Researching more effective and safer viral vaccines

Virulent gene deletion and virulent protein complementation as a mechanism for viral vaccine production

Most viral vaccines fall into two categories: self-replicating ("live" vaccine) or non-replicating vaccines. Non-replicating vaccines typically contain antigenic proteins and no self-replicating viral DNA. Live virus vaccination generally results in the most effective immunity against a particular disease in most individuals, because it best mimics the natural course of immune priming through natural infection: Further, live virus vaccines don't require the addition of dangerous adjuvant, an immunogenic stimulator that is often necessary to prime the immune system when only purified viral protein is used as the vaccine component. Because they have been implicated in autoimmunity, adjuvants have often been referred to as immunologists' "dirty little secret."

Despite their superiority for inducing an immune response in most people, live attenuated viral vaccines can be very dangerous for a small percentage of the population. My research on Varicella Zoster virus vaccine indicates that this is largely because the mechanism of attenuation of live virus vaccines makes them attenuated only for the vast majority of individuals and not for all. This is because attenuated vaccines have often become "safe" merely by acquiring mutations in the promoter for one of their genes that contributes to viral virulence. When this attenuated virus infects most individuals, its mutation is sufficient to reduce the transcription of the virulent gene to nonvirulent levels. Certain individuals, however, may have increased expression of these virulent genes because they have altered gene regulation. Such increased expression is typically associated with certain genetic variations in transcription factors, pregnancy, leukemia, fetal development, or corticosteroid or desmopressin use.
My class investigated the possibility of creating a viral vaccine that would have its virulence gene (or genes) completely deleted from the genome. Since the protein products of many virulence genes are likely to be structurally important for the creation of a high titer viral vaccine, simply deleting the virulence gene frequently leads to ineffective vaccination, resulting in the need to increase the vaccine dosages greatly in order to ensure that an immune response develops. However, by simply: PCR amplifying the virulent gene of a viral vaccine; engineering the correct sticky ends around the gene using site directed mutagenesis; and ligating the gene into a plasmid bearing the CMV promoter element upstream of our virulence gene, we can develop a human cell expression vector that can be transfected into human cells.

To illustrate the concept we introduced the gene for green fluorescent protein into human cells using calcium phosphate transfection. The plasmid provides eukaryotic cells resistance to Geneticin. The resulting transfected human cell line produced the desired protein along the pathway that viral capsids have been shown to traffic. An infection of such cells, with a viral vaccine that had been attenuated by deleting a virulent gene, should produce harvestable virus with extremely high titer. Vaccination with such a virus would lead to the replication of the viral genome within the host, but not the replication of the virulent protein within the host--thus limiting exposure to the virulent protein for all vaccine recipients, even those usually contraindicated for receiving currently used vaccines. Using this method, the vaccinee would receive inoculation with virions that would be complete at the protein level but incomplete at the genetic level. Because the virulent gene would be physically absent from the viral genome, the dangers associated with uncontrolled virulent gene expression in exceptional vaccinees would be eliminated.

Background to vaccine induction of immune response 


How monoclonal antibodies neutralize influenza virus

Developing an immune response to influenza

First Experiment: Engineering complementary cell lines for the production of safer live virus vaccine

Viral vaccines must be grown in cells

Human viruses require cells to replicate--frequently human cells. Most current live viral vaccines are not well characterized with respect to their mechanism of attenuation. Some viruses become attenuated simply by random mutations to the promoters that regulate expression of their virulent genes. In individuals with abnormal gene regulation due to altered transcription factor activity, occasional problems and even death can result from vaccination with live "attenuated" virus. Thus, we investigated how better, safer vaccines can be developed by completely deleting the virulent genes from the viral genome. Genetically deficient viral genomes can be packaged into complete viral particles by growing them in a cell line that expresses the protein in which they are deficient. Thus, our class investigated how one would go about engineering these complementing cell lines from which safer recombinant viral vaccines can be harvested. Because the virulent proteins will be on the viral particles found in the vaccine, it will have a high concentration of viral titre, better stability, and an ability to induce protective antibody production against the most virulent proteins, yet it will be completely unable to produce more of the virulent proteins after it is inoculated into the vaccine recipient.

Types of Cells Used

Skin cell culture

For viruses which infect human skin, primary cells can be obtained through biopsy or circumcision. Alternatively, an immortalized cell line, such as the malignant melanoma cells (shown above), can be used for testing of concept.

White blood cells 

One of the most difficult types of viral infections to clear is the type in which immune cells become infected. To grow lymphotropic (white blood cell infecting) viruses, lymphocytes are typically isolated from human blood or an immortalized lymphocyte cell line is used. 

Note: In the production of the ideal vaccine, all human cellular material and nutrients (such as Bovine Casein, etc) that would produce an undesirable immune response (allergy, autoimmunity, etc) should be removed-leaving only cell free virus vaccine in an osmotically appropriate saline or perhaps saline/sugar solution.

Foreign genes were vectored into human cells with plasmids

Plasmids are frequently used to vector genes into various types of cells. Students grew large quantities of a mammalian expression vector in bacteria and purified the plasmid from those bacteria. Students isolated millions of plasmids. Above: Student prepares his plasmid DNA for transfection of human cells. Here, the student compares his calcium phosphate precipitate with a control to identify precipitated DNA.

Measuring transfection efficiency, cell survival, and foreign gene expression in human cells

The fluorescent microscope was used to visualize the expression of the green fluorescent protein in our cells and test for the success of the delivery.

 Immortalized melanocyte transfections

Above: Transfected malignant melanoma cells were labeled with DAPI nuclear stain. 
Above:The green channel was added, showing the expression of transfected enhanced green fluorescent protein in the melanoma cells.


Second Experiment: Creating safer viral vaccines using DNA vaccines: preliminary research

Staying ahead of the curve is the name of the game in for vaccine geneticists. That is why, even before pandemic fears arose, our class was looking into how vaccine technology could help address the need to develop more rapidly a vaccine against any given pandemic virus. With DNA vaccine technology, a workable vaccine could theoretically be produced in only two weeks, not three months, and a billion doses of the vaccine could be made in weeks, not months, thanks to the reproductive power of bacteria such as E. coli, which are used to replicate the virus in high copy number. While the "ancient" egg based vaccines are a mixture of many different things (including eggs) and the processing could alter the antigenic determinants, DNA vaccines produce pure viral antigens in the host just as they would be produced if one were infected by the virus. Unlike the highly effective, but potentially dangerous live "attenuated" viruses used as vaccines, with a DNA vaccine there is no viral genome, and therefore no ability for the vaccine to produce live virus or for unforeseen recombination events to create a more dangerous virus. Using green fluorescent protein as a test marker to substitute for swine flu hemagglutinin protein H1, the genetics class went through the steps necessary to produce large quantities of DNA vaccine and tested the production of the marker protein in human cells.

To produce large quantities of special circularized DNA containing genes coding for both kanamycin resistance and green fluorescent protein, the plasmid was incubated with E. coli cells. Using the heat shock method, Genetics students transformed their bacterial cells, introducing a plasmid containing gfp as a stand in marker for a hypothetical virulent protein against which a neutralizing antibody can be produced. The bacterial cells were then plated on LB Kan agar plates. These plates only allowed the replication of those bacteria that had taken up the plasmid which, in addition to gfp, also contained a kanamycin resistance cassette. The resulting kanamycin resistant colonies were picked and used to inoculate LB Kan broth cultures.  Each student then isolated high concentrations of purified, endotoxin-free plasmid from their bacterial cultures. These plasmids were introduced into two human cells, resulting in their expression of the green fluorescent protein. This fluorescence was micrographed using a state of the art fluorescent microscopes.
Above: Enhanced green fluorescent protein was expressed by the cell machinery after we transfected the cell with our DNA plasmid containing the GFP gene. Cell nuclei were labeled with DAPI (blue) approximately 16 hours after replating trypsinized transfected melanocytes onto coverslips.

Gene vectoring into student white blood cells

Plasmid delivery into cells, especially immune cells, is the basis for DNA vaccines. Further, because numerous viruses infect white blood cells, they are a logical cell in which to grow vaccine. Further, HIV clinical research suggests that vectoring genes into a patient's own isolated white blood cells that would protect those cells against infection, and then reintroducing them into the patient, could allow for some recovery from--or even a cure for--HIV.

Above: Dr. Storlie demonstrates how to isolate white blood cells from (his own) human blood.
We tested the ability of isolated student lymphocytes to express a foreign gene by introducing the gene coding for green fluorescent protein into student lymphocytes.
Students generated and purified large quantities of an enhanced GFP containing plasmid. This plasmid was then transfected into cells. After two days of incubation, the student lymphocytes were analyzed for expression of the foreign gene using fluorescent microscopy. Live lymphocytes were found to express GFP.
 Success! A Genetics student's white blood cells express the foreign gene. (DIC image left), EGFP in green and DAPI in blue (middle), and merged image (right).

The experiments we did in class demonstrated that viral antigens can be successfully produced in human cells using DNA vectors. Taking this one step further, we tested if cells engineered to produce foreign proteins could produce viral vaccine that most closely replicated the initial infection by wild-type virus, complete with virulent proteins, while not carrying the genes for those proteins into the vaccine recipient. The possibility of engineering viral vaccines that would have their virulence gene or genes deleted from the viral vaccine genome while still carrying the virulence protein on the viral vaccine particles delivered in the first inoculation appears to be very possible. In this preliminary test, two different human cell lines were engineered to express a fluorescent protein (in lieu of an actual virulence gene). These cell lines were then used to grow viral vaccine. The protein expressed by the cell was then incorporated into the nascent viral particles. The vaccine produced from this should allow the vaccine recipient exposure to a limited amount of an important antigen that is often missing in current vaccines and further, allow much lower levels of the vaccine to be delivered. While most vaccines may be considered safe, there are many documented cases of individuals who, due to their unique genetic differences, unique medications, or immune-compromised state have experienced complications and sometimes death as a result of vaccines considered to be safe.

Subpages (1): Transfections