Mucosal vaccination with a live recombinant rhinovirus followed by intradermal DNA administration elicits potent and protective HIV-specific immune responses

Published: 17 November 2016Mucosal immunity is deemed crucial to control sexual transmission of human immunodeficiency virus (HIV). Herein we report the efficacy of a mucosal HIV vaccine strategy comprising intranasal (IN) vaccination with a cocktail of live recombinant human rhinoviruses (HRVs) encoding overlapping fragments of HIV Gag and full length Tat (rHRV-Gag/Tat) followed by intradermal (ID) vaccination with DNA vaccines encoding HIV Gag and Tat (pVAX-Gag-Tat). This heterologous prime-boost strategy will be referred to hereafter as rHRV-DNA. As a control, IN vaccination with wild type (wt)-HRV-A1 followed by a single ID dose of pVAX (wt-HRV-A1/pVAX vaccination) was included. rHRV-DNA vaccination elicited superior multi-functional CD8(+)T cell responses in lymphocytes harvested from mesenteric lymph nodes and spleens, and higher titres of Tat-specific antibodies in blood and vaginal lavages, and reduced the viral load more effectively after challenge with EcoHIV, a murine HIV challenge model, in peritoneal macrophages, splenocytes and blood compared compared with wt-HRV-A1/pVAX vaccination or administration of 3 ID doses of pVAX-Gag-Tat (3X pVAX-Gag-Tat vaccination). These data provide the first evidence that a rHRV-DNA vaccination regimen can induce HIV-specific immune responses in the gut, vaginal mucosa and systemically, and supports further testing of this regimen in the development of an effective mucosally-targeted HIV-1 vaccine.Khamis Tomusange, Danushka Wijesundara, Jason Gummow, Steve Wesselingh, Andreas Suhrbier, Eric J. Gowans, Branka Grubor-Bau

Increase in DNA vaccine efficacy by virosome delivery and co-expression of a cytolytic protein

The potential of DNA vaccines has not been realised due to suboptimal delivery, poor antigen expression and the lack of localised inflammation, essential for antigen presentation and an effective immune response to the immunogen. Initially, we examined the delivery of a DNA vaccine encoding a model antigen, luciferase (LUC), to the respiratory tract of mice by encapsulation in a virosome. Virosomes that incorporated influenza virus haemagglutinin effectively delivered DNA to cells in the mouse respiratory tract and resulted in antigen expression and systemic and mucosal immune responses to the immunogen after an intranasal (IN) prime/intradermal (ID) boost regimen, whereas a multidose ID regimen only generated systemic immunity. We also examined systemic immune responses to LUC after ID vaccination with a DNA vaccine, which also encoded one of the several cytolytic or toxic proteins. Although the herpes simplex virus thymidine kinase, in the presence of the prodrug, ganciclovir, resulted in cell death, this failed to increase the humoral or cell-mediated immune responses. In contrast, the co-expression of LUC with the rotavirus non-structural protein 4 (NSP4) protein or a mutant form of mouse perforin, proteins which are directly cytolytic, resulted in increased LUC-specific humoral and cell-mediated immunity. On the other hand, co-expression of LUC with diphtheria toxin subunit A or overexpression of perforin or NSP4 resulted in a lower level of immunity. In summary, the efficacy of DNA vaccines can be improved by targeted IN delivery of DNA or by the induction of cell death in vaccine-targeted cells after ID delivery.Tessa Gargett, Branka Grubor-Bauk, Darren Miller, Tamsin Garrod, Stanley Yu, Steve Wesselingh, Andreas Suhrbier, and Eric J Gowan

Safety profile of a multi-antigenic DNA vaccine against hepatitis C virus

Despite direct acting antivirals (DAAs) curing >95% of individuals infected with hepatitis C (HCV), in order to achieve the World Health Organization HCV Global Elimination Goals by 2030 there are still major challenges that need to be overcome. DAAs alone are unlikely to eliminate HCV in the absence of a vaccine that can limit viral transmission. Consequently, a prophylactic HCV vaccine is necessary to relieve the worldwide burden of HCV disease. DNA vaccines are a promising vaccine platform due to their commercial viability and ability to elicit robust T-cell-mediated immunity (CMI). We have developed a novel cytolytic DNA vaccine that encodes non-structural HCV proteins and a truncated mouse perforin (PRF), which is more immunogenic than the respective canonical DNA vaccine lacking PRF. Initially we assessed the ability of the HCV pNS3-PRF and pNS4/5-PRF DNA vaccines to elicit robust long-term CMI without any adverse side-effects in mice. Interferon-γ (IFN-γ) enzyme-linked immunosorbent spot (ELISpot) assay was used to evaluate CMI against NS3, NS4 and NS5B in a dose-dependent manner. This analysis showed a dose-dependent bell-curve of HCV-specific responses in vaccinated animals. We then thoroughly examined the effects associated with reactogenicity of cytolytic DNA vaccination with the multi-antigenic HCV DNA vaccine (pNS3/4/5B). Hematological, biochemical and histological studies were performed in male Sprague Dawley rats with a relative vaccine dose 10–20-fold higher than the proposed dose in Phase I clinical studies. The vaccine was well tolerated, and no toxicity was observed. Thus, the cytolytic multi-antigenic DNA vaccine is safe and elicits broad memory CMI.Jason Gummow, Makutiro G. Masavuli, Zelalem A. Mekonnen, Yanrui Li, Danushka K. Wijesundara, Ashish C. Shrestha, Ilia Voskoboinik, Eric J. Gowans and Branka Grubor-Bau

Viral vector and route of administration determine the ILC and DC profiles responsible for downstream vaccine-specific immune outcomes

This study demonstrates that route and viral vector can significantly influence the innate lymphoid cells (ILC) and dendritic cells (DC) recruited to the vaccination site, 24 h post delivery. Intranasal (i.n.) vaccination induced ST2/IL-33R+ ILC2, whilst intramuscular (i.m.) induced IL-25R+ and TSLPR+ (Thymic stromal lymphopoietin protein receptor) ILC2 subsets. However, in muscle a novel ILC subset devoid of the known ILC2 markers (IL-25R- IL-33R- TSLPR-) were found to express IL-13, unlike in lung. Different viral vectors also influenced the ILC-derived cytokines and the DC profiles at the respective vaccination sites. Both i.n. and i.m. recombinant fowlpox virus (rFPV) priming, which has been associated with induction of high avidity T cells and effective antibody differentiation exhibited low ILC2-derived IL-13, high NKp46+ ILC1/ILC3 derived IFN-γ and low IL-17A, together with enhanced CD11b+ CD103- conventional DCs (cDC). In contrast, recombinant Modified Vaccinia Ankara (rMVA) and Influenza A vector priming, which has been linked to low avidity T cells, induced opposing ILC derived-cytokine profiles and enhanced cross-presenting DCs. These observations suggested that the former ILC/DC profiles could be a predictor of a balanced cellular and humoral immune outcome. In addition, following i.n. delivery Rhinovirus (RV) and Adenovius type 5 (Ad5) vectors that induced elevated ILC2-derived IL-13, NKp46+ ILC1/ILC3-derived-IFN-γ and no IL-17A, predominantly recruited CD11b- B220+ plasmacytoid DCs (pDC). Knowing that pDC are involved in antibody differentiation, we postulate that i.n. priming with these vectors may favour induction of effective humoral immunity. Our data also revealed that vector-specific replication status and/or presence or absence of immune evasive genes can significantly alter the ILC and DC activity. Collectively, our findings suggest that understanding the route- and vector-specific ILC and DC profiles at the vaccination site may help tailor/design more efficacious viral vector-based vaccines, according to the pathogen of interest.S. Roy, M.I. Jaeson, Z. Li, S. Mahboob, R.J. Jackson, B. Grubor-Bauk, D.K. Wijesundara, E.J. Gowans, C. Ranasingh

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

Pre-clinical evaluation of a quadrivalent HCV VLP vaccine in pigs following microneedle delivery

The introduction of directly acting antiviral agents (DAAs) has produced significant improvements in the ability to cure chronic hepatitis C infection. However, with over 2% of the world's population infected with HCV, complications arising from the development of cirrhosis of the liver, chronic hepatitis C infection remains the leading indication for liver transplantation. Several modelling studies have indicated that DAAs alone will not be sufficient to eliminate HCV, but if combined with an effective vaccine this regimen would provide a significant advance towards achieving this critical World Health Organisation goal. We have previously generated a genotype 1a, 1b, 2a, 3a HCV virus like particle (VLP) quadrivalent vaccine. The HCV VLPs contain the core and envelope proteins (E1 and E2) of HCV and the vaccine has been shown to produce broad humoral and T cell immune responses following vaccination of mice. In this report we further advanced this work by investigating vaccine responses in a large animal model. We demonstrate that intradermal microneedle vaccination of pigs with our quadrivalent HCV VLP based vaccine produces long-lived multi-genotype specific and neutralizing antibody (NAb) responses together with strong T cell and granzyme B responses and normal Th1 and Th2 cytokine responses. These responses were achieved without the addition of adjuvant. Our study demonstrates that our vaccine is able to produce broad immune responses in a large animal that, next to primates, is the closest animal model to humans. Our results are important as they show that the vaccine can produce robust immune responses in a large animal model before progressing the vaccine to human trials.D. Christiansen, L. Earnest-Silveira, B. Grubor-Bauk, D. K. Wijesundara, I. Boo, P. A. Ramsland, E. Vincan, H. E. Drummer, E. J. Gowans and J. Torres

Immunological responses following administration of a genotype 1a/1b/2/3a quadrivalent HCV VLP vaccine

The significant public health problem of Hepatitis C virus (HCV) has been partially addressed with the advent of directly acting antiviral agents (DAAs). However, the development of an effective preventative vaccine would have a significant impact on HCV incidence and would represent a major advance towards controlling and possibly eradicating HCV globally. We previously reported a genotype 1a HCV viral-like particle (VLP) vaccine that produced neutralizing antibodies (NAb) and T cell responses to HCV. To advance this approach, we produced a quadrivalent genotype 1a/1b/2a/3a HCV VLP vaccine to produce broader immune responses. We show that this quadrivalent vaccine produces antibody and NAb responses together with strong T and B cell responses in vaccinated mice. Moreover, selective neutralizing human monoclonal antibodies (HuMAbs) targeting conserved antigenic domain B and D epitopes of the E2 protein bound strongly to the HCV VLPs, suggesting that these critical epitopes are expressed on the surface of the particles. Our findings demonstrate that a quadrivalent HCV VLP based vaccine induces broad humoral and cellular immune responses that will be necessary for protection against HCV. Such a vaccine could provide a substantial addition to highly active antiviral drugs in eliminating HCV.D. Christiansen, L. Earnest-Silveira, B. Chua, P. Meuleman, I. Boo, B. Grubor-Bauk, D.C. Jackson, Z.Y. Keck, S.K.H. Foung, H.E. Drummer, E.J. Gowans, J. Torres

Cytolytic DNA vaccine encoding lytic perforin augments the maturation of- and antigen presentation by- dendritic cells in a time-dependent manner

The use of cost-effective vaccines capable of inducing robust CD8+ T cell immunity will contribute significantly towards the elimination of persistent viral infections and cancers worldwide. We have previously reported that a cytolytic DNA vaccine encoding an immunogen and a truncated mouse perforin (PRF) protein significantly augments anti-viral T cell (including CD8+ T cell) immunity. Thus, the current study investigated whether this vaccine enhances activation of dendritic cells (DCs) resulting in greater priming of CD8+ T cell immunity. In vitro data showed that transfection of HEK293T cells with the cytolytic DNA resulted in the release of lactate dehydrogenase, indicative of necrotic/lytic cell death. In vitro exposure of this lytic cell debris to purified DCs from naïve C57BL/6 mice resulted in maturation of DCs as determined by up-regulation of CD80/CD86. Using activation/proliferation of adoptively transferred OT-I CD8+ T cells to measure antigen presentation by DCs in vivo, it was determined that cytolytic DNA immunisation resulted in a time-dependent increase in the proliferation of OT-I CD8+ T cells compared to canonical DNA immunisation. Overall, the data suggest that the cytolytic DNA vaccine increases the activity of DCs which has important implications for the design of DNA vaccines to improve their translational prospects.Danushka K. Wijesundara, Wenbo Yu, Ben J. C. Quah, Preethi Eldi, John D. Hayball, Kerrilyn R. Diener, Ilia Voskoboinik, Eric J. Gowans, and Branka Grubor-Bau

Maintenance of broad neutralizing antibodies and memory B cells 1 year post-infection is predicted by SARS-CoV-2-specific CD4+ T cell responses

Understanding the long-term maintenance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immunity is critical for predicting protection against reinfection. In an age- and gender-matched cohort of 24 participants, the association of disease severity and early immune responses on the maintenance of humoral immunity 12 months post-infection is examined. All severely affected participants maintain a stable subset of SARS-CoV-2 receptor-binding domain (RBD)-specific memory B cells (MBCs) and good neutralizing antibody breadth against the majority of the variants of concern, including the Delta variant. Modeling these immune responses against vaccine efficacy data indicate a 45%–76% protection against symptomatic infection (variant dependent). Overall, these findings indicate durable humoral responses in most participants after infection, reasonable protection against reinfection, and implicate baseline antigen-specific CD4+ T cell responses as a predictor of maintenance of antibody neutralization breadth and RBD-specific MBC levels at 12 months post-infection.Harikrishnan Balachandran, Chansavath Phetsouphanh, David Agapiou, Anurag Adhikari, Chaturaka Rodrigo, Mohamed Hammoud, Lok Bahadur Shrestha, Elizabeth Keoshkerian, Money Gupta, Stuart Turville, Daniel Christ, Cecile King, Sarah C. Sasson, Adam Bartlett, Branka Grubor-Bauk, William Rawlinson, Anupriya Aggarwal, Alberto Ospina Stella, Vera Klemm, Michael M. Mina, Jeffrey J. Post, Bernard Hudson, Nicky Gilroy, Pam Konecny, Golo Ahlenstiel, Dominic E. Dwyer, Tania C. Sorrell, Anthony Kelleher, Nicodemus Tedla, Andrew R. Lloyd, Marianne Martinello, Rowena A. Bull, and on behalf of the COSIN Study Grou