Chemical-free inactivated whole influenza virus vaccine prepared by ultrashort pulsed laser treatment

Abstract

There is an urgent need for rapid methods to develop vaccines in response to emerging viral pathogens. Whole inactivated virus (WIV) vaccines represent an ideal strategy for this purpose; however, a universal method for producing safe and immunogenic inactivated vaccines is lacking. Conventional pathogen inactivation methods such as formalin, heat, ultraviolet light, and gamma rays cause structural alterations in vaccines that lead to reduced neutralizing antibody specificity, and in some cases, disastrous T helper type 2-mediated immune pathology. We have evaluated the potential of a visible ultrashort pulsed (USP) laser method to generate safe and immunogenic WIV vaccines without adjuvants. Specifically, we demonstrate that vaccination of mice with laser-inactivated H1N1 influenza virus at about a 10-fold lower dose than that required using conventional formalin-inactivated influenza vaccines results in protection against lethal H1N1 challenge in mice. The virus, inactivated by the USP laser irradiation, has been shown to retain its surface protein structure through hemagglutination assay. Unlike conventional inactivation methods, laser treatment did not generate carbonyl groups in protein, thereby reducing the risk of adverse vaccine-elicited T helper type 2 responses. Therefore, USP laser treatment is an attractive potential strategy to generate WIV vaccines with greater potency and safety than vaccines produced by current inactivation techniques.

Fig. 1

Body weight changes in H1N1-challenged mice. Groups of BALB/c mice ($n=8$) were vaccinated twice with $2\times {10}^8$ ${\mathrm{TCID}}_{50}/\mathrm{ml}$ ($20\mu \mathrm{l}$) of laser-inactivated H1N1 virus by intranasal administration at a 2-week interval, or received no treatment. 21 days later, all mice were challenged with a lethal dose of $6\times {10}^2$ ${\mathrm{TCID}}_{50}/\mathrm{mL}$ of A/PR/8/34 influenza virus. Following lethal challenge, mice were monitored for percent change of initial weight for 9 days. Each point represents the mean and bars represent standard deviation. (Student’s $t$ test comparing the final weights of each group of mice, $P=0.026$).

Fig. 2

$\mathrm{CD}8+$ T cell induction following vaccination. Splenocytes were isolated from vaccinated and untreated BALB/c mice and then the cells were incubated overnight in the presence of $2\mu \mathrm{g}/\mathrm{ml}$ of NP peptide. After washing with FACScan buffer, the cells were stained with phycoerythrin-conjugated anti-mouse CD8a antibody, followed by fixing/permeabilizing and staining with FITC-conjugated anti-mouse IFN-$\gamma$ antibody. The splenocytes were then analyzed by flow cytometry on a FACSCalibur with CellQuest software, and gated on the lymphocyte area. (a) Representative flow cytometry analysis. The upper right-hand quadrant shows the percentage of NP-specific, IFN-$\gamma$ secreting $\mathrm{CD}8+$ T cells among lymphocytes. (b) Bar graph depicting the percentage of activated $\mathrm{CD}8+$ T cells among splenocytes in vaccinated and unvaccinated mice ($P<0.001$).

Fig. 3

Neutralizing antibodies detected by microneutralization assay. Serum from vaccinated ($n=5$) and unvaccinated ($n=4$) groups of mice was extracted 20 days after vaccination and then added to Madin-Darby canine kidney (MDCK) cells in a 96-well plate, serially diluted, and incubated for 3 days. Subsequently, a constant H1N1 concentration of $1.75\times {10}^5$ ${\mathrm{TCID}}_{50}/\text{well}$ was used for each plate. The virus and serum were incubated at 25°C for 2 h and then added to the 96-well plate with MDCK cells. The plates were stored for three nights in an incubator at 37°C and 5% ${\mathrm{CO}}_2$. Formaldehyde and naphthol blue–black was added to visualize the results of the reaction. The presence of neutralizing antibodies was determined by cell survival. Neutralization titers were calculated using the Reed–Muench method. Bar graph depicts neutralization titer of untreated and vaccinated mice. ($P=0.021$).

Fig. 4

Transmission electron microscopy (TEM) images of influenza virus. Samples of laser-inactivated and active A/PR/8/34 influenza virus were fixed with glutaraldehyde and visualized using TEM. (a) Fixed sample of laser-inactivated virus at $\mathrm{93,000}\times$ magnification. (b) Fixed sample of the active virus at $\mathrm{93,000}\times$ magnification. These TEM images demonstrate the retention of capsid and global structure of the influenza virus after the ultrashort pulsed (USP) laser irradiation and provide support for the laser-induced protein aggregation through ISRS process for the most likely inactivation mechanism.

Fig. 5

Carbonyl content of laser-treated bovine serum albumin (BSA) protein. The carbonyl concentrations in untreated BSA, USP laser-treated BSA, or UV-treated BSA were assessed using a dinitrophenylhydrazine (DNPH) protein carbonyl colorimetric assay kit. Untreated BSA served as negative control; UV-treated BSA served as positive control.

Fig. 6

Body weight changes in H1N1-challenged mice vaccinated with conventional formalin-inactivated vaccine. (a) One control group vaccinated with two doses of conventional formalin-inactivated H1N1 vaccine at $\approx 2.76\times {10}^6\mathrm{pfu}/\text{dose}$; (b) the other control group vaccinated with two doses of conventional formalin-inactivated H1N1 vaccine at $\approx 2.76\times {10}^7\mathrm{pfu}/\text{dose}$. The mice of the control group in (a) all died (the weights decreased more than 30% of their initial ones) 10 days after challenging; whereas the mice of the control group in (b) achieved 75% protection.