2.2.4. Rat Endocarditis Model

The vaccine was tested in two rat endocarditis models, a standard model [23] and a refined model that more accurately represents clinical disease.

Groups of female Sprague Dawley rats (5–6–week–old, 20/group, Charles River Laboratories) were vaccinated at weeks 0, 3, and 6, with SA4Ag in AlPO₄ or with placebo. At week 8, two weeks after the final vaccination, catheter placement surgery was performed to generate sterile valvular vegetations [23]. Due to the complex nature of the model and surgery required, a proportion (<10%) of animals succumbed before infectious challenge due to the surgery and were thus not included in the analysis. Two days after surgery, animals were challenged intravenously with ~4 × 10⁶ CFU of S. aureus PFESA0158 in 0.1 mL PBS. Two days post-challenge, the rats were euthanized and hearts collected. Bacteria in homogenized heart tissue were enumerated (CFU/mL), and the arithmetic mean and 95% confidence interval (CI) were calculated for each treatment group.

In order to reflect the low challenge inoculum that has been reported in human infections [24], the endocarditis model was refined. Groups of female Sprague Dawley rats (5–6–week–old, 14/group; Charles River Laboratories) were vaccinated at weeks 0, 3, and 6, with SA4Ag in AlPO₄ or with AlPO₄ alone, and catheter placement surgery was conducted on week 8 as described above. Two days after surgery, animals were challenged intravenously with ~4 × 10³ CFU of S. aureus PFESA0186. Two days post-challenge, the rats were euthanized and hearts and kidneys collected. Tissues were plated and the presence or absence of bacterial CFU scored.