A pinboard by
Elena Mitsi

Phd student, Liverpool School of Tropical Medicine


Design and development of a universal vaccine against pneumococcal pneumonia and systemic disease

Pneumonia is a life-threatening disease with high mortality and morbidity among children under 5 years of age, immunocompromised and elderly worldwide. Such an acute disease, as well as otitis media, septicaemia and meningitis are caused by a gram positive bacterium known as Streptococcus pneumoniae (“the pneumococcus”). In the community, there are currently 94 different capsules (the pneumococcus “sugar coat”), resulting to 94 pneumococcal serotypes. Their prevalence differs by geographic region and the period studied. The current licenced vaccines have been designed to protect against a handful of pneumococcal serotypes and they show poor matching with the circulating serotypes in developing countries.

My research project is focused on the development of an innovative vaccine formation against pneumonia. The vaccine will be administered through the airway to the lung airspaces, where it will gradually release immunogenic factors (conserved among the pneumococcal serotypes) to the lung microenvironment, initiating the local immune responses and boosting the host’s immunity. The innovative idea of such a vaccine formation is the usage of nanoparticles (“microspheres”) as an adjuvant. These nanoparticles trigger non-antigen specific immune responses by themselves and in the current research project are coated with the protein-antigens layer by layer.

In our group, we study the interaction between pneumococcus and the human host immunity. We think that small numbers of pneumococci present in the nose (“nasal carriage”) can may protect people against disease. Small numbers of these bacteria are often found in the nose of healthy adults (at least once per year) and more often in children. Therefore, we annually recruit over a hundred healthy volunteers and we instill a safe dose of live bacteria into their nose. For the purposes of our research we use innovative techniques to collect unique samples, such as nasosampling, nasal biopsies and bronchoscopies.


Experimental human pneumococcal carriage.

Abstract: Experimental human pneumococcal carriage (EHPC) is scientifically important because nasopharyngeal carriage of Streptococcus pneumoniae is both the major source of transmission and the prerequisite of invasive disease. A model of carriage will allow accurate determination of the immunological correlates of protection, the immunizing effect of carriage and the effect of host pressure on the pathogen in the nasopharyngeal niche. Further, methods of carriage detection useful in epidemiologic studies, including vaccine studies, can be compared.We aim to develop an EHPC platform that is a safe and useful reproducible method that could be used to down-select candidate novel pneumococcal vaccines with prevention of carriage as a surrogate of vaccine induced immunity. It will work towards testing of candidate vaccines and descriptions of the mechanisms underlying EHPC and vaccine protection from carriage. Current conjugate vaccines against pneumococcus protect children from invasive disease although new vaccines are urgently needed as the current vaccine does not confer optimal protection against non-bacteraemic pneumonia and there has been evidence of serotype replacement with non-vaccine serotypes.We inoculate with S. pneumoniae suspended in 100 μl of saline. Safety is a major factor in the development of the EHPC model and is achieved through intensive volunteer screening and monitoring. A safety committee consisting of clinicians and scientists that are independent from the study provides objective feedback on a weekly basis. The bacterial inoculum is standardized and requires that no animal products are inoculated into volunteers (vegetable-based media and saline). The doses required for colonization (10(4)-10(5)) are much lower than those used in animal models (10(7)). Detecting pneumococcal carriage is enhanced by a high volume (ideally > 10 ml) nasal wash that is relatively mucus free. This protocol will deal with the most important parts of the protocol in turn. These are (a) volunteer selection, (b) pneumococcal inoculum preparation, (c) inoculation, (d) follow-up and (e) carriage detection.Our current protocol has been safe in over 100 volunteers at a range of doses using two different bacterial serotypes. A dose ranging study using S. pneumoniae 6B and 23F is currently being conducted to determine the optimal inoculation dose for 50% carriage. A predicted 50% rate of carriage will allow the EHPC model to have high sensitivity for vaccine efficacy with small study numbers.

Pub.: 27 Feb '13, Pinned: 13 Oct '17

Experimental human pneumococcal carriage augments IL-17A-dependent T-cell defence of the lung.

Abstract: Pneumococcal carriage is both immunising and a pre-requisite for mucosal and systemic disease. Murine models of pneumococcal colonisation show that IL-17A-secreting CD4(+) T-cells (Th-17 cells) are essential for clearance of pneumococci from the nasopharynx. Pneumococcal-responding IL-17A-secreting CD4(+) T-cells have not been described in the adult human lung and it is unknown whether they can be elicited by carriage and protect the lung from pneumococcal infection. We investigated the direct effect of experimental human pneumococcal nasal carriage (EHPC) on the frequency and phenotype of cognate CD4(+) T-cells in broncho-alveolar lavage and blood using multi-parameter flow cytometry. We then examined whether they could augment ex vivo alveolar macrophage killing of pneumococci using an in vitro assay. We showed that human pneumococcal carriage leads to a 17.4-fold (p = 0.007) and 8-fold (p = 0.003) increase in the frequency of cognate IL-17A(+) CD4(+) T-cells in BAL and blood, respectively. The phenotype with the largest proportion were TNF(+)/IL-17A(+) co-producing CD4(+) memory T-cells (p<0.01); IFNγ(+) CD4(+) memory T-cells were not significantly increased following carriage. Pneumococci could stimulate large amounts of IL-17A protein from BAL cells in the absence of carriage but in the presence of cognate CD4(+) memory T-cells, IL-17A protein levels were increased by a further 50%. Further to this we then show that alveolar macrophages, which express IL-17A receptors A and C, showed enhanced killing of opsonised pneumococci when stimulated with rhIL-17A (p = 0.013). Killing negatively correlated with RC (r = -0.9, p = 0.017) but not RA expression. We conclude that human pneumococcal carriage can increase the proportion of lung IL-17A-secreting CD4(+) memory T-cells that may enhance innate cellular immunity against pathogenic challenge. These pathways may be utilised to enhance vaccine efficacy to protect the lung against pneumonia.

Pub.: 05 Apr '13, Pinned: 13 Oct '17

Polysaccharide-specific Memory B-cells Predict Protection Against Experimental Human Pneumococcal Carriage.

Abstract: We have previously demonstrated that experimental pneumococcal carriage enhances immunity and protects healthy adults against carriage reacquisition following re-challenge with homologous strain. Here we have used a heterologous challenge model to investigate the role of naturally acquired pneumococcal protein and polysaccharide (PS)-specific immunity in protection against carriage acquisition.We identified healthy volunteers that were naturally colonised with pneumococcus and, following clearance of their natural carriage episode, challenged them with a heterologous 6B strain. In another cohort of volunteers we assessed 6BPS-specific, PspA-specific and PspC-specific IgG and IgA plasma and memory B-cell populations prior to and 7, 14 and 35 days following experimental pneumococcal inoculation.Heterologous challenge with 6B resulted in 50% carriage among volunteers with previous natural pneumococcal carriage. Protection from carriage was associated with a high number of circulating 6BPS IgG-secreting memory B-cells at baseline. There were no associations between protection from carriage and baseline levels of 6BPS IgG in serum or nasal wash, PspA-specific or PspC-specific memory B-cells or plasma cells. In volunteers who did not develop carriage, the number of circulating 6BPS memory B-cells decreased and the number of 6BPS plasma cells 7 days post inoculation.Our data indicate that naturally acquired polysaccharide-specific memory B-cells, but not levels of circulating IgG at time of pneumococcal exposure, are associated with protection against carriage acquisition.

Pub.: 13 Jul '16, Pinned: 13 Oct '17

Agglutination by anti-capsular polysaccharide antibody is associated with protection against experimental human pneumococcal carriage.

Abstract: The ability of pneumococcal conjugate vaccine (PCV) to decrease transmission by blocking the acquisition of colonization has been attributed to herd immunity. We describe the role of mucosal immunoglobulin G (IgG) to capsular polysaccharide (CPS) in mediating protection from carriage, translating our findings from a murine model to humans. We used a flow cytometric assay to quantify antibody-mediated agglutination demonstrating that hyperimmune sera generated against an unencapsulated mutant was poorly agglutinating. Passive immunization with this antiserum was ineffective to block acquisition of colonization compared to agglutinating antisera raised against the encapsulated parent strain. In the human challenge model, samples were collected from PCV and control-vaccinated adults. In PCV-vaccinated subjects, IgG levels to CPS were increased in serum and nasal wash (NW). IgG to the inoculated strain CPS dropped in NW samples after inoculation suggesting its sequestration by colonizing pneumococci. In post-vaccination NW samples pneumococci were heavily agglutinated compared with pre-vaccination samples in subjects protected against carriage. Our results indicate that pneumococcal agglutination mediated by CPS-specific antibodies is a key mechanism of protection against acquisition of carriage. Capsule may be the only vaccine target that can elicit strong agglutinating antibody responses, leading to protection against carriage acquisition and generation of herd immunity.Mucosal Immunology advance online publication, 31 August 2016; doi:10.1038/mi.2016.71.

Pub.: 01 Sep '16, Pinned: 13 Oct '17