Multiplex Real-Time Reverse-Transcription Polymerase Chain Reaction Assays for Diagnostic Testing of Severe Acute Respiratory Syndrome Coronavirus 2 and Seasonal Influenza Viruses: A Challenge of the Phase 3 Pandemic Setting.

BACKGROUND: Pandemic coronavirus disease 2019 (COVID-19) disease represents a challenge for healthcare structures. The molecular confirmation of samples from infected individuals is crucial and therefore guides public health decision making. Clusters and possibly increased diffuse transmission could occur in the context of the next influenza season. For this reason, a diagnostic test able to discriminate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from influenza viruses is urgently needed. METHODS: A multiplex real-time reverse-transcription polymerase chain reaction (PCR) assay was assessed using 1 laboratory protocol with different real-time PCR instruments. Overall, 1000 clinical samples (600 from samples SARS-CoV-2-infected patients, 200 samples from influenza-infected patients, and 200 negative samples) were analyzed. RESULTS: The assay developed was able to detect and discriminate each virus target and to intercept coinfections. The limit of quantification of each assay ranged between 5 and 10 genomic copy numbers, with a cutoff value of 37.7 and 37.8 for influenza and SARS-CoV-2 viruses, respectively. Only 2 influenza coinfections were detected in COVID-19 samples. CONCLUSIONS: This study suggests that multiplex assay is a rapid, valid, and accurate method for the detection of SARS-CoV-2 and influenza viruses in clinical samples. The test may be an important diagnostic tool for both diagnostic and surveillance purposes during the seasonal influenza activity period.

Co-circulation of the two influenza B lineages during 13 consecutive influenza surveillance seasons in Italy, 2004-2017.

BACKGROUND: Since 1985, two antigenically distinct lineages of influenza B viruses (Victoria-like and Yamagata-like) have circulated globally. Trivalent seasonal influenza vaccines contain two circulating influenza A strains but a single B strain and thus provide limited immunity against circulating B strains of the lineage not included in the vaccine. In this study, we describe the characteristics of influenza B viruses that caused respiratory illness in the population in Italy over 13 consecutive seasons of virological surveillance, and the match between the predominant influenza B lineage and the vaccine B lineage, in each season. METHODS: From 2004 to 2017, 26,886 laboratory-confirmed influenza cases were registered in Italy, of which 18.7% were type B. Among them, the lineage of 2465 strains (49%) was retrieved or characterized in this study by a real-time RT-PCR assay and/or sequencing of the hemagglutinin (HA) gene. RESULTS: Co-circulation of both B lineages was observed each season, although in different proportions every year. Overall, viruses of B/Victoria and B/Yamagata lineages caused 53.3 and 46.7% of influenza B infections, respectively. A higher proportion of infections with both lineages was detected in children, and there was a declining frequency of B/Victoria detections with age. A mismatch between the vaccine and the predominant influenza B lineage occurred in eight out of thirteen influenza seasons under study. Considering the seasons when B accounted for > 20% of all laboratory-confirmed influenza cases, a mismatch was observed in four out of six seasons. Phylogenetic analysis of the HA1 domain confirmed the co-circulation of both lineages and revealed a mixed circulation of distinct evolutionary viral variants, with different levels of match to the vaccine strains. CONCLUSIONS: This study contributes to the understanding of the circulation of influenza B viruses in Italy. We found a continuous co-circulation of both B lineages in the period 2004-2017, and determined that children were particularly vulnerable to Victoria-lineage influenza B virus infections. An influenza B lineage mismatch with the trivalent vaccine occurred in about two-thirds of cases.

A heat-inactivated H7N3 vaccine induces cross-reactive cellular immunity in HLA-A2.1 transgenic mice.

BACKGROUND: Cross-reactive immunity against heterologous strains of influenza virus has the potential to provide partial protection in individuals that lack the proper neutralizing antibodies. In particular, the boosting of memory CD8+ T cell responses to conserved viral proteins can attenuate disease severity caused by influenza virus antigenic variants or pandemic strains. However, little is yet known about which of these conserved internal antigens would better induce and/or recall memory CD8+ T cells after in vivo administration of an inactivated whole virus vaccine. METHODS: We explored the CD8 + T cell responses to selected epitopes of the internal proteins of an H7N3 influenza virus that were cross-reactive with A/PR/8/34 virus in HLA-A2.1 transgenic (AAD) mice. RESULTS: CD8+ T cells against dominant and subdominant epitopes were detected upon infection of mice with live H7N3 virus, whereas immunization with non-replicating virus elicited CD8+ T cell responses against mostly immunodominant epitopes, which were rapidly recalled following infection with A/PR/8/34 virus. These vaccine-induced T cell responses were able to reduce the lung viral load in mice challenged intranasally with the heterologous influenza virus. CONCLUSIONS: A single immunization with non-replicating influenza virus vaccines may be able to elicit or recall cross-reactive CD8+ T cell responses to conserved immunodominant epitopes and, to some extent, counteract an infection by heterologous virus.

Characterization of an influenza B virus isolated from a fatal case of myocarditis in a pediatric patient in Italy.

Influenza B is one of the infective agents that can cause rapid and fatal myocarditis in children. Here, we describe a fatal case of myocarditis in a previously healthy child, after infection with an influenza B/Victoria-lineage virus during the 2022-23 epidemic season in Italy. Influenza B virus was isolated also in a second case, a younger family member showing only a mild influenza-like illness. Genotypic and phenotypic analyses have been performed on both virus samples and results showed that HA1 sequences were identical and genetically and antigenically related to other B viruses circulating in 2022-23 season in Italy. However, a D129N substitution was found in the receptor binding domain of the HA of the two viruses, not detected in other circulating viruses in Italy but only in a proportion of those circulating in other European countries. Phenotypic analyses assessed the susceptibility towards either neuraminidase inhibitors and baloxavir. Annual influenza vaccination remains one of the best interventions to prevent complications such as myocarditis, particularly in children.

Notch3 and canonical NF-kappaB signaling pathways cooperatively regulate Foxp3 transcription.

Notch3 overexpression has been previously shown to positively regulate the generation and function of naturally occurring regulatory T cells and the expression of Foxp3, in cooperation with the pTα/pre-TCR pathway. In this study, we show that Notch3 triggers the trans activation of Foxp3 promoter depending on the T cell developmental stage. Moreover, we discovered a novel CSL/NF-κB overlapping binding site within the Foxp3 promoter, and we demonstrate that the activation of NF-κB, mainly represented by p65-dependent canonical pathway, plays a positive role in Notch3-dependent regulation of Foxp3 transcription. Accordingly, the deletion of protein kinase C, which mediates canonical NF-κB activation, markedly reduces regulatory T cell number and per cell Foxp3 expression in transgenic mice with a constitutive activation of Notch3 signaling. Collectively, our data indicate that the cooperation among Notch3, protein kinase C, and p65/NF-κB subunit modulates Foxp3 expression, adding new insights in the understanding of the molecular mechanisms involved in regulatory T cell homeostasis and function.

Analysis of Genomic Characteristics of SARS-CoV-2 in Italy, 29 January to 27 March 2020.

We performed next-generation sequencing (NGS), phylogenetic analysis, gene flows, and N- and O-glycosylation prediction on SARS-CoV-2 genomes collected from lab-confirmed cases from different Italian regions. To this end, a total of 111 SARS-CoV-2 genomes collected in Italy between 29 January and 27 March 2020 were investigated. The majority of the genomes belonged to lineage B.1, with some descendant lineages. The gene flow analysis showed that the spread occurred mainly from the north to the center and to the south of Italy, as confirmed by epidemiological data. The mean evolutionary rate estimated here was 8.731 × 10-4 (95% highest posterior density, HPD intervals 5.809 × 10-4 to 1.19 × 10-3), in line with values reported by other authors. The dated phylogeny suggested that SARS-CoV-2 lineage B.1 probably entered Italy between the end of January and early February 2020. Continuous molecular surveillance is needed to trace virus circulation and evolution.

Immunogenicity of a recombinant influenza virus bearing both the CD4+ and CD8+ T cell epitopes of ovalbumin.

Recombinant influenza viruses that bear the single immunodominant CD8+ T cell epitope OVA(257-264) or the CD4+ T cell epitope OVA323₋339 of the model antigen ovalbumin (OVA) have been useful tools in immunology. Here, we generated a recombinant influenza virus, WSN-OVA(I/II), that bears both OVA-specific CD8+ and CD4+ epitopes on its hemagglutinin molecule. Live and heat-inactivated WSN-OVA(I/II) viruses were efficiently presented by dendritic cells in vitro to OT-I TCR transgenic CD8+ T cells and OT-II TCR transgenic CD4+ T cells. In vivo, WSN-OVA(I/II) virus was attenuated in virulence, highly immunogenic, and protected mice from B16-OVA tumor challenge in a prophylactic model of vaccination. Thus, WSN-OVA(I/II) virus represents an additional tool, along with OVA TCR transgenic mice, for further studies on T cell responses and may be of value in vaccine design.

Strategies to obtain multiple recombinant modified vaccinia Ankara vectors. Applications to influenza vaccines.

As a vaccination vector, MVA has been widely investigated both in animal models and humans. The construction of recombinant MVA (rMVA) relies on homologous recombination between an acceptor virus and a donor plasmid in infected/transfected permissive cells. Our construction strategy "Red-to-Green gene swapping" - based on the exchange of two fluorescent markers within the flanking regions of MVA deletion ΔIII, coupled to fluorescence activated cell sorting - is here extended to a second insertion site, within the flanking regions of MVA deletion ΔVI. Exploiting this strategy, both double and triple rMVA were constructed, expressing as transgenes the influenza A proteins HA, NP, M1, and PB1. Upon validation of the harbored transgenes co-expression, double and triple recombinants rMVA(ΔIII)-NP-P2A-M1 and rMVA(ΔIII)-NP-P2A-M1-(ΔVI)-PB1 were assayed for in vivo immunogenicity and protection against lethal challenge. In vivo responses were identical to those obtained with the reported combinations of single recombinants, supporting the feasibility and reliability of the present improvement and the extension of Red-to-Green gene swapping to insertion sites other than ΔIII.

Protective immunity against influenza in HLA-A2 transgenic mice by modified vaccinia virus Ankara vectored vaccines containing internal influenza proteins.

BACKGROUND: The emergence of novel strains of influenza A viruses with hemagglutinins (HAs) that are antigenically distinct from those circulating in humans, and thus have pandemic potential, pose concerns and call for the development of more broadly protective influenza vaccines. In the present study, modified vaccinia virus Ankara (MVA) encoding internal influenza antigens were evaluated for their immunogenicity and ability to protect HLA-A2.1 transgenic (AAD) mice from infection with influenza viruses. METHODS: MVAs expressing NP (MVA-NP), M1 (MVA-M1) or polymerase PB1 (MVA-PB1) of A/California/4/09 (CA/09) virus were generated and used to immunize AAD mice. Antibodies and CD8+T cell responses were assessed by ELISA and ELISPOT, respectively, and challenge experiments were performed by infecting vaccinated mice with CA/09 virus. RESULTS: CD8+T cells specific to immunodominant and subdominant epitopes on the internal influenza proteins were elicited by MVA-based vectors in AAD mice, whereas influenza-specific antibodies were detected only in MVA-NP-immunized mice. Both M1- and NP-based MVA vaccines, regardless of whether they were applied individually or in combination, conferred protection against lethal influenza virus challenge. CONCLUSION: Our data further emphasize the promising potential of MVA vector expressing internal antigens toward the development of a universal influenza vaccine.

Immunogenicity of modified vaccinia virus Ankara expressing the hemagglutinin stalk domain of pandemic (H1N1) 2009 influenza virus.

BACKGROUND: Vaccination offers protection against influenza, although current vaccines need to be reformulated each year. The development of a broadly protective influenza vaccine would guarantee the induction of heterosubtypic immunity also against emerging influenza viruses of a novel subtype. Vaccine candidates based on the stalk region of the hemagglutinin (HA) have the potential to induce broad and persistent protection against diverse influenza A viruses. METHODS: Modified vaccinia virus Ankara (MVA) expressing a headless HA (hlHA) of A/California/4/09 (CA/09) virus was used as a vaccine to immunize C57BL/6 mice. Specific antibody and cell-mediated immune responses were determined, and challenge experiments were performed by infecting vaccinated mice with CA/09 virus. RESULTS: Immunization of mice with CA/09-derived hlHA, vectored by MVA, was able to elicit influenza-specific broad cross-reactive antibodies and cell-mediated immune responses, but failed to induce neutralizing antibodies and did not protect mice against virus challenge. CONCLUSION: Although highly immunogenic, our vaccine was unable to induce a protective immunity against influenza. A misfolded and unstable conformation of the hlHA molecule may have affected its capacity of inducing neutralizing antiviral, conformational antibodies. Design of stable hlHA-based immunogens and their delivery by recombinant MVA-based vectors has the potential of improving this promising approach for a universal influenza vaccine.

PKC theta mediates pre-TCR signaling and contributes to Notch3-induced T-cell leukemia.

Protein kinase (PK)C theta is a critical regulator of mature T-cell activation and proliferation, being implicated in TCR-triggered nuclear factor (NF)-kappa B activation and providing important survival signals to leukemic T cells. We previously showed that overexpression of pT alpha/pre-TCR and constitutive activation of NF-kappa B characterize the T-cell leukemia/lymphoma developing in Notch3-IC transgenic mice. We report here that PKC theta is a downstream target of Notch3 signaling and that its activation and membrane translocation require a functional pre-TCR in order to trigger NF-kappa B activation in thymocytes and lymphoma cells of transgenic mice. Furthermore, deletion of PKC theta in Notch3-IC transgenic mice reduces the incidence of leukemia, correlating with decreased NF-kappa B activation. This paper therefore suggests that PKC theta mediates the activation of NF-kappa B by pre-TCR in immature thymocytes and contributes to the development of Notch3-dependent T-cell lymphoma.

Exploring mucosal immunization with a recombinant influenza virus carrying an HIV-polyepitope in mice with pre-existing immunity to influenza.

HIV-1 vaccines based on recombinant vectors have been developed to elicit immune responses; however, the failure of the STEP HIV-1 vaccine trial has caused concern regarding the impact on vaccine efficacy of pre-existing vector seropositivity in humans. By using a mouse model of infection, we evaluated the immune responses elicited by intranasal and vaginal immunization with the recombinant influenza virus WSN/CKG carrying the PCLUS3-P18 peptide and a Gag epitope in its hemagglutinin, and the impact of pre-existing vector immunity on protection against recombinant vaccinia virus challenge. We found that despite the protective immunity induced in naïve mice by the WSN/CKG virus via either route, the vaginal immunization of mice with pre-existing influenza immunity restricted vPE16 replication more significantly in the ovaries than intranasal immunization. Thus, successful vaccination strategies under limiting conditions, such as pre-existing vector immunity, require the local induction of mucosal immunity at the site of virus infection.

Modified vaccinia virus Ankara expressing the hemagglutinin of pandemic (H1N1) 2009 virus induces cross-protective immunity against Eurasian 'avian-like' H1N1 swine viruses in mice.

OBJECTIVES: To examine cross-reactivity between hemagglutinin (HA) derived from A/California/7/09 (CA/09) virus and that derived from representative Eurasian "avian-like" (EA) H1N1 swine viruses isolated in Italy between 1999 and 2008 during virological surveillance in pigs. DESIGN: Modified vaccinia virus Ankara (MVA) expressing the HA gene of CA/09 virus (MVA-HA-CA/09) was used as a vaccine to investigate cross-protective immunity against H1N1 swine viruses in mice. SAMPLE: Two classical swine H1N1 (CS) viruses and four representative EA-like H1N1 swine viruses previously isolated during outbreaks of respiratory disease in pigs on farms in Northern Italy were used in this study. SETTING: Female C57BL/6 mice were vaccinated with MVA/HA/CA/09 and then challenged intranasally with H1N1 swine viruses. MAIN OUTCOME MEASURES: Cross-reactive antibody responses were determined by hemagglutination- inhibition (HI) and virus microneutralizing (MN) assays of sera from MVA-vaccinated mice. The extent of protective immunity against infection with H1N1 swine viruses was determined by measuring lung viral load on days 2 and 4 post-challenge. RESULTS AND CONCLUSIONS: Systemic immunization of mice with CA/09-derived HA, vectored by MVA, elicited cross-protective immunity against recent EA-like swine viruses. This immune protection was related to the levels of cross-reactive HI antibodies in the sera of the immunized mice and was dependent on the similarity of the antigenic site Sa of H1 HAs. Our findings suggest that the herd immunity elicited in humans by the pandemic (H1N1) 2009 virus could limit the transmission of recent EA-like swine HA genes into the influenza A virus gene pool in humans.

Enhancement of T cell-mediated immune responses to whole inactivated influenza virus by chloroquine treatment in vivo.

Current influenza vaccines induce poor cross-reactive CD8+ T cell responses. Cellular immunity is generally specific for epitopes that are remarkably conserved among different subtypes, suggesting that strategies to improve the cross-presentation of viral antigens by dendritic cells (DC) could elicit a broadly protective immune response. Previous studies have shown that limited proteolysis within the endocytic pathway can favorably influence antigen processing and thus immune responses. Herein, we demonstrate that chloroquine improves the cross-presentation of non-replicating influenza virus in vitro and T cell responses in mice following a single administration of inactivated HI-X31 virus. CD8+ T cells were also recruited to lymph nodes draining the site of infection and able to reduce viral load following pulmonary challenge with the heterologous PR8 virus. These findings may have implications for vaccination strategies aimed at improving the cross-presentation capacity of DCs and thus the size of effector and memory CD8+ T cells against influenza vaccines.

Cross-talk between tumor and endothelial cells involving the Notch3-Dll4 interaction marks escape from tumor dormancy.

The Notch ligand Dll4 has a recognized role during both physiologic and tumor angiogenesis, as it contributes to regulate Notch activity in endothelial cells (EC). The effects of Dll4 on Notch signaling in tumor cells expressing Notch receptors remain, however, largely unknown. Here, we report that escape of human T-cell acute lymphoblastic leukemia (T-ALL) cells or colorectal cancer cells from dormancy is associated with Dll4 expression in the tumor microenvironment and increased Notch3 signaling in tumor cells. Dll4 was expressed at early time points during the angiogenic process, and its expression preceded perfusion of the newly established vessels. Treatment of EC with angiogenic factors induced Dll4 expression and increased Notch3 activation in cocultured T-ALL cells. Neutralization of Dll4 greatly reduced EC-mediated activation of Notch 3 signaling in T-ALL cells and blocked tumorigenesis. Moreover, silencing Notch3 by RNA interference had marked antiproliferative and proapoptotic effects on T-ALL cells in vitro and reduced tumorigenicity in vivo. Our results elucidate a novel mechanism by which a direct interplay between endothelial and tumor cells promotes survival and triggers tumor growth.

Notch3 and pre-TCR interaction unveils distinct NF-kappaB pathways in T-cell development and leukemia.

Notch signaling plays a critical role in T-cell differentiation and leukemogenesis. We previously demonstrated that, while pre-TCR is required for thymocytes proliferation and leukemogenesis, it is dispensable for thymocyte differentiation in Notch3-transgenic mice. Notch3-transgenic premalignant thymocytes and T lymphoma cells overexpress pTalpha/pre-TCR and display constitutive activation of NF-kappaB, providing survival signals for immature thymocytes. We provide genetic and biochemical evidence that Notch3 triggers multiple NF-kappaB activation pathways. A pre-TCR-dependent pathway preferentially activates NF-kappaB via IKKbeta/IKKalpha/NIK complex, resulting in p50/p65 heterodimer nuclear entry and recruitment onto promoters of Cyclin D1, Bcl2-A1 and IL7-receptor-alpha genes. In contrast, upon pTalpha deletion, Notch3 binds IKKalpha and maintains NF-kappaB activation through an alternative pathway, depending on an NIK-independent IKKalpha homodimer activity. The consequent NF-kappaB2/p100 processing allows nuclear translocation of p52/RelB heterodimers, which only trigger transcription from Bcl2-A1 and IL7-receptor-alpha genes. Our data suggest that a finely tuned interplay between Notch3 and pre-TCR pathways converges on regulation of NF-kappaB activity, leading to differential NF-kappaB subunit dimerization that regulates distinct gene clusters involved in either cell differentiation or proliferation/leukemogenesis.