Effective early antiretroviral therapy in perinatal-HIV infection reduces subsequent plasma inflammatory profile.

BACKGROUND: The long-term immunologic effects of antiretroviral therapy (ART) in children with perinatally-acquired HIV (PHIV) have not been fully elucidated. Here, we investigated how the timing of ART initiation affects the long-term immune profile of children living with PHIV by measuring immunomodulatory plasma cytokines, chemokines, and adenosine deaminases (ADAs). METHODS: 40 PHIV participants initiated ART during infancy. 39 participant samples were available; 30 initiated ART ≤6 months (early-ART treatment); 9 initiated ART >6 months and <2 years (late-ART treatment). We compared plasma cytokine and chemokine concentrations and ADA enzymatic activities between early-ART and late-ART treatment 12.5 years later and measured correlation with clinical covariates. RESULTS: Plasma concentrations of 10 cytokines and chemokines (IFNγ, IL-12p70, IL-13, IL-17A, IL-IRA, IL-5, IL-6, and IL-9 as well as CCL7, CXCL10), ADA1, and ADA total were significantly higher in late-ART compared to early-ART treatment. Furthermore, ADA1 was significantly positively correlated with IFNγ, IL-17A, and IL-12p70. Meanwhile, total ADA was positively correlated with IFNγ, IL-13, IL-17A, IL-1RA, IL-6, and IL-12p70 as well as CCL7. CONCLUSIONS: Elevation of several pro-inflammatory plasma analytes in late-ART despite 12.5 years of virologic suppression compared to early-ART treatment suggests that early treatment dampens the long-term plasma inflammatory profile in PHIV participants. IMPACT: This study examines differences in the plasma cytokine, chemokine, and ADA profiles 12.5 years after treatment between early (≤6months) and late (>6 months and <2 years) antiretroviral therapy (ART) treatment initiation in a cohort of European and UK study participants living with PHIV. Several cytokines and chemokines (e.g., IFNγ, IL-12p70, IL-6, and CXCL10) as well as ADA-1 are elevated in late-ART treatment in comparison to early-ART treatment. Our results suggest that effective ART treatment initiated within 6 months of life in PHIV participants dampens a long-term inflammatory plasma profile as compared to late-ART treatment.

Perinatally Human Immunodeficiency Virus-Infected Adolescents and Young Adults Demonstrate Distinct BNT162b2 Messenger RNA Coronavirus Disease 2019 Vaccine Immunogenicity.

BACKGROUND: Immunization of vulnerable populations with distinct immunity often results in suboptimal immunogenicity, durability, and efficacy. METHODS: Safety and immunogenicity profiles of BNT162b2 messenger RNA coronavirus disease 2019 (COVID-19) vaccine, among people living with human immunodeficiency virus (HIV), were evaluated in 28 perinatally HIV-infected patients under antiretroviral therapy (ART) and 65 healthy controls (HCs) with no previous history of COVID-19. Thus, we measured severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific humoral and CD4+ T cell responses. Samples were collected before vaccination (baseline, day [D] 0), at the second dose (D21), and at 4 weeks (D28) and 6 months (D180) after D0. Proteomic profiles at D0 and D28 were assessed with a multiplexed proximity extension assay (Olink) on plasma samples. RESULTS: All HIV-infected patients mounted similar anti-SARS-CoV-2 humoral responses to those of HCs, albeit with lower titers of anti-trimeric S at D28 (P = .01). Only peripheral blood mononuclear cells of HIV-infected patients demonstrated at D28 an impaired ability to expand their specific (CD40L+) CD4+ T-cell populations. Similar humoral titers were maintained between the 2 groups at 6-months follow-up. We additionally correlated baseline protein levels to either humoral or cellular responses, identifying clusters of molecules involved in immune response regulation with inverse profiles between the 2 study groups. CONCLUSIONS: Responses of ART-treated HIV-infected patients, compared to those of HCs, were characterized by distinct features especially within the proteomic compartment, supporting their eligibility to an additional dose, similarly to the HC schedule.

Determinants of B-Cell Compartment Hyperactivation in European Adolescents Living With Perinatally Acquired HIV-1 After Over 10 Years of Suppressive Therapy.

Background: Despite a successful antiretroviral therapy (ART), adolescents living with perinatally acquired HIV (PHIV) experience signs of B-cell hyperactivation with expansion of 'namely' atypical B-cell phenotypes, including double negative (CD27-IgD-) and termed age associated (ABCs) B-cells (T-bet+CD11c+), which may result in reduced cell functionality, including loss of vaccine-induced immunological memory and higher risk of developing B-cells associated tumors. In this context, perinatally HIV infected children (PHIV) deserve particular attention, given their life-long exposure to chronic immune activation. Methods: We studied 40 PHIV who started treatment by the 2nd year of life and maintained virological suppression for 13.5 years, with 5/40 patients experiencing transient elevation of the HIV-1 load in the plasma (Spike). We applied a multi-disciplinary approach including immunological B and T cell phenotype, plasma proteomics analysis, and serum level of anti-measles antibodies as functional correlates of vaccine-induced immunity. Results: Phenotypic signs of B cell hyperactivation were elevated in subjects starting ART later (%DN T-bet+CD11c+ p=0.03; %AM T-bet+CD11c+ p=0.02) and were associated with detectable cell-associated HIV-1 RNA (%AM T-bet+CD11c+ p=0.0003) and transient elevation of the plasma viral load (spike). Furthermore, B-cell hyperactivation appeared to be present in individuals with higher frequency of exhausted T-cells, in particular: %CD4 TIGIT+ were associated with %DN (p=0.008), %DN T-bet+CD11c+ (p=0.0002) and %AM T-bet+CD11c+ (p=0.002) and %CD4 PD-1 were associated with %DN (p=0.048), %DN T-bet+CD11c+ (p=0.039) and %AM T-bet+CD11c+ (p=0.006). The proteomic analysis revealed that subjects with expansion of these atypical B-cells and exhausted T-cells had enrichment of proteins involved in immune inflammation and complement activation pathways. Furthermore, we observed that higher levels of ABCs were associated a reduced capacity to maintain vaccine-induced antibody immunity against measles (%B-cells CD19+CD10- T-bet+, p=0.035). Conclusion: We identified that the levels of hyperactivated B cell subsets were strongly affected by time of ART start and associated with clinical, viral, cellular and plasma soluble markers. Furthermore, the expansion of ABCs also had a direct impact on the capacity to develop antibodies response following routine vaccination.

Higher PIK3C2B gene expression of H1N1+ specific B-cells is associated with lower H1N1 immunogenicity after trivalent influenza vaccination in HIV infected children.

Perinatally HIV-infected children (PHIV), despite successful antiretroviral therapy, present suboptimal responses to vaccinations compared to healthy-controls (HC). Here we investigated phenotypic and transcriptional signatures of H1N1-specific B-cells (H1N1-Sp) in PHIV, differentially responding to trivalent-influenza-vaccine (TIV), and HC. Patients were categorized in responders (R) and non-responders (NR) according to hemagglutination-inhibition-assay at baseline and 21 days after TIV. No differences in H1N1-Sp frequencies were found between groups. H1N1-Sp transcriptional analysis revealed a distinct signature between PHIV and HC. NR presented higher PIK3C2B and NOD2 expression compared to R, confirmed by downregulation of PIK3C2B in resting-memory of R after H1N1 in-vitro stimulation. In conclusion this study confirms that qualitative rather than quantitative analyses are needed to characterize immune responses in PHIV. These results further suggest that higher PIK3C2B in H1N1-Sp of NR is associated with lower H1N1 immunogenicity and may be targeted by future modulating strategies to improve TIV responses in PHIV.

Enhancing the Secretion of a Glyco-Engineered Anti-CD20 scFv-Fc Antibody in Hairy Root Cultures.

Hairy root (HR) cultures represent an attractive platform for the production of heterologous proteins, due to the possibility of secreting the molecule of interest in the culture medium. The main limitation is the low accumulation yields of heterologous proteins. The aim of this study is to enhance the accumulation of a tumor-targeting antibody with a human-compatible glycosylation profile in HR culture medium. To this aim, the authors produce Nicotiana benthamiana HR cultures expressing the red fluorescent protein (RFP) to easily screen for different auxins able to induce heterologous protein secretion in the medium. The hormone 2,4-dichlorophenoxyacetic acid (2,4-D) is found to induce high accumulation levels (334 mg L-1 ) of RFP in the culture medium. The same protocol is used to improve the secretion of the tumor-targeting, CD20-specific 2B8-FcΔXF recombinant antibody from glyco-engineered ΔXTFT N. benthamiana HR cultures. The addition of 2,4-D determine a 28-fold increase of the accumulation of fully functional 2B8-FcΔXF in the culture medium, at levels of ≈16 mg L-1 . Antibody N-glycosylation profiling reveal the prominent occurrence of GnGn structures and low levels of xylose- and fucose-containing counterparts. This result is the first example of the expression of an engineered anti-CD20 antibody with a scFv-Fc format at high levels in HR.

N-glycan engineering of a plant-produced anti-CD20-hIL-2 immunocytokine significantly enhances its effector functions.

Anti-CD20 recombinant antibodies are among the most promising therapeutics for the treatment of B-cell malignancies such as non-Hodgkin lymphomas. We recently demonstrated that an immunocytokine (2B8-Fc-hIL2), obtained by fusing an anti-CD20 scFv-Fc antibody derived from C2B8 mAb (rituximab) to the human interleukin 2 (hIL-2), can be efficiently produced in Nicotiana benthamiana plants. The purified immunocytokine (IC) bearing a typical plant protein N-glycosylation profile showed a CD20 binding activity comparable to that of rituximab and was efficient in eliciting antibody-dependent cell-mediated cytotoxicity (ADCC) of human PBMC against Daudi cells, indicating its fuctional integrity. In this work, the immunocytokine devoid of the typical xylose/fucose N-glycosylation plant signature (IC-ΔXF) and the corresponding scFv-Fc-ΔXF antibody not fused to the cytokine, were obtained in a glyco-engineered ΔXylT/FucT N. benthamiana line. Purification yields from agroinfiltrated plants amounted to 20-35 mg/kg of leaf fresh weight. When assayed for interaction with FcγRI and FcγRIIIa, IC-ΔXF exhibited significantly enhanced binding affinities if compared to the counterpart bearing the typical plant protein N-glycosylation profile (IC) and to rituximab. The glyco-engineered recombinant molecules also exhibited a strongly improved ADCC and complement-dependent cytotoxicity (CDC). Notably, our results demonstrate a reduced C1q binding of xylose/fucose carrying IC and scFv-Fc compared to versions that lack these sugar moieties. These results demonstrate that specific N-glycosylation alterations in recombinant products can dramatically affect the effector functions of the immunocytokine, resulting in an overall improvement of the biological functions and consequently of the therapeutic potential.