Application of built-in adjuvants for epitope-based vaccines

Several studies have shown that epitope vaccines exhibit substantial advantages over conventional vaccines. However, epitope vaccines are associated with limited immunity, which can be overcome by conjugating antigenic epitopes with built-in adjuvants (e.g., some carrier proteins or new biomaterials) with special properties, including immunologic specificity, good biosecurity and biocompatibility, and the ability to vastly improve the immune response of epitope vaccines. When designing epitope vaccines, the following types of built-in adjuvants are typically considered: (1) pattern recognition receptor ligands (i.e., toll-like receptors); (2) virus-like particle carrier platforms; (3) bacterial toxin proteins; and (4) novel potential delivery systems (e.g., self-assembled peptide nanoparticles, lipid core peptides, and polymeric or inorganic nanoparticles). This review primarily discusses the current and prospective applications of these built-in adjuvants (i.e., biological carriers) to provide some references for the future design of epitope-based vaccines

Vaccines for COVID-19

Since the emergence of COVID-19, caused by the SARS-CoV-2 virus, at the end of 2019 there has been an explosion of vaccine development. By the 1st September 2020, a staggering number of vaccines (over 200) had started pre-clinical development of which 39 had entered clinical trials, including some approaches that have not previously been licensed for human vaccines. Vaccines have been widely considered as part of the exit strategy to enable the return to previous patterns of working, schooling and socialising. Importantly, to effectively control the COVID-19 pandemic, production needs to be scaled up from a small number of pre-clinical doses to enough filled vials to immunise the world's population, which requires close engagement with manufacturers and regulators. It will require a global effort to control the virus, necessitating equitable access for all countries to effective vaccines. This review explores the immune responses required to protect against SARS-CoV-2 and the potential for vaccine-induced immunopathology. It describes the profile of the different platforms and the advantages and disadvantages of each approach. The review also addresses the critical steps between promising pre-clinical leads and manufacturing at scale. The issues faced during this pandemic and the platforms being developed to address it will be invaluable for future outbreak control. Nine months after the outbreak began, we are at a point where pre-clinical and early clinical data is being generated for the vaccines, an overview of this important area will help our understanding of the next phases

The cyanobacterial rhomboid protease is a regulator of the CCM

Cyanobacteria are aquatic photosynthetic bacteria and useful models for study of the chloroplast and photosynthesis. We are studying a ‘rhomboid’ membrane-located proteases in Synechocystis sp. PCC 6803, which appears to function as a previously undiscovered regulator of the carbon concentrating mechanism (CCM) of this phototroph. Rhomboids are almost ubiquitous across evolution, and are known to activate diverse cellular processes via proteolysis of their specific, membrane-sequestered substrates. Although this well-conserved family has solved crystal structures of bacterial enzymes such as Escherichia coli GlpG, ironically, most work has been carried out on eukaryotic representatives. Following our study of the Arabidopsis thaliana chloroplast RBL10 protease, we identified cyanobacterial orthologues with the aim of discovering if roles might be conserved between these and organellar rhomboids. Molecular biology and reverse-genetics studies were made on slr1461, a mutant in the single rhomboid protease of Synechocystis. When photosynthetic parameters were investigated, it could be seen that inactivation of slr1461 did not affect nonphotochemical quenching, unlike the chloroplast rbl10 mutant, but Slr1461 was required for reduction of photosynthetic activity in mixotrophic conditions. This reduction allows cyanobacteria to avoid expending energy on the uptake of CO2 when an organic carbon source can be utilised: as might be expected, therefore, Slr1461 transcription was linked with downregulation of genes encoding proteins facilitating high-affinity CO2 import under high CO2 and mixotrophic conditions. Quantitative RT-PCR of CCM network genes suggested that Slr1461 is located upstream of known regulators, including another membrane protease, the Slr0228 FtsH, and a central, controlling transcription factor NdhR

Imaging polyphenolic therapeutic compounds in a eukaryotic model microbe

Flavonoids are polyphenolic metabolites that have a range of physiological and developmental functions in plants. They are the focus of much work as potential therapeutics, although investigation of specific mode of action remains a notably under-researched area. Monitoring transport and location of flavonoids in cells is difficult because, despite a role in UV-absorption in plants, they emit only low levels of fluorescence. Visualising them in plants is possible using the Naturstoff reagent (NA), reported historically to be a polyphenol-fluorescence-enhancing stain. We explored therefore whether this agent was effective during preclinical assessment of polyphenolic therapeutics in a microbial-model. The eukaryote Dictyostelium discoideum has been shown to be a useful model when identifying novel drug targets for treating various diseases. For example, in the case of polycystic kidney disease, naringenin decreased Dictyostelium cell division whereas a polycystin-2-null Dictyostelium line was resistant to the flavonoid, and, subsequently, naringenin treatment proved to reduce cyst-formation in mammalian-kidney model cell lines1. To monitor transport and site of action of the drugs investigated in such studies, we developed a method using NA-staining in this model organism. A range of polyphenolics were assayed in cells, cell-extracts and the cell-washes, and NA-enhanced imaging was evaluated in parallel with LCMS-quantification. NA-enhanced fluorescence of compounds at therapeutically relevant concentrations proved an effective and qualitative measure of transport and localisation in Dictyostelium, and could be used in concert with localisation dyes. Fluorescence-enhancement is limited to a subset of flavonoids, however, and not more widely applicable in our studies to date

Promiscuous Coxiella burnetii CD4 Epitope Clusters Associated With Human Recall Responses Are Candidates for a Novel T-Cell Targeted Multi-Epitope Q Fever Vaccine

Coxiella burnetii, the causative agent of Q fever, is a Gram-negative intracellular bacterium transmitted via aerosol. Regulatory approval of the Australian whole-cell vaccine Q-VAX® in the US and Europe is hindered by reactogenicity in previously exposed individuals. The aim of this study was to identify and rationally select C. burnetii epitopes for design of a safe, effective, and less reactogenic T-cell targeted human Q fever vaccine. Immunoinformatic methods were used to predict 65 HLA class I epitopes and 50 promiscuous HLA class II C. burnetii epitope clusters, which are conserved across strains of C. burnetii. HLA binding assays confirmed 89% of class I and 75% of class II predictions, and 11 HLA class II epitopes elicited IFNγ responses following heterologous DNA/DNA/peptide/peptide prime-boost immunizations of HLA-DR3 transgenic mice. Human immune responses to the predicted epitopes were characterized in individuals naturally exposed to C. burnetii during the 2007–2010 Dutch Q fever outbreak. Subjects were divided into three groups: controls with no immunological evidence of previous infection and individuals with responses to heat-killed C. burnetii in a whole blood IFNγ release assay (IGRA) who remained asymptomatic or who experienced clinical Q fever during the outbreak. Recall responses to C. burnetii epitopes were assessed by cultured IFNγ ELISpot. While HLA class I epitope responses were sparse in this cohort, we identified 21 HLA class II epitopes that recalled T-cell IFNγ responses in 10–28% of IGRA+ subjects. IGRA+ individuals with past asymptomatic and symptomatic C. burnetii infection showed a comparable response pattern and cumulative peptide response which correlated with IGRA responses. None of the peptides elicited reactogenicity in a C. burnetii exposure-primed guinea pig model. These data demonstrate that a substantial proportion of immunoinformatically identified HLA class II epitopes show long-lived immunoreactivity in naturally infected individuals, making them desirable candidates for a novel human multi-epitope Q fever vaccine

Tools for Studying Human Enteroviruses and the Serological Immune Responses They Elicit

Enterovirukset ovat joukko vaipattomia yksijuosteisia RNA-viruksia, jotka aiheuttavat sekä lieviä että vakavia sairauksia. Poliovirukset ovat tunnetuimpia ihmisten enteroviruksia, mutta onnistuneiden rokotekampanjoiden ansiosta, uudet tapaukset ovat harvinaisia. Enterovirukset tuottavat vuositasolla merkittäviä kuluja yhteiskunnallisesti, sillä ne aiheuttavat flunssaa ja pahentavat hengitystieoireita astmaatikoilla. Enterovirusinfektiot on myös liitetty tyypin 1 diabetekseen ja muihin autoimmuunitauteihin. Enterovirusten ja kroonisten tautien yhteyksien tutkiminen on kuitenkin haastavaa ja uusia immunologisia ja serologisia työkaluja tarvitaan. Tämän tutkielman osana tuotettiin rekombinanttisia proteiineja ja vasta-aineita, joita voidaan käyttää enterovirusten ja niiden aiheuttamien tautien tutkimisessa. Rotan hybridoomasoluissa tuotettava 3A6 anti-enterovirus vasta-aine on osoittautunut laajaa enteroviruskirjoa tunnistavaksi yleistyökaluksi viruksen suoraa osoitusta varten. Työn tuloksena syntyneet VP1 proteiinit vuorostaan ovat osoittautuneet toimiviksi koettimiksi enterovirusvasta-aineille serologisissa näytteissä aiemmin sairastettujen enterovirusinfektioiden tutkimisessa. Tuotettuja antigeeneja käytettiin vasta-ainevasteiden tutkimiseen hiirten ja eri ikäisten ihmisten seeruminäytteistä. Löydösten perusteella vasta-ainevasteet ei- rakenteellisille 2A ja 3C proteaaseille ovat aikuisissa hyviä osoittajia akuutille enterovirusinfektiolle. Havaitsimme myös, että vasta-ainevasteet VP1-proteiineille ovat vähemmän ristiinreagoivia lapsissa, kuin aikuisissa, mutta niiden käyttö enterovirusten serodiagnostiikassa alle puolenvuoden ikäisille lapsille ei ole järkevää korkeiden äidin vasta-ainetasojen vuoksi. Antigeenien toimivuutta enteorvirusvasta- aineiden havaitsemiseen testattiin suurempiin kohortteihin skaalautuvassa multiplex- asetelmassa. Tämä on tärkeää, sillä taudinaiheuttajien ja (kroonisten) tautien yhteyksien tutkimiseen tarvitaan usein suuria näytemääriä riittävän statistisen voiman saavuttamiseksi.Enteroviruses are a group of non-enveloped single stranded-stranded positive RNA viruses that cause illnesses both mild and severe. Polioviruses are the most well- known and studied human enteroviruses, but due to successful vaccination campaigns new cases are exceedingly rare. Enteroviruses have significant societal costs yearly as they cause common cold and asthma exacerbations. Enteroviruses have also been linked to type 1 diabetes and other autoimmune diseases. Studying the link between enteroviruses and chronic diseases is challenging. Thus, new immunological and serological tools are needed. Recombinant proteins and monoclonal antibodies for studying enteroviruses and associated diseases were produced during this Ph.D project. The 3A6 anti- enterovirus monoclonal antibody produced in rat hybridoma cells has proven to be a good tool for directly detecting a broad range of enteroviruses. The recombinant VP1-proteins on the other hand have proven effective tools in detecting antibodies against enteroviruses to show prior enterovirus infections from serological samples. Antibody responses to enteroviruses were studied in serum samples from both mice and human subjects of different ages. Antibody responses to non-structural proteins 2A and 3C were found to be good indicators for an acute enterovirus infection, especially in adults. Cross-reactivities towards VP1 proteins from different enterovirus species were found to be less extensive in young children than adults, but the waning levels of maternal antibodies were determined to be a confounding factor in studying sera from children under 6 months of age, limiting the usefulness of serological assays for that age group. The performance of the antigens was tested in a multiplex setting that can be adapted to testing larger cohorts for antibodies against enteroviruses. This is crucial as high number of samples is often required for sufficient statistical power to show linkage between pathogens and (chronic) diseases

Measles virus as vaccine platform against highly pathogenic emerging viruses

Highly pathogenic viruses are a significant global danger since they can be spread by worldwide travel and trade almost without restriction. One particular threat comes from emerging infections, for which no adequate treatment options currently exist. To guard against local or global outbreaks of these viruses, the development of protective vaccines at an early stage is therefore a desirable form of intervention. Vector-based vaccine platforms, such as that of replication-competent recombinant measles virus (rMV), constitute good prospective vaccine candidates, since they have the potential to allow for an easy exchange of antigen-encoding genes, thereby enabling rapid vaccine production after standardisation. To assess their suitability as a potential vaccine platform against highly infectious viral pathogens, rMVs were generated as part of the practical element of this thesis. These encoded for antigens of the following emerging pathogens: Middle East respiratory syndrome coronavirus (MERS-CoV), influenza virus H7N9 or Crimean-Congo haemorrhagic fever virus (CCHFV). Insertions of antigen-encoding genes resulted in the detectable expression of the MERS-CoV spike glycoprotein in both membrane-bound (MERS-S) and soluble form (MERS-solS), the MERS-CoV nucleocapsidprotein (MERS-N), haemagluttinin or neuraminidase of H7N9 (H7 or N9), the CCHFV glycoprotein Gc (CCHFV-Gc); and the CCHFV-nucleocapsid protein (CCHFV-N), in cells infected with respective vaccines. Immunisation of MV susceptible mice with MERS-S-, MERS-solS-, H7-, or N9-encoding vaccines also resulted in the induction of humoral immune responses. These included virus-neutralising antibodies (nAbs), if mice were vaccinated with MV-MERS-S, MV-MERS-solS or MV-H7. Generation of syngeneic for the respective antigens' transgenic dendritic cell (DC) cell lines, moreover, enabled an efficient re-stimulation of antigen-specific T cells without knowledge of immunogenic epitopes or the availability of antigens as proteins. When using these transgenic DC cell lines, MV-MERS-S-, MV-MERS-solS-, MV-MERS-N-, and MV-H7-induced cellular immune responses were demonstrated in an IFN-γ-ELISpot. Moreover, MERS-S specific CD8+T cells of immunised mice responded to respective re-stimulation by MERS-S-dependent proliferation and MERS-S-specific cytotoxicity. A reduction of viral loads, as well as virus-induced inflammation of lung tissue, was observed in MV-MERS-S- or MV-MERS-solS-vaccinated mice within a MERS-CoV challenge model. This impressively demonstrated the protective efficacy of an MV-based vaccine against MERS-CoV. In the second part of this thesis, MERS-CoV-induced innate immune responses in human and murine antigen-presenting cells (APCs) were analysed. As a result, human plasmoid DCs (pDCs) were identified as a source of significant amounts of antiviral type I (IFN-α, IFN-β) and Typ III (IFN-λ) interferons (IFNs), which were secreted upon infection with MERS-CoV. As a so far exclusively-identified source of type I and III IFNs pDC might hence play a significant role in MERS-CoV-induced pathogenesis in humans. Thus, by using MERS-CoV as an example, this thesis identified several key interactions between an emerging pathogen and defined immune cells, which might prove to be of clinical significance, particularly in the future development of antiviral drugs. As potential vaccine candidate, an MV-based vaccine platform was generated as part of this thesis; and its protection efficacy was demonstrated. A rapidly conducted production of MV-based vaccine platforms against three different viral pathogens, an efficient induction of humoral and cellular immunity as well as protection efficacy in a challenge model indicated the potential of recombinant MV to be used as an effective vaccine platform to protect against emerging viral pathogens