Expression of foreign LpxA acyltransferases in Neisseria meningitidis results in modified lipid A with reduced toxicity and retained adjuvant activity

Abstract

SummaryA major problem in the development of vaccines against Gram-negative bacteria is the endotoxic activity of lipopolysaccharide (LPS), which is determined by its lipid A moiety. Nevertheless, LPS would be an interesting vaccine component because of its immune-stimulating properties. In the present study, we have changed the fatty acid composition of Neisseria meningitidis LPS by replacing the lpxA gene of strain H44/76 with the Escherichia coli or Pseudomonas aeruginosa homologue. The majority of the Olinked 3-OH C12 in N. meningitidis lipid A was replaced by 3-OH C14 (strain HA01E) and 3-OH C10 (strain HA25P) respectively. Both strains, but most notably strain HA01E, had reduced amounts of LPS compared with the wild-type strain. In addition, growth was severely impaired for HA01E. The major outer membrane proteins were expressed normally. Outer membrane complexes of both strains normalized on their LPS content showed a 10-fold reduction in their ability to induce tumour necrosis factor (TNF)-α α α α . Immunogenicity studies in BALB/c mice revealed that the adjuvant activity of the LPS was not affected. Thus, the replacement of the O -linked fatty acids in meningococcal lipid A results in immunogenic outer membranes with reduced endotoxic activity, more suitable for use in outer membrane vesicle vaccines.

Crystal structure and catalytic mechanism of the LPS 3-O-deacylase PagL fromPseudomonas aeruginosa

“…Unfortunately, a broad-scale use of this adjuvant quality is not possible because of the endotoxic activity. Recently, it has been shown that changing the physico-chemical properties of LPS influences its endotoxic activity (1,22,23). Thus, LPS-modifying enzymes may be valuable tools for detoxification of LPS.…”

Expression of the Lipopolysaccharide-Modifying Enzymes PagP and PagL Modulates the Endotoxic Activity ofBordetella pertussis

“…Previous studies have shown that both the phosphate groups and the number and length of the acyl chains are important determinants of the endotoxic properties of lipid A (31,44,54). Recognition of LPS is based upon its binding to LBP and subsequent transfer to CD14, followed by interaction with the TLR4/MD2 complex.…”

“…The endotoxic activity of LPS is determined entirely by the composition of the lipid A moiety (23). Previous studies have shown that the phosphate groups as well as the number and length of the acyl chains are critical determinants of the endotoxic activity of lipid A (31,44,54). Besides its endotoxic activity, LPS also has a powerful adjuvant activity and in that respect is potentially an interesting vaccine component.…”

TLR4-dependent adjuvant activity of Neisseria meningitidis lipid A

“…LOSspecific antibodies were not induced in mice sera even when higher doses of meningococcal LOS (5 µg) were used as the adjuvant. Steeghs et al, using various LPSs as adjuvants, demonstrated that the composition and length of the carbohydrate chain of LPS do not influence the induction of bactericidal antibodies against PorA proteins [6,36]. Further, the influence of various adjuvants on the immune response towards LPS-deficient OMPs indicates that 5 µg meningococcal LOS is at least equivalent to 20 µg MPL (monophoshoryl lipid A), 500 µg AIPO 4 (aluminium hydroxide), 20 µg Quil A or 20 µg MF59 [6].…”