Malaria Vaccines: The Continuing Quest

The Continuing Quest

Irwin W Sherman

• Publisher: World Scientific Publishing Company
• Year: 2016
• eBook ISBN: 9781786340061
• Topic: Medicine
• Subtopic: Diseases & Allergies

In 2013, 200 million people were infected with malaria, resulting in over 584,000 deaths, with the potential to affect over half the world's population. Such is the widespread nature of malaria that it is increasingly believed only a vaccine will lead to its eradication.

Although the first attempt at a vaccine was made a century ago, it is only in the last 30 years that real progress in testing has been made, in the hope of discovering a molecule that can provide long-lasting protection against the disease. In July 2015, GlaxoSmithKline (GSK) announced that after 30 years of research it had received the green light from the European Medicines Agency for the world's first malaria vaccine, RTS, S, for use in African children aged 6 weeks to 17 months.

This book chronicles the development of RTS, S — done in collaboration with the Walter Reed Army Institute for Research, the PATH Malaria Vaccine Initiative (MVI) and funded in part by the Bill and Melinda Gates Foundation — as well as previous candidate vaccines. It also focusses on the continuing quest to find more effective vaccines against this continuing health crisis. Finally, it provides an easily understood background on recombinant DNA and monoclonal antibodies and places them in perspective to their contributions to malaria vaccine development.

This book serves as a convenient and easily accessible source of information for students, teachers, microbiologists, parasitologists, physicians, clinicians and research funders.

Contents:

• The Three Lives of the Malaria Parasite
• A Personal Scientific Odyssey
• Taming the Malaria Parasite
• The Quest for a Blood Stage Vaccine Begins
• Malaria Vaccines and Malfeasance
• Dreaming of A Nobel Prize
• Molecular Biology Assists in Vaccine Development
• Developing Vaccines Against Blood Stages
• PfEMP1, pfalhesin and DBR
• Vaccines to Halt Transmission
• Sporozoite Invasion and The Path to RTS, S
• Attenuated Plasmodial Vaccines
• Viruses and Plasmodial Vaccines
• Why the Quest Continues

Readership: Graduate students, teachers, microbiologists, parasitologists, physicians, clinicians, funders of research and readers interested in the research area.
Malaria;Plasmodium;Vaccine;Falciparum;Disease Key Features:

• Provides an easily understood background on recombinant DNA and monoclonal antibodies and places them in perspective to their contributions to malaria vaccine development
• Provides an up-to-date account of developments and insight into the creative scientific process
• Short biographical sketches tell the value of the contributions made by numerous investigators including the author
• Celebrates the successes and failure and places the reasons for optimism (or pessimism) in perspective

Chapter 1

The Three Lives of the Malaria Parasite

Historically, the most common experience with malaria was that it was a sickness associated with wetlands. Indeed, the French name paludisme is from the Latin “palus” meaning swamp.² The name of the disease, literally ‘bad air’ comes from the belief that it resulted from human exposure to the poisonous vapors (miasma) that had seeped in from the polluted soil. But by the middle of the 19^th century — a time when Louis Pasteur’s germ theory was in full flower — there were various reports of a sighting of the causative agent of malaria. In 1879 two investigators in Italy, Edwin Klebs and Corrado Tommasi-Crudeli, found a bacillus in the mud and waters of the marshes in the highly malarious region of the Roman Campagna.² These bacteria when injected into rabbits resulted in fever, and the spleen was enlarged. They named the bacillus Bacillus malariae.

In the United States, the U.S. Board of Health commissioned Major George Sternberg, a well-trained bacteriologist, to try to repeat the experiments of Klebs and Tommasi-Crudeli in the malaria affected area around New Orleans.² He found bacteria similar to B. malariae in the mud from the Mississippi delta; however, after the bacteria were injected into rabbits the fevers produced were not typical of malaria in that they were not periodic. Sternberg was also able to produce the disease in rabbits by injecting them with his own saliva. He concluded that the disease was septicemia, not malaria, and suggested the bacteria were contaminants. What then was the cause?

i. Laveran’s Animalcule

One of the signal characteristics of malaria in cadavers is the enlarged and blackened spleen and liver. This discoloration is the result of the accumulation of a brownish-black pigment. In 1847 the German pathologist– psychiatrist Heinrich Meckel, after observing the accumulation of pigment in the blood of a malaria patient with an enlarged spleen, proposed that the pigment itself, called hemozoin, was the cause of the disease.³ L. F. Achille Kelsch (1841–1911), a pathologist working in malaria-ridden Algeria had been drawn to the pigment, and like Meckel he observed that hemozoin was almost always contained in the white blood cells but occasionally was enclosed in a clear body. In the main, Kelsch studied malaria in autopsied material but on occasion was able to examine blood from patients living with malaria and in almost all cases there was the telltale pigment. Although he recognized the presence of pigment as diagnostic for malaria and even found that the pigment appeared in the blood at the time of fever, Kelsch did not discover the causal agent itself.² Why? As a pathologist Kelsch looked at only dead material, and even freshly drawn blood would routinely be ‘fixed’ before microscopic examination: hence his failure.

Charles-Louis Alphonse Laveran (1845–1922) studied at the Public Health School in Strasbourg where he received his medical degree (1867). In 1874 after a competitive examination he was appointed Chair of Military Diseases and Epidemics at the Ecole du Val-de-Grace in Paris.⁴ At Val-de-Grace Laveran became well acquainted with several physicians who had previously worked in the French territory of Algeria studying malaria. One in particular, L. F. Achille Kelsch exercised a profound influence on the young Laveran teaching him about the characteristics of malaria. Laveran has been described as “bespectacled with sharp features and a small trim beard.” He was reputed to be extraordinarily precise, meticulous, singularly sharp-minded, incisive, and self-opinionated.⁵ In short, he did not suffer fools gladly. Initially, in Algeria, Laveran spent much of his time looking at autopsy material (as did Kelsch) but he also examined fresh specimens. His microscope was not a good one and he used no stains but he was patient and determined. On November 6, 1880 while examining a drop of fresh blood, liquid and unstained from a feverish artilleryman he saw several transparent mobile filaments — flagella — emerging from a clear spherical body. He recognized that these bodies were alive, and that he was looking at an animal, not a bacterium or a fungus. Subsequently he examined blood samples from 192 malaria patients: in 148 of these he found telltale hemozoin-containing crescents.⁶ Where there were no crescents, there were no symptoms of malaria. Laveran also found spherical bodies in or on the blood cells of those suffering with malaria all containing malaria pigment. He named the parasite Oscillaria malariae and communicated his findings to the Société Médicale des Hôspitaux on December 24, 1880. The drawings in his paper provide convincing evidence that, without use of stains or a microscope fitted with an oil immersion lens, Laveran had seen the development of the malaria “animalcule.”

Laveran was anxious to confirm his observations on malaria parasites in other parts of the world, and so he traveled to the Santo Spirito Hospital in Rome where he met with two Italian malariologists (one of whom was Ettore Marchiafava, Tommasi-Crudeli’s assistant and the other Angelo Celli, Professor of Hygiene) and showed them his slides. The Italians, whose chief interest was B. malariae, were unconvinced and told him that the spherical bodies he had seen were nothing more than degenerating red blood cells caused by B. malariae or some other cause.²

The vast majority of the medical world remained skeptical of Laveran’s findings. In 1883 Marchiafava and Celli claimed to have seen the same bodies as described by Laveran but without any pigment granules. They also denied the visit by Laveran two years earlier. The Italians were unsuccessful in growing the bodies outside the body of the malaria patient. This lack of consensus on the causative agent was due not only to differences in interpretation of what was seen with the microscope, it was a matter of focus: the Italians emphasized the smallest forms in the blood, whereas Laveran concentrated on the hemozoin-laden crescents and the whip-like filaments.² While the search for the parasite itself occupied most research during this period, there was a serendipitous finding of great significance: malaria was an infectious disease, but not one that could be contracted by simply being exposed to a patient with a fever, as would be the case in persons with influenza. In 1882, C. Gerhardt deliberately induced malaria for therapeutic purposes in two patients with tertiary syphilis by injection of blood from another patient suffering with intermittent fever, and then cured them all with quinine. A year later Marchiafava and Celli, working on the wards of Rome’s Santo Spirito Hospital, gave multiple injections intravenously and subcutaneously to five healthy subjects. Parasites were recovered from three of the five who came down with malaria; all recovered after quinine treatment. Clearly, it was the blood of a malaria patient that was infectious, not his breath.

In late 1884 or early 1885, Marchaifava and Celli abandoned their use of fixed stained smears and began to study, as had Laveran, fresh blood. Examining drops of liquid blood they observed the ameba-like movements of the parasite within the red blood cell and hence they called it Plasmodium (from the Latin ‘plasmo’ meaning ‘mold’). They also witnessed emerging whip-like filaments (called flagella) from the clear spherical bodies within the red blood cell although they questioned the significance of the flagella in the disease. The differences between the interpretations of Laveran and Marchiafava and Celli are now evident: for several years the Italians examined only dried stained specimens and so did not see any movement of the parasite that had caused Laveran to give it the name Oscillaria.²

Later, using both fixed, stained, and liquid preparations Marchaifava and Celli were able to trace the development of the small non-pigmented bodies within the red cell; they also described the hemozoin as the by-product of the destruction of the red cell’s hemoglobin by the growing parasite. In 1886 during a visit to Europe Major George Sternberg visited Celli at the Santo Spirito Hospital. Celli drew a drop of blood from the finger of a malaria patient and was able to show Sternberg the ameba-like movement of the parasite and the emergence of flagella.² Sternberg returned to the United States and working with blood taken from a malaria patient in the Bay View Hospital in Baltimore was able to find Laveran’s parasite in Welch’s laboratory at Johns Hopkins University. A year later Welch separated the two kinds of malarias with 48 hour fever peaks; one would be named P. vivax and the other he named Plasmodium falciparum because it had sickle shaped crescents (and ‘falcip’ in Latin means ‘sickle or scythe’).

In 1885, Camillo Golgi (1843–1926) of the University of Pavia convinced by the Italian confirmation of Laveran’s observations felt malaria parasites deserved further study.⁶ Examining the blood of 22 patients with a 72 hour fever cycle (later named P. malariae) he traced the tiny, unpigmented bodies of Marchiafava over three days until they grew to fill the red cell, and on the day of the fever paroxysm he found the pigment to concentrate in the center of the parasite as it divided. Golgi discovered that the parasite reproduced asexually by fission and correlated the clinical course of fever with destruction of the red blood cell to release the parasite. In 1886 when he noted that in both the 48 hour and the 72 hour fevers there were no crescents he effectively had distinguished the three kinds (species) of malaria based on fever symptoms. Marchiafava and his student, Amico Bignami, took Golgi’s studies of malaria a step further. Although cases of 48 hour and 72 hour fever cycle malarias occurred throughout the year in Italy, in the autumn and summer they were outnumbered by a much more severe 48 hour-type, called aestivo-autumnal or malignant malaria. Only later, would it be recognized that the malignant disease was caused by the malaria parasite with crescents, P. falciparum.

Clinicians in the United States, however, continued to be skeptical of the significance of Laveran’s discovery to malaria as a disease. William Osler, the premier blood specialist of his day, on hearing a paper presented at the inaugural meeting of the American Association of Physicians (June 1886) by W. T. Councilman, working in Welch’s Johns Hopkins University laboratory, in which he found flagellated parasites in 80 attempts, challenged his findings. Osler also questioned the role of the flagellated bodies of Laveran, finding them improbable and contrary to all past experience of flagellated organisms occurring in the blood. By late 1886, however, after verifying the existence of the parasites with his own eyes — and postponing his Canadian vacation to examine the blood of every malaria patient he could find — Osler became a convert to the doctrine of “Laveranity.” Osler published his findings on October 28, 1886 and in his 1889 treatise Hematozoa of Malaria. Henry Van Dyke Carter, a pathologist working at the Grant Medical College in India read Osler’s paper. Previously, VanDyke Carter had been unable to find the malaria parasite in the blood, but with Osler’s guidance he succeeded. Van Dyke Carter published his findings of three kinds of malaria parasites in India but for more than a decade his report received little notice among his colleagues in the Indian Medical Service. Despite this, malaria parasites were now being identified elsewhere: in Russia by Metchnikoff, by Morado and Coronado in Cuba, Anderson in Mauritius, and Atkinson in Hong Kong. By 1890 almost all the world believed in both the existence of Laveran’s “animalcules” as well as in their being the cause of the disease malaria. The significance of Laveran’s observation of the release of motile filaments (flagella) called exflagellation — would remain unappreciated, however, until William MacCallum and Eugene Opie of Johns Hopkins University made some critical observations.²

MacCallum and Opie were both medical students at Johns Hopkins when they followed up Laveran’s observations using the malaria-like parasites found in the blood of birds. In 1897, Opie described some of these parasites in wild caught birds. During that same summer MacCallum, on vacation outside of Toronto, Canada studied one of these “malarias” named Haemoproteus where the male and female sex cells (gametocytes) in the blood are clearly different from one another even in unstained preparations. This is unlike human malarias where the gametocytes are very similar in appearance. One type, the hyaline or clear form put out Laveran’s flagella, whereas the granular forms freed themselves from the red cell and remained quiescent. Observing the two forms in the same field under the microscope, he found the released flagella to invade and unite with the hyaline form to produce a wormlike gliding form. MacCallum immediately recognized that the flagella were sperm-like, that the granular forms were egg-like, and that he had witnessed fertilization to form a vermicule (later called the ookinete). On his return to Baltimore MacCallum confirmed his discovery in a woman suffering from subtertian (falciparum) malaria. In his 1898 publication MacCallum described the ookinete: “the movement is slow and even … with the pointed end forward. It can move in any direction readily … Often it is seen to rotate continually along its long axis. The forward progression … occurs with considerable force … pushing directly through the obstacle. The ultimate fate and true significance of these forms is difficult to determine.” But, then he incorrectly concluded: “it is reasonable to suppose … it is the much sought resistant stage.”⁷

ii. Miasma to Mosquito

Although Ronald Ross, a Surgeon-Major in the Indian Medical Service, was a most unlikely person to solve the puzzle of how humans “catch” malaria, he did so.⁵ Ross was born on Friday, May 13, 1857, in the foothills of the Himalayas where his father was an officer in the British Army stationed in India, which, at that time, was a part of the British Empire. As a boy of eight, his parents shipped him to England to receive a proper British education. He was a dreamer and, although he liked mathematics, he preferred wandering around the countryside, observing and collecting plants, and animals. At Springhill Boarding School, which he attended from the age of 12, he began to write poetry, painted watercolors and thought of becoming an artist. But, his father insisted him to study medicine in preparation for entry into the Indian Medical Service. Therefore, at age 17, young Ronald began his medical studies. He was not a good student, not because of laziness, but because he had so many other interests and could not concentrate on medicine. He preferred composing music to learning anatomy, and wrote epic dramas rather than writing prescriptions. Publishers rejected these “great works,” and so he had them printed at his own expense. He eventually did pass his medical examination (after failing the first time), worked as a ship’s doctor and then entered the Indian Medical Service. Although India was rife with disease — there was malaria, plague, and cholera — Ross busied himself writing mathematical equations, took long walks, wrote poetry, played the violin, and studied languages. Occasio nally he used his microscope to look at the blood of soldiers ill with malaria, but he found nothing. He shouted to all who could hear: “Laveran is wrong. There is no germ of malaria.”⁸

In 1894 Ross returned to England on leave. By that time, he had spent 13 years in India and had few scientific accomplishments: he wrote a few papers on malaria for the Indian Medical Gazette and claimed (without any real evidence) that malaria was primarily an intestinal infection. His hunt for the way malaria was transmitted from person-to-person began on April 9, when the 37-year old Ross visited with the 50-year old Patrick Manson at his home at 21 Queen Street in London.

Manson (1844–1922) received his medical training at the University of Aberdeen (1866) and then served as Medical Officer (1871–1873) to the British-run Chinese Imperial Maritime Customs Office in Amoy, a subtropical port in China.⁵ There he studied the transmission via mosquito of the worm that causes elephantiasis. Returning to England (1889) he developed a lucrative consulting practice and was also appointed Medical Advisor to the Colonial Office in London. At the time of Ross’ visit Manson was physician to the Seaman’s Hospital Society at Greenwich and a lecturer on Tropical Diseases at St. George’s Hospital and Charing Cross Hospital Medical Schools where he had access to malaria contracted by sailors and others in the tropical regions of West Africa and India. Manson was a dedicated and experienced clinician as well as an expert microscopist and he had been shown Laveran’s “animalcule” by H.G. Plimmer (1856–1918) of the University...

Table of contents

• Cover
• Halftitle
• Title
• Copyright
• Contents
• Preface
• Chapter 1. The Three Lives of the Malaria Parasite
• Chapter 2. A Personal Scientific Odyssey
• Chapter 3. Taming the Malaria Parasite
• Chapter 4. The Quest for a Blood Stage Vaccine Begins
• Chapter 5. Malaria Vaccines and Malfeasance
• Chapter 6. Dreaming of a Nobel Prize
• Chapter 7. Molecular Biology Assists in Vaccine Development
• Chapter 8. Developing Vaccines against Blood Stages
• Chapter 9. PfEMP1, pfalhesin, and DBR
• Chapter 10. Vaccines to Halt Transmission
• Chapter 11. Sporozoite Invasion and the Path to RTS, S
• Chapter 12. Attenuated Plasmodial Vaccines
• Chapter 13. Viruses and Plasmodial Vaccines
• Chapter 14. Why the Quest Continues
• References
• Index