Cassandra M. James

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.

Critical Role for the Oligoadenylate Synthetase/RNase L Pathway in Response to IFN-β during Acute Ocular Herpes Simplex Virus Type 1 Infection

We previously demonstrated that IFN-β transgene treatment protects mouse trigeminal ganglia (TG) cells from acute HSV-1 infection in vitro. However, IFN-α6 transgene treatment does not provide protection against acute HSV-1 infection in vitro, even though equivalent levels of IFN are expressed with both transgene treatments. In the present study we show that IFN-β transgene treatment before acute ocular HSV-1 infection protects mice from HSV-1-mediated mortality, whereas IFN-α6 transgene treatment does not reduce mortality. Treatment with the IFN-β and IFN-α6 transgenes was associated with increased expression of oligoadenylate synthetase (OAS)1a mRNA in the eye. However, protein kinase R mRNA was not up-regulated in the eye. In TG, only IFN-β transgene treatment reduced infectious virus levels. Furthermore, in the absence of a functional OAS pathway, corneal HSV-1 Ag expression was more widespread, and the ability of IFN-β transgene treatment to reduce infectious HSV-1 in eyes and TG was lost. Along with selective up-regulation of OAS1a mRNA expression in TG from IFN-β transgene-treated mice, we found increased levels of phospho-STAT1. Likewise, p38 MAPK phosphorylation was increased in TG from IFN-β transgene-treated mice, compared with both IFN-α6 and vector-treated mice. We also observed a time-dependent increase in JNK phosphorylation in TG from IFN-β transgene-treated vs IFN-α6 and vector-treated mice. Our results demonstrate that IFN-β is a potent antiviral cytokine that exerts protection against ocular HSV-1 infection via selective up-regulation of OAS1a mRNA in TG and by altering the phosphorylation of proteins in antiviral signaling cascades.

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.

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‐α subtypes but the biological functions of IFN‐α subtypes in retroviral infections are unknown. We have used the Friend retrovirus model to determine the anti‐viral effects of IFN‐α subtypes in vitro and in vivo. IFN‐α subtypes α1, α4, α6 or α9 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‐α subtypes α1, α4 or α9, but not α6 led to a significant reduction in viral loads. Decreased splenic viral load after IFN‐α1 treatment correlated with an expansion of activated FV‐specific CD8+ T cells and NK cells into the spleen, whereas in IFN‐α4‐ and ‐α9‐treated mice it exclusively correlated with the activation of NK cells. The results demonstrate the distinct anti‐retroviral effects of different IFN‐α subtypes, which may be relevant for new therapeutic approaches.

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.

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.

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.

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-α1, -α 5, and -β 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- α 4, -α 5, -α 6, and -α 9) are non effectual against HSV-2. We further show that the efficacy of IFN-1 transgene treatment is independent of CD4+ T lymphocytes. However, in mice depleted of CD8+ T lymphocytes, the ability of IFN-α 1 transgene treatment to antagonize HSV-2 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.