Biodegradable Polyester Nanoparticle Vaccines Deliver Self-Amplifying mRNA in Mice at Low Doses

Delivery of self-amplifying mRNA (SAM) has high potential for infectious disease vaccination due to its self-adjuvanting and dose-sparing properties. Yet a challenge is the susceptibility of SAM to degradation and the need for SAM to reach the cytosol fully intact to enable self-amplification. Lipid nanoparticles are successfully deployed at incredible speed for mRNA vaccination, but aspects such as cold storage, manufacturing, efficiency of delivery, and the therapeutic window can benefit from further improvement. To investigate alternatives to lipid nanoparticles, a class of >200 biodegradable end-capped lipophilic poly(beta-amino ester)s (PBAEs) that enable efficient delivery of SAM in vitro and in vivo as assessed by measuring expression of SAM encoding reporter proteins is developed. The ability of these polymers to deliver SAM intramuscularly in mice is evaluated, and a polymer-based formulation that yields up to 37-fold higher intramuscular (IM) expression of SAM compared to injected naked SAM is identified. Using the same nanoparticle formulation to deliver a SAM encoding rabies virus glycoprotein, the vaccine elicits superior immunogenicity compared to naked SAM delivery, leading to seroconversion in mice at low RNA injection doses. These biodegradable nanomaterials may be useful in the development of next-generation RNA vaccines for infectious diseases.Copyright © 2023 The Authors. Advanced Therapeutics published by Wiley-VCH GmbH.

Fast de novo antibody structure prediction with atomic accuracy

Background: In the field of antibody engineering, an essential task is to design a novel antibody whose paratopes bind to a specific antigen with correct epitopes. Understanding antibody structure and its paratope can facilitate a mechanistic understanding of its function. Therefore, antibody structure prediction from its sequence alone has always been a highly valuable problem for de novo antibody design. AlphaFold2 (AF2), a breakthrough in the field of structural biology, provides a solution to this protein structure prediction problem by learning a deep learning model. However, the computational efficiency and undesirable prediction accuracy on antibody, especially on the complementarity-determining regions limit its applications in de novo antibody design. Method(s): To learn informative representation of antibodies, we trained a deep antibody language model (ALM) on curated sequences from observed antibody space database via a well-designed transformer model. We also developed a novel model named xTrimoABFold++ to predict antibody structure from antibody sequence only based on the pretrained ALM as well as efficient evoformers and structural modules. The model was trained end-to-end on the antibody structures in PDB by minimizing the ensemble loss of domain-specific focal loss on CDR and the frame aligned point loss. Result(s): xTrimoABFold++ outperforms AF2 and OmegaFold, HelixFold-Single with 30+% improvement on RMSD. Also, it is 151 times faster than AF2 and predicts antibody structure in atomic accuracy within 20 seconds. In recently released antibodies, for example, cemiplimab of PD1 (PDB: 7WVM) and cross-neutralizing antibody 6D6 of SARS-CoV-2 (PDB: 7EAN), the RMSD of xTrimoABFold++ are 0.344 and 0.389 respectively. Conclusion(s): To the best of our knowledge, xTrimoABFold++ achieved the state-of-the-art in antibody structure prediction. Its improvement on both accuracy and efficiency makes it a valuable tool for de novo antibody design, and could make further improvement in immuno-theory.

SARS-CoV-2 spike S1 mediated lung tumor regression in a mouse model

Lung cancer is the leading cause of cancer related deaths worldwide, with a relatively low 5-year survival rate. Although there are some therapies against lung cancer, new effective treatment options are urgently required. Recently during the COVID-19 pandemic, we have seen that SARSCoV-2 binds to its receptor angiotensin-converting enzyme 2 (ACE2) via spike S1 to enter the cells. This study underlines the importance of SARS-CoV-2 spike S1 in inducing death in human lung cancer cells. Interestingly, we have seen that recombinant spike S1 treatment at very low doses led to death of human A549 lung cancer cells. On the other hand, boiled recombinant SARS-CoV-2 spike S1 remained unable to induce death, suggesting that the induction of cell death in A549 cells was due to native SARS-CoV-2 spike S1 protein. SARS-CoV-2 spike S1-induced A549 cell death was also inhibited by neutralizing antibodies against spike S1 and ACE2. Moreover, our newly designed wild type ACE2-interacting domain of SARS-CoV-2 (wtAIDS), but not mAIDS, peptide also attenuated SARS-CoV-2 spike S1-induced cell death, suggesting that SARS-CoV-2 spike S1- induced death in lung cancer cells depends on its interaction with ACE2 receptor. Similarly, recombinant spike S1 treatment also led to death of H1299 and H358 human lung cancer cells. Finally, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) intoxication led to the formation tumors in lungs of A/J mice and alternate day intranasal treatment with low dose of recombinant SARS-CoV-2 spike S1 from 22-weeks of NNK insult (late stage) led to induced apoptosis and tumor regression in the lungs. These studies indicate that recombinant SARS-CoV-2 Spike S1 protein may have implications in the treatment of lung cancer.

Mesenchymal Stem/Stromal Cells: HUMAN MESENCHYMAL STEM CELL THERAPY IN SEVERE COVID-19 PATIENTS: 2-YEAR FOLLOW-UP RESULTS OF A RANDOMIZED, DOUBLE-BLIND, PLACEBO-CONTROLLED TRIAL

Background & Aim: The long-term effects of human mesenchymal stem cell (MSC) treatment on COVID-19 patients have not been fully characterized. The aim of this study was to evaluate the safety and efficacy of a MSC treatment administered to severe COVID-19 patients enrolled in a randomized, double-blind, placebo-controlled clinical trial (NCT 04288102). Methods, Results & Conclusion(s): A total of 100 patients experiencing severe COVID-19 received either MSC treatment (n = 65, 4x107 cells per infusion) or a placebo (n = 35) combined with standard of care on days 0, 3, and 6. Patients were subsequently evaluated 18 and 24 months after treatment to evaluate the long-term safety and efficacy of the MSC treatment. The outcomes measured included: 6-minute walking distance (6-MWD), lung imaging, quality of life according to the Short Form 36 questionnaire, COVID-19-related symptoms, titers of SARS-CoV-2 neutralizing antibodies, MSC-related adverse events (AEs), and tumor markers. Two years after treatment, a marginally smaller proportion of patients had a 6-MWD below the lower limit of the normal range in the MSC group than in the placebo group (OR = 0.19, 95% CI: 0.04-0.80, Fisher's exact test, p = 0.015). On the SF-36 questionnaire, a marginally higher general health score was received by the MSC group at month 18 compared with the placebo group (50.00 vs. 35.00;95% CI: 0.00-20.00, Wilcoxon rank sum test, p = 0.016). In contrast, there were no differences in the total severity score of lung imaging or the titer of neutralizing antibodies between the two groups. Meanwhile, there were no MSC-related AEs reported at the 18- or 24-month follow-ups. The serum levels of most of the tumor markers examined remained within normal ranges and were similar between the MSC and placebo groups. Long-term safety was observed for the COVID-19 patients who received MSC treatment. Yet few sustained efficacy of MSC treatment was observed at the end of the 2-year follow-up period. Funding(s): The National Key Research and Development Program of China (2022YFA1105604, 2020YFC0860900), the specific research fund of The Innovation Platform for Academicians of Hainan Province (YSPTZX202216) and the Fund of National Clinical Center for Infectious Diseases, PLA General Hospital (NCRCID202105,413FZT6). [Figure presented]Copyright © 2023 International Society for Cell & Gene Therapy

COVID-19 pre-exposure prophylaxis with tixagevimab/cilgavimab in patients with haematological malignancy: A single-centre experience

Background: COVID-19 pandemic still pose a substantial threat worldwide despite increasing vaccine availability. Patients with haematological malignancies have been shown to have increased risk of contracting COVID-19 and are more susceptible to develop severe illness from SARS-CoV- 2 infection. The immune response to vaccines is impaired in patients with haematological malignancy due to underlying disease or antineoplastic therapies. The monoclonal-antibody combination, Evusheld is composed of tixagevimab and cilgavimab, two neutralising antibodies against SARS-CoV- 2. It has been shown to be safe and have efficacy for the prevention of COVID-19. Our aim of study is to describe the incidence and outcome of breakthrough COVID-19 infection among patients who received Evusheld in our centre and analyse the factors that possibly increase the risk of breakthrough infection. Material(s) and Method(s): A retrospective review of all adult patients with haematological malignancy who received tixagevimab/ cilgavimab 150/150 mg injection in Hospital Pulau Pinang from 1 July 2022 to 31 August 2022 with a follow-up period to 30 November 2022 was conducted. Demographic data, clinical characteristics and outcome will be retrieved from patient's medical records. Data were analysed using Statistical Package for Social Sciences software (version 21.0). Result(s): A total of 96 patients (50 males and 46 females) received tixagevimab/cilgavimab injection during the study period with a median age of 61 years (range 19-82). Majority of them were diagnosed with multiple myeloma (42.7%), followed by lymphoma (33.3%) and leukaemia (24%). One third of them had history of therapy with monoclonal antibody and 20% had haematopoietic stem cell transplantation. No major adverse effects of tixagevimab/cilgavimab injection were noted among the study population. Of the 12 patients (12.5%) who had COVID-19 infection, all of them had mild infection;three were asymptomatic and six patients received Paxlovid antiviral therapy. The median time from tixagevimab/cilgavimab to the onset of COVID-19 infection was 35 days (range 5-97 days). The mean age of patients with breakthrough COVID-19 infection were older compared to those without breakthrough infection but was not statistically significant. The incidence of breakthrough COVID-19 infection was not affected by type of haematological malignancy, history of monoclonal antibody therapy or COVID-19 vaccination. Discussion and Conclusion(s): Our findings showed that tixagevimab/cilgavimab was safe and effective in preventing COVID-19- related morbidity and mortality among patients with haematological malignancy during the study period. However, the limitation is the lack of access to whole genome sequencing for detection of resistant variants for breakthrough infections.

Safety of Pharmacotherapy in COVID-19 Patients: A Literature Review.

The safety of COVID-19 pharmacotherapy is a relevant issue, first of all, because of the current lack of experience with using particular medicinal products and with off-label prescribing. The aim of the study was to analyse information on potential adverse drug reactions (ADRs) and their predictors in etiology- and pathogenesis-oriented COVID-19 therapy. According to literature data, the main clinically significant risk factors for COVID-19 patients to develop an ADR are the duration of their hospital stay, combined use of antivirals, polypharmacy, and their history of drug allergies. The most common adverse reactions to antivirals, to virus-neutralising antibodies, and to human anti-COVID-19 immunoglobulin and convalescent plasma are, respectively, gastrointestinal and hepatobiliary disorders;gastrointestinal disorders, neurological disorders, and allergic reactions;and transfusion reactions (fever, chills, etc.). For pathogenesis-oriented therapy with systemic glucocorticosteroids, the most characteristic ADR is hyperglycaemia. Janus kinase inhibitors and interleukin inhibitors are most often associated with gastrointestinal disorders and hypertransaminasemia;neutropenia is also characteristic of a number of interleukin inhibitors. Haemostatic adverse reactions to anticoagulants depend on the patient's dosing regimen and condition. Drug-drug interactions are a common problem in COVID-19 treatment, with the combination of nirmatrelvir and ritonavir showing the largest number of significant interactions attributed to their pharmacokinetics. Currently, there is data on the role of pharmacogenetic biomarkers in the safety and clinical outcomes of COVID-19 therapy. Thus, to improve the safety of COVID-19 therapy, an integrated approach is needed that will take into account both the clinical, demographic, and pharmacogenetic predictors of ADRs and the risk of drug-drug interactions.Copyright © 2023 Safety and Risk of Pharmacotherapy. All rights reserved.

The Impact of Sputnik SARS-CoV-2 Vaccines on Antibody Response in the Egyptian Population

BACKGROUND: The coronavirus disease 2019 (COVID-2019) causes the severe contagious acute respiratory syndrome. Therefore, massive vaccination campaign is mandatory to control the spread. Sputnik COVID-19 vaccines induce immunity through different mechanisms involving antibody response that bind to the spike protein to neutralize the viral entry into the cells. AIM: This study aims to compare the titers of specific antibodies in the pre-and post-vaccination sera in the vaccinated Egyptian population to evaluate the efficacy of the sputnik vaccine. METHOD(S): Samples were collected from 205 adult volunteers receiving the Sputnik vaccine in the Reference Laboratory of Egyptian University Hospitals. Samples were collected before vaccination and within 1, 2, or 3 months after receiving two doses of Sputnik SARS-CoV-2 vaccines from August to October 2021, serum samples collected were tested by quantitative chemiluminescent immunoassay using (Mindray CL-960i chemiluminescence analyzer, India) at the Reference laboratory of Egyptian University Hospitals for neutralizing antibodies, anti-spike antibodies, and total antibody levels before and after vaccination. RESULT(S): The results of the 205 paired samples illustrated that there was a statistically significant difference between pre-and post-vaccination antibody levels with a p-value of (< 0.001) indicating that the vaccine produced significantly high levels of antibodies. CONCLUSION(S): COVID-19 Sputnik vaccines induce immunity through an antibody response that binds to the virus to neutralize its entry into cells. Our study showed a significant increase in the measured post-vaccination levels of the three antibodies among the enrolled volunteers compared to the basal pre-vaccination level and thus sputnik vaccine protects against SARS-CoV-2 infections.Copyright © 2023 Ghada Ismail, Dalia Abdelhamid, Marwa Salah Mostafa, Noha Alaa Eldin Fahim, Ahmed Elshafei, Hossam Abdelghaffar, Nashwa Naguib, Omnia Taher, Menna Asker.

Animal Pre-Clinical Study of an Inactivated SARS-CoV-2 Vaccine candidate (Osvid-19 ): Immunogenicity, Protective, and Safety Aspects

Background: This study aimed to evaluate the outcomes of preclinical studies on the safety and immunogenicity of an inactivated COVID-19 vaccine candidate to warrant further clinical evaluation. Method(s): SARS-CoV-2 positive nasopharyngeal swab specimens were confirmed by real-time polymerase chain reaction and next-generation sequencing. The safety and immunogenicity tests of the COVID-19 vaccine were carried out in rats and Rhesus monkeys, and Balb/C mice and Rhesus monkeys, respectively. Result(s): The candidate vaccine was well tolerated and induced promising levels of SARS-CoV-2- specific IgG1, IgG2a, and Granzyme B in Balb/C mice, and anti-SARS-CoV-2 spike IgG and neutralizing antibodies in Rhesus monkeys. Based on cVNT results, the inactivated vaccine in 0.5 and 1 microg/100 microL doses was able to induce a neutralizing effect against the SARS-CoV-2 virus up to a dilution of 1:512 and 1:1000. The protective efficacy of the vaccine candidate was challenged with 2 x108 PFU of live viruses and confirmed by lung CT scan and histopathological evaluations compared to the control group. Repeated intramuscular injection of the candidate vaccine was generally well-tolerated in Rats and Rhesuses. No significant side effects were observed in rats injected with ten full human doses and in the Rhesus monkeys with three full human doses. Conclusion(s): Based on the findings presented in this study, it is recommended that this vaccine be moved into human testing commencing with a phase I clinical trial.Copyright © 2021 Muslim OT et al.

Production and characterisation of a SARSCoV-2 S-protein RBD homodimer with increased avidity for specific antibodies.

Monitoring of the proportion of immune individuals and the effectiveness of vaccination in a population involves evaluation of several important parameters, including the level of virus-neutralising antibodies. In order to combat the COVID-19 pandemic, it is essential to develop approaches to detecting SARS-CoV-2 neutralising antibodies by safe, simple and rapid methods that do not require live viruses. To develop a test system for enzyme-linked immunosorbent assay (ELISA) that detects potential neutralising antibodies, it is necessary to obtain a highly purified recombinant receptor-binding domain (RBD) of the spike (S) protein with high avidity for specific antibodies. The aim of the study was to obtain and characterise a SARSCoV-2 S-protein RBD homodimer and a recombinant RBD-expressing cell line, as well as to create an ELISA system for detecting potential neutralising antibodies. Material(s) and Method(s): the genetic construct was designed in silico. To generate a stable producer cell line, the authors transfected CHO-S cells, subjected them to antibiotic pressure, and selected the optimal clone. To isolate monomeric and homodimeric RBD forms, the authors purified the recombinant RBD by chromatographic methods. Further, they analysed the activity of the RBD forms by Western blotting, bio-layer interferometry, and indirect ELISA. The analysis involved monoclonal antibodies GamXRH19, GamP2C5, and h6g3, as well as serum samples from volunteers vaccinated with Gam-COVID-Vac (Sputnik V) and unvaccinated ones. Result(s): the authors produced the CHO-S cell line for stable expression of the recombinant SARS-CoV-2 S-protein RBD. The study demonstrated the recombinant RBD's ability to homodimerise after fed-batch cultivation of the cell line for more than 7 days due to the presence of unpaired cysteines. The purified recombinant RBD yield from culture broth was 30-50 mg/L. Monomeric and homodimeric RBD forms were separated using gel-filtration chromatography and characterised by their ability to interact with specific monoclonal antibodies, as well as with serum samples from vaccinated volunteers. The homodimeric recombinant RBD showed increased avidity for both monoclonal and immune sera antibodies. Conclusion(s): the homodimeric recombinant RBD may be more preferable for the analysis of levels of antibodies to the receptor-binding domain of the SARS-CoV-2 S protein.Copyright © 2023 Authors. All rights reserved.

Anti-interferon-alpha autoantibodies in patients with inborn errors of Immunity and rheumatic diseases before and during the COVID-19 pandemic

Introduction: Type 1 interferon (IFN) autoantibodies, such as anti-IFNalpha, have pathogenic significance in life-threatening COVID-19 pneumonia. Ten to twenty percent of severe COVID cases are associated with type I IFN autoantibodies. These autoantibodies likely pre-exist while others arise de novo relative to SARS-CoV-2 infection. It is unclear to what extent type I anti-IFN autoantibodies are induced by SARS-CoV-2 infection and contribute to COVID-19 severity. We investigated these phenomena in those with inborn errors of immunity (IEI) and rheumatic disease (RHE). Aim(s): We aim to compare the prevalence and neutralization ability of anti-IFNalpha autoantibodies in IEI and RHE patients using archived blood samples before and after the COVID-19 pandemic began. Method(s): We determined the presence of autoantibodies against IFNalpha in plasma samples by enzyme linked immunosorbent assay in 453 patients with IEI or RHE who were testing either before or after the COVID-19 pandemic began in March 2020. Using flow cytometry, we determined the function of IFNalpha autoantibodies in plasma to block CD4T cell activation by inhibiting STAT-1 phosphorylation. Result(s): We found that 25 patients with IEI or RHE were positive for anti-IFNalpha autoantibodies. 10 out of 229 patient samples collected before the pandemic (4.2%) tested positive whereas 15 out of 224 patient samples collected after the pandemic began (7.0%) were positive. Seven of the 25 patients (28%) who tested positive had neutralizing antibodies in plasma, which prevented STAT-1 phosphorylation in CD4T cells;all of these patients had partial recombination activating gene deficiency (pRD) except for one patient with autoimmunity, leukemia and selective IgA deficiency. One pRD patient had anti-IFNalpha autoantibodies with neutralization capacity before the pandemic, which persisted after hematopoietic stem cell transplantation (HSCT) with full immune reconstitution. The patient was immunized for SARS-CoV-2 before and after HSCT and acquired COVID-19 infection a year after HSCT. The patient was symptomatic but never hospitalized and fully recovered despite having anti-IFNalpha autoantibodies. Conclusion(s): Anti-IFNalpha autoantibody levels were comparable before and after the start of the COVID-19 pandemic in IEI and RHE patients but only 28% of cases were neutralizing. The clinical implications of these autoantibodies are yet to be determined.Copyright © 2023 Elsevier Inc.

Sequential Administration of SARS-CoV-2 Strains-Based Vaccines Effectively Induces Potent Immune Responses against Previously Unexposed Omicron Strain.

In the past few years, the continuous pandemic of COVID-19 caused by SARS-CoV-2 has placed a huge burden on public health. In order to effectively deal with the emergence of new SARS-CoV-2 variants, it becomes meaningful to further enhance the immune responses of individuals who have completed the first-generation vaccination. To understand whether sequential administration using different variant sequence-based inactivated vaccines could induce better immunity against the forthcoming variants, we tried five inactivated vaccine combinations in a mouse model and compared their immune responses. Our results showed that the sequential strategies have a significant advantage over homologous immunization by inducing robust antigen-specific T cell immune responses in the early stages of immunization. Furthermore, the three-dose vaccination strategies in our research elicited better neutralizing antibody responses against the BA.2 Omicron strain. These data provide scientific clues for finding the optimal strategy within the existing vaccine platform in generating cross-immunity against multiple variants including previously unexposed strains.

Humoral and cellular immune response to SARS-CoV-2 mRNA BNT162b2 vaccine in pediatric kidney transplant recipients compared with dialysis patients and healthy children.

BACKGROUND: Compared with the general population, the immune response to COVID-19 mRNA vaccines is lower in adult kidney transplant recipients (KTRs). However, data is limited for pediatric KTRs. In this study, we aimed to assess humoral and cellular immune responses to the COVID-19 mRNA vaccine in pediatric KTRs. METHODS: This multicenter, prospective, case-control study included 63 KTRs (37 male, aged 12-21 years), 19 dialysis patients, and 19 controls. Humoral (anti-SARS-CoV2 IgG, neutralizing Ab (nAb)) and cellular (interferon-gamma release assay (IGRA)) immune responses were assessed at least one month after two doses of BNT162b2 mRNA vaccine. RESULTS: Among COVID-19 naïve KTRs (n = 46), 76.1% tested positive for anti-SARS-CoV-2 IgG, 54.3% for nAb, and 63% for IGRA. Serum levels of anti-SARS-CoV-2 IgG and nAb activity were significantly lower in KTRs compared to dialysis and control groups (p < 0.05 for all). Seropositivity in KTRs was independently associated with shorter transplant duration (p = 0.005), and higher eGFR (p = 0.007). IGRA titer was significantly lower than dialysis patients (p = 0.009). Twenty (43.4%) KTRs were positive for all immune parameters. Only four of 11 seronegative KTRs were IGRA-positive. COVID-19 recovered KTRs had significantly higher anti-SARS-CoV-2 IgG and nAb activity levels than COVID-19 naïve KTRs (p = 0.018 and p = 0.007, respectively). CONCLUSIONS: The humoral and cellular immune responses to SARS-CoV-2 mRNA BNT162b2 vaccine are lower in pediatric KTRs compared to dialysis patients. Further prospective studies are required to demonstrate the clinical efficacy of the mRNA vaccine in KTRs. This prospective study was registered in ClinicalTrials.gov (NCT05465863, registered retrospectively at 20.07.2022). A higher resolution version of the Graphical abstract is available as Supplementary information.

Diminished Neutralization Capacity of SARS-CoV-2 Omicron BA.1 in Donor Plasma Collected from January to March 2021.

The 50% plaque reduction neutralization assay (PRNT50) has been previously used to assess the neutralization capacity of donor plasma against wild-type and variant of concern (VOC) severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Emerging data suggest that plasma with an anti-SARS-CoV-2 level of ≥2 × 104 binding antibody units/mL (BAU/mL) protects against SARS-CoV-2 Omicron BA.1 infection. Specimens were collected using a cross-sectional random sampling approach. For PRNT50 studies, 63 previously analyzed specimens by PRNT50 versus SARS-CoV-2 wild-type, Alpha, Beta, Gamma, and Delta were analyzed by PRNT50 versus Omicron BA.1. The 63 specimens plus 4,390 specimens (randomly sampled regardless of serological evidence of infection) were also tested using the Abbott SARS-CoV-2 IgG II Quant assay (anti-spike [S]; Abbott, Chicago, IL, USA; Abbott Quant assay). In the vaccinated group, the percentages of specimens with any measurable PRNT50 versus wild-type or VOC were wild type (21/25 [84%]), Alpha (19/25 [76%]), Beta (18/25 [72%]), Gamma (13/25 [52%]), Delta (19/25 [76%]), and Omicron BA.1 (9/25 [36%]). In the unvaccinated group, the percentages of specimens with any measurable PRNT50 versus wild type or VOC were wild-type SARS-CoV-2 (16/39 [41%]), Alpha (16/39 [41%]), Beta (10/39 [26%]), Gamma (9/39 [23%]), Delta (16/39 [41%]), and Omicron BA.1 (0/39) (Fisher's exact tests, vaccinated versus unvaccinated for each variant, P < 0.05). None of the 4,453 specimens tested by the Abbott Quant assay had a binding capacity of ≥2 × 104 BAU/mL. Vaccinated donors were more likely than unvaccinated donors to neutralize Omicron when assessed by a PRNT50 assay. IMPORTANCE SARS-CoV-2 Omicron emergence occurred in Canada during the period from November 2021 to January 2022. This study assessed the ability of donor plasma collected earlier (January to March 2021) to generate any neutralizing capacity against Omicron BA.1 SARS-CoV-2. Vaccinated individuals, regardless of infection status, were more likely to neutralize Omicron BA.1 than unvaccinated individuals. This study then used a semiquantitative binding antibody assay to screen a larger number of specimens (4,453) for individual specimens that might have high-titer neutralizing capacity against Omicron BA.1. None of the 4,453 specimens tested by the semiquantitative SARS-CoV-2 assay had a binding capacity suggestive of a high-titer neutralizing capacity against Omicron BA.1. These data do not imply that Canadians lacked immunity to Omicron BA.1 during the study period. Immunity to SARS-CoV-2 is complex, and there is still no wide consensus on correlation of protection to SARS-CoV-2.

SARS-CoV-2 spike S2-specific neutralizing antibodies.

Since the onset of the coronavirus disease 2019 (COVID-19), numerous neutralizing antibodies (NAbs) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been developed and authorized for emergency use to control the pandemic. Most COVID-19 therapeutic NAbs prevent the S1 subunit of the SARS-CoV-2 spike (S) protein from binding to the human host receptor. However, the emergence of SARS-CoV-2 immune escape variants, which possess frequent mutations on the S1 subunit, may render current NAbs ineffective. In contrast, the relatively conserved S2 subunit of the S protein can elicit NAbs with broader neutralizing potency against various SARS-CoV-2 variants. In this review, the binding specificity and functional features of SARS-CoV-2 NAbs targeting different domains of the S2 subunit are collectively discussed. The knowledge learned from the investigation of the S2-specific NAbs provides insights and potential strategies for developing antibody cocktail therapy and next-generation coronavirus vaccine.

The Impact of Vaccination Time on the Antibody Response to an Inactivated Vaccine against SARS-CoV-2 (IMPROVE-2): A Randomized Controlled Trial.

There is still controversy about whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination at different times of day will induce a stronger immune response. Therefore, a randomized controlled trial (ChiCTR2100045109) is conducted to investigate the impact of vaccination time on the antibody response to the inactivated vaccine against SARS-CoV-2 from April 15 to 28, 2021. Participants are randomly assigned in a 1:1 ratio to receive inactivated SARS-CoV-2 vaccine in the morning or afternoon. The primary endpoint is the change of neutralizing antibody between baseline and 28 days after the second dose. In total, 503 participants are randomized, and 469 participants (238 in the morning group and 231 in the afternoon group) complete the follow-up. There is no significant difference in the change of neutralizing antibody between baseline and 28 days after the second dose between the morning and afternoon groups (22.2 [13.2, 45.0] AU mL-1  vs 22.0 [14.4, 40.7] AU mL-1 , P = 0.873). In prespecified age and sex subgroup analyses, there is also no significant difference in the morning and afternoon group (all P > 0.05). This study demonstrates that the vaccination time does not affect the antibody response of two doses of inactivated SARS-CoV-2 vaccine.

Long COVID and Hybrid Immunity among Children and Adolescents Post-Delta Variant Infection in Thailand.

This study aimed to assess long COVID, and describe immunogenicity against Omicron variants following BNT162b2 vaccination. A prospective cohort study was conducted among children (aged 5-11) and adolescents (aged 12-17) who had SARS-CoV-2 infection from July to December 2021 (Delta predominant period). Long COVID symptoms were assessed by questionnaires at 3 months after infection. Immunogenicity was evaluated by using a surrogate virus-neutralizing antibody test (sVNT) against the Omicron variant. We enrolled 97 children and 57 adolescents. At 3 months, 30 children (31%) and 34 adolescents (60%) reported at least one long COVID symptom, with respiratory symptoms prevailing (25% children and 32% adolescents). The median time from infection to vaccination was 3 months in adolescents and 7 months in children. At 1 month following vaccination, in children who received one-dose and two-dose BNT162b2 vaccines, the median (IQR) sVNT against Omicron was 86.2% inhibition (71.1-91.8) and 79.2% inhibition (61.5-88.9), respectively (p = 0.26). Among adolescents who received one-dose and two-dose BNT162b2 vaccines, the median (IQR) sVNT against Omicron was 64.4% inhibition (46.8-88.8) and 68.8% inhibition (65.0-91.2) (p = 0.64). Adolescents had a higher prevalence of long COVID than children. Immunogenicity against the Omicron variant after vaccination was high and did not vary between one or two doses of the vaccine in either children or adolescents.

A Novel Conserved Linear Neutralizing Epitope on the Receptor-Binding Domain of the SARS-CoV-2 Spike Protein.

The continuous emergence of new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has made it challenging to develop broad-spectrum prophylactic vaccines and therapeutic antibodies. Here, we have identified a broad-spectrum neutralizing antibody and its highly conserved epitope in the receptor-binding domain (RBD) of the spike protein (S) S1 subunit of SARS-CoV-2. First, nine monoclonal antibodies (MAbs) against the RBD or S1 were generated; of these, one RBD-specific MAb, 22.9-1, was selected for its broad RBD-binding abilities and neutralizing activities against SARS-CoV-2 variants. An epitope of 22.9-1 was fine-mapped with overlapping and truncated peptide fusion proteins. The core sequence of the epitope, 405D(N)EVR(S)QIAPGQ414, was identified on the internal surface of the up-state RBD. The epitope was conserved in nearly all variants of concern of SARS-CoV-2. MAb 22.9-1 and its novel epitope could be beneficial for research on broad-spectrum prophylactic vaccines and therapeutic antibody drugs. IMPORTANCE The continuous emergence of new variants of SARS-CoV-2 has caused great challenge in vaccine design and therapeutic antibody development. In this study, we selected a broad-spectrum neutralizing mouse monoclonal antibody which recognized a conserved linear B-cell epitope located on the internal surface of RBD. This MAb could neutralize all variants until now. The epitope was conserved in all variants. This work provides new insights in developing broad-spectrum prophylactic vaccines and therapeutic antibodies.

Humoral Response after a Fourth Dose with mRNA-1273 in Healthcare Workers with and without a History of SARS-CoV-2 Infection and Previously Vaccinated with Two Doses of BBIBP-CorV Plus BNT162b2 Vaccine.

There is limited information on the kinetics of the humoral response elicited by a fourth dose with a heterologous mRNA1273 booster in patients who previously received a third dose with BNT162b2 and two doses of BBIBP-CorV as the primary regimen. We conducted a prospective cohort study to assess the humoral response using Elecsys® anti-SARS-CoV-2 S (anti-S-RBD) of 452 healthcare workers (HCWs) in a private laboratory in Lima, Peru at 21, 120, 210, and 300 days after a third dose with a BNT162b2 heterologous booster in HCW previously immunized with two doses of BBIBP-CorV, depending on whether or not they received a fourth dose with the mRNA1273 heterologous vaccine and on the history of previous SARS infection -CoV-2. Of the 452 HCWs, 204 (45.13%) were previously infected (PI) with SARS-CoV-2, and 215 (47.57%) received a fourth dose with a heterologous mRNA-1273 booster. A total of 100% of HCWs presented positive anti-S-RBD 300 days after the third dose. In HCWs receiving a fourth dose, GMTs 2.3 and 1.6 times higher than controls were observed 30 and 120 days after the fourth dose. No statistically significant differences in anti-S-RBD titers were observed in those HCWs PI and NPI during the follow-up period. We observed that HCWs who received a fourth dose with the mRNA1273 and those previously infected after the third dose with BNT162b2 (during the Omicron wave) presented higher anti-S-RBD titers (5734 and 3428 U/mL, respectively). Further studies are required to determine whether patients infected after the third dose need a fourth dose.

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.

Evaluation of the Diagnostic Performance of Two Automated SARS-CoV-2 Neutralization Immunoassays following Two Doses of mRNA, Adenoviral Vector, and Inactivated Whole-Virus Vaccinations in COVID-19 Naïve Subjects.

BACKGROUND: Limited data are available on humoral responses determined by automated neutralization tests following the administration of the three different types of COVID-19 vaccinations. Thus, we here evaluated anti-SARS-CoV-2 neutralizing antibody titers via two different neutralization assays in comparison to total spike antibody levels. METHODS: Healthy participants (n = 150) were enrolled into three subgroups who were tested 41 (22-65) days after their second dose of mRNA (BNT162b2/mRNA-1273), adenoviral vector (ChAdOx1/Gam-COVID-Vac) and inactivated whole-virus (BBIBP-CorV) vaccines, with no history or serologic evidence of prior SARS-CoV-2 infection. Neutralizing antibody (N-Ab) titers were analyzed on a Snibe Maglumi® 800 instrument and a Medcaptain Immu F6® Analyzer in parallel to anti-SARS-CoV-2 S total antibody (S-Ab) levels (Roche Elecsys® e602). RESULTS: Subjects who were administered mRNA vaccines demonstrated significantly higher SARS-CoV-2 N-Ab and S-Ab levels compared to those who received adenoviral vector and inactivated whole-virus vaccinations (p < 0.0001). N-Ab titers determined by the two methods correlated with each other (r = 0.9608; p < 0.0001) and S-Ab levels (r = 0.9432 and r = 0.9324; p < 0.0001, respectively). Based on N-Ab values, a new optimal threshold of Roche S-Ab was calculated (166 BAU/mL) for discrimination of seropositivity showing an AUC value of 0.975 (p < 0.0001). Low post-vaccination N-Ab levels (median value of 0.25 µg/mL or 7.28 AU/mL) were measured in those participants (n = 8) who were infected by SARS-CoV-2 within 6 months after immunizations. CONCLUSION: Both SARS-CoV-2 N-Ab automated assays are effective to evaluate humoral responses after various COVID-19 vaccines.