Seroprevalence of Unidentified SARS-CoV-2 Infection in Hong Kong During 3 Pandemic Waves.

Importance: Seroprevalence studies inform the extent of infection and assist evaluation of mitigation strategies for the COVID-19 pandemic. Objective: To estimate the prevalence of unidentified SARS-CoV-2 infection in the general population of Hong Kong. Design, Setting, and Participants: A prospective cross-sectional study was conducted in Hong Kong after each major wave of the COVID-19 pandemic (April 21 to July 7, 2020; September 29 to November 23, 2020; and January 15 to April 18, 2021). Adults (age ≥18 years) who had not been diagnosed with COVID-19 were recruited during each period, and their sociodemographic information, symptoms, travel, contact, quarantine, and COVID-19 testing history were collected. Main Outcomes and Measures: The main outcome was prevalence of SARS-CoV-2 infection. SARS-CoV-2 IgG antibodies were detected by an enzyme-linked immunosorbent assay based on spike (S1/S2) protein, followed by confirmation with a commercial electrochemiluminescence immunoassay based on the receptor binding domain of spike protein. Results: The study enrolled 4198 participants (2539 [60%] female; median age, 50 years [IQR, 25 years]), including 903 (22%), 1046 (25%), and 2249 (53%) during April 21 to July 7, 2020; during September 29 to November 23, 2020; and during January 15 to April 18, 2021, respectively. The numbers of participants aged 18 to 39 years, 40 to 59 years, and 60 years or older were 1328 (32%), 1645 (39%), and 1225 (29%), respectively. Among the participants, 2444 (58%) stayed in Hong Kong since November 2019 and 2094 (50%) had negative SARS-CoV-2 RNA test results. Only 170 (4%) reported ever having contact with individuals with confirmed cases, and 5% had been isolated or quarantined. Most (2803 [67%]) did not recall any illnesses, whereas 737 (18%), 212 (5%), and 385 (9%) had experienced respiratory symptoms, gastrointestinal symptoms, or both, respectively, before testing. Six participants were confirmed to be positive for anti-SARS-CoV-2 IgG; the adjusted prevalence of unidentified infection was 0.15% (95% CI, 0.06%-0.32%). Extrapolating these findings to the whole population, there were fewer than 1.9 unidentified infections for every recorded confirmed case. The overall prevalence of SARS-CoV-2 infection in Hong Kong before the roll out of vaccination was less than 0.45%. Conclusions and Relevance: In this cross-sectional study of participants from the general public in Hong Kong, the prevalence of unidentified SARS-CoV-2 infection was low after 3 major waves of the pandemic, suggesting the success of the pandemic mitigation by stringent isolation and quarantine policies even without complete city lockdown. More than 99.5% of the general population of Hong Kong remain naive to SARS-CoV-2, highlighting the urgent need to achieve high vaccine coverage.

Osteopontin Fragments with Intact Thrombin-Sensitive Site Circulate in Cervical Cancer Patients.

We investigated whether circulating osteopontin (OPN) could be used as a biomarker for cervical cancer. We employed a monoclonal antibody (mAb 659) specific for the unique and intact thrombin-sensitive site in OPN using an inhibition ELISA. We found significantly higher levels of OPN in 33 cervical cancer patients in both the plasma (mean +/- SD, 612 +/- 106 ng/mL) and serum (424 +/- 121 ng/mL) compared to healthy subjects [409 +/- 56 ng/mL, from 31 plasma samples (P < 0.0001), and 314 +/- 98 ng/mL, from 32 serum samples (P = 0.0002), respectively]. Similar results were obtained when the plasma from a bigger group (147 individuals) of cervical cancer patients (560 +/- 211 ng/mL) were compared with the same plasma samples of the healthy individuals (P = 0.0014). More significantly, the OPN level was highest in stage III-IV disease (614 +/- 210 ng/mL, from 52 individuals; P = 0.0001) and least and non-discriminatory in stage I (473 +/- 110 ng/mL, from 40 individuals; P = 0.5318). No such discrimination was found when a mAb of a different specificity (mAb 446) was used in a similar inhibition ELISA to compare the two groups in the first study; a commercial capture ELISA also failed. The possibility that the target epitope recognized by the antibody probe in these assays was absent from the circulating OPN due to protein truncation was supported by gel fractionation of the OPN found in patients' plasma: 60-64 kDa fragments were found instead of the presumably full-length OPN (68 kDa) seen in healthy people. How these fragments are generated and what possible role they play in cancer biology remain interesting questions.

Modification of the TUBEX typhoid test to detect antibodies directly from haemolytic serum and whole blood.

The TUBEX test for typhoid fever detects serum antibodies in a simple and rapid assay system based on the inhibition of binding between two types of reagent particles - magnetic particles coated with an antigen (Salmonella O9 LPS) and coloured indicator particles coated with an anti-O9 mAb. A magnet is used to separate the colour indicator particles bound to the magnetic particles from the unbound indicator particles. Specific colour changes following magnetic separation are indicative of antibodies in the patient's serum; however, because results are interpreted based on changes in the colour red, haemolytic or icteric specimens cannot be used. This study describes a simple modification of the protocol to accommodate such specimens, including whole blood. This involves the addition of a quick and simple washing step after mixing the specimen with the antigen-bound magnetic particles. This modification has the advantage of allowing larger sample volumes to be used, thus enhancing the assay sensitivity, and also enables cases considered to be borderline positive by the original method to be re-assessed.

New rapid test for paratyphoid a fever: usefulness, cross-detection, and solution.

We described a 5-min colorimetric test for paratyphoid A fever, which detects anti-Salmonella O2 antibodies by inhibiting the binding between 2 types of reagent particles. This test (TUBEX-PA) is based on that (TUBEX-TF) used for typhoid fever, which detects anti-O9 antibodies. TUBEX-PA showed a sensitivity of 81.0% (47/58 culture-confirmed patients) to 93.3% (14/15) and was 98.1% (52/53) specific for healthy subjects. However, TUBEX-PA also detected 50% (7/14) to 81.8% (9/11) of typhoid patients, and conversely, TUBEX-TF detected 46.7% (7/15) to 73.3% (11/15) of paratyphoid A cases. This cross-detection could be abrogated in both tests by adding a blocker (heterologous antigen) to remove the antibodies responsible, which presumably bind to a common antigen (O12) located close to O2 and O9. The presence of anti-O12 antibodies in typhoid (9/12 or 75.0% sensitive) and paratyphoid A (22/33 or 66.7%) patients was demonstrated directly using a prototypic TUBEX test designed specifically to detect these antibodies. Thus, using TUBEX-PA and TUBEX-TF together can increase the diagnostic accuracy of detecting both typhoid and paratyphoid A fever, while the further use of differential tests allows possible immediate discrimination between these diseases.

Cells that produce deleterious autoreactive antibodies are vulnerable to suicide.

It is puzzling how autoreactive B cells that escape self-tolerance mechanisms manage to produce Abs that target vital cellular processes without succumbing themselves to the potentially deleterious effects of these proteins. We report that censorship indeed exists at this level: when the Ab synthesis in the cell is up-regulated in IL-6-enriched environments (e.g., adjuvant-primed mouse peritoneum), the cell dies of the increased intracellular binding between the Ab and the cellular autoantigen. In the case in which telomerase is the autoantigen, mouse hybridoma cells synthesizing such an autoantibody, which appeared to grow well in culture, could not grow in syngeneic BALB/c mice to form ascites, but grew nevertheless in athymic siblings. Culture experiments demonstrated that peritoneal cell-derived IL-6 (and accessory factors) affected the growth and functions of the hybridoma cells, including the induction of mitochondria-based apoptosis. Electron microscopy revealed an abundance of Abs in the nuclear chromatin of IL-6-stimulated cells, presumably piggy-backed there by telomerase from the cytosol. This nuclear presence was confirmed by light microscopy analysis of isolated nuclei. In two other cases, hybridoma cells synthesizing an autoantibody to GTP or osteopontin also showed similar growth inhibition in vivo. In all cases, Ab function was crucial to the demise of the cells. Thus, autoreactive cells, which synthesize autoantibodies to certain intracellular Ags, live delicately between life and death depending on the cytokine microenvironment. Paradoxically, IL-6, which is normally growth-potentiating for B cells, is proapoptotic for these cells. The findings reveal potential strategies and targets for immunotherapy.

The TUBEX test detects not only typhoid-specific antibodies but also soluble antigens and whole bacteria.

TUBEX (IDL Biotech) is a 5 min semiquantitative colorimetric test for typhoid fever, a widely endemic disease. TUBEX detects anti-Salmonella O9 antibodies from a patient's serum by the ability of these antibodies to inhibit the binding between an indicator antibody-bound particle and a magnetic antigen-bound particle. Herein, we report that TUBEX could also be used to specifically detect soluble O9 lipopolysaccharide in antigen-spiked buffer by the ability of the antigen to inhibit the same binding between the particles. Sensitivity of antigen detection was improved (8-31 mug ml(-1)) by using a modified protocol in which the test sample was mixed with the indicator particles first, rather than with the magnetic particles as for antibody detection. The antigen was also detectable in spiked serum and urine samples, albeit less well (2-4-fold) than in buffer generally. However, no antigen was detected from six typhoid sera examined, all of which had anti-O9 antibodies. In addition, whole organisms of Salmonella Typhi (15 strains) and Salmonella Enteritidis (6 strains) (both O9(+) Salmonella), grown in simulated blood broths or on MacConkey agar, were also detectable by TUBEX when suspended at >9 x 10(8) organisms ml(-1). Expectedly, Salmonella Paratyphi A (7 strains), Salmonella Typhimurium (1 strain) and Escherichia coli (2 strains) were negative in the test. Thus, the same TUBEX kit may be used in several ways both serologically and microbiologically for the rapid diagnosis of typhoid fever. However, validation of the newer applications will require the systematic examination of real patient and laboratory materials.

Carrier-specificity of a phosphorylcholine-binding antibody requires the presence of the constant domains and is not dependent on the unique VH49 glycine or VH30 threonine residues.

Bacterially-produced antibody fragments, such as single-chain Fv (scFv) which comprises the variable regions of the light (VL) and heavy (VH) chains joined together by a short flexible linker, are useful as diagnostic and therapeutic agents. We previously constructed a scFv fragment from a hybridoma antibody (Mab2) but it unexpectedly lacked the unique carrier specificity of the native antibody. Thus, it bound indiscriminately to various phosphorylcholine (PC)-associated antigens, whereas the hybridoma antibody recognized the PC epitope only in the context of the immunizing antigen. Here, we investigated whether the problem was linker-related by changing the linker composition or by deleting it, but these attempts proved futile. Instead, we have constructed a recombinant Fab fragment of the antibody in bacteria that was carrier-specific. This suggests that constant regions are required for the carrier specificity, which presumably helps to mould the fine structure of the antibody combining site or in stabilizing such a structure. Consistent with this global effect is the finding that replacing specific residues in VH with germ-line residues, namely, VH49 glycine and VH30 threonine, both thought previously to be important for the carrier specificity, had no effect on the carrier specificity of the recombinant Fab.

Antibody avidity maturation during severe acute respiratory syndrome-associated coronavirus infection.

The maturation of virus-specific immunoglobulin G avidity during severe acute respiratory syndrome-associated coronavirus infection was examined. The avidity indices were low (mean +/- SD, 30.8% +/- 11.6%) among serum samples collected < or =50 days after fever onset, intermediate (mean +/- SD, 52.1% +/- 14.1%) among samples collected between days 51 and 90, and high (mean +/- SD, 78.1% +/- 8.0%) among samples collected after day 90. Avidity indices of 40% and 55% could be considered as cutoff values for determination of recent (< or =50 days) and past (>65 days) infection, respectively. Measurement of antibody avidity can be used to differentiate primary infection from reexposure and to assess humoral responses to candidate vaccines.

Evaluation of a recombinant nucleocapsid protein-based assay for anti-SARS-CoV IgG detection.

A high throughput accurate assay for anti-SARS-CoV IgG detection is needed for large-scale epidemiological studies. The evaluation of a commercial recombinant nucleocapsid protein-based microtitre plate enzyme immunoassay, ELISARS is described. The results on 150 sera from SARS patients and 450 sera from non-SARS controls showed that this assay had a high level of sensitivity (96.2% for late serum samples) and specificity (97.8%). The performance and setup of this assay fulfills the requirement as a screening test for large-scale studies. A vast majority of SARS patients developed antibodies against the nucleocapsid protein. In some patients (10/45), a high level of anti-nucleocapsid antibody appeared very early in the course of the illness. In contrast, a minority (4 of 105 patients) never developed these antibodies. The implication of differences in antibody response to the nucleocapsid protein deserves further investigation.