Time- and cost-effective production of untagged recombinant MVA by flow virometry and direct virus sorting.

Background: Recombinant MVAs (rMVAs) are widely used both in basic and clinical research. Our previously developed Red-to-Green Gene Swapping Method (RGGSM), a cytometry-based Cell-Sorting protocol, revolves around the transient expression of a green fluorescent cytoplasmic marker, to subsequently obtain purified untagged rMVA upon loss of that marker by site-specific recombination. The standard RGSSM is quite costly in terms of bench work, reagents, and Sorting Facility fees.

A Genetic Vaccine Encoding Shared Cancer Neoantigens to Treat Tumors with Microsatellite Instability.

Tumors with microsatellite instability (MSI) are caused by a defective DNA mismatch repair system that leads to the accumulation of mutations within microsatellite regions. Indels in microsatellites of coding genes can result in the synthesis of frameshift peptides (FSP). FSPs are tumor-specific neoantigens shared across patients with MSI.

The importance of naturally attenuated SARS-CoV-2in the fight against COVID-19.

The current SARS-CoV-2 pandemic is wreaking havoc throughout the world and has rapidly become a global health emergency. A central question concerning COVID-19 is why some individuals become sick and others not. Many have pointed already at variation in risk factors between individuals.

Multi-antigen Vaccination With Simultaneous Engagement of the OX40 Receptor Delays Malignant Mesothelioma Growth and Increases Survival in Animal Models.

Malignant Mesothelioma (MM) is a rare and highly aggressive cancer that develops from mesothelial cells lining the pleura and other internal cavities, and is often associated with asbestos exposure. To date, no effective treatments have been made available for this pathology. Herein, we propose a novel immunotherapeutic approach based on a unique vaccine targeting a series of antigens that we found expressed in different MM tumors, but largely undetectable in normal tissues.

Corrigendum: HIV-1 Env associates with HLA-C free-chains at the cell membrane modulating viral infectivity.

This corrects the article DOI: 10.1038/srep40037.

A Combined NMR-Computational Study of the Interaction between Influenza Virus Hemagglutinin and Sialic Derivatives from Human and Avian Receptors on the Surface of Transfected Cells.

The development of small-molecule inhibitors of influenza virus Hemagglutinin could be relevant to the opposition of the diffusion of new pandemic viruses. In this work, we made use of Nuclear Magnetic Resonance (NMR) spectroscopy to study the interaction between two derivatives of sialic acid, Neu5Ac-α-(2,6)-Gal-β-(1⁻4)-GlcNAc and Neu5Ac-α-(2,3)-Gal-β-(1⁻4)-GlcNAc, and hemagglutinin directly expressed on the surface of recombinant human cells. We analyzed the interaction of these trisaccharides with 293T cells transfected with the H5 and H1 variants of hemagglutinin, which thus retain their native trimeric conformation in such a realistic environment.

NMR interaction studies of Neu5Ac-α-(2,6)-Gal-β-(1-4)-GlcNAc with influenza-virus hemagglutinin expressed in transfected human cells.

The emergence of escape-mutants of influenza hemagglutinin (HA) following vaccination compels the yearly re-formulation of flu vaccines. Since binding the sialic acid receptor remains in all cases essential for infection, small-molecule inhibitors of HA binding to sialic acid could be interesting therapeutic complements or alternatives to immuno-prophylaxis in the control of flu epidemics. In this work, we made use of NMR spectroscopy to study the interaction between a derivative of sialic acid (the Neu5Ac-α-(2,6)-Gal-β-(1-4)-GlcNAc trisaccharide) and HAs (H1 and H5) from human and avian strains of influenza virus, directly expressed on the surface of stable transfected 293 T human cells.

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.

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.

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.

HIV-1 Env associates with HLA-C free-chains at the cell membrane modulating viral infectivity.

HLA-C has been demonstrated to associate with HIV-1 envelope glycoprotein (Env). Virions lacking HLA-C have reduced infectivity and increased susceptibility to neutralizing antibodies. Like all others MHC-I molecules, HLA-C requires β-microglobulin (βm) for appropriate folding and expression on the cell membrane but this association is weaker, thus generating HLA-C free-chains on the cell surface.

Cutting Edge: IgE Plays an Active Role in Tumor Immunosurveillance in Mice.

Exogenous IgE acts as an adjuvant in tumor vaccination in mice, and therefore a direct role of endogenous IgE in tumor immunosurveillance was investigated. By using genetically engineered mice, we found that IgE ablation rendered mice more susceptible to the growth of transplantable tumors. Conversely, a strengthened IgE response provided mice with partial or complete resistance to tumor growth, depending on the tumor type.

Preclinical development of HIvax: Human survivin highly immunogenic vaccines.

Our previous work involved the development of a recombinant fowlpox virus encoding survivin (FP-surv) vaccine that was evaluated for efficacy in mesothelioma mouse models. Results showed that FP-surv vaccination generated significant immune responses, which led to delayed tumor growth and improved animal survival. We have extended those previous findings in the current study, which involves the pre-clinical development of an optimized version of FP-surv designed for human immunization (HIvax).

Mucosal delivery of a vectored RSV vaccine is safe and elicits protective immunity in rodents and nonhuman primates.

Respiratory Syncytial Virus (RSV) is a leading cause of severe respiratory disease in infants and the elderly. No vaccine is presently available to address this major unmet medical need. We generated a new genetic vaccine based on chimpanzee Adenovirus (PanAd3-RSV) and Modified Vaccinia Ankara RSV (MVA-RSV) encoding the F, N, and M2-1 proteins of RSV, for the induction of neutralizing antibodies and broad cellular immunity.

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.

Fowlpox-based survivin vaccination for malignant mesothelioma therapy.

Survivin protein is an attractive candidate for cancer immunotherapy since it is abundantly expressed in most common human cancers and mostly absent in normal adult tissues. Malignant mesothelioma (MM) is a deadly cancer associated with asbestos or erionite exposure for which no successful therapies are currently available. In this study, we evaluated the therapeutic efficacy of a novel survivin-based vaccine by subcutaneous or intraperitoneum injection of BALB/c mice with murine fiber-induced MM tumor cells followed by vaccination with recombinant Fowlpox virus replicons encoding survivin.

A human monoclonal antibody with neutralizing activity against highly divergent influenza subtypes.

The interest in broad-range anti-influenza A monoclonal antibodies (mAbs) has recently been strengthened by the identification of anti-hemagglutinin (HA) mAbs endowed with heterosubtypic neutralizing activity to be used in the design of "universal" prophylactic or therapeutic tools. However, the majority of the single mAbs described to date do not bind and neutralize viral isolates belonging to highly divergent subtypes clustering into the two different HA-based influenza phylogenetic groups: the group 1 including, among others, subtypes H1, H2, H5 and H9 and the group 2 including, among others, H3 subtype. Here, we describe a human mAb, named PN-SIA28, capable of binding and neutralizing all tested isolates belonging to phylogenetic group 1, including H1N1, H2N2, H5N1 and H9N2 subtypes and several isolates belonging to group 2, including H3N2 isolates from the first period of the 1968 pandemic.

An antitumor cellular vaccine based on a mini-membrane IgE.

The IgE-mediated immune system activation can be redirected to combat tumors. Mouse and human IgE have been shown to provide a potent adjuvant effect in antitumor vaccination, with a crucial role played by FcεRI. This effect results from T cell-mediated adaptive immune response.

Joint production of prime/boost pairs of Fowlpox Virus and Modified Vaccinia Ankara recombinants carrying the same transgene.

Pairs of recombinant MVA (Modified Vaccinia Ankara) and FPV (Fowlpox Virus) expressing the same transgene are reasonable candidates for prime/boost regimens, because cross-reacting immune responses between the two vectors, both non-replicative in mammalian hosts, are very limited. The acceptor virus FPD-Red, a derivative of FPV, carrying a red fluorescent protein gene flanked by the homology regions of MVA deletion III, was constructed. The same MVA Transfer Plasmid Green, designed to insert transgenes into the MVA deletion III locus, can therefore be used to transfer transgenes into both acceptor viruses MVA-Red and FPD-Red with the described recently Red-to-Green gene swapping method.

The combination of marker gene swapping and fluorescence-activated cell sorting improves the efficiency of recombinant modified vaccinia virus Ankara vaccine production for human use.

Modified vaccinia virus Ankara (MVA) is employed as a human vaccine vector for the high expression of heterologous genes and the lack of replication in mammalian cells. This study demonstrates that cells infected by recombinant viruses can be obtained by fluorescence-activated cell sorting. Recombinant viruses are generated by a swapping event between a red fluorescent protein gene in the acceptor virus and a plasmid cassette coding for both a green fluorescent marker and a transgene.

HLA-C is necessary for optimal human immunodeficiency virus type 1 infection of human peripheral blood CD4 lymphocytes.

The hypothesis that open conformers of HLA-C on target cells might directly exert an effect on their infectability by human immunodeficiency virus (HIV) has been suggested previously. This was tested by exploiting the peculiar specificity of monoclonal antibody (mAb) L31 for HLA-C open conformers to show that normal levels of Env-driven fusion were restored in HLA-C transfectants of a major histocompatibility complex-deleted (fusion-incompetent) cell line. The physiological relevance of this finding is now confirmed in this report, where small interfering RNA (siRNA) technology was used to silence HLA-C expression in peripheral blood lymphocytes (PBLs) from 11 healthy donors.

Antitumor IgE adjuvanticity: key role of Fc epsilon RI.

Working with C57BL/6 mouse tumor models, we had previously demonstrated that vaccination with IgE-coated tumor cells can protect against tumor challenge, an observation that supports the involvement of IgE in antitumor immunity. The adjuvant effect of IgE was shown to result from eosinophil-dependent priming of the T cell-mediated adaptive immune response. The protective effect is likely to be mediated by the interaction of tumor cell-bound IgE with receptors, which then trigger the release of mediators, recruitment of effector cells, cell killing and tumor Ag cross-priming.

Marker gene swapping facilitates recombinant Modified Vaccinia Virus Ankara production by host-range selection.

Modified Vaccinia Virus Ankara (MVA) is employed widely as an experimental and human vaccine vector for its lack of replication in mammalian cells and high expression of heterologous genes. Recombinant MVA technology can be improved greatly by combining transient host-range selection (based on the restoration in MVA of the deleted vaccinia gene K1L) with the differential expression of fluorescent proteins. Recombinant virus results from swapping a red protein gene (in the acceptor virus) with a cassette of the transfer plasmid comprising the transgene and the green marker K1Lgfp (a chimeric gene comprising K1L and EGFP).