Responses of N fluxes and pools to elevated atmospheric CO2 in model forest ecosystems with acidic and calcareous soils

The objectives of this study were to estimate how soil type, elevated N deposition (0.7 vs. 7 g N m^–2y^–1) and tree species influence the potential effects of elevated CO₂ (370 vs. 570 mol CO₂ mol^–1) on N pools and fluxes in forest soils. Model spruce-beech forest ecosystems were established on a nutrient-rich calcareous sand and on a nutrient-poor acidic loam in large open-top chambers. In the fourth year of treatment, we measured N concentrations in the soil solution at different depths, estimated N accumulation by ion exchange resin (IER) bags, and quantified N export in drainage water, denitrification, and net N uptake by trees. Under elevated CO₂, concentrations of N in the soil solution were significantly reduced. In the nutrient-rich calcareous sand, CO₂ enrichment decreased N concentrations in the soil solution at all depths (–45 to –100%). In the nutrient-poor acidic loam, the negative CO₂ effect was restricted to the uppermost 5 cm of the soil. Increasing the N deposition stimulated the negative impact of CO₂ enrichment on soil solution N in the acidic loam at 5 cm depth from –20% at low N inputs to –70% at high N inputs. In the nutrient-rich calcareous sand, N additions did not influence the CO₂ effect on soil solution N. Accumulation of N by IER bags, which were installed under individual trees, was decreased at high CO₂ levels under spruce in both soil types. Under beech, this decrease occurred only in the calcareous sand. N accumulation by IER bags was negatively correlated with current-years foliage biomass, suggesting that the reduction of soil N availability indices was related to a CO₂-induced growth enhancement. However, the net N uptake by trees was not significantly increased by elevated CO₂. Thus, we suppose that the reduced N concentrations in the soil solution at elevated CO₂ concentrations were rather caused by an increased N immobilisation in the soil. Denitrification was not influenced by atmospheric CO₂ concentrations. CO₂ enrichment decreased nitrate leaching in drainage by 65%, which suggests that rising atmospheric CO₂ potentially increases the N retention capacity of forest ecosystems.     

Capacity of Broadly Neutralizing Antibodies to Inhibit HIV-1 Cell-Cell Transmission Is Strain- and Epitope-Dependent

An increasing number of broadly neutralizing antibodies (bnAbs) are considered leads for HIV-1 vaccine development and novel therapeutics. Here, we systematically explored the capacity of bnAbs to neutralize HIV-1 prior to and post-CD4 engagement and to block HIV-1 cell-cell transmission. Cell-cell spread is known to promote a highly efficient infection with HIV-1 which can inflict dramatic losses in neutralization potency compared to free virus infection. Selection of bnAbs that are capable of suppressing HIV irrespective of the transmission mode therefore needs to be considered to ascertain their in vivo activity in therapeutic use and vaccines. Employing assay systems that allow for unambiguous discrimination between free virus and cell-cell transmission to T cells, we probed a panel of 16 bnAbs for their activity against 11 viruses from subtypes A, B and C during both transmission modes. Over a wide range of bnAb-virus combinations tested, inhibitory activity against HIV-1 cell-cell transmission was strongly decreased compared to free virus transmission. Activity loss varied considerably between virus strains and was inversely associated with neutralization of free virus spread for V1V2- and V3-directed bnAbs. In rare bnAb-virus combinations, inhibition for both transmission modes was comparable but no bnAb potently blocked cell-cell transmission across all probed virus strains. Mathematical analysis indicated an increased probability of bnAb resistance mutations to arise in cell-cell rather than free virus spread, further highlighting the need to block this pathway. Importantly, the capacity to efficiently neutralize prior to CD4 engagement correlated with the inhibition efficacy against free virus but not cell-cell transmitted virus. Pre-CD4 attachment activity proved strongest amongst CD4bs bnAbs and varied substantially for V3 and V1V2 loop bnAbs in a strain-dependent manner. In summary, bnAb activity against divergent viruses varied depending on the transmission mode and differed depending on the window of action during the entry process, underscoring that powerful combinations of bnAbs are needed for in vivo application.     

Virus isolates during acute and chronic human immunodeficiency virus type 1 infection show distinct patterns of sensitivity to entry inhibitors

We studied the effect of entry inhibitors on 58 virus isolates derived during acute and chronic infection to validate these inhibitors in vitro and to probe whether viruses at early and chronic disease stages exhibit general differences in the interaction with entry receptors. We included members of all types of inhibitors currently identified: (i) agents that block gp120 binding to CD4 (CD4-IgG2 and monoclonal antibody [MAb] IgG1b12), (ii) compounds that block the interaction with CCR5 (the chemokine RANTES/CCL5, the small-molecule inhibitor AD101, and the anti-CCR5 antibody PRO 140), (iii) the fusion inhibitor enfuvirtide (T-20), and (iv) neutralizing antibodies directed against gp120 (MAb 2G12) and gp41 (MAbs 2F5 and 4E10). No differences between viruses from acute and chronic infections in the susceptibility to inhibitors targeting the CD4 binding site, CCR5, or fusion or to MAb 2G12 were apparent, rendering treatment with entry inhibitors feasible across disease stages. The notable exceptions were antibodies 2F5 and 4E10, which were more potent in inhibiting viruses from acute infection (P = 0.0088 and 0.0005, respectively), although epitopes of these MAbs were equally well preserved in both groups. Activities of these MAbs correlated significantly with each other, suggesting that common features of the viral envelope modulate their potencies.     

Full-length haplotype reconstruction to infer the structure of heterogeneous virus populations

Next-generation sequencing (NGS) technologies enable new insights into the diversity of virus populations within their hosts. Diversity estimation is currently restricted to single-nucleotide variants or to local fragments of no more than a few hundred nucleotides defined by the length of sequence reads. To study complex heterogeneous virus populations comprehensively, novel methods are required that allow for complete reconstruction of the individual viral haplotypes. Here, we show that assembly of whole viral genomes of ∼8600 nucleotides length is feasible from mixtures of heterogeneous HIV-1 strains derived from defined combinations of cloned virus strains and from clinical samples of an HIV-1 superinfected individual. Haplotype reconstruction was achieved using optimized experimental protocols and computational methods for amplification, sequencing and assembly. We comparatively assessed the performance of the three NGS platforms 454 Life Sciences/Roche, Illumina and Pacific Biosciences for this task. Our results prove and delineate the feasibility of NGS-based full-length viral haplotype reconstruction and provide new tools for studying evolution and pathogenesis of viruses.     

Estimating the Stoichiometry of Human Immunodeficiency Virus Entry

To enter target cells, human immunodeficiency virus (HIV) first attaches to the cells and fuses with the cell membrane. Attachment and fusion involve envelope glycoprotein trimers on the surface of the virion and the CD4 receptor and chemokine coreceptors on the surface of the target cell. The stoichiometry of entry, that is, the number of bonds between such trimers and CD4 that are required for infection, is unknown. Pseudotyped virions that express mixed trimers consisting of functional and nonfunctional envelope proteins have been used to study how many trimer-receptor interactions are required for virus entry. However, to extract information on the stoichiometry of entry from data generated in in vitro infectivity assays with such viruses, mathematical models are required. Here, we describe mathematical models that can be used to infer the stoichiometry of entry. By fitting our simplest model to previously published data (X. Yang, S. Kurteva, X. Ren, S. Lee, and J. Sodroski, J. Virol. 79: 12132-12147, 2005), we estimated that the number of trimer-receptor interactions required for HIV to infect a target cell is approximately eight, which is higher than previous estimates. We also consider model extensions that explain some systematic deviations of the data from the prediction of the simplest model. However, these extended models yield very different estimates of the stoichiometry of entry ranging from 2 to 19. These results strongly suggest that, based on our present knowledge of HIV entry, the stoichiometry of this process cannot be reliably estimated. Our study identifies parameters that need to be defined to render the estimation of the stoichiometry of HIV entry possible.     

CD4-specific designed ankyrin repeat proteins are novel potent HIV entry inhibitors with unique characteristics

Here, we describe the generation of a novel type of HIV entry inhibitor using the recently developed Designed Ankyrin Repeat Protein (DARPin) technology. DARPin proteins specific for human CD4 were selected from a DARPin DNA library using ribosome display. Selected pool members interacted specifically with CD4 and competed with gp120 for binding to CD4. DARPin proteins derived in the initial selection series inhibited HIV in a dose-dependent manner, but showed a relatively high variability in their capacity to block replication of patient isolates on primary CD4 T cells. In consequence, a second series of CD4-specific DARPins with improved affinity for CD4 was generated. These 2nd series DARPins potently inhibit infection of genetically divergent (subtype B and C) HIV isolates in the low nanomolar range, independent of coreceptor usage. Importantly, the actions of the CD4 binding DARPins were highly specific: no effect on cell viability or activation, CD4 memory cell function, or interference with CD4-independent virus entry was observed. These novel CD4 targeting molecules described here combine the unique characteristics of DARPins-high physical stability, specificity and low production costs-with the capacity to potently block HIV entry, rendering them promising candidates for microbicide development.     

In vivo and in vitro escape from neutralizing antibodies 2G12, 2F5, and 4E10

Recently, passive immunization of human immunodeficiency virus (HIV)-infected individuals with monoclonal antibodies (MAbs) 2G12, 2F5, and 4E10 provided evidence of the in vivo activity of 2G12 but raised concerns about the function of the two membrane-proximal external region (MPER)-specific MAbs (A. Trkola, H. Kuster, P. Rusert, B. Joos, M. Fischer, C. Leemann, A. Manrique, M. Huber, M. Rehr, A. Oxenius, R. Weber, G. Stiegler, B. Vcelar, H. Katinger, L. Aceto, and H. F. Gunthard, Nat. Med. 11:615-622, 2005). In the light of MPER-targeting vaccines under development, we performed an in-depth analysis of the emergence of mutations conferring resistance to these three MAbs to further elucidate their activity. Clonal analysis of the MPER of plasma virus samples derived during antibody treatment confirmed that no changes in this region had occurred in vivo. Sequence analysis of the 2G12 epitope relevant N-glycosylation sites of viruses derived from 13 patients during the trial supported the phenotypic evaluation, demonstrating that mutations in these sites are associated with resistance. In vitro selection experiments with isolates of four of these individuals corroborated the in vivo finding that virus strains rapidly escape 2G12 pressure. Notably, in vitro resistance mutations differed, in most cases, from those found in vivo. Importantly, in vitro selection with 2F5 and 4E10 demonstrated that resistance to these MAbs can be difficult to achieve and can lead to selection of variants with impaired infectivity. This remarkable vulnerability of the virus to interference within the MPER calls for a further evaluation of the safety and efficacy of MPER-targeting therapeutic and vaccination strategies.     

Low human immunodeficiency virus envelope diversity correlates with low in vitro replication capacity and predicts spontaneous control of plasma viremia after treatment interruptions

Genetic diversity of viral isolates in human immunodeficiency virus (HIV)-infected individuals varies substantially. However, it remains unclear whether HIV-related disease progresses more rapidly in patients harboring virus swarms with low or high diversity and, in the same context, whether high or low diversity is required to induce potent humoral and cellular immune responses. To explore whether viral diversity predicts virologic control, we studied HIV-infected patients who received antiretroviral therapy (ART) for years before undergoing structured treatment interruptions (STI). Viral diversity before initiation of ART and the ability of the patients to contain viremia after STI and final cessation of treatment was evaluated. Seven out of 21 patients contained plasma viremia at low levels after the final treatment cessation. Clonal sequences encompassing the envelope C2V3C3 domain derived from plasma prior to treatment, exhibited significantly lower diversity in these patients compared to those derived from patients with poor control of viremia. Viral diversity pre-ART correlated with the viral replication capacity of rebounding virus isolates during STI. Neutralizing antibody activity against autologous virus was significantly higher in patients who controlled viremia and was associated with lower pretreatment diversity. No such association was found with binding antibodies directed to gp120. In summary, lower pretreatment viral diversity was associated with spontaneous control of viremia, reduced viral replication capacity and higher neutralizing antibody titers, suggesting a link between viral diversity, replication capacity, and neutralizing antibody activity.     

Different Infectivity of HIV-1 Strains Is Linked to Number of Envelope Trimers Required for Entry

HIV-1 enters target cells by virtue of envelope glycoprotein trimers that are incorporated at low density in the viral membrane. How many trimers are required to interact with target cell receptors to mediate virus entry, the HIV entry stoichiometry, still awaits clarification. Here, we provide estimates of the HIV entry stoichiometry utilizing a combined approach of experimental analyses and mathematical modeling. We demonstrate that divergent HIV strains differ in their stoichiometry of entry and require between 1 to 7 trimers, with most strains depending on 2 to 3 trimers to complete infection. Envelope modifications that perturb trimer structure lead to an increase in the entry stoichiometry, as did naturally occurring antibody or entry inhibitor escape mutations. Highlighting the physiological relevance of our findings, a high entry stoichiometry correlated with low virus infectivity and slow virus entry kinetics. The entry stoichiometry therefore directly influences HIV transmission, as trimer number requirements will dictate the infectivity of virus populations and efficacy of neutralizing antibodies. Thereby our results render consideration of stoichiometric concepts relevant for developing antibody-based vaccines and therapeutics against HIV.     

Cell-Cell Transmission Enables HIV-1 to Evade Inhibition by Potent CD4bs Directed Antibodies

HIV is known to spread efficiently both in a cell-free state and from cell to cell, however the relative importance of the cell-cell transmission mode in natural infection has not yet been resolved. Likewise to what extent cell-cell transmission is vulnerable to inhibition by neutralizing antibodies and entry inhibitors remains to be determined. Here we report on neutralizing antibody activity during cell-cell transmission using specifically tailored experimental strategies which enable unambiguous discrimination between the two transmission routes. We demonstrate that the activity of neutralizing monoclonal antibodies (mAbs) and entry inhibitors during cell-cell transmission varies depending on their mode of action. While gp41 directed agents remain active, CD4 binding site (CD4bs) directed inhibitors, including the potent neutralizing mAb VRC01, dramatically lose potency during cell-cell transmission. This implies that CD4bs mAbs act preferentially through blocking free virus transmission, while still allowing HIV to spread through cell-cell contacts. Thus providing a plausible explanation for how HIV maintains infectivity and rapidly escapes potent and broadly active CD4bs directed antibody responses in vivo.