Vaccine 33 (2015) 108–116
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Cross-protection against H7N9 influenza strains using a live-attenuated H7N3 virus vaccine Donald M. Carter a , Chalise E. Bloom a , Greg A. Kirchenbaum a , Vadim Tsvetnitsky b,1 , Irina Isakova-Sivak c , Larisa Rudenko c , Ted M. Ross a,∗ a
Vaccine and Gene Therapy Institute of Florida, Port St. Lucie, FL, USA PATH, Washington, DC, USA c Department of Virology, Institute of Experimental Medicine, Saint Petersburg, Russia b
a r t i c l e
i n f o
Article history: Received 24 August 2014 Received in revised form 18 October 2014 Accepted 6 November 2014 Available online 18 November 2014 Keywords: Influenza H7N9 Live-attenuated Ferrets Antibodies
a b s t r a c t In 2013, avian H7N9 influenza viruses were detected infecting people in China resulting in high mortality. Influenza H7 vaccines that provide cross-protection against these new viruses are needed until specific H7N9 vaccines are ready to market. In this study, an available H7N3 cold-adapted, temperature sensitive, live attenuated influenza vaccine (LAIV) elicited protective immune responses in ferrets against H7N9 viruses. The H7N3 LAIV administered alone (by intranasal or subcutaneous administration) or in a prime-boost strategy using inactivated H7N9 virus resulted in high HAI titers and protected 100% of the animals against H7N9 challenge. Naïve ferrets passively administered immune serum from H7N3 LAIV infected animals were also protected. In contrast, recombinant HA protein or inactivated viruses did not protect ferrets against challenge and elicited lower antibody titers. Thus, the H7N3 LAIV vaccine was immunogenic in healthy seronegative ferrets and protected these ferrets against the newly emerged H7N9 avian influenza virus. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction A new avian-origin influenza virus emerged near Shanghai in February 2013, and by the end of June 2014 it had caused 450 human infections and 165 deaths [1]. Human-to-human transmission of avian-origin H7N9 influenza A has been limited to a few family clusters, but the high case mortality rate (over 35%) associated with human infection has raised concern about the potential for this virus to become a significant human pathogen. Early results from human studies evaluating various forms of vaccine against H7N9 demonstrated poor immunogenicity [2–8]. Should H7N9 develop pandemic potential, novel strategies for improving vaccine immunogenicity for this unique low-immunogenicity strain of avian-origin influenza will be needed. In the event of a pandemic threat, the use of live attenuated cold-adapted reassortant influenza vaccine (LAIV) may have an advantage over inactivated vaccines due to its ability to provide a
∗ Corresponding author. Tel.: +1 772 345 5693; fax: +1 772 345 0625. E-mail address: tross@vgtifl.org (T.M. Ross). 1 Current address: Vaccine Development, International AIDS Vaccine Initiative (IAVI), 125 Broad St, New York, NY 10004, USA. Tel.: +1 212 847 1070/+1 646 287 9127.
[email protected] http://dx.doi.org/10.1016/j.vaccine.2014.11.008 0264-410X/© 2014 Elsevier Ltd. All rights reserved.
broader and more long-lasting cross protective immune response [9,10]. The LAIVs have been used extensively in Russia over 40 years and in the USA since 2003 to control seasonal influenza and shown to be safe, immunogenic, and highly protective among all populations, as well as providing a significant level of community-wide herd immunity [11–16]. Therefore, development of live attenuated influenza vaccines would increase preparedness against newly emerging pandemic influenza strains. The candidates for Russian live attenuated influenza A vaccines are generated by methods of classical reassortment of epidemiologically relevant strains with comprehensively studied attenuated (att) temperature-sensitive (ts), cold-adapted (ca) A/Leningrad/134/17/57 (H2N2) master donor virus (MDV-L17). MDV-L17 was derived from A/Leningrad/134/57 (H2N2) (Lwt) the wild type virus-precursor as a result of 17 passages in eggs at reduced (25 ◦ C) temperature. It differs from Lwt by eight coding mutations in internal genes. Mutations in the polymerase genes of MDV-L17 were found to be responsible for its att/ts/ca phenotype. Creation of 6:2 vaccine reassortant by replacement of internal genes of wild type (wt) virus with the appropriate internal genes of MDV is a reliable and reproducible method of attenuation of wt viruses [17]. Internal viral proteins of MDV provide attenuation of vaccine reassortant ensuring its safety [9], and surface glycoproteins, hemagglutinin (HA) and neuraminidase
D.M. Carter et al. / Vaccine 33 (2015) 108–116
(NA), of the wild type virus provide the targets for an adequate immune response in vaccinated host. Our group has developed live attenuated influenza vaccine(s) (LAIV) against several strains of influenza, including H7N3 [18–20]. These vaccine candidates use the A/Leningrad/cold-adapted backbone in 6:2 or 7:1 gene constellation reassortant viruses [21]. The H7N3 6:2 reassortant expresses the hemagglutinin and neuraminidase from low pathogenic A/mallard/Netherlands/12/2000 strain that is closely related to the H7N7 viruses responsible for highly pathogenic avian influenza outbreaks in the Netherlands and Germany in 2003. The resulting A/17/mallard/Netherlands/00/95 (H7N3) LAIV vaccine was safe and immunogenic in both pre-clinical [18] and Phase 1 clinical studies [20]. Given the sequence similarities between hemagglutinin (HA) molecule in the H7N3 live-attenuated vaccine and the HA sequence of the H7N9 strains isolated from humans in Shanghai in 2013 and other lines of evidence, we conducted animal studies to determine whether the H7N3 LAIV vaccine could offer cross-protection against these novel H7N9 viruses. 2. Materials and methods 2.1. Viruses Pre-pandemic vaccine candidate A/17/mallard/Netherlands/00/95 (H7N3) is a live attenuated, cold–adapted (ca), temperaturesensitive (ts) influenza reassortant virus generated in embryonated chicken eggs by classical reassortment between the avian influenza wt A/mallard/Netherlands/12/2000 (H7N3) virus (CDC, Atlanta, GA) in humans and A/Leningrad/134/17/57 (H2N2) ca/ts Russian MDV-L17 [17,18,20]. Briefly, embryonated chicken eggs (CE) were co-infected with MDV-L17 and H7N3 virus as previously described [17]. Five rounds of selective propagation were performed, three of which occurred at low temperature (25–26 ◦ C). The production and selection of reassortants was carried out in the presence of rabbit antiserum to MDV-L17. The virus contains six gene segments encoding the internal proteins from the MDV and the HA and NA genes from the wtH7N3 virus (6:2 genomic composition). Influenza virus wt isolates representing both subtypes H7N9 and H7N3 were used in this study. H7N9 and H7N3 viruses were obtained from CDC (Atlanta, GA). Abbreviations for the H7N9 viral isolates were A/Anhui/1/2013 (Anh/13). For the H7N3 isolate, A//mallard/Netherlands/12/2000, the abbreviation NL/00 was used. In order to inactivate the Anh/13 virus, pre-titrated virus was inactivated with 0.1% beta-propriolactone to make a whole virus inactivated preparation (inactivated H7N9 virus) [22]. To ensure complete inactivation, an aliquot of inactivated virus was used to infect MDCK cells to verify the lack of cytopathic effect (data not shown). All viruses were propagated in 10–11 day old embryonated chicken eggs. LAIV reassortant and parental viruses were grown at optimum temperatures for ts, ca viruses (33 ◦ C), low (25 ◦ C) or elevated (38 ◦ C, 39 ◦ C and 40 ◦ C) temperatures as determined by titration in CE. The log10 EID50 /ml calculation was based on the Reed and Muench method [23]. Viruses were considered as possessing ts phenotype if the titer at temperature 38 C and above was ≤4.2 log10 EID50 /ml when titrated in 10–11 day old embryonated CE. Viruses were considered as having a ca phenotype if the titer at low temperature of 25C was ≥5.7 log10 EID50 /ml. 2.2. Animals and vaccinations Fitch ferrets (Mustela putorius furo, male, 6–12-months of age), influenza naïve and descented were purchased from Triple F Farms (Sayre, PA, USA). Ferrets were pair housed in stainless steel cages (Shoreline, Kansas City, KS, USA) containing Sani-chips
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Laboratory Animal Bedding (P.J. Murphy Forest Products, Montville, NJ, USA). Ferrets were provided with Teklad Global Ferret Diet (Harlan Teklad, Madison, WI, USA) and fresh water ad libitum. Ferrets (n = 6 vaccine/challenge group) were vaccinated with one of six vaccine regimens listed in Table 1. One group of ferrets was administered the H7N3 LAIV vaccine (1 × 10e + 7 plaque forming units (pfu) in 0.5 ml) twice intranasally on day 0 and 28. Additional groups of ferrets were vaccinated intramuscularly with recombinant HA (derived from H7N9) (Protein Sciences, Meriden, CT) or an inactivated H7N9 virus with or without adjuvant; Emulsigen (MVP Technologies, Omaha, NE, USA). Adjuvant and antigen were mixed according to manufacturer’s instructions just prior to vaccination. For passive immunization, serum samples were pooled (1 ml per each of the 6 ferret serum samples) from ferrets previously vaccinated with the H7N3 LAIV. Each individual antiserum collected from vaccinated ferrets recognized all three H7N9 strains with titers that varied from 1:40 to 1:320. Serum from 6 ferrets was pooled together and the pooled serum had an HAI titer of 1:128. Blood was collected from anesthetized ferrets via the anterior vena cava. Blood was transferred to a tube containing a serum separator and clot activator and allowed to clot at room temperature. Tubes were centrifuged and sera was removed and frozen at −80 ± 5 ◦ C. All procedures were in accordance with the NRC Guide for the Care and Use of Laboratory Animals, the Animal Welfare Act, and the CDC/NIH Biosafety in Microbiological and Biomedical Laboratories. On day 35, ferrets were challenged intranasally with 1 × 106 pfu/ml in 1 ml of 0.9% saline of the H7N9 virus Anh/13 or H7N3 virus NL/00 in a volume of 0.5 ml. Following infection, ferrets were monitored daily for weight loss, disease signs and death for 14 days. Nasal washes were collected in a volume of 3 ml of sterile 0.9% saline at days 1, 2, 3, 5, 7 and 9 post-infection. All H7N9 influenza virus studies were performed under high-containment biosafety level 3 enhanced conditions (BSL3+). 2.3. Hemagglutination inhibition (HAI) assay The HAI assay was used to assess functional, HA-specific antibodies able to inhibit agglutination of turkey erythrocytes. The protocol was adapted from the CDC laboratory-based influenza surveillance manual [24]. To inactivate non-specific inhibitors, sera were treated with receptor destroying enzyme (RDE; Denka Seiken, Co., Japan) prior to being tested [25–32]. Briefly, three parts RDE was added to one part sera and incubated overnight at 37 ◦ C. RDE was inactivated by incubation at 56 ◦ C for ∼30 min. RDE treated sera was two-fold serially diluted in v-bottom microtiter plates. An equal volume of reassortant virus, adjusted to approximately 8 HAU/50 l, was added to each well. The plates were covered and incubated at room temperature for 20 min followed by the addition of 1% turkey erythrocytes (TRBC) (Lampire Biologicals, Pipersville, PA, USA) in PBS. TRBC were stored at 4 ◦ C and used within 72 h of preparation. The plates were mixed by agitation, covered, and the RBCs were allowed to settle for 1 h at room temperature [33]. The HAI titer was determined by the reciprocal dilution of the last well that contained non-agglutinated TRBC. Positive and negative serum controls were included for each plate. All ferrets were negative (HAI ≤1:10) for pre-existing antibodies to currently circulating human seasonal influenza viruses prior to vaccination. 2.4. Plaque assay Madin-Darby canine kidney (MDCK) cells were plated (5 × 105 ) in each well of a 6-well plate. Samples were diluted (final dilution factors of 100 to 10−6 ) and overlayed onto the cells in 1−l of DMEM supplemented with penicillin-streptomycin and incubated for 1 h. Samples were removed, cells were washed twice and media was replaced with 2 ml of L15 medium plus 0.8% agarose (Cambrex;
110
Group
Vaccine
Route of administration
Dose
Adjuvant
Bleeds (Days)
Challenge (D35)
1A 1B 2A 2B 3A 3B 4A 4B 5A 5B 6A 6B
LAIV H7N3
IN (two doses) on D0 and D28
1.00E + 08
None
Day 0, 27 and 35
LAIV H7N3
SC (one dose) on D0
1.00E + 08
None
H7N9 HA1 protein
IM (two doses) on D0 and D28
15 g
Emulsigen
H7N9 H7N3 H7N9 H7N3 H7N9
Inactivated H7N9 virus
IM (two doses) on D0 and D28
15 g
Emulsigen
H7N9
LAIV H7N3/Inactivated H7N9 virus
IN prime with LAIV on D0 and IM boost on D28 with WIV
10e + 7/15 g
None/emulsigen
PBS
IM (two doses) on D0 and D28
0
None
H7N9 H7N3 H7N9 H7N3
D.M. Carter et al. / Vaccine 33 (2015) 108–116
Table 1 Experimental groups and study design.
D.M. Carter et al. / Vaccine 33 (2015) 108–116
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East Rutherford, NJ, USA) and incubated for 72 h at 37 ◦ C with 5% CO2 . Agarose was removed and discarded. Cells were fixed with 10% buffered formalin, and then stained with 1% crystal violet for 15 min. Following thorough washing in dH2 O to remove excess crystal violet, plates were allowed to dry, plaques counted, and the pfu/ml were calculated. 2.5. ELISA Immulon 4HBX ELISA plates (Thermo) were coated overnight at 4 ◦ C with full-length Anh/13 rHA (Protein Sciences) or cH4/7 protein (kindly provided by Florian Krammer) in carbonate buffer (pH 9.4). The chimeric HA (cH4/7) used as the coating antigen in the ELISA assays described herein our manuscript consists of an exotic globular head domain, derived from A/duck/Czech/1956 (H4), which was grafted on to the stalk domains (encoded by both HA1 and HA2 domains) of the WT H7 (A/Anhui/1/2013). Plates were treated with blocking buffer containing 2% w/v bovine serum albumin (BSA) (Equitec-Bio, Kerrville, TX), 1% w/v bovine gelatin (Sigma, St. Louis MO, USA) and 0.05% v/v Tween 20 (Sigma) in PBS pH 7.5 at 37 ◦ C for 1 h. Serum samples were diluted in blocking buffer and plates incubated overnight at 4 ◦ C. After thorough washing with PBS to remove unbound antibody, biotinylated goat anti-ferret IgG secondary antibody (Sigma) was added and plates incubated at 37 ◦ C for 1 h. Plates were washed with PBS, SA-HRP added and plates incubated at 37 ◦ C for 35 min. After washing with PBS, 2,2 -azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) substrate (Amresco, Solon, OH, USA) diluted to 1 mg/ml in McIlvain’s buffer pH 5.0 containing 0.5% v/v H2 O2 was added and plates incubated at 37 ◦ C for 35 min. Colorimetric conversion was terminated with 5% sodium dodecyl sulfate solution (Sigma) and optical density (O.D. 414 nm) was measured using the PowerWave XS microplate spectrophotometer (Biotek, Winooski, VT, USA). 2.6. Statistical analysis Statistical significance of the antibody data was determined using a two-way analysis of variance (ANOVA) with Bonferroni’s post-test to analyze differences between each vaccine group for the different test antigens (multiparametric). Differences in weight loss, sickness score, and viral titers were analyzed by two-way ANOVA, followed by Bonferroni’s post-test for each vaccine group at multiple time points. Significance was defined as p < 0.05. Statistical analyses were done using GraphPad Prism software.
Fig. 1. Hemagglutination-inhibition (HAI) serum antibody titers induced by single H7N3 LAIV vaccination of ferrets. HAI titers were determined using of H7N3 LAIV vaccinated ferrets against A/Anhui/1/2013 (H7N9) influenza virus collected at day 14 post-vaccination. (A). Individual ferret geometric mean titers are listed as a dot on the y-axis and the average of the 11 ferrets is shown as a line with +S.E.M. (B) Pooled serum samples from the same 11 ferrets was tested in an HAI assays against the same H7N3 influenza virus. Bars represents the geometric mean titer (+S.E.M.) from antisera.
had a rapid drop in viral titers (Fig. 2C) and little to no weight loss (Fig. 2D). 3.2. Active vaccination In order to determine if the H7N3 LAIV vaccines would elicit a protective immune responses following active vaccination, ferrets were immunized with the H7N3 LAIV vaccine either intranasally or subcutaneously (Table 1). This vaccine was compared to three prime-boost vaccine regimens: rHA, Inactivated H7N9 virus, or a combination of LAIV prime and an inactivated H7N9 virus boost. Emulsigen (MVP Technologies, Omaha, NE, USA) was used with the rHA and inactivated virus vaccines to enhance vaccine efficacy. At day 35 post-vaccination, almost all ferrets had HAI titers against the H7N9 virus, but unexpectedly titers against the H7N3 virus were lower (Fig. 3). Ferrets infected with H7N3 LAIV by either route, including single subcutaneous administration, had a range of HAI titer against the H7N9 virus between 1:256 and 1:400, which was similar to ferrets vaccinated twice with rHA plus Emulsigen, the inactivated H7N9 virus plus Emulsigen, or the LAIV prime/inactivated virus boost vaccine (Fig. 3). Ferrets vaccinated with rHA or inactivated virus without Emulsigen adjuvant elicited low HAI titers (1:40) in ∼50% of the vaccinated animals.
3. Results 3.3. ELISA 3.1. Passive vaccination Previously, as part of pre-clinical development of a live-attenuated H7N3 virus vaccine our group vaccinated ferrets intranasally with this vaccinated four weeks postvaccination collected serum samples with high HAI titer against A/17/mallard/Netherlands/00/95 (unpublished data). In this study, we tested this antiserum against the H7N9 virus, Anh/13, isolated in 2013 (Fig. 1). The pooled serum from LAIV H7N3 positive or immunologically influenza-negative ferrets was passively transferred to six immunologically naïve ferrets, which then were challenged 24 h later with Anh/13. Ferrets administered control serum from anti-influenza negative ferrets had high H7N9 viral titers over 7 days of observation (Fig. 2A) with a 15–20% weight loss by day 8–10 post-infection (Fig. 2B). One ferret had severe signs of morbidity and was euthanized at day 5 post-infection. In contrast, ferrets administered sera from ferrets previously infected with H7N3 LAIV vaccine and then challenged with the H7N9 virus
Since HA stalk-specific antibodies are not detected in the HAI assay, we performed ELISAs using full-length H7N9 rHA or a rHA chimeric protein (cH4/7) possessing the globular head domain of A/duck/Czech/1956 (H4) and the stalk domain of the H7N9 virus, A/Anhui/1/2013. Ferrets infected with H7N3 LAIV or vaccinated with inactivated virus, had antibodies (IgG+ ) that recognized both the full-length H7 and the cH4/7 proteins (Fig. 4). Moreover, we also detected a significant increase in the antibody titer against full-length H7 in ferrets boosted with inactivated virus plus the Emulsigen adjuvant (* p < 0.05). Furthermore, regardless of prior treatment, all ferrets exhibited a significant increase in antibody titer to the full-length H7 rHA 14 days following the H7N9 challenge (Anh/13) (*** p < 0.001). When we evaluated the sera for evidence of H7 stalk-specific antibodies using the cH4/7 rHA, we observed similar titers (IgG+ ) among all groups 3 weeks following either LAIV infection or inactivated virus vaccination. We also detected a slight, but still significant, increase in the stalk-specific antibody
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Fig. 2. Infection of ferrets with H7N9 influenza virus following passive transfer of ferret antiserum. Antiserum from 11 ferrets was pooled with equal volumes and 15 ml of the pooled antiserum was administered intravenously to 6 ferrets. Twenty-four hours later, ferrets were infected with 10e + 6 pfu of A/Anhui/1/2013 (H7N9) and observed for 14 days. Naïve ferrets (no passive transfer of antiserum) are shown in panel (A) nasal viral titers and (B) Percent original weight is shown +S.E.M. Ferrets passively administered passive antiserum from H7N3 LAIV vaccinated ferrets are shown in panel (C) nasal viral titers and (D) Percent original weight is shown +S.E.M.
titer in ferrets vaccinated twice with inactivated virus plus Emulsigen adjuvant relative to the other treatment groups at week 5 (* p < 0.05). Despite the increased HA stalk-specific titer in these animals prior to challenge, H7N9 challenge did not further boost the titer. In contrast, ferrets previously infected with LAIV (IN or SC) exhibited a significant increase in the magnitude of the H7 stalk-specific antibody response following H7N9 challenge relative to their week 5 titer (* p < 0.05). 3.4. H7 Influenza virus challenge All groups of ferrets were challenged with the 2013 Anh/13 strain of the H7N9 virus. Ferrets vaccinated with LAIV by intranasal HAI Titers
256
LAIV H7N3-IN LIAVH7N3-SC rHA rHA + Emulsigen
128
Inactivated H7N9
1024
HAI Titer
512
Inactivated H7N9 + Emulsigen LAIV H7N3/Inactivated H7N9
64 32
Mock
16 8 H7N3
H7N9
Vaccine
Fig. 3. Hemagglutination-inhibition (HAI) serum antibody titers induced by direct vaccination of ferrets. HAI titers were determined for each group using of vaccinated ferrets against A/Anhui/1/2013 (H7N9) influenza virus. (A). Bars represent the geometric mean titer (±S.E.M.) from antisera collected at day 35.
or subcutaneous routes had little weight loss (Fig. 5) and no signs of morbidity. Similar results were observed in ferrets primed with LAIV and boosted with Inactivated H7N9 virus. In contrast, ferrets vaccinated with Inactivated H7N9 virus quickly lost weight and as an average had ∼85% of their original body weight by day 8 postinfection. These results were almost identical to mock vaccinated and virus challenged ferrets (Fig. 5A). These ferrets did not recover their body weight during the two weeks of observation, regardless of the use of adjuvant. Interestingly, even though ferrets vaccinated with the rHA vaccine and mixed with adjuvant lost weight, they recovered ∼10% of their weight back by day 14, whereas the ferrets administered the Inactivated H7N9 virus vaccine did not (Fig. 5A). Groups of ferrets vaccinated with LAIV vaccines or placebo were also challenged with the H7N3 virus (Fig. 5B) as control. All vaccinated ferrets had no weight loss and no signs of morbidity. Only Mock vaccinated ferrets lost ∼15% weight, as expected, with signs of lethargy and dehydration. Viral titers following challenge were assessed from ferret nasal washes (Fig. 6). Mock-vaccinated ferrets that were challenged with the H7N9 virus (1 X10e + 6 pfu) had a rise in viral titers during the first three days post-infection (Fig. 6A). The viral titers declined slowly to ∼10e + 4 at day 9 post-infection. Ferrets vaccinated with H7N3 LAIV vaccine had a more rapid decline in viral titers and were below the level of detection between days 7 and 9 post-infection. Ferrets vaccinated with any of the other vaccines had viral titers that were similar to mock vaccinated ferrets. Similar trends were observed in LAIV and mock vaccinated ferrets that were challenged with the H7N3 virus (Fig. 6B).
D.M. Carter et al. / Vaccine 33 (2015) 108–116
B
C
Wk 3 - A/Anhui/1/2013 rHA
Wk 5 - A/Anhui/1/2013
2
+
9
ac tiv
at
Em
ed
ul
H
si
7N
ge n
C 3 7N 9
In
7N
-- >
H d
7N
3
at e IV
H
ct iv
LA
In a
9 7N H d te
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ed
- ->
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7N
In a
9
ct
+
iv a
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H
ti v In ac
7N H IV LA
n
3
3 N H7 IV LA
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ed at ti v
Em ul si ge
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ct In a
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ed
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9
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+
te
H 7N IV
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Em ul si ge
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-I N 3 7N LA In ac
2
0
0 n
0
2
4
IV
2
*
* 4
LA
4
14 dpi - cH4/7 6
* Log End-Point Titerr L
Log End-Point Titerr L
6
H
-S
-I 3 N H7 IV LA
Wk 5 - cH4/7
6
IV
N
9 7N H ed at
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3 7N H IV LA
F
Wk 3 - cH4/7 Log End-Point Titerr L
ge n
C
N
9 H ed at
ac tiv In -- >
H d at e ct iv In a
E
D
2
0
7N
ge n si ul Em +
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9
LA
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H
7N
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3
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Log E End-Point Titer
4
LA
14 dpi - A/Anhui/1/2013
6
Log E End-Point Titer
Log E End-Point Titer
6
LA
A
113
Fig. 4. Determination by ELISA of elicited antibodies that detect the HA stem region. Antisera collected from ferrets vaccinated with H7N3 LAIV by IN (red bars) with H7N3 LAIV by SC (blue bars), inactivated H7N9 virus vaccine (purple bars), or a prime-boost of LAIV/inactivated H7N9 virus vaccine (white bars) were used in an ELISA to detect the stem region of HA. Full-length HA from the H7N9 virus, A/Anhui/1/2013, was used as a detection protein for the elicited antisera collected from vaccinated ferrets (Panels A–C). The same serum samples were used to probe a chimeric HA protein with an HA globular head from A/duck/Czech/1956 (H4) and the stalk domain of the H7N9 virus, A/Anhui/1/2013 (Panels D–F). Serum samples were tested collected at week 3 (Panels A and D), week 5 (Panels B and E), and 14 days post-infection with A/Anhui/1/2013 (Panels C and F). The error bars represent +S.E.M. Significance was determined using a 2-tailed paired t-test (* p < 0.05).
4. Discussion In early 2013, H7N9 avian influenza viruses emerged into human population. As of July, 2014, there have been 541 human H7N9 influenza virus infections, resulting in 145 deaths [1,34,35]. Genetic analyses identified that the new virus is a reassortant with HA from H7N3, NA from H7N9, and 6 internal genes from H9N2 avian influenza viruses and it carries amino acids associated with mammalian receptor binding. [1,34–37]. These newly isolated 2013 viruses have acquired an ability to break the species barrier to establish infection in humans. This enhanced virulence and possession of multiple mammalian adaptation markers, as well as the persistence of the virus in avian reservoir, suggest that H7N9 viruses have pandemic potential. To prevent infection by H7N9 viruses, vaccines are needed. But prior to the release of any specific H7N9 vaccines, all candidate vaccines have to pass a number of necessary safety tests that can take many months to years to complete. In order to prepare for a potential H7N9 pandemic, existing H7 vaccines can be used to prime the population and allow for rapid and protective immune responses. A number of H7 influenza vaccines have been developed in response to multiple outbreaks of H7 influenza viruses in
poultry and sporadic human infections. The majority of these vaccines are inactivated whole virion or split vaccines formulated with or without adjuvant. In this study, we determined if an H7N3 live-attenuated influenza virus vaccine could elicit cross-reactive antibodies to 2013 H7N9 isolates in ferrets and protect animals against challenge with these viruses. Earlier clinical trial using this LAIV H7N3 vaccine showed that it was safe and immunogenic against H7 avian influenza viruses [20]. This vaccine elicited cross-reactive HAI antibodies in the blood of volunteers against 2013 H7N9 viruses. This vaccine was prepared using low pathogenicity avian influenza virus A/mallard/Netherlands/12/2000 (H7N3), which is closely related to the H7N7 viruses responsible for highly pathogenic avian influenza outbreaks in the Netherlands and Germany in 2003. The H7N3 LAIV candidate A/17/mallard/Netherlands/00/1995 elicited measurable antibody responses in mice [18,20]. The attenuated nature of H7N3 LAIV was confirmed in naïve ferrets, in which the vaccine elicited immune responses that protected the animals from subsequent infection with wild-type (wt) H7N3 influenza virus challenge. One of the advantages to using LAIV vaccines is that they do not need an adjuvant to elicit high titer immune responses. Using HAI as a correlate of protection is not considered to be valid for LAIV, but
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D.M. Carter et al. / Vaccine 33 (2015) 108–116
A. LAIV H7N3-IN LIAV H7N3-SC rHA rHA + Emulsigen Inactivated H7N9 Inactivated H7N9 + Emulsigen LAIV H7N3/Inactivated H7N9 Mock
100 95 90 85 80 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Days Post Infection
B. Pe ercent Original Body Weig ght
H7N3 110
LAIV H7N3-IN H7N3 IN LIAV H7N3-SC LAIV H7N3/Inactivated H7N9 Mock
105 100 95 90 85 80 0
1
2
3
4
5
6
7
8
9 10 11 12 13 14
Days Post Infection
Fig. 5. Infection of ferrets infected with H7N9 or H7N3 influenza virus. Vaccinated ferrets were infected with 10e + 6 pfu of (A) A/Anhui/1/2013 (H7N9) or (B) A/NL//2005 and observed for 14 days for weight loss. Percent original weight is shown +S.E.M.
in this study recombinant HA or inactivated H7N9 virus elicited similar HAI titers as LAIV, but only when administered with an adjuvant (Fig. 3). Vaccination with rHA or inactivated virus without an adjuvant did not elicit detectable titers. Interestingly, the inactivated virus vaccine group had marked weight loss, morbidity, and viral titers in the nasal washes relative to animals vaccinated with LAIV either IN or SC. Both the full H7 HA and H7 HA stalk-specific ELISA showed that inactivated virus vaccine with Emulsigen elicited the highest titers to both antigens prior to H7N9 challenge (Fig. 4). This suggests that the presence of stalk-specific antibodies did not correlate with protective efficacy. However, the enhanced stalk-specific IgG titers elicited by inactivated virus vaccines may correlate with enhanced disease. Swine vaccinated with whole inactivated H1N2 virus vaccine had enhanced pneumonia and disease after challenge with pandemic
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H1N1 virus [38]. Epitope mapping identified antibodies to the stalk-region of HA correlated, in the absence of receptor-binding antibodies, enhanced viral infection and disease [38]. Live-attenuated influenza vaccines delivered via the nose elicit both humoral and cellular immune responses with cytokine response in the respiratory tract of vaccinated animals [10,39–43]. Confirming our hypothesis, ferrets in this study were protected against a 2013 H7N9 virus following direct vaccination with H7N3 LAIV (Fig. 4). Even though the HAI titers were similar between ferrets vaccinated with LAIV and other protein based vaccines in this study, there was a difference in weight loss between these groups (Fig. 5). LAIV vaccinated ferrets lost little or no weight, but the other vaccinated groups lost significant weight over the period of observation. Therefore, something other than HAI titers by IgG, such as sIgA, were necessary to achieve full protection against weight loss and disease. These LAIV vaccines could have elicited robust cellular response, particularly cytotoxic CD8+ T cell (CTL) response, but these responses are unlikely to be sterilizing, since cellular responses are more important for clearance of virally infected cells [44–46]. In mouse studies, CD8+ T cell responses alone were able to protect against influenza virus induced morbidity [45]. In passive transfer experiments (Fig. 2), sera from previously vaccinated ferrets protected immunologically naïve ferrets, indicating that antibodies alone are sufficient for protection. If HAI antibodies are not sufficient, then there must be another component in the sera elicited by LAIV, and not rHA or inactivated virus vaccines, which elicits sterilizing protection of ferrets to challenge. To address this possibility, sera from vaccinated ferrets was tested in HA stalk binding assays to determine if antibodies targeting other regions of the HA molecule, besides the globular head, were elicited by the LAIV vaccines and provided additional protection against the H7N9 challenge (Figs. 4 and 5). Live attenuated influenza vaccines are typically less expensive to produce and can be manufactured quicker and with higher yields than inactivated influenza vaccines. Through intranasal administration of LAIV, which mimics natural infection and engages humoral, cellular and mucosal immunity, it naturally lends itself to mass use in case of a pandemic, thus providing clear benefits for its use. However, persistent, yet never proven, concerns exist that attenuated LAIV may reassort with circulating virus during a pandemic and cause greater damage. Back in 1936, Shope described the use of subcutaneous administration of influenza virus for robust immunization without pathological effects typically associated
Days Post Infection
Fig. 6. Nasal wash viral titers from ferrets infected with H7N9 or H7N3 influenza virus. Vaccinated ferrets were infected with 10e + 6 pfu of (A) A/Anhui/1/2013 (H7N9) or (B) A/NL//2005 (H7N3) and nasal washes were collected. The amount of virus (pfu/ml) is shown +S.E.M.
D.M. Carter et al. / Vaccine 33 (2015) 108–116
with influenza infection [47]. We hypothesized that an attenuated and temperature-sensitive LAIV delivered subcutaneously would negate the risk of such reassortment and that is why we wanted to see whether a single dose of H7N3 LAIV delivered subcutaneously can be protective. Using subcutaneous (or related intradermal) route for respiratory viruses is an accepted and proven vaccination approach to generate robust immunity with attenuated viruses used for smallpox, measles and mumps, herpes zoster and yellow fever. Our data from this study demonstrate that this unconventional route of LAIV administration resulted in full protection against the challenge, based on viral titers and weight loss data, and high immunogenicity reflected in levels of HAI titers. In humans HAI titers elicited by intranasally delivered LAIV do not correlate with protection (in contrast to inactivated influenza vaccines) so it would be interesting to demonstrate whether other LAIVs (seasonal or based on H5N1) can produce the immunogenic response we observed in this study. Provided no safety issues emerge, this approach could be extended to human studies to see whether a meaningful correlation could be established between HAI and protection for subcutaneously administered LAIV. H5N1 LAIV studies in humans showed this type of vaccine to be poorly immunogenic and little or no HAI titers were detected in those people [48], as well as our own data (manuscript in preparation). However, upon boosting years later with a recombinant heterologous H5 protein vaccine, robust HAI titers were recalled from people previously vaccinated with the H5N1 LAIV vaccine, but no HAI activity was detected in non-LAIV, naïve volunteers [39]. Similar approach could be true for H7N9, since our LAIV is based upon H7N3 and low HAI titers were present in ferrets following a single intranasal vaccination, but rose sharply following boosting with inactivated virus vaccine similar to two vaccinations with LAIV or other H7N9 recombinant vaccines. Therefore, the H7naïve human population could be primed with the available H7N3 LAIV vaccines that have already been through human safety clinical trial [20] to give some protection to H7N9 isolates now. Then later, when H7N9 specific vaccines have received regulatory approval, they may be used to boost protective H7N9 immunity regardless of the platform(s) used (live or inactivated). Unfortunately, in silico prediction models indicate that there are few T cell epitopes in the HA sequences and therefore there is a poor potential for cross-reactivity with T cells specific for currently circulating influenza strains. Coupled with the absence of any prior exposure to H7 in human populations, these findings suggest that the H7N9 influenza virus, should it acquire the ability to be transmitted from person to person, has the potential to have a much more serious impact on human populations than did pandemic H1N1 influenza in 2009. Previous studies of influenza vaccines have clearly demonstrated the link between T cell response and antibody titers [49], thus the absence of T helper epitopes in the HA antigen of the current circulating strain of H7N9 can be expected to lead to very poor immunogenicity, particularly for subunit vaccines containing HA alone. Prediction models suggest that H7N9 HA could bind both mammalian and avian receptors [50], which is in agreement with measurements of H7N9 virus binding to sialic acid receptors [51]. This ability of H7N9 to bind both human and avian receptors is important and is also supported by H7N9 replication in the upper and lower respiratory tracts of non-human primates and nasal turbinates of ferrets. H7N9 viruses transmit by respiratory droplet in some ferret pairs [52,53] or via direct contact [35]. H7N9 strains readily transmitted to naive ferrets, replicate to higher viral titers in human airway epithelial cells and in the respiratory tract of ferrets, and show greater infectivity and lethality in mice compared to genetically related H7N9 and H9N2 viruses [54–56]. Therefore, H7N9 viruses currently circulating in China have demonstrated pandemic potential and may already be transmissible or could be
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a mutation away from being readily human transmissible. Recent data from Chinese sentinel sites suggest that the small number of H7N9 severe clinical cases may be just a reflection of a much larger number of undetected subclinical cases [57]. Therefore, not only are H7N9 vaccines needed, new innovative designs could be employed to enable development of vaccines against future influenza viruses, as opposed to the current practice of making vaccines retrospectively against previously circulating viruses.
Acknowledgements This work was supported by a sponsored research award from PATH Vaccine Solutions to TMR. The authors would like to thank Corey Crevar and Bradford Lefoley for technical assistance. We would like to thank the Centers for Disease Control and Prevention for providing the H7N9 and H7N3 influenza viruses and Dr. Florian Krammer at Icahn School of Medicine at Mt. Sinai for providing the chimeric HA (cH4/7) protein.
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