This is a small summary for the course Advanced Medical Microbiology from the master Biomedical Sciences at the UvA. It includes all the information you need for the first test of this course including lectures from Joppe Hovius, Abijeet Nayak, Esther de Jong, Bas Zaat, Constance Schultz, Aldert Ba...
Borrelia ticks and tricks
Introduction
Lyme borreliosis comes from Ixodes ticks. In USA you have the deer tick, Ixodes scapularis and in
Europe you have Ixodes Ricinus. With the naked eye you cannot see the difference and also on DNA
level there is a small difference. However, there is difference
in the pathogens they transmit. The USA ticks transmit B.
burgdorferi. In Europe there are 3 species: B. burgdorferi
(arthritis), B. afzelli (main, skin modifications), and B. garinii
(infection central nervous system). Ticks are also able to
transmit other pathogens like intracellular bacteria, parasites
or viruses (tick borne encephalitis).
When you are bitten by a tick and you acquire the pathogen erythema migrans (1-2 weeks after
tick bites). The edges of the circle are sharp and red and in the middle there is central clearance. If it
is not noticed disease can transmit and infect other organs affect joints (arthritis), heart
(carditis), central nerves system (meningitis/paralysis), other parts of the skin (legs or arms).
There is no human Lyme vaccine available, there was one in the past.
Tick tricks
More than passive carriers of disease. In their saliva its full of proteins. The tick spits and introduces
pathogen and tick proteins with a variety of functions:
Anti complement (TSLPI): Prevents killing of borrelia by the complement system. TSLPI
inhibits the lectin pathway. The lectin pathway is activated when carbohydrate recognition
proteins recognise MBL/ficolins on the pathogen. When TSLPI is present carbohydrate
recognition proteins are saturated with TSLPI inhibiting binding to MBL/ficolins no
downstream pathway.
Anti coagulant (TIX-5): Used a thrombin generation assay. In presence of TIX-5, thrombin
(factor 2a) formation is very delayed. Thrombin is very important in early phases/initiation
of coagulation systems. Early in coagulation, activation of factor 5 by factor 10 is important.
TIX-5 interferes with this activation leads to delay (but once activated it is normal).
Immunosuppressive (Salp15!): If a tick feeds it introduces saliva with salp15. Salp15 binds to
CD4 (T cell) inhibiting activation naive T cells. There are not a lot of naive T cells in the
skin look at dendritic cells (bridge to T cells).
o They took PBMCs from healthy volunteers cultivated monocytes differentiate
into monocyte derived dendritic cells (immature, not activated). Activated DCs in
absence or presence of Salp15. Salp15 is able to inhibit production of pro-
inflammatory cytokines (IL-12) by DCs.
o How does this work? Normally TLR signals
via NF-kb to express genes. Salp15 binds to
C type lectin receptor (DC-SIGN)
triggers phosphorylation raf-1
activation MEK1/2 nucleosome not
unfolded IL-12 promotor not accessible
for transcription factors no Il-12.
, Immunoprotective (Salp15): Flat tick is not eating a meal. Borrelia is in the gut of this flat
tick signal from feeding penetrate gut borrelia goes to salivary glands stimulates
tick to make more salp15.
o Beneficial because salp15 binds to surface of the borrelia (to outer surface protein
C/ OspC). The spirochete is covered by OspC and Salp15 binds this. Antibodies
cannot reach the bacterium, it shields bacterium from antibodies (invisibility cloak).
Tick needs to stay put for several days, without it can’t eat since blood will clot. It would also be
attacked by immune system if it wouldn’t have these proteins.
How to trick a tick
There is tick immunity. Certain animals after multiple infections mount an immune response against
proteins (saliva proteins mostly) of the tick. If this happens, the tick is less able to feed on the host.
Host is also less susceptible for pathogens from tick (protected against borrelia). There is
inflammation going on at the site where the tick binds. Normally after 5 days ticks drop off (done
with feeding), when there is immunity they drop off much sooner and sometimes die.
If you take serum of tick immune host and transfer to a naïve host immunity by
antibodies. This is the rational for anti tick vaccines mimic this. Might also protect against
other pathogens besides borrelia.
Targeting tick feeding: Experiment on adult ticks (humans are bitten by younger ones): Ticks that
drop off from animals immunized with TIX-5 (anti coagulant) ticks are much lighter than others.
Targeting pathogen transmission: There is normally first expression of TSLPI and then Salp15. Using
RNA interference silencing salp15 significant reduction of ability of ticks to transmit borrelia to
the mice suggests role Salp15 in transmission. Same thing true for TSLPI reduction in borrelia
transmission suggests role TSLPI in transmission.
You cannot do RNAi in nature. You want an immune response to these proteins more
modest effects. To have good effect combine with tick vaccines.
In mice there is no immune response to TSLPI.
New candidates? ANTIDotE project. Using transcriptomics and proteomics they found a whole
bunch of proteins used by the tick for feeding important for feeding process so also for
transmitting pathogens maybe. Top 25 from transcriptomics validated 20 tick proteins left (very
important in tick feeding process). Vaccinated mice with protein challenged mice with borrelia
infected ticks not infected & lower borrelia DNA load.
Looked at encephalitis because ticks also transmit TBEV (lethal model, survival curves) challenged
mice that got vaccine lived. Vaccinated with the recombinant protein half survived.
Tick proteins can be used as vaccine and can protect against borrelia or tick borne encephalitis virus.
Conclusions
Ticks introduce a cocktail of functional TSGPs into host skin that
o Facilitate tick feeding
o Facilitate pathogen transmission
Interfering with TSGPs by anti-tick vaccines can inhibit tick feeding, pathogen transmission or
both
Targeting TGPs could be an alternative approach to prevent Lyme borreliosis
, Multivalent OspA vaccine against Lyme borreliosis
Introduction
Lyme borreliosis: Most prevalent vector borne disease in
northern hemisphere.
Cycle: There are 3 stages of the tick. First stage is the larval
stage, feeds on small mammals nymphal stage, feeds on
second small mammal adults, feeds on larger animals like
deers lay eggs. From nymphal stage they can feed on
humans. Humans are not part of the cycle (dead end hosts).
Can also feed large mammals like dogs.
Borrelia has no LPS on the surface but they have multiple
different lipoproteins. They have a segmented genome consisting of a linear chromosome and
circular/linear plasmids.
LYMErix vaccine: Vaccine based on outer surface protein A used in 1998-2002. It only targets B.
burgdorferi which is the only species in USA. The species complexity is higher in Europe. Now we
don’t have human vaccines available. We need a multivalent vaccine that protects to multiple
species of borrelia.
Outer surface protein A: 31 kDa membrane lipoprotein. Expressed in the mid-gut of unfed tick
once starts to feed OspA down regulation migration to salivary gland host. C terminal part
exposed on the outside (immunodominant), N terminal in membrane.
Mechanism of action OspA vaccine: Antibodies against OspA (really high titers) ingested
by tick go to gut neutralises borrelia blocking transmission.
VLA15: Second generation OspA vaccine which is a combination of 3 proteins. They took C terminal
parts of different serotypes/species combined with other OspA proteins using linker sequences
(and disulphide bonds for stabilizing) fusion protein. Serotype 1+2, 3+4 and 5+6 = 3 proteins.
Serotype 1 till 6 are most important for causing disease. They adjuvanted the vaccine with Alum
(epitope removed). There is broad protection for 4 genospecies representing 5 subtypes. Phase 2
clinical trials.
New vaccine
Objectives: Create a single antigen vaccine using OspA (1 protein) because this is economically good.
You need protection against the 6 major serotypes.
Create a single antigen and test immune response and protection in mouse models.
Surface shaping approach: You want multiple epitopes on one protein.
Idea from vaccine against Neisseria meningitidis where they used variant 1 backbone with
immunogenic epitopes variant 2 & 3. This 1 protein induced antibodies against all
meningococcus B strains tested.
It is important to know the sequence and folding of the proteins you want to use. You also need
knowledge of immunogenic epitopes. They want to use OspA.
1. Sequence conservation analysis: High sequence similarity was observed between the
serotypes.
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