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
CONTROL MEASURES
Vaccines and Vectors
Medical Parasitology
CBIO4500
April 17, 2012
Silvia N J Moreno
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
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
• Anti-parasite/pathogen therapies
• Vaccines
Parasite/Pathogen
• Blocking the parasite
acquisition or transmission by
arthropods: Vaccine
• Insect immune regulators
Examples of novel control strategies developed based on arthropod genome
(smart
sprays)
resources (red shaded text boxes) and the parasite
or human
host genome
resources (yellow text box) are shown. Nature reviews Microbiology 3:262 (2005).
• Insecticides
• Attractants/repellants and other
behavioural modifiers
• Vector longevity curtailers
• Genetically modified vectors
incapable of reproduction or
pathogen transmission
Arthropod Vector
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 ?
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
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
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.
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 Tdependent 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.
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).
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.
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 : multiresponses
(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
Protozoal vaccines
With a few exceptions protozoal vaccines are live vaccines.
Parasite strains selected for:
1.
Complete but shortened life cycles (Eimeria strains)
2.
Truncated life cycle (Toxoplasma gondii S48 strain which does
not form cysts)
3.
Vaccine with inactivated Neospora caninum tachyzoites. Vaccination
with Bovilis Neoguard reduces the incidence of abortion due to
Neosporosis in cattle
4.
Attenuated virulence by repeated passage through splenectomized
calves (Babesia bovis and Babesia bigemina or by in vitro culture
(Theileria annulata)
5.
Other kinds of live vaccine are low dose infections and use of
chemotherapy to control the infection
"Livacox" vaccine was
also introduced in the
late 1980's and
comprises attenuated
("precocious") lines
except for an eggadapted line of E.
tenella.
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.
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.
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
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.
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
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.
The vaccine effect
Disease prevention
Neutralizing antibody
Sporozoite
Cell mediated immunity
Liver Stage
(Limited boosting)
Intra-vascular
3-5 minutes
Intra-hepatocytic
1-2 weeks
Transfer antibody
to mosquito
Sexual Stages
Blood Stage
Merozoite
Neutralization
+/- Complement
Activation
Pre-erythrocytic Stage
Intra-mosquito
(mostly)
10-14 days
(Limited boosting)
Intra-erythrocytic
2+ days/cycle
+/- Complement
lysis
(Boosting possible)
Th1 response
CD4:INF gamma
CD8:CTL
+/- NK Cells
(Boosting possible)
Antibody
dependent
protection
No MHCI or MHCII on
RBCs so ADCC
(Antibody Dependent
cell-mediated
cytotoxicty)
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
Animal models
PHASE 0
Preclinical
Non-immune human volunteers in
non-malarious areas.
Clinical setting
PHASE 1
Human volunteers. Experimental
challenge with infected mosquitos.
Clinical setting
PHASE II
Semi-immune residents of malarious
areas (all endemicities). Small target
population, special groups.
Natural challenge
Semi-immune residents of malarious
areas.Large target population, whole
communities.
Natural Challenge
Clinical
Clinical
Safety, immunogenicity,
tolerability, efficacy
Safety, immunogenicity,
tolerability
Phase IIa: non-immune volunteers
Phase IIb: Immune volunteers
Vaccine efficacy, safety, tolerability,
acceptance
PHASE III
Vaccine efficacy, safety,
tolerability, acceptance
PHASE IV
Vaccine efficacy, safety,
tolerability, acceptance,
vaccination strategy,
effectiveness
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).
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%.
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
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 preerythrocytic 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?