Hongshuo Song

Cooperation of B Cell Lineages in Induction of HIV-1-Broadly Neutralizing Antibodies

Development of strategies for induction of HIV-1 broadly neutralizing antibodies (bnAbs) by vaccines is a priority. Determining the steps of bnAb induction in HIV-1-infected individuals who make bnAbs is a key strategy for immunogen design. Here, we study the B cell response in a bnAb-producing individual and report cooperation between two B cell lineages to drive bnAb development. We isolated a virus-neutralizing antibody lineage that targeted an envelope region (loop D) and selected virus escape mutants that resulted in both enhanced bnAb lineage envelope binding and escape mutant neutralization-traits associated with increased B cell antigen drive. Thus, in this individual, two B cell lineages cooperated to induce the development of bnAbs. Design of vaccine immunogens that simultaneously drive both helper and broadly neutralizing B cell lineages may be important for vaccine-induced recapitulation of events that transpire during the maturation of neutralizing antibodies in HIV-1-infected individuals.

Maturation Pathway from Germline to Broad HIV-1 Neutralizer of a CD4-Mimic Antibody

Antibodies with ontogenies from VH1-2 or VH1-46-germline genes dominate the broadly neutralizing response against the CD4-binding site (CD4bs) on HIV-1. Here, we define with longitudinal sampling from time-of-infection the development of a VH1-46-derived antibody lineage that matured to neutralize 90% of HIV-1 isolates. Structures of lineage antibodies CH235 (week 41 from time-of-infection, 18% breadth), CH235.9 (week 152, 77%), and CH235.12 (week 323, 90%) demonstrated the maturing epitope to focus on the conformationally invariant portion of the CD4bs. Similarities between CH235 lineage and five unrelated CD4bs lineages in epitope focusing, length-of-time to develop breadth, and extraordinary level of somatic hypermutation suggested commonalities in maturation among all CD4bs antibodies. Fortunately, the required CH235-lineage hypermutation appeared substantially guided by the intrinsic mutability of the VH1-46 gene, which closely resembled VH1-2. We integrated our CH235-lineage findings with a second broadly neutralizing lineage and HIV-1 co-evolution to suggest a vaccination strategy for inducing both lineages.

Diversion of HIV-1 vaccine–induced immunity by gp41-microbiota cross-reactive antibodies

Microbiota can mislead antibodies Unlike the response to many viral infections, most people do not produce antibodies capable of clearing HIV-1. Non-neutralizing antibodies that target HIV-1s envelope glycoprotein (Env) typically dominate the response, which is generated by B cells that cross-react with Env and the intestinal microbiota. Williams et al. analyzed samples from individuals who had received a vaccine containing the Env protein, including the gp41 subunit. Most of the antibodies were non-neutralizing and targeted gp41. The antibodies also reacted to intestinal microbiota, suggesting that preexisting immunity to microbial communities skews vaccineinduced immune responses toward an unproductive target. Science, this issue 10.1126/science.aab1253. The antibody response to an HIV-1 vaccine is dominated by preexisting immunity to microbiota. INTRODUCTION Inducing protective antibodies is a key goal in HIV-1 vaccine development. In acute HIV-1 infection, the dominant initial plasma antibody response is to the gp41 subunit of the envelope (Env) glycoprotein of the virus. These antibodies derive from polyreactive B cells that cross-react with Env and intestinal microbiota (IM) and are unable to neutralize HIV-1. However, whether a similar gp41-IM cross-reactive antibody response would occur in the setting of HIV-1 Env vaccination is unknown. RATIONALE We studied antibody responses in individuals who received a DNA prime vaccine, with a recombinant adenovirus serotype 5 (rAd5) boost (DNA prime–rAd5 boost), a vaccine that included HIV-1 gag, pol, and nef genes, as well as a trivalent mixture of clade A, B, and C env gp140 genes containing both gp120 and gp41 components. This vaccine showed no efficacy. Thus, study of these vaccinees provided an opportunity to determine whether the Env-reactive antibody response in the setting of Env vaccination was dominated by gp41-reactive antibodies derived from Env-IM cross-reactive B cells. RESULTS We found that vaccine-induced antibodies to HIV-1 Env dominantly focused on gp41 compared with gp120 by both serologic analysis and by vaccine-Env memory B cells sorted by flow cytometry (see the figure). Remarkably, the majority of HIV-1 Env-reactive memory B cells induced by the vaccine produced gp41-reactive antibodies, and the majority of gp41-targeted antibodies used restricted immunoglobulin heavy chain variable genes. Functionally, none of the gp41-reactive antibodies could neutralize HIV, and the majority could not mediate antibody-dependent cellular cytotoxicity. Most of the vaccine-induced gp41-reactive antibodies cross-reacted with host and IM antigens. Two of the candidate gp41-intestinal cross-reactive antigens were bacterial RNA polymerase and pyruvate-flavodoxin oxidoreductase, which shared sequence similarities with the heptad repeat 1 region of HIV gp41. Next-generation sequencing of vaccinee B cells demonstrated a prevaccination antibody that was reactive to both IM and the vaccine–Env gp140, which demonstrated the presence of a preexisting pool of gp41-IM cross-reactive B cells from which the vaccine gp41-reactive antibody response was derived. CONCLUSION In this study, we found that the DNA prime–rAd5 boost HIV-1 vaccine induced a gp41-reactive antibody response that was mainly non-neutralizing and derived from an IM-gp41 cross-reactive B cell pool. These findings have important implications for HIV-1 vaccine design. Because IM antigens shape the B cell repertoire from birth, our data raise the hypothesis that neonatal immunization with HIV-1 envelope may be able to imprint the B cell repertoire to respond to envelope antigenic sites that may otherwise be subdominant or disfavored, such as Env broadly neutralizing antibody epitopes. Our data also suggest that deleting or modifying amino acids in the gp41 heptad repeat 1 region of Env-containing vaccine immunogens may avoid IM-gp41 cross-reactivity. Thus, an obstacle that may need to be overcome for development of a successful HIV vaccine is diversion of potentially protective HIV-1 antibody responses by preexisting envelope-IM cross-reactive pools of B cells. Diversion of HIV-1 vaccine–induced immunity by Env gp41–microbiota cross-reactive antibodies. Immunization of humans with a vaccine containing HIV-1 Env gp120 and gp41 components, including the membrane-proximal external region (MPER) of Env, induced a dominant B cell response primarily from a preexisting pool of gp41-IM cross-reactive B cells. This response diverted the vaccine-stimulated antibody response away from smaller subdominant B cell pools capable of reacting with potentially protective epitopes on HIV-1 Env. An HIV-1 DNA prime vaccine, with a recombinant adenovirus type 5 (rAd5) boost, failed to protect from HIV-1 acquisition. We studied the nature of the vaccine-induced antibody (Ab) response to HIV-1 envelope (Env). HIV-1–reactive plasma Ab titers were higher to Env gp41 than to gp120, and repertoire analysis demonstrated that 93% of HIV-1–reactive Abs from memory B cells responded to Env gp41. Vaccine-induced gp41-reactive monoclonal antibodies were non-neutralizing and frequently polyreactive with host and environmental antigens, including intestinal microbiota (IM). Next-generation sequencing of an immunoglobulin heavy chain variable region repertoire before vaccination revealed an Env-IM cross-reactive Ab that was clonally related to a subsequent vaccine-induced gp41-reactive Ab. Thus, HIV-1 Env DNA-rAd5 vaccine induced a dominant IM-polyreactive, non-neutralizing gp41-reactive Ab repertoire response that was associated with no vaccine efficacy.

Thermal stability and inactivation of hepatitis C virus grown in cell culture

BackgroundHepatitis C virus (HCV) is a blood-borne flavivirus that infects many millions of people worldwide. Relatively little is known, however, concerning the stability of HCV and reliable procedures for inactivating this virus.MethodsIn the current study, the thermostability of cell culture-derived HCV (HCVcc, JFH-1 strain) under different environmental temperatures (37°C, room temperature, and 4°C) and the ability of heat, UVC light irradiation, and aldehyde and detergent treatments to inactivate HCVcc were evaluated. The infectious titers of treated viral samples were determined by focus-forming unit (FFU) assay using an indirect immunofluorescence assay for HCV NS3 in hepatoma Huh7-25-CD81 cells highly permissive for HCVcc infection. MTT cytotoxicity assay was performed to determine the concentrations of aldehydes or detergents at which they were no longer cytotoxic.ResultsHCVcc in culture medium was found to survive 37°C and room temperature (RT, 25 ± 2°C) for 2 and 16 days, respectively, while the virus was relatively stable at 4°C without drastic loss of infectivity for at least 6 weeks. HCVcc in culture medium was sensitive to heat and could be inactivated in 8 and 4 min when incubated at 60°C and 65°C, respectively. However, at 56°C, 40 min were required to eliminate HCVcc infectivity. Addition of normal human serum to HCVcc did not significantly alter viral stability at RT or its susceptibility to heat. UVC light irradiation (wavelength = 253.7 nm) with an intensity of 450 μW/cm2 efficiently inactivated HCVcc within 2 min. Exposures to formaldehyde, glutaraldehyde, ionic or nonionic detergents all destroyed HCVcc infectivity effectively, regardless of whether the treatments were conducted in the presence of cell culture medium or human serum.ConclusionsThe results provide quantitative evidence for the potential use of a variety of approaches for inactivating HCV. The ability of HCVcc to survive ambient temperatures warrants precautions in handling and disposing of objects and materials that may have been contaminated with HCV.

Primary Infection by a Human Immunodeficiency Virus with Atypical Coreceptor Tropism

ABSTRACT The great majority of human immunodeficiency virus type 1 (HIV-1) strains enter CD4+ target cells by interacting with one of two coreceptors, CCR5 or CXCR4. Here we describe a transmitted/founder (T/F) virus (ZP6248) that was profoundly impaired in its ability to utilize CCR5 and CXCR4 coreceptors on multiple CD4+ cell lines as well as primary human CD4+ T cells and macrophages in vitro yet replicated to very high titers (>80 million RNA copies/ml) in an acutely infected individual. Interestingly, the envelope (Env) glycoprotein of this clade B virus had a rare GPEK sequence in the crown of its third variable loop (V3) rather than the consensus GPGR sequence. Extensive sequencing of sequential plasma samples showed that the GPEK sequence was present in virtually all Envs, including those from the earliest time points after infection. The molecularly cloned (single) T/F virus was able to replicate, albeit poorly, in cells obtained from ccr5 Δ32 homozygous donors. The ZP6248 T/F virus could also infect cell lines overexpressing the alternative coreceptors GPR15, APJ, and FPRL-1. A single mutation in the V3 crown sequence (GPEK->GPGK) of ZP6248 restored its infectivity in CCR5+ cells but reduced its ability to replicate in GPR15+ cells, indicating that the V3 crown motif played an important role in usage of this alternative coreceptor. These results suggest that the ZP6248 T/F virus established an acute in vivo infection by using coreceptor(s) other than CCR5 or CXCR4 or that the CCR5 coreceptor existed in an unusual conformation in this individual.

Impact of immune escape mutations on HIV-1 fitness in the context of the cognate transmitted/ founder genome

BackgroundA modest change in HIV-1 fitness can have a significant impact on viral quasispecies evolution and viral pathogenesis, transmission and disease progression. To determine the impact of immune escape mutations selected by cytotoxic T lymphocytes (CTL) on viral fitness in the context of the cognate transmitted/founder (T/F) genome, we developed a new competitive fitness assay using molecular clones of T/F genomes lacking exogenous genetic markers and a highly sensitive and precise parallel allele-specific sequencing (PASS) method.ResultsThe T/F and mutant viruses were competed in CD4+ T-cell enriched cultures, relative proportions of viruses were assayed after repeated cell-free passage, and fitness costs were estimated by mathematical modeling. Naturally occurring HLA B57-restricted mutations involving the TW10 epitope in Gag and two epitopes in Tat/Rev and Env were assessed independently and together. Compensatory mutations which restored viral replication fitness were also assessed. A principal TW10 escape mutation, T242N, led to a 42% reduction in replication fitness but V247I and G248A mutations in the same epitope restored fitness to wild-type levels. No fitness difference was observed between the T/F and a naturally selected variant carrying the early CTL escape mutation (R355K) in Env and a reversion mutation in the Tat/Rev overlapping region.ConclusionsThese findings reveal a broad spectrum of fitness costs to CTL escape mutations in T/F viral genomes, similar to recent findings reported for neutralizing antibody escape mutations, and highlight the extraordinary plasticity and adaptive potential of the HIV-1 genome. Analysis of T/F genomes and their evolved progeny is a powerful approach for assessing the impact of composite mutational events on viral fitness.

Preexisting compensatory amino acids compromise fitness costs of a HIV-1 T cell escape mutation

BackgroundFitness costs and slower disease progression are associated with a cytolytic T lymphocyte (CTL) escape mutation T242N in Gag in HIV-1-infected individuals carrying HLA-B*57/5801 alleles. However, the impact of different context in diverse HIV-1 strains on the fitness costs due to the T242N mutation has not been well characterized. To better understand the extent of fitness costs of the T242N mutation and the repair of fitness loss through compensatory amino acids, we investigated its fitness impact in different transmitted/founder (T/F) viruses.ResultsThe T242N mutation resulted in various levels of fitness loss in four different T/F viruses. However, the fitness costs were significantly compromised by preexisting compensatory amino acids in (Isoleucine at position 247) or outside (glutamine at position 219) the CTL epitope. Moreover, the transmitted T242N escape mutant in subject CH131 was as fit as the revertant N242T mutant and the elimination of the compensatory amino acid I247 in the T/F viral genome resulted in significant fitness cost, suggesting the fitness loss caused by the T242N mutation had been fully repaired in the donor at transmission. Analysis of the global circulating HIV-1 sequences in the Los Alamos HIV Sequence Database showed a high prevalence of compensatory amino acids for the T242N mutation and other T cell escape mutations.ConclusionsOur results show that the preexisting compensatory amino acids in the majority of circulating HIV-1 strains could significantly compromise the fitness loss due to CTL escape mutations and thus increase challenges for T cell based vaccines.

Extensive recombination due to heteroduplexes generates large amounts of artificial gene fragments during PCR.

Artificial recombinants can be generated during PCR when more than two genetically distinct templates coexist in a single PCR reaction. These recombinant amplicons can lead to the false interpretation of genetic diversity and incorrect identification of biological phenotypes that do not exist in vivo. We investigated how recombination between 2 or 35 genetically distinct HIV-1 genomes was affected by different PCR conditions using the parallel allele-specific sequencing (PASS) assay and the next generation sequencing method. In a standard PCR condition, about 40% of amplicons in a PCR reaction were recombinants. The high recombination frequency could be significantly reduced if the number of amplicons in a PCR reaction was below a threshold of 1013–1014 using low thermal cycles, fewer input templates, and longer extension time. Heteroduplexes (each DNA strand from a distinct template) were present at a large proportion in the PCR products when more thermal cycles, more templates, and shorter extension time were used. Importantly, the majority of recombinants were identified in heteroduplexes, indicating that the recombinants were mainly generated through heteroduplexes. Since prematurely terminated extension fragments can form heteroduplexes by annealing to different templates during PCR amplification, recombination has a better chance to occur with samples containing different genomes when the number of amplicons accumulate over the threshold. New technologies are warranted to accurately characterize complex quasispecies gene populations.

Recombination-mediated escape from primary CD8+ T cells in acute HIV-1 infection

BackgroundA major immune evasion mechanism of HIV-1 is the accumulation of non-synonymous mutations in and around T cell epitopes, resulting in loss of T cell recognition and virus escape.ResultsHere we analyze primary CD8+ T cell responses and virus escape in a HLA B*81 expressing subject who was infected with two T/F viruses from a single donor. In addition to classic escape through non-synonymous mutation/s, we also observed rapid selection of multiple recombinant viruses that conferred escape from T cells specific for two epitopes in Nef.ConclusionsOur study shows that recombination between multiple T/F viruses provide greater options for acute escape from CD8+ T cell responses than seen in cases of single T/F virus infection. This process may contribute to the rapid disease progression in patients infected by multiple T/F viruses.

Reversion and T cell escape mutations compensate the fitness loss of a CD8+ T cell escape mutant in their cognate transmitted/founder virus.

Immune escape mutations that revert back to the consensus sequence frequently occur in newly HIV-1-infected individuals and have been thought to render the viruses more fit. However, their impact on viral fitness and their interaction with other immune escape mutations have not been evaluated in the background of their cognate transmitted/founder (T/F) viral genomes. To precisely determine the role of reversion mutations, we introduced reversion mutations alone or together with CD8+ T cell escape mutations in their unmodified cognate T/F viral genome and determined their impact on viral fitness in primary CD4+ T cells. Two reversion mutations, V247I and I64T, were identified in Gag and Tat, respectively, but neither had measurable effect on the fitness of their cognate T/F virus. The V247I and G248A mutations that were detected before and concurrently with the potent T cell escape mutation T242N, respectively, were selected by early T cell responses. The V247I or the G248A mutation alone partially restored the fitness loss caused by the T242N mutation. Together they could fully restore the fitness of the T242N mutant to the T/F level. These results demonstrate that the fitness loss caused by a T cell escape mutation could be compensated by preexisting or concurrent reversion and other T cell escape mutations. Our findings indicate that the overall viral fitness is modulated by the complex interplay among T cell escape, compensatory and reversion mutations to maintain the balance between immune escape and viral replication capacity.