ABSTRACT
Background: The recently emerged SARS-CoV-2 Omicron variant exhibits several mutations on the spike protein, enabling it to escape the immunity elicited by natural infection or vaccines. Avidity is the strength of binding between an antibody and its specific epitope. The SARS-CoV-2 spike protein binds to its cellular receptor with high affinity, and is the primary target of neutralizing antibodies. Therefore, protective antibodies should show high avidity. This study aimed at investigating the avidity of receptor-binding domain (RBD) binding antibodies and their neutralizing activity against the Omicron variant in COVID-19 patients and vaccinees. Methods: . Samples collected from COVID-19 patients and from subjects who received homologous or heterologous vaccination were tested for the avidity of RBD-binding IgG and neutralizing antibodies against the wild-type SARS-CoV-2 virus and the Omicron variant. Results: . In patients, RBD-binding IgG titres against the wild-type virus increased with time, but remained low. High neutralizing titres against the wild-type virus were not matched by high avidity or neutralizing activity against the Omicron variant. Vaccinees showed higher avidity than patients. Two vaccine doses elicited the production of neutralizing antibodies, but low avidity for the wild-type virus; antibody levels against the Omicron variant were even lower. Conversely, 3 doses of vaccine elicited high avidity and high neutralizing antibodies against both the wild-type virus and the Omicron variant. Conclusions: . Repeated vaccination increases antibody avidity against the spike protein of the Omicron variant, suggesting that antibodies with high avidity and high neutralizing potential increase cross-protection against variants that carry several mutations on the RBD.
Subject(s)
COVID-19ABSTRACT
BackgroundThe highly contagious SARS-CoV-2 is mainly transmitted by respiratory droplets and aerosols. Consequently, people are required to wear masks and maintain a social distance to avoid spreading of the virus. Despite the success of the commercially available vaccines, the virus is still uncontained globally. Given the tropism of SARS-CoV-2, a mucosal immune reaction would help to reduce viral shedding and transmission locally. Only seven out of hundreds of ongoing clinical trials are testing the intranasal delivery of COVID-19 vaccines. MethodsIn the current study, we tested in murine model the immunogenicity of a conventional vaccine platform based on virus-like particles (VLPs) displaying RBD of SARS-CoV-2 for intranasal vaccination. The candidate vaccine, CuMVTT-RBD, has been immunologically optimized to incorporate tetanus-toxin and is self-adjuvanted with TLR7/8 ligands. ResultsCuMVTT-RBD elicited strong RBD- and spike- specific systemic IgG and IgA antibody responses of high avidity. Local immune responses were assessed and results demonstrate strong mucosal antibody and plasma cell production in lung tissue. The induced systemic antibodies could efficiently recognize and neutralize different Variants of Concerns of mutated SARS-CoV-2 RBDs. ConclusionIn summary, intranasal vaccination with CuMVTT-RBD shows high immunogenicity and induces protective systemic and local specific antibody response against SARS-CoV-2 and its variants. One sentence summaryEvaluation of an intransal administrated conventional VLP-based vaccine against COVID-19 in a murine model.
Subject(s)
COVID-19 , Tetanus , Severe Acute Respiratory SyndromeABSTRACT
The massive emergence of COVID19 cases in the first phase of pandemic within an extremely short period of time suggest that an undetected earlier circulation of SARS-CoV-2 might have occurred, as documented by several papers in different countries, including a few that reported positive cases even earlier the first cases identified in Wuhan. Given the importance of this evidence, an independent evaluation was recommended. Here we report the results of SARS-CoV-2 antibodies blind retesting of blood samples collected in the prepandemic period in Italy, and in control samples collected one year before, by two independent centers. Results suggest the presence of SARS-CoV-2 antibodies in some samples collected in the prepandemic period, though the detection of IgM and/or IgG binding and neutralizing antibodies is strongly dependent on the different serological assays and thresholds employed, while being absent in control samples collected one year before. These findings highlight the importance of harmonizing serological assays for testing SARS-CoV-2 virus spreading and may contribute to a better understanding the future virus dynamics. Article Summary LineWe report the results of an independent retesting of SARS-CoV-2 antibodies in blood samples collected in prepandemic period in Italy and in matched samples collected one year before. The findings indicate the presence of IgM and/or IgG antibodies in selected samples of the prepandemic period only with different performance of serological assays used by the two centers. The results could give highlights on SARS-CoV-2 circulation in the pre-pandemic period and contribute to better predict future virus dynamic.
Subject(s)
COVID-19ABSTRACT
To control future epidemics, discovery platforms are urgently needed, for the rapid development of diagnostic assays. Molecular diagnostic tests for COVID-19 emerged shortly after the isolation of SARS-CoV-2, however, serological tests based on antiviral antibody detection, revealing previous exposure to the virus, required longer developmental phases, due to the need for correctly folded and glycosylated antigens. The delay between the identification of a new virus and the development of reliable serodiagnostic tools limits our readiness for the control of a future epidemic. In this context, we propose the protozoan Leishmania tarentolae as an easy-to-handle micro-factory for the rapid production of viral antigens, to be used at the forefront of emerging epidemics. As a study model, we engineered L. tarentolae to express the SARS-CoV-2 Receptor Binding Domain (RBD) and report the ability of the purified RBD antigen to detect SARS-CoV-2 infection, with a sensitivity and reproducibility comparable to that of a reference antigen produced in human cells. This is the first application of an antigen produced in L. tarentolae for the serodiagnosis of a Coronaviridae infection. Based on our results, we propose L. tarentolae as an effective system for viral antigen production, even in countries that lack high-tech cell factories.
Subject(s)
COVID-19 , Coronaviridae InfectionsABSTRACT
SARS-CoV-2 pandemic is causing high morbidity and mortality burden worldwide with unprecedented strain on health care systems. To elucidate the mechanism of infection, protection, or rapid evolution until fatal outcome of the disease we performed a study in hospitalized COVID-19 patients to investigate the time course of the antibody response in relation to the outcome. In comparison we investigated the time course of the antibody response in SARS-CoV-2 asymptomatic subjects. Study results show that patients produce a strong antibody response to SARS-CoV-2 with high correlation between different viral antigens (spike protein and nucleoprotein) and among antibody classes (IgA, IgG, and IgM and neutralizing antibodies). The peak is reached by 3 weeks from hospital admission followed by a sharp decrease. No difference was observed in any parameter of the antibody classes, including neutralizing antibodies, between subjects who recovered or with fatal outcome. Only few asymptomatic subjects developed antibodies at detectable levels.
Subject(s)
COVID-19ABSTRACT
To investigate the evolution of SARS-CoV-2 in the immune population, we co-incubated authentic virus with a highly neutralizing plasma from a COVID-19 convalescent patient. The plasma fully neutralized the virus for 7 passages, but after 45 days, the deletion of F140 in the spike N-terminal domain (NTD) N3 loop led to partial breakthrough. At day 73, an E484K substitution in the receptor-binding domain (RBD) occurred, followed at day 80 by an insertion in the NTD N5 loop containing a new glycan sequon, which generated a variant completely resistant to plasma neutralization. Computational modeling predicts that the deletion and insertion in loops N3 and N5 prevent binding of neutralizing antibodies. The recent emergence in the United Kingdom and South Africa of natural variants with similar changes suggests that SARS-CoV-2 has the potential to escape an effective immune response and that vaccines and antibodies able to control emerging variants should be developed.
Subject(s)
COVID-19ABSTRACT
Due to the global spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), there is an urgent need for reliable high-throughput serological assays in order to evaluate the immunological responses against SARS-COV-2 virus and to enable population screening, as well as vaccines and drugs efficacy testing. Several serological assays for SARS-CoV-2 are now becoming available in the market. However, it has also become extremely important to have well-established assays with desirable high sensitivity and specificity. To date, the micro-neutralization (MN) assay, is currently considered the gold-standard being capable of evaluating and detecting, functional neutralizing antibodies (nAbs). Several protocols exist for micro-neutralization assays which vary in several steps of the protocol: cell seeding conditions, number of cells seeded, virus amount used in the infection step, virus-serum-cells incubation period etc. These potential differences account for a high degree of variability and inconsistency of the results and using a harmonized protocol for the micro-neutralization assay could potentially solve this. Given this situation, the main aim of our study was to carry out SARS-CoV-2 wild type virus MN assay in order to investigate which optimal tissue culture infective dose 50 (TCID50) infective dose in use is the most adequate choice for implementation in terms of reproducibility, standardization possibilities and comparability of results. Therefore, we assessed the MN by using two different viral infective doses: a standard dose of 100 TCID50/well and a lower dose of 25 TCID50/well. The results obtained, yielded by MN on using the lower infective dose (25 TCID50), were in line with those obtained with the standard infective dose; in some cases, however, we detected a titre that was one or two dilution steps higher, which maintained all negative samples negative. This suggesting that the lower dose can potentially have a positive impact on the detection and estimation of neutralizing antibodies present in a given sample, showing higher sensitivity but similar specificity and therefore, it would require a more accurate assessment and cross-laboratories standardisation especially when MN is employed as serological assay of choice for pre-clinical and clinical studies.
ABSTRACT
SARS-CoV-2 has emerged as pandemic all over the world since late 2019. In this study, we investigated the diversity of the virus in the context of SARS-CoV-2 spread in India. Full-length SARS-CoV-2 genome sequences of the circulating viruses from all over India were collected from GISAID, an open data repository, until 25thJuly, 2020. We have focused on the non-synonymous changes across the genome that resulted in amino acid substitutions. Analysis of the genomic signatures of the non-synonymous mutations demonstrated a strong association between the time of sample collection and the accumulation of genetic diversity. Most of these isolates from India belonged to the A2a clade (63.4%) which has overcome the selective pressure and is spreading rapidly across several continents. Interestingly a new clade I/A3i has emerged as the second-highest prevalent type among the Indian isolates, comprising 25.5% of the Indian sequences. Emergence of new mutations in the S protein was observed. Major SARS-CoV-2 clades in India have defining mutations in the RdRp. Maximum accumulation of mutations was observed in ORF1a. Other than the clade-defining mutations, few representative non-synonymous mutations were checked against the available crystal structures of the SARS-CoV-2 proteins in the DynaMut server to assess their thermodynamic stability. We have observed that SARS-CoV-2 genomes contain more uracil than any other nucleotide. Furthermore, substitution of nucleotides to uracil was highest among the non-synonymous mutations observed. The A+U content in SARS-CoV-2 genome is much higher compared to other RNA viruses, suggesting that the virus RdRp has a propensity towards uracil incorporation in the genome. This implies that thymidine analogues may have a better chance to competitively inhibit SARS-CoV-2 RNA replication than other nucleotide analogues.
ABSTRACT
Human monoclonal antibodies are safe, preventive and therapeutic tools, that can be rapidly developed to help restore the massive health and economic disruption caused by the Covid-19 pandemic. By single cell sorting 4277 SARS-CoV-2 spike protein specific memory B cells from 14 Covid-19 survivors, 453 neutralizing antibodies were identified and 220 of them were expressed as IgG. Up to 65,9% of monoclonals neutralized the wild type virus at a concentration of >500 ng/mL, 23,6% neutralized the virus in the range of 100 - 500 ng/mL and 9,1% had a neutralization potency in the range of 10 - 100 ng/mL. Only 1,4% neutralized the authentic virus with a potency of 1-10 ng/mL. We found that the most potent neutralizing antibodies are extremely rare and recognize the RBD, followed in potency by antibodies that recognize the S1 domain, the S-protein trimeric structure and the S2 subunit. The three most potent monoclonal antibodies identified were able to neutralize the wild type and D614G mutant viruses with less than 10 ng/mL and are good candidates for the development of prophylactic and therapeutic tools against SARS-CoV-2. One Sentence SummaryExtremely potent neutralizing human monoclonal antibodies isolated from Covid-19 convalescent patients for prophylactic and therapeutic interventions.
Subject(s)
COVID-19ABSTRACT
A newly identified coronavirus, named SARS-CoV-2, emerged in December 2019 in Hubei Province, China, and quickly spread throughout the world; so far, it has caused more than 18 million cases of disease and 700,000 deaths. The diagnosis of SARS-CoV-2 infection is currently based on the detection of viral RNA in nasopharyngeal swabs by means of molecular-based assays, such as real-time RT-PCR. Furthermore, serological assays aimed at detecting different classes of antibodies constitute the best surveillance strategy for gathering information on the humoral immune response to infection and the spread of the virus through the population, in order to evaluate the immunogenicity of novel future vaccines and medicines for the treatment and prevention of COVID-19 disease. The aim of this study was to determine SARS-CoV-2-specific antibodies in human serum samples by means of different commercial and in-house ELISA kits, in order to evaluate and compare their results first with one another and then with those yielded by functional assays using wild-type virus. It is important to know the level of SARS-CoV-2-specific IgM, IgG and IgA antibodies in order to predict population immunity and possible cross-reactivity with other coronaviruses and to identify potentially infectious subjects. In addition, in a small sub-group of samples, we performed a subtyping Immunoglobulin G ELISA. Our data showed an excellent statistical correlation between the neutralization titer and the IgG, IgM and IgA ELISA response against the receptor-binding domain of the spike protein, confirming that antibodies against this portion of the virus spike protein are highly neutralizing and that the ELISA Receptor-Binding Domain-based assay can be used as a valid surrogate for the neutralization assay in laboratories which do not have Biosecurity level-3 facilities.
Subject(s)
COVID-19ABSTRACT
In the absence of approved drugs or vaccines, there is a pressing need to develop tools for therapy and prevention of Covid-19. Human monoclonal antibodies have very good probability of being safe and effective tools for therapy and prevention of SARS-CoV-2 infection and disease. Here we describe the screening of PBMCs from seven people who survived Covid-19 infection to isolate human monoclonal antibodies against SARS-CoV-2. Over 1,100 memory B cells were single-cell sorted using the stabilized prefusion form of the spike protein and incubated for two weeks to allow natural production of antibodies. Supernatants from each cell were tested by ELISA for spike protein binding, and positive antibodies were further tested for neutralization of spike binding to receptor(s) on Vero E6 cells and for virus neutralization in vitro. From the 1,167 memory B specific for SARS-CoV-2, we recovered 318 B lymphocytes expressing human monoclonals recognizing the spike protein and 74 of these were able to inhibit the binding of the spike protein to the receptor. Finally, 17 mAbs were able to neutralize the virus when assessed for neutralization in vitro. Lead candidates to progress into the drug development pipeline will be selected from the panel of neutralizing antibodies identified with the procedure described in this study. One Sentence SummaryNeutralizing human monoclonal antibodies isolated from Covid-19 convalescent patients for therapeutic and prophylactic interventions.