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High yield production process for Shigella outer membrane particles.

Berlanda Scorza F, Colucci AM, Maggiore L, Sanzone S, Rossi O, Ferlenghi I, Pesce I, Caboni M, Norais N, Di Cioccio V, Saul A, Gerke C - PLoS ONE (2012)

Bottom Line: Yields of approximately 100 milligrams of membrane-associated proteins per liter of fermentation were obtained from cultures of S. sonnei ΔtolR ΔgalU at optical densities of 30-45 in a 5 L fermenter.These were highly immunogenic in mice.Furthermore, we demonstrate the feasibility of using this process with other genetic manipulations e.g. abolition of O antigen synthesis and modification of the lipopolysaccharide structure in order to modify the immunogenicity or reactogenicity of the particles.

View Article: PubMed Central - PubMed

Affiliation: Novartis Vaccines Institute for Global Health, Siena, Italy.

ABSTRACT
Gram-negative bacteria naturally shed particles that consist of outer membrane lipids, outer membrane proteins, and soluble periplasmic components. These particles have been proposed for use as vaccines but the yield has been problematic. We developed a high yielding production process of genetically derived outer membrane particles from the human pathogen Shigella sonnei. Yields of approximately 100 milligrams of membrane-associated proteins per liter of fermentation were obtained from cultures of S. sonnei ΔtolR ΔgalU at optical densities of 30-45 in a 5 L fermenter. Proteomic analysis of the purified particles showed the preparation to primarily contain predicted outer membrane and periplasmic proteins. These were highly immunogenic in mice. The production of these outer membrane particles from high density cultivation of bacteria supports the feasibility of scaling up this approach as an affordable manufacturing process. Furthermore, we demonstrate the feasibility of using this process with other genetic manipulations e.g. abolition of O antigen synthesis and modification of the lipopolysaccharide structure in order to modify the immunogenicity or reactogenicity of the particles. This work provides the basis for a large scale manufacturing process of Generalized Modules of Membrane Antigens (GMMA) for production of vaccines from gram-negative bacteria.

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ELISA analysis of sera reactivity against GMMA.Groups 1–6 received 2 µg of GMMA with or without Freund’s adjuvant (FA), group 1) GMMA from S. sonnei ΔtolR ΔgalU (grown in HTMC, 37°C), 2) GMMA of group 1 plus FA, 3) GMMA S. sonnei –pSS ΔtolR (defined medium, 37°C), 4) GMMA of group 3 plus FA, 5) GMMA from S. sonnei –pSS ΔtolR ΔmsbB (defined medium, 30°C), 6) GMMA of group 5 plus FA. Group 7 received 0.2 µg of GMMA from S. sonnei –pSS ΔtolR ΔmsbB. Control groups were immunized with PBS alone (group 8) or FA alone (group 9). Sera from individual mice obtained 14 days after the third immunization and pooled preimmune sera from each group respectively were assayed in dilutions of 1∶1000, 1∶10,000, and 1∶100,000 on GMMA from S. sonnei 53G –pSS ΔtolR as coating and arbitrary units were calculated. Data are presented as scatter plots of ELISA units determined in individual mice (groups 1–9) or of the pooled preimmune sera (pre). The horizontal lines represent the geometric mean. ELISA units of groups 1–6 receiving 2 µg of GMMA were analyzed using the non-parametric Kruskal-Wallis test to compare the immunogenicity of the different GMMA to each other and with and without FA. No statistically significant differences were found (n.s.). Reduction of the immunization dosage of S. sonnei –pSS ΔtolR ΔmsbB GMMA to 0.2 µg (group 7) resulted in statistically significant reduction of ELISA units in the sera of the immunized animals compared to sera of mice immunized with 2 µg of the same GMMA (group 5) as determined by Mann-Whitney test (p = 0.0047). All groups receiving GMMA showed higher S. sonnei –pSS ΔtolR-specific antibody responses than groups immunized with PBS or FA alone (Mann-Whitney, p≤0.003). For all comparisons a p value smaller than 0.05 was considered to be significant.
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pone-0035616-g006: ELISA analysis of sera reactivity against GMMA.Groups 1–6 received 2 µg of GMMA with or without Freund’s adjuvant (FA), group 1) GMMA from S. sonnei ΔtolR ΔgalU (grown in HTMC, 37°C), 2) GMMA of group 1 plus FA, 3) GMMA S. sonnei –pSS ΔtolR (defined medium, 37°C), 4) GMMA of group 3 plus FA, 5) GMMA from S. sonnei –pSS ΔtolR ΔmsbB (defined medium, 30°C), 6) GMMA of group 5 plus FA. Group 7 received 0.2 µg of GMMA from S. sonnei –pSS ΔtolR ΔmsbB. Control groups were immunized with PBS alone (group 8) or FA alone (group 9). Sera from individual mice obtained 14 days after the third immunization and pooled preimmune sera from each group respectively were assayed in dilutions of 1∶1000, 1∶10,000, and 1∶100,000 on GMMA from S. sonnei 53G –pSS ΔtolR as coating and arbitrary units were calculated. Data are presented as scatter plots of ELISA units determined in individual mice (groups 1–9) or of the pooled preimmune sera (pre). The horizontal lines represent the geometric mean. ELISA units of groups 1–6 receiving 2 µg of GMMA were analyzed using the non-parametric Kruskal-Wallis test to compare the immunogenicity of the different GMMA to each other and with and without FA. No statistically significant differences were found (n.s.). Reduction of the immunization dosage of S. sonnei –pSS ΔtolR ΔmsbB GMMA to 0.2 µg (group 7) resulted in statistically significant reduction of ELISA units in the sera of the immunized animals compared to sera of mice immunized with 2 µg of the same GMMA (group 5) as determined by Mann-Whitney test (p = 0.0047). All groups receiving GMMA showed higher S. sonnei –pSS ΔtolR-specific antibody responses than groups immunized with PBS or FA alone (Mann-Whitney, p≤0.003). For all comparisons a p value smaller than 0.05 was considered to be significant.

Mentions: Groups of 8 CD1 mice were immunized 3 times with GMMA (2 µg of total protein) obtained from S. sonnei 53G –pSS ΔtolR and S. sonnei 53G –pSS ΔtolR ΔmsbB, both grown in defined medium with 2 µM iron, and S. sonnei 53G ΔtolR ΔgalU grown in HTMC. GMMA from S. sonnei 53G –pSS ΔtolR ΔgalU, S. sonnei 53G –pSS ΔtolR and S. sonnei 53G –pSS ΔtolR ΔmsbB were also administered in combination with Freund’s adjuvant (FA). Freund’s complete adjuvant was used in the first immunization and Freund’s incomplete adjuvant was used in the second and third immunization. In addition, a lower dosage of 0.2 µg of GMMA from S.sonnei 53G –pSS ΔtolR ΔmsbB was tested. Serum samples were obtained 2 weeks after the second and third doses and analyzed individually. Mice immunized with GMMA showed very high IgG responses to all 3 types of GMMA that were tested. No difference was found between groups immunized with different GMMA or between groups receiving the same GMMA with or without FA (Fig. 6). Adsorption of GMMA onto alum as adjuvant also did not have an effect on the IgG response (data not shown). Control mice immunized with PBS or FA alone had very low levels of anti-GMMA antibodies (Fig. 6). The 10-fold lower dosage of GMMA from S. sonnei 53G –pSS ΔtolR ΔmsbB (0.2 µg) resulted in a statistically significant, approximately 3-fold reduction in the IgG response compared to the group immunized with 2 µg of the same GMMA. However, the IgG response to the lower dosage still showed an approximately 8000-fold increase compared to preimmune sera (Fig. 6).


High yield production process for Shigella outer membrane particles.

Berlanda Scorza F, Colucci AM, Maggiore L, Sanzone S, Rossi O, Ferlenghi I, Pesce I, Caboni M, Norais N, Di Cioccio V, Saul A, Gerke C - PLoS ONE (2012)

ELISA analysis of sera reactivity against GMMA.Groups 1–6 received 2 µg of GMMA with or without Freund’s adjuvant (FA), group 1) GMMA from S. sonnei ΔtolR ΔgalU (grown in HTMC, 37°C), 2) GMMA of group 1 plus FA, 3) GMMA S. sonnei –pSS ΔtolR (defined medium, 37°C), 4) GMMA of group 3 plus FA, 5) GMMA from S. sonnei –pSS ΔtolR ΔmsbB (defined medium, 30°C), 6) GMMA of group 5 plus FA. Group 7 received 0.2 µg of GMMA from S. sonnei –pSS ΔtolR ΔmsbB. Control groups were immunized with PBS alone (group 8) or FA alone (group 9). Sera from individual mice obtained 14 days after the third immunization and pooled preimmune sera from each group respectively were assayed in dilutions of 1∶1000, 1∶10,000, and 1∶100,000 on GMMA from S. sonnei 53G –pSS ΔtolR as coating and arbitrary units were calculated. Data are presented as scatter plots of ELISA units determined in individual mice (groups 1–9) or of the pooled preimmune sera (pre). The horizontal lines represent the geometric mean. ELISA units of groups 1–6 receiving 2 µg of GMMA were analyzed using the non-parametric Kruskal-Wallis test to compare the immunogenicity of the different GMMA to each other and with and without FA. No statistically significant differences were found (n.s.). Reduction of the immunization dosage of S. sonnei –pSS ΔtolR ΔmsbB GMMA to 0.2 µg (group 7) resulted in statistically significant reduction of ELISA units in the sera of the immunized animals compared to sera of mice immunized with 2 µg of the same GMMA (group 5) as determined by Mann-Whitney test (p = 0.0047). All groups receiving GMMA showed higher S. sonnei –pSS ΔtolR-specific antibody responses than groups immunized with PBS or FA alone (Mann-Whitney, p≤0.003). For all comparisons a p value smaller than 0.05 was considered to be significant.
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Related In: Results  -  Collection

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pone-0035616-g006: ELISA analysis of sera reactivity against GMMA.Groups 1–6 received 2 µg of GMMA with or without Freund’s adjuvant (FA), group 1) GMMA from S. sonnei ΔtolR ΔgalU (grown in HTMC, 37°C), 2) GMMA of group 1 plus FA, 3) GMMA S. sonnei –pSS ΔtolR (defined medium, 37°C), 4) GMMA of group 3 plus FA, 5) GMMA from S. sonnei –pSS ΔtolR ΔmsbB (defined medium, 30°C), 6) GMMA of group 5 plus FA. Group 7 received 0.2 µg of GMMA from S. sonnei –pSS ΔtolR ΔmsbB. Control groups were immunized with PBS alone (group 8) or FA alone (group 9). Sera from individual mice obtained 14 days after the third immunization and pooled preimmune sera from each group respectively were assayed in dilutions of 1∶1000, 1∶10,000, and 1∶100,000 on GMMA from S. sonnei 53G –pSS ΔtolR as coating and arbitrary units were calculated. Data are presented as scatter plots of ELISA units determined in individual mice (groups 1–9) or of the pooled preimmune sera (pre). The horizontal lines represent the geometric mean. ELISA units of groups 1–6 receiving 2 µg of GMMA were analyzed using the non-parametric Kruskal-Wallis test to compare the immunogenicity of the different GMMA to each other and with and without FA. No statistically significant differences were found (n.s.). Reduction of the immunization dosage of S. sonnei –pSS ΔtolR ΔmsbB GMMA to 0.2 µg (group 7) resulted in statistically significant reduction of ELISA units in the sera of the immunized animals compared to sera of mice immunized with 2 µg of the same GMMA (group 5) as determined by Mann-Whitney test (p = 0.0047). All groups receiving GMMA showed higher S. sonnei –pSS ΔtolR-specific antibody responses than groups immunized with PBS or FA alone (Mann-Whitney, p≤0.003). For all comparisons a p value smaller than 0.05 was considered to be significant.
Mentions: Groups of 8 CD1 mice were immunized 3 times with GMMA (2 µg of total protein) obtained from S. sonnei 53G –pSS ΔtolR and S. sonnei 53G –pSS ΔtolR ΔmsbB, both grown in defined medium with 2 µM iron, and S. sonnei 53G ΔtolR ΔgalU grown in HTMC. GMMA from S. sonnei 53G –pSS ΔtolR ΔgalU, S. sonnei 53G –pSS ΔtolR and S. sonnei 53G –pSS ΔtolR ΔmsbB were also administered in combination with Freund’s adjuvant (FA). Freund’s complete adjuvant was used in the first immunization and Freund’s incomplete adjuvant was used in the second and third immunization. In addition, a lower dosage of 0.2 µg of GMMA from S.sonnei 53G –pSS ΔtolR ΔmsbB was tested. Serum samples were obtained 2 weeks after the second and third doses and analyzed individually. Mice immunized with GMMA showed very high IgG responses to all 3 types of GMMA that were tested. No difference was found between groups immunized with different GMMA or between groups receiving the same GMMA with or without FA (Fig. 6). Adsorption of GMMA onto alum as adjuvant also did not have an effect on the IgG response (data not shown). Control mice immunized with PBS or FA alone had very low levels of anti-GMMA antibodies (Fig. 6). The 10-fold lower dosage of GMMA from S. sonnei 53G –pSS ΔtolR ΔmsbB (0.2 µg) resulted in a statistically significant, approximately 3-fold reduction in the IgG response compared to the group immunized with 2 µg of the same GMMA. However, the IgG response to the lower dosage still showed an approximately 8000-fold increase compared to preimmune sera (Fig. 6).

Bottom Line: Yields of approximately 100 milligrams of membrane-associated proteins per liter of fermentation were obtained from cultures of S. sonnei ΔtolR ΔgalU at optical densities of 30-45 in a 5 L fermenter.These were highly immunogenic in mice.Furthermore, we demonstrate the feasibility of using this process with other genetic manipulations e.g. abolition of O antigen synthesis and modification of the lipopolysaccharide structure in order to modify the immunogenicity or reactogenicity of the particles.

View Article: PubMed Central - PubMed

Affiliation: Novartis Vaccines Institute for Global Health, Siena, Italy.

ABSTRACT
Gram-negative bacteria naturally shed particles that consist of outer membrane lipids, outer membrane proteins, and soluble periplasmic components. These particles have been proposed for use as vaccines but the yield has been problematic. We developed a high yielding production process of genetically derived outer membrane particles from the human pathogen Shigella sonnei. Yields of approximately 100 milligrams of membrane-associated proteins per liter of fermentation were obtained from cultures of S. sonnei ΔtolR ΔgalU at optical densities of 30-45 in a 5 L fermenter. Proteomic analysis of the purified particles showed the preparation to primarily contain predicted outer membrane and periplasmic proteins. These were highly immunogenic in mice. The production of these outer membrane particles from high density cultivation of bacteria supports the feasibility of scaling up this approach as an affordable manufacturing process. Furthermore, we demonstrate the feasibility of using this process with other genetic manipulations e.g. abolition of O antigen synthesis and modification of the lipopolysaccharide structure in order to modify the immunogenicity or reactogenicity of the particles. This work provides the basis for a large scale manufacturing process of Generalized Modules of Membrane Antigens (GMMA) for production of vaccines from gram-negative bacteria.

Show MeSH
Related in: MedlinePlus