Background Monitoring the adaptive immune responses during the natural course of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection provides useful information for the development of vaccination strategies against this virus and its emerging variants. We thus profiled the serum anti-SARS-CoV-2 antibody levels and specific memory B- and T-cell responses in convalescent coronavirus disease-2019 (COVID-19) patients. Methods Altogether 119 samples from 88 convalescent donors who experienced mild to critical disease were tested for the presence of elevated anti-spike and anti-receptor binding domain antibody levels over a period of eight months. In addition, level of SARS-CoV-2 neutralizing antibodies, specific memory B- and T-cell responses were tested in a subset of samples. Findings Anti-SARS-CoV-2 antibodies were present in 85% of the samples collected within 4 weeks after onset of symptoms in COVID-19 patients. Levels of specific IgM/IgA antibodies declined after 1 month while levels of specific IgG antibodies and plasma neutralizing activities remained relatively stable up to 6 months after diagnosis. Anti-SARS-CoV-2 IgG antibodies were still present, though at a significantly lower level, in 80% of the samples collected at 6-8 months after symptom onset. SARS-CoV-2-specific memory B- and T-cell responses developed with time and were persistent in all patients followed up till 6-8 months. Conclusions Our data suggest that protective adaptive immunity following natural infection of SARS-CoV-2 might persist for at least 6-8 months, regardless of disease severity. Development of medium or long-term protective immunity through vaccination might thus be possible. Funding EU-ATAC consortium, the Italian Ministry of Health and SciLife/KAW.

The recent emergence of the Omicron variant has raised concerns on vaccine efficacy and the urgent need to study more efficient vaccination strategies. Here we observed that an mRNA vaccine booster in individuals vaccinated with two doses of inactivated vaccine significantly increased the plasma level of specific antibodies that bind to the receptor-binding domain (RBD) or the spike (S) ectodomain (S1 + S2) of both the G614 and the Omicron variants, compared to two doses of homologous inactivated vaccine. The level of RBD- and S-specific IgG antibodies and virus neutralization titers against variants of concern in the heterologous vaccination group were similar to that in individuals receiving three doses of homologous mRNA-vaccine or a boost of mRNA vaccine after infection, but markedly higher than that in individuals receiving three doses of a homologous inactivated vaccine. This heterologous vaccination regime furthermore significantly enhanced the RBD-specific memory B cell response and S1-specific T cell response, compared to two or three doses of homologous inactivated vaccine. Our study demonstrates that mRNA vaccine booster in individuals vaccinated with inactivated vaccines can be highly beneficial, as it markedly increases the humoral and cellular immune responses against the virus, including the Omicron variant.

Background: The longevity of the immune response against SARS-CoV-2 is currently debated. We thus profiled the serum anti-SARS-CoV-2 antibody levels and virus specific memory B- and T-cell responses over time in convalescent COVID-19 patients. Methods: A cohort of COVID-19 patients from the Lombardy region in Italy who experienced mild to critical disease and Swedish volunteers with mild symptoms, were tested for the presence of elevated anti-spike and anti-receptor binding domain antibody levels over a period of eight months. In addition, specific memory B- and T-cell responses were tested in selected patient samples. Results: Anti-SARS-CoV-2 antibodies were present in 85% samples collected within 4 weeks after onset of symptoms in COVID-19 patients. Levels of specific IgM or IgA antibodies declined after 1 month while levels of specific IgG antibodies remained stable up to 6 months after diagnosis. Anti-SARS-CoV-2 IgG antibodies were still present, though at a significantly lower level, in 80% samples collected at 6-8 months after symptom onset. SARS-CoV-2-specific memory B- and T-cell responses were developed in vast majority of the patients tested, regardless of disease severity, and remained detectable up to 6-8 months after infection. Conclusions: Although the serum levels of anti-SARS-CoV-2 IgG antibodies started to decline, virus-specific T and/or memory B cell responses increased with time and maintained during the study period (6-8 months after infection).

Background The COVID-19 pandemic is caused by the betacoronavirus SARS-CoV-2. In November 2021, the Omicron variant was discovered and immediately classified as a variant of concern (VOC), since it shows substantially more mutations in the spike protein than any previous variant, especially in the receptor-binding domain (RBD). We analyzed the binding of the Omicron RBD to the human angiotensin-converting enzyme-2 receptor (ACE2) and the ability of human sera from COVID-19 patients or vaccinees in comparison to Wuhan, Beta, or Delta RBD variants. Methods All RBDs were produced in insect cells. RBD binding to ACE2 was analyzed by ELISA and microscale thermophoresis (MST). Similarly, sera from 27 COVID-19 patients, 81 vaccinated individuals, and 34 booster recipients were titrated by ELISA on RBDs from the original Wuhan strain, Beta, Delta, and Omicron VOCs. In addition, the neutralization efficacy of authentic SARS-CoV-2 wild type (D614G), Delta, and Omicron by sera from 2× or 3× BNT162b2-vaccinated persons was analyzed. Results Surprisingly, the Omicron RBD showed a somewhat weaker binding to ACE2 compared to Beta and Delta, arguing that improved ACE2 binding is not a likely driver of Omicron evolution. Serum antibody titers were significantly lower against Omicron RBD compared to the original Wuhan strain. A 2.6× reduction in Omicron RBD binding was observed for serum of 2× BNT162b2-vaccinated persons. Neutralization of Omicron SARS-CoV-2 was completely diminished in our setup. Conclusion These results indicate an immune escape focused on neutralizing antibodies. Nevertheless, a boost vaccination increased the level of anti-RBD antibodies against Omicron, and neutralization of authentic Omicron SARS-CoV-2 was at least partially restored. This study adds evidence that current vaccination protocols may be less efficient against the Omicron variant.

Information concerning the longevity of immunity to SARS-CoV-2 following natural infection may have considerable implications for durability of immunity induced by vaccines. Here, we monitored the SARS-CoV-2 specific immune response in COVID-19 patients followed up to 15 months after symptoms onset. Following a peak at day 15-28 postinfection, the IgG antibody response and plasma neutralizing titers gradually decreased over time but stabilized after 6 months. Compared to G614, plasma neutralizing titers were more than 8-fold lower against variants Beta, Gamma, and Delta. SARS-CoV-2-specific memory B and T cells persisted in the majority of patients up to 15 months although a significant decrease in specific T cells, but not B cells, was observed between 6 and 15 months. Antiviral specific immunity, especially memory B cells in COVID-19 convalescent patients, is long-lasting, but some variants of concern may at least partially escape the neutralizing activity of plasma antibodies.

Diphtheria is an infectious disease caused by Corynebacterium diphtheriae. the bacterium primarily infects the throat and upper airways and the produced diphtheria toxin (Dt), which binds to the elongation factor 2 and blocks protein synthesis, can spread through the bloodstream and affect organs, such as the heart and kidneys. For more than 125 years, the therapy against diphtheria has been based on polyclonal horse sera directed against Dt (diphtheria antitoxin; DAt). Animal sera have many disadvantages including serum sickness, batch-to-batch variation in quality and the use of animals for production. In this work, 400 human recombinant antibodies were generated against DT from two different phage display panning strategies using a human immune library. A panning in microtiter plates resulted in 22 unique in vitro neutralizing antibodies and a panning in solution combined with a functional neutralization screening resulted in 268 in vitro neutralizing antibodies. 61 unique antibodies were further characterized as scFv-Fc with 35 produced as fully human IgG1. The best in vitro neutralizing antibody showed an estimated relative potency of 454 IU/mg and minimal effective dose 50% (MED50%) of 3.0 pM at a constant amount of DT (4x minimal cytopathic dose) in the IgG format. The targeted domains of the 35 antibodies were analyzed by immunoblot and by epitope mapping using phage display. All three Dt domains (enzymatic domain, translocation domain and receptor binding domain) are targets for neutralizing antibodies. When toxin neutralization assays were performed at higher toxin dose levels, the neutralizing capacity of individual antibodies was markedly reduced but this was largely compensated for by using two or more antibodies in combination, resulting in a potency of 79.4 IU/mg in the in vivo intradermal challenge assay. these recombinant antibody combinations are candidates for further clinical and regulatory development to replace equine DAt.Diphtheria is an infectious disease and caused by Corynebacterium diphtheriae. The bacterium primarily infects the throat and upper airways but can also affect other body sites. The produced toxin can spread through the bloodstream and affect organs, such as the heart and kidneys. In severe cases, diphtheria toxin (DT) may cause myocarditis or peripheral neuropathy. Due to a membrane of dead tissue over the throat and tonsils, swallowing and breathing can be difficult. The disease is spread through direct physical contact or by coughing or sneezing of infected individuals 1-3 . Diphtheria is fatal in 5-10% of cases, but children under the age of five have a mortality rate of up to 20%. Treatment involves antibiotics to kill the bacteria (erythromycin or penicillin for 14 days) and administering of diphtheria antitoxin (DAT) to neutralize the effects of the toxin 4-6 . C. diphtheriae was identified as the causative agent of diphtheria in 1883 and in 1888 the diphtheria toxin was first described in the culture medium of C. diphtheriae 7 . The gene for DT is encoded on a coryn...

The novel betacoronavirus SARS-CoV-2 causes a form of severe pneumonia disease, termed COVID-19. To develop human neutralizing anti-SARS-CoV-2 antibodies, antibody gene libraries from convalescent COVID-19 patients were constructed and recombinant antibody fragments (scFv) against the receptor binding domain (RBD) of the spike protein were selected by phage display. The antibody STE90-C11 shows a sub nM IC50 in a plaque-based live SARS-CoV-2 neutralization assay. The in vivo efficacy of the antibody is demonstrated in the Syrian hamster and in the hACE2 mice model. The crystal structure of STE90-C11 Fab in complex with SARS-CoV-2-RBD is solved at 2.0 Å resolution showing that the antibody binds at the same region as ACE2 to RBD. The binding and inhibition of STE90-C11 is not blocked by many known emerging RBD mutations. STE90-C11 derived human IgG1 with FcγR silenced Fc (COR-101) is currently undergoing Phase Ib/II clinical trials for the treatment of moderate to severe COVID-19.

COVID-19 is a severe acute respiratory disease caused by SARS-CoV-2, a novel betacoronavirus discovered in December 2019 and closely related to the SARS coronavirus (CoV). Both viruses use the human ACE2 receptor for cell entry, recognizing it with the Receptor Binding Domain (RBD) of the S1 subunit of the viral spike (S) protein.The S2 domain mediates viral fusion with the host cell membrane. Experience with SARS and MERS coronavirus has shown that potent monoclonal neutralizing antibodies against the RBD can inhibit the interaction with the virus cellular receptor (ACE2 for SARS) and block the virus cell entry. Assuming that a similar strategy would be successful against SARS-CoV-2, we used phage display to select from the human naïve universal antibody gene libraries HAL9/10 anti SARS2 spike antibodies capable of inhibiting interaction with ACE2. 309 unique fully human antibodies against S1 were identified. 17 showed more than 75% inhibition of spike binding to cells expressing ACE2, assessed by flow cytometry and several antibodies showed even an 50% inhibition at a molar ratio of the antibody to spike protein or RBD of 1:1. Furthermore, these antibodies neutralized active SARS-Cov-2 virus infection of VeroE6 cells. All 17 were all able to bind the isolated RBD, four of them with sub-nanomolar EC50. Epitope analysis of the antibodies revealed that six bind at the RBD-ACE2 interface and two on the opposite side of the domain. Universal libraries from healthy donors offer the advantage that antibodies can be generated quickly and independent from the availability of material from recovered patients in a pandemic situation. 4/34 Main textIn 2015 Menachery et al. wrote: "Our work suggests a potential risk of SARS-CoV reemergence from viruses currently circulating in bat populations." 1 . Four years later, a novel coronavirus causing a severe pneumonia was discovered and later named SARS-CoV-2. The outbreak started on a sea food market in Wuhan, Hubei province (China) at the end of 2019. The disease was named COVID-19 (coronavirus disease 2019) by the World Health Organization (WHO). Sequencing showed high identity to bat corona viruses (CoV, in particular RaTG13), beta-CoV virus causing human diseases like SARS and MERS and, to a lesser extent, the seasonal CoV hCoV-OC43 and HCov-HKU1 2,3 . The spike (S) protein of SARS-CoV-2, as well as SARS-CoV, binds to the human zinc peptidase angiotensin-converting enzyme 2 (ACE2) which is expressed on lung cells, heart, kidney and intestine cells and acts as receptor for virus entry. S protein consists of the N-terminal S1 subunit, which includes the receptor binding domain (RBD), and the Cterminal S2 subunit which is anchored to the viral membrane and is required for trimerization and fusion of the virus and host membrane 4-6 . The membrane bound host protease TMPRSS2 is responsible for S protein priming by cleavage of specific sites between S1 and S2. In addition to proteolytic activation of the S2' site, conformational changes and viral entry 7-10 .Antibodies against the...