Immunological Characterization of novel Adjuvants for Vaccines against Pathogenic Escherichia coli strains

Abstract

Escherichia coli (E. coli) is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of humans and animals as part of the commensal microbiota. However pathogenic E. coli strains have been isolated and these are usually classified as intestinal or extraintestinal pathogenic E. coli (InPEc or ExPEc) according to the disease location and the clinical outcomes. The extent of multidrug-resistance to last-line antibiotics in both InPEc and ExPEc, together with the recent rising incidence of hypervirulent strains and the emergence of new sequence types among E. coli pathotypes are becoming a growing concern since diseases caused by these pathogens are associated with significant human suffering and high costs for the healthcare system. A broad spectrum E. coli vaccine could be a promising alternative to prevent the spread of such diseases while offering the potential for covering against several pathovars at once. A critical goal of new generation vaccines against E. coli is to increase the breadth, quality and efficiency of protection and immune response and this could be achieved using adjuvants. Vaccine adjuvants enhance T and B cell responses by engaging components of the innate immune system. Either acting as delivery systems or as immune-potentiators, these compounds enhance antigen uptake by APCs triggering their maturation or activation, thus promoting immune modulatory cytokines production that, in turn, elicits local inflammation and cellular recruitment. Between the APCs, macrophages can acquire distinct functional phenotypes, with different functions and transcriptional profiles: classical or M1-activated macrophages, induced by microbial products in the presence or absence of IFN-γ, are microbicidal, tumoricidal and pro-inflammatory; in contrast, alternative or M2 activation by IL-4 and/or IL-13 gives rise to anti-inflammatory and immuno-tolerant cells. These subtypes are thought to represent extremes of a continuum of activation states; However, different polarization phenotypes have been shown to differently affect the adaptive immune response with M1 macrophages promoting Th1 and Th17 responses and M2 cells being related to Th2 differentiation. GSK has identified a series of new adjuvants, called SMIPs (Wu et al., 2014), that trigger members of the TLRs family expressed on a variety of APCs. The aim of the work described in this thesis was to characterize the in vitro and in vivo effect of GSK TLR2 and TLR-7 agonist adjuvants, named SMIP.2-7, SMIP.7-10 and SMIP.7-11, on macrophage polarization at early time-points and also to verify if the obtained activation state correlates coherently with the adaptive immune response observed after a complete immunization protocol. On in vitro experiments with murine macrophage cell line RAW 264.7, SMIP.2-7, SMIP.7-10 and SMIP.7-11 stimulated a very clear M1 phenotype which was confirmed also on ex vivo SMIP stimulated murine peritoneal macrophages. However, when the SMIPs (TLR7 agonists only) were intraperitoneally injected in mice, either in their soluble forms or formulated with Alum, a mixed polarization phenotype was elicited, with apparent up-regulation of M2 marker genes as well as pro-inflammatory M1-typical citokines. M1 and M2 cells are thought to have antagonistic roles in the immune response with M1 macrophages being involved in the inflammatory response and M2 macrophages serving to limit excessive Nitric Oxide production and support healing. The ability of these vaccine adjuvants to elicit the expression of both phenotypes’ markers at different time-points could play an important positive role in the immune response to a SMIP-containing vaccine, creating an inflammatory-responsive environment within few hours from injection that could favor other immune cells recruitment and vaccine antigen uptake and presentation while other cells would be involved in restoring the system to healing-homeostatic conditions at later time-points. Vaccine adjuvants can induce immunologic memory to vaccine-antigens through local activation of the innate immune system; since muscle constitutes the preferred site of injection of most human vaccines, the kinetics of cell recruitment into adjuvants (Alum-SMIP.7-10, Alum and SMIP.7-10)- or PBS-treated mice was determined directly in quadriceps muscles at early time-points post injection. At 24h, Alum-SMIP.7-10 showed to induce recruitment of CD11b+ myeloid cells, in particular monocytes and macrophages, into the injected quadriceps muscles as compared to PBS treated muscle; also B and T cells showed a slight, but still significant, increase in the Alum-SMIP.7- 10-treated muscles as compared to the PBS and the SMIP.7-10 alone-injected animals. After two days from injection, also a strong eosinophils influx was observed in the Alum-SMIP.7-10-treated muscles, together with a new wave of monocytes infiltration and a further massive increase in neutrophils and macrophages numbers as compared to the PBS and SMIP.7-10-administered groups; the latter could be presumably addressed to monocyte–macrophage differentiation rather than to a new recruitment of these cells. Monocytes, macrophages and neutrophils resulted the most abundant populations of the Alum-SMIP.7-10 treated muscles up to three days post-injection, meaning that these innate cells are likely involved in antigen “capture” and presentation in the presence of this adjuvant. Once assessed which are the immune cells involved in the first response to an Alum-SMIP.7-10- containing vaccine, the ability of this adjuvant to increase the immunogenicity of a candidate E. coli vaccine antigen was evaluated. E. coli considerable antigenic diversity and virulence factor redundancy has undoubtedly hampered the release of a broadly protective vaccine against pathogenic strains. Using the Reverse Vaccinology approach, nine antigens were identified as protective against a mouse sepsis model and among these, the Secreted Surface-associated Lipoprotein of E. coli (SslE) was the most promising candidate. Functionally, SslE is a zinc-metallo peptidase involved in mucins degradation; such mucinase activity plays an essential role in E. coli colonization and virulence. SslE already showed to be protective against other ExPEc models as well as against InPEcs strains. Vaccines based on purified antigens normally require multiple doses to achieve protective antibody levels and high cost, which makes their use in the developing world problematic. Alum-SMIP.7-10 showed to strongly increase immunogenicity of SslE antigen already after a single dose, inducing a significant increase in antigen-specific IgG titres. Alum-SMIP.7-10- SslE vaccine was also the sole formulation in which specific anti-SslE IgG2a and IgG2b subclasses were detectable, indicating that this adjuvant is able to enhance isotype switching. In addition to triggering a humoral response, SslE-Alum-SMIP.7-10 formulation was able to induce the higher antigen-specific T cells frequencies than Alum-SslE or SslE alone. After a complete three-doses immunization schedule, antigen-specific T cells in the Alum-SMIP.7-10 group were predominantly Th1 and, to a lower extent, Th17 polarized, although a good portion of SslE-specific CD4+ cells resulted activated, but yet in a Th0 phenotype. To finally assess the role of macrophages in SslE antigen-presentation and the effect of SMIPs on this process, SMIPs-pretreated and in vitro SslE-loaded Bone Marrow Derived Macrophages (BMDMs) were used as “Trojan Horse” for the antigen into naïve recipient mice. Adoptivelly- transferred cells successfully presented the antigen, being able to greatly induce SslE immunogenicity by promoting a both a sistemic and humoral response even after a sole immunization. SMIPs pretreatment stimulated up-regulation of co-stimulatory molecules that are important for APC-induced T cell activation and overall increased antigen-specific CD4+ T cell expansion above the level reached by SslE-loaded but SMIP-untreated macrophages. Overall, the findings of this thesis emphasize that SMIP adjuvants affect macrophages functionality, but also other cells at the injection site, leading to a rapid selective cellular recruitment that have a strong impact on the stimulation and the success of the following adaptive immune response. In the perspective of the urgent need for development of a broadly protective vaccine against pathogenic E. coli strains, Alum-SMIP.7-10 adjuvant can greatly increase the immunogenicity of the candidate antigen SslE and would deserve further investigation. However, SslE does not cover all known pathogenic strains: a recent study has identified antigen YncE, present in >99% of all E. coli genomes available, as potential vaccine candidate, showing already protection against a bacteremia model of infection and being recognized by antibodies present in the sera of convalescent urosepsis patients. The idea of a multi-component broad-spectrum vaccine including candidates such as SslE and YncE formulated with Alum-SMIP.7-10 would need attentive consideration in future E. coli vaccine research strategies.