Characterization of a lipopolysaccharide-targeted monoclonal antibody and its variable fragments as candidates for prophylaxis against the obligate intracellular bacterial pathogen Coxiella burnetii

Abstract

Our previous study demonstrated that treatment of Coxiella burnetii with the phase I lipopolysaccharide (PI-LPS)-targeted monoclonal antibody (MAb) 1E4 significantly inhibited C. burnetii infection in mice, suggesting that 1E4 is a protective MAb. To determine whether passive transfer of antibodies (Abs) can provide protection against C. burnetii natural infection, we examined if passive transfer of 1E4 would protect SCID mice against C. burnetii aerosol infection. The results indicated that 1E4 conferred significant protection against aerosolized C. burnetii, suggesting that 1E4 may be useful for preventing C. burnetii natural infection. To further understand the mechanisms of 1E4-mediated protection and to test the possibility of using humanized 1E4 to prevent C. burnetii infection, we examined whether the Fab fragment of 1E4 (Fab1E4), a recombinant murine single-chain variable fragment (muscFv1E4), and a humanized single-chain variable fragment (huscFv1E4) retained the ability of 1E4 to inhibit C. burnetii infection. The results indicated that Fab1E4, muscFv1E4, and huscFv1E4 were able to inhibit C. burnetii infection in mice but that their ability to inhibit C. burnetii infection was lower than that of 1E4. In addition, treatment of C. burnetii with Fab1E4, muscFv1E4, or huscFv1E4 can block C. burnetii infection of macrophages. Interestingly, treatment of C. burnetii with huscFv1E4 can significantly reduce C. burnetii infectivity in human macrophages. This report provides the first evidence to demonstrate that the humanized variable fragments of an LPS-specific MAb can neutralize C. burnetii infection and appears to be a promising step toward the potential use of a humanized MAb as emergency prophylaxis against C. burnetii exposure.

FIG 1

Evaluation of the ability of 1E4 to confer protection in naive recipient SCID mice against C. burnetii aerosol infection by comparing splenomegaly, bacterial burden in the spleen and lung, and pathological changes in the lung with control mice at 14 days postinfection. (A) Splenomegaly was measured by spleen weight as a percentage of body weight. (B) Bacterial burden in the spleen was determined by real-time PCR and reported as log₁₀ of C. burnetii com1 gene copy numbers. (C) Bacterial burden in the lung. (D) Pathological changes in the lung. (E) Inflammation score in the lung. Lungs (1 section per mouse) were scored for interstitial inflammation (macrophages and neutrophils in interalveolar septum and alveolar spaces) according to the following scale: 0, none (no accumulations of neutrophils and macrophages); 1, few small scattered accumulations of neutrophils and macrophages; 2, mild to moderate accumulations of macrophages and neutrophils which affects less than 10% of lung parenchyma; 3, moderate to large accumulations of macrophages and neutrophils which affects ≥10% of lung parenchyma. The data presented in each group are the averages with standard deviations from four mice. **, P < 0.01; ***, P < 0.001.

FIG 2

Comparison of the CDR amino acid sequence and structure of muscFv1E4 with huscFv1E4. (A) Alignment of deduced amino acid sequences of muscFv1E4 and huscFv1E4. Nonidentical residues are shown. Dots indicate identical residues. The definitions of CDR residues according to the Kabat database are indicated in boxes. H, heavy chain; L, light chain; CDR, complementarity-determining region; FR, framework region. The retained murine amino acids (back mutation) are indicated by asterisks. PelB leader sequence, HFR2 loop, linker, and 6×His tag are underlined. (B) Structure alignment models of muscFv1E4 (black) and huscFv1E4 (gray). The cartoon graph displays the loop structure of CDR domains and HFR2.

FIG 3

Characterization of the Fab fragment of 1E4 (Fab1E4), recombinant murine Fab fragments (muscFv1E4), and recombinant humanized Fab fragments (huscFv1E4). (A) Analysis of the purity of purified Fab1E4, muscFv1E4, and huscFv1E4 by SDS-PAGE. Lane M, molecular mass protein markers; lane 1, MAb 1E4 (IgG); lane 2, Fab1E4; lane 3, muscFv1E4; lane 4, huscFv1E4. (B) Analysis of the specificity of purified Fab1E4, muscFv1E4, and huscFv1E4 by Western blotting. Anti-6×His MAb was used as a primary antibody, and HRP-conjugated rabbit anti-mouse IgG was used as the second antibody. The samples used in lanes 1 to 4 are the same as the samples in panel A. (C) Evaluation of the ability of 1E4, Fab1E4, muscFv1E4, and huscFv1E4 to bind with the mimetic peptide m1E41920 by ELISA. Microtiter polystyrene plates were coated with 50 μg/ml m1E41920 and incubated with a 2-fold serial dilution of 1E4, Fab1E4, muscFv1E4, or huscFv1E4. Anti-6×His MAb was used as the primary antibody, and HRP-conjugated rabbit anti-mouse IgG was used as the second antibody in the ELISA. The values represent the average absorbance at 490 nm of quadruplicate wells. (D) Evaluation of the ability of 1E4, Fab1E4, muscFv1E4, and huscFv1E4 to bind with C. burnetii native antigen (NMI whole-cell antigen [WCA]) and mimetic peptide by ELISA. Microtiter polystyrene plates were coated with 5 μg/ml NMI whole-cell antigen, 50 μg/ml m1E41920, or a negative mimetic peptide control, LPS6. The ELISA was performed in the same manner as described for panel C, and the values represent the average absorbance at 490 nm of duplicate wells.

FIG 4

Evaluation of the ability of 1E4, Fab1E4, muscFv1E4, and huscFv1E4 to inhibit C. burnetii infection in vivo by comparing splenomegaly, bacterial burden, and pathological changes in the spleen with control mice at 14 days postinfection. (A) Analysis of the ability of 1E4 to bind live virulent C. burnetii by IFA. (B) Splenomegaly was measured by spleen weight as a percentage of body weight. (C) Bacterial burden in the spleen was determined by real-time PCR and reported as the log₁₀ of C. burnetii com1 gene copy numbers. (D) Pathological changes in the spleen at 14 days postinfection. (E) Inflammation score in the spleen. Spleens (1 section per mouse) were scored for histiocytic inflammation in red pulp of spleen according to the following scale: 0, none (no accumulations of macrophages); 1, few small accumulations of macrophages; 2, few small to moderate accumulations of macrophages; 3, large numbers of moderate to large accumulations of macrophages. The data presented in each group are the averages with standard deviations from four mice. **, P < 0.01; ***, P < 0.001.

FIG 5

Evaluation of the ability of 1E4, Fab1E4, muscFv1E4, and huscFv1E4 to inhibit C. burnetii infection in mouse bone marrow-derived macrophages (BMDM) by comparing infection rate and C. burnetii genomic copy numbers with PBS and IgG2a isotype controls at 1 day postinfection. (A) IFA staining of BMDM infected with PBS-, IgG2a isotype-, 1E4-, Fab1E4-, muscFv1E4-, or huscFv1E4-treated C. burnetii. Host nuclei were stained by DAPI fluorescence (blue); intracellular C. burnetii was stained with rabbit anti-Nine Mile phase II/NMI polyclonal antibodies, followed by incubation with 10 μg/ml FITC-labeled goat anti-rabbit IgG (green, FITC-labeled NMI cells). (B) C. burnetii infection rate was determined by IFA, represented as percentage of cells containing more than 5 organisms. A total of 200 cells were counted per sample to determine the infection rate. (C) C. burnetii genomic number was determined by real-time PCR. The data presented in each group in panels B and C are the averages with standard deviations from duplicate samples. **, P < 0.01; ***, P < 0.001.

FIG 6

Evaluation of the ability of huscFv1E4 to inhibit C. burnetii infection in THP-1 differentiated human macrophages by comparing infection rate and C. burnetii genomic numbers with values of PBS and IgG2a isotype control treatment at 1 day postinfection (dpi). (A) IFA staining of human macrophages infected with PBS-, IgG2a isotype-, or huscFv1E4-treated C. burnetii. Host nuclei were stained by DAPI fluorescence (blue); intracellular C. burnetii was stained with rabbit anti-Nine Mile phase II/NMI polyclonal antibodies, followed by incubation with 10 μg/ml FITC-labeled goat anti-rabbit IgG (green, FITC-labeled NMI cells). (B) C. burnetii infection rate was determined by IFA, represented as a percentage of cells containing more than 5 organisms. A total of 200 cells were counted per sample to determine the infection rate. (C) C. burnetii genomic number was determined by real-time PCR. The data presented in each group in panels B and C are the averages with standard deviations from duplicate samples. ***, P < 0.001.

FIG 7

BacLight assay staining of viable and nonviable C. burnetii cells after treatment with PBS, IgG2a isotype, 1E4, Fab1E4, muscFv1E4, or huscFv1E4 at 4°C overnight. (A) 1E4-treated C. burnetii was stained by a BacLight assay. Viable bacteria were stained by SYTO9 (green) while nonviable bacteria were stained by propidium iodide (red). (B) EDTA-treated C. burnetii was stained by a BacLight assay and used as a dead bacteria staining control. (C) Evaluation of the ability of 1E4, Fab1E4, muscFv1E4, and huscFv1E4 to directly kill C. burnetii. A total of 100 bacteria were counted per slide at a magnification of ×1,000 using a fluorescence microscope, and numbers of dead bacteria were recorded in duplicate experiments. The data presented in each group are the averages with standard deviations from duplicate numbers of dead bacteria. ***, P < 0.001.