The HIV-1 antibody response: a footprint of the viral reservoir in children vertically infected with HIV.

Several assays have been developed to measure and characterise the replication-competent HIV-1 reservoir, which constitutes the barrier to cure. To date, the application of these assays to studies in children and in limited-resource settings has been minimal, primarily because of their expense, the large required blood volumes, and labour-intensive technologies. For children vertically infected with HIV-1 who initiated suppressive antiretroviral therapy (ART) regimens in infancy, HIV-1-specific antibody concentrations are associated with viral persistence and could be used to estimate the size of the residual latent reservoir on ART. This strategy could be particularly useful for screening children on suppressive ART for enrolment into therapeutic vaccine trials and other protocols aimed at achieving HIV-1 remission.

Early antiretroviral therapy-treated perinatally HIV-infected seronegative children demonstrate distinct long-term persistence of HIV-specific T-cell and B-cell memory.

OBJECTIVE: To investigate long-term persistence of HIV-specific lymphocyte immunity in perinatally HIV-infected children treated within the first year of life. DESIGN: Twenty perinatally HIV-infected children who received ART therapy within the first year of life (early treated) and with stable viral control (>5 years) were grouped according to their serological response to HIV. METHODS: Western blot analysis and ELISA defined 14 HIV-seropositive and six seronegative patients. Frequencies of gp140-specific T-cell and B-cell, and T-cell cytokine production were quantified by flow cytometry in both seronegatives and seropositives. Transcriptional signatures in purified gp140-specific B-cell subsets, in response to in-vitro stimulation with HIV peptides was evaluated by multiplex RT-PCR. RESULTS: Gp140-specific T cells and B cells persist at similar levels in both groups. A higher production of IL-21 in gp140-specific T cells was found in seropositives vs. seronegatives (P = 0.003). Gene expression in switched IgM-IgD- gp140-specific memory B cells after stimulation with HIV peptides in vitro demonstrated a differential expression of genes involved in signal transduction and activation after BCR/TLR triggering and B-cell activation. Genes relating to antibody production (PRDM1) and T-B cognate stimulation (CXCR4, IL21R) were differentially induced after in-vitro stimulation in seronegatives vs. seropositives suggesting a truncated process of B-cell maturation. CONCLUSION: HIV-specific memory B and T cells persist in early treated regardless their serological status. Seronegatives and seropositives are distinguished by gp140-specific T-cell function and by distinct transcriptional signatures of gp140-specific B cells after in-vitro stimulation, presumably because of a different antigen exposure. Such qualitative insights may inform future immunotherapeutic interventions.

Impact of Early Antiretroviral Therapy Initiation on HIV-Specific CD4 and CD8 T Cell Function in Perinatally Infected Children.

Early initiation of antiretroviral therapy (ART) in vertically HIV-infected children limits the size of the virus reservoir, but whether the time of treatment initiation (TI) can durably impact host immune responses associated with HIV infection is still unknown. This study was conducted in PBMC of 20 HIV-infected virally suppressed children on ART (mean age 9.4 y), classified as early treated (ET; age at ART initiation ≤0.5 y, n = 14) or late treated (LT; age at ART initiation 1-10 y, n = 6). Frequencies and functions of Ag-specific CD4 (CD40L+) and CD8 (CD69+) T cells were evaluated by intracellular IL-2, IFN-γ, and TNF-α production with IL-21 in CD4 or CD107a, granzyme B and perforin in CD8 T cells following stimulation with HIV gp140 protein (ENV) or GAG peptides by multiparameter flow cytometry. ET showed a higher proportion of cytokine-producing ENV- and GAG-specific CD4 and CD8 T cells compared with LT. In particular, ET were enriched in polyfunctional T cells. RNA sequencing analysis showed upregulation of immune activation pathways in LT compared with ET. Our results suggest that timing of TI in HIV-infected children has a long-term and measurable impact on the quality of the HIV-specific T cell immune responses and transcriptional profiles of PBMC, reinforcing the importance of early TI.

Prolonged Impairment of Immunological Memory After Anti-CD20 Treatment in Pediatric Idiopathic Nephrotic Syndrome.

Anti-CD20 therapy is effective in idiopathic nephrotic syndrome (INS). However, transient or sustained hypogammaglobulinemia predisposing to an increased risk of infectious diseases can follow treatment in some patients. We analyzed the long-term effects of anti-CD20 therapy on immunological memory in 27 frequently-relapsing/steroid-dependent INS pediatric patients after more than 4 years from the first and at least 2 years from the last anti-CD20 infusion. Twenty-one INS children, never treated with anti-CD20 and under an intense oral immunosuppression with prednisone, mycophenolate mofetil, and calcineurin inhibitors were also included as control group. Levels of circulating B-cell subpopulations, total serum immunoglobulins and IgG and memory B cells directed against hepatitis B virus (HBV) and tetanus were determined and correlated with clinical characteristics. Nine patients never relapsed after more than 2 years from the last anti-CD20 administration (5 after the first, 3 after the second, and 1 after the fifth infusion). At last follow-up, most patients showed a complete recovery and normalization of total (27/27), transitional (27/27), and mature-naïve B cells (25/27). However, a sustained and significant reduction of total memory (20/27) and switched memory (21/27) B cells was found in most patients. 11/27 patients showed hypogammaglobulinemia at last follow-up and, among these, four presented with a severe hypogammaglobulinemia (IgG < 160 mg/dl). In contrast, no patient in the control group developed a severe hypogammaglobulinemia. Age at the time of first anti-CD20 administration was positively associated with IgG levels at last follow-up (p = 0.008); accordingly, younger patients had an increased risk of hypogammaglobulinemia (p = 0.006). Furthermore, severe hypogammaglobulinemia and delayed switched memory B-cell reconstitution were more frequent in non-relapsing patients. Reduced IgG levels against HBV and tetanus were observed at baseline and further declined at last follow-up. Antigen-specific memory B-cells were induced by re-immunization, but specific IgG titers remained low. In conclusion, anti-CD20 therapy can be disease-modifying in some INS patients. However, a prolonged impairment of immunological memory occurs frequently, independently from the number of anti-CD20 infusions, particularly in younger patients. Re-immunization may be necessary in these patients.

OMIC Technologies and Vaccine Development: From the Identification of Vulnerable Individuals to the Formulation of Invulnerable Vaccines.

Routine vaccination is among the most effective clinical interventions to prevent diseases as it is estimated to save over 3 million lives every year. However, the full potential of global immunization programs is not realised because population coverage is still suboptimal. This is also due to the inadequate immune response and paucity of informative correlates of protection upon immunization of vulnerable individuals such as newborns, preterm infants, pregnant women, and elderly individuals as well as those patients affected by chronic and immune compromising medical conditions. In addition, these groups are undervaccinated for a number of reasons, including lack of awareness of vaccine-preventable diseases and uncertainty or misconceptions about the safety and efficacy of vaccination by parents and healthcare providers. The presence of these nonresponders/undervaccinated individuals represents a major health and economic burden to society, which will become particularly difficult to address in settings with limited public resources. This review describes innovative and experimental approaches that can help identify specific genomic profiles defining nonresponder individuals for whom specific interventions might be needed. We will provide examples that show how such information can be useful to identify novel biomarkers of safety and immunogenicity for future vaccine trials. Finally, we will discuss how system biology "OMICs" data can be used to design bioinformatic tools to predict the vaccination outcome providing genetic and molecular "signatures" of protective immune response. This strategy may soon enable identification of signatures highly predictive of vaccine safety, immunogenicity, and efficacy/protection thereby informing personalized vaccine interventions in vulnerable populations.

Immune Activation, Inflammation, and Non-AIDS Co-Morbidities in HIV-Infected Patients under Long-Term ART.

Despite effective antiretroviral therapy (ART), people living with HIV (PLWH) still present persistent chronic immune activation and inflammation. This condition is the result of several factors including thymic dysfunction, persistent antigen stimulation due to low residual viremia, microbial translocation and dysbiosis, caused by the disruption of the gut mucosa, co-infections, and cumulative ART toxicity. All of these factors can create a vicious cycle that does not allow the full control of immune activation and inflammation, leading to an increased risk of developing non-AIDS co-morbidities such as metabolic syndrome and cardiovascular diseases. This review aims to provide an overview of the most recent data about HIV-associated inflammation and chronic immune exhaustion in PLWH under effective ART. Furthermore, we discuss new therapy approaches that are currently being tested to reduce the risk of developing inflammation, ART toxicity, and non-AIDS co-morbidities.

Human Immunodeficiency Virus (HIV)-Antibody Repertoire Estimates Reservoir Size and Time of Antiretroviral Therapy Initiation in Virally Suppressed Perinatally HIV-Infected Children.

BACKGROUND: Assays to estimate human immunodeficiency virus (HIV) reservoir size require large amounts of blood, which represents a drawback especially in pediatric settings. We investigated whether HIV-antibody repertoire could estimate the viral reservoir size. Moreover, we assessed the magnitude of HIV-antibody response as a predictor of time of antiretroviral therapy (ART) initiation. METHODS: Human immunodeficiency virus-antibody responses to 10 different viral proteins were evaluated by HIV Western blot (WB) kit and a WB score was assigned to each patient. Patients were classified in 2 subgroups based on the timing of ART initiation (early treated [ET], 0-24 weeks and late treated [LT], >24 weeks). Human immunodeficiency virus-deoxyribonucleic acid (DNA) was quantified using real-time quantitative polymerase chain reaction on total peripheral blood mononuclear cells. Logistic regression and principal component analysis were built on these data to test the ability of WB score to predict the expected value of HIV-DNA and the timing of ART initiation. RESULTS: Sixty-nine perinatally HIV-infected children were evaluated. Reduced HIV-specific antibody responses and lower size of HIV-DNA were observed in ET compared with LT patients (P < .001 and P = .02, respectively). We found that WB score correlates with HIV-DNA (P = .032) and timing of ART initiation (P < .001). Based on the logistic regression analysis, we found that WB score can predict the HIV-DNA size and the timing of ART initiation with an Akaike information criterion of -118.13 and -151.51, respectively. CONCLUSIONS: Western blot score can estimate HIV-DNA size and timing of ART initiation in long-term virally suppressed children. This rapid, inexpensive, and easily reproducible tool can provide useful information to identify potential candidates for HIV remission studies.

Quantitative Multiplexed Imaging Analysis Reveals a Strong Association between Immunogen-Specific B Cell Responses and Tonsillar Germinal Center Immune Dynamics in Children after Influenza Vaccination.

Generation of Ag-specific humoral responses requires the orchestrated development and function of highly specialized immune cells in secondary lymphoid organs. We used a multiparametric approach combining flow cytometry, confocal microscopy, and histocytometry to analyze, for the first time to our knowledge in children, tonsils from seasonal influenza-vaccinated children. We used these novel imaging assays to address the mucosal immune dynamics in tonsils investigating the spatial positioning, frequency, and phenotype of immune cells after vaccination. Vaccination was associated with a significantly higher frequency of follicular helper CD4 T cells compared with the unvaccinated control group. The imaging analysis revealed that potential suppressor (FOXP3hi) CD4 T cells are mainly located in extrafollicular areas. Furthermore, a significantly reduced frequency of both follicular and extrafollicular FOXP3hi CD4 T cells was found in the vaccine group compared with the control group. Levels of circulating CXCL13 were higher in those vaccinated compared with controls, mirroring an increased germinal center reactivity in the tonsils. Notably, a strong correlation was found between the frequency of tonsillar T follicular helper cells and tonsillar Ag-specific Ab-secreting cells. These data demonstrate that influenza vaccination promotes the prevalence of relevant immune cells in tonsillar follicles and support the use of tonsils as lymphoid sites for the study of germinal center reactions after vaccination in children.

Waning of vaccine-induced immunity to measles in kidney transplanted children.

Vaccine-preventable diseases are a significant cause of morbidity and mortality in solid organ transplant recipients who undergo immunosuppression after transplantation. Data on immune responses and long-term maintenance after vaccinations in such population are still limited.We cross-sectionally evaluated the maintenance of immune response to measles vaccine in kidney transplanted children on immunosuppressive therapy. Measles-specific enzyme-linked immunosorbent assay and B-cell enzyme-linked immunosorbent spot were performed in 74 kidney transplant patients (Tps) and in 23 healthy controls (HCs) previously vaccinated and tested for humoral protection against measles. The quality of measles antibody response was measured by avidity test. B-cell phenotype, investigated via flow cytometry, was further correlated to the ability of Tps to maintain protective humoral responses to measles over time.We observed the loss of vaccine-induced immunity against measles in 19% of Tps. Nonseroprotected children showed signs of impaired B-cell distribution as well as immune senescence and lower antibody avidity. We further reported as time elapsed between vaccination and transplantation, as well as the vaccine administration during dialysis are clinical factors affecting the maintenance of the immune memory response against measles.Tps present both quantitative and qualitative alterations in the maintenance of protective immunity to measles vaccine. Prospective studies are needed to optimize the vaccination schedules in kidney transplant recipients in order to increase the immunization coverage over time in this population.

Sesquiterpene compounds from Inula viscosa.

Two new compounds, 2,5-dihydroxyisocostic acid and 2,3-dihydroxycostic acid together with three known sesquiterpene compounds, Isocostic acid, Carabrone and Tomentosin, have been isolated from the acetone extract of Inula viscosa (L.) Aiton. The structures of all new compounds were determined by spectroscopic methods, in particular 1D and 2D (1)H- and (13)C-NMR. The (13)C-NMR spectra of Isocostic acid and of Tomentosin are reported here for the first time.