Rivaroxaban Versus Vitamin K Antagonist in Antiphospholipid Syndrome: A Randomized Noninferiority Trial.

Background: The potential role of new oral anticoagulants in antiphospholipid antibody syndrome (APS) remains uncertain. Objective: To determine whether rivaroxaban is noninferior to dose-adjusted vitamin K antagonists (VKAs) for thrombotic APS. Design: 3-year, open-label, randomized noninferiority trial. (EU Clinical Trials Register: EUDRA [European Union Drug Regulatory Authorities] code 2010-019764-36). Setting: 6 university hospitals in Spain. Participants: 190 adults (aged 18 to 75 years) with thrombotic APS. Intervention: Rivaroxaban (20 mg/d or 15 mg/d, according to renal function) versus dose-adjusted VKAs (target international normalized ratio, 2.0 to 3.0, or 3.1 to 4.0 in patients with a history of recurrent thrombosis). Measurements: The primary efficacy outcome was the proportion of patients with new thrombotic events; the primary safety outcome was major bleeding. The prespecified noninferiority margin for risk ratio (RR) was 1.40. Secondary outcomes included time to thrombosis, type of thrombosis, changes in biomarker levels, cardiovascular death, and nonmajor bleeding. Results: After 3 years of follow-up, recurrent thrombosis occurred in 11 patients (11.6%) in the rivaroxaban group and 6 (6.3%) in the VKA group (RR in the rivaroxaban group, 1.83 [95% CI, 0.71 to 4.76]). Stroke occurred more commonly in patients receiving rivaroxaban (9 events) than in those receiving VKAs (0 events) (corrected RR, 19.00 [CI, 1.12 to 321.9]). Major bleeding occurred in 6 patients (6.3%) in the rivaroxaban group and 7 (7.4%) in the VKA group (RR, 0.86 [CI, 0.30 to 2.46]). Post hoc analysis suggested an increased risk for recurrent thrombosis in rivaroxaban-treated patients with previous arterial thrombosis, livedo racemosa, or APS-related cardiac valvular disease. Limitation: Anticoagulation intensity was not measured in the rivaroxaban group. Conclusion: Rivaroxaban did not show noninferiority to dose-adjusted VKAs for thrombotic APS and, in fact, showed a non-statistically significant near doubling of the risk for recurrent thrombosis. Primary Funding Source: Bayer Hispania.

Factors Leading to the Loss of Natural Elite Control of HIV-1 Infection.

HIV-1 elite controllers (EC) maintain undetectable viral loads (VL) in the absence of antiretroviral treatment. However, these subjects have heterogeneous clinical outcomes, including a proportion that loses HIV-1 control over time. In this work, we compared, in a longitudinal design, transient EC, analyzed before and after the loss of virological control, with persistent EC. The aim was to identify factors leading to the loss of natural virological control of HIV-1 infection with a longitudinal retrospective study design. Gag-specific T-cell responses were assessed by in vitro intracellular polycytokine production quantified by flow cytometry. Viral diversity determinations and sequence dating were performed in proviral DNA by PCR amplification at limiting dilution of env and gag genes. The expression profile of 70 serum cytokines and chemokines was assessed by multiplex immunoassays. We identified transient EC as subjects with low Gag-specific T-cell polyfunctionality, high viral diversity, and high proinflammatory cytokine levels before the loss of control. Gag-specific T-cell polyfunctionality was inversely associated with viral diversity in transient controllers before the loss of control (r = -0.8; P = 0.02). RANTES was a potential biomarker of transient control. This study identified virological and immunological factors, including inflammatory biomarkers associated with two different phenotypes within EC. These results may allow a more accurate definition of EC, which could help in better clinical management of these individuals and in the development of future curative approaches.IMPORTANCE There is a rare group of HIV-infected patients who have the extraordinary capacity to maintain undetectable viral load levels in the absence of antiretroviral treatment, the so-called HIV-1 elite controllers (EC). However, there is a proportion within these subjects that eventually loses this capability. In this work, we found differences in virological and immune factors, including soluble inflammatory biomarkers, between subjects with persistent control of viral replication and EC that will lose virological control. The identification of these factors could be a key point for a right medical care of those EC who are going to lose natural control of viral replication and for the design of future immunotherapeutic strategies using as a model the natural persistent control of HIV infection.

Loading dendritic cells with gold nanoparticles (GNPs) bearing HIV-peptides and mannosides enhance HIV-specific T cell responses.

Gold nanoparticles (GNPs) decorated with glycans ameliorate dendritic cells (DC) uptake, antigen-presentation and T-cells cross-talk, which are important aspects in vaccine design. GNPs allow for high antigen loading, DC targeting, lack of toxicity and are straightforward prepared and easy to handle. The present study aimed to assess the capacity of DC to process and present HIV-1-peptides loaded onto GNPs bearing high-mannoside-type oligosaccharides (P1@HM) to autologous T-cells from HIV-1 patients. The results showed that P1@HM increased HIV-specific CD4+ and CD8+ T-cell proliferation and induced highly functional cytokine secretion compared with HIV-peptides alone. P1@HM elicits a highly efficient secretion of pro-TH1 cytokines and chemokines, a moderate production of pro-TH2 and significant higher secretion of pro-inflammatory cytokines such as TNF-α and IL-1ß. Thus, co-delivery of HIV-1 antigens and HM by GNPs is an excellent vaccine delivery system inducing HIV-specific cellular immune responses in HIV+ patients, being a promising approach to improve anti-HIV-1 vaccines.

The HIV-1 integrase-LEDGF allosteric inhibitor MUT-A: resistance profile, impairment of virus maturation and infectivity but without influence on RNA packaging or virus immunoreactivity.

BACKGROUND: HIV-1 Integrase (IN) interacts with the cellular co-factor LEDGF/p75 and tethers the HIV preintegration complex to the host genome enabling integration. Recently a new class of IN inhibitors was described, the IN-LEDGF allosteric inhibitors (INLAIs). Designed to interfere with the IN-LEDGF interaction during integration, the major impact of these inhibitors was surprisingly found on virus maturation, causing a reverse transcription defect in target cells. RESULTS: Here we describe the MUT-A compound as a genuine INLAI with an original chemical structure based on a new type of scaffold, a thiophene ring. MUT-A has all characteristics of INLAI compounds such as inhibition of IN-LEDGF/p75 interaction, IN multimerization, dual antiretroviral (ARV) activities, normal packaging of genomic viral RNA and complete Gag protein maturation. MUT-A has more potent ARV activity compared to other INLAIs previously reported, but similar profile of resistance mutations and absence of ARV activity on SIV. HIV-1 virions produced in the presence of MUT-A were non-infectious with the formation of eccentric condensates outside of the core. In studying the immunoreactivity of these non-infectious virions, we found that inactivated HIV-1 particles were captured by anti-HIV-specific neutralizing and non-neutralizing antibodies (b12, 2G12, PGT121, 4D4, 10-1074, 10E8, VRC01) with efficiencies comparable to non-treated virus. Autologous CD4+ T lymphocyte proliferation and cytokine induction by monocyte-derived dendritic cells (MDDC) pulsed either with MUT-A-inactivated HIV or non-treated HIV were also comparable. CONCLUSIONS: Although strongly defective in infectivity, HIV-1 virions produced in the presence of the MUT-A INLAI have a normal protein and genomic RNA content as well as B and T cell immunoreactivities comparable to non-treated HIV-1. These inactivated viruses might form an attractive new approach in vaccine research in an attempt to study if this new type of immunogen could elicit an immune response against HIV-1 in animal models.

Cyclosporine A in addition to standard ART during primary HIV-1 infection: pilot randomized clinical trial.

Background: Initiating ART during acute/recent HIV-1 infection reduces viral reservoir formation. It has been proposed that, during this phase, the size of the viral reservoir could be further reduced by the association of immunomodulatory therapy with ART. Contradictory results have emerged, however, from two trials evaluating the impact on immune recovery and the viral reservoir of adding cyclosporine A to ART during primary HIV-1 infection. Patients and methods: Twenty patients with acute/recent HIV-1 infection were randomized to receive ART alone (tenofovir, emtricitabine and lopinavir/ritonavir) or associated with 8 weeks of cyclosporine A (0.3-0.6 mg/kg twice daily). The impact on viral load, immune response and integrated and non-integrated DNA viral reservoir at 0, 8 and 36 weeks of treatment was evaluated. Results: The estimated median time from HIV-1 infection to ART onset was 63 days (IQR 53; 79.5) with 90% of patients at Fiebig V stage. No significant differences were observed in viral load decay, CD4 T cell recovery, immune response markers or the evolution of integrated DNA at week 8 (end of cyclosporine A) and week 36 between groups. However, non-integrated DNA significantly increased in the cyclosporine A arm between weeks 0 and 36. Cyclosporine A was well tolerated. Conclusions: Adding cyclosporine A to ART during acute/recent infection did not improve immune recovery. However, unintegrated DNA increased in the cyclosporine A group, suggesting an anti-integration effect, a point warranting further research (ClinicalTrials.gov Identifier: NCT00979706).

Enteric-coated mycophenolate sodium versus azathioprine in patients with active systemic lupus erythematosus: a randomised clinical trial.

OBJECTIVE: To compare the efficacy and safety of enteric-coated mycophenolate sodium (EC-MPS) versus azathioprine (AZA) in patients with active systemic lupus erythematosus (SLE) disease. METHODS: A multicentre, 24-month, superiority, open-label, randomised controlled trial (NCT01112215) was conducted with 240 patients (120 per arm) receiving either EC-MPS (target dose: 1440 mg/day) or AZA (target dose: 2 mg/kg/day) in addition to prednisone and/or antimalarials. The primary endpoint was the proportion of patients achieving clinical remission, assessed by SLE Disease Activity Index 2000 (SLEDAI-2K) and British Isles Lupus Assessment Group (BILAG), at 3 and 24 months. Secondary endpoints included time to clinical remission, BILAG A and B flare rates, time to flare, corticosteroid reduction and adverse events (AEs). RESULTS: Proportion of patients achieving clinical remission (clinical SLEDAI=0) was higher in the EC-MPS group at 3 (32.5% vs 19.2%; treatment difference, 13.3 (CI 2.3 to 24), p=0.034) and 24 months (71.2% vs 48.3%; treatment difference, 22.9 (CI 10.4 to 34.4), p<0.001). EC-MPS was superior with respect to time to clinical remission (HR 1.43; 95% CI 1.07 to 1.91; p=0.017). BILAG A/B and B flares occurred more frequently in the AZA group (71.7% vs 50%, p=0.001 and 21.67% vs 8.3%, p=0.004, respectively). EC-MPS was superior with respect to time to first BILAG A/B (HR 1.81; 95% CI 1.3 to 2.56; p=0.0004) and BILAG A flare (HR 2.84; 95% CI 1.37 to 5.89; p=0.003). AEs were similar in both groups except for leucopenia that occurred more frequently with AZA. CONCLUSIONS: EC-MPS was superior to AZA in treating SLE and preventing further relapses. TRIAL REGISTRATION NUMBER: NCT01112215; Results.

PD-L1 Blockade Differentially Impacts Regulatory T Cells from HIV-Infected Individuals Depending on Plasma Viremia.

Blocking the PD-1/PD-L1 pathway has emerged as a potential therapy to restore impaired immune responses in human immunodeficiency virus (HIV)-infected individuals. Most reports have studied the impact of the PD-L1 blockade on effector cells and neglected possible effects on regulatory T cells (Treg cells), which play an essential role in balancing immunopathology and antiviral effector responses. The aim of this study was to define the consequences of ex vivo PD-L1 blockade on Treg cells from HIV-infected individuals. We observed that HIV infection led to an increase in PD-1+ and PD-L1+ Treg cells. This upregulation correlated with disease progression and decreased under antiretroviral treatment. Treg cells from viremic individuals had a particularly high PD-1 expression and impaired proliferative capacity in comparison with Treg cells from individuals under antiretroviral treatment. PD-L1 blockade restored the proliferative capacity of Treg cells from viremic individuals but had no effect on its suppressive capacity. Moreover, it increased the viral production in cell cultures from viremic individuals. This increase in viral production correlated with an increase in Treg cell percentage and a reduction in the CD4/Treg and CD8/Treg cell ratios. In contrast to the effect of the PD-L1 blockade on Treg cells from viremic individuals, we did not observe a significant effect on the proliferative capacity of Treg cells from individuals in whom viremia was controlled (either spontaneously or by antiretroviral treatment). However, PD-L1 blockade resulted in an increased proliferative capacity of HIV-specific-CD8 T cells in all subjects. Taken together, our findings suggest that manipulating PD-L1 in vivo can be expected to influence the net gain of effector function depending on the subject's plasma viremia.

Neurological involvement in patients with acute/recent HIV-1 infection. A case-control study.

Primary HIV-1 infection is a relevant period for its virological and epidemiological consequences. Most patients present a symptomatic disease that can be potentially serious, but neurological involvement during primary HIV-1 infection has been poorly studied. The aim of this study was to describe the characteristics and outcomes of primary HIV-1 infection patients presenting neurological symptoms and to compare them with primary HIV-1 infection patients without neurological involvement. Retrospective case-control study (1:3) comparing primary HIV-1 infection patients with and without neurological involvement enrolled in the Acute/Recent Hospital Clinic PHI Cohort between 1997 and 2016. Matching criteria included age (±10 years), gender, year of diagnosis (±4 years), and Fiebig stage. The conditional logit model was used for comparisons. Fourteen out of 463 patients (3.02%) enrolled in the Acute/Recent Hospital Clinic PHI Cohort between 1997 and 2016 presented neurological symptoms. 28.5% of cases presented as meningitis and 71.5% as meningoencephalitis. Cerebrospinal fluid showed non-specific findings, including pleocytosis with lymphocyte predominance and increased protein levels. All cases required hospitalisation, whereas only 19% of the controls did. No other pathogen was identified in any case, but five patients initiated empirically antimicrobial treatment for other aetiologies until diagnosis was confirmed. CD4/CD8 ratio was significantly lower (p = 0.039) and plasmatic viral load significantly higher in the case group, compared to controls (p = 0.028). Risk factors, HIV-1 tropism, subtype distribution, and prescribed ART regimens were comparable between cases and controls. After 6 months on ART, 92% of cases had undetectable viral load, similar to controls, and CD4/CD8 ratio became also comparable between groups. All cases recovered rapidly with ART and were discharged without sequels. Neurological involvement during primary HIV-1 infection is unusual but serious, always requiring hospitalisation. Diagnosis is difficult because of the wide range of symptoms and similarities with other viral aetiologies. Neurological manifestations during primary HIV-1 infection are associated with a lower CD4/CD8 ratio and with a higher viral load than controls. Immediate ART initiation and rapid viral load decrease are required, allowing complete clinical recovery.

Increased expression with differential subcellular location of cytidine deaminase APOBEC3G in human CD4(+) T-cell activation and dendritic cell maturation.

APOBEC3G (apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3G; A3G) is an innate defense protein showing activity against retroviruses and retrotransposons. Activated CD4(+) T cells are highly permissive for HIV-1 replication, whereas resting CD4(+) T cells are refractory. Dendritic cells (DCs), especially mature DCs, are also refractory. We investigated whether these differences could be related to a differential A3G expression and/or subcellular distribution. We found that A3G mRNA and protein expression is very low in resting CD4(+) T cells and immature DCs, but increases strongly following T-cell activation and DC maturation. The Apo-7 anti-A3G monoclonal antibody (mAb), which was specifically developed, confirmed these differences at the protein level and disclosed that A3G is mainly cytoplasmic in resting CD4(+) T cells and immature DCs. Nevertheless, A3G translocates to the nucleus in activated-proliferating CD4(+) T cells, yet remaining cytoplasmic in matured DCs, a finding confirmed by immunoblotting analysis of cytoplasmic and nuclear fractions. Apo-7 mAb was able to immunoprecipitate endogenous A3G allowing to detect complexes with numerous proteins in activated-proliferating but not in resting CD4(+) T cells. The results show for the first time the nuclear translocation of A3G in activated-proliferating CD4(+) T cells.

Consequences of HLA-B*13-Associated Escape Mutations on HIV-1 Replication and Nef Function.

UNLABELLED: HLA-B*13 is associated with superior in vivo HIV-1 viremia control. Protection is thought to be mediated by sustained targeting of key cytotoxic T lymphocyte (CTL) epitopes and viral fitness costs of CTL escape in Gag although additional factors may contribute. We assessed the impact of 10 published B*13-associated polymorphisms in Gag, Pol, and Nef, in 23 biologically relevant combinations, on HIV-1 replication capacity and Nef-mediated reduction of cell surface CD4 and HLA class I expression. Mutations were engineered into HIV-1NL4.3, and replication capacity was measured using a green fluorescent protein (GFP) reporter T cell line. Nef-mediated CD4 and HLA-A*02 downregulation was assessed by flow cytometry, and T cell recognition of infected target cells was measured via coculture with an HIV-specific luciferase reporter cell line. When tested individually, only Gag-I147L and Gag-I437L incurred replicative costs (5% and 17%, respectively), consistent with prior reports. The Gag-I437L-mediated replication defect was rescued to wild-type levels by the adjacent K436R mutation. A novel B*13 epitope, comprising 8 residues and terminating at Gag147, was identified in p24(Gag) (GQMVHQAIGag140-147). No other single or combination Gag, Pol, or Nef mutant impaired viral replication. Single Nef mutations did not affect CD4 or HLA downregulation; however, the Nef double mutant E24Q-Q107R showed 40% impairment in HLA downregulation with no evidence of Nef stability defects. Moreover, target cells infected with HIV-1-NefE24Q-Q107R were recognized better by HIV-specific T cells than those infected with HIV-1NL4.3 or single Nef mutants. Our results indicate that CTL escape in Gag and Nef can be functionally costly and suggest that these effects may contribute to long-term HIV-1 control by HLA-B*13. IMPORTANCE: Protective effects of HLA-B*13 on HIV-1 disease progression are mediated in part by fitness costs of CTL escape mutations in conserved Gag epitopes, but other mechanisms remain incompletely known. We extend our knowledge of the impact of B*13-driven escape on HIV-1 replication by identifying Gag-K436R as a compensatory mutation for the fitness-costly Gag-I437L. We also identify Gag-I147L, the most rapidly and commonly selected B*13-driven substitution in HIV-1, as a putative C-terminal anchor residue mutation in a novel B*13 epitope. Most notably, we identify a novel escape-driven fitness defect: B*13-driven substitutions E24Q and Q107R in Nef, when present together, substantially impair this protein's ability to downregulate HLA class I. This, in turn, increases the visibility of infected cells to HIV-specific T cells. Our results suggest that B*13-associated escape mutations impair HIV-1 replication by two distinct mechanisms, that is, by reducing Gag fitness and dampening Nef immune evasion function.

HIV-1 Reservoir Dynamics after Vaccination and Antiretroviral Therapy Interruption Are Associated with Dendritic Cell Vaccine-Induced T Cell Responses.

UNLABELLED: HIV-1-specific immune responses induced by a dendritic cell (DC)-based therapeutic vaccine might have some effect on the viral reservoir. Patients on combination antiretroviral therapy (cART) were randomized to receive DCs pulsed with autologous HIV-1 (n = 24) (DC-HIV-1) or nonpulsed DCs (n = 12) (DC-control). We measured the levels of total and integrated HIV-1 DNA in CD4 T cells isolated from these patients at 6 time points: before any cART; before the first cART interruption, which was at 56 weeks before the first immunization to isolate virus for pulsing DCs; before and after vaccinations (VAC1 and VAC2); and at weeks 12 and 48 after the second cART interruption. The vaccinations did not influence HIV-1 DNA levels in vaccinated subjects. After the cART interruption at week 12 postvaccination, while total HIV-1 DNA increased significantly in both arms, integrated HIV-1 DNA did not change in vaccinees (mean of 1.8 log10 to 1.9 copies/10(6) CD4 T cells, P = 0.22) and did increase in controls (mean of 1.8 log10 to 2.1 copies/10(6) CD4 T cells, P = 0.02) (P = 0.03 for the difference between groups). However, this lack of increase of integrated HIV-1 DNA observed in the DC-HIV-1 group was transient, and at week 48 after cART interruption, no differences were observed between the groups. The HIV-1-specific T cell responses at the VAC2 time point were inversely correlated with the total and integrated HIV-1 DNA levels after cART interruption in vaccinees (r [Pearson's correlation coefficient] = -0.69, P = 0.002, and r = -0.82, P < 0.0001, respectively). No correlations were found in controls. HIV-1-specific T cell immune responses elicited by DC therapeutic vaccines drive changes in HIV-1 DNA after vaccination and cART interruption. (This study has been registered at ClinicalTrials.gov under registration no. NCT00402142.) IMPORTANCE: There is an intense interest in developing strategies to target HIV-1 reservoirs as they create barriers to curing the disease. The development of therapeutic vaccines aimed at enhancing immune-mediated clearance of virus-producing cells is of high priority. Few therapeutic vaccine clinical trials have investigated the role of therapeutic vaccines as a strategy to safely eliminate or control viral reservoirs. We recently reported that a dendritic cell-based therapeutic vaccine was able to significantly decrease the viral set point in vaccinated patients, with a concomitant increase in HIV-1-specific T cell responses. The HIV-1-specific T cell immune responses elicited by this therapeutic dendritic cell vaccine drove changes in the viral reservoir after vaccinations and significantly delayed the replenishment of integrated HIV-1 DNA after cART interruption. These data help in understanding how an immunization could shift the virus-host balance and are instrumental for better design of strategies to reach a functional cure of HIV-1 infection.

Safety and immunogenicity of a modified vaccinia Ankara-based HIV-1 vaccine (MVA-B) in HIV-1-infected patients alone or in combination with a drug to reactivate latent HIV-1.

OBJECTIVES: The safety, immunogenicity, impact on the latent reservoir and rebound of viral load after therapeutic HIV-1 vaccination with recombinant modified vaccinia Ankara-based (MVA-B) HIV-1 vaccine expressing monomeric gp120 and the fused Gag-Pol-Nef polyprotein of clade B with or without a drug to reactivate latent HIV-1 (disulfiram) were assessed. METHODS: HIV-1-infected patients were randomized to receive three injections of MVA-B (n = 20) or placebo (n = 10). Twelve patients (eight who received vaccine and four who were given placebo) received a fourth dose of MVA-B followed by 3 months of disulfiram. Combined ART (cART) was discontinued 8 weeks after the last dose of MVA-B. Clinical Trials.gov identifier: NCT01571466. RESULTS: MVA-B was safe and well tolerated. A minor, but significant, increase in the T cell responses targeting vaccine inserts of Gag was observed [a median of 290, 403 and 435 spot-forming-cells/10(6) PBMCs at baseline, after two vaccinations and after three vaccinations, respectively; P = 0.02 and P = 0.04]. After interruption of cART, a modest delay in the rebound of the plasma viral load in participants receiving vaccine but not disulfiram was observed compared with placebo recipients (P = 0.01). The dynamics of the viral load rebound did not change in patients receiving MVA-B/disulfiram. No changes in the proviral reservoir were observed after disulfiram treatment. CONCLUSIONS: MVA-B vaccination was a safe strategy to increase Gag-specific T cell responses in chronically HIV-1-infected individuals, but it did not have a major impact on the latent reservoir or the rebound of plasma viral load after interruption of cART when given alone or in combination with disulfiram.

Direct interrogation of viral peptides presented by the class I HLA of HIV-infected T cells.

UNLABELLED: Identification of CD8(+) cytotoxic T lymphocyte (CTL) epitopes has traditionally relied upon testing of overlapping peptide libraries for their reactivity with T cells in vitro. Here, we pursued deep ligand sequencing (DLS) as an alternative method of directly identifying those ligands that are epitopes presented to CTLs by the class I human leukocyte antigens (HLA) of infected cells. Soluble class I HLA-A*11:01 (sHLA) was gathered from HIV-1 NL4-3-infected human CD4(+) SUP-T1 cells. HLA-A*11:01 harvested from infected cells was immunoaffinity purified and acid boiled to release heavy and light chains from peptide ligands that were then recovered by size-exclusion filtration. The ligands were first fractionated by high-pH high-pressure liquid chromatography and then subjected to separation by nano-liquid chromatography (nano-LC)-mass spectrometry (MS) at low pH. Approximately 10 million ions were selected for sequencing by tandem mass spectrometry (MS/MS). HLA-A*11:01 ligand sequences were determined with PEAKS software and confirmed by comparison to spectra generated from synthetic peptides. DLS identified 42 viral ligands presented by HLA-A*11:01, and 37 of these were previously undetected. These data demonstrate that (i) HIV-1 Gag and Nef are extensively sampled, (ii) ligand length variants are prevalent, particularly within Gag and Nef hot spots where ligand sequences overlap, (iii) noncanonical ligands are T cell reactive, and (iv) HIV-1 ligands are derived from de novo synthesis rather than endocytic sampling. Next-generation immunotherapies must factor these nascent HIV-1 ligand length variants and the finding that CTL-reactive epitopes may be absent during infection of CD4(+) T cells into strategies designed to enhance T cell immunity. IMPORTANCE: HIV-1 epitopes catalogued by the Los Alamos National Laboratory (LANL) have yielded limited success in vaccine trials. Because the HLA of infected cells have not previously been assessed for HIV-1 ligands, the objective here was to directly characterize the viral ligands that mark infected cells. Recovery of HLA-presented peptides from HIV-1-infected CD4(+) T cells and interrogation of the peptide cargo by mass spectrometric DLS show that typical and atypical viral ligands are efficiently presented by HLA and targeted by human CTLs. Nef and Gag ligands dominate the infected cell's antigenic profile, largely due to extensive ligand sampling from select hot spots within these viral proteins. Also, HIV-1 ligands are often longer than expected, and these length variants are quite antigenic. These findings emphasize that an HLA-based view of HIV-1 ligand presentation to CTLs provides previously unrealized information that may enhance the development of immune therapies and vaccines.

Modulating the immune response to SARS-CoV-2 by different nanocarriers delivering an mRNA expressing trimeric RBD of the spike protein: COVARNA Consortium.

Vaccines based on mRNA technology have revolutionized the field. In fact, lipid nanoparticles (LNP) formulated with mRNA are the preferential vaccine platform used in the fight against SARS-CoV-2 infection, with wider application against other diseases. The high demand and property right protection of the most potent cationic/ionizable lipids used for LNP formulation of COVID-19 mRNA vaccines have promoted the design of alternative nanocarriers for nucleic acid delivery. In this study we have evaluated the immunogenicity and efficacy of different rationally designed lipid and polymeric-based nanoparticle prototypes against SARS-CoV-2 infection. An mRNA coding for a trimeric soluble form of the receptor binding domain (RBD) of the spike (S) protein from SARS-CoV-2 was encapsulated using different components to form nanoemulsions (NE), nanocapsules (NC) and lipid nanoparticles (LNP). The toxicity and biological activity of these prototypes were evaluated in cultured cells after transfection and in mice following homologous prime/boost immunization. Our findings reveal good levels of RBD protein expression with most of the formulations. In C57BL/6 mice immunized intramuscularly with two doses of formulated RBD-mRNA, the modified lipid nanoparticle (mLNP) and the classical lipid nanoparticle (LNP-1) were the most effective delivery nanocarriers at inducing binding and neutralizing antibodies against SARS-CoV-2. Both prototypes fully protected susceptible K18-hACE2 transgenic mice from morbidity and mortality following a SARS-CoV-2 challenge. These results highlight that modulation of mRNAs immunogenicity can be achieved by using alternative nanocarriers and support further assessment of mLNP and LNP-1 prototypes as delivery vehicles for mRNA vaccines.

The Combination of an mRNA Immunogen, a TLR7 Agonist and a PD1 Blocking Agent Enhances In-Vitro HIV T-Cell Immune Responses.

The development of new strategies to achieve a functional cure for HIV remains a priority. We tested a novel HIV therapeutic vaccine using unmodified mRNA (TMEP-B) and mRNA modified by 1-methyl-3'-pseudouridylyl (TMEP-Bmod) expressing both a multiepitopic sequences from Gag, Pol, and Nef proteins, including different CD4 and CD8 T-cell epitopes functionally associated with HIV control in transfected monocyte-derived dendritic cells (MDDCs) obtained from HIV infected patients. In vitro assays were used to test the mRNAs alone and in combination with immunomodulator agents, such as the TLR-7 agonist Vesatolimod and the PD-1 antagonist Nivolumab to try to improve HIV-specific cellular immune responses. Combining the mRNAs with the immunomodulators enhanced HIV-specific T-cell responses, together with the secretion of IFNγ, IP10, MIP-1α, and MIP-1ß, which are fundamental mediators of viral control. Our data suggest that the mRNA vaccine prototypes TMEP-B and TMEP-Bmod, when combined with Vesatolimod and/or Nivolumab, could achieve functional cure for patients with HIV.

Pharmacokinetics, the Immunological Impact, and the Effect on HIV Ex-Vivo Infectivity of Maraviroc, Raltegravir, and Lopinavir in Men Who Have Sex with Men Using Postexposure Prophylaxis.

Most of the studies using the colorectal tissue explants challenge model have been conducted after one single dose and before reaching a steady state. We consider that longer exposure as in 28-day postexposure prophylaxis (PEP) course and in an at-risk setting, such as after a sexual risk exposure to HIV could give us valuable information about these drugs. In a substudy we assessed pharmacokinetics, changes on immune system and ex-vivo rectal mucosal susceptibility to HIV-1 infection after taking maraviroc (MVC), raltegravir (RAL), and ritonavir-boosted lopinavir (LPV/r) PEP-based regimens in 30 men who have sex with men. Participants received 28 days of twice-daily MVC (n = 11), RAL (n = 10) or LPV/r (n = 9) all with tenofovir/emtricitabine (TDF/FTC) backbone. Blood, rectal fluid, and rectal tissue samples were collected at days 7, 28, and 90 after starting PEP. The samples obtained at day 90 were considered baseline. All studied antiretrovirals were quantifiable at 7 and 28 days in all tissues. Activation markers were increased in CD4 mucosal mononuclear cells (MMCs) after 28 days of MVC: CD38 + 68.5 versus 85.1, p = .008 and CD38+DR +16.1 versus 26.7, p = .008. Exposure to MVC at both endpoints (7 and 28 days) was associated with significant suppression of HIV-1BAL (p = .005 and p = .028), but we did not observe this effect with RAL or LPV/r. Merging together changes in MMC in all arms, we found a positive correlation in the CD8 T cell lineage between the infectivity at day 7 and activation (CD38+ r = 0.43, p = .025, DR + r = 0.547, p = .003 and 38+DR+ r = 0.526, p = .05), senescence (CD57+CD28- r = 0.479, p = .012), naive cells (RA+CCR7+ r = 0.484, p = .01), and CCR5 expression (r = 0.593, p = .001). We conclude that MVC in combination with TDF/FTC was associated with viral suppression in rectal explants and that overall ex-vivo HIV infectivity correlated with activation and senescence in CD8 MMCs.

Immunological and virological findings in a patient with exceptional post-treatment control: a case report.

BACKGROUND: Although antiretroviral therapy (ART) is effective in suppressing viral replication, HIV-1 persists in reservoirs and rebounds after ART has been stopped. However, a very few people (eg, elite and post-treatment controllers) are able to maintain viral loads below detection limits without ART, constituting a realistic model for long-term HIV remission. Here, we describe the HIV control mechanisms of an individual who showed exceptional post-treatment control for longer than 15 years. METHODS: We report the case of a Hispanic woman aged 59 years with sexually acquired acute HIV infection, who was included in an immune-mediated primary HIV infection trial involving a short course of ciclosporine A, interleukin-2, granulocyte macrophage colony-stimulating factor, and pegylated interferon alfa, followed by analytical treatment interruption. We did the following viral assays: total and integrated HIV-1 DNA in CD4 T cells and rectal tissue, quantitative viral outgrowth assay, HIV-1 infectivity in peripheral blood mononuclear cells and CD4 T-cell cultures and viral inhibitory activity by natural killer (NK) and CD8 T cells. NK and T-cell phenotypes were determined by flow cytometry. HLA, killer cell immunoglobulin-like receptors, Δ32CCR5, and NKG2C alleles were genotyped. FINDINGS: After ART and immunomodulatory treatment, the person maintained undetectable plasma viral load for 15 years. HIV-1 subtype was CFR_02AG, CCR5-tropic. We found progressive reductions in viral reservoir during the 15-year treatment interruption: total HIV DNA (from 4573·50 copies per 106 CD4 T cells to 95·33 copies per 106 CD4 T cells) and integrated DNA (from 85·37 copies per 106 CD4 T cells to 5·25 copies per 106 CD4 T cells). Viral inhibition assays showed strong inhibition of in vitro HIV replication in co-cultures of CD4 T cells with autologous NK or CD8 T cells at 1:2 ratio (75% and 62%, respectively). Co-cultures with NK and CD8 T cells resulted in 93% inhibition. We detected higher-than-reference levels of both NKG2C-memory-like NK cells (46·2%) and NKG2C γδ T cells (64·9%) associated with HIV-1 control. INTERPRETATION: We described long-term remission in a woman aged 59 years who was treated during primary HIV infection and has maintained undetectable viral load for 15 years without ART. Replication-competent HIV-1 was isolated. NKG2C-memory-like NK cells and γδ T cells were associated with the control viral replication. Strategies promoting these cells could bring about long-term HIV remission. FUNDING: Fondo Europeo para el Desarrollo Regional (FEDER), SPANISH AIDS Research Network (RIS), Fondo de Investigación Sanitaria (FIS), HIVACAT, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), CERCA Programme/Generalitat de Catalunya, la Caixa Foundation, and Centro de Investigación Biomédica en Red de Enfermedades Infecciosas (CIBERINFEC). TRANSLATION: For the Spanish translation of the abstract see Supplementary Materials section.

Assessment of Human SARS CoV-2-Specific T-Cell Responses Elicited In Vitro by New Computationally Designed mRNA Immunogens (COVARNA).

The COVID-19 pandemic has brought significant changes and advances in the field of vaccination, including the implementation and widespread use of encapsidated mRNA vaccines in general healthcare practice. Here, we present two new mRNAs expressing antigenic parts of the SARS-CoV-2 spike protein and provide data supporting their functionality. The first mRNA, called RBD-mRNA, encodes a trimeric form of the virus spike protein receptor binding domain (RBD). The other mRNA, termed T-mRNA, codes for the relevant HLA I and II spike epitopes. The two mRNAs (COVARNA mRNAs) were designed to be used for delivery to cells in combination, with the RBD-mRNA being the primary source of antigen and the T-mRNA working as an enhancer of immunogenicity by supporting CD4 and CD8 T-cell activation. This innovative approach substantially differs from other available mRNA vaccines, which are largely directed to antibody production by the entire spike protein. In this study, we first show that both mRNAs are functionally transfected into human antigen-presenting cells (APCs). We obtained peripheral blood mononuclear cell (PBMC) samples from three groups of voluntary donors differing in their immunity against SARS-CoV-2: non-infected (naïve), infected-recovered (convalescent), and vaccinated. Using an established method of co-culturing autologous human dendritic cells (hDCs) with T-cells, we detected proliferation and cytokine secretion, thus demonstrating the ability of the COVARNA mRNAs to activate T-cells in an antigen-specific way. Interestingly, important differences in the intensity of the response between the infected-recovered (convalescent) and vaccinated donors were observed, with the levels of T-cell proliferation and cytokine secretion (IFNγ, IL-2R, and IL-13) being higher in the vaccinated group. In summary, our data support the further study of these mRNAs as a combined approach for future use as a vaccine.

Safety and immunogenicity of a recombinant protein RBD fusion heterodimer vaccine against SARS-CoV-2.

In response to COVID-19 pandemic, we have launched a vaccine development program against SARS-CoV-2. Here we report the safety, tolerability, and immunogenicity of a recombinant protein RBD fusion heterodimeric vaccine against SARS-CoV-2 (PHH-1V) evaluated in a phase 1-2a dose-escalation, randomized clinical trial conducted in Catalonia, Spain. 30 young healthy adults were enrolled and received two intramuscular doses, 21 days apart of PHH-1V vaccine formulations [10 µg (n = 5), 20 µg (n = 10), 40 µg (n = 10)] or control [BNT162b2 (n = 5)]. Each PHH-1V group had one safety sentinel and the remaining participants were randomly assigned. The primary endpoint was solicited events within 7 days and unsolicited events within 28 days after each vaccination. Secondary endpoints were humoral and cellular immunogenicity against the variants of concern (VOCs) alpha, beta, delta and gamma. All formulations were safe and well tolerated, with tenderness and pain at the site of injection being the most frequently reported solicited events. Throughout the study, all participants reported having at least one mild to moderate unsolicited event. Two unrelated severe adverse events (AE) were reported and fully resolved. No AE of special interest was reported. Fourteen days after the second vaccine dose, all participants had a >4-fold change in total binding antibodies from baseline. PHH-1V induced robust humoral responses with neutralizing activities against all VOCs assessed (geometric mean fold rise at 35 days p < 0.0001). The specific T-cell response assessed by ELISpot was moderate. This initial evaluation has contributed significantly to the further development of PHH-1V, which is now included in the European vaccine portfolio.ClinicalTrials.gov Identifier NCT05007509EudraCT No. 2021-001411-82.