Phase 1 trial to model primary, secondary, and tertiary dengue using a monovalent vaccine.

BACKGROUND: The four co-circulating and immunologically interactive dengue virus serotypes (DENV1-4) pose a unique challenge to vaccine design because sub-protective immunity can increase the risk of severe dengue disease. Existing dengue vaccines have lower efficacy in DENV seronegative individuals but higher efficacy in DENV exposed individuals. There is an urgent need to identify immunological measures that are strongly associated with protection against viral replication and disease following sequential exposure to distinct serotypes. METHODS/DESIGN: This is a phase 1 trial wherein healthy adults with neutralizing antibodies to zero (seronegative), one non-DENV3 (heterotypic), or more than one (polytypic) DENV serotype will be vaccinated with the live attenuated DENV3 monovalent vaccine rDEN3Δ30/31-7164. We will examine how pre-vaccine host immunity influences the safety and immunogenicity of DENV3 vaccination in a non-endemic population. We hypothesize that the vaccine will be safe and well tolerated, and all groups will have a significant increase in the DENV1-4 neutralizing antibody geometric mean titer between days 0 and 28. Compared to the seronegative group, the polytypic group will have lower mean peak vaccine viremia, due to protection conferred by prior DENV exposure, while the heterotypic group will have higher mean peak viremia, due to mild enhancement. Secondary and exploratory endpoints include characterizing serological, innate, and adaptive cell responses; evaluating proviral or antiviral contributions of DENV-infected cells; and immunologically profiling the transcriptome, surface proteins, and B and T cell receptor sequences and affinities of single cells in both peripheral blood and draining lymph nodes sampled via serial image-guided fine needle aspiration. DISCUSSION: This trial will compare the immune responses after primary, secondary, and tertiary DENV exposure in naturally infected humans living in non-endemic areas. By evaluating dengue vaccines in a new population and modeling the induction of cross-serotypic immunity, this work may inform vaccine evaluation and broaden potential target populations. TRIAL REGISTRATION: NCT05691530 registered on January 20, 2023.

A Longitudinal Study of COVID-19 Sequelae and Immunity: Baseline Findings.

BACKGROUND: A substantial proportion of persons who develop COVID-19 report persistent symptoms after acute illness. Various pathophysiologic mechanisms have been implicated in the pathogenesis of postacute sequelae of SARS-CoV-2 infection (PASC). OBJECTIVE: To characterize medical sequelae and persistent symptoms after recovery from COVID-19 in a cohort of disease survivors and controls. DESIGN: Cohort study. (ClinicalTrials.gov: NCT04411147). SETTING: National Institutes of Health Clinical Center, Bethesda, Maryland. PARTICIPANTS: Self-referred adults with laboratory-documented SARS-CoV-2 infection who were at least 6 weeks from symptom onset were enrolled regardless of presence of PASC. A control group comprised persons with no history of COVID-19 or serologic evidence of SARS-CoV-2 infection, recruited regardless of their current health status. Both groups were enrolled over the same period and from the same geographic area. MEASUREMENTS: All participants had the same evaluations regardless of presence of symptoms, including physical examination, laboratory tests and questionnaires, cognitive function testing, and cardiopulmonary evaluation. A subset also underwent exploratory immunologic and virologic evaluations. RESULTS: 189 persons with laboratory-documented COVID-19 (12% of whom were hospitalized during acute illness) and 120 antibody-negative control participants were enrolled. At enrollment, symptoms consistent with PASC were reported by 55% of the COVID-19 cohort and 13% of control participants. Increased risk for PASC was noted in women and those with a history of anxiety disorder. Participants with findings meeting the definition of PASC reported lower quality of life on standardized testing. Abnormal findings on physical examination and diagnostic testing were uncommon. Neutralizing antibody levels to spike protein were negative in 27% of the unvaccinated COVID-19 cohort and none of the vaccinated COVID-19 cohort. Exploratory studies found no evidence of persistent viral infection, autoimmunity, or abnormal immune activation in participants with PASC. LIMITATIONS: Most participants with COVID-19 had mild to moderate acute illness that did not require hospitalization. The prevalence of reported PASC was likely overestimated in this cohort because persons with PASC may have been more motivated to enroll. The study did not capture PASC that resolved before enrollment. CONCLUSION: A high burden of persistent symptoms was observed in persons after COVID-19. Extensive diagnostic evaluation revealed no specific cause of reported symptoms in most cases. Antibody levels were highly variable after COVID-19. PRIMARY FUNDING SOURCE: Division of Intramural Research, National Institute of Allergy and Infectious Diseases.

Investigation of the Importance of Protein 3D Structure for Assessing Conservation of Lysine Acetylation Sites in Protein Homologs.

Acetylation is a protein post-translational modification (PTM) that can affect a variety of cellular processes. In bacteria, two PTM Nε-acetylation mechanisms have been identified: non-enzymatic/chemical acetylation via acetyl phosphate or acetyl coenzyme A and enzymatic acetylation via protein acetyltransferases. Prior studies have shown that extensive acetylation of Nε-lysine residues of numerous proteins from a variety of bacteria occurs via non-enzymatic acetylation. In Escherichia coli, new Nε-lysine acetyltransferases (KATs) that enzymatically acetylate other proteins have been identified, thus expanding the repertoire of protein substrates that are potentially regulated by acetylation. Therefore, we designed a study to leverage the wealth of structural data in the Protein Data Bank (PDB) to determine: (1) the 3D location of lysine residues on substrate proteins that are acetylated by E. coli KATs, and (2) investigate whether these residues are conserved on 3D structures of their homologs. Five E. coli KAT substrate proteins that were previously identified as being acetylated by YiaC and had 3D structures in the PDB were selected for further analysis: adenylate kinase (Adk), isocitrate dehydrogenase (Icd), catalase HPII (KatE), methionyl-tRNA formyltransferase (Fmt), and a peroxide stress resistance protein (YaaA). We methodically compared over 350 protein structures of these E. coli enzymes and their homologs; to accurately determine lysine residue conservation requires a strategy that incorporates both flexible structural alignments and visual inspection. Moreover, our results revealed discrepancies in conclusions about lysine residue conservation in homologs when examining linear amino acid sequences compared to 3D structures.

Generating enzyme and radical-mediated bisubstrates as tools for investigating Gcn5-related N-acetyltransferases.

Gcn5-related N-acetyltransferases (GNATs) are found in all kingdoms of life and catalyze important acyl transfer reactions in diverse cellular processes. While many 3D structures of GNATs have been determined, most do not contain acceptor substrates in their active sites. To expand upon existing crystallographic strategies for improving acceptor-bound GNAT structures, we synthesized peptide substrate analogs and reacted them with CoA in PA4794 protein crystals. We found two separate mechanisms for bisubstrate formation: (a) a novel X-ray induced radical-mediated alkylation of CoA with an alkene peptide and (b) direct alkylation of CoA with a halogenated peptide. Our approach is widely applicable across the GNAT superfamily and can be used to improve the success rate of obtaining liganded structures of other acyltransferases.

Structural and Biochemical Characterization of Acinetobacter spp. Aminoglycoside Acetyltransferases Highlights Functional and Evolutionary Variation among Antibiotic Resistance Enzymes.

Modification of aminoglycosides by N-acetyltransferases (AACs) is one of the major mechanisms of resistance to these antibiotics in human bacterial pathogens. More than 50 enzymes belonging to the AAC(6') subfamily have been identified in Gram-negative and Gram-positive clinical isolates. Our understanding of the molecular function and evolutionary origin of these resistance enzymes remains incomplete. Here we report the structural and enzymatic characterization of AAC(6')-Ig and AAC(6')-Ih from Acinetobacter spp. The crystal structure of AAC(6')-Ig in complex with tobramycin revealed a large substrate-binding cleft remaining partially unoccupied by the substrate, which is in stark contrast with the previously characterized AAC(6')-Ib enzyme. Enzymatic analysis indicated that AAC(6')-Ig and -Ih possess a broad specificity against aminoglycosides but with significantly lower turnover rates as compared to other AAC(6') enzymes. Structure- and function-informed phylogenetic analysis of AAC(6') enzymes led to identification of at least three distinct subfamilies varying in oligomeric state, active site composition, and drug recognition mode. Our data support the concept of AAC(6') functionality originating through convergent evolution from diverse Gcn5-related-N-acetyltransferase (GNAT) ancestral enzymes, with AAC(6')-Ig and -Ih representing enzymes that may still retain ancestral nonresistance functions in the cell as provided by their particular active site properties.

Eosinophil-associated processes underlie differences in clinical presentation of loiasis between temporary residents and those indigenous to Loa-endemic areas.

BACKGROUND: Loa loa has emerged as an important public health problem due to the occurrence of immune-mediated severe posttreatment reactions following ivermectin distribution. Also thought to be immune-mediated are the dramatic differences seen in clinical presentation between infected temporary residents (TR) and individuals native to endemic regions (END). METHODS: All patients diagnosed with loiasis at the National Institutes of Health between 1976 and 2012 were included. Patients enrolled in the study underwent a baseline clinical and laboratory evaluation and had serum collected and stored. Stored pretreatment serum was used to measure filaria-specific antibody responses, eosinophil-related cytokines, and eosinophil granule proteins. RESULTS: Loa loa infection in TR was characterized by the presence of Calabar swelling (in 82% of subjects), markedly elevated eosinophil counts, and increased filaria-specific immunoglobulin G (IgG) levels; these findings were thought to reflect an unmodulated immune response. In contrast, END showed strong evidence for immune tolerance to the parasite, with high levels of circulating microfilariae, few clinical symptoms, and diminished filaria-specific IgG. The striking elevation in eosinophil counts among the TR group was accompanied by increased eosinophil granule protein levels (associated with eosinophil activation and degranulation) as well as elevated levels of eosinophil-associated cytokines. CONCLUSIONS: These data support the hypothesis that differing eosinophil-associated responses to the parasite may be responsible for the marked differences in clinical presentations between TR and END populations with loiasis.

Quiescent innate response to infective filariae by human Langerhans cells suggests a strategy of immune evasion.

Filarial infection is initiated by mosquito-derived third-stage larvae (L3) deposited on the skin that transit through the epidermis, which contains Langerhans cells (LC) and keratinocytes (KC), among other cells. This earliest interaction between L3 and the LC likely conditions the priming of the immune system to the parasite. To determine the nature of this interaction, human LC (langerin(+) E-cadherin(+) CD1a(+)) were generated in vitro and exposed to live L3. LC exposed to live L3 for 48 h showed no alterations in the cell surface markers CD14, CD86, CD83, CD207, E-cadherin, CD80, CD40, and HLA-DR or in mRNA expression of inflammation-associated genes, such as those for interleukin 18 (IL-18), IL-18BP, and caspase 1. In contrast to L3, live tachyzoites of Toxoplasma gondii, an intracellular parasite, induced production of CXCL9, IP-10, and IL-6 in LC. Furthermore, preexposure of LC to L3 did not alter Toll-like receptor 3 (TLR3)- or TLR4-mediated expression of the proinflammatory cytokines IL-1ß, gamma interferon (IFN-γ), IL-6, or IL-10. Interestingly, cocultures of KC and LC produced significantly more IL-18, IL-1α, and IL-8 than did cultures of LC alone, although exposure of the cocultures to live L3 did not result in altered cytokine production. Microarray examination of ex vivo LC from skin blisters that were exposed to live L3 also showed few significant changes in gene expression compared with unexposed blisters, further underscoring the relatively muted response of LC to L3. Our data suggest that failure by LC to initiate an inflammatory response to the invasive stage of filarial parasites may be a strategy for immune evasion by the filarial parasite.

Filariasis in travelers presenting to the GeoSentinel Surveillance Network.

BACKGROUND: As international travel increases, there is rising exposure to many pathogens not traditionally encountered in the resource-rich countries of the world. Filarial infections, a great problem throughout the tropics and subtropics, are relatively rare among travelers even to filaria-endemic regions of the world. The GeoSentinel Surveillance Network, a global network of medicine/travel clinics, was established in 1995 to detect morbidity trends among travelers. PRINCIPAL FINDINGS: We examined data from the GeoSentinel database to determine demographic and travel characteristics associated with filaria acquisition and to understand the differences in clinical presentation between nonendemic visitors and those born in filaria-endemic regions of the world. Filarial infections comprised 0.62% (n = 271) of all medical conditions reported to the GeoSentinel Network from travelers; 37% of patients were diagnosed with Onchocerca volvulus, 25% were infected with Loa loa, and another 25% were diagnosed with Wuchereria bancrofti. Most infections were reported from immigrants and from those immigrants returning to their county of origin (those visiting friends and relatives); the majority of filarial infections were acquired in sub-Saharan Africa. Among the patients who were natives of filaria-nonendemic regions, 70.6% acquired their filarial infection with exposure greater than 1 month. Moreover, nonendemic visitors to filaria-endemic regions were more likely to present to GeoSentinel sites with clinically symptomatic conditions compared with those who had lifelong exposure. SIGNIFICANCE: Codifying the filarial infections presenting to the GeoSentinel Surveillance Network has provided insights into the clinical differences seen among filaria-infected expatriates and those from endemic regions and demonstrated that O. volvulus infection can be acquired with short-term travel.

Relapse following discontinuation of imatinib mesylate therapy for FIP1L1/PDGFRA-positive chronic eosinophilic leukemia: implications for optimal dosing.

Although imatinib is clearly the treatment of choice for FIP1L1/PDGFRA-positive chronic eosinophilic leukemia (CEL), little is known about optimal dosing, duration of treatment, and the possibility of cure in this disorder. To address these questions, 5 patients with FIP1L1/PDGFRA-positive CEL with documented clinical, hematologic, and molecular remission on imatinib (400 mg daily) and without evidence of cardiac involvement were enrolled in a dose de-escalation trial. The imatinib dose was tapered slowly with close follow-up for evidence of clinical, hematologic, and molecular relapse. Two patients with endomyocardial fibrosis were maintained on imatinib 300 to 400 mg daily and served as controls. All 5 patients who underwent dose de-escalation, but neither of the control patients, experienced molecular relapse (P < .05). None developed recurrent symptoms, and eosinophil counts, serum B12, and tryptase levels remained suppressed. Reinitiation of therapy at the prior effective dose led to molecular remission in all 5 patients, although 2 patients subsequently required increased dosing to maintain remission. These data are consistent with suppression rather than elimination of the clonal population in FIP1L1/PDGFRA-positive CEL and suggest that molecular monitoring may be the most useful method in determining optimal dosing without the risk of disease exacerbation. This trial was registered at http://www.clinicaltrials.gov as no. NCT00044304.

Rebound eosinophilia after treatment of hypereosinophilic syndrome and eosinophilic gastroenteritis with monoclonal anti-IL-5 antibody SCH55700.

BACKGROUND: Hypereosinophilic syndrome and eosinophilic gastroenteritis with peripheral eosinophilia are characterized by sustained eosinophilia and eosinophil-mediated tissue damage. Although treatment with the humanized monoclonal anti-IL-5 antibody SCH55700 resulted in improvement of eosinophilia and clinical symptoms in 6 of 8 of patients with hypereosinophilic syndrome or eosinophilic gastroenteritis with peripheral eosinophilia for as long as 12 weeks, eosinophil counts subsequently rose above baseline levels, accompanied by an exacerbation of symptoms. OBJECTIVE: To identify the mechanism underlying this rebound eosinophilia. METHODS: Purified eosinophils from patients or normal donors were cultured with IL-5, patient serum, and/or anticytokine antibodies, and eosinophil survival was assessed by flow cytometry. Serum and intracellular cytokine levels were measured by multiplex sandwich ELISA and flow cytometry, respectively. RESULTS: Before treatment with SCH55700, in vitro eosinophil survival in media and in response to recombinant IL-5 was similar in patients and normal donors. At 1 month posttreatment, the eosinophil survival curves were unchanged in 4 of 5 patients in media and in all 5 patients in response to recombinant IL-5. Normal eosinophil survival was prolonged in cultures containing posttreatment but not pretreatment sera (pretreatment vs posttreatment, 10.74% vs 73.02% live cells; P = .01). This posttreatment serum effect on eosinophil survival was reversed by the addition of the monoclonal anti-IL-5 antibody TRFK5. Although increased levels of serum IL-5 were observed at 1 month compared with 2 to 3 days posttreatment in 5 of 6 patients ( P = .04), intracellular cytokine analysis did not reveal increased production of IL-5 by peripheral blood mononuclear cells. CONCLUSIONS: The rebound eosinophilia after SCH55700 treatment is a result of a serum factor that enhances eosinophil survival. Reversal of this effect by the addition of antibody to IL-5 suggests that this factor may be IL-5 itself.

Filaria-induced immune evasion: suppression by the infective stage of Brugia malayi at the earliest host-parasite interface.

To assess the physiologic interactions between the infective stage of Brugia malayi--one of the extracellular parasites responsible for lymphatic filariasis in humans--and the APC with which they come in contact during their development and routes of travel, we have investigated the interaction between the infective stage (L3) of B. malayi and human Langerhans cells (LC) in the skin. Our data indicate that live L3 result in increased migration of LC from the epidermis without affecting the viability of these cells and up-regulation of the IL-18 cytokine involved in LC migration. Live L3 also result in down-regulation of MHC class I and II on the LC cell surface. Additionally, microarray data indicate that live L3 significantly down-regulated expression of IL-8 as well as of multiple genes involved in Ag presentation, reducing the capacity of LC to induce CD4(+) T cells in allogeneic MLR, and thus resulting in a decreased ability of LC to promote CD4(+) T cell proliferation and production of IFN-gamma and IL-10. These data suggest that L3 exert a down-regulatory response in epidermal LC that leads to a diminished capacity of these cells to activate CD4(+) T cells.

Safety and efficacy of the monoclonal anti-interleukin-5 antibody SCH55700 in the treatment of patients with hypereosinophilic syndrome.

Four patients with hypereosinophilic syndrome (HES) refractory to or intolerant of treatment with conventional therapy were treated with a single 1 mg/kg dose of SCH55700. SCH55700 was extremely well tolerated. Two of the 4 patients responded with a fall in eosinophil counts to within the normal range within 48 hours of receiving the drug, accompanied by marked improvement in clinical signs and symptoms. Response was not predicted by serum interleukin-5 (IL-5) levels or presence of the FIP1L1/PDGFRA mutation. Eosinophil counts remained suppressed for up to 12 weeks after treatment; however, exacerbation of symptoms and eosinophilia above baseline levels occurred as drug levels waned. Reinstitution of treatment with monthly SCH55700 led to decreased eosinophilia and symptomatic improvement, albeit to a lesser degree than that seen after the initial dose. These data suggest that anti-IL-5 therapy may be useful in the treatment of HES irrespective of the underlying etiology, although the observed rebound eosinophilia and attenuation of response require further study.

Familial eosinophilia: a benign disorder?

Familial eosinophilia (FE) is an autosomal dominant disorder characterized by marked eosinophilia and progression to end organ damage in some, but not all, affected family members. To better define the pathogenesis of FE, 13 affected and 11 unaffected family members (NLs) underwent a detailed clinical evaluation at the National Institutes of Health (NIH). No clinical abnormalities were more frequent in the family members with FE compared with the NLs. There was, however, a decreased prevalence of asthma in family members with FE compared with unaffected family members. Eosinophil morphology as assessed by either light or transmission electron microscopy was normal in family members with and without FE. Although levels of eosinophil-derived neurotoxin (EDN) and major basic protein (MBP) were elevated in patients with FE compared with NL, levels of both granule proteins were lower than in nonfamilial hypereosinophilic syndrome (HES). Similarly, increased surface expression of the activation markers CD69, CD25, and HLA-DR was detected by flow cytometry on eosinophils from patients with FE compared with NL, albeit less than that seen in HES. These data suggest that, despite prolonged marked eosinophilia, FE can be distinguished from HES by a more benign clinical course that may be related to a relative lack of eosinophil activation.

Molecular remission and reversal of myelofibrosis in response to imatinib mesylate treatment in patients with the myeloproliferative variant of hypereosinophilic syndrome.

We recently described a subset of patients with a myeloproliferative variant of hypereosinophilic syndrome (MHES) characterized by elevated serum tryptase levels, increased atypical mast cells in the bone marrow, tissue fibrosis, and the presence of the fusion tyrosine kinase, FIP1L1-PDGFRalpha, which is a therapeutic target of imatinib mesylate. Seven patients with MHES were treated with imatinib mesylate (300-400 mg daily). Clinical improvement and resolution of eosinophilia was observed in all patients, although cardiac dysfunction, when present, was not altered by therapy. Reversal of bone marrow pathology, including increased cellularity, the presence of spindle-shaped mast cells, and myelofibrosis, was evident in all patients at 4 to 8 weeks following initiation of therapy. This was accompanied by a decrease in activated eosinophils and mast cells in the peripheral blood and bone marrow, respectively. Serum tryptase levels declined rapidly to normal levels in all patients and remained in the normal range throughout therapy. Molecular remission, with disappearance of detectable FIP1L1/PDGFRA (F/P) transcripts, was achieved in 5 of 6 patients tested. The lack of reversal of cardiac abnormalities and persistence of the F/P mutation in some patients suggests that early intervention with higher doses of imatinib mesylate may be desirable in the treatment of patients with MHES.

Elevated serum tryptase levels identify a subset of patients with a myeloproliferative variant of idiopathic hypereosinophilic syndrome associated with tissue fibrosis, poor prognosis, and imatinib responsiveness.

Since serum tryptase levels are elevated in some patients with myeloproliferative disorders, we examined their utility in identifying a subset of patients with hypereosinophilic syndrome (HES) and an underlying myeloproliferative disorder. Elevated serum tryptase levels (> 11.5 ng/mL) were present in 9 of 15 patients with HES and were associated with other markers of myeloproliferation, including elevated B12 levels and splenomegaly. Although bone marrow biopsies in these patients showed increased numbers of CD25+ mast cells and atypical spindle-shaped mast cells, patients with HES and elevated serum tryptase could be distinguished from patients with systemic mastocytosis and eosinophilia by their clinical manifestations, the absence of mast cell aggregates, the lack of a somatic KIT mutation, and the presence of the recently described fusion of the Fip1-like 1 (FIP1L1) gene to the platelet-derived growth factor receptor alpha gene (PDGFRA). Patients with HES and elevated serum tryptase were more likely to develop fibroproliferative end organ damage, and 3 of 9 died within 5 years of diagnosis in contrast to 0 of 6 patients with normal serum tryptase levels. All 6 patients with HES and elevated tryptase treated with imatinib demonstrated a clinical and hematologic response. In summary, elevated serum tryptase appears to be a sensitive marker of a myeloproliferative variant of HES that is characterized by tissue fibrosis, poor prognosis, and imatinib responsiveness.