Conjugate vaccines represent one of the most effective means for controlling the occurrence of bacterial diseases. Although nanotechnology has been greatly applied in the field of vaccines, it is ...seldom used for conjugate vaccine research because it is very difficult to connect polysaccharides and nanocarriers. In this work, an orthogonal and modular biosynthesis method was used to produce nanoconjugate vaccines using the SpyTag/SpyCatcher system. When SpyTag/SpyCatcher system is combined with protein glycosylation technology, bacterial O-polysaccharide obtained from
Shigela flexneri
2a can be conjugated onto the surfaces of different virus-like particles (VLPs) in a biocompatible and controlled manner. After confirming the excellent lymph node targeting and humoral immune activation abilities, these nanoconjugate vaccines further induced efficient prophylactic effects against infection in a mouse model. These results demonstrated that natural polysaccharide antigens can be easily connected to VLPs to prepare highly efficient nanoconjugate vaccines. To the best of the researchers’ knowledge, this is the first time VLP-based nanoconjugate vaccines are produced efficiently, and this strategy could be applied to develop various pathogenic nanoconjugate vaccines.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Summary
Acinetobacter baumannii poses a serious threat to human health, mainly because of its widespread distribution and severe drug resistance. However, no licensed vaccines exist for this ...pathogen. In this study, we created a conjugate vaccine against A. baumannii by introducing an O‐linked glycosylation system into the host strain. After demonstrating the ability of the vaccine to elicit Th1 and Th2 immune responses and observing its good safety in mouse a model, the strong in vitro bactericidal activity and prophylactic effects of the conjugate vaccine against infection were further demonstrated by evaluating post‐infection tissue bacterial loads, observing suppressed serum pro‐inflammatory cytokine levels. Additionally, the broad protection from the vaccine was further proved via lethal challenge with A. baumannii. Overall, these results indicated that the conjugate vaccine could elicit an efficient immune response and provide good protection against A. baumannii infection in murine sepsis models. Thus, the conjugate vaccine can be considered as a promising candidate vaccine for preventing A. baumannii infection.
We created a conjugate vaccine against A. baumannii by introducing an O‐linked glycosylation system into the host strain. After demonstrating the safety of the conjugate vaccine in a mouse model, animal experiments revealed that the vaccine could elicit robust antibody responses that reduced post‐infection bacterial loads in tissues and protected against lethal challenge by A. baumannii in murine sepsis models.
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FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK
As next-generation pathogen detection methods, CRISPR-Cas-based detection methods can perform single-nucleotide polymorphism (SNP) level detection with high sensitivity and good specificity. They do ...not require any particular equipment, which opens up new possibilities for the accurate detection and identification of Bacillus anthracis. In this study, we developed a complete detection system for B. anthracis based on Cas12a. We used two chromosomally located SNP targets and two plasmid targets to identify B. anthracis with high accuracy. The CR5 target is completely new. The entire detection process can be completed within 90 min without electrical power and with single-copy level sensitivity. We also developed an unaided-eye visualization system based on G4-DNAzyme for use with our CRISPR-Cas12a detection system. This visualization system has good prospects for deployment in field-based point-of-care detection. We used the antisense nucleic acid CatG4R as the detection probe, which showed stronger resistance to interference from components of the solution. CatG4R can also be designed as an RNA molecule for adaptation to Cas13a detection, thereby broadening the scope of the detection system.
Genome editing is an effective tool for the functional examination of bacterial genes and for live attenuated vaccine construction. Here, we report a method to edit the genomic DNA of
Bacillus ...anthracis
and
Bacillus cereus
using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas)9 system. Using two prophages in
B. anthracis
as targets, large-fragment deletion mutants were achieved with rates of 100 or 20%. In
B. cereus
, we successfully introduced precise point mutations into
plcR
, with phenotypic assays showing that the resulting mutants lost hemolytic and phospholipase enzyme activities similar to
B. anthracis
, which is a natural
plcR
mutant. Our study indicates that CRISPR/Cas9 is a powerful genetic tool for genome editing in the
Bacillus cereus
group, and can efficiently modify target genes without the need for residual foreign DNA such as antibiotic selection markers. This system could be developed for use in the generation of marker-free live anthrax vaccines or for safer construction of microbiological candidate-based recombinant
B. cereus
.
The CRISPR-Cas system has been widely applied in prokaryotic genome editing with its high efficiency and easy operation. We constructed some “scissors plasmids” via using the temperature-sensitive ...pJOE8999 shuttle plasmid, which carry the different 20nt (N20) guiding the Cas9 nuclease as a scissors to break the target DNA. We successfully used scissors plasmids to eliminate native plasmids from
Bacillus anthracis
and
Bacillus cereus
, and specifically killed
B. anthracis
. When curing pXO1 and pXO2 virulence plasmids from
B. anthracis
A16PI2 and A16Q1, respectively, we found that the plasmid elimination percentage was slightly higher when the sgRNA targeted the replication initiation region (96–100%), rather than the non-replication initiation region (88–92%). We also tried using a mixture of two scissors plasmids to simultaneously eliminate pXO1 and pXO2 plasmids from
B. anthracis
, and the single and double plasmid-cured rates were 29 and 14%, respectively. To our surprise, when we used the scissor plasmid containing two tandem sgRNAs to cure the target plasmids pXO1 and pXO2 from wild strain
B. anthracis
A16 simultaneously, only the second sgRNA could guide Cas9 to cleave the target plasmid with high efficiency, while the first sgRNA didn't work in all the experiments we designed. When we used the CRISPR/cas9 system to eliminate the pCE1 mega-virulence plasmid from
B. cereus
BC307 by simply changing the sgRNA, we also obtained a plasmid-cured isogenic strain at a very high elimination rate (69%). The sterilization efficiency of
B. anthracis
was about 93%, which is similar to the efficiency of plasmid curing, and there was no significant difference in the efficiency of among the scissors plasmids containing single sgRNA, targeting multi-sites, or single-site targeting and the two tandem sgRNA. This simple and effective curing method, which is applicable to
B. cereus
group strains, provides a new way to study these bacteria and their virulence profiles.
is a Gram-positive bacterium that causes the zoonotic disease anthrax. Here, we studied the characteristic phenotype and virulence attenuation of the putative No. II vaccine strain, PNO2, which was ...reportedly introduced from the Pasteur Institute in 1934. Characterization of the strain showed that, compared with the control strain, A16Q1, the attenuated PNO2 (PNO2D1) was phospholipase-positive, with impaired protein hydrolysis and significantly reduced sporulation. Additionally, PNO2D1 significantly extended the survival times of anthrax-challenged mice. An evolutionary tree analysis revealed that PNO2D1 was not a Pasteur strain but was more closely related to a Tsiankovskii strain. A database comparison revealed a seven-base insertion mutation in the
gene. Although it did not block
transcription, the insertion mutation resulted in the premature termination of protein translation.
deletion of A16Q1 resulted in a nonproteolytic phenotype that could not sporulate. The database comparison revealed that the
gene is also prone to mutation, and the
promoter activity was much lower in PNO2D1 than in A16Q1. Low
expression may be an important reason for the decreased virulence of PNO2D1.
The large plasmid pXO1 encoding the anthrax toxin is important for the virulence of Bacillus anthracis. It is essential to cure pXO1 from B. anthracis to evaluate its role in the pathogenesis of ...anthrax infection. Because conventional methods for curing plasmids (e.g., curing agents or growth at elevated temperatures) can induce mutations in the host chromosomal DNA, we developed a specific and reliable method to eliminate pXO1 from B. anthracis using plasmid incompatibility. Three putative replication origins of pXO1 were inserted into a temperature-sensitive plasmid to generate three incompatible plasmids. One of the three plasmids successfully eliminated the large plasmid pXO1 from B. anthracis vaccine strain A16R and wild type strain A16. These findings provided additional information about the replication/partitioning of pXO1 and demonstrated that introducing a small incompatible plasmid can generate plasmid-cured strains of B. anthracis without inducing spontaneous mutations in the host chromosome.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Brucellosis is a major zoonotic public health threat worldwide, causing veterinary morbidity and major economic losses in endemic regions. However, no efficacious brucellosis vaccine is yet ...available, and live attenuated vaccines commonly used in animals can cause human infection.
- and
-linked glycosylation systems have been successfully developed and exploited for the production of successful bioconjugate vaccines. Here, we applied an
-linked glycosylation system to a low-pathogenicity bacterium,
serotype O:9 (
O:9), which has repeating units of O-antigen polysaccharide (OPS) identical to that of
(
), to develop a bioconjugate vaccine against
. The glycoprotein we produced was recognized by both anti-
and anti-
O:9 monoclonal antibodies. Three doses of bioconjugate vaccine-elicited
OPS-specific serum IgG in mice, significantly reducing bacterial loads in the spleen following infection with the
hypovirulent smooth strain A19. This candidate vaccine mitigated
infection and prevented severe tissue damage, thereby protecting against lethal challenge with A19. Overall, the results indicated that the bioconjugate vaccine elicited a strong immune response and provided significant protection against brucellosis. The described vaccine preparation strategy is safe and avoids large-scale culture of the highly pathogenic
.
Bacillus anthracis is a spore-forming bacterium that causes life-threatening infections in animals and humans and has been used as a bioterror agent. Rapid and reliable detection and identification ...of B. anthracis are of primary interest for both medical and biological threat-surveillance purposes. Few chromosomal sequences provide enough polymorphisms to clearly distinguish B. anthracis from closely related species. We analyzed 18 loci of the chromosome of B. anthracis and discovered eight novel single-nucleotide polymorphism (SNP) sites that can be used for the specific identification of B. anthracis. Using these SNP sites, we developed software-named AGILE V1.1 (
nthracis
enome-based
dentification with high-fide
ity
-probe)-for easy, user-friendly identification of B. anthracis from whole-genome sequences. We also developed a recombinase polymerase amplification-Cas12a-based method that uses nucleic acid extracts for the specific, rapid, in-the-field identification of B. anthracis based on these SNPs. Via this method and B. anthracis-specific CRISPR RNAs for the target CR5_2, CR5_1, and Ba813 SNPs, we clearly detected 5 aM genomic DNA. This study provides two simple and reliable methods suitable for use in local hospitals and public health programs for the detection of B. anthracis.
Bacillus anthracis is the etiologic agent of anthrax, a fatal disease and a potential biothreat. A specific, accurate, and rapid method is urgently required for the identification of B. anthracis. We demonstrate the potential of using eight novel SNPs for the rapid and accurate detection of B. anthracis via
and laboratory-based testing methods. Our findings have important implications for public health responses to disease outbreaks and bioterrorism threats.
Immunoproteomics was used to screen the immunogenic spore and vegetative proteins of Bacillus anthracis vaccine strain A16R. The spore and vegetative proteins were separated by 2D gel electrophoresis ...and transferred to polyvinylidene difluoride membranes, and then western blotting was performed with rabbit immune serum against B.anthracis live spores. Immunogenic spots were cut and digested by trypsin. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry was performed to identify the proteins. As a result, 11 and 45 immunogenic proteins were identified in the spores and vegetative cells, respectively; 26 of which have not been reported previously. To verify their immunogenicity, 12 of the identified proteins were selected to be expressed, and the immune sera from the mice vaccinated by the 12 expressed proteins, except BA0887, had a specific western blot band with the A16R whole cellular lytic proteins. Some of these immunogenic proteins might be used as novel vaccine candidates themselves or for enhancing the protective efficacy of a protective-antigen-based vaccine.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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