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and L.B. BA.2 sub-lineage. KEYWORDS: Cross immunity, mRNA booster, neutralizing antibodies, Omicron BA.1, Omicron BA.2, SARS-CoV-2, SARS-CoV-2 vaccines Brief report Despite the effectiveness of spike-based vaccines and monoclonal antibodies, the SARS-CoV-2 pandemic continues more than two and a half years after its onset. Relentless investigation has defined a causal dynamic between host-derived antibodies and reciprocal viral subversion. Integrating this paradigm into the architecture of next-generation antiviral strategies will become critical for successfully fighting the growing SARS-CoV-2 variants.1 Since the 1st isolation of SARS-CoV-2 in January 2020 in China, several viral variants have been detected. The Omicron B.1.1.529 variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first reported to the World Health Corporation (WHO) in November 2021.2 Compared to the ancestral disease isolated in Wuhan in 2019, the Omicron BA.1 sub-lineage bears a total of 60 mutations within the nonstructural proteins, spike (S) protein, envelope protein, membrane protein, and nucleocapsid (NP) protein. Of these mutations, 32 are on the S protein, 15 of which impact the receptor-binding website (RBD). The BA.2 sub-lineage shares 32 mutations with BA.1 but has 28 distinct ones.3 The S protein plays an essential role in SARS-CoV-2 infection and constitutes the main target of neutralizing antibodies.4 The current vaccine formulations are designed to target the S protein of the wild-type (wt) virus, derived from the original Wuhan strain, and have proven to offer a high degree of safety. Currently, six vaccines have been authorized in Europe5: the BNT162b2 (Pfizer-BioNTech) and mRNA-1273 (Moderna) vaccines were developed by using the mRNA vaccine platform, Ad26.COV2.S (Janssen/Johnson & Johnson) and ChAdOx1-S (AstraZeneca) are adenovirus vectored vaccines, NVX-CoV2373 (Novavax) is a recombinant SARS-CoV-2 nanoparticle vaccine and VLA2001 (Valneva) is an inactivated disease vaccine. The very rapid substitute of Omicron BA.1 by BA.2 during the period FebruaryCMarch 2022 suggests that Omicron BA.2 is inherently more transmissible than BA.1 and capable of vaccine breakthrough.6,7 Furthermore, approved antibodies with neutralizing activity against Omicron BA.1 are ineffective against a subset of Omicron subvariants containing a R346K substitution, BA.1.1 and BA.2, demonstrating the continued need to discover and characterize candidate therapeutic antibodies with the breadth and potency of neutralizing activity required to treat future SARS-CoV-2 variants.8 The purpose of this study was to compare the neutralizing antibody Herbacetin response toward the two Omicron sub-lineages BA.1 and BA.2 by analyzing COVID-19 individuals, subjects who had received two doses of mRNA vaccine, subjects naturally infected with SARS-CoV-2 who had received two doses of mRNA, who Herbacetin had received three doses of mRNA vaccine, and who had received two doses of an adenovirus vectored vaccine plus a booster dose of mRNA vaccine. Neutralizing titers were determined by live disease microneutralization (MN) assay. Serum samples were grouped into five different cohorts. Thirty-seven (37) serum samples from COVID-19 individuals hospitalized at Humanitas Gavazzeni (Italy) during the 1st pandemic wave (MarchCMay 2020) were included in the present study. Herbacetin Subjects characteristics and study methods have been explained in detail elsewhere.9 This study was authorized by the Ethics Committee of the University of Siena (approval number 17373) and by the Ethics Committee of Humanitas Gavazzeni (approval number 236). Fifty (50) serum samples were collected in the Bari correctional facility (Italy) approximately 20 days after the second vaccination with mRNA vaccine (mRNA-273 and BNT162b2). These samples were bad to antibodies against the NP protein (IDScreenSARS-CoV-2 Double Antigen Multi-species ELISA, ID.vet, DNM1 France). Twenty-three (23) serum samples were collected in the Bari correctional facility (Italy) a mean of 20?days after the second dose of mRNA vaccine (mRNA-1273 and BNT162b2). These samples were positive to antibodies against the NP protein (as explained above). Forty-four (44) serum samples were collected in the Bari correctional facility (Italy), a mean of 21?days after the third mRNA vaccine dose (mRNA-1273 and BNT162b2). Thirty-five (35) serum samples were collected from employees of the University or college of Bari, a mean of 42?days after the third dose of mRNA vaccine. These subjects received two doses of adenovirus vectored vaccine and a booster dose with mRNA vaccine (mRNA-1273 or BNT162b2). The research protocol was authorized Herbacetin by the Ethics Herbacetin Committee of the University or college Hospital of Bari (n.6955, prot.N.0067544-02082021) and subject matter provided informed consent to participate in the study. African green monkey kidney Vero E6 cells ([ATCC] #CRL-1586/Vero C1008) were cultivated in Dulbeccos Revised Eagles Medium (DMEM) high Glucose with 2?mM L-Glutamine, 100?U/mL of penicillin?100?g/mL streptomycin (complete DMEM), and 10% Fetal Bovine Serum (FBS). Cells were managed at 37C inside a humidified 5%CO2 environment and passaged every 3C4?days; 18C24?hours before execution of the microneutralization (MN) assay, plates were seeded with 100?L/well of cells (1.5 105cell/mL) diluted in complete DMEM with 2% FBS and incubated at.