2016 Pipeline Report HIV and TB

HIV and TB: Drugs, Diagnostics, Vaccines, Preventive Technologies, Research Toward a Cure, and Immune-Based and Gene Therapies in Developmen

Latency, Expression and Splicing During Infection With HIV

Over 35 million people are living with human immunodeficiency virus (HIV-1). The mechanisms causing integrated provirus to become latent, the diversity of spliced viral transcripts and the cellular response to infection are not fully characterized and hinder the eradication of HIV-1. We applied high-throughput sequencing to investigate the effects of host chromatin on proviral latency and variation of expression and splicing in both the host and virus during infection. To evaluate the link between host chromatin and proviral latency, we compared genomic and epigenetic features to HIV-1 integration site data for latent and active provirus from five cell culture models. Latency was associated with chromosomal position within individual models. However, no shared mechanisms of latency were observed between cell culture models. These differences suggest that cell culture models may not completely reflect latency in patients. We carried out two studies to explore mRNA populations during HIV infection. Single-molecule amplification and sequencing revealed that the clinical isolate HIV89.6 produces at least 109 different spliced mRNAs. Viral message populations differed between cell types, between human donors and longitudinally during infection. We then sequenced mRNA from control and HIV89.6-infected primary human T cells. Over 17 percent of cellular genes showed altered activity associated with infection. These gene expression patterns differed from HIV infection in cell lines but paralleled infections in primary cells. Infection with HIV89.6 increased intron retention in cellular genes and abundance of RNA from human endogenous retroviruses. We also quantified the frequency and location of chimeric HIV-host RNAs. These two studies together provided a detailed accounting of both HIV89.6 and host expression and alternative splicing. A more cost-effective method of detecting viral load would aid patients with poor access to healthcare. We developed improved methods for assaying HIV-1 RNA using loop-mediated isothermal amplification based on primers targeting regions of the HIV-1 genome conserved across subtypes. Combined with lab-on-a-chip technology, these techniques allow quantitative measurements of viral load in a point-of-care device targeted to resource-limited settings. This work disclosed novel HIV-host interactions and developed techniques and knowledge that will aid in the study and management of HIV-1 infection

Oncolytic Virus Immunotherapy

Dear Readers, Oncolytic Viruses (OV) are self-propagating agents that can selectively induce the lysis of cancer cells while sparing normal tissues. OV-mediated cancer cell death is often immunogenic and triggers robust anticancer immune responses and immunoconversion of tumor microenvironments. This makes oncolytic virotherapy a promising new form of immunotherapy and OVs ideal candidates for combination therapy with other anticancer agents, including other immunotherapeutics. There are more than 40 OVs from nine different families in clinical development and many more at the preclinical stage. Each OV has its own unique characteristics, its pros and cons. Although herpes simplex virus is currently the lead clinical agent, a real champion among the OVs has not yet emerged, justifying the continuous development and optimization of these agents. This book, “Oncolytic Virus Immunotherapy”, summarizes the state-of-the-art and gives a comprehensive overview of the OV arena with a particular focus on new trends, directions, challenges, and opportunities

A Novel Methodology for Isolating Broadly Neutralizing HIV-1 Human Monoclonal Antibodies

Abstract also published in AIDS Research and Human Retroviruses. November 2013, 29(11): A-53. doi:10.1089/aid.2013.1500Poster presentationpublished_or_final_versio

Investigation of azithromycin analogues and proteasome-like inhibitors as quick-killing antimalarials

Malaria is caused by mosquito-borne parasites of the genus Plasmodium which were responsible for ~435,000 of deaths annually, with >90% caused by the deadliest species, P. falciparum. Over the last two decades, global implementation of vector control and artemisinin combination therapies have resulted in significant reductions in the global burden of malaria. Of current concern is the spread of multi-drug resistant parasites that have severely limited the efficacy of antimalarials, including front-line artemisinins, highlighting the urgent need to identify new antimalarials for use as treatments. The aim of this thesis was to investigate novel antimalarial development avenues and identify new chemotypes that could be used in the near future as treatments. The macrolide antibiotic azithromycin is known to target the malaria parasites remnant plastid organelle (the apicoplast’s) bacterial-like ribosome and causes slow-killing ‘delayed death’, where the parasite dies in the second replication cycle (4 days). Azithromycin has also been shown to inhibit invading merozoites and kill blood stages within the first replication cycle (2 days) via an unidentified mechanism, proposed to be independent of delayed death. Thus, we hypothesised that azithromycin could be redeveloped into an antimalarial with two different mechanisms of action against parasites: delayed death and quick-killing. Over 100 azithromycin analogues that featured a high proportion of different structural profiles were obtained, leading to improved quick-killing activities over azithromycin. Quick-killing was also confirmed to be completely unrelated to delayed death, as blood stage parasites lacking the apicoplast were equally susceptible to quick-killing of azithromycin and analogues. Two different avenues were also confirmed for azithromycin’s antimalarial re-development: delayed death and quick-killing or quick-killing only, which could be modulated depending on the location of added functional groups. Azithromycin and analogues were found to be active across blood stage development, with only short treatments required to kill parasites. The metabolomics signatures of parasites treated with azithromycin and analogues suggested that quick-killing acts multi-factorially, with the parasite’s food vacuole and mitochondria being likely targets. Finally, in vitro activities of two subtypes of tri-peptide proteasome-like inhibitors, vinyl sulfone and aldehydes, were addressed against P. falciparum and the zoonotic malaria parasite P. knowlesi. All compounds exhibited low-nanomolar activities against both Plasmodium spp. and showed excellent selectivity for parasites over human cells, suggesting these inhibitors provide viable chemical scaffolds for optimisation. There was no evidence of increased protein ubiquitination upon treating parasites with these compounds, suggesting they do not target the proteasome. We also investigated whether hypoxia inducible pro-drug proteasome-like inhibitors could be used to reduce host toxicity of antimalarials. However, these pro-drugs could be not activated in in vitro culture conditions and there was limited evidence suggesting this strategy would be applicable in malaria. These studies build on previous findings on the drug-killing efficacy, mechanism of action and possible application of redeveloping azithromycin analogues as new and improved antimalarials. I also identified new proteasome inhibitor-like scaffolds as starting points for further development. This body of work provides thorough biological characterisation of a panel of compounds that could lead to new avenues for antimalarial development.Thesis (Ph.D.) -- University of Adelaide, School of Biololgical Sciences, 202

Elicitation of broadly neutralizing HIV-1 antibodies by guiding the immune responses using primary and secondary immunogens

Abstract also published in AIDS Research and Human Retroviruses. November 2013, 29(11): A-44. doi:10.1089/aid.2013.1500Poster presentationpublished_or_final_versio

In vitro expanded human CD4+CD25+ regulatory T cells suppress effector T cell proliferation.

Regulatory T cells (Tregs) have been shown to be critical in the balance between autoimmunity and tolerance and have been implicated in several human autoimmune diseases. However, the small number of Tregs in peripheral blood limits their therapeutic potential. Therefore, we developed a protocol that would allow for the expansion of Tregs while retaining their suppressive activity. We isolated CD4+CD25 hi cells from human peripheral blood and expanded them in vitro in the presence of anti-CD3 and anti-CD28 magnetic Xcyte Dynabeads and high concentrations of exogenous Interleukin (IL)-2. Tregs were effectively expanded up to 200-fold while maintaining surface expression of CD25 and other markers of Tregs: CD62L, HLA-DR, CCR6, and FOXP3. The expanded Tregs suppressed proliferation and cytokine secretion of responder PBMCs in co-cultures stimulated with anti-CD3 or alloantigen. Treg expansion is a critical first step before consideration of Tregs as a therapeutic intervention in patients with autoimmune or graft-versus-host disease

Pharmacotherapy

The intent of this book is to provide an overview of current conceptualizations of Pharmacotherapy. The book focuses on three major areas; diagnosis, treatment, and prevention for a wide array of diseases; Cognitive and Psychological disorders (Schizophrenia and Nicotine addiction), Inflammatory disorders (New Chemical anti-inflammatory and Immunotherapy), updated antihypertensive therapy and healing of ulcers with venous origin. A separate chapter is dedicated to the rationality of drug use in earthquake injuries. The last chapter deals with Imaging of potential therapeutic or diagnostic agents in animal models in the early stage of research. We hope this book is useful to a wide range of people, from students first learning about Pharmacotherapy, to advanced clinicians and researchers