Hidden in tropical regions, there exists an ancient plague that can “… eat away your nose and lips until they fall off and leave your face an open weeping sore” (1). This plague is Leishmaniasis, a thousand-year-old human disease that has repeatedly proven its resilience to effective treatment or prevention. In the present day, Leishmaniasis rates have increased worldwide, with 12 to 15 million people infected and 1.5 to 2 million new cases every year. With the advent of modern gene technology, scientists are currently focusing on finding a vaccine to Leishmaniasis, yet the road to such a cure has had a winding path that has not yet ended.
Leishmaniasis is a disease caused by the protozoan parasite Leishmania and is endemic to hot areas with its sandfly vector. The parasite, which comes embedded in the sandfly’s guy, goes into the bloodstream of its human victim and rapidly replicates inside of host macrophages. It comes in three forms: visceral, cutaneous and mucocutaneous forms with the most common and mild form being cutaneous Lesishmaniasis. They all begin with skin lesions that cause scarring, yet the other two less common forms are more destructive and cause more extreme symptoms to humans since they involve the parasite’s spread to other areas of the body. Taking a reactive approach to the disease by treating cases when they appear rather than preventing them is not ideal Current treatments consist of toxic, expensive and often ineffective chemotherapeutic drugs, and even with treatment, the scars or facial disfiguration caused by Leishmaniasis will still leave a person with “serious disability or stigma” (2;3). Since the consequences of contracting such a disease are high, a proactive approach – one that utilizes vaccination or effective prevention – is currently being focused upon.
A particularly intriguing route of vaccination research followed the path of disabling a surface virulence factor of Leishmania. Scientists have known about a suspected surface virulence factor, the lipophosphoglycan molecule (LPG), for many decades. Among other factors, the LPG helps Leishmania survive the sandflies digestive enzymes, modulate the immune response of the vertebrate host, and protect the parasite from lysosomes (4). LPG was confirmed to be a distinct virulence factor in 2000 when it was demonstrated that removing the LPG would remove virulence, and re-adding the LPG would restore virulence (5). The discovery of the LPG and its seemingly ubiquitous function in its ability to persist in the sandfly and macrophage inspired some researchers to take a different approach towards vaccine development.
The early attempts at a Leishmaniasis vaccine involved the standard inoculation and training of the secondary immune system using heat-killed parasites or proteins, but no evidence that the doses offered any more protective immunity than the control was found (6). With that line of research towards a vaccine proven to be not fruitful, researchers began to look into a live vaccine. Such a vaccine has been hypothesized since 1993, with the discovery that virulent Leishmania persisted in miscellaneous cells inside the body after infection and is associated with lifelong immunity (7). However, a live vaccine was not possible at the time, since injecting live Leishmania into humans is dangerous and the dormant Leishmania has been known to resurface in immunocompromised individuals (7). Therefore, any effective live vaccine must be able to stay dormant in the miscellaneous cells in the body and not resurface by rapid replication within macrophages. Around 1998, researchers had identified four genes involved in LPG synthesis by creating lines of LPG-deficient L.major (the most common strain of Leishmania) via mutagenesis (8). It seemed plausible that knocking out some combination of these genes would inhibit LPG synthesis, and theoretically, create an avirulent strain that could serve as a live vaccine.
The breakthrough came in late 2003 when researchers found that when one of the genes, LPG2, was knocked out in L.major, the knockout mutant was able to stay dormant and confer immunity to the host while being unable to survive in macrophages (9). Even better, in 2004, it was found that when this knockout mutant was already present in mice, it conferred “dramatic protection” from any attempted infection with wild-type virulent L.major without any strong immune response from the host(10). This seemed like a promising candidate for a live vaccine, since the removal of the virulence factor should theoretically create an avirulent strain. Unfortunately, the road to a vaccine took a turn.
Later in the same year, a population of knockout mutants was identified as able to partially revert back to the wild-type virulent strain by some unknown “compensatory mutation” that allowed for the L. major to survive in macrophages and replicate, which after some delay, created symptoms of Leishmaniasis in the lab rats (11). The presence of a population of LPG2 knockouts that could revert back to its virulent behavior means that LPG2 knockouts cannot be used as a safe live vaccine. While this result may seem like a setback, it could also open up an entire line of scientific inquiry into this “compensatory mutation” that can help scientists obtain a better grasp on Leishmania and may contribute to future vaccine efforts.
The three-decade long attempt of manipulating the LPG to create a live vaccine shows the winding and unpredictable nature of vaccine research. Who would have guessed that some LPG2 knockout mutants would revert back to wild-type? In the more recent past, many more potential avenues towards vaccine development have emerged including immunotherapy and DNA vaccines, but as of 2018, none have shown effective in humans (12). Genome modification has advanced greatly since 2003, with the more efficient CRISPR-Cas9 system supplanting homologous gene replacement for creating knockout mutants, which may allow for more findings in the near future. However, Leishmania still is not fully understood and oftentimes, as we could see in the case of the revertant LPG2 knockouts, there are factors influencing its virulence that are still unknown. Even in very promising cases with testing animals, the human testing process is long, expensive and risky. As a result, the timeline for a Leishmaniasis vaccine is difficult to predict, since future discoveries and irregularities might simply yield more questions.
Edited by: Katrianna Urrea
Illustrated by: Eugenia Yoh