Structural basis of TMPRSS2 zymogen activation and recognition by the HKU1 seasonal coronavirus.

The human seasonal coronavirus HKU1-CoV, which causes common colds worldwide, relies on the sequential binding to surface glycans and transmembrane serine protease 2 (TMPRSS2) for entry into target cells. TMPRSS2 is synthesized as a zymogen that undergoes autolytic activation to process its substrates. Several respiratory viruses, in particular coronaviruses, use TMPRSS2 for proteolytic priming of their surface spike protein to drive membrane fusion upon receptor binding. We describe the crystal structure of the HKU1-CoV receptor binding domain in complex with TMPRSS2, showing that it recognizes residues lining the catalytic groove. Combined mutagenesis of interface residues and comparison across species highlight positions 417 and 469 as determinants of HKU1-CoV host tropism. The structure of a receptor-blocking nanobody in complex with zymogen or activated TMPRSS2 further provides the structural basis of TMPRSS2 activating conformational change, which alters loops recognized by HKU1-CoV and dramatically increases binding affinity.

TMPRSS2 is a functional receptor for human coronavirus HKU1.

Four endemic seasonal human coronaviruses causing common colds circulate worldwide: HKU1, 229E, NL63 and OC43 (ref. 1). After binding to cellular receptors, coronavirus spike proteins are primed for fusion by transmembrane serine protease 2 (TMPRSS2) or endosomal cathepsins2-9. NL63 uses angiotensin-converting enzyme 2 as a receptor10, whereas 229E uses human aminopeptidase-N11. HKU1 and OC43 spikes bind cells through 9-O-acetylated sialic acid, but their protein receptors remain unknown12. Here we show that TMPRSS2 is a functional receptor for HKU1. TMPRSS2 triggers HKU1 spike-mediated cell-cell fusion and pseudovirus infection. Catalytically inactive TMPRSS2 mutants do not cleave HKU1 spike but allow pseudovirus infection. Furthermore, TMPRSS2 binds with high affinity to the HKU1 receptor binding domain (Kd 334 and 137 nM for HKU1A and HKU1B genotypes) but not to SARS-CoV-2. Conserved amino acids in the HKU1 receptor binding domain are essential for binding to TMPRSS2 and pseudovirus infection. Newly designed anti-TMPRSS2 nanobodies potently inhibit HKU1 spike attachment to TMPRSS2, fusion and pseudovirus infection. The nanobodies also reduce infection of primary human bronchial cells by an authentic HKU1 virus. Our findings illustrate the various evolution strategies of coronaviruses, which use TMPRSS2 to either directly bind to target cells or prime their spike for membrane fusion and entry.

NK Cell Responses in Zika Virus Infection Are Biased towards Cytokine-Mediated Effector Functions.

Zika virus (ZIKV) is a mosquito-borne flavivirus that has emerged as a global concern because of its impact on human health. ZIKV infection during pregnancy can cause microcephaly and other severe brain defects in the developing fetus and there have been reports of the occurrence of Guillain-Barré syndrome in areas affected by ZIKV. NK cells are activated during acute viral infections and their activity contributes to a first line of defense because of their ability to rapidly recognize and kill virus-infected cells. To provide insight into NK cell function during ZIKV infection, we have profiled, using mass cytometry, the NK cell receptor-ligand repertoire in a cohort of acute ZIKV-infected female patients. Freshly isolated NK cells from these patients contained distinct, activated, and terminally differentiated, subsets expressing higher levels of CD57, NKG2C, and KIR3DL1 as compared with those from healthy donors. Moreover, KIR3DL1+ NK cells from these patients produced high levels of IFN-γ and TNF-α, in the absence of direct cytotoxicity, in response to in vitro stimulation with autologous, ZIKV-infected, monocyte-derived dendritic cells. In ZIKV-infected patients, overproduction of IFN-γ correlated with STAT-5 activation (r = 0.6643; p = 0.0085) and was mediated following the recognition of MHC class 1-related chain A and chain B molecules expressed by ZIKV-infected monocyte-derived dendritic cells, in synergy with IL-12 production by the latter cells. Together, these findings suggest that NK cells contribute to the generation of an efficacious adaptive anti-ZIKV immune response that could potentially affect the outcome of the disease and/or the development of persistent symptoms.

An intranasal lentiviral booster reinforces the waning mRNA vaccine-induced SARS-CoV-2 immunity that it targets to lung mucosa.

As the coronavirus disease 2019 (COVID-19) pandemic continues and new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern emerge, the adaptive immunity initially induced by the first-generation COVID-19 vaccines starts waning and needs to be strengthened and broadened in specificity. Vaccination by the nasal route induces mucosal, humoral, and cellular immunity at the entry point of SARS-CoV-2 into the host organism and has been shown to be the most effective for reducing viral transmission. The lentiviral vaccination vector (LV) is particularly suitable for this route of immunization owing to its non-cytopathic, non-replicative, and scarcely inflammatory properties. Here, to set up an optimized cross-protective intranasal booster against COVID-19, we generated an LV encoding stabilized spike of SARS-CoV-2 Beta variant (LV::SBeta-2P). mRNA vaccine-primed and -boosted mice, with waning primary humoral immunity at 4 months after vaccination, were boosted intranasally with LV::SBeta-2P. A strong boost effect was detected on cross-sero-neutralizing activity and systemic T cell immunity. In addition, mucosal anti-spike IgG and IgA, lung-resident B cells, and effector memory and resident T cells were efficiently induced, correlating with complete pulmonary protection against the SARS-CoV-2 Delta variant, demonstrating the suitability of the LV::SBeta-2P vaccine candidate as an intranasal booster against COVID-19. LV::SBeta-2P vaccination was also fully protective against Omicron infection of the lungs and central nervous system, in the highly susceptible B6.K18-hACE2IP-THV transgenic mice.

LED-pumped Cr:LiSAF laser system operating at 100†Hz based on a multipass amplifier.

The LED-pumping technology is used for the first time, to the best of our knowledge, to develop a complete master oscillator power amplifier (MOPA) system including a multipass amplifier. A pumping head using an original slab architecture is developed integrating a Cr:LiSAF slab pumped by 2112 blue LEDs via a Ce:YAG luminescent concentrator. The slab configuration enables the reaching of a large number of passes-up to 22-together with access to efficient cooling, allowing for a repetition rate scale up. For 22 passes, the amplifier delivers pulses with energy up to 2.4 mJ at 10-Hz repetition rate with a gain of 4.36 at 825 nm. A complete study of the MOPA is described, concluding in nearly constant performances versus the repetition rate, up to 100 Hz.

Improvement in the temporal contrast in the tens of ps range of the multi-PW Apollon laser front-end.

We demonstrate the impact of the optics roughness in Öffner stretchers used in chirped pulse amplification laser chains and how it is possible to improve the temporal contrast ratio in the temporal range of 10-100 ps by adequately choosing the optical quality of the key components. Experimental demonstration has been realized in the front-end source of the multi-petawatt (PW) laser facility Apollon, resulting in an enhancement of the contrast ratio by two to three orders of magnitude.

Pain Intensity in the First 96 Hours After Abdominal Surgery: A Prospective Cohort Study.

OBJECTIVE: Multimodal pain management strategies aim to improve postoperative pain control. The purpose of this study was to analyze pain scores and risk factors for acute postoperative pain after various abdominal surgery procedures. METHODS: Data on 11 different abdominal surgery procedures were prospectively recorded. Pain intensity (rest, mobilization) and patient satisfaction at discharge were assessed using a visual analog scale (VAS; 0-10), and analgesic consumption was recorded until 96 hours postoperation. Demographic, surgery-related, and pain management-related univariate risk factors for insufficient pain control (VAS ≥ 4) were entered in a multivariate logistic regression model. RESULTS: A total of 1,278 patients were included. Overall, mean VAS scores were <3 at all time points, and scores at mobilization were consistently higher than at rest (P < 0.05). Thirty percent of patients presented a prolonged VAS score ≥4 at mobilization at 24 hours, significantly higher than at rest (14%, P < 0.05). High pain scores correlated with high opioid consumption, whereas a variability of pain scores was observed in patients with low opioid consumption. The only independent risk factor for moderate and severe pain (VAS ≥ 4) was younger age (<70 years, P = 0.001). The mean satisfaction score was 8.18 ± 1.29. CONCLUSIONS: Among 1,278 patients, pain was controlled adequately during the first four postoperative days, resulting in high levels of patient satisfaction. Pain levels were higher at mobilization. Younger age was the only independent risk factor for insufficient pain control. Preventive treatment in patients <70 years old and before mobilization could be evaluated for potential improvement.

The Cuticle Mutant eca2 Modifies Plant Defense Responses to Biotrophic and Necrotrophic Pathogens and Herbivory Insects.

We isolated previously several Arabidopsis thaliana mutants with constitutive expression of the early microbe-associated molecular pattern-induced gene ATL2, named eca (expresión constitutiva de ATL2). Here, we further explored the interaction of eca mutants with pest and pathogens. Of all eca mutants, eca2 was more resistant to a fungal pathogen (Botrytis cinerea) and a bacterial pathogen (Pseudomonas syringae) as well as to a generalist herbivorous insect (Spodoptera littoralis). Permeability of the cuticle is increased in eca2; chemical characterization shows that eca2 has a significant reduction of both cuticular wax and cutin. Additionally, we determined that eca2 did not display a similar compensatory transcriptional response, compared with a previously characterized cuticular mutant, and that resistance to B. cinerea is mediated by the priming of the early and late induced defense responses, including salicylic acid- and jasmonic acid-induced genes. These results suggest that ECA2-dependent responses are involved in the nonhost defense mechanism against biotrophic and necrotrophic pathogens and against a generalist insect by modulation and priming of innate immunity and late defense responses. Making eca2 an interesting model to characterize the molecular basis for plant defenses against different biotic interactions and to study the initial events that take place in the cuticle surface of the aerial organs.

Postoperative urinary retention in colorectal surgery within an enhanced recovery pathway.

BACKGROUND: Enhanced recovery after surgery (ERAS) guidelines for colorectal surgery suggest routine transurethral bladder drainage with early removal to prevent urinary tract infection (UTI). The aim of this study was to identify risk factors for urinary retention (UR). METHODS: This retrospective analysis included all colorectal patients since ERAS implementation in May 2011-November 2014. From the prospective ERAS database, over 100 items related to demographics, surgery, compliance, and outcome were analyzed. Risk factors for UR were identified by multiple logistic regressions; then, UR was correlated to functional outcomes and UTI and acute kidney injury rates. RESULTS: The study cohort consisted of 513 consecutive patients. Of these, 73 patients (14%) presented with UR. Multivariate analysis identified male gender (odds ratio 1.4; 95% CI, 1-1.8; P = 0.045) and postoperative thoracic epidural analgesia (EDA; odds ratio 2.6; 95% CI, 1.6-4.3; P ≤ 0.001) as independent risk factors for postoperative UR. Functional recovery was impeded in patients with UR, who were less mobile (mobilization day 1 >4 h: 57% versus 70%, P = 0.024) and gained more weight (2.8 ± 2.5 kg versus 1.6 ±3 kg on day 1, P = 0.001) due to fluid overload. Furthermore, patients with urinary catheters reported more pain (visual analog scales day 3: 3.1 ± 2.5 versus 2.2 ± 2.4, P = 0.002) and depended longer on intravenous fluid administration (termination of intravenous fluids later than day 1: 53% versus 39%, P = 0.021). Ten of 73 patients (14%) developed UTI in patients with UR and 42 of 440 (10%) in patients without UR (P = 0.276). Six of 73 patients (8%) developed acute kidney injury in patients with UR and 36 of 440 (8%) in patients without UR (P = 0.991). CONCLUSIONS: Male gender and EDA were independent risk factors for postoperative UR which appeared to be a significant impediment for functional recovery.

Enhanced Recovery after Elective Colorectal Surgery - Reasons for Non-Compliance with the Protocol.

BACKGROUND/AIMS: Enhanced recovery after surgery (ERAS) protocols for elective colorectal surgery reduce the intensity of postoperative complications, hospital stays and costs. Improvements in clinical outcome are directly proportional to the adherence to the recommended pathway (compliance). The aim of the present study was to analyze reasons for the non-compliance of colorectal surgeries with the ERAS protocol. METHODS: A consecutive cohort of patients undergoing elective colorectal surgery was prospectively analyzed with regards to the surgery's compliance with the ERAS protocol. The reason for every single protocol deviation was documented and the decision was categorized based on whether it was medically justified or not. RESULTS: During the 8-month study period, 76 patients were included. The overall compliance with 22 ERAS items was 76% (96% in the preoperative, 82% in the perioperative, and 63% in the postoperative period). The decision to deviate from the clinical pathway was mainly a medical decision, while patients and nurses were responsible in 26 and 14% of the cases, respectively. However, reasons for non-compliance were medically justified in 78% of the study participants. CONCLUSION: 'Non-compliance' with the ERAS protocol was observed mostly in the postoperative period. Most deviations from the pathway were decided by doctors and in a majority of cases it appeared that they were due to a medical necessity rather than non-compliance. However, almost a quarter of deviations that were absolutely required are still amenable to improvement.

A new human mast cell line expressing a functional IgE receptor converts to tumorigenic growth by KIT D816V transfection.

In systemic mastocytosis (SM), clinical problems arise from factor-independent proliferation of mast cells (MCs) and the increased release of mediators by MCs, but no human cell line model for studying MC activation in the context of SM is available. We have created a stable stem cell factor (SCF) -dependent human MC line, ROSA(KIT WT), expressing a fully functional immunoglobulin E (IgE) receptor. Transfection with KIT D816V converted ROSA(KIT WT) cells into an SCF-independent clone, ROSA(KIT D816V), which produced a mastocytosis-like disease in NSG mice. Although several signaling pathways were activated, ROSA(KIT D816V) did not exhibit an increased, but did exhibit a decreased responsiveness to IgE-dependent stimuli. Moreover, NSG mice bearing ROSA(KIT D816V)-derived tumors did not show mediator-related symptoms, and KIT D816V-positive MCs obtained from patients with SM did not show increased IgE-dependent histamine release or CD63 upregulation. Our data show that KIT D816V is a disease-propagating oncoprotein, but it does not activate MCs to release proinflammatory mediators, which may explain why mediator-related symptoms in SM occur preferentially in the context of a coexisting allergy. ROSA(KIT D816V) may provide a valuable tool for studying the pathogenesis of mastocytosis and should facilitate the development of novel drugs for treating SM patients.

Implementation of Enhanced Recovery (ERAS) in Colorectal Surgery Has a Positive Impact on Non-ERAS Liver Surgery Patients.

BACKGROUND: Enhanced recovery after surgery (ERAS) reduces complications and hospital stay in colorectal surgery. Thereafter, ERAS principles were extended to liver surgery. Previous implementation of an ERAS program in colorectal surgery may influence patients undergoing liver surgery in a non-ERAS setting, on the same ward. This study aimed to test this hypothesis. METHODS: Retrospective analysis based on prospective data of the adherence to the institutional ERAS-liver protocol (compliance) in three cohorts of consecutive patients undergoing elective liver surgery, between June 2010 and July 2014: before any ERAS implementation (pre-ERAS n = 50), after implementation of ERAS in colorectal (intermediate n = 50), and after implementation of ERAS in liver surgery (ERAS-liver n = 74). Outcomes were functional recovery, postoperative complications, hospital stay, and readmissions. RESULTS: The three groups were comparable for demographics; laparoscopy was more frequent in ERAS-liver (p = 0.009). Compliance with the enhanced recovery protocol increased along the three periods (pre-ERAS, intermediate, and ERAS-liver), regardless of the perioperative phase (pre-, intra-, or postoperative). ERAS-liver group displayed the highest overall compliance rate with 73.8 %, compared to 39.9 and 57.4 % for pre-ERAS and intermediate groups (p = 0.072/0.056). Overall complications were unchanged (p = 0.185), whereas intermediate and ERAS-liver groups showed decreased major complications (p = 0.034). Consistently, hospital stay was reduced by 2 days (p = 0.005) without increased readmissions (p = 0.158). CONCLUSIONS: The previous implementation of an ERAS protocol in colorectal surgery may induce a positive impact on patients undergoing non-ERAS-liver surgery on the same ward. These results suggest that ERAS is safely applicable in liver surgery and associated with benefits.

Cost-Benefit Analysis of the Implementation of an Enhanced Recovery Program in Liver Surgery.

BACKGROUND: Enhanced recovery after surgery (ERAS) programs have been shown to ease the postoperative recovery and improve clinical outcomes for various surgery types. ERAS cost-effectiveness was demonstrated for colorectal surgery but not for liver surgery. The present study aim was to analyze the implementation costs and benefits of a specific ERAS program in liver surgery. METHODS: A dedicated ERAS protocol for liver surgery was implemented in our department in July 2013. The subsequent year all consecutive patients undergoing liver surgery were treated according to this protocol (ERAS group). They were compared in terms of real in-hospital costs with a patient series before ERAS implementation (pre-ERAS group). Mean costs per patient were compared with a bootstrap T test. A cost-minimization analysis was performed. RESULTS: Seventy-four ERAS patients were compared with 100 pre-ERAS patients. There were no significant pre- and intraoperative differences between the two groups, except for the laparoscopy number (n = 18 ERAS, n = 9 pre-ERAS, p = 0.010). Overall postoperative complications were observed in 36 (49 %) and 64 patients (64 %) in the ERAS and pre-ERAS groups, respectively (p = 0.046). The median length of stay was significantly shorter for the ERAS group (8 vs. 10 days, p = 0.006). The total mean costs per patient were €38,726 and €42,356 for ERAS and pre-ERAS (p = 0.467). The cost-minimization analysis showed a total mean cost reduction of €3080 per patient after ERAS implementation. CONCLUSIONS: ERAS implementation for liver surgery induced a non-significant decrease in cost compared to standard care. Significant decreased complication rate and hospital stay were observed in the ERAS group.

Enhanced recovery implementation in colorectal surgery-temporary or persistent improvement?

PURPOSE: Enhanced recovery after surgery (ERAS) implementation has proven to reduce complication rate and length of hospital stay. Little is known about the sustainability of these results over time. The study aim was to assess the application of ERAS pathway over the first 4 years after initial implementation. METHODS: This retrospective study analyzed data collected prospectively from 482 consecutive elective colorectal patients operated in 2011 during the ERAS implementation process (n = 66), and after initial implementation in 2012 (n = 136), 2013 (n = 152), and 2014 (n = 128). Auditing ERAS was appraised from different perspectives: adherence with the ERAS protocol, clinical outcomes, and functional recovery. Patients were compared by year. RESULTS: The groups were similar in terms of demographics and surgical strategies. Length of stay remained stable at 7 days without significant variation over time (p = 0.741). The 30-day complications and readmission rates were not statistically different (respectively p = 0.068 and p = 0.639). There was also no difference in functional recovery, more particularly, first flatus occurred at day 2 (p = 0.177), pain was adequately controlled with oral analgesics at day 2 (p = 0.111), and patients were mobilized more than 4 h the first postoperative day in more than 60 % of cases (p = 0.343). Overall adherence with the ERAS pathway was 73 % during implementation and, respectively, 73 and 77 % for 2012 and 2013 after initial implementation (p = 0.614). A significant decrease between 2013 (77 %) and 2014 (73 %) was observed (p = 0.032). CONCLUSIONS: Application of the institutional ERAS pathway could be maintained in the first 4 years after implementation leading to sustained improved functional recovery and clinical outcome.

Is standardized care feasible in the emergency setting? A case matched analysis of patients undergoing laparoscopic cholecystectomy.

BACKGROUND: Immediate laparoscopic cholecystectomy is the accepted standard for the treatment of acute cholecystitis. The aim of the present study was to evaluate the feasibility of a standardized approach with tailored care maps for pre- and postoperative care by comparing pain, nausea and patient satisfaction after elective and emergent laparoscopic cholecystectomy. METHODS: From January 2014 until April 2015, data on pain and nausea management were prospectively recorded for all elective and emergency procedures in the department of visceral surgery. This prospective observational study compared consecutive laparoscopic elective vs. emergency cholecystectomies. Visual analogue scales (VAS) were used to measure pain, nausea, and satisfaction from recovery room until 96 hours postoperatively. RESULTS: Final analysis included 168 (79%) elective cholecystectomies and 44 (21%) emergent procedures. Demographics (Age, gender, BMI and ASA-scores) were comparable between the 2 groups. In the emergency group, patients did not receive anxiolytic medication (0% vs.13%, p = 0.009) and less postoperative nausea and vomiting (PONV) prophylaxis (77% vs. 97% p = <0.001). Perioperative pain management was similar in terms of opioid consumption (median amount of fentanyl 450ug [IQR 350-500] vs. 450ug [375-550], p = 0.456) and wound infiltration rates (24% vs. 25%, p = 0.799). Postoperative consumption of paracetamol, metamizole and opiod medications were similar between the 2 groups. VAS scores for pain (p = 0.191) and nausea (p = 0.392) were low for both groups. Patient satisfaction was equally high in both clinical settings (VAS 8.5 ± 1.1 vs. 8.6 ± 1.1, p = 0.68). CONCLUSIONS: A standardized pathway allows equally successful control of pain and nausea after both elective and emergency laparoscopic cholecystectomy. This study was retrospectively registered by March 01, 2016 in the following trial register: www.researchregistry.com (UIN researchregistry993).

[Chronic postoperative pain]. / Douleur chronique postopératoire.

Chronic postoperative pain remains a frequent pathology whose global impact approximates 20 and 30% and accounts for 20% of the consultations in a pain center. Risk factors consider firstly each patient's feature and comorbidity and also different surgical procedures with their technical approach. Neuropathic pain compared to nociceptive pain is a great component in the postoperative period and needs to be recognized by specific tests (DN4). Pain prevention involves risk factors' detection, appropriate anesthetic support and effective postoperative pain management. Treatment is based on the type of pain and includes a multimodal analgesia with interventional pain therapy.

Randomized clinical trial on epidural versus patient-controlled analgesia for laparoscopic colorectal surgery within an enhanced recovery pathway.

OBJECTIVE: To compare epidural analgesia (EDA) to patient-controlled opioid-based analgesia (PCA) in patients undergoing laparoscopic colorectal surgery. BACKGROUND: EDA is mainstay of multimodal pain management within enhanced recovery pathways [enhanced recovery after surgery (ERAS)]. For laparoscopic colorectal resections, the benefit of epidurals remains debated. Some consider EDA as useful, whereas others perceive epidurals as unnecessary or even deleterious. METHODS: A total of 128 patients undergoing elective laparoscopic colorectal resections were enrolled in a randomized clinical trial comparing EDA versus PCA. Primary end point was medical recovery. Overall complications, hospital stay, perioperative vasopressor requirements, and postoperative pain scores were secondary outcome measures. Analysis was performed according to the intention-to-treat principle. RESULTS: Final analysis included 65 EDA patients and 57 PCA patients. Both groups were similar regarding baseline characteristics. Medical recovery required a median of 5 days (interquartile range [IQR], 3-7.5 days) in EDA patients and 4 days (IQR, 3-6 days) in the PCA group (P = 0.082). PCA patients had significantly less overall complications [19 (33%) vs 35 (54%); P = 0.029] but a similar hospital stay [5 days (IQR, 4-8 days) vs 7 days (IQR, 4.5-12 days); P = 0.434]. Significantly more EDA patients needed vasopressor treatment perioperatively (90% vs 74%, P = 0.018), the day of surgery (27% vs 4%, P < 0.001), and on postoperative day 1 (29% vs 4%, P < 0.001), whereas no difference in postoperative pain scores was noted. CONCLUSIONS: Epidurals seem to slow down recovery after laparoscopic colorectal resections without adding obvious benefits. EDA can therefore not be recommended as part of ERAS pathways in laparoscopic colorectal surgery.

Combined ART started during acute HIV infection protects central memory CD4+ T cells and can induce remission.

BACKGROUND: Therapeutic control of HIV replication reduces the size of the viral reservoir, particularly among central memory CD4+ T cells, and this effect might be accentuated by early treatment. METHODS: We examined the effect of ART initiated at the time of the primary HIV infection (early ART), lasting 2 and 6 years in 11 and 10 patients, respectively, on the HIV reservoir in peripheral resting CD4+ T cells, sorted into naive (TN), central memory (TCM), transitional memory (TTM) and effector memory (TEM) cells, by comparison with 11 post-treatment controllers (PTCs). RESULTS: Between baseline and 2 years, CD4+ T cell subset numbers increased markedly (P < 0.004) and HIV DNA levels decreased in all subsets (P < 0.009). TTM cells represented the majority of reservoir cells at both timepoints, T cell activation status normalized and viral diversity remained stable over time. The HIV reservoir was smaller after 6 years of early ART than after 2 years (P < 0.019), and did not differ between PTCs and patients treated for 6 years. One patient, who had low reservoir levels in all T cell subsets after 2 years of treatment similar to the levels in PTCs, spontaneously controlled viral replication during 18 months off treatment. CONCLUSIONS: Early prolonged ART thus limits the size of the HIV reservoir, protects long-lived cells from persistent infection and may enhance post-treatment control.