Hepatitis B Virus

This small, extraordinary virus causes liver diseases and

a common form of cancer. New vaccines produced by genetic
engineering hold the promise of eventually eradicating both

by Pierre Tiollais and Marie-Annick Buendia

T
he liver disease called hepati­ delphia, was studying certain proteins In some patients, however, the high
tis B is a widespread and seri­ in blood serum. In a sample from a levels of viral antigen in the blood per­
ous health problem in its own patient with hemophilia, he observed sist for several years or even for a life­
right, but it conceals a greater men­ an antibody that reacted with an anti­ time, yet no antibodies against HBsAg
ace: the virus that causes hepatitis B is gen present in the blood of an Austra­ ever appear. The virus survives in the
second in importance only to tobacco lian aborigine infected with hepatitis. liver, and the patient becomes a chron­
as a known human carcinogen. Hun­ In 1968 Blumberg identified it as the ic carrier. The mechanism by which
dreds of millions of people, most of HBV surface antigen (HBsAg). such a chronic state establishes itself
them in regions with poor medical care, For the next decade, the absence of is not well understood, but it seems to
are chronically infected with the virus cell-culture systems for propagating be related to a weak immune response.
and face an elevated risk of acquiring HBV hampered investigators. Then in That explanation may account for why
liver cancer. Moreover, although many 1978 our laboratory and others began the infection becomes chronic in about
chronic carriers appear healthy, they to apply recombinant DNA technolo­ 80 percent of infants, whose immune
can still transmit the hepatitis B virus gy to the problem. Today the work on systems are immature, but in only 5 to
to those with whom they have close HBV stands as the most successful ap­ 10 percent of adults.
contact, thereby starting the cycle of plication of recombinant DNA technol­ Chronic infections can assume sever­
disease anew. ogy to medical virology. al different forms. Some chronic carri­
Fortunately, the prospects for inter­ ers are healthy: they experience limit­

P
rupting that cycle have brightened con­ eople infected with HBV are often ed liver damage and have no function­
siderably during the past decade. Re­ unaware of it. After a two- to six­ al deficiencies. Others acquire chronic
combinant DNA technology, or genetic month incubation period, HBV in­ persistent hepatitis, which is general-
engineering, has unlocked many of the fection can lead to acute hepatitis and
secrets of the hepatitis B virus (HBV). liver damage, which cause abdominal
Today researchers understand the pe­ pain, jaundice, elevated levels of cer­
culiar life history of HBV and are prob­ tain enzymes in the blood and other
ably closing in on answers to how HBV symptoms. At this stage, HBV infection
causes cancer. Moreover, vaccines pro­ can be diagnosed by detecting HBsAg
duced by genetic engineering can pre­ in a patient's blood serum. More fre­
vent HBV from spreading. quently, the disease remains perma­
The first step in the identification of nently asymptomatic.
the hepatitis B virus was made in 1963, In very rare cases, the HBV infection
when Baruch S. Blumberg, then at the causes fulminant hepatitis-a rapidly
Institute for Cancer Research in Phila- progressive, often fatal form of the dis­
ease in which massive sections of the
liver are destroyed. The liver damage is
PIERRE TIOLLAIS and MARIE-ANNICK not indicative of a more virulent form
BUENDIA are pioneers in the use of of the virus but is instead the conse­
recombinant DNA technology to study quence of a stronger immune response
hepatitis viruses. Tiollais is professor by the host: white blood cells called
and director of an INSERM research unit
"killer" T lymphocytes attack infected
at the Pasteur Institute and professor of
cells bearing the viral antigens.
biochemistry at the University of Paris 7.
After receiving his medical degree from
Usually a patient with acute hepati­
tis will recover completely. The clinical HEPATIllS B VIRUSES have a double­
the University of Paris in 1968, he began
and biological symptoms of the infec­ walled structure consisting of an outer
studies of nucleic acid synthesis. He was
envelope and an inner shell, or capsid,
the first to clone the human hepatitis tion gradually disappear as more anti­
B virus. Buendia, a director of research that contains the viral DNA. Many in­
bodies against the virus are produced.
of the eNRS at the Pasteur Institute, re­ complete viral particles also appear in
After recovery, the patient continues to
ceived her doctorate in biochemistry the blood of infected persons, as shown
produce low levels of those antibodies,
from the University of Paris in 1977. She in the micrograph (above). These parti­
which maintain immunity to HBV for cles are small spheres or long filaments
has played a particularly important role
in illuminating how the woodchuck hep­ several years. In the event of a new HBV without an internal structure, and only
atitis virus may induce liver cancers. attack , the antibody level rises quickly two of the three characteristic viral en­
and neutralizes the virus. velope proteins appear on their surface.

116 SCIENTIFIC AMERICAN April 1991

© 1991 SCIENTIFIC AMERICAN, INC
ly asymptomatic but sometimes caus­ studies have also found a consistent In the Third World the virus is most
es fatigue. In the worst cases, chronic and specific causal association between often transmitted from an infected
active hepatitis develops and can lead HEY and HCC. HEY is therefore one of mother to her infant in the baby's first
to cirrhosis of the liver and finally to the few viruses known to cause a spe­ month, primarily during birth. If an
hepatocellular carcinoma (HCC), a pri­ cific cancer in humans. infant is female and infected, then she
mary liver cancer. Usually the cancer will probably become a chronic carri­

T
does not develop until after a 30- to he gravity of the link between er and transmit HEY to her own off­
5 0-year latency period, but a few cases HEY and cancer becomes clear spring when she reaches childbearing
of HCC have been observed in children. when the huge numbers of in­ age. Mother-to-child transmission is not
Today the link between chronic HEY fected persons are considered. World­ the only mechanism: because the virus
infection and the development of HCC wide, chronic HEY infections affect al­ can be found in blood, saliva and se­
is very clear. The percentage of chronic most 300 million people, three quar­ men, any intimate or sexual contact can
carriers is higher among patients with ters of whom are in Asia. The incidence pass on the disease. That fact explains
HCC than among the rest of the pop­ of the condition varies greatly among the easy spread of hepatitis B within a
ulation. Moreover, in an epidemiologi­ different regions. In southeast Asia and family or small community.
cal study performed in Taiwan, R. Pal­ tropical Africa, chronic carriers of the In Western industrialized countries,
mer Beasley of the University of Texas virus represent 10 percent or more of other mechanisms of HEY transmis­
at Houston has shown that for chronic the population, whereas they make up sion become much more important.
carriers the risk of acquiring HCC is less than 1 percent in North America High-risk populations consist of per­
100 times higher than normal. Other and most of western Europe. sons in direct contact with chronic car-

© 1991 SCIENTIFIC AMERICAN, INC

 the St. Louis Hospital in Paris, subse­
quently determined the complete nu­
cleotide sequence of the HEY genome,
thereby providing the first available in­
formation about the genetic organiza­
tion of the virus.
The HEY genome is a miracle of com­
pactness. It consists of only four poten­
tial genes, termed S, C, P and X, which
overlap extensively. The regulatory se­
quences that control the production of
." the viral proteins and the replication
cycle are also embedded in these cod­
ing sequences.
PERCENTAGE Gene S encodes, or carries the blue­
OF POPULATION prints for, the major envelope protein
D 0.1-1 and includes all the specifications for
D 1 -5 HbsAg. A sequence of about 500 nu­
_ 5-20 cleotides preceding the S gene can also
be transcribed along with it. As Wol­
fram H. Gerlich of the University of
Chronic HBY carriers are most common in developing countries where these in­ Gbttingen has shown, this "upstream"
fections are endemic. About 225 million chronic carriers of HBY are in Asia alone. sequence can be divided into two re­
gions, pre-S1 and pre-S2, that are in­
volved in the synthesis of the other en­
riers or with their blood samples (such cles leave the cell and can infect neigh­ velope proteins: the middle protein is
as nurses, physicians and dentists), the boring cells. encoded by pre-S2 and the S gene, and
recipients of blood or blood products HBY has a double-walled structure the large protein is encoded by pre-S1,
(such as hemophiliacs and patients who consisting of two concentric protein pre-S2 and the S gene. The pre-S1 re­
receive transfusions or dialysis treat­ coats. The envelope, or outer coat, con­ gion also plays an important role in the
ments), intravenous drug abusers, ho­ tains three proteins that are deSignat­ entry of the virus into a liver cell, as
mosexuals and persons with multiple ed major, middle and large. HEsAg, the shown by A . Robert Neurath of the
sex partners. For the population out­ surface antigen, is found on all three of New York Blood Center.
side those groups, the risk of contract­ the proteins. The capsid, or inner coat, Gene C encodes the protein of the
ing hepatitis is low. In fact, the epide­ consists of a single core protein that capsid. Like S, the C gene is preceded
miology of hepatitis B is very similar to surrounds and interacts with the vi­ by a short pre-C region that encodes a
that of acquired immunodefiCiency syn­ ral DNA . hydrophobic peptide involved with the
drome (AIDS)-which explains why hep­ The HEY genome, which was first assembly of the viral particle. Gene P,
atitis B infection is common among isolated in 1974 by William S. Robinson which is so long that it includes parts
patients with AIDS or the AIDS-related of the Stanford University School of of all the other genes, encodes enzymes
complex . Hepatitis is far more conta­ MediCine, is a circular DNA molecule essential to the viral replicative cycle.
gious than AIDS, however. composed of only about 3,200 nucleo­ Gene X covers the cohesive ends of the
Hepatitis B is therefore primarily a tide subunits. It is the smallest-known viral DNA strands. Its protein product
disease of infants in developing na­ animal virus genome-the genome of stimulates the expression of all the vi­
tions, whereas in Western countries the common herpesvirus, for example, ral genes by interacting with a specific
it is mostly confined to adults. This is 50 times larger. Like the DNA mole­ DNA sequence found in the the HEY
distinction has practical ramifications cules in most organisms, the genome genome.
for future vaccination strategies. In the of HEY consists of two base-paired In the life cycle of HBY the synthe­
Third World, mass vaccinations would strands, but it has an unusual feature: sis of viral proteins is tightly regu­
be necessary, but in Western countries one strand is longer than the other. lated at the transcriptional and trans­
only the high-risk population needs to The short "plus" strand, which can vary lational level. Two types of messenger

�
be protected. in length, is only 50 to 80 percent as RNAs (mRNAs) copied from the viral
long as its mate, the "minus" strand. genome are known. The smaller one,
tiviral vaccines work by sensitiz­ ( As we shall explain later, this unusu­ about 2,100 nucleotides long, encodes
ing a person's immune system al structure is a consequence of the the major and the middle envelope pro­
against viral molecules. Much of unique replication mechanism used by teins. The larger mRNA , which is about
the early work on HBY therefore con­ the virus.) The circular structure of the 3,500 nucleotides long, is surprising: it
centrated on deciphering the structure genome is maintained by base pairing is actually longer than the complete ge­
and life cycle of the virus to identify of the strands at one end. nome and contains a terminal repeat of
good vaccine targets. Using recombinant DNA techniques, about 100 nucleotides. The large mRNA
All viruses are cellular parasites made in the late 1970s our research group encodes the capsid protein and the
up of an RNA or DNA chromosome (ge­ succeeded in clOning, or copying, the products of gene P, as shown by Har­
nome) packed in a protein coat. To pro­ HEY genome in Escherichia coli bacte­ old E. Yarmus of the University of Cali­
liferate, a virus must penetrate the cell ria. This breakthrough made it possible fornia at San Francisco and Heinz Schal­
and use the cellular machinery to syn­ to produce large quantities of the virus ler of the University of Heidelberg. It
thesize the proteins of the coat and the and its components for further study. also represents an intermediate in the
viral genome. After a genome is pack­ Patrick Charnay of our laboratory, in replication of the viral DNA , as we shall
aged inside its coat, the new viral parti- collaboration with Francis Galibert of soon describe.

118 SCIENTIFIC AMERICAN April 1991

© 1991 SCIENTIFIC AMERICAN, INC
 Transcriptional "enhancer" elements man HEV carriers, which have shown cules, and almost all of them have iden­
in the HEV genome activate the expres­ that men (whose natural steroid hor­ tical genetic organizations.
sion of all viral genes and operate pref­ mone levels are higher than women's) Hepadnaviruses reproduce by an ex­
erentially in liver cells. Other regulato­ run a greater risk for chronic HEV in­ traordinary mechanism. Most DNA vi­
ry elements modulate the levels of in­ fection, liver damage and HCC. ruses directly copy their genomes with
dividual proteins. A good illustration In other strains of transgenic mice, vi­ enzymes called polymerases: the poly­
of this complex mechanism is provid­ ral replicative forms were observed by merases use the original DNA strands
ed by the variations observed in the Pourcel in kidney and heart cells as well as templates for assembling new com­
amounts of large, middle and major as in the liver. These results, too, mir­ plementary strands. Yet hepadnavirus­
proteins of the envelope made in in­ ror the affinity of HEV for different tis­ es use an indirect method, involving a
fected liver cells. The large proteins are sues in humans: although HEV is most strand of RNA as an intermediate. The
produced in small quantities and are abundant in the liver, the viral DNA and mechanism was first identified in 1982
found only on the surface of complete, proteins have also been found in the by Jesse W. Sununers and William S.
infectious viral particles. In contrast, kidney, spleen, pancreas, skin, bone Mason of the Fox Chase Cancer Center
the major proteins (and, to a lesser marrow and circulating blood cells. The in Philadelphia, who were working with
extent, the middle proteins) are made blood cells might be the first targets the Peking duck virus.
in great excess and secreted in small­ of HEV infection; viruses persisting in The mechanism works as follows: Af­
er (22-nanometer) particles, which are these sites might also be able to re­ ter a hepadnavirus genome penetrates
more abundant than complete particles emerge and attack the new livers of a cell, it migrates to the nucleus, where
in the serum. hepatitis patients who have received cellular polymerases transcribe it into a
transplants. The infection of certain long RNA molecule. This 3,500-nucle­

O
ne important question we sought white blood cells may be directly in­ otide RNA molecule is called the prege­
to answer was how the various volved in some other diseases, such as nome. The pregenome and a viral DNA
HEV genes are expressed. We aplastic anemia and polyarthritis nodo­ polymerase are packed into a newly
have found that transgenic mice-ani­ sa, and in the development of AIDS formed capsid and pass back into the
mals that have had part or all of the and AIDS-related complex. cell 's cytoplasm. There the polymer­
HEV genome added to their own DNA­ Some understanding of HEV has ase goes to work , reverse transcribing
are powerful tools for studying the ex­ come from studies of similar viruses, the pregenome into a new minus DNA
pression of the viral genes in normal called hepadnaviruses, that cause hep­ strand. As soon as the minus strand
host tissues. atitislike conditions in animals. Hepad­ forms, the pregenome is destroyed by
Working with transgenic mice, Chris­ naviruses that infect the woodchuck , enzymes. DNA polymerases then begin
tine Pourcel of our laboratory has stud­ the ground squirrel, the Peking duck , to reconstruct the plus strand, using
ied why HEY preferentially infects cer­ the tree squirrel and the heron have the minus strand as a template. The
tain hosts and host tissues. She has been discovered in recent years. The capsid and the viral DNA are finally en­
shown that gene S is expressed at very viral particles of these animal viruses closed in a new outer envelope as the
high levels only in liver tissue and un­ strongly resemble HEY particles. Like virus leaves the liver cell. When the vi­
der the control of steroid hormones. HEV, they have genomes that are circu­ rus exits, however, the elongation of the
These discoveries clarify studies of hu- lar, partially single-strand DNA mole- plus strand stops immediately. Conse-

LARGE PROTEIN pRE-S2

MAJOR PROTEIN j
\
MIDDLE PROTEIN

\�
I
CAPSID

VIRAL PROTEINS in the hepatitis B infectious particle (left) The color code shown here matches proteins to their genes.
are encoded by overlapping genes in the viral DNA (right). Other genes make products essential to the viral life cycle.

SCIENTIFIC AMERICAN April 1991 119

© 1991 SCIENTIFIC AMERICAN, INC

 quently, the length of the plus strand
How the Hepatitis B Virus Replicates is variable.
In its broadest outline, the amazing
The hepatitis B virus infects a liver cell. replication mechanism of the hepad­
naviruses is the mirror image of the
2 Enzymes extend the short DNA strand of the viral genome. one used by the retroviruses, such as
the AIDS virus, which have an RN A
3 The viral DNA migrates to the cell nucleus, where it is copied into genome and use a DNA molecule as
RNA. The 3.5-kilobase strand of RNA becomes the pregenome an intermediate. Hepadnaviruses and
intermediate for further replication. retroviruses have several other analo­
gous features as well. Both types can
4 The pregenome is packaged in a freshly made capsid. infect cells chronically without destroy­
Polymerase enzymes begin to make a DNA copy of the ing them . The order and function of
pregenome. the retroviral genes gag, pol and env
are directly comparable to those of the
5 The new DNA strand is a duplicate of the original long strand in hepadnaviral genes C, P and S. (Gag
the viral genome. The pregenome disintegrates as the new DNA encodes the retroviral capsid protein,
is completed. pol encodes a transcriptional enzyme
and envencodes the envelope proteins.)
6 Polymerases begin reconstructing a complementary DNA strand Both families of viruses can also cause
from the long-strand template. some cancers.
Epidemiological studies clearly indi­
7 The viral DNA may persist in the cell long enough to become fully cate that chronic infection with HEY
double stranded. It can then return to the nucleus for another or the similar hepadnaviruses is suffi­
round of replication. cient to induce liver malignancies. Mam­
mals chronically infected with hepad­
8 If the new viral particle exits the cell instead of replicating again, naviruses, for example, show a high in­
the capsid is enclosed in a fresh envelope. The extension of the cidence of liver tumors: more than 80
short DNA strand stops immediately. percent of infected woodchucks devel­
op primary liver cancers within two
years. Hans Popper of Cornell Univer­
sity, John L. Gerin of Georgetown Uni­
�:::=�Io.. ....ENVELOPE
....-- versity and their colleagues have also
shown that HCC could be experimen­
CAPSID
tally induced in woodchucks through
VIRAL GENOME
inoculation with the hepatitis virus for
that species.
Those experiments confirmed the

21
cancer-causing properties of a hepati­
tis virus and ruled out the need for car­
cinogenic cofactors. In the human dis­
�... ease, other influences may also be at
work: the period of latency preceding
CELL MEMBRANE

Q�
the development of HCC is much long­
er in people than in woodchucks, and
the tumors frequently appear in cir­
rhotic livers. Environmental carcino­
DOUBLE-STRAND DNA gens and excessive consumption of al­
cohol may still be involved in the gene­
sis of human HCC.
The question of how HEY induces
-- cancer is still open to debate. HEY might

�CELL NUCLEUS
3 directly trigger tumor development. Al­
ternatively, the liver tumor may arise in­
directly because of the chronic inflam­
mation, cirrhosis and cell regeneration
taking place in the diseased tissues.
Many tumor viruses carry oncogenes,
genes that can directly transform in­
fected body cells. (Similar oncogenes
are also found naturally in cells, where
they seem to control growth and devel­
opment.) The HEY genome, however,
does not apparently carry an oncogene.
Moreover, the long latency period be­
tween an HEY infection and the ap­
�________________________ � t� ________ �
pearance of a tumor is not compatible
with the idea that a viral oncogene

120 SCIENTIFIC AMERICAN April 1991

© 1991 SCIENTIFIC AMERICAN, INC
 READER SERVICE DIRECTORY
The advertisers listed below are making additional information available, free. Technical
narratives, as noted, are also available at no charge. Just circle the corresponding number on
the adjacent card, detach and mail.

NEW JERSEY, THE INVENTION STATE:

TECHNOLOGY FOR ECONOMIC GROWTH

Technical Narratives, by Title:

l. ACE Software 15. Prosthetic Limbs and Joints
2. Cold Extrusion 16. Fiber Optics Sensors
3. Salicylic Acid 17. PVC Sorting
4. X-Ray Laser Microscopes 18. Sol-Gel Membranes
5. Stable Isotope Assay 19. Spray Conversion Processing
6. Desorption System 20. Automatic Circuit Testing
7. Photonic Switches 2l. Computer Chip Interconnects
8. Flux Method for Producing Crystals 22. Seashell Waste Reduction
9. Chloroplast Engineering 23. Anti-Fouling Coating
10. Microvacuum Tube 24. Biopolymer Systems
11. Vaccine Vectors 25. Bone Lead Detection
12. Removing Soil Contaminants 26. Respiration Monitor
13. Psoralens 27. Reduction Photo-Dechlorination
14. Fluid Simulation Software 28. Infrared Detector Array

Advertisers:
29. American Cyanamid Co.
30. AT&T 38. Public Service Electric & Gas Co.
3l. Bellcore 39. R&D Council of New Jersey
32. CIBA-GEIGY Corp. 40. Sandoz Research Corp.
33. Hoechst Celanese Corp. 4l. David Sarnoff Research Center
34. Hoffman-La Roche Inc. 42. Schering-Plough Corp.
35. Mobil Corp. 43. Unilever Research, Inc.
36. N.J. Dept. of Commerce & Economic Development 44. U.S. Army: CECOM
37. Port Authority of New York & New Jersey 45. U.s. Army: LABCOM

46. 3-D Visions-GRAFTOOL (For information)

47. 3-D Visions-GRAFTOOL (To have a representative call) 53. Levenger
48. ARC Software 54. Lufthansa German Airlines
49. Audio-Forum 55. MathType
50. Avis Rent-A-Car Systems, Inc. 56. Microprose Software
5l. CompuServe 57. Polyhedron Software Ltd.
52. Indium Corporation of America 58. Textron Lycoming

Should the card be missing, write on a postcard the names and corresponding numbers of
the advertisers and technical narratives about which you would like additional informa­
tion, and send to:
Scientific American
April 1991
P.O. Box 5147
Pittsfield, MA 01203-9827

© 1991 SCIENTIFIC AMERICAN, INC

 liferation, and excesses of it during ear­
ly pregnancy have been linked to birth
defects. It can also make many cultured,
abnormal cells return to their normal
form and so is used to cure some leu­
kemias. A mutation in a retinoic acid
receptor gene caused by the insertion
of HBV DNA could play a part in the
\ genesis of human HCC.
CELLULAR

1;
Brechot, who is now at the Necker
GROWTH
GENE Hospital in Paris, observed the similar
VIRAL integration of HBV DNA into the cyclin
IN N A gene in another human HCC. Cyclin
A is involved in cellular growth, and
its abnormal expression can deregulate
the normal control of cell proliferation.
Much more information about the fre­
HBV CELLULAR
quency of such viral insertions into cel­
GROWTH
HBV-INFECTED LIVER CELL L.-
� GENE lular genomes is needed.
�======L TUMOR We have obtained further clues to the
=j= CELL role of viral integration in HCC from a
HBV DNA
study of woodchuck tumors induced by
woodchuck hepatitis virus. Viral DNA
LIV ER REGENERATION invariably integrates itself in these tu­
TRANSFORMATION
mors, and in about 30 percent of the
cases, we have identified the target sites
for viral integration as two members
of the cellular myc oncogene family.
Those genes are normally involved in
the control of cell growth and differen­
tiation, but they can also contribute to
ACTIVATED CELLULAR GROWTH GENE
the formation of lymphoid tumors and
various carcinomas.
THREE EXPLANATIONS for how hepatitis B infections could transform liver cells
into tumor cells have been proposed. Viral DNA may turn on cellular growth genes A part of the viral genome harboring
by integrating itself adjacent to those genes (top pathway) or elsewhere in the the enhancer element inserts itself near
genome (middle pathway). Another possibility is that the cells regenerating after the myc oncogene without modifying
the infection may erroneously activate their own growth genes (bottom pathway). the gene's code. The synthesis of nor­
mal Myc proteins, now triggered by
viral information, escapes its normal
causes HCC. It is nonetheless conceiv­ chronic HBsAg carriers, they were also cellular control. Consequently, the cell
able that the gene-activating protein found in tumors from HBsAg-negative overproduces the Myc proteins that
produced by HBV gene X might be in­ patients. Integrated viral DNA was also induce unregulated cell growth. This
volved at some early stage of the tu­ occasionally detected in the livers of mechanism is basically identical to the
morigenic process. chronic HBV carriers, which suggests way in which retroviruses are known
A virus can also transform a host cell that the DNA integration may occur be­ to cause lymphoid tumors in rodents
by introducing its DNA into the cel­ fore or at a very early stage of tumor and birds.

�
lular chromosomes. The insertion of vi­ formation. There is as yet no evidence that a
ral sequences adjacent to cellular onco­ similar pathway leads to the develop­
genes can deregulate their expression alYSeS of tumors and cell lines ment of human HCC: extensive studies
and lead to uncontrolled growth. That have shown that HBV can insert have failed to reveal insertional muta­
strategy is typical of some retroviruses its DNA at many different sites tions in myc oncogenes caused by HBV
that induce leukemias and carcinomas in human chromosomes. It often induc­ DNA . Nevertheless, other human onco­
in mammals and birds after a laten­ es substantial genetic rearrangements, genes might be activated either direct­
cy period. Although HBV does not nor­ such as chromosomal deletions, trans­ ly or indirectly by the HBV insertion.
mally need to integrate genetic materi­ locations or amplifications-abnormal­ Peter Hans Hofschneider of the Max
al into its host 's genome to complete ities common in human tumors and Planck Institute in Munich has shown
its life cycle, such integrated forms cancers. The consequences of those ge­ that integrated HBV DNA can produce
might be incidental by-products. netic events are still unknown. modified proteins that stimulate the
Christian Brechot, while working in In one early human HCC, Anne De­ expression of both viral and cellular
our laboratory, David A. Shafritz of the jean of our laboratory found a single genes, including potent oncogenes. A
Albert Einstein College of Medicine and insertion of HBV DNA into one mem­ more indirect role for the virus was re­
William J. Rutter of the University of ber of a gene family that makes recep­ ported by Francis V. Chisari of the Re­
California at San Francisco have ana­ tors for thyroid and steroid hormones. search Institute of Scripps Clinic: in
lyzed DNA molecules from HCCs and She was able to isolate and character­ transgenic mice, overproduction of the
found that the viral sequences were in­ ize that gene and identify its speCific HBV large protein leads to chronic liver
tegrated into the chromosomes of the product as a receptor for retinoic acid. cell injury and finally to HCC.
tumor cells. Although such sequences Retinoic acid exerts a powerful in­ In DNA transfer experiments, Keni­
were mainly observed in tumors from fluence on cell differentiation and pro- chi Matsubara of Osaka University has

122 SCIENTIFIC AMERICAN April 1991

© 1991 SCIENTIFIC AMERICAN, INC

demonstrated the activation of two oth­ empty envelopes bearing HEsAg-pu­ Another intriguing recombinant DNA
er cellular oncogenes: /ca, a gene that rified from the blood of chronic car­ approach would be to construct a live
has so far been implicated only in HCC, riers and treated to eliminate all in­ vaccine using the vaccinia virus or the
and hst-1, which is also involved in the fectious particles. It was the only anti­ adenovirus as vectors. Genetic engi­
formation of stomach cancers. Other viral vaccine ever developed from the neering could make these harmless vi­
genetic alterations that are not obvi­ blood of patients. Maupas and his col­ ruses express HEV antigens. This tech­
ously linked with HEV infection have leagues later clearly demonstrated the nique has not been applied to vaccines
also been observed in human HCC tu­ safety and efficacy of the vaccine for a for humans until now. It should also be
mors. Further studies are needed to very large group of recipients, includ­ possible to chemically synthesize ami­
understand the viral role in carcinogen­ ing many infants in regions where HEV no acid sequences that would duplicate
esis at the molecular level. is endemic and high-risk populations the antigenic determinants of HEsAg,
where it is not endemic. (The vaccine and these could provide an inexpensive -

T
he preceding experiments dem­ was less effective in patients on dialy­ HEV vaccine. Unfortunately, such an ap­
onstrate how instrumental re­ sis, probably because of their weakened proach is not practical today, because
combinant DNA technology has immune systems.) the immunogenicity of such artificial
been for understanding the basic biol­ Although the serum-derived vaccine peptides, like those made by bacteria,
ogy of HEV. The technology also has is effective, producing it poses several is extremely low.
practical applications in the develop­ problems: the supply of human serum In addition to producing vaccines
ment of new diagnostic techniques and from chronic carriers is limited, and that can prevent HEV infection, recom­
vaccines. the purification procedure is long and binant DNA technology may also be on
Diagnostic tests for hepatitis B have expensive. Moreover, each batch of vac­ the verge of finding a cure for chronic
been improved by the technique called cine prepared from a different stock of carriers. In August 1990 Robert P. Pe­
molecular hybridization, which capi­ infected human serum must be tested rillo of Washington University and his
talizes on the high affinity that short on chimpanzees to ensure its safety. colleagues announced that daily treat­
strands of cloned DNA have for related For all these reasons, the idea of ap­ ments with alpha interferon, a genet­
complementary DNA . Cloned, labeled plying genetic engineering to produce a ically engineered protein that boosts
viral DNA can serve as a sensitive, re­ hepatitis B vaccine was a very attractive immune responses, had eliminated or
liable probe for detecting infectious vi­ one to researchers. inhibited infection in more than a third
ral particles in blood serum. Current­ of 85 chronic HEV carriers. Further re­

S
ly molecular hybridization using HEV everal genetic engineering tech­ search on the use of interferon against
DNA is routinely used to identify re­ niques can be used to develop vac­ HEV promises to refine and improve
cent hepatitis B infections. Because the cines, but the choices are more such treatments.
identification of viral DNA also allows restricted in practice because HEsAg During the past decade, the evolv­
an accurate estimation of how much is not fully immunogenic unless it re­ ing understanding of the molecular bi­
viral replication is taking place, mo­ tains its complete natural structure. Ge­ ology of HEV has found medical ap­
lecular hybridization is also suitable netically engineered bacteria can mass­ plications, particularly for preventing
for following the progress of antiviral produce HEsAg, but the antigen does the infection. Many of the benefits have
treatments. not have its correct chemical form or barely been tapped. In developing coun­
The sensitivity of the method can structure. Yeast and manunalian cells, tries, mass vaccinations with the re­
be further improved by using the poly­ however, can produce useful HEsAg combinant vaccine-the first vaccine
merase chain reaction technique, which proteins. for human use ever developed by re­
is a simple enzymatic procedure for du­ HEV vaccines produced in yeast or combinant DNA technology-will go far
plicating even traces of specific DNA se­ mammalian cells are now on the mar­ in controlling hepatitis B. Such a cam­
quences at exponential rates [see "The ket. Rutter obtained a vaccine by pro­ paign would bring twofold relief: the
Unusual Origin of the Polymerase Chain ducing one in Saccharomyces cerevi­ vaccinations would prevent not only
Reaction, " by Kary B. Mullis; SCIENTIFIC siae (baker's yeast). In 1984 Marie Lou­ the acute liver illness but also the as­
AMERICAN, April 1990). With this tech­ ise Michel of our laboratory genetically sociated cancer. We can hope that it
nique, hybridization-based diagnostic constructed a line of Chinese hamster would usher in an era of better medical
tests can increase their sensitivity more ovary cells that produce HEV antigens. care based on prudent applications of
than 1, 000-fold: they can detect as few The recombinant HEV DNA that she in­ genetic engineering.
as 100 viral particles in a milliliter of serted contained both the S gene and
serum, which is considered to be the the pre-S2 region. The resulting parti­
minimum infectious concentration . cles therefore carry both the major and FURTHER READING
Recombinant DNA technology is also the middle envelope proteins and bear ADVANCES IN HEPATITIS RESEARCH. Edit­
developing new vaccines. Such progress both the HEsAg and pre-S2 antigenic ed by Francis V. Chisari. Masson Pub­
began in 1970, when Saul Krugman determinants. lishing USA , 1984.
and his co-workers of New York Univer­ That fact may be significant because HEPATITIS B. Edited by Robert J. Gerety.
Academic Press, 1985.
sity demonstrated that the heat-inacti­ David R . Milich of the Scripps Clinic
THE HEPATITIS B VIRUS. Pierre Tio\lais,
vated serum of a chronic carrier could has shown in mice that the pre-S2 de­
Christine Pourcel and Anne Dejean in
protect susceptible persons against terminant elicits a stronger immune re­ Nature, Vol. 3 17, No. 6037, pages 489-
HEV. Later, several groups showed the sponse than HEsAg. Moreover, in mouse 495; October 10, 1985.
protective effect of purified viral parti­ strains that do not produce antibodies THE MOLECULAR BIOLOGY OF TIlE HEP­
cles carrying HEsAg in chimpanzees. against HEsAg, immunization with Mi­ ATITIS B VIRUS ES . Don Ganem and H. E .
In 1976 Philippe Maupas and his chel's recombinant particles can circum­ Varmus in Annual Review of Biochem­
co-workers of the University of Tours vent the nonresponsiveness. This prop­ istry, Vol. 56, pages 651-693; 1987.
VIRAL HEPATITIS AND l.IVER DISEASE. Ed­
reported the first results of vaccina­ erty could be important for the vacci­
ited by Arie J. Zuckerman. Alan R. Liss,
tion in humans. Their vaccine consisted nation of people who are unresponsive lne., 1988.
of defective viral particles-essentially, to that antigen.

SCIENTIFIC AMERICAN April 1991 123

© 1991 SCIENTIFIC AMERICAN, INC