11-11-2020
Lyme Borrelia: Ticks & Tricks
Introduction
(Ixodes) ticks are able to transmit bacteria that cause lyme disease: B.burgdorferi
Europe also: B.afzelii & B.garinii
→ Diagnosis & treatment is the same & able to transmit other pathogens → other bacteria,
parasites & viruses
Lyme borreliosis:
Divided into 3 stages
1) Acute lyme borreliosis: where thick bites → inoculate bacteria → skin rash =
erythema migrans
2) Early disseminated lyme borreliosis: rash is not treatment or not recognized →
disease progress → to deeper organs (central nervous system & hart etc)
→ weeks to months
3) Late disseminated lyme borreliosis
→ months to years
There is no humane vaccine available to treat Lyme disease !
Tick Tricks
Ixodes ticks are more than passive carriers of pathogens
- tick penetrating to human skin → attach to human skin → sucks some blood &
spits saliva (all sort of proteins) : important for transmission of pathogens from the
thick to humans
- Thick saliva proteins with:
- Anti-complement
- Anti coagulant: not clotting
- Immunosuppressive
- Immuno Protective
The complement system
3 pathways: classical, lectin & alternative pathway
Leading to C3 convertase → MAC (lysis), chemotaxis (immune cells are attracted) &
opsonization
Anti-complement tick protein: able to inhibit host complement = P8 = TSLP1
Experiment: Human serum with active complement & inactive complement
- active complement → spirochetes dies
- inactive complement → spirochetes stay alive
- thick protein → spirochetes stay alive → thus protection against complement
Anti-complement tick protein interferes with lectin pathway: MBL with CRDs → thick
protein binds CRD → prevent MBL binding: you don’t have opsonization, less chemotaxis
& less MAC → Borellia spirochetes stay alive
,Anticoagulant:
New anticoagulant thick protein: P23 → TIX-5 : inhibitor of factor 10 through factor 5
- add TIX5 → thrombin (clotting) formation is delayed
TIX5: prevent activated 10a to activate factor 5 → inhibits downstream coagulation events
: thick do not want clotting otherwise he cannot eat.
Immunosuppressive thick proteins:
Thick feeds for several days → immune response: would inhibit feeding → so thick want to
inhibit immune response
- Salp15: thick protein was able to inhibit naive T-cell activation
- Influence DC? → present in human cells: bridge to innate & adaptive
immune system
Study: we obtained human PBMC → differentiate into monocyte DC → activate immature
DC with LPS but also with the lyme bacterium: look at the difference with and without Salp15
- thick protein inhibits pro-inflammatory cytokine production (e.g. IL-12 & IL-6) by DCs
Salp15 signaling:
Normal: add TLR ligand → binds to TLR on DC → NFkb to the nucleus → nucleosome (with
IL-12 promotor) → nucleosome unfold → promotor accessible for TF → IL-12 production
now add Salp15: Salp15 binds to different receptor DC-sign → phosphorylation of Raf-1 →
activation of Mek1/2 → impaires unfolding of nucleosome: more tight bole → not accessible
for TF → no transcription of IL-12 → inhibition of IL-12 = immunosuppression
Exam: Salp15!!!!
Immuno Protection:
Flat thick: spirochetes (bacteria) are in the thick gut → feeding → spirochete going from the
gut to the salivary glands → spirochete in the gut → express OspA protein is
downregulated and OspC are upregulated: spirochete that enter salivary glands → more
expression of salp15 → Salp15 binds OspC of borrelia protein → whole surface of
spirochete is covered by Salp15 when it enters the human host: protect the spirochetes
from being killed by antibodies of the host: it makes the spirochete invisible for the host
antibodies
Salp15 can be beneficial for borrelia and the thick in 2 ways:
1) immunosuppression by the host
2) directly interact with the spirochete: protecting the spirochete directly from killing by
antibodies from the host
How to trick a tick?
Hope: targeting thick proteins through vaccination prevent tick feeding &
transmission of bacteria:
Tick immunity: thick immune animals → put thicks → thicks die & largely protected against
tick-borne diseases
You can develop immune response by natural thick infection → can we mimic this with
a vaccine antigen/protein ?
→ rabbits vaccinated with Anticoagulant protein → thicks cannot feed properly: there is
some effect on thick feeding process
,Salp15 & TSLP1 vaccines
- Salp15: immunosuppressive & protective
- TSLPI: anti complement
Borrelia infections thicks → silence expression of gene of interest in the thick (Salp15 or
SLP1) → knock out: no transmission of borrelia to the host
Vaccinate with Salp15: also protection, but phenotype is not that strong
Looking for new candidates for anti-tick vaccines:
Fed thicks with different time proteins → salivary glands → Transcriptomics & proteomics:
looking for proteins that are expressed
Early-Feeding induced salivary gland proteins → top 20 → some of these thick proteins are
interesting candidates for anti-tick vaccine:
- rContig11: vaccinate mice → challenge with borrelia → protected against Borrelia
- FSME vaccine: targeting the virus transmitted by the thick → mice do not die
- rContig16: half of the mice survived
- rContig22: some survival in lethal infection
→ give the antigens in a cocktail → more efficacy: come close to commercial available
vaccin
Conclusion
- thicks introduce a cocktail of functional TSGPs (thick salivary gland proteins) into
host skin that:
- facilitate tick feeding
- facilitate pathogen transmission (Salp15)
- immunosuppression (no IL-12) & immune protection (invisible for Ag)
- interfering with TSGP by anti-tick vaccines can inhibit tick feeding & pathogen
transmission or both
- targeting TGPs is an attractive alternative approach to prevent Lyme borreliosis &
more diseases
Broadly Protective Multivalent OspA Vaccine against Lyme Borreliosis
We need new LB vaccines, because LB is increasing, the infection is preventable with
vaccination & is cost-effective, species complexity is higher in europa (different OspA
serotypes) and a multivalent vaccine is required to protect against different Borrelia
OspA = outer surface protein A
- expressed by Borrelia in the mid-gut of an unfed thick
- thick feeding: OspA downregulation → spirochetes migrate to salivary gland & host
There was a vaccin, but they thought that it perhaps can lead to autoimmunity → rejected
We want to create a single recombinant vaccine with the potential to confer a
comprehensive immune response and protection with significant economic advantage
→ surface shaping approach to the C-terminal domain of OspA
Surface shaping = multiple epitopes of OspA in 1 protein → 1 vaccine for all targets
1) sequence conservation research of OspA 2) homology modeling 3) surface partitioning
based on mAb 4) make layout of protein via combination of patches 5) test coverage of
protein for serotypes
, Staphylococcus aureus: innate immune evasion
S.aureus introduction:
S.aureus:
- colonies are gold (aureus)
- beta-hemolytic
- grape-like microscopic appearance (druiventros)
30% of humans are colonized with S.aureus: skin, GI tract → no problem, but it can cause
infections: sepsis, osteomyelitis and infections of surgical wounds or prosthetic material
Problem: we do not have a good immune response against S.aureus (you do not become
immune & problem for vaccination)
- neutrophils are main effective agents against S.aureus, bso when you have a
neutrophil defect → very susceptible for S.aureus
S.aureus virulence regulation by Agr QS (quorum sensing)
Virulence is depending on bacteria concentration present: quorum-sensing
- sense the quorum: sense peptide by each individual cell → threshold of peptide is
detected → high enough concentration → genes are switched ‘on’
Quorum sensing circuit
AIP (auto inducing peptide):
made by AgrD → exported by
membrane protein ArgB→
getting out of the cell → sensed
by AgrC → phosphorylate AgrA
: TF activator of the genes
- 2 promoters in AgrA
operon are activated:
1) P2 promoter:
upregulation of entire QS
system (Agr): positive
feedback loop
2) P3 promoter: RNA3 ORF
→ partial messenger →
delta toxin & RNA3
molecule can bind to UTR
→ down regulation or
upregulation of translation
(positive or negative
regulator)
What does RNA3 presence do?
- activation of translation:
hemolysin A (toxin)
- inhibition of translation:
→ adhesins & Protein A
(single proteins that have
to adhere)
Lyme Borrelia: Ticks & Tricks
Introduction
(Ixodes) ticks are able to transmit bacteria that cause lyme disease: B.burgdorferi
Europe also: B.afzelii & B.garinii
→ Diagnosis & treatment is the same & able to transmit other pathogens → other bacteria,
parasites & viruses
Lyme borreliosis:
Divided into 3 stages
1) Acute lyme borreliosis: where thick bites → inoculate bacteria → skin rash =
erythema migrans
2) Early disseminated lyme borreliosis: rash is not treatment or not recognized →
disease progress → to deeper organs (central nervous system & hart etc)
→ weeks to months
3) Late disseminated lyme borreliosis
→ months to years
There is no humane vaccine available to treat Lyme disease !
Tick Tricks
Ixodes ticks are more than passive carriers of pathogens
- tick penetrating to human skin → attach to human skin → sucks some blood &
spits saliva (all sort of proteins) : important for transmission of pathogens from the
thick to humans
- Thick saliva proteins with:
- Anti-complement
- Anti coagulant: not clotting
- Immunosuppressive
- Immuno Protective
The complement system
3 pathways: classical, lectin & alternative pathway
Leading to C3 convertase → MAC (lysis), chemotaxis (immune cells are attracted) &
opsonization
Anti-complement tick protein: able to inhibit host complement = P8 = TSLP1
Experiment: Human serum with active complement & inactive complement
- active complement → spirochetes dies
- inactive complement → spirochetes stay alive
- thick protein → spirochetes stay alive → thus protection against complement
Anti-complement tick protein interferes with lectin pathway: MBL with CRDs → thick
protein binds CRD → prevent MBL binding: you don’t have opsonization, less chemotaxis
& less MAC → Borellia spirochetes stay alive
,Anticoagulant:
New anticoagulant thick protein: P23 → TIX-5 : inhibitor of factor 10 through factor 5
- add TIX5 → thrombin (clotting) formation is delayed
TIX5: prevent activated 10a to activate factor 5 → inhibits downstream coagulation events
: thick do not want clotting otherwise he cannot eat.
Immunosuppressive thick proteins:
Thick feeds for several days → immune response: would inhibit feeding → so thick want to
inhibit immune response
- Salp15: thick protein was able to inhibit naive T-cell activation
- Influence DC? → present in human cells: bridge to innate & adaptive
immune system
Study: we obtained human PBMC → differentiate into monocyte DC → activate immature
DC with LPS but also with the lyme bacterium: look at the difference with and without Salp15
- thick protein inhibits pro-inflammatory cytokine production (e.g. IL-12 & IL-6) by DCs
Salp15 signaling:
Normal: add TLR ligand → binds to TLR on DC → NFkb to the nucleus → nucleosome (with
IL-12 promotor) → nucleosome unfold → promotor accessible for TF → IL-12 production
now add Salp15: Salp15 binds to different receptor DC-sign → phosphorylation of Raf-1 →
activation of Mek1/2 → impaires unfolding of nucleosome: more tight bole → not accessible
for TF → no transcription of IL-12 → inhibition of IL-12 = immunosuppression
Exam: Salp15!!!!
Immuno Protection:
Flat thick: spirochetes (bacteria) are in the thick gut → feeding → spirochete going from the
gut to the salivary glands → spirochete in the gut → express OspA protein is
downregulated and OspC are upregulated: spirochete that enter salivary glands → more
expression of salp15 → Salp15 binds OspC of borrelia protein → whole surface of
spirochete is covered by Salp15 when it enters the human host: protect the spirochetes
from being killed by antibodies of the host: it makes the spirochete invisible for the host
antibodies
Salp15 can be beneficial for borrelia and the thick in 2 ways:
1) immunosuppression by the host
2) directly interact with the spirochete: protecting the spirochete directly from killing by
antibodies from the host
How to trick a tick?
Hope: targeting thick proteins through vaccination prevent tick feeding &
transmission of bacteria:
Tick immunity: thick immune animals → put thicks → thicks die & largely protected against
tick-borne diseases
You can develop immune response by natural thick infection → can we mimic this with
a vaccine antigen/protein ?
→ rabbits vaccinated with Anticoagulant protein → thicks cannot feed properly: there is
some effect on thick feeding process
,Salp15 & TSLP1 vaccines
- Salp15: immunosuppressive & protective
- TSLPI: anti complement
Borrelia infections thicks → silence expression of gene of interest in the thick (Salp15 or
SLP1) → knock out: no transmission of borrelia to the host
Vaccinate with Salp15: also protection, but phenotype is not that strong
Looking for new candidates for anti-tick vaccines:
Fed thicks with different time proteins → salivary glands → Transcriptomics & proteomics:
looking for proteins that are expressed
Early-Feeding induced salivary gland proteins → top 20 → some of these thick proteins are
interesting candidates for anti-tick vaccine:
- rContig11: vaccinate mice → challenge with borrelia → protected against Borrelia
- FSME vaccine: targeting the virus transmitted by the thick → mice do not die
- rContig16: half of the mice survived
- rContig22: some survival in lethal infection
→ give the antigens in a cocktail → more efficacy: come close to commercial available
vaccin
Conclusion
- thicks introduce a cocktail of functional TSGPs (thick salivary gland proteins) into
host skin that:
- facilitate tick feeding
- facilitate pathogen transmission (Salp15)
- immunosuppression (no IL-12) & immune protection (invisible for Ag)
- interfering with TSGP by anti-tick vaccines can inhibit tick feeding & pathogen
transmission or both
- targeting TGPs is an attractive alternative approach to prevent Lyme borreliosis &
more diseases
Broadly Protective Multivalent OspA Vaccine against Lyme Borreliosis
We need new LB vaccines, because LB is increasing, the infection is preventable with
vaccination & is cost-effective, species complexity is higher in europa (different OspA
serotypes) and a multivalent vaccine is required to protect against different Borrelia
OspA = outer surface protein A
- expressed by Borrelia in the mid-gut of an unfed thick
- thick feeding: OspA downregulation → spirochetes migrate to salivary gland & host
There was a vaccin, but they thought that it perhaps can lead to autoimmunity → rejected
We want to create a single recombinant vaccine with the potential to confer a
comprehensive immune response and protection with significant economic advantage
→ surface shaping approach to the C-terminal domain of OspA
Surface shaping = multiple epitopes of OspA in 1 protein → 1 vaccine for all targets
1) sequence conservation research of OspA 2) homology modeling 3) surface partitioning
based on mAb 4) make layout of protein via combination of patches 5) test coverage of
protein for serotypes
, Staphylococcus aureus: innate immune evasion
S.aureus introduction:
S.aureus:
- colonies are gold (aureus)
- beta-hemolytic
- grape-like microscopic appearance (druiventros)
30% of humans are colonized with S.aureus: skin, GI tract → no problem, but it can cause
infections: sepsis, osteomyelitis and infections of surgical wounds or prosthetic material
Problem: we do not have a good immune response against S.aureus (you do not become
immune & problem for vaccination)
- neutrophils are main effective agents against S.aureus, bso when you have a
neutrophil defect → very susceptible for S.aureus
S.aureus virulence regulation by Agr QS (quorum sensing)
Virulence is depending on bacteria concentration present: quorum-sensing
- sense the quorum: sense peptide by each individual cell → threshold of peptide is
detected → high enough concentration → genes are switched ‘on’
Quorum sensing circuit
AIP (auto inducing peptide):
made by AgrD → exported by
membrane protein ArgB→
getting out of the cell → sensed
by AgrC → phosphorylate AgrA
: TF activator of the genes
- 2 promoters in AgrA
operon are activated:
1) P2 promoter:
upregulation of entire QS
system (Agr): positive
feedback loop
2) P3 promoter: RNA3 ORF
→ partial messenger →
delta toxin & RNA3
molecule can bind to UTR
→ down regulation or
upregulation of translation
(positive or negative
regulator)
What does RNA3 presence do?
- activation of translation:
hemolysin A (toxin)
- inhibition of translation:
→ adhesins & Protein A
(single proteins that have
to adhere)
