Announcements

1. Announcements • Thursday April 19, 2012: Lecture by Dr. Don Harn on Schistosome vaccines • Tuesday April 24, 2012: Lecture on Vector control • Monday April 30, 2012: Review session • Thursday May 3, 2012: FINAL EXAM at Pharmacy School, Room 338: 8-11 AM

2. CONTROL MEASURES Vaccines and Vectors Medical Parasitology CBIO4500 April 17, 2012 Silvia N J Moreno

3. PARASITE CONTROL Control methods should be integrated with the parasite life cycle: Direct life cycles with no IH (monoxenous): Only the DH and the environments to be considered. For example, safe sewage disposal will give satisfactory control of fecally transmitted monoxenous parasites like Ascaris. One or more Intermediate hosts (heteroxenous): parasites with an intermediate host often undergo asexual reproduction in the intermediate host. This increase in biotic potential can make control more difficult. For the control of digeneans such as schistosomes, prevention of fecal contamination of snail habitats has to be almost perfect, since a single infected snail can shed thousands of cercariae. Vector control measures: for the control of parasites with an arthropod definitive host

4. Typical transmission cycle of a vector-borne parasite or pathogen between a human host and an arthropod vector, and potential steps for intervention. Human Host • Insecticides • Attractants/repellants and other behavioural modifiers • Vector longevity curtailers • Genetically modified vectors incapable of reproduction or pathogen transmission • Anti-parasite/pathogen therapies • Vaccines Parasite/Pathogen Arthropod Vector • Blocking the parasite acquisition or transmission by arthropods: Vaccine • Insect immune regulators (smart sprays) Examples of novel control strategies developed based on arthropod genome resources (red shaded text boxes) and the parasite or human host genome resources (yellow text box) are shown. Nature reviews Microbiology 3:262 (2005).

5. PARASITE VACCINES What does a vaccine do ? • Stimulates normal protective immune response of host to fight invading pathogen. What knowledge is needed to produce a vaccine ? 1. Understand life–cycle of parasite → find best target stage. 2. Understand immune mechanisms stimulated by parasite. → humoral /cellular response ?

6. What does a vaccine needs to do to work? • Vaccines contain antigens that serve as targets for the immune system • Antigens must produce protective response: Protection against illness resulting from exposure to live pathogen and ideally sustained protection • Vaccine must stimulate good response → without adjuvant* is best. (adjuvant is an agent that may stimulate the immune system and increase the response to a vaccine, without having any specific antigenic effect in itself). • Good level of protection without boosting→ using simple delivery system. • Safe:Vaccine must not itself cause illness or death • PRACTICAL CONSIDERATIONS:Low cost per dose. Biological stability. Ease of administration. Few side-effects

7. Types of Vaccines 1. Whole pathogens killed prior to inoculation. 2. Attenuated live or low virulence vaccines 3. Protein Subunit vaccines. → Natural tissue purified proteins. → Recombinant protein antigens. → Chemical small peptide vaccines. 4. Nucleic acid vaccines

8. Killed and attenuated organisms • Killed organisms: • These have been destroyed with chemicals, heat, radioactivity or antibiotics. Examples are vaccines against influenza, cholera, bubonic plague, polio, hepatitis A and rabies. • Attenuated: • Live but attenuated microorganisms. • Live viruses cultured under conditions that disable their virulent properties. Also could be less virulent strains to produce a broad immune response. • Attenuated vaccines have some advantages and disadvantages: they have the capacity of transient growth so they give prolonged protection, and no booster dose is required. But they may get reverted to the virulent form and cause the disease. • Examples: yellow fever, measles, rubella and mumps and the bacterial disease typhoid.

9. Subunit vaccines: natural antigens or recombinant proteins • These vaccines consist of subcomponents of the pathogenic organisms, usually proteins or polysaccharides. Polysaccharides are made more immunogenic and T-dependent by conjugation with proteins (e.g., haemophilus, meningococcus, pneumococcus, etc.). • Hepatitis-B consist of antigenic proteins cloned into a suitable vector. These subunit have less problems of toxicity and risk of infection. • When the pathogenic mechanism of an agent involves a toxin, a modified form of the toxin (toxoid) is used as vaccine(e.g., diphtheria, tetanus, etc.). Toxoids remains immunogenic.

10. DNA VACCINES DNA plasmid vector carry the genetic information for an antigen, which is made inside a host cell and leads to a cell-mediated immune response via the MHC I pathway. The plasmid DNA vaccine carries the gene for an antigenic pathogen protein. The plasmid vector is taken up and transcribed in the nucleus. The mRNA is translated into protein. The protein antigen is degraded by proteosomes into peptides. The derived-peptide binds MHC class I molecules. Peptide antigen/MHC I complexes are presented on the cell surface, binding cytotoxic CD 8+ lymphocytes, and inducing a cell-mediated immune response. Some of this protein is released, and it could be bound by antibody molecules on B cells or phagocytosed by macrophages. Protein is digested into small peptides and placed in the binding groove of a cell surface protein MHC II. The peptides are bound and recognized as foreign by the TCR, the helper T cell releases interleukins (IL) to stimulate both arms of the immune system (humoral and cellular).

11. DNA VACCINES • Advantages of DNA vaccines: • Expression of antigens in their native form, improved processing and presentation to the immune system • Induction of cytotoxic T cells (in addition, protective antibody and CD4+ responses in the same individual) • Easy to produce and purify • Easy to modify and combine vaccines • Induction of long-lived immunity • Prolonged antigen expression • Disadvantages • Introduced (foreign) DNA may become incorporated into host chromosomes and subsequent potential for a transformation event (DNA triplex formation) • Introduced DNA may become incorporated into germ line cells • The DNA may stimulate anti-DNA antibodies • Unexpected and untoward consequences of the persistent expression of a foreign antigen therefore difficult to proceed to clinical trials • There are no DNA vaccines currently on market for use in humans. In 2005, a DNA vaccine that protects against West Nile virus was approved for use in horses.

12. Why limited success in parasite vaccine development ? • Parasites avoid, deflect & confuse host immune system. • Right parasite antigens not identified yet: complicated life cycles. (maybe 20,000 proteins in nematodes). • Protective host responses not understood in target species : multi-responses (most research in rodent models) • Ten antigenic targets • Of which only two induce protective antibodies • Four life stages • Many potential targets at each stage • Over 5000 potential antigenic targets in total • Adapts to the immune system

13. Protozoal vaccines With a few exceptions protozoal vaccines are live vaccines. Parasite strains selected for: Complete but shortened life cycles (Eimeria strains) Truncated life cycle (Toxoplasma gondii S48 strain which does not form cysts) Vaccine with inactivated Neospora caninum tachyzoites. Vaccination with Bovilis Neoguard reduces the incidence of abortion due to Neosporosis in cattle Attenuated virulence by repeated passage through splenectomized calves (Babesia bovis and Babesia bigemina or by in vitro culture (Theileria annulata) Other kinds of live vaccine are low dose infections and use of chemotherapy to control the infection Eimeriavax 4m is a live precocious Eimeria vaccine. It is a 4 strain breeder and layer product to prevent coccidiosis reducing the dependence on chemical control of this disease. Eimeriavax 4m aids in the control of coccidiosis in chickens caused by E. acervulina, E. maxima, E. necatrix and E. tenella. Eimeriavax 4m was first registered in 2003. "Livacox" vaccine was also introduced in the late 1980's and comprises attenuated ("precocious") lines except for an egg-adapted line of E. tenella.

14. A vaccine against ovine toxoplasmosis Toxovax • Toxoplasmosis causes a disease in sheep when infection occurs for the first time during pregnancy. The parasite invades and sometimes kill the fetus. • In 1988, a live vaccine (T gondii tachyzoites of the s48 “incomplete strain”; toxovax) was marketed for the control of ovine toxoplasmosis in New Zealand. • In 1992 was launched in the UK and Edire (Toxovax, Mycofarm UK Ltd.) as a tissue-culture grown vaccine. • These tachyzoites have lost the ability to form tissue cysts. • It increases the % of lambs born live and viable when the pregnant ewe is infected. Placental pathology in vaccinated ewes is much less frequent and/or severe. • Protective immunity induced by the S48 vaccine is likely to involve both CD4+ and CD8+ T cells and the cytokine IFN- This photograph shows the effect of toxoplasmosis on several pairs of twin lambs – one of each pair is relatively normal and the other is smaller and mummified.

15. Is a vaccine against malaria possible? • Individuals continually exposed to infection by the parasite develop immunity to the disease. In endemic areas with increasing age • Decreased mortality • Decreased severe disease • Decreased level of parasitemia • Decreased prevalence of parasitemia • Sera from immune adults transfer resistance to malaria to children • Inoculation of live attenuated parasites can protect naïve volunteers against infection • Immunization with whole killed organisms can protect in animal models • Subunit vaccines including one or just a few antigens could be developed to evoke an IR. • The genome has provided tools for advancement

16. Scientific challenges to malaria vaccine development • The parasite • Genome • Life cycle (stage specific expression) • Variability • Allelic and antigenic variation • Human response based on genetics • Who is not at risk (e.g. sickle cell trait)? • Who is at risk (1-3 million/27 million)? • Human response based on transmission dynamics • Intense year long transmission (severe anemia, young) • Less intense seasonal transmission (cerebral malaria, 3-5 year olds) • Other host factors: age, nutritional status, genetics, coexisting disease, prior exposure to agent, maternal antibodies. • Modern Subunit Vaccinology • Subunit recombinant protein, synthetic peptide, recombinant virus or bacteria, DNA vaccines • None on world market since hepatitis B introduced in 1986 • Multi-immune response subunit vaccine against multiple antigens • None • Adjuvants:do not have an specific antigenic effect in itself but stimulate the immune system, increasing the response to a vaccine. Aluminum salts are used in some human vaccines.

17. WHY DO WE NEED A VACCINE AGAINST MALARIA For children in the developing world a vaccine holds the greatest promise for protecting them against malaria For travelers (military) a vaccine holds the greatest promise for protecting them against malaria

18. Vaccine strategies against malaria. Sporozoites are carried through the blood to the liver, invade hepatocytes and undergo asexual (mitotic) replication (exoerythrocytic schizont). After seven days, the liver schizonts rupture to release merozoites into the blood. Merozoites invades erythrocytes and divides mitotically to form an schizont, containing up to 20 daughter merozoites. These merozoites can re-infect erythrocytes. A subset of merozoites differentiate into male and female gametocytes, which, when taken up by a feeding mosquito, give rise to gametes. In the mosquito mid-gut, the gametes fuse to form a zygote (ookinete), which penetrates the mid-gut wall and forms an oocyst, within sporozoites develop.

19. The vaccine effect Disease prevention Cell mediated immunity Neutralizing antibody Th1 response CD4:INF gamma CD8:CTL +/- NK Cells Liver Stage Sporozoite Intra-hepatocytic 1-2 weeks Intra-vascular 3-5 minutes (Limited boosting) (Boosting possible) Pre-erythrocytic Stage Transfer antibody to mosquito Antibody dependent protection Blood Stage Merozoite Sexual Stages Intra-mosquito (mostly) 10-14 days Intra-erythrocytic 2+ days/cycle No MHCI or MHCII on RBCs so ADCC (Antibody Dependent cell-mediated cytotoxicty) Neutralization +/- Complement Activation (Limited boosting) +/- Complement lysis (Boosting possible) Disease reduction Transmission blocking

20. Clinical trials of malaria vaccines Vaccine clinical trials are long term studies aimed at assessing the safety, efficacy and immunogenicity of a new vaccine product PHASE 0 Preclinical Safety, immunogenicity, tolerability, efficacy Animal models Non-immune human volunteers in non-malarious areas. Clinical setting PHASE 1 Clinical Safety, immunogenicity, tolerability Phase IIa: non-immune volunteers Phase IIb: Immune volunteers Vaccine efficacy, safety, tolerability, acceptance Human volunteers. Experimental challenge with infected mosquitos. Clinical setting PHASE II Clinical Semi-immune residents of malarious areas (all endemicities). Small target population, special groups. Natural challenge Vaccine efficacy, safety, tolerability, acceptance PHASE III Vaccine efficacy, safety, tolerability, acceptance, vaccination strategy, effectiveness Semi-immune residents of malarious areas.Large target population, whole communities. Natural Challenge PHASE IV

21. RTS,S/AS02 an anti sporozoite vaccine A protein particle vaccine in a complex adjuvant R: central repeat of the csp protein TS: The entire c-terminus of CS protein (containing known T cell epitopes) S: hepatitis B virus surface antigen. Several viral antigens, such as the surface and core antigens of HBV, spontaneously form particles, and it has been found to enhance their uptake by antigen-presenting cells, and immunogenicity. To achieve particle formation co-expression of an excess of non-hybrid HBV S antigen was required to form RTS,S. Rip Ballou giving himself malaria in 1987. He had been injected with a vaccine candidate a year earlier and he was testing the immunity developed by challenging with malaria parasites. Schematic representation of RTS,S particles. A, RTS and S proteins; B, RTS, S particles. HBsAg, Hepatitis B surface antigen (S antigen).

22. RTS,S a preerythrocytic vaccine • Hybrid containing the central repeats and most of the C-terminal of the CSP fused with hepatitis B surface antigen • Complex adjuvant mixture AS02 • Completely protected six out of seven volunteers against SP infection • Field study in The Gambia showed good short-term protection • A clinical trial in Mozambique showed delay of infection and reduction in incidence of severe malaria in young children • The vaccine was advanced to Phase III trial in 2009. Preliminary reports published in 2011 shows protection against clinical and severe malaria in African children of approximately 50%.

23. OTHER PARASITE VACCINES • Leishmania: whole killed parasites combined with BCG was tested in Iran against CL and VL. Limited efficacy. • Various subunit recombinant candidates have been tested in mice and provided some degree of protection. These were based on: gp63, LPG, LACK and a 46 kDa Ag • Schistosomes: radiation-attenuated cercaria to laboratory animals provided protection against S. mansoni infection. A phase I and II clinical trial using a 28 kDa S. haematobium GST was safe and showed good immunogenicity in human volunteers.The schistosomiasis vaccine Development Programme has focused on two S. mansoni antigens: paramyosin and a synthetic peptide construct containing multiple antigen epitopes (MAP). Only partial reduction in challenge-derived worm burdens. • Hookworms: use of live irradiated L3 larvae was successful against canine hookworm infection. The Human Hookworm vaccine initiative (HHVI) have identified , isolated, cloned and expressed the major L3 antigens and tested as recombinant vaccine

24. SUMMARY • What does a vaccine do and what kind of knowledge is needed to create one? • Why it is difficult to make a vaccine against a protozoan parasite? • What kind of evidences are telling us that a vaccine against malaria is possible? • Name some potential targets for the development of a pre-erythrocytic vaccine • The effect of the vaccine depends on the antigen(s) selected: which antigens would trigger an immune response that will prevent malaria disease, reduce disease or block transmission? • Which are the phases of vaccine trials?