Dealing with SARS-CoV-2 Mutants: Their Achilles Heal and More

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.

JM109/pIC20RNR1.1 E. coli inoculated at a billion organisms following growth for 24 hours in 3-AT broth containing the various 10-fold dilutions of the mutagen 2-chloroethylethyl sulfide then transferred to ampicillin LB agar or sheep blood agar for colony counts after 24 hours of growth. The higher to lower mutagen concentrations from left to right show a negative linear correlation with colony counts. The general survivability on blood agar was more sensitive to the mutagen than was the specific antibiotic resistance amongst the survivors, although a substantial number of survivors lost ampicillin resistance. The DALM formation in the 3-AT broth killed off many of those bacteria still maintaining the plasmid with ampicillin resistance. The overall mutation rate increased with increasing mutagen concentration from right to left on the x axis.

**Separating sensitivity to 3-AT (and nitrate) and resistance to ampicillin by increasing mutations, when they are genetically linked on the same plasmid, seems counterintuitive:** At the lower concentrations of mutagen (log -6 to log -8), the survival on blood and ampicillin correlate and show the same trend of response for the spontaneous natural mutation at the lower concentrations; growth in 3-AT medium is lethal because of DALM polymer formation and the ampicillin resistance on the plasmid that enhances that production selects for its expression. To escape this lethal effect while maintaining ampicillin resistance requires very selective mutations. The more mutations the less fit is the surviving bacteria until they become uniformly lethal. This effect is demonstrated in the first rise in survival on blood agar but not on ampicillin agar (loss of ampicillin resistance first). The latter lags but costs in overall fitness as the mutations increase to select for ampicillin resistance separated from 3-AT sensitivity until the mutations increase to lethal. Therefore, it is very difficult to achieve mutations that select for survival of two eliminating treatments, especially if they are genetically and expression linked.

Simple diagram of paradoxical selection pressure of Ampicillin/nitrate/3-amino-L-tyrosine growth medium on JM109/pIC20RNR1.1 E. coli

The decline in thermochemilumescence of bacteria grown in 3-AT medium with increasing mutagen concentrations, as an indirect measure of DALM polymer production. This correlation with increased survivability is not as great as expected, but this is probably because polymer production requires many genes as opposed to ampicillin resistance which requires just one. Complete inactivation of the polymer production requires accumulation of too many mutations, leading to death.

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).