The global emergency caused by COVID-19 makes the discovery of drugs capable of inhibiting SARS-CoV-2 a priority, to reduce the mortality and morbidity of this disease. Repurposing approved drugs can provide therapeutic alternatives that promise rapid and ample coverage because they have a documented safety record, as well as infrastructure for large-scale production. The main protease of SARS-CoV-2 (Mpro) is an excellent therapeutic target because it is critical for viral replication; however, Mpro has a highly flexible active site that must be considered when performing computer-assisted drug discovery. In this work, potential inhibitors of the main protease (Mpro) of SARS-Cov-2 were identified through a docking-assisted virtual screening procedure. A total of 4384 drugs, all approved for human use, were screened against three conformers of Mpro. The ligands were further studied through molecular dynamics simulations and binding free energy analysis. A total of nine currently approved molecules are proposed as potential inhibitors of SARS-CoV-2. These molecules can be further tested to speed the development of therapeutics against COVID-19.

The 2009 H1N1 pandemic highlights the need to better understand influenza A infectivity and antigenicity. Relative to other recent seasonal H1N1 influenza strains, the 2009 H1N1 virus grew less efficiently in eggs, which hindered efforts to rapidly supply vaccine. Using lentiviral pseudotypes bearing influenza hemagglutinin (HA-pseudotypes) we evaluated a glutamine to arginine mutation at position 223 (Q223R) and glycosylation at residue 276 in HA for their effects on infectivity and neutralization. Q223R emerged during propagation in eggs and lies in the receptor binding site. We found that the Q223R mutation greatly enhanced infectivity of HA-pseudotypes in human cells, which was further augmented by inclusion of the viral neuraminidase (NA) and M2 proteins. Loss of glycosylation at residue 276 did not alter infectivity. None of these modifications affected neutralization. These findings provide information for increasing 2009 H1N1HA-pseudotype titers without altering antigenicity and offer insights into receptor use.

The last influenza A pandemic provided an excellent opportunity to study the adaptation of the influenza A(H1N1)pdm09 virus to the human host. Particularly, due to the availability of sequences taken from isolates since the beginning of the pandemic until date, we could monitor amino acid changes that occurred in the hemagglutinin (HA) as the virus spread worldwide and became the dominant H1N1 strain. HA is crucial to viral infection because it binds to sialidated cell-receptors and mediates fusion of cell and viral membranes; because antibodies that bind to HA may block virus entry to the cell, this protein is subjected to high selective pressure. Multiple alignment analysis of sequences of the HA from isolates taken since 2009 to date allowed us to find amino acid changes that were positively selected as the pandemic progressed. We found nine changes that became prevalent: HA1 subunits D104N, K166Q, S188T, S206T, A259T, and K285E; and HA2 subunits E47K, S124N, and E172K. Most of these changes were located in areas involved in inter- and intrachain interactions, while only two (K166Q and S188T) were located in known antigenic sites. We conclude that selective pressure on HA was aimed to improve its functionality and hence virus fitness, rather than at avoidance of immune recognition.

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We report that a toxin neutralization assay (TNA) can detect a decrease in the immunogenicity of anthrax vaccines as a consequence of brief exposure to elevated temperature. This attribute of TNA may help in adopting immunogenicity as a replacement of the current potency test, which involves protection from lethal challenge.All anthrax vaccine that enters the Strategic National Stockpile must maintain its potency through protracted storage periods. Thus, robust, practical, and meaningful potency tests are essential to evaluate vaccine manufacturing consistency and stability. The stability of protective antigen (PA), a major antigen included in anthrax vaccines, is critical to establish the suitability of a formulation for long-term storage. PA is denatured at temperatures as low as 40°C (4, 7, 9), which can jeopardize vaccine potency if the product is heated at any time prior to its administration, even during manufacturing. The current potency assay for anthrax vaccines is an active protection test that consumes many animals and requires security and biosafety measures because of the use of virulent Bacillus anthracis. To circumvent these drawbacks, immunogenicity assays have been developed in which lethal challenge is replaced with the measurement of differential antibody induction using in vitro tests.We assessed whether a toxin neutralization assay (TNA) can detect changes in antibody response as a consequence of the exposure of an experimental recombinant PA vaccine (rPAV) and BioThrax, a commercial vaccine, to high temperatures for brief periods. The number of possible time/temperature combinations to which a vaccine can be exposed before use is very high. Therefore, we selected a few combinations to model the possibility of using murine immunogenicity to detect anthrax vaccine exposure to nonideal storage conditions.We used a published method (3), slightly modified, to measure neutralizing activity in mouse sera. The reference serum and samples were prediluted with Dulbecco's modified Eagle medium (DMEM) and serially diluted (1:2) in a 96-well microtiter plate. Predilution was made to achieve full neutralization curves, i.e., to obtain upper and lower asymptotes. Lethal toxin (LT; 100 ng/ml of PA plus 80 ng/ml of lethal factor in DMEM) was added, and mixtures were incubated (37°C and 5% CO 2 ) for 30 min. One well in each column contained only the sample at the lowest dilution tested (sample control [SC]). One column contained normal mouse serum diluted 1:25 in DMEM. Toxin activity was confirmed by the addition of LT to four wells (LT control), while four wells were used to verify cell viability (reagent control). Toxin-serum mixtures were added to J774A.1 cells seeded in a second 96-well plate (40% to 60% confluence) and incubated (37°C and 5% CO 2 ) for 4 h. Cell viability was estimated with a vital dye, MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] (2). The absorbance per well (determined as the change in optical density [⌬OD] at 570 and 690 nm) was transformed to the percentag...