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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 #{149} Reduced cost of production #{149} Safety of manufacture #{149} 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: #{149} in diagnostic assays #{149} in antibody induction #{149} as affinity chromatography ligands for antibody purification #{149} in competition assays #{149} as reagents to map epitopes #{149} 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: #{149} Post-translational modifications #{149} Conformational epitope requirements #{149} 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. References 1. Darnell J, Lodish H, Baltimore D. Molecular cell biology. New York: WH Freeman & Co., 1986. 2. Atassi MZ. Antigenic structures of proteins. Eur J Biochem 1984;145: 1-20. 3. Hood LE, Weissman JL, Wood WB. Immunology. Menlo Park, CA: Benjamin/Cummings Publishing Co., 1978. 4. Kieney MP, Lathe R, Riviere Y, et al. Improved antigenicity of the HW e,w protein by cleavage site removal. Protein Eng 1988;2:219-25. 5. Chang TW, Kate I, McKinney S. et al. Detection of antibodies to human T-cell lymphotropic virus-ifi (HTLV-ffl) with an immunoassay employing a recombinant Escherichia coli-derived viral antigenic peptide. Bio/Technology 1985;3:905-9. 6. Sternberger LA. Immunocytochemistry, 2nd ed. New York: John Wiley & Sons, 1979. 7. Flexner C, Murphy BR, RooneyJF, et al. Successful vaccination with a polyvalent live vector despite existing immunity to an CLINICAL CHEMISTRY, Vol. 35, No. 9, 1989 1841 expressed antigen. Nature (London) 1988;335:259-62. 8. Mimms L, Staller J, Mushawhar 1K, et al. Production, purification, and immunological characterization of a recombinant DNA-derived hepatitis B e antigen. In: Viral hepatitis and liver disease. New York: Alan R Liss, Inc., 1988:248-51. 9. Decker RH, Kuhns MC, Brawner TA, et al. Future advanced diagnostic techniques for hepatitis B. Ibid.:231-6. 10. Decker RH, Dawson GJ. 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