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Comparison of surface labeling of CXCR4/CCR7 in unfixed or fixed cells (S1); CXCL12/CCL21 do not interfere with binding of the anti-CXCR4/CCR7 antibodies used in this study (S2); Co-localization analysis of internalized CXCR4 and CCR7 with Rab11 and Rab7 in normal and CLL B cells following antibody-mediated downregulation (S3); The imbalance between the plasma membrane and endosomal CXCR4/CCR7 pools in CLL cells can be normalized by targeting the microtubule cytoskeleton (S4); B-cell treatment with CXCL12 or CCL21 results in an increase in the surface levels of the respective receptors (S5); Enhanced adhesion to fibronectin of CXCL12- or CCL21-treated CLL cells compared to normal B cells (S6); CCR7 and S1P1 expression in B-CLL bone marrow biopsy samples (S7).

Supplemental Figure Legends from Enhanced Chemokine Receptor Recycling and Impaired S1P1 Expression Promote Leukemic Cell Infiltration of Lymph Nodes in Chronic Lymphocytic Leukemia

List of the primers used in this study.

For the first time we have defined naïve, central memory, effector memory and differentiated effector porcine CD8 T cells and analyzed their distribution in lymphoid and respiratory tissues after influenza infection or immunization, using peptide-MHC tetramers of three influenza nucleoprotein (NP) epitopes. The hierarchy of response to the three epitopes changes during the response in different tissues. Most NP-specific CD8 T cells in broncho-alveolar lavage (BAL) and lung are tissue resident memory cells (TRM) that express CD69 and downregulate CD45RA and CCR7. NP-specific cells isolated from BAL express genes characteristic of TRM, but gene expression differs at 7, 21 and 63 days post infection. In all tissues the frequency of NP-specific CD8 cells declines over 63 days almost to background levels but is best maintained in BAL. The kinetic of influenza specific memory CD8 T cell in this natural host species differs from that in small animal models.

Background: Increasing evidence suggests a link between T-cell senescence and tumour prognosis. In particular, high levels of circulating senescent T-lymphocytes have been also correlated with a worse response to anti-cancer treatments. In this perspective, a therapeutic approach aimed at T-cell senescence clearance and restoring is regarded as an innovative strategy in cancer treatment and is currently under investigation in pre-clinical and clinical models. The purpose of the present study is to characterize the impact of T-cell senescence as a predictive factor of response in patients with operable breast cancer (BC) treated with NeoAdjuvant Therapy (NAT), according to biological subtypes. Methods: Fifty-four patients have been enrolled so far. CD3+ T cells were isolated from peripheral blood (PB) by Ficoll stratification at baseline, before start of treatment, and after NAT, before surgery. The relative expression of two cyclin-dependent kinase inhibitors (CDKi) p16 and/or p21 was used to characterize T-cell senescence, by RT-qPCR. RPLP-0 gene was used as housekeeping gene and data were normalized on normal controls (2-ΔΔCt). Association with type of response to NAT was performed by using the Wilcoxon signed-rank test. Results: At baseline, 19 patients have been tested for T-cell senescence so far: of these, 7 (37%) were Triple Negative (TN), 8 (42%) Luminal B and 4 (21%) HER2+. A higher expression of p16 and p21 was observed in TN BC versus Luminal B and HER2+. p16: 2.78±6.87 vs 1.21±2.10 vs 0.09±0.07 (p=0.05); p21: 2.09±2.79 vs 1.58±1.95 vs 0.86±0.61 (p=0.63). At present surgery has been performed in 16 patients, with an overall pCR rate of 56% (9/16). Patients with a pCR after NAT had a significantly lower expression of p16 at baseline as compared to patients with residual disease: 0.87±2.006 vs 3.07±6.76; p=0.02. Conversely, p21 expression level was not significantly associated: 1.42±1.00 vs 0,81±0.41; p=0.29). Expression of p16 and p21 was assessed in 13 patients at baseline and after the end of NAT, before surgery: overall, p16 expression resulted significantly increased from baseline to end of treatment (p=0,01), whereas p21 did not show the same trend (p=0.13). Conclusions: These preliminary results suggest that the presence of circulating senescent T cells, identified by a p16 low expression level, might represent a novel biomarker to identify those patients with operable BC who are most likely to achieve a pCR at NAT. Updated analyses will be presented. Citation Format: Veronica Martini, Chiara Saggia, Francesca D'Avanzo, Valentina Rossi, Ivan Dodaro, Eleonora Gallarotti, Tania Pretato, Feba Mariam Varughese, Ajay Ram Vachanaram, Martina Cavalleri, Arianna Stella, Simone Gobbato, Andrea Tassone, Anna Gambaro, Carmen Branni, Paola Maria Maggiora, Alessia Rua, Renzo Boldorini, Eleonora Ferrara, Marco Krengli, Alessandra Gennari. Peripheral T-lymphocytes senescence and response to neoadjuvant therapy (NAT) in operable breast cancer (BC) [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-07-06.

BackgroundEvaluation of extracellular vescicles (EVs) has been identified as a potential predictive biomarker in patients treated with IO. In particular, Glypican-1 (GPC-1), was reported to be abnormally expressed in BC tissues and GPC-1+ EVs resulted significantly higher in BC patients than in healthy controls. Triple negative BC (TNBC) is characterized by poor prognosis, due to the lack of effective treatments; in this context, Immune Checkpoint Inhibitors (ICIs) are regarded as promising agents; however, their impact is not the same as in other cancers, such as melanoma and lung. With these premises, the aim of the IRIS project is to evaluate the prognostic and predictive role of tumor and leukocyte-derived EVs in patients with early and advanced TNBC, treated with ICIs.MethodsTwenty consecutive patients have been enrolled so far: 10 with TN MBC PD-L1+ treated with Ist line Nab-Paclitaxel + Atezolizumab and 10 with high-risk TN early BC treated with neo-adjuvant pembrolizumab (KEYNOTE-522 schedule). In all patients, 5 ml of peripheral blood in Na-Citrate were drawn at baseline. An optimized flow cytometry method was used to identify and subtype circulating EVs. EVs were identified as LCD+/phalloidin−events, falling in the scatter area with physical parameters lower than platelets. CD45, GPC-1, EpCAM and PD-L1 were chosen as markers for EVs characterization. Data acquisition and analyses were performed with flow cytometry (FACS Symphony) and FACSDiva v9.0.2. Volumetric count was applied to calculate EV concentrations. Statistical analysis was performed with GraphPad software, and a non-parametric t-test was utilized for EVs characterization, with a p-value < 0.05.ResultsOverall, a significantly lower concentration of circulating tumor-derived EVs (CD45-EpCAM+GPC-1+) was detected as compared to leukocyte-derived EVs (CD45+): Mean±SD = 95.16 ± 219.3 vs 1623 ± 1582; p Citation Format: Veronica Martini, Arianna Stella, Rahma Ben Ayed, Carmen Branni, Andrea Tassone, Nazanin Keivan, Chiara Saggia, Francesca D'Avanzo, Valentina Rossi, Francesca Platini, Incoronata Romaniello, Elena Giacobino, Valentina Guarneri, Renzo Luciano Boldorini, Francesca Mercalli, Ivan Dodaro, Anna Gambaro, David James Pinato, Alessandra Gennari. Evaluation of extracellular vesicles (EVs) as potential predictors of response to immunotherapy (IO) in triple negative early and advanced breast cancer (BC): preliminary evidence from the IRIS project [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2174.

There is need for effective and affordable vaccines against SARS-CoV-2 to tackle the ongoing pandemic. In this study, we describe a protein nanoparticle vaccine against SARS-CoV-2. The vaccine is based on the display of coronavirus spike glycoprotein receptor-binding domain (RBD) on a synthetic virus-like particle (VLP) platform, SpyCatcher003-mi3, using SpyTag/SpyCatcher technology. Low doses of RBD-SpyVLP in a prime-boost regimen induce a strong neutralising antibody response in mice and pigs that is superior to convalescent human sera. We evaluate antibody quality using ACE2 blocking and neutralisation of cell infection by pseudovirus or wild-type SARS-CoV-2. Using competition assays with a monoclonal antibody panel, we show that RBD-SpyVLP induces a polyclonal antibody response that recognises key epitopes on the RBD, reducing the likelihood of selecting neutralisation-escape mutants. Moreover, RBD-SpyVLP is thermostable and can be lyophilised without losing immunogenicity, to facilitate global distribution and reduce cold-chain dependence. The data suggests that RBD-SpyVLP provides strong potential to address clinical and logistic challenges of the COVID-19 pandemic., Vaccines for SARS-COV-2 are needed in the ongoing pandemic. Here the authors characterize a vaccine candidate that presents the receptor-binding domain (RBD) of SARS-CoV-2 spike protein on a synthetic VLP platform using SpyTag/SpyCatcher technology and show immunogenicity of a prime-boost regimen in mice and pigs.

Key Points Neutralizing mAb 2–12C reduces influenza viral load and lung pathology in pigs. DNA plasmid–encoded 2–12C reduces lung pathology. The pig is a useful preclinical model for testing mAbs and mAb delivery platforms., mAbs are a possible adjunct to vaccination and drugs in treatment of influenza virus infection. However, questions remain whether small animal models accurately predict efficacy in humans. We have established the pig, a large natural host animal for influenza, with many physiological similarities to humans, as a robust model for testing mAbs. We show that a strongly neutralizing mAb (2–12C) against the hemagglutinin head administered prophylactically at 15 mg/kg reduced viral load and lung pathology after pandemic H1N1 influenza challenge. A lower dose of 1 mg/kg of 2–12C or a DNA plasmid–encoded version of 2–12C reduced pathology and viral load in the lungs but not viral shedding in nasal swabs. We propose that the pig influenza model will be useful for testing candidate mAbs and emerging delivery platforms prior to human trials.

There is a critical need to develop superior influenza vaccines that provide broader protection. Influenza vaccines are traditionally tested in naïve animals, although humans are exposed to influenza in the first years of their lives, but the impact of prior influenza exposure on vaccine induced immune responses has not been well studied. Pigs are an important natural host for influenza, are a source of pandemic viruses, and are an excellent model for human influenza. Here we investigated the immunogenicity of the ChAdOx2 viral vectored vaccine, expressing influenza nucleoprotein, matrix protein 1 and neuraminidase in H1N1pdm09 pre-exposed pigs. We evaluated the importance of route of administration by comparing intra-nasal, aerosol and intra-muscular immunizations. Aerosol delivery boosted the local lung T cell and antibody responses, while intra-muscular immunization boosted systemic immunity. These results will inform how best to deliver vaccines in order to harness optimal protective immunity.

We defined naïve, central memory, effector memory and terminally differentiated porcine CD8 T cells and analyzed their phenotype in lymphoid and respiratory tissues after influenza infection or immunization using peptide-MHC tetramers of three influenza nucleoprotein (NP) epitopes. The hierarchy of response to the three epitopes changes during the response in different tissues. Most NP-specific CD8 T cells in broncho-alveolar lavage (BAL) and lung are tissue resident memory cells (TRM), that express CD69 and have an effector memory or terminally differentiated phenotype. NP-specific cells isolated from BAL express genes characteristic of TRM, but gene expression differs at 7, 21 and 63 days post infection. The frequency of NP-specific cells declines over 63 days in all tissues but is best maintained in BAL. The pig is a powerful model for understanding how best to induce and harness local immunity to respiratory viruses.One sentence summaryInfluenza NP-specific porcine tissue resident memory CD8 T cells persist in the lung with major changes in gene expression.

[This corrects the article DOI: 10.1371/journal.ppat.1009330.].

A single-center cross-sectional study was conducted to describe the use of ceftaroline in a large teaching hospital in Northern Italy, during a period also including the first months of the coronavirus disease 2019 (COVID-19) pandemic. The primary objective was to describe the use of ceftaroline in terms of indications and characteristics of patients. A secondary objective was to describe the rate of favorable clinical response in patients with bloodstream infections (BSI) due to methicillin-resistant Staphylococcus aureus (MRSA-BSI) receiving ceftaroline. Overall, 200 patients were included in the study. Most of them had COVID-19 (83%, 165/200) and were hospitalized in medical wards (78%, 155/200). Included patients with COVID-19 pneumonia were given empirical ceftaroline in the suspicion of bacterial co-infection or superinfection. Among patients with MRSA-BSI, ceftaroline was used as a first-line therapy and salvage therapy in 25% (3/12) and 75% (9/12) of cases, respectively, and as a monotherapy or in combination with daptomycin in 58% (7/12) and 42% (5/12) of patients, respectively. A favorable response was registered in 67% (8/12) of patients. Improving etiological diagnosis of bacterial infections is essential to optimize the use of ceftaroline in COVID-19 patients. The use of ceftaroline for MRSA-BSI, either as a monotherapy or in combination with other anti-MRSA agents, showed promising rates of favorable response.

Background: The combination of an anti-Programmed Cell Death-Ligand-1 (anti-PD-L1) agent, atezolizumab, plus chemotherapy (CT) as first line treatment in triple negative (TN) advanced breast cancer (ABC), resulted into a significant improvement in Progression Free Survival (PFS) as compared to Nab-paclitaxel alone, and a significant improvement in Overall Survival (OS) in the PD-L-1 positive subgroup (> 1% by IHC). With these results, the IMpassion 130 study met its primary endpoint, although its impact on the prognosis of TN ABC is less than expected, suggesting that innovative approaches are needed to maximize the role of immunotherapy in this disease subset. In this perspective, the potential role of addressing innate immunity as opposed to adaptive immunity has not yet been evaluated. It has been shown that Tumor-Associated Macrophages (TAMs) may express the immune checkpoint inhibitor PD-L1, thus contributing to immunosuppression. In addition, in response to microenvironmental signals they acquire a tumor-promoting M2-like phenotype, that supports angiogenesis, tissue remodeling and metastasis formation. They also express the Signal-regulatory protein (SIRPα) which provides a “don’t eat” me signal that impairs phagocytosis of CD47 positive cancer cells. CD47 is expressed in all human solid tumor cells; its role is to protect cancer cells from being recognized by innate immune surveillance. With these premises, the aim of this study is to evaluate the expression and prognostic value of different biomarkers related to innate and adaptive immunity, by using publicy available gene expression datasets, to assess if double immune co-targeting (i.e. innate and adaptive) might represent a viable strategy to optimise immunotherapy in ABC. Methods: We used previously published gene expression datasets by two online tools: GOBO and GEPIA. GOBO is an ultrafast tool including 1881 early BC patients generated on Affymetrix U133A microarrays, with a median follow up of 120 months. Expression of CD47, SIRP, CD163 and CD204 (M2-like phenotype) mRNA were evaluated as continuous variables. Kaplan Meier survival curves were used to assess the prognostic ability of the identified biomarkers. GEPIA is a web server for analysing the RNA sequencing expression data of tumours and normal samples from the TCGA and the GTEx projects, by a standard processing pipeline. Results: CD47 and CD204 mRNAs were significantly overexpressed in BC samples with respect to normal tissues (p < 0.05). Furthermore, CD47, SIRPalpha and CD163 were differentially modulated in the different BC subtypes (p Conclusions: These results indicate that innate immunity is involved in BC prognosis, and might be considered a target for therapy, in particular in those subtypes such as the Luminal A group, characterised by a favourable outcome; conversely, no effect was observed in subtypes characterised by a worse prognosis such as the basal-like tumors. Further investigation on the role of innate immunity in BC is warranted. Citation Format: Veronica Martini, Francesco Favero, Davide Corà, Feba Varughese, Antonio Sica, Alessandra Gennari. mRNA expression level and prognostic significance of different immune-related biomarkers in early breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-05-09.

Designing effective and safe tuberculosis (TB) subunit vaccines for inhalation requires identification of appropriate antigens and adjuvants and definition of the specific areas to target in the lungs. Magnetic resonance imaging (MRI) enables high spatial resolution, but real-time anatomical and functional MRI of lungs is challenging. Here, we describe the design of a novel gadoteridol-loaded cationic adjuvant formulation 01 (CAF01) for MRI-guided vaccine delivery of the clinically tested TB subunit vaccine candidate H56/CAF01. Gadoteridol-loaded CAF01 liposomes were engineered by using a quality-by-design approach to (i) increase the mechanistic understanding of formulation factors governing the loading of gadoteridol and (ii) maximize the loading of gadoteridol in CAF01, which was confirmed by cryotransmission electron microscopy. The encapsulation efficiency and loading of gadoteridol were highly dependent on the buffer pH due to strong attractive electrostatic interactions between gadoteridol and the cationic lipid component. Optimal gadoteridol loading of CAF01 liposomes showed good in vivo stability and safety upon intrapulmonary administration into mice while generating 1.5-fold MRI signal enhancement associated with approximately 30% T1 relaxation change. This formulation principle and imaging approach can potentially be used for other mucosal nanoparticle-based formulations, species, and lung pathologies, which can readily be translated for clinical use.

BackgroundAge is considered as one of the most important risk-factor for many types of solid and hematological cancers, as their incidence increases with age in parallel to the ever-growing elderly population. Moreover, cancer incidence is constantly increasing as a consequence of the increase in life expectancy that favors the process of cellular senescence. Geriatric assessment has been increasingly recognized as predictive and prognostic instrument to detect frailty in older adults with cancer. In particular, the G8 score is a simple and reproducible instrument to identify elderly patients who should undergo full geriatric evaluation. Due to their frailty, elderly patients may be often under-treated and a therapeutic choice based also on a comprehensive geriatric assessment (CGA) is recommended. With these premises, we aim to test the impact of the CGA based interventions on the quality of life (QoL) of frail elderly onco-hematological patients, identified by the G8 screening, candidate for innovative target directed drugs or treatments including the combination of radiotherapy and chemotherapy (RT + CT).MethodsPatients aged > 65 years, candidate to target directed agents or to RT + CT treatments are screened for frailty by the G8 test; those patients classified as frail (G8 ≤ 14) are randomized to receive a CGA at baseline or to conventional care. The primary endpoint is QoL, assessed by EORTC QLQ-C30C. As collateral biological study, the potential prognostic/predictive role of T-cell senescence and myeloid derived suppressor cells (MDSC) are evaluated on plasma samples.DiscussionThis trial will contribute to define the impact of CGA on the management of frail elderly onco-hematologic patients candidate to innovative biological drugs or to integrated schedules with the association of RT + CT. Furthermore, the use of plasma samples to assess the potential prognostic value of imbalance of immune-competent cells is expected to contribute to the individualized care of elderly patients, resulting into a fine tuning of the therapeutic strategies.Trial registrationClinicalTrials.gov ID:NCT04478916. registered July 21, 2020 – retrospectively registered.

We have used the pig, a large natural host animal for influenza with many physiological similarities to humans, to characterize αβ, γδ T cell and antibody (Ab) immune responses to the 2009 pandemic H1N1 virus infection. We evaluated the kinetic of virus infection and associated response in inbred Babraham pigs with identical MHC (Swine Leucocyte Antigen) and compared them to commercial outbred animals. High level of nasal virus shedding continued up to days 4 to 5 post infection followed by a steep decline and clearance of virus by day 9. Adaptive T cell and Ab responses were detectable from days 5 to 6 post infection reaching a peak at 9 to 14 days. γδ T cells produced cytokines ex vivo at day 2 post infection, while virus reactive IFNγ producing γδ T cells were detected from day 7 post infection. Analysis of NP tetramer specific and virus specific CD8 and CD4 T cells in blood, lung, lung draining lymph nodes, and broncho-alveolar lavage (BAL) showed clear differences in cytokine production between these tissues. BAL contained the most highly activated CD8, CD4, and γδ T cells producing large amounts of cytokines, which likely contribute to elimination of virus. The weak response in blood did not reflect the powerful local lung immune responses. The immune response in the Babraham pig following H1N1pdm09 influenza infection was comparable to that of outbred animals. The ability to utilize these two swine models together will provide unparalleled power to analyze immune responses to influenza.

Key Points Simultaneous aerosol and i.m. immunization reduces viral load and pathology. Simultaneous immunization induces strong protective local and systemic responses. The hierarchy of NP-specific T cells in blood does not reflect local lung responses., A vaccine providing both powerful Ab and cross-reactive T cell immune responses against influenza viruses would be beneficial for both humans and pigs. In this study, we evaluated i.m., aerosol (Aer), and simultaneous systemic and respiratory immunization (SIM) by both routes in Babraham pigs, using the single cycle candidate influenza vaccine S-FLU. After prime and boost immunization, pigs were challenged with H1N1pdm09 virus. i.m.-immunized pigs generated a high titer of neutralizing Abs but poor T cell responses, whereas Aer induced powerful respiratory tract T cell responses but a low titer of Abs. SIM pigs combined high Ab titers and strong local T cell responses. SIM showed the most complete suppression of virus shedding and the greatest improvement in pathology. We conclude that SIM regimes for immunization against respiratory pathogens warrant further study.

Malignant pleural mesothelioma (MPM) is a highly aggressive cancer with a long latency period and dismal prognosis. Recently, tazemetostat (EPZ-6438), an inhibitor of the histone methyltransferase EZH2, has entered clinical trials due to the antiproliferative effects reported on MPM cells. However, the direct and indirect effects of epigenetic reprogramming on the tumor microenvironment are hitherto unexplored. To investigate the impact of tumor-associated macrophages (TAMs) on MPM cell responsiveness to tazemetostat, we developed a three-dimensional MPM spheroid model that recapitulates in vitro, both monocytes’ recruitment in tumors and their functional differentiation toward a TAM-like phenotype (Mo-TAMs). Along with an increased expression of genes for monocyte chemoattractants, inhibitory immune checkpoints, immunosuppressive and M2-like molecules, Mo-TAMs promote tumor cell proliferation and spreading. Prolonged treatment of MPM spheroids with tazemetostat enhances both the recruitment of Mo-TAMs and the expression of their protumor phenotype. Therefore, Mo-TAMs profoundly suppress the antiproliferative effects due to EZH2 inhibition in MPM cells. Overall, our findings indicate that TAMs are a driving force for MPM growth, progression, and resistance to tazemetostat, therefore, strategies of TAM depletion might be evaluated to improve the therapeutic efficacy of pharmacological inhibition of EZH2.

Background: Breast cancer (BC) is the most common neoplasm in women. Many clinical and preclinical studies investigated the possible relationship between host metabolism and BC. Significant differences among BC subtypes have been reported for glucose metabolism. Insulin can promote tumorigenesis through a direct effect on epithelial tissues or indirectly by affecting the levels of other modulators, such as the insulin-like growth factor (IGF) family of receptors, sex hormones, and adipokines. The potential anti-cancer activity of metformin is based on two principal effects: first, its capacity for lowering circulating insulin levels with indirect endocrine effects that may impact on tumor cell proliferation; second, its direct influence on many pro-cancer signaling pathways that are key drivers of BC aggressiveness. Methods: In the present review, the interaction between BC, host metabolism, and patients’ prognosis has been reviewed across available literature evidence. Conclusions: Obesity, metabolic syndrome, and insulin resistance are all involved in BC growth and could have a relevant impact on prognosis. All these factors act through a pro-inflammatory state, mediated by cytokines originated in fat tissue, and seem to be related to a higher risk of BC development and worse prognosis.

We have used the pig, a large natural host animal for influenza with many physiological similarities to humans, to characterize αβ, γδ T cell and antibody (Ab) immune responses to the 2009 pandemic H1N1 virus infection. We evaluated the kinetic of virus infection and associated response in inbred Babraham pigs with identical MHC (Swine Leucocyte Antigen) and compared them to commercial outbred animals. High level of nasal virus shedding continued up to day 4-5 post infection followed by a steep decline and clearance of virus by day 9. Adaptive T cell and Ab responses were detectable from day 5-6 post infection reaching a peak at 9-14 days. γδ cells produced cytokines ex vivo at day 2 post infection, while virus specific IFNγ producing γδ T cells were detected from day 7 post infection. Analysis of NP tetramer specific and virus specific CD8 and CD4 T cells in blood, lung, lung draining lymph nodes and broncho-alveolar lavage (BAL) showed clear differences in cytokine production between these tissues. BAL contained the most highly activated CD8, CD4 and γδ cells producing large amounts of cytokines, which likely contribute to elimination of virus. The weak response in blood did not reflect the powerful local lung immune responses. The immune response in the Babraham pig following H1N1pdm09 influenza infection was comparable to that of outbred animals. The ability to utilize these two swine models together will provide unparalleled power to analyse immune responses to influenza.

The antiparasitic drug nitazoxanide is widely available and exerts broad-spectrum antiviral activity in vitro. However, there is no evidence of its impact on SARS-CoV-2 infection.In a multicenter, randomized, double-blind, placebo-controlled trial, adult patients who presented up to 3 days after onset of Covid-19 symptoms (dry cough, fever, and/or fatigue) were enrolled. After confirmation of SARS-CoV2 infection by RT-PCR on nasopharyngeal swab, patients were randomized 1:1 to receive either nitazoxanide (500 mg) or placebo, TID, for 5 days. The primary outcome was complete resolution of symptoms. Secondary outcomes were viral load, general laboratory tests, serum biomarkers of inflammation, and hospitalization rate. Adverse events were also assessed.From June 8 to August 20, 2020, 1,575 patients were screened. Of these, 392 (198 placebo, 194 nitazoxanide) were analyzed. Median time from symptom onset to first dose of study drug was 5 (4-5) days. At the 5-day study visit, symptom resolution did not differ between the nitazoxanide and placebo arms. However, at the 1-week follow-up, 78% in the nitazoxanide arm and 57% in the placebo arm reported complete resolution of symptoms (p=0.048). Swabs collected were negative for SARS-CoV-2 in 29.9% of patients in the nitazoxanide arm versus 18.2% in the placebo arm (p=0.009). Viral load was also reduced after nitazoxanide compared to placebo (p=0.006). No serious adverse events were observed.In patients with mild Covid-19, symptom resolution did not differ between the nitazoxanide and placebo groups after 5 days of therapy. However, early nitazoxanide therapy was safe and reduced viral load significantly.Take home messageThis was the first study to evaluate the effect of early nitazoxanide therapy in mild Covid-19. Nitazoxanide did not accelerate symptom resolution after 5 days of therapy; however, reduced viral load significantly with no serious adverse events.

A vaccine providing both powerful antibody and cross-reactive T cell immune responses against influenza viruses would be beneficial for both humans and pigs. Here we evaluated intramuscular (IM), aerosol (Aer) and simultaneous immunization (SIM) by both routes in pigs, using the single cycle candidate influenza vaccine S-FLU. After prime and boost immunization pigs were challenged with H1N1pdm09 virus. IM immunized pigs generated high titer of neutralizing antibodies but poor T cell responses, while Aer induced powerful respiratory tract T cell responses, but a low titer of antibodies. SIM pigs combined high antibody titers and strong local T cell responses. SIM pigs showed the most complete suppression of virus shedding and the greatest improvement in pathology. We conclude that SIM regimes for immunization against respiratory pathogens warrant further study.

Pigs are natural hosts for the same subtypes of influenza A viruses as humans and integrally involved in virus evolution with frequent interspecies transmissions in both directions. The emergence of the 2009 pandemic H1N1 virus illustrates the importance of pigs in evolution of zoonotic strains. Here we generated pig influenza-specific monoclonal antibodies (mAbs) from H1N1pdm09 infected pigs. The mAbs recognized the same two major immunodominant haemagglutinin (HA) epitopes targeted by humans, one of which is not recognized by post-infection ferret antisera that are commonly used to monitor virus evolution. Neutralizing activity of the pig mAbs was comparable to that of potent human anti-HA mAbs. Further, prophylactic administration of a selected porcine mAb to pigs abolished lung viral load and greatly reduced lung pathology but did not eliminate nasal shedding of virus after H1N1pdm09 challenge. Hence mAbs from pigs, which target HA can significantly reduce disease severity. These results, together with the comparable sizes of pigs and humans, indicate that the pig is a valuable model for understanding how best to apply mAbs as therapy in humans and for monitoring antigenic drift of influenza viruses in humans, thereby providing information highly relevant to making influenza vaccine recommendations., Author summary Antibodies (Ab) are increasingly used to treat human infectious diseases. Pigs are large animals, natural hosts for influenza viruses and very similar to humans. We generated monoclonal Abs from influenza infected pigs and show that they recognize the same sites of the virus as humans. One of these sites was not recognized by ferret anti-sera, which are commonly used to predict the evolution of the virus and inform vaccine design. We also show that prophylactic administration of one of these mAb to pigs abolished lung viral load and prevented lung damage following infection with influenza. We conclude that the pig is a useful model to test how best to use Abs for therapy and to inform vaccine recommendations for humans.

There is dire need for an effective and affordable vaccine against SARS-CoV-2 to tackle the ongoing pandemic. In this study, we describe a modular virus-like particle vaccine candidate displaying the SARS-CoV-2 spike glycoprotein receptor-binding domain (RBD) using SpyTag/SpyCatcher technology (RBD-SpyVLP). Low doses of RBD-SpyVLP in a prime-boost regimen induced a strong neutralising antibody response in mice and pigs that was superior to convalescent human sera. We evaluated antibody quality using ACE2 blocking and neutralisation of cell infection by pseudovirus or wild-type SARS-CoV-2. Using competition assays with a monoclonal antibody panel, we showed that RBD-SpyVLP induced a polyclonal antibody response that recognised all key epitopes on the RBD, reducing the likelihood of selecting neutralisation-escape mutants. The induction of potent and polyclonal antibody responses by RBD-SpyVLP provides strong potential to address clinical and logistic challenges of the COVID-19 pandemic. Moreover, RBD-SpyVLP is highly resilient, thermostable and can be lyophilised without losing immunogenicity, to facilitate global distribution and reduce cold-chain dependence.

Clinical development of the COVID-19 vaccine candidate ChAdOx1 nCoV-19, a replication-deficient simian adenoviral vector expressing the full-length SARS-CoV-2 spike (S) protein was initiated in April 2020 following non-human primate studies using a single immunisation. Here, we compared the immunogenicity of one or two doses of ChAdOx1 nCoV-19 in both mice and pigs. Whilst a single dose induced antigen-specific antibody and T cells responses, a booster immunisation enhanced antibody responses, particularly in pigs, with a significant increase in SARS-CoV-2 neutralising titres.

Recent evidence indicates that local immune responses and tissue resident memory T cells (TRM) are critical for protection against respiratory infections but there is little information on the contributions of upper and lower respiratory tract (URT and LRT) immunity. To provide a rational basis for designing methods for optimal delivery of vaccines to the respiratory tract in a large animal model, we investigated the distribution of droplets generated by a mucosal atomization device (MAD) and two vibrating mesh nebulizers (VMNs) and the immune responses induced by delivery of influenza virus by MAD in pigs. We showed that droplets containing the drug albuterol, a radiolabel (99mTc-DTPA) or a model influenza virus vaccine (S-FLU) have similar aerosol characteristics. 99mTc-DTPA scintigraphy showed that VMNs deliver droplets with uniform distribution throughout the lungs as well as the URT. Surprisingly MAD administration (1ml/nostril) also delivered a high proportion of the dose to the lungs, albeit concentrated in a small area. After MAD administration of influenza virus, antigen specific T cells were found at high frequency in nasal turbinates, trachea, broncho-alveolar lavage, lungs, tracheobronchial nodes and blood. We conclude that the pig is useful for investigating optimal targeting of vaccines to the respiratory tract.

Introduction: Insulin and the insulin-like growth factor (IGF) family play a key role in breast cancer (BC). Objective: In this study, we evaluated on a genomic scale the potential prognostic value of insulin signaling in early BC. Methods: Candidate genes were selected from the published literature and gene expression profiling experiments. Three publicly available BC datasets, containing gene expression data on 502 cases, were used to test the prognostic ability of the score. The gene signature was developed on GSE1456, containing microarray data from 159 patients, split into a training set (102 breast tumors) and a validation set (n = 57). GSE3494 and GSE2990 (350 patients) were used for external validation. Univariate Mann-Whitney test was used to identify genes differentially expressed between relapsed and nonrelapsed patients. Expression of genes significantly correlated with relapse was combined in a linear score. Patients were classified as low or high risk with respect to the median value. Results: On the training set, 15 genes turned out to be differentially expressed: 8-year disease-free survival (DFS) was 51 and 91% in the high- and low-risk group (p < 0.001), respectively. In the validation set, DFS was 97 and 54% (p = 0.009), respectively. External validation: 8-year DFS was 72 and 61%, respectively, in GSE3494 (p = 0.03) and 74 and 55% in GSE2990 (p = 0.03). By multivariate analyses, the insulin signature was significantly associated with DFS, independently of age, hormone receptor status, nodal status, and grade. Conclusions: Our findings indicate that the insulin pathway is involved in BC prognosis at a genomic level and provide a window of selectivity for preventive and treatment strategies targeting the insulin/IGF pathway in BC patients.