Barbara Niederöst

Versican V2 and the central inhibitory domain of Nogo-A inhibit neurite growth via p75NTR/NgR-independent pathways that converge at RhoA.

Myelin is a major obstacle for regenerating nerve fibers of the adult mammalian central nervous system (CNS). Several proteins including Nogo-A, myelin-associated glycoprotein (MAG), oligodendrocyte myelin glycoprotein (OMgp) and the chondroitin-sulfate proteoglycan (CSPG) Versican V2 have been identified as inhibitory components present in CNS myelin. MAG, OMgp as well as the Nogo specific domain Nogo-66 exert their inhibitory activity by binding to a neuronal receptor complex containing the Nogo-66 receptor NgR and the neurotrophin receptor p75(NTR). While this suggests a converging role of the p75(NTR)/NgR receptor complex for myelin-derived neurite growth inhibitors, we show here that NgR/p75(NTR) is not required for mediating the inhibitory activity of the two myelin components NiG, unlike Nogo-66 a distinct domain of Nogo-A, and Versican V2. Primary neurons derived from a complete null mutant of p75(NTR) are still sensitive to NiG and Versican V2. In line with this result, neurite growth of p75(NTR) deficient neurons is still significantly blocked on total bovine CNS myelin. Furthermore, modulation of RhoA and Rac1 in p75(NTR)-/- neurons persists with NiG and Versican V2. Finally, we demonstrate that neither NiG nor Versican V2 interact with the p75(NTR)/NgR receptor complex and provide evidence that the binding sites of NiG and Nogo-66 are physically distinct from each other on neural tissue. These results indicate not only the existence of neuronal receptors for myelin inhibitors independent from the p75(NTR)/NgR receptor complex but also establish Rho GTPases as a common point of signal convergence of diverse myelin-induced regeneration inhibitory pathways.

Complement lysis activity in autologous plasma is associated with lower viral loads during the acute phase of HIV-1 infection.

Background To explore the possibility that antibody-mediated complement lysis contributes to viremia control in HIV-1 infection, we measured the activity of patient plasma in mediating complement lysis of autologous primary virus. Methods and Findings Sera from two groups of patients—25 with acute HIV-1 infection and 31 with chronic infection—were used in this study. We developed a novel real-time PCR-based assay strategy that allows reliable and sensitive quantification of virus lysis by complement. Plasma derived at the time of virus isolation induced complement lysis of the autologous virus isolate in the majority of patients. Overall lysis activity against the autologous virus and the heterologous primary virus strain JR-FL was higher at chronic disease stages than during the acute phase. Most strikingly, we found that plasma virus load levels during the acute but not the chronic infection phase correlated inversely with the autologous complement lysis activity. Antibody reactivity to the envelope (Env) proteins gp120 and gp41 were positively correlated with the lysis activity against JR-FL, indicating that anti-Env responses mediated complement lysis. Neutralization and complement lysis activity against autologous viruses were not associated, suggesting that complement lysis is predominantly caused by non-neutralizing antibodies. Conclusions Collectively our data provide evidence that antibody-mediated complement virion lysis develops rapidly and is effective early in the course of infection; thus it should be considered a parameter that, in concert with other immune functions, steers viremia control in vivo.

In Vivo and In Vitro Escape from Neutralizing Antibodies 2G12, 2F5, and 4E10

ABSTRACT 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.

Characterization of Human Immunodeficiency Virus Type 1 (HIV-1) Diversity and Tropism in 145 Patients With Primary HIV-1 Infection

BACKGROUND In the context of sexual transmission of human immunodeficiency virus type 1 (HIV-1), current findings suggest that the mucosal barrier is the major site of viral selection, transforming the complex inoculum to a small, homogeneous founder virus population. We analyzed HIV-1 transmission in relation to viral and host characteristics within the Zurich primary HIV-1 infection study. METHODS Clonal HIV-1 envelope sequences (on average 16 clones/patient) were isolated from the first available plasma samples during the early phase of infection from 145 patients with primary HIV-1 infection. Phylogenetic and tropism analyses were performed. Differences of viral diversities were investigated in association with several parameters potentially influencing HIV-1 transmission, eg, concomitant sexually transmitted infections (STIs) and mode of transmission. RESULTS Median viral diversity within env C2-V3-C3 region was 0.39% (range 0.04%-3.23%). Viral diversity did not correlate with viral load, but it was slightly correlated with the duration of infection. Neither transmission mode, gender, nor STI predicted transmission of more heterogeneous founder virus populations that were found in 16 of 145 patients (11%; diversity >1%). Only 2 patients (1.4%) were assuredly infected with CXCR4-tropic HIV-1 within a R5/X4-tropic--mixed population, as revealed and confirmed using several genotypic prediction algorithms and phenotypic assays. CONCLUSIONS Our findings suggest that transmission of multiple HIV-1 variants might be a complex process that is not dependent on mucosal factors alone. CXCR4-tropic viruses can be sexually transmitted in rare instances, but their clinical relevance remains to be determined.

Productive Human Immunodeficiency Virus Type 1 Infection in Peripheral Blood Predominantly Takes Place in CD4/CD8 Double-Negative T Lymphocytes

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) transcription is subject to substantial fluctuation during the viral life cycle. Due to the low frequencies of HIV-1-infected cells, and because latently and productively infected cells collocate in vivo, little quantitative knowledge has been attained about the range of in vivo HIV-1 transcription in peripheral blood mononuclear cells (PBMC). By combining cell sorting, terminal dilution of intact cells, and highly sensitive, patient-specific PCR assays, we divided PBMC obtained from HIV-1-infected patients according to their degree of viral transcription activity and their cellular phenotype. Regardless of a patients treatment status, the bulk of infected cells exhibited a CD4+ phenotype but transcribed HIV-1 provirus at low levels, presumably insufficient for virion production. Furthermore, the expression of activation markers on the surface of these CD4+ T lymphocytes showed little or no association with enhancement of viral transcription. In contrast, HIV-infected T lymphocytes of a CD4−/CD8− phenotype, occurring exclusively in untreated patients, exhibited elevated viral transcription rates. This cell type harbored a substantial proportion of all HIV RNA+ cells and intracellular viral RNAs and the majority of cell-associated virus particles. In conjunction with the observation that the HIV quasispecies in CD4+ and CD4−/CD8− T cells were phylogenetically closely related, these findings provide evidence that CD4 expression is downmodulated during the transition to productive infection in vivo. The abundance of viral RNA in CD4−/CD8− T cells from viremic patients and the almost complete absence of viral DNA and RNA in this cell type during antiretroviral treatment identify HIV+ CD4−/CD8 T cells as the major cell type harboring productive infection in peripheral blood.

Tailored enrichment strategy detects low abundant small noncoding RNAs in HIV-1 infected cells.

BackgroundThe various classes of small noncoding RNAs (sncRNAs) are important regulators of gene expression across divergent types of organisms. While a rapidly increasing number of sncRNAs has been identified over recent years, the isolation of sncRNAs of low abundance remains challenging. Virally encoded sncRNAs, particularly those of RNA viruses, can be expressed at very low levels. This is best illustrated by HIV-1 where virus encoded sncRNAs represent approximately 0.1-1.0% of all sncRNAs in HIV-1 infected cells or were found to be undetected. Thus, we applied a novel, sequence targeted enrichment strategy to capture HIV-1 derived sncRNAs in HIV-1 infected primary CD4+ T-lymphocytes and macrophages that allows a greater than 100-fold enrichment of low abundant sncRNAs.ResultsEight hundred and ninety-two individual HIV-1 sncRNAs were cloned and sequenced from nine different sncRNA libraries derived from five independent experiments. These clones represent up to 90% of all sncRNA clones in the generated libraries. Two hundred and sixteen HIV-1 sncRNAs were distinguishable as unique clones. They are spread throughout the HIV-1 genome, however, forming certain clusters, and almost 10% show an antisense orientation. The length of HIV-1 sncRNAs varies between 16 and 89 nucleotides with an unexpected peak at 31 to 50 nucleotides, thus, longer than cellular microRNAs or short-interfering RNAs (siRNAs). Exemplary HIV-1 sncRNAs were also generated in cells infected with different primary HIV-1 isolates and can inhibit HIV-1 replication.ConclusionsHIV-1 infected cells generate virally encoded sncRNAs, which might play a role in the HIV-1 life cycle. Furthermore, the enormous capacity to enrich low abundance sncRNAs in a sequence specific manner highly recommends our selection strategy for any type of investigation where origin or target sequences of the sought-after sncRNAs are known.

Biphasic decay kinetics suggest progressive slowing in turnover of latently HIV-1 infected cells during antiretroviral therapy

BackgroundMathematical models based on kinetics of HIV-1 plasma viremia after initiation of combination antiretroviral therapy (cART) inferred HIV-infected cells to decay exponentially with constant rates correlated to their strength of virus production. To further define in vivo decay kinetics of HIV-1 infected cells experimentally, we assessed infected cell-classes of distinct viral transcriptional activity in peripheral blood mononuclear cells (PBMC) of five patients during 1 year after initiation of cARTResultsIn a novel analytical approach patient-matched PCR for unspliced and multiply spliced viral RNAs was combined with limiting dilution analysis at the single cell level. This revealed that HIV-RNA+ PBMC can be stratified into four distinct viral transcriptional classes. Two overlapping cell-classes of high viral transcriptional activity, suggestive of a virion producing phenotype, rapidly declined to undetectable levels. Two cell classes expressing HIV-RNA at low and intermediate levels, presumably insufficient for virus production and occurring at frequencies exceeding those of productively infected cells matched definitions of HIV-latency. These cells persisted during cART. Nevertheless, during the first four weeks of therapy their kinetics resembled that of productively infected cells.ConclusionWe have observed biphasic decays of latently HIV-infected cells of low and intermediate viral transcriptional activity with marked decreases in cell numbers shortly after initiation of therapy and complete persistence in later phases. A similar decay pattern was shared by cells with greatly enhanced viral transcriptional activity which showed a certain grade of levelling off before their disappearance. Thus it is conceivable that turnover/decay rates of HIV-infected PBMC may be intrinsically variable. In particular they might be accelerated by HIV-induced activation and reactivation of the viral life cycle and slowed down by the disappearance of such feedback-loops after initiation of cART.

Equal Amounts of Intracellular and Virion‐Enclosed Hepatitis C Virus RNA Are Associated with Peripheral‐Blood Mononuclear Cells In Vivo

BACKGROUND Hepatitis C virus (HCV) replicating in peripheral-blood mononuclear cells (PBMCs) may represent an extrahepatic viral reservoir. Quantitation of HCV RNA with regard to its subcellular distribution and longitudinal course is needed for better understanding of the largely unexplored in vivo dynamics and potential pathogenetic significance of HCV in PBMCs. METHODS Plasma and PBMCs from 30 patients coinfected with HCV and human immunodeficiency virus were evaluated in cross-sectional and longitudinal analyses, for up to 40 months. Differential extraction of virion-enclosed HCV RNA associated with cells was performed in parallel with extraction of total cellular HCV RNA. HCV RNA of either orientation was quantified by real-time polymerase chain reaction. RESULTS HCV RNA was detected only in PBMCs from patients with viremia and at relatively stable quantities over time. Intracellular HCV RNA corresponding to ~60% of total cellular HCV RNA was strongly correlated with virion-enclosed HCV RNA but was only weakly associated with viral loads in plasma. In contrast, the ratio of HCV RNA load in PBMCs versus that in plasma was patient specific and stable over time. CONCLUSIONS The substantial and patient-specific amounts of intracellular HCV RNA found by the present study support a concept of low-level replication in PBMCs. There was no evidence for persistent HCV infection in PBMCs after clearance of viremia in plasma.

Tracing HIV-1 transmission: envelope traits of HIV-1 transmitter and recipient pairs

BackgroundMucosal HIV-1 transmission predominantly results in a single transmitted/founder (T/F) virus establishing infection in the new host despite the generally high genetic diversity of the transmitter virus population. To what extent HIV-1 transmission is a stochastic process or driven by selective forces that allow T/F viruses best to overcome bottlenecks in transmission has not been conclusively resolved. Building on prior investigations that suggest HIV-1 envelope (Env) features to contribute in the selection process during transmission, we compared phenotypic virus characteristics of nine HIV-1 subtype B transmission pairs, six men who have sex with men and three male-to-female transmission pairs.ResultsAll recipients were identified early in acute infection and harbored based on extensive sequencing analysis a single T/F virus allowing a controlled analysis of virus properties in matched transmission pairs. Recipient and transmitter viruses from the closest time point to transmission showed no signs of selection for specific Env modifications such as variable loop length and glycosylation. Recipient viruses were resistant to circulating plasma antibodies of the transmitter and also showed no altered sensitivity to a large panel of entry inhibitors and neutralizing antibodies. The recipient virus did not consistently differ from the transmitter virus in terms of entry kinetics, cell–cell transmission and replicative capacity in primary cells. Our paired analysis revealed a higher sensitivity of several recipient virus isolates to interferon-α (IFNα) which suggests that resistance to IFNα cannot be a general driving force in T/F establishment.ConclusionsWith the exception of increased IFNα sensitivity, none of the phenotypic virus properties we investigated clearly distinguished T/F viruses from their matched transmitter viruses supporting the notion that at least in subtype B infection HIV-1 transmission is to a considerable extent stochastic.

Minority K65R variants and early failure of antiretroviral therapy in HIV-1-infected Eritrean Immigrant

To the Editor: Genotypic drug resistance testing before initiation of first-line antiretroviral therapy (ART) is recommended to detect drug-resistant viruses and to avoid treatment failure caused by preexisting drug-resistant viruses (1). However, standard resistance testing cannot detect drug-resistant HIV-1 minority variants unless they represent 20%–25% of the population (2). Approximately 15% of those who underwent seroconversion in the acute phase in industrialized settings harbor drug-resistant HIV-1 minority variants, while standard resistance testing did not detect drug-resistant viruses in those patients (3). We report the case of a treatment-naive HIV-1–infected patient with early treatment failure because of preexisting minority K65R-harboring HIV-1 variants. A 32-year-old immigrant to Switzerland from Eritrea with a recently diagnosed HIV-1 subtype C infection was seen at University Hospital, Zurich. On the basis of the low CD4+ T-cell count of 69 cells/µL (15%) and high HIV-1 viral load of 980,000 copies/mL plasma, we started directly observed ART with tenofovir and emtricitabine plus nevirapine. Genotypic resistance testing showed no evidence of resistance. Within the first 4 weeks of ART, the viral load decreased to 540; however, 4 weeks later it increased to 15,000, and then 12 days later to 71,000 HIV-1 RNA copies/mL (Figure). Resistance testing at this time revealed the reverse transcriptase (RT) mutations K65R, K103N, and M184V, which confer resistance to all prescribed drugs. ART was changed to lamivudine/zidovudine, darunavir/ritonavir, and etravirine, and subsequently viremia decreased and remained undetectable. Figure Kinetics of viremia, CD4+ T-cell count, and drug resistance mutations in a treatment-naive person from Eritrea, infected with HIV-1 subtype C, who was experiencing early antiretroviral therapy (ART) failure. Viral load (circles) was measured by using ... We hypothesized that preexisting drug-resistant HIV-1 minority variants might have caused this early treatment failure. Thus, we performed clonal analysis of the RT gene before and during ART. At baseline, 17/222 clones (7.7%) carried the K65R mutation, synonymous to ≈51,000 HIV-1 RNA copies. Later, the K65R mutation was comprised in all clones. Further preexisting drug-resistance mutations have been detected in single, K65 wild-type, separate clones: K70R, V106A, and V108I. However, neither the K103N nor the M184V mutation was detected in any of those 222 clones, but both mutations were rapidly selected during early treatment failure. Thus, the presence of the K65R mutation in a substantial fraction of the virus population and the rapid acquisition of the K103N and M184V mutations led eventually to early treatment failure. We cannot formally rule out that K103N mutants were present before ART at a very low frequency <0.5% and that K65R/K103N double mutants were potentially selected. The M184V mutation was acquired later because only 12.5% of K65R/K103N viruses carried the M184V mutation at the first time point during treatment failure. Because of the considerable absolute number of less replication-competent K65R-harboring viruses in the patient, we assume that this variant has been transmitted. The additional presence of isolated, nonnucleoside RT inhibitor (NNRTI)/nucleoside RT inhibitor (NRTI) resistance mutations is another indication for the transmission of drug-resistant viruses, although they and the K65R mutation were not found in the same viral genomes. Presumably, the index patient was treated with nevirapine, stavudine, and lamivudine, the commonly prescribed first-line ART in resource-limited settings (4). The prevalence of K65R-harboring drug-resistant HIV-1 minority variants is not negligible in treatment-naive patients. We have shown that 2.7% of HIV-1–infected patients, mainly those infected with HIV-1 subtype B, harbor the K65R mutation as a minority variant (5), which is comparable with the prevalence of minority K65R-harboring variants (4%) in patients from South Africa who are infected with HIV-1 subtype C (6). A meta-analysis showed that limited or unavailable HIV-1 RNA monitoring in combination with ART regimens with a low genetic barrier to resistance in resource-limited settings is associated with high rates of NNRTI/NRTI resistance in patients for whom ART fails (7). Although ritonavir-boosted protease inhibitors containing ART regimens have a higher genetic barrier to resistance and would be preferable in such settings, they are generally not part of first-line therapy in resource-limited settings. Thus, proper monitoring and drug-resistance testing would be desirable when using NNRTI-based ART regimens. Moreover, it could be beneficial to apply sensitive assays for the detection of drug-resistant HIV-1 minority variants in clinical practice. However, clinical cut-off levels for those minority variants need to be defined. Immigration from resource-limited settings is increasing, and data suggest that minority variants harboring the K65R mutation are quite prevalent in those infected with HIV-1 subtype C, who are treatment naive (6). The case described here demonstrates that these drug-resistant HIV-1 minority variants can quickly accumulate further drug-resistance mutations and lead to early treatment failure, especially in the context of an ART regimen with low genetic barriers to resistance. This case was the first among immigrants treated in our center, but, given the potential for considerable transmission rates of resistance in countries that lack virologic monitoring, this mutation could become a larger problem.