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Analyzing Encapsulated mRNA with LC, MS, and Calorimetry

By Matt Lauber, Joe Fredette, Colette Quinn, and Nick Zampa   Sponsored content brought to you by   The Emergence of mRNA for Vaccines and Therapy mRNA vaccines have emerged as perhaps the most promising means to protecting the world’s population…

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By Matt Lauber, Joe Fredette, Colette Quinn, and Nick Zampa

 

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The Emergence of mRNA for Vaccines and Therapy

lipid nanoparticle
Figure 1. A schematic representation of a lipid nanoparticle encapsulating mRNA.

mRNA vaccines have emerged as perhaps the most promising means to protecting the world’s population from SARS-CoV-2 and future viruses.1 In these vaccines, genetic material coding for a viral protein is encapsulated within a lipid nanoparticle (LNP), which protects it from destructive enzymes and facilitates its delivery into the human body. For the recent Pfizer-BioNTech and Moderna vaccines, the mRNA encodes a harmless piece of the SARS-CoV-2 spike protein (Figure 1). Because the spike protein is unique to the SARS-CoV-2 virus, the human immune system recognizes it as a foreign antigen, thereby resulting in the production of antibodies and T cells against the SARS-CoV-2 virus. This inoculation primes the immune system to better deal with future exposure. Clinical trials have shown that the Pfizer-BioNTech and Moderna mRNA vaccines are highly efficacious at preventing both mild and severe symptoms of COVID-19. Additionally, there have not been any reports of serious safety concerns.2

Beyond their clinical efficacy and safety, mRNA vaccines are advantageous for other reasons. Because mRNA vaccine manufacturing is scalable and cell-free, clinical batches can be manufactured within weeks of discovering an antigen-encoding sequence.3 Furthermore, the development  of mRNA vaccines can be much shorter than that of comparable protein-based vaccines because complex cell culture processes are not required for nonclinical research, clinical research, and development studies.4 mRNA vaccine manufacturing (including formulation) is also highly standardized such that a wide variety of vaccines can be rapidly prepared using shared processes and shared facilities.4 The manufacturing firm need only insert or change the coding region of the mRNA to adapt the platform for a new protein or antigen. This means that mRNA vaccines may prove useful in combating new viral variants and to more effectively control seasonal influenza strains.5

With the emergency authorization of mRNA COVID-19 vaccines, there is more momentum than ever for mRNA to be used as a modality for other therapeutic approaches, like treating rare genetic disorders in both gene replacement and gene editing capacities. In one case, mRNA encoding an enzyme implicated in lysosomal storage disorders is being studied.6 And in another example, mRNA for Cas proteins is being paired with guide RNA to provide in vivo CRISPR gene editing.7 There is a significant wave of progress at hand, and it has been unlocked by advances in lipid nanoparticles as well as pseudouridine and methyl-pseudouridine residues that have helped unlock high levels of intracellular translation.3

As this new form of biotechnology makes a leap of progress, so too do the analytical approaches supporting its development. From an analytical perspective, LNP-encapsulated mRNA molecules need to be characterized and QC tested to ensure safety and efficacy. Modern techniques including liquid chromatography (LC), mass spectrometry (MS), and differential calorimetry (DSC) are therefore being applied to analyze important lipid and mRNA attributes. Here we will touch on three key analytical strategies for ensuring that safe and efficacious encapsulated mRNA drugs are developed and manufactured:

  1. LNP purity and composition by LC
  2. Differential scanning calorimetry for LNP formulation stability
  3. mRNA Analysis by LC and LC-MS methods

 

Lipid Nanoparticles Are the Key to Delivery

In and of themselves, LNPs represent a breakthrough technology. LNPs are the preferred delivery vessel for mRNA therapies and vaccines. For vaccines, they have the added benefit of stimulating the innate immune system without inducing immunogenicity. LNPs are engineered with a specific pKa and size which facilitates optimal protein expression and immunogenicity. These nanoparticles are approximately 50 to 100 nm in diameter and are created through the mixing of the four different types of lipids (Figure 2).8 Each component is combined at a specific stoichiometric ratio, and nanoparticle formation is initiated by the controlled mixing of aqueous mRNA with an alcohol solution of the lipids. These lipids include an ionizable, cationic lipid (such as MC3), a zwitterionic phospholipid (such as DSPC), cholesterol, and a pegylated lipid (such as PEG-DMG).9,10

lipid nanoparticle
Figure 2. Four example lipids used in the formulation of a lipid nanoparticle.

There is an important role for each lipid to play. The ionizable cationic lipid is electrostatically attracted to the negatively charged backbone of the mRNA, and it initiates the formation of an inverted micelle. Meanwhile, the cholesterol and zwitterionic phospholipid serve as bulking agent, and the pegylated lipid stabilizes an energetically favorable surface layer. Progress on lipid chemistry continues, but these lipids have served as the formulation for patisiran, a transthyretin-directed small interfering RNA,11 and for some of Moderna’s earliest clinical studies.12 Novel lipids and new mixtures are in development and can now even be found in the Pfizer-BioNTech COVID-19 vaccine, but the general architecture of the LNP is likely to persist regardless of new developments in lipid technology, such as the use of alternative cationic lipids.8

 

Chromatography Checks on Purity and Lipid Composition

As one might predict, the purity of the lipids used in the LNP matters, and this is an important attribute to check during preclinical, clinical, and product stages of a new vaccine or therapy. Where needed, lab-scale chromatographic purification can be applied to further purify lipid raw materials. Interestingly, there is an opportunity to consider supercritical fluid chromatography for this purpose and to create a green chemistry purification scheme with carbon dioxide as the only chemical waste, not excess volumes of organic solvents.13 Preparative-scale LC with up to 50-mm internal diameter columns and up to 150-mL/min flow rates can also be employed.

With the lipid reagents in hand, purity can be assayed by gas chromatography (GC) and MS. When these techniques are used together, in GC-MS analysis, it is often possible to comprehensively identify and quantitate lipid impurities. LC paired with MS offers potential advantages for impurity identification as well. LC instrumentation can be applied with ESI-MS detection, using method insights first developed for lipidomic studies.14 Impurities corresponding to degradation, oxidation, and synthesis side reactions can be identified according to accurate mass information, and structural information can be deciphered from the application of various types of gas-phase fragmentation and subsequent MS/MS analysis.

ACQUITY PREMIER LC System
Figure 3A. ACQUITY PREMIER LC System

Yet another way that LC is being applied to the analysis of lipids is in the determination of each lipid’s relative abundance in a formulated LNP-encapsulated mRNA. In one important analysis, the lipids are chromatographically separated using a form of reversed-phase chromatography and then detected by evaporative light-scattering detection and potentially even MS. This assay has been used through stages of development as well batch release. Preliminary work suggests that a Waters ACQUITY PREMIER LC system, with its low-adsorption hybrid surfaces,15 might prove useful to establish more robust relative quantitation on account of it facilitating improved recovery of phospholipids (Figure 3).16 Initial work on these separations has also shown that PREMIER columns packed with a charged surface hybrid stationary phase provide advantageous selectivity and peak shape with the low ionic strength formic acid/formate-based mobile phases preferred for evaporative light-scattering detection.17 With these LC approaches being more widely adopted, it is foreseeable that MS detection will also become more routine. It might soon be possible to implement regulatory-compliant MS along with new peak detection for a comprehensive analytical approach akin to peptide-level multi-attribute monitoring on monoclonal antibodies.18

Modern stationary-phase technology
Figure 3B. Modern stationary-phase technology: BEH technology, CSH technology, and high-strength silica (HSS).

 

DSC for LNP Formulation Studies

TA Instruments’ Nano DSC with autosample
Figure 4. TA Instruments’ Nano DSC with autosampler.

DSC is a thermal analysis technique that measures changes as a function of temperature by either heating and/or cooling a sample to induce a transition (Figure 4). For complex lipids, such as LNPs, the transitions of interest include phase changes, confirmational changes in their packing, and melting temperatures. These events can be observed in both the raw material for investigation of contaminants and on the colloidally dispersed state for general characterization. A colloid solution is formed through the self-assembly of LNPs. This is a spontaneous event, and the larger complex is held together by noncovalent interactions where these weak intermolecular forces allow structural changes as the complex moves toward equilibrium.19 The state initially adopted is typically a “metastable state.” Upon reheating the sample, they change their configuration into a more stable form that persists upon additional heat cycles in a DSC. This can be problematic especially when considering the impact of longer-term storage if the metastable state is the preferred configuration. Due to a structure-activity relationship (SAR) of an LNP, a different form could have deleterious effects as it will preferentially change cellular or organ uptake.19

An ideally formulated LNP contains multiple components, each responsible for a different attribute and each impacting the higher-order structure (HOS). One example is the addition of cholesterol added to enhance LNP stability and promote membrane fusion.20 In the DSC, the addition of cholesterol presents itself as a broad endotherm, and it often decreases the enthalpy of the transition without changing the Tmax. Other lipid combinations will impact the transition temperatures, for example, the increase of the Tmax of a complex event versus its individual components is attributed to the formation of strong electrostatic interactions between the positively charged ammonium ion from the cationic lipid and the zwitterionic phospholipid.21 The other element that will change the phase transition temperature is the addition of the mRNA. The structure will be perturbed upon the addition of the highly charged molecule, especially when considering the involvement of so many forces, like electrostatic interactions, hydrophobic interactions, and van der Waals forces. The composition of the LNP, which is related to function and potency, can be correlated to HOS alterations and changes in transition temperatures through the use of a DSC instrument.

 

mRNA Analysis by LC and LC-MS

The messenger RNA that is manufactured for a vaccine or therapy is produced through a cell-free process known as in vitro transcription (IVT) synthesis, which produces millions of copies of the molecule from an initial plasmid DNA template. After mRNA has been IVT-synthesized, it is processed by enzymes that add a guanine nucleotide to its 5’ end (5’ cap), and a chain of several adenine nucleotides to its 3’ end. These post-transcriptional modifications are considered critical quality attributes as they help protect the IVT-synthesized mRNA from degradation and have an impact on translation efficiency as well.22

Waters BioAccord LC-MS System
Figure 5A. Waters BioAccord LC-MS System for compliance-ready LC-UV-MS analysis.

In the development of mRNA-based therapeutics and vaccines, LC and LC-MS workflows are used to ensure proper addition of the 5’ cap and polyA tail, as well as to measure capping efficiency and confirm 5’ cap identity and polyA tail length. For the determination of 5’ capping efficiency and identity, Beverly and co-workers from the Novartis Institutes of Biomedical Research devised a method utilizing specific RNase H probes to cleave off 50 bases from the 5’ end of the mRNA. With a biotin-streptavidin-enriched sample, they were then able to perform LC-MS analysis of the cleaved fragments.23 In another paper, Beverly and colleagues combined an RNase digestion with polydT affinity enrichment to collect polyA tail oligonucleotides from an mRNA, and they then subjected the sample to LC-MS analysis.24 In this more recent work, MS detection was shown to be capable of directly measuring multiple oligonucleotide sequences with single-nucleotide resolution. Consequently, it was demonstrated that the approach was suited to the study of mRNA poly A tail length. Permutations of these mRNA fragment analyses are being considered for both characterization and QC testing. Advances in analytical technology are making it possible to robustly deploy these assays across the world. New mobile-phase systems based on hexylammonium acetate and diisopropylethylamine afford high peak capacity along with unprecedented MS sensitivity.25,26 Also, because it was designed for regulatory-compliant work, the Waters BioAccord LC-MS system makes it possible to carry through the use of one instrument from development and preclinical testing to batch release QC testing (Figure 5). With respect to chromatography, the recent commercialization of PREMIER Oligonucleotide BEH C18 columns has proven to be fortuitous. PREMIER columns, with their hybrid surface column hardware, alleviate the well- known concerns of oligonucleotides adsorbing to metal hardware, which means that right-the-first time results can be achieved without concerns over column-to-column reproducibility and without needing to suffer through a process of conditioning new columns with repeat sample injections.27

ACQUITY_Premier_BEH_C18_VanGuard_Fit_186009455_angle left
Figure 5B. ACQUITY Premier Oligonucleotide BEH C18 Column

In addition to these examples of mRNA fragment analysis, some researchers are exploring an orthogonal method for confirming mRNA identity. Hua and co-workers have explored a so-called oligo-mapping approach that is somewhat analogous to peptide mapping methods for protein-based therapeutics. Here too, the mRNA is digested, and the resulting small oligonucleotide fragments are separated via ion pairing reversed-phase LC to generate an oligonucleotide map that can serve as a fingerprint to verify mRNA ID and sequence.28 Lastly, there is interest in studying the mRNA at its intact level, which is typically 2,000 to 5,000+ nucleotides long. With the pace of advances in MS, it is quite likely that we are on the cusp of such an analytical approach. If not by MS, an analyst might choose to apply size-exclusion chromatography or an ion-exchange separation29 for an intact level analysis of the molecule.

 

Learn more Waters.com/CGT

 

References
1. Alkandari, D.; Herbert, J. A.; Alkhalaf, M. A.; Yates, C.; Panagiotou, S., SARS-CoV-2 vaccines: fast track versus efficacy. The Lancet Microbe 2021.
2. Maragakis, L. L.; Kelen, G. D. Is the COVID-19 Vaccine Safe?
3. Jackson, N. A. C.; Kester, K. E.; Casimiro, D.; Gurunathan, S.; DeRosa, F., The promise of mRNA vaccines: a biotech and industrial perspective. NPJ Vaccines 2020, 5 (1), 11.
4. The Advantages of mRNA Vaccines
5. Einstein, M. For mRNA vaccines, COVID-19 is just the beginning.
6. Zhu, X.; Yin, L.; Theisen, M.; Zhuo, J.; Siddiqui, S.; Levy, B.; Presnyak, V.; Frassetto, A.; Milton, J.; Salerno, T.; Benenato, K. E.; Milano, J.; Lynn, A.; Sabnis, S.; Burke, K.; Besin, G.; Lukacs, C. M.; Guey, L. T.; Finn, P. F.; Martini, P. G. V., Systemic mRNA Therapy for the Treatment of Fabry Disease: Preclinical Studies in Wild-Type Mice, Fabry Mouse Model, and Wild-Type Non-human Primates. Am J Hum Genet 2019, 104 (4), 625-637.
7. Idrus, A. A. Using gene editing to keep cholesterol down and heart attacks at bay.
8. Buschmann, M. D.; Carrasco, M. J.; Alishetty, S.; Paige, M.; Alameh, M. G.; Weissman, D., Nanomaterial Delivery Systems for mRNA Vaccines. Vaccines (Basel) 2021, 9 (1).
9. Hassett, K. J.; Benenato, K. E.; Jacquinet, E.; Lee, A.; Woods, A.; Yuzhakov, O.; Himansu, S.; Deterling, J.; Geilich, B. M.; Ketova, T.; Mihai, C.; Lynn, A.; McFadyen, I.; Moore, M. J.; Senn, J. J.; Stanton, M. G.; Almarsson, O.; Ciaramella, G.; Brito, L. A., Optimization of Lipid Nanoparticles for Intramuscular Administration of mRNA Vaccines. Mol Ther Nucleic Acids 2019, 15, 1-11.
10. Wadhwa, A.; Aljabbari, A.; Lokras, A.; Foged, C.; Thakur, A., Opportunities and Challenges in the Delivery of mRNA-based Vaccines. Pharmaceutics 2020, 12 (2).
11. Adams, D.; Gonzalez-Duarte, A.; O’Riordan, W. D.; Yang, C. C.; Ueda, M.; Kristen, A. V.; Tournev, I.; Schmidt, H. H.; Coelho, T.; Berk, J. L.; Lin, K. P.; Vita, G.; Attarian, S.; Plante-Bordeneuve, V.; Mezei, M. M.; Campistol, J. M.; Buades, J.; Brannagan, T. H., 3rd; Kim, B. J.; Oh, J.; Parman, Y.; Sekijima, Y.; Hawkins, P. N.; Solomon, S. D.; Polydefkis, M.; Dyck, P. J.; Gandhi, P. J.; Goyal, S.; Chen, J.; Strahs, A. L.; Nochur, S. V.; Sweetser, M. T.; Garg, P. P.; Vaishnaw, A. K.; Gollob, J. A.; Suhr, O. B., Patisiran, an RNAi Therapeutic, for Hereditary Transthyretin Amyloidosis. N Engl J Med 2018, 379 (1), 11-21.
12. Bahl, K.; Senn, J. J.; Yuzhakov, O.; Bulychev, A.; Brito, L. A.; Hassett, K. J.; Laska, M. E.; Smith, M.; Almarsson, O.; Thompson, J.; Ribeiro, A. M.; Watson, M.; Zaks, T.; Ciaramella, G., Preclinical and Clinical Demonstration of Immunogenicity by mRNA Vaccines against H10N8 and H7N9 Influenza Viruses. Mol Ther 2017, 25 (6), 1316-1327.
13. Beginner’s Guide to Preparative SFC. Waters 2017.
14. Isaac, G.; McDonald, S.; Astarita, G., Lipid Separation using UPLC with Charged Surface Hybrid Technology. Waters Application Note 720004107 2011.
15. T. H. Walter, M. T., J. Simeone, P. Rainville, A. V. Patel, M. A. Lauber, J. Kellett, M. DeLano, K. Brennan, C. Boissel, R. Birdsall, K. BertheletteWaters Corporation, Milford, MA, USA, Low Adsorption UPLC Systems and Columns Based on MaxPeak High Performance Surfaces: The ACQUITY PREMIER Solution. Waters White Paper 720007128 2021.
16. Isaac, G.; Plumb, R., ACQUITY PREMIER LC Technology Significantly Improves Sensitivity, Peak Shape, and Recovery for Phosphorylated and Carboxylate Lipids Waters Application Note 720007092 2021.
17. Iraneta, P. c.; Kevin D. wyndham; Mccabe, D. R.; walter, T. H., Charged surface Hybrid (csH) technology and its Use in liquid chromatography. Waters White Paper 20003929EN 2011.
18. Ranbaduge, N.; Yu, Y. Q., A Streamlined Compliant Ready Workflow for Peptide-Based Multi-Attribute Method (MAM). Waters Application Note 720007094 2021.
19. Kim, J.; Eygeris, Y.; Gupta, M.; Sahay, G., Self-assembled mRNA vaccines. Adv Drug Deliv Rev 2021, 170, 83-112.
20. Kauffman, K. J.; Dorkin, J. R.; Yang, J. H.; Heartlein, M. W.; DeRosa, F.; Mir, F. F.; Fenton, O. S.; Anderson, D. G., Optimization of Lipid Nanoparticle Formulations for mRNA Deliver in Vivo with fractional factorial and definitive screening designs. Nano Lett. 2015, (15), 7300-7306.
21. Ziller, A.; Nogueira, S. S.; Huhn, E.; Funari, S. S.; Brezesinski, G.; Hartmann, H.; Sahin, U.; Haas, H.; Langguth, P., Incorporation of mRNA in Lamellar Lipid Matrices for Parenteral Administration. Mol Pharm 2018, 15 (2), 642-651.
22. Pardi, N.; Hogan, M. J.; Porter, F. W.; Weissman, D., mRNA vaccines – a new era in vaccinology. Nat Rev Drug Discov 2018, 17 (4), 261-279.
23. Beverly, M.; Dell, A.; Parmar, P.; Houghton, L., Label-free analysis of mRNA capping efficiency using RNase H probes and LC-MS. Analytical and Bioanalytical Chemistry 2016, (408), 5021–5030
24. Beverly, M.; Hagen, C.; Slack, O., Poly A tail length analysis of in vitro transcribed mRNA by LC-MS. Anal Bioanal Chem 2018, 410 (6), 1667-1677.
25. Gong, L.; McCullagh, J. S., Comparing ion-pairing reagents and sample dissolution solvents for ion-pairing reversed-phase liquid chromatography/electrospray ionization mass spectrometry analysis of oligonucleotides. Rapid Commun Mass Spectrom 2014, 28 (4), 339-50.
26. McCarthy, S.; Gilar, M., Hexylammonium Acetate as an Ion-pairing Agent for IP-RP LC Analysis of Oligonucleotides. Waters Application Note 720003361 November 2016.
27. Brennan, K.; Trudeau, M.; Rainville, P. D., Utilization of the ACQUITY PREMIER System and Column for Improved Oligonucleotide Bioanalytical Chromatographic Performance. Waters Application Note 720007119 2011.
28. Jiang, T.; Yu, N.; Kim, J.; Murgo, J. R.; Kissai, M.; Ravichandran, K.; Miracco, E. J.; Presnyak, V.; Hua, S., Oligonucleotide Sequence Mapping of Large Therapeutic mRNAs via Parallel Ribonuclease Digestions and LC-MS/MS. Anal Chem 2019, 91 (13), 8500-8506.
29. Kanavarioti, A., HPLC methods for purity evaluation of man-made single-stranded RNAs. Sci Rep 2019, 9 (1), 1019.

The post Analyzing Encapsulated mRNA with LC, MS, and Calorimetry appeared first on GEN - Genetic Engineering and Biotechnology News.

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Four Years Ago This Week, Freedom Was Torched

Four Years Ago This Week, Freedom Was Torched

Authored by Jeffrey Tucker via The Brownstone Institute,

"Beware the Ides of March,” Shakespeare…

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Four Years Ago This Week, Freedom Was Torched

Authored by Jeffrey Tucker via The Brownstone Institute,

"Beware the Ides of March,” Shakespeare quotes the soothsayer’s warning Julius Caesar about what turned out to be an impending assassination on March 15. The death of American liberty happened around the same time four years ago, when the orders went out from all levels of government to close all indoor and outdoor venues where people gather. 

It was not quite a law and it was never voted on by anyone. Seemingly out of nowhere, people who the public had largely ignored, the public health bureaucrats, all united to tell the executives in charge – mayors, governors, and the president – that the only way to deal with a respiratory virus was to scrap freedom and the Bill of Rights. 

And they did, not only in the US but all over the world. 

The forced closures in the US began on March 6 when the mayor of Austin, Texas, announced the shutdown of the technology and arts festival South by Southwest. Hundreds of thousands of contracts, of attendees and vendors, were instantly scrapped. The mayor said he was acting on the advice of his health experts and they in turn pointed to the CDC, which in turn pointed to the World Health Organization, which in turn pointed to member states and so on. 

There was no record of Covid in Austin, Texas, that day but they were sure they were doing their part to stop the spread. It was the first deployment of the “Zero Covid” strategy that became, for a time, official US policy, just as in China. 

It was never clear precisely who to blame or who would take responsibility, legal or otherwise. 

This Friday evening press conference in Austin was just the beginning. By the next Thursday evening, the lockdown mania reached a full crescendo. Donald Trump went on nationwide television to announce that everything was under control but that he was stopping all travel in and out of US borders, from Europe, the UK, Australia, and New Zealand. American citizens would need to return by Monday or be stuck. 

Americans abroad panicked while spending on tickets home and crowded into international airports with waits up to 8 hours standing shoulder to shoulder. It was the first clear sign: there would be no consistency in the deployment of these edicts. 

There is no historical record of any American president ever issuing global travel restrictions like this without a declaration of war. Until then, and since the age of travel began, every American had taken it for granted that he could buy a ticket and board a plane. That was no longer possible. Very quickly it became even difficult to travel state to state, as most states eventually implemented a two-week quarantine rule. 

The next day, Friday March 13, Broadway closed and New York City began to empty out as any residents who could went to summer homes or out of state. 

On that day, the Trump administration declared the national emergency by invoking the Stafford Act which triggers new powers and resources to the Federal Emergency Management Administration. 

In addition, the Department of Health and Human Services issued a classified document, only to be released to the public months later. The document initiated the lockdowns. It still does not exist on any government website.

The White House Coronavirus Response Task Force, led by the Vice President, will coordinate a whole-of-government approach, including governors, state and local officials, and members of Congress, to develop the best options for the safety, well-being, and health of the American people. HHS is the LFA [Lead Federal Agency] for coordinating the federal response to COVID-19.

Closures were guaranteed:

Recommend significantly limiting public gatherings and cancellation of almost all sporting events, performances, and public and private meetings that cannot be convened by phone. Consider school closures. Issue widespread ‘stay at home’ directives for public and private organizations, with nearly 100% telework for some, although critical public services and infrastructure may need to retain skeleton crews. Law enforcement could shift to focus more on crime prevention, as routine monitoring of storefronts could be important.

In this vision of turnkey totalitarian control of society, the vaccine was pre-approved: “Partner with pharmaceutical industry to produce anti-virals and vaccine.”

The National Security Council was put in charge of policy making. The CDC was just the marketing operation. That’s why it felt like martial law. Without using those words, that’s what was being declared. It even urged information management, with censorship strongly implied.

The timing here is fascinating. This document came out on a Friday. But according to every autobiographical account – from Mike Pence and Scott Gottlieb to Deborah Birx and Jared Kushner – the gathered team did not meet with Trump himself until the weekend of the 14th and 15th, Saturday and Sunday. 

According to their account, this was his first real encounter with the urge that he lock down the whole country. He reluctantly agreed to 15 days to flatten the curve. He announced this on Monday the 16th with the famous line: “All public and private venues where people gather should be closed.”

This makes no sense. The decision had already been made and all enabling documents were already in circulation. 

There are only two possibilities. 

One: the Department of Homeland Security issued this March 13 HHS document without Trump’s knowledge or authority. That seems unlikely. 

Two: Kushner, Birx, Pence, and Gottlieb are lying. They decided on a story and they are sticking to it. 

Trump himself has never explained the timeline or precisely when he decided to greenlight the lockdowns. To this day, he avoids the issue beyond his constant claim that he doesn’t get enough credit for his handling of the pandemic.

With Nixon, the famous question was always what did he know and when did he know it? When it comes to Trump and insofar as concerns Covid lockdowns – unlike the fake allegations of collusion with Russia – we have no investigations. To this day, no one in the corporate media seems even slightly interested in why, how, or when human rights got abolished by bureaucratic edict. 

As part of the lockdowns, the Cybersecurity and Infrastructure Security Agency, which was and is part of the Department of Homeland Security, as set up in 2018, broke the entire American labor force into essential and nonessential.

They also set up and enforced censorship protocols, which is why it seemed like so few objected. In addition, CISA was tasked with overseeing mail-in ballots. 

Only 8 days into the 15, Trump announced that he wanted to open the country by Easter, which was on April 12. His announcement on March 24 was treated as outrageous and irresponsible by the national press but keep in mind: Easter would already take us beyond the initial two-week lockdown. What seemed to be an opening was an extension of closing. 

This announcement by Trump encouraged Birx and Fauci to ask for an additional 30 days of lockdown, which Trump granted. Even on April 23, Trump told Georgia and Florida, which had made noises about reopening, that “It’s too soon.” He publicly fought with the governor of Georgia, who was first to open his state. 

Before the 15 days was over, Congress passed and the president signed the 880-page CARES Act, which authorized the distribution of $2 trillion to states, businesses, and individuals, thus guaranteeing that lockdowns would continue for the duration. 

There was never a stated exit plan beyond Birx’s public statements that she wanted zero cases of Covid in the country. That was never going to happen. It is very likely that the virus had already been circulating in the US and Canada from October 2019. A famous seroprevalence study by Jay Bhattacharya came out in May 2020 discerning that infections and immunity were already widespread in the California county they examined. 

What that implied was two crucial points: there was zero hope for the Zero Covid mission and this pandemic would end as they all did, through endemicity via exposure, not from a vaccine as such. That was certainly not the message that was being broadcast from Washington. The growing sense at the time was that we all had to sit tight and just wait for the inoculation on which pharmaceutical companies were working. 

By summer 2020, you recall what happened. A restless generation of kids fed up with this stay-at-home nonsense seized on the opportunity to protest racial injustice in the killing of George Floyd. Public health officials approved of these gatherings – unlike protests against lockdowns – on grounds that racism was a virus even more serious than Covid. Some of these protests got out of hand and became violent and destructive. 

Meanwhile, substance abuse rage – the liquor and weed stores never closed – and immune systems were being degraded by lack of normal exposure, exactly as the Bakersfield doctors had predicted. Millions of small businesses had closed. The learning losses from school closures were mounting, as it turned out that Zoom school was near worthless. 

It was about this time that Trump seemed to figure out – thanks to the wise council of Dr. Scott Atlas – that he had been played and started urging states to reopen. But it was strange: he seemed to be less in the position of being a president in charge and more of a public pundit, Tweeting out his wishes until his account was banned. He was unable to put the worms back in the can that he had approved opening. 

By that time, and by all accounts, Trump was convinced that the whole effort was a mistake, that he had been trolled into wrecking the country he promised to make great. It was too late. Mail-in ballots had been widely approved, the country was in shambles, the media and public health bureaucrats were ruling the airwaves, and his final months of the campaign failed even to come to grips with the reality on the ground. 

At the time, many people had predicted that once Biden took office and the vaccine was released, Covid would be declared to have been beaten. But that didn’t happen and mainly for one reason: resistance to the vaccine was more intense than anyone had predicted. The Biden administration attempted to impose mandates on the entire US workforce. Thanks to a Supreme Court ruling, that effort was thwarted but not before HR departments around the country had already implemented them. 

As the months rolled on – and four major cities closed all public accommodations to the unvaccinated, who were being demonized for prolonging the pandemic – it became clear that the vaccine could not and would not stop infection or transmission, which means that this shot could not be classified as a public health benefit. Even as a private benefit, the evidence was mixed. Any protection it provided was short-lived and reports of vaccine injury began to mount. Even now, we cannot gain full clarity on the scale of the problem because essential data and documentation remains classified. 

After four years, we find ourselves in a strange position. We still do not know precisely what unfolded in mid-March 2020: who made what decisions, when, and why. There has been no serious attempt at any high level to provide a clear accounting much less assign blame. 

Not even Tucker Carlson, who reportedly played a crucial role in getting Trump to panic over the virus, will tell us the source of his own information or what his source told him. There have been a series of valuable hearings in the House and Senate but they have received little to no press attention, and none have focus on the lockdown orders themselves. 

The prevailing attitude in public life is just to forget the whole thing. And yet we live now in a country very different from the one we inhabited five years ago. Our media is captured. Social media is widely censored in violation of the First Amendment, a problem being taken up by the Supreme Court this month with no certainty of the outcome. The administrative state that seized control has not given up power. Crime has been normalized. Art and music institutions are on the rocks. Public trust in all official institutions is at rock bottom. We don’t even know if we can trust the elections anymore. 

In the early days of lockdown, Henry Kissinger warned that if the mitigation plan does not go well, the world will find itself set “on fire.” He died in 2023. Meanwhile, the world is indeed on fire. The essential struggle in every country on earth today concerns the battle between the authority and power of permanent administration apparatus of the state – the very one that took total control in lockdowns – and the enlightenment ideal of a government that is responsible to the will of the people and the moral demand for freedom and rights. 

How this struggle turns out is the essential story of our times. 

CODA: I’m embedding a copy of PanCAP Adapted, as annotated by Debbie Lerman. You might need to download the whole thing to see the annotations. If you can help with research, please do.

*  *  *

Jeffrey Tucker is the author of the excellent new book 'Life After Lock-Down'

Tyler Durden Mon, 03/11/2024 - 23:40

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Fauci Deputy Warned Him Against Vaccine Mandates: Email

Fauci Deputy Warned Him Against Vaccine Mandates: Email

Authored by Zachary Stieber via The Epoch Times (emphasis ours),

Mandating COVID-19…

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Fauci Deputy Warned Him Against Vaccine Mandates: Email

Authored by Zachary Stieber via The Epoch Times (emphasis ours),

Mandating COVID-19 vaccination was a mistake due to ethical and other concerns, a top government doctor warned Dr. Anthony Fauci after Dr. Fauci promoted mass vaccination.

Coercing or forcing people to take a vaccine can have negative consequences from a biological, sociological, psychological, economical, and ethical standpoint and is not worth the cost even if the vaccine is 100% safe,” Dr. Matthew Memoli, director of the Laboratory of Infectious Diseases clinical studies unit at the U.S. National Institute of Allergy and Infectious Diseases (NIAID), told Dr. Fauci in an email.

“A more prudent approach that considers these issues would be to focus our efforts on those at high risk of severe disease and death, such as the elderly and obese, and do not push vaccination on the young and healthy any further.”

Dr. Anthony Fauci, ex-director of the National Institute of Allergy and Infectious Diseases (NIAID. in Washington on Jan. 8, 2024. (Madalina Vasiliu/The Epoch Times)

Employing that strategy would help prevent loss of public trust and political capital, Dr. Memoli said.

The email was sent on July 30, 2021, after Dr. Fauci, director of the NIAID, claimed that communities would be safer if more people received one of the COVID-19 vaccines and that mass vaccination would lead to the end of the COVID-19 pandemic.

“We’re on a really good track now to really crush this outbreak, and the more people we get vaccinated, the more assuredness that we’re going to have that we’re going to be able to do that,” Dr. Fauci said on CNN the month prior.

Dr. Memoli, who has studied influenza vaccination for years, disagreed, telling Dr. Fauci that research in the field has indicated yearly shots sometimes drive the evolution of influenza.

Vaccinating people who have not been infected with COVID-19, he said, could potentially impact the evolution of the virus that causes COVID-19 in unexpected ways.

“At best what we are doing with mandated mass vaccination does nothing and the variants emerge evading immunity anyway as they would have without the vaccine,” Dr. Memoli wrote. “At worst it drives evolution of the virus in a way that is different from nature and possibly detrimental, prolonging the pandemic or causing more morbidity and mortality than it should.”

The vaccination strategy was flawed because it relied on a single antigen, introducing immunity that only lasted for a certain period of time, Dr. Memoli said. When the immunity weakened, the virus was given an opportunity to evolve.

Some other experts, including virologist Geert Vanden Bossche, have offered similar views. Others in the scientific community, such as U.S. Centers for Disease Control and Prevention scientists, say vaccination prevents virus evolution, though the agency has acknowledged it doesn’t have records supporting its position.

Other Messages

Dr. Memoli sent the email to Dr. Fauci and two other top NIAID officials, Drs. Hugh Auchincloss and Clifford Lane. The message was first reported by the Wall Street Journal, though the publication did not publish the message. The Epoch Times obtained the email and 199 other pages of Dr. Memoli’s emails through a Freedom of Information Act request. There were no indications that Dr. Fauci ever responded to Dr. Memoli.

Later in 2021, the NIAID’s parent agency, the U.S. National Institutes of Health (NIH), and all other federal government agencies began requiring COVID-19 vaccination, under direction from President Joe Biden.

In other messages, Dr. Memoli said the mandates were unethical and that he was hopeful legal cases brought against the mandates would ultimately let people “make their own healthcare decisions.”

“I am certainly doing everything in my power to influence that,” he wrote on Nov. 2, 2021, to an unknown recipient. Dr. Memoli also disclosed that both he and his wife had applied for exemptions from the mandates imposed by the NIH and his wife’s employer. While her request had been granted, his had not as of yet, Dr. Memoli said. It’s not clear if it ever was.

According to Dr. Memoli, officials had not gone over the bioethics of the mandates. He wrote to the NIH’s Department of Bioethics, pointing out that the protection from the vaccines waned over time, that the shots can cause serious health issues such as myocarditis, or heart inflammation, and that vaccinated people were just as likely to spread COVID-19 as unvaccinated people.

He cited multiple studies in his emails, including one that found a resurgence of COVID-19 cases in a California health care system despite a high rate of vaccination and another that showed transmission rates were similar among the vaccinated and unvaccinated.

Dr. Memoli said he was “particularly interested in the bioethics of a mandate when the vaccine doesn’t have the ability to stop spread of the disease, which is the purpose of the mandate.”

The message led to Dr. Memoli speaking during an NIH event in December 2021, several weeks after he went public with his concerns about mandating vaccines.

“Vaccine mandates should be rare and considered only with a strong justification,” Dr. Memoli said in the debate. He suggested that the justification was not there for COVID-19 vaccines, given their fleeting effectiveness.

Julie Ledgerwood, another NIAID official who also spoke at the event, said that the vaccines were highly effective and that the side effects that had been detected were not significant. She did acknowledge that vaccinated people needed boosters after a period of time.

The NIH, and many other government agencies, removed their mandates in 2023 with the end of the COVID-19 public health emergency.

A request for comment from Dr. Fauci was not returned. Dr. Memoli told The Epoch Times in an email he was “happy to answer any questions you have” but that he needed clearance from the NIAID’s media office. That office then refused to give clearance.

Dr. Jay Bhattacharya, a professor of health policy at Stanford University, said that Dr. Memoli showed bravery when he warned Dr. Fauci against mandates.

“Those mandates have done more to demolish public trust in public health than any single action by public health officials in my professional career, including diminishing public trust in all vaccines.” Dr. Bhattacharya, a frequent critic of the U.S. response to COVID-19, told The Epoch Times via email. “It was risky for Dr. Memoli to speak publicly since he works at the NIH, and the culture of the NIH punishes those who cross powerful scientific bureaucrats like Dr. Fauci or his former boss, Dr. Francis Collins.”

Tyler Durden Mon, 03/11/2024 - 17:40

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Trump “Clearly Hasn’t Learned From His COVID-Era Mistakes”, RFK Jr. Says

Trump "Clearly Hasn’t Learned From His COVID-Era Mistakes", RFK Jr. Says

Authored by Jeff Louderback via The Epoch Times (emphasis ours),

President…

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Trump "Clearly Hasn't Learned From His COVID-Era Mistakes", RFK Jr. Says

Authored by Jeff Louderback via The Epoch Times (emphasis ours),

President Joe Biden claimed that COVID vaccines are now helping cancer patients during his State of the Union address on March 7, but it was a response on Truth Social from former President Donald Trump that drew the ire of independent presidential candidate Robert F. Kennedy Jr.

Robert F. Kennedy Jr. holds a voter rally in Grand Rapids, Mich., on Feb. 10, 2024. (Mitch Ranger for The Epoch Times)

During the address, President Biden said: “The pandemic no longer controls our lives. The vaccines that saved us from COVID are now being used to help beat cancer, turning setback into comeback. That’s what America does.”

President Trump wrote: “The Pandemic no longer controls our lives. The VACCINES that saved us from COVID are now being used to help beat cancer—turning setback into comeback. YOU’RE WELCOME JOE. NINE-MONTH APPROVAL TIME VS. 12 YEARS THAT IT WOULD HAVE TAKEN YOU.”

An outspoken critic of President Trump’s COVID response, and the Operation Warp Speed program that escalated the availability of COVID vaccines, Mr. Kennedy said on X, formerly known as Twitter, that “Donald Trump clearly hasn’t learned from his COVID-era mistakes.”

“He fails to recognize how ineffective his warp speed vaccine is as the ninth shot is being recommended to seniors. Even more troubling is the documented harm being caused by the shot to so many innocent children and adults who are suffering myocarditis, pericarditis, and brain inflammation,” Mr. Kennedy remarked.

“This has been confirmed by a CDC-funded study of 99 million people. Instead of bragging about its speedy approval, we should be honestly and transparently debating the abundant evidence that this vaccine may have caused more harm than good.

“I look forward to debating both Trump and Biden on Sept. 16 in San Marcos, Texas.”

Mr. Kennedy announced in April 2023 that he would challenge President Biden for the 2024 Democratic Party presidential nomination before declaring his run as an independent last October, claiming that the Democrat National Committee was “rigging the primary.”

Since the early stages of his campaign, Mr. Kennedy has generated more support than pundits expected from conservatives, moderates, and independents resulting in speculation that he could take votes away from President Trump.

Many Republicans continue to seek a reckoning over the government-imposed pandemic lockdowns and vaccine mandates.

President Trump’s defense of Operation Warp Speed, the program he rolled out in May 2020 to spur the development and distribution of COVID-19 vaccines amid the pandemic, remains a sticking point for some of his supporters.

Vice President Mike Pence (L) and President Donald Trump deliver an update on Operation Warp Speed in the Rose Garden of the White House in Washington on Nov. 13, 2020. (Mandel Ngan/AFP via Getty Images)

Operation Warp Speed featured a partnership between the government, the military, and the private sector, with the government paying for millions of vaccine doses to be produced.

President Trump released a statement in March 2021 saying: “I hope everyone remembers when they’re getting the COVID-19 Vaccine, that if I wasn’t President, you wouldn’t be getting that beautiful ‘shot’ for 5 years, at best, and probably wouldn’t be getting it at all. I hope everyone remembers!”

President Trump said about the COVID-19 vaccine in an interview on Fox News in March 2021: “It works incredibly well. Ninety-five percent, maybe even more than that. I would recommend it, and I would recommend it to a lot of people that don’t want to get it and a lot of those people voted for me, frankly.

“But again, we have our freedoms and we have to live by that and I agree with that also. But it’s a great vaccine, it’s a safe vaccine, and it’s something that works.”

On many occasions, President Trump has said that he is not in favor of vaccine mandates.

An environmental attorney, Mr. Kennedy founded Children’s Health Defense, a nonprofit that aims to end childhood health epidemics by promoting vaccine safeguards, among other initiatives.

Last year, Mr. Kennedy told podcaster Joe Rogan that ivermectin was suppressed by the FDA so that the COVID-19 vaccines could be granted emergency use authorization.

He has criticized Big Pharma, vaccine safety, and government mandates for years.

Since launching his presidential campaign, Mr. Kennedy has made his stances on the COVID-19 vaccines, and vaccines in general, a frequent talking point.

“I would argue that the science is very clear right now that they [vaccines] caused a lot more problems than they averted,” Mr. Kennedy said on Piers Morgan Uncensored last April.

“And if you look at the countries that did not vaccinate, they had the lowest death rates, they had the lowest COVID and infection rates.”

Additional data show a “direct correlation” between excess deaths and high vaccination rates in developed countries, he said.

President Trump and Mr. Kennedy have similar views on topics like protecting the U.S.-Mexico border and ending the Russia-Ukraine war.

COVID-19 is the topic where Mr. Kennedy and President Trump seem to differ the most.

Former President Donald Trump intended to “drain the swamp” when he took office in 2017, but he was “intimidated by bureaucrats” at federal agencies and did not accomplish that objective, Mr. Kennedy said on Feb. 5.

Speaking at a voter rally in Tucson, where he collected signatures to get on the Arizona ballot, the independent presidential candidate said President Trump was “earnest” when he vowed to “drain the swamp,” but it was “business as usual” during his term.

John Bolton, who President Trump appointed as a national security adviser, is “the template for a swamp creature,” Mr. Kennedy said.

Scott Gottlieb, who President Trump named to run the FDA, “was Pfizer’s business partner” and eventually returned to Pfizer, Mr. Kennedy said.

Mr. Kennedy said that President Trump had more lobbyists running federal agencies than any president in U.S. history.

“You can’t reform them when you’ve got the swamp creatures running them, and I’m not going to do that. I’m going to do something different,” Mr. Kennedy said.

During the COVID-19 pandemic, President Trump “did not ask the questions that he should have,” he believes.

President Trump “knew that lockdowns were wrong” and then “agreed to lockdowns,” Mr. Kennedy said.

He also “knew that hydroxychloroquine worked, he said it,” Mr. Kennedy explained, adding that he was eventually “rolled over” by Dr. Anthony Fauci and his advisers.

President Donald Trump greets the crowd before he leaves at the Operation Warp Speed Vaccine Summit in Washington on Dec. 8, 2020. (Tasos Katopodis/Getty Images)

MaryJo Perry, a longtime advocate for vaccine choice and a Trump supporter, thinks votes will be at a premium come Election Day, particularly because the independent and third-party field is becoming more competitive.

Ms. Perry, president of Mississippi Parents for Vaccine Rights, believes advocates for medical freedom could determine who is ultimately president.

She believes that Mr. Kennedy is “pulling votes from Trump” because of the former president’s stance on the vaccines.

“People care about medical freedom. It’s an important issue here in Mississippi, and across the country,” Ms. Perry told The Epoch Times.

“Trump should admit he was wrong about Operation Warp Speed and that COVID vaccines have been dangerous. That would make a difference among people he has offended.”

President Trump won’t lose enough votes to Mr. Kennedy about Operation Warp Speed and COVID vaccines to have a significant impact on the election, Ohio Republican strategist Wes Farno told The Epoch Times.

President Trump won in Ohio by eight percentage points in both 2016 and 2020. The Ohio Republican Party endorsed President Trump for the nomination in 2024.

“The positives of a Trump presidency far outweigh the negatives,” Mr. Farno said. “People are more concerned about their wallet and the economy.

“They are asking themselves if they were better off during President Trump’s term compared to since President Biden took office. The answer to that question is obvious because many Americans are struggling to afford groceries, gas, mortgages, and rent payments.

“America needs President Trump.”

Multiple national polls back Mr. Farno’s view.

As of March 6, the RealClearPolitics average of polls indicates that President Trump has 41.8 percent support in a five-way race that includes President Biden (38.4 percent), Mr. Kennedy (12.7 percent), independent Cornel West (2.6 percent), and Green Party nominee Jill Stein (1.7 percent).

A Pew Research Center study conducted among 10,133 U.S. adults from Feb. 7 to Feb. 11 showed that Democrats and Democrat-leaning independents (42 percent) are more likely than Republicans and GOP-leaning independents (15 percent) to say they have received an updated COVID vaccine.

The poll also reported that just 28 percent of adults say they have received the updated COVID inoculation.

The peer-reviewed multinational study of more than 99 million vaccinated people that Mr. Kennedy referenced in his X post on March 7 was published in the Vaccine journal on Feb. 12.

It aimed to evaluate the risk of 13 adverse events of special interest (AESI) following COVID-19 vaccination. The AESIs spanned three categories—neurological, hematologic (blood), and cardiovascular.

The study reviewed data collected from more than 99 million vaccinated people from eight nations—Argentina, Australia, Canada, Denmark, Finland, France, New Zealand, and Scotland—looking at risks up to 42 days after getting the shots.

Three vaccines—Pfizer and Moderna’s mRNA vaccines as well as AstraZeneca’s viral vector jab—were examined in the study.

Researchers found higher-than-expected cases that they deemed met the threshold to be potential safety signals for multiple AESIs, including for Guillain-Barre syndrome (GBS), cerebral venous sinus thrombosis (CVST), myocarditis, and pericarditis.

A safety signal refers to information that could suggest a potential risk or harm that may be associated with a medical product.

The study identified higher incidences of neurological, cardiovascular, and blood disorder complications than what the researchers expected.

President Trump’s role in Operation Warp Speed, and his continued praise of the COVID vaccine, remains a concern for some voters, including those who still support him.

Krista Cobb is a 40-year-old mother in western Ohio. She voted for President Trump in 2020 and said she would cast her vote for him this November, but she was stunned when she saw his response to President Biden about the COVID-19 vaccine during the State of the Union address.

I love President Trump and support his policies, but at this point, he has to know they [advisers and health officials] lied about the shot,” Ms. Cobb told The Epoch Times.

“If he continues to promote it, especially after all of the hearings they’ve had about it in Congress, the side effects, and cover-ups on Capitol Hill, at what point does he become the same as the people who have lied?” Ms. Cobb added.

“I think he should distance himself from talk about Operation Warp Speed and even admit that he was wrong—that the vaccines have not had the impact he was told they would have. If he did that, people would respect him even more.”

Tyler Durden Mon, 03/11/2024 - 17:00

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