Global Biosurveillance

Further damage to global biosurveillance participation of US agencies and other essential Federal government services is likely to occur if Schedule F, created by an executive order last month, an administrative overhaul of the competitive Civil Service, is allowed to proceed. It makes senior agency positions, policymaking positions, scientific and evidence-gathering personnel and other key professional positions subject to new hiring and termination rules which eliminate Civil Service rules that protect these jobs from political influence, opening them up to performing contrary to facts, or professional or scientific principles. The order instructs agencies to submit a preliminary review of positions to the Office of Personnel Management and petition for reclassification on or before Jan. 19, 2021, one day before the presidential inauguration. https://1105direct.com/portal/wts/ucmcmQego0%7Cbb-yQbEmT%5Ec8vrO2-a.

However, this is not the first time political influence and unwarranted fear of professionals, based on politics, in the Federal Civil Service and Senior Executive Service, have led to damage to their effectiveness to defend this Nation. First, let us see what independence has yielded on behalf of National Defense and Security, within the laws and regulations of these United States, before we started to suspect those very people who lay down and risk their lives, without acknowledgement, for this country.

Interference in the operations of the Brooks Counterproliferation Team (by DHHS, Department of Human Health Services) began on 13 Dec 2002, when DHHS published 42 CFR Parts 73 rules in Federal Register to implement the USA PATRIOT ACT and the Public Health Security and Bioterrorism Preparedness and Response Act of 2002. The USDA published 7 CFR Part 331 and 9 CFR Part 121 in the same issue and required a USDA export/import permit to transfer, even within the USA, not only Select Agents, but all isolated infectious, disease-causing microbes. Even more rules started flowing from Washington on 4 Feb 2003; the Office of the Secretary of Defense issued an Interim Policy for Safeguarding Biological Select Agents and Toxins, and on 24 Feb 2003, our lab and all other Select Agent Labs were notified to file under new CDC rules by 12 Mar 2003, and that they must have prior approval to ship BSATs (Biological Select Agents and Toxins) after that date. I sent two of my team to Ames, Iowa, again, on 8 Jul 2003, to transfer a second USDA anthrax collection (Dr Schuman’s collection from 1969: 32 isolates) found hidden in a freezer when the Ames lab was inventorying their collection for the new Select Agent rules. They wanted them out or they would destroy them. I had to get emergency permission from Dr Ellis at CDC to save the collection. In 1986, Dr Knudson, of USAMRIID, had published a vaccine study using the “Ames strain,” which was unusually hardy, deadly and fast-growing. Researchers shared the strain with as many as 20 labs in the United States, Canada and Britain. His mistake arose from Dr Whitford’s (the State Bacteriologist of Texas State Veterinary Diagnostic Laboratory) sending Dr Knudson a box containing the anthrax with a pre-paid mailing label provided by the National Veterinary Diagnostic Lab at Ames, Iowa, containing their return address rather than Dr Whitford’s. The technician that received the box simply removed the sample and labeled it the “Ames strain” based on that label. Dr Bruce Ivins, who has been attributed as the source of the “Ames strain” used in the Amerithrax attacks, revealed in his papers on his vaccine work that he was completely unaware of the real source of the strain and considered it to be from the USDA Ames Lab. In his 1995 paper in the Journal Vaccine, he stated, “The virulent Ames strain of B. anthracis, obtained from the US Department of Agriculture, Ames, IA, was cultured….” The first transfer of a historical anthrax collection from USDA, Ames, Iowa, had occurred on October 2002. Bringing back anthrax samples from Ames took 16 hours of driving and one overnight in a motel. I never let the box or the car containing the box out of our sight. I even slept with it on my night stand by my bed in the motel. When we got the box back to the lab at Brooks, we carefully unpacked it under biosafety lab conditions and swabbed and cultured the surface of each layer of the box from the outside in to check for anthrax contamination. We found it on the second layer and called the isolate the “hitch hiker” isolate. Fortunately, it was missing one of the two plasmids (small pieces of circular DNA in addition to the main chromosome) that make anthrax dangerous.

In March 2003, Dr Holwitt brought back K strain anthrax from the Bacterial Research Laboratory, Zoonoses Unit, of the Baghdad Veterinary College, which was originally a gift from Al-Kindi Co. for Production of Veterinary Vaccines & Drugs to the Vet College and collected by him as part of his duties with the United Nations Monitoring, Verification and Inspection Commission (UNMOVIC). He had to depart in a hurry. He was the last American on the ground in Baghdad before the Second Gulf War began and when he left the bombs were about to fall. The Brooks CP Team’s mission was nearly ended because of the FBI’s response to Dr Holwitt’s bringing back the last anthrax from an Iraqi lab, by raiding our lab, unannounced of course, on 5 Sept 2003. They provided a handwritten receipt for the anthrax samples they took (duplicates). It was signed by Dr Douglas J. Beecher of the FBI Laboratory, Hazardous Materials Response Unit, 2501 Investigation Parkway, Quantico, Virginia. I was interrogated by FBI agents for three hours in my office. They especially wanted to talk to Dr Holwitt who was not present because he was on leave at the time. On 6 August 2008, the US Attorney declared Bruce Ivins to be the sole perpetrator in the Amerithrax case, and later, on 19 February 2010, the FBI formally closed the case. In the meantime on 15 August 2008, Lieutenant General Darnell, USAF, DCS, Operations, Plans and Requirements, officially closed my CP Team’s BSL-3 at Brooks and declared that the transportation or shipment of USAF Biological Select Agents and Toxins (BSATS) and working with BSATS was to immediately cease until the personnel so engaged were properly enrolled in and monitored by a Biological Personnel Reliability Program (BPRP). Of course, this amounted to a total lock out, in violation of the CDC regulations and Federal Statute requiring the Responsible Official (RO) to have complete access and control over the BSATS. The RO was I. There had never been a BPRP program and one was not yet implemented. To avoid complete default on the Air Force Program and potential abandonment of the BSATS, the Detachment 5 Commander quickly appointed an Alternate RO (ARO) from the ranks of the military to take over. Finally, on 30 March 2009, Lt Gen Darnell sent out a letter resuming the BSAT operations at Brooks. The facility had been down for 8 months and the effect was nearly fatal. New rules were implemented including the two-man rule of entry and working with BSATS in the lab (which I had had in force since 2000), that at least one person had to be BPRP certified, and both had to be on the CDC Select Agent registration as approved for access to Select Agents. This delay and the closing of the lab under the Base Realignment and Closure Act, ostensibly to save money and increase Federal efficiency, destroyed the USAF AFRL mission of global biosurveillance and development of science and technology for that purpose to serve the greater mission of Counterproliferation. It was ended.

“Thanks to you all. Goodbye.” Remember us; for one moment in history, we were the Titans.”— The Black Dragon Trilogy by JOHNATHAN KIEL
https://a.co/bdT2NXF

In a recent article, Robert F. Kennedy, Jr. (an American environmental lawyer and activist, author, and anti-vaccinationist) states–what sounds likes scientific evidence and reasons why we should not take the new mRNA vaccines. For those with limited scientific knowledge, they sound plausible. All vaccines have caveats around them and careful steps that must be taken to assure benefits out weigh risks. Eliminating risk is not absolute, as assuring total safety can never be achieved regardless of the amount of favorable data collected. The argument of absolutes has always been used to deny the benefits of new technology, rather than to manage the risks and weigh the costs vs benefits through scientific evidence.

“The great “democratization” of information (don’t confuse this with “Knowledge” or “Wisdom”) has led to nefarious consequences which resemble an infectious disease with pathological consequences, even affecting the susceptibility and spread of actual biological infectious disease in the world or generating disease without an agent through suggestion and brain “plasticity”. It is the ultimate “divorce” of the definition of a “pathogen” from the biological into the concept, idea, the Pattern, being the “pathogen”. Although pseudoscience may be literally defined as “false” science, it is much more. It grows out of the need to promote and popularize a myth which is accepted based on some authoritarian view or bias, religious, political, or philosophical, and the need to justify that view with “facts” which are carefully selected to support the view and presented in a way to mimic the scientific method.”

“One of the more notorious of these was Peter Duesberg’s campaign against HIV causing AIDS. This example shows that scientists can also be lured into supporting pseudoscience if they do not manage their biases and remain true to the scientific method even when it contradicts their most favorite hypotheses (I did not say “theories”, which are often confused with the term “theoretical”). Scientific hypotheses are just that, they remain to be tested with well-designed experimentation, while scientific theories, like evolution, or the theory of relativity, are supported by many generations of observations and experimentation and predictive science before being generally accepted as a scientific consensus. Another major problem, which is pervasive even in “real” science, is the bias against the publication of negative data in scientific journals. This bias results in amplification of a relatively few positive reports against large numbers of negative ones, or ones in which results, of the positive one, are unable to be repeated. The popular press addresses the positive one as absolute, whereas the negative ones are ignored, or at worst, even disparaged. Some scientists are trying to remove this systemic problem by publishing negative data in journals of their own. These defenders of negative data state that scientists have the responsibility to study Nature, and based on what Einstein has stated, “The right to search for truth implies also a duty; one must not conceal any part of what one has recognized to be true,” to not hide the truth. They assert this includes documenting negative studies in peer-reviewed, acceptable scientific publications. This action is even more imperative because the research projects have most likely been funded by national, public-supported (funded) agencies. These funding agencies have the obligation to publish all results, especially negative results, not just positive ones to maintain the greatest transparency and disclosure of publicly funded research.”— The Black Dragon Trilogy by JOHNATHAN KIEL
https://a.co/dIFEnFy.

Therefore, it is essential that these COVID vaccine trials have full disclosure of results to assure safety, efficacy and an acceptable cost/benefit ratio.

Kennedy’s destructive comments go beyond merely another internet opinion to a display of medical scientific authority and, more so, knowledge he does not possess. “….next Covid-19 vaccination. For the first time in the history of vaccination, the so-called last generation mRNA vaccines intervene directly in the genetic material of the patient and therefore alter the individual genetic material, which represents the genetic manipulation, something that was already forbidden and until then considered criminal.”….”Dear patients, after an unprecedented mRNA vaccine, you will no longer be able to treat the vaccine symptoms in a complementary way. They will have to live with the consequences, because they can no longer be cured simply by removing toxins (SARS-CoV-2 does not make toxins that need clearing from the body—pseudoscience) from the human body, just as a person with a genetic defect (mRNA is not a genetic defect nor a change in the genome which would be necessary for a permanent change) like Down syndrome, Klinefelter syndrome, Turner syndrome, genetic cardiac arrest, hemophilia, cystic fibrosis, Rett syndrome, etc.), because the genetic defect is forever!” https://principia-scientific.com/robert-f-kennedy-jr-covid19-vaccine-should-be-avoided-at-all-cost/?fbclid=IwAR13qqTYiHOTzhRDlaVngMtJHdWl0j-VUVEcQmJlcUvMqWMgSzxGeIFqdCI. This is completely wrong and endangers effective vaccination necessary to bring the pandemic under control. It encourages vaccine hesitancy in the midst of the uncontrollable rise in COVID in the US. We are now facing the onslaught of pseudoscience from people like Robert F. Kennedy Jr. https://apple.news/Act3bYCTrQ0GRWfRYshPprg.

SARS-CoV-2 will not reach herd immunity without vaccination for reasons discussed in an earlier post. A smoldering endemic form will persist with periodic epidemic and even pandemic outbreaks. With this persistence, the potential for escape mutations from any vaccine effort will become more likely statistically as the number of persistently replicating viruses within a population increases. Again, mRNA administration is NOT genetic engineering; mRNA does not change the genome and does not persist forever. The opposite is true. His comparison of genetic errors and chromosomal issues in the human genome to mRNA vaccine demonstrates his ignorance or intentional spreading of malicious misinformation. As I discussed in a previous post, it is difficult to sustain RNA in a cell due to natural frequent turnover. This introduction of mRNA is transient transfection; defined as the introduction of nucleic acid in the cell only for a limited period of time and not integrating the nucleic acid into the genome. Therefore, transiently transfected genetic material is not passed from generation to generation during cell division, and it can be lost by environmental factors or diluted out during cell division. However, the high copy number of the initial transfected genetic material leads to high levels of expressed protein within the period that it exists in the cell. Needed for effective vaccination. The mRNA (with added appropriate sequences) would need reverse transcriptase from a retrovirus to turn it into DNA and insert it into the genome. mRNA vaccination, at a minimum, is no different from SARS-CoV-2 infection, but without the pathogenic parts. If what he said were true, then every person who recovered from COVID would be genetically engineered. This man is a dangerous not-medically- qualified quack, who uses equivocation and nonsequiturs to make GMO=mRNA vaccines=genetic engineering of the genome, Technobabble. The official Pfizer protocol shows that mRNA vaccine is transient: it uses 2 shots (A PHASE 1/2/3, PLACEBO-CONTROLLED, RANDOMIZED, OBSERVER-BLIND, DOSE-FINDING STUDY TO EVALUATE THE SAFETY, TOLERABILITY, IMMUNOGENICITY, AND EFFICACY OF SARS-COV-2 RNA VACCINE CANDIDATES AGAINST COVID-19 IN HEALTHY INDIVIDUALS, PF-07302048 (BNT162 RNA-Based COVID-19 Vaccines) Protocol C4591001).

Even true examples of gene transfer to the genome, like our original DALM Nanobes, described in my earlier posts, are genome modifying technology which has limited capability to transfer that genetic material horizontally to naive cells. The ability is more limited in human cells than bacterial cells, even though the Nanobes that transfected them contained DNA. The subsequent transfected human cells only produce empty DALM nanoparticles or those which contain RNA, which, in turn, can be transferred to progeny cells. New Nanobes made in human cells , which could only make RNA that could be transferred to naive cells, could only mediate transient transfection at most.

No one can deny the great achievements of Pfizer and Moderna in developing vaccines that provide protective immunity to COVID that appear, based on the data they have presented, to be more than 90% and 94% effective, respectively. They both involve the delivery of mRNA which translate into in situ in vivo SARS-CoV-2 spike protein immunogens. Both vaccines because of the inherent instability of RNA need “cold chains” that are more demanding than for current vaccines for other viral diseases. Moderna’s vaccine remains stable in conventional refrigerators (4C) for a month and ordinary freezers (-20C) for six months. The Pfizer–BioNTech vaccine must be stored at -70C. The latter requires an ultra low freezer. This is because RNA is sensitive to environmentally abundant RNAase enzymes and oxidative damage, which break it down or renders it unusable. How can this be, if viruses like coronaviruses, contain RNA which does not suffer this fate as easily? There are even infectious agents that are composed entirely of RNA (viroids) which survive ordinary environmental conditions and the onslaught of host cell ribonucleases. In fact, many viruses produce their own ribonucleases to breakdown host cell messenger RNA (mRNA). Are there lessons to be learned from these agents that would lead to more stable vaccines, even to those that would not need a cold chain at all ? BioNTech/Pfizer and Moderna encapsulated their mRNA vaccines within lipid nanoparticles while the University of Oxford/Astrazeneca and CanSino incorporated antigen-encoding sequences within the DNA carried by adenovirus to yield mRNA to produce the spike protein immunogen. These physical barriers protect the mRNA until it has to be exposed while being translated into protein by host ribosomes, Novavax placed external recombinant S proteins of SARS-CoV-2 onto their proprietary virus like particle nanoparticles, providing direct stimulation to the immune system. All these to provide stability to the RNA and some direct immunogens, and consistency in dose delivery. Nanoparticles can codeliver adjuvants to help prime the desired immune responses, as well. Adjuvants are immunostimulatory molecules administered together with the vaccine to help boost immune responses mainly by activating additional molecular receptors that predominantly recognize pathogens or danger signals, but are inherently, in themselves, nonspecific. While the vaccine stimulates recognition and response of lymphocytes, in preference to innate cells, the activation of the innate immune cells is required to activate the lymphocytes, both B and T-cell responses. Encapsulation and/or conjugation of both the adjuvant and antigen or mRNA within the same nanoparticle enables targeted, synchronous delivery to the same antigen presenting cells (APC). Uptake of the antigen and adjuvant by APCs at separate times, can sometimes lead to autoimmunity against host proteins because the adjuvant can activate APCs that are not primed against the immunogen, but rather against self-antigens. BioNTech/Pfizer, composed their SARS-CoV-2 receptor binding domain (RBD) immunogens onto a T4 fibritin-derived “foldon” trimerization base to resemble the trimeric natural structure of the spike protein. Fibritin is a structural protein of bacteriophage T4, which catalyzes a phage-assembly process. It promotes the assembly of the long tail fibers of the bacteriophage and their attachment to the tail baseplate which attaches to host bacteria; it is also a sensing device that controls the retraction of the long tail fibers in adverse environments and prevents infection of target bacteria under those unfavorable conditions https://pubs.acs.org/doi/pdf/10.1021/acsnano.0c07197.

All this containment and, as mentioned in a previous post, certain sequence changes to discourage adverse innate or off target adverse effects, have only limited effect on stability. Viruses have developed several strategies to protect the stability of their RNA including inherent RNA shields, appropriating host RNA stability factors, incapacitating host RNA degradation and changing the general host RNA stability in cells using virally encoded nucleases. Viral RNA with poly(A) tails of the transcript, form an inhibitory structure for ribonucleases. Another common RNA structure that provides protection from a class of RNA degradation enzymes is the stable terminal stem loop found at the 3’ end of all viruses that lack a poly(A) tail. These structures prevent exosome-associated exoribonuclease from attacking viral RNAs, thereby keeping the 3’ end intact and blocking the 3’to-5’ degradation of the transcript.

Some viruses encode proteins that specifically protect viral RNAs from degradation during infection. An example of this is the nucleocapsid protein of negative sense RNA viruses such as vesicular stomatitis virus which assembles on the viral genome and antigenomic RNAs cotranscriptionally and renders the RNA resistant to RNases. Another example is the KSHV ORF57 protein, which binds to a specific viral element and protects intronless transcripts from degradation in the nucleus. In summary, these inherent defense mechanisms seem to be used by numerous viruses as a general way of protecting non-coding RNAs or RNAs, that are not translated, from degradation by the cellular RNA degradation machinery. This assists in the accumulation of viral templates for replication, transcription and packaging. Viral mRNAs, however, do not seem to employ this strategy for stabilization because it might be largely incompatible with efficient translation by cellular ribosomes. Instead, using host cell RNA stability factors is preferable for the latter. One attractive strategy for viruses to confound host cell RNAase enzymes is for their mRNAs to use cellular factors whose natural function is to stabilize cellular transcripts. The two most widely studied cellular RNA stability factors are HuR and poly(C)-binding protein 2 (PCBP2). The ubiquitously expressed HuR protein has been demonstrated to bind and stabilize well over 50 independent mRNAs by interacting with uridine-rich or adenine-uridine-rich elements. Alphaviruses such as Sindbis virus have been shown to contain high-affinity U-rich HuR protein-binding sites in their 3’ untranslated regions (UTRs) and to use HuR protein to stabilize viral mRNAs and promote a productive infection. Segmented RNA viruses remove the caps along with a small portion of the 5’ UTR from cellular transcripts and incorporate them into their own transcripts to facilitate the stability and translation of viral mRNAs. RNA viruses also encode their own ribonucleases or factors that can stimulate RNA degradation. Cap-stealing endonucleases encoded by many segmented RNA viruses, for example, selectively target cellular mRNAs for decay by 5’-to-3’ exonu- cleases and this dramatically changes the regulation of mRNA stability in infected cells. Segmented RNA viruses use the capped oligomers generated by endonuclease cleavage to initiate transcription of their own mRNAs. The nucleocapsid protein of Lassa fever virus, a member of the Arenaviridae, has recently been shown to possess 3’-to-5’ exonuclease activity. This exonuclease is essential for the virus to interfere with aspects of the interferon and other innate immune responses. The only other RNA virus known to date to encode an exonuclease is the Nsp14 protein of the SARS coronaviruses. This protein is similar to that of the Lassa fever virus nucleocapsid protein, thereby, also probably attacking RNAs from the 3’ end using a catalytic mechanism with two metal ions. Small RNA from viral genomic transcripts can also aid in protecting viral RNA. All insect-borne flaviviruses use the cellular Xrn1 5’-to-3’ exonuclease to generate a small flavivirus RNA (sfRNA) from the 3’ UTR of the virus. This small RNA is generated because of a set of pseudoknot structures capable of stopping the exonuclease. The small sfRNA generation is important for viral replication and cytopathology in West Nile Virus infection. Furthermore, instead of avoiding the RNAi (interference RNA) machinery, HCV, another member of the Flaviviridae, uses the abundant liver miRNA-122 to increase the accumulation of its mRNA during infection. miRNA-122 binds to two sites in the 5’ UTR of HCV and forms a unique structure required for efficient viral replication. Finally, viruses use nucleases to increase the rate of viral RNA evolution. Mutations in the Nsp14 exonuclease of independent coronaviruses result in a 15–21-fold decrease in replication fidelity. Therefore, the presence of this exonuclease can clearly influence viral RNA mutation rates and the generation of quasispecies https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3123725/pdf/main.pdf.

Viroids are small circular RNAs that mostly infect plants, mimic DNA in secondary structure in order to use host DNA-dependent RNA polymerase to replicate and do so by a rolling circle method that does not require the ring of RNA to open. The linear replicates are then ligated into new circles by a host ligase. This process avoids open ends that would be attacked by cell exonucleases and the necessity for start/stop sequences. They encode a hammerhead ribozyme which can cut and splice RNAs. The replication cycles of viroids include, for members of Pospiviroidae, replication in an asymmetric cycle. DNA-dependent RNA polymerase (polII) transcribes mature circular RNA into oligomeric (−) intermediates and these into oligomeric (+) intermediates. The (+) intermediates are enzymatically cleaved into monomers and ligated with DNA ligase 1 into mature circles. Members of Avsunviroidae replicate in a symmetric cycle where circular RNA is transcribed into oligomeric intermediates by nuclear-encoded polymerase (NEP). The intermediates are cleaved into monomeric units by the internal hammerhead ribozyme and ligated by host tRNA (transfer RNA) ligase into circles. The presence of thermodynamically stable as well as metastable structures of RNA are essential through the entire process so RNA structure determines fitness for replication. Virusoids are essentially viroids that have been encapsulated by a helper virus coat protein (providing a viral physical barrier to protect stabilize its RNA). They are thus similar to viroids in their means of replication (rolling circle replication) and due to the lack of expressible genes (into protein) but they differ in that viroids do not possess a protein coat https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6237808/.

Artificial stability can be built into synthetic RNAs. This has been necessary for diagnostic and especially therapeutic use of aptamers. These are small synthetic nucleic acids selected in vitro from a large library of sequences for their ability to bind to proteins and other targets specifically and strongly and then to be amplified by PCR. Derivatives or replacements of the phosphate and sugar backbone while maintaining base integrity allows them in many cases to still replicate. Some of these modifications are phosphorothioate (PS) bond substituting a sulfur atom for a non-bridging oxygen in the phosphate backbone of an oligonucleotide; and 2′-O-Methyl (2’OMe), a naturally occurring post-transcriptional modification of RNA, 2’OMe found in tRNA and other small RNAs. Oligonucleotides can be directly synthesized to contain 2’OMe. This modification increases the Tm (melting temperature) of RNA:RNA duplexes, but results in only small changes in RNA:DNA stability. It prevents attack by single-stranded endonucleases, but not exonuclease digestion. 2′-fluoro bases have a fluorine-modified ribose which increases binding affinity (T_m) and also confers some relative nuclease resistance compared to native RNA. Inverted dT can be incorporated at the 3′ end of an oligonucleotide, leading to a 3′-3′ linkage that will inhibit degradation by 3′ exonucleases and extension by DNA polymerases. In addition, placing an inverted, 2′,3′ dideoxy-dT base (5′ Inverted ddT) at the 5′ end of an oligonucleotide prevents spurious ligations and may protect against some types of enzymatic degradation. Phosphorylation of the 3′ end of oligonucleotides will inhibit degradation by some 3′-exonucleases. Phosphoramidite C3 Spacer can be incorporated internally, or at either end of an oligo to introduce a long hydrophilic spacer arm for the attachment of fluorophores or other pendent groups. The C3 spacer also can be used to inhibit degradation by 3′ exonucleases. https://www.idtdna.com/pages/education/decoded/article/modification-highlight-modifications-that-block-nuclease-degradation. Other modifications include the replacement of the phosposaccharide backbone with a polypeptide. For aptamers and Nanobes some of these modifications and others have been employed:

In Nanobes, we chose double-stranded DNA aptamers to provide the most stable unmodified synthetic nucleic acids. However, these are still vulnerable to nucleases of certain types. Also, DALM, in Nanobes, chelates metals that are necessary to certain nucleases, inhibiting them, but not all non-metal nucleases. The final modification in Nanobes that has yet to be tried is replacement of circular RNA or DNA with micro linear nucleic acid capped at the ends with carbon nanotubes and/or nanoparticles:

Nanobes in general do not require lipids to enter cells and can have aptamers on their surfaces which direct them to certain cells to maximize therapeutics or vaccines to optimal effects.

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The Black Dragon Trilogy collection tells the story behind this blog and the principles upon which it is based as truthfully as I can. It is for those who wish to explore the subjects of this blog more deeply and thoughtfully. It has become most relevant in these turbulent times, this time of COVID.

How it began:

.

The simplest mathematical description of Ro: Infectious contacts β per unit time, all assumed infected, and disease with infectious (latent incubation) period of 1/γ, (frequency), then the basic reproduction number is R0 = β/γ. Diseases have multiple latency periods; therefore, the reproduction number for the disease is the sum of reproduction numbers for each transition time (Rt). The effective reproduction number, Re, changes with time, affected populations, and circumstances. The reproduction number, as widely used and referred to, appropriately and inappropriately, hides that transmission is stochastic not deterministic, often dominated by a small number of individuals, and heavily influenced by superspreading events https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3000897. The complexities of tracking, and therefore, mitigating infectious diseases, when they get out of hand, like COVID, limit the usefulness and predictability of mathematical models. Coronaviruses, as a group and SARS-CoV-2 in particular, confound Ro values; they can be easily blocked by barriers (masks, etc), distance, quarantine, and other environmental factors but are highly efficient in infecting hosts exposed to an infectious dose; this keeps overall % of infection low, but sustains virus in the population indefinitely. All this calculating gives one the sense of potentially great precision and accuracy in detecting an infectious disease in the environment, in reservoir or primary hosts, whether the infection is apparent or not, if one only has a test that is sensitive enough, has a low false positive rate, and one collects enough samples. In practice, many infectious agents, including SARS-CoV-2, are not so homogeneously distributed or even distributed normally. They favor certain niches and circumstances. Most of what one collects are samples of “empty space, volume” in respect to the agent, where the probability of detecting it in spite of the size of the sampled volume is zero. This condition is what defeats the models and mathematics. The math and models can be beautiful and sophisticated and the calculations perfect but “garbage in, garbage out” or “empty space in, empty space out!”Testing must give the idea of prevalence of active cases, recovered resistant individuals, and their respective density distribution in a population to have any idea of the probability of effective or failed transmission or growth rate of a local epidemic or global pandemic. The Ro (Re, more realtime) value is not very valuable for predicting absolute numbers of a population which will be infected but better for predicting how to stop an epidemic or pandemic, and it is not trivial to accurately calculate https://royalsociety.org/-/media/policy/projects/set-c/set-covid-19-R-estimates.pdf. So in the simplest terms what does the Ro value tells us, especially since it continuously changes into different Rt and Re subsets? Does it tell us, without resorting to history, what conditions have to be met over time? When a disease will move from endemic to epidemic? Ideally, the answer is yes to the second question, and how it is done, the answer to the first question, theoretically, has been made quite simple and clear, as math is supposed to do after, what seems to those of us who are not mathematicians, very much a confusing and esoteric series of symbolic manipulations and calculations. To put the answer most succinctly, R0, R naught, the basic reproduction number, the average number of other people each infected person must infect in a population, who are susceptible to the disease, in order to maintain the disease endemically (without any outside introduction or change which influences the number of infected to grow) or for it to go epidemic (exponentially in number of infected people or animals), or eventually pandemic (super exponential because of the interaction in space and time of many populations). For the endemic case:

For an infectious disease to die out after introduction:

For an infectious disease to become epidemic:

For it to become pandemic:

Where n is the number of separate susceptible populations which may be connected by the infectious agent being transported from one susceptible population to another through space and p is the exponential multiplication of the original source contacts which will become sources for spread to new populations. For an infectious disease to remain in the endemic state, the basic reproduction number and the proportion of the population susceptible must be inversely related, otherwise it will disappear or become epidemic and, perhaps, pandemic. This all assumes the susceptible hosts subject to exposure are exposed under the same circumstances with the highest probability of infection. Here is where the simple math gets complicated and I will not pursue it here but will just say it brings up the problem of incident versus absorbed dose (seen with toxins and radiation effects) which is somewhat analogous in infectious disease to the infectious dose, or multiplicity of infection (MOI).

Viruses cannot be seen (usually except for a few very large ones at the limits of light microscopy) with a visible light microscope and will not grow independently in cell-free medium. Their effect on a lawn of target cells in which they have replicated is seen as the formation of plaques (clear “holes”) in an otherwise solid opaque or translucent lawn of animal or human cells or, in the case of bacteriophages, a bacterial parasitizing virus, in a lawn of bacteria on agar. Some animal and human viruses like retroviruses do not kill the cells they infect but transform them, so they do not stop growing when they form a monolayer in culture but continue to “pile up” into transforming foci representing a “viral colony”. Other effects, cytopathic effects (CPE), are even more subtle and take more training to observe and count as “colonies”. Even though cells may be shedding virus without dying or showing observable cytopathic effect, they cannot be observed or counted. The dilution to zero plaque or CPE is an inverse measure of the number of virions found in the original volume of inoculums and should be extrapolatable to zero virus particles, and thus, theoretically, one viral particle can be detected without ever seeing it. Like the bacteria this is a fiction and only theoretical. Otherwise, if one million virions are added to one million cells, the MOI should be one. If ten million virions are added, the MOI should be 10. If you add 100,000 virions, then the MOI is 0.1. However, this doesn’t happen because every target cell does not actually come in contact with a single virion. If we use k to represent the number of viral particles per cell, then we let P( k) equal the fraction of cells infected by that number of viral particles and m be the MOI. Then the fraction of target cells which remain uninfected (0 viral particles) is equal to the following:

Where e is the natural log base, approximately equal to 2.71828182846. For MOI of 1 or more, the calculation is:

In many cultures of viruses, in order to infect most of the cells a MOI of 10 is used because for a million of target cells, less than a hundred should remain uninfected, a trivial number. In fact, in a culture of one million cells, 999,500 cells receive more than one virion:

In other words, it takes a certain number of microbes to cause an infection, or an obvious apparent infection such as illness or death. For instance, the infectious agent of Q Fever, the most infectious agent in the world, requires a single organism to cause disease, and for comparison, anthrax requires about 10,000 spores to kill a human. However, they are not uniformly applied to susceptible populations and many other factors control and confound their outbreaks as detectable disease. Although it is difficult to test this simple model in an uncontrolled population like humans or animals in the wild, it can and has been demonstrated and predictive in at least one case that supports the validity of the model: the eradication of smallpox in 1977. This global experiment was based on the assumption that If the proportion of the population that is immune exceeds the “herd immunity” for the infectious disease, then the disease can no longer be sustained in that population. In the case of smallpox, this level was exceeded by vaccination and the disease was consequently eliminated. It is hoped that this can be done with other diseases by vaccination such as with polio or by isolation and depopulation in the case of animals as with brucellosis in cattle in the United States or as rabies was in the United Kingdom. The reverse has been demonstrated by refusal to vaccinate children or by inadequate immunization because of changes in the vaccine or virulence of the pathogen, as in the cases of measles and whooping cough, respectively. How would we write this in mathematical terms to determine the number of individuals which should be vaccinated to protect the population? We would use the following formula:

Where q equals the herd immunity, the portion of a population (or herd), which provides protection for individuals who have not developed immunity. As you can derive from the simple algebraic formula, the larger the reproduction number of the infectious disease the more the population has to be covered by vaccination.

A mathematical approach to increase the precision, accuracy and predictability of epidemiology, is the application of the “ROC curve” to determine if a disease can be “detected” in a population by infection and subsequent testing for that infection by some assay or observation of signs and symptoms. The “ROC curve”, Receiver Operating Characteristic was first used during World War II for the interpretation of radar signals before it was used in other types of sensors, including “sentinel” animals or humans who would indicate the presence of infectious diseases. Following the attack on Pearl Harbor in 1941, the United States Army began new research to increase the prediction of correctly detected Japanese aircraft from their radar signals, so was born the “ROC curve”. DARPA decided that the ROC curve would be the “gold standard” of detectability for biological agents. In the most general terms, the ROC curve is the fraction of true positives out of the total actual positives vs. the fraction of false positives out of the total actual negatives at various threshold settings. The ROC curve which applies to the ability of a sample to detect a positive for the presence of an infectious agent is an empirical plot of the number of infectious particles (bacteria or viruses) necessary to infect, or be “detected”, by the human or animal acting as a detector (actually measured by a clinical assay for the specific presence of the agent or the appearance of signs or symptoms which meets the case definition of the particular infectious disease) versus the false detection of such a particle. As the sensitivity of the detector is increased (equal to the number of true positives), the number of false positives increases sometimes linearly sometimes asymptotically or non-linearly depending on the characteristics of the detector. One has to decide the threshold of sensitivity vs acceptable false positives. The ROC curve is used to do this. The idea is to determine what you would expect for a given set of characteristics of the infection to be able to detect an infection. This approach tells the minimum number of samples from hosts, or the number of infected individuals in a given population that must be collected in the former and examined in the latter to detect an infection in a population, especially if it is not apparent or is endemic. In a sense, testing should tell the prevalence of the infection in the population in order to predict its Ro and growth rate of infection in that population, at least in theory. Bottom line is Ro is always being re-calculated from these data and testing results in addition to following numbers of cases over time (in realtime). It is more a result, an effect, an assessment, of where we stand in the fight against COVID and if we are approaching its end.

A new Spotted fever was seen in 2018 and 2019 in the United States in 3 dogs which had fever and hematological abnormalities; blood samples contained Rickettsia reactive antibody and Rickettsia DNA sequences by PCR amplification. These dogs came from 3 separate states: Tennessee, Illinois (signs developed 3 days after returning from a tick-infested area in Arkansas), and Oklahoma. The tickborne Rickettsia parkeri, R. philipii (Rickettsia 364D), and R. rickettsii cause Rocky Mountain spotted fever (RMSF), human spotted fever group (SFG) rickettsioses (mortality as high as 30% without treatment, but historically, a death rate as high as 80%; however, with doxycycline treatment, that rate drops to 0.5 %). R. rickettsii is the only known cause of SFG rickettsiosis in dogs. SFG Rickettsia seroprevalence is high in dogs in the United States and Mexico. In this study, they examined 3 genes (gltA, htrA, and ompA) and 2 intergenic spacer regions (23S-5S and mmpA-purC) in Rickettsia sp. by PCR. These targetted genes of Rickettsia were 100% identical in the 3 dogs. Multilocus genetic analysis placed this new Rickettsia sp. in a clade among SFG Rickettsia between human pathogens R. heilongjiangensis and R. massiliae. They failed to culture and isolate the new Rickettsia sp. from whole blood. The tick species, in these cases, were not identified, but ticks common to the states involved include Amblyomma americanum, Dermacentor variabilis, and Rhipicephalus sanguineus sensu lato, all of which are known to transmit Rickettsia. Haemophysalis longicornis, an invasive tick species recently found in the United States, including in Tennessee and Arkansas, may also be a possible vector of Rickettsia spp. Wilson JM, Breitschwerdt EB, Juhasz NB, Marr HS, de Brito Galvão JF, Pratt CL, et al. Novel Rickettsia species infecting dogs, United States. Emerg Infect Dis. 2020 Dec. https://doi.org/10.3201/eid2612.200272. The appearance of a new tick-borne spotted fever in dogs should not be unexpected; we have seen this before.

Flinders Island Spotted Fever, caused by Rickettsia honei, first discovered on Flinders Island Australia, is transmitted by Aponomma hydrosauri (a reptile-associated tick, now Bothriocroton hydrosauri). It was first identified on Flinders Island in 1991 by Dr Robert Stewart, who suspected the clinical symptoms of his human patients indicated a rickettsial infection. Symptoms included fever, headache, myalgia, a mild cough, and a maculopapular rash. Serological analysis, including the Weil-Felix agglutination and rickettsial-specific immunofluorescence tests, indicated a member of the Spotted Fever Group (SFG) was responsible for the disease. The reptilian vertebrate hosts for the tick on Flinders Island are tiger snakes (Notechis scutatus), copperhead snakes (Austrelaps superbus), and blue tongue lizards (Tiliqua nigrolutea). In 1998, a similar, if not identical, rickettsia was found in ticks in South Texas. In 2008, our entomologists at Brooks, also found the rickettsia in Amblyomma cajennense ticks co-infected with Coxiella burnetii (Q Fever). R. honei has been found in Ixodes and Rhipicephalus ticks in Asia and in Amblyomma cajennense, as we observed, in North America. Its worldwide spread is hard to explain.

A Spotted fever type Rickettsia from Ghana, West Africa, entered the US in ticks and imported snakes through Florida in 2002. The disease was associated with the Gulf Coast Tick, Amblyomma maculatum, introduced into a snake collection after importation, and the imported Snake Tick Aponomma latum, on the premises. The latter is the most common tick parasite of large snakes in most of sub-Saharan Africa and in the pet trade worldwide. It frequently arrives in the USA on imported pet pythons especially from West African countries such as Ghana. After many snakes died, the outbreak was finally stopped by tick control on the snakes and premises and tetracycline treatment of snakes in prodromal or asymptomatic phases of the disease (otherwise it was 100% lethal after neurological signs appeared even with treatment). Its responses to tetracycline and our inability to consistently culture any free-living microbes from the blood and tissues of acute cases supported it being a Rickettsia or Ehrlichia. Various Rickettsia, Ehrlichia ruminantium (cause of Heartwater), and Orientia tsutsugamushi (cause of Scrub Typhus) target vascular endothelium (Heartwater especially targets brain endothelium) as their main tissue to be infected as did Viper Plague. It was isolated, by blind passage for 4 years, from the livers of snakes, and cultured and isolated in snake and turtle cell lines, and then, the isolates were used to infect various other mammalian cell lines, including mouse, human, and bovine. Because of clinical signs of accumulated peritoneal straw-colored fluid, pneumonitis, gastroenteritis, neurological signs, seizures and sudden death as well as a positive PCR for the genetic marker, it was assumed to be an aberrant Heartwater. The putative causative agent was isolated in viper cells and propagated in turtle cells, but also infected bovine endothelial cells, and human cells (HeLa) as well.

The Heartwater agent is of such concern that it is forbidden, even for research purposes, to be maintained on the US mainland. Snakes belonging to Viperid, Colubrid and Elapid families succumbed to this new disease from July 2002-February 2003 in a private collection into which the snakes imported from Africa through Florida were introduced along with the ticks they carried. However, in spite of being intermingled in the collection, no western diamond-backed rattlesnakes (Crotalus atrox) showed signs of infection or died. This agent(s) may have already appeared in the US as a zoonosis combined with or diagnosed as Q Fever (MMWR, September 1, 1978, V o l. 27, No. 3, 5 May 25, 1978, New York State, the Suffolk County Department of Health Services). During this 1978 outbreak, a total of 11 persons became symptomatic. All were involved in unpacking and de-ticking (Amblyomma nuttalli, Aponomma latum, and Aponomma flavomaculatum) or came in contact with a shipment of 500 ball pythons (Python regius), imported on May 3 from Accra, Ghana. The vipers, which were involved with the 2002 outbreak, were shipped to Florida from Ghana along with ball pythons (some may have been co-mingled in transport bags) from the same vendor(s). Characteristic lesions of abundant straw-colored fluid in the common body cavity, inflammation with abundant mucus in the lungs and intestines, and accompanying diarrhea as well as per acute deaths in many cases with little or no signs beforehand were observed. This unknown Rickettsia tested positive with antibody against the OX19 Proteus vulgaris antigen, the same antigen used in the Weil–Felix Test. This test uses antibody against the OX19 antigen of the Gram-negative, free-living bacterium Proteus vulgaris which cross reacts with spotted fever and typhus groups of Rickettsia. It was the first serological test for the diagnosis of these rickettsial diseases (Edmund Weil and Arthur Felix in 1916). As I have already stated, emerging spotted fever group Rickettsia has already been seen as a zoonosis from reptiles, so except for the unusual genetics, this result should not have been a surprise. What was a surprise was that a Rickettsia could have appropriated genetic material from an Ehrlichia, probably co-infecting the same ticks. Species lines in these types of microbes have become very blurred. We were just very lucky that this worked because overall sensitivity can be as low as 33% and specificity as low as 46%.

DARPA is planning for an airborne COVID detector — Defense Systems https://defensesystems.com/articles/2020/11/11/darpa-sensars-covid-detection.aspx?s=ds_121120&oly_enc_id=&m=1. Once again DARPA has chosen to ignore or is totally unaware of what they already had in-house in the DoD. Trying to get antibody or other anti-ligands to interact in the air is extremely difficult unless one uses nanoparticles. The molecular interaction of ligand and anti-ligand (antigen and antibody) in air was attempted in the past at U.S. Army Edgewood Chemical Biological Center and AFRL; it failed but nanoparticles carrying a water coating had promise spiedigitallibrary.org/conference-pro…

“Dr Andy Ellington of the University of Texas, one of the co-inventors of SELEX, unbeknownst to us, had received substantially more support than we had, from DARPA, to develop aptamer sensors for biological warfare agents. DARPA was not under the constraint of “forced collaboration” suffered by the Service Labs and their contractors. He went on to receive more money for developing aptamer “beacons”, with a consortium of other institutions and contractors, that were supposed to interact with the agents on the fly in a biological “attack” cloud and cause them to “light up” by fluorescence (from interaction with laser light) that would reveal their presence remotely. This project was doomed from the beginning for two reasons: (1) the Army had tried the approach with antibody in the early 1990’ s at Aberdeen and failed; and (2) As Dr Eric Holwitt always said, “Most of the volume of air that we are looking in for agents is empty space; it is not that the interaction is not sensitive enough, but that there is no agent with which to interact.” This would later also be a problem with standoff detectors that depended on lasers only; the cross section of the microbes diluted in a cloud was just too small to give a decent return signal, much less any natural spectra that were specific for the agent. They were lucky to just tell the difference between a dust cloud or sand storm and a biological, which just as well have been pollen or fungal spores.—-The Black Dragon Trilogy.https://a.co/e38es8z.

Nano droplets for in air interactions:

Recently, it was reported in Kaiser Health News (https://khn.org/news/search-for-a-snakebite-drug-might-lead-to-a-covid-treatment-too/) and the AVMA Animal Health SmartBrief that a new small molecule inhibitor of phospholipase A2, Varespladib, (https://www.nature.com/articles/s41598-019-53755-5.pdf) of snake venom for treatment of snake bite might treat COVID-19 lung disease as well because another PLA2 enzyme in human tissue is in the inflammatory cascade. The Brooks Counterproliferation Team made neutralizing aptamers against AB binary toxins: Botox, Shiga toxin, phospholipase A2 of South American rattlesnake venom, and anthrax toxins; United States Patent US 9,273,345 B2, Mar.1,2016. The snake antivenin aptamers were going to be pursued for this snake (Crotalus durissus terrificus) as well as other significantly dangerous snakes in collaboration with Administración Nacional De Laboratorios E Institutos De Salud (National Administration of Laboratories and Institutes of Health (ANLIS) ) but because of the closure of Brooks in 2011, this never happened.

Many bacterial toxins are AB toxins, that is, they contain a trans-cell membrane cell transporting component B and an affector or toxic effect generator A. Oddly, this motif is seen in the venoms of snakes. The most famous of the AB toxins is produced by the anaerobic bacterium Clostridium botulinum. It is the number one biotoxin on the Select Agent, potential biowarfare/ bioterrorism, list with an estimated human median lethal dose (LD-50) of 1.3–2.1 ng/ kg by injection or 10–13 ng/kg by inhalation. Other species of this genera also produce AB toxins which cause neurological toxicity, tetanus (tetanus toxin produced in wounds by Clostridium tetani), or necrotizing effects (iota toxin of Clostridium perfringens), gangrene. Botulinum toxin is composed of a heavy chain (equivalent to the B chain) and a light chain (equivalent to the A chain) linked together by a single disulfide bond (as many of the snake venoms and related polypeptide hormones such as insulin and nerve growth factor are linked by more than one disulfide linkage). The toxin is produced by the bacteria as an inactive pre-toxin (approximately 150k) which is activated by proteolytic cleavage into the 100kD heavy chain and the 50kD light chain. Botulinum neurotoxin exists as 7 different serotypes, A, B, C, D, E, F and G. All these various serotypes inhibit acetylcholine release from nerve endings, but by targeting different intracellular neuronal protein components of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) acetylcholine transport complex. However, their potencies vary substantially, with some being more toxic and more prevalent clinically in some host species than others. Some are more likely to be associated with human disease like Type A (Western North America) and B (Eastern North America) and others with animal disease, in waterfowl Type C and Type E sporadically among fish-eating birds, such as common loons (Gavia immer) and gulls. Even though botulinum toxicity is usually considered only a toxicity, it can be produced in infants by the consumption of honey contaminated with C. botulinum spores which grow into vegetative forms and produce toxin in vivo, in situ. Occasionally this is seen in adults. An even rarer form is wound botulism related to C. botulinum which grows and produces Type A and Type B toxins, but in at least one case, Type E, in place. Another prominent AB toxin which has seen much press is Shiga (from Shigella dysenteriae shiga toxigenic group of Escherichia coli (STEC), which includes serotypes O157:H7, O104:H4, and other enterohemorrhagic E. coli (EHEC)), which produce related Shiga-like toxin, which is composed of Shiga-like toxin 1 and 2 (SLT-1 and 2 or Stx-1 and 2), with Stx-1 differing from Stx by only 1 amino acid and Stx-2 sharing 56% of its sequences with Stx-1. Shiga toxin inhibits protein synthesis by a mechanism similar to ricin toxin produced by the castor bean, Ricinus communis. After entering a cell, the affector portion of the toxin acts as a N-glycosidase, cleaving a specific adenine from the 28S RNA of the 60S subunit of the eukaryotic ribosome, stopping protein synthesis. Besides causing the gastroenteritis of O157 infection, it also is neurotoxic. The glycosphingolipid, Gb3, is the receptor for Shiga and Shiga-like toxins. It is present in great amounts on renal epithelial cells leading to renal toxicity. Gb3-type receptors are also found in central nervous system neurons and endothelium, which may explain the toxin’s neurotoxicity. Stx-2 increases the expression of its receptor Gb3 and causes neuronal dysfunctions through this positive feedback mechanism. Our group of researchers at AFRL, Brooks City-Base, discovered that curcumin, if given before exposure to the toxin, decreases the Gb3 on target cells. We also developed small synthetic oligonucleotides which bind to the toxin and prevent its action in cell culture. Therefore, Shiga toxicity is a toxigenic disease which is infectious, but not only for the human host. Its genes are encoded on a latent, temperate lambdoid prophage in Escherichia coli. The phage regulatory network is a significant contributor to toxin production and release by this pathogenic E. coli and allows the phage to be released from lysogenic E. coli to co-opt other normally non-hemorrhagic enteric E. coli. This opens another variant from Koch’s Postulates, the conversion of resident microbes which are non-pathogenic into pathogenic ones, violating the first hypothesis by having all hosts carrying potentially pathogenic microbes. This mechanism of toxin genes being conveyed to a susceptible co-opted microbial host does not end with E. coli. It exists in one of the most common bacteria which we vaccinate against in childhood with the classic DPT shot, the toxin of the diphtheria bacterium, Corynebacterium diphtheria. Diphtheria toxin was discovered in 1890 by Emil Adolf von Behring. In 1951, it was discovered that the toxin gene was not encoded on the bacterial chromosome, but by a latent temperate phage infecting all toxigenic lysogenic strains of the diphtheria bacteria. The toxin inhibits protein synthesis as does Shiga and other bacterial toxins and some antibiotics. It does this by acting as the enzyme NAD-ADP-ribosyltransferase (EC 2.4.2.36). It catalyzes the transfer of nicotinamide adenine dinucleotide to the eukaryotic cell elongation factor-2 (eEF2), inactivating this protein so that it cannot participate in the protein synthesizing function of the ribosomes. It ADP-ribosylates the unusual amino acid diphthamide in eEF2. The toxin, like Shiga toxin and snake venoms, is an AB toxin. It is structurally a single polypeptide chain of 535 amino acids composed of two subunits linked by disulfide bridges. Cancer drugs were eventually developed using the toxin, Denileukin Diftitox, which uses diphtheria toxin as an anti-neoplastic pharmaceutical, and Resimmune ™ which is an immunotoxin for cutaneous T cell lymphoma. The latter uses diphtheria toxin (truncated by the cell binding domain) coupled to anti-CD3 antibody. Cholera, which Robert Koch investigated and used the observation of its carrier state in people to disregard his first postulate, is caused by the bacterium Vibrio cholerae, which produces an AB toxin similar to diphtheria toxin and is also co-opted by a bacteriophage which conveys the toxin genes. The cholera toxin is an oligomeric protein made up of six subunits, a single copy of the A (enzymatic “affector” subunit) and five copies of the B subunit (receptor binding), AB5. Subunit B binds and delivers subunit A to the cell where it activates the G protein which activates adenylate cyclase. The five B subunits have a mass of 11 kDa each and form a five-membered ring. The A subunit is 28 kDa and has two functional substructures, the A1 portion of the chain: a globular ADP-G protein-ribosylase and the A2 chain, an extended alpha helix which sits in the center of the B subunit ring. The toxin is similar in structure and mechanism to the heat-labile enterotoxin of Escherichia coli. The subunit A enzyme activates, with different specificity, but essentially the same catalytic activity as subunit A of diphtheria toxin. All these toxins are potential targets for therapeutic neutralizing aptamers which can be selected from a large library of DNA sequences and amplified for clinical use https://patentimages.storage.googleapis.com/4d/9f/d0/ce71a48231f7c5/US9273345.pdf.

This blog is their legacy and the book Notes from the Shadows in The Black Dragon Trilogy their history (see Amazon eBooks).

In an earlier post, I discussed autoantibodies against Interferon I and how this interfered with the first line of defense against SARS-CoV-2. Now recent data has revealed that the virus induces other autoantibodies against other targets which explains the variety of symptoms and perhaps chronic disease following initial COVID recovery. Autoantibodies are not unprecedented in viral diseases. The exclusion of cells in the immune system that produce antibody or cellular immune responses directly against self is a central tenet of immunology. Sir Frank McFarlane Burnet was awarded the Nobel Prize for Medicine and Physiology in 1960 for his work on the immunological recognition distinguishing non-self from self. He coined the term for immune cells’ generating antibodies against self the “forbidden clone”, and formulated the hypothesis, of the same name, that in autoimmunity there exist immune cells able to make antibodies against a variety of self-antigens. He proposed autoimmune disease develops because of the escape of self-reactive clones of lymphocytes’, which are normally deleted during development to generate immune tolerance of self. These autoreactive clones can multiply and cause immune-mediated organ damage through peripheral self-antigen immune recognition and direction of effector cells to kill targets cell in a variety of tissues and organs. He thought these clones might arise as a result of somatic mutation early in lymphoid development. The proposal pre-dated knowledge of the roles of T and B lymphocytes. During his time, the role of autoantibodies was recognized in diseases such as Grave’s Disease, associated with thyroid stimulating antibodies, directed against the human thyroid stimulating hormone receptor. Later in the 20th century, T cells came to be central in theories of autoimmunity and B cells fell off the map. The truth lies somewhere in between. https://pmj.bmj.com/content/postgradmedj/88/1038/226.full.pdf.

However, autoimmunity may persist not only because of detrimental mutation, but also because it serves an evolutionary, beneficial purpose. It has been counterbalanced by apoptosis, which suppresses immune signals while still getting rid of unwanted or unneeded cells, without triggering inflammation. When necrotic tissue infected by pathogens or cells undergoing disease processes contain intracellular pathogens hidden from antibodies or immune surveillance, the tiniest departure from normal surface structure or exposure of internal cell structures to the immune response may be essential to elimination of the pathogen. Various forms of programmed cell death may lead to various levels of immune response exposure: autophagic cell death, necroptosis, elimination by shedding, keratinocyte death by cornification,and cell–cell cannibalism by entosis https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2994033/. These various self- killing mechanisms and autoimmunity may also be important in preventing the development of cancer, or allowing it to proceed, when these countermeasures are out of balance.

The autoimmune response is not new to viral infection https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6723519/pdf/viruses-11-00762.pdf. Examples of infections that yield autoantibodies are those with enteric viruses such as Coxsackie B virus and rotavirus and other types such as influenza A viruses, herpesviruses, measles, mumps, and rubella. Autoimmune B cell responses contribute to mixed cryoglobulinemia, glomerulonephritis, and vasculitis, of hepatitis B and C virus infection. Another example in animals is antibody to feline oncornavirus-associated cell membrane antigen (FOCMA) on the surface of feline lymphosarcoma cells in sera of cats relatively resistant to development of FeLV (feline leukemia virus), Lymphosarcoma, and FeSV (feline sarcoma virus) fibrosarcoma. The antibodies do not exclusively attack viral antigens but consistently bind to 70-kDa proteins on the surface of lymphosarcoma cells https://pubmed.ncbi.nlm.nih.gov/6318434/. Therefore, resistance to cancer, caused by these viruses, is associated with this anti-host cell antibody, not necessarily direct antiviral antibody.

Other chronic disease conditions triggered by viruses and caused by autoimmunity include Type 1 juvenile diabetes and Type 1.5 adult onset autoimmune diabetes. Type 1 diabetes is a lifelong chronic disease characterized by the loss of or severely reduced number of insulin-producing β cells in the islet of Langerhans, presence of islet autoantibodies, and, especially in younger individuals, insulitis consisting of infiltration of the islets predominantly by CD8+ T cytotoxic cells and macrophages. In explanted islets cultured from coxsackievirus B-1-infected islets, the expression of the coxsackie–adenovirus receptor gene was increased and was 10-fold higher in endocrine compared with exocrine cells of the pancreas. There is high suspicion of viral causality because of these observed experimental results https://drc.bmj.com/content/4/1/e000219.

Another autoimmune disease where virus is suspected but not immediately linked is multiple sclerosis (MS). The comparison of chronic canine distemper, which leads to spinal cord and CNS nerve process demyelination, has led to this suspicion by analogy, but incrimination of the canine distemper virus being the cause in human MS has not withstood scientific examination. T cells have been considered the main (MS) protagonists in the pathogenesis of multiple sclerosis. Only recently has evidence arose supporting B cells as major contributors. Rather than merely producing autoantibodies, they collaborate with T cells by priming and regulating T cells, and mediate both pro- and anti-inflammatory interactions. However, despite intensive searching, the target antigen(s) of B cells in MS have not been identified https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4519967/pdf/ijms-16-16576.pdf. The original viral instigator may have only left a ghost antigen behind long after it was eliminated.

Such examples make the autoimmune pathogenesis of COVID not unexpected. Autoantibodies which recognize cell membrane phospholipids cause an autoimmune disease called antiphospholipid syndrome (APS). In APS patients, autoantibodies activate clot-forming cells, putting those patients at higher risk of blood clot formation. These antibodies may also be present in a variety of bacterial or viral infections. However, cause and effect of blood clotting during infection is difficult to prove. Severely ill COVID-19 patients can have high levels of neutrophils, and some have phospholipid-binding antibodies in their blood. Of the patients studied, of those who developed blood clots, only half had the auto antibodies https://stm.sciencemag.org/content/scitransmed/early/2020/11/02/scitranslmed.abd3876.full.pdf. Additionally, the immunological response in severe COVID-19 infection, including TLR7 activation by SARS-CoV2 single-stranded RNA, is sufficient to drive new autoimmunity without the patient’s response against a variety of self-antigens being genetically pre-determined. https://www.medrxiv.org/content/10.1101/2020.10.21.20216192v2.full.pdf. All this being said, the immune system has several possible pathways to follow in COVID-19, some of which I have discussed in earlier posts. The good and bad news is that the progression has these choices. This means that chemically programmable immunity using antigen-aptamer conjugates has a significant probability of re-directing the response down one such pathway that effectively eliminates the SARS-CoV-2 virus without triggering pathogenic autoimmunity or chronic autoimmune disease, which would persist after the virus is gone. There is even a possibility that redirection can ameliorate such residual autoimmunity or even, under ideal conditions, end it. However, until research is supported toward testing these hypotheses, we will never know.

Viruses are such good vectors of engineered genetic material because they are near perfect deliverers of nucleic acids from durable, protective packages into host cells. The nucleic acids delivered take over the replicative machinery of the host cells to reproduce themselves and express proteins which automatically re-package the new nucleic acids. The new virions have the means, by virtue of their coat proteins, to extrude from the cell by fusing with the outer cell membrane, enveloping with cell membrane lipids, by passing through pores formed by transmembrane proteins, or by bursting the cell to be released. How could these wonderfully elegant devices be duplicated without protein to assemble nucleic acids to direct the host cells to build more vectors? Polymers or lipid containers or combinations thereof could duplicate the delivery of nucleic acids across cell membranes to provide expressible genetic elements, but the vectors could not be biosynthetically reproduced, like viruses, to carry on the process to new host cells. This was an advantage to prevent inadvertent uncontrollable transfer, but limited the number of cells producing the new trait or protein. How could both be achieved without loss of control? Could a polymer be made from existing biological feedstock synthetically as well as biosynthetically? Could sufficient but minimal genetic information be transferred to drive host cells to make more vector as well as transfer the designed expressible genes included? Diazoluminomelanin (DALM) is key to the answers. The whole story of this wonderful, amazing material is told in 4 books I wrote and published between 1994 and 2018, inclusive, and previous posts. The three main reasons for the discovery of DALM was (1) to replace the enzyme peroxidase in the hydrogen peroxide and luminol interaction in the chemiluminescent reaction with a single component, (2) find a chemical indicator of radio frequency radiation and microwave absorption, thermochemiluminescence, (3) find a potential application(s) that would justify funding the basic research. The latter made for a moving target of potential applications. The polymer was first synthesized by the following simple reaction:

Solving the structure of the resulting polymer and explaining it’s extraordinary properties would be far more difficult and take much longer.

What was most remarkable was that by cloning a subunit of a plant (barley) nitrate reductase gene through plasmids into bacterial to even human cells, endogenous nitrate reductase in bacteria and nitric oxide synthase in animal and human cells could be greatly enhanced in their production of nitrite. They also became capable of endogenously synthesizing DALM when fed nitrate, 3-amino-L-tyrosine and luminol. They produced it in a spheroid nanoparticle form.

In a previous post, I showed how our last experiments at Brooks showed plasmids containing the nitrate reductase gene subunit could significantly inhibit the replication of vaccinia virus (close vaccine relative of smallpox) and surprisingly how certain arrangements of the gene, with one that produces iRNA, which blocks a viral gene that inhibits apoptosis, actually increases host cell lytic plaques (following viral infection), but did not necessarily increase the subsequent yield of virus. Unfortunately, because of the closing of Brooks further confirmation was not possible, particularly of the latter. What has been confirmed over and over again with many experiments is that DALM produced by these cells could be activated by pulsed microwaves which will destroy cells bearing DALM nanoparticles, external or internal, as a fail safe against transfected or transformed cells, and destroy viral or bacterial pathogens contained within or as bystanders, but spare cells that are not in the immediate vicinity. This would have been a remarkable achievement that makes DALM nanoparticles superior to any gene vector currently available for research or therapy or for vaccine delivery.

mRNA vaccines, like those being clinically tested, and possibly alternate DNA vaccines, discussed in an earlier post, not only must lead to the synthesis of the correct protein in situ to yield the appropriate immune response, but also not trigger innate immune responses directly that are pathological. We saw this in our previous research on artificial nucleic acids for treatment of infectious diseases. The toxicity of aptamers noted in our earlier studies may have been influenced by their interactions with the innate immune system, either TLR-9 receptors or other unknown ones that trigger adverse responses. In vitro studies have demonstrated that, in autoimmune-prone mice, dual signaling via the B cell receptor and non-CpG (cytosine/guanine base coupled) DNA results in synergistic B cell activation in a TLR9-independent manner. These results suggest that engagement of a TLR9-independent DNA activation pathway may trigger autoimmunity. TLR9 can mediate either efficient Th1- or Th2-dominated effects depending on whether innate immunity is stimulated by CpG or other certain non-CpG oligodeoxynucleotides (ODN).

Lymphocyte produced Th1-type cytokines produce pro-inflammatory responses responsible for killing intracellular parasites and for promoting autoimmune responses. Interferon gamma is the main Th1 cytokine. Excessive proinflammatory responses can lead to uncontrolled tissue damage. The Th2-type cytokines, including interleukins 4, 5, and 13, promote IgE and eosinophilic responses in atopy, and in addition, interleukin-10, as an anti-inflammatory response. IL-10, produced by CD4 + lymphocytes of the Th-2 subset, not only inhibits synthesis of gamma-interferon by both T cells and NK cells, but also inhibits the synthesis of NOS by mouse macrophages. Th2 responses counteract Th1 mediated anti-microbicidal actions. The optimal scenario is well balanced Th1 and Th2 responses, appropriate for the immune challenge. Allergy may be a Th2 weighted imbalance with Th-1. Immunologists have been seeking ways to redirect allergic Th2 responses toward Th1 responses to try to reduce the occurrence of atopy.

The non-CpG sequences that can be found in aptamers could cause this Th1 to Th2 shift, the latter is usually considered less inflammatory. Therefore, subsets of sequences must be carefully selected in therapeutic or vaccine nucleic acids to avoid these untoward undesirable effects. The following chart shows that some of the aptamers in our studies had these non-CpG sequences:

The following types of Toll-like receptors control the off-target consequences of nucleic acid therapeutics and vaccines; the secret is finding a combination with the targeted function of the nucleic acids that is mutually beneficial: TLR3 against Double-stranded RNA of Viruses; Poly (I:C) Synthetic analog of double-stranded RNA ; TLR7 and TLR8 against Single-stranded RNA from Viruses; and TLR9 against Unmethylated CpG DNA of Bacteria, Protozoa, Viruses, and Mitochondrial DNA (possibly pathogenic).

In addition to insights into how the immune system might respond to synthetic artificial nucleic acids, we must also consider how off-target effects might manipulate host nucleic acid functions, protein expression and physiology, in general. There are natural examples (viroids) of such manipulation, some pathological. However, their discovery also led to new approaches to gene therapy such as RNA interference to effect gene expression changes (translation into functional proteins by affecting other genes but not being transcribed themselves). RNA interference (RNAi) inhibits gene expression or translation by neutralizing targeted mRNA molecules. Historically, RNAi has also been known as co-suppression, post-transcriptional gene silencing (PTGS), and quelling.

“Viroids are plant pathogens and one human pathogen, Hepatitis D “virus”, which are made solely of nucleotides composing a single-stranded RNA. Viroid genomes range in size from 246 to 467 nucleotide bases, smaller than any known virus. They were discovered by Theodor Otto Diener, plant pathologist at the Agricultural Research Service in Maryland, in 1971. They do not code for protein and they use RNA polymerase II, a host cell enzyme which mediates messenger RNA (mRNA) synthesis from DNA, for replication, using the viroid RNA for a template. Some viroids are ribozymes, being catalysts which self-cleavage and ligate viroid genomes from larger replication intermediates. The first viroid identified was Potato Spindle Tuber Viroid. Approximately 33 “species” are known. The viroids replicate through a double-stranded intermediate RNA. They are then cleaved by a dicer enzyme into siRNAs (small interfering RNA), which are then incorporated into an RNA-induced silencing complex. The viroid siRNAs hybridize with the plant’s mRNA and cause disease by degradation or inhibition of translation of the mRNA into plant proteins, causing viroid diseases.”— The Black Dragon Trilogy by JOHNATHAN KIEL
https://a.co/3iXtFwv

“Human disease caused by a viroid is hepatitis D, or Delta Agent. Delta Agent is a viroid contained in the Hepatitis B Virus capsid. So, this virus and the Delta Agent simultaneous infect the patient. The hepatitis D viroid RNA complements and hybridizes with human liver cell 7S RNA, a small cytoplasmic RNA component of the signal recognition particle, the structure involved in the translocation of secretory and membrane-associated proteins. The hepatitis D viroid causes liver cell death by binding to and preventing the function of the 7S RNA and perhaps cleaving it.”— The Black Dragon Trilogy by JOHNATHAN KIEL
https://a.co/iUKBaeW

This examination is not meant to discourage taking advantage of nucleic acids for vaccine or therapeutic development but only to encourage the appropriate care needed before proceeding to the clinic.