scholarly journals Use of mchI Encoding Immunity to the Antimicrobial Peptide Microcin H47 as a Plasmid Selection Marker in Attenuated Bacterial Live Vectors

2008 ◽  
Vol 76 (10) ◽  
pp. 4422-4430 ◽  
Author(s):  
Chee-Mun Fang ◽  
Jin Yuan Wang ◽  
Magaly Chinchilla ◽  
Myron M. Levine ◽  
William C. Blackwelder ◽  
...  
Keyword(s):  
Antimicrobial Peptide ◽  
Fluorescent Protein ◽  
Vaccine Development ◽  
Shigella Flexneri ◽  
Antibody Responses ◽  
Selection Marker ◽  
Human Pathogens ◽  
Bacterial Strains ◽  
Igg Antibody ◽  
Expression Plasmids

ABSTRACTLive attenuated bacterial strains expressing heterologous antigens represent an attractive vaccine development strategy. However, the use of drug resistance genes for the selection of expression plasmids introduced into live vectors poses theoretical health risks. Therefore, we developed a novel approach for plasmid selection based on immunity to the antimicrobial peptide microcin H47 (MccH47). Two expression plasmids encoding the reporter green fluorescent protein (GFPuv) were constructed; selection markers comprised eithermchI, conferring immunity to MccH47 (pGEN222I), orbla(encoding β-lactamase), conferring conventional resistance to ampicillin (pGEN222). GFPuv-specific serum immunoglobulin G (IgG) antibody responses were analyzed in mice immunized intranasally either withSalmonella entericaserovar Typhi CVD 908-htrAorShigella flexneri2a CVD 1208S live vector and were boosted parenterally with purified GFPuv. Similar IgG antibody responses were observed for both pGEN222 and pGEN222I when either CVD 1208S or CVD 908-htrA(pGEN222I) was used as the carrier. Interestingly, CVD 908-htrA(pGEN222I) elicited a significantly higher IgG response than CVD 908-htrA(pGEN222). We also compared the priming potential of homologous priming either with CVD 908-htrA(pGEN222I) or CVD 1208S(pGEN222I) to heterologous priming first with CVD 908-htrA(pGEN222I) and then with CVD 1208S(pGEN222I) and vice versa. Immunization with two unrelated live vectors significantly enhanced the IgG responses compared to responses engendered by homologous CVD 908-htrA(pGEN222I) but not to those of CVD 1208S(pGEN222I). MccH47 offers an alternate system for plasmid selection in bacterial live vectors that greatly improves their clinical acceptability. Furthermore, the success of the heterologous priming strategy supports the feasibility of the future development of multivalent live vector-based immunization strategies against multiple human pathogens.

scholarly journals A New Generation of Stable, Nonantibiotic, Low-Copy-Number Plasmids Improves Immune Responses to Foreign Antigens in Salmonella enterica Serovar Typhi Live Vectors

2009 ◽  
Vol 78 (1) ◽  
pp. 337-347 ◽  
Author(s):  
James E. Galen ◽  
Jin Yuan Wang ◽  
Magaly Chinchilla ◽  
Christopher Vindurampulle ◽  
Jeffrey E. Vogel ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Copy Number ◽  
Protective Antigen ◽  
Antibody Responses ◽  
Human Pathogens ◽  
Foreign Antigen ◽  
Low Copy Number ◽  
New Generation ◽  
Export Protein ◽  
Expression Plasmids

ABSTRACTWe hypothesized that adequately engineered attenuatedSalmonella entericaserovar Typhi strains can serve as multivalent mucosal live vector vaccines to immunize against unrelated human pathogens. Toward this ultimate goal, we have developed a novel genetic stabilization system for antigen-expressing plasmids, engineered to encode the single-stranded binding protein (SSB), an essential protein involved in DNA metabolism which was deleted from the live vector chromosome. We utilized full-length protective antigen (PA83) of anthrax toxin fromBacillus anthracisas a foreign antigen and expressed PA83 as a fusion with the ClyA export protein, which allows export of ClyA-PA83 to the surface ofS. Typhi live vectors. A series of SSB-encoding multicopy expression plasmids were introduced into reengineeredS. Typhi strains previously tested in clinical trials, i.e., CVD 908-htrAand its less attenuated parent CVD 908. Immunogenicity was examined using a mouse model of intranasal immunization with live vector, followed by parenteral boosting with purified PA83. PA-specific antibody responses markedly improved as the copy number of the SSB-encoding plasmids decreased, and this effect was dramatically enhanced when the foreign antigen was delivered by the less attenuated live vector CVD 908ssb. These results suggest that antibody responses to antigens delivered byS. Typhi live vectors are inversely related to the metabolic burden imposed by expression of the foreign antigen and that these responses can be improved when antigens are expressed from low-copy-number plasmids and exported out of the cytoplasm of less attenuated live vectors.

scholarly journals Interactions between Food-Borne Pathogens and Protozoa Isolated from Lettuce and Spinach

2008 ◽  
Vol 74 (8) ◽  
pp. 2518-2525 ◽  
Author(s):  
Poornima Gourabathini ◽  
Maria T. Brandl ◽  
Katherine S. Redding ◽  
John H. Gunderson ◽  
Sharon G. Berk
Keyword(s):  
Fluorescent Protein ◽  
Tetrahymena Pyriformis ◽  
Enteric Bacteria ◽  
Enteric Pathogens ◽  
Leafy Vegetables ◽  
Human Pathogens ◽  
Bacterial Strains ◽  
E Coli ◽  
Green Fluorescent

ABSTRACT The survival of Salmonella enterica was recently shown to increase when the bacteria were sequestered in expelled food vacuoles (vesicles) of Tetrahymena. Because fresh produce is increasingly linked to outbreaks of enteric illness, the present investigation aimed to determine the prevalence of protozoa on spinach and lettuce and to examine their interactions with S. enterica, Escherichia coli O157:H7, and Listeria monocytogenes. Glaucoma sp., Colpoda steinii, and Acanthamoeba palestinensis were cultured from store-bought spinach and lettuce and used in our study. A strain of Tetrahymena pyriformis previously isolated from spinach and a soil-borne Tetrahymena sp. were also used. Washed protozoa were allowed to graze on green fluorescent protein- or red fluorescent protein-labeled enteric pathogens. Significant differences in interactions among the various protist-enteric pathogen combinations were observed. Vesicles were produced by Glaucoma with all of the bacterial strains, although L. monocytogenes resulted in the smallest number per ciliate. Vesicle production was observed also during grazing of Tetrahymena on E. coli O157:H7 and S. enterica but not during grazing on L. monocytogenes, in vitro and on leaves. All vesicles contained intact fluorescing bacteria. In contrast, C. steinii and the amoeba did not produce vesicles from any of the enteric pathogens, nor were pathogens trapped within their cysts. Studies of the fate of E. coli O157:H7 in expelled vesicles revealed that by 4 h after addition of spinach extract, the bacteria multiplied and escaped the vesicles. The presence of protozoa on leafy vegetables and their sequestration of enteric bacteria in vesicles indicate that they may play an important role in the ecology of human pathogens on produce.

scholarly journals Activity of the Novel Peptide Arminin against Multiresistant Human Pathogens Shows the Considerable Potential of Phylogenetically Ancient Organisms as Drug Sources

2009 ◽  
Vol 53 (12) ◽  
pp. 5245-5250 ◽  
Author(s):  
René Augustin ◽  
Friederike Anton-Erxleben ◽  
Stephanie Jungnickel ◽  
Georg Hemmrich ◽  
Björn Spudy ◽  
...  
Keyword(s):  
Antimicrobial Peptide ◽  
Multidrug Resistant ◽  
Proteolytic Processing ◽  
Erythrocyte Membranes ◽  
Resistant Bacteria ◽  
Minimal Bactericidal Concentration ◽  
Human Pathogens ◽  
Bacterial Strains ◽  
Multiresistant Bacteria ◽  
High Level

ABSTRACT The emergence of multidrug-resistant bacteria highlights the need for new antibacterial agents. Arminin 1a is a novel antimicrobial peptide discovered during investigations of the epithelial defense of the ancient metazoan Hydra. Following proteolytic processing, the 31-amino-acid-long positively charged C-terminal part of arminin 1a exhibits potent and broad-spectrum activity against bacteria, including multiresistant human pathogenic strains, such as methicillin-resistant Staphylococcus aureus (MRSA) strains (minimal bactericidal concentration, 0.4 μM to 0.8 μM). Ultrastructural observations indicate that bacteria are killed by disruption of the bacterial cell wall. Remarkably, the antibacterial activity of arminin 1a is not affected under the physiological salt conditions of human blood. In addition, arminin 1a is a selective antibacterial agent that does not affect human erythrocyte membranes. Arminin 1a shows no sequence homology to any known antimicrobial peptide. Because of its high level of activity against multiresistant bacterial strains pathogenic for humans, the peptide arminin 1a is a promising template for a new class of antibiotics. Our data suggest that ancient metazoan organisms such as Hydra hold promise for the detection of novel antimicrobial molecules and the treatment of infections caused by multiresistant bacteria.

scholarly journals FnCas12a/crRNA-Mediated Genome Editing in Eimeria tenella

2021 ◽  
Vol 12 ◽  
Author(s):  
Peipei Cheng ◽  
Zhihao Zhang ◽  
Fayu Yang ◽  
Shuo Cai ◽  
Lina Wang ◽  
...  
Keyword(s):  
Genome Editing ◽  
Fluorescent Protein ◽  
Vaccine Development ◽  
Yellow Fluorescent Protein ◽  
Ectopic Expression ◽  
Eimeria Tenella ◽  
Selection Marker ◽  
Dna Double Strand Breaks ◽  
Enhanced Yellow Fluorescent Protein

Eimeria species are intracellular parasites residing inside the intestinal epithelial cell, which cause poultry coccidiosis and result in significant financial losses in the poultry industry. Genome editing of Eimeria is of immense importance for the development of vaccines and drugs. CRISPR/Cas9 has been utilized for manipulating the genome of Eimeria tenella (E. tenella). Ectopic expression of Cas9, i.e., via plasmids, would introduce transgene, which substantially limits its application, especially for vaccine development. In this study, we initially optimized the condition of the transfection protocol. We demonstrated that with the optimized condition, the transfection of FnCas12a (also known as “FnCpf1”) protein and crRNA targeting EtHistone H4 triggered DNA double-strand breaks in vivo. We then used this strategy to knock-in a coding cassette for an enhanced yellow fluorescent protein (EYFP) and dihydrofolate reductase–thymidylate synthase gene (DHFR) as a selection marker to tag endogenous EtActin. The engineered E. tenella parasite possesses EYFP expression in its entire life cycle. Our results demonstrated that FnCas12a could trigger genome editing in E. tenella, which augments the applicability of the dissection of gene function and the development of anticoccidial drugs and vaccines for Eimeria species.

scholarly journals Flavivirus NS1 and Its Potential in Vaccine Development

Vaccines ◽  
2021 ◽  
Vol 9 (6) ◽  
pp. 622
Author(s):  
Kassandra L. Carpio ◽  
Alan D. T. Barrett
Keyword(s):  
Vaccine Development ◽  
Virus Assembly ◽  
Ns1 Protein ◽  
Human Pathogens ◽  
Unusual Structure ◽  
Replication Complex ◽  
Virus Challenge ◽  
Humoral Immune ◽  
Tick Borne Encephalitis ◽  
Flavivirus Infection

The Flavivirus genus contains many important human pathogens, including dengue, Japanese encephalitis (JE), tick-borne encephalitis (TBE), West Nile (WN), yellow fever (YF) and Zika (ZIK) viruses. While there are effective vaccines for a few flavivirus diseases (JE, TBE and YF), the majority do not have vaccines, including WN and ZIK. The flavivirus nonstructural 1 (NS1) protein has an unusual structure–function because it is glycosylated and forms different structures to facilitate different roles intracellularly and extracellularly, including roles in the replication complex, assisting in virus assembly, and complement antagonism. It also plays a role in protective immunity through antibody-mediated cellular cytotoxicity, and anti-NS1 antibodies elicit passive protection in animal models against a virus challenge. Historically, NS1 has been used as a diagnostic marker for the flavivirus infection due to its complement fixing properties and specificity. Its role in disease pathogenesis, and the strong humoral immune response resulting from infection, makes NS1 an excellent target for inclusion in candidate flavivirus vaccines.

scholarly journals Insights into Antibody-Mediated Alphavirus Immunity and Vaccine Development Landscape

2021 ◽  
Vol 9 (5) ◽  
pp. 899
Author(s):  
Anthony Torres-Ruesta ◽  
Rhonda Sin-Ling Chee ◽  
Lisa F.P. Ng
Keyword(s):  
Diagnostic Tests ◽  
Inflammatory Arthritis ◽  
Chikungunya Virus ◽  
Vaccine Development ◽  
Antibody Responses ◽  
Chikv Infection ◽  
Wide Range ◽  
Susceptible Populations ◽  
Humoral Responses

Alphaviruses are mosquito-borne pathogens distributed worldwide in tropical and temperate areas causing a wide range of symptoms ranging from inflammatory arthritis-like manifestations to the induction of encephalitis in humans. Historically, large outbreaks in susceptible populations have been recorded followed by the development of protective long-lasting antibody responses suggesting a potential advantageous role for a vaccine. Although the current understanding of alphavirus antibody-mediated immunity has been mainly gathered in natural and experimental settings of chikungunya virus (CHIKV) infection, little is known about the humoral responses triggered by other emerging alphaviruses. This knowledge is needed to improve serology-based diagnostic tests and the development of highly effective cross-protective vaccines. Here, we review the role of antibody-mediated immunity upon arthritogenic and neurotropic alphavirus infections, and the current research efforts for the development of vaccines as a tool to control future alphavirus outbreaks.

scholarly journals Polyclonal antibody responses to HIV Env immunogens resolved using cryoEM

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Aleksandar Antanasijevic ◽  
Leigh M. Sewall ◽  
Christopher A. Cottrell ◽  
Diane G. Carnathan ◽  
Luis E. Jimenez ◽  
...  
Keyword(s):  
High Resolution ◽  
Polyclonal Antibody ◽  
Vaccine Development ◽  
Mutational Analysis ◽  
Rhesus Macaques ◽  
Antibody Responses ◽  
Glycosylation Sites ◽  
High Resolution Analysis ◽  
Resolution Analysis ◽  
Resolution Mapping

AbstractEngineered ectodomain trimer immunogens based on BG505 envelope glycoprotein are widely utilized as components of HIV vaccine development platforms. In this study, we used rhesus macaques to evaluate the immunogenicity of several stabilized BG505 SOSIP constructs both as free trimers and presented on a nanoparticle. We applied a cryoEM-based method for high-resolution mapping of polyclonal antibody responses elicited in immunized animals (cryoEMPEM). Mutational analysis coupled with neutralization assays were used to probe the neutralization potential at each epitope. We demonstrate that cryoEMPEM data can be used for rapid, high-resolution analysis of polyclonal antibody responses without the need for monoclonal antibody isolation. This approach allowed to resolve structurally distinct classes of antibodies that bind overlapping sites. In addition to comprehensive mapping of commonly targeted neutralizing and non-neutralizing epitopes in BG505 SOSIP immunogens, our analysis revealed that epitopes comprising engineered stabilizing mutations and of partially occupied glycosylation sites can be immunogenic.

scholarly journals Influence of Bacterial Competitors on Salmonella enterica and Enterohemorrhagic Escherichia coli Growth in Microbiological Media and Attachment to Vegetable Seeds

Foods ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 285
Author(s):  
Da Liu ◽  
Ronald Walcott ◽  
Kevin Mis Solval ◽  
Jinru Chen
Keyword(s):  
Escherichia Coli ◽  
Foodborne Pathogens ◽  
Salmonella Enterica ◽  
Lactobacillus Rhamnosus ◽  
Probiotic Bacterium ◽  
Enterohemorrhagic Escherichia Coli ◽  
Biocontrol Agents ◽  
Human Pathogens ◽  
Bacterial Strains ◽  
Pathogen Populations

Interests in using biological agents for control of human pathogens on vegetable seeds are rising. This study evaluated whether probiotic bacterium Lactobacillus rhamnosus GG, bacterial strains previously used as biocontrol agents in plant science, as well as a selected plant pathogen could compete with foodborne human pathogens, such as Salmonella enterica and enterohemorrhagic Escherichia coli (EHEC), for growth in microbiological media and attachment to vegetable seeds; and to determine whether the metabolites in cell-free supernatants of competitive bacterial spent cultures could inhibit the growth of the two pathogens. The results suggest that the co-presence of competitive bacteria, especially L. rhamnosus GG, significantly (p < 0.05) inhibited the growth of Salmonella and EHEC. Cell-free supernatants of L. rhamnosus GG cultures significantly reduced the pathogen populations in microbiological media. Although not as effective as L. rhamnosus GG in inhibiting the growth of Salmonella and EHEC, the biocontrol agents were more effective in competing for attachment to vegetable seeds. The study observed the inhibition of human bacterial pathogens by competitive bacteria or their metabolites and the competitive attachment to sprout seeds among all bacteria involved. The results will help strategize interventions to produce vegetable seeds and seed sprouts free of foodborne pathogens.

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