Passive Immunization to Outer Membrane Proteins MLP and PAL Does Not Protect Mice from Sepsis

Abstract

Multiple older studies report that immunoglobulin directed to rough mutant bacteria, such as E. coli J5, provides broad protection against challenge with heterologous strains of Gram-negative bacteria. This protection was initially believed to occur through binding of immunoglobulin to bacterial lipopolysaccharide (LPS). However, hundreds of millions of dollars have been invested in attempting to develop clinically-effective anti-LPS monoclonal antibodies without success, and no study has shown that IgG from this antiserum binds LPS. Identification of the protective mechanism would facilitate development of broadly protective human monoclonal antibodies for treating sepsis. IgG from this antiserum binds 2 bacterial outer membrane proteins: murein lipoprotein (MLP) and peptidoglycan-associated lipoprotein (PAL). Both of these outer membrane proteins are highly conserved, have lipid domains that are anchored in the bacterial membrane, are shed from bacteria in blebs together with LPS, and activate cells through Toll-like receptor 2. Our goal in the current work was to determine if passive immunization directed to MLP and PAL protects mice from Gram-negative sepsis. Neither monoclonal nor polyclonal IgG directed to MLP or PAL conferred survival protection in 3 different models of sepsis: cecal ligation and puncture, an infected burn model, and an infected fibrin clot model mimicking peritonitis. Our results are not supportive of the hypothesis that either anti-MLP or anti-PAL IgG are the protective antibodies in the previously described anti-rough mutant bacterial antisera. These studies suggest that a different mechanism of protection is involved.

Hemopexin in severe inflammation and infection: mouse models and human diseases

“…The mouse burn wound infection model was performed as described previously [ 15 ], followed immediately by subcutaneous injection of E. coli O4 at log phase into the burned area as described. Blood was collected from the tail vein at different times, except the terminal bleed, which was done by cardiac puncture.…”

“…This model of sepsis due to peritonitis was adapted from Ahrenholz and Simmons as described previously [ 15 , 16 ]. Mice were implanted with a fibrin clot with or without E. coli O4 as desired or with 250 μl of blood in the peritoneum.…”

Bacterial Lipoprotein TLR2 Agonists Broadly Modulate Endothelial Function and Coagulation Pathways In Vitro and In Vivo

“…PAL and MLP were prepared from E. coli bacteria as described, and were confirmed to contain a single protein band by gold staining (29, 34). Pam3CysSKKK was purchased (EMC Microcollections, Tubingen, Germany).…”

Peptidoglycan-associated lipoprotein (Pal) of Gram-negative bacteria: function, structure, role in pathogenesis and potential application in immunoprophylaxis

“…The effect of passive immunization with mono‐ and polyclonal anti‐Pal antibodies has been tested in three different mouse models of sepsis (cecal ligation and puncture, an infected burn model, and an infected fibrin clot model mimicking peritonitis). However, these trials indicated that this type of strategy does not have any protective/therapeutic effect (Valentine et al , 2006).…”