109 research outputs found

    Improving Vaccine Design For Viral Diseases Using Modified Antigens And Vectors

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    Two of the principal challenges facing vaccine design today are how to generate protective antibody responses against viruses that have evolved sophisticated strategies to evade the humoral immune system and how to more rapidly and effectively produce vaccines to address emerging epidemics. In this regard, we explored multiple strategies to improve vaccine design for HIV-1 and Zika virus. In one approach, we derived CD4-independent variants of HIV-1 envelope (Env) with the hypothesis that such Envs would expose conserved epitopes that may be targets of protective, non-neutralizing antibodies. We characterized the biological and structural properties of two CD4-independent Env clones and found that they exhibited significantly greater exposure of a relatively conserved, linear epitope in the second variable loop (V2) that had previously been associated with decreased risk of infection in a clinical HIV-1 vaccine trial. This epitope was significantly more immunogenic in mice and nonhuman primates and, intriguingly, was associated with more rapid development of antibody-dependent cell-mediated cytotoxicity. In another approach, we designed mutations in the cytoplasmic tail of HIV-1 Env that were predicted to increase its cell surface expression and thus its immunogenicity in a vaccinia prime-protein boost vaccine protocol. We found that the highest level of surface expression was mediated by Envs with truncated cytoplasmic tails, and this was associated with higher levels of binding and neutralizing antibodies after vaccinia primes and protein boosts, respectively. These two studies revealed that modifications to HIV-1 Env immunogens are able to influence both the quality and magnitude of desirable antibody responses. Finally, we used a newly developed vaccine platform based on nucleoside-modified mRNA to design a vaccine against Zika virus. This vaccine, encoding the surface prM and E proteins, was potently immunogenic and elicited high and sustained titers of neutralizing antibodies in mice and nonhuman primates following a single intradermal immunization. We observed rapid and durable protection from Zika virus infection in mice and a high level of protection in monkeys challenged five weeks after vaccination. This vaccine thus represents a promising candidate for clinical use in controlling the spread of Zika virus

    Viral Gene Therapy

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    The development of technologies that allow targeting of specific cells has progressed substantially in recent years for several types of vectors, particularly viral vectors, which have been used in 70% of gene therapy clinical trials. Particular viruses have been selected as gene delivery vehicles because of their capacities to carry foreign genes and their ability to efficiently deliver these genes associated with efficient gene expression. This book is designed to present the most recent advances in viral gene therap

    Oncolytic adenoviruses for treatment of ovarian cancer

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    Virotherapy, the use of oncolytic properties of viruses for eradication of tumor cells, is an attractive strategy for treating cancers resistant to traditional modalities. Adenoviruses can be genetically modified to selectively replicate in and destroy tumor cells through exploitation of molecular differences between normal and cancer cells. The lytic life cycle of adenoviruses results in oncolysis of infected cells and spreading of virus progeny to surrounding cells. In this study, we evaluated different strategies for improving safety and efficacy of oncolytic virotherapy against human ovarian adenocarcinoma. We examined the antitumor efficacy of Ad5/3-Δ24, a serotype 3 receptor-targeted pRb-p16 pathway-selective oncolytic adenovirus, in combination with conventional chemotherapeutic agents. We observed synergistic activity in ovarian cancer cells when Ad5/3-Δ24 was given with either gemcitabine or epirubicin, common second-line treatment options for ovarian cancer. Our results also indicate that gemcitabine reduces the initial rate of Ad5/3-Δ24 replication without affecting the total amount of virus produced. In an orthotopic murine model of peritoneally disseminated ovarian cancer, combining Ad5/3-Δ24 with either gemcitabine or epirubicin resulted in greater therapeutic benefit than either agent alone. Another useful approach for increasing the efficacy of oncolytic agents is to arm viruses with therapeutic transgenes such as genes encoding prodrug-converting enzymes. We constructed Ad5/3-Δ24-TK-GFP, an oncolytic adenovirus encoding the thymidine kinase (TK) green fluorescent protein (GFP) fusion protein. This novel virus replicated efficiently on ovarian cancer cells, which correlated with increased GFP expression. Delivery of prodrug ganciclovir (GCV) immediately after infection abrogated viral replication, which might have utility as a safety switch mechanism. Oncolytic potency in vitro was enhanced by GCV in one cell line, and the interaction was not dependent on scheduling of the treatments. However, in murine models of metastatic ovarian cancer, administration of GCV did not add therapeutic benefit to this highly potent oncolytic agent. Detection of tumor progression and virus replication with bioluminescence and fluorescence imaging provided insight into the in vivo kinetics of oncolysis in living mice. For optimizing protocols for upcoming clinical trials, we utilized orthotopic murine models of ovarian cancer to analyze the effect of dose and scheduling of intraperitoneally delivered Ad5/3-Δ24. Weekly administration of Ad5/3-Δ24 did not significantly enhance antitumor efficacy over a single treatment. Our results also demonstrate that even a single intraperitoneal injection of only 100 viral particles significantly increased the survival of mice compared with untreated animals. Improved knowledge of adenovirus biology has resulted in creation of more effective oncolytic agents. However, with more potent therapy regimens an increase in unwanted side-effects is also possible. Therefore, inhibiting viral replication when necessary would be beneficial. We evaluated the antiviral activity of chlorpromazine and apigenin on adenovirus replication and associated toxicity in fresh human liver samples, normal cells, and ovarian cancer cells. Further, human xenografts in mice were utilized to evaluate antitumor efficacy, viral replication, and liver toxicity. Our data suggest that these agents can reduce replication of adenoviruses, which could provide a safety switch in case of replication-associated side-effects. In conclusion, we demonstrate that Ad5/3-Δ24 is a useful oncolytic agent for treatment of ovarian cancer either alone or in combination with conventional chemotherapeutic drugs. Insertion of genes encoding prodrug-converting enzymes into the genome of Ad5/3-Δ24 might not lead to enhanced antitumor efficacy with this highly potent oncolytic virus. As a safety feature, viral activity can be inhibited with pharmacological substances. Clinical trials are however needed to confirm if these preclinical results can be translated into efficacy in humans. Promising safety data seen here, and in previous publications suggest that clinical evaluation of the agent is feasible.Ei saatavill

    Machine-learning-based identification of factors that influence molecular virus-host interactions

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    Viruses are the cause of many infectious diseases such as the pandemic viruses: acquired immune deficiency syndrome (AIDS) and coronavirus disease 2019 (COVID-19). During the infection cycle, viruses invade host cells and trigger a series of virus-host interactions with different directionality. Some of these interactions disrupt host immune responses or promote the expression of viral proteins and exploitation of the host system thus are considered ‘pro-viral’. Some interactions display ‘pro-host’ traits, principally the immune response, to control or inhibit viral replication. Concomitant pro-viral and pro-host molecular interactions on the same host molecule suggests more complex virus-host conflicts and genetic signatures that are crucial to host immunity. In this work, machinelearning-based prediction of virus-host interaction directionality was examined by using data from Human immunodeficiency virus type 1 (HIV-1) infection. Host immune responses to viral infections are mediated by interferons(IFNs) in the initial stage of the immune response to infection. IFNs induce the expression of many IFN-stimulated genes (ISGs), which make the host cell refractory to further infection. We propose that there are many features associated with the up-regulation of human genes in the context of IFN-α stimulation. They make ISGs predictable using machine-learning models. In order to overcome the interference of host immune responses for successful replication, viruses adopt multiple strategies to avoid being detected by cellular sensors in order to hijack the machinery of host transcription or translation. Here, the strategy of mimicry of host-like short linear motifs (SLiMs) by the virus was investigated by using the example of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The integration of in silico experiments and analyses in this thesis demonstrates an interactive and intimate relationship between viruses and their hosts. Findings here contribute to the identification of host dependency and antiviral factors. They are of great importance not only to the ongoing COVID-19 pandemic but also to the understanding of future disease outbreaks

    Labeling HIV-1 nucleic acids during infection and effect of MOV10 in HBV replication

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    Technical limitations in simultaneous microscopic visualization of RNA, DNA, and proteins of HIV have curtailed progress in this field. To address this need we have developed a microscopy approach, multiplex immunofluorescent cell-based detection of DNA, RNA and Protein (MICDDRP), which is based on branched DNA in situ hybridization technology. MICDDRP enables simultaneous single-cell visualization of HIV spliced and unspliced RNA, cytoplasmic and nuclear DNA, and Gag. We use MICDDRP to visualize incoming capsid cores containing RNA and/or nascent DNA and follow reverse transcription kinetics. We have also reported transcriptional "bursts" of nascent RNA from integrated proviral DNA. The major barrier towards a cure is the ability of the virus to remain dormant in long-lived cells known as the latent HIV reservoirs; however, the mechanisms that regulate latency are poorly understood. HIV-1 integration is directed to regions of the genome by the chromatin binding protein lens epitheliumderived growth factor (LEDGF/p75, referred to here as LEDGF). In the absence of LEDGF, HIV-1 infection is markedly less efficient. We report for the first time that knocking out LEDGF results in upregulation of antisense HIV-1 RNA transcription. Antisense HIV-1 transcripts are abundant in a higher proportion of cells when the Integrase-LEDGF interaction is lost, whether by knocking out LEDGF or by pharmacological inhibition. We also demonstrate that intriguingly, antisense RNA is rapidly lost from infected cells. Using a dual reporter virus to investigate the association between HIV-1 antisense RNA and latency, we have determined that antisense RNA has low expression in “latently” infected cells. Hepatitis B virus infection is a major health problem, there are more than 350 million people living with chronic HBV. Immune system can clear infection during the acute phase in immune-competent patients. A better understanding of the immune response against HBV infection may lead to new insights a find a cure. RNA helicases contribute to the immune response and some also have antiviral effects. MOV10 is a helicase RNA protein that has detrimental effect in HIV infection and HCV replication under MOV10 depletion or overexpression. Considering that HBV replication rely on reverse transcription to form DNA from pre-genomic RNA, we study the effect of MOV10 in HBV replication. Our data suggest that while MOV10 overexpression leads to HBV reduction, suppression of MOV10 enhances HBV replication.Includes bibliographical reference

    Mechanism of Alphavirus Restriction by the Interferon-Induced Exonuclease, ISG20

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    Type I interferon-stimulated genes (ISGs) have critical roles in inhibiting virus replication and dissemination. Despite advances in understanding of the molecular basis of restriction by ISGs, the antiviral mechanisms of many remain unclear. The 20 kDa ISG, ISG20, is a nuclear 3’-5’ exonuclease with preference for ssRNA, which has been implicated in the IFN-mediated restriction of several RNA viruses. While the exonuclease activity of ISG20 has been shown to degrade viral RNA in vitro, these findings have not been reconciled with proposed effects of ISG20 against RNA viruses that replicate in the cell cytoplasm. In the present study, we utilize a combination of an inducible, overexpression system for murine ISG20 and Isg20-/- mice to investigate mechanisms and consequences of ISG20-mediated restriction of alphaviruses. Overexpressed ISG20 primarily localized to Cajal bodies in the nucleus and potently restricted chikungunya virus and Venezuelan equine encephalitis virus replication by inhibiting the translation of infecting genomic RNA. However, degradation of viral RNAs was not observed. Translation inhibition was associated with an ISG20-induced upregulation of over 100 other gene products, many of which possess known antiviral activity. ISG20-responsive gene upregulation correlated with IRF3 activity among other transcription factors. Importantly, ISG20 modulated the production of IFIT1, an ISG that suppresses translation of RNAs possessing the type-0 5’ cap structure such as the alphavirus genome. Consistent with this, the replication and virulence of IFIT1-sensitive alphaviruses was significantly increased in Isg20-/- compared to congenic wild-type mice. Our findings establish an indirect role for ISG20 in the early restriction of RNA virus replication by altering regulation of other ISGs that inhibit virus translation and possibly other viral activities in the replication cycle

    Genotypic and phenotypic variation in the human immunodeficiency viruses.

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    Despite the involvement of multiple genetic variants of HIV in the causation of disease worldwide, most research has focussed on subtype B, the prevalent subtype in the western world. As a consequence, it is currently not clear whether genetically distinct HIV strains have different biological properties that cause differences in in vivo transmission, disease progression, replication capacity or sensitivity to antiretroviral drugs. In order to increase the ease of classification of viral diversity and thus aid studies into its importance, a novel genotyping tool for classifying HIV-1 subtype based on pol sequence, produced routinely during drug resistance monitoring, has been developed in this thesis. A dataset of 187 full-length HIV-1 sequences was used to generate Gag, Pol, Protease-Reverse Transcriptase (PR-RT) and Env protein sequence alignments. Phylogenetic analyses enabled generation of subtype specific alignments and, whilst sequence variation in the PR-RT dataset was low, this variation was adequate for PR-RT subtype assignment. The subtyping tool, named STAR, utilises position specific scoring matrices (PSSMs) derived from these subtype specific multiple sequence alignments and results in highly accurate reclassification of the subtype alignment sequences, with 98.6% of sequences being accurately assigned a subtype. Subsequent to the development of STAR the importance of HIV genetic variation classified as subtype, was addressed. A comparison of the relative growth capacity of HIV-1 primary isolates of subtypes A, B, C, D, F, group O and HIV-2 was performed in two T-cell environments. A novel reporter cell line was developed specifically to facilitate this work. Clear and consistent differences in in vitro growth phenotype in terms of rate and cytopathogenicity were detected, indicative of intrinsic differences between the HIV-1 types and subtypes. This work was extended by the utilisation of microarray technology which offers the possibility to analyse, at any given time point, the transcriptome of a virus-infected cell. A comparison of the transcriptional responses within T-cells to infection with HIV-1 subtype B, Group O and HIV-2 enabled identification of both core and diverging transcriptional response programs. Whilst the core response program provides insight into the most essential interactions between virus and host during HIV infection of T-cells, analysis of the diverging responses provide evidence that genetically divergent strains of HIV may interact differently with the host. It is proposed that these differences may have the potential to influence disease outcome

    the role of lipids in the pathogenesis of neuroHIV

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    A critical comorbidity of HIV infection is HIV-associated neurocognitive disorders (HAND). Although combined antiretroviral therapy (cART) is an effective treatment in blocking systemic viral replication, it is unsuccessful in reducing the incidence of HAND. HIV CNS damage, in the current cART era, can be associated to the presence of latently HIV-infected cells including microglia/macrophages and a small population of astrocytes. This Ph.D. thesis focuses on identifying, localizing, and quantifying viral reservoirs using an improved staining and microscopy technique. Although in low amount, our data confirmed that microglia/macrophages and a small population of astrocytes are still infected. These cells synthetize and secrete viral proteins generating a bystander damage in the CNS. Viral proteins are also involved in lipid dysregulation. We demonstrated by Mass Spectrometry Imaging (MSI) that in the brain of HIV-infected individuals with HAND lipids including sulfatide play a key role in bystander damage. Sulfatide is dysregulated in several neurocognitive diseases such as Alzheimer’s disease and Parkinson’s disease. Thus, we propose that sulfatide as a potential biomarker of neurocognitive disorders. We demonstrated that sulfatide secretion can be regulated by HIV proteins and we evaluated sulfatide effects in vitro, focusing on cell-to-cell communication and mitochondrial metabolism, all parameters altered in NeuroHIV. Therefore, this thesis provides specific tools and unique data to a better understanding the neuropathogenesis of HAND in the current cART-era and may lead to the identification of new molecular targets for preventing or curing HIV neurological decline

    Pathophysiology of Arbroviral Encephalitides in Laboratory Rodents

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    Western equine encephalitis virus (WEEV) is an arboviral pathogen naturally found in North America. The primary disease phenotype associated with WEEV infection in susceptible hosts is a relatively long prodromal period followed by viral encephalitis. By contrast, in the current work, experimental inoculation of WEEV into the peritoneum of Syrian golden hamsters produced rapid death within approximately 96 h. It was determined that direct virus killing of lymphoid cells leads to death in WEEV-infected Syrian golden hamsters, and that inflammatory cytokines have the potential to enhance virus-induced lymphoid cell destruction. It was further concluded that WEEV retains its ability to cause encephalitis in Syrian golden hamsters, if hamsters survive the early stages of virus infection or if virus is introduced directly into the CNS. Death in WEEV-infected hamsters is associated with lymphonecrotic lesions in the absence of pathological lesions in the central nervous system (CNS). Few clinical parameters were altered by WEEV infection, with the exception of circulating lymphocyte numbers. Circulating lymphocyte numbers decreased dramatically during WEEV infection, and lymphopenia was identified as a consistent indicator of eventual death. Virus infection also increased serum concentrations of the cytokines interferon and tumor necrosis factor-alpha (TNF-alpha). Hamster peritoneal macrophages exposed to WEEV expressed TNF-alpha in a dose-responsive manner. Macrophage expression of TNF-alpha could be significantly inhibited by treatment of cells with anti-inflammatory agents flunixin meglumine (FM) or dexamethasone (Dex). Anti-inflammatory treatment also protected macrophages from cytotoxicity associated with exposure to WEEV. Treatment of WEEV-infected hamsters with either FM or Dex significantly improved survival compared to placebo-treated controls. WEEV induced cytotoxicity in hamster splenocytes exposed to WEEV in a virus dose-responsive manner. Supernatant from WEEV-exposed macrophages significantly enhanced WEEV killing of splenocytes. Hamsters that survived the early stages of WEEV infection occasionally developed signs of neurological disease and died approximately 6 to 9 d after virus inoculation. These animals had histopathological lesions in the CNS consistent with alphavirus-induced encephalitis. Inoculation of WEEV directly into the CNS caused apparent encephalitic disease. Death following CNS inoculation of WEEV was rapid and concurrent with histopathological lesions in the CNS similar to lesions seen in encephalitic hamsters following peripheral inoculation
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