Using viral diversity to identify HIV-1 variants under HLA-dependent selection in a systematic viral genome-wide screen.

The pathogenesis of HIV-1 infection is governed by a highly dynamic, time-dependent interaction between the host and the viral genome. In this study, we developed a novel systematic approach to assess the host-virus interaction, using average pairwise viral diversity as a proxy for time since infection, and applied this method to nearly whole viral genome sequences (n = 4,464), human leukocyte antigen (HLA) genotyping data (n = 1,044), and viral RNA load (VL) measurements during the untreated chronic phase (n = 829) of Swiss HIV Cohort Study participants. Our systematic genome-wide screen revealed for 98 HLA/viral-variant pairs a signature of immune-driven selection in the form of an HLA-dependent effect of infection time on the presence of HIV amino acid variants. Of these pairs, 12 were found to have an effect on VL. Furthermore, 28/58 pairs were validated by time-to-event analyses and 48/92 by computational HLA-epitope predictions. Our diversity-based approach allows a powerful and systematic investigation of the interaction between the virus and cellular immunity, revealing a notable subset of such interaction effects. From an evolutionary perspective, these observations underscore the complexity of HLA-mediated selection pressures on the virus that shape viral evolution and pathogenesis.

Genetic Diversity From Proviral DNA as a Proxy for Time Since HIV-1 Infection.

HIV-1 RNA genetic diversity predicts time since infection, which is important for clinical care and research. It is unclear, however, whether proviral DNA genetic diversity sampled under suppressive antiretroviral therapy can be used for this purpose. We tested whether proviral genetic diversity from next-generation sequencing predicts time since infection and recency in 221 people with HIV-1 with known infection time. Proviral diversity was significantly associated with time since infection (P < 5×10-7, R2 up to 25%) and predictive of treatment initiation during recent infection (area under the curve-receiver operating characteristic up to 0.85). This shows the utility of proviral genetic diversity as a proxy for time since infection.

Absence of Proviral Human Immunodeficiency Virus (HIV) Type 1 Evolution in Early-Treated Individuals With HIV Switching to Dolutegravir Monotherapy During 48 Weeks.

Human immunodeficiency virus type 1 (HIV-1) infection is treated with antiretroviral therapy (ART), usually consisting of 2-3 different drugs, referred to as combination ART (cART). Our recent randomized clinical trial comparing a switch to dolutegravir monotherapy with continuation of cART in early-treated individuals demonstrated sustained virological suppression over 48 weeks. Here, we characterize the longitudinal landscape of the HIV-1 reservoir in these participants, with particular attention to potential differences between treatment groups regarding evidence of evolution as a proxy for low-level replication. Near full-length HIV-1 proviral polymerase chain reaction and next-generation sequencing was applied to longitudinal peripheral blood mononuclear cell samples to assess proviral evolution and the potential emergence of drug resistance mutations (DRMs). Neither an increase in genetic distance nor diversity over time was detected in participants of both treatment groups. Single proviral analysis showed high proportions of defective proviruses and low DRM numbers. No evidence for evolution during dolutegravir monotherapy was found in these early-treated individuals.

Sustained Viral Suppression With Dolutegravir Monotherapy Over 192 Weeks in Patients Starting Combination Antiretroviral Therapy During Primary Human Immunodeficiency Virus Infection (EARLY-SIMPLIFIED): A Randomized, Controlled, Multi-site, Noninferiority Trial.

BACKGROUND: Starting combination antiretroviral therapy (cART) during primary human immunodeficiency virus type 1 (HIV-1) infection results in a smaller HIV-1 latent reservoir, reduced immune activation, and less viral diversity compared to starting cART during chronic infection. We report results of a 4-year study designed to determine whether these properties would allow sustained virological suppression after simplification of cART to dolutegravir (DTG) monotherapy. METHODS: EARLY-SIMPLIFIED is a randomized, open-label, noninferiority trial. People with HIV (PWH) who started cART <180 days after a documented primary HIV-1 infection with suppressed viral load were randomized (2:1) to DTG monotherapy with 50 mg daily or continuation of cART. The primary endpoints were the proportion of PWH with viral failure at 48, 96, 144, and 192 weeks; noninferiority margin was 10%. After 96 weeks, randomization was lifted and patients were permitted to switch treatment groups as desired. RESULTS: Of 101 PWH randomized, 68 were assigned to DTG monotherapy and 33 to cART. At week 96 in the per-protocol population, 64/64 (100%) showed virological response in the DTG monotherapy group versus 30/30 (100%) in the cART group (difference, 0.00%; upper bound of 95% confidence interval 6.22%). This demonstrated noninferiority of DTG monotherapy at the prespecified level. At week 192, the study end, no virological failure occurred in either group during 13 308 and 4897 person weeks of follow-up for the DTG monotherapy (n = 80) and cART groups, respectively. CONCLUSIONS: This trial suggests that early cART initiation during primary HIV infection allows sustained virological suppression after switching to DTG monotherapy.

Prevalence of HIV-1 drug resistance mutations in proviral DNA in the Swiss HIV Cohort Study, a retrospective study from 1995 to 2018.

BACKGROUND: Genotypic resistance testing (GRT) is routinely performed upon diagnosis of HIV-1 infection or during virological failure using plasma viral RNA. An alternative source for GRT could be cellular HIV-1 DNA. OBJECTIVES: A substantial number of participants in the Swiss HIV Cohort Study (SHCS) never received GRT. We applied a method that enables access to the near full-length proviral HIV-1 genome without requiring detectable viraemia. METHODS: Nine hundred and sixty-two PBMC specimens were received. Our two-step nested PCR protocol was applied to generate two overlapping long-range amplicons of the HIV-1 genome, sequenced by next-generation sequencing (NGS) and analysed by MinVar, a pipeline to detect drug resistance mutations (DRMs). RESULTS: Six hundred and eighty-one (70.8%) of the samples were successfully amplified, sequenced and analysed by MinVar. Only partial information of the pol gene was contained in 82/681 (12%), probably due to naturally occurring deletions in the proviral sequence. All common HIV-1 subtypes were successfully sequenced. We detected at least one major DRM at high frequency (≥15%) in 331/599 (55.3%) individuals. Excluding APOBEC-signature (G-to-A mutation) DRMs, 145/599 (24.2%) individuals carried at least one major DRM. RT-inhibitor DRMs were most prevalent. The experienced time on ART was significantly longer in DRM carriers (P = 0.001) independent of inclusion or exclusion of APOBEC-signature DRMs. CONCLUSIONS: We successfully applied a reliable and efficient method to analyse near full-length HIV-1 proviral DNA and investigated DRMs in individuals with undetectable or low viraemia. Additionally, our data underscore the need for new computational tools to exclude APOBEC-related hypermutated NGS sequence reads for reporting DRMs.

The Interplay Between Replication Capacity of HIV-1 and Surrogate Markers of Disease.

BACKGROUND: HIV-1 replication capacity (RC) of transmitted/founder viruses may influence the further course of HIV-1 infection. METHODS: RCs of 355 whole-genome primary HIV-1 isolates derived from samples acquired during acute and recent primary HIV-1 infection (PHI) were determined using a novel high-throughput infection assay in primary cells. The RCs were used to elucidate potential factors that could be associated with RC during PHI. RESULTS: Increased RC was found to be associated with increased set point viral load (VL), and significant differences in RCs among 13 different HIV-1 subtypes were discerned. Notably, we observed an increase in RCs for primary HIV-1 isolates of HIV-1 subtype B over a 17-year period. Associations were not observed between RC and CD4 count at sample date of RC measurement, CD4 recovery after initiation of antiretroviral treatment, CD4 decline in untreated individuals, and acute retroviral syndrome severity scores. CONCLUSIONS: These findings highlight that RCs of primary HIV-1 isolates acquired during the acute and recent phase of infection are more associated with viral factors, that is set point VL, than with host factors. Furthermore, we observed a temporal increase in RC for HIV-1 subtype B viruses over a period of 17 years. CLINICAL TRIALS REGISTRATION: NCT00537966.

Noninferiority of Simplified Dolutegravir Monotherapy Compared to Continued Combination Antiretroviral Therapy That Was Initiated During Primary Human Immunodeficiency Virus Infection: A Randomized, Controlled, Multisite, Open-label, Noninferiority Trial.

BACKGROUND: Patients who start combination antiretroviral therapy (cART) during primary human immunodeficiency virus type 1 (HIV-1) infection show a smaller HIV-1 latent reservoir, less immune activation, and less viral diversity compared to patients who start cART during chronic infection. We conducted a pilot study to determine whether these properties would allow sustained virological suppression after simplification of cART to dolutegravir monotherapy. METHODS: EARLY-SIMPLIFIED is a randomized, open-label, noninferiority trial. Patients who started cART <180 days after a documented primary HIV-1 infection and had an HIV-1 RNA <50 copies/mL plasma for at least 48 weeks were randomized (2:1) to monotherapy with dolutegravir 50 mg once daily or to continuation of cART. The primary efficacy endpoint was the proportion of patients with <50 HIV-1 RNA copies/mL on or before week 48; noninferiority margin 10%. RESULTS: Of the 101 patients randomized, 68 were assigned to simplification to dolutegravir monotherapy and 33 to continuation of cART. At week 48 in the per-protocol population, 67/67 (100%) had virological response in the dolutegravir monotherapy group vs 32/32 (100%) in the cART group (difference, 0.00%; 95% confidence interval, -100%, 4.76%). This showed noninferiority of the dolutegravir monotherapy at the prespecified level. CONCLUSION: In this pilot study consisting of patients who initiated cART during primary HIV-1 infection and had <50 HIV-1 RNA copies/mL for at least 48 weeks, monotherapy with once-daily dolutegravir was noninferior to cART. Our results suggest that future simplification studies should use a stratification according to time of HIV infection and start of first cART. CLINICAL TRIALS REGISTRATION: NCT02551523.

Mycobacterium tuberculosis infection associated immune perturbations correlate with antiretroviral immunity.

Infection with Mycobacterium tuberculosis (MTB) remains one of the most important opportunistic infections in people with HIV-1 (PWH). While active Tuberculosis (TB) leads to rapid progression of immunodeficiency in PWH, the interaction between MTB and HIV-1 during the asymptomatic phase of both infections remains poorly understood. In a cohort of individuals with HIV (PWH) with and without suppressed HIV-1 viral load, the transcriptomic profiles of peripheral blood mononuclear cells (PBMC) clustered in individuals infected with Mycobacterium tuberculosis (MTB) compared to carefully matched controls. Subsequent functional annotation analysis disclosed alterations in the IL-6, TNF, and KRAS pathways. Notably, MTB-associated genes demonstrated an inverse correlation with HIV-1 viremia, evident at both on individual gene level and when employed as a gene score. In sum, our data show that MTB infection in PWH is associated with a shift in the activation state of the immune system, displaying an inverse relationship with HIV-1 viral load. These results could provide an explanation for the observed increased antiretroviral control associated with MTB infection in PWH.

Self-reported neurocognitive complaints in the Swiss HIV Cohort Study: a viral genome-wide association study.

People with HIV may report neurocognitive complaints, with or without associated neurocognitive impairment, varying between individuals and populations. While the HIV genome could play a major role, large systematic viral genome-wide screens to date are lacking. The Swiss HIV Cohort Study biannually enquires neurocognitive complaints. We quantified broad-sense heritability estimates using partial 'pol' sequences from the Swiss HIV Cohort Study resistance database and performed a viral near full-length genome-wide association study for the longitudinal area under the curve of neurocognitive complaints. We performed all analysis (i) restricted to HIV Subtype B and (ii) including all HIV subtypes. From 8547 people with HIV with neurocognitive complaints, we obtained 6966 partial 'pol' sequences and 2334 near full-length HIV sequences. Broad-sense heritability estimates for presence of memory loss complaints ranged between 1% and 17% (Subtype B restricted 1-22%) and increased with the stringency of the phylogenetic distance thresholds. The genome-wide association study revealed one amino acid (Env L641E), after adjusting for multiple testing, positively associated with memory loss complaints (P = 4.3 * 10-6). Other identified mutations, while insignificant after adjusting for multiple testing, were reported in other smaller studies (Tat T64N, Env *291S). We present the first HIV genome-wide association study analysis of neurocognitive complaints and report a first estimate for the heritability of neurocognitive complaints through HIV. Moreover, we could identify one mutation significantly associated with the presence of memory loss complaints. Our findings indicate that neurocognitive complaints are polygenetic and highlight advantages of a whole genome approach for pathogenicity determination.

A Novel High Throughput, Parallel Infection Assay for Determining the Replication Capacities of 346 Primary HIV-1 Isolates of the Zurich Primary HIV-1 Infection Study in Primary Cells.

HIV-1 replication capacity is an important characteristic to understand the replication competence of single variants or virus populations. It can further aid in the understanding of HIV-1 pathogenicity, disease progression, and drug resistance mutations. To effectively study RC, many assays have been established. However, there is still demand for a high throughput replication capacity assay using primary cells which is robust and reproducible. In this study, we established such an assay and validated it using 346 primary HIV-1 isolates from patients enrolled in the Zurich Primary HIV Infection study (ZPHI) and two control viruses, HIV-1 JR-CSFWT and HIV-1 JR-CSFK65R_M184V. Replication capacity was determined by measuring the viral growth on PBMCs over 10 days by longitudinally transferring cell culture supernatant to TZM-bl reporter cells. By utilizing the TZM-bl luciferase reporter assay, we determined replication capacity by measuring viral infectivity. The simplicity of the experimental setup allowed for all 346 primary HIV-1 isolates to be replicated at one time. Although the infectious input dose for each virus was normalized, a broad range of replication capacity values over 4 logs was observed. The approach was confirmed by two repeated experiments and we demonstrated that the reproducibility of the replication capacity values is statistically comparable between the two separate experiments. In summary, these results endorse our high throughput replication capacity assay as reproducible and robust and can be utilized for large scale HIV-1 replication capacity experiments in primary cells.

HIV-1 integration sites in CD4+ T cells during primary, chronic, and late presentation of HIV-1 infection.

HIV-1 is capable of integrating its genome into that of its host cell. We examined the influence of the activation state of CD4+ T cells, the effect of antiretroviral therapy (ART), and the clinical stage of HIV-1 infection on HIV-1 integration site features and selection. HIV-1 integration sites were sequenced from longitudinally sampled resting and activated CD4+ T cells from 12 HIV-1-infected individuals. In total, 589 unique HIV-1 integration sites were analyzed: 147, 391, and 51 during primary, chronic, and late presentation of HIV-1 infection, respectively. As early as during primary HIV-1 infection and independent of the activation state of CD4+ T cells collected on and off ART, HIV-1 integration sites were preferentially detected in recurrent integration genes, genes associated with clonal expansion of latently HIV-1-infected CD4+ T cells, cancer-related genes, and highly expressed genes. The preference for cancer-related genes was more pronounced at late stages of HIV-1 infection. Host genomic features of HIV-1 integration site selection remained stable during HIV-1 infection in both resting and activated CD4+ T cells. In summary, characteristic HIV-1 integration site features are preestablished as early as during primary HIV-1 infection and are found in both resting and activated CD4+ T cells.

HIV-1 promoter is gradually silenced when integrated into BACH2 in Jurkat T-cells.

BACKGROUND: The persistence of the latent HIV-1 reservoir is a major obstacle to curing HIV-1 infection. HIV-1 integrates into the cellular genome and some targeted genomic loci are frequently detected in clonally expanded latently HIV-1 infected cells, for instance, the gene BTB domain and CNC homology 2 (BACH2). METHODS: We investigated HIV-1 promoter activity after integration into specific sites in BACH2 in Jurkat T-cells. The HIV-1-based vector LTatCL[M] contains two fluorophores: (1) Cerulean, which reports the activity of the HIV-1 promoter and (2) mCherry driven by a constitutive promotor and flanked by genetic insulators. This vector was inserted into introns 2 and 5 of BACH2 of Jurkat T-cells via CRISPR/Cas9 technology in the same and convergent transcriptional orientation of BACH2, and into the genomic safe harbour AAVS1. Single cell clones representing active (Cerulean+/mCherry+) and inactive (Cerulean-/mCherry+) HIV-1 promoters were characterised. RESULTS: Upon targeted integration of the 5.3 kb vector LTatCL[M] into BACH2, the HIV-1 promoter was gradually silenced as reflected by the decrease in Cerulean expression over a period of 162 days. Silenced HIV-1 promoters could be reactivated by TNF-α and Romidepsin. This observation was independent of the targeted intron and the transcriptional orientation. BACH2 mRNA and protein expression was not impaired by mono-allelic integration of LTatCL[M]. CONCLUSION: Successful targeted integration of the HIV-1-based vector LTatCL[M] allows longitudinal analyses of HIV-1 promoter activity.

Heritability of the HIV-1 reservoir size and decay under long-term suppressive ART.

The HIV-1 reservoir is the major hurdle to curing HIV-1. However, the impact of the viral genome on the HIV-1 reservoir, i.e. its heritability, remains unknown. We investigate the heritability of the HIV-1 reservoir size and its long-term decay by analyzing the distribution of those traits on viral phylogenies from both partial-pol and viral near full-length genome sequences. We use a unique nationwide cohort of 610 well-characterized HIV-1 subtype-B infected individuals on suppressive ART for a median of 5.4 years. We find that a moderate but significant fraction of the HIV-1 reservoir size 1.5 years after the initiation of ART is explained by genetic factors. At the same time, we find more tentative evidence for the heritability of the long-term HIV-1 reservoir decay. Our findings indicate that viral genetic factors contribute to the HIV-1 reservoir size and hence the infecting HIV-1 strain may affect individual patients' hurdle towards a cure.

Host Genomics of the HIV-1 Reservoir Size and Its Decay Rate During Suppressive Antiretroviral Treatment.

BACKGROUND: The primary hurdle for the eradication of HIV-1 is the establishment of a latent viral reservoir early after primary infection. Here, we investigated the potential influence of human genetic variation on the HIV-1 reservoir size and its decay rate during suppressive antiretroviral treatment. SETTING: Genome-wide association study and exome sequencing study to look for host genetic determinants of HIV-1 reservoir measurements in patients enrolled in the Swiss HIV Cohort Study, a nation-wide prospective observational study. METHODS: We measured total HIV-1 DNA in peripheral blood mononuclear cells from study participants, as a proxy for the reservoir size at 3 time points over a median of 5.4 years, and searched for associations between human genetic variation and 2 phenotypic readouts: the reservoir size at the first time point and its decay rate over the study period. We assessed the contribution of common genetic variants using genome-wide genotyping data from 797 patients with European ancestry enrolled in the Swiss HIV Cohort Study and searched for a potential impact of rare variants and exonic copy number variants using exome sequencing data generated in a subset of 194 study participants. RESULTS: Genome-wide and exome-wide analyses did not reveal any significant association with the size of the HIV-1 reservoir or its decay rate on suppressive antiretroviral treatment. CONCLUSIONS: Our results point to a limited influence of human genetics on the size of the HIV-1 reservoir and its long-term dynamics in successfully treated individuals.

EBV renders B cells susceptible to HIV-1 in humanized mice.

HIV and EBV are human pathogens that cause a considerable burden to worldwide health. In combination, these viruses are linked to AIDS-associated lymphomas. We found that EBV, which transforms B cells, renders them susceptible to HIV-1 infection in a CXCR4 and CD4-dependent manner in vitro and that CXCR4-tropic HIV-1 integrates into the genome of these B cells with the same molecular profile as in autologous CD4+ T cells. In addition, we established a humanized mouse model to investigate the in vivo interactions of EBV and HIV-1 upon coinfection. The respective mice that reconstitute human immune system components upon transplantation with CD34+ human hematopoietic progenitor cells could recapitulate aspects of EBV and HIV immunobiology observed in dual-infected patients. Upon coinfection of humanized mice, EBV/HIV dual-infected B cells could be detected, but were susceptible to CD8+ T-cell-mediated immune control.

Determinants of HIV-1 reservoir size and long-term dynamics during suppressive ART.

The HIV-1 reservoir is the major hurdle to a cure. We here evaluate viral and host characteristics associated with reservoir size and long-term dynamics in 1,057 individuals on suppressive antiretroviral therapy for a median of 5.4 years. At the population level, the reservoir decreases with diminishing differences over time, but increases in 26.6% of individuals. Viral blips and low-level viremia are significantly associated with slower reservoir decay. Initiation of ART within the first year of infection, pretreatment viral load, and ethnicity affect reservoir size, but less so long-term dynamics. Viral blips and low-level viremia are thus relevant for reservoir and cure studies.

Spontaneous reactivation of latent HIV-1 promoters is linked to the cell cycle as revealed by a genetic-insulators-containing dual-fluorescence HIV-1-based vector.

Long-lived latently HIV-1-infected cells represent a barrier to cure. We developed a dual-fluorescence HIV-1-based vector containing a pair of genetic insulators flanking a constitutive fluorescent reporter gene to study HIV-1 latency. The protective effects of these genetic insulators are demonstrated through long-term (up to 394 days) stable fluorescence profiles in transduced SUP-T1 cells. Analysis of 1,941 vector integration sites confirmed reproduction of HIV-1 integration patterns. We sorted monoclonal cells representing latent HIV-1 infections and found that both vector integration sites and integrity of the vector genomes influence the reactivation potentials of latent HIV-1 promoters. Interestingly, some latent monoclonal cells exhibited a small cell subpopulation with a spontaneously reactivated HIV-1 promoter. Higher expression levels of genes involved in cell cycle progression are observed in these cell subpopulations compared to their counterparts with HIV-1 promoters that remained latent. Consistently, larger fractions of spontaneously reactivated cells are in the S and G2 phases of the cell cycle. Furthermore, genistein and nocodazole treatments of these cell clones, which halted cells in the G2 phase, resulted in a 1.4-2.9-fold increase in spontaneous reactivation. Taken together, our HIV-1 latency model reveals that the spontaneous reactivation of latent HIV-1 promoters is linked to the cell cycle.

The Hydra viridis/Chlorella symbiosis. Growth and sexual differentiation in polyps without symbionts.

To investigate interactions between the basal metazoan Hydra viridis and its symbiotic Chlorella algae, we generated aposymbiotic hydra lacking algae and compared them to symbiotic ones with regard to growth and sexual differentiation. Under standard feeding conditions aposymbiotic polyps proliferated similarly to symbiotic polyps. Under moderate and low feeding conditions asexual growth was reduced in polyps lacking algae, indicating that the symbionts supply nutrients to their hosts. In addition, the Chlorella symbionts had a strong influence on the sexual reproduction of Hydra viridis: in most cases female gonads were produced only when symbiotic algae were present. Spermatogenesis proceeded similarly in symbiotic and aposymbiotic polyps. Since during oogenesis symbionts are actively transferred from endodermal epithelial cells to the ectodermal oocytes, this oogenesis promoting role could indicate that the symbionts are critically involved in the control of sexual differentiation in green hydra.

Increasing prevalence of Addison's disease in German females: health insurance data 2008-2012.

OBJECTIVE: Our objective was to investigate the epidemiology of autoimmune Addison's disease (AD) in Germany. DESIGN: Routine data were analyzed from the Statutory Health Insurance (SHI) database of the Techniker Krankenkasse (TK) for an observation period from 01/01/2008 to 31/12/2012. The TK is one of the largest German health care insurance providers covering more than 10% of the German population. SUBJECTS AND METHODS: Between 2008 and 2012, a total of 2477 diagnoses of primary adrenal failure were recorded in the SHI database. After exclusion of secondary, iatrogenic or other non-idiopathic forms and after adjustment for incomplete data sets, 1364 diagnoses of autoimmune-mediated AD remained. RESULTS: The prevalence of AD in our cohort showed a steady increase from 82 per million in 2008 to 87 per million in 2012. On average, the prevalence rose about 1.8% per year, and due to a pronounced increase (2.7%) in females. The prevalence was lower in men (63-68 per million) than in women (96-108 per million). Autoimmune comorbidities were found in 46.5% of AD patients. Adrenal crises were documented with a frequency of 14-17/100 patient years. CONCLUSIONS: These data provide a first epidemiological profile of this rare and perilous endocrine disease in Germany. Although the prevalence of AD appears lower than in the Scandinavian countries, the increasing figures in females over the last 5 years warrant further investigations. Furthermore, adrenal crises pose a considerable burden. Hereby, we can show that health insurance data provide a valuable tool for epidemiological studies in the absence of national registries.