Vaccines 2020: The era of the digital vaccine is here
Reproduced for educational purposes.
ABSTRACT
The SARS-CoV-2 pandemic has generated a renaissance in vaccinology, with COVID-19 mRNA vaccines delivering a “digital code” of the viral antigen with no need to purify proteins or inactivate pathogens.
INTRODUCTION
The essence of vaccination consists of exposing the human body to a surrogate of a pathogenic microorganism so that the human immune system can learn how to recognize and fight the foreign invader (1). Vaccination dates back to ancient China and the Ottoman empire. Smallpox virus, harvested from the pustules of sick people, was inoculated into healthy people with a procedure known as variolation. People were fully protected by this procedure, but the treatment often caused severe disease resulting in death of some of the inoculated individuals. Vaccination in the modern era began when Edward Jenner (2) made the astute observation that inoculating people with cowpox, a nonvirulent form of the smallpox virus isolated from cows, could educate the immune system to prevent smallpox disease. The new procedure was named “vaccination,” as the word cow is “vacca” in Latin. A century later, Louis Pasteur rationally attenuated pathogens, such as the rabies virus and the bacterium causing chicken cholera, to present nonvirulent surrogates of real pathogens to the host immune system. In the late 19th century, Emil von Behring and Shibasaburo Kitasato (3) realized that it was sufficient to deliver killed bacteria or even crude bacterial cultures containing diphtheria toxin to elicit serum antibodies that inhibited bacterial replication or neutralized the toxin. New technologies allowed the production of inactivated diphtheria and tetanus toxins, rational attenuation of the Mycobacterium tuberculosis bacterium to produce the Bacille Calmette-Guérin vaccine, and rational attenuation of the yellow fever virus to yield the 17D vaccine. Killed bacteria have been used in vaccines against typhoid fever and pertussis. The production of vaccines based on surrogate pathogens was accelerated by the discovery that viruses could be grown in chicken eggs or cell cultures. This allowed the development of killed vaccines for influenza; killed and attenuated vaccines against poliomyelitis; and live attenuated vaccines against measles, mumps, rubella, and varicella zoster. Purified components of bacteria such as capsular polysaccharides were then used as vaccines against meningococcus and pneumococcus. With the genetic engineering revolution of the 1980s, recombinant antigens, that is, immunogenic proteins expressed in a different cell rather than the original pathogen, began to be used in vaccines. Indeed, yeast was engineered to express the hepatitis B virus antigen (4). Other recombinant antigens were produced in yeast; Escherichia coli; or mammalian, insect, or plant cells, enabling production of vaccines against papillomavirus, meningococcus B, Borrelia burgdorferi (the bacterial pathogen causing Lyme disease), and herpes zoster. Vaccines using recombinant antigens of respiratory syncytial virus, cytomegalovirus, and coronaviruses have been brought to clinical development. Although recombinant antigens made the design and development of many vaccines possible, they have downsides. For example, discovery is a long and complex process, and scale-up and manufacturing take even longer because new production processes must be set up for each antigen. In many instances, vaccines took 10 to 15 years to be developed and approved (Fig. 1).
THE ERA OF DIGITAL VACCINES
In the early 1990s, it was discovered that just injecting the genetic code of antigens as DNA or RNA rather than using the purified surrogate antigen was sufficient to make a vaccine because host cells would synthesize the antigen and deliver it to the immune system. Injecting genetic information instead of purified antigen was an attractive approach that resulted in a race to develop DNA-based vaccines, with RNA thought to be too unstable. Unfortunately, DNA vaccines were not sufficiently potent in humans, so vaccine developers turned their attention to RNA technologies, which, in the meantime, had made substantial progress. In animal models, purified mRNA-based vaccines were at least one order of magnitude more potent than DNA-based vaccines. However, initially large amounts of mRNA were required, rendering the approach costly and not scalable. One way to reduce the RNA dose required was to use self-amplifying RNA, that is, mRNA encoding the antigen of interest that is amplified by the replication machinery of alphaviruses such as Venezuelan equine encephalitis virus and Sindbis virus. A 2005 study by Karikó et al. (5) demonstrated that mRNA itself was an immunostimulatory molecule and that replacing the uridine with pseudouridine resulted in decreased host proinflammatory cytokine production. This mRNA sequence optimization, together with improved mRNA purification strategies, was later used to enhance antigen expression in vivo. Last, the discovery that mRNA encapsulated in lipid nanoparticles increased vaccine potency by an additional two or three orders of magnitude made mRNA vaccines very attractive. The immunogenicity of the modified lipid nanoparticle-encapsulated mRNA could be tweaked, an important property with many therapeutic applications. One example is the induction of immune tolerance in a mouse model of multiple sclerosis using mRNA encoding myelin oligodendrocyte glycoprotein to induce antigen-specific regulatory T cells, which is the opposite of what is done to induce protection against pathogens (6).
As mRNA vaccine technology was slowly progressing, the avian H1N1 influenza virus caused a flu pandemic in 2009. Vaccine manufacturers rushed to make vaccines as quickly as possible. However, manufacturers had to wait for the vaccine viral strain to be adapted to growth in chicken eggs before large-scale production could start. Finally, the vaccine was produced in a record time of 10 months but, unfortunately, by then, the peak of the pandemic had already hit most of the world and the vaccine turned out to be too late to make an impact. Clearly, we needed much faster ways to make vaccines for emerging infections, and such an opportunity occurred on Easter Day in 2013. The Chinese Center for Disease Control uploaded online the genome sequence of H7N9, a new potentially pandemic avian influenza virus that had already killed three people in China (7). This time, while other vaccine makers were shipping the virus across the world and adapting the virus to grow in chicken eggs to make a conventional vaccine, we and other groups decided to act differently. The next day, scientists at the J. Craig Venter Institute in San Diego downloaded the H7N9 influenza virus genome sequence from the internet and synthesized the viral hemagglutinin and neuraminidase genes using an enzymatic isothermal assembly method with a self-error correction system (8). The synthetic genes were shipped overnight to Boston and were used by the Novartis Vaccines team to make a self-amplifying RNA vaccine within 1 week for preclinical testing. The vaccine had been produced without the need to grow and manipulate the virus in chicken eggs. Indeed, sending viral genetic code information over the internet rather than shipping a dangerous virus across borders heralded the arrival of the so-called digital vaccine era (Fig. 1) (9).
On 10 January 2020, the Chinese Center for Disease Control shared the genome sequence of SARS-CoV-2 online, prompting several hundred laboratories worldwide to download the gene sequence encoding the SARS-CoV-2 spike protein, generate a synthetic gene, and use it to make COVID-19 vaccines without the need to ship the virus. The synthetic SARS-CoV-2 gene encoding spike protein was used to make mRNA vaccines; was spliced into human or chimpanzee adenovirus vectors; or was used to genetically engineer mammalian, insect, or plant cells to make viral spike protein that was delivered in a vaccine with adjuvant (10). Out of all these digital approaches, the mRNA vaccines won the race. In just 66 days, the Moderna mRNA vaccine entered clinical testing, and 10 months later, mRNA vaccines from Moderna and Pfizer/BioNTech received emergency use authorization from the US Food and Drug Administration (FDA).
Traditionally, making vaccines involved growing bacteria, viruses, or parasites and using them to produce live attenuated, killed, or subunit vaccines, so-called analog vaccines. Digital vaccines are not based on any component derived from the live pathogen. Instead, they are based on genetic information downloaded from the internet with synthesis of the gene or RNA that then delivers the genetic code to host cells. Host cells then synthesize the antigen that activates the host immune response.
THE NEXT FRONTIER FOR VACCINES
The speed of making fully synthetic digital vaccines has allowed rapid development of COVID-19 vaccines to combat the current pandemic. However, emergency use authorization within 10 months would not have been possible without complete transformation of vaccine development (Fig. 1). In the 1980s, vaccine development was lengthy, and sometimes vaccines could not be made because the technology was not available. However, vaccine discovery and development accelerated with the advent of new technologies such as recombinant DNA, polysaccharide conjugation, sequencing of microorganism genomes, reverse vaccinology (the identification of new antigens by computational analyses of pathogen genomes), and high-throughput methods that enabled parallel instead of sequential analysis. By 2010, vaccine discovery and development had become much shorter and reverse vaccinology enabled development of the complex meningococcus B vaccine. The next acceleration of vaccine development occurred in 2020 with digital vaccines, which enabled the design of antigens using their molecular structure, often in a complex with the antibody (reverse vaccinology 2.0) (11), and made it possible to make synthetic genes and deliver them as mRNAs.
Vaccine development was relatively simple in the 1980s but later became longer, more complicated, and very expensive. There were several reasons for this, including higher international standards and Good Manufacturing Practices, which often required building a dedicated manufacturing facility for each vaccine, and Good Clinical Practices, which standardized clinical development and required large efficacy and safety clinical trials. The overall cost of developing one vaccine started to exceed $1 billion, and therefore, it became standard practice to de-risk the investment by performing each step of vaccine development sequentially (Fig. 1). Thus, industry would not invest in early vaccine development unless the discovery phase had been successfully completed. In addition, industry would not invest in late vaccine development and would not build a manufacturing plant unless the early development phase had been successfully completed. In 2011, we predicted that by 2020, new technologies would enable many development steps to be performed in parallel, resulting in an acceleration of vaccine development (12). However, this still had not happened by 2019, despite new technologies that shortened the long and expensive timelines of vaccine development (13). As soon as the COVID-19 pandemic emerged, the public sector asked companies to accelerate vaccine development and provided financial incentives to do so. The US government’s Operation Warp Speed made $13.5 billion available to companies that had technologies for making vaccines; the Coalition for Epidemic Preparedness Innovation (CEPI) and other governments soon followed. The message to the manufacturers was clear: The public sector was taking the financial risk to enable companies to perform the different phases of vaccine development in parallel instead of sequentially and to build manufacturing plants. Thanks to this financial incentive and to the flexibility and collaboration of the regulatory agencies, vaccine development was transformed in a few months: Phase 1 clinical trials were performed immediately after discovery. Phase 2 trials were started as soon as initial data on safety and immunogenicity were available from phase 1 trials; phase 3 trials were started as soon as phase 2 data were available, and phase 1 and 2 trials were often merged. COVID-19 vaccine development required an enormous financial risk but was accomplished in less than 8 months and without skipping any of the steps required to assess the safety and efficacy of these vaccines. It has been calculated that the financial risk taken by Operation Warp Speed would have paid for itself if it accelerated vaccine development by just 12 hours (14).
Digital vaccines have not only brought new vaccines to people very quickly but also have launched a revolution in vaccine manufacturing. So far, vaccines have been classified by regulators as “biologics,” and each vaccine requires a dedicated manufacturing plant, which can take 3 to 5 years to build and validate, requiring hundreds of millions of dollars of investment. Vaccines comprising multiple components such as that for meningococcus B may require more than one manufacturing plant. By contrast, the manufacture of RNA vaccines requires the same process for every vaccine. Moreover, the differences among RNA vaccines reside in the gene sequence encoding the antigen and not in the antigen itself. Thus, a manufacturing plant for RNA vaccines can be built and scaled up much faster than a manufacturing plant producing a biologic vaccine. Indeed, 18 months after the COVID-19 pandemic began, more than 1 billion doses of the two COVID-19 mRNA vaccines have been produced and delivered, whereas none of the vaccines based on recombinant proteins have been licensed yet, principally because of manufacturing complexities. An additional advantage of RNA vaccines is that multiple vaccines can be manufactured in the same manufacturing plant, as they share the same process. Last, the most interesting advantage of RNA vaccines is that, theoretically, their design and production can be fully automated, and instructions can be provided even from a remote location. We can imagine a future scenario where a pandemic vaccine is designed and developed in 1 week by a single research group and produced globally by a series of robotic stations distributed across different continents (Fig. 1) (15).
THE RENAISSANCE OF VACCINOLOGY
We are living in a unique moment, which can be considered a renaissance of vaccinology. The COVID-19 pandemic continues to devastate the health, economy, and freedom of people worldwide. However, it has provided opportunities, including not only validating new vaccine technologies such as mRNA but also reshaping and hopefully changing forever the way that vaccines and possibly drugs will be developed. By taking a financial risk, vaccine development can be accelerated without compromising on safety and efficacy. Will we be able to take such financial risks in the future? And who should take them? Clearly, the return on investment for the public sector is huge, and being an area of public interest, the public sector should be accountable for the investment. However, companies have also learned that development can be accelerated by taking this kind of financial risk and might be willing to invest to bring new vaccines to market. Several initiatives have already been pushed forward as a result of the COVID-19 pandemic. The US government has launched H-ARPA, an advanced research project agency to improve the health of its population (www.harpa.org/). Meanwhile, CEPI plans to turn the tide against infectious diseases by compressing vaccine development timelines to 100 days. The time to debate and act is clearly now.
So what will vaccine development look like in 2030? We can imagine that protein antigens will require very little experimental effort because they could be fully designed using machine learning, artificial intelligence, and neural networks. Such efforts are being pioneered by the groups of DeepMind and David Baker, enabling, with atomic accuracy, the prediction of the structure and behavior of antigens and antigen complexes starting from the protein sequence alone (16, 17). Sequences of potential vaccine antigens from viruses, bacteria, parasites, and fungi will be identified by reverse vaccinology. By then, the genome sequences of most microorganisms and their diversity will be available in databases, and sequences of protein antigens will be optimized to capture experimental and predicted diversity, antigenic drift, and stability. Development will be fast, as toxicology, safety and immunogenicity analyses could be conducted on organoids on chips. These organoids on chips combined with artificial intelligence will capture an enormous amount of data that can be translated to predict vaccine formulation, dose, safety, schedule, and possibly efficacy.
Vaccines that are difficult or impossible to develop today could become possible tomorrow, and therefore, vaccines for combating antimicrobial resistance, chronic diseases, cancer, and neurodegenerative diseases potentially could be made. We could develop better malaria vaccines, redesigned by reverse vaccinology, machine learning, artificial intelligence, and neural networks. We will revisit and possibly solve the puzzle of an HIV vaccine by predicting what would be the optimal immunization strategy to elicit broadly neutralizing antibodies in humans (18).
CONCLUSION
Clearly, digital vaccines, and particularly mRNA vaccines, represent a new vaccine class with vast prophylactic and therapeutic potential, combining speed of development with simple manufacturing. However, mRNA vaccines are far from mature as the formulation is only stable at very low temperatures and is expensive, which limits their use (19). One area where mRNA could be revolutionary is passive immunization. Today, development and manufacturing of human monoclonal antibodies is still heavily analog because it requires production in cell lines, fermentation, purification, and long and expensive efficacy testing before the product can be delivered to patients. Timelines and costs of development and manufacturing could be reduced by delivering the sequence of the antibody as mRNA and letting the human body produce the antibody. Initial promising results of such an approach have been reported in preclinical animal studies and in one clinical trial (#NCT03829384) (20). However, like all new technologies, mRNA will solve some but not all problems. For instance, it is difficult to imagine that some of the bacterial vaccines such as glycoconjugate vaccines will be replaced by mRNA vaccines or that all bacterial and fungal proteins will be effectively produced by our own cells. Last, we should consider that the SARS-CoV-2 mRNA vaccine was relatively straightforward to make and that we may not be so lucky with other pathogens that may be less amenable to mRNA vaccine technology. Therefore, it is imperative to continue to nurture this digital vaccine revolution and to bring it to all biotechnologies to ensure a renaissance era that will extend to all health interventions.
Acknowledgments
We thank G. Corsi for the artwork and C. Mallia for editorial assistance.
Competing interests: M.P., S.P., and R.R. are full-time employees of the GSK group of companies.
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