International Journal of Obesity (2014) 38, 1470–1474 © 2014 Macmillan Publishers Limited All rights reserved 0307-0565/14 www.nature.com/ijo
SHORT COMMUNICATION
Proof-of-concept for a virus-induced obesity vaccine; vaccination against the obesity agent adenovirus 36 H-N Na and J-H Nam Human adenovirus 36 (Ad36) is positively associated with obesity in humans and animals. Ad36 infection is characterized by increased adiposity and inflammation. To investigate the possibility that a prophylactic vaccine candidate might protect against Ad36-induced obesity and inflammation, we purified Ad36 and ultraviolet-irradiated virus to obtain a vaccine candidate. After immunizing the mice with the vaccine candidate (vaccinated group), live Ad36 was injected into mice as a challenge test. Unvaccinated mice (control group) were immunized with phosphate-buffered saline and then challenged with live Ad36. Fourteen weeks after challenge, we compared adiposity and inflammation in vaccinated and control mice. The control group showed 17% greater body weight and 20% more epididymal fats compared with the vaccinated group. In addition, the vaccinated group had decreased serum levels of pro-inflammatory cytokines, and infiltrated immune cells, especially M1 macrophages, in fat tissue. Therefore, the vaccine candidate for Ad36 was able to protect against Ad36-increased body weight and fat as well as inflammatory states after challenge. These results provide proof-of-concept for prophylactic vaccination against virus-induced adiposity. International Journal of Obesity (2014) 38, 1470–1474; doi:10.1038/ijo.2014.41 Keywords: adenovirus 36; vaccine; inflammation; M1 macrophage
INTRODUCTION Obesity is associated with increased risk of type 2 diabetes, cardiovascular disease, gastrointestinal disorders and several types of cancers.1 As obesity has become one of the leading health problems in modern society, there is a need to develop effective strategies for weight management. Currently approved antiobesity drugs have only limited efficacy, generally facilitating no more than a 5–10% reduction of body weight, and are often associated with side effects.2 Thus, there is a major medical need for the development of new antiobesity drugs. Recent studies have shown an association between human adenovirus 36 (Ad36) infection and increased adiposity in humans, mice, chickens and monkeys.3–5 Ad36 infection in animals is associated with increased body weight, epididymal fats, visceral fats and inguinal fats.4,5 Moreover, Ad36 infection induces inflammation in fat tissues through inflammatory cytokines and infiltrated immune cells.6 Therefore, an antiobesity vaccine is needed to control Ad36 infection. In this study, we inactivated purified Ad36 by ultraviolet (UV) irradiation. Mice injected with the inactivated virus were protected from Ad36-induced adiposity and inflammation. Thus, our results provide proof-of-concept for prophylactic vaccination against virus-induced adiposity.
MATERIALS AND METHODS Purification and inactivation of adenovirus 36 Human adenovirus 36 (Ad36) was purchased from American Type Culture Collection (Manassas, VA, USA). The A549 cell line (adenocarcinomic human alveolar basal epithelial cells), was used for Ad36 growth. Ad36 was harvested from the supernatant of infected A549 cells, obtained by
freeze-thawing three times. The virus was then purified by the CsCl method.7 Purified virus was inactivated by irradiation using the UV crosslinker 115 VAC (2500 mJ cm − 2; GE Healthcare Life Sciences, Piscataway, NJ, USA).8 Inactivation of purified UV-irradiated virus was confirmed by re-infection to A549 cells (data not shown).
Animal care Four-week-old C57BL/6 mice were purchased from Orient Bio Inc. (Seongnam, Korea). The mice were adapted to laboratory conditions (18–23 °C, 55–60% humidity, 12 h light/dark cycle with the lights on at 0700 h). The mice were fed a normal diet and water ad libitum. Food and water intake as well as body weight were monitored in mice every week. All mice were handled according to the guidelines and regulations of the Korean Association for Laboratory Animals. The protocol was approved by the International Animal Care and Use Committee, Sungsim Campus, Catholic University of Korea (#2012-017).
Vaccination and challenge Mice (n = 10) were injected intraperitoneally with 5 μg of the vaccine candidate and complete adjuvant (1:1 ratio, Freund’s complete adjuvant, # F5881; Sigma-Aldrich, St Louis, MO, USA). Two weeks after the first immunization, mice were injected again with 5 μg of the vaccine candidate and incomplete adjuvant (Freund’s incomplete adjuvant, #F5506; Sigma-Aldrich). One week after the last immunization, mice were infected intraperitoneally with live Ad36 (1 × 107 plaque forming units per mouse) for the challenge test. The body weights of mice were monitored for 14 weeks after challenge. The control mouse group (n = 10) was injected with phosphate-buffered saline mixed with same adjuvant, and the injection and challenge schedules were exactly the same as the vaccinated group. In that, both the vaccinated and control groups were equally challenged with live Ad36. The average weight of the 4-week-old mice in both the vaccination and control groups was 10–12 g, and their weight was 14–15 g at the beginning of the challenge test. Both groups were fed a
Department of Biotechnology, The Catholic University of Korea, Bucheon, Korea. Correspondence: Professor J-H Nam, Department of Biotechnology, The Catholic University of Korea, Bucheon 420-743, Korea. E-mail: [email protected] Received 20 October 2013; revised 23 February 2014; accepted 4 March 2014; accepted article preview online 11 March 2014; advance online publication, 8 April 2014
Virus-induced obesity vaccine H-N Na and J-H Nam
1471 normal diet during the immunization with the vaccination candidate and challenged with live Ad36.
Statistical analysis All the data were analyzed with the Student’s t-test using the SAS 9.1 program (SAS Institute Inc., Cary, NC, USA). The data are presented as means ± s.e. Multiple comparisons were analyzed by analysis of variance test. Differences between means were considered statistically significant at Po0.05. See the Supplementary Information Materials and Methods for details and other information.
RESULTS Vaccine candidate for Ad36 reduces weight gain and fat Ad36 infection increases body weight and fat in most animals.4,5 In the present study, we investigated whether vaccination could prevent Ad36-induced adiposity. Mice were immunized twice every 2 weeks with the vaccine candidate (vaccinated group) or phosphate-buffered saline (control group), then challenged with live Ad36. Food and water intake as well as body weight were monitored in mice for 14 weeks after challenge. Although food and water intake were similar in all groups (data not shown), the vaccinated group showed less total body weight compared with the control group at 14 weeks after challenge (Figure 1a). Weight gain was calculated as the difference in weight at the time of challenge and the time of sacrifice. The vaccinated group showed less weight gain compared with the control group (Figure 1b; P = 0.041). In addition, the vaccinated group also showed less epididymal fat than the control group (Figure 1c; P = 0.036). The vaccinated group (Ad36 vaccine candidate injection) showed increased neutralizing antibody against Ad36 as the control group (live Ad36 injection). The mock (PBS injection) did not show the antibody, but control group and vaccinated group increased five times compared with mock (Figure 1d). Therefore, the vaccine candidate was able to protect mice from increased body weight and fat caused by Ad36 infection. Vaccine candidate for Ad36 reduces inflammation Ad36 infection increases infiltration of immune cells such as macrophages into fat tissues.6 Immune cells infiltrate adipose tissue and secrete cytokines leading to an increased inflammatory state and adiposity.6,9,10 In this study, we observed that the vaccine candidate protected mice from infiltration of immune cells into epididymal fats. Fourteen weeks after the challenge injection, epididymal fats were collected from mice and stained with hematoxylin and eosin. Infiltrated immune cells were reduced in the vaccinated group compared with the control group (Figure 2a). Moreover, the sliced fats were also stained with F4/80 antibody to confirm infiltrated macrophages. Infiltration of macrophages was also decreased in the vaccinated group compared with the control group (Figure 2a). Previously, it was reported that Ad36-induced obesity increases inflammation through pro-inflammatory cytokines such as MCP 1 and TNF α.6 Here, serum levels of these two cytokines were measured by an immunoassay 14 weeks after challenge. MCP 1 serum levels were decreased by four times in the vaccinated group compared with the control group (Figure 2b; P = 0.0002). TNF α level in the vaccinated group were also reduced compared with the control group (Figure 2b; P = 0.022). Moreover, levels of MCP 1 mRNA in epididymal fats of the vaccinated group were reduced compared with the control group (Figure 2c; P = 0.0003). However, expression of TNF α mRNA in epididymal fats was not significantly different between the vaccinated and control groups (Figure 2c; P = 0.446). Macrophages can be divided into two subtypes based on immune response: M1 macrophages (pro-inflammatory © 2014 Macmillan Publishers Limited
macrophages, CD64) and M2 macrophages (anti-inflammatory macrophages, CD206).6 In the present study, the vaccinated group had decreased M1 macrophages (P = 0.014) unlike M2 macrophages (Figure 2d). According to these results, the vaccine candidate reduced production of serum pro-inflammatory cytokines as well as infiltration of immune cells after live Ad36 challenge. DISCUSSION Ad36 is associated with human obesity in the USA, Korea and Italy.3,11,12 In addition to humans, virus infection induces adiposity in animals including chickens, mice, rats and monkeys.4,5 Moreover, Ad36 infection induces inflammation in adipose tissue and maintains an inflammatory state through pro-inflammatory cytokines.6 Eventually, increased inflammation is able to sustain obesity. Therefore, it is necessary to develop therapeutic drugs or prophylactic vaccines that can regulate infection of the virus. In the present study, we inactivated Ad36 by UV irradiation to create an Ad36 vaccine candidate. UV irradiation can inactivate viruses without harmful effects on the antigenic structure and biological function of proteins.13 In a previous study, UV-irradiated Ad36 was also evaluated.8 However, changes in body weight and fat were not observed 4 days after infection.8 Actually, 4 days is not enough time to identify changes in body weight or fat after Ad36 infection. Therefore, we monitored body weight for 14 weeks after live-Ad36 challenge. The vaccine candidate provided protection against increase in body weight and fat after Ad36 challenge (Figure 1). Moreover, the vaccine candidate diminished the induction of pro-inflammatory cytokines as well as M1 macrophages in adipose tissue by Ad36 infection (Figure 2); thus, indicating that the vaccine ameliorates Ad36-induced inflammation. Previous papers showed that infecting with Ad36 improves the liver condition and glycemic control in mice and human.14,15 Thus, we also analyzed the liver condition and lipid profiles after vaccination by hematoxylin and eosin staining and by measuring the glucose and nonesterified fatty acid levels in serum. However, in the liver, we did not observe any differences between vaccinated group and control group (in Supplementary Figure 1), because we used normal diet and this normal diet may mask the Ad36-benefit effects, which indicate the improving glycemic control and hepatic-lipid content. Actually, these Ad36-benefit effects seem to make unnecessary the developing vaccine for protecting Ad36 infection. However, other paper also showed that infecting with Ad36 may associate with the reduction of bone strength in late-adolescent females.16 Moreover, Ad36 can trigger the inflammation in murine and human,6,14 even in this inflammation seems to be required to improve glycemic control. Interestingly, E4orf1, one of the Ad36 viral proteins, can stimulate the glucose uptake through independent insulin-signaling pathway.17 By the way, we observed that E4orf1 expression induced different signaling pathways compared with Ad36 (unpublished data). Thus, E4orf1 may have the development potential as an antidiabetic drug. However, Ad36 itself may be not used as a therapeutic agent as compared with oncolytic viruses, which is involved in the selective lysis of cancer cells, because the mechanism and etiological function of Ad36 is yet to be completely understood. Therefore, we think we still need the development of Ad36 vaccine, apart from the development of antidiabetic drug by E4orf1. Three types of virus vaccines have been used clinically; inactivated virus (purified whole viruses inactivated by chemical or UV irradiation), live-attenuated vaccine (live virus, but attenuated), and subunit vaccine (viral structural proteins, sometimes constructed as a virus-like particle without the viral genome).18 Although live-attenuated vaccines can trigger highimmune responses, safe use of such vaccines is a concern.19 International Journal of Obesity (2014) 1470 – 1474
Virus-induced obesity vaccine H-N Na and J-H Nam
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Figure 1. The vaccine candidate for Ad36 decreased body weight and fat (n = 10 per group). (a) Mice were injected with the Ad36 vaccine candidate (vaccination group) or phosphate-buffered saline (control group). One week after the second immunization, mice were injected with live Ad36 (1 × 107 pfu per mouse) for the challenge test. The body weights of mice were monitored for 14 weeks after challenge (**P o0.01, ***Po0.001; t-test). (b) Fourteen weeks after the challenge, weight gain was calculated as the difference in body weight at the time of challenge and the time of sacrifice (*Po 0.05; t-test). (c) Fourteen weeks after challenge, mice were killed and epididymal fats were extracted. The weights of the epididymal fats were measured (*Po0.05; t-test). The amount of epididymal fats in the control and vaccination groups was quantified (The size of scale bar, 1 cm). (d) The amount of Ad36-specific antibody is indicated by absorbance. Serum was obtained from mice 2 weeks after the secondary immunization. The wells of plates were coated with purified Ad36 (10, 100 or 1000 ng per well), serum was added, and the absorbance was measured at 450 nm using a spectrophotometer to confirm the amount of neutralizing antibody (Control, live Ad36 injection; Vaccination, Ad36 vaccine candidate injection; Mock, PBS injection; *P o0.05, ANOVA).
Recently, subunit vaccines, such as the human papilloma virus vaccine, have been developed. However, the manufacturing of this vaccine can be technically difficult and can be time consuming. Therefore, we chose to develop a prophylactic Ad36 vaccine of the inactivated type by UV irradiation. UV irradiation was used for inactivation of the virus because it does not modify the conformation of the virus-capsid proteins20 and induces International Journal of Obesity (2014) 1470 – 1474
proper neutralizing antibody against Ad36. Thus, virus inactivation by UV irradiation is suitable for early-stage development of a Ad36 vaccine. However, to develop a Ad36 vaccine to be used in a clinic, other methods must also be considered because of issues such as the licensing-approval system, economical efficiency, and so on. Moreover, further study is needed for the cross-protection against other adenoviruses induced by Ad36-specific-neutralizing antibodies. © 2014 Macmillan Publishers Limited
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Figure 2. The vaccine candidate for Ad36 reduced inflammation in mice (n = 10 per group). (a) Epididymal fats were extracted from mice 14 weeks after challenge. The fats were fixed with 1% paraformaldehyde, and then stained with hematoxylin and eosin. In addition, fats were added with F4/80 antibody to confirm infiltration of macrophages. Adipocytes were observed with AxioVision version 4.8 (Carl Zeiss, Oberkochen, Germany). (b) Fourteen weeks after challenge, sera were collected from mice. The protein concentration of MCP 1 and TNF α was measured by an immunoassay (*Po0.05, **Po0.01; t-test). (c) RNA was isolated from the extracted epididymal fats with TRIzol reagent and synthesized into complementary DNA using reverse transcriptase. mRNA expression of MCP 1 and TNF α was determined by quantitative real-time PCR (*Po0.05; t-test). (d) mRNA expression of M1 (CD64) and M2 (CD206) macrophage markers was confirmed in epididymal fats by quantitative real-time PCR (*Po0.05; t-test).
Further experiments should include an Ad2-injected group as a negative control because, unlike Ad36, Ad2 does not increase adiposity.14
© 2014 Macmillan Publishers Limited
To our knowledge, the results of this study are the first proof-of-concept for the development of a vaccine against Ad36.
International Journal of Obesity (2014) 1470 – 1474
Virus-induced obesity vaccine H-N Na and J-H Nam
1474 CONFLICT OF INTEREST The authors declare no conflict of interest.
ACKNOWLEDGEMENTS This work was supported by a grant from the GRRC of the Catholic University of Korea and by the Basic Science Research Program of the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2012R1A1A2039819).
REFERENCES 1 Reaven GM. All obese individuals are not created equal: insulin resistance is the major determinant of cardiovascular disease in overweight/obese individuals. Diab Vasc Dis Res 2005; 2: 105–112. 2 Hainer V. Comparative efficiency and safety of pharmacological approaches to the management of obesity. Diabetes Care 2011; 34: S349–S354. 3 Atkinson RL, Dhurandhar NV, Allison DB, Bowen RL, Israel BA, Augustus AS et al. Human adenovirus-36 is associated with increased body weight and paradoxical reduction of serum lipids. Int J Obes (Lond) 2005; 29: 281–286. 4 Dhurandhar NV, Israel BA, Kolesar JM, Mayhew GF, Cook ME, Atkinson RL. Increased adiposity in animals due to a human virus. Int J Obes Relat Metab Disord 2000; 24: 989–996. 5 Dhurandhar NV, Whigham LD, Abbott DH, Schultz-Darken NJ, Israel BA, Atkinson RL et al. Human adenovirus Ad-36 promotes weight gain in male rhesus and marmoset monkeys. J Nutr 2002; 132: 3155–3160. 6 Na HN, Nam JH. Adenovirus 36 as an obesity agent maintains the obesity state by increasing MCP-1 and inducing inflammation. J Infect Dis 2012; 205: 914–922. 7 Tollefson AE, Kuppuswamy M, Shashkova EV, Doronin K, Wold WS. Preparation and titration of CsCl-banded adenovirus stocks. Methods Mol Med 2007; 130: 223–235.
8 Pasarica M, Loiler S, Dhurandhar NV. Acute effect of infection by adipogenic human adenovirus Ad36. Arch Virol 2008; 153: 2097–2102. 9 Lumeng CN, Saltiel AR. Inflammatory links between obesity and metabolic disease. J Clin Invest 2011; 121: 2111–2117. 10 Sun S, Ji Y, Kersten S, Qi L. Mechanisms of inflammatory responses in obese adipose tissue. Annu Rev Nutr 2012; 32: 261–286. 11 Na HN, Kim J, Lee HS, Shim KW, Kimm H, Nam JH et al. Association of human adenovirus-36 in overweight Korean adults. Int J Obes (Lond) 2012; 36: 281–285. 12 Trovato GM, Castro A, Tonzuso A, Garozzo A, Martines GF, Catalano D et al. Human obesity relationship with Ad36 adenovirus and insulin resistance. Int J Obes (Lond) 2009; 33: 1402–1409. 13 Raviv Y, Viard M, Bess JW, Chertova E, Blumenthal R. Inactivation of retroviruses with preservation of structural integrity by targeting the hydrophobic domain of the viral envelope. J Virol 2005; 79: 12394–12400. 14 Krishnapuram R, Dhurandhar EJ, Dubuisson O, Kirk-Ballard H, Bajpeyi S, Butte N et al. Template to improve glycemic control without reducing adiposity or dietary fat. Am J Physiol Endocrinol Metab 2011; 300: E779–E789. 15 Trovato GM, Martines GF, Trovato FM, Pirri C, Pace P, Garozzo A et al. Adenovirus36 seropositivity enhances effects of nutritional intervention on obesity, bright liver, and insulin resistance. Dig Dis Sci 2012; 57: 535–544. 16 Laing EM, Tripp RA, Pollock NK, Baile CA, Della-Fera MA, Rayalam S et al. Adenovirus 36, adiposity, and bone strength in late-adolescent females. J Bone Miner Res 2013; 28: 489–496. 17 Dhurandhar NV. Insulin sparing action of adenoirus 36 and its E4orf1 protein. J Diabetes Complications 2013; 27: 191–199. 18 Choi Y, Chang J. Viral vectors for vaccine applications. Clin Exp Vaccine Res 2013; 2: 97–105. 19 Burchill MA, Tamburini BA, Pennock ND, White JT, Kurche JS, Kedl RM. T cell vaccinology: exploring the known unknowns. Vaccine 2013; 31: 297–305. 20 Lee SG, Jin JY, Kwon BM, Park SW, Paik SY. Inactivation of 2009 pandemic human influenza A virus H1N1 by photocatalyst under UV irradiation. J Bac Virol 2012; 42: 83–85.
Supplementary Information accompanies this paper on International Journal of Obesity website (http://www.nature.com/ijo)
International Journal of Obesity (2014) 1470 – 1474
© 2014 Macmillan Publishers Limited
SHORT COMMUNICATION
Proof-of-concept for a virus-induced obesity vaccine; vaccination against the obesity agent adenovirus 36 H-N Na and J-H Nam Human adenovirus 36 (Ad36) is positively associated with obesity in humans and animals. Ad36 infection is characterized by increased adiposity and inflammation. To investigate the possibility that a prophylactic vaccine candidate might protect against Ad36-induced obesity and inflammation, we purified Ad36 and ultraviolet-irradiated virus to obtain a vaccine candidate. After immunizing the mice with the vaccine candidate (vaccinated group), live Ad36 was injected into mice as a challenge test. Unvaccinated mice (control group) were immunized with phosphate-buffered saline and then challenged with live Ad36. Fourteen weeks after challenge, we compared adiposity and inflammation in vaccinated and control mice. The control group showed 17% greater body weight and 20% more epididymal fats compared with the vaccinated group. In addition, the vaccinated group had decreased serum levels of pro-inflammatory cytokines, and infiltrated immune cells, especially M1 macrophages, in fat tissue. Therefore, the vaccine candidate for Ad36 was able to protect against Ad36-increased body weight and fat as well as inflammatory states after challenge. These results provide proof-of-concept for prophylactic vaccination against virus-induced adiposity. International Journal of Obesity (2014) 38, 1470–1474; doi:10.1038/ijo.2014.41 Keywords: adenovirus 36; vaccine; inflammation; M1 macrophage
INTRODUCTION Obesity is associated with increased risk of type 2 diabetes, cardiovascular disease, gastrointestinal disorders and several types of cancers.1 As obesity has become one of the leading health problems in modern society, there is a need to develop effective strategies for weight management. Currently approved antiobesity drugs have only limited efficacy, generally facilitating no more than a 5–10% reduction of body weight, and are often associated with side effects.2 Thus, there is a major medical need for the development of new antiobesity drugs. Recent studies have shown an association between human adenovirus 36 (Ad36) infection and increased adiposity in humans, mice, chickens and monkeys.3–5 Ad36 infection in animals is associated with increased body weight, epididymal fats, visceral fats and inguinal fats.4,5 Moreover, Ad36 infection induces inflammation in fat tissues through inflammatory cytokines and infiltrated immune cells.6 Therefore, an antiobesity vaccine is needed to control Ad36 infection. In this study, we inactivated purified Ad36 by ultraviolet (UV) irradiation. Mice injected with the inactivated virus were protected from Ad36-induced adiposity and inflammation. Thus, our results provide proof-of-concept for prophylactic vaccination against virus-induced adiposity.
MATERIALS AND METHODS Purification and inactivation of adenovirus 36 Human adenovirus 36 (Ad36) was purchased from American Type Culture Collection (Manassas, VA, USA). The A549 cell line (adenocarcinomic human alveolar basal epithelial cells), was used for Ad36 growth. Ad36 was harvested from the supernatant of infected A549 cells, obtained by
freeze-thawing three times. The virus was then purified by the CsCl method.7 Purified virus was inactivated by irradiation using the UV crosslinker 115 VAC (2500 mJ cm − 2; GE Healthcare Life Sciences, Piscataway, NJ, USA).8 Inactivation of purified UV-irradiated virus was confirmed by re-infection to A549 cells (data not shown).
Animal care Four-week-old C57BL/6 mice were purchased from Orient Bio Inc. (Seongnam, Korea). The mice were adapted to laboratory conditions (18–23 °C, 55–60% humidity, 12 h light/dark cycle with the lights on at 0700 h). The mice were fed a normal diet and water ad libitum. Food and water intake as well as body weight were monitored in mice every week. All mice were handled according to the guidelines and regulations of the Korean Association for Laboratory Animals. The protocol was approved by the International Animal Care and Use Committee, Sungsim Campus, Catholic University of Korea (#2012-017).
Vaccination and challenge Mice (n = 10) were injected intraperitoneally with 5 μg of the vaccine candidate and complete adjuvant (1:1 ratio, Freund’s complete adjuvant, # F5881; Sigma-Aldrich, St Louis, MO, USA). Two weeks after the first immunization, mice were injected again with 5 μg of the vaccine candidate and incomplete adjuvant (Freund’s incomplete adjuvant, #F5506; Sigma-Aldrich). One week after the last immunization, mice were infected intraperitoneally with live Ad36 (1 × 107 plaque forming units per mouse) for the challenge test. The body weights of mice were monitored for 14 weeks after challenge. The control mouse group (n = 10) was injected with phosphate-buffered saline mixed with same adjuvant, and the injection and challenge schedules were exactly the same as the vaccinated group. In that, both the vaccinated and control groups were equally challenged with live Ad36. The average weight of the 4-week-old mice in both the vaccination and control groups was 10–12 g, and their weight was 14–15 g at the beginning of the challenge test. Both groups were fed a
Department of Biotechnology, The Catholic University of Korea, Bucheon, Korea. Correspondence: Professor J-H Nam, Department of Biotechnology, The Catholic University of Korea, Bucheon 420-743, Korea. E-mail: [email protected] Received 20 October 2013; revised 23 February 2014; accepted 4 March 2014; accepted article preview online 11 March 2014; advance online publication, 8 April 2014
Virus-induced obesity vaccine H-N Na and J-H Nam
1471 normal diet during the immunization with the vaccination candidate and challenged with live Ad36.
Statistical analysis All the data were analyzed with the Student’s t-test using the SAS 9.1 program (SAS Institute Inc., Cary, NC, USA). The data are presented as means ± s.e. Multiple comparisons were analyzed by analysis of variance test. Differences between means were considered statistically significant at Po0.05. See the Supplementary Information Materials and Methods for details and other information.
RESULTS Vaccine candidate for Ad36 reduces weight gain and fat Ad36 infection increases body weight and fat in most animals.4,5 In the present study, we investigated whether vaccination could prevent Ad36-induced adiposity. Mice were immunized twice every 2 weeks with the vaccine candidate (vaccinated group) or phosphate-buffered saline (control group), then challenged with live Ad36. Food and water intake as well as body weight were monitored in mice for 14 weeks after challenge. Although food and water intake were similar in all groups (data not shown), the vaccinated group showed less total body weight compared with the control group at 14 weeks after challenge (Figure 1a). Weight gain was calculated as the difference in weight at the time of challenge and the time of sacrifice. The vaccinated group showed less weight gain compared with the control group (Figure 1b; P = 0.041). In addition, the vaccinated group also showed less epididymal fat than the control group (Figure 1c; P = 0.036). The vaccinated group (Ad36 vaccine candidate injection) showed increased neutralizing antibody against Ad36 as the control group (live Ad36 injection). The mock (PBS injection) did not show the antibody, but control group and vaccinated group increased five times compared with mock (Figure 1d). Therefore, the vaccine candidate was able to protect mice from increased body weight and fat caused by Ad36 infection. Vaccine candidate for Ad36 reduces inflammation Ad36 infection increases infiltration of immune cells such as macrophages into fat tissues.6 Immune cells infiltrate adipose tissue and secrete cytokines leading to an increased inflammatory state and adiposity.6,9,10 In this study, we observed that the vaccine candidate protected mice from infiltration of immune cells into epididymal fats. Fourteen weeks after the challenge injection, epididymal fats were collected from mice and stained with hematoxylin and eosin. Infiltrated immune cells were reduced in the vaccinated group compared with the control group (Figure 2a). Moreover, the sliced fats were also stained with F4/80 antibody to confirm infiltrated macrophages. Infiltration of macrophages was also decreased in the vaccinated group compared with the control group (Figure 2a). Previously, it was reported that Ad36-induced obesity increases inflammation through pro-inflammatory cytokines such as MCP 1 and TNF α.6 Here, serum levels of these two cytokines were measured by an immunoassay 14 weeks after challenge. MCP 1 serum levels were decreased by four times in the vaccinated group compared with the control group (Figure 2b; P = 0.0002). TNF α level in the vaccinated group were also reduced compared with the control group (Figure 2b; P = 0.022). Moreover, levels of MCP 1 mRNA in epididymal fats of the vaccinated group were reduced compared with the control group (Figure 2c; P = 0.0003). However, expression of TNF α mRNA in epididymal fats was not significantly different between the vaccinated and control groups (Figure 2c; P = 0.446). Macrophages can be divided into two subtypes based on immune response: M1 macrophages (pro-inflammatory © 2014 Macmillan Publishers Limited
macrophages, CD64) and M2 macrophages (anti-inflammatory macrophages, CD206).6 In the present study, the vaccinated group had decreased M1 macrophages (P = 0.014) unlike M2 macrophages (Figure 2d). According to these results, the vaccine candidate reduced production of serum pro-inflammatory cytokines as well as infiltration of immune cells after live Ad36 challenge. DISCUSSION Ad36 is associated with human obesity in the USA, Korea and Italy.3,11,12 In addition to humans, virus infection induces adiposity in animals including chickens, mice, rats and monkeys.4,5 Moreover, Ad36 infection induces inflammation in adipose tissue and maintains an inflammatory state through pro-inflammatory cytokines.6 Eventually, increased inflammation is able to sustain obesity. Therefore, it is necessary to develop therapeutic drugs or prophylactic vaccines that can regulate infection of the virus. In the present study, we inactivated Ad36 by UV irradiation to create an Ad36 vaccine candidate. UV irradiation can inactivate viruses without harmful effects on the antigenic structure and biological function of proteins.13 In a previous study, UV-irradiated Ad36 was also evaluated.8 However, changes in body weight and fat were not observed 4 days after infection.8 Actually, 4 days is not enough time to identify changes in body weight or fat after Ad36 infection. Therefore, we monitored body weight for 14 weeks after live-Ad36 challenge. The vaccine candidate provided protection against increase in body weight and fat after Ad36 challenge (Figure 1). Moreover, the vaccine candidate diminished the induction of pro-inflammatory cytokines as well as M1 macrophages in adipose tissue by Ad36 infection (Figure 2); thus, indicating that the vaccine ameliorates Ad36-induced inflammation. Previous papers showed that infecting with Ad36 improves the liver condition and glycemic control in mice and human.14,15 Thus, we also analyzed the liver condition and lipid profiles after vaccination by hematoxylin and eosin staining and by measuring the glucose and nonesterified fatty acid levels in serum. However, in the liver, we did not observe any differences between vaccinated group and control group (in Supplementary Figure 1), because we used normal diet and this normal diet may mask the Ad36-benefit effects, which indicate the improving glycemic control and hepatic-lipid content. Actually, these Ad36-benefit effects seem to make unnecessary the developing vaccine for protecting Ad36 infection. However, other paper also showed that infecting with Ad36 may associate with the reduction of bone strength in late-adolescent females.16 Moreover, Ad36 can trigger the inflammation in murine and human,6,14 even in this inflammation seems to be required to improve glycemic control. Interestingly, E4orf1, one of the Ad36 viral proteins, can stimulate the glucose uptake through independent insulin-signaling pathway.17 By the way, we observed that E4orf1 expression induced different signaling pathways compared with Ad36 (unpublished data). Thus, E4orf1 may have the development potential as an antidiabetic drug. However, Ad36 itself may be not used as a therapeutic agent as compared with oncolytic viruses, which is involved in the selective lysis of cancer cells, because the mechanism and etiological function of Ad36 is yet to be completely understood. Therefore, we think we still need the development of Ad36 vaccine, apart from the development of antidiabetic drug by E4orf1. Three types of virus vaccines have been used clinically; inactivated virus (purified whole viruses inactivated by chemical or UV irradiation), live-attenuated vaccine (live virus, but attenuated), and subunit vaccine (viral structural proteins, sometimes constructed as a virus-like particle without the viral genome).18 Although live-attenuated vaccines can trigger highimmune responses, safe use of such vaccines is a concern.19 International Journal of Obesity (2014) 1470 – 1474
Virus-induced obesity vaccine H-N Na and J-H Nam
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Figure 1. The vaccine candidate for Ad36 decreased body weight and fat (n = 10 per group). (a) Mice were injected with the Ad36 vaccine candidate (vaccination group) or phosphate-buffered saline (control group). One week after the second immunization, mice were injected with live Ad36 (1 × 107 pfu per mouse) for the challenge test. The body weights of mice were monitored for 14 weeks after challenge (**P o0.01, ***Po0.001; t-test). (b) Fourteen weeks after the challenge, weight gain was calculated as the difference in body weight at the time of challenge and the time of sacrifice (*Po 0.05; t-test). (c) Fourteen weeks after challenge, mice were killed and epididymal fats were extracted. The weights of the epididymal fats were measured (*Po0.05; t-test). The amount of epididymal fats in the control and vaccination groups was quantified (The size of scale bar, 1 cm). (d) The amount of Ad36-specific antibody is indicated by absorbance. Serum was obtained from mice 2 weeks after the secondary immunization. The wells of plates were coated with purified Ad36 (10, 100 or 1000 ng per well), serum was added, and the absorbance was measured at 450 nm using a spectrophotometer to confirm the amount of neutralizing antibody (Control, live Ad36 injection; Vaccination, Ad36 vaccine candidate injection; Mock, PBS injection; *P o0.05, ANOVA).
Recently, subunit vaccines, such as the human papilloma virus vaccine, have been developed. However, the manufacturing of this vaccine can be technically difficult and can be time consuming. Therefore, we chose to develop a prophylactic Ad36 vaccine of the inactivated type by UV irradiation. UV irradiation was used for inactivation of the virus because it does not modify the conformation of the virus-capsid proteins20 and induces International Journal of Obesity (2014) 1470 – 1474
proper neutralizing antibody against Ad36. Thus, virus inactivation by UV irradiation is suitable for early-stage development of a Ad36 vaccine. However, to develop a Ad36 vaccine to be used in a clinic, other methods must also be considered because of issues such as the licensing-approval system, economical efficiency, and so on. Moreover, further study is needed for the cross-protection against other adenoviruses induced by Ad36-specific-neutralizing antibodies. © 2014 Macmillan Publishers Limited
Virus-induced obesity vaccine H-N Na and J-H Nam
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Figure 2. The vaccine candidate for Ad36 reduced inflammation in mice (n = 10 per group). (a) Epididymal fats were extracted from mice 14 weeks after challenge. The fats were fixed with 1% paraformaldehyde, and then stained with hematoxylin and eosin. In addition, fats were added with F4/80 antibody to confirm infiltration of macrophages. Adipocytes were observed with AxioVision version 4.8 (Carl Zeiss, Oberkochen, Germany). (b) Fourteen weeks after challenge, sera were collected from mice. The protein concentration of MCP 1 and TNF α was measured by an immunoassay (*Po0.05, **Po0.01; t-test). (c) RNA was isolated from the extracted epididymal fats with TRIzol reagent and synthesized into complementary DNA using reverse transcriptase. mRNA expression of MCP 1 and TNF α was determined by quantitative real-time PCR (*Po0.05; t-test). (d) mRNA expression of M1 (CD64) and M2 (CD206) macrophage markers was confirmed in epididymal fats by quantitative real-time PCR (*Po0.05; t-test).
Further experiments should include an Ad2-injected group as a negative control because, unlike Ad36, Ad2 does not increase adiposity.14
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To our knowledge, the results of this study are the first proof-of-concept for the development of a vaccine against Ad36.
International Journal of Obesity (2014) 1470 – 1474
Virus-induced obesity vaccine H-N Na and J-H Nam
1474 CONFLICT OF INTEREST The authors declare no conflict of interest.
ACKNOWLEDGEMENTS This work was supported by a grant from the GRRC of the Catholic University of Korea and by the Basic Science Research Program of the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2012R1A1A2039819).
REFERENCES 1 Reaven GM. All obese individuals are not created equal: insulin resistance is the major determinant of cardiovascular disease in overweight/obese individuals. Diab Vasc Dis Res 2005; 2: 105–112. 2 Hainer V. Comparative efficiency and safety of pharmacological approaches to the management of obesity. Diabetes Care 2011; 34: S349–S354. 3 Atkinson RL, Dhurandhar NV, Allison DB, Bowen RL, Israel BA, Augustus AS et al. Human adenovirus-36 is associated with increased body weight and paradoxical reduction of serum lipids. Int J Obes (Lond) 2005; 29: 281–286. 4 Dhurandhar NV, Israel BA, Kolesar JM, Mayhew GF, Cook ME, Atkinson RL. Increased adiposity in animals due to a human virus. Int J Obes Relat Metab Disord 2000; 24: 989–996. 5 Dhurandhar NV, Whigham LD, Abbott DH, Schultz-Darken NJ, Israel BA, Atkinson RL et al. Human adenovirus Ad-36 promotes weight gain in male rhesus and marmoset monkeys. J Nutr 2002; 132: 3155–3160. 6 Na HN, Nam JH. Adenovirus 36 as an obesity agent maintains the obesity state by increasing MCP-1 and inducing inflammation. J Infect Dis 2012; 205: 914–922. 7 Tollefson AE, Kuppuswamy M, Shashkova EV, Doronin K, Wold WS. Preparation and titration of CsCl-banded adenovirus stocks. Methods Mol Med 2007; 130: 223–235.
8 Pasarica M, Loiler S, Dhurandhar NV. Acute effect of infection by adipogenic human adenovirus Ad36. Arch Virol 2008; 153: 2097–2102. 9 Lumeng CN, Saltiel AR. Inflammatory links between obesity and metabolic disease. J Clin Invest 2011; 121: 2111–2117. 10 Sun S, Ji Y, Kersten S, Qi L. Mechanisms of inflammatory responses in obese adipose tissue. Annu Rev Nutr 2012; 32: 261–286. 11 Na HN, Kim J, Lee HS, Shim KW, Kimm H, Nam JH et al. Association of human adenovirus-36 in overweight Korean adults. Int J Obes (Lond) 2012; 36: 281–285. 12 Trovato GM, Castro A, Tonzuso A, Garozzo A, Martines GF, Catalano D et al. Human obesity relationship with Ad36 adenovirus and insulin resistance. Int J Obes (Lond) 2009; 33: 1402–1409. 13 Raviv Y, Viard M, Bess JW, Chertova E, Blumenthal R. Inactivation of retroviruses with preservation of structural integrity by targeting the hydrophobic domain of the viral envelope. J Virol 2005; 79: 12394–12400. 14 Krishnapuram R, Dhurandhar EJ, Dubuisson O, Kirk-Ballard H, Bajpeyi S, Butte N et al. Template to improve glycemic control without reducing adiposity or dietary fat. Am J Physiol Endocrinol Metab 2011; 300: E779–E789. 15 Trovato GM, Martines GF, Trovato FM, Pirri C, Pace P, Garozzo A et al. Adenovirus36 seropositivity enhances effects of nutritional intervention on obesity, bright liver, and insulin resistance. Dig Dis Sci 2012; 57: 535–544. 16 Laing EM, Tripp RA, Pollock NK, Baile CA, Della-Fera MA, Rayalam S et al. Adenovirus 36, adiposity, and bone strength in late-adolescent females. J Bone Miner Res 2013; 28: 489–496. 17 Dhurandhar NV. Insulin sparing action of adenoirus 36 and its E4orf1 protein. J Diabetes Complications 2013; 27: 191–199. 18 Choi Y, Chang J. Viral vectors for vaccine applications. Clin Exp Vaccine Res 2013; 2: 97–105. 19 Burchill MA, Tamburini BA, Pennock ND, White JT, Kurche JS, Kedl RM. T cell vaccinology: exploring the known unknowns. Vaccine 2013; 31: 297–305. 20 Lee SG, Jin JY, Kwon BM, Park SW, Paik SY. Inactivation of 2009 pandemic human influenza A virus H1N1 by photocatalyst under UV irradiation. J Bac Virol 2012; 42: 83–85.
Supplementary Information accompanies this paper on International Journal of Obesity website (http://www.nature.com/ijo)
International Journal of Obesity (2014) 1470 – 1474
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