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Xinyu Liu, Sibylle Siegrist, Mario Amacker, Rinaldo Zurbriggen, Gerd Pluschke and Peter H. Seeberger ACS Chem. Biol., 2006 1 (3), 161-164

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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

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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.

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Question: Researchers expect the area susceptible to Leishmaniasis to increase over time, why?

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Tropical scourge is transmitted to humans via the bite of a female phlebotomine sandfly.

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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.

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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.

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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

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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.

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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

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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

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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.

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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).

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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%.

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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.

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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

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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.

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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).

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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)

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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

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Dr. Case Students of Chem258