Neutralizing Antibody Activity Research Articles

The Equal Neutralizing Effectiveness of BNT162b2, ChAdOx1 nCoV-19, and Sputnik V Vaccines in the Palestinian Population.

Since the beginning of the COVID-19 pandemic, different viral vector-based and mRNA vaccines directed against the SARS-CoV-2 "S" spike glycoprotein have been developed and have shown a good profile in terms of safety and efficacy. Nevertheless, an unbiased comparison of vaccination efficiency, including post-vaccination neutralizing activity, between the different vaccines remains largely unavailable. This study aimed to compare the efficacy of one mRNA (BNT162b2) and two non-replicating adenoviral vector vaccines (ChAdOx1 nCoV-19 and Sputnik V) in a cohort of 1120 vaccinated Palestinian individuals who received vaccines on an availability basis and which displayed a unique diversity of genetic characteristics. We assessed the level of anti-S antibodies and further determined the antibody neutralizing activity in 261 of those individuals vaccinated with BNT162b2a (121), ChAdOx1 (72) or Sputnik V (68). Our results showed no significant difference in the distribution of serum-neutralizing activity or S-antibody serum levels for the three groups of vaccines, proving equivalence in efficacy for the three vaccines under real-life conditions. In addition, none of the eight demographic parameters tested had an influence on vaccination efficacy. Regardless of the vaccine type, the vaccination campaign ultimately played a pivotal role in significantly reducing the morbidity and mortality associated with COVID-19 in Palestine.

Isolation and characterization of Hc-targeting chimeric heavy chain antibodies neutralizing botulinum neurotoxin type B.

Botulinum neurotoxin (BoNT) produced by Clostridium botulinum is one of the most potent known toxins. Moreover, BoNT is classified as one of the most important biological warfare agents that threatens the biosafety of the world. Currently, the approved treatment for botulism in humans is the use of polyvalent horse serum antitoxins. However, they are greatly limited because of insufficient supply and adverse reactions. Thus, treatment of human botulism requires the development of effective toxin-neutralizing antibodies. Considering their advantages, neutralizing nanobodies will play an increasing role as BoNTs therapeutics. Herein, neutralizing nanobodies binding to the heavy chain (Hc) domain of BoNT/B (BHc) were screened from a phage display library. Then, BoNT/B-specific clones were identified and fused with the human Fc fragment (hFc) to form chimeric heavy chain antibodies. Finally, the affinity, specificity, and neutralizing activity of antibodies against BoNT/B in vivo were evaluated. The B5-hFc, B9-hFc and B12-hFc antibodies demonstrated high affinity for BHc in the nanomolar range. The three antibodies were proven to have potent neutralizing activity against BoNT/B in vivo. The results demonstrate that inhibiting toxin binding to the host receptor is an efficient strategy and the three antibodies could be used as candidates for the further development of drugs to prevent and treat botulism.

Immunogenicity of Monovalent mRNA-1273 and BNT162b2 Vaccines in Children Less Than 5 Years of Age.

The mRNA-based COVID-19 vaccines approved for use in children less than 5 years of age have different antigen doses and administration schedules that could affect vaccine immunogenicity and effectiveness. We sought to compare the strength and breadth of serum binding and neutralizing antibodies to SARS-CoV-2 elicited by monovalent mRNA-based COVID-19 vaccines in young children. We conducted a prospective cohort study of children 6 months to 4 years of age who completed primary series vaccination with monovalent mRNA-1273 or BNT162b2 vaccines. Serum was collected one month after primary vaccine series completion for measurement of SARS-CoV-2-specific humoral immune responses, including antibody binding responses to Spike proteins from an ancestral strain (D614G) and major variants of SARS-CoV-2 and antibody neutralizing activity against D614G and Omicron subvariants (BA.1, BA.4/5). Of 75 participants, 40 (53%) received mRNA-1273 and 35 (47%) received BNT162b2. Children receiving either primary vaccine series developed robust and broad SARS-CoV-2-specific binding and neutralizing antibodies, including to Omicron subvariants. Children with a prior history of SARS-CoV-2 infection developed significantly higher antibody binding responses and neutralization titers to Omicron subvariants, consistent with the occurrence of identified infections during the circulation of Omicron subvariants in the region. Monovalent mRNA-1273 and BNT162b2 elicited similar antibody responses one month after vaccination in young children. Further, prior infection significantly enhanced the strength of antibody responses to Omicron subvariants. Future studies should evaluate incorporation of these vaccines into the standard childhood immunization schedule.

Identification of an IGHV3-53-Encoded RBD-Targeting Cross-Neutralizing Antibody from an Early COVID-19 Convalescent.

Since November 2021, Omicron has emerged as the dominant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant, and its sublineages continue to appear one after another, significantly reducing the effectiveness of existing therapeutic neutralizing antibodies (NAbs). It is urgent to develop effective NAbs against circulating Omicron variants. Here, we isolated receptor binding domain (RBD)-specific single memory B cells via flow cytometry from a COVID-19 convalescent. The antibody variable region genes of the heavy chain (VHs) and light chain (VLs) were amplified and cloned into expression vectors. After antibody expression, ELISA screening and neutralizing activity detection, we obtained an IGHV3-53-encoded RBD-targeting cross-neutralizing antibody D6, whose VL originated from the IGKV1-9*01 germlines. D6 could potently neutralize circulating Omicron variants (BA.1, BA.2, BA.4/5 and BF.7), with IC50 values of less than 0.04 μg/mL, and the neutralizing ability against XBB was reduced but still effective. The KD values of D6 binding with RBD of the prototype and BA.1 were both less than 1.0 × 10-12 M. The protein structure of the D6-RBD model indicates that D6 interacts with the RBD external subdomain and belongs to the RBD-1 community. The sufficient contact and deep interaction of D6 HCDR3 and LCDR3 with RBD may be the crucial reason for its cross-neutralizing activity. The sorting and analysis of mAb D6 will provide important information for the development of anti-COVID-19 reagents.

Robust and sensitive amplicon-based whole-genome sequencing assay of respiratory syncytial virus subtype A and B.

In this paper, we report an improved high-throughput respiratory syncytial virus (RSV) whole-genome sequencing (WGS) assay performed directly on clinical samples, using a 4-primer-pool, short-amplicon PCR-tiling approach that is suitable for short-read sequencing platforms. The RSV WGS approach described in this study has increased sensitivity compared to previous approaches and can be applied to clinical specimens without the requirement for enrichment. The updated approach produces sequences of high quality consistently and cost-effectively, suitable for implementation to underpin national and global programs for the surveillance of RSV genomic variation. The quality of sequence produced is essential for preparedness for new interventions in monitoring antigenic escape, where a single point mutation might lead to a reduction in antibody binding effectiveness and neutralizing activity, or indeed in the monitoring of retaining susceptibility to neutralization by existing and new interventions.

Immunogenicity and safety of RAZI recombinant spike protein vaccine (RCP) as a booster dose after priming with BBIBP-CorV: a parallel two groups, randomized, double blind trial

BackgroundThe immunity induced by primary vaccination is effective against COVID-19; however, booster vaccines are needed to maintain vaccine-induced immunity and improve protection against emerging variants. Heterologous boosting is believed to result in more robust immune responses. This study investigated the safety and immunogenicity of the Razi Cov Pars vaccine (RCP) as a heterologous booster dose in people primed with Beijing Bio-Institute of Biological Products Coronavirus Vaccine (BBIBP-CorV).MethodsWe conducted a randomized, double-blind, active-controlled trial in adults aged 18 and over primarily vaccinated with BBIBP-CorV, an inactivated SARS-CoV-2 vaccine. Eligible participants were randomly assigned (1:1) to receive a booster dose of RCP or BBIBP-CorV vaccines. The primary outcome was neutralizing antibody activity measured by a conventional virus neutralization test (cVNT). The secondary efficacy outcomes included specific IgG antibodies against SARS-CoV-2 spike (S1 and receptor-binding domain, RBD) antigens and cell-mediated immunity. We measured humoral antibody responses at 2 weeks (in all participants) and 3 and 6 months (a subgroup of 101 participants) after the booster dose injection. The secondary safety outcomes were solicited and unsolicited immediate, local, and systemic adverse reactions.ResultsWe recruited 483 eligible participants between December 7, 2021, and January 13, 2022. The mean age was 51.9 years, and 68.1% were men. Neutralizing antibody titers increased about 3 (geometric mean fold increase, GMFI = 2.77, 95% CI 2.26–3.39) and 21 (GMFI = 21.51, 95% CI 16.35–28.32) times compared to the baseline in the BBIBP-CorV and the RCP vaccine groups. Geometric mean ratios (GMR) and 95% CI for serum neutralizing antibody titers for RCP compared with BBIBP-CorV on days 14, 90, and 180 were 6.81 (5.32–8.72), 1.77 (1.15–2.72), and 2.37 (1.62–3.47) respectively. We observed a similar pattern for specific antibody responses against S1 and RBD. We detected a rise in gamma interferon (IFN-γ), tumor necrosis factor (TNF-α), and interleukin 2 (IL-2) following stimulation with S antigen, particularly in the RCP group, and the flow cytometry examination showed an increase in the percentage of CD3 + /CD8 + lymphocytes. RCP and BBIBP-CorV had similar safety profiles; we identified no vaccine-related or unrelated deaths.ConclusionsBBIBP-CorV and RCP vaccines as booster doses are safe and provide a strong immune response that is more robust when the RCP vaccine is used. Heterologous vaccines are preferred as booster doses.Trial registrationThis study was registered with the Iranian Registry of Clinical Trial at www.irct.ir, IRCT20201214049709N4. Registered 29 November 2021.

Intranasal vaccine for Lyme disease provides protection against tick transmitted Borrelia burgdorferi beyond one year

Strategies for disease control are necessary to reduce incidence of Lyme Disease (LD) including development of safe vaccines for human use. Parainfluenza virus 5 (PIV5) vector has an excellent safety record in animals and PIV5-vectored vaccines are currently under clinical development. We constructed PIV5-vectored LD vaccine candidates expressing OspA from B. burgdorferi (OspAB31) and a chimeric protein containing sequences from B. burgdorferi and B. afzelii (OspABPBPk). Immunogenicity and vaccine efficacy were analyzed in C3H-HeN mice after prime-boost intranasal vaccination with live PIV5-OspAB31 or PIV5-OspABPBPk, subcutaneous (s.c.) vaccination with rOspAB31+Alum, and the respective controls. Mice vaccinated intranasally with live PIV5-AB31 or PIV5-ABPBPk had higher endpoint titers of serum antibody against OspAB31 at 6- and 12- months post vaccination, compared to mice vaccinated s.c. with rOspAB31. Neutralization activity of antibody was maintained up to 18-months post-immunization, with the response greater in live PIV5-delivered OspA vaccines, than that induced by s.c. rOspAB31. Challenge with infected ticks carrying 10-19 strains of B. burgdorferi performed at 4-, 9- or 15-months post-immunization showed increased breakthrough infections in mice vaccinated with s.c. rOspAB31 compared to intranasal PIV5-AB31 or PIV5-ABPBPk at 9- and 15-months, as determined by quantification of serologic antibodies to B. burgdorferi proteins as well as flaB DNA in tissues, and by visualization of motile B. burgdorferi in culture of tissues under dark field microscope. These findings indicate that immunization of mice with PIV5 delivered OspA generates immune responses that produce longer-lasting protection ( > 1 year) against tick-transmitted B. burgdorferi than a parenteral recombinant OspA vaccine.

Potent induction of humoral and cellular immunity after bivalent BA.4/5 mRNA vaccination in dialysis patients

Knowledge on immunogenicity of the bivalent Omicron BA.4/5 vaccine in dialysis patients and the effect of a previous infection is limited. Therefore, vaccine-induced humoral and cellular immunity was analyzed in dialysis patients and immunocompetent controls with and without prior infection. In an observational study, 33 dialysis patients and 58 controls matched for age, sex and prior infection status were recruited. Specific IgG, neutralizing antibody activity and cellular immunity towards the spike-antigen from parental SARS-CoV-2 and Omicron-subvariants BA.1, BA.2 and BA.4/5 were analyzed before and 13-18 days after vaccination. The bivalent vaccine led to a significant induction of IgG, neutralizing titers, and specific CD4+ and CD8+ T-cell levels. Neutralizing activity towards the parental strain was higher than towards the Omicron-subvariants, whereas specific T-cell levels towards parental spike and Omicron-subvariants did not differ indicating substantial cross-reactivity. Dialysis patients with prior infection had significantly higher spike-specific CD4+ T-cell levels with lower CTLA-4 expression compared to infection-naive patients. When compared to controls, no differences were observed between infection-naive individuals. Among convalescent individuals, CD4+ T-cell levels were higher in patients and neutralizing antibodies were higher in controls. Vaccination was overall well tolerated in both dialysis patients and controls with significantly less adverse events among patients. In conclusion, our study did not provide any evidence for impaired immunogenicity of the bivalent Omicron BA.4/5 vaccine in dialysis patients. Unlike in controls, previous infection of patients was even associated with higher levels of spike-specific CD4+ T cells, which may reflect prolonged encounter with antigen during infection.

A Gamma-adapted subunit vaccine induces broadly neutralizing antibodies against SARS-CoV-2 variants and protects mice from infection.

In the context of continuous emergence of SARS-CoV-2 variants of concern (VOCs), one strategy to prevent the severe outcomes of COVID-19 is developing safe and effective broad-spectrum vaccines. Here, we present preclinical studies of a RBD vaccine derived from the Gamma SARS-CoV-2 variant adjuvanted with Alum. The Gamma-adapted RBD vaccine is more immunogenic than the Ancestral RBD vaccine in terms of inducing broader neutralizing antibodies. The Gamma RBD presents more immunogenic B-cell restricted epitopes and induces a higher proportion of specific-B cells and plasmablasts than the Ancestral RBD version. The Gamma-adapted vaccine induces antigenspecific T cell immune responses and confers protection against Ancestral and Omicron BA.5 SARS-CoV-2 challenge in mice. Moreover, theGammaRBD vaccine induces higher and broader neutralizing antibody activity than homologous booster vaccination in mice previously primed with different SARS-CoV-2 vaccine platforms. Our study indicates that the adjuvanted Gamma RBD vaccine is highly immunogenic and a broad-spectrum vaccine candidate.

Dynamics of humoral and cellular response to three doses of anti-SARS-CoV-2 BNT162b2 vaccine in patients with hematological malignancies and older subjects.

Few data are available about the durability of the response, the induction of neutralizing antibodies, and the cellular response upon the third dose of the anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine in hemato-oncological patients. To investigate the antibody and cellular response to the BNT162b2 vaccine in patients with hematological malignancy. We measured SARS-CoV-2 anti-spike antibodies, anti-Omicron neutralizing antibodies, and T-cell responses 1 month after the third dose of vaccine in 93 fragile patients with hematological malignancy (FHM), 51 fragile not oncological subjects (FNO) aged 80-92, and 47 employees of the hospital (healthcare workers, (HW), aged 23-66 years. Blood samples were collected at day 0 (T0), 21 (T1), 35 (T2), 84 (T3), 168 (T4), 351 (T pre-3D), and 381 (T post-3D) after the first dose of vaccine. Serum IgG antibodies against S1/S2 antigens of SARS-CoV-2 spike protein were measured at every time point. Neutralizing antibodies were measured at T2, T3 (anti-Alpha), T4 (anti-Delta), and T post-3D (anti-Omicron). T cell response was assessed at T post-3D. An increase in anti-S1/S2 antigen antibodies compared to T0 was observed in the three groups at T post-3D. After the third vaccine dose, the median antibody level of FHM subjects was higher than after the second dose and above the putative protection threshold, although lower than in the other groups. The neutralizing activity of antibodies against the Omicron variant of the virus was tested at T2 and T post-3D. 42.3% of FHM, 80,0% of FNO, and 90,0% of HW had anti-Omicron neutralizing antibodies at T post-3D. To get more insight into the breadth of antibody responses, we analyzed neutralizing capacity against BA.4/BA.5, BF.7, BQ.1, XBB.1.5 since also for the Omicron variants, different mutations have been reported especially for the spike protein. The memory T-cell response was lower in FHM than in FNO and HW cohorts. Data on breakthrough infections and deaths suggested that the positivity threshold of the test is protective after the third dose of the vaccine in all cohorts. FHM have a relevant response to the BNT162b2 vaccine, with increasing antibody levels after the third dose coupled with, although low, a T-cell response. FHM need repeated vaccine doses to attain a protective immunological response.

Immunoglobulin replacement products protect against SARS-CoV-2 infection in vivo despite poor neutralizing activity.

Immunoglobulin (IG) replacement products are used routinely in patients with immune deficiency and other immune dysregulation disorders who have poor responses to vaccination and require passive immunity conferred by commercial antibody products. The binding, neutralizing, and protective activity of intravenously administered IG against SARS-CoV-2 emerging variants remains unknown. Here, we tested 198 different IG products manufactured from December 2019 to August 2022. We show that prepandemic IG had no appreciable cross-reactivity or neutralizing activity against SARS-CoV-2. Anti-spike antibody titers and neutralizing activity against SARS-CoV-2 WA1/2020 D614G increased gradually after the pandemic started and reached levels comparable to vaccinated healthy donors 18 months after the diagnosis of the first COVID-19 case in the United States in January 2020. The average time between production to infusion of IG products was 8 months, which resulted in poor neutralization of the variant strain circulating at the time of infusion. Despite limited neutralizing activity, IG prophylaxis with clinically relevant dosing protected susceptible K18-hACE2-transgenic mice against clinical disease, lung infection, and lung inflammation caused by the XBB.1.5 Omicron variant. Moreover, following IG prophylaxis, levels of XBB.1.5 infection in the lung were higher in FcγR-KO mice than in WT mice. Thus, IG replacement products with poor neutralizing activity against evolving SARS-CoV-2 variants likely confer protection to patients with immune deficiency disorders through Fc effector function mechanisms.

Utility of an In-Vitro Micro-Neutralizing Test in Comparison to a Plaque Reduction Neutralization Test for Dengue Virus, Japanese Encephalitis Virus, and Zika Virus Serology and Drug Screening.

Flavivirus infections, including dengue virus (DENV), Japanese encephalitis virus (JEV), and Zika virus (ZIKV), present significant global public health challenges. For successful vaccine design, the assessment of neutralizing antibody activity requires reliable and robust methodologies for determining antibody titers. Although the plaque reduction neutralization test (PRNT) is commonly acknowledged as the gold standard, it has limitations in terms of time and cost, and its usage may be limited in resource-limited settings. To address these challenges, we introduced the micro-neutralization test (MNT) as a simplified alternative to the PRNT. The MNT employs a 96-well plate format, conducts microscale neutralization assays, and assesses cell viability by dissolving cells to create a uniform color solution, which is measured with a spectrometer. In this study, we evaluated the utility of the MNT by contrasting the end-point titers of the MNT and PRNT using 4 monoclonal antibodies, 15 non-human primate serum samples, and 2 therapeutic drug candidates across flaviviruses. The results demonstrated a strong correlation between the MNT and PRNT titers, affirming the robustness and reproducibility of the MNT for evaluating control measures against flaviviruses. This research contributes valuable insights toward the development of a cost-effective antibody titer testing approach that is particularly suitable for resource-limited settings.

SARSCoV-2 antibody prevalence and titers in persons living with HIV cared for at a large tertiary reference center in Mexico City

ObjectiveTo assess SARS-CoV-2 antibody prevalence and titers in people living with HIV (PLWHIV) on antiretroviral treatment (ART) enrolled at a tertiary reference hospital in Mexico.MethodsTwo plasma aliquots per person, used for HIV viral load follow-up between 01/2020 and 09/2021, were used to assess total anti-N and neutralizing SARS-CoV-2 antibodies. Sociodemographic, clinical, and SARS-CoV-2 exposure risk information were collected. The risk associated with SARS-CoV-2 exposure and associations with antibody titers were analyzed with logistic, Cox, and linear multivariable models.Results803 PLWHIV participated; 233 had detectable SARS-CoV-2 antibodies (prevalent cases), and 132 seroconverted (incident cases). Overall, the adjusted prevalence was 46.45%, with an incidence rate of 3.78 cases/100 person-months. Factors associated with prevalent cases included lower age, location (western zone of Mexico City and the neighboring Mexico State), use of public transport, attendance at meetings without social distancing, and higher CD4 + T cell counts (p < 0.05; multivariable logistic model). BNT162b2 vaccination reduced incident cases (Cox adjusted HR = 0.4; p = 0.013). Notably, previously infected and vaccinated individuals showed maximization of neutralizing activity (p < 0.001). No associations between SARS-CoV-2 neutralization and HIV-related variables (CD4 + T cell counts, viral load, number of years in viral suppression, ART regimen) were found in multivariable analysis.ConclusionsSARS-CoV-2 infection was associated with community risk rather than HIV-associated variables in PLWH on ART and clinical follow-up. Antibody neutralization activity in vaccinated participants was maximized with previous SARS-CoV-2 infection.

Sars-Cov-2 Antibody and T-Cell Responses in Patients with Paroxysmal Nocturnal Hemoglobinuria and Aplastic Anemia after Four COVID-19 Vaccinations

Introduction: There is limited data on SARS-CoV-2 vaccination responses in the rare hematological disorders paroxysmal nocturnal hemoglobinuria (PNH) and aplastic anemia (AA). These patients were expected to have more severe COVID-19 infection and reduced vaccination responses due to their underlying disease and immunosuppressive treatment. We previously reported limited anti-spike IgA/G/M responses after first COVID-19 vaccination which improved following second vaccination (Pike et al, Lancet Haematology 2022). Here we report spike-specific IgG, in vitro viral neutralization and T-cell responses in 244 patients with PNH and/or AA and in 49 healthy volunteers, following the first four vaccinations. Methods: In 2021, the United Kingdom National PNH service centre based in Leeds initiated a prospective observational non-interventional study evaluating immune responses to COVID-19 vaccinations in patients with PNH and/or AA. All patients were consented to the Leeds PNH Research Tissue Bank. At baseline, the patient cohort comprised 94 patients with classic PNH, 75 with AA-PNH overlap and 75 with AA with asymptomatic PNH clones &amp;lt;50%. Serological humoral responses were evaluated in blood samples using a quantitative spike-specific IgG SARS-CoV-2 ELISA (EuroImmun). Samples were further analyzed using a high-throughput live-virus neutralization assay performed at the Francis Crick Institute, London, against wild-type SARS-CoV-2 and against omicron B.1.1.529 and delta B.1.617.2 variants. Adaptive T-cell immune responses were assessed by incubating cryopreserved peripheral blood mononuclear cells with SARS-CoV-2 spike and nucleocapsid peptides in an ELISpot interferon-gamma release assay (Oxford Immunotec). Results: Prior to vaccination (baseline) in-vitro anti-spike IgG response was not significantly different between patients and healthy volunteers (p&amp;gt;0.99). After first vaccination, 2/40 (5%) healthy volunteers and 66/155 (42.6%) PNH and AA patients failed to mount a detectible spike-specific IgG. The antibody titre was also significantly reduced (median spike-specific IgG titre in patients 37.9 BAU/ml [IQR 11.56-120.7] versus 289.4 BAU/ml in healthy volunteers [IQR 177.7-1326], p&amp;lt;0.0001). The magnitude of IgG response improved after second vaccination but remained lower than healthy volunteers (median IgG titre in patients 408.6 BAU/ml [IQR 184.1-942.6] versus healthy volunteers 2639 BAU/ml [IQR 894.0-3706], p&amp;lt;0.001). However, responses improved sequentially following repeated vaccinations and after the third dose were equivalent to healthy volunteers (p&amp;gt;0.99). Viral neutralizing antibody activity elicited by vaccination showed similar results, with significantly reduced titres post first vaccination against wild-type SARS-CoV-2 in patients compared with healthy volunteers (p=0.0001). After second vaccination, neutralizing antibody titres in patients versus healthy volunteers remained significantly reduced against wild-type (p=0.0088) and the delta variant (p=0.0001) but not the omicron variant (p=0.078). Reassuringly, responses improved after 3 rd dose of vaccine following which there was no significant difference detected for all three strains (p&amp;gt;0.99). Of the evaluable samples, anti-spike T-cell responses were reduced at all timepoints in patients compared with healthy volunteers but improved with each vaccination. After first vaccination, a reactive result was seen in 32.1% (n=27/84) versus 40% in healthy volunteers (n=16/40) and remained low after second vaccination (35.0% (n=43/123) in patients versus 64.1% (n=24/39) in healthy volunteers). However, after third vaccination, responses had improved to 58.7% (n=44/75) in patients versus 75% (n=24/32) in healthy volunteers. After 4 th vaccination, spike-specific responses were detectable in 67.6% (n=25/37) of patients. There was limited reactivity to nucleocapsid antigens in both groups at all timepoints highlighting that the observed responses were due to vaccination rather than COVID-19 infection. To date there have been no deaths due to COVID-19 infection in patients in this study or in our wider service population since the introduction of the vaccination programme. Conclusion: This study demonstrates the importance of repeated SARS-Cov-2 vaccinations in patients with PNH and/or AA in order to improve humoral and cellular immune responses.

RBD class 1 and 2 antibody epitopes elicit around 70% neutralizing capacity against SARS-CoV-2 virus following boosting with inactivated virus vaccine

A third dose of inactivated virus vaccine (IVV) boosts neutralizing antibodies, reducing SARS-CoV-2 transmission rate and COVID-19 severity. However, the impact of RBD-elicited antibodies and their neutralizing activity by the boost of IVV is unknown. We investigated the impact of IVV's boost shot on RBD-elicited antibodies and their neutralizing activity in 18 subjects receiving the second and third IVV doses. Using an RBD antibodies depletion assay, we assessed the neutralizing activity of RBD-elicited antibodies. After the second dose, RBD-antigen elicitation accounted for ∼60% of neutralizing activity, which increased to 82% after the IVV boost against ancestral SARS-CoV-2. Depleting class 3 and class 4-specific antibodies with the Beta-RBD protein revealed that NAbs targeting RBD class 1 and class 2 subdomains increased from 57% to 75% post-boost. These findings highlight the significant enhancement of RBD-specific antibodies, especially against RBD class 1 and class 2, with IVV booster doses. Our study offers valuable insights for optimizing COVID-19 vaccine strategies.

Safety and immunogenicity of an inactivated SARS-CoV-2 vaccine (FAKHRAVAC®) in healthy adults aged 18–55 years: Randomized, double-blind, placebo-controlled, phase I clinical trial

BackgroundThe FAKHRAVAC®, an inactivated SARS-CoV-2 vaccine, was assessed for safety and immunogenicity. Methods and findingsIn this double-blind, placebo-controlled, phase I trial, we randomly assigned 135 healthy adults between 18 and 55 to receive vaccine strengths of 5 or 10 μg/dose or placebo (adjuvant only) in 0–14 or 0–21 schedules. This trial was conducted in a single center in a community setting. The safety outcomes in this study were reactogenicity, local and systemic adverse reactions, abnormal laboratory findings, and Medically Attended Adverse Events (MAAE). Immunogenicity outcomes include serum neutralizing antibody activity and specific IgG antibody levels.The most frequent local adverse reaction was tenderness (28.9%), and the most frequent systemic adverse reaction was headache (9.6%). All adverse reactions were mild, occurred at a similar incidence in all six groups, and were resolved within a few days. In the 10-µg/dose vaccine group, the geometric mean ratio for neutralizing antibody titers at two weeks after the second injection compared to the placebo group was 9.03 (95% CI: 3.89–20.95) in the 0–14 schedule and 11.77 (95% CI: 2.77–49.94) in the 0–21 schedule. The corresponding figures for the 5-µg/dose group were 2.74 (1.2–6.28) and 5.2 (1.63–16.55). The highest seroconversion rate (four-fold increase) was related to the 10-µg/dose group (71% and 67% in the 0–14 and 0–21 schedules, respectively). ConclusionsFAKHRAVAC® is safe and induces a strong humoral immune response to the SARS-CoV-2 virus at 10-µg/dose vaccine strength in adults aged 18–55. This vaccine strength was used for further assessment in the phase II trial.Trial registrationThis study is registered with https://www.irct.ir; IRCT20210206050259N1.

Phase II, Safety and Immunogenicity of RAZI Cov Pars (RCP) SARS Cov-2 Vaccine in Adults Aged 18–70 Years; A Randomized, Double-Blind Clinical Trial

BackgroundThis study explores the safety and immunogenicity of the Razi-Cov-Pars (RCP) SARS Cov-2 recombinant spike protein vaccine. MethodIn a randomized, double-blind, placebo-controlled trial, adults aged 18–70 were randomly allocated to receive selected 10 µg/200 µl vaccine strengths or placebo (adjuvant). It included two intramuscular injections at days 0 and 21, followed by an intranasal dose at day 51. Immediate and delayed solicited local and systemic adverse reactions after each dose up to a week, and specific IgG antibodies against SARS Cov-2 spike antigens two weeks after the 2nd dose were assessed as primary outcomes. Secondary safety outcomes were abnormal laboratory findings and medically attended adverse events (MAAE) over six months follow up. Secondary immunogenicity outcomes were neutralizing antibody activity and cell-mediated immune response. ResultBetween May 27th and July 15th, 2021, 500 participants were enrolled. Participants' mean (SD) age was 37.8 (9.0), and 67.0 % were male. No immediate adverse reaction was observed following the intervention. All solicited local and systemic adverse events were moderate (Grade I-II). Specific IgG antibody response against S antigen in the vaccine group was 5.28 times (95 %CI: 4.02–6.94) the placebo group with a 75 % seroconversion rate. During six months of follow-up, 8 SAEs were reported, unrelated to the study intervention. The participants sustained their acquired humoral responses at the end of the sixth month. The vaccine predominantly resulted in T-helper 1 cell-mediated immunity, CD8+ cytotoxic T-cell increase, and no increase in inflammatory IL-6 cytokine. ConclusionRCP vaccine is safe and creates strong and durable humoral and cellular immunity. Trial registration(IRCT20201214049709N2).

Pseudotyping Improves the Yield of Functional SARS-CoV-2 Virus-like Particles (VLPs) as Tools for Vaccine and Therapeutic Development.

Virus-like particles (VLPs) have been proposed as an attractive tool in SARS-CoV-2 vaccine development, both as (1) a vaccine candidate with high immunogenicity and low reactogenicity and (2) a substitute for live virus in functional and neutralization assays. Though multiple SARS-CoV-2 VLP designs have already been explored in Sf9 insect cells, a key parameter ensuring VLPs are a viable platform is the VLP spike yield (i.e., spike protein content in VLP), which has largely been unreported. In this study, we show that the common strategy of producing SARS-CoV-2 VLPs by expressing spike protein in combination with the native coronavirus membrane and/or envelope protein forms VLPs, but at a critically low spike yield (~0.04-0.08 mg/L). In contrast, fusing the spike ectodomain to the influenza HA transmembrane domain and cytoplasmic tail and co-expressing M1 increased VLP spike yield to ~0.4 mg/L. More importantly, this increased yield translated to a greater VLP spike antigen density (~96 spike monomers/VLP) that more closely resembles that of native SARS-CoV-2 virus (~72-144 Spike monomers/virion). Pseudotyping further allowed for production of functional alpha (B.1.1.7), beta (B.1.351), delta (B.1.617.2), and omicron (B.1.1.529) SARS-CoV-2 VLPs that bound to the target ACE2 receptor. Finally, we demonstrated the utility of pseudotyped VLPs to test neutralizing antibody activity using a simple, acellular ELISA-based assay performed at biosafety level 1 (BSL-1). Taken together, this study highlights the advantage of pseudotyping over native SARS-CoV-2 VLP designs in achieving higher VLP spike yield and demonstrates the usefulness of pseudotyped VLPs as a surrogate for live virus in vaccine and therapeutic development against SARS-CoV-2 variants.

Assessment of antibody dynamics and neutralizing activity using serological assay after SARS-CoV-2 infection and vaccination.

The COVID-19 antibody test was developed to investigate the humoral immune response to SARS-CoV-2 infection. In this study, we examined whether S antibody titers measured using the anti-SARS-CoV-2 IgG II Quant assay (S-IgG), a high-throughput test method, reflects the neutralizing capacity acquired after SARS-CoV-2 infection or vaccination. To assess the antibody dynamics and neutralizing potency, we utilized a total of 457 serum samples from 253 individuals: 325 samples from 128 COVID-19 patients including 136 samples from 29 severe/critical cases (Group S), 155 samples from 71 mild/moderate cases (Group M), and 132 samples from 132 health care workers (HCWs) who have received 2 doses of the BNT162b2 vaccinations. The authentic virus neutralization assay, the surrogate virus neutralizing antibody test (sVNT), and the Anti-N SARS-CoV-2 IgG assay (N-IgG) have been performed along with the S-IgG. The S-IgG correlated well with the neutralizing activity detected by the authentic virus neutralization assay (0.8904. of Spearman's rho value, p < 0.0001) and sVNT (0.9206. of Spearman's rho value, p < 0.0001). However, 4 samples (2.3%) of S-IgG and 8 samples (4.5%) of sVNT were inconsistent with negative results for neutralizing activity of the authentic virus neutralization assay. The kinetics of the SARS-CoV-2 neutralizing antibodies and anti-S IgG in severe cases were faster than the mild cases. All the HCWs elicited anti-S IgG titer after the second vaccination. However, the HCWs with history of COVID-19 or positive N-IgG elicited higher anti-S IgG titers than those who did not have it previously. Furthermore, it is difficult to predict the risk of breakthrough infection from anti-S IgG or sVNT antibody titers in HCWs after the second vaccination. Our data shows that the use of anti-S IgG titers as direct quantitative markers of neutralizing capacity is limited. Thus, antibody tests should be carefully interpreted when used as serological markers for diagnosis, treatment, and prophylaxis of COVID-19.

Acquisition of Humoral Immune Responses in Convalescent Japanese People with SARS-CoV-2 (COVID-19) Infection in 2021.

We investigated humoral immune responses in 222 unvaccinated Japanese people after recovery from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in 2021. Anti-spike-protein IgG antibody levels and neutralizing antibody titers were measured in serum samples obtained within 20-180 days after diagnosis. The geometric mean of antibody titers was 1555 ELU/mL (95% confidence interval (CI) = 1257-1923), and the neutralizing activity (50% inhibitory dilution) was 253 (95% CI = 204-313). The antibody titer and neutralizing activity both increased with increasing disease severity, and both values were approximately fourfold higher for hospitalized patients than for non-hospitalized patients. However, these differences were smaller in older patients. The humoral immune response, which increased with increasing disease severity, gradually decreased over time after SARS-CoV-2 infection. Most patients with mild or moderate symptoms sustained neutralizing activity for up to 180 days after the infection; the decay of the neutralizing activity in the asymptomatic patients was rather faster than in the other groups. Around 11.7% (26/222) of patients had very low neutralizing activity, and half of these were aged in their 20s. Our study's results show the importance of measuring the neutralizing activity to confirm the immune status and to estimate the timing of vaccines.