INFECTION CONTROL AND HOSPITAL EPIDEMIOLOGY
494
May 2005
DURATION OF VIRUS SHEDDING AFTER
TRIVALENT INTRANASAL LIVE ATTENUATED
INFLUENZA VACCINATION IN ADULTS
Thomas R. Talbot, MD, MPH; Deidre D. Crocker, MD; Jody Peters, BS; Jennifer K. Doersam, BS; Mine R. Ikizler, BS;
Edith Sannella, MA; Peter F. Wright, MD; Kathryn M. Edwards, MD
ABSTRACT
OBJECTIVE: To characterize the probability and duration of viral shedding among adults given trivalent live attenuated influenza vaccine (LAIV).
DESIGN: Prospective sur veillance study.
METHODS: Nasal wash samples were collected from
adult volunteers at baseline and on days 3, 7, and 10 and between days 17 and 21 following intranasal LAIV vaccination. The
presence, titer, and identification of each specific strain of influenza virus shed were determined by standard methodology.
RESULTS: Twenty subjects received LAIV. No samples
were positive for influenza virus at baseline. After LAIV vaccination, influenza virus was recovered from 10 of 20 vaccinees on
day 3, from 1 of 18 vaccinees on day 7, and from none of the
samples on days 10 or 17 through 21. Vaccinees who shed vaccine virus were significantly younger than those who did not
(mean age, 26.4 vs 38.6 years; P < .01). Although the presence
of specific mucosal immunoglobulin A to influenza B was associated with significantly less shedding of influenza B after vaccination (P = .02), associations of shedding with other measures of
immunity were not detected.
CONCLUSION: The duration of shedding of vaccine virus after LAIV in adults is limited and may be associated with
an individual’s prior influenza vaccination histor y (Infect Control
Hosp Epidemiol 2005;26:494-500).
Vaccination with inactivated influenza virus has been
a cornerstone of efforts to reduce the burden of disease
due to influenza for many years.1 However, live attenuated
influenza vaccines targeted to strains of influenza A (monovalent H1N1 or H3N2 or bivalent H1N1/H3N2 vaccines),
influenza B (monovalent vaccines), or both (trivalent H1N1/
H3N2/B vaccines) have also been extensively evaluated as
an attractive method to prevent influenza infection.2 In 2003,
the trivalent vaccine FluMist (MedImmune Vaccines, Inc.,
Gaithersburg, MD) became the first live attenuated influenza vaccine approved for use in the United States. Administered intranasally, the trivalent live attenuated influenza
vaccine has been shown to be efficacious against influenza
infection, leading to a reduction in the incidence of cultureconfirmed influenza in children3,4 and a decrease in febrile
upper respiratory tract episodes and days of work lost due
to illness in adults.5
However, one concern with the use of the live attenuated influenza vaccines has been the potential risk of
secondar y transmission of the vaccine virus from recent
vaccinees to non-immune, high-risk individuals. As a result, FluMist was not recommended for use in immunocompromised individuals,6 and vaccine recipients were
strongly advised to avoid close contact with immunocompromised individuals for at least 21 days after vaccination.6 Due to this advisor y, some hospitals furloughed
employees who had been vaccinated with live attenuated
influenza vaccine from direct patient care duties for several weeks.7
Specific data regarding mucosal shedding of vaccine
virus following intranasal administration of live attenuated
influenza vaccine are limited. Studies in children noted that
vaccinees could shed vaccine virus for up to 21 days after
vaccination,6,8 but data from adults suggested the degree
and duration of shedding was substantially lower.9 Thus,
we conducted a prospective study of mucosal shedding of
vaccine virus at various intervals in adults following vaccination.
Dr. Talbot is from the Division of Infectious Diseases, Departments of Medicine and Preventive Medicine; Drs. Crocker, Wright, and Edwards, Ms.
Peters, Ms. Doersam, Ms. Ikizler, and Ms. Sannella are from the Division of Infectious Diseases, Department of Pediatrics; and Dr. Edwards is also from
the Pediatric Clinical Research Office, Vanderbilt University School of Medicine, Nashville, Tennessee.
Address reprint requests to Thomas R. Talbot, MD, MPH, A-2200 Medical Center North, 1161 21st Avenue South, Vanderbilt University Medical
Center, Nashville, TN 37232. tom.talbot@vanderbilt.edu
Supported in part by National Institute of Allergy and Infectious Diseases, Division of Microbiology and Infectious Diseases, contract #N01-AI25462 and General Clinical Research Center grant #M01-RR-00095.
The authors thank all of the members of the Vanderbilt Pediatric Clinical Research Office, especially Alice O’Shea, RN; Nancy Olsen, MD; the
Vanderbilt General Clinical Research Center; and colleagues at the National Institute of Allergy and Infectious Diseases, Division of Microbiology and
Infectious Diseases, particularly Linda Lambert, Mamodikoe Makhene, and Holli Hamilton, for their support of and guidance with this project. They also
thank Henry Hsu and Chiron Biocine for providing antigens for the humoral immunity studies.
Presented in part at the 14th Annual Meeting of the Society for Healthcare Epidemiology of America; April 18-20, 2004; Philadelphia, PA.
SHEDDING AFTER LIVE INFLUENZA VACCINE
Vol. 26 No. 5
495
TABLE 1
RECOVERY OF INFLUENZA VIRUS FROM NASAL WASH SPECIMENS IN ADULTS FOLLOWING INTRANASAL VACCINATION WITH THE
TRIVALENT LIVE ATTENUATED INFLUENZA VACCINE
No. of Subjects
Tested
No. of Specimens
Positive
Proportion of
Subjects With
Shedding
0
20
0
0%
0–16.8%
None
3
20
10
50%
27.2–72.8%
A H1N1 alone (1),
A H3N2 alone (1),
both A strains (1),
B alone (6),
all three strains (1)
7
18
1
5.5%
0.1–27.3%
B (1)
10
19
0
0%
0–17.6%
None
17–21
20
0
0%
0–16.8%
None
Post-Vaccination
Day
Exact CI95
Influenza
Strain(s) Shed
(No. of Subjects)
CI95 = 95% confidence interval.
METHODS
Study Subjects
Healthy adults 18 to 49 years old were recruited for
this study. Individuals with a current or past history of egg
or egg product allergy, allergic reactions to prior influenza
vaccinations, immunodeficiency due to underlying conditions or medications, a previous history of Guillain–Barré
syndrome, or any medical or psychiatric condition that precluded their compliance were excluded. Individuals with an
acute respiratory tract infection or immunocompromised
household contacts were also excluded. This study was reviewed and approved by the Vanderbilt University Institutional Review Board. All subjects provided written informed
consent.
Vaccination
All enrolled subjects were vaccinated with trivalent
live attenuated influenza vaccine (FluMist), administered
intranasally (0.25 mL in each nostril). Each 0.5-mL total
dose was formulated to contain 106.5–7.5 50% tissue culture infectious doses (TCID50) of each of the live attenuated influenza vaccine reassortants of the influenza strains
recommended by the U.S. Public Health Service for the
2003–2004 season: A/New Caledonia/20/99 (H1N1), A/
Panama/2007/99 (H3N2) (A/Moscow/10/00-like), and B/
Hong Kong/330/2001.
Patient Follow-Up and Specimen Collection
Subjects were obser ved during scheduled clinic
visits on days 3, 7, and 10 and between days 17 and 21 after vaccination. At each visit, subjects were assessed for
histor y of symptoms (including headache, sore throat,
rhinorrhea, myalgias, fatigue, cough, or fever). Nasal
wash specimens were collected from each subject immediately prior to vaccination and at each visit after vaccination. Specifically, 15 mL of lactated Ringer’s solution
was instilled into one nostril using a bulb syringe, and
the resulting nasal wash was collected from the opposite
FIGURE. Quantitative influenza B mucosal IgA at baseline in subjects vaccinated with live attenuated influenza vaccine, by presence or absence of
influenza B shedding after vaccination. Gray horizontal bars denote mean
baseline nasal wash influenza B IgA enzyme-linked immunosorbent assay
(ELISA) units corrected for total mucosal IgA in each group (as described
in reference 13). The comparison of mean baseline IgA values in shedders
versus non-shedders yielded a P value of .03.
nostril. The specimens were then inoculated into two
tissue culture tubes containing rhesus monkey kidney
cells. After incubation at room temperature for 1 hour to
allow for virus adsorption, the cultures were incubated
in serum-free Eagle/199 medium (Invitrogen, Carlsbad,
CA) supplemented with antibiotics. Cultures were read
at regular inter vals for cytopathic effect. On days 5 and
10 following inoculation, the rhesus monkey kidney cells
were hemadsorbed with 0.1% guinea pig red blood cells
in phosphate-buffered saline.
Specimens were considered positive for influenza
if they exhibited the presence of cytopathic effect or hemadsorption. Positive specimens were initially characterized as influenza A or B using indirect immunofluorescent assay analysis. Positive tissue culture harvests were
then plaqued in Madin–Darby canine kidney cells, and
the plaques were stained with immunoperoxidase using
MVB
18–40 y
26
Gruber et al.
King et al.25
Vesikari et al.8
a) 44
c) 47
Yes
HIV negative: 25
HIV positive: 24
98
19
20
b) 19
a) 15
32
b) Dose 6.6: 14
a) Dose 7.6: 29
b) 21
a) 38
HIV negative: 27
HIV positive: 28
20
No. of
Subjects
c) BVA
6.2
No
Yes
No
No
Yes
Yes
Yes
Yes
Yes
No
Only Studied
Seronegative
Subjects?
b) 45
a) MV H1N1A
6–18 mo
7.0
Not noted
7.8
6.2–7.2
7.5
7.5
6.6–7.6
7.1
7.0
6.5–7.5
Vaccine
Dose†
6, 11
3–5, 7–10,
28–35
Approximately
3 times/wk
for 3 wk
Daily days 1–5
Daily days 1–5
Daily days 1–4
Daily days 1–3
1, 2, 3, 4, 5, 6,
7, 28
Daily days 1–7
3–5, 7–10,
28–35
0, 3, 7, 10,
17–21
c) 55
b) 36
a) 16
HIV negative: 28
HIV positive: 13
80
11
10
21
Not measured
Not measured
7.6
0
Seropositive: 0
2.2
2.0–2.8
b) 0.6 ± 1.1
a) 1.9 ± 1.7
0.6 ± 0.2
b) 2.7 ± 1.4
a) 2.4 ± 1.1
b) 2.7–3.4
a) 3.8–4.2
Not measured
Not measured
Average
Duration of
Shedding (d)
4.0
Not noted
4
Not noted
Not noted
Not noted
Not noted
5
7
Last Day
Shedding
Detected
Equivocal: 14
Seronegative: 50
85
b) 26
a) 60
28
b) 79
a) 100
b) 95
a) 92
HIV negative: 0
HIV positive: 4
50
Subjects With
Viral Shedding
(%)
Not measured
2.7
1.2
0
1.7
1.7
2.5–3.0
b) 0.7 ± 0.3
a) 1.8 ± 1.4
0.8 ± 0.1
b) 0.7 ± 0.3
a) 0.9 ± 0.2
b) 0.5–1.6
a) 0.5–2.1
Peak Titer of
Shed Virus‡
INFECTION CONTROL AND HOSPITAL EPIDEMIOLOGY
b) MV H3N2A
TV
TV
1–7 y
8–36 mo
Mortiz et al.24
Studies of children
MVB
MV H3N2A
20–24 y
Not noted
Reeve et al.23§
b) MV H3N2A
a) MV H1N1A
18–40 y
b) TV
a) MVB or BVA
Sears et al.22
Clements et al.
18–35 y
Keitel et al.20
21
MVB (varying
doses‡)
18–40 y
Keitel et al.11§
TV
18–58 y
King et al.9
TV
19–49 y
Age Range
Current
Studies of adults
Study
Vaccine
Studied*
Days of
Specimen
Collection
PostVaccination
TABLE 2
SUMMARY OF STUDIES OF NASAL MUCOSAL SHEDDING OF INFLUENZA VIRUS AFTER VACCINATION WITH A LIVE ATTENUATED INFLUENZA VACCINE
496
May 2005
SHEDDING AFTER LIVE INFLUENZA VACCINE
Not measured
a) 3.6 ± 0.4
b) 3.5 ± 0.3
Not measured
a) 9.8 ± 0.9
b) 6.9 ± 0.9
b) 100
b) 10
Yes
a) 11
Daily days
1–15
a) 73
6
Wright et al.29
b) MV H3N2A
7.8
a) MV H1N1A
1.5–4.5 y
a) 31
b) 29
0, 3, 6
a) 29
b) 28
Yes
7.5–7.8
a) MV H1N1A
b) MV H3N2A
Seronegative: 91
1–7 y
Feldman et al.
28
497
monoclonal antibodies specific for the hemagglutinin of
the H1N1 influenza A, H3N2 influenza A, and B influenza viruses to further characterize the specific influenza
strains shed.10,11 Viral quantification was also performed
using serial 1:10 dilutions of aliquots of positive samples
that had been frozen directly in the lactated Ringer’s solution, as described previously.12
HIV = human immunodeficiency virus; TV = trivalent vaccine; MV H1N1A = monovalent H1N1 A vaccine; MV H3N2A = monovalent H3N2 A vaccine; MVB = monovalent B vaccine; BVA = bivalent H1N1 and H3N2 A vaccine.
*All vaccines studied were cold adapted.
†
Expressed as log10 50% tissue culture infectious doses (TCID50).
‡
Expressed as TCID50 log10/mL.
§
Only data following primary vaccination are included.

Seronegative studies conducted in ages 6 to 48 months only.
3.4
9.0
Not noted
0.9
MVB
Edwards et al.27
6 mo–9 y
7.2
No
24
Daily days
1–10
Seropositive: 54
10
5.0
Vol. 26 No. 5
Immunity Assays
Hemagglutination inhibition antibody titers to each
of the three influenza strains included in the live attenuated influenza vaccine were determined at baseline prior
to vaccination and between days 17 and 21 after vaccination, as described previously.13-16 Mucosal and serum IgA
antibodies were detected using a kinetic enzyme-linked
immunosorbent assay run in triplicate, as described previously.17 Those subjects with a hemagglutination inhibition
titer greater than 1:32 or the presence of detectable serum
or mucosal IgA prior to vaccination were noted and correlations with vaccine shedding were evaluated.18,19 Cell-mediated immunity assays were not performed.
Analysis
Comparisons between subjects with and without evidence of nasal shedding were made using Fisher’s exact
test for categorical data and the Mann–Whitney U test for
nonparametric continuous data. Analyses were conducted
using Stata software (version 7.0; StataCorp, College Station, TX).
RESULTS
Twenty subjects underwent live attenuated influenza vaccine vaccination. The mean age of the cohort was
31.6 years (standard deviation, ± 8.9 years; range, 19 to 49
years). The majority of the subjects were women (11 of 20;
55%) and white (18 of 20; 90%). All subjects completed the
study, although three missed one of the four scheduled follow-up visits (two subjects missed the day 7 visit and one
missed the day 10 visit).
Influenza was not detected in any of the nasal wash
specimens obtained at baseline (0 of 20; 95% confidence
interval, 0 to 16.8%; Table 1). In contrast, half of the specimens obtained on day 3 after vaccination (10 of 20; 95% confidence interval, 27.2% to 72.8%) were positive for influenza
virus. By day 7 after vaccination, the degree of mucosal
shedding had markedly declined, with only 1 of 18 nasal
wash specimens obtained (5.5%; 95% confidence interval,
0.1% to 27.3%) being culture positive for influenza virus. Influenza was not recovered from any of the subjects on days
10 (95% confidence interval, 0 to 17.6%) or 17 to 21 (95%
confidence interval, 0 to 16.8%) after vaccination. In total,
virus was recovered from 10 subjects; 1 subject shed on day
3 and day 7 after vaccination.
The specific influenza strain shed varied: one subject
shed influenza A H1N1 alone, one shed influenza A H3N2
alone, one shed both influenza A strains, six shed influenza
B alone, and one shed all three strains (Table 1). The subject who shed on multiple visits had influenza B isolated
498
INFECTION CONTROL AND HOSPITAL EPIDEMIOLOGY
on day 3 and day 7 after vaccination. Characterization of
preimmunization status in that subject revealed a baseline
hemagglutination inhibition titer to influenza B of 1:64, suggesting exposure to that strain through previous influenza
vaccination. However, she did not have any detectable mucosal IgA to influenza B at baseline. Quantitative titrations
were performed on all positive specimens. Only 1 of the 11
positive samples contained a detectable quantity of virus
with a peak titer of 5  100 plaque-forming units (PFU)/mL
(the lowest level of detection).
Shedding of vaccine virus was significantly associated with younger age (mean age, 26.4 years for those with
shedding vs 38.6 years for those without; P < .01). Although
those who did not shed vaccine virus were more likely to
report prior influenza vaccination (8 of 10) than those who
did (3 of 10), this did not achieve statistical significance
(P = .07). Absence of shedding of influenza B vaccine virus
was significantly associated with influenza B–specific mucosal IgA antibody at baseline (Figure; P = .03). However, no
associations were noted between the shedding of influenza
A vaccine virus and baseline mucosal IgA antibody titers
nor with any baseline strain-specific serum IgG or IgA antibody titers to any of the vaccine strains (data not shown).
DISCUSSION
The licensure of trivalent live attenuated influenza
vaccine has raised questions and concerns regarding the
use of this vaccine in some populations.7 In particular, the
potential for inadvertent secondary transmission of vaccine
virus led to an initial advisory in the package insert that individuals recently vaccinated with live attenuated influenza
vaccine avoid close contact with immunocompromised individuals for at least 21 days after vaccination,6 a duration
based on the recovery of influenza in one transmission
study from a single child 21 days after vaccination.8 However, data regarding mucosal shedding after live attenuated
influenza vaccine vaccination in adults have been limited,
with investigations rarely assessing shedding past the first
week after vaccination. The current study clearly demonstrated that the degree and duration of viral shedding in
adults markedly declined within the first week after live attenuated influenza vaccine vaccination and that shedding
could not be documented after that time.
Previous studies of nasal shedding of vaccine virus
after administration of live attenuated influenza vaccine are
summarized in Table 2.8,9,11,20-29 These studies vary regarding type of live attenuated influenza vaccine studied (monovalent or bivalent influenza A, monovalent influenza B, or
trivalent vaccines), study population (adults or children),
and number of specimens collected and the timing of that
collection after vaccination, impeding inter-trial comparisons. In general, the duration of shedding after vaccination
has been greater in children compared with adults (range
of duration in children, 5.0 to 9.8 days; range of duration in
adults, 0 to 4.0 days). In addition, prior serologic immunity
to the vaccine strains was associated with a reduced frequency and duration of viral shedding.24,27 In the current
study, the finding of an association between baseline muco-
May 2005
sal immunity to influenza B and the shedding of influenza
B after vaccination and the absence of baseline nasal IgA in
the one subject who shed virus on two visits further support the importance of prior mucosal immunity for shedding risk. Although no correlation between baseline serum
immunity and detection of shedding was found in the current investigation, such analyses may have been limited by
our small sample size.
Studies specifically investigating the degree of mucosal shedding after use of the newly licensed trivalent live
attenuated influenza vaccine are limited. In one study of human immunodeficiency virus–infected and healthy adults,
only one human immunodeficiency virus–positive subject
(1 of 28; 3.6%) shed influenza virus, detected on day 5 after vaccination. Shedding was not documented in any human immunodeficiency virus–negative subjects.9 A similar
study of children 1 to 7 years old noted a greater duration
(up to 10 days after vaccination) and frequency (13% to 28%)
of shedding.25 In contrast, all 11 healthy adults vaccinated
with the trivalent live attenuated influenza vaccine in another study shed virus during the first week after vaccination,
with an average of shedding of 3.4 days.11 In the largest investigation of shedding after trivalent live attenuated influenza vaccine vaccination, a randomized, placebo-controlled
trial conducted in Finland using a cohort of 197 day care
attendees, 78% (76 of 98) of vaccinated children shed virus
for an average of 7.6 days.8 Shedding was detected up to 21
days after vaccination in one child.
Even fewer studies have directly examined the transmission of vaccine-based virus from vaccinated subjects. In
the Finnish day care trial mentioned above, transmission of
a vaccine-type influenza B virus from a vaccinee to one of
99 placebo recipients was documented.6,8 Four other subjects receiving placebo shed influenza A, but these isolates
could not be characterized further to determine their origin
(wild type vs vaccine). In this study, the probability of acquiring vaccine virus after close contact with a single live
attenuated influenza vaccine vaccinee was estimated to be
between 0.58% and 2.4%.8 Despite the transmission to one
unvaccinated subject in the Finnish study, the transmitted
virus retained the attenuated phenotype and did not lead to
clinical disease.8 In another study in which 31 adult subjects
recently vaccinated with monovalent avian–human influenza A H3N2 live attenuated influenza virus were housed for
7 to 9 days after vaccination with 6 placebo recipients, no
episodes of transmission were detected.30
A larger issue regarding secondary transmission of
vaccine-based influenza virus is whether such transmission
is harmful to the exposed individual. Modification of the
vaccine virus to restrict replication above 37°C (similar to
the temperature of the lower airways) as well as attenuation
to non-virulent viral reassortants markedly restricts the potential for clinical infection following inadvertent transmission of vaccine virus.6 Although reversion of the vaccine
virus to a wild genotype is a concern, an investigation of
individuals vaccinated with a trivalent live attenuated influenza vaccine who exhibited mucosal shedding after vaccination found that the shed viruses were genotypically stable,
Vol. 26 No. 5
SHEDDING AFTER LIVE INFLUENZA VACCINE
retaining the attenuated vaccine genotype in all instances.31
Transmission of vaccine virus may, in fact, be beneficial, as
transmission of the attenuated strain of influenza may result
in a de facto vaccination of the exposed subject, providing
protection from circulating wild-type influenza.
Effective secondary transmission of vaccine virus
from vaccinees also requires a significant quantity of virus
to be shed from the nasal mucosa. Based on prior investigations with live attenuated influenza vaccine, the dose of
vaccine virus required to effectively infect an individual is
approximately 105 to 107 PFU/mL.21,22,32 From our investigation and from other studies of mucosal shedding after
live attenuated influenza vaccine use, the quantity of virus
shed after live attenuated influenza vaccine vaccination was
exponentially lower (from 100 to 102 PFU/mL) and likely
would not have been able to infect other individuals. In our
study, although 50% of subjects shed at day 3 after vaccination, only one subject shed virus above the lower limit of
quantification. Studies further evaluating the magnitude of
shedding after live attenuated influenza vaccine vaccination
are now needed to assist with assessment of the risk of secondary transmission of the vaccine virus.
Healthcare workers have been among those most
affected by the recommendations restricting contact with
immunocompromised individuals following live attenuated
influenza vaccine vaccination. Following live attenuated influenza vaccine licensure, some facilities instituted policies
requiring a 21-day furlough of any healthcare worker vaccinated with live attenuated influenza vaccine.7 Such restrictions created staffing shortages during influenza season, a
time of year typically associated with high patient volume.
Some vaccine experts speculate that intranasal live attenuated influenza vaccine is a more acceptable method of delivery than the intramuscular vaccine, an idea supported by
recent investigations.33,34 Although annual influenza vaccination is recommended for all healthcare workers to reduce
work absenteeism and prevent the nosocomial transmission of influenza, only 37% of surveyed healthcare workers
reported having received influenza vaccination in 2001.1
Expanding the use of live attenuated influenza vaccine to
healthcare workers and reducing or removing post-vaccination work restrictions may improve vaccine acceptance and
coverage, limiting the risk of nosocomial transmission of
wild influenza.
The licensure of the live attenuated influenza vaccine provides an additional tool in the armamentarium
against influenza infection. As with other live viral vaccines, secondar y transmission to those at risk for clinical
infection from transmitted virus is a concern; however,
previous guidelines on restriction of contacts after live
attenuated influenza vaccine vaccination were too restrictive. In this investigation, we found that mucosal shedding in 20 adults vaccinated with the trivalent live attenuated influenza vaccine was markedly diminished within 1
week after vaccination.
Although the sample size of this investigation was
small, such results have provided a more detailed description of the duration of shedding after vaccination,
499
leading to a modification of recommendations for live
attenuated influenza vaccine use in healthcare workers
that reduces the period of separation from patients after
live attenuated influenza vaccine vaccination to at most 7
days.35 In addition, the amount of virus shed was much
lower than the estimated dose needed for effective infection with vaccine virus in immunocompetent individuals, suggesting that the risk of infection from secondar y
transmission of vaccine virus is low. Additional studies
refining estimates of the degree of mucosal shedding after live attenuated influenza vaccine vaccination and the
risk of secondar y transmission of the attenuated vaccine
virus to immunocompromised individuals are needed to
further explore the necessity for healthcare workers to
be restricted from patients after live attenuated influenza
vaccine vaccination.
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