Original Paper Received: September 5, 2014 Accepted after revision: November 24, 2014 Published online: March 11, 2015
Neuroimmunomodulation DOI: 10.1159/000371354
Suppressive Effects of Chronic Stress on Influenza Virus Protection after Vaccination with Plasmid DNA-Encoded Nucleoprotein Fatemeh Sadat Nezam a, b Seyed Masoud Hosseini a Masoumeh Tavassoti Kheiri b Asghar Abdoli c Arash Memarnejadian c Mohammad Shenagari d Shima Gholami b Hesam Sohani b Hamidreza Rahmatollahi b Abbas Jamali b
Department of Microbiology, Faculty of Biological Sciences, Shahid-Beheshti University, and b Influenza Research Laboratory and c Hepatitis and AIDS Department, Pasteur Institute of Iran, Tehran, and d Department of Microbiology, Faculty of Medicine, Guilan University of Medical Science, Rasht, Iran
Key Words Influenza · DNA vaccine · Chronic stress
Abstract Background: Influenza is a highly infectious and acute respiratory disease caused by an infection of the host respiratory tract mucosa by the influenza virus. The use of DNA vaccines that express conserved genes such as nucleoprotein (NP) represents a new method of vaccination against influenza. In this study, the effect of chronic stress on the efficiency of this type of vaccine has been evaluated in a mouse model. Methods: The NP DNA vaccine was administered intradermally 3 times on days 0, 3 and 6 to stressed and nonstressed male BALB/c mice. Two weeks after the last immunization, half of these mice were challenged with A/Puerto Rico/8/34 (PR8) influenza virus and were weighed for 12 days, and their mortality rate was assessed during this period. The cellular immune response of the other half of the mice was evaluated by cytotoxicity assay. Results: The results indicate a significant reduction in the cytotoxic T-lymphocyte response of stressed mice in comparison with unstressed mice. Also, the percentage of weight loss and mortality after the challenge in stressed mice was significantly increased compared to the
© 2015 S. Karger AG, Basel 1021–7401/15/0000–0000$39.50/0 E-Mail [email protected] www.karger.com/nim
other group. Conclusion: These results indicate that the NP DNA vaccine is not able to induce any effective cytotoxic T-lymphocyte response against influenza virus in stressed mice and cannot induce protective immunity against influenza infection in this group of mice. © 2015 S. Karger AG, Basel
Introduction
Influenza is an acute and infectious respiratory disease that results in significant extra yearly morbidity and mortality rates. Current influenza vaccines contain hemagglutinin and nuraminidase antigens. Although these antigens are prone to antigenic drift and shift, the annual evaluation of vaccine strains and annual vaccination are required [1–3]. One strategy to overcome this disadvantage includes utilizing plasmid DNA (pDNA) encoding conserved influenza virus antigens [4]. Vaccination with conserved influenza vaccines can provide protection against different influenza A strains and subtypes. The highly conserved nucleoprotein (NP) of influenza virus is a candidate for a DNA vaccine that can induce immune responses against heterosubtype viruses [5–11]. Abbas Jamali, PhD Influenza Research Laboratory Pasteur Institute of Iran Tehran (Iran) E-Mail jamali @ pasteur.ac.ir
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a
Material and Methods Plasmid A pcDNA3-NP plasmid, which was constructed in the previous study [23], was amplified in Escherichia coli Top10F and purified using an endotoxin-free plasmid purification kit (Qiagen). Animals Forty male BALB/c mice aged 6–8 weeks were obtained from Iran Pasteur Institute, Karaj Branch. Mice were given free access to food and water and kept on a 12-hour light-dark cycle, lights on at 6.00 a.m. Five animals were kept in each cage and were adapted to the above conditions for at least 1 week before experiments. They were divided into 5 experimental groups: (1) stressed, vaccinated with pcDNA3-NP plasmid group (RST-pNP); (2) nonstressed, vaccinated with pcDNA3-NP plasmid group (non-RSTpNP); (3) group with no-stress treatment that was injected with PBS (PBS); (4) group with no-stress treatment that was injected with influenza H1N1 A/Puerto Rico/8/34 virus (PR8); (5) stressed mice injected with PBS (RST-PBS). Stress Model Mice were restraint stressed for 6 h in 50 ml conical tubes with holes to keep air moving inside, beginning at 9.00 a.m. [24, 25]. After completion of the stress cycle, mice had free access to food and water. Stress was started 2 weeks before immunization. Immunization Two weeks after induction of stress, stressed and nonstressed vaccinated mice were injected intradermally with 50 μg NP DNA
2
Neuroimmunomodulation DOI: 10.1159/000371354
vaccine. PBS mice were injected intradermally with 50 μl sterile PBS and the fourth group was injected intraperitoneally with 100 μl H1N1 (PR8) influenza virus with 104/100 μl 50% tissue culture infection dose titer. All injections were repeated 3 times at intervals of 3 days. Virus Challenge Two weeks after the last immunization, ketamine/xylazineanesthetized mice were challenged intranasally with two 50% minimum lethal doses (MLD50) of mouse-adapted H1N1 (PR8) influenza virus. After viral challenge, mice were monitored for 10 days for weight changes and mortality [26]. Cytotoxic T-Lymphocyte Assay Splenocytes were isolated from naïve mice, and their red blood cells were removed with red blood cell lysis buffer. Then these cells were divided into two parts. Half were labeled with 5 μM 5-(and 6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) (high CFSE) and the other half with 0.5 μM CFSE (low CFSE).Then, they were incubated for 15 min at 37 ° C. After labeling, high CFSE cells were stimulated with 10 μg/ml stimulating peptide (NP). High and low CFSE cells were mixed (1:1) with a final concentration of 5–6 × 106 cells/100 μl. Then 100 μl of these cells were injected intravenously to control and immunized mice. Twenty hours later, splenocytes from control and immunized mice were isolated, and CFSE intensities were assessed by flow cytometry. Lysis of target cells (high CFSE) was calculated as:
¬ high CFSE Lysis % ¡¡1 immunized mice ® low CFSE ¡¢ low CFSE ¬¯ q naïve mice°° q100. ®° high CFSE ±
Statistical Analysis Cytotoxicity response (cytotoxic T-lymphocyte assay), and weight change were analyzed by 1-way analysis of variance followed by Tukey’s test. Survival rates were analyzed by KaplanMeier and log rank tests. Differences were considered statistically significant when p < 0.05.
Results
Effect of Chronic Stress on Cytotoxicity Responses Induced by NP DNA Vaccine The cytotoxicity response in the 4 groups was examined using the CFSE cytotoxicity assay 14 days after the last immunization. As shown in figure 1, the cytotoxicity response was significantly lower in the RST-pNP mouse group compared to the non-RST-pNP mouse group (p = 0.002). Although a higher cytotoxicity response was observed in the RST-pNP group than the PBS group, this difference was not significant (p = 0.479).
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Stress is a state of change in homeostasis resulting from either an external or internal stimulus [12]. It has been shown that psychological stress can reduce immune responses and increase intensity and sensitivity of infectious diseases [13–16]. In different studies, the effects of stress on influenza virus infections and the immune responses after vaccination with current influenza vaccines have been investigated. These studies show that chronic stress has a negative effect on immune responses against influenza infection and vaccination [12, 17– 22]. Since vaccination against different diseases including influenza has become a routine procedure, the investigation of the efficiency of the proper induction of immune responses of these vaccines in different people with different physiological conditions is highly recommended. In our previous study [23] the efficiency of this type of vaccine in diabetic mice was investigated. In this study, the influenza DNA vaccine encoding NP as a candidate for vaccination against influenza virus infection was utilized to immunize BALB/c mice with chronic stress, and its efficiency in induction of cellular immunity was evaluated.
120
PBS
*
PR8
Non-RST-pNP vs. RST-pNP p < 0.01) 100
80
60
Weight change (%)
Lysis (%)
Non-RST-pNP
110
100
40
20
0
RST-pNP
90
80
70 PBS
RST-pNP
Non-RST-pNP
PR8 60
stressed mice. Fourteen days after induction of stress, stressed and nonstressed mice were immunized intradermally with NP DNA vaccine (RST-pNP and non-RST-pNP groups, respectively), PBS mice were injected intradermally with sterile PBS and those of the fourth group were immunized intraperitoneally with PR8/H1N1 influenza virus. All injections were repeated 3 times on days 0, 3 and 6. Fourteen days after the last immunization, splenocytes of 3 naïve mice were stained with CFSE and stimulated with the NP peptide for 20 h. After that, splenocytes from control and immunized mouse groups (each containing 5 mice) were isolated, and CFSE intensities were assessed by flow cytometry. Lysis of target cells (high CFSE) was calculated, and the average percent of lysis was calculated for each group. The cytotoxicity activity of the nonRST-pNP mice was significantly higher than that of the RST-pNP mice (* p = 0.002). There was no significant difference between the cytotoxicity responses of the RST-pNP and PBS groups. Error bars reflect the standard error of the data.
1
2
3
4
5 6 7 8 9 10 11 12 13 14 Days after challenge
Fig. 2. Percentage of weight loss after influenza challenge. Two
weeks after the induction, stress RST-pNP and non-RST-pNP mice were immunized with NP DNA vaccine. PBS and PR8 mice were injected with PBS and PR8 influenza virus, respectively. Fourteen days after the last immunization, 5 mice of each group were challenged with 2 MLD50 doses of mouse-adapted H1N1 (PR8) influenza virus, and weight changes were measured for 12 days after the challenge. RST-pNP mice had significantly more weight loss after the challenge compared to non-RST-pNP mice (p = 0.003). Error bars reflect the standard error of the data.
Effect of Chronic Stress on the Efficiency of NP DNA Vaccine against Weight Loss after Influenza Virus Challenge Fourteen days after the last immunization, the efficiency of NP DNA vaccine to protect mice against weight loss after influenza challenge was evaluated by weighing the mice daily for 12 days. As shown in figure 2, non-RSTpNP mice had less weight loss after challenge than RSTpNP mice, and this difference was significant from the eighth day (p = 0.003). PBS mice had more weight loss than RST-pNP mice but this difference was not significant. The smallest weight loss was observed in the PR8 group (5.1%) and this recovered after 7 days.
Effect of Chronic Stress on the Efficiency of NP DNA Vaccine to Protect Mice against Lethal Challenge The NP DNA vaccine was tested for the ability to protect stressed mice from death after a lethal challenge (fig. 3). To this effect, 14 days after the last immunization, mice were challenged with influenza virus, and survival was assessed for 12 days. The non-RST-pNP and PR8 groups had no death in this time period. All mice that received RST-PBS succumbed within 10 days after challenge. RST-pNP mice survived significantly less compared to non-RST-pNP mice (p < 0.05). RST-pNP mice showed partial protection against challenge but there was no significant survival rate difference compared with the RST-PBS group.
Chronic Stress and Plasmid DNA Vaccination
Neuroimmunomodulation DOI: 10.1159/000371354
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Fig. 1. Cytotoxicity responses induced by NP DNA vaccine in
Non-RST-pNP RST-PBS
RST-pNP
120
*
100
Survival (%)
80
60
40
20
0
1
2
3
4
5 6 7 8 9 Days after challenge
10
11
12
Fig. 3. Survival of mice after lethal challenge. Two weeks after the induction of stress, RST-pNP and non-RST-pNP mice were immunized with NP DNA vaccine, PBS mice were injected with PBS and PR8 mice were immunized with PR8 influenza virus. All injections were done 3 times with intervals of 3 days. Fourteen days after the last immunization, 5 mice of each group were challenged with 2 MLD50 doses of mouse-adapted H1N1 (PR8) influenza virus, and their survival was evaluated for 12 days after the challenge. Non-RST-pNP and PR8 mice had 100% survival. RST-pNP mice and PBS mice had 40 and 20% survival rates, respectively. The RST-PBS group did not show any survival after challenge. * p < 0.05, RST-pNP versus non-RST-pNP.
Discussion
Stressors can modulate the immune function, and the extent of these effects depends on their type, duration and intensity [22]. In this study we have shown that influenza DNA vaccine that encoded NP gene cannot induce an adequate protection in mice with chronic stress. Since the first demonstration of a protective efficacy against influenza disease in 1993, DNA vaccines have attracted a lot of interest in many studies [11]. These vaccines do not have the disadvantages of killed and inactivated vaccines [27], and due to their ability to induce both 4
Neuroimmunomodulation DOI: 10.1159/000371354
Nezam et al.
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PR8 PBS
humoral and cellular immunity [28] have been investigated for many different diseases, such as HIV, HSV-1, hepatitis B or rabies [29–33]. Due to antigenic changes in the influenza virus, a DNA vaccine containing a conserved viral gene might induce active responses against a broad range of influenza viruses. In different studies [6, 11, 27, 34] it has been shown that influenza NP DNA vaccine can induce both antibody and cytotoxic T-lymphocyte responses and has a protective efficacy in mice against a heterosubtype virus challenge. Data presented in this article are consistent with the results of previous studies and showed that the influenza NP DNA vaccine induces protective immunity against viral challenge in an NP DNA-vaccinated group. Skin and mucous membranes are rich in keratinocytes, and the abundance of Langerhans cells in the skin is higher than in other areas. Both of these cells can facilitate the initiation of immune responses, even at very low antigen concentrations. Results of a study [35], in which the effects of different injection sites of NP DNA vaccine on the immune response were evaluated, suggest that the density and state of activation of antigen-presenting macrophages and dendritic cells at the injection site strongly affect the subsequent immune responses. Therefore, in this study we have used intradermal immunization. The results suggest that this type of immunization can induce a protective immune response against influenza challenge in the control group. In other studies [3, 6, 7] intramuscular injection was used, and there is no difference between these results and ours. Many studies [14, 24, 36–40] have shown that stress can alter the immune function. The central nervous system, the endocrine system and the immune system are complex systems that interact with each other, and stressors can change immune responses by affecting the interaction of these systems [39]. Former studies [41, 42] show that acute stress increases the immune responses, in contrast to the chronic stress which decreases them. Previous studies [12, 17, 38, 43, 44] explained that chronic stress reduces both humoral and cellular immune responses against influenza infections. Additionally, it has been shown that this type of stress decreases the efficiency of current influenza vaccines in the induction of protective immune responses in vaccinated groups [17, 19, 20, 38]. Most of these experiments have studied the humoral responses, and cellular immune responses have not been investigated as much. The results of our investigation indicate that an NP DNA vaccine cannot induce an effective cytotoxic T-lymphocyte response in mice with chronic stress in contrast to unstressed mice and controls.
In former studies in which the effect of chronic stress on the efficiency of current influenza vaccine has been tested, the survival rate and percentage of weight loss of vaccinated groups were not investigated after an influenza virus challenge. In this study we have shown that chronically stressed mice survive less and their weights decrease much more than those of unstressed mice and controls. In conclusion, the present data suggests that the NP DNA vaccine with the protocol of immunization described here cannot induce protective responses in chronically stressed mice. This information is new in the field of DNA immunization for infectious diseases in a person with chronic stress. Inclusion of other conserved influ-
enza virus genes, especially M1 and M2, and also Th1 cytokine genes in DNA constructs, employment of other DNA immunization protocols and using an adjuvant may develop vaccine potency and will be investigated in future studies. In addition further studies are needed to test the effects of chronic stress on the DNA vaccination to induce humoral immune responses.
Acknowledgment We would like to thank Mrs. Pyvand Biglary and Mr. Ali Torabi of Pasteur Institute of Iran, for their technical assistance.
References
Chronic Stress and Plasmid DNA Vaccination
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38 Vedhara K, McDermott MP, Evans TG, Treanor JJ, Plummer S, Tallon D, et al: Chronic stress in nonelderly caregivers: psychological, endocrine and immune implications. J Psychosom Res 2002;53:1153–1161. 39 Glaser R, Kiecolt-Glaser JK: Stress-induced immune dysfunction: implications for health. Nat Rev Immunol 2005;5:243–251. 40 Padgett DA, Glaser R: How stress influences the immune response. Trends Immunol 2003; 24:444–448. 41 Dragos D, Tanasescu MD: The effect of stress on the defense systems. J Med Life 2010;3:10– 18. 42 Herbert TB, Cohen S: Stress and immunity in humans: a meta-analytic review. Psychosom Med 1993;55:364–379. 43 Feng N, Pagniano R, Tovar CA, Bonneau RH, Glaser R, Sheridan JF: The effect of restraint stress on the kinetics, magnitude, and isotype of the humoral immune response to influenza virus infection. Brain Behav Immun 1991; 5: 370–382. 44 Sheridan JF, Feng N, Bonneau RH, Allen CM, Huneycutt BS, Glaser R: Restraint stress differentially affects anti-viral cellular and humoral immune responses in mice. J Neuroimmunol 1991;31:245–255.
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27 Bender B, Ulmer J, DeWitt C, Cottey R, Taylor S, Ward A, et al: Immunogenicity and efficacy of DNA vaccines encoding influenza A proteins in aged mice. Vaccine 1998;16:1748– 1755. 28 Giese M: DNA-antiviral vaccines: new developments and approaches – a review. Virus Genes 1998;17:219–232. 29 Boyer JD, Ugen KE, Wang B, Agadjanyan M, Gilbert L, Bagarazzi ML, et al: Protection of chimpanzees from high-dose heterologous HIV-1 challenge by DNA vaccination. Nat Med 1997;3:526–532. 30 Xiang ZQ, Spitalnik S, Tran M, Wunner WH, Cheng J, Ertl HCJ: Vaccination with a plasmid vector carrying the rabies virus glycoprotein gene induces protective immunity against rabies virus. Virology 1994;199:132–140. 31 Davis HL, Michel M-L, Whalen RG: DNAbased immunization induces continuous secretion of hepatitis B surface antigen and high levels of circulating antibody. Hum Mol Genet 1993;2:1847–1851. 32 Ghiasi H, Cai S, Slanina S, Nesburn AB, Wechsler SL: Vaccination of mice with herpes simplex virus type 1 glycoprotein D DNA produces low levels of protection against lethal HSV-1 challenge. Antiviral Res 1995; 28: 147–157.
Neuroimmunomodulation DOI: 10.1159/000371354
Suppressive Effects of Chronic Stress on Influenza Virus Protection after Vaccination with Plasmid DNA-Encoded Nucleoprotein Fatemeh Sadat Nezam a, b Seyed Masoud Hosseini a Masoumeh Tavassoti Kheiri b Asghar Abdoli c Arash Memarnejadian c Mohammad Shenagari d Shima Gholami b Hesam Sohani b Hamidreza Rahmatollahi b Abbas Jamali b
Department of Microbiology, Faculty of Biological Sciences, Shahid-Beheshti University, and b Influenza Research Laboratory and c Hepatitis and AIDS Department, Pasteur Institute of Iran, Tehran, and d Department of Microbiology, Faculty of Medicine, Guilan University of Medical Science, Rasht, Iran
Key Words Influenza · DNA vaccine · Chronic stress
Abstract Background: Influenza is a highly infectious and acute respiratory disease caused by an infection of the host respiratory tract mucosa by the influenza virus. The use of DNA vaccines that express conserved genes such as nucleoprotein (NP) represents a new method of vaccination against influenza. In this study, the effect of chronic stress on the efficiency of this type of vaccine has been evaluated in a mouse model. Methods: The NP DNA vaccine was administered intradermally 3 times on days 0, 3 and 6 to stressed and nonstressed male BALB/c mice. Two weeks after the last immunization, half of these mice were challenged with A/Puerto Rico/8/34 (PR8) influenza virus and were weighed for 12 days, and their mortality rate was assessed during this period. The cellular immune response of the other half of the mice was evaluated by cytotoxicity assay. Results: The results indicate a significant reduction in the cytotoxic T-lymphocyte response of stressed mice in comparison with unstressed mice. Also, the percentage of weight loss and mortality after the challenge in stressed mice was significantly increased compared to the
© 2015 S. Karger AG, Basel 1021–7401/15/0000–0000$39.50/0 E-Mail [email protected] www.karger.com/nim
other group. Conclusion: These results indicate that the NP DNA vaccine is not able to induce any effective cytotoxic T-lymphocyte response against influenza virus in stressed mice and cannot induce protective immunity against influenza infection in this group of mice. © 2015 S. Karger AG, Basel
Introduction
Influenza is an acute and infectious respiratory disease that results in significant extra yearly morbidity and mortality rates. Current influenza vaccines contain hemagglutinin and nuraminidase antigens. Although these antigens are prone to antigenic drift and shift, the annual evaluation of vaccine strains and annual vaccination are required [1–3]. One strategy to overcome this disadvantage includes utilizing plasmid DNA (pDNA) encoding conserved influenza virus antigens [4]. Vaccination with conserved influenza vaccines can provide protection against different influenza A strains and subtypes. The highly conserved nucleoprotein (NP) of influenza virus is a candidate for a DNA vaccine that can induce immune responses against heterosubtype viruses [5–11]. Abbas Jamali, PhD Influenza Research Laboratory Pasteur Institute of Iran Tehran (Iran) E-Mail jamali @ pasteur.ac.ir
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a
Material and Methods Plasmid A pcDNA3-NP plasmid, which was constructed in the previous study [23], was amplified in Escherichia coli Top10F and purified using an endotoxin-free plasmid purification kit (Qiagen). Animals Forty male BALB/c mice aged 6–8 weeks were obtained from Iran Pasteur Institute, Karaj Branch. Mice were given free access to food and water and kept on a 12-hour light-dark cycle, lights on at 6.00 a.m. Five animals were kept in each cage and were adapted to the above conditions for at least 1 week before experiments. They were divided into 5 experimental groups: (1) stressed, vaccinated with pcDNA3-NP plasmid group (RST-pNP); (2) nonstressed, vaccinated with pcDNA3-NP plasmid group (non-RSTpNP); (3) group with no-stress treatment that was injected with PBS (PBS); (4) group with no-stress treatment that was injected with influenza H1N1 A/Puerto Rico/8/34 virus (PR8); (5) stressed mice injected with PBS (RST-PBS). Stress Model Mice were restraint stressed for 6 h in 50 ml conical tubes with holes to keep air moving inside, beginning at 9.00 a.m. [24, 25]. After completion of the stress cycle, mice had free access to food and water. Stress was started 2 weeks before immunization. Immunization Two weeks after induction of stress, stressed and nonstressed vaccinated mice were injected intradermally with 50 μg NP DNA
2
Neuroimmunomodulation DOI: 10.1159/000371354
vaccine. PBS mice were injected intradermally with 50 μl sterile PBS and the fourth group was injected intraperitoneally with 100 μl H1N1 (PR8) influenza virus with 104/100 μl 50% tissue culture infection dose titer. All injections were repeated 3 times at intervals of 3 days. Virus Challenge Two weeks after the last immunization, ketamine/xylazineanesthetized mice were challenged intranasally with two 50% minimum lethal doses (MLD50) of mouse-adapted H1N1 (PR8) influenza virus. After viral challenge, mice were monitored for 10 days for weight changes and mortality [26]. Cytotoxic T-Lymphocyte Assay Splenocytes were isolated from naïve mice, and their red blood cells were removed with red blood cell lysis buffer. Then these cells were divided into two parts. Half were labeled with 5 μM 5-(and 6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) (high CFSE) and the other half with 0.5 μM CFSE (low CFSE).Then, they were incubated for 15 min at 37 ° C. After labeling, high CFSE cells were stimulated with 10 μg/ml stimulating peptide (NP). High and low CFSE cells were mixed (1:1) with a final concentration of 5–6 × 106 cells/100 μl. Then 100 μl of these cells were injected intravenously to control and immunized mice. Twenty hours later, splenocytes from control and immunized mice were isolated, and CFSE intensities were assessed by flow cytometry. Lysis of target cells (high CFSE) was calculated as:
¬ high CFSE Lysis % ¡¡1 immunized mice ® low CFSE ¡¢ low CFSE ¬¯ q naïve mice°° q100. ®° high CFSE ±
Statistical Analysis Cytotoxicity response (cytotoxic T-lymphocyte assay), and weight change were analyzed by 1-way analysis of variance followed by Tukey’s test. Survival rates were analyzed by KaplanMeier and log rank tests. Differences were considered statistically significant when p < 0.05.
Results
Effect of Chronic Stress on Cytotoxicity Responses Induced by NP DNA Vaccine The cytotoxicity response in the 4 groups was examined using the CFSE cytotoxicity assay 14 days after the last immunization. As shown in figure 1, the cytotoxicity response was significantly lower in the RST-pNP mouse group compared to the non-RST-pNP mouse group (p = 0.002). Although a higher cytotoxicity response was observed in the RST-pNP group than the PBS group, this difference was not significant (p = 0.479).
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Stress is a state of change in homeostasis resulting from either an external or internal stimulus [12]. It has been shown that psychological stress can reduce immune responses and increase intensity and sensitivity of infectious diseases [13–16]. In different studies, the effects of stress on influenza virus infections and the immune responses after vaccination with current influenza vaccines have been investigated. These studies show that chronic stress has a negative effect on immune responses against influenza infection and vaccination [12, 17– 22]. Since vaccination against different diseases including influenza has become a routine procedure, the investigation of the efficiency of the proper induction of immune responses of these vaccines in different people with different physiological conditions is highly recommended. In our previous study [23] the efficiency of this type of vaccine in diabetic mice was investigated. In this study, the influenza DNA vaccine encoding NP as a candidate for vaccination against influenza virus infection was utilized to immunize BALB/c mice with chronic stress, and its efficiency in induction of cellular immunity was evaluated.
120
PBS
*
PR8
Non-RST-pNP vs. RST-pNP p < 0.01) 100
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70 PBS
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stressed mice. Fourteen days after induction of stress, stressed and nonstressed mice were immunized intradermally with NP DNA vaccine (RST-pNP and non-RST-pNP groups, respectively), PBS mice were injected intradermally with sterile PBS and those of the fourth group were immunized intraperitoneally with PR8/H1N1 influenza virus. All injections were repeated 3 times on days 0, 3 and 6. Fourteen days after the last immunization, splenocytes of 3 naïve mice were stained with CFSE and stimulated with the NP peptide for 20 h. After that, splenocytes from control and immunized mouse groups (each containing 5 mice) were isolated, and CFSE intensities were assessed by flow cytometry. Lysis of target cells (high CFSE) was calculated, and the average percent of lysis was calculated for each group. The cytotoxicity activity of the nonRST-pNP mice was significantly higher than that of the RST-pNP mice (* p = 0.002). There was no significant difference between the cytotoxicity responses of the RST-pNP and PBS groups. Error bars reflect the standard error of the data.
1
2
3
4
5 6 7 8 9 10 11 12 13 14 Days after challenge
Fig. 2. Percentage of weight loss after influenza challenge. Two
weeks after the induction, stress RST-pNP and non-RST-pNP mice were immunized with NP DNA vaccine. PBS and PR8 mice were injected with PBS and PR8 influenza virus, respectively. Fourteen days after the last immunization, 5 mice of each group were challenged with 2 MLD50 doses of mouse-adapted H1N1 (PR8) influenza virus, and weight changes were measured for 12 days after the challenge. RST-pNP mice had significantly more weight loss after the challenge compared to non-RST-pNP mice (p = 0.003). Error bars reflect the standard error of the data.
Effect of Chronic Stress on the Efficiency of NP DNA Vaccine against Weight Loss after Influenza Virus Challenge Fourteen days after the last immunization, the efficiency of NP DNA vaccine to protect mice against weight loss after influenza challenge was evaluated by weighing the mice daily for 12 days. As shown in figure 2, non-RSTpNP mice had less weight loss after challenge than RSTpNP mice, and this difference was significant from the eighth day (p = 0.003). PBS mice had more weight loss than RST-pNP mice but this difference was not significant. The smallest weight loss was observed in the PR8 group (5.1%) and this recovered after 7 days.
Effect of Chronic Stress on the Efficiency of NP DNA Vaccine to Protect Mice against Lethal Challenge The NP DNA vaccine was tested for the ability to protect stressed mice from death after a lethal challenge (fig. 3). To this effect, 14 days after the last immunization, mice were challenged with influenza virus, and survival was assessed for 12 days. The non-RST-pNP and PR8 groups had no death in this time period. All mice that received RST-PBS succumbed within 10 days after challenge. RST-pNP mice survived significantly less compared to non-RST-pNP mice (p < 0.05). RST-pNP mice showed partial protection against challenge but there was no significant survival rate difference compared with the RST-PBS group.
Chronic Stress and Plasmid DNA Vaccination
Neuroimmunomodulation DOI: 10.1159/000371354
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Fig. 1. Cytotoxicity responses induced by NP DNA vaccine in
Non-RST-pNP RST-PBS
RST-pNP
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*
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0
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5 6 7 8 9 Days after challenge
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Fig. 3. Survival of mice after lethal challenge. Two weeks after the induction of stress, RST-pNP and non-RST-pNP mice were immunized with NP DNA vaccine, PBS mice were injected with PBS and PR8 mice were immunized with PR8 influenza virus. All injections were done 3 times with intervals of 3 days. Fourteen days after the last immunization, 5 mice of each group were challenged with 2 MLD50 doses of mouse-adapted H1N1 (PR8) influenza virus, and their survival was evaluated for 12 days after the challenge. Non-RST-pNP and PR8 mice had 100% survival. RST-pNP mice and PBS mice had 40 and 20% survival rates, respectively. The RST-PBS group did not show any survival after challenge. * p < 0.05, RST-pNP versus non-RST-pNP.
Discussion
Stressors can modulate the immune function, and the extent of these effects depends on their type, duration and intensity [22]. In this study we have shown that influenza DNA vaccine that encoded NP gene cannot induce an adequate protection in mice with chronic stress. Since the first demonstration of a protective efficacy against influenza disease in 1993, DNA vaccines have attracted a lot of interest in many studies [11]. These vaccines do not have the disadvantages of killed and inactivated vaccines [27], and due to their ability to induce both 4
Neuroimmunomodulation DOI: 10.1159/000371354
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PR8 PBS
humoral and cellular immunity [28] have been investigated for many different diseases, such as HIV, HSV-1, hepatitis B or rabies [29–33]. Due to antigenic changes in the influenza virus, a DNA vaccine containing a conserved viral gene might induce active responses against a broad range of influenza viruses. In different studies [6, 11, 27, 34] it has been shown that influenza NP DNA vaccine can induce both antibody and cytotoxic T-lymphocyte responses and has a protective efficacy in mice against a heterosubtype virus challenge. Data presented in this article are consistent with the results of previous studies and showed that the influenza NP DNA vaccine induces protective immunity against viral challenge in an NP DNA-vaccinated group. Skin and mucous membranes are rich in keratinocytes, and the abundance of Langerhans cells in the skin is higher than in other areas. Both of these cells can facilitate the initiation of immune responses, even at very low antigen concentrations. Results of a study [35], in which the effects of different injection sites of NP DNA vaccine on the immune response were evaluated, suggest that the density and state of activation of antigen-presenting macrophages and dendritic cells at the injection site strongly affect the subsequent immune responses. Therefore, in this study we have used intradermal immunization. The results suggest that this type of immunization can induce a protective immune response against influenza challenge in the control group. In other studies [3, 6, 7] intramuscular injection was used, and there is no difference between these results and ours. Many studies [14, 24, 36–40] have shown that stress can alter the immune function. The central nervous system, the endocrine system and the immune system are complex systems that interact with each other, and stressors can change immune responses by affecting the interaction of these systems [39]. Former studies [41, 42] show that acute stress increases the immune responses, in contrast to the chronic stress which decreases them. Previous studies [12, 17, 38, 43, 44] explained that chronic stress reduces both humoral and cellular immune responses against influenza infections. Additionally, it has been shown that this type of stress decreases the efficiency of current influenza vaccines in the induction of protective immune responses in vaccinated groups [17, 19, 20, 38]. Most of these experiments have studied the humoral responses, and cellular immune responses have not been investigated as much. The results of our investigation indicate that an NP DNA vaccine cannot induce an effective cytotoxic T-lymphocyte response in mice with chronic stress in contrast to unstressed mice and controls.
In former studies in which the effect of chronic stress on the efficiency of current influenza vaccine has been tested, the survival rate and percentage of weight loss of vaccinated groups were not investigated after an influenza virus challenge. In this study we have shown that chronically stressed mice survive less and their weights decrease much more than those of unstressed mice and controls. In conclusion, the present data suggests that the NP DNA vaccine with the protocol of immunization described here cannot induce protective responses in chronically stressed mice. This information is new in the field of DNA immunization for infectious diseases in a person with chronic stress. Inclusion of other conserved influ-
enza virus genes, especially M1 and M2, and also Th1 cytokine genes in DNA constructs, employment of other DNA immunization protocols and using an adjuvant may develop vaccine potency and will be investigated in future studies. In addition further studies are needed to test the effects of chronic stress on the DNA vaccination to induce humoral immune responses.
Acknowledgment We would like to thank Mrs. Pyvand Biglary and Mr. Ali Torabi of Pasteur Institute of Iran, for their technical assistance.
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