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.

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.

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.

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).

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.

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.

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.

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