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The Curiosities of Hepatitis B Virus

Prevention, Sex Ratio, and Demography

Fox Chase Cancer Center, Philadelphia, Pennsylvania

Correspondence and requests for reprints should be addressed to Baruch S. Blumberg, M.D., Ph.D., Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111. E-mail: baruch.blumberg{at}fccc.edu

Viruses have been feared as major pathologic agents, a scourge of humans and other species. However, they have a more textured relation to their hosts. This article focuses on hepatitis B virus (HBV), one of the most common and deadly agents that infect humans and that has a powerful selective effect on survival, which also has complex and subtle interactions with its host that are not pathologic. HBV affects populations and their evolution in an imaginative manner that is a model for understanding how other microorganisms can both plague and assist their hosts at different times and in different environmental and genomic contexts. The host responses to infection with HBV are affected, in large part, by a series of polymorphic alleles at several loci on the human genome, and these loci are also related to other infections. HBV has an effect on the sex ratio in populations, both at birth and later in life. The effects of these dynamic systems on the demography, population biology, and microevolution can be profound.

CLINICAL ASPECTS OF HEPATITIS B INFECTION

The most common outcome after infection is the development of antibody against the surface antigen of HBV (Table 1). The infected person experiences no symptoms and ordinarily has lifelong protection against further infection. A significant number of those infected will develop acute hepatitis. This is ordinarily a self-limited disease that includes flulike symptoms, fever, joint pains, malaise, severe loss of appetite, and, in time, jaundice. Most patients recover in several weeks or months with no further symptoms, but a small percentage may develop fulminant hepatitis, which has a high mortality rate. About 10% of patients with acute hepatitis will retain the infection and develop chronic hepatitis. They can remain ill for months or years and this may result in liver dysfunction, liver failure, and early death. Some of those chronically infected individuals may proceed to primary cancer of the liver, which is difficult to treat and life shortening. In some cases, chronic liver disease may occur without an antecedent acute phase.

PRIMARY CANCER OF THE LIVER

Primary cancer of the liver is one of the most common cancers in the world, and particularly so in sub-Saharan Africa, China, and East Asia. It is the third most common cause of death from cancer in males and the seventh most common cause in females. Its toll is about one million deaths a year. HBV is believed to cause about 70 to 85% of the cases; many of the rest are caused by hepatitis C virus (HCV). Other factors contribute to the etiology of primary cancer of the liver, including liver toxins such as alcohol. Treatments are available, but they are far from ideal.

OTHER HEPATITIS VIRUSES

There are other hepatitis viruses and they are shown Table 2. They are different from each other and several are in separate families. Although the clinical manifestations may be similar, the epidemiology, method of spread, chronicity, and other features are different. Hepatitis A virus is transmitted by the fecal–oral route and is rarely chronic. As noted, HBV causes acute and chronic disease and the carrier state. It is transmitted from infected mothers to their children, by sexual transmission, and by transfer of blood from one person to another (as in blood transfusion). HCV is transmitted in the same way as HBV, but it is less infectious. Hepatitis D virus is an unusual, very small virus that is "wrapped" in the surface antigen of HBV. It can only infect people who are already infected with HBV, or who are infected at the same time with both viruses. Hepatitis E virus is transmitted in the same manner as hepatitis A virus and can occur in large and devastating epidemics.

WORLDWIDE DISTRIBUTION AND PATHOLOGIC IMPACT

It is difficult to estimate the total number of deaths due to infection with HBV; an estimate of over a million is often used. An estimate of the expected number of deaths due to prevalent HBV carriers compared with deaths due to prevalent cases of HIV was made in 1999. (The number of HIV prevalent infections has increased since that time.) Using a high estimate for deaths due to HBV, the total expected deaths is 92.8 million; using a low estimate, the number is 55.7 million. The equivalent number for deaths associated with HIV was somewhat less; estimates for HIV deaths in 2005 would be higher.

It is estimated that two billion people alive today, one-third of the world's population, have been infected with HBV. Most of these would have developed anti-HBs and remained well, but many have developed clinical hepatitis and some 375 million worldwide are currently carriers of HBV. Humans now have and have had in the past a highly significant interaction with HBV. Mortality by sex, age, and carrier status in Haimen City, China, is shown in Figure 1. Male carriers have a higher mortality than noncarrier males, and female carriers have a higher mortality than noncarrier females. Male carriers and noncarriers have a much higher mortality than females. These differences increase with age. Therefore, HBV kills more males, particularly older males, than females. An effective vaccination program (see below) will increase the male-to-female sex ratio, especially in the older age groups. As shown later, chronic infection with HBV appears to have the opposite effect in newborns. The worldwide distribution of the approximately 375 million carriers of HBV is shown in Figure 2. The prevalence is extremely high in Asia, the Pacific, Africa (particularly sub-Saharan Africa), in the Amazon River basin of South America, and, strangely, among the Inuits of the Arctic.

View larger version (18K): [in this window] [in a new window]   Figure 1. Mortality by age, sex, and hepatitis B virus (HBV) response in Haimen City, China. Reprinted by permission from Chen G, Lin W, Shen F, Iloeje UH, London WT, Evans AA. Chronic hepatitis B virus infection and mortality from non-liver causes: results from the Haimen City cohort study. Int J Epidemiol 2005;34:132–137.

View larger version (70K): [in this window] [in a new window]   Figure 2. Global distribution of chronic HBV infection. Courtesy of the Centers for Disease Control and Prevention.

The research that resulted in the discovery of HBV and the invention of the HBV vaccine began as a basic, nondirected study of human genetic polymorphisms and differential susceptibility to disease. My colleagues and I did not start off to discover the virus; as is often the case in science, during the course of this "pure," nonapplied research, the path we were following led to an unexpected and practical outcome. In the search for antigenic polymorphic variation in human serum proteins, we used the serum of patients who had received many transfusions. Our hypothesis was that transfused patients would develop antibodies against an antigenic protein variant inherited by the donor and present in his or her blood but that had not been inherited by the recipient of the blood. Using this method, we found a complex and medically important polymorphism of the serum lipoproteins. When we continued the studies, we found another antibody, which we subsequently found to be directed to the outer coat of the HBV. In time, this became a widely used clinical laboratory test that could detect HBV in blood donors. Widespread use of this test soon eliminated post-transfusion hepatitis due to HBV. Subsequently, a test for HCV in the blood was introduced that further decreased the incidence of post-transfusion hepatitis.

The electron microscope image shows the three particles that constitute the HBV complex (Figure 3). The large 42-nm particle is the whole virus. It includes the enveloping surface antigen (HBsAg) and the core antigen (HBcAg) that surrounds the circular, partially double-stranded DNA. HBV is an extremely small virus—smaller than polio virus—with only 3,200 base pairs. In addition to the 42-nm whole virus particles that are pathogenic and infectious, there are small, circular particles that are 22 nm in circumference, and elongated particles of the same diameter and of varying length. They consist only of the surface antigen HBsAg and are not infectious or pathogenic. In 1969, my colleague Irving Millman and I invented a vaccine that was made by separating the small HBsAg particles from the whole virus particles, discarding the infectious virus, treating the resulting surface antigen particles to kill any remaining virus, and adding adjuvants as necessary. This was a unique method of manufacturing vaccine that had not been used before or since. This vaccine was field tested, approved by the U.S. Food and Drug Administration (FDA) for safety and efficacy, and has been used in tens of millions of people.

View larger version (145K): [in this window] [in a new window]   Figure 3. HBV morphology. Nucleic acid: DNA; classification: hepadnavirus type 1; serotypes: multiple; in vivo replication: reverse transcription in liver and other tissues; in vitro propagation: primary hepatocyte culture and transfection by cloned HBV DNA.

View larger version (34K): [in this window] [in a new window]   Figure 4. HBV genes and gene products. Striped arrow, HBsAg; solid arrow, DNA polymerase; checkered arrow, HBxAg; dotted arrow, HBcAg/HBeAg.

National vaccination programs have been in place since the early 1980s. The effect on the prevalence of HBV carriers and the incidence of acute hepatitis is impressive. In a regional study in China, the prevalence of HBV carriers dropped from 16 to 1.4%. In the United States, new infections with HBV have dropped from 260,000 in the 1980s to about 78,000 in 2001. In Alaska, acute hepatitis B infection dropped from 215 cases per 100,000 population before the vaccination program to 7 to 14 cases in 1993 after the program was in place. In 1995, no cases were reported. In Gambia, West Africa, the prevalence of chronic infection in the young had dropped from 10.0 to 0.6%.

In Afragola, Italy, a community with very high rates of HBV infection and of morbidity and mortality from liver disease, the prevalence of HBsAg in males up to 12 yr old dropped from 10.5% in 1978 to 0.8% in 1993 after the vaccination program was in place for 10 yr. The prevalence of anti-HBc dropped from 52.6 to 1.2%. There was also a drop in the prevalence of HBsAg in the mostly unvaccinated population—that is, males aged 13 to 60 yr. HBsAg dropped from 18% in 1978 to 5.5% in 1989. This implies that the reduction of the carrier prevalence in the vaccinated group has an indirect effect on unvaccinated carriers and susceptible individuals, and suggests that there may an amplification characteristic to the vaccination program. Similar observations have been made in other population-based studies.

Perhaps the most remarkable effect of the program has been in the decrease in primary cancer of the liver in vaccine-impacted populations. In Taiwan, the average annual incidence in the age range of 6 to 14 yr fell from 0.7/100,000 in 1981–1986 (before vaccination) to 0.36/100,000 in 1990–1994 (after vaccination). The annual incidence in children aged 6 to 9 yr fell from 0.52/100,000 in those born in 1974–1984 (pre–vaccination program) to 0.13/100,00 in those born in 1986–1988 (post–vaccination program). In South Korea, the study cohort included 370,285 males aged 30 yr or older. Of these, 18,914 (5%) were HBsAg positive and 78,094 (21%) anti-HBs positive. A total of 273,277 had no markers. Of these, 35,934 (13.2%) were vaccinated during 1985. The prevalence of primary cancer of the liver was determined from 1986 to 1989. The unvaccinated and chronic carriers of HBsAg had an incidence of 18.1 per 100,000 population. The incidence of cancer of the liver in those who were vaccinated dropped to 0.58 and that in those with "natural" anti-HBs dropped to 0.34.

In both of these studies, the drop was dramatic. If these findings are confirmed in subsequent studies, the HBV vaccine is the first preventive cancer vaccine and the vaccination program is the major medical intervention program for the prevention of cancer. These results give hope that other cancer vaccination programs will be instituted. The recent announcement of the successful field trial of a vaccine to prevent papilloma virus infection—a major cause of cancer of the cervix—is very promising.

As of May 2003, 151 (79%) of 192 national members of the World Health Organization (WHO) had universal childhood vaccination programs. There are 89 member states that have been designated as having a high prevalence of HBV carriers. Sixty-four (72%) have universal infant vaccination programs. It is the goal of the WHO to have vaccination programs in all countries by 2007. The worldwide vaccination program is proceeding very well; it has saved millions of people from infection, illness, and death.

SEX EFFECTS

There are some unusual aspects of HBV infection related to sex differences. In general, males when infected with HBV are more likely to become carriers of the virus and females are more likely to develop anti-HBs (Table 3). Curiously, in areas of high HBV prevalence, the response of parents to infection with HBV is related to the sex of their offspring. In a study in Greece, families in which either parent was a carrier of HBV had a higher ratio of boys to girls than in families where the parents (particularly the mother) had anti-HBs (Table 4). Families with unaffected families had an intermediate ratio. Similar studies were done in five other populations; they were consistent with the initial results.

View larger version (17K): [in this window] [in a new window]   Figure 5. Sex ratio and hepatitis throughout the world. Sex ratio is number of boys for each girl. Only countries with more than 15,000 people used to calculate HBV prevalence are included. Reproduced by permission from Oster E. Hepatitis B and the case of the missing women (presentation on October 12, 2004).

GENETICS

We discovered HBV during the course of research on serum protein polymorphisms. Family genetic studies of the "Australia antigen"—that is, the carrier state for HBV identified by its surface antigen HbsAg—were done even before we were aware that we had discovered a virus. A summary of the family data analyses done on families in Cebu, the Philippines, and Bougainville, Solomon Islands, was consistent with the segregation of an autosomal recessive allele designated Au¹. When present in double dose, it increased susceptibility to becoming a carrier of HBV (HBsAg positive) when the individual was exposed to the virus. Later, extensive investigations by Adrian Hill and others identified multiple human polymorphisms in which alleles segregating at the polymorphic locus increased the susceptibility to become a carrier of HBV. Many of these identifications were based on population studies. Alleles at the polymorphic locus, which were significantly more common in HBV carriers than in noncarriers, were inferred to increase susceptibility to the carrier state.

Often, alleles at the same susceptibility locus are related to response to several infections. These are arranged in Table 5 using published data available in the late 1990s. For example, individuals homozygous for the t allele at the vitamin D receptor locus (VDR) are more likely to become carriers of HBV and they also are more susceptible to pulmonary tuberculosis. Those who are homozygous for the alternate T allele, when exposed to the leprosy bacillus, are more likely to develop the lepromatous form, whereas those homozygous for t are more likely to develop the tuberculoid form of the disease. Another example is the tumor necrosis factor (TNF) locus. TNF is a cytokine (a protein or glycoprotein involved in the regulation of cellular proliferation and function) that has many roles, including the control of inflammation and the stimulation of the proliferation and destruction of cancer cells. There are several polymorphic sites on the TNF gene. They are related to susceptibility to HBV chronicity, cerebral malaria, lepromatous leprosy, and a form of the tropical disease caused by Leishmania braziliensis.

HBV CARRIERS AND IRON STORAGE

The alteration in sex ratio associated with (and probably due to) HBV infections is an example of a nonpathologic effect of the virus on its human host populations. Another example is the relation between HBV and iron storage. Patients with Down's syndrome who are HBV carriers have higher hemoglobin, hematocrit, and serum iron levels than those who are not (Table 6). In other populations, there is also increased serum iron levels in the carriers (Table 7). In populations with low iron intake in their diets, HBV carriers could be at an advantage compared with noncarriers in that they could retain more iron for metabolic use. This could be a positive selective factor, particularly in premodern times when populations had a shorter life expectancy and carriers would die before there were any clinical effects of the HBV infection.

The events and times for discovery and application are shown in Tables 8 and 9. The finding of Australia antigen in 1963 can be taken as the beginning of the research. By 1997, we had identified it as a hepatitis virus and confirmation from other laboratories followed quickly. (This was, in part, a result of our distributing the necessary reagents to those who requested them.) We invented diagnostic methods for identifying the virus in 1968, and subsequently (1975) were issued a patent for a radioactive method to greatly increase sensitivity. By 1969, donor blood was being tested for HBV and within the next few years, with the development of readily available commercial reagents, post-transfusion hepatitis due to HBV had largely been controlled in the United States and many other countries. We invented the vaccine in 1969 and the patent was issued in 1972. We soon (in 1975) licensed a nearby pharmaceutical company (Merck) to develop and produce the vaccine. The field trials for the vaccine were completed by Wolf Szmuness and his colleagues in New York City in the early 1980s, and the vaccine was approved rapidly (in 1982) by the U.S. FDA. National vaccination programs were in place by 1982; by 2003, vaccination was in use worldwide.

Similar but not exactly comparable dates for the polio vaccination program were supplied by Professor Francois Gros in the World Life Science Forum Newsletter for 2005. There was an interval of 70 yr from the basic science to the current status of near eradication of polio.

CONCLUSIONS

Hepatitis B vaccination is one of the largest worldwide disease-prevention programs. It has decreased the spread of HBV, particularly in China and East Asia. It has significantly decreased morbidity from liver disease and prevented the death of millions. HBV vaccination appears to prevent primary cancer of the liver; it is the first widely used preventive cancer vaccine.

There are important nonpathologic interactions of HBV with humans. Parents who are carriers of HBV have a higher ratio of males to females among their offspring than parents who developed antibody against the surface antigen. This may account for the high sex ratios seen in China and in other areas with a high prevalence of HBV infection. The apparent "loss" of females in these populations may be ascribed, at least in part, to HBV infection. HBV vaccination has decreased the ratio of males to females among newborns. If confirmed, this may have important biological, demographic, and economic effects. Another nonpathologic interaction is the relation of HBC to iron storage. HBV carriers have higher hemoglobin and hematocrit levels and appear to retain more iron from their diet than do noncarriers.

The response of the host to HBV infection is related to a series of polymorphic loci, which are, in turn, related to other disease-causing agents. Any changes in the prevalence of HBV may have an effect on the epidemiology of the other infectious agents.

FOOTNOTES

A similar paper was presented by the author at the World Life Sciences Forum in Lyon, France, April 12, 2005.

Supported by Fox Chase Cancer Center and the Ascherman/Blumberg Fund for Basic Research.

Conflict of Interest Statement: B.S.B. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

(Received in original form October 19, 2005; accepted in final form November 8, 2005)

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