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Negative-stranded RNA viruses (NSVs) are important human pathogens, including emerging and reemerging viruses that cause respiratory, hemorrhagic and other severe illnesses. Vaccine design traditionally relies on the viral surface glycoproteins. However, surface glycoproteins rarely elicit effective long-term immunity due to high variability. Therefore, an alternative approach is to include conserved structural proteins such as nucleoprotein (NP). NP is engaged in myriad processes in the viral life cycle: coating and protection of viral RNA, regulation of transcription/replication processes and induction of immunosuppression of the host. A broad heterosubtypic T-cellular protection was ascribed very early to this protein. In contrast, the understanding of the humoral immunity to NP is very limited in spite of the high titer of non-neutralizing NP-specific antibodies raised upon natural infection or immunization. In this review, the data with important implications for the understanding of the role of NP in the immune response to human NSVs are revisited. Major implications of the elicited T-cell immune responses to NP are evaluated, and the possible multiple mechanisms of the neglected humoral response to NP are discussed. The intention of this review is to remind that NP is a very promising target for the development of future vaccines.

BackgroundThe pathophysiology of long-COVID remains unknown, and information is particularly limited for symptoms of very long duration. We aimed to assess the serological, T-cell immune responses and ANA titers of patients with long-COVID-19 syndrome of 1-year duration.MethodsProspective, longitudinal study of hospitalized COVID-19 patients followed-up for 12 months. Sequential blood samples and COVID-19 symptom questionnaires (CSQ) were obtained, and humoral and cellular immune responses, antinuclear antibodies (ANA) and inflammation biomarkers were analyzed.ResultsOf 154 patients discharged from hospital, 72 non-vaccinated with available CSQ in all visits were included. Of them, 14 (19.4%) reported persistent symptoms both at 6-months and 12-months, mainly asthenia (15.3%), myalgia (13.9%), and difficulty concentrating/memory loss (13.9%). Symptomatic patients were more frequently women, smokers, showed higher WHO severity score, and a trend to higher ICU admission. In the adjusted analysis, long-COVID syndrome was associated with lower frequency of detectable neutralizing antibodies (adjusted hazard ratio [aHR] 0.98; 95% confidence interval [CI], 0.97-0.99) and lower SARS-CoV-2-S1/S2 titers (aHR [95%CI] 0.14 [0.03–0.65]). T-cell immune response measured with a SARS-CoV-2-interferon-γ release assay was not different between groups. There was a higher frequency of positive ANA titers (≥160) in symptomatic patients (57.1% vs 29.3%, p=0.04), that was attenuated after adjustment aHR [95% CI] 3.37 [0.84-13.57], p=0.087. Levels of C-reactive protein and D-dimer were higher during follow-up in symptomatic patients, but with no differences at 12 months.ConclusionPatients with 1-year duration long-COVID-19 syndrome exhibit a distinct immunologic phenotype that includes a poorer SARS-CoV-2 antibody response, low-degree chronic inflammation that tends to mitigate, and autoimmunity.

Macrophages have an affinity to developing tumors and have been shown to play a role in tumor combat and immune surveillance. However, the exact mechanism by which macrophages participate in the anti-tumor immune response remains unclear. Hence, the current study aimed to identify the effect of macrophages on gastric cancer (GC) cells via exosomes. Paired cancerous, tumor-adjacent, and non-cancerous stomach tissues were initially from 68 GC patients. T cells were isolated from peripheral blood mononuclear cells (PBMCs) obtained from both the GC patients as well as the healthy donors. Next, the exosomes were isolated from LPS and IFN-γ-induced PBMCs (M1 macrophages) and co-cultured with human GC cells. Another co-culture system comprised of CD3 + T cells and exosomes-treated GC cells was then performed. BALB/c mice and NOD/SCID nude mice were prepared for effects of exosomal miR-16-5p on tumor growth and anti-tumor immune response in GC in vivo . A relationship between M1 macrophages and the poor survival of GC patients was identified, while they secreted exosomes to inhibit GC development and activate a T cell-dependent immune response. Our results revealed that miR-16-5p was transferred intercellularly from M1 macrophages to GC cells via exosomes and targeted PD-L1. M1 macrophage-derived exosomes containing miR-16-5p were found to trigger a T cell immune response which inhibited tumor formation both in vitro and in vivo by decreasing the expression of PD-L1. Taken together, the key findings of the current study suggest that M1 macrophage-derived exosomes carrying miR-16-5p exert an inhibitory effect on GC progression through activation of T cell immune response via PD-L1. Our study highlights the promise of M1 macrophages as a potential cell-based therapy for GC treatment by increasing miR-16-5p in exosomes.

Immunomodulatory/immunosuppressive activity of multiple sclerosis (MS) disease modifying therapies (DMTs) might affect immune responses to SARS-CoV-2 exposure or vaccination in patients with MS (PwMS). We evaluated the effect of DMTs on humoral and cell-mediated immune responses to 2 and 3 vaccinations and the longevity of SARS-Cov-2 IgG levels in PwMS. 522 PwMS and 68 healthy controls vaccinated with BNT162b2-Pfizer mRNA vaccine against SARS-CoV-2, or recovering from COVID-19, were recruited in a nation-wide multi-center study. Blood was collected at 3 time-points: 2-16 weeks and ~6 months post 2 vaccination and 1-16 weeks following 3 vaccination. Serological responses were measured by quantifying IgG levels against the spike-receptor-binding-domain of SARS-CoV-2, and cellular responses (in a subgroup analysis) by quantifying IFNγ secretion in blood incubated with COVID-19 spike-antigen. 75% PwMS were seropositive post 2 or 3 vaccination. IgG levels decreased by 82% within 6 months from vaccination (p<0.0001), but were boosted 10.3 fold by the 3 vaccination (p<0.0001), and 1.8 fold compared to ≤3m post 2 vaccination (p=0.025). Patients treated with most DMTs were seropositive post 2 and 3 vaccinations, however only 38% and 44% of ocrelizumab-treated patients and 54% and 46% of fingolimod-treated patients, respectively, were seropositive. Similarly, in COVID-19-recovered patients only 54% of ocrelizumab-treated, 75% of fingolimod-treated and 67% of cladribine-treated patients were seropositive. A time interval of ≥5 months between ocrelizumab infusion and vaccination was associated with higher IgG levels (p=0.039 post-2 vaccination; p=0.036 post-3 vaccination), and with higher proportions of seropositive patients. Most fingolimod- and ocrelizumab-treated patients responded similarly to 2 and 3 vaccination. IFNγ-T-cell responses were detected in 89% and 63% of PwMS post 2 and 3 vaccination, however in only 25% and 0% of fingolimod-treated patients, while in 100% and 86% of ocrelizumab-treated patients, respectively. PwMS treated with most DMTs developed humoral and T-cell responses following 2 and 3 mRNA SARS-CoV-2 vaccinations. Fingolimod- or ocrelizumab-treated patients had diminished humoral responses, and fingolimod compromised the cellular responses, with no improvement after a 3 booster. Vaccination following >5 months since ocrelizumab infusion was associated with better sero-positivity. These findings may contribute to the development of treatment-stratified vaccination guidelines for PwMS.

Here, we demonstrate that moesin (MSN) phosphorylation by Rho‐associated protein kinase (ROCK) stabilizes PD‐L1 protein levels by blocking SPOP mediating PD‐L1 degradation. Administration of ROCK inhibitor Y‐27632 into immunocompetent mice bearing breast tumors suppressed tumor progression and triggers antitumor T‐cell activity. Expression of programmed cell death ligand (PD‐L1) is associated with poor prognosis in breast cancer. Understanding the regulation of PD‐L1 expression in breast cancer could provide a new strategy for breast cancer treatment. Here, we demonstrate that moesin (MSN) phosphorylation by Rho‐associated protein kinase (ROCK) stabilizes PD‐L1 protein levels. Our results indicate that phosphorylated MSN may compete with the E3 ubiquitin ligase SPOP for binding PD‐L1. ROCK inhibition via the Y‐27632 inhibitor or MSN silencing decreased PD‐L1 expression, resulting in T‐cell activation both in vitro and in vivo. Administration of Y‐27632 into immunocompetent Balb/c mice bearing breast tumors suppressed tumor progression and enhanced CD4+ and CD8+ T‐cell infiltration. RNA‐seq analysis of Y‐27632‐treated mouse tumors revealed that ROCK inhibition upregulated several immune response genes. However, the combination of Y‐27632 and an anti‐PD‐1 antibody did not show additive or synergistic effects due to reduced PD‐L1 in the presence of Y‐27632. Our study unravels a previously unappreciated mechanism of PD‐L1 regulation through the ROCK‐MSN pathway. Moreover, we found that ROCK inhibitors could be combined with breast cancer immunotherapy.

The adaptive immune response plays a crucial role in resolution of viral infections; however, there is limited data on the T cell response to SARS-CoV-2 vaccines such as BBIBP-CorV, Gam-COVID-Vac, and especially for the heterologous combination Gam-COVID-Vac/mRNA-1273 scheme. Furthermore, emerging variants may compromise the adaptive immune response established in the population. To evaluate the T cell immune response induced by the BBIBP-CorV, Gam-COVID-Vac and Gam-COVID-Vac/mRNA-1273, against the ancestral wild type (WT) virus and the Gamma and Omicron variants. Serum levels of IgG antibodies were assessed by CMIA (Abbott Diagnostics, Abbott Park, Illinois). T cell responses against WT virus, Gamma and Omicron were evaluated through an activation-induced marker assay. Sixty individuals, evenly distributed across the BBIBP-CorV, Gam-COVID-Vac, and Gam-COVID-Vac/mRNA-1273 groups, were included. The median age of participants was 48 years (IQR: 34–56), with 58.3 % (n = 35) being female. Seventeen (28.3 %) individuals had a prior confirmed COVID-19 infection. Gam-COVID-Vac/mRNA-1273 scheme elicited significantly higher humoral responses (p < 0.001). However, the three vaccination platforms showed consistent T cell responses across the three stimuli tested. Particularly, all three schemes induced stronger CD8+ responses against the WT virus. In addition, more responders to WT were observed in the BBIBP-CorV and Gam-COVID-Vac groups, while the Gam-COVID-Vac/mRNA-1273 group showed a greater proportion of responders to Omicron. This study provides new data on effective humoral and cellular immune responses against the ancestral WT virus as well as the Gamma and Omicron variants. Moreover, memory CD4+ and CD8+ T cell responses remain protective, and the heterologous scheme may elicit stronger T cell responses against emerging variants. •Strong adaptive T cell responses were observed across all vaccination schemes and stimuli tested.•Higher frequency of activated CD8+ lymphocytes was detected compared to CD4+ in response to SARS-CoV-2•BBIBP-CorV and Gam-COVID-Vac showed a response biased toward wild type virus.•Gam-COVID-Vac / mRNA-1273 recipients showed higher T cell responses to Omicron.•The Gam-COVID-Vac /mRNA-1273 vaccination scheme induced significantly enhanced humoral immune responses.

Vaccines against porcine circovirus type 2 (PCV2) and Mycoplasma hyopneumoniae (Mhp) are routinely used by intramuscular injection. However, since intramuscular vaccination causes stress and increases the risk of cross-contamination among pigs, research on intradermal vaccination is currently being actively conducted. This study was designed to evaluate the efficacy of intradermally administered inactivated vaccines against PCV2 and Mhp in pigs. Three-week-old specific pathogen-free pigs were divided into three groups (5 pigs per group). Pigs in the two groups were intradermally vaccinated with the PCV2 or Mhp vaccine using a needle-free injector. Pigs in the third group were kept as nonvaccinated controls. At 21 days post-vaccination, pigs in one of these vaccinated groups and the nonvaccinated group were intranasally challenged with PCV2b and Mhp, while the other vaccinated group pigs were maintained as vaccine controls. Vaccine efficacy was evaluated by observing weight gain, pathogen load, pathological changes, and humoral or cellular immune responses. As a result, vaccinated pigs revealed significantly higher body weight gain, with lower clinical scores. Vaccinated pigs also showed higher antibody responses but lower PCV2b or Mhp loads in sera, nasal swabs, or lungs than nonvaccinated pigs. Intriguingly, vaccinated pigs upregulated cytotoxic T cells (CTLs), helper T type 1 cells (Th1 cells), and helper T type 17 cells (Th17 cells) after immunization and showed significantly higher levels of CTLs, Th1 and Th17 cells at 14 days post-challenge than nonvaccinated and challenged pigs. This study demonstrated that protective immune responses against PCV2 and Mhp could be efficiently induced in pigs using a relatively small volume of intradermal vaccines, probably due to effective antigen delivery to antigen-presenting cells in the dermis.

Background Cancer-testis antigens (CTAs) and tumour-associated antigens (TAAs) are frequently expressed in hepatocellular carcinoma (HCC); however, the role of tumour-antigen-specific T cell immunity in HCC progression is poorly defined. We characterized CTA- and TAA-specific T cell responses in different HCC stages and investigated their alterations during HCC progression. Methods Fifty-eight HCC patients, 15 liver cirrhosis patients, 15 chronic hepatitis B patients and 10 heathy controls were enrolled in total. IFN-γ ELSPOT using CTAs, including MAGE-A1, MAGE-A3, NY-ESO-1, and SSX2, and two TAAs, SALL4 and AFP, was performed to characterize the T-cell immune response in the enrolled individuals. The functional phenotype of T cells and the responsive T cell populations were analyzed using short-term T-cell culture. Results T cell responses against CTAs and TAAs were specific to HCC. In early-stage HCC patients, the SALL4-specific response was the strongest, followed by MAGE-A3, NY-ESO-1, MAGE-A1 and SSX2. One-year recurrence-free survival after transcatheter arterial chemoembolization plus radiofrequency ablation treatment suggested the protective role of CTA-specific responses. The four CTA- and SALL4-specific T cell responses decreased with the progression of HCC, while the AFP-specific T cell response increased. A higher proportion of CD4+ T cells specific to CTA/SALL4 was observed than AFP-specific T cell responses. Conclusions The IFN-γ ELISPOT assay characterized distinct profiles of tumour-antigen-specific T cell responses in HCC patients. CTA- and SALL4-specific T cell responses may be important for controlling HCC in the early stage, whereas AFP-specific T cell responses might be a signature of malignant tumour status in the advanced stage. The application of immunotherapy at an early stage of HCC development should be considered.

Xenotransplantation offers a promising alternative to circumvent the lack of donated human organs available for transplantation. Different attempts to improve the survival of xenografts led to the generation of transgenic pigs expressing various combinations of human protective genes or knocked out for specific antigens. Currently, testing the efficiency of porcine organs carrying different genetic modifications in preventing xenogeneic immune responses completely relies on in vitro assays, humanized mouse models, or non-human primate transplantation models. However, these tests are often associated with major concerns due to reproducibility and generation of insufficient data as well as they raise ethical, logistical, and economic issues. In this study, we investigated the feasibility of specifically assessing the strength of human T-cell responses towards the kidneys of wild-type (WT) or transgenic pigs overexpressing human programmed death-1 ligand 1 (hPD-L1) during ex vivo kidney perfusion (EVKP). Human T cells were shown to adhere to the endothelium and transmigrate into WT and hPD-L1 kidneys. However, transcript levels of TNF-a and IFN-y as well as cytotoxic molecules such as granzyme B and perforin secreted by human T cells were significantly decreased in the tissue of hPD-L1 kidneys in comparison to WT kidneys. These results were confirmed via in vitro assays using renal endothelial cells (ECs) isolated from WT and hPD-L1 transgenic pigs. Both CD4 + and CD8 + T cells showed significantly lower proliferation rates after exposure to hPD-L1 porcine renal ECs in comparison to WT ECs. In addition, the secretion of pro-inflammatory cytokines was significantly reduced in cultures using hPD-L1 ECs in comparison to WT ECs. Remarkably, hPD-L1 EC survival was significantly increased in cytotoxic assays. This study demonstrates the feasibility of evaluating the human response of specific immune subsets such as human T cells towards the whole xenograft during EVKP. This may represent a robust strategy to assess the potency of different genetic modifications to prevent xenogeneic immune responses and thereby predict the risk of immune rejection of new genetically engineered xenografts.

With this article, the authors aim to honor the memory of Serafino Zappacosta, who had been their mentor during the early years of their career in science. The authors discuss how the combination of Serafino Zappacosta's extraordinary commitment to teaching and passion for science created a fostering educational environment that led to the creation of the \" .\" The review also illustrates how the research on the MHC and the inspirational scientific context in the Zappacosta's laboratory influenced the authors' early scientific interests, and subsequent professional work as immunologists.