Most of what we know about adaptive immunity has come from inbred mouse studies, using methods that are often difficult or impossible to confirm in humans. In addition, vaccine responses in mice are ...often poorly predictive of responses to those same vaccines in humans. Here we use human tonsils, readily available lymphoid organs, to develop a functional organotypic system that recapitulates key germinal center features in vitro, including the production of antigen-specific antibodies, somatic hypermutation and affinity maturation, plasmablast differentiation and class-switch recombination. We use this system to define the essential cellular components necessary to produce an influenza vaccine response. We also show that it can be used to evaluate humoral immune responses to two priming antigens, rabies vaccine and an adenovirus-based severe acute respiratory syndrome coronavirus 2 vaccine, and to assess the effects of different adjuvants. This system should prove useful for studying critical mechanisms underlying adaptive immunity in much greater depth than previously possible and to rapidly test vaccine candidates and adjuvants in an entirely human system.
The current practice for diagnosis of COVID-19, based on SARS-CoV-2 PCR testing of pharyngeal or respiratory specimens in a symptomatic patient at high epidemiologic risk, likely underestimates the ...true prevalence of infection. Serologic methods can more accurately estimate the disease burden by detecting infections missed by the limited testing performed to date. Here, we describe the validation of a coronavirus antigen microarray containing immunologically significant antigens from SARS-CoV-2, in addition to SARS-CoV, MERS-CoV, common human coronavirus strains, and other common respiratory viruses. A comparison of antibody profiles detected on the array from control sera collected prior to the SARS-CoV-2 pandemic versus convalescent blood specimens from virologically confirmed COVID-19 cases demonstrates near complete discrimination of these two groups, with improved performance from use of antigen combinations that include both spike protein and nucleoprotein. This array can be used as a diagnostic tool, as an epidemiologic tool to more accurately estimate the disease burden of COVID-19, and as a research tool to correlate antibody responses with clinical outcomes.
Despite largely successful control efforts, malaria remains a significant public health problem in Thailand. Based on microscopy, the northwestern province of Tak, once Thailand's highest burden ...area, is now considered a low-transmission region. However, microscopy is insensitive to detect low-level parasitaemia, causing gross underestimation of parasite prevalence in areas where most infections are subpatent. The objective of this study was to assess the current epidemiology of malaria prevalence using molecular and serological detection methods, and to profile the antibody responses against Plasmodium as it relates to age, seasonal changes and clinical manifestations during infection. Three comprehensive cross-sectional surveys were performed in a sentinel village and from febrile hospital patients, and whole blood samples were collected from infants to elderly adults. Genomic DNA isolated from cellular fraction was screened by quantitative-PCR for the presence of Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale and Plasmodium knowlesi. Plasma samples were probed on protein microarray to obtain antibody response profiles from the same individuals.
Within the studied community, 90.2 % of Plasmodium infections were submicroscopic and asymptomatic, including a large number of mixed-species infections. Amongst febrile patients, mixed-species infections comprised 68 % of positive cases, all of which went misdiagnosed and undertreated. All samples tested showed serological reactivity to Plasmodium antigens. There were significant differences in the rates of antibody acquisition against P. falciparum and P. vivax, and age-related differences in species-specific immunodominance of response. Antibodies against Plasmodium increased along the ten-month study period. Febrile patients had stronger antibody responses than asymptomatic carriers.
Despite a great decline in malaria prevalence, transmission is still ongoing at levels undetectable by traditional methods. As current surveillance methods focus on case management, malaria transmission in Thailand will not be interrupted if asymptomatic submicroscopic infections are not detected and treated.
Whether tectonic convergence at subduction zones is accommodated predominantly through seismic or aseismic deformation, the former potentially generating large earthquakes, varies considerably ...between subduction margins. This margin‐scale variability has previously been linked to overriding plate deformation, trench migration, and their influence on the plate interface stress state. While these processes are linked to mantle‐scale dynamics, it is unclear how such dynamics influence interface stress. We systematically analyze the interface stress state in a suite of 2‐D thermo‐mechanical subduction models, where slabs display a range of morphologies that arise from diverse multiscale interactions with adjacent mantle and the overriding plate. We demonstrate that the thickness of the interface layer varies dynamically, in response to Poiseuille flow induced by slab bending or unbending, leading to associated effects on interface shear stress at typical seismogenic depth. Lower shear stress occurs when slab unbending is significant, which is commonly associated with trench retreat and draping of the slab as it impinges on the higher‐viscosity lower‐mantle. Conversely, higher shear stress is associated with limited slab unbending, which is promoted by negligible trench migration and vertically subducting slabs. We conclude that the diversity of slab dynamics may cause large variations in interface stress state between and maybe within margins. This is an additional variable that potentially controls seismogenic behavior, and we compare broad stress estimates for Circum‐Pacific margins to previous studies. Although predicted shear stress varies with observed seismogenic behavior, more detailed constraints on stress state are needed to test for correlation.
Key Points
Modeled subduction interface thickness and shear stress are controlled by slab bending, pull, and rollback
Lower shear stress is associated with significant slab unbending, promoted by trench retreat
Higher shear stress is associated with limited slab unbending and high slab pull, promoted by low slab‐lower‐mantle interaction
The effects of adjuvants for increasing the immunogenicity of influenza vaccines are well known. However, the effect of adjuvants on increasing the breadth of cross-reactivity is less well ...understood. In this study we have performed a systematic screen of different toll-like receptor (TLR) agonists, with and without a squalene-in-water emulsion on the immunogenicity of a recombinant trimerized hemagglutinin (HA) vaccine in mice after single-dose administration. Antibody (Ab) cross-reactivity for other variants within and outside the immunizing subtype (homosubtypic and heterosubtypic cross-reactivity, respectively) was assessed using a protein microarray approach. Most adjuvants induced broad IgG profiles, although the response to a combination of CpG, MPLA and AddaVax (termed 'IVAX-1') appeared more quickly and reached a greater magnitude than the other formulations tested. Antigen-specific plasma cell labeling experiments show the components of IVAX-1 are synergistic. This adjuvant preferentially stimulates CD4 T cells to produce Th1>Th2 type (IgG2c>IgG1) antibodies and cytokine responses. Moreover, IVAX-1 induces identical homo- and heterosubtypic IgG and IgA cross-reactivity profiles when administered intranasally. Consistent with these observations, a single-cell transcriptomics analysis demonstrated significant increases in expression of IgG1, IgG2b and IgG2c genes of B cells in H5/IVAX-1 immunized mice relative to naïve mice, as well as significant increases in expression of the IFNγ gene of both CD4 and CD8 T cells. These data support the use of adjuvants for enhancing the breath and durability of antibody responses of influenza virus vaccines.
•Cell-free, cell-mimicking platform tested for COVID-19 vaccination.•Presentation of SARS-CoV-2 spike (S) protein by DC-derived extracellular blebs (EBs)•Efficient antibody production and virus ...neutralization by S-presenting, DC-derived EBs.
COVID-19 has caused significant morbidity and mortality worldwide but also accelerated the clinical use of emerging vaccine formulations. To address the current shortcomings in the prevention and treatment of SARS-CoV-2 infection, this study developed a novel vaccine platform that closely mimics dendritic cells (DCs) in antigen presentation and T-cell stimulation in a cell-free and tunable manner. Genetically engineered DCs that express the SARS-CoV-2 spike protein (S) were chemically converted into extracellular blebs (EBs). The resulting EBs elicited potentially protective humoral immunity in vivo, indicated by the production of antibodies that potently neutralized S-pseudotyped virus, presenting EBs as a promising and safe vaccine.
Humans and other animals with Lyme borreliosis produce antibodies to a number of components of the agent Borrelia burgdorferi, but a full accounting of the immunogens during natural infections has ...not been achieved. Employing a protein array produced in vitro from 1,292 DNA fragments representing ~80% of the genome, we compared the antibody reactivities of sera from patients with early or later Lyme borreliosis to the antibody reactivities of sera from controls. Overall, ~15% of the open reading frame (ORF) products (Orfs) of B. burgdorferi in the array detectably elicited an antibody response in humans with natural infections. Among the immunogens, 103 stood out on the basis of statistical criteria. The majority of these Orfs were also immunogenic with sera obtained from naturally infected Peromyscus leucopus mice, a major reservoir. The high-ranking set included several B. burgdorferi proteins hitherto unrecognized as immunogens, as well as several proteins that have been established as antigens. The high-ranking immunogens were more likely than nonreactive Orfs to have the following characteristics: (i) plasmid-encoded rather than chromosome-encoded proteins, (ii) a predicted lipoprotein, and (iii) a member of a paralogous family of proteins, notably the Bdr and Erp proteins. The newly discovered antigens included Orfs encoded by several ORFs of the lp36 linear plasmid, such as BBK07 and BBK19, and proteins of the flagellar apparatus, such as FliL. These results indicate that the majority of deduced proteins of B. burgdorferi do not elicit antibody responses during infection and that the limited sets of immunogens are similar for two different host species.
High-throughput and rapid screening testing is highly desirable to effectively combat the rapidly evolving COVID-19 pandemic co-presents with influenza and seasonal common cold epidemics. Here, we ...present a general workflow for iterative development and validation of an antibody-based microarray assay for the detection of a respiratory viral panel: (a) antibody screening to quickly identify optimal reagents and assay conditions, (b) immunofluorescence assay design including signal amplification for low viral titers, (c) assay characterization with recombinant proteins, inactivated viral samples and clinical samples, and (d) multiplexing to detect a panel of common respiratory viruses. Using RT-PCR-confirmed SARS-CoV-2 positive and negative pharyngeal swab samples, we demonstrated that the antibody microarray assay exhibited a clinical sensitivity and specificity of 77.2% and 100%, respectively, which are comparable to existing FDA-authorized antigen tests. Moreover, the microarray assay is correlated with RT-PCR cycle threshold (Ct) values and is particularly effective in identifying high viral titers. The multiplexed assay can selectively detect SARS-CoV-2 and influenza virus, which can be used to discriminate these viral infections that share similar symptoms. Such protein microarray technology is amenable for scale-up and automation and can be broadly applied as a both diagnostic and research tool.
Sustained signaling through the B cell antigen receptor (BCR) is thought to occur only when antigen(s) crosslink or disperse multiple BCR units, such as by multimeric antigens found on the surfaces ...of viruses or bacteria. B cell-intrinsic Toll-like receptor (TLR) signaling synergizes with the BCR to induce and shape antibody production, hallmarked by immunoglobulin (Ig) class switch recombination (CSR) of constant heavy chains from IgM/IgD to IgG, IgA or IgE isotypes, and somatic hypermutation (SHM) of variable heavy and light chains. Full B cell differentiation is essential for protective immunity, where class switched high affinity antibodies neutralize present pathogens, memory B cells are held in reserve for future encounters, and activated B cells also serve as semi-professional APCs for T cells. But the rules that fine-tune B cell differentiation remain partially understood, despite their being essential for naturally acquired immunity and for guiding vaccine development. To address this in part, we have developed a cell culture system using splenic B cells from naive mice stimulated with several biotinylated ligands and antibodies crosslinked by streptavidin reagents. In particular, biotinylated lipopolysaccharide (LPS), a Toll-like receptor 4 (TLR4) agonist, and biotinylated anti-IgM were pre-assembled (multimerized) using streptavidin, or immobilized on nanoparticles coated with streptavidin, and used to active B cells in this precisely controlled, high throughput assay. Using B cell proliferation and Ig class switching as metrics for successful B cell activation, we show that the stimuli are both synergistic and dose-dependent. Crucially, the multimerized immunoconjugates are most active over a narrow concentration range. These data suggest that multimericity is an essential requirement for B cell BCR/TLRs ligands, and clarify basic rules for B cell activation. Such studies highlight the importance in determining the choice of single vs multimeric formats of antigen and PAMP agonists during vaccine design and development.
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