Vaccine 32 (2014) 1897–1900
Contents lists available at ScienceDirect
Vaccine journal homepage: www.elsevier.com/locate/vaccine
Brief report
Intranasal vaccination with a replication-deficient influenza virus induces heterosubtypic neutralising mucosal IgA antibodies in humans A. Morokutti a,b , T. Muster a , B. Ferko a,∗ a b
AVIR Green Hills Biotechnology AG, Vienna, Austria University of Natural Resources and Life Sciences, Vienna, Austria
a r t i c l e
i n f o
Article history: Received 20 October 2013 Received in revised form 12 January 2014 Accepted 7 February 2014 Available online 22 February 2014 Keywords: Nasal wash IgA Cross-reactive antibodies Heterologous immunity Single intranasal immunisation delNS1 influenza virus vaccine
a b s t r a c t We investigated the cross-neutralising potential of serum and nasal wash samples from volunteers who were intranasally immunised once with a monovalent replication-deficient delNS1-H1N1 influenza virus vaccine (7.7 log10 TCID50 /volunteer). Eight out of twelve (8/12) vaccinees responded to vaccination with a significant increase of antibody levels in serum IgG ELISA, mucosal IgA ELISA, MNA or HAI. Four responders showed delNS1-specific ELISA IgA increases and revealed excellent homosubtypic neutralising activity in serum and mucosal washings (4/4). However, 0/4 of the sera but 3/4 of the nasal washings neutralised also heterosubtypic H3N2 and H5N1 influenza viruses. Depletion experiments proved that IgA but not IgG is responsible for the cross-neutralising activity of the nasal wash sample. Our findings indicate that the induction of virus-neutralising IgA may represent a valuable correlate of cross-protection of intranasal influenza vaccines and that the delNS1 concept constitutes a promising approach to protect humans from seasonal and pandemic influenza threats. Clinical trial registration: NCT00724997. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Influenza is a highly infectious respiratory disease, causing significant morbidity and mortality each year. Continuous antigenic changes enable influenza viruses to escape pre-existing immunity [1]. So, to achieve an optimal protection against influenza regular vaccination with adapted vaccine compositions is recommended. At present, intramuscularly administered inactivated vaccines and intranasal cold-adapted live attenuated influenza virus (LAIV) vaccines are licensed [2–4]. A novel attenuation approach for LAIV vaccines is the deletion of the NS1 gene encoding the multifunctional non-structural protein (delNS1), which counteracts the interferon-mediated antiviral response [5,6]. Hence, delNS1 viruses are replication deficient in interferon-competent systems and, unlike wildtype viruses, are unable to overcome the cellular antiviral defence. The host rapidly reacts to the delNS1 intruder by releasing high systemic levels of antiviral IFN-␣/, local pro-inflammatory cytokines and by inducing fast apoptosis thus stimulating also a pronounced Th1-type immune response [7–9].
∗ Corresponding author. Tel.: +43 699 17017836. E-mail address: [email protected] (B. Ferko). http://dx.doi.org/10.1016/j.vaccine.2014.02.009 0264-410X/© 2014 Elsevier Ltd. All rights reserved.
Immunity induced by inactivated influenza vaccines is predominantly virus subtype-specific whereas wildtype influenza virus infection and LAIV vaccines are known to induce mucosal and systemic heterosubtypic immunity (HSI) [10,11]. Animal and human studies with intranasal vaccines prove that mucosal antibodies play a prominent role in the defence against influenza virus infection, even when T-cells are depleted [12,13]. The locally produced and actively secreted s-IgA is the predominant antibody isotype at mucosal surfaces of the upper respiratory tract, preventing viral entry and intracellular viral replication [14–16]. In this work we show that mucosal IgA induced by delNS1 virus vaccines neutralises both homologous and heterologous influenza viruses [5,6]. Thus, single intranasal vaccination of humans with the safe replication-deficient delNS1-H1N1 vaccine candidate induces heterosubtypic mucosal immunity comparable to wildtype virus infection. 2. Materials and methods 2.1. Volunteers and samples Samples originated from a randomised, double-blind, placebocontrolled, dose-escalation study (trial registration: NCT00724997) with H1N1 A/New Caledonia/20/99-like delNS1 virus vaccine
1898
A. Morokutti et al. / Vaccine 32 (2014) 1897–1900
Table 1 Individual pre- to postvaccination (day)- 29/1 titres and increases of delNS1-H1N1-specific antibodies in serum or nasal wash samples of a cohort of volunteers, who were intranasally immunised once with a monovalent replication-deficient live attenuated delNS1-H1N1 vaccine candidate (7.7 log10 TCID50 ) or formulation buffer (placebo), determined by ELISA, MNA and HAI (summary geometric mean titres for all vaccine cohorts were presented by Wacheck et al. [5]). Group
Vaccine recipients
ID
Day
a
277-34
a
290-38
293-43a 274-36a 241-33a 289-42 291-45 278-46 295-40 299-47 266-37 254-41 Placebo
273-35 294-39 305-44 311-48a
IgA-ELISA nasal wash
IgG-ELISA serum
MNA serum
HAI serum
AU (g IgA)
AU (ml)
x-Fold increase
Titre
x-Fold increase
Titre
x-Fold increase
32 175.4 25.1 163.1 33.2 79.3 29 73.4 56.7 76.1 35.1 82.3 24.4 67.6 1.79 9.58 23.9 43.6 22.5 42 75.8 94.6 51.1 82.1
5.5
16 256 32 512 16 64 32 512 16 64 32 128 16 128 8 32 16 32 16 32 16 16 32 32
16
Contents lists available at ScienceDirect
Vaccine journal homepage: www.elsevier.com/locate/vaccine
Brief report
Intranasal vaccination with a replication-deficient influenza virus induces heterosubtypic neutralising mucosal IgA antibodies in humans A. Morokutti a,b , T. Muster a , B. Ferko a,∗ a b
AVIR Green Hills Biotechnology AG, Vienna, Austria University of Natural Resources and Life Sciences, Vienna, Austria
a r t i c l e
i n f o
Article history: Received 20 October 2013 Received in revised form 12 January 2014 Accepted 7 February 2014 Available online 22 February 2014 Keywords: Nasal wash IgA Cross-reactive antibodies Heterologous immunity Single intranasal immunisation delNS1 influenza virus vaccine
a b s t r a c t We investigated the cross-neutralising potential of serum and nasal wash samples from volunteers who were intranasally immunised once with a monovalent replication-deficient delNS1-H1N1 influenza virus vaccine (7.7 log10 TCID50 /volunteer). Eight out of twelve (8/12) vaccinees responded to vaccination with a significant increase of antibody levels in serum IgG ELISA, mucosal IgA ELISA, MNA or HAI. Four responders showed delNS1-specific ELISA IgA increases and revealed excellent homosubtypic neutralising activity in serum and mucosal washings (4/4). However, 0/4 of the sera but 3/4 of the nasal washings neutralised also heterosubtypic H3N2 and H5N1 influenza viruses. Depletion experiments proved that IgA but not IgG is responsible for the cross-neutralising activity of the nasal wash sample. Our findings indicate that the induction of virus-neutralising IgA may represent a valuable correlate of cross-protection of intranasal influenza vaccines and that the delNS1 concept constitutes a promising approach to protect humans from seasonal and pandemic influenza threats. Clinical trial registration: NCT00724997. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Influenza is a highly infectious respiratory disease, causing significant morbidity and mortality each year. Continuous antigenic changes enable influenza viruses to escape pre-existing immunity [1]. So, to achieve an optimal protection against influenza regular vaccination with adapted vaccine compositions is recommended. At present, intramuscularly administered inactivated vaccines and intranasal cold-adapted live attenuated influenza virus (LAIV) vaccines are licensed [2–4]. A novel attenuation approach for LAIV vaccines is the deletion of the NS1 gene encoding the multifunctional non-structural protein (delNS1), which counteracts the interferon-mediated antiviral response [5,6]. Hence, delNS1 viruses are replication deficient in interferon-competent systems and, unlike wildtype viruses, are unable to overcome the cellular antiviral defence. The host rapidly reacts to the delNS1 intruder by releasing high systemic levels of antiviral IFN-␣/, local pro-inflammatory cytokines and by inducing fast apoptosis thus stimulating also a pronounced Th1-type immune response [7–9].
∗ Corresponding author. Tel.: +43 699 17017836. E-mail address: [email protected] (B. Ferko). http://dx.doi.org/10.1016/j.vaccine.2014.02.009 0264-410X/© 2014 Elsevier Ltd. All rights reserved.
Immunity induced by inactivated influenza vaccines is predominantly virus subtype-specific whereas wildtype influenza virus infection and LAIV vaccines are known to induce mucosal and systemic heterosubtypic immunity (HSI) [10,11]. Animal and human studies with intranasal vaccines prove that mucosal antibodies play a prominent role in the defence against influenza virus infection, even when T-cells are depleted [12,13]. The locally produced and actively secreted s-IgA is the predominant antibody isotype at mucosal surfaces of the upper respiratory tract, preventing viral entry and intracellular viral replication [14–16]. In this work we show that mucosal IgA induced by delNS1 virus vaccines neutralises both homologous and heterologous influenza viruses [5,6]. Thus, single intranasal vaccination of humans with the safe replication-deficient delNS1-H1N1 vaccine candidate induces heterosubtypic mucosal immunity comparable to wildtype virus infection. 2. Materials and methods 2.1. Volunteers and samples Samples originated from a randomised, double-blind, placebocontrolled, dose-escalation study (trial registration: NCT00724997) with H1N1 A/New Caledonia/20/99-like delNS1 virus vaccine
1898
A. Morokutti et al. / Vaccine 32 (2014) 1897–1900
Table 1 Individual pre- to postvaccination (day)- 29/1 titres and increases of delNS1-H1N1-specific antibodies in serum or nasal wash samples of a cohort of volunteers, who were intranasally immunised once with a monovalent replication-deficient live attenuated delNS1-H1N1 vaccine candidate (7.7 log10 TCID50 ) or formulation buffer (placebo), determined by ELISA, MNA and HAI (summary geometric mean titres for all vaccine cohorts were presented by Wacheck et al. [5]). Group
Vaccine recipients
ID
Day
a
277-34
a
290-38
293-43a 274-36a 241-33a 289-42 291-45 278-46 295-40 299-47 266-37 254-41 Placebo
273-35 294-39 305-44 311-48a
IgA-ELISA nasal wash
IgG-ELISA serum
MNA serum
HAI serum
AU (g IgA)
AU (ml)
x-Fold increase
Titre
x-Fold increase
Titre
x-Fold increase
32 175.4 25.1 163.1 33.2 79.3 29 73.4 56.7 76.1 35.1 82.3 24.4 67.6 1.79 9.58 23.9 43.6 22.5 42 75.8 94.6 51.1 82.1
5.5
16 256 32 512 16 64 32 512 16 64 32 128 16 128 8 32 16 32 16 32 16 16 32 32
16
