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

New quick method for isolating RNA from laser captured cells stained by immunofluorescent immunohistochemistry; RNA suitable for direct use in fluorogenic TaqMan one-step real-time RT-PCR

We describe a new approach for reliably isolating one-step real-time quantitative RT-PCR-quality RNA from laser captured cells retrieved from frozen sections previously subjected to immunofluorescent immunohistochemistry (IF-IHC) and subsequently subjected to fluorogenic one-step real-time RT-PCR analysis without the need for costly, time-consuming linear amplification. One cell’s worth of RNA can now be interrogated with confidence. This approach represents an amalgam of technologies already offered commercially by Applied Biosystems, Arcturus and Invitrogen. It is the primary focus of this communication to expose the details and execution of an important new LCM RNA isolation technique, but also provide a detailed account of the IF-IHC procedure preceding RNA isolation, and provide information regarding our approach to fluorogenic one-step real-time RT-PCR in general. Experimental results shown here are meant to supplement the primary aim and are not intended to represent a complete scientific study. It is important to mention, that since LCM-RT-PCR is still far less expensive than micro-array analysis, we feel this approach to isolating RNA from LCM samples will be of continuing use to many researchers with limited budgets in the years ahead

PROBER: oligonucleotide FISH probe design software

PROBER is an oligonucleotide primer design software application that designs multiple primer pairs for generating PCR probes useful for fluorescence in situ hybridization (FISH). PROBER generates Tiling Oligonucleotide Probes (TOPs) by masking repetitive genomic sequences and delineating essentially unique regions that can be amplified to yield small (100-2000 bp) DNA probes that in aggregate will generate a single, strong fluorescent signal for regions as small as a single gene. TOPs are an alternative to bacterial artificial chromosomes (BACs) that are commonly used for FISH but may be unstable, unavailable, chimeric, or non-specific to small (10-100 kb) genomic regions. PROBER can be applied to any genomic locus, with the limitation that the locus must contain at least 10 kb of essentially unique blocks. To test the software, we designed a number of probes for genomic amplifications and hemizygous deletions that were initially detected by Representational Oligonucleotide Microarray Analysis of breast cancer tumors. AVAILABILITY: http://prober.cshl.ed

Emerging targets for developing T cell-mediated vaccines for human immunodeficiency virus (HIV)-1

Human immunodeficiency virus (HIV)-1 has infected >75 million individuals globally, and, according to the UN, is responsible for ~2.1 million new infections and 1.1 million deaths each year. Currently, there are ~37 million individuals with HIV infection and the epidemic has already resulted in 35 million deaths. Despite the advances of anti-retroviral therapy (ART), a cost-effective vaccine remains the best long-term solution to end the HIV-1 epidemic especially given that the vast majority of infected individuals live in poor socio-economic regions of the world such as Sub-Saharan Africa which limits their accessibility to ART. The modest efficacy of the RV144 Thai trial provides hope that a vaccine for HIV-1 is possible, but as markers for sterilizing immunity are unknown, the design of an effective vaccine is empirical, although broadly cross-reactive neutralizing antibodies (bNAb) that can neutralize various quasispecies of HIV-1 are considered crucial. Since HIV-1 transmission often occurs at the genito-rectal mucosa and is cell-associated, there is a need to develop vaccines that can elicit CD8+ T cell immunity with the capacity to kill virus infected cells at the genito-rectal mucosa and the gut. Here we discuss the recent progress made in developing T cell-mediated vaccines for HIV-1 and emphasize the need to elicit mucosal tissue-resident memory CD8+ T (CD8+ Trm) cells. CD8+ Trm cells will likely form a robust front-line defense against HIV-1 and eliminate transmitter/founder virus-infected cells which are responsible for propagating HIV-1 infections following transmission in vast majority of cases.From the National Health and Medical Research Council (NHMRC): grants APP1026293 (EG), APP525431 (CR), APP543139 (EG), and APP543143 (EG). From the Australian Centre for HIV and Hepatitis Virology Research, CR received an EOI gran

Inferring tumor progression from genomic heterogeneity

Cancer progression in humans is difficult to infer because we do not routinely sample patients at multiple stages of their disease. However, heterogeneous breast tumors provide a unique opportunity to study human tumor progression because they still contain evidence of early and intermediate subpopulations in the form of the phylogenetic relationships. We have developed a method we call Sector-Ploidy-Profiling (SPP) to study the clonal composition of breast tumors. SPP involves macro-dissecting tumors, flow-sorting genomic subpopulations by DNA content, and profiling genomes using comparative genomic hybridization (CGH). Breast carcinomas display two classes of genomic structural variation: (1) monogenomic and (2) polygenomic. Monogenomic tumors appear to contain a single major clonal subpopulation with a highly stable chromosome structure. Polygenomic tumors contain multiple clonal tumor subpopulations, which may occupy the same sectors, or separate anatomic locations. In polygenomic tumors, we show that heterogeneity can be ascribed to a few clonal subpopulations, rather than a series of gradual intermediates. By comparing multiple subpopulations from different anatomic locations, we have inferred pathways of cancer progression and the organization of tumor growth. © 2010 by Cold Spring Harbor Laboratory Press

Three phase eulerian-granular model applied on numerical simulation of non-conventional liquid fuels combustion in a bubbling fluidized bed

The paper presents a 2-D CFD model of liquid fuel combustion in bubbling fluidized bed. The numerical procedure is based on the two-fluid Euler-Euler approach, where the velocity field of the gas and particles are modeled in analogy to the kinetic gas theory. The model is taking into account also the third liquid phase, as well as its interaction with the solid and gas phase. The proposed numerical model comprise energy equations for all three phases, as well as the transport equations of chemical components with source terms originated from the component conversion. In the frame of the proposed model, user sub-models were developed for heterogenic fluidized bed combustion of liquid fuels, with or without water. The results of the calculation were compared with experiments on a pilot-facility (power up to 100 kW), combusting, among other fuels, oil. The temperature profiles along the combustion chamber were compared for the two basic cases: combustion with or without water. On the basis of numerical experiments, influence of the fluid-dynamic characteristics of the fluidized bed on the combustion efficiency was analyzed, as well as the influence of the fuel characteristics (reactivity, water content) on the intensive combustion zone

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

Two Distinct Categories of Focal Deletions in Cancer Genomes

One of the key questions about genomic alterations in cancer is whether they are functional in the sense of contributing to the selective advantage of tumor cells. The frequency with which an alteration occurs might reflect its ability to increase cancer cell growth, or alternatively, enhanced instability of a locus may increase the frequency with which it is found to be aberrant in tumors, regardless of oncogenic impact. Here we've addressed this on a genome-wide scale for cancer-associated focal deletions, which are known to pinpoint both tumor suppressor genes (tumor suppressors) and unstable loci. Based on DNA copy number analysis of over one-thousand human cancers representing ten different tumor types, we observed five loci with focal deletion frequencies above 5%, including the A2BP1 gene at 16p13.3 and the MACROD2 gene at 20p12.1. However, neither RNA expression nor functional studies support a tumor suppressor role for either gene. Further analyses suggest instead that these are sites of increased genomic instability and that they resemble common fragile sites (CFS). Genome-wide analysis revealed properties of CFS-like recurrent deletions that distinguish them from deletions affecting tumor suppressor genes, including their isolation at specific loci away from other genomic deletion sites, a considerably smaller deletion size, and dispersal throughout the affected locus rather than assembly at a common site of overlap. Additionally, CFS-like deletions have less impact on gene expression and are enriched in cell lines compared to primary tumors. We show that loci affected by CFS-like deletions are often distinct from known common fragile sites. Indeed, we find that each tumor tissue type has its own spectrum of CFS-like deletions, and that colon cancers have many more CFS-like deletions than other tumor types. We present simple rules that can pinpoint focal deletions that are not CFS-like and more likely to affect functional tumor suppressors

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

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