothelial cells.

Elevated ALK4/5 ligands including TGF-ß and activins have been linked to cardiovascular remodeling and heart failure. Doxorubicin (Dox) is commonly used as a model of cardiomyopathy, a condition that often precedes cardiovascular remodeling and heart failure. In 7-8-week-old C57Bl/6 male mice treated with Dox we found decreased capillary density, increased levels of ALK4/5 ligand and Smad2/3 transcripts, and increased expression of Smad2/3 transcriptional targets. Human cardiac microvascular endothelial cells (HCMVEC) treated with Dox also showed increased levels of ALK4/5 ligands, Smad2/3 transcriptional targets, a decrease in proliferation and suppression of vascular network formation in a HCMVEC and human cardiac fibroblasts co-culture assay. Our hypothesis is that the deleterious effects of Dox on endothelial cells are mediated in part by the activation of the TGF-ß pathway. We used the inhibitor of ALK4/5 kinases SB431542 (SB) in concert with Dox to ascertain the role of TGF-ß pathway activation in doxorubicin induced endothelial cell defects. SB prevented the suppression of HCMVEC proliferation in the presence of TGF-ß2 and activin A, and alleviated the inhibition of HCMVEC proliferation by Dox. SB also prevented the suppression of vascular network formation in co-cultures of HCMVEC and human cardiac fibroblasts treated with Dox. Our results show that the inhibition of the TGF-ß pathway alleviates the detrimental effects of Dox on endothelial cells in vitro.

The Inhibitory Receptor NKG2A Sustains Virus-Specific CD8⁺ T Cells in Response to a Lethal Poxvirus Infection.

CD8(+) T cells and NK cells protect from viral infections by killing virally infected cells and secreting interferon-γ. Several inhibitory receptors limit the magnitude and duration of these anti-viral responses. NKG2A, which is encoded by Klrc1, is a lectin-like inhibitory receptor that is expressed as a heterodimer with CD94 on NK cells and activated CD8(+) T cells. Previous studies on the impact of CD94/NKG2A heterodimers on anti-viral responses have yielded contrasting results and the in vivo function of NKG2A remains unclear. Here, we generated Klrc1(-/-) mice and found that NKG2A is selectively required for resistance to ectromelia virus (ECTV). NKG2A functions intrinsically within ECTV-specific CD8(+) T cells to limit excessive activation, prevent apoptosis, and preserve the specific CD8(+) T cell response. Thus, although inhibitory receptors often cause T cell exhaustion and viral spreading during chronic viral infections, NKG2A optimizes CD8(+) T cell responses during an acute poxvirus infection.

EVM005: an ectromelia-encoded protein with dual roles in NF-κB inhibition and virulence.

Poxviruses contain large dsDNA genomes encoding numerous open reading frames that manipulate cellular signalling pathways and interfere with the host immune response. The NF-κB signalling cascade is an important mediator of innate immunity and inflammation, and is tightly regulated by ubiquitination at several key points. A critical step in NF-κB activation is the ubiquitination and degradation of the inhibitor of kappaB (IκBα), by the cellular SCFß-TRCP ubiquitin ligase complex. We show here that upon stimulation with TNFα or IL-1ß, Orthopoxvirus-infected cells displayed an accumulation of phosphorylated IκBα, indicating that NF-κB activation was inhibited during poxvirus infection. Ectromelia virus is the causative agent of lethal mousepox, a natural disease that is fatal in mice. Previously, we identified a family of four ectromelia virus genes (EVM002, EVM005, EVM154 and EVM165) that contain N-terminal ankyrin repeats and C-terminal F-box domains that interact with the cellular SCF ubiquitin ligase complex. Since degradation of IκBα is catalyzed by the SCFß-TRCP ubiquitin ligase, we investigated the role of the ectromelia virus ankyrin/F-box protein, EVM005, in the regulation of NF-κB. Expression of Flag-EVM005 inhibited both TNFα- and IL-1ß-stimulated IκBα degradation and p65 nuclear translocation. Inhibition of the NF-κB pathway by EVM005 was dependent on the F-box domain, and interaction with the SCF complex. Additionally, ectromelia virus devoid of EVM005 was shown to inhibit NF-κB activation, despite lacking the EVM005 open reading frame. Finally, ectromelia virus devoid of EVM005 was attenuated in both A/NCR and C57BL/6 mouse models, indicating that EVM005 is required for virulence and immune regulation in vivo.

Co-administration of the broad-spectrum antiviral, brincidofovir (CMX001), with smallpox vaccine does not compromise vaccine protection in mice challenged with ectromelia virus.

Natural orthopoxvirus outbreaks such as vaccinia, cowpox, cattlepox and buffalopox continue to cause morbidity in the human population. Monkeypox virus remains a significant agent of morbidity and mortality in Africa. Furthermore, monkeypox virus's broad host-range and expanding environs make it of particular concern as an emerging human pathogen. Monkeypox virus and variola virus (the etiological agent of smallpox) are both potential agents of bioterrorism. The first line response to orthopoxvirus disease is through vaccination with first-generation and second-generation vaccines, such as Dryvax and ACAM2000. Although these vaccines provide excellent protection, their widespread use is impeded by the high level of adverse events associated with vaccination using live, attenuated virus. It is possible that vaccines could be used in combination with antiviral drugs to reduce the incidence and severity of vaccine-associated adverse events, or as a preventive in individuals with uncertain exposure status or contraindication to vaccination. We have used the intranasal mousepox (ectromelia) model to evaluate the efficacy of vaccination with Dryvax or ACAM2000 in conjunction with treatment using the broad spectrum antiviral, brincidofovir (BCV, CMX001). We found that co-treatment with BCV reduced the severity of vaccination-associated lesion development. Although the immune response to vaccination was quantifiably attenuated, vaccination combined with BCV treatment did not alter the development of full protective immunity, even when administered two days following ectromelia challenge. Studies with a non-replicating vaccine, ACAM3000 (MVA), confirmed that BCV's mechanism of attenuating the immune response following vaccination with live virus was, as expected, by limiting viral replication and not through inhibition of the immune system. These studies suggest that, in the setting of post-exposure prophylaxis, co-administration of BCV with vaccination should be considered a first response to a smallpox emergency in subjects of uncertain exposure status or as a means of reduction of the incidence and severity of vaccine-associated adverse events.

Initial characterization of vaccinia virus B4 suggests a role in virus spread.

Currently, little is known about the ankyrin/F-box protein B4. Here, we report that B4R-null viruses exhibited reduced plaque size in tissue culture, and decreased ability to spread, as assessed by multiple-step growth analysis. Electron microscopy indicated that B4R-null viruses still formed mature and extracellular virions; however, there was a slight decrease of virions released into the media following deletion of B4R. Deletion of B4R did not affect the ability of the virus to rearrange actin; however, VACV811, a large vaccinia virus deletion mutant missing 55 open reading frames, had decreased ability to produce actin tails. Using ectromelia virus, a natural mouse pathogen, we demonstrated that virus devoid of EVM154, the B4R homolog, showed decreased spread to organs and was attenuated during infection. This initial characterization suggests that B4 may play a role in virus spread, and that other unidentified mediators of actin tail formation may exist in vaccinia virus.

Expression of a non-coding RNA in ectromelia virus is required for normal plaque formation.

Poxviruses are dsDNA viruses with large genomes. Many genes in the genome remain uncharacterized, and recent studies have demonstrated that the poxvirus transcriptome includes numerous so-called anomalous transcripts not associated with open reading frames. Here, we characterize the expression and role of an apparently non-coding RNA in orthopoxviruses, which we call viral hairpin RNA (vhRNA). Using a bioinformatics approach, we predicted expression of a transcript not associated with an open reading frame that is likely to form a stem-loop structure due to the presence of a 21 nt palindromic sequence. Expression of the transcript as early as 2 h post-infection was confirmed by northern blot and analysis of publicly available vaccinia virus infected cell transcriptomes. The transcription start site was determined by RACE PCE and transcriptome analysis, and early and late promoter sequences were identified. Finally, to test the function of the transcript we generated an ectromelia virus knockout, which failed to form plaques in cell culture. The important role of the transcript in viral replication was further demonstrated using siRNA. Although the function of the transcript remains unknown, our work contributes to evidence of an increasingly complex poxvirus transcriptome, suggesting that transcripts such as vhRNA not associated with an annotated open reading frame can play an important role in viral replication.

Investigations of TB vaccine-induced mucosal protection in mice.

A better understanding of mucosal immunity is required to develop more protective vaccines against Mycobacterium tuberculosis. We developed a murine aerosol challenge model to investigate responses capable of protecting against mucosal infection. Mice received vaccinations intranasally with CpG-adjuvanted antigen 85B (Ag85B/CpG) and/or Bacillus Calmette-Guerin (BCG). Protection against aerosol challenge with a recombinant GFP-expressing BCG was assessed. Mucosal prime/boost vaccinations with Ag85B/CpG and BCG were protective, but did not prevent lung infection indicating more efficacious mucosal vaccines are needed. Our novel finding that protection correlated with increased airway dendritic cells early post-challenge could help guide the development of enhanced mucosal vaccines.

Use of a recombinant vaccinia virus expressing interferon gamma for post-exposure protection against vaccinia and ectromelia viruses.

Post-exposure vaccination with vaccinia virus (VACV) has been suggested to be effective in minimizing death if administered within four days of smallpox exposure. While there is anecdotal evidence for efficacy of post-exposure vaccination this has not been definitively studied in humans. In this study, we analyzed post-exposure prophylaxis using several attenuated recombinant VACV in a mouse model. A recombinant VACV expressing murine interferon gamma (IFN-γ) was most effective for post-exposure protection of mice infected with VACV and ectromelia virus (ECTV). Untreated animals infected with VACV exhibited severe weight loss and morbidity leading to 100% mortality by 8 to 10 days post-infection. Animals treated one day post-infection had milder symptoms, decreased weight loss and morbidity, and 100% survival. Treatment on days 2 or 3 post-infection resulted in 40% and 20% survival, respectively. Similar results were seen in ECTV-infected mice. Despite the differences in survival rates in the VACV model, the viral load was similar in both treated and untreated mice while treated mice displayed a high level of IFN-γ in the serum. These results suggest that protection provided by IFN-γ expressed by VACV may be mediated by its immunoregulatory activities rather than its antiviral effects. These results highlight the importance of IFN-γ as a modulator of the immune response for post-exposure prophylaxis and could be used potentially as another post-exposure prophylaxis tool to prevent morbidity following infection with smallpox and other orthopoxviruses.

Immunoproteasome Activation During Early Antiviral Response in Mouse Pancreatic ß-cells: New Insights into Auto-antigen Generation in Type I Diabetes?

Type 1 diabetes results from autoimmune destruction of the insulin producing pancreatic ß-cells. The immunoproteasome, a version of the proteasome that collaborates with the 11S/PA28 activator to generate immunogenic peptides for presentation by MHC class I molecules, has long been implicated in the onset of the disease, but little is known about immunoproteasome function and regulation in pancreatic ß-cells. Interesting insight into these issues comes from a recent analysis of the immunoproteasome expressed in pancreatic ß-cells during early antiviral defenses mediated by interferon ß (IFNß), a type I IFN implicated in the induction of the diabetic state in human and animal models. Using mouse islets and the MIN6 insulinoma cell line, Freudenburg et al. found that IFNß stimulates expression of the immunoproteasome and the 11S/PA28 activator in a manner fundamentally similar to the classic immuno-inducer IFNγ, with similar timing of mRNA accumulation and decline; similar transcriptional activation mediated primarily by the IRF1 and similar mRNA and protein levels. Furthermore, neither IFNß nor IFNγ altered the expression of regular proteolytic subunits or prevented their incorporation into proteolytic cores. As a result, immunoproteasomes had stochastic combinations of immune and regular proteolytic sites, an arrangement that would likely increase the probability with which unique immunogenic peptides are produced. However, immunoproteasomes were activated by the 11S/PA28 only under conditions of ATP depletion. A mechanism that prevents the activation of immunoproteasome at high ATP levels has not been reported before and could have a major regulatory significance, as it could suppress the generation of immunogenic peptides as cell accumulate immunoproteasome and 11S/PA28, and activate antigen processing only when ATP levels drop. We discuss implications of these new findings on the link between early antiviral response and the onset of type 1 diabetes.

RNA helicase signaling is critical for type i interferon production and protection against Rift Valley fever virus during mucosal challenge.

Rift Valley fever virus (RVFV) is an emerging RNA virus with devastating economic and social consequences. Clinically, RVFV induces a gamut of symptoms ranging from febrile illness to retinitis, hepatic necrosis, hemorrhagic fever, and death. It is known that type I interferon (IFN) responses can be protective against severe pathology; however, it is unknown which innate immune receptor pathways are crucial for mounting this response. Using both in vitro assays and in vivo mucosal mouse challenge, we demonstrate here that RNA helicases are critical for IFN production by immune cells and that signaling through the helicase adaptor molecule MAVS (mitochondrial antiviral signaling) is protective against mortality and more subtle pathology during RVFV infection. In addition, we demonstrate that Toll-like-receptor-mediated signaling is not involved in IFN production, further emphasizing the importance of the RNA cellular helicases in type I IFN responses to RVFV.

Reduction in ATP levels triggers immunoproteasome activation by the 11S (PA28) regulator during early antiviral response mediated by IFNß in mouse pancreatic ß-cells.

Autoimmune destruction of insulin producing pancreatic ß-cells is the hallmark of type I diabetes. One of the key molecules implicated in the disease onset is the immunoproteasome, a protease with multiple proteolytic sites that collaborates with the constitutive 19S and the inducible 11S (PA28) activators to produce immunogenic peptides for presentation by MHC class I molecules. Despite its importance, little is known about the function and regulation of the immunoproteasome in pancreatic ß-cells. Of special interest to immunoproteasome activation in ß-cells are the effects of IFNß, a type I IFN secreted by virus-infected cells and implicated in type I diabetes onset, compared to IFNγ, the classic immunoproteasome inducer secreted by cells of the immune system. By qPCR analysis, we show that mouse insulinoma MIN6 cells and mouse islets accumulate the immune proteolytic ß1(i), ß2(i) and ß5(i), and 11S mRNAs upon exposure to IFNß or IFNγ. Higher concentrations of IFNß than IFNγ are needed for similar expression, but in each case the expression is transient, with maximal mRNA accumulation in 12 hours, and depends primarily on Interferon Regulatory Factor 1. IFNs do not alter expression of regular proteasome genes, and in the time frame of IFNß-mediated response, the immune and regular proteolytic subunits co-exist in the 20S particles. In cell extracts with ATP, these particles have normal peptidase activities and degrade polyubiquitinated proteins with rates typical of the regular proteasome, implicating normal regulation by the 19S activator. However, ATP depletion rapidly stimulates the catalytic rates in a manner consistent with levels of the 11S activator. These findings suggest that stochastic combination of regular and immune proteolytic subunits may increase the probability with which unique immunogenic peptides are produced in pancreatic ß-cells exposed to IFNß, but primarily in cells with reduced ATP levels that stimulate the 11S participation in immunoproteasome function.

Soft-X-ray-enhanced electrostatic precipitation for protection against inhalable allergens, ultrafine particles, and microbial infections.

Protection of the human lung from infectious agents, allergens, and ultrafine particles is difficult with current technologies. High-efficiency particulate air (HEPA) filters remove airborne particles of >0.3 µm with 99.97% efficiency, but they are expensive to maintain. Electrostatic precipitation has been used as an inexpensive approach to remove large particles from airflows, but it has a collection efficiency minimum in the submicrometer size range, allowing for a penetration window for some allergens and ultrafine particles. Incorporating soft X-ray irradiation as an in situ component of the electrostatic precipitation process greatly improves capture efficiency of ultrafine particles. Here we demonstrate the removal and inactivation capabilities of soft-X-ray-enhanced electrostatic precipitation technology targeting infectious agents (Bacillus anthracis, Mycobacterium bovis BCG, and poxviruses), allergens, and ultrafine particles. Incorporation of in situ soft X-ray irradiation at low-intensity corona conditions resulted in (i) 2-fold to 9-fold increase in capture efficiency of 200- to 600-nm particles and (ii) a considerable delay in the mean day of death as well as lower overall mortality rates in ectromelia virus (ECTV) cohorts. At the high-intensity corona conditions, nearly complete protection from viral and bacterial respiratory infection was afforded to the murine models for all biological agents tested. When optimized for combined efficient particle removal with limited ozone production, this technology could be incorporated into stand-alone indoor air cleaners or scaled for installation in aircraft cabin, office, and residential heating, ventilating, and air-conditioning (HVAC) systems.

Mousepox, a small animal model of smallpox.

Ectromelia virus infections in the laboratory mouse have emerged as a valuable model to investigate human orthopoxvirus infections to understand the progression of disease, to discover and characterize antiviral treatments, and to study the host-pathogen relationship as it relates to pathogenesis and the immune response. Here we describe how to safely work with the virus and protocols for common procedures for the study of ectromelia virus in the laboratory mouse including the preparation of virus stocks, the use of various routes of inoculation, and collection of blood and tissue from infected animals. In addition, several procedures are described for assessing the host response to infection: for example, measurement of virus-specific CD8 T cells and the use of ELISA and neutralization assays to measure orthopoxvirus-specific antibody titers.

Evaluation of disease and viral biomarkers as triggers for therapeutic intervention in respiratory mousepox - an animal model of smallpox.

The human population is currently faced with the potential use of natural or recombinant variola and monkeypox viruses as biological weapons. Furthermore, the emergence of human monkeypox in Africa and its expanding environs poses a significant natural threat. Such occurrences would require therapeutic and prophylactic intervention with antivirals to minimize morbidity and mortality of exposed populations. Two orally-bioavailable antivirals are currently in clinical trials; namely CMX001, an ether-lipid analog of cidofovir with activity at the DNA replication stage and ST-246, a novel viral egress inhibitor. Both of these drugs have previously been evaluated in the ectromelia/mousepox system; however, the trigger for intervention was not linked to a disease biomarker or a specific marker of virus replication. In this study we used lethal, intranasal, ectromelia virus infections of C57BL/6 and hairless SKH1 mice to model human disease and evaluate exanthematous rash (rash) as an indicator to initiate antiviral treatment. We show that significant protection can be provided to C57BL/6 mice by CMX001 or ST-246 when therapy is initiated on day 6 post infection or earlier. We also show that significant protection can be provided to SKH1 mice treated with CMX001 at day 3 post infection or earlier, but this is four or more days before detection of rash (ST-246 not tested). Although in this model rash could not be used as a treatment trigger, viral DNA was detected in blood by day 4 post infection and in the oropharyngeal secretions (saliva) by day 2-3 post infection - thus providing robust and specific markers of virus replication for therapy initiation. These findings are discussed in the context of current respiratory challenge animal models in use for the evaluation of poxvirus antivirals.

The attenuated NYCBH vaccinia virus deleted for the immune evasion gene, E3L, completely protects mice against heterologous challenge with ectromelia virus.

The New York City Board of Health (NYCBH) vaccinia virus (VACV) vaccine strain was deleted for the immune evasion gene, E3L, and tested for its pathogenicity and ability to protect mice from heterologous challenge with ectromelia virus (ECTV). NYCBHΔE3L was found to be highly attenuated for pathogenicity in a newborn mouse model and showed a similar attenuated phenotype as the NYVAC strain of vaccinia virus. Scarification with one or two doses of the attenuated NYCBHΔE3L was able to protect mice equally as well as NYCBH from death, weight loss, and viral spread to visceral organs. A single dose of NYCBHΔE3L resulted in low poxvirus-specific antibodies, and a second dose increased levels of poxvirus-specific antibodies to a level similar to that seen in animals vaccinated with a single dose of NYCBH. However, similar neutralizing antibody titers were observed following one or two doses of NYCBHΔE3L or NYCBH. Thus, NYCBHΔE3L shows potential as a candidate for a safer human smallpox vaccine since it protects mice from challenge with a heterologous poxvirus.

Poxvirus interleukin-4 expression overcomes inherent resistance and vaccine-induced immunity: pathogenesis, prophylaxis, and antiviral therapy.

In 2001, Jackson et al. reported that murine IL-4 expression by a recombinant ectromelia virus caused enhanced morbidity and lethality in resistant C57BL/6 mice as well as overcame protective immune memory responses. To achieve a more thorough understanding of this phenomenon and to assess a variety of countermeasures, we constructed a series of ECTV recombinants encoding murine IL-4 under the control of promoters of different strengths and temporal regulation. We showed that the ECTV-IL-4 recombinant expressing the highest level of IL-4 was uniformly lethal for C57BL/6 mice even when previously immunized. The lethality of the ECTV-IL-4 recombinants resulted from virus-expressed IL-4 signaling through the IL-4 receptor but was not due to IL-4 toxicity. A number of treatment approaches were evaluated against the most virulent IL-4 encoding virus. The most efficacious therapy was a combination of two antiviral drugs (CMX001(®) and ST-246(®)) that have different mechanisms of action.

VennVax, a DNA-prime, peptide-boost multi-T-cell epitope poxvirus vaccine, induces protective immunity against vaccinia infection by T cell response alone.

The potential for smallpox to be disseminated in a bioterror attack has prompted development of new, safer smallpox vaccination strategies. We designed and evaluated immunogenicity and efficacy of a T-cell epitope vaccine based on conserved and antigenic vaccinia/variola sequences, identified using bioinformatics and immunological methods. Vaccination in HLA transgenic mice using a DNA-prime/peptide-boost strategy elicited significant T cell responses to multiple epitopes. No antibody response pre-challenge was observed, neither against whole vaccinia antigens nor vaccine epitope peptides. Remarkably, 100% of vaccinated mice survived lethal vaccinia challenge, demonstrating that protective immunity to vaccinia does not require B cell priming.