Immunogenicity and protective activity of recombinant influenza viruses expressing fragments of ScaAB lipoprotein of group B streptococci in a mouse model

2020 ◽  
Vol 20 (3) ◽  
pp. 33-42
Author(s):  
E. A. Stepanova ◽  
I. N. Isakova-Sivak ◽  
V. A. Matyushenko ◽  
A. S. Matushkina ◽  
T. A. Smolonogina ◽  
...  
Keyword(s):  
Amino Acids ◽  
Bacterial Infections ◽  
Reverse Genetics ◽  
Mdck Cells ◽  
Influenza Viruses ◽  
Effective Vaccine ◽  
Temperature Sensitive ◽  
Protective Activity ◽  
Group B Streptococci ◽  
Group B

Group B streptococci (GBS) cause a number of serious diseases in humans. The development of an effective vaccine against GBS requires special approaches. In the present study, three recombinant influenza viruses were constructed on the backbone of H7N9 live attenuated influenza vaccine (LAIV) strain expressing fragments of the ScaAB lipoprotein of Streptococcus agalactiae, fused to the surface protein of the virus, hemagglutinin, using a flexible linker. Recombinant viruses with ScaAB inserts of 85, 141, and 200 amino acids were successfully rescued by the means of reverse genetics. The recombinant strains were able to grow in developing chicken embryos and MDCK cells and retained the temperature-sensitive phenotype attributable to the LAIV viruses. Studies of immunogenicity and protective activity of the vaccine candidates in BALB/c mice revealed that the most promising strain was a strain with an insert of 141 amino acids: this variant had optimal immunogenicity against influenza and GBS and had a protective effect against both pathogens. These data indicate that further studies of the recombinant vectored vaccine H7-ScaAB-141 as a combined viral-bacterial vaccine capable of protection against both influenza virus and bacterial infections caused by group B streptococci are warranted.

Comparative Protective Activity of Human Monoclonal and Hyperimmune Polyclonal Antibody against Group B Streptococci

1991 ◽  
Vol 163 (4) ◽  
pp. 792-798 ◽  
Author(s):  
H. R. Hill ◽  
L. A. Gonzales ◽  
W. A. Knappe ◽  
G. W. Fischer ◽  
D. K. Kelsey ◽  
...  
Keyword(s):  
Polyclonal Antibody ◽  
Protective Activity ◽  
Group B Streptococci ◽  
Group B

scholarly journals Screening for Neuraminidase Inhibitor Resistance Markers among Avian Influenza Viruses of the N4, N5, N6, and N8 Neuraminidase Subtypes

2017 ◽  
Vol 92 (1) ◽  
Author(s):  
Won-Suk Choi ◽  
Ju Hwan Jeong ◽  
Jin Jung Kwon ◽  
Su Jeong Ahn ◽  
Khristine Kaith S. Lloren ◽  
...  
Keyword(s):  
Avian Influenza ◽  
Reverse Genetics ◽  
Mdck Cells ◽  
Random Mutagenesis ◽  
Influenza Viruses ◽  
Human Pathogens ◽  
Avian Influenza Viruses ◽  
Oseltamivir Carboxylate ◽  
Resistance Markers ◽  
Na Gene

ABSTRACTSeveral subtypes of avian influenza viruses (AIVs) are emerging as novel human pathogens, and the frequency of related infections has increased in recent years. Although neuraminidase (NA) inhibitors (NAIs) are the only class of antiviral drugs available for therapeutic intervention for AIV-infected patients, studies on NAI resistance among AIVs have been limited, and markers of resistance are poorly understood. Previously, we identified unique NAI resistance substitutions in AIVs of the N3, N7, and N9 NA subtypes. Here, we report profiles of NA substitutions that confer NAI resistance in AIVs of the N4, N5, N6, and N8 NA subtypes using gene-fragmented random mutagenesis. We generated libraries of mutant influenza viruses using reverse genetics (RG) and selected resistant variants in the presence of the NAIs oseltamivir carboxylate and zanamivir in MDCK cells. In addition, two substitutions, H274Y and R292K (N2 numbering), were introduced into each NA gene for comparison. We identified 37 amino acid substitutions within the NA gene, 16 of which (4 in N4, 4 in N5, 4 in N6, and 4 in N8) conferred resistance to NAIs (oseltamivir carboxylate, zanamivir, or peramivir) as determined using a fluorescence-based NA inhibition assay. Substitutions conferring NAI resistance were mainly categorized as either novel NA subtype specific (G/N147V/I, A246V, and I427L) or previously reported in other subtypes (E119A/D/V, Q136K, E276D, R292K, and R371K). Our results demonstrate that each NA subtype possesses unique NAI resistance markers, and knowledge of these substitutions in AIVs is important in facilitating antiviral susceptibility monitoring of NAI resistance in AIVs.IMPORTANCEThe frequency of human infections with avian influenza viruses (AIVs) has increased in recent years. Despite the availability of vaccines, neuraminidase inhibitors (NAIs), as the only available class of drugs for AIVs in humans, have been constantly used for treatment, leading to the inevitable emergence of drug-resistant variants. To screen for substitutions conferring NAI resistance in AIVs of N4, N5, N6, and N8 NA subtypes, random mutations within the target gene were generated, and resistant viruses were selected from mutant libraries in the presence of individual drugs. We identified 16 NA substitutions conferring NAI resistance in the tested AIV subtypes; some are novel and subtype specific, and others have been previously reported in other subtypes. Our findings will contribute to an increased and more comprehensive understanding of the mechanisms of NAI-induced inhibition of influenza virus and help lead to the development of drugs that bind to alternative interaction motifs.

scholarly journals 993. Combining Key Residues of the Russian and US Live-Attenuated Influenza Viruses for a More Attenuated Virus

2018 ◽  
Vol 5 (suppl_1) ◽  
pp. S294-S295
Author(s):  
Andrew Smith ◽  
Laura Rodriguez-Garcia ◽  
Maya El Ghouayel ◽  
Luis Martinez-Sobrido ◽  
Stephen Dewhurst
Keyword(s):  
United States ◽  
Influenza Virus ◽  
Growth Curve ◽  
Mdck Cells ◽  
The United States ◽  
Polymerase Activity ◽  
Influenza Viruses ◽  
Renilla Luciferase ◽  
Temperature Sensitive

Abstract Background The Live Attenuated Influenza Virus (LAIV) used in the United States is based on the cold-passaged A/AnnArbor/6/60 strain (AA). An alternative LAIV (Len), developed from the cold-passaged A/Leningrad/134/17/57 strain, has also been used in some countries outside the United States. Recent concerns with the efficacy and safety of the current US LAIV warrant the development of an improved LAIV. Methods We used in vitro minireplicon and multicycle viral growth assays to analyze the combined effects of polymerase mutations from LAIV (AA) and LAIV (Len) on the phenotype of PR8. Mini-replicon assays were performed in HEK-293T cells with firefly luciferase under the control of the influenza virus NP promoter; we controlled for cell density with a constitutively active Renilla luciferase. Multicycle growth curve experiments were performed at 33°C, 37°C, and 39°C in MDCK cells with an m.o.i. of 0.001. Mean values for triplicate infections at 12, 24, 48, and 72 hours were plotted as TCID50/mL. Results Control experiments showed replication of PR8 (AA) and PR8 (Len) in MDCK cells was significantly decreased as compared with WT PR8 at 37°C and 39°C at 24–48 hour time points, but not at 33C (the temperature of nasal passages). We found that polymerase activity was up to 3 logs more temperature-sensitive (ts) at 37°C and 39°C with the combined Len and AA mutations using the mini-replicon assay. In the growth curve experiments, the combined Len and AA mutations conferred up to a 4-log decrease in replication levels at 37°C as compared with PR8 (Len) and an even greater decrease compared with PR8 (AA). Conclusion Our findings suggest combining the AA and Len LAIV polymerase mutations decreases LAIV replication at body temperature (37°C), as compared with either LAIV alone. This could be useful in developing an improved LAIV that is safer in vulnerable hosts (e.g., children under the age of 2 who may be vulnerable to wheezing), while also permitting dose escalation that might result in greater efficacy. Minireplicon assay. Polymerase activity of combination mutants. Multicycle replication kinetics of combination mutants (red) against PR8 Len (black) at 33C (solid), 37C (dashed) and 39C (dotted). Disclosures All authors: No reported disclosures.

scholarly journals Characterization of a porcine intestinal epithelial cell line for influenza virus production

2012 ◽  
Vol 93 (9) ◽  
pp. 2008-2016 ◽  
Author(s):  
Zhi Sun ◽  
Victor C. Huber ◽  
Kara McCormick ◽  
Radhey S. Kaushik ◽  
Adrianus C. M. Boon ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Cell Line ◽  
Influenza A ◽  
Reverse Genetics ◽  
Mdck Cells ◽  
Influenza Viruses ◽  
Epithelial Cell Line ◽  
Influenza B ◽  
Specific Cell ◽  
Influenza A Viruses

We have developed a porcine intestine epithelial cell line, designated SD-PJEC for the propagation of influenza viruses. The SD-PJEC cell line is a subclone of the IPEC-J2 cell line, which was originally derived from newborn piglet jejunum. Our results demonstrate that SD-PJEC is a cell line of epithelial origin that preferentially expresses receptors of oligosaccharides with Sia2-6Gal modification. This cell line is permissive to infection with human and swine influenza A viruses and some avian influenza viruses, but poorly support the growth of human-origin influenza B viruses. Propagation of swine-origin influenza viruses in these cells results in a rapid growth rate within the first 24 h post-infection and the titres ranged from 4 to 8 log10 TCID50 ml−1. The SD-PJEC cell line was further tested as a potential alternative cell line to Madin–Darby canine kidney (MDCK) cells in conjunction with 293T cells for rescue of swine-origin influenza viruses using the reverse genetics system. The recombinant viruses A/swine/North Carolina/18161/02 (H1N1) and A/swine/Texas/4199-2/98 (H3N2) were rescued with virus titres of 7 and 8.25 log10 TCID50 ml−1, respectively. The availability of this swine-specific cell line represents a more relevant substrate for studies and growth of swine-origin influenza viruses.

scholarly journals Mutation of Neuraminidase Cysteine Residues Yields Temperature-Sensitive Influenza Viruses

1999 ◽  
Vol 73 (10) ◽  
pp. 8095-8103 ◽  
Author(s):  
Christopher F. Basler ◽  
Adolfo García-Sastre ◽  
Peter Palese
Keyword(s):  
Influenza Virus ◽  
Disulfide Bonds ◽  
Reverse Genetics ◽  
Expression System ◽  
Influenza Viruses ◽  
Surface Glycoprotein ◽  
H3n2 Virus ◽  
Cysteine Residues ◽  
Temperature Sensitive ◽  
Virus Surface

ABSTRACT The influenza virus neuraminidase (NA) is a tetrameric, virus surface glycoprotein possessing receptor-destroying activity. This enzyme facilitates viral release and is a target of anti-influenza virus drugs. The NA structure has been extensively studied, and the locations of disulfide bonds within the NA monomers have been identified. Because mutation of cysteine residues in other systems has resulted in temperature-sensitive (ts) proteins, we asked whether mutation of cysteine residues in the influenza virus NA would yield ts mutants. The ability to rationally design tight and stable ts mutations could facilitate the creation of efficient helper viruses for influenza virus reverse genetics experiments. We generated a series of cysteine-to-glycine mutants in the influenza A/WSN/33 virus NA. These were assayed for neuraminidase activity in a transient expression system, and active mutants were rescued into infectious virus by using established reverse genetics techniques. Mutation of two cysteines not involved in intrasubunit disulfide bonds, C49 and C146, had modest effects on enzymatic activity and on viral replication. Mutation of two cysteines, C303 and C320, which participate in a single disulfide bond located in the β5L0,1 loop, produced ts enzymes. Additionally, the C303G and C320G transfectant viruses were found to be attenuated and ts. Because both the C303G and C320G viruses exhibited stable ts phenotypes, they were tested as helper viruses in reverse genetics experiments. Efficiently rescued were an N1 neuraminidase from an avian H5N1 virus, an N2 neuraminidase from a human H3N2 virus, and an N7 neuraminidase from an H7N7 equine virus. Thus, these cysteine-to-glycine NA mutants allow the rescue of a variety of wild-type and mutant NAs into influenza virus.

scholarly journals Molecular Characterization of Nonhemolytic and Nonpigmented Group B Streptococci Responsible for Human Invasive Infections

2015 ◽  
Vol 54 (1) ◽  
pp. 75-82 ◽  
Author(s):  
Anne Six ◽  
Arnaud Firon ◽  
Céline Plainvert ◽  
Camille Caplain ◽  
Gérald Touak ◽  
...  
Keyword(s):  
Bacterial Infections ◽  
Clinical Laboratory ◽  
Developed Countries ◽  
Pigment Production ◽  
Group B Streptococci ◽  
Gene Encoding ◽  
Content Type ◽  
Invasive Infections ◽  
Group B ◽  
Routine Clinical Laboratory

Group BStreptococcus(GBS) is a common commensal bacterium in adults, but is also the leading cause of invasive bacterial infections in neonates in developed countries. The β-hemolysin/cytolysin (β-h/c), which is always associated with the production of an orange-to-red pigment, is a major virulence factor that is also used for GBS diagnosis. A collection of 1,776 independent clinical GBS strains isolated in France between 2006 and 2013 was evaluated on specific medium for β-h/c activity and pigment production. The genomic sequences of nonhemolytic and nonpigmented (NH/NP) strains were analyzed to identify the molecular basis of this phenotype. Gene deletions or complementations were carried out to confirm the genotype-phenotype association. Sixty-three GBS strains (3.5%) were NH/NP, and 47 of these (74.6%) originated from invasive infections, including bacteremia and meningitis, in neonates or adults. The mutations are localized predominantly in thecyloperon, encoding the β-h/c pigment biosynthetic pathway and, in theabx1gene, encoding a CovSR regulator partner. In conclusion, although usually associated with GBS virulence, β-h/c pigment production is not absolutely required to cause human invasive infections. Caution should therefore be taken in the use of hemolysis and pigmentation as criteria for GBS diagnosis in routine clinical laboratory settings.

Neonatal Cellular and Humoral Immunity to Group B Streptococci

PEDIATRICS ◽  
1979 ◽  
Vol 64 (5) ◽  
pp. 787-794
Author(s):  
Harry R. Hill ◽  
Ann O. Shigeoka ◽  
Robert T. Hall ◽  
Val G. Hemming
Keyword(s):  
Bacterial Infections ◽  
Metabolic Activation ◽  
Minor Role ◽  
Complement Pathway ◽  
Group B Streptococci ◽  
Opsonic Activity ◽  
Bacterial Uptake ◽  
Group B ◽  
Major Factors ◽  
A Minor

The mechanisms of host resistance to group B streptococci have not been defined precisely. In the studies reported here we have assessed the contributions of both humoral and cellular factors in protection against strains of this group. With assays of specific opsonic activity based upon the production of polymorphonuclear leukocyte chemiluminescence and radiolabeled bacterial uptake, we have demonstrated that specific heat-stable antibody and the classic complement pathway are major factors in opsonization of these organisms. In the absence of specific antibody, fresh serum resulted in markedly reduced bacterial uptake indicating, at best, a minor role for the alternative complement pathway. Additional studies have indicated that strain-specific antiphagocytic factors as well as type-specific ones may play a role in the virulence of these organisms. Neonates who developed group B streptococcal sepsis usually lacked opsonic activity in their infecting strain. In addition, polymorphonuclear leukocytes from normal term and stressed neonates showed impaired metabolic activation as measured in the chemiluminescence assay following exposure to opsonized group B streptococci. These results suggest that neonates who develop group B streptococcal disease may have defects in both the humoral and cellular aspects of their acute inflammatory response which may contribute to the high mortality observed in this most fulminant of bacterial infections.

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