From killing 19th century chickens to protecting today’s humans.
In the late 19th century, Louis Pasteur (1822-1895) – a French microbiologist – performed experiments that changed the world of vaccines as we know it today. In 1879, he started his first experiment, which was initially designed to test the fatal mechanisms of fowl cholera. Pasteur often examined the virulence (ability to cause a disease) of his laboratory grown cholera bacteria by inserting them into chickens every few days. The insertion of these carefully cultivated microorganisms was generally devastating for the birds. Until he went on holidays, because Pasteur did not refresh the bacterial cultures during that time.
When he came back, he resumed his experiments with the grown-old bacteria cultures, but unlike before his break, the chickens survived easily. When he subsequently inoculated the chickens with a fresh, virulent strain, they miraculously survived, because they had become immune to the disease causing bacteria. So the first lab-grown vaccine was found by accident.
This vaccine was an important follow-up of the first ever vaccination, which was performed by physicist Edward Jenner in 1796. Jenner successfully vaccinated an eight-year-old boy against smallpox by inoculating him with the less virulent cowpox from a young dairymaid. Largely based on Jenner’s findings, Pasteur explained that the virulence of the cholera microbe – and any pathogen – was diminished by a different mode of culturing. He confirmed this hypothesis multiple times, using both other bacteria (anthrax, in sheep) (1) and viruses (rabies, in human subjects).
Louis Pasteur’s experiments even helped develop a promising malaria vaccine…
What is malaria?
Malaria is a prevalent infectious disease that affects more than 200 million people worldwide. The disease is mainly caused by the transmission of a particularly dangerous malaria species, the Plasmodium falciparum protozoa. This protozoa parasite is carried by infected Anopheles species and consequently can infect humans through mosquito bites. The process of infection is shortly described below, with the protozoa taking on different names during different phases ( sporozoites, merozoites and gametocytes). When a person is bitten, falciparum protozoa in the form of sporozoites enter the bloodstream (see figure below), travel to the liver to develop into merozoites, go back to the blood stream and infect the red blood cells. The infection of these cells cause terrible symptoms (as mentioned below). After development in the red blood cells, some falciparum falciparum protozoa develop into gametocytes and are sucked up by another Anopheles mosquito, where they induce the development of new sporozoites to close the cycle.
Shortly after infection, falciparum malaria can cause several symptoms, including breathing problems, liver dysfunction, shock and even death. In fact, this terrible parasite accounts for 650’000 deaths each year, which is more than 90% of global malaria deaths. In order to prevent the spread of the disease, several options are currently used, such as mosquito nets, rapid diagnostic tests and antimalarials. However, to significantly reduce death toll of this disease, vaccine development seems one of the most promising options for the near future. More about the epidemiology, disease mechanisms and common treatments are described in Africa vs. malaria: the tables are turning.
Malaria: A variety of vaccines
Vaccines prepare the body for a real infection. They instruct the immune system how to combat specific parasites. Especially the immunological memory is important, for this can elicit quick and profound immune responses against specific parasitic molecules. However, in malaria it has been difficult to design functional vaccines. Currently, the two most promising malaria vaccines (which are discussed later) will be one of the following types:
(i) The subunit vaccine: This is the most commonly used vaccine. It contains up to 20 molecules (or antigens) that can be found on the surface of the parasite. A common example is the hepatitis B vaccine.
(ii) The second variant is the live, attenuated vaccine, which is a whole parasite vaccine that most closely resembles the real infection. This kind of vaccine is a weakened version of the virulent microorganism as was discovered by Pasteur.
Fresh findings about a promising malaria vaccine
Recently, a small-scale clinical trial by Seder and colleagues tested a safe and promising vaccine against malaria. This Plasmodium falciparum sporozoites (PfSPZ) vaccine is directed against malaria parasites, which are in the pre-erythrocytic phase (before red blood cell infection (see figure above)). The vaccine aims to prevent the early development and symptoms of the disease. This whole parasite vaccine is derived from parasite carrying Anopheles mosquitoes that are exposed to radiation. This weakens the virulence of the Plasmodium falciparum parasites. After the acquisition of radiation-attenuated falciparum sporozoites, they were purified, sterilized and preserved at very low temperatures. After melting, the researchers injected the live, attenuated vaccine in the veins (intravenous) of human subjects.
The safety and efficacy of the vaccine was measured by inoculating human subjects several times with different doses (from 7’500 to 135’000) harmless falciparum sporozoites. Three weeks after the latest vaccination (or immunization), subjects were infected by a so-called controlled human malaria infection (CHMI) to test the strength of the vaccines. The vaccines themselves were relatively safe, only a few subjects showed adverse reactions. However, most tested vaccines did not protect the subjects against the actual malaria infection. Multiple inoculations of 135’000 irradiated sporozoites, which protected 6 out of 10 patients (60%) after four immunizations and, amazingly, 6 out of 6 patients (100%) after five immunizations. In the remainder of the Seder’s article, groups receiving immunizations of 135’000 sporozoites are considered as protected.
Compared to unprotected subjects, the protected subjects produced more immediate immunological responses, as indicated by the increased presence and activity of naive γδ T cells. These T cells presumably contribute to protection by encountering falciparum sporozoites in the liver, since both the naïve T cells and the sporozoites prefer this organ. After stimulation by CHMI, these naïve T cells can activate other components of the immune system by producing large amounts of IFNγ. This is a crucial molecule in the immunologic battle against falciparum sporozoites.
Another characteristic of the successful PfSPZ-specific vaccines is the induced immunological memory, which is indicated by the formation of PfSPZ-specific memory T cells. In protected subjects, these specific lymphocytes are formed after multiple inoculations with attenuated sporozoites (see dark green T cells in figure below). After CHMI, these memory cells quickly produce many PfSPZ-specific effector (CD8) T cells or PfSPZ-specific helper (CD4) T cells, depending on the nature of the memory cell. CD8 T cells specifically attack sporozoites, whilst the CD4 T cells assist antibody-producing B cells and CD8 T cells. Both CD4 T cells and CD8 T cells produce a great amount of IFNγ.
PfSPZ vaccine vs. other malaria vaccines
Together, this small-scale clinical trial shows that five vaccinations with 135.000 attenuated Plasmodium falciparum sporozoites (PfSPZ-vaccine) can bring about the very high unbelievable protection rates of 100% against malaria infections in this small example. This protection is coincided with the increase of naive γδ T cells and PfSPZ-specific T cells, which all produce large amounts of a crucial molecule that helps the immune system protect against malaria; IFNγ.
In comparison, the lead candidate malaria vaccine is currently the so-called RTS,S vaccine, for it has been tested in large-scale clinical trials (with thousands of human subjects). Unfortunately, this vaccine only protects 30 to 50% of the subjects. Because the RTS,S vaccine is a subunit vaccine, it only trains the immune system against a limited number of infectious molecules (or antigens) on the falciparum sporozoite. The plasmodium sporozoites, however, display a wide array of antigens, which suggests that some variants can escape RTS,S-induced immunity. This complexity also explains why it is difficult to design a functional malaria vaccine. This is a major problem. The PfSPZ-vaccine, however, is an interesting solution, since it is a whole parasite vaccine that trains the immune system to combat many more antigens.
Challenges of the Pf-SPZ-vaccine
A number of challenges remain for the PfSPZ-vaccine. Firstly, to induce full protection against malaria in human subjects, almost 700’000 sporozoites are necessary. Sporozoites are derived from mosquitoes and deliver less than 100 sporozoites per bite, so it takes a lot of effort to collect enough attenuated sporozoites to vaccinate one patient. Therefore, either a method that lowers the required number of sporozoites to immunize, or a method that can obtain lab-grown sporozoites, is required. Secondly, the timespan of the protection is only measured after three weeks. More experiments are needed to uncover the effectiveness of this vaccine in the long term. Thirdly, it would be interesting to test the antibody responses against CHMI. These responses, which are caused by B cells, are essential in the battle against parasites. Finally, experiments should be repeated, using a large number of human subjects. When these challenges are overcome, this Pasteurian approach for a new malaria vaccine could save many lives in the near future.
Note 1: Pasteur tested his new hypothesis by conducting a large-scale experiment with 70 sheep: 35 sheep received two lab-grown attenuated anthrax vaccines, which consisted of bacteria that were less virulent than wild anthrax bacteria. The other 35 farm animals were control sheep and did not receive a vaccine. After a few weeks, all sheep received a virulent strain of anthrax: all control sheep died, whereas all vaccinated sheep survived. Based on these incredibly straight-forward results, successful attenuated vaccines were made against devastating diseases like Rabies and yellow fever.
References:
Seder RA, Chang LJ, Enama ME, Zephir KL, Sarwar UN, Gordon IJ, Holman LA, James ER, Billingsley PF, Gunasekera A, Richman A, Chakravarty S, Manoj A, Velmurugan S, Li M, Ruben AJ, Li T, Eappen AG, Stafford RE, Plummer SH, Hendel CS, Novik L, Costner PJ, Mendoza FH, Saunders JG, Nason MC, Richardson JH, Murphy J, Davidson SA, Richie TL, Sedegah M, Sutamihardja A, Fahle GA, Lyke KE, Laurens MB, Roederer M, Tewari K, Epstein JE, Sim BK, Ledgerwood JE, Graham BS, Hoffman SL, & VRC 312 Study Team (2013). Protection against malaria by intravenous immunization with a nonreplicating sporozoite vaccine. Science (New York, N.Y.), 341 (6152), 1359-65 PMID: 23929949
Good MF (2013). Immunology. Pasteur approach to a malaria vaccine may take the lead. Science (New York, N.Y.), 341 (6152), 1352-3 PMID: 24052298
Smith, K. (2012). Louis Pasteur, the Father of Immunology? Frontiers in Immunology, 3 DOI: 10.3389/fimmu.2012.00068
Hill A.V.S. (2011). Vaccines against malaria. Philosophical transactions of the royal society B, 366: 2806-2814
Breman J.G., Daily J. and Baron E.L. (2014). Epidemiology, prevention, and control of malaria in endemic areas. UpToDate
Pictures:
Breman J.G., Daily J. and Baron E.L. (2014). Epidemiology, prevention, and control of malaria in endemic areas. UpToDate
tcells.org
malaria, vaccine, pasteur, protect, infection, tcells, protozoa, falciparum, parasite
