JVI Accepts, published online ahead of print on 31 December 2014 J. Virol. doi:10.1128/JVI.02636-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved.
1
Development of a mouse-adapted, live-attenuated influenza virus that permits in
2
vivo analysis of enhancements to the safety of LAIV.
3
Andrew Cox1, Steven F. Baker1, Aitor Nogales, Luis Martínez-Sobrido* and Stephen
4
Dewhurst*
5 6
Department of Microbiology and Immunology, University of Rochester, 601 Elmwood
7
Avenue, Rochester, New York 14642
8 1
These authors contributed equally to this work
11
*
To whom correspondence should be addressed:
12
Luis Martínez-Sobrido
13
Department of Microbiology and Immunology
14
University of Rochester School of Medicine and Dentistry
15
601 Elmwood Avenue, Rochester, NY 14642
16
Phone: +1 585 276 4733; fax: +1 585 473 9573
17
e-mail: [email protected]
9 10
18 19
Stephen Dewhurst
20
Department of Microbiology and Immunology
21
University of Rochester School of Medicine and Dentistry
22
601 Elmwood Avenue, Rochester, NY 14642
23
Phone: +1 585 275 3216; fax: +1 585 473 9573
24
e-mail: [email protected]
25 26 27
Running title: Mouse-adapted LAIV
28
ABSTRACT
29 30
The live attenuated influenza vaccine is preferentially recommended for use in
31
persons 2 through 49 years of age, but not approved for children under 2 or asthmatics
32
due to safety concerns. Therefore, increasing safety is desirable. Here we describe a
33
murine LAIV with reduced pathogenicity that retains lethality at high doses and further
34
demonstrate that we can enhance safety in vivo through mutations within NS1. This
35
model may permit preliminary safety analysis of improved LAIVs.
36
37
BODY
38 39
Influenza A virus is a respiratory pathogen that infects through the upper airway,
40
and leads to pathology via replication in the lower airway (1). The temperature gradient
41
between these two areas in people enabled the development of the cold-adapted, live
42
attenuated influenza vaccine (LAIV, FluMist) that replicates in the cooler upper
43
respiratory tract to trigger a protective immune response, but cannot damage the lower
44
respiratory tract due to the elevated temperatures restricting replication (2). This
45
temperature sensitive (ts), attenuated (att) phenotype is imparted by five mutations within
46
the viral replicative machinery: namely PB2 N265S; PB1 K391E, D581G and A661T;
47
NP D34G (3, 4). Though this vaccine has an overall acceptable safety profile, it is not
48
approved for use in children under two due to concerns of elevated hospitalizations due to
49
wheezing (5, 6). For this reason it is also not approved for use in asthmatics. Therefore,
50
development of vaccines with increased safety over LAIV is desirable. Currently, no
51
mouse model exists for the adequate assessment of the safety of the LAIV. In
52
experimental animal studies with LAIV, elevated doses of LAIV do not elicit pathology,
53
rendering determination of safety impossible (7-10). Here, we describe a model with
54
which we can assess alterations in vaccine safety.
55
The parental strain for our vaccine is a well-characterized, murine-lethal strain of
56
influenza A virus (A/Puerto Rico/8/34 H1N1, PR8). We introduced four ts, att mutations
57
from LAIV into PR8 (NP D34G is natively present) via site directed mutagenesis
58
(Agilent) and rescued this virus using plasmid-based reverse genetics techniques (11).
59
This ts, att virus (referred to henceforth as PR8 LAIV) has been previously characterized
60
in cell culture, but its phenotype in mice was not demonstrated (8). PR8 wild-type (WT)
61
virus has a 50% lethal dose (LD50) in C57BL/6 (B6) mice of 10 – 25 plaque forming
62
units (12, 13). Thus we sought to ascertain the LD50 of PR8 LAIV (Fig. 1). Groups of
63
mice (n = 5) were intranasally inoculated with 10-fold serial dilutions of PR8 LAIV (106
64
– 103 focus forming units [FFU]/mouse), and signs of morbidity (percent loss in body
65
weight) were monitored daily, sacrificing animals that lost greater than 25% of their
66
initial weight (Fig. 1A). While PR8 LAIV was indeed lethal at doses ≥ 105 FFU, it
67
exhibited no lethality in this experiment below 104 FFU (Fig. 1B). Therefore, by
68
introducing the four remaining ts, att mutations of LAIV into PR8, the LD50 shifted to
69
3.16 x 104 FFU (using the method of Reed & Muench, (14)), >1,000 fold greater than
70
WT. Additionally, consistent with FluMist in humans (10, 15), PR8 LAIV replicated in
71
the airways, albeit to lower levels than PR8 WT (Fig. 1C). It is important to note that,
72
unlike humans, mice show a lower body temperature upon influenza infection (16).
73
Therefore, the replication of PR8 LAIV in mouse lungs is fully consistent with the ts
74
phenotype of virus as the lung temperature would drop upon infection– and it also
75
suggests that temperature sensitivity is not likely to be the sole mechanism of attenuation
76
of PR8 LAIV, at least in mice.
77
To evaluate the protection conferred by PR8 LAIV vaccination, mice were primed
78
with PBS or the highest dose that showed no overt weight loss (103 FFU), and 14 days
79
later challenged with 10 LD50 of homologous PR8 (n = 9-11) (Figs. 2A-C). Whereas
80
mice mock-immunized with PBS all rapidly lost weight and succumbed by day seven
81
postchallenge, PR8 LAIV-primed mice maintained body weight and survived (Figs. 2A
82
& 2B). The ability for PR8 LAIV-primed mice to overcome homologous challenge was
83
not surprising, as one day prior to challenge, the sera contained high titers of PR8
84
hemagglutination inhibition (HAI) activity (Table 1), indicative of the induction of virus-
85
neutralizing humoral immunity. This is also exemplified by the lack of detectable
86
challenge virus in the lungs of immunized mice at three and six days postchallenge, while
87
mock-immunized animals showed challenge virus replication of up to 106 FFU/ml lung
88
tissue (Fig. 2C).
89
Current influenza vaccines are reformulated each year due to the changing
90
antigenicity of the virus, where virus-neutralizing humoral immunity typically only
91
confers protection against matched influenza strains (17). Thus it is desirable to develop
92
vaccines that target conserved viral epitopes. To ascertain whether PR8 LAIV confers
93
protection against heterologous challenge, mice were vaccinated as above prior to
94
challenge with 10 LD50 of X31, a recombinant influenza virus that contains the HA and
95
NA genes derived from A/Hong Kong/1/1968 (H3N2), and the remaining six segments
96
from PR8 (18) (Figs. 2 D-F). Antibodies generated from H1N1 viral infections do not
97
typically neutralize H3N2 isolates (19-22), allowing us to evaluate cross-protective
98
immunogenicity. After challenge with X31 virus, mice from both mock and PR8 LAIV
99
immunized cohorts rapidly lost weight postchallenge, likely due to an absence of X31
100
neutralizing antibodies (Table 1). The loss of body weight in mock-immunized animals
101
progressed to fatality, but all the PR8 LAIV animals regained weight on day 4,
102
completely recovered by day 7 postchallenge, and survived (Figs. 2D & 2E). Although
103
the onset of disease was similar for both groups, viral lung burden was significantly
104
reduced (>3.5 logs) in PR8 LAIV compared to PBS immunized mice on day three
105
postchallenge (Fig. 2F). This heterosubtypic immunity is consistent with the previously
106
reported ability of LAIV to induce flu-specific, lung-tropic CD8 cytotoxic T cells (23-
107
26).
108
FluMist is now preferentially recommended over inactivated vaccines for children
109
aged 2-8 (27), but contraindications and safety concerns in children under 2 years of age
110
prevent universal licensure. As a result, it is highly desirable to increase the tolerability of
111
FluMist in order to broaden the target population for this effective and needle-free
112
vaccine. Similarly, development of improved LAIV strains requires analyses to ensure
113
comparable safety with FluMist. However, to the best of our knowledge, no reliable
114
animal models have been created thus far to efficiently evaluate the safety of LAIV. As a
115
proof of concept, we sought to further attenuate PR8 LAIV by introducing three
116
previously described mutations into NS1 (11C) that had been shown to affect viral egress
117
and budding in a temperature sensitive manner (Fig. 3A) (28) . We introduced the three ts
118
mutations into the NS1 gene of WT PR8 (11C) or PR8 LAIV (LAIV-11C) via site
119
directed mutagenesis and rescued these viruses as described above. We then confirmed
120
the ts phenotype of these viruses by plaque assay and growth kinetics in MDCK cells
121
(Figs. 3B & 3C). As shown in Fig. 3B, the plaque size of LAIV-11C was reduced
122
compared to LAIV; this can likely be attributed to the 11C mutations affecting virus
123
egress/budding (28).
124
Consistent with previous results, 11C virus was most drastically attenuated at
125
39C compared to WT PR8 (28), whereas LAIV showed severe, slight, and absent
126
attenuation at 39, 37, and 33C, respectively (8). When 11C mutations were introduced in
127
the context of LAIV (PR8 LAIV-11C), a moderate growth defect compared to PR8 LAIV
128
was observed at 72 h postinfection in MDCK cells at 37C (P = 0.02; two-tailed
129
Student’s t test).
130
We next sought to evaluate if the ts phenotype of 11C could further attenuate PR8
131
LAIV in vivo. Mice were infected as above with serial dilutions of 11C or LAIV-11C
132
(107 – 103 FFU) (Fig. 4) and body weight and survival were then measured over a 14-day
133
period. As expected, PR8 11C demonstrated an attenuated phenotype (LD50
1
Development of a mouse-adapted, live-attenuated influenza virus that permits in
2
vivo analysis of enhancements to the safety of LAIV.
3
Andrew Cox1, Steven F. Baker1, Aitor Nogales, Luis Martínez-Sobrido* and Stephen
4
Dewhurst*
5 6
Department of Microbiology and Immunology, University of Rochester, 601 Elmwood
7
Avenue, Rochester, New York 14642
8 1
These authors contributed equally to this work
11
*
To whom correspondence should be addressed:
12
Luis Martínez-Sobrido
13
Department of Microbiology and Immunology
14
University of Rochester School of Medicine and Dentistry
15
601 Elmwood Avenue, Rochester, NY 14642
16
Phone: +1 585 276 4733; fax: +1 585 473 9573
17
e-mail: [email protected]
9 10
18 19
Stephen Dewhurst
20
Department of Microbiology and Immunology
21
University of Rochester School of Medicine and Dentistry
22
601 Elmwood Avenue, Rochester, NY 14642
23
Phone: +1 585 275 3216; fax: +1 585 473 9573
24
e-mail: [email protected]
25 26 27
Running title: Mouse-adapted LAIV
28
ABSTRACT
29 30
The live attenuated influenza vaccine is preferentially recommended for use in
31
persons 2 through 49 years of age, but not approved for children under 2 or asthmatics
32
due to safety concerns. Therefore, increasing safety is desirable. Here we describe a
33
murine LAIV with reduced pathogenicity that retains lethality at high doses and further
34
demonstrate that we can enhance safety in vivo through mutations within NS1. This
35
model may permit preliminary safety analysis of improved LAIVs.
36
37
BODY
38 39
Influenza A virus is a respiratory pathogen that infects through the upper airway,
40
and leads to pathology via replication in the lower airway (1). The temperature gradient
41
between these two areas in people enabled the development of the cold-adapted, live
42
attenuated influenza vaccine (LAIV, FluMist) that replicates in the cooler upper
43
respiratory tract to trigger a protective immune response, but cannot damage the lower
44
respiratory tract due to the elevated temperatures restricting replication (2). This
45
temperature sensitive (ts), attenuated (att) phenotype is imparted by five mutations within
46
the viral replicative machinery: namely PB2 N265S; PB1 K391E, D581G and A661T;
47
NP D34G (3, 4). Though this vaccine has an overall acceptable safety profile, it is not
48
approved for use in children under two due to concerns of elevated hospitalizations due to
49
wheezing (5, 6). For this reason it is also not approved for use in asthmatics. Therefore,
50
development of vaccines with increased safety over LAIV is desirable. Currently, no
51
mouse model exists for the adequate assessment of the safety of the LAIV. In
52
experimental animal studies with LAIV, elevated doses of LAIV do not elicit pathology,
53
rendering determination of safety impossible (7-10). Here, we describe a model with
54
which we can assess alterations in vaccine safety.
55
The parental strain for our vaccine is a well-characterized, murine-lethal strain of
56
influenza A virus (A/Puerto Rico/8/34 H1N1, PR8). We introduced four ts, att mutations
57
from LAIV into PR8 (NP D34G is natively present) via site directed mutagenesis
58
(Agilent) and rescued this virus using plasmid-based reverse genetics techniques (11).
59
This ts, att virus (referred to henceforth as PR8 LAIV) has been previously characterized
60
in cell culture, but its phenotype in mice was not demonstrated (8). PR8 wild-type (WT)
61
virus has a 50% lethal dose (LD50) in C57BL/6 (B6) mice of 10 – 25 plaque forming
62
units (12, 13). Thus we sought to ascertain the LD50 of PR8 LAIV (Fig. 1). Groups of
63
mice (n = 5) were intranasally inoculated with 10-fold serial dilutions of PR8 LAIV (106
64
– 103 focus forming units [FFU]/mouse), and signs of morbidity (percent loss in body
65
weight) were monitored daily, sacrificing animals that lost greater than 25% of their
66
initial weight (Fig. 1A). While PR8 LAIV was indeed lethal at doses ≥ 105 FFU, it
67
exhibited no lethality in this experiment below 104 FFU (Fig. 1B). Therefore, by
68
introducing the four remaining ts, att mutations of LAIV into PR8, the LD50 shifted to
69
3.16 x 104 FFU (using the method of Reed & Muench, (14)), >1,000 fold greater than
70
WT. Additionally, consistent with FluMist in humans (10, 15), PR8 LAIV replicated in
71
the airways, albeit to lower levels than PR8 WT (Fig. 1C). It is important to note that,
72
unlike humans, mice show a lower body temperature upon influenza infection (16).
73
Therefore, the replication of PR8 LAIV in mouse lungs is fully consistent with the ts
74
phenotype of virus as the lung temperature would drop upon infection– and it also
75
suggests that temperature sensitivity is not likely to be the sole mechanism of attenuation
76
of PR8 LAIV, at least in mice.
77
To evaluate the protection conferred by PR8 LAIV vaccination, mice were primed
78
with PBS or the highest dose that showed no overt weight loss (103 FFU), and 14 days
79
later challenged with 10 LD50 of homologous PR8 (n = 9-11) (Figs. 2A-C). Whereas
80
mice mock-immunized with PBS all rapidly lost weight and succumbed by day seven
81
postchallenge, PR8 LAIV-primed mice maintained body weight and survived (Figs. 2A
82
& 2B). The ability for PR8 LAIV-primed mice to overcome homologous challenge was
83
not surprising, as one day prior to challenge, the sera contained high titers of PR8
84
hemagglutination inhibition (HAI) activity (Table 1), indicative of the induction of virus-
85
neutralizing humoral immunity. This is also exemplified by the lack of detectable
86
challenge virus in the lungs of immunized mice at three and six days postchallenge, while
87
mock-immunized animals showed challenge virus replication of up to 106 FFU/ml lung
88
tissue (Fig. 2C).
89
Current influenza vaccines are reformulated each year due to the changing
90
antigenicity of the virus, where virus-neutralizing humoral immunity typically only
91
confers protection against matched influenza strains (17). Thus it is desirable to develop
92
vaccines that target conserved viral epitopes. To ascertain whether PR8 LAIV confers
93
protection against heterologous challenge, mice were vaccinated as above prior to
94
challenge with 10 LD50 of X31, a recombinant influenza virus that contains the HA and
95
NA genes derived from A/Hong Kong/1/1968 (H3N2), and the remaining six segments
96
from PR8 (18) (Figs. 2 D-F). Antibodies generated from H1N1 viral infections do not
97
typically neutralize H3N2 isolates (19-22), allowing us to evaluate cross-protective
98
immunogenicity. After challenge with X31 virus, mice from both mock and PR8 LAIV
99
immunized cohorts rapidly lost weight postchallenge, likely due to an absence of X31
100
neutralizing antibodies (Table 1). The loss of body weight in mock-immunized animals
101
progressed to fatality, but all the PR8 LAIV animals regained weight on day 4,
102
completely recovered by day 7 postchallenge, and survived (Figs. 2D & 2E). Although
103
the onset of disease was similar for both groups, viral lung burden was significantly
104
reduced (>3.5 logs) in PR8 LAIV compared to PBS immunized mice on day three
105
postchallenge (Fig. 2F). This heterosubtypic immunity is consistent with the previously
106
reported ability of LAIV to induce flu-specific, lung-tropic CD8 cytotoxic T cells (23-
107
26).
108
FluMist is now preferentially recommended over inactivated vaccines for children
109
aged 2-8 (27), but contraindications and safety concerns in children under 2 years of age
110
prevent universal licensure. As a result, it is highly desirable to increase the tolerability of
111
FluMist in order to broaden the target population for this effective and needle-free
112
vaccine. Similarly, development of improved LAIV strains requires analyses to ensure
113
comparable safety with FluMist. However, to the best of our knowledge, no reliable
114
animal models have been created thus far to efficiently evaluate the safety of LAIV. As a
115
proof of concept, we sought to further attenuate PR8 LAIV by introducing three
116
previously described mutations into NS1 (11C) that had been shown to affect viral egress
117
and budding in a temperature sensitive manner (Fig. 3A) (28) . We introduced the three ts
118
mutations into the NS1 gene of WT PR8 (11C) or PR8 LAIV (LAIV-11C) via site
119
directed mutagenesis and rescued these viruses as described above. We then confirmed
120
the ts phenotype of these viruses by plaque assay and growth kinetics in MDCK cells
121
(Figs. 3B & 3C). As shown in Fig. 3B, the plaque size of LAIV-11C was reduced
122
compared to LAIV; this can likely be attributed to the 11C mutations affecting virus
123
egress/budding (28).
124
Consistent with previous results, 11C virus was most drastically attenuated at
125
39C compared to WT PR8 (28), whereas LAIV showed severe, slight, and absent
126
attenuation at 39, 37, and 33C, respectively (8). When 11C mutations were introduced in
127
the context of LAIV (PR8 LAIV-11C), a moderate growth defect compared to PR8 LAIV
128
was observed at 72 h postinfection in MDCK cells at 37C (P = 0.02; two-tailed
129
Student’s t test).
130
We next sought to evaluate if the ts phenotype of 11C could further attenuate PR8
131
LAIV in vivo. Mice were infected as above with serial dilutions of 11C or LAIV-11C
132
(107 – 103 FFU) (Fig. 4) and body weight and survival were then measured over a 14-day
133
period. As expected, PR8 11C demonstrated an attenuated phenotype (LD50
