Bactericidal fully human single-chain fragment variable antibodies protect mice against methicillin-resistant Staphylococcus aureus bacteraemia

Abstract

Objectives: The increasing prevalence of antibiotic-resistant Staphylococcus aureus, besides the inadequate numbers of effective antibiotics, emphasises the need to find new therapeutic agents against this lethal pathogen.

Methods: In this study, to obtain antibody fragments against S. aureus, a human single-chain fragment variable (scFv) library was enriched against living methicillin-resistant S. aureus (MRSA) cells, grown in three different conditions, that is human peripheral blood mononuclear cells with plasma, whole blood and biofilm. The antibacterial activity of scFvs was evaluated by the growth inhibition assay in vitro. Furthermore, the therapeutic efficacy of anti-S. aureus scFvs was appraised in a mouse model of bacteraemia.

Results: Three scFv antibodies, that is MEH63, MEH158 and MEH183, with unique sequences, were found, which exhibited significant binding to S. aureus and reduced the viability of S. aureus in in vitro inhibition assays. Based on the results, MEH63, MEH158 and MEH183, in addition to their combination, could prolong the survival rate, reduce the bacterial burden in the blood and prevent inflammation and tissue destruction in the kidneys and spleen of mice with MRSA bacteraemia compared with the vehicle group (treated with normal saline).

Conclusion: The combination therapy with anti-S. aureus scFvs and conventional antibiotics might shed light on the treatment of patients with S. aureus infections.

Keywords: bacteraemia; bactericidal antibodies; fully human antibody; methicillin‐resistant Staphylococcusaureus; single‐chain fragment variable.

Figure 1

scFv antibodies were expressed in E. coli HB2151. (a) The successful expression of scFv antibody (molecular weight, 27 kDa) in E. coli HB2151 was examined by SDS‐PAGE (12%). Lane M, protein marker; lane HB2151, the periplasmic extract of normal E. coli HB2151 induced by 0.1 mM IPTG. (b) Western blot analysis. A single band of approximately 27 kDa, corresponding to scFv, was found by probing with mouse anti‐human scFv antibody, followed by goat anti‐mouse IgG‐horseradish peroxidase (HRP) antibody, and visualised by DAB. Lane M, protein marker. DAB, diaminobenzidine; IPTG, isopropyl β‐d‐1‐thiogalactopyranoside.

Figure 2

MEH63, MEH158 and MEH183 showed significant binding to S. aureus. The purity and binding ability of MEH63, MEH158 and MEH183 were analysed by reducing SDS‐PAGE (12%) and dot‐blot assay respectively. (a) SDS‐PAGE. A single band was observed at ~ 27 kDa. Lane M, protein marker. (b) The binding ability of MEH63, MEH158 and MEH183 to S. aureus S.a.124 (S.a.), S. epidermidis ATCC 12228 (S.e.), S. pyogenes ATCC 10403 (S.p.) and A. baumannii A.b.56 (A.b.) was appraised by the dot‐blot assay. The controls included the spotted bacteria incubated with BSA (5 mg mL^‐1), followed by mouse anti‐human scFv antibody (MoAb) and then goat anti‐mouse IgG‐horseradish peroxidase (HRP) antibody (GoAb); MEH63 and then mouse anti‐human scFv antibody; MEH63 and then goat anti‐mouse IgG‐HRP; mouse anti‐human scFv antibody and then goat anti‐mouse IgG‐HRP; and DAB. (c) The cross‐reactivity of scFvs with PBMCs was examined using the dot‐blot assay. The controls included the cells incubated with MEH63 and then mouse anti‐human scFv antibody; MEH63 and then goat anti‐mouse IgG‐HRP; mouse anti‐human scFv antibody and then goat anti‐mouse IgG‐HRP; and DAB. BSA, bovine serum albumin; DAB, diaminobenzidine; IgG, immunoglobulin G; LC, light chain; HC, heavy chain; PBMCs, peripheral blood mononuclear cells.

Figure 3

MEH63, MEH158 and MEH183 exhibited antibacterial activities. The antimicrobial activity of MEH63, MEH158 and MEH183 was evaluated by the agar plate assay. (a) The inhibitory activity of MEH63, MEH158 and MEH183 against S. aureus S.a.48, S.a.61, S.a.124 and ATCC 6538 (after 4 h of incubation). Vancomycin was used as the positive control. (b) Different concentrations of MEH63, MEH158 and MEH183, and (c) the scFv combination could significantly reduce the colony‐forming unit count of S. aureus S.a.48 and S.a.124. (d) MEH63, MEH158 and MEH183 (200 µg mL^‐1) exhibited antibacterial activity against S. aureus S.a.48, S. epidermidis ATCC 12228 and S. pyogenes ATCC 10403, but not against A. baumannii A.b.56. Data are representative of three independent experiments, and error bars correspond to the mean ± SEM. * P‐value < 0.05, ** P‐value = 0.01 and *** P‐value < 0.01.

Figure 4

MEH63, MEH158 and MEH183 reacted with some cell wall proteins of S. aureus, S. epidermidis and S. pyogenes. The crude cell wall extracts (CW) of S. aureus S.a.124 (S.a.), S. epidermidis ATCC 12228 (S.e.), S. pyogenes ATCC 10403 (S.p) and A. baumannii A.b.56 (A.b.), as well as non‐covalent bond cell wall proteins (NB) of S. aureus S.a.124, were assessed by SDS‐PAGE and Western blot analysis. (a) SDS‐PAGE (12%). Lane M, protein marker. (b) Western blot analysis. The crude cell wall extracts and non‐covalent bond cell wall proteins, separated by SDS‐PAGE, were blotted onto the PVDF membranes. The membranes were incubated with MEH63, MEH158 or MEH183. After incubation with mouse anti‐human scFv antibody and then goat anti‐mouse IgG‐horseradish peroxidase (HRP) antibody, the bands were visualised with DAB and H₂O₂. Lane M, protein marker. DAB, diaminobenzidine.

Figure 5

MEH63, MEH158 and MEH183 showed negligible haemolytic activities against rabbit erythrocytes and no cytotoxic activity in vivo. The toxic activity of MEH63, MEH158 and MEH183 was assessed in vitro (haemolysis assay) and in vivo. (a) The treatment of rabbit erythrocytes with MEH63, MEH158 and MEH183 (400 µg mL^‐1) led to 0.9%, 0.7% and 0.7% haemolysis respectively. The incubation of rabbit erythrocytes with normal saline and 0.1% Triton X‐100 resulted in 0% and 100% haemolysis respectively. Data are representative of three independent experiments, and error bars correspond to the mean ± SEM. The histopathological evaluation of (b) kidneys and (c) liver of mice receiving 8 μg per gram of MEH63, MEH158 and MEH183 (alone and in combination) was conducted every 12 h for three days. Furthermore, the mice receiving normal saline or 20 μg per gram of vancomycin every 12 h for three days served as the controls. No toxic activity or tissue damage was observed in the kidneys or liver. Yellow arrowhead, renal corpuscle; red arrowhead, hepatocytes; red arrow, sinusoids; and V, central vein.

Figure 6

MEH63, MEH158 and MEH183 exhibited therapeutic efficacy in a mouse model of MRSA bacteraemia. To assess the therapeutic activity of MEH63, MEH158 and MEH183, the mice (n = 8) were intravenously infected with MRSA S.a.124 (~ 10⁸ CFU). Two h after the challenge, the infected mice were intraperitoneally treated with MEH63, MEH158, MEH183, the scFv combination, vancomycin, EB211 (an unrelated scFv antibody) or normal saline (vehicle) every 12 h (BID) for three days. The scFv combination was also administrated every 24 h (QD) for three days. (a) Mortality was recorded daily for two weeks, and the survival rate was calculated using the log‐rank test (Mantel–Cox test). Data are representative of three independent experiments. * P‐value = 0.024. (b) The bacterial colony‐forming unit count in the blood of infected mice (n = 6), treated with MEH63, MEH158, MEH183, vancomycin, EB211 or normal saline BID for three days. The scFv combination was also administrated QD for three days. Data are representative of three independent experiments, and error bars correspond to the mean ± SEM. * P‐value < 0.05, ** P‐value < 0. 01 and *** P‐value < 0.001.

Figure 7

MEH63, MEH158 and MEH183 attenuated S. aureus‐induced inflammation and prevented tissue damage in kidneys. Mice (n = 6) were intravenously infected with MRSA S.a.124 (~ 10⁸ CFU). Two h after the challenge, the infected mice were intraperitoneally treated with MEH63, MEH158, MEH183, the scFv combination, vancomycin or normal saline (vehicle) every 12 h (BID) for three days. The kidneys were harvested from the uninfected mice, the vehicle group and infected mice receiving scFvs or vancomycin. The H & E‐stained sections of tissues were histopathologically examined. Bacterial foci and inflammatory infiltration of neutrophils and macrophages (yellow arrows) at 24, 48 and 72 h after the challenge, besides necrosis of the lining epithelium of renal tubules (asterisks) at 48 and 72 h after the challenge, are marked pathological events in the kidneys of the vehicle group, but not in the groups treated with either anti‐S. aureus scFvs or vancomycin. Yellow arrowhead, renal corpuscle.

Figure 8

MEH63, MEH158 and MEH183 attenuated S. aureus‐induced inflammation and prevented tissue damage in the spleen. Mice (n = 6) were intravenously infected with MRSA S.a.124 (~ 10⁸ CFU). Two h after the challenge, the infected mice were intraperitoneally treated with MEH63, MEH158, MEH183, the scFv combination, vancomycin or normal saline (vehicle) every 12 h (BID) for three days. The spleens were harvested from the uninfected mice, the vehicle group and infected mice receiving scFvs or vancomycin. The H & E‐stained sections of tissues were histopathologically examined. Basophilic granular materials and macrophages (white arrows) and widened sinusoids (black arrows) were observed after 24, 48 and 72 h of infection in the spleen of mice in the vehicle group. Also, the excessive accumulation and congestion of blood (black arrowheads) and hyperplastic Malpighian follicles with fragmented cells were observed after 48 and 72 h of infection in the vehicle group. At 24, 48 and 72 h after infection, no significant pathological symptoms were found in mice treated with MEH63, MEH158, MEH183, the scFv combination or vancomycin. White arrowhead, megakaryocyte; R, red pulp; and W, white pulp.