A novel S2-derived peptide-based ELISA for broad detection of antibody against infectious bronchitis virus.

Avian infectious bronchitis (IB) is a highly contagious disease caused by infectious bronchitis virus (IBV). Vaccination is an effective approach for controlling IBV. Therefore, reliable immune monitoring for IB is critical for poultry. In this study, a novel peptide derived from S2 protein was used to develop an enzyme-linked immunosorbent assay (ELISA) for the detection of broadly cross-reactive antibodies against IBV. The peptide-based ELISA (pELISA) showed good specificity and sensitivity in detecting IBV antibodies against different serotypes. A semilogarithmic regression method for determining IBV antibody titers was also established. Antibody titers detected by pELISA and calculated with this equation were statistically similar to those evaluated by indirect fluorescence assay (IFA). Moreover, the comparison analysis showed a 96.07% compatibility between the pELISA and IDEXX ELISA. All these data demonstrate that the pELISA generated here can be as a rapid and reliable serological surveillance tool for monitoring IBV infection or vaccination.

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

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

HR121 targeting HR2 domain in S2 subunit of spike protein can serve as a broad-spectrum SARS-CoV-2 inhibitor via intranasal administration.

The continuously emerging SARS-CoV-2 variants pose a great challenge to the efficacy of current drugs, this necessitates the development of broad-spectrum antiviral drugs. In the previous study, we designed a recombinant protein, heptad repeat (HR) 121, as a variant-proof vaccine. Here, we found it can act as a fusion inhibitor and demonstrated broadly neutralizing activities against SARS-CoV-2 and its main variants. Structure analysis suggested that HR121 targets the HR2 domain in SARS-CoV-2 spike (S) 2 subunit to block virus-cell fusion. Functional experiments demonstrated that HR121 can bind HR2 at serological-pH and endosomal-pH, highlighting its inhibition capacity when SARS-CoV-2 enters via either cellular membrane fusion or endosomal route. Importantly, HR121 can effectively inhibit SARS-CoV-2 and Omicron variant pseudoviruses entering the cells, as well as block authentic SARS-CoV-2 and Omicron BA.2 replications in human pulmonary alveolar epithelial cells. After intranasal administration to Syrian golden hamsters, it can protect hamsters from SARS-CoV-2 and Omicron BA.2 infection. Together, our results suggest that HR121 is a potent drug candidate with broadly neutralizing activities against SARS-CoV-2 and its variants.

The Highly Conserved Stem-Loop II Motif Is Dispensable for SARS-CoV-2.

The stem-loop II motif (s2m) is an RNA structural element that is found in the 3' untranslated region (UTR) of many RNA viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Though the motif was discovered over 25 years ago, its functional significance is unknown. In order to understand the importance of s2m, we created viruses with deletions or mutations of the s2m by reverse genetics and also evaluated a clinical isolate harboring a unique s2m deletion. Deletion or mutation of the s2m had no effect on growth in vitro or on growth and viral fitness in Syrian hamsters in vivo. We also compared the secondary structure of the 3' UTR of wild-type and s2m deletion viruses using selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) and dimethyl sulfate mutational profiling and sequencing (DMS-MaPseq). These experiments demonstrate that the s2m forms an independent structure and that its deletion does not alter the overall remaining 3'-UTR RNA structure. Together, these findings suggest that s2m is dispensable for SARS-CoV-2. IMPORTANCE RNA viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), contain functional structures to support virus replication, translation, and evasion of the host antiviral immune response. The 3' untranslated region of early isolates of SARS-CoV-2 contained a stem-loop II motif (s2m), which is an RNA structural element that is found in many RNA viruses. This motif was discovered over 25 years ago, but its functional significance is unknown. We created SARS-CoV-2 with deletions or mutations of the s2m and determined the effect of these changes on viral growth in tissue culture and in rodent models of infection. Deletion or mutation of the s2m element had no effect on growth in vitro or on growth and viral fitness in Syrian hamsters in vivo. We also observed no impact of the deletion on other known RNA structures in the same region of the genome. These experiments demonstrate that s2m is dispensable for SARS-CoV-2.

Identification of nurse shark VNAR single-domain antibodies targeting the spike S2 subunit of SARS-CoV-2.

SARS-CoV-2 is the etiological agent of the COVID-19 pandemic. Antibody-based therapeutics targeting the spike protein, specifically the S1 subunit or the receptor binding domain (RBD) of SARS-CoV-2, have gained attention due to their clinical efficacy in treating patients diagnosed with COVID-19. An alternative to conventional antibody therapeutics is the use of shark new antigen variable receptor domain (VNAR ) antibodies. VNAR s are small (<15 kDa) and can reach deep into the pockets or grooves of the target antigen. Here, we have isolated 53 VNAR s that bind to the S2 subunit by phage panning from a naïve nurse shark VNAR phage display library constructed in our laboratory. Among those binders, S2A9 showed the best neutralization activity against the original pseudotyped SARS-CoV-2 virus. Several binders, including S2A9, showed cross-reactivity against S2 subunits from other ß coronaviruses. Furthermore, S2A9 showed neutralization activity against all variants of concern (VOCs) from alpha to omicron (including BA1, BA2, BA4, and BA5) in both pseudovirus and live virus neutralization assays. Our findings suggest that S2A9 could be a promising lead molecule for the development of broadly neutralizing antibodies against SARS-CoV-2 and emerging variants. The nurse shark VNAR phage library offers a novel platform that can be used to rapidly isolate single-domain antibodies against emerging viral pathogens.

Humoral Response after SARS-CoV-2 Vaccination in Prostate Cancer Patients.

Cancer is an important public health problem. Prostate cancer is one of the most common cancers among men. In Poland, the incidence of this type of cancer is constantly growing. Considering the appearance of a new coronavirus in December 2019 (SARS-CoV-2) and the fact that oncology patients, including those with prostate cancer, are particularly vulnerable to infection, it is recommended to get vaccinated against COVID-19. In our study, we determined the level and prevalence of antibodies against SARS-CoV-2 IgG in patients with prostate cancer compared to the control group and whether the patients' ages affected the level of antibodies. PCa patients and controls were divided into two age groups: 50-59 years and 60-70 years. We also analyzed the level of antibodies in patients belonging to the relevant risk groups for prostate cancer (the European Society of Urology risk group classification of prostate cancer). For the study, we used the Microblot-Array COVID-19 IgG test to detect antibodies against the three main SARS-CoV-2 antigens: NCP, RBD, and S2. Our results showed that prostate cancer patients had significantly lower levels of anti-SARS-CoV-2 IgG antibodies compared to controls. In addition, age also affected the decrease in the number of IgG antibodies. The level of antibodies in the intermediate/high-risk group was lower compared to the low-risk group.

SARS-CoV-2 Spike-Mediated Entry and Its Regulation by Host Innate Immunity.

The constantly evolving severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOC) fuel the worldwide coronavirus disease (COVID-19) pandemic. The spike protein is essential for the SARS-CoV-2 viral entry and thus has been extensively targeted by therapeutic antibodies. However, mutations along the spike in SARS-CoV-2 VOC and Omicron subvariants have caused more rapid spread and strong antigenic drifts, rendering most of the current antibodies ineffective. Hence, understanding and targeting the molecular mechanism of spike activation is of great interest in curbing the spread and development of new therapeutic approaches. In this review, we summarize the conserved features of spike-mediated viral entry in various SARS-CoV-2 VOC and highlight the converging proteolytic processes involved in priming and activating the spike. We also summarize the roles of innate immune factors in preventing spike-driven membrane fusion and provide outlines for the identification of novel therapeutics against coronavirus infections.

Monitoring of SARS-CoV-2 Infection in Ragusa Area: Next Generation Sequencing and Serological Analysis.

The coronavirus disease 19 (COVID-19) post pandemic evolution is correlated to the development of new variants. Viral genomic and immune response monitoring are fundamental to the surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Since 1 January to 31 July 2022, we monitored the SARS-CoV-2 variants trend in Ragusa area sequencing n.600 samples by next generation sequencing (NGS) technology: n.300 were healthcare workers (HCWs) of ASP Ragusa. The evaluation of anti-Nucleocapside (N), receptor-binding domain (RBD), the two subunit of S protein (S1 and S2) IgG levels in 300 exposed vs. 300 unexposed HCWs to SARS-CoV-2 was performed. Differences in immune response and clinical symptoms related to the different variants were investigated. The SARS-CoV-2 variants trend in Ragusa area and in Sicily region were comparable. BA.1 and BA.2 were the most representative variants, whereas the diffusion of BA.3 and BA.4 affected some places of the region. Although no correlation was found between variants and clinical manifestations, anti-N and anti-S2 levels were positively correlated with an increase in the symptoms number. SARS-CoV-2 infection induced a statistically significant enhancement in antibody titers compared to that produced by SARS-CoV-2 vaccine administration. In post-pandemic period, the evaluation of anti-N IgG could be used as an early marker to identify asymptomatic subjects.

Identification of a conserved S2 epitope present on spike proteins from all highly pathogenic coronaviruses.

To address the ongoing SARS-CoV-2 pandemic and prepare for future coronavirus outbreaks, understanding the protective potential of epitopes conserved across SARS-CoV-2 variants and coronavirus lineages is essential. We describe a highly conserved, conformational S2 domain epitope present only in the prefusion core of ß-coronaviruses: SARS-CoV-2 S2 apex residues 980-1006 in the flexible hinge. Antibody RAY53 binds the native hinge in MERS-CoV and SARS-CoV-2 spikes on the surface of mammalian cells and mediates antibody-dependent cellular phagocytosis and cytotoxicity against SARS-CoV-2 spike in vitro. Hinge epitope mutations that ablate antibody binding compromise pseudovirus infectivity, but changes elsewhere that affect spike opening dynamics, including those found in Omicron BA.1, occlude the epitope and may evade pre-existing serum antibodies targeting the S2 core. This work defines a third class of S2 antibody while providing insights into the potency and limitations of S2 core epitope targeting.

Dynamics of SARS-CoV-2 Spike-IgG throughout Three COVID-19 Vaccination Regimens: A 21-Month Longitudinal Study of 82 Norwegian Healthcare Workers.

To facilitate interpretation of clinical SARS-CoV-2 anti-spike IgG analyses post-vaccination, 82 healthcare workers were followed through three vaccination-regimens: two regimens were comprised of two doses of BNT162b2 three or six weeks apart, followed by a dose of mRNA-vaccine, and in the other regimen, the first dose was replaced by ChAdOx1 nCov-19. After each dose, anti-spike IgG was compared between regimens. As many participants became infected, anti-spike IgG persistence was compared between infected and uninfected participants. Thirteen to twenty-one days after the first dose, seroconversion, and the median anti-spike IgG level in the ChAdOx1 group was significantly lower than in the BNT162b2 groups (23 versus 68 and 73 AU/mL). The second dose caused a significant increase in anti-spike IgG, but the median level was lower in the BNT162b2-short-interval group (280 AU/mL), compared to the BNT162b2-long-interval (1075 AU/mL) and ChAdOx1 (1160 AU/mL) group. After the third dose, all groups showed increases to similar anti-spike IgG levels (2075-2390 AU/mL). Over the next half year, anti-spike IgG levels declined significantly in all groups, but appeared to persist longer after post-vaccination infection. This is the first three-dose study with one dose of ChAdOx1. Despite initial differences, all vaccine regimens gave similarly high antibody levels and persistence after the third dose.

Broadly neutralizing anti-S2 antibodies protect against all three human betacoronaviruses that cause deadly disease.

Pan-betacoronavirus neutralizing antibodies may hold the key to developing broadly protective vaccines against novel pandemic coronaviruses and to more effectively respond to SARS-CoV-2 variants. The emergence of Omicron and subvariants of SARS-CoV-2 illustrates the limitations of solely targeting the receptor-binding domain (RBD) of the spike (S) protein. Here, we isolated a large panel of broadly neutralizing antibodies (bnAbs) from SARS-CoV-2 recovered-vaccinated donors, which targets a conserved S2 region in the betacoronavirus spike fusion machinery. Select bnAbs showed broad in vivo protection against all three deadly betacoronaviruses, SARS-CoV-1, SARS-CoV-2, and MERS-CoV, which have spilled over into humans in the past two decades. Structural studies of these bnAbs delineated the molecular basis for their broad reactivity and revealed common antibody features targetable by broad vaccination strategies. These bnAbs provide new insights and opportunities for antibody-based interventions and for developing pan-betacoronavirus vaccines.

Deletion of the s2m RNA Structure in the Avian Coronavirus Infectious Bronchitis Virus and Human Astrovirus Results in Sequence Insertions.

Coronaviruses infect a wide variety of host species, resulting in a range of diseases in both humans and animals. The coronavirus genome consists of a large positive-sense single-stranded molecule of RNA containing many RNA structures. One structure, denoted s2m and consisting of 41 nucleotides, is located within the 3' untranslated region (3' UTR) and is shared between some coronavirus species, including infectious bronchitis virus (IBV), severe acute respiratory syndrome coronavirus (SARS-CoV), and SARS-CoV-2, as well as other pathogens, including human astrovirus. Using a reverse genetic system to generate recombinant viruses, we investigated the requirement of the s2m structure in the replication of IBV, a globally distributed economically important Gammacoronavirus that infects poultry causing respiratory disease. Deletion of three nucleotides predicted to destabilize the canonical structure of the s2m or the deletion of the nucleotides corresponding to s2m impacted viral replication in vitro. In vitro passaging of the recombinant IBV with the s2m sequence deleted resulted in a 36-nucleotide insertion in place of the deletion, which was identified to be composed of a duplication of flanking sequences. A similar result was observed following serial passage of human astrovirus with a deleted s2m sequence. RNA modeling indicated that deletion of the nucleotides corresponding to the s2m impacted other RNA structures present in the IBV 3' UTR. Our results indicated for both IBV and human astrovirus a preference for nucleotide occupation in the genome location corresponding to the s2m, which is independent of the specific s2m sequence. IMPORTANCE Coronaviruses infect many species, including humans and animals, with substantial effects on livestock, particularly with respect to poultry. The coronavirus RNA genome consists of structural elements involved in viral replication whose roles are poorly understood. We investigated the requirement of the RNA structural element s2m in the replication of the Gammacoronavirus infectious bronchitis virus, an economically important viral pathogen of poultry. Using reverse genetics to generate recombinant IBVs with either a disrupted or deleted s2m, we showed that the s2m is not required for viral replication in cell culture; however, replication is decreased in tracheal tissue, suggesting a role for the s2m in the natural host. Passaging of these viruses as well as human astrovirus lacking the s2m sequence demonstrated a preference for nucleotide occupation, independent of the s2m sequence. RNA modeling suggested deletion of the s2m may negatively impact other essential RNA structures.

Age and Cytokine Gene Variants Modulate the Immunogenicity and Protective Effect of SARS-CoV-2 mRNA-Based Vaccination.

The introduction of anti-SARS-CoV-2 vaccines in late 2020 substantially changed the pandemic picture, inducing effective protection in the population. However, individual variability was observed with different levels of cellular response and neutralizing antibodies. We report data on the impact of age, gender, and 16 single nucleotide polymorphisms (SNPs) of cytokine genes on the anti-SARS-CoV-2 IgG titers measured 31 and 105 days after administration of the second dose of BNT162b2 vaccine to 122 healthy subjects from the health care staff of the Palermo University Hospital, Italy. The higher titers at 31 days were measured in the younger subjects and in subjects bearing T-positive genotypes of IL-1R1 rs2234650 or the GG homozygous genotype of IL-6 rs1800795 SNP. T-positive genotypes are also significantly more common in subjects with higher titers at day 105. In addition, in this group of subjects, the frequency of the CT genotype of IL-4 rs2243250 is higher among those vaccinated with higher titers. Moreover, these SNPs and TNFA rs1800629 are differently distributed in a group of subjects that were found infected by SARS-CoV-2 at day 105 of evaluation. Finally, subjects that were found to be infected by SARS-CoV-2 at day 105 were significantly older than the uninfected subjects. Taken together, these data seem to suggest that age and polymorphisms of key cytokines, which regulate inflammation and humoral immune response, might influence the magnitude of the antibody response to vaccination with BNT162B2, prompting speculation about the possible benefit of a genetic background-based assessment of a personalized approach to the anti-COVID vaccination schedule.

A Retrospective Study of the Safety and Immunogenicity of MVC-COV1901 Vaccine for People Living with HIV.

BACKGROUND: This study aimed to assess the safety and immunogenicity of MVC-COV1901, a recombinant COVID-19 protein vaccine, containing S-2P protein adjuvanted with CpG 1018 and aluminum hydroxide, for people living with HIV (PWH). METHODS: A total of 57 PWH of ≥20 years of age who are on stable antiretroviral therapy were compared with 882 HIV-negative participants. Participants received two doses of MVC-COV1901 28 days apart. RESULTS: No vaccine-related serious adverse events (SAEs) were recorded. Seroconversion rates (SCRs) of 100% and 99.8% were achieved in PWH and comparators, respectively, 28 days after the second dose. After adjusting for sex, age, BMI category, and comorbidity, the adjusted GMT ratio of comparator/PWH was 3.2 (95% CI 2.5-4). A higher CD4/CD8 ratio was associated with a higher GMT (R = 0.27, p = 0.039). MVC-COV1901 has shown robust safety but elicited weaker immune responses in PWH. CONCLUSIONS: Further investigations may be needed to determine whether PWH require distinct immunization strategies with improved immunogenicity. The main study is registered at ClinicalTrials.gov (NCT04695652).

Antibody-mediated immunity to SARS-CoV-2 spike.

Despite effective spike-based vaccines and monoclonal antibodies, the SARS-CoV-2 pandemic continues more than two and a half years post-onset. Relentless investigation has outlined a causative dynamic between host-derived antibodies and reciprocal viral subversion. Integration of this paradigm into the architecture of next generation antiviral strategies, predicated on a foundational understanding of the virology and immunology of SARS-CoV-2, will be critical for success. This review aims to serve as a primer on the immunity endowed by antibodies targeting SARS-CoV-2 spike protein through a structural perspective. We begin by introducing the structure and function of spike, polyclonal immunity to SARS-CoV-2 spike, and the emergence of major SARS-CoV-2 variants that evade immunity. The remainder of the article comprises an in-depth dissection of all major epitopes on SARS-CoV-2 spike in molecular detail, with emphasis on the origins, neutralizing potency, mechanisms of action, cross-reactivity, and variant resistance of representative monoclonal antibodies to each epitope.

Epitope-directed anti-SARS-CoV-2 scFv engineered against the key spike protein region could block membrane fusion.

The newly emerged SARS-CoV-2 causing coronavirus disease (COVID-19) resulted in >500 million infections. A great deal about the molecular processes of virus infection in the host is getting uncovered. Two sequential proteolytic cleavages of viral spike protein by host proteases are prerequisites for the entry of the virus into the host cell. The first cleavage occurs at S1/S2 site by the furin protease, and the second cleavage at a fusion activation site, the S2' site, by the TMPRSS2 protease. S2' cleavage site is present in the S2 domain of spike protein followed by a fusion peptide. Given the S2' site to be conserved among all the SARS-CoV-2 variants, we chose an S2' epitope encompassing the S2' cleavage site and generated single-chain antibodies (scFvs) through an exhaustive phage display library screening. Crystal structure of a scFv in complex with S2' epitope was determined. Incidentally, S2' epitope in the scFv bound structure adopts an alpha-helical conformation equivalent to the conformation of the epitope in the spike protein. Furthermore, these scFvs can bind to the spike protein expressed either in vitro or on the mammalian cell surface. We illustrate a molecular model based on structural and biochemical insights into the antibody-S2' epitope interaction emphasizing scFvs mediated blocking of virus entry into the host cell by restricting the access of TMPRSS2 protease and consequently inhibiting the S2' cleavage competitively.

Structural Analysis and Epitope Prediction of S2 Domain of SARS-CoV-2, Conservation Analysis Among Major Variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic. There are four structural proteins of the virus: spike, envelope, membrane, and nucleocapsid proteins. Various vaccines were designed and are effectively being used against the spike protein of the virus. However, several vaccine-related complications have been reported worldwide. Assuming that the structural integrity of the whole protein might be contributing to these complications, this study was performed to design epitopes using the S2 domain of the spike protein, which could trigger a strong immune response. We have also predicted antigenic and allergenic properties of the selected epitopes. A total of 49 B cell epitopes passing antigenicity and other assessment filters were found using three methods. Among them, RDLICAQ had the highest antigenicity score (1.1443). However, only one cytotoxic T lymphocyte epitope, RSFIEDLLF, passed the essential filters with an antigenicity score of 0.5782 to show an appropriate immune response for T cells, while among 21 helper T cell lymphocyte epitopes that were filtered, FAMQMAYRFNGIGVT showed the highest (1.3688) antigenicity score. Conservation analysis revealed that the S2 domain is significantly conserved, thus making it an ideal candidate for vaccine development. We have also designed a vaccine construct based on the best suiting components found during the whole study. This construct and S2 domain solely can be future subjects of interest or might be included in a subunit cocktail formulation for attaining unabridged immunogenicity.

4-Aminoquinolines modulate RNA structure and function: Pharmacophore implications of a conformationally restricted polyamine.

RNA structure plays an important role in regulating cellular function and there is a significant emerging interest in targeting RNA for drug discovery. Here we report the identification of 4-aminoquinolines as modulators of RNA structure and function. Aminoquinolines have a broad range of pharmacological activities, but their specific mechanism of action is often not fully understood. Using electrophoretic mobility shift assays and enzymatic probing we identified 4-aminoquinolines that bind the stem-loop II motif (s2m) of SARS-CoV-2 RNA site-specifically and induce dimerization. Using fluorescence-based RNA binding and T-box riboswitch functional assays we identified that hydroxychloroquine binds the T-box riboswitch antiterminator RNA element and inhibits riboswitch function. Based on its structure and riboswitch dose-response activity we identified that the antagonist activity of hydroxychloroquine is consistent with it being a conformationally restricted analog of the polyamine spermidine. Given the known role that polyamines play in RNA function, the identification of an RNA binding ligand with the pharmacophore of a conformationally restricted polyamine has significant implications for further elucidation of RNA structure-function relationships and RNA-targeted drug discovery.

SARS-CoV-2 Inhibitors Identified by Phenotypic Analysis of a Collection of Viral RNA-Binding Molecules.

Antiviral agents are needed for the treatment of SARS-CoV-2 infections and to control other coronavirus outbreaks that may occur in the future. Here we report the identification and characterization of RNA-binding compounds that inhibit SARS-CoV-2 replication. The compounds were detected by screening a small library of antiviral compounds previously shown to bind HIV-1 or HCV RNA elements with a live-virus cellular assay detecting inhibition of SARS-CoV-2 replication. These experiments allowed detection of eight compounds with promising anti-SARS-CoV-2 activity in the sub-micromolar to micromolar range and wide selectivity indexes. Examination of the mechanism of action of three selected hit compounds excluded action on the entry or egress stages of the virus replication cycle and confirmed recognition by two of the molecules of conserved RNA elements of the SARS-CoV-2 genome, including the highly conserved S2m hairpin located in the 3'-untranslated region of the virus. While further studies are needed to clarify the mechanism of action responsible for antiviral activity, these results facilitate the discovery of RNA-targeted antivirals and provide new chemical scaffolds for developing therapeutic agents against coronaviruses.

Antibody Response after SARS-CoV-2 Infection with the Delta and Omicron Variant.

The SARS-CoV-2 virus caused a worldwide COVID-19 pandemic. So far, 6,120,834 confirmed cases of COVID-19 with 116,773 deaths have been reported in Poland. According to WHO, a total of 54,662,485 vaccine doses have been administered. New variants emerge that become dominant. The aim of this study was a comparison of antibody level after infection caused by Delta and Omicron variants. The study included 203 persons who underwent mild COVID-19 despite two doses of vaccine. The obtained results indicate that a significantly lower titer was observed in patients with the Omicron variant infection. Therefore, these patients may be at risk of reinfection with new strains of the Omicron variant. Due to the possibility of reinfection, booster vaccinations are necessary. Further epidemiological and clinical studies are necessary to develop new prevention strategies.