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1.
Vaccines (Basel) ; 10(5)2022 May 19.
Article in English | MEDLINE | ID: covidwho-1928676

ABSTRACT

BACKGROUND: Protozoa of the genus Leishmania are characterized by their capacity to target macrophages and Dendritic Cells (DCs). These microorganisms could thus be exploited for the delivery of antigens to immune cells. Leishmania tarentolae is regarded as a non-pathogenic species; it was previously used as a biofactory for protein production and has been considered as a candidate vaccine or as an antigen delivery platform. However, results on the type of immune polarization determined by L. tarentolae are still inconclusive. METHODS: DCs were derived from human monocytes and exposed to live L. tarentolae, using both the non-engineered P10 strain, and the same strain engineered for expression of the spike protein from SARS-CoV-2. We then determined: (i) parasite internalization in the DCs; and (ii) the capacity of the assayed strains to activate DCs and the type of immune polarization. RESULTS: Protozoan parasites from both strains were effectively engulfed by DCs, which displayed a full pattern of maturation, in terms of MHC class II and costimulatory molecule expression. In addition, after parasite infection, a limited release of Th1 cytokines was observed. CONCLUSIONS: Our results indicate that L. tarentolae could be used as a vehicle for antigen delivery to DCs and to induce the maturation of these cells. The limited cytokine release suggests L. tarentolae as a neutral vaccine vehicle that could be administered in association with appropriate immune-modulating molecules.

2.
Viruses ; 14(7)2022 06 30.
Article in English | MEDLINE | ID: covidwho-1917788

ABSTRACT

Italy was the second country affected by the SARS-CoV-2 pandemic; the virus spread mainly in Northern Italy with a subsequent diffusion to the center and southern part of the country. In this study, we aimed to assess the prevalence of antibodies against SARS-CoV-2 in the general population of the Siena province in the Tuscany region (Central Italy) during 2020. A total of 2480 serum samples collected from January to December 2020 were tested for IgM and IgG antibodies against SARS-CoV-2 by a commercial ELISA. Positive and borderline samples were further tested for the presence of anti-receptor-binding domain (RBD) IgM and IgG antibodies by an in-house ELISA and by a micro-neutralization assay. Out of the 2480 samples tested by the commercial ELISA, 81 (3.3%) were found to be positive or borderline for IgG and 58 (2.3%) for IgM in a total of 133 samples (5.4%) found to be positive or borderline for at least one antibody class. When the commercial ELISA and in-house ELISA/micro-neutralization assay results were combined, 26 samples (1.0%) were positive for RBD IgG, 11 (0.4%) for RBD IgM, and 23 (0.9%) for a neutralizing antibody. An increase in seroprevalence was observed during the year 2020, especially from the end of summer, consistent with the routine epidemiological surveillance of COVID-19 cases.


Subject(s)
COVID-19 , SARS-CoV-2 , Antibodies, Viral , COVID-19/epidemiology , Humans , Immunoglobulin G , Immunoglobulin M , Pandemics , Seroepidemiologic Studies
3.
EuropePMC; 2022.
Preprint in English | EuropePMC | ID: ppcovidwho-335709

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.

4.
Front Public Health ; 10: 847384, 2022.
Article in English | MEDLINE | ID: covidwho-1792872

ABSTRACT

Background: Immunity and clinical protection induced by mRNA vaccines against SARS-CoV-2 have been shown to decline overtime. To gather information on the immunity profile deemed sufficient in protecting against hospitalization, we tested IgG levels, interferon-gamma (IFN-γ) secretion, and neutralizing antibodies 180 days (d180) after the second shot of BNT162b vaccine, in HW. Methods: A total of 392 subjects were enrolled. All received BioNTech/Pfizer from February 2020 to April 2021. The vaccine-specific humoral response was quantitatively determined by testing for IgG anti-S1 domain of SARS-CoV-spike protein. Live virus microneutralization (MN) was evaluated by an assay performing incubation of serial 2-fold dilution of human serum samples, starting from 1:10 to 1:5120, with an equal volume of Wuhan strain and Delta VOC viral solution and assessing the presence/absence of a cytopathic effect. SARS-CoV-2-spike protein-specific T-cell response was determined by a commercial IFN-γ release assay. Results: In 352 individuals, at d180, IgG levels decreased substantially but no results below the assay's positivity threshold were observed. Overall, 22 naive (8.1%) had values above the highest threshold. Among COVID-naive, the impact of age, which was observed at earlier stages, disappeared at d180, while it remained significant for 81 who had experienced a previous infection. Following the predictive model of protection by Khoury, we transformed the neutralizing titers in IU/ml and used a 54 IU/ml threshold to identify subjects with 50% protective immunity. Overall, live virus MN showed almost all subjects with previous exposure to SARS-CoV-2 neutralized the virus as compared to 33% of naive double-dosed subjects (p < 0.0001). All previously exposed subjects had strong IFN-γ secretion (>200 mIU/ml); among 271 naive, 7 (2.58%) and 17 (6.27%) subjects did not show borderline or strong secretion, respectively. Conclusions: In naive subjects, low IgG titers are relatively long-lasting. Only a third of naive subjects maintain neutralizing responses. After specific stimulation, a very limited number of naive were unable to produce IFN-γ. The results attained in the small group of subjects with breakthrough infection suggest that simultaneous neutralizing antibody titers <20, binding antibody levels/ml <200, and IFN-γ <1,000 mIU/ml in subjects older than 58 may identify at-risk groups.


Subject(s)
COVID-19 Vaccines , COVID-19 , Antibodies, Neutralizing/pharmacology , Antibodies, Viral , COVID-19/prevention & control , Health Personnel , Humans , Immunity, Humoral , Immunoglobulin G , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/pharmacology
5.
Allergy ; 77(8): 2446-2458, 2022 Aug.
Article in English | MEDLINE | ID: covidwho-1784580

ABSTRACT

BACKGROUND: The 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 a vaccine against COVID-19. METHODS: In the current study, we evaluated the immunogenicity of a traditional vaccine platform based on virus-like particles (VLPs) displaying RBD of SARS-CoV-2 for intranasal administration in a murine model. The candidate vaccine platform, CuMVTT -RBD, has been optimized to incorporate a universal T helper cell epitope derived from tetanus-toxin and is self-adjuvanted with TLR7/8 ligands. RESULTS: CuMVTT -RBD vaccine elicited a strong systemic RBD- and spike-IgG and IgA antibodies of high avidity. Local immune response was assessed, and our results demonstrate a strong mucosal antibody and plasma cell production in lung tissue. Furthermore, the induced systemic antibodies could efficiently recognize and neutralize different variants of concern (VOCs). CONCLUSION: Our data demonstrate that intranasal administration of CuMVTT -RBD induces a protective systemic and local specific antibody response against SARS-CoV-2 and its VOCs.

6.
Hum Vaccin Immunother ; 18(5): 2047582, 2022 Nov 30.
Article in English | MEDLINE | ID: covidwho-1740707

ABSTRACT

In March 2020, the first pandemic caused by a coronavirus was declared by the World Health Organization. Italy was one of the first and most severely affected countries, particularly the northern part of the country. The latest evidence suggests that the virus could have been circulating, at least in Italy, before the first autochthonous SARS-COV-2 case was detected in February 2020. The present study aimed to investigate the presence of antibodies against SARS-CoV-2 in human serum samples collected in the last months of 2019 (September-December) in the Apulia region, Southern Italy. Eight of 455 samples tested proved positive on in-house receptor-binding-domain-based ELISA. Given the month of collection of the positive samples, these findings may indicate early circulation of SARS-CoV-2 in Apulia region in the autumn of 2019. However, it cannot be completely ruled out that the observed sero-reactivity could be an unknown antigen specificity in another virus to which subjects were exposed containing an epitope adventitiously cross-reactive with an epitope of SARS-CoV-2.


Subject(s)
COVID-19 , SARS-CoV-2 , Antibodies, Viral , COVID-19/epidemiology , Epitopes , Humans , Italy/epidemiology , Pandemics
7.
Viruses ; 14(1)2021 12 30.
Article in English | MEDLINE | ID: covidwho-1580400

ABSTRACT

The massive emergence of COVID-19 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. Given the importance of this evidence, an independent evaluation was recommended by the World Health Organization (WHO) to test a subset of samples selected on the level of positivity in ELISA assays (positive, low positive, negative) detected in our previous study of prepandemic samples collected in Italy. SARS-CoV-2 antibodies were blindly retested by two independent centers in 29 blood samples collected in the prepandemic period in Italy, 29 samples collected one year before and 11 COVID-19 control samples. The methodologies used included IgG-RBD/IgM-RBD ELISA assays, a qualitative micro-neutralization CPE-based assay, a multiplex IgG protein array, an ELISA IgM kit (Wantai), and a plaque-reduction neutralization test. The results suggest the presence of SARS-CoV-2 antibodies in some samples collected in the prepandemic period, with the oldest samples found to be positive for IgM by both laboratories collected on 10 October 2019 (Lombardy), 11 November 2019 (Lombardy) and 5 February 2020 (Lazio), the latter with neutralizing antibodies. The detection of IgM and/or IgG binding and neutralizing antibodies was strongly dependent on the different serological assays and thresholds employed, and they were not detected in control samples collected one year before. These findings, although gathered in a small and selected set of samples, highlight the importance of harmonizing serological assays for testing the spread of the SARS-CoV-2 virus and may contribute to a better understanding of future virus dynamics.


Subject(s)
COVID-19/epidemiology , SARS-CoV-2/isolation & purification , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , Asymptomatic Infections/epidemiology , COVID-19/diagnosis , COVID-19 Serological Testing/standards , Humans , Immunoglobulin G/blood , Immunoglobulin M/blood , Italy/epidemiology , SARS-CoV-2/immunology , Time Factors
8.
Front Microbiol ; 12: 736530, 2021.
Article in English | MEDLINE | ID: covidwho-1581277

ABSTRACT

To detect and prevent emerging epidemics, discovery platforms are urgently needed, for the rapid development of diagnostic assays. Molecular diagnostic tests for COVID-19 were developed shortly after the isolation of SARS-CoV-2. However, serological tests based on antiviral antibody detection, revealing previous exposure to the virus, required longer testing phases, due to the need to obtain correctly folded and glycosylated antigens. The delay between the identification of a new virus and the development of reliable serodiagnostic tools limits our readiness to tackle future epidemics. We suggest that the protozoan Leishmania tarentolae can be used as an easy-to-handle microfactory for the rapid production of viral antigens to face emerging epidemics. We engineered L. tarentolae to express the SARS-CoV-2 receptor-binding domain (RBD) and we recorded the ability of the purified RBD antigen to detect SARS-CoV-2 infection in human sera, 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. On the basis of our results, we propose L. tarentolae as an effective system for viral antigen production, even in countries that lack high-technology cell factories.

9.
J Immunol Methods ; 500: 113197, 2022 01.
Article in English | MEDLINE | ID: covidwho-1536657

ABSTRACT

Since the first detection of a novel Coronavirus (SARS-CoV-2) in December 2019 in Wuhan (China), it has become crucial to assess and quantize the human humoral immune response after SARS-CoV-2 natural infection and/or vaccination. Having well standardized and reliable serological assays able to accurately measure the total IgG antibodies response as well as the neutralization dynamics, play a pivotal role for the evaluation of "second" and "third" vaccines generation and in monitoring the effect in case of reinfection in the human population caused by the original strains or new SARS-CoV-2 variants. In the present study we reported that both symptomatic convalescent and vaccinated donors showed the presence of different levels of neutralizing antibodies. In addition, vaccinated subjects presented high levels of anti-S antibodies, whereas the complete absence of anti-N antibodies, whereas convalescent patients presented high levels of both anti-S and anti-N antibodies. The evaluation of the correlation between SARS-CoV-2 neutralizing and binding antibodies in convalescent and vaccinated subjects revealed that the IgG anti-S log-values were significantly higher in the vaccinated group respect to convalescent subjects. In addition, the level of binding antibodies recognizing the S protein shows a positive linear regression when compared to neutralizing titres in both the two groups evaluated.


Subject(s)
COVID-19 Serological Testing/methods , COVID-19 Vaccines/immunology , COVID-19/immunology , SARS Virus/physiology , SARS-CoV-2/genetics , Antibodies, Neutralizing/immunology , Antibodies, Neutralizing/metabolism , Antibodies, Viral/immunology , Antibodies, Viral/metabolism , COVID-19/diagnosis , Convalescence , Humans , Immunization, Secondary , Protein Binding , Vaccination
10.
Proc Natl Acad Sci U S A ; 118(36)2021 09 07.
Article in English | MEDLINE | ID: covidwho-1366850

ABSTRACT

To investigate the evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the immune population, we coincupi bated the authentic virus with a highly neutralizing plasma from a COVID-19 convalescent patient. The plasma fully neutralized the virus for seven passages, but, after 45 d, 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, South Africa, Brazil, and Japan 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)
Amino Acid Substitution , Angiotensin-Converting Enzyme 2/immunology , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/immunology , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/immunology , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/genetics , Animals , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/genetics , Antibodies, Neutralizing/pharmacology , Antibodies, Viral/chemistry , Antibodies, Viral/genetics , Antibodies, Viral/pharmacology , Binding Sites , COVID-19/genetics , COVID-19/virology , Chlorocebus aethiops , Convalescence , Gene Expression , Humans , Immune Evasion , Immune Sera/chemistry , Models, Molecular , Mutation , Neutralization Tests , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , SARS-CoV-2/drug effects , SARS-CoV-2/immunology , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Vero Cells
11.
J Virol Methods ; 297: 114261, 2021 11.
Article in English | MEDLINE | ID: covidwho-1356335

ABSTRACT

The neutralization assays are considered the gold-standard being capable of evaluating and detecting, functional antibodies. To date, many different protocols exist for micro-neutralization (MN) assay which varies in several steps: cell number and seeding conditions, virus amount used in the infection step, virus-serum-cells incubation time and read out. The aim of the present preliminary study was to carry out SARS-CoV-2 wild type 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 viral infective doses: the "standard" dose of 100 TCID50/well and a reduced dose of 25 TCID50/well. The results obtained, yielded by MN on using the lower infective dose (25 TCID50), were higher respect to those obtained with the standard infective dose. This suggests that the lower dose can potentially have a positive impact on the detection and estimation of real amount of neutralizing antibodies present in a given sample, showing higher sensitivity maintaining high specificity.


Subject(s)
COVID-19 , SARS-CoV-2 , Antibodies, Neutralizing , Antibodies, Viral , Humans , Neutralization Tests , Reproducibility of Results
12.
Viruses ; 13(7)2021 07 12.
Article in English | MEDLINE | ID: covidwho-1308455

ABSTRACT

The recent spreading of new SARS-CoV-2 variants, carrying several mutations in the spike protein, could impact immune protection elicited by natural infection or conferred by vaccination. In this study, we evaluated the neutralizing activity against the viral variants that emerged in the United Kingdom (B.1.1.7), Brazil (P.1), and South Africa (B.1.351) in human serum samples from hospitalized patients infected by SARS-CoV-2 during the first pandemic wave in Italy in 2020. Of the patients studied, 59.5% showed a decrease (≥2 fold) in neutralizing antibody titer against B.1.1.7, 83.3% against P.1, and 90.5% against B.1.351 with respect to the original strain. The reduction in antibody titers against all analyzed variants, and in particular P.1 and B.1.351, suggests that previous symptomatic infection might be not fully protective against exposure to SARS-CoV-2 variants carrying a set of relevant spike mutations.


Subject(s)
Antibodies, Neutralizing/pharmacology , COVID-19/drug therapy , COVID-19/immunology , SARS-CoV-2/drug effects , Animals , Antibodies, Neutralizing/blood , Antibodies, Neutralizing/immunology , Brazil/epidemiology , COVID-19/epidemiology , COVID-19/virology , COVID-19 Vaccines/administration & dosage , Chlorocebus aethiops , Mutation , Pandemics , SARS-CoV-2/genetics , SARS-CoV-2/immunology , South Africa/epidemiology , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , United Kingdom/epidemiology , Vaccination , Vero Cells
13.
PLoS One ; 16(7): e0253977, 2021.
Article in English | MEDLINE | ID: covidwho-1295521

ABSTRACT

SARS-CoV-2 pandemic is causing high morbidity and mortality burden worldwide with unprecedented strain on health care systems. To investigate the time course of the antibody response in relation to the outcome we performed a study in hospitalized COVID-19 patients. As comparison we also 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 antibody 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)
Antibodies, Neutralizing/biosynthesis , Antibodies, Viral/biosynthesis , Asymptomatic Infections , COVID-19/immunology , SARS-CoV-2/immunology , Aged , Antibodies, Neutralizing/blood , Antibodies, Neutralizing/immunology , Antibodies, Viral/blood , Antibodies, Viral/immunology , COVID-19/mortality , Comorbidity , Female , Hospitalization , Humans , Immunoglobulin A/biosynthesis , Immunoglobulin A/blood , Immunoglobulin A/immunology , Immunoglobulin G/biosynthesis , Immunoglobulin G/blood , Immunoglobulin G/immunology , Immunoglobulin M/biosynthesis , Immunoglobulin M/blood , Immunoglobulin M/immunology , Length of Stay , Male , Middle Aged , Patient Admission , Retrospective Studies
14.
BMJ Open ; 11(3): e046800, 2021 03 24.
Article in English | MEDLINE | ID: covidwho-1150238

ABSTRACT

OBJECTIVES: In Italy, the pandemic of COVID-19 resulted in congestion of hospitals and laboratories and probably determined an underestimation of the number of infected subjects, as the molecular diagnosis of SARS-CoV-2 infection was mainly performed on hospitalised patients. Therefore, limited data are available about the number of asymptomatic/paucisymptomatic subjects in the general population across time. To understand SARS-CoV-2 infection in the general population, we have developed a cross-sectional study (the 'UNIversity against CORoNavirus study') to investigate infection trends in asymptomatic/paucisymptomatic subjects in Milan (Italy), between March and June 2020. PARTICIPANTS: The study population included 2023 subjects asymptomatic at the enrolment. PRIMARY OUTCOME MEASURES: A nasal mid-turbinate swab for the detection of SARS-CoV-2 RNA and blood specimen for testing serum antibodies (immunoglobulin M (IgM) and IgG) were collected. RESULTS: Subjects showing positivity for the SARS-CoV-2 RNA and/or for anti-SARS-CoV-2 Ig is 237 (11.7%). Only 1.2% (n=25) of the total population had a positive nasal swab for SARS-CoV-2 and the large majority (21/25) of them were observed in March. A total of 226 subjects (11%) had IgM (n=19; 0.9%), IgG (n=155; 7.7%) or both (n=52; 2.6%) against SARS-CoV-2. Subjects with a present or past SARS-CoV-2 infection did not differ from other subjects as regards the number of cohabiting family members, travels, fever and upper and lower respiratory infection episodes. CONCLUSIONS: Results from the present study support the hypothesis that the actual spread of the virus in Lombardy was underestimated in the official records. However, as it is not known how long Ig persist, numbers should be taken cautiously.


Subject(s)
Antibodies, Viral/isolation & purification , COVID-19 Serological Testing , COVID-19/diagnosis , Immunoglobulin G/isolation & purification , Immunoglobulin M/isolation & purification , RNA, Viral/isolation & purification , Adult , COVID-19/blood , Cross-Sectional Studies , Female , Humans , Italy/epidemiology , Male , Middle Aged , SARS-CoV-2
15.
Cell ; 184(7): 1821-1835.e16, 2021 04 01.
Article in English | MEDLINE | ID: covidwho-1095899

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 coronavirus disease 2019 (COVID-19) pandemic. By single-cell sorting 4,277 SARS-CoV-2 spike protein-specific memory B cells from 14 COVID-19 survivors, 453 neutralizing antibodies were identified. The most potent neutralizing antibodies recognized the spike protein receptor-binding domain, followed in potency by antibodies that recognize the S1 domain, the spike protein trimer, and the S2 subunit. Only 1.4% of them neutralized the authentic virus with a potency of 1-10 ng/mL. The most potent monoclonal antibody, engineered to reduce the risk of antibody-dependent enhancement and prolong half-life, neutralized the authentic wild-type virus and emerging variants containing D614G, E484K, and N501Y substitutions. Prophylactic and therapeutic efficacy in the hamster model was observed at 0.25 and 4 mg/kg respectively in absence of Fc functions.


Subject(s)
Antibodies, Monoclonal/administration & dosage , Antibodies, Neutralizing/administration & dosage , Antibodies, Viral/administration & dosage , B-Lymphocytes/immunology , COVID-19 , Convalescence , 3T3 Cells , Animals , Antibodies, Monoclonal/isolation & purification , Antibodies, Neutralizing/isolation & purification , Antibodies, Viral/isolation & purification , B-Lymphocytes/cytology , COVID-19/immunology , COVID-19/prevention & control , COVID-19/therapy , Chlorocebus aethiops , Disease Models, Animal , Female , HEK293 Cells , Humans , Immunoglobulin Fc Fragments/immunology , Male , Mice , Spike Glycoprotein, Coronavirus/immunology , Vero Cells
16.
J Immunol Methods ; 489: 112937, 2021 02.
Article in English | MEDLINE | ID: covidwho-947285

ABSTRACT

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 49.7 million cases of disease and 1,2 million 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 detecting different classes of antibodies constitute an excellent surveillance strategy for gathering information on the humoral immune response to infection and the spread of the virus through the population. In addition, it can contribute 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 identify the level of SARS-CoV-2-specific IgM, IgG and IgA antibodies in order to predict human population immunity, possible cross-reactivity with other coronaviruses and to identify potentially infectious subjects. In addition, in a small sub-group of samples, a subtyping IgG ELISA has been performed. Our findings showed a notable statistical correlation between the neutralization titers and the IgG, IgM and IgA ELISA responses against the receptor-binding domain of the spike protein. Thus 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 that do not have biosecurity level-3 facilities.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/blood , COVID-19/blood , COVID-19/immunology , Immunoglobulin A/immunology , Immunoglobulin G/immunology , Immunoglobulin M/immunology , SARS-CoV-2/immunology , Animals , Antibodies, Neutralizing/blood , Antibodies, Viral/immunology , Cells, Cultured , Chlorocebus aethiops , Enzyme-Linked Immunosorbent Assay , Humans , Immunity, Humoral , Immunoglobulin A/blood , Immunoglobulin G/blood , Immunoglobulin M/blood , Vero Cells
17.
Tumori ; 107(5): 446-451, 2021 Oct.
Article in English | MEDLINE | ID: covidwho-920972

ABSTRACT

There are no robust data on the real onset of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and spread in the prepandemic period worldwide. We investigated the presence of SARS-CoV-2 receptor-binding domain (RBD)-specific antibodies in blood samples of 959 asymptomatic individuals enrolled in a prospective lung cancer screening trial between September 2019 and March 2020 to track the date of onset, frequency, and temporal and geographic variations across the Italian regions. SARS-CoV-2 RBD-specific antibodies were detected in 111 of 959 (11.6%) individuals, starting from September 2019 (14%), with a cluster of positive cases (>30%) in the second week of February 2020 and the highest number (53.2%) in Lombardy. This study shows an unexpected very early circulation of SARS-CoV-2 among asymptomatic individuals in Italy several months before the first patient was identified, and clarifies the onset and spread of the coronavirus disease 2019 (COVID-19) pandemic. Finding SARS-CoV-2 antibodies in asymptomatic people before the COVID-19 outbreak in Italy may reshape the history of pandemic.


Subject(s)
Antibodies, Viral/blood , COVID-19/epidemiology , SARS-CoV-2/immunology , Aged , Asymptomatic Infections , Female , Humans , Italy/epidemiology , Male , Middle Aged , Prospective Studies
18.
Viruses ; 12(9)2020 09 10.
Article in English | MEDLINE | ID: covidwho-769396

ABSTRACT

The recent outbreak of a novel Coronavirus (SARS-CoV-2) and its rapid spread across the continents has generated an urgent need for assays to detect the neutralising activity of human sera or human monoclonal antibodies against SARS-CoV-2 spike protein and to evaluate the serological immunity in humans. Since the accessibility of live virus microneutralisation (MN) assays with SARS-CoV-2 is limited and requires enhanced bio-containment, the approach based on "pseudotyping" can be considered a useful complement to other serological assays. After fully characterising lentiviral pseudotypes bearing the SARS-CoV-2 spike protein, we employed them in pseudotype-based neutralisation assays in order to profile the neutralising activity of human serum samples from an Italian sero-epidemiological study. The results obtained with pseudotype-based neutralisation assays mirrored those obtained when the same panel of sera was tested against the wild type virus, showing an evident convergence of the pseudotype-based neutralisation and MN results. The overall results lead to the conclusion that the pseudotype-based neutralisation assay is a valid alternative to using the wild-type strain, and although this system needs to be optimised and standardised, it can not only complement the classical serological methods, but also allows serological assessments to be made when other methods cannot be employed, especially in a human pandemic context.


Subject(s)
Betacoronavirus/genetics , Coronavirus Infections/virology , Lentivirus/genetics , Neutralization Tests/methods , Pandemics , Pneumonia, Viral/virology , Animals , Antibodies, Neutralizing , Antibodies, Viral/immunology , Betacoronavirus/immunology , COVID-19 , Cell Line , Coronavirus Infections/epidemiology , Humans , Immune Sera/immunology , Italy/epidemiology , Plasmids/genetics , Pneumonia, Viral/epidemiology , SARS-CoV-2 , Seroepidemiologic Studies , Spike Glycoprotein, Coronavirus/biosynthesis , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/physiology , Transfection , Vesiculovirus/genetics , Viral Load
19.
J Med Virol ; 92(10): 2096-2104, 2020 10.
Article in English | MEDLINE | ID: covidwho-379986

ABSTRACT

The micro-neutralization assay is a fundamental test in virology, immunology, vaccine assessment, and epidemiology studies. Since the SARS-CoV-2 outbreak at the end of December 2019 in China, it has become extremely important to have well-established and validated diagnostic and serological assays for this new emerging virus. Here, we present a micro-neutralization assay with the use of SARS-CoV-2 wild type virus with two different methods of read-out. We evaluated the performance of this assay using human serum samples taken from an Italian seroepidemiological study being performed at the University of Siena, along with the human monoclonal antibody CR3022 and some iper-immune animal serum samples against Influenza and Adenovirus strains. The same panel of human samples have been previously tested in enzyme-linked immunosorbent assay (ELISA) as a pre-screening. Positive, borderline, and negative ELISA samples were evaluated in neutralization assay using two different methods of read-out: subjective (by means of an inverted optical microscope) and objective (by means of a spectrophotometer). Our findings suggest that at least 50% of positive ELISA samples are positive in neutralization as well, and that method is able to quantify different antibody concentrations in a specific manner. Taken together, our results confirm that the colorimetric cytopathic effect-based microneutralization assay could be used as a valid clinical test method for epidemiological and vaccine studies.


Subject(s)
Antibodies, Neutralizing/blood , Antibodies, Viral/blood , COVID-19/diagnosis , Colorimetry/standards , Microscopy/standards , Neutralization Tests/standards , SARS-CoV-2/immunology , Animals , Antibodies, Monoclonal/analysis , COVID-19/immunology , COVID-19/virology , Cell Line, Tumor , Chlorocebus aethiops , Colorimetry/methods , Enzyme-Linked Immunosorbent Assay , Hepatocytes/immunology , Hepatocytes/virology , Humans , Immune Sera/chemistry , Microscopy/methods , Spectrophotometry , Vero Cells , Viral Load/immunology
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