The elusive source of HIV-1 rebound after treatment interruption

Identifying the source of viral rebound during a monitored analytical treatment interruption (ATI) would reveal potential targets for cure strategies. Therefore, we examined the genetic composition of proviral DNA in different subsets from participants on antiretroviral therapy and compared this to rebounding virus after an ATI. Eleven participants underwent a monitored ATI and were sampled from different anatomical sites prior to and after the ATI. From the peripheral blood, Naïve (TNA), central (TCM), transitional (TTM) and effector (TEM) memory CD4+ T cells were sorted as were CD45 cells from gut-associated lymphoid tissue (GALT). Using single-genome sequencing (SGS) the env region of HIV DNA and plasma-derived RNA was sequenced. In an ongoing study, Full-Length Individual Proviral Sequencing (FLIPS) and Integration Site Loop Amplification (ISLA) assays were performed on the T cell subsets from 2 participants. For participant STAR10, 87 integration sites (IS) and 113 proviral genomes were sequenced while only 3 unique intact proviruses (3%) were identified. A cluster of 17 identical defective proviruses were linked to an IS (9% of all IS) in STAT5B located in TCM, TNA, TEM and TTM. When comparing the FLIPS to SGS env sequences a 100% match was found between one defective provirus and one plasma HIV RNA sequence after rebound. For participant STAR11, 37 IS and 105 proviral genomes were sequenced yielding 14 intact proviruses (13%) with the highest proportion found predominantly in the TEM subset (n=13, 45%). Four different clusters of identical sequences could be identified of which 2 (n=3 and n=9) consisted of intact TEM sequences with the smaller cluster linked to an IS in ZNF274. A 99% match between 2 env from rebounding plasma RNA and this smaller cluster of intact proviral genomes was identified. Comparing proviral sequences and their IS to plasma-derived RNA sequences after an ATI reveals additional information in terms of the source of viral rebound. However, this comparison is complicated by multiple factors. For example, we found a plasma-derived RNA sequence obtained during viral rebound matched a defective proviral sequence which highlights the problem of using one HIV RNA subgenomic region for identifying replication-competent virus. In addition, ongoing viral replication during rebound may prevent a 100% match with genetically intact proviral sequences making it challenging to determine the absolute source of rebound

Evaluating predictive markers for viral rebound and safety assessment in blood and lumbar fluid during HIV-1 treatment interruption

Background: Validated biomarkers to evaluate HIV-1 cure strategies are currently lacking, therefore requiring analytical treatment interruption (ATI) in study participants. Little is known about the safety of ATI and its long-term impact on patient health. Objectives: ATI safety was assessed and potential biomarkers predicting viral rebound were evaluated. Methods: PBMCs, plasma and CSF were collected from 11 HIV-1-positive individuals at four different timepoints during ATI (NCT02641756). Total and integrated HIV-1 DNA, cell-associated (CA) HIV-1 RNA transcripts and restriction factor (RF) expression were measured by PCR-based assays. Markers of neuroinflammation and neuronal injury [neurofilament light chain (NFL) and YKL-40 protein] were measured in CSF. Additionally, neopterin, tryptophan and kynurenine were measured, both in plasma and CSF, as markers of immune activation. Results: Total HIV-1 DNA, integrated HIV-1 DNA and CA viral RNA transcripts did not differ pre- and post-ATI. Similarly, no significant NFL or YKL-40 increases in CSF were observed between baseline and viral rebound. Furthermore, markers of immune activation did not increase during ATI. Interestingly, the RFs SLFN11 and APOBEC3G increased after ATI before viral rebound. Similarly, Tat-Rev transcripts were increased preceding viral rebound after interruption. Conclusions: ATI did not increase viral reservoir size and it did not reveal signs of increased neuronal injury or inflammation, suggesting that these well-monitored ATIs are safe. Elevation of Tat-Rev transcription and induced expression of the RFs SLFN11 and APOBEC3G after ATI, prior to viral rebound, indicates that these factors could be used as potential biomarkers predicting viral rebound

Myosins 1 and 6, myosin light chain kinase, actin and microtubules cooperate during antibody-mediated internalisation and trafficking of membrane-expressed viral antigens in feline infectious peritonitis virus infected monocytes

International audienceMonocytes infected with feline infectious peritonitis virus, a coronavirus, express viral proteins in their plasma membranes. Upon binding of antibodies, these proteins are quickly internalised through a new clathrin- and caveolae-independent internalisation pathway. By doing so, the infected monocytes can escape antibody-dependent cell lysis. In the present study, we investigated which kinases and cytoskeletal proteins are of importance during internalisation and subsequent intracellular transport. The experiments showed that myosin light chain kinase (MLCK) and myosin 1 are crucial for the initiation of the internalisation. With co-localisation stainings, it was found that MLCK and myosin 1 co-localise with antigens even before internalisation started. Myosin 6 co-localised with the internalising complexes during passage through the cortical actin, were it might play a role in moving or disintegrating actin filaments, to overcome the actin barrier. One minute after internalisation started, vesicles had passed the cortical actin, co-localised with microtubules and association with myosin 6 was lost. The vesicles were further transported over the microtubules and accumulated at the microtubule organising centre after 10 to 30 min. Intracellular trafficking over microtubules was mediated by MLCK, myosin 1 and a small actin tail. Since inhibiting MLCK with ML-7 was so efficient in blocking the internalisation pathway, this target can be used for the development of a new treatment for FIPV

Characterization of antigenic regions in the porcine reproductive and respiratory syndrome virus by the use of peptide-specific serum antibodies

AbstractThe porcine reproductive and respiratory syndrome virus (PRRSV) is an RNA virus that causes reproductive failure in sows and boars, and respiratory disease in pigs of all ages. Antibodies against several viral envelope proteins are produced upon infection, and the glycoproteins GP4 and GP5 are known targets for virus neutralization. Still, substantial evidence points to the presence of more, yet unidentified neutralizing antibody targets in the PRRSV envelope proteins. The current study aimed to identify and characterize linear antigenic regions (ARs) within the entire set of envelope proteins of the European prototype PRRSV strain Lelystad virus (LV).Seventeen LV-specific antisera were tested in pepscan analysis on GP2, E, GP3, GP4, GP5 and M, resulting in the identification of twenty-one ARs that are capable of inducing antibodies upon infection in pigs. A considerable number of these ARs correspond to previously described epitopes in different European- and North-American-type PRRSV strains. Remarkably, the largest number of ARs was found in GP3, and two ARs in the GP3 ectodomain consistently induced antibodies in a majority of infected pigs. In contrast, all remaining ARs, except for a highly immunogenic epitope in GP4, were only recognized by one or a few infected animals.Sensitivity to antibody-mediated neutralization was tested for a selected number of ARs by in vitro virus-neutralization tests on alveolar macrophages with peptide-purified antibodies. In addition to the known neutralizing epitope in GP4, two ARs in GP2 and one in GP3 turned out to be targets for virus-neutralizing antibodies. No virus-neutralizing antibody targets were found in E, GP5 or M. Since the neutralizing AR in GP3 induced antibodies in a majority of infected pigs, the immunogenicity of this AR was studied more extensively, and it was demonstrated that the corresponding region in GP3 of virus strains other than LV also induces virus-neutralizing antibodies. This study provides new insights into PRRSV antigenicity, and contributes to the knowledge on protective immunity and immune evasion strategies of the virus

Long-read sequencing assay allows accurate characterization of the HIV-1 reservoir

Background The advent of near full-length (NFL) HIV-1 proviral genome sequencing greatly expanded our understanding of the quality of the viral reservoir, revealing that only 2-5% of the persistent proviruses in ART-treated individuals can be considered genome-intact. However, current NFL assays are based on labor-intensive and costly principles of repeated PCRs at limiting dilution, restricting their scalability. We developed a long-read sequencing assay to characterize many proviral genomes in parallel from bulk DNA. Methods The sensitivity of the long-read assay was determined on a DNA dilution series of J-Lat in uninfected Jurkat ranging from 80,000 to <8 HIV-1 copies. Next, the assay was performed on 15 chronic ART-suppressed individuals, using a fixed input of 500 ng DNA extracted from peripheral blood CD4 T cells (reservoir sizes ranging from 321 to 6581 total HIV-1 DNA copies/million CD4 T cells). Individual proviruses were tagged with a different unique molecular identifier (UMI) at each end during a single reaction, followed by NFL PCR amplification and long-read sequencing on an Oxford Nanopore MinION. UMI-based demultiplexing allowed for the construction of highly accurate consensus genomes, while excluding aberrant chimeric PCR artefacts. In addition, Full-Length Individual Provirus Sequencing (FLIPS) was performed on 2 individuals. Data from both assays were compared through phylogenetic analyses. Results The lower limit of the long-read assay was found to be <8 HIV-1 copies. The long-read assay yielded an average of 14 distinct HIV-1 proviruses per participant (range: 3-42). Across all participants, 213 distinct proviruses were retrieved of which 8% were considered putatively intact. In terms of reservoir composition, data obtained with FLIPS showed an overall agreement with data obtained with the long-read assay. In an individual with limited clonality (6% clonality of FLIPS data, n=1 clone) only 1 overlapping provirus was found, while an overlap of 3 proviruses was observed in an individual with higher clonality (91% clonality of FLIPS data, n=4 clones). Comparing the 4 overlapping proviral consensus genomes to their matching FLIPS counterparts showed an average sequence accuracy of 99,97%. Conclusion The long-read assay offers a high-throughput NFL sequencing method which enables an accurate characterization of the proviral landscape while retaining sequencing accuracy comparable to current gold standard NFL assays

Linking inducible HIV-1 reservoir to rebound after treatment interruption

BACKGROUND: The origin of viral rebound remains elusive as only a few links between proviral sequences and rebound plasma viruses have been described. Here we characterized the translation- and replication-competent reservoirs of three individuals under ART and compared it to rebound plasma viruses detected during analytical treatment interruption (ATI). METHODS: Peripheral blood CD4 T cells were collected from 3 ART-treated individuals right before ATI. P24-expressing cells were single-cell sorted following latency reversal and near full-length proviral sequencing was performed. Replication-competent viral sequences were isolated from supernatant of positive quantitative viral outgrowth assay (qVOA) wells. During ATI, plasma was collected at first detectable viral load (>1000 copies/mL) and either 5’- or 3’-half viral RNA genomes were isolated through single genome amplification. RESULTS: We retrieved 20 sequences from positive qVOA wells, 30 proviral genomes from p24+ cells, 89 (median=32) 5’-half and 94 (median=32) 3’-half rebound plasma sequences acquired during ATI. Among distinct sequences retrieved from p24+ cells, 88% displayed defects in the packaging signal (PSI) region and/or major splice donor (MSD) site. Among qVOA and rebound sequences, none had PSI/MSD defects, suggesting their minor role in viral rebound and replication. One overlap between the translation- and replication-competent reservoir was observed, notably between the only p24+ cell with an intact PSI/MSD and one genome-intact qVOA sequence. Moreover, two overlaps were observed between viruses from qVOA wells and 5’-half rebound plasma sequences. CONCLUSIONS: The direct origin of rebounding plasma virus remains hard to identify as only few overlaps were detected by comparing the translation and replication-competent fractions to plasma viruses collected during ATI, with no overlap between the three datasets. Yet, we report an overlap between an intact qVOA sequence and a provirus from a p24-expressing cell, confirming that some of the translation-competent proviruses are capable of inducing new cycles of replication

HIV-PULSE : a long-read sequencing assay for high-throughput near full-length HIV-1 proviral genome characterization

A deep understanding of the composition of the HIV-1 reservoir is necessary for the development of targeted therapies and the evaluation of curative efforts. However, current near full-length (NFL) HIV-1 proviral genome sequencing assays are based on labor-intensive and costly principles of repeated PCRs at limiting dilution, restricting their scalability. To address this, we developed a high-throughput, long-read sequencing assay called HIV-PULSE (HIV Proviral UMI-mediated Long-read Sequencing). This assay uses unique molecular identifiers (UMIs) to tag individual HIV-1 genomes, allowing for the omission of the limiting dilution step and enabling long-range PCR amplification of many NFL genomes in a single PCR reaction, while simultaneously overcoming poor single-read accuracy. We optimized the assay using HIV-infected cell lines and then applied it to blood samples from 18 individuals living with HIV on antiretroviral therapy, yielding a total of 1308 distinct HIV-1 genomes. Benchmarking against the widely applied Full-Length Individual Proviral Sequencing assay revealed similar sensitivity (11 vs 18%) and overall good concordance, although at a significantly higher throughput. In conclusion, HIV-PULSE is a cost-efficient and scalable assay that allows for the characterization of the HIV-1 proviral landscape, making it an attractive method to study the HIV-1 reservoir composition and dynamics

In-depth single-cell analysis of translation-competent HIV-1 reservoirs identifies cellular sources of residual viremia and rebound viruses

C

HIV-PULSE: A long-read sequencing assay for high-throughput near full-length HIV-1 proviral genome characterization

A deep understanding of the composition of the HIV-1 reservoir is necessary for the development of targeted therapies and the evaluation of curative efforts. However, current near full-length (NFL) HIV-1 proviral genome sequencing assays are based on labor-intensive and costly principles of repeated PCRs at limiting dilution, restricting their scalability. To address this, we developed a high-throughput, long-read sequencing assay called HIV-PULSE (HIV Proviral UMI-mediated Long-read Sequencing). This assay uses unique molecular identifiers (UMIs) to tag individual HIV-1 genomes, allowing for the omission of the limiting dilution step and enabling long-range PCR amplification of many NFL genomes in a single PCR reaction, while simultaneously overcoming poor single-read accuracy. We optimized the assay using HIV-infected cell lines and then applied it to blood samples from 18 individuals living with HIV on antiretroviral therapy, yielding a total of 1,308 distinct HIV-1 genomes. Benchmarking against the widely applied Full-Length Individual Proviral Sequencing assay revealed similar sensitivity (11% vs 18%) and overall good concordance, though at a significantly higher throughput. In conclusion, HIV-PULSE is a cost-efficient and scalable assay that allows for the characterization of the HIV-1 proviral landscape, making it an attractive method to study the HIV-1 reservoir composition and dynamics.</jats:p