M A J O R A R T I C L E
Protein Energy Malnutrition Decreases
Immunity and Increases Susceptibility to
Influenza Infection in Mice
Andrew K. Taylor,1Weiping Cao,1Keyur P. Vora,1Juan De La Cruz,1Wun-Ju Shieh,2Sherif R. Zaki,2
Jacqueline M. Katz,1Suryaprakash Sambhara,1and Shivaprakash Gangappa1
1Influenza Division, National Center for Immunization and Respiratory Diseases and2Divison of High Consequence Pathogens and Pathology, National
Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia
dren, is associated with an increased risk of infections. Very few studies have addressed the relevance of PEM as a
risk factor for influenza.
Methods. We investigated the influence of PEM on susceptibility to, and immune responses following, influ-
enza virus infection using isocaloric diets providing either adequate protein (AP; 18%) or very low protein (VLP;
2%) in a mouse model.
Results.We found that mice maintained on the VLP diet, when compared to mice fed with the AP diet,
exhibited more severe disease following influenza infection based on virus persistence, trafficking of inflammatory
cell types to the lung tissue, and virus-induced mortality. Furthermore, groups of mice maintained on the VLP
diet showed significantly lower virus-specific antibody response and a reduction in influenza nuclear protein-spe-
cific CD8+T cells compared with mice fed on the AP diet. Importantly, switching diets for the group maintained
on the VLP diet to the AP diet improved virus clearance, as well as protective immunity to viral challenge.
Conclusions.Our results highlight the impact of protein energy on immunity to influenza infection and suggest
that balanced protein energy replenishment may be one strategy to boost immunity against influenza viral infections.
Protein energy malnutrition (PEM), a common cause of secondary immune deficiency in chil-
Keywords.Nutrition; protein energy; malnutrition; influenza; immunity.
Influenza viruses cause seasonal epidemics and occa-
sional pandemics of highly contagious, acute respirato-
ry illness that result in a substantial global public
health burden [1, 2]. Although the recent 2009 H1N1
pandemic was relatively less severe than the 3 prior
pandemics, the global spread of the pandemic virus
highlighted the explosive nature of pandemic influenza
. The continued circulation of highly pathogenic
avian influenza A (H5N1) viruses in domestic poultry
and their occasional introduction into humans under-
score the concern regarding the emergence of a pan-
demic virus that could result in high morbidity and
fatality rates . Therefore, it is critical to understand
the risk factors that lead to increased disease severity
of influenza infections, so that appropriate counter-
measures are identified for all age groups worldwide.
Malnutrition, a major risk factor for a number of
infectious diseases, including influenza, is widely
prevalent in developing countries . Therefore, it is
malnutrition on morbidity and mortality associated
with influenza infection. Although studies using the
murine model have addressed the effects of some
aspects of malnutrition on respiratory viral pathogens
[6–8], to our knowledge no studies to date have ad-
dressed the effects of protein energy malnutrition
(PEM) on influenza infection. PEM, a common cause
of secondary immune deficiency in children, is
Received 23 January 2012; accepted 14 March 2012; electronically published 4
This work was presented at 2 scientific meetings: 1. Options for the Control of
Influenza VII, September, 2010, Hong Kong, SAR China; 2. American Association
of Immunologists, May 2011, San Francisco, California.
Correspondence. Shivaprakash Gangappa, PhD, Immunology and Pathogenesis
Branch, Influenza Division, 1600 Clifton Rd, Bldg 15/Rm SSB611, MS G47,
Centers for Disease Control and Prevention, Atlanta, GA 30333 (sgangappa@cdc.
The Journal of Infectious Diseases2013;207:501–510
Published by Oxford University Press on behalf of the Infectious Diseases Society of
Protein Energy Malnutrition and Influenza Infection • JID 2013:207 (1 February) • 501
by guest on October 21, 2015
defined as an imbalance between food intake (protein and
energy) and the body’s requirement to ensure the most favor-
able growth [9, 10]. PEM is the most fatal form of malnutri-
tion in developing countries, with more than 150 million
children <5 years suffering worldwide . There are 2 major
forms of PEM—kwashiorkar and marasmus. Although inade-
quate protein in the diet, even with an adequate caloric intake,
is the major cause of kwashiorkar, consumption of insufficient
protein and calories are known to be responsible for maras-
mus . Few studies, however, have linked these 2 major
forms of PEM to higher incidence of influenza infection and
mortality [13, 14].
In this study, we used a mouse model to address the impact
of PEM on influenza A virus infection. We evaluated the effect
of 2 isocaloric diets supplementing distinct levels of protein
energy (18%, adequate protein [AP]; 2%, very low protein
[VLP]) on infection of weanling mice with either a laboratory
strain (A/PR/8/34 [A/PR8]) or a 2009 H1N1 pandemic influ-
enza virus (A/Mexico/4108/2009 [A/Mex]). Our findings dem-
onstrate the deleterious impact of PEM on influenza virus
disease and subsequent immune responses and furthermore
show that supplementing protein energy can restore immune
function and improve the outcome of influenza infections in
the mouse model.
MATERIALS AND METHODS
Mice and Diets
Four week-old female C57BL/6 mice (Jackson Laboratory, Maine)
were randomly assigned to isocaloric diets (Harlan Laboratories,
Indianapolis, Indiana) containing either 18% (AP diet; TD09530)
or 2% (VLP diet; TD09532) protein. Establishing the isocaloric
diets, containing comparable levels of micronutrients, required
balancing the energy source with carbohydrate levels in the VLP
diet. The composition and caloric content for the diets used in the
to confirm comparable feed consumption. For protein resupple-
mentation experiments, after 3 weeks of feeding respective experi-
mental diets (AP and VLP) to mice, we supplemented an
additional VLP group of mice with the AP diet (hereafter referred
to as supplemented protein [SP] diet) and, 3 weeks later, investi-
gated their response to influenza A virus infection. All animal
IACUC and was conducted in an AAALAC-accredited facility.
Viruses and Infection
Influenza viruses A/PR8 and A/Mex were propagated in 10-
day-old embryonated chicken eggs and stored at –80°C. The
mice were deeply anesthetized with 2,2,2-tribromoethanol in
tert-amyl alcohol before intranasal inoculation with virus (25,
50, or 100 mouse infectious dose [MID50]) diluted in phos-
phate-buffered saline (PBS). The 50% MID50was determined
as described elsewhere . As per CDC-IACUC guidelines,
any mouse that lost >25% of its preinfection body weight was
Lung Virus Titer Estimation
Lungs collected at days 3, 6, 9, and 12 postinfection were ho-
mogenized in PBS and titrated in eggs to determine virus in-
fectivity (limit of detection, 101.5EID50/mL). Allantoic fluid
from the inoculated eggs was added to wells containing 0.5%
turkey red blood cells (RBCs) in PBS. Virus titers were calcu-
lated by the Reed and Muench method and are expressed as
the mean log10EID50/mL ±SEM .
Hemagglutination Inhibition Assay
Serum samples were treated with receptor-destroying enzyme
(RDE) and tested for reactivity to viruses by the standard
hemagglutination inhibition (HI) assay with 0.5% turkey
RBCs as described elsewhere . Briefly, 25 µL of PBS was
added to a 96 well plate containing 25 µL of RDE-treated
serum samples. Eight hemagglutination units of virus was
added and incubated at room temperature for 30 minutes.
Finally, 50 µL of 0.5% turkey RBCs in PBS was added and
incubated at room temperature for 30 minutes. The serial di-
lution of serum showing complete inhibition of hemagglutina-
tion was recorded as the HI titer.
On day 6 postinfection, 3 mice per group were killed, and
lungs were collected in 10% neutral buffered formalin. After
72 hours, the samples were transferred to 70% ethanol, sec-
tioned, and stained with hematoxylin and eosin .
Single cell suspensions were prepared from mouse tissues
following influenza infection. Spleen and lung tissue were
digested by treatment with type-1 collagenase (1 : 4 dilution)
and made into a single cell suspension in MACS buffer
(500 mL phosphate-buffered saline, 2.5 mL fetal bovine
serum, 2 mL EDTA), using a cell strainer (VWR, California).
For analysis of NK cell and neutrophil infiltration in lung
tissue, Alexa Fluor 700-anti-CD45, Pacific Blue-CD11b, FITC-
anti-Ly6G, PerCP-Cy5.5-anti-CD3, and APC-Cy7-anti-NK1.1
(BD Biosciences, California) antibodies were used. Virus-
specific CD8+T cells and intracellular interferon γ (IFN-γ),
after in vitro stimulation of 106splenocytes/well with 0.1
MOI of virus for 3 days, were analyzed using pentameric com-
plexes of the PE-H-2Db-influenza A (A/PR8) NP 366–374
ASNENMETM (Proimmune, UK), Alexa Fluor 700-anti-
CD8, PE-Cy7-anti-CD4, PerCP-Cy5.5-anti-IFN-γ following
the manufacturer’s recommendation. Approximately 105cells
were acquired and analyzed on an LSRII flow cytometer (BD
Bioscience, California). Data were analyzed with FlowJo soft-
ware (Treestar, Oregon).
502 • JID 2013:207 (1 February) • Taylor et al
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Following the manufacturer’s protocol, serum leptin concen-
trations were measured using a mouse enzyme-linked immu-
nosorbent assay (ELISA) kit (GenWay Biotech, California).
Briefly, samples and standards were added to antibody-coated
strips, followed by a biotinylated-labeled antibody, streptavi-
din-HRP conjugate and, finally, a substrate and stop solution.
The plate was read using a BioTek Synergy plate reader
(BioTek, Vermont). Measured values were then converted to
concentrations (pg/mL) based on standard curves.
Statistical analysis was performed using GraphPad Prism 5.0
software (GraphPad Software, California). The Student t test
was used to analyze differences among treatments for serum
leptin concentrations, virus titer, and flow cytometry. A 2-way
analysis of variance (ANOVA) was used, in conjunction with
the Bonferonni post-test, on all virus-induced morbidity data;
data were presented as mean± SEM. The Mann–Whitney U
test was used to determine significance among antibody (HI)
titers. The Logrank (Mantel-Cox) test was used to compare
percent survival among groups of mice. All differences were
considered statistically significant when the P value was ≤.05.
Low Protein Diet Enhances Virus-Induced Mortality in Mice
Infected With Either a Laboratory Strain or 2009 H1N1
Because PEM is associated with an increased risk of infections
in children [5, 19], we used weanling mice to examine the
effects of diet-induced PEM on influenza virus infection. First,
on beginning the feeding regimen with either the AP or VLP
diet, we found that feed consumption was comparable
between AP and VLP groups of mice (data not shown). Inabil-
ity to gain body weight and lower levels of serum leptin, 2
markers of PEM [20–22], were assessed over 4 weeks of
feeding custom diets. As shown in Figure 1A, although the AP
diet group of mice gained body weight, the VLP group of
mice failed to achieve similar growth. The mean serum leptin
concentration was significantly lower in the VLP group of
mice compared with mice maintained on the AP diet
Next, to determine the outcome of PEM on susceptibility to
influenza virus infection, we infected mice maintained on
either the AP or VLP diet with sublethal doses of influenza A
viruses (A/PR8, 50 or 100 MID50; A/Mex, 100 MID50) and
assessed for virus-induced mortality and morbidity. Following
viral infection, the feed consumption, despite transient decline
for both the AP and VLP groups, was comparable (data not
shown). Also, compared with preinfection, the leptin levels
did not vary after infection of mice with either virus (data not
shown). Interestingly, as shown in Figure 1C (left and middle
panels), the VLP group of mice, in response to infection with
A/PR8, showed higher mortality when compared with mice
maintained on the AP diet. Similarly, when compared with
mice fed with the AP diet, the VLP group of mice showed
higher mortality in response to infection with A/Mex virus
(Figure 1C, right panel).
Low Protein Diet Leads to Impairment in Virus Clearance and
Enhanced Inflammatory Cell Recruitment in Lungs of Influenza
The consequence of PEM on virus-induced mortality follow-
ing administration of a sublethal dose of influenza-A virus
prompted us to investigate its effects on virus clearance and
antiviral response in the lungs. To address this, we examined
virus titer in lung tissue homogenates of mice maintained on
AP or VLP diets. As shown in Figure 2, virus titer, for both
viruses, in the AP and VLP groups of mice was comparable at
days 3 and 6 postinfection. Furthermore, mice maintained on
the AP diet demonstrated a decline in virus titer by day 9
postinfection and efficiently cleared virus by day 12 postinfec-
tion. However, mice on the VLP diet, when compared to
those on the AP diet, showed significantly higher virus titer
for A/Mex at day 9 postinfection, and for both viruses at day
To address the influence of PEM on inflammation in the
lungs, we performed histological analysis of lung tissues har-
vested on day 6 postinfection from the AP and VLP groups of
mice. As shown in Figure 3A, both AP and VLP groups of
mice demonstrated inflammatory lesions following infection
with either A/PR8 or A/Mex viruses compared with PBS-
treated mice. To quantitatively assess the differences in lung
inflammation, we measured infiltration of neutrophils, a key
cell type known to orchestrate inflammation in influenza-
infected hosts [23, 24] and found that groups of mice main-
tained on the VLP diet had a significantly higher percentage
of neutrophils in response to infection with either influenza
virus compared with mice fed the AP diet (Figure 3B; left
panel). The defect in virus clearance in lungs of mice main-
tained on the VLP diet also prompted us to investigate
changes in lung natural killer (NK) cells, an innate immune
cell type known to secrete interferon γ (IFN-γ) key antiviral
cytokine in effector tissues during the early stage of infection
[25, 26]. We found a significant reduction in the percentage of
NK cells in lungs of the VLP diet group of mice when com-
pared with the AP diet group (Figure 3B, right panel).
Low Protein Diet Leads to Decrease in Virus-Specific
Antibody and NP-Specific CD8+T Cells in Influenza AVirus-
Because we observed higher mortality, as well as impairment in
viral clearance, we investigated the effects of PEM on adaptive
Protein Energy Malnutrition and Influenza Infection • JID 2013:207 (1 February) • 503
by guest on October 21, 2015
protein (AP) and VLP diets were assessed daily for percent change in original body weight (A) and serum leptin concentration (B) at 5 weeks after
beginning the feeding regimen. A, Results are shown from 1 of 2 independent experiments and consists of 5 mice per group. B, Serum leptin concentra-
tion is shown from 6 mice in each group. Error bars represent mean ±SEM. The differences in mean body weights between the VLP and AP groups
were statistically significant as follows: P<.001 for day 9 and P<.0001 for days 10–38. C, Mice on the VLP diet show increased susceptibility to
influenza infection. Mice maintained on the AP or VLP diets were infected with either A/PR8 or A/Mex influenza and assessed for virus-induced
mortality (percent survival). Data represent results from 3 independent experiments.
A, B, Very low protein (VLP) diet leads to reduced growth and a decrease in serum leptin concentration. Mice maintained on the adequate
harvested from mice on the adequate protein (AP) or VLP diets, on days 3, 6, 9, and 12 postinfection (A/PR8, 50 MID50; A/Mex, 100 MID50), were
homogenized and assayed for virus titer as described in Materials and Methods. Data represent values from 2 independent experiments with each
experiment consisting of n=6 lung tissues per group at each time point. Values represent mean±SEM.
Influenza A virus–infected mice maintained on a very low protein (VLP) diet show a defect in viral clearance in the lungs. Lung tissues
504 • JID 2013:207 (1 February) • Taylor et al
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immunity to influenza infection. As shown in Figure 4A, in
response to either A/PR8 or A/Mex infection, serum HI titer
in the VLP diet fed mice, was significantly less than the AP
diet fed group of mice at 30 days postinfection. To assess the
effects of PEM on virus-specific CD8+T-cell responses, we ex-
amined the kinetics (days 8, 15, and 30 postinfection) of influ-
enza NP-specific CD8+T cells in the spleen of AP and VLP
diet fed mice infected with A/PR8. First, flow cytometric anal-
ysis of splenocytes harvested from naive mice maintained on
VLP diet revealed a reduction in the total number of spleno-
cytes (leukocyte, B-cell, and T-cell subsets) compared with the
AP diet-fed group (Supplementary Table 2). Upon A/PR8 in-
fection, the total numbers of splenocytes were increased in AP
diet group of mice on day 30 postinfection compared with
naive mice (Supplementary Tables 2 and 3). In contrast, mice
fed the VLP diet and infected with A/PR8 failed to show any
increase in either the splenocyte numbers or the proportion of
B and T cells (Supplementary Tables 2 and 3). Notably,
evaluation of B-cell and T-cell subsets in A/PR8 infected mice
showed comparable percentages of T-cell and T-cell subsets
between the AP and VLP diet fed mice but a significant de-
crease in the percentage of B cells (Supplementary Table 3).
Importantly, splenocytes harvested from the A/PR8-infected
VLP group of mice, showed a lower percentage of influenza
NP-specific CD8+T cells at days 8, 15, and 30 postinfection,
when compared with mice maintained on the AP diet
(Figure 4B) despite comparable percentages of T-cell subsets
(Supplementary Table 3).
Supplementing Protein Energy-Malnourished Hosts with the
Diet Containing Higher Protein Level Modulates PEM-
Associated Immune Deficits and Decreases Incidence of
Influenza Virus-Induced Mortality
Nutritional supplementation can be an effective strategy,
either as a supportive or adjunct approach, for the promotion
of disease prevention [27, 28]. Provision of adequately
adequate protein (AP) and VLP diet fed mice were harvested at 6 days postinfection (A/PR8, 50 MID50; A/Mex, 100 MID50) and analyzed using histo-
chemical staining (hematoxylin and eosin) as described in Materials and Methods. Representative images (magnification, 10×) from an experiment
consisting of 3 mice per group are shown. B, Lung tissues from mice fed the AP and VLP diets, harvested at days 3 and 6 postinfection, were analyzed
for percent neutrophils (CD11b+Ly6G+) (left panel) and NK cells (CD3−NK1.1+) (right panel) as described in Materials and Methods. Values in panel B
represent mean±SEM, n=6/group, from 2 independent experiments.
Lung tissues from mice fed the very low protein (VLP) diet show increased inflammation following influenza infection. A, Lung tissues from
Protein Energy Malnutrition and Influenza Infection • JID 2013:207 (1 February) • 505
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balanced nutrition may help with modulation of immunity
against infections [29, 30]. To test this hypothesis in our ex-
perimental findings of PEM-induced immune deficits and en-
hanced influenza disease outcome, after 3 weeks of feeding
respective experimental diets to mice, we supplemented an ad-
ditional VLP group of mice with the AP diet (SP diet;
Figure 5A) and, 3 weeks later, investigated their response to
influenza A virus infection. Because infection with both A/
PR8 and A/Mex led to increased morbidity, mortality, and
changes in markers of innate and adaptive immune responses,
only A/PR8 virus infection was used for the following protein
supplementation experiments. As shown in Figure 5B, the SP
group of mice began to gain body weight as soon as their diet
was changed. More importantly, after infection, the SP group
showed a significant reduction in morbidity and improved
survival when compared with the VLP group of mice
(Figures 5C and 5D). At day 9 postinfection, lung virus titer in
the SP group was significantly reduced compared with the
VLP group of mice and, in fact, was comparable to that ob-
served in the AP diet-fed mice (Figure 6A). Moreover, analysis
of lung homogenates harvested from the 3 groups of mice on
day 6 postinfection for production of IFN-γ showed that the
SP group of mice had significantly higher levels when com-
pared with the VLP group of mice (Figure 6B).
Next, we investigated the effects of protein supplementation
on adaptive immune responses to influenza virus infection. As
shown in Figure 7A (left panel), we found that numbers of
influenza virus NP-specific splenic CD8+T cells harvested on
days 8 and 15 postinfection and restimulated in vitro, were
significantly higher in the SP group compared with mice fed
with the VLP diet. Both CD4+and CD8+T-cell subsets har-
vested from the SP group of mice on days 8, 15, and 30 days
postinfection and restimulated in vitro secreted significantly
higher level of IFN-γ compared with the VLP group of mice
(Figure 7A; middle and right panels). Finally, the serum HI
titer in the SP group of mice on day 30 postinfection was sig-
nificantly higher compared to that of mice fed the VLP diet
(Figure 7B) but was comparable to that observed for the mice
fed the AP diet. Taken together, these data indicate that
protein supplementation of mice that had initially received a
protein-deficient diet, restored the ability to elicit appropriate
adaptive immune responses.
NP-specific CD8+T cells. A, Serum samples harvested from mice fed the adequate protein (AP) and VLP diets, at 30 days postinfection (A/PR8, 50
MID50; A/Mex, 100 MID50), were analyzed for HI titer by HI assay as described in Materials and Methods. B, Splenocytes harvested at days 8, 15, 30
days postinfection (A/PR8, 50 MID50) were analyzed for influenza NP-specific CD8 T cells using flow cytometry as described in Materials and Methods.
B, Shown is the percent of NP-specific CD8+T cells. Values in panel A are combined from 2 independent experiments. Values in panel B represent
mean±SEM, n≥3 per group.
Mice maintained on the very low protein (VLP) diet show a decrease in influenza-specific hemagglutination inhibition (HI) antibody titer and
506 • JID 2013:207 (1 February) • Taylor et al
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PEM, particularly in developing countries, represents one of
the most common forms of childhood malnutrition [5, 31].
PEM increases susceptibility to multiple infectious diseases
[10, 32]. In this article, we examined the consequences of
PEM on influenza A virus infection in mice. We found that
infection of mice manifesting signs of diet (VLP)-induced
PEM, with either a laboratory strain or a 2009 H1N1 pandemic
virus, resulted in higher rates of virus-induced morbidity
and mortality compared with mice that received adequate nu-
trition. The VLP group of mice also showed impaired virus
clearance, as well as an increase in inflammatory cells in
the lungs. In addition, the VLP group of mice demonstrated
a significant decreasein adaptive
including production of virus-specific HI antibodies, influenza
(NP)-specific CD8+T cells, and IFN-γ–producing T cells.
Importantly, these effects could be reversed by supplementing
additional protein in the diet of the VLP group of mice, which
resulted in significantly improved immunity to influenza virus
challenge and enhanced host survival. Taken together, our
results demonstrate multiple immune deficits associated with
PEM and emphasize an immune stimulatory role for protein
supplementation during influenza virus infection.
Although other studies have established the impact of PEM
on immunity to microbial infections [5, 6, 20, 32], very few
studies have addressed the relevance of malnutrition, PEM espe-
cially, as a risk factor for seasonal influenza [33–35]. Bellei et al
 found that influenza vaccine-induced antibody responses
were poor in elderly subjects that presented malnutrition. Using
an energy restriction diet in the mouse model of influenza infec-
tion, Ritz et al  showed that mice maintained on an energy-
restricted diet exhibit increased morbidity, and a reduction in
NK cell numbers and function. In our study, similar to the
protein (SP) group of mice. Groups of mice were fed either adequate protein (AP) or very low protein (VLP) diets. Three weeks later, a subgroup of mice
fed the VLP diet was switched to the AP diet and is referred to as the SP group. Three weeks later, all 3 groups of mice were infected with influenza
virus (A/PR8) and assessed for markers of susceptibility to infection and immunity. B–D, Supplementing the mice fed the VLP diet with the diet
containing higher protein energy modifies immune deficits and decreases susceptibility to A/PR8 infection. Mice fed with the AP, VLP, or SP diets were
examined for change in body weight prior to switching diet (B) and after infection with A/PR8 (25 MID50) (C). Dotted line indicates the time point when
the VLP diet was switched to the AP diet for the SP group of mice. Mice fed with the AP, VLP, or SP diets were examined for mortality following
infection with A/PR8 (25 MID50) (D). Data represent results from 2 to 3 independent experiments. Values in panels A represent mean ±SEM. The
differences in mean body weights between the VLP and SP groups for preinfection phase (A, left panel) were statistically significant as follows:
P<.0001 for days 27, 30, 33, 36, 39, and 42. The differences in mean body weights between the VLP and SP groups for postinfection phase (A, right
panel) were statistically significant as follows: day 12, P=.03; day 13, P=.03; day 14, P=.004; day 15, P=.001; day 16, P=.0004; day 17, P=.0001;
and day 18, P<.0001.
A, Schematic representation of diets used, virus infection, and assessments for markers of infection and immunity in the supplemental
Protein Energy Malnutrition and Influenza Infection • JID 2013:207 (1 February) • 507
by guest on October 21, 2015
virus clearance. Groups of mice fed with the AP, VLP, or supplemental protein (SP) diet regimen were either infected with influenza virus (A/PR8) or
administered with phosphate-buffered saline (PBS), as described in Materials and Methods. Lung homogenates were assayed for virus titer on days 6
and 9 postinfection (A) and interferon γ (IFN-γ) on day 6 postinfection (B), as described in Materials and Methods. Data in panels A and B represent
results from 2 independent experiments and consists of lung tissue harvested from 6 mice at each time point. Values represent mean±SEM.
Switching from the very low protein (VLP) diet to the adequate protein (AP) diet enhances antiviral innate immune response and promotes
with the AP, VLP, or supplemental protein (SP) diet were either infected with influenza virus (A/PR8) or administered with phosphate-buffered saline
(PBS), as described in Materials and Methods. Percent NP-specific CD8+T cells, percent interferon γ (IFN-γ)+CD4+T cells, and percent IFN-γ+CD8+T
cells on days 8, 15, and 30 postinfection in the spelenocytes (A) and hemagglutination inhibition (HI) antibody titer on day 30 postinfection in the serum
(B) were analyzed as described in Materials and Methods. A, Data represent results from 2 independent experiments with 3–6 mice per group. B, Each
symbol represents an individual mouse from 2 independent experiments consisting of 8 mice per group, and the horizontal line indicates the mean
value for the group.
Switching from very low protein (VLP) diet to adequate protein (AP) diet modulates virus-specific adaptive immunity. Groups of mice fed
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experimental approach previously described for studying effects
of PEM on LCMV infection , we used experimental diets to
address the impact of PEM on influenza infection. Of note, the
protein level in the AP diet used in our study is 18%, which is
within the range recommended by the US Department of Agri-
culture and the US Department of Health and Human Services
for children up to 3 years of age .
Previous studies have demonstrated an increased viral
burden in clinical settings and experimental models of malnu-
trition. After initiating highly active antiretroviral therapy,
Wang et al  found a higher baseline HIV load and an asso-
ciation with malnutrition. In a mouse model of PEM, Pena-
Cruz et al  found that, compared with a 20% protein diet,
mice fed a 2% protein energy diet had significantly increased
virus titer in the lungs following Sendai virus infection. We
found that although the group of mice fed with the AP diet had
essentially cleared virus by day 12 postinfection, the group
maintained on the VLP diet still exhibited substantial levels (ap-
proximately 104EID50/mL) of virus in the lungs. This delay in
virus clearance in the lung tissue of the VLP group of mice may
be responsible for the increase in neutrophil infiltration in our
studies, as also suggested by other studies [38,39].
Previous studies have established roles for several cell types
and soluble mediators in bridging non-specific innate re-
sponse with pathogen-specific adaptive immunity by priming
antigen specific B and T lymphocytes [40–42]. In our studies,
we found a significant reduction in both virus-specific anti-
body titer and percent NP-specific CD8+T cells in the VLP
group of mice infected with A/PR8 virus. Mutations in the
peptide sequence corresponding to H-2Db
epitope (amino acid sequence; A/PR8-ASNENMETM and A/
Mex, ASNENVEIM) (J.P. Patel and S. Gangappa, et al unpub-
lished observations) of the NP protein in A/Mex virus limited
the application of commercially available influenza NP-specific
pentamer for tracking NP-specific CD8+T cells in A/Mex in-
fected groups of mice. However, similar to our results,
Chatraw et al  found a reduction in frequency of LCMV-
specific CD8+T cells and CD8+IFN-γ +T cells in spleens
from groups of mice fed with a low protein diet. It is possible
that, in the face of diet-induced PEM, antigen processing and/
or dendritic cell subsets responsible for priming antigen-spe-
cific T cells, could be defective in number, trafficking, or func-
tion [41, 43]. Alternatively, in our model, in the VLP group of
mice, CD4+T cells required for either facilitating virus-specific
antibodies or cytotoxic CD8+T cells [44, 45] could be impact-
ed by altered proliferation, function, or survival. Although the
CD4+T cells primed in mice fed the VLP diet showed a
relative decrease in IFN-γ production, our experiments to de-
termine possible defects in subsets of dendritic cells (conven-
tional DC and plasmacytoid DC) in the lymph nodes did not
show any striking differences between the AP and VLP groups
of mice (data not shown).
What are the challenges for improving immunity to influ-
enza in a PEM population? Similar to micronutrient deficien-
cies [46, 47], PEM impacts overall growth and development of
individuals . As a result, a number of immune deficits, as
evident from our studies, could ensue and pose a threat by
limiting an individual’s ability to mount appropriate host re-
sponses to influenza infection important for efficient viral
clearance and recovery. Therefore, studies to define the non-
compromising limits for PEM (moderate vs severe PEM) and
assessment of immune responses to influenza infection could
aid in designing appropriate interventions for overcoming
PEM-specific immune deficits. Our studies, using an experi-
mental model of “severe” PEM and influenza infection, identify
multiple virus-specific immune deficits, and, more important-
ly, underscore the benefit of a nutritional intervention strategy
for dealing with influenza in certain malnourished popula-
tions. Studies focused on influenza infection in a mild to
moderate range of PEM, as well as evaluations of vaccine-
specific responses in dietary protein supplemented hosts,
remain promising areas for future investigations.
Supplementary materials are available at The Journal of Infectious Diseases
online (http://jid.oxfordjournals.org/). Supplementary materials consist of
data provided by the author that are published to benefit the reader. The
posted materials are not copyedited. The contents of all supplementary
data are the sole responsibility of the authors. Questions or messages re-
garding errors should be addressed to the author.
ogenesis Branch in the Influenza Division, Centers for Disease Control
and Prevention, Vic Veguilla and Jessica Belser in particular, for providing
reagents and constructive comments on this research.
A. T. was supported by Emerging and Infectious
Potential conflicts of interest.
All authors: no reported conflicts.
All authors have submitted the ICMJE Form for Disclosure of Potential
Conflicts of Interest. Conflicts that the editors consider relevant to the
content of the manuscript have been disclosed.
We thank members of the Immunology and Path-
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