scholarly journals Mechanistic Analysis of the Effect of Deamidation on the Immunogenicity of Anthrax Protective Antigen

2016 ◽  
Vol 23 (5) ◽  
pp. 396-402 ◽  
Author(s):  
Anita Verma ◽  
Miriam M. Ngundi ◽  
Drusilla L. Burns
Keyword(s):  
T Cell ◽  
Antibody Production ◽  
Protective Antigen ◽  
Primary Component ◽  
Wild Type ◽  
Immunological Mechanisms ◽  
Anthrax Protective Antigen ◽  
T Cell Help ◽  
New Generation ◽  
Anthrax Vaccines

ABSTRACTThe spontaneous modification of proteins, such as deamidation of asparagine residues, can significantly affect the immunogenicity of protein-based vaccines. Using a “genetically deamidated” form of recombinant protective antigen (rPA), we have previously shown that deamidation can decrease the immunogenicity of rPA, the primary component of new-generation anthrax vaccines. In this study, we investigated the biochemical and immunological mechanisms by which deamidation of rPA might decrease the immunogenicity of the protein. We found that loss of the immunogenicity of rPA vaccine was independent of the presence of adjuvant. We assessed the effect of deamidation on the immunodominant neutralizing B-cell epitopes of rPA and found that these epitopes were not significantly affected by deamidation. In order to assess the effect of deamidation on T-cell help for antibody production elicited by rPA vaccine, we examined the ability of the wild-type and genetically deamidated forms of rPA to serve as hapten carriers. We found that when wild-type and genetically deamidated rPA were modified to similar extents with 2,4-dinitrophenyl hapten (DNP) and then used to immunize mice, higher levels of anti-DNP antibodies were elicited by wild-type DNP-rPA than those elicited by the genetically deamidated DNP-rPA, indicating that wild-type rPA elicits more T-cell help than the genetically deamidated form of the protein. These results suggest that a decrease in the ability of deamidated rPA to elicit T-cell help for antibody production is a possible contributor to its lower immunogenicity.

scholarly journals Genetic Construction of Bacillus subtilis Recombinant Strain, Producing Anthrax Protective Antigen

2018 ◽  
Vol 2 (2) ◽  
pp. 51-60
Keyword(s):  
Bacillus Subtilis ◽  
Recombinant Strain ◽  
Protective Antigen ◽  
Shuttle Vector ◽  
Subtilis Strain ◽  
Anthrax Vaccine ◽  
Anthrax Protective Antigen ◽  
Recombinant Bacillus Subtilis ◽  
New Generation ◽  
Anthrax Vaccines

Anthrax is a serious infectious disease with high mortality. The epidemiological security depends on the vaccination of susceptible animals and population at risk. But many of the existing anthrax vaccine strains possess low levels of protective antigen production and high reactogenicity. One of the most promising trends in production of new generation of vaccines is the cloning of particular determinants of immunogenicity of anthrax microbe for the creation of highly effective producers of Bacillus anthracis protective antigen. The aim of the article is to present the results of the study on the construction of recombinant Bacillus subtilis strain, producing B.anthracis protective antigen, promising for use in chemical anthrax vaccines technology. The pHT43PA plasmid containing the gene pag, providing the synthesis of protective antigen of the anthrax microbe and functioning stably in the cells of the recombinant strain Amy21(pHT43РА) of B. subtilis, was constructed on the basis of the shuttle vector pHT43. It is found out during the research, that the microbial cells of the recombinant strain Amy21(pHT43РА) of B. subtilis provide the production of immunologically active protective antigen in quantities, not inferior than anthrax vaccine strains. These data, as well as safety and simplicity of В. subtilis make it possible to continue the research of this recombinant strain as a producer of anthrax protective antigen, promising for use in vaccines production

scholarly journals Use of Site-Directed Mutagenesis To Model the Effects of Spontaneous Deamidation on the Immunogenicity of Bacillus anthracis Protective Antigen

2012 ◽  
Vol 81 (1) ◽  
pp. 278-284 ◽  
Author(s):  
Anita Verma ◽  
Beth McNichol ◽  
Rocío I. Domínguez-Castillo ◽  
Juan C. Amador-Molina ◽  
Juan L. Arciniega ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Large Scale ◽  
Protective Antigen ◽  
Cd Spectroscopy ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Vaccine Formulation ◽  
Wild Type ◽  
Vaccine Immunogenicity ◽  
Anthrax Vaccines

Long-term stability is a desired characteristic of vaccines, especially anthrax vaccines, which must be stockpiled for large-scale use in an emergency situation; however, spontaneous deamidation of purified vaccine antigens has the potential to adversely affect vaccine immunogenicity over time. In order to explore whether spontaneous deamidation of recombinant protective antigen (rPA)—the major component of new-generation anthrax vaccines—affects vaccine immunogenicity, we created a “genetically deamidated” form of rPA using site-directed mutagenesis to replace six deamidation-prone asparagine residues, at positions 408, 466, 537, 601, 713, and 719, with either aspartate, glutamine, or alanine residues. We found that the structure of the six-Asp mutant rPA was not significantly altered relative to that of the wild-type protein as assessed by circular dichroism (CD) spectroscopy and biological activity. In contrast, immunogenicity of aluminum-adjuvanted six-Asp mutant rPA, as measured by induction of toxin-neutralizing antibodies, was significantly lower than that of the corresponding wild-type rPA vaccine formulation. The six-Gln and six-Ala mutants also exhibited lower immunogenicity than the wild type. While the wild-type rPA vaccine formulation exhibited a high level of immunogenicity initially, its immunogenicity declined significantly upon storage at 25°C for 4 weeks. In contrast, the immunogenicity of the six-Asp mutant rPA vaccine formulation was low initially but did not change significantly upon storage. Taken together, results from this study suggest that spontaneous deamidation of asparagine residues predicted to occur during storage of rPA vaccines would adversely affect vaccine immunogenicity and therefore the storage life of vaccines.

scholarly journals Critical Role for Mouse Marginal Zone B Cells in PF4/Heparin Antibody Production

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1175-1175
Author(s):  
Yongwei Zheng ◽  
Mei Yu ◽  
Andrew Podd ◽  
Debra K. Newman ◽  
Renren Wen ◽  
...  
Keyword(s):  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Antibody Production ◽  
Marginal Zone ◽  
Critical Role ◽  
Wild Type ◽  
Platelet Factor ◽  
Specific Antibodies ◽  
Deficient Mice

Abstract Abstract 1175 Heparin-induced thrombocytopenia (HIT) is an immune-mediated disorder that can cause fatal arterial or venous thrombosis/thromboembolism. Immune complexes consisting of platelet factor 4 (PF4), heparin and PF4/heparin-reactive antibodies are central to the pathogenesis of HIT. However, the B-cell origin of HIT antibody production is not known. Here we show that upon challenge with PF4/heparin complexes, anti-PF4/heparin antibody production is severely impaired in B cell-specific Notch2-deficient mice (CD19CreNotch2fl/fl) that specifically lack marginal zone (MZ) B cells, and that antibody production is readily generated in wild-type mice (CD19CreNotch2+/+). As expected, Notch2-deficient mice responded normally to challenge with T cell-dependent antigen NP-CGG but not T cell-independent antigen TNP-Ficoll, in agreement with the lack of MZ B cells in the mutant mice. PF4/heparin-specific antibodies produced by wild-type mice on a C57BL/6 background were IgG2b and IgG3 isotypes. An in vitro class-switching assay showed that MZ B cells from wild-type C57BL/6 mice were capable of producing antibodies of IgG2b and IgG3 isotypes. Lastly, MZ, but not follicular (FO), B cells adoptively transferred into B cell-deficient muMT mice responded to PF4/heparin complex challenge by producing PF4/heparin-specific antibodies of IgG2b and IgG3 isotypes. Taken together, these data demonstrate that MZ B cells play a critical role in production of PF4/heparin-specific antibodies. Disclosures: Arepally: Teva Pharmaceuticals: Research Funding.

scholarly journals Critical Role of T Cells in PF4/Heparin Antibody Production

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1554-1554
Author(s):  
Yongwei Zheng ◽  
Mei Yu ◽  
Anand Padmanabhan ◽  
Richard H. Aster ◽  
Renren Wen ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Antibody Production ◽  
Cd4 T Cells ◽  
Wild Type ◽  
Specific Antibodies ◽  
Deficient Mice

Abstract Heparin-induced thrombocytopenia (HIT) is an antibody-mediated disorder that can cause arterial or venous thrombosis/thromboembolism, and platelet factor 4 (PF4)/ heparin-reactive antibodies are essential to the pathogenesis of HIT. Our recent studies have demonstrated that marginal zone (MZ) B cells play a major role in production of PF4/heparin-specific antibodies. However, the role of T cells in production of these pathogenic antibodies is not clear. Here we showed that PF4/heparin complex-induced production of PF4/heparin-specific antibodies was markedly impaired in mice, in which CD4 T cells were depleted by administration of GK1.5 anti-CD4 monoclonal antibody. As expected, the CD4 T cell-depleted mice responded normally to T cell-independent antigen TNP-Ficoll but not T cell-dependent antigen NP-CGG, in agreement with the lack of CD4 T cells in these GK1.5-treated mice. Further, following adoptive transfer of a mixture of wild-type splenic B cells and splenocytes from B cell-deficient μMT mice, T and B cell-deficient Rag1 knockout mice responded to PF4/heparin complex challenge to produce PF4/heparin-specific antibodies. In contrast, Rag1-deficient mice that received a mixture of wild-type splenic B cells and splenocytes from Rag1-deficient mice barely produced PF4/heparin-specific antibodies upon PF4/heparin complex challenge. These data suggest that T cells are required for production of PF4/heparin-specific antibodies. Consistent with this concept, mice with B cells lacking CD40 molecule, a B cell costimulatory molecule that helps T cell-dependent B cell responses, displayed a marked reduction of PF4/heparin-specific antibody production following PF4/heparin complex challenge. Also as expected, mice with CD40-deficient B cells were able to respond to T cell-independent antigen TNP-Ficoll but not T cell-dependent antigen NP-CGG, consistent with the lack of T-cell help in these mice. Taken together, these findings demonstrate that T cells play an essential role in production of PF4/heparin-specific antibodies by MZ B cells. Disclosures No relevant conflicts of interest to declare.

scholarly journals MicroRNA-155 is essential for the optimal proliferation and survival of plasmablast B cells

2019 ◽  
Vol 2 (3) ◽  
pp. e201800244 ◽  
Author(s):  
Giuseppina Arbore ◽  
Tom Henley ◽  
Laura Biggins ◽  
Simon Andrews ◽  
Elena Vigorito ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Comparative Analysis ◽  
T Cell ◽  
B Cells ◽  
Antibody Response ◽  
Antibody Production ◽  
Metabolic Process ◽  
Cellular Processes ◽  
T Cell Help

A fast antibody response can be critical to contain rapidly dividing pathogens. This can be achieved by the expansion of antigen-specific B cells in response to T-cell help followed by differentiation into plasmablasts. MicroRNA-155 (miR-155) is required for optimal T-cell–dependent extrafollicular responses via regulation of PU.1, although the cellular processes underlying this defect are largely unknown. Here, we show that miR-155 regulates the early expansion of B-blasts and later on the survival and proliferation of plasmablasts in a B-cell–intrinsic manner, by tracking antigen-specific B cells in vivo since the onset of antigen stimulation. In agreement, comparative analysis of the transcriptome of miR-155–sufficient and miR-155–deficient plasmablasts at the peak of the response showed that the main processes regulated by miR-155 were DNA metabolic process, DNA replication, and cell cycle. Thus, miR-155 controls the extent of the extrafollicular response by regulating the survival and proliferation of B-blasts, plasmablasts and, consequently, antibody production.

scholarly journals Short Vi-polysaccharide abrogates T-independent immune response and hyporesponsiveness elicited by long Vi-CRM197 conjugate vaccine

2020 ◽  
Vol 117 (39) ◽  
pp. 24443-24449
Author(s):  
Francesca Micoli ◽  
Stefania P. Bjarnarson ◽  
Melissa Arcuri ◽  
Audur Anna Aradottir Pind ◽  
Gudbjorg J. Magnusdottir ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
B Cells ◽  
Short Chain ◽  
Wild Type ◽  
Protein Conjugates ◽  
T Cell Help ◽  
The Impact ◽  
Deficient Mice ◽  
Long Chain

Polysaccharide-protein conjugates have been developed to overcome the T-independent response, hyporesponsiveness to repeated vaccination, and poor immunogenicity in infants of polysaccharides. To address the impact of polysaccharide length, typhoid conjugates made with short- and long-chain fractions of Vi polysaccharide with average sizes of 9.5, 22.8, 42.7, 82.0, and 165 kDa were compared. Long-chain-conjugated Vi (165 kDa) induced a response in both wild-type and T cell-deficient mice, suggesting that it maintains a T-independent response. In marked contrast, short-chain Vi (9.5 to 42.7 kDa) conjugates induced a response in wild-type mice but not in T cell-deficient mice, suggesting that the response is dependent on T cell help. Mechanistically, this was explained in neonatal mice, in which long-chain, but not short-chain, Vi conjugate induced late apoptosis of Vi-specific B cells in spleen and early depletion of Vi-specific B cells in bone marrow, resulting in hyporesponsiveness and lack of long-term persistence of Vi-specific IgG in serum and IgG+ antibody-secreting cells in bone marrow. We conclude that while conjugation of long-chain Vi generates T-dependent antigens, the conjugates also retain T-independent properties, leading to detrimental effects on immune responses. The data reported here may explain some inconsistencies observed in clinical trials and help guide the design of effective conjugate vaccines.

scholarly journals Vaccination against Anthrax with Attenuated Recombinant Strains of Bacillus anthracis That Produce Protective Antigen

1999 ◽  
Vol 67 (2) ◽  
pp. 562-567 ◽  
Author(s):  
John P. Barnard ◽  
Arthur M. Friedlander
Keyword(s):  
Bacillus Anthracis ◽  
Protective Antigen ◽  
Plasmid Stability ◽  
Protective Efficacy ◽  
Live Vaccines ◽  
Antibody Titers ◽  
Anthrax Protective Antigen ◽  
Anthrax Vaccines

ABSTRACT The protective efficacy of several live, recombinant anthrax vaccines given in a single-dose regimen was assessed with Hartley guinea pigs. These live vaccines were created by transforming ΔANR and ΔSterne, two nonencapsulated, nontoxinogenic strains of Bacillus anthracis, with four different recombinant plasmids that express the anthrax protective antigen (PA) protein to various degrees. This enabled us to assess the effect of the chromosomal background of the strain, as well as the amount of PA produced, on protective efficacy. There were no significant strain-related effects on PA production in vitro, plasmid stability in vivo, survival of the immunizing strain in the host, or protective efficacy of the immunizing infection. The protective efficacy of the live, recombinant anthrax vaccine strains correlated with the anti-PA antibody titers they elicited in vivo and the level of PA they produced in vitro.

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