Burkholderia pseudomallei is the etiological agent of human melioidosis, a disease with a broad spectrum of clinical manifestations ranging from fatal septicemia to chronic localized infection or ...asymptomatic latent infection. Most clinical and immunological studies to date have focused on the acute disease process; however, little is known about pathology and immune response in chronic melioidosis. Here, we have developed a murine model of chronic disease by challenging C57BL/6 mice intranasally with a low dose of B. pseudomallei and monitoring them up to 100 days postinfection. Bacterial burdens were heterogeneous in different animals at all time points, consistent with the spectrum of clinical severity observed in humans. Proinflammatory cytokines such as gamma interferon (IFN-γ), interleukin-6 (IL-6), monocyte chemotactic protein-1 (MCP-1), and tumor necrosis factor-α (TNF-α) were induced during chronic infection, and histopathological analysis showed features in common with human melioidosis. Interestingly, many of these features were similar to those induced by Mycobacterium tuberculosis in humans, such as development of a collagen cord that encapsulates the lesions, the presence of multinucleated giant cells, and granulomas with a caseous necrotic center, which may explain why chronic melioidosis is often misdiagnosed as tuberculosis. Our model now provides a relevant and practical tool to define the immunological features of chronic melioidosis and aid in the development of more effective treatment of this disease in humans.
Enzyme-linked immunosorbent assays (ELISAs) remain the gold standard for measuring antibodies, but are time-consuming and use significant amounts of precious sample and reagents. Protein microarrays ...represent an appealing alternative, particularly for studies focused on large gene families such as those encoding variant surface antigens in the malaria parasite Plasmodium falciparum. Such microarrays represent an ideal high-throughput platform to study antibody responses to hundreds of malaria parasite variant surface antigens at once, providing critical insights into the development of natural immunity to malaria. We describe the essential background and approach to run an assay using a P. falciparum microarray populated with variant surface antigens. This allows the user to define serologic profiles and identify serodominant antigens that represent promising targets for vaccine or therapeutic development.
Current seasonal influenza virus vaccines engender antibody-mediated protection that is hemagglutinin (HA) subtype specific and relatively short-lived. Coverage for other subtypes or even variants ...within a subtype could be improved from a better understanding of the factors that promote HA-specific antibody cross-reactivity. Current assays to evaluate cross-reactivity, such as the ELISA, require a separate test for each antigen and are neither high-throughput nor sample-sparing. To address this need, we produced an array of 283 purified HA proteins from influenza A virus subtypes H1 to H16 and H18 and influenza B virus. To evaluate performance, arrays were probed with sera from individuals before and after a booster dose of inactivated heterologous H5N1 vaccine and naturally infected cases at presentation and follow-up during the 2010 to 2011 influenza season, when H3N2 was prevalent. The response to the H5 vaccine boost was IgG only and confined to H5 variants. The response to natural H3N2 infection consisted of IgG and IgA and was reactive with all H3 variants displayed, as well as against other group 2 HA subtypes. In both groups, responses to HA1 proteins were subtype specific. In contrast, baseline signals were higher, and responses broader, against full-length HA proteins (HA1+HA2) compared to HA1 alone. We propose that these elevated baseline signals and breadth come from the recognition of conserved epitopes in the stalk domain by cross-reactive antibodies accumulated from previous exposure(s) to seasonal influenza virus. This array is a valuable high-throughput alternative to the ELISA for monitoring specificity and cross-reactivity of HA antibodies and has many applications in vaccine development.
Seasonal influenza is a serious public health problem because the viral infection spreads easily from person to person and because of antigenic drift in neutralizing epitopes. Influenza vaccination is the most effective way to prevent the disease, although challenging because of the constant evolution of influenza virus subtypes. Our high-throughput protein microarrays allow for interrogation of subunit-specific IgG and IgA responses to 283 different HA proteins comprised of HA1 and HA2 domains as well as full-length HA proteins. This provides a tool that allows for novel insights into the response to exposure to influenza virus antigens. Data generated with our technology will enhance our understanding of the factors that improve the strength, breadth, and durability of vaccine-mediated immune responses and develop more effective vaccines.
Background
Efficacy of donated COVID‐19 convalescent plasma (dCCP) is uncertain and may depend on antibody titers, neutralizing capacity, timing of administration, and patient characteristics.
Study ...Design and Methods
In a single‐center hypothesis‐generating prospective case–control study with 1:2 matched dCCP recipients to controls according to disease severity at day 1, hospitalized adults with COVID‐19 pneumonia received 2 × 200 ml pathogen‐reduced treated dCCP from 2 different donors. We evaluated severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) antibodies in COVID‐19 convalescent plasma donors and recipients using multiple antibody assays including a Coronavirus antigen microarray (COVAM), and binding and neutralizing antibody assays. Outcomes were dCCP characteristics, antibody responses, 28‐day mortality, and dCCP ‐related adverse events in recipients.
Results
Eleven of 13 dCCPs (85%) contained neutralizing antibodies (nAb). PRT did not affect dCCP antibody activity. Fifteen CCP recipients and 30 controls (median age 64 and 65 years, respectively) were enrolled. dCCP recipients received 2 dCCPs from 2 different donors after a median of one hospital day and 11 days after symptom onset. One dCCP recipient (6.7%) and 6 controls (20%) died (p = 0.233). We observed no dCCP‐related adverse events. Transfusion of unselected dCCP led to heterogeneous SARS CoV‐2 antibody responses. COVAM clustered dCCPs in 4 distinct groups and showed endogenous immune responses to SARS‐CoV‐2 antigens over 14–21 days post dCCP in all except 4 immunosuppressed recipients.
Discussion
PRT did not impact dCCP anti‐virus neutralizing activity. Transfusion of unselected dCCP did not impact survival and had no adverse effects. Variable dCCP antibodies and post‐transfusion antibody responses indicate the need for controlled trials using well‐characterized dCCP with informative assays.
The eradication of smallpox by vaccination with vaccinia virus was probably one of the greatest achievements of vaccinology. However, the immunological basis of this protection is not fully ...understood. To this end, we have used protein microarrays of the vaccinia (Western Reserve, WR) proteome to profile antibody reactivities after primary infection or boosting with the licensed smallpox vaccine, Dryvax®, and with archival convalescent smallpox sera. Some 25 antigens were consistently recognized by Dryvax® sera, of which half were envelope proteins (notably, H3, A13, B5, and D8). The remainder consisted mainly of core proteins (e.g. A10, L4, and I1), proteins involved in intracellular morphogenesis (A11, D13), and the A‐type inclusion protein, WR148. Convalescent smallpox sera also detected vaccinia antigens on the array, consistent with the notion that there is serological cross‐reactivity between these two orthopox species that underlies protection. Moreover, the profiles of immunodominant antigens recognized by variola‐infected individuals and Dryvax® vaccinees were indistinguishable. This is the first description of antibody‐specificity profiles induced after smallpox infection. The array data indicate that a significant component of the antibody response is not involved in virus neutralization, although these antigens should be considered alongside the envelope proteins as potential candidates for diagnostic and vaccine applications.
Background
COVID‐19 convalescent plasma (CCP), from donors recovered from severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) infection, is one of the limited therapeutic options currently ...available for the treatment of critically ill patients with COVID‐19. There is growing evidence that CCP may reduce viral loads and disease severity; and reduce mortality. However, concerns about the risk of transfusion‐transmitted infections (TTI) and other complications associated with transfusion of plasma, remain. Amotosalen/UVA pathogen reduction treatment (A/UVA‐PRT) of plasma offers a mitigation of TTI risk, and when combined with pooling has the potential to increase the diversity of the polyclonal SARS‐CoV‐2 neutralizing antibodies.
Study design and methods
This study assessed the impact of A/UVA‐PRT on SARS‐CoV‐2 antibodies in 42 CCP using multiple complimentary assays including antigen binding, neutralizing, and epitope microarrays. Other mediators of CCP efficacy were also assessed.
Results
A/UVA‐PRT did not negatively impact antibodies to SARS‐CoV‐2 and other viral epitopes, had no impact on neutralizing activity or other potential mediators of CCP efficacy. Finally, immune cross‐reactivity with other coronavirus antigens was observed raising the potential for neutralizing activity against other emergent coronaviruses.
Conclusion
The findings of this study support the selection of effective CCP combined with the use of A/UVA‐PRT in the production of CCP for patients with COVID‐19.
The hallmark of vaccines is their ability to prevent the spread of infectious pathogens and thereby serve as invaluable public health tool. Despite their medical relevance, there is a gap in our ...understanding of the physiological factors that mediate innate and adaptive immune response to vaccines. The endocannabinoid (eCB) system is a critical modulator of homeostasis in vertebrates. Our results indicate that macrophages and dendritic cells produce the endocannabinoid, 2-arachidonoyl-sn-glycerol (2-AG) upon antigen activation. We have also established that 2-AG levels are upregulated in the serum and in the lymph node of mice during vaccination. We hypothesized that the intrinsic release of eCBs from immune cells during activation by pathogenic antigens mitigate inflammation, but also suppress overall innate and adaptive immune response. Here we demonstrate, for the first time, that transient administration of the cannabinoid receptor 2 antagonist AM630 (10 mg/kg) or inverse agonist JTE907 (3 mg/kg) during immunization heightens the intensity and breadth of antigen-specific immune responses in young and aged mice through the upregulation of immunomodulatory genes in secondary lymphoid tissues.
Abstract
Circumsporozoite protein (CSP) coats the Plasmodium falciparum sporozoite surface and is a major malaria subunit vaccine target. We measured epitope-specific reactivity to field-derived CSP ...haplotypes in serum samples from Malian adults and children on a custom peptide microarray. Compared to children, adults showed greater antibody responses and responses to more variants in regions proximal to and within the central repeat region. Children acquired short-lived immunity to an epitope proximal to the central repeat region but not to the central repeat region itself. This approach has the potential to differentiate immunodominant from protective epitope-specific responses when combined with longitudinal infection data.
On a custom peptide microarray that measured naturally acquired, epitope-specific antibody reactivity to field-derived Plasmodium falciparum circumsporozoite protein haplotypes, Malian adult sera had immunoglobulin G (IgG) antibodies to the Region 1-NANP junction and children had short-lived IgG responses to the same region.
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.
A next-generation proteome array for Schistosoma mansoni de Assis, Rafael Ramiro; Ludolf, Fernanda; Nakajima, Rie ...
International journal for parasitology,
June 2016, 2016-06-00, 20160601, Volume:
46, Issue:
7
Journal Article
Peer reviewed
Open access
Display omitted
•A proteome microarray consisting of 992 Schistosoma mansoni proteins was produced.•We describe the methods used to derive the gene list for the array, which represents 10% of the ...predicted proteome.•The array with pilot array using sera from S. mansoni naive individuals and S. mansoni infected individuals was validated.•We compare the sera from non-infected individuals with sera from S. mansoni acute and chronically infected individuals.
A proteome microarray consisting of 992 Schistosoma mansoni proteins was produced and screened with sera to determine antibody signatures indicative of the clinical stages of schistosomiasis and the identification of subunit vaccine candidates. Herein, we describe the methods used to derive the gene list for this array (representing approximately 10% of the predicted S. mansoni proteome). We also probed a pilot version of the microarray with sera from individuals either acutely or chronically infected with S. mansoni from endemic areas in Brazil and sera from individuals resident outside the endemic area (USA) to determine if the array is functional and informative.
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