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While the capsule can shield the pathogen from host immune defense mechanisms , it also presents a vulnerability to the pathogen since anti-capsular antibodies elicited by the host can opsonize the pathogen , facilitating uptake and killing by neutrophils [ 16 ] . | PMC6331205 | -1 | [] |
Several highly successful vaccines to other bacterial pathogens target CP , including those against Haemophilus influenzae type b , S. pneumoniae and N. meningitidis , serogroups A , C , W , and Y [ 25 β 27 ] . | PMC6331205 | -1 | [] |
Given these successes , S. aureus vaccine candidates based on CP have been tested clinically . | PMC6331205 | -1 | [] |
To increase the immunogenicity of the CP and to induce T-cell memory , S. aureus CP5 and CP8 were conjugated to carrier proteins for use in preclinical and early clinical evaluations [ 28 β 30 ] . | PMC6331205 | -1 | [] |
Preclinical studies with CP5 and CP8 antibodies or vaccination with CP conjugates showed some evidence of protection in animal models of S. aureus infection and that polyclonal antibodies to staphylococcal CP are opsonic for encapsulated S. aureus [ 28 , 31 β 34 ] . | PMC6331205 | -1 | [] |
Staphvax ( by Nabi Biopharmaceutical ) , a first generation vaccine containing CP5 and CP8 coupled to detoxified recombinant Pseudomonas aeruginosa exotoxin A was the first to enter clinical efficacy phase 3 trials but failed to demonstrate efficacy in end-stage renal disease patients [ 35 , 36 ] . | PMC6331205 | -1 | [] |
The lack of efficacy of Staphvax raised concerns that S. aureus isolates may be deficient in CP expression , or that the patient population targeted was not competent in eliciting the appropriate protective immune responses . | PMC6331205 | -1 | [] |
In addition , the vaccine didn β t consistently generate functional , bacterial killing responses in that patient population [ 35 ] . | PMC6331205 | -1 | [] |
Furthermore , Scully et al. ( 2018 ) demonstrated that O-acetylation of the S. aureus capsular polysaccharide has to be maintained in the CP5 and 8 conjugates for them to induce bacterial killing antibodies [ 37 ] . | PMC6331205 | -1 | [] |
Passive immunization has also been evaluated to prevent S. aureus infections . | PMC6331205 | -1 | [] |
A Phase II trial of a polyclonal immune globulin ( Ig ) of anti-CP5 and anti-CP8 antibodies ( Altastaph ; by Nabi Biopharmaceutical ) failed to show a significant difference in clinical outcomes among patients with complicated S. aureus bacteremia [ 38 ] . | PMC6331205 | -1 | [] |
However , in another Phase II trial enrolled patients with documented S. aureus bacteremia who received Altastaph in addition to standard therapy or placebo , the vaccine induced CP antibodies were insufficient to significantly reduce S. aureus bacteremia [ 39 ] . | PMC6331205 | -1 | [] |
Liu et al. ( 2017 ) attributed the inconsistency of protective efficacy of passive CP8 immunization to the release of solube CP8 by some S. aureus isolates , which can possibly interfere with antibody binding to the encapsulated bacterial surface [ 40 β 42 ] . | PMC6331205 | -1 | [] |
Using lessons learned from previous clinical trials , the inclusion of multiple staphylococcal antigens would likely result in a more effective vaccine given the complexity of S. aureus and its myriad of virulence factors [ 43 β 45 ] . | PMC6331205 | -1 | [] |
Thus , several next-generation multi-antigen vaccines containing CP conjugates are currently in development . | PMC6331205 | -1 | [] |
The most advanced vaccine in clinical development is a 4 antigen S. aureus vaccine ( SA4Ag ) candidate that is being evaluated in a phase 2b / 3 efficacy study [ 31 , 46 ] . | PMC6331205 | -1 | [] |
SA4Ag is composed of CP5 and CP8 individually conjugated to to the nontoxic mutant form of diphtheria toxin ( cross-reactive material 197 [ CRM197 ] ) , recombinant form of clumping factor A ( rmClfA ) and recombinant non lipidated form of manganese transport protein C ( MntC ) [ 31 , 47 , 48 ] . | PMC6331205 | -1 | [] |
Confirmation of antigen expression is important to support the inclusion of these antigens in a prophylactic vaccine . | PMC6331205 | -1 | [] |
Due to the difficulty of studying bacterial gene expression in vivo , many studies have been performed under in vitro conditions to mimic the host environment , including nutrient limitation and low oxygen conditions [ 49 , 50 ] . | PMC6331205 | -1 | [] |
These studies have provided valuable information ; however , the full complexity of the in vivo environment cannot be adequately mimicked . | PMC6331205 | -1 | [] |
It has been reported that a significant proportion of clinical isolates do not express CP when grown in vitro [ 51 β 53 ] . | PMC6331205 | -1 | [] |
This observation was first noted for isolates belonging to the USA300 lineage , and remained to be evaluated in other lineages [ 54 , 55 ] . | PMC6331205 | -1 | [] |
CP expression is highly regulated by complex genetic mechanisms and subject to in vitro and in vivo physiological signals , including those encountered in the host [ 56 β 63 ] . | PMC6331205 | -1 | [] |
Recent data have emerged showing that a subset of S. aureus isolates that do not produce capsule in vitro , express the capsular polysaccharide when harvested from mice infected with these isolates [ 21 , 31 , 64 ] . | PMC6331205 | -1 | [] |
To expand upon this observation , a prospective analysis of disease causing isolates collected in the US either in 2004 or 2009 β 10 as part of the T.E.S.T surveillance trial was performed to investigate CP genotype distribution and expression in the context of S. aureus epidemiology in the US . | PMC6331205 | -1 | [] |
Materials and methods . | PMC6331205 | -1 | [] |
Ethics statement . | PMC6331205 | -1 | [] |
For the experiments with blood from subjects vaccinated with Sa3Ag ( 3-antigen S. aureus vaccine , ClinicalTrials.gov NCT01018641 ) , the protocol , informed consent , and all associated documents were approved by the Human Research Ethics Committees for each of the participating study sites : Site 001 , Alfred Research and Ethics Unit ; Site 002 , Bellberry Human Research Ethics Committee ; Site 003 , Children 's , Youth and Women 's Health Services Human Research Ethics Committee ; Site 004 , Princess Margaret Hospital for Children Ethics Committee ; Site 005 , Royal Children 's Hospital and Health Services District Ethics Committee . | PMC6331205 | -1 | [] |
Written informed consent was obtained from all subjects prior to study entry . | PMC6331205 | -1 | [] |
The clinical study was conducted in compliance with Good Clinical Practice ( GCP ) guidelines of the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use , the International Ethical Guidelines for Biomedical Research Involving Human Subjects , and the principles of the Declaration of Helsinki.All S. aureus Isolates including those obtained from the CDC and the global collection of Tigecycline Evaluation and Surveillance Trial were not linked to their associated patient data.All animal studies were conducted according to Pfizer local and approved global Institutional Animal Care and Use Committee ( IACUC ) guidelines . | PMC6331205 | -1 | [] |
The animal protocol is PRL-2011-00102 and it was approved by the Pfizer , Inc ., Pearl River IACUC . | PMC6331205 | -1 | [] |
Pfizer , Inc ., Pearl River utilizes the Guide for the Care and Use of Laboratory Animals ( Guide , NRC , 2011 ) as the primary standard for all animals under its institutional animal care and use program and also complies with the United States Department of Agriculture β s Animal Welfare Act ( AWA ) and Animal Welfare Regulations β Blue Book β ( USDA / APHIS , November 2013 ) for regulated species ( this does not apply to this protocol ) . | PMC6331205 | -1 | [] |
In all instances where Guide recommendations are different from AWA regulations for covered species , the mandated governing standard shall apply . | PMC6331205 | -1 | [] |
Female , CD1 mice 8 to 12 weeks old ( Charles River Laboratories ) were used and were humanely euthanized 6 h post infection by slow fill inhalation of CO followed by cervical dislocation or thoracotomy as a confirmatory method . | PMC6331205 | -1 | [] |
No adverse events occurred and animals did not experience more than momentary pain or distress . | PMC6331205 | -1 | [] |
Isolate selection . | PMC6331205 | -1 | [] |
We obtained 516 S. aureus isolates that were collected as part of the global Tigecycline Evaluation and Surveillance Trial ( T.E.S.T ) , now ATLAS ( Antimicrobial Testing Leadership And Surveillance ) . | PMC6331205 | -1 | [] |
For the purpose of conducting a longitudinal assessment of S. aureus isolates associated with disease in the USA , we prospectively selected healthcare centers across 6 US census regions ( East North Central , East South Central , Middle Atlantic , South Atlantic , West North Central , West South Central ) that were participating between 2004 and 2010 ( five sites ) . | PMC6331205 | -1 | [] |
Additional seven sites active at the time of our study were included . | PMC6331205 | -1 | [] |
A total of 516 S. aureus clinical isolates derived from 2004 ( n = 117 ) , 2009 ( n = 162 ) and 2010 ( n = 237 ) were collected and analyzed . | PMC6331205 | -1 | [] |
In line with T.E.S.T requirements , the participating US hospital sites collected consecutive isolates from patients with a documented infection of nosocomial or community origin . | PMC6331205 | -1 | [] |
Only one isolate per patient was permitted and all body sites were considered as acceptable clinical sources . | PMC6331205 | -1 | [] |
S. aureus isolates were cultured from specimens of patients aged 0 to 95 in various clinical settings , with samples broadly defined as derived from either inpatient ( medicine general , medicine ICU , pediatric general , pediatric ICU , surgery general , and surgery ICU ) or outpatient wards ( clinic , office , emergency room and nursing home-rehab ) [ 65 ] . | PMC6331205 | -1 | [] |
Culture identification and data management were coordinated by a single reference laboratory ( Laboratories International for Microbiology Studies , International Health Management Associates [ IHMA ] , Schaumburg , IL ) . | PMC6331205 | -1 | [] |
For the purpose of our study , all US isolates were grouped into HA-SA ( obtained from inpatient wards ) and community-associated ( CA-SA , obtained from outpatient wards ) infections and categorized into four main types defined as wound , invasive ( organism is cultured from synovial and pleural fluids , blood or bone ) , respiratory or other infections.For the in vivo expression experiments , additional isolates from a contemporary US collection were also tested . | PMC6331205 | -1 | [] |
These isolates were obtained from the Centers for Disease Control and Prevention ( CDC ) from a reference collection of 1,984 MRSA isolates collected in 2005 β 2006 by the Active Bacterial Core surveillance system [ 66 ] . | PMC6331205 | -1 | [] |
The clinical and epidemiologic characteristics of the these isolates are described in Murphy et al. [ 67 ] . | PMC6331205 | -1 | [] |
Construction of a PFESA0119 Deltacap5HIJK ermC strain . | PMC6331205 | -1 | [] |
All PCRs were performed with iProof High-Fidelity DNA polymerase ( Bio-Rad , Hercules , CA ) , and all restriction endonucleases were purchased from New England Biolabs ( Ipswich , MA ) . | PMC6331205 | -1 | [] |
A Ξ cap5HIJK ermC knockout cassette , consisting of two ~ 1.1 kb fragments ( cap-1 and cap-2 ) flanking the erythromycin resistance gene , ermC , was constructed . | PMC6331205 | -1 | [] |
The primer sets ( with relevant restriction sites in bold font ) and DNA templates used to amplify each of the fragments were : ( i ) cap-1 : oLH291 ( GCT GCT CCC GGG GAC AAT AGT TGG TAC AAG GCC ) / oLH277 ( GTA GCA TGT CTC ATT CAA TTG CTA TCC TCA TCG TCA TTT CC ) , S. aureus strain Newman genomic DNA ; ( ii ) ermC : oLH278 ( GGA AAT GAC GAT GAG GAT AGC AAT TGA ATG AGA CAT GCT AC ) / oLH279 ( CGA CGT CCT TTT TAT TAA TGA AAA CTG GTT TAA GCC GAC ) , pE194 ; ( iii ) cap-2 : oLH280 ( GTC GGC TTA AAC CAG TTT TCA TTA ATA AAA AGG ACG TCG ) / oLH281 ( GCT GCT GTC GAC TCT CGT GCA ATT CCT TAC TCG ) , S. aureus strain Newman genomic DNA . | PMC6331205 | -1 | [] |
These three products were spliced together by amplification with oLH291 and oLH281 , and the knockout cassette was cloned into pSPT181 [ 68 ] at the XmaI and SalI sites . | PMC6331205 | -1 | [] |
After electroporation and chromosomal integration into S. aureus strain RN4220 , the final Ξ cap5HIJK ermC mutant strain was obtained after plasmid excision by secondary recombination . | PMC6331205 | -1 | [] |
This mutation was transduced into S. aureus isolate PFESA0119 essentially as described elsewhere [ 69 ] . | PMC6331205 | -1 | [] |
In the resulting erythromycin resistant transductants , deletion of cap5HIJK was confirmed by PCR . | PMC6331205 | -1 | [] |
Mutants were shown to be of the expected strain lineage with a Qualicon RiboPrinter ( DuPont , Wilmington , DE ) . | PMC6331205 | -1 | [] |
Whole genome sequencing of S. aureus isolates . | PMC6331205 | -1 | [] |
Isolated colonies of pure cultures of S. aureus were cultured in TSB in 2 mL 96-well blocks . | PMC6331205 | -1 | [] |
Genomic DNA was isolated using the Qiagen QIAcube system according to manufacturer β s protocol . | PMC6331205 | -1 | [] |
Tagged DNA libraries were created using a method adapted from a standard Illumina Indexing protocol [ 70 ] . | PMC6331205 | -1 | [] |
Whole genome sequencing was performed on the Illumina HiSeq 2000 platform with 100 bp paired-end reads . | PMC6331205 | -1 | [] |
The Illumina sequence data were submitted to the European Nucleotide Archive ( ENA ) under accession numbers ERS234133 - ERS432212 ( S1 Table ) . | PMC6331205 | -1 | [] |
Whole genome sequence data analysis . | PMC6331205 | -1 | [] |
Annotated assemblies were produced as previously described [ 71 ] . | PMC6331205 | -1 | [] |
Briefly , de novo assembly of whole genome sequences was performed using Velvet v1.2 [ 72 ] with Velvet Optimiser v2.2.5 ( http://www.vicbioinformatics.com/software.velvetoptimiser.shtml ) ) . | PMC6331205 | -1 | [] |
Contigs were scaffolded with SSPACE [ 73 ] and sequence gaps closed using GapFiller [ 74 ] . | PMC6331205 | -1 | [] |
The draft assemblies have been submitted to the ENA with accession numbers ( ERS234133 - ERS432212 ) provided in S1 Table . | PMC6331205 | -1 | [] |
The assembled contigs were annotated using Prokka v1.11 [ 75 ] and S. aureus specific database from RefSeq [ 76 ] . | PMC6331205 | -1 | [] |
In silico PCR was used to determine the SCCmec type amongst mecA positive MRSA isolates [ 77 , 78 ] . | PMC6331205 | -1 | [] |
Isolate typing . | PMC6331205 | -1 | [] |
S. aureus isolates were characterized by MLST [ 79 ] and spa typing ( based on DNA sequence analysis of the S. aureus-specific staphylococcal protein A ( spa ) gene ) [ 80 ] . | PMC6331205 | -1 | [] |
For the MLST , the allelic profiles were extracted from the assembled genomes using an in-house developed pipeline , MLST check ( https://github.com/sanger-pathogens/mlst_check ) . | PMC6331205 | -1 | [] |
Novel allelic profiles and novel allele sequences were submitted to MSLT database curator ( http://saureus.beta.mlst.net ) and new STs were assigned . | PMC6331205 | -1 | [] |
For the spa typing , the spa gene was extracted by an in silico PCR using previously described primers ( http://www.ridom.de/doc/Ridom_spa_sequencing.pdf ) and the spa types were assigned using the spaTyper application ( http://spatyper.fortinbras.us ) . | PMC6331205 | -1 | [] |
Presence of genes of interest ( mecA , pvl , cap operon ) was analyzed using the Short Read Sequence Typer 2 [ 81 ] . | PMC6331205 | -1 | [] |
The capsular polysaccharide type was determined based on the presence of the type-specific capHIJK genes . | PMC6331205 | -1 | [] |
In silico PCR was used to determine the SCCmec type amongst mecA positive MRSA isolates [ 77 , 78 ] . | PMC6331205 | -1 | [] |
Opsonophagocytic assays ( OPA ) . | PMC6331205 | -1 | [] |
OPA were performed as previously described [ 16 ] . | PMC6331205 | -1 | [] |
OPA titers of sera from ten subjects vaccinated with Sa3Ag ( pre-vaccination bleed and 29 days post-vaccination bleed ) [ 82 ] were determined against CP5 and CP8 isolates representing the four most prevalent clonal complexes . | PMC6331205 | -1 | [] |
OPA titers are defined as the reciprocal of the highest serum dilution that kills 50 % of the bacteria in the test . | PMC6331205 | -1 | [] |
Each sample was tested in replicate and geometric mean titers with 95 % confidence interval were calculated using data from the ten subjects and from two independent experiments . | PMC6331205 | -1 | [] |
Negative samples were arbitrarily assigned a titer of 50 , which is half of the lowest serum dilution tested in the OPA . | PMC6331205 | -1 | [] |
The specificity of the OPA was determined by preabsorbing immune serum with homologous capsular polysaccharides CP5 ( 140 ΞΌ g / mL ) or CP8 ( 320 ΞΌ g / mL ) . | PMC6331205 | -1 | [] |
After overnight incubation at 4 Β° C , anti-CP antibody depleted sera were used in the OPA described above . | PMC6331205 | -1 | [] |
Due to limited availability of serum volume , the PFESA2069 isolate was tested with depleted sera from 7 out of 10 vaccinated subjects . | PMC6331205 | -1 | [] |
Detection of capsule production in vitro by Luminex-based assays . | PMC6331205 | -1 | [] |
As not all 516 isolates were tested in this assay , the proportion that may not express capsule in vitro was estimated by calculating the % CP-negative isolates within each spa / CC group tested and then extrapolating to the % of the total population ( n = 516 ) based on the distribution of each given CC . | PMC6331205 | -1 | [] |
For example , 47 isolates of CC8 ( 37 % of the total S. aureus population ) were tested for in vitro capsule expression and since 41 CC8 isolates ( 87 % of CC8 ) were CP-negative , the % CP-negative CC8 isolates was estimated to be 32 % of the total population . | PMC6331205 | -1 | [] |
The Luminex assays used to detect capsule expression in vitro are based on several steps including polysaccharide sample preparation , generation of monoclonal antibodies , coupling of CP5 and CP8 antigens to Luminex MagPlex microspheres , and analysis of samples by singleplex liquid array system , Luminex 200 . | PMC6331205 | -1 | [] |
Polysaccharide sample preparation . | PMC6331205 | -1 | [] |
S. aureus isolates were grown overnight on Columbia salt agar ( CSA ) plates and isolated colonies were then resuspended in Dulbecco β s PBS ( DPBS ) buffer without Ca / Mg to one OD . | PMC6331205 | -1 | [] |
The cell suspension was then autoclaved at 121 Β° C for 30 min as previously described [ 83 ] . | PMC6331205 | -1 | [] |
Cells were pelleted by centrifugation ( 3,000 x g , 15 min ) and supernatants were used in the Luminex assays . | PMC6331205 | -1 | [] |
Monoclonal antibodies ( mAbs ) . | PMC6331205 | -1 | [] |
CP5 and CP8 specific mAbs were generated by immunizing mice ( Swiss Webster , BALB / c , or SJL ) with polysaccharides conjugated to the CRM197 carrier protein . | PMC6331205 | -1 | [] |
These polysaccharides were produced according to the methods as described in Fattom et al. [ 84 ] . | PMC6331205 | -1 | [] |
The CP were conjugated to cross-reacting CRM197 ( cross-reacting material 197 ) , a nontoxic recombinant mutant of diphtheria toxin , in a conjugation process using 1,1 β² - carbonyldiimidazole / 1,1 β² - carbonyl-di - ( 1,2,4 - triazole ) ( described in US patent US 8568735 B2 ) . | PMC6331205 | -1 | [] |
Splenocytes were fused with the non secreting myeloma cell line X63Ag8.653 to generate hybridomas that were screened for the appropriate serotype specificity . | PMC6331205 | -1 | [] |
The CP5-specific mAb CP5-5-1 is of the IgG1 subclass and the CP8-specific mAb CP8-119-11 is of the IgG2b subclass . | PMC6331205 | -1 | [] |