Adeno-Associated Virus Delivery of Anti-HIV Monoclonal Antibodies Can Drive Long-Term Virologic Suppression.

Long-term delivery of anti-HIV monoclonal antibodies (mAbs) using adeno-associated virus (AAV) vectors holds promise for the prevention and treatment of HIV infection. We describe a therapy trial in which four rhesus monkeys were infected with SHIV-AD8 for 86 weeks before receiving the AAV-encoded mAbs 3BNC117, 10-1074, and 10E8. Although anti-drug antibody (ADA) responses restricted mAb delivery, one monkey successfully maintained 50-150 µg/mL of 3BNC117 and 10-1074 for over 2 years. Delivery of these two mAbs to this monkey resulted in an abrupt decline in plasma viremia, which remained undetectable for 38 successive measurements over 3 years. We generated two more examples of virologic suppression using AAV delivery of a cocktail of four mAbs in a 12-monkey study. Our results provide proof of concept for AAV-delivered mAbs to produce a "functional cure." However, they also serve as a warning that ADAs may be a problem for practical application of this approach in humans.

Abnormal biomarkers predict complex FAS or FADD defects missed by exome sequencing.

BACKGROUND: Elevated TCRαß+CD4-CD8- double-negative T cells (DNT) and serum biomarkers help identify FAS mutant patients with autoimmune lymphoproliferative syndrome (ALPS). However, in some patients with clinical features and biomarkers consistent with ALPS, germline or somatic FAS mutations cannot be identified on standard exon sequencing (ALPS-undetermined: ALPS-U). OBJECTIVE: We sought to explore whether complex genetic alterations in the FAS gene escaping standard sequencing or mutations in other FAS pathway-related genes could explain these cases. METHODS: Genetic analysis included whole FAS gene sequencing, copy number variation analysis, and sequencing of FAS cDNA and other FAS pathway-related genes. It was guided by FAS expression analysis on CD57+DNT, which can predict somatic loss of heterozygosity (sLOH). RESULTS: Nine of 16 patients with ALPS-U lacked FAS expression on CD57+DNT predicting heterozygous "loss-of-expression" FAS mutations plus acquired somatic second hits in the FAS gene, enriched in DNT. Indeed, 7 of 9 analyzed patients carried deep intronic mutations or large deletions in the FAS gene combined with sLOH detectable in DNT; 1 patient showed a FAS exon duplication. Three patients had reduced FAS expression, and 2 of them harbored mutations in the FAS promoter, which reduced FAS expression in reporter assays. Three of the 4 ALPS-U patients with normal FAS expression carried heterozygous FADD mutations with sLOH. CONCLUSION: A combination of serum biomarkers and DNT phenotyping is an accurate means to identify patients with ALPS who are missed by routine exome sequencing.

A Generic Approach for Miniaturized Unbiased High-Throughput Screens of Bispecific Antibodies and Biparatopic Antibody-Drug Conjugates.

The toolbox of modern antibody engineering allows the design of versatile novel functionalities exceeding nature's repertoire. Many bispecific antibodies comprise heterodimeric Fc portions recently validated through the approval of several bispecific biotherapeutics. While heterodimerization methodologies have been established for low-throughput large-scale production, few approaches exist to overcome the bottleneck of large combinatorial screening efforts that are essential for the identification of the best possible bispecific antibody. This report presents a novel, robust and miniaturized heterodimerization process based on controlled Fab-arm exchange (cFAE), which is applicable to a variety of heterodimeric formats and compatible with automated high-throughput screens. Proof of applicability was shown for two therapeutic molecule classes and two relevant functional screening read-outs. First, the miniaturized production of biparatopic anti-c-MET antibody-drug conjugates served as a proof of concept for their applicability in cytotoxic screenings on tumor cells with different target expression levels. Second, the automated workflow enabled a large unbiased combinatorial screening of biparatopic antibodies and the identification of hits mediating potent c-MET degradation. The presented workflow utilizes standard equipment and may serve as a facile, efficient and robust method for the discovery of innovative therapeutic agents in many laboratories worldwide.

Structure and dynamics of UBA5-UFM1 complex formation showing new insights in the UBA5 activation mechanism.

Ubiquitin fold modifier 1 (UFM1) is an ubiquitin-like protein (Ubl) involved especially in endoplasmic stress response. Activation occurs via a three-step mechanism like other Ubls. Data obtained reveal that UFM1 regulates the oligomeric state of ubiquitin activating enzyme 5 (UBA5) to initiate the activation step. Mixtures of homodimers and heterotrimers are observed in solution at the equilibrium state, demonstrating that the UBA5-UFM1 complex undergoes several concentration dependent oligomeric translational states to form a final functional complex. The oligomerization state of unbound UBA5 is also concentration dependent and shifts from the monomeric to the dimeric state. Data describing different oligomeric states are complemented with binding studies that reveal a negative cooperativity for the complex formation and thereby provide more detailed insights into the complex formation mechanism.

Hydraulic variability of three temperate broadleaf tree species along a water availability gradient in central Europe.

Plant hydraulic traits are key for understanding and predicting tree drought responses. Information about the degree of the traits' intra-specific variability may guide the selection of drought-resistant genotypes and is crucial for trait-based modelling approaches. For the three temperate minor broadleaf tree species Acer platanoides, Carpinus betulus and Tilia cordata, we measured xylem embolism resistance (P50 ), leaf turgor loss point (PTLP ), specific hydraulic conductivity (KS ), Huber values (HVs), and hydraulic safety margins in adult trees across a precipitation gradient. We further quantified trait variability on different organizational levels (inter-specific to within-canopy variation), and analysed its relationship to climatic and soil water availability. Although we observed a certain intra-specific trait variability (ITV) in safety-related traits (P50 , PTLP ) with higher within-tree and between-tree than between populations variability, the magnitude was small compared to inter-specific differences, which explained 78.4% and 58.3% of the variance in P50 and PTLP , respectively. In contrast, efficiency-related traits (KS , HV) showed a high ITV both within populations and within the crowns of single trees. Surprisingly, the observed ITV of all traits was neither driven by climatic nor soil water availability. In conclusion, the high degree of conservatism in safety-related traits highlights their potential for trait-based modelling approaches.

AAV-expressed eCD4-Ig provides durable protection from multiple SHIV challenges.

Long-term in vivo expression of a broad and potent entry inhibitor could circumvent the need for a conventional vaccine for HIV-1. Adeno-associated virus (AAV) vectors can stably express HIV-1 broadly neutralizing antibodies (bNAbs). However, even the best bNAbs neutralize 10-50% of HIV-1 isolates inefficiently (80% inhibitory concentration (IC80) > 5 µg ml(-1)), suggesting that high concentrations of these antibodies would be necessary to achieve general protection. Here we show that eCD4-Ig, a fusion of CD4-Ig with a small CCR5-mimetic sulfopeptide, binds avidly and cooperatively to the HIV-1 envelope glycoprotein (Env) and is more potent than the best bNAbs (geometric mean half-maximum inhibitory concentration (IC50) < 0.05 µg ml(-1)). Because eCD4-Ig binds only conserved regions of Env, it is also much broader than any bNAb. For example, eCD4-Ig efficiently neutralized 100% of a diverse panel of neutralization-resistant HIV-1, HIV-2 and simian immunodeficiency virus isolates, including a comprehensive set of isolates resistant to the CD4-binding site bNAbs VRC01, NIH45-46 and 3BNC117. Rhesus macaques inoculated with an AAV vector stably expressed 17-77 µg ml(-1) of fully functional rhesus eCD4-Ig for more than 40 weeks, and these macaques were protected from several infectious challenges with SHIV-AD8. Rhesus eCD4-Ig was also markedly less immunogenic than rhesus forms of four well-characterized bNAbs. Our data suggest that AAV-delivered eCD4-Ig can function like an effective HIV-1 vaccine.

High-dimensional single-cell proteomics analysis identifies immune checkpoint signatures and therapeutic targets in ulcerative colitis.

Immune checkpoints are regulators of immune cells and play key roles in the modulation of immune responses. The role of checkpoints in autoimmune disease is poorly understood but likely to be central since checkpoint inhibition during cancer treatment can cause autoimmunity. We generated a high-dimensional single-cell proteomics data set from PBMCs of healthy individuals and patients with ulcerative colitis (UC) by mass cytometry, enabling systems-wide analyses of immune cell frequencies and cell type-specific expression patterns of 12 immune checkpoints. Subtle but significant changes in immune cell frequencies and checkpoint expression were observed between UC patients on different treatment regimens and between patients and healthy controls. Most strikingly, UC patients showed a reduced number of peripheral NK-cells and those cells showed an altered phenotype including increased TIGIT expression. Based on these results, we modulated NK-cell function ex vivo through targeting of TIGIT pathway members. In summary, we describe a pattern of changes in immune cell abundance and checkpoint expression as a basis for UC patient stratification and we show modulation of a corresponding immune cell subset through checkpoint targeting. Our approach can be used for the identification of pathogenic immune cell subsets and guide target selection in autoimmunity and chronic inflammation.

Anti-drug Antibody Responses Impair Prophylaxis Mediated by AAV-Delivered HIV-1 Broadly Neutralizing Antibodies.

Adeno-associated virus (AAV) delivery of potent and broadly neutralizing antibodies (bNAbs is a promising approach for the prevention of HIV-1 infection. The immunoglobulin G (IgG)1 subtype is usually selected for this application, because it efficiently mediates antibody effector functions and has a somewhat longer half-life. However, the use of IgG1-Fc has been associated with the generation of anti-drug antibodies (ADAs) that correlate with loss of antibody expression. In contrast, we have shown that expression of the antibody-like molecule eCD4-Ig bearing a rhesus IgG2-Fc domain showed reduced immunogenicity and completely protected rhesus macaques from simian-HIV (SHIV)-AD8 challenges. To directly compare the performance of the IgG1-Fc and the IgG2-Fc domains in a prophylactic setting, we compared AAV1 expression of rhesus IgG1 and IgG2 forms of four anti-HIV bNAbs: 3BNC117, NIH45-46, 10-1074, and PGT121. Interestingly, IgG2-isotyped bNAbs elicited significantly lower ADA than their IgG1 counterparts. We also observed significant protection from two SHIV-AD8 challenges in macaques expressing IgG2-isotyped bNAbs, but not from those expressing IgG1. Our data suggest that monoclonal antibodies isotyped with IgG2-Fc domains are less immunogenic than their IgG1 counterparts, and they highlight ADAs as a key barrier to the use of AAV1-expressed bNAbs.

Hypomorphic caspase activation and recruitment domain 11 (CARD11) mutations associated with diverse immunologic phenotypes with or without atopic disease.

BACKGROUND: Caspase activation and recruitment domain 11 (CARD11) encodes a scaffold protein in lymphocytes that links antigen receptor engagement with downstream signaling to nuclear factor κB, c-Jun N-terminal kinase, and mechanistic target of rapamycin complex 1. Germline CARD11 mutations cause several distinct primary immune disorders in human subjects, including severe combined immune deficiency (biallelic null mutations), B-cell expansion with nuclear factor κB and T-cell anergy (heterozygous, gain-of-function mutations), and severe atopic disease (loss-of-function, heterozygous, dominant interfering mutations), which has focused attention on CARD11 mutations discovered by using whole-exome sequencing. OBJECTIVES: We sought to determine the molecular actions of an extended allelic series of CARD11 and to characterize the expanding range of clinical phenotypes associated with heterozygous CARD11 loss-of-function alleles. METHODS: Cell transfections and primary T-cell assays were used to evaluate signaling and function of CARD11 variants. RESULTS: Here we report on an expanded cohort of patients harboring novel heterozygous CARD11 mutations that extend beyond atopy to include other immunologic phenotypes not previously associated with CARD11 mutations. In addition to (and sometimes excluding) severe atopy, heterozygous missense and indel mutations in CARD11 presented with immunologic phenotypes similar to those observed in signal transducer and activator of transcription 3 loss of function, dedicator of cytokinesis 8 deficiency, common variable immunodeficiency, neutropenia, and immune dysregulation, polyendocrinopathy, enteropathy, X-linked-like syndrome. Pathogenic variants exhibited dominant negative activity and were largely confined to the CARD or coiled-coil domains of the CARD11 protein. CONCLUSION: These results illuminate a broader phenotypic spectrum associated with CARD11 mutations in human subjects and underscore the need for functional studies to demonstrate that rare gene variants encountered in expected and unexpected phenotypes must nonetheless be validated for pathogenic activity.

Fabclavine diversity in Xenorhabdus bacteria.

The global threat of multiresistant pathogens has to be answered by the development of novel antibiotics. Established antibiotic applications are often based on so-called secondary or specialized metabolites (SMs), identified in large screening approaches. To continue this successful strategy, new sources for bioactive compounds are required, such as the bacterial genera Xenorhabdus or Photorhabdus. In these strains, fabclavines are widely distributed SMs with a broad-spectrum bioactivity. Fabclavines are hybrid SMs derived from nonribosomal peptide synthetases (NRPS), polyunsaturated fatty acid (PUFA), and polyketide synthases (PKS). Selected Xenorhabdus and Photorhabdus mutant strains were generated applying a chemically inducible promoter in front of the suggested fabclavine (fcl) biosynthesis gene cluster (BGC), followed by the analysis of the occurring fabclavines. Subsequently, known and unknown derivatives were identified and confirmed by MALDI-MS and MALDI-MS2 experiments in combination with an optimized sample preparation. This led to a total number of 22 novel fabclavine derivatives in eight strains, increasing the overall number of fabclavines to 32. Together with the identification of fabclavines as major antibiotics in several entomopathogenic strains, our work lays the foundation for the rapid fabclavine identification and dereplication as the basis for future work of this widespread and bioactive SM class.

Hematopoietic stem cell transplantation in patients with gain-of-function signal transducer and activator of transcription 1 mutations.

BACKGROUND: Gain-of-function (GOF) mutations in signal transducer and activator of transcription 1 (STAT1) cause susceptibility to a range of infections, autoimmunity, immune dysregulation, and combined immunodeficiency. Disease manifestations can be mild or severe and life-threatening. Hematopoietic stem cell transplantation (HSCT) has been used in some patients with more severe symptoms to treat and cure the disorder. However, the outcome of HSCT for this disorder is not well established. OBJECTIVE: We sought to aggregate the worldwide experience of HSCT in patients with GOF-STAT1 mutations and to assess outcomes, including donor engraftment, overall survival, graft-versus-host disease, and transplant-related complications. METHODS: Data were collected from an international cohort of 15 patients with GOF-STAT1 mutations who had undergone HSCT using a variety of conditioning regimens and donor sources. Retrospective data collection allowed the outcome of transplantation to be assessed. In vitro functional testing was performed to confirm that each of the identified STAT1 variants was in fact a GOF mutation. RESULTS: Primary donor engraftment in this cohort of 15 patients with GOF-STAT1 mutations was 74%, and overall survival was only 40%. Secondary graft failure was common (50%), and posttransplantation event-free survival was poor (10% by 100 days). A subset of patients had hemophagocytic lymphohistiocytosis before transplant, contributing to their poor outcomes. CONCLUSION: Our data indicate that HSCT for patients with GOF-STAT1 mutations is curative but has significant risk of secondary graft failure and death.

T+ NK+ IL-2 Receptor γ Chain Mutation: a Challenging Diagnosis of Atypical Severe Combined Immunodeficiency.

PURPOSE: All reported patients with hypomorphic X-linked severe combined immunodeficiency (X-SCID) due to c.664C>T (p.R222C) mutations in the gene (IL2RG) encoding the common γ chain (γc) have presented with opportunistic infections within the first year of life, despite the presence of nearly normal NK and T cell numbers. Reporting five children of one extended family with hemizygous mutations in IL2RG, we explore potential diagnostic clues and extend our comprehension of the functional impact of this mutation. METHODS: Whole exome sequencing (WES); detailed immune phenotyping; cytokine-induced STAT phosphorylation; B, T, and NK cell activation; and quantification of sjTRECs in five Arab children with c.664C>T (p.R222C) IL2RG mutation. RESULTS: The mean age at clinical presentation with respiratory tract infection or diarrhea was 6.8 (range: 2-12) months. None of the children presented with opportunistic infections. Diagnostic clues were early onset in the first year of life, and a suggestive family history associated with reduced naïve CD4 T cells and absent switched memory B cells. Number and phenotype of NK cells and innate-like lymphocytes were normal. The diagnosis was made by WES and corroborated by absent STAT phosphorylation and reduced functional response after IL-2 and IL-21 stimulation. Four patients underwent successful hematopoietic stem cell transplantation. CONCLUSIONS: As early diagnosis and treatment are important, a high index of suspicion in the diagnosis of c.664C>T (p.R222C) X-SCID is needed. This requires prompt genetic testing by next generation sequencing in order to avoid unnecessary delays in the definite diagnosis since immunological work up may not be discriminating. Assays directly testing cytokine signaling or cytokine-dependent functions are helpful in confirming the functional impact of the identified hypomorphic variants.

CD57 identifies T cells with functional senescence before terminal differentiation and relative telomere shortening in patients with activated PI3 kinase delta syndrome.

Premature T-cell immunosenescence with CD57+ CD8+ T-cell accumulation has been linked to immunodeficiency and autoimmunity in primary immunodeficiencies including activated PI3 kinase delta syndrome (APDS). To address whether CD57 marks the typical senescent T-cell population seen in adult individuals or identifies a distinct population in APDS, we compared CD57+ CD8+ T cells from mostly pediatric APDS patients to those of healthy adults with similarly prominent senescent T cells. CD57+ CD8+ T cells from APDS patients were less differentiated with more CD27+ CD28+ effector memory T cells showing increased PD1 and Eomesodermin expression. In addition, transition of naïve to CD57+ CD8+ T cells was not associated with the characteristic telomere shortening. Nevertheless, they showed the increased interferon-gamma secretion, enhanced degranulation and reduced in vitro proliferation typical of senescent CD57+ CD8+ T cells. Thus, hyperactive PI3 kinase signaling favors premature accumulation of a CD57+ CD8+ T-cell population, which shows most functional features of typical senescent T cells, but is different in terms of differentiation and relative telomere shortening. Initial observations indicate that this specific differentiation state may offer the opportunity to revert premature T-cell immunosenescence and its potential contribution to inflammation and immunodeficiency in APDS.

Inspiratory muscle training does not improve clinical outcomes in 3-week COPD rehabilitation: results from a randomised controlled trial.

The value of inspiratory muscle training (IMT) in pulmonary rehabilitation in chronic obstructive pulmonary disease (COPD) is unclear. The RIMTCORE (Routine Inspiratory Muscle Training within COPD Rehabilitation) randomised controlled trial examined the effectiveness of IMT added to pulmonary rehabilitation.In total, 611 COPD patients (Global Initiative for Chronic Obstructive Lung Disease stage II-IV) received a 3-week inpatient pulmonary rehabilitation, of which 602 patients were included in the intention-to-treat analyses. The intervention group (n=300) received highly intensive IMT and the control group (n=302) received sham IMT. The primary outcome was maximal inspiratory pressure (PImax). The secondary outcomes were 6-min walk distance, dyspnoea, quality of life and lung function. Outcomes were assessed pre- and post-pulmonary rehabilitation. ANCOVA was used.The intervention group showed higher effects in PImax (p<0.001) and forced inspiratory volume in 1 s (p=0.013). All other outcomes in both study groups improved significantly, but without further between-group differences. Sex and pulmonary rehabilitation admission shortly after hospitalisation modified quality of life effects.IMT as an add-on to a 3-week pulmonary rehabilitation improves inspiratory muscle strength, but does not provide additional benefits in terms of exercise capacity, quality of life or dyspnoea. A general recommendation for COPD patients to add IMT to a 3-week pulmonary rehabilitation cannot be made.

Potent Plasmablast-Derived Antibodies Elicited by the National Institutes of Health Dengue Vaccine.

Exposure to dengue virus (DENV) is thought to elicit lifelong immunity, mediated by DENV-neutralizing antibodies (nAbs). However, Abs generated by primary infections confer serotype-specific protection, and immunity against other serotypes develops only after subsequent infections. Accordingly, the induction of these nAb responses acquired after serial DENV infections has been a long-sought-after goal for vaccination. Nonetheless, it is still unclear if tetravalent vaccines can elicit or recall nAbs. In this study, we have characterized the responses from a volunteer who had been previously exposed to DENV and was immunized with the live attenuated tetravalent vaccine Butantan-DV, developed by the NIH and Butantan Institute. Eleven days after vaccination, we observed an ∼70-fold expansion of the plasmablast population. We generated 21 monoclonal Abs (MAbs) from singly sorted plasmablasts. These MAbs were the result of clonal expansions and had significant levels of somatic hypermutation (SHM). Nineteen MAbs (90.5%) neutralized at least one DENV serotype at concentrations of 1 µg/ml or less; 6 of the 21 MAbs neutralized three or more serotypes. Despite the tetravalent composition of the vaccine, we observed a neutralization bias in the induced repertoire: DENV3 was targeted by 18 of the 19 neutralizing MAbs (nMAbs). Furthermore, the P3D05 nMAb neutralized DENV3 with extraordinary potency (concentration to achieve half-maximal neutralization [Neut50] = 0.03 µg/ml). Thus, the Butantan-DV vaccine engendered a mature, antigen-selected B cell repertoire. Our results suggest that preexisting responses elicited by a previous DENV3 infection were recalled by immunization.IMPORTANCE The dengue epidemic presents a global public health challenge that causes widespread economic burden and remains largely unchecked by existing control strategies. Successful control of the dengue epidemic will require effective prophylactic and therapeutic interventions. Several vaccine clinical efficacy trials are approaching completion, and the chances that one or more live attenuated tetravalent vaccines (LATVs) will be introduced worldwide is higher than ever. While it is widely accepted that dengue virus (DENV)-neutralizing antibody (nAb) titers are associated with protection, the Ab repertoire induced by LATVs remain uncharacterized. Here, we describe the isolation of potent (Neut50 < 0.1 µg/ml) nAbs from a DENV-seropositive volunteer immunized with the tetravalent vaccine Butantan-DV, which is currently in phase III trials.

Mutations in AP3D1 associated with immunodeficiency and seizures define a new type of Hermansky-Pudlak syndrome.

Genetic disorders affecting biogenesis and transport of lysosome-related organelles are heterogeneous diseases frequently associated with albinism. We studied a patient with albinism, neutropenia, immunodeficiency, neurodevelopmental delay, generalized seizures, and impaired hearing but with no mutation in genes so far associated with albinism and immunodeficiency. Whole exome sequencing identified a homozygous mutation in AP3D1 that leads to destabilization of the adaptor protein 3 (AP3) complex. AP3 complex formation and the degranulation defect in patient T cells were restored by retroviral reconstitution. A previously described hypopigmented mouse mutant with an Ap3d1 null mutation (mocha strain) shares the neurologic phenotype with our patient and shows a platelet storage pool deficiency characteristic of Hermansky-Pudlak syndrome (HPS) that was not studied in our patient because of a lack of bleeding. HPS2 caused by mutations in AP3B1A leads to a highly overlapping phenotype without the neurologic symptoms. The AP3 complex exists in a ubiquitous and a neuronal form. AP3D1 codes for the AP3δ subunit of the complex, which is essential for both forms. In contrast, the AP3ß3A subunit, affected in HPS2 patients, is substituted by AP3ß3B in the neuron-specific heterotetramer. AP3δ deficiency thus causes a severe neurologic disorder with immunodeficiency and albinism that we propose to classify as HPS10.

Nonadditivity of Optical and Casimir-Polder Potentials.

An atom irradiated by an off-resonant laser field near a surface is expected to experience the sum of two fundamental potentials, the optical potential of the laser field and the Casimir-Polder potential of the surface. Here, we report a new nonadditive potential, namely, the laser-induced Casimir-Polder potential, which arises from a correlated coupling of the atom with both the laser and the quantum vacuum. We apply this result to an experimentally realizable scenario of an atomic mirror with an evanescent laser beam leaking out of a surface. We show that the nonadditive term is significant for realistic experimental parameters, transforming potential barriers into potential wells, which can be used to trap atoms near surfaces.

Dengue Virus Evades AAV-Mediated Neutralizing Antibody Prophylaxis in Rhesus Monkeys.

Development of vaccines against mosquito-borne Flaviviruses is complicated by the occurrence of antibody-dependent enhancement (ADE), which can increase disease severity. Long-term delivery of neutralizing antibodies (nAbs) has the potential to effectively block infection and represents an alternative to vaccination. The risk of ADE may be avoided by using prophylactic nAbs harboring amino acid mutations L234A and L235A (LALA) in the immunoglobulin G (IgG) constant region. Here, we used recombinant adeno-associated viruses (rAAVs) to deliver the anti-dengue virus 3 (DENV3) nAb P3D05. While the administration of rAAV-P3D05-rhesus immunoglobulin G1 (rhIgG1)-LALA to rhesus macaques engendered DENV3-neutralizing activity in serum, it did not prevent infection. The emergence of viremia following DENV3 challenge was delayed by 3-6 days in the rAAV-treated group, and replicating virus contained the envelope mutation K64R. This neutralization-resistant variant was also confirmed by virus outgrowth experiments in vitro. By delivering P3D05 with unmutated Fc sequences, we further demonstrated that DENV3 also evaded wild-type nAb prophylaxis, and serum viral loads appeared to be higher in the presence of low levels of unmutated P3D05-rhIgG1. Our study shows that a vectored approach for long-term delivery of nAbs with the LALA mutations is promising, but prophylaxis using a single nAb is likely insufficient at preventing DENV infection and replication.

Missense mutation in immunodeficient patients shows the multifunctional roles of coiled-coil domain 3 (CC3) in STIM1 activation.

Store-operated Ca(2+) entry (SOCE) is a universal Ca(2+) influx pathway that is important for the function of many cell types. SOCE occurs upon depletion of endoplasmic reticulum (ER) Ca(2+) stores and relies on a complex molecular interplay between the plasma membrane (PM) Ca(2+) channel ORAI1 and the ER Ca(2+) sensor stromal interaction molecule (STIM) 1. Patients with null mutations in ORAI1 or STIM1 genes present with severe combined immunodeficiency (SCID)-like disease. Here, we describe the molecular mechanisms by which a loss-of-function STIM1 mutation (R429C) in human patients abolishes SOCE. R429 is located in the third coiled-coil (CC3) domain of the cytoplasmic C terminus of STIM1. Mutation of R429 destabilizes the CC3 structure and alters the conformation of the STIM1 C terminus, thereby releasing a polybasic domain that promotes STIM1 recruitment to ER-PM junctions. However, the mutation also impairs cytoplasmic STIM1 oligomerization and abolishes STIM1-ORAI1 interactions. Thus, despite its constitutive localization at ER-PM junctions, mutant STIM1 fails to activate SOCE. Our results demonstrate multifunctional roles of the CC3 domain in regulating intra- and intermolecular STIM1 interactions that control (i) transition of STIM1 from a quiescent to an active conformational state, (ii) cytoplasmic STIM1 oligomerization, and (iii) STIM1-ORAI1 binding required for ORAI1 activation.

A prospective study on the natural history of patients with profound combined immunodeficiency: An interim analysis.

BACKGROUND: Absent T-cell immunity resulting in life-threatening infections provides a clear rationale for hematopoetic stem cell transplantation (HSCT) in patients with severe combined immunodeficiency (SCID). Combined immunodeficiencies (CIDs) and "atypical" SCID show reduced, not absent T-cell immunity. If associated with infections or autoimmunity, they represent profound combined immunodeficiency (P-CID), for which outcome data are insufficient for unambiguous early transplant decisions. OBJECTIVES: We sought to compare natural histories of severity-matched patients with/without subsequent transplantation and to determine whether immunologic and/or clinical parameters may be predictive for outcome. METHODS: In this prospective and retrospective observational study, we recruited nontransplanted patients with P-CID aged 1 to 16 years to compare natural histories of severity-matched patients with/without subsequent transplantation and to determine whether immunologic and/or clinical parameters may be predictive for outcome. RESULTS: A total of 51 patients were recruited (median age, 9.6 years). Thirteen of 51 had a genetic diagnosis of "atypical" SCID and 14 of 51 of CID. About half of the patients had less than 10% naive T cells, reduced/absent T-cell proliferation, and at least 1 significant clinical event/year, demonstrating their profound immunodeficiency. Nineteen patients (37%) underwent transplantation within 1 year of enrolment, and 5 of 51 patients died. Analysis of the HSCT decisions revealed the anticipated heterogeneity, favoring an ongoing prospective matched-pair analysis of patients with similar disease severity with or without transplantation. Importantly, so far neither the genetic diagnosis nor basic measurements of T-cell immunity were good predictors of disease evolution. CONCLUSIONS: The P-CID study for the first time characterizes a group of patients with nontypical SCID T-cell deficiencies from a therapeutic perspective. Because genetic and basic T-cell parameters provide limited guidance, prospective data from this study will be a helpful resource for guiding the difficult HSCT decisions in patients with P-CID.