biotechnological

1. MALARIA VACCINE
Presented by: DEEPTI SINGH
Ph.D. Biotechnology
DUVASU, Mathura

2.  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.

8. 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

9. 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

10. 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.

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

12. 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

13. LABORATORY DIAGNOSIS OF MALARIA
Rapid diagnostic tests detect malaria
antigens

14. Plastic cassette format of RDT
RAPID DIAGNOSTIC TESTS DETECT MALARIA
ANTIGENS

15. 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. 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.

17. 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

18. 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.

19. 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

20. 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

21. 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

22. 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

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

25. 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

26. 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

27. 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

28. 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
1. sporozoite (CSP and sporozoite surface protein 2),
2. Liver (liver stage antigen 1),
3. blood (merozoite surface protein 1, serine repeat antigen, and apical
membrane 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 antigens
were characterized during 1a clinical trial.

29. 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

30. 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.

31. 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%.

32.  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)

33. 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.

34. REASON FOR INCOMPLETE PROTECTION
 Polymorphism and clonal variation in antigens of plasmodium
 Parasite induced immuno-suppression
 Intracellular parasites

35. 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

36. 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

37. BIBLIOGRAPHY
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