A hepatitis C virus DNA vaccine encoding a secreted, oligomerized form of envelope proteins is highly immunogenic and elicits neutralizing antibodies in vaccinated mice

Hepatitis C virus (HCV) persistently infects approximately 71 million people globally. To prevent infection a vaccine which elicits neutralizing antibodies against the virus envelope proteins (E1/E2) which are required for entry into host cells is desirable. DNA vaccines are cost-effective to manufacture globally and despite recent landmark studies highlighting the therapeutic efficacy of DNA vaccines in humans against cervical cancer, DNA vaccines encoding E1/E2 developed thus far are poorly immunogenic. We now report a novel and highly immunogenic DNA vaccination strategy that incorporates secreted E1 and E2 (sE1 and sE2) into oligomers by fusion with the oligomerization domain of the C4b-binding protein, IMX313P. The FDA approved plasmid, pVax, was used to encode sE1, sE2, or sE1E2 with or without IMX313P, and intradermal prime-boost vaccination studies in BALB/c mice showed that vaccines encoding IMX313P were the most effective in eliciting humoral and cell-mediated immunity against the envelope proteins. Further boosting with recombinant E1E2 proteins but not DNA nor virus-like particles (VLPs) expressing E1E2 increased the immunogenicity of the DNA prime-boost regimen. Nevertheless, the antibodies generated by the homologous DNA prime-boost vaccinations more effectively inhibited the binding of VLPs to target cells and neutralized transduction with HCV pseudoparticles (HCVpp) derived from different genotypes including genotypes 1, 2, 3, 4, 5, and 6. This report provides the first evidence that IMX313P can be used as an adjuvant for E1/E2-based DNA vaccines and represents a translatable approach for the development of a HCV DNA vaccine.Makutiro Ghislain Masavuli, Danushka K. Wijesundara, Alexander Underwood, Dale Christiansen, Linda Earnest-Silveira, Rowena Bull, Joseph Torresi, Eric J. Gowans and Branka Grubor-Bau

Enhanced T cell responses induced by a necrotic dendritic cell vaccine, expressing HCV NS3

A vaccine that induces potent, broad and sustained cell-mediated immunity, resulting in effective memory has the potential to restrict hepatitis C (HCV) virus infection. Early, multi-functional CD4+ and CD8+ T cell responses against non-structural protein 3 (NS3) have been associated with HCV clearance. Necrotic cells generate strong immune responses and represent a major antigenic source used by dendritic cells (DC) for processing and presentation, but there is conflicting evidence as to their immunogenicity in vaccination. Immunization with DC loaded with viral antigens has been done in the past, but to date the immunogenicity of live vs. necrotic DC vaccines has not been investigated. We developed a DC2.4 cell line stably expressing HCV NS3, and compared the NS3-specific responses of live vs. necrotic NS3 DC. Vaccination of mice with necrotic NS3 DC increased the breadth of T-cell responses and enhanced the production of IL-2, TNF-α, and IFN-γ by effector memory CD4+ and CD8+T cells, compared to mice vaccinated with live NS3 DC. A single dose of necrotic NS3 DC vaccine induced a greater influx and activation of cross-presenting CD11c+ CD8α+ DC and necrosis-sensing Clec9A+ DC in the draining lymph nodes. Furthermore, using a hydrodynamic challenge model necrotic NS3 DC vaccination resulted in enhanced clearance of NS3-positive hepatocytes from the livers of vaccinated mice. Taken together, the data demonstrate that necrotic DC represent a novel and exciting vaccination strategy capable of inducing broad and multifunctional T cell memory.Zelalem A. Mekonnen, Makutiro G. Masavuli, Wenbo Yu, Jason Gummow, Dawn M. Whelan, Zahraa Al-Delfi ... et al

Toward DNA-based T-cell mediated vaccines to target HIV-1 and hepatitis C virus: approaches to elicit localized immunity for protection

Human immunodeficiency virus (HIV)-1 and hepatitis C virus (HCV) are major contributors to the global disease burden with many experts recognizing the requirement of an effective vaccine to bring a durable end to these viral epidemics. The most promising vaccine candidates that have advanced into pre-clinical models and the clinic to eliminate or provide protection against these chronic viruses are viral vectors [e.g., recombinant cytomegalovirus, Adenovirus, and modified vaccinia Ankara (MVA)]. This raises the question, is there a need to develop DNA vaccines against HIV-1 and HCV? Since the initial study from Wolff and colleagues which showed that DNA represents a vector that can be used to express transgenes durably in vivo, DNA has been regularly evaluated as a vaccine vector albeit with limited success in large animal models and humans. However, several recent studies in Phase I-IIb trials showed that vaccination of patients with recombinant DNA represents a feasible therapeutic intervention to even cure cervical cancer, highlighting the potential of using DNA for human vaccinations. In this review, we will discuss the limitations and the strategies of using DNA as a vector to develop prophylactic T cell-mediated vaccines against HIV-1 and HCV. In particular, we focus on potential strategies exploiting DNA vectors to elicit protective localized CD8+ T cell immunity in the liver for HCV and in the cervicovaginal mucosa for HIV-1 as localized immunity will be an important, if not critical component, of an efficacious vaccine against these viral infections.Zelalem A. Mekonnen, Branka Grubor-Bauk, Makutiro G. Masavuli, Ashish C. Shrestha, Charani Ranasinghe, Rowena A. Bull, Andrew R. Lloyd, Eric J. Gowans, and Danushka K. Wijesundar

Single-dose vaccination with a hepatotropic adeno-associated virus efficiently localizes T cell immunity in the liver with the potential to confer rapid protection against Hepatitis C virus

Hepatitis C virus (HCV) is a significant contributor to the global disease burden and development of an effective vaccine is required to eliminate HCV infections worldwide. CD4+ and CD8+ T cell immunity correlate with viral clearance in primary HCV infection, and intrahepatic CD8+ tissue-resident memory T (TRM) cells provide lifelong and rapid protection against hepatotropic pathogens. Consequently, we aimed to develop a vaccine to elicit HCV-specific CD4+ and CD8+ T cells, including CD8+ TRM cells in the liver given that HCV primarily infects hepatocytes. To achieve this we vaccinated wild-type BALB/c mice with a highly immunogenic cytolytic DNA vaccine encoding a model HCV (genotype 3a) non-structural protein 5B (NS5B) and a mutant perforin (pVAX-NS5B-PRF) as well as a recombinant adeno-associated virus (AAV) encoding NS5B (rAAV-NS5B). A novel fluorescent target array (FTA) was used to map immunodominant CD4+ T helper (TH) cell and cytotoxic CD8+ T cell epitopes of NS5B in vivo which were subsequently used to design a KdNS5B451-459 tetramer and analyse NS5B-specific T cell responses in vaccinated mice in vivo The data showed that intradermal prime/boost vaccination with pVAX-NS5B-PRF was effective in eliciting TH and cytotoxic CD8+ T cell responses, and intrahepatic CD8+ TRM cells, but a single intravenous dose of hepatotropic rAAV-NS5B was significantly more effective. As a T-cell based vaccine against HCV should ideally result in localised T cell responses in the liver this study describes primary observations in the context of HCV vaccination which can be used to achieve this goal. Importance: There are currently at least 71 million individuals with chronic HCV worldwide, and almost two million new infections annually. Although the advent of direct-acting antivirals (DAAs) offer highly effective therapy, considerable remaining challenges argue against reliance on DAAs for HCV elimination including high drug cost, poorly developed health infrastructure, low screening rates, and significant reinfection rates. Accordingly, development of an effective vaccine is crucial to HCV elimination. A HCV vaccine which elicits T cell immunity in the liver will be highly protective for the following reasons: 1) T cell responses against non-structural proteins of the virus are associated with clearance of primary infection, and 2) long-lived liver-resident T cells alone can protect against malaria infection of hepatocytes. Thus, in this study we exploit promising vaccination platforms to highlight strategies that can be used to evoke highly functional and long-lived T cell responses in the liver for protection against HCV.Zelalem A. Mekonnen, Branka Grubor-Bauk, Kieran English, Preston Leung, Makutiro G. Masavuli, Ashish C. Shrestha, Patrick Bertolino, David G. Bowen, Andrew R. Lloyd, Eric J. Gowans, Danushka K. Wijesundar

Myeloperoxidase creates a permissive microenvironmental niche for the progression of multiple myeloma

OnlinePublExpression of myeloperoxidase (MPO), a key inflammatory enzyme restricted to myeloid cells, is negatively associated with the development of solid tumours. Activated myeloid cell populations are increased in multiple myeloma (MM); however, the functional consequences of myeloid-derived MPO within the myeloma microenvironment are unknown. Here, the role of MPO in MM pathogenesis was investigated, and the capacity for pharmacological inhibition of MPO to impede MM progression was evaluated. In the 5TGM1-KaLwRij mouse model of myeloma, the early stages of tumour development were associated with an increase in CD11b+ myeloid cell populations and an increase in Mpo expression within the bone marrow (BM). Interestingly, MM tumour cell homing was increased towards sites of elevated myeloid cell numbers and MPO activity within the BM. Mechanistically, MPO induced the expression of key MM growth factors, resulting in tumour cell proliferation and suppressed cytotoxic T-cell activity. Notably, tumour growth studies in mice treated with a small-molecule irreversible inhibitor of MPO (4-ABAH) demonstrated a significant reduction in overall MM tumour burden. Taken together, our data demonstrate that MPO contributes to MM tumour growth, and that MPO-specific inhibitors may provide a new therapeutic strategy to limit MM disease progression.Connor M. D. Williams, Jacqueline E. Noll, Alanah L. Bradey, Jvaughn Duggan, Vicki J. Wilczek, Makutiro G. Masavuli, Branka Grubor-Bauk, Romana A. Panagopoulos, Duncan R. Hewett, Krzysztof M. Mrozik, Andrew C. W. Zannettino, Kate Vandyke, Vasilios Panagopoulo