Improved OMV vaccine against Neisseria meningitidis using genetically engineered strains and a detergent-free purification process

The use of detergent-extracted outer membrane vesicles (OMVs) is an established approach for development of a multivalent PorA vaccine against N. meningitidis serogroup B. Selective removal of lipopolysaccharide (LPS) decreases toxicity, but promotes aggregation and narrows the immune response. Detergent-free OMV vaccines retain all LPS, which preserves the native vesicle structure, but result in high toxicity and lower yield. The present study assessed the effects of gene mutations that attenuated LPS toxicity (lpxL1) or improved OMV yield (rmpM) in combination with the available OMV purification processes. The results substantiate that OMVs from a strain with both mutations, produced with a detergent-free process provide better vaccine characteristics than the traditional detergent-based approach. With comparable toxicity and yield, no aggregation and cross-protection against other PorA subtypes, these OMV vaccines are potentially safe and effective for parenteral use in human

Improved OMV vaccine against Neisseria meningitidis using genetically engineered strains and a detergent-free purification process

The use of detergent-extracted outer membrane vesicles (OMVs) is an established approach for development of a multivalent PorA vaccine against N. meningitidis serogroup B. Selective removal of lipopolysaccharide (LPS) decreases toxicity, but promotes aggregation and narrows the immune response. Detergent-free OMV vaccines retain all LPS, which preserves the native vesicle structure, but result in high toxicity and lower yield. The present study assessed the effects of gene mutations that attenuated LPS toxicity (lpxL1) or improved OMV yield (rmpM) in combination with the available OMV purification processes. The results substantiate that OMVs from a strain with both mutations, produced with a detergent-free process provide better vaccine characteristics than the traditional detergent-based approach. With comparable toxicity and yield, no aggregation and cross-protection against other PorA subtypes, these OMV vaccines are potentially safe and effective for parenteral use in human

Meningococcal outer membrane vesicle composition-dependent activation of the innate immune response

Meningococcal outer membrane vesicles (OMVs) have been extensively investigated and successfully implemented as vaccines. They contain pathogen associated molecular patterns including lipopolysaccharide (LPS), capable of triggering innate immunity. However, Neisseria meningitidis contains an extremely potent hexa-acylated LPS, leading to adverse effects when its OMVs are applied as vaccines. To create safe OMV vaccines detergent treatment is generally used to reduce LPS content. While effective, this method also leads to loss of protective antigens such as lipoproteins. Alternatively, genetic modification of LPS can reduce its toxicity. In the present study, we have compared standard OMV isolation methods using detergent or EDTA with genetic modifications of LPS to yield a penta-acylated lipid A (lpxL1 and pagL), on the in vitro induction of innate immune responses. The use of detergent decreased both TLR4 and TLR2 activation by OMVs, while the LPS modifications only reduced TLR4 activation. Mutational removal of PorB or fHbp, two proteins known to trigger TLR2 signaling, had no effect indicating that multiple TLR2 ligands are removed by detergent treatment. Detergent treated OMV and lpxL1 OMV showed similar reduction of cytokine profiles in the human monocytic cell line MM6 and human DCs. OMVs with the alternative penta-acylated LPS structure obtained after PagL-mediated deacylation showed reduced induction of pro-inflammatory cytokines IL-6 and IL-1β but not of IP-10, a typical TRIF dependent chemokine. Taken together, these data show that lipid A modification can be used to obtain OMVs with reduced activation of innate immunity, similar to what is found after detergent treatment

Quality of sOMV and eOMV vaccines.

<p>In addition to yield, quality of the sOMV and eOMV vaccines is compared. It was previously demonstrated that both vaccine types provide low toxicity and high functional immunogenicity in mice <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054314#pone.0054314-vandeWaterbeemd1" target="_blank">[11]</a>. (A) Protein composition of eOMV reference vaccines is comparable to sOMV vaccines after cysteine depletion (time points I, K, M). Each lane contains 4 µg total protein, except sOMV at time points D and F (low protein concentration due to a low yield; maximal sample volume is loaded). PorA antigen (∼41 kD) has a major contribution to total protein content (>60%) in all vaccines. (B) Dynamic light scattering analysis reveals that sOMV vaccines have a slightly broader size distribution and minor aggregation compared to the eOMV references, indicating that the purification procedure is not yet fully consistent. X-axis represents vesicle size distribution (nm).</p

Implications for vaccine development.

<p>A novel approach for the production of sOMV vaccine against N. meningitidis serogroup B is explored by utilizing the effect of cysteine depletion. (A) Biomass concentration (closed circles) is monitored in bioreactor cultivations (time points A to N). Time point G marks onset of stationary growth, caused by depletion of cysteine (open circles). (B) Yield of purified sOMV vaccine (black bars) is compared with eOMV reference vaccine, which uses detergent-free biomass extraction to improve yield (white bars). Several time points before (D, F) and after (I, K, M) cysteine depletion are included. After cysteine depletion, sOMV yield increases gradually to quantities that are comparable to the eOMV reference (no significant difference at time point M). Significant yield differences are indicated with asterikses (p<0.05). ‘NS’ indicates a non-significant difference.</p