JVI Accepts, published online ahead of print on 19 November 2014 J. Virol. doi:10.1128/JVI.02449-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved.
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JVI02449-14
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Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America
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A live attenuated equine H3N8 influenza vaccine is highly immunogenic and efficacious in mice and ferrets
Mariana Baz1, Myeisha Paskel1, Yumiko Matsuoka1, James Zengel, Xing Cheng, John J. Treanor2, Hong Jin3 and Kanta Subbarao1#
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Laboratory of Infectious Diseases, NIAID, NIH, Bethesda, MD 20892, United States Department of Medicine, University of Rochester Medical Center, Rochester, New York, United States 3 MedImmune LLC, Mountain View, CA 94043, United States 2
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Running title: Equine H3 influenza vaccine in mice and ferrets
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Abstract count: 227 Text count: 4556 Inserts: 4 figures, 2 tables
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Corresponding author: Kanta Subbarao, MD, MPH Emerging Respiratory Viruses Section Laboratory of Infectious Diseases, NIAID, NIH Bldg 33, Room 3E13C.1, 33 North Drive, MSC 3203 Bethesda, MD 20892-3203 Phone: 301-451-3839 Fax: 301-480-4749 Email:
[email protected]
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ABSTRACT
48
respiratory disease in horses. Although natural infection of humans with EIV have not
49
been reported, experimental inoculation of humans with these viruses can lead to a
50
productive infection and elicit a neutralizing antibody response. Moreover, EIV have
51
crossed the species barrier to infect dogs, pigs and camels and therefore may also pose a
52
threat to humans. Based on serologic cross-reactivity of H3N8 EIV from different
53
lineages and sublineages, A/equine/Georgia/1/1981 (eq/GA/81) was selected to produce a
54
live attenuated candidate vaccine by reverse genetics with the hemagglutinin and
55
neuraminidase genes of the eq/GA/81 wild-type (wt) virus and the six internal protein
56
genes of the cold-adapted (ca) A/Ann Arbor/6/60 ca (H2N2) vaccine donor virus, which
57
is the backbone of the licensed seasonal live attenuated influenza vaccine. In both mice
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and ferrets, intranasal administration of a single dose of the eq/GA/81 ca vaccine virus
59
induced neutralizing antibodies and conferred complete protection from homologous wt
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virus challenge in the upper respiratory tract. One dose of the eq/GA/81 ca vaccine also
61
induced neutralizing antibodies and conferred complete protection in mice and nearly
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complete protection in ferrets upon heterologous challenge with the H3N8
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(eq/Newmarket/03) wt virus. These data support further evaluation of the eq/GA/81 ca
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vaccine in humans for use in the event of transmission of an equine H3N8 influenza virus
65
to humans.
Equine influenza viruses (EIV) are responsible for rapidly spreading outbreaks of
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IMPORTANCE
Equine influenza viruses have crossed the species barrier to infect other mammals such as
74
dogs, pigs and camels and therefore may also pose a threat to humans. We believe that it
75
is important to develop vaccines against equine influenza viruses in the event that an EIV
76
evolves, adapts and spreads in humans causing disease. We generated a live attenuated
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H3N8 vaccine candidate and demonstrated that the vaccine was immunogenic and
78
protected mice and ferrets against homologous and heterologous EIV.
79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107
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108 109 110 111
INTRODUCTION
112
respiratory disease in horses for centuries. Influenza A viruses contain a single-stranded,
113
negative-sense RNA genome, consisting of 8 gene segments and are further classified
114
into subtypes on the basis of the antigenicity of the two major surface glycoproteins:
115
hemagglutinin (HA) and neuraminidase (NA) (1). Two subtypes of EIV have been
116
isolated from horses: H7N7 and H3N8 viruses. The prototype equine H7N7 virus
117
(A/equine/Prague/56) virus emerged in 1956 (2) but has not been isolated since the late
118
1970s (3), although serological evidence suggest that this virus subtype circulated among
119
horses in Europe and the Americas before 1956 (4, 5); its circulation in unvaccinated
120
horses was recorded in the 1980s in India (6) and the beginning of the 1990s in Europe
121
and USA (7, 8). Equine H3N8 viruses were first isolated during a major epidemic in
122
Miami in 1963 (A/eq/Miami/1/63) (9) and since then have circulated enzootically in
123
horses, causing significant disease and economic burden worldwide (10). These viruses
124
have continued to evolve and have diverged into two antigenically and genetically
125
distinct American and the European lineages since 1986. The American lineage further
126
evolved into Kentucky, South American and Florida sublineages. Subsequent evolution
127
within the Florida sublineage has resulted in the emergence of two distinct clades (clades
128
1 and 2) (11).
129
Influenza A viruses can transmit between species and this characteristic of interspecies
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transmission allows the emergence of reassortant influenza viruses (12). The H3N8 EIV
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has crossed the species barrier and transmitted to racing greyhounds that shared a racing
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track with horses in Florida in January 2004 (13) although retrospective serological
Equine influenza viruses (EIV) have been responsible for rapidly spreading outbreaks of
4
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analysis suggests that H3N8 influenza viruses were circulating in racing greyhounds
134
since 1999 (14). Subsequently, canine H3N8 influenza viruses spread to pet dogs and
135
became enzootic in the USA (15). Canine H3N8 infections have also been reported in the
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United Kingdom, Australia and Algeria (16-19). Studies on the distribution of the
137
sialoreceptors in the respiratory tract of horses and dogs have shown that both horses and
138
dogs have a predominance of SAα2,3-gal receptors (13, 18, 20). Pecorano et al., have
139
recently shown by binding assays that canine and equine influenza isolates have a higher
140
affinity for SAα2,3-gal compared to SAα2,6-gal receptors (20). These data may explain
141
the natural transmission of equine influenza virus to dogs.
142
In addition, two H3N8 influenza viruses were isolated from pigs in central China during
143
surveillance for swine influenza in 2004-2006. Sequence and phylogenetic analyses of
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the eight gene segments revealed that the two swine isolates were of equine origin and
145
were most closely related to European H3N8 EIV from the early 1990s (21). Recently, an
146
EIV (H3N8) was isolated from a Bactrian camel in Mongolia highlighting a novel
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interspecies transmission (22).
148
While natural transmission of EIV to humans has not been documented, experimental
149
challenge studies done in the 1960s indicate that the influenza A/equi 2/Miami/1/63 virus
150
was able to infect 64% of 33 human volunteers who received an intranasal dose of
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between 104.6 and 105.3 fifty percent tissue culture infectious doses (TCID50) of virus.
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However, illness only occurred in 12% of the volunteers, suggesting that the virus was
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more virulent for horses than for humans (23-25). Human birth cohorts from the late 19th
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century, particularly individuals born before 1890, demonstrated serologic reactivity with
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equine H3N8 viruses many decades later (26). However, a recent study reported by
5
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Burnell et al., showed sparse evidence for H3N8 infection in 100 subjects enrolled during
157
equine events in Australia (27). In this study, only nine subjects showed serologic
158
reactivity against EIV antigens and although eight of the subjects reported horse
159
exposure, antibody titers were low except for one subject who had a titer of 1:80 by
160
microneutralization assay. Another study done by Khurelbaatar et al., also showed sparse
161
evidence for EIV infection in 439 subjects ≥18 years of age (28) though 76% of the
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participants reported exposure to horses.
163
The social and economic impact of widespread disease caused by EIV in humans could
164
be devastating since people in different regions of the world still rely heavily upon horses
165
for recreation, communication, military or general transport, and EIV has already crossed
166
the species barrier to dogs. We believe it is important to develop vaccines against animal
167
influenza viruses of the H3 subtype. The ongoing circulation of seasonal H3N2 viruses
168
does not preclude the possibility of a pandemic caused by an antigenically distinct animal
169
H3 virus as demonstrated by the unexpected emergence of a swine-origin H1N1
170
influenza virus as a pandemic strain in 2009 despite the ongoing circulation of seasonal
171
H1N1 viruses.
172
Seasonal live attenuated influenza vaccines (LAIV) have been licensed in the United
173
States since 2003 and they elicit both systemic and local mucosal immunity (29, 30). Our
174
laboratory has previously generated live attenuated H1N1, H2N2, H5N1, H6N1, H7N3,
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H7N7, H7N9 and H9N2 viruses and found that these candidate vaccines were safe and
176
efficacious in conferring protection against wild-type (wt) viruses in mice and ferrets (31-
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36) and several of these vaccines have been evaluated in phase 1 clinical trials (33, 37,
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38).
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We analyzed the antigenic relatedness and replicative capacity of H3N8 EIV from the
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pre-divergent and American lineage (sublineage Florida Clade 1 and 2) viruses using
181
post-infection mouse and ferret sera (39). We selected A/equine/Georgia/1/1981
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(eq/GA/81) for vaccine development because it induced the most broadly cross-
183
neutralizing antibodies (NtAb) and replicated to a high titer in the upper respiratory tract
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of mice and ferrets (39). We used reverse genetics to generate a live attenuated cold-
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adapted (ca) H3N8 virus bearing wild-type (wt) HA and NA genes from the eq/GA/81wt
186
virus, and the six internal protein gene segments from the ca influenza A vaccine donor
187
strain, A/Ann Arbor/6/60 ca (AA ca) (H2N2). The immunogenicity and protective
188
efficacy against challenge with the homologous wt eq/GA/81 and heterologous
189
A/equine/Newmarket/5/2003 (eq/Newm/03) viruses were evaluated in mice and ferrets.
190 191
MATERIALS AND METHODS
192 193
Viruses.
194
H3N8 EIV isolates were provided by Richard Webby, St. Jude Children’s Research
195
Hospital, Memphis, TN (A/equine/Georgia/1/1981 [H3N8]) and Debra Elton, Animal
196
Health Trust, Newmarket, UK (A/equine/Newmarket/5/2003 [H3N8]). The HA amino
197
acid sequence identity between eq/GA/81 and eq/Newm/03 is 97.3 % (39). Eq/GA/81
198
belongs to the “Florida Clade 1” sub-lineage and eq/Newm/03 belongs to the “American
199
lineage” “Florida Clade 2” (40). We have previously reported (39) that sera from ferrets
200
infected with eq/GA/81 showed cross-reactivity against the homologous virus (NtAb titer
201
≥1280) as well as eq/Newm/03 (NtAb titer ≥640). Virus stocks were propagated in the
7
202
allantoic cavity of 9- to 11-day-old embryonated specific-pathogen-free hen’s eggs
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(Charles River Laboratories, North Franklin, CT) at 35°C. The allantoic fluid was
204
harvested at 72 h postinoculation (p.i.), tested for hemagglutinating activity using 0.5%
205
turkey red blood cells (Lampire Biological Laboratories, Pipersville, PA), pooled,
206
aliquoted, and stored at -80°C until use. Virus titers were determined in Madin-Darby
207
canine kidney (MDCK) cells (ATCC, Manassas, VA) and calculated using the Reed and
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Muench method (41).
209 210
Generation of reassortant eq/GA/81 ca vaccine virus by reverse genetics.
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The HA and NA gene segments of eq/GA/81 (H3N8) were amplified from vRNA by
212
reverse transcription-polymerase chain reaction (RT-PCR) using primers that are
213
universal to the HA and NA genes, sequenced and cloned into the plasmid vector
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pAD3000 (42). The 6:2 reassortant vaccine virus was generated by co-transfecting eight
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plasmids encoding the HA and NA of the eq/GA/81 virus and the 6 internal protein gene
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segments of the AA ca virus into co-cultured 293T and MDCK cells. At 3 to 5 days post-
217
transfection, the transfected cell supernatant was inoculated into the allantoic cavity of 10
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to 11 day old embryonated chicken eggs (Charles River Laboratories, Franklin, CT) and
219
incubated at 33°C for 2 days. Virus titer was determined by immunostaining plaques
220
using an anti-NP monoclonal antibody and expressed as log10 PFU (plaque-forming
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units)/ml as previously described (43). The HA and NA sequences of the rescued virus
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were verified by sequencing the genes amplified from viral RNA by RT-PCR.
223 224
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225 226
Serologic assays.
227
Anti-influenza antibody titers in serum samples were measured by hemagglutination
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inhibition (HAI) according to standard protocols (44) or microneutralization (MN) assay
229
as previously described (45). For the HAI assay, nonspecific inhibitors were removed
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from serum by overnight treatment with receptor-destroying enzyme (Denka Seiken,
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Tokyo, Japan). Sera were 2-fold serially diluted in 96-well V-bottom plates starting at a
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dilution of 1:10, and 4 HA units of virus was added. Control wells received phosphate-
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buffered saline (PBS) alone. Virus and sera were incubated together for 30 min at room
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temperature and 50 μl of a 0.5% (vol/vol) suspension of turkey erythrocytes was added.
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The virus-serum mixture and erythrocytes were gently mixed, and the results were
236
recorded after incubation for 45 min at room temperature. HAI titers were recorded as the
237
inverse of the highest antibody dilution that inhibited hemagglutination. A cross-reactive
238
antibody response was defined as a ≤4-fold difference between the homologous HAI titer
239
and the titer generated against the heterologous virus. For the MN assay, serial 2-fold
240
dilutions of heat-inactivated serum were prepared starting from a 1:20 dilution. Equal
241
volumes of serum and virus were mixed and incubated for 60 minutes at room
242
temperature. The residual infectivity of the virus-serum mixture was determined in
243
MDCK cells in 4 replicates for each dilution of serum. The neutralizing antibody (NtAb)
244
titer was defined as the reciprocal of the serum dilution that completely neutralized the
245
infectivity of 100 TCID50 of the virus as determined by the absence of cytopathic effect
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on MDCK cells at day 4. A cross-reactive antibody response was defined as a ≤4-fold
247
difference between the homologous NtAb titer and the titer generated against the
9
248
heterologous virus.
249
Immunogenicity and protective efficacy of the H3N8 ca virus in mice.
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Six- to 8-week-old female BALB/c mice (Taconic Farms, Inc., Germantown, NY) were
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used in all mouse experiments. Animal studies were conducted in biosafety level 2
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laboratories (BSL-2) at the National Institutes of Health (NIH) and protocols were
253
approved by the National Institutes of Health Animal Care and Use Committee.
254
Groups of eight mice were lightly anesthetized and inoculated intranasally (i.n.) with 50
255
µl containing 106 PFU of the H3N8 ca vaccine virus in one or two doses. Mock-
256
inoculated controls received Leibovitz-15 (L15) medium alone. Neutralizing antibody
257
responses to homologous (eq/GA/81) and heterologous (eq/Newm/03) H3N8 wt viruses
258
were determined from sera collected prior to inoculation (prebleed) and at 38 days after
259
the first or second immunization by MN assay (45).
260
On day 38 after the first or second dose of vaccine, groups of eight mice were challenged
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i.n. with 105 TCID50 of the H3N8 equine wt viruses, eq/GA/81 or eq/Newm/03. Four
262
mice per challenge virus were sacrificed on days 2 and 4 post-challenge (p.c.), and lungs
263
and nasal turbinates (NTs) were harvested and stored at -80°C. We chose these time
264
points based on previous observations in our laboratory that equine wt viruses replicate to
265
high titers in the NTs and lungs of mice from days 2 to 4 post-infection (39). Organs were
266
weighed and homogenized in L15 medium containing 2X antibiotic-antimycotic
267
(penicillin, streptomycin, and amphotericin B) (Invitrogen-GIBCO) to make 10% and 5%
268
(wt/vol) tissue homogenates of lung and NT, respectively. Tissue homogenates clarified
269
by centrifugation at 1,500 rpm for 10 min were titered in 24 and 96-well tissue culture
270
plates containing MDCK cell monolayers The virus titer for each organ was determined
10
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by Reed and Muench method (41) and was expressed as log10 TCID50/gram of tissue.
272
Replication of H3N8 equine wt and ca viruses in the respiratory tract.
273
Ten- to 12-week-old ferrets (Triple F Farms, Sayre, PA) were used in these ferret
274
experiments. Animals were seronegative for antibodies to circulating human H3N2,
275
H1N1 and B influenza viruses. Ferret studies were conducted in a BSL-2 at MedImmune
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and NIH and protocols were approved by the MedImmune and NIH Animal Care and
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Use Committees.
278 279
Groups of ferrets were lightly anesthetized with isoflurane and inoculated i.n. with 500 μl
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containing 107 PFU of wt or ca viruses. At 3 and 5 dpi, ferrets were euthanized, and right
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middle and the caudal portion of the left cranial lobe of the lungs and the NTs were
282
harvested and stored at -80°C. Organs were thawed, weighed and homogenized in L-15
283
medium as described above to make a 10% (wt/vol) suspension and titers were
284
determined by plaque assay on MDCK cells using an anti-NP monoclonal antibody and
285
expressed as log10 PFU/gram of tissue.
286 287
Immunogenicity and protective efficacy of the H3N8 ca virus.
288
Groups of ferrets were inoculated i.n. with one or two doses 28 days apart of 500 μl
289
containing 107 PFU of eq/GA/81 ca or L15 medium (mock immunized) and serum
290
samples were collected on days 0 (pre-immunization), 28 or 56 p.i. Antibody titers in pre
291
and post-infection ferret sera were determined by MN and HAI assays, as described
292
above.
293
On day 28 or 56 p.i. ferrets were challenged i.n. with 107 PFU of each of the H3N8
11
294
equine wt viruses, eq/GA/81 or eq/Newm/03. Three ferrets per challenge virus were
295
euthanized on days 3 and 5 p.c., and lungs and NTs were harvested and stored at -80°C.
296
We chose these time points based on previous observations in our laboratory that wt EIV
297
are detected at high titers in the NTs of ferrets from days 1 to 5 p.i. (39). Challenge virus
298
titers were determined in MDCK cells and expressed as log10TCID50 per gram of NT or
299
lung tissue as described above.
300 301
Human sera.
302
Sera collected during a study in 2009 from healthy adult men and nonpregnant women
303
before vaccination with the monovalent inactivated 2009 pH1N1 vaccine were provided
304
by John Treanor (University of Rochester) (46). Subjects were enrolled in 3 age cohorts:
305
18 to 32 years (n = 19), 60 to 69 years (n = 19), and ≥70 years (n = 18). The study was
306
conducted under a protocol approved by the University of Rochester Research Subjects
307
Review Board. Informed written consent was obtained from each participant.
308 309
RESULTS
310 311 312 313 314
Immunogenicity of the eq/GA/81 ca virus in mice.
A single dose of eq/GA/81 ca virus induced a robust neutralizing antibody response
315
against the homologous virus, with a geometric mean titer (GMT) of 418 (range 57 to
316
1016) (Table 1). The NtAb titers against the heterologous virus, eq/Newm/03, were
317
similar (GMT of 490; range 160 to 1613). When two doses of the eq/GA/81 ca vaccine
318
were administered to mice, the GMT achieved was 264 (range 57 to 905) following the
319
first dose, with a further increase (GMT 2426; range 640 to 7241) 38 days after the
12
320
second dose (Table 1). A similar profile was observed against the heterologous virus
321
(Table 1). As expected, mock-immunized mice did not develop detectable NtAb
322
antibodies. These results demonstrate that the eq/GA/81 ca vaccine candidate is highly
323
immunogenic in mice and serum antibodies cross-reacted well with the heterologous
324
eq/Newm/03 wt virus.
325 326 327 328 329
Efficacy of the eq/GA/81 ca virus in mice.
330
To determine whether immunization with the eq/GA/81 ca virus induced protection, we
331
inoculated mice with either a single dose or two doses of the vaccine candidate. Thirty-
332
eight days after the final vaccination, mice were challenged with 106 PFU/50μl of the
333
homologous (eq/GA/81) or the heterologous (eq/Newm/03) wt viruses. In mock-
334
immunized mice, the mean titers on days 2 and 4 p.c. in the NTs after challenge with the
335
eq/GA/81 wt virus were 105.5 and 105.25 TCID50/g, respectively, and the mean titers in the
336
lungs were 106.1 and 104.6 TCID50/g, respectively. In mock-immunized mice, the mean
337
titers on days 2 and 4 p.c. in the NTs after challenge with the heterologous eq/Newm/03
338
wt virus were 106.8 and 104.9 TCID50/g, respectively, and the mean titers in the lungs were
339
106.4 and 104.95 TCID50/g on days 2 and 4 p.c., respectively (Fig. 1). A single dose of the
340
eq/GA/81 ca virus provided complete protection against challenge with homologous and
341
heterologous wt viruses in both the upper and lower respiratory tract (Fig.1). A similar
342
pattern was observed for mice vaccinated with two doses of the eq/GA/81 ca virus (Fig.
343
1). We had previously observed that the eq/GA/81 and eq/Newm/03 wt viruses did not
344
cause weight loss or mortality in mice, so we did not assess protection from clinical
13
345
illness in mice (39). Thus, the eq/GA/81 ca vaccine candidate offered complete
346
protection against homologous and heterologous H3N8 wt virus challenge in mice.
347 348 349
Level of replication of the eq/GA/81 wt and ca viruses in ferrets.
350
To determine whether the eq/GA/81 ca vaccine virus was attenuated in ferrets, the level
351
of replication in the NT and lungs 3 and 5 days following i.n. administration were
352
compared with those of the eq/GA/81 wt virus. The mean titers on days 3 and 5 in the
353
NTs of ferrets inoculated with the eq/GA/81 wt virus were 107.2 and 106.9 PFU/g,
354
respectively, and 104.8 and 105.7 PFU/g in ferrets inoculated with the eq/GA/81 ca virus.
355
Thus, the replication of the eq/GA/81 ca virus was 16 to 250-fold lower than the
356
corresponding wt virus in the URT of ferrets. The eq/GA/81 wt virus did not replicate
357
well in the lower respiratory tract of ferrets, as previously reported (39) and replication of
358
the vaccine candidate was not detected in the lungs of ferrets (Fig. 2). No notable signs of
359
disease were observed in ferrets infected with the equine ca or wt viruses. These data
360
indicate that the eq/GA/81 ca virus was attenuated in ferrets.
361 362 363
Immunogenicity of the eq/GA/81 ca virus in ferrets.
364
Ferrets that received a single dose of eq/GA/81 ca virus developed neutralizing and HAI
365
antibodies to the homologous wt virus at titers that ranged from 320 to 905 (GMT=538)
366
and from 40 to 160 (GMT=85), respectively (Table 2). One dose of the eq/GA/81 ca
367
virus elicited cross-reactive neutralizing and HAI antibodies to the heterologous
368
eq/Newm/03 wt virus at titers that ranged from 113 to 453 (GMT=196) and from 160 to
369
640 (GMT=226), respectively. In animals that received two doses of the vaccine, the first
14
370
dose of the eq/GA/81 ca vaccine induced homologous NtAb and HAI response at titers
371
that ranged from 226 to 1280 (GMT=559) and 40 to 160 (GMT=90), respectively. Titers
372
increased after the second dose and ranged from 453 to 3620 (GMT=932) and 80 to 1280
373
(GMT=302), respectively. One dose of the eq/GA/81 ca virus elicited cross-reactive
374
neutralizing and HAI antibodies to the heterologous wt virus at titers that ranged from
375
320 to 1280 (GMT=512) and 80 to 640 (GMT=151), respectively. Again, titers increased
376
after the second dose and ranged from 320 to 5120 (GMT=1243) and 160 to 2560
377
(GMT=678), respectively (Table 2). Consistent with findings from the study in mice,
378
these data indicate that a single dose of the eq/GA/81 ca virus was immunogenic in
379
ferrets and that serum antibodies cross-reacted with a heterologous H3N8 virus. Sera
380
from vaccinated ferrets failed to neutralize an older (A/Port Chalmers/1973) and a recent
381
(A/Texas/50/2012) human H3N2 virus (data not shown).
382 383 384
Efficacy of the eq/GA/81 ca virus in ferrets.
385
To determine whether immunization with the eq/GA/81 ca virus induced protection in
386
ferrets, we inoculated animals intranasally with either a single dose or two doses of the
387
vaccine candidate and challenged them 28 days later with 107 PFU of the homologous
388
(eq/GA/81) or the heterologous (eq/Newm/03) wt viruses. In mock-immunized ferrets the
389
titers of eq/GA/81 wt challenge virus on days 3 and 5 p.c. in the NTs were 107.1 and 105.0
390
TCID50/g, respectively, and the mean virus titers in the lungs were 102.3 and 102.5
391
TCID50/g on days 3 and 5 p.c. In mock-immunized ferrets the mean titers of the
392
heterologous eq/Newm/03 wt virus on days 3 and 5 p.c. in the NTs were 108.1 and 107.2
393
TCID50/g, respectively, and the mean virus titers in the lungs were 102.5 and 101.7
15
394
TCID50/g on days 3 and 5 p.c. (Fig. 3). A single dose of the eq/GA/81 ca virus provided
395
complete protection against challenge (no detectable replication) with homologous wt
396
virus in the upper respiratory tract of the ferrets and restricted replication and early
397
clearance of the heterologous wt challenge virus (only one ferret out of 3 had 102
398
TCID50/g on day 5 p.c.) (Fig.3). Similar results were observed in ferrets that received a
399
second dose of the vaccine. Because the eq/GA/81 and the eq/Newm/03 wt viruses did
400
not replicate well in the lower respiratory tract of ferrets, the protection conferred by the
401
eq/GA/81 ca virus in the lungs could not be evaluated (Fig.3).
402 403 404 405 406 407
Testing for presence of cross-reactive antibodies in human sera representing three age cohorts.
408
whether prior exposure to seasonal H3N2 viruses induced cross-reactive Ab against EIV.
409
We assessed the presence of antibodies that cross-reacted with eq/GA/81 (H3N8) virus in
410
human sera collected in 2009. As a control we assayed the levels of antibodies against the
411
seasonal influenza virus A/Wisconsin/67/2005 (H3N2) that was circulating at the time
412
the sera were collected. Subjects from three age groups, 18-32 years old (n=19), 60-69
413
years old (n=19), and 70 years or older (n=18), were enrolled in a clinical trial of a
414
monovalent 2009 H1N1pdm vaccine that has been reported previously (46).
415
The 18-32 year old subjects failed to show detectable NtAb against the equine H3N8
416
virus (Fig.4A). Interestingly, subjects from the other two cohorts, 60-69 and ≥70 years
417
old, had a GMT of 22 (range 10 to 57) and 20 (range 10 to 113), respectively. In subjects
418
older than 60 years of age (n=37), seven had NtAb titers of 40, one of 50, three of 57 and
419
one of 113. Nine subjects showed lower levels of NtAb (between 20 and 28). In total, 22
Because human H3N2 viruses have circulated since 1968, we sought to determine
16
420
out of 37 individuals had NtAb titers ≥20 against the equine H3N8 virus. Unfortunately,
421
we did not enroll subjects who were between 33 and 59 years of age in the study, so we
422
cannot comment on the level of cross-reactive antibody in this age group. However, the
423
study was conducted in 2009-2010 and therefore subjects born after 1968, when H3N2
424
viruses emerged and became established in humans, would have been 42 years of age or
425
younger. Therefore, we would expect that people over 42 years of age would have been
426
exposed to H3N2 viruses and could have some cross reactive antibody. The GMT in the
427
18-32 year old, 60-69 year old and ≥70 year old subjects against the
428
A/Wisconsin/67/2005 (H3N2) virus was 248 (range 10 to 3620), 232 (range 10 to 3620)
429
and 134.5 (range 10 to 1613), respectively (Fig.4A).
430
Interestingly, none of the subjects had detectable HAI antibody against the equine H3N8
431
virus (Fig. 4B). The GMT of HAI antibodies in the 18-32 year old, 60-69 year old and
432
≥70 year old subjects against the A/Wisconsin/67/2005 (H3N2) virus was 46 (range 10 to
433
1280), 67 (range 10 to 640) and 27 (range 10 to 640), respectively (Fig.4B). The
434
detection of cross-reactive NtAb in the absence of HAI antibodies in 59% of subjects
435
over 60 years of age suggests that the antibody was induced by prior or repeated exposure
436
by infection or vaccination with older seasonal H3N2 viruses and that the Abs could be
437
directed at the HA stalk.
438 439 440 441
DISCUSSION
442
Although direct transmission of EIV to humans has not been reported, experimental
443
infection of humans with EIV can lead to a productive infection and elicit a significant
444
NtAb response (24, 25). The fact that EIV can infect humans and these viruses have
17
445
crossed the species barrier and infected dogs (13, 16, 18), pigs (21) and camels (21)
446
underscores the potential threat posed to human health by viruses of this subtype. The
447
emergence and pandemic spread of the swine-origin H1N1 influenza virus in 2009,
448
despite the ongoing circulation of human H1N1 viruses suggests that an antigenically
449
distant animal origin H3 virus may pose a pandemic threat despite the circulation of
450
H3N2 viruses in humans since 1968.
451
The purpose of our study was to generate and evaluate a vaccine candidate to be used in
452
humans in the event that an EIV evolves, adapts and spreads in humans causing disease.
453
To this end, we previously evaluated three H3N8 equine influenza viruses from different
454
lineages and selected the eq/GA/81 virus for vaccine development because it elicited
455
cross-reactive antibodies against heterologous EIV (39). We generated an eq/GA/81
456
candidate LAIV by plasmid-based reverse genetics on the backbone of the AA ca donor
457
virus that is used to produce the licensed seasonal live attenuated influenza vaccine.
458
In mice, a single dose of the eq/GA/81 ca vaccine virus induced robust neutralizing
459
antibody titers against the homologous and heterologous wt challenge viruses and
460
conferred full protection against homologous and heterologous virus challenge in both
461
the upper and lower respiratory tract. In ferrets, as in mice, one dose of the vaccine was
462
highly immunogenic and conferred complete protection against homologous challenge
463
virus and near complete protection against the heterologous challenge virus. We observed
464
a direct correlation between serum antibody response and protection against challenge in
465
mice and ferrets that received one dose of the eq/GA/81 ca vaccine virus, although
466
contributions from other arms of the immune system such as the cellular or mucosal
467
immune response cannot be excluded.
18
468
In 2009, when the novel H1N1pdm virus emerged, it was assumed that two doses of
469
vaccine would be needed to immunize the human population against the pandemic virus
470
because studies in 1977 had demonstrated the need for two doses of vaccine in a naïve
471
population; preclinical evaluation of 2009 H1N1pdm vaccines in influenza-naïve animal
472
models supported this conclusion. However, when clinical trials of the inactivated 2009
473
H1N1pdm vaccine were undertaken, a single dose of vaccine was sufficient in all except
474
children younger than 3 years of age, indicating that most of the population had been
475
primed by prior exposure or vaccination with seasonal H1N1 viruses. We evaluated this
476
phenomenon in a mouse model and demonstrated that priming was achieved by infection
477
with seasonal H1N1 influenza virus or seasonal LAIV but not by seasonal inactivated
478
influenza vaccine (47). In the present study, we evaluated sera from 56 subjects from
479
three age groups, 18-32 years old, 60-69 years old, and 70 years and older, who were
480
enrolled in a previous study for cross-reactive H3 antibodies. Most subjects in each
481
cohort reported receiving the 2009-2010 seasonal trivalent influenza vaccine 2 to 4
482
months before their blood sample was collected (46). We do not know if the participants
483
had been exposed to horses. We observed reactivity against the eq/GA/81 virus in
484
individuals over 60 years of age and speculate that the detectable cross-reactive NtAb
485
titers may be explained by cross-reactivity due to previously circulating human influenza
486
A H3N2 viruses. In the event of a pandemic caused by a related virus, a large portion of
487
the human population may be immunologically primed because of previous exposure to
488
seasonal H3N2 influenza viruses and therefore one dose of the H3N8 vaccine may be
489
sufficient to confer protection.
490
In summary, we generated a candidate LAIV against an EIV and demonstrated that a
19
491
single dose of the vaccine was highly immunogenic and efficacious in protecting mice
492
and ferrets from challenge with the homologous and an antigenically distinct
493
heterologous H3N8 virus from a different sub-lineage. Based on these promising
494
preclinical data, careful clinical evaluation of the eq/GA/81 (H3N8) ca vaccine is
495
warranted as part of pandemic preparedness efforts. We found evidence of cross-reactive
496
antibodies in subjects >60 years of age that could be directed at the stalk domain of the
497
HA protein. Although data from persons between 32-60 years of age are lacking, it
498
appears that a proportion of the human population may be previously primed for a robust
499
response to an equine influenza H3N8 vaccine.
500 501
ACKNOWLEDGMENTS
502
This research was supported by the Intramural Research Program of the NIAID, NIH and
503
was performed as part of a Cooperative Research and Development Agreement between
504
the Laboratory of Infectious Diseases, NIAID, and MedImmune, LLC.
505
We thank Dr. Ian Moore, the staff of the Comparative Medicine Branch, NIAID, and the
506
staff at MedImmune’s Animal Care Facility for technical support for animal studies. We
507
are grateful to Drs. Richard Webby and Debra Elton for providing the viruses used in this
508
study and Dr. JoAnn Suzich for reviewing the manuscript.
509 510 511 512 513 514 515 516 517 518
20
519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564
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24
704 705 706 707
FIGURE LEGENDS
708
heterologous challenge in the upper (A) or lower (B) respiratory tract of mice. Mice were
709
intranasally inoculated with either L-15 (mock) or 1 or 2 doses of 106 PFU/mouse of
710
eq/GA/81 ca vaccine and challenged 38 days following the last vaccine dose with 106
711
PFU/mouse of the indicated challenge virus. Virus titers were determined on days 2 and 4
712
postchallenge. The dashed horizontal line represents the lower limit of detection.
Fig 1. Protection conferred by the eq/GA/81 ca vaccine against homologous and
713 714
Fig 2. Level of replication of the eq/GA/81 ca vaccine virus compared with the
715
corresponding wt virus in the upper (A) and lower (B) respiratory tracts of ferrets. Lightly
716
anesthetized ferrets were inoculated intranasally with 107 PFU/ferret and tissues were
717
harvested on days 3 and 5 postinfection. The dashed horizontal line represents the lower
718
limit of detection.
719 720
Fig 3. Protection conferred by the eq/GA/81 ca vaccines against homologous and
721
heterologous challenge in ferrets. Animals were intranasally inoculated with either L-15
722
(mock) or 1 or 2 doses of 107 PFU/ferret of eq/GA/81 ca vaccine and challenged 28 days
723
following the last vaccine administration with 107 PFU/ferrets of the indicated challenge
724
virus. Virus titers were determined on days 3 and 5 postchallenge. Levels of replication
725
of the indicated challenge viruses in the upper (A) or lower (B) respiratory tract of ferrets
726
that were challenged following 1 dose of the ca vaccine. The dashed horizontal line
727
represents the lower limit of detection.
25
728
Fig 4. Serum neutralizing antibody (A) and hemagglutination inhibiting antibody (B)
729
titers in individuals of different age groups. The serum antibody titer against eq/GA/81
730
(circles) and A/WI/67/05 (triangles) are shown for individual subjects. Bars identify
731
geometric mean titer of the group. The dashed horizontal line represents the lower limit
732
of detection.
733 734 735
26
736 737 738 739
Table 1. Serum neutralizing antibody response to the eq/GA/81 ca vaccine in micea.
Test antigen
eq/GA/81 wt eq/Newm/03 wt 740 741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756 757 758 759 760 761 762 763 764 765 766 767 768 769 770 771 772 773 774 775 776 777 778
Geometric mean titer of serum NtAb achieved at indicated days post-immunization in mice: 1 dose (D38)b 2 doses (D28/D66b) 418 264/2426 490 225/1540
a
Groups of eight mice were inoculated i.n. with 106 PFU of the eq/GA/81 ca vaccine. Serum was collected at the indicated days after the first immunization. Homologous antibody titers are in bold. b Mice were bled on day 38 after the first or second immunization because of technical reasons.
27
779 780 781 782
Table 2. Serum antibody responses to the eq/GA/81 ca vaccine in ferretsa. Test antigen
eq/GA/81 wt eq/Newm/03 wt 783 784 785 786 787 788 789 790 791 792 793 794 795 796
Geometric mean serum HAI or NtAb titers achieved at indicated days post-immunization in ferrets: Assay 1 dose (D28) 2 doses (D28/D56) MN 538 559/932 HAI 85 90/302 MN 196 512/1243 HAI 226 151/678
a
Groups of twelve ferrets were inoculated i.n. with 107 PFU of the eq/GA/81 ca vaccine. Serum was collected at the indicated days after immunization. Homologous antibody titers are in bold.
28
Fig. 1
Virus Titer (log10 TCID50/g)
A: NTs 8 7 6 5 4 3 2 1
1 dose
Challenged with:
1 dose
2 doses
eq/GA/81 wt
d4
d2
ca
/8 1
G A
eq /
eq /
G A
/8 1
ca
d4
d2
ca
/8 1
G A
eq /
eq /
G A
/8 1
ca
L1 5d L1 2 5d4
d4
d2
ca
/8 1
G A
eq /
eq /
G A
/8 1
ca
d4
ca
/8 1
G A
eq /
eq /
G A
/8 1
ca
L1 5d L1 2 5d4
Immunized with:
d2
0
2 doses
eq/Newm/03 wt
Virus Titer (log10 TCID50/g)
B: Lungs 8 7 6 5 4 3 2 1
1 dose
Challenged with:
2 doses
eq/GA/81 wt
d4 A eq /81 /G A ca d2 /8 1 ca d4 eq /G A eq /81 /G A ca d /8 2 1 ca d4
d2 5-
L1
L1
5-
/G
eq
eq
/G
A eq /81 /G A ca d /8 2 1 ca d4 eq /G A eq /81 /G A ca d2 /8 1 ca d4
d2
5-
L1
5L1
d4
0
Immunized with:
1 dose
2 doses
eq/Newm/03 wt
Fig 1. Protection conferred by the eq/GA/81 ca vaccines against homologous and heterologous challenge in the upper (A) or lower (B) respiratory tract of mice. Mice were intranasally inoculated with either L-15 (mock) or 1 or 2 doses of 106 PFU/mouse of eq/GA/81 ca vaccine and challenged 38 days following the last vaccine dose with 106 PFU/mouse of the indicated challenge virus. Virus titers were determined on days 2 and 4 postchallenge. The dashed horizontal line represents the lower limit of detection.
Fig. 2
Virus Titer (log10 PFU/g)
eq/GA/81 wt 8
eq/GA/81 ca
A: NTs
7 6 5 4 3 2 1 5
3
Days post-inoculation
B: Lungs
Virus Titer (log10 PFU/g)
8
5
3
0
7 6 5 4 3 2 1 5
3
5
3
0 Days post-inoculation
Fig 2. Level of replication of the eq/GA/81 ca vaccine virus compared with the corresponding wt virus in the upper (A) and lower (B) respiratory tract of ferrets. Lightly anesthetized ferrets were inoculated intranasally with 107 PFU/ferret and tissues were harvested on days 3 and 5 postinfection. The dashed horizontal line represents the lower limit of detection.
Virus Titer (log10 TCID50/g)
Fig. 3 9
A: NTs
8 7 6 5 4 3 2 1
/G eq A / 8 /G 1 A ca /8 1 d3 ca eq d5 /G eq A / 8 /G 1 A ca /8 1 d3 ca d5
5L1 d3 5d5
1 dose
eq
L1
L1 d3 5d5 eq
L1
5-
Immunized with:
/G eq A / 8 /G 1 A ca /8 1 d3 ca eq d5 /G eq A / 8 /G 1 A ca /8 1 d3 ca d5
0
1 dose
2 doses
eq/Newm/03 wt
eq/GA/81 wt
Challenged with:
2 doses
B: Lungs Virus Titer (log10 TCID50/g)
9 8 7 6 5 4 3 2 1
Challenged with:
2 doses
eq/GA/81 wt
eq A/8 /G 1 A ca /8 1 d3 ca eq d5 /G A eq /8 /G 1 A ca /8 1 d3 ca d5
d5
5-
G
L1
5-
d3
eq /
G eq /
1 dose
L1
L1 d3 5d5
5L1
eq A/8 /G 1 A ca /8 1 d3 ca eq d5 /G eq A/8 /G 1 A ca /8 1 d3 ca d5
0 Immunized with:
1 dose
2 doses
eq/Newm/03 wt
Fig 3. Protection conferred by the eq/GA/81 ca vaccines against homologous and heterologous challenge in the upper (A) or lower (B) respiratory tract of ferrets. Ferrets were intranasally inoculated with either L-15 (mock) or 1 or 2 doses of 107 PFU/ferret of eq/GA/81 ca vaccine and challenged 28 days following the last vaccine administration with 107 PFU/ferrets of the indicated challenge virus. Virus titers were determined on days 3 and 5 postchallenge. The dashed horizontal line represents the lower limit of detection.
Fig. 4
A 4096
NtAb titers
1024 256 64 16 4
18-32 yr
60-69 yr
³70 yr
n=19
n=19
n=18
Age groups
B
HAI antibody titers
4096 1024 256 64 16 4
18-32 yr
60-69 yr
³70 yr
n=19
n=19
n=18
Age groups
Fig 4. Serum neutralizing antibody (A) and hemagglutination inhibiting antibody (B) titers in individuals of different age groups. The serum antibody titer against eq/GA/81 (circles) and A/WI/67/05 (triangles) are shown for individual subjects. Bars identify geometric mean titer of the group. The dashed horizontal line represents the lower limit of detection.
