Xinyu Liu, Sibylle Siegrist, Mario Amacker, Rinaldo Zurbriggen,
Gerd Pluschke and Peter H. Seeberger ACS Chem. Biol., 2006 1 (3),
161-164
Slide 2
Leishmaniasis (LEASH-ma-NIGH-a-sis ) Carbohydrate vaccines
Relevant immunological concepts Virosomes Synthesis of
tetrasaccharide antigen/conjugation to lipid Preliminary evaluation
of vaccines efficacy Conclusions/Future work
Slide 3
In 1901 Sir William Boog Leishman, a pathologist and British
Army medical officer examines spleens of those killed by kala azar
and published his account implicating the protozoan parasite
Leishmania donovani. Dum Dum fever is a serious global health
problem! Visceral leishmaniasis threatens more than 350 million
people worldwide, infects 12 million, and kills an estimated 70,
000 yearly. The four main forms of Leishmaniasis Visceral: infects
the vital organs; most fatal. Cutaneous: most common form; causes
sores at bite site. Diffuse cutaneous: shows up as widespread skin
legions akin to leprosy and is difficult to treat. Mucocutaneous:
ends up as skin ulcers and preferentially attacks the mouth and
nose.
Slide 4
Question: Researchers expect the area susceptible to
Leishmaniasis to increase over time, why?
Slide 5
Tropical scourge is transmitted to humans via the bite of a
female phlebotomine sandfly.
Slide 6
Symptoms begin swiftly in children, but progress more slowly in
adults. Newly infected adults usually have non-specific symptoms
that begin to show up after about 2 weeks. Between 2 weeks and 2
months the symptoms remain non-specific (prolonged fever, cough,
diarrhea, fatigue). Cough (children) Diarrhea (children) Fever that
persists for weeks; may come and go in cycles Night sweats Scaly,
gray, dark, ashen skin Thinning hair Vague belly area (abdominal)
discomfort Vomiting (children) Weight loss Key to diagnosis is a
bone-marrow or spleen biopsy that reveals via microscopy the
distinct morphology of the protozoa.
Slide 7
Leishmaniasis spreads rapidly in urban settings. Leishmaniasis
accelerates the transition of HIV to AIDS via immunosuppression and
stimulation of viral replication. Currently, co-infection with
leishmaniasis and HIV has been reported in 34 countries in Africa,
Asia, Europe, and South America. In Mediterranean Europe, for
example, up to 70% of adult cases of visceral leishmaniasis are
associated with HIV infection. HIV infection actually stimulates
the progression of leishmaniasis thereby making those with LM more
infectious to be around.
Slide 8
Pentavalent Antimony (Sb; metalloid) drugs (detailed mechanism
by which these work just elucidated recently and presented just two
weeks ago J. Med. Chem. March 24, 2009). PA drugs have significant
side effects; therefore, new approaches are being looked at for
treatment. Paromomycin, an antibiotic known since the 1960s but not
produced en masse for economic reasons (Leishmaniasis predominantly
threatens the poorer tropical regions), is claimed to be effective
and was approved for use by the Indian govt in 2006. A vaccine,
unlike an antibiotic, would have the advantage of preventing the
infection from even progressing in the first place
post-administration. Thus, paromomycin does not render the
development of a carbohydrate vaccine as described in this study
obsolete. Paromomycin
Slide 9
Not a new idea, but a powerful one. Basically a weakened or
attenuated form of pathogen that stimulates antibodies or immune
cells to combat a disease. Vaccine (Lat. vaccn-us, means cow)
concept comes from Edward Jenners late 1700s use of cowpox virus to
help humans fight smallpox. Vaccination, although not without its
critics, is widespread and taken for granted throughout the rich-
world. We all benefit from early-life vaccinations against polio,
measles, mumps, rubella, Hepatitis B, etc.
Slide 10
Carbohydrate: hydrates of carbon; basically molecules
containing carbon skeleton with lots of OH groups. Exist in
straight chains, rings, and polysaccharides (sugar polymers). Main
energy source for living creatures on Earth. Photosynthesis is
essentially the process by which carbohydrates are created; living
plants and bacteria use photons of light from the Sun to create
these energy rich food stuffs (often tasty!). Used in cell to cell
communication; antigenic. In the context of this paper, a
tetrasaccharide cap has been implicated as a key player in the
invasion of macrophages by leishmania donovani.
GlucoseMannoseGalactose
Slide 11
Synthetic carbohydrate vaccines, relative to their natural
analogues which are essentially impure heterogeneous mixtures,
would be close to homogeneous mixtures. They can be made, via
medicinal chemistry techniques, to be multiantigenic. Thus, a
single carbohydrate vaccine moiety could be synthesized to include
several antigens thereby priming the immune system to launch a
multi-pronged attack which a disease causing organism would have a
hard time surviving. Again, carbohydrates are ubiquitous in nature
and are basically just sugar molecules. Thus, toxicity of an
ultra-pure synthetic carbohydrate vaccine should theoretically be
quite low. Because carbohydrates are found on the surface of many
types of cells, vaccines for a variety of ailments (malaria,
cancers, and HIV) exist in carbohydrate form. A multiantigenic
carb. vaccine Danishefsky et al, Columbia University
Slide 12
The immune system distinguishes between self and non-self.
There is humoral immunity and cell mediated immunity. Humoral
immunity is antibody (IgG) based and specific. Specific antigens
are targeted with specific types of antibodies. Cell-mediated
immunity does not involve antibodies and involves neutral killer
cells, macrophages, and Cytotoxic T-Lymphocytes. Immune system
reacts to non-self bits of foreign matter referred to as antigens.
Antigens are sizeable molecules of polysaccharide or protein, or,
less commonly, lipids or nucleic acids. Small molecules by
themselves do not make for good antigens b/c they are not attracted
to macrophages. Immune response is not aimed at the entire antigen
molecule. Rather, it is directed toward the small molecule portions
of the antigen molecule called the epitope. Most antigens contain
many different epitopes. Homo sapiens immune system has two central
parts. One main part consists of B lymphocytes, the other consists
T lymphocytes. B cells act in a type of immunity known as
antibody-mediated immunity; T cells act in cell mediated immunity.
When a vaccine is administered very large protein molecules called
antibodies are produced via humoral immunity. Special memory B
cells remember this vaccine antigen and speed up immune response
when the organism contracts the disease.
Slide 13
Slide 14
The infectious surface Leishmania donovani lipophosphoglycan
(LPG) has been isolated. We know the substances chemical structure.
Scientific goal: Based on what we know about the LPG structure, can
we use synthetic organic chemistry to make a molecule very similar
in terms of structure which could be used to prime the immune
system (hopefully without harming the organism) of an individual in
a susceptible region so that when the real LPG presents itself,
human antibodies produced by memory B cells quickly and efficiently
block the real LPG thereby preventing LM infection and its hideous
symptoms? General structure of the leishmania lipophosphoglycan
(LPG).
Slide 15
Synthesis of the thiol-tethered LPG tetrasaccharide epitope.
(i) 1, TBSOTf (cat.), Et2O/CH2Cl2, 0 C, 92%; (ii) Et3SiH, TfOH, 4
MS, CH2Cl2, 78 C, 63%; (iii) 2, TMSOTf, CH2Cl2, 40 C; then AcCl in
THF/MeOH, 0 C to rt, 76% 2 steps; (iv) 3, TMSOTf (cat.),
Et2O/CH2Cl2, 0 C, 69%; (v) AcCl in THF/MeOH, 0 C to RT, 90%; (vi)
3, TMSOTf (cat.), Et2O/CH2Cl2, 0 C, 53%; (vii) Na, NH3, THF/MeOH,
78 C, 81%.
Slide 16
A Liposome is a submicroscopic spherically shaped entity
consisting of a phospholipid bilayer shell. Many viruses, including
influenza, have a liposomal-like cap enclosing their nucleic acid
core that is aptly referred to as a virosome. Typically, virosomes
are approximately 150 nm in diameter. Virosomes do not contain the
genetic material of the virus. Unlike liposomes, however, virosomes
do have glycoproteins (carbohydrate-protein conjugates) on their
surfaces that confer immunological properties to them. For example,
in (Seeberger et al 2006) an influenza virosome is used as an
adjuvant (adjuvant: substance used to prime the IS of animal test
subjects sera prior to vaccination. Then it is used as a carrier of
the synthetic oligosaccharide itself for administration to the
mouse sera.
Slide 17
Slide 18
Slide 19
Enzyme-linked Immunosorbent Assay. Antibodies are attached to
an enzyme as opposed to a fluorescent dye or radioactive substance.
The antibodies attached to the enzymes are anti-antibodies (e.g.
goat-anti-mouse antibodies). The antibodies in the mouse sera
attach to the goat-anti-mouse antibodies. After incubation,
O-phenylendiamine substrate molecules are added to the micro-titer
plates. If the substrate molecules react with the enzyme on the
anti-antibody, then a color change should occur. If the color
change fails to occur this implies that the suspected antibodies
were not present in the mouse sera. The authors did in fact observe
a color change thereby confirming that antibodies to KLH-glycan
were created. Chemical conjugation of LPG tetrasaccharide for
immunological studies
Slide 20
Used to detect antibodies and determine their concentration in
a serum sample. The authors want to be able to say that their
carbohydrate vaccine produces antibodies to the leishmania LPG in
mouse sera. Leishmania donovani were incubated with mouse sera.
They were in turn incubated with goat-anti-mouse IgG, Fcgamma
fragment specific antibodies for immunofluorescence. Test comprises
of a mouse antibody joining with the fluorescing goat-anti-mouse
IgG. If a reaction between antibodies in the sera and the
dye-tagged antibodies occurs, the sera antibodies can be
pin-pointed. If no reaction takes place, then its hard to reconcile
with the existence of mouse sera antibodies. Indeed, when placed
under a fluorescence microscope and subjected to UV-light, the
slides start in this experiment show colored beads representing the
aforementioned reaction. The serial dilutions are used to make the
interpretation of the slides more manageable since the
concentration of fluorescing antibodies increases with time (like a
bacteria colony) and would dominate the entire picture. All of the
mice sera that were loaded with IRIVs ended up fluorescing.
Conclusion : glycan loaded IRIVs did elicit a specific immune
response.
Slide 21
Generation of Leishmania donovani cross-reactive IgG by
immunization with glycan 11 loaded IRIVs a) Immunofluorescence
staining of parasites by serial dilution (1:100 to 1:1600) of serum
from mice immunized three times with HA-glycan (left panel) or
PE-glycan (right panel) loaded IRIV b) Lack of immunofluorescence
staining (left panel) of parasites by pre-immune sera (dilution
1:100) and by serum (dilution 1:100) of a mouse immunized three
times with unloaded IRIV. Presence of parasites on the slides was
demonstrated by staining of parasite DNA with Hoechst dye 33258
(right panel).
Slide 22
This is the first study demonstrating the preliminary efficacy
of a LM vaccine candidate: a switch from IgM (the first antibodies
to arrive at an infection) to the longer lived IgG antibodies most
important in the specific immune response was observed. Although
toxicity should be minimal, tests of toxicity/side effects will
need to be part of the future animal challenge studies. As
scientists, we should constantly be asking: is there another,
perhaps better way? Thus, although great improvements in
carbohydrate synthesis have occurred in the last decade alone, can
the oligosaccharide synthesis be further improved upon---fewer
steps, greater purity, greener reagents, lower cost, etc.? Perhaps
some type of enzymatic synthesis (last talk) could be employed to
ensure even greater purity of a carbohydrate epitope. Investigate
the cell surface molecules of other protozoan parasites, as
Leishmaniasis is by no means the only threat to human health caused
by protists. Leishmania donovani in human bone marrow cells
(biopsy)
Slide 23
LineageSpeciesDisease DinoflagettesA variety of species
implicated Toxins let loose during red tides accumulate in mussels
and clams causing shell fish poisoning Diplomonads Giardia Diarrhea
Trichomonads Reproductive tract infections Entamoebae Entamoeba
histolytica Amoebic dysentery Oomocytes Phytophthora infestans
Implicated in the 1845- 1847 Irish potatoe famine that killed 1
million people Microsporidians Encephalitozoon Diseases of
respiratory tract, nervous system, and digestive tract.
Kinetoplastids Trypanosoma cruzi Chagas disease