Molecular Biotechnology: Principles and Applications - Glick B., Pasternak J. 2002

Molecular Biotechnology of Microbial Systems
Vaccines
Subunit Vaccines

Generally, Vaccines contain intact but non-living or attenuated pathogenic microorganisms. Antibodies produced in response to their administration bind to the surface Proteins of the pathogen and trigger an Immune Response. This raises the question: should a vaccine contain whole Cells or only specific surface components? In the case of Viruses, it has been shown that purified viral surface proteins (capsid or envelope proteins) are sufficient to induce Antibody production in the host Organism in response to viral infection (Fig. 11.1). Vaccines containing only individual components of a pathogen are called "subunit" vaccines; Recombinant DNA technology is successfully used for their development.

Subunit vaccines have their Advantages and disadvantages. The advantages are that a preparation containing a purified immunogenic protein is stable and safe, its chemical properties are known, and it lacks additional proteins and Nucleic Acids that could cause undesirable side effects in the host. The disadvantages are that purifying a specific protein is expensive, and the conformation of the isolated protein may differ from its in situ state (i.e., within the viral capsid or envelope), which can alter its antigenic properties. The decision to produce a subunit vaccine is made considering all relevant biological and economic factors.

Class="center">Table 11.1. Pathogenic microorganisms for which vaccines are currently under development

Fig. 11.1. Structure of an animal virus. The viral genome is typically represented by a relatively small nucleic acid molecule (single- or double-stranded DNA or RNA ranging from 3 to 200 kb) enclosed in a protein capsid. In some viruses, the capsid is further surrounded by a protein envelope.

Herpes vaccines

Herpes simplex virus (HSV) causes systemic or localized infectious diseases (severe eye damage, encephalitis, urogenital infections, etc.). In addition, it is oncogenic, so vaccination with killed or attenuated virus carries a certain risk of Cancer development. To protect against HSV infection, a non-oncogenic subunit vaccine can be used.

To develop any subunit vaccine, one must first identify the Components of the pathogen that induce antibody production. In the case of HSV type 1 (HSV-1), this component is the envelope glycoprotein D (gD). In response to the administration of this glycoprotein, mice produce antibodies that neutralize intact HSV. The HSV-1 gD Gene was isolated, cloned into a mammalian expression vector, and introduced into Chinese hamster Ovary (CHO) cells, which, unlike E. coli, glycosylate foreign proteins. The full-length gD gene encodes a protein that normally binds to the mammalian Cell membrane (Fig. 11.2, A). Such a protein is harder to purify than a soluble one, so the gD gene was modified by removing the portion encoding the C-terminal transmembrane domain (Fig. 11.2, B). CHO cells were then transformed with the modified gene; these cells glycosylated the protein product and secreted it into the extracellular medium, as it could not integrate into The Cell membrane. Laboratory tests showed that antibodies produced in response to the modified gD protein are effective against both HSV-1 and HSV-2.

FOOT-and-Mouth disease vaccines

Foot-and-mouth disease virus (FMDV) is highly virulent and causes mass mortality in cattle and swine. To protect against FMDV infection, a vaccine containing formalin-inactivated virus is used. Approximately 1 billion doses of this vaccine are produced worldwide annually.

Fig. 11.2. A. HSV-1 gD molecule with its transmembrane domain embedded in The Plasma Membrane. B. Soluble gD protein lacking the transmembrane domain.

The major antigenic determinant inducing antibody production is viral capsid protein 1 (VP1). Although it is a weaker antigen than intact Viral Particles, it still induces antibody production and provides protection against infection in animals. Therefore, attempts were made to clone the VP1 gene.

The FMDV genome is a single-stranded RNA. Therefore, a full-length double-stranded cDNA of approximately 8000 bp was first synthesized. It was then digested with Restriction Endonucleases, and the resulting fragments were cloned into an E. coli expression vector. The product of the VP1 gene coding sequence was immunologically identified as part of a fusion (chimeric) protein, whose synthesis is controlled by the p^L promoter-cI repressor system. The protein consists of 396 amino acid residues and contains a portion of the bacteriophage MS2 replicase molecule along with the full-length FMDV VP1 protein, which enables it to induce The production of FMDV-neutralizing antibodies.

Obtaining regulatory approval for a vaccine containing a chimeric protein is very difficult, so the VP1 sequence will likely need to be subcloned into another expression vector. In any case, a subunit vaccine against foot-and-mouth disease will soon be ready for preclinical trials.

Tuberculosis Vaccines Tuberculosis is a systemic infectious disease widespread worldwide. Its causative agent is the bacterium Mycobacterium tuberculosis. It infects various Tissues and Organs (most commonly the Lungs) and leads to cell death. Patients experience fever and weight loss, and without Treatment, the disease is fatal. It is estimated that about 2 billion people are infected with this pathogen, and tuberculosis claims approximately 3 million lives annually. For the past 50 years, Antibiotics have been used to treat tuberculosis, but many drug-resistant strains of M. tuberculosis have already emerged, so

the disease, which once seemed conquered, has again become a serious problem.

Currently, several countries use a strain of Mycobacterium bovis, Bacillus Calmette–Guérin (BCG), as a tuberculosis vaccine. However, the efficacy of this approach is questionable for two reasons: 1) live BCG cells can cause serious illness in immunocompromised individuals (e.g., AIDS patients); 2) individuals who have received the BCG vaccine test positive in routine screening for tuberculosis-causing bacteria, making it impossible to distinguish them from active tuberculosis patients. Consequently, the BCG vaccine is not approved for use in some countries, including the United States. In attempts to develop a safer and more effective subunit tuberculosis vaccine, the immunoprotective properties of purified extracellular proteins of M. tuberculosis were investigated. Six major proteins out of 100 secreted proteins were isolated and purified from liquid bacterial culture, and each of them individually, as well as in various combinations, was used to immunize guinea pigs. The animals were challenged with an aerosol of approximately 200 live M. tuberculosis cells, which is a very high dose for them. After 9–10 weeks, the animals were euthanized, and their lungs and spleens were examined for the presence of the pathogen. With certain combinations of purified proteins, weight loss, lung and Spleen damage, and mortality rates were comparable to those observed with live BCG immunization. The next step is to compare the efficacy of M. tuberculosis proteins produced via recombinant DNA technology with that of natural secretory proteins, and to develop a safe and effective vaccine for the Prevention of human tuberculosis.

Peptide vaccines

This raises the next question: can a small region of a protein molecule (a domain) serve as an effective subunit vaccine and induce antibody production? Intuitively, it seems that domains accessible to antibodies (i.e., those on the viral surface) possess immunogenic properties, while internal domains are non-essential unless they affect the conformation of the immunogenic domain (Fig. 11.3). If this assumption is correct, short Peptides mimicking epitopes (antigenic determinants) could be used to design vaccines.

Fig. 11.3. Generalized membrane-bound protein, whose external epitopes (1–5) can induce an immune response.

With this in mind, VP1 domains of FMDV were chemically synthesized, and the feasibility of developing peptide vaccines based on them was tested. Peptides corresponding to amino acid residues 141–160, 151–160, and 200–213 of the C-terminal region of VP1, and residues 9–24, 17–32, and 25–41 of the N-terminal region, were individually conjugated to an inert carrier protein (keyhole limpet hemocyanin) to prevent degradation, and administered to guinea pigs (Fig. 11.4). Antibody synthesis sufficient to protect the animals from subsequent FMDV infection was observed only with the administration of peptide 141–160. In contrast, administration of the entire VP1 or peptides 9–24, 17–32, and 25–41 induced antibody synthesis in lower amounts.

Fig. 11.4. Short peptides conjugated to a carrier protein, serving as the basis for a peptide vaccine.

A longer peptide consisting of amino acid residues 141–158 and 200–213, linked by two Proline residues, induced effective antibody synthesis in guinea pigs even when not conjugated to a carrier protein. This "di-peptide" molecule proved more effective than any isolated peptide and blocked FMDV proliferation in cattle and guinea pigs.

These results are highly promising; however, the amount (dose) of peptide material required to induce an immune response is approximately 1,000-fold higher than that for the killed FMDV vaccine. To address this issue, a DNA fragment encoding a peptide of amino acid residues 142–160 of FMDV VP1 was fused to the gene encoding the hepatitis B core antigen (HBcAg). When this chimeric gene was expressed in E. coli or ANIMAL CELL CULTURE, its protein products self-assembled into stable "27-nm particles" displaying the FMDV VP1 peptide on their surface. These particles were highly immunogenic. Thus, HBcAg can be used as an effective carrier molecule for synthetic peptides. A comparison of the immunogenicity of various peptide FMDV vaccines containing the 142–160 domain of the VP1 protein in guinea pigs showed that the immunogenicity of the chimeric protein consisting of HBcAg and this domain was 10-fold lower than that of inactivated FMDV particles, 35-fold higher than that of a chimeric protein containing E. coli ß-galactosidase and the 137–162 domain of FMDV VP1, and 500-fold higher than that of the free synthetic peptide consisting of amino acid residues 142–160. Since the synthetic peptide fused to HBcAg forms 27-nm particles resembling hepatitis B virus, and these possess nearly the same immunogenicity as the intact virus from which the synthetic peptide was derived, this approach could become a primary method for delivering peptide vaccines to their Site of Action.

Nevertheless, there are several limitations to using short peptides as vaccines.

✵ The epitope used to create an effective peptide vaccine must be a short but continuous segment of the protein molecule, which is not always the case.

✵ The conformation of the peptide must be identical to that of the epitope in the intact viral particle.

✵ An isolated epitope may lack sufficient immunogenicity. In the future, synthetic peptide vaccines could become a highly specific, relatively inexpensive, safe, and effective alternative to traditional vaccines, though much research remains to be done to achieve this.

Genetic Immunization

A novel approach that induces an immune response without introducing an antigen is based on introducing a gene encoding the antigen protein into the Cells of the target animal. In the first experiments of this kind, an E. coli plasmid containing the cloned antigen gene, under the transcriptional control of an animal virus promoter, was conjugated to gold microparticles and bombarded into mouse ear cells. Subsequently, it was discovered that cloned cDNA could also be introduced into cells via intramuscular injection of a solution containing a large amount of the plasmid carrying the corresponding DNA. This requires 10³—10⁴ times more DNA than Microparticle Bombardment. In one experiment, the gene was incorporated into mouse cells in over 75% of cases, and the synthesized antigen protein induced antibody production. This approach avoids the time-consuming and costly purification of Antigens or The Use of recombinant DNA technology to produce vaccines. Furthermore, the proteins produced this way are more likely to undergo correct post-translational modification than proteins synthesized by heterologous host organisms. This method, termed genetic immunization, can be used for vaccinating domestic animals.

The Prospects of genetic immunization have been thoroughly investigated. In one series of experiments, mice were injected in the quadriceps of both hind limbs with a solution containing an E. coli plasmid carrying the influenza A virus nucleoprotein cDNA, which was transcribed under the control of either the Rous Sarcoma virus or cytomegalovirus promoter. Although the expression level of the nucleoprotein gene was too low to be detected, antibodies against it were found in the Blood of the mice 2 weeks after immunization. The survival rate of the immunized mice was significantly higher than that of the control group (Fig. 11.5). Moreover, they were also resistant to a different strain of influenza virus. Such cross-protection is not elicited by traditional influenza vaccines derived from viral surface antigens, which makes each vaccine specific to only a single viral strain. Furthermore, traditional vaccines remain effective only as long as the surface antigens remain unchanged. Unfortunately, surface antigen genes have a high mutation rate, leading to The Emergence of significantly different viral strains. In contrast, core proteins, such as the nucleoprotein, are relatively stable and activate The Immune System through a different mechanism than surface antigens.

DNA immunization not only avoids the purification of protein antigens but also induces an immune response directed specifically against the plasmid-encoded protein rather than the plasmid itself. Therefore, the same vector can be used to deliver different proteins or for repeated administration of the same gene.

The Fate of the DNA introduced into the cell remains unknown. In principle, it could integrate into the host genome with highly serious consequences if an essential gene is disrupted or malignant cell transformation occurs. However, such an outcome is considered extremely unlikely. Most likely, this DNA will persist in the cell for some time as a non-replicating extrachromosomal element and then degrade. Genetic immunization is currently used to induce Immunity against certain pathogens (influenza A virus, human immunodeficiency virus type 1, bovine herpesvirus, rabies virus, Plasmodium sp. causing malaria, hepatitis B virus) in animals, but not yet in humans.

Fig. 11.5. Survival of mice immunized with viral DNA. Test mice were immunized with an E. coli plasmid carrying the influenza A virus nucleoprotein cDNA under the control of the Rous sarcoma virus promoter. Control mice received only plasmid DNA. The x-axis represents the time elapsed after the animals were challenged with the influenza virus.

To facilitate DNA delivery into animal cells during genetic immunization, a modified strain of Shigella flexneri was engineered. This bacterium enters animal epithelial cells via phagocytosis, and its plasmid DNA is released into the host Cell Cytoplasm, where METABOLISM/31.html">Transcription and Introduction/27.html">Translation of the delivered gene occur under the control of a eukaryotic promoter. Shigella is a pathogen and, as such, cannot be used for DNA delivery. A non-pathogenic strain can be obtained by introducing a deletion into the asd gene, which encodes the enzyme aspartate-ß-semialdehyde dehydrogenase involved in the synthesis of diaminopimelic acid, a Cell wall component. Strains with a mutation in the asd gene grow only in the presence of diaminopimelic acid and can be used to deliver plasmid DNA into animal epithelial cells because they do not proliferate within them.

Experiments using Shigella as a vector for DNA delivery into cells were conducted in guinea pigs, and although they were successful, the safety of this system can only be assessed after clinical trials. A major advantage of this approach is the potential for oral vaccine administration.