Emerging Cerebrospinal Fluid Biomarkers of Disease Activity and Progression in Multiple Sclerosis.

Importance: Biomarkers distinguishing nonrelapsing progressive disease biology from relapsing biology in multiple sclerosis (MS) are lacking. Cerebrospinal fluid (CSF) is an accessible fluid that most closely reflects central nervous system biology. Objective: To identify CSF biological measures associated with progressive MS pathobiology. Design, Setting, and Participants: This cohort study assessed data from 2 prospective MS cohorts: a test cohort provided serial CSF, clinical, and imaging assessments in a multicenter study of patients with relapsing MS (RMS) or primary progressive MS (PPMS) who were initiating anti-CD20 treatment (recruitment: 2016-2018; analysis: 2020-2023). A single-site confirmation cohort was used to assess CSF at baseline and long-term (>10 year) clinical follow-up (analysis: 2022-2023). Exposures: Test-cohort participants initiated standard-of-care ocrelizumab treatment. Confirmation-cohort participants were untreated or received standard-of-care disease-modifying MS therapies. Main Outcomes and Measures: Twenty-five CSF markers, including neurofilament light chain, neurofilament heavy chain, and glial fibrillary acid protein (GFAP); 24-week confirmed disability progression (CDP24); and brain magnetic resonance imaging measures reflecting focal injury, tissue loss, and progressive biology (slowly expanding lesions [SELs]). Results: The test cohort (n = 131) included 100 patients with RMS (mean [SD] age, 36.6 [10.4] years; 68 [68%] female and 32 [32%] male; Expanded Disability Status Scale [EDSS] score, 0-5.5), and 31 patients with PPMS (mean [SD] age, 44.9 [7.4] years; 15 [48%] female and 16 [52%] male; EDSS score, 3.0-6.5). The confirmation cohort (n = 68) included 41 patients with RMS and 27 with PPMS enrolled at diagnosis (age, 40 years [range, 20-61 years]; 47 [69%] female and 21 [31%] male). In the test cohort, GFAP was correlated with SEL count (r = 0.33), greater proportion of T2 lesion volume from SELs (r = 0.24), and lower T1-weighted intensity within SELs (r = -0.33) but not with acute inflammatory measures. Neurofilament heavy chain was correlated with SEL count (r = 0.25) and lower T1-weighted intensity within SELs (r = -0.28). Immune markers correlated with measures of acute inflammation and, unlike GFAP, were impacted by anti-CD20. In the confirmation cohort, higher baseline CSF GFAP levels were associated with long-term CDP24 (hazard ratio, 2.1; 95% CI, 1.3-3.4; P = .002). Conclusions and Relevance: In this study, activated glial markers (in particular GFAP) and neurofilament heavy chain were associated specifically with nonrelapsing progressive disease outcomes (independent of acute inflammatory activity). Elevated CSF GFAP was associated with long-term MS disease progression.

Development of an LC-MS/MS Method to Measure Sphingolipids in CSF from Patients with Multiple Sclerosis.

Multiple sclerosis is an inflammatory and degenerative disease characterized by different clinical courses including relapsing multiple sclerosis (RMS) and primary progressive multiple sclerosis (PPMS). A hallmark of patients with multiple sclerosis (pwMS) includes a putative autoimmune response, which results in demyelination and neuroaxonal damage in the central nervous system. Sphingolipids in cerebrospinal fluid (CSF) have been proposed as potential biomarkers reflective of disease activity in pwMS. Hence, sensitive methods to accurately quantify sphingolipids in CSF are needed. In this study, we report the development of a sensitive high-throughput multiplexed liquid chromatography coupled to a tandem mass spectrometry method to perform quantitation on 14 species of sphingolipids in human CSF. We applied this method to measure CSF sphingolipids in healthy controls (n = 10), PPMS (n = 27), and RMS (n = 17) patients before and after ocrelizumab treatment. The median CSF levels of the 14 sphingolipids measured herein was higher in PPMS (17.2 ng/mL) and RMS (17.6 ng/mL) when compared with the healthy controls (13.8 ng/mL). Levels of sphingolipids were decreased by 8.6% at week 52 after treatment with ocrelizumab in RMS patients but not in PPMS patients. Specifically, C16 glucosylceramide (-26%; P = 0.004) and C18 ceramides (-13%; P = 0.042) decreased from baseline in RMS patients. Additionally, in PPMS patients C16 glucosylceramide levels correlated with CSF neurofilament heavy levels at baseline (Rho =0.532; P = 0.004) and after treatment (Rho =0.424; P = 0.028). Collectively, these results indicate that CSF sphingolipid levels are altered in pwMS and treatment with ocrelizumab results in significant shifts in the sphingolipid profile that may reflect a reduction in disease activity supporting further investigation into sphingolipids as tools to monitor disease state. SIGNIFICANCE STATEMENT: This study describes the development of a new method to measure 14 sphingolipid species in CSF. These results demonstrate that sphingolipids levels are elevated in CSF from pwMS compared to healthy controls. Distinct sphingolipid signatures were observed between patients with different clinical disease courses, and these lipid signatures changed after treatment with ocrelizumab, especially in RMS patients. This method enables further investigation into the role of sphingolipids as candidate biomarkers in pwMS and other central nervous system disorders.

Validation of a murine proteome-wide phage display library for identification of autoantibody specificities.

Autoimmunity is characterized by loss of tolerance to tissue-specific as well as systemic antigens, resulting in complex autoantibody landscapes. Here, we introduce and extensively validate the performance characteristics of a murine proteome-wide library for phage display immunoprecipitation and sequencing (PhIP-seq) in profiling mouse autoantibodies. This library was validated using 7 genetically distinct mouse lines across a spectrum of autoreactivity. Mice deficient in antibody production (Rag2-/- and µMT) were used to model nonspecific peptide enrichments, while cross-reactivity was evaluated using anti-ovalbumin B cell receptor-restricted OB1 mice as a proof of principle. The PhIP-seq approach was then utilized to interrogate 3 distinct autoimmune disease models. First, serum from Lyn-/- IgD+/- mice with lupus-like disease was used to identify nuclear and apoptotic bleb reactivities. Second, serum from nonobese diabetic (NOD) mice, a polygenic model of pancreas-specific autoimmunity, was enriched in peptides derived from both insulin and predicted pancreatic proteins. Lastly, Aire-/- mouse sera were used to identify numerous autoantigens, many of which were also observed in previous studies of humans with autoimmune polyendocrinopathy syndrome type 1 carrying recessive mutations in AIRE. These experiments support the use of murine proteome-wide PhIP-seq for antigenic profiling and autoantibody discovery, which may be employed to study a range of immune perturbations in mouse models of autoimmunity profiling.

Validation of a murine proteome-wide phage display library for the identification of autoantibody specificities.

Autoimmunity is characterized by loss of tolerance to tissue-specific as well as systemic antigens, resulting in complex autoantibody landscapes. Here, we introduce and extensively validate the performance characteristics of a murine proteome-wide library for phage display immunoprecipitation and sequencing (PhIP-seq), to profile mouse autoantibodies. This system and library were validated using seven genetic mouse models across a spectrum of autoreactivity. Mice deficient in antibody production (Rag2-/- and µMT) were used to model non-specific peptide enrichments, while cross-reactivity was evaluated using anti-ovalbumin B cell receptor (BCR)-restricted OB1 mice as a proof of principle. The PhIP-seq approach was then utilized to interrogate three distinct autoimmune disease models. First, serum from Lyn-/- IgD+/- mice with lupus-like disease was used to identify nuclear and apoptotic bleb reactivities, lending support to the hypothesis that apoptosis is a shared origin of these antigens. Second, serum from non-obese diabetic (NOD) mice, a polygenic model of pancreas-specific autoimmunity, enriched peptides derived from both insulin and predicted pancreatic proteins. Lastly, Aire-/- mouse sera were used to identify numerous auto-antigens, many of which were also observed in previous studies of humans with autoimmune polyendocrinopathy syndrome type 1 (APS1) carrying recessive mutations in AIRE. Among these were peptides derived from Perilipin-1, a validated autoimmune biomarker of generalized acquired lipodystrophy in humans. Autoreactivity to Perilipin-1 correlated with lymphocyte infiltration in adipose tissue and underscores the approach in revealing previously unknown specificities. These experiments support the use of murine proteome-wide PhIP-seq for antigenic profiling and autoantibody discovery, which may be employed to study a range of immune perturbations in mouse models of autoimmunity.

Metagenomic characterization of swine slurry in a North American swine farm operation.

Modern day large-scale, high-density farming environments are inherently susceptible to viral outbreaks, inadvertently creating conditions that favor increased pathogen transmission and potential zoonotic spread. Metagenomic sequencing has proven to be a useful tool for characterizing the microbial burden in both people, livestock, and environmental samples. International efforts have been successful at characterizing pathogens in commercial farming environments, especially swine farms, however it is unclear whether the full extent of microbial agents have been adequately captured or is representative of farms elsewhere. To augment international efforts we performed metagenomic next-generation sequencing on nine swine slurry and three environmental samples from a United States of America (U.S.A.) farm operation, characterized the microbial composition of slurry, and identified novel viruses. We assembled a remarkable total of 1792 viral genomes, of which 554 were novel/divergent. We assembled 1637 Picobirnavirus genome segments, of which 538 are novel. In addition, we discovered 10 new viruses belonging to a novel taxon: porcine Statoviruses; which have only been previously reported in human, macaques, mouse, and cows. We assembled 3 divergent Posaviruses and 3 swine Picornaviruses. In addition to viruses described, we found other eukaryotic genera such as Entamoeba and Blastocystis, and bacterial genera such as Listeria, Treponema, Peptoclostridium and Bordetella in the slurry. Of these, two species Entamoeba histolytica and Listeria monocytogenes known to cause human disease were detected. Further, antimicrobial resistance genes such as tetracycline and MLS (macrolide, lincosamide, streptogramin) were also identified. Metagenomic surveillance in swine fecal slurry has great potential for novel and antimicrobial resistant pathogen detection.

Use of unbiased metagenomic and transcriptomic analyses to investigate the association between feline calicivirus and feline chronic gingivostomatitis in domestic cats.

OBJECTIVE: To identify associations between microbes and host genes in cats with feline chronic gingivostomatitis (FCGS), a debilitating inflammatory oral mucosal disease with no known cause, compared with healthy cats and cats with periodontitis (control cats). ANIMALS: 19 control cats and 23 cats with FCGS. PROCEDURES: At least 1 caudal oral mucosal swab specimen was obtained from each cat. Each specimen underwent unbiased metatranscriptomic next-generation RNA sequencing (mNGS). Filtered mNGS reads were aligned to all known genetic sequences from all organisms and to the cat transcriptome. The relative abundances of microbial and host gene read alignments were compared between FCGS-affected cats and control cats and between FCGS-affected cats that did and did not clinically respond to primary treatment. Assembled feline calicivirus (FCV) genomes were compared with reverse transcription PCR (RT-PCR) primers commonly used to identify FCV. RESULTS: The only microbe strongly associated with FCGS was FCV, which was detected in 21 of 23 FCGS-affected cats but no control cats. Problematic base pair mismatches were identified between the assembled FCV genomes and RT-PCR primers. Puma feline foamy virus was detected in 9 of 13 FCGS-affected cats that were refractory to treatment and 5 healthy cats but was not detected in FCGS-affected cats that responded to tooth extractions. The most differentially expressed genes in FCGS-affected cats were those associated with antiviral activity. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that FCGS pathogenesis has a viral component. Many FCV strains may yield false-negative results on RT-PCR-based assays. Coinfection of FCGS-affected cats with FCV and puma feline foamy virus may adversely affect response to treatment.

Gut microbiota-specific IgA+ B cells traffic to the CNS in active multiple sclerosis.

Changes in gut microbiota composition and a diverse role of B cells have recently been implicated in multiple sclerosis (MS), a central nervous system (CNS) autoimmune disease. Immunoglobulin A (IgA) is a key regulator at the mucosal interface. However, whether gut microbiota shape IgA responses and what role IgA+ cells have in neuroinflammation are unknown. Here, we identify IgA-bound taxa in MS and show that IgA-producing cells specific for MS-associated taxa traffic to the inflamed CNS, resulting in a strong, compartmentalized IgA enrichment in active MS and other neuroinflammatory diseases. Unlike previously characterized polyreactive anti-commensal IgA responses, CNS IgA cross-reacts with surface structures on specific bacterial strains but not with brain tissue. These findings establish gut microbiota-specific IgA+ cells as a systemic mediator in MS and suggest a critical role of mucosal B cells during active neuroinflammation with broad implications for IgA as an informative biomarker and IgA-producing cells as an immune subset to harness for therapeutic interventions.

A pathogenic and clonally expanded B cell transcriptome in active multiple sclerosis.

Central nervous system B cells have several potential roles in multiple sclerosis (MS): secretors of proinflammatory cytokines and chemokines, presenters of autoantigens to T cells, producers of pathogenic antibodies, and reservoirs for viruses that trigger demyelination. To interrogate these roles, single-cell RNA sequencing (scRNA-Seq) was performed on paired cerebrospinal fluid (CSF) and blood from subjects with relapsing-remitting MS (RRMS; n = 12), other neurologic diseases (ONDs; n = 1), and healthy controls (HCs; n = 3). Single-cell immunoglobulin sequencing (scIg-Seq) was performed on a subset of these subjects and additional RRMS (n = 4), clinically isolated syndrome (n = 2), and OND (n = 2) subjects. Further, paired CSF and blood B cell subsets (RRMS; n = 7) were isolated using fluorescence activated cell sorting for bulk RNA sequencing (RNA-Seq). Independent analyses across technologies demonstrated that nuclear factor kappa B (NF-κB) and cholesterol biosynthesis pathways were activated, and specific cytokine and chemokine receptors were up-regulated in CSF memory B cells. Further, SMAD/TGF-ß1 signaling was down-regulated in CSF plasmablasts/plasma cells. Clonally expanded, somatically hypermutated IgM+ and IgG1+ CSF B cells were associated with inflammation, blood-brain barrier breakdown, and intrathecal Ig synthesis. While we identified memory B cells and plasmablast/plasma cells with highly similar Ig heavy-chain sequences across MS subjects, similarities were also identified with ONDs and HCs. No viral transcripts, including from Epstein-Barr virus, were detected. Our findings support the hypothesis that in MS, CSF B cells are driven to an inflammatory and clonally expanded memory and plasmablast/plasma cell phenotype.

Genomic and serologic characterization of enterovirus A71 brainstem encephalitis.

OBJECTIVE: In 2016, Catalonia experienced a pediatric brainstem encephalitis outbreak caused by enterovirus A71 (EV-A71). Conventional testing identified EV in the periphery but rarely in CSF. Metagenomic next-generation sequencing (mNGS) and CSF pan-viral serology (VirScan) were deployed to enhance viral detection and characterization. METHODS: RNA was extracted from the CSF (n = 20), plasma (n = 9), stool (n = 15), and nasopharyngeal samples (n = 16) from 10 children with brainstem encephalitis and 10 children with meningitis or encephalitis. Pathogens were identified using mNGS. Available CSF from cases (n = 12) and pediatric other neurologic disease controls (n = 54) were analyzed with VirScan with a subset (n = 9 and n = 50) validated by ELISA. RESULTS: mNGS detected EV in all samples positive by quantitative reverse transcription polymerase chain reaction (qRT-PCR) (n = 25). In qRT-PCR-negative samples (n = 35), mNGS found virus in 23% (n = 8, 3 CSF samples). Overall, mNGS enhanced EV detection from 42% (25/60) to 57% (33/60) (p-value = 0.013). VirScan and ELISA increased detection to 92% (11/12) compared with 46% (4/12) for CSF mNGS and qRT-PCR (p-value = 0.023). Phylogenetic analysis confirmed the EV-A71 strain clustered with a neurovirulent German EV-A71. A single amino acid substitution (S241P) in the EVA71 VP1 protein was exclusive to the CNS in one subject. CONCLUSION: mNGS with VirScan significantly increased the CNS detection of EVs relative to qRT-PCR, and the latter generated an antigenic profile of the acute EV-A71 immune response. Genomic analysis confirmed the close relation of the outbreak EV-A71 and neuroinvasive German EV-A71. A S241P substitution in VP1 was found exclusively in the CSF.

Unbiased Metagenomic Sequencing for Pediatric Meningitis in Bangladesh Reveals Neuroinvasive Chikungunya Virus Outbreak and Other Unrealized Pathogens.

The burden of meningitis in low-and-middle-income countries remains significant, but the infectious causes remain largely unknown, impeding institution of evidence-based treatment and prevention decisions. We conducted a validation and application study of unbiased metagenomic next-generation sequencing (mNGS) to elucidate etiologies of meningitis in Bangladesh. This RNA mNGS study was performed on cerebrospinal fluid (CSF) specimens from patients admitted in the largest pediatric hospital, a World Health Organization sentinel site, with known neurologic infections (n = 36), with idiopathic meningitis (n = 25), and with no infection (n = 30), and six environmental samples, collected between 2012 and 2018. We used the IDseq bioinformatics pipeline and machine learning to identify potentially pathogenic microbes, which we then confirmed orthogonally and followed up through phone/home visits. In samples with known etiology and without infections, there was 83% concordance between mNGS and conventional testing. In idiopathic cases, mNGS identified a potential bacterial or viral etiology in 40%. There were three instances of neuroinvasive Chikungunya virus (CHIKV), whose genomes were >99% identical to each other and to a Bangladeshi strain only previously recognized to cause febrile illness in 2017. CHIKV-specific qPCR of all remaining stored CSF samples from children who presented with idiopathic meningitis in 2017 (n = 472) revealed 17 additional CHIKV meningitis cases, exposing an unrecognized meningitis outbreak. Orthogonal molecular confirmation, case-based clinical data, and patient follow-up substantiated the findings. Case-control CSF mNGS surveys can complement conventional diagnostic methods to identify etiologies of meningitis, conduct surveillance, and predict outbreaks. The improved patient- and population-level data can inform evidence-based policy decisions.IMPORTANCE Globally, there are an estimated 10.6 million cases of meningitis and 288,000 deaths every year, with the vast majority occurring in low- and middle-income countries. In addition, many survivors suffer from long-term neurological sequelae. Most laboratories assay only for common bacterial etiologies using culture and directed PCR, and the majority of meningitis cases lack microbiological diagnoses, impeding institution of evidence-based treatment and prevention strategies. We report here the results of a validation and application study of using unbiased metagenomic sequencing to determine etiologies of idiopathic (of unknown cause) cases. This included CSF from patients with known neurologic infections, with idiopathic meningitis, and without infection admitted in the largest children's hospital of Bangladesh and environmental samples. Using mNGS and machine learning, we identified and confirmed an etiology (viral or bacterial) in 40% of idiopathic cases. We detected three instances of Chikungunya virus (CHIKV) that were >99% identical to each other and to a strain previously recognized to cause systemic illness only in 2017. CHIKV qPCR of all remaining stored 472 CSF samples from children who presented with idiopathic meningitis in 2017 at the same hospital uncovered an unrecognized CHIKV meningitis outbreak. CSF mNGS can complement conventional diagnostic methods to identify etiologies of meningitis, and the improved patient- and population-level data can inform better policy decisions.

Pan-viral serology implicates enteroviruses in acute flaccid myelitis.

Since 2012, the United States of America has experienced a biennial spike in pediatric acute flaccid myelitis (AFM)1-6. Epidemiologic evidence suggests non-polio enteroviruses (EVs) are a potential etiology, yet EV RNA is rarely detected in cerebrospinal fluid (CSF)2. CSF from children with AFM (n = 42) and other pediatric neurologic disease controls (n = 58) were investigated for intrathecal antiviral antibodies, using a phage display library expressing 481,966 overlapping peptides derived from all known vertebrate and arboviruses (VirScan). Metagenomic next-generation sequencing (mNGS) of AFM CSF RNA (n = 20 cases) was also performed, both unbiased sequencing and with targeted enrichment for EVs. Using VirScan, the viral family significantly enriched by the CSF of AFM cases relative to controls was Picornaviridae, with the most enriched Picornaviridae peptides belonging to the genus Enterovirus (n = 29/42 cases versus 4/58 controls). EV VP1 ELISA confirmed this finding (n = 22/26 cases versus 7/50 controls). mNGS did not detect additional EV RNA. Despite rare detection of EV RNA, pan-viral serology frequently identified high levels of CSF EV-specific antibodies in AFM compared with controls, providing further evidence for a causal role of non-polio EVs in AFM.

Isolation and Structure of an Antibody that Fully Neutralizes Isolate SIVmac239 Reveals Functional Similarity of SIV and HIV Glycan Shields.

HIV- and SIV-envelope (Env) trimers are both extensively glycosylated, and antibodies identified to date have been unable to fully neutralize SIVmac239. Here, we report the isolation, structure, and glycan interactions of antibody ITS90.03, a monoclonal antibody that completely neutralized the highly neutralization-resistant isolate, SIVmac239. The co-crystal structure of a fully glycosylated SIVmac239-gp120 core in complex with rhesus CD4 and the antigen-binding fragment of ITS90.03 at 2.5-Å resolution revealed that ITS90 recognized an epitope comprised of 45% glycan. SIV-gp120 core, rhesus CD4, and their complex could each be aligned structurally to their human counterparts. The structure revealed that glycans masked most of the SIV Env protein surface, with ITS90 targeting a glycan hole, which is occupied in ∼83% of SIV strains by glycan N238. Overall, the SIV glycan shield appears to functionally resemble its HIV counterpart in coverage of spike, shielding from antibody, and modulation of receptor accessibility.

Metagenomic next-generation sequencing of samples from pediatric febrile illness in Tororo, Uganda.

Febrile illness is a major burden in African children, and non-malarial causes of fever are uncertain. In this retrospective exploratory study, we used metagenomic next-generation sequencing (mNGS) to evaluate serum, nasopharyngeal, and stool specimens from 94 children (aged 2-54 months) with febrile illness admitted to Tororo District Hospital, Uganda. The most common microbes identified were Plasmodium falciparum (51.1% of samples) and parvovirus B19 (4.4%) from serum; human rhinoviruses A and C (40%), respiratory syncytial virus (10%), and human herpesvirus 5 (10%) from nasopharyngeal swabs; and rotavirus A (50% of those with diarrhea) from stool. We also report the near complete genome of a highly divergent orthobunyavirus, tentatively named Nyangole virus, identified from the serum of a child diagnosed with malaria and pneumonia, a Bwamba orthobunyavirus in the nasopharynx of a child with rash and sepsis, and the genomes of two novel human rhinovirus C species. In this retrospective exploratory study, mNGS identified multiple potential pathogens, including 3 new viral species, associated with fever in Ugandan children.

Longitudinally persistent cerebrospinal fluid B cells can resist treatment in multiple sclerosis.

B cells are key contributors to chronic autoimmune pathology in multiple sclerosis (MS). Clonally related B cells exist in the cerebrospinal fluid (CSF), meninges, and CNS parenchyma of MS patients. We sought to investigate the presence of clonally related B cells over time by performing Ig heavy chain variable region repertoire sequencing on B cells from longitudinally collected blood and CSF samples of MS patients (n = 10). All patients were untreated at the time of the initial sampling; the majority (n = 7) were treated with immune-modulating therapies 1.2 (±0.3 SD) years later during the second sampling. We found clonal persistence of B cells in the CSF of 5 patients; these B cells were frequently Ig class-switched and CD27+. Specific blood B cell subsets appear to provide input into CNS repertoires over time. We demonstrate complex patterns of clonal B cell persistence in CSF and blood, even in patients on immune-modulating therapy. Our findings support the concept that peripheral B cell activation and CNS-compartmentalized immune mechanisms can in part be therapy resistant.

Structure and Diversity of the Rhesus Macaque Immunoglobulin Loci through Multiple De Novo Genome Assemblies.

The rhesus macaque is a critically important animal model in biomedical research, most recently playing a key role in the development of vaccines against human immunodeficiency virus-1. Nevertheless, the immunoglobulin (Ig) loci of macaques are as yet incompletely determined and our understanding of differences between human and macaque humoral immunity remains deficient. We completed a high-coverage, high-quality whole genome sequencing and assembly project with a single rhesus macaque of Indian origin, and partial genome assemblies using genomic molecular targeting of the Ig loci in nine other rhesus macaques of Indian origin. These data indicate that the macaque Ig loci are substantially more diverse than those in humans, including greater sequence diversity and copy-number variation between individuals. It appears likely that such copy-number variation even occurs between allelic loci within individuals. Different Ig gene families in the macaque show distinct relationships to the corresponding human gene families and appear to evolve under different mechanisms. These results raise intriguing questions about the evolution of antigen receptors in primates but also have important practical implications for the design and interpretation of biomedical studies.

Initiation of HIV neutralizing B cell lineages with sequential envelope immunizations.

A strategy for HIV-1 vaccine development is to define envelope (Env) evolution of broadly neutralizing antibodies (bnAbs) in infection and to recreate those events by vaccination. Here, we report host tolerance mechanisms that limit the development of CD4-binding site (CD4bs), HCDR3-binder bnAbs via sequential HIV-1 Env vaccination. Vaccine-induced macaque CD4bs antibodies neutralize 7% of HIV-1 strains, recognize open Env trimers, and accumulate relatively modest somatic mutations. In naive CD4bs, unmutated common ancestor knock-in mice Env+B cell clones develop anergy and partial deletion at the transitional to mature B cell stage, but become Env- upon receptor editing. In comparison with repetitive Env immunizations, sequential Env administration rescue anergic Env+ (non-edited) precursor B cells. Thus, stepwise immunization initiates CD4bs-bnAb responses, but immune tolerance mechanisms restrict their development, suggesting that sequential immunogen-based vaccine regimens will likely need to incorporate strategies to expand bnAb precursor pools.

Analysis of immunoglobulin transcripts and hypermutation following SHIV(AD8) infection and protein-plus-adjuvant immunization.

Developing predictive animal models to assess how candidate vaccines and infection influence the ontogenies of Envelope (Env)-specific antibodies is critical for the development of an HIV vaccine. Here we use two nonhuman primate models to compare the roles of antigen persistence, diversity and innate immunity. We perform longitudinal analyses of HIV Env-specific B-cell receptor responses to SHIV(AD8) infection and Env protein vaccination with eight different adjuvants. A subset of the SHIV(AD8)-infected animals with higher viral loads and greater Env diversity show increased neutralization associated with increasing somatic hypermutation (SHM) levels over time. The use of adjuvants results in increased ELISA titres but does not affect the mean SHM levels or CDR H3 lengths. Our study shows how the ontogeny of Env-specific B cells can be tracked, and provides insights into the requirements for developing neutralizing antibodies that should facilitate translation to human vaccine studies.

Population genetics of the filarial worm wuchereria bancrofti in a post-treatment region of Papua New Guinea: insights into diversity and life history.

BACKGROUND: Wuchereria bancrofti (Wb) is the primary causative agent of lymphatic filariasis (LF). Our studies of LF in Papua New Guinea (PNG) have shown that it is possible to reduce the prevalence of Wb in humans and mosquitoes through mass drug administration (MDA; diethylcarbamazine with/without ivermectin). While MDAs in the Dreikikir region through 1998 significantly reduced prevalence of Wb infection, parasites continue to be transmitted in the area. METHODS: We sequenced the Wb mitochondrial Cytochrome Oxidase 1 (CO1) gene from 16 people infected with Wb. Patients were selected from 7 villages encompassing both high and moderate annual transmission potentials (ATP). We collected genetic data with the objectives to (i) document contemporary levels of genetic diversity and (ii) distinguish between populations of parasites and hosts across the study area. PRINCIPLE FINDINGS: We discovered 109 unique haplotypes currently segregating in the Wb parasite population, with one common haplotype present in 15 out of 16 infections. We found that parasite diversity was similar among people residing within the same village and clustered within transmission zones. For example, in the high transmission area, diversity tended to be more similar between neighboring villages, while in the moderate transmission area, diversity tended to be less similar. CONCLUSIONS: In the Dreikikir region of PNG there are currently high levels of genetic diversity in populations of Wb. High levels of genetic diversity may complicate future MDAs in this region and the presence of dominant haplotypes will require adjustments to current elimination strategies.

The complete mitochondrial genome sequence of the filarial nematode Wuchereria bancrofti from three geographic isolates provides evidence of complex demographic history.

Mitochondrial (mt) genome sequences have enabled comparison of population genetics and evolution for numerous free-living and parasitic nematodes. Here we define the complete mt genome of Wuchereria bancrofti through analysis of isolates from Papua New Guinea, India and West Africa. Sequences were assembled for each isolate and annotated with reference to the mt genome sequence for Brugia malayi. The length of the W. bancrofti mt genome is approximately 13,637 nucleotides, contains 2 ribosomal RNAs (rrns), 22 transfer RNAs (trns), 12 protein-coding genes, and is characterized by a 74.6% AT content. The W. bancrofti mt gene order is identical to that reported for Onchocerca volvulus, Dirofilaria immitis, Setaria digitata and B. malayi. In addition to using translational start codons identified previously in the mt protein-coding genes of other filarial nematodes, W. bancrofti appears to be unique in using TGT as a translational start codon. Similarly, use of incomplete stop codons in mt protein-coding genes appears to be more common in W. bancrofti than in other human filarial parasites. The complete mt genome sequence reported here provides new genetic markers for investigating phylogenetic and geographic relationships between isolates, and assessing population diversity within endemic regions. The sequence polymorphism enables new strategies to monitor the progress of public health interventions to control and eliminate this important human parasite. We illustrate the utility of this sequence and single nucleotide polymorphisms by inferring the divergence times between the three W. bancrofti isolates, suggesting predictions into their origin and migration.