JVI Accepts, published online ahead of print on 12 March 2014 J. Virol. doi:10.1128/JVI.03101-13 Copyright © 2014, American Society for Microbiology. All Rights Reserved.
1
Mucosal immunization with a candidate universal influenza vaccine
2
reduces virus transmission in a mouse model
3 4
Graeme E. Price1, Chia-Yun Lo1, Julia A. Misplon1 and Suzanne L. Epstein1
5 6
1
7
Food and Drug Administration, Rockville, Maryland, USA.
Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research,
8 9
Address correspondence to Graeme E. Price, [email protected]
10 11
Running Title:
12
NP+M2 vaccine reduces influenza transmission in mice
13
1
14
Word Counts:
15
Running Title (with spaces):
52 characters
17
Abstract:
208 words
18
Main Text:
5179 words
19
References:
64
20
Display Items:
4 tables, 4 figures
21
Supplementary Material:
None
16
22
2
23
ABSTRACT
24
Pandemic influenza is a major public health concern, but conventional strain-matched vaccines
25
are unavailable early in a pandemic. Candidate “universal” vaccines targeting viral antigens
26
nucleoprotein (NP) and matrix 2 (M2), which are conserved among all influenza A virus strains
27
and subtypes, could be manufactured in advance for use at the onset of a pandemic. These
28
vaccines do not prevent infection, but can reduce disease severity, deaths, and virus titers in the
29
respiratory tract. We hypothesized that such immunization may reduce virus transmission from
30
vaccinated, infected animals. To investigate this hypothesis, we studied mouse models for direct
31
contact and airborne transmission of H1N1 and H3N2 influenza viruses. We established
32
conditions under which virus transmission occurs, and showed that transmission efficiency is
33
determined in part at the level of host susceptibility to infection. Our findings indicate that virus
34
transmission between mice has both airborne and direct contact components. Finally, we
35
demonstrated that immunization with recombinant adenovirus vectors expressing NP and M2
36
significantly reduced transmission of virus to co-housed, unimmunized mice in comparison to
37
controls. These findings have broad implications for the impact of conserved antigen vaccines,
38
not only in protecting the vaccinated individual but also in protecting others by limiting
39
influenza virus transmission and potentially reducing the size of epidemics.
40 41
IMPORTANCE
42
Using a mouse model of influenza A virus transmission, we demonstrate that a candidate
43
“universal” influenza vaccine both protects vaccinated animals from lethal infection and reduces
44
transmission of virus from vaccinated to non-vaccinated mice. This vaccine induces immunity
45
against proteins conserved between all known influenza A virus strains and subtypes, so it could 3
46
be used early in a pandemic before conventional strain-matched vaccines are available and
47
could potentially reduce spread of infection in the community.
4
48
INTRODUCTION
49
The possibility that newly emergent influenza viruses may become efficiently transmissible
50
among humans is a major public health concern. Thus, studies of influenza virus transmission
51
and development of medical countermeasures, including antiviral drugs and vaccines, are of
52
high priority. Current influenza vaccines generate strain-specific antibodies against the highly
53
variable viral hemagglutinin and neuraminidase. However, production of strain-matched
54
vaccines requires several months, limiting their utility against new, rapidly spreading viruses. In
55
contrast, cross-protective vaccines targeting conserved antigens such as nucleoprotein (NP) and
56
matrix 2 (M2) do not rely on strain identification and could be used off the shelf early in an
57
outbreak. They protect animals from lethal challenge with a broad range of influenza A virus
58
strains and subtypes, including H1N1, H3N2 and H5N1 (1-8). While these vaccines do not
59
prevent infection, they significantly reduce morbidity, mortality, and viral titers in the
60
respiratory tract (6,7). A potential concern about infection-permissive vaccines is that although
61
they would protect the vaccinee, they might not control transmission to others.
62 63
A recent epidemiological modeling study suggests that reducing virus transmission could
64
dramatically reduce the size of influenza pandemics or seasonal epidemics, and slow the
65
antigenic evolution of seasonal influenza viruses (9). Thus a critical question to test empirically is
66
whether cross-protective vaccines provide that reduction in transmission.
67 68
Animal models for influenza virus transmission include ferrets and guinea pigs (10-13), which
69
efficiently transmit infection to either co-housed animals (direct contact) or across a perforated 5
70
barrier (either aerosol or respiratory droplet transmission). While experiments in these species
71
have elucidated viral and environmental factors affecting host-range and transmission (14-17),
72
studies have been limited by space requirements, expense, and lack of reagents for
73
immunological studies. For practical reasons it is generally not possible to use sufficient
74
numbers of ferrets or guinea pigs for statistical analyses in transmission experiments (18).
75 76
In contrast, mice have been critical for defining host defenses against influenza viruses, and
77
many immunological reagents are available. A mouse influenza transmission system was
78
developed by Schulman and Kilbourne 50 years ago (19-24). Mice have since been seldom used
79
to study influenza virus transmission, although a recent report described contact-dependent
80
transmission in mice (25). Here we have established a model allowing transmission experiments
81
with H1N1 and H3N2 influenza viruses, and accommodating group sizes that permit statistical
82
analyses. We demonstrate that transmission efficiency is in part dependent on the susceptibility
83
of contact animals, and show that limiting exposure to the airborne route alone results in a
84
significantly lower frequency of transmission than when mice are co-housed. We then use this
85
model to demonstrate that immunization with a combination of recombinant adenovirus
86
vectors expressing NP and M2, a candidate universal influenza vaccine, reduces viral
87
transmission from vaccinated to co-housed, unvaccinated animals in the absence of neutralizing
88
antibodies.
6
89
MATERIALS AND METHODS
90
Experimental animals and animal housing. CFW mice [Crl:CFW(SW)] were obtained from
91
Charles River Laboratories, DBA/2J mice were purchased from the Jackson Laboratory (Bar
92
Harbor, ME) and BALB/cAnNCR mice were purchased from the National Cancer Institute
93
(Frederick, MD). All animals were females aged 8-15 weeks at time of use. For experiments using
94
mechanically ventilated housing, mice were housed 5 per cage in standard shoebox cages with
95
microisolator lids (191 × 292 × 127 mm), in a Smart Bio-Pak rack (model SB4100, Allentown
96
Caging Co., Allentown, NJ) with water supplied by an automated system and positive pressure
97
ventilation rate set at a factory-defined 60 air changes per hour. For non-ventilated housing,
98
mice were housed in the same sized shoebox cages with water bottles and microisolator lids on
99
an open-sided shelf with no exhaust system, just room ventilation.
100 101
For experiments examining airborne transmission (defined here as transmission by either
102
aerosols or droplets), contact and donor mice were separated using dividers manufactured to
103
our specifications by Ancare (Belmore, NY), and further modified to ensure a tight fit to the sides
104
of the cage (D.P. Cook Construction, Gaithersburg, MD). Each divider consisted of two
105
perforated stainless steel sheets held 5 mm apart by spacers, with angled flanges at either end
106
of the divider to prevent direct contact between mice at the corners. Each sheet had 532
107
perforations 3.5 mm in diameter and 71 perforations 7 mm in diameter allowing air to exchange
108
between the two compartments. The 7 mm diameter perforations were arranged so as to be
109
inaccessible to mice when the cages were assembled. The divider runs the length of a standard
110
mouse, separating it into two compartments of ~90 × 295 mm. Dividers were held in place with
7
111
a wire grid (Ancare) providing water bottles and feed for each of the compartments. Cages were
112
topped with standard microisolator lids and further secured with elastic bands.
113 114
As a humane end point, mice losing >25% of body weight were euthanized. All experiments used
115
standard mouse bedding (Paperchip, Shepherd Specialty Papers, Watertown, TN), which was
116
not changed during the transmission period. Food and water were provided ad libitum. All
117
animal experiments were conducted under temperature- and humidity-controlled (mean daily
118
values: 22.3 ± 0.1 °C, 51.02 ± 0.1 % humidity) ABSL-2 conditions in animal facilities accredited by
119
the Association for Assessment and Accreditation of Laboratory Animal Care International. All
120
animal protocols and procedures were approved by Institutional Animal Care and Use
121
Committees at the Center for Biologics Evaluation and Research
122 123
Influenza viruses. Mouse-adapted A/FM/1/47-ma (H1N1) [A/FM] originally provided by Earl
124
Brown (University of Ottawa, Canada) (26), the mouse-adapted A/Philippines/2/82 × A/PR/8/34
125
(H3N2) reassortant [X-79] (27), A/PR/8/34 (H1N1) [PR8], and A/Udorn/307/72 (H3N2) [A/Udorn]
126
were prepared as previously described (28). Mice were infected intranasally (i.n.) under
127
isoflurane anesthesia in a 50 µl volume with a dose of 104 50% Tissue Culture Infectious Doses
128
(TCID50), unless stated otherwise.
129 130
rAd vaccines. Production and titration of recombinant adenovirus (rAd) vectors expressing
131
influenza A nucleoprotein (NP), influenza B nucleoprotein (B/NP) or consensus M2 has been 8
132
described previously (3,4). For immunization experiments, a dose of 5×109 particles each of NP-
133
rAd and M2-rAd, or 1×1010 particles of B/NP-rAd in a 50 µl volume were given i.n. under
134
isoflurane anesthesia.
135 136
Lung sampling, BAL, and nasal washing. Mice were euthanized with ketamine/xylazine. Nasal
137
washes were obtained by inserting an intravenous catheter (24 gauge × ¾”, Terumo Medical
138
Corp., Elkton MD) into the nasopharynx via a slit in the trachea and injecting PBS into the nasal
139
cavity until 200 µl of flow-through was collected from the nares. Lungs were then harvested. To
140
prevent cross-contamination at the harvest stage, dissections were performed on contact
141
animals before donor animals in each cage. Instruments were cleaned and sterilized by
142
immersion of contact surfaces in 70% ethanol between each animal. Mucosal sampling for lung
143
cells and bronchoalvelolar lavage (BAL) was otherwise as described (6).
144 145
Virologic analyses. Virus titers in lung tissues and nasal washes were determined by TCID50
146
assay on MDCK cells (29), read out by hemagglutination with 0.5% chicken erythrocytes as
147
described (6). Titers were calculated using the method of moving averages and Weil’s tables
148
(30,31). Assay limits of detection were 102.68 TCID50/organ and 102.19 TCID50/ml for lung and
149
nasal washes, respectively. Contact animals were considered positive for virus transmission if
150
virus was detected by TCID50 assay in either lung homogenate, nasal wash, or both.
151
9
152
50% Mouse Infectious Dose (MID50) determination. Groups of 5 mice were intranasally
153
inoculated with each dilution of virus in a 50 µl volume under isoflurane anesthesia and
154
euthanized 3 days later for collection of lung and nasal wash samples. Animals were considered
155
infected if virus was detected by TCID50 assay in either lung homogenate, nasal wash, or both.
156
MID50 with 95% confidence intervals were calculated using the method of moving averages as
157
described above.
158 159
Immunologic analyses. ELISAs for antibody against the M2 ectodomain (M2e) and recombinant
160
NP have been previously described (5,6). Quantitation of interferon-γ (IFN-γ) secreting cells by
161
ELISPOT was performed as described (4), except that peptide pools covering the M2 and NP
162
sequences of PR8 were used. The M2 peptide pool was a series of 13-15-mer peptides with 11
163
amino acid overlap. The NP peptides (15-17-mer with 11-13 amino acid overlap) were divided
164
into 3 pools of 27-29 peptides each. Peptides were synthesized by Genscript (Piscataway, NJ).
165 166
Hemagglutination inhibition assay. Sera were incubated overnight at 37ºC with 3 volumes of
167
receptor-destroying enzyme (RDE; Denka Seiken Co. Ltd, Tokyo, Japan), and heat inactivated at
168
56ºC for 30 minutes before addition of 6 volumes 0.85% saline. Two-fold dilutions of sera were
169
made in microtiter plates for a final volume of 25 μl and an equal volume of A/FM diluted to 4
170
HAU/25 µl was added to each well and mixed thoroughly. One hour later, 50 µl of 0.5% chicken
171
erythrocytes were added and mixed well. Plates were scored after 45 min, and
172
hemagglutination inhibition (HAI) titers were calculated. Polyclonal anti-A/Fort Monmouth/1/47
10
173
(H1N1) antiserum (NR-3099) obtained through the NIAID, NIH Biodefense and Emerging
174
Infections Research Resources Repository was used as a positive control.
175 176
Microneutralization Assay. Serial two-fold dilutions of RDE treated sera in PBS were made in a
177
96 well U-bottom plate. An equal volume of A/FM at 1×104 TCID50/ml was added to each well,
178
gently mixed, and incubated at 37 ºC. After 2 hours, 20 µl of the virus-antibody mixture was
179
transferred to plates of MDCK cells plus virus growth medium, so as to give 100 TCID50 per well.
180
Plates were then incubated for 48 hours and read out by hemagglutination with 0.5% chicken
181
erythrocytes.
182 183
Statistical Analyses. SigmaPlot 12 (Systat Software, Point Richmond, CA) was used for statistical
184
analyses as follows: Survival analysis used the log rank method, virus titer comparisons used the
185
t- test, transmission rate comparisons used the Chi-Square test.
11
186
RESULTS
187
Establishment of transmission system. In preliminary experiments, CFW outbred mice (as used
188
by Schulman and Kilbourne (21)) were infected with a virulent mouse-adapted influenza H1N1
189
strain, A/FM. Twenty-four hours later, 2 infected donor mice were placed in the same cage as 3
190
uninfected contact animals. 3 days later, all mice were euthanized and lungs and nasal washes
191
collected for virus titration. During the contact period, donors but not contacts progressively
192
lost weight. While all donors had virus recoverable from lungs and most from nasal wash, A/FM
193
was detectable in only 2/18 (11%) contacts when cages were held on a mechanically ventilated
194
cage rack (data not shown), but this rose to 6/18 (33%) of contacts when cages were housed on
195
an open-sided shelf with room ventilation only (Table 1). There was no obvious correlation in
196
either experiment between individual donor lung or nasal wash virus titers on day 4 and the
197
number of co-housed contacts that became infected. This suggests that virus shedding from
198
donors earlier than day 4 is more important for virus transmission, and/or that other factors are
199
involved. Because of the higher rate of transmission observed, all further experiments used
200
cages housed under room ventilation conditions.
201 202
In a further experiment using CFW mice, contacts were monitored for weight loss and survival.
203
All A/FM infected donors died between days 3 and 5 (Fig. 1). Significant weight loss was seen in
204
2 contacts (both in the same cage), but all survived. One month post contact, 4/18 contacts
205
(including the two that lost weight) had A/FM-specific antibody responses with HAI titers of 160-
206
640. Using seroconversion as a marker for transmission, only half the transmitted infections
207
caused clinically apparent disease and insufficient virus was transmitted to initiate a lethal
12
208
infection. This experiment also suggests that secondary transmission (i.e. between infected and
209
uninfected contacts) is either inefficient or absent in the case of A/FM.
210 211
Effect of mouse and virus strain. As different mouse strains vary in their susceptibility to
212
influenza viruses, we extended our studies to investigate direct contact transmission in two
213
additional mouse strains: DBA/2J, which are highly susceptible to lethal influenza virus infection,
214
and BALB/c, which are relatively resistant (32,33). Transmission experiments in these two mouse
215
strains using 104 TCID50 of A/FM were conducted as described above. Transmission of A/FM
216
between DBA/2J mice occurred to a similar extent as seen between CFW mice, but transmission
217
between BALB/c mice was not seen (Table 1).
218 219
To investigate whether the lack of transmission of A/FM in BALB/c mice was due to decreased
220
susceptibility to infection or because of an impaired ability of BALB/c mice to transmit the virus,
221
a “criss-cross” transmission experiment was performed, in which BALB/c mice were used as
222
donors with CFW mice as recipients, and vice versa. Limited transmission of A/FM was seen for
223
CFW donors with BALB/c contacts, but significantly higher levels of transmission were seen for
224
BALB/c donors with CFW contacts (Table 1). A similar experiment examining transmission
225
between the two susceptible stains, DBA/2J and CFW, showed similar rates of transmission with
226
both donor/contact combinations (Table 1). Taken together, these findings suggest that
227
establishment of infection in contact animals is at least in part dependent on the relative
228
sensitivity of the contact animal. However, donor traits also seem to be involved, as
229
transmission of A/FM is more efficient from BALB/c donors to CFW contacts than from CFW 13
230
donors to CFW contacts. Interestingly, virus titers in lungs and nasal washes of infected BALB/c
231
and CFW mice were very similar over the first 4 days of infection (data not shown).
232 233
Limited transmission (2/18 contacts becoming infected; data not shown) between BALB/c mice
234
occurred for two different mouse-adapted viruses (PR8 and X-79). These viruses were also
235
inefficiently transmitted between CFW mice (1/18 contacts becoming infected; data not shown).
236 237
Taken in combination, our data suggest that transmission efficiency is multifactorial and
238
dependent on sensitivity of contacts, donor characteristics, and biological properties of the
239
specific virus strain.
240 241
Mode of transmission. To investigate whether transmission was via a direct contact-dependent
242
route or via the airborne route (by either droplet or aerosol), we developed a removable
243
perforated divider running the length of the cage, as described in Materials and Methods. This
244
prevents direct contact between donor and contact mice while permitting air exchange between
245
the two cage compartments. Using CFW mice and the non-mouse adapted strain A/Udorn,
246
which has been reported to efficiently transmit between BALB/c mice by direct contact but not
247
via the airborne route (25), we found that direct contact resulted in efficient transmission (Table
248
2, Experiment 1). However, when donor and contact mice were physically separated by the
249
perforated divider, a significantly lower proportion of contacts became infected (approximately
250
6-fold, P
1
Mucosal immunization with a candidate universal influenza vaccine
2
reduces virus transmission in a mouse model
3 4
Graeme E. Price1, Chia-Yun Lo1, Julia A. Misplon1 and Suzanne L. Epstein1
5 6
1
7
Food and Drug Administration, Rockville, Maryland, USA.
Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research,
8 9
Address correspondence to Graeme E. Price, [email protected]
10 11
Running Title:
12
NP+M2 vaccine reduces influenza transmission in mice
13
1
14
Word Counts:
15
Running Title (with spaces):
52 characters
17
Abstract:
208 words
18
Main Text:
5179 words
19
References:
64
20
Display Items:
4 tables, 4 figures
21
Supplementary Material:
None
16
22
2
23
ABSTRACT
24
Pandemic influenza is a major public health concern, but conventional strain-matched vaccines
25
are unavailable early in a pandemic. Candidate “universal” vaccines targeting viral antigens
26
nucleoprotein (NP) and matrix 2 (M2), which are conserved among all influenza A virus strains
27
and subtypes, could be manufactured in advance for use at the onset of a pandemic. These
28
vaccines do not prevent infection, but can reduce disease severity, deaths, and virus titers in the
29
respiratory tract. We hypothesized that such immunization may reduce virus transmission from
30
vaccinated, infected animals. To investigate this hypothesis, we studied mouse models for direct
31
contact and airborne transmission of H1N1 and H3N2 influenza viruses. We established
32
conditions under which virus transmission occurs, and showed that transmission efficiency is
33
determined in part at the level of host susceptibility to infection. Our findings indicate that virus
34
transmission between mice has both airborne and direct contact components. Finally, we
35
demonstrated that immunization with recombinant adenovirus vectors expressing NP and M2
36
significantly reduced transmission of virus to co-housed, unimmunized mice in comparison to
37
controls. These findings have broad implications for the impact of conserved antigen vaccines,
38
not only in protecting the vaccinated individual but also in protecting others by limiting
39
influenza virus transmission and potentially reducing the size of epidemics.
40 41
IMPORTANCE
42
Using a mouse model of influenza A virus transmission, we demonstrate that a candidate
43
“universal” influenza vaccine both protects vaccinated animals from lethal infection and reduces
44
transmission of virus from vaccinated to non-vaccinated mice. This vaccine induces immunity
45
against proteins conserved between all known influenza A virus strains and subtypes, so it could 3
46
be used early in a pandemic before conventional strain-matched vaccines are available and
47
could potentially reduce spread of infection in the community.
4
48
INTRODUCTION
49
The possibility that newly emergent influenza viruses may become efficiently transmissible
50
among humans is a major public health concern. Thus, studies of influenza virus transmission
51
and development of medical countermeasures, including antiviral drugs and vaccines, are of
52
high priority. Current influenza vaccines generate strain-specific antibodies against the highly
53
variable viral hemagglutinin and neuraminidase. However, production of strain-matched
54
vaccines requires several months, limiting their utility against new, rapidly spreading viruses. In
55
contrast, cross-protective vaccines targeting conserved antigens such as nucleoprotein (NP) and
56
matrix 2 (M2) do not rely on strain identification and could be used off the shelf early in an
57
outbreak. They protect animals from lethal challenge with a broad range of influenza A virus
58
strains and subtypes, including H1N1, H3N2 and H5N1 (1-8). While these vaccines do not
59
prevent infection, they significantly reduce morbidity, mortality, and viral titers in the
60
respiratory tract (6,7). A potential concern about infection-permissive vaccines is that although
61
they would protect the vaccinee, they might not control transmission to others.
62 63
A recent epidemiological modeling study suggests that reducing virus transmission could
64
dramatically reduce the size of influenza pandemics or seasonal epidemics, and slow the
65
antigenic evolution of seasonal influenza viruses (9). Thus a critical question to test empirically is
66
whether cross-protective vaccines provide that reduction in transmission.
67 68
Animal models for influenza virus transmission include ferrets and guinea pigs (10-13), which
69
efficiently transmit infection to either co-housed animals (direct contact) or across a perforated 5
70
barrier (either aerosol or respiratory droplet transmission). While experiments in these species
71
have elucidated viral and environmental factors affecting host-range and transmission (14-17),
72
studies have been limited by space requirements, expense, and lack of reagents for
73
immunological studies. For practical reasons it is generally not possible to use sufficient
74
numbers of ferrets or guinea pigs for statistical analyses in transmission experiments (18).
75 76
In contrast, mice have been critical for defining host defenses against influenza viruses, and
77
many immunological reagents are available. A mouse influenza transmission system was
78
developed by Schulman and Kilbourne 50 years ago (19-24). Mice have since been seldom used
79
to study influenza virus transmission, although a recent report described contact-dependent
80
transmission in mice (25). Here we have established a model allowing transmission experiments
81
with H1N1 and H3N2 influenza viruses, and accommodating group sizes that permit statistical
82
analyses. We demonstrate that transmission efficiency is in part dependent on the susceptibility
83
of contact animals, and show that limiting exposure to the airborne route alone results in a
84
significantly lower frequency of transmission than when mice are co-housed. We then use this
85
model to demonstrate that immunization with a combination of recombinant adenovirus
86
vectors expressing NP and M2, a candidate universal influenza vaccine, reduces viral
87
transmission from vaccinated to co-housed, unvaccinated animals in the absence of neutralizing
88
antibodies.
6
89
MATERIALS AND METHODS
90
Experimental animals and animal housing. CFW mice [Crl:CFW(SW)] were obtained from
91
Charles River Laboratories, DBA/2J mice were purchased from the Jackson Laboratory (Bar
92
Harbor, ME) and BALB/cAnNCR mice were purchased from the National Cancer Institute
93
(Frederick, MD). All animals were females aged 8-15 weeks at time of use. For experiments using
94
mechanically ventilated housing, mice were housed 5 per cage in standard shoebox cages with
95
microisolator lids (191 × 292 × 127 mm), in a Smart Bio-Pak rack (model SB4100, Allentown
96
Caging Co., Allentown, NJ) with water supplied by an automated system and positive pressure
97
ventilation rate set at a factory-defined 60 air changes per hour. For non-ventilated housing,
98
mice were housed in the same sized shoebox cages with water bottles and microisolator lids on
99
an open-sided shelf with no exhaust system, just room ventilation.
100 101
For experiments examining airborne transmission (defined here as transmission by either
102
aerosols or droplets), contact and donor mice were separated using dividers manufactured to
103
our specifications by Ancare (Belmore, NY), and further modified to ensure a tight fit to the sides
104
of the cage (D.P. Cook Construction, Gaithersburg, MD). Each divider consisted of two
105
perforated stainless steel sheets held 5 mm apart by spacers, with angled flanges at either end
106
of the divider to prevent direct contact between mice at the corners. Each sheet had 532
107
perforations 3.5 mm in diameter and 71 perforations 7 mm in diameter allowing air to exchange
108
between the two compartments. The 7 mm diameter perforations were arranged so as to be
109
inaccessible to mice when the cages were assembled. The divider runs the length of a standard
110
mouse, separating it into two compartments of ~90 × 295 mm. Dividers were held in place with
7
111
a wire grid (Ancare) providing water bottles and feed for each of the compartments. Cages were
112
topped with standard microisolator lids and further secured with elastic bands.
113 114
As a humane end point, mice losing >25% of body weight were euthanized. All experiments used
115
standard mouse bedding (Paperchip, Shepherd Specialty Papers, Watertown, TN), which was
116
not changed during the transmission period. Food and water were provided ad libitum. All
117
animal experiments were conducted under temperature- and humidity-controlled (mean daily
118
values: 22.3 ± 0.1 °C, 51.02 ± 0.1 % humidity) ABSL-2 conditions in animal facilities accredited by
119
the Association for Assessment and Accreditation of Laboratory Animal Care International. All
120
animal protocols and procedures were approved by Institutional Animal Care and Use
121
Committees at the Center for Biologics Evaluation and Research
122 123
Influenza viruses. Mouse-adapted A/FM/1/47-ma (H1N1) [A/FM] originally provided by Earl
124
Brown (University of Ottawa, Canada) (26), the mouse-adapted A/Philippines/2/82 × A/PR/8/34
125
(H3N2) reassortant [X-79] (27), A/PR/8/34 (H1N1) [PR8], and A/Udorn/307/72 (H3N2) [A/Udorn]
126
were prepared as previously described (28). Mice were infected intranasally (i.n.) under
127
isoflurane anesthesia in a 50 µl volume with a dose of 104 50% Tissue Culture Infectious Doses
128
(TCID50), unless stated otherwise.
129 130
rAd vaccines. Production and titration of recombinant adenovirus (rAd) vectors expressing
131
influenza A nucleoprotein (NP), influenza B nucleoprotein (B/NP) or consensus M2 has been 8
132
described previously (3,4). For immunization experiments, a dose of 5×109 particles each of NP-
133
rAd and M2-rAd, or 1×1010 particles of B/NP-rAd in a 50 µl volume were given i.n. under
134
isoflurane anesthesia.
135 136
Lung sampling, BAL, and nasal washing. Mice were euthanized with ketamine/xylazine. Nasal
137
washes were obtained by inserting an intravenous catheter (24 gauge × ¾”, Terumo Medical
138
Corp., Elkton MD) into the nasopharynx via a slit in the trachea and injecting PBS into the nasal
139
cavity until 200 µl of flow-through was collected from the nares. Lungs were then harvested. To
140
prevent cross-contamination at the harvest stage, dissections were performed on contact
141
animals before donor animals in each cage. Instruments were cleaned and sterilized by
142
immersion of contact surfaces in 70% ethanol between each animal. Mucosal sampling for lung
143
cells and bronchoalvelolar lavage (BAL) was otherwise as described (6).
144 145
Virologic analyses. Virus titers in lung tissues and nasal washes were determined by TCID50
146
assay on MDCK cells (29), read out by hemagglutination with 0.5% chicken erythrocytes as
147
described (6). Titers were calculated using the method of moving averages and Weil’s tables
148
(30,31). Assay limits of detection were 102.68 TCID50/organ and 102.19 TCID50/ml for lung and
149
nasal washes, respectively. Contact animals were considered positive for virus transmission if
150
virus was detected by TCID50 assay in either lung homogenate, nasal wash, or both.
151
9
152
50% Mouse Infectious Dose (MID50) determination. Groups of 5 mice were intranasally
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inoculated with each dilution of virus in a 50 µl volume under isoflurane anesthesia and
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euthanized 3 days later for collection of lung and nasal wash samples. Animals were considered
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infected if virus was detected by TCID50 assay in either lung homogenate, nasal wash, or both.
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MID50 with 95% confidence intervals were calculated using the method of moving averages as
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described above.
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Immunologic analyses. ELISAs for antibody against the M2 ectodomain (M2e) and recombinant
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NP have been previously described (5,6). Quantitation of interferon-γ (IFN-γ) secreting cells by
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ELISPOT was performed as described (4), except that peptide pools covering the M2 and NP
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sequences of PR8 were used. The M2 peptide pool was a series of 13-15-mer peptides with 11
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amino acid overlap. The NP peptides (15-17-mer with 11-13 amino acid overlap) were divided
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into 3 pools of 27-29 peptides each. Peptides were synthesized by Genscript (Piscataway, NJ).
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Hemagglutination inhibition assay. Sera were incubated overnight at 37ºC with 3 volumes of
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receptor-destroying enzyme (RDE; Denka Seiken Co. Ltd, Tokyo, Japan), and heat inactivated at
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56ºC for 30 minutes before addition of 6 volumes 0.85% saline. Two-fold dilutions of sera were
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made in microtiter plates for a final volume of 25 μl and an equal volume of A/FM diluted to 4
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HAU/25 µl was added to each well and mixed thoroughly. One hour later, 50 µl of 0.5% chicken
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erythrocytes were added and mixed well. Plates were scored after 45 min, and
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hemagglutination inhibition (HAI) titers were calculated. Polyclonal anti-A/Fort Monmouth/1/47
10
173
(H1N1) antiserum (NR-3099) obtained through the NIAID, NIH Biodefense and Emerging
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Infections Research Resources Repository was used as a positive control.
175 176
Microneutralization Assay. Serial two-fold dilutions of RDE treated sera in PBS were made in a
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96 well U-bottom plate. An equal volume of A/FM at 1×104 TCID50/ml was added to each well,
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gently mixed, and incubated at 37 ºC. After 2 hours, 20 µl of the virus-antibody mixture was
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transferred to plates of MDCK cells plus virus growth medium, so as to give 100 TCID50 per well.
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Plates were then incubated for 48 hours and read out by hemagglutination with 0.5% chicken
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erythrocytes.
182 183
Statistical Analyses. SigmaPlot 12 (Systat Software, Point Richmond, CA) was used for statistical
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analyses as follows: Survival analysis used the log rank method, virus titer comparisons used the
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t- test, transmission rate comparisons used the Chi-Square test.
11
186
RESULTS
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Establishment of transmission system. In preliminary experiments, CFW outbred mice (as used
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by Schulman and Kilbourne (21)) were infected with a virulent mouse-adapted influenza H1N1
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strain, A/FM. Twenty-four hours later, 2 infected donor mice were placed in the same cage as 3
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uninfected contact animals. 3 days later, all mice were euthanized and lungs and nasal washes
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collected for virus titration. During the contact period, donors but not contacts progressively
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lost weight. While all donors had virus recoverable from lungs and most from nasal wash, A/FM
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was detectable in only 2/18 (11%) contacts when cages were held on a mechanically ventilated
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cage rack (data not shown), but this rose to 6/18 (33%) of contacts when cages were housed on
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an open-sided shelf with room ventilation only (Table 1). There was no obvious correlation in
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either experiment between individual donor lung or nasal wash virus titers on day 4 and the
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number of co-housed contacts that became infected. This suggests that virus shedding from
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donors earlier than day 4 is more important for virus transmission, and/or that other factors are
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involved. Because of the higher rate of transmission observed, all further experiments used
200
cages housed under room ventilation conditions.
201 202
In a further experiment using CFW mice, contacts were monitored for weight loss and survival.
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All A/FM infected donors died between days 3 and 5 (Fig. 1). Significant weight loss was seen in
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2 contacts (both in the same cage), but all survived. One month post contact, 4/18 contacts
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(including the two that lost weight) had A/FM-specific antibody responses with HAI titers of 160-
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640. Using seroconversion as a marker for transmission, only half the transmitted infections
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caused clinically apparent disease and insufficient virus was transmitted to initiate a lethal
12
208
infection. This experiment also suggests that secondary transmission (i.e. between infected and
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uninfected contacts) is either inefficient or absent in the case of A/FM.
210 211
Effect of mouse and virus strain. As different mouse strains vary in their susceptibility to
212
influenza viruses, we extended our studies to investigate direct contact transmission in two
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additional mouse strains: DBA/2J, which are highly susceptible to lethal influenza virus infection,
214
and BALB/c, which are relatively resistant (32,33). Transmission experiments in these two mouse
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strains using 104 TCID50 of A/FM were conducted as described above. Transmission of A/FM
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between DBA/2J mice occurred to a similar extent as seen between CFW mice, but transmission
217
between BALB/c mice was not seen (Table 1).
218 219
To investigate whether the lack of transmission of A/FM in BALB/c mice was due to decreased
220
susceptibility to infection or because of an impaired ability of BALB/c mice to transmit the virus,
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a “criss-cross” transmission experiment was performed, in which BALB/c mice were used as
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donors with CFW mice as recipients, and vice versa. Limited transmission of A/FM was seen for
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CFW donors with BALB/c contacts, but significantly higher levels of transmission were seen for
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BALB/c donors with CFW contacts (Table 1). A similar experiment examining transmission
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between the two susceptible stains, DBA/2J and CFW, showed similar rates of transmission with
226
both donor/contact combinations (Table 1). Taken together, these findings suggest that
227
establishment of infection in contact animals is at least in part dependent on the relative
228
sensitivity of the contact animal. However, donor traits also seem to be involved, as
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transmission of A/FM is more efficient from BALB/c donors to CFW contacts than from CFW 13
230
donors to CFW contacts. Interestingly, virus titers in lungs and nasal washes of infected BALB/c
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and CFW mice were very similar over the first 4 days of infection (data not shown).
232 233
Limited transmission (2/18 contacts becoming infected; data not shown) between BALB/c mice
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occurred for two different mouse-adapted viruses (PR8 and X-79). These viruses were also
235
inefficiently transmitted between CFW mice (1/18 contacts becoming infected; data not shown).
236 237
Taken in combination, our data suggest that transmission efficiency is multifactorial and
238
dependent on sensitivity of contacts, donor characteristics, and biological properties of the
239
specific virus strain.
240 241
Mode of transmission. To investigate whether transmission was via a direct contact-dependent
242
route or via the airborne route (by either droplet or aerosol), we developed a removable
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perforated divider running the length of the cage, as described in Materials and Methods. This
244
prevents direct contact between donor and contact mice while permitting air exchange between
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the two cage compartments. Using CFW mice and the non-mouse adapted strain A/Udorn,
246
which has been reported to efficiently transmit between BALB/c mice by direct contact but not
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via the airborne route (25), we found that direct contact resulted in efficient transmission (Table
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2, Experiment 1). However, when donor and contact mice were physically separated by the
249
perforated divider, a significantly lower proportion of contacts became infected (approximately
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6-fold, P
