Fluorescence- Activated Cell Sorter is a powerful technique used in cell sorting, cell-cycle analysis etc.
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Antigen processing and presentation by Dr K.Geetha, Associate Professor, Department of Biotechnology, Kamaraj College of Engineering & Technology, Near Virudhunagar, Madurai Dist.
Exosomes biomarkers mediating important biological process,especially in the systemic disease
diagnostics and therapeutics,yet the protective exosomal vesicle structure hinders rapid,simple detection of the harbored molecules.
Fluorescence- Activated Cell Sorter is a powerful technique used in cell sorting, cell-cycle analysis etc.
The presentation gives a basic understanding of the principle of FACS, instrumentation, interpretation of results, applications, how to do cell-cycle analysis using FACS and various troubleshooting tips.
Antigen processing and presentation by Dr K.Geetha, Associate Professor, Department of Biotechnology, Kamaraj College of Engineering & Technology, Near Virudhunagar, Madurai Dist.
Exosomes biomarkers mediating important biological process,especially in the systemic disease
diagnostics and therapeutics,yet the protective exosomal vesicle structure hinders rapid,simple detection of the harbored molecules.
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Multiple Alignment Sequence using Clustal Omega/ Shumaila RiazShumailaRiaz6
Alignment of three or more biological sequences of Protein, DNA, RNA of similar length
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Comparing Residual Integration Levels of Some IntegrationDeficient Lentiviral...inventionjournals
Lentiviral vectors (LVs) have many advantageous characteristics making them a good choice in the field of gene therapy. Nevertheless, their integration may lead to detrimental effects. To overcome this problem, lentiviral integration can be targeted through using integration-deficient lentiviral vectors (IDLVs). In this study, an integration-proficient lentiviral vector (IPLV) and a battery of IDLVs with single or multiple mutations affecting integration were produced and their integration levels were compared. eGFP time-course experiment and clonogenic assay were used to make these comparisons. It was found that there was not any significant difference between the residual integration of any of the IDLVs used in this study and that of the standard IDLV; D64V-IDLV. It can be concluded that most IDLV integration is mediated by integraseindependent mechanisms.
IMMUNOINFORMATICS , MICROARRAY and Machine Learning - All about Immunology, I...Mekhla Diwan
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Multiple Alignment Sequence using Clustal Omega/ Shumaila RiazShumailaRiaz6
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Clustal Omega is tool for analyzing the Multiple sequence alignments of proteins
Comparing Residual Integration Levels of Some IntegrationDeficient Lentiviral...inventionjournals
Lentiviral vectors (LVs) have many advantageous characteristics making them a good choice in the field of gene therapy. Nevertheless, their integration may lead to detrimental effects. To overcome this problem, lentiviral integration can be targeted through using integration-deficient lentiviral vectors (IDLVs). In this study, an integration-proficient lentiviral vector (IPLV) and a battery of IDLVs with single or multiple mutations affecting integration were produced and their integration levels were compared. eGFP time-course experiment and clonogenic assay were used to make these comparisons. It was found that there was not any significant difference between the residual integration of any of the IDLVs used in this study and that of the standard IDLV; D64V-IDLV. It can be concluded that most IDLV integration is mediated by integraseindependent mechanisms.
INTRODUCTION
KEY ELEMENTS IN TISSUE ENGINEERING
- Progenitor cells
- Scaffold
- Signalling molecules
DESIRED PROPERTIES AND WAYS TO ENHANCE THE REGENERATIVE CAPACITY OF SCAFFOLDS
4. GENE THERAPY IN PERIODONTAL TISSUE ENGINEERING
5. RECENT DEVELOPMENTS
6. CRITICAL ANALYSIS OF PRESENT STATUS OF TISSUE ENGINEERING FOR PERIODONTICS.
4. CONCLUSION
5. REFERENCES
"A Nano-In-Micro System for Enhanced Stem Cell Therapy of Ischemic Diseases" is a research article published in ACS central science http://pubs.acs.org/journal/acscii
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ID
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For more information, visit-www.vavaclasses.com
3. INTRODUCTION
• Nowadays, vaccines must combine the lowest possible adverse effects with a
high efficacy.
• The development of a highly safe and effective vaccine requires a lot of resources
and time. At the same time the upcoming post-antibiotic era may require the
development of more vaccines that also need to be developed in a short period
(WHO 2014).
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4. INTRODUCTION
• Outer membrane vesicles (OMV) are non replicative, spherical outer
membrane products of gram negative and gram positive bacteria.
• OMV are produced by both pathogenic and non pathogenic bacteria and
are involved in diverse functions such as mediating bacterial intercellular
communication, promoting virulence, elimination of unwanted components
and modulating host immune response.
• The diverse functions performed by OMVs are conferred by the different
cargoes such as LPS, Phospholipids, Peptidoglycan, OMPs, cell wall
components, proteins, nucleic acids, ion metabolites and signaling
molecules.
10/20/2018 Department of Plant Biotechnology, UAS, GKVK, Bengaluru -65 4
5. 10/20/2018 Department of Plant Biotechnology, UAS, GKVK, Bengaluru -65 5
STRUCTURE OF OMV
Anand and Choudari, 2016
6. 10/20/2018 Department of Plant Biotechnology, UAS, GKVK, Bengaluru -65 6
STRUCTURE OF OMV
The bacterial OM is unique for its asymmetrical distribution of lipid moieties,
wherein the exterior leaflet is enriched in lipopolysaccharides (LPS), and the inner
leaflet predominantly contains glycerophospholipid
Endotoxin or lipid-A, a component of LPS, decorates the OM and is responsible
for the immune modulation during pathogenic invasions
The spherical structure of OMVs provides a higher surface to- volume ratio,
therefore allowing the residence of LPS in high density. LPS plays a major role in
host pathogen interplay and modulation of host immunity.
The enzymes involved in LPS synthesis extend between the IM and OM of the
bacterial cell wall. LPS synthesis is initiated by a group of Lpx enzymes, present on
the cytoplasmic face of the IM by the formation of a precursor called kdo2-lipid A
The major population of lipid A moieties are further modified by ligation of O-
antigen units of various lengths. Finally, the LPS moiety is exported to the OM
Out of these steps, the ligation of O antigen confers variability to the structure of
LPS, and these differential modifications cater to the need of the bacterium to
adapt to its local environment
Anand and Choudari, 2016
7. 10/20/2018 Department of Plant Biotechnology, UAS, GKVK, Bengaluru -65 7
Outer membrane vesicles are produced by the expansion and bulging of the OM
followed by pinching of the OM to form spherical vesicles.
Anand and Choudari, 2016
8. REGULATORY MECHANISMS OF OMV FORMATION
10/20/2018 Department of Plant Biotechnology, UAS, GKVK, Bengaluru -65 8
9. CONT…
10/20/2018 Department of Plant Biotechnology, UAS, GKVK, Bengaluru -65 9
Membrane proteins like Lpp and OmpA, located in the periplasmic space, span the
gap between the OM and PG. Loss of association between OM and PG by a
reduction in synthesis of Lpp or OmpA, or alteration in PG structure (reducing the
binding of these proteins to PG layer), enhances OMV formation.
An IM lipoprotein gene nlpA deletion was found to reduce OMV production.
However, the deletion of ompA or lpp genes enhanced the production of OMVs.
Outer
membrane
and PG cross
linking
&
Alteration in
PG structure
Anand and Choudari, 2016
10. 10/20/2018 Department of Plant Biotechnology, UAS, GKVK, Bengaluru -65 10
CONT…
Stress inducing factors like osmotic-shock brought about by high saline conditions,
dehydration caused by long chain alcohol, heat-shock, antibiotic stress and
accumulation of misfolded proteins or components of PG are potent inducers of
OMV production.
Short fragments of PG are produced from interrupted biosynthesis or imbalance
between synthesis and degradation. Local accumulation of these unwanted products
interacts with the membrane and causes bulging that eventually turns into vesicles.
Envelope
stress
response
Anand and Choudari, 2016
11. 10/20/2018 Department of Plant Biotechnology, UAS, GKVK, Bengaluru -65 11
CONT…
The Tol-Pal is an IM protein complex involved in the uptake of iron siderophores,
type-A colicins and single stranded DNA phages. The Tol-Pal system consists of 5
proteins. The tolA, tolR and tolQ genes are part of an operon, and their deletion
causes a significant increase in vesiculation.
Tol-Pal system
Anand and Choudari, 2016
12. 10/20/2018 Department of Plant Biotechnology, UAS, GKVK, Bengaluru -65 12
CONT…
Another proposed mechanism for inducing OMV production involves the
phospholipid transporter VacJ-Yrb ABC (ATP-binding cassette) system located in
the bacterial IM. Low levels of VacJ-Yrb components were shown to increase OMV
production by increased accumulation of curvature-inducing phospholipids in the
bacterial OM.
VacJ/Yrb ABC
– phospholipid
transporter
Anand and Choudari, 2016
13. 10/20/2018 Department of Plant Biotechnology, UAS, GKVK, Bengaluru -65 13
CONT…
Thermoregulation
• Cells growing at a lower temperature (22 or 30 C) produce higher levels of
OMVs compared to the cells grown at 37 C. Enhancement in OMV production
under thermal stress was due to reduced production of Enterobacterial common
antigen (ECA), a glycerophospholipid found in the outer leaflet of OM.
• This reduction in the ECA was linked to RcsB phosphorelay signaling. Thermal
stress causes RcsB signaling activation and reduced transcription of the ECA
gene. Eventually, reduced membrane integrity leads to increased OMV
production.
OM remodeling
Lipid-A modifications play an important role in OMV production. The
enzyme lipid-A deacylase called PagL modifies lipid-A by deacylation. The
accumulation of deacylated lipid-A on the outer leaflet creates positive curvature
leading to increased vesiculation.
14. High-throughput detection of OMVs
10/20/2018 Department of Plant Biotechnology, UAS, GKVK, Bengaluru -65 14
• Recently, a novel method has been used for determination of the size and
concentration of extracellular vesicles using tunable resistive pulse sensing.
• which employs extrapolation of scanning ion occlusion spectroscopy (SIOS)
which is based on the property of scalable membrane pores (i.e. screening by
stretchability of membrane pores of different sizes) to analyze nano-sized
particles.
• As the particles of different size pass through the membrane pore, it induces
changes in the current across the membrane by interfering with the open pore
conductivity, which can be detected with high sensitivity and accuracy.
Anand and Choudari, 2016
15. FUNCTIONS OF OMV
10/20/2018 Department of Plant Biotechnology, UAS, GKVK, Bengaluru -65 15
• Outer Membrane Vesicles have characteristics that enable them to mediate
transfer of DNA fragments, autolysins, cytotoxins, virulence factors and a
variety of other biomolecules.
• Their secretion helps bacteria in establishing inter- and intraspecies
communication and also strengthens the interaction with the host.
• Among the prominent roles in diverse physiological and pathological functions,
OMVs have been recognized for their role in acquisition of nutrients, stress
responses and delivery of toxins, adhesion and virulence factors to evade host
defense system.
Jan, 2017
16. Designing the OMV
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Heterologous antigens on OMVs can be presented with or without surface
exposure, attached to the vesicle or non-attached and directly produced by the
bacterium or combined in a later production stage.
• Endogenous loading of surface exposed antigens
• Endogenous loading of antigens to the OMV lumen
• Exogenous loading of surface exposed antigens
• Exogenous loading of antigens to the OMV lumen
• Surface exposed antigens are accessible for antigen-specific B cell binding,
while the inside of the vesicle is shielded from these cells.
• Luminal antigens may be skewed towards cytotoxic T-cell responses, hence the
desired immune response determines the design of the OMV.
17. Endogenous loading of surface exposed antigens
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• To express larger proteins on the surface of the outer membrane, Kim et al.
fused several heterologous protein including GFP with a five residue glycine
linker to the C-terminus of the pore-forming cytotoxin ClyA.
• These fusion constructs were efficiently transported across the inner membrane
to the outer membrane of E. coli.
• Recently it was shown by their group that surface expressed M2e antigens on E.
coli OMVs provided protection against influenza A infection in a mice study
(Rappazzo et al. 2016).
• Besides single M2e- ClyA fusion constructs, the authors also successfully
constructed a nearly 50 kDa multimeric variant containing the serine analogs of
human, swine and two avian M2e variants.
Surface exposed expression of heterologous lipoproteins has been
achieved by fusion of the heterologous lipoprotein to a membrane
anchoring second lipoprotein.
18. Endogenous loading of antigens to the OMV lumen
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• Antigens have been bioengineered to be targeted to the lumen of OMVs.
• Kesty and Kuehn fused GFP to the twin-arginine (Tat) signal sequence to
produce E. coli OMVs with GFP in their lumen.
• This Tat pathway transfers folded proteins over the cytoplasmic membrane.
• To transfer unfolded proteins over the cytoplasmic membrane the Sec pathway
could be used.
• The OMVs, containing GFP in their lumen, showed to shield GFP from
proteinases, which indicates the stability of proteins inside the vesicles.
• Heterologous antigens can be expressed in the lumen of OMVs by
fusion of the antigen to secretion signals or periplasmic proteins.
• These fusion based methods are relatively straight forward and have
been successfully used to express full size antigens in a
conformational correct manner and antibody have been found
against these antigens.
19. Exogenous loading of surface exposed antigens
• Antigens can be added afterwards by introducing affinity to the antigen on the
OMV.
• This method of exogenous loading was used by adding a SpyTag to OmpA.
• The SpyTag peptide is designed to form a covalent bond with the SpyCatcher
protein to ease conjugation of proteins.
• The SpyTag-OmpA fusion was expressed on OMVs and can be coupled to
SpyCatcher fused to any protein.
• By this method, OMVs can be produced in mass production followed by adding
antigens on the OMV.
10/20/2018 Department of Plant Biotechnology, UAS, GKVK, Bengaluru -65 19
20. Exogenous loading of antigens to the OMV lumen
• Loading of antigens to the vesicle lumen, after the vesicles and antigens have
separately been produced in bulk, can be an attractive approach
• The concentration of the active components may be better controllable when
compared to endogenous loading.
• To load antigens in their native conformation to the OMV lumen in vitro, the
vesicle should be opened and closed again without permanent damage.
• The exogenous loading of proteins to the vesicle lumen has not been described
although several methods of loading smaller molecules into extracellular vesicles
(EVs) are explored.
10/20/2018 Department of Plant Biotechnology, UAS, GKVK, Bengaluru -65 20
• EVs can be loaded by simple incubation (passive loading) or by active treatment
of the EVs (active loading).
• Methods of loading EVs by electroporation, saponin-treatment, extrusion or
dialysis
23. INTRODUCTION
• Each year, 5–10% of adults and 20–30% of children are infected with seasonal
influenza, leading to 3–5 million severe cases and 250,000–500,000 deaths
worldwide. (WHO, 2016)
• Current influenza vaccines primarily target the immune response against the
immuno dominant—but hyper variable—glycoproteins hemagglutinin (HA), and
to a lesser extent, neuraminidase (NA), necessitating annual development of
strain-specific vaccines.
• Target antigen for a universal vaccine is M2e, the ectodomain of the influenza A
matrix protein 2 (M2), a homo tetrameric ion channel with roles in viral entry and
budding,
• First explored as a vaccine target by Richardson et al. in 1985
10/20/2018 Department of Plant Biotechnology, UAS, GKVK, Bengaluru -65 23
24. • M2e demonstrates remarkable conservation of its 23 amino acid sequence.
• This high conservation has been attributed to M2e’s genetic relationship with M1
– an integral structural protein – and the lack of selective pressure against M2e
due to M2e’s low virion presence and immunogenicity.
• It has been previously shown that IgG2a isotype antibody levels, associated with
a Th1 type immune response in mice, correlate significantly with enhanced
survival against influenza challenge for M2e-based vaccines.
• Therefore, M2e-based vaccines have aimed to generate Th1-biased responses in
an effort to improve protective efficacy.
10/20/2018 Department of Plant Biotechnology, UAS, GKVK, Bengaluru -65 24
25. • OMVs can be engineered to display recombinant heterologous proteins by
transforming hyper vesiculating (ΔnlpI) strains of Escherichia coli with a plasmid
encoding the transmembrane protein ClyA followed by the antigen of interest.
• Demonstrated that the display of M2e-based peptides on the surface of OMVs
derived from EcN generate high levels of IgG2a antibodies indicative of a Th1
bias and protect against a lethal challenge with influenza A virus.
10/20/2018 Department of Plant Biotechnology, UAS, GKVK, Bengaluru -65 25
26. Methods
• Plasmid and expression vector design
Three M2e-based genetic sequences with a terminal histidine-tag were
designed and codon optimized for E. Coli.
Gene blocks were inserted into a pBAD18-CM vector directly 3’ terminal to
the ClyA-encoding region.
• OMV production and characterization
E. coli Nissle 1917 ΔnlpI (EcN) cultures transformed with appropriate
plasmid were grown overnight, sub cultured to OD600= 0.08 in Miller LB broth,
grown to mid-log phase.
o Total protein concentration - Pierce BCA Protein Assay.
o Antigenic content - Western Blot with anti-His6x primary antibody
10/20/2018 Department of Plant Biotechnology, UAS, GKVK, Bengaluru -65 26
27. • Immunization
Female BALB/c mice were immunized subcutaneously with 100µL of
vaccine at age 8-weeks. Treatment groups M2e1xC-OMV, M2e1xS-OMV, and
M2e4xHet-OMV received 40µg total protein of OMVs containing ∼0.2µg total
M2e antigen in PBS.
• Influenza challenge
A/Puerto Rico/8/1934 (PR8) virus was diluted in PBS and administered via
intranasal injection of 50µL PBS under isoflurane anesthesia.
Mice were immunized via PR8 virus, all groups received lethal dose of
50FFU at 10 weeks post initial vaccination.
10/20/2018 Department of Plant Biotechnology, UAS, GKVK, Bengaluru -65 27
28. • Viral load quantification via FFU assay
Day 6 following PR8 challenge, mice were sacrificed from sham,
M2e4xHet-OMV, and pre-exposed groups (n = 3/group) and their lungs and trachea
isolated and homogenized. Homogenates were centrifuged (5000 × g, 10 min, 4◦C),
filtered (0.2µm) and stored at −80◦C.
For FFU assay, sample aliquots were diluted into MEM media containing
0.2% BSA and activated with TPCK Trypsin at 37◦C (1µg/mL) for 20 min then
deactivated with soybean trypsin inhibitor (8.4µg/mL).
Samples were diluted, 100µL loaded into 96-well plates with confluent
layers of MDCK cells, and incubated for 24 h.
• Enzyme-linked immuno sorbent assay (ELISA)
plates were coated with2 g/mL M2e1xC peptide in PBS at 37◦C overnight.
10/20/2018 Department of Plant Biotechnology, UAS, GKVK, Bengaluru -65 28
• Cells were stained with mouse anti-nucleoprotein from hybridoma H16-L10-495
and Hoechst.
• FFU/Ml was back calculated from the no of infected cells at the lowest infected
dilution.
29. RESULTS
• Production of OMVs containing M2e derived peptides.
10/20/2018 Department of Plant Biotechnology, UAS, GKVK, Bengaluru -65 29
Western blot of M2e-OMV constructs
demonstrates that M2e1xC, M2e1xS,
and M2e4xHet are present in OMVs.
TEM images of OMVs show
polydispersed particles ranging
approximately 50–100 nm in diameter
30. • M2e-OMV vaccination leads to high IgG titers
10/20/2018 Department of Plant Biotechnology, UAS, GKVK, Bengaluru -65 30
• Serum from vaccinated mice was
further assessed to compare the
IgG2a:IgG1 ratio.
• IgG2a isotype antibodies,
associated with aTh1 type
immune response,
• are the proposed correlate of
protection for M2e-based
vaccines and are necessary and
sufficient for protection
• M2e1xC-OMV, M2e1xS-OMV,
and M2e4xHet-OMV vaccinated
mice all developed high and
statistically equivalent anti-M2e
IgG titers 8 weeks post initial
vaccination that were all
significantly higher than those of
the M2e1xS alum group.
• the M2e1xS peptide adsorbed to
alum was unable to generate a
robust immune response.
31. • M2e-OMV vaccines protect BALB/c mice from influenza A challenge
10/20/2018 Department of Plant Biotechnology, UAS, GKVK, Bengaluru -65 31
• All pre-exposed mice survived challenge and all sham vaccinated (PBS) mice died.
• Mice that received the M2e4xHet-OMV vaccine fared best, with the highest survival
rate (100%) and the least weight loss.
• Mice vaccinated with M2e1xS with alum fared worst, 25% survival, and M2e1xS-
OMV and M2e1xC OMV vaccinated mice fared intermediately, 50% and 40%
survival respectively
• The average lung and trachea viral titer of M2e4xHet-OMV vaccinated mice
was approximately 10-fold lower than that of the sham (PBS) control at day 6
post challenge
32. • Passive transfer of serum protects against influenza A challenge
Mortality (A) and morbidity (B) of mice immunized through passive transfer
of 100µL of serum (Ab) from PR8 pre-exposed, M2e4xHet-OMV vaccinated, or
sham vaccinated (PBS) mice 8 weeks following initial vaccination or exposure.
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• Mice were vaccinated with serum 24 h
prior to challenge with 50 FFU of PR8.
• Cohorts that received serum from the pre-
exposed or M2e4xHet-OMV vaccinated
mice had 100%survival during challenge.
• All mice that received PBS serum required
euthanasia during challenge.
33. CONCLUSION
• Demonstrated that engineering EcN to display M2e pep-tides upon OMVs
resulted in significantly elevated anti-M2e IgG2atiters vs. IgG1 titers and
protection against influenza challenge without supplemental adjuvants.
• Additionally, the IgG2a:IgG1 ratio of 3:1 is comparable to that of M2e vaccines
supplemented with viral components.
• And it has been shown that IgG2a antibodies are both necessary and sufficient to
provide protection in mice.
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34. • M2e antigens presented on E. coli Nissle 1917 OMVs demonstrate protection
against influenza A strain PR8.
• The three OMV vaccines tested, M2e1xC, M2e1xS, and M2e4xHet, all yielded
high anti-M2e IgG titers with significantly elevated IgG2a:IgG1 ratios, and had
improved survival over the peptide M2e1xS with alum formulation.
• The heterologous M2e4xHet vaccine was most protective, resulting in 100%
survival following challenge.
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35. SUMMARY
• OMV’s ability to elicit an immunogenic response has been utilized for vaccine
development by engineering pathogenic bacteria to secrete OMVs with desired
immunogenic properties.
• Heterologous proteins and glycan antigens can be easily added to the vesicle, the
immune stimulating properties of the vesicle can be engineered, and the toxicity
can be reduced.
• Future research should address the immune response against the platform
backbone, the heterogeneity of the vesicle product and the relatively
underexplored effects of luminal OMV components.
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36. • Besides the use of OMVs as prophylactic vaccines, vast possibilities of OMVs as
therapeutic vaccines exist.
• With the approval of human use of several OMV based meningococcal vaccines,
the future of OMV vaccines is bright, paving the way to designer and tailor made
OMV vaccines.
• The use of OMV based vaccine platforms will facilitate enhanced vaccine design
and will speed up the introduction of new much needed vaccines.
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37. REFERENCES:
GERRITZEN, M.J.H. MARTENS, D.E. WIJFFELS, R.H. POL, L. AND STORK,
M. 2017. Bioengineering bacterial outer membrane vesicles as vaccine
platform, Biotechn. Adv. 35: 565–574.
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The structure (A) and production of OMVs from gram-negative bacteria (B).
(A) The composition of the lipid bilayer of OMVs retains the asymmetry observed in bacterial OM, with complex lipids in outermost layer mounted on a uniform phospholipid inner layer. The outermost layer of OMVs consists of the LPS layer (made up of lipid A, LPS core, and O-antigen) derived from the bacterial OM. OMVs host a large repertoire of cargoes such as nucleic acids, enzymes, toxins, fragments of peptidoglycans, and fluid from the periplasmic space.
(B) Formation of OMV starts with the bulging of the outer membrane at very small region followed by progress in the expansion of the membrane. The increased expansion is established with the spherical structure of blebs. Eventually vesicle pinches off at the joint neck and gets separated from the cell.
This suggests that the function of these proteins as crosslinkers between OM and PG restrict the initiation of bulging of OM to produce OMVs.
This suggests the important role of TolA, TolQ, and TolR in regulating OMV formation