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

Cysteine is the growth-limiting medium component.

<p>Previous work demonstrated that vesicle release by <i>N. meningitidis</i> increased during the stationary growth phase <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054314#pone.0054314-vandeWaterbeemd2" target="_blank">[23]</a>. Nutrient analysis of the chemically defined medium indicated that only arginine and cysteine were depleted during early stationary growth. Therefore, concentrations of both components were systematically lowered to identify the growth-limiting nutrient. Black lines and black intersected lines represent growth curves on media with normal and low arginine concentration, respectively. Normal and low cysteine concentrations are marked with circles and triangles. Cultivation time t = 0 represents the expected onset of stationary growth on reference medium with normal amounts of cysteine and arginine. The results indicate that cysteine, not arginine is the growth-limiting component. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054314#pone.0054314.s002" target="_blank">Figure S2</a> for corresponding nutrient data.</p

Prevalence and Clinical Course in Invasive Infections with Meningococcal Endotoxin Variants

<div><h3>Background</h3><p>Meningococci produce a penta-acylated instead of hexa-acylated lipid A when their <em>lpxL1</em> gene is inactivated. Meningococcal strains with such lipid A endotoxin variants have been found previously in adult meningitis patients, where they caused less blood coagulopathy because of decreased TLR4 activation.</p> <h3>Methods</h3><p>A cohort of 448 isolates from patients with invasive meningococcal disease in the Netherlands were screened for the ability to induce IL-6 in monocytic cell Mono Mac 6 cells. The <em>lpxL1</em> gene was sequenced of isolates, which show poor capacity to induce IL-6.. Clinical characteristics of patients were retrieved from hospital records.</p> <h3>Results</h3><p>Of 448 patients, 29 (6.5%) were infected with meningococci expressing a lipid A variant strain. Lipid A variation was not associated with a specific serogroup or genotype. Infections with lipid A variants were associated with older age (19.3 vs. 5.9 (median) years, p = 0.007) and higher prevalence of underlying comorbidities (39% vs. 17%; p = 0.004) compared to wild-type strains. Patients infected with lipid A variant strains had less severe infections like meningitis or shock (OR 0.23; 95%CI 0.09–0.58) and were less often admitted to intensive care (OR 0.21; 95%CI 0.07–0.60) compared to wild-type strains, independent of age, underlying comorbidities or strain characteristics.</p> <h3>Conclusions</h3><p>In adults with meningococcal disease lipid A variation is rather common. Infection with penta-acylated lipid A variant meningococci is associated with a less severe disease course.</p> </div

Antibody titers of mice after immunization with <i>N. meningitidis</i> P1.5-1,2-2 OMVs.

<p>Mice were immunized with <i>N. meningitidis</i> P1.5-1,2-2 OMVs and antigen-specific titers of IgG, IgG1, IgG2a/c, IgG2b, and IgG3 in sera were determined with ELISA. Also total IgE in sera of mice immunized with PBS or <i>N. meningitidis</i> P1.5-1,2-2 OMVs was measured with ELISA. Results for C57BL/6, TLR2−/−, and TRIF-deficient (TRIFdef) mice are shown in panel A, results for C3H/HeOuJ and C3H/HeJ mice are shown in panel B. Levels of serum bactericidal antibodies after immunization with <i>N. meningitidis</i> P1.5-1,2-2 OMVs are shown in panel C. Data are expressed as means of log₁₀ titers for 6 mice per group, except levels of serum bactericidal antibodies in C57BL/6 and TLR2−/− mice (n = 12). Error bars represent S.E.M. An asterisk indicates a significant difference compared to the wild type group, * indicates p<0.05, ** indicates p<0.01.</p

Cytokines produced by spleen cells after restimulation with FHA.

<p>Spleen cells from C57BL/6, TLR2−/−, TRIF-deficient (TRIFdef) mice (A), or C3H/HeOuJ and C3H/HeJ mice (B) immunized with PBS or whole cell pertussis vaccine were incubated for 4 days with 500 ng/ml FHA Cytokines IL-2, IL-4, IL-5, IL-10, IL-13, IL-17, and IFN-γ were determined in the supernatant with Luminex. Data are expressed as means of three mice per group (PBS), or six mice per group (whole cell pertussis vaccine), error bars represent S.E.M. An asterisk indicates a significant difference compared to the wild type group, * indicates p<0.05, ** indicates p<0.01.</p

Antibody titers of mice after immunization with whole cell pertussis vaccine.

<p>Mice were immunized with whole cell pertussis vaccine (<i>Bordetella pertussis</i>) and antigen-specific titers of IgG, IgG1, IgG2a/c, IgG2b, and IgG3 in sera were determined with ELISA. Also total IgE in sera of mice immunized with PBS or whole cell pertussis vaccine was measured with ELISA. Results for C57BL/6, TLR2−/−, and TRIF-deficient (TRIFdef) mice are shown in panel A, results for C3H/HeOuJ and C3H/HeJ mice are shown in panel B. Data are expressed as means of log₁₀ titers for 6 mice per group. An asterisk indicates a significant difference compared to the wild type group, * indicates p<0.05, ** indicates p<0.01.</p

Multivariable analyses for impact of infection with lipid A variants on different outcome variables.

a<p>Odd ratio adjusted for age, gender, underlying comorbidities, serogroup and clonal complex.</p

Cytokines produced by spleen cells after restimulation with P1.5-1,2-2 PorA peptides.

<p>Spleen cells from C57BL/6, TLR2−/−, or TRIF-deficient (TRIFdef) mice immunized with PBS or <i>N. meningitidis</i> P1.5-1,2-2 OMVs were incubated for 4 days with peptide 11–24/25 (A) or peptide 4–52/53 (B). Spleen cells from C3H/HeOuJ and C3H/HeJ mice immunized with PBS or <i>N. meningitidis</i> P1.5-1,2-2 OMVs were incubated for 4 days with peptide pool 6 (C). Cytokines IL-4, IL-5, IL-10, IL-13, IL-17, and IFN-γ were determined in the supernatant with Luminex. Data are expressed as means of three mice per group (PBS), or six mice per group (<i>N. meningitidis</i> OMVs), error bars represent S.E.M. An asterisk indicates a significant difference compared to the wild type group, * indicates p<0.05, ** indicates p<0.01.</p

Serum cytokine levels shortly after vaccination.

<p>Two and four hours after immunization blood samples were taken from all mice and cytokine levels in the sera were analyzed with Luminex. Results for <i>N. meningitidis</i> OMVs are shown in panel A, results for whole cell pertussis vaccine are shown in panel B. There were 3 mice per group for the animals that received PBS and 6 mice per group for <i>N. meningitidis</i> OMVs and whole cell pertussis vaccine. The data are expressed as means, error bars represent S.E.M. An asterisk indicates a significant difference compared to the wild type group, * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001.</p