Chapter one: HEPATITIS A and B----Viral Hepatitis A,B,C,D,and E.

A few years ago a friend developed hepatitis shortly after she returned from India. She was fatigued, had no appetite and her eyeballs were the color of ripe lemons. The exhaustion lasted a month. Then she gradually improved. She had been vaccinated for hepatitis A and was careful about what she ate, but she must been served food prepared by a local who had a mild case of the infection. The strain of hepatitis that attacked her liver isn’t often seen in the U.S. or Europe, and there’s no U.S. vaccine. Called hepatitis E, it’s a common ailment in China and India and, according to Jay Hoofnagle of the NIH, it is “probably the most common strain of infectious hepatitis in the world.” I’ll talk about it in a later blog.

Mankind has known about epidemic hepatitis for over 5000 years. Archeologists found a description of the illness on a Sumerian clay tablet that was written 3000 B.C., and Epidemics of an illness that caused yellow skin and eyes and lethargy afflicted 10s of thousands of soldiers who fought in the American Civil War and World War 2.

But our understanding of the infectious illnesses that attacked us was extremely limited in late 1700 when a Dutchman named Van Leuwenhoek created and looked through powerful magnifying lenses and wrote about a previously unknown world of tiny creatures. In the 1800s Carl Zeiss and others developed quality microscopes and Pasteur, Koch, and a slew of doctors and scientists studied the organisms that lived in the invisible world. They identified bacteria and parasites that make us sick, but the cause of some illnesses remained a mystery. We now know that many of them were caused by "agents" that could not be seen using powerful microscopes, "creatures" that were smaller than the wave length of light.

In 1882 a German named Adolf Mayer investigated a disease afflicting tobacco plants--tobacco mosaic disease. and he proved the disease was caused by an infectious agent. He extracted “juice from a diseased plant and passed the liquid through a bacteria retaining filter. Then he poured the juice on healthy tobacco plants and they got sick.” (1)

In the 20^th century German physicist, Ernst Ruska and others developed the electron microscope, a device that “used beams of electrons as a source of illumination.” It allowed scientists to see the “life” form that the “we call viruses.

Viruses are infectious agents that are smaller than the wavelength of light, thus submicroscopic. They are structurally a chain of DNA or RNA nucleotides surrounded by a protein coat. They don’t reproduce in the wild but after their protein coat gets them into a specific kind of living cell they force that cell “to produce thousands of copies of the original virus."

A small number of the thousands of viruses that are present on our planet invade an animal or a human Once inside a body they travel to the liver and enter hepatocytes(liver cells). The body's immune system tries to get rid of the intruders and is often successful.

The liver is the largest organ in the body and we can’t live without it. Among other things, it filters the blood that drains the small intestine, dissolves toxins in bile, and pours the bile into the gut. In some people it is damaged because the people drink too much alcohol, take certain drugs, or have too much body fat. In millions of others it is harmed by a virus.

In 1945 investigators learned that the serum of people who had recovered from an attack of infectious jaundice contained antibodies that protected them and others. Then in 1950 a prominent physician decided to find out if hepatitis was caused by more than one family of viruses. When he published his findings the study was praised by many. Years later a leading vaccinologist named Maurice Hilleman called it “the most unethical medical experiment ever performed on children in the United States.” –

The pediatrician, Saul Krugman, worked at Willowbrook, the largest state-run institution for people with mental disabilities in the United States. Located on Staten Island, New York, the facility opened in 1947 and was designed to house 4000 kids. Krugman believed the study was ethical because each kid who entered the school eventually developed hepatitis. Suspecting more than one virus was responsible, he collected the feces from children who were jaundiced and fed a bit of it to incoming students. They all got sick. During the subsequent 15 years he kept track of how long it took before the new kid turned yellow. His published study showed that some kids developed hepatitis within a few weeks; others didn’t get sick until a month or months had passed. He concluded that one strain of virus incubated for a long time before the child got sick and the other strain’s incubation was briefer. (3)

The cause or causes of epidemic jaundice were still unknown in the late 1950s when a doctor named Baruch Blumberg gave up clinical medicine and became a researcher at the NIH. At the time researchers knew that each gene is the blue print for one specific protein. We didn’t know how to examine DNA and identify individual genes. Some thought we might be able to indirectly name and categorize genes by identifying the proteins they mothered. Blumberg’s team studied the blood of indigenous people in various parts of the world and hoped to find proteins that were genetically significant. At one point they found a distinctive protein in an Australian aborigine and called it the Australia antigen. Then Blumberg realized it couldn’t have a genetic source because the protein was also present in the blood of an American with leukemia who had received multiple transfusions. After Blumberg reported his finding, he and many others tried to understand the protein’s significance.

In the 1960s his team found the protein was present in the blood of a number of institutionalized kids with Down’s syndrome and it was not present in newborns with Downs. Then in the 1960s “a virologist named Alfred Prince found Blumberg’s protein in the blood of a person with hepatitis. Samples of blood that had been drawn before the person got sick did not contain the protein. The protein, they realized, was part of a virus that caused hepatitis.”

Blumberg, the man whose discovery revolutionized our approach to hepatitis, was the son of a lawyer who struggled during the Great Depression. Blumberg was 16 when the Japanese bombed Pearl Harbor and he joined the navy and became a midshipman. When the war ended, he used the G.I. Bill to pay for his schooling. He planned to study math but his father, who usually didn’t say much, said, “I think you should go to medical school.” Blumberg liked being a student and he said OK. He met his future wife, Jean, when he was a medical intern at Bellevue hospital. She worked in the hospital research lab and their dates were dinners in the hospital cafeteria and strolls through the streets of New York. “A child of the depression,” Blumberg was paid $25 a week and managed to save most of the money. He later spoke of his days as a young doctor “before widespread health insurance." Many of the city’s poor were hospitalized at Bellevue, including many formerly middle class people impoverished by the expenses of chronic illness. The wards were crowded, often with beds in the halls.” In the late 1950s Blumberg left blood-and-guts medicine and became a researcher at the NIH.

The protein he discovered turned out to be part of the protein shell of the hepatitis B virus. It is one of our current markers for the presence or absence of disease that, in its chronic form is estimated to chronically infect 296 million people of whom 221 million live in low- and middle-income countries.^{” (2)}

We now know that when new born infants are infected by a chronically infected mother, they develop an infection that is usually present for the rest of the newborn's life. When the virus enters liver cells during the first 5 years of life, 70% of those infected shake the virus and 30% don’t. In the people with chronic disease the viral DNA has integrated into the DNA of liver cells at early stages of the viral replication cycle. Blocking viral replication at stages past virus entry does not affect integration rates. When someone is infected as an adult he or she is usually able to eradicate the virus completely; only 5% develop a chronic form of the disease.

In California newborns are routinely encouraged to take a hepatitis B vaccine when they are born.

Most chronic hepatitis B carriers don’t have active liver disease. Their immune systems don’t seem to notice the virus and their antibodies don’t attack its cells. Most make antibodies that quietly destroy the billions of new viruses that are made by the liver daily. At some point some people’s immune system starts attacking their infected liver cells, causing inflammation. Sometimes the flare up is short lived. On other occasions part of the liver is destroyed. Scar tissue is formed and it surrounds groups of liver cells. That’s what cirrhosis is. Some estimate that 25% of people with Chronic Hepatitis B are cirrhotic by the time they are 50.

Over time those who carry the virus have a heightened risk of developing liver cancer or Hepatoma. Cancer of the liver is the third most common lethal cancer in the world and hepatitis B and C play a major role in many of those afflicted.

For most of my career there wasn’t much doctors could do for people whose Hepatitis B was acting up. There were no anti-virals. Then the HIV epidemic exploded and researchers and pharmaceutical companies started creating anti-virals. Drugs that suppressed the HIV virus became available, and some of them worked for Hepatitis B. People having a flare took them, and had dramatically decreased the numbers of viruses in their blood stream. When the viral load is low antibodies usually stop attacking the liver and the inflammation cools. We now have several potent anti viral drugs that suppress Hepatitis B.

After studies showed that people who developed antibodies to the protein viral envelope were immune to the disease, scientists in France and Maurice Hilleman, the head of vaccine development for the pharmaceutical company Merck, decided to develop a protective vaccine.

The son of a Montana farmer and a mother who died shortly after he was born, Hilleman claims he was raised by the childless couple down the road and was put to “work as soon as he was able to tell a weed from a seed. “Everyone in Montana had to earn their keep.” He grew up picking berries and watering the animals. As a teenager he worked at J.C. Penney in the nearby town and “helped cowpokes pick out chenille bathrobes for their girlfriends.”

He attended college, was an incredible student, and eventually became the most important volcanologist of the 20^th century. His life is the subject of the book Vaccinated by Paul Offit.

Hilleman’s group obtained samples of the B virus by collecting blood from people in the New York Bowery--- from people who lived in flop houses or occupied stairwells and fire escapes.” His team isolated the protein from the gathered blood and “purified” it. First they treated the blood with pepsin; that reduced the viral particles 100,000 fold. Then they treated the antibody with urea—a chemical that destroys proteins and prions. Finally they poured in formaldehyde and filtered the fluid 15 times. In the end it was thought to be “virtually100 percent pure, and non infectious." Some samples had contained the HIV virus and that, too, was destroyed.

When some questioned the vaccine’s safety, Hilleman forced mid level executives at Merck who wanted to keep their jobs to be “voluntarily” immunized. On one occasion Hilleman learned the company was accelerating the production of the vaccine. His killing process was effective, but it was slow. Hilleman feared steps were being skipped and it could have affected the vaccine’s safety.

He told the production crew he suspected someone was “changing the fucking process so he can get more yield and earn a bonus. Meatheads are everywhere.”

Unfortunately many were reluctant to take the vaccine, in part as a result of the “Cutter incident,” a dramatic epidemic that shook the medical world:

It happened in 1955, shortly after the Salk polio Vaccine was FDA approved. One day the news was full of stories about kids who had received the polio vaccine, then developed polio. Some kids were paralyzed and a few died. Half of the 760,000 doses made by Cutter in Berkeley, California, were immediately recalled. In the end, “40,000 kids who received the vaccine developed polio, two hundred had at least partial paralysis and ten died.” The formaldehyde in the vats had failed to completely inactivate the virus. (4)

A few years after the Hilleman vaccine hit the market Bill Rutter and others decided they had to make a vaccine that did not contain viral material.

Rutter was the chief of biochemistry at the University of California in San Francisco. The son of a British Quaker, he was born and raised in Malad, a small town in southern Idaho. His grandfather had been a British Army officer in India and had told young William about the poverty and exotic parasitic diseases he had witnessed. After hearing the stories, young William thought he would someday go to the school of tropical medicine in Calcutta.

During the Second World War, Rutter lied about his age, and when he was 16 he joined the navy. When the war ended, he went to Harvard and was drawn to science. He was accepted to Harvard medical school, but after he attended a few medical school classes with a cousin, he realized he wanted to be a scientist. He earned a PhD at the University of Illinois and spent a decade as a researcher and professor at the University of Illinois, Stanford, and the University of Washington.

In 1968, after refusing the job offer three times, he became the chief of the biochemistry department at the University of California in San Francisco. The unit had been leaderless for six years and (he once quipped) “every good scientist in the United States had probably been asked to take that job and turned it down.” He claimed he accepted the post because twenty faculty positions were open, and that was a “bonanza for recruiting.”

In academia, investigators don’t always cooperate and share. Rutter knew science was competitive. “Everyone is trying to beat you and will use every trick in the book. You try to cover your bets in many different ways.” Rutter gathered top-notch researchers and got them to work collaboratively. In his department, associates shared their knowledge.

While he was chief Herbert Boyer, one of the researchers in his department, learned how to insert genes into plasmids. Plasmids are small, circular, molecules of double-stranded DNA that are present in the cytoplasm of most bacteria. Boyer would eventually leave academics and help form Genentech, but when he was a young researcher at the university he attended a conference in Hawaii, and conferred with a researcher who worked at Stanford.

Herbert Boyer,” had grown up in a small railroad town near Pittsburgh and, as a college student, had at times hitchhiked to classes at a nearby college. Majoring in biology and chemistry, he was “really taken with the Watson-Crick structure of DNA.” He earned his PhD in bacteriology, and at age 37 he was a researcher at the University of California in San Francisco. While he was there, one of his graduate students isolated an important enzyme. It sliced DNA at a specific position. The raw exposed nucleotide ends were sticky and other lengths of DNA---genes that instructed a cell to make a certain protein-- could be attached.

The man he met in Hawaii, Stan Cohen, was a hematologist at Stanford University. He was 36-year-old “trim, bald, and bearded,” and he was studying circular collections of DNA called plasmids. In vertebrates DNA is only present in the nucleus of cells but small collections of DNA are normally present in the cytoplasm of most bacteria. They can be passed from one bacterium to another by direct contact. It’s one way antibiotic resistance is spread. The two investigators wondered if it was possible to use Boyer’s enzyme to hook a gene (a known chain of DNA) onto the sticky ends of a plasmid’s DNA. If the plasmid was then inserted into the cytoplasm of a bacterium or yeast cell, would the gene direct the cell to make a specific protein? Would the transformed plasmid survive and clone itself?

The idea of using implanted genes to make proteins frightened some politicians. “Some portion of scientists was genuinely concerned. Others enjoyed the debate and the public controversy.”

There was a Senate hearing on the topic of genetic engineering and Rutter attended. Margaret Mead arrived wearing a huge long robe and carrying a shepherd’s staff. “Adlai Stevenson, a Senator and a lawyer who would later run for the presidency of the U.S., ran the proceeding. He introduced Margaret Mead as a world-renowned scientist who could give guidance on these issues.” Mead was an anthropologist who became famous after she spent nine months in Samoa. She learned that “adolescence on the island was not a stressful time for girls because their cultural patterns were different than those in the U.S.”

At the hearing she stood and repeatedly said something like, “You’re going to hear today from these scientists that this (genetic engineering) is not dangerous. I’m here to tell you it is dangerous.” After each repetition of her statement, she pounded the floor with her staff for emphasis. “Boom! Boom!” To Rutter, “a social anthropologist with her shepherd’s staff giving advice on molecular, microbiological, and physiological science” was troubling. Observing the theatrics and attitudes, Rutter realized that if he wanted to make the hepatitis B vaccine using genes inserted in plasmids he would have to give up his job as chief of biochemistry and go private. In 1981, Rutter and a few colleagues acquired venture funding, hired great researchers, formed a pharmaceutical company and named it Chiron."

Merck gave Rutter’s group the genome--hepatitis B DNA and one of Chiron founders, Pablo Valenzuela, isolated the section of the viral genome that coded (was the blueprint) for the outer membrane of the hepatitis B virus. In 1981, after a number of unsuccessful tries in bacteria, Rutter collaborated with Benjamin Hall and colleagues, of the University of Washington. Using yeast cells they got the organism to make a significant amount of Hepatitis B surface antigen.

“At Merck, Hilleman used the recombinant yeast-derived hepatitis B surface antigen-- rather than blood plasma-derived antigen, and he made an improved version of a hepatitis B vaccine.” When the particles were injected into people, their bodies produced antibodies that attacked the outer membrane of the virus, and the vaccine prevented hepatitis B. It was the first effective human vaccine made of viral sub-units but it was not the last. (5)

In 2019 the world health organization reported that WHO estimates that 296 million people were living with chronic hepatitis B infection and 1.5 million acquired the infection each year. That year the virus caused and estimated 820 000 deaths, mostly from cirrhosis and hepatocellular carcinoma (primary liver cancer).

Progress has been made, but in chronic carriers it’s near impossible to completely clear the virus from a person’s body. When a person’s immunity slips the virus can start replicating. (6)

Hepatitis A

After he graduated from Johns Hopkins Medical School in the early 1970s John Feinstone had to choose between being a doctor in Viet Nam or becoming a researcher at the NIH. He chose the latter. At some point that year he was asked to “take a crack at finding the virus responsible for hepatitis A in stools collected from prisoners at the penitentiary in Joliet Illinois who were voluntarily infected. Viruses grow and proliferate inside appropriate cells and in the 1960s cell culture was a tremendous boost to viral research. In 1972 immunoelectron microscopy became available. The electron microscopy providee structural information and the immune electron microscope allowed researchers to use the interaction of viruses and their respective antibodies to identify viruseS or a molecule of interest. Researchers attached a particular antibody to the molecule and the reaction between the antigen and antibody, caused specific viruses to clump together and form a visible mass.

When Feinstein and others examined the stools of prison volunteers they noted that the stools had “multiple particulate forms." Many resembled viruses. Others were bacteriophages or breakdown products of food. The researchers tested the serum before and after volunteers were infected with the stool from people who had short incubation hepatitis. They found no antibody in the pre inoculation sample and they easily detected antibody in the convalescent sample. All six tested patients showed either seroconversion or antibody increases on the 4-point scale. The researchers found the reaction also occurred when they used serum taken from people who were infected with hepatitis during an outbreak in Massachusetts and in American Samoa. When they added the antibody to the stool of a patient with acute hepatitis Feinstone “saw the hepatitis A conglomeration. It immediately popped out as different. It almost glowed as a result of the antibody covering the particles.”

“Feinstein ran upstairs and told his supervisor. When his boss saw the virus he was excited and the researchers performed the tests needed to prove this was the virus responsible for hepatitis A.” The viruses were “spherical 27-nanometer particles”

Then, in 1970 Provost and Hilleman at Merck successfully cultivated hepatitis A virus in flask containing a layer of human cells. Once they could grow quantities of the pure virus Hilleman and others developed live and killed hepatitis A vaccines humans. .

Single-antigen HepA vaccine was licensed for use in the United States in 1995 (Havrix) and 1996 (Vaqta), and doctors started using it. By 2004 the incidence of hepatitis A in the United States had decreased significantly, but “the majority of the world’s population remained at moderate-to-high risk for hepatitis A virus infection” In 2004 Allen Craig at Vanderbilt reported that, “the rates of hepatitis A infection in the United States were gradually decreasing but 9 percent of children 4 to 11 and 75 % of individuals over 70 had serological evidence of a prior disease.

Sporadic outbreaks of food borne hepatitis A continue to occur, and insurers are quick to point out that the cost of universal vaccination is not inconsequential; in response researchers point out that childhood hepatitis A vaccination prevents infection in adolescents and adults and that makes it cost effective. One researcher estimated that in 1997, the economic burden of symptomatic hepatitis A infection in adolescents and adults was $488.8 million.

After hepatitis A and B were identified the world’s blood supply was safer, but--they discovered--transfused blood sometime contained at least one more virus. They called the entity non A non B hepatitis.

SOME REFERENCES:

1. https://www.apsnet.org/edcenter/apsnetfeatures/Documents/1998/ZaitlinDiscoveryCausalAgentTobaccoMosaicVirus.pdf

2. https://www.nejm.org/doi/full/10.1056/NEJMra2211764

3. https://www.qcc.cuny.edu/socialsciences/ppecorino/medical_ethics_text/chapter_7_human_experimentation/case_study_willowbrook_experiments.htm

4. https://www.washingtonpost.com/history/2020/04/14/cutter-polio-vaccine-paralyzed-children-coronavirus/

5. https://www.nasonline.org/publications/beyond-discovery/hepatitis-b-story.pdf

6. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10170306/

https://en.wikipedia.org/wiki/Immune_electron_microscopy

7. History of the Discovery of Hepatitis A Virus Stephen M. Feinstone

8. ELECTRON MICROSCOPE THE RUSKA BROTHERS https://www.sciencedirect.com/science/article/pii/S1076567008701551

https://www.nobelprize.org/prizes/physics/1986/perspectives/

https://en.wikipedia.org/wiki/Ernst_Ruska https://authors.library.caltech.edu/5456/1/hrst.mit.edu/hrs/materials/public/ElectronMicroscope/EM_HistOverview.htm#:~:text=1.,the%20limitations%20of%20visible%20light.