GRAd-COV2 vaccine provides potent and durable humoral and cellular immunity to SARS-CoV-2 in randomized placebo-controlled phase 2 trial.

The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic and heterologous immunization approaches implemented worldwide for booster doses call for diversified vaccine portfolios. GRAd-COV2 is a gorilla adenovirus-based COVID-19 vaccine candidate encoding prefusion-stabilized spike. The safety and immunogenicity of GRAd-COV2 is evaluated in a dose- and regimen-finding phase 2 trial (COVITAR study, ClinicalTrials.gov: NCT04791423) whereby 917 eligible participants are randomized to receive a single intramuscular GRAd-COV2 administration followed by placebo, or two vaccine injections, or two doses of placebo, spaced over 3 weeks. Here, we report that GRAd-COV2 is well tolerated and induces robust immune responses after a single immunization; a second administration increases binding and neutralizing antibody titers. Potent, variant of concern (VOC) cross-reactive spike-specific T cell response peaks after the first dose and is characterized by high frequencies of CD8s. T cells maintain immediate effector functions and high proliferative potential over time. Thus, GRAd vector is a valuable platform for genetic vaccine development, especially when robust CD8 response is needed.

The Many Faces of Immune Activation in HIV-1 Infection: A Multifactorial Interconnection.

Chronic immune activation has a significant role in HIV-1 disease pathogenesis and CD4+ T-cell depletion. The causes of chronic inflammation and immune activation are incompletely understood, but they are likely multifactorial in nature, involving both direct and indirect stimuli. Possible explanations include microbial translocation, coinfection, and continued presence of competent replicating virus. In fact, long-term viral suppression treatments are unable to normalize elevated markers of systemic immune activation. Furthermore, high levels of pro-inflammatory cytokines increase susceptibility to premature aging of the immune system. The phenomenon of "inflammaging" has begun to be evident in the last decades, as a consequence of increased life expectancy due to the introduction of cART. Quality of life and survival have improved substantially; however, PLWH are predisposed to chronic inflammatory conditions leading to age-associated diseases, such as inflammatory bowel disease, neurocognitive disorders, cardiovascular diseases, metabolic syndrome, bone abnormalities, and non-HIV-associated cancers. Several approaches have been studied in numerous uncontrolled and/or randomized clinical trials with the aim of reducing immune activation/inflammatory status in PLWH, none of which have achieved consistent results.

Lipopolysaccharide induces platelet activation in HIV patients: the role of different viral load patterns.

OBJECTIVES: This study aimed to assess whether gut-derived lipopolysaccharide (LPS) could affect platelet function in HIV-1 patients with residual viral load. METHODS: In 23 HIV-1 patients on effective antiretroviral treatment, 10 treatment-naïve HIV-1 subjects and 20 healthy subjects (HS), LPS, zonulin, markers of platelet activation and oxidative stress were evaluated. In vitro, platelets from HS were exposed to plasma from HIV-1-infected treated and untreated patients. RESULTS: Compared with HS, LPS was higher in treated and treatment-naïve subjects with HIV-1 (7.7 ± 2.9, 80.9 ± 13.7 and 75.3 ± 22.6 pg/mL, P < 0.001 vs. HS) as well as serum zonulin (1.3 ± 0.5, 6.1 ± 1.5 and 5.3 ± 1.7 ng/mL, P < 0.001 vs. HS). LPS and zonulin were correlated in HIV patients (Spearman correlation coefficient (rS) = 0.73, P < 0.0001). Levels of soluble CD40 ligand (sCD40L), soluble P-selectin (sP-selectin) and thromboxane B2 (TxB2 ) were higher in HIV-1-treated and treatment-naïve subjects compared with HS as well as NADPH oxidase 2 (NOX2) activation and hydrogen peroxide (H2 O2 ) production. In vitro, sCD40L, sP-selectin and TxB2 production, NOX2 activation and p47phox phosphorylation were higher in platelets exposed to plasma from HIV-1 patients with different viral load compared with the exposure to plasma from HS. This effect was blunted in platelets pre-treated with TLR4 or TLR7 inhibitors. CONCLUSIONS: Low-grade endotoxaemia and persistent viraemia increase platelet function with a mechanism mediated by NOX2 in patients with HIV-1 infection.

Physical Activity and HIV: Effects on Fitness Status, Metabolism, Inflammation and Immune-Activation.

Several studies evidenced that a sedentary lifestyle is related with higher levels of systemic inflammation and highlighted that physical activity can trigger anti-inflammatory effects. To evaluate the impact of self-prescribed physical activity on fitness status, metabolism, inflammation and immune-activation in people living with HIV, an interim analysis of the results of the clinical trial PRIMO (NCT03392805) was performed. Patients enrolled were divided in 2 groups on the basis of self-prescribed physical activity: a physically active group (self-prescribed physical activity) and a sedentary group. Physical fitness was evaluated by sport medicine specialists and related to nutritional status, anthropometric variables, adipokines levels (adiponectin, leptin, resistin), peripheral immune-activation (CD38, HLA-DR on CD4 and CD8), and plasma inflammatory markers (IL-6 and TNF-α). The physically active group had a better profile in anthropometric measures and aerobic fitness but did not show lower levels of immune-activation compared to sedentary group. Also serum IL-6, TNF-α, and adipokines levels showed no statistical differences. On the basis of these data, a regular self-organized physical activity seems useful to improve cardio-respiratory fitness, but unable to control HIV-related immune-activation.

IFN-stimulated gene expression is independent of the IFNL4 genotype in chronic HIV-1 infection.

This study aimed to evaluate the association between the IFNL4 rs368234815 (ΔG/TT) dinucleotide polymorphism and the IFN response during chronic HIV-1 infection. We carried out genotyping analysis and measured the expression of IFN-stimulated genes (ISGs) (myxovirus resistance protein A [MxA], ISG15, ISG56, apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like [APOBEC] 3F and APOBEC3G) on peripheral blood mononuclear cells collected from naïve and HAART-treated HIV-1-infected patients. There were no statistically significant differences in endogenous ISGs mRNA levels among HIV-1-positive patients bearing different IFNL4 genotypes, suggesting that ISG expression is independent of the IFNL4 genotype in HIV-1 infection.

Reduced Plasma Levels of sCD14 and I-FABP in HIV-infected Patients with Mesalazine-treated Ulcerative Colitis.

BACKGROUND: Microbial translocation (MT) is a shared feature of HIV infection and inflammatory bowel disease (IBD). AIMS: This study was conducted to assess the impact of IBD (and particularly ulcerative colitis, UC) on plasma markers of MT and immune activation in HIV+ subjects. METHODS: A cross-sectional study was conducted in 3 groups of patients: HIV+/UC+(group HIV/UC); HIV+/UC- (group HIV); HIV-/UC+(group UC). Plasma levels of soluble CD14 (sCD14), intestinal fatty acid-binding protein (I-FABP), and endotoxin core antibodies (endoCAB) were measured as plasma markers of MT. Inflammation and immune activation were evaluated by measuring plasma levels of IL-6, IL-21, TNF-alpha, and high-sensitivity C-reactive protein (hs-CRP). T- and B-cells subpopulations were characterized by FACS analysis. RESULTS: Seven patients were enrolled in group HIV/UC, 9 in HIV, and 10 in UC. All HIV-positive patients had plasma values of HIV-1 RNA<37 copies/mL for at least 12 months and good immunological recovery. All patients with UC were treated with oral mesalazine. Markers of MT, immune activation, and inflammation were not increased in subjects with HIV/UC. In fact, they had lower levels of I-FABP (p=0.001) and sCD14 (p=0.007) when compared to other patients groups. Positive correlations were found between I-FABP and sCD14 (r=.355, p=0.076). Frequency of T- and B-cell subsets did not differ among groups. CONCLUSIONS: Our results suggest that UC does not worsen MT, inflammation, or immune activation in HIV-infected subjects. The anti-inflammatory activity of chronic mesalazine administration on intestinal mucosa may contribute to this finding.

ISG15 expression correlates with HIV-1 viral load and with factors regulating T cell response.

Given the multifactorial nature of action of type I interferon (IFN) in HIV-1 infection and the need to firmly establish the action of key components of IFN pathways, we compared the IFN stimulated gene (ISG)15 expression with that of other well-characterized ISGs, evaluating its relationship with immunosuppressive factors regulating T-cell response in HIV-1 patients. PBMC from 225 subjects were included: healthy donors (n=30), naïve (n=93) and HAART treated HIV-1 subjects (n=102). Levels of ISG15-mRNA, ISG56-mRNA, APOBEC3G/3F-mRNA, TRAIL-mRNA, IDO-mRNA, proviral load andISG15 (rs15842 and rs1921) SNPs were evaluated by using TaqMan assays. We found that ISG15, ISG56, APOBEC3G/3F levels were increased in untreated HIV-1 patients compared to healthy donors, being ISG15 the highest ISG expressed. The amount of ISG15 correlated with viral load and with CD4+ T cell counts whereas no relationship was found between all ISGs analyzed and proviral load or HIV-1 tropism. ISG15 expression was reduced following long-term antiretroviral therapy. In addition, ISG15 levels were correlated with those of TRAIL and IDO in HIV-1 viremic patients. Lastly, ISG15 SNPs had no influence on ISG15 levels. We demonstrates that ISG15 is elevated in viremic HIV-1 patients and is associated with high TRAIL and IDO levels.

HIV-1 induces in vivo platelet activation by enhancing platelet NOX2 activity.

OBJECTIVES: HIV-1 patients show increased platelet activation, but the mechanisms involved are not completely clarified. We speculated that HIV-1 might induce in vivo platelet activation by enhancing platelet NOX2-related oxidative stress. METHODS: We measured soluble CD40 Ligand (sCD40L), a systemic marker of platelet activation, in 36 HIV-1 patients under effective combined antiretroviral therapy (cART) and in 10 naïve HIV-1 subjects. As control, 20 healthy subjects (HS) were included. Platelet oxidative stress was measured by platelet NOX2-derived peptide (sNOX2-dp), p47(phox) translocation to platelet membrane and platelet prostaglandin F2α (8-iso-PGF2α). RESULTS: sCD40L was increased both in HIV-1 naïve and cART patients compared to HS (p < 0.001). Platelet sNOX2-dp and 8-iso-PGF2α were significantly higher in HIV-1 naïve subjects compared to those on cART and to HS, and both were mutually correlated (R = 0.734, p < 0.001). A stepwise multivariable linear regression analysis showed that platelet sNOX2-dp (ß: 0.803, p < 0.001), HIV-1 infection (ß: 0.146, p = 0.014) and age (ß: 0.166, p = 0.001) were independently associated to sCD40L levels. CONCLUSIONS: HIV-1 infection is associated with increased platelet oxidative stress, which was related to the activation of NOX2. The independent association between platelet NOX2 activation and plasma levels of sCD40L suggest that in vivo platelet activation may be dependent upon platelet oxidative stress.

Autoantibodies to estrogen receptor α in systemic sclerosis (SSc) as pathogenetic determinants and markers of progression.

Systemic sclerosis (SSc) is a multisystem autoimmune disease of unknown etiology characterized by inflammation, autoantibody production, and fibrosis. It predominantly affects women, this suggesting that female sex hormones such as estrogens may play a role in disease pathogenesis. However, up to date, the role of estrogens in SSc has been scarcely explored. The activity of estrogens is mediated either by transcription activity of the intracellular estrogen receptors (ER), ERα and ERß, or by membrane-associated ER. Since the presence of autoantibodies to ERα and their role as estrogen agonists interfering with T lymphocyte homeostasis were demonstrated in other autoimmune diseases, we wanted to ascertain whether anti-ERα antibodies were detectable in sera from patients with SSc. We detected anti-ERα antibody serum immunoreactivity in 42% of patients with SSc (30 out of 71 analyzed). Importantly, a significant association was found between anti-ERα antibody values and key clinical parameters of disease activity and severity. Fittingly, anti-ERα antibody levels were also significantly associated with alterations of immunological features of SSc patients, including increased T cell apoptotic susceptibility and changes in T regulatory cells (Treg) homeostasis. In particular, the percentage of activated Treg (CD4(+)CD45RA(-) FoxP3(bright)CD25(bright)) was significantly higher in anti-ERα antibody positive patients than in anti-ERα antibody negative patients. Taken together our data clearly indicate that anti-ERα antibodies, probably via the involvement of membrane-associated ER, can represent: i) promising markers for SSc progression but, also, ii) functional modulators of the SSc patients' immune system.

Altered clonogenic capability and stromal cell function characterize bone marrow of HIV-infected subjects with low CD4+ T cell counts despite viral suppression during HAART.

BACKGROUND: Inflammatory cytokines in bone marrow may impair hematolymphopoiesis in human immunodeficiency virus (HIV)-infected subjects who do not experience reconstitution of CD4(+) T cells despite suppression of virus replication while receiving highly active antiretroviral therapy (HAART) (immunological nonresponders). METHODS: Bone marrow samples from 12 immunological nonresponders receiving HAART were studied and compared with samples from 11 immunological responders. The mean CD4(+) T cell count (+/- standard deviation) was 174 +/- 68 cells/mm(3) and plasma HIV RNA levels had been <50 copies/mL for at least 1 year for individuals enrolled in the study. The clonogenic capability of bone marrow samples was evaluated using the colony forming cell assay and the long-term culture-initiating cell assay. CD34(+) cells from the colony forming cell assay were pooled for real-time polymerase chain reaction analysis of Fas and Fas ligand. Bone marrow cytokine production (interleukin-2 and tumor necrosis factor-alpha) and stromal interleukin-7 levels were analyzed by enzyme-linked immunosorbent assay in both groups. Flow cytometric analysis of CD4(+) and CD8(+) T cell subsets was performed. RESULTS: A reduced clonogenic capability and a decrease in the level of more primitive progenitor cells were observed in parallel with lower production of interleukin-2 and increased tumor necrosis factor-alpha levels. A significant upregulation of Fas and Fas ligand on CD34(+) cells and a higher stromal interleukin-7 production were observed. Impairment of the naive T cell compartment and persistent T cell activation were observed in peripheral blood. CONCLUSIONS: Samples from immunological nonresponders show reduced growth of in vitro colonies and an altered cytokine production in bone marrow. The cytokine pattern observed and the altered Fas and Fas ligand pathway may determine stem cell apoptosis and low CD4(+) cell recovery. These features, which are similar to those observed in HIV-infected subjects before starting therapy, persist despite treatment.

Comparison of a rule-based algorithm with a phenotype-based algorithm for the interpretation of HIV genotypes in guiding salvage regimens in HIV-infected patients by a randomized clinical trial: the mutations and salvage study.

BACKGROUND: There is still considerable uncertainty as to the best algorithm for interpreting human immunodeficiency virus (HIV) genotyping results. METHODS: A total of 318 subjects with HIV RNA levels of >1000 copies/mL were enrolled in 41 centers throughout Italy from 2001 through 2003, stratified on the basis of their drug history, randomized (1:1) to 2 arms to have their treatments modified on the basis of the results of HIV genotyping (as interpreted by virtual phenotype analysis or with use of a rule-based interpretation system), and followed up for 48 weeks. At least 1 nucleoside reverse-transcriptase inhibitor and 1 protease inhibitor had to be included in any new regimen; nonnucleoside reverse-transcriptase inhibitor-naive patients were also prescribed a nonnucleoside reverse-transcriptase inhibitor. Only drugs licensed in Italy were allowed. The primary end point was a decrease in HIV RNA level to <400 copies/mL by week 12 according to on-treatment analysis. RESULTS: The mean (+/- standard deviation) values at baseline were as follows: HIV RNA level, 4.1+/-0.74 log(10) copies/mL; CD4(+) T lymphocyte count, 410+/-262 cells/microL; reverse-transcriptase mutations, 4.8+/-2.9; and protease mutations, 2.8+/-2.5. There were 133 patients (41.8%) who were nonnucleoside reverse-transcriptase inhibitor naive and protease inhibitor experienced, 63 patients (19.8%) who were nonnucleoside reverse-transcriptase inhibitor experienced and protease inhibitor naive, and 122 patients (38.4%) who were 3-class experienced. A total of 192 patients completed 12 weeks of the treatment regimen assigned at baseline; at 12 weeks, 66.3% of patients in the virtual phenotype arm and 71.3% of patients in the rule-based interpretation arm had HIV RNA levels of <400 copies/mL (P = .46). No statistically significant difference between arms was observed by intention-to-treat analysis. CONCLUSION: Both the virtual phenotype and rule-based interpretation methods of HIV genotyping can guide the selection of effective antiretroviral drugs for a salvage regimen.

Immunodysregulation of HIV disease at bone marrow level.

Hematological abnormalities frequently occur in patients infected with HIV-1. Increasing evidence indicates that bone marrow (BM) suppression results from viral infection of accessory cells, with impaired stromal function and alteration of hematopoietic growth factor network. We investigated the effects of antiretroviral therapy on cytokine and chemokine production by BM cells and stromal cells, in a group of HIV-1-infected subjects before and during treatment. Compared with uninfected controls, an altered cytokine and chemokine production by BM cells has been observed before treatment, characterised by decreased IL-2 and elevated TNF-alpha, MIP-1alpha, MIP-1beta, and RANTES levels, along with a defective BM clonogenic activity. Antiretroviral therapy determined an amelioration of stem cell activity, a restoration of stromal cell pattern and functions, and an increased IL-2 production at BM level and a decrease of Fas expression on progenitor cells, in parallel with the diminution of TNF-alpha levels. HIV-1 protease inhibitors (PIs) may improve hematopoietic functions owing to their direct effects on the BM progenitor cells. Ritonavir and indinavir increased the colony growth of BM obtained either from HIV-1-infected patients or from normal individuals, in parallel with the normalization of functional and morphologic characteristics of stromal cells.

Bone marrow clonogenic capability, cytokine production, and thymic output in patients with common variable immunodeficiency.

In patients with primary Ab deficiencies, hematological and immunological abnormalities are frequently observed. A regenerative failure of hemopoietic stem/progenitor cells has been hypothesized. We evaluated in the bone marrow (BM) of 11 patients with common variable immunodeficiency, the phenotype of BM progenitors and their in vitro growth by colony-forming cell (CFC) and long-term culture (LTC) assays. A significant decrease in erythroid and mixed CFC and, to a greater extent, in primitive LTC-CFC progenitors was observed in patients compared with healthy controls. The frequency of BM pre-B and pro-B cells correlated directly with the absolute number of CD19+ lymphocytes. BM cells cultured in vitro produced spontaneously lower amounts of IL-2 and elevated levels of TNF-alpha compared with controls, indicating a skewing toward a proapoptotic cytokine pattern. In addition, stromal cells generated after BM LTC secreted less IL-7 and displayed by immunohistochemistry an altered phenotype. These findings were associated with a significant decrease in naive Th cells coexpressing CD31 in the peripheral blood. These results indicate an impaired growth and differentiation capacity of progenitor cells in patients with common variable immunodeficiency.

Idiopathic CD4+ lymphocytopenia may be due to decreased bone marrow clonogenic capability.

BACKGROUND: Idiopathic CD4+ lymphocytopenia is defined by a stable decrease of CD4+ T cells in the absence of any known cause of immune deficiency. The mechanisms responsible for the immunological impairment are still unknown, but a regenerative failure of hematopoietic stem/progenitor cells has been hypothesized. METHODS: We evaluated in the bone marrow (BM) of 5 patients with idiopathic CD4+ lymphocytopenia the phenotype of BM progenitor cells, their differentiation capacity with colony-forming cells and long-term culture-initiating cell assays, in parallel with the spontaneous IL-7 production in the patient sera. RESULTS: Compared with controls, a regenerative failure of hematopoietic stem cells has been observed, both in 'committed' and in 'uncommitted' progenitor cells, despite high IL-7 serum levels. The percentage of phenotypically primitive CD34+CD38-DR+ cells (this includes the lymphoid precursor cells) was decreased, suggesting an involvement of the more primitive BM compartment in the de novo T cell generation. CONCLUSIONS: Despite the low number of patients, due to the low incidence of the disease, the decrease of primitive precursors sustains the possibility that diminished stem cell precursors might contribute to the development of CD4+ T cell depletion.

HIV type 1 protease inhibitors enhance bone marrow progenitor cell activity in normal subjects and in HIV type 1-infected patients.

HIV-1 protease inhibitors (PIs) may improve hematopoietic functions owing to their direct effects on bone marrow (BM) progenitor cells. In this study we investigated this hypothesis evaluating the effect of adding ritonavir (RTV) and indinavir (IND) on hematopoietic colony formation assays by colony-forming cell (CFC) and long-term culture-initiating cell (LTC-IC) assays, on apoptosis, on cytokine production and stromal cells, in subjects with HIV-1 infection, and in seronegative controls. After PI addition, CFC and LTC-IC assays in HIV-1-infected patients showed levels of colony growth significantly higher than those observed at baseline; the same PI activity on colony formation was observed in healthy subjects. No significant modifications on Fas, the membrane form of Fas (mFas) and Fas-ligand (FasL) expression, and on cytokine production were observed at BM level after the addition of PIs. At baseline, in HIV-1-infected patients, the majority of the stromal cells appeared as large and rounded, whereas after the addition of RTV or IND the stromal cells exhibited a "fibroblast-like" morphology and produced higher stem cell factor (SCF) and lower MIP-1alpha levels when compared with the stromal production without the addition of IND. RTV and IND increased colony growth of BM obtained either from HIV-1-infected patients or from normal individuals, in parallel with the normalization of functional and morphological characteristics of stromal cells.

Decreased apoptosis of bone marrow progenitor cells in HIV-1-infected patients during highly active antiretroviral therapy.

Impaired haematopoiesis during HIV-1 infection may be caused by the overproduction of inflammatory cytokines by immune cells at the bone marrow level inducing Fas-mediated apoptosis of stem progenitors. In this study, we evaluated the effects of highly active antiretroviral therapy on apoptosis of CD34+ stem cells derived from the bone marrow of HIV-1-infected patients, and observed decreased Fas expression on progenitor cells, in parallel with the diminution of TNF-alpha levels and the amelioration of clonogenic parameters.

Improvement of interleukin 2 production, clonogenic capability and restoration of stromal cell function in human immunodeficiency virus-type-1 patients after highly active antiretroviral therapy.

Haematological abnormalities frequently occur in patients infected by human immunodeficiency virus-type 1 (HIV-1). Increasing evidence indicates that bone marrow suppression (BM) results from viral infection of accessory cells, with impaired stromal function and alteration of haematopoietic growth factor network. We have investigated the effects of antiretroviral therapy on cytokine and chemokine production by BM cells and stromal cells in a group of HIV-1-infected subjects before and during treatment. Compared with uninfected controls, an altered cytokine and chemokine production by BM cells was observed before treatment, characterized by decreased interleukin 2 (IL-2) and elevated tumour necrosis factor-alpha, macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, and RANTES (regulated on activation, normal T cell-expressed and secreted) levels, along with a defective BM clonogenic activity. Antiretroviral therapy showed increased BM clonogenic capability, associated with normalization of IL-2 production and chemokine receptors expression on CD34+ cells. Pre-therapy, BM accessory cells were represented by macrophage-like cells, in some cases positive for HIV-1 DNA, suggesting that these cells are the main target of HIV-1 infection. During therapy, the stromal cells became predominantly fibroblastoid-like, as observed in normal controls, and were negative for HIV-1 DNA. Controlling HIV-1 replication may produce amelioration of stem cell activity, and restoration of stromal cell pattern and functions, with increased IL-2 production at BM level.