Professor Earl Brown is specialized in virology and microbiology. His main activities are on viral genetics and evolution, mainly directed at understanding how viruses cause disease (pathogenesis) or become adapted to new hosts; and the mechanism used by influenza virus to control the antiviral interferon response. He has experience with several viruses, including hepatitis C, reovirus, mumps virus and Torque Tino virus (TTV).
The public are concerned that the mRNA vaccines may interfere with human DNA, and misinformation about their capacity to transcribe or modify human DNA is circulating online. How can the general public be ensured that mRNA vaccines do not interfere with human DNA and have no impacts on human cells other than their original purpose?
Earl Brown: The Covid-19 mRNA vaccines consist of a biosynthetic RNA molecule that encodes all (Moderna) or part (Pfizer BioNtech) of the SARS-CoV-2 spike subunit protein, encapsulated within lipid shells. When the vaccine is injected into muscle, the lipid particles fuse to the lipid membranes of cells to deliver the mRNA into the cytoplasm where it is translated into spike protein. The protein is then cut into pieces and presented on the surface of the cells to be sensed by the immune system, thus initiating immune reactions. mRNA vaccines also stimulate the innate immune system, which is designed to inhibit virus infections. This occurs largely through detection of viral RNA to trigger production of immune system cytokines, which are activators of the immune system, that then recruit immune cells to the site of infection or vaccination. An advantage of mRNA and other expression-vectored vaccines is that production of protein within cells results in the activation of both cellular and antibody dependent immunity, whereas protein-based vaccines generally induce weaker T-cell responses. Antibodies target virus particles to neutralise them and prevent infection, whereas killer T-cells destroy infected cells. Both are important for eliminating the SARS-CoV-2 infection.
Earl Brown: The spike mRNA will function to synthesise protein for a short period of time (usually hours) before being degraded into subunits and ceasing to function. The spike mRNA vaccines will not change the genetic makeup of cells because they are not delivered into the nucleus – where DNA resides – but into the cytoplasm. In addition, these vaccine mRNA molecules possess cytoplasmic localisation signals which prevent transport into the nucleus and are also unstable, being degraded in a matter of hours. RNA has not been found to modify the DNA when introduced into the nucleus or cytoplasm. This is consistent with the observation that infections with viruses (both DNA and RNA genome viruses) does not result in genetic changes to DNA, even though all infected cells are expressing large amounts of viral mRNA.
Some viruses are obvious exceptions, such as retroviruses, which insert their genome into the DNA strands of all the cells they infect through specific functions of their genes. Although mRNA vaccines are relatively new, such vaccines have been used clinically in humans for cancer treatment and have been found to be safe and not toxic to genes. In addition, human mRNA vaccines for influenza, rabies, and zika viruses are now in late phase trials for licensing with no observed safety problems.
What is the biggest challenge during the development of mRNA vaccines?
Earl Brown: While the function of mRNA as the information-carrying molecule that directs protein synthesis has been known since the dawn of molecular biology, its instability has hampered its application to produce proteins in cells. Protein synthesis by introducing synthetic mRNA into cells is normally inefficient for two reasons: loss thorough degradation and excessive reactogenicity (seen as inflammation). The introduction of mRNA into cells therefore results in its breakdown by degradative enzymes and it stimulates a strong innate immune response. The innate immune response functions to sense the molecular features of pathogens, many of which focus on the RNA, with detection both on the cell surface and within the cytoplasm of infected cells. On activation by RNA, cells secrete immune regulatory molecules termed cytokines, such as interferon, which then cause inflammation and make cells resistant to virus infection. Interferon induces further enzymes that degrade mRNA or decrease protein synthesis.
Earl Brown: Having observed that non-mRNA types that are more stable possessed modified ribonucleotides such as those of U (uridine), the same modifications have been used to make mRNA more stable and less reactogenic for application into vaccines since 2005. The lipid coating of the mRNA vaccine further protects the mRNA and delivers it into cytoplasm by fusing with cellular lipid membranes.
Thus, the biggest breakthrough in solving the problems of the stability and reactogenicity of mRNA has been made by using modified ribonucleotides, in addition to protection within lipid nanoparticles.
Can the current mRNA-based Covid vaccines provide cross-reactivity against SARS-CoV variants or other respiratory viruses? If so, can we expect them to be effective against new coronaviruses and other diseases in the future?
Earl Brown: Although the SARS-CoV of 2003 is the closest know human coronavirus relative to SARS-CoV-2, they only share 80% of the sequence identity between their RNA genomes. This makes them distant cousins that would be expected to have diverged from their common ancestor over 100 years ago. Studies comparing SARS-CoV antibodies show a lack of neutralisation of SARS-CoV-2 virus because the surface spike proteins are too structurally dissimilar at their neutralising antibody binding sites. Some cross-reactive antibody binding was seen for other internal proteins that evolve more slowly.
The low cross-reactivity between the SARS-CoV-2 viruses is also see with antibody responses to other garden-variety human coronaviruses such as HCoV-229E, HKCoV-HKU1, HCoV-OC43 and HCoV-NL63. This low cross-reactivity does not protect cells against infection, however, it is speculated that it may help decrease disease severity and may be a factor in regulating the difference in severity seen in different populations, especially comparing the young to the elderly.
It is expected that other novel viruses, including novel coronaviruses that enter humans in the future, will require their own dedicated vaccines.
What is the main advantage of RNA vaccines compared to vector vaccines?
Earl Brown: The advantages of RNA vaccines are their speed of design and implementation due in large part to the uniformity of mRNA properties. These benefits are embodied in a reliable vaccine platform where any mRNA can be designed into a vaccine as long as the mRNA sequence is known. mRNA has uniform chemical properties and an established biomanufacturing platform that relies on DNA templates (made in bacteria) where T7 DNA-dependent RNA polymerase subunits transcribe mRNA using ribonucleotide subunits. RNA behaves in predictable ways, whereas proteins are highly variable and thus less predictable in their solubility properties, such as clumping or falling out of solution. You also have to make protein subunits in cell-based systems, which are very exacting systems to maintain and operate.
mRNA vaccine design is both fast and flexible. So much so that Moderna was able to design the prototype within two weeks (of receiving the sequence of the spike protein) and a vaccine candidate for trail in 63 days. In the future, mRNA vaccines will be an obvious first choice for any emerging infection.
mRNA vaccines will not immediately be usable for diseases to which we cannot currently develop a vaccination approach, such as Hepatitis C virus or HIV. If a novel pathogen emerges in the future that cannot be prevented by conventional vaccination approaches, then it would likewise not be solved by using the mRNA vaccine approach.
mRNA stability will be improved in the future, so I expect that the low temperature freezer requirement for mRNA stability will soon be replaced with conventional cold-chain requirements.
Vectored vaccines such as those of AstraZeneca (Chimpanzee adenovirus), Johnson and Johnson (Adenovirus type 26), CanSinoBIO-Beijing Institute of Biotechnology vaccine (Adenovirus type 5) and Russia’s Sputnik V (Adenovirus type 26 and type 5) all use defective adenovirus vectors to deliver the spike protein gene as a DNA copy, which is then transcribed into mRNA for translation into protein in the cytoplasm of cells. The virus vector contains a defective genome of adenovirus that lacks the regulatory genes that are required to express its virus genes, but instead makes mRNA for the SARS-CoV-2 spike protein. Consequently, adenovirus vectored Covid-19 vaccines give intracellular protein synthesis via mRNA, so the benefits of the expression approach are quite similar when it comes to inducing a good T-cell and antibody response.
Earl Brown: However, the virus vector also stimulates an immune response against its own constituent proteins. This becomes important for the two-dose vaccines because anti-adenovirus antibodies resulting from the primer injection will now interfere with the adenovirus vectored vaccine given as a booster, functioning to bind to the vectored adenovirus particle and decrease its delivery to cells. So vectored vaccines have to balance the effectiveness of the delivered primer and boost vaccines for the spike protein versus the immune responses to the adenovirus vector, which serves to block immunisation via the booster shot. Sputnik V uses two different adenoviruses for the prime (Ad26) and the boost (Ad5) so that immune inhibition of the second vector shot by the immune response to the primer shot does not occur. The Johnson and Johnson Covid-19 vaccine approach solves this anti-adenovirus vector response by using a single shot of Ad26-vectored vaccine (to achieve 67% protection against disease) and AstraZeneca has used dosing regimens that allow an effective booster response with the same chimpanzee adenovirus vector.
A further problem of adenovirus vectors that use human adenoviruses, such as the CanSinoBio vaccine, which uses type Ad5 as a vector, is the presence of pre-existing antibodies that are present in about 25% of the human population, and that function to inhibit the vaccine vector. This can reduce the overall effectiveness of Ad5-vectored vaccines in some populations. Although the Sputnik V vaccine uses a less prevalent Ad26 vector for the priming inoculation followed by Ad5 as the booster vector, it has been reported to be 9 % effective in published phase I and II trials. Although the reported protection provided by adenovirus-vectored vaccines is generally not as high as that provided by mRNA vaccines against mild infections, they do offer 90% protection against severe disease. Unlike mRNA vaccines, their storage does not require ultra-low temperatures, making them more practical for general use.
The recent emergence of SARS-CoV-2 variants such as B.1.1.7, which can partially escape immunity, has stimulated the development of second-generation vaccines directed against them. Vectored vaccines will require the production of modified replication defective adenovirus genomes for production in packaging cell lines, which is an additional step compared to mRNA vaccines, but should not impede their timeliness by more than a few weeks longer than the six weeks needed to start producing a novel mRNA vaccine.
