Improvements in the detection of biomarkers indicative of disease continue to be vital to human health. Toward this end, this paper presents (a) the development and characterization of a biomarker ...detection strategy based on the inherently stronger signals generated by surface‐enhanced resonance Raman scattering (SERRS), as opposed to surface‐enhanced Raman scattering (SERS); (b) the application of this approach to the detection of mannose‐capped lipoarabinomannan (ManLAM), an antigenic marker indicative of active tuberculosis infection; and (c) a comparison of readout for this SERRS platform to that of the more frequently used approach based on SERS. More specifically, the work detailed herein describes the design and testing of a SERRS immunoassay that incorporates a resonance Raman‐enhanced adlayer of cyanine 5 on a smooth gold capture surface and its application to biomarker detection when “turned on” by the tagging of captured ManLAM with gold nanoparticle labels. The results of these experiments demonstrated an improvement in the detection of ManLAM spiked into human serum in terms of limit of detection by 10× and analytical sensitivity of almost 40× when compared with SERS. Findings also indicate that these improvements arise primarily from the intrinsic increase in signal strength due to the resonance Raman effect and a small but measurable increase in nanoparticle label density. Potential routes to further improve the performance of this approach to immunoassay signal generation are briefly discussed.
We apply turn‐on surface‐enhanced resonance Raman scattering (SERRS) as a novel detection strategy for a disease biomaker. This paper includes a detailed characterization of this novel sensor and a head‐to‐head performance comparison with a more conventional assay system. Ultimately we realized a 10× improvement in limit of detection and a ~40× improvement in analytical sensitivity.
This analysis of correlates of the risk of HIV-1 infection in the RV144 vaccine trial identifies hypotheses for improving efficacy. The data indicate key roles of V1V2 (variable regions 1 and 2) IgG ...antibodies and envelope protein (Env) IgA antibodies in modulating infection risk.
In clinical trials that show the efficacy of a vaccine, the identification of immune responses that are predictive of trial outcomes generates hypotheses about which of those responses are responsible for protection.
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The RV144 phase 3 trial in Thailand (ClinicalTrials.gov number, NCT00223080) was an opportunity to perform such a hypothesis-generating analysis for a human immunodeficiency virus type 1 (HIV-1) vaccine.
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Studies involving patients with HIV-1 infection in whom the disease did not progress in the long term have shown that cellular responses control the disease,
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and passive infusion of neutralizing antibodies prevents infection with chimeric simian–human immunodeficiency virus (SHIV). . . .
BACKGROUNDInadequate tuberculosis (TB) diagnostics are a major hurdle in the reduction of disease burden, and accurate point-of-care tests (POCTs) are urgently needed. We assessed the diagnostic ...accuracy of Fujifilm SILVAMP TB lipoarabinomannan (FujiLAM) POCT for TB diagnosis in HIV-negative outpatients and compared it with Alere Determine TB LAM Ag (AlereLAM) POCT and a laboratory-based ultrasensitive electrochemiluminescence LAM research assay (EclLAM).METHODSIn this multicenter diagnostic test accuracy study, we recruited HIV-negative adults with symptoms suggestive of pulmonary TB presenting to outpatient health care centers in Peru and South Africa. Urine samples were tested using FujiLAM, AlereLAM, and EclLAM, and the diagnostic accuracy was assessed against a microbiological reference standard (MRS) and a composite reference standard.RESULTSThree hundred seventy-two HIV-negative participants were included and the prevalence of microbiologically confirmed TB was 30%. Compared with the MRS, the sensitivities of AlereLAM, FujiLAM, and EclLAM were 10.8% (95% confidence interval CI 6.3%-18.0%), 53.2% (95% CI 43.9%-62.1%), and 66.7% (95% CI 57.5%-74.7%), respectively. The specificities of AlereLAM, FujiLAM, and EclLAM were 92.3% (95% CI 88.5%-95.0%), 98.9% (95% CI 96.7%-99.6%), and 98.1% (95% CI 95.6%-99.2%), respectively. Positive likelihood ratios of AlereLAM, FujiLAM, and EclLAM were 1.4, 46.2, and 34.8, respectively, and positive predictive values were 37.5%, 95.2%, and 93.7%, respectively.CONCLUSIONCompared with AlereLAM, FujiLAM detected 5 times more patients with TB in HIV-negative participants, had a high positive predictive value, and has the potential to improve rapid diagnosis of TB at the point-of-care. EclLAM demonstrated that additional sensitivity gains are possible, which highlights LAM's potential as a biomarker. Additional research is required to assess FujiLAM's performance in prospective cohorts, its cost-effectiveness, and its impact in real-world clinical settings.FUNDINGGlobal Health Innovative Technology Fund, the UK Department for International Development, the Dutch Ministry of Foreign Affairs, the Bill and Melinda Gates Foundation, the Australian Department of Foreign Affairs and Trade, the German Federal Ministry of Education and Research through Kreditanstalt für Wiederaufbau, and the NIH and National Institute of Allergy and Infectious Diseases.
HIV-1-specific immunoglobulin G (IgG) subclass antibodies bind to distinct cellular Fc receptors. Antibodies of the same epitope specificity but of a different subclass therefore can have different ...antibody effector functions. The study of IgG subclass profiles between different vaccine regimens used in clinical trials with divergent efficacy outcomes can provide information on the quality of the vaccine-induced B cell response. We show that HIV-1-specific IgG3 distinguished two HIV-1 vaccine efficacy studies (RV144 and VAX003 clinical trials) and correlated with decreased risk of HIV-1 infection in a blinded follow-up case-control study with the RV144 vaccine. HIV-1-specific IgG3 responses were not long-lived, which was consistent with the waning efficacy of the RV144 vaccine. These data suggest that specific vaccine-induced HIV-1 IgG3 should be tested in future studies of immune correlates in HIV-1 vaccine efficacy trials.
As a result of the SARS-CoV-2 pandemic numerous scientific groups have generated antibodies against a single target: the CoV-2 spike antigen. This has provided an unprecedented opportunity to compare ...the efficacy of different methods and the specificities and qualities of the antibodies generated by those methods. Generally, the most potent neutralizing antibodies have been generated from convalescent patients and immunized animals, with non-immune phage libraries usually yielding significantly less potent antibodies. Here, we show that it is possible to generate ultra-potent (IC
< 2 ng/ml) human neutralizing antibodies directly from a unique semisynthetic naïve antibody library format with affinities, developability properties and neutralization activities comparable to the best from hyperimmune sources. This demonstrates that appropriately designed and constructed naïve antibody libraries can effectively compete with immunization to directly provide therapeutic antibodies against a viral pathogen, without the need for immune sources or downstream optimization.
In the RV144 HIV-1 vaccine efficacy trial, IgG antibody (Ab) binding levels to variable regions 1 and 2 (V1V2) of the HIV-1 envelope glycoprotein gp120 were an inverse correlate of risk of HIV-1 ...infection. To determine if V1V2-specific Abs cross-react with V1V2 from different HIV-1 subtypes, if the nature of the V1V2 antigen used to asses cross-reactivity influenced infection risk, and to identify immune assays for upcoming HIV-1 vaccine efficacy trials, new V1V2-scaffold antigens were designed and tested. Protein scaffold antigens carrying the V1V2 regions from HIV-1 subtypes A, B, C, D or CRF01_AE were assayed in pilot studies, and six were selected to assess cross-reactive Abs in the plasma from the original RV144 case-control cohort (41 infected vaccinees, 205 frequency-matched uninfected vaccinees, and 40 placebo recipients) using ELISA and a binding Ab multiplex assay. IgG levels to these antigens were assessed as correlates of risk in vaccine recipients using weighted logistic regression models. Levels of Abs reactive with subtype A, B, C and CRF01_AE V1V2-scaffold antigens were all significant inverse correlates of risk (p-values of 0.0008-0.05; estimated odds ratios of 0.53-0.68 per 1 standard deviation increase). Thus, levels of vaccine-induced IgG Abs recognizing V1V2 regions from multiple HIV-1 subtypes, and presented on different scaffolds, constitute inverse correlates of risk for HIV-1 infection in the RV144 vaccine trial. The V1V2 antigens provide a link between RV144 and upcoming HIV-1 vaccine trials, and identify reagents and methods for evaluating V1V2 Abs as possible correlates of protection against HIV-1 infection.
ClinicalTrials.gov NCT00223080.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The RV144 Thai trial HIV-1 vaccine of recombinant poxvirus (ALVAC) and recombinant HIV-1 gp120 subtype B/subtype E (B/E) proteins demonstrated 31% vaccine efficacy. Here we design an ...ALVAC/Pentavalent B/E/E/E/E vaccine to increase the diversity of gp120 motifs in the immunogen to elicit a broader antibody response and enhance protection. We find that immunization of rhesus macaques with the pentavalent vaccine results in protection of 55% of pentavalent-vaccine-immunized macaques from simian-human immunodeficiency virus (SHIV) challenge. Systems serology of the antibody responses identifies plasma antibody binding to HIV-infected cells, peak ADCC antibody titres, NK cell-mediated ADCC and antibody-mediated activation of MIP-1β in NK cells as the four immunological parameters that best predict decreased infection risk that are improved by the pentavalent vaccine. Thus inclusion of additional gp120 immunogens to a pox-prime/protein boost regimen can augment antibody responses and enhance protection from a SHIV challenge in rhesus macaques.
Abstract
Since human infection with coronavirus SARS-CoV-2 was first detected in December 2019, we are still developing an understanding of the nature and duration of protection against this ...infection. Most neutralizing antibodies, which are a key component of the protective response against SARS-CoV-2 infection, target the Receptor Binding Domain (RBD) of the Spike glycoprotein and critically prevent binding of the virus to the host cell receptor, and viral entry. Thus, it is of vital importance to monitor the presence of neutralizing antibodies and RBD-specific B cells that are key for rapid production of protective antibodies upon reinfection with SARS-COV2 infection. In this study, we developed a multicolor flow cytometric assay to enumerate the RBD-specific memory B cell and memory B cell subsets. We collected peripheral blood cells and plasma from 22 subjects 1–2 months since COVID-19 diagnosis (early time point - ET) and then again after 5–7 months (late time point – LT). Comparing the data collected from these two time points, we observed a significant decrease in plasma blasts and double-negative memory B cell and an increase in the IgG+ switched-memory B cells and decrease in the IgM+ switched-memory B cells at LT relative to ET. We concurrently observed a trend toward decreased anti-RBD IgG titers over time. When we tested plasma neutralizing activity employing ACE2-expressing HeLa cell lines infected with mNeonGreen(mNG)SARS-CoV-2, we also observed reduced neutralizing antibody titers over time. Thus, a correlation exists between titers of RBD-specific IgG antibody and neutralizing titers. In addition, the presence of RBD-specific B-cell memory in circulating blood is a strong indication of a durable protective response.
The RV144 HIV-1 trial of the canary pox vector (ALVAC-HIV) plus the gp120 AIDSVAX B/E vaccine demonstrated an estimated efficacy of 31%, which correlated directly with antibodies to HIV-1 envelope ...variable regions 1 and 2 (V1-V2). Genetic analysis of trial viruses revealed increased vaccine efficacy against viruses matching the vaccine strain at V2 residue 169. Here, we isolated four V2 monoclonal antibodies from RV144 vaccinees that recognize residue 169, neutralize laboratory-adapted HIV-1, and mediate killing of field-isolate HIV-1-infected CD4+ T cells. Crystal structures of two of the V2 antibodies demonstrated that residue 169 can exist within divergent helical and loop conformations, which contrasted dramatically with the β strand conformation previously observed with a broadly neutralizing antibody PG9. Thus, RV144 vaccine-induced immune pressure appears to target a region that may be both sequence variable and structurally polymorphic. Variation may signal sites of HIV-1 envelope vulnerability, providing vaccine designers with new options.
► mAbs recognize HIV-1 envelope V2 region that is a site of vaccine-induce immune pressure ► The V2 antibodies target the regions shared partially with broad neutralizing HIV-1 mAbs ► Vaccine-induced V2 antibodies share a light chain signature that is critical for binding ► V2 antibodies bind to field HIV-1 isolate Envs expressed on CD4+ infected T cells
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