Maternal transfer of anti-GAPDH IgG prevents neonatal infections caused by Staphylococcus aureus and group B Streptococcus

Abstract

Group B Streptococcus (GBS) and Staphylococcus aureus cause 200.000 neonatal deaths every year and no vaccine has been developed yet. Here, we described that extracellular glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from S. aureus is an immunomodulatory protein. Antibody mediated neutralization of S. aureus extracellular GAPDH promotes a protective inflammatory response by inhibiting an early and abnormal production of IL-10 in infected neonatal mice. As an immunomodulatory role for extracellular GAPDH was already described for GBS, we selected peptides exposed on bacterial GAPDH from both bacteria but completely absent from human GAPDH. These peptides were chemically synthesized and conjugated to a carrier protein. Maternal vaccination with these conjugated peptides induced an increased survival of mouse pups from infection with GBS or S. aureus, when compared to controls. The addition of anti-bacterial GAPDH IgG into infected human cord-blood cells caused a significant reduction in bacterial replication, suggesting a putative efficacy for humans.

Keywords: Immunology; Microbiology.

Graphical abstract

Figure 1

Antibody-mediated neutralization of extracellular bacterial GAPDH confers protection to the offspring against MRSA challenging infection (A) Extracellular products (ExtP) from MRSA were separated by SDS-PAGE and analyzed by western blot using anti-GAPDH IgG purified from rGAPDH-immunized mice serum. Recombinant (Rec) GAPDH from S. aureus, GBS or GBS ExtP were used as positive controls. (B) C57BL/6 females (6–8 weeks) were immunized sc with 20 μg of S. aureus rGAPDH in a saline suspension containing 0.125 mg of Alum as adjuvant, or with the adjuvant alone (sham-immunized). The animals received two administrations before mating, with a three-week interval between administrations. A boost administration was performed at gestational day 14. Neonates were sc infected 48 h after birth with 10⁶ CFU of MRSA and survival was assessed for 12 days (C and D) Neonates were sc immunized (24 h after birth) with 150 μg of anti-GAPDH or control IgG and 24 h later, were infected sc with 10⁶ CFU of MRSA. (B-C) In parentheses is shown the number of animals that survived versus the number of animals infected. Log rank test was used to determine differences between groups. p values are indicated in the graphs. (D) At indicated time points, bacterial load was assessed in the liver, lungs and blood. Results are presented as mean ± SEM. The differences between anti-GAPDH IgG and control IgG were analyzed using T-test (Mann-Whitney) and a p ≤ 0.05 was considered significant. (B–D) Results show data from at least three independent experiments.

Figure 2

Antibody-mediated neutralization of S. aureus GAPDH inhibits early IL-10 production and promotes inflammatory response C57BL/6 neonatal mice were sc immunized (24 h after birth) with 150 μg of anti-GAPDH IgG or control IgG, and 24 h later were infected sc with 10⁶ CFU of MRSA. (A–C) At the indicated time points after infection, liver, lung and blood were collected. (A-B) mRNA expression of the indicated genes in the (A) liver and (B) lung. The fold change in gene expression was obtained through the calculation of 2^−ΔΔCT and relative to β-actin mRNA. (C) Serum cytokines were quantified by ELISA. (D–F) Lung inflammatory macrophages (IM) were analyzed by flow cytometry. (D) Gating strategy for the analysis of CD45⁺CD11b⁺ cells that correspond to the following phenotype: CD11c⁺F4/80⁺Ly6G^negCXCR2⁺Ly6C⁺. (E) Representative example of the frequency of activated IM in the lung of infected mice treated with anti-GAPDH IgG or with control IgG, at 18 h post infection. (F) Total numbers of IM are presented as mean ± SEM. The differences between anti-GAPDH IgG and control IgG were analyzed using T-test (Mann-Whitney) and a p ≤ 0.05 was considered significant.

Figure 3

IMTP_vac1804 peptides are present at the surface of bacterial GAPDH and absent in the human homologue (A) Alignment of protein sequences of GAPDH from human (Uniprot ID: P04406), GBS (Uniprot ID: Q9ALW2) and S. aureus (Uniprot ID: P0A038), obtained using Clustal Omega at EMBL-EBI. The IMTP_vac1804 peptides are highlighted in bold and underlined. An “∗” (asterisk) indicates positions which have a single, fully conserved residue; a “:” (colon) indicates conservation between groups of residues with strongly similar properties, and a “.” (period) indicates conservation between groups of residues with weakly similar properties. (B and C) 3D representation of GBS (PDB: 5Y37) and S. aureus (PDB: 3LC2) GAPDH, respectively. Peptide 1 is represented in yellow and peptide 2 in purple. (D) Reactivity of mouse anti-bGAPDH IgG (purified from IMTP_vac1804 immunized mice serum) against GBS, S. aureus or huGAPDH from pre-immune (PI) and immune sera (IMNZ), collected 1 week after the last immunization. Absorbance was determined at 450 nm. At least, four independent experiments were performed. Results are presented as mean ± SEM. (E) Recombinant proteins from GBS, S. aureus and human GAPDH, human erythrocytes (Eryt) GAPDH and proteins from human cell lines (THP1 and A549) were separated by SDS-PAGE. Analysis was done by western blot using anti-bGAPDH IgG purified from IMTP_vac1804-immunized mice. (F) Western blot analysis of blotted extracellular products (ExtP) of GBS and S. aureus. Recombinant (Rec) proteins were used as positive controls. Anti-bGAPDH IgG of pooled sera collected from IMTP_vac1804-immunized mice were used as developing Ab.

Figure 4

Maternal immunization with IMTP_vac1804 protects neonatal mice against GBS and S. aureus infection C57BL/6 females (6–8 weeks) were sc immunized with IMTP_vac1804 or with the adjuvant alone (sham-immunized). (A and B) The animals received two administrations before mating, with a three-week interval between administrations. A boost immunization was administered at gestational day 14. Neonates were infected sc 48 h after birth with (A) 4 × 10⁵ CFU of GBS, or with (B) 10⁶ CFU of MRSA. At least three independent experiments were performed. In parentheses is shown the number of animals that survived vs. the number of animals infected. Log rank test was used to determine differences between groups. p values are indicated in the graphs. (C and D) Serum anti-bGAPDH IgG titers of females and neonates determined by ELISA. Blood was collected from IMTP_vac1804-or sham-immunized females 1 week after 2nd dose (2nd immunization) and 12 days post infection of the newborns (12 days after nb infection). Blood was collected from the offspring before infection (Before infection) or 12 days after infection of the newborn (12 days after infection). ELISA plates were coated with rGAPDH from (C) GBS or (D) S. aureus. IgG titers were calculated as the first value of serum dilution where OD 450 nm ≤ 0.1. Titers below 90 were considered non-detectable (ND). Results are presented as mean ± SEM.

Figure 5

IMTP_vac1804 does not interfere with host microbiome C57BL/6 females were immunized two times sc with IMTP_vac1804 or the adjuvant alone, with a three-week interval between administrations. Feces were collected before immunization, and two and thirty days after the last administration. At least, sixteen animals were used in each condition. (A) PCoA of IMTP_vac1804 versus sham-immunized females. Each dot represents a sample. Statistical analysis of group distance was performed using the ANOSIM test. Family microbial relative abundances in (B) IMTP_vac1804 and (C) sham-immunized group.

Figure 6

IMTP_vac1804-elicited anti-bGAPDH IgG can control bacterial replication in human cord blood Total fresh human cord blood was diluted 1:2 in RPMI medium supplemented with 1% HEPES and 50 μM 2-Mercaptoethanol. Diluted blood was incubated with 200 μg of anti-bGAPDH IgG (purified from goats immunized with IMTP_vac1804), or with goat anti-KLH IgG and infected for 3 h with 10⁷ CFU of (A) GBS or (B) MRSA. Percentage of bacterial CFU in each indicated condition relative to bacterial CFU in conditions with no IgG for GBS and MRSA (100%) are indicated in the graphs. Results are presented as box-plot and at least seven independent experiments were conducted. The dashed line in the box-plot representation corresponds to the same cord blood sample treated with anti-bGAPDH IgG (purified from the sera of goat immunized with IMTP_vac1804) or anti-KLH IgG (Control). The differences between groups were analyzed using a T-test (Mann-Whitney) and a p ≤ 0.05 was considered significant.