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1.  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/mm3, 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.
doi:10.1093/infdis/jit425
PMCID: PMC3864385  PMID: 23922373
HIV-1 Subtype; subtype A; subtype D; disease progression; polymerase; replication capacity; amino acid polymorphisms
2.  Differential Specificity of HIV Incidence Assays in HIV Subtypes A and D-Infected Individuals from Rakai, Uganda 
AIDS Research and Human Retroviruses  2013;29(8):1146-1150.
Abstract
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.
doi:10.1089/aid.2012.0105
PMCID: PMC3715796  PMID: 23641870
3.  The Role of Viral Introductions in Sustaining Community-Based HIV Epidemics in Rural Uganda: Evidence from Spatial Clustering, Phylogenetics, and Egocentric Transmission Models 
PLoS Medicine  2014;11(3):e1001610.
Using different approaches to investigate HIV transmission patterns, Justin Lessler and colleagues find that extra-community HIV introductions are frequent and likely play a role in sustaining the epidemic in the Rakai community.
Please see later in the article for the Editors' Summary
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.
Please see later in the article for the Editors' Summary
Editors' Summary
Background
About 35 million people (25 million of whom live in sub-Saharan Africa) are currently infected with HIV, the virus that causes AIDS, and about 2.3 million people become newly infected every year. HIV destroys immune system cells, leaving infected individuals susceptible to other infections. HIV infection can be controlled by taking antiretroviral drugs (antiretroviral therapy, or ART) daily throughout life. Although originally available only to people living in wealthy countries, recent political efforts mean that 9.7 million people in low- and middle-income countries now have access to ART. However, ART does not cure HIV infection, so prevention of viral transmission remains extremely important. Because HIV is usually transmitted through unprotected sex with an infected partner, individuals can reduce their risk of infection by abstaining from sex, by having one or a few partners, and by using condoms. Male circumcision also reduces HIV transmission. In addition to reducing illness and death among HIV-positive people, ART also reduces HIV transmission.
Why Was This Study Done?
Effective HIV control requires an understanding of how HIV spreads through sexual networks. These networks include sexual partnerships between individuals in households, between community members in different households, and between individuals from different communities. Local sexual networks (household and intra-community sexual partnerships) are sometimes assumed to be the dominant driving force in HIV spread in sub-Saharan Africa, but are viral introductions from sexual partnerships with individuals outside the community also important? This question needs answering because the effectiveness of interventions such as ART as prevention partly depends on how many new infections in an intervention area are attributable to infection from partners residing in that area and how many are attributable to infection from partners living elsewhere. Here, the researchers use three analytical methods—spatial clustering statistics, viral phylogenetics, and egocentric transmission modeling—to ask whether HIV transmission in rural Uganda is driven predominantly by intra-community sexual networks. Spatial clustering analysis uses the geographical coordinates of households to measure the tendency of HIV-infected people to cluster spatially at scales consistent with community transmission. Viral phylogenetic analysis examines the genetic relatedness of viruses; if transmission is through local networks, viruses in newly infected individuals should more closely resemble viruses in other community members than those in people outside the community. Egocentric transmission modelling uses information on the locations of recent sexual partners to estimate the proportions of new transmissions from household, intra-community, and extra-community partners.
What Did the Researchers Do and Find?
The researchers applied their three analytical methods to data collected from 14,594 individuals living in 46 communities (governmental administrative units) in Rakai District, Uganda. Spatial clustering analysis indicated that individuals who lived in households with individuals with incident HIV (newly diagnosed) or prevalent HIV (previously diagnosed) were 3.2 times more likely than the general population to be HIV-positive themselves. Spatial clustering outside households was relatively weak, however, and was confined to distances of less than half a kilometer. Viral phylogenetic analysis indicated that 44% of phylogenetic clusters (viruses with related genetic sequences found in more than one individual) were within households, but that 40% of clusters crossed community borders. Finally, analysis of the locations of self-reported sexual partners indicated that 39% of new viral transmissions occurred within stable household partnerships, but that among people newly infected by extra-household partners, nearly two-thirds were infected by partners from outside their community.
What Do These Findings Mean?
The results of all three analyses suggest that HIV introductions into communities are frequent and are likely to play an important role in sustaining HIV transmission in the Rakai District. Specifically, within this rural HIV-endemic region (a region where HIV infection is always present), viral introductions combined with intra-household transmission account for the majority of new infections, although community-based sexual networks also play a critical role in HIV transmission. These findings may not be generalizable to the broader Ugandan population or to other regions of Africa, and their accuracy is likely to be limited by the use of self-reported sexual partner data. Nevertheless, these findings indicate that the dynamics of HIV transmission in rural Uganda (and probably elsewhere) are complex. Consequently, to halt the spread of HIV, prevention efforts will need to be implemented at spatial scales broader than individual communities, and key populations that are likely to introduce HIV into communities will need to be targeted.
Additional Information
Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001610.
Information is available from the US National Institute of Allergy and Infectious Diseases on HIV infection and AIDS
NAM/aidsmap provides basic information about HIV/AIDS, and summaries of recent research findings on HIV care and treatment
Information is available from Avert, an international AIDS charity, on many aspects of HIV/AIDS, including information on HIV and AIDS in Uganda and on HIV prevention strategies (in English and Spanish)
The UNAIDS Report on the Global AIDS Epidemic 2013 provides up-to-date information about the AIDS epidemic and efforts to halt it
The Center for AIDS Prevention Studies (University of California, San Francisco) has a fact sheet about sexual networks and HIV prevention
Wikipedia provides information on spatial clustering analysis (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
A PLOS Computational Biology Topic Page (a review article that is a published copy of record of a dynamic version of the article as found in Wikipedia) about viral phylodynamics is available
Personal stories about living with HIV/AIDS are available through Avert, NAM/aidsmap, and Healthtalkonline
doi:10.1371/journal.pmed.1001610
PMCID: PMC3942316  PMID: 24595023
4.  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.
doi:10.3389/fimmu.2014.00170
PMCID: PMC4001017  PMID: 24795717
somatic hypermutation; experimental influenza infection; antibody selection; antibody affinity maturation; phylogenetics
5.  Laser Captured Hepatocytes Show Association of BCHE Loss and Fibrosis Progression in Hepatitis C Infected Drug Users 
Hepatology (Baltimore, Md.)  2012;56(2):544-554.
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 pre-cirrhotic 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 pre-cirrhotic 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 were already decreased at study inception in 19 fibrosis progressors compared to 20 fibrosis non-progressors (p<0.05). Non-progressors also had decreased BCHE activity over time compared to 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 to hepatocytes but pre-cirrhotic livers lost this enrichment.
Conclusion
Overall, we found that 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.
doi:10.1002/hep.25655
PMCID: PMC3388175  PMID: 22331678
butyrylcholinesterase; Hepacivirus; chronic HCV infection; laser capture microdissection; portal tract
6.  Constraints on Viral Evolution during Chronic Hepatitis C Virus Infection Arising from a Common-Source Exposure 
Journal of Virology  2012;86(23):12582-12590.
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.
doi:10.1128/JVI.01440-12
PMCID: PMC3497661  PMID: 22973048
7.  Immunogenicity and cross-reactivity of a representative ancestral sequence in HCV infection1 
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 vaccine sequences generated using three rational approaches: 1) combining epitopes with predicted tight binding to the major histocompatibility complex (MHC), 2) consensus sequence (most common amino acid at each position), and 3) 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 interferon-gamma T cell response in human leukocyte antigen-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 T cell expanded with either consensus sequence or naturally occurring sequence variants. These data support the use of representative sequence in HCV vaccine design.
doi:10.4049/jimmunol.1103008
PMCID: PMC3345099  PMID: 22508927
Animals-human; T cells; Infections-viral; Processes-vaccination
8.  Comparison of a High-Resolution Melting Assay to Next-Generation Sequencing for Analysis of HIV Diversity 
Journal of Clinical Microbiology  2012;50(9):3054-3059.
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.
doi:10.1128/JCM.01460-12
PMCID: PMC3421787  PMID: 22785188
9.  Analysis of Genetic Linkage of HIV From Couples Enrolled in the HIV Prevention Trials Network 052 Trial 
The Journal of Infectious Diseases  2011;204(12):1918-1926.
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.
doi:10.1093/infdis/jir651
PMCID: PMC3209811  PMID: 21990420
10.  Computational Reconstruction of Bole1a, a Representative Synthetic Hepatitis C Virus Subtype 1a Genome 
Journal of Virology  2012;86(10):5915-5921.
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.
doi:10.1128/JVI.05959-11
PMCID: PMC3347276  PMID: 22438535
11.  Molecular Epidemiology of HIV Type 1 in Singapore and Identification of Novel CRF01_AE/B Recombinant Forms 
AIDS Research and Human Retroviruses  2011;27(10):1135-1137.
Abstract
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).
doi:10.1089/aid.2010.0364
PMCID: PMC3186691  PMID: 21235306
12.  Initial antibodies binding to HIV-1 gp41 in acutely infected subjects are polyreactive and highly mutated 
The Journal of Experimental Medicine  2011;208(11):2237-2249.
Many HIV-1 envelope-reactive antibodies shortly after HIV-1 transmission may arise from crow-reactive memory B cells previously stimulated by non-HIV-1 host or microbial antigens
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.
doi:10.1084/jem.20110363
PMCID: PMC3201211  PMID: 21987658
13.  H3N2 Influenza Infection Elicits More Cross-Reactive and Less Clonally Expanded Anti-Hemagglutinin Antibodies Than Influenza Vaccination 
PLoS ONE  2011;6(10):e25797.
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.
doi:10.1371/journal.pone.0025797
PMCID: PMC3198447  PMID: 22039424
14.  High-throughput isolation of immunoglobulin genes from single human B cells and expression as monoclonal antibodies 
Journal of virological methods  2009;158(1-2):171-179.
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 (VH and VL) 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 VH and VL 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 VH or VL genes. The utility of these Ig gene expression cassettes was established using synthetic VH or VL genes from an anti-HIV-1 gp41 mAb 2F5 as a model system, and validated further using VH and VL 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.
doi:10.1016/j.jviromet.2009.02.014
PMCID: PMC2805188  PMID: 19428587
Monoclonal antibody; single B cells; immunoglobulin gene; RT-PCR; linear gene expression cassette
15.  SoDA2: a Hidden Markov Model approach for identification of immunoglobulin rearrangements 
Bioinformatics  2010;26(7):867-872.
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.
Contact: kepler@duke.edu
doi:10.1093/bioinformatics/btq056
PMCID: PMC2844993  PMID: 20147303

Results 1-15 (15)