Cloning, expression and purification of M2e.Hsp70c constructs were performed as previously described (Ebrahimi S M, et al., 2010).
One hundred and thirty 6-week-old female BALB/c mice were obtained from the Animal Rearing Department of the Pasteur Institute, Iran. All of the mice were randomly divided into ten equal groups of 13 at 7 weeks old. The animals were maintained in a controlled-temperature environment with 12h-light/12h-dark cycles and given food and water ad libitum. Mice were kept for 1 week in the animal room for adaption and immunized when they reached 8 weeks of age (Table 1).
Group Vaccination Challenge virus Total no. of mice 1 M2e-Hsp70 PR/8(H1N1) 13 2 M2e PR/8(H1N1) 13 3 Hsp70 PR/8(H1N1) 13 4 Split vaccine PR/8(H1N1) 13 5 No(PBS) PR/8(H1N1) 13 6 M2e-Hsp70 H9N2 13 7 M2e H9N2 13 8 Hsp70 H9N2 13 9 Split vaccine H9N2 13 10 No(PBS) H9N2 13
Table 1. Experimental design, vaccination status and number of mice in control and treatment groups.
All experiments were performed in accordance with conditions stated by law and authorized by the Institutional Ethical Committee on Experimental Animals, Razi Vaccine and Serum Research Institute of Iran.
Iranian isolates of influenza viruses H1N1 were a kind gift from Dr Mokhtari (Virus Research Dept, Tehran University of Medical Science, Tehran, Iran), and propagated in Madin-Darby canine kidney (MDCK) cells. Avian influenza virus, H9N2, was a kind gift from the Marand branch of Iran's Razi Vaccine and Serum Research Institute, and passaged in allantoic fluid of 10-day-old specific-pathogen-free (SPF) embryonated chicken eggs.
Experimental mice were immunized at the age of 8 weeks and boosted 2 weeks later by the (s.c.) route without endotoxin. As shown in Table 1, five groups were allocated for experimental study of each of the H1N1 and H9N2 influenza A viruses. In the treatment groups, 30 μg of M2e-Hsp70c, M2e peptide and Hsp70c were used for immunization in groups one to three, respectively; the fourth group was used for split vaccine and the fifth for negative control (only buffer).
In order to synthesize the M2e peptide (23 amino acids, 2-24, SLLTEVETPIRNEWGCRCNDSSD) based on the consensus sequence of A/Chicken/Iran/101/1998 (H9N2) influenza virus, solid-phase technology (GL Biochem, China) with a minimum purity of 95% was used. Subsequently, the peptide was employed in the anti-M2-specific ELISA (enzyme-linked immunosorbent assay).
Blood samples were taken from a ventral tail vein in all groups before—and 2 weeks after—immunization. Samples were kept at 37℃ for 60 min and then placed on ice; serum was separated after two successive centrifugations.
The ELISA was performed with some modification as described previously (Zhao G, et al., 2010). Briefly, 96-well plates (MaxiSorp, Nunc, Denmark) were coated with 100 µL of 10 μg/mL M2e synthetic peptide solution in 50 mmol/L sodium bicarbonate buffer, pH 9.6, and incubated overnight at 4℃. The plates were then washed with washing buffer and blocked for 1 h with PBS-BSA 2% at room temperature. The plates were loaded with 1/200 dilution of experimental sera and incubated for 2 h at 37℃ and, after washing, 100 µL of 1:10000 rabbit anti mouse IgG-HRP conjugates (Sigma-Aldrich, St. Louis, USA) were added to the wells and incubated for 2 h at 37℃. The color reaction was developed with 3, 3', 5, 5'-tetramethylbenzidine, TMB (Pishtaz Teb, Tehran, Iran) at optical density (OD) 450 nm.
The ODs of the experimental groups at 1/200 dilution were compared with each other and with those of the control groups.
Interferon γ(IFN-γ) ELISPOT (enzyme-linked immunospot) assay was performed as described (Tompkins S M, et al., 2007) on mouse spleen cells by stimulation with M2e peptides. A total of 2×105 spleen cells were placed in each well of a 96-well plate and in vitro stimulation carried out with 10 μg/mL of M2e peptide, as positive control cells stimulated with PHA (phytohemaglutinin), and wells containing un-stimulated cells and RPMI-1640 (Gibco®, USA) were used as negative controls. The number of cells secreting M2e-specific IFN-γ in the mice were counted using commercial ELISPOT assay kits (Mabtech AB, Sweden). This procedure was conducted according to the instruction manual. The visible spots of IFN-γ-secreting cells were enumerated using Cellular Technology Ltd (USA) software.
To detect the cytotoxicity of the candidate vaccine, the carboxyfluorescein succinimidyl ester (CFSE, Invitrogen, Carlsbad, CA, USA) method was used in this study (Bijker M S, et al., 2007). Splenocytes from naive mice were stained with 5 μmol/L (high intensity) or 0.5 μmol/L (low intensity) of CFSE. The high-intensity CFSE-labeled cells were loaded with M2e peptide 10 μg/mL while the low-intensity CFSE-stained cells (0.5 μmol/L) were used as a peptide un-pulsed control. The cells were then transferred intravenously (3×106 cells of each) into the experimental groups of mice. After 20 h, the lymphocytes were isolated from spleen and resuspended, and RBCs were lysed and flowcytometry analysis was then carried out to highlight the difference in separation pattern based on intensity of CFSE staining. The percent killing of the high-and low-intensity CFSE-labeled cells was calculated and the specific killing of M2e pulsed (CFSE high) target cells was calculated as follows:(1-((CFSE high/CFSE low) vaccinated ×(CFSE low/CFSE high) naive))× 100%.
At 2 weeks after the last immunization, the mice were challenged intranasally (i.n.) with a lethal dose (1×107 pfu) of A/PR8 and H9N2 in a total volume of 50 μL under mild anesthesia with ketamin/xylasine solution.
Three mice were selected randomly in each group to represent lung viral titration on days 3, 6 and 9 post challenge, and for 2 weeks other mice were monitored daily in each group for mortality and morbidity following monitoring of body weight and observation of clinical symptoms at 2-day intervals.
To verify the impact of M2e-based vaccines on viral replication in the respiratory system of mice, viral shedding was evaluated by taking a throat swab from the mice on different days after challenge. Three mice from each group on days 3, 6 and 9 died following challenge via cervical dislocation under deep anesthesia with ketamin/xylasine.
After removal, the lungs were aseptically homogenized for evaluation of viral load. Next, the lungs were titrated by inoculation of decimal dilutions in Madin-Darby canine kidney (MDCK) cell cultures, as described previously (Rimmelzwaan G F, et al., 1998).
Viral titers of the lung homogenates are shown as log10 geometric mean titer of TCID50/mL 7SEM, and infectious titers were established according to the method described by Reed and Muench (Reed L J, et al., 1983) using BPL-treated Iran/H1N1, or Iran/H9N2 antigens.
All negative samples for the virus titration test were set at 0.9 log10 TCID50/mL.
The lungs of infected mice were fixed in 10% neutralbuffered formalin for histopathologic examination on day 7 after challenge for evidence of cellular inflammation and necrosis.
Five-micrometer paraffin-embedded sections were used for histopathologic examination following staining with hematoxylin and eosin (H & E).
SPSS 16.0 software (SPSS Inc., Chicago, IL, USA) was used to statistically compare the results of the treatment and control groups. Data were analyzed for significance (P ≤ 0.05) via one-way ANOVA (analysis of variance) when the variance between groups was homogeneous and distribution of the data was normal, and when the normality test or homogeneity of variance test was unsuccessful, a nonparametric (Kruskal-Wallis) test was used.