Identification of infection biomarkers in a murine model of pneumonia by Streptococcus pneumoniae

Streptococcus pneumoniae infections remain a public health problem despite the availability of vaccines. In order to study alternative preventive strategies it is essential to have a reliable animal model of the infection. To date, the majority of vaccine efficacy evaluation studies still rely on direct read-outs such as the survival rate after challenge and the CFU counts in blood and in the lungs. A murine model of lung infection was developed in which animal death was not the endpoint: clinical parameters, cellular lung infiltrate and systemic immune response were evaluated in (i) infected mice, (ii) vaccinated mice, and (iii) vaccinated and challenged mice. All these parameters were used to identify biomarkers of sublethal pneumonia and protection following vaccination with the pneumococcal conjugate vaccine. Female C57BL/6 mice were infected with different doses of S. pneumoniae strain TIGR4, the dose of 1.6 x 107 CFUs was capable to induce measurable signs of the disease with a 100% survival and was therefore used for subsequent experiments. Histological examination and flow cytometry analysis of the lung tissue were performed at different time-points after bacterial inoculation. Data revealed that absolute numbers of the cell populations evaluated through flow cytometry were significantly different 7 days after the infection from those of uninfected mice, and histological evaluation of the lungs at this time-point also revealed the presence of leukocyte infiltrate. The presence of the infiltrate represented a biomarker of infection. When mice were vaccinated prior to pneumococcal challenge, they showed no weight loss and a mild infiltrate in the lungs. The absence of the infiltrate in relation with mild clinical signs and a good humoral response defined the protected phenotype. The type of immune response mounting following interaction of the immune system with S. pneumoniae was evaluated using mice splenocytes re-stimulated in vitro with inactivated TIGR4. The production of 23 cytokines was measured, revealing that splenocytes started to react to in vitro re-stimulation at 4 days after infection and peaking 7 days after infection. A predominant production of IFN-γ and IL-17 was observed in infected mice, while this profile was skewed to Th2 characteristic cytokines such as IL-4 and IL-5 when mice had received the vaccine. These features, together with cellular infiltrates observed in the lungs, can be considered biomarkers of protection that could be used to study the protection induced by a vaccine candidate using parameters other than the survival rates and the CFU counts. We identified measurable and reliable biomarkers of pneumococcal pneumonia that could be investigated in vaccine efficacy studies and could become tools to develop new immunization strategies against S. pneumoniae.