IgG ELISA Assay Research Articles

Safety and immunogenicity of a self-amplifying RNA vaccine against COVID-19: COVAC1, a phase I, dose-ranging trial.

SummaryBackgroundLipid nanoparticle (LNP) encapsulated self-amplifying RNA (saRNA) is a novel technology formulated as a low dose vaccine against COVID-19.MethodsA phase I first-in-human dose-ranging trial of a saRNA COVID-19 vaccine candidate LNP-nCoVsaRNA, was conducted at Imperial Clinical Research Facility, and participating centres in London, UK, between 19th June to 28th October 2020. Participants received two intramuscular (IM) injections of LNP-nCoVsaRNA at six different dose levels, 0.1-10.0μg, given four weeks apart. An open-label dose escalation was followed by a dose evaluation. Solicited adverse events (AEs) were collected for one week from enrolment, with follow-up at regular intervals (1-8 weeks). The binding and neutralisation capacity of anti-SARS-CoV-2 antibody raised in participant sera was measured by means of an anti-Spike (S) IgG ELISA, immunoblot, SARS-CoV-2 pseudoneutralisation and wild type neutralisation assays. (The trial is registered: ISRCTN17072692, EudraCT 2020-001646-20).Findings192 healthy individuals with no history or serological evidence of COVID-19, aged 18-45 years were enrolled. The vaccine was well tolerated with no serious adverse events related to vaccination. Seroconversion at week six whether measured by ELISA or immunoblot was related to dose (both p<0.001), ranging from 8% (3/39; 0.1μg) to 61% (14/23; 10.0μg) in ELISA and 46% (18/39; 0.3μg) to 87% (20/23; 5.0μg and 10.0μg) in a post-hoc immunoblot assay. Geometric mean (GM) anti-S IgG concentrations ranged from 74 (95% CI, 45-119) at 0.1μg to 1023 (468-2236) ng/mL at 5.0μg (p<0.001) and was not higher at 10.0μg. Neutralisation of SARS-CoV-2 by participant sera was measurable in 15% (6/39; 0.1μg) to 48% (11/23; 5.0μg) depending on dose level received.InterpretationEncapsulated saRNA is safe for clinical development, is immunogenic at low dose levels but failed to induce 100% seroconversion. Modifications to optimise humoral responses are required to realise its potential as an effective vaccine against SARS-CoV-2.FundingThis study was co-funded by grants and gifts from the Medical Research Council UKRI (MC_PC_19076), and the National Institute Health Research/Vaccine Task Force, Partners of Citadel and Citadel Securities, Sir Joseph Hotung Charitable Settlement, Jon Moulton Charity Trust, Pierre Andurand, Restore the Earth.

Immunogenicity of an inactivated SARS-CoV-2 vaccine in people living with HIV-1: a non-randomized cohort study.

BackgroundInactivated COVID-19 vaccines are safe and effective in the general population with intact immunity. However, their safety and immunogenicity have not been demonstrated in people living with HIV (PLWH).Methods42 HIV-1 infected individuals who were stable on combination antiretroviral therapy (cART) and 28 healthy individuals were enrolled in this open-label two-arm non-randomized study at Hubei Provincial Center for Disease Control and Prevention, China. Two doses of an inactivated COVID-19 vaccine (BBIBP-CorV) were given on April 22, 2021 and May 25, 2021, respectively. The reactogenicity of the vaccine were evaluated by observing clinical adverse events and solicited local and systemic reactions. Humoral responses were measured by anti-spike IgG ELISA and surrogate neutralization assays. Cell-mediated immune responses and vaccine induced T cell activation were measured by flow cytometry.FindingsAll the HIV-1 infected participants had a CD4+ T cell count >200 cells/μL both at baseline (659·0 ± 221·9 cells/μL) and 4 weeks after vaccination (476·9 ± 150·8 cells/μL). No solicited adverse reaction was observed among all participants. Similar binding antibody, neutralizing antibody and S protein specific T cell responses were elicited in PLWH and healthy individuals. PLWH with low baseline CD4+/CD8+ T cell ratios (<0·6) generated lower antibody responses after vaccination than PLWH with medium (0·6∼1·0) or high (≥1·0) baseline CD4+/CD8+ T cell ratios (P<0·01). The CD3+, CD4+ and CD8+ T cell counts of PLWH decreased significantly after vaccination (P<0·0001), but it did not lead to any adverse clinical manifestation. Moreover, we found that the general HIV-1 viral load among the PLWH cohort decreased significantly after vaccination (P=0·0192). The alteration of HIV-1 viral load was not significantly associated with the vaccine induced CD4+ T cell activation (P>0·2).InterpretationOur data demonstrated that the inactivated SARS-CoV-2 vaccine was safe, immunogenic in PLWH who are stable on cART with suppressed viral load and CD4+ T cell count > 200 cells/μL. However, the persistence of the vaccine-induced immunities in PLWH need to be further investigated.

EVALUACIÓN DE LA CAPACIDAD ADYUVANTE DEL GINSENÓSIDO RG1 EN RATONES BALB/C INMUNIZADOS CON UN LISADO DE Staphylococcus aureus

Rg1 ginsenoside, the main active compound of Panax ginseng extract, could be a promising vaccine adjuvant against Staphylococcus aureusbovine mastitis. The aim was to evaluate the adjuvant effects of Rg1 combined with liposomes or alone against S. aureus lysate from bovine mastitis in mice. Female Balb/c mice were divided into 4 groups: S. aureus Lysate + IFA (Group I), S. aureus lysate + liposomes (Group II), S. aureus lysate + Rg1 (Group III) and S. aureus lysate + liposomes + Rg1 (Group IV). All mice were subcutaneously immunized with all formulations 3 times at 2-weeks intervals. Humoral response was evaluated by indirect IgG ELISA assay in plasma collected at the end of the assay. Cellular response was evaluated by MTT kit assay in spleen cells from immunized mice. Groups I and IV showed higher IgG values than groups II and III (p &lt; 0.05). Groups I and II showed higher proliferation values than groups III and IV (p &lt; 0.05). The Rg1-liposome combination increased S. aureus-specific IgG production. However, Rg1 was not able to stimulate lymphocyte proliferation from immunized mice, possibly due to the short S. aureus-specific stimulation time. New assays with different experimental conditions are needed to elucidate adjuvant effects of Rg1 in a murine model.

Long-term analysis of antibodies elicited by SPUTNIK V: A prospective cohort study in Tucumán, Argentina

BackgroundGam-COVID-Vac (SPUTNIK V) has been granted emergency use authorization in 70 nations and has been administered to millions worldwide. However, there are very few peer-reviewed studies describing its effects. Independent reports regarding safety and effectiveness could accelerate the final approval by the WHO. We aimed to study the long-term humoral immune response in naïve and previously infected volunteers who received SPUTNIK V.MethodsHumoral immune responses, assayed by anti-SARS-CoV-2-spike-RBD IgG ELISA and neutralization assays, were measured in 602 healthcare workers at 0, 14, 28, 60 and 180 days after receiving SPUTNIK V between December 2020 and July 2021 in Tucumán, Argentina.FindingsSeroconversion was detected in 97% of individuals after 28 days post-vaccination (dpv) (N = 405). Anti-RBD titers began to decrease after 60 dpv (N = 328), but remained detectable in 94% at 90 dpv (N = 224). At 180 dpv, anti-RDB titers persisted in 31% (N = 146). Previous infection triggered an increased immune response to the first dose and increased neutralization activity against variants of concern (VOC). Second doses in previously infected individuals further increased titers, even 90 dpv (N = 75). Basal antibody titers had more influence on post-vaccination anti-RBD responses than the time elapsed between diagnosis and vaccination (N = 274).InterpretationData presented herein provides essential knowledge regarding the kinetics of antibodies induced by SPUTNIK V up to six months after immunization, and suggests that when considering one-dose vaccination policies for individuals with previous SARS-CoV-2 infection, serological studies to determine basal titers may be important, independent of when diagnosis occurred.FundingTucumán Public Health System (SIPROSA), Argentinean National Research Council (CONICET), National University of Tucumán (UNT).

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) seroprevalence: Navigating the absence of a gold standard.

BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) seroprevalence studies bridge the gap left from case detection, to estimate the true burden of the COVID-19 pandemic. While multiple anti-SARS-CoV-2 immunoassays are available, no gold standard exists.MethodsThis serial cross-sectional study was conducted using plasma samples from 8999 healthy blood donors between April-September 2020. Each sample was tested by four assays: Abbott SARS-Cov-2 IgG assay, targeting nucleocapsid (Abbott-NP) and three in-house IgG ELISA assays (targeting spike glycoprotein, receptor binding domain, and nucleocapsid). Seroprevalence rates were compared using multiple composite reference standards and by a series of Bayesian Latent Class Models.ResultWe found 13 unique diagnostic phenotypes; only 32 samples (0.4%) were positive by all assays. None of the individual assays resulted in seroprevalence increasing monotonically over time. In contrast, by using the results from all assays, the Bayesian Latent Class Model with informative priors predicted seroprevalence increased from 0.7% (95% credible interval (95% CrI); 0.4, 1.0%) in April/May to 0.7% (95% CrI 0.5, 1.1%) in June/July to 0.9% (95% CrI 0.5, 1.3) in August/September. Assay characteristics varied over time. Overall Spike had the highest sensitivity (93.5% (95% CrI 88.7, 97.3%), while the sensitivity of the Abbott-NP assay waned from 77.3% (95% CrI 58.7, 92.5%) in April/May to 64.4% (95% CrI 45.6, 83.0) by August/September.DiscussionOur results confirmed very low seroprevalence after the first wave in Canada. Given the dynamic nature of this pandemic, Bayesian Latent Class Models can be used to correct for imperfect test characteristics and waning IgG antibody signals.

Immune Responses in 101HEMB01, a Phase 1/2 Open-Label, Single Ascending Dose-Finding Trial of DTX101 (AAVrh10FIX) in Patients with Severe Hemophilia B

▪Background: Humoral and cellular immune responses to adeno-associated virus (AAV) serotype rh10 (AAVrh10 vector) and to the human FIX transgene were analyzed in blood and serum samples from hemophilia B subjects enrolled in a phase 1/2 open-label, single-arm, multicenter, dose-finding study (101HEMB01; See also ASH 2017 Abstract: Pipe et al., 101HEMB01 is a phase 1/2 open-label, single ascending dose finding trial of DTX101 (AAVrh10FIX) in patients with moderate/severe hemophilia B that demonstrated meaningful but transient expression of human factor IX (hFIX)).Methods: T cell responses in blood PBMCs were analyzed by IFNγ ELISPOT and polychromatic flow cytometry. In addition, a multi-analyte profiling of secreted cytokines, chemokines, and growth factors in serum was performed. Furthermore, we performed a computational HLA class I affinity prediction analysis for each subject's FIX mutations. Humoral immune response in serum to AAVrh10 capsid was analyzed by IgG ELISA and by an in vitro neutralizing antibody (NAb) assay.Results: A total of 6 subjects were dosed. Subjects in Cohort 1 (n=3) and Cohort 2 (n=3) received a dose of DTX101 of 1.6x1012 GC/kg and 5.0x1012 GC/kg, respectively. Five out of six subjects had asymptomatic elevations in ALT of 40 U/L or higher. Subject 6 had a peak ALT of 914 U/L. IFNγ ELISPOT analysis showed a limited response to the AAVrh10 capsid 6-8 weeks after vector administration in subjects 3, 4, 5, and 6. The magnitude of the response was low and did not persist in subjects 3, 5, and 6. Intracellular cytokine analysis also showed low magnitude T cell responses to both the AAVrh10 capsid and FIX transgene. Subject 6 was the only subject with a strong CD4+ T cell IL-2+ IFNγ+ response to FIX that did not persist. HLA class I affinity prediction analysis revealed that subject 6 had high binding affinity for a single 9-mer peptide spanning the FIX mutation. Multi-analyte analysis in serum showed an increase in TGFa, IL-10, IL-7, and PDGF-AB/BB only observed in subject 6. IgG ELISA and NAb assay analyses showed strong antibody responses to AAVrh10 after vector administration in all six subjects. The antibody response remained stable for at least 52 weeks.Conclusion: Low magnitude and non-persisting T cell responses to AAVrh10 and FIX were detected in the peripheral blood of many patients, although there is no clear etiology for the higher elevation of transaminases observed in subject 6. Several aspects of his immune response do suggest that it differed qualitatively from that of the others, such as an exaggerated humoral response to the AAV capsid, a CD4 IL2 response against FIX, increases in serum growth factors associated with cell proliferation, differentiation and angiogenesis, and an increase of the anti-inflammatory cytokine IL10. Studies in nonhuman primates indicate that the T cell compartment in peripheral blood does not reflect that of the target of AAV, or in this case the liver, which could complicate the analysis of host immune responses to gene therapy in humans. DisclosuresWilson:REGENXBIO: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; University of Pennsylvania: Patents & Royalties; Dimension Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Solid Gene Therapy: Membership on an entity's Board of Directors or advisory committees.

A recombinant protein (MyxoTLm) for the serological diagnosis of acute and chronic Trypanosoma vivax infection in cattle

Human trypanosomiases and animal trypanosomoses are caused by distinct protozoan parasites of the genus Trypanosoma. The etiological agents of bovine trypanosomosis (BT) are T. vivax, T. congolense, or T. brucei, whose acute infections are initially characterized by hyperthermia, following moderate to severe anemia, subcutaneous edema, lethargy, reduced milk production, progressive weight loss, enlarged lymph nodes, reproductive disorders and death. Animals that survive the acute phase might recover and progress to the chronic, often asymptomatic, phase of infection. Despite their low sensitivity due to the characteristic low parasitemia, simple and costless direct parasitological examinations are the preferred diagnostic methods for animals. Thus, most of the epidemiological studies of BT are based on serological techniques using crude antigen. In this study, we describe the use of the MyxoTLm recombinant protein as an antigen on serological assays. Anti-T. vivax IgM and anti-T. vivax IgG ELISA assays using purified MyxoTLm revealed specificity rates of 91.30 % and 95.65 % and sensitivity rates of 82.35 % and 88.23 %, respectively, being higher than reported for crude antigens. Also, MyxoTLm demonstrated a good performance to detect IgM (ROC curve area = 0.8568) and excellent performance to detect IgG (ROC curve area = 0.9565) when compared to a crude antigen. T. evansi crude antigen used in the indirect anti-T. vivax IgM ELISA reached 70.58 % sensitivity and 78.26 % specificity, and had a lower test performance (ROC curve area = 0.7363). When applied to the anti-T. vivax IgG ELISA, the crude antigen reached 82.35 % sensitivity and 69.56 % specificity, also presenting a low performance with area under the ROC curve of 0.7570. Therefore, the use of MyxoTLm as an antigen on serological diagnosis of BT revealed to increase the sensitivity and the specificity if compared to crude antigens.

Serology- and Blood-PCR-Based Screening for Schistosomiasis in Pregnant Women in Madagascar-A Cross-Sectional Study and Test Comparison Approach.

This work was conducted as a cross sectional study to define the disease burden of schistosomiasis in pregnant Madagascan women and to evaluate serological and molecular diagnostic assays. A total of 1154 residual EDTA blood samples from pregnant Madagascan women were assessed. The nucleic acid extractions were subjected to in-house real-time PCRs specifically targeting S. mansoni complex, S. haematobium complex, and African Schistosoma spp. on genus level, while the EDTA plasma samples were analyzed using Schistosoma-specific IgG and IgM commercial ELISA and immunofluorescence assays. The analyses indicated an overall prevalence of schistosomiasis in Madagascan pregnant women of 40.4%, with only minor regional differences and differences between serology- and blood PCR-based surveillance. The S. mansoni specific real-time PCR showed superior sensitivity of 74% (specificity 80%) compared with the genus-specific real-time PCR (sensitivity 13%, specificity 100%) in blood. The laborious immunofluorescence (sensitivity IgM 49%, IgG 87%, specificity IgM 85%, IgG 96%) scored only slightly better than the automatable ELISA (sensitivity IgM 38%, IgG 88%, specificity IgM 78%, IgG 91%). Infections with S. mansoni were detected only. The high prevalence of schistosomiasis recorded here among pregnant women in Madagascar calls for actions in order to reduce the disease burden.

MO283IMPACT OF VIS649, AN APRIL-NEUTRALIZING IGG2 MONOCLONAL ANTIBODY, ON TETANUS- AND DIPHTHERIA-TOXOID VACCINATION-ELICITED IMMUNE RESPONSES IN HEALTHY VOLUNTEERS: PHASE 1, RANDOMIZED, DOUBLE-BLIND, PLACEBO-CONTROLLED STUDY

Abstract Background and Aims VIS649, a humanized immunoglobulin G (IgG2) monoclonal antibody that binds to and blocks the biological actions of a proliferation-inducing ligand (APRIL), is in clinical development as a potential treatment for immunoglobulin A (IgA) nephropathy (IgAN). In a Phase 1 study, VIS649 was associated with dose-dependent reductions in serum IgA, IgG and IgM, which were reversible and showed a dose-response effect with respect to time-to-recovery. The aim of the present analysis was to determine if VIS649 suppression of APRIL influences antibody responses to tetanus and diphtheria toxoid vaccination. Method This was a Phase 1, randomized, double-blind, placebo-controlled, single ascending dose study of VIS649 in healthy adult male and female volunteers (ClinicalTrials.gov identifier: NCT03719443). In one cohort within the study, participants were randomized in a 2:1 ratio to receive intravenous administration of VIS649 6.0 mg/kg or placebo, followed by a vaccine composed of tetanus and diphtheria toxoids (TENIVAC®, Sanofi Pasteur Limited), in order to evaluate the effect of VIS649 on recipients’ ability to generate a vaccine booster response (exploratory endpoint). Participants received intravenous administration of study drug on Day 1, were discharged from the institution on Day 2, received a single intramuscular dose of vaccine at the Week 4 visit, and were followed for 16 weeks in total on an outpatient basis. Blood samples were taken at regular intervals, and anti-tetanus toxoid and anti-diphtheria toxoid IgG, IgM and IgA quantitative ELISA assays were performed. Tetanus and diphtheria anti-toxoid IgG titers ≥0.1 IU/mL are generally considered to be protective. Results In the vaccination cohort, 15 participants were randomized and dosed with study drug or placebo, of whom 14 completed the study, and one participant who received VIS649 was lost to follow-up prior to receiving the vaccine. Both groups (placebo and VIS649) demonstrated increased tetanus anti-toxoid IgG titers following immunization, with a mean 7.9-fold increase in IU/mL at Week 6 for placebo recipients and a mean 6.4-fold increase in IU/mL for VIS649 recipients (Figure). At visits after Week 6, tetanus anti-toxoid IgG titers declined faster in the VIS649 group than in the placebo group (consistent with the reduction in total IgG associated with VIS649 administration) but remained above the protective threshold of 0.1 IU/mL for all participants throughout the study. Similar trends were observed for diphtheria anti-toxoid IgG titers, with a mean 5.5-fold increase in IU/mL at the Week 6 visit for placebo recipients and a mean 5.1-fold increase for VIS649 recipients (Figure). There was no evidence of tetanus- or diphtheria-toxoid elicited IgM responses in either the placebo or VIS649 groups, consistent with the recall nature of the vaccination. In a post hoc analysis, pre-existing serum tetanus/diphtheria anti-toxoid IgA titers fell between Day 1 and Week 4 in the VIS649 group, consistent with the overall suppression of total serum IgA, were boosted after vaccination in both groups, and declined faster in the VIS649 recipients thereafter. Conclusion VIS649 treatment did not interfere with participants’ ability to mount an antigen-specific serum IgG or IgA boost response to tetanus and diphtheria toxoid vaccination. There was no evidence of tetanus- or diphtheria-specific IgM responses in either the placebo or VIS649 groups, consistent with recall vaccination exposure. These data indicate that qualitative antibody responses are preserved during APRIL suppression.

Incidence of Heparin Induced Thrombocytopenia in Patients Undergoing Implantation of Durable and Non-Durable Mechanical Circulatory Support Devices

Purpose Unfractionated heparin exposure in patients undergoing mechanical circulatory support device (MCSD) implantation increases the risk for developing heparin-induced-thrombocytopenia (HIT). The false positive rate of HIT in this population using current standard testing is unknown. Postoperative and device related thrombocytopenia complicate the HIT diagnosis. Methods A retrospective analysis of patients undergoing MCSD pre-implantation assessment or implantation of MCSD (October 2014 - August 2020) was performed. The primary objective was to assess the incidence of HIT with positive PF4 IgG ELISA (PF4) and serotonin release (SRA) assays. False positive HIT was defined as PF4 ≥0.4 optical density (OD) and negative SRA. Argatroban usage was also assessed. Results 366 patients with 512 tests were analyzed. PF4 tests were performed in 128 patients (158 tests) undergoing pre-implantation assessment, 136 patients (185 tests) with non-durable devices (primarily Impella®), and 102 (169 tests) patients with durable devices (72 ventricular assist devices, 30 total artificial hearts). 25% (92/366) of patients had positive PF4 ≥0.4 OD and had subsequent SRA performed (92 patients, 122 tests). True positive HIT was documented in 13% (12/92) of PF4 positive patients consisting of 1 pre-implantation, 9 non-durable MCSD, and 2 durable MCSD patients. The mean PF4 for true positive HIT was 2.2±0.7 OD. False positive HIT was documented in 15% (19/128) of pre-implantation patients (mean PF4 0.9±0.6 OD), 14% (19/136) of non-durable MCSD patients (mean PF4 0.8±0.5 OD), and 41% (42/102) of durable MCSD patients (mean PF4 1.1±0.6 OD). Argatroban was administered in 40% (37/92) of patients with a positive PF4 and 67% (25/37) of patients with a negative SRA. Conclusion Without clinical suspicion of HIT, preoperative PF4 testing is not recommended. Patients with non-durable MCSD devices are at the highest risk of developing HIT (7%). Although a PF4 ≥0.4 OD is considered a positive screening test for HIT, a higher threshold should be considered for MCSD patients. To our knowledge, this is the first in depth assessment of the incidence of HIT in patients undergoing pre-implantation evaluation for both non-durable and durable MCS devices.

Analytical evaluation and critical appraisal of early commercial SARS-CoV-2 immunoassays for routine use in a diagnostic laboratory

ABSTRACT Background: The objective of this study was to evaluate the performance characteristics of early commercial SARS-CoV-2 antibody assays in mild and asymptomatic subjects to enable the selection of suitable immunoassays for routine diagnostic use. Methods: We used serum samples from a pre-COVID era patient cohort (n = 50, pre-December 2019), designated SARS-CoV-2 negative, and serum samples from a SARS-CoV-2 RT-PCR-positive cohort (n = 90) taken > 14 days post-symptom onset (April–May 2020). Six ELISA assays were evaluated, including one confirmation assay to investigate antibody specificity. We also evaluated one point-of-care lateral flow device (LFIA) and one high throughput electrochemiluminescence immunoassay (CLIA). Results: The ELISA specificities ranged from 84% to 100%, with sensitivities ranging from 75.3% to 90.0%. The LFIA showed 100% specificity and 80% sensitivity using smaller sample numbers. The Roche CLIA immunoassay showed 100% specificity and 90.7% sensitivity. When used in conjunction, the Euroimmun nucleocapsid (NC) and spike-1 (S1) IgG ELISA assays had a sensitivity of 95.6%. The confirmation Dia.Pro IgG assay showed 92.6% of samples tested contained both NC and S1 antibodies, 32.7% had NC, S1 and S2 and 0% had either S1 or S2 only. Conclusions: The Roche assay and the Euroimmun NC and S1 assays had the best sensitivity overall. Combining the assays detecting NC and S1/S2 antibody increased diagnostic yield. These first-generation assays were not calibrated against reference material and the results were reported qualitatively. A portfolio of next-generation SARS-CoV-2 immunoassays will be necessary to investigate herd and vaccine-induced immunity.

Prevalence of syphilis and associated factors in the adult population treated at healthcare facilities in Vitória (ES), Brazil

Introduction: There are few population-sampling studies on the prevalence of syphilis in Brazil. Objectives: We aim to determine the seroprevalence of syphilis and identify factors associated with the infection in adult patients observed at six regional healthcare facilities in Vitória city, state of Espírito Santo, Brazil. Methods: A cross-sectional study was conducted between September 2010 and December 2011. For individuals included in the study, a Venereal Disease Research Laboratory (VDRL) test and two treponemal tests (immunochromatographic and IgG ELISA assays) were performed. Demographic data, history of sexually transmitted infections, and behavioral data were collected. Results: Of the 1,502 subjects included in the study, 47% were men and 53% were women. The mean age was 41.63±14.57 years. The prevalence of syphilis was (0.9%; 95%CI 0.4–1.3) when a diagnosis of syphilis was considered with VDRL titers equal to or greater than 1:8. However, the prevalence was higher (2.8%) when a positive VDRL test, regardless of the titer, was considered. A multivariate analysis showed a significant association between syphilis and homosexual or bisexual behavior [OR=6.80; 95%CI 1.00–46.20], prior history of sexually transmitted infection [OR=16.30; 95%CI 3.61–73.41], the presence of a tattoo [OR=6.21; 95%CI 1.49–25.84], and cocaine use [OR=6.80; 95%CI 1.15–40.30]. The prevalence of positive treponemal test was 10.4%. Conclusion: The seroprevalence of active syphilis in this population was similar to that observed in other populational studies in Brazil. The high prevalence of positive treponemal tests may be due to the positive serological memory of a cured infection, but the results may also be due to cases of early or late syphilis that were not detected by the VDRL test.

Immunogenicity and Safety of an Inactivated SARS-CoV-2 Vaccine in People Living with HIV-1

Background: It has been proven that inactivated COVID-19 vaccines are safe and effective in general population with intact immunity. However, their safety and immunogenicity have not been demonstrated in people living with HIV (PLWH).Methods: 42 HIV-1 infected individuals who were stable on cART and 28 healthy individuals were enrolled in this study. Two doses of an inactivated COVID-19 vaccine (BIBP-CorV) were given 4 weeks apart. The safety and reactogenicity of the vaccine were evaluated by observing clinical adverse events and solicited local and systemic reactions. Humoral responses were measured by anti-spike IgG ELISA and surrogate neutralization assays. Cell-mediated immune responses and vaccine induced T cell activation were measured by flow cytometry.Findings: All the HIV-1 infected participants had a CD4+ T cell count of above 200 cells/μL both at baseline and 4 weeks after vaccination. No solicited adverse reaction was observed among all participants. Similar binding antibody, neutralizing antibody and S protein specific T cell responses were elicited in PLWH and healthy individuals. Further analyses showed that PLWH with low baseline CD4+/CD8+ T cell ratios (+/CD8+ T cell ratios (P+, CD4+ and CD8+ T cell counts of PLWH decreased significantly after vaccination, but it did not lead to any adverse clinical manifestation. Moreover, we found that the general burden of HIV-1 among the PLWH cohort decreased significantly (P=0·0192) after vaccination. And the alteration of HIV-1 viral load was not significantly associated with the vaccine induced CD4+ T cell activation.Interpretation: Our data demonstrate that the inactivated COVID-19 vaccine is safe and immunogenic in PLWH who are stable on cART with unsuppressed CD4 counts.Funding: This work was funded by the National Natural Science Foundation of China (Grant No. 81971559, 82041010).Declaration of Interest: None to declare. Ethical Approval: Written informed consent was obtained from all participants,and the study was approved by the Research Ethics Committee of Hubei CDC (approval reference number: HBCDC-AF/SC-08/02.0).

Safety and Immunogenicity of a Self-Amplifying RNA Vaccine Against COVID-19: COVAC1, a Phase I, Dose-Ranging Trial

Summary: Lipid nanoparticle (LNP) encapsulated self-amplifying RNA (saRNA) is a novel technology formulated as a low dose vaccine against COVID-19.Methods: A phase I first-in-human dose-ranging trial of a saRNA COVID-19 vaccine candidate LNP-nCoVsaRNA, was conducted at Imperial Clinical Research Facility, and participating centres in London, UK. Participants received two intramuscular (IM) injections of LNP-nCoVsaRNA at six different dose levels, 0·1-10·0mg, given four weeks apart. An open-label dose escalation was followed by a dose evaluation. Solicited adverse events (AEs) were collected for one week from enrolment, with follow-up at regular intervals (1-8 weeks). The binding and neutralisation capacity of anti-SARS-CoV-2 antibody raised in participant sera was measured by means of an anti-Spike (S) IgG ELISA, immunoblot, SARS-CoV-2 pseudoneutralisation and wild type neutralisation assays.Findings: 192 healthy individuals with no history or serological evidence of COVID-19, aged 18-45 years were enrolled. The vaccine was well tolerated with no serious adverse events related to vaccination. Seroconversion at week six whether measured by ELISA or immunoblot was related to dose (both pConcurrent anti-S IgG ranged from GM concentration (95% CI) 74 (45-119) at 0·1mg to 1023 (468-2236) ng/ml at 5·0mg (pInterpretation: Encapsulated saRNA is safe for clinical development and is immunogenic at low dose levels. Modifications to optimise humoral responses are required to realise its potential as an effective vaccine against SARS-CoV-2.Trial Registration: (ISRCTN17072692, EudraCT 2020-001646-20)Funding Statement: Medical Research Council UKRI (MC_PC_19076 and MC_UU_12023/23), National Institute for Health Research, Partners of Citadel and Citadel Securities, Sir Joseph Hotung Charitable Settlement, Jon Moulton Charity Trust.Declaration of Interests: P.F.M. and R.J.S. are co-inventors on a patent application covering this SARS-CoV-2 saRNA vaccine. All other authors have nothing to declare. Ethics Approval Statement: This study was approved in the UK by the Medicines and Healthcare products Regulatory Agency and the North East - York Research Ethics Committee (reference 20/SC/0145).

Clinical performance of a semi-quantitative assay for SARS-CoV2 IgG and SARS-CoV2 IgM antibodies

BackgroundWhile the diagnosis of SARS-CoV-2 infection is primarily based on detection of viral RNA, the detection of SARS-CoV-2 antibodies is useful for assessing past prevalence of the disease, and in corroborating a current infection in challenging cases. Sensitive and specific immunoassays provide the ability to identify exposure to SARS-CoV-2, to determine seroconversion, to confirm eligibility for donation of convalescent plasma as well as play an essential part in epidemiological studies. We report on the validation of the Ansh Laboratories SARS-CoV-2 IgG and SARS-CoV-2 IgM ELISA immunoassays. These assays were evaluated for detection of anti-SARS-CoV-2 IgG and IgM antibodies for clinical use in our hospital as part of an orthogonal testing algorithm recommended by the CDC. MethodsDiagnostic specificity and sensitivity of the IgG and IgM ELISA assays were tested using samples confirmed to be negative or positive for COVID-19 by RT-PCR. We also evaluated precision, analytical interference, and cross-reactivity with known cases of infection with other viruses. Additionally, we validated concordance with molecular and other serological testing and evaluated seroconversion in our patient population. ResultsThe IgG and IgM ELISA assays showed acceptable precision, were robust to analytical interference and did not exhibit cross reactivity with specimens positive for common respiratory viruses. Both assays exhibited 95% agreement with a primary screening serological assay utilized at our institution as well as with a reference laboratory semi-quantitative method. Concordance with RT-PCR was excellent > 6 days after symptom onset (100%). ConclusionsThe Ansh SARS-CoV-2 ELISA assays have good analytical performance suitable for clinical use.

Quantification of SARS-CoV-2 antibodies with eight commercially available immunoassays.

Since the emergence of SARS-CoV-2 numerous antibody assays have become available, demonstrating different performance characteristics. This study focused on a quantitative correlation between different commercial assays and a neutralization test (NT). Comparative data is needed as a basis for the production of convalescent plasma and potential interpretations COVID-19 immunity.Sera of 100 SARS-CoV-2 convalescent plasma donors were collected and SARS-CoV-2 antibodies were characterized using three different IgG-ELISAs (EUROIMMUN IgG and NCP-IgG ELISA, Wantai ELISA), two CLIA (Elecsys, LIAISON) and two lateral flow tests (MEDsan IgM/IgG-Rapid-Test, Wantai Rapid Test) and subsequently correlated to neutralization titers. The Wantai ELISA and the Elecsys provide the highest sensitivities in this sample (98 and 95 percent respectively). Titers with the best overall quantitative correlation to the NT titer were obtained with the Euroimmun IgG ELISA assay (Rho=0.759) and the Wantai ELISA assay (Rho=0.729).An infection without fever and negative or weakly positive reactions in the Wantai Rapid test were negative predictive factors for NT titers >1:200 (negative predictive value of 92 % and 92 % respectively, combination of both 100 %).The Wantai ELISA titer could be a suitable substitute for NT. An adequate pooling strategy of plasma units additionally could compensate deviations of individual antibody titers.

Detection of SARS-CoV-2 antibodies using commercial assays and seroconversion patterns in hospitalized patients

ObjectivesSARS-CoV-2 antibody assays are needed for serological surveys and as a complement to molecular tests to confirm COVID-19. However, the kinetics of the humoral response against SARS-CoV-2 remains poorly described and relies on the performance of the different serological tests. MethodsIn this study, we evaluated the performance of six CE-marked point-of-care tests (POC) and three ELISA assays for the diagnosis of COVID-19 by exploring seroconversions in hospitalized patients who tested positive for SARS-CoV-2 RNA. ResultsBoth the ELISA and POC tests were able to detect SARS-CoV-2 antibodies in at least half of the samples collected seven days or more after the onset of symptoms. After 15 days, the rate of detection rose to over 80% but without reaching 100%, irrespective of the test used. More than 90% of the samples collected after 15 days tested positive using the iSIA and Accu-Tell® POC tests and the ID.Vet IgG ELISA assay. Seroconversion was observed 5 to 12 days after the onset of symptoms. Three assays suffer from a specificity below 90% (EUROIMMUN IgG and IgA, UNscience, Zhuhai Livzon). ConclusionsThe second week of COVID-19 seems to be the best period for assessing the sensitivity of commercial serological assays. To achieve an early diagnosis of COVID-19 based on antibody detection, a dual challenge must be met: the immunodiagnostic window period must be shortened and an optimal specificity must be conserved.

Toxocara Prevalence in Soil and Humans in Vladivostok: A Long-term Study

Background: Toxocariasis is a zoonotic parasitosis with worldwide distribution. Environmental contamination with Toxocara eggs is a main source of human infections. In Russia, particular in Vladivostok, knowledge regarding the current situation, including distribution and environmental contamination by Toxocara spp., is largely unknown. Objectives: The present study investigated environmental contamination with Toxocara spp. eggs, in soil samples collected from sandpits and public parks in Vladivostok, Russia during 23-month period. Methods: A total of 260 soil samples from sandpits and 155 samples from public parks were collected during July 2015 to December 2017. Soil samples were tested by sedimentation/flotation technique. Seroprevalence of Toxocara antibody in human was analyzed using Toxocara canis IgG ELISA assay. Results: Overall, the annual prevalence of Toxocara spp. in sandpits varied between 20% - 23% the annual prevalence of Toxocara spp. in public parks varied between 25% - 42% in different years. The average number of Toxocara spp. varied from 0 to 220 eggs per 1 kg over the 3-years study period. The highest prevalence was registered in summer period. The significant correlation appeared between Toxocara spp. contamination rates in sandboxes and parks and the Toxocara prevalence in humans in 2016 year. Conclusions: Current study demonstrates that it is necessary to promote preventive public health measures to avoid soil contamination.

Development of an Improved Epstein-Barr Virus (EBV) Neutralizing Antibody Assay to Facilitate Development of a Prophylactic gp350-Subunit EBV Vaccine.

No licensed vaccine is available for prevention of EBV-associated diseases, and robust, high-throughput bioanalytical assays are needed to evaluate immunogenicity of gp350 subunit-based candidate EBV vaccines. Here we have developed an improved EBV-GFP based neutralization assay for such a vaccine’s pre-clinical and clinical validation to measure EBV specific neutralizing antibodies in human donors. The supplementation of guinea pig complement of our previously published high-throughput EBV-GFP fluorescent focus (FFA)-based neutralization assay allowed the detection of complement-dependent neutralizing antibodies using a panel of heat-inactivated healthy human sera. Anti-gp350 antibody titers, which were evaluated using a previously optimized anti-gp350 IgG ELISA assay, were moderately correlated to the FFA-based neutralization titers. Overall, this improved high-throughput neutralization assay is capable of characterizing the serologic neutralizing antibody response to natural EBV infection, with applications in evaluating EBV antibody status in epidemiologic studies and immunogenicity of candidate gp350-subunit EBV vaccines in clinical studies.

Engineering and characterisation of BCG-loaded polymeric microneedles

The aim of this study was to fabricate Bacillus Calmette–Guérin (BCG)-loaded microneedle patches using micromould casting technique and compare their efficacy with the injectable counterparts. The microneedle patches were formulated using sodium alginate (10% w/v) and trehalose (20% of polymer). The patches were characterised using optical microscopy, scanning electron microscopy and folding endurance. Serum IgG, TLC, granulocyte count, lymphocyte count and CRP were assessed and results were compared to that of intradermal injections alongside controls. The results showed that polymeric patches had a thickness of 0.8 mm, microneedle projections of 272 ± 12 µm and folding endurance of more than 300. Based on haematological and IgG ELISA assays, microneedle-based BCG administration significantly activated the immune cells and induced production of lymphocytes, granulocytes and peptide-specific IgG in immunised rats that were comparable to injectable counterparts. There was an increase in IgG antibodies from 3 g/L to 5.98 g/L and an increase in leucocytes from 2.6 × 109/L to 18.45 × 109/L. There was also an increase in granulocytes from 14.4% to 29.15% and lymphocyte count from 58.75% to 85.3%. It was concluded that BCG-coated polymeric microneedle patches are suitable for the transdermal delivery of vaccine without inducing discomfort usually observed with injections.