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Live Attenuated Influenza Vaccine Enhances Colonization of Streptococcus pneumoniae and Staphylococcus aureus in Mice

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Unlabelled: Community interactions at mucosal surfaces between viruses, like influenza virus, and respiratory bacterial pathogens are important contributors toward pathogenesis of bacterial disease. What has not been considered is the natural extension of these interactions to live attenuated immunizations, and in particular, live attenuated influenza vaccines (LAIVs). Using a mouse-adapted LAIV against influenza A (H3N2) virus carrying the same mutations as the human FluMist vaccine, we find that LAIV vaccination reverses normal bacterial clearance from the nasopharynx and significantly increases bacterial carriage densities of the clinically important bacterial pathogens Streptococcus pneumoniae (serotypes 19F and 7F) and Staphylococcus aureus (strains Newman and Wright) within the upper respiratory tract of mice. Vaccination with LAIV also resulted in 2- to 5-fold increases in mean durations of bacterial carriage. Furthermore, we show that the increases in carriage density and duration were nearly identical in all aspects to changes in bacterial colonizing dynamics following infection with wild-type (WT) influenza virus. Importantly, LAIV, unlike WT influenza viruses, had no effect on severe bacterial disease or mortality within the lower respiratory tract. Our findings are, to the best of our knowledge, the first to demonstrate that vaccination with a live attenuated viral vaccine can directly modulate colonizing dynamics of important and unrelated human bacterial pathogens, and does so in a manner highly analogous to that seen following wild-type virus infection. Importance: Following infection with an influenza virus, infected or recently recovered individuals become transiently susceptible to excess bacterial infections, particularly Streptococcus pneumoniae and Staphylococcus aureus. Indeed, in the absence of preexisting comorbidities, bacterial infections are a leading cause of severe disease during influenza epidemics. While this synergy has been known and is well studied, what has not been explored is the natural extension of these interactions to live attenuated influenza vaccines (LAIVs). Here we show, in mice, that vaccination with LAIV primes the upper respiratory tract for increased bacterial growth and persistence of bacterial carriage, in a manner nearly identical to that seen following wild-type influenza virus infections. Importantly, LAIV, unlike wild-type virus, did not increase severe bacterial disease of the lower respiratory tract. These findings may have consequences for individual bacterial disease processes within the upper respiratory tract, as well as bacterial transmission dynamics within LAIV-vaccinated populations.
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Live Attenuated Influenza Vaccine Enhances Colonization of
Streptococcus pneumoniae and Staphylococcus aureus in Mice
Michael J. Mina,
a,b,c
Jonathan A. McCullers,
c,d
Keith P. Klugman
b
Medical Scientist Training Program, Emory University School of Medicine, Atlanta, Georgia, USA
a
; Hubert Department of Global Health, Rollins School of Public Health,
Emory University, Atlanta, Georgia, USA
b
; Department of Infectious Diseases, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA
c
; Department of Pediatrics,
University of Tennessee Health Sciences Center, Memphis, Tennessee, USA
d
ABSTRACT Community interactions at mucosal surfaces between viruses, like influenza virus, and respiratory bacterial patho-
gens are important contributors toward pathogenesis of bacterial disease. What has not been considered is the natural extension
of these interactions to live attenuated immunizations, and in particular, live attenuated influenza vaccines (LAIVs). Using a
mouse-adapted LAIV against influenza A (H3N2) virus carrying the same mutations as the human FluMist vaccine, we find that
LAIV vaccination reverses normal bacterial clearance from the nasopharynx and significantly increases bacterial carriage densi-
ties of the clinically important bacterial pathogens Streptococcus pneumoniae (serotypes 19F and 7F) and Staphylococcus aureus
(strains Newman and Wright) within the upper respiratory tract of mice. Vaccination with LAIV also resulted in 2- to 5-fold in-
creases in mean durations of bacterial carriage. Furthermore, we show that the increases in carriage density and duration were
nearly identical in all aspects to changes in bacterial colonizing dynamics following infection with wild-type (WT) influenza vi-
rus. Importantly, LAIV, unlike WT influenza viruses, had no effect on severe bacterial disease or mortality within the lower re-
spiratory tract. Our findings are, to the best of our knowledge, the first to demonstrate that vaccination with a live attenuated
viral vaccine can directly modulate colonizing dynamics of important and unrelated human bacterial pathogens, and does so in a
manner highly analogous to that seen following wild-type virus infection.
IMPORTANCE Following infection with an influenza virus, infected or recently recovered individuals become transiently suscepti-
ble to excess bacterial infections, particularly Streptococcus pneumoniae and Staphylococcus aureus. Indeed, in the absence of
preexisting comorbidities, bacterial infections are a leading cause of severe disease during influenza epidemics. While this syn-
ergy has been known and is well studied, what has not been explored is the natural extension of these interactions to live attenu-
ated influenza vaccines (LAIVs). Here we show, in mice, that vaccination with LAIV primes the upper respiratory tract for in-
creased bacterial growth and persistence of bacterial carriage, in a manner nearly identical to that seen following wild-type
influenza virus infections. Importantly, LAIV, unlike wild-type virus, did not increase severe bacterial disease of the lower respi-
ratory tract. These findings may have consequences for individual bacterial disease processes within the upper respiratory tract,
as well as bacterial transmission dynamics within LAIV-vaccinated populations
Received 6 January 2014 Accepted 14 January 2014 Published 18 February 2014
Citation Mina MJ, McCullers JA, Klugman KP. 2014. Live attenuated influenza vaccine enhances colonization of Streptococcus pneumoniae and Staphylococcus aureus in mice.
mBio 5(1):e01040-13. doi:10.1128/mBio.01040-13.
Editor Larry McDaniel, University of Mississippi Medical Center
Copyright © 2014 Mina et al. This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license,
which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Address correspondence to Michael J. Mina, mmina@emory.edu.
The conventional view of pathogen dynamics posits that patho-
gen species act independently of one another. More recently,
however, community interactions between pathogens have been
recognized as necessary to modulate both health and disease (1–
7). These interactions might be expected to be most prevalent
within gut, respiratory, and other mucosal surfaces that harbor
complex populations of commensal and, occasionally, pathogenic
microbes. In the respiratory tract, for example, viral infections are
known to predispose to secondary bacterial invasive disease and
pneumonia from pathogens that are most commonly benign but
occasionally become virulent, particularly following a viral infec-
tion (8–10). A well-known example is the often lethal synergy
between influenza virus and pneumococcal or staphylococcal bac-
terial secondary infections.
Infection with influenza viruses increases susceptibility to se-
vere lower and upper respiratory tract (LRT and URT, respec-
tively) bacterial infections resulting in complications, such as
pneumonia, bacteremia, sinusitis, and acute otitis media (11).
Bacterial infections may be a primary cause of mortality associated
with influenza virus infection in the absence of preexisting comor-
bidity (12, 13). Primary influenza virus infection increases acqui-
sition, colonization, and transmission of bacterial pathogens (14),
most notably the pneumococcus Streptococcus pneumoniae and
Staphylococcus aureus (11, 15).
Although the underlying mechanisms, while well studied, are
not entirely defined, they likely include a combination of influ-
enza virus-mediated cytotoxic breakdown of mucosal and epithe-
lial barriers (16–18) and aberrant innate immune responses to
RESEARCH ARTICLE
January/February 2014 Volume 5 Issue 1 e01040-13 ®mbio.asm.org 1
bacterial invaders in the immediate postinfluenza state, character-
ized by uncontrolled pro- and anti-inflammatory cytokine pro-
duction, excessive leukocyte recruitment, and extensive immuno-
pathology (11, 19–22). When coupled with diminished epithelial
and mucosal defenses, such an environment becomes increasingly
hospitable for bacterial pathogens to flourish and invade in the
days and first few weeks following influenza virus infection.
Increasingly, evidence is linking the early innate immune re-
sponse triggered by infection or vaccination to sustained adaptive
immunity (23). Thus, a broad goal of vaccination is to elicit an
immune response analogous to that of the pathogen itself, without
subsequent disease (24). The intranasally administered live atten-
uated influenza vaccine (LAIV) contains temperature-sensitive
and attenuated virus designed to replicate efficiently in the cooler
temperatures of the upper respiratory tract (URT) but which fails
to do so in the warmer temperatures of the lower respiratory tract
(LRT) (25, 26). Through selective replication in the URT, LAIV
proteins are exposed to the host immune system in their native
conformation, eliciting highly robust (IgA), serum (IgG), and cel-
lular immune responses mimicking those of the pathogenic virus
itself (27).
Although an innate immune response to vaccination is bene-
ficial for long-term protection from influenza virus (28) and in-
fluenza virus-bacterial (29) coinfections, the direct consequences
of such a response to a viral vaccine, with respect to secondary
colonization and disease due to entirely unrelated bacterial patho-
gen species, are unknown. As increased susceptibility to and trans-
mission of bacterial pathogens following influenza are due in large
part to the innate immune response and breakdowns of the epi-
thelial barriers of the URT, it is important to understand whether
similar effects, elicited by live attenuated virus replication, may
also predispose to bacterial infection. We sought here to deter-
mine the effects of a live attenuated influenza vaccine on URT and
LRT bacterial infections. In particular, we ask whether LAIV vac-
cination alters bacterial colonization dynamics of the upper respi-
ratory tract or disease in the lower respiratory tract of mice.
RESULTS
Using a live attenuated influenza A virus vaccine, HK/Syd 6:1:1
(LAIV), which contains many of the same mutations and demon-
strates similar growth dynamics to those in the commercially
available human FluMist vaccine (MedImmune, Gaithersburg,
MD) (see reference 30 and Fig. S1 in the supplemental material for
vaccine details), we evaluated the effects of LAIV and its wild-type
(WT) HK/Syd parent strain (referred to as WT virus) on Strepto-
coccus pneumoniae (the pneumococcus) and Staphylococcus au-
reus replication and disease.
LAIV virus is restricted in growth at 37°C but not at 33°C. To
determine whether LAIV virus grows efficiently at temperatures
seen within the nasopharynx (NP) while remaining restricted in
growth at warmer temperatures of the LRT, WT influenza virus
and its LAIV derivative were grown in MDCK cells at 37°C. As
expected (30), a 3-log decrease in viral titers was measured for
LAIV relative to the WT parent strain (P0.001) (Fig. 1A). How-
ever, when LAIV was propagated at 33°C, a temperature often
associated with the nasopharyngeal environment (31), viral repli-
cation was no different from that of WT virus titers measured at
37°C.
HK/Syd 1:1:6 LAIV vaccination is safe and effective in mice.
Although LAIV is attenuated, inoculation with very high doses
may cause morbidity and weight loss. Via a series of dosing exper-
iments (data not shown), a vaccinating dose of 2e6 tissue culture
infective doses (TCID
50
) of LAIV in 40
l phosphate-buffered
saline (PBS) vehicle was determined to be safe, with no weight loss
or other detectable signs of morbidity in mice (Fig. 1B). This dose
is in agreement with previous studies (28, 30). Inoculation with
the same dose of the WT parent virus led to significant morbidity
and mortality (5/12 mice succumbed by day 7 postinfection)
(Fig. 1B), demonstrating the attenuated nature of the LAIV.
The vaccine efficacy and antibody response using this LAIV
strain were described previously (30). To phenotypically confirm
efficacy here, groups of 8 4-week-old mice were inoculated with
LAIV or the PBS control and 4 weeks later with a lethal dose of the
WT virus. Early vaccination with LAIV conferred complete pro-
tection from any detectable morbidity or weight loss due to infec-
tion with the WT strain, versus 100% mortality in unvaccinated
control mice (Fig. 1C).
LAIV is restricted in growth in the lower but not the upper
respiratory tract. To determine whether the differences in repli-
cation seen in vitro also occur in vivo in the upper (~33°C) versus
lower (~37°C) respiratory tract, groups of 5 mice were vaccinated
with LAIV, and viral titers were measured in whole lung and whole
NP homogenates (Fig. 1D). By 3 days postvaccination, NP titers
were 10,000-fold greater than in the lungs (1.3e6 versus 1.2e2
TCID
50
;P0.001). In contrast, the WT virus grew to high viral
titers in both the NP and lungs (5e5 TCID
50
) (data not shown),
in agreement with previous reports (32), which led to significant
morbidity and mortality, as demonstrated in the controls in
Fig. 1B. Overall, maximal NP titers occurred earlier and were
nearly 400-fold greater than maximum lung titers (1.3e6 versus
3.4e3 TCID
50
;P0.001). Importantly, these NP viral dynamics
are in agreement with viral shedding in NP aspirates from human
subjects following vaccination with the FluMist vaccine (33).
LAIV cytokine response in the nasopharynx and lungs. While
LAIV replication in the NP induces a robust systemic inflamma-
tory response (34, 35), the cytokine response in the NP has, to our
knowledge, not been observed. Nasopharyngeal homogenates and
bronchoalveolar lavage (BAL) specimen cytokines were measured
in groups of 5 mice each at days 0, 3, 5, and 7 postvaccination
(Fig. 1E). Of particular interest, the type I interferon (IFN-
) was
significantly increased in the NP and BAL specimens following
LAIV vaccination, and this cytokine has been demonstrated to
play a pivotal role in excess bacterial colonization of the nasophar-
ynx following WT influenza virus infection (36). As well, macro-
phage inflammatory protein 1
(MIP-1
) was also significantly
upregulated following LAIV, similar to what was seen following
influenza virus-pneumococcal coinfections of human middle ear
epithelial cells (37). In general, the responses measured here in the
NP are similar to those measured from nasopharyngeal washes in
humans infected naturally with seasonal influenza A viruses (38).
LAIV enhances pneumococcal bacterial dynamics in the
URT in a manner highly analogous to WT influenza virus. Nu-
merous previous investigations have demonstrated that replica-
tion of WT influenza virus within the URT predisposes to excess
bacterial replication and colonization within the NP, particularly
by Streptococcus pneumoniae (36, 39, 40). Because, as demon-
strated above, LAIV replicates to near WT levels when in the
cooler temperatures of the URT, we sought to study effects of
LAIV on bacterial carriage density within the NP of mice and
compared them to the changes in bacterial carriage following WT
Mina et al.
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virus infection. LAIV vaccination or sublethal infection with the
WT parent strain was delivered 7 days following inoculation with
a common nasopharynx-colonizing strain of pneumococcus type
19F (Fig. 2A to C) included in the current pneumococcal conju-
gate vaccine (41). Following vaccination, normal bacterial clear-
ance from the NP was halted, and bacteria reverted to exponential
growth within 3 days postvaccination (Fig. 2B). Receipt of LAIV
significantly increased the density of bacterial carriage and ex-
tended the mean duration of colonization from 35 to 57 days
(Fig. 2C). Of particular importance, these effects were nearly iden-
tical in all aspects to the effects of the WT influenza virus on
bacterial carriage density and duration (Fig. 2B and C). Although
no detectable morbidity was associated with vaccination alone
(Fig. 1B), vaccination in the presence of bacterial colonization
resulted in very mild, though sustained weight loss (~3 to 5%; P
0.05) relative to colonized, unvaccinated controls (see Fig. S2 in
the supplemental material) that corresponded with time of great-
est excess bacterial proliferation.
To test whether order and timing of vaccination relative to
bacterial acquisition are important, LAIV or WT virus was admin-
istered 7 days before (rather than after) 19F colonization (Fig. 2D
to F). Early vaccination or infection with WT virus led to imme-
diate excess bacterial outgrowth following pneumococcal inocu-
lation relative to that in mice pretreated with PBS vehicle (Fig. 2E).
This increase was generally more pronounced following LAIV
vaccination relative to WT virus infection, but the difference only
reached statistical significance at day 1 post-bacterial infection.
Increases in mean durations of carriage were also demonstrated
and were similar between the two groups, with duration extending
from 38 days following treatment with PBS to 63 or 65 days fol-
lowing LAIV or WT virus infection, respectively (Fig. 2F).
To further define the temporal nature of these interactions and
simultaneously test whether this response is strain specific, vacci-
nation was given at either 1 or 7 days prior to infection with a
slightly more invasive type 7F pneumococcus (Fig. 3A). The max-
imum bacterial density in both groups of vaccinated mice reached
a near 100-fold increase versus that in PBS controls. When inoc-
ulation with bacteria followed only 1 day (versus 7 days) postvac-
cination, similar but delayed dynamics (Fig. 3A) and cumulative
bacterial titers (Fig. 3B) were measured. Interestingly, the delay
FIG 1 LAIV is safe, effective, replicates well within the URT, and elicits a robust cytokine response. (A) WT and LAIV HK/Syd viruses were grown in MDCK
cells at 37°C and LAIV virus was grown at 33°C, and viral titers were measured via the median TCID50 (n 3 per group). (B) Groups of 12 to 14 8-week-old
BALB/c mice were inoculated with 2e6 TCID50 LAIV, WT HK/Syd virus, or PBS and monitored for weight loss. Three of 12 mice and 2/12 mice died at 4 and
7 days postinfection with WT HK/Syd virus, respectively, while no mice died following LAIV or PBS inoculation. (C) Groups of 8 4-week-old BALB/c mice were
inoculated with 2e6 TCID50 of LAIV (2 of the 3 groups) or PBS and 4 weeks later infected with a lethal dose (5e7 TCID50) of WT HK/Syd virus or the PBS control.
Infection was considered lethal if body weight fell below 70% of the initial body weight. (D) Four groups of 5 mice each were vaccinated with LAIV, and whole
lung and NP viral titers were measured at 1, 3, 5, and 7 days postvaccination. (E) Four groups of 5 mice were vaccinated with LAIV, and NP and BAL specimen
cytokines were measured at day 0 (unvaccinated mice) and days 3, 5, and 7 following vaccination. Error bars represent standard errors (SE) of the mean. Asterisks
indicate statistically significant differences from controls by two-sided Student’s t test. *, P 0.05; **, P 0.001; ***, P 0.0001. NS, not significant (no
difference between groups).
LAIV Enhances Bacterial Carriage Dynamics
January/February 2014 Volume 5 Issue 1 e01040-13 ®mbio.asm.org 3
was consistent with the difference in times from vaccination to
bacterial inoculation between the two groups.
We sought to understand whether these effects of LAIV vacci-
nation on bacterial proliferation would continue over a longer
duration. Mice were infected with pneumococcus 28 days follow-
ing LAIV vaccination—well after viral clearance from the NP was
complete (~7 days postvaccination). Despite the 28-day lag be-
tween LAIV and pneumococcal infection, LAIV continued to
yield immediate excess bacterial proliferation relative to PBS con-
trols (Fig. 3C); however, the effect was modest and short-lived,
with only 2- to 4-fold increases over PBS controls measured be-
tween days 1 and 3 postinfection, respectively. By day 4, bacterial
density in the NP returned to control levels, and the duration of
colonization was not increased.
LAIV enhances Staphylococcus aureus dynamics in the URT.
We next sought to test the effects of LAIV on carriage of an entirely
distinct but important Gram-positive bacterium, Staphylococcus
aureus. LAIV was administered 7 days prior to infection with
S. aureus strain Wright (Fig. 4A and B) or Newman (Fig. 4C and
D). Similar to the previous experiments using two strains of pneu-
mococcus, the density of these two strains of S. aureus following
vaccination was increased at all measured time points for both the
Wright and Newman strains (Fig. 4A and C), and duration of
colonization was significantly extended 3- to 5-fold over that in
the PBS controls (Fig. 4B and D).
LAIV does not increase morbidity or mortality from bacte-
rial LRT infections. Given the severe and often lethal interaction
seen between circulating influenza virus strains and bacterial
FIG 2 LAIV and WT influenza virus infection similarly enhance 19F pneumococcal carriage density and duration of colonization. Groups of 12 to 14 mice were
vaccinated with LAIV and infected with WT influenza virus or PBS vehicle at 7 days following colonization with 19F pneumococcus (A to C) or 7 days prior to
colonization with 19F (D to F). Bacterial strains constitutively expressed luciferase, and nasopharyngeal carriage density was measured via in vivo imaging (IVIS)
at 12 h postbacterial infection and daily thereafter (B and E). Duration of colonization (C and F) was measured via bacterial plating of nasal washes taken daily
after carriage density decreased below the limit of detection for IVIS imaging (~1e4 CFU/ml). Asterisks indicate significant differences between vaccinated (black
asterisks in panels B and E) or WT influenza virus-infected (white asterisks in panels B and E) versus control groups (P 0.05 by Students t test), and error bars
represent standard errors around the mean.
Mina et al.
4®mbio.asm.org January/February 2014 Volume 5 Issue 1 e01040-13
lower respiratory tract infections (LRIs) (11, 42), we assessed the
effects of LAIV on bacterial LRIs and mortality and compared
these effects to those seen following WT influenza virus-bacterial
coinfection and single infections with bacteria. Mice received
LAIV, WT influenza virus, or PBS control and 7 days later (a time
known to maximize the lethal effects of influenza virus-bacterial
coinfections [43]) were inoculated with a sublethal dose of either
of the highly invasive type 2 or 3 pneumococcal serotypes D39 or
A66.1, respectively (Fig. 5A to C).
In contrast to the 100% mortality observed when sublethal
inoculation with D39 or A66.1 followed pretreatment with wild-
type influenza virus, bacterial inoculation following pretreatment
with LAIV demonstrated no increases in morbidity (i.e., weight
loss; data not shown) or mortality (Fig. 5B and C) relative to
bacterial infection alone.
DISCUSSION
The potent and often lethal effects of an antecedent influenza virus
infection on secondary bacterial disease have been reported pre-
viously (11, 21, 44–46). Viral replication induced epithelial and
mucosal degradation, and the ensuing innate immune response
yield diminished capacity to avert secondary bacterial infections.
FIG 3 LAIV enhancement of pneumococcal density is time dependent and long lasting. Groups of 12 to 14 mice were vaccinated with LAIV or PBS vehicle at
1 or 7 days prior to colonization with pneumococcal (pneumo) serotype 7F. Bacterial strains constitutively expressed luciferase, and bacterial NP density was
measured via IVIS in vivo imaging (A and B). Mean cumulative bacterial titers in panel B were calculated by first calculating the cumulative bacterial titers per
individual mouse NP at each time point and then calculating the average and SE across the individual cumulative titers per time point, rather than simply
averaging the areas under the mean density curves shown in panel A. Asterisks indicate significant differences in bacterial densities between the vaccinated and
PBS control groups (dark green indicates LAIV given 7 days prior and red indicates LAIV given 1 day prior to 7F inoculation; P0.05 by two-tailed Student’s
ttest). (C) Groups of mice were vaccinated with LAIV (n20) or PBS vehicle control (n30), respectively, at 28 days prior to colonization with 19F
pneumococcus. Fold differences per day between mean bacterial densities measured in mice treated 28 days prior with LAIV versus PBS are reported. Error bars
indicate standard errors of the mean and asterisks indicate significant differences (P0.05) from PBS controls (by two-tailed single-sample ttest).
LAIV Enhances Bacterial Carriage Dynamics
January/February 2014 Volume 5 Issue 1 e01040-13 ®mbio.asm.org 5
Recent clinical and experimental data suggest that influenza virus
infection may exert its influence beginning in the URT by enhanc-
ing susceptibility to bacterial colonization (14, 47, 48) and in-
creasing NP carriage density (36).
Although vaccination with LAIV, in the longer term, thwarts
secondary bacterial infections by inhibiting primary infections
with influenza virus (29, 49), the immediate effects of LAIV on
bacterial replication and disease have never before been described.
Indeed, although vaccines are among our greatest achievements in
the constant battle against microbial pathogens, the effects of vac-
cination on distinct pathogen species unrelated to vaccine-
targeted pathogens have, until now, remained entirely unex-
plored. LAIV viruses selectively replicate in the URT, partially
denude the epithelium (50), and induce robust innate immune
responses that ultimately contribute to long-term protective im-
munity (28). In so doing, LAIV viruses may, like WT influenza
viruses, condition the site of replication for enhanced secondary
bacterial colonization.
Here, we demonstrated that vaccination with LAIV, like a WT
influenza virus, induces swift increases in bacterial density within
the URT, with no discernible differences in effects on bacterial
dynamics in the NP between the two virus strains. A lag between
viral inoculation and excess bacterial replication of at least 3 to
5 days was consistently measured, no matter the bacterial strain.
Of particular interest, the type I interferon, IFN-
, known to play
a pivotal role in excess pneumococcal colonization following WT
influenza virus infections (36), was maximally upregulated at
3 days post-LAIV vaccination, coincident with commencement of
excess bacterial proliferation. After the 3- to 5-day threshold fol-
lowing vaccination was met, the murine NP remained condi-
tioned for excess pneumococcal replication for at least 28 days
(our furthest time point out) post-vaccination. However, as the
delay between vaccination and bacterial infection was increased,
the magnitude of the effects of vaccination on bacterial dynamics
became considerably more modest, although statistically signifi-
cant excess growth was measured even when acquisition followed
28 days post-vaccination.
While the studies described here are limited in scope to murine
models, enhanced bacterial load in the URT following LAIV may
agree with human data (51), where LAIV has been associated with
increases in adverse upper respiratory tract symptoms. Although
adverse URT symptoms following administration of FluMist are
considered to be of viral etiology, they are most evident in children
5 years of age, where rates of bacterial carriage are greatest (52).
Potentially corroborating this are data from a large prospective
double-blind trial of FluMist (trial no. MI-CP111 [53]) that as-
sessed reactogenicity and adverse URT events within the first
28 days following vaccination in ~3,000 children between the ages
of 6 and 59 months. This trial demonstrated a bimodal increase in
URT symptoms following FluMist vaccination, the first between
days 2 and 4 post-vaccination and the second between days 5 and
10 post-vaccination (53). While these increased URT events (rel-
ative to controls receiving trivalent inactivated influenza vaccine)
were considered normal reactions to the live vaccine, the bimodal
nature of the increased symptoms suggests that two distinct mech-
anisms may be in place. In the context of the current findings, the
first peak may correspond with viral replication, while the second,
more sustained peak may, at least in part, be driven by symptoms
due to excess bacterial carriage.
Perhaps the most important finding from our study, with re-
gard to the health of the public and potential concerns regarding
vaccination, is that LAIV did not enhance lower respiratory tract
infections, morbidity, or mortality following bacterial infections,
which are, by most accounts, the most significant issues to be
FIG 4 LAIV enhances bacterial load and duration of staphylococcal carriage. Groups of 12 to 14 mice were vaccinated with LAIV or PBS vehicle 7 days prior to
colonization with S. aureus (S.A.) strain Wright (A and B) or Newman (C and D). S. aureus constitutively expressed luciferase, and bacterial density was measured
via IVIS in vivo imaging. Duration of colonization (B and D) was measured via bacterial plating of nasal washes taken daily after the carriage density decreased
below the limit of detection for IVIS imaging. Asterisks indicate significant differences between vaccinated and control groups (P0.05 by two-sided Student’s
ttest), and error bars represent standard errors around the mean.
Mina et al.
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concerned with in terms of respiratory tract bacterial disease. In-
deed, this finding is consistent with numerous epidemiological
reports all failing to detect any serious adverse sequelae of LAIV
vaccination in humans (51, 54). Furthermore, this finding is con-
sistent with significantly diminished LAIV virus replication within
the lower respiratory tract, suggesting that viral replication is a
requirement for the synergistic response seen between WT influ-
enza viruses and bacterial LRT infections.
While care should be taken to not overgeneralize the data de-
scribed here to all vaccines, the broad implications suggest that
live attenuated viral vaccines may have unintended consequences
on important human bacterial pathogens unrelated to the vaccine
target species. Furthermore, our findings suggest a role for labo-
ratory models of multispecies interactions with vaccine strains to
inform future vaccine monitoring and evaluation programs
aimed at identifying thus far entirely unrealized “unconventional”
effects, both beneficial and detrimental, of live attenuated viral
vaccines and cross-species microbial dynamics.
MATERIALS AND METHODS
Infectious agents and vaccines. Viral infections were carried out with an
H3N2 1:1:6 reassortant virus developed as described previously (30), con-
taining the surface glycoproteins hemagglutinin (HA) and neuraminidase
(NA) from A/Hong Kong/1/68 (HK68) and A/Sydney/5/97 (Syd97) iso-
lates, respectively, and the six internal protein gene segments from
A/Puerto Rico/8/34 (or PR8; referred to here as WT influenza virus).
LAIV vaccinations consisted of a temperature-sensitive (ts) attenuated
variant of HK/Syd, HK/Syd
att/ts
(LAIV) that contains site-specific muta-
tions in the PB1 and PB2 RNA segments of the genome (see Fig. S1 in the
supplemental material) as described previously (30). These are the same
mutations found in the attenuated A/Ann Arbor/6/60 master donor strain
used to produce the influenza A virus strains found in the commercial
product FluMist (30). WT and LAIV viruses were propagated in 10-day-
old embryonated chicken eggs at 37 and 33°C, respectively) and charac-
terized in Madin-Darby canine kidney cells to determine the 50% infec-
tive tissue culture dose (TCID
50
) in wells. The pneumococcal carrier
isolates ST425 (serotype 19F) and ST191 (serotype 7F), chosen based on
their colonizing potential as previously described (14), were used for col-
onization experiments. The highly invasive type 2 and type 3 pneumococ-
cal isolates D39 and A66.1, respectively, were used for pneumonia and
survival studies. The 19F and 7F strains were engineered to express lu-
ciferase, as described previously (14). Staphylococcus aureus strains
Wright (ATCC 49525) and Newman (ATCC 25905) were engineered to
express luciferase by Caliper Life Sciences (Alameda, CA).
Ethics statement. All experimental procedures were approved by the
Institutional Animal Care and Use Committee (protocol no. 353) at St.
Jude Children’s Research Hospital (SJCRH) under relevant institutional
FIG 5 LAIV does not increase severe bacterial disease or mortality. Groups of mice received intranasal LAIV vaccination (solid red curves), sublethal infection
with WT influenza virus (broken black curves), or PBS (broken blue curves) 7 days prior to inoculation with a sublethal dose of Streptococcus pneumoniae type
2 (1e5 CFU D39; n20 per group) (B) or type 3 (1e3 CFU A66.1; n12 to 15 per group) (C), and body weight and mortality were observed at least every 12
h for the first 4 days postpneumococcal inoculation and daily thereafter. Kaplan-Meier survival curves with 95% confidence intervals (CI) were constructed, and
asterisks indicate statistically significant differences (P0.05 by log rank test) between LAIV- or WT virus-infected groups versus PBS controls.
LAIV Enhances Bacterial Carriage Dynamics
January/February 2014 Volume 5 Issue 1 e01040-13 ®mbio.asm.org 7
and American Veterinary Medical Association guidelines and were per-
formed in a biosafety level 2 facility that is accredited by the American
Association for Laboratory Animal Science (AALAS).
Animal and infection models. Eight-week-old BALB/c mice (Jackson
Laboratories, Bar Harbor, ME) were used in all experiments, with the
exception of mice treated with early vaccination to demonstrate vaccine
efficacy and effectiveness. In these cases, 4-week-old BALB/c mice were
vaccinated or administered PBS and monitored for 4 weeks before further
inoculation. All inoculations and vaccinations were via the intranasal
route under general anesthesia with inhaled 2.5% isoflurane (Baxter
Healthcare, Deerfield, IL). LAIV vaccination consisted of 2e6 TCID
50
HK/
Syd
att/ts
LAIV in 40
l PBS. The lethal and sublethal doses of WT HK/Syd
were 5e7 and 1e5 TCID
50
in 50
l PBS, respectively. Pneumococcal infec-
tions with 19F and 7F were performed as described previously (14), except
inoculation was in 40
l PBS. Infection with S. aureus strains Wright and
Newman contained 1e7 CFU in 40
l PBS. Mortality studies were per-
formed as described previously (43) with sublethal doses of the invasive
type 2 and type 3 pneumococcal serotype D39 and A66.1 isolates, consist-
ing of 1e5 and 1e3 CFU in 100
l PBS (to ensure bacterial entry into the
lower lungs), respectively. Animals were monitored for body weight and
mortality at least once per day for all survival studies. Mice were sacrificed
if body weight fell below 70% initial weight.
Bacterial CFU titers for duration studies. Bacterial CFU titers were
measured in nasal washes using 12
l of PBS administered and retrieved
from each nare and quantitated by serial dilution plating on blood agar
plates. Washes were performed daily only after the pneumococcal density
fell below the limit of detection for IVIS imaging (~1e4 CFU/ml).
Determination of bacterial and viral titers in lungs and nasopharyn-
geal homogenates. Viral and bacterial titers were measured in whole lung
and nasopharyngeal (NP) homogenates. Whole lungs were harvested and
homogenized using a gentleMACS system (Miltenyi Biotech), as per the
manufacturer’s protocol. NP was isolated via careful dissection dorsally
across the frontal bones, laterally via removal of the zygomatic bone, pos-
teriorly by dislocation of the upper jaw from the mandible, and inferiorly
just posterior to the soft palate. Isolated NP was homogenized via plung-
ing in 1.5 ml PBS through a 40-
m-mesh strainer. Bacterial titers were
measured via plating of serial dilutions, and viral titers were measured by
determining the TCID
50
as previously described (30).
Determination of cytokine levels in the NP and BAL specimens by
enzyme-linked immunosorbent assay. Nasopharyngeal isolates and BAL
specimens were collected as described above, and cytokines were mea-
sured using commercially available kits from R&D systems (macrophage
inflammatory protein 1
[MIP-1
], transforming growth factor
[TGF-
], and beta interferon [IFN-
]) or eBiosciences (interleukin-4 [IL-4],
IL-6, IL-10, IL-17, IL-23, and gamma interferon [IFN-
]).
Bioluminescent imaging. Mice were imaged using an IVIS charge-
coupled device (CCD) camera (Xenogen) as described previously (14,
29). Nasopharyngeal bacterial density was measured as total photons/s/
cm
2
in prespecified regions covering the NP, and background (calculated
for each mouse on a region of equal area over the hind limb) was sub-
tracted. Each NP measurement represents an average of two pictures, one
for each side of the mouse head. Quantitation was performed using Liv-
ingImage software (version 3.0; Caliper Life Sciences) as described previ-
ously (14).
Statistical analyses. All statistical analyses were performed within the
R statistical computing environment (version 2.14R; R Foundation for
Statistical Computing, R Development Core Team, Vienna, Austria). The
specific statistical tests used are as indicated in the legend to each figure.
The R package Survival was used for all survival analyses, Kaplan-Meier
(KM) plots, and KM log rank tests. All other statistical tests were per-
formed using R base functions.
SUPPLEMENTAL MATERIAL
Supplemental material for this article may be found at http://mbio.asm.org
/lookup/suppl/doi:10.1128/mBio.01040-13/-/DCSupplemental.
Figure S1, TIFF file, 0.2 MB.
Figure S2, TIFF file, 0.3 MB.
ACKNOWLEDGMENTS
This work was funded by ALSAC-St. Jude Children’s Research Hospital to
J.A.M. and a Burroughs Wellcome Fund “Molecules to Mankind” grant to
M.J.M.
We thank Amy R. Iverson for assistance in carrying out laboratory
experiments. As well, we thank Bruce R. Levin, Rustom Antia, Veronika
Zarnitsyna, and Jaap de Roode for assistance in reviewing the manuscript.
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... 9,10 These effects are similar to wild-type influenza infection, but without progression to clinical symptoms or disease. 9,11 These data highlight the potential indirect consequences of LAIV on S pneumoniae density in the URT, as well as the potential importance of paucisymptomatic or asymptomatic viral infection on colonisation dynamics of residing nasopharyngeal bacteria such as S pneumoniae. ...
... Although these findings are consistent with the only other such study among children in the UK, 10 the increase in density we observed was less (1·78-times the original magnitude by day 21) than the six-times increase (at day 28) in genome copies per mL reported by Thors and colleagues. 22 Furthermore, the increase in our study was mainly seen between day 0 and 7 (1·58-times), consistent with the peak seen in a mouse model, 9,10 and appeared to be driven by higher viral shedding and co-infection with asymptomatic respiratory viruses at baseline. ...
... We did an individually randomised study, which theoretically might underestimate the effect on children in the control group as higher pneumococcal densities in vaccinated children might increase transmission in shared environments. Although this might be an issue when recruiting from day care centres, 9,10 it is less likely to be relevant in our community-based study in which young children spend most days within and around their own households. We recruited 20 sibling pairs, with only six pairs in which one sibling was in the LAIV group and the other in the control group during the same year. ...
Article
Full-text available
Background Influenza and other respiratory viruses promote Streptococcus pneumoniae proliferation in the upper respiratory tract. We sought to investigate for what we believe is the first time, the effect of intranasal live attenuated influenza vaccine (LAIV) on nasopharyngeal S pneumoniae density in a low-income to middle-income country population with high pneumococcal carriage rates. Methods In an open-label, randomised, controlled trial in The Gambia, 330 healthy children aged 24–59 months were randomly assigned 2:1 to receive one trivalent LAIV dose at enrolment (day 0, intervention) or at the end of active follow-up (day 21, control). The investigator team were initially masked to block size and randomisation sequence to avoid allocation bias. Group allocation was later revealed to the investigator team. The primary outcome was PCR-quantified day 7 and 21 pneumococcal density. Asymptomatic respiratory viral infection at baseline and LAIV strain shedding were included as covariates in generalised mixed-effects models, to assess the effect of LAIV and other variables on pneumococcal densities. The study is registered at ClinicalTrials.gov, NCT02972957, and is closed to recruitment. Findings Between Feb 8 and April 12, 2017, and Jan 15 and March 28, 2018, of 343 children assessed for eligibility, 213 in the intervention group and 108 in the control group completed the study and were included in the final analysis. Although no significant differences were seen in pneumococcal carriage or density at each timepoint when comparing groups, changes from baseline were observed in the LAIV group. The baseline S pneumoniae carriage prevalence was high in both LAIV and control groups (75%) and increased by day 21 in the LAIV group (85%, p=0·0037), but not in the control group (79%, p=0·44). An increase in pneumococcal density from day 0 amounts was seen in the LAIV group at day 7 (+0·207 log10 copies per μL, SE 0·105, p=0·050) and day 21 (+0·280 log10 copies per μL, SE 0·105, p=0·0082), but not in the control group. Older age was associated with lower pneumococcal density (−0·015 log10 copies per μL, SE 0·005, p=0·0030), with the presence of asymptomatic respiratory viruses at baseline (+0·259 log10 copies per μL, SE 0·097, p=0·017), and greater LAIV shedding at day 7 (+0·380 log10 copies per μL, SE 0·167, p=0·024) associated with higher pneumococcal density. A significant increase in rhinorrhoea was reported in the LAIV group compared with the control group children during the first 7 days of the study (103 [48%] of 213, compared with 25 [23%] of 108, p<0·0001), and between day 7 and 21 (108 [51%] of 213, compared with 28 [26%] of 108, p<0·0001). Interpretation LAIV was associated with a modest increase in nasopharyngeal pneumococcal carriage and density in the 21 days following vaccination, with the increase in density lower in magnitude than previously described in the UK. This increase was accelerated when LAIV was administered in the presence of pre-existing asymptomatic respiratory viruses, suggesting that nasopharyngeal S pneumoniae proliferation is driven by cumulative mixed-viral co-infections. The effect of LAIV on pneumococcal density is probably similar to other respiratory viral infections in children. Our findings provide reassurance for the use of LAIV to expand influenza vaccine programmes in low-income to middle-income country populations with high pneumococcal carriage. Funding Wellcome Trust.
... monia. Influenza vaccination with live attenuated virus increases the amounts of both pneumococci and staphylococci in the upper airway (16), suggesting that the immune response activated by influenza virus affects the factors that normally regulate the upper airway microbiome. ...
Article
Full-text available
Much of the morbidity and mortality associated with influenza virus respiratory infection is due to bacterial coinfection with pathogens that colonize the upper respiratory tract such as methicillin-resistant Staphylococcus aureus (MRSA) and Streptococcus pneumoniae. A major component of the immune response to influenza virus is the production of type I and III interferons. Here we show that the immune response to infection with influenza virus causes an increase and restructuring of the upper respiratory microbiota in wild-type (WT) mice but not in Il28r−/− mutant mice lacking the receptor for type III interferon. Mice lacking the IL-28 receptor fail to induce STAT1 phosphorylation and expression of its regulator, SOCS1. Il28r−/− mutant mice have increased expression of interleukin-22 (IL-22), as well as Ngal and RegIIIγ, in the nasal cavity, the source of organisms that would be aspirated to cause pneumonia. Proteomic analysis reveals changes in several cytoskeletal proteins that contribute to barrier function in the nasal epithelium that may contribute to the effects of IL-28 signaling on the microbiota. The importance of the effects of IL-28 signaling in the pathogenesis of MRSA pneumonia after influenza virus infection was confirmed by showing that WT mice nasally colonized before or after influenza virus infection had significantly higher levels of infection in the upper airways, as well as significantly greater susceptibility to MRSA pneumonia than Il28r−/− mutant mice did. Our results suggest that activation of the type III interferon in response to influenza virus infection has a major effect in expanding the upper airway microbiome and increasing susceptibility to lower respiratory tract infection. IMPORTANCE S. aureus and influenza virus are important respiratory pathogens, and coinfection with these organisms is associated with significant morbidity and mortality. The ability of influenza virus to increase susceptibility to S. aureus infection is less well understood. We show here that influenza virus leads to a change in the upper airway microbiome in a type III interferon-dependent manner. Mice lacking the type III interferon receptor have altered STAT1 and IL-22 signaling. In coinfection studies, mice without the type III interferon receptor had significantly less nasal S. aureus colonization and subsequent pneumonia than infected WT mice did. This work demonstrates that type III interferons induced by influenza virus contribute to nasal colonization and pneumonia due to S. aureus superinfection.
... There is a complex interaction between respiratory pathogens such as influenza and bacterial co-infections like S. pneumoniae, that leads to more severe disease (12). Influenza A has been associated with an increase in the colonization and transmission of secondary bacterial infections, including S. pneumoniae and S. aureus possibly through a mechanism of viral priming (13,14). Bacterial pneumococcal colonization was 100,000X times higher in the nasopharynx in patients with influenza A infection compared to those without influenza. ...
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The current COVID-19 pandemic has highlighted a need to further understand lung mucosal immunity to reduce the burden of community acquired pneumonia, including that caused by the SARS-CoV-2 virus. Local mucosal immunity provides the first line of defence against respiratory pathogens, however very little is known about the mechanisms involved, with a majority of literature on respiratory infections based on the examination of peripheral blood. The mortality for severe community acquired pneumonia has been rising annually, even prior to the current pandemic, highlighting a significant need to increase knowledge, understanding and research in this field. In this review we profile key mediators of lung mucosal immunity, the dysfunction that occurs in the diseased lung microenvironment including the imbalance of inflammatory mediators and dysbiosis of the local microbiome. A greater understanding of lung tissue-based immunity may lead to improved diagnostic and prognostic procedures and novel treatment strategies aimed at reducing the disease burden of community acquired pneumonia, avoiding the systemic manifestations of infection and excess morbidity and mortality.
... Although licensed LAVs are confirmed for their safety by clinical trials, new concerns may arise when vaccines are applied for different uses. Superinfection of LAVs and virulent pathogens is unavoidable for interference and may advertently enhance the pathology of secondary infections (80,81). Therefore, to assure the safety of non-conventional LAV use, it is essential to determine the underlying mechanisms of vaccineinduced NSEs (Figure 1). ...
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Live vaccines use attenuated microbes to acquire immunity against pathogens in a safe way. As live attenuated vaccines (LAVs) still maintain infectivity, the vaccination stimulates diverse immune responses by mimicking natural infection. Induction of pathogen-specific antibodies or cell-mediated cytotoxicity provides means of specific protection, but LAV can also elicit unintended off-target effects, termed non-specific effects. Such mechanisms as short-lived genetic interference and non-specific innate immune response or long-lasting trained immunity and heterologous immunity allow LAVs to develop resistance to subsequent microbial infections. Based on their safety and potential for interference, LAVs may be considered as an alternative for immediate mitigation and control of unexpected pandemic outbreaks before pathogen-specific therapeutic and prophylactic measures are deployed.
... For IAV infection, even the inactivated IAV vaccine (LAIV) was found to enhance colonization by S. pneumoniae and S. aureus in a mouse model (Mina et al. 2014). Multiple mechanisms have been suggested to support bacterial superinfection after IAV infection. ...
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With the discovery of the lung microbiota, its study in both pulmonary health and disease has become a vibrant area of emerging research interest. Thus far, most studies have described the lung microbiota composition in lung disease quite well, and some of these studies indicated alterations in lung microbial communities related to the onset and development of lung disease and vice versa. However, the underlying mechanisms, particularly the cellular and molecular links, are still largely unknown. In this review, we highlight the current progress in the complex cellular and molecular mechanisms by which the lung microbiome interacts with immune homeostasis and pulmonary disease pathogenesis to advance our understanding of the elaborate function of the lung microbiota in lung disease. We hope that this work can attract more attention to this still-young yet very promising field to facilitate the identification of new therapeutic targets and provide more innovative therapies. Additional accurate standard-based methodologies and technological breakthroughs are critical to propel the field forward to ultimately achieve the goal of maintaining respiratory health.
... Alternatively, IL-6 is also known to confer epithelial repair and promote proliferation (Kuhn et al., 2014), which may inhibit pneumococcal adherence to the epithelium. For example, coinfection with Influenza A increases susceptibility to S. pneumoniae in both adult and older mice and in younger adults, characterized by increased bacterial burden in the URT (Mina et al., 2014;Jochems et al., 2018;Gou et al., 2019). In the murine study, IL-6 production was required to maintain barrier function and macrophage phagocytic function, which played a role in pneumococcal control and clearance (Gou et al., 2019). ...
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In humans, nasopharyngeal carriage of Streptococcus pneumoniae is common and although primarily asymptomatic, is a pre-requisite for pneumonia and invasive pneumococcal disease (IPD). Together, these kill over 500,000 people over the age of 70 years worldwide every year. Pneumococcal conjugate vaccines have been largely successful in reducing IPD in young children and have had considerable indirect impact in protection of older people in industrialized country settings (herd immunity). However, serotype replacement continues to threaten vulnerable populations, particularly older people in whom direct vaccine efficacy is reduced. The early control of pneumococcal colonization at the mucosal surface is mediated through a complex array of epithelial and innate immune cell interactions. Older people often display a state of chronic inflammation, which is associated with an increased mortality risk and has been termed ‘Inflammageing’. In this review, we discuss the contribution of an altered microbiome, the impact of inflammageing on human epithelial and innate immunity to S. pneumoniae, and how the resulting dysregulation may affect the outcome of pneumococcal infection in older individuals. We describe the impact of the pneumococcal vaccine and highlight potential research approaches which may improve our understanding of respiratory mucosal immunity during pneumococcal colonization in older individuals.
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Objective To determine whether the prior usage of the flu vaccine is a risk factor for bacterial co-infection in patients with severe influenza. Design This was a retrospective observational cohort study of subjects admitted to the ICU. A propensity score matching, and logistic regression adjusted for potential confounders were carried out to evaluate the association between prior influenza vaccination and bacterial co-infection. Settings 184 ICUs in Spain due to severe influenza. Patients Patients included in the Spanish prospective flu registry. Interventions Flu vaccine prior to the hospital admission. Results A total of 4175 subjects were included in the study. 489 (11.7%) received the flu vaccine prior to develop influenza infection. Prior vaccinated patients were older 71 [61–78], and predominantly male 65.4%, with at least one comorbid condition 88.5%. Prior vaccination was not associated with bacterial co-infection in the logistic regression model (OR: 1.017; 95%CI 0.803–1.288; p = 0.885). After matching, the average treatment effect of prior influenza vaccine on bacterial co-infection was not statistically significant when assessed by propensity score matching (p = 0.87), nearest neighbor matching (p = 0.59) and inverse probability weighting (p = 0.99). Conclusions No association was identified between prior influenza vaccine and bacterial coinfection in patients admitted to the ICU due to severe influenza. Post influenza vaccination studies are necessary to continue evaluating the possible benefits.
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Parasites can mediate competition among host species in an ecological community by differentially affecting key parameters that normally give one species a competitive edge. In nature, however, coinfecting parasites that antagonize or facilitate each other-for example, by altering cross-protective host immune responses-can modulate host infection outcomes and parasite transmission relative to a single infection. Under what conditions is coinfection likely to interfere with parasite-mediated apparent competition among hosts? To address this question, we created a model of two coinfected host species. Parasites could interact indirectly by affecting host reproduction or directly by modulating recovery and disease-induced mortality of each host species to a focal infection. We grounded our model with parameters from a classic apparent competition system but allowed for multiple parasite transmission modes and interaction scenarios. Our results suggest that infection-induced mortality has an outsized effect on competition outcomes relative to recovery but that coinfection-mediated modulation of mortality can produce a range of coexistence or competitive exclusion outcomes. Moreover, while infection prevalence is sensitive to variation in parasite transmission mode, host competitive outcomes are not. Our generalizable model highlights the influence of immunological variation and parasite ecology on community ecology.
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Objective: To determine whether the prior usage of the flu vaccine is a risk factor for bacterial co-infection in patients with severe influenza. Design: This was a retrospective observational cohort study of subjects admitted to the ICU. A propensity score matching, and logistic regression adjusted for potential confounders were carried out to evaluate the association between prior influenza vaccination and bacterial co-infection. Settings: 184 ICUs in Spain due to severe influenza. Patients: Patients included in the Spanish prospective flu registry. Interventions: Flu vaccine prior to the hospital admission. Results: A total of 4175 subjects were included in the study. 489 (11.7%) received the flu vaccine prior to develop influenza infection. Prior vaccinated patients were older 71 [61-78], and predominantly male 65.4%, with at least one comorbid condition 88.5%. Prior vaccination was not associated with bacterial co-infection in the logistic regression model (OR: 1.017; 95%CI 0.803-1.288; p=0.885). After matching, the average treatment effect of prior influenza vaccine on bacterial co-infection was not statistically significant when assessed by propensity score matching (p=0.87), nearest neighbor matching (p=0.59) and inverse probability weighting (p=0.99). Conclusions: No association was identified between prior influenza vaccine and bacterial coinfection in patients admitted to the ICU due to severe influenza. Post influenza vaccination studies are necessary to continue evaluating the possible benefits.
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Pathogen transmission is a key point not only for infection control and public health interventions but also for understanding the selective pressures in pathogen evolution. The “success” of a pathogen lies not in its ability to cause signs and symptoms of illness but in its ability to be shed from the initial hosts, survive between hosts, and then establish infection in a new host. Recent insights have shown the importance of the interaction between the pathogen and both the commensal microbiome and coinfecting pathogens on shedding, environmental survival, and acquisition of infection. Pathogens have evolved in the context of cooperation and competition with other microbes, and the roles of these cooperations and competitions in transmission can inform novel preventative and therapeutic strategies. IMPORTANCE Transmission of pathogens from one host to another is an essential event in pathogenesis. Transmission is driven by factors intrinsic to the host and to the pathogen. In addition, transmission is altered by interactions of the pathogen with the commensal microbiota of the host and coinfecting pathogens. Recent insights into these interactions have shown both enhanced and reduced transmission efficiencies depending on the makeup of the polymicrobial community. This review will discuss polymicrobial interactions during shedding from the initial host, time in the environment, and acquisition by the new host.
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Background: In the United States, live attenuated influenza vaccine (LAIV) was initially approved for use in individuals aged 5-49 years in 2003, which was extended to individuals aged 2-49 years in 2007. At that time, a postlicensure commitment was made to describe the safety of LAIV within a cohort of eligible children aged 2-5 years. Methods: A prospective observational postmarketing study was conducted to evaluate the safety of LAIV. Rates of medically attended events (MAEs) and serious adverse events (SAEs) in eligible children aged 24-59 months receiving LAIV as part of routine care from October 2007 to March 2010 were compared with rates in a within-cohort self-control, as well as matched unvaccinated and matched trivalent inactivated influenza vaccine (TIV)-vaccinated controls. Children with asthma and other high-risk medical conditions before vaccination were excluded. All MAEs and SAEs through 42 days postvaccination and all hospitalizations and deaths through 6 months postvaccination were analyzed. Statistical significance was declared without multiplicity adjustment. Results: A total of 28,226 unique LAIV recipients were matched with similar numbers of TIV-vaccinated and unvaccinated children. Of 4696 MAE incidence rate comparisons, 83 (1.8%) were statistically significantly higher and 221 (4.7%) were statistically significantly lower in LAIV recipients versus controls. No pattern of MAE rate differences suggested a safety signal with LAIV. Asthma/wheezing MAEs were not statistically increased in LAIV recipients. No anaphylaxis events occurred within 3 days postvaccination. Rates of SAEs were similar between LAIV and control groups. Conclusions: Results of this postlicensure evaluation of LAIV safety in US children are consistent with preapproval clinical studies and Vaccine Adverse Event Reporting System reports, both of which demonstrated no significant increase in asthma/wheezing events or other adverse outcomes among eligible children aged 24-59 months who received LAIV.
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Bacterial coinfection complicated nearly all influenza deaths in the 1918 influenza pandemic and up to 34% of 2009 pandemic influenza A(H1N1) infections managed in intensive care units worldwide. More than 65,000 deaths attributable to influenza and pneumonia occur annually in the United States. Data from 683 critically ill patients with 2009 pandemic influenza A(H1N1) infection admitted to 35 intensive care units in the United States reveal that bacterial coinfection commonly occurs within the first 6 days of influenza infection, presents similarly to influenza infection occurring alone, and is associated with an increased risk of death. Pathogens that colonize the nasopharynx, including Staphylococcus aureus, Streptococcus pneumoniae, and Streptococcus pyogenes, are most commonly isolated. Complex viral, bacterial, and host factors contribute to the pathogenesis of coinfection. Reductions in morbidity and mortality are dependent on prevention with available vaccines as well as early diagnosis and treatment.
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Influenza A virus (IAV) predisposes individuals to secondary infections with the bacterium Streptococcus pneumoniae (the pneumococcus). Infections may manifest as pneumonia, sepsis, meningitis, or otitis media (OM). It remains controversial as to whether secondary pneumococcal disease is due to the induction of an aberrant immune response or IAV-induced immunosuppression. Moreover, as the majority of studies have been performed in the context of pneumococcal pneumonia, it remains unclear how far these findings can be extrapolated to other pneumococcal disease phenotypes such as OM. Here, we used an infant mouse model, human middle ear epithelial cells, and a series of reverse-engineered influenza viruses to investigate how IAV promotes bacterial OM. Our data suggest that the influenza virus HA facilitates disease by inducing a proinflammatory response in the middle ear cavity in a replication-dependent manner. Importantly, our findings suggest that it is the inflammatory response to IAV infection that mediates pneumococcal replication. This study thus provides the first evidence that inflammation drives pneumococcal replication in the middle ear cavity, which may have important implications for the treatment of pneumococcal OM.
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While the importance of changes in host biodiversity for disease risk continues to gain empirical support, the influence of natural variation in parasite diversity on epidemiological outcomes remains largely overlooked. Here, we combined field infection data from 2,191 amphibian hosts representing 158 parasite assemblages with mechanistic experiments to evaluate the influence of parasite richness on both parasite transmission and host fitness. Using a guild of larval trematode parasites (six species) and an amphibian host, our experiments contrasted the effects of parasite richness vs. composition, observed vs. randomized assemblages, and additive vs. replacement designs. Consistent with the dilution effect hypothesis extended to intrahost diversity, increases in parasite richness reduced overall infection success, including infections by the most virulent parasite. However, the effects of parasite richness on host growth and survival were context dependent; pathology increased when parasites were administered additively, even when the presence of the most pathogenic species was held constant, but decreased when added species replaced or reduced virulent species, emphasizing the importance of community composition and assembly. These results were similar or stronger when community structures were weighted by their observed frequencies in nature. The field data also revealed the highly nested structure of parasite assemblages, with virulent species generally occupying basal positions, suggesting that increases in parasite richness and antagonism in nature will decrease virulent infections. Our findings emphasize the importance of parasite biodiversity and coinfection in affecting epidemiological responses and highlight the value of integrating research on biodiversity and community ecology for understanding infectious diseases.
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Secondary bacterial infections due to Streptococcus pneumoniae and Staphylococcus aureus, responsible for excess morbidity and mortality during influenza epidemics, are often preceded by excess bacterial density within the upper respiratory tract. Influenza and pneumococcal vaccines reduce secondary infections within the lungs; however, their effects on upper respiratory tract carriage remain unknown. We demonstrate that a live attenuated influenza vaccine significantly reduces pneumococcal growth and duration of carriage during subsequent influenza to levels seen in influenza-naive controls. No benefit was seen after pneumococcal conjugate vaccine. Our results suggest that live attenuated influenza vaccines may significantly reduce bacterial disease during influenza epidemics.
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Influenza and respiratory bacterial synergy, a term often used when describing the relationship between these two highly divergent phyla of respiratory tract pathogens, has been known and documented for nearly a century (1-3).…
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: Viral upper respiratory tract infections have been described as an important factor in the development of otitis media (OM), although it is unclear whether they facilitate bacterial OM or can directly cause OM. To clarify the role of viral infections in OM, we compared the relative contribution of viruses and bacteria with the induction of inflammatory cytokine responses in the middle ear of children suffering from OM. : Children up to 5 years of age, with recurrent or chronic episodes of OM and scheduled for ventilation tube insertion were enrolled in a prospective study. Middle ear fluids (n = 116) were collected during surgery, and quantitative polymerase chain reaction was performed to detect bacterial and viral otopathogens, that is, Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis and 15 respiratory viruses. Finally, concentrations of the inflammatory mediators interleukin (IL)-1β, IL-6, IL-8, IL-10, IL-17a and tumor necrosis factor-α were determined. : Middle ear fluids were clustered into 4 groups, based on the detection of viruses (28%), bacteria (27%), both bacteria and viruses (27%) or no otopathogens (19%). Bacterial detection was associated with significantly elevated concentrations of cytokines compared with middle ear fluids without bacteria (P < 0.001 for all cytokines tested) in a bacterial load-dependent and species-dependent manner. In contrast, the presence of viruses was not associated with changes in cytokine values, and no synergistic effect between viral-bacterial coinfections was observed. : The presence of bacteria, but not viruses, is associated with an increased inflammatory response in the middle ear of children with recurrent or chronic OM.