The public health world has been abuzz recently with the results of the Phase I clinical trial of a malaria vaccine that proved 100% effective in protecting vaccinated people against Plasmodium falciparum infection when they were bitten by infected mosquitoes [1, 2]. P. falciparum is the species of malaria parasite that causes the most severe cases of disease – multiplying quickly in the blood and sometimes causing infected blood cells to clog blood vessels, which can lead to cerebral malaria and death [3]. As such, it is the most important species to vaccinate against. This new vaccine consists of inactivated P. falciparum administered intravenously five times. It is the culmination of years of research including previous iterations that were similar to this formulation, but ultimately less successful.
Sporozoites and the Malarial Life Cycle
The new vaccine consists of P. falciparum parasites at the sporozoite stage of development, but to know why that’s important we first need to understand P. falciparum’s somewhat complicated lifecycle. Despite the fact that each parasite is made of only one cell, Plasmodium goes through multiple stages of development as it moves through different steps of infection in both human and mosquito hosts.
Malaria transmission from mosquitoes to humans occurs when sporozoites are injected by the biting mosquito (Fig 1a) [4, 5]. The sporozoites then travel through the blood to infect the liver (Fig 1b). In the liver, each sporozoite multiplies into thousands of merozoites that are released into the bloodstream (Fig 1c). Merozoites are the parasite stage responsible for the nasty side effects of malarial infection – namely the high fevers. Merozoites infect red blood cells and replicate, bursting those blood cells and releasing many more merozoites into the bloodstream, which causes waves of fever and chills during infection. Some merozoites produce gametocytes when they multiply – this is the stage necessary for sexual reproduction, and each gametocyte contains only one set of genetic material instead of the two sets that the other parasite stages have (just as human egg or sperm contain only one set, whereas the other cells of our body have two sets, one from each parent). It is these gametocytes that are taken up by a mosquito (Fig 1d) and eventually produce new sporozoites that can cause another human infection.
Figure 1. Plasmodium lifecycle. (A) Infection takes place when the mosquito bites the human host, injecting sporozoites. (B) Sporozoites infect liver cells, where they proliferate and become merozoites, which in turn infect red blood cells. (C) In the red blood cells, the merozoites proliferate, bursting the cell and allowing infection of new cells. (D) Gametocytes are produced from the merozoites, and can be transmitted to mosquitoes that bite the human host. Over the course of 10-14 days, the gametocytes in the mosquito will produce sporozoites, which can cause a new infection once they localize to the mosquito’s salivary glands. Image from: Yannis Michalakis and François Renaud. “Malaria: Evolution in vector control” Nature 462, 298-300. Used with permission.
The contribution of inactivated sporozoites to produce a successful vaccine where other strategies have failed may be two-fold. First, since sporozoites are the first malarial stage that the immune system would see upon infection, developing a response to this stage of the parasite would be most effective in preventing an infection. Secondly, it’s functionally very helpful to have the whole parasite in a vaccine – our immune system senses danger through a combination of signals, and the more danger signals are present at once, the stronger that immune response may become. Another recent malaria vaccine that contained only one protein piece of the parasite, instead of the entire organism, was much less effective in preventing infection [6]. But of course when using whole parasites in vaccines, they must be weakened to prevent the vaccine itself from causing infection.
Inactivation by Irradiation
In this vaccine, sporozoites are attenuated, or weakened, by exposing malaria-infected mosquitoes to radiation. Radiation damages the DNA of the sporozoites so that they are alive, but they can no longer develop normally. They are able to go through the first steps of infection to infect a liver cell, but once there they can’t produce the burst of merozoites that would cause the symptoms of malaria infection. Interestingly, vaccinating with heat-killed sporozoites is not effective in protecting mice from malaria infection [7], indicating that it’s not only the whole parasite, but also the fact that it’s alive, that helps to generate a protective immune response.
Generating enough irradiated parasites for a vaccine is quite a production. Malaria parasites are grown in human red blood cells from donated blood in the lab. The mosquitoes are then infected by taking up the gametocytes from this blood culture of parasites about two weeks before the sporozoites will be harvested from the mosquito salivary glands. Once sporozoites have developed, the mosquitoes are exposed to radiation, and their salivary glands are surgically removed under a microscope. Of course all of this must be done under sterile conditions. Once the salivary gland material is purified to remove the contaminating mosquito bits from the sporozoites, the parasites are frozen in liquid nitrogen to later be used in vaccination.
Straight to the Vein
Notably, in this successful vaccine trial, fully-protected patients received five doses of the vaccine intravenously. Of the vaccinees who only received four doses, three of nine became infected, suggesting that all five doses are necessary for optimal protection. Perhaps more astonishingly, the protection offered by the vaccine only happens when it is injected intravenously, or straight into the bloodstream. When the same vaccine was injected subcutaneously or intradermally – into the skin instead of directly into a blood vessel – only two of 44 vaccinated people were protected from malaria infection [8].
In many of the developing areas where malaria is endemic and where many people may not have frequent access to healthcare, delivery of a vaccine in five separate doses is a difficult task. Moreover, delivery of that vaccine intravenously requires well-trained medical personnel and is far more time consuming to carry out than skin injections or the oral vaccination campaign that is currently waging the war against polio. Yet perhaps the greatest difficulty in rolling out this vaccine strategy will be the cold-chain that is required: sporozoites must remain frozen in liquid nitrogen prior to delivery, as they’re not stable at room temperature. Liquid nitrogen is very volatile and evaporates quickly, especially under hot conditions, which would obviously be an issue in warmer climates. Once the liquid nitrogen evaporates, the vaccine would warm up and become useless in short order.
A Thousand Mosquito Bites?
But there is good news! This protective vaccine, in only five doses, is far more pleasant to have administered than a previously studied treatment in which vaccine subjects were bitten by 1000-3000 mosquitoes over the course of biweekly immunization sessions (aka. irradiated mosquito feasts) [9]. It was this study that inspired the current successful trial, and if we can move from over 1000 mosquito bites to merely 5 injections, certainly the field of malaria vaccine development can overcome the current shortcomings of this very promising vaccine strategy.
Jamie Schafer is a graduate student in the Harvard Virology Program.
References:
[1] Declan Butler. “Zapped Malaria Parasite Raises Vaccine Hopes” Nature News. Aug 8, 2013. http://www.nature.com/news/zapped-malaria-parasite-raises-vaccine-hopes-1.13536
[2] Robert A. Seder, et al. “Protection Against Malaria by Intravenous Immunization with a Nonreplicating Sporozoite Vaccine” Science. Epub Aug 8, 2013.
[3] CDC. “About Malaria: Disease” Feb 8, 2010. http://www.cdc.gov/malaria/about/disease.html
[4] Yannis Michalakis and François Renaud. “Malaria: Evolution in vector control” Nature 462: 298-300.
[5] National Institute of Allergy and Infectious Diseases. “Life Cycle of the Malaria Parasite” Apr 3, 2012. http://www.niaid.nih.gov/topics/malaria/pages/lifecycle.aspx
[6] Cassandra Willyard. “Malaria Vaccine Results Present Infant Immunization Quandary” Nature Medicine: Spoonful of Medicine. Nov 16, 2012. http://blogs.nature.com/spoonful/2012/11/malaria-vaccine-results-present-infant-immunization-quandary.html#wpn-more-3910
[7] George L. Spitalny and Ruth Nussenzweig. “Effects of various routes of immunization and methods of parasite attenuation on the development of protection against sporozoite-induced rodent malaria” Proc. Helminthol. Soc. 39: 506–514.
[8] Judith E. Epstein, et al., “Live attenuated malaria vaccine designed to protect through hepatic CD8⁺ T cell immunity” Science 334: 475–480.
[9] Stephen L. Hoffman, et al. “Protection of humans against malaria by immunization with radiation-attenuated Plasmodium falciparum sporozoites. J. Infect. Dis. 185: 1155–1164.