PARASITE VACCINES

 

There is a strong argument in support of developing vaccines for parasitic diseases. Vaccines have a number of advantages over chemotherapy, in theory a single treatment gives life long protection. Chemotherapy requires repeated doses and for something lie malaria you may be taking the drug continuously for years: so there are worries about effects on the host of long term treatment and the accumulation of drug residues in tissues and milk. Long term drug treatment can also be expensive and drug resistance is widespread.

 

Initially the success of anti-viral and anti-bacterial vaccines suggested that vaccine development for parasitic diseases would be straight forward. However, to date there has only been very limited success in producing effective anti-parasite vaccines. Using a conventional approach lve attenuated vaccines have been produced for the protozoan Theileria and for the nematode Dictyocaulus. Live attenuated larvae are not very popular:

·        The stages have to be isolated form natural sources

·        Limited shelf life

·        Attenuation is critical

 

A vaccine against the dog hookworm, Ancylostoma caninum was developed in the USA in the 1970's, based on X-ray attenuated infective larvae. It was eventually withdrawn because of the small number of infections arising from the attenuated larvae. Subunit vaccines (based on a single recombinant protein have been developed against ticks (tickGARD). Initially it was found that you could vaccinate cattle using whole tick homogenates, the protective antigen was narrowed down to the gut, and eventually a single protein Bm86. It was released in Australia in 1994 after 12 years of development and trials involving 18,000 cattle. Production has now stopped because it was no longer a commercial proposition. Another sub-unit vaccine was developed against Taenia ovis in sheep. The adult tapeworm occurs in dogs, sheep are the intermediate host . In sheep the parasite develops into a cystic stage in the muscles, carcasses are condemned.. Once infected sheep show a strong resistance to re-infection. A recombinant vaccine gave 90+% protection in lambs, but it was never developed commercially because of the difficulty of stabilizing the recombinant protein.

 

What do you need to know in order to develop a successful vaccine?

·        First you need to understand the life cycle of the parasite to identify which is the best target stage. For example in malaria an anti-sporozoite vaccine would block infection and be very attractive An anti-gametocyte vaccine would block transmission, but not do much for the host.

·        Secondly you need to understand the immune mechanisms stimulated by the parasite. Is the effective response an antibody mediated response or is it primarily a cel mediated response or does it involve the innate immune response?

 

Despite the huge investment in time and money there is no commercial vaccine for a human parasitic infection. There are a number of reasons for this failure:

·        Parasites have strategies to avoid and confuse the host immune response - including things like non specific activation of B-cells, so the host produces large amounts of irrelevant antibodies, many parasites produce factors (possibly cytokine mimics) which down regulate the cellular response. Protein polymorphism, particularly of surface proteins so a vaccine against one serotype is ineffective against others. (fortunate in tickGARD that Bm86 showed very little polymorphism).

 

·        Much vaccine development is done using laboratory strains of parasites which may have become atypical and have lost their natural degree of polymorphism, laboratory passage itself selects for certain phenotypes (trypanosomes which have been kept in culture for many years loose the ability to develop in the tsetse fly.) You can also get 'founder effects' when the population is grown from a few individuals.

 

·        Parasite antigens are complex and difficult to characterise. A nematode may contain 20,000 different proteins, added to which may be post-translational modifications. Carbohydrate side chains are often important in determining antgenicity but such proteins are extremely difficult to analyse. The traditional approach to identifying potential antigens for vaccine development is Western Blotting. The parasite proteins are separated on a gel, blotted onto a membrane and the membrane probed with hyper-immune serum. Bands which give a strong  response are subsequently isolated and cloned. The problem is that whilst many proteins are immunogenic few of the antibodies are protective. Quite a lot of the proteins identified by Western Blotting turn out to be internal proteins which are probably released when the parasite die and have nothing to do with immunity

 

So the identification of protective antigens is extremely difficult. The other classical approach is to start by immunising with whole parasite extracts, if protection is achieved then fractionate the extract until the key protective antigen is found. This approach worked for tickGARD, but often it is the synergy between proteins that leads to protection and fractionation leads to a loss of efficiency.

 

Much of the research on parasite immunology is done with rodent models and the immune response of rodents are very different from those of man. For example in the rodent, immune killing of schistosomulae takes place primarily in the lungs, in man it takes place in the skin.

 

The immune reponse to parasites is often multi-faceted and involves a range of mechanisms, so it may be necessary to activate several different pathways. Different breeds of animals may respond differently. In sheep, for example, the Indonseaian thin tailed show a high resistance to Fasciola gigantica compared to European breeds, but is equally susceptible to Fasciola hepatica. It has been found that in Thin tailed sheep the oxidative burst of macrophages and neutrophils is much more intense than in other breeds. But F. hepatica has much higher levels of protective enzymes, particularly glutathione transferase than F. gigantica. So F. hepatica can cope with the oxidative burst but F. gigantica succumbs.

Vaccine development is expensive, registration, particularly for use in humans can be time consuming and difficult, so there has to be a sufficient market to cover this. With human vaccines there is always concern if anything goes wrong or is perceived to go wrong you may be sued.

 

Type of Vaccines

1. Killed whole organisms. In general killed organisms do not seem to work with parasites, although tickGARD started as whole homogenates.

2.Attenuated organisms. The key seems to be that the attenuated organism folows the same migration route in the host, but does not mature. (attenuated vaccines exist for Dictyocaulus, Theileria, Ancylostoma, T. ovis, Eimeria)

Interestingly infection with irradiated malarial sporozoites or with irradiated schistosome cercariae induces strong resistance, whereas natural infections do not (although there may be a slow acquired immunity). Why irradiation should increase immunogenicity is completely unknown, but probably involves changes in surface molecules.

3. Defined vaccines. These can be based on purified parasite proteins, or more usefully on recombinant proteins. Naturally isolated parasite antigens require infected animals to produce the parasites, natural antigens often show batch variation and cannot be easily produced in large amounts. In contrast, recombinant antigens are well characterised and can be produced consistently, in large quantities and relatively cheaply. A possible problem with recombinant antigens is if the target protein turns out to be polymorphic so the vaccines is ineffective against some strains of parasite. In addition a simple defined antigen may not stimulate all of the host immune components needed to give an effective response. There are possible ways round this:

·        Peptide vaccines. These are short synthetic sequences which cover the immuno-dominant epitopes . Being synthetic there is no danger of contamination with other proteins. An approach being tried with malaria is to incorporate a series of such epitopes, from different proteins, into a single construct. By choosing the epitopes it is possible to bias the immune response towards antibody production or a cell mediated response.

·        Adjuvants. A current area of intense interest is trying to manipulate the response to the vaccine. In particular trying to increase the response and also trying to steer it towards a cell mediated Th1 response rather than an antibody response. Adjuvants are used when the antigen is only weakly immunogenic or there are only small amounts of antigen available. It is not entirely clear how they work. In part they may act as depositories and slowly release the antigen over a period of time. Some antigens are known to bind to Toll like receptors on dendritic cells and macrophages. Activated dendritic cells and macrophages are more phagocytic than their unstimulated counterparts and express higher levels of the molecules that trigger co-stimulation and enhancement of the T-cell response. So in the presence of adjuvant antigen presentation and co-stimulaton signals are increased. Only one adjuvant is widely used for human use- alum (aluminium potassium sulphate) which probably primarily acts as a depot. Much more efficient adjuvants are used in animals, these are usually water in oil mixtures (Freund's). Many of these exerimental or veterinary adjuvants have side effects which make them unsuitable for human use.

 

4. Recombinant vector vaccines. Cell mediated responses occur in particular in response to intercellular antigens which are processed and presented on the cell surface in association with Class 1 MHC proteins. This MHC 1 pathway is usually activated by intracellular viruses or bacteria. Extracellularly applied vaccine antigens do not normally enter the cell and so di not enter the MHC 1 pathway, and so tend to produce an antibody rather than a cellular response. In recombinant vector vaccines, DNA encoding the major parasite antigenic determinants is introduced into the genome of an attenuated virus or bacterium which will replicate inside host cells and express the required gene product.

 

The most promising vector is attenuated vaccinia virus (cow pox). The vaccinia virus can be engineered to express several recombinant genes. Another possible vector is attenuated Salmonella, this is useful because it can induce mucosal immunity.

 

The advantage of recombinant vector vaccines is that they induce both a cellular and an antibody response. The disadvantage is that they may not be safe to use with immunocompromised individuals and you could develop immunity to the vector, so could not have a second vaccinations with the same vector.

 

5. DNA Vaccines. The principal behind DNA vaccines is in some way the same as recombinant vestor vaccines, by introducing a plasmid containing the immunodominant sequence into a cell, the antigen is produced intracellularly and enters the MHC 1 pathway thus provoking a strong cellular response. Prolonged expression of the plasmid encoded peptide means there should be no need for boosters.

 

DNA vaccines are usually circular plasmids encoding the target antigen  (or antigens) under the control of a promoter active in human cells. Delivery of DNA vaccines:

Direct injection of DNA into the muscle and the muscle cells take it up, often get poor results.

·        Gene gun. The plasmid is coated onto gold particles which are 'fired' into the skin by compressed air (uses much less DNA).

·        Complexed DNA. The plasmid is complexed with a carrier such as a liposome or a specific ligand which can target the DNA to a specific tissue or cell type.

·        Electroporation, using electric current to make cells permeable, used widely in bacteria, experimental stage in animals.

 

Advantages of DNA vaccines:

·        Safe, can be used in immuno-compromised individuals, no danger of contamination with foreign protein.

·        Protection should be long lived as the infected cells produce antgens continuously thus boosting to immune system.

·        DNA vaccines inexpensive to make.

·        DNA vaccines can generate a cell mediated as well as an antibody response.

 

But there are also potential problems with DNA vaccines:

·        Danger that immune cells will become sensitized to DNA and could result in auto-immune disease.

·        Foreign DNA could be incorporated into the host genome and could cause cancer.

·        Antibiotic resistance genes used in the generation of plasmids might 'escape'.

 

However all these methods -subunit vaccines, recombinant vector vaccines, DNA vaccines all depend on the identification of the key protective antigens and this remains a major challenge.