IntroductionActivated neutrophils kill microbes intracellularly after phagocytosis and by extracellular mechanisms, including neutrophil extracellular traps (NETs), which are composed of chromatin decorated with granular proteins. 1 NETs bind bacteria 1 and fungi 2 and expose antimicrobial molecules. Generation of NETs requires reactive oxygen species produced by the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. 3 Chronic granulomatous disease (CGD) is caused by mutations in genes encoding NADPH oxidase subunits. CGD patients do not produce reactive oxygen species, kill microbes poorly, and are susceptible to recurrent life-threatening infections. 4 Aspergillus spp infections cause pneumonia and disseminated disease and are the leading cause of death in these patients. [4][5][6] It is unclear how Aspergillus infections are controlled in healthy persons. [7][8][9][10][11][12][13] In CGD patients, these infections are frequently refractory to antifungal therapy, treatment with interferon-␥, or granulocyte transfusions. 5 Here we show that the recently discovered NADPH oxidase-dependent microbicidal pathway through NETs 1-3 is efficient against Aspergillus nidulans conidia and hyphae in vitro and that restoration of NET formation by GT of X-CGD aided clearing severe invasive A nidulans infection in vivo. Methods Gene therapyWe treated an 8.5-year-old boy with X-linked gp91 phox -deficient CGD and therapy refractory A nidulans lung infection with a monocistronic long terminal repeat-driven gamma-retroviral SF71gp91 phox vector (see supplemental data, available on the Blood website; see the Supplemental Materials link at the top of the online article). The protocol for the patient's treatment was approved by the ethics review board of the University Children's Hospital Zurich and the Swiss Expert Committee for Bio-Safety, after written informed consent from his parents in accordance with the Declaration of Helsinki. For follow-up monitoring, gp91 phox expression was measured by fluorescence-activated cell sorter (FACS) on peripheral neutrophils after 30 minutes of staining at room temperature with 10 g/mL gp91 phox -fluorescein isothiocyanate (FITC) antibody (Anti-Flavocytochrome b558, clone 7D5, MBL). NADPH oxidase activity was measured by standard dihydrorhodamine and nitroblue tetrazolium tests (supplemental materials). Bone marrow colony assays and determination of proviral gp91 phox sequences in genomic DNA were performed as described. 14 NET inductionNET formation was visualized as described (supplemental materials) and quantified after stimulation of 5 ϫ 10 4 neutrophils for 3 hours with 40 nM phorbol 12-myristate 13-acetate (PMA) and staining the NET-DNA with 1 M Sytox green (Invitrogen) in a black 96-well plate (BD Biosciences). The plates were read in a fluorescence microplate reader (Victor, 3 PerkinElmer Life and Analytical Sciences) with a filter setting of 485 nm/535 nm (excitation/emission). NET antifungal activityThe A nidulans strain used was isolated from bronchoalveolar lavage fluid ...

SummaryCharacterizing polyclonal antibody responses via currently available methods is inherently complex and difficult. Mapping epitopes in an immune response is typically incomplete, which creates a barrier to fully understanding the humoral response to antigens and hinders rational vaccine design efforts. Here, we describe a method of characterizing polyclonal responses by using electron microscopy, and we applied this method to the immunization of rabbits with an HIV-1 envelope glycoprotein vaccine candidate, BG505 SOSIP.664. We detected known epitopes within the polyclonal sera and revealed how antibody responses evolved during the prime-boosting strategy to ultimately result in a neutralizing antibody response. We uncovered previously unidentified epitopes, including an epitope proximal to one recognized by human broadly neutralizing antibodies as well as potentially distracting non-neutralizing epitopes. Our method provides an efficient and semiquantitative map of epitopes that are targeted in a polyclonal antibody response and should be of widespread utility in vaccine and infection studies.

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SummaryPassive administration of HIV neutralizing antibodies (nAbs) can protect macaques from hard-to-neutralize (tier 2) chimeric simian-human immunodeficiency virus (SHIV) challenge. However, conditions for nAb-mediated protection after vaccination have not been established. Here, we selected groups of 6 rhesus macaques with either high or low serum nAb titers from a total of 78 animals immunized with recombinant native-like (SOSIP) Env trimers. Repeat intrarectal challenge with homologous tier 2 SHIVBG505 led to rapid infection in unimmunized and low-titer animals. High-titer animals, however, demonstrated protection that was gradually lost as nAb titers waned over time. An autologous serum ID50 nAb titer of ∼1:500 afforded more than 90% protection from medium-dose SHIV infection. In contrast, antibody-dependent cellular cytotoxicity and T cell activity did not correlate with protection. Therefore, Env protein-based vaccination strategies can protect against hard-to-neutralize SHIV challenge in rhesus macaques by inducing tier 2 nAbs, provided appropriate neutralizing titers can be reached and maintained.

Highlights d Electron microscopy polyclonal epitope mapping of immunized and challenged macaques d Diversity of epitopes and specific angles of approach are hallmarks of protection d Neutralization breadth is due in part to fusion peptidespecific antibodies d Cryo-EM analysis of a protected animal details a putatively neutralizing paratope

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SummaryImmunoglobulin A (IgA) plays an important role in protecting our mucosal surfaces from viral infection, in maintaining a balance with the commensal bacterial flora, and in extending maternal immunity via breast feeding. Here, we report an additional innate immune effector function of human IgA molecules in that we demonstrate that the C-terminal tail unique to IgA molecules interferes with cell-surface attachment of influenza A and other enveloped viruses that use sialic acid as a receptor. This antiviral activity is mediated by sialic acid found in the complex N-linked glycans at position 459. Antiviral activity was observed even in the absence of classical antibody binding via the antigen binding sites. Our data, therefore, show that the C-terminal tail of IgA subtypes provides an innate line of defense against viruses that use sialic acid as a receptor and the role of neuraminidases present on these virions.

Rational immunogen design aims to focus antibody responses to vulnerable sites on the primary antigens. Given the size of these antigens there is however potential for eliciting unwanted, off-target responses. Here, we used our electron microscopy polyclonal epitope mapping approach to describe the antibody specificities elicited by immunization of non-human primates with soluble HIV envelope trimers and subsequent repeated viral challenge. An increased diversity of epitopes recognized, and the approach angle by which these antibodies bound, constituted a hallmark of the humoral response in most protected animals. We also show that fusion peptide-specific antibodies are responsible for some neutralization breadth. Moreover, cryoEM analysis of a fully-protected animal revealed a high degree of clonality within a subset of putatively neutralizing antibodies, enabling a detailed molecular description of the antibody paratope. Our results provide important insights into the immune response against a vaccine candidate that entered into clinical trials earlier this year.