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1.
PLoS One ; 17(2): e0263419, 2022.
Article in English | MEDLINE | ID: covidwho-1674014

ABSTRACT

Mucosal immunity plays a crucial role in controlling upper respiratory infections, including influenza. We established a quantitative ELISA to measure the amount of influenza virus-specific salivery IgA (sIgA) and salivary IgG (sIgG) antibodies using a standard antibody broadly reactive to the influenza A virus. We then analyzed saliva and serum samples from seven individuals infected with the A(H1N1)pdm09 influenza virus during the 2019-2020 flu seasons. We detected an early (6-10 days post-infection) increase of sIgA in five of the seven samples and a later (3-5 weeks) increase of sIgG in six of the seven saliva samples. Although the conventional parenteral influenza vaccine did not induce IgA production in saliva, vaccinated individuals with a history of influenza infection had higher basal levels of sIgA than those without a history. Interestingly, we observed sIgA and sIgG in an asymptomatic individual who had close contact with two influenza cases. Both early mucosal sIgA secretion and late systemically induced sIgG in the mucosal surface may protect against virus infection. Despite the small sample size, our results indicate that the saliva test system can be useful for analyzing upper mucosal immunity in influenza.


Subject(s)
Immunity, Mucosal/physiology , Influenza, Human/immunology , Saliva/immunology , Adult , Aged , Antibodies, Viral/analysis , Antibodies, Viral/metabolism , Antibody Formation , Cohort Studies , Female , History, 21st Century , Humans , Immunoglobulin A/analysis , Immunoglobulin A/metabolism , Immunoglobulin A, Secretory/analysis , Immunoglobulin A, Secretory/metabolism , Immunoglobulin G/analysis , Immunoglobulin G/metabolism , Influenza A Virus, H1N1 Subtype/immunology , Influenza Vaccines/therapeutic use , Influenza, Human/diagnosis , Influenza, Human/prevention & control , Japan , Longitudinal Studies , Male , Predictive Value of Tests , Prognosis , Saliva/chemistry , Saliva/metabolism , Young Adult
2.
J Immunol ; 208(5): 1001-1005, 2022 03 01.
Article in English | MEDLINE | ID: covidwho-1674946

ABSTRACT

Advanced age is a main risk factor for severe COVID-19. However, low vaccination efficacy and accelerated waning immunity have been reported in this age group. To elucidate age-related differences in immunogenicity, we analyzed human cellular, serological, and salivary SARS-CoV-2 spike glycoprotein-specific immune responses to the BNT162b2 COVID-19 vaccine in old (69-92 y) and middle-aged (24-57 y) vaccinees compared with natural infection (COVID-19 convalescents, 21-55 y of age). Serological humoral responses to vaccination excee-ded those of convalescents, but salivary anti-spike subunit 1 (S1) IgA and neutralizing capacity were less durable in vaccinees. In old vaccinees, we observed that pre-existing spike-specific CD4+ T cells are associated with efficient induction of anti-S1 IgG and neutralizing capacity in serum but not saliva. Our results suggest pre-existing SARS-CoV-2 cross-reactive CD4+ T cells as a predictor of an efficient COVID-19 vaccine-induced humoral immune response in old individuals.


Subject(s)
Aging/immunology , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , CD4-Positive T-Lymphocytes/immunology , SARS-CoV-2/immunology , Adult , Age Factors , Aged , Aged, 80 and over , COVID-19/immunology , Female , Humans , Immunoglobulin A/blood , Immunoglobulin A/immunology , Immunoglobulin G/blood , Immunoglobulin G/immunology , Male , Middle Aged , Nursing Homes , Saliva/immunology , Spike Glycoprotein, Coronavirus/immunology , Vaccination , Young Adult
3.
J Am Geriatr Soc ; 70(3): 659-668, 2022 03.
Article in English | MEDLINE | ID: covidwho-1626892

ABSTRACT

BACKGROUND: SARS-CoV-2 circulating variants coupled with waning immunity pose a significant threat to the long-term care (LTC) population. Our objective was to measure salivary IgG antibodies in residents and staff of an LTC facility to (1) evaluate IgG response in saliva post-natural infection and vaccination and (2) assess its feasibility to describe the seroprevalence over time. METHODS: We performed salivary IgG sampling of all residents and staff who agreed to test in a 150-bed skilled nursing facility during three seroprevalence surveys between October 2020 and February 2021. The facility had SARS-CoV-2 outbreaks in May 2020 and November 2020, when 45 of 138 and 37 of 125 residents were infected, respectively; they offered two Federal vaccine clinics in January 2021. We evaluated quantitative IgG in saliva to the Nucleocapsid (N), Spike (S), and Receptor-binding domain (RBD) Antigens of SARS-CoV-2 over time post-infection and post-vaccination. RESULTS: One hundred twenty-four residents and 28 staff underwent saliva serologic testing on one or more survey visits. Over three surveys, the SARS-CoV-2 seroprevalence at the facility was 49%, 64%, and 81%, respectively. IgG to S, RBD, and N Antigens all increased post infection. Post vaccination, the infection naïve group did not have a detectable N IgG level, and N IgG levels for the previously infected did not increase post vaccination (p < 0.001). Fully vaccinated subjects with prior COVID-19 infection had significantly higher RBD and S IgG responses compared with those who were infection-naïve prior to vaccination (p < 0.001 for both). CONCLUSIONS: Positive SARS-COV-2 IgG in saliva was concordant with prior infection (Anti N, S, RBD) and vaccination (Anti S, RBD) and remained above positivity threshold for up to 9 months from infection. Salivary sampling is a non-invasive method of tracking immunity and differentiating between prior infection and vaccination to inform the need for boosters in LTC residents and staff.


Subject(s)
Antibodies, Viral/immunology , COVID-19 Vaccines/immunology , COVID-19/immunology , COVID-19/prevention & control , Immunoglobulin G/immunology , Saliva/immunology , Aged , COVID-19/epidemiology , COVID-19 Vaccines/administration & dosage , Female , Humans , Male , Nursing Homes , SARS-CoV-2 , Seroepidemiologic Studies , United States/epidemiology
4.
Front Immunol ; 12: 759688, 2021.
Article in English | MEDLINE | ID: covidwho-1605844

ABSTRACT

Antibodies (Abs) are essential for the host immune response against SARS-CoV-2, and all the vaccines developed so far have been designed to induce Abs targeting the SARS-CoV-2 spike. Many studies have examined Ab responses in the blood from vaccinated and infected individuals. However, since SARS-CoV-2 is a respiratory virus, it is also critical to understand the mucosal Ab responses at the sites of initial virus exposure. Here, we examined plasma versus saliva Ab responses in vaccinated and convalescent patients. Although saliva levels were significantly lower, a strong correlation was observed between plasma and saliva total Ig levels against all SARS-CoV-2 antigens tested. Virus-specific IgG1 responses predominated in both saliva and plasma, while a lower prevalence of IgM and IgA1 Abs was observed in saliva. Antiviral activities of plasma Abs were also studied. Neutralization titers against the initial WA1 (D614G), B.1.1.7 (alpha) and B.1.617.2 (delta) strains were similar but lower against the B.1.351 (beta) strain. Spike-specific antibody-dependent cellular phagocytosis (ADCP) activities were also detected and the levels correlated with spike-binding Ig titers. Interestingly, while neutralization and ADCP potencies of vaccinated and convalescent groups were comparable, enhanced complement deposition to spike-specific Abs was noted in vaccinated versus convalescent groups and corresponded with higher levels of IgG1 plus IgG3 among the vaccinated individuals. Altogether, this study demonstrates the detection of Ab responses after vaccination or infection in plasma and saliva that correlate significantly, although Ig isotypic differences were noted. The induced plasma Abs displayed Fab-mediated and Fc-dependent functions with comparable neutralization and ADCP potencies, but a greater capacity to activate complement was elicited upon vaccination.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19 Vaccines/immunology , COVID-19/immunology , SARS-CoV-2/immunology , Saliva/immunology , Spike Glycoprotein, Coronavirus/immunology , Adult , Aged , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , Antibody Formation/immunology , COVID-19/blood , COVID-19/virology , Female , Humans , Immunoglobulin G/immunology , Male , Middle Aged , SARS-CoV-2/metabolism , SARS-CoV-2/physiology , Saliva/virology , Vaccination
5.
Front Immunol ; 12: 777858, 2021.
Article in English | MEDLINE | ID: covidwho-1581332

ABSTRACT

Background: Developing an understanding of the antibody response, seroprevalence, and seroconversion from natural infection and vaccination against SARS-CoV-2 will give way to a critical epidemiological tool to predict reinfection rates, identify vulnerable communities, and manage future viral outbreaks. To monitor the antibody response on a larger scale, we need an inexpensive, less invasive, and high throughput method. Methods: Here we investigate the use of oral mucosal fluids from individuals recovered from SARS-CoV-2 infection to monitor antibody response and persistence over a 12-month period. For this cohort study, enzyme-linked immunosorbent assays (ELISAs) were used to quantify anti-Spike(S) protein IgG antibodies in participants who had prior SARS-CoV-2 infection and regularly (every 2-4 weeks) provided both serum and oral fluid mucosal fluid samples for longitudinal antibody titer analysis. Results: In our study cohort (n=42) with 17 males and 25 females with an average age of 45.6 +/- 19.3 years, we observed no significant change in oral mucosal fluid IgG levels across the time course of antibody monitoring. In oral mucosal fluids, all the participants who initially had detectable antibodies continued to have detectable antibodies throughout the study. Conclusions: Based on the results presented here, we have shown that oral mucosal fluid-based assays are an effective, less invasive tool for monitoring seroprevalence and seroconversion, which offers an alternative to serum-based assays for understanding the protective ability conferred by the adaptive immune response from viral infection and vaccination against future reinfections.


Subject(s)
Antibodies, Viral/immunology , COVID-19/immunology , Immunoglobulin G/immunology , Saliva/immunology , Adult , Aged , Aged, 80 and over , Cohort Studies , Female , Humans , Longitudinal Studies , Male , Middle Aged , Mouth Mucosa/immunology , SARS-CoV-2 , Seroconversion , Spike Glycoprotein, Coronavirus/immunology
6.
Front Immunol ; 12: 798859, 2021.
Article in English | MEDLINE | ID: covidwho-1581315

ABSTRACT

SARS-CoV-2 antibodies in saliva serve as first line of defense against the virus. They are present in the mucosa, more precisely in saliva, after a recovered infection and also following vaccination. We report here the antibody persistence in plasma and in saliva up to 15 months after mild COVID-19. The IgG antibody response was measured every two months in 72 participants using an established and validated in-house ELISA assay. In addition, the virus inhibitory activity of plasma antibodies was assessed in a surrogate virus neutralization test before and after vaccination. SARS-CoV-2-specific antibody concentrations remained stable in plasma and saliva and the response was strongly boosted after one dose COVID-19 vaccination.


Subject(s)
Antibodies, Viral/immunology , COVID-19 Vaccines/immunology , COVID-19/immunology , Immunoglobulin G/immunology , Saliva/immunology , Adult , Aged , Aged, 80 and over , Antibodies, Neutralizing/immunology , Female , Humans , Male , Middle Aged , SARS-CoV-2
7.
EBioMedicine ; 75: 103788, 2022 Jan.
Article in English | MEDLINE | ID: covidwho-1587926

ABSTRACT

BACKGROUND: Although the BNT162b2 COVID-19 vaccine is known to induce IgG neutralizing antibodies in serum protecting against COVID-19, it has not been studied in detail whether it could generate specific immunity at mucosal sites, which represent the primary route of entry of SARS-CoV-2. METHODS: Samples of serum and saliva of 60 BNT162b2-vaccinated healthcare workers were collected at baseline, two weeks after the first dose and two weeks after the second dose. Anti-S1-protein IgG and IgA total antibodies titres and the presence of neutralizing antibodies against the Receptor Binding Domain in both serum and saliva were measured by quantitative and by competitive ELISA, respectively. FINDINGS: Complete vaccination cycle generates a high serum IgG antibody titre as a single dose in previously infected seropositive individuals. Serum IgA concentration reaches a plateau after a single dose in seropositive individuals and two vaccine doses in seronegative subjects. After the second dose IgA level was higher in seronegative than in seropositive subjects. In saliva, IgG level is almost two orders of magnitude lower than in serum, reaching the highest values after the second dose. IgA concentration remains low and increases significantly only in seropositive individuals after the second dose. Neutralizing antibody titres were much higher in serum than in saliva. INTERPRETATION: The mRNA BNT162b2 vaccination elicits a strong systemic immune response by drastically boosting neutralizing antibodies development in serum, but not in saliva, indicating that at least oral mucosal immunity is poorly activated by this vaccination protocol, thus failing in limiting virus acquisition upon its entry through this route. FUNDING: This work was funded by the Department of Medicine and Surgery, University of Insubria, and partially supported by Fondazione Umberto Veronesi (COVID-19 Insieme per la ricerca di tutti, 2020).


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/immunology , Immunity, Mucosal/drug effects , Immunization, Secondary , Adult , COVID-19/prevention & control , Female , Health Personnel , Humans , Immunoglobulin A/immunology , Immunoglobulin G/immunology , Male , Middle Aged , Saliva/immunology
8.
Sci Rep ; 11(1): 21768, 2021 11 05.
Article in English | MEDLINE | ID: covidwho-1505016

ABSTRACT

Rapid design, screening, and characterization of biorecognition elements (BREs) is essential for the development of diagnostic tests and antiviral therapeutics needed to combat the spread of viruses such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To address this need, we developed a high-throughput pipeline combining in silico design of a peptide library specific for SARS-CoV-2 spike (S) protein and microarray screening to identify binding sequences. Our optimized microarray platform allowed the simultaneous screening of ~ 2.5 k peptides and rapid identification of binding sequences resulting in selection of four peptides with nanomolar affinity to the SARS-CoV-2 S protein. Finally, we demonstrated the successful integration of one of the top peptides into an electrochemical sensor with a clinically relevant limit of detection for S protein in spiked saliva. Our results demonstrate the utility of this novel pipeline for the selection of peptide BREs in response to the SARS-CoV-2 pandemic, and the broader application of such a platform in response to future viral threats.


Subject(s)
COVID-19/immunology , Combinatorial Chemistry Techniques , Peptides/chemistry , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/chemistry , COVID-19/virology , Computational Biology , Electrochemistry/methods , Enzyme-Linked Immunosorbent Assay , Humans , Interferometry , Kinetics , Peptide Library , Protein Array Analysis , Protein Engineering , Saliva/immunology
9.
JAMA Netw Open ; 4(11): e2132563, 2021 11 01.
Article in English | MEDLINE | ID: covidwho-1499193

ABSTRACT

Importance: Although several studies have provided information on short-term clinical outcomes in children with perinatal exposure to SARS-CoV-2, data on the immune response in the first months of life among newborns exposed to the virus in utero are lacking. Objective: To characterize systemic and mucosal antibody production during the first 2 months of life among infants who were born to mothers infected with SARS-CoV-2. Design, Setting, and Participants: This prospective cohort study enrolled 28 pregnant women who tested positive for SARS-CoV-2 infection and who gave birth at Policlinico Umberto I in Rome, Italy, from November 2020 to May 2021, and their newborns. Maternal and neonatal systemic immune responses were investigated by detecting spike-specific antibodies in serum, and the mucosal immune response was assessed by measuring specific antibodies in maternal breastmilk and infant saliva 48 hours after delivery and 2 months later. Exposures: Maternal infection with SARS-CoV-2 in late pregnancy. Main Outcomes and Measures: The systemic immune response was evaluated by the detection of SARS-CoV-2 IgG and IgA antibodies and receptor binding domain-specific IgM antibodies in maternal and neonatal serum. The mucosal immune response was assessed by measuring spike-specific antibodies in breastmilk and in infant saliva, and the presence of antigen-antibody spike IgA immune complexes was investigated in breastmilk samples. All antibodies were detected using an enzyme-linked immunosorbent assay. Results: In total, 28 mother-infant dyads (mean [SD] maternal age, 31.8 [6.4] years; mean [SD] gestational age, 38.1 [2.3] weeks; 18 [60%] male infants) were enrolled at delivery, and 21 dyads completed the study at 2 months' follow-up. Because maternal infection was recent in all cases, transplacental transfer of virus spike-specific IgG antibodies occurred in only 1 infant. One case of potential vertical transmission and 1 case of horizontal infection were observed. Virus spike protein-specific salivary IgA antibodies were significantly increased (P = .01) in infants fed breastmilk (0.99 arbitrary units [AU]; IQR, 0.39-1.68 AU) vs infants fed an exclusive formula diet (0.16 AU; IQR, 0.02-0.83 AU). Maternal milk contained IgA spike immune complexes at 48 hours (0.53 AU; IQR, 0.25-0.39 AU) and at 2 months (0.09 AU; IQR, 0.03-0.17 AU) and may have functioned as specific stimuli for the infant mucosal immune response. Conclusions and Relevance: In this cohort study, SARS-CoV-2 spike-specific IgA antibodies were detected in infant saliva, which may partly explain why newborns are resistant to SARS-CoV-2 infection. Mothers infected in the peripartum period appear to not only passively protect the newborn via breastmilk secretory IgA but also actively stimulate and train the neonatal immune system via breastmilk immune complexes.


Subject(s)
COVID-19/immunology , Immunoglobulin A/immunology , Milk, Human/immunology , Pregnancy Complications, Infectious/immunology , Adult , COVID-19/blood , COVID-19/transmission , COVID-19 Serological Testing , Female , Humans , Immunoglobulin A/isolation & purification , Immunoglobulin G/immunology , Immunoglobulin G/isolation & purification , Infant , Infant, Newborn , Infectious Disease Transmission, Vertical/prevention & control , Male , Pregnancy , Pregnancy Complications, Infectious/blood , Prospective Studies , SARS-CoV-2 , Saliva/immunology , Spike Glycoprotein, Coronavirus/immunology
10.
Front Immunol ; 12: 753435, 2021.
Article in English | MEDLINE | ID: covidwho-1485058

ABSTRACT

Saliva is a body fluid with hitherto unused potential for the assessment of SARS-CoV-2 antibodies. Specific antibodies can indicate a past SARS-CoV-2 infection and allow to estimate the proportion of individuals with a potential protective immunity. First, we carefully characterized plasma samples obtained from adult control groups with and without prior SARS-CoV-2 infection using certified reference ELISAs. Simultaneously collected saliva samples of confirmed convalescent and negative individuals where then used to validate the herein newly developed ELISA for the detection of SARS-CoV-2 IgG antibodies in saliva. The saliva ELISA was applied to assess SARS-CoV-2 exposure in young children (N = 837) in the age between 1 and 10 years in Tübingen, Germany, towards the end of the first pandemic year 2020. Sensitivity and specificity of the new saliva ELISA was 87% and 100%, respectively. With 12% of all Tübingen children sampled via their respective educational institutions, estimates of SARS-CoV-2 antibody prevalence was 1.6%. Interestingly, only 0.4% preschool kids were positive compared to 3.0% of primary school children. Less than 20% of positive children self-reported symptoms within two months prior to saliva sampling that could be associated - but not exclusively - with a SARS-CoV-2 infection. The saliva ELISA is a valid and suitable protocol to enable population-based surveys for SARS-CoV-2 antibodies. Using non-invasive sampling and saliva ELISA testing, we found that prevalence of SARS-CoV-2 antibodies was significantly lower in young children than in primary school children.


Subject(s)
Antibodies, Viral/immunology , COVID-19 Serological Testing , COVID-19 , SARS-CoV-2/immunology , Saliva/immunology , Adult , COVID-19/diagnosis , COVID-19/immunology , Child , Child, Preschool , Enzyme-Linked Immunosorbent Assay , Female , Germany , Humans , Infant , Male , Prospective Studies
11.
Front Immunol ; 12: 701411, 2021.
Article in English | MEDLINE | ID: covidwho-1430694

ABSTRACT

In March 2020, the World Health Organization (WHO) declared a global health emergency-the coronavirus disease 2019 (COVID-19) pandemic. Since then, the development and implementation of vaccines against the virus amidst emerging cases of re-infection has prompted researchers to work towards understanding how immunity develops and is sustained. Serological testing has been instrumental in monitoring the development and persistence of antibodies against SARS-CoV-2 infection, however inconsistencies in detection have been reported by different methods. As serological testing becomes more commonplace, it is important to establish widespread and repeatable processes for monitoring vaccine efficacy. Therefore, we present enzyme linked immunosorbent assays (ELISAs) compatible for antibody detection in saliva as highly accurate, efficacious, and scalable tools for studying the immune response in individuals vaccinated against SARS-CoV-2.


Subject(s)
Antibodies, Neutralizing/analysis , Antibodies, Viral/analysis , COVID-19/immunology , SARS-CoV-2/immunology , Saliva/immunology , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19 Vaccines/immunology , Enzyme-Linked Immunosorbent Assay , Humans
12.
Microbiol Spectr ; 9(2): e0073121, 2021 10 31.
Article in English | MEDLINE | ID: covidwho-1410324

ABSTRACT

COVID-19 patients produce circulating and mucosal antibodies. In adults, specific saliva antibodies have been detected. Nonetheless, seroprevalence is routinely investigated, while little attention has been paid to mucosal antibodies. We therefore assessed SARS-CoV-2-specific antibody prevalence in serum and saliva in children in the Netherlands. We assessed SARS-CoV-2 antibody prevalence in serum and saliva of 517 children attending medical services in the Netherlands (irrespective of COVID-19 exposure) from April to October 2020. The prevalence of SARS-CoV-2 spike (S), receptor binding domain (RBD), and nucleocapsid (N)-specific IgG and IgA were evaluated with an exploratory Luminex assay in serum and saliva and with the Wantai SARS-CoV-2 RBD total antibody enzyme-linked immunosorbent assay in serum. Using the Wantai assay, the RBD-specific antibody prevalence in serum was 3.3% (95% confidence interval [CI]. 1.9 to 5.3%). With the Luminex assay, we detected heterogeneity between antibodies for S, RBD, and N antigens, as IgG and IgA prevalence ranged between 3.6 and 4.6% in serum and between 0 and 4.4% in saliva. The Luminex assay also revealed differences between serum and saliva, with SARS-CoV-2-specific IgG present in saliva but not in serum for 1.5 to 2.7% of all children. Using multiple antigen assays, the IgG prevalence for at least two out of three antigens (S, RBD, or N) in serum or saliva can be calculated as 3.8% (95% CI, 2.3 to 5.6%). Our study displays the heterogeneity of the SARS-CoV-2 antibody response in children and emphasizes the additional value of saliva antibody detection and the combined use of different antigens. IMPORTANCE Comprehending humoral immunity to SARS-CoV-2, including in children, is crucial for future public health and vaccine strategies. Others have suggested that mucosal antibody measurement could be an important and more convenient tool to evaluate humoral immunity compared to circulating antibodies. Nonetheless, seroprevalence is routinely investigated, while little attention has been paid to mucosal antibodies. We show the heterogeneity of SARS-CoV-2 antibodies, in terms of both antigen specificity and differences between circulating and mucosal antibodies, emphasizing the additional value of saliva antibody detection next to detection of antibodies in serum.


Subject(s)
Antibodies, Viral/blood , COVID-19 Serological Testing/methods , Coronavirus Nucleocapsid Proteins/immunology , SARS-CoV-2/immunology , Saliva/immunology , Spike Glycoprotein, Coronavirus/immunology , Adolescent , COVID-19/diagnosis , Child , Child, Preschool , Enzyme-Linked Immunosorbent Assay , Female , Humans , Immunity, Humoral/immunology , Immunoglobulin A/blood , Immunoglobulin G/blood , Male , Phosphoproteins/immunology , Prevalence , Sensitivity and Specificity , Seroepidemiologic Studies
13.
Microbiol Spectr ; 9(2): e0069321, 2021 10 31.
Article in English | MEDLINE | ID: covidwho-1410323

ABSTRACT

Noninvasive salivary antibody immunoassays can enable low-cost epidemiological surveillance of infections. This study involved developing and validating a multiplex suspension immunoassay on the Luminex platform to measure immunoglobulin G (IgG) responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid and spike (S) proteins, and the spike protein's S1 and S2 subunits and receptor binding domain. Multiple versions of these recombinant proteins acquired from commercial and noncommercial sources were evaluated. Assay development and validation utilized saliva and serum samples from coronavirus disease 2019 (COVID-19) cases procured from commercial sources and negative controls from a prepandemic survey. Saliva was also collected in a demonstration survey by mail involving adult individuals in the United States who were diagnosed with SARS-CoV-2 infection 15 to 80 days prior to sample collection. The survey had an 83% valid sample return rate (192 samples from 38 states). Most COVID-19 cases (93%) reported mildly symptomatic or asymptomatic infections. The final salivary assay based on the best-performing spike and nucleocapsid proteins had a sensitivity of 87.1% (95% bootstrap confidence interval, 82.1 to 91.7%) and specificity of 98.5% (95.0 to 100%) using 227 and 285 saliva samples, respectively. The same assay had 95.9% (92.8 to 98.9%) sensitivity and 100% (98.4 to 100%) specificity in serum (174 and 285 serum samples, respectively). Salivary and serum antibody responses to spike and nucleocapsid proteins were strongly correlated in 22 paired samples (r = 0.88 and r = 0.80, respectively). Antibody responses peaked at approximately 50 days postonset; greater illness severity was associated with stronger responses. This study demonstrated that a salivary antibody assay can be used in large-scale population surveys by mail to better characterize public health impacts of COVID-19. IMPORTANCE Given the enormous impacts of the COVID-19 pandemic, developing tools for population surveillance of infection is of paramount importance. This article describes the development of a multiplex immunoassay on a Luminex platform to measure salivary immunoglobulin G responses to the spike protein, its two subunits and receptor binding domain, and the nucleocapsid protein of SARS-CoV-2. The assay validation utilized serum and saliva samples from prepandemic controls and recent COVID-19 cases. A survey by mail targeting recent COVID-19 cases across the United States also demonstrated the utility of safe, at-home self-collection of saliva. By incorporating multiple SARS-CoV-2 proteins, this assay may differentiate responses to natural SARS-CoV-2 infections from responses to most vaccines. Application of this noninvasive immunoassay in COVID-19 surveillance can help provide estimates of cumulative incidence rates of symptomatic and asymptomatic infections in various communities and subpopulations, temporal patterns of antibody responses, and risk factors for infection.


Subject(s)
Antibodies, Viral/analysis , COVID-19 Serological Testing/methods , COVID-19/diagnosis , Immunoglobulin G/analysis , SARS-CoV-2/immunology , Saliva/immunology , Adolescent , Adult , Aged , Coronavirus Nucleocapsid Proteins/immunology , Female , Humans , Male , Middle Aged , Phosphoproteins/immunology , Postal Service , Sensitivity and Specificity , Spike Glycoprotein, Coronavirus/immunology , Young Adult
15.
PLoS One ; 16(7): e0251342, 2021.
Article in English | MEDLINE | ID: covidwho-1290172

ABSTRACT

Amperial™ is a novel assay platform that uses immobilized antigen in a conducting polymer gel followed by detection via electrochemical measurement of oxidation-reduction reaction between H2O2/Tetrametylbenzidine and peroxidase enzyme in a completed assay complex. A highly specific and sensitive assay was developed to quantify levels of IgG antibodies to SARS-CoV-2 in saliva. After establishing linearity and limit of detection we established a reference range of 5 standard deviations above the mean. There were no false positives in 667 consecutive saliva samples obtained prior to 2019. Saliva was obtained from 34 patients who had recovered from documented COVID-19 or had documented positive serologies. All of the patients with symptoms severe enough to seek medical attention had positive antibody tests and 88% overall had positive results. We obtained blinded paired saliva and plasma samples from 14 individuals. The plasma was analyzed using an EUA-FDA cleared ELISA kit and the saliva was analyzed by our Amperial™ assay. All 5 samples with negative plasma titers were negative in saliva testing. Eight of the 9 positive plasma samples were positive in saliva and 1 had borderline results. A CLIA validation was performed as a laboratory developed test in a high complexity laboratory. A quantitative non-invasive saliva based SARS-CoV-2 antibody test was developed and validated with sufficient specificity to be useful for population-based monitoring and monitoring of individuals following vaccination.


Subject(s)
Antibodies, Viral/immunology , COVID-19 Serological Testing/methods , COVID-19/diagnosis , Immunoglobulin G/immunology , SARS-CoV-2/immunology , Saliva/immunology , Antibodies, Viral/analysis , COVID-19/immunology , COVID-19/virology , Electrochemical Techniques/methods , Humans , Immunoglobulin G/analysis , Limit of Detection , SARS-CoV-2/isolation & purification , Saliva/virology
16.
Nat Commun ; 12(1): 3109, 2021 05 25.
Article in English | MEDLINE | ID: covidwho-1243298

ABSTRACT

SARS-CoV-2 is evolving with mutations in the receptor binding domain (RBD) being of particular concern. It is important to know how much cross-protection is offered between strains following vaccination or infection. Here, we obtain serum and saliva samples from groups of vaccinated (Pfizer BNT-162b2), infected and uninfected individuals and characterize the antibody response to RBD mutant strains. Vaccinated individuals have a robust humoral response after the second dose and have high IgG antibody titers in the saliva. Antibody responses however show considerable differences in binding to RBD mutants of emerging variants of concern and substantial reduction in RBD binding and neutralization is observed against a patient-isolated South African variant. Taken together our data reinforce the importance of the second dose of Pfizer BNT-162b2 to acquire high levels of neutralizing antibodies and high antibody titers in saliva suggest that vaccinated individuals may have reduced transmission potential. Substantially reduced neutralization for the South African variant further highlights the importance of surveillance strategies to detect new variants and targeting these in future vaccines.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/prevention & control , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Vaccination , Adult , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , Antibody Formation , COVID-19/blood , Female , Gene Expression , Humans , Immunoglobulin G/blood , Immunoglobulin G/immunology , Male , Middle Aged , Mutation , Neutralization Tests , Protein Binding , Protein Domains/genetics , Receptors, Coronavirus/metabolism , Recombinant Proteins , SARS-CoV-2/genetics , Saliva/immunology , Saliva/virology
18.
J Infect Dis ; 224(3): 407-414, 2021 08 02.
Article in English | MEDLINE | ID: covidwho-1225629

ABSTRACT

BACKGROUND: Declining humoral immunity in coronavirus disease 2019 (COVID-19) patients and possible reinfection have raised concern. Mucosal immunity, particularly salivary antibodies, may be short lived although long-term studies are lacking. METHODS: Using a multiplex bead-based array platform, we investigated antibodies specific to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins in 256 saliva samples from convalescent patients 1-9 months after symptomatic COVID-19 (n = 74, cohort 1), undiagnosed individuals with self-reported questionnaires (n = 147, cohort 2), and individuals sampled prepandemic (n = 35, cohort 3). RESULTS: Salivary IgG antibody responses in cohort 1 (mainly mild COVID-19) were detectable up to 9 months postrecovery, with high correlations between spike and nucleocapsid specificity. At 9 months, IgG remained in blood and saliva in most patients. Salivary IgA was rarely detected at this time point. In cohort 2, salivary IgG and IgA responses were significantly associated with recent history of COVID-19-like symptoms. Salivary IgG tolerated temperature and detergent pretreatments. CONCLUSIONS: Unlike SARS-CoV-2 salivary IgA that appeared short lived, specific saliva IgG appeared stable even after mild COVID-19, as for blood serology. This noninvasive saliva-based SARS-CoV-2 antibody test with home self-collection may be a complementary alternative to conventional blood serology.


Subject(s)
Antibodies, Viral/immunology , COVID-19/immunology , Immunoglobulin G/immunology , SARS-CoV-2/immunology , Saliva/immunology , Adult , Aged , Antibodies, Viral/blood , Enzyme-Linked Immunosorbent Assay , Female , Humans , Immunoglobulin G/blood , Male , Middle Aged , Time Factors , Young Adult
19.
Anal Chem ; 93(13): 5365-5370, 2021 04 06.
Article in English | MEDLINE | ID: covidwho-1147379

ABSTRACT

Tests for COVID-19 generally measure SARS-CoV-2 viral RNA from nasal swabs or antibodies against the virus from blood. It has been shown, however, that both viral particles and antibodies against those particles are present in saliva, which is more accessible than both swabs and blood. We present methods for highly sensitive measurements of both viral RNA and antibodies from the same saliva sample. We developed an efficient saliva RNA extraction method and combined it with an ultrasensitive antibody test based on single molecule array (Simoa) technology. We apply our test to the saliva of patients who presented to the hospital with COVID-19 symptoms, some of whom tested positive with a conventional RT-qPCR nasopharyngeal swab test. We demonstrate that combining viral RNA detection by RT-qPCR with antibody detection by Simoa identifies more patients as infected than either method alone. Our results demonstrate the utility of combining viral RNA and antibody testing from saliva, a single easily accessible biofluid.


Subject(s)
Antibodies, Viral/analysis , COVID-19 Testing/methods , COVID-19/diagnosis , RNA, Viral/analysis , SARS-CoV-2/genetics , Saliva/immunology , COVID-19/virology , Female , Humans , Limit of Detection , Male , Real-Time Polymerase Chain Reaction , Reproducibility of Results , SARS-CoV-2/immunology
20.
J Clin Microbiol ; 59(2)2021 01 21.
Article in English | MEDLINE | ID: covidwho-1043035

ABSTRACT

Current commercially available methods for reliably detecting antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remain expensive and inaccessible due to the need for whole-blood collection by highly trained phlebotomists using personal protective equipment (PPE). We have evaluated an antibody detection approach using the OraSure Technologies oral antibody collection device (OACD) and their proprietary SARS-CoV-2 total antibody detection enzyme-linked immunosorbent assay (ELISA). We found that the OraSure test for total antibody detection in oral fluid had comparable sensitivity and specificity to commercially available serum-based ELISAs for SARS-CoV-2 antibody detection while allowing for a more accessible form of specimen collection with the potential for self-collection.


Subject(s)
Antibodies, Viral/analysis , COVID-19 Serological Testing/methods , COVID-19/diagnosis , SARS-CoV-2/isolation & purification , Specimen Handling/instrumentation , COVID-19 Serological Testing/instrumentation , COVID-19 Serological Testing/standards , Enzyme-Linked Immunosorbent Assay , Humans , Immunoglobulin G/analysis , SARS-CoV-2/immunology , Saliva/immunology , Sensitivity and Specificity , Specimen Handling/methods , Specimen Handling/standards
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