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

Mario, Giuseppina Di; Soprana, Elisa; Gubinelli, Francesco; Panigada, Maddalena; Facchini, Marzia; Fabiani, Concetta; Garulli, Bruno; Basileo, Michela; Cassone, Antonio; Siccardi, Antonio G.; Donatelli, Isabella; Castrucci, Maria Rita

doi:10.1080/20477724.2016.1275464

Cited by 3 publications

(3 citation statements)

References 32 publications

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“…It was inserted in the MCS of both TPs, yielding TP-G- M1-V5 and TP-HAG- M1-V5. The same transgene, in a previous construct (here called rMVA-M1-V5-Rome) was tested as an experimental vaccine [ 12 ]. In this paper, rMVA-M1-V5-Rome DNA is used as a positive control for PCR and Western Blot analyses.…”

Section: Methodsmentioning

confidence: 99%

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

Boselli,

Panigada,

Di Terlizzi

et al. 2023

J Transl Med

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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. Although faster than other methods to obtain recombinant MVAs, the standard RGSSM still is time-consuming, taking at least 25 days to yield the final product. Methods The direct sorting of fluorescent virions is made amenable by the marker HAG, a flu hemagglutinin/EGFP fusion protein, integrated into the external envelope of extracellular enveloped virions (EEVs). Fluorescent EEVs-containing supernatants of infected cultures are used instead of purified virus. Direct Virus-Sorting was performed on BD FACSAria Fusion cell sorter equipped with 4 lasers and a 100-mm nozzle, with 20 psi pressure and a minimal flow rate, validated using Megamix beads. Results Upon infection of cells with recombinant EEVs, at the first sorting step virions that contain HAG are harvested and cloned, while the second sorting step yields EEVs that have lost HAG, allowing to clone untagged rMVA. Because only virion-containing supernatants are used, no virus purification steps and fewer sortings are necessary. Therefore, the final untagged rMVA product can be obtained in a mere 8 days. Conclusions Altogether, we report that the original RGSSM has been markedly improved in terms of time- and cost efficiency by substituting Cell-Sorting with direct Virus-Sorting from the supernatants of infected cells. The improved virometry-based RGGSM may find wide applicability, considering that rMVAs hold great promise to serve as personalized vaccines for therapeutic intervention against cancer and various types of infectious diseases. Graphical Abstract

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Section: Methodsmentioning

confidence: 99%

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

Boselli,

Panigada,

Di Terlizzi

et al. 2023

J Transl Med

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“…Following this protocol, from colorless infected cells untagged rMVAs are obtained, that are suitable for both research [9][10][11][12][13][14] and biotechnological purposes [5,15]. The RGSM offers an elegant method to produce untagged rMVA, nevertheless the procedure is laborious, time-consuming, and costly.…”

Section: Introductionmentioning

confidence: 99%

Time- and Cost-effective Production of Untagged Recombinant Mva by Flow Virometry and Direct Virus Sorting

Daniela,

Panigada,

Terlizzi

et al. 2023

Preprint

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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. Although faster than other methods to obtain recombinant MVAs, the standard RGSSM still is time-consuming, taking at least 25 days to yield the final product. Methods The direct sorting of fluorescent virions is made amenable by the marker HAG, a flu hemagglutinin/ EGFP fusion protein, integrated into the external envelope of extracellular enveloped virions (EEVs). Fluorescent EEVs-containing supernatants of infected cultures are used instead of purified virus. Direct Virus-Sorting was performed on BD FACSAria Fusion cell sorter equipped with 4 lasers and a 100-mm nozzle, with 20 psi pressure and a minimal flow rate, validated using Megamix beads. Results Upon infection of cells with recombinant EEVs, at the first sorting step virions that contain HAG are harvested and cloned, while the second sorting step yields EEVs that have lost HAG, allowing to clone untagged rMVA. Because only virion-containing supernatants are used, no virus purification steps and fewer sortings are necessary. Therefore, the final untagged rMVA product can be obtained in a mere 8 days. Conclusions Altogether, we report that the original RGSSM has been markedly improved in terms of time- and cost efficiency by substituting Cell-Sorting with direct Virus-Sorting from the supernatants of infected cells. The improved virometry-based RGGSM may find wide applicability, considering that rMVAs hold great promise to serve as personalized vaccines for therapeutic intervention against cancer and various types of infectious diseases.

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“…To target the more conserved immunosubdominant epitopes in HA, several different strategies of vaccine antigen design have been attempted based on influenza A H1 HA, H3 HA, or influenza B HA, such as chimeric HA (cHA) or “mosaic” HA composed of the immunodominant head domain or amino acid residues derived from exotic HA subtypes ( 1 - 6 ), hyper-glycosylated HAs to shield immunodominant epitopes ( 7 , 8 ), and headless HAs ( 9 - 16 ). Here, based on H3 HA, we devised another strategy to design a universal vaccine candidate with the idea of “diluting out” the immunodominancy of the head domain: scrambled HA (scrHA), which has a variety of amino acids at the antigenically dominant loci in the HA head domain.…”

Section: Introductionmentioning

confidence: 99%

Influenza H3 hemagglutinin vaccine with scrambled immunodominant epitopes elicits antibodies directed toward immunosubdominant head epitopes

Chiba

Kong

Kawaoka

2023

mBio

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Vaccination is the most effective countermeasure to reduce the severity of influenza. Current seasonal influenza vaccines mainly elicit humoral immunity targeting hemagglutinin (HA). In particular, the amino acid residues around the receptor-binding site in the HA head domain are predominantly targeted by humoral immunity as “immunodominant” epitopes. However, mutations readily accumulate in the head domain due to high plasticity, resulting in antigenic drift and vaccine mismatch, particularly with influenza A (H3N2) viruses. A vaccine strategy that targets more conserved immunosubdominant epitopes is required to attain a universal vaccine. Here, we designed an H3 HA vaccine antigen with various amino acids at immunodominant epitopes of the HA head domain, termed scrambled HA (scrHA). In ferrets, scrHA vaccination induced lower serum neutralizing antibody levels against homologous virus compared with wild-type (WT) HA vaccination; however, similar levels of moderately neutralizing titers against antigenically distinct H3N2 viruses were observed. Ferrets vaccinated with scrHA twice and then challenged with homologous or heterologous virus showed the same level of reduced virus shedding in nasal swabs as WT HA-vaccinated animals but reduced body temperature increase, whereas WT HA-vaccinated ferrets exhibited body temperature increases similar to those of mock-vaccinated animals. scrHA elicited antibodies against HA immunodominant and -subdominant epitopes at lower and higher levels, respectively, than WT HA vaccination, whereas antistalk antibodies were induced at the same level for both groups, suggesting scrHA-induced redirection from immunodominant to immunosubdominant head epitopes. scrHA vaccination thus induced broader coverage than WT HA vaccination by diluting out the immunodominancy of HA head epitopes. IMPORTANCE Current influenza vaccines mainly elicit antibodies that target the immunodominant head domain, where strain-specific mutations rapidly accumulate, resulting in frequent antigenic drift and vaccine mismatch. Targeting conserved immunosubdominant epitopes is essential to attain a universal vaccine. Our findings with the scrHA developed in this study suggest that designing vaccine antigens that “dilute out” the immunodominancy of the head epitopes may be an effective strategy to induce conserved immunosubdominant epitope-based immune responses.

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

Cited by 3 publications

References 32 publications

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

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

Time- and Cost-effective Production of Untagged Recombinant Mva by Flow Virometry and Direct Virus Sorting

Influenza H3 hemagglutinin vaccine with scrambled immunodominant epitopes elicits antibodies directed toward immunosubdominant head epitopes

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