Respiratory mucosal vaccination of peptide-poloxamine-DNA nanoparticles provides complete protection against lethal SARS-CoV-2 challenge.

The ongoing SARS-CoV-2 pandemic represents a brutal reminder of the continual threat of mucosal infectious diseases. Mucosal immunity may provide robust protection at the predominant sites of SARS-CoV-2 infection. However, it remains unclear whether respiratory mucosal administration of DNA vaccines could confer protective immune responses against SARS-CoV-2 challenge due to insurmountable barriers posed by the airway. Here, we applied self-assembled peptide-poloxamine nanoparticles with mucus-penetrating properties for pulmonary inoculation of a COVID-19 DNA vaccine (pSpike/PP-sNp). The pSpike/PP-sNp not only displays superior gene transfection and favorable biocompatibility in the mouse airway, but also promotes a tripartite immunity consisting of systemic, cellular, and mucosal immune responses that are characterized by mucosal IgA secretion, high levels of neutralizing antibodies, and resident memory phenotype T-cell responses in the lungs of mice. Most importantly, immunization with pSpike/PP-sNp completely eliminates SARS-CoV-2 infection in both upper and lower respiratory tracts and enables 100% survival rate of mice following lethal SARS-CoV-2 challenge. Our findings indicate PP-sNp is a promising platform in mediating DNA vaccines to elicit all-around mucosal immunity against SARS-CoV-2.

Efficient Transfection of In vitro Transcribed mRNA in Cultured Cells Using Peptide-Poloxamine Nanoparticles.

In vitro transcribed messenger RNA (mRNA) vaccines have displayed enormous potential in fighting against the coronavirus disease 2019 (COVID-19) pandemic. Efficient and safe delivery systems must be included in the mRNA vaccines due to the fragile properties of mRNA. A self-assembled peptide-poloxamine nanoparticle (PP-sNp) gene delivery system is specifically designed for the pulmonary delivery of nucleic acids and displays promising capabilities in mediating successful mRNA transfection. Here, an improved method for preparing PP-sNp is described to elaborate on how the PP-sNp encapsulates Metridia luciferase (MetLuc) mRNA and successfully transfects cultured cells. MetLuc-mRNA is obtained by an in vitro transcription process from a linear DNA template. A PP-sNp is produced by mixing synthetic peptide/poloxamine with mRNA solution using a microfluidic mixer, allowing for the self-assembly of PP-sNp. The charge of PP-sNp is subsequently evaluated by measuring the zeta potential. Meanwhile, the polydispersity and hydrodynamic size of PP-sNp nanoparticles are measured using dynamic light scattering. The mRNA/PP-sNp nanoparticles are transfected into cultured cells, and supernatants from the cell culture are assayed for luciferase activity. The representative results demonstrate their capacity for in vitro transfection. This protocol may shed light on developing next-generation mRNA vaccine delivery systems.

A "Plug-and-Display" Nanoparticle Vaccine Platform Based on Outer Membrane Vesicles Displaying SARS-CoV-2 Receptor-binding Domain.

Biomimetic nanoparticles obtained from bacteria or viruses have attracted substantial interest in vaccine research and development. Outer membrane vesicles (OMVs) are mainly secreted by gram-negative bacteria during average growth, with a nano-sized diameter and self-adjuvant activity, which may be ideal for vaccine delivery. OMVs have functioned as a multifaceted delivery system for proteins, nucleic acids, and small molecules. To take full advantage of the biological characteristics of OMVs, bioengineered Escherichia coli-derived OMVs were utilized as a carrier and SARS-CoV-2 receptor-binding domain (RBD) as an antigen to construct a "Plug-and-Display" vaccine platform. The SpyCatcher (SC) and SpyTag (ST) domains in Streptococcus pyogenes were applied to conjugate OMVs and RBD. The Cytolysin A (ClyA) gene was translated with the SC gene as a fusion protein after plasmid transfection, leaving a reactive site on the surface of the OMVs. After mixing RBD-ST in a conventional buffer system overnight, covalent binding was formed between the OMVs and RBD. Thus, a multivalent-displaying OMV vaccine was achieved. By replacing with diverse antigens, the OMVs vaccine platform can efficiently display a variety of heterogeneous antigens, thereby potentially rapidly preventing infectious disease epidemics. This protocol describes a precise method for constructing the OMV vaccine platform, including production, purification, bioconjugation, and characterization.

Novel Synthetic Lipopeptides as Potential Mucosal Adjuvants Enhanced SARS-CoV-2 rRBD-Induced Immune Response.

As TLR2 agonists, several lipopeptides had been proved to be candidate vaccine adjuvants. In our previous study, lipopeptides mimicking N-terminal structures of the bacterial lipoproteins were also able to promote antigen-specific immune response. However, the structure-activity relationship of lipopeptides as TLR2 agonists is still unclear. Here, 23 synthetic lipopeptides with the same lipid moiety but different peptide sequences were synthesized, and their TLR2 activities in vitro and mucosal adjuvant effects to OVA were evaluated. LP1-14, LP1-30, LP1-34 and LP2-2 exhibited significantly lower cytotoxicity and stronger TLR2 activity compared with Pam2CSK4, the latter being one of the most potent TLR2 agonists. LP1-34 and LP2-2 assisted OVA to induce more profound specific IgG in sera or sIgA in BALF than Pam2CSK4. Furthermore, the possibility of LP1-34, LP2-2 and Pam2CSK4 as the mucosal adjuvant for the SARS-CoV-2 recombinant RBD (rRBD) was investigated. Intranasally immunized with rRBD plus either the novel lipopeptide or Pam2CSK4 significantly increased the levels of specific serum and respiratory mucosal IgG and IgA, while rRBD alone failed to induce specific immune response due to its low immunogenicity. The novel lipopeptides, especially LP2-2, significantly increased levels of rRBD-induced SARS-CoV-2 neutralizing antibody in sera, BALF and nasal wash. Finally, Support vector machine (SVM) results suggested that charged residues in lipopeptides might be beneficial to the agonist activity, while lipophilic residues might adversely affect the agonistic activity. Figuring out the relationship between peptide sequence in the lipopeptide and its TLR2 activity may lay the foundation for the rational design of novel lipopeptide adjuvant for COVID-19 vaccine.

Progress and Prospects on Vaccine Development against SARS-CoV-2.

In December 2019, the outbreak of pneumonia caused by a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to a serious pandemic in China and other countries worldwide. So far, more than 460,000 confirmed cases were diagnosed in nearly 190 countries, causing globally over 20,000 deaths. Currently, the epidemic is still spreading and there is no effective means to prevent the infection. Vaccines are proved to be the most effective and economical means to prevent and control infectious diseases. Several countries, companies, and institutions announced their programs and progress on vaccine development against the virus. While most of the vaccines are under design and preparation, there are some that have entered efficacy evaluation in animals and initial clinical trials. This review mainly focused on the progress and our prospects on field of vaccine development against SARS-CoV-2.

α-Hemolysin-Aided Oligomerization of the Spike Protein RBD Resulted in Improved Immunogenicity and Neutralization Against SARS-CoV-2 Variants.

The increase in confirmed COVID-19 cases and SARS-CoV-2 variants calls for the development of safe and broad cross-protective vaccines. The RBD of the spike protein was considered to be a safe and effective candidate antigen. However, the low immunogenicity limited its application in vaccine development. Herein, we designed and obtained an RBD heptamer (mHla-RBD) based on a carrier protein-aided assembly strategy. The molecular weight of mHla-RBD is up to 450 kDa, approximately 10 times higher than that of the RBD monomer. When formulated with alum adjuvant, mHla-RBD immunization significantly increased the immunogenicity of RBD, as indicated by increased titers of RBD-specific antibodies, neutralizing antibodies, Th2 cellular immune response, and pseudovirus neutralization activity, when compared to RBD monomer. Furthermore, we confirmed that RBD-specific antibodies predominantly target conformational epitopes, which was approximately 200 times that targeting linear epitopes. Finally, a pseudovirus neutralization assay revealed that neutralizing antibodies induced by mHla-RBD against different SARS-CoV-2 variants were comparable to those against the wild-type virus and showed broad-spectrum neutralizing activity toward different SARS-CoV-2 variants. Our results demonstrated that mHla-RBD is a promising candidate antigen for development of SARS-CoV-2 vaccines and the mHla could serve as a universal carrier protein for antigen design.

The efficacy assessment of convalescent plasma therapy for COVID-19 patients: a multi-center case series.

Convalescent plasma (CP) transfusion has been indicated as a promising therapy in the treatment for other emerging viral infections. However, the quality control of CP and individual variation in patients in different studies make it rather difficult to evaluate the efficacy and risk of CP therapy for coronavirus disease 2019 (COVID-19). We aimed to explore the potential efficacy of CP therapy, and to assess the possible factors associated with its efficacy. We enrolled eight critical or severe COVID-19 patients from four centers. Each patient was transfused with 200-400 mL of CP from seven recovered donors. The primary indicators for clinical efficacy assessment were the changes of clinical symptoms, laboratory parameters, and radiological image after CP transfusion. CP donors had a wide range of antibody levels measured by serology tests which were to some degree correlated with the neutralizing antibody (NAb) level. No adverse events were observed during and after CP transfusion. Following CP transfusion, six out of eight patients showed improved oxygen support status; chest CT indicated varying degrees of absorption of pulmonary lesions in six patients within 8 days; the viral load was decreased to a negative level in five patients who had the previous viremia; other laboratory parameters also tended to improve, including increased lymphocyte counts, decreased C-reactive protein, procalcitonin, and indicators for liver function. The clinical efficacy might be associated with CP transfusion time, transfused dose, and the NAb levels of CP. This study indicated that CP might be a potential therapy for severe patients with COVID-19.