Malaria vaccine

biotechnological

1. Malaria vaccine Presented by: DEEPTI SINGH Ph.D. Biotechnology DUVASU, Mathura

2. OUTLINE DEFINITION AETIOLOGY & TAXONOMY EPIDEMIOLOGY LIFE CYCLE CLINICAL SIGNS DIAGNOSIS MALARIA VACCINE INITIATIVE CLASSIFICATION OF MALARIA VACCINES PROBLEMS IN VACCINE DEVELOPMENT CHALLENGES FOR MALARIA VACCINE

3. Definition A protozoan disease caused by Plasmodium species of the phylum Apicomplexa. Transmitted by the bite of infected female anopheline mosquitoes. It is characterized by periodic paroxysm with shaking chills, high fever, heavy sweating. Anemia and splenomegaly may also occur in cases.

4. aetiology Four species of Plasmodium cause malaria in human. P. vivax ( benign tertian malaria) P. ovale ( benign tertian malaria) P. malariae ( quartan malaria) P. falciparum ( malignant tertian malaria) Each species has its own morphologic, biologic, pathogenic, and clinical characteristics.

5. TAXONOMY Kingdom: Protista Sub-Kingdom: Protozoa Phylum: Apicomplexa Class: Sporozoasida Order: Eucoccidiorida Family: Plasmodiidae Genus: Plasmodium Specie: P. falciparum

6. EPIDEMIOLOGY Malaria is the third leading cause of death due to infectious disease. It affects 300- 500 million people annually worldwide and accounts for over 100million deaths, mainly in African children under the age of 5yrs. A child in Africa dies every 30 seconds of malaria. Endemic around the tropics and sub-tropics although it is world wide in distribution.

7. CLINICAL SIGNS AND SYMPTOMS C Cold stage feeling of intense cold vigorous shivering lasts 15-60 minutes Hot stage intense heat dry burning skin throbbing headache lasts 2-6 hours   Sweating stage profuse sweating declining temperature exhausted and weak →sleep lasts 2-4 hours

8. ANTIGENIC VARIATION Malaria has many tools to evade the immune system. P. falciparum has a very high degree of antigenic variation, making it difficult for the immune system to recognize malaria. P. falciparum has two different ways in which to vary which antigens it expresses. The first way in which this might occur is during the sexually reproducing stage in the lifecycle when P. Falciparum recombines genetic material. This has unlimited potential to change the genome of P. Falciparum . The second way in which antigenic variation can occur is through variable genes and point mutations during asexually reproducing stages of the lifecycle. P. Falciparum o has several families of variable antigenic genes. These are var family, the rosettin / rif family, and the p60 family. With such a large amount of variability available to malaria it is no wonder that it can successfully evade the immune system and cause many recurring infections if not properly treated. Information for the following slides adapted from: Chen, Q., M. Schlichtherle , and M. Wahlgren . 2000. Molecular aspects of severe malaria. Clin . Microbiol . Rev. 13: 439-450

9. Var Family There are ~40-50 genes in the var family with a few exception they are extremely variable. The var genes are scattered throughout the chromosomes, but concentrated on the 4, 7, and 12 chromosomes. Using the high variability in these regions at least 2% of individuals vary their antigenic expression each generation. These genes are thought to be involved with resistance to chloroquine and to help P. falciparum evade the host’s immune system. Mutations at this sight are found in 100% of all resistant strains of P. falciparum . The efficacy of the resistance is greater when a mutation also occurs at a sight known as pfmdr1 ( P. falciparum multidrug resistance gene). Information for the following slides adapted from: Chen, Q., M. Schlichtherle , and M. Wahlgren . 2000. Molecular aspects of severe malaria. Clin . Microbiol . Rev. 13: 439-450 Dorsey, G., M. R. Kamya , A. Singh, and P. J. Rosenthal . 2001. Polymorphisms in the Plasmodium falciparum pfcrt and pfmdr-1 genes and clinical response to chloroquine in Kampala, Uganda . J. Infect. Dis . 183: 1417-1420.

10. DIAGNOSIS LIGHT MICROSCOPY RAPID DIAGNOSTIC TEST SEROLOGY: ELISA KITS MOLECULAR TECHNIQUES: PCR

11. Laboratory diagnosis of malaria Plasmodium falciparum Diagnostic Points: Small, regular, fine to fleshy cytoplasm Infected RBCs not enlarged Numerous, multiple infection is common Ring, comma, marginal are seen; often have double chromatin dots CCMOVBD Multiple infection Marginal form Double chromatin

12. Laboratory diagnosis of malaria Rapid diagnostic tests detect malaria antigens

13. Plastic cassette format of R DT RAPID DIAGNOSTIC TESTS DETECT MALARIA ANTIGENS

14. Malaria vaccine initiative (MVI) MVI is working with the International Centre for Genetic Engineering and Biotechnology (ICGEB) in New Delhi, India, to develop a vaccine against Plasmodium vivax. This development effort includes Bharat Biotech International Ltd. (Hyderabad), which will manufacture the vaccine for preclinical testing followed by initial safety trials in adults.

15. 16 MVI mission, vision, and goal Mission: To accelerate the development of malaria vaccines and ensure their availability and accessibility in the developing world Vision: A world free from malaria Goal: To develop by 2025 a malaria vaccine with 80% or greater efficacy that lasts for at least four years MVI was established in 1999 as a program of PATH, an international nonprofit organization that creates sustainable, culturally relevant solutions, enabling communities worldwide to break longstanding cycles of poor health.

16. RECENT LANDMARKS IN Malaria genomes - Sequencing 2002: Complete genome sequence of P. falciparum A partial sequence of rodent parasite, P. berghei 2005: sequences of several other rodent parasites P. vivax (a human malaria parasite) P. knowlesi (primarily a monkey parasite) + sequence of: Human genome Anopheles mosquito New Candidates for drug and vaccine pipeline

17. Ideal malarial vaccine prevent the infection at the first instance and if this is not possible, should decrease the intensity of infection and should be successful in preventing malaria transmission. Reduce the clinical disease severity. Reduce the transmission.

18. Classification of malaria vaccine Stage of plasmodium Antigens Salient features Pre- erythrocytic Irradiated sporozoites , Circum Sporozoite Protein (CSP) or peptides, Liver stage Antigens -1 (LSA-1) Stage/species specific; antibody blocks infection of liver; large immunising dose required; can abort an infection Merozoite and Erythrocytes Erythrocyte Binding Antigen (EBA-175), Merozoite Surface Antigen 1&2 (MSA-1&2) ; Ring Infected Erythrocyte Surface Antigen (RESA); Serine Repeat Antigen (SERA); Rhoptry Associated Protein (RAP); Histidine Rich Protein (HRP); Apical Membrane Antigen-1 (AMA-1) Specific for species and stage; Cannot abort an infection; Prevents invasion of erythrocytes, thus reducing severity of infection Gametocytes & gametes Pfs 25 , 48/45k, Pfs 230 Prevents infection of mosquitoes; antibody to this antigen prevents either fertilization or maturation of gametocytes, zygotes or ookinetes ; is of use in endemic areas but not suited for travelers; antibody blocks transmission cycle Combined vaccine (cocktail) SPf 66 (based on pre- erythrocytic and asexual blood stage proteins of Pf) Based on incorporation of antigens from different stages into one vaccine to produce an immune response, blocking all stages of the parasite development

19. Pre- Erythrocytic Stage Vaccines How they work: Generates Ab response against sporozoites and prevents them from invading the liver Prevents intra-hepatic multiplication by killing parasite-infected hepatocytes Intended Use: Ideal for travelers - protects against malaria infection Pre- erythrocytic stage:that is the stage that takes place shortly after being bitten by an infected mosquito up to and including the liver stage .

20. Asexual Erythrocytic Stage Vaccines How they work: Elicit antibodies that will inactivate merozoites and/or target malarial Ag expressed on RBC surface Inhibit development of parasite in RBCs Intended Use: Morbidity reduction in endemic countries

21. Sexual Stage Vaccines How they work: Induces Ab against sexual stage Ag Prevents development of infectious sporozoites in salivary glands of mosquitoes Prevent or decrease transmission of parasite to new hosts Intended Use: Decreased malaria transmission

22. OTHER VACCINES FOR MALARIA SPf66 AdCh63/MVA MSP1 PfSPZ MSP3 GMZ2 AMA1-C1/ Alhydrogel +CPG 7909 FMP1AS02A

23. other vaccine avenues Several antigens expressed during the blood stream and liver stage of P. falciparum have been shown to elicit an immune response in humans. The study showed that liver stage antigen 3 was highly immunogenic and a good candidate for use in a vaccine to prevent the invasion of RBC by P. falciparum. Immune memory of the antigens (especially LSA3) lasted up to 9 months when tested in chimpanzees. Information for this slide from: Pouniotis DS, Proudfoot O, Minigo G, Hanley JC, Plebanski M. Long-Term Multiepitopic Cytotoxic-T-Lymphocyte Responses Induced in Chimpanzees by Combinations of Plasmodium falciparum Liver-Stage Peptides and Lipopeptides Infection and Immunity, August 2004, p. 4376-4384, Vol. 72, No. 8

24. spf66 The first vaccine developed that has undergone field trials Developed by Manuel Elkin Patarroyo in 1987. It presents a combination of antigens from the sporozoite (using CS repeats) and merozoite parasites. During phase I trials a 75% efficacy rate was demonstrated and the vaccine appeared to be well tolerated by subjects and immunogenic. The phase IIb and III trials were less promising, with the efficacy falling to between 38.8% and 60.2%. Despite the relatively long trial periods and the number of studies carried out, it is still not known how the SPf66 vaccine confers immunity; it therefore remains an unlikely solution to malaria

25. Csp Based on the circumsporoziote protein, but additionally has the recombinant protein covalently bound to a purified Pseudomonas aeruginosa toxin (A9). A complete lack of protective immunity was demonstrated in those inoculated at early stage. The study group used in Kenya had an 82% incidence of parasitaemia whilst the control group only had an 89% incidence. Elicits a cellular response enabling the destruction of infected hepatocytes

26. NYVAC - Pf. 7 Blocks transmission of the parasite from vertebrate host to mosquitoes. The highly attenuated NYVAC vaccinia virus strain has been utilized to develop a multiantigen , multistage vaccine candidate for malaria. Genes encoding seven Pf antigens derived from the sporozoite (CSP and sporozoite surface protein 2), Liver (liver stage antigen 1), blood ( merozoite surface protein 1, serine repeat antigen, and apical membrane antigen 1), sexual (25-kDa sexual-stage antigen) inserted into a single NYVAC genome to generate NYVAC-Pf7. safe and well tolerated. Specific antibody responses against four of the P. falciparum antigens were characterized during 1a clinical trial.

27. Rts,s /as02 Most recently developed recombinant vaccine The RTS,S attempted by fusing the protein CPS with a surface antigen from Hepatitis B , hence creating a more potent and immunogenic vaccine. When tested in trials an emulsion of oil in water and the added adjuvants of monophosphoryl A the vaccine gave 7 out of 8 volunteers challenged with P. falciparum protective immunity

28. VACCINATING MOSQUITOES In mosquitoes, there are proteins on the surface of gametes and ookinets that may prove useful in formulating a vaccine that protects mosquitoes from infection. Antibodies to these proteins prevent the parasite from taking up residence in the mid-gut of mosquitoes and forming oocysts. However, in order for such vaccines to reach mosquitoes they must be combined with efforts to vaccinate people living in endemic areas.

29. PARATRANSGENESIS Paratransgenesis is the manipulation of symbiotic bacteria such as E. coli to make the host immune to a pathogen. Bacteria are engineered to produce proteins or peptides that either block binding of or kill parasites. Several bacteria known to live in the anopheles midgut including Escherichia, Pseudomonas , and bacillus . When fed with E. coli that produced antibodies to P. berghei , Anopheles mosquitoes showed a reduction in oocyst formation of 95%.

30. Transgenic mosquitoes expressing bee venom known as Phospholipidase A2 have also been shown to resist oocyst formation by up to 87% . Synthetic molecules have also been studied as ways of reducing susceptibility. Anopheles mosquitoes with a synthetic gene expressing SM1 peptide were found to have 82% reduction in formation of oocysts . Information on this slides from Michael A. Riehle , Prakash Srinivasan , Cristina K. Moreira and Marcelo Jacobs-Lorena . Towards genetic manipulation of wild mosquito populations to combat malaria: advances and challenges. The Journal of Experimental Biology 206, 3809-3816 (2003)

31. Other control methods Biological Control Mosquito fishes ( Gambusia affinis) have been found to be predatory on the anopheles larvae. Chemical Control Spray insecticides: DDVP and so on. Use mosquito nets, screen, or mosquito repellents to protect the person from mosquito bites. Physical Control : Eradicate the breeding places of mosquitoes.

32. Reason for incomplete protection Polymorphism and clonal variation in antigens of plasmodium Parasite induced immuno-suppression Intracellular parasites

33. Problems in vaccine development Difficulty of evaluation Parasites’ ingenious ways of avoiding hosts’ immune response Complexity of conducting clinical and field trials Mutation of the parasites Antigenic variations e.g. MSA-I has 8 variants, MSA-2 has 10 and CSP has 6 variants Multiple antigens, specific to species and stage

34. Challenges for Malaria Vaccine Four antigenetically distinct malaria species Each has ~6,000 genes First gene only identified in 1983 Immunity in malaria is complex and immunological responses and correlates of protection are incompletely understood. Identifying and assessing vaccine candidates takes time and is expensive There is no clear ‘best approach’ for designing a malaria vaccine

35. Bibliography Regules , J., Cummings, J., & Ockenhouse , C. (2011). The RTS,S Vaccine Candidate for Malaria. Expert Reviews , 10 (5). Agnandji , S., & Lell , B. (2011). First Results of Phase 3 Trial of RTS,S/AS01 Malaria Vaccine in African Children. The New England Journal of Medicine , 365 . L, Schwartz and B, Graham.(2012). A Review of Malaria Vaccine Clinical Projects Based on the WHO Rainbow Table. Malaria Journal 11.11. "PATH Malaria Vaccine Initiative: The need for a vaccine." PATH Malaria Vaccine Initiative . N.p ., n.d . Web. 28 Nov. 2012. Geoffrey, T., & Greenwood, B. (2008). Malaria vaccines and their potential role in the elimination of malaria. Malaria Journal , 7 . Mutabingwa , T. (2005). Artemisinin -based combination therapies (ACTs): best hope for malaria treatment but inaccessible to the needy! Acta Trop , 95 (3). WHO ( n.d .). Malaria Transmission Blocking Vaccine: an ideal public good. Special Programme for Research & Training in Tropical Disease . PATH Malaria Vaccine Initiative . ( n.d .). Retrieved from http://www.malariavaccine.org/files/MVI-brief-RandD-strategy-FINAL-web.pdf Moorthy , V., & Ballou , R. (2009). Immunological Mechanisms Underlying Protection Mediated by RTS,S: a review of the available data. Malaria Journal , 8 (312). Milstein, J., & Cárdenas, V. (2010). WHO policy development processes for a new vaccine: case study of malaria vaccines. Malaria Journal , 9 . PATH Malaria Vaccine Initiative: Advocacy fellowship. ( n.d .). PATH Malaria Vaccine Initiative . Retrieved from http://www.malariavaccine.org/preparing-mvaf.php WHO | Malaria. ( n.d .). Retrieved from http://www.who.int/mediacentre/factsheets/fs094/en/ The role of vaccine in the prevention of malaria « HCDCP. ( n.d .). ΚΕΕΛΠΝΟ . Retrieved from http://www2.keelpno.gr/blog/?p=2178&lang=en

36. Thank you.