Viral proteins structure is slightly affected by HHP treatment.

<p>(A) The changes in spectral center of mass (●) and light scattering (○) were followed as a function of the pressure at 289.6 MPa over 6 h. For tryptophan fluorescence emission, the sample was excited at 280 nm, and the emission was measured at 300 to 420 nm. (B) The influenza virus was pre-incubated for 10 min with 15 mM of bis-ANS probe and then exposed to 289.6 MPa for 3 h, and the intensity of the probe was measured every 10 min.</p

HHP treatment preserves viral fusogenic activity.

<p>Virus samples were pressurized for 3, 6, or 12 h at 289.6 MPa. (A) Viruses were incubated with DiD and monitored for their fusogenic properties. Mock (cells incubated with PBS), control (influenza viruses kept for 12 h at 25°C), and pressurized influenza virus. (B) Fusogenic activity relative to the control. The asterisks (***) mark a significant difference (***p<0.0001 by Tukey´s post-test).</p

Viral glycoproteins remain functional after pressurization.

<p>(A) Hemagglutination assay titer of viruses pressurized at pH 7.4 for 3, 6, or 12 h at 289.6 MPa. Hemagglutination units were given by the reciprocal of the highest dilution where total hemagglutination was observed. (B) X-31 NA activity. Virus particles were pressurized at pH 7.4 for 3 h at 289.6 MPa. Enzymatic activity was determined with the MUNANA substrate, as described in the Materials and Methods. The NA activity was calculated by normalizing the NA activity of the pressurized virus to the NA activity of the native virus. </p

Intranasal Immunization with Pressure Inactivated Avian Influenza Elicits Cellular and Humoral Responses in Mice

<div><p>Influenza viruses pose a serious global health threat, particularly in light of newly emerging strains, such as the avian influenza H5N1 and H7N9 viruses. Vaccination remains the primary method for preventing acquiring influenza or for avoiding developing serious complications related to the disease. Vaccinations based on inactivated split virus vaccines or on chemically inactivated whole virus have some important drawbacks, including changes in the immunogenic properties of the virus. To induce a greater mucosal immune response, intranasally administered vaccines are highly desired as they not only prevent disease but can also block the infection at its primary site. To avoid these drawbacks, hydrostatic pressure has been used as a potential method for viral inactivation and vaccine production. In this study, we show that hydrostatic pressure inactivates the avian influenza A H3N8 virus, while still maintaining hemagglutinin and neuraminidase functionalities. Challenged vaccinated animals showed no disease signs (ruffled fur, lethargy, weight loss, and huddling). Similarly, these animals showed less Evans Blue dye leakage and lower cell counts in their bronchoalveolar lavage fluid compared with the challenged non-vaccinated group. We found that the whole inactivated particles were capable of generating a neutralizing antibody response in serum, and IgA was also found in nasal mucosa and feces. After the vaccination and challenge we observed Th1/Th2 cytokine secretion with a prevalence of IFN-γ. Our data indicate that the animals present a satisfactory immune response after vaccination and are protected against infection. Our results may pave the way for the development of a novel pressure-based vaccine against influenza virus.</p></div

Analysis of residual virus infectivity after pressure-induced inactivation^a.

<p>^a The residual infectivity of the pressurized virus samples (282.7 MPa for 12 h at 25°C) was assayed for three sequential serial passages in embryonated hens eggs and in MDCK cell monolayers. For each blind passage, the samples revealed the absence of infectivity by a hemagglutination assay or a TCID₅₀.</p><p>^b No residual infectivity was detected in MDCK cells.</p><p>Analysis of residual virus infectivity after pressure-induced inactivation<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128785#t001fn001" target="_blank">^a</a>.</p

Virus titer measured in lung homogenates after challenge^a.

<p>^a BALB/c mice were vaccinated with pressurized virus and challenged intranasally with native virus.</p><p>^b The results are expressed as the means of six animals.</p><p>^c No infectivity was detected in MDCK cells.</p><p>Virus titer measured in lung homogenates after challenge<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128785#t003fn001" target="_blank">^a</a>.</p

Vaccinated mice are protected against challenge.

<p>(A) Study design: BALB/c mice were vaccinated intranasally with the pressurized H3N8 at the indicated time points, followed by an intranasal challenge with the native virus. (B) The vaccinated and further challenged animals were monitored daily for body weight changes. The results are expressed as the mean of 10 animals. The mice were monitored for 28 days. (C) Total cell counts in the bronchoalveolar lavage (BAL) fluid are shown. At days 21 and 35 of the study, BAL was performed on the animals (n = 6) to analyze cellularity. The bars represent the averages and respective standard deviations of the individual results. (D) The measurement of Evans Blue dye in the lungs is shown. The animals (n = 9) were anesthetized and injected with an endovenous solution and 2% Evans Blue. After 1 hour, the animals were euthanized and perfused through the right atrium with physiological saline. Next, the lung was removed and placed in 5 mL of formamide for 18 h at 37°C. The supernatant was then collected and the absorbance of the Evans Blue dye was measured. The result was evaluated in mg/mL Evans Blue by making a standard curve. The bars represent the means and respective standard deviations of the individual results. (E) Histological sections of the lung parenchyma of the mice vaccinated and further challenged are shown. I: vaccinated challenged BALB/c, II: non-vaccinated challenged mice. The lungs were collected 7 days after challenge. The blocks were cut on a microtome to a thickness of 5 μm, and the slides were stained with hematoxylin and eosin for the morphological analysis. The slides were photographed using an Olympus BX41 microscope coupled to a photographic system at 100X magnification. Sal = Saline, Vir = Pressure-inactivated virus, PV = Post vaccination. P.I. = Post-infection with native virus. The asterisk (*) marks a significant difference (* p <0.05, ** p <0.01, *** p <0.0001 and a Student-Newman-Keuls post-test).</p

Induction of humoral immune responses and neutralizing antibodies by pressurized H3N8 after vaccination and challenge.

<p>(A) The influenza-specific IgG1, IgG2a, and IgA levels in serum at days zero, 21, and 35 according to the study design are shown. (B) Influenza-specific IgA in the nasal wash and feces at days zero and 21, according to the study design, are shown. After the third vaccination, followed one week later by the viral challenge, the antibodies were measured by ELISA using 2 μg of the antigen per well and goat anti-mouse IgG1, IgG2a, or IgA horseradish peroxidase-conjugated antibodies. The results are expressed as the mean of the absorbance values (492 nm) of the 1/1,000 diluted serum of each animal. (C) The response of the H3N8 neutralizing antibodies in sera is shown. The serum of the vaccinated and further challenged mice was assessed by a hemagglutination inhibition assay. The titer of the antibodies is referred to as the reciprocal of the highest serum dilution that resulted in the complete inhibition of the cytopathic effect. The dotted line represents the detection limit of the assay at a dilution of 10¹⁰. The symbols represent the result for each individual animal. Sal = Saline, Vir = Pressure-inactivated virus, P.I. = Post-infection with native virus (challenge). The asterisk (*) marks a significant difference (* p <0.05, ** p <0.01, p <0.0001 and a Student-Newman-Keuls post-test).</p

Virus titer measured in nasal wash and lung after vaccination^a.

<p>^a BALB/c mice were vaccinated with pressurized virus. Samples were collected 3 and 6 days after vaccination.</p><p>^b The results are expressed as the means of six animals.</p><p>Virus titer measured in nasal wash and lung after vaccination<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128785#t002fn001" target="_blank">^a</a>.</p

Activity of the surface glycoproteins in the pressure-inactivated influenza virus.

<p>(A) The hemagglutinating activity of H3N8 was tested as a function of the incubation time under pressure (282.7 MPa) at room temperature. The virus was pressurized for 12 hours at pH 7.4. For the assay, 50 μL of virus and 50 μL of 0.01 M phosphate buffered saline (PBS) were added to the wells. The contents of the wells in column 1 were serially diluted (1:2) through column 12, resulting in dilutions ranging from 1 to 1/1024. Fifty microliters of a 1% human blood cell suspension was added to all of the wells. The hemagglutination units (HAU) were calculated by the reciprocal of the highest dilution where complete hemagglutination was observed. (B) The virus was pressurized for 12 hours (282.7 MPa) at pH 7.4. The virus particles were diluted in 32.5 mM MES, pH 6.5, 4.0 mM CaCl₂ prior to the assay such that the final concentration of virus was 20 μg/mL. N-acetylneuraminic acid (Neu5Ac) was used as negative control. All of the reactions were carried out in triplicate. The mean values of these replicates were used in the analysis of the data.</p