MALARIA VACCINE

Presented by: DEEPTI SINGH

Ph.D. Biotechnology

DUVASU, Mathura

DEFINITION

AETIOLOGY & TAXONOMY

EPIDEMIOLOGY

LIFE CYCLE

CLINICAL SIGNS

DIAGNOSIS

MALARIA VACCINE INITIATIVE

CLASSIFICATION OF MALARIA VACCINES

PROBLEMS IN VACCINE DEVELOPMENT

CHALLENGES FOR MALARIA VACCINE

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.

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, andclinical characteristics.

TAXONOMY

Kingdom: Protista

Sub-Kingdom: Protozoa

Phylum: Apicomplexa

Class: Sporozoasida

Order: Eucoccidiorida

Family: Plasmodiidae

Genus: Plasmodium

Specie: P. falciparum

EPIDEMIOLOGY

Malaria is the third leading cause of death due toinfectious disease.

It affects 300- 500 million people annually worldwideand accounts for over 100million deaths, mainly inAfrican children under the age of 5yrs. A child in Africadies every 30 seconds of malaria.

Endemic around the tropics and sub-tropics although itis world wide in distribution.

CLINICAL SIGNS AND SYMPTOMS

C

Cold stagefeeling 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

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 torecognize malaria. P. falciparum has two different ways in which to vary whichantigens it expresses.

The first way in which this might occur is during the sexually reproducing stagein the lifecycle when P. Falciparum recombines genetic material. This hasunlimited potential to change the genome of P. Falciparum.

The second way in which antigenic variation can occur is through variablegenes 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 wonderthat it can successfully evade the immune system and cause many recurringinfections if not properly treated.

Information for the following slides adapted from: Chen, Q., M. Schlichtherle, and M. Wahlgren. 2000. Molecularaspects of severe malaria. Clin. Microbiol. Rev. 13:439-450

VAR FAMILY

There are ~40-50 genes in the var family with a few exception they are extremelyvariable. The var genes are scattered throughout the chromosomes, butconcentrated on the 4, 7, and 12 chromosomes.

Using the high variability in these regions at least 2% of individuals vary theirantigenic expression each generation. These genes are thought to be involvedwith resistance to chloroquine and to help P. falciparum evade the host’s immunesystem.

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 sightknown as pfmdr1 (P. falciparum multidrug resistance gene).

Information for the following slides adapted from: Chen, Q., M. Schlichtherle, and M. Wahlgren. 2000. Molecular aspectsof 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 andpfmdr-1 genes and clinical response to chloroquine in Kampala, Uganda. J. Infect. Dis. 183:1417-1420.

DIAGNOSIS

LIGHT MICROSCOPY

RAPID DIAGNOSTIC TEST

SEROLOGY: ELISA KITS

MOLECULAR TECHNIQUES: PCR

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 dotsCCMOVBD

Multiple infection

Marginal form

Double chromatin

LABORATORY DIAGNOSIS OF MALARIA

Rapid diagnostic tests detect malaria

antigens

Plastic cassette format of RDT

RAPID DIAGNOSTIC TESTS DETECT MALARIA ANTIGENS

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.

16

MVI MISSION, VISION, AND GOAL

Mission: To accelerate thedevelopment of malariavaccines and ensure theiravailability and accessibility inthe developing world

Vision: A world free frommalaria

Goal: To develop by 2025 amalaria vaccine with 80% orgreater efficacy that lasts for atleast 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.

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

IDEAL MALARIAL VACCINE

prevent the infection at the first instance and if this is notpossible, should decrease the intensity of infection and should besuccessful in preventing malaria transmission.

Reduce the clinical disease severity.

Reduce the transmission.

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

PRE- ERYTHROCYTIC STAGE VACCINES

How they work:

Generates Ab response against sporozoites and prevents themfrom invading the liver

Prevents intra-hepatic multiplication by killing parasite-infected hepatocytes

Intended Use:

Ideal for travelers - protects against malaria infection

ASEXUAL ERYTHROCYTIC STAGE VACCINES

How they work:

Elicit antibodies that will inactivate merozoites and/or targetmalarial Ag expressed on RBC surface

Inhibit development of parasite in RBCs

Intended Use:

Morbidity reduction in endemic countries

SEXUAL STAGE VACCINES

How they work:

Induces Ab against sexual stage Ag

Prevents development of infectious sporozoites insalivary glands of mosquitoes

Prevent or decrease transmission of parasite to newhosts

Intended Use:

Decreased malaria transmission

SPf66 AdCh63/MVA MSP1 PfSPZ MSP3 GMZ2 AMA1-C1/Alhydrogel +CPG 7909 FMP1AS02A

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-LymphocyteResponses Induced in Chimpanzees by Combinations of Plasmodium falciparum Liver-Stage Peptides and Lipopeptides Infection andImmunity, August 2004, p. 4376-4384, Vol. 72, No. 8

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 CSrepeats) and merozoite parasites.

During phase I trials a 75% efficacy rate was demonstrated and thevaccine appeared to be well tolerated by subjects and immunogenic.

The phase IIb and III trials were less promising, with the efficacy fallingto between 38.8% and 60.2%.

Despite the relatively long trial periods and the number of studiescarried out, it is still not known how the SPf66 vaccine confersimmunity; it therefore remains an unlikely solution to malaria

CSP

Based on the circumsporoziote protein, but additionally has therecombinant protein covalently bound to a purifiedPseudomonas aeruginosa toxin (A9).

A complete lack of protective immunity was demonstrated inthose inoculated at early stage.

The study group used in Kenya had an 82% incidence ofparasitaemia whilst the control group only had an 89% incidence.

Elicits a cellular response enabling the destruction of infectedhepatocytes

NYVAC - PF. 7

Blocks transmission of the parasite from vertebrate host to mosquitoes.

The highly attenuated NYVAC vaccinia virus strain has been utilized todevelop a multiantigen , multistage vaccine candidate for malaria.

Genes encoding seven Pf antigens derived from the

1. sporozoite (CSP and sporozoite surface protein 2),

2. Liver (liver stage antigen 1),

3. blood (merozoite surface protein 1, serine repeat antigen, and apicalmembrane antigen 1),

4. 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 antigenswere characterized during 1a clinical trial.

RTS,S /AS02

Most recently developed recombinant vaccine

The RTS,S attempted by fusing the protein CPS with a surfaceantigen from Hepatitis B, hence creating a more potent andimmunogenic vaccine. When tested in trials an emulsion of oil inwater and the added adjuvants of monophosphoryl A the vaccinegave 7 out of 8 volunteers challenged with P. falciparumprotective immunity

VACCINATING MOSQUITOES

In mosquitoes, there are proteins on the surface ofgametes and ookinets that may prove useful informulating a vaccine that protects mosquitoes frominfection.

Antibodies to these proteins prevent the parasite fromtaking up residence in the mid-gut of mosquitoes andforming oocysts. However, in order for such vaccines toreach mosquitoes they must be combined with effortsto vaccinate people living in endemic areas.

PARATRANSGENESIS

Paratransgenesis is the manipulation of symbiotic bacteria such asE. coli to make the host immune to a pathogen.

Bacteria are engineered to produce proteins or peptides that eitherblock binding of or kill parasites.

Several bacteria known to live in the anopheles midgut includingEscherichia, Pseudomonas , and bacillus .

When fed with E. coli that produced antibodies to P. berghei,Anopheles mosquitoes showed a reduction in oocyst formation of95%.

Transgenic mosquitoes expressing bee venom known asPhospholipidase A2 have also been shown to resist oocystformation by up to 87%. Synthetic molecules have also beenstudied as ways of reducing susceptibility.

Anopheles mosquitoes with a synthetic gene expressing SM1peptide were found to have 82% reduction in formation ofoocysts.

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. TheJournal of Experimental Biology 206, 3809-3816 (2003)

OTHER CONTROL METHODS

Biological Control

Mosquito fishes (Gambusia affinis) have been found to be predatoryon the anopheles larvae.

Chemical Control

Spray insecticides: DDVP and so on.

Use mosquito nets, screen, or mosquito repellents to protect theperson from mosquito bites.

Physical Control:

Eradicate the breeding places of mosquitoes.

REASON FOR INCOMPLETE PROTECTION

Polymorphism and clonal variation in antigens of plasmodium

Parasite induced immuno-suppression

Intracellular parasites

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 andCSP has 6 variants

Multiple antigens, specific to species and stage

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 responsesand correlates of protection are incompletely understood.

Identifying and assessing vaccine candidates takes time and isexpensive

There is no clear ‘best approach’ for designing a malaria vaccine

BIBLIOGRAPHY1. Regules, J., Cummings, J., & Ockenhouse, C. (2011). The RTS,S Vaccine Candidate for Malaria. Expert Reviews,

10(5).

2. 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.

3. L, Schwartz and B, Graham.(2012). A Review of Malaria Vaccine Clinical Projects Based on the WHO Rainbow

Table. Malaria Journal 11.11.

4. "PATH Malaria Vaccine Initiative: The need for a vaccine." PATH Malaria Vaccine Initiative. N.p., n.d. Web. 28

Nov. 2012.

5. Geoffrey, T., & Greenwood, B. (2008). Malaria vaccines and their potential role in the elimination of malaria.

Malaria Journal, 7.

6. Mutabingwa , T. (2005). Artemisinin-based combination therapies (ACTs): best hope for malaria treatment but

inaccessible to the needy! Acta Trop, 95(3).

7. WHO (n.d.). Malaria Transmission Blocking Vaccine: an ideal public good. Special Programme for Research &

Training in Tropical Disease.

8. PATH Malaria Vaccine Initiative. (n.d.). Retrieved from http://www.malariavaccine.org/files/MVI-brief-RandD-

strategy-FINAL-web.pdf

9. Moorthy, V., & Ballou, R. (2009). Immunological Mechanisms Underlying Protection Mediated by RTS,S: a

review of the available data. Malaria Journal, 8(312).

10. Milstein, J., & Cárdenas, V. (2010). WHO policy development processes for a new vaccine: case study of

malaria vaccines. Malaria Journal, 9.

11. PATH Malaria Vaccine Initiative: Advocacy fellowship. (n.d.). PATH Malaria Vaccine Initiative. Retrieved from

http://www.malariavaccine.org/preparing-mvaf.php

12. WHO | Malaria. (n.d.). Retrieved from http://www.who.int/mediacentre/factsheets/fs094/en/

13. The role of vaccine in the prevention of malaria « HCDCP. (n.d.). ΚΕΕΛΠΝΟ. Retrieved from

http://www2.keelpno.gr/blog/?p=2178&lang=en