Table 1.
Bacterial strains, Antibodies and Plasmids.
Table 2.
Primers used for the amplification of the variable domains.
Table 3.
Sequences of heavy and light chain of the 4 antibodies isolated after the 4th round of selection.
Fig 1.
Opsonic killing of sera from healthy human volunteers.
Opsonophagocytic killing against E. faecalis 12030 was assessed in sera at a dilution of 1:100. Statistical analysis was done by ANOVA with Dunnett post test. * p<0.05, ** p<0.01, *** p<0.001. Error bars represent standard error of the mean.
Fig 2.
Determination of the target of VH4E and VH8.
(A) Opsonophagocytic Inhibition Assay after absorbing mAbs with E. faecalis 12030 treated with Proteinase K or NaIO4 (B) Binding of VH4E and VH8 to LTA is shown in an ELISA coating wells with S. aureus LTA (C) Osonophagocytic Inhibition Assay absorbing monoclonal antibodies with LTA from S. aureus. (A) Killing of VH4E and VH8 against E. faecalis 12030 is about 65% (first white or grey column) and killing is completely abolished when bacteria are treated with proteinase K before absorption. Opsonic activity of VH4E and VH8 is not inhibited when mAbs are absorbed with E. faecalis treated with NaIO4 indicating that a polysaccharide is the target of the mAbs. (B) Binding of VH8 and the mouse anti-lipoteichoic monoclonal antibody to LTA was tested in an ELISA. As coating antigen LTA from S. aureus was used and serum dilutions are indicated in the X axis. Each point represents the average of two measurements. (C) An Opsonophagytosis assay after absorbing monoclonal antibodies with purified LTA confirmed the results of the ELISA. Opsonophagocytic killing was compared to controls from which leukocytes were obtained. Statistical analysis was done by ANOVA with Dunnett post test. * p<0.05, ** p<0.01, *** p<0.001. Error bars represent standard error of the mean.
Fig 3.
Measurement of opsonophagocytic killing of monoclonal antibodies VH4E and VH8 and the mouse anti-lipoteichoic monoclonal antibody against (A) MRSA strain LAC and (B) E. faecalis 12030.
The opsonophagocytic assay was performed using baby rabbit serum as complement source and rabbit sera raised against purified LTA from E. faecalis 12030. VH4E and VH8 show high opsonic killing against both strains in a dose dependent manner and are clearly more efficient than the mouse anti-lipoteichoic monoclonal antibody, which showed modest killing against LAC at very high concentrations (i.e. 1,000x). Opsonophagocytic killing activity was compared to controls from which leukocytes were omitted. Statistical analysis was done by ANOVA with Dunnett post test. * p<0.05, ** p<0.01, *** p<0.001. Error bars represent standard error of the mean.
Fig 4.
Investigating efficacy of the monoclonal antibodies in two independent animal models.
A mouse bacterial sepsis model was tested for the MRSA strain LAC (A) and E. faecium (B). A rat endocarditis model was confirms the protection of VH4E (C and D). In the mouse bacteremia model E. faecium (1.8 x 108 cfu) or S. aureus (5.0 x 107 cfu) was injected i.v. into the tail vein. Cfu in the liver and the kidney was assessed after 24h. Statistical analysis was done by ANOVA with Tukey post test (a and b). * p<0.05, ** p<0.01, *** p<0.001. Error bars represent standard error of the mean. A rat endocarditis model confirmed protection against E. faecalis 12030 by VH4E (at a total concentration of 10 pg). Inoculation of bacteria was done 48 h after catheter placement via injection into the tail vein. Bacterial counts in the vegetations (cfu per mg vegetation) are shown in (C), and (D) shows the absolute weights of explanted vegetation. Differences in mg per vegetations are significant (p < 0.05) and were tested by unparied t-test (c and d). * p<0.05, ** p<0.01, *** p<0.001. Error bars represent standard error of the mean.
Fig 5.
Protection against S. aureus LAC infection with VH8 and mouse anti-lipoteichoic monoclonal antibody.
A dose of 12ng/mL VH8, 3000ng/mL mouse anti-lipoteichoic monoclonal antibody and 200 μl of Normal Rabbit Serum were given to animals 24 hours before bacterial challenge. Strain S. aureus LAC was used at a challenge dose of 2x109 cfu/mouse (6 mice per group). Protection by different sera was observed for VH8 (5/6 mice survived) while the mouse anti-lipoteichoic monoclonal antibody and the NRS groupdid not result in protection.