A potent, broadly protective vaccine against SARS-CoV-2 variants of concern.

Since the first outbreak in December 2019, SARS-CoV-2 has been constantly evolving and five variants have been classified as Variant of Concern (VOC) by the World Health Organization (WHO). These VOCs were found to enhance transmission and/or decrease neutralization capabilities of monoclonal antibodies and vaccine-induced antibodies. Here, we successfully designed and produced a recombinant COVID-19 vaccine in CHO cells at a high yield. The vaccine antigen contains four hot spot substitutions, K417N, E484K, N501Y and D614G, based on a prefusion-stabilized spike trimer of SARS-CoV-2 (S-6P) and formulated with an Alum/CpG 7909 dual adjuvant system. Results of immunogenicity studies showed that the variant vaccine elicited robust cross-neutralizing antibody responses against SARS-CoV-2 prototype (Wuhan) strain and all 5 VOCs. It further, stimulated a TH1 (T Helper type 1) cytokine profile and substantial CD4+ T cell responses in BALB/c mice and rhesus macaques were recorded. Protective efficacy of the vaccine candidate was evaluated in hamster and rhesus macaque models of SARS-CoV-2. In Golden Syrian hamsters challenged with Beta or Delta strains, the vaccine candidate reduced the viral loads in nasal turbinates and lung tissues, accompanied by significant weight gain and relieved inflammation in the lungs. In rhesus macaque challenged with prototype SARS-CoV-2, the vaccine candidate decreased viral shedding in throat, anal, blood swabs over time, reduced viral loads of bronchus and lung tissue, and effectively relieved the lung pathological inflammatory response. Together, our data demonstrated the broadly neutralizing activity and efficacy of the variant vaccine against both prototype and current VOCs of SARS-CoV-2, justifying further clinical development.

Efficacy of heterologous boosting against SARS-CoV-2 using a recombinant interferon-armed fusion protein vaccine (V-01): a randomized, double-blind and placebo-controlled phase III trial.

Waning of neutralizing titres along with decline of protection efficacy after the second dose of COVID-19 vaccines was observed, including China-made inactivated vaccines. Efficacy of a heterologous boosting using one dose of a recombinant SARS-CoV-2 fusion protein vaccine (V-01) in inactivated vaccine-primed population was studied, aimed to restore the immunity. A randomized, double-blind and placebo-controlled phase III trial was conducted in healthy people aged 18 years or older in Pakistan and Malaysia. Each eligible participant received one dose of the V-01 vaccine developed by Livzon Mabpharm Inc. or placebo within the 3-6 months after the two-dose primary regimen, and was monitored for safety and efficacy. The primary endpoint was protection against confirmed symptomatic SARS-CoV-2 infection. A total of 10,218 participants were randomly assigned to receive a vaccine or placebo. Virus-neutralizing antibodies were assessed in 419 participants. A dramatic increase (11.3-fold; 128.3-1452.8) of neutralizing titres was measured in the V-01 group at 14 days after the booster. Over two months of surveillance, vaccine efficacy was 47.8% (95%CI: 22.6-64.7) according to the intention-to-treat principle. The most common adverse events were transient, mild-to-moderate pain at the injection site, fever, headache, and fatigue. Serious adverse events occurred almost equally in V-01 (0.12%) and placebo (0.16%) groups. The heterologous boosting with the V-01 vaccine was safe and efficacious, which could elicit robust humoral immunity under the epidemic of the Omicron variant.Trial registration: ClinicalTrials.gov identifier: NCT05096832.

Immunogenicity and safety of a recombinant fusion protein vaccine (V-01) against coronavirus disease 2019 in healthy adults: a randomized, double-blind, placebo-controlled, phase II trial.

BACKGROUND: Innovative coronavirus disease 2019 (COVID-19) vaccines, with elevated global manufacturing capacity, enhanced safety and efficacy, simplified dosing regimens, and distribution that is less cold chain-dependent, are still global imperatives for tackling the ongoing pandemic. A previous phase I trial indicated that the recombinant COVID-19 vaccine (V-01), which contains a fusion protein (IFN-PADRE-RBD-Fc dimer) as its antigen, is safe and well tolerated, capable of inducing rapid and robust immune responses, and warranted further testing in additional clinical trials. Herein, we aimed to assess the immunogenicity and safety of V-01, providing rationales of appropriate dose regimen for further efficacy study. METHODS: A randomized, double-blind, placebo-controlled phase II clinical trial was initiated at the Gaozhou Municipal Centre for Disease Control and Prevention (Guangdong, China) in March 2021. Both younger (n = 440; 18-59 years of age) and older (n = 440; ≥60 years of age) adult participants in this trial were sequentially recruited into two distinct groups: two-dose regimen group in which participants were randomized either to follow a 10 or 25 µg of V-01 or placebo given intramuscularly 21 days apart (allocation ratio, 3:3:1, n = 120, 120, 40 for each regimen, respectively), or one-dose regimen groups in which participants were randomized either to receive a single injection of 50 µg of V-01 or placebo (allocation ratio, 3:1, n = 120, 40, respectively). The primary immunogenicity endpoints were the geometric mean titers of neutralizing antibodies against live severe acute respiratory syndrome coronavirus 2, and specific binding antibodies to the receptor binding domain (RBD). The primary safety endpoint evaluation was the frequencies and percentages of overall adverse events (AEs) within 30 days after full immunization. RESULTS: V-01 provoked substantial immune responses in the two-dose group, achieving encouragingly high titers of neutralizing antibody and anti-RBD immunoglobulin, which peaked at day 35 (161.9 [95% confidence interval [CI]: 133.3-196.7] and 149.3 [95%CI: 123.9-179.9] in 10 and 25 µg V-01 group of younger adults, respectively; 111.6 [95%CI: 89.6-139.1] and 111.1 [95%CI: 89.2-138.4] in 10 and 25 µg V-01 group of older adults, respectively), and remained high at day 49 after a day-21 second dose; these levels significantly exceed those in convalescent serum from symptomatic COVID-19 patients (53.6, 95%CI: 31.3-91.7). Our preliminary data show that V-01 is safe and well tolerated, with reactogenicity predominantly being absent or mild in severity and only one vaccine-related grade 3 or worse AE being observed within 30 days. The older adult participants demonstrated a more favorable safety profile compared with those in the younger adult group: with AEs percentages of 19.2%, 25.8%, 17.5% in older adults vs. 34.2%, 23.3%, 26.7% in younger adults at the 10, 25 µg V-01 two-dose group, and 50 µg V-01 one-dose group, respectively. CONCLUSIONS: The vaccine candidate V-01 appears to be safe and immunogenic. The preliminary findings support the advancement of the two-dose, 10 µg V-01 regimen to a phase III trial for a large-scale population-based evaluation of safety and efficacy. TRIAL REGISTRATION: http://www.chictr.org.cn/index.aspx (No. ChiCTR2100045107, http://www.chictr.org.cn/showproj.aspx?proj=124702).

Nasal delivery of broadly neutralizing antibodies protects mice from lethal challenge with SARS-CoV-2 delta and omicron variants.

Multiple new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have constantly emerged, as the delta and omicron variants, which have developed resistance to currently gained neutralizing antibodies. This highlights a critical need to discover new therapeutic agents to overcome the variants mutations. Despite the availability of vaccines against coronavirus disease 2019 (COVID-19), the use of broadly neutralizing antibodies has been considered as an alternative way for the prevention or treatment of SARS-CoV-2 variants infection. Here, we show that the nasal delivery of two previously characterized broadly neutralizing antibodies (F61 and H121) protected K18-hACE2 mice against lethal challenge with SARS-CoV-2 variants. The broadly protective efficacy of the F61 or F61/F121 cocktail antibodies was evaluated by lethal challenge with the wild strain (WIV04) and multiple variants, including beta (B.1.351), delta (B.1.617.2), and omicron (B.1.1.529) at 200 or 1000 TCID50, and the minimum antibody administration doses (5-1.25 â€‹mg/kg body weight) were also evaluated with delta and omicron challenge. Fully prophylactic protections were found in all challenged groups with both F61 and F61/H121 combination at the administration dose of 20 â€‹mg/kg body weight, and corresponding mice lung viral RNA showed negative, with almost all alveolar septa and cavities remaining normal. Furthermore, low-dose antibody treatment induced significant prophylactic protection against lethal challenge with delta and omicron variants, whereas the F61/H121 combination showed excellent results against omicron infection. Our findings indicated the potential use of broadly neutralizing monoclonal antibodies as prophylactic and therapeutic agent for protection of current emerged SARS-CoV-2 variants infection.

Suppression and Activation of Intracellular Immune Response in Initial Severe Acute Respiratory Syndrome Coronavirus 2 Infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently the most important emerging pathogen worldwide, but its early transcriptional dynamics and host immune response remain unclear. Herein, the expression profiles of viral interactions with different types of hosts were comprehensively dissected to shed light on the early infection strategy of SARS-CoV-2 and the host immune response against infection. SARS-CoV-2 was found to exhibit a two-stage transcriptional strategy within the first 24 h of infection, comprising a lag phase that ends with the virus being paused and a log phase that starts when the viral load increases rapidly. Interestingly, the host innate immune response was found not to be activated (latent period) until the virus entered the log stage. Noteworthy, when intracellular immunity is suppressed, SARS-CoV-2 shows a correlation with dysregulation of metal ion homeostasis. Herein, the inhibitory activity of copper ions against SARS-CoV-2 was further validated in in vitro experiments. Coronavirus disease 2019-related genes (including CD38, PTX3, and TCN1) were also identified, which may serve as candidate host-restricted factors for interventional therapy. Collectively, these results confirm that the two-stage strategy of SARS-CoV-2 effectively aids its survival in early infection by regulating the host intracellular immunity, highlighting the key role of interferon in viral infection and potential therapeutic candidates for further investigations on antiviral strategies.

Transcriptomic characteristics and impaired immune function of patients who retest positive for SARS-CoV-2 RNA.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, has become a global public health crisis. Some patients who have recovered from COVID-19 subsequently test positive again for SARS-CoV-2 RNA after discharge from hospital. How such retest-positive (RTP) patients become infected again is not known. In this study, 30 RTP patients, 20 convalescent patients, and 20 healthy controls were enrolled for the analysis of immunological characteristics of their peripheral blood mononuclear cells. We found that absolute numbers of CD4+ T cells, CD8+ T cells, and natural killer cells were not substantially decreased in RTP patients, but the expression of activation markers on these cells was significantly reduced. The percentage of granzyme B-producing T cells was also lower in RTP patients than in convalescent patients. Through transcriptome sequencing, we demonstrated that high expression of inhibitor of differentiation 1 (ID1) and low expression of interferon-induced transmembrane protein 10 (IFITM10) were associated with insufficient activation of immune cells and the occurrence of RTP. These findings provide insight into the impaired immune function associated with COVID-19 and the pathogenesis of RTP, which may contribute to a better understanding of the mechanisms underlying RTP.

Safety and immunogenicity of a recombinant interferon-armed RBD dimer vaccine (V-01) for COVID-19 in healthy adults: a randomized, double-blind, placebo-controlled, Phase I trial.

Safe and effective vaccines are still urgently needed to cope with the ongoing COVID-19 pandemic. Recently, we developed a recombinant COVID-19 vaccine (V-01) containing fusion protein (IFN-PADRE-RBD-Fc dimer) as antigen verified to induce protective immunity against SARS-CoV-2 challenge in pre-clinical study, which supported progression to Phase I clinical trials in humans. A Randomized, double-blind, placebo-controlled Phase I clinical trial was initiated at the Guangdong Provincial Center for Disease Control and Prevention (Gaozhou, China) in February 2021. Healthy adults aged between 18 and 59 years and over 60 years were sequentially enrolled and randomly allocated into three subgroups (1:1:1) either to receive the vaccine (10, 25, and 50 µg) or placebo (V-01: Placebo = 4:1) intramuscularly with a 21-day interval by a sentinel and dose escalation design. The data showed a promising safety profile with approximately 25% vaccine-related overall adverse events (AEs) within 30 days and no grade 3 or worse AEs. Besides, V-01 provoked rapid and strong immune responses, elicited substantially high-titre neutralizing antibodies and anti-RBD IgG peaked at day 35 or 49 after first dose, presented with encouraging immunogenicity at low dose (10 µg) subgroup and elderly participants, which showed great promise to be used as all-aged (18 and above) vaccine against COVID-19. Taken together, our preliminary findings indicate that V-01 is safe and well tolerated, capable of inducing rapid and strong immune responses, and warrants further testing in Phase II/III clinical trials.

Chemiluminescence Immunoassay Based Serological Immunoassays for Detection of SARS-CoV-2 Neutralizing Antibodies in COVID-19 Convalescent Patients and Vaccinated Population.

The development of rapid serological detection methods re urgently needed for determination of neutralizing antibodies in sera. In this study, four rapid methods (ACE2-RBD inhibition assay, S1-IgG detection, RBD-IgG detection, and N-IgG detection) were established and evaluated based on chemiluminescence technology. For the first time, a broadly neutralizing antibody with high affinity was used as a standard for the quantitative detection of SARS-CoV-2 specific neutralizing antibodies in human sera. Sera from COVID-19 convalescent patients (N = 119), vaccinated donors (N = 86), and healthy donors (N = 299) confirmed by microneutralization test (MNT) were used to evaluate the above methods. The result showed that the ACE2-RBD inhibition assay calculated with either ACE2-RBD binding inhibition percentage rate or ACE2-RBD inhibiting antibody concentration were strongly correlated with MNT (r ≥ 0.78, p < 0.0001) and also highly consistent with MNT (Kappa Value ≥ 0.94, p < 0.01). There was also a strong correlation between the two evaluation indices (r ≥ 0.99, p < 0.0001). Meanwhile, S1-IgG and RBD-IgG quantitative detection were also significantly correlated with MNT (r ≥ 0.73, p < 0.0001), and both methods were highly correlated with each other (r ≥ 0.95, p < 0.0001). However, the concentration of N-IgG antibodies showed a lower correlation with the MNT results (r < 0.49, p < 0.0001). The diagnostic assays presented here could be used for the evaluation of SARS-CoV-2 vaccine immunization effect and serological diagnosis of COVID-19 patients, and could also have guiding significance for establishing other rapid serological methods to surrogate neutralization tests for SARS-CoV-2.

Methyltransferase-like 3 Modulates Severe Acute Respiratory Syndrome Coronavirus-2 RNA N6-Methyladenosine Modification and Replication.

The coronavirus disease 2019 pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is an ongoing global public crisis. Although viral RNA modification has been reported based on the transcriptome architecture, the types and functions of RNA modification are still unknown. In this study, we evaluated the roles of RNA N6-methyladenosine (m6A) modification in SARS-CoV-2. Our methylated RNA immunoprecipitation sequencing (MeRIP-Seq) and Nanopore direct RNA sequencing (DRS) analysis showed that SARS-CoV-2 RNA contained m6A modification. Moreover, SARS-CoV-2 infection not only increased the expression of methyltransferase-like 3 (METTL3) but also altered its distribution. Modification of METTL3 expression by short hairpin RNA or plasmid transfection for knockdown or overexpression, respectively, affected viral replication. Furthermore, the viral key protein RdRp interacted with METTL3, and METTL3 was distributed in both the nucleus and cytoplasm in the presence of RdRp. RdRp appeared to modulate the sumoylation and ubiquitination of METTL3 via an unknown mechanism. Taken together, our findings demonstrated that the host m6A modification complex interacted with viral proteins to modulate SARS-CoV-2 replication. IMPORTANCE Internal chemical modifications of viral RNA play key roles in the regulation of viral replication and gene expression. Although potential internal modifications have been reported in SARS-CoV-2 RNA, the function of the SARS-CoV-2 N6-methyladenosine (m6A) modification in the viral life cycle is unclear. In the current study, we demonstrated that SARS-CoV-2 RNA underwent m6A modification by host m6A machinery. SARS-CoV-2 infection altered the expression pattern of methyltransferases and demethylases, while the expression level of methyltransferase-like 3 (METTL3) and fat mass and obesity-associated protein (FTO) was linked to the viral replication. Further study showed that METTL3 interacted with viral RNA polymerase RNA-dependent RNA polymerase (RdRp), which influenced not only the distribution but also the posttranslational modification of METTL3. Our study provided evidence that host m6A components interacted with viral proteins to modulate viral replication.

Antibody Cocktail Exhibits Broad Neutralization Activity Against SARS-CoV-2 and SARS-CoV-2 Variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has precipitated multiple variants resistant to therapeutic antibodies. In this study, 12 high-affinity antibodies were generated from convalescent donors in early outbreaks using immune antibody phage display libraries. Of them, two RBD-binding antibodies (F61 and H121) showed high-affinity neutralization against SARS-CoV-2, whereas three S2-target antibodies failed to neutralize SARS-CoV-2. Following structure analysis, F61 identified a linear epitope located in residues G446-S494, which overlapped with angiotensin-converting enzyme 2 (ACE2) binding sites, while H121 recognized a conformational epitope located on the side face of RBD, outside from ACE2 binding domain. Hence the cocktail of the two antibodies achieved better performance of neutralization to SARS-CoV-2. Importantly, these two antibodies also showed efficient neutralizing activities to the variants including B.1.1.7 and B.1.351, and reacted with mutations of N501Y, E484K, and L452R, indicated that it may also neutralize the recent India endemic strain B.1.617. The unchanged binding activity of F61 and H121 to RBD with multiple mutations revealed a broad neutralizing activity against variants, which mitigated the risk of viral escape. Our findings revealed the therapeutic basis of cocktail antibodies against constantly emerging SARS-CoV-2 variants and provided promising candidate antibodies to clinical treatment of COVID-19 patients infected with broad SARS-CoV-2 variants.

A human antibody of potent efficacy against SARS-CoV-2 in rhesus macaques showed strong blocking activity to B.1.351.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), which causes coronavirus disease-2019 (COVID-19), interacts with the host cell receptor angiotensin-converting enzyme 2 (hACE2) via its spike 1 protein during infection. After the virus sequence was published, we identified two potent antibodies against the SARS-CoV-2 receptor binding domain (RBD) from antibody libraries using a phage-to-yeast (PtY) display platform in only 10 days. Our lead antibody JMB2002, now in a Phase 1 clinical trial (ChiCTR2100042150), showed broad-spectrum in vitro blocking activity against hACE2 binding to the RBD of multiple SARS-CoV-2 variants, including B.1.351 that was reportedly much more resistant to neutralization by convalescent plasma, vaccine sera and some clinical-stage neutralizing antibodies. Furthermore, JMB2002 has demonstrated complete prophylactic and potent therapeutic efficacy in a rhesus macaque disease model. Prophylactic and therapeutic countermeasure intervention of SARS-CoV-2 using JMB2002 would likely slow down the transmission of currently emerged SARS-CoV-2 variants and result in more efficient control of the COVID-19 pandemic.

Profiling of the immune repertoire in COVID-19 patients with mild, severe, convalescent, or retesting-positive status.

Forty-seven samples of peripheral blood mononuclear cells from four groups of coronavirus disease (COVID)-19 patients (mild, severe, convalescent, retesting-positive) and healthy controls were applied to profile the immune repertoire of COVID-19 patients in acute infection or convalescence by transcriptome sequencing and immune-receptor repertoire (IRR) sequencing. Transcriptome analyses showed that genes within principal component group 1 (PC1) were associated with infection and disease severity whereas genes within PC2 were associated with recovery from COVID-19. A "dual-injury mechanism" of COVID-19 severity was related to an increased number of proinflammatory pathways and activated hypercoagulable pathways. A machine-learning model based on the genes associated with inflammatory and hypercoagulable pathways had the potential to be employed to monitor COVID-19 severity. Signature analyses of B-cell receptors (BCRs) and T-cell receptors (TCRs) revealed the dominant selection of longer V-J pairs (e.g., IGHV3-9-IGHJ6 and IGHV3-23-IGHJ6) and continuous tyrosine motifs in BCRs and lower diversity of TCRs. These findings provide potential predictors for COVID-19 outcomes, and new potential targets for COVID-19 treatment.

Contribution of Temperature Increase to Restrain the Transmission of COVID-19.

The COVID-19 outbreak has already become a global pandemic and containing this rapid worldwide transmission is of great challenge. The impacts of temperature and humidity on the COVID-19 transmission rate are still under discussion. Here, we elucidated these relationships by utilizing two unique scenarios, repeated measurement and natural experiment, using the COVID-19 cases reported from January 23 - February 21, 2020, in China. The modeling results revealed that higher temperature was most strongly associated with decreased COVID-19 transmission at a lag time of 8 days. Relative humidity (RH) appeared to have only a slight effect. These findings were verified by assessing SARS-CoV-2 infectivity under the relevant conditions of temperature (4°C-37°C) and RH (> 40%). We concluded that temperature increase made an important, but not determined, contribution to restrain the COVID-19 outbreak in China. It suggests that the emphasis of other effective controlling polices should be strictly implemented to restrain COVID-19 transmission in cold seasons.