Global Biosurveillance

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.”— The Black Dragon Trilogy by JOHNATHAN KIEL https://a.co/hnHWBsg. This kind of propaganda now depends on the internet and social media full of testimonials and posting unsubstantiated reports supporting the emotional assertions. Testimonials are not science and neither are uncontrolled observations leading to causal conclusions based on association, especially time related causality. This is not science. It is not based on the scientific method. People also confuse the unedited collection of adverse reactions data by the FDA on medicines and vaccines as causal proof, but it is only to consider all possible adverse reactions for future rigorous scientific investigation using the scientific method (https://www.fda.gov/files/vaccines,%20blood%20&%20biologics/published/Understanding-the-Vaccine-Adverse-Event-Reporting-System-(VAERS).pdf. and https://www.nature.com/articles/d41586-021-00290-x?utm_source=Nature+Briefing&utm_campaign=5eb3714930-briefing-dy-20210217&utm_medium=email&utm_term=0_c9dfd39373-5eb3714930-43804265).

The most notorious example of pseudoscience perpetrated by a scientist involved AIDS in South Africa. UC Berkeley professor Dr. Peter Duesberg, a University of California at Berkeley, tenured professor in the Department of Molecular and Cell Biology, believed that HIV does not cause AIDS. In 1987, he first questioned the link between HIV and AIDS in the journal Cancer Research (“Retroviruses as carcinogens and pathogens: Expectations and reality”. Cancer Research 47 (5): 1199–220, 1987).” In 2000, Duesberg was a prominent member of the panel which advised President Thabo Mbeki of South Africa on the cause of the AIDS epidemic which was exploding out of control in South Africa. This “scientific support” led President Mbeki to deny AIDS was caused by a virus and denied anti-viral treatments in his country. Between 2000 and 2005, more than 330,000 deaths and an estimated 35,000 infant HIV infections occurred. Nicoli Nattrass of the University of Cape Town estimated 343,000 additional AIDS-related deaths and 171,000 infections occurred because of President Mbeki’s administration’s policies. According to Peter Mandelson in 2002, a British Labour Party politician and President of the international think tank Policy Network, it was a “genocide by sloth”. Duesberg recently still asserted his views in a paper first published in 2009, then withdrawn and republished in a revised form, in the peer-reviewed journal The Italian Journal of Anatomy and Embryology (IJAE) in December 2011. 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.

In respect to vaccination hesitancy and resistance, the consequences can be swift and devastating, with diseases almost never seen anymore, erupting abruptly seemingly out of nowhere. From 2011 to date, measles has become a problem for public health officials in the US. There were 220 cases in 2011, just 55 in 2012 and 186 in 2013. The illnesses have appeared in clusters for the most part, although single cases have also appeared in many states. In 2014, there have been three large measles outbreaks. Southern California saw an outbreak from January through May 2014 at 59 cases. New York City had an outbreak that stopped at 26 cases. Ohio has had an outbreak through 16 May 2014, with 83 people infected In 2013 through 2014, in the US, three outbreaks accounted for most of the measles cases. These included clusters of measles cases: in Texas tied to the Kenneth Copeland televangelism ministry and his mega-church; in North Carolina linked to a Hindu religious community and shrine; and in New York City, in 2014, in the Hasidic Orthodox Jewish community in Brooklyn. The national measles report from the Centers for Disease Control 1 Jan through 9 May 2014, released 12 May 2014, showed 187 measles cases from 17 states. Since that report, Ohio reported an additional 23 cases, with new cases in Tennessee, Pennsylvania, Massachusetts, and other states. The measles cases in both Ohio and California, in 2014, were linked to an ongoing measles epidemic in the Philippines. Both outbreaks in the States were results of travelers returning from the Philippines who had not been vaccinated and who brought back incubating measles. Six cases in Washington State were in patients without immunizations with ties to the Dutch Reformed Church, in British Columbia, which was the center of a 400-case outbreak of measles. The church opposes the use of all vaccines, including the measles vaccine. These examples illustrate the effects of misinformation, particularly on the internet and social media, where its removal is problematic, and perhaps, where it remains eternal. A study of the correlation of the dissemination of such information, the decline of vaccination, and the occurrence of cases should be made. My hypothesis is that the correlation would be high, and the pattern would resemble the spread of an infectious disease itself, with the computer being the vector and the electronic misinformation being the infectious agent (at least a surrogate for measles). Now imagine the consequences of anti-vaccination campaigns against SARS-CoV-2 and the devastation which has already occurred at this writing to date, continuing indefinitely because herd immunity can never be reached through “natural means” and large populations of unfettered virus allows for probable continuous emergence and “ natural selection” of resistant variants. This is, as I have posted earlier, the common nature of coronaviruses. Finally, as noted in a previous post, development of immunity after widespread dissemination of the virus in organs and tissues sets them up for devastating immune mediated complement interactions and innate immune cell (neutrophil and other granulocyte) mediated collateral destruction of blood vessels and other tissues.

All this being said, potential detrimental effects as well as beneficial ones of treatments and vaccines must be reported and investigated by the scientific method with even handedness. This includes herbal and indigenous peoples’ remedies. Historically, pharmaceuticals and pharmacology are deeply rooted in botany of medicinal plants; many which originated in folk remedies but which stood scientific scrutiny. Examples include Belladona (resulted in atropine and scopolamine), digitalis (digoxin), quinine, and lastly, the very important antimalarial drug Artemisinin (Chinese remedy). Belladonna (Atropa belladonna) is a plant which has been used as a medicine since ancient times. “Belladonna” means “beautiful women” used by the ladies of Renaissance Italy to enlarge their pupils, which they found alluring. But because it can be a lethal poison, the plant of origin also goes by the more sinister name deadly nightshade. Digoxin and digitalis are cardiac glycosides derived from the plant, foxglove, used to treat mild to moderate congestive heart failure and abnormally rapid atrial rhythms (atrial fibrillation, atrial flutter, and atrial tachycardia). The quinine mentioned above, and its present day Chinese successor, Artemisinin (from Wormwood), are actually very old remedies. Quinine, from the bark of the cinchona tree, and which can now be made synthetically, was originally discovered by the Quechua, indigenous people of Peru and Bolivia. Jesuit Missionaries were the first to introduce cinchona to Europe in the 17th century. Chinese herbalist’s use of Artemisia annua (Wormwood), which predated quinine, was first described in a 4th-century Chinese text, the source of the modern day antimalarial drug arteminisin. Quinine was commonly used for treatment of malaria until the 1940s, when chloroquine and other drugs were developed because they had fewer side effects. Except for vitamin D3 (addressed in a previous post) and some ongoing studies on the microbiome, I know of no other natural remedies being scientifically examined for prevention or treatment of COVID at this time. However, recent studies don’t support the expectations for Vitamin D3. In a study of hospitalized COVID-19 patients, a single high dose of vitamin D3 did not significantly shorten hospital lengths of stay. These findings do not support the use of high dose vitamin D3 for treatment of moderate to severe COVID-19. Criticism of this study suggesting that it did not look at subpopulations of different severity independently or early treatment suggests that the treatment should not be rejected before it has been further studied and then found effective or not (https://jamanetwork.com/journals/jama/articlepdf/2776738/jama_murai_2021_oi_200145_1613509376.92008.pdf and https://jamanetwork.com/journals/jama/articlepdf/2776736/jama_leaf_2021_ed_200126_1613509372.9982.pdf). Vaccination, in spite of variants, (https://www.researchsquare.com/article/rs-226857/v1 and https://jamanetwork.com/journals/jama/articlepdf/2776739/jama_walensky_2021_vp_210031_1613509382.71695.pdf.) and sanitary precautions are still our best defense against COVID. What is certain, COVID can be lethal and can have persistent long term debilitating effects which are lacking with vaccination and can be prevented by it.

Out of my frustration with ignored proposals for support and failure of acceptance by institutions, I have decided to fully disclose my concepts and ideas of my last, and believe most important, contribution to medical science. This is the briefest and most complete description I can give in an effort to engage others more competent to take up the pursuit, with I hope, greater success than I. In discussing the limitations of CRISPR, two major points come to mind: 1) how to deliver it and 2) how to prevent off target effects. Francisco Mojica discovered CRISPR in 1993. He researched these sequences and their products throughout the 1990s, and into 2000. He recognized that the various repeat sequences shared a common set of features, the CRISPR sequences (a term he created in correspondence with Ruud Jansen, who first published the term in 2002). In 2005, he reported that these sequences matched subset sequences from the genomes of bacteriophages (Mojica et al., 2005). This led to the hypothesis, which was later proven, that CRISPR is an adaptive immune system to prevent further invasion and replication of bacteriophages in the host bacteria. The other group to independently discover this fact published similar findings in 2005 (Pourcel et al., 2005). The Nobel Prize in chemistry was awarded in 2020 to Jennifer Doudna and Emmanuelle Charpentier “for the development of a method for genome editing”, developing CRISPR. A recent Science Magazine article discusses the hope and difficulties of delivering CRISPR to correct a rare but devastating genetic disease. CRISPR research shows promise for correcting the faulty wolframin gene but it affects so many tissues, researchers will have to figure out how to deliver CRISPR components to most cells in large organs such as the brain and liver, a “pretty daunting task”. It might take 10 to 20 years to accomplish. The syndrome named for Donald Wolfram, a physician at the Mayo Clinic in Rochester, Minnesota, in the 1930’s, and an ophthalmologist colleague, ruled out malnutrition as the cause of the puzzling condition and discovered it was hereditary. Recessive mutations in the gene for a protein called wolframin are responsible for most cases, with involvement of a second gene causing the remainder. However, the pair was wrong to think the defect lies only in the brain. Instead, the symptoms stem from widespread cell death. The first sign of the illness, appearing in children, is usually diabetes mellitus, because of loss of insulin-secreting beta cells in the pancreas. Most patients also develop diabetes insipidus, in which the pituitary gland does not produce the hormone vasopressin (AVP), which is also known as antidiuretic hormone (ADH), that helps control the body’s fluid balance, causing the kidneys to produce huge amounts of urine. Blindness will also develop; patients usually go blind within 10 years of their first visual symptoms https://t.co/DCWpJuCJAI.

Using CRISPR has other problems associated with introducing foreign protein. Editas Medicine and Allergan announced human in vivo CRISPR-therapy trials for an inherited blindness, but have run into a potential hurdle to therapeutic CRISPR, the human immune responses to its bacterial components. For instance, a majority of tested blood samples showed existing immune responses to Cas9, which is commonly taken from Staphylococcus or Streptococcus bacteria. It is needed to do the double-stranded cutting of the DNA to remove the offending gene to be replaced. Correcting multiple genes is also a problem, but may have an answer in CRISPR. The solution is in multiplexing, changing more than one gene at a time, requiring only the introduction of a single Cas enzyme and of gRNAs (guide RNAs which direct the cutting of the DNA by hybridization of the specific RNAs to the DNA to be excised), and template DNAs for each targeted gene. CRISPR multiplexing to tag multiple genes in the same cell is achievable, but in practice, gets increasingly complicated with each added gene https://www.sciencemag.org/features/2019/09/beyond-crispr-what-s-current-and-upcoming-genome-editing and https://www.frontiersin.org/articles/10.3389/fbioe.2019.00459/full.

Our solutions, including a delivery system for CRISPR, grew out of USAF Air Force Research Laboratory Brooks Counterproliferation Team’s research to answer requirements of Special Forces for biodefense Counterproliferation. We needed methods and equipment to immediately detect, identify and safely collect and isolate highly pathogenic microbes rapidly in non-permissive territories for their safe return to the laboratory, with chain of custody, for further culture, analysis and attribution. As a fortuitous consequence, these methods and equipment could be used with little or no support and with simple to operate and maintain equipment which would be very useful in the most remote areas of developing countries. Even the reagent manufacturing could be accomplished in place with portable fermentation miniature plants by microbial biosynthesis. Additional mechanisms of electromagnetic radiation interaction, allowed for raising the impact of the detection system to the level of specific antimicrobial biocidal effects on the microbes in the environment. The process also had the potential of inhibiting, killing or genetically altering the microbes in vivo. The last step before closing the program demonstrated that the nanotechnology was operational in animal and human cells without being lethal, suggesting utility in genetic therapy in animals and humans. These experiments, preliminary applied science, and concepts were not developed and fully exploited for field and medical applications. What follows are step by step progression of the development and concepts of the technology I had hoped would be applied to Global Biosurveillance, Counterproliferation, and infectious disease control as close to the source of the outbreak as possible. The nanoparticle material would have to be carefully selected to avoid toxicity. For instance, carbon nanotubes can cause lung inflammation and fibrosis if inhaled but are broken down by myeloperoxidase and other oxidative enzymes (http://www.particleandfibretoxicology.com/content/3/1/15, Franco Cataldo and Tatiana Da Ros, Editors, Medicinal Chemistry and Pharmacological Potential of Fullerenes and Carbon Nanotubes, and Prabakaran Ravichandran et al, PULMONARY BIOCOMPATIBILITY ASSESSMENT OF INHALED SINGLE-WALL AND MULTI- WALL CARBON NANOTUBES IN BALB/C MICE, JBC Published on June 24, 2011) and may even be useful as immune adjuvants to stimulate immunity induced by vaccines. The synthetic Nanobes may simply be used to induce biosynthetic Nanobes in the manufacturing process and avoid all toxicity problems all together. Could these technologies along with CRISPR be brought to bear against SARS-CoV-2 and all its variants? New emerging or old re-emerging pathogens?

Recently, DARPA has sponsored an inhalable CRISPR nano construct which can attack viruses like influenza and SARS-CoV-2, at least in mice and hamsters. The researchers used mRNA coding for the protein Cas13a, which chops up parts of the viral genome required for replication. This treatment is delivered via polymer nanoparticles (https://www.nature.com/articles/s41587-021-00822-w). Before there was CRISPR plug and play by DARPA, there was the Nanobe inhalable plug and play by AFRL, “Chapter 54 Plug and Play..how a component-based design can be used in nanotechnology to produce a highly versatile new set of diagnostics to therapeutic tools and, for the first time, to make a material which is also practical for treatment.” The Black Dragon Trilogy, 2018.

Footnote: Perhaps why this was never developed and I have been excluded from developing it further: “By adding specific aptamer sequences to the surface of the Nanobes, they could be directed to bind to specific neurons with those surface markers to deliver “nano-level stimulation”. This application would meet the nefarious goal of behavior control of specific adversaries by external application of electromagnetic fields which would not affect anyone else exposed. However, this approach could also provide relief for those suffering from various neurological or mental disease and brain injuries, including counter acting addiction, the dual use dilemma.” “Chapter 50 Why is Nanowarfare at War and Not Peace? This chapter discusses the duplicity of bionanotechnology, the “political science” and economics behind the funding of this research and its dual use applications revealed in the open literature and by government policy.”“A high-ranking colleague in the Department of Defense at the Pentagon once called me the Most Dangerous Man Alive. But now, as of this writing, I am the most helpless as a scientist and technologist. The only resource I have left is knowledge. Now I have shared it with you. I only hope you have the wisdom to use it appropriately.”—-The Black Dragon Trilogy by JOHNATHAN KIEL
https://a.co/hhbwuTE

I thought the potential for nefarious use was the reason no one supported the development of Nanobe technology, but recent pursuits starting around 2013 have proven it just depends on who you are and where you come from, Spherical Nucleic Acid Nanoparticle Conjugates as an RNAi-Based Therapy for Glioblastoma, https://stm.sciencemag.org/content/scitransmed/5/209/209ra152.full.pdf ;https://www.scientificamerican.com/article/the-power-of-spheres/; https://www.feinberg.northwestern.edu/_rwd/head.php; and https://www.news-medical.net/news/20200702/Developing-Vaccines-and-Therapeutics-by-Exploiting-Structure-in-Spherical-Nucleic-Acids.aspx.

Above is a notional ensemble of spherical Nanobes associated with carbon nanotubes followed by pictures of cells penetrated with these actual structures and traced with fluorescence into the nucleus of a human cell in the last photograph.

With the rise of many mutations and variants, the questions arise: 1) will the current vaccines protect and stem the tide of the pandemic? 2) Will the vaccines drive, by selection pressure, the establishment of new more resistant, transmissible and/or pathogenic variants? 3) Will masks and social distancing still be required after vaccination? Variants which emerged in Brazil (known as P.1.), Britain (known as 20I/501Y.V1 or B.1.1.7) and South Africa (known as 20I/501Y.V2 or B.1.351) are now the globally dominant strains because of their apparently enhanced infectivity and transmissibility.

Current vaccines work against the UK B.1.1.7 variant without the E484K mutation. However, recent clinical trials by Novavax and Johnson & Johnson showed that their new vaccines were less effective in South Africa compared with the UK or US, probably because of the high level of virus carrying the E484K mutation. However, Novavax vaccine was reported to have a 60% efficacy in South Africa which is a good response, when compared to influenza vaccine responses of from 40-60 %. Studies have shown that people who have been infected with SARS-CoV-2 generate T cells that target at least 15–20 different fragments of coronavirus proteins. Because of natural variability, a population will generate a large variety of T cells that could attack the virus (https://reader.elsevier.com/reader/sd/pii/S1074761320305379?token=3775A68CFC8B8609E4A6753EF03F560FAF6F4B30D3B821B81F3A1CBF077E6DB8C53232C5B16ACCA1E569F0AE6C5368F3). As I mentioned in an earlier post, it is very hard for the CoVID variant viruses to mutate to escape cell recognition because of this T cell variation, The specificity of antibodies works in favor of variants. A recent pre-print shows a single mutation can defeat monoclonal antibodies against the spike protein of the virus and increased cell receptor binding, unfortunately a rare converging of properties in a single mutation. The mutation has been observed in a recent variant in California, CAL.20C viral variant from clade 20C, lineage B.1.429, that since November 2020 has generated multiple outbreaks and is undergoing massive expansion in California. This L452R mutation allows SARS-CoV-2 positive selection toward strong viral adaptation against containment measures that work on less contagious variants and against increasing population immunity against previous forms. The L452R mutation is at the leucine-452 position in the receptor-binding area of RBD (receptor binding domain) at the interface with the ACE2 receptor. Replacing it with arginine is anticipated to result in both a much stronger receptor binding and escape from neutralizing antibodies https://www.biorxiv.org/content/10.1101/2021.02.22.432189v1.full.pdf. Escape from two monoclonal antibodies has been predicted in a recent Science article https://science.sciencemag.org/content/sci/371/6531/850.full.pdf.

In a preprint published on 9 February, researchers found most T-cell responses to coronavirus vaccination or previous infection do not target regions mutated in two recently discovered variants, including 501Y.V2 (https://www.researchsquare.com/article/rs-226857/v1). Also, the vaccine made by Novavax of Gaithersburg, Maryland, a single-shot vaccine made by Johnson & Johnson of New Brunswick, New Jersey, and a vaccine made by AstraZeneca of Cambridge, UK, and the University of Oxford, UK are less effective at protecting against mild COVID-19 in South Africa, where the 501Y.V2 variant dominates, than in countries where it is now less common. AstraZeneca’s vaccine was only 22% effective against mild COVID-19 in a study of 2,000 people in South Africa, but the trial was too small and participants too young to draw any conclusions about any loss of benefit in protecting against severe disease. However, another recent paper compared the vaccine – induced neutralization of a less contagious version of the SARS-CoV-2 virus from last year (USA-WA1/2020), to the new variants Brazilian P1 and UK variant B.1.1.7 which gave results “roughly equivalent.” Geometric mean titers (GMT) of neutralizing antibodies against the USA-WA1/2020 virus, the UK B.1.1.7 variant, and the P1 variant were 532, 633, and 437, respectively. The GMT against three versions of the South African B.1.3.5 variant were less at 195, 485, and 331, respectively (https://www.medscape.com/viewarticle/947064).

A recent, yet to be peer-reviewed, study of men from 70-84 years of age, who died from COVID-19, increased from 5% of those who tested positive for the older variant, to more than 6%, if confirmed positive for variant B.1.1.7. For men 85 years or older, the risk of dying increased from 17% to 22% of those confirmed positive for the new variant. Therefore, the apparent increase in severity of this new variant has been limited in scope. It may reflect an increased viral load rather than a normalized increase in pathogenicity. There is little evolutionary advantage for a virus to become more pathogenic in a host in preference to transmissibility. This may happen only if there is a maintenance reservoir host in which the pathogenicity is less and from which the virus periodically emerges entering the host more vulnerable to lethality.

As to the third question, until the pandemic has subsided, even vaccinated people, including those who may have gotten upgraded vaccine for new variants, should continue to use masks, social distance, and, of course, continue to use good hand washing practices. Another early treatment to block viral replication has been discovered; a single pill of the investigational drug molnupiravir, when taken twice a day for 5 days, has been shown to eliminate SARS-CoV-2 from the nasopharynx of 49 participants in a new study (https://www.medscape.com/viewarticle/947061). Phase 2/3 efficacy and safety studies of the drug have started. The drug creates viral error catastrophe by inducing replication and mutations until the virus can’t effectively produce viable copies. This new early treatment and the non-pharmaceutical practices are necessary to decrease the total number of viruses in circulation, to greatly decrease the probability of establishing new variants even if they are more transmissible, and especially if those infected shed larger amounts of virus asymptomatically or symptomatically.

As stated in an earlier post, it is much more difficult to mutate past many different antiviral antibodies or different simultaneous multiple drug therapies. For example, in 1995, a combination drug treatment known as the “AIDS cocktail” was introduced which made AIDS a survivable, chronic disease. This type of therapy is known as highly active antiretroviral therapy (HAART), also called combination antiretroviral therapy (cART). HIV is a virus much more prone to mutations than SARS-CoV-2. Our own research, demonstrated that a mutagen (2-chloroethylethylsulfide) could make bacteria resistant to one but not two antibiotics at the same time in the same microbes without substantial mutations that largely proved lethal for the bacteria (ampicillin and 3-amino-L-tyrosine: Patent #: 5,902,728; date: May11,1999). As the number of mutations increased in the microbial population in order to achieve both ampicillin resistance and 3-amino-L-tyrosine/nitrate resistance, the bacteria became less robust and sacrificed their competitiveness to less resistant subpopulations. The JM109/pIC20RNR1.1 E. coli, genetically modified bacteria by a plasmid, was designed to have conflicting selection pressures when grown in 3-AT medium containing both ampicillin and 3-amino-L-tyrosine/nitrate. The biosynthesis of the DALM polymer was driven by the ampicillin resistance expressed from the same plasmid which contained the gene for enhanced DALM biosynthesis. However, driving DALM synthesis led to lethality. Loss of the ampicillin resistance would spare the bacteria from DALM lethality, but lead to their death by the ampicillin. Therefore, there needed to be a set of simultaneous mutations to spare the bacteria from both these diametrically opposed lethal effects. A similar response would prevent SARS-CoV-2 from escape mutations if hit by several different antiviral approaches simultaneously. The number of mutations in SARS-CoV-2 increase by chance as the circulating population of viruses increases and they become available for natural selection for traits that increase their survivability and transmission. Conversely, limiting their spread and numbers by both pharmaceutical (vaccines and antivirals) and non-pharmaceutical means (masks and distancing) greatly reduce these possibilities. Finally, I illustrate below how the appearance of variants, which evade vaccines and treatments, can be countered by aptamer adapters that restore the effectiveness of these vaccines and treatments.

Re-Directing an old vaccine against a new variant of a virus. The blue virus was vaccinated against, then the circulating virus mutated into the red virus which is not protected against by the previous vaccine. The red virus is isolated, then nucleic acid aptamers are selected in vitro against the new virus. Another set of aptamers are selected against ligands on existing antibody against the original blue virus, or against ligands on cells in the immune system to recruit them to attack the virus (NK=Natural Killer cells, cytotoxic T cells, helper T cells and associated B cells). These aptamer sequences can be fused together to provide an adapter (>~~<) which links the binding of the variant virus to mediator (APC, Antigen Presenting Cells; dendrocytes) and effector cells in the immune system. The linkage to pre-existing antibody to the predecessor virus (>~~) allows neutralization of the variant virus and recruitment of Antibody Dependent Cell Cytotoxicity (ADCC; macrophages and granulocytes). The artificial evolution of aptamers, to go into such adapters, against new viral variants will prevent a vaccine from ever becoming obsolete. Double-faced artificial polypeptides, like aptamers, have been computationally designed to re-direct humoral vaccine immunity from existing antibodies against other virus targets to SARS-CoV-2 by having one end of the polypeptide mimicking ACE2 viral receptor and the other a Hepatitis B antigen to bind the previous Hepatitis B vaccine induced antibody to the SARS-CoV-2 virus through the ACE2 viral receptor mimic (https://pubs.acs.org/doi/pdf/10.1021/acs.jpclett.0c03615).

Bats and children 9 years and under may have something in common in their immune responses to COVID which spares them from severe symptoms— immunotolerance. Bats have several mechanisms to prevent over responsiveness to viruses and limit collateral non-specific damage by inflammation. Humans express minimal baseline levels of type I interferons (IFNs), and they are highly inducible upon stimulation, but many bat species, such as the black flying fox (Pteropus alecto), constitutively express some baseline IFNα, and several IFN-stimulated genes before stimulation. Therefore, they are prepared to inhibit viral replication before it can spread to many tissues and prime an overt response with a heavy viral load. The restricted induction of type I IFNs minimizes production of inflammatory cytokines and reactive oxygen species by innate immune inflammatory and phagocytic cells. The kinetics of the IFN response in bats also differs from those of other mammals, in that it has a faster decline for some bat interferon-stimulated genes post-stimulation. Bats have a reduced production of reactive oxygen species compared to similar-sized non-flying mammals, but retain intact activity of the important antioxidant enzymes like superoxide dismutase. These findings suggest either a more effective scavenging of reactive oxygen species or a lower production of reactive oxygen species by bats, or a combination of both.

Immune tolerance in bats is also expressed in dampening of the STING-dependent type I IFN response in several bat species, resulting from a point mutation in the Stimulator of Interferon Genes (STING) https://www.frontiersin.org/articles/10.3389/fimmu.2020.00615/full. STING is an important pattern recognition receptor which triggers cytosolic-DNA-induced signalling and has a key role in infection, inflammation, and cancer. This mutation has the effect of dampening STING mediated responses to infection with bat-borne RNA viruses, which activate STING by inducing host DNA damage. A more recent study has revealed another key mechanism by which bats naturally dampen host inflammation in response to ‘sterile’ danger signals and infections with RNA viruses. NLR-family pyrin (NLRP3), a key inflammasome sensor that recognizes various cellular stresses and pathogen invasion, is dampened at both the transcription and protein translation level in bats. Importantly, reduced NLRP3-mediated inflammatory responses to RNA viruses have no, or minimal, effect on viral loads https://www.nature.com/articles/s41586-020-03128-0.pdf.

In children, which I have discussed in earlier posts, there is a rapid early interferon response to SARS-CoV-2. Community acquired pneumonia in children younger than 5 years old, with severe and life-threatening symptoms, have significantly increased interleukin-17 (IL-17) production. Immune profiling of these children showed that mucosal-associated invariant T (MAIT) cells from the lungs, but not blood, actively produced IL-17 (MAIT17) https://www.nature.com/articles/s41385-020-0273-y.pdf. Previously, I posted that the IL-17/Th17 pathway plays a major role in regulating the severity of COVID and turning it down by removing viruses early with interferon, reducing the viral load and distribution, limiting the reactive oxygen response of this pathway and, therefore, the collateral damage, as seen in most children exposed to COVID who are less than 9 years old. Th17 lymphocytes promote signals typical of early inflammatory events, and serve as a bridge between innate and adaptive immunity https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2821718/pdf/nihms-152030.pdf. Therefore, they represent the pivot point between a severe and mild antiviral innate immune response and an effective long term protective adaptive immune response. In children, they produce a mild but virus-eliminating early response, and are less likely to yield a sustained adaptive immune memory as in adults, from the infection, a form of immunotolerance. They sacrifice long term immunity for the lower severity of the disease. Stopping the virus before it systemically disseminates is critical because it can otherwise damage blood vessels in many tissues and organs causing retiform purpura, gangrene and vascular permeability, swelling to bleeding, as well as widespread deposition and activation of neutrophils by activating the complement system. It induces a hypercoagulable state with micro- and macroangiopathy. The activation occurs by forming complement cascade activating antibody-antigen complexes or by the alternate complement pathway activated by the spike glycoprotein acting as a lectin (https://www.translationalres.com/action/showPdf?pii=S1931-5244%2820%2930070-0). However, to achieve long term immune memory one might have to first endure a “baptism of fire”.

The loss of smell (anosmia) and disruption or loss of taste (dysguesia or ageusia) are unique symptoms of COVID-19. The sensations of hot peppers and mint can also be lost, but these are transmitted by different neurons, those for pain. The former 2 lack ACE2 viral receptors but some of the pain sensor neurons have them. Most recover smell and taste within 6 months, but some require 2 years or fail to recover the senses at all. During recovery, parosmia may occur, which is the sensing of irrelevant smells unrelated to the objects’ odors. Irrelevant tastes, often unpleasant ones, may also develop. These effects are postulated to be from errors in re-established neuronal connections, which can be corrected, in time, due to the plasticity of the nervous system. Influenza has produced anosmia in the past, and provides some understanding of the symptomology and prognosis. The primary mechanism for the loss of smell and taste with COVID is not infection of neurons but supporting cells that help maintain neuronal function. For smell, these are sustentacular cells, and different supporting cells for taste. The neurons per se are spared, but in the case of the attack on sustentacular cells, the finger-like cilia on the neurons, which contain the smell receptors, are lost because of the loss of the supporting sustentacular cells. Since the neurons are not attacked directly, the loss of smell and taste are not usually irreversible. The pain sensation associated with sensing aromatics like mint and plant capsaicinoids like capsaicin (8-methyl-N-vanillyl-6-none amid) is another matter since these neurons have ACE2 viral receptors https://www.scientificamerican.com/article/mysteries-of-covid-smell-loss-finally-yield-some-answers1/.

A more ominous effect on the Central Nervous System (CNS) of some virus infections and chronic neurodegenerative diseases, that resemble the response to a virus but have no known virus such as Multiple Sclerosis (MS) associated with them, is demyelination. It, like the effects of SARS-CoV-2 on the sustentacular cells, involves supporting cells of neurons, the oligodendrocytes. In MS, the demyelination occurs in the Central Nervous System (CNS), the white matter of the brain and spinal cord, leading to pain, tremors, speech difficulties, loss of balance, vision difficulties, sensation loss and paralysis to various degrees, with multiple isolated episodes of recurrence. The myelin is a fatty coating of the nerve processes that resembles insulation on an electrical wire, strip it away and nerve conduction fails. It has been associated with viral infections, Epstein-Barr virus most recently (https://www.science.org/doi/10.1126/science.abj8222), and genetic pre-disposition leading to this autoimmune disease. Previously, measles, and even closely related canine distemper viruses, were suspected of causing MS. The latter because it commonly causes CNS demyelination in dogs, especially adult dogs. In puppies it starts as a respiratory virus with skin lesions, causing footpads to thicken and harden, leading to its nickname, “hard pad disease” (measles also causes hypersensitivity in the skin causing damage to small blood vessels and a rash, Koplik’s spots). Measles can also attack the CNS directly or secondarily, probably by triggering autoimmunity. CNS involvement, even in uncomplicated measles, is common. Transient EEG abnormalities are detected in 50% of patients. Measles virus is rarely isolated in acute measles panencephalitis. Therefore, it is considered the result of autoimmunity. Distemper does not kill the oligodendrocytes, which make myelin, but inhibits its synthesis. Sterile demyelination can be induced in animals (in guinea pigs, experimental allergic encephalomyelitis (EAE) induced by guinea pig myelin basic protein) by immunization against their own basic protein, a component of myelin. Other viruses like alphaviruses, encephalitis viruses, do not use this mechanism, but open the CNS blood vessels (blood brain barrier) to penetration by lymphocytes and other inflammatory cells causing collateral damage to the brain. SARS-CoV-2 has the potential of following all these neurological pathogenic pathways: damaging blood vessels in the brain to let in inflammatory cells; damaging or altering cells that support neurons (glia); and causing autoimmune responses in the CNS. With as many people as have been infected, all of these are likely to be seen, at one time or another, no matter how rare. Oddly enough, the CNS may provide a way to truncate the disease so mild COVID-19 does not turn into severe to lethal or chronic disease. The answer may be in a class of drugs, Selective Serotonin Re-uptake Inhibitor (SSRI), for anti-depression and anti-anxiety. New data shows fluvoxamine (FLV; Luvox), one such drug, could keep mild COVID-19 from worsening. In a preliminary study, 65 patients who received fluvoxamine (50 mg twice daily) and 48 who did not, the results were the incidence of hospitalization being 0% (0/65) with fluvoxamine and 12.5% (6/48) with no treatment. At 14 days, residual symptoms persisted in 0% (0/65) with fluvoxamine and 60% (29/48) with no treatment, (David Seftel and David R Boulware, Prospective cohort of fluvoxamine for early treatment of COVID-19, published by Oxford University Press on behalf of Infectious Diseases Society of America, 2021, and https://jamanetwork.com/journals/jama/articlepdf/2773108/jama_lenze_2020_pc_200006_1607119471.77989.pdf). Fluvoxamine has a well-established mechanism of action as an anti-inflammatory (Dorian A. Rosen et al, Modulation of the sigma-1 receptor−IRE1 pathway is beneficial in preclinical models of inflammation and sepsis, Sci Transl Med 11, eaau5266. DOI: 10.1126/scitranslmed.aau5266, 2019; https://stm.sciencemag.org/content/scitransmed/11/478/eaau5266.full.pdf). The related SSRI drug Fluoxetine (Prozac) has a long history of anti-inflammatory action against stress-induced oxidative cell damage; https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4876141/pdf/PRP2-4-e00231.pdf.

The ER stress sensing protein IRE1alpha, and the closely related protein IRE1beta, are able to powerfully affect the inflammatory behavior of both immune and non-immune cells. A major ER stress sensor, inositol-requiring enzyme (IRE1) is selectively activated by the Toll-like receptor 4 (TLR4) for the ligand lipopolysaccharide (LPS), bacterial endotoxins. IRE1 regulates inflammatory cytokine production via both its endonuclease activity and transcriptional regulation. IRE1 activity is required for cytokine production, likely via XBP1-mediated transactivation of interleukin-6 (IL-6) and tumor necrosis factor–alpha (TNF-alpha). FLV, an antidepressant drug, has low-nanomolar affinity for S1R, ER-resident protein sigma-1 receptor, which has also been reported to have anti-inflammatory properties. In one set of experiments, a high dose of LPS (6 mg/kg) was administered with FLV (20 mg/kg) at the same time. FLV treatment protected mice from mortality and reduced serum IL-6. S1R can also dampen inflammation in human cells. Heparinized peripheral blood from healthy donors was stimulated in the test tube with LPS (10 ng/ml) in the presence or absence of FLV (20 micromolar), and the production of inflammatory mediators was measured. FLV significantly reduced LPS-induced IL-6, IL-1beta and IL-12, and decreased IL-8 production in cells from all donors. These data indicated that the anti-inflammatory action of the FLV S1R ligand is conserved across species. Taken together, these anti- inflammatory effects and the benefits to COVID treatments, correlate with what I have said about the inflammatory effects and mechanisms of COVID in earlier posts and provides hope these off-the-shelf drugs will provide relief to both acutely severely affected patients as well as long haulers with neurological symptoms.

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With the reported rise of mutants with increased transmissibility and potential immune escape supported by epidemiological and clinical data, the search for a panacea which will cover all strains of SARS-CoV-2 has become a frenzied pursuit. Such haste and grasping any favorable observations, no matter how tenuous, have inundated social media, the press, and placed undue pressure on medical institutions to modify and scale back their warnings about such unsubstantiated treatments and prophylactics, https://www.nytimes.com/interactive/2021/health/coronavirus-mutations-B117-variant.html. The problem is balancing the specific with the non-specific. The former decreases adverse side effects, such as anaphylaxis and Guillain-Barré syndrome (GBS; neurological weakness and paralysis), but is subject to mutation leading to neutralization of effectiveness. The 1976 flu vaccine, I took while in the military and which was falsely associated with GBS, rose public concern and misconceptions and fears because of a historic small increase in GBS risk associated with the vaccine, which was later found to be lower than initially thought. Concerns about vaccine-related GBS risk persist despite comprehensive surveillance of influenza vaccine-related increases showing only one additional case of GBS per million flu vaccinations. In contrast, the risk of flu-related complications is 17 per million cases. A recent study showed no link between COVID-19 and Guillain-Barré syndrome https://www.cidrap.umn.edu/news-perspective/2020/12/study-finds-no-link-between-covid-19-guillain-barr-syndrome and https://academic.oup.com/brain/advance-article/doi/10.1093/brain/awaa433/6031905. The most common trigger for GBS is infection with Campylobacter jejuni, a bacterium that causes gastroenteritis, or infection of the digestive tract, the most common cause of food borne illness. Therefore, benefits of vaccination far outweigh the risks. The use of broad spectrum, non-specific potential treatments have more adverse side effects associated with them, and diluted effectiveness.

What are we up against? Three mutants V367F, W436R, and D364Y, have arisen in Wuhan, Shenzhen, Hong Kong, and France, which have demonstrated higher human ACE2 affinity. This is because of enhanced stabilization of the receptor binding protein (receptor binding domain, RBD). High frequencies of RBD mutations have been identified: V367F with five mutations from France and one Hong Kong mutant, V483A, and G476S mutant from the USA. Lineage B.1.1.7 (also called 501Y.V1) is rapidly spreading in southeastern England. It has 17 mutations which developed prior to its detection in early September 2020, which indicates prior evolution, possibly in a chronically infected patient. B.1.1.7 expanded when SARS-CoV-2 cases were widespread and achieved dominance by outcompeting an existing population of circulating virus, natural selection of virus more transmissible. Dr. Joel Ernst, an infectious disease expert at UCSF, has stated the B.1.1.7 UK variant has been found in 22 states so far, including 72 cases in California, according to the Centers for Disease Control and Prevention.

The variant in South Africa was first detected in Nelson Mandela Bay in samples dating back to the beginning of October 2020. South Africa showed a sharp rise in the number of infected during November 2020, and reported 501Y.V2 to the World Health Organization on 18 Dec 2020. More than 1.3 million people have been infected with the virus in South Africa, highest of any African nation, more than 37,000 South Africans have died from COVID-19. Most new infections are caused by the 501Y.V2 variant.

Mutations have significantly changed the South African virus’ spike protein; the spike protein has now rotated about twenty degrees and can therefore get more deeply into the binding site on cells; thus, the virus can bind better to human cells, enabling it to become a more efficient virus for transmission. The South Africa variant is about 50% more contagious than the original strain. The Western Cape region took 107 days to reach 100,000 cases in the first wave compared to 54 days in the second wave, when the variant appeared. The South African variant has spread to 14 countries. The director of the Centers for Disease Control and Prevention said, 29 Jan 2021, that the South African Covid-19 variant, which was just detected in two people in South Carolina, had already reached the point of community spread in the U.S. On 9 Jan 2021, Japan notified WHO of a new SARS-CoV-2 variant B.1.1.28 detected in 4 travelers from Brazil. This variant has 12 mutations to the spike protein, including 3 mutations in common with 501Y.V2, ie, K417N/T, E484K and N501Y, which potentially can change transmissibility and host immune responses. Brazil has also reported emergence of a similar variant, with E484K mutation, which evolved independently of the variant from the travelers.

By mid January 2021, Dr. Charles Chiu, director of the UCSF-Abbott Viral Diagnostics and Discovery Center, from sequencing results from his lab at UCSF, found a rare variant, that Chiu had only seen in a handful of samples, suddenly jump up to 25% in his samples. Chiu’s team was the first to report the new variant, now called L452R, but within days, diagnosticians at Cedars-Sinai reported the same variant was now making up more than a third of cases in Los Angeles.

These variants suggest specific antiviral measures may be sidestepped by their changes if the measures are targeted too specifically to an antigen. I stated this in an earlier post about monoclonal antibody driving escape mutations by selection pressure. This has been borne out by recent clinical studies. Treatment with bamlanivimab and etesevimab, monoclonal anti-spike antibodies to different epitope targets, of nonhospitalized patients with mild to moderate COVID-19 illness, compared with placebo, showed statistically significant decrease in SARS-CoV-2 viral load at day 11; however, no significant reduction in viral load was seen with bamlanivimab monotherapy. The South African strain has also demonstrated escape from such treatment. It has been observed to completely escape from three monoclonal antibodies, reported by scientists from three South African universities, with the National Institute for Communicable Diseases (NICD), in a paper published on the bioRxiv website (https://bit.ly/2Y0lHEt). In addition, 501Y.V2 showed substantial or complete escape from neutralizing antibodies from COVID-19 convalescent plasma. They concluded reinfection could occur, and the differences in the variant may lead to reduced efficacy of current spike-based vaccines (New COVID-19 Variant Defeats Plasma Treatment, MABs, May Reduce Vaccine Efficacy – Medscape – Jan 20, 2021). Such Dior predictions, have led individuals and physicians to pursue broad-spectrum treatments which make such variations irrelevant.

Didier Raoult’s tarnished claim hydroxychloroquine and azithromycin were effective against COVID-19 has not completely died because of this need for a generalized treatment, even in the scientific community. In a recent ACS paper, the investigators used hydroxychloroquine (HCQ) as a reference template to screen for structural similarity against a library of approximately 4000 approved drugs. The top-ranked drugs, based on structural similarity to HCQ, selected for in vitro antiviral assessment, were zuclopenthixol and nebivolol, which blocked SARS-CoV-2 in vitro infection with EC₅₀ values in the low micromolar range. The anti-SARS-CoV-2 potential of ambroxol, amodiaquine, and its active metabolite (N-monodesethyl amodiaquine) were discussed. They concluded both the pK_a (acidity) and the HCQ aromatic core play a role in antiviral activity. These are such broad mechanisms that such studies are academic and may have too many in vivo variable interactions to be clinically relevant (Giovanni Bocci et al. ACS Pharmacol. Transl. Sci. 2020, 3, 6, 1278–1292; publication date: October 14, 2020, https://doi.org/10.1021/acsptsci.0c00131). Unfortunately, another anti-parasitic drug, Ivermectin, has been revisited as a general antiviral after 50 years of study that have not translated into such clinical use https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7251046/pdf/210_2020_Article_1902.pdf and https://www.nature.com/articles/s41429-020-0336-z.pdf. Among the many mechanisms of action of Ivermectin, the most relevant to antiviral activity is as an inhibitor of nuclear transport mediated by the importin α/β1 heterodimer, responsible for the translocation of various viral species proteins. Unfortunately, most of the viruses it is supposed to effect are not inhibited in replication or infection, or at least consistently. For HIV type 1, a study showed that Ivermectin failed to control the nuclear accumulation of telomer repeat factor-1 (GFP-TRF) which can only be transferred by IMPβ1 to the cell nucleus disputing the proposed mechanism. Researchers concluded that Ivermectin is not a specific inhibitor for IN-IMP α/β interaction, but it appears to be a specific inhibitor of cargos that are dependent on the heterodimer, making its predicted antiviral activity in a host even more suspicious. Law of diminishing returns seems to come into play with Ivermectin treatment. Virus out runs capacity. In pigs with circovirus 2, in the first 24 hrs, Ivermectin reduced viral load by 41% and 28.2%, However, in 48 hrs, it reduced viral load 28.8% and 15.7%, at the same treatment concentrations. Evidently, it decreases in drug efficacy at later time points of infection. In a recent study among adults with mild COVID-19, a 5-day course of ivermectin, compared with placebo, did not significantly improve the time to resolution of symptoms. The findings did not support the use of ivermectin for treatment of mild COVID-19 https://jamanetwork.com/journals/jama/articlepdf/2777389/jama_lpezmedina_2021_oi_210022_1614808764.76432.pdf. In spite of all its years of use in Veterinary Medicine, to prevent heartworms in dogs and other worms in livestock, I have never heard of a veterinarian using it to treat viral infections. Also, I don’t see Ivermectin listed in the Overview of Antiviral Agents, Pharmacology, in the Merck Veterinary Manual. For Ivermectin to be effective against SARS-CoV-2, in cell culture, the concentrations had to be in the microgram per ml range, blood levels of Ivermectin are at safe therapeutic doses in the 20–80 ng/ml range.

The National Institutes of Health (NIH), under pressure from clinicians, has dropped its recommendation against the inexpensive antiparasitic drug Ivermectin for treatment of COVID-19, and the agency now states it can’t recommend for or against its use, leaving the decision to physicians and their patients. “Results from adequately powered, well-designed, and well-conducted clinical trials are needed to provide more specific, evidence-based guidance on the role of ivermectin for the treatment of COVID-19,” according to new NIH guidance released recently. Some clinical studies showed no benefits or worsening of disease after its use; however, others reported shorter time to resolution of disease. This may be tied to viral load or just an unreliable, unpredictable response.

Some have already failed clinical trials (such as hydroxychloroquine) and some seem counterintuitive (like the immunosuppressant cyclosporine) so caution must be taken in extrapolating these results and they only be considered for suggesting possible clinical testing candidates. They found nine drugs with antiviral activity in lung epithelial cells: 7 of the drugs have been used in humans, 3 FDA approved in the United States (cyclosporine, dacomitinib, and salinomycin), and ebastine (an anti-histamine) approved outside the United States (https://www.cell.com/action/showPdf?pii=S2211-1247%2821%2900273-4). Some of the immunosuppressive and ant-histamine anti-SARS-CoV-2 activities may be explained by the linkage of the host response to the virus to the peculiar anti-parasite, anti-tumor immunity and its adverse effects in COVID.

Other non-immune-mediated drugs are being considered. The government is paying Merck & Co about 356 million USD to fast-track production of one of its potential treatments under Operation Warp Speed. The funding will allow the company to deliver up to 100,000 doses by 30 June 2021, if successful and FDA clears the treatment for emergency use. It is called MK-7110, designed to minimize damaging effects of an overactive immune response to COVID-19. Another promising drug is plitidepsin (aplidin), 27.5 times more potent than remdesivir in cell culture. The mechanism of action against SARS-CoV-2 is through inhibiting eEF1A, a key cellular protein in proliferation and growth (the eukaryotic elongation factor 1 α, eEF1A). During protein translation, eEF1A recruits aminoacyl t-RNA to the ribosome and translocates the growing polypeptide from the ribosomal A site to the P site. Therefore, the drug inhibits viral protein production and, subsequently, viral replication. It’s potential toxicity, because of this non-specificity, is on protein synthesis, growth and healing. The viral production was inhibited at 8-12 hours but was not significant after 24 hours of infection. Therefore, it must be used early in infection based on these in vitro and animal studies https://science.sciencemag.org/content/sci/early/2021/01/22/science.abf4058.full.pdf. Recently modeling efforts and in vitro cell culture studies have examined repurposing other FDA approved drugs as antivirals to treat COVID. These include the old anti-tapeworm drug niclosamide. It has shown broad efficacy against viruses including SARS-CoV-2. This salicylanilide’s antiviral activity is based on its ability to prevent acidification of endocytotic compartments, blocking membrane fusion and infection of the host cell. Early viral studies showed that the efficacy of salicylanilides was derived from the anilide ring, the important role of the N− H group in shuttling protons into the endosome. Niclosamide’s reduction of infection in cells without interacting directly with any viral components presents the possibility of anti-COVID-19 therapeutics with broad activity against current and future variants of the virus, but also may dilute out its effectiveness as seen with antihelmintics like ivermectin and increase the possibility of side effects (Steven Blake et al., Salicylanilides Reduce SARS-CoV‐2 Replication and Suppress Induction of Inflammatory Cytokines in a Rodent Model, https://doi.org/10.1021/acsinfecdis.1c00253
A, ACS Infect. Dis.).

How do we reconcile broad-spectrum, non-specific treatments to counter natural selection of viruses resistant to specific antibody or cellular immunity against requirements to avoid off-target and diluted-out effects of non-specific treatments ? First, the current vaccines may induce broader coverage immunity than illustrated by monoclonal antibody or convalescent plasma responses to infection. Even the difference between male and female immune responses, females having more effective T cell cellular immune responses than males’ B cell dominated antibody response, indicates broader immune coverage from the same SARS-CoV-2 antigens https://science.sciencemag.org/content/sci/371/6527/347.full.pdf. The Th17 response to COVID-19 also indicates a more generalized polyclonal immune response. So don’t write off the current vaccines yet. Second, I have alluded to, in earlier posts, that aptamers which can be selected synthetically to many targets on the virus and be changed rapidly, can also link these anti-ligands to specific pre-existing antibody or immune cells to rapidly mobilize the immune system against the virus and even convert the pathogenic virus into an autogenous endogenous vaccine after directly neutralizing it. The original route of administration was by inhalation to enable self-application of the treatment/prevention. However, these approaches are yet to get out of the basic research laboratory.

HeLa-NR1cells producing and extruding biosynthetic clusters of polymer nanoparticles (white) capable of carrying cell-synthesized RNA

January 31 is the anniversary of my receiving my PhD and embarking on my One Health career. The following excerpts are from my book collection, The Black Dragon Trilogy. They are not just history. They are for now, especially in the time of COVID along with all my previous posts, and show how relevant my studies have been to COVID-19 and more to come. Acts 2:17 : “In the last days,’ God says, ‘I will pour out my Spirit upon all people. … Your young men will see visions, and your old men will dream dreams.” Everyone eventually faces their last days.

I give my own professional life story not to win your sympathy, admiration, or brag, but to show that there are different, no less authentic, paths than the academic one to great discoveries and these should not be discarded by society but judged on their stand-alone-merit. After all great things have come out of a little patent office in Switzerland. One Health is my story. The acceptance of a message should not be based on the authority of the messenger but the content of the message.

“I was eager to return to academia, to research. Because of my financial limitations, I decided to commit longer to the USAF by entering the Air Force Institute of Technology Civilian Institutions Program to obtain my PhD. I wanted one in biochemistry because I thought the molecular approach was the future of microbiology and the response to infectious agents, immunology. The USAF wanted me trained as a field microbiologist to go to Indonesia or Egypt and serve in one of the Navy field infectious disease surveillance laboratories for encephalitis virus or blood flukes (schistosomiasis) in those countries respectively. The Navy does not have veterinarians or veterinary specialists so at that time the USAF provided them and later the Army. I convinced the USAF program manager that I could get a double interdisciplinary PhD in both biochemistry and microbiology in 3 years (the limit on the length of the program for each student). I entered Texas Tech University Health Science Center, School of Medicine, Graduate School, in the Departments of Biochemistry and Microbiology, as one of their first PhD students, in the summer of 1977 at Lubbock, Texas. My dissertation research was on the cytotoxic mechanisms of peroxidases, a principle antimicrobial and anti-parasitic enzyme in granulocytes and macrophages of animals and humans. I wanted to study the effects of eosinophilic peroxidase on blood flukes (an animal counterpart of Schistosoma called Heterobilharzia americana) but my graduate committee and the safety officer would not approve of my keeping my infected snails in an aquarium in the laboratory. Therefore, I turned to peroxidase killing cancer cells. Most of the research at that time, in the laboratories there, was focused on cancer treatments. I found the Novikoff hepatocarcinoma in rats an available and convenient model already being studied in the Biochemistry Department. My biochemistry professor was studying immobilized enzymes, so I decided to immobilize peroxidase and glucose oxidase on implants in rats with this cancer. The usually fatal cancer was cured in most cases if the implants were placed at the right time and place in the rats. This led to a patent with the USAF and university. However, except for legal action and hard feelings over the patent rights, not much ever came of this laboratory “curiosity”. In 1979, just prior to me finishing my last year of my PhD, I was commissioned as a regular officer Captain in the USAF. Soon after, the Vet Corps of the USAF was dissolved, leaving the only Vet Corps being in the Army. I was transferred to the USAF Biomedical Science Corps. The powers to be tried to get me to transfer to the Army Vet Corps and gave me the tour of USAMRIID, the US Army Medical Research Institute of Infectious Disease, to entice me to transfer, but I decided not to transfer. I had to go to my next assignment at Brooks AFB, Texas, the School of Aerospace Medicine, at San Antonio, Texas. I had not finished writing my dissertation at the end of the 3-year term at Texas Tech so I requested a 6 month extension because I knew that I might not be given the time to complete it at my new assignment and get my degree. I finished my dissertation and graduated from the TTHSC in Jan 1981 with my interdisciplinary PhD in biochemistry and microbiology. My new research assignment was not in microbiology or biochemistry. I was to become a veterinary research officer in radiobiology, specifically non-ionizing radiation bioeffects, the bioeffects of radar, microwaves and radiofrequency radiation. I spent the first few weeks trying to find out what that was by reading Professor Ted (E H) Grant’s book. “The Dielectric Behaviour of Biological Molecules in Solution” (Oxford University Press, 1979). I worked in this area from 1981 until about 2000, sponsored primarily by the Air Force Office of Scientific Research. In 1994, I received the Air Force Basic Research Award for work on the mechanisms of radio frequency radiation bioeffects. I stayed involved in Veterinary Microbiology and passed the board exams for the American College of Veterinary Microbiologists, with prior tutoring from some of my professors and colleagues at Texas A& M Vet School, Dr Syed Naqi and Dr Richard Hidalgo (most recently at the Louisiana State University School of Veterinary Medicine at Baton Rouge. LA). I became a consultant to the Surgeon General of the USAF in Veterinary Microbiology (I believe the last one). This involvement was put to use in 1989, just in time, to develop a field assay for determining the viability of anthrax spores, for use in the first Iraqi War. In 2002, I became the Senior Scientist for Counterproliferation and Electromagnetic Effects of the then Air Force Research Laboratory, Brooks AFB, Tx. I retired 6 May 2011, forced retirement, after I decided not to move to Wright Patterson AFB, Ohio, as part of the Base Realignment and Closure Act implementation closing Brooks City-Base (formerly Brooks AFB). So, ended the tenure of the last Senior Scientist for Counterproliferation and Electromagnetics Bioeffects in the USAF. After retirement, I became an independent consultant serving the Defense Threat Reduction Agency in Biological Agent Defeat and in the Cooperative Biological Engagement Program in Azerbaijan (until a disagreement in approach with the prime contractor forced me to resign) and Kazakhstan until 2015. I was also accepted as a Charter Diplomate of the newly boarded specialty of Veterinary Parasitology in April 2011. I will not go into the details, but since my retirement I tried on many occasions to get employed by the USAF electromagnetic research group as a contractor or annuitant temporary employee and contacted many of my old colleagues in academia looking for a job in research, but no one would even respond or give me an interview.”— The Black Dragon Trilogy by JOHNATHAN KIEL
https://a.co/e2PwEwK

My professional mission was to stop biological agents, whether nefarious or natural, in non-permissive territory, not letting them enter the US or its territories, and to protect our military, and by extension, civilian populations, by developing detection, isolation and identification methods and neutralization and decontamination technologies and ways to confirm the kill. COVID has become my worst case scenario of an uncontrolled strategic biological agent (weapon). I had hoped “I would be welcomed back to the fight” and that “This time I know our side will win,” but my participation looks like it is not to be, even for a patriot.

My final guidance written so it may not be forgotten: “These books approach the same place, the convergence, where Pathogenic Ecology intends to be, where nature has maintained these pathogens in sylvatic and wild cycles for millennia awaiting the disturbances which lead to epidemics and pandemics among humankind and their domestic animals.”— The Black Dragon Trilogy by JOHNATHAN KIEL
https://a.co/barCq6l.

This post is a summary in lay, non-technical terms, of what we know about COVID and the virus that causes it, amongst its kind. I will couch this description in military terms and as if the virus enemy is a sentient being because of my military background. The analogy is only for illustration and not proof of what I say. Our story, in late 2019, early 2020, began with a foreboding of the consequences of our happy hurried ignorance of larger life and the cost of practicing ignorance of it when confronting it head on : “Violent Delights, Violent Ends: Fire and Powder, Kiss and Consume”, “I fear too early, for my mind misgives some consequence yet hanging in the stars shall bitterly begin this fearful date with this night’s revels, and expire the term of ….. life ….. by some vile forfeit of untimely death….” William Shakespeare, Romeo and Juliet, Act 1, Scene 4”. There is an elegance in design in what we have faced and experienced when we look past the undeniable tragedy, which should never be forgotten as a great battle is remembered, which we must admit we have lost to learn from it, in our ongoing war with microbes, and the missteps that led to it. We started with underestimating our enemy. After all, most physicians’ experience with coronaviruses is the cold-causing, self-limiting, take-an-aspirin-and-call-me-in-morning ones successfully treated by benign neglect. Even the serious ones like SARS 1 (Severe Acute Respiratory Syndrome Virus, from civet cats, originally from bats), which appeared in 2003 and disappeared in 6 months, and MERS (Middle East Respiratory Syndrome Virus, from camels, originally from bats), which has only spread slowly in the Middle East and Northern Africa, were largely ignored as harbingers of something worse to come. In our arrogance and ignorance, we thought they disappeared or were restricted because of expert human interference; we ignored the warning of “some consequence yet hanging in the stars” of something much worse. Not only physicians fed this false security (besides the inevitable politicians at the top), but also veterinarians, who thought coronaviruses were under control. We have seen coronaviruses pervasively in dogs, cats, cattle and birds, but largely of the young, from self-limiting, and diminishing as agents necessitating prevention by vaccination, especially in dogs, to vaccination effective, but not fully optimal control (focusing on the pregnant females or newborns to produce immunity even if transient or against disease but not infection), in preventing large economic losses in intensive bovine, porcine, and poultry production. This requirement makes their use routine and necessary in agriculture, especially in preventing respiratory disease losses in poultry, and losses in calves and pigs from hemorrhagic diarrhea and shipping fever complex in finishing out beef cattle. However, in birds, live attenuated vaccine viruses have been detected circulating in wild bird populations, leading to potential recombination with or mutation to pathogenic strains. Only in the virus that causes Feline Infectious Peritonitis (FIP), which is usually 100% fatal in young cats showing signs but showing few signs, if any, and self-limiting when the kittens are infected with a less lethal strain of the virus (most often), was there evidence of something persistent and on occasion much more lethal. Presently, veterinarians are attempting to treat FIP with the same antiviral drugs as COVID over very protracted times, prolonging life but struggling to get cures. Not even this foreshadowing of the enemy becoming capable of new more insidious and dangerous strategies was heeded. One health appeared to learn nothing from these experiences until the stealthy tragedy of COVID was upon the world. The FIP lesson of a virus that is highly infectious and widespread in close collections of cats, which could largely be ignored and not even worth vaccination, could on occasion, when symptomatic, kill with such lethality, was attributed solely to a rare emergence of a selected highly lethal strain. However, although it appears again and again, it never seems to get an epizootic foot hold, killing only a few or one cat in a close knit group. Where does it go? Why does it keep coming back in such a limited way? No very satisfactory answers. But it surely appears to have an uncanny, but not as well developed, resemblance to the COVID modus operandi—myth of a largely non-symptomatic disease, low overall mortality, until in reality it becomes severely symptomatic and then lethality jumps sky high. These characteristics feed the denial and false claims of inflated lethality for COVID. Unfortunately, this feeds more careless spread of the virus. Could this be its evolutionary “intent” ? The major strategy change of the COVID virus from the others, especially animal ones (although all coronaviruses are zoonotic, the cold ones appearing to have originated in rodents, cattle and some unknown animal eons ago), has been to sparing the young of disease, although still being asymptomatic in most age groups, except the old and infirm. Its infectivity in cats, minks and ferrets, says SARS-CoV-2 (originating in bats) is still very much an animal virus. It still holds in common with all coronaviruses the goal of not burning through a host population so as to prevent herd immunity and always to have susceptible individuals in that population to maintain them indefinitely. Even in the most densely populated and numerous mammals in nature, perhaps only second to humans, bats, coronaviruses only infect about 10% of a given group at a time. The viruses, including SARS-CoV-2, continue to evolve by mutation and selection toward being more efficient at infection and transmission within close, tight social groups but falling off quickly when moderate barriers, like masks and good ventilation, and social distancing are implemented. Pathogenicity and lethality tend to be held in check except in individuals in a population that do not contribute to maintenance of the virus in the population, like the elderly and infirm who do not reproduce individuals vulnerable to infection and transmission. Why is COVID different from animals in these respects of populations? The answer may lie in that animal viruses establish carriers by preferring gastrointestinal infections that are not easily cleared and chronically shed transmissible virus. In other viruses, like influenza, especially in migratory birds and water fowl, it is predominantly a GI rather than respiratory virus, which most effectively maintains the birds as a reservoir rather than incapacitating or killing the birds, which would limit their ability to spread the viruses. The COVID virus has chosen a more unorthodox method to assure maintenance and transmission in the human population, although with time I believe we will find more and more asymptomatic GI shedders (by monitoring sewage which has begun) https://onlinelibrary.wiley.com/doi/full/10.1002/jmv.25825 and https://www.medrxiv.org/content/10.1101/2021.01.08.21249379v1. It has chosen to enhance anti-parasite immunity like that used by the hosts to control parasitic worm, pathogenic protozoan and fungal infestations and infections. This response often controls the number of parasites to a tolerable, but not immediately lethal, level and prevents further infestation (a process called premunition), but does not totally eliminate the parasite. In children, this form of innate non-specific immunity often rapidly clears an infection without inducing long term immunity. Therefore, they are allowed to survive, grow and reproduce more naive vulnerable individuals after spreading the virus in the population. They are also vulnerable to re-infection as they grow older. Adults can mount this type of immunity but then develop specific adaptive long term immunity, but unfortunately also “memory” of the initial innate response with a vengeance. If this “remembered” non-specific response becomes overt or is activated, as it is highly vulnerable to do, by non-viral stimuli, it can lead to autoimmunity and pro-inflammatory nonspecific disease, which has been observed in “long haulers”. The fatal severe COVID is a continuum of the infection from inapparent widespread tissue infection to a late activation of this parasite immunity to the extreme, death, without a break. This type of incomplete immunity is beneficial for resisting parasites and in tumor immunology because it is highly effective locally, but suppressed systemically to avoid collateral damage to normal self tissues.

I have observed an artificial reproduction, perhaps enhancement, or at least a mimic, of this type of reactive oxygen species innate immunity early in my research career. I described it in my first book . “Even though weaker, nonspecific cytotoxicity was expected with glucose oxidase and horseradish peroxidase, they were used. Very malignant Novikoff hepatocarcinoma (including metastases) in rats, spontaneous mammary adenocarcinomas in rats, L1210 leukemia in mice, B16 melanomas in mice, and carcinogen-induced squamous cell carcinomas in hamster cheek pouches all responded to regional or local injections of the cross-linked enzymes in saline and glucose solution. The Novikoff hepatocarcinoma responses were 50 to 100% cures in animals treated. Lactoperoxidase could be substituted for horseradish peroxidase, and similar results were obtained. However, galactose oxidase substitution or the absence of either the glucose oxidase or the respective peroxidase in the preparation failed to bring about tumor regression. L1210 leukemia in outbred white mice showed 50% cures. Treatment of B16 melanoma prolonged life from 23 days postinoculation of controls to 37 days with treatment. However, no simple dose-response curve was demonstrated..Also, smaller squamous cell carcinoma tumors responded better (some with cures) than did larger cheek pouch tumors, which did not respond at all..No direct injury to normal tissue was observed in histopathological sections of treated animals. However, some animals with heavy tumor loads (P388 lymphoma, L1210 leukemia, some with Novikoff hepatocarcinoma) died more quickly when treated than did untreated controls due to an apparent “necrotic crisis” from dying tumor tissue.”— Type-B Cytochromes: Sensors and Switches by J.L. Kiel. https://a.co/aJEAVsl. I was acutely aware of the relationship between oxidase/peroxidase innate non-specific immunity and resistance to parasites and cancer because I entered Texas Tech University Health Science Center, School of Medicine, Graduate School, in the Departments of Biochemistry and Microbiology, as one of their first PhD students, in the summer of 1977 at Lubbock, Texas, with the intent to study this anti-parasitic mechanism. My dissertation research was on the cytotoxic mechanisms of peroxidases, a principle antimicrobial and anti-parasitic enzyme in granulocytes and macrophages of animals and humans. I wanted to study the effects of eosinophilic peroxidase on blood flukes (an animal counterpart of Schistosoma called Heterobilharzia americana) but my graduate committee and the safety officer would not approve of my keeping my infected snails in an aquarium in the laboratory. Therefore, I turned to peroxidase killing of cancer cells.

This highly localized approach is necessary because parasites and cancers disguise themselves as normal host tissue and this type of immunity must attack them without confusing them with normal tissue, which could be fatal or at least highly damaging to normal tissues and organs. It is a very delicate balance that could go either way. Therefore, any treatments or vaccines must direct a response that limits early replication of the virus and do not trigger an overt systemic parasite immune response. Hopefully, the current vaccines and treatments currently being evaluated will do this.

As a final personal note, I dealt, in my professional lifetime, with trying to address these mechanisms in other infectious zoonotic diseases and where infectious agents come from, go to between outbreaks, and how to stop them from re-emerging and spreading. By my measure, what has happened with COVID-19 would have been a total failure if it had been in my purview of investigative and applied functions and responsibilities. I spent the first third of my career investigating the non-specific responses to chemical, biological, and radiation insults, especially non-ionizing electromagnetic radiation, microwaves and radiowaves, and the second two thirds investigating means to detect hidden infectious agents and to stop re-emergence of infectious agents from between outbreaks. You may question my authority and knowledge in this area and assume I should have some honored and respected position in academia or industry after my retirement from serving the military and nation. I do not. It appears the arts and sciences of attribution and Counterproliferation of biological agents, natural or nefarious, are so specialized that there is no venerable honored academic or industrial positions or no longer governmental positions that meet with my qualifications. Since my retirement in 2011 and being turned down many times for academic positions, I have only been called on by the federal government when emergency situations required my services and was summarily dismissed when the crisis passed. However, when the crisis occurs, as with COVID, is when my talents are least but albeit still critically effective if heeded. I thought that even at 71, I might be valuable in sharing my experience, knowledge and insights with those who need to follow me in these pursuits, which I did in the military, but am not allowed to do now except by my books, past publications and currently this blog. I will continue as long as one person reads this blog and am able to continue.

How can symptoms of COVID-19 persist after the virus is gone? The answer may lie in the type of immune response triggered by SARS-CoV-2, which resembles one more familiar to those studying the immune response to worm to protozoan parasites. I mentioned this in earlier posts citing observations of clinical characteristics of COVID and how they resembled the response to a parasite rather than a virus. Parasite immunology more closely resembles tumor immunology than that of responses to many viruses and bacteria. This cross reactivity between tumor immunology and the immune response to SARS-CoV-2 has been dramatically illustrated in a recent case of a rare remission of advance Hodgkin lymphoma in conjunction with COVID (https://onlinelibrary.wiley.com/doi/10.1111/bjh.17116). The host must mount a different kind of response to parasites because of their many strategies to evade the immune system, the innate and adaptive components. Therefore, animals and humans have developed a different series of cellular and humoral components unique to parasites, but often with potentially severe collateral damage to normal tissues and organs. There is a delicate balance between controlling a parasite infestation so that it does not consume the host and so completely destroying the parasites that the host “burns down the house to get rid of the rats”, auto destruction of the host in the process of eliminating the parasite and confusion between parasite antigen and self antigens (autoimmunity) https://downloads.hindawi.com/journals/iji/2014/651503.pdf. The remaining “memory” of this response, after the virus is gone, is continuously reactivated by a broad range of stimuli https://jlb.onlinelibrary.wiley.com/doi/epdf/10.1189/jlb.0313113. This primitive process, discussed in my book Type B Cytochromes: Sensors and Switches and cited in previous posts, is built on anticipatory responding to potential pathogens, toxins and physical insults before those insults are actually known or specifically identified. This is good for evolutionary survival but can lead to unnecessary overt nonspecific responses and collateral damage. What is the evidence for such a hypothesis in regards to COVID-19 and how might it lead to treatments, especially for long haulers? As stated in a previous post, the immune response of hospitalized COVID-19 patients falls across a spectrum of immune responses in 3 categories: (1) antiviral (some macrophages and dendritic cells, natural killer cells, cytotoxic T cells, helper and suppressor T cells, antibody making B cells and plasma cells, and gamma interferon; other interferons made by tissue cells: alpha and beta); (2) anti-parasitic granulocyte white blood cells (some special B cells that make IgE, eosinophils, basophils (in tissue mast cells), and all kinds of biochemical mediators: histamine, Prostaglandins, leukotrienes, and kinins); and (3) anti-bacterial and fungal (granulocyte: neutrophils, and monocytes and macrophages). COVID-19 can move through all three types. The worst and most likely cause of the long hauler syndrome is type 2 which was meant for worms and other multicellular localized parasites and can be locally severe and if generalized, fatal. The serum concentrations of IL-17A (a protein that recruits monocytes and neutrophils to an inflammatory site produced by Th17 lymphocytes) and IFN-γ, but not TNF-α or IL-6 (the pro-inflammatory culprits), decrease with age of patients (this might argue against Th17 memory cells being the source of long haulers); therefore, gamma interferon’s ability to prevent viral replication but not induce oxidative damage to host cells, as with TNF-α, is more important in children’s responses. However, as stated in a previous post, evidence supports decline in soluble IL17A receptors, which block TNF-alpha and interferon gamma production by competing with the cellular T17 cellular receptors, leads to more severe COVID19 (supporting Th17 cell involvement). Adults show a more robust T cell response to the viral spike protein compared to pediatric patients shown by increased expression of CD25+ on CD4+ T cells and the frequency of IFN-γ+CD4+ T cells. Also, serum neutralizing antibody titers and antibody-dependent cellular phagocytosis are higher in adults than in pediatric COVID-19 patients. The neutralizing antibody titer increases with age while the IL-17A and IFN-γ serum concentrations decrease. Children seem to be resistant to the severe effects, except in some instances when they display multiple organ inflammation, Kawasaki-like syndrome, because they produce a lot of the anti-inflammatory cytokine IL10. The responses of neonates compared to adults change from increased IL-10 as neonates to balanced IL-10/T helper type 1 (Th1)/Th2/Th17 cytokine levels early in life. This allows protection from pathogens but reduces the chances of severe inflammatory reaction. Th17 cells are the most probable mediators in many human autoimmune and chronic inflammatory disorders, including psoriasis, Crohnˈs disease, rheumatoid arthritis, Multiple Sclerosis, and uveitis https://www.frontiersin.org/articles/10.3389/fimmu.2018.01112/full. The development of pathogenic Th17 cells relies on signals from multiple cytokines: IL‐1, IL‐23, and IL‐6. TGF‐β (transforming growth factor) can promote Th17 development and induces a Th17 response which promotes intestinal muscle hypercontractility that drives worm expulsion. TGFβ-activation by dendritic cells yields this Th17 induction and intestinal contractility and facilitates the expulsion of the parasite Trichinella spiralis in mice. This unique immune response to intestinal helminth expulsion beyond that of classical Th2 driven immunity, highlights the importance of IL-17 in parasitic disease. Th17 protection against parasites is very broad spectrum extending to Th17 cells’ providing robust protection against Trypanosoma cruzi, the intracellular protozoan parasite that causes Chagas disease. Parasites that establish long term chronic infections suppress Th17 immunity by modulation of antigen-specific CD4+ T responses. This is seen in human infection with the worm parasite Strongyloides stercoralis https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4546867/pdf/nihms-704134.pdf. Th17 cells of infected people showed significantly decreased responses upon antigen-stimulation, but following treatment, significantly increased the antigen-specific responses of Th17 cells. Th17 cells produce pro-inflammatory cytokines, including IL-17A, IL-17F, and IL-22. This cytokine production not only promotes accumulation of immune cells, such as macrophages, neutrophils and lymphocytes, at inflammatory sites, but also can cause collateral tissue damage. Other Th17 cells can negatively regulate immune responses by secreting immunosuppressive factors, such as IL-10 as noted in the response of children to COVID. Th17 cells are required for vaccine-induced protection from pulmonary infection with the pathogenic fungi Coccidioides posadasii, Histoplasma capsulatum, and Blastomyces dermatitidis, fungal pathogens causing systemic mycoses in humans. IL-17 is required for neutrophil and macrophage recruitment to the lungs. This balance is evidently what helps dispose of or suppress parasites and perhaps SARS-CoV-2 virus, or, if out of balance, severe acute or chronic persistent long hauler pathologies https://www.thermofisher.com/us/en/home/life-science/cell-analysis/cell-analysis-learning-center/immunology-at-work/t-helper-17-cell-overview.html. Some in vitro experiments suggest a benefit from Vitamin D because of its effects on Th17 cells. These cells are characterized by the chemokine receptor CCR6, RORC expression, and production of IL-17A, IFNγ, and TNFα. Using rheumatoid arthritis (RA) as a model of autoimmune disease, investigators demonstrated that pro-inflammatory memory CCR6+ Th cells can switch into anti-inflammatory cells by treating them with the active vitamin D metabolite 1,25(OH)₂D₃. Memory CCR6+ Th cells were sorted from healthy controls or treatment-naïve patients with early rheumatoid arthritis (RA) and cultured with or without 1,25(OH)₂D₃. Vitamin D₃ inhibited pro-inflammatory cytokine production, IL-17A, IL-17F, IL-22 and IFNγ, in memory CCR6+ Th cells from both healthy controls and RA patients. This inhibition was accompanied by induction of anti-inflammatory factors, IL-10 and CTLA4 https://www.frontiersin.org/articles/10.3389/fimmu.2019.01504/full. Perhaps, this approach provides a means to test the hypothesis that Th17 memory cells are the root cause of long haulers. Also, antioxidants may help because the pro-inflammatory cytokines enhance the production of reactive oxygen species which are mediators of the anti-parasitic effector mechanism and collateral pathology. These benefits remain to be seen.

The anti-parasitic immune response leading to long haul and neurological disease has gained further support in a new paper showing the involvement of the cytokine CCL11, eosinophil chemotactic protein and eotaxin-1 (https://www.biorxiv.org/content/10.1101/2022.01.07.475453v1.full.pdf).

Concerns over the safety of vaccines and effectiveness of non-pharmaceutical control measures have led to and will lead to worse outcomes for COVID, especially in the US, where such resistance has led to tragic devastation. Therefore, any over interpretation of worst case scenarios will lead to self-fulfilling apocalyptic outcomes. This can only be prevented by open and honest assessment of risk/cost/benefits of any such approaches https://www.medrxiv.org/content/10.1101/2021.01.29.21250653v1.full.pdf. The latest most concerning challenges to vaccines are their potential to instigate autoimmunity, allergic reactivity, their ability to enhance infectivity, and their loss of effectiveness because of the appearance and rapid spread of mutant strains of SARS-CoV-2. Another more common place and older concern for attenuated vaccines is their possible reversion to pathogenic forms. I will start with this last concern first because it has some well-documented clinical examples. The earliest was with the first vaccine, smallpox vaccine, cowpox or now vaccinia (DNA from an early 1902 sample of smallpox vaccine showed that it was 99.7% similar to horsepox virus, now extinct). This vaccine is a close attenuated relative of smallpox virus, usually only causing localized lesions, skin vesicles, followed by erosions and ulceration, at most resulting in a single scar. However, it can cause generalized vaccinia, with widespread reddening, inflammation and pealing of the skin and on very rare occasion heart damage and even death (eczema vaccinatum, accidental inoculation, and progressive vaccinia). The spread of the virus from inoculation sites during active infections to others, especially amongst children, was common in the past. But we must never forget that without this vaccine, smallpox could have never been eradicated. We would still be living with its up to 30% mortality and life-long disfigurement.

A lesser known reversion was seen with Venezuelan Equine Encephalitis modified live virus vaccine, TC-83 virus, was developed as an anti-biological warfare vaccine in the early 1960’s at USAMRIID (US Army Medical Research Institute of Infectious Diseases) and transitioned to a commercial horse vaccine in the 1970’s and eventually was replaced with a killed vaccine. VEEV was attenuated into TC-83 by 83 serial passages in guinea pig heart cells. It was first tested extensively in horses during the 1971 Texas VEE epizootic. Although the vaccine produces viremia, fever and leucopenia in horses, neutralizing antibodies are generated with protection from VEEV challenge. TC-83 produces seroconversion in about 80% of humans (neutralizing antibody titers ≥20) but mild to moderate flu-like symptoms. TC-83 is the result of only 2 attenuating mutations among the 12 mutations that accompanied in vitro passage of the Trinidad donkey strain. These mutations could revert in vaccinees, which also presented a risk of epizootic amplification. To produce a more stably attenuated VEEV vaccine candidate, the virulent Trinidad donkey genome was cloned in cDNA and attenuated by inserting either point mutations or a PE2 cleavage-signal mutation combined with an E1 gene resuscitating mutation. The latter strain, called V3526, is safe and immunogenic for mice and nonhuman primates, and appears superior to TC-83 in rodents. V3526 has a lower risk for environmental transmission and distribution. TC-83 was transmitted by mosquito vectors and was isolated in mosquitoes collected in Louisiana during the 1971 VEE epizootic/epidemic. Efforts were made to improve its safety by genetic modification to prevent its ability to infect insects. This recombinant virus further attenuated TC-83, but was completely unable to infect mosquito cells or mosquitoes in vivo. Currently, commercial equine vaccines marketed in the U.S. consist of inactivated TC-83, often combined with inactivated eastern and western equine encephalitis viruses.

“The small and simple VEEV has one more trick which it shares with the plant Tobacco Mosaic Virus; it can resurrect itself from broken pieces. The nsP4 sequence in VEEV is very similar to the replicases of three plant viruses, including Tobacco Mosaic Virus. The Brooks Lab performed a critical experiment using the RetroXpress (TM) system of Clontech derived from a retrovirus which could transfer genes but could not infect and replicate, my group at Brooks constructed a new virus that contained the nsP4 sequence inserted into this platform and expected that the new virus could infect cells but not replicate. It not only infected (transfected) marker genes into the target mammalian cells but also formed plaques of transformed cells (those which resembled cancer cells and formed uninhibited growth mounds of cells). These grew and multiplied in the cell culture. A new complete infectious virus had formed. How? Tobacco Mosaic Virus has been shown to repair and reactivate after exposure to UV with the help of an unknown plant nucleic acid repair system (Multiplicity Reactivation: Recombination). Since we inserted only the nsP4 protein into the host cells co-infected with the defective retrovirus, we proposed that the new virus used cellular RNA dependent RNA polymerase in conjunction with the expressed nsP4 to form a repair complex which constructed a chimera virus from the retroviral pieces and other unknown viral nucleic acid components inherently present. Perhaps, the infections by killed virus from killed vaccines previously reported were not just because of contamination with whole live virus but with components which survived the formalin damage and were reassembled into whole restored infectious virus, albeit at a less effective infectious dose of original broken pieces. Intact VEEV will produce a transmissible infection with as little as 12 PFU (plaque forming units equivalent to 10 virions for a multiplicity of infection of 1 but more likely 10X10 = 100 virions). I have speculated that these broken viruses, if reconstituted from broken virions or free RNA would require 10e6 units to make one intact virion.” — The Black Dragon Trilogy by JOHNATHAN KIEL. https://a.co/gtIO9aR

The third example of a problematic attenuated live vaccine still causing trouble is Sabin modified live polio vaccine. The first, vaccine-associated paralytic poliomyelitis (VAPP) was recognized in the early 1960s. VAPP is a very rare and occurs sporadically when a dose of oral live polio vaccine reverts to neurovirulence and causes paralysis in the vaccine recipient or a nonimmune contact. VAPP can be in immunologically normal vaccine recipients and their contacts as well as among persons with immunodeficiencies, defects in antibody production. There were live vaccine-associated outbreaks in Madagascar and South Sudan in 2014. Eight newly identified persons in six countries were found to excrete vaccine virus, and a patient in the United Kingdom was still excreting the virus in 2014 after more than 28 years. Reported by ProMed on 15 Dec 2020 were three new polio cases in Sierra Leone for the first time in 10 years. The cases were recorded in Kambia, Tonkolili, and Western Area Rural District. The cases were not from the wild poliovirus strain, but were vaccine derived. Only 2 countries worldwide continue to have wild poliovirus transmission: Pakistan and Afghanistan; thanks to worldwide polio vaccination. Because of violent resistance to polio vaccination in these two countries, polio persists and cannot be eradicated. Killed Salk polio vaccination is now once again being used with no chance of reversion because it is a killed vaccine, as the risk of live Sabin vaccine now out weighs its benefits.

The severe reactions to vaccines (anaphylaxis) are extremely rare for humans and animals and are expected to be no worse for COVID vaccines. They are often, when they do occur associated with adjuvants and additives, rather than the primary immunogen, except for perhaps some vaccines in animals, like that for leptospirosis (actually a bacterin).

Autoimmunity is related to allergic reaction but distinct, especially in regards to SARS-CoV-2 pathology. I have discussed the immune response to the virus in detail in previous posts. As a reminder, the virus has the unusual association of autoantibody against interferons as a marker of severe disease. Also, unlike other animal coronaviruses, SARS-CoV-2 is more severe in adults than children, in general. The animal ones are more severe in the young than old. What is a plausible explanation, or hypothesis? Children have a more robust innate immune response, including interferon, than an adaptive immune (antibody) response than adults, but they have higher amounts of circulating soluble interleukin 17 receptors which help reduce the cellular innate pro-inflammatory response mediated in immune cells through membrane-bound IL-17 receptors. I have pointed out, in an earlier post, that the uncontrollable innate immune response yields reactive oxygen species that cause considerable local collateral host cell damage and death. Therefore, taking out interferon, and the subsequent wide distribution of infectious virus, then leads to antibody against virally infected cells which directs antibody-dependent cell cytotoxicity, causing a whole host inflammatory response, and collateral cellular, tissue to whole organ damage and failure. The pro-inflammatory activity of whole virus leads to a by-stander adjuvant effect which can activate forbidden B cells to produce the self antibody, i.e. anti-interferon. In adult immune systems, having seen more related viruses than the naive immune systems of children, this is more likely to happen. A good humoral and cellular immune response, triggered by a vaccine directed against specific antigens that direct neutralizing antibody response avoids this outcome because it takes out virus before it can spread widely in the body.

This line of thinking leads to a more recent disconcerting response to the virus that involves a minor antibody directed to a critical part of the binding portion of the viral spike protein (S1) https://www.biorxiv.org/content/10.1101/2020.12.18.423358v1?fbclid=IwAR3D1MJ-SyW9wLeqrRV6pdSP6ToUwpaLjbB5LK2oA4B0b9iYjJ4RTvYs_PQ. The paper provides evidence that minor antibodies from COVID patients’ serum bind to part of the S1 spike protein that enhances the binding to ACE2 viral receptors and uptake of infectious virus in cell culture in established cell lines. They further raise issue that a vaccine could enhance viral uptake and disease by extension of this observation to the whole human. Unfortunately, such a stretch will provide support to those who oppose vaccination. This leap may be moot from the get go because patients have circulating soluble ACE2, derived from proteolytically cleaved membrane bound ACE2. First, there is no evidence that any vaccine enhances viral infectivity in vivo. Second, these soluble receptors would first see and soak up systemic virus, especially if its binding were enhanced by the antibody. Then, immune cells would take up these immune complexes and dispose of the virus. In addition, the antigens selected for the vaccines are specifically selected to optimize induction of neutralizing antibody.

The vaccines released for use so far have had exceptional efficacy in generating neutralizing antibody. The fear of a mutation in SARS-CoV-2 has come to fruition but those so far such as in mink, have increased efficacy of transmission and infection but not pathogenicity. Coronaviruses have a large RNA genome and can therefore undergo more mutations without significant functional change than smaller genome RNA viruses. They also have a proof reading polymerase to reduce replication mistakes (mutations) unlike other error prone RNA viruses like HIV. However, they can undergo selection pressure for advantageous mutants, when necessary, generated by mutations mediated by Nsp14 exonuclease in independent coronaviruses, resulting 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 quasi species when needed. Therefore, there is a continuous tension balance between the polymerase and the exonuclease to only produce mutations that are favored by environmental selection pressure. The latest of greatest concern, VOC 202012/01 variant, occurred in the UK. This variant has a mutation in the receptor binding domain (RBD) of the spike protein at position 501, where the amino acid asparagine (N) has been replaced with tyrosine (Y). The shorthand of the mutation is N501Y, also designated as S:N501Y, because it is in the spike protein. This variant carries many other mutations, 14 amino acid (AA) mutations, 6 non-AA altering mutations, and 3 deletions, notably including 69/70 deletion: this double deletion has occurred spontaneously many times, and likely leads to a change in the shape of the spike protein; P681H: near the S1/S2 furin cleavage site, a site with high variability in coronaviruses, which has also emerged spontaneously multiple times; ORF8 stop codon (Q27stop), a mutation not in the spike protein but in a different gene (in open reading frame 8), the function of which is unknown. Similar mutations have occurred in the past such as in Singapore, in which one strain with this type of mutation emerged and disappeared. Preliminary analysis in the UK indicates this variant is significantly more transmissible than previously circulating variants, with an estimated potential to increase the reproductive number (R) by 0.4 or greater with an estimated increased transmissibility of up to 70%. There is no indication, at this point, of increased disease severity associated with the new variant. A few cases of this new variant have to date been reported in Denmark and the Netherlands. Another similar mutant has arisen in South Africa, with structural changes in the spike protein, which is still being evaluated for impact. However, in a recently released study, pending peer review, the Pfizer vaccine has been shown to still induce neutralizing protection to the mutants https://www.biorxiv.org/content/10.1101/2021.01.07.425740v1.full.pdf. One must remember that mutations in the spike protein are not limitless. They must conserve essential structure that allows binding and entry of the virus https://science.sciencemag.org/content/sci/371/6526/284.full.pdf. The ACE2 viral receptors do not mutate as fast as the virus so they limit what variations in the virus are still tolerable for infection. If coronavirus behavior is to hold true, the most efficiently transmitted virus with the lowest mortality necessary to eliminate less than ideal virus replicating hosts, and that which does not preferentially infect much more than 10% of the susceptible population, especially when simple non-pharmaceutical methods as distancing, good ventilation and wearing of masks, are so effective, will prevail to maintain itself in the general population indefinitely. This persistence at a low percentage of the susceptible population is the nature of coronaviruses in animals.

One last note, the Brooks USAF AFRL CPT groups’ seeking of aptamer-based anti-pathogens to bind toxic components, inhibit target cell binding and uptake, and re-direct existing immunity, natural or vaccine induced, rapidly toward new, emerging and re-emerging pathogens, including viruses, was designed to address outbreaks like COVID without always having to resort to the Herculean efforts that have been necessary to develop COVID vaccines. The failure or inability to apply the same efforts, as we have had to to rapidly develop COVID vaccines, to the insidious smoldering problem of antimicrobial (antibiotic) resistance was also the motivation for developing this new technology, as well as to relieve the costs and socioeconomic impacts of addressing these problems by conventional, albeit modern technological, versions of biomedical approaches.