Vanessa S. Cull

Differential Effect of Murine Alpha/Beta Interferon Transgenes on Antagonization of Herpes Simplex Virus Type 1 Replication

ABSTRACT Alpha/beta interferons (IFN-α/β) are potent, endogenous antiviral cytokines that suppress the replication of RNA and DNA viruses, including herpes simplex virus type 1 (HSV-1). The present study compared the efficacies of IFN-α/β transgenes, including IFN-α1, -α4, -α5, -α6, -α9, and -β, against HSV-1 infection. L929 cells transfected with the IFN-α/β transgenes produced similar levels of IFN, as measured by bioassay and enzyme-linked immunosorbent assay. In addition, transfected cells were less susceptible to HSV-1 infection than were cells transfected with a plasmid vector control. The murine IFN-β plasmid construct exhibited the greatest reduction, while the murine IFN-α5 transgene showed a modest inhibitory effect in viral titers recovered from the supernatants of transfected, infected L929 cultures. Consistent with this observation, the IFN-β transgene antagonized viral transcript levels, including infected cell protein 27, thymidine kinase, and glycoprotein B, to a greater extent than did the IFN-α transgenes at 6 to 10 h postinfection as determined by real-time PCR. Cells transfected with the IFN-α4, IFN-α9, or IFN-β transgenes showed the greatest reduction in viral protein expression relative to the other transfected cells, which was associated with increased STAT1 expression. The absence of the IFN-responsive protein kinase R (PKR) gene completely abrogated the antiviral induction by all IFN-α/β against HSV-1. In the absence of RNase L, viral yields were increased 10-fold, but the antiviral effect of IFN was either unaffected or enhanced. These results suggest that the predominant IFN-mediated, antiviral pathway during HSV-1 infection taken by IFN-α/β in L929 cells utilizes PKR.

Type I interferon gene therapy protects against cytomegalovirus-induced myocarditis

Type I interferons (IFNs) are produced early in response to viral infection and modulate adaptive immunity. Previously we demonstrated localized protection against murine cytomegalovirus (MCMV) infection in IFN DNA‐inoculated mice. Here we examine the effect of seven IFN subtypes (IFNA1, A2, A4, A5, A6, A9 and B), administered by DNA inoculation, on systemic MCMV infection and myocarditis. IFN transgene expression altered the pathogenesis of MCMV infection with regard to virus titre and myocarditis. IFNA6 treatment reduced MCMV replication whilst IFNA5 and A2 enhanced virus replication. IFNA6, A9, and B treatment inhibited acute myocarditis. A T helper type 1‐like, antibody and cytokine, response correlated with decreased virus titre and myocarditis. In addition, IFNA6 was able to reduce chronic cardiac inflammation. This research into the effectiveness of seven type I IFNs, using DNA gene therapy, highlights the need for correct subtype usage in the treatment of disease. We demonstrate effective subtypes for treatment in both the acute and chronic phases of MCMV infection and the resultant development of myocarditis.

Coimmunisation with type I IFN genes enhances protective immunity against cytomegalovirus and myocarditis in gB DNA-vaccinated mice.

Viral DNA vaccines encoding the glycoprotein B (gB) of cytomegalovirus provide partial protective immunity upon challenge with infectious virus. Although it is known that type I IFN can stimulate the adaptive immune response, their direct use in vaccines has been limited. Here we show that coimmunisation of type I IFN and gB CMV DNA constructs enhances protective immunity in mice. In vivo expression of IFN transgenes ranged from 1.2 to 2.0 × 104 IU/g tibialis anterior muscle. Viral titre in major target organs and the severity of acute CMV-induced myocarditis was reduced preferentially with either IFN-alpha 9 or IFN-beta, but not with IFN-alpha 6, coimmunisation. However, all IFN subtypes investigated markedly reduced chronic myocarditis in gB-vaccinated mice. The early antiviral IgG1 and IgG2a titres were enhanced with IFN-beta coimmunisation. TNF and IL-10 was increased in response to MCMV infection in mice coimmunised with IFN subtypes and viral gB DNA. Indeed T cells from IFN-inoculated mice reduced myocarditis upon in vivo transfer. These results suggest that select type I IFNs may act as a natural adjuvant for the immune response against CMV infection. Type I IFN DNA coimmunisation may provide increased efficacy for viral vaccines and subsequently modulate post-viral chronic inflammatory disorders.

Differential regulation of SOCS genes in normal and transformed erythroid cells

The SOCS family of genes are negative regulators of cytokine signalling with SOCS-1 displaying tumor suppressor activity. SOCS-1, CIS and SOCS-3 have been implicated in the regulation of red blood cell production. In this study, a detailed examination was conducted on the expression patterns of these three SOCS family members in normal erythroid progenitors and a panel of erythroleukemic cell lines. Unexpectedly, differences in SOCS gene expression were observed during maturation of normal red cell progenitors, viz changes to CIS were inversely related to the alterations of SOCS-1 and SOCS-3. Similarly, these SOCS genes were differentially expressed in transformed erythoid cells – erythroleukemic cells immortalized at an immature stage of differentiation expressed SOCS-1 and SOCS-3 mRNA constitutively, whereas in more mature cell lines SOCS-1 and CIS were induced only after exposure to erythropoietin (Epo). Significantly, when ectopic expression of the tyrosine kinase Lyn was used to promote differentiation of immature cell lines, constitutive expression of SOCS-1 and SOCS-3 was completely suppressed. Modulation of intracellular signalling via mutated Epo receptors in mature erythroleukemic lines also highlighted different responses by the three SOCS family members. Close scrutiny of SOCS-1 revealed that, despite large increases in mRNA levels, the activity of the promoter did not alter after erythropoietin stimulation; in addition, erythroid cells from SOCS-1−/− mice displayed increased sensitivity to Epo. These observations indicate complex, stage-specific regulation of SOCS genes during normal erythroid maturation and in erythroleukemic cells.

Transient transfection of mouse fibroblasts with type I interferon transgenes provides various degrees of protection against herpes simplex virus infection.

Type I interferons (IFN) constitute one of the initial and most potent components of the innate immune response against viral infections. While there is only one IFN-beta gene, there are several IFN-alpha genes whose products act through the same receptor calling into question the role of these gene products against viral infection. The focus of the present study was to compare the anti-viral state of cells transiently transfected with different murine type I IFN transgenes including IFN-alpha1, -alpha4, -alpha5, -alpha6, -alpha9, and IFN-beta. Transfected cells produced biologically active IFN ranging from 6 to 46 units/ml. L929 and 3T12.3 cells transfected with the IFN-beta transgene consistently showed a 2-4 fold reduction in herpes simplex virus type 1 (HSV-1) and HSV-2 viral titers compared with cells transfected with the IFN-alpha transgenes which were much less consistent based on HSV species and cell type. Parallel with the reduction in viral titers, cells transfected with the IFN-beta transgene showed the complete absence or significant reduction in viral immediate early, early, and late gene expression. Collectively, the results suggest that the IFN-beta transgene is superior to IFN-alpha transgenes against HSV infection in vitro in part due to a reduction in viral gene expression. These results indicate events downstream of the type I IFN receptor distinguish between the subtypes of IFN-alpha species relative to the activation of genes ultimately responsible for the establishment of the anti-HSV state.

Type I IFN-β gene therapy suppresses cardiac CD8 + T-cell infiltration during autoimmune myocarditis

Gene therapy using DNA encoding type I IFN subtypes IFNA6, IFNA9 and IFNB suppresses murine cytomegalovirus (MCMV)‐myocarditis, a predominantly cell‐mediated disease in BALB/c mice. CD8+ T cells are the principal cell type within the inflamed myocardium. As such, we investigated the effects of IFN subtype treatment on this T‐cell subset and other cell types in the cardiac infiltrate. In the acute phase of disease, IFNA6 and IFNA9 treatments significantly reduced the number of CD8+ T cells within the foci of cellular infiltration in the heart. During the chronic phase, which is primarily autoimmune in nature, IFNB treatment significantly reduced CD8+ T cells. B‐cell and neutrophil numbers in the cardiac infiltrate were also reduced following IFNB immunotherapy. Although early inflammatory responses are important for resolution of virus infection, high numbers of lymphocytes persisting in the myocardium may lead to exacerbation of disease. Our data suggests that type I IFN DNA therapy regulates cardiac cellular infiltration. Thus, treatment with IFN‐β administered prophylactically to high‐risk patients in acquiring CMV infection may reduce the development of chronic autoimmune myocarditis.

Cytokine expression in murine cytomegalovirus-induced myocarditis: modulation with interferon-α therapy

Cytomegalovirus-induced myocarditis is largely immune-mediated. BALB/c mice produced higher levels of IL-4 in the heart indicative of a Th2-like response. Although IL-6, IL-10, IL-18, and TNF-alpha were produced in the heart during acute infection, BALB/c mice lacked a substantial IL-2 and IFN-gamma response. Conversely, C57BL/6 mice produced significant levels of IFN-gamma in the heart with no significant levels of IL-4 or IL-6, suggestive of a dominant Th1-like response to virus infection. IFN-alpha/beta immunotherapy is known to suppress the development of MCMV-myocarditis. Cytokine secretion in IFN-stimulated MCMV-infected BALB/c myocytes was found to be IFN subtype-dependent with elevation of IL-6 and IL-18 levels. During the chronic phase of disease, IFNA6 DNA treatment in vivo increased IL-18 production in the heart. These results suggest that IFN subtype therapy may have immunomodulating effects in reducing disease severity in BALB/c mice via regulation of cytokine production in the heart.

Dominant action of mutated erythropoietin receptors on differentiation in vitro and erythroleukemia development in vivo

J2E cells produce rapid, fatal erythroleukemias in vivo but still respond to erythropoietin (epo) in vitro by differentiating, proliferating and remaining viable in the absence of serum. Mutant epo receptors were introduced into these cells to determine whether they could influence the different biological responses to epo in vitro and the development of erythroleukemias. Three mutant receptors were used as cytoplasmic truncation mutants Δ257 and Δ321 (above box 1 and below box 2 respectively), and the cytoplasmic point mutant W282R (defective for JAK2 activation). Strikingly, the Δ321 mutation produced a hyper-sensitive response in vitro to epo-induced differentiation and viability, but not to proliferation. In contrast with the Δ321 receptor, the Δ257 and W282R mutants inhibited all biological responses to epo due to impaired JAK2 phosphorylation. Significantly, erythroleukemias took almost twice as long to develop with cells containing the W282R mutation, indicating that JAK2 plays an important role in the emergence of these leukemias. These data demonstrate that mutant epo receptors dominantly altered responses of J2E cells to epo in culture and the development of erythroleukemias.

Optimization of naked DNA delivery for interferon subtype immunotherapy in cytomegalovirus infection

Type I interferon (IFN) gene therapy modulates the immune response leading to inflammatory heart disease following cytomegalovirus (CMV) infection in a murine model of post-viral myocarditis. Efficacy of different immunisation protocols for the IFN constructs was influenced by the dose of DNA, subtype choice, combination use, pre-medication, and timing of DNA administration. Optimal efficacy was found with bupivacaine treatment prior to DNA inoculation of 200µgIFN DNA 14 days prior to virus challenge. Maximal antiviral and antimyocarditic effects were achieved with this vaccination schedule. Furthermore, inoculation of synergistic IFN subtypes demonstrated enhanced efficacy when delivered either alone or with CMVgB DNA vaccination in the CMV model. Thus naked DNA delivery of IFN provides an avenue of immunotherapy for regulating herpesvirus-induced diseases.

Interferon Subtype Gene Therapy for Regulating Cytomegalovirus Disease

Delivery of type I interferon (IFN) subtypes by intramuscular inoculation of mice with a recombinant mammalian expression vector encoding IFN stimulates the immune response. Such immunomodulation drives towards a Th1-like response. The degree of stimulation of the immune response was influenced by several parameters of the naked deoxyribonucleic acid (DNA) vaccination protocol. Pretreatment of mice with bupivacaine increased transgene expression in situ. The specific subtype gene of type I IFN, the DNA concentration, the combined use of two or more subtypes, and the timing of the DNA immunisations were all found to influence the level of efficacy of IFN gene therapy in a mouse model for cytomegalovirus (CMV) infection and disease. In addition, adjuvant therapy, using type I IFN genes, for DNA virus vaccination (CMV glycoprotein B) enhanced viral-specific immunity and reduced the severity of myocarditis in mice. Thus, type I IFN gene therapy has potent adjuvant properties when delivered as DNA and can be used to regulate virus infection and disease via pleiotropic actions in the stimulation of immune responses.