Fighting back

The immune response to Leishmania and vaccine development

Different people respond very differently to the Leishmania parasite. Understanding why will greatly aid vaccine development.

Leishmaniasis comes in many forms. These range from a spontaneously healing skin ulcer to a visceral condition that kills within one or two years. The big question is, what decides the severity of the outcome – the host or the parasite?

Whether the parasite is killed or not depends on the tussle between the host's immune response and the parasite's virulence. And while a strong immune response may seem like the best course of action, it can actually be just as dangerous as the parasite.

Face off

The remote Andes and the jungles of Peru and Bolivia are the spectacular backdrops for a research project led by geneticist Dr Marie-Anne Shaw of the University of Leeds. Her research is aimed at cracking the mystery of mucocutaneous leishmaniasis, a grossly disfiguring condition. "The nasal and pharyngeal mucosa suffer serious, potentially irreversible, long-term damage," says Dr Shaw. "It is expensive and difficult to treat."

The causal agent is the South American species L. brasiliensis. Initially, it provokes a localised lesion that may self-heal or may need treatment. Oddly, only 10 per cent of people with an infection go on to develop mucocutaneous disease. "What causes the damage," explains Dr Shaw, "is an exaggerated immune response – very high levels of cytokines."

Cytokines are the messenger molecules that orchestrate the body's immune response. They coordinate the activities of a wide range of immune cells, in response to a threat from an external agent such as a parasite.

But why do some people launch an immune attack sufficient to clear the parasite while others mobilise the proverbial sledgehammer? Part of the answer may lie in people's differing genetic make-ups. Dr Shaw has piggybacked on fieldwork carried out by scientists at the London School of Hygiene and Tropical Medicine at three sites in Peru and Bolivia. From the thousands of people included in the surveys, Dr Shaw selected more than 200 that fulfilled the criteria for mucocutaneous leishmaniasis. Her intention is to find if there are genetic factors linked to the development of mucosal disease.

The genes that govern the immune response are the obvious first choice, and Dr Shaw has found some associations with particular alleles, which she believes could be worth pursuing. "Testing a limited number of single nucleotide polymorphisms is only the beginning. An initial association is only a lead to follow up," she adds. If it turns out that there are major genes controlling susceptibility to mucocutaneous leishmaniasis, however, those individuals who are at risk could be treated at the time of the initial lesion, cutting short its insidious progression.

Sorting the cytokines

Dr Ingrid Müller, at Imperial College in London, is also keen to solve the riddle of disease severity. Rather than people, she is studying the disease in mice.

Inbred mouse strains vary in their susceptibility to Leishmania, and Dr Müller has adopted the 'healer' and 'non-healer' strains that originally helped to expose the immune system as the guilty party. What is known is that resistant mice show a good T helper 1 response, while susceptible mice veer towards a Th2 response (see Puppy dog tales).

Dr Müller set out to track – and eventually stop – these damaging Th2 cells. Her strategy is to follow immune cells that have the T1/ST2 molecule on their surface, as these have been implicated in the control of Th2 responses. As she suspected, Th2 cells were heavily involved in the non-healing process. But a first attempt to neutralise these cells with antibodies did not turn them into 'healers'. A cleaner answer will emerge from looking at mice genetically engineered to lack Th2 cells. And indeed, Dr Müller's preliminary results are encouraging: with no Th2 cells milling around, Leishmania infection is mild.

"After establishing these animal models, our plan is to look into human leishmaniasis to find if there are cells expressing this marker. Because if Th2 cells are contributing to the non-healing form, counteracting these cells could manipulate the negative aspects of the disease. But there's a long way to go," Dr Müller stresses.

Professor Jennie Blackwell, a Wellcome-funded scientist at the Cambridge Institute for Medical Research, is also on the hunt for genes affecting human responses to Leishmania. By looking at affected families from Brazil and Sudan, she has found that polymorphisms in the SLC11A1 (formerly Nramp1) and IL4 genes influence susceptibility to disease. Genome scans of these families identified major regions on chromosomes 1, 6 and 17 that carry susceptibility genes for this pathogen, as well as genes that regulate the host's immune responses against them.

The IL-4 enigma

If a cure depends on crushing the Th2 response, one would assume that targeting IL-4, a key cytokine made by these cells, would be a good idea. Yet for some time, immunologists have puzzled over IL-4 – no one could agree whether it really was the 'bad guy'. In a bid to resolve this conundrum, Dr Frank Brombacher, a Wellcome International Senior Research Fellow in South Africa, has studied a stream of increasingly sophisticated gene-deficient mice.

In a collaboration funded by a Wellcome Trust grant, he genetically engineered a mouse that lacked the IL-4 gene. "We showed that IL-4 is a bad factor," says Dr Brombacher. "If we knock out IL-4, the mice become resistant to disease."

But pigeonholing IL-4 as the villain was not full story. Under different circumstances IL-4 can protect the host. "IL-4 has a dual function, both positive and negative," Dr Brombacher confirms. For example, for a vaccine to induce an effective immune response against visceral leishmaniasis, it must stimulate IL-4 production. (In fact, what is needed is a cocktail of CD8 T cells primed by IL-4, acting in concert with macrophages, complement and natural antibodies.) Dr Brombacher has now produced other IL-4-deficient mice, for example animals in which only T cells lack it, to try to decipher once and for all how IL-4 plays its dual role.

Genome to vaccine

Leishmania has so far resisted every effort to develop an effective vaccine. With the genome sequences of both human and parasite now available, this is an exciting time, as the choice of leads for potential vaccines has rocketed. The good news is that there are several vaccine preparations currently being tested.

Professor Debbie Smith, whose group recently moved from Imperial College in London to the University of York, has been working on one promising candidate, in collaboration with professor Paul Kaye. It is a protein called HASPB (hydrophilic acylated surface protein B) found on the parasite's external coat. Crucially, it prompts an infected animal to produce protective antibodies.

Professor Smith arrived at this molecule by screening different gene libraries, pulling out only those proteins unique to the infective stages – the obvious stage to intercept. So far, the HASPB protein has protected mice from infection and the vaccine is now being tested in dogs. "We still don't know exactly what the protein is doing, but we do know that it is an excellent vaccine candidate for leishmaniasis," comments Professor Smith.

The Blackwell group has used genomic approaches to identify new vaccine candidates for leishmaniasis. Using microarrays to analyse the expression of more than 1000 genes, the group has identified 147 new genes coding for proteins that are expressed in the stage of the parasite that multiplies in macrophages in humans. Each of these 147 proteins is a potential new vaccine candidate. By screening 100 of these candidates using DNA vaccination in mice, the group has identified 14 new protective vaccines for leishmaniasis. Professor Blackwell plans to take these candidates into vaccine trials in humans and reservoirs of disease.

As a result of the genome projects, researchers now have the information to shed light on the different types of disease. "The outcome of infection is an interplay between parasite factors, host factors and host genetics," says Dr Müller. "Now [with the sequencing of the Leishmania genome] we are on the cusp of being able to answer what the parasite contributes to all that. It's complex but it's exciting."

Adventure travel

Holidaymakers travelling to the Mediterranean could be taking home more than a tan – they could be harbouring a Leishmania parasite. Leishmaniasis is not only a tropical disease: it is still endemic in Mediterranean countries.

The soaring popularity of adventure travel, moreover, is also bumping up the number of tropical infections in the UK. In October 2004, the BMJ reported the cases of young travellers who developed chronic mucocutaneous leishmaniasis after spending time in the Amazonian rainforest. A prompt diagnosis ensured they received antimonial, drugs clearing the infection and avoiding any disfiguring lesions.

Also at risk are US troops based overseas. In Colombia, where soldiers are fighting the Revolutionary Armed Forces of Colombia, about 2500 US soldiers were hospitalised with leishmaniasis in 2004, up more than threefold from 2003 and more than double the 1150 injured in combat. Hundreds of troops in Iraq were shipped home, suffering from the notorious ‘Baghdad boil’.

Curing cattle

Sleeping sickness is not solely a human disease – it afflicts cattle too. The trypanosome has a devastating effect on communities in sub-Saharan Africa, where it infects as much as a third of livestock. With no vaccines and few drugs on the horizon, a group of researchers is focusing on a breed of African cow that is naturally tolerant to the parasite. Their aim is to reveal why these animals resist infection and to use these findings to breed ‘trypanotolerant’ cattle.

Sleeping sickness in cattle (or nagana) is fatal. Without treatment, 99 per cent of infected cows die. But it has long been known that a few indigenous African cow breeds, such as the N’dama breed, tolerate the parasite’s presence remarkably well. However, these cows are not popular with farmers because they grow slowly and are small. Farmers prefer the Boran cattle, which are more beefy but susceptible to sleeping sickness.

In a collaborative project with the International Livestock Research Institute in Kenya, Wellcome Trust-funded geneticist Professor Steven Kemp and colleagues in Liverpool are using functional genomics tools to discover why T. congolense, the main trypanosome infecting cattle, is lethal to some animals and not others.

The project began in 1989 when two cattle breeds were crossed in conventional genetic studies. Eventually, 127 animals and their parents and grandparents were genotyped. The scientists applied a genetic analysis called quantitative trait loci (QTL) mapping to identify areas of a chromosome that might be contributing to a positive trypanotolerant effect.

They found several QTL on different chromosomes that contribute to the three major tolerance indicators: anaemia, body weight, and parasite load.

This is hardly the end of their genetic pilgrimage. “From knowing the region, we still need to identify the genes,” says Dr Harry Noyes, who is part of the Liverpool team. Each of the regions identified by QTL mapping contains thousands of genes, but only 20 or so are likely to control the response to infection. Although the study began in 1989, results were published in 2003: “That gives you an idea how long it takes to do an experiment with farm animals,” Dr Noyes points out.

Breeding is faster with smaller mammals, and Professor Kemp and collaborators have been performing parallel experiments in mice to single out critical trypanotolerance genes – strains of mice also differ in their susceptibility to infection with T. congolense. In their first set of results, they identified three chromosomal areas that account for most of the genetic variation in susceptibility.

“We use mice not because we think the same genes are important, but because the same mechanisms may be at work in cattle,” Dr Noyes points out. Unexpectedly, the results have hinted at mechanisms that are related to oxidative stress and not under the control of the immune system.

The team is now zooming in, to identify single nucleotide polymorphisms (SNPs) – places in the DNA where the two breeds differ by one base pair – that may be associated with parasite tolerance.