human parasite vaccines

Human Parasite Vaccines

Dr Abhijit Chaudhury MD, DNB, D(ABMM)

SVIMS, Tirupati.

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.

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 protectionVaccine 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.

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)

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.


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.


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.


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.


Adjuvants Wrong choice of adjuvant may lead to

misleading experimental outcomes. Thus good candidate antigens may be

rejected.

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.

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.

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.

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.

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

MAJOR HUMAN PARASITIC DISEASES

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.

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.

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.

Malaria Vaccines- multiple approaches

1. Sporozoite subunit vaccination, especially with the CS protein: e.g. RTS,S in adjuvant.

2. Irradiated or genetically attenuated sporozoites

3. Immunization with DNA and/or viral vectors against the liver-stage parasites, or other life cycle stages.

4. Use of whole blood-stage malaria parasites .

5. Immunization with parasite adhesion ligands such as PfEMP1.

6. Use of peptide-based vaccines, mainly against blood-stage parasites—e.g. SPf66, PEV3a.

7. Development of anti-disease vaccines based on parasite toxins—e.g. GPI-based.

8. Immunization with sexual stage parasite antigens as transmission-blocking vaccines.

9. Use of mosquito antigens as transmission-blocking vaccines.

10. Use of protein in adjuvant vaccines to reduce the growth rate of blood-stage parasites

Clinical trials of malaria vaccines

Animal modelsSafety, immunogenicity, tolerability, efficacy

Safety, immunogenicity, tolerability

Non-immune human volunteers in non-malarious areas. Clinical setting

Human volunteers. Experimental challenge with infected mosquitos.Clinical setting

Phase IIa: non-immune volunteersPhase IIb: Immune volunteersVaccine efficacy, safety, tolerability, acceptance

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

Vaccine efficacy, safety, tolerability, acceptance

Vaccine efficacy, safety, tolerability, acceptance, vaccination strategy, effectiveness

Vaccine clinical trials are long term studies aimed at assessing the safety, efficacy and immunogenicity of a new vaccine product

PHASE 0Preclinical

PHASE 1Clinical

PHASE IIClinical

PHASE III

PHASE IV

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 C-terminal 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.


• 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.


• 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.


• 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%.


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 follow-up period

• Before vaccination, 34.3% of infants were positive for anti-circumsporozoite 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)


• 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).

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.

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.

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.

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.

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.

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.

Leishmania VaccinesSecond 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.

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.

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.

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.

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.


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.


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 .


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.

HELMINTHIC VACCINES: HOOKWORMPROBLEMS WITH HOOKWORM (+other HELMINTHS) VACCINE

DEVELOPMENT1. 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

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.

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.

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.

Present Focus

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.

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.


• 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.


• 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).

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 .

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 (PD-PPP) 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.

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.

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human parasite vaccines

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