Age differentially impacts adaptive immune responses induced by adenoviral versus mRNA vaccines against COVID-19.

Adenoviral and mRNA vaccines encoding the viral spike (S) protein have been deployed globally to contain severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Older individuals are particularly vulnerable to severe infection, probably reflecting age-related changes in the immune system, which can also compromise vaccine efficacy. It is nonetheless unclear to what extent different vaccine platforms are impacted by immunosenescence. Here, we evaluated S protein-specific immune responses elicited by vaccination with two doses of BNT162b2 or ChAdOx1-S and subsequently boosted with a single dose of BNT162b2 or mRNA-1273, comparing age-stratified participants with no evidence of previous infection with SARS-CoV-2. We found that aging profoundly compromised S protein-specific IgG titers and further limited S protein-specific CD4+ and CD8+ T cell immunity as a probable function of progressive erosion of the naive lymphocyte pool in individuals vaccinated initially with BNT162b2. Our results demonstrate that primary vaccination with ChAdOx1-S and subsequent boosting with BNT162b2 or mRNA-1273 promotes sustained immunological memory in older adults and potentially confers optimal protection against coronavirus disease 2019.

New Scaffolds of Proteasome Inhibitors: Boosting Anticancer Potential by Exploiting the Synergy of In Silico and In Vitro Methodologies.

Cancer is a complex multifactorial disease whose pathophysiology involves multiple metabolic pathways, including the ubiquitin-proteasome system, for which several proteasome inhibitors have already been approved for clinical use. However, the resistance to existing therapies and the occurrence of severe adverse effects is still a concern. The purpose of this study was the discovery of novel scaffolds of proteasome inhibitors with anticancer activity, aiming to overcome the limitations of the existing proteasome inhibitors. Thus, a structure-based virtual screening protocol was developed using the structure of the human 20S proteasome, and 246 compounds from virtual databases were selected for in vitro evaluation, namely proteasome inhibition assays and cell viability assays. Compound 4 (JHG58) was shortlisted as the best hit compound based on its potential in terms of proteasome inhibitory activity and its ability to induce cell death (both with IC50 values in the low micromolar range). Molecular docking studies revealed that compound 4 interacts with key residues, namely with the catalytic Thr1, Ala20, Thr21, Lys33, and Asp125 at the chymotrypsin-like catalytic active site. The hit compound is a good candidate for additional optimization through a hit-to-lead campaign.

Ageing Curtails the Diversity and Functionality of Nascent CD8+ T Cell Responses against SARS-CoV-2.

Age-related changes in the immune system are thought to underlie the vulnerability of elderly individuals to emerging viral diseases, such as coronavirus disease 2019 (COVID-19). In this study, we used a fully validated in vitro approach to determine how age impacts the generation of de novo CD8+ T cell responses against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of COVID-19. Our data revealed a generalized deficit in the ability of elderly individuals to prime the differentiation of naïve precursors into effector CD8+ T cells defined by the expression of interferon (IFN)-γ and the transcription factor T-bet. As a consequence, there was an age-related decline in the diversity of newly generated CD8+ T cell responses targeting a range of typically immunodominant epitopes derived from SARS-CoV-2, accompanied by an overall reduction in the expression frequency of IFN-γ. These findings have potential implications for the development of new strategies to protect the elderly against COVID-19.

Effects of the age of vaccination on the humoral responses to a human papillomavirus vaccine.

Adult vaccination programs are receiving increasing attention however, little is known regarding the impact of age on the maintenance of the immune response. We investigated this issue in the context of a human papillomavirus (HPV) vaccination program collecting real-world data on the durability of humoral immunity in 315 female subjects stratified according to vaccination age (adolescents and adults) and sampled at early or late time points after the last vaccine dose. HPV-specific IgGs, but not memory B cells, were induced and maintained at higher levels in subjects vaccinated during adolescence. Nonetheless, antibody functions waned over time to a similar degree in adolescents and adults. To shed light on this phenomena, we analyzed quantitative and qualitative properties of lymphocytes. Similar biochemical features were observed between B-cell subsets from individuals belonging to the two age groups. Long term humoral responses toward vaccines administered at an earlier age were comparably maintained between adolescents and adults. The percentages of naïve B and CD4+ T cells were significantly higher in adolescents, and the latter directly correlated with IgG titers against 3 out of 4 HPV types. Our results indicate that age-specific HPV vaccine responsiveness is mostly due to quantitative differences of immune cell precursors rather than qualitative defects in B cells. In addition, our results indicate that adults also have a good humoral immunogenic profile, suggesting that their inclusion in catch-up programmes is desirable.

Altered Basal Lipid Metabolism Underlies the Functional Impairment of Naive CD8+ T Cells in Elderly Humans.

Aging is associated with functional deficits in the naive T cell compartment, which compromise the generation of de novo immune responses against previously unencountered Ags. The mechanisms that underlie this phenomenon have nonetheless remained unclear. We found that naive CD8+ T cells in elderly humans were prone to apoptosis and proliferated suboptimally in response to stimulation via the TCR. These abnormalities were associated with dysregulated lipid metabolism under homeostatic conditions and enhanced levels of basal activation. Importantly, reversal of the bioenergetic anomalies with lipid-altering drugs, such as rosiglitazone, almost completely restored the Ag responsiveness of naive CD8+ T cells. Interventions that favor lipid catabolism may therefore find utility as adjunctive therapies in the elderly to promote vaccine-induced immunity against targetable cancers and emerging pathogens, such as seasonal influenza viruses and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

Design, Synthesis and Evaluation of New Multifunctional Benzothiazoles as Photoprotective, Antioxidant and Antiproliferative Agents.

A current trend of research in the health field is toward the discovery of multifunctional compounds, capable of interacting with multiple biological targets, thus simplifying multidrug therapies and improving patient compliance. The aim of this work was to synthesize new multifunctional chemical entities bearing a benzothiazole nucleus, a structure that has attracted increasing interest for the great variety of biological actions that it can perform, and already used as a scaffold in several multifunctional drugs. Compounds are reported, divided into two distinct series, synthetized and tested in vitro for the antioxidant, and include UV-filtering and antitumor activities. DPPH and FRAP tests were chosen to outline an antioxidant activity profile against different radical species. The UV-filtering activity was investigated, pre- and post-irradiation, through evaluation of a O/W sunscreen standard formulation containing 3% of the synthetic compounds. The antitumor activity was investigated both on human melanoma cells (Colo-38) and on immortalized human keratinocytes as a control (HaCat). A good antiproliferative profile in terms of IC50 was chosen as a mandatory condition to further investigate apoptosis induction as a possible cytotoxicity mechanism through the Annexin V test. Compound BZTcin4 was endowed with excellent activity and a selectivity profile towards Colo-38, supported by a good antioxidant capacity and an excellent broad-spectrum photoprotective profile.

Impaired Priming of SARS-CoV-2-Specific Naive CD8+ T Cells in Older Subjects.

Advanced age is associated with severe symptoms and death upon SARS-CoV-2 infection. Virus-specific CD8+ T-cell responses have shown to be protective toward critical COVID-19 manifestations, suggesting that suboptimal cellular immunity may contribute to the age-pattern of the disease. The induction of a CD8+ T-cell response against an emerging pathogen like SARS-CoV-2 relies on the activation of naive T cells. To investigate whether the primary CD8+ T-cell response against this virus is defective in advanced age, we used an in vitro approach to prime SARS-CoV-2-specific naive CD8+ T cells from healthy, unexposed donors of different age groups. Compared to younger adults, older individuals display a poor SARS-CoV-2-specific T-cell priming capacity in terms of both magnitude and quality of the response. In addition, older subjects recognize a lower number of epitopes. Our results implicate that immune aging is associated with altered primary SARS-CoV-2-specific CD8+ T-cell responses.

Use of a Novel Peptide Welding Technology Platform for the Development of B- and T-Cell Epitope-Based Vaccines.

Peptide vaccines incorporating B- and T-cell epitopes have shown promise in the context of various cancers and infections. These vaccines are relatively simple to manufacture, but more immunogenic formulations are considered a priority. We developed tetrabranched derivatives for this purpose based on a novel peptide welding technology (PWT). PWTs provide molecular scaffolds for the efficient synthesis of ultrapure peptide dendrimers, which allow the delivery of multiple ligands within a single macromolecular structure. Peptide vaccines incorporating T-cell epitopes derived from melanoma and B-cell epitopes derived from human immunodeficiency virus, synthesized using this approach, elicited primary immune responses in vitro and in vivo. Subcutaneous administration of the B-cell epitope-based vaccines also elicited more potent humoral responses than subcutaneous administration of the corresponding peptides alone. Highly immunogenic peptide epitope-based vaccines can therefore be generated quickly and easily using a novel PWT.

The Tat Protein of HIV-1 Prevents the Loss of HSV-Specific Memory Adaptive Responses and Favors the Control of Viral Reactivation.

The development of therapeutic strategies to control the reactivation of the Herpes Simplex Virus (HSV) is an unaddressed priority. In this study, we evaluated whether Tat, a HIV-1 protein displaying adjuvant functions, could improve previously established HSV-specific memory responses and prevent viral reactivation. To this aim, mice were infected with non-lethal doses of HSV-1 and, 44 days later, injected or not with Tat. Mice were then monitored to check their health status and measure memory HSV-specific cellular and humoral responses. The appearance of symptoms associated with HSV-reactivation was observed at significantly higher frequencies in the control group than in the Tat-treated mice. In addition, the control animals experienced a time-dependent decrease in HSV-specific Immunoglobulin G (IgG), while the Tat-treated mice maintained antibody titers over time. IgG levels were directly correlated with the number of HSV-specific CD8+ T cells, suggesting an effect of Tat on both arms of the adaptive immunity. Consistent with the maintenance of HSV-specific immune memory, Tat-treated mice showed a better control of HSV-1 re-infection. Although further studies are necessary to assess whether similar effects are observed in other models, these results indicate that Tat exerts a therapeutic effect against latent HSV-1 infection and re-infection by favoring the maintenance of adaptive immunity.

HPV-Specific Systemic Antibody Responses and Memory B Cells are Independently Maintained up to 6 Years and in a Vaccine-Specific Manner Following Immunization with Cervarix and Gardasil in Adolescent and Young Adult Women in Vaccination Programs in Italy.

: Human papillomavirus (HPV) persistent infections are associated with cervical cancer and other HPV-related diseases and tumors. Thus, the characterization of long lasting immunity to currently available HPV vaccines is important. A total of 149 female subjects vaccinated with Cervarix or Gardasil participated to the study and they were stratified according to age (10-12-year-old and 16-20-year-old). Humoral immune responses (IgG and neutralizing antibody titers, antibody avidity) and circulating memory B cells were analyzed after an average of 4-6 years from the third immunization. The humoral responses against HPV-16 and HPV-18 (and HPV-6 and HPV-11 for Gardasil) were high in both age groups and vaccines up to six years from the third dose. However, Cervarix induced significantly higher and more persistent antibody responses, while the two vaccines were rather equivalent in inducing memory B cells against HPV-16 and HPV-18. Moreover, the percentage of subjects with vaccine-specific memory B cells was even superior among Gardasil vaccinees and, conversely, Cervarix vaccinated individuals with circulating antibodies, but undetectable memory B cells were found. Finally, a higher proportion of Cervarix-vaccinated subjects displayed cross-neutralizing responses against non-vaccine types HPV-31 and HPV-45. Gardasil and Cervarix may, thus, differently affect long-lasting humoral immunity from both the quantitative and qualitative point of view.

Synthesis and Biological Activity of Peptide α-Ketoamide Derivatives as Proteasome Inhibitors.

Proteasome activity affects cell cycle progression as well as the immune response, and it is largely recognized as an attractive pharmacological target for potential therapies against several diseases. Herein we present the synthesis of a series of pseudodi/tripeptides bearing at the C-terminal position different α-ketoamide moieties as pharmacophoric units for the interaction with the catalytic threonine residue that sustains the proteolytic action of the proteasome. Among these, we identified the 1-naphthyl derivative 13c as a potent and selective inhibitor of the ß5 subunit of the 20S proteasome, exhibiting nanomolar potency in vitro (ß5 IC50 = 7 nM, ß1 IC50 = 60 µM, ß2 IC50 > 100 µM). Furthermore, it significantly inhibited proliferation and induced apoptosis of the human colorectal carcinoma cell line HCT116.

The HIV-1 Tat protein affects human CD4+ T-cell programing and activation, and favors the differentiation of naïve CD4+ T cells.

OBJECTIVE: HIV infection is characterized by several immune dysfunctions, such as chronic activation of the immune system, premature aging and loss of CD4 T cells, in particular within the naïve compartment. The Tat protein of HIV is released extracellularly and enters neighboring cells affecting their functionality, for instance impacting on CD8 T-cell programs and activity. As the presence and/or induction of anti-Tat immune responses is associated with reduced T-cell dysfunction and CD4 T-cell loss, we investigated whether Tat impacts human resting or activated CD4 T cells. METHODS: Purified CD4 T cells were activated by T cell receptor engagement in the presence or absence of Tat. Cytokine production, surface phenotype and expression of transcription factors important for T-cell programing were measured. Purified naïve CD4 T cells were cultured in nonpolarizing conditions in the presence or absence of Tat and their proliferation and differentiation was evaluated. RESULTS: Tat favors the secretion of IL2, IFNγ and TNFα in CD4 T cells, as well as the upregulation of T-bet and Eomes expression. Naïve CD4 T cells cultured in the presence of Tat showed enhanced expansion and differentiation toward memory phenotype, showing in particular recruitment into the effector memory T-cell pool. CONCLUSION: Tat affects the programing and functionality of CD4 T lymphocytes favoring the differentiation of naïve CD4 T cells.

Rational Design of Nucleoside-Bile Acid Conjugates Incorporating a Triazole Moiety for Anticancer Evaluation and SAR Exploration.

Herein we report a study on the synthesis and biological evaluation of a library of nucleoside-bile acid conjugates prepared by combining 2'-deoxyadenosine, 2'-deoxyguanosine, 2'-deoxyuridine as well as adenosine and guanosine derivatives with cheno-, urso-, nor-cheno-, nor-urso- and taurourso-desoxycholic acid derivatives by means of the click reaction. The new nucleoside-bile acid conjugates incorporating a triazole moiety were tested in vitro against leukemic K562 and HCT116 colon carcinoma, as well as on normal fibroblast cells. Six compounds displayed interesting anti-proliferative activity against the selected cancer lines and no cytotoxic effects against normal fibroblasts. A possible structure activity relationship was also investigated.

Solid lipid nanoparticles for the delivery of 1,3,5-triaza-7-phosphaadamantane (PTA) platinum (II) carboxylates.

The use of solid lipid nanoparticles (SLN) is a promising route for the delivery of platinum complexes aimed to anticancer activity. This paper describes the production and characterization of SLN suitable for the loading of Pt complexes containing the biocompatible phosphine 1,3,5-triaza-7-phosphaadamantane (PTA) as neutral ligand. After a screening of several lipidic phases, stearic acid-based SLN were identified as the most appropriate for the purpose. They were produced by emulsion-dilution method and then characterized in terms of dimension, polydispersity, time stability, pH balance and morphological aspect. Stearic acid SLN are designed as a system able to coordinate to platinum, acting as anionic carboxylic ligands, replacing the base carbonate of the Pt synthon [PtCO3(DMSO)2], where also DMSO can subsequently be substituted by phosphinic ligands, namely PTA. SLN functionalised with Pt-PTA were produced and characterized by this synthetic route. The toxicity of plain SLN and the antiproliferative effect of SLN functionalised with Pt-PTA were evaluated on two human cancer cell lines K562 and A2780. The results indicate that SLN can be exploited as a delivery system for Pt complexes with potential anticancer activity.

NO Photoreleaser-Deoxyadenosine and -Bile Acid Derivative Bioconjugates as Novel Potential Photochemotherapeutics.

This contribution reports the synthesis of some novel bioconjugates with anticancer activity and able to release nitric oxide (NO) under visible light excitation. The 4-nitro-2-(trifluoromethyl)aniline derivative, a suitable NO photodonor, was conjugated with 2'-deoxyadenosine and urso- and cheno-deoxycholic acid derivatives, through a thioalkylic chain or the 4-alkyl-1,2,3-triazole moiety. Photochemical experiments demonstrated the effective release of NO from 2'-deoxyadenosine and ursodeoxycholic acid conjugates under the exclusive control of visible light inputs. Studies for the in vitro antiproliferative activity against leukemic K562 and colon carcinoma HCT116 cell lines are reported for all the compounds as well as a case study of photocytotoxicity against HCT116.

AGHLDDLPGALSAL: A hemoglobin fragment potentially competing with albumin to bind transition metal ions.

Protein degradation leads to the formation of endogenous peptides, the biological activity of which is most often unknown. The peptide AGHLDDLPGALSAL, recently isolated from mouse brain homogenates, has been recognized as a fragment of the α-chain of hemoglobin. AGHLDDLPGALSAL has the ability of inhibiting the peripheral hyperalgesic inflammatory responses through the indirect activation of the µ-opioid receptors. A peculiarity of AGHLDDLPGALSAL is the presence, at its N-terminus of a strong binding site for divalent transition metal ions, similar to that characterizing the human albumin and called "ATCUN motif". The consequential metal binding ability of AGHLDDLPGALSAL can be connected to its biological activity. For this reason, we decided to investigate the coordination properties of AGHLDDLPGALSAL towards Cu(II), Ni(II) and Zn(II) ions, reported here for the first time. The results confirm that AGHLDDLPGALSAL is a strong ligand for those metals: it can even compete with albumin under suitable conditions. In vitro assays on the inhibition of Cu(II) toxicity towards different cell lines confirmed that the binding ability of AGHLDDLPGALSAL can imply relevant biological consequences.

Systemic immunodominant CD8 responses with an effector-like phenotype are induced by intravaginal immunization with attenuated HSV vectors expressing HIV Tat and mediate protection against HSV infection.

Mucosal HSV infection remains a public health issue in developing and developed world. However, an effective vaccine is still missing, partly because of the incomplete knowledge of correlates of protection. In this study we have investigated the kinetics and quality of immunity elicited by an attenuated HSV1 vector expressing the immunomodulatory Tat protein of HIV-1 (HSV1-Tat). Animals were immunized by intravaginal (IVag) or intradermal (ID) route with HSV1-Tat or with a control HSV1 vector expressing the LacZ gene (HSV1-LacZ) and immune responses were characterized in different anatomical districts. IVag immunization with HSV1-Tat enhanced both expansion and memory phases of HSV-specific immunodominant CD8 responses at systemic, but not local, level and induced short- and long-term protection against mucosal challenge. Conversely, ID immunization with HSV1-Tat favored HSV-subdominant CD8 responses, which protected mice only at early time points after immunization. IVag immunization, in particular with HSV1-Tat, compared to ID immunization, induced the differentiation of CD8(+) T lymphocytes into short-lived effector (SLEC) and effector memory (Tem) cells, generating more robust recall responses associated with increased control of virus replication. Notably, systemic SLEC and Tem contributed to generate protective local secondary responses, demonstrating their importance for mucosal control of HSV. Finally, IgG responses were observed mostly in IVag HSV1-Tat immunized animals, although seemed dispensable for protection, which occurred even in few IgG negative mice. Thus, HSV1 vectors expressing Tat induce protective anti-HSV1 immune responses.

Studies of C-terminal naphthoquinone dipeptides as 20S proteasome inhibitors.

The ubiquitin proteasome pathway is crucial in regulating many processes in the cell. Modulation of proteasome activities has emerged as a powerful strategy for potential therapies against much important pathologies. In particular, specific inhibitors may represent a useful tool for the treatment of tumors. Here, we report studies of a new series of peptide-based analogues bearing a naphthoquinone pharmacophoric unit at the C-terminal position. Some derivatives showed inhibition in the µM range of the post-acidic-like and chymotrypsin-like active sites of the proteasome.

Development and characterization of K562 cell clones expressing BCL11A-XL: Decreased hemoglobin production with fetal hemoglobin inducers and its rescue with mithramycin.

Induction of fetal hemoglobin (HbF) is considered a promising strategy in the treatment of ß-thalassemia, in which production of adult hemoglobin (HbA) is impaired by mutations affecting the ß-globin gene. Recent results indicate that B-cell lymphoma/leukemia 11A (BCL11A) is a major repressor of γ-globin gene expression. Therefore, disrupting the binding of the BCL11A transcriptional repressor complex to the γ-globin gene promoter provides a novel approach for inducing expression of the γ-globin genes. To develop a cellular screening system for the identification of BCL11A inhibitors, we produced K562 cell clones with integrated copies of a BCL11A-XL expressing vector. We characterized 12 K562 clones expressing different levels of BCL11A-XL and found that a clear inverse relationship does exist between the levels of BCL11A-XL and the extent of hemoglobinization induced by a panel of HbF inducers. Using mithramycin as an inducer, we found that this molecule was the only HbF inducer efficient in rescuing the ability to differentiate along the erythroid program, even in K562 cell clones expressing high levels of BCL11A-XL, suggesting that BCL11A-XL activity is counteracted by mithramycin.

Incorporation of Naked Peptide Nucleic Acids into Liposomes Leads to Fast and Efficient Delivery.

The delivery of peptide nucleic acids (PNAs) to cells is a very challenging task. We report here that a liposomal formulation composed of egg PC/cholesterol/DSPE-PEG2000 can be loaded, according to different encapsulation techniques, with PNA or fluorescent PNA oligomers. PNA loaded liposomes efficiently and quickly promote the uptake of a PNA targeting the microRNA miR-210 in human erythroleukemic K562 cells. By using this innovative delivery system for PNA, down-regulation of miR-210 is achieved at a low PNA concentration.