Cross-reactive immune responses between enteroviruses and islet cell autoantigens

Ohjaaja Merja Roivainen/MIBO/ENV

Immune response against <i>Mycobacterium avium</i> subsp. <i>paratuberculosis</i>, Epstein-Barr virus, HERV-K and IRF5 in rheumatoid arthritis

Rheumatoid arthritis (RA) is an inflammatory disease characterized by synovitis, systemic inflammation, autoantibodies that causes joint damage, disability, decreased quality of life, and cardiovascular and other comorbidities. Its aetiology as well the exact etiopathogenetic mechanisms are not well clear so far. RA is triggered by an interplay between genes and environmental factors. Several studies showed that microorganisms play an important role in triggering autoimmunity through different mechanisms of action. Viral and bacterial infections, such as those caused by Epstein-Barr virus (EBV), Human Endogenous Retrovirus (HERVs) and mycobacteria, may play a pathogenetic role in RA through immunological cross-reactivity or molecular mimicry. Sardinians have a peculiar genetic background resulting from a long lasting geographical isolation with a strong incidence and prevalence of different autoimmune disease such as RA, multiple sclerosis (MS) and diabetes. During this PhD course, I have studied the role of EBV, HERV-K and Mycobacterium avium subsp. paratuberculosis (MAP) in RA pathogenesis and other rheumatic diseases for a better understanding of how these infectious agents can lead to a deregulation of transcription factors such as Interferon Regulatory Factor 5 (IRF5) that is important in the regulation of different cells type like macrophages and neutrophils. Finally, in order to better understand the etiopathogenesis of RA, an animal model has been used to study the molecular mechanisms involved in the above-mentioned diseases and to better understand the link between the environment and genes in RA with an objective to develop new therapeutic strategies

Development of Novel Zika and Anthrax Viral Nanoparticle Vaccines

Vaccines protect against numerous infectious diseases and prevent millions of deaths annually, but there are still many infectious diseases for which no licensed vaccine exists. Developing a new vaccine requires balancing safety and efficacy, and viral nanoparticle (VNP) vaccines possess both of these characteristics. The work herein demonstrates how tobacco mosaic virus (TMV) nanoparticles can serve as a platform to create candidate vaccines for Zika virus (ZIKV) and anthrax. In the first study, a ZIKV-specific epitope was genetically fused to TMV to create a safe and inexpensive vaccine that proved highly immunogenic in mice and led to the discovery of ZIKV-specific neutralizing antibodies that may have applications in therapeutics and diagnostics. In the second study, anthrax toxin domains were expressed, purified, and conjugated to the outer surface of modified TMV nanoparticles. These VNPs were readily recognized by anthrax immune serum, but further studies will be necessary to ascertain their ability to induce a protective immune response. As demonstrated in these studies, genetic fusions and chemical conjugations to TMV each have distinct benefits and limitations. However, both methods result in the production of TMV-based VNPs, in which the TMV virion acts as both a scaffold and delivery mechanism, ensuring that the foreign antigens are taken up by DCs, transported to lymph nodes, and stimulate robust, antigen-specific B and T cell responses. In summation, this work shows how TMV VNPs displaying exogenous antigens can be used to create novel vaccines against both viral and bacterial pathogens

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

Studies on the extracellular envelope glycoprotein of maedi-visna virus

The extracellular envelope glycoprotein (gpl35) of maedi-visna virus interacts with cellular receptor molecules and is the major target of neutralising antisera in vivo. Antigenic drift of gpl35 may have an important role in viral persis tence.In order to begin to investigate the roles of different regions of gpl35, yeast and bacterial expression systems were used to generate recombinant protein and gpl35 was expressed as 3 overlapping fragments. There have been no published reports of expression of recombinant gpl35 proteins.By using the proteins to screen sera from infected sheep it was shown that sheep vary in the regions of gpl35 to which they mount an antibody response detectable in this system. At least 3 epitopes on gpl35 are recognised by sera from infected sheep.The recombinant proteins were used to investigate interactions of gpl35 with cellular molecules, and as immunogens to raise gpl35-specific sera. Possible future experiments using these reagents are suggested.gpl35 fragments derived from different viral stocks of the British isolate of maedi-visna virus were sequenced, to obtain a preliminary estimate of the extent of the variability of the gene. The data suggested the presence of both relatively conserved and variable regions in gpl35

The role of Human Endogenous Retrovirus K10 (HERV-K10) in the pathogenesis of rheumatoid arthritis via molecular mimicry

Rheumatoid arthritis (RA) is a chronic joint disease of unknown aetiology. The autoimmune nature of RA is underlined by abundant generation of rheumatoid factor (RF) autoantibodies to IgG1 Fc, and anti-citrullinated protein antibodies (ACPA) to citrullinated autoantigens such as fibrinogen. Although RA pathogenesis has not been elucidated, genetic predisposition, environmental insults and viral pathogens are considered contributory factors. Human endogenous retrovirus K10 (HERV-K10) is one such virus as it retained the capacity to produce viral particles in RA synovium. This study set out to explore how HERV-K10 Gag matrix region could contribute to RA pathogenesis and perpetuation, with particular emphasis on its ability to mimic host autoantigens. We showed that Gag region exhibits high levels of sequence and structural homology to IgG1 Fc and it could provide a key epitope important for auto-reactivity in RA. Analysis of how HERV-K10 may evoke immune responses in RA was broadened by investigation of serological cross-reactivity of novel anti-K10 polyclonal antibody (PAbMAG) with IgG1 Fc. We showed that PAbMAG cross-reacted with linear and conformational epitopes on IgG1 Fc. In a further development, we showed a significantly elevated mean IgG response to HERV-K10 epitopes in serum samples from RA patients when compared to other arthritides. These data suggest that molecular mimicry between viral and host proteins has the potential to lead to antigen-driven high-affinity RF IgG immunological reactivity in RA. Finally, we broadened our study of mimicry in RA by the investigation of citrullinated autoantigens. Structural studies demonstrated high levels of homology between citrullinated fibrinogen, IgG1 Fc and HERV. We further explored how protein citrullination affects the cross-reactivity of autoantibody responses in RA. These experiments revealed that generation of neoepitopes through citrullination of HERV-K10 and autoantigens IgG1 Fc and fibrinogen enhanced the reactivity of RA sera to these targets. Moreover, we showed that RF autoantibodies could mediate responses to a classical ACPA target fibrinogen, only when it is citrullinated, in the absence of ACPAs. These data provide a new insight into the initiation and propagation of immunological responses in RA and how viral/host molecular mimics and citrullination could modulate serum cross-reactivity profiles in RA.South Staffordshire Medical Foundation, Rotha Abraham Bequest, The Royal Wolverhampton Hospital Charity, New Cross Kidney Patients Association, James Beattie Charitable Trust.A thesis submitted in partial fulfilment of the requirements of the University of Wolverhampton for the degree of Doctor of Philosoph

B cell epitopes in fish nodavirus

Three epitope-mapping procedures were used to identify B-cell epitopes on Betanodaviruses: neutralisation escape mutant sequence analysis, phage display, and pepscan. Betanodaviruses have emerged as major pathogens of marine fish. These viruses are the aetiological agents of a disease referred to as viral nervous necrosis (VNN), which affects many species of fish that are economically valuable to the aquaculture industry. The identification of betanodavirus B-cell epitopes will facilitate the rational development of vaccines to counter VNN. A panel of mouse monoclonal antibodies (MAbs) was produced using hybridoma methodology for use in each of the epitope mapping procedures. These antibodies were characterised in Western blotting, ELISA, and virus neutralisation tests. Rabbit polyclonal sera, and serum samples from nodavirus-infected fish were also used for pepscan analyses. Attempts to produce betanodavirus neutralisation escape mutants, using plaque assay or limiting dilution based methods, were not successful. Two phage libraries expressing random peptides of seven (Ph.D.7™) or twelve (Ph.D.12™) amino acids in length as fusions to the coat protein were used to identify the ligands recognised by MAbs directed against betanodavirus. Neither of these phage libraries yielded conclusive results. Phage clones containing tandem inserts were obtained after MAb selection from library Ph.D.7™. Extensive screening and nucleotide sequence analysis of MAb-selected clones from library Ph.D.12™) failed to yield a consensus sequence. Pepscan analyses were performed using the recently developed suspension array technology (SAT). This was used to map the recognition sites of MAbs and serum samples onto a panel of overlapping synthetic peptides (12mers) that mimicked the betanodavirus coat protein. The results of pepscan analyses required careful interpretation due to the binding of antibodies and serum samples to multiple peptides. However, three regions of the nodavirus coat protein were identified as containing B-cell epitopes: amino acids 1-50, 141-162, and 181-212. These results are discussed in relation to previous studies of immune responses to betanodaviruses, and to the future development of betanodavirus vaccines and diagnostic reagents