Use of a Mobile App to Augment Psychotherapy in a Community Psychiatric Clinic: Feasibility and Fidelity Trial.

BACKGROUND: Even though 1 in 5 Americans experience some form of mental illness each year, 80% have been shown to discontinue psychotherapy prematurely. The traditional psychotherapy service delivery model, consisting of isolated clinical sessions, lacks the ability to keep patients engaged outside clinical sessions. Newer digital mental health platforms can address the clinical need for a robust tool that tracks mental well-being and improves engagement in patients with depressive symptoms. OBJECTIVE: The primary goals of this feasibility study were to (1) assess compliance among providers and their patients with a digital mental health platform protocol, and (2) examine the usability and fidelity of a mobile app through structured participant feedback. METHODS: A sample of 30 participants was recruited for a 5-week study from a community-based mental health clinic in Baltimore, Maryland, USA. Inclusion criteria were: aged 18 years or older, having access to a smartphone, and having at least mild-to-moderate depression and/or anxiety as measured by the Patient Health Questionnaire-9 (PHQ-9) and Generalized Anxiety Disorder-7 (GAD-7) scales, respectively. Eligible participants were randomized into one of two study arms: (1) the intervention arm or (2) the waitlist control arm. Participants in the intervention arm were asked to download the Rose app and were prompted to complete clinical assessments (PHQ-9 and GAD-7) every other week, daily mood and anxiety Likert scales, and daily journal entries. The participants in the waitlist arm served as controls for the study and completed the clinical assessments only. Both arms engaged in weekly psychotherapy sessions, with participant in-app input informing the psychotherapy process of the intervention arm, while those in the waitlist control arm continued their standard care. Outcomes of interest included adherence to completion of in-app assessments and usability of the Rose mobile app assessed through the modified Mobile Application Rating Scale. RESULTS: Over the study period, a sample of 30 participants used the Rose app 2834 times to complete clinical assessments. On average, 70% (21; 95% CI 61.14%-77.41%) of participants completed mood and anxiety daily check-ins and journal entries 5 days per week. Nearly all participants (29/30, 97%) completed all PHQ-9 and GAD-7 in-app scales during the study. Subjective impressions showed that 73% (22/30) of participants found the mobile app to be engaging and in line with their needs, and approximately 70% (21/30) of participants reported the app functionality and quality of information to be excellent. Additionally, more than two-thirds of the participants felt that their knowledge and awareness of depression and anxiety management improved through using the app. CONCLUSIONS: Steady compliance and high app ratings showcase the utility of the Rose mobile mental health app in augmenting the psychotherapy process for patients with mood disorders and improving mental health knowledge. Future studies are needed to further examine the impact of Rose on treatment outcomes. TRIAL REGISTRATION: ClinicalTrials.gov NCT04200170; https://clinicaltrials.gov/ct2/show/NCT04200170.

A Hepatitis C Virus Envelope Polymorphism Confers Resistance to Neutralization by Polyclonal Sera and Broadly Neutralizing Monoclonal Antibodies.

UNLABELLED: Hepatitis C virus (HCV) infection is a global health problem, with millions of chronically infected individuals at risk for cirrhosis and hepatocellular carcinoma. HCV vaccine development is vital in the effort toward disease control and eradication, an undertaking aided by an increased understanding of the mechanisms of resistance to broadly neutralizing antibodies (bNAbs). In this study, we identified HCV codons that vary deep in a phylogenetic tree of HCV sequences and showed that a polymorphism at one of these positions renders Bole1a, a computationally derived, ancestral genotype 1a HCV strain, resistant to neutralization by both polyclonal-HCV-infected plasma and multiple broadly neutralizing monoclonal antibodies with unique binding epitopes. This bNAb resistance mutation reduces replicative fitness, which may explain the persistence of both neutralization-sensitive and neutralization-resistant variants in circulating viral strains. This work identifies an important determinant of bNAb resistance in an ancestral, representative HCV genome, which may inform HCV vaccine development. IMPORTANCE: Worldwide, more than 170 million people are infected with hepatitis C virus (HCV), the leading cause of hepatocellular carcinoma and liver transplantation in the United States. Despite recent significant advances in HCV treatment, a vaccine is needed. Control of the HCV pandemic with drug treatment alone is likely to fail due to limited access to treatment, reinfections in high-risk individuals, and the potential for resistance to direct-acting antivirals (DAAs). Broadly neutralizing antibodies (bNAbs) block infection by diverse HCV variants and therefore serve as a useful guide for vaccine development, but our understanding of resistance to bNAbs is incomplete. In this report, we identify a viral polymorphism conferring resistance to neutralization by both polyclonal plasma and broadly neutralizing monoclonal antibodies, which may inform HCV vaccine development.

Reconstructing a B-Cell Clonal Lineage. II. Mutation, Selection, and Affinity Maturation.

Affinity maturation of the antibody response is a fundamental process in adaptive immunity during which B-cells activated by infection or vaccination undergo rapid proliferation accompanied by the acquisition of point mutations in their rearranged immunoglobulin (Ig) genes and selection for increased affinity for the eliciting antigen. The rate of somatic hypermutation at any position within an Ig gene is known to depend strongly on the local DNA sequence, and Ig genes have region-specific codon biases that influence the local mutation rate within the gene resulting in increased differential mutability in the regions that encode the antigen-binding domains. We have isolated a set of clonally related natural Ig heavy chain-light chain pairs from an experimentally infected influenza patient, inferred the unmutated ancestral rearrangements and the maturation intermediates, and synthesized all the antibodies using recombinant methods. The lineage exhibits a remarkably uniform rate of improvement of the effective affinity to influenza hemagglutinin (HA) over evolutionary time, increasing 1000-fold overall from the unmutated ancestor to the best of the observed antibodies. Furthermore, analysis of selection reveals that selection and mutation bias were concordant even at the level of maturation to a single antigen. Substantial improvement in affinity to HA occurred along mutationally preferred paths in sequence space and was thus strongly facilitated by the underlying local codon biases.

The role of viral introductions in sustaining community-based HIV epidemics in rural Uganda: evidence from spatial clustering, phylogenetics, and egocentric transmission models.

BACKGROUND: It is often assumed that local sexual networks play a dominant role in HIV spread in sub-Saharan Africa. The aim of this study was to determine the extent to which continued HIV transmission in rural communities--home to two-thirds of the African population--is driven by intra-community sexual networks versus viral introductions from outside of communities. METHODS AND FINDINGS: We analyzed the spatial dynamics of HIV transmission in rural Rakai District, Uganda, using data from a cohort of 14,594 individuals within 46 communities. We applied spatial clustering statistics, viral phylogenetics, and probabilistic transmission models to quantify the relative contribution of viral introductions into communities versus community- and household-based transmission to HIV incidence. Individuals living in households with HIV-incident (n = 189) or HIV-prevalent (n = 1,597) persons were 3.2 (95% CI: 2.7-3.7) times more likely to be HIV infected themselves compared to the population in general, but spatial clustering outside of households was relatively weak and was confined to distances <500 m. Phylogenetic analyses of gag and env genes suggest that chains of transmission frequently cross community boundaries. A total of 95 phylogenetic clusters were identified, of which 44% (42/95) were two individuals sharing a household. Among the remaining clusters, 72% (38/53) crossed community boundaries. Using the locations of self-reported sexual partners, we estimate that 39% (95% CI: 34%-42%) of new viral transmissions occur within stable household partnerships, and that among those infected by extra-household sexual partners, 62% (95% CI: 55%-70%) are infected by sexual partners from outside their community. These results rely on the representativeness of the sample and the quality of self-reported partnership data and may not reflect HIV transmission patterns outside of Rakai. CONCLUSIONS: Our findings suggest that HIV introductions into communities are common and account for a significant proportion of new HIV infections acquired outside of households in rural Uganda, though the extent to which this is true elsewhere in Africa remains unknown. Our results also suggest that HIV prevention efforts should be implemented at spatial scales broader than the community and should target key populations likely responsible for introductions into communities.

HIV type 1 polymerase gene polymorphisms are associated with phenotypic differences in replication capacity and disease progression.

BACKGROUND: Determinants of intersubtype differences in human immunodeficiency virus type 1 (HIV-1) clinical disease progression remain unknown. METHODS: HIV-1 subtype was independently determined for 5 separate genomic regions in 396 HIV-1 seroconverters from Rakai, Uganda, using a multiregion hybridization assay. Replication capacities (RC) in samples from a subset of 145 of these subjects were determined. HIV-1 genomic regions and pol RC were examined for association with disease progression. Amino acid polymorphisms were examined for association with pol RC. RESULTS: In multivariate analyses, the hazard for progression to the composite end point (defined as a CD4(+) T-cell count <250 cells/mm(3), antiretroviral therapy initiation, or death) among patients with subtype D pol infection was 2.4 times the hazard for those infected with subtype A pol infection (P = .001). Compared with subtype A pol (the reference group), the hazard for progression to the composite end point for subtype D pol infection with a pol RC >67% (ie, the median pol RC) was significantly greater (HR, 4.6; 95% confidence interval [CI], 1.9-11.0; P = .001), whereas the hazard for progression to the composite end point for subtype D pol infection with a pol RC ≤67% was not significantly different (HR, 2.2; 95% CI, 1.0-4.9; P = .051). Amino acid substitutions at protease positions 62 and 64 and at reverse transcriptase position 272 were associated with significant differences in pol RC. CONCLUSIONS: HIV-1 pol gene intersubtype and RC differences are associated with disease progression and may be influenced by amino acid polymorphisms.

Differential specificity of HIV incidence assays in HIV subtypes A and D-infected individuals from Rakai, Uganda.

Assays to determine HIV incidence from cross-sectional surveys have exhibited a high rate of false-recent misclassification in Kenya and Uganda where HIV subtypes A and D predominate. Samples from individuals infected with HIV for at least 2 years with known infecting subtype (133 subtype A, 373 subtype D) were tested using the BED-CEIA and an avidity assay. Both assays had a higher rate of false-recent misclassification for subtype D compared to subtype A (13.7% vs. 6.0%, p=0.02 for BED-CEIA; 11.0% vs. 1.5%, p<0.001 for avidity). For subtype D samples, false-recent misclassification by the BED-CEIA was also more frequent in women than men (15.0% vs. 5.6%, p=0.002), and for samples where that had an amino acid other than lysine at position 12 in the BED-CEIA peptide coding region (p=0.002). Furthermore in subtype D-infected individuals, samples misclassified by one assay were 3.5 times more likely to be misclassified by the other assay. Differential misclassification by infecting subtype of long-term infected individuals as recently infected makes it difficult to use these assays individually to estimate population level incidence without precise knowledge of the distribution of these subtypes within populations where subtype A and D cocirculate. The association of misclassification of the BED-CEIA with the avidity assay in subtype D-infected individuals limits the utility of using these assays in combination within this population.

Constraints on viral evolution during chronic hepatitis C virus infection arising from a common-source exposure.

Extraordinary viral sequence diversity and rapid viral genetic evolution are hallmarks of hepatitis C virus (HCV) infection. Viral sequence evolution has previously been shown to mediate escape from cytotoxic T-lymphocyte (CTL) and neutralizing antibody responses in acute HCV infection. HCV evolution continues during chronic infection, but the pressures driving these changes are poorly defined. We analyzed plasma virus sequence evolution in 5.2-kb hemigenomes from multiple longitudinal time points isolated from individuals in the Irish anti-D cohort, who were infected with HCV from a common source in 1977 to 1978. We found phylogenetically distinct quasispecies populations at different plasma time points isolated late in chronic infection, suggesting ongoing viral evolution and quasispecies replacement over time. We saw evidence of early pressure driving net evolution away from a computationally reconstructed common ancestor, known as Bole1b, in predicted CTL epitopes and E1E2, with balanced evolution toward and away from the Bole1b amino acid sequence in the remainder of the genome. Late in chronic infection, the rate of evolution toward the Bole1b sequence increased, resulting in net neutral evolution relative to Bole1b across the entire 5.2-kb hemigenome. Surprisingly, even late in chronic infection, net amino acid evolution away from the infecting inoculum sequence still could be observed. These data suggest that, late in chronic infection, ongoing HCV evolution is not random genetic drift but rather the product of strong pressure toward a common ancestor and concurrent net ongoing evolution away from the inoculum virus sequence, likely balancing replicative fitness and ongoing immune escape.

Comparison of a high-resolution melting assay to next-generation sequencing for analysis of HIV diversity.

Next-generation sequencing (NGS) has recently been used for analysis of HIV diversity, but this method is labor-intensive, costly, and requires complex protocols for data analysis. We compared diversity measures obtained using NGS data to those obtained using a diversity assay based on high-resolution melting (HRM) of DNA duplexes. The HRM diversity assay provides a single numeric score that reflects the level of diversity in the region analyzed. HIV gag and env from individuals in Rakai, Uganda, were analyzed in a previous study using NGS (n = 220 samples from 110 individuals). Three sequence-based diversity measures were calculated from the NGS sequence data (percent diversity, percent complexity, and Shannon entropy). The amplicon pools used for NGS were analyzed with the HRM diversity assay. HRM scores were significantly associated with sequence-based measures of HIV diversity for both gag and env (P < 0.001 for all measures). The level of diversity measured by the HRM diversity assay and NGS increased over time in both regions analyzed (P < 0.001 for all measures except for percent complexity in gag), and similar amounts of diversification were observed with both methods (P < 0.001 for all measures except for percent complexity in gag). Diversity measures obtained using the HRM diversity assay were significantly associated with those from NGS, and similar increases in diversity over time were detected by both methods. The HRM diversity assay is faster and less expensive than NGS, facilitating rapid analysis of large studies of HIV diversity and evolution.

Immunogenicity and cross-reactivity of a representative ancestral sequence in hepatitis C virus infection.

Vaccines designed to prevent or to treat hepatitis C viral infection must achieve maximum cross-reactivity against widely divergent circulating strains. Rational approaches for sequence selection to maximize immunogenicity and minimize genetic distance across circulating strains may enhance vaccine induction of optimal cytotoxic T cell responses. We assessed T cell recognition of potential hepatitis C virus (HCV) vaccine sequences generated using three rational approaches: combining epitopes with predicted tight binding to the MHC, consensus sequence (most common amino acid at each position), and representative ancestral sequence that had been derived using bayesian phylogenetic tools. No correlation was seen between peptide-MHC binding affinity and frequency of recognition, as measured by an IFN-γ T cell response in HLA-matched HCV-infected individuals. Peptides encoding representative, consensus, and natural variant sequences were then tested for the capacity to expand CD8 T cell populations and to elicit cross-reactive CD8 T cell responses. CD8(+) T cells expanded with representative sequence HCV generally more broadly and robustly recognized highly diverse circulating HCV strains than did T cells expanded with either consensus sequence or naturally occurring sequence variants. These data support the use of representative sequence in HCV vaccine design.

Computational reconstruction of Bole1a, a representative synthetic hepatitis C virus subtype 1a genome.

Hepatitis C virus (HCV) research is hampered by the use of arbitrary representative isolates in cell culture and immunology. The most replicative isolate in vitro is a subtype 2a virus (JFH-1); however, genotype 1 is more prevalent worldwide and represents about 70% of infections in the United States, and genotypes differ from one another by 31% to 33% at the nucleotide level. For phylogenetic and immunologic analyses, viruses H77 and HCV-1 (both subtype 1a) are commonly used based on their historic importance. In an effort to rationally design a representative subtype 1a virus (Bole1a), we used Bayesian phylogenetics, ancestral sequence reconstruction, and covariance analysis on a curated set of 390 full-length human HCV 1a sequences from GenBank. By design, Bole1a contains variations present in widely circulating strains and matches more epitope-sized peptides in a full-genome comparison to subtype 1a isolates than any other sequence studied. Parallel analyses confirm that selected epitopes from the Bole1a genome were able to elicit a robust T cell response. In a proof of concept for infectivity, the envelope genes (E1 and E2) of Bole1a were expressed in an HIV pseudoparticle system containing HCV envelope genes and HIV nonenvelope genes with luciferase expression. The resulting Bole1a pseudoparticle robustly infected Hep3B cells. In this study, we demonstrate that a rationally designed, fully synthetic HCV genome contains representative epitopes and envelope genes that assemble properly and mediate entry into target cells.

An outbreak of human parainfluenza virus 3 infection in an outpatient hematopoietic stem cell transplantation clinic.

BACKGROUND: Parainfluenza viruses cause respiratory tract infections in adults and children, with peak activity during the spring and summer months. Human parainfluenza virus type 3 (hPIV-3) can contribute to significant morbidity and mortality in patients undergoing hematopoietic stem cell transplantation (HSCT). METHODS: Automated surveillance software was used to identify an hPIV-3 outbreak in an HSCT clinic. Active surveillance for respiratory illness and infection control measures were instituted. A retrospective molecular investigation of outbreak viral strains was performed by direct sequencing. RESULTS: Twelve of 196 HSCT recipients attending the clinic during the outbreak period had hPIV-3; one of these patients died. Sequencing demonstrated highly related strains in 9 of 10 patients studied. Despite the ongoing presence of hPIV-3 outside the inpatient/outpatient care continuum clinic, only 2 cases were observed after institution of respiratory season infection control measures. CONCLUSIONS: This investigation demonstrates the utility of surveillance software in the identification of respiratory virus outbreaks and the importance of rapid implementation of infection control/prevention measures for containment of outbreaks.

Laser captured hepatocytes show association of butyrylcholinesterase gene loss and fibrosis progression in hepatitis C-infected drug users.

UNLABELLED: Chronic hepatitis C virus (HCV) infection is complicated by hepatic fibrosis. Hypothesizing that early fibrogenic signals may originate in cells susceptible to HCV infection, hepatocyte gene expression was analyzed from persons with chronic HCV at different stages of liver fibrosis. Four HCV-infected subjects with precirrhosis liver fibrosis (Ishak fibrosis 3-5) were matched for age, race, and gender to five HCV-infected subjects with no evidence of fibrosis (Ishak fibrosis 0). Hepatocytes from each subject were isolated from liver biopsies using laser capture microdissection. Transcriptome profiling was performed on hepatocyte RNA using hybridization arrays. We found that hepatocytes in precirrhosis fibrosis were depleted for genes involved in small molecule and drug metabolism, especially butyrylcholinesterase (BCHE), a gene involved in the metabolism of drugs of abuse. Differential expression of BCHE was validated in the same tissues and cross-sectionally in an expanded cohort of 143 HCV-infected individuals. In a longitudinal study, serum BCHE activity was already decreased at study inception in 19 fibrosis progressors compared with 20 fibrosis nonprogressors (P < 0.05). Nonprogressors also had decreased BCHE activity over time compared with initial values, but these evolved a median (range) 8.6 (7.8-11.4) years after the study period inception (P < 0.05). Laser captured portal tracts were enriched for immune related genes when compared with hepatocytes but precirrhosis livers lost this enrichment. CONCLUSION: Chronic HCV is associated with hepatocyte BCHE loss years before hepatic synthetic function is impaired. These results indicate that BCHE may be involved in the pathogenesis of HCV-related fibrosis among injection drug users.

H3N2 influenza infection elicits more cross-reactive and less clonally expanded anti-hemagglutinin antibodies than influenza vaccination.

BACKGROUND: During the recent H1N1 influenza pandemic, excess morbidity and mortality was seen in young but not older adults suggesting that prior infection with influenza strains may have protected older subjects. In contrast, a history of recent seasonal trivalent vaccine in younger adults was not associated with protection. METHODS AND FINDINGS: To study hemagglutinin (HA) antibody responses in influenza immunization and infection, we have studied the day 7 plasma cell repertoires of subjects immunized with seasonal trivalent inactivated influenza vaccine (TIV) and compared them to the plasma cell repertoires of subjects experimentally infected (EI) with influenza H3N2 A/Wisconsin/67/2005. The majority of circulating plasma cells after TIV produced influenza-specific antibodies, while most plasma cells after EI produced antibodies that did not react with influenza HA. While anti-HA antibodies from TIV subjects were primarily reactive with single or few HA strains, anti-HA antibodies from EI subjects were isolated that reacted with multiple HA strains. Plasma cell-derived anti-HA antibodies from TIV subjects showed more evidence of clonal expansion compared with antibodies from EI subjects. From an H3N2-infected subject, we isolated a 4-member clonal lineage of broadly cross-reactive antibodies that bound to multiple HA subtypes and neutralized both H1N1 and H3N2 viruses. This broad reactivity was not detected in post-infection plasma suggesting this broadly reactive clonal lineage was not immunodominant in this subject. CONCLUSION: The presence of broadly reactive subdominant antibody responses in some EI subjects suggests that improved vaccine designs that make broadly reactive antibody responses immunodominant could protect against novel influenza strains.

Analysis of genetic linkage of HIV from couples enrolled in the HIV Prevention Trials Network 052 trial.

BACKGROUND: The HIV Prevention Trials Network (HPTN) 052 trial demonstrated that early initiation of antiretroviral therapy (ART) reduces human immunodeficiency virus (HIV) transmission from HIV-infected adults (index participants) to their HIV-uninfected sexual partners. We analyzed HIV from 38 index-partner pairs and 80 unrelated index participants (controls) to assess the linkage of seroconversion events. METHODS: Linkage was assessed using phylogenetic analysis of HIV pol sequences and Bayesian analysis of genetic distances between pol sequences from index-partner pairs and controls. Selected samples were also analyzed using next-generation sequencing (env region). RESULTS: In 29 of the 38 (76.3%) cases analyzed, the index was the likely source of the partner's HIV infection (linked). In 7 cases (18.4%), the partner was most likely infected from a source other than the index participant (unlinked). In 2 cases (5.3%), linkage status could not be definitively established. CONCLUSIONS: Nearly one-fifth of the seroconversion events in HPTN 052 were unlinked. The association of early ART and reduced HIV transmission was stronger when the analysis included only linked events. This underscores the importance of assessing the genetic linkage of HIV seroconversion events in HIV prevention studies involving serodiscordant couples.

Initial antibodies binding to HIV-1 gp41 in acutely infected subjects are polyreactive and highly mutated.

The initial antibody response to HIV-1 is targeted to envelope (Env) gp41, and is nonneutralizing and ineffective in controlling viremia. To understand the origins and characteristics of gp41-binding antibodies produced shortly after HIV-1 transmission, we isolated and studied gp41-reactive plasma cells from subjects acutely infected with HIV-1. The frequencies of somatic mutations were relatively high in these gp41-reactive antibodies. Reverted unmutated ancestors of gp41-reactive antibodies derived from subjects acutely infected with HIV-1 frequently did not react with autologous HIV-1 Env; however, these antibodies were polyreactive and frequently bound to host or bacterial antigens. In one large clonal lineage of gp41-reactive antibodies, reactivity to HIV-1 Env was acquired only after somatic mutations. Polyreactive gp41-binding antibodies were also isolated from uninfected individuals. These data suggest that the majority of gp41-binding antibodies produced after acute HIV-1 infection are cross-reactive responses generated by stimulating memory B cells that have previously been activated by non-HIV-1 antigens.

Molecular epidemiology of HIV type 1 in Singapore and identification of novel CRF01_AE/B recombinant forms.

To investigate HIV-1 molecular epidemiology in Singapore, we sequenced portions of three regions of the HIV-1 genome (protease HXB2: 2163 to 2620, gp120 HXB2: 6904 to 7628, and gp41 HXB2: 7817 to 8264) from 212 plasma samples collected between February 2008 and August 2009. From these samples, 109 (51.4%) generated interpretable data in all regions. Sixty-one (56.0%) were identified as CRF01_AE, 26 (23.9%) as subtype B and 14 (12.8%) as possible novel recombinant forms. The main novel recombinant pattern, detected in 13 sequences, had subtype B in protease and gp41 and CRF01_AE in gp120. There was intermixing of subtypes within transmission risk groups. However, 85% of subjects infected with the novel recombinant forms self-identified as men who have sex with men or bisexuals compared with only 41% of individuals infected with CRF01_AE and 62% infected with subtype B (p = 0.001).

SoDA2: a Hidden Markov Model approach for identification of immunoglobulin rearrangements.

MOTIVATION: The inference of pre-mutation immunoglobulin (Ig) rearrangements is essential in the study of the antibody repertoires produced in response to infection, in B-cell neoplasms and in autoimmune disease. Often, there are several rearrangements that are nearly equivalent as candidates for a given Ig gene, but have different consequences in an analysis. Our aim in this article is to develop a probabilistic model of the rearrangement process and a Bayesian method for estimating posterior probabilities for the comparison of multiple plausible rearrangements. RESULTS: We have developed SoDA2, which is based on a Hidden Markov Model and used to compute the posterior probabilities of candidate rearrangements and to find those with the highest values among them. We validated the software on a set of simulated data, a set of clonally related sequences, and a group of randomly selected Ig heavy chains from Genbank. In most tests, SoDA2 performed better than other available software for the task. Furthermore, the output format has been redesigned, in part, to facilitate comparison of multiple solutions. AVAILABILITY: SoDA2 is available online at https://hippocrates.duhs.duke.edu/soda. Simulated sequences are available upon request.

High-throughput isolation of immunoglobulin genes from single human B cells and expression as monoclonal antibodies.

Defining human B cell repertoires to viral pathogens is critical for design of vaccines that induce broadly protective antibodies to infections such as HIV-1 and influenza. Single B cell sorting and cloning of immunoglobulin (Ig) heavy- and light-chain variable regions (V(H) and V(L)) is a powerful technology for defining anti-viral B cell repertoires. However, the Ig-cloning step is time-consuming and prevents high-throughput analysis of the B cell repertoire. Novel linear Ig heavy- and light-chain gene expression cassettes were designed to express Ig V(H) and V(L) genes isolated from sorted single B cells as IgG1 antibody without a cloning step. The cassettes contain all essential elements for transcriptional and translational regulation, including CMV promoter, Ig leader sequences, constant region of IgG1 heavy- or Ig light-chain, poly(A) tail and substitutable V(H) or V(L) genes. The utility of these Ig gene expression cassettes was established using synthetic V(H) or V(L) genes from an anti-HIV-1 gp41 mAb 2F5 as a model system, and validated further using V(H) and V(L) genes isolated from cloned EBV-transformed antibody-producing cell lines. Finally, this strategy was successfully used for rapid production of recombinant influenza mAbs from sorted single human plasmablasts after influenza vaccination. These Ig gene expression cassettes constitute a highly efficient strategy for rapid expression of Ig genes for high-throughput screening and analysis without cloning.

An information-theoretic method for the treatment of plural ancestry in phylogenetics.

In the presence of recombination and gene conversion, a given genomic segment may inherit information from 2 distinct immediate ancestors. The importance of this type of molecular inheritance has become increasingly clear over the years, and the potential for erroneous inference when it is not accounted for in the statistical model is well documented. Yet, the inclusion of plural ancestry (PA) in phylogenetic analysis is still not routine. This omission is due to the greater difficulty of phylogenetic inference on general acyclic graphs compared that on with trees and the accompanying computational burden. We have developed a technique for phylogenetic inference in the presence of PA based on the principle of minimum description length, which assigns a cost--the description length--to each network topology given the observed sequence data. The description length combines the cost of poor data fit and model complexity in terms of information. This device allows us to search through network topologies to minimize the total description length. By comparing the best models obtained with and without PA, one can determine whether or not recombination has played an active role in the evolution of the genes under investigation, identify those genes that appear to be mosaic, and infer the phylogenetic network that best represents the history of the alignment. We show that the method performs well on simulated data and demonstrate its application on HIV env gene sequence data from 8 human subjects. The software implementation of the method is available upon request.