Peri-infection titers of neutralizing and binding antibodies as a predictor of COVID-19 breakthrough infections in vaccinated healthcare professionals: importance of the timing.

OBJECTIVES: The BNT162b2 messenger RNA vaccine is highly effective in reducing COVID-19 infection, hospitalization and death. However, many subjects developed a breakthrough infection despite a full vaccination scheme. Since the waned efficacy of mRNA vaccines is correlated with the decrease of antibodies occurring over time, we aimed at evaluating whether lower levels of antibodies were associated with an increased risk of breakthrough infection in a cohort of breakthrough subjects that received three vaccine doses. METHODS: Total binding antibodies against the RBD of the S1 subunit (Roche Diagnostics, Machelen, Belgium) and neutralizing antibodies using the Omicron B.1.1.529 variant pseudovirus were measured. Based on individual kinetic curves, the antibody titer of each subject was interpolated just before the breakthrough infection and compared to a matched-control group that did not develop a breakthrough infection. RESULTS: Lower levels of total binding and neutralizing antibodies were observed compared to the control group (6.900 [95% CI; 5.101-9.470] vs. 11.395 BAU/mL [8.627-15.050] [p=0.0301] and 26.6 [18.0-39.3] vs. 59.5 dilution titer-1 [32.3-110] [p=0.0042], respectively). The difference between breakthrough and control subjects was mostly observed for neutralizing antibodies before three months after the homologous booster administration (46.5 [18.2-119] vs. 381 [285-509] [p=0.0156]). Considering the measurement of total binding antibodies before 3 months, there was no significant difference (p=0.4375). CONCLUSIONS: In conclusion, our results showed that subjects that developed a breakthrough infection had lower levels of neutralizing and total binding antibodies compared to controls. The difference was mostly noticeable considering neutralizing antibodies, especially for infections occurring before 3 months after the booster administration.

Antibody titer levels and the effect on subsequent SARS-CoV-2 infection in a large US-based cohort.

Despite a growing amount of data around the kinetics and durability of the antibody response induced by vaccination and previous infection, there is little understanding of whether or not a given quantitative level of antibodies correlates to protection against SARS-CoV-2 infection or reinfection. In this study, we examine SARS-CoV-2 anti-spike receptor binding domain (RBD) antibody titers and subsequent SARS-CoV-2 reverse transcription polymerase chain reaction (RT-PCR) tests in a large cohort of US-based patients. We analyzed antibody test results in a cohort of 22,204 individuals, 6.8% (n = 1,509) of whom eventually tested positive for SARS-CoV-2 RNA, suggesting infection or reinfection. Kaplan-Meier curves were plotted to understand the effect of various levels of anti-spike RBD antibody titers (classified into discrete ranges) on subsequent RT-PCR positivity rates. Statistical analyses included fitting a Cox proportional hazards model to estimate the age-, sex- and exposure-adjusted hazard ratios for S antibody titer, using zip-code positivity rates by week as a proxy for COVID-19 exposure. It was found that the best models of the temporally associated infection risk were those based on log antibody titer level (HR = 0.836 (p < 0.05)). When titers were binned, the hazard ratio associated with antibody titer >250 Binding Antibody Units (BAU) was 0.27 (p < 0.05, 95% CI [0.18, 0.41]), while the hazard ratio associated with previous infection was 0.20 (p < 0.05, 95% CI [0.10, 0.39]). Fisher exact odds ratio (OR) for Ab titers <250 BAU showed OR = 2.84 (p < 0.05; 95% CI: [2.30, 3.53]) for predicting the outcome of a subsequent PCR test. Antibody titer levels correlate with protection against subsequent SARS-CoV-2 infection or reinfection when examining a cohort of real-world patients who had the spike RBD antibody assay performed.

Immunity against Delta and Omicron variants elicited by homologous inactivated vaccine booster in kidney transplant recipients.

Background: A third mRNA vaccine booster is recommended to improve immunity against SARS-CoV-2 in kidney transplant recipients (KTRs). However, the immunity against SARS-CoV-2 Ancestral strain and Delta and Omicron variants elicited by the third dose of inactivated booster vaccine in KTRs remains unknown. Methods: The blood parameters related to blood cells count, hepatic function, kidney function, heart injury and immunity were explored clinically from laboratory examinations. SARS-CoV-2 specific antibody IgG titer was detected using an enzyme-linked immunosorbent assay. Cellular immunity was analyzed using interferon-γ enzyme-linked immunospot assay. Results: The results showed that there were no severe adverse effects and apparent changes of clinical laboratory biomarkers in KTRs and healthy volunteers (HVs) after homologous inactivated vaccine booster. A third dose of inactivated vaccine booster significantly increased anti-Ancestral-spike-trimer-IgG and anti-Ancestral-receptor binding domain (RBD)-IgG titers in KTRs and HVs compared with the second vaccination. However, the anti-Delta-RBD-IgG and anti-Omicron-RBD-IgG titers were significantly lower than anti-Ancestral-RBD-IgG titer in KTRs and HVs after the third dose. Notably, only 25.6% (10/39) and 10.3% (4/39) of KTRs had seropositivity for anti-Delta-RBD-IgG and anti-Omicron-RBD-IgG after booster, which were significantly lower than HVs (anti-Delta-RBD-IgG: 100%, anti-Omicron-RBD-IgG: 77.8%). Ancestral strain nucleocapsid protein and spike specific T cell frequency after booster was not significantly increased in KTRs compared with the second dose, significantly lower than that in HVs. Moreover, 33.3% (12/36), 14.3% (3/21) and 14.3% (3/21) of KTRs were positive for the Ancestral strain and Delta and Omicron spike-specific T cells, which were significantly lower than HVs (Ancestral: 80.8%, Delta: 53.8%, and Omicron: 57.7%). Conclusions: A third dose of inactivated booster vaccine may significantly increase humoral immunity against the Ancestral strain in KTRs, while humoral and cellular immunity against the Delta and Omicron variants were still poor in KTRs.

Omicron variants breakthrough infection elicited higher specific memory immunity than third dose booster in healthy vaccinees.

Homologous booster, heterologous booster, and Omicron variants breakthrough infection (OBI) could improve the humoral immunity against Omicron variants. Questions concerning about memory B cells (MBCs) and T cells immunity against Omicron variants, features of long-term immunity, after booster and OBI, needs to be explored. Here, comparative analysis demonstrate antibody and T cell immunity against ancestral strain, Delta and Omicron variants in Omicron breakthrough infected patients (OBIPs) are comparable to that in Ad5-nCoV boosted healthy volunteers (HVs), higher than that in inactivated vaccine (InV) boosted HVs. However, memory B cells (MBCs) immunity against Omicron variants was highest in OBIPs, followed by Ad5-nCoV boosted and InV boosted HVs. OBIPs and Ad5-nCoV boosted HVs have higher classical MBCs and activated MBCs, and lower naïve MBCs and atypical MBCs relative to both vaccine boosted HVs. Collectively, these data indicate Omicron breakthrough infection elicit higher MBCs and T cells against SARS-CoV-2 especially Omicron variants relative to homologous InV booster and heterologous Ad5-nCoV booster.

Comparison of SARS-CoV-2 Antibody Levels after a Third Heterologous and Homologous BNT162b2 Booster Dose.

This study aimed to determine the anti-S (receptor binding protein) RBD IgG antibody titers formed against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2) and the neutralizing antibody inhibition percentages (nAb IH%) in blood samples taken after two doses of inactive or mRNA-based vaccine and a booster dose. Volunteers with two doses of inactivated CoronaVac (heterologous group; n = 75) and BioNTech (BNT)162b2 mRNA vaccine (homologous group; n = 75) were included in this study. All participants preferred the BNT162b2 vaccine as a booster dose. First, peripheral blood samples were taken 3 months after the second vaccine dose. Second, peripheral blood samples were taken 1 month after the booster dose. Anti-S-RBD IgG titers were determined by CMIA (SARS-CoV-2 IgG II Quant). Neutralizing antibodies were detected by a surrogate neutralization assay (SARS-CoV-2 NeutraLISA, Euroimmun, Lübeck, Germany). The median age of the volunteers was 40 (IQR 29-47) years old. After the heterologous booster dose, anti-S-RBD IgG levels and neutralizing antibodies increased approximately 50-fold and 9-fold, respectively. Anti-S-RBD IgG titers increased by 9 and 57 times, respectively, while nAb IH% increased by 1.5 and 16 times, respectively, among those with heterologous reminder doses and those with and without a prior history of coronavirus disease (COVID-19). This study showed that after the administration of a heterologous booster dose with BNT162b2 to those whose primary vaccination was with inactivated CoronaVac, the binding and neutralizing antibody levels were similar to those who received a homologous BNT162b2 booster dose. It was observed that the administration of heterologous and homologous booster doses resulted in the development of similar levels of neutralizing antibodies, independently from a prior history of COVID-19.

Heterologous Prime-boost of SARS-CoV-2 inactivated vaccine and mRNA BNT162b2 among Healthy Thai Adolescents.

Background: Heterologous prime-boost SARS-CoV-2 vaccination is a widely accepted strategy during the COVID-19 pandemic, which generated a superior immune response than homologous vaccination strategy. Objective: To describe immunogenicity of heterologous prime-boost vaccination with inactivated vaccine, CoronaVac, followed by BNT162b2 and 5-month booster dose with BNT162b2 in healthy Thai adolescents. Methods: Adolescents aged 12-18 years were randomized 1:1:1:1 to receive CoronaVac (SV) followed by BNT162b2 (PZ) 30 or 20 µg at either 3- or 6-week interval (SV3w/PZ30µg, SV3w/PZ20µg, SV6w/PZ30µg or SV6w/PZ20µg). During the Omicron-predominant period, participants were offered a BNT162b2 booster dose 30, 15, or 10 µg. Immunogenicity was determined using IgG antibody against spike-receptor-binding domain of wild type(anti-S-RBD IgG) and surrogate virus neutralization test(sVNT) against Delta variant at 14 days and 5 months after the 2nd dose. Neutralization tests(sVNT and pseudovirus neutralization test; pVNT) against Omicron strain were tested pre- and 14 days post-booster dose. Results: In October 2021, 76 adolescents with a median age of 14.3 years (IQR 12.7-16.0) were enrolled: 20 in SV3w/PZ30µg; 17 in SV3w/PZ20µg; 20 in SV6w/PZ30µg; 19 in SV6w/PZ20µg. At day 14, the geometric mean(GM) of anti-S-RBD IgG in SV3w/PZ30µg was 4713 (95 %CI 4127-5382) binding-antibody unit (BAU)/ml, while geometric mean ratio(GMR) was 1.28 (1.09-1.51) in SV6w/PZ30µg. The GMs of sVNT against Delta variants at day 14 among participants in SV3w/PZ30µg and SV6wk/PZ30µg arm were 95.3 % and 99.7 %inhibition, respectively. At 5 months, GMs of sVNT against Delta variants in SV3w/PZ30µg were significantly declined to 47.8 % but remained at 89.0 % inhibition among SV6w/PZ30µg arm. In April 2022, 52 adolescents received a BNT162b2 booster dose. Proportion of participants with sVNT against Omicron strain > 80 %inhibition was significantly increased from 3.8 % pre-booster to 67 % post-booster. Proportion of participants with pVNT ID50 > 185 was 42 % at 14 days post 2nd dose and 88 % post booster, respectively. Conclusions: Heterologous prime-boost vaccination with CoronaVac followed by BNT162b2 induced high neutralizing titer against SARS-CoV-2 Delta strain. After 5-month interval, booster with BNT162b2 induced high neutralizing titer against Omicron strain.Thai Clinical Trials Registry (thaiclinicaltrials.org): TCTR20210923012.

Humoral Immunogenicity of the mRNA-1273 Vaccine in the Phase 3 Coronavirus Efficacy (COVE) Trial.

BACKGROUND: Messenger RNA (mRNA)-1273 vaccine demonstrated 93.2% efficacy against coronavirus disease 2019 (COVID-19) in the Coronavirus Efficacy (COVE) trial. The humoral immunogenicity results are now reported. METHODS: Participants received 2 mRNA-1273 (100 µg) or placebo injections, 28 days apart. Immune responses were evaluated in a prespecified, randomly selected per-protocol immunogenicity population (n = 272 placebo; n = 1185 mRNA-1273). Serum binding antibodies (bAbs) and neutralizing antibodies (nAbs) to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-spike protein were assessed at days 1, 29, and 57 by baseline SARS-CoV-2-negative (n = 1197) and SARS-CoV-2-positive (n = 260) status, age, and sex. RESULTS: SARS-CoV-2-negative vaccinees had bAb geometric mean AU/mL levels of 35 753 at day 29 that increased to 316 448 at day 57 and nAb inhibitory dilution 50% titers of 55 at day 29 that rose to 1081 at day 57. In SARS-CoV-2-positive vacinees, the first mRNA-1273 injection elicited bAb and nAb levels that were 11-fold (410 049) and 27-fold (1479) higher than in SARS-CoV-2-negative vaccinees, respectively, and were comparable to levels after 2 injections in uninfected participants. Findings were generally consistent by age and sex. CONCLUSIONS: mRNA-1273 elicited robust serologic immune responses across age, sex, and SARS-CoV-2 status, consistent with its high COVID-19 efficacy. Higher immune responses in those previously infected support a booster-type effect. Clinical Trials Registration. NCT04470427.

Lasting SARS-CoV-2 specific IgG Antibody response in health care workers from Venezuela, 6 months after vaccination with Sputnik V.

BACKGROUND: Scarce information is available regarding the long-term immunogenicity of the Sputnik V vaccine. Here Sputnik V vaccinated subjects were evaluated 6 months after receiving the 2-dose prime-boost schedule. METHODS: Eighty-six hospital workers from Venezuela, 32 with a previous COVID-19 infection and 54 SARS-CoV-2 naïve subjects, were enrolled. IgG antibodies levels against the wild-type Receptor Binding Domain (RBD) were measured in an ELISA and with an in vitro ACE2-surrogate RBD binding inhibition assay at day 42 and day 180 after receiving the second dose. IgG levels were expressed in BAU/ml. Binding inhibition antibodies were expressed in IU/ml. RESULTS: On average, RBD-IgG levels decreased by approximately 50% between the two time-points in the COVID-19 naïve cohort (geometric mean concentration (GMC) 675 BAU/mL vs. 327 BAU/ml) and decreased by approximately 25% in the previously infected cohort (GMC 1209 BAU/mL vs 910 BAU/ml). Within our cohort, 94% showed a "good to excellent" neutralizing activity measured with the in vitro test 6 months after vaccination. CONCLUSIONS: The Sputnik V vaccine provided long-term and durable humoral immunity in our cohort specially if a person has been both vaccinated and had a previous infection with SARS-CoV-2.

Performance of Severe Acute Respiratory Syndrome Coronavirus 2 Serological Diagnostic Tests and Antibody Kinetics in Coronavirus Disease 2019 Patients.

Serological testing is recommended to support the detection of undiagnosed coronavirus disease 2019 (COVID-19) cases. However, the performance of serological assays has not been sufficiently evaluated. Hence, the performance of six severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) binding antibody assays [three chemiluminescence (CLIAs) and three lateral flow immunoassays (LFIAs)] and a surrogate virus neutralization test (sVNT) was analyzed in a total of 988 serum samples comprising 389 COVID-19-positives and 599 COVID-19-negatives. The overall diagnostic sensitivities of CLIAs and LFIAs ranged from 54.2 to 56.6% and from 56.3 to 64.3%, respectively. The overall diagnostic specificities of CLIAs and LFIAs ranged from 98.2 to 99.8% and from 97.3 to 99.0%, respectively. In the symptomatic group (n = 321), the positivity rate increased by over 80% in all assays > 14 days after symptom onset. In the asymptomatic group (n = 68), the positivity rate increased by over 80% in all assays > 21 days after initial RT-PCR detection. In LFIAs, negatively interpreted trace bands accounted for the changes in test performance. Most false-positive results were weak or trace reactions and showed negative results in additional sVNT. For six binding antibody assays, the overall agreement percentages ranged from 91.0 to 97.8%. The median inhibition activity of sVNT was significantly higher in the symptomatic group than in the asymptomatic group (50.0% vs. 29.2%; p < 0.0001). The median times to seropositivity in the symptomatic group were 9.7 days for CLIA-IgG, 9.2 and 9.8 days for two CLIAs-Total (IgM + IgG), 7.7 days for LFIA-IgM, 9.2 days for LFIA-IgG, and 8.8 days for sVNT-IgG, respectively. There was a strong positive correlation between the quantitative results of the four binding antibody assays and sVNT with Spearman ρ-values ranging from 0.746 to 0.854. In particular, when using LFIAs, we recommend using more objective interpretable assays or establishing a band interpretation system for each laboratory, accompanied by observer training. We also anticipate that sVNT will play an essential role in SARS-CoV-2 antibody testing and become the practical routine neutralizing antibody assay.

Impaired SARS-CoV-2-specific T-cell reactivity in patients with cirrhosis following mRNA COVID-19 vaccination.

Background & Aims: Cirrhosis entails elevated risk of COVID-19-associated mortality. This study determined T cell-mediated and antibody reactivity against the spike 1 (S1) protein of SARS-CoV-2 among 48 patients with cirrhosis and 39 healthy controls after mRNA COVID-19 vaccination. Methods: SARS-CoV-2-specific T-cell reactivity was measured by induced level of T cell-derived interferon-γ (IFN-γ) in blood cells stimulated ex vivo with multimeric peptides spanning the N-terminal portion of S1. S1-induced IFN-γ was quantified before and after the 1st and 2nd vaccination (BNT162b2, Pfizer-BioNTech or mRNA-1273, Moderna) alongside serum IgG against the receptor-binding domain (RBD) within S1 (anti-RBD-S1 IgG). Results: T-cell reactivity against S1 was reduced in patients with cirrhosis after the 1st (p <0.001 vs. controls) and 2nd (p <0.001) vaccination. Sixty-eight percent of patients lacked detectable S1-specific T-cell reactivity after the 1st vaccination vs. 19% in controls (odds ratio 0.11, 95% CI 0.03-0.48, p = 0.003) and 36% remained devoid of reactivity after the 2nd vaccination vs. 6% in controls (odds ratio 0.12, 95% CI 0.03-0.59, p = 0.009). T-cell reactivity in cirrhosis remained significantly impaired after correction for potential confounders in multivariable analysis. Advanced cirrhosis (Child-Pugh class B) was associated with absent or lower T-cell responses (p <0.05 vs. Child-Pugh class A). The deficiency of T-cell reactivity was paralleled by lower levels of anti-RBD-S1 IgG after the 1st (p <0.001 vs. controls) and 2nd (p <0.05) vaccination. Conclusions: Patients with cirrhosis show deficient T-cell reactivity against SARS-CoV-2 antigens along with diminished levels of anti-RBD-S1 IgG after dual COVID-19 vaccination, highlighting the need for vigilance and additional preventative measures. Clinical trial registration: EudraCT 2021-000349-42. Lay summary: T cells are a pivotal component in the defence against viruses. We show that patients with cirrhosis have impaired SARS-CoV-2-specific T-cell responses and lower antibody levels after mRNA vaccination against COVID-19 compared with healthy controls. Patients with more advanced liver disease exhibited particularly inferior vaccine responses. These results call for additional preventative measures in these patients.

Serum IgG anti-SARS-CoV-2 Binding Antibody Level Is Strongly Associated With IgA and Functional Antibody Levels in Adults Infected With SARS-CoV-2.

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first reported in December 2019 in Wuhan, China, and then rapidly spread causing an unprecedented pandemic. A robust serological assay is needed to evaluate vaccine candidates and better understand the epidemiology of coronavirus disease (COVID-19). Methods: We used the full-length spike (S) protein of SARS-CoV-2 for the development of qualitative and quantitative IgG and IgA anti-S enzyme linked immunosorbent assays (ELISA). A total of 320 sera used for assay development were comprised of pandemic sera from SARS-CoV-2 infected adults (n=51) and pre-pandemic sera (n=269) including sera from endemic human coronavirus infected adults. Reverse cumulative curves and diagnostic test statistics were evaluated to define the optimal serum dilution and OD cutoff value for IgG anti-S and IgA anti-S ELISAs. The IgG and IgA anti-S, and three functional antibodies (ACE-2 receptor blocking antibody, lentipseudovirus-S neutralizing antibody, and SARS-CoV-2 neutralizing antibody) were measured using additional SARS-CoV-2 PCR positive sera (n=76) and surveillance sera (n=25). Lastly, the IgG and IgA anti-S levels were compared in different demographic groups. Results: The optimal serum dilution for the qualitative IgG anti-S ELISA was at 1:1024 yielding a 99.6% specificity, 92.2% sensitivity, 92.9% positive predictive value (PPV), and 99.6% negative predictive value (NPV) at a SARS-CoV-2 seroprevalence of 5%. The optimal serum dilution for the qualitative IgA anti-S ELISA was at 1:128 yielding a 98.9% specificity, 76.5% sensitivity, 78.3% PPV, and 98.8% NPV at the same seroprevalence. Significant correlations were demonstrated between the IgG and IgA (r=0.833 for concentrations, r=0.840 for titers) as well as between IgG and three functional antibodies (r=0.811-0.924 for concentrations, r=0.795-0.917 for titers). The IgG and IgA anti-S levels were significantly higher in males than females (p<0.05), and in adults with moderate/severe symptoms than in adults with mild/moderate symptoms (p<0.001). Conclusion: We developed a highly specific and sensitive IgG anti-S ELISA assay to SARS-CoV-2 using full length S protein. The IgG anti-S antibody level was strongly associated with IgA and functional antibody levels in adults with SARS-CoV-2 infection. Gender and disease severity, rather than age, play an important role in antibody levels.

Quantitative SARS-CoV-2 Spike Antibody Response in COVID-19 Patients Using Three Fully Automated Immunoassays and a Surrogate Virus Neutralization Test.

Quantitative SARS-CoV-2 antibody assays against the spike (S) protein are useful for monitoring immune response after infection or vaccination. We compared the results of three chemiluminescent immunoassays (CLIAs) (Abbott, Roche, Siemens) and a surrogate virus neutralization test (sVNT, GenScript) using 191 sequential samples from 32 COVID-19 patients. All assays detected >90% of samples collected 14 days after symptom onset (Abbott 97.4%, Roche 96.2%, Siemens 92.3%, and GenScript 96.2%), and overall agreement among the four assays was 91.1% to 96.3%. When we assessed time-course antibody levels, the Abbott and Siemens assays showed higher levels in patients with severe disease (p < 0.05). Antibody levels from the three CLIAs were correlated (r = 0.763-0.885). However, Passing-Bablok regression analysis showed significant proportional differences between assays and converting results to binding antibody units (BAU)/mL still showed substantial bias. CLIAs had good performance in predicting sVNT positivity (Area Under the Curve (AUC), 0.959-0.987), with Abbott having the highest AUC value (p < 0.05). SARS-CoV-2 S protein antibody levels as assessed by the CLIAs were not interchangeable, but showed reliable performance for predicting sVNT results. Further standardization and harmonization of immunoassays might be helpful in monitoring immune status after COVID-19 infection or vaccination.

Longitudinal analysis shows durable and broad immune memory after SARS-CoV-2 infection with persisting antibody responses and memory B and T cells.

Ending the COVID-19 pandemic will require long-lived immunity to SARS-CoV-2. Here, we evaluate 254 COVID-19 patients longitudinally up to 8 months and find durable broad-based immune responses. SARS-CoV-2 spike binding and neutralizing antibodies exhibit a bi-phasic decay with an extended half-life of >200 days suggesting the generation of longer-lived plasma cells. SARS-CoV-2 infection also boosts antibody titers to SARS-CoV-1 and common betacoronaviruses. In addition, spike-specific IgG+ memory B cells persist, which bodes well for a rapid antibody response upon virus re-exposure or vaccination. Virus-specific CD4+ and CD8+ T cells are polyfunctional and maintained with an estimated half-life of 200 days. Interestingly, CD4+ T cell responses equally target several SARS-CoV-2 proteins, whereas the CD8+ T cell responses preferentially target the nucleoprotein, highlighting the potential importance of including the nucleoprotein in future vaccines. Taken together, these results suggest that broad and effective immunity may persist long-term in recovered COVID-19 patients.