A DNA vaccine-encoded nucleoprotein of influenza virus fails to induce cellular immune responses in a diabetic mouse model.
ABSTRACT Influenza virus infections cause yearly epidemics and are a major cause of lower respiratory tract illnesses in humans worldwide. Influenza virus has long been recognized to be associated with higher morbidity and mortality in diabetic patients. Vaccination is an effective tool to prevent influenza virus infection in this group of patients. Vaccines employing recombinant-DNA technologies are an alternative to inactivated virus and live attenuated virus vaccines. Internal highly conserved viral nucleoprotein (NP) can be delivered as a DNA vaccine to provide heterosubtypic immunity, offering resistance against various influenza virus strains. In this study, we investigated the efficacy of an NP DNA vaccine for induction of cell-mediated immune responses and protection against influenza virus infection in a mouse model of diabetes. Healthy and diabetic BALB/c mice were immunized on days 0, 14, and 28 by injection of NP DNA vaccine. Two weeks after the last immunization, the cellular immune response was evaluated by gamma interferon (IFN-gamma), lymphocyte proliferation, and cytotoxicity assays. The mice were challenged with influenza virus, and the viral titers in the lungs were measured on day 4. Diabetic mice showed significantly smaller amounts of IFN-gamma production, lymphocyte proliferation, and cytotoxicity responses than nondiabetic mice. Furthermore, higher titers of the influenza virus were detected after challenge in the lungs of the diabetic mice. The present data suggest that the NP DNA vaccine with the protocol of immunization described here is not able to induce efficient cellular immune responses against influenza virus infection in diabetic mice.
Article: Infections in diabetes.[show abstract] [hide abstract]
ABSTRACT: Diabetics are predisposed to infections because of various immune deficiencies, including neutrophil and monocyte dysfunction. Some of these immune deficiencies are improved by tight glucose control. This article is a review of the immune deficiencies seen in diabetes and an overview of selected infections that are commonly or predominantly seen in diabetics.Infectious Disease Clinics of North America 07/2001; 15(2):407-21, viii. · 2.63 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Influenza continues to have a major worldwide impact, resulting in considerable human suffering and economic burden. The regular recurrence of influenza epidemics is thought to be caused by antigenic drift, and a number of studies have shown that sufficient changes can accumulate in the virus to allow influenza to reinfect the same host. To address this, influenza vaccine content is reviewed annually to ensure protection is maintained, despite the emergence of drift variants; however, it is not always possible to capture every significant drift, partly due to the timing of the recommendations. Vaccine mismatch can impact on vaccine effectiveness, and has significant epidemiological and economical consequences, as was seen most apparently in the 1997-1998 influenza season. To meet the challenge of antigenic drift, vaccines that confer broad protection against heterovariant strains are needed against seasonal, epidemic and pandemic influenza. In addition to the use of vaccine adjuvants, emerging research areas include development of a universal vaccine and the use of vaccines that exploit mechanisms of cross-protective immunity.Vaccine 10/2007; 25(39-40):6852-62. · 3.49 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The antibody response and delayed type hypersensitivity reaction to commercially available trivalent influenza vaccine in 159 patients with diabetes mellitus was compared with response and reaction in 28 healthy volunteers. A correction for prevaccination titres was made. No differences were found between diabetic patients and control subjects with respect to antibody response to the three vaccine strains as measured by the difference between geometric mean titres of post- and prevaccination sera. In Type 1 (insulin-dependent) diabetic patients the incidence of non-responders to two vaccine components was significantly increased (p less than 0.05). The delayed type hypersensitivity reaction to influenza antigen was significantly decreased in patients with high concentrations of glycosylated haemoglobin (p less than 0.01). These findings suggest a role for impaired immune response in the increased influenza morbidity and mortality in patients with diabetes mellitus. Implications for therapy and vaccination strategy are discussed.Diabetologia 07/1987; 30(6):397-401. · 6.49 Impact Factor
CLINICAL AND VACCINE IMMUNOLOGY, Apr. 2010, p. 683–687
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 17, No. 4
A DNA Vaccine-Encoded Nucleoprotein of Influenza Virus Fails To
Induce Cellular Immune Responses in a Diabetic Mouse Model?
Abbas Jamali,1Farzaneh Sabahi,1* Taravat Bamdad,1Hamidreza Hashemi,1
Fereidoun Mahboudi,2and Masume Tavasoti Kheiri3*
Department of Virology, School of Medical Sciences, Tarbiat Modares University, Tehran, Iran1; Biotechnology Research Center,
Pasteur Institute of Iran, Tehran, Iran2; and Influenza Unit, Pasteur Institute of Iran, Tehran, Iran3
Received 1 November 2009/Returned for modification 8 December 2009/Accepted 8 February 2010
Influenza virus infections cause yearly epidemics and are a major cause of lower respiratory tract
illnesses in humans worldwide. Influenza virus has long been recognized to be associated with higher
morbidity and mortality in diabetic patients. Vaccination is an effective tool to prevent influenza virus
infection in this group of patients. Vaccines employing recombinant-DNA technologies are an alternative
to inactivated virus and live attenuated virus vaccines. Internal highly conserved viral nucleoprotein (NP)
can be delivered as a DNA vaccine to provide heterosubtypic immunity, offering resistance against various
influenza virus strains. In this study, we investigated the efficacy of an NP DNA vaccine for induction of
cell-mediated immune responses and protection against influenza virus infection in a mouse model of
diabetes. Healthy and diabetic BALB/c mice were immunized on days 0, 14, and 28 by injection of NP DNA
vaccine. Two weeks after the last immunization, the cellular immune response was evaluated by gamma
interferon (IFN-?), lymphocyte proliferation, and cytotoxicity assays. The mice were challenged with
influenza virus, and the viral titers in the lungs were measured on day 4. Diabetic mice showed signifi-
cantly smaller amounts of IFN-? production, lymphocyte proliferation, and cytotoxicity responses than
nondiabetic mice. Furthermore, higher titers of the influenza virus were detected after challenge in the
lungs of the diabetic mice. The present data suggest that the NP DNA vaccine with the protocol of
immunization described here is not able to induce efficient cellular immune responses against influenza
virus infection in diabetic mice.
Influenza viruses cause highly contagious respiratory dis-
eases with potentially fatal outcomes (29). The current licensed
vaccine is only partially efficacious in healthy adults, and due to
the impact of antigenic drift on vaccine effectiveness, compo-
nents have to be reevaluated each year (12). Because of the
significant impact that antigenic drift has on vaccine effective-
ness, it is evident that new vaccines are needed to induce
cross-reactive immunity against epidemic or pandemic influ-
enza (2, 13). Vaccines employing recombinant-DNA technol-
ogies are an alternative to inactivated virus and live attenuated
virus vaccines (4, 10). Many influenza DNA vaccine experi-
ments have successfully demonstrated that DNA vaccines are
effective for the induction of immune responses against the
influenza virus (5, 7, 8, 27).
Influenza virus nucleoprotein (NP), a core antigen of influ-
enza virus, is a more conserved protein than its membrane
glycoproteins (7, 19). DNA vaccination with influenza virus NP
has been studied in animal models, and heterosubtypic immu-
nity offering resistance against different influenza virus strains
has been demonstrated (7, 22, 23, 26). On the other hand,
Influenza usually causes high morbidity and mortality in pa-
tients with diabetes and is associated with a loss of metabolic
control, development of ketoacidosis, and compromise of col-
lectin-mediated host defense of the lung by glucose (1, 3, 21).
Hence, vaccination against influenza infection is an important
public health intervention for reducing morbidity and mortality
among persons with diabetes (28). Although some experimen-
tal and clinical studies have investigated the capacity of inac-
tivated-influenza vaccines in diabetic patients or animal mod-
els (3, 31), the potency of DNA immunization in induction of
cellular immune responses of diabetic animal models has not
been studied. In the present study, the DNA construct encod-
ing influenza virus NP as a candidate for vaccination against
influenza virus infection was used to immunize diabetic
BALB/c mice, and its effectiveness in induction of cellular
immunity was evaluated.
MATERIALS AND METHODS
Construction of pcDNA3-NP. To construct the DNA vaccine, the NP gene of
influenza A/New Caledonia?20?99 H1N1 virus (Obtained from the National In-
stitute for Biological Standards and Control from United Kingdom) was ampli-
fied from total RNA of infected Madin-Darby canine kidney (MDCK) cells by
reverse transcription-PCR (RT-PCR) with specific primers. The NP gene was
then inserted into the pcDNA3 plasmid to generate pcDNA3-NP. Expression of
the NP protein was confirmed by transient transfection into BHK-21 cells and
subsequent immunofluorescence staining. BHK-21 cells were transfected with 1
?g of the pcDNA3-NP or pcDNA3 vector using Lipofectamine 2000 (Invitro-
gen). At 2 days posttransfection, the cells were fixed with 4% formaldehyde-
phosphate-buffered saline (PBS). Next, the cells were treated with Triton X-100
and then anti-NP monoclonal antibody (Ab) (Serotec), followed by incubation
with anti-mouse IgG-rhodamine conjugate (Invitrogen). pcDNA3-NP was am-
plified in Escherichia coli DH5? and purified using an endotoxin-free plasmid
purification kit (Qiagen).
* Corresponding author. Mailing address for Farzaneh Sabahi: De-
partment of Virology, School of Medical Sciences, Tarbiat Modares
University, P.O. Box 14115-111, Tehran, Iran. Phone: 982182883880.
Fax: 982182884555. E-mail: email@example.com. Mailing address
for Masume Tavasoti Kheiri: Influenza Unit, Pasteur Institute of Iran,
P.O. Box 1316943551, Tehran, Iran. Phone and fax: 982166953315, ext.
2131. E-mail: firstname.lastname@example.org.
?Published ahead of print on 17 February 2010.
Establishment of animal model of diabetes. Six- to eight-weeks-old male
BALB/c mice were obtained from the animal facilities of the Pasteur Institute
(Karaj, Iran). Mice were housed for 1 week before the experiment, given free
access to food and water, and maintained in a light/dark cycle with lights on from
6:00 to 18:00 h. All experiments were done according to the Animal Care and
Use Protocol of the Pasteur institute of Iran. An animal model of diabetes was
successfully established as described previously (30). Briefly, foods were forbid-
den to mice 12 h before the test. Streptozocin (STZ) was injected intraperito-
neally (i.p.) into mice at a dose of 150 mg/kg body weight. After injection with
STZ, the plasma samples were collected by tail bleeding for determination of
plasma glucose with a digital blood sugar apparatus. The nonfasting plasma
glucose concentrations in diabetic mice at 1 week after injection were more than
20 mmol/liter, while nonfasting glucose levels were kept at less than 10.2 mmol/
liter in control group of mice.
Immunization. On day 14 of STZ administration, BALB/c mice were injected
intradermally with 50 ?g of pcDNA3-NP or with PBS (the groups are called
pNP-Dia and PBS-Dia, respectively). Healthy control groups were injected with
the same protocol (pNP and PBS groups) as well. The vaccination was repeated
twice at intervals of 2 weeks in all groups.
Cytotoxicity assay. Two weeks after the last immunization, single-cell
splenocyte suspensions were prepared as effector cells without in vitro stim-
ulation (8). The P815 target cells were infected with influenza A/New Cale-
donia?20?99 H1N1 virus at a multiplicity of infection (MOI) of 5 overnight
and washed three times with assay medium. The cytotoxicity activity was
measured with the lactate dehydrogenase (LDH) release assay. Supernatants
(50 ?l/well) were transferred to 96-well flat-bottom plates, and lysis of target
cells was determined by the LDH assay kit (Takara, Japan) according to the
manufacturer’s instructions. Blank PBS buffer and a solution of 0.1% Triton
X-100 in PBS were used as controls. The LDH-mediated conversion of the
tetrazolium salt into red formazan product was measured at 490 nm after
incubation at room temperature for 30 min. The percentage of specific cy-
tolysis was determined by the following formula.
Specific cytolysis (%) ? (optical density [OD] of experimental LDH release ?
OD of spontaneous LDH release of effector cells ? OD of spontaneous LDH
release from target cells)/(maximum LDH release of target cells ? OD of
spontaneous LDH release of target cells) ? 100%. All experiments were per-
formed in triplicate.
Lymphocyte proliferation assay. The lymphocyte proliferation rate was mea-
sured by using a 3-(4,5-dimethylthiazol-2-thiazolyl)-2,5-diphenyltetrazolium bro-
mide, thiazolyl-blue (MTT) dye assay. Under sterile conditions, spleens were
removed and a single-cell suspension was prepared in phenol red-free RPMI
1640 (Gibco, United Kingdom). Red blood cells (RBCs) were lysed using 0.75%
NH4Cl in Tris buffer (0.02%; pH 7.2). The concentration was adjusted to 1 ? 106
cells/ml in phenol red-free RPMI 1640 supplemented with 10% fetal calf serum
(FCS), 2 mM L-glutamine, and 25 mM HEPES. One hundred microliters of
diluted cell suspensions were dispensed into 96-well flat-bottom culture plates.
The mitogen phytohemagglutinin A (PHA) at 5 ?g/ml (final concentration)
(positive control) or UV-inactivated influenza A/New Caledonia?20?99 H1N1
virus at an MOI of 5 was added to each well, and the volume was adjusted to 0.2
ml. After incubation for 72 h at 37°C in a 5%-CO2humid incubator, cell
proliferation was determined by MTT assay (16). Briefly, 20 ?l MTT was added
in each well, and plates were further incubated at 37°C for 4 h. Following
incubation, the supernatant from each well was aspirated carefully, and formazan
crystals were solubilized by adding 100 ?l dimethyl sulfoxide (DMSO) to each
well. The absorbance of each well was then determined at a wavelength of 540
nm. The stimulation index (SI) was calculated as the ratio of the average OD
value of wells containing antigen-stimulated cells to the average OD value of
wells containing only cells with medium.
IFN-? assay. Two weeks after the last immunization, spleens of individual
mice were removed aseptically and homogenized in RPMI 1640 medium
(Gibco-BRL, Germany) supplemented with 10% FCS and antibiotics. Red
blood cells were osmotically lysed using ammonium chloride buffer (NH4Cl [0.16
M], Tris [0.17 M]). Cells were washed twice with RPMI 1640 and counted, and
viability was determined by trypan blue (0.4% [wt/vol]) exclusion. A nominal
total of 1 ? 106spleen cells was plated on each well of 24-well plates using RPMI
1640 supplemented with 10% FCS, 100 IU/ml penicillin, 100 ?g/ml streptomycin,
and 5 ? 10?5M 2-mercaptoethanol. The experiment was performed in dupli-
cate. The cells were restimulated in vitro with UV-inactivated influenza A/New
Caledonia?20?99 H1N1 virus at an MOI of 5. Plates were incubated at 37°C in 5%
CO2, and 48 h after stimulation, supernatants were removed and kept at ?70°C
for evaluation of secreted gamma interferon (IFN-?) levels. The concentration of
IFN-? in the supernatants was estimated using a commercial enzyme-linked
immunosorbent assay (ELISA) kit (R&D systems).
Virus challenge. Two weeks after the last immunization, BALB/c mice were
challenged intranasally with 100 50% mouse infectious doses (MID50) of influ-
enza A/New Caledonia?20?99 H1N1 under anesthesia with diethyl ether. Since
influenza A/New Caledonia?20?99 H1N1 is not fatal in mice, the virus titers were
measured in mouse lungs at 4 days after challenge. Lungs were collected from
five mice of each group, and the viral titers were measured as the 50% tissue
culture infectious dose (TCID50) by inoculation of decimal dilutions (10?2to
10?7) of lung in MDCK cells.
Statistical analysis. Cytotoxicity response (LDH assay), MTT assay, IFN-?
production, and virus titers in the lungs were analyzed by one-way analysis of
variance (ANOVA) followed by Tukey’s test. A P value less than 0.05 was
Expression of NP gene in vitro. To confirm the expression of
NP, BHK-21 cells were transfected with the pcDNA3-NP plas-
mid, and expression of NP was confirmed by immunofluores-
cence (Fig. 1).
Effect of diabetes on cytotoxicity responses induced by pNP
immunization. The cytotoxicity response in immunized mice
was examined using the LDH release assay. As shown in Fig. 2,
the cytotoxicity response was significantly lower in the pNP-
Dia immunized mouse group than in pNP-immunized healthy
mice (P ? 0.001).
Effect of diabetes on lymphocyte proliferation responses in-
duced by pNP immunization. Splenocytes from all four
groups of mice were tested for their lymphocyte prolifera-
tive response. The results (Fig. 3) indicate a significant de-
crease in the proliferation index of the lymphocytes in the
pNP-Dia group compared to that of the pNP group (P ?
Effect of diabetes on IFN-? production induced by pNP
immunization. Spleen cells were collected 10 days after the last
immunization. The cultured cells were restimulated with UV-
inactivated influenza A/New Caledonia?20?99 H1N1 at an MOI
of 5, and supernatants were harvested after 48 h. As shown in
Fig. 4, IFN-? production was significantly lower in the pNP-
Dia immunized mouse group than in pNP-immunized healthy
mice (P ? 0.001).
FIG. 1. pcDNA3-NP expression in BHK-21 cells. NP expression in
BHK-21 cells was detected by immunofluorescence. BHK-21 cells were
transfected with 1 ?g of pcDNA3-NP or pcDNA3 vector using Lipo-
fectamine 2000. At 2 days posttransfection, the cells were fixed with
4% formaldehyde-PBS. Next, the cells were treated with Triton X-100
and then anti-NP monoclonal Ab, followed by incubation with anti-
mouse IgG-rhodamine conjugate. (A) BHK-21 cells were transfected
with pcDNA3-NP plasmid. (B) BHK-21 cells were transfected with
pcDNA3 plasmid as a control.
684 JAMALI ET AL.CLIN. VACCINE IMMUNOL.
Effect of immunization of diabetic mice on viral clearance
from the lungs. To assess the effect of pNP DNA vaccination
on the virus clearance rate in diabetic mice, they were chal-
lenged with 100 MID50of influenza A/New Caledonia?20?99
H1N1. As shown in Fig. 5, the influenza virus titer was
significantly lower in the pNP-immunized healthy mouse
group than in the PBS-immunized healthy group (P ?
0.006), but there were no significant differences between
virus titers in the pNP-Dia and PBS-Dia groups. Further-
more, the influenza virus titer was significantly higher in
pNP-Dia immunized mouse group than in pNP-immunized
healthy mice (P ? 0.001).
FIG. 2. Cytotoxicity responses induced by pNP immunization in
diabetic mice. Healthy and diabetic mice were immunized three times
intradermally with pcDNA3-NP (pNP and pNP-Dia groups, respec-
tively) or PBS (PBS and PBS-Dia groups, respectively) on days 0, 14,
and 28. Two weeks after final immunization, splenocytes from immu-
nized and mock-immunized mice were prepared as described in Ma-
terials and Methods. LDH release assays were performed in triplicate
with splenocytes as effector cells and A/New Caledonia?20?99 H1N1
p815 cells as target cells. All experiments were performed more than
three times, and each group consisted of five mice. The cytotoxicity
activity of the pNP group was significantly higher than that of the
pNP-Dia group (???, P ? 0.001). There was no significant difference
between the cytotoxicity responses of the pNP-Dia and PBS-Dia
groups. E/T, effector-to-target-cell ratio.
FIG. 4. IFN-? production induced by pNP immunization in dia-
betic mice. Healthy and diabetic mice were immunized three times
intradermally with pcDNA3-NP (pNP and pNP-Dia groups, respec-
tively) or PBS (PBS and PBS-Dia groups, respectively) on days 0, 14,
and 28. Two weeks after final immunization, spleens of individual mice
(five per group) were removed, and IFN-? production was measured
with an ELISA kit. IFN-? production of the pNP group was signifi-
cantly higher than that of the pNP-Dia group (???, P ? 0.001). There
was no significant difference between the IFN-? production of the
pNP-Dia and PBS-Dia groups.
FIG. 3. Lymphocyte proliferation responses induced by pNP im-
munization in diabetic mice. Healthy and diabetic mice were im-
munized three times intradermally with pcDNA3-NP (pNP and
pNP-Dia groups, respectively) or PBS (PBS and PBS-Dia groups,
respectively) on days 0, 14, and 28. Two weeks after final immuni-
zation, spleens of individual mice (five per group) were removed,
and lymphocyte proliferation was evaluated using the MTT method.
Values are the means ? standard errors of the means for five
experiments. The lymphocyte proliferation of the pNP group was
significantly higher than that of the pNP-Dia group (???, P ?
0.001). There was no significant difference between the lymphocyte
proliferation of the pNP-Dia and PBS-Dia groups.
FIG. 5. Replication of influenza A viruses in the lungs of diabetic
and healthy immunized mice. Healthy and diabetic mice were immu-
nized three times intradermally with pcDNA3-NP (pNP and pNP-Dia
groups, respectively) or PBS (PBS and PBS-Dia groups, respectively)
on days 0, 14, and 28. Two weeks after final immunization, virus titers
in the lungs of diabetic and healthy mice (five per group) were mea-
sured 4 days after intranasal inoculation with influenza virus. Values
are the means ? standard errors of the means for three experiments.
The horizontal line shows the minimum inoculation of decimal dilu-
tions (10?2) of lung in MDCK cells.***, lung virus titers for pNP
group were significantly lower than those for the pNP-Dia group (P ?
0.001), PBS group (P ? 0.006), and PBS-Dia group (P ? 0.001).**,
lung virus titers of PBS group were significantly lower than those of the
PBS-Dia group (P ? 0.009). There was no significant difference be-
tween the lung virus titers of the pNP-Dia and PBS-Dia groups.
VOL. 17, 2010DNA IMMUNIZATION AND DIABETES 685
The present study is the first to examine the effects of dia-
betes on the cell-mediated immune response induced by ge-
netic immunization against influenza virus infection. Patients
with diabetes belong to a high-risk population of influenza
virus infection (1, 28). However, the current immunization
schedule of influenza vaccination for patients with diabetes is
the same as that for healthy persons. Diepersloot et al. showed
that the patients with diabetes had a poor immune response
after immunization with inactivated influenza virus vaccine (3).
Furthermore, due to a major concern with the emergence of
lethal influenza pandemics, such as that of avian-flu A/H5N1
virus, it is evident that new vaccines are needed to ensure
optimal protection against all strains of influenza viruses (18,
25). Cell-mediated immunity has a very important role in in-
hibiting viral replication and clearance of influenza A virus
infections (8, 29). Vaccines designed to induce cellular im-
mune responses are being investigated to create a universal
vaccine (13, 18). DNA vaccination with influenza virus NP has
been studied in animal models and induced cell-mediated im-
mune responses (7, 12). Data presented in this article show
that diabetes reduces the capacity of the pNP DNA vaccine to
induce cytotoxicity responses, lymphocyte proliferation, and
IFN-? production. In previous studies, it was demonstrated
that to be effective, DNA vaccines need to induce optimal
functional activities of many components of the immune sys-
tem, including T CD4?, T CD8?, and B cells (11, 17). On the
other hand, several studies have shown that diabetes causes an
abnormality in many immunologic parameters, including cyto-
kine production, such as s decrease of interleukin-2, interleu-
kin-12, and IFN-? production, aberrancies in the maturation of
dendritic cells (DCs), and impaired function of CD4?T cells
(6, 9, 14, 15, 20, 24, 30). Clinical studies have suggested the
importance of higher vaccination coverage in diabetic patients
to reduce the impact of influenza virus infection on morbidity
and mortality of infected patients (3, 28). Furthermore, Zhu et
al. showed that antibody responses in diabetic mice were re-
duced and only immunization with higher doses of inactivated
influenza vaccine provided protection against lethal influenza
virus challenge in diabetic mice (31). Another study showed
that influenza replication increased in lungs of diabetic mice
Cellular responses are very important in clearing influenza A
virus infections in mice (7, 27). In our experiment, reduced
viral clearance from lungs of pNP-immunized diabetic mice in
comparison to healthy mice may be due to suppression of the
In summary, the present data suggest that the pNP DNA
vaccine with the protocol of immunization described here is
not able to induce optimal cell-mediated immunity in diabetic
mice. These results provide new insights into the field of DNA
immunization for infectious diseases in diabetes. Inclusion of
Th1 cytokine genes in DNA constructs and employment of
other DNA immunization protocols may improve vaccine po-
tency and will be investigated in future studies. Furthermore,
follow-up studies are needed to test the effects of diabetes on
the potency of genetic immunization in induction of humoral
We thank all members of the Influenza Unit, Pasteur Institute of
Iran, for their advice and assistance with the experiments.
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