Time to establish an international vaccine candidate pool for potential highly infectious respiratory disease: a community's view.

In counteracting highly infectious and disruptive respiratory diseases such as COVID-19, vaccination remains the primary and safest way to prevent disease, reduce the severity of illness, and save lives. Unfortunately, vaccination is often not the first intervention deployed for a new pandemic, as it takes time to develop and test vaccines, and confirmation of safety requires a period of observation after vaccination to detect potential late-onset vaccine-associated adverse events. In the meantime, nonpharmacologic public health interventions such as mask-wearing and social distancing can provide some degree of protection. As climate change, with its environmental impacts on pathogen evolution and international mobility continue to rise, highly infectious respiratory diseases will likely emerge more frequently and their impact is expected to be substantial. How quickly a safe and efficacious vaccine can be deployed against rising infectious respiratory diseases may be the most important challenge that humanity will face in the near future. While some organizations are engaged in addressing the World Health Organization's "blueprint for priority diseases", the lack of worldwide preparedness, and the uncertainty around universal vaccine availability, remain major concerns. We therefore propose the establishment of an international candidate vaccine pool repository for potential respiratory diseases, supported by multiple stakeholders and countries that contribute facilities, technologies, and other medical and financial resources. The types and categories of candidate vaccines can be determined based on information from previous pandemics and epidemics. Each participant country or region can focus on developing one or a few vaccine types or categories, together covering most if not all possible potential infectious diseases. The safety of these vaccines can be tested using animal models. Information for effective candidates that can be potentially applied to humans will then be shared across all participants. When a new pandemic arises, these pre-selected and tested vaccines can be quickly tested in RCTs for human populations.

Efficacy of Grignard Pure to Inactivate Airborne Phage MS2, a Common SARS-CoV-2 Surrogate.

Grignard Pure (GP) is a unique and proprietary blend of triethylene glycol (TEG) and inert ingredients designed for continuous antimicrobial treatment of air. TEG has been designated as a ″Safer Chemical" by the US EPA. GP has already received approval from the US EPA under its Section 18 Public Health Emergency Exemption program for use in seven states. This study characterizes the efficacy of GP for inactivating MS2 bacteriophage─a nonenveloped virus widely used as a surrogate for SARS-CoV-2. Experiments measured the decrease in airborne viable MS2 concentration in the presence of different concentrations of GP from 60 to 90 min, accounting for both natural die-off and settling of MS2. Experiments were conducted both by introducing GP aerosol into air containing MS2 and by introducing airborne MS2 into air containing GP aerosol. GP is consistently able to rapidly reduce viable MS2 bacteriophage concentration by 2-3 logs at GP concentrations of 0.04-0.5 mg/m3 (corresponding to TEG concentrations of 0.025 to 0.287 mg/m3). Related GP efficacy experiments by the US EPA, as well as GP (TEG) safety and toxicology, are also discussed.

An alternative approach-combination of lockdown and open in fighting COVID-19 pandemics.

As the COVID-19 pandemic enters its third year and the omicron variant becomes dominant, we propose an alternative strategy for dealing with COVID-19, called hybrid lockdown, that is, the combination of lockdown (the centralized and organized lockdown of the high-risk population) and free mobility (normal mobility) of the low-risk population. Such an approach will enable a country or region, especially with a high population density, to achieve significant prevention and control the effects of the COVID-19 pandemic at the least cost.

Lamarck redux and other false arguments against SARS-CoV-2 vaccination: Pseudoscientific arguments against vaccination do not stand up to scrutiny.

The COVID-19 pandemic has triggered a new bout of anti-vaccination propaganda. These are often grounded in pseudoscience and misinterpretation of evolutionary biology.

Higher Concentrations of Bacterial Enveloped Virus Phi6 Can Protect the Virus from Environmental Decay.

Phage Phi6 is an enveloped virus considered a possible nonpathogenic surrogate for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other viral pathogens in transmission studies. Larger input amounts of bacteriophage Phi6 are shown to delay and protect the phage from environmental decay, both when the phages are dried in plastic tubes and when they are stored in saline solution at 4°C. In contrast, when bacteriophage Phi6 is placed in LB (Luria-Bertani) growth medium (instead of saline) prior to placement on the plastic surface, the influence of the starting concentration on viral recovery is negligible. Protection is reflected in the phage half-lives at higher concentrations being longer than the half-lives at lower concentrations. Because experiments supporting the possibility of fomite transmission of SARS-CoV-2 and other viruses rely upon the survival of infectious virus following inoculation onto various surfaces, large initial amounts of input virus on a surface may generate artificially inflated survival times compared to realistic lower levels of virus that a subject would normally encounter. This is not only because there are extra half-lives to go through at higher concentrations but also because the half-lives themselves are extended at higher virus concentrations. It is important to design surface drying experiments for pathogens with realistic levels of input virus and to consider the role of the carrier and matrix if the results are to be clinically relevant. IMPORTANCE During the coronavirus disease 2019 (COVID-19) pandemic, much attention has been paid to the environmental decay of SARS-CoV-2 due to the proposed transmission of the virus via fomites. However, published experiments have commenced with inocula with very high virus titers, an experimental design not representative of real-life conditions. The study described here evaluated the impact of the initial virus titer on the environmental decay of an enveloped virus, using a nonpathogenic surrogate for the transmission of SARS-CoV-2, enveloped bacteriophage Phi6. We establish that higher concentrations of virus can protect the virus from environmental decay, depending on conditions. This has important implications for stability studies of SARS-CoV-2 and other viruses. Our results point to a limitation in the fundamental methodology that has been used to attribute fomite transmission for almost all respiratory viruses.

SARS Wars: the Fomites Strike Back.

Controversy continues about the significance of fomite transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Recent papers continue to advocate concern. However, designs of studies showing virus survival on surfaces under laboratory conditions are unsuitable for extrapolation to real life. Although viral RNA is frequently found on real-life surfaces, actual tests for infectious virus are almost entirely negative, even in hospitals with COVID-19 patients. Fomite transmission should be regarded as no more than a very minor component of this pandemic.

COVID-19 vaccine: Call for employees in international transportation industries and international travelers as the first priority in global distribution.

While countries are in a hurry to obtain SARS-CoV-2 vaccine, we are concerned with the availability of vaccine and whether a vaccine will be available to all in need. We predicted three possible scenarios for vaccine distributions and urge an international united action on the worldwide equitable access. In case the international community does not reach a consensus on how to distribute the vaccine to achieve worldwide equitable access, we call for a distribution plan that includes the employees in international transportation industries and international travelers to halt the disease transmission and promote the recovery of the global economy.

A cost-effective plan for global testing - an infection rate stratified, algorithm guided, multiple-level, continuously pooled testing strategy.

The most effective measure to prevent or stop the spread of infectious diseases is the early identification and isolation of infected individuals through comprehensive screening. At present, in the COVID-19 pandemic, such screening is often limited to isolated regions as determined by local governments. Screening of potentially infectious individuals should be conducted through coordinated national or global unified actions. Our current research focuses on using resources to conduct comprehensive national and regional regular testing with a risk rate based, algorithmic guided, multiple-level, pooled testing strategy. Here, combining methodologies with mathematical logistic models, we present an analytic procedure of an overall plan for coordinating state, national, or global testing. The proposed plan includes three parts 1) organization, resource allocation, and distribution; 2) screening based on different risk levels and business types; and 3) algorithm guided, multiple level, continuously screening the entire population in a region. This strategy will overcome the false positive and negative results in the polymerase chain reaction (PCR) test and missing samples during initial tests. Based on our proposed protocol, the population screening of 300,000,000 in the US can be done weekly with between 15,000,000 and 6,000,000 test kits. The strategy can be used for population screening for current COVID-19 and any future severe infectious disease when drugs or vaccines are not available.

Engineered EF-Tu and tRNA-Based FRET Screening Assay to Find Inhibitors of Protein Synthesis in Bacteria.

Antibiotic-resistant infections that do not respond to available drugs are becoming more common. Methicillin-resistant Staphylococcus aureus, carbapenem-resistant enterobacteria ("superbugs"), and many others pose a continuous threat to public health. To provide tools to combat such deadly infections, we present in this study a homogeneous assay focused on an insufficiently addressed molecular interaction linked to ribosomal translation. We show that a fluorescence resonance energy transfer (FRET) based screening assay can identify antibiotic molecules that inhibit ternary complex (EF-Tu:tRNA:GTP complex) formation, and therefore, protein synthesis in bacteria. Specifically engineered Escherichia coli EF-Tu and tRNAPhe are used to prepare two key components of this assay: (1) Cy5-EF-Tu:GTP and (2) Cy3-Phe-tRNAPhe. When mixed and Cy3 is excited at 532 nm, increased Cy5 fluorescence intensity is observed at 665 nm due to ternary complex formation and FRET. If the same assay is carried out in presence of an inhibitor, such as GE2270A (a known inhibitor of the EF-Tu-tRNA interaction), fluorescence intensity is significantly diminished. To establish proof of principle and to show the adaptability of this assay to high throughput screening (HTS), we analyzed the effect of different classes of antibiotics, including beta-lactams, quinolone compounds, and protein synthesis inhibitors, on fluorescence. The assay was done in a 96-well microplate. We observed inhibition by GE2270A, and no effect of nineteen other tested antibiotics, confirming the ability of this FRET assay to serve as a screen for potential inhibitor molecules of ternary complex formation from libraries of compounds.

Labeled EF-Tus for rapid kinetic studies of pretranslocation complex formation.

The universally conserved translation elongation factor EF-Tu delivers aminoacyl(aa)-tRNA in the form of an aa-tRNA·EF-Tu·GTP ternary complex (TC) to the ribosome where it binds to the cognate mRNA codon within the ribosomal A-site, leading to formation of a pretranslocation (PRE) complex. Here we describe preparation of QSY9 and Cy5 derivatives of the variant E348C-EF-Tu that are functional in translation elongation. Together with fluorophore derivatives of aa-tRNA and of ribosomal protein L11, located within the GTPase associated center (GAC), these labeled EF-Tus allow development of two new FRET assays that permit the dynamics of distance changes between EF-Tu and both L11 (Tu-L11 assay) and aa-tRNA (Tu-tRNA assay) to be determined during the decoding process. We use these assays to examine: (i) the relative rates of EF-Tu movement away from the GAC and from aa-tRNA during decoding, (ii) the effects of the misreading-inducing antibiotics streptomycin and paromomycin on tRNA selection at the A-site, and (iii) how strengthening the binding of aa-tRNA to EF-Tu affects the rate of EF-Tu movement away from L11 on the ribosome. These FRET assays have the potential to be adapted for high throughput screening of ribosomal antibiotics.

Design and properties of efficient tRNA:EF-Tu FRET system for studies of ribosomal translation.

Formation of the ternary complex between GTP-bound form of elongation factor Tu (EF-Tu) and aminoacylated transfer RNA (aa-tRNA) is a key event in protein biosynthesis. Here we show that fluorescently modified Escherichia coli EF-Tu carrying three mutations, C137A, C255V and E348C, and fluorescently modified Phe-tRNA(Phe) form functionally active ternary complex that has properties similar to those of the naturally occurring (unmodified) complex. Similarities include the binding and binding rate constants, behavior in gel retardation assay, as well as activities in tRNA protection and in vitro translation assays. Proper labeling of EF-Tu was demonstrated in MALDI mass spectroscopy experiments. To generate the mutant EF-Tu, a series of genetic constructions were performed. Two native cysteine residues in the wild-type EF-Tu at positions 137 and 255 were replaced by Ala and Val, respectively, and an additional cysteine was introduced either in position 324 or 348. The assembly FRET assay showed a 5- to 7-fold increase of Cy5-labeled EF-Tu E348C mutant fluorescence upon formation of ternary complex with charged tRNA(Phe)(Cy3-labeled) when the complex was excited at 532 nm and monitored at 665 nm. In a control experiment, we did not observe FRET using uncharged tRNA(Phe)(Cy3), nor with wild-type EF-Tu preparation that was allowed to react with Cy5 maleimide, nor in the absence of GTP. The results obtained demonstrate that the EF-Tu:tRNA FRET system described can be used for investigations of ribosomal translation in many types of experiments.

Properties of Escherichia coli EF-Tu mutants designed for fluorescence resonance energy transfer from tRNA molecules.

Here we describe the design, preparation and characterization of 10 EF-Tu mutants of potential utility for the study of Escherichia coli elongation factor Tu (EF-Tu) interaction with tRNA by a fluorescence resonance energy transfer assay. Each mutant contains a single cysteine residue at positions in EF-Tu that are proximal to tRNA sites within the aminoacyl-tRNA.EF-Tu.GTP ternary complex that have previously been labeled with fluorophores. These positions fall in the 323-326 and 344-348 regions of EF-Tu, and at the C terminus. The EF-Tus were isolated as N-terminal fusions to glutathione S-transferase (GST), which was cleaved to yield intact EF-Tus. The mutant EF-Tus were tested for binding to GDP, binding to tRNA in gel retardation and protection assays, and activity in poly-U translation in vitro. The results indicate that at least three EF-Tu mutants, K324C, G325C and E348C, are suitable for further studies. Remarkably, GST fusions that were not cleaved were also active in the various assays, despite the N-terminal fusion.

Inhibition of translation by consecutive rare leucine codons in E. coli: absence of effect of varying mRNA stability.

Consecutive homologous codons that are rarely used in E. coli are known to inhibit translation to varying degrees. As few as two consecutive rare arginine codons exhibit a profound inhibition of translation when they are located in the 5' portion of a gene in E. coli. We have previously shown that nine consecutive rare CUA leucine codons cause almost complete inhibition of translation when they are placed after the 13th codon of a test message (although they do not inhibit translation when they are placed in the middle of the message). In the present work, we report that five consecutive rare CUA leucine codons exhibit approximately a threefold inhibition of translation when they are similarly placed after the 13th codon of a test message, compared to five consecutive common CUG leucine codons, in a T7 RNA polymerase-driven system. Further, by removing RNase III processing sites at the 3' ends of the mRNAs, we have manipulated the stability of the mRNAs encoding the test and control messages to see if decreasing mRNA stability might have an effect on the extent of translation inhibition by the rare leucine codons. However, the inhibition with the less stable mRNAs was similar to that with the stable mRNAs, approximately 3.4-fold, indicating that mRNA stability per se does not have a major influence on the effects of rare codons in this system.

CCC CGA is a weak translational recoding site in Escherichia coli.

Previously published experiments had indicated unexpected expression of a control vector in which a beta-galactosidase reporter was in the +1 reading frame relative to the translation start. This control vector contained the codon pair CCC CGA in the zero reading frame, raising the possibility that ribosomes rephased on this sequence, with peptidyl-tRNA(Pro) pairing with CCC in the +1 frame. This putative rephasing might also be exacerbated by the rare CGA Arg codon in the second position due to increased vacancy of the ribosomal A-site. To test this hypothesis, a series of site-directed mutants was constructed, including mutations in both the first and second codons of this codon pair. The results show that interrupting the continuous run of C residues with synonymous codon changes essentially abolishes the frameshift. Further, changing the rare Arg codon to a common Arg codon also reduces the frequency of the frameshift. These results provide strong support for the hypothesis that CCC CGA in the zero frame is indeed a weak translational frameshift site in Escherichia coli, with a 1-2% efficiency. Because the vector sequence also contains another CCC triplet in the +1 reading frame starting within the next codon after the CGA, our data also support possible contribution to expression of a +7 nucleotide ribosome hop into the same +1 reading frame. We also confirm here a previous report that CCC UGA is a translational frameshift site, in these experiments, with about 5% efficiency.

Expression of non-open reading frames isolated from phage display due to translation reinitiation.

An unusual 38 codon sequence was previously isolated from a random peptide library by binding to growth hormone binding protein in phage display. This sequence, H10, and several variants did not contain open reading frames, but expressed a beta-galactosidase reporter 10-40% as well as control in both the original reading frame from phage display and the frame -1 to it. Inspection of the sequence suggested that expression in the -1 frame resulted from initiation at a downstream ATG in that frame, present in H10 and its variants, subsequently confirmed by site-directed mutagenesis. Unexpectedly, mutagenesis of that out-of-frame downstream ATG also increased expression in the original non-open reading frame by two- to threefold, creating a TTG codon adjacent to an existing in-frame TTG codon, suggesting downstream translational reinitiation at a putative TTG start. We undertook an extensive site-directed mutagenesis approach and report that this hypothesis is almost certainly correct. Features required for this reinitiation include an upstream translation start and a stop that can even be a suppressed amber codon 22 nucleotides further downstream from the restart. Replacing the TTG with ATG increases expression only twofold. Reinitiation occurs in either of two reading frames in this sequence.

Genetic analysis of the basis of translation in the -1 frame of an unusual non-ORF sequence isolated from phage display.

An unusual peptide-encoding sequence, called H10, and several derivatives of this sequence were previously isolated from a random peptide library screened by phage display during drug discovery protocols. The H10 family of sequences had the unusual property of being expressed despite the absence of an open reading frame. When these sequences were fused to a reporter lacZ gene in all three frames, beta-galactosidase was expressed not only from the parental non-open reading frame, consistent with the original isolations, but also from the frame -1 to the parental. This unexpected translation in a second reading frame could result from either a recoding event or from an internal translation initiation event. In order to elucidate which type of event, a genetic approach was selected to eliminate a potential downstream initiator site within the H10 sequence. This report provides strong evidence that translation in the -1 frame in this family of sequences is indeed originating from a downstream translation initiation event. Unexpectedly, the mutation eliminating the downstream initiation event in the -1 frame simultaneously elevated expression in the original non-open reading frame.