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

Molecular Biotechnology of Microbial Systems
Vaccines
Attenuated Vaccines

In some cases, genetically modified (recombinant) microorganisms (Bacteria or Viruses) can be used as live Vaccines. Such vaccines contain either non-pathogenic microorganisms that synthesize the antigenic determinants of a specific pathogen, or strains of pathogenic microorganisms in which virulence genes have been modified or deleted. In these instances, the primary antigenic determinants are integral Components of the bacterial or Viral Particles and retain the same conformation they adopt in the pathogen. In contrast, an isolated antigen often loses its native conformation and elicits only a weak Immune Response.

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Cholera vaccines

Live vaccines are generally much more effective than inactivated or Subunit Vaccines. The primary requirement for them is the complete absence of virulent Microorganisms in the inoculum. This requirement was also taken into account when developing the live cholera vaccine. Cholera is a rapidly progressing intestinal infection characterized by fever, diarrhea, abdominal pain, and dehydration; it is transmitted through drinking Water contaminated with feces. In developing countries, where water purification and sewage disposal systems are underdeveloped, the threat of cholera remains very real.

The CAUSATIVE AGENT OF cholera is Vibrio cholerae. The bacterium multiplies in the Small Intestine and secretes large amounts of enterotoxin, which is responsible for the pathogenic effect. The enterotoxin is a hexameric protein: it consists of one A subunit, which possesses ADP-ribosylating activity and stimulates adenylate cyclase, and five B subunits, which specifically bind to The Cell receptor of the intestinal mucosa. The A subunit has two functional domains: A₁, which has toxic activity, and A₂, which is responsible for binding to the B subunits. Currently, a cholera vaccine containing phenol-killed cholera vibrios is used; it provides only partial protection against infection and only for 3 to 6 months. Therefore, attempts have been made to develop Other types of cholera vaccines.

As previous studies have shown, a subunit vaccine containing inactivated cholera enterotoxin does not elicit full Immunity. Since V. cholerae colonizes the intestinal mucosa, it was reasonable to assume that an oral cholera vaccine would be the most effective. With this in mind, a strain of V. cholerae was engineered in which a portion of The nucleotide sequence encoding the A₁ peptide was deleted from The Genome. This strain does not synthesize enterotoxin, is therefore non-pathogenic, and is suitable for developing a live vaccine.

The experiment was designed as follows. A tetracycline resistance Gene was inserted into the A₁-peptide gene of V. cholerae. This disrupted the reading frame for the A₁-peptide, but rendered the strain resistant to tetracycline. However, it could not be used as a vaccine because, over time, spontaneous loss of the tetracycline gene occurred, restoring enterotoxin synthesis. To circumvent this problem, a strain was engineered with a defective nucleotide sequence encoding the A₁-peptide that could not be restored (Fig. 11.6). The following approach was used for this purpose.

1. A plasmid containing a DNA segment encoding the A₁-peptide was digested with the Restriction Endonucleases ClaI and XbaI, each of which cleaved only the insert sequence encoding the A₁-peptide.

2. To circularize the plasmid, an XbaI linker was ligated to the ClaI site, and the plasmid was digested with the restriction enzyme XbaI.

3. Using T4 DNA ligase, the XbaI sites of the plasmid were joined. As a result, a 550 bp segment corresponding to amino acid residues 183-194 was deleted from the middle of the sequence encoding the A₁-peptide.

4. The plasmid was transferred via conjugation into a V. cholerae strain carrying a tetracycline resistance gene in the locus encoding the A₁-peptide.

5. As a result of recombination between the remaining plasmid-borne portion of the sequence encoding the A₁-peptide and the chromosomal A₁-peptide gene interrupted by the Tet^r gene, the chromosomal sequence encoding the A₁-peptide was replaced by the homologous plasmid segment containing the deletion.

6. The extrachromosomal plasmid could not persist long in the cholera vibrio and was lost after several generations.

7. Cells with the integrated defective A₁-encoding sequence were selected based on their sensitivity to tetracycline.

The stable strain obtained in this manner, with the deleted sequence encoding the A₁-peptide, did not synthesize active enterotoxin while retaining all other biochemical Properties of the pathogenic form of V. cholerae. Ongoing clinical trials evaluating the efficacy of this strain as a cholera vaccine have not yet yielded definitive results. The vaccine provides nearly 90% protection against cholera, but some subjects experience side effects. It may be necessary to modify another chromosomal locus of this strain to make it suitable for use as a vaccine.

Salmonella vaccines

Another method for obtaining non-pathogenic strains suitable for live vaccines involves deleting chromosomal regions responsible for independent, essential Functions from the genome of pathogenic bacteria. It is preferable to delete at least two such regions, as the probability of their simultaneous restoration is extremely low. It is assumed that a double-deletion strain will have limited proliferative capacity and reduced pathogenicity, yet still elicit an immune response. Various strains of Salmonella cause acute intestinal infections, postnatal infection, typhoid fever, and foodborne illness. An effective vaccine is absolutely essential for the Prevention of all these diseases. To obtain attenuated Salmonella strains, deletions were introduced into the aro genes, which encode Enzymes for The Biosynthesis of Aromatic Compounds, and the pur genes, which encode enzymes for purine METABOLISM. Such double-deletion strains cause a mild form of infection and are 10⁶-fold less virulent. Effective oral vaccines based on these strains have already been developed for mice, sheep, cattle, chickens, and, most recently, humans.

Fig. 11.6. Construction of a V. cholerae strain with a deletion in a portion of the nucleotide sequence encoding the cholera toxin A₁ peptide.

Leishmania vaccines

Protozoan parasites of the genus Leishmania can also elicit an immune response in humans; however, developing effective vaccines against them is a challenging task. Attenuated Leishmania lines can be used for this purpose, but they frequently revert to virulence and, furthermore, can persist asymptomatically for long periods in the human host—acting as an infection reservoir—and be transmitted to others. To address these issues, attempts were made to construct an attenuated, non-reverting Leishmania line by deleting a metabolically essential gene (for example, the Dihydrofolate Reductase-thymidylate synthase gene). In one such parasite, Leishmania major E10-5A3, both dihydrofolate reductase-thymidylate synthase genes were replaced with genes conferring resistance to the Antibiotics G-418 and hygromycin. Unlike wild-type parasites, when L. major E10-5A3 is grown in standard culture or macrophage culture, thymidine must be added to the medium (Fig. 11.7). In BALB/c mice, the parasites remained viable for several days, which is sufficient to induce robust immunity in the animals (Fig. 11.8) but insufficient for disease development. Neither persistent infection nor disease occurred even in the most susceptible mouse strains, making this line highly suitable for vaccine development. Following additional animal experiments, its efficacy for human immunization can be evaluated.

Fig. 11.7. Proliferation of wild-type and attenuated L. major in mouse macrophages. For infection, equal numbers of stationary-phase L. major were used in both cases. (Modified from Titus et al., Proc. Natl. Acad. Sci. USA 92: 10267—10268, 1995.)

"Vector" vaccines

Antiviral vaccines. Vaccinia virus (VV), belonging to the genus Orthopoxvirus, is widely used as an effective live smallpox vaccine. The genome of this virus has been fully sequenced; it consists of a double-stranded DNA of 187 kb, encoding approximately 200 different Proteins. VV DNA replicates in the Cytoplasm of infected cells rather than in The Nucleus, due to the presence of viral genes encoding DNA polymerase, RNA polymerase, and enzymes responsible for mRNA capping, methylation, and polyadenylation. Therefore, if a foreign gene is inserted into the VV genome under the control of a VV promoter, it will be expressed independently of the host's regulatory and enzymatic systems.

Fig. 11.8. Immunity to the virulent parasite L. major developing in BALB/c mice after inoculation with the attenuated parasite L. major. At time 0, mice previously vaccinated with the attenuated parasite were challenged with the virulent parasite, and the mean size of leishmanial lesions was subsequently determined. Control mice were not vaccinated. (Modified from Titus et al., Proc. Natl. Acad. Sci. USA 92: 10267—10268, 1995.)

Vaccinia virus (VV) has a broad host range (both vertebrates and invertebrates), remains viable for many years after lyophilization (freeze-drying), and is non-oncogenic, making it suitable for developing so-called vector vaccines. These vaccines are used to deliver and express cloned genes encoding antigenic proteins that induce The production of protective Antibodies in the host Organism. The VV genome is large and lacks unique restriction sites, which prevents the direct insertion of additional nucleotide sequences. However, target genes can be introduced into the VV genome via in vivo Homologous Recombination as follows.

1. A DNA segment encoding a specific antigen (e.g., HBcAg) is inserted into a plasmid vector immediately downstream of a cloned VV promoter that has been inserted into a nonessential VV gene, such as the thymidine kinase gene (Fig. 11.9, A).

2. This plasmid is used to transfect a culture of thymidine kinase-deficient animal cells, typically chick embryo fibroblasts, previously infected with wild-type VV, which synthesizes functional thymidine kinase.

3. As a result of recombination between The nucleotide sequences flanking the promoter and the protective antigen gene, and the homologous sequences of the viral genome, the cloned gene is integrated into the viral DNA (Fig. 11.9, B). Although the frequency of such recombination events is low, the cell population containing the recombinant VV can be enriched using a selective medium containing bromodeoxyuridine. In the absence of thymidine kinase, this toxic thymidine analog is not incorporated into the synthesizing DNA and does not exert a toxic effect. Thymidine kinase-deficient host cells containing wild-type VV die in the presence of bromodeoxyuridine, whereas cells harboring recombinant VV with a disrupted thymidine kinase gene become resistant to its toxic effect.

Fig. 11.9. Insertion of a gene into VV DNA whose protein product (usually a viral antigen) induces an immune response. A. A plasmid carrying an expressible antigenic protein gene. B. Double crossover resulting in the integration of this gene into the VV DNA.

4. Final Selection is performed using a DNA probe that hybridizes with the antigenic protein gene.

Since thymidine kinase-deficient VV mutants arise spontaneously at a relatively high frequency (approximately 1 in 10³—10⁴ viral particles), cells are often cotransfected with a selective marker and the target gene. This facilitates the differentiation between spontaneous mutants and those obtained via homologous recombination. The neo gene, which encodes the enzyme neomycin phosphotransferase II and confers resistance to the kanamycin analog G-418, is commonly used as a selective marker. Unlike other selective markers, this gene remains stable when integrated into the VV genome.

A special system has been developed to avoid disrupting the reading frame of VV genes when inserting a foreign gene. This eliminates The Need for selective markers, as every plaque-forming recombinant virus will contain and express the target gene. Wild-type VV DNA carries the vp37 gene, which is responsible for plaque formation during viral growth in monolayer Animal Cell Cultures (Fig. 11.10, A). If this gene is replaced with an E. coli marker gene, a mutant VV is generated that does not form plaques when grown for 2—3 days in ANIMAL CELL CULTURE (Fig. 11.10, B). The target gene is introduced into this mutant virus via homologous recombination of its DNA with a vector carrying both the vp37 gene and the target gene (Fig. 11.10, C). The mutant VV that receives the vp37 gene regains The ability to form plaques; meanwhile, the target gene is integrated into its genome, and the marker gene is lost. The mutant virus with the deleted vp37 gene cannot revert to the wild type, so every plaque-forming viral particle contains the desired construct. This method is simple, applicable for the transfer and expression of any target gene, requires no additional marker genes, and does not disrupt the reading frame of VV genes.

Several genes encoding antigenic proteins have already been successfully integrated into the VV genome and expressed in animal cell culture: the rabies virus G protein, hepatitis B surface antigen, Sindbis virus surface proteins, Influenza virus NP and HA proteins, vesicular stomatitis virus N and G proteins, and Herpes simplex virus Glycoproteins. Some of the recombinant vectors obtained based on VV can be used to develop effective vaccines. For instance, a recombinant VV expressing the herpes simplex virus type 1 glycoprotein D gene prevents herpes infections in mice, while a recombinant VV expressing the rabies virus surface antigen gene induces the production of protective antibodies in foxes, the main vectors of rabies in Europe.

Fig. 11.10. A. Wild-type VV DNA. B. Mutant VV DNA. C. VV-based vector. Left flank and right flank are the sequences flanking the vp37 gene on the left and right, respectively, in the wild-type VV genome. The vp37 promoter is not shown. p7.5 is a strong 'early' (or 'late') VV promoter. The target gene is inserted into the polylinker. Homologous recombination between the recombinant vector and the mutant viral DNA results in the replacement of the E. coli marker gene with the vp37 gene and the target gene.

VV-based vector vaccines allow immunization against several diseases simultaneously. For this purpose, a recombinant VV carrying multiple genes encoding different Antigens can be used.

Depending on the VV promoter used, the foreign protein can be synthesized during the early or late phase of the infectious cycle, with its yield determined by the strength of the promoter. Typically, 'late' VV promoters are used to achieve high expression levels: p11 (the promoter of the gene responsible for the synthesis of an 11-kDa protein) or pCAE (the promoter of the cowpox virus type A inclusion body protein gene). When integrating multiple foreign genes into a single VV DNA, each gene is placed under the control of a separate VV promoter to prevent homologous recombination between different Regions of the viral DNA, which could lead to the loss of the inserted genes.

A live recombinant viral vaccine offers several advantages over inactivated viral and subunit vaccines: 1) the presentation of the authentic antigen is virtually identical to that during a natural infection; 2) the virus can replicate in the host cell and increase The amount of antigen, which activates antibody production by B cells (humoral immunity) and stimulates T-cell production (cellular immunity); 3) the insertion of antigenic protein genes into one or more sites of the VV genome further reduces its virulence.

A disadvantage of live recombinant viral vaccines is that their administration to immunocompromised individuals (e.g., AIDS patients) can lead to severe viral infection. To address this issue, a gene encoding human interleukin-2 can be integrated into the viral vector, which stimulates a T-cell response and limits viral proliferation.

Undesirable side effects of VV proliferation can be prevented by inactivating the virus after vaccination. To achieve this, an interferon-sensitive virus was developed (wild-type VV is relatively resistant to its action), whose proliferation can be regulated in the event of vaccination-related complications.

The Mechanism of VV resistance to interferon remained unknown until the Discovery of the K3L Open Reading Frame, which encodes a 10.5-kDa protein. This protein contains an Amino Acid Sequence homologous to the N-terminal region of the 36.1-kDa eukaryotic initiation factor eIF-2a. The N-terminal regions of both proteins contain 87 virtually identical amino acid residues, with Serine located at position 51 in both cases; in eIF-2a, this serine is phosphorylated by an interferon-activated P1 kinase, leading to the inhibition of Protein Synthesis in interferon-treated cells. The K3L protein acts as a competitive inhibitor of eIF-2a phosphorylation, conferring interferon resistance to VV; if the K3L gene or a portion of it is deleted from the VV genome, the virus becomes sensitive to interferon. By using PCR mutagenesis of the plasmid-borne K3L gene followed by homologous recombination between VV DNA and the plasmid to replace the wild-type K3L sequence with a modified variant, a mutant VV K3L strain was constructed. This strain proved to be 10- to 15-fold more sensitive to interferon than the wild-type strain (Fig. 11.11). This work represents an important milestone toward developing safer VV vectors. Sequences similar to K3L may also be present in other interferon-resistant viruses, allowing the generation of interferon-sensitive strains through gene deletions.

Fig. 11.11. Interferon sensitivity of wild-type (K3L⁺) and mutant (K3L^-) VV strains. Prior to viral inoculation, murine L929 cells were treated for 24 h with a mixture of murine alpha and beta interferons. Approximately 3∙10⁷ plaques were present on each plate not treated with interferon. (Modified from Paoletti, Tartaglia, U.S. patent 5,378,657, 1995).

Most work on developing live viral vaccines has focused on VV; however, other viruses, such as adenovirus, poliovirus, and varicella-zoster virus, are also being considered as potential vaccine vectors. A vector based on a live attenuated poliovirus (research on which is in its early stages) is attractive because it allows for oral vaccination. Such mucosal vaccines (vaccines whose components bind to receptors in the Lungs or gastrointestinal tract) are suitable for preventing A wide variety of diseases: cholera, typhoid fever, influenza, Pneumonia, mononucleosis, rabies, AIDS, and Lyme disease. However, prior to any clinical trials of any seemingly harmless virus used as a delivery and expression system for a target gene, its absolute safety must be ensured. For example, the widely used VV causes complications in humans at a frequency of approximately 3.0∙10^-6. Therefore, it is desirable to delete sequences responsible for virulence from the genome of any recombinant virus intended for human vaccination.

Antibacterial Vaccines

Antibiotics are widely used to treat bacterial diseases, and work on developing antibacterial vaccines has begun only recently. There were very compelling reasons driving this effort.

✵ Not all bacterial infections are treatable with antibiotics.

✵ The widespread use of antibiotics over the last 40 years has led to The Emergence of A large number of resistant bacterial strains.

✵ Many tropical countries lack proper storage conditions for antibiotics.

✵ Patients often stop Treatment earlier than prescribed by their doctor, or take antibiotics in insufficient doses.

To ensure that a newly developed antibacterial vaccine is sufficiently effective, choosing the right strategy is crucial. If a pathogenic bacterium grows poorly in culture, making it difficult to obtain an attenuated strain, alternative approaches must be used. For example, Rickettsia rickettsii, a Gram-negative obligate intracellular bacterium that causes Rocky Mountain spotted fever, does not grow in culture. To overcome this challenge, a subunit vaccine was developed containing the major surface antigen of R. rickettsii, a protein with a molecular mass of 155 kDa. It effectively protected mice against this pathogen.

Bacteria as antigen delivery systems

Antigens located on the outer surface of a bacterial cell exhibit higher immunogenicity than those localized in the cytoplasm. Therefore, one approach used in vaccine development is to display the protective antigen of a pathogenic bacterium On the surface of a live non-pathogenic bacterium. Many bacteria possess flagella composed of the protein flagellin; under a Microscope, they appear as filaments extending from the bacterial cell. By engineering the flagella of a non-pathogenic microorganism to carry a specific epitope of a pathogen, it is possible to induce the production of protective antibodies.

This exact approach was used to develop a cholera vaccine. A synthetic oligonucleotide encoding an epitope of the cholera toxin B subunit was inserted into the hypervariable region of the Salmonella flagellin gene, and the resulting construct was introduced into a flagellin-deficient Salmonella strain. It was already known that the epitope comprising amino acid residues 50–64 of the cholera toxin B subunit induces the production of antibodies against the intact cholera toxin. The chimeric flagellin functioned normally, and the cholera toxin epitope was displayed on The surface of the flagella. Immunization of mice via intraperitoneal injection of approximately 5∙10⁶ live or formalin-killed bacteria carrying the modified flagellin induced high titers of antibodies against both the peptide (amino acid residues 50–64) and the intact cholera toxin molecule. Similarly, two or even three different epitopes can be inserted into a single Salmonella flagellin gene to create a multivalent antibacterial vaccine.

Oral administration of attenuated Salmonella strains can be used to deliver various bacterial, viral, and parasitic antigens to the host organism. In this process, the choice of promoter controlling the Transcription of the foreign gene plays a crucial role. If an excessively strong promoter is used, a metabolic burden may occur, hindering bacterial proliferation. Unlike a bioreactor, the host animal's body is not a closed system, and foreign Gene Expression cannot be regulated by changing the Temperature or adding specific metabolites. Only a promoter that responds to specific environmental cues can play a regulatory role. For example, the E. coli nirB promoter can be regulated by varying nitrite and oxygen levels in the medium, and it is most active under anaerobic conditions. In one experiment, the nirB promoter was used to control the expression of the gene for the non-toxic, immunogenic fragment C of tetanus toxin in an attenuated Salmonella strain. In developing countries, Clostridium tetani infections claim more than 1 million lives annually. If the genetically modified Salmonella strain is grown under aerobic conditions, the tetanus toxin fragment C is not synthesized. However, upon oral administration of this bacterium to test mice, fragment C is synthesized, and the animals produce antibodies against it. Thus, the Salmonella strain containing the tetanus toxin fragment C under the control of the nirB promoter can be used as a live oral tetanus vaccine. Further studies are required to determine the efficacy of this approach for human vaccination.