Broadly neutralizing humanized SARS-CoV-2 antibody binds to a conserved epitope on Spike and provides antiviral protection through inhalation-based delivery in non-human primates.

The COVID-19 pandemic represents a global challenge that has impacted and is expected to continue to impact the lives and health of people across the world for the foreseeable future. The rollout of vaccines has provided highly anticipated relief, but effective therapeutics are required to further reduce the risk and severity of infections. Monoclonal antibodies have been shown to be effective as therapeutics for SARS-CoV-2, but as new variants of concern (VoC) continue to emerge, their utility and use have waned due to limited or no efficacy against these variants. Furthermore, cumbersome systemic administration limits easy and broad access to such drugs. As well, concentrations of systemically administered antibodies in the mucosal epithelium, a primary site of initial infection, are dependent on neonatal Fc receptor mediated transport and require high drug concentrations. To reduce the viral load more effectively in the lung, we developed an inhalable formulation of a SARS-CoV-2 neutralizing antibody binding to a conserved epitope on the Spike protein, ensuring pan-neutralizing properties. Administration of this antibody via a vibrating mesh nebulization device retained antibody integrity and resulted in effective distribution of the antibody in the upper and lower respiratory tract of non-human primates (NHP). In comparison with intravenous administration, significantly higher antibody concentrations can be obtained in the lung, resulting in highly effective reduction in viral load post SARS-CoV-2 challenge. This approach may reduce the barriers of access and uptake of antibody therapeutics in real-world clinical settings and provide a more effective blueprint for targeting existing and potentially emerging respiratory tract viruses.

Seven classes of antiviral agents.

The viral epidemics and pandemics have stimulated the development of known and the discovery of novel antiviral agents. About a hundred mono- and combination antiviral drugs have been already approved, whereas thousands are in development. Here, we briefly reviewed 7 classes of antiviral agents: neutralizing antibodies, neutralizing recombinant soluble human receptors, antiviral CRISPR/Cas systems, interferons, antiviral peptides, antiviral nucleic acid polymers, and antiviral small molecules. Interferons and some small molecules alone or in combinations possess broad-spectrum antiviral activity, which could be beneficial for treatment of emerging and re-emerging viral infections.

Phosphorylation Sites in the Hypervariable Domain in Chikungunya Virus nsP3 Are Crucial for Viral Replication.

Chikungunya virus (CHIKV, family Togaviridae) is a mosquito-transmitted alphavirus. The positive-sense RNA genome of CHIKV encodes four nonstructural proteins (nsP1 to nsP4) that are virus-specific subunits of the RNA replicase. Among nsP functions, those of nsP3 are the least understood. The C-terminal hypervariable domain (HVD) in nsP3 is disordered and serves as a platform for interactions with multiple host proteins. For Sindbis virus (SINV) and Semliki Forest virus (SFV), the nsP3 HVD has been shown to be phosphorylated. Deletion of phosphorylated regions has a mild effect on the growth of SFV and SINV in vertebrate cells. Using radiolabeling, we demonstrated that nsP3 in CHIKV and o'nyong-nyong virus is also phosphorylated. We showed that the phosphorylated residues in CHIKV nsP3 are not clustered at the beginning of the HVD. The substitution of 20 Ser/Thr residues located in the N-terminal half of the HVD or 26 Ser/Thr residues located in its C-terminal half with Ala residues reduced the activity of the CHIKV replicase and the infectivity of CHIKV in mammalian cells. Furthermore, the substitution of all 46 potentially phosphorylated residues resulted in the complete loss of viral RNA synthesis and infectivity. The mutations did not affect the interaction of the HVD in nsP3 with the host G3BP1 protein; interactions with CD2AP, BIN1, and FHL1 proteins were significantly reduced but not abolished. Thus, CHIKV differs from SFV and SINV both in the location of the phosphorylated residues in the HVD in nsP3 and, significantly, in their effect on replicase activity and virus infectivity.IMPORTANCE CHIKV outbreaks have affected millions of people, creating a need for the development of antiviral approaches. nsP3 is a component of the CHIKV RNA replicase and is involved in interactions with host proteins and signaling cascades. Phosphorylation of the HVD in nsP3 is important for the virulent alphavirus phenotype. Here, we demonstrate that nsP3 in CHIKV is phosphorylated and that the phosphorylation sites in the HVD are distributed in a unique pattern. Furthermore, the abrogation of some of the phosphorylation sites results in the attenuation of CHIKV, while abolishing all the phosphorylation sites completely blocked its replicase activity. Thus, the phosphorylation of nsP3 and/or the phosphorylation sites in nsP3 have a major impact on CHIKV infectivity. Therefore, they represent promising targets for antiviral compounds and CHIKV attenuation. In addition, this new information offers valuable insight into the vast network of virus-host interactions.

Semliki Forest Virus Chimeras with Functional Replicase Modules from Related Alphaviruses Survive by Adaptive Mutations in Functionally Important Hot Spots.

Alphaviruses (family Togaviridae) include both human pathogens such as chikungunya virus (CHIKV) and Sindbis virus (SINV) and model viruses such as Semliki Forest virus (SFV). The alphavirus positive-strand RNA genome is translated into nonstructural (ns) polyprotein(s) that are precursors for four nonstructural proteins (nsPs). The three-dimensional structures of nsP2 and the N-terminal 2/3 of nsP3 reveal that these proteins consist of several domains. Cleavage of the ns-polyprotein is performed by the strictly regulated protease activity of the nsP2 region. Processing results in the formation of a replicase complex that can be considered a network of functional modules. These modules work cooperatively and should perform the same task for each alphavirus. To investigate functional interactions between replicase components, we generated chimeras using the SFV genome as a backbone. The functional modules corresponding to different parts of nsP2 and nsP3 were swapped with their counterparts from CHIKV and SINV. Although some chimeras were nonfunctional, viruses harboring the CHIKV N-terminal domain of nsP2 or any domain of nsP3 were viable. Viruses harboring the protease part of nsP2, the full-length nsP2 of CHIKV, or the nsP3 macrodomain of SINV required adaptive mutations for functionality. Seven mutations that considerably improved the infectivity of the corresponding chimeric genomes affected functionally important hot spots recurrently highlighted in previous alphavirus studies. These data indicate that alphaviruses utilize a rather limited set of strategies to survive and adapt. Furthermore, functional analysis revealed that the disturbance of processing was the main defect resulting from chimeric alterations within the ns-polyprotein. IMPORTANCE Alphaviruses cause debilitating symptoms and have caused massive outbreaks. There are currently no approved antivirals or vaccines for treating these infections. Understanding the functions of alphavirus replicase proteins (nsPs) provides valuable information for both antiviral drug and vaccine development. The nsPs of all alphaviruses consist of similar functional modules; however, to what extent these are independent in functionality and thus interchangeable among homologous viruses is largely unknown. Homologous domain swapping was used to study the functioning of modules from nsP2 and nsP3 of other alphaviruses in the context of Semliki Forest virus. Most of the introduced substitutions resulted in defects in the processing of replicase precursors that were typically compensated by adaptive mutations that mapped to determinants of polyprotein processing. Understanding the principles of virus survival strategies and identifying hot spot mutations that permit virus adaptation highlight a route to the rapid development of attenuated viruses as potential live vaccine candidates.

Identification of Natural Molecular Determinants of Ross River Virus Type I Interferon Modulation.

Ross River virus (RRV) belongs to the genus Alphavirus and is prevalent in Australia. RRV infection can cause arthritic symptoms in patients and may include rash, fever, arthralgia, and myalgia. Type I interferons (IFN) are the primary antiviral cytokines and trigger activation of the host innate immune system to suppress the replication of invading viruses. Alphaviruses are able to subvert the type I IFN system, but the mechanisms used are ill defined. In this study, seven RRV field strains were analyzed for induction of and sensitivity to type I IFN. The sensitivities of these strains to human IFN-ß varied significantly and were highest for the RRV 2548 strain. Compared to prototype laboratory strain RRV-T48, RRV 2548 also induced higher type I IFN levels both in vitro and in vivo and caused milder disease. To identify the determinants involved in type I IFN modulation, the region encoding the nonstructural proteins (nsPs) of RRV 2548 was sequenced, and 42 amino acid differences from RRV-T48 were identified. Using fragment swapping and site-directed mutagenesis, we discovered that substitutions E402A and R522Q in nsP1 as well as Q619R in nsP2 were responsible for increased sensitivity of RRV 2548 to type I IFN. In contrast, substitutions A31T, N219T, S580L, and Q619R in nsP2 led to induction of higher levels of type I IFN. With exception of E402A, all these variations are common for naturally occurring RRV strains. However, they are different from all known determinants of type I IFN modulation reported previously in nsPs of alphaviruses.IMPORTANCE By identifying natural Ross River virus (RRV) amino acid determinants for type I interferon (IFN) modulation, this study gives further insight into the mechanism of type I IFN modulation by alphaviruses. Here, the crucial role of type I IFN in the early stages of RRV disease pathogenesis is further demonstrated. This study also provides a comparison of the roles of different parts of the RRV nonstructural region in type I IFN modulation, highlighting the importance of nonstructural protein 1 (nsP1) and nsP2 in this process. Three substitutions in nsP1 and nsP2 were found to be independently associated with enhanced type I IFN sensitivity, and four independent substitutions in nsP2 were important in elevated type I IFN induction. Such evidence has clear implications for RRV immunobiology, persistence, and pathology. The identification of viral proteins that modulate type I IFN may also have importance for the pathogenesis of other alphaviruses.

SARS-CoV-2 neutralising antibody testing in Europe: towards harmonisation of neutralising antibody titres for better use of convalescent plasma and comparability of trial data.

We compared the performance of SARS-CoV-2 neutralising antibody testing between 12 European laboratories involved in convalescent plasma trials. Raw titres differed almost 100-fold differences between laboratories when blind-testing 15 plasma samples. Calibration of titres in relation to the reference reagent and standard curve obtained by testing a dilution series reduced the inter-laboratory variability ca 10-fold. The harmonisation of neutralising antibody quantification is a vital step towards determining the protective and therapeutic levels of neutralising antibodies.

Basic insights into Zika virus infection of neuroglial and brain endothelial cells.

Zika virus (ZIKV) has recently emerged as an important human pathogen due to the strong evidence that it causes disease of the central nervous system, particularly microcephaly and Guillain-Barré syndrome. The pathogenesis of disease, including mechanisms of neuroinvasion, may include both invasion via the blood-brain barrier and via peripheral (including cranial) nerves. Cellular responses to infection are also poorly understood. This study characterizes the in vitro infection of laboratory-adapted ZIKV African MR766 and two Asian strains of (1) brain endothelial cells (hCMEC/D3 cell line) and (2) olfactory ensheathing cells (OECs) (the neuroglia populating cranial nerve I and the olfactory bulb; both human and mouse OEC lines) in comparison to kidney epithelial cells (Vero cells, in which ZIKV infection is well characterized). Readouts included infection kinetics, intracellular virus localization, viral persistence and cytokine responses. Although not as high as in Vero cells, viral titres exceeded 104 plaque-forming units (p.f.u.) ml-1 in the endothelial/neuroglial cell types, except hOECs. Despite these substantial titres, a relatively small proportion of neuroglial cells were primarily infected. Immunolabelling of infected cells revealed localization of the ZIKV envelope and NS3 proteins in the cytoplasm; NS3 staining overlapped with that of dsRNA replication intermediate and the endoplasmic reticulum (ER). Infected OECs and endothelial cells produced high levels of pro-inflammatory chemokines. Nevertheless, ZIKV was also able to establish persistent infection in hOEC and hCMEC/D3 cells. Taken together, these results provide basic insights into ZIKV infection of endothelial and neuroglial cells and will form the basis for further study of ZIKV disease mechanisms.

Reverse genetic system, genetically stable reporter viruses and packaged subgenomic replicon based on a Brazilian Zika virus isolate.

Zika virus (ZIKV, genus Flavivirus) has emerged as a major mosquito-transmitted human pathogen, with recent outbreaks associated with an increased incidence of neurological complications, particularly microcephaly and the Guillain-Barré syndrome. Because the virus has only very recently emerged as an important pathogen, research is being hampered by a lack of reliable molecular tools. Here we report an infectious cDNA (icDNA) clone for ZIKV isolate BeH819015 from Brazil, which was selected as representative of South American ZIKV isolated at early stages of the outbreak. icDNA clones were assembled from synthetic DNA fragments corresponding to the consensus sequence of the BeH819015 isolate. Virus rescued from the icDNA clone had properties identical to a natural ZIKV isolate from South America. Variants of the clone-derived virus, expressing nanoluciferase, enhanced green fluorescent or mCherry marker proteins in both mammalian and insect cells and being genetically stable for multiple in vitro passages, were obtained. A ZIKV subgenomic replicon, lacking a prM- and E glycoprotein encoding region and expressing a Gaussia luciferase marker, was constructed and shown to replicate both in mammalian and insect cells. In the presence of the Semliki Forest virus replicon, expressing ZIKV structural proteins, the ZIKV replicon was packaged into virus-replicon particles. Efficient reverse genetic systems, genetically stable marker viruses and packaged replicons offer significant improvements for biological studies of ZIKV infection and disease, as well as for the development of antiviral approaches.

Stress granule components G3BP1 and G3BP2 play a proviral role early in Chikungunya virus replication.

UNLABELLED: Stress granules (SGs) are protein-mRNA aggregates that are formed in response to environmental stresses, resulting in translational inhibition. SGs are generally believed to play an antiviral role and are manipulated by many viruses, including various alphaviruses. GTPase-activating protein (SH3 domain)-binding protein 1 (G3BP1) is a key component and commonly used marker of SGs. Its homolog G3BP2 is a less extensively studied SG component. Here, we demonstrate that Chikungunya virus (CHIKV) infection induces cytoplasmic G3BP1- and G3BP2-containing granules that differ from bona fide SGs in terms of morphology, composition, and behavior. For several Old World alphaviruses it has been shown that nonstructural protein 3 (nsP3) interacts with G3BPs, presumably to inhibit SG formation, and we have confirmed this interaction in CHIKV-infected cells. Surprisingly, CHIKV also relied on G3BPs for efficient replication, as simultaneous depletion of G3BP1 and G3BP2 reduced viral RNA levels, CHIKV protein expression, and viral progeny titers. The G3BPs colocalized with CHIKV nsP2 and nsP3 in cytoplasmic foci, but no colocalization with nsP1, nsP4, or dsRNA was observed. Furthermore, G3BPs could not be detected in a cellular fraction enriched for CHIKV replication/transcription complexes, suggesting that they are not directly involved in CHIKV RNA synthesis. Depletion of G3BPs did not affect viral entry, translation of incoming genomes, or nonstructural polyprotein processing but resulted in severely reduced levels of negative-stranded (and consequently also positive-stranded) RNA. This suggests a role for the G3BPs in the switch from translation to genome amplification, although the exact mechanism by which they act remains to be explored. IMPORTANCE: Chikungunya virus (CHIKV) causes a severe polyarthritis that has affected millions of people since its reemergence in 2004. The lack of approved vaccines or therapeutic options and the ongoing explosive outbreak in the Caribbean underline the importance of better understanding CHIKV replication. Stress granules (SGs) are cytoplasmic protein-mRNA aggregates formed in response to various stresses, including viral infection. The RNA-binding proteins G3BP1 and G3BP2 are essential SG components. SG formation and the resulting translational inhibition are generally considered an antiviral response, and many viruses manipulate or block this process. Late in infection, we and others have observed CHIKV nonstructural protein 3 in cytoplasmic G3BP1- and G3BP2-containing granules. These virally induced foci differed from true SGs and did not appear to represent replication complexes. Surprisingly, we found that G3BP1 and G3BP2 were also needed for efficient CHIKV replication, likely by facilitating the switch from translation to genome amplification early in infection.

The combination of pleconaril, rupintrivir, and remdesivir efficiently inhibits enterovirus infections in vitro, delaying the development of drug-resistant virus variants.

Enteroviruses are a significant global health concern, causing a spectrum of diseases from the common cold to more severe conditions like hand-foot-and-mouth disease, meningitis, myocarditis, pancreatitis, and poliomyelitis. Current treatment options for these infections are limited, underscoring the urgent need for effective therapeutic strategies. To find better treatment option we analyzed toxicity and efficacy of 12 known broad-spectrum anti-enterovirals both individually and in combinations against different enteroviruses in vitro. We identified several novel, synergistic two-drug and three-drug combinations that demonstrated significant inhibition of enterovirus infections in vitro. Specifically, the triple-drug combination of pleconaril, rupintrivir, and remdesivir exhibited remarkable efficacy against echovirus (EV) 1, EV6, EV11, and coxsackievirus (CV) B5, in human lung epithelial A549 cells. This combination surpassed the effectiveness of single-agent or dual-drug treatments, as evidenced by its ability to protect A549 cells from EV1-induced cytotoxicity across seven passages. Additionally, this triple-drug cocktail showed potent antiviral activity against EV-A71 in human intestinal organoids. Thus, our findings highlight the therapeutic potential of the pleconaril-rupintrivir-remdesivir combination as a broad-spectrum treatment option against a range of enterovirus infections. The study also paves the way towards development of strategic antiviral drug combinations with virus family coverage and high-resistance barriers.

Antiviral efficacy of nanomaterial-treated textiles in real-life like exposure conditions.

Due to the growing interest towards reducing the number of potentially infectious agents on critical high-touch surfaces, the popularity of antimicrobially and antivirally active surfaces, including textiles, has increased. The goal of this study was to create antiviral textiles by spray-depositing three different nanomaterials, two types of CeO2 nanoparticles and quaternary ammonium surfactant CTAB loaded SiO2 nanocontainers, onto the surface of a knitted polyester textile and assess their antiviral activity against two coronaviruses, porcine transmissible gastroenteritis virus (TGEV) and severe acute respiratory syndrome virus (SARS CoV-2). Antiviral testing was carried out in small droplets in semi-dry conditions and in the presence of organic soiling, to mimic aerosol deposition of viruses onto the textiles. In such conditions, SARS CoV-2 stayed infectious at least for 24 h and TGEV infected cells even after 72h of semi-dry deposition suggesting that textiles exhibiting sufficient antiviral activity before or at 24 h, can be considered promising. The antiviral efficacy of nanomaterial-deposited textiles was compared with the activity of the same nanomaterials in colloidal form and with positive control textiles loaded with copper nitrate and CTAB. Our results indicated that after deposition onto the textile, CeO2 nanoparticles lost most of their antiviral activity, but antiviral efficacy of CTAB-loaded SiO2 nanocontainers was retained also after deposition. Copper nitrate deposited textile that was used as a positive control, showed relatively high antiviral activity as expected. However, as copper was effectively washed away from the textile already during 1 h, the use of copper for creating antiviral textiles would be impractical. In summary, our results indicated that antiviral activity of textiles cannot be predicted from antiviral efficacy of the deposited compounds in colloid and attention should be paid on prolonged efficacy of antivirally coated textiles.

Boosted production of antibodies that neutralized different SARS-CoV-2 variants in a COVID-19 convalescent following messenger RNA vaccination - a case study.

Vaccinated convalescents do not develop severe COVID-19 after infection with new SARS-CoV-2 variants. We questioned how messenger RNA (mRNA) vaccination of convalescents provides protection from emerging virus variants. From the cohort of 71 convalescent plasma donors, we identified a patient who developed immune response to infection with SARS-CoV-2 variant of 20A clade and who subsequently received mRNA vaccine encoding spike (S) protein of strain of 19A clade. We showed that vaccination increased the production of immune cells and anti-S antibodies in the serum. Serum antibodies neutralized not only 19A and 20A, but also 20B, 20H, 21J, and 21K virus variants. One of the serum antibodies (100F8) completely neutralized 20A, 21J, and partially 21K strains. 100F8 was structurally similar to published Ab188 antibody, which recognized non-conserved epitope on the S protein. We proposed that 100F8 and other serum antibodies of the patient which recognized non- and conserved epitopes of the S protein, could have additive or synergistic effects to neutralize various virus variants. Thus, mRNA vaccination could be beneficial for convalescents because it boosts production of neutralizing antibodies with broad-spectrum activity.

Antibacterial and Antiviral Effects of Ag, Cu and Zn Metals, Respective Nanoparticles and Filter Materials Thereof against Coronavirus SARS-CoV-2 and Influenza A Virus.

Due to the high prevalence of infectious diseases and their concurrent outbreaks, there is a high interest in developing novel materials with antimicrobial properties. Antibacterial and antiviral properties of a range of metal-based nanoparticles (NPs) are a promising means to fight airborne diseases caused by viruses and bacteria. The aim of this study was to test antimicrobial metals and metal-based nanoparticles efficacy against three viruses, namely influenza A virus (H1N1; A/WSN/1933) and coronaviruses TGEV and SARS-CoV-2; and two bacteria, Escherichia coli and Staphylococcus aureus. The efficacy of ZnO, CuO, and Ag NPs and their respective metal salts, i.e., ZnSO4, CuSO4, and AgNO3, was evaluated in suspensions, and the compounds with the highest antiviral efficacy were chosen for incorporation into fibers of cellulose acetate (CA), using electrospinning to produce filter materials for face masks. Among the tested compounds, CuSO4 demonstrated the highest efficacy against influenza A virus and SARS-CoV-2 (1 h IC50 1.395 mg/L and 0.45 mg/L, respectively), followed by Zn salt and Ag salt. Therefore, Cu compounds were selected for incorporation into CA fibers to produce antiviral and antibacterial filter materials for face masks. CA fibers comprising CuSO4 decreased SARS-CoV-2 titer by 0.38 logarithms and influenza A virus titer by 1.08 logarithms after 5 min of contact; after 1 h of contact, SARS-COV-2 virus was completely inactivated. Developed CuO- and CuSO4-based filter materials also efficiently inactivated the bacteria Escherichia coli and Staphylococcus aureus. The metal NPs and respective metal salts were potent antibacterial and antiviral compounds that were successfully incorporated into the filter materials of face masks. New antibacterial and antiviral materials developed and characterized in this study are crucial in the context of the ongoing SARS-CoV-2 pandemic and beyond.

Novel Synergistic Anti-Enteroviral Drug Combinations.

Background: Enterovirus infections affect people around the world, causing a range of illnesses, from mild fevers to severe, potentially fatal conditions. There are no approved treatments for enterovirus infections. Methods: We have tested our library of broad-spectrum antiviral agents (BSAs) against echovirus 1 (EV1) in human adenocarcinoma alveolar basal epithelial A549 cells. We also tested combinations of the most active compounds against EV1 in A549 and human immortalized retinal pigment epithelium RPE cells. Results: We confirmed anti-enteroviral activities of pleconaril, rupintrivir, cycloheximide, vemurafenib, remdesivir, emetine, and anisomycin and identified novel synergistic rupintrivir-vemurafenib, vemurafenib-pleconaril and rupintrivir-pleconaril combinations against EV1 infection. Conclusions: Because rupintrivir, vemurafenib, and pleconaril require lower concentrations to inhibit enterovirus replication in vitro when combined, their cocktails may have fewer side effects in vivo and, therefore, should be further explored in preclinical and clinical trials against EV1 and other enterovirus infections.

Antiviral efficacy of cerium oxide nanoparticles.

Nanomaterials are prospective candidates for the elimination of viruses due to their multimodal mechanisms of action. Here, we tested the antiviral potential of a largely unexplored nanoparticle of cerium dioxide (CeO2). Two nano-CeO2 with opposing surface charge, (+) and (-), were assessed for their capability to decrease the plaque forming units (PFU) of four enveloped and two non-enveloped viruses during 1-h exposure. Statistically significant antiviral activity towards enveloped coronavirus SARS-CoV-2 and influenza virus was registered already at 20 mg Ce/l. For other two enveloped viruses, transmissible gastroenteritis virus and bacteriophage φ6, antiviral activity was evidenced at 200 mg Ce/l. As expected, the sensitivity of non-enveloped viruses towards nano-CeO2 was significantly lower. EMCV picornavirus showed no decrease in PFU until the highest tested concentration, 2000 mg Ce/l and MS2 bacteriophage showed slight non-monotonic response to high concentrations of nano-CeO2(-). Parallel testing of antiviral activity of Ce3+ ions and SiO2 nanoparticles allows to conclude that nano-CeO2 activity was neither due to released Ce-ions nor nonspecific effects of nanoparticulates. Moreover, we evidenced higher antiviral efficacy of nano-CeO2 compared with Ag nanoparticles. This result along with low antibacterial activity and non-existent cytotoxicity of nano-CeO2 allow us to propose CeO2 nanoparticles for specific antiviral applications.

Protective antibodies and T cell responses to Omicron variant after the booster dose of BNT162b2 vaccine.

The high number of mutations in the Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes its immune escape. We report a longitudinal analysis of 111 vaccinated individuals for their antibody levels up to 6 months after the third dose of the BNT162b2 vaccine. After the third dose, the antibody levels decline but less than after the second dose. The booster dose remarkably increases the serum ability to block wild-type or Omicron variant spike protein's receptor-binding domain (RBD) interaction with the angiotensin-converting enzyme 2 (ACE2) receptor, and these protective antibodies persist 3 months later. Three months after the booster dose, memory CD4+ and CD8+ T cells to the wild-type and Omicron variant are detectable in the majority of vaccinated individuals. Our data show that the third dose restores the high levels of blocking antibodies and enhances T cell responses to Omicron.

Mono- and combinational drug therapies for global viral pandemic preparedness.

Broadly effective antiviral therapies must be developed to be ready for clinical trials, which should begin soon after the emergence of new life-threatening viruses. Here, we pave the way towards this goal by reviewing conserved druggable virus-host interactions, mechanisms of action, immunomodulatory properties of available broad-spectrum antivirals (BSAs), routes of BSA delivery, and interactions of BSAs with other antivirals. Based on the review, we concluded that the range of indications of BSAs can be expanded, and new pan- and cross-viral mono- and combinational therapies can be developed. We have also developed a new scoring algorithm that can help identify the most promising few of the thousands of potential BSAs and BSA-containing drug cocktails (BCCs) to prioritize their development during the critical period between the identification of a new virus and the development of virus-specific vaccines, drugs, and therapeutic antibodies.

Bovine colostrum-derived antibodies against SARS-CoV-2 show great potential to serve as prophylactic agents.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to impose a serious burden on health systems globally. Despite worldwide vaccination, social distancing and wearing masks, the spread of the virus is ongoing. One of the mechanisms by which neutralizing antibodies (NAbs) block virus entry into cells encompasses interaction inhibition between the cell surface receptor angiotensin-converting enzyme 2 (ACE2) and the spike (S) protein of SARS-CoV-2. SARS-CoV-2-specific NAb development can be induced in the blood of cattle. Pregnant cows produce NAbs upon immunization, and antibodies move into the colostrum immediately before calving. Here, we immunized cows with SARS-CoV-2 S1 receptor binding domain (RBD) protein in proper adjuvant solutions, followed by one boost with SARS-CoV-2 trimeric S protein and purified immunoglobulins from colostrum. We demonstrate that this preparation indeed blocks the interaction between the trimeric S protein and ACE2 in different in vitro assays. Moreover, we describe the formulation of purified immunoglobulin preparation into a nasal spray. When administered to human subjects, the formulation persisted on the nasal mucosa for at least 4 hours, as determined by a clinical study. Therefore, we are presenting a solution that shows great potential to serve as a prophylactic agent against SARS-CoV-2 infection as an additional measure to vaccination and wearing masks. Moreover, our technology allows for rapid and versatile adaptation for preparing prophylactic treatments against other diseases using the defined characteristics of antibody movement into the colostrum.

Novel functions of the alphavirus nonstructural protein nsP3 C-terminal region.

The functions of the alphavirus-encoded nonstructural protein nsP3 during infection are poorly understood. In contrast, nsP1, nsP2, and nsP4 have known enzymatic activities and functions. A functional analysis of the C-terminal region of nsP3 of Semliki Forest virus revealed the presence of a degradation signal that overlaps with a sequence element located between nsP3 and nsP4 that is required for proteolytic processing. This element was responsible for the short half-life (1 h) of individually expressed nsP3, and it also was functionally transferable to other proteins. Inducible cell lines were used to express native nsP3 or truncated mutants. The removal of 10 C-terminal amino acid (aa) residues from nsP3 increased the half-life of the protein approximately 8-fold. While the deletion of 30 C-terminal aa residues resulted in a similar stabilization, this deletion also changed the cellular localization of nsP3. This truncated mutant no longer exhibited a punctate localization in the cytoplasm, but instead filamentous stretches could be formed around the nuclei of induced cells, suggesting the existence of an additional functional element upstream of the degradation signal. C-terminally truncated uncleavable polyprotein P12(CA)3del30 was localized diffusely, which is in contrast to P12(CA)3, which is known to be associated with vesicle membranes. The induction of nsP3 or its truncated forms reduced the efficiency of virus multiplication in corresponding cells by affecting different steps of the infection cycle. The expression of nsP3 or a mutant lacking the 10 C-terminal aa residues repressed the establishment of infection, while the expression of nsP3 lacking 30 C-terminal aa residues led to the reduced synthesis of subgenomic RNA.

HIV Replication Is Increased by RNA Methylation METTL3/METTL14/WTAP Complex Activators.

The N6-methyladenosine (m6A) modifications in both viral and host cell RNAs play an important role in HIV-1 virus genome transcription and virus replication. We demonstrate here that activators of the METTL3/METTL14/WTAP RNA methyltransferase complex enhance the production of virus particles in cells harboring HIV-1 provirus. In parallel, the amount of m6A residues in the host cell mRNA was increased in the presence of these activator compounds. Importantly, the m6A methylation of the HIV-1 RNA was also enhanced significantly (about 18%). The increase of virus replication by the small-molecule activators of the METTL3/METTL14/WTAP complex excludes them as potential anti-HIV-1 drug candidates. However, the compounds may be of large interest as activators for the latent HIV-1 provirus copies deposited in host cells' genome and the subsequent virus eradication by an antiviral compound.