Download Antigens Produced by Recombinant DNA

CLIN. CHEM. 35/9, 1838-1842 (1989)
Antigens Produced by Recombinant DNA Technology
J. Lawrence Fox and MIchael KIa8s
Some of the greatest beneficiariesof the revolutionaryadvances in biotechnology over the past 15 years have been
producers of diagnostic reagents, especially for the cloning
and expressionof antigens,primarilyof viral origin.Recombinant DNA technology provides methods for producing
antigensfor diagnosticassays that are more highlypurified,
more specific,and safer to producethan viralcultureand that
are significantlyless expensive to manufacture.Antigensso
produced can be used for productionof antibodiesor antisera for competition assays, as reagents for mapping
epitopes, as affinity-chromatographyligandsfor purification
of antibodies or protein, and as research reagents. Their
initial use in some hepatitis B assays may be primarilya
cost-reduction application, but in otherapplications(e.g., HIV
diagnostic tests) they present the first opportunity to commercially produce an otherwise very expensive antigen.
Recombinant-DNA-producedantigensare alsobeingusedto
develop safer vaccines, but not, however, without some
consideration of the structural nature of immunodominant
epitopes and the adequacy of the immune response.
The emergence
of recombinant
DNA technology
has had
far-reaching
consequences
for diagnostic
products, not only
affecting production
methodology
but, perhaps more importantly,
also opening many new doors for research efforts.
Robert Gallo (personal communication,
1988) has stated
that without
recombinant
DNA technology it would have
taken at least two decades to understand
the human
immunodeficiency
virus (HIV), with consequences
much
more disastrous than they in fact are.’
Molecular
BIology
Background
Central to an understanding
of recombinant antigens is
an understanding
of recombinant DNA technology (the
reader may want to consult a more extensive presentation
of this technology; Weatherahl’s The New Genetics and
Clinical Practice is recommended).
The flow of information
in biological systems is generally DNA
RNA - protein.
DNA serves as the genetic repository for all living orgamama, but must be transcribed
into a related molecule,
RNA, to finally find its expression through translation into
protein sequences.
The linear sequence of the nucleic acid bases in DNA
molecules
codes for each of the 20 amino acids. When the
DNA sequence is known, the amino acid it encodes is
unambiguously
identified.
If the amino acid is known, one
can work backwards
to the series of three nucleic acid bases
in the DNA (a codon) that encodes it; however, this “reverse” translation
process is not unambiguous.
The nature of the process of expressing information
-,
Corporate Molecular Biology, Abbott Laboratories, Abbott Park,
IL 60064.
‘Nonstandard
abbreviations: HIV, human immunodeficiency
virus; mRNA, messenger RNA; cDNA, complementary DNA;
MAb, monoclonal antibody; and CEA, carcinoembryonic antigen.
Received April 13, 1989; accepted June 16, 1989.
1838
CLINICALCHEMISTRY,Vol. 35, No. 9, 1989
contained in DNA into a protein sequence of amino acids is
different for bacteria, which are prokaryotes (cells without
nuclei), and organisms with eukaryotic (nucleated) cells,
e.g., humans. In bacteria, the process of RNA transcription
is directly coupled to protein translation. In higher plants
and animals, the process is complicated by the presence of
introns, noncoding regions interspersed within the aminoacid-coding
regions (exons) of the gene (see Figure 1). The
synthesis of RNA is contained within the nuclei of eukaryotic cells and produces an RNA species that is much larger
than the finally processed messenger RNA (mRNA), typically by an order of magnitude, because it is a transcript of
both introns and exons. This primary RNA transcript must
undergo maturation processing, during which the introns
are spliced out. Only after this step is a mature RNA
molecule formed, mRNA, that can direct the synthesis of a
protein.
Recombinant DNA Background
Recombinant DNA technology is based on the manipulation of DNA molecules. Because working with DNA,
which contains introns, increases the complexity of the
task by an order of magnitude, an alternative
approach was
adopted. If one isolates mature mRNA and uses a viral
enzyme that can use RNA as a template on which to
reverse-transcribe
DNA, then a complementary
DNA
(cDNA) can be made. This cDNA is a direct copy of the
mRNA and does not contain introns. This simplifies the
problem of DNA manipulation.
To utilize the DNA isolated from an organism or the
cDNA generated from an organism’s mRNA, it must be
inserted into another piece of DNA. This recipient DNA,
called a vector, is typically a circular DNA double helix
(Figure 1). To open the circle to insert the new DNA, the
vector must be cut. A series of enzymes, restriction endonucleases, have been isolated that recognize and cut at
specific sequences of bases in double-stranded DNA. Endonucleases are ordinarily found in bacteria (their names are
derived from the strains of bacteria from which they are
isolated) and serve to restrict the kinds of DNA that can be
taken up and used by a bacterial cell. To the bacterial cell,
they are a defense mechanism against foreign DNA, but to
the molecular biologist, they are the tools required to
engineer DNA.
Once the vector DNA is cut, any desired DNA can be
inserted in the vector. The result of combining vector DNA
with a DNA insert creates a recombinant DNA. Bacterial
cells can now be made to take up exogenous DNA, usually
by treating them with calcium ions. The efficiency of this
bacterial transformation process (see Figure 2), the giving
of new genetic information to a recipient or host cell, is
about 1 in iO.
Thus the major remaining problem is how to identify
those bacterial cells that contain the DNA insert of interest
from a large number of cells that contain either nothing or,
perhaps, other inserts. This screening process may be
accomplished by DNA probe screening or antibody expression screening. The remaining task is to insert the DNA
Expression
Eukaryote
cDNA
Piasmid
1
Secreted Proleln
Bacterium
Fig. 1. Expressionand insertionof geneticinformationin recombinantDNA techniques
0
The genetic information is maintained in eukaryotic cells in the chromosomes contained in the cell’s nucleus.The process ofRNA transcription initially creates a
“very large”heteronuclear
(hn) RNA molecule that must undergo processing to evolve into the messenger ANA (mRNA), which ultimately codes for protein
synthesis. The process includes
joining theexons(expressedregions,shown in bold vertical line)bysplicingouttheintrons(interveningregions, the loops). Protein
tranalation occurs outside of the nucleusin the cytoplasm of the cell. For recombinant DNA manipulationsof eukaryotic proteins, it is most useful to isolate the
mRNA,whichnolongerhas the lntronspresent,and use it as a templatewiththeviralenzyme“reversetranscriptase”to prepare complementary DNA(cONA).
The cDNAcan then be inserted into a bacterialplasmid and used totransforma bacterium(theinserts are highlightedby cross-hatching). Once insidea bacterium,
a plasmid is typically replicatedinto50-100 copies
sequence of interest
into a suitable expression system and
produce large quantities of protein. For a review see Darnell et al. (1).
Producing Antigens by Recombinant
DNA Technology
General Advantages
and vaccines.
Such antigens offer several advantages:
#{149}
More abundanticonsistent
supply
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Reduced cost of production
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Safety of manufacture
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Potential
By definition, an antigen is a foreign protein, i.e., virtually any nonhuman
protein recognized as foreign by our
immune system (2, 3). Practically, however, antigens tend
to be surface proteins of viral, bacterial,
or protozoan
origin. In nature, they exist in relatively minute quantities, which, in the past, made them intractable to experimentation or investigation and precluded any practical, let
alone industrial,
use. Recombinant DNA technology has of
course changed this entirely. Recombinant-DNA-produced
antigens have become a powerful tool in both diagnostics
for genetic
manipulation
The general advantages
of recombinant
antigens are
many. Today, even average-size (200 L) bacterial fermenter
vessels can produce gram quantities of an antigen (protein)
free from nonhost proteins. For example, p41, the envelope
protein of HIV-1, can be expressed
in Escherichia
coli so
that hundreds of milligrams of purified p41 can be produced
free from any other HIV-1 or non-E. coli proteins. Furthermore, the yield of recombinant
antigen is consistent; i.e.,
repetition of the same protocol will yield identical product,
thus reducing the variability observed when protein is
Repllcator
Replicator
Eco RI
Foreign
oo
DNA
oOC’c
Translormed
E. coil
Translormallon
Chromoeome
Fig. 2. Production of a recombinant DNA molecule by cutting a vectorwithan endonucleaseand insertingthe foreignDNA
Combiningthe two DNAsand covalentlyligatingthemtogetherproducesa recombinantDNAmolecule,whichcan be used to transforma bacterialcell. The vector
(here it is a plasmid) can multiply many times in a bacterial cell, typically achieving a 50- to 100-foldamplification
CLINICALCHEMISTRY, Vol.35, No. 9, 1989
1839
isolated from poorly controllable biological sources, e.g.,
tissue, sera, etc.
Not only can consistent, pure, abundant quantities be
obtained, but also the cost of this is significantly less than
that for product isolated from natural sources, e.g., tissue
culture, human tissues, or live animals. Although each
antigen is different, a general rule of thumb is that the
production in E. coli and yeast (or equivalent prokaryote) is
1/100 to 1/1000 the cost of production in tissue culture. This
is especially true for some particularly virulent pathogens,
e.g., HIV antigens, which previously had to be isolated
from intact virus grown in tissue culture in special containment facilities by specially trained technicians-all
of
which significantly increased the cost.
Safety is another general advantage of recombinant
antigens. Because one is working with only one, or a few, of
the genes of the intact infectious agent, the danger of
infection, which requires a complete gene complement, is
eliminated.
Contrast this with the isolation of live, intact,
infectious H1V virus from tissue culture in strictly controlled biological and physical containment facilities. Although it has been proposed that working with recombinant-DNA-produced
antigens might lead to seroconversion, no incidents have been observed to date.
Finally, cloning the gene for the desired antigen empowers the investigator with all the tools of modern molecular
biology for making any desired modifications such as insertions, fusions, and (or) deletions to the recombinant antigen. Such modifications may actually improve the antigenicity of the recombinant antigens. For example, removal
of a cleavage site in the HW enu protein appears to increase
its antigenicity (4). Furthermore,
specific deletions allow us
to obtain antibodies to whatever region may be deemed
most important for a diagnostic assay, e.g., the main
immunogenic
region for H1V-1 (5). In addition, if a recombinant-DNA-produced
antigen is less immunogenic than
desired, it can be genetically fused with a protein of high
immunogenicity
(6). Further,
this technique
allows the
production of polyvalent antigens to induce immunity to
multiple infectious agents simultaneously
(7).
Applications
Recombinant-DNA-produced
antigens
have many
impor-
tant uses in medical research:
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in diagnostic assays
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in antibody induction
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as affinity chromatography ligands for antibody purification
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in competition assays
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as reagents to map epitopes
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as basic research tools
Recombinant-DNA-produced
antigens are beginning to
be used in an ever-increasing number of diagnostic assays.
For example, recombinant-DNA-based
diagnostic assays
for hepatitis B (8, 9), HIV (10, 11), carcinoembryonic
antigen (CEA) (12), and Haemophilus
influenzae
type b P2
protein (13) are in various stages of use or development. A
search of patent applications worldwide revealed that in
the past decade more than 87 applications have been filed
for diagnostic tests based on the use of recombinantDNA-produced antigens.
As diagnostic reagents, these antigens are extremely
valuable for the induction of both polyclonal and monoclonal antibodies. Once the gene encoding the desired antigen
is cloned, virtually any region of the protein can be ex1840
CLINICALCHEMISTRY,Vol. 35, No. 9, 1989
pressed and used to obtain region-specific antibodies (14).
In addition, any part of the antigen may be expressed as a
fusion protein, similar to the practice of conjugating a
hapten to a larger, highly immunogenic protein (6). Such
recombinant fusion proteins can be important
for stimulating an immune response. Fusions between different proteins have also been used to construct polyvalent antigens.
For example, a Vaccinia virus that expresses the surface
proteins of influenza A virus hemagglutinin
and herpes
simplex type 1 (HSV-1) glycoprotein D induced antibody
response to both proteins, producing immunity in mice (7).
Another
use for recombinant-DNA-produced
antigens is
in the purificationlisolation
of specific antibodies. Antigen
can be bound to a chromatography
matrix to produce an
affinity column (15), through which antibody preparations
are passed. Depending on the part or domain of the antigen
used, this affinity chromatography
can yield preparations
of mono-epitopic antibodies. Such techniques have been
used to obtain both high- and low-affinity antibodies from
polyclonal sera, depending on the elution salts used on the
column.
In some diagnostic assays, the antibody used often crossreacts with similar antigens that are usually present. For
example, in diagnostic assays for CEA, a marker for colon
cancer, many anti-CEA antibody preparations
cross-react
with related members
of the CEA family (13). Such crossreactivity increases the background response and may lead
to false-positive results. One key to the solution of this
problem lies in detecting regions of amino acid sequence of
the desired antigen that are unique and not found in the
ordinarily
cross-reactive antigens. The DNA encoding this
unique region is then cloned and expressed separately to
yield a mono-epitopic antigen capable of inducing noncross-reacting antibodies.
If this antigen is used to affinity-purify
polyclonal sera,
then only antibodies that bind this specific region of the
antigen will be isolated. Similarly, such subdomains
of
recombinant-produced
antigens can by themselves be used
to obtain monoclonal
antibodies
(MAbs). The polyclonal
approach has the advantage of consistently giving highaffinity antibodies, whereas most MAbs display low aflinities.
Recombinant
antigens are also used in diagnostic assay
configurations.
They can provide superior quantities
of
reagent to serve as positive controls, and they are used
extensively in antibody capture assays. For example, Abbott’s Second Generation HIV diagnostic assays are configured with recombinant antigens (HIV p41 and p24) bound
to polystyrene beads. The sample is added and any antiHIV antibodies top41 or p24 are bound to the recombinant
antigens on the beads. These human antibodies are then
detected with an anti-human IgG conjugated to horseradish peroxidase (EC 1.11.1.7). A strong color reaction from a
substrate cleaved by horseradish peroxidase indicates the
presence of anti-H1V antibodies in the patient’s serum.
The HIV diagnostic assay EnvaCor (Abbott Labs.) is
configured with human IgG anti-fflV bound to polystyrene
beads. The sample to be tested is mixed with a limited
amount of recombinant antigen (p41 envelope or p24 core).
If there is no anti-HIV antibody in the patient’s serum, all
the recombinant antigen will bind to the anti-HIV antibody
on the bead. This bound antigen will then be detected by
the addition of horseradish peroxidase-conjugated
MAb to
lilY (p41 or p24). A strong color reaction caused by the
peroxidase is indicative of a sample negative for anti-HIV
antibodies. If the patient’s sample does contain anti-HJV
antibodies, they will compete for the recombinant antigen;
this is indicated by a loss of color. Results with recombinant
antigens have been comparable with (and are generally
superior to) those with native antigens from viral lysates
(10, 11).
Another advantage
to using recombinant antigens in
diagnostic assays is the ability to independently
assay
different antigens, each of which may be diagnostic for a
different disease state. For example, Decker and Dawson
(10) found that anti-core antibody titers decreased
dramatically when persons infected with HIV entered the symptomatic state of AIDS. Thus, the ratio of anti-core to anti-enu
could probably be used to follow disease progression (1618).
Caveats
Despite the many advantages
recombinant
antigens
have as diagnostic reagents, it is important
to recognize
that there are a number of caveats:
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Post-translational
modifications
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Conformational
epitope requirements
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Oversimplification
of epitopes
Recombinant
antigens expressed in prokaryotic organisms lack most, if not all, of the post-translational
modifications typically found in eukaryotic proteins. Modifications such as glycosylation,
phosphorylation,
etc., if they
constitute immunologically
important epitopes, would not
occur, so epitopes important for diagnostic assays may be
missing.
Furthermore, recombinant
antigens expressed in prokaryotic hosts will often not possess the same conformational epitopes as the native antigen. If conformational
or
discontinuous epitopes constitute the major immunodominant region of an infectious agent, then use of recombinant
antigens from prokaryotic hosts may not detect antibodies
to the infectious agent. For example, normally
protective
MAbs to the Bordetella
pertussis
Si toxin will only bind to
the recombinant antigen expressed in E. coli after refolding
of the protein to allow the formation of a discontinuous
epitope (19). Conformational
or discontinuous
epitopes are
also a major concern for the infectious agents causing
hepatitis A (20-22),
polio (23), and foot and mouth disease
(24), to name but a few cases.
Recombinant
antigens produced in prokaryotes
do not
reconstitute these epitopes. In such cases, special efforts are
required
to achieve the identical conformation and posttranslational
modifications found in eukaryotic proteins.
These efforts include cloning and expression in appropriate
tissue culture systems capable of the same post-translational modifications. In the case of hepatitis A virus this is
done by growing the virus in tissue culture (25, 26).
Unfortunately,
as mentioned above, expression in tissue
culture
is more
expensive
than
in bacteria,
and
working
with live infectious agents is not desirable.
Finally, in configuring diagnostic assays that seek to
determine
in the patient the presence of antibody directed
to an infectious agent, one must always determine how
many epitopes are sufficient to adequately detect all infected individuals. For example, in an HN diagnostic test
is it sufficient to use only p41 as the recombinant antigen or
are p24 or other HIV proteins required? This question
unfortunately must be answered by the thorough screening
of thousands of confinned samples.
Recombinant
Recombinant
Antigens for Vaccines
antigens
are becoming
increasingly
impor-
tant in the development of vaccines. Recombinant
DNA
technology can provide abundant quantities of antigen
required for the development of vaccines that were previously intractable owing to the scarcity of reagents. Not only
can sufficient immune response be induced, but this can be
accomplished without the risk associated with the use of
live infectious agents. As such, recombinant antigens provide a relatively inexpensive,
abundant source of consistently pure material for vaccination. Vaccines for many
infectious agents, e.g., cholera toxin B (27), hepatitis B
(28-30),
lilY
(31),
influenza A (32), malaria
(33), etc.,
prepared by using the respectively cloned genes and the
resulting recombinant antigens, are in various stages of
development.
Furthermore,
because of the flexibility of
recombinant
antigens, polyvalent antigens can be obtained
and used to develop immunity to multiple infectious agents
at the same time (8).
Although recombinant
antigens possess important
advantages for the development
of vaccines, they are not
necessarily
an automatic success. For example, HW vaccine based on env expressed from recombinant
Vaccinia
virus in whole animals has elicited low titers of lilYreactive antibodies (4).
Obviously, more factors must also be considered. First,
the recombinant antigen must possess a sufficient number
of epitopes to induce adequate immunity. If such immunity
requires the presence of post-translational
modifications or
conformational
epitopes, then vaccination with recombinant antigens produced in bacteria will most likely not
succeed. In addition, infectious agents with rapidly changing or highly variable epitopes-e.g.,
common cold (Rhinovirus) (34), malaria
(Plasmodium
falciparum)
(35, 36),
sleeping sickness (Trypanosoma
brucei) (37, 38), group A
streptococci (39), and, most likely, lilY-i (31, 40)-will
probably remain intractable
to immunization
by a single
recombinant antigen. Newer, more clever and complex
immunization schemes mimicking the natural variation of
these infectious agents or blocking their cell receptor sites,
as in the case of CD4 for AIDS, will have to be developed.
Current efforts involving vaccination
with live recombinant Vaccinia virus provide hope for circumventing
this
problem. Recombinant
Vaccinia expressing
the desired
antigens in the actual host facilitates the natural
posttranslational modifications and conformational folding (7,
41). This is, perhaps, the most promising expression system
for recombinant vaccines we possess today.
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