A Polysaccharide-RBD-Fc-Conjugated COVID-19 Vaccine, SCTV01A, Showed High Immunogenicity and Low Toxicity in Animal Models.

We previously developed a polysaccharide--RBD-conjugated nanoparticle vaccine which induced protective efficacy against SARS-CoV-2 in a mouse model. Here, we newly developed a vaccine, SCTV01A, by chemically conjugating recombinant SARS-CoV-2 RBD-Fc and PPS14 (Streptococcus pneumoniae serotype type 14 capsular polysaccharide). The immunogenicity and toxicity of SCTV01A were evaluated in animal models. The PPS14 conjugation enhanced the immunogenicity of RBD-Fc in C57BL/6 mice whether formulated with SCT-VA02B or Alum adjuvant. SCTV01A also induced high opsonophagocytic activity (OPA) against S. pneumoniae serotype 14. In addition, SCTV01A stimulated potent neutralizing titers in rhesus macaques and effectively reduced lung inflammation after SARS-CoV-2 infection with neither antibody-dependent enhancement (ADE) nor vaccine-enhanced diseases (VED) phenomenon. Importantly, the long-term toxicity study of SCTV01A in rhesus macaques did not cause any abnormal toxicity and was tolerated at the highest tested dose (120 µg). The existing immunogenicity and toxicological evaluation results have demonstrated the safety and efficacy of SCTV01A, which will be a promising and feasible vaccine to protect against SARS-CoV-2 infection.

An antibody cocktail with broadened mutational resistance and effective protection against SARS-CoV-2.

Neutralizing antibodies have been proven to be highly effective in treating mild and moderate COVID-19 patients, but continuous emergence of SARS-CoV-2 variants poses significant challenges. Antibody cocktail treatments reduce the risk of escape mutants and resistance. In this study, a new cocktail composed of two highly potent neutralizing antibodies (HB27 and H89Y) was developed, whose binding epitope is different from those cocktails that received emergency use authorization. This cocktail showed more potent and balanced neutralizing activities (IC50 0.9-11.3 ng mL-1) against a broad spectrum of SARS-CoV-2 variants over individual HB27 or H89Y antibodies. Furthermore, the cocktail conferred more effective protection against the SARS-CoV-2 Beta variant in an aged murine model than monotherapy. It was shown to prevent SARS-CoV-2 mutational escape in vitro and effectively neutralize 61 types of pseudoviruses harbouring single amino acid mutation originated from variants and escape strains of Bamlanivimab, Casirivimab and Imdevimab with IC50 of 0.6-65 ng mL-1. Despite its breadth of variant neutralization, the HB27+H89Y combo and EUA cocktails lost their potencies against Omicron variant. Our results provide important insights that new antibody cocktails covering different epitopes are valuable tools to counter virus mutation and escape, highlighting the need to search for more conserved epitopes to combat Omicron.

Safety and immunogenicity of a bivalent SARS-CoV-2 protein booster vaccine, SCTV01C, in adults previously vaccinated with mRNA vaccine: A randomized, double-blind, placebo-controlled phase 1/2 clinical trial.

BACKGROUND: Booster vaccination is an efficient way to address the waning protection of vaccines and immune escape of SARS-CoV-2 variants. We aimed to assess the safety and immunogenicity of SCTV01C, a novel bivalent protein vaccine as a booster for people who previously received two doses of mRNA vaccine. METHODS: In this randomized, phase 1/2 trial, adults fully vaccinated with mRNA vaccines 3-24 month earlier were enrolled. Participants received SCTV01C at 20 µg, 40 µg or placebo. The primary endpoints were adverse reactions within 7 days and immunogenicity on Day 28 after vaccination. This trial was registered with ClinicalTrials.gov (NCT05043311). FINDINGS: Between January 27 and April 28, 2022, 234 adults were randomly assigned to receive SCTV01C or placebo. The most common solicited adverse events (AEs) were Grade 1 injection-site pain (10.7%) and pyrexia (6.3%). There were no reports of Grade 3 or above solicited AE, serious AEs or AEs of special interests. On Day 28 post the booster, the geometric mean concentrations (GMCs) of the specific binding IgG antibodies to spike protein for placebo, 20 µg and 40 µg SCTV01C were 1649, 4153 and 5354 BAU/mL, with fold of increase from baseline of 1.0, 2.8 and 3.4-fold, respectively. GMTs of neutralizing antibodies against live Delta variant were 1280, 3542, and 4112, with fold of increase of 1.1, 3.9 and 4.1-fold, respectively; GMTs of neutralizing antibodies against live Omicron variant were 218, 640, and 1083, with fold of increase of 1.1, 4.4 and 5.1-fold, respectively. Participants with low neutralizing antibody titers at baseline (below the lower limit of quantitation) had 64.0 and 49.4-fold of increase in GMTs for Delta and Omicron, respectively. INTERPRETATION: The heterologous booster of SCTV01C was safe, and induced uniformly high cross-neutralization antibody responses against Delta and Omicron variants. FUNDING: Beijing Science and Technology Plan Project (Z221100007922012) and the National Key Research and Development Program of China (2022YFC0870600) supported this study.

A spike-trimer protein-based tetravalent COVID-19 vaccine elicits enhanced breadth of neutralization against SARS-CoV-2 Omicron subvariants and other variants.

Multivalent vaccines combining crucial mutations from phylogenetically divergent variants could be an effective approach to defend against existing and future SARS-CoV-2 variants. In this study, we developed a tetravalent COVID-19 vaccine SCTV01E, based on the trimeric Spike protein of SARS-CoV-2 variants Alpha, Beta, Delta, and Omicron BA.1, with a squalene-based oil-in-water adjuvant SCT-VA02B. In the immunogenicity studies in naïve BALB/c and C57BL/6J mice, SCTV01E exhibited the most favorable immunogenic characteristics to induce balanced and broad-spectrum neutralizing potencies against pre-Omicron variants (D614G, Alpha, Beta, and Delta) and newly emerging Omicron subvariants (BA.1, BA.1.1, BA.2, BA.3, and BA.4/5). Booster studies in C57BL/6J mice previously immunized with D614G monovalent vaccine demonstrated superior neutralizing capacities of SCTV01E against Omicron subvariants, compared with the D614G booster regimen. Furthermore, SCTV01E vaccination elicited naïve and central memory T cell responses to SARS-CoV-2 ancestral strain and Omicron spike peptides. Together, our comprehensive immunogenicity evaluation results indicate that SCTV01E could become an important COVID-19 vaccine platform to combat surging infections caused by the highly immune evasive BA.4/5 variants. SCTV01E is currently being studied in a head-to-head immunogenicity comparison phase 3 clinical study with inactivated and mRNA vaccines (NCT05323461).

A highly efficient needle-free-injection delivery system for mRNA-LNP vaccination against SARS-CoV-2.

Despite the various vaccines that have been developed to combat the coronavirus disease 2019 (COVID-19) pandemic, the persistent and unpredictable mutations of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) require innovative and unremitting solutions to cope with the resultant immune evasion and establish a sustainable immune barrier. Here we introduce a vaccine-delivery system with a combination of a needle-free injection (NFI) device and a SARS-CoV-2-Spike-specific mRNA-Lipid Nanoparticle (LNP) vaccine. The benefits are duller pain and a significant increase of immunogenicity compared to the canonical needle injection (NI). From physicochemical and bioactivity analyses, the structure of the mRNA-LNP maintains stability upon NFI, contradictory to the belief that LNPs are inclined towards destruction under the high-pressure conditions of NFI. Moreover, mRNA-LNP vaccine delivered by NFI induces significantly more binding and neutralizing antibodies against SARS-CoV-2 variants than the same vaccine delivered by NI. Heterogeneous vaccination of BA.5-LNP vaccine with NFI enhanced the generation of neutralizing antibodies against Omicron BA.5 variants in rabbits previously vaccinated with non-BA.5-specific mRNA-LNP or other COVID-19 vaccines. NFI parameters can be adjusted to deliver mRNA-LNP subcutaneously or intramuscularly. Taken together, our results suggest that NFI-based mRNA-LNP vaccination is an effective substitute for the traditional NI-based mRNA-LNP vaccination.

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.

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.

Feasibility Studies of Nebulized SARS-CoV-2 Neutralizing Antibody in Mice and Cynomolgus Monkeys.

PURPOSE: Neutralizing antibodies, administrated through intravenous infusion, have shown to be highly efficacious in treating mild and moderate COVID-19 caused by SARS-CoV-2 infection in the lung. However, antibodies do not transport across the plasma-lung barrier efficiently, and up to 100 mg/kg dose was used in human causing significant supply and cost burdens. This study was to explore the feasibility of nebulized antibodies inhalation delivery as an alternative route. METHODS: HB27, a potent RBD-specific humanized monoclonal antibody (Zhu et al. in National Sci Rev. 8:nwaa297, 2020), showed excellent protection against SARS-CoV-2 in animal model and good safety profile in clinical studies. The pharmacokinetics and preliminary safety of HB27 administrated through the respiratory tract were studied in mice and cynomolgus monkeys here. RESULTS: At a single 5 mg/kg dose, the peak HB27 concentration in mice pulmonary epithelial lining fluid (ELF) reached 857.8 µg/mL, 670-fold higher than the PRNT90 value of 1.28 µg/mL, and maintained above PRNT90 over 240 h. In contrast, when administrated by intravenous injection at a 5 mg/kg dose, the antibody concentrations in mice ELF were below PRNT90 value throughout, and were about 50-fold lower than that in the serum. In cynomolgus monkeys administrated with a single dose through inhalation, the antibody concentration in ELF remained high within 3 days. No drug-related safety concerns were observed in the studies. CONCLUSIONS: The study demonstrated that nebulized neutralizing antibody delivery though inhalation could be a more efficient and efficacious alternative approach for treating COVID-19 and other respiratory infectious diseases, and warrants further evaluation in clinical studies.

Rational Development of a Polysaccharide–Protein-Conjugated Nanoparticle Vaccine Against SARS-CoV-2 Variants and Streptococcus pneumoniae (Adv. Mater. 21/2022)

Nanoparticle Vaccines In article number 2200443, Liangzhi Xie, Chengfeng Qin, and co-workers develop a novel bivalent nanoparticle vaccine that confers protection against infection of multiple SARS-CoV-2 variants and Streptococcus pneumoniae. This universal polysaccharide?protein-conjugated vaccine platform provides a powerful tool to fight against cocirculating viral and bacterial pathogens worldwide.

A Bivalent COVID-19 Vaccine Based on Alpha and Beta Variants Elicits Potent and Broad Immune Responses in Mice against SARS-CoV-2 Variants.

With the emergence and rapid spread of new pandemic variants, especially variants of concern (VOCs), the development of next-generation vaccines with broad-spectrum neutralizing activities is of great importance. In this study, SCTV01C, a clinical stage bivalent vaccine based on trimeric spike extracellular domain (S-ECD) of SARS-CoV-2 variants Alpha (B.1.1.7) and Beta (B.1.351) with a squalene-based oil-in-water adjuvant was evaluated in comparison to its two corresponding (Alpha and Beta) monovalent vaccines in mouse immunogenicity studies. The two monovalent vaccines induced potent neutralizing antibody responses against the antigen-matched variants, but drastic reductions in neutralizing antibody titers against antigen-mismatched variants were observed. In comparison, the bivalent vaccine SCTV01C induced relatively higher and broad-spectrum cross-neutralizing activities against various SARS-CoV-2 variants, including the D614G variant, VOCs (B.1.1.7, B.1.351, P.1, B.1.617.2, B.1.1.529), variants of interest (VOIs) (C.37, B.1.621), variants under monitoring (VUMs) (B.1.526, B.1.617.1, B.1.429, C.36.3) and other variants (B.1.618, 20I/484Q). All three vaccines elicited potent Th1-biased T-cell immune responses. These results provide direct evidence that variant-based multivalent vaccines could play important roles in addressing the critical issue of reduced protective efficacy against the existing and emerging SARS-CoV-2 variants.

Inhibitor screening using microarray identifies the high capacity of neutralizing antibodies to Spike variants in SARS-CoV-2 infection and vaccination.

Rationale: Mutations of SARS-CoV-2, which is responsible for coronavirus disease 2019 (COVID-19), could impede drug development and reduce the efficacy of COVID-19 vaccines. Here, we developed a multiplexed Spike-ACE2 Inhibitor Screening (mSAIS) assay that can measure the neutralizing effect of antibodies across numerous variants of the coronavirus's Spike (S) protein simultaneously. Methods: The SARS-CoV-2 spike variant protein microarrays were prepared by printing 72 S variants onto a chemically-modified glass slides. The neutralization potential of purified anti-S antibodies and serum from convalescent COVID-19 patients and vaccinees to S variants were assessed with the mSAIS assay. Results: We identified new S mutations that are sensitive and resistant to neutralization. Serum from both infected and vaccinated groups with a high titer of neutralizing antibodies (NAbs) displayed a broader capacity to neutralize S variants than serum with low titer NAbs. These data were validated using serum from a large vaccinated cohort (n = 104) with a tiled S peptide microarray. In addition, similar results were obtained using a SARS-CoV-2 pseudovirus neutralization assay specific for wild-type S and five prevalent S variants (D614G, B.1.1.7, B.1.351, P.1, B.1.617.2), thus demonstrating that high antibody diversity is associated with high NAb titers. Conclusions: Our results demonstrate the utility of the mSAIS platform in screening NAbs. Moreover, we show that heterogeneous antibody populations provide a more protective effect against S variants, which may help direct COVID-19 vaccine and drug development.

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.

Rapid and Quantitative In Vitro Evaluation of SARS-CoV-2 Neutralizing Antibodies and Nanobodies.

Neutralizing monoclonal antibodies and nanobodies have shown promising results as potential therapeutic agents for COVID-19. Identifying such antibodies and nanobodies requires evaluating the neutralization activity of a large number of lead molecules via biological assays, such as the virus neutralization test (VNT). These assays are typically time-consuming and demanding on-lab facilities. Here, we present a rapid and quantitative assay that evaluates the neutralizing efficacy of an antibody or nanobody within 1.5 h, does not require BSL-2 facilities, and consumes only 8 µL of a low concentration (ng/mL) sample for each assay run. We tested the human angiotensin-converting enzyme 2 (ACE2) binding inhibition efficacy of seven antibodies and eight nanobodies and verified that the IC50 values of our assay are comparable with those from SARS-CoV-2 pseudovirus neutralization tests. We also found that our assay could evaluate the neutralizing efficacy against three widespread SARS-CoV-2 variants. We observed increased affinity of these variants for ACE2, including the ß and γ variants. Finally, we demonstrated that our assay enables the rapid identification of an immune-evasive mutation of the SARS-CoV-2 spike protein, utilizing a set of nanobodies with known binding epitopes.

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.

Safety, Tolerability, Pharmacokinetics, and Immunogenicity of a Monoclonal Antibody (SCTA01) Targeting SARS-CoV-2 in Healthy Adults: a Randomized, Double-Blind, Placebo-Controlled, Phase I Study.

SCTA01 is a novel monoclonal antibody with promising prophylactic and therapeutic potential for COVID-19. This study aimed to evaluate the safety, tolerability, pharmacokinetics (PK) and immunogenicity of SCTA01 in healthy adults. This was a randomized, double-blind, placebo-controlled, dose escalation phase I clinical trial. Healthy adults were randomly assigned to cohort 1 (n = 5; 3:2), cohort 2 (n = 8; 6:2), cohort 3, or cohort 4 (both n = 10; 8:2) to receive SCTA01 (5, 15, 30, and 50 mg/kg, respectively) versus placebo. All participants were followed up for clinical, laboratory, PK, and immunogenicity assessments for 84 days. The primary outcomes were the dose-limiting toxicity (DLT) and maximal tolerable dose (MTD), and the secondary outcomes included PK parameters, immunogenicity, and adverse events (AE). Of the 33 participants, 18 experienced treatment-related AEs; the frequency was 52.0% (13/25) in participants receiving SCTA01 and 62.5% (5/8) in those receiving placebo. All AEs were mild. There was no serious AE or death. No DLT was reported, and the MTD of SCTA01 was not reached. SCTA01 with a dose range of 5 to 50 mg/kg had nearly linear dose-proportional increases in Cmax and AUC parameters. An antidrug antibody response was detected in four (16.0%) participants receiving SCTA01, with low titers, between the baseline and day 28, but all became negative later. In conclusion, SCTA01 up to 50 mg/kg was safe and well-tolerated in healthy participants. Its PK parameters were nearly linear dose-proportional. (This study has been registered at ClinicalTrials.gov under identifier NCT04483375.).

SARS-CoV-2 501Y.V2 variants lack higher infectivity but do have immune escape.

The 501Y.V2 variants of SARS-CoV-2 containing multiple mutations in spike are now dominant in South Africa and are rapidly spreading to other countries. Here, experiments with 18 pseudotyped viruses showed that the 501Y.V2 variants do not confer increased infectivity in multiple cell types except for murine ACE2-overexpressing cells, where a substantial increase in infectivity was observed. Notably, the susceptibility of the 501Y.V2 variants to 12 of 17 neutralizing monoclonal antibodies was substantially diminished, and the neutralization ability of the sera from convalescent patients and immunized mice was also reduced for these variants. The neutralization resistance was mainly caused by E484K and N501Y mutations in the receptor-binding domain of spike. The enhanced infectivity in murine ACE2-overexpressing cells suggests the possibility of spillover of the 501Y.V2 variants to mice. Moreover, the neutralization resistance we detected for the 501Y.V2 variants suggests the potential for compromised efficacy of monoclonal antibodies and vaccines.

Double lock of a potent human therapeutic monoclonal antibody against SARS-CoV-2.

Receptor recognition and subsequent membrane fusion are essential for the establishment of successful infection by SARS-CoV-2. Halting these steps can cure COVID-19. Here we have identified and characterized a potent human monoclonal antibody, HB27, that blocks SARS-CoV-2 attachment to its cellular receptor at sub-nM concentrations. Remarkably, HB27 can also prevent SARS-CoV-2 membrane fusion. Consequently, a single dose of HB27 conferred effective protection against SARS-CoV-2 in two established mouse models. Rhesus macaques showed no obvious adverse events when administrated with 10 times the effective dose of HB27. Cryo-EM studies on complex of SARS-CoV-2 trimeric S with HB27 Fab reveal that three Fab fragments work synergistically to occlude SARS-CoV-2 from binding to the ACE2 receptor. Binding of the antibody also restrains any further conformational changes of the receptor binding domain, possibly interfering with progression from the prefusion to the postfusion stage. These results suggest that HB27 is a promising candidate for immuno-therapies against COVID-19.