Article
Immunogenicity and efficacy of a recombinant adenovirus expressing hemagglutinin from the H5N1 subtype of swine influenza virus in mice Yunpu Wu, Chuanling Qiao, Huanliang Yang, Yan Chen, Xiaoguang Xin, Hualan Chen
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.
Résumé Les virus de l’influenza de type H5N1 infectent une grande variété d’espèces aviaires et ont récemment été isolés des humains et des porcs. Dans la présente étude nous avons généré un adénovirus recombinant défectueux pour la réplication (rAd-H5HA-EGFP) exprimant le gène de l’hémagglutinine (HA) du virus H5N1 A/Porc/Fujian/1/2001 (SW/FJ/1/01) et évalué son immunogénicité et son activité protectrice chez des souris BALB/c. Le virus recombinant induisit des titres élevés d’anticorps inhibant l’hémagglutination (HI) à une dose médiane infectant les cultures cellulaires de 108 ou 107. Comparativement aux souris des groupes témoins, les souris vaccinées avec rAd-H5HA-EGFP n’ont pas montré de perte de poids apparente après une infection défi avec soit le virus homologue SW/FJ/1/01 ou le virus H5N1 hétérologue A/ Poulet/Hunan/77/2005 (CK/HuN/77/05). La réplication du virus de l’infection défi fut partiellement ou complètement inhibée, et les virus furent détectés à une charge virale significativement inférieure dans les organes des souris vaccinées, qui ont toutes survécu à l’infection défi avec le virus CK/HuN/77/05, ce qui ne fut pas le cas de la majorité des souris témoins. Ces résultats indiquent que rAd-H5HA-EGFP peut fournir une protection efficace contre des virus H5N1 hautement pathogènes chez les souris et est ainsi un nouveau vaccin candidat prometteur contre l’influenza H5N1 chez les animaux. (Traduit par Docteur Serge Messier)
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
Animal Influenza Laboratory of the Ministry of Agriculture, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, 427 Maduan Street, Harbin 150001, People’s Republic of China. M.S. Yunpu Wu’s current address is Liaoning Provincial Center for Animal Disease Control and Prevention, Shenyang 110164, People’s Republic of China. Address all correspondence to Dr. Chuanling Qiao or Dr. Hualan Chen; telephone: 86 451 51997168; fax: 86 451 51997166; e-mail: [email protected] or [email protected] Received November 6, 2012. Accepted March 15, 2013. 2014;78:117–126
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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 pBHGloxDE1,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 (59-AGCAAAAGCAGG-39). The HA gene was amplified from the cDNA with the primers HAF (59-CCCGTCGACATGGAGAAAATAGTGCTTCTTCTTGC-39) and HAR (59-TCCCCCGGGTTAAATGCAAATTCTGCATTGTAACG-39). 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
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adenovirus backbone plasmid (pBHGloxDE1,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 mL of Oligo (dT)18 (500 mg/mL), 4.0 mL of 53 buffer, 0.5 mL of deoxynucleotide triphosphate (10 mM) (Promega), and 14.5 mL of mRNA. After incubation of the reaction mixture at 65°C for 10 min, 0.5 mL of reverse transcriptase M-MLV (200 U/mL) (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 23 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.
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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.
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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-H5HAEGFP 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.
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, 120
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and the 22 mice in the other control group were inoculated intramuscularly with 50 mL 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 3 108 TCID50 and 2 3 107 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
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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.
5.3-kb band (Figure 1B, lane 3). The rAd shuttle plasmid pDC315H5HA-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).
Hemagglutination inhibition (HI) assay
Recombinant adenovirus shuttle plasmid pDC315-H5HA-EGFP and backbone plasmid pBHGloxDE1,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. 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-H5HAEGFP (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 rAdH5HA-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.
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-IRESEGFP, 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
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Generation of the rAd expressing the HA gene (rAd-H5HA-EGFP)
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 3 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
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, 0.5 , 0.5 , 0.5 , 0.5
, 0.5 , 0.5 , 0.5 , 0.5
, 0.5 , 0.5 , 0.5 , 0.5
, 0.5 , 0.5 0.58 6 1.01 1.50 6 1.30
, 0.5 , 0.5 , 0.5 , 0.5
8/8 8/8 5/5 5/5
6 d after challenge Proportion Kidney Brain Spleen Lung Kidney surviving
Titer of virus in organ (log10 EID50/gc)
CK/HuN/77/05 rAd-H5HA-EGFP, 108 20 , 0.5 , 0.5 1.67 6 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 6 2.59 , 0.5 , 0.5 , 0.5 3.25 6 1.09 , 0.5 8/8 rAd5-EGFP, 108 , 10 , 0.5 1.50 6 0 5.50 6 0 0.50 6 0.87 2.83 6 0.58 2.00 6 1.80 4.83 6 0.58 2.00 6 1.80 1/5 PBS, 50 mL , 10 , 0.5 1.33 6 1.26 4.50 6 0 1.00 6 0.87 3.17 6 0.58 3.42 6 0.14 5.88 6 0.14 2.25 6 0.43 0/5 a Groups of 6-wk-old BALB/c mice were vaccinated with a 50% tissue culture infective dose (TCID ) of 108 or 107 of rAd5-H5HA-EGFP or were inoculated as controls with 108 TCID 50 50 of rAd5-EGFP or 50 mL of phosphate-buffered saline (PBS) by intramuscularly administration. Three weeks later the vaccinated mice were given booster injections of rAd5-H5HA-EGFP, 2 3 108 TCID50 and 2 3 107 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. b Blood 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. c Three 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.
Challenge virus; Mean initial inoculum, HI Ab 4 d after challenge dose (TCID50 or mL)a titerb Brain Spleen Lung SW/FJ/1/01 rAd-H5HA-EGFP, 108 120 , 0.5 , 0.5 , 0.5 rAd-H5HA-EGFP, 107 92 , 0.5 , 0.5 , 0.5 rAd5-EGFP, 108 , 10 , 0.5 , 0.5 0.58 6 1.01 PBS, 50 mL , 10 , 0.5 , 0.5 1.33 6 1.26
Table I. Protective efficacy of the recombinant adenovirus rAd-H5HA-EGFP against challenge with highly pathogenic H5N1 viruses in mice
A
108 TCID50 rAd-H5HA-EGFP 107 TCID50 rAd-H5HA-EGFP rAd-EGFP PBS
B
108 TCID50 rAd-H5HA-EGFP 107 TCID50 rAd-H5HA-EGFP rAd-EGFP PBS
C 108 TCID50 rAd-H5HA-EGFP 107 TCID50 rAd-H5HA-EGFP rAd-EGFP
PBS
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 3 108 TCID50 or 2 3 107 TCID50, respectively, and in control groups of mice inoculated intramuscularly with rAd5-EGFP at a dose of 108 TCID50 (triangles) or with 50 mL 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 (10 6 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 EID 50 of 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.
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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. 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. 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 rAdH5HA-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
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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 3 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 rAdH5HA-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 103.7 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 replicationdefective 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
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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.
Acknowledgments This study was supported by the Harbin Municipal S&T Plan (2009AA6BN078) and the Scientific Research Program of the State Key Laboratory of Veterinary Biotechnology (NKLVBP200818), Harbin, People’s Republic of China. We thank Dr. Yanbing Li and Dr. Jianzhong Shi, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, for preparing the virus used in the animal challenge study.
References 1. Fouchier RA, Munster V, Wallensten A, et al. Characterization of a novel influenza A virus hemagglutinin subtype (H16) obtained from black-headed gulls. J Virol 2005;79:2814–2822. 2. Kuiken T, Rimmelzwaan G, van Riel D, et al. Avian H5N1 influenza in cats. Science 2004;306:241. 3. Mushtaq MH, Juan H, Jiang P, et al. Complete genome analysis of a highly pathogenic H5N1 influenza A virus isolated from a tiger in China. Arch Virol 2008;153:1569–1574. 4. World Health Organization (L’Organisation mondiale de la santé,[WHO]). Cumulative number of confirmed human cases of avian influenza A(H5N1) reported to WHO,2003–2013. http:// www.who.int/entity/influenza/human_animal_interface/ EN_GIP_20130215CumulativeNumberH5N1cases.pdf, WHO, 2013. 5. Choi YK, Nguyen TD, Ozaki H, et al. Studies of H5N1 influenza virus infection of pigs by using viruses isolated in Vietnam and Thailand in 2004. J Virol 2004;79:10821–10825. 6. Lipatov AS, Kwon YK, Sarmento LV, et al. Domestic pigs have low susceptibility to H5N1 highly pathogenic avian influenza viruses. PLoS Pathog 2008;4(7):e1000102. 7. Li H, Yu K, Yang H, et al. [Isolation and identification of H5N1 and H9N2 subtypes of avian influenza virus from Chinese pigs]. Chin J Prev Vet Med 2004;26:1–6. 8. Zhu Q, Yang H, Chen W, et al. A naturally occurring deletion in its NS gene contributes to the attenuation of an H5N1 swine influenza virus in chickens. J Virol 2008;82:220–228. 9. Shi WF, Gibbs MJ, Zhang Y, et al. Genetic analysis of four porcine avian influenza viruses isolated from Shandong, China. Arch Virol 2008;153:211–217. 10. Hoelscher MA, Garg S, Bangari DS, et al. Development of adenoviral-vector-based pandemic influenza vaccine against antigenically distinct human H5N1 strains in mice. Lancet 2006; 367:475–481. 11. Tatsis N, Ertl HC. Adenoviruses as vaccine vectors. Mol Ther 2004;10:616–629. 12. Juillard V, Villefroy P, Godfrin D, et al. Long-term humoral and cellular immunity induced by a single immunization with replication-defective adenovirus recombinant vector. Eur J Immunol 1995;25:3467–3473.
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13. Pacheco JM, Brum MC, Moraes MP, et al. Rapid protection of cattle from direct challenge with foot-and-mouth disease virus (FMDV) by a single inoculation with an adenovirus-vectored FMDV subunit vaccine. Virology 2005;337:205–209. 14. Reed LJ, Muench H. A simple method of estimating fifty percent endpoints. Am J Hyg 1938;27:493–497. 15. Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd ed. [Translated by Jing D and Li M]. Beijing: Chinese Science Press, 1989:891–897. 16. Chen H, Deng G, Li Z, et al. The evolution of H5N1 influenza viruses in ducks in southern China. Proc Natl Acad Sci U S A 2004;101:10452–10457. 17. World Organisation for Animal Health (Organisation mondiale de la santé animale [OIE]). Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. Paris, France: OIE, 2013. Chapter 2.8.8, Swine influenza:6. 18. Ito T, Couceiro JN, Kelm S, et al. Molecular basis for the generation in pigs of influenza A viruses with pandemic potential. J Virol 1998;72:7367–7373. 19. Holman DH, Wang D, Raja NU, et al. Multi-antigen vaccines based on complex adenovirus vectors induce protective immune responses against H5N1 avian influenza viruses. Vaccine 2008;6:2627–2639. 20. Treanor JJ, Wilkinson BE, Masseoud F, et al. Safety and immuno genicity of a recombinant hemagglutinin vaccine for H5 influenza in humans. Vaccine 2001;19:1732–1737. 21. Kodihalli S, Goto H, Kobasa DL, et al. DNA vaccine encoding hemagglutinin provides protective immunity against H5N1 influenza virus infection in mice. J Virol 1999;73:2094–2098. 22. Perrone LA, Ahmad A, Veguilla V, et al. Intranasal vaccination with 1918 influenza virus-like particles protects mice and ferrets from lethal 1918 and H5N1 influenza virus challenge. J Virol 2009;83:5726–5734. 23. Gao WT, Soloff AC, Lu X, et al. Protection of mice and poultry from lethal H5N1 avian influenza virus through adenovirusbased immunization. J Virol 2006;80:1959–1964. 24. Roy S, Kobinger GP, Lin J, et al. Partial protection against H5N1 influenza in mice with a single dose of a chimpanzee adenovirus vector expressing nucleoprotein. Vaccine 2007;25:6845–6851. 25. Davies MV, Kaufman RJ. The sequence context of the initiation codon in the encephalomyocarditis virus leader modulates efficiency of internal translation initiation. J Virol 1992;66:1924–1932. 26. Gubareva LV, McCullers JA, Bethell RC, et al. Characterization of influenza A/HongKong/156/97 (H5N1) virus in a mouse model and protective effect of zanamivir on H5N1 infection in mice. J Infect Dis 1998;178:1592–1596. 27. McDermott JE, Shankaran H, Eisfeld AJ, et al. Conserved host response to highly pathogenic avian influenza virus infection in human cell culture, mouse and macaque model systems. BMC Syst Biol 2011;5:190. 28. Uraki R, Kiso M, Shinya K, et al. Virulence determinants of pandemic A(H1N1)2009 influenza virus in a mouse model. J Virol 2013;87:2226–2233. 29. Tompkins SM, Zhao ZS, Lo CY, et al. Matrix protein 2 vaccination and protection against influenza viruses, including subtype H5N1. Emerg Infect Dis 2007;13:426–435.
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30. Zhou D, Wu TL, Emmer KL, et al. Hexon-modified recombinant E1-deleted adenovirus vectors as dual specificity vaccine carriers for influenza virus. Mol Ther 2013;21:696–706. 31. Patel A, Tikoo S, Kobinger G. A porcine adenovirus with low human seroprevalence is a promising alternative vaccine vector to human adenovirus 5 in an H5N1 virus disease model. PLoS One 2010;5(12):e15301.
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32. Chen H, Xiang ZQ, Li Y, et al. Adenovirus-based vaccines: Comparison of vectors from three species of adenoviridae. J Virol 2010;84:10522–10532.
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Immunogenicity and efficacy of a recombinant adenovirus expressing hemagglutinin from the H5N1 subtype of swine influenza virus in mice Yunpu Wu, Chuanling Qiao, Huanliang Yang, Yan Chen, Xiaoguang Xin, Hualan Chen
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.
Résumé Les virus de l’influenza de type H5N1 infectent une grande variété d’espèces aviaires et ont récemment été isolés des humains et des porcs. Dans la présente étude nous avons généré un adénovirus recombinant défectueux pour la réplication (rAd-H5HA-EGFP) exprimant le gène de l’hémagglutinine (HA) du virus H5N1 A/Porc/Fujian/1/2001 (SW/FJ/1/01) et évalué son immunogénicité et son activité protectrice chez des souris BALB/c. Le virus recombinant induisit des titres élevés d’anticorps inhibant l’hémagglutination (HI) à une dose médiane infectant les cultures cellulaires de 108 ou 107. Comparativement aux souris des groupes témoins, les souris vaccinées avec rAd-H5HA-EGFP n’ont pas montré de perte de poids apparente après une infection défi avec soit le virus homologue SW/FJ/1/01 ou le virus H5N1 hétérologue A/ Poulet/Hunan/77/2005 (CK/HuN/77/05). La réplication du virus de l’infection défi fut partiellement ou complètement inhibée, et les virus furent détectés à une charge virale significativement inférieure dans les organes des souris vaccinées, qui ont toutes survécu à l’infection défi avec le virus CK/HuN/77/05, ce qui ne fut pas le cas de la majorité des souris témoins. Ces résultats indiquent que rAd-H5HA-EGFP peut fournir une protection efficace contre des virus H5N1 hautement pathogènes chez les souris et est ainsi un nouveau vaccin candidat prometteur contre l’influenza H5N1 chez les animaux. (Traduit par Docteur Serge Messier)
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
Animal Influenza Laboratory of the Ministry of Agriculture, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, 427 Maduan Street, Harbin 150001, People’s Republic of China. M.S. Yunpu Wu’s current address is Liaoning Provincial Center for Animal Disease Control and Prevention, Shenyang 110164, People’s Republic of China. Address all correspondence to Dr. Chuanling Qiao or Dr. Hualan Chen; telephone: 86 451 51997168; fax: 86 451 51997166; e-mail: [email protected] or [email protected] Received November 6, 2012. Accepted March 15, 2013. 2014;78:117–126
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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 pBHGloxDE1,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 (59-AGCAAAAGCAGG-39). The HA gene was amplified from the cDNA with the primers HAF (59-CCCGTCGACATGGAGAAAATAGTGCTTCTTCTTGC-39) and HAR (59-TCCCCCGGGTTAAATGCAAATTCTGCATTGTAACG-39). 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
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adenovirus backbone plasmid (pBHGloxDE1,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 mL of Oligo (dT)18 (500 mg/mL), 4.0 mL of 53 buffer, 0.5 mL of deoxynucleotide triphosphate (10 mM) (Promega), and 14.5 mL of mRNA. After incubation of the reaction mixture at 65°C for 10 min, 0.5 mL of reverse transcriptase M-MLV (200 U/mL) (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 23 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.
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A
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H5HA
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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.
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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-H5HAEGFP 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.
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, 120
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and the 22 mice in the other control group were inoculated intramuscularly with 50 mL 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 3 108 TCID50 and 2 3 107 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
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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.
5.3-kb band (Figure 1B, lane 3). The rAd shuttle plasmid pDC315H5HA-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).
Hemagglutination inhibition (HI) assay
Recombinant adenovirus shuttle plasmid pDC315-H5HA-EGFP and backbone plasmid pBHGloxDE1,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. 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-H5HAEGFP (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 rAdH5HA-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.
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-IRESEGFP, 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
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Generation of the rAd expressing the HA gene (rAd-H5HA-EGFP)
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 3 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
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, 0.5 , 0.5 , 0.5 , 0.5
, 0.5 , 0.5 , 0.5 , 0.5
, 0.5 , 0.5 , 0.5 , 0.5
, 0.5 , 0.5 0.58 6 1.01 1.50 6 1.30
, 0.5 , 0.5 , 0.5 , 0.5
8/8 8/8 5/5 5/5
6 d after challenge Proportion Kidney Brain Spleen Lung Kidney surviving
Titer of virus in organ (log10 EID50/gc)
CK/HuN/77/05 rAd-H5HA-EGFP, 108 20 , 0.5 , 0.5 1.67 6 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 6 2.59 , 0.5 , 0.5 , 0.5 3.25 6 1.09 , 0.5 8/8 rAd5-EGFP, 108 , 10 , 0.5 1.50 6 0 5.50 6 0 0.50 6 0.87 2.83 6 0.58 2.00 6 1.80 4.83 6 0.58 2.00 6 1.80 1/5 PBS, 50 mL , 10 , 0.5 1.33 6 1.26 4.50 6 0 1.00 6 0.87 3.17 6 0.58 3.42 6 0.14 5.88 6 0.14 2.25 6 0.43 0/5 a Groups of 6-wk-old BALB/c mice were vaccinated with a 50% tissue culture infective dose (TCID ) of 108 or 107 of rAd5-H5HA-EGFP or were inoculated as controls with 108 TCID 50 50 of rAd5-EGFP or 50 mL of phosphate-buffered saline (PBS) by intramuscularly administration. Three weeks later the vaccinated mice were given booster injections of rAd5-H5HA-EGFP, 2 3 108 TCID50 and 2 3 107 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. b Blood 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. c Three 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.
Challenge virus; Mean initial inoculum, HI Ab 4 d after challenge dose (TCID50 or mL)a titerb Brain Spleen Lung SW/FJ/1/01 rAd-H5HA-EGFP, 108 120 , 0.5 , 0.5 , 0.5 rAd-H5HA-EGFP, 107 92 , 0.5 , 0.5 , 0.5 rAd5-EGFP, 108 , 10 , 0.5 , 0.5 0.58 6 1.01 PBS, 50 mL , 10 , 0.5 , 0.5 1.33 6 1.26
Table I. Protective efficacy of the recombinant adenovirus rAd-H5HA-EGFP against challenge with highly pathogenic H5N1 viruses in mice
A
108 TCID50 rAd-H5HA-EGFP 107 TCID50 rAd-H5HA-EGFP rAd-EGFP PBS
B
108 TCID50 rAd-H5HA-EGFP 107 TCID50 rAd-H5HA-EGFP rAd-EGFP PBS
C 108 TCID50 rAd-H5HA-EGFP 107 TCID50 rAd-H5HA-EGFP rAd-EGFP
PBS
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 3 108 TCID50 or 2 3 107 TCID50, respectively, and in control groups of mice inoculated intramuscularly with rAd5-EGFP at a dose of 108 TCID50 (triangles) or with 50 mL 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 (10 6 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 EID 50 of 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.
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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. 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. 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 rAdH5HA-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
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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 3 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 rAdH5HA-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 103.7 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 replicationdefective 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
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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.
Acknowledgments This study was supported by the Harbin Municipal S&T Plan (2009AA6BN078) and the Scientific Research Program of the State Key Laboratory of Veterinary Biotechnology (NKLVBP200818), Harbin, People’s Republic of China. We thank Dr. Yanbing Li and Dr. Jianzhong Shi, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, for preparing the virus used in the animal challenge study.
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