Global Biosurveillance

The initial vaccine candidates from Pfizer and Moderna are both mRNA vaccines, if approved they would be the first mRNA vaccines ever approved. mRNA is an unstable molecule that breaks down when exposed to significant temperature fluctuations, even at ultra-low temperatures. mRNA vaccines require ultra-low temperatures of -70˚ to -80 for storage and shipment. In vaccine form, the importance of mRNA’s temperature stability is greatly amplified. This is an example of advanced biotechnology leading medical practicability: a demanding cold chain difficult to achieve even in the US and advanced countries but impossible in developing countries, not even considering that these requirements will drive the expected cost through the roof. https://www.laboratoryequipment.com/569702-Hurdle-1-Develop-COVID-19-Vaccine-Hurdle-2-Deliver-it-at-Ultra-low-Temperatures/.

Because of the mRNA vaccine ULT demands, Pfizer designed temperature-controlled shipping containers which use dry ice to keep the vaccine vials at -75˚C for 10 days. These are roughly the size of a suitcase and weigh about 70 pounds when fully loaded. However, a shortage of ultra-low temperature (ULT) freezers is anticipated because they are not usually available in most clinics, creating an added expense and probable loss, waste of vaccine, especially since federal distribution of cold-storage vaccines is anticipated to be sent in 1,000-dose shipments. The rush to purchase ULT freezers could lead to a national shortage, hampering distribution of such vaccines. AstraZenca and Oxford’s vaccine, which may be released next, needs -20˚C temperatures to ensure efficacy, a little better.

The developing world will need vaccine volume provided by 2 to 3 approved vaccines. They may even have to wait for a liquid vaccine, or at least a -20˚C vaccine that is widely available (still problematic in many countries and remote regions). The logistics of the frozen mRNA vaccines are nearly impossible for developing countries. The Associated Press estimates vaccine storage issues could leave 3 billion people in developing countries without access to a coronavirus vaccine. Are there other subunit nucleic acid vaccines that are more stable? Yes. DNA vaccines https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1986720/.Messenger RNA (mRNA) molecules provide the templates in the cytoplasm of a cell for translation by the ribosome and tRNA (amino acid transfer RNA) into protein, making multiple copies of the protein from each mRNA template. This amplification provides a multiplication of the vaccine immunogen per molecule compared to providing individual protein molecules. However, offsetting that advantage, in addition to the instability of mRNA, only about one of 10,000 molecules of mRNA will escape into the cytoplasm after being taken up by a cell. Amplification by translation of mRNA into protein has to overcome the losses and the inefficiencies of degradation and the translation to protein. Unlike plasmid DNA, which must enter the nucleus of the cell, mRNA only needs to be present in the cytoplasm, which eliminates the additional barrier, the nuclear membrane, that plasmid DNA has to cross. However, plasmid DNA is more stable than mRNA, and each DNA molecule results in the production of multiple mRNA molecules, thus the theoretical advantages of one over the other is found in the net stability of plasmid DNA versus mRNA, as well as differences in efficiencies of targeting the proper cells, movement to the cytoplasm or nucleus followed by transcription of the plasmid DNA (transcription from DNA to mRNA), and the translation of mRNA, whether transcribed from DNA or translated from mRNA, to protein https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6631684/. The Brooks Counterproliferation Team had to address these problems and others to achieve field durability for DNA aptamers, their vectors, and resistance to degradation in transit in the field and even methods to manufacture them in place in the field as necessary on demand.

In the military, we always considered durability and survivability of all technology used against biological agents so it could be used under adverse conditions with little or no logistical tail, minimal technical skill to operate or maintain safely under the most adverse conditions. Of particular concern was the area of NBC (Nuclear, Biological, Chemical) defense, aircraft and WMD (Weapons of Mass Destruction) neutralization (that is decontamination of aircraft and preventing the Special Forces aircraft from becoming contaminated in the first place by using appropriate NBC neutralization, and NBC sensor, monitoring, technologies). All these detection, isolation and identification technologies had to answer these questions: Is the agent present? What might it be? And is it safe to bring back a sample without contaminating the Special Forces aircraft so that it can’t land in permissive territory after exiting non-permissive territory with the agent in hand? Aptamers, used to isolate, identify and neutralize biological agents in place of antibodies and to turn infectious agents into autogenous vaccines by neutralizing their pathological mechanisms in an infected individual therapeutically, could be self-delivered by inhalation. This approach would have extreme operational utility when an attack occurred and could save many military by safe administration of the aptamers, quickly, that required no special handling or even cold storage. All the technologies were measured by their ease of use under adverse conditions, an approach which seems to be lost on SARS-CoV-2 vaccine developers, but which is absolutely necessary to distribute and effectively apply vaccines quickly worldwide to quell this pandemic.

A “system of systems”: The following are examples of military ruggidized technologies for use against pathogens under adverse, limited field conditions:

Controlling the spread of SARS-CoV-2 within buildings is tied to air quality, flow, dilution with outside air, filtration, and active decontamination. A recent webinar was held discussing how buildings can be made safer in the presence of SARS-CoV-2: Heating, Ventilation, and Air Conditioning Strategies for COVID-19 by Federal Facilities Council, Wed, October 28, 2020, 1:00-2:30 pm CST. The bottom line was that dilution of internal air with frequent external air intake exchanges is the “gold standard” to reduce levels of indoor infectious virus, but depending on outside air temperature and humidity characteristics, conditioning could prove expensive, especially for old systems in aging buildings. Measures which achieve comparable air quality to dilution were discussed. Filtration is the next best and cost effective, if the proper filter is chosen, installed in the right part of the HVAC system and installed properly so air doesn’t go around it. It effectively reduces numbers of airborne infectious particles which, in turn, reduces the exponential decay effect and increases the efficacy of active decontamination methods. The most cost effective filter is rated MERV 13, as long as it has not achieved this rating by being electrostaticly charged (when the charge is lost as with condensation, the filter rating drops to 6-8). Particles that were of most concern were around 500 nm because, as I discussed in the post on aerosol vs airborne, these are not subject to the described mechanisms of deposition and can enter deep into the respiratory system. The presentation stated these made up 90% of the air suspended particles for up to 41 hours, but did not indicate what portion of original expelled particles these represented (since the range in absolute numbers is indeterminate). Previously reported averages in that post were around 800 nm aerodynamic diameter.

According to the American Society of Heating, Refrigerating and Air-Conditioning Engineers (ASHRAE), this filtration helps provide healthier indoor air quality.

Minimum Efficiency Reporting Values, or MERVs (Minimum Efficiency Reporting Values)

UVC (254 nm wavelength) was considered the most effective active decontamination technology. The presenters stated that UVC should be positioned downstream of the cooling coils, to kill 90 % of the virus and to keep clean condensation pans from Legionella bacteria. UV lamps were also recommended to be placed in ceilings out of eye and skin damage range (radiation drops off 1/re2 with distance from the source). My experience with such lamps is they continue to visibly shine even after they have lost much of their UVC output, and that output is drastically reduced by dust accumulated on the bulbs. Besides the hazard and that not enough UVC for the required dwell time may reach infectious particles to fully inactivate a dose in transit, in spite of controlled optimized laboratory experiments demonstrating high efficacy, they fall short in prolonged field use unless closely monitored and maintained.

Examining UV dose of disinfection in a precise way, the inactivation ratio is determined by the applied UV dose through the disinfecting unit. The UV dose (mJ cm−2) is calculated by the delivered irradiance or fluence rate to microbial cells (mW cm−2) multiplied by the exposure time (s). Therefore, for UV- induced reactions, the most accurate report of the kinetic data is related to UV dose rather than time. If there were no confounding factors, the disinfection of biological agent contaminated air and surfaces with UVC could be straightforward and predictable. The required UV dose for 90% viral inactivation is increased by 1.5−2 times for a surface compared with air due to microscopic aggregation on surfaces. Other surfaces have microscopic irregularities, crevices, and roughness which counter the line of sight killing of the virus or microbe on that surface. A similar shadowing effect in air treatment can be caused by the presence of larger particles and dust in the air stream. In addition, the microbial cells may be attached and agglomerated on dust, which require higher UV doses for inactivation. One study indicated 120 mJ cm−2 of UVC dose is required to reduce the tissue culture infective dose (TCID) from 3.8 × 10e7 to 180 TCID50/mL, which is a more than 99.999% reduction. Based on this observation, SARS-CoV-1 (and most likely SARS-CoV-2) is similar to influenza virus and hepatitis A virus in being eliminated by UV radiation https://pubs.acs.org/doi/pdf/10.1021/acsphotonics.0c01245.

Another active decontamination method discussed at the webinar was Photocatalytic Oxidation (PCO), commercially available hydroxy-free radical generating systems that are 95% effective and “smoke eaters” which destroy VOCs, volatile organic compounds. in general. At Brooks, we tested our own in-lab-manufactured device under field conditions to efficiently destroy biological warfare stimulants.

Since hydroxyls result from water vapor, hydroxyl generation requires significant relative humidity in the air. One such commercial system, HYDRA PRO XL solves the low-humidity problem by generating and transferring water vapor to a direct injection manifold inside the machine which evenly distributes the vapor over UV lamps to maximize hydroxyl production https://www.boie.us/hydra-pro-xl.php.

In addition to this PCO mode, the HYDRA PRO XL’s has three AOP modes which can double to triple hydroxyl free radical production. They are low concentration Hydrogen Peroxide Vapor (HPV), low concentration UV Ozone (O3/UV) and a combination process. In the PCO mode (Safe Mode), the HYDRA PRO XL uses water, UV light and a catalyst, titanium oxide, to safely generate hydroxyl free radicals which remove organics causing odors, kill bacterial spores and pathogens. PCO mode can be used with or without the included water vapor injection system, depending on site conditions. The resulting byproducts are harmless carbon dioxide and water.

PCO hydroxyl generation, originally developed by NASA, is ozone-free and safe to use indoors for continuous operation in unventilated spaces for as long as it takes to remove odors and break down contaminants.

Contaminated air is drawn through an odor-neutralizing pre-filter and a HEPA filter to trap most particulates. Then, the unit uses six germicidal (253-nm UVC) lamps in a 2-layer crosshatch pattern along with 3 layers of nano-catalyst to produce hydroxyl radicals which breakdown volatile organic compounds (VOCs) and other contaminants that pass through the unit’s air flow. The next mode, AOP Mode 1, uses vaporous hydrogen peroxide in a direct injection system instead of pure water. The resulting low-concentration hydrogen peroxide vapor (HPV) breaks down into hydroxyl free radicals when exposed to the unit’s germicidal UV light without requiring the catalyst. To initiate AOP Mode 1, one adds a commercially-available hydrogen peroxide solution to the unit’s direct injection reservoir and runs the Advance in PCO mode. PhotoCatalytic reactions will occur simultaneously. No ozone is generated in Safe Mode, however, HPV levels in the air must be monitored if workers are present. This AOP is the simplest method to generate hydroxyl radicals using UV light: Hydrogen Peroxide / UV Light:
H₂O₂ + UV —› 2 •OH

To maintain safe air quality they must be tested routinely for ozone production. Ionizing air systems from producing airborne ions to plasma-induced active (singlet) oxygen and monoatomic oxygen species are also effective but also must not produce ozone. Ozone is a poor decontamination component in the output of these systems when compared with hydroxyl radicals, singlet oxygen or monoatomic oxygen or thermal electrons generated at the plasma source.

As noted in an earlier post, the initial amount of an infectious agent greatly influences significantly surviving amounts of the agent, regardless of method of active decontamination, based on the conversion of the deactivation of the agent from linear to exponential decay. The following graphs show such a conversion, not with amount of anthrax spores, although this has been seen with large amounts of anthrax spores exposed to ionizing radiation, but with the addition of other wavelengths of light (visible) to the UV exposure. This is not biological repair but chemical passive repair which could occur in viruses or by the viral genome being reassembled from damaged pieces into infectious virus once they enter host cells. The damage by UV or higher energy electromagnetic radiation, like X-rays and gamma radiation, is dependent on unobscured line of sight and intensity, partially dependent on the particulate nature of all electromagnetic radiation, photons. Also, target theory plays a role; the smaller the RNA or DNA genome the more photons, intensity, required to destroy or damage individual viral genomes, to inactivate potential infectious doses in the environment.

In our investigations at Brooks, we discovered some viruses’ ability to reconstruct themselves from broken pieces, albeit from high numbers of original virus, even following UV damage. “The small and simple VEEV (Venezuelan Equine Encephalitis Virus, a previous biological warfare agent) 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. 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.”

As discussed in an earlier post, cold plasma devices, which generate active oxygen species from air, require no additional chemicals only energy, but at present, are not as developed for commercial use (Sloan, M.A., Vivekananda, J., Holwitt, E.A., and Kiel J.L. U.S. Patent 7,892,484. Methods and Compositions for Neutralizing Anthrax and Other Bioagents, 22 Feb 2011).Water vapor or humidity does favor plasma gas production from the nanoparticle/pulsed microwave version. The humidity levels, which favor PCO and certain cold plasma generation, have also been shown to shorten the environmental life of SARS-CoV-2 , alone, (40-60% relative humidity), but humidifying a building holds its own unique set of problems—big challenges are the condensation problem, thermal breaks and vapor barrier.

Much confusion has arisen about when to test, what are the best tests, what do the results mean for SARS-CoV-2. The biggest lie is that they are the reason the numbers have risen so fast lately. If we don’t see it, we can’t report it, nor can we even begin to control it. The highest level of separation of what tests tell us is the distinction between incidence and prevalence of infection. The frequency (number of health events in a population and it’s relationship of that number to the size of the population is prevalence) and pattern (the occurrence of health-related events by time, place, and person is incidence) of health events in a population. COVID is a dynamic moving target for diagnostics and when target levels are measured in the course of the infection is critical. When the USAF Biological Counterproliferation Team at Brooks was still active, we told commanders and general officers that the only way to make an informed decision about a biological attack was to run three orthogonal tests (measuring different aspects of a biological agent by different methods). If only two were run, then they should have a coin to flip to make a decision. The third test was a tie breaker in case one of the other tests were positive and the other negative. The fundamental tests are (1) isolation, culture and identification, (2) PCR (polymerase chain reaction, for SARS-CoV-2, real time reverse transcriptase PCR, RNA to DNA to complete the reactions), (3) antigen testing (measuring parts of virus other than RNA with specific indicator labeled antibody), and finally, (4) measuring antibody levels in serum with a specific viral antigen, and sometimes, measuring type of antibodies with second labeled antibody https://www.nature.com/articles/d41586-020-02661-2. The last one is the least useful for early diagnosis, except if IgA or IgM antibody is measured. As noted in an earlier post, IgA, and/or IgM decline within 3 months. It is the IgG, particularly IgG3, that lasts longer: 7 months and the memory B cells even longer.

Culture is dependent on taking samples from actively shedding sites and blocking contamination, especially bacterial with antibiotics, and isolating specific plaques showing cytopathic effects (CPE) on at least partially selective host cells. However, such CPE may vary from very subtle host cellular changes to out right lysis, leaving holes in a single continuous mono layer of host cells in culture. The difficulties are obvious, and multiplying virus in the lab is hazardous https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7036342/pdf/jkms-35-e84.pdf and https://www.protocols.io/view/culture-of-the-severe-acute-respiratory-syndrome-c-bcduis6w.

PCR is easier and only requires isolation of the RNA in this case, removing inhibitors or performing viral lysis and release of intact RNA in the process. However, it measures genomic equivalents of virus, if quantitative, but for most commercially available tests, only threshold of detection, but not necessarily infectious virus. Fortunately, most viral RNA is resistant to ubiquitous environmental RNA degrading enzymes. The threshold for a positive is the number of PCR replication cycles to produce detectable DNA copies, usually less than 35 to 30 cycles (Ct, cycle threshold) of the PCR machine. Cts < 29 are strong positive reactions indicative of abundant target nucleic acid in the sample. Cts of 30-37 are positive reactions indicative of moderate amounts of target nucleic acid. Cts of 38-40 are weak reactions indicative of minimal amounts of target nucleic acid which could represent an infection state or environmental contamination.

In a PCR study of 396 patients, time until negative conversion was significantly shorter in the asymptomatic group than in the symptomatic group: median 14.5 days (11.0–21.0 days) and 18.0 days (15.0– 22.0 days), respectively. Rebound of thermal cycle threshold of detection values (Ct) was seen in 78 patients (19.7%). Time until negative conversion is shorter in asymptomatic COVID-19 than in symptomatic COVID-19. Rebound of Ct values was common https://www.ijidonline.com/action/showPdf?pii=S1201-9712%2820%2930620-2.

The antigen tests measure the outer proteins of the virus and must be sampled, isolated from binding inhibitors, and detected in a tube or chromatographic surface, by trapping in a precipitate or along a diffusion path and labeling it specifically, usually with a second antibody in addition to the trapping one, with a colorimetric or fluorescent reagent. This test can be quantitative in a micro titer well plate (replacing tubes) by making dilutions to extinction, or below threshold of detection. The bed-side immunodiffusion rapid tests are only positive or negative tests. The course of an infection or the quality of the sample and its quantitative viral content control the outcomes https://www.ijidonline.com/action/showPdf?pii=S1201-9712%2820%2930658-5. One study showed a high sensitivity and specificity in samples mainly obtained during the first week of symptoms and with high viral loads. The rapid antigen assay has the potential to become important for early diagnosis of SARS-CoV-2, especially in situations with limited access to molecular methods. Sensitivity and specificity were 93.9% (86.5–97.4%) and 100% (92.1– 100%), respectively, with a diagnostic accuracy of 96.1%. Sensitivity was significantly higher in samples with high viral loads https://www.ijidonline.com/action/showPdf?pii=S1201-9712%2820%2930405-7.

Seroprevalence is most useful in determining the vulnerability of a population as opposed to the incidence, a rate, showing the most immediate infection progression.

To show how difficult the gold standard for viruses, isolation and culture, and converting these numbers into quantitative PCR is, I give work we performed at Brooks on adenovirus, a human virus causing everything from mild, cold-like symptoms to death and with asymptomatic human carriers, depending on the strain, as an example. It is a much more durable virus than SARS-CoV-2 and can easily be spread by fomites and airborne secretions. We investigated its transmission by shared gas masks used for training and supposedly disinfected, because of some deaths from the virus amongst recruit trainees (Establishment of Optimal Parameters for United States Air Force MCU-2/P Mask Sanitization Procedures to Effectively Neutralize Adenovirus Serotype 14).

From the initial copy numbers generated during the RT-PCR reaction, the titer (ge/mL: genomic equivalents per milliliter) of each unknown sample was calculated as shown below:
Copy Number 1 X 10e3 μL Titer = 9 μL x 10 x Dilution Factor x mL
Titration methods fall into one of two categories: determination of either the infectious or the particle (infectious plus noninfectious) titer. To overcome this problem, the current RT-PCR assay was developed to detect only encapsidated viral genomes. Nonencapsidated and/or incomplete genomes were eliminated during the DNase treatment (to destroy free DNA not extracted from intact virus) step, more accurately reflecting the infectious titer.

Adenoviral genome equivalents/mL following elution from mask pieces exposed to 181 ppm of bleach and/or detergent. M=mask, V=virus, D=detergent, B=bleach, W=water

How bad is prevalence of SARS-CoV-2, COVID, in highly vulnerable populations? The kidney hemodialysis population has a higher proportion of older people, men, and people living in majority Black and Hispanic neighbourhoods than in the US adult population. Seroprevalence of SARS-CoV-2 was 8.0% , 8.3% when standardized to the US dialysis population, and 9.3% (8.8–9.9) when standardized to the US adult population https://www.thelancet.com/action/showPdf?pii=S0140-6736%2820%2932009-2. Even for the most vulnerable, the highest prevalence argues against natural herd immunity and more for the typical “animal” coronavirus endemic sustainment strategies that these viruses have evolved with their hosts. For the least at risk populations, the samples examined in a recent study, showed 250 were positive of 33,041 children (age of 0-18 years) without symptoms who were tested at 28 hospitals for SARS-CoV-2 through May 29, 2020. Across the hospitals represented by these children, prevalence varied from 0% to 2.2%, with a pooled prevalence of 0.65% (0.47%-0.83%). Asymptomatic pediatric prevalence was associated with weekly incidence of COVID-19 in the general population during the 6-week period over which testing of asymptomatic children occurred. No other factor (population, number of tests performed, region, testing indication, or sample collection site) demonstrated a significant association with prevalence in asymptomatic infected children. Later data from 15,612 children were compared with prevalence in an asymptomatic pediatric population calculated from concurrent Johns Hopkins University weekly incidence data using a best-fit equation derived from this correlation, and the results showed the correlation persisted at this later time (coefficient, 0.86). Therefore, the incidence in the local general population and contacts should drive testing in children.https://jamanetwork.com/journals/jamapediatrics/fullarticle/2769878.

The general US population incidence has been determined by various methods. As of April 4, 2020, the estimated case count was 5 to 50 times higher than the official positive test counts across the different states. Nationally, the estimates of COVID-19 symptomatic cases as of April 4 were likely in the range of 2.2 to 4.9 million, with possibly as many as 8.1 million cases, up to 26 times greater than the cumulative confirmed cases of about 311,000. When these methods were extended to May 16, 2020, it was estimated that cumulative symptomatic incidence ranged from 6.0 to 10.3 million, as opposed to 1.5 million positive test counts https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7310656/pdf/nihpp-2020.04.18.20070821.pdf. How tests are applied is critical to the value of the data to making true numbers and distribution of the virus and cases of COVID-19.

“What would the simplest and purest form of a Nanobe which could take over another life form and make it a new, different one? The following is such a model of such a transforming synthetic life form which could be made from basic chemicals:”— The Black Dragon Trilogy by JOHNATHAN KIEL
https://a.co/136glXE

Multiple potential applications:

1) Simulants for microbial detection and neutralization, using the Nanobes’ “internal” diagnostics. (2) Test platform for nanotoxicological research. (3) Transfection vector for biomedical research and therapeutics (superior to just re-directing the immune response, as we had previously tried, because Nanobes can be turned “on” and “off” with nonionizing electromagnetic radiation (NEMR)). (4) Against antibiotic resistant bacteria, especially intracellular bacteria (in vivo and ex vivo, in self–sterilizing materials). (5) Against viruses and other parasites (in vivo and ex vivo, self-sterilizing materials). (6) Against cancer (7) RFR nano-directed ablation surgery (8) RFR controlled gene expression (9) Transformation vector for agriculture (10) RFR (and other NEMR) controlled pesticides (11) Transient persistent transformation of crops (For one season; self-limiting desirable trait transfer) (12) Transformation vector for bio energy (13) Enhancing cyanobacterial production of biofuels (14) Enhancing microbial production of materials and feedstocks.

Although CRISPR (clustered regularly interspaced short palindromic repeats) gene editing technology has come like a storm with great promise for therapeutic gene editing ( 2020 Nobel Prize in Chemistry: Emmanuelle Charpentier, at the Max Planck Unit for the Science of Pathogens in Berlin, and Jennifer Doudna, at the University of California, Berkeley), it still has delivery, off- target effect, and control problems (can’t readily be turned “on and off”). Many researchers are vigorously pursuing solutions. Nanobes provide an alternative that is electromagnetically controlled with a fail-safe system. Nanobes could even provide a fail-safe delivery system for CRISPR.

There are also plant agricultural applications

“We tried to address a major problem in agriculture, Greening Disease of Citrus Fruit caused by the bacterium Candidatus Liberibacter asiaticus transmitted by the insect vector the Asian Citrus Psyllid, which has invaded citrus farms of Florida and threatens those of Texas.”… “Aside from using Nanobes to deliver CRISPR in the field, which might be quite complicated, the alternative was to use the microwave and radio frequency absorbing properties of Nanobes with DALM to do the job, combining physics and engineering with biology. Heat treatment eliminates Candidatus Liberibacter asiaticus from infected citrus trees under controlled conditions. This approach would have provided wireless control (using light, microwaves, or magnetic induction to activate the Nanobe heating) which could have been turned on and off with an external switch controlling the energy source. Even if transformation was used as a method as well, if something went wrong, the transformed cells could be killed by raising the energy level to a lethal level, but not high enough to destroy surrounding non-transformed cells in the plant.”— The Black Dragon Trilogy by JOHNATHAN KIEL. https://a.co/0ApfKej

Non-biological applications of Nanobes:

(1) Nano-plasma cleaning of surfaces by E field focusing (2) Nano-plasma etching of surfaces for device construction (3) Ultrawide-band-emitting nano-transmitters/ receivers for RFIDs (4) Nano-magnetically controlled switches and circuits (5) Taggants to track materials (in conjunction with people?)”

“Nanobes not only facilitate this biohacking but provide a convenient delivery system for the products. No longer will this type of biotechnology be confined to highly sophisticated and elite well-funded and institutionally supported labs of the Northeast or California coast of the USA, but they will also be available to all as the personal computer made computer technology and software development availabe to all. There is inherent freedom, progress and danger in such accessibility.”— The Black Dragon Trilogy by JOHNATHAN KIEL
https://a.co/fLu1BHE

• Project summary. 1.Project Title
  Chemically Programmed Immunity and Adaptive Vaccinology
  
  This One Health proposal is based on an invention by the Nobel Laureate Dr Kary Mullis (December 28, 1944-August 7, 2019). The platform consists of nucleic acids (RNA, DNA, or single-stranded or double- stranded of either or both) chemically modified to provide a linker through a nanoparticle or nanofiber with nuclease resistance; the multivalent aptamers bind pre-existing immune cell receptors or natural or induced antibodies through the nanostructures to other aptamers pre-selected in vitro to bind to a moiety on the pathogen of interest. The pre-existing, anti-ligand activity is intended to re-direct an effective immediate immune response (including pre-existing successful vaccination technology) against the pathogen targeted by the in vitro selected nucleic acid binding sequence. This linker construct is in some instances intended to trigger further immune memory for future antibody, cell immune, toll-like receptor, or other adaptive and innate immune responses through a natural cascade, providing for autogenous vaccine development. The second part of this proposed platform consists of a vector delivery system consisting of the linker synthetic nanoparticles or nanofibers and the synthetic nucleic acid aptamers linked in such a way to retain the above activity upon release of the aptamer that will facilitate dermal, oral, nasal, respiratory, or parenteral delivery of the effective nucleic acid. In the best example of this complete platform, the composite will transform or transfect a target cell for the mass production of a biosynthetic counterpart of the fully synthetic composite, which performs the same anti-pathogen, anti-parasite function, allowing for scale-up by fermentation for manufacture or in vivo amplification. Parasite pathogens have been selected for the targets under this proposal because of the difficulty and underserved need for developing vaccines against them and because animal models, and in some cases side-by-side in field zoonotic disease, allow for animal field testing under conditions which are most appropriate for predicting a human response from animal data. This composite structure also allows for tracer technology to be incorporated while maintaining function in order to determine the biological fate of such composites in vivo and in situ.
  2. Platform Description and Rationale:
  The platform has been described in detail in enabling US and international patents and peer reviewed scientific papers. The platform and examples will be briefly described here. The details will be given by reference to save space in this expression of interest. The technology is presented in two complementary parts: (1) the nucleic acid aptamer with a chemically conjugated ligand to recruit an existing immune response to natural or vaccine-derived immunogen attached to an aptamer which links the sequences previously selected for DNA binding in vitro to the target pathogen or parasite; and (2) the synthetic nanoparticle/nanofiber vector to carry the aptamers into the host and target tissues by a convenient port of entry, releasing them at the nidus of infection or infestation. Also, a selection process and ruggedized prototype device for use under field conditions to collect target pathogens and isolate an appropriate binding aptamer for amplification and therapeutic use has been designed and undergone preliminary tests. A nanosprayer for topical and respiratory/mucous membrane delivery has also been developed; however, several types of nanospray delivery systems are now commercially available (for cosmetics). The same aptamers and vectors designed for therapeutics have been initially designed for diagnostics using fluorescence dequenching, thermochemiluminescence, magnetic capture, electron microscopy contrast media, and visible light microscopy staining and marking and diagnostically demonstrated against spotted fever type rickettsia, Bacillus anthracis, botulinum toxin, Francisella tularensis (in the field to confirm collection and isolation), and Shiga toxin. Furthermore, the synthetic vectors have been made which transfect and transform bacterial, animal and human cells, traceable by PCR, gene expression, fluorescence, and staining. The bacterial hosts have shown not only the physical and biological evidence of genetic transfer, but also subsequent production of transferable genetic material and vector biosynthetic polymer and transfer of the genetic material to naïve bacterial hosts and their transfer in the same way for three iterations. This biosynthetic form was also transferable to human and animal cells and detected by PCR. The transfer polymer made synthetically or biosynthetically was diazoluminomelanin, a poly (hydroxy) ortho phenylene, made from 3-amino-tyrosine or tyrosine by nitration, reduction and diazotization. This polymer when activated absorbs a wide range of electromagnetic radiation from UV to visible light to microwave and radiofrequency radiation. The polymer has a high affinity for DNA, protects it from nucleases and radiation damage until saturated (high energy pulsed radiation) then can cut or destructively damage DNA or RNA by free radical generation. The microwave killing mechanism has been demonstrated against anthrax and other bacilli spores. Biosynthetic polymer containing the transferable genetic material is readily transferred to bacteria, animal and human cells.
  
  Phenotypic consequences of nanoparticle composite transformation or transfection and fail-safe system for its destruction have been demonstrated.
  Data for treatment of anthrax in mice using Sterne and Ames strain Bacillus anthracis has been collected. The data for immediate recruitment of the immune system in mice were very robust because although survival of the more pathogenic strain was limited, one must keep in mind that the anthrax was delivered by inhalation at very high multiple lethal doses and the aptamer was delivered most effectively after a 3 hour delay by inhalation and was only against the protective antigen of the anthrax toxin and had to mobilize the immune system within 3 days with total lethality at 4 days. Alpha 1,3 galactose aptamer was the immune linker, but the mice do not make antibody against this epitope and human serum, containing natural anti-gal antibody had to be used to direct the mouse immune system after immunization of the mice with human serum. Independent experiments at the US Army Medical Institute of Infection Disease showed that lung clearance of spores was superior in the presence of aptamer: approximately 67 CFU (colony forming units) of bacilli per gram of lung of a treated mouse vs an estimated 44,600 CFU bacilli per gram of lung of an untreated mouse, a 665-benefit ratio.
  No human in vivo toxicity data are available, only in vitro data with human cells. However, experiments were also performed using the FETAX (Frog Embryo Teratogenesis Assay: Xenopus) procedure, which showed DALM to be non-toxic. Clinical trials with polymeric nanoparticles and DNA vaccines which are very similar to these agents have been performed with favourable results and some are still underway (see references). DNA vaccines have been shown to be well tolerated and safe.
  3. Research and Technical Objectives – Statement of work
  Objectives
  The objectives of this proposal are to demonstrate the effectiveness of the platform technology and delivery systems described in this proposal in appropriate in vitro and in vivo models, safety and efficacy of such a demonstrably effective form against a clinically significant parasitic disease with both natural animal and human correlates, first in clinical animal models then in human clinically derived samples, and finally in human subjects.
  4. Endpoints
  The endpoints are to produce a highly reproducible and clinically relevant, for safety and efficacy, composite nanoparticle aptamer platform which can be readily re-directed toward many infectious and parasitic targets by mere substitution of components without extensive re-testing and field trials.
  5. Target pathogens
  Pathogen 1: Malaria (Plasmodium falciparum and others): There is no current effective vaccine for this disease and resistance to the most effective treatment with artemisinin is spreading. However, natural resistance has been demonstrated in endemic areas in children, but the effective response requires IgM and not IgG and the target antigen galactose-alpha-1,3-galactose. Vaccines that lead to IgG response development will not be as effective as those that direct the immune system toward an IgM response to alpha gal.
  Pathogen 2: Heartworms (Dirofilaria immitis): this disease is not only pervasive in canines across the world, but the current treatment uses the same macrocyclic lactones, to which resistance is developing and spreading, used in human filariasis; macrocyclic lactones are the only class of drugs currently available to treat these filarial diseases, which means the development of resistance would eliminate any effective treatment, and they involve altering or blocking the release of an antigen which interferes with the immune response to these filaria and which is effectively activated upon treatment to eliminate microfilaria. There is a robust antibody response which is interfered with by soluble antigen/antibody complex formation which could be overcome by the appropriate redirection or facilitation of immune targeting.
  Pathogen 3: Dracunculus medinensis, Guinea worm; the hope to eradicate this parasite, now only in Africa, has recently shown signs of failure after finding that the once thought to be rare infection in dogs (over a thousand infected dogs discovered) is now commonly present in them in Chad. This infection of dogs in areas of re-emerging human infection allows for studies of the platform response in dogs under field conditions which would be most relevant to concurrent human disease.
  6. Pre-Clinical Approach:
  The worst case non-clinical/pre-clinical development approach has already been established by previous Department of Defence research on anthrax spore protection using a mouse/Sterne/Ames anthrax spore inhalation and immediate mobilization of the immune system by inhalation aptamer composites and looking for prolonged survival and clearing of inhaled anthrax spores. The dose ranges have been pre-determined by in vitro neutralization molar ratio data (for anthrax toxins but of course not the proposed targets). The general toxicity issues are the activation of toll-like receptor innate immunity and direction toward Th1 vs Th2 immune pathways (i.e., avoiding alpha gal meat allergy-like responses by appropriate cellular immune cell targeting along with parasite targeting) which can be adjusted by making alterations in the nucleic acid sequences of the aptamers. Since the immune response can be recruited during concomitant infections, the reduction in microfilaria or other circulating parasitic forms (i.e., in malaria), we can directly assess the response in an individual human or animal as its own control.
• 7. Clinical development plan and regulatory strategy:
  The strategy for clinical development through Phase I of the proposed pathogens uses constructs which are essentially variations of the same form and these lead to certain derived, in-depth analyses of immunological mechanisms of action of the platform and toxicity. Each pathogen/parasite addressed will take a similar approach by substitution: the anti-malarial aptamer composite will contain a bifunctional nanoparticle coated with a nucleic acid with galactose-alpha-1,3-galactose moiety at the exposed end to engage natural human antibody against the alpha gal and an anti-B1 lymphocyte receptor(s) aptamer, and/or including anti-IgM aptamers, on the same nanoparticle surface to directly stimulate IgM production for the removal of the malarial parasites. These composites can be tested against peripheral human monocytic/lymphocytic cells first (binding and stimulation assays) and antibody in human serum for binding. The Phase I test would look at the toxicity of the alpha gal/anti-B1 lymphocyte aptamer nanoparticle composite. Initially, a model form will be tested (for toxicity and efficacy) in the avian model of malaria infection by using poultry immunized against alpha gal and a nanoparticle with an alpha gal nucleic acid moiety and an anti-avian IgY aptamer. The poultry will be infected with avian malaria and the various component controls and complete platform. The anti-heartworm platform, which can be extended to human filarial parasites by substitution of appropriate aptamer specific parasite or general cross-reacting filarial antigen moieties, will consist of a bifunctional nanoparticle coated with an aptamer selected against the endosymbiont Wolbachia and another aptamer against one or more excretory/secretory antigens (currently used as diagnostic antigens in dogs). Domestic (pet) dogs with and without concurrent heartworm infections from endemic heartworm areas will be recruited for treatment/vaccination with the preparation following in laboratory testing of efficacy and toxicity in dogs and monitored for microfilaria and immunological responses indicating adult female heartworm presence. The anti-dracunculid composite will be composed of the same nanoparticle types as tested above, species specific anti-excretory/secretory antigens aptamer(s) for the parasite (many have already been cloned) and an anti-measles/distemper virus or anti-measles/distemper antibody epitope aptamer. Cross reacting anti-measles/distemper aptamers will allow the same composite prep to be used in both dogs and humans, who have been immunized against these viruses and will either be challenged with the currently available vaccines at time of treatment or those demonstrating high antibody titers against these viruses at the time. The planned licensure pathway for approval of a phase 1 clinical trial will be through a San Antonio clinical trial firm to be hired.
• 8. Mechanism of action:
  Because the mechanism involves re-directing an immune response from an ineffective but pre-existing abundant one, the reduction in numbers of immature circulating forms of the parasites or adult parasite fertility and viability will be used to assess the effectiveness of the mechanism of action. Furthermore, levels of isotypes and idiotypes of antibodies and specific committed cellular immunity will be assessed by quantity and specificity of response to the effective antigens/immunogens. The reappearance of disease in an endemic area in individuals (animals or humans) treated and cleared in this way, by adaptive vaccinology, will be assessed for reappearance of infection over time and the extent of signs and symptomology of apparent disease. The effectiveness of re-treatment will be evaluated and the necessity to further re-direct the response.
• 9. Chemistry, Manufacturing & Control (CMC) Development
  The large-scale manufacture of the platform involves methods already being used in commercial production of aptamers (SELEX) and newer methods described in the publications and patents referenced, including an automated method, and a field diagnostic system for finding binding sequences and cloning them for fermentation production.
• 10. Timeline and Key milestones:
  Timeline – Decision/key milestones
  From initiation of project: 3-6 months to select aptamers for potential molecular targets; starting after selection/conjugation of aptamers to nanoparticles and testing of binding activity, determining quality and quantity reliability (first milestone that must be passed): 6 months; testing in in vitro cellular models, animal and human, and laboratory models if necessary (mice and chickens or other poultry for malarial responses; dogs for microfilaria and adult heartworm responses; dracunculid responses will have to be done in the field in Chad in dogs); at least one, and preferably, the first two laboratory animal models must be passed without overt toxicity to proceed. By 2.5 years from the initiation, at least phase 1 trials should begin for one of the specific anti-parasite composites. Phase 2 and 3 will most likely exceed the 3-year limit of this funding proposal.
• 11. Vaccine Development Timeline: Because changing the target for vaccination only requires a substitution into the platform, human trials could occur within 3 months of identifying a target. The approval of prior tests of the DNA/nucleic acid and nanoparticle platforms for use in humans as safe and the concurrent use of similar preparations in domestic animals (under USDA/APHIS biologicals rules) or in-country animals would greatly accelerate the process.
  The aptamer platform composite approach does not require the prior development of immunity against the target organism but employs pre-existing or concomitant immunity to provide protection. In anthrax trials with naïve mice at multiple lethal doses of anthrax spores, immune protection against lethal anthrax toxin was initiated as early as 2 hours after exposure and maintained in an advancing infection at periods of 24 hours until 14 days when primary immunity took over. This worst case would not be expected with the parasites described since they are not uniformly fatal at 4 days as the overwhelming anthrax spore challenge would be. Also, the parasites themselves, when present as initial larval forms or subsequent adults maintain an active but ineffectual immune response, which the aptamer composite platform would re-direct for the elimination of the parasites. The presence of the nanoparticle-based aptamer composite platform is anticipated to continue for at least a year and it remains to be seen if the re-directed immune pathway will persist after the composite platform has broken down, but this is possible if not probable and will be explored as part of the experimental phase with subsequent re-challenges with parasites. Potentially no boosting will be required.
• 12. Scale up to target doses
  The estimated time of release, if anti-ligand targets are already available for aptamer binding selection, and if no clinical trials are required before such an emergency response (except for safety), then the best time would be 3 months and worst 6 months, starting from the beginning of the process. If the aptamer sequence is already known, production and delivery could occur in 2-4 weeks. The platform is designed for single dose use at best, but can be used, as it was with anthrax, in multiple serial doses for severe overwhelming potential lethal disease. The questions about manufacture process have been addressed elsewhere in this pre-proposal.
• 13. Budget
  Total budget estimate
  The initial pre-clinical development approximate cost will be at least $1.8M per year, which includes all the manufacturing technology development for scale, initial in vitro cell culture and animal testing. It will also include testing for toxicity of the nanoparticle and nucleic acid constructs unless data can be accepted on similar DNA and nanoparticle constructs already being used in human and animal clinical trials corresponding to this purpose (if not this may raise the cost to $3M yearly). The production of the scaled-up end-product for each parasite for use in testing, clinical trials and/or emergency response would be at least $550,000 for the custom made (not final manufacturing cost) for 100,000 doses. The cost objective for the final product for distribution for medical and veterinary use is 16 to 25 cents a dose at today’s costs of intermediate scale up manufacturing of nucleic acids (of bifunctional target length aptamers) and nanoparticles, or as close to this cost as possible. The major pass through costs would be for clinical trials, if they are to be funded all or in part, about $55M. Otherwise work up to that time (over a minimum of 3 years) would be $5.9M to $6.55M.
  Additional funding not applicable.
• 14. Application organization/consortium
  Experience and track record of applicant organization and cooperating partner(s)
  John G. Bruno: B.S., Microbiology with Chemistry Minor, Univ. of Arizona (1985) , Ph.D., Microbiology with emphasis on Immunology, Univ. of Arizona, Tucson (1991), Jan 2003-August 2018 Senior Scientist, Vice President and Chief Science Officer- Operational Technologies Corporation (OpTech), OTC Biotechnologies, LLC, and Pronucleotein Biotechnologies, Inc., San Antonio, TX., September 2018 – Present, Principal Scientist and Director of Biotechnology – Nanohmics, Inc., Austin, TX., 68 Grants/Contracts; Funding > $16M; 104 Peer-Reviewed Journal Articles; 16 Total U.S. and WIPO/PCT/EU Patents .
  Ronald M. Cook: UC Berkeley College of Chemistry (BS 1969); University of Washington, Seattle, Department of Chemistry, Ph.D. 1974; postdoctoral, University of California, San Francisco, (Dept of Microbiology and Immunology), 1974-76; founded Biosearch, Inc in 1977 to supply synthetic peptides (enkephalins) and protein bioconjugates for research; 1980, synthesis of nucleic acids, Biosearch became leading innovator in synthetic DNA oligonucleotide chemistry; 1982, the company developed first commercially viable Oligo synthesizer (SAM I); one of the first SAM units was placed with Kary Mullis, at Cetus, enabling the invention of PCR in 1983; 1990-1993, Principal Consultant to Beckman Instruments; 1993- 2015, Biosearch Technologies, Inc.: President/CEO, Chief Technical Officer; Chairman, Light Speed Genomics, a Santa Clara company; Chairman, DNA Technology, a Danish oligo-manufacturing company; Chairman, Vitra Bio, a German manufacturer of specialty glass for solid phase chemistry; consultant and/or served on Scientific Advisory Boards of Beckman Instruments, Solexa (Cambridge, England, RMC was a co-founder), developing next generation DNA sequencing technology; and Mosaic (Cambridge, Ma), developing solid phase PCR and contingent diagnostic applications; 2015-2019. LGC-Genomics, Chief Scientific Officer, Chairman, Co-Founder Optical Biosystems.
  George W. Irving III; DVM (1965), MS (1970), Diplomate American College of Laboratory Animal Medicine (1972), and American College of Veterinary Preventive Medicine (1977); served 30 years as USAF Military Officer, and 23 Years as VP at Conceptual MindWorks, Inc., directing research in Chemical & Bio Warfare Agent defense, Non-Lethal Weapons Bio Effects, and Pharmaceutical/Vaccine Development.
  Johnathan L. Kiel: DVM (1974, Texas A&M); PhD (Microbiology and Biochemistry, 1981, Texas Tech); Diplomate American College of Veterinary Microbiologists (1984), Charter Diplomate in Veterinary Parasitology (2011); served 38 years total, as a military officer, as a Civil Servant (GS14-15) research veterinarian, and Senior Scientist (Brigadier General Equivalent) in the Senior Executive Service in electromagnetic bioeffects and biosurveillance and counterproliferation of biological warfare and other infectious agents; 106 peer-reviewed articles; 30 patents. The fundamental form of this technology was invented by Dr Kary Mullis (deceased 7 August 2019), the recipient of the Nobel Prize, for inventing, PCR in 1993; with his collaboration, the platform was extended to other applications and delivery systems by the researchers of the United States Air Force at the Air Force Research Laboratory.
• 15. Dissemination plan of study results and data sharing:
  Peer-reviewed journal papers will be used when applicable to share data. Clinical trial registration and studies will be managed by a San Antonio clinical trials firm (such as Clinical Trials of Texas, Inc.). Because the targets for this project involve tropical diseases as well, we will reach out to public health and medical authorities of potential target countries such as Chad and other African countries through the World Health Organization and the OIE, World Organisation for Animal Health. All patient data will be protected in accord with US and international laws. Patent applications, both US and international, will be used to disclose the processes, but non-exclusive licensing will be used as much as possible to make the technology globally available. Procedures in accord with the USDA/APHIS will be followed to license the resulting animal biologicals produced from the project platform.
• 16. References.
• 1. Mullis, K.B., Vivekananda. J., Kiel, J.L., Cook, R.M. Chemically Programmable Immunity, US Patent 8,604,184 B2, December 10, 2013.
• 2. Kiel, J.L., Tijerina, A., Holwitt, E.A., Sloan, M.A., Woitaske, M., and Fan, M. Compositions, Methods and Uses for Biosynthetic Plasmid Integrated Capture Elements, US Patent 8,628,955 B2, Jan. 14, 2014.
• 3. Kiel, Johnathan L., Holwitt, Eric A., Fan, Michael (Maomian), Roper, Shelly D., Methods and Compositions for Rapid Selection and Production of Nucleic Acid Aptamers, US Patent 9,273,345 B2, March 1, 2016.
• 4.Vivekananda, J., and Kiel, J. L. Anti-Francisella tularensis DNA aptamers detect tularemia antigen from different subspecies by Aptamer-Linked Immobilized Sorbent Assay, Laboratory Investigation 86: 610-618, 2006.
• 5. Fan, M., McBurnett, S. R., Andrews, C. J., Allman, A. M., Bruno, J. G., and Kiel, J. L. Aptamer Selection Express: A Novel Method for Rapid Single-Step Selection and Sensing of Aptamers. J Biomol Tech 19(5), 311–319 (December 2008).
• 6. Bruno, J G. Aptamer–biotin–streptavidin–C1q complexes can trigger the classical complement pathway to kill cancer cells In Vitro Cell.Dev.Biol.—Animal (2010) 46:107–113.
• 7. Mutapi, F., Billingsley, P.F., and Secor, W.E. Infection and treatment immunizations for successful parasite vaccines. Trends in Parasitology March 2013, Vol. 29, No. 3, 135-141.
• 8. Bruno, J.B., Richarte, A.M., Savage, A.A., and Sivils, J.C. Development and Characterization of DNA Aptamers Which Bind Kinesins from Leishmania Promastigotes. J. Bionanosci. 8, 1–12, 2014.
• 9. Aguilar, R., et al. Antibody responses to α-Gal in African children vary with age and site and are associated with malaria protection. Scientific Reports. 3 July 2018, DOI:10.1038/s41598-018-28325-w.
• 10. Kristian, S.A., Hwang, J.H., Hall, B., Leire, E., Iacomini, J., Old, R., Galili, U., Roberts, C., Mullis, K.B., Westby, M., and Nizet, V. Retargeting pre-existing human antibodies to a bacterial pathogen with an alpha-Gal conjugated aptamer. J Mol Med (Berl). 2015 June; 93(6): 619–631.
• 11.Slatko, B.E.,Taylor, M.J., and Foster, J.M. The Wolbachia endosymbiont as an anti-filarial nematode target. Symbiosis (2010) 51:55–65.
• 12. Pati, R., Shevtsov, M., and Sonawane, A. Nanoparticle Vaccines Against Infectious Diseases. Frontiers in Immunology (9), October 2018, Article 2224.
  
• Don’t lose sight of the versatility of this approach (just because the original proposal involves its use against parasites),especially for COVID-19, where the path the immune response takes is the difference between recovery or death or long term chronic disease.

The new definition of sepsis is as life-threatening organ dysfunction caused by a dysregulated host response to infection more closely fits severe COVID-19. It abandons use of host inflammatory response syndrome criteria (SIRS) in identification of sepsis and eliminated the term severe sepsis. It is usually associated with bacterial rather than viral infections, but this new definition fits severe COVID better than cytokine storm. This latter description suggests a simple positive correlation and causation with pro-inflammatory cytokines. One should expect as their systemic levels go up, the symptoms get worse. The measurable level should correlate with the severity. The alternative is, if this doesn’t happen, the severity must be related to direct viral effects and immunosuppression, allowing the virus to greatly increase in number and spread to many organs, reeking havoc. At first glance these mechanisms may appear diametrically opposed. However, measurements can reflect the dynamics of cytokine levels verses progression of symptoms and exhaustion of the immune system not being synchronized. A recent study illustrates this paradox. https://www.thelancet.com/action/showPdf?pii=S2213-2600%2820%2930404-5. In spite of clinically resembling other classic syndromes associated with cytokine storms, meta-analysis of COVID-19 studies, published or posted preprints, between Nov 1, 2019, and April 14, 2020, in which interleukin-6 concentrations in patients with severe or critical disease were determined (25 COVID-19 studies of 1245 patients were included) showed lower than expected cytokine levels measured. The study groups included four each in sepsis (n=5320), cytokine release syndrome (n=72), and acute respiratory distress syndrome unrelated to COVID-19 (n=2767). Cytokine release syndrome may occur after treatment with some types of immunotherapy, such as monoclonal antibodies and CAR-T cells. Cytokine release syndrome is caused by a large, rapid release of cytokines into the blood from immune cells affected by the immunotherapy. I had seen this with intraperitoneally injected immobilized oxidase-peroxidase causing the rapid experimental destruction of large Novikoff abdominal hepatocarcinoma cancers in rats. In patients with severe or critical COVID-19, the pooled mean serum interleukin-6 concentration was 36.7 pg/ml. Mean interleukin-6 concentrations were nearly 100 times higher in patients with cytokine release syndrome (3110.5 pg/mL), 27 times higher in patients with sepsis (983.6 pg/mL), and 12 times higher in patients with acute respiratory distress syndrome unrelated to COVID-19 (460 pg/ml). Other cytokines gave similar levels with a mean TNFα (tumor necrosis factor alpha) concentration of 5.0 pg/mL (2.3–10.7 pg/mL) in patients with COVID-19, mean concentration of 34.6 pg/mL (20.0–59.9 pg/mL) in patients with sepsis and 52.2 pg/mL (2.0–1390 pg/mL) in patients with cytokine release syndrome. All but one COVID-19 study of the studies analyzed had mean TNFα concentrations lower than 10 pg/mL. IFNγ (gamma interferon) concentrations were not elevated in patients with COVID-19, at an average of 10.8 pg/mL, but were highly elevated in patients with cytokine release syndrome, averaging 3722.1 pg/mL (624–21838 pg/ml). Mean sIL-2R (serum soluble interleukin 2 receptor) was elevated in patients with COVID-19, but much less than in patients with cytokine release syndrome comparison (506 pg/mL vs 12396 pg/ml). The disparity between studies in TNF alpha level reported in an earlier post suggests that when it is measured in the course of the disease is important, but where it is measured is also important. Innate immunity is often very localized, and therefore, not measurable accurately in the serum. CRP (C reactive protein) concentrations were similar in COVID-19 and sepsis patients, and higher in patients with cytokine release syndrome. The studies examined indicated that patients with COVID-19 had higher D-dimer elevations (local fibrin formation and lysis are part of the inflammatory response, and fibrin degradation products, including D-dimer, have been shown to have diverse effects on inflammatory processes and acute phase responses, including hepatic synthesis of acute-phase proteins, such as CRP and fibrinogen) than did patients with sepsis. Mean ferritin and lactate dehydrogenase (measures of cell damage) concentrations were much higher in patients with cytokine release syndrome than in those with COVID-19, but still highly elevated in patients with COVID-19. Again, this indicates that pro-inflammatory processes were in play somewhere other than reflected in the blood measurements. In other highly lethal infectious disease which should resemble sepsis and septic shock syndrome, surprises have been found in cytokine stochastic measurements https://www.ncbi.nlm.nih.gov/pmc/articles/PMC303127/. Edema toxin (ET) of anthrax bacteria and lipopolysaccharide of Gram negative bacteria (LPS) each induced human monocytes to secrete approximately the same amounts of IL-6. ET did not inhibit and, in most experiments, modestly enhanced LPS-induced IL-6 production. In contrast to this stimulation of IL-6 production, ET yielded little or no TNF-alpha production, which is associated with septic shock. Moreover, ET profoundly inhibited LPS-induced TNF-alpha synthesis. Monocytes treated with dibutyryl cAMP, an active analog agonist of cAMP, produced cytokines in a pattern identical to that of cells treated with ET. The disruption of cytokine networks as a consequence of unregulated, ET-mediated cAMP accumulation in human monocytes may impair cellular antimicrobial responses and contribute to clinical signs and symptoms of anthrax. Lethal toxin of anthrax proteolytically inhibits MAP kinase kinase, an essential enzyme in the activation pathways of lymphocytes (MAPKK1 and MAPKK2 are cleaved which inactivates MAPKK1 and inhibits the MAPK signal transduction pathway). Another study with anthrax, that I was involved in, examined these two toxins that cause an intense systemic inflammatory response, edema, shock, and eventually death and showed similar dysfunction and dysregulation of the immune system not expected based on the effects on cytokine function. https://europepmc.org/article/med/11890668. Parent strain mice and knockouts were challenged with LD95 of anthrax spores (5 x 10exp6). administered subcutaneously. The results showed that all genetic knockouts succumbed to anthrax infection as frequently as the parent. TNF antibody delayed death but TNF receptor 1 knockout genetic modification had no effect. IL-1 receptor or iNOS (inducible nitric oxide synthase) knockouts died sooner. Anthrax was more abundant in the injection site of TNF-alpha and iNOS knockouts compared to parent suggesting that localized attenuated inflammatory cellular response increased the disease progression. The deaths were so rapid that, with the exception of edema and necrosis at the injection sites, no pathological changes were observed in internal organs, indicating a lethal per acute shock syndrome. Therefore, the dysregulation leading to dysfunction of the immune system in severe COVID-19 is not unheard of. The general distribution of an otherwise localized response may be the real culprit in severe COVID-19. As mentioned in a previous post, when anti-parasitic immune responses are suddenly generalized, shock and death can rapidly and irreversibly ensue. This is seen with crushing of parasitic fly larvae in a wound (myiasis) or rapid disintegration of filarial or other parasitic worms in a host.

To summarize. the possible sequence of events in COVID-19 are to first infect nasal and oral epithelial cells, trigger interferon and mucosal immunity; if this does not clear the virus, it descends into the lungs, widely infecting pneumocytes, leading to low grade pneumonitis that may trigger an effective adaptive response or not. If the adaptive response fails or results in autoantibody to interferons, then viremia spreads the virus systemically and causes lymphocytopenia (decrease in lymphocyte counts in blood, with removal of lymphocyte regulation of the immune response, typical of viremia). Multi-organ infection and generalized infection of the lungs, followed by immune-mediated cellular (antibody-dependent) inflammatory response against all the infected sites leading to sudden, generalized collateral damage that would otherwise be local, but because of the widespread multiple sites leads to severe disease and even multiple organ failure and death.

A rapid diagnostic method for SARS-CoV-2, which provides results in about 5 minutes, based on DNA oligonucleitides labeled with fluorescent dyes binding to viruses, which, in turn, undergo microscopic image analysis for identification has been reported https://www.medrxiv.org/content/10.1101/2020.10.13.20212035v2.full.pdf. Aptamers are synthetic oligonucleotides selected for specifically binding to infectious agents or many other targets. They have been labeled with fluorescent dyes for tracing the binding and further developed into double-stranded DNA probes, where a complementary DNA strand has a fluorescent quencher that when displaced allows the fluorescence to be observed https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2628068/pdf/JBT-05-311.pdf . Therefore, the Oxford method could be enhanced by the rapid aptamer method.

It appears protective immunity to SARS-CoV-2 may last at least 6 months and up to more than a year. Although the strain changes recently seen might be the reason for the rare re-infections recently reported, I believe it is more. The evidence for protective cross immunity with other CoVs and even bacterial antigens (pneumonia vaccines) rules (see earlier post) against the small genetic changes (D614G in the spike protein) in the spike protein inhibiting immunity. New data indicates that the mutation actually increases antibody neutralization https://www.medrxiv.org/content/10.1101/2020.07.22.20159905v2.full.pdf. I suspect that it is because the first viral infection was cleared early by interferon 1, IgA, and/or IgM, the latter which decline within 3 months. It is the IgG, particularly IgG3, that lasts longer: 7 months and the memory B cells even longer. But if infection response clears virus in less than 2 weeks, the IgG response never happens—opening up for reinfection. Researchers at Massachusetts General Hospital found IgG levels remained elevated for four months in those who recovered from COVID-19, and were protective neutralizing antibodies, which decreased over time. The researchers also showed that those infected with SARS-CoV-2 had immunoglobulin A (IgA) and immunoglobulin M (IgM) responses that were relatively short-lived, declining within about two and a half months or less, on average. https://immunology.sciencemag.org/content/immunology/5/52/eabe0367.full.pdf. Researchers at the University of Toronto discovered IgG antibodies were detectable for up to 115 days (approximately 4 months) in those recovered from COVID-19 https://immunology.sciencemag.org/content/immunology/5/52/eabe5511.full.pdf. University of Arizona focused on antibodies that bind to two different parts of the SARS-CoV-2 virus, most antibody tests look for antibodies at S1, which includes the receptor-binding domain by which the spike protein binds to a protein receptor to infect cells. But they also looked for antibody to the S2 region of the spike protein. They found SARS-CoV-2 antibodies in blood for at least 5 to 7 months, although they believe immunity actually lasts longer. They knew that people who were infected with SARS coronavirus-1, which is the closest relative to SARS-CoV-2, still show immunity 17 years after infection. If SARS-CoV-2 is anything like the first one, they expect antibodies to last at least two years, and it would be unlikely for anything less https://www.cell.com/immunity/pdf/S1074-7613(20)30445-3.pdf?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS1074761320304453%3Fshowall%3Dtrue.

https://news.yahoo.com/until-coronavirus-vaccine-ready-pneumonia-191844263.htmlThe higher the rate of pneumococcal vaccination, the lower COVID-19 morbidity and mortality. Vaccination rates with Bacillus Calmette–Guerin, poliovirus, and other vaccines do not affect these COVID-19 numbers, nor do COVID-19 case or death rates follow the numbers of people with diabetes, obesity, or who are over 65. Infant protection may be due to maternal antibodies and antiviral proteins in milk such as lactoferrin that are known to protect against coronavirus infections. Protection might be further provided by rates of Haemophilus influenzae type B (Hib) (universal in infants) and pneumococcal vaccination, the latter varying widely among nations for infants, at-risk adults, and elderly.
The huge differences in the incidence of severe COVID-19 cases and deaths from one nation to another might be due to population density, number of elderly, how and when social distancing restrictions or other protective measures are implemented. It is difficult to explain differences in COVID-19 death rates that vary from between 650 per million to 65 per million in neighboring European nations or death rates as low as 0.5 per million in some Asian nations. The rate of vaccination against Haemophilus influenzae type B and pneumococcal infections by Streptococcus pneumoniae may make the difference in preventing fatal COVID infection outcomes. https:onlinelibrary.wiley.com/doi/epdf/10.1002/bies.202000076. One last caveat, association does not prove causation. This beneficial effect could be extended or enhanced by using aptamer linkers with the current vaccine antigen at one end and the aptamer, selected in vitro for binding to the SARS-CoV-2 (spike) antigens, at the other end of the aptamer, Chemically Programmable Immunity.

A recent carefully done laboratory experiment has been published https://virologyj.biomedcentral.com/articles/10.1186/s12985-020-01418-7 . The information needs to be put in context to assess real field environmental survival of SARS-CoV-2. Viruses, in some sense, survive better environmentally than vegetative bacteria but less well than bacterial spores. The rule of thumb is the “dirtier the virus, the longer it survives inactivation”. The cleaner it is, the less it survives. However, as stated in my earlier post, the initial amount can lead to even longer or shorter apparent survival due to exponential decay and the threshold of the detection methods: viral culture in vitro or true infectious dose in a susceptible host. The paper to be discussed used the in vitro method to assess virus viability and vigorous extraction methods (worst case transmission) not touch transfer or re-aerosolization (which are more hazardous to do in the lab). They also used a standard biological matrix (mimic of human expelled biological material) and maximum inoculum dose (based on assumed human expelled dose) which is appropriate to produce optimal survival data in the laboratory. Real life conditions are messier and UV light (all light) was excluded from the experimental conditions. They observed half lives of between 1.7 and 2.7 days at 20 °C, decreasing to a few hours when temperature was elevated to 40 °C. Using initial viral loads approximately equivalent to the highest titers excreted by infectious patients and in a simulated biological matrix, viable virus was isolated for up to 28 days at 20 °C from common surfaces such as glass, stainless steel and both paper and polymer banknotes. In contrast, infectious virus survived less than 24 h at 40 °C (104 F) on some surfaces. Fomite transmission is highly efficient for transmission (efficiencies of 33%) for both fomite to hand and fingertip to mouth transfer for bacteria and phages (no data for CoV). The inoculum was 10 microliters of the suspension (final concentration of 3.38 × 10⁵/10 µL). A single relative humidity set point (50%) was maintained for experiments at 20 °C, 30 °C, 40 °C, respectively, although dew point would have been a more accurate measure since relative humidity changes with temperature. Three replicates of each surface type were inoculated and sampled at the following time points; 1 h, 1 day, 3 days, 7 days, 14 days, 21 days and 28 days post inoculation. For the 40 °C experiment, triplicate samples were inoculated for the following time points; 1 h, 1 day, 2 days, 3 days, 4 days, and 7 days. For non-porous surfaces, for each replicate, virus was eluted in 2 × 115 µL volumes of DMEM (cell culture medium) with repeated pipetting then titrated individually, in quadruplicate wells on a 96-well plate. The recovery from porous surfaces, such as cotton cloth, which are likely to inhibit transfer and recovery under field conditions, was accomplished by soaking swatches of the cloth in 500 µL DMEM and pipetting repeatedly for at least 1 min before 230 µL of the recovered eluent from each swatch was titrated separately, in quadruplicate. Suspensions of Vero E6 cells (3 × 10⁵/mL) were added to the wells and the plates were incubated for 3 days at 37 °C with 5% CO2 to look for cell damage (cytopathic effect) from viral replication in the cells. The majority of virus reduction on cotton occurred very soon after application of virus, suggesting an immediate adsorption effect. The calculated 90%, 1 log, inactivation of virus for surfaces at 20 °C (room temperature = 25 degrees C) ranged from 5.5 days for cotton to 9.1 days for paper notes. At 30 °C, infectious virus was recoverable for 7 days from stainless steel, polymer notes and glass, and 3 days for vinyl and cotton cloth. For paper notes, infectious virus was detected for 21 days, although there was less than 1 log of virus recovered for both 14 day and 21 day time points. At 40 °C, virus recovery was reduced compared to both 20 °C and 30 °C experiments. Infectious SARS-CoV-2 was not recovered past 24 h for cotton cloth and 48 h for all remaining surfaces tested. Greater than 4-log reduction (99.99% reduction from starting titer) was observed in less than 24 h at 40 °C on all surfaces. All calculations were based on linear decay fits, not exponential decay. Again, as stated in an earlier post, the latter model is what is observed under field conditions for other biological agents. Also, CoV is less durable than other viruses ( non-enveloped) such as adenovirus, picornaviruses (can persist for up to a year in the environment) and Norovirus (diverse group of single-stranded positive-sense RNA, non-enveloped viruses belonging to the family Caliciviridae). What needs to be kept in mind here is that incident surface contamination does not equal transferable host absorbed infectious dose. Smooth non-porous surfaces are more likely to transfer virus than porous surfaces that adsorb and bind virus. This is borne out by the low transmission rate (approximately 10% infected) for most exposed populations. However, the paper indicates that we should err on the side of caution and rigorously disinfect potentially contaminated surfaces.

I discussed in earlier posts that immunity can be incomplete, even with recovery; inappropriate, leading to severe disease; long lasting with cellular mediated immunity; or side-stepped or even made to make things worse when infected by a different strain of the same virus. Also, viruses and other microbes can hide in sequestered privileged sites in the body to re-emerge to spread to other sites before the immune system kicks in again or be shed and spread to other susceptible hosts. Herpes viruses such as those that cause cold sores, genital lesions and chicken pox, followed years later by shingles, hide in nerve endings, only to re-emerge when adaptive immunity declines. Bacterial diseases like brucellosis recur producing undulant fever over years by hiding in cells where immunity or antibiotics have a hard time getting. Leptospira hide in the kidneys and shed in the urine to infect humans and other animals, giving them active leptospirosis, after the shedding host has recovered from acute disease and is generally immune but not in the kidneys. Syphilis moves through 4 phases as immunity to this microbe develops: acute genital form; secondary systemic rash form (skin rashes and/or mucous membrane lesions: sores in the mouth, vagina, or anus; symptoms of secondary syphilis may include fever, swollen lymph glands, sore throat, patchy hair loss, headaches, weight loss, muscle aches, and fatigue); latent (hidden) stage of syphilis, a period of time when there are no visible signs or symptoms of syphilis; and the tertiary fatal central nervous system form (can appear 10–30 years after infection was first acquired; affects multiple organ systems, including the brain, nerves, eyes, heart, blood vessels, liver, bones, and joints). Each step it becomes more and more hidden from the immune system or exhausts the immune responses in the final stage. Perhaps, albeit to a much lesser extent, CoV uses some of these strategies to maintain infections in or among individuals. The change in strain internally could also occur if there is sufficient immune selection pressure to accelerate mutation of the virus (see earlier post on monoclonal antibody treatments). No matter which above mechanism has come to play, re-infection with or re-activation of SARS-CoV-2 has occurred https://www.medrxiv.org/content/10.1101/2020.09.22.20192443v1.full.pdf. In Brazil, two cases of “re-infection” has occurred with two different variants, with increased severity, in one case, associated with increased viral load https://www.preprints.org/manuscript/202101.0132/v2. One additional note, as public health fails and more and more virus is present and carelessness allows larger viral doses to be transmitted, these mechanisms, no matter how rare, become more likely to occur and are amplified.

In recent studies of COVID patients, of the inflammatory mediators analyzed in serum, IL-6, IL-10, monocyte chemoattractant protein-1 (MCP-1), and interferon-gamma induced protein 10 (IP-10) were the ones significantly increased and correlated with disease severity. No significant changes in other cytokines or chemokines measured at the time of hospitalization with symptoms, including IFN-gamma, IL-1-beta, IL-8, or tumor necrosis factor-alpha (TNF alpha) were seen in these patients. High neutrophilia (numbers of blood neutrophilic granulocytes) appeared in severe COVID-19, because of their role in acute respiratory distress syndrome. Elevated C Reactive Protein (CRP) and IL-6 in patients, when entering hospital, later resulted in death, and increased IP-10 was seen in those who later developed severe disease. IP-10 is an interferon- inducible chemokine that aids directed migration of many immune cells. https://immunology.sciencemag.org/content/immunology/5/51/eabd6197.full.pdf.

Buying time until adaptive immunity develops appears to be where things go wrong in COVID-19. In very lethal diseases, like inhalation anthrax, it progresses too rapidly, 2-4 days, to lethality, for any adaptive immunity to develop, even though vaccination against anthrax is very effective but the initial vaccination response takes about 2 weeks and the anamnestic (booster) response requires 3 days. This latter is just enough to stop a lethal response if the dose of spores inhaled is not too high. If it is, it requires antibiotics as well as prior vaccination over as long as a couple of months. Although anthrax is far more lethal than COVID-19, it appears if the innate immunity (interferon and perhaps monocytic responses) fail too quickly before adaptive cellular and appropriate antibodies kick in, the disease becomes severe and even lethal. As was learned from treating secondary bacterial infections with HIV, antibiotics don’t cure anything without an immune system, they only buy time, or until they fail from bacteria developing antibiotic resistance. The similar failure with COVID-19, is a result of both an inadequate innate immunity and an inappropriate adaptive immunity which accelerate the previous failure by making autoantibodies against interferons, which otherwise block viral replication. The initial dose of virus and the failure to inhibit its replication with antivirals can lead to the same end. IL-6, IP-10, and MCP-1 levels were generally the highest in patients around the time of entering the hospital but decreased rapidly if they proceeded to intensive care, which probably indicates exhaustion of innate immunity.

The good news is that if adaptive immunity is given the time to appropriately develop, it becomes enduring. Preinfection and postinfection patients developed IgG and IgG memory B cells (MBCs) reactive to SARS-CoV-2 proteins. Most importantly, the studies demonstrated that infection generates both IgG and IgG MBCs against the SARS-CoV-2 unique receptor binding domain and the conserved S2 subunit of the SARS-CoV-2 spike protein, shared by other human coronaviruses. This means even after antibody levels decline or become undetectable, long-lived MBCs remain to bring about rapid anamnestic antibody production. The study results also suggested that SARS-CoV-2 infection strengthens preexisting broad immunity to coronavirus, in general, through S2-binding antibody and MBC formation. https://mbio.asm.org/content/mbio/11/5/e01991-20.full.pdf.