1. Human Parasite Vaccines
Dr Abhijit Chaudhury MD, DNB,
D(ABMM)
SVIMS, Tirupati.

2. Introduction
• 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.

3. Introduction
• 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: ideally
sustained protection
Vaccine must stimulate good response → without adjuvant
is best.
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.

4. Introduction
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)

5. Introduction: Problems
The Protective Immune Response:
Lack of definition of the exact immune
effectors and responsible parasitic antigens.
 Lack of consensus on the type of immune
response required to eliminate infections.

6. Introduction: Problems
Antigen Expression and Variation:
 Parasites present a variety of temporally
expressed antigens to the host immune
system, which also shows stage-specificity.
 Some may be associated with protective
immune response, and not all.
 Most parasites also show great antigenic
variation of surface proteins.

7. Introduction: Problems
Animal models
 Animal models are essential to define
immune response, protective efficacy, and
safety prior to clinical trials.
 Candidate antigens may show efficacy in
animals but not in humans.
 Animals may not be fully permissive so that
infection outcomes may be different from
those in humans.

8. Introduction: Problems
Single/Multiple antigens:
 Parasites are complex organisms and
protective response may not be elicited by a
single protein antigen.
 Antigen combinations are generally more
effective , and possibly targeting more than
one stage of the parasite.

9. Introduction: Problems
Adjuvants
 Wrong choice of adjuvant may lead to
misleading experimental outcomes.
 Thus good candidate antigens may be
rejected.

10. Other Problems
Antigen discovery:
 Complicated genetic structures of pathogens
 Absence of genome databases or bioinformatic
algorithms for selecting candidate antigens of promise.
Process development
 Necessity to scale up production of vaccine at
adequate yields and at low cost.
 Failure of many bacterial expression systems to
produce properly folded recombinant proteins and the
requirement for eukaryotic or other less common
expression vectors.

11. Other Problems
Preclinical development
 Difficulty in maintaining different life cycle stages
of parasites in vitro
 Paucity of laboratory animal models permissive
to the parasites.
Clinical development
 Clinical trials in resource-poor setting difficult.
 Highly modulated immune response from
infection with many parasites, especially
helminths , present some dangers for vaccination.

12. Historical Aspects
Leishmanization
• It has been known since antiquity that individuals who
had healed cutaneous leishmaniasis skin lesions were
protected from further infections.
• Bedouin or some Kurdistani tribal societies
traditionally expose their babies' bottoms to sandfly
bites in order to protect them from facial lesions.
• Another ancient technique practised in the Middle
East has been the use of a thorn to transfer infectious
material from lesions to uninfected individuals.

13. Neglected Tropical Diseases
• The core group of 13 neglected tropical diseases
includes 7 helminth infections—ascariasis, trichuriasis,
hookworm, lymphatic filariasis , onchcocerciasis,
dracunculiasis, schistosomiasis; 3 protozoan
infections—Chagas disease, human African
trypanosomiasis, and leishmaniasis; and 3 bacterial
infections—trachoma, leprosy, and Buruli ulcer
• These are ancient conditions that have plagued
humankind for centuries, and are sometimes also
referred to as the “biblical diseases.
• Most are confined to economically deprived countries.
• Anti-poverty vaccines.

14. MAJOR HUMAN PARASITIC DISEASES
DISEASE
Population at Risk
(106 )
Cases
(106)
Mortality
(103)
MALARIA
> 2100
270-400
1120
Sleeping Sickness
>60
0.3-0.5
49
Chagas Disease
120
17
13
Leishmaniasis
350
12
57
Intestinal Protozoa
3500
450
65
Schistosomiasis
600
>200
15
Onchocerciasis
120
18
0
Lymphatic filariasis
1000
120
0
Geohelminths
4500
3000
17

15. Protozoan Vaccines:
1. MALARIA VACCINE
 Modern malaria vaccine development stems
from immunization studies of mice with
Irradiated sporozoites, conducted in the 1960s
 Challenge studies by Clyde in 1970s in
humans demonstrated that a high level of
protection could be induced in volunteers but
required large numbers of bites by irradiated
infectious mosquitoes.

16. Malaria Vaccines
 The emergence of a peptide-based candidate
vaccine from Colombia, called SPf66, with
apparent efficacy in new world monkeys and
humans generated enormous interest and
controversy but eventually disappointment as
successive, independent field efficacy trials in
Africa and Asia failed to demonstrate
protection.

17. Malaria Vaccine
• The difficulty of developing a highly effective
malaria vaccine has led to the design and
assessment of a very wide range of new
approaches.

19. Malaria Vaccines- multiple approaches
1.
2.
3.
4.
5.
Sporozoite subunit
vaccination, especially with
the CS protein: e.g. RTS,S in
adjuvant.
Irradiated or genetically
attenuated sporozoites
Immunization with DNA
and/or viral vectors against
the liver-stage parasites, or
other life cycle stages.
Use of whole blood-stage
malaria parasites .
Immunization with parasite
adhesion ligands such as
PfEMP1.
6.
7.
8.
9.
10.
Use of peptide-based
vaccines, mainly against
blood-stage parasites—e.g.
SPf66, PEV3a.
Development of anti-disease
vaccines based on parasite
toxins—e.g. GPI-based.
Immunization with sexual
stage parasite antigens as
transmission-blocking
vaccines.
Use of mosquito antigens as
transmission-blocking
vaccines.
Use of protein in adjuvant
vaccines to reduce the growth
rate of blood-stage parasites

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
Animal models
Non-immune human volunteers in
non-malarious areas.
Clinical setting
Human volunteers. Experimental
challenge with infected mosquitos.
Clinical setting
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
PHASE 0
Preclinical
PHASE 1
Clinical
PHASE II
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

22. RTS,S Vaccine: The Leading Candidate
• Work started in 1980 (Walter Reed Army
Institute and GSK Biologicals).
• Pre-erythrocytic vaccine
• Consists of the central repeat (‘R’) of
circumsporozoite protein fused to the Cterminal region known to contain T cell
epitopes (hence ‘T’) fused in turn to the
hepatitis B surface antigen (‘S’) yielded a
yeast-expressed protein RTS.

23. RTS,S Vaccine: The Leading Candidate
• To generate immunogenic particles, the RTS
protein needed to be co-expressed with large
amounts of the unfused S protein to yield
RTS,S.
• Adjuvant contains the immunostimulants,
mono-phosphoryl lipid A (MPL, a toll-like
receptor 4 agonist) and QS21 (a derivative of
Quill A) . A related adjuvant AS01, which
contains liposomes is used in this vaccine.

24. RTS,S Vaccine: The Leading Candidate
• This vaccine to induce a very high
concentration of antibodies, often of
hundreds of micrograms per millilitre, that
target the conserved central repeat region of
the circumsporozoite protein.
• The level of these antibodies correlates with
protection against infection or disease.

25. RTS,S Vaccine: The Leading Candidate
• It has demonstrated 51% efficacy in reducing the rate
of all episodes of clinical malaria over fifteen months of
follow-up in a Phase 2 trial in children aged 5-17
months in Kenya.
• The ongoing Pivotal Phase 3 trial started in May 2009
and has enrolled 15,460 children.
• The first of 3 sets of results from the Phase 3 trial was
published in Oct 2011 and was in line with
expectations from the Phase 2 trials .(NEJM, 2011,365:1863-75)
• The trial, conducted at 11 sites in seven countries
across sub-Saharan Africa, reported that RTS,S reduced
the incidence of all episodes of clinical malaria by 55%.

26. RTS,S Vaccine: The Leading Candidate
Second set of results: 2012
• The incidence of a first or only episode of clinical malaria meeting
the primary case definition during 12 months of follow-up was 0.37
per person-year in the RTS,S/AS01 group and 0.48 per person-year
in the control group, for a vaccine efficacy of 31.3% .
• Efficacy was higher at the beginning than at the end of the followup period
• Before vaccination, 34.3% of infants were positive for anticircumsporozoite antibodies but at low titers. After vaccination,
99.7% were positive for anti-circumsporozoite antibodies, with a
geometric mean titer of 209 EU per milliliter.
(The RTS,S Clinical Trials Partnership. A Phase 3 Trial of RTS,S/AS01 Malaria
Vaccine in African Infants. NEJM 2012,367:2284-95)

27. RTS,S Vaccine: The Leading Candidate
• The full trial results are expected in 2014 .
• The aim of licensure and deployment is in
2015.
• This vaccine will be evaluated as a potential
addition to, not a replacement for, integrated
approaches of existing preventive, diagnostic
and treatment measures tailored to a given
endemic setting .(Malaria Policy Advisory Committee to
the WHO: conclusions and recommendations of March 2013
meeting).

28. Leishmania Vaccines
 Evidence that most individuals who were
once infected with Leishmania are resistant to
clinical infections when later exposed provides
the justification for vaccine development.
 The leishmaniases are unique among parasitic
diseases because a single vaccine could
successfully prevent and treat disease and has
the potential to protect against more than one
Leishmania parasite species.

29. Leishmania Vaccines
(i) Live Leishmania vaccine (Leishmanization, LZ)
(ii) First generation vaccines consisting of whole
killed Leishmania or fractions of the parasite
(iii) Second generation vaccines including all
defined vaccines, i.e., recombinant proteins,
DNA vaccines and combinations
(iv) Live-attenuated Leishmania vaccines.

30. Leishmania Vaccines
LEISHMANIZATION:
 Not licensed, but used in Uzbekistan, former USSR,
Iran, and Israel.
 Live virulent L. major promastigotes are harvested
from cultures and used.
 At present, there is only one prophylactic live vaccine
in use. This is a mixture of live virulent L. major mixed
with killed parasite registered in Uzbekistan.
 Adverse side effects, include development of large
persistent lesions, psoriasis and immunosuppression.

31. Leishmania Vaccines
First generation Vaccines: Whole killed/Fractions
 New World: Mayrink’s vaccine(L.amazonensis)
; Convit’s Vaccine( L.mexicana+BCG).
Incidence rate amongst the Montenegro Skin
Test (MST) converted individuals in the
vaccine group was significantly lower than
those in the control (unvaccinated) group or
vaccinated but MST nonconverted individuals.

32. Leishmania Vaccines
 Old World: Autoclaved L.major+BCG (ALM+BCG)
 Two doses of the vaccine reduced the incidence
by 43% in Leishmanin Skin Test converted
volunteers in Sudan against Visceral
Leishmaniasis involving 2306 volunteers.
 To enhance the immunogenicity of the ALM+BCG
vaccine, ALM was adsorbed to alum and the
resulting alum-ALM was mixed with BCG just
prior to injection.
 Appears to constitute a safe vaccine and an
appropriate candidate for further development.

33. Leishmania Vaccines
Fractionated Leishmania vaccine: Fucose
Mannose ligand antigen; Soluble L. donovani
antigens ; soluble exogenous antigens of
L.major; L.donovani promastigote soluble
antigen.
 Canine, hamster, and murine experimental
stages.

34. Leishmania Vaccines
Second Generation Vaccines: Recombinant protein
vaccines.
 A variety of Leishmania vaccines consist of recombinant
proteins; poly-proteins produced by DNA cloning.
 More recent efforts aim at increasing the immunogenicity
of DNA cloned vaccines, including the use of genetic
adjuvants and plasmid-based expression of viral replicons.
 Some of the important recombinant protein candidate
vaccines include: surface expressed glycoprotein
leishmaniolysin (gp63), Leishmania activated C kinase
(LACK), parasite surface antigen (PSA), Leishmania derived
recombinant polyprotein (Leish-111f) and serine proteases.

35. Leishmania Vaccines
•
Leish-111f is a single polyprotein composed of three
molecules fused in tandem: the L. major homologue of
eukaryotic thiol-specific antioxidant, TSA; the L. major
stress-inducible protein-1, LmSTI1; and the L.
braziliensis elongation and initiation factor, LeIF.
• The Leish-111f product is the first defined vaccine for
leishmaniasis in human clinical trials and has
completed phase 1 and 2 safety and immunogenicity
testing in normal, healthy human subjects.
• Efficacious against cutaneous or mucosal
leishmaniasis.

36. Leishmania Vaccines
DNA Vaccine:
 DNA vaccines can be used therapeutically to
treat CL caused by L. major as well
prophylactically.
Live-attenuated Leishmania vaccines:
 The live-attenuated anti-leishmanial vaccine
is still at its early stages of development.

37. Leishmania Vaccines
• Dihydrofolate reductase thymidylate synthase
(dhfr-ts) knockout parasites led to protection in a
mouse model but not in monkeys.
• Deletion of serine protease in L. mexicana gave
partial protection.
• Recently, use of L. donovani centrin null mutants
(LdCEN-/-) in mice showed clearance of virulent
challenge parasites in 10 weeks after challenge,
with significantly reduced parasite burden in the
spleen and no parasites in the liver.

38. Other Protozoan Vaccines
AMOEBIASIS:
THE SERINE-RICH E. HISTOLYTICA PROTEIN:
 Mediates the binding of trophozoites of E.
histolytica to the mammalian cells.
 85% of the vaccinated gerbils in a total of 3
trials were completely protected from
developing amebic liver abscess.
 The safety and immunogenicity has been
well-documented in African green
monkeys.

39. Other Protozoan Vaccines
THE N-ACETYLGALACTOSAMINE-INHIBITABLE E.
HISTOLYTICA LECTIN (GAL / GALNAC): Mediates
adherence of trophozoites
 Although the vaccination has been protective in
66% of the animals, in the remaining there has
been evidence of a significant increase in liver
abscess size.
OTHERS: 29-kDa cysteine-rich protein (peroxiredoxin) ,
lipophosphoglycan, oral/intranasal administration of
lectins.

40. Other Protozoan Vaccines
GIARDIASIS:
 Vaccines against giardiasis is potentially complicated by the
fact that the parasite can undergo antigenic variation.
 Cyst wall proteins are candidate antigens.
CRYPTOSPORIDIUM:
 Vaccination against C. parvum is focused on
immunodominant antigens expressed on the surface of
sporozoites .
 Additional antigens, to which invasion-neutralizing
antibody responses are directed, included CpMuc4 and
CpMuc5 which are mucin-like glycoproteins.
 Recent approaches have used salmonella as a vaccine
delivery vehicle .

41. Other Protozoan Vaccines
Chagas Disease:
Bivalent therapeutic vaccine for the treatment
of chronic Chagas disease.
The vaccine is comprised of two Trypanosoma
cruzi recombinant proteins formulated on
alum. One of the antigens is a unique T. cruzi
24 kDa antigen (Tc24) and the other is a
unique T. cruzi surface transialidase (TSA-1).
 Pre-clinical work.

42. HELMINTHIC VACCINES: HOOKWORM
PROBLEMS WITH HOOKWORM (+other HELMINTHS) VACCINE
DEVELOPMENT
1. The difficulty of maintaining human hookworms in animal models
and the cost of maintaining the hookworm in laboratory-canine
model.
2. The absence of an laboratory animal that is permissive to human
hookworms and can accurately reproduce human disease (anemia).
3. Paucity of in vitro functional tests to determine the effectiveness of
the immune response induced by an experimental hookworm
vaccine.
4. The lack of a protective immune response in humans and the
consequent absence of Correlates of Protection that can guide the
discovery of vaccine antigens and be used to assess their
effectiveness in preclinical and clinical trials.
5. No model of an effective immune response in humans to determine
the biological effect of the vaccine in humans

45. HELMINTHIC VACCINES: HOOKWORM
• Human Hookworm Vaccine Initiative (HHVI) is
the only group currently working on vaccines
targeting this parasite.
• Ancylostoma Secreted Protein-2 of N.
americanus (Na-ASP-2) is a 21 kDa protein
that is secreted by infective hookworm larvae
upon entry into the host.
• Na-ASP-2 was chosen as a lead hookworm
vaccine candidate in 2007-8.

46. HELMINTHIC VACCINES: HOOKWORM
• In a phase 1 study in hookworm-naïve adults living in the
US, Na-ASP-2 adjuvanted with Alhydrogel (wet gel
suspension of alum) was well-tolerated and immunogenic.
• However, a phase 1 safety and immunogenicity trial of this
vaccine in healthy adults from a hookworm endemic area in
rural Brazil has to be halted when 3 participants developed
immediate, generalized urticarial reaction.
• The urticarial reactions were associated with elevated
levels of IgE antibodies specific for Na-ASP-2, which were
present before immunization most likely due to previous
hookworm infection.

47. Present Focus
APR: Aspartic Protease (Haemoglobinase): Antibodies will block
haemoglobinase lining the digestive tract of parasites.
GST: Glutathione S Transferase: Antibodies will block detoxification of
host heme.

48. HELMINTHIC VACCINES : HOOKWORM
• Sabin Vaccine Institute announced the start of
Part II of its Phase I clinical trial of the Na-GST-1
vaccine candidate in November, 2012.
• Part II of the trial commenced in Americaninhas,
Brazil, following successful vaccinations in Part I
of the study, which began in Belo Horizonte,
Brazil in late 2011.
• Ultimately, Na -GST-1 and Na -APR-1 would be
used together a bivalent vaccine.
• Aim of the vaccine will be to reduce moderate to
heavy infections in the host.

49. HELMINTHIC VACCINES:
SCHISTOSOMA
• Only one schistosome antigen has entered into clinical
trials.
• The Institut Pasteur has taken a recombinant 28 kDa
GST cloned from S. haematobium through both phase
1 and 2 clinical testing in Europe and West Africa
(Senegal and Niger).
• Sh28-GST (Bilhvax) is a recombinant protein formulated
with an aluminum hydroxide adjuvant .
• Bilhvax appears to be immunogenic and well-tolerated
in healthy adults from non-endemic (France) and S.
haematobium endemic areas in Africa.

50. HELMINTHIC VACCINES:
SCHISTOSOMA
• The most important vaccine target of the
schistosome is the tegument.
• The tegument is thought to be involved in several
key physiologic processes: parasite nutrition,
osmoregulation, and the evasion of host
immunity.
• Tetraspanins found in outer tegument play
important role in maintaining the integrity of the
tegument.
• Sm-TSP-2 has been selected by the HHVI for
development as a human vaccine antigen.

51. HELMINTHIC VACCINES:
SCHISTOSOMA
• The Sm -TSP-2 recombinant schistosomiasis
vaccine would be intended primarily for
school-aged children living in the S. mansoni
endemic regions of sub-Saharan Africa and
Brazil.
• The vaccine ideally would prevent the
reacquisition of schistosomes in the blood
stream following initial treatment with
Praziquantel (vaccine-linked chemotherapy).

52. Veterinary (transmission-blocking)
vaccines
• Field trials of the EG95 vaccine against echinococcosis
in sheep are currently underway in the Patagonian
region of Argentina.
• Independent vaccine trials for Taenia solium carried
out in pigs with the TSOL18 antigen in Mexico, Peru,
Honduras, and Cameroon have all achieved 99–100%
protection.
• Results were published of the first field trial of the
TSOL18 vaccine, which was carried out in north
Cameroon. The vaccine completely eliminated the
transmission of T. solium by the pigs involved in the
trial .

53. CONCLUSION
• In addition to the technological hurdles, the economic
challenges have until very recently discouraged the
multinational pharmaceutical companies from
embarking on parasite vaccine R&D.
• Product development- Public Private Partnership (PDPPP) are non-profit organizations that use industry
practices or partner with industry for purposes of
developing, manufacturing, and clinically testing
vaccines . Because of their non-profit status, they
attract private and public donor support
• International Vaccine Institute (S.Korea), Sabin
Institute, MSD Wellcome Trust Hilleman Lab (India) etc.

54. CONCLUSION
• The availability of funds has speeded up the
process of vaccine development for many
neglected diseases.
• To pursue vaccine manufacture through
partnerships with innovative developing
countries (IDC). IDCs are middle-income
countries, such as Brazil, Cuba, China, and India,
with modest economic productivity but which
have achieved a high level of innovation in
biotechnonology.