Emmalene J. Bartlett

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.

Attenuation and efficacy of human parainfluenza virus type 1 (HPIV1) vaccine candidates containing stabilized mutations in the P/C and L genes

BackgroundTwo recombinant, live attenuated human parainfluenza virus type 1 (rHPIV1) mutant viruses have been developed, using a reverse genetics system, for evaluation as potential intranasal vaccine candidates. These rHPIV1 vaccine candidates have two non-temperature sensitive (non-ts) attenuating (att) mutations primarily in the P/C gene, namely CR84GHNT553A (two point mutations used together as a set) and CΔ170 (a short deletion mutation), and two ts att mutations in the L gene, namely LY942A (a point mutation), and LΔ1710–11 (a short deletion), the last of which has not been previously described. The latter three mutations were specifically designed for increased genetic and phenotypic stability. These mutations were evaluated on the HPIV1 backbone, both individually and in combination, for attenuation, immunogenicity, and protective efficacy in African green monkeys (AGMs).ResultsThe rHPIV1 mutant bearing the novel LΔ1710–11 mutation was highly ts and attenuated in AGMs and was immunogenic and efficacious against HPIV1 wt challenge. The rHPIV1-CR84G/Δ170HNT553ALY942A and rHPIV1-CR84G/Δ170HNT553ALΔ1710–11 vaccine candidates were highly ts, with shut-off temperatures of 38°C and 35°C, respectively, and were highly attenuated in AGMs. Immunization with rHPIV1-CR84G/Δ170HNT553ALY942A protected against HPIV1 wt challenge in both the upper and lower respiratory tracts. In contrast, rHPIV1-CR84G/Δ170HNT553ALΔ1710–11 was not protective in AGMs due to over-attenuation, but it is expected to replicate more efficiently and be more immunogenic in the natural human host.ConclusionThe rHPIV1-CR84G/Δ170HNT553ALY942A and rHPIV1-CR84G/Δ170HNT553ALΔ1710–11 vaccine candidates are clearly highly attenuated in AGMs and clinical trials are planned to address safety and immunogenicity in humans.

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.

The C Proteins of Human Parainfluenza Virus Type 1 (HPIV1) Control the Transcription of a Broad Array of Cellular Genes That Would Otherwise Respond to HPIV1 Infection

ABSTRACT Human parainfluenza virus type 1 (HPIV1) is an important respiratory pathogen in children and the most common cause of viral croup. We performed a microarray-based analysis of gene expression kinetics to examine how wild-type (wt) HPIV1 infection altered gene expression in human respiratory epithelial cells and what role beta interferon played in this response. We similarly evaluated HPIV1-P(C−), a highly attenuated and apoptosis-inducing virus that does not express any of the four C proteins, and HPIV1-CF170S, a less attenuated mutant that contains a single point mutation in C and, like wt HPIV1, does not efficiently induce apoptosis, to examine the role of the C proteins in controlling host gene expression. We also used these data to investigate whether the phenotypic differences between the two C mutants could be explained at the transcriptional level. Mutation or deletion of the C proteins of HPIV1 permitted the activation of over 2,000 cellular genes that otherwise would be repressed by HPIV1 infection. Thus, the C proteins profoundly suppress the response of human respiratory cells to HPIV1 infection. Cellular pathways targeted by the HPIV1 C proteins were identified and their transcriptional control was analyzed using bioinformatics. Transcription factor binding sites for IRF and NF-κB were overrepresented in some of the C protein-targeted pathways, but other pathways were dominated by less-known factors, such as forkhead transcription factor FOXD1. Surprisingly, the host responses to the P(C−) and CF170S mutants were very similar, and only subtle differences in the expression kinetics of caspase 3 and TRAIL receptor 2 were observed. Thus, changes in host cell transcription did not reflect the striking phenotypic differences observed between these two viruses.

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.