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mRNA vaccines instruct cells to produce spike protein that will trigger an immune response. But which types of cells will it work on? and which cells will it not work on?
I'm assuming the question refers to the Pfizer/BioNTech and Moderna vaccines in which Lipid NanoParticles (LNP) are used and to the injection to be intramuscular (i.m.). According to a blog by Derek Lowe , the parts of your body producing the coronavirus Sprike protein antigen are:
the muscle tissue at the site of injection, the lymphatic tissue downstream in your armpit on that side, your spleen, and (for the first day or two) your liver.
In other words, it is not only your muscle cells that become the antigen factory.
 Lowe, Derek (2021) “mRNA Vaccines: What happens”, Science Translational Medicine, AAAS, https://blogs.sciencemag.org/pipeline/archives/2021/01/21/mrna-vaccines-what-happens .
According to this paper written by Emily Bettini and Michela Locci, mRNA-LNPs and locally produced antigens are taken up by dendritic cells via endocytosis . Within the cell, mRNA is translated into the antigenic protein. These locally produced antigens are either degraded by proteasome in the cytoplasm or secreted from the host cell, leading to 2 different immune response activation pathways .
When antigenic proteins undergone degradation by proteasome, the generated antigenic peptide epitopes are loaded onto MHC class I molecules within the endoplasm reticulum, then transported to and expressed at the plasma membrane . When dendritic cells transfer to the lymph nodes, these loaded MHC class I peptide epitope complexes are recognized by CD8+ T cells. This pathway induces the formation of cytotoxic T cells which are capable of directly killing the infected cells .
Figure 1. Immune responses elicited by SARS-CoV-2 mRNA vaccines 
When antigenic proteins are secreted from the host cell, they are recognized and taken up by dendritic cells again. They are then degraded in endosome, loaded onto MHC class II molecules, followed by expression at the cell surface . At the lymph nodes, these loaded MHC class II peptide epitope complexes are recognized by CD4+ T cells. This pathway induces the formation of memory B cells (MBCs) and the antibody-secreting long-lived plasma cells (LLPCs) .
1)Bettini E, Locci M. SARS-CoV-2 mRNA Vaccines: Immunological Mechanism and Beyond. Vaccines (Basel). 2021;9(2):147. Published 2021 Feb 12. doi:10.3390/vaccines9020147
2)Wadhwa A, Aljabbari A, Lokras A, Foged C, Thakur A. Opportunities and Challenges in the Delivery of mRNA-Based Vaccines. Pharmaceutics. 2020; 12(2):102. https://doi.org/10.3390/pharmaceutics12020102
Yes, some COVID vaccines use genetic engineering. Get over it.
We’ve all heard the conspiracy theories about COVID-19. Now a whole new set is emerging around COVID vaccines — and spreading as virulently as the pandemic they are meant to control.
Though the public health community tends to resort to reassurances about some of the more reasonable concerns — yes, the vaccines have been developed incredibly quickly and short-term side effects can occur — this post aims to do something different.
We’re going right to the heart of the matter. So no, COVID-19 vaccines aren’t delivery vehicles for government microchips. They aren’t tainted by material from aborted fetuses. And they won’t turn us into GMOs — though some of them do use genetic engineering, and all of them use genetics more broadly.
We think this is way cool — something to celebrate, not shy away from. So, we’re doing the deep reveal on exactly how genetics and biotechnology have been a central component of the vaccine effort. Because we know the conspiracists don’t care about evidence, anyway.
MRNA — BioNTech/Pfizer and Moderna vaccines
First up: mRNA. It won’t reprogram your brain. But it does reprogram some of your cells, in a manner of speaking. And that’s not a defect — it’s intentional.
To get your head around this you need to understand what mRNA is for. Basically, it’s a single-stranded nucleic acid molecule that carries a genetic sequence from the DNA in the cell’s nucleus into the protein factories — called ribosomes — that sit outside the nucleus in the cellular cytoplasm.
That’s what the “m” in mRNA stands for: messenger. Messenger RNA just carries instructions for the assembly of proteins from the DNA template to the ribosomes. (Proteins do almost everything that matters in the body.) That’s it.
This is useful for vaccines because scientists can easily reconstruct specific genetic sequences that encode for proteins that are unique to the invading virus. In the COVID case, this is the familiar spike protein that enables the coronavirus to enter human cells.
What mRNA vaccines do is prompt a few of your cells near the injection site to produce the spike protein. This then primes your immune system to build the antibodies and T-cells that will fight off the real coronavirus infection when it comes.
It’s not hugely different from how traditional vaccines work. But instead of injecting a weakened live or killed virus, the mRNA approach trains your immune system directly with a single protein.
Contrary to assertions made by opponents, it won’t turn you or anyone else into a GMO. mRNA stays in the cytoplasm, where the ribosomes are. It does not enter the nucleus and cannot interact with your DNA or cause any changes to the genome. No Frankencure here, either.
A variant of the mRNA approach is to go one step back in the process and construct a vaccine platform out of DNA instead. This DNA template — constructed by scientists to encode for the coronavirus spike protein — gets into cells where it is read into mRNA and… well the rest is the same.
You might ask whether this DNA can genetically engineer your cells. Once again, the answer is no. DNA is injected in little circular pieces called “plasmids” — not to be confused with plastics — and while these do enter the nucleus, the new DNA does not integrate into your cellular genome. Got it?
Adenovirus — the Oxford vaccine
This one really is genetically engineered. But what does that actually mean?
The Oxford vaccine uses what is called a “viral vector” approach. The scientific team took an adenovirus — a type of pathogen that causes a common cold — and spliced in the same spike protein genetic sequence from the coronavirus.
The adenovirus simply serves as the vehicle to get the genetic sequence into your cells. That’s why it’s called a “viral vector” after all. Viruses have been designed by billions of years of evolution precisely to figure out ways to sneak into host cells.
Note that genetic engineering is an essential part of the development process. Firstly, vector viruses are stripped of any genes that might harm you and actually cause disease. Genes that cause replication are also removed, so the virus is harmless and cannot replicate.
Then the coronavirus spike protein genes are added — a classic use of recombinant DNA. So yes, the Oxford/AstraZeneca vaccine does actually mean a genetically engineered virus is injected into your body.
And that’s a good thing. In the past, for example with the polio vaccine, live viruses in the vaccine can sometimes mutate and revert to being pathogenic, causing vaccine-derived polio. You can see it’s far better to use a GM virus that cannot cause any such harm!
As we have reported before at the Alliance for Science, the anti-GMO and anti-vaccine movements substantially overlap. These groups tend to share an ideology that is suspicious of modern science and fetishsize “natural” approaches instead. Whatever “natural” means.
Note that these groups are not always marginalized to the fringe where they belong. In Europe, anti-GMO regulations have stymied any substantial use of crop biotechnology for nearly two decades, hindering efforts to to make agriculture more sustainable.
And back in July, the European Parliament actually had to suspend the EU’s anti-GMO rules in order to allow the unimpeded development of COVID vaccines. Very embarrassing for Brussels!
Will the anti-GMO and anti-vaxxer movements use their usual scaremongering tactics to drum up fear, increase vaccine hesitancy and thereby prolong the hell of the COVID-19 pandemic? That remains to be seen. If they do succeed, then tragically many more people will die and our economies will continue to suffer. It’s up to all of us — the grassroots pro-science movement — to stop them.
Do COVID-19 RNA-based vaccines put at risk of immune-mediated diseases? In reply to “potential antigenic cross-reactivity between SARS-CoV-2 and human tissue with a possible link to an increase in autoimmune diseases”
I read with great interest the article by Vojdani et al. [ 1 ], concerning the hypothesis of a molecular mimicry mechanism between the nucleoprotein/spike protein of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and self-antigens. Viruses are notoriously involved in the pathogenesis of autoimmune diseases [ 2 ], and the authors reasonably conclude that such a cross-reactivity might lead to the development of immune-mediated disorders in COronaVirus Disease-19 (COVID-19) patients in the long term. The authors also suggest that a similar scenario might take place following COVID-19 vaccination.
Vaccine-associated autoimmunity is a well-known phenomenon attributed to either the cross-reactivity between antigens or the effect of adjuvant [ 3 ]. When coming to COVID-19 vaccine, this matter is further complicated by the nucleic acid formulation and the accelerated development process imposed by the emergency pandemic situation [ 4 ]. Currently, lipid nanoparticle-formulated mRNA vaccines coding for the SARS-CoV-2 full-length spike protein have shown the highest level of evidence according to the efficacy and safety profile in clinical trials, being therefore authorized and recommended for use in the United States and Europe. Although the results from phase I and II/III studies have not raised serious safety concerns [ 5 ], the time of observation was extremely short and the target population not defined. Reported local and systemic adverse events seemed to be dose-dependent and more common in participants aged under 55 years. These results presumably depend on the higher reactogenicity occurring in younger people that may confer greater protection towards viral antigens but also predispose to a higher burden of immunological side effects.
The reactogenicity of COVID-19 mRNA vaccine in individuals suffering from immune-mediated diseases and having therefore a pre-existent dysregulation of the immune response has not been investigated. It may be hypothesized that immunosuppressive agents prescribed to these patients mitigate or even prevent side effects related to vaccine immunogenicity.
Besides the mechanism of molecular mimicry, mRNA vaccines may give rise to a cascade of immunological events eventually leading to the aberrant activation of the innate and acquired immune system.
RNA vaccines have been principally designed for cancer and infectious diseases. This innovative therapeutic approach is based on the synthesis of RNA chains coding for desired antigenic proteins and exploits the intrinsic immunogenicity of nucleic acids. In order to avoid degradation by RNases, RNA can be encapsulated in nanoparticles or liposomes, which deliver the cargo inside target cells following a process of endocytosis. mRNA is then translated into immunogenic proteins by cell ribosomal machinery [ 6 ].
However, prior to the translation, mRNA may bind pattern recognition receptors (PRRs) in endosomes or cytosol. Toll-like receptor (TLR)3, TLR7 and TLR8 are able to recognize chains of double-stranded (ds)RNA or single-stranded (ss)RNA in endosomes, while retinoic acid-inducible gene-I (RIG-I) and melanoma differentiation-associated protein 5 (MDA5) may detect short and long filaments of dsRNA in the cytosol. The final result is the activation of several pro-inflammatory cascades, including the assembly of inflammasome platforms, the type I interferon (IFN) response and the nuclear translocation of the transcription factor nuclear factor (NF)-kB [ 7 ].
Importantly, the up-regulation of these immunological pathways is widely considered to be at the basis of several immune-mediated diseases, especially in genetically predisposed subjects who have an impaired clearance of nucleic acids [ 8 ]. This could particularly hold true in young female individuals, due to the over-expression of X-linked genes presiding over the antiviral response and the stimulatory effect played by estrogens on the immune system. The X chromosome hosts several genes involved in the immune response, including TLR7 and TLR8 genes, and about 10% of microRNAs indirectly controlling the activation of the immune system [ 9 ].
Therefore, young and female patients who are already affected or predisposed (e.g. immunological and serological abnormalities in absence of clinical symptoms, familiarity for immune-mediated diseases) to autoimmune or autoinflammatory disorders should be carefully evaluated for the benefits and risks of COVID-19 mRNA vaccination. According to epidemiological data, these subjects may develop the infection asymptomatically or pauci-symptomatically and it is worth noting that, in line with the article of Vojdani et al. [ 1 ], the presence of autoreactive cells and autoantibodies cross-reacting against SARS-CoV-2 epitopes may even turn naturally protective towards the infection. Until proven otherwise, the administration of a nucleic acid vaccine may instead put these individuals at risk of unwanted immunological side effects by either sensitizing the PRRs or generating cross-reactive cell clones and antibodies. Moreover, COVID-19 mRNA vaccine might differently stimulate myeloid or plasmacytoid dendritic cells (DCs), generating an unbalance in the downstream cytokine pathways that play a crucial role in autoimmunity and autoinflammation [ 3 ].
Modifications in nucleoside and nanoparticle composition through a proper manufacturing may help to prevent some of these drawbacks. For instance, the substitution of uridine with pseudouridine was shown to reduce immunogenicity and type I IFN production while enhancing the synthesis of viral antigenic proteins [ 10 ]. A strong type I IFN response may, in fact, negatively affect the vaccine efficacy by suppressing the process of mRNA translation [ 10 ]. However, type I IFNs play a beneficial role in strengthening the antiviral response, as they favor the maturation of DCs, the CD8+ T cell-mediated cytotoxicity and the secretion of several cytokines, like interleukin (IL)-12 and IL-23 [ 11 ]. Notably, polymorphisms in the genes encoding these cytokines or their receptors have been associated with the susceptibility to autoimmune diseases [ 12 ]. Additionally, an excessive production of type I IFNs may result in the breakdown of the immunological tolerance and, therefore, in autoimmunity [ 10 ].
Lipid components may also dictate the type and intensity of the immune response, by enhancing the production of IFN-γ, IL-2 and tumor necrosis factor (TNF)-α with the subsequent activation of both CD4+ and CD8+ T lymphocytes. Although this is not the case of the authorized COVID-19 mRNA vaccines, future formulations containing adjuvant like TLR agonists [ 13 ] may exacerbate pre-existing autoimmune or autoinflammatory disorders and should therefore be discouraged in this cohort of patients.
Given the current state of the art, my view is that individuals with a dysfunctional immune response should receive the COVID-19 mRNA vaccine only if the benefits of this approach clearly outweigh any risks and after a careful evaluation case by case.
How Do mRNA Vaccines Work?
Scientists working today are usually a part of a team, and many advancements and discoveries that are now made don't trace neatly back to a single person. The vaccines that have been developed in record time for COVID-19 are examples of that researchers working together, building on previous efforts, have engineered these desperately needed medicines.
The Pfizer and Moderna vaccines that the FDA has approved for use are both mRNA (messenger RNA) vaccines.
Since our immune system has a memory, it can (usually) fight a pathogen more effectively when it encounters that pathogen for a second time. So vaccines are used to 'teach' your immune system about an infection, giving it the first exposure, without making the body sick. Many traditional vaccines use inactivated or weakened forms of pathogens to trigger an immune response and create the immunological memory of the infection. But mRNA vaccines work in a different way.
Animal cells keep the genome, or DNA in the nucleus. Cellular machines can transcribe active genes from the DNA into another molecule called mRNA. The mRNA is then used in another part of the cell to generate proteins that are required for carrying out biological functions.
Viruses are different. Some, like the SARS-CoV-2 virus that causes COVID-19, are RNA viruses their genomes are made of RNA. When they infect cells, their RNA genome is dumped into a host animal cell. These RNA molecules are then transcribed by the host cell's machinery into viral proteins that take the host cell over and spread more infectious particles to other cells.
In the case of mRNA vaccines, the body is exposed to a piece of viral mRNA that encodes for a harmless viral protein - the mRNA that's injected as the vaccine is contained inside of lipid nanoparticles that help it get into cells. But once that viral protein is made by cells, the immune system will mount a response against it. Because the piece of mRNA encodes for only one portion of a viral protein that's ineffective at causing an infection on its own or creating more viral particles, it cannot cause an illness. But it's sufficient to train the immune system. It takes a few days after the vaccine is administered for the body to generate viral proteins from the mRNA and then respond. Once that happens, however, a vaccinated person's immune system will have been trained to attack the COVID-19 virus in the case of infection.
Cells break down mRNA quickly once it's used, so these molecules will not linger in the body. They also do not enter the nucleus so they will not impact a person's DNA.
Scientist Katalin Karikó has been studying mRNA vaccines for years, and now works at BioNTech, a German research company. She spent years getting grant rejections for her ideas about mRNA vaccines and left the University of Pennsylvania after being demoted. But now she may be on track to win the Nobel Prize, and her efforts are helping to end a global pandemic.
Karikó and her co-authors from a 2014 review on mRNA vaccines that was published in Nature Review Drug Discovery, Ugur Sahin and Özlem Türeci, are the leaders of the work behind the Pfizer vaccine.
The authors noted in that review that this concept originated with a team including physician-scientist Jon A. Wolff, reporting in Science in 1990. Wolff and co-authors showed that when mRNA was injected into mouse skeletal muscle, it would be transcribed into protein. Later efforts showed that this was a feasible approach for vaccination.
Synthetic mRNA for therapy is in general designed following the blueprint of eukaryotic mRNA. Cap and poly(A) tail are essential elements because they are required for efficient translation. 2 , 15 , 21 Positioned at the very 5′- and 3′-end of mRNA, Cap and poly(A) tail are also required to stabilize mRNA in the cytosol, where decay is catalyzed predominantly by exonucleases. 22 , 23
However, to further increase both translation and stability, mRNA requires 5′ and 3′ untranslated regions (UTRs) to flank the ORF. 24 - 27 UTRs have to be carefully chosen because they may also impair translation or mRNA stability. 28 In particular, specific cis-acting destabilizing sequences like AU-rich elements 29 and miRNA binding sites 30 , 31 mostly reside in UTRs, although they may also be found in ORFs. 32 Care must be taken to avoid such destabilizing signals.
Following these considerations, efforts have been made to identify beneficial mRNA elements in order to improve translation and stability of synthetic mRNA molecules inside the cell. Improved mRNA formats thus identified will likely also yield better mRNA vaccines, as it is widely assumed that the efficacy of an mRNA vaccine will rise as protein expression is increased and prolonged.
mRNA may be capped during transcription by including a cap analog in the reaction. However, it has been found that the regular cap analog is often incorporated in the reverse orientation so that the m 7 G nucleotide does not constitute the cap but is instead the first transcribed nucleotide. As a result, about one third of mRNA molecules are not methylated at their cap. 33 Such mRNA lacking methylation of the cap base is not translated. 34
In order to avoid unmethylated cap by reverse orientation, mRNA may be transcribed without cap analog and subsequently capped using the vaccinia virus capping complex. 35 This complex with triphosphatase, guanylyltransferase and (guanine-7-)methyltransferase activity adds a natural cap to the 5′-triphosphate of an RNA molecule. However, an additional enzymatic step may complicate production, particularly at an industrial scale.
Alternatively, a cap exclusively in the correct orientation is obtained with the use of 𠇊nti-reverse” cap analogs (ARCAs). In the most common ARCA, 3′-O-methylation of the base-methylated guanosine only allows addition of a nucleotide at the non-methylated guanosine. ARCA-capped mRNA was translated at more than doubled efficiency in rabbit reticulocyte lysate compared with mRNA capped by regular cap analog. 34 In addition, it has been shown that mRNA transcribed in vitro with ARCA also has a longer half-life in cultured cells. 36 In an independent study, ARCA-capped mRNA has been reported to both increase and prolong protein expression in cultured cells. 37
Protein expression from in vitro transcribed, enzymatically capped mRNA can be further increased by enzymatic 2’-O-methylation of the first transcribed nucleotide, resulting in protein expression comparable to that from mRNA capped with ARCA co-transcriptionally. 38
ARCAs have been further modified within the triphosphate linkage in order to inhibit decapping of the corresponding mRNA and increase binding of eukaryotic initiation factor 4E involved in the recruitment of ribosomes. Modifications either substituted for a bridging oxygen (e.g., (methylenebis)phosphonate and imidodiphosphate) or a non-bridging oxygen (e.g., phosphorothioate, phosphoroselenoate and boranophosphate). 39 Phosphorothioate-modified ARCAs yielded mRNA with both further increased translation efficiency and elongated half-life in cultured cells compared with ARCA. 40 However, phosphorothioate-modified ARCAs are obtained as a mixture of two diastereomers that must be separated after synthesis because of their different biological activity.
When the poly(A) tail was unveiled to enhance translation initiation, it was noted that the efficiency of polysome formation increased with increasing length of the poly(A) tail up to 68 residues. 15 Translation of in vitro transcribed mRNA transfected into cultured cells still increased slightly by lengthening the poly(A) tail from 54 to 98 residues. 41 This study was further extended by investigating the effect of even longer poly(A) tails on protein expression. 38 The peak protein level, reached one day after electroporation of mRNA into cells, was doubled when the poly(A) tail was extended from 64 to 150 residues. Further extension of the poly(A) tail by enzymatic polyadenylation led to an additional moderate increase in peak expression. By contrast, upon transfection of UMR-106 cells, protein levels 16 h post transfection increased with increasing length of the poly(A) tail only up to 60 residues, but declined with further increasing poly(A) tail length. 42 In practical terms, it is noteworthy that maintenance of long poly(d(A/T)) sequences is demanding and strongly dependent on the bacterial strain. 42
Already early on, in vitro transcribed mRNA contained 5‘- and 3‘-UTRs, specifically those of the β globin gene of Xenopus. 11 Both the Xenopus β globin 5′- and 3′-UTRs were demonstrated to impart much greater translational efficiency on heterologous mRNA in the mouse NIH 3T3 fibroblast cell line. 2 A combination of the β globin 5′-UTR, improving translation and the α globin 3′-UTR, known to stabilize mRNA, 27 has been used in the construction of a library from amplified tumor-derived cRNA for use as vaccines against metastatic melanomas. 43 Globin UTRs are still in widespread use in in vitro transcribed mRNA including RNA for immune therapy. 38 , 44 , 45
UTRs from non-globin genes have also been included in in vitro transcribed mRNAs used for investigations of the therapeutic value of mRNA. The 5′-UTR of tobacco etch virus enhances translation of in vitro transcribed mRNA in mammalian cells 46 and has been included in mRNA expressing erythropoietin in different cell types 20 and mice. 47 Furthermore, a structure of the 5′-UTR of human heat shock protein 70 enhanced translation of mRNA in mammalian cells and was predicted to be valuable in the context of genetic vaccination. 48
Inclusion of an internal ribosomal entry site (IRES) in in vitro transcribed mRNA can be an alternative and/or complementary means to achieve expression of therapeutic proteins. For instance, the EMCV IRES was included in mRNAs coding for four transcription factors used to reprogram fibroblasts to pluripotent stem cells. 49 The EMCV IRES has even been used successfully to direct protein expression from mRNA lacking a cap. 50 Vaccination with dendritic cells transfected with such IRES-containing, cap-less mRNA protected mice from metastasis upon intravenous injection of melanoma cells.
Completely novel UTRs may be provided by screening whole transcriptomes for sequence elements that either increase translation or mRNA stability. 51
Codon usage is also considered as a factor affecting the efficiency of translation in many species. However, in humans codon usage bias does not correlate with tRNA levels and gene expression. 52 , 53 In conclusion, codon optimization cannot be expected to (generally) improve mRNA translation in humans, particularly if the ORF is already of human (or even mammalian) origin.
Obviously, the start codon should be part of a Kozak sequence 54 and the sequence surrounding the stop codon may be optimized. 55 In addition, no upstream start codons, preceding the correct start codon, should be present in the mRNA.
In order to obtain effective vaccine platforms, different beneficial mRNA elements have been joined.
Capping with ARCA has been combined with a long transcribed poly(A) tail of 100 residues. Such luciferase-encoding mRNA was tested in immortalized cell lines (JawsII, HepG2, HeLa) as well as immature and mature human dendritic cells. 56 Compared with mRNA capped with regular cap analog and ending with a shorter A64 poly(A) tail, a very substantial improvement in protein expression was seen in all tested cell types. The magnitude of the rise in protein level afforded by either element alone or their combination was strongly dependent on the cell type.
Sahin and coworkers combined two consecutive β globin 3‘-UTRs, a rather long transcribed poly(A) tail of 120 residues 57 and a phosphorothioate modified anti-reverse cap. 58 This resulted in increased and prolonged protein expression in transfected dendritic cells. Upon injection of mRNA into the lymph node, protein expression peaked at 8 h and was demonstrated up to 72 h after mRNA injection.
Using our proprietary mRNA technology modifying coding and noncoding parts of the molecule, we were able to improve both the level and duration of expression, increasing total protein expression by several orders of magnitude. 59 Upon intradermal mRNA injection, strongly prolonged translation gives maximum protein levels 24 to 48 h after mRNA injection and lasts for many more days ( Fig.ਁ ).
Figureਁ. Protein expression in vivo is strongly prolonged using CureVac’s proprietary mRNA technology and lasts for many days. Firefly luciferase-encoding mRNA, optimized for translation and stability, was injected intradermally in a BALB/c mouse (4 injection sites). At various time points after mRNA injection, luciferase expression was visualized in the living animal by optical imaging. (A) Visualization of luciferase expression at selected time points, showing maximal protein levels 24 to 48 h after mRNA injection. (B) Time course of luciferase expression until 9 d after mRNA injection. Background signal was set to 1.
A Deep Dive On mRNA Vaccines
February 11, 2021 | Messenger RNA (mRNA) vaccines were the focus of a symposium on COVID-19 vaccines held during last week&rsquos COVID-19 and Cancer virtual meeting of the American Association for Cancer Research. To date, only two vaccines have received Emergency Use Authorization by the U.S. Food and Drug Administration and both are mRNA vaccines&mdashone developed by Moderna and the other by Pfizer and BioNTech.
Speaking on development of the Moderna vaccine was Randall N. Hyer, M.D., Ph.D., the company&rsquos senior vice president of global medical affairs. Michela Locci, Ph.D., assistant professor of microbiology at the University of Pennsylvania, presented on the ability of mRNA vaccines to elicit potent germinal center (GC) responses associated with neutralizing antibody generation. This was followed by a lively Q&A session moderated by Deepta Bhattacharya, Ph.D., a member of the cancer biology program at the University of Arizona Cancer Center.
Moderna&rsquos COVID-19 vaccine development program utilizes an advanced mRNA technology platform also being used to develop other medicines and vaccines, says Hyer. The approach uses DNA to make mRNA that instructs cells to make a harmless piece of the spike protein found on the surface of the virus, triggering an immune response and the production of antibodies.
The vaccine does not alter DNA, he says. It also does not signal for nuclear access or reverse transcription. It contains no adjuvant at all, says Hyer. Rather, the vaccine appears to trigger the innate immune system.
At the lymph node, B cells (derived from the bone marrow) and T (thymus) cells interact with the spike protein and develop an adaptive immune response, he explains. Once the mRNA and protein it produces have done their job, they degrade after a day or two.
The vaccine has been produced in large quantity and formulated with lipid nanoparticles that are 100 nanometers in diameter, Hyer continues. In animal models, it demonstrated robust COVID-19 neutralizing antibody response and prevented the replication of the virus in the airways.
Adaptive Trial Design
Phase 1, 2, and 3 clinical studies overlapped to accelerate the traditional vaccine timeline, says Hyer. Convalescent serum from 41 individuals diagnosed with COVID-19 was used as a comparator, and participants ranged in age from 20 to 77 years. Antibody levels were detected and &ldquodeclined very little,&rdquo and remained elevated for three months after a second booster shot.
The geometric mean titer and T-cell response &ldquolooked good&rdquo after the first dose, he says. &ldquoBut it took a second dose to get above the levels of convalescent serum, so the booster is important.&rdquo
In trials, the second dose was administered about 28 days after the first but the &ldquoimmunization window&rdquo extended out another 10 days. The maximum acceptable delay in receiving the second dose is unknown, says Hyer. Other multi-dose vaccines typically have a firm minimum interval, he adds, &ldquobut we&rsquore in new territory here.&rdquo
In the phase 3 trial with 30,000 U.S. participants, randomized 1:1 to two shots of the vaccine or placebo, Moderna&rsquos mRNA vaccine showed 94.1% efficacy overall with tight confidence intervals, says Hyer. Participants included individuals at risk of developing severe COVID-19 based on their age and comorbid conditions. Individuals 18-65 without comorbid conditions comprised 17% of the study population, people 18-65 with comorbid condition 17%, and those 65 and up (with and without comorbid conditions) 25%.
Vaccine efficacy remained &ldquovery high&rdquo for different racial groups, but case numbers were too small to calculate conclusively, Hyer adds. Importantly, there was no evidence of vaccine-associated enhanced respiratory disease. Cases of severe COVID-19 numbered 30 in the placebo group compared to zero for those in the vaccine group.
Mortality was lower among individuals in the placebo group than the population at large and, most notably, the elderly. The reason, Hyer says, is that study participants were &ldquomore sensitive to basic public health precautions, and we followed up on them.&rdquo
Systemic reactions to the vaccine&mdashprimarily pain at the injection site and flu-like symptoms&mdashwere higher after the second injection and each was more common among younger participants, reports Hyer. Some of those same ill effects were seen in the placebo group. Unsolicited adverse events were roughly the same in the placebo and vaccine groups.
As recently announced by Moderna, in vitro neutralization studies indicate that the vaccine retains its neutralizing action against emerging variants, including those from South Africa (B.1.351), the U.K. (B.1.1.7), and Brazil (P.1), adds Hyer, but new studies are planned. It provides a similar level of immune protection against these new variants as it does the original Wuhan strain.
The company is also testing two different booster vaccines aimed at the concerning B.1.351 strain, Hyer says. Antibody levels produced by the Moderna vaccine, while above levels expected to be protective, were about six times lower with B.1.351 than prior variants.
If a variant emerges that becomes the dominant strain in circulation, using a mix of vaccines might be considered. &ldquoAll options are on the table,&rdquo says Hyer. Unlike the 1918 pandemic, the second and faster moving wave of infection with SARS-CoV-2 is unlikely to end after a few months, even if COVID-19 and influenza are both mRNA viruses.
Moving forward, Moderna also plans to collect additional data specific to children, cancer patients, and pregnant women, all of whom were excluded from the initial round of clinical trials, he says. For the effects in cancer patients, Moderna is partnering on a study with the National Cancer Institute.
&ldquoWe felt it was important to have as clean a look [as possible] at immunogenicity offered by the vaccine, says Hyer, noting that immunosuppressed people are typically not enrolled in vaccine trials. However, the Moderna trial included 176 people with HIV, among whom no unusual safety concerns were reported.
Memory B Cells
The race to come up with a safe and efficacious COVID-19 vaccine has had many contenders, including 173 candidates in preclinical studies and 63 vaccine candidates in clinical development, says Locci. Among them are RNA and DNA vaccines, recombinant protein vaccines, inactivated vaccines, and live attenuated vaccines.
The spike protein is the target of both the Moderna and Pfizer/BioNTech mRNA vaccines, and the receptor-binding domain (RBD) it contains is an ideal candidate for vaccine development, Locci says. While the immune cellular responses to mRNA vaccination is known to be good, she has endeavored to fill the information gap on the magnitude and quantity of memory B cells they generate.
As detailed in a recently published study in Immunity (DOI: 10.1016/j.immuni.2020.11.009), two SARS-CoV-2 mRNA vaccines&mdashbut not a recombinant protein vaccine formulated with the MF59-like adjuvant AddaVax&mdashpromote robust GC-derived immune responses in mice, she says. These include GC B and T follicular helper (Tfh) cell responses as well as long-lived plasma cells and memory B cells&mdashall of which strongly correlated with neutralizing antibody production.
RBD-specific GC B cells peaked at seven days and fell a week later and induced the potent GC reaction. For at least 60 days post-immunization, immunoglobulin G (IgG) antibodies that are expressed by B cells were also significantly higher. The Tfh cells elevated by the mRNA vaccines also had stronger Th2 polarization, says Locci, which plays a key role in maintaining the delicate balance between antigen responsiveness and tolerance.
Locci speculates that memory B cells drive a secondary GC response with the second shot of the mRNA vaccines since they tend not to re-enter the germinal center upon boost.
The two hot topics discussed during the final Q&A session were transmissibility of the virus post-vaccination and how long to wait between the first and second doses.
To answer the transmission question, researchers will need to look for penetration of IgG nodes in the respiratory airways of larger animal models, says Locci. But she suspects this is the case, given the vaccine&rsquos cellular presence is transient.
Up to 30% of infected individuals are asymptomatic, which would explain the rapid spread of COVID-19, Hyer says. With the SARS-CoV-1 in 2003, it was clear who was infected so they could be isolated. &ldquoWe could shut it down after 840 cases. Now we see 840 cases before lunch, or more.&rdquo
An interesting finding from clinical trials for the Moderna vaccine was that 39 individuals in the placebo group and 15 in the vaccine group who were PCR negative for SARS-CoV-2 at baseline had nasopharyngeal swabs that were positive for the virus at the second dose but had no evidence of COVID-19 symptoms, says Hyer.
The implication is that even one dose of the vaccine could potentially reduce asymptomatic cases, which will need to be confirmed by larger follow-up studies. &ldquoIf we can get a grip on any of the effects vaccination has on transmission of asymptomatic infection, that would be huge,&rdquo says Hyer.
As to the mechanism of early protection seen with the Moderna vaccine at around 14 days after the first dose, Locci points to antibodies as a contributing force. In mice as early as day seven, the vaccine produced robust (if short-lived) plasma cells, she says.
Hyer&rsquos thought is that the mRNA technique has a &ldquomore pronounced effect upstream, turning the body into an in vivo vaccine antigen production [factory].&rdquo Some innate effects may simultaneously be taking place, he adds. &ldquoWe don&rsquot know.&rdquo
Interferon may be an &ldquoimportant player,&rdquo says Locci, indicating that this will be studied shortly.
&ldquoThe mRNA vaccine may just be a potent inducer of innate immunity,&rdquo adds Hyer.
&ldquoThe mRNA component is important, but the real star is lipid nanoparticles,&rdquo Locci says. A soon-to-publish study will show that they are &ldquoalmost as good&rdquo as vaccine themselves at prompting an immune response.
In reply to questions regarding the interval between first and second vaccine doses of the Moderna vaccine, Hyer says it would be &ldquofolly&rdquo to assume knowledge from previous vaccines could be applied to this one. &ldquoIt may very well be the second dose needs to occur within that [10-day] window&rdquo used in clinical trials.
&ldquoWhat&rsquos striking is that two different efforts to develop prophylactic vaccination using a similar strategy but a different vaccine, different manufacturer, different lipids, and different populations had their point efficacy within 1% [of each other],&rdquo Hyer says.
In the absence of clinical data on immunocompromised patients on therapies for cancer, &ldquoit is up to individual doctors&rdquo to decide if the benefit of vaccination outweighs the risks, says Hyer. The only contraindication to the Moderna vaccine is anaphylaxis to the vaccine itself, he adds.
Even if patients&rsquo antibody response is expected to be suboptimal, adds Locci, odds are good that an mRNA vaccine will produce CD4 (helper) and CD8 T (cytotoxic) cells. While both the Moderna and Pfizer vaccines trigger strong CD4 T-cell responses, Pfizer&rsquos vaccine has been shown to elicit a more robust CD8 T-cell response, she says.
An advantage of vaccine delivery via lipid nanoparticles, relative to traditional approaches, may be that mRNA expresses the full-length spike protein and is a &ldquonatural process in the antigen-presenting cell,&rdquo says Hyer. The mRNA platform endeavors to &ldquomimic natural infection processes, so I assume that has something to do with triggering innate response and other aspects of the immune system.&rdquo
Viral vector approaches face the possibility of anti-vector cellular immunity diminishing vaccine effectiveness, Hyer says, in reply to a question about their disadvantages.
Synthetic mRNA: Production, Introduction into Cells, and Physiological Consequences
Recent advances have made it possible to synthesize mRNA in vitro that is relatively stable when introduced into mammalian cells, has a diminished ability to activate the innate immune response against exogenous (virus-like) RNA, and can be efficiently translated into protein. Synthetic methods have also been developed to produce mRNA with unique investigational properties such as photo-cross-linking, fluorescence emission, and attachment of ligands through click chemistry. Synthetic mRNA has been proven effective in numerous applications beneficial for human health such as immunizing patients against cancer and infections diseases, alleviating diseases by restoring deficient proteins, converting somatic cells to pluripotent stem cells to use in regenerative medicine therapies, and engineering the genome by making specific alterations in DNA. This introductory chapter provides background information relevant to the following 20 chapters of this volume that present protocols for these applications of synthetic mRNA.
Keywords: Cap analogs Cationic lipids Electroporation Immunotherapy Innate immunity Nucleoporation Poly(A) Protein expression Translational efficiency mRNA stability.
Vaccines and prion disease
Before I discuss all these claims, I have to answer the question: What are prions? Prion diseases are more properly referred to as transmissible spongiform encephalopathies (TSEs) and represent a rare form of progressive neurodegenerative disorders that can affect both humans and animals. The most famous prion diseases in animals are bovine spongiform encephalopathy (BSE, also known as “Mad Cow Disease”), scrapie, and chronic wasting disease in humans, Creutzfeldt-Jakob Disease (CJD) and kuru. These diseases have a long incubation period and produce in the brain characteristic spongiform changes associated with loss of neurons with no inflammatory response. The causative agents for these diseases are prions. According to the CDC:
The term “prions” refers to abnormal, pathogenic agents that are transmissible and are able to induce abnormal folding of specific normal cellular proteins called prion proteins that are found most abundantly in the brain. The functions of these normal prion proteins are still not completely understood. The abnormal folding of the prion proteins leads to brain damage and the characteristic signs and symptoms of the disease. Prion diseases are usually rapidly progressive and always fatal.
Basically, prions are believed to be misfolded proteins that can transmit their misfolded shape onto normal variants of the same protein. I don’t need to go into the details for you to understand the very basics of prion disease, other than to note that the protein that makes up prions (PrP) is found in many places in the body in healthy people and animals, and its normal function in biology and physiology is poorly understood.
The claim by antivaxxers that vaccines can result in prion disease is one that I’ve seen far less frequently than the claim that they cause Alzheimer’s disease, so much so that a review of SBM found that we’ve only touched on it briefly in John Snyder’s review of Dr. Bob Sears’ book touting his “alternative” vaccine schedule. The origin of the claim appears to derive from the observation that the media used to support the growth of some of the cell lines used to grow virus stock from which vaccines are made contains fetal bovine serum (FBS), a common ingredient in cell culture media. Antivaxxers use that observation to claim a risk of “prion contamination” of vaccines. Never mind that such contamination of a vaccine has never been demonstrated.
Like many antivaccine myths, the myth that vaccines might become contaminated with prions originates from a grain of truth. Decades ago, during the height of the mad cow disease outbreak in the UK, there was indeed concern about using FBS from British herds of cattle to manufacture vaccines. A news article in Nature from 2000 reported on the findings of an inquiry into Britain’s BSE epidemic and even specifically asked the question: “Were some CJD victims infected by vaccines?” The report basically concluded that the answer to that question was unknown, but:
The BSE inquiry’s report (see above) calls for vaccines to be investigated as a possible route of transmission. But it concedes that this will be hampered by the fact that “systematic records of the action taken in response to BSE in respect of individual medical products are lacking”.
Alarmingly, vaccines produced after the point at which the BSE epidemic had been identified — possibly using British bovine material — were still in use as recently as November 1993. According to the inquiry’s report, the chief medical officer of the day, Donald Acheson, decided to phase out the existing stocks because new batches of vaccines take time to grow, and medical experts considered that the benefit of maintaining a continuous national immunization programme outweighed the risk of interrupting it.
An article in MPR from 2010 by pediatrician Dr. Gary S. Marshall, author of The Vaccine Handbook: A Practical Guide for Clinicians (The Purple Book) , is more reassuring. Noting that the FDA convened a meeting in 2000 to discuss the very question of whether vaccines could transmit BSE, Dr. Marshall characterizes the conclusions of the FDA thusly:
Although the risk of transmission of vCJD to humans from such vaccines was considered theoretic and remote, the recommendation was made that vaccines use bovine materials originating from countries without endogenous MCD. Mathematical models suggest that the agent of MCD first entered cattle feed in the United Kingdom around 1980 since the vast majority of initial cases of vCJD were born well before then, childhood vaccines were not likely to be the cause.
Maternal-fetal transmission of prions has never been documented in animals and fetal blood is not known to contain prions. Moreover, the fetal bovine serum used in vaccine manufacture is highly diluted and eventually removed from cells during purification of vaccine viruses. It should be pointed out as well that prions propagate in mammalian brain but not in cell culture.
Final reassurance comes from the fact that transmission of prions occurs from eating the brains of infected animals or from directly inoculating preparations of brains of infected animals into the brains of experimental animals. Transmission of prions has not been documented after inoculation into the muscles or under the skin, which are the routes used for vaccination. Taken together, the chances that currently licensed vaccines contain prions and represent a risk to humans is essentially zero.
So, basically, although the claim that vaccines could transmit prions entered the antivaccine lexicon of disinformation in the 1990s, after billions upon billions of doses of vaccines for which the viral stock was grown in FBS-containing media, there remains no good evidence that vaccines have ever been contaminated with prions or ever caused a single case of prion disease in humans or animals.
Killing Infected Cells
The antigen-presenting cells can also activate another type of immune cell called a killer T cell to seek out and destroy any coronavirus-infected cells that display the spike protein fragments on their surfaces.
An new spin on an old antivaccine trope
The bottom line is that the fear mongering by Drs. Madej and Mercola is nothing new. The lie that vaccines somehow permanently alter your DNA is not a new one the rise of mRNA-based vaccines simply makes it easier for antivaxxers to spin a convincing yarn making such false claims. Indeed, I saw the very same claim, namely that vaccines are transhumanism, eight years ago, when Sayer Ji wrote a hilariously silly piece claiming that vaccines are transhumanism in the service of subverting evolution because they interfere with how we have co-evolved with pathogens. (I certainly hope they interfere with our co-evolution with pathogens! That’s rather the point, I’d say!) A few years later, antivaxxer Sherri Tenpenny was making the same sort of nonsensical argument, but by then antivaxxers had started pointing to DNA vaccines as a hopelessly unnatural corruption of our genes. Throughout it all, antivaxxers were also making ridiculous claims about how trace amounts of contaminating DNA from the cell lines used to grow viral antigens for some vaccines can somehow get into the brain, express “non-self” proteins, and trigger an autoimmune response causing autism. Truly, to antivaxxers, DNA and RNA are magic!
When you come right down to it, this new spin on an old antivaccine trope is nothing more than an appeal to “nature” as being somehow always superior to anything humans can do. Indeed, Dr. Madej’s video is nothing new. Months before, a “natural healing consultant” named Dr. Andrew Kaufmann was featured in a video in which he made very similar claims, including that a future COVID-19 vaccine would provide a vessel to “inject genes” into humans, first by a procedure known as “electroporation”, in which an electric current “create[s] little holes in our cells that allow the DNA to go into our own cells” and then through the insertion of “foreign proteins that supposedly generate immunity”. Kaufman even concluded that such an mRNA vaccine, like the results of biotechnology in agriculture, will turn humans into “genetically modified organisms”.
Of course, as I mentioned before, biology is more complex than the central dogma, which was based on an understanding of molecular biology that is now 60 years old. That’s why I, as I enter the conclusion of this post, I will link to an article by Edward Nirenberg, which gets into the weeds of the exceptions to the central dogma as he refutes the nonsensical idea that mRNA vaccines permanently alter your DNA and concludes, quite reasonably:
There is no feasible means by which an mRNA vaccine could end up in the nucleus of a cell, nor prime a reverse transcription reaction, nor give you a mitochondrial disease.
There is no reasonable possibility based on the totality of our knowledge of cell biology, reverse transcriptases, human genetics, and the immune system that mRNA vaccines can affect your DNA.
We should await the detailed safety data, but, a priori, a segment of RNA encoding the spike protein RBD of SARS-CoV-2 with no replicative potential, and no ability to form whole virus, nor even whole ability to form an ENTIRE spike protein, should be expected to be a safe vaccine that isn’t going to cause these insane pie-in-the-sky science fiction scenarios.
If you are worried about the mRNA vaccines, then don’t get them. The data suggest that there will soon be other kinds of vaccines with good efficacy as well. I, however, am content to roll up my sleeves for one of them.
As I likely will be, once they are available.
On a final note, let me just conclude with a thought about the ridiculous claim that mRNA vaccines permanently alter your DNA or somehow make you “transhuman”. You have to remember that antivaxxers view vaccines as somehow “unnatural” to the point of altering what human beings are. They’ve been making that clear ever since I started paying attention to antivaccine pseudoscience two decades ago, and were doing so long before that. Of course, just because something is natural does not make it good, benign, or even just neutral. Nature is harsh, and the battle for survival brutal, and it’s completely “natural” for all manner of animals to be eaten by bigger, faster, and hungrier animals, and it’s just as natural for humans do die horrible deaths from infectious diseases. Indeed, just look at how horrible the deaths suffered by over 260K of my fellow Americans have been, and COVID-19 is entirely “natural”. Yet the mindset behind so much of “alternative” medicine and antivaccine views is that natural is always good and that anything synthetic should be viewed with extreme suspicion. (Come to think of it, that’s why COVID-19 denialists go to such enormous lengths to falsely portray SARS-CoV-2, the coronavirus that causes the disease, as somehow “unnatural” and bioengineered in a laboratory, with the pandemic being a “plandemic” initiated by global elites to control and subjugate the population.) It’s silly, because even “natural” nutrients and medicines are just as much chemicals as any synthetic nutrient or chemical. We have to judge whether such chemicals are harmful based on science and where the evidence leads us, not based on whether the chemical is “natural” or not. When considering claims about a novel disease such as COVID-19 and vaccines against it, we must also consider the totality of what we know about biology, especially molecular biology, and how a potential vaccine works in assessing the plausibility of alarmist claims about vaccines like those developed by Moderna and Pfizer. Claims that mRNA vaccines like these can “permanently alter your DNA” (or make you “transhuman”) fail miserably on that score.
Think of the claims about mRNA vaccines this way. They are very much of a piece with how quacks glom on to any new finding in molecular biology, particularly the way quacks have hijacked the new science of epigenetics to claim that you can use mind-body interventions to reprogram your own DNA. In quackworld, RNA and DNA are magic, and the disinformation about mRNA vaccines against COVID-19 reflects that belief in magic.