bff8ceda83ac6aaf2457a018256d71cb pmc_11209067.pdf ece4989d5c8b01b291b1174a0e24a7ea4c7b235e pmc_11209067.pdf 344909fd1d94fa7910c90a98dea4e3cc429fb3a7232450b114d54f649d868fb8 pmc_11209067.pdf Title: Development of a Fully Protective Pandemic Avian Influenza Subunit Vaccine in Insect Pupae Subject: In this study, we pioneered an alternative technology for manufacturing subunit influenza hemagglutinin (HA)-based vaccines. This innovative method involves harnessing the pupae of the Lepidoptera Trichoplusia ni (T. ni) as natural biofactories in combination with baculovirus vectors (using CrisBio® technology). We engineered recombinant baculoviruses encoding two versions of the HA protein (trimeric or monomeric) derived from a pandemic avian H7N1 virus A strain (A/chicken/Italy/5093/99). These were then used to infect T. ni pupae, resulting in the production of the desired recombinant antigens. The obtained HA proteins were purified using affinity chromatography, consistently yielding approximately 75 mg/L of insect extract. The vaccine antigen effectively immunized poultry, which were subsequently challenged with a virulent H7N1 avian influenza virus. Following infection, all vaccinated animals survived without displaying any clinical symptoms, while none of the mock-vaccinated control animals survived. The CrisBio®-derived antigens induced high titers of HA-specific antibodies in the vaccinated poultry, demonstrating hemagglutination inhibition activity against avian H7N1 and human H7N9 viruses. These results suggest that the CrisBio® technology platform has the potential to address major industry challenges associated with producing recombinant influenza subunit vaccines, such as enhancing production yields, scalability, and the speed of development, facilitating the global deployment of highly effective influenza vaccines. Keywords: pandemic influenza; baculovirus; Trichoplusia ni; insect pupae; subunit vaccine Author: Ana Falcón, Susana Martínez-Pulgarín, Sergi López-Serrano, Edel Reytor, Miguel Cid, Maria del Carmen Nuñez, Lorena Córdoba, Ayub Darji and José M. Escribano Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25 CreationDate: Thu May 23 11:45:50 2024 CEST ModDate: Thu May 23 11:48:45 2024 CEST Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 14 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 2629312 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- XWOCXD+VnURWPalladioL-Bold Type 1 Custom yes yes yes 10 0 BWRUVA+URWPalladioL-Bold Type 1 Custom yes yes yes 16 0 ZZBRTY+URWPalladioL-Roma Type 1 Custom yes yes yes 22 0 EAVYGE+URWPalladioL-Ital Type 1 Custom yes yes yes 27 0 NBMIUS+VnURWPalladioL Type 1 Custom yes yes yes 32 0 HOWKDX+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 48 0 CAMXTK+EURM10 Type 1 Builtin yes yes yes 65 0 PDYDVH+CMSY10 Type 1 Builtin yes yes yes 73 0 GGAKBH+Symbol Type 1C Custom yes yes yes 91 0 GGAJIF+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 96 0 GGAJIH+PalatinoLinotype TrueType WinAnsi yes yes no 99 0 GGAJJJ+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 102 0 GGAKBH+Symbol Type 1C Custom yes yes yes 130 0 GGAJIF+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 135 0 GGAJIH+PalatinoLinotype TrueType WinAnsi yes yes no 138 0 GGAJJJ+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 141 0 GGAKBH+Symbol Type 1C Custom yes yes yes 158 0 GGAJIF+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 163 0 GGAJIH+PalatinoLinotype TrueType WinAnsi yes yes no 166 0 GGAJJJ+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 169 0 GGAKBH+Symbol Type 1C Custom yes yes yes 196 0 GGAJIF+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 201 0 GGAJIH+PalatinoLinotype TrueType WinAnsi yes yes no 204 0 GGAJJJ+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 207 0 GGAKBH+Symbol Type 1C Custom yes yes yes 224 0 GGAJIF+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 229 0 GGAJIH+PalatinoLinotype TrueType WinAnsi yes yes no 232 0 GGAJJJ+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 235 0 GGAPDK+Calibri CID TrueType Identity-H yes yes yes 238 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2025-02-27 02:10:17 CET RepresentationInformation: pmc_11209067.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2025-02-27 02:03:32 CET Size: 2629312 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 389 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Materials and Methods Destination: section.2 Children: Item: Title: Cell Culture Destination: subsection.2.1 Item: Title: Baculovirus Generation Destination: subsection.2.2 Children: Item: Title: Mel-H7-His Destination: subsubsection.2.2.1 Item: Title: Mel-H7-FD4-His Destination: subsubsection.2.2.2 Item: Title: Pupae Production Process Destination: subsection.2.3 Item: Title: Infecting Pupae and Expressing Recombinant Proteins Destination: subsection.2.4 Item: Title: Protein Extraction and Analysis Destination: subsection.2.5 Item: Title: Western Blot Destination: subsection.2.6 Item: Title: Virus Destination: subsection.2.7 Item: Title: Animals and Experimental Design Destination: subsection.2.8 Item: Title: Disease Monitoring Destination: subsection.2.9 Item: Title: Real-Time Quantitative PCR (RT-qPCR) Destination: subsection.2.10 Item: Title: H7-Specific Enzyme-Linked Immunosorbent Assays (ELISA) Destination: subsection.2.11 Item: Title: Hemagglutination Inhibition (HI) Assay Destination: subsection.2.12 Item: Title: Statistical Analysis Destination: subsection.2.13 Item: Title: Results Destination: section.3 Children: Item: Title: Production of Monomeric and Trimeric Versions of HA Using CrisBio® Technology Destination: subsection.3.1 Item: Title: Protection of Vaccinated Poultry against a Highly Virulent H7N1 Influenza Virus Destination: subsection.3.2 Item: Title: Antibody Response in Vaccinated Poultry Destination: subsection.3.3 Item: Title: Discussion Destination: section.4 Item: Title: References Destination: section.5 Info: Title: Development of a Fully Protective Pandemic Avian Influenza Subunit Vaccine in Insect Pupae Author: Ana Falcón, Susana Martínez-Pulgarín, Sergi López-Serrano, Edel Reytor, Miguel Cid, Maria del Carmen Nuñez, Lorena Córdoba, Ayub Darji and José M. Escribano Subject: In this study, we pioneered an alternative technology for manufacturing subunit influenza hemagglutinin (HA)-based vaccines. This innovative method involves harnessing the pupae of the Lepidoptera Trichoplusia ni (T. ni) as natural biofactories in combination with baculovirus vectors (using CrisBio® technology). We engineered recombinant baculoviruses encoding two versions of the HA protein (trimeric or monomeric) derived from a pandemic avian H7N1 virus A strain (A/chicken/Italy/5093/99). These were then used to infect T. ni pupae, resulting in the production of the desired recombinant antigens. The obtained HA proteins were purified using affinity chromatography, consistently yielding approximately 75 mg/L of insect extract. The vaccine antigen effectively immunized poultry, which were subsequently challenged with a virulent H7N1 avian influenza virus. Following infection, all vaccinated animals survived without displaying any clinical symptoms, while none of the mock-vaccinated control animals survived. The CrisBio®-derived antigens induced high titers of HA-specific antibodies in the vaccinated poultry, demonstrating hemagglutination inhibition activity against avian H7N1 and human H7N9 viruses. These results suggest that the CrisBio® technology platform has the potential to address major industry challenges associated with producing recombinant influenza subunit vaccines, such as enhancing production yields, scalability, and the speed of development, facilitating the global deployment of highly effective influenza vaccines. 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