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ScienceDirect Production of live attenuated influenza vaccines against seasonal and potential pandemic influenza viruses Hong Jin and Zhongying Chen Vaccination remains the most effective means to prevent morbidity and mortality caused by influenza epidemics and pandemics. Live attenuated influenza vaccine (LAIV) has been proven to be effective in preventing influenza with broad cross reactivity to drifted strains. Owing to the sophisticated nature of the influenza vaccine production process, the time needed to develop high yield LAIV strains for vaccine production and product release remains a constant challenge. This review summarizes LAIV production process with highlights on the experiences gained during the past decade generating seasonal and pandemic LAIV seeds by reverse genetics strategy. Addresses MedImmune LLC, 319 North Bernardo Avenue, Mountain View, CA 94043, United States Corresponding author: Jin, Hong ([email protected])
Current Opinion in Virology 2014, 6:34–39 This review comes from a themed issue on Vaccines Edited by Shan Lu
1879-6257/$ – see front matter, Published by Elsevier B.V. http://dx.doi.org/10.1016/j.coviro.2014.02.008
Introduction Constant antigenic drift of human influenza viruses requires annual update of the seasonal vaccine to prevent influenza epidemics. Three types of influenza vaccines are commercially available in the US: inactivated influenza vaccines (IIV) and recently approved recombinant HA protein subunit vaccines for adults [1] administered intramuscularly, and live attenuated influenza vaccines (LAIV) administered intranasally. LAIVs are produced annually as 6:2 reassortant viruses with the six internal protein gene segments derived from the cold adapted master donor viruses, A/Ann Arbor/6/60 for influenza A strains and B/Ann Arbor/1/66 for influenza B strains, and the surface hemagglutinin (HA) and neuraminidase (NA) protein gene segments from wild type strains. A number of genetic loci distributed in multiple segments of the Ann Arbor donor strains have been identified to confer the cold adapted, temperature sensitive and attenuated phenotypes [2–4], explaining the observed phenotypic stability of the vaccine viruses. The 2013–2014 influenza season marks the first time that quadrivalent LAIV is used in the United States, which contains four vaccine strains with antigens from circulating human influenza subtype A Current Opinion in Virology 2014, 6:34–39
(A/H1N1, A/H3N2) and B (B/Victoria and B/Yamagata lineages) viruses. The quadrivalent LAIV provides coverage for an additional influenza B strain compared to trivalent LAIV which only contained one B virus from either the B/Yamagata or the B/Victoria lineage. Concomitant circulation of influenza viruses in humans and animal reservoirs combined with antigenic drift and/or antigenic shift (genetic reassortment) can result in new influenza viruses that can infect humans and cause human to human transmission. Such viruses can rapidly spread in humans with little or no preexisting immunity and lead to a pandemic [5,6]. In the past, three subtypes of influenza A viruses caused influenza pandemics: H1N1 (the 1918 Spanish flu and the 2009 pandemic), H2N2 (the 1957 pandemic), and H3N2 (the 1968 pandemic). The avian H5N1, H7 (H7N7, H7N3, H7N9), H9N2 viruses and swine H3N2v viruses have caused sporadic human infections. The high mortality rate caused by the highly pathogenic avian H5N1 virus (60%) and the new H7N9 virus (30%) that emerged in China in 2013 [7,8] have heightened concerns about influenza pandemics and have highlighted the need to develop pandemic vaccines before a pandemic is declared. LAIVs can prime influenza specific immune responses in naı¨ve populations and elicit broad cross reactivity to drifted strains [9], both of which are important attributes of candidate vaccines against pandemic influenza. In addition to the induction of protective serum neutralizing antibodies, LAIV also elicits innate and local mucosal IgA antibodies and cell-mediated immune responses that are important for viral clearance [10,11,12]. These attributes may explain why LAIV is more efficacious in young children for both antigenically matched and drifted viruses than IIV that mainly induces neutralizing antibodies [13,14]. Furthermore, its high production yield in eggs and ‘needle-free’ nasal administration make LAIV a desirable vaccine for use in a pandemic setting. A number of LAIVs have been produced and evaluated in preclinical and small Phase I clinical studies (Table 1). These vaccines had safety and tolerability profiles in humans similar to seasonal LAIV. These pre-made pandemic vaccine seeds could be used for commercial-scale manufacturing if they match a newly emerged pandemic strain; alternatively they could be used as a priming dose in humans before the precisely matched vaccine seed is available.
Influenza vaccine production process Influenza vaccine strain composition is updated every year to match the circulating influenza viruses. This www.sciencedirect.com
Influenza vaccine Jin and Chen 35
Generation of high yield LAIV seeds that are immunogenic and antigenically accurate
Table 1 Pandemic vaccines evaluated in Phase I clinical trials. Vaccine strains
References
A/Ann Arbor/6/60 (H2N2) A/swine/MO/2006 (H2N3) A/Vietnam/1203/2004 (H5N1) A/HK/213/2003 (H5N1) A/teal/HK/W312/97 (H6N1) A/chicken/BC/CN-6/2004 (H7N3) A/Netherland/219/2003 (H7N7) A/Anhui/1/2013 (H7N9) A/chicken/HK/G9/97 (H9N2)
[35] Unpublished [36] [36] [37] [38] Unpublished Unpublished [39]
Subtype H2 H5 H6 H7
H9
requires rapid seed development and manufacture to ensure timely delivery of both LAIV and IIV. Each year, vaccine manufacturers have to go through a lengthy process from making reassortant vaccine seeds to bulk production, filling, packaging and lot release testing for the upcoming season: this process normally takes at least 4.5 months for LAIV (Table 2). Several key challenges or hurdles can significantly delay vaccine delivery especially for pandemic vaccines where the growth of a new strain in embryonated chicken eggs (the main vaccine manufacture substrate) is unpredictable and new reagents have to be rapidly produced for product lot release testing. For example, the H3N2 A/Fujian/411/ 2002 strain selected by the WHO for the 2003–2004 Northern Hemisphere season could not be incorporated into the seasonal vaccine due to its poor growth characteristics. The A/Moscow/10/1999 or A/Panama/20/1999 vaccine from the previous season was less effective against the drifted strains during the 2003 H3N2 epidemic [15,16]. Similarly, the poor growth of the 2009 H1N1pdm strain also caused delays in vaccine availability. Furthermore, to produce vaccine for influenza strains that have the potential to cause pandemics, a USDA permit is required to handle the animal-origin viruses; this can prolong the vaccine manufacture and release testing schedule.
Table 2 Key activities and timeline required for LAIV production and release. LAIV production activities Generation of 6:2 reassortant vaccine virus by reverse genetics Vaccine growth evaluation and preclinical studies a Production of GMP seed and master seed virus (MVS) Production of monovalent bulk virus Antigen and antiserum production for lot release test a Lot release test of MVS and monovalent bulk vaccine a Vaccine filling, package and release test Vaccine safety trials Minimal time required to release vaccine for distribution a
Conducted concurrently with other activities.
www.sciencedirect.com
Time 2 weeks 3 weeks 3 weeks 1 week 10 weeks 7 weeks 4 weeks 4 weeks 22 weeks
Influenza viruses isolated from humans often do not replicate well in embryonated chicken eggs, the substrate for production of both IIV and LAIV. Replication of influenza viruses in eggs, and occasionally in MDCK cells, often results in the generation of mutations at or near the HA receptor binding region and at the antigenic sites. Therefore, to select an appropriate vaccine strain, a number of 6:2 LAIV reassortant variants from a single strain or other like-strains are often generated each year. Each HA variant is carefully examined for its immunogenicity in ferrets and the antigenicity of each variant is compared to the parental wild type influenza virus. The application of reverse genetics [3,4] not only advances our understanding of influenza biology and understanding of the impact of viral egg adaptation sites on viral growth, antigenicity, and immunogenicity, but also enables the rapid generation of high quality vaccine strains for manufacture. Reverse genetics technology also enables modification of the HA protein cleavage site to generate safe vaccines against highly pathogenic H5 and H7 viruses [17–19]. Examples of how specific HA sequence changes can alter the biological properties of seasonal and pandemic vaccine strains are described below. Influenza A/H3N2
The mutations frequently identified in the HA gene of the H3N2 viruses are shown in Fig. 1. The majority of the egg adaptation changes do not affect viral antigenicity and immunogenicity, such as the HA 183, 186, 188, 189, 190, 193, 195, 196, 219 and 226 residues. However, the HA 156 residue change [20] alters viral antigenicity which could affect vaccine efficacy, while the L194P change reduces LAIV immunogenicity [21] due to its reduced replication in the upper respiratory tract. Therefore, changes to the HA 156 and 194 residues are generally avoided when possible. As an example, the A/Victoria/361/2011 vaccine strain recommended for the 2012–2013 season contained an H156Q change from egg adaptation and did not maintain the same antigenicity as either the cell-derived A/Victoria/361/2011 strain, or the majority of field isolates which had the H156 residue. Thus, the A/Texas/50/2012 strain, which did not have the H156Q change, was recommended for the following year by the WHO to replace the A/Victoria/361/2011 strain. We have found that three egg adaptation related sequence changes, G186V, S219F and I226N, in the HA segment of A/ Texas/50/2012 LAIV are important for improving viral growth in eggs without affecting the strain’s immunogenicity and antigenicity (unpublished data). Influenza A/H1N1
The seasonal H1N1 viruses before 2009 tended to have amino acid changes in the receptor binding region, such as the HA 226 (H3#, H1# 223) residue reported for A/ Solomon Island/3/2006 vaccine virus, that also affected Current Opinion in Virology 2014, 6:34–39
36 Vaccines
Figure 1
190 helix
A196T N246H
Y195H
D188Y
H156Q D190E S219F/Y G186V L194P H183L I226N
6’SLN 130 loop
220 loop A138S
K140I
Influenza B R142G
Current Opinion in Virology
The locations of the egg adaptations changes of H3N2 strains on the model of the 3D structure of HA in complex with human receptor analog 6SLN (PDB# 2YP8, A/HK/4443/2005 HA monomer structure). Only the receptor binding region is shown. The N246H change causes the loss of the N246 glycosylation site. The H156Q change (red) affected viral antigenicity. The L194P (yellow) significantly reduced immunogenicity of the vaccine virus.
viral antigenicity [22]. The previous seasonal H1N1 viruses have now been replaced by the H1N1pdm virus that emerged in 2009 and that has since been circulating worldwide. Developing a high yield 2009 H1N1pdm vaccine virus was a significant challenge due to the low yield of reassortant viruses derived from egg adapted wild viruses. Although the variants with sequence changes at the HA residues 156–158 (H3#) had high yield in eggs, they altered viral antigenicity. Through cell culture adaptation, a high yield vaccine virus with the K119E and A186D changes in the HA was identified and used for vaccine production [23]. We also found that the amino acids substitutions of N125D, D127E or K209E could also improve H1N1pdm growth without affecting antigenicity and immunogenicity [24]. The original H1N1pdm vaccine virus from 2009 also appeared to be less thermally stable than previous seasonal H1N1 vaccine viruses and more recently produced H1N1pdm strains. The E47 residue in the HA2 stalk region of A/ California/7/2009 (H1N1pdm) was identified as being responsible for the structural instability resulting in a higher fusion pH (pH 5.4). Recent H1N1pdm strains Current Opinion in Virology 2014, 6:34–39
containing the K47 residue have a fusion pH of 5.0 [25]. The E47 residue also made the HA protein insensitive to bromelain cleavage [26]; bromelain cleavage is required to produce HA antigens suitable for generation of antiserum used in vaccine potency and adventitious viral agents (AVA) testing. The A/California/7/2009 HA could be efficiently cleaved by bromelain when the HA2 portion was replaced with that of seasonal H1N1 A/South Dakota/6/2007 or a single HA2-E47K substitution was introduced. Interestingly, the H1N1pdm have evolved to have the N369K change in the NA protein in humans and this N369K site appears to have some impact on viral growth in the eggs [24]. The H1N1pdm viruses isolated in 2013 have the same poor growth characteristics in eggs as A/California/7/2009, the identified residues that have been shown to improve viral growth in eggs could be introduced into the future H1N1 vaccine strain to shorten vaccine seed development time.
Type B influenza viruses almost always lose the Nlinked glycosylation site at the N196 (B/Yamagata lineage) or N197 (B/Victoria lineage) residue of the HA when passaged in eggs, resulting in sequence variations at the residues 196 and 198 (B/Yamagata lineage) or 197 and 199 (B/Victoria lineage viruses) [27]. These mutations can result in different viral growth phenotypes and impact viral antigenicity, therefore, multiple variants have to be evaluated to select an appropriate vaccine that grows well and is immunogenic and antigenically accurate. In both lineages, the N to D substitution at this N-glycosylation site (N196D for B/ Yamagata lineage and N197D for B/Victoria lineage) normally leads to high viral growth in eggs compared to other changes while maintains immunogenicity and antigenicity in ferrets. Pandemic LAIV
LAIVs representing the H2, H5, H6, H7, and H9 subtypes have been developed since 1997 when the first human infection of H5N1 virus was reported (Table 1). This experience has proven to be very valuable for the generation of vaccines against both the 2009 H1N1pdm as well as the 2013 H7N9 strains. An H7N7 A/Netherland/219/2003 vaccine is antigenically related to the H7N9 A/Anhui/1/2013 virus. Thus, this vaccine could have been used as an initial, albeit incomplete, preventative measure against the H7N9 A/Anhui/1/2013 virus while vaccines designed specifically for this virus were going through the commercial manufacturing process [28]. The development of a high yield H7N9 A/Anhui/ 1/2013 vaccine was also very challenging. Six vaccine variants that were initially generated had titers < 8.0 log10 fluorescence focus units (FFU)/mL in eggs or altered viral antigenicity. Further adaptation of these variants in eggs led to two changes, N133D and G198E, in the HA head region that improved vaccine www.sciencedirect.com
Influenza vaccine Jin and Chen 37
virus growth by 10-fold [29]. The H7N9 LAIV with these two changes in the HA is immunogenic in ferrets and reacts well with wt H7N9 virus-infected ferret serum, and was therefore chosen for the production of the clinical trial material. The H5 and H7 LAIV derived from highly pathogenic vaccine viruses do not contain multi-basic amino acids in the HA cleavage site and have been consistently proven to be attenuated and non-pathogenic in chicken [17–19]. These data should provide scientific justification to remove the requirement of conducting chicken pathogenicity test before progressing pandemic LAIV manufacture for the emerging H5 and H7 pandemic threat in the future.
Methods to improve vaccine virus immunogenicity Most avian influenza viruses preferentially bind to the a2,3-linked sialic acid receptor (avian receptor), enabling them to replicate well in embryonated chicken eggs but less well in the human upper airway which predominantly contains a2,6-linked sialic acid receptors [30,31]. Modification of the HA receptor binding site from Q226-G228 to L226-S228 increases viral binding to a2,6-linked sialic acid and improves the immunogenicity of H2, H6 and H7 subtype vaccine viruses in ferrets while maintaining high growth in eggs [28,32]. In addition, removal of the N158glycosylation site improves H5N1 A/Vietnam/2004 vaccine virus immunogenicity in ferrets [33]. Additional studies are needed to further understand vaccine mediated protective immune responses in order to optimize vaccine virus production.
Vaccine release test Each LAIV is tested to ensure that it retains the cold adapted (ca), temperature sensitive (ts) and attenuated (att) phenotypes before the vaccine virus seed can be released for further manufacturing [4]. The ca phenotype is defined as the viral titer reduction at 25 8C 100-fold compared to its replication at 33 8C. The att phenotype is assessed in a ferret model, in which ferrets are intranasally inoculated with the vaccine and vaccine viral replication is assessed in both the upper respiratory tract (nose) and the lower respiratory tract (lungs). Decreased levels of replication in the lower respiratory tract (but not the upper tract) reflect viral attenuation. Each vaccine lot must be extensively tested to ensure that it does not contain any adventitious agents. For these tests, complete neutralization of the vaccine virus is required. Timely production of the HA-specific highly potent virus-neutralizing antiserum for lot release testing can be extremely challenging. The antiserum must completely neutralize 9.0 log10 FFU/mL of vaccine virus at a concentration of
ScienceDirect Production of live attenuated influenza vaccines against seasonal and potential pandemic influenza viruses Hong Jin and Zhongying Chen Vaccination remains the most effective means to prevent morbidity and mortality caused by influenza epidemics and pandemics. Live attenuated influenza vaccine (LAIV) has been proven to be effective in preventing influenza with broad cross reactivity to drifted strains. Owing to the sophisticated nature of the influenza vaccine production process, the time needed to develop high yield LAIV strains for vaccine production and product release remains a constant challenge. This review summarizes LAIV production process with highlights on the experiences gained during the past decade generating seasonal and pandemic LAIV seeds by reverse genetics strategy. Addresses MedImmune LLC, 319 North Bernardo Avenue, Mountain View, CA 94043, United States Corresponding author: Jin, Hong ([email protected])
Current Opinion in Virology 2014, 6:34–39 This review comes from a themed issue on Vaccines Edited by Shan Lu
1879-6257/$ – see front matter, Published by Elsevier B.V. http://dx.doi.org/10.1016/j.coviro.2014.02.008
Introduction Constant antigenic drift of human influenza viruses requires annual update of the seasonal vaccine to prevent influenza epidemics. Three types of influenza vaccines are commercially available in the US: inactivated influenza vaccines (IIV) and recently approved recombinant HA protein subunit vaccines for adults [1] administered intramuscularly, and live attenuated influenza vaccines (LAIV) administered intranasally. LAIVs are produced annually as 6:2 reassortant viruses with the six internal protein gene segments derived from the cold adapted master donor viruses, A/Ann Arbor/6/60 for influenza A strains and B/Ann Arbor/1/66 for influenza B strains, and the surface hemagglutinin (HA) and neuraminidase (NA) protein gene segments from wild type strains. A number of genetic loci distributed in multiple segments of the Ann Arbor donor strains have been identified to confer the cold adapted, temperature sensitive and attenuated phenotypes [2–4], explaining the observed phenotypic stability of the vaccine viruses. The 2013–2014 influenza season marks the first time that quadrivalent LAIV is used in the United States, which contains four vaccine strains with antigens from circulating human influenza subtype A Current Opinion in Virology 2014, 6:34–39
(A/H1N1, A/H3N2) and B (B/Victoria and B/Yamagata lineages) viruses. The quadrivalent LAIV provides coverage for an additional influenza B strain compared to trivalent LAIV which only contained one B virus from either the B/Yamagata or the B/Victoria lineage. Concomitant circulation of influenza viruses in humans and animal reservoirs combined with antigenic drift and/or antigenic shift (genetic reassortment) can result in new influenza viruses that can infect humans and cause human to human transmission. Such viruses can rapidly spread in humans with little or no preexisting immunity and lead to a pandemic [5,6]. In the past, three subtypes of influenza A viruses caused influenza pandemics: H1N1 (the 1918 Spanish flu and the 2009 pandemic), H2N2 (the 1957 pandemic), and H3N2 (the 1968 pandemic). The avian H5N1, H7 (H7N7, H7N3, H7N9), H9N2 viruses and swine H3N2v viruses have caused sporadic human infections. The high mortality rate caused by the highly pathogenic avian H5N1 virus (60%) and the new H7N9 virus (30%) that emerged in China in 2013 [7,8] have heightened concerns about influenza pandemics and have highlighted the need to develop pandemic vaccines before a pandemic is declared. LAIVs can prime influenza specific immune responses in naı¨ve populations and elicit broad cross reactivity to drifted strains [9], both of which are important attributes of candidate vaccines against pandemic influenza. In addition to the induction of protective serum neutralizing antibodies, LAIV also elicits innate and local mucosal IgA antibodies and cell-mediated immune responses that are important for viral clearance [10,11,12]. These attributes may explain why LAIV is more efficacious in young children for both antigenically matched and drifted viruses than IIV that mainly induces neutralizing antibodies [13,14]. Furthermore, its high production yield in eggs and ‘needle-free’ nasal administration make LAIV a desirable vaccine for use in a pandemic setting. A number of LAIVs have been produced and evaluated in preclinical and small Phase I clinical studies (Table 1). These vaccines had safety and tolerability profiles in humans similar to seasonal LAIV. These pre-made pandemic vaccine seeds could be used for commercial-scale manufacturing if they match a newly emerged pandemic strain; alternatively they could be used as a priming dose in humans before the precisely matched vaccine seed is available.
Influenza vaccine production process Influenza vaccine strain composition is updated every year to match the circulating influenza viruses. This www.sciencedirect.com
Influenza vaccine Jin and Chen 35
Generation of high yield LAIV seeds that are immunogenic and antigenically accurate
Table 1 Pandemic vaccines evaluated in Phase I clinical trials. Vaccine strains
References
A/Ann Arbor/6/60 (H2N2) A/swine/MO/2006 (H2N3) A/Vietnam/1203/2004 (H5N1) A/HK/213/2003 (H5N1) A/teal/HK/W312/97 (H6N1) A/chicken/BC/CN-6/2004 (H7N3) A/Netherland/219/2003 (H7N7) A/Anhui/1/2013 (H7N9) A/chicken/HK/G9/97 (H9N2)
[35] Unpublished [36] [36] [37] [38] Unpublished Unpublished [39]
Subtype H2 H5 H6 H7
H9
requires rapid seed development and manufacture to ensure timely delivery of both LAIV and IIV. Each year, vaccine manufacturers have to go through a lengthy process from making reassortant vaccine seeds to bulk production, filling, packaging and lot release testing for the upcoming season: this process normally takes at least 4.5 months for LAIV (Table 2). Several key challenges or hurdles can significantly delay vaccine delivery especially for pandemic vaccines where the growth of a new strain in embryonated chicken eggs (the main vaccine manufacture substrate) is unpredictable and new reagents have to be rapidly produced for product lot release testing. For example, the H3N2 A/Fujian/411/ 2002 strain selected by the WHO for the 2003–2004 Northern Hemisphere season could not be incorporated into the seasonal vaccine due to its poor growth characteristics. The A/Moscow/10/1999 or A/Panama/20/1999 vaccine from the previous season was less effective against the drifted strains during the 2003 H3N2 epidemic [15,16]. Similarly, the poor growth of the 2009 H1N1pdm strain also caused delays in vaccine availability. Furthermore, to produce vaccine for influenza strains that have the potential to cause pandemics, a USDA permit is required to handle the animal-origin viruses; this can prolong the vaccine manufacture and release testing schedule.
Table 2 Key activities and timeline required for LAIV production and release. LAIV production activities Generation of 6:2 reassortant vaccine virus by reverse genetics Vaccine growth evaluation and preclinical studies a Production of GMP seed and master seed virus (MVS) Production of monovalent bulk virus Antigen and antiserum production for lot release test a Lot release test of MVS and monovalent bulk vaccine a Vaccine filling, package and release test Vaccine safety trials Minimal time required to release vaccine for distribution a
Conducted concurrently with other activities.
www.sciencedirect.com
Time 2 weeks 3 weeks 3 weeks 1 week 10 weeks 7 weeks 4 weeks 4 weeks 22 weeks
Influenza viruses isolated from humans often do not replicate well in embryonated chicken eggs, the substrate for production of both IIV and LAIV. Replication of influenza viruses in eggs, and occasionally in MDCK cells, often results in the generation of mutations at or near the HA receptor binding region and at the antigenic sites. Therefore, to select an appropriate vaccine strain, a number of 6:2 LAIV reassortant variants from a single strain or other like-strains are often generated each year. Each HA variant is carefully examined for its immunogenicity in ferrets and the antigenicity of each variant is compared to the parental wild type influenza virus. The application of reverse genetics [3,4] not only advances our understanding of influenza biology and understanding of the impact of viral egg adaptation sites on viral growth, antigenicity, and immunogenicity, but also enables the rapid generation of high quality vaccine strains for manufacture. Reverse genetics technology also enables modification of the HA protein cleavage site to generate safe vaccines against highly pathogenic H5 and H7 viruses [17–19]. Examples of how specific HA sequence changes can alter the biological properties of seasonal and pandemic vaccine strains are described below. Influenza A/H3N2
The mutations frequently identified in the HA gene of the H3N2 viruses are shown in Fig. 1. The majority of the egg adaptation changes do not affect viral antigenicity and immunogenicity, such as the HA 183, 186, 188, 189, 190, 193, 195, 196, 219 and 226 residues. However, the HA 156 residue change [20] alters viral antigenicity which could affect vaccine efficacy, while the L194P change reduces LAIV immunogenicity [21] due to its reduced replication in the upper respiratory tract. Therefore, changes to the HA 156 and 194 residues are generally avoided when possible. As an example, the A/Victoria/361/2011 vaccine strain recommended for the 2012–2013 season contained an H156Q change from egg adaptation and did not maintain the same antigenicity as either the cell-derived A/Victoria/361/2011 strain, or the majority of field isolates which had the H156 residue. Thus, the A/Texas/50/2012 strain, which did not have the H156Q change, was recommended for the following year by the WHO to replace the A/Victoria/361/2011 strain. We have found that three egg adaptation related sequence changes, G186V, S219F and I226N, in the HA segment of A/ Texas/50/2012 LAIV are important for improving viral growth in eggs without affecting the strain’s immunogenicity and antigenicity (unpublished data). Influenza A/H1N1
The seasonal H1N1 viruses before 2009 tended to have amino acid changes in the receptor binding region, such as the HA 226 (H3#, H1# 223) residue reported for A/ Solomon Island/3/2006 vaccine virus, that also affected Current Opinion in Virology 2014, 6:34–39
36 Vaccines
Figure 1
190 helix
A196T N246H
Y195H
D188Y
H156Q D190E S219F/Y G186V L194P H183L I226N
6’SLN 130 loop
220 loop A138S
K140I
Influenza B R142G
Current Opinion in Virology
The locations of the egg adaptations changes of H3N2 strains on the model of the 3D structure of HA in complex with human receptor analog 6SLN (PDB# 2YP8, A/HK/4443/2005 HA monomer structure). Only the receptor binding region is shown. The N246H change causes the loss of the N246 glycosylation site. The H156Q change (red) affected viral antigenicity. The L194P (yellow) significantly reduced immunogenicity of the vaccine virus.
viral antigenicity [22]. The previous seasonal H1N1 viruses have now been replaced by the H1N1pdm virus that emerged in 2009 and that has since been circulating worldwide. Developing a high yield 2009 H1N1pdm vaccine virus was a significant challenge due to the low yield of reassortant viruses derived from egg adapted wild viruses. Although the variants with sequence changes at the HA residues 156–158 (H3#) had high yield in eggs, they altered viral antigenicity. Through cell culture adaptation, a high yield vaccine virus with the K119E and A186D changes in the HA was identified and used for vaccine production [23]. We also found that the amino acids substitutions of N125D, D127E or K209E could also improve H1N1pdm growth without affecting antigenicity and immunogenicity [24]. The original H1N1pdm vaccine virus from 2009 also appeared to be less thermally stable than previous seasonal H1N1 vaccine viruses and more recently produced H1N1pdm strains. The E47 residue in the HA2 stalk region of A/ California/7/2009 (H1N1pdm) was identified as being responsible for the structural instability resulting in a higher fusion pH (pH 5.4). Recent H1N1pdm strains Current Opinion in Virology 2014, 6:34–39
containing the K47 residue have a fusion pH of 5.0 [25]. The E47 residue also made the HA protein insensitive to bromelain cleavage [26]; bromelain cleavage is required to produce HA antigens suitable for generation of antiserum used in vaccine potency and adventitious viral agents (AVA) testing. The A/California/7/2009 HA could be efficiently cleaved by bromelain when the HA2 portion was replaced with that of seasonal H1N1 A/South Dakota/6/2007 or a single HA2-E47K substitution was introduced. Interestingly, the H1N1pdm have evolved to have the N369K change in the NA protein in humans and this N369K site appears to have some impact on viral growth in the eggs [24]. The H1N1pdm viruses isolated in 2013 have the same poor growth characteristics in eggs as A/California/7/2009, the identified residues that have been shown to improve viral growth in eggs could be introduced into the future H1N1 vaccine strain to shorten vaccine seed development time.
Type B influenza viruses almost always lose the Nlinked glycosylation site at the N196 (B/Yamagata lineage) or N197 (B/Victoria lineage) residue of the HA when passaged in eggs, resulting in sequence variations at the residues 196 and 198 (B/Yamagata lineage) or 197 and 199 (B/Victoria lineage viruses) [27]. These mutations can result in different viral growth phenotypes and impact viral antigenicity, therefore, multiple variants have to be evaluated to select an appropriate vaccine that grows well and is immunogenic and antigenically accurate. In both lineages, the N to D substitution at this N-glycosylation site (N196D for B/ Yamagata lineage and N197D for B/Victoria lineage) normally leads to high viral growth in eggs compared to other changes while maintains immunogenicity and antigenicity in ferrets. Pandemic LAIV
LAIVs representing the H2, H5, H6, H7, and H9 subtypes have been developed since 1997 when the first human infection of H5N1 virus was reported (Table 1). This experience has proven to be very valuable for the generation of vaccines against both the 2009 H1N1pdm as well as the 2013 H7N9 strains. An H7N7 A/Netherland/219/2003 vaccine is antigenically related to the H7N9 A/Anhui/1/2013 virus. Thus, this vaccine could have been used as an initial, albeit incomplete, preventative measure against the H7N9 A/Anhui/1/2013 virus while vaccines designed specifically for this virus were going through the commercial manufacturing process [28]. The development of a high yield H7N9 A/Anhui/ 1/2013 vaccine was also very challenging. Six vaccine variants that were initially generated had titers < 8.0 log10 fluorescence focus units (FFU)/mL in eggs or altered viral antigenicity. Further adaptation of these variants in eggs led to two changes, N133D and G198E, in the HA head region that improved vaccine www.sciencedirect.com
Influenza vaccine Jin and Chen 37
virus growth by 10-fold [29]. The H7N9 LAIV with these two changes in the HA is immunogenic in ferrets and reacts well with wt H7N9 virus-infected ferret serum, and was therefore chosen for the production of the clinical trial material. The H5 and H7 LAIV derived from highly pathogenic vaccine viruses do not contain multi-basic amino acids in the HA cleavage site and have been consistently proven to be attenuated and non-pathogenic in chicken [17–19]. These data should provide scientific justification to remove the requirement of conducting chicken pathogenicity test before progressing pandemic LAIV manufacture for the emerging H5 and H7 pandemic threat in the future.
Methods to improve vaccine virus immunogenicity Most avian influenza viruses preferentially bind to the a2,3-linked sialic acid receptor (avian receptor), enabling them to replicate well in embryonated chicken eggs but less well in the human upper airway which predominantly contains a2,6-linked sialic acid receptors [30,31]. Modification of the HA receptor binding site from Q226-G228 to L226-S228 increases viral binding to a2,6-linked sialic acid and improves the immunogenicity of H2, H6 and H7 subtype vaccine viruses in ferrets while maintaining high growth in eggs [28,32]. In addition, removal of the N158glycosylation site improves H5N1 A/Vietnam/2004 vaccine virus immunogenicity in ferrets [33]. Additional studies are needed to further understand vaccine mediated protective immune responses in order to optimize vaccine virus production.
Vaccine release test Each LAIV is tested to ensure that it retains the cold adapted (ca), temperature sensitive (ts) and attenuated (att) phenotypes before the vaccine virus seed can be released for further manufacturing [4]. The ca phenotype is defined as the viral titer reduction at 25 8C 100-fold compared to its replication at 33 8C. The att phenotype is assessed in a ferret model, in which ferrets are intranasally inoculated with the vaccine and vaccine viral replication is assessed in both the upper respiratory tract (nose) and the lower respiratory tract (lungs). Decreased levels of replication in the lower respiratory tract (but not the upper tract) reflect viral attenuation. Each vaccine lot must be extensively tested to ensure that it does not contain any adventitious agents. For these tests, complete neutralization of the vaccine virus is required. Timely production of the HA-specific highly potent virus-neutralizing antiserum for lot release testing can be extremely challenging. The antiserum must completely neutralize 9.0 log10 FFU/mL of vaccine virus at a concentration of
