Characterization of Adaptive Immune Responses to SARS-CoV-2 Updated Bivalent Booster

Bivalent COVID-19 vaccines that contain two mRNAs encoding Wuhan-1 and Omicron BA.4/5 spike proteins are successful in preventing infection from the original strain and Omicron variants, but the quality of adaptive immune responses is still not well documented. This study aims at characterizing adaptive immune responses to the bivalent booster vaccination in 46 healthy participants. Plasma and PBMC were collected prior and three weeks after bivalent booster. We measured anti-N, anti-S, and RBD IgM, IgA, IgG plasma titers against original, Omicron BA.1, and BA.5 variants (pending) as well as total anti-S IgG titers and surrogate Virus Neutralization capacity against the Alpha, Delta, and BA.1 variant. With spectral flow-cytometry we identified peripheral blood B-cells specific for the RBD of the S-protein of the original and BA.1 variants. T-cell-specific responses were assessed by cytokine release assay after stimulation with SARS-CoV-2 peptides from the original, BA.1, BA.4, and BA.5 variants (pending). Finally, we performed TRB and IGH repertoire studies on sorted CD4+, CD8+, CD19+ lymphocytes, to study breadth of SARS-CoV-2 specific clonotypes (pending). 27/46 participants were analyzed;9 had SARS-CoV-2 infection (COVID+), while 18 are infection naive (COVID-). In both groups, median time since last dose of SARS-CoV-2 vaccine (3rd or 4th) was 11 months. All subjects were positive for anti-S IgG prior to bivalent booster. The COVID + group displayed anti-S IgG pre-booster levels and neutralization against BA.1 higher than the COVID- group. Significant increase post-boost of total anti-S IgG and BA.1 neutralizing activity was detected in the COVID- but not in the COVID+ group;however, no difference in neutralization activity post-boost was detected between the two groups. Furthermore, the COVIDgroup showed significant increase in the frequency of CD19+ and CD27+ switched memory B-cells specific for BA.1 RBD in post-boost compared to pre-boost samples. However, post-boost frequencies of the same B-cells were higher in the COVID+ compared to the COVID- group. These preliminary findings confirm that among individual immunized with the original COVID-19 mRNAvaccine, prior COVID infection provides increased protection against SARS-CoV-2 variants. They also demonstrate that booster immunization with the bivalent vaccine induces robust adaptive immune responses against Omicron variant.[Formula presented][Formula presented]Copyright © 2023 Elsevier Inc.

Determination of antibodies to SARS-CoV-2 in patients with coronavirus infection

COVID-19, caused by the novel SARS-CoV-2 virus, poses major challenges for global public health. The detection of antibodies in blood serum is one of the important methods for diagnostics of COVID-19 patients. The main aim was to study the dynamics of the appearance of neutralizing antibodies and antibodies to the SARS-CoV-2 proteins in COVID-19 patients sera. Material and methods. The blood sera of four groups of people were studied: "intact" donors (blood sera were collected in 2016-2019);patients with a laboratory-confirmed diagnosis of acute respiratory viral infection;patients with influenza (antibodies to the influenza virus have been identified) and patients with a PCR confirmed diagnosis of COVID-19. Blood sera were analyzed in ELISA with commercial kits for detection of IgG to SARS-CoV-2 (N, S) proteins and total antibodies to RBD of protein S and in neutralization test (NT). Results and discussion. Antibodies to SARS-CoV-2 were not detected in paired blood sera of people from groups 1-3 by ELISA and NT. At the time of hospitalization of patients with COVID-19 in the sera of 12 (19%) patients antibodies to SARS-CoV-2 were absent when they were determined by NT and ELISA. In blood sera taken 4-9 days after hospitalization, neutralizing antibodies and antibodies to at least one viral protein were detected in ELISA. Conclusion. At the time of hospitalization, the overwhelming majority of patients had a humoral immune response to the SARS-CoV-2. In the dynamics of observation, the levels of antibodies to SARS-CoV-2 proteins increased, to a greater extent to RBD.Copyright © 2022 Geotar Media Publishing Group

Persistence and Dynamicity of Sars-Cov-2 Antibody Response after Double Dose of Bbv-152 and Azd1222 Vaccines: A Prospective, Longitudinal, Year-Long Cohort Study

Intro: Different vaccines against COVID-19 have been approved by the World Health Organization (WHO) at different stages, however, limited data is available on long-term kinetics of antibodies induced by vaccines. This study was performed to investigate the persistence and dynamicity of BBV-152 (Covaxin)- and AZD1222 (Covishield)-induced immunoglobulin-G (IgG) antibodies over the year and neutralizing antibodies' status after the one-month post booster dose. Method(s): This 52-week longitudinal cohort study documented antibody persistence and neutralizing antibody status among 278 health-care workers (HCWs) from four different healthcare and research facilities in Odisha, enrolled in January 2021 and continued until March 2022. An automated chemiluminescence immune assay (CLIA) platform from Abbott Diagnostics was used to quantify IgG antibodies against SARS-CoV-2's spike receptor-binding domain (RBD) and a surrogate virus neutralization test (sVNT) was performed by enzyme-linked immunosorbent assay (ELISA). If any participants developed any symptoms of COVID-19, nasopharyngeal swabs were collected and sent to ICMR- RMRC, Bhubaneswar for RT-PCR confirmation. Finding(s): Among the 243 participants, 119 HCWs (48.97%) were Covaxin recipients and the remaining 124 (51.02%) were Covishield recipients. During the seven follow- ups, 104 participants (42.79%) were identified as vaccine breakthrough cases. In 139 non-infected HCWs, the median antibody titer significantly waned after ten months of double dose, both for Covaxin (342.7 AU/mL at DD1 vs 43.9 AU/mL at DD10) and Covishield (2325.8 AU/mL at DD3 vs 595.2 AU/mL at DD10). No statistically significant differences in antibody titers were observed based on age, gender, comorbidities, and blood groups. The median inhibition activity of sVNT was increased significantly for Covaxin and Covishield booster recipients. Among the booster dose recipients, 24 had breakthrough cases by the Omicron variant. Conclusion(s): Results of this longitudinal cohort study can be used to implement vaccination strategies and could also aid in tracking and designing vaccine mandates to minimize vaccine escape.Copyright © 2023

Phase 3 Study of Safety and Immunogenicity of Gbp510 Vaccine Adjuvanted with As03 in Adults

Intro: GBP510 contains the self-assembling recombinant nanoparticle displaying SARS-CoV-2 Spike protein receptor binding domain and is adjuvanted with AS03. We report interim Phase 3 study (NCT05007951) results up to 4 weeks post-dose 2 (Data-cut: March-18-2022), where immunogenicity to the D614G ancestral strain and safety of 25mug GBP510/AS03 candidate was compared to ChAdOx1-S (Vaxzevria). Method(s): This Phase 3 randomized, active-controlled, observer-blind, parallel- group study in adults was conducted in 6 countries. Cohort1: 1,895 subjects (naive to COVID-19 vaccination and infection) randomized at 2:1 ratio (GBP510/AS03:ChAdOx1-S) to assess immunogenicity and safety;Cohort 2: 2,141 subjects at 5:1 ratio, regardless of their serostatus at screening for safety assessment. Subjects were vaccinated twice at a 4-week interval with 0.5 mL of the test vaccine (GBP510/AS03) or active control (ChAdOx1-S) in deltoid muscle. The primary objective was to demonstrate the superiority of geometric mean titer (GMT) and non-inferiority in seroconversion rate (SCR: >=4-fold rise from baseline) of neutralizing antibodies over ChAdOx1-S by live-virus neutralization assay (FRNT). Finding(s): At 2 weeks post-dose 2, GMT ratio of the two groups (Test vaccine/Active control) was 2.93 [95% CI: 2.63, 3.27], satisfying the hypothesis of superiority (95% CI lower limit> 1). The SCR difference (Test vaccine - Active control) was 10.76% [95% CI: 7.68, 14.32], satisfying the hypothesis of non- inferiority (95% CI lower limit> -5%). Good cell-mediated immune responses for Th1 cytokines were also observed with the test vaccine (FluoroSpot). The AE incidence rate for the test vaccine was higher than the active control for solicited local AEs (56.69% vs 49.20%), and comparable for solicited systemic AEs (51.21% vs 53.51%) and unsolicited AEs (13.34% vs 14.66%) after any vaccination. Conclusion(s): Higher immune responses were observed with GBP510/AS03 compared to ChAdOx1-S against D614G strain after 2 weeks post-dose 2. GBP510/AS03 showed a clinically acceptable safety profile;no safety concerns were identified during the study period.Copyright © 2023

Detection of SARS-CoV-2 Antibodies: Comparison of Enzyme Immunoassay, Surrogate Neutralization and Virus Neutralization Test.

BACKGROUND: Since sensitivity and specificity vary widely between tests, SARS-CoV-2 serology results should be interpreted with caution. METHODS: The study included serum samples from patients who had recovered from COVID-19 (n = 71), individuals vaccinated against SARS-CoV-2 (n = 84), and asymptomatic individuals (n = 33). All samples were tested for the presence of binding antibodies (enzyme immunoassay; EIA), neutralizing (NT) antibodies (virus neutralization test; VNT), and surrogate NT (sNT) antibodies (surrogate virus neutralization test; sVNT) of SARS-CoV-2. RESULTS: SARS-CoV-2-binding antibodies were detected in 71 (100%) COVID-19 patients, 77 (91.6%) vaccinated individuals, and 4 (12.1%) control subjects. Among EIA-positive samples, VNT was positive (titer ≥ 8) in 100% of COVID-19 patients and 63 (75.0%) of the vaccinated individuals, while sVNT was positive (>30% inhibition) in 62 (87.3%) patients and 59 (70.2%) vaccinated individuals. The analysis of antibody levels showed a significant moderate positive correlation between EIA and VNT, a moderate positive correlation between EIA and sVNT, and a strong positive correlation between VNT and sVNT. The proportion of positive sVNT detection rate was associated with VNT titer. The lowest positivity (72.4%/70.8%) was detected in samples with low NT titers (8/16) and increased progressively from 88.2% in samples with titer 32 to 100% in samples with titer 256. CONCLUSIONS: sVNT appeared to be a reliable method for the assessment COVID-19 serology in patients with high antibody levels, while false-negative results were frequently observed in patients with low NT titers.

INVESTIGATING ANTIGENIC FEATURES OF THE SARS-CoV-2 ISOLATED IN RUSSIAN FEDERATION IN 2021-2022 BY HYPERIMMUNE MOUSE SERUM NEUTRALISATION.

Introduction. The rapid spread of a new coronavirus infection among populations in many countries worldwide has contributed to the genetic evolution of the virus, resulting in the emergence of multiple genetic variants of the SARSCoV-2 coronavirus. Mutations in the viral genome can affect the ability of the virus to bypass the immune system and complicate development of diagnostic and prophylactic drugs. Data on the neutralizing activity of the sera obtained against previously circulating genetic variants of the virus in relation to current SARS-CoV-2 strains may serve as a scientific basis for the selection of the antigens in vaccine development. The aim of this work was to study cross-reactivity of SARSCoV-2 coronavirus strains belonging to different genetic variants, which were isolated in the territory of the Russian Federation during 2020-2022 in the neutralization reaction using mouse hyperimmune sera. Materials and methods. Ten strains of SARS-CoV-2 coronavirus belonging to different genetic variants were used (three non-VOC strains, alpha, beta, gamma, delta, delta+AY, omicron 1 and omicron 2). The hCoV-19/Australia/VIC01/2020 strain (Wuhan) was included in the study as a prototypical variant. BALBc mice were immunized with inactivated concentrated antigen mixed with a 1:1 adjuvant, which was a virus-like immunostimulatory complex based on Quillaja saponaria (Quillaja saponaria). The antibody titer was determined in the neutralization reaction. Results. Essential decrease of neutralizing ability of antibodies specific to non-vOC genetic variants of SARS-CoV-2 coronavirus was revealed against beta VOC and to a lesser degree against alpha and gamma VOC variants. The differences in the neutralizing activity level of antibodies for alpha and beta VOC variants are not significant among themselves, and with gamma VOC variants - there are no significant differences. Neutralizing ability of antibodies specific to delta VOC against alpha and beta VOC variants decreased 4-fold. Neutralizing activity of sera obtained to omicron 1 and 2 variants in relation to the prototype coronavirus variant was reduced 18-fold, to the gamma variant - 12-fold, to delta variants - more than 30-fold;for other variants it was even lower. Conclusions. The results obtained testify to the presence of cross-reactivity between strains of coronavirus belonging to genetic lines Wuhan, alpha, beta, gamma;it is weaker for delta variants. Mutations in the genome of VOC omicron variants led to a significant decrease in antigenic cross-links with earlier genetic variants of the coronavirus. These findings explain the low efficacy of vaccines based on the Wuhan strain, synthetic immunogens, and recombinant proteins based on it against omicron VOC variants, which have caused a rise in morbidity since early 2022, as well as cases of re-infection of humans with new genetic variants of the coronavirus.Copyright © 2023 Saint Petersburg Pasteur Institute. All rights reserved.

Improved virus inhibition by microencapsulated monoclonal antibody against porcine epidemic diarrhea virus

Porcine epidemic diarrhea virus (PEDV) has been affecting the swine industry, especially in suckling pigs in with a high mortality rate. Among all the strategies to overcome PEDV, boosting mucosal immunity in pig intestine via oral administration appears to be more efficient than other routes. However, there are biological obstacles such as acidic environment that could damage biologics, a product from organisms often used for PEDV treatment. The plant-derived 2C10 monoclonal antibody (mAb) from Nicotiana benthamiana produced by transient expression was revealed as one of the potential candidates against PEDV through oral delivery. Herein, we demonstrated the calcium-alginate microencapsulation system to protect the 2C10 mAb from the harsh condition in the stomach and to be released the 2C10 mAb when arriving in the intestine. The pH-responsive encapsulated 2C10 mAb microbeads were constructed from the calcium-alginate system. The microbeads were well-tolerated under the acidic environment of simulated gastric fluid (SGF) and were digested under the alkaline condition of simulated intestinal fluid (SIF). The encapsulated 2C10 mAb in the SPF-treated microbeads exhibited high virus neutralization efficiency in Vero cells when compared to the unencapsulated 2C10 mAb treated by SPF that cannot neutralize the virus. For these reasons, calcium-alginate microencapsulation system is an attractive platform to be considered as a candidate for the next generation of oral vaccine development.

Quantification of functional antibody titers in a mouse model of SARS-CoV-2 infection

Towards the end of 2019 a novel severe acute respiratory syndrome (SARS)-like coronavirus (SARS-CoV-2) caused the ongoing global pandemic. The virus surface consists of spike proteins that mediate SARS-CoV-2 entry into cells through its receptor-binding domain (RBD) that attaches to the human receptor Angiotensin- Converting Enzyme 2 (ACE2). Upon infection with foreign material, like viruses and bacteria, the human immune system responds by producing a humoral response specific to the viral antigen. Cells from the innate immune system and antibodies generated in the humoral response work to destroy and block infectious antigens from causing damage to the human cells. The S protein of SARSCoV- 2 is the key protein that stimulates the immune system to generate neutralizing antibodies. To safely test and investigate SARS-CoV-2 in BSL-2 lab setting, we propagated a surrogate pseudo typed virus to evaluate the ability of antibodies to reduce viral cell entry and replication in SARS-CoV-2 infected mice model. Quantifying the functional ability of neutralizing antibodies would help us understand how they influence reinfection in recovered individuals. We hypothesize that antibodies generated in SARS-CoV-2 infected mice models will induce a protective immune response against the SARSCoV- 2 infection. To detect and quantify the protective immune response generated in mice, we performed two different serological assays and identified antibodies endpoint titers. Mice were infected with Delta and Beta at time points Day 3 and Day 4. We performed a SARS-CoV-2 Spike pseudo virus neutralization assay and measured luminescence to determine the percentage neutralization of functional antibodies induced in mice serum samples upon infection. Utilizing indirect ELISAs,' we measured absorbance for IgA antibodies in Bronchoalveolar lavage fluid (BALF) serum and total IgG antibodies in cardiac bleeds. Our results showed we did not obtain neutralizing activity of antibodies in mice serum samples taken at early time points, 24 hrs and 4 days, after infection with the Delta variant of SARS CoV2 virus using both the pseudo viruses Omicron andWA spike.We obtained 100% neutralizing activity in mice serum samples taken at day 21 and infected with Beta variant of SARS CoV2 virus using both the pseudo viruses Omicron and WA spike demonstrating that there is cross-neutralization against various variants of concern. Antibodies (IgA, IgM, IgG) generated in mice 3 weeks post infection with SARS CoV2 (Beta) virus are capable of neutralizing and inhibiting the entry of WA spike and Omicron pseudo viruses in human HEK293 T Ace2 cells. Moving forward utilizing samples with timepoints surpassing 3 weeks could possibly yield higher concentrations of IgA and IgM antibodies that can neutralize the SARS-CoV-2 pseudo virus. Thank you to Dr. Rhea Coler, the entire Coler lab, National Institutes of Health (NIH), and Seattle Children's Research Institute.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

Targeting Spike Glycans to Inhibit SARS-CoV-2 Viral Entry

SARS-CoV-2 Spike harbors glycans which function as ligands for lectins. Therefore, it should be possible to exploit lectins to target SARS-CoV-2 and inhibit cellular entry by binding glycans on the Spike protein. Burkholderia oklahomensis agglutinin (BOA) is an antiviral lectin that interacts with viral glycoproteins via N-linked high mannose glycans. Here, we show that BOA binds to the Spike protein and is a potent inhibitor of SARS-CoV-2 viral entry at nanomolar concentrations. Using a variety of biophysical tools such as SEC chromatography, dynamics light scattering, fluorescence binding assays, and electron microscopy, we demonstrate that the interaction is avidity driven and that BOA crosslinks the Spike protein into soluble aggregates. Furthermore, using virus neutralization assays, we demonstrate that BOA effectively inhibits all tested variants of concern as well as SARS-CoV-1, establishing that glycan-targeting molecules have the potential to be pan-coronavirus inhibitors.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

BROAD ANTIBODIES TO FUNCTIONALLY CONSTRAINED REGIONS OF SARS-CoV-2 SPIKE

Background: While remarkable and rapid progress was made in fighting the SARS-CoV-2 pandemic with vaccines and therapeutic antibodies, these approaches were quickly compromised by viral evolution. Therapeutic monoclonal antibodies (mAbs) that were once authorized for clinical use, which all target the receptor binding domain (RBD), are no longer effective against current variants of concern (VOCs) due to mutations in this region of Spike. Thus, to achieve durable protection against SARS-CoV-2, novel mAbs need to show breadth and potency across VOCs and target epitopes that are more constrained. Method(s): mAbs from an individual who had a breakthrough Delta VOC infection after vaccination were isolated from Spike-specific memory B cells. mAbs were assessed for binding affinity and neutralization potency using Spike-pseudotyped lentivirus (PSV) and live SARS-CoV-2 virus neutralization assays. Epitopes were mapped using deep mutational sequencing (DMS) and structural-based methods. Result(s): Three novel mAbs (C68.3, C68.13, C68.59) demonstrated binding breadth to Spikes from various VOCs including Omicron VOCs despite that C68 had not yet been exposed to Omicron. These mAbs potently neutralized the Wuhan-Hu-1 vaccine and Delta strains (IC50 = 9-61ng/mL), and early Omicron strains BA.1, BA.2, BA.5 (IC50 = 12-149 ng/mL). C68.3 and C68.59 retained potency against recent VOCs BQ.1.1 and XBB (IC50 = 121-122 ng/mL and 56-82 ng/mL, respectively) in the PSV assay. Similar neutralization activity was observed in the live virus assay. The potency of these mAbs was greater against Omicron VOCs than all but one of the mAbs previously authorized for treatment and they showed greater breadth. The mAbs target distinct epitopes on the Spike glycoprotein, two in the RBD (C68.3, C68.13) and one in an invariant region downstream of RBD in subdomain 1 (SD1) (C68.59). Structural analysis of C68.59 Fab binding to Spike trimer revealed significant allosteric changes to regions of Spike outside of the epitope in the S2 unit. Finally, DMS escape pathways showed these mAbs target regions highly conserved across VOCs that are also functionally constrained, suggesting escape could incur a fitness cost. Conclusion(s): Overall, these mAbs are novel in their breadth across VOCs and include a potent mAb targeting a rare epitope outside of the RBD in SD1. These mAbs focus on diverse, functionally constrained regions in Spike making them candidates for development as combination therapeutics with good durability against future VOCs.

PROLONGED SARS-CoV-2 VIRAL BURDEN IN SIV-SARS-CoV-2 COINFECTED MACAQUES

Background: Individuals living with HIV are at increased risk of morbidity and mortality from COVID-19. Furthermore, SARS-CoV-2 infection in immunocompromised HIV infected individuals poses a risk to prolonged infection and viral shedding and the emergence of new variants of concern (VOCs). Using the SIV macaque model for AIDS, we are investigating the hypothesis that immune dysfunction during HIV infection will prolong SARSCoV- 2 viral infection, promote enhanced COVID-19 disease, and accelerate viral evolution. Here, we report the impact of SIV-CoV-2 co-infection on immune responses and pathogenesis. Method(s): Eight female rhesus macaques (aged 7-15 years, 5.5-9.9kg) were infected with SIVmac251 via low dose intravaginal challenge and then inoculated with 6.5x105 TCID50/mL SARS-CoV-2 (WA-1) at 17-34 weeks post-SIV infection via combined intranasal and intratracheal routes. Blood, bronchoalveolar lavage (BAL), stool, and nasal, oral, and rectal swabs were collected pre-infection through 14 days post-infection (DPI) to measure immune responses and viremia. ELISAs, ELISPOT, qRT-PCR, lung pathology, cytokine multiplex, and virus neutralization assays were performed to measure viral loads, pathogenesis, and immune responses. Result(s): Three days post-SARS-CoV-2 infection, we observed a transient decrease in CD4 counts, but there were no changes in clinical symptoms or plasma SIV viral loads. However, SARS-CoV-2 replication persisted in the upper respiratory tract, but not the lower respiratory tract. In addition, SARS-CoV-2 IgG seroconversion was delayed and antigen-specific T-cell responses were dampened. Notably, viral RNA levels in nasal swabs were significantly higher 7-14 DPI in SIV+ compared to previously published results using the same SARS-CoV-2 challenge virus in SIV- rhesus (PMCID: PMC8462335, PMC8829873). In addition, SIV/CoV-2 co-infected animals exhibited elevated levels of myeloperoxidase (MPO), a marker of neutrophil activation and increased lung inflammation. Conclusion(s): Here we provide evidence for the utility of the rhesus macaque in modeling human HIV-SARS-CoV-2 co-infection. Our results suggest that immunosuppression during SIV infection impairs de novo generation of anti-SARS-CoV-2 immunity, that may contribute to prolonged SARS-CoV-2 viral shedding, increased transmission windows, altered disease pathogenesis, and lower protection against subsequent SARS-CoV-2 exposures. Studies in progress will determine if SARS-CoV-2 viral evolution is accelerated in SIV-infected macaques.

SARS-CoV-2 LIVE VIRUS NEUTRALIZATION AFTER FOUR COVID-19 VACCINE DOSES IN PWH ON ART

Background: Limited data exist regarding the immune benefits of fourth COVID-19 vaccine doses in people with HIV (PWH) receiving antiretroviral therapy (ART), particularly given that most have now experienced SARS-CoV-2 infection. We measured the effect of fourth doses on SARS-CoV-2 neutralization in 63 PWH, including 19 SARS-CoV-2-naive and 44 SARS-CoV-2-experienced participants. Method(s): Wild-type (WT)-, Omicron-BA.5 and Omicron-BQ.1-specific neutralization activities were longitudinally quantified using live virus assays up to one month post-fourth vaccine dose. Multiple linear regression was used to investigate the relationship between sociodemographic, health and vaccinerelated variables and SARS-CoV-2 neutralization. Result(s): Participants (54 male;9 female) received monovalent (44%) or bivalent (56%) mRNA fourth doses. In COVID-19-naive PWH, a fourth dose enhanced WT- and BA.5-specific neutralization modestly above three-dose levels (p=0.1). In COVID-19-experienced PWH, a fourth dose enhanced WT neutralization modestly (p=0.1) and BA.5 neutralization significantly (p=0.002). Consistent with the humoral benefits of 'hybrid' immunity, the highest neutralization was observed in COVID-19-experienced PWH after a fourth dose. Of note, PWH with Omicron-era infections exhibited higher WT-specific (p=0.04), but comparable BA.5- or BQ.1-specific neutralization, compared to PWH with pre-Omicron-era infections. Overall, BA.5 neutralization was significantly lower than WT in all participants regardless of COVID-19 experience, and BQ.1 neutralization was significantly lower than BA.5 (all p< 0.0001). In multivariable analyses, fourth dose valency did not significantly affect neutralization magnitude, nor did sex, age nor CD4+ T-cell count (neither recent nor nadir). Rather, an mRNA-1273 fourth dose (versus a BNT162b2 one) was the strongest correlate of WT-specific neutralization, while prior COVID-19, regardless of infection era, was the strongest correlate of BA.5 and BQ.1-specific neutralization post-fourth dose. Conclusion(s): Fourth COVID-19 vaccine doses, irrespective of valency, benefit PWH regardless of prior SARS-CoV-2 infection, but the highest neutralization of Omicron-BA.5 and BQ.1 variants post-fourth dose occurred in PWH with hybrid immunity. These results support existing recommendations that all adults receive a fourth immunization within 6 months of their third vaccine dose (or their most recent SARS-CoV-2 infection). (Figure Presented).

SARS-CoV-2 Seroprevalence and Cross-Variant Antibody Neutralization in Cats, United Kingdom.

Anthropogenic transmission of SARS-CoV-2 to pet cats highlights the importance of monitoring felids for exposure to circulating variants. We tested cats in the United Kingdom for SARS-CoV-2 antibodies; seroprevalence peaked during September 2021-February 2022. The variant-specific response in cats trailed circulating variants in humans, indicating multiple human-to-cat transmissions over a prolonged period.

Plasma after both SARS-CoV-2 boosted vaccination and COVID-19 potently neutralizes BQ.1.1 and XBB.1.

Recent 2022 SARS-CoV-2 Omicron variants, have acquired resistance to most neutralizing anti-Spike monoclonal antibodies authorized, and the BQ.1.* sublineages are notably resistant to all authorized monoclonal antibodies. Polyclonal antibodies from individuals both vaccinated and recently recovered from Omicron COVID-19 (VaxCCP) could retain new Omicron neutralizing activity. Here we reviewed BQ.1.* virus neutralization data from 920 individual patient samples from 43 separate cohorts defined by boosted vaccinations (Vax) with or without recent Omicron COVID-19, as well as infection without vaccination (CCP) to determine level of BQ.1.* neutralizing antibodies and percent of plasma samples with neutralizing activity. More than 90 % of the plasma samples from individuals in the recently (within 6 months) boosted VaxCCP study cohorts neutralized BQ.1.1, and BF.7 with 100 % neutralization of WA-1, BA.4/5, BA.4.6 and BA.2.75. The geometric mean of the geometric mean 50 % neutralizing titres (GM (GMT50) were 314, 78 and 204 for BQ.1.1, XBB.1 and BF.7, respectively. Compared to VaxCCP, plasma sampled from COVID-19 naïve subjects who also recently (within 6 months) received at least a third vaccine dose had about half of the GM (GMT50) for all viral variants. Boosted VaxCCP characterized by either recent vaccine dose or infection event within 6 months represents a robust, variant-resilient, neutralizing antibody source against the new Omicron BQ.1.1, XBB.1 and BF.7 variants.

Antibody response of heterologous vs homologous mRNA vaccine boosters against the SARS-CoV-2 Omicron variant: interim results from the PRIBIVAC study, A Randomized Clinical Trial.

BACKGROUND: Waning antibody levels post-vaccination and the emergence of variants of concern (VOCs) capable of evading protective immunity has raised the need for booster vaccinations. However, which combination of COVID-19 vaccines offers the strongest immune response against Omicron variant is unknown. METHODS: This randomized, subject-blinded, controlled trial assessed the reactogenicity and immunogenicity of different COVID-19 vaccine booster combinations. 100 BNT162b2-vaccinated individuals were enrolled and randomized 1: 1 to either homologous (BNT162b2 + BNT162b2 + BNT162b2; 'BBB') or heterologous mRNA booster vaccine (BNT162b2 + BNT162b2 + mRNA-1273; 'BBM'). Primary endpoint was the level of neutralizing antibodies against SARS-CoV-2 wild-type and VOCs at Day 28. RESULTS: 51 participants were allocated to BBB and 49 to BBM; 50 and 48 respectively were analyzed for safety and immunogenicity outcomes. At Day 28 post-boost, mean SARS-CoV-2 spike antibody titers were lower with BBB (22,382  IU/mL 95% CI, 18,210 to 27,517) vs BBM (29,751  IU/mL 95% CI, 25,281 to 35,011, p = 0.034) as was the median level of neutralizing antibodies: BBB 99.0% (IQR 97.9 to 99.3%) vs BBM 99.3% (IQR 98.8 to 99.5%, p = 0.021). On sub-group analysis, significant differences in mean spike antibody titer and live Omicron neutralization titer was only observed in older adults. Median surrogate neutralizing antibody level against all VOCs was also significantly higher with BBM in older adults, and against Omicron was BBB 72.8% (IQR 54.0 to 84.7%) vs BBM 84.3% (IQR 78.1 to 88.7%, p = 0.0073). Both vaccines were well tolerated. CONCLUSIONS: Heterologous mRNA-1273 booster vaccination induced a stronger neutralizing response against the Omicron variant in older individuals compared with homologous BNT123b2.

Comparison of the approaches to evaluation the virus-neutralizing activity of sera from COVID-19 recovered individuals

Background: Serum virus-neutralization (VNT)capacity is an important parameter of immunological response in COVID-19 recovered individuals and it can also be used to predict the effectiveness of therapy with COVID-19 convalescent plasma. The most commonly used tests to assess virus-neutralization are those that use native SARS-CoV- 2 (cVNT), SARS-CoV- 2 spike pseudotyped lentivirus-like particles (pVNT), and also inhibition of recombinant RBD binding to ACE2 receptor in vitro (surrogate VNT, sVNT). The aim of this study was to determine the interchangeability of different approaches in the evaluation of the virus-neutralization activity of convalescent serum samples. Method(s): Serum samples (n = 111) were collected 10-36 days after recovery between May and September 2020. The SARS-CoV- 2 strain PMVL-12/ 2020 was used in cVNT at 100 doses of TCID50. In pVNT SARS-CoV- 2 pseudotyped HIV-1 based virus-like particles with GFP reporter gene were used. sVNT was performed with a kit from Xema Co., Russia. Result(s): A very strong correlation was observed between cVNT and pVNT results (Spearman's r = 0.841). The correlation of cVNT and pVNT with the sVNT was only moderate (r = 0.674 and 0.696, respectively). This is consistent with the fact that sVNT detects only RBD blocking antibodies, which are the main but not the only inhibitors of viral infection. Serum samples were also tested for RBD-specific IgG, IgM, IgA antibodies by ELISA. A good correlation was found between the cVNT, pVNT, sVNT results and the RBD-specific IgG (r = 0.669, 0.620, and 0.643, respectively), that confirms a crucial role of specific IgG antibodies in virus-neutralization. RBD-specific IgA showed a moderate correlation with the neutralization capacity of sera from recovered individuals (r = 0.563, 0.583, 0.544). Correlation of approximately the same level was observed between cVNT, pVNT, and RBD-specific IgM (r = 0.663, 0.615), but not sVNT and RBD-specific IgM (r = 0.395). Conclusion(s): This study demonstrated the possibility of using the safe and relatively simple pseudotyped virus-neutralization test instead of cVNT to assess the sera virus-neutralizing capacity. sVNT may be efficiently used in screening studies.

Development of SARS-CoV-2 neutralizing antibody detection assay by using recombinant plant-produced proteins.

Detecting immunity against SARS-CoV-2 is vital for evaluating vaccine response and natural infection, but conventional virus neutralization test (cVNT) requires BSL3 and live viruses, and pseudo-virus neutralization test (pVNT) needs specialized equipment and trained professionals. The surrogate virus neutralization test (sVNT) was developed to overcome these limitations. This study explored the use of angiotensin converting enzyme 2 (ACE2) produced from Nicotiana benthamiana for the development of an affordable neutralizing antibodies detection assay. The results showed that the plant-produced ACE2 can bind to the receptor binding domain (RBD) of the SARS-CoV-2, and was used to develop sVNT with plant-produced RBD protein. The sVNT developed using plant-produced proteins showed high sensitivity and specificity when validated with a group of 30 RBD vaccinated mice sera and the results were correlated with cVNT titer. This preliminary finding suggests that the plants could offer a cost-effective platform for producing diagnostic reagents.

Kinetics and ability of binding antibody and surrogate virus neutralization tests to predict neutralizing antibodies against the SARS-CoV-2 Omicron variant following BNT162b2 booster administration.

OBJECTIVES: To assess the long-term humoral immunity induced by booster administration, as well as the ability of binding antibody and surrogate virus neutralization tests (sVNT) to predict neutralizing antibodies (NAbs) against the SARS-CoV-2 Omicron variant. METHODS: A total of 269 sera samples were analyzed from 64 healthcare workers who had received a homologous booster dose of BNT162b2. Neutralizing antibodies assessed by sVNT and anti-RBD IgG measured with the sCOVG assay (Siemens Healthineers®) were analyzed at five timepoints; before and up to 6 months following the booster. Antibody titers were correlated with neutralizing antibodies against the Omicron BA.1 variant obtained by pseudovirus neutralization test (pVNT) as a reference method. RESULTS: While Wild-type sVNT percentage of inhibition (POI) remained above 98.6% throughout the follow-up period after booster administration, anti-RBD IgG and NAbs assessed by Omicron BA.1 pVNT showed respectively a 3.4-fold and 13.3-fold decrease after 6 months compared to the peak reached at day 14. NAbs assessed by Omicron sVNT followed a steady decline until reaching a POI of 53.4%. Anti-RBD IgG and Omicron sVNT assays were strongly correlated (r=0.90) and performed similarly to predict the presence of neutralizing antibodies with Omicron pVNT (area under the ROC: 0.82 for both assays). In addition, new adapted cut-off values of anti-RBD IgG (>1,276 BAU/mL) and Omicron sVNT (POI>46.6%) were found to be better predictors of neutralizing activity. CONCLUSIONS: This study showed a significant drop in humoral immunity 6 months after booster administration. Anti-RBD IgG and Omicron sVNT assays were highly correlated and could predict neutralizing activity with moderate performance.

Immunogenicity and Safety of Homologous Chadox1 Ncov-19 and Heterologous Prime-Boost of Coronavac and Chadox1 Ncov-19 among Kidney Transplant Recipients: An Observational Prospective Cohort Study

Introduction: Kidney transplant recipients (KTRs) are at risk for substantial morbidity and mortality during COVID-19 infection. Vaccination for this group of patients is reccommended. However, immunogenicity and safety data after COVID-19 vaccination among KTRs remains limited. Method(s): We conducted an observational prospective trial involving KTRs at Chiang Mai University hospital, Chiang Mai, Thailand. The participants were received homologous ChAdOx1 nCoV-19 (AZ-AZ), or the heterologous prime-boost of CoronaVac,followed by AZ (SV-AZ). The immunogenicity was assessed by measuring antibodies against the S1 receptor-binding domain (anti-RBD), and SARS-CoV-2 surrogate virus neutralization test (sVNT) at specific timepoints. The primary outcome was the seroconversion rate of sVNT at day 28 after complete vaccination. The secondary outcomes were the seroconversion rate of sVNT at day 28 after the first dose of vaccination, the level of sVNT and anti-RBD at specific timepoints, and the adverse events of each vaccine regimen. Result(s): A total of 18 KTRs were recruited. Among those, 13 (72.2%), and 5 (27.8%) patients were received AZ-AZ, and SV-AZ regimen, respectively. The seroconversion rate of sVNT at day 28 after the second dose were 23.1%, and 20.0% for AZ-AZ, and SV-AZ, respectively (P>0.99). The level of sVNT and the level of anti-RBD at day 28 after the first and at day 28 after the second dose were not different between groups (Figure 1). There were no serious adverse events reported in any vaccine groups. However, AZ-AZ showed sign of tubular dysfunction demonstrated by increasing of fractional excretion of magnesium after complete course of vaccination which correlated to the trend of urine albumin and urine protein creatinine ratio (r=0.720, P=0.013;and r=0.726, P=0.011, respectively). [Formula presented] [Formula presented] Figure 1 Percentage of neutralization inhibition (a) and level of anti-RBD antibody (b) at each visit of homologous ChAdOx1 nCoV-19 (AZ-AZ), and heterologous prime-boost of CoronaVac, followed by AZ (SV-AZ) regimen Conclusion(s): Immunogenicity after COVID-19 vaccination with either homologous or heterologous prime-boost regimen among KTRs was compromised. Homologous replication-defective viral vectors vaccine regimen seemed to affect renal tubular function, and further follow-up should be warranted. No conflict of interestCopyright © 2023