Association of fat-soluble vitamins (A, D, and E) status with humoral immune response to COVID-19 inactivated vaccination.

Background: Fat-soluble vitamins (A, D, and E) are essential for the proper functioning of the immune system and are of central importance for infection risk in humans. Vitamins A, D, and E have been reported to be associated with the immune response following vaccination; however, their effects on the immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination remain unknown. Methods: We measured the neutralizing antibody titers against wild type and omicron within 98 days after the third homologous boosting shot of inactivated SARS-CoV-2 vaccine (BBIBP-CorV or CoronaVac) in 141 healthy adults in a prospective, open-label study. High-performance liquid chromatography-tandem mass spectroscopy was used to determine the concentrations of plasma vitamins A, D, and E. Results: We found that the anti-wide-type virus and anti-omicron variant antibody levels significantly increased compared with baseline antibody levels (P < 0.001) after the third vaccination. 25(OH)D3 was significantly negatively associated with the baseline anti-wide-type virus antibody concentrations [beta (95% CI) = -0.331 (-0.659 ~ -0.003)] after adjusting for covariates. A potentially similar association was also observed on day 98 after the third vaccination [beta (95% CI) = -0.317 (-0.641 ~ 0.007)]. After adjusting for covariates, we also found that 25(OH)D3 was significantly negatively associated with the seropositivity of the anti-omicron variant antibody at day 98 after the third vaccination [OR (95% CI) = 0.940 (0.883 ~ 0.996)]. The association between plasma 25(OH)D3 with anti-wild-type virus antibody levels and seropositivity of anti-omicron variant antibodies were persistent in subgroup analyses. We observed no association between retinol/α-tocopherol and anti-wide-type virus antibody levels or anti-omicron variant antibody seropositive in our study. Conclusion: The third inactivated SARS-CoV-2 vaccination significantly improved the ability of anti-SARS-CoV-2 infection in the human body. Higher vitamin D concentrations could significantly decrease the anti-wide-type virus-neutralizing antibody titers and anti-omicron variant antibody seropositive rate after the inactivated SARS-CoV-2 vaccination in people with adequate levels of vitamin D, better immune status, and stronger immune response; further studies comprising large cohorts of patients with different nutritional status are warranted to verify our results.

Synergistic Immunity and Protection in Mice by Co-Immunization with DNA Vaccines Encoding the Spike Protein and Other Structural Proteins of SARS-CoV-2.

The emergence of new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has generated recurring worldwide infection outbreaks. These highly mutated variants reduce the effectiveness of current coronavirus disease 2019 (COVID-19) vaccines, which are designed to target only the spike (S) protein of the original virus. Except for the S of SARS-CoV-2, the immunoprotective potential of other structural proteins (nucleocapsid, N; envelope, E; membrane, M) as vaccine target antigens is still unclear and worthy of investigation. In this study, synthetic DNA vaccines encoding four SARS-CoV-2 structural proteins (pS, pN, pE, and pM) were developed, and mice were immunized with three doses via intramuscular injection and electroporation. Notably, co-immunization with two DNA vaccines that expressed the S and N proteins induced higher neutralizing antibodies and was more effective in reducing the SARS-CoV-2 viral load than the S protein alone in mice. In addition, pS co-immunization with either pN or pE + pM induced a higher S protein-specific cellular immunity after three immunizations and caused milder histopathological changes than pS alone post-challenge. The role of the conserved structural proteins of SARS-CoV-2, including the N/E/M proteins, should be investigated further for their applications in vaccine design, such as mRNA vaccines.

External quality assessment of nucleic acid testing in provincial or municipal CDC laboratories in the COVID-19 surveillance network in China

Objective: To understand the RNA detection performance of SARS-CoV-2 Variants of Concern (VOCs) in 32 provincial or municipal CDC laboratories in COVID-19 surveillance network in China through an external quality assessment (EQA), and evaluate the clinical performance of the current SARS-CoV-2 nucleic acids detection kits used and provided by the participating laboratories.

Factors associated with neutralizing antibody levels induced by two inactivated COVID-19 vaccines for 12 months after primary series vaccination.

Background: BBIBP-CorV and CoronaVac inactivated COVID-19 vaccines are widely-used, World Health Organization-emergency-listed vaccines. Understanding antibody level changes over time after vaccination is important for booster dose policies. We evaluated neutralizing antibody (nAb) titers and associated factors for the first 12 months after primary-series vaccination with BBIBP-CorV and CoronaVac. Methods: Our study consisted of a set of cross-sectional sero-surveys in Zhejiang and Shanxi provinces, China. In 2021, we enrolled 1,527 consenting 18-59-year-olds who received two doses of BBIBP-CorV or CoronaVac 1, 3, 6, 9, or 12 months earlier and obtained blood samples and demographic and medical data. We obtained 6-month convalescent sera from 62 individuals in Hebei province. Serum nAb titers were measured by standard micro-neutralization cytopathic effect assay in Vero cells with ancestral SARS-CoV-2 strain HB01. We used the first WHO International Standard (IS) for anti-SARS-CoV-2 immunoglobulin (NIBSC code 20/136) to standardized geometric mean concentrations (IU/mL) derived from the nAb geometric mean titers (GMT over 1:4 was considered seropositive). We analyzed nAb titer trends using Chi-square and factors related to nAb titers with logistic regression and linear models. Results: Numbers of subjects in each of the five month-groupings ranged from 100 to 200 for each vaccine and met group-specific target sample sizes. Seropositivity rates from BBIBP-CorV were 98.0% at 1 month and 53.5% at 12 months, and GMTs were 25.0 and 4.0. Respective seropositivity rates from CoronaVac were 90.0% and 62.5%, and GMTs were 20.2 and 4.1. One-, three-, six-, nine-, and twelve-month GMCs were 217.2, 84.1, 85.7, 44.6, and 10.9 IU/mL in BBIBP-CorV recipients and 195.7, 94.6, 51.7, 27.6, and 13.4 IU/mL in CoronaVac recipients. Six-month convalescent seropositivity was 95.2%; GMC was 108.9 IU/mL. Seropositivity and GMCs were associated with age, sex, and time since vaccination. Conclusions: Neutralizing Ab levels against ancestral SARS-CoV-2 from BBIBP-CorV or CoronaVac vaccination were similar and decreased with increasing time since vaccination; over half of 12-month post-vaccination subjects were seropositive. Seropositivity and GMCs from BBIBP-CorV and CoronaVac six and nine months after vaccination were similar to or slightly lower than in six-month convalescent sera. These real-world data suggest necessity of six-month booster doses.

Safety, immunogenicity, and immune persistence of two inactivated COVID-19 vaccines replacement vaccination in China: An observational cohort study.

BACKGROUND: To mitigate a national shortage of WIBP-CorV COVID-19 vaccine, China's regulator approved administering BBIBP-CorV after WIBP-CorV for completion of a primary series. In a pragmatic observational study, we compared immunogenicity and safety of a primary series of WIBP-CorV followed by BBIBP-CorV with a primary series of two doses of BBIBP-CorV. METHODS: We invited healthy 18-59-years-old adults who had already received either WIBP-CorV or BBIBP-CorV as their first dose in a primary series to participate in this observational cohort study. Subjects who had received WIBP-CorV as their first dose became the observation group; subjects who had received BBIBP-CorV as their first dose became the control group. All participants received BBIBP-CorV as their second dose. We obtained sera 1, 2, and 6 months after second doses for nAb titer measurement by micro-neutralization cytopathic effect assay with SARS-CoV-2 strain HB01, standardized with WHO International Standard for anti-SARS-CoV-2 immunoglobulin. Safety was assessed for the 7 days after administration of second doses. RESULTS: Between March and December 2021, 275 subjects were included in the observation group and 133 in the control group. Neutralizing seropositivity (≥1:4) rates were 98.91 % and 99.25 % at 1 month and 53.16 % and 70.69 % at 6 months. One-month geometric mean titers (GMTs) were 21.33 and 22.45; one-month geometric mean concentrations (GMCs) were 227.71 IU/mL and 273.27 IU/mL. One to two months after vaccination, observation group seropositivity rates and titers were not significantly different to the control group's. Adverse reaction rates were 11.27 % and 18.80 %, all mild or moderate in severity. CONCLUSIONS: Both primary series were immunogenic; immunogenicity of WIBP-CorV followed by BBIBP-CorV was not different than immunogenicity following two doses of BBIBP-CorV for two months after vaccination; safety profiles were acceptable for both regimens. BBIBP-CorV can be used to complete a primary series that started with WIBP-CorV.

Novaferon Effectively Inhibits Ancestral SARS-CoV-2 and Omicron Variant in Vitro, 2022.

Introduction: To identify Novaferon (Nova), a novel recombinant protein of interferon (IFN)-α, antiviral activity against ancestral severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Omicron variant in vitro. Methods: Vero cells were infected with SARS-CoV-2 and Omicron variant in a biosafety level-3 laboratory. And viral replications were accessed using quantitative real-time reverse transcription polymerase chain reaction (RT-PCR). Results: Results demonstrated that Nova has effective inhibition against ancestral SARS-CoV-2 and Omicron variant in vitro. Discussion: The in vivo effects of Nova need to be further tested in animal models. And large-scale randomized double-blind clinical trials are needed to reveal its potentially clinical application.

DNA Vaccines Expressing the Envelope and Membrane Proteins Provide Partial Protection Against SARS-CoV-2 in Mice.

The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a public health emergency of international concern, and an effective vaccine is urgently needed to control the pandemic. Envelope (E) and membrane (M) proteins are highly conserved structural proteins among SARS-CoV-2 and SARS-CoV and have been proposed as potential targets for the development of cross-protective vaccines. Here, synthetic DNA vaccines encoding SARS-CoV-2 E/M proteins (called p-SARS-CoV-2-E/M) were developed, and mice were immunised with three doses via intramuscular injection and electroporation. Significant cellular immune responses were elicited, whereas no robust humoral immunity was detected. In addition, novel H-2d-restricted T-cell epitopes were identified. Notably, although no drop in lung tissue virus titre was detected in DNA-vaccinated mice post-challenge with SARS-CoV-2, immunisation with either p-SARS-CoV-2-E or p-SARS-CoV-2-M provided minor protection and co-immunisation with p-SARS-CoV-2-E+M increased protection. Therefore, E/M proteins should be considered as vaccine candidates as they may be valuable in the optimisation of vaccination strategies against COVID-19.

Immunogenicity and Safety of a 3-Dose Regimen of a SARS-CoV-2 Inactivated Vaccine in Adults: A Randomized, Double-Blind, Placebo-Controlled Phase 2 Trial.

BACKGROUND: Control of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic needs effective vaccines. METHODS: In a phase 2 randomized, double-blind, placebo-controlled trial, 500 adults aged 18-59 years or ≥60 years were randomized in 2:2:1 ratio to receive 3 doses of 5 µg or 10 µg of a SARS-CoV-2 inactivated vaccine, or placebo separated by 28 days. Adverse events (AEs) were recorded through day 28 after each dosing. Live virus or pseudovirus neutralizing antibodies, and receptor binding domain immunoglobulin G (RBD-IgG) antibody were tested after the second and third doses. RESULTS: Two doses of the vaccine elicited geometric mean titers (GMTs) of 102-119, 170-176, and 1449-1617 for the 3 antibodies in younger adults. Pseudovirus neutralizing and RBD-IgG GMTs were similar between older and younger adults. The third dose slightly (<1.5 fold) increased GMTs. Seroconversion percentages were 94% or more after 2 doses, which were generally similar after 3 doses. The predominant AEs were injection-site pain. All the AEs were grade 1 or 2 in intensity. No serious AE was deemed related to study vaccination. CONCLUSIONS: Two doses of this vaccine induced robust immune response and had good safety profile. A third dose given 28 days after the second dose elicited limited boosting antibody response.

A core-shell structured COVID-19 mRNA vaccine with favorable biodistribution pattern and promising immunity.

Although inoculation of COVID-19 vaccines has rolled out globally, there is still a critical need for safe and effective vaccines to ensure fair and equitable supply for all countries. Here, we report on the development of a highly efficacious mRNA vaccine, SW0123 that is composed of sequence-modified mRNA encoding the full-length SARS-CoV-2 Spike protein packaged in core-shell structured lipopolyplex (LPP) nanoparticles. SW0123 is easy to produce using a large-scale microfluidics-based apparatus. The unique core-shell structured nanoparticle facilitates vaccine uptake and demonstrates a high colloidal stability, and a desirable biodistribution pattern with low liver targeting effect upon intramuscular administration. Extensive evaluations in mice and nonhuman primates revealed strong immunogenicity of SW0123, represented by induction of Th1-polarized T cell responses and high levels of antibodies that were capable of neutralizing not only the wild-type SARS-CoV-2, but also a panel of variants including D614G and N501Y variants. In addition, SW0123 conferred effective protection in both mice and non-human primates upon SARS-CoV-2 challenge. Taken together, SW0123 is a promising vaccine candidate that holds prospects for further evaluation in humans.

Non-invasive bioluminescence imaging of HCoV-OC43 infection and therapy in the central nervous system of live mice.

Human coronaviruses (HCoVs) are important pathogens that cause upper respiratory tract infections and have neuroinvasive abilities; however, little is known about the dynamic infection process of CoVs in vivo, and there are currently no specific antiviral drugs to prevent or treat HCoV infection. Here, we verified the replication ability and pathogenicity of a reporter HCoV-OC43 strain expressing Renilla luciferase (Rluc; rOC43-ns2DelRluc) in mice with different genetic backgrounds (C57BL/6 and BALB/c). Additionally, we monitored the spatial and temporal progression of HCoV-OC43 through the central nervous system (CNS) of live BALB/c mice after intranasal or intracerebral inoculation with rOC43-ns2DelRluc. We found that rOC43-ns2DelRluc was fatal to suckling mice after intranasal inoculation, and that viral titers and Rluc expression were detected in the brains and spinal cords of mice infected with rOC43-ns2DelRluc. Moreover, viral replication was initially observed in the brain by non-invasive bioluminescence imaging before the infection spread to the spinal cord of BALB/c mice, consistent with its tropism in the CNS. Furthermore, the Rluc readout correlated with the HCoV replication ability and protein expression, which allowed quantification of antiviral activity in live mice. Additionally, we validated that chloroquine strongly inhibited rOC43-ns2DelRluc replication in vivo. These results provide new insights into the temporal and spatial dissemination of HCoV-OC43 in the CNS, and our methods provide an extremely sensitive platform for evaluating the efficacy of antiviral therapies to treat neuroinvasive HCoVs in live mice.

Development and effectiveness of pseudotyped SARS-CoV-2 system as determined by neutralizing efficiency and entry inhibition test in vitro.

With the development of the COVID-19 epidemic, there is an urgent need to establish a system for determining the effectiveness and neutralizing activity of vaccine candidates in biosafety level 2 (BSL-2) facilities. Previously, researchers had developed a pseudotyped virus system for SARS-CoV and MERS-CoV, based on HIV-1 core, bearing virus spike protein. During the development of a pseudotyped SARS-CoV-2 system, a eukaryotic expression plasmid expressing SARS-CoV-2 spike (S) protein was constructed and then co-transfected with HIV-1 based plasmid which containing the firefly luciferase reporter gene, into HEK293T cells to prepare the pseudotyped SARS-CoV-2 virus (ppSARS-2). We have successfully established the pseudotyped SARS-CoV-2 system for neutralization and entry inhibition assays. Huh7.5 cell line was found to be the most susceptible to our pseudotyped virus model. Different levels of neutralizing antibodies were detected in convalescent serum samples of COVID-19 patients using ppSARS-2. The recombinant, soluble, angiotensin-converting enzyme 2 protein was found to inhibit the entry of ppSARS-2 in Huh7.5 cells effectively. Furthermore, the neutralization results for ppSARS-2 were consistent with those of live SARS-CoV-2 and determined using the serum samples from convalescent patients. In conclusion, we have developed an easily accessible and reliable tool for studying the neutralizing efficiency of antibodies against SARS-CoV-2 and the entry process of the virus in a BSL-2 laboratory.

Morphogenesis and cytopathic effect of SARS-CoV-2 infection in human airway epithelial cells.

SARS-CoV-2, a ß-coronavirus, has rapidly spread across the world, highlighting its high transmissibility, but the underlying morphogenesis and pathogenesis remain poorly understood. Here, we characterize the replication dynamics, cell tropism and morphogenesis of SARS-CoV-2 in organotypic human airway epithelial (HAE) cultures. SARS-CoV-2 replicates efficiently and infects both ciliated and secretory cells in HAE cultures. In comparison, HCoV-NL63 replicates to lower titers and is only detected in ciliated cells. SARS-CoV-2 shows a similar morphogenetic process as other coronaviruses but causes plaque-like cytopathic effects in HAE cultures. Cell fusion, apoptosis, destruction of epithelium integrity, cilium shrinking and beaded changes are observed in the plaque regions. Taken together, our results provide important insights into SARS-CoV-2 cell tropism, replication and morphogenesis.

Lack of antibody-mediated cross-protection between SARS-CoV-2 and SARS-CoV infections.

BACKGROUND: The novel coronavirus (SARS-CoV-2) shares approximately 80% whole genome sequence identity and 66% spike (S) protein identity with that of SARS-CoV. The cross-neutralization between these viruses is currently not well-defined. METHODS: Here, by using the live SARS-CoV-2 virus infection assay as well as HIV-1 based pseudotyped-virus carrying the spike (S) gene of the SARS-CoV-2 (ppSARS-2) and SARS-CoV (ppSARS), we examined whether infections with SARS-CoV and SARS-CoV-2 can induce cross-neutralizing antibodies. FINDINGS: We confirmed that SARS-CoV-2 infects cells via angiotensin converting enzyme 2 (ACE2), the functional receptor for SARS-CoV, and we also found that the recombinant receptor binding domain (RBD) of the S protein of SARS-CoV effectively inhibits ppSARS-2 entry in Huh7.5 cells. However, convalescent sera from SARS-CoV and SARS-CoV-2 patients showed high neutralizing activity only against the homologous virus, with no or limited cross-neutralization activity against the other pseudotyped virus. Similar results were also observed in vaccination studies in mice. INTERPRETATION: Our study demonstrates that although both SARS-CoV and SARS-CoV-2 use ACE2 as a cellular receptor, the neutralization epitopes are not shared by these two closely-related viruses, highlighting challenges towards developing a universal vaccine against SARS-CoV related viruses. FUNDING: This work was supported by the National Key Research and Development Program of China, the National Major Project for Control and Prevention of Infectious Disease in China, and the One Belt and One Road Major Project for infectious diseases.