Analysis of the mechanism by which BALB/c mice having prior immunization with nucleocapsid protein of SARS-CoV develop severe pneumonia after SARS-CoV infection

The precise mechanism of severe acute respiratory syndrome (SARS), which is caused by SARS-associated coronavirus (SARS-CoV), is still unclear. We generated recombinant vaccinia virus (rVV) LC16m8 strain which simultaneously expresses four structural proteins of SARS-CoV, including nucleocapsid (N), membrane (M), envelop (E), spike (S) proteins (rVV-NMES) and reported that old BALB/c mice having prior immunization with rVV-NMES develop severe pneumonia similar to those of control mice though rVV-NMES-immunized mice showed lower pulmonary viral titer than in the control mice. Furthermore, we determined which SARS-CoV structural protein for the prior rVV-immunization is responsible for the severe pneumonia after the SARS-CoV infection as observed in the rVV-NMES-immunized mice. Old BALB/c mice were inoculated intradermally with rVV that expressed each structural proteins of SARS-CoV (rVV-N, -M, -E, or -S) with or without rVV-S and then infected intranasally with SARS-CoV more than 4 weeks later. At 9 days after SARS-CoV infection, the rVV-N-immunized mice show more severe pneumonia than in other groups. Furthermore, significant up-regulation of Th1 (IL-2)- and Th2 (IL-4 and IL-5)-bias cytokines and down-regulation of anti-inflammatory cytokine (IL-10 and TGF-β) were observed in rVV-N-immunized mice, resulting in the intensive infiltration of immunocompetent cells into the lung. In contrast, rVV-S-immunized mice showed only low pulmonary viral tier and slight pneumonia. However, the mice having co-immunization with both rVV-N and rVV-S showed severe pneumonia though their pulmonary viral titer was low. These results suggest that an excessive host immune response against the N protein of SARS-CoV is involved in severe pneumonia caused by SARS-CoV infection. These findings increase our understanding of the pathogenesis of SARS.