4 research outputs found
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
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
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
Proteome Analysis Is a Valuable Tool to Monitor Antigen Expression during Upstream Processing of Whole-Cell Pertussis Vaccines
Physicochemical
and immunochemical assays were applied to substantiate
the relation between upstream processing and the quality of whole-cell
pertussis vaccines. <i>Bordetella pertussis</i> bacteria
were cultured on a chemically defined medium using a continuous cultivation
process in stirred tank reactors to obtain uniform protein expression.
Continuous culture favors the consistent production of proteins known
as virulence factors. Magnesium sulfate was added during the steady
state of the culture in order to diminish the expression of virulence
proteins. Changes in gene expression and antigen composition were
measured by microarrays, mass spectrometry and ELISA. Transcriptome
and proteome data revealed high similarity between the biological
triplicates demonstrating consistent cultivation of <i>B. pertussis</i>. The addition of magnesium sulfate resulted in an instant downregulation
of the virulence genes in <i>B. pertussis</i>, but
a gradual decrease of virulence proteins. The quantity of virulence
proteins concurred highly with the potency of the corresponding whole-cell
pertussis vaccines, which were determined by the Kendrick test. In
conclusion, proteome analysis provided detailed information on the
composition and proportion of virulence proteins present in the whole-cell
preparations of <i>B. pertussis</i>. Moreover, proteome
analysis is a valuable method to monitor the production process of
whole-cell biomass and predict the product quality of whole-cell pertussis
vaccines