Influenza-like illness symptoms due to endemic human coronavirus reinfections are not influenced by the length of the interval separating reinfections.

After 3 years of its introduction to humans, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been declared as endemic. Little is known about the severity of the disease manifestation that future infections may cause, especially when reinfections occur after humoral immunity from a previous infection or vaccination has waned. Such knowledge could inform policymakers regarding the frequency of vaccination. Reinfections by endemic human coronaviruses (HCoVs) can serve as a model system for SARS-CoV-2 endemicity. We monitored 44 immunocompetent male adults with blood sampling every 6 months (for 17 years), for the frequency of HCoV (re-)infections, using rises in N-antibodies of HCoV-NL63, HCoV-29E, HCoV-OC43, and HCoV-HKU1 as markers of infection. Disease associations during (re-)infections were examined by comparison of self-reporting records of influenza-like illness (ILI) symptoms, every 6 months, by all participants. During 8,549 follow-up months, we found 364 infections by any HCoV with a median of eight infections per person. Symptoms more frequently reported during HCoV infection were cough, sore throat, and myalgia. Two hundred fifty-one of the 364 infections were species-specific HCoV-reinfections, with a median interval of 3.58 (interquartile range 1.92-5.67) years. The length of the interval between reinfections-being either short or long-had no influence on the frequency of reporting ILI symptoms. All HCoV-NL63, HCoV-229E, HCoV-OC43, and HCoV-HKU1 (re-)infections are associated with the reporting of ILIs. Importantly, in immunocompetent males, these symptoms are not influenced by the length of the interval between reinfections. IMPORTANCE: Little is known about the disease following human coronavirus (HCoV) reinfection occurring years after the previous infection, once humoral immunity has waned. We monitored endemic HCoV reinfection in immunocompetent male adults for up to 17 years. We found no influence of reinfection interval length in the disease manifestation, suggesting that immunocompetent male adults are adequately protected against future HCoV infections.

Determinants of epidemic size and the impacts of lulls in seasonal influenza virus circulation.

During the COVID-19 pandemic, levels of seasonal influenza virus circulation were unprecedentedly low, leading to concerns that a lack of exposure to influenza viruses, combined with waning antibody titres, could result in larger and/or more severe post-pandemic seasonal influenza epidemics. However, in most countries the first post-pandemic influenza season was not unusually large and/or severe. Here, based on an analysis of historical influenza virus epidemic patterns from 2002 to 2019, we show that historic lulls in influenza virus circulation had relatively minor impacts on subsequent epidemic size and that epidemic size was more substantially impacted by season-specific effects unrelated to the magnitude of circulation in prior seasons. From measurements of antibody levels from serum samples collected each year from 2017 to 2021, we show that the rate of waning of antibody titres against influenza virus during the pandemic was smaller than assumed in predictive models. Taken together, these results partially explain why the re-emergence of seasonal influenza virus epidemics was less dramatic than anticipated and suggest that influenza virus epidemic dynamics are not currently amenable to multi-season prediction.

Robust Vaccine-Induced as Well as Hybrid B- and T-Cell Immunity across SARS-CoV-2 Vaccine Platforms in People with HIV.

Few studies have comprehensively compared severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine-induced and hybrid B- and T-cell responses in people with HIV (PWH) to those in comparable controls without HIV. We included 195 PWH and 246 comparable controls from the AGEhIV COVID-19 substudy. A positive nucleocapsid antibody (INgezim IgA/IgM/IgG) or self-reported PCR test defined prior SARS-CoV-2 infection. SARS-CoV-2 anti-spike (anti-S) IgG titers and anti-S IgG production by memory B cells were assessed. Neutralizing antibody titers were determined in a subset of participants. T-cell responses were assessed by gamma interferon (IFN-γ) release and activation-induced marker assay. We estimated mean differences in postvaccination immune responses (ß) between levels of determinants. Anti-S IgG titers and anti-S IgG production by memory B cells were not different between PWH and controls. Prior SARS-CoV-2 infection (ß = 0.77), receiving mRNA vaccine (ß = 0.56), female sex (ß = 0.24), fewer days between last vaccination and sampling (ß = 0.07), and a CD4/CD8 ratio of <1.0 (ß = -0.39) were independently associated with anti-S IgG titers, but HIV status was not. Neutralization titers against the ancestral and Delta and Omicron SARS-CoV-2 variants were not different between PWH and controls. IFN-γ release was higher in PWH. Prior SARS-CoV-2 infection (ß = 2.39), HIV-positive status (ß = 1.61), and fewer days between last vaccination and sampling (ß = 0.23) were independently associated with higher IFN-γ release. The percentages of SARS-CoV-2-reactive CD4+ and CD8+ T cells, however, were not different between PWH and controls. Individuals with well-controlled HIV generally mount robust vaccine-induced as well as hybrid B- and T-cell immunity across SARS-CoV-2 vaccine platforms similar to controls. Determinants of a reduced vaccine response were likewise largely similar in both groups and included a lower CD4/CD8 ratio. IMPORTANCE Some studies have suggested that people with HIV may respond less well to vaccines against SARS-CoV-2. We comprehensively compared B- and T-cell responses to different COVID-19 vaccines in middle-aged persons with well-treated HIV and individuals of the same age without HIV, who were also highly comparable in terms of demographics and lifestyle, including those with prior SARS-CoV-2 infection. Individuals with HIV generally mounted equally robust immunity to the different vaccines. Even stronger immunity was observed in both groups after prior SARS-CoV-2 infection. These findings are reassuring with respect to the efficacy of SARS-Cov-2 vaccines for the sizable and increasing global population of people with HIV with access and a good response to HIV treatment.

Anellovirus evolution during long-term chronic infection.

Human anelloviruses (AVs) are extremely genetically diverse, are widespread in the human population, and cause chronic infections. However, the evolutionary dynamics of AVs within single hosts is currently unknown, and it is unclear whether these changes have an implication on the long-term persistence of AVs in the host. Here, we assessed the evolutionary dynamics of six AV lineages during 30 years of chronic infection at single host resolution. The total number of substitutions and the number of variable sites increased over time. However, not all substitutions reached population fixation, showing that AV lineages form heterogeneous swarms within the host. Most substitutions occurred within a hypervariable region (HVR) located between nucleotide positions 800 and 1,300 of ORF1, which is known to be located within the spike domain. Different regions of the ORF1 gene undergo either positive or negative selection pressure. Sites under strong diversifying selection pressure were detected in the HVR, while the majority of the sites under purifying selection were detected outside this region. The HVR may play the role of an immunological decoy that prevents antibodies from binding to more vulnerable parts of ORF1. Moreover, the frequent substitutions in this region may increase the chances of AV particles escaping immune recognition.

Control of Human Anelloviruses by Cytosine to Uracil Genome Editing.

Anelloviruses are the most common viruses infecting humans. Every human carries a nonpathogenic personal anellovirus virome (anellome), yet it is unknown which mechanisms contribute to its stability. Here, we assessed the dynamics and impact of a host antiviral defense mechanism-cytidine deaminase activity leading to C to U editing in anelloviruses-on the stability of the anellome. We investigated anellome sequence data obtained from serum samples collected every 6 months from two healthy subjects followed for more than 30 years. The subjects were infected by a total of 64 anellovirus lineages. Minus-stranded C to U editing was observed in lineages belonging to the Alpha-, Beta-, and Gammatorquevirus genera. The edited genomes were present within virus particles, therefore editing must have occurred at the late stages of the virus life cycle. Editing was favored by 5'-TC contexts in the virus genome, indicating that apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like, catalytic subunit 3 or A3 (APOBEC3) proteins are involved. Within a lineage, mutational dynamics varied over time and few fixations of mutations were detected, indicating that C to U editing is a dead end for a virus genome. We detected an editing coldspot in the GC-rich regions, suggesting that the GC-rich region is crucial for genome packaging, since only packaged virus particles were included in the analysis. Finally, we noticed a lineage-specific reduced concentration after an editing event, yet no clearance. In conclusion, cytidine deaminase activity does not clear anelloviruses, nor does it play a major role in virus evolution, but it does contribute to the stability of the anellome. IMPORTANCE Despite significant attention on anellovirus research, the interaction between the anellovirus virome and the human host remains unknown. We show the dynamics of APOBEC3-mediated cytidine deaminase activity on anelloviruses during a 30-year period of chronic infection and postulate that this antiviral mechanism controls anelloviruses. These results expand our knowledge of anellovirus-host interactions, which may be important for the design of gene therapies.

Potential impacts of prolonged absence of influenza virus circulation on subsequent epidemics.

Background: During the first two years of the COVID-19 pandemic, the circulation of seasonal influenza viruses was unprecedentedly low. This led to concerns that the lack of immune stimulation to influenza viruses combined with waning antibody titres could lead to increased susceptibility to influenza in subsequent seasons, resulting in larger and more severe epidemics. Methods: We analyzed historical influenza virus epidemiological data from 2003-2019 to assess the historical frequency of near-absence of seasonal influenza virus circulation and its impact on the size and severity of subsequent epidemics. Additionally, we measured haemagglutination inhibition-based antibody titres against seasonal influenza viruses using longitudinal serum samples from 165 healthy adults, collected before and during the COVID-19 pandemic, and estimated how antibody titres against seasonal influenza waned during the first two years of the pandemic. Findings: Low country-level prevalence of influenza virus (sub)types over one or more years occurred frequently before the COVID-19 pandemic and had relatively small impacts on subsequent epidemic size and severity. Additionally, antibody titres against seasonal influenza viruses waned negligibly during the first two years of the pandemic. Interpretation: The commonly held notion that lulls in influenza virus circulation, as observed during the COVID-19 pandemic, will lead to larger and/or more severe subsequent epidemics might not be fully warranted, and it is likely that post-lull seasons will be similar in size and severity to pre-lull seasons. Funding: European Research Council, Netherlands Organization for Scientific Research, Royal Dutch Academy of Sciences, Public Health Service of Amsterdam. Research in context: Evidence before this study: During the first years of the COVID-19 pandemic, the incidence of seasonal influenza was unusually low, leading to widespread concerns of exceptionally large and/or severe influenza epidemics in the coming years. We searched PubMed and Google Scholar using a combination of search terms (i.e., "seasonal influenza", "SARS-CoV-2", "COVID-19", "low incidence", "waning rates", "immune protection") and critically considered published articles and preprints that studied or reviewed the low incidence of seasonal influenza viruses since the start of the COVID-19 pandemic and its potential impact on future seasonal influenza epidemics. We found a substantial body of work describing how influenza virus circulation was reduced during the COVID-19 pandemic, and a number of studies projecting the size of future epidemics, each positing that post-pandemic epidemics are likely to be larger than those observed pre-pandemic. However, it remains unclear to what extent the assumed relationship between accumulated susceptibility and subsequent epidemic size holds, and it remains unknown to what extent antibody levels have waned during the COVID-19 pandemic. Both are potentially crucial for accurate prediction of post-pandemic epidemic sizes.Added value of this study: We find that the relationship between epidemic size and severity and the magnitude of circulation in the preceding season(s) is decidedly more complex than assumed, with the magnitude of influenza circulation in preceding seasons having only limited effects on subsequent epidemic size and severity. Rather, epidemic size and severity are dominated by season-specific effects unrelated to the magnitude of circulation in the preceding season(s). Similarly, we find that antibody levels waned only modestly during the COVID-19 pandemic.Implications of all the available evidence: The lack of changes observed in the patterns of measured antibody titres against seasonal influenza viruses in adults and nearly two decades of epidemiological data suggest that post-pandemic epidemic sizes will likely be similar to those observed pre-pandemic, and challenge the commonly held notion that the widespread concern that the near-absence of seasonal influenza virus circulation during the COVID-19 pandemic, or potential future lulls, are likely to result in larger influenza epidemics in subsequent years.

Transmission of anelloviruses to HIV-1 infected children.

Anelloviruses (AVs) are widespread in the population and infect humans at the early stage of life. The mode of transmission of AVs is still unknown, however, mother-to-child transmission, e.g., via breastfeeding, is one of the likely infection routes. To determine whether the mother-to-child transmission of AVs may still occur despite the absence of natural birth and breastfeeding, 29 serum samples from five HIV-1-positive mother and child pairs were Illumina-sequenced. The Illumina reads were mapped to an AV lineage database "Anellometrix" containing 502 distinct ORF1 sequences. Although the majority of lineages from the mother were not shared with the child, the mother and child anellomes did display a significant similarity. These findings suggest that AVs may be transmitted from mothers to their children via different routes than delivery or breastfeeding.

Early-Life Colonization by Anelloviruses in Infants.

Anelloviruses (AVs) are found in the vast majority of the human population and are most probably part of a healthy virome. These viruses infect humans in the early stage of life, however, the characteristics of the first colonizing AVs are still unknown. We screened a collection of 107 blood samples from children between 0.4 and 64.8 months of age for the presence of three AV genera: the Alpha-, Beta- and Gammatorquevirus. The youngest child that was positive for AV was 1.2 months old, and a peak in prevalence (100% of samples positive) was reached between the twelfth and eighteenth months of life. Intriguingly, the beta- and gammatorqueviruses were detected most at the early stage of life (up to 12 months), whereas alphatorqueviruses, the most common AVs in adults, increased in prevalence in children older than 12 months. To determine whether that order of colonization may be related to oral transmission and unequal presence of AV genera in breast milk, we examined 63 breast milk samples. Thirty-two percent of the breast milk samples were positive in a qPCR detecting beta- and gammatorqueviruses, while alphatorqueviruses were detected in 10% of the samples, and this difference was significant (p = 0.00654). In conclusion, we show that beta- and gammatorqueviruses colonize humans in the first months of life and that breastfeeding could play a role in AV transmission.

Diversity and Long-Term Dynamics of Human Blood Anelloviruses.

Anelloviruses (AVs) are commensal members of the human blood virome. Even though it was estimated that over 90% of the human population carries AVs, the dynamics of the AV virome ("anellome") are unknown. We investigated the dynamics of blood anellomes in two healthy people followed up for more than 30 years. Both subjects were positive for AVs in the majority of samples. Alphatorquevirus (torque teno virus [TTV]) was the most common genus in both subjects, followed by Betatorquevirus (torque teno minivirus [TTMV]) and Gammatorquevirus (torque teno midivirus [TTMDV]). Almost five times more lineages were found in subject 1 than in subject 2, and the anellomes differed phylogenetically. Both anellomes remained compositionally stable, and 9 out of 64 AV lineages were detected in over half of the time points. We confirmed the long-term and short-term persistence of 13 lineages by specific quantitative PCR (qPCR). AV lineages were detected in blood for over 30 years. Noticeable differences in anellome richness were found between the tested subjects, but both anellomes remained compositionally stable over time. These findings demonstrate that the human blood anellome is personal and that AV infection is chronic and potentially commensal. IMPORTANCE Knowledge of the persistence of AVs in humans is crucial to our understanding of the nature of AV infection (chronic or acute) and the role of AV in the host. We therefore investigated the dynamics of anellovirus infection in two healthy people followed up for 30 years. Our findings suggest that the human blood anellovirus virome (anellome) remains stable and personal for decades.

Phylogenetic estimation of the viral fitness landscape of HIV-1 set-point viral load.

Set-point viral load (SPVL), a common measure of human immunodeficiency virus (HIV)-1 virulence, is partially determined by viral genotype. Epidemiological evidence suggests that this viral property has been under stabilising selection, with a typical optimum for the virus between 104 and 105 copies of viral RNA per ml. Here we aimed to detect transmission fitness differences between viruses from individuals with different SPVLs directly from phylogenetic trees inferred from whole-genome sequences. We used the local branching index (LBI) as a proxy for transmission fitness. We found that LBI is more sensitive to differences in infectiousness than to differences in the duration of the infectious state. By analysing subtype-B samples from the Bridging the Evolution and Epidemiology of HIV in Europe project, we inferred a significant positive relationship between SPVL and LBI up to approximately 105 copies/ml, with some evidence for a peak around this value of SPVL. This is evidence of selection against low values of SPVL in HIV-1 subtype-B strains, likely related to lower infectiousness, and perhaps a peak in the transmission fitness in the expected range of SPVL. The less prominent signatures of selection against higher SPVL could be explained by an inherent limit of the method or the deployment of antiretroviral therapy.

A highly virulent variant of HIV-1 circulating in the Netherlands.

We discovered a highly virulent variant of subtype-B HIV-1 in the Netherlands. One hundred nine individuals with this variant had a 0.54 to 0.74 log10 increase (i.e., a ~3.5-fold to 5.5-fold increase) in viral load compared with, and exhibited CD4 cell decline twice as fast as, 6604 individuals with other subtype-B strains. Without treatment, advanced HIV-CD4 cell counts below 350 cells per cubic millimeter, with long-term clinical consequences-is expected to be reached, on average, 9 months after diagnosis for individuals in their thirties with this variant. Age, sex, suspected mode of transmission, and place of birth for the aforementioned 109 individuals were typical for HIV-positive people in the Netherlands, which suggests that the increased virulence is attributable to the viral strain. Genetic sequence analysis suggests that this variant arose in the 1990s from de novo mutation, not recombination, with increased transmissibility and an unfamiliar molecular mechanism of virulence.

Similar Risk of Severe Acute Respiratory Syndrome Coronavirus 2 Infection and Similar Nucleocapsid Antibody Levels in People With Well-Controlled Human Immunodeficiency Virus (HIV) and a Comparable Cohort of People Without HIV.

BACKGROUND: Within the ongoing AGEhIV Cohort Study in Amsterdam, we prospectively compared the incidence of and risk factors for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection between human immunodeficiency virus (HIV)-positive and HIV-negative participants. Moreover, we compared SARS-CoV-2 nucleocapsid antibody levels between participants with incident infection from both groups. METHODS: Starting in September 2020, consenting HIV-positive and HIV-negative participants were assessed every 6 months for incident SARS-CoV-2 infection, using combined immunoglobulin (Ig) A/IgM/IgG SARS-CoV-2 nucleocapsid antibody assay. Cumulative incidence of SARS-CoV-2 infection and associated risk factors were assessed from 27 February 2020 through 30 April 2021, using complementary log-log regression. In those with incident SARS-CoV-2 infection, nucleocapsid (N) antibody levels were compared between groups using linear regression. RESULTS: The study included 241 HIV-positive (99.2% virally suppressed) and 326 HIV-negative AGEhIV participants. The cumulative SARS-CoV-2 incidence by April 2021 was 13.4% and 11.6% in HIV-positive and HIV-negative participants, respectively (P = .61). Younger age and African origin were independently associated with incident infection. In those with incident infection, only self-reported fever, but not HIV status, was associated with higher N antibody levels. CONCLUSIONS: HIV-positive individuals with suppressed viremia and adequate CD4 cell counts had similar risk of SARS-CoV-2 acquisition and similar SARS-CoV-2 N antibody levels after infection compared with a comparable HIV-negative cohort. CLINICAL TRIAL REGISTRATION: NCT01466582.

Non-nucleoside reverse transcriptase inhibitor-based combination antiretroviral therapy is associated with lower cell-associated HIV RNA and DNA levels compared to protease inhibitor-based therapy.

Background: It remains unclear whether combination antiretroviral therapy (ART) regimens differ in their ability to fully suppress human immunodeficiency virus (HIV) replication. Here, we report the results of two cross-sectional studies that compared levels of cell-associated (CA) HIV markers between individuals receiving suppressive ART containing either a non-nucleoside reverse transcriptase inhibitor (NNRTI) or a protease inhibitor (PI). Methods: CA HIV unspliced RNA and total HIV DNA were quantified in two cohorts (n = 100, n = 124) of individuals treated with triple ART regimens consisting of two nucleoside reverse transcriptase inhibitors (NRTIs) plus either an NNRTI or a PI. To compare CA HIV RNA and DNA levels between the regimens, we built multivariable models adjusting for age, gender, current and nadir CD4+ count, plasma viral load zenith, duration of virological suppression, NRTI backbone composition, low-level plasma HIV RNA detectability, and electronically measured adherence to ART. Results: In both cohorts, levels of CA HIV RNA and DNA strongly correlated (rho = 0.70 and rho = 0.54) and both markers were lower in NNRTI-treated than in PI-treated individuals. In the multivariable analysis, CA RNA in both cohorts remained significantly reduced in NNRTI-treated individuals (padj = 0.02 in both cohorts), with a similar but weaker association between the ART regimen and total HIV DNA (padj = 0.048 and padj = 0.10). No differences in CA HIV RNA or DNA levels were observed between individual NNRTIs or individual PIs, but CA HIV RNA was lower in individuals treated with either nevirapine or efavirenz, compared to PI-treated individuals. Conclusions: All current classes of antiretroviral drugs only prevent infection of new cells but do not inhibit HIV RNA transcription in long-lived reservoir cells. Therefore, these differences in CA HIV RNA and DNA levels by treatment regimen suggest that NNRTIs are more potent in suppressing HIV residual replication than PIs, which may result in a smaller viral reservoir size. Funding: This work was supported by ZonMw (09120011910035) and FP7 Health (305522).

Cell-Associated HIV-1 Unspliced-to-Multiply-Spliced RNA Ratio at 12 Weeks of ART Predicts Immune Reconstitution on Therapy.

Incomplete restoration of CD4+ T-cell counts on antiretroviral therapy (ART) is a major predictor of HIV-related morbidity and mortality. To understand the possible mechanisms behind this poor immunological response despite viral suppression, we longitudinally measured more than 50 virological and immunological biomarkers in a cohort of HIV-infected individuals at several time points during the first 96 weeks of virologically suppressive ART. No baseline virological or immunological marker was predictive of the degree of immune reconstitution. However, the cell-associated HIV-1 unspliced-to-multiply-spliced (US/MS) RNA ratio at 12 weeks of ART positively correlated with markers of CD4+ T-cell activation and apoptosis and negatively predicted both the absolute and relative CD4+ T-cell counts at 48 and 96 weeks. A higher US/MS RNA ratio may reflect the higher frequency of productively infected cells that could exert pressure on the immune system, contributing to persistent immune activation and apoptosis and subsequently to a poor immunological response to ART.IMPORTANCE Human immunodeficiency virus (HIV) infection is currently managed by antiretroviral drugs, which block virus replication and promote immune restoration. However, the latter effect is not universal, with a proportion of infected individuals failing to sufficiently reconstitute their immune function despite a successful virological response to antiretroviral therapy (ART). No reliable predictive markers of immunological failure have been identified, and there is still no efficient therapeutic strategy, apart from ART itself, to facilitate immune reconstitution. Here, we measured more than 50 viral and host biomarkers at five time points during the first 2 years of ART and identified the cell-associated HIV-1 unspliced-to-multiply-spliced RNA ratio at 12 weeks of ART as a predictive factor for the immunological response to therapy. Moreover, the same marker positively correlated with markers of CD4+ T-cell activation and apoptosis. The fact that a virological biomarker performed better than any immunological biomarker in predicting an immunological outcome highlights the importance of considering the residual HIV activity on ART as a correlate and a possible cause of the residual immune dysfunction that frequently occurs despite virologically suppressive ART.

Seasonal coronavirus protective immunity is short-lasting.

A key unsolved question in the current coronavirus disease 2019 (COVID-19) pandemic is the duration of acquired immunity. Insights from infections with the four seasonal human coronaviruses might reveal common characteristics applicable to all human coronaviruses. We monitored healthy individuals for more than 35 years and determined that reinfection with the same seasonal coronavirus occurred frequently at 12 months after infection.

CD32+CD4+ T Cells Are Highly Enriched for HIV DNA and Can Support Transcriptional Latency.

The HIV latent reservoir forms the major hurdle to an HIV cure. The discovery of CD32 as marker of this reservoir has aroused much interest, but subsequent reports have challenged this finding. Here, we observe a positive correlation between the percentages of CD32+ cells among CD4+ T cells of aviremic cART-treated, HIV-infected individuals and their HIV DNA loads in peripheral blood. Moreover, optimization of the CD32+CD4+ T cell purification protocol reveals prominent enrichment for HIV DNA (mean, 292-fold) in these cells. However, no enrichment for HIV RNA is observed in CD32+CD4+ cells, yielding significantly reduced HIV RNA/DNA ratios. Furthermore, HIV proviruses in CD32+CD4+ cells can be reactivated ex vivo to produce virus, strongly suggesting that these cells support HIV transcriptional latency. Our results underscore the importance of isolating pure, bona fide CD32+CD4+ T cells for future studies and indicate that CD32 remains a promising candidate marker of the HIV reservoir.

Cell-associated HIV-1 RNA predicts viral rebound and disease progression after discontinuation of temporary early ART.

Plasma viral load (VL) and CD4+ T cell count are widely used as biomarkers of HIV type 1 (HIV-1) replication, pathogenesis, and response to antiretroviral therapy (ART). However, the clinical potential of cell-associated (CA) HIV-1 molecular markers is much less understood. Here, we measured CA HIV-1 RNA and DNA in HIV-infected individuals treated with temporary ART initiated during primary HIV-1 infection. We demonstrate substantial predictive value of CA RNA for (a) the virological and immunological response to early ART, (b) the magnitude and time to viral rebound after discontinuation of early ART, and (c) disease progression in the absence of treatment. Remarkably, when adjusted for CA RNA, plasma VL no longer appeared as an independent predictor of any clinical endpoint in this cohort. The potential of CA RNA as an HIV-1 clinical marker, in particular as a predictive biomarker of virological control after stopping ART, should be explored in the context of HIV-1 curative interventions.

Viral Metagenomics on Cerebrospinal Fluid.

Identifying the causative pathogen in central nervous system (CNS) infections is crucial for patient management and prognosis. Many viruses can cause CNS infections, yet screening for each individually is costly and time-consuming. Most metagenomic assays can theoretically detect all pathogens, but often fail to detect viruses because of their small genome and low viral load. Viral metagenomics overcomes this by enrichment of the viral genomic content in a sample. VIDISCA-NGS is one of the available workflows for viral metagenomics, which requires only a small input volume and allows multiplexing of multiple samples per run. The performance of VIDISCA-NGS was tested on 45 cerebrospinal fluid (CSF) samples from patients with suspected CNS infections in which a virus was identified and quantified by polymerase chain reaction. Eighteen were positive for an RNA virus, and 34 for a herpesvirus. VIDISCA-NGS detected all RNA viruses with a viral load >2 × 104 RNA copies/mL (n = 6) and 8 of 12 of the remaining low load samples. Only one herpesvirus was identified by VIDISCA-NGS, however, when withholding a DNase treatment, 11 of 18 samples with a herpesvirus load >104 DNA copies/mL were detected. Our results indicate that VIDISCA-NGS has the capacity to detect low load RNA viruses in CSF. Herpesvirus DNA in clinical samples is probably non-encapsidated and therefore difficult to detect by VIDISCA-NGS.

Dendritic cells potently purge latent HIV-1 beyond TCR-stimulation, activating the PI3K-Akt-mTOR pathway.

BACKGROUND: The latent HIV-1 reservoir in treated patients primarily consists of resting memory CD4+ T cells. Stimulating the T-cell receptor (TCR), which facilitates transition of resting into effector T cells, is the most effective strategy to purge these latently infected cells. Here we supply evidence that TCR-stimulated effector T cells still frequently harbor latent HIV-1. METHODS: Primary HIV-1 infected cells were used in a latency assay with or without dendritic cells (DCs) and reversion of HIV-1 latency was determined, in the presence or absence of specific pathway inhibitors. FINDINGS: Renewed TCR-stimulation or subsequent activation with latency reversing agents (LRAs) did not overcome latency. However, interaction of infected effector cells with DCs triggered further activation of latent HIV-1. When compared to TCR-stimulation only, CD4+ T cells from aviremic patients receiving TCR + DC-stimulation reversed latency more frequently. Such a "one-two punch" strategy seems ideal for purging the reservoir. We determined that DC contact activates the PI3K-Akt-mTOR pathway in CD4+ T cells. INTERPRETATION: This insight could facilitate the development of a novel class of potent LRAs that purge latent HIV beyond levels reached by T-cell activation.

Easy and accurate reconstruction of whole HIV genomes from short-read sequence data with shiver.

Studying the evolution of viruses and their molecular epidemiology relies on accurate viral sequence data, so that small differences between similar viruses can be meaningfully interpreted. Despite its higher throughput and more detailed minority variant data, next-generation sequencing has yet to be widely adopted for HIV. The difficulty of accurately reconstructing the consensus sequence of a quasispecies from reads (short fragments of DNA) in the presence of large between- and within-host diversity, including frequent indels, may have presented a barrier. In particular, mapping (aligning) reads to a reference sequence leads to biased loss of information; this bias can distort epidemiological and evolutionary conclusions. De novo assembly avoids this bias by aligning the reads to themselves, producing a set of sequences called contigs. However contigs provide only a partial summary of the reads, misassembly may result in their having an incorrect structure, and no information is available at parts of the genome where contigs could not be assembled. To address these problems we developed the tool shiver to pre-process reads for quality and contamination, then map them to a reference tailored to the sample using corrected contigs supplemented with the user's choice of existing reference sequences. Run with two commands per sample, it can easily be used for large heterogeneous data sets. We used shiver to reconstruct the consensus sequence and minority variant information from paired-end short-read whole-genome data produced with the Illumina platform, for sixty-five existing publicly available samples and fifty new samples. We show the systematic superiority of mapping to shiver's constructed reference compared with mapping the same reads to the closest of 3,249 real references: median values of 13 bases called differently and more accurately, 0 bases called differently and less accurately, and 205 bases of missing sequence recovered. We also successfully applied shiver to whole-genome samples of Hepatitis C Virus and Respiratory Syncytial Virus. shiver is publicly available from https://github.com/ChrisHIV/shiver.