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1.
Commun Med (Lond) ; 1: 38, 2021.
Article in English | MEDLINE | ID: covidwho-1860417

ABSTRACT

Background: The antibody response to SARS-CoV-2 mRNA vaccines in individuals with waning immunity generated by a previous SARS-CoV-2 infection, as well as the patterns of IgA and IgM responses in previously infected and in naïve individuals are still poorly understood. Methods: We performed a serology study in a cohort of BTN162b2 mRNA vaccine recipients who were immunologically naïve (N, n = 50) or had been previously infected with SARS-CoV-2 (P.I., n = 51) during the first (n = 25) or second (n = 26) pandemic waves in Italy, respectively. We measured IgG, IgM and IgA antibodies against the SARS-CoV-2 Spike (S) and IgG against the nucleocapsid (N) proteins, as well as the neutralizing activity of sera collected before vaccination, after the first and second dose of vaccine. Results: Most P.I. individuals from the first pandemic wave who showed declining antibody titres responded to the first vaccine dose with IgG-S and pseudovirus neutralization titres that were significantly higher than those observed in N individuals after the second vaccine dose. In all recipients, a single dose of vaccine was sufficient to induce a potent IgA response that was not associated with serum neutralization titres. We observed an unconventional pattern of IgM responses that were elicited in only half of immunologically naïve subjects even after the second vaccine dose. Conclusions: The response to a single dose of vaccine in P.I. individuals is more potent than that observed in N individuals after two doses. Vaccine-induced IgA are not associated with serum neutralization.

2.
EBioMedicine ; 77: 103888, 2022 Mar.
Article in English | MEDLINE | ID: covidwho-1701663

ABSTRACT

BACKGROUND: Currently, evaluation of the IgG antibodies specific for the SARS-CoV-2 Spike protein following vaccination is used worldwide to estimate vaccine response. Limited data are available on vaccine-elicited IgM antibodies and their potential implication in immunity to SARS-CoV-2. METHODS: We performed a longitudinal study to quantify anti-S SARS-CoV-2 IgG and IgM (IgG-S and IgM-S) in health care worker (HCW) recipients of the BNT162b2 vaccine. Samples were collected before administration (T0), at the second dose (T1) and three weeks after T1 (T2). The cohort included 1584 immunologically naïve to SARS-CoV-2 (IN) and 289 with history of previous infection (PI). FINDINGS: IN showed three patterns of responses: (a) IgG positive/IgM negative (36.1%), (b) coordinated IgM-S/IgG-S responses appearing at T1 (37.4%) and (c) IgM appearing after IgG (26.3%). Coordinated IgM-S/IgG-S responses were associated with higher IgG titres. In IgM-S positive PI, 64.5% were IgM-S positive before vaccination, whereas 32% and 3.5% developed IgM-S after the first and second vaccine dose, respectively. IgM-S positive sera had higher pseudovirus neutralization titres compared to the IgM-S negative. INTERPRETATION: Coordinated expression of IgG-S and IgM-S after vaccination was associated with a significantly more efficient response in both antibody levels and virus-neutralizing activity. The unconventional IgG-S positive/IgM-S negative responses may suggest a recruitment of cross coronaviruses immunity by vaccination, warranting further investigation. FUNDING: Italian Ministry of Health under "Fondi Ricerca Corrente"- L1P5 and "Progetto Ricerca Finalizzata COVID-2020-12371675"; FUR 2020 Department of Excellence 2018-2022, MIUR, Italy; The Brain Research Foundation Verona.


Subject(s)
COVID-19 , SARS-CoV-2 , Antibodies, Viral , COVID-19/prevention & control , COVID-19 Vaccines , Humans , Immunoglobulin M , Longitudinal Studies , Spike Glycoprotein, Coronavirus , Vaccination
3.
EuropePMC; 2021.
Preprint in English | EuropePMC | ID: ppcovidwho-309294

ABSTRACT

Background: The reference test for SARS-CoV-2 detection is the reverse transcriptase real time PCR (real time RT-PCR). However, evidences reported that real time RT-PCR has a lower sensitivity compared with the droplet digital PCR (ddPCR) leading to possible false negative in low viral load cases. Methods: We used ddPCR for viral genes N1 and N2 on 20 negative (no detection) samples from symptomatic hospitalized COVID-patients presenting fluctuating real time RT-PCR results and 10 suspected samples (Ct value>35) from asymptomatic not hospitalized subjects. Results: ddPCR performed on RNA revealed 65% of positivity for at least one viral target in the hospitalized patients group of samples (35% for N1 and N2, 10% only for N1 and 20% only for N2) and 50% in the suspected cases (30% for N1 and N2, while 20% only for N2). On hospitalized patients’samples, we applied also a direct ddPCR approach on the swab material, achieving an overall positivity of 83%. Conclusion: ddPCR, in particular the direct quantitation on swabs, shows a sensitivity advantage for the SARS-CoV-2 identification and may be useful to reduce the false negative diagnosis, especially for low viral load suspected samples.

4.
EuropePMC; 2020.
Preprint in English | EuropePMC | ID: ppcovidwho-309293

ABSTRACT

Droplet digital PCR (ddPCR) is a sensitive and reproducible technology widely used for quantitation of several different viruses. The aim of this study was to compare the 2019-nCoV CDC ddPCR Triplex Probe Assay (BioRad) performance on the direct quantitation of SARS-CoV-2 on nasopharyngeal swab respect to the procedure applied to the extracted RNA. Moreover, the two widely used swab types were compared (UTM 3mL and ESwab 1mL, COPAN). A total of 50 nasopharyngeal swabs (n=25 UTM 3mL and n=25 ESwab 1mL) from SARS-CoV-2 patients collected during the pandemic from IRCCS Sacro Cuore Don Calabria Hospital (Veneto Region, North-East Italy) were used for our purpose. After heat inactivation, an aliquot of swab medium was used in order to perform the direct quantitation. Then, we compared the direct method with the quantitation performed on the RNA purified from nasopharyngeal swab by automated extraction. We observed that the direct approach achieved generally equal RNA copies compared to the RNA extracted. The results with the direct quantitation were more accurate on ESwab with a sensitivity of 93.33% [95% CI, 68.05 to 99.83] and specificity of 100.00% for both N1 and N2. On the other hand, on UTM we observed a higher rate of discordant results for N1 and N2. The human internal amplification control (RPP30) showed 100% of both sensitivity and specificity independent of swabs and approaches.In conclusion, we described a simple and fast approach for the quantitation of SARS-CoV-2 in nasopharyngeal swab. Our approach resulted in an efficient quantitation, without automated RNA extraction and purification. However, special care needs to be taken on the potential bias due to the conservation of samples as we used thawed material and to the heating treatment. Further studies on a larger cohort of samples are warranted to evaluate the clinical value of this direct approach.

5.
EuropePMC; 2021.
Preprint in English | EuropePMC | ID: ppcovidwho-306877

ABSTRACT

Background: the devastating outbreak of COVID-19 poses serious challenges for the diagnostics laboratories, which are often facing global shortage of reagents and equipment. With the aim of increasing the diagnostic throughput for SARS-CoV-2 molecular test, the purpose of this study was to validate an additional RNA extraction method respect to those already recommended by WHO and the US Centers for Disease Control and Prevention (CDC). Methods: a new protocol for RNA extraction from nasopharyngeal swab was set up, adapting the Qiagen RNeasy 96 plate and validated on a set of 100 clinical samples analyzed in parallel by Roche-Magnapure method (already recommended by CDC guidelines). Results: the internal control and target genes analysis showed a good agreement between the two extraction methods indicating that the two methods can be considered equivalent and that the RNeasy-adapted method can be applied for the SARS-CoV-2 diagnostics. The addition of this new extraction method resulted in a throughput increase for SARS-CoV-2 molecular test of about 2000 samples/month during the initial months of the pandemic emergency in which the lack of reagents for the extraction led to an insufficient sample processing throughput of the analysis of the swabs.

6.
EuropePMC; 2021.
Preprint in English | EuropePMC | ID: ppcovidwho-325484

ABSTRACT

We profiled antibody responses in a cohort of recipients of the BTN162b2 mRNA vaccine who were either immunologically naïve (n=50) or had been previously infected with SARS-CoV-2 (n=51). Of the previously infected, 25 and 26 were infected during the first and second pandemic waves in Italy, respectively;the majority of those from the first wave had corresponding waning immunity with low to undetectable levels of anti-S antibodies and low anti-N antibodies. We observed in recipients who had been previously infected that spike-specific IgG and pseudovirus neutralization titers were rapidly recalled by a single vaccine dose to higher levels than those in naïve recipients after the second vaccine dose, irrespective of waning immunity. In all recipients, a single vaccine dose was sufficient to induce a potent IgA response that was not associated with serum neutralization titers.

7.
Front Immunol ; 13: 834851, 2022.
Article in English | MEDLINE | ID: covidwho-1686489

ABSTRACT

Understanding the cause of sex disparities in COVID-19 outcomes is a major challenge. We investigate sex hormone levels and their association with outcomes in COVID-19 patients, stratified by sex and age. This observational, retrospective, cohort study included 138 patients aged 18 years or older with COVID-19, hospitalized in Italy between February 1 and May 30, 2020. The association between sex hormones (testosterone, estradiol, progesterone, dehydroepiandrosterone) and outcomes (ARDS, severe COVID-19, in-hospital mortality) was explored in 120 patients aged 50 years and over. STROBE checklist was followed. The median age was 73.5 years [IQR 61, 82]; 55.8% were male. In older males, testosterone was lower if ARDS and severe COVID-19 were reported than if not (3.6 vs. 5.3 nmol/L, p =0.0378 and 3.7 vs. 8.5 nmol/L, p =0.0011, respectively). Deceased males had lower testosterone (2.4 vs. 4.8 nmol/L, p =0.0536) and higher estradiol than survivors (40 vs. 24 pg/mL, p = 0.0006). Testosterone was negatively associated with ARDS (OR 0.849 [95% CI 0.734, 0.982]), severe COVID-19 (OR 0.691 [95% CI 0.546, 0.874]), and in-hospital mortality (OR 0.742 [95% CI 0.566, 0.972]), regardless of potential confounders, though confirmed only in the regression model on males. Higher estradiol was associated with a higher probability of death (OR 1.051 [95% CI 1.018, 1.084]), confirmed in both sex models. In males, higher testosterone seems to be protective against any considered outcome. Higher estradiol was associated with a higher probability of death in both sexes.


Subject(s)
COVID-19/blood , Gonadal Steroid Hormones/blood , Sex Characteristics , Aged , Aged, 80 and over , Cohort Studies , Female , Hospital Mortality , Hospitalization , Humans , Italy , Male , Middle Aged , Retrospective Studies , Risk Factors , SARS-CoV-2
8.
J Transl Med ; 20(1): 22, 2022 01 08.
Article in English | MEDLINE | ID: covidwho-1637829

ABSTRACT

BACKGROUND: COVID-19 vaccines have demonstrated effectiveness in reducing SARS-CoV-2 mild and severe outcomes. In vaccinated subjects with SARS-CoV-2 history, RBD-specific IgG and pseudovirus neutralization titers were rapidly recalled by a single BTN162b2 vaccine dose to higher levels than those in naïve recipients after the second dose, irrespective of waning immunity. In this study, we inspected the long-term kinetic and neutralizing responses of S-specific IgG induced by two administrations of BTN162b2 vaccine in infection-naïve subjects and in subjects previously infected with SARS-CoV-2. METHODS: Twenty-six naïve and 9 previously SARS-CoV-2 infected subjects during the second wave of the pandemic in Italy were enrolled for this study. The two groups had comparable demographic and clinical characteristics. By means of ELISA and pseudotyped-neutralization assays, we investigated the kinetics of developed IgG-RBD and their neutralizing activity against both the ancestral D614G and the SARS-CoV-2 variants of concern emerged later, respectively. The Wilcoxon matched pair signed rank test and the Kruskal-Wallis test with Dunn's correction for multiple comparison were applied when needed. RESULTS: Although after 15 weeks from vaccination IgG-RBD dropped in all participants, naïve subjects experienced a more dramatic decline than those with previous SARS-CoV-2 infection. Neutralizing antibodies remained higher in subjects with SARS-CoV-2 history and conferred broad-spectrum protection. CONCLUSIONS: These data suggest that hybrid immunity to SARS-CoV-2 has a relevant impact on the development of IgG-RBD upon vaccination. However, the rapid decay of vaccination-elicited antibodies highlights that the administration of a third dose is expected to boost the response and acquire high levels of cross-neutralizing antibodies.


Subject(s)
Antibody Formation , COVID-19 , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/immunology , Humans , SARS-CoV-2 , Vaccination
10.
Front Immunol ; 12: 789735, 2021.
Article in English | MEDLINE | ID: covidwho-1581322

ABSTRACT

Background: The host immune response has a prominent role in the progression and outcome of SARS-CoV-2 infection. Lymphopenia has been described as an important feature of SARS-CoV-2 infection and has been associated with severe disease manifestation. Lymphocyte dysregulation and hyper-inflammation have been shown to be associated with a more severe clinical course; however, a T cell subpopulation whose dysfunction correlate with disease progression has yet to be identify. Methods: We performed an immuno-phenotypic analysis of T cell sub-populations in peripheral blood from patients affected by different severity of COVID-19 (n=60) and undergoing a different clinical evolution. Clinical severity was established based on a modified WHO score considering both ventilation support and respiratory capacity (PaO2/FiO2 ratio). The ability of circulating cells at baseline to predict the probability of clinical aggravation was explored through multivariate regression analyses. Results: The immuno-phenotypic analysis performed by multi-colour flow cytometry confirmed that patients suffering from severe COVID-19 harboured significantly reduced circulating T cell subsets, especially for CD4+ T, Th1, and regulatory T cells. Peripheral T cells also correlated with parameters associated with disease severity, i.e., PaO2/FiO2 ratio and inflammation markers. CD4+ T cell subsets showed an important significant association with clinical evolution, with patients presenting markedly decreased regulatory T cells at baseline having a significantly higher risk of aggravation. Importantly, the combination of gender and regulatory T cells allowed distinguishing between improved and worsened patients with an area under the ROC curve (AUC) of 82%. Conclusions: The present study demonstrates the association between CD4+ T cell dysregulation and COVID-19 severity and progression. Our results support the importance of analysing baseline regulatory T cell levels, since they were revealed able to predict the clinical worsening during hospitalization. Regulatory T cells assessment soon after hospital admission could thus allow a better clinical stratification and patient management.


Subject(s)
COVID-19/epidemiology , COVID-19/immunology , Hospitalization , Lymphocyte Count , SARS-CoV-2/immunology , T-Lymphocytes, Regulatory/immunology , Biomarkers , COVID-19/diagnosis , COVID-19/virology , COVID-19 Serological Testing , Cytokines/blood , Cytokines/metabolism , Disease Progression , Humans , Immunophenotyping , Inflammation Mediators/blood , Inflammation Mediators/metabolism , Prognosis , Public Health Surveillance , ROC Curve , Severity of Illness Index , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , T-Lymphocytes, Regulatory/metabolism
11.
SSRN; 2021.
Preprint in English | SSRN | ID: ppcovidwho-292038

ABSTRACT

Background: IgG antibodies specific for the SARS-CoV-2 Spike protein are under intensive investigation for the urgent need to identify a correlate of protective immunity in individuals vaccinated with licensed and candidate COVID-19 vaccines. Limited data are available on vaccine-elicited IgM antibodies and their potential implication in immunity to SARS-CoV-2. Methods: We performed a longitudinal study to quantify IgG and IgM antibodies specific for the SARS-CoV-2 spike protein (IgG-S and IgM-S) in 1873 health care worker (HCW) recipients of the BNT162b2 (Comirnaty) vaccine. Samples were collected before administration (T0), at the second dose (T1) and three weeks after the second dose (T2). The cohort included 1584 immunologically naïve to SARS-CoV-2 (IN) and 289 had a history of previous infection (PI). Findings: In IN we identified three patterns of responses: (a) IgG positive/IgM negative (36.1%), (b) coordinated IgM-S/IgG-S responses appearing three weeks after the first dose (37.4%) and (c) delayed IgM appearing after IgG (26.3%). Coordinated IgM-S/IgG-S responses were associated with higher IgG titers. Of the 289 PI vaccinees, 64.5% were IgM-S positive before vaccination, whereas 32% and 3.5% developed IgM-S after the first and second vaccine dose respectively. IgM-S positive sera had higher pseudovirus neutralization titers compared to the IgM-S negative. Similar results were observed in individuals who received either Moderna or Astrazeneca. Interpretation: Coordinated expression of IgG-S and IgM-S after vaccination was associated with a significantly more efficient response in both antibody titers and virus-neutralizing activity, representing a potential correlate of protection. Instead, the unconventional IgG-S positive/IgM-S negative responses may be suggestive of a recruitment of cross coronaviruses immunity by vaccination, warranting further investigation. Funding Information: This work was supported by the Italian Ministry of Health under “Fondi Ricerca Corrente”- L1P5 and “Progetto Ricerca Finalizzata COVID-2020-12371675” to IRCCS Sacro Cuore Don Calabria Hospital, by FUR 2020 Department of Excellence 2018-2022, MIUR, Italy and by The Brain Research Foundation Verona.

12.
SSRN; 2021.
Preprint in English | SSRN | ID: ppcovidwho-291897

ABSTRACT

Background: Understanding the cause of sex disparities in COVID-19 outcomes is a major challenge. We investigate sex hormone levels and their association with outcomes in COVID-19 patients, stratified by sex and age. Methods: This observational, retrospective, cohort study included 138 patients aged 18 years or older with COVID-19, hospitalized in Italy between February 1 and May 30, 2020. The association between sex hormones (testosterone, estradiol, progesterone, dehydroepiandrosterone) and outcomes (ARDS, severe COVID-19, in-hospital mortality) was explored in 120 patients aged 50 years and over. STROBE checklist was followed. Findings: The median age was 73·5 years [IQR 61, 82];55·8% were male. In older males, testosterone was lower if ARDS and severe COVID-19 were reported than if not (3·6 vs. 5·3 nmol/L, p =0·0378 and 3·7 vs. 8·5 nmol/L, p =0·0011, respectively). Deceased males had lower testosterone (2·4 vs. 4·8 nmol/L, p =0·0536) and higher estradiol than survivors (40 vs. 24 pg/mL, p = 0·0006). Testosterone was negatively associated with ARDS (OR 0·849 [95% CI 0·734, 0·982]), severe COVID-19 (OR 0·691 [95% CI 0·546, 0·874]), and in-hospital mortality (OR 0·742 [95% CI 0·566, 0·972]), regardless of potential confounders, though confirmed only in the regression model on males. Higher estradiol was associated with a higher probability of death (OR 1·051 [95% CI 1·018, 1.084]), confirmed in both sex models. Interpretation: In males, higher testosterone seems to be protective against any considered outcome. Higher estradiol was associated with a higher probability of death in both sexes.

13.
Front Med (Lausanne) ; 8: 745665, 2021.
Article in English | MEDLINE | ID: covidwho-1497091

ABSTRACT

Following the SARS-CoV-2 pandemic, several clinical trials have been approved for the investigation of the possible use of mAbs, supporting the potential of this technology as a therapeutic approach for infectious diseases. The first monoclonal antibody (mAb), Muromonab CD3, was introduced for the prevention of kidney transplant rejection more than 30 years ago; since then more than 100 mAbs have been approved for therapeutic purposes. Nonetheless, only four mAbs are currently employed for infectious diseases: Palivizumab, for the prevention of respiratory syncytial virus (RSV) infections, Raxibacumab and Obiltoxaximab, for the prophylaxis and treatment against anthrax toxin and Bezlotoxumab, for the prevention of Clostridium difficile recurrence. Protozoan infections are often neglected diseases for which effective and safe chemotherapies are generally missing. In this context, drug resistance and drug toxicity are two crucial problems. The recent advances in bioinformatics, parasite genomics, and biochemistry methodologies are contributing to better understand parasite biology, which is essential to guide the development of new therapies. In this review, we present the efforts that are being made in the evaluation of mAbs for the prevention or treatment of leishmaniasis, Chagas disease, malaria, and toxoplasmosis. Particular emphasis will be placed on the potential strengths and weaknesses of biological treatments in the control of these protozoan diseases that are still affecting hundreds of thousands of people worldwide.

14.
J Med Virol ; 2021 Oct 07.
Article in English | MEDLINE | ID: covidwho-1453611

ABSTRACT

We assessed the performance of the Panbio rapid antigen detection (RAD) test for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and we compared it with the routine reverse transcriptase-polymerase chain reaction (RT-PCR)-based molecular test in a population of 4167 unselected patients admitted to IRCCS Sacro Cuore Don Calabria Hospital. Analysis stratified by cycling threshold (Ct ) value of SARS-CoV-2 gene targets indicated that antigen (Ag)-positive Ct values were significantly lower compared to Ag-negative values (p < 0.0001). Overall, we found discordance in 140, tested negative by RAD and positive by RT-PCR, and in 4 resulted positive by RAD and negative by RT-PCR. RAD test achieved a sensitivity and specificity of 66.82% and 99.89%, respectively. The positive predictive value was shown to be 97.87% while the negative predictive value was shown to be 97.62%. In our context, the RAD test showed a reliable diagnostic response in subjects that displayed high Ct values, corresponding to high viral load, while low ability was displayed to identify positive cases with medium-low Ct values, thus presenting low viral load and where confirmatory RT-PCR was needed. Our finding supports the use of the RAD test in real-life settings where a high volume of swabs is being processed but with caution when interpreting a positive test result in a low prevalence setting.

15.
Genomics ; 113(4): 1628-1638, 2021 07.
Article in English | MEDLINE | ID: covidwho-1386752

ABSTRACT

Sequencing the SARS-CoV-2 genome from clinical samples can be challenging, especially in specimens with low viral titer. Here we report Accurate SARS-CoV-2 genome Reconstruction (ACoRE), an amplicon-based viral genome sequencing workflow for the complete and accurate reconstruction of SARS-CoV-2 sequences from clinical samples, including suboptimal ones that would usually be excluded even if unique and irreplaceable. The protocol was optimized to improve flexibility and the combination of technical replicates was established as the central strategy to achieve accurate analysis of low-titer/suboptimal samples. We demonstrated the utility of the approach by achieving complete genome reconstruction and the identification of false-positive variants in >170 clinical samples, thus avoiding the generation of inaccurate and/or incomplete sequences. Most importantly, ACoRE was crucial to identify the correct viral strain responsible of a relapse case, that would be otherwise mis-classified as a re-infection due to missing or incorrect variant identification by a standard workflow.


Subject(s)
COVID-19/genetics , Genome, Viral/genetics , Reinfection/genetics , SARS-CoV-2/genetics , COVID-19/pathology , COVID-19/virology , Genetic Variation/genetics , Humans , Reinfection/pathology , Reinfection/virology , SARS-CoV-2/pathogenicity , Whole Genome Sequencing
16.
Microorganisms ; 9(8)2021 Aug 14.
Article in English | MEDLINE | ID: covidwho-1355014

ABSTRACT

SARS-CoV-2 infection was monitored in 1898 health care workers (HCWs) after receiving full vaccination with BNT162b2. Untill 30 June 2021, 10 HCWs tested positive for SARS-CoV-2 using real time RT-PCR, resulting in a 4-month cumulative incidence of 0.005%. The infection was mildly symptomatic in six (60%) and asymptomatic in four (40%) individuals. Among the infected HCWs, eight consenting individuals provided paired NPS and saliva during the course of infection, for the purpose of the analysis performed in the present study. Genomic and subgenomic viral RNAs were investigated using real-time RT-PCR in both biological specimens. The temporal profile of viral load was measured using ddPCR. Viral mutations were also analysed. Subgenomic viral RNA was detected in 8/8 (100%) NPS and in 6/8 (75%) saliva specimens at the baseline. The expression of subgenomic RNA was observed for up to 7 days in 3/8 (38%) symptomatic cases. Moreover, concordance was observed between NPS and saliva in the detection of viral mutations, and both N501Y and 69/70del (associated with the B.1.1.7 variant) were detected in the majority 6/8 (75%) of subjects, while the K417T mutation (associated with the P.1-type variants) was detected in 2/8 (25%) individuals. Overall, our findings report a low frequency of infected HCWs after full vaccination. It is, therefore, important to monitor the vaccinees in order to identify asymptomatic infected individuals. Saliva can be a surrogate for SARS-CoV-2 surveillance, particularly in social settings such as hospitals.

17.
Clin Microbiol Infect ; 27(12): 1845-1850, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1330709

ABSTRACT

OBJECTIVES: To assess the antibody response to BNT162b2 mRNA COVID-19 vaccine in a cohort of health-care workers (HCW), comparing individuals with previous severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and SARS-CoV-2-naive individuals. METHODS: HCW were tested at T0 (day of first dose), T1 (day of second dose) and T2 (2-3 weeks after second dose) for IgG anti-nucleocapsid protein, IgM anti-spike protein and IgG anti-receptor binding domain (IgG-RBD-S). The antibody response was compared between four main groups: group A, individuals with previous infection and positive antibodies at baseline; group B, individuals with the same history but negative antibodies; group C, individuals with no infection history but positive antibodies; group D, naive individuals. Repeated measures analysis was used to compare results over time-points. RESULTS: A total of 1935 HCW were included. Median IgG-RBD-S titre was significantly higher for group A (232 individuals) than for group B (56 individuals) both at T1 (A: 22 763 AU/mL, interquartile range (IQR) 14 222-37 204 AU/mL; B: 1373 AU/mL, IQR 783-3078 AU/mL, p 0.0003) and T2 (A: 30 765 AU/mL, IQR 19 841-42 813 AU/mL; B: 13 171 AU/mL, IQR 2324-22 688 AU/mL, p 0.0038) and for group D (1563 individuals; 796 AU/mL, IQR 379-1510 AU/mL at T1; 15 494 AU/mL, IQR 9122-23 916 AU/mL at T2, p < 0.0001 for both time-points). T1 values of group A were also significantly higher than T2 values of group D (p < 0.0001). Presence of symptoms, younger age and being female were associated with stronger antibody response. HCW infected in March showed a significantly stronger response (T1: 35 324 AU/mL, IQR 22 003-44 531 AU/mL; T2: 37 648 AU/mL, IQR 27 088-50 451 AU/mL) than those infected in November (T1: 18 499 AU/mL, IQR 11 492-27 283 AU/mL; T2: 23 210 AU/mL, IQR 18 074-36 086 AU/mL, p < 0.0001 for both time-points. CONCLUSIONS: Individuals with past SARS-CoV-2 infection had a strong antibody response after one single vaccine shot. A single dose might be sufficient for this group, regardless of the time elapsed since infection; however, the clinical correlation with antibody response needs to be studied.


Subject(s)
Antibodies, Viral/blood , Antibody Formation , COVID-19 , COVID-19/prevention & control , Health Personnel , Humans , Immunoglobulin G/blood , Prospective Studies , RNA, Messenger
18.
Viruses ; 13(3)2021 03 05.
Article in English | MEDLINE | ID: covidwho-1167750

ABSTRACT

Although antibody levels progressively decrease following SARS-CoV-2 infection, the immune memory persists for months. Thus, individuals who naturally contracted SARS-CoV-2 are expected to develop a more rapid and sustained response to COVID-19 vaccines than naïve individuals. In this study, we analyzed the dynamics of the antibody response to the BNT162b2 mRNA COVID-19 vaccine in six healthcare workers who contracted SARS-CoV-2 in March 2020, in comparison to nine control subjects without a previous infection. The vaccine was well tolerated by both groups, with no significant difference in the frequency of vaccine-associated side effects, with the exception of local pain, which was more common in previously infected subjects. Overall, the titers of neutralizing antibodies were markedly higher in response to the vaccine than after natural infection. In all subjects with pre-existing immunity, a rapid increase in anti-spike receptor-binding domain (RBD) IgG antibodies and neutralizing antibody titers was observed one week after the first dose, which seemed to act as a booster. Notably, in previously infected individuals, neutralizing antibody titers 7 days after the first vaccine dose were not significantly different from those observed in naïve subjects 7 days after the second vaccine dose. These results suggest that, in previously infected people, a single dose of the vaccine might be sufficient to induce an effective response.


Subject(s)
Antibodies, Viral/immunology , COVID-19 Vaccines/immunology , COVID-19/immunology , RNA, Messenger/immunology , RNA, Viral/immunology , SARS-CoV-2/immunology , Adult , Antibody Formation , COVID-19/prevention & control , COVID-19/virology , COVID-19 Vaccines/administration & dosage , COVID-19 Vaccines/genetics , Female , Humans , Immunoglobulin G/immunology , Male , Middle Aged , RNA, Messenger/administration & dosage , RNA, Messenger/genetics , RNA, Viral/administration & dosage , RNA, Viral/genetics , SARS-CoV-2/genetics
19.
Vaccines (Basel) ; 9(1)2021 Jan 11.
Article in English | MEDLINE | ID: covidwho-1022025

ABSTRACT

A workflow for rapid SARS-CoV-2 epitope discovery on peptide microarrays is herein reported. The process started with a proteome-wide screening of immunoreactivity based on the use of a high-density microarray followed by a refinement and validation phase on a restricted panel of probes using microarrays with tailored peptide immobilization through a click-based strategy. Progressively larger, independent cohorts of Covid-19 positive sera were tested in the refinement processes, leading to the identification of immunodominant regions on SARS-CoV-2 spike (S), nucleocapsid (N) protein and Orf1ab polyprotein. A summary study testing 50 serum samples highlighted an epitope of the N protein (region 155-71) providing good diagnostic performance in discriminating Covid-19 positive vs. healthy individuals. Using this epitope, 92% sensitivity and 100% specificity were reached for IgG detection in Covid-19 samples, and no cross-reactivity with common cold coronaviruses was detected. Likewise, IgM immunoreactivity in samples collected within the first month after symptoms onset showed discrimination ability. Overall, epitope 155-171 from N protein represents a promising candidate for further development and rapid implementation in serological tests.

20.
Sci Rep ; 10(1): 18764, 2020 10 30.
Article in English | MEDLINE | ID: covidwho-894422

ABSTRACT

Droplet digital PCR (ddPCR) is a sensitive and reproducible technology widely used for quantitation of several viruses. The aim of this study was to evaluate the 2019-nCoV CDC ddPCR Triplex Probe Assay (BioRad) performance, comparing the direct quantitation of SARS-CoV-2 on nasopharyngeal swab with the procedure applied to the extracted RNA. Moreover, two widely used swab types were compared (UTM 3 mL and ESwab 1 mL, COPAN). A total of 50 nasopharyngeal swabs (n = 25 UTM 3 mL and n = 25 ESwab 1 mL) from SARS-CoV-2 patients, collected during the pandemic at IRCCS Sacro Cuore Don Calabria Hospital (Veneto Region, North-East Italy), were used for our purpose. After heat inactivation, an aliquot of swab medium was used for the direct quantitation. Then, we compared the direct method with the quantitation performed on the RNA purified from nasopharyngeal swab by automated extraction. We observed that the direct approach achieved generally equal RNA copies compared to the extracted RNA. The results with the direct quantitation were more accurate on ESwab with a sensitivity of 93.33% [95% CI, 68.05 to 99.83] and specificity of 100.00% for both N1 and N2. On the other hand, on UTM we observed a higher rate of discordant results for N1 and N2. The human internal amplification control (RPP30) showed 100% of both sensitivity and specificity independent of swabs and approaches. In conclusion, we described a direct quantitation of SARS-CoV-2 in nasopharyngeal swab. Our approach resulted in an efficient quantitation, without automated RNA extraction and purification. However, special care needs to be taken on the potential bias due to the conservation of samples and to the heating treatment, as we used thawed and heat inactivated material. Further studies on a larger cohort of samples are warranted to evaluate the clinical value of this direct approach.


Subject(s)
Clinical Laboratory Techniques/methods , Multiplex Polymerase Chain Reaction/methods , Betacoronavirus/genetics , Betacoronavirus/pathogenicity , COVID-19 Testing , Clinical Laboratory Techniques/standards , Coronavirus Infections/diagnosis , Humans , Limit of Detection , Multiplex Polymerase Chain Reaction/standards , Reproducibility of Results , Respiratory Mucosa/virology , SARS-CoV-2
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