Protective properties of vaccinia virus-based vaccines: skin scarification promotes a nonspecific immune response that protects against orthopoxvirus disease.

The process of vaccination introduced by Jenner generated immunity against smallpox and ultimately led to the eradication of the disease. Procedurally, in modern times, the virus is introduced into patients via a process called scarification, performed with a bifurcated needle containing a small amount of virus. What was unappreciated was the role that scarification itself plays in generating protective immunity. In rabbits, protection from lethal disease is induced by intradermal injection of vaccinia virus, whereas a protective response occurs within the first 2 min after scarification with or without virus, suggesting that the scarification process itself is a major contributor to immunoprotection. importance: These results show the importance of local nonspecific immunity in controlling poxvirus infections and indicate that the process of scarification should be critically considered during the development of vaccination protocols for other infectious agents.

Modulation of NKp30- and NKp46-mediated natural killer cell responses by poxviral hemagglutinin.

Natural killer (NK) cells are an important element in the immune defense against the orthopox family members vaccinia virus (VV) and ectromelia virus (ECTV). NK cells are regulated through inhibitory and activating signaling receptors, the latter involving NKG2D and the natural cytotoxicity receptors (NCR), NKp46, NKp44 and NKp30. Here we report that VV infection results in an upregulation of ligand structures for NKp30 and NKp46 on infected cells, whereas the binding of NKp44 and NKG2D was not significantly affected. Likewise, infection with ectromelia virus (ECTV), the mousepox agent, enhanced binding of NKp30 and, to a lesser extent, NKp46. The hemagglutinin (HA) molecules from VV and ECTV, which are known virulence factors, were identified as novel ligands for NKp30 and NKp46. Using NK cells with selectively silenced NCR expression and NCR-CD3ζ reporter cells, we observed that HA present on the surface of VV-infected cells, or in the form of recombinant soluble protein, was able to block NKp30-triggered activation, whereas it stimulated the activation through NKp46. The net effect of this complex influence on NK cell activity resulted in a decreased NK lysis susceptibility of infected cells at late time points of VV infection when HA was expression was pronounced. We conclude that poxviral HA represents a conserved ligand of NCR, exerting a novel immune escape mechanism through its blocking effect on NKp30-mediated activation at a late stage of infection.

Roles of vaccinia virus genes E3L and K3L and host genes PKR and RNase L during intratracheal infection of C57BL/6 mice.

The importance of the 2'-5' oligoadenylate synthetase (OAS)/RNase L and double-stranded RNA (dsRNA)-dependent protein kinase (PKR) pathways in host interferon induction resulting from virus infection in response to dsRNA has been well documented. In poxvirus infections, the interactions between the vaccinia virus (VV) genes E3L and K3L, which target RNase L and PKR, respectively, serve to prevent the induction of the dsRNA-dependent induced interferon response in cell culture. To determine the importance of these host genes in controlling VV infections, mouse single-gene knockouts of RNase L and PKR and double-knockout mice were studied following intratracheal infection with VV, VVΔK3L, or VVΔE3L. VV caused lethal disease in all mouse strains. The single-knockout animals were more susceptible than wild-type animals, while the RNase L(-/-) PKR(-/-) mice were the most susceptible. VVΔE3L infections of wild-type mice were asymptomatic, demonstrating that E3L plays a critical role in controlling the host immune response. RNase L(-/-) mice showed no disease, whereas 20% of the PKR(-/-) mice succumbed at a dose of 10(8) PFU. Lethal disease was routinely observed in RNase L(-/-) PKR(-/-) mice inoculated with 10(8) PFU of VVΔE3L, with a distinct pathology. VVΔK3L infections exhibited no differences in virulence among any of the mouse constructs, suggesting that PKR is not the exclusive target of K3L. Surprisingly, VVΔK3L did not disseminate to other tissues from the lung. Hence, the cause of death in this model is respiratory disease. These results also suggest that an unanticipated role of the K3L gene is to facilitate virus dissemination.

Web-based assessment and brief intervention for alcohol use in women of childbearing potential: a report of the primary findings.

BACKGROUND: There is a need for more effective assessment and primary prevention programs aimed at accurately measuring and reducing alcohol consumption among women before conception in underserved, high-risk populations. Health information technology may serve this purpose; however, the effectiveness of such tools within this population is not known. METHODS: We conducted a small-scale randomized controlled trial to test the effectiveness of an adapted Web-based alcohol assessment and intervention tool among low-income, nonpregnant women of reproductive age who were receiving Women Infant and Children (WIC) services in San Diego County and who reported currently drinking at a moderate risk level. A total of 150 risky drinking participants completed a Web-based assessment and were randomly assigned to either receive a personalized feedback intervention or general health information about alcohol consumption and fetal alcohol syndrome. Follow-up assessments on reported alcohol consumption were conducted via telephone at 1- and 2-months postbaseline. Participants ranged in age from 18 to 44 and were predominately Hispanic/Latina (44%). RESULTS: At baseline, all respondents reported consuming ≥3 standard drinks on ≥1 occasion in the previous month. Outcome data were available for 131 participants. The main outcome measure was reduction in the number of risky drinking occasions, which did not differ significantly between treatment conditions (odds ratio 1.200, 95% CI 0.567 to 2.539, p=0.634). Over 70% of the participants, however, reported a reduction in risky drinking occasions regardless of treatment condition (control 43/63, 68%; experimental 49/68, 72%). CONCLUSIONS: The results of this study demonstrate that web-based assessment of alcohol consumption among low-income women of reproductive age, as represented by WIC clients, is feasible and acceptable. The findings also suggest that detailed and interactive assessments of alcohol consumption may be sufficient for the reduction of risky drinking within this population without personalized feedback.

Aptamers recognizing glycosylated hemagglutinin expressed on the surface of vaccinia virus-infected cells.

Traditional methods for detection and identification of pathogenic viruses or bacteria tend to be slow and cumbersome. We have developed aptamer probes with the capacity to rapidly detect the presence of viral infection with specificity and sensitivity. Vaccinia virus (VV) was chosen as the model because it is closely related to variola virus that causes smallpox. A method known as cell-SELEX (systematic evolution of ligands by exponential enrichment) was used to generate very selective and highly specific aptamers designed to recognize proteins expressed on the surface of VV-infected cells. Characterization of the aptamers showed that the virus-encoded hemagglutinin, a protein expressed on the surface of infected cells, is the preferential binding target. These studies show the feasibility of generating aptamers against a given specific infectious agent and will enable further development of aptamers as diagnostic and/or therapeutic tools against a broad range of infectious agents.

Cowpox virus expresses a novel ankyrin repeat NF-kappaB inhibitor that controls inflammatory cell influx into virus-infected tissues and is critical for virus pathogenesis.

Many pathogenic orthopoxviruses like variola virus, monkeypox virus, and cowpox virus (CPXV), but not vaccinia virus, encode a unique family of ankyrin (ANK) repeat-containing proteins that interact directly with NF-kappaB1/p105 and inhibit the NF-kappaB signaling pathway. Here, we present the in vitro and in vivo characterization of the targeted gene knockout of this novel NF-kappaB inhibitor in CPXV. Our results demonstrate that the vCpx-006KO uniquely induces a variety of NF-kappaB-controlled proinflammatory cytokines from infected myeloid cells, accompanied by a rapid phosphorylation of the IkappaB kinase complex and subsequent degradation of the NF-kappaB cellular inhibitors IkappaBalpha and NF-kappaB1/p105. Moreover, the vCpx-006KO virus was attenuated for virulence in mice and induced a significantly elevated cellular inflammatory process at tissue sites of virus replication in the lung. These results indicate that members of this ANK repeat family are utilized specifically by pathogenic orthopoxviruses to repress the NF-kappaB signaling pathway at tissue sites of virus replication in situ.

Deriving Immune Modulating Drugs from Viruses-A New Class of Biologics.

Viruses are widely used as a platform for the production of therapeutics. Vaccines containing live, dead and components of viruses, gene therapy vectors and oncolytic viruses are key examples of clinically-approved therapeutic uses for viruses. Despite this, the use of virus-derived proteins as natural sources for immune modulators remains in the early stages of development. Viruses have evolved complex, highly effective approaches for immune evasion. Originally developed for protection against host immune responses, viral immune-modulating proteins are extraordinarily potent, often functioning at picomolar concentrations. These complex viral intracellular parasites have "performed the R&D", developing highly effective immune evasive strategies over millions of years. These proteins provide a new and natural source for immune-modulating therapeutics, similar in many ways to penicillin being developed from mold or streptokinase from bacteria. Virus-derived serine proteinase inhibitors (serpins), chemokine modulating proteins, complement control, inflammasome inhibition, growth factors (e.g., viral vascular endothelial growth factor) and cytokine mimics (e.g., viral interleukin 10) and/or inhibitors (e.g., tumor necrosis factor) have now been identified that target central immunological response pathways. We review here current development of virus-derived immune-modulating biologics with efficacy demonstrated in pre-clinical or clinical studies, focusing on pox and herpesviruses-derived immune-modulating therapeutics.

Generating aptamers for recognition of virus-infected cells.

BACKGROUND: The development of molecular probes capable of recognizing virus-infected cells is essential to meet the serious clinical, therapeutic, and national-security challenges confronting virology today. We report the development of DNA aptamers as probes for the selective targeting of virus-infected living cells. METHODS: To create aptamer probes capable of recognizing virus-infected cells, we used cell-SELEX (systematic evolution of ligands via exponential enrichment), which uses intact infected live cells as targets for aptamer selection. In this study, vaccinia virus-infected and -uninfected lung cancer A549 cells were chosen to develop our model probes. RESULTS: A panel of aptamers has been evolved by means of the infected cell-SELEX procedure. The results demonstrate that the aptamers bind selectively to vaccinia virus-infected A549 cells with apparent equilibrium dissociation constants in the nanomolar range. In addition, these aptamers can specifically recognize a variety of target infected cell lines. The aptamers' target is most likely a viral protein located on the cell surface. CONCLUSIONS: The success of developing a panel of DNA-aptamer probes capable of recognizing virus-infected cells via a whole living cell-SELEX selection strategy may increase our understanding of the molecular signatures of infected cells. Our findings suggest that aptamers can be developed as molecular probes for use as diagnostic and therapeutic reagents and for facilitating drug delivery against infected cells.

The serpin saga; development of a new class of virus derived anti-inflammatory protein immunotherapeutics.

Serine proteinase inhibitors, also called serpins, are an ancient grouping of proteins found in primitive organisms from bacteria, protozoa and horseshoe crabs and thus likely present at the time of the dinosaurs, up to all mammals living today. The innate or inflammatory immune system is also an ancient metazoan regulatory system, providing the first line of defense against infection or injury. The innate inflammatory defense response evolved long before acquired, antibody dependent immunity. Viruses have developed highly effective stratagems that undermine and block a wide variety of host inflammatory and immune responses. Some of the most potent of these immune modifying strategies utilize serpins that have also been developed over millions of years, including the hijacking by some viruses for defense against host immune attacks. Serpins represent up to 2-10 percent of circulating plasma proteins, regulating actions as wide ranging as thrombosis, inflammation, blood pressure control and even hormone transport. Targeting serpin-regulated immune or inflammatory pathways makes evolutionary sense for viral defense and many of these virus-derived inhibitory proteins have proven to be highly effective, working at very low concentrations--even down to the femptomolar to picomolar range. We are studying these viral anti-inflammatory proteins as a new class of immunomodulatory therapeutic agents derived from their native viral source. One such viral serpin, Serp-1 is now in clinical trial (conducted by VIRON Therapeutics, Inc.) for acute unstable coronary syndromes (unstable angina and small heart attacks), representing a 'first in class' therapeutic study. Several other viral serpins are also currently under investigation as anti-inflammatory or anti-immune therapeutics. This chapter describes these original studies and the ongoing analysis of viral serpins as a new class of virus-derived immunotherapeutic.

Serp-2, a virus-derived apoptosis and inflammasome inhibitor, attenuates liver ischemia-reperfusion injury in mice.

BACKGROUND: Ischemia-reperfusion injury (IRI) is an antigen-independent, innate immune response to arterial occlusion and ischemia with subsequent paradoxical exacerbation after reperfusion. IRI remains a critical problem after vessel occlusion and infarction or during harvest and surgery in transplants. After transplant, liver IRI (LIRI) contributes to increased acute and chronic rejection and graft loss. Tissue loss during LIRI has been attributed to local macrophage activation and invasion with excessive inflammation together with hepatocyte apoptosis and necrosis. Inflammatory and apoptotic signaling are key targets for reducing post-ischemic liver injury.Myxomavirus is a rabbit-specific leporipoxvirus that encodes a suite of immune suppressing proteins, often with extensive function in other mammalian species. Serp-2 is a cross-class serine protease inhibitor (serpin) which inhibits the inflammasome effector protease caspase-1 as well as the apoptotic proteases granzyme B and caspases 8 and 10. In prior work, Serp-2 reduced inflammatory cell invasion after angioplasty injury and after aortic transplantation in rodents. In this report, we explore the potential for therapeutic treatment with Serp-2 in a mouse model of LIRI. METHODS: Wildtype (C57BL/6 J) mice were subjected to warm, partial (70%) hepatic ischemia for 90 min followed by treatment with saline or Serp-2 or M-T7, 100 ng/g/day given by intraperitoneal injection on alternate days for 5 days. M-T7 is a Myxomavirus-derived inhibitor of chemokine-GAG interactions and was used in this study for comparative analysis of an unrelated viral protein with an alternative immunomodulating mechanism of action. Survival, serum ALT levels and histopathology were assessed 24 h and 10 days post-LIRI. RESULTS: Serp-2 treatment significantly improved survival to 85.7% percent versus saline-treated wildtype mice (p = 0.0135), while M-T7 treatment did not significantly improve survival (p = 0.2584). Liver viability was preserved by Serp-2 treatment with a significant reduction in serum ALT levels (p = 0.0343) and infarct scar thickness (p = 0.0016), but with no significant improvement with M-T7 treatment. Suzuki scoring by pathologists blinded with respect to treatment group indicated that Serp-2 significantly reduced hepatocyte necrosis (p = 0.0057) and improved overall pathology score (p = 0.0046) compared to saline. Immunohistochemistry revealed that Serp-2 treatment reduced macrophage infiltration into the infarcted liver tissue (p = 0.0197). CONCLUSIONS: Treatment with Serp-2, a virus-derived inflammasome and apoptotic pathway inhibitor, improves survival after liver ischemia-reperfusion injury in mouse models. Treatment with a cross-class immune modulator provides a promising new approach designed to reduce ischemia-reperfusion injury, improving survival and reducing chronic transplant damage.

Vaccinia virus lacking the deoxyuridine triphosphatase gene (F2L) replicates well in vitro and in vivo, but is hypersensitive to the antiviral drug (N)-methanocarbathymidine.

BACKGROUND: The vaccinia virus (VV) F2L gene encodes a functional deoxyuridine triphosphatase (dUTPase) that catalyzes the conversion of dUTP to dUMP and is thought to minimize the incorporation of deoxyuridine residues into the viral genome. Previous studies with with a complex, multigene deletion in this virus suggested that the gene was not required for viral replication, but the impact of deleting this gene alone has not been determined in vitro or in vivo. Although the crystal structure for this enzyme has been determined, its potential as a target for antiviral therapy is unclear. RESULTS: The F2L gene was replaced with GFP in the WR strain of VV to assess its effect on viral replication. The resulting virus replicated well in cell culture and its replication kinetics were almost indistinguishable from those of the wt virus and attained similar titers. The virus also appeared to be as pathogenic as the WR strain suggesting that it also replicated well in mice. Cells infected with the dUTPase mutant would be predicted to affect pyrimidine deoxynucleotide pools and might be expected to exhibit altered susceptibility to pyrimidine analogs. The antiviral activity of cidofovir and four thymidine analogs were evaluated both in the mutant and the parent strain of this virus. The dUTPase knockout remained fully susceptible to cidofovir and idoxuridine, but was hypersensitive to the drug (N)-methanocarbathymidine, suggesting that pyrimidine metabolism was altered in cells infected with the mutant virus. The absence of dUTPase should reduce cellular dUMP pools and may result in a reduced conversion to dTMP by thymidylate synthetase or an increased reliance on the salvage of thymidine by the viral thymidine kinase. CONCLUSION: We confirmed that F2L was not required for replication in cell culture and determined that it does not play a significant role on virulence of the virus in intranasally infected mice. The recombinant virus is hypersensitive to (N)-methanocarbathymidine and may reflect metabolic differences in the mutant virus.

Isolation and characterization of cidofovir resistant vaccinia viruses.

BACKGROUND: The emergence of drug resistant viruses, together with the possibility of increased virulence, is an important concern in the development of new antiviral compounds. Cidofovir (CDV) is a phosphonate nucleotide that is approved for use against cytomegalovirus retinitis and for the emergency treatment of smallpox or complications following vaccination. One mode of action for CDV has been demonstrated to be the inhibition of the viral DNA polymerase. RESULTS: We have isolated several CDV resistant (CDVR) vaccinia viruses through a one step process, two of which have unique single mutations within the DNA polymerase. An additional resistant virus isolate provides evidence of a second site mutation within the genome involved in CDV resistance. The CDVR viruses were 3-7 fold more resistant to the drug than the parental viruses. The virulence of the CDVR viruses was tested in mice inoculated intranasally and all were found to be attenuated. CONCLUSION: Resistance to CDV in vaccinia virus can be conferred individually by at least two different mutations within the DNA polymerase gene. Additional genes may be involved. This one step approach for isolating resistant viruses without serial passage and in the presence of low doses of drug minimizes unintended secondary mutations and is applicable to other potential antiviral agents.

Viral chemokine-binding proteins inhibit inflammatory responses and aortic allograft transplant vasculopathy in rat models.

BACKGROUND: Both CC and CXC chemokines direct monocyte and T-cell migration and activation at sites of vascular injury, but the relative contributions of each chemokine class to transplant vasculopathy development have not been defined. The nonselective C, CC, and CXC chemokine binding protein, M-T7, inhibits vasculopathy development after angioplasty and after renal transplant. We have assessed the effects of three viral chemokine-binding proteins with differing ranges of chemokine inhibition on plaque growth in rats after aortic allograft transplant. METHODS: One of two myxomaviral chemokine binding proteins, (1). M-T1, a selective CC chemokine inhibitor, or (2). M-T7, a nonselective chemokine-binding protein, was given immediately after transplant. A separate group was treated with the gamma68-herpesvirus protein, M3, a C, CC, CXC, and CX3C binding protein, with preferential CC binding. RESULTS: Intimal hyperplasia was significantly reduced at late times posttransplant after infusion of each chemokine-binding protein (P <0.05). Early inhibition of macrophage and T-cell invasion was associated with a late decrease in vasculopathy development. Infusion of an inactive myxomavirus protein did not inhibit plaque growth. Combined high-dose M-T1 and M-T7 did not reduce plaque growth or early cell invasion to a greater extent than either protein alone. Coinfusion of the CC chemokines macrophage chemoattractant protein-1 and macrophage inflammatory protein-1alpha neutralized M-T1 and M-T7 inhibition of monocyte invasion, respectively, suggesting a key role for CC chemokine-mediated cellular influx. CONCLUSION: Viral chemokine-modulating proteins effectively reduce aortic allograft vasculopathy, acting predominantly through inhibition of a CC chemokine-mediated response.

Poxvirus immune modulators: functional insights from animal models.

Poxviruses express several different classes of immune modulators that suppress the host response to infection, including soluble cytokine binding proteins, serpins, chemokine binding proteins, a complement control protein, and members of the semaphorin and Toll/IL-1 receptor families. Biochemical activity of these proteins has been demonstrated by many in vitro studies. Conservation in evolution of poxvirus immune modulators implies that these genes are functional in vivo, but the results of infecting animals with knockout viruses have not always been clear cut. Studies involving different animal models are reviewed, and the criteria for suitable models are discussed. Challenges include finding an appropriate animal host, and using an inoculation route that resembles the process of natural infection. The fact that multiple immune modulators can target the same pathway at different steps may explain why single knockout mutants are not always attenuated in animals.

Anthocyanins, phenolics, and antioxidant capacity in diverse small fruits: vaccinium, rubus, and ribes.

Fruits from 107 genotypes of Vaccinium L., Rubus L., and Ribes L., were analyzed for total anthocyanins (ACY), total phenolics (TPH), and antioxidant capacities as determined by oxygen radical absorbing capacity (ORAC) and ferric reducing antioxidant power (FRAP). Fruit size was highly correlated (r = 0.84) with ACY within Vaccinium corymbosum L., but was not correlated to ACY across eight other Vaccinium species, or within 27 blackberry hybrids. Certain Vaccinium and Ribes fruits with pigmented flesh were lower in ACY, TPH, ORAC, and FRAP compared to those values in berries with nonpigmented flesh. ORAC values ranged from 19 to 131 micromol Trolox equivalents/g in Vaccinium, from 13 to 146 in Rubus, and from 17 to 116 in Ribes. Though ACY may indicate TPH, the range observed in ACY/TPH ratios precludes prediction of ACY from TPH and vice versa for a single genotype. In general, TPH was more highly correlated to antioxidant capacity than ACY was. This study demonstrates the wide diversity of phytochemical levels and antioxidant capacities within and across three genera of small fruit.

Scoring the collective effects of SNPs: association of minor alleles with complex traits in model organisms.

It has long been assumed that most parts of a genome and most genetic variations or SNPs are non-functional with regard to reproductive fitness. However, the collective effects of SNPs have yet to be examined by experimental science. We here developed a novel approach to examine the relationship between traits and the total amount of SNPs in panels of genetic reference populations. We identified the minor alleles (MAs) in each panel and the MA content (MAC) that each inbred strain carried for a set of SNPs with genotypes determined in these panels. MAC was nearly linearly linked to quantitative variations in numerous traits in model organisms, including life span, tumor susceptibility, learning and memory, sensitivity to alcohol and anti-psychotic drugs, and two correlated traits poor reproductive fitness and strong immunity. These results suggest that the collective effects of SNPs are functional and do affect reproductive fitness.

The serpin solution; targeting thrombotic and thrombolytic serine proteases in inflammation.

Serpins in the mammalian body are highly potent serine protease inhibitors which modulate both thrombotic and thrombolytic pathway activation, with direct and indirect crosstalk with immune and inflammatory pathways. In this review, we discuss mammalian and viral serpins as regulators of coagulation and inflammation. We focus first on the thrombotic and thrombolytic serine proteases and known interactions between these protease cascades and elements of the innate immune response. Serpin-mediated regulation of the thrombotic pathway is then discussed, with emphasis on those serpins that have been evaluated as potential new drugs. Finally the potential of viral serpins that target the coagulation and thrombolytic cascades as potential therapeutics for anti-inflammatory properties is discussed from basic molecular activity to studies in animal models. The studies discussed range from thrombosis and hemorrhage to vascular disease and transplant rejection and finally to sepsis and clinical studies in humans. In conclusion, these unique proteins, the serpin family, now have demonstrated therapeutic potential for a wide variety of inflammatory diseases in both animal and human studies and represent a new approach for drug development.

Viral cross-class serpin inhibits vascular inflammation and T lymphocyte fratricide; a study in rodent models in vivo and human cell lines in vitro.

Poxviruses express highly active inhibitors, including serine proteinase inhibitors (serpins), designed to target host immune defense pathways. Recent work has demonstrated clinical efficacy for a secreted, myxomaviral serpin, Serp-1, which targets the thrombotic and thrombolytic proteases, suggesting that other viral serpins may have therapeutic application. Serp-2 and CrmA are intracellular cross-class poxviral serpins, with entirely distinct functions from the Serp-1 protein. Serp-2 and CrmA block the serine protease granzyme B (GzmB) and cysteine proteases, caspases 1 and 8, in apoptotic pathways, but have not been examined for extracellular anti-inflammatory activity. We examined the ability of these cross-class serpins to inhibit plaque growth after arterial damage or transplant and to reduce leukocyte apoptosis. We observed that purified Serp-2, but not CrmA, given as a systemic infusion after angioplasty, transplant, or cuff-compression injury markedly reduced plaque growth in mouse and rat models in vivo. Plaque growth was inhibited both locally at sites of surgical trauma, angioplasty or transplant, and systemically at non-injured sites in ApoE-deficient hyperlipidemic mice. With analysis in vitro of human cells in culture, Serp-2 selectively inhibited T cell caspase activity and blocked cytotoxic T cell (CTL) mediated killing of T lymphocytes (termed fratricide). Conversely, both Serp-2 and CrmA inhibited monocyte apoptosis. Serp-2 inhibitory activity was significantly compromised either in vitro with GzmB antibody or in vivo in ApoE/GzmB double knockout mice. Conclusions The viral cross-class serpin, Serp-2, that targets both apoptotic and inflammatory pathways, reduces vascular inflammation in a GzmB-dependent fashion in vivo, and inhibits human T cell apoptosis in vitro. These findings indicate that therapies targeting Granzyme B and/or T cell apoptosis may be used to inhibit T lymphocyte apoptosis and inflammation in response to arterial injury.

An efficient method for generating poxvirus recombinants in the absence of selection.

The use of selectable markers (ecogpt) and selection pressures to aid in detection of poxvirus (Vaccinia, VV) recombinants has been implicated in the unintended introduction of second site mutations. We have reinvestigated the use of the helper virus system described by Scheiflinger et al. and adapted by Yao and Evans which produces recombinants at a high frequency in the absence of any selection, at a rate of 6­100%. Our system uses fowlpox virus (FPV) as the infectious helper virus which in infected cells provides the enzymatic apparatus for transcription and replication of a purified, transfected VV genome and for recombination with a second transfected PCR generated DNA fragment. To optimize the system, a PCR DNA fragment was generated that contained poxvirus promoter driven gfp and lacZ genes inserted within the coding sequences of the viral thymidine kinase gene. This PCR fragment was co-transfected together with VV genomic DNA. Recombinant VV was identified by plaquing the mixture on cells non-permissive for FPV and selection of green fluorescent or LacZ positive recombinant vaccinia plaques. The system was optimized using FPV permissive cells (CEF) and non-permissive cells (A549, CV-1) for both the initial infection/transfection and the subsequent selection. Up to 70% of the progeny vaccinia virus contained the gfp/LacZ insertion. In order to test for the presence of FPV/VV intertypic recombinants or other unintended mutations, recombinant wtVV (RwtVV) was regenerated from the gfp/LacZ viruses and evaluated by RFLP analysis and pathogenesis in animals. While all RwtVVs were viable in cell culture, in many of the RwtVV isolates, RFLP differences were noted and while some recombinant viruses exhibited wild type behavior in mice, a wide range of virulence indicative of unintended changes suggests that mutants created by "rescue" systems require careful analysis particularly before use for in vivo studies employing animal models.

Efficacy of CMX001 as a prophylactic and presymptomatic antiviral agent in New Zealand white rabbits infected with rabbitpox virus, a model for orthopoxvirus infections of humans.

CMX001, a lipophilic nucleotide analog formed by covalently linking 3-(hexdecyloxy)propan-1-ol to cidofovir (CDV), is being developed as a treatment for smallpox. CMX001 has dramatically increased potency versus CDV against all dsDNA viruses and, in contrast to CDV, is orally available and has shown no evidence of nephrotoxicity in healthy volunteers or severely ill transplant patients to date. Although smallpox has been eliminated from the environment, treatments are urgently being sought due to the risk of smallpox being used as a bioterrorism agent and for monkeypox virus, a zoonotic disease of Africa, and adverse reactions to smallpox virus vaccinations. In the absence of human cases of smallpox, new treatments must be tested for efficacy in animal models. Here we first review and discuss the rabbitpox virus (RPV) infection of New Zealand White rabbits as a model for smallpox to test the efficacy of CMX001 as a prophylactic and early disease antiviral. Our results should also be applicable to monkeypox virus infections and for treatment of adverse reactions to smallpox vaccination.