Immunotherapy with a combination of intravenous immune globulin and p4 peptide rescues mice from postinfluenza pneumococcal pneumonia

Abstract

Alternate therapies are needed for treatment of secondary bacterial pneumonia following influenza. The immunomodulatory peptide P4 has shown promise in mouse models of primary pneumococcal infection. Mice infected with influenza virus and then challenged with Streptococcus pneumoniae were treated with a combination of P4 peptide and intravenous immune globulin. Survival was improved from 20% to 80% in treated mice relative to controls. Clinical cure correlated with increased clearance of bacteria and decreased lung consolidation. Greater trafficking of professional phagocytic cells to the site of pneumococcal infection coupled with enhanced opsonophagocytosis as manifest by decreased surface display of Fcγ receptors (FcγR) on neutrophils and macrophages were associated with P4 peptide treatment. This suggests that the mechanism of action for improved clearance of bacteria engendered by P4 is through improved uptake by phagocytes mediated by IgG Fc-Fcγ receptor interactions following antibody-mediated opsonophagocytosis of bacteria. Antibody-based therapies, when coupled with immune modulators, such as P4 peptide, may be an effective tool together with antibiotics in our armamentarium against severe pneumonia.

Fig. 1.

P4 peptide combination therapy rescues mice from bacterial pneumonia following influenza. (A) Mice were mock infected with PBS (A66.1 group; n = 4) or infected with a sublethal dose of influenza A virus strain PR8 (PR8 group [n = 4] and PR8 + A66.1 group [n = 10]) and then challenged 7 days later with S. pneumoniae strain A66.1 (A66.1 and PR8 + A66.1 groups) or PBS (PR8 group). (B) Forty-eight and seventy-two hours after bacterial challenge, groups of virus-and-bacterium-coinfected mice (n = 10) were mock treated with PBS or treated with IVIG alone, P4 peptide alone, or IVIG followed by P4 peptide 20 min later. Morbidity represented by weight loss (C) and survival (B) were followed for 21 days. An asterisk indicates a significant (P < 0.05) difference in survival compared to that of the other groups by the log-rank test on the Kaplan-Meier data. In panel C, values indicate the mean and error bars the standard deviation of the measurements.

Fig. 2.

In vivo bioluminescent imaging of secondary bacterial pneumonia. Representative mice coinfected with influenza virus and pneumococci and either mock treated with PBS or treated with IVIG and P4 48 h and 72 h after bacterial challenge were imaged daily for localization and quantitation of bioluminescent bacteria.

Fig. 3.

Histopathology of lungs from mice treated with IVIG and P4 peptide. Lungs were removed from mice coinfected with influenza virus and pneumococci (n = 4 per group per time point). Group treated with IVIG and P4 peptide (A and C); PBS control group (B and D); single-blind, histopathology scores by a veterinary pathologist using a semiquantitative grading scheme (E). Representative photomicrographs taken 5 days after bacterial challenge at a magnification of ×4 (A and B) and ×40 (C and D) are presented. The extensive neutrophilic infiltrate observed with mock-treated mice (D) caused near-complete consolidation of the lungs (B), while the relatively mild inflammatory infiltrate found in IVIG-and-P4-treated mice (C) was limited to specific foci (A). In panel E, horizontal lines indicate the mean pathology score.

Fig. 4.

Bacterial lung load from mice treated with PBS or IVIG and P4. Lung titers were determined for groups of influenza-virus-and-pneumococcus-coinfected mice (n = 5) either mock treated with PBS or treated with IVIG and P4 peptide 48 and 72 h after bacterial challenge. Values indicate the mean, and error bars the standard deviation of the measurements. An asterisk indicates a significant (P < 0.05) difference in bacterial titer compared to mock-treated animals at that time point.

Fig. 5.

Airway inflammatory cells in mice treated with PBS or IVIG and P4. BALF was obtained from groups of mice (n = 8 to 10) coinfected with influenza virus and pneumococci and mock-treated with PBS or treated with IVIG and P4 peptide 48 and 72 h after bacterial challenge. The relative numbers of total white blood cells (A), macrophages (B), and neutrophils (C) were determined by flow cytometry. The proportion of either macrophages (D) or neutrophils (E) displaying CD16/32 was determined by flow cytometry. Arrows indicate the timing of treatment relative to BALF collection. An asterisk indicates a significant (P < 0.05) difference in cell number or proportion compared to mock-treated animals at that time point.