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Indian Institute of Science Education and Research (IISER), Pune, India

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Experiments & Protocols

Background:

In April 2013, human infections with a novel avian influenza (H7N9) virus emerged in China. The laboratory-confirmed cases have been reported from 13 provinces/municipalities in eastern mainland China, Hong Kong, and Special Administrative Region of China.[1]
It has caused serious concerns for public health throughout the world. However, there is presently no effective treatment, and an A (H7N9) H7 subtype influenza vaccine is not available. More specifically, as of 28 January 2014, the case fatality rate of all confirmed cases is 22%, but many cases are still hospitalized. Of all cases, 67% were male. The median age of reported cases is 58 years and that of fatal cases is 66 years.[2]
As with other types of influenza viruses, vaccination shall be considered to be the most effective measure to control the pandemic of the disease. However, most experts believe that there would be great difficulty in providing adequate supplies of a vaccine if the virus were to develop into a pandemic. Even with extensive antigenic drift and vaccine manufacturing capacity, the global public health community is concerned with the effectiveness of the traditional vaccines, particularly in persons older than 65 years.[3] Besides, biosafety-level 3 (BSL-3) containment facilities should be equipped to manufacture such vaccines.
In order to address these obstacles, new strategies for rapid production of H7N9 influenza vaccines are a priority for pandemic preparedness. Influenza VLPs are produced by a self-assembly process when matrix protein 1 (M1), hemagglutinin (HA) and neuraminidase (NA) proteins are co-expressed VLPs.[4] These VLPs are similar to infectious virions in the morphological and structural features, yet are non-infectious particles and have advantages in safety and manufacturing.[5]

Experiment:

1.Construction of VLP Expression Plasmids
The QIAamp Viral RNA kit (Qiagen, Santa Clarita, CA, USA) was used to extract genomic RNA of H7N9 virus from allantoic fluid of infected chicken eggs. The extracted RNA was subjected to reverse transcription PCR (RT-PCR) using Transcriptor High Fidelity cDNA Synthesis Kit (Roche,Penzberg, Germany) to generate cDNA.
The specific information about HA,NA,M1 genes are provided below: The PCR segments were digested with specific enzymes and ligated with linearized pcDNA5/FRT vector. The nucleotide sequences of the HA-pcDNA5, NA-pcDNA5, and M1-pcDNA5 genes were confirmed by sequencing.

2.Transient Transfection of 293T Cells with H7N9 VLP Expression Plasmids
293T cells were seeded in 6-well plates with a density of 3×10^5 per well one day before transfection.Recombinant plasmids containing HA-pcDNA5, NA-pcDNA5, and M1-pcDNA5 were co-transfected to cells by X-tremeGENE HP DNA Transfection Reagent (Roche) following manufacturer’s instruction.48 h post-transfection, cells and supernatants were harvested for future use. 10 cm cell culture dishes were used for large-scale production of VLPs.
3.Western Blot Analysis
Culture supernatants were harvested without concentration, then electrophoresed in NuPAGE
4%–12% Bis-Tris gradient gel and transferred to nitrocellulose membrane (GE Healthcare, Piscataway,NJ, USA). Membranes were blocked by 10% skimmed milk for 1 h at room temperature (RT), and incubated with diluted H7N9 virus-specific mouse or human sera for another hour at RT. After intensive washing, the membranes were incubated with HRP- conjugated second antibodies, respectively. Protein bands were visualized by ECL plus Western blotting detection system kit (GE Healthcare). Radiographs were exposed, developed, and fixed according to the manufacturer’s instructions.

As shown in Figure A, three bands with sizes of 75 kD, 68 kD, and 28 kD were confirmed by Western blot. It demonstrated that HA, NA, and M1 of VLPs were successfully expressed as expected.
4.Electron Microscope
To further confirm the formation of self-assembled VLPs, the supernatant of transfected 293T cells was characterized by electron microscopy. Seventy-two hours post-transfection, 293T cells were fixed with 0.25% glutaraldehyde and 1% osmium tetraoxide, dehydrated with ethanol, and then embedded in epon resin. Thin sections were stained with lead citrate and uranyl acetate and observed byelectron microscope.

As shown in Figure 1B, the morphology of VLPs resembles the morphology of influenza virus particles with spikes on their surfaces, characteristic of influenza virus HA proteins on virions. Particle sizes ranged from approximately 100 to 120 nm.
5.Purification of H7N9-Derived VLPs
The culture supernatants containing extracellular VLPs were harvested and clarified by centrifugation at 10,000 g for 30 min followed by concentration using ultra-filtration system Startocon slice 200(Sartorius stedim biotech, Aubagne, France). VLPs were pelleted for 4 h at 100,000 g at 4℃ using SW41 rotor (Beckman Coulter Inc., Fullerton, CA, USA). The pellets were re-suspended in 5 mL of PBS(pH 7.2), and loaded onto a 20%–60% (w/v) discontinuous sucrose step density gradient, and sedimented by ultracentrifugation for 6 h at 100,000 g. About 1 mL fractions were collected and pelleted by a second ultracentrifugation and then re-suspended in PBS. Total protein concentrations of VLPs were determined by Pierce BCA Protein Assay Kit.

6.Mice Immunization
Six-week-old female BALB/c mice were purchased from Model Animal Research Center of Nanjing University, China. Mice were randomly divided into three groups (8 mice per group). Mice were immunized intramuscularly with 40 µg (total protein) of VLPs or purified H7N9 whole influenza virions(WIV) in Freund’s complete adjuvant (Sigma) for priming and in Freund’s incomplete adjuvant for two times of boosting at an interval of 2 weeks. Negative control mice were immunized with PBS in the same adjuvant. On days 0, 14, and 28, blood samples were collected through the tails in for antibody detection. Two weeks after the last immunization, mice were sacrificed to collect serum for the neutralizing antibodies assay and hemagglutination inhibition (HAI) test. All experimental procedures were conducted in conformity with the National Institutes of Health Guide for Care and Use of Laboratory Animals (Publication No. 85-23, revised 1985). The protocol was approved by the animal care committee of JSCDC. Mice were sacrificed by cervical dislocation, and all efforts were made to minimize suffering.
7.Antibody Responses Induced by Immunizations
To evaluate humoral responses induced by recombinant VLPs, BALB/c mice were immunized with 40 µg of VLPs three times, at two-week intervals. As shown in Figure 2, in comparison to the control, which immunized with PBS, VLPs elicited significant increase in antibody titer with immunization in mice. At week 6, the average antibody endpoint dilution titer (>1:60,000), and the magnitude of humoral immune responses induced by VLPs was similar to those induced by whole influenza virions (WIV).

Result: As shown in the antibody responses experiment, the amount of antibody induced by H7N9 virions and VLPs is very similar. This has proved that H7N9 VLPs could efficiently induce cellular and humoral immunoresponse specific to H7N9 virus. In the future, with more developed experiments and testings, VLPs may potentially become a popular method of countering flu and other diseases.