One of the major challenges of vaccine development is the conservation of immunogenicity and protective efficacy through the stages of design, production, formulation and delivery. The critical issue is that how and in what form an antigen is taken up by antigen presenting cells for proteolytic processing and presentation to the immune system bound to MHC can have dramatic effects on the activation of Th cells to drive clonal responses and induction of immunological memory. Nontypeable Haemophilus influenzae (NTHi) is a pathogenic commensal of the human respiratory tract that causes diseases with enormous socioeoconomic burdens. There is no licensed vaccine, although the potential for vaccination with outer membrane components to reduce the incidence of disease caused by NTHi has recently been demonstrated in clinical trials. The issue of immunomodulation was explored in this thesis in the context of the further evaluation of a leading NTHi vaccine candidate, the outer membrane protein OMP26. The efficacy of recombinant OMP26 (rOMP26) against NTHi challenge has been previously demonstrated in mice, rats and chinchillas. In rats, efficacy was shown to be restricted to the precursor form (containing the signal peptide) and not the mature form of rOMP26. The immunodulatory effects of changes to the rOMP26 structure were further investigated in this thesis. A range of structural variants of rOMP26 were constructed in view of reducing extraneous plasmid-derived sequence from the antigen and to introduce a unique cysteine residue as a potential conjugate site for multivalent vaccine development (Chapter 2). It was demonstrated that minor structural changes to rOMP26 such as the addition, deletion, modification or relative positioning of a single amino acid or bulky group, designed to increase the efficiency of production or introduce (cysteine) conjugation sites, altered the expression of the protein in E. coli and the immunogenicity in Balb/C mice. Furthermore, in contradiction to the published report (El-Adhami et al. 1999) and a new study in rats (Chapter 3), there was no positive effect of the signal peptide in mice, with precursor and mature forms of rOMP26 equally immunogenic (Chapter 2). Following confirmation of the need to retain the signal peptide for the immunogenicity of rOMP26 in rats, a precursor form (rOMP26VTAL) in which the conserved n-region of the signal peptide was deleted, and shown to reduce the efficiency of the cleavage of the signal peptide by signal peptidase during protein overexpression in E. coli (Chapter 3). Not only did this deletion result in an increase the yield and stability of the purified precursor protein, but rOMP26VTAL was highly immunogenic and enhanced the clearance of NTHi from the lungs of challenged rats. The potential for signal peptides to be exploited as an immune-enhancing moiety in a proteinaceous vaccine is discussed. Following the development of rOMP26VTAL as a production optimised variant of rOMP26, the next step was to test the feasibility of rOMP26VTAL as a component of a multivalent vaccine (Chapter 4). Two chimeras were constructed with LB1(f)2,1,3, a trivalent synthetic B-cell epitope from the extracellular loop 3 region of the P5 fimbrin protein of NTHi, positioned at the N- or C-terminus of rOMP26VTAL. The solubility of rOMP26VTAL was affected by the fusion, with both chimera constructs expressed only in the insoluble fraction, thus requiring a denaturing protocol for purification. Although rLB1(f)2,1,3-OMP26VTAL was expressed and purified as a more stable protein and in greater yield than rOMP26VTAL-LB1(f)2,1,3, the relative positioning of the fusion was important and rOMP26VTAL-LB1(f)2,1,3 was significantly more immunogenic in rats than rLB1(f)2,1,3-OMP26VTAL. In addition, rOMP26VTALLB1( f)2,1,3, but not rLB1(f)2,1,3-OMP26VTAL induced a significant degree of bacterial clearance following pulmonary challenge with NTHi, in levels comparable to the highly efficacious rOMP26VTAL construct. In the third part of the thesis, bacterial ghosts were evaluated as a novel mucosal delivery technology for rOMP26VTAL and rOMP26VTAL-LB1(f)2,1,3, (Chapter 5). To mimic the natural presentation of OMP26 and P5 fimbrin antigens on the cell surface of NTHi, an OmpA� sandwich fusion surface display system was developed for the outer membrane expression of the OMP26 constructs in E. coli ghosts. Following gut immunisation, but not intranasal immunisation even when co-administered with the cholera toxin�derived adjuvant CTA1-DD, bacterial ghosts were successful at presenting OMP26VTAL and rOMP26VTAL-LB1(f)2,1,3 to the immune system for the induction of enhanced clearance of NTHi in the rat pulmonary challenge model. Although this study was the first to demonstrate enhanced bacterial clearance induced by heterologous antigens expressed in the outer membrane of bacterial ghosts, future studies with ghosts will require optimisation of the expression levels of the OmpA� fusion proteins possibly to avoid cross-reactive responses related to high doses of ghosts in the inoculum. This thesis presents data that both supports the further evaluation of rOMP26 constructs for clinical trials, and has demonstrated the significant effects of structural changes, method of production and delivery system can have on the immunogenicity of a candidate vaccine. Such knowledge will contribute to and provide some new approaches for enhancing the efficiency of vaccine development against a range of diseases including those caused by NTHi. Major Outcomes: 1. Demonstration that the immunogenicity of rOMP26 antigen constructs is affected by structural modifications and their positioning within the construct, and by the delivery system. 2. Development of rOMP26VTAL, an rOMP26 construct with the KNIAK sequence deletion of the signal peptide n-region. This protein retains the immunogenicity and protective efficacy of rOMP26, but is produced with reduced cleavage of the signal peptide, resulting in higher yields and a stable protein. Lacks extraneous plasmidderived multiple cloning site sequence, and is produced in high yield as a stable protein. 3. Construction of a NTHi rOMP26VTAL-LB1(f)2,1,3 chimera antigen that induced enhanced clearance of NTHi in an acute pulmonary challenge model in rats. 4. Development of an OmpA� surface display system for the expression of rOMP26 antigen constructs in the outer membrane of E. coli/bacterial ghosts 5. Bacterial ghosts were successful as delivery vehicles for rOMP26 candidate vaccine constructs when delivered in the gut.

Hepatitis C virus (HCV) has chronically infected an estimated 170million people worldwide. There are many impediments to the development of an effective vaccine for HCV infection. Dendritic cells (DC) remain the most important antigen-presenting cells for host immune responses, and are capable of either inducing productive immunity or maintaining the state of tolerance to self and non-self antigens. Researchers have recently explored the mechanisms by which DC function is regulated during HCV infection, leading to impaired antiviral T-cell responses and so to persistent viral infection. Recently, DC-based vaccines against HCV have been developed. This review summarizes the current understanding of DC function during HCV infection and explores the prospects of DC-based HCV vaccine. In particular, it describes the biology of DC, the phenotype of DC in HCV-infected patients, the effect of HCV on DC development and function, the studies on new DC-based vaccines against HCV infection, and strategies to improve the efficacy of DC-based vaccines.

Malaria is a leading cause of death in some developing countries. The malaria parasite has been around for over a century, and has coevolved with humans. Coming up with an effective vaccine for P. falciparum will save millions of lives and reduce the morbidity and mortality of malaria globally. Understanding the role of exported parasite proteins i.e PfEMP1 a virulence factor and major cause of malarial pathogenesis, has been of great interest to vaccine researchers in the last decade. The focus of this review is to provide a literature review on PfEMP1s, their interaction with the human immune system, and their role in helping P. falciparum parasite to evade the immune system. This review will primarily focus on the intra-erythrocytic stage, which is the stage that results in the symptoms of malaria. A review is necessary to understand the antigenic variation of PfEMP1s, and how PfEMP1s challenge the different arms of the immune response, both the innate and adaptive. This review is unique in touching on the major parts of the immune system's interaction with the PfEMP1 antigen. Furthermore, the review explores the discussion of future research and therapeutic opportunities based on our knowledge of PfEMP1 antigens.

Please read the abstract in the 00front part of this document / Dissertation (MSc Agric (Microbiology))--University of Pretoria, 2007. / Microbiology and Plant Pathology / unrestricted

Porcine epidemic diarrhea Virus (PEDV) is a virus effecting swine. It is the cause of disease that manifests with symptoms ranging from depression, to severe dehydration and death. Young piglets are particularly susceptible to the virus, which can reach mortality rates of 100%. Presence of the virus on a swine farm can therefore cause severe economic losses. Treatments currently exist for PEDV, but are mostly generated from the virus itself. There has recently been renewed interest in a vaccine that is made from a different source, which might help eliminate some of the side effects of those that currently exist on the market. This project outlines three experiments performed in animals. During the first experiment, a structural protein from the Hepatitis B virus was genetically altered to include important structural portions of PEDV. This new protein is generated in E. coli and purified. After purification, the protein assembles into a virus-like particle (VLP). VLPs are structural proteins of existing viruses that are expressed and assembled to mimic the virus. By doing so, the immune system recognizes the protein as a potential threat, and launches a response in the form of antibodies. Manipulations of the VLPs as describe herein allow the new vaccine to generate antibodies toward other diseases such as PEDV. Although all five of the vaccines used in the first experiment were able to generate appropriate antibodies, only two of them were effective at preventing PEDV from entering susceptible cells (virus neutralization). A second experiment, with three newly designed vaccines was therefore performed. This experiment, like the first, was successful in producing antibodies to several of the included PEDV protein sections, but none were able to neutralize the virus. These results led to a third experiment, during which further design improvements were made to the basic vaccine structure in an attempt to increase the neutralization capabilities of the vaccines. The results from the third experiment indicated that several changes to the vaccine increased the immune response to the structural portions of PEDV, providing a better overall vaccine candidate. This also led to the conclusion that one specific sequence from PEDV has a better ability to neutralize the virus than the other sections. / PHD

Viruses in the Flavivirus genus (e.g., Zika, yellow fever, dengue, West Nile, and Japanese encephalitis viruses) are arthropod-borne, globally distributed, and can cause a range of neurological or hemorrhagic diseases. The ongoing epidemics of flaviviral disease consistently demonstrate the need for new vaccines capable of outbreak control. However, safe, effective, and easy-to-produce vaccines remain relatively elusive due to limitations of conventional vaccine development that make it difficult to balance safety and efficacy. Insect-specific flaviviruses (ISFVs) are emerging as a novel method to overcome this challenge. Herein, we develop a new flavivirus vaccine platform based on the novel insect-specific flavivirus called Aripo virus, which we used to create a preclinical Zika virus (ZIKV) vaccine named Aripo/Zika virus (ARPV/ZIKV). ARPV/ZIKV is a live recombinant virus consisting of the ZIKV pre-membrane and envelope protein genes expressed on an Aripo virus backbone. In this work, we quantify the safety and efficacy of ARPV/ZIKV in multiple murine models, and begin to elucidate the mechanisms of humoral and cell-mediated immune induction for this new platform. Overall, the vaccine showed no evidence of pathogenicity in immunocompromised or suckling mice, and demonstrated a complete inability to replicate in various vertebrate cell lines. Despite this lack of replication, a single dose of live, unadjuvanted ARPV/ZIKV completely prevented ZIKV disease in mice and prevented in utero ZIKV transmission in gravid mice. Direct protection post-ZIKV challenge appears to be primarily mediated by neutralizing antibodies based on passive transfer, adoptive transfer, and T-cell depletion studies. However, vaccination studies in Rag1 KO, Tcra KO, and muMt- mice demonstrate the critical role of T-cell responses in developing immunity post-vaccination. In summary, ARPV/ZIKV is a promising vaccine candidate that induces robust adaptive immune responses, and this success is a positive indication of ARPV's potential as a new resource for flavivirus vaccine development. This body of work contributes to the rapidly expanding field of insect-specific virus-based vaccines and generates new insights into their optimization. Ultimately, this work may help protect the health of millions of people worldwide that are currently at risk of flavivirus infection. / MPH / Arthropod-borne viruses (especially flaviviruses such as Zika virus (ZIKV), yellow fever virus, West Nile virus) represent a major global health threat and a significant burden on human life. Vaccination is a critical tool for controlling the often unpredictable outbreaks of flavivirus diseases. However, licensed flavivirus vaccines remain relatively elusive. This is, in part, because the same characteristics of traditional live-attenuated vaccines that make them highly effective can also reduce their safety. Insect-specific flaviviruses (ISFVs) are emerging as a novel method to overcome this challenge. ISFVs only replicate in insects and thus are safe in humans. They do not cause disease in vertebrates, eliminating the need for the chemical or physical inactivation methods required by traditional whole inactivated vaccines and which can result in reduced efficacy. Herein, we develop a new flavivirus vaccine platform based on a novel ISFV called Aripo virus (ARPV). As proof of concept, we used ARPV to create a preclinical ZIKV vaccine named Aripo/Zika virus (ARPV/ZIKV). ARPV/ZIKV expresses immune system-stimulating ZIKV structural proteins on its virus particle. However, it remains highly safe because the genetic material from ARPV makes it incompatible for replication in human cells. Here, we demonstrate the safety and protective ability of ARPV/ZIKV, and begin to elucidate its mechanisms of protection. Overall, ARPV/ZIKV shows promise as a ZIKV vaccine candidate, which supports the potential of ARPV as a platform for new flavivirus vaccines and the potential to protect the health of the millions of people currently at risk of flavivirus infection.

Yes / Blood clotting was reported in April 2020 [1] as another serious symptom due to COVID-19, but also came other reports such as young adults dying due to strokes and heart attacks [2]. The currently known head-to-toe symptoms of COVID-19, seem to indicate vascular as well as respiratory diseases and that 40% of related death are due to cardiovascular complications [2]. In a recently published journal paper in Lancet [3], the authors found that SARS-CoV-2 virus can infect the endothelial cells that line the inside of blood vessels noting that endothelial cells protect the cardiovascular system and release proteins that influence everything from blood clotting to the immune response. In the same paper, the authors showed damage to endothelial cells in the lungs, heart, kidneys, liver, and intestines in patients with Covid-19. Therefore, the emerging belief is that the novel coronavirus is a respiratory illness to begin with, but as it spreads further into blood vessels it becomes a vascular illness that is capable of killing patients via vascular system.

Staphylococcus aureus remains an important pathogen, which, due to its capability to develop antimicrobial resistance, imposes an increasing threat to human health. Developing preventive means to decrease disease burden is a major aim. However, the development of an S. aureus vaccine, which would be one strategy to achieve such goals, has been complicated through limited understanding of the bacterium's pathogenic mechanisms. This work uses four approaches to address these limitations: Firstly, a reproducible RNA sequencing based method for the determination of gene transcription by S. aureus in vivo during mammalian infection. Secondly, examination of the impact of the bacterial transcription regulator 'Rsp' on the bacterium, which shows that mutations in this gene have profound functional and transcriptional impacts. Thirdly, by examining the in vivo transcription of multiple S. aureus strains during infection, proposing a 'core in vivo transcriptome' of induced genes under the conditions tested. Some of these genes are known to be involved in pathogenesis, others are not completely characterised and may represent suitable vaccine antigens. Finally, this work addresses limited understanding of S. aureus pathogenesis through defining transcriptional changes in vivo, which are induced by an altered immune response in immunised hosts. Together, this body of work contributes to the understanding of S. aureus pathogenesis and provides candidate antigens for future vaccine development.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Ying Fang / Porcine reproductive and respiratory syndrome virus (PRRSV) employs multiple functions to modulate host’s innate immune response, and several viral nonstructural proteins (nsps) are major players. In this dissertation, the research was mainly focused on identification and functional dissection of ORF1a-encoded nsps. PRRSV replicase polyproteins encoded by ORF1a region are predicted to be processed into at least ten nonstructural proteins. In chapter 2, these predictions were verified by using a panel of newly established antibodies specific to ORF1a-encoded nsps. Most predicted nsps (nsp1β, nsp2, nsp4, nsp7α, nsp7β and nsp8) were identified, and observed to be co-localized with de novo-synthesized viral RNA in the perinuclear region of the cell. Among all PRRSV proteins screened, nsp1β is the strongest type I interferon antagonist. In chapter 3, mutagenesis analysis of nsp1β was performed to knock down nsp1β’s IFN antagonist function. A highly conserved motif, GKYLQRRLQ, was determined to be critical for nsp1β’s ability to suppress IFN-β and reporter gene expression. Double mutations introduced in this motif, K130A/R134A (type 1 PRRSV) or K124A/R128A (type 2 PRRSV), improved PRRSV’s ability to stimulate the expression of IFN-α, IFN-β and ISG15. In addition to its critical roles involving in modulating host innate immune response, in the studies of Chapter 4, we demonstrated that PRRSV nsp1β functions as a transactivator to induce the -2/-1 ribosomal frameshifting in nsp2, which results in expression of two novel PRRSV proteins, nsp2TF and nsp2N. The conserved motif GKYLQRRLQ is also determined to be critical for the transactivation function of nsp1β. In chapter 5, the interferon antagonist, de-Ub and de-ISGylation activity of newly identified nsp2TF and nsp2N were evaluated. In vitro and in vivo characterization of three nsp2TF-deficient recombinant viruses indicated that all mutant viruses have improved ability to stimulate the innate immune response and provide improved protection in mutant virus-vaccinated animals. In summary, this study verified the previously predicted PRRSV pp1a processing products, further evaluated the function of nsp1β and nsp2-related proteins. These data obtained here will provide basic knowledge for future development of vaccines and control measurements.

Die Transplantation von porzinen Organen könnte eine Lösung des akuten Mangels von Allotransplantaten in der Transplantationsmedizin darstellen. Bevor die Xenotransplantation klinische Realität werden kann, sind jedoch zahlreiche Hürden zu überwinden. Insbesondere die mögliche Übertragung porziner endogener Retroviren (PERVs), die integraler Bestandteil des porzinen Genoms sind, stellt ein mikrobiologisches Risiko dar. PERVs können humane Zellen in vitro produktiv infizieren. Mögliche Strategien zur Abwehr von Xenosen sind die Verwendung von PERV-knockout Tieren oder die Entwicklung eines effektiven Impfstoffes, durch den der Transplantatrezipient vor einer möglichen Übertragung geschützt werden kann. Dazu wurden Antiseren gegen die Hauptstrukturproteine von PERV generiert. Es konnte gezeigt werden, dass in Ziegen und Ratten durch Immunisierung mit der rekombinant generiereten Ektodomäne des transmembranen Hüllprotein p15E neutralisierende Antikörper induziert werden konnten. Die Epitopkartierung der induzierten Antikörper zeigt eine Bindung an eine Domäne im N-terminalen, nahe des Fusionspeptids (E1, GPQQLEK) und eine Domäne im C-terminalen, membranproximalen (E2, FEGWFN) Bereich der p15E-Ektodomäne. Diese Sequenzen sind in allen PERVs identisch und innerhalb der Gammaretroviren hochkonserviert. Aus AIDS-Patienten isolierte neutralisierende Antikörper (mAb2F5: ELDKWA, mAb4E10: LWNWFN) binden ebenfalls an den C-terminalen Bereich der Ektodomäne des Transmembranproteins gp41. Der Bindungsmechannismus dieser Antikörper wurde in ELISA-Experimenten und in vitro-Inhibitionsassays analysiert. Die Ergebnisse legen die Bindung eines Konformationsepitopes nahe, das aus der E1 und der E2 Domäne gebildet wird. Die Aufklärung des Bindungsmechnismus breit neutralisierender Antikörper gegen Transmembranproteine von Retroviren könnte die Basis für neue Impfstoffansätze darstellen. / Porcine xenotransplants may offer a potential solution to the problem posed by the limited supply of allotransplants. However, xenotransplantation may be associated with the risk of transmission of microorganisms, in particular of porcine endogenous retroviruses (PERVs) that are an integral part of the porcine genome and able to infect human cells in vitro. Possible strategies to prevent virus transmission include the development of PERV knockout animals or of effective vaccines. When antisera prepared against the main structural proteins of PERV were screened, goat and rat antisera against the recombinant ectodomain of the transmembrane envelope protein p15E were found to neutralize PERV infectivity. Epitope mapping using overlapping peptides revealed two epitopes, one near the fusion peptide (E1, GPQQLEK) and the other near the transmembrane domain (E2, FEGWFN). These sequences are identical for all PERVs and are highly conserved among all gammaretroviruses. Interestingly, neutralizing antibodies isolated from AIDS patients that recognize regions partially homologous with E2 (mAb4E10, LWNWFN) or located in close proximity to E2 (mAb2F5, ELDKWA) are known to neutralize a broad range of HIV-1 strains. The binding mechanisms of these HIV neutralizing antibodies were analyzed in ELISA experiments and in vitro inhibition assays. The results indicate that the two most broadly reactive HIV-1 envelope gp41 human mAbs are specific for a discontinuous epitope composed of the E1 and the E2 domain. If so, these two transmembrane protein domains in different retroviruses act as effective targets for neutralizing antibodies and may provide the basis for effective antiretroviral vaccines.