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How are vaccines mass-produced?

How are vaccines mass-produced?


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I have a background in product design and so am familiar with with how most things are mass-produced - food, machines, etc. But I've been able to find very little information on how vaccines are mass-produced.

It looks like there are 4 types of vaccines, all of which include pieces or byproducts of the virus they're intended to counteract.

If you're producing billions of vaccines, I imagine you need a tremendous amount of the virus.

How is such a mass of virus obtained? Do they just fill up tanks with a culturing agent and a sample of the virus and wait for it to grow, like a giant petri dish? Are there big vats of Coronavirus sitting in factories somewhere?


Per Wikipedia, typically when one needs a lot of virus, it is grown in a controlled cell environment. This used to be eggs, but is moving toward cell cultures instead. So basically yes, factories full of virus (though more like in nice discrete bioreactors than big Joker-friendly vats).

Synthetic vaccines, such as the mRNA vaccines for COVID, do not need this step at all, since they do not actually use the virus, but can be done through cell-free biochemical reactions that replicate the mRNA directly.


Vaccines Are Important—But What Are They and How Do They Work?

To say that the COVID-19 pandemic has disrupted our lives would be an understatement. Since March, when stay-at-home orders were first put in place, many of us have spent our time in our homes, avoiding crowds, wearing masks, social distancing, and working remotely, often while taking care of or homeschooling children.

In spite of all of this, the virus continues to spread and our lives remain far from normal. And while research into therapies and treatments for COVID-19 is advancing, there are still none that can prevent severe illness.

We’ve been told that a vaccine that provides full protection against SARS-CoV-2, the virus that causes COVID-19, offers the best chance for a return to normalcy. Getting one would allow people to go about their daily lives, free from worry of getting infected, as well as the need to social distance and wear masks.

Ideally, one or more of the many COVID-19 vaccines in development will prove to be effective. But even a vaccine that offers less than complete immunity would slow the virus’s spread and could reduce the severity of the disease. Along these lines, the Food and Drug Administration (FDA) announced in July that it would approve any vaccines that demonstrated at least 50% efficacy against the coronavirus.

All of this may be confusing—and frustrating—to those of us who are simply trying to put one foot in front of the other during this pandemic. And the confusion surrounding COVID-19 vaccines may be compounded by the uptick in reporting about them—the trials, their efficacy, and their safety.

With this in mind, Yale Medicine offers this primer on vaccine basics—how they work, their different platforms, and the approval process—and what that means for a COVID-19 vaccine.


The Promise of Cell Culture in Vaccine Development

Hopes of growing poliovirus in the lab without the use of live animals drove many of the researchers in the 1930s and 1940s. Cell cultures involve growing cells in a culture dish, often with a supportive growth medium like collagen. They offer a level of control that was unavailable using live animals, and can also support large-scale virus production. (For more about cell cultures and cell lines, as well as cell lines made using human cells, see our article “ Human Cell Strains in Vaccine Development .”) Early efforts to grow poliovirus in culture, however, repeatedly ended in failure.

In 1936, Albert Sabin and Peter Olitsky at the Rockefeller Institute successfully grew poliovirus in a culture of brain tissue from a human embryo. The virus grew quickly, which was promising, but Sabin and Olitsky were concerned about using this as starting material for a vaccine, fearing nervous system damage for vaccine recipients. They tried to grow poliovirus in cultures using tissue that had been taken from other sources, but were unsuccessful.


Examples of Vaccine Production

Inactivated Virus (Influenza)

Influenza virus vaccine for intramuscular use is a sterile suspension prepared from influenza viruses propagated in chicken embryos. This vaccine is the primary method for preventing influenza and its more severe complications. 13

Typically, influenza vaccine contains two strains of influenza A viruses (H1N1 and H3N2) and a single influenza B virus. An additional strain of the influenza B virus was added, with the first four-antigen-containing-vaccine licensed in 2012. 14 The two type A viruses are identified by their subtypes of hemagglutinin and neuraminidase. The hemagglutinin and neuraminidase glycoproteins of influenza A virus comprise the major surface proteins and the principal immunizing antigens of the virus. These proteins are inserted into the viral envelopes as spike-line projections in a ratio of approximately 4 :𠂑. 15

The trivalent subunit vaccine is the predominant influenza vaccine used today. This vaccine is produced from viral strains that are identified early each year by the World Health Organization, the Centers for Disease Control and Prevention (CDC), and CBER. For U.S.-licensed manufacturers, the viral strains are normally acquired from CBER or CDC. European strains are typically provided by the National Institute for Biological Standards and Control, and Southern Hemisphere strains by the Therapeutic Goods Administration of Australia. These viral strains are used to prepare cells banks at each manufacturer, which cell banks are ultimately used as the inoculums for vaccine production.

The substrate most commonly used by producers of influenza vaccine is the 11-day-old embryonated chicken egg. A monovalent virus (suspension) is received from CBER or the CDC. The monovalent virus suspension is passed in eggs. The inoculated eggs are incubated for a specific time and temperature regimen under controlled relative humidity and then harvested. In the European Union, the number of passages from the original sample is limited. The harvested allantoic fluids, which contain the live virus, are tested for infectivity, titer, specificity, and sterility. These fluids are then stored wet frozen at extremely low temperatures to maintain the stability of the monovalent seed virus (MSV). 16 This MSV is also certified by CBER.

Once the MSV is introduced into the egg by automated inoculators, the virus is grown at incubated temperatures, and then the allantoic fluid is harvested and purified by high-speed centrifugation on a sucrose gradient or by chromatography. The purified virus is often split using a detergent before final filtration. The virus is inactivated using formaldehyde before or after the primary purification step, depending on the manufacturer. This is repeated for three or four strains of virus, and the individually tested and released inactivated viral concentrates are combined and diluted to final vaccine strength. Fig. 5.2 outlines the overall process.

Egg-based influenza vaccine manufacturing process flow.

CBER, Center for Biologics Evaluation and Research (of the U.S. Food and Drug Administration) QA, quality assurance QC, quality control.

The inactivated virus vaccine described above is used for the majority of flu vaccine produced and sold today. In recent years, the inactivated influenza vaccine produced on mammalian cell culture has been approved in a number of countries. The process replaces the egg-based virus expansion with a certified cell line the downstream processes are similar, but focused on removing the host cell protein and DNA to below designated thresholds. A recombinant influenza vaccine, produced in insect cells infected with a recombinant baculovirus to express the hemagglutinin protein has also been approved in the United States.

Recombinant Protein (Hepatitis B)

In July 1986, a recombinant hepatitis B vaccine was licensed in the United States. This vaccine built on the knowledge that heat-inactivated serum containing hepatitis B virus (HBV) and hepatitis B surface antigen (HBsAg) was not infectious, but was immunogenic and partially protective against subsequent exposure to HBV. 17 HBsAg was the component that conferred protection to HBV on immunization. 18 To produce this vaccine, the gene coding for HBsAg, or “S” gene, was inserted into an expression vector that was capable of directing the synthesis of large quantities of HBsAg in Saccharomyces cerevisiae. The HBsAg particles expressed by and purified from the yeast cells have been demonstrated to be equivalent to the HBsAg derived from the plasma of the blood of hepatitis B chronic carriers. 17 , 19 , 20

The recombinant S. cerevisiae cells expressing HBsAg are grown in stirred tank fermenters. The medium used in this process is a complex fermentation medium that consists of an extract of yeast, soy peptone, dextrose, amino acids, and mineral salts. In-process testing is conducted on the fermentation product to determine the percentage of host cells with the expression construct. 7 At the end of the fermentation process, the HBsAg is harvested by lysing the yeast cells. It is separated by hydrophobic interaction and size-exclusion chromatography. The resulting HBsAg is assembled into 22-nm𠄽iameter lipoprotein particles. The HBsAg is purified to greater than 99% for protein by a series of physical and chemical methods. The purified protein is treated in phosphate buffer with formaldehyde, sterile filtered, and then coprecipitated with alum (potassium aluminum sulfate) to form bulk vaccine adjuvanted with amorphous aluminum hydroxyphosphate sulfate. The vaccine contains no detectable yeast DNA but may contain not more than 1% yeast protein. 7 , 19 , 21 In a second recombinant hepatitis B vaccine, the surface antigen expressed in S. cerevisiae cells is purified by several physiochemical steps and formulated as a suspension of the antigen absorbed on aluminum hydroxide. The procedures used in its manufacturing result in a product that contains no more than 5% yeast protein. No substances of human origin are used in its manufacture. 20 Vaccines against hepatitis B prepared from recombinant yeast cultures are noninfectious 20 and are free of association with human blood and blood products. 19

Each lot of hepatitis B vaccine is tested for safety, in mice and guinea pigs, and for sterility. 19 QC product testing for purity and identity includes numerous chemical, biochemical, and physical assays on the final product to assure thorough characterization and lot-to-lot consistency. Quantitative immunoassays using monoclonal antibodies can be used to measure the presence of high levels of key epitopes on the yeast-derived HBsAg. A mouse potency assay is also used to measure the immunogenicity of hepatitis B vaccines. The effective dose capable of seroconverting 50% of the mice (ED50) is calculated. 21

Hepatitis B vaccines are sterile suspensions for intramuscular injection. The vaccine is supplied in four formulations: pediatric, adolescent/high-risk infant, adult, and dialysis.

All formulations contain approximately 0.5 mg of aluminum (provided as amorphous aluminum hydroxyphosphate sulfate) per milliliter of vaccine. 19 Table 5.2 summarizes the QC testing requirements for the release of recombinant hepatitis B vaccine.

TABLE 5.2

Testing Requirements for the Release of Recombinant Hepatitis B Vaccine

Type of TestStage of Production
Plasmid retentionFermentation production
Purity and identityBulk-adsorbed product or nonadsorbed bulk product
SterilityFinal bulk product
SterilityFinal container
General safetyFinal container
PyrogenFinal container
PurityFinal container
PotencyFinal container

Most vaccines are still released by CBER on a lot-by-lot basis but for several extensively characterized vaccines, such as hepatitis B and human papillomavirus (HPV) vaccines, which are manufactured using recombinant DNA processes, this requirement has been eliminated.. Their manufacturing process includes significant purification, and they are extensively characterized by their analytical methods. In addition, hepatitis B vaccine had to demonstrate a “track record” of continued safety, purity, and potency to qualify for this exemption. 7 , 22

Conjugate Vaccine (Haemophilus influenzae Type B)

The production of Haemophilus influenzae type b (Hib) conjugate includes the separate production of capsular polysaccharide from Hib and a carrier protein such as tetanus protein from Clostridium tetani (i.e., purified tetanus toxoid), CRM protein from Corynebacterium diphtheriae, or outer membrane protein complex of Neisseria meningitidis.

The capsular polysaccharide is produced in industrial bioreactors using approved seeds of Hib. A crude intermediate is recovered from fermentation supernatant, using a cationic detergent. The resulting material is harvested by continuous-flow centrifugation. The paste is then resuspended in buffer, and the polysaccharide is selectively dissociated from disrupted paste by increasing the ionic strength. The polysaccharide is then further purified by phenol extraction, ultrafiltration, and ethanol precipitation. The final material is precipitated with alcohol, dried under vacuum, and stored at �ଌ for further processing.

Tetanus protein is prepared in bioreactors using approved seeds of C. tetani. The crude toxin is recovered from the culture supernatant by continuous-flow centrifugation and diafiltration. Crude toxin is then purified by a combination of fractional ammonium sulfate precipitation and ultrafiltration. The resulting purified toxin is detoxified using formaldehyde, concentrated by ultrafiltration, and stored at between 2ଌ and 8ଌ for further processing.

The industrial conjugation process was initially developed using tetanus toxoid by a team headed by J.B. Robbins at the National Institute of Allergy and Infectious Diseases (NIAID), Bethesda, Maryland. 23

Conjugate preparation is a two-step process that involves: (a) activation of the Hib capsular polysaccharide and (b) conjugation of activated polysaccharide to tetanus protein through a spacer. Activation includes chemical fragmentation of the native polysaccharide to a specified molecular weight target and covalent linkage of adipic acid dihydrazide. The activated polysaccharide is then covalently linked to the purified tetanus protein by carbodiimide-mediated condensation using 1-ethyl-3(3-dimethylaminopropyl)carbodiimide. Purification of the conjugated material is performed to obtain high-molecular-weight conjugate molecules devoid of chemical residues and free protein and polysaccharide. Conjugate bulk is then diluted in an appropriate buffer, filled into unit-dose and/or multidose vials, and lyophilized.

Live Attenuated Vaccine (Measles)

The measles virus, isolated in 1954, is part of the genus Morbillivirus in the family Paramyxoviridae. Current vaccines are derived from Edmonston, Moraten, or Schwarz strains. Such vaccines have been on the market since the 1960s and in combination (measles, mumps, rubella [MMR]) since the 1970s. The final vaccine is a live attenuated viral vaccine inducing immunity in more than 90% of recipients.

For one measles vaccine, the manufacture of the vaccine starts with specific pathogen-free embryonated chicken eggs that are incubated several days. The embryos are collected and treated with trypsin to prepare the chick embryo fibroblasts for cell culture. All of the operations are done under strict aseptic conditions, performed by well-trained operators.

Cell culture are grown in roller bottles using fetal calf sera and M199 Hanks media for optimal cell growth. Chick embryo fibroblast cells are further infected by the viral working seed and incubated several days for viral culture. At the end of the viral culture, the cells are disrupted by mechanical lysis to release the virus. The virus is purified by centrifugation and filtration and stored frozen. After release of all QC tests, the vaccine is formulated alone or with mumps and rubella vaccines and lyophilized to obtain the stable product. The vaccine is reconstituted just before use.

Other manufacturers use different cell substrates for example, the Serum Institute of India uses human diploid cells to manufacture their measles vaccine (see http://www.seruminstitute.com/content/products/product_mvac.htm).

Virus-Like Particle�sed Vaccines

Traditional viral vaccines rely on attenuated virus strains or inactivation of infectious virus. Subunit vaccines based on viral proteins expressed in heterologous systems have been effective for some pathogens, but have often had poor immunogenicity because of incorrect folding or modification. 24 Virus-like particles (VLPs) are designed to mimic the overall structure of virus particles and, thus, preserve the native antigenic conformation of the immunogenic proteins. VLPs have been produced for a wide range of taxonomically and structurally distinct viruses and have unique potential advantages in terms of safety and immunogenicity over previous approaches. 1 Attenuation or inactivation of the VLP is not required this is particularly important as epitopes are commonly modified by inactivation treatments. 25 However, if a viral vector (e.g., baculovirus) is used as the expression system, inactivation may be required if the purification process cannot eliminate residual viral activity.

For a VLP to be a realistic vaccine candidate, it needs to be produced in a safe expression system that is easy to scale up to large-scale production 1 and by an accompanying purification and inactivation process that will maintain native structure and immunogenicity and will meet the requirements of today's global regulatory authorities. A number of expression systems manufacture multimeric VLPs, including the baculovirus expression system (BVES) in Sf9 and High Five cells, Escherichia coli, Aspergillus niger, Chinese hamster ovary cells, human function liver cells, baby hamster kidney cells, transgenic plants (potato, tobacco, soybean), S. cerevisiae, Pichia pastoris, human embryonic kidney 293 (HEK293) cells, and lupin callus (a plant-cell production system) with yields ranging from 0.3 to 10 µg/mL or as high as 300 to 500 µg/mL with E. coli and HEK293 (purified). 2

The BVES has proven quite versatile, demonstrating the capability of preparing vaccine candidates for papillomavirus, feline calicivirus, hepatitis E virus, porcine parvovirus, chicken anemia virus, porcine circovirus, SV40 (simian virus 40), poliovirus, bluetongue virus, rotavirus, hepatitis C virus, HIV, simian immunodeficiency virus, feline immunodeficiency virus, Newcastle disease virus, severe acute respiratory syndrome (SARS) coronavirus, Hantaan virus, influenza A virus, and infectious bursal disease virus. 1

Many pathogenic viruses, such as influenza, HIV, and hepatitis C, are surrounded by an envelope, a membrane that consists of a lipid bilayer derived from the host cell, inserted with virus glycoprotein spikes. These proteins are targets of neutralizing antibodies and are essential components of a vaccine. Owing to inherent properties of the lipid envelope, assembly of VLPs in insect cells for these viral vaccines is a different type of technical challenge to those produced viruses with multiple capsids. 1 For these targets, production of VLPs is a challenging task because the synthesis and assembly of one or more recombinant proteins may be required. This is the case for VLPs of rotavirus, which is an RNA virus with capsids formed by 1860 monomers of four different proteins. In addition, the production of most VLPs requires the simultaneous expression and assembly of several recombinant proteins, which, in the case of RLP, needs to occur in a single host cell. 26 Purification of VLPs also constitutes a particularly challenging task. VLPs are structures of several nanometers in diameter and of molecular weights in the range of 10 6 �. Also, for guaranteeing the quality of the product, it is not sufficient to demonstrate the absence of contaminant proteins it is also necessary to show that proteins are correctly assembled into VLPs.

Production of HPV VLPs represents another challenge. The HPV type 16 major 55-kDa capsids protein, L1, when produced in certain recombinant expression systems such as S. cerevisiae, can form irregularly shaped VLPs with a broad size distribution. These HPV VLPs are inherently unstable and tend to aggregate in solution. The primary challenge of HPV vaccine formulation development was the preparation of aqueous HPV VLP solutions that are stable under a variety of purification, processing, and storage conditions. By treating the HPV VLPs through a process of disassembly and reassembly, the stability and in vitro potency of the vaccine are enhanced significantly. In addition, the in vivo immunogenicity of the vaccine was also improved by as much as approximately 10-fold, as shown in mouse potency studies. 27 The disassembly and reassembly of particles may also be important to remove residual proteins from the expression system or host cells used in the production and is a serious processing challenge, particularly for enveloped VLPs.


GE Research Developing DNA Vaccine Technology to Enable Rapid Response to Infectious Diseases

Niskayuna, NY – A team of GE scientists at GE Research in Upstate New York have been awarded a two-year, $4.7 MM program from DTRA, an agency within the U.S. Department of Defense (DOD), to develop and demonstrate DNA vaccine technology that would enable the agency and medical community to more rapidly respond to new or emerging biothreats.

“Every hour, day, week and month we can save when developing a vaccine in response to a new or emerging biothreat could mean the difference between saving a life, hundreds of lives, or more,” said John Nelson, a Senior Principal Scientist in the Biology and Applied Physics group at GE Research and principal investigator on the DTRA program. “The use of our special kind of DNA in making vaccines holds a lot of promise, but still needs to be proven. We have high hopes that we can address the technical gaps required to pave the way for its future use.”

Through the efforts of Nelson and others in the field of DNA technology, dramatic improvements have been made over the past decade to speed up DNA genome sequencing to make the technology more viable for use in vaccine development. DNA sequencing technology is improving rapidly. “10 years ago, it cost $50,000 and took several weeks to sequence an entire human genome. Today for a few thousand dollars, it can be done in under two weeks,” Nelson said. “Currently, in only a few days we can obtain the complete sequence of any new pathogen for a few hundred dollars”. This allows us to design a new DNA vaccine and have the DNA synthesized.

How Vaccines are made today

The processes for creating vaccines for viruses such as the flu are complex and can take from several months to a year. This long timeframe, in turn, can make it difficult to create an effective vaccine. Nelson said, “Viruses like the flu mutate or change in their characteristics as they are transferred from person-to person. Today, vaccines for the flu are developed almost a year ahead of the next flu season.” Nelson said, “Scientists do their best to predict what the flu will look like and then engineer a version of the killed virus to inject in people’s arms. The issue is that by the time it is administered, the profile of the flu virus may have changed so much the vaccine won’t be as effective in suppressing the virus. That’s why you hear about people who received the flu vaccine, but still got the flu.”

GE’s New DNA Vaccine Technology

DNA vaccine technology has been developed over the last two decades and is currently in human clinical trials. However, the DNA used in the current version is made in bacterial cells and is very complicated and expensive to purify. GE has taken the cellular process used to make DNA out of the cells and moved it to a cell-free method to create the DNA enzymatically.

The process starts by simply taking a swab from someone’s nose to capture sample that contains the virus. The next step is to send the sample to the lab to sequence the virus and identify the genes for the surface proteins of the virus. Once you have those genes, you then create a piece of DNA that will provide instructions on how to express the genes into proteins. This section of DNA is then mass-produced using the GE method, and formulated into vaccines. This DNA vaccine is then put into a few human cells (just like a normal vaccine in the skin or muscle) and expressed over a few days to produce the foreign proteins that prime the body’s immune response against the virus.

Together with its research partners, Albany Medical Center, the University of Washington, Profectus Biosciences and the United States Army Medical Research Institute of Infectious Diseases (USAMRIID), the team will test different vaccine delivery mechanisms to prove the effectiveness of this new form of DNA vaccine. One novel approach is where the vaccine is delivered through the skin using GE’s Ultrasound technology.

“The ability to develop vaccines in days, not months, would be a game-changer in the way the medical community responds to newly emerging contagious diseases,” Nelson concluded. “GE’s synthetic approach to produce DNA could also open up new opportunities in other areas, including the treatment of cancer and autoimmune diseases.”

About GE Research

GE Research is GE’s innovation powerhouse where research meets reality. We are a world-class team of 1,000+ scientific, engineering and marketing minds (600+ Ph. Ds), working at the intersection of physics and markets, physical and digital technologies, and across a broad set of industries to deliver world-changing innovations and capabilities for our customers.


10.2 Biotechnology in Medicine and Agriculture

It is easy to see how biotechnology can be used for medicinal purposes. Knowledge of the genetic makeup of our species, the genetic basis of heritable diseases, and the invention of technology to manipulate and fix mutant genes provides methods to treat diseases. Biotechnology in agriculture can enhance resistance to disease, pests, and environmental stress to improve both crop yield and quality.

Genetic Diagnosis and Gene Therapy

The process of testing for suspected genetic defects before administering treatment is called genetic diagnosis by genetic testing. In some cases in which a genetic disease is present in an individual’s family, family members may be advised to undergo genetic testing. For example, mutations in the BRCA genes may increase the likelihood of developing breast and ovarian cancers in women and some other cancers in women and men. A woman with breast cancer can be screened for these mutations. If one of the high-risk mutations is found, her female relatives may also wish to be screened for that particular mutation, or simply be more vigilant for the occurrence of cancers. Genetic testing is also offered for fetuses (or embryos with in vitro fertilization) to determine the presence or absence of disease-causing genes in families with specific debilitating diseases.

Concepts in Action

See how human DNA is extracted for uses such as genetic testing.

Gene therapy is a genetic engineering technique that may one day be used to cure certain genetic diseases. In its simplest form, it involves the introduction of a non-mutated gene at a random location in the genome to cure a disease by replacing a protein that may be absent in these individuals because of a genetic mutation. The non-mutated gene is usually introduced into diseased cells as part of a vector transmitted by a virus, such as an adenovirus, that can infect the host cell and deliver the foreign DNA into the genome of the targeted cell (Figure 10.8). To date, gene therapies have been primarily experimental procedures in humans. A few of these experimental treatments have been successful, but the methods may be important in the future as the factors limiting its success are resolved.

Production of Vaccines, Antibiotics, and Hormones

Traditional vaccination strategies use weakened or inactive forms of microorganisms or viruses to stimulate the immune system. Modern techniques use specific genes of microorganisms cloned into vectors and mass-produced in bacteria to make large quantities of specific substances to stimulate the immune system. The substance is then used as a vaccine. In some cases, such as the H1N1 flu vaccine, genes cloned from the virus have been used to combat the constantly changing strains of this virus.

Antibiotics kill bacteria and are naturally produced by microorganisms such as fungi penicillin is perhaps the most well-known example. Antibiotics are produced on a large scale by cultivating and manipulating fungal cells. The fungal cells have typically been genetically modified to improve the yields of the antibiotic compound.

Recombinant DNA technology was used to produce large-scale quantities of the human hormone insulin in E. coli as early as 1978. Previously, it was only possible to treat diabetes with pig insulin, which caused allergic reactions in many humans because of differences in the insulin molecule. In addition, human growth hormone (HGH) is used to treat growth disorders in children. The HGH gene was cloned from a cDNA (complementary DNA) library and inserted into E. coli cells by cloning it into a bacterial vector.

Transgenic Animals

Although several recombinant proteins used in medicine are successfully produced in bacteria, some proteins need a eukaryotic animal host for proper processing. For this reason, genes have been cloned and expressed in animals such as sheep, goats, chickens, and mice. Animals that have been modified to express recombinant DNA are called transgenic animals (Figure 10.9).

Several human proteins are expressed in the milk of transgenic sheep and goats. In one commercial example, the FDA has approved a blood anticoagulant protein that is produced in the milk of transgenic goats for use in humans. Mice have been used extensively for expressing and studying the effects of recombinant genes and mutations.

Transgenic Plants

Manipulating the DNA of plants (creating genetically modified organisms, or GMOs) has helped to create desirable traits such as disease resistance, herbicide, and pest resistance, better nutritional value, and better shelf life (Figure 10.10). Plants are the most important source of food for the human population. Farmers developed ways to select for plant varieties with desirable traits long before modern-day biotechnology practices were established.

Transgenic plants have received DNA from other species. Because they contain unique combinations of genes and are not restricted to the laboratory, transgenic plants and other GMOs are closely monitored by government agencies to ensure that they are fit for human consumption and do not endanger other plant and animal life. Because foreign genes can spread to other species in the environment, particularly in the pollen and seeds of plants, extensive testing is required to ensure ecological stability. Staples like corn, potatoes, and tomatoes were the first crop plants to be genetically engineered.

Transformation of Plants Using Agrobacterium tumefaciens

In plants, tumors caused by the bacterium Agrobacterium tumefaciens occur by transfer of DNA from the bacterium to the plant. The artificial introduction of DNA into plant cells is more challenging than in animal cells because of the thick plant cell wall. Researchers used the natural transfer of DNA from Agrobacterium to a plant host to introduce DNA fragments of their choice into plant hosts. In nature, the disease-causing A. tumefaciens have a set of plasmids that contain genes that integrate into the infected plant cell’s genome. Researchers manipulate the plasmids to carry the desired DNA fragment and insert it into the plant genome.

The Organic Insecticide Bacillus thuringiensis

Bacillus thuringiensis (Bt) is a bacterium that produces protein crystals that are toxic to many insect species that feed on plants. Insects that have eaten Bt toxin stop feeding on the plants within a few hours. After the toxin is activated in the intestines of the insects, death occurs within a couple of days. The crystal toxin genes have been cloned from the bacterium and introduced into plants, therefore allowing plants to produce their own crystal Bt toxin that acts against insects. Bt toxin is safe for the environment and non-toxic to mammals (including humans). As a result, it has been approved for use by organic farmers as a natural insecticide. There is some concern, however, that insects may evolve resistance to the Bt toxin in the same way that bacteria evolve resistance to antibiotics.

FlavrSavr Tomato

The first GM crop to be introduced into the market was the FlavrSavr Tomato produced in 1994. Molecular genetic technology was used to slow down the process of softening and rotting caused by fungal infections, which led to increased shelf life of the GM tomatoes. Additional genetic modification improved the flavor of this tomato. The FlavrSavr tomato did not successfully stay in the market because of problems maintaining and shipping the crop.


Rapid-response technology could produce billions of vaccine doses fast enough to stop the next pandemic

James Swartz operating a bioreactor that his lab uses to grow cells from which cell extracts used for CFPS are prepared. Credit: Andrew Brodhead

Ever since the COVID-19 pandemic began more than a year ago, public health officials, scientists and policy leaders have struggled to contain the viral contagion that has claimed more than 2.4 million lives worldwide and caused global economic upheaval.

This should never happen again, says Stanford bioengineer James Swartz, who has spent more than a dozen years laying the groundwork for a novel vaccine technology designed to stop viral outbreaks by inoculating millions, indeed billions, of people within weeks.

Swartz praised the current COVID-19 vaccines as unprecedented scientific and medical achievements, developed as they were with unparalleled haste and global collaboration, but what he's proposing now is even more ambitious: a radically new vaccine design and ultrafast biomanufacturing process so effective that global herd immunity could be established before a pandemic could even start.

To make good on this promise, Swartz envisions a two-stage program. Stage one would involve making bioparticles designed to carry the active ingredient for the new vaccine, testing these delivery agents for safety and then stockpiling the bioparticles without a medical payload until a pandemic threatened. The beginning of stage two would resemble the process used to create current COVID-19 vaccines, with scientists racing to identify unique molecular fingerprints, or antigens, that can be used to target the dangerous virus. Only this time, there will be a rapid-response biomanufacturing system poised to load the antigens onto the bioparticles. That could make all the difference, Swartz said, and allow a rapid-response vaccine to potentially be tested for efficacy and transformed into billions of injection-ready doses within weeks.

But two big obstacles stand in the way. First, Swartz has based his approach on an only partially-proven technology called cell-free protein synthesis that represents a complete break with the bio-processing techniques that have been used to make protein medicines for the last 40 years. Second, his radical idea faces the harsh, economic realities of pharmaceutical development: though the rewards for success could prove extraordinary, the costs of taking the risky project from conception to injection have so far proven insurmountable. Swartz figures he needs $10 million now to fund more extensive animal experiments, that build on the preliminary work he has already done, in order to establish the likelihood of eventual success. Should those animal experiments provide a tentative green light, at least another $30 million would be required to carry out human clinical trials to test the safety and efficacy of trial vaccines. And should all of this go well over the next four or five years, Swartz would then have to convince pharmaceutical manufacturers to invest $250 million or more to build sufficient bio-processing capability to make good his plan to inoculate the world in a hurry when threats emerge.

"I've kept this project alive with my own personal money at times, but I've taken it about as far as I can alone," said Swartz. "I know my proposal is expensive and faces many unknowns, but the question we should ask is what will happen if we don't do this, or something like it, and the next pandemic catches us unprepared?"

Swartz's approach hearkens back to the 1960s when molecular biologists started conducting early DNA experiments to figure out how genes made proteins, the complex biomolecules that perform multiple functions inside cells. The experimental technique they used was a process called cell-free protein synthesis, or CFPS. Scientists identified the basic bio-machinery that cells use to make proteins, extracted these bare-bones components from cells and put them into test tubes. A CFPS system includes three components: a gene to direct the protein-making process bio-machines called ribosomes and chaperone molecules that have the dual purpose of assembling amino acids, like chains, to form proteins and then folding these protein chains into whatever shape the gene dictated and, finally, the CFPS process requires the bio-fuels ATP and GTP to provide power. By the 1970s and 1980s, as CFPS revealed more about how proteins are made, scientists learned how to splice genes into living cells to give their biomachinery the blueprints for making medicinal proteins. CFPS continued as a research tool, and biotech startups focused on turning live cells into medicine-making biofactories.

A cross-sectional illustration of stockpiled bioparticle without a medical payload (left) and a bioparticle that has been “activated” (right) by attaching antigens that mirror parts of a dangerous virus that the vaccine will protect against. Credit: Farrin Abbott

It was at this critical juncture, in 1981, that Swartz joined a fledgling firm called Genentech and learned how to make protein medicines in cells. His first project was helping the then-startup company produce human growth hormone (HGH), a protein secreted by the pituitary gland to stimulate the growth of bone and cartilage. Over the next 17 years, Swartz became adept at cell-based biotechnology, which involved splicing bits of human DNA into fast-growing bacterial or, sometimes, mammalian cells that were grown in large vats. As the gene-spliced cells multiplied, they made copies of medicinal proteins that could be harvested and purified for use. But Swartz also came to learn what could go wrong, particularly with the crucial step of folding proteins, origami style, into the precise shape needed to achieve their therapeutic purpose. "We had to control a chemical assembly process inside cells that weren't built to accommodate what we wanted to make," Swartz said. "If something went wrong in our process, we would end up with a vat of proteins that weren't folded properly and were useless."

He left Genentech to join the Stanford faculty in 1998 to reinvent biomanufacturing by, paradoxically, taking it back to the CFPS style of protein making, by putting the bare-bones protein-making machinery into vats rather than petri dishes. In 2003, Swartz's lab showed how industrial-scale CFPS systems could make and fold proteins more reliably and cost-effectively than prevailing cell-based technologies. He then co-founded a biotech startup that has licensed the CFPS process from Stanford and has used it to make four protein-based, cancer-fighting therapies that are in early-stage human clinical trials. The trials are a partial vindication for CFPS, but still shy of the full validation that would occur if or when the U.S. Food and Drug Administration approves bio-medicines made using his new approach.

Meanwhile, another event in 2003—the first SARS outbreak in China—got Swartz wondering whether CFPS might be useful for mass-producing vaccines. In 2008, he and former Stanford graduate student Brad Bundy co-authored a paper postulating that CFPS was "well suited for producing versatile protein-based nanoparticles"—VLPs (virus-like particles) for short—providing the intellectual framework for the two-stage, rapid response vaccine technology for which he now hopes to garner support. In a 2015 paper, his lab showed how to remodel and repurpose the inner shell of a common virus making a VLP that resembles a tiny soccer ball with spikes. The spikes are convenient attachment points for antigens and other molecular bells and whistles, making the VLP so obnoxious that the immune system regards any virus resembling it as an enemy, and creates antibodies to render the infectious invader incapable of attacking our cells.

Swartz has already conducted small-scale animal tests on the rapid response technology and had produced promising results when the new coronavirus caused the COVID-19 pandemic. Now his hope is to get the funding in place to test his approach in more animals, and then in humans, loading the VLPs with antigens to known viral infections for which no vaccine currently exists. One such candidate would be chikungunya, a mosquito-borne viral infection prevalent in Africa, Asia and India that causes fever and joint pain. These human trials would be designed to prove the safety of VLP delivered vaccines for people in general and demonstrate that this approach would be efficacious. Pending a successful outcome, Swartz would still have to persuade pharmaceutical companies to build CFPS production plants to stockpile billions of doses of VLPs ready for activation when it became necessary.

Swartz estimates all of that will take about six years. But with luck, that could still be enough time for his rapid-response technology to be ready before the next pandemic-grade virus hits. Things could proceed swiftly after that: Immunologists could identify an effective antigen within a couple of weeks. Biotech engineers could retrieve the stockpiled VLPs and hook the newly produced antigens onto the spikes. Since the prior clinical trials would have already proven the safety of VLP vaccines produced by CFPS, the new, pandemic-stopping vaccine could be given on a trial basis to high-risk individuals at the epicenter of the contagion, to further confirm safety and begin testing the efficacy of the antigen. In a best-case scenario, Swartz estimates that billions of doses could be produced within six weeks. Even if the response took twice as long as projected, he says it would still be at least five times faster than current COVID-19 vaccine development and production processes.

Swartz knows it's premature for biotech firms to undertake a project facing so many hurdles, and a stretch even for funding agencies to underwrite the considerable upfront costs of validating or negating his approach. But as he sees it, the current pandemic has proven the need for this new approach. Now is the time for bioengineers to retool the 40-year-old technology for making protein-based therapies. He is eager to complete the mission that brought him to Stanford more than two decades ago.

"If we have the will, this could be how we make sure that the world never has to suffer a pandemic like COVID-19 again," he said.


How are vaccines mass-produced? - Biology

Houston, TX 77204-5017 Fax: 713.743.8199

FOR IMMEDIATE RELEASE
December 22, 2004

Contact : Lisa Merkl
713.743.8192 (office)
713.605.1757 (pager)

INEXPENSIVE, MASS-PRODUCED GENES CORE OF SYNTHETIC BIOLOGY ADVANCES AT UH
Professor Xiaolian Gao’s Research Unlocks Potential for New Medications, Vaccines and Diagnostics

HOUSTON, Dec. 22, 2004 – Devices the size of a pager now have greater capabilities than computers that once occupied an entire room. Similar advances are being made in the emerging field of synthetic biology at the University of Houston, now allowing researchers to inexpensively program the chemical synthesis of entire genes on a single microchip.

Xiaolian Gao, a professor in the department of biology and biochemistry at UH, works at the leading edge of this field. Her recent findings on how to mass produce multiple genes on a single chip are described in a paper titled “Accurate multiplex gene synthesis from programmable DNA microchips,” appearing in the current issue of Nature, the weekly scientific journal for biological and physical sciences research.

“Synthetic genes are like a box of Lego building blocks,” Gao said. “Their organization is very complex, even in simple organisms. By making programmed synthesis of genes economical, we can provide more efficient tools to aid the efforts of researchers to understand the molecular mechanisms that regulate biological systems. There are many potential biochemical and biomedical applications.”

Most immediately, examples include understanding the regulation of gene function. Down the road, these efforts will improve health care, medicine and the environment at a fundamental level.

Using current methods, programmed synthesis of a typical gene costs thousands of dollars. Thus, the prospect of creating the most primitive of living organisms, which requires synthesis of several thousand genes, would be prohibitive, costing millions of dollars and years of time. The system developed by Gao and her partners employs digital technology similar to that used in making computer chips and thereby reduces cost and time factors drastically. Gao’s group estimates that the new technology will be about one hundred times more cost- and time-efficient than current technologies.

With this discovery, Gao and her colleagues have developed a technology with the potential to make complete functioning organisms that can produce energy, neutralize toxins and make drugs and artificial genes that could eventually be used in gene therapy procedures. Gene therapy is a promising approach to the treatment of genetic disorders, debilitating neurological diseases such as Parkinson’s and endocrine disorders such as diabetes. This technology may therefore yield profound benefits for human health and quality of life.

“The technology developed by Dr. Gao and her collaborators has the potential to make research that many of us could only dream about both plausible and cost effective,” said Stuart Dryer, chair of the department of biology and biochemistry at UH. “In my own research on neurological diseases, we’ve often wished we could rapidly synthesize many variations of large naturally occurring genes. The costs of current technology have prevented us from doing this, but Dr. Gao’s research will break down that barrier.”

This technology offers tremendous potential benefits, as synthetic genes could allow for development and production of safer, less toxic proteins that are currently used in disease treatment. It also could allow for production of large molecules that do not occur naturally, but that are needed for new generations of vaccines and therapeutic agents, including vaccines for HIV and other viral diseases. This technology also will facilitate development of new medications through the creation of humanized yet synthetic antibodies that could be especially useful in detection and treatment of infectious organisms that could be used by terrorists.

Gao’s co-authors include Erdogan Gulari and Xiaochuan Zhou from the University of Michigan and George Church of Harvard University. Gao, Gulari and Zhou are partners in Atactic Technologies, a company that produces and markets products for life sciences research. Atactic Technologies currently holds the license to this breakthrough technology, called picoarray gene synthesis. UH and the University of Michigan are co-holders of the patents to these DNA microchip technologies.

Prior to coming to UH in 1992, Gao was a senior investigator at Glaxo Research Laboratory and received her postdoctoral training at Columbia University, her doctorate from Rutgers University and bachelor of science from the Beijing Institute of Chemical Technology. She is an expert in nucleic acid chemistry, biomolecular nuclear magnetic resonance technology, structural biological chemistry and combinatorial chemistry. Research in her lab involves the interface of chemistry and biological sciences. Holding patents in biochip technologies, Gao is currently focusing on understanding the relationships of function and structure of complex genomes of humans and other species. Gao’s research has been funded by the National Institutes of Health, the Welch Foundation, the Texas Higher Education Coordinating Board, the National Foundation for Cancer Research, the Merck Genomic Research Institute and the Defense Advanced Research Projects Agency.

About the University of Houston
The University of Houston, Texas’ premier metropolitan research and teaching institution, is home to more than 40 research centers and institutes and sponsors more than 300 partnerships with corporate, civic and governmental entities. UH, the most diverse research university in the country, stands at the forefront of education, research and service with more than 35,000 students.


Inexpensive, Mass-produced Genes At Core Of Synthetic Biology Advances At UH

HOUSTON, Dec. 22, 2004 &ndash Devices the size of a pager now have greater capabilities than computers that once occupied an entire room. Similar advances are being made in the emerging field of synthetic biology at the University of Houston, now allowing researchers to inexpensively program the chemical synthesis of entire genes on a single microchip.

Xiaolian Gao, a professor in the department of biology and biochemistry at UH, works at the leading edge of this field. Her recent findings on how to mass produce multiple genes on a single chip are described in a paper titled "Accurate multiplex gene synthesis from programmable DNA microchips," appearing in the current issue of Nature, the weekly scientific journal for biological and physical sciences research.

"Synthetic genes are like a box of Lego building blocks," Gao said. "Their organization is very complex, even in simple organisms. By making programmed synthesis of genes economical, we can provide more efficient tools to aid the efforts of researchers to understand the molecular mechanisms that regulate biological systems. There are many potential biochemical and biomedical applications."

Most immediately, examples include understanding the regulation of gene function. Down the road, these efforts will improve health care, medicine and the environment at a fundamental level.

Using current methods, programmed synthesis of a typical gene costs thousands of dollars. Thus, the prospect of creating the most primitive of living organisms, which requires synthesis of several thousand genes, would be prohibitive, costing millions of dollars and years of time. The system developed by Gao and her partners employs digital technology similar to that used in making computer chips and thereby reduces cost and time factors drastically. Gao's group estimates that the new technology will be about one hundred times more cost- and time-efficient than current technologies.

With this discovery, Gao and her colleagues have developed a technology with the potential to make complete functioning organisms that can produce energy, neutralize toxins and make drugs and artificial genes that could eventually be used in gene therapy procedures. Gene therapy is a promising approach to the treatment of genetic disorders, debilitating neurological diseases such as Parkinson's and endocrine disorders such as diabetes. This technology may therefore yield profound benefits for human health and quality of life.

"The technology developed by Dr. Gao and her collaborators has the potential to make research that many of us could only dream about both plausible and cost effective," said Stuart Dryer, chair of the department of biology and biochemistry at UH. "In my own research on neurological diseases, we've often wished we could rapidly synthesize many variations of large naturally occurring genes. The costs of current technology have prevented us from doing this, but Dr. Gao's research will break down that barrier."

This technology offers tremendous potential benefits, as synthetic genes could allow for development and production of safer, less toxic proteins that are currently used in disease treatment. It also could allow for production of large molecules that do not occur naturally, but that are needed for new generations of vaccines and therapeutic agents, including vaccines for HIV and other viral diseases. This technology also will facilitate development of new medications through the creation of humanized yet synthetic antibodies that could be especially useful in detection and treatment of infectious organisms that could be used by terrorists.

Gao's co-authors include Erdogan Gulari and Xiaochuan Zhou from the University of Michigan and George Church of Harvard University. Gao, Gulari and Zhou are partners in Atactic Technologies, a company that produces and markets products for life sciences research. Atactic Technologies currently holds the license to this breakthrough technology, called picoarray gene synthesis. UH and the University of Michigan are co-holders of the patents to these DNA microchip technologies.

Prior to coming to UH in 1992, Gao was a senior investigator at Glaxo Research Laboratory and received her postdoctoral training at Columbia University, her doctorate from Rutgers University and bachelor of science from the Beijing Institute of Chemical Technology. She is an expert in nucleic acid chemistry, biomolecular nuclear magnetic resonance technology, structural biological chemistry and combinatorial chemistry. Research in her lab involves the interface of chemistry and biological sciences. Holding patents in biochip technologies, her current focus is to understand the relationships of function and structure of complex genomes of humans and other species. Gao's research has been funded by the National Institutes of Health, the Welsh Foundation, the Texas Higher Education Coordinating Board, the National Foundation for Cancer Research, the Merck Genomic Research Institute and the Defense Advanced Research Projects Agency.

About the University of Houston

The University of Houston, Texas' premier metropolitan research and teaching institution, is home to more than 40 research centers and institutes and sponsors more than 300 partnerships with corporate, civic and governmental entities. UH, the most diverse research university in the country, stands at the forefront of education, research and service with more than 35,000 students.

Story Source:

Materials provided by University Of Houston. Note: Content may be edited for style and length.


Lyme Disease Vaccine Proteins Patented

Scientists at the U.S. Department of Energy's Brookhaven National Laboratory and collaborators at Stony Brook University have received U.S. Patent Number 7,179,448 for developing chimeric, or "combination," proteins that may advance the development of vaccines and diagnostic tests for Lyme disease.

The genetically engineered proteins combine pieces of two proteins that are normally present on the surface of the bacterium that causes Lyme disease, but at different parts of the organism&rsquos life cycle. "Combining pieces of these two proteins into one chimeric protein should trigger a 'one-two-punch' immune response more capable of fending off the bacterium than either protein alone," says Brookhaven biologist John Dunn, a researcher on the BNL Lyme disease team. "These chimeric proteins could also be used as diagnostic reagents that distinguish disease-causing strains of bacteria from relatively harmless ones, and help assess the severity of an infection," Dunn said.

Lyme disease is the most common vector-borne disease in the U.S., causing approximately 25,000 new cases each year &mdash a rate that is expected to increase by at least a third from 2002 to 2012, according to a new study. Early symptoms of the disease, which is spread by the bite of an infected deer tick, include a bull's-eye rash at the site of the bite and flu-like symptoms. If not promptly treated with antibiotics, it can lead to more serious symptoms, including joint and neurological complications.

Scientists have been working on vaccines based on the structures of proteins found on the outer surface of Borrelia burgdoferi, the bacterium that causes Lyme disease. Dunn and colleagues deciphered the atomic level structures of these proteins, known as outer surface proteins A and C (OspA and OspC), at the National Synchrotron Light Source (NSLS) at Brookhaven Lab. The OspA protein, which was used to make the original vaccine against Lyme disease, is only present in the bacteria while they are in the cold-blooded deer tick&rsquos stomach, and not in the host. After the tick bites a warm-blooded mammalian host, the injected bacteria produce OspC on their surface.

With the aim of developing a vaccine that would trigger an immune response against both these life cycle stages, Dunn's team used methods of recombinant DNA to create new proteins that combine the most immunogenic portions of OspA and OspC &mdash that is, the regions of the two proteins that are most likely to trigger an immune response.

The researchers have demonstrated that the new combination proteins retain the ability to trigger an immune response to at least one or both of the antigens, and can trigger the production of antibodies that inhibit growth of and/or kill Borrelia bacteria in laboratory cultures. They've also shown that the chimeric proteins can be mass-produced in E. coli bacteria, a common laboratory technique for making proteins, and easily purified for possible use in vaccines or diagnostic assays.

"This could lead to a vaccine that is effective at different stages of the organism&rsquos life cycle," said Dunn. Moreover, by incorporating unique protein fragments from various pathogenic families of Borrelia, these chimeric proteins could be used to distinguish clinically important exposure to disease-causing Borrelia from exposures to other non-pathogenic families of Borrelia.

The patent covers the development of the chimeric proteins themselves, the nucleic acids (genetic material) used to generate the proteins, the methods used to make the proteins, the methods used to deliver either the proteins or nucleic acids, the use of the proteins in diagnostic assays or kits, and their use in animals and humans as vaccines against Lyme disease.