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1. Generation of Recombinant Live Attenuated Influenza Vaccines by Reverse Genetics
NOTE: The detailed protocol for the generation of recombinant influenza viruses by reverse genetics has been described by previous studies&#160;and is out of the scope of this report. Briefly, rescue of cold-adapted influenza vaccines (CAV) is done in a biosafety class II cabinet under biosafety level 2 (BSL2) containment, and involves steps detailed below.
<ol>
	<li>Generate a reassortant cold-adapted influenza vaccine using as background the cold-adapted strain A/Ann Arbor/6/60 (H2N2), as well as the hemagglutinin (HA) and a modified neuraminidase (NA) of the A/PR/8/34 (H1N1) strain (Figure 1A). The modification in the NA gene consists of a PCR-based replacement of a conserved sequence in the protein stalk (residues 65 to 72) by a short cDNA sequence encoding the chicken ovalbumin (OVA)-derived peptide SIINFEKL (Figure 1B). The SIINFEKL peptide is a highly immunodominant peptide in the context of the H-2b MHC class I-restricted response of C57BL/6 mice.</li>
	<li>Plate 106&#160;293T cells per well in 6-well plates using DMEM 10% fetal bovine serum (FBS) supplemented with 1% penicillin/streptomycin (P/E).</li>
	<li>Prepare plasmid transfection mixture: Add 1 &#181;g of each one of the plasmids encoding cDNAs for PB2, PB1, PA, NP, M, NS from the influenza A/Ann Arbor/6/60 strain and 1 &#181;g of the plasmids encoding for the NA-SIINFEKL and HA from influenza A/PR/8/34. Add pre-warmed Opti- minimal essential medium (Opti-MEM) (15 &#181;l for a final volume of 100 &#181;l/well).</li>
	<li>Incubate transfection mix for 30 min at RT.</li>
	<li>Add 100 &#181;l of transfection mix/well and incubate for 16 hr&#160;at 37 &#176;C and 5% CO2.</li>
	<li>Change cell media by DMEM 0.3% BSA 1% P/E at 33 &#176;C and 5% CO2&#160;for 5 hr.</li>
	<li>Add 106&#160;influenza permissive Madin-Darby Canine Kidney (MDCK) cells in dimethyl sulfoxide (DMSO) containing 1 &#181;g/ml of trypsin from bovine pancreas treated with L-1-Tosylamide-2-phenylethyl chloromethyl ketone (TPCK-trypsin). Maintain the 293T-MDCK co-cultures for 2 - 3 days at 33 &#176;C and 5% CO2.</li>
	<li>Collect supernatants of MDCK-293T cell co-cultures and clear by centrifugation at 260 x g for 5 min.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM 1X)</td>
			<td>Gibco RL-Life Technologies</td>
			<td>41965-039</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti MEM</td>
			<td>Gibco RL-Life Technologies</td>
			<td>31985-047</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Invitrogen-Life Technologies</td>
			<td>11668-027</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10.000 U/ml)</td>
			<td>PAA</td>
			<td>p11-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr>
			<td>Dymethil-Sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-TPCK</td>
			<td>Sigma-Aldrich</td>
			<td>T1426</td>
			<td></td>
		</tr>
	</tbody>
</table>

generating  recombinant live-attenuated influenza viruses for vaccines

Source: P&#233;rez-Gir&#243;n, J. V. et al, <a href="https://app.jove.com/v/52803">Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity.</a>&#160;J. Vis. Exp. (2015)
The video showcases a method to create cold-adapted recombinant influenza viruses. It involves transfecting human cells with bi-directional plasmids, followed by incubation at lower temperature incubation to yield viruses suitable for vaccine production, characterized by reduced replication in warmer conditions.

<img alt="Figure 1" src="/files/ftp_upload/22254/22254_Figure1.jpg" /> 
Figure 1.&#160;Engineering of traceable LAIVs.&#160;(A)&#160;Influenza segments cloned in ambisense plasmids were transfected into 293T cells using Lipofectamine 2000. 24 hr after transfection, supernatants obtained from 293T cells were used to coat influenza-permissive Madin-Darby canine kidney (MDCK) cells in the presence of 1% of TPCK trypsin. All the protocol was performed at...

Generating  Recombinant Live-Attenuated Influenza Viruses for Vaccines

1. Generation of Recombinant Live Attenuated Influenza Vaccines by Reverse Genetics
NOTE: The detailed protocol for the generation of recombinant ...

Begin with bi-directional plasmids carrying cDNA encoding certain viral essential proteins of a cold-adapted influenza strain.
Introduce a new set of bi-directional plasmids containing hemagglutinin and modified neuraminidase cDNA from a different influenza strain.
Introduce a transfection reagent to this mix and incubate to form a complex with the plasmids.
Transfer these complexes onto cultured human epithelial-like cells to enable plasmid entry into the cell.
The bi-directional plasmids with two promoters in opposite orientations enable the production of viral proteins and their corresponding RNA.
Incubate the cells at a lower temperature.
The viral proteins and the viral RNAs self-assemble to form cold-adapted recombinant influenza viruses with reduced replication in warmer conditions.
Overlay with sialic acid-overexpressing MDCK cells and modified trypsin enzymes that allow viral attachment and entry inside cells.
Incubate at the same temperature to induce replication and production of new viruses.
Centrifuge and harvest the recombinant live-attenuated influenza viruses ready for a vaccine preparation.&#160;

generating-recombinant-live-attenuated-influenza-viruses-for-vaccines

Research

JoVE Encyclopedia of Experiments

Immunology

Immunotherapy

Immunogenics and Vaccine Development

91 Views. Fonte: Pérez-Girón, J. V. et al, Somministrazione intranasale di vaccini antinfluenzali ricombinanti in modelli murini chimerici per studiare l'immunità della mucosa.J. Vis. Exp. (2015) Il video mostra un metodo per creare virus influenzali ricombinanti adattati al freddo. Comporta la trasfezione di cellule umane con plasmidi bidirezionali, seguita dall'incubazione a temperatura più bassa per produrre virus adatti alla produzione di vaccini, caratterizzati da una ridotta replicazione in con...

Video: Generazione di virus influenzali vivi attenuati ricombinanti per vaccini - Concept

Measuring Influenza Neutralizing Antibody Responses to A(H3N2) Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for influenza infections. Microneutralization (MN) assays are often used to measure neutralizing antibody responses in human sera after influenza vaccination or infection. Madine Darby Canine Kidney (MDCK) cells are the commonly used cell substrate for MN assays. However, currently circulating 3C.2a and 3C.3a A(H3N2) influenza viruses have acquired altered receptor binding specificity. The MDCK-SIAT1 cell line with increased &#945;-2,6 sialic galactose moieties on the surface has proven to provide improved infectivity and more faithful replications than conventional MDCK cells for these contemporary A(H3N2) viruses. Here, we describe a MN assay using MDCK-SIAT1 cells that has been optimized to quantify neutralizing antibody titers to these contemporary A(H3N2) viruses. In this protocol, heat inactivated sera containing neutralizing antibodies are first serially diluted, then incubated with 100 TCID50/well of influenza A(H3N2) viruses to allow antibodies in the sera to bind to the viruses. MDCK-SIAT1 cells are then added to the virus-antibody mixture, and incubated for 18 - 20 h at 37 &#176;C, 5% CO2 to allow A(H3N2) viruses to infect MDCK-SIAT1 cells. After overnight incubation, plates are fixed and the amount of virus in each well is quantified by an enzyme-linked immunosorbent assay (ELISA) using anti-influenza A nucleoprotein (NP) monoclonal antibodies. Neutralizing antibody titer is defined as the reciprocal of the highest serum dilution that provides &#8805;50% inhibition of virus infectivity.

Measuring Influenza Neutralizing Antibody Responses to AH3N2 Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Influenza neutralizing antibodies correlate with protection of influenza infections. Microneutralization assays measure neutralizing antibodies in human sera and are often used for influenza human serology. We describe a microneutralization assay using MDCK-SIAT1 cells to measure neutralizing antibody titers to contemporary 3C.2a and 3C.3a A(H3N2) viruses following influenza vaccination or infection.

Neutralizzando le risposte dell'anticorpo al virus A(H3N2) in sieri umani di Microneutralization l'Influenza di misurazione analisi utilizzando cellule MDCK-SIAT1

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for ...

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JoVE Journal

Immunologia e infezioni

Production of High-Titer Infectious Influenza Pseudotyped Particles with Envelope Glycoproteins from Highly Pathogenic H5N1 and Avian H7N9 Viruses

The occasional direct transmission of the highly pathogenic avian influenza A virus H5N1 (HPAI H5N1) and H7N9 to humans and their lethality are serious public health issues and suggest the possibility of an epidemic. However, our molecular understanding of the virus is rudimentary, and it is necessary to study the biological properties of its envelope proteins as therapeutic targets and to develop strategies to control infection. We developed a solid viral pseudotyped particle (pp) platform to study avian influenza virus, including the functional analysis of its hemagglutinin (HA) and neuraminidase (NA) envelope glycoproteins, the reassortment characteristics of the HAs and NAs, receptors, tropisms, neutralizing antibodies, diagnosis, infectivity, for the purposes of drug development and vaccine design. Here, we describe an experimental procedure to establish pps with the envelope glycoproteins (HA, NA) from two influenza A strains (HAPI H5N1 and 2013 avian H7N9). Their generation is based on the capacity of some viruses, such as murine leukemia virus (MLV), to incorporate envelope glycoproteins into a pp. In addition, we also detail how these pps are quantified with RT-qPCR, and the infectivity detection of native and mismatched virus pps depending on the origin of the HAs and NAs. This system is highly flexible and adaptable and can be used to establish viral pps with envelope glycoproteins that can be incorporated in any other type of enveloped virus. Thus, this viral particle platform can be used to study wild viruses in many research investigations.

This protocol describes an experimental process to produce high-titer infectious viral pseudotyped particles (pp) with envelope glycoproteins from two influenza A strains and how to determine their infectivity. This protocol is highly adaptable to develop pps of any other type of enveloped viruses with different envelope glycoproteins.

Produzione di particelle pseudotioniche di influenza ad alto livello con le legiole di busta dei virus altamente patogeni H5N1 e Avian H7N9

The occasional direct transmission of the highly pathogenic avian influenza A virus H5N1 (HPAI H5N1) and H7N9 to humans and their lethality are serious ...

Expression and Purification of Virus-like Particles for Vaccination

Virus-like particles (VLPs) and subviral particles (SVPs) are an alternative approach to viral vaccine design that offers the advantages of increased biosafety and stability over use of live pathogens. Non-infectious and self-assembling, VLPs are used to present structural proteins as immunogens, bypassing the need for live pathogens or recombinant viral vectors for antigen delivery. In this article, we demonstrate the different stages of VLP design and development for future applications in preclinical animal testing. The procedure includes the following stages: selection of antigen, expression of antigen in cell line of choice, purification of VLPs/SVPs, and quantification for antigen dosing. We demonstrate use of both mammalian and insect cell lines for expression of our antigens and demonstrate how methodologies differ in yield. The methodology presented may apply to a variety of pathogens and can be achieved by substituting the antigens with immunogenic structural proteins of the user&#39;s microorganism of interest. VLPs and SVPs assist with antigen characterization and selection of the best vaccine candidates.

Here, we present a protocol for synthesizing virus-like particles using either baculovirus or mammalian expression systems, and ultracentrifugation purification. This highly customizable approach is used to identify viral antigens as vaccine targets in a safe and flexible manner.

Espressione e purificazione di particelle virus-simili per la vaccinazione

Virus-like particles (VLPs) and subviral particles (SVPs) are an alternative approach to viral vaccine design that offers the advantages of increased ...

Generating Recombinant Live-Attenuated Influenza Viruses for Vaccines

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Generating Recombinant Live-Attenuated Influenza Viruses for Vaccines