Anti-retroviral effects of type I IFN subtypes in vivo.

Type I IFN play a very important role in immunity against viral infections. Murine type I IFN belongs to a multigene family including 14 IFN-alpha subtypes but the biological functions of IFN-alpha subtypes in retroviral infections are unknown. We have used the Friend retrovirus model to determine the anti-viral effects of IFN-alpha subtypes in vitro and in vivo. IFN-alpha subtypes alpha1, alpha4, alpha6 or alpha9 suppressed Friend virus (FV) replication in vitro, but differed greatly in their anti-viral efficacy in vivo. Treatment of FV-infected mice with the IFN-alpha subtypes alpha1, alpha4 or alpha9, but not alpha6 led to a significant reduction in viral loads. Decreased splenic viral load after IFN-alpha1 treatment correlated with an expansion of activated FV-specific CD8(+) T cells and NK cells into the spleen, whereas in IFN-alpha4- and -alpha9-treated mice it exclusively correlated with the activation of NK cells. The results demonstrate the distinct anti-retroviral effects of different IFN-alpha subtypes, which may be relevant for new therapeutic approaches.

Contraceptive potential of porcine and feline zona pellucida A, B and C subunits in domestic cats.

Feral cat populations are a major problem in many urban regions throughout the world, threatening biodiversity. Immunocontraception is considered as an alternative and a more humane means to control overpopulation of pest animals than current methods including trapping, poisoning and shooting. In this study, we evaluate porcine zona pellucida (ZP) polypeptide (55 kDa) and feline ZP A, B and C subunits expressed by plasmid vectors as candidate vaccines against fertility in the female domestic cat. Cats were injected subcutaneously with three doses of the ZP vaccines. Vaccinated cats were compared with naïve cats for ZP-antibody response, ovarian histology and fertility after mating. Vaccination with native porcine ZP 55 kDa polypeptide induced anti-porcine ZP antibodies detected by ELISA. However, these antibodies did not cross-react with feline ZP as assessed by immunohistochemistry and no effect on fertility in vivo was observed after mating. However, vaccination of cats with feline ZPA or feline ZPB+C DNA vectors elicited circulating antibodies specific for feline ZP as assessed by ELISA, with reactivity to native feline ZP in ovarian follicles in situ. Vaccination with feline ZPA and ZPB+C DNA did not elicit changes in ovarian histology. Although sample sizes were small, conception rates in mated females were 25 and 20% in the ZPA and ZPB+C vaccinated groups respectively, compared with 83% in the control group. We conclude that feline ZPA and ZPB+C subunits are potential candidate antigens for immunocontraceptive vaccines in the domestic cat.

Direct application of plasmid DNA containing type I interferon transgenes to vaginal mucosa inhibits HSV-2 mediated mortality.

The application of naked DNA containing type I interferon (IFN) transgenes is a promising potential therapeutic approach for controlling chronic viral infections. Herein, we detail the application of this approach that has been extensively used to restrain ocular HSV-1 infection, for antagonizing vaginal HSV-2 infection. We show that application of IFN-alpha1, -alpha5, and -beta transgenes to vaginal mouse lumen 24 hours prior to HSV-2 infection reduces HSV-2 mediated mortality by 2.5 to 3-fold. However, other type I IFN transgenes (IFN- alpha4, -alpha5, -alpha6, and -alpha9) are non effectual against HSV-2. We further show that the efficacy of IFN-alpha1 transgene treatment is independent of CD4+ T lymphocytes. However, in mice depleted of CD8+ T lymphocytes, the ability of IFN-alpha1 transgene treatment to antagonize HSV-2 was lost.

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.

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 200mg IFN 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 CMV gB DNA vaccination in the CMV model. Thus naked DNA delivery of IFN provides an avenue of immunotherapy for regulating herpesvirus-induced diseases.

Interferon-beta suppresses herpes simplex virus type 1 replication in trigeminal ganglion cells through an RNase L-dependent pathway.

The induction of an antiviral state by type I interferons (IFN) was evaluated in primary trigeminal ganglion cell cultures using herpes simplex virus type 1 (HSV-1). Cells treated with mouse IFN-beta consistently showed the greatest resistance to HSV-1 infection in comparison to cells treated with IFN-alpha1, IFN-alpha4, IFN-alpha5, IFN-alpha6, or IFN-alpha9. The antiviral efficacy was dose-dependent and correlated with the induction of the IFN-inducible, antiviral genes, 2'-5' oligoadenylate synthetase (OAS) and double-stranded RNA-dependent protein kinase. In trigeminal ganglion cells deficient in the downstream effector molecule of the OAS pathway, RNase L, the antiviral state induced by IFN-beta was lost.

Evaluation of a Subunit H5 Vaccine and an Inactivated H5N2 Avian Influenza Marker Vaccine in Ducks Challenged with Vietnamese H5N1 Highly Pathogenic Avian Influenza Virus.

The protective efficacy of a subunit avian influenza virus H5 vaccine based on recombinant baculovirus expressed H5 haemagglutinin antigen and an inactivated H5N2 avian influenza vaccine combined with a marker antigen (tetanus toxoid) was compared with commercially available inactivated H5N2 avian influenza vaccine in young ducks. Antibody responses, morbidity, mortality, and virus shedding were evaluated after challenge with a Vietnamese clade 1 H5N1 HPAI virus [A/VN/1203/04 (H5N1)] that was known to cause a high mortality rate in ducks. All three vaccines, administered with water-in-oil adjuvant, provided significant protection and dramatically reduced the duration and titer of virus shedding in the vaccinated challenged ducks compared with unvaccinated controls. The H5 subunit vaccine was shown to provide equivalent protection to the other two vaccines despite the H5 antibody responses in subunit vaccinated ducks being significantly lower prior to challenge. Ducks vaccinated with the H5N2 marker vaccine consistently produced antitetanus toxoid antibody. The two novel vaccines have attributes that would enhance H5N1 avian influenza surveillance and control by vaccination in small scale and village poultry systems.

Evaluation of a positive marker of avian influenza vaccination in ducks for use in H5N1 surveillance.

Control measures for H5N1 avian influenza involve increased biosecurity, monitoring, surveillance and vaccination. Subclinical infection in farmed ducks is important for virus persistence. In major duck rearing countries, homologous H5N1 vaccines are being used in ducks, so sero-surveillance using H5- or N1-specific antibody testing cannot identify infected flocks. An alternative is to include a positive marker for vaccination. Testing for an antibody response to the marker would confirm approved vaccine use. Concurrent testing for H5 antibody responses would determine levels adequate for protection or indicate recent infection, with an anamnestic H5 antibody response requiring further virological investigation. In this study, we have evaluated the use of a TT marker in ducks given avian influenza vaccination. Wild or domestic ducks were tested for antibodies against TT and all 463 ducks were negative. High levels of TT-specific antibodies, produced in twice-TT vaccinated Muscovy ducks, persisted out to 19 weeks. There was no interference by inclusion of TT in an inactivated H6N2 vaccine for H6- or TT-seroconversion. Thus TT is a highly suitable exogenous marker for avian influenza vaccination in ducks and allows sero-surveillance in countries using H5N1 vaccination.

Use of tetanus toxoid as a differentiating infected from vaccinated animals (DIVA) strategy for sero-surveillance of avian influenza virus vaccination in poultry.

Strategies for differentiating infected from vaccinated animals (DIVA) require improvement for increased surveillance of avian influenza (AI), where vaccination is employed to control disease. We propose a novel DIVA approach for chickens using tetanus toxoid (TT) as an exogenous marker independent of serotype and relatedness of circulating and vaccine strains. Of 1779 chickens tested from Australia, Hong Kong and China, 100% were seronegative for TT-specific antibodies without vaccination. Tetanus toxoid adjuvanted to mineral oil was immunogenic in chickens. Co-delivery of both TT and inactivated LPAI (H6N2) vaccines in chickens elicited strong TT and influenza-specific antibody responses, which persisted to 53 weeks post-vaccination. Furthermore, immunization with a combined vaccine composed of TT and AI induced high levels of antibodies to both antigens. We conclude that TT is a highly suitable exogenous marker for AI vaccination in chickens allowing simple and effective monitoring of AI vaccination status.

Differential activities of alpha/beta IFN subtypes against influenza virus in vivo and enhancement of specific immune responses in DNA vaccinated mice expressing haemagglutinin and nucleoprotein.

Vaccines are urgently needed to elicit immunity to different influenza virus strains. DNA vaccines can elicit partial protective immunity, however their efficacy requires improvement. We assessed the capacity of individual type I IFN multigene family members as subtype transgenes to abrogate influenza virus replication in a vaccination/challenge mouse model. Differences in antiviral efficacy were found among the subtypes with IFNA5 and IFNA6 being most effective, while IFNA1 was the least effective in reducing lung virus replication. Mice vaccinated with combinatorial HA/IFNA6 or NP/IFNA6 showed reduced lung viral titres, clinical score, body weight loss, and pulmonary tissue damage compared to IFNA6, HA, or NP viral vaccination alone. In addition, IFNA6 increased IgG2a titres with upregulation of IFN-gamma response in the respiratory tract. We conclude that IFN-alpha 6 has antiviral and immunomodulatory effects, which improve efficacy of DNA vaccines for enhanced control of influenza.

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.

Cytokine expression in murine cytomegalovirus-induced myocarditis: modulation with interferon-alpha 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.

Type I interferon differential therapy for erythroleukemia: specificity of STAT activation.

Type I interferons (IFNs), pleiotropic cytokines with antiviral, antiproliferative, apoptotic, and immunoregulatory functions, are efficacious in the treatment of malignancies, viral infections, and autoimmune diseases. Binding of these cytokines to their cognate receptor leads to activation of the Jak-signal transducers and activators of transcription (STAT) signaling pathway and altered gene expression. This signal pathway has been intensely studied using human IFN-alpha 2 and IFN-beta. However, there are over 14 human IFN-alpha subtypes and over 10 murine IFN-alpha subtypes, with a single IFN-beta subtype in both species. J2E cells are immortalized at the proerythroblast stage of development and produce a rapid and fatal erythroleukemia in vivo. These cells retain the ability to respond to erythropoietin in vitro by proliferating, differentiating, and remaining viable in the absence of serum. Here, we show that J2E cells are also functionally regulated differentially by IFN subtype treatment in vitro. A novel finding was the selective activation of STAT and mitogen-activated protein kinase (MAPK) molecules by different subtypes binding the IFN receptor. These findings indicate distinct effects for individual type I IFN subtypes, which are able to differentially activate members of the STAT and MAPK family. Finally, we investigated the efficacy of IFN naked DNA therapy in treating J2E-induced erythroleukemia in athymic nude mice. IFN subtypes differentially regulated the onset of erythroleukemia with delayed onset and increased survival, possibly via a reduction in cell viability, and enhanced antiproliferative and apoptotic effects observed for IFNA6 and IFNA9 treatment, respectively. Moreover, these data highlight the necessity to choose the best IFN subtype in disease treatment.

Synergy of type I interferon-A6 and interferon-B naked DNA immunotherapy for cytomegalovirus infection.

Delivery of type I IFN transgenes by naked DNA immunization can protect against cytomegalovirus infection and myocarditis. Here, we investigate IFN transgene expression, antiviral efficacy, and immunomodulation of myocarditis using various treatment regimes in a mouse CMV model. In vivo expression of the IFN transgene was observed in the sera for 35 days post-DNA inoculation. Prophylactic IFN-A6 and IFN-B DNA treatment for 14 days prior to murine cytomegalovirus (MCMV) infection was more efficacious in significantly reducing viral titres, than 2 days prior to or 2 days post-virus infection. Similarly, IFN-A6 DNA treatment commencing 14 days prior to virus infection was superior in suppressing both acute and chronic myocarditis. Furthermore, reduction of autoantibody titres was more pronounced when IFN was administered 14 days prior to viral infection. Combinational IFN gene therapy was assessed for synergy between IFN subtypes. Combination treatment with either IFN-A6/A9 or IFN-A6/B greatly reduced spleen viral titres while IFN-A6/B and IFN-A9/B reduced virus replication in the liver. Only IFN-A6/A9 and IFN-A9/B reduced acute viral myocarditis, whereas IFNA6/B treatment was most efficacious for autoimmune chronic myocarditis. Finally, treatment with IFN-A6 DNA 2 weeks post-MCMV infection proved effective at inhibiting the development of chronic autoimmune myocarditis. These findings suggest that immunomodulation of both antiviral and autoimmune responses by IFN DNA immunization may be an avenue for improved viral immunotherapy.

Type I IFN-beta 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-beta administered prophylactically to high-risk patients in acquiring CMV infection may reduce the development of chronic autoimmune myocarditis.