Serotype-Independent Protection Against Invasive Pneumococcal Infections Conferred by Live Vaccine With lgt Deletion

Abstract

Streptococcus pneumoniae is the most common respiratory bacterial pathogen among cases of community-acquired infection in young children, older adults, and individuals with underlying medical conditions. Although capsular polysaccharide-based pneumococcal vaccines have contributed to significant decrease in invasive pneumococcal infections, these vaccines have some limitations, including limited serotype coverage, lack of effective mucosal antibody responses, and high costs. In this study, we investigated the safety and immunogenicity of a live, whole-cell pneumococcal vaccine constructed by deleting the gene for prolipoprotein diacylglyceryl transferase (lgt) from the encapsulated pneumococcal strain TIGR4 (TIGR4Δlgt) for protection against heterologous pneumococcal strains. Pneumococcal strain TIGR4 was successfully attenuated by deletion of lgt, resulting in the loss of inflammatory activity and virulence. TIGR4Δlgt colonized the nasopharynx long enough to induce strong mucosal IgA and IgG2b-dominant systemic antibody responses that were cross-reactive to heterologous pneumococcal serotypes. Finally, intranasal immunization with TIGR4Δlgt provided serotype-independent protection against pneumococcal challenge in mice. Taken together, our results suggest that TIGR4Δlgt is an avirulent and attractive broad-spectrum pneumococcal vaccine candidate. More broadly, we assert that modulation of such "master" metabolic genes represents an emerging strategy for developing more effective vaccines against numerous infectious agents.

Keywords: Lgt; Streptococcus pneumoniae; lipoprotein; live attenuated vaccine; mucosal immunity.

Figure 1

Biochemical and Immunological Characteristics of TIGR4Δlgt. (A) Expression of PsaA protein in cell lysates and culture supernatants from wild-type (WT) and lgt-deficient (Δlgt) TIGR4 strains. (B,C) Nitric oxide (B) and TNF-α (C) production in culture supernatants of RAW264.7 cells treated with WT or Δlgt strain for 12 h. (D) TNF-α production in the culture supernatants of peritoneal cells from wild-type (WT), TLR2-deficient (ΔTLR2), and TLR4-deficient (ΔTLR4) mice infected with WT or Δlgt strain for 12 h. Data in (B,C), and (D) represent mean values ± S.D. of triplicate results. ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001 compared to the WT treatment group. (E) LTA expression levels in 3-fold serial dilutions of culture supernatants from WT (W) or Δlgt (M) strains. (F) Quantification of LTA isolated from the pellets of WT and Δlgt strains. ^*P < 0.05 compared to the WT group.

Figure 2

Durable Colonization of TIGR4Δlgt in the Nasopharynx. (A–C) Pneumococcal colonization of the nasopharynx in mice (n = 5 per group) inoculated intranasally (i.n.) with either wild-type (WT) or Δlgt TIGR4 (10⁷ CFU) at 24 h (A), 48 h (B), and 72 h (C) after inoculation. (D) Survival of mice (n = 10 per group) inoculated i.n. with either WT (10⁵ CFU) or Δlgt TIGR4 (10⁵, 10⁶, or 10⁷ CFU). (E,F) Pneumococcal numbers in serially diluted lung tissue homogenates (E) and blood (F) from mice (n = 5 per group) inoculated i.n. with either WT or Δlgt TIGR4 (10⁷ CFU) at 3 days after infection. (G) Hematoxylin & eosin (H&E) staining of lungs from mice inoculated with WT or Δlgt TIGR4 (10⁷ CFU). ^*P < 0.05 and ^**P < 0.01 compared with the WT-infected mouse group.

Figure 3

Humoral and Mucosal Protective Immune Responses Induced by Live Δlgt Vaccination. (A–D) Levels of Sp-specific serum IgM (A), serum IgG (B), and BALF IgA (C) and ratios of serum IgG subclasses (D) determined 10 days after second immunization in mice (n = 5 per group) i.n. immunized twice with killed Δlgt or live Δlgt at 14-day intervals. Data in (A–C) indicate mean values ± S.D. n.s.; not significant, ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001 compared to PBS-inoculated controls. (E) Survival of mice (n = 5 per group) i.n. immunized twice with killed Δlgt or live Δlgt at 14-day intervals, followed by intraperitoneal (i.p.) challenge with 10⁷ CFU of wild-type TIGR4. ^*P < 0.05 and ^***P < 0.001 compared to control groups immunized with PBS. ^#P < 0.05 compared to the live Δlgt-vaccinated group.

Figure 4

Cross-reactive antibody production following immunization with TIGR4Δlgt. Serum levels of IgM and IgG reactive to serotypes 2 (D39; A), 3 (wu2; B), 6B (C), 9V (D), 19F (E), and 23F (F) and serum levels of LytA-, PspA-, and cell wall PS-specific IgG (G,H) were determined at 10 days after the second immunization in mice (n = 5 per group) i.n. immunized twice with Δlgt (10⁶ CFU) at 14-day intervals. ^*P < 0.05, ^**P < 0.01 and ^***P < 0.001 compared with control groups immunized with PBS.

Figure 5

Serotype-independent protection conferred by TIGR4Δlgt vaccination. (A,B) Survival of mice (n = 5) i.n. immunized with Δlgt (10⁶ CFU) twice at 14-day intervals, followed by i.n. challenge with 10⁸ CFU of wu2 (A) or D39 (B) strains. (C–H) Pneumococcal numbers counted within serially diluted nasal washes (C,F), lung tissue homogenates (D,G), and blood (E,H) from Δlgt (10⁶ CFU)-immunized mice challenged with wu2 (C–E) or D39 (F–H). ^*P < 0.05 and ^***P < 0.001 compared to the control group immunized with PBS.