Immunogenicity and efficacy of a recombinant adenovirus expressing hemagglutinin from the H5N1 subtype of swine influenza virus in mice

Abstract

The H5N1 influenza viruses infect a range of avian species and have recently been isolated from humans and pigs. In this study we generated a replication-defective recombinant adenovirus (rAd-H5HA-EGFP) expressing the hemagglutinin (HA) gene of H5N1 A/Swine/Fujian/1/2001 (SW/FJ/1/01) and evaluated its immunogenicity and protective efficacy in BALB/c mice. The recombinant virus induced high levels of hemagglutination inhibition (HI) antibody at a median tissue culture infective dose of 10⁸ or 10⁷. Compared with mice in the control groups, the mice vaccinated with rAd-H5HA-EGFP did not show apparent weight loss after challenge with either the homologous SW/FJ/1/01 or the heterologous H5N1 A/Chicken/Hunan/77/2005 (CK/HuN/77/05). Replication of the challenge virus was partially or completely inhibited, and viruses were detected at significantly lower numbers in the organs of the vaccinated mice, all of which survived the challenge with CK/HuN/77/05, whereas most of the control mice did not. These results indicate that rAd-H5HA-EGFP can provide effective immune protection from highly pathogenic H5N1 viruses in mice and is therefore a promising new candidate vaccine against H5N1 influenza in animals.

Introduction

Influenza A viruses are divided into 16 subtypes on the basis of the hemagglutinin (HA) protein and 9 subtypes on the basis of the neuraminidase protein (1). The highly pathogenic (HP) subtype of influenza virus, subtype H5N1, not only causes outbreaks of influenza in a variety of birds but can also fatally infect humans, cats, and other mammals (2,3). From the end of 2003 to February 2013, HP H5N1 viruses caused 620 confirmed cases of influenza in humans, 367 fatal (4). Pigs are an intermediate host of influenza viruses and have been shown to be susceptible to HP H5N1 viruses under experimental conditions. Pigs inoculated with H5N1 viruses showed moderate levels of virus shedding in nasal swabs, mild or no symptoms, and no virus transmission to in-contact animals (5,6). In China in 2003 and 2005, H5N1 viruses were isolated from pigs, although none of the animals died or showed evidence of related illness (7–9). However, taken together, these findings highlight the potential risk of swine influenza to public health.

The HA protein is one of the most important antigens for the induction of protective immunity against influenza viruses. It can induce predominantly subtype-specific antibodies that can neutralize infecting virus (10). Human adenovirus type 5 (Ad5) is an ideal expression vector for viral antigens because it is easily manipulated and produced under conditions of good manufacturing practice. The recombinant adenovirus (rAd) can elicit robust cellular and humoral immunity against the encoded antigen (11), and vaccination with rAd has produced effective immunity in preclinical models (12,13).

In the present study, we generated a replication-defective rAd, rAd-H5HA-EGFP (EGFP = enhanced green fluorescent protein), using Ad5 as the vector and the HA gene from the H5N1 subtype of swine influenza virus (SIV) as the donor. To evaluate the immuno-genicity and efficacy of the recombinant virus, we inoculated BALB/c mice with different doses.

Materials and methods

Viruses, plasmids, and cells

The H5N1 SIV A/Swine/Fujian/1/2001 (SW/FJ/1/01; accession number AY747617, which has been characterized previously (8), and the H5N1 avian influenza virus A/Chicken/Hunan/77/2005 (CK/HuN/77/05), kindly provided by Dr. Yanbing Li, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, People’s Republic of China, were propagated in the allantoic cavity of 10-day-old embryonated eggs from chickens free of specific pathogens. The viruses were stored at −70°C before RNA extraction or challenge studies and were titrated to determine the 50% egg infective dose (EID₅₀) by the Reed and Muench method (14). The adenovirus shuttle plasmid pDC315 and the backbone plasmid pBHGloxΔE1,E3Cre of the Ad-Max adenovirus system was purchased from Microbix Biosystems (Mississauga, Ontario). Plasmid pIRES2-EGFP (Clontech Laboratories, Mountain View, California, USA) was kindly provided by Professor Zhigao Bu, Harbin Veterinary Research Institute.

Human embryonic kidney (HEK293) cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum.

Construction of the rAd shuttle plasmid

Viral RNA was extracted with an RNeasy Mini Kit (Qiagen, Valencia, California, USA), and then cDNA encoding the HA protein of SW/FJ/1/01 was reverse-transcribed from the RNA with the Uni12 primer (5′-AGCAAAAGCAGG-3′). The HA gene was amplified from the cDNA with the primers HAF (5′-CCCGTCGACATGGAGAAAATAGTGCTTCTTCTTGC-3′) and HAR (5′-TCCCCCGGGTTAAATGCAAATTCTGCATTGTAACG-3′). Artificial SalI and SmaI restriction sites (underlined) permitted direct cloning of the amplification product into plasmid pIRES2-EGFP, the resulting plasmid being designated pHA-IRES-EGFP (IRES = internal ribosome entry site). The rAd shuttle plasmid was constructed by inserting DNA fragments containing the HA and EGFP genes, connected by an IRES nucleotide sequence, into pDC315. The resultant plasmid was designated pDC315-H5HA-EGFP.

Generation of the rAd

With use of the transfection reagent Lipofectamine 2000 (Invitrogen, Carlsbad, California, USA), HEK293 cells were cotransfected with the rAd shuttle plasmid (pDC315-H5HA-EGFP) and the adenovirus backbone plasmid (pBHGloxΔE1,E3Cre) at an appropriate ratio. Adenovirus vector pFG140 was used as a control in the transfection experiments. Sixteen hours after transfection the cells were checked under a fluorescence microscope. When the fluorescence intensity increased in the infected cells and typical cytopathic effects appeared, the cells were harvested for rAd screening. After further empty plaque screening, the rAd (rAd-H5HA-EGFP) was stored before its use in other experiments.

Genetic stability of rAd-H5HA-EGFP

To evaluate the genetic stability of the foreign gene after integration into the rAd, rAd-H5HA-EGFP was passaged 14 times in HEK293 cells. DNA was extracted from the rAd from the 5th to the 14th passage (10 batches) with the Wizard SV Genomic DNA Purification System (Promega, Madison, Wisconsin, USA), and the inserted gene was detected by polymerase chain reaction (PCR) as follows.

Detection of HA gene expression by the rAd

The total RNA was extracted from HEK293 cells infected with the rAd by conventional methods. Any remaining DNA contamination was removed by treatment of the total RNA with DNase I at 37°C for 30 min, then inactivation at 75°C for 10 min. Reverse transcription was carried out with RNA as the template and Oligo (dT)-18 as the primer. The reaction mixture contained 0.5 μL of Oligo (dT)-18 (500 μg/mL), 4.0 μL of 5× buffer, 0.5 μL of deoxynucleotide triphosphate (10 mM) (Promega), and 14.5 μL of mRNA. After incubation of the reaction mixture at 65°C for 10 min, 0.5 μL of reverse transcriptase M-MLV (200 U/μL) (Promega) was added and well mixed. The reaction was then continued at 37°C for 60 min. To analyze transcription of the target gene at the mRNA level, the HA gene was amplified as described above.

To examine the biologic activity of the HA protein expressed by rAd-H5HA-EGFP, we harvested HEK293 cells infected with the rAd or with the vector virus rAd-EGFP and suspended them in phosphate-buffered saline (PBS). An equal volume of 2× sodium dodecyl sulfate (SDS) electrophoresis sample buffer (100 mM Tris-HCl, pH 6.8; 200 mM dithiothreitol; 4% SDS; 0.2% bromophenol blue; and 20% glycerol) was then added. The samples were boiled in water for 10 min for protein denaturation and then loaded onto a 10% polyacrylamide gel for electrophoresis. Separated proteins were probed for the HA protein with serum from chickens that had been infected with the H5N1 subtype of influenza virus, prepared in our laboratory, and rabbit anti-chicken IgG antibody labeled with IRDye 700DX (LI-COR Biosciences, Lincoln, Nebraska, USA). Western blot analysis was done according to the method of Sambrook, Fritsch, and Maniatis (15).

Vaccination and challenge of mice

The rAd was prepared for vaccination by proliferation in HEK293 cells, purification, and concentration with a Vivapure AdenoPACK 100 RT kit (Sartorius AG, Hannover, Germany). The 50% tissue culture infective dose (TCID₅₀) of the purified virus was titrated in HEK293 cells with the Rapid Test Kit for Adenovirus Titer [VGTC (Vector Gene Technology Company), Beijing, People’s Republic of China], according to the manufacturer’s instructions.

Six-week-old female BALB/c mice (n = 100), purchased from Beijing Vital River Experimental Animal Company, Beijing, were housed in a clean chamber under negative pressure. The mice were randomly divided into 4 groups and observed for a week before vaccination. Two groups of mice (28 per group) were vaccinated intramuscularly with rAd5-H5HA-EGFP at a dose of 10⁸ TCID₅₀ and 10⁷ TCID₅₀, respectively. The 22 mice in 1 control group were inoculated intramuscularly with rAd5-EGFP at a dose of 10⁸ TCID₅₀, and the 22 mice in the other control group were inoculated intra-muscularly with 50 μL of PBS. Three weeks after the 1st vaccination the mice in the 2 rAd5-H5HA-EGFP-inoculated groups were given another intramuscular dose of rAd5-H5HA-EGFP, 2 × 10⁸ TCID₅₀ and 2 × 10⁷ TCID₅₀, respectively, to boost the immunization. Three weeks after the 2nd vaccination the mice in each group were divided into 2 subgroups, which were challenged intranasally with 10⁶ EID₅₀ of SW/FJ/1/01 and CK/HuN/77/05, respectively. The challenge experiments were conducted in a biosafety level 3 laboratory at Harbin Veterinary Research Institute.

All the mice were monitored daily by clinical examination, and their body weight was measured each day after challenge until the end of the experiment. Four and six days after challenge 3 mice from each subgroup were euthanized, and tissues, including brain, lung, kidney, and spleen, were removed for virus titration in eggs, as previously described (16). The remaining animals were kept for clinical observation and euthanized 14 d after challenge.

Hemagglutination inhibition (HI) assay

Blood was collected from all of the mice at 3 time points: 2 wk and 3 wk after the 1st vaccination and 3 wk after the booster vaccination. Antibodies to the HA protein of influenza viruses (including SW/FJ/1/01 and CK/HuN/77/05) were assayed in the serum according to the procedure described in the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals of the World Organisation for Animal Health (17). Briefly, serum samples were treated with receptor-destroying enzyme, heat-inactivated, and centrifuged. Supernatant samples were then serially diluted in V-shaped well microtiter plates with an equal volume of a virus suspension containing 4 hemagglutination units of virus, and the plates were incubated at room temperature before the addition of 0.5% chicken erythrocytes. The virus titer was defined as the reciprocal of the maximum dilution at which hemagglutination was inhibited.

Virus titration

Virus titration in eggs was carried out using 10% (weight/volume) clarified tissue homogenates in PBS. Different 0.1-mL dilutions of each sample were used to inoculate 10-day-old embryonated eggs via the allantoic cavity. The allantoic fluids were tested for HA activity after incubation at 37°C for 72 h. The titer of virus in each sample, expressed in EID₅₀/mL, was calculated by the method of Reed and Muench (14).

Statistical analysis

The results from each experimental group were compared by analysis of variance and the t-test. A P-value of < 0.05 was considered statistically significant.

Results

Construction of the rAd shuttle plasmid

The cDNA of the HA gene derived from the SW/FJ/1/01 virus was amplified with use of the primers HAF and HAR. As expected, the size of the PCR products was about 1.7 kilo base pairs (kb) (Figure 1A, lane 2). The resultant intermediate plasmid, pHA-IRES-EGFP, was constructed by inserting the HA fragment into plasmid pIRES2-EGFP. When PCR was carried out to amplify the HA gene using pHA-IRES-EGFP as the template and HAF and HAR as the primers, the size of the PCR products was again about 1.7 kb (Figure 1B, lane 4), as expected. The plasmid pHA-IRES-EGFP was confirmed by SalI/SmaI double digestion, which resulted in 2 bands, at sizes of 1.7 and 5.3 kb, respectively (Figure 1B, lane 2). The plasmid pIRES2-EGFP was also digested by SalI, which generated a 5.3-kb band (Figure 1B, lane 3). The rAd shuttle plasmid pDC315-H5HA-EGFP was generated by subcloning the fragments containing the HA and EGFP genes, connected by an IRES, into pDC315. The shuttle plasmid was similarly confirmed by PCR, and double digestion with SalI/SmaI or NheI/SmaI resulted in 2 fragments, at sizes of 5.3 and 1.7 kb, respectively (Figure 1C, lanes 2 and 3, respectively).

Figure 1.

A — Amplification by polymerase chain reaction (PCR) of the hemagglutinin (HA) gene of the H5N1 swine influenza virus (SIV) A/Swine/Fujian/1/2001 (SW/FJ/1/01). Lane 1 — DNA marker DL2000. Lane 2 — PCR product of the HA gene. Lane 3 — negative control. Bp — base pairs. B — Identification of the recombinant plasmid pHA-IRES-EGFP. Lane 1 — DNA marker DL15 000. Lane 2 — Products of digestion of pHA-IRES-EGFP by SalI and SmaI. Lane 3 — Product of digestion of the plasmid pIRES2-EGFP by SalI. Lane 4 — PCR product of pHA-IRES-EGFP. Lane 5 — DNA marker DL2000. IRES — internal ribosome entry site; EGFP — enhanced green fluorescent protein. C — Restriction analysis of the recombinant adenovirus (rAd) shuttle plasmid pDC315-H5HA-EGFP. Lane 1 — DNA marker DL15 000. Lane 2 — Products of digestion of the plasmid by SalI and SmaI. Lane 3 — Products of digestion of the plasmid by NheI and SmaI. D — Diagrammatic representation of the rAd rAd-H5HA-EGFP and the vector virus rAd-EGFP. ITR — inverted terminal repeat.

Generation of the rAd expressing the HA gene (rAd-H5HA-EGFP)

Recombinant adenovirus shuttle plasmid pDC315-H5HA-EGFP and backbone plasmid pBHGloxΔE1,E3Cre were used to cotransfect HEK293 cells. Seven days after cotransfection the HEK293 cells became swollen and rounded. The cells were then harvested and passaged on HEK293 cells. Typical adenovirus-induced cytopathic effects — grape-cluster-like cells (Figure 2A) — and intensive fluorescence signals (Figure 2B) were observed by fluorescence microscopy.

Figure 2.

A — Cytopathic effects displayed by human embryonic kidney cells (HEK293) infected by rAd-H5HA-EGFP. B — Intense fluorescence signals of rAd-H5HA-EGFP expressing the EGFP gene. C — Results of Western blot analysis of lysates of HEK293 cells infected with rAd-EGFP or rAd-H5HA-EGFP and incubated with chicken antiserum specific for H5 SIV HA that was generated by H5HA gene vaccination. Lane 1 — Lysates of HEK293 cells infected with rAd-EGFP. Lane 2 — Molecular weight protein. Lane 3 — lysates of HEK293 cells infected with rAd-H5HA-EGFP.

The presence of the HA gene was confirmed by PCR, and expression of the HA protein was confirmed by reverse-transcription PCR (RT-PCR) and Western blot analysis. As expected, a band corresponding to the HA gene was amplified by RT-PCR only when the RNA isolated from the HEK293 cells infected with rAd-H5HA-EGFP was used as a template. In the Western blot analysis the expressed HA protein was specifically recognized by chicken polyclonal antiserum to SW/FJ/1/01 from the HEK293 cells infected with rAd-H5HA-EGFP (Figure 2C). Figure 1D is a diagrammatic representation of rAd-EGFP and rAd-H5HA-EGFP expressing the HA gene from SW/FJ/1/01 and the EGFP gene connected by an IRES. In contrast, the HEK293 cells infected with rAd-EGFP gave negative results in the Western blot analysis (Figure 2C).

The presence of the HA gene in the recombinant virus rAd-H5HA-EGFP was confirmed by PCR in the 5th to 14th passages (10 batches) in HEK293 cells, and expression of the HA protein in the infected HEK293 cells of each passage was confirmed by Western blot analysis.

Antibody responses induced by rAd-H5HA-EGFP in mice

The immunogenicity of rAd-H5HA-EGFP was determined by measuring the HI antibody titers in serum from the vaccinated mice. The TCID₅₀ of the rAd was found to be 2.26 × 10¹⁰/mL. Two weeks after the 1st vaccination, HI antibody (mean titer 1:36) was detected in the 2 groups of vaccinated mice. Three weeks after the 1st vaccination the titers reached up to 1:80 in the mice inoculated with 10⁸ TCID₅₀ of rAd-H5HA-EGFP and 1:66 in the mice vaccinated with 10⁷ TCID₅₀. Three weeks after the booster vaccination the mean titers had increased again, up to 1:120 and 1:92, respectively. In the control mice inoculated with rAd-EGFP or PBS, HI antibody was not detected.

Protective efficacy of rAd-H5HA-EGFP against H5N1 influenza virus

Three weeks after the 2nd vaccination, we challenged the mice with 10⁶ EID₅₀ of SW/FJ/1/01 or CK/HuN/77/05. On days 4 and 6 after challenge, 3 mice from each subgroup were euthanized, and brain, lung, kidney and spleen tissue were removed for virus titration. After challenge with SW/FJ/1/01 the virus was not detected in any of the organs from the mice vaccinated with rAd-H5HA-EGFP at either dose (Table I). Viruses were detected at lower titers in the lungs alone in the mice from both control groups. After challenge with CK/HuN/77/05, high titers of virus were detected at both 4 and 6 d after challenge in the lungs of the mice in both control groups. Furthermore, the challenge virus was also isolated from the spleen and kidney at 4 and 6 d after challenge and from the brain at 6 d. Among the rAd-H5HA-EGFP-inoculated mice the virus titers in the lungs were significantly lower (P < 0.05) than those in the control groups, and no virus was detected in the brain, spleen, or kidney. These results indicate that vaccination with rAd-H5HA-EGFP significantly inhibits the replication of homologous and heterologous H5N1 virus in mice.

Table I.

Protective efficacy of the recombinant adenovirus rAd-H5HA-EGFP against challenge with highly pathogenic H5N1 viruses in mice

Challenge virus; initial inoculum, dose (TCID50 or μL)a	Mean HI Ab titerb	Titer of virus in organ (log10 EID50/g c)								Proportion surviving
		4 d after challenge				6 d after challenge
		Brain	Spleen	Lung	Kidney	Brain	Spleen	Lung	Kidney
SW/FJ/1/01
rAd-H5HA-EGFP, 108	120	< 0.5	< 0.5	< 0.5	< 0.5	< 0.5	< 0.5	< 0.5	< 0.5	8/8
rAd-H5HA-EGFP, 107	92	< 0.5	< 0.5	< 0.5	< 0.5	< 0.5	< 0.5	< 0.5	< 0.5	8/8
rAd5-EGFP, 108	< 10	< 0.5	< 0.5	0.58 ± 1.01	< 0.5	< 0.5	< 0.5	0.58 ± 1.01	< 0.5	5/5
PBS, 50 μL	< 10	< 0.5	< 0.5	1.33 ± 1.26	< 0.5	< 0.5	< 0.5	1.50 ± 1.30	< 0.5	5/5
CK/HuN/77/05
rAd-H5HA-EGFP, 108	20	< 0.5	< 0.5	1.67 ± 1.75	< 0.5	< 0.5	< 0.5	< 0.5	< 0.5	8/8
rAd-H5HA-EGFP, 107	< 10	< 0.5	< 0.5	1.50 ± 2.59	< 0.5	< 0.5	< 0.5	3.25 ± 1.09	< 0.5	8/8
rAd5-EGFP, 108	< 10	< 0.5	1.50 ± 0	5.50 ± 0	0.50 ± 0.87	2.83 ± 0.58	2.00 ± 1.80	4.83 ± 0.58	2.00 ± 1.80	1/5
PBS, 50 μL	< 10	< 0.5	1.33 ± 1.26	4.50 ± 0	1.00 ± 0.87	3.17 ± 0.58	3.42 ± 0.14	5.88 ± 0.14	2.25 ± 0.43	0/5

^aGroups of 6-wk-old BALB/c mice were vaccinated with a 50% tissue culture infective dose (TCID₅₀) of 10⁸ or 10⁷ of rAd5-H5HA-EGFP or were inoculated as controls with 10⁸ TCID₅₀ of rAd5-EGFP or 50 μL of phosphate-buffered saline (PBS) by intramuscularly administration. Three weeks later the vaccinated mice were given booster injections of rAd5-H5HA-EGFP, 2 × 10⁸ TCID₅₀ and 2 × 10⁷ TCID₅₀, respectively, and the control groups were given the same doses of inocula as given previously. The challenge experiment was conducted with the H5N1 viruses A/Swine/Fujian/1/2001 (SW/FJ/1/01) and A/Chicken/Hunan/77/2005 (CK/HuN/77/05) at a 50% egg infective dose (EID₅₀) of 10⁶ 3 wk after the 2nd inoculation.

^bBlood was collected from the mice 3 wk after the 2nd inoculation to assay hemagglutination inhibition (HI) antibody (Ab) in the serum. The titers are means of titers of antibody against the specific challenge virus.

^cThree mice from each group were euthanized on days 4 and 6, and the organs were collected for virus titration in specific-pathogen-free chicken embryos; < 0.5 — no virus was isolated from the undiluted sample.

The rest of the mice in each group were kept for clinical observation for a further 2 wk after challenge. Five days after challenge with SW/FJ/1/01 the mice inoculated with rAd-EGFP or PBS started to show ruffled fur and weight loss (Figure 3A). The 2 groups of mice vaccinated with rAd-H5HA-EGFP remained healthy and showed no weight loss after this challenge with SW/FJ/1/01. None of the mice died during the observation period after this challenge.

Figure 3.

A — Weight change (mean and standard deviation) in mice vaccinated intramuscularly with rAd-H5HA-EGFP at a 50% tissue culture infective dose (TCID₅₀) of 10⁸ (diamonds) or 10⁷ (squares) and then 3 wk later given a booster dose of 2 × 10⁸ TCID₅₀ or 2 × 10⁷ TCID₅₀, respectively, and in control groups of mice inoculated intramuscularly with rAd5-EGFP at a dose of 10⁸ TCID₅₀ (triangles) or with 50 μL of phosphate-buffered saline (PBS; crosses) on 2 occasions 3 wk apart, and challenged by intranasal inoculation with SW/FJ/1/01 at a 50% egg infective dose (10⁶ EID₅₀) of 10⁶ 3 wk after the 2nd vaccination or the inoculation of rAd5-EGFP or PBS. B — Weight change in mice vaccinated or inoculated identically but challenged by intranasal inoculation with the H5N1 avian influenza virus A/Chicken/Hunan/77/2005 (CK/HuN/77/05) at an EID₅₀of 10⁶ 3 wk after the 2nd vaccination or the inoculation of rAd5-EGFP or PBS. C — Survival rate of the mice challenged with 10⁶ EID₅₀ of CK/HuN/77/05 3 wk after the 2nd vaccination or the inoculation of rAd5-EGFP or PBS.

The control mice showed significant weight loss beginning 2 d after challenge with CK/HuN/77/05 (P < 0.05). Four mice inoculated with rAd-EGFP died within 9 to 11 d after challenge, and 5 PBS-inoculated mice died within 8 to 11 d after challenge (Figures 3B and 3C; Table I). All of the mice that received rAd-H5HA-EGFP remained healthy during the observation period after challenge with CK/HuN/77/05; slight weight loss was observed within the 1st week after challenge, followed by weight gain in the 2nd week (Figure 3B).

Discussion

Since their re-emergence in 2003, influenza A (H5N1) viruses have become endemic in some countries and continue to cause outbreaks in poultry and sporadic human infections (4). Current data suggest that H5N1 influenza viruses can be isolated from pigs but do not become established in swine populations (5–9). Pigs have been suggested to be “mixing vessels” capable of generating reassorted influenza viruses with pandemic potential (18), which suggests the urgent need for a vaccine to prevent swine H5N1 influenza.

Inactivated whole-virus vaccines have been widely used to control influenza outbreaks. However, the production of this kind of vaccine is highly dependent on chicken eggs, which limits production capacity and can create a bottleneck in the comprehensive prevention and control of H5N1 influenza pandemics (19). Several approaches have been used to generate vaccines to supplement the current egg-based production systems, such as recombinant subunit vaccines using baculovirus (20), plasmid DNA vaccines (21), virus-like particle vaccines (22), and replication-incompetent rAd vectors (10,23,24).

In this study we generated an rAd that expressed the HA gene of H5N1 SIV using the Ad-Max adenovirus system. To overcome the difficulties in screening for the rAd, we ligated the HA gene with the EGFP gene via an IRES and then inserted the HA-IRES-EGFP fragment into an adenovirus vector, ensuring independent expression of nonrelated genes connected by an IRES (25). The rAd was confirmed by PCR, RT-PCR, and Western blot analysis. The HA gene was accurately expressed by rAd-H5HA-EGFP, producing HA protein with good biologic activity, and the gene was highly stable after continuous passage. The TCID₅₀ of the purified rAd was determined to be 2.26 × 10¹⁰/mL.

The mouse is widely accepted as a relevant model of human and other mammalian influenza virus infections (26–28). Although H5N1 influenza viruses have been isolated from pigs (8,9), the pig infection model is not well established under laboratory conditions. Therefore, the immunogenicity and efficacy of rAd-H5HA-EGFP was evaluated in the mouse model in this study. Our findings indicated that rAd-H5HA-EGFP was immunogenic, inducing high HI antibody titers, and efficacious in challenge with homologous and heterologous H5N1 influenza viruses. The HP H5N1 virus CK/HuN/77/05 was used as a challenge virus in this study because preliminary investigations had shown that it could replicate systemically in vivo and cause death in mice. The protective efficacy of rAd-H5HA-EGFP against virus challenge was positively correlated with the antibody levels induced by the vaccine in mice. The vaccinated mice produced HI antibodies against homologous H5N1 strains, but there was little or no cross-reactivity with heterologous H5N1 viruses. The rAd-H5HA-EGFP vaccine offered protection against challenge with SW/FJ/1/01 or CK/HuN/77/05, preventing disease and reducing virus replication after challenge.

Previously reported adenovirus-based experimental influenza vaccines have also shown protective efficacy in mouse models of influenza challenge (10,19,23,29). It is difficult to make direct comparisons between the results of our study and those referenced above owing to differences in experimental design, vaccine dose, vaccination route, the challenge virus strain used, and the dose of the challenge virus. For example, adenovirus-based vaccines carrying HP avian influenza HA genes (10,23) protected BALB/c mice from challenge with lethal HP avian influenza virus. However, the vaccinated mice in those studies were all challenged with lower doses of HP avian influenza virus than the mice in our study: our mice vaccinated with rAd-H5HA-EGFP were challenged with 10⁶ EID₅₀ of SW/FJ/1/01 or CK/HuN/77/05, whereas the animals in the previous studies were challenged with 10^3.7 EID₅₀ of A/VN/1203/04. Moreover, our mice were vaccinated with a lower dose of vaccine, 10⁸ or 10⁷ TCID₅₀, than the mice in the other studies. A chimpanzee-derived replication-defective adenovirus vector of serotype SAd-V25 that expressing a linear B-cell epitope of the ectodomain of matrix 2 (M2e) within variable region 1 (VR1) induced M2e-specific antibody responses of higher magnitude and avidity than that of vectors carrying M2e within variable region 4 (VR4) or expressing the M2e as part of a transgene product (30). Compared with an AdHu5-HA control, a replication-incompetent porcine adenovirus 3 vector expressing an optimized A/Hanoi/30408/2005 H5N1 HA antigen (PAV3-HA) elicited robust humoral and/or cellular immune responses in vaccinated mice; thus, a PAV3-based vector is capable of mediating swift, strong immune responses and offers a promising alternative to AdHu5 (31). The substantial progress in molecular engineering of rAd vectors has indicated that AdHu26 as a vaccine vector may suffer from limitations similar to those found for vectors based on other prevalent human adenoviruses (32).

In summary, rAd-H5HA-EGFP induced high levels of HI antibodies and provided protection against challenge with 2 different H5N1 influenza viruses. It is therefore a promising vaccine candidate for protection against animal influenza. Further studies now need to be conducted to evaluate the immune efficacy of the vaccine in pigs.