Comprehensive performance evaluation of six bioaerosol samplers based on an aerosol wind tunnel.

Choosing a suitable bioaerosol sampler for atmospheric microbial monitoring has been a challenge to researchers interested in environmental microbiology, especially during a pandemic. However, a comprehensive and integrated evaluation method to fully assess bioaerosol sampler performance is still lacking. Herein, we constructed a customized wind tunnel operated at 2-20 km/h wind speed to systematically and efficiently evaluate the performance of six frequently used samplers, where various aerosols, including Arizona test dust, bacterial spores, gram-positive and gram-negative bacteria, phages, and viruses, were generated. After 10 or 60 min of sampling, the physical and biological sampling efficiency and short or long-term sampling capabilities were determined by performing aerodynamic particle size analysis, live microbial culturing, and a qPCR assay. The results showed that AGI-30 and BioSampler impingers have good physical and biological sampling efficiencies for short-term sampling. However, their ability to capture aerosols at low concentrations is restricted. SASS 2300 and BSA-350 wet-wall cyclones had excellent enrichment ratios and high microbial cultivability in both short-term and long-term sampling; however, they were not suitable for quantitative studies of aerosols. Polycarbonate filter samplers showed outstanding performance in physical and long-term sampling but lacked the ability to maintain microbial activity, which can be improved by gelatin filter samplers. However, limitations remain for some fragile microorganisms, such as E. coli phage PhiX174 and coronavirus GX_P2V. In addition, the effects of wind speed and direction should be considered when sampling particles larger than 4 µm. This study provides an improved strategy and guidance for the characterization and selection of a bioaerosol sampler for better measurement and interpretation of collected ambient bioaerosols.

An Engineered IgG-VHH Bispecific Antibody against SARS-CoV-2 and Its Variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) neutralizing antibodies are shown to be effective therapeutics for providing coronavirus disease 2019 (COVID-19) protection. However, recurrent variants arise and facilitate significant escape from current antibody therapeutics. Bispecific antibodies (bsAbs) represent a unique platform to increase antibody breadth and to reduce neutralization escape. Herein, a novel immunoglobulin G-variable domains of heavy-chain-only antibody (IgG-VHH) format bsAb derived from a potent human antibody R15-F7 and a humanized nanobody P14-F8-35 are rationally engineered. The resulting bsAb SYZJ001 efficiently neutralizes wild-type SARS-CoV-2 as well as the alpha, beta, gamma, and delta variants, with superior efficacy to its parental antibodies. Cryo-electron microscopy structural analysis reveals that R15-F7 and P14-F8-35 bind to nonoverlapping epitopes within the RBD and sterically hindered ACE2 receptor binding. Most importantly, SYZJ001 shows potent prophylactic and therapeutic efficacy against SARS-CoV-2 in three established mouse models. Collectively, the current results demonstrate that the novel bsAb format is feasible and effective, suggesting great potential as an inspiring antiviral strategy.

Development of a thermostable SARS-CoV-2 variant-based bivalent protein vaccine with cross-neutralizing potency against Omicron subvariants.

SARS-CoV-2 variants have posed significant challenges to the hopes of using ancestral strain-based vaccines to address the risk of breakthrough infection by variants. We designed and developed a bivalent vaccine based on SARS-CoV-2 Alpha and Beta variants (named SCTV01C). SCTV01C antigens were stable at 25 oC for at least 6 months. In the presence of a squalene-based oil-in-water adjuvant SCT-VA02B, SCTV01C showed significant protection efficacy against antigen-matched Beta variant, with favorable safety profiles in rodents. Notably, SCTV01C exhibited cross-neutralization capacity against Omicron subvariants (BA.1, BA.1.1, BA.2, BA.3, and BA.4/5) in mice, superior to a WT (D614G)-based vaccine, which reinforced our previously published findings that SCTV01C exhibited broad-spectrum neutralizing potencies against over a dozen pre-Omicron variants and the Omicron BA.1 variant. In summary, variant-based multivalent protein vaccine could be a platform approach to address the challenging issues of emerging variants, vaccine hesitancy and the needs of affordable and thermal stable vaccines.

Aerosolized Zika virus infection in Guinea pigs.

Zika virus (ZIKV) is primarily transmitted through mosquito bites and sexual contact, and vertical transmission of ZIKV has also been observed in humans. In addition, ZIKV infection via unknown transmission routes has been frequently reported in clinical settings. However, whether ZIKV can be transmitted via aerosol routes remains unknown. In this study, we demonstrated that aerosolized ZIKV is fully infectious in vitro and in vivo. Remarkably, intratracheal (i.t.) inoculation with aerosolized ZIKV led to rapid viremia and viral secretion in saliva, as well as robust humoral and innate immune responses in guinea pigs. Transcriptome analysis further revealed that the expression of genes related to viral processes, biological regulation and the immune response was significantly changed. Together, our results confirm that aerosolized ZIKV can result in systemic infection and induce both innate and adaptive immune responses in guinea pigs, highlighting the possibility of ZIKV transmission via aerosols.

Rational development of a combined mRNA vaccine against COVID-19 and influenza.

As the world continues to experience the COVID-19 pandemic, seasonal influenza remain a cause of severe morbidity and mortality globally. Worse yet, coinfection with SARS-CoV-2 and influenza A virus (IAV) leads to more severe clinical outcomes. The development of a combined vaccine against both COVID-19 and influenza is thus of high priority. Based on our established lipid nanoparticle (LNP)-encapsulated mRNA vaccine platform, we developed and characterized a novel mRNA vaccine encoding the HA antigen of influenza A (H1N1) virus, termed ARIAV. Then, ARIAV was combined with our COVID-19 mRNA vaccine ARCoV, which encodes the receptor-binding domain (RBD) of the SARS-CoV-2 S protein, to formulate the final combined vaccine, AR-CoV/IAV. Further characterization demonstrated that immunization with two doses of AR-CoV/IAV elicited robust protective antibodies as well as antigen-specific cellular immune responses against SARS-CoV-2 and IAV. More importantly, AR-CoV/IAV immunization protected mice from coinfection with IAV and the SARS-CoV-2 Alpha and Delta variants. Our results highlight the potential of the LNP-mRNA vaccine platform in preventing COVID-19 and influenza, as well as other respiratory diseases.

The infectivity and pathogenicity of hepatitis A virus live-attenuated vaccine strain H2 in type I interferon receptor-deficient mice.

Hepatitis A virus (HAV) live-attenuated vaccine H2 strain has been approved for clinical use for decades with ideal safety profiles in nonhuman primate models and humans. Recently, type I interferon (IFN) receptor-deficient mice were shown to be susceptible to HAV infection. Herein, we sought to determine the infection and replication dynamics of the H2 in Ifnar-/- mice that lack type I IFN receptor. Following intravenous injection, the H2 failed to cause obvious clinical symptoms in Ifnar-/- mice, and no significant upregulation in serum alanine aminotransferase (ALT) levels was observed. Notably, the histopathological examination showed that there were significant focal infiltrations of lymphocytes and neutrophils in the portal area, but no focal necrosis was observed in liver tissues. Viral RNAs sustained in the liver, and the infectious virus could be recovered from the liver tissue until 42 days post-infection. More importantly, H2 infection induced obvious viremia and persistent viral shedding in feces. In addition, robust HAV-specific humoral immune responses were induced in Ifnar-/- mice. Overall, our study revealed the safety profile of H2 in Ifnar-/- mice, which not only helps understand the attenuation mechanism of H2, but also expands the application of the Ifnar-/- mouse model for HAV studies.

An experimental method for efficiently evaluating the size-resolved sampling efficiency of liquid-absorption aerosol samplers.

Aerosol samplers are critical tools for studying indoor and outdoor aerosols. Development and evaluation of samplers is often labor-intensive and time-consuming due to the need to use monodisperse aerosols spanning a range of sizes. This study develops a rapid experimental methodology using polydisperse solid aerosols to evaluate size-resolved aerosol-to-aerosol (AtoA) and aerosol-to-hydrosol (AtoH) sampling efficiencies. Arizona Test Dust (diameter 0.5-20 µm) was generated and dispersed into an aerosol test chamber and two candidate samplers were tested. For the AtoA test, aerosols upstream and downstream of a sampler were measured using an online aerodynamic particle sizer. For the AtoH test, aerosols collected in sampling medium were mixed with a reference sample and then measured by the laser diffraction method. The experimental methodology were validated as an impressive time-saving procedure, with reasonable spatial uniformity and time stability of aerosols in the test chamber and an acceptable accuracy of absolute mass quantification of collected particles. Evaluation results showed that the AGI-30 and the BioSampler sampler had similar size-resolved sampling efficiencies and that efficiencies decreased with decreasing sampling flow rate. The combined evaluation of AtoA and AtoH efficiency provided more comprehensive performance indicators than either test alone. The experimental methodology presented here can facilitate the design and choice of aerosol sampler.

Rational Development of a Polysaccharide-Protein-Conjugated Nanoparticle Vaccine Against SARS-CoV-2 Variants and Streptococcus pneumoniae.

The ongoing COVID-19 pandemic caused by SARS-CoV-2 has led to millions of deaths worldwide. Streptococcus pneumoniae (S. pneumoniae) remains a major cause of mortality in underdeveloped countries. A vaccine that prevents both SARS-CoV-2 and S. pneumoniae infection represents a long-sought "magic bullet". Herein, a nanoparticle vaccine, termed SCTV01B, is rationally developed by using the capsular polysaccharide of S. pneumoniae serotype 14 (PPS14) as the backbone to conjugate with the recombinant receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. The final formulation of conjugated nanoparticles in the network structure exhibits high thermal stability. Immunization with SCTV01B induces potent humoral and Type 1/Type 2 T helper cell (Th1/Th2) cellular immune responses in mice, rats, and rhesus macaques. In particular, SCTV01B-immunized serum not only broadly cross-neutralizes all SARS-CoV-2 variants of concern (VOCs), including the most recent Omicron variant, but also shows high opsonophagocytic activity (OPA) against S. pneumoniae serotype 14. Finally, SCTV01B vaccination confers protection against challenges with the SARS-CoV-2 mouse-adapted strain and the original strain in established murine models. Collectively, these promising preclinical results support further clinical evaluation of SCTV01B, highlighting the potency of polysaccharide-RBD-conjugated nanoparticle vaccine platforms for the development of vaccines for COVID-19 and other infectious diseases.

A highly immunogenic live-attenuated vaccine candidate prevents SARS-CoV-2 infection and transmission in hamsters.

The highly pathogenic and readily transmissible SARS-CoV-2 has caused a global coronavirus pandemic, urgently requiring effective countermeasures against its rapid expansion. All available vaccine platforms are being used to generate safe and effective COVID-19 vaccines. Here, we generated a live-attenuated candidate vaccine strain by serial passaging of a SARS-CoV-2 clinical isolate in Vero cells. Deep sequencing revealed the dynamic adaptation of SARS-CoV-2 in Vero cells, resulting in a stable clone with a deletion of seven amino acids (N679SPRRAR685) at the S1/S2 junction of the S protein (named VAS5). VAS5 showed significant attenuation of replication in multiple human cell lines, human airway epithelium organoids, and hACE2 mice. Viral fitness competition assays demonstrated that VAS5 showed specific tropism to Vero cells but decreased fitness in human cells compared with the parental virus. More importantly, a single intranasal injection of VAS5 elicited a high level of neutralizing antibodies and prevented SARS-CoV-2 infection in mice as well as close-contact transmission in golden Syrian hamsters. Structural and biochemical analysis revealed a stable and locked prefusion conformation of the S trimer of VAS5, which most resembles SARS-CoV-2-3Q-2P, an advanced vaccine immunogen (NVAX-CoV2373). Further systematic antigenic profiling and immunogenicity validation confirmed that the VAS5 S trimer presents an enhanced antigenic mimic of the wild-type S trimer. Our results not only provide a potent live-attenuated vaccine candidate against COVID-19 but also clarify the molecular and structural basis for the highly attenuated and super immunogenic phenotype of VAS5.

Lipid nanoparticle-encapsulated mRNA antibody provides long-term protection against SARS-CoV-2 in mice and hamsters.

Monoclonal antibodies represent important weapons in our arsenal to against the COVID-19 pandemic. However, this potential is severely limited by the time-consuming process of developing effective antibodies and the relative high cost of manufacturing. Herein, we present a rapid and cost-effective lipid nanoparticle (LNP) encapsulated-mRNA platform for in vivo delivery of SARS-CoV-2 neutralization antibodies. Two mRNAs encoding the light and heavy chains of a potent SARS-CoV-2 neutralizing antibody HB27, which is currently being evaluated in clinical trials, were encapsulated into clinical grade LNP formulations (named as mRNA-HB27-LNP). In vivo characterization demonstrated that intravenous administration of mRNA-HB27-LNP in mice resulted in a longer circulating half-life compared with the original HB27 antibody in protein format. More importantly, a single prophylactic administration of mRNA-HB27-LNP provided protection against SARS-CoV-2 challenge in mice at 1, 7 and even 63 days post administration. In a close contact transmission model, prophylactic administration of mRNA-HB27-LNP prevented SARS-CoV-2 infection between hamsters in a dose-dependent manner. Overall, our results demonstrate a superior long-term protection against SARS-CoV-2 conferred by a single administration of this unique mRNA antibody, highlighting the potential of this universal platform for antibody-based disease prevention and therapy against COVID-19 as well as a variety of other infectious diseases.

A proof of concept for neutralizing antibody-guided vaccine design against SARS-CoV-2.

Mutations and transient conformational movements of the receptor binding domain (RBD) that make neutralizing epitopes momentarily unavailable present immune escape routes for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To mitigate viral escape, we developed a cocktail of neutralizing antibodies (NAbs) targeting epitopes located on different domains of spike (S) protein. Screening of a library of monoclonal antibodies generated from peripheral blood mononuclear cells of COVID-19 convalescent patients yielded potent NAbs, targeting the N-terminal domain (NTD) and RBD domain of S, effective at nM concentrations. Remarkably, a combination of RBD-targeting NAbs and NTD-binding NAbs, FC05, enhanced the neutralization potency in cell-based assays and an animal model. Results of competitive surface plasmon resonance assays and cryo-electron microscopy (cryo-EM) structures of antigen-binding fragments bound to S unveil determinants of immunogenicity. Combinations of immunogens, identified in the NTD and RBD of S, when immunized in rabbits and macaques, elicited potent protective immune responses against SARS-CoV-2. More importantly, two immunizations of this combination of NTD and RBD immunogens provided complete protection in macaques against a SARS-CoV-2 challenge, without observable antibody-dependent enhancement of infection. These results provide a proof of concept for neutralization-based immunogen design targeting SARS-CoV-2 NTD and RBD.

SARS-CoV-2 infection in the mouse olfactory system.

SARS-CoV-2 infection causes a wide spectrum of clinical manifestations in humans, and olfactory dysfunction is one of the most predictive and common symptoms in COVID-19 patients. However, the underlying mechanism by which SARS-CoV-2 infection leads to olfactory disorders remains elusive. Herein, we demonstrate that intranasal inoculation with SARS-CoV-2 induces robust viral replication in the olfactory epithelium (OE), not the olfactory bulb (OB), resulting in transient olfactory dysfunction in humanized ACE2 (hACE2) mice. The sustentacular cells and Bowman's gland cells in the OE were identified as the major target cells of SARS-CoV-2 before invasion into olfactory sensory neurons (OSNs). Remarkably, SARS-CoV-2 infection triggers massive cell death and immune cell infiltration and directly impairs the uniformity of the OE structure. Combined transcriptomic and quantitative proteomic analyses revealed the induction of antiviral and inflammatory responses, as well as the downregulation of olfactory receptor (OR) genes in the OE from the infected animals. Overall, our mouse model recapitulates olfactory dysfunction in COVID-19 patients and provides critical clues for understanding the physiological basis for extrapulmonary manifestations of COVID-19.

Rational development of a human antibody cocktail that deploys multiple functions to confer Pan-SARS-CoVs protection.

Structural principles underlying the composition and synergistic mechanisms of protective monoclonal antibody cocktails are poorly defined. Here, we exploited antibody cooperativity to develop a therapeutic antibody cocktail against SARS-CoV-2. On the basis of our previously identified humanized cross-neutralizing antibody H014, we systematically analyzed a fully human naive antibody library and rationally identified a potent neutralizing antibody partner, P17, which confers effective protection in animal model. Cryo-EM studies dissected the nature of the P17 epitope, which is SARS-CoV-2 specific and distinctly different from that of H014. High-resolution structure of the SARS-CoV-2 spike in complex with H014 and P17, together with functional investigations revealed that in a two-antibody cocktail, synergistic neutralization was achieved by S1 shielding and conformational locking, thereby blocking receptor attachment and viral membrane fusion, conferring high potency as well as robustness against viral mutation escape. Furthermore, cluster analysis identified a hypothetical 3rd antibody partner for further reinforcing the cocktail as pan-SARS-CoVs therapeutics.

A Thermostable mRNA Vaccine against COVID-19.

There is an urgent need for vaccines against coronavirus disease 2019 (COVID-19) because of the ongoing SARS-CoV-2 pandemic. Among all approaches, a messenger RNA (mRNA)-based vaccine has emerged as a rapid and versatile platform to quickly respond to this challenge. Here, we developed a lipid nanoparticle-encapsulated mRNA (mRNA-LNP) encoding the receptor binding domain (RBD) of SARS-CoV-2 as a vaccine candidate (called ARCoV). Intramuscular immunization of ARCoV mRNA-LNP elicited robust neutralizing antibodies against SARS-CoV-2 as well as a Th1-biased cellular response in mice and non-human primates. Two doses of ARCoV immunization in mice conferred complete protection against the challenge of a SARS-CoV-2 mouse-adapted strain. Additionally, ARCoV is manufactured as a liquid formulation and can be stored at room temperature for at least 1 week. ARCoV is currently being evaluated in phase 1 clinical trials.

Mixing at double-Tee junctions with unequal pipe sizes in water distribution systems.

Pipe flow mixing with various solute concentrations and flow rates at pipe junctions is investigated. The degree of mixing affects contaminant spread in a water distribution system, and many studies have focused on mixing at the cross junctions; however, only a few have focused on double-Tee junctions of unequal pipe diameters. To investigate the solute mixing at such junctions, a series of experiments was conducted in a turbulent regime (Re = 12,500-50,000) with different Reynolds number ratios and connecting pipe lengths. Dimensionless outlet concentrations were found to depend on mixing mechanism at the impinging interface of junctions, where junctions with a larger pipe diameter ratio were associated with more complete mixing. Further, the inlet Reynolds number ratio affected mixing more strongly than the outlet Reynolds number ratio. Finally, the dimensionless connecting pipe length in a double-Tee played an important and complicated role in the flow mixing. The results were used to develop two-dimensional isopleth maps for the calculation of normalized north outlet concentrations.

Effects of Danshen Injection () on inhibiting proliferation and inducing apoptosis through down-regulation of mutant JAK2 gene and its protein phosphorylation in human erythroid leukemic cells.

OBJECTIVE: To explore the effects of Danshen Injection () on inhibition proliferation, inducing apoptosis and its possible mechanisms on human erythroid leukemic (HEL) cells. METHODS: The commercial Chinese patent medicine of Danshen Injection was extracted and isolated from Chinese herb of Salvia miltiorrhiza bung. The inhibition effects of proliferation were assayed by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) method in HEL cells treated by Danshen Injection at various concentrations for 48 h. The cellular apoptosis was observed in morphology, analyzed by flow cytometry with annexin V and propidium iodide (PI) staining, and examined by DNA degradation ladder on agarose gel electrophoresis. Meanwhile, the expression levels of mutant Janus kinasez (JAK2) gene and phosphorylation-JAK2 (P-JAK2) protein were detected by allele specific-polymerase chain reaction and Western blot. RESULTS: The proliferation of HEL cells was effectively inhibited by Danshen Injection in a dose-dependent manner, with suppression rates from 19.46±2.31% to 50.20±5.21%. Typical apoptosis cells was observed in Danshen Injection treated HEL cells, the rates of annexin V positive cells increased obviously in a dose-dependent manner, as well as the DNA degradation ladder of apoptosis revealed on gel electrophoresis. The expression levels of mutant JAK2 gene and P-JAK2 protein reduced gradually with increasing dosage of Danshen injection. CONCLUSION: Danshen Injection could not only significantly inhibit the proliferation, but also induce apoptosis in HEL cells; down-regulation of the mutant JAK2 gene and P-JAK2 protein expressions are probably one of its molecular mechanisms.