An alternative method for preparation of pandemic influenza strain-specific antibody for vaccine potency determination

Vaccine 28 (2010) 2442–2449

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An alternative method for preparation of pandemic influenza strain-specific antibody for vaccine potency determination Falko Schmeisser a , Galina M. Vodeiko a , Vladimir Y. Lugovtsev a , Richard R. Stout b , Jerry P. Weir a,∗ a b

Division of Viral Products, Center for Biologics Evaluations and Research, Food and Drug Administration, 8800 Rockville Pike, Bethesda, MD 20892, United States Bioject Inc., Tualatin, OR, United States

a r t i c l e

i n f o

Article history: Received 13 November 2009 Received in revised form 22 December 2009 Accepted 25 December 2009 Available online 12 January 2010 Keywords: Pandemic influenza Vaccine potency Single-radial immunodiffusion assay

a b s t r a c t The traditional assay used to measure potency of inactivated influenza vaccines is a single-radial immunodiffusion (SRID) assay that utilizes an influenza strain-specific antibody to measure the content of virus hemagglutinin (HA) in the vaccine in comparison to a homologous HA reference antigen. Since timely preparation of potency reagents by regulatory authorities is challenging and always a potential bottleneck in influenza vaccine production, it is extremely important that additional approaches for reagent development be available, particularly in the event of an emerging pandemic influenza virus. An alternative method for preparation of strain-specific antibody that can be used for SRID potency assay is described. The approach does not require the presence or purification of influenza virus, and furthermore, is not limited by the success of the traditional technique of bromelain digestion and purification of virus HA. Multiple mammalian expression vectors, including plasmid and modified vaccinia virus Ankara (MVA) vectors expressing the HAs of two H5N1 influenza viruses and the HA of the recently emerging pandemic H1N1 (2009) virus, were developed. An immunization scheme was designed for the sequential immunization of animals by direct vector injection followed by protein booster immunization using influenza HA produced in vitro from MVA vector infection of cells in culture. Each HA antibody was highly specific as shown by hemagglutination inhibition assay and the ability to serve as a capture antibody in ELISA. Importantly, each H5N1 antibody and the pandemic H1N1 (2009) antibody preparation were suitable for use in SRID assays for determining the potency of pandemic influenza virus vaccines. The results demonstrate a feasible approach for addressing one of the potential bottlenecks in inactivated pandemic influenza vaccine production and are particularly important in light of the difficulties in preparation of potency reagent antibody for pandemic H1N1 (2009) virus vaccines. Published by Elsevier Ltd.

1. Introduction To facilitate the development, manufacture, and standardization of inactivated influenza vaccines, potency reagents are typically prepared and distributed by public health agencies including the World Health Organization (WHO) Essential Regulatory Laboratories (ERL) (National Institutes of Biological Standards and Control [NIBSC], UK; Center for Biologics Evaluation and Research [CBER] of the Food and Drug Administration, USA; National Institute of Infectious Diseases [NIID], Japan; Therapeutic Goods Administration [TGA], Australia). These reagents are used by manufacturers and regulatory agencies to determine the potency of licensed inactivated influenza vaccines, and allow standardization of vaccines made by various manufacturers. The traditional assay used to measure potency of inactivated influenza vaccines is a single-radial

∗ Corresponding author. Tel.: +1 301 827 2935; fax: +1 301 496 1810. E-mail address: [email protected] (J.P. Weir). 0264-410X/$ – see front matter Published by Elsevier Ltd. doi:10.1016/j.vaccine.2009.12.079

immunodiffusion (SRID) assay that utilizes a strain-specific antibody to measure the content of virus hemagglutinin (HA) in the vaccine in comparison to a homologous HA reference antigen [1,2]. The annual composition of influenza vaccines is routinely evaluated and changed as necessary to maintain effectiveness against currently circulating strains of virus. The entire process from virus surveillance, strain recommendation, generation of suitable vaccine candidates and potency reagents, manufacture and licensure is complex and critically time-sensitive [3]. There is little flexibility in these timelines if vaccine is to be available in time for the peak of influenza season. Although preparation of potency reagents rarely delays the production and availability of seasonal influenza vaccines, timely reagent production is challenging and always a potential bottleneck in influenza vaccine production. Obviously, in the event of a novel influenza virus emerging into the human population, the time frame for development and expanded manufacturing would be compressed to an even greater extent. Potency reagents would need to be developed and distributed as quickly as possible to expedite the characterization, formulation, and release

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Fig. 1. Expression of H5N1 HA from DNA and MVA vectors in Western blot analysis. (A) Expression of HA in whole cell lysates from Vero cells transfected with 1 ␮g of plasmid DNA carrying the HA gene from strain A/Vietnam/1203/04 (lane 1) or A/Indonesia/5/05 (lane 2). Sheep polyclonal antibody to HA derived from A/Vietnam/1203/04 (CBER potency reagent, diluted 1:2000) was used for detection. (B) Expression of HA in whole cell lysates from Vero cells infected with recombinant MVA carrying the HA gene from strains A/Vietnam/1203/04 (lanes 2 and 5) or A/Indonesia/5/05 (lanes 3 and 6). Samples from infection with a control recombinant MVA were run in lanes 1 and 4. Infection was performed at a moi of 2. Samples were harvested 48 h after infection. Sheep polyclonal antibodies to HA derived from A/Vietnam/1203/04 (CBER potency reagent, diluted 1:2000, lanes 1–3) and A/Indonesia/5/05 (CBER potency reagent, diluted 1:2000, lanes 4–6) were used for detection. (C) Expression of HA in supernatants from Vero cells infected with recombinant MVA carrying HA from strain A/Vietnam/1203/04 at 24 and 48 h post-infection. Supernatants from uninfected (ui) and infected (i) cells were harvested at either 24 or 48 h post-infection and pelleted through a 25% sucrose cushion. Infection was performed at a moi of 2. Sheep polyclonal antibodies to HA derived from A/Vietnam/1203/04 (CBER potency reagent, diluted 1:2000) was used for detection. Molecular weight markers (M) are shown in kDa.

of vaccine. Consequently, it is extremely important that several approaches for the development of vaccine potency reagents be available, especially if unexpected problems in virus growth or vaccine production be encountered. For production of the strain-specific reference antibody, HA is usually prepared for immunization by treatment of either the wildtype or vaccine candidate virus strain with bromelain to remove the HA from the virus particles [4,5]. The cleaved HA, which is missing the hydrophobic membrane anchor, is then purified from remaining virus proteins. Due to the quantities needed, production of the antibody is typically prepared in sheep and this process usually takes several weeks depending on the immunization schedule. Antiserum produced by immunization of this bromelain-HA is tested for specificity to HA in the SRID assay. While this procedure is usually reliable for HA production and immunization, the characteristics of bromelain digestion vary with different viruses and it is sometimes necessary to establish optimal conditions for HA purification experimentally. For example, recent experience revealed the pandemic H1N1 (2009) virus, as well as the related vaccine reassortants, showed a particular sensitivity to bromelain digestion that made purification of the virus HA for potency antibody production exceedingly difficult. Because of such problems, we have investigated alternative methods that might be suitable for generating strain-specific antibody. Ideally, such reagent preparation would begin as soon as a pandemic strain is identified so that the reagents and the potency assay would already be in place when manufacturers produce the first lots of vaccine. Here, we describe an alternative method for generating strainspecific potency antibody for use in potency assays for inactivated influenza virus vaccines. We use a combination of mammalian expression vectors for direct immunization of animals, as well as for the production of influenza hemagglutinin in vitro that can be used to boost the antibody titer. We show the suitability of this approach to generate strain-specific antibody that can be used to assay the potency of two H5N1 virus vaccines. We further demonstrate the use of this method to produce a strain-specific vaccine potency reagent antibody to the recently emerged swine-origin H1N1 virus [pandemic (H1N1) 2009] [6]. 2. Results Expression of H5N1 hemagglutinin using plasmid and modified vaccinia virus Ankara vectors. Since DNA expression vectors can be produced relatively quickly in the laboratory, we constructed plasmid vectors expressing the hemagglutinin (HA) genes of 2 H5N1 strains, and used these vectors as an initial “priming” immunization for the produc-

tion of strain-specific antibody. The genes encoding the HA of A/Vietnam/1203/04 and A/Indonesia/5/05 from the vaccine reference strains were inserted into plasmid expression vectors. Each of these genes was previously modified to remove the polybasic amino acid region at the cleavage site between HA1 and HA2 that is responsible for virulence of H5N1 viruses. Expression of authentic HA was verified by transfection of Vero cells and Western blot analysis using a polyclonal sheep antibody to HA. Fig. 1A shows that each plasmid vector expressed HA in lysates of transfected cells and that the predominant species in each case was the full-length hemagglutinin protein (HA0). Although construction of modified vaccinia virus Ankara (MVA) vectors is more time-consuming than construction of plasmid expression vectors, the ability to express high levels of recombinant antigen makes such vectors attractive as for immunization. We reasoned that construction of an MVA expression vector could take place in parallel with plasmid expression vector construction and then be used to boost the animals already primed with plasmid expression vectors. In addition, the MVA expression vectors could be used to produce sufficient amounts of recombinant protein in vitro for additional boosting to generate a high-titer hyperimmune antiserum. Therefore, we constructed MVA expression vectors expressing the HA of A/Vietnam/1203/04 and A/Indonesia/5/05. Viruses were plaque purified, expanded, and used to infect cells in culture to verify HA expression. As shown in Fig. 1B, each MVA expression vector expressed authentic HA in lysates of infected cells. As for the plasmid expression vectors, the predominant species of HA expressed was HA0. Since it has been shown in at least one in vitro system that expressed influenza HA, either in the presence of exogenously added neuraminidase (NA) or coexpressed neuraminidase, can be released from mammalian cells into the supernatant [7], we looked for HA in the supernatants of cells infected with the MVA expression vectors in the presence of exogenously added NA (Fig. 1C). By 48 h post-infection, supernatants contained authentic HA. This HA was contained in high molecular weight particles (data not shown) and the size of the HA was full-length (HA0) as shown in Fig. 1C. This supernatant derived HA was concentrated for use as a booster immunization. Antibody production to H5N1 hemagglutinin following recombinant vector immunization. The plasmid DNA and MVA vectors expressing the HA of A/Vietnam/1203/04 and A/Indonesia/5/05 were used in a sequential series to immunize rabbits in order to test the feasibility of the recombinant approach to generate a high-titer polyclonal antibody suitable for use as a potency reagent. For the A/Vietnam/1203/04 and A/Indonesia/5/05 vectors, rabbits were immunized with plasmid DNA vectors using a needle-free injector device (Biojector,

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Fig. 2. Specificity of polyclonal antibody preparations for H5N1 HA in Western blot analysis. A/Vietnam/1203/04 (lanes 1 and 3) and A/Indonesia/5/05 (lanes 2 and 4) virus samples (2 ␮g) were separated by SDS-PAGE, transferred to nitrocellulose and blotted with rabbit polyclonal antibodies to HA derived from A/Indonesia/5/05 (diluted 1:2500, lanes 1 and 2) or A/Vietnam/1203/04 (diluted 1:2500, lanes 3 and 4). Molecular weight markers (M) are shown in kDa.

Inc.) followed by direct subcutaneous injections of the corresponding MVA expression vector, and finally by 2 immunizations with HA produced by MVA vector infection of tissue culture cells in vitro. One week following the final immunization, sera was collected and antibody was purified by Protein A affinity chromatography. Fig. 2 shows that each polyclonal antibody preparation recognized HA from H5N1 viruses in western blot analysis. The A/Vietnam/1203/04 antibody preparation recognized A/Indonesia/05/05 virus, and vice versa, in such an analysis. Antibody preparations recognized both HA1 and HA2 forms of the virus as might be expected since the immunizing antigen expressed in the recombinant systems was full-length HA. Specificity of each H5N1 HA antibody prepared by the recombinant expression vector method was analyzed by hemaggutination inhibition (HI) analysis, and compared to control antiserum produced in the traditional manner by immunization of sheep with bromelain-cleaved and purified HA (Table 1). In this analysis, each polyclonal antibody had a high HI titer to the homologous reference virus or reference antigen. The sera to the H5N1 hemagglutinin had a low HI titer to the H1N1 antigen used as a specificity control and vice versa, but there was cross-reactivity of each H5N1 antibody with the heterologous H5N1 antigen. In general each rabbit polyclonal antibody preparation made to recombinant HA was highly specific for H5N1 hemagglutinin and inhibited hemagluti-

Fig. 3. Specificity of polyclonal antibody preparations in antigen-specific ELISA. ELISA plates were coated with A/Indonesia/5/05 polyclonal antibody (10 ␮g/ml) and incubated with the indicated reference antigens (inactivated virus) at dilutions starting at 15 ␮g/ml. Binding of each influenza antigen subtype was detected using a homologous type-matched secondary sheep antibody.

nation as well as did reference antiserum made by immunization of sheep with bromelain-cleaved and purified HA. Interestingly, there was a low level of inhibition of the control A/Brisbane/59/07 antigen observed with the traditional reference antiserum, suggesting some differences in the two types of antibody preparations. The specificity of each recombinant vector-generated antiserum was also demonstrated by the high affinity for H5N1 HA when the antibody was used as a capture antibody in an antigen-specific ELISA. Fig. 3 shows that the A/Indonesia/5/05 antibody bound both the homologous and heterologous H5N1 HA with high affinity, but bound weakly the HA from other influenza subtypes such as H1N1 (A/New Caledonia/20/99) or H3N2 (A/Wisconsin/67/05). Similar results were observed using the A/Vietnam/1203/04 antibody preparation (data not shown). Finally, the ability of each recombinant HA-generated polyclonal antibody to substitute as a reference reagent in an SRID potency assay was evaluated by comparison to the designated reference antiserum for each strain. As shown in Fig. 4A and B, the size of the precipitin ring is linear over the concentration range of 5.4–21.7 ␮g for both A/Vietnam/1203/04 and A/Indonesia/5/05 reference antigens when the standard sheep reference antiserum is used in the SRID assay. For each reference antigen, easily measurable precipitin rings were observed when the homologous rabbit antibody was substituted for the sheep reference antiserum, with a similar linear relationship between ring size and antigen concentration. The

Table 1 HA inhibition titers of traditional and alternative H5N1 potency antibody. HA inhibition titers Potency antibodya

A/Vietnam (traditional)

A/Vietnam (alternative)

A/Indonesia (traditional)

Antigen A/Vietnam/1203/2004 (H5N1) Reference antigen Reference virus

160 320

160 320–640

160 160

A/Indonesia/5/2005 (H5N1) Reference antigen Reference virus

160 320

160 160–320

640 1280

A/Brisbane/59/2007 (H1N1) Reference virus

40

<16

64–128

a

A/Indonesia (alternative)

A/Brisbane/59/07 (traditional)

80 160

<16 <16

640 1280

<16 <16

16

1024

Antiserum used in the HAI assay was made by the traditional method of immunization with bromelain-cleaved and purified HA; alternative antibody was made by immunization with recombinant vectors and protein as described in Section 2; values in bold indicate potency antibody was homologous with the tested antigen.

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Table 2 HA content (␮g/ml) in vaccine lots determined by SRID using traditional and alternative potency antibody. HA (␮g/ml) Potency antibodya

A/Vietnam (traditional)

A/Vietnam (alternative)

A/Indonesia (traditional)

A/Indonesia (alternative)

A/California (traditional)

A/California (alternative)

Vaccine A/Vietnam/1203/2004 (H5N1) 116 ± 11 Lot 1c Lot 2 116 ± 20

100 ± 11 112 ± 30

NDb 124 ± 21

ND 164 ± 4

ND ND

ND ND

A/Indonesia/5/2005 (H5N1) Lot 1 ND Lot 2 664 ± 86

ND 716 ± 68

185 ± 18 664 ± 87

303 ± 24 646 ± 50

ND ND

ND ND

A/California/7/2009 (H1N1) Lot 1 ND Lot 2 ND Lot 3 ND

ND ND ND

ND ND ND

ND ND ND

79 ± 9 925 ± 137 70 ± 8

72 ± 2 1017 ± 34 70 ± 9

a Traditional potency antiserum used in the SRID assay was made by the immunization with bromelain-cleaved and purified HA; alternative antibody was made by immunization with recombinant vectors and protein as described; values in bold indicate potency antibody was homologous with the tested antigen. b ND—not done c Lots 1, 2, and 3 for each indicated vaccine were produced by different manufacturers

results indicated that the HA antibody prepared by the recombinant expression vector method was functional in the traditional SRID assay and suggested that such an antibody preparation could be used to determine the potency of inactivated influenza vaccines. To formally test this possibility, lots of both A/Vietnam/1203/04 and A/Indonesia/5/05 vaccine were assayed for their HA antigen content by SRID using either the traditional reference antiserum or the

Fig. 4. SRID analysis of H5N1 potency reference antigen using reference antiserum and antiserum prepared by the alternative method. Dilutions of A/Vietnam/1203/04 (A) or A/Indonesia/5/05 (B) reference antigens were analyzed by SRID using either the homologous reference antiserum (closed circles) or the alternative antiserum (open circles). Precipitin rings were measured in two directions to the nearest 0.1 mm for determination of diameter.

rabbit recombinant HA-generated polyclonal antibody (Table 2). In most cases, there was good agreement between the values determined using either antibody, although for one vaccine lot of A/Indonesia/5/05 there was a significant difference in the potency value obtained using the two types of antibody. The reason for the discrepancy in assay results for this lot is not known, although it is unlikely to be manufacturer specific since other vaccine lots from the same manufacturer were assayed in the A/Vietnam/1203/04 and the A/California/7/2009 analysis. In addition, one lot of each H5N1 vaccine was assayed with the heterologous H5N1 antibody; the HA values obtained were similar to those obtained with the homologous antibody, suggesting the feasibility of using heterologous H5N1 antibody for potency determination in at least some circumstances. Overall, the results strongly suggested the feasibility of the recombinant vector method to produce a strain-specific antibody that was capable of being used as a reagent for potency determination of inactivated influenza vaccines. Specific antibody production to pandemic H1N1 hemagglutinin following recombinant vector immunization. The emergence of a novel swine-related H1N1 into the human population in the spring of 2009 presented formidable challenges for vaccine manufacturers and public health agencies [6]. For example, the pandemic H1N1 (2009) viruses, including vaccine reassortants, were peculiarly sensitive to digestion with bromelain which made purification of the virus HA for potency antibody production exceedingly difficult. Fig. 5A shows the results of one of many attempts to digest a pandemic H1N1 (2009) virus reference strain (A/California/07/2009) with bromelain to obtain HA for animal immunization. Within an hour after bromelain addition, HA was cleaved from the virus, but only small soluble molecular weight fragments of HA were observed. Since successful bromelain digestion would have cleaved HA near the hydrophobic membrane anchor, a soluble full-length HA1 fragment would have been expected upon reducing SDS-PAGE analysis. We were routinely unsuccessful in our attempts to isolate HA by this method in spite of modifications to enzyme concentration, time of digestion, temperature, and concentration of reducing agents present. Although one of the WHO Essential Reference Laboratories (NIBSC, UK) was able to obtain enough HA by bromelain digestion for animal immunization, other laboratories and vaccine manufacturers were not successful in this process. As an alternative, we applied the strategy described above to generate a pandemic H1N1 (2009) antibody and evaluate its suitability for potency determination in an SRID assay. Fig. 5B and C show the expression of A/California/04/2009H1N1 HA from plasmid and MVA expression vectors. As for the H5N1

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Fig. 5. Pandemic H1N1 (2009) HA. (A) Bromelain treatment of A/California/7/2009 reference virus. Twenty micrograms bromelain was added to 10 mg of purified virus at room temperature. At 1, 2, and 4 h, aliquots of the reaction were removed and adjusted to 0.1 M NaCl to stop the bromelain reaction. Virus pellets (p) and supernatants (s) were separated by ulracentrifugation (∼100,000 × g), and analyzed by SDS-PAGE and Western blot. (B) Expression of pandemic H1N1 (2009) HA from DNA vector in Western blot analysis. (C) Expression of pandemic H1N1 (2009) HA from MVA vector in Western blot analysis. Table 3 HA inhibition titers of traditional and alternative pandemic H1N1 (2009) potency antibody. HA inhibition titers Potency antibodya

A/California (traditional)

A/California #1 (alternative)b

A/California#2 (alternative)

A/Brisbane/59/07 (traditional)

A/Uruguay/716/2007 (traditional)

Antigen A/California/07/2009 (H1N1) Reference antigen Reference virus

2048 2048

1024 1024

2048 1024-2048

1024 256–512

1024 256–512

A/Brisbane/59/2007 (H1N1) Reference virus A/Uruguay/716/2007 (H3N2) reference virus

64–128 128

32 64

4096 256

128 4096

<8 <8

a Traditional antiserum used in the HAI assay was made by immunization with bromelain-cleaved and purified HA; alternative antiserum and antibody was made by immunization with recombinant vectors and protein as described; values in bold indicate potency antibody was homologous with the tested antigen. b A/California #1 was purified rabbit IgG; California #2 was sheep antiserum.

Fig. 6. SRID analysis of pandemic H1N1 (2009) potency reference antigen using reference antiserum and antiserum prepared by the alternative method. Dilutions of A/California/7/2009 reference antigen were analyzed by SRID using either the homologous reference antiserum (closed circles) or the alternative antiserum (open circles). Precipitin rings were measured in two directions to the nearest 0.1 mm for determination of diameter.

hemagglutinin expression vectors, the predominant HA species expressed from both types of vectors was HA0. Following a similar strategy as used for H5N1 HA antibody production, rabbits were immunized with plasmid DNA vector using a needle-free injector device (Biojector, Inc.) followed by a subcutaneous injection of the

MVA expression vector, and finally by 2 immunizations with HA produced by MVA vector infection of tissue culture cells in vitro. Serum was obtained following the MVA immunization and each of the two protein boosts and was found to be suitable for use in an SRID assay using the pandemic H1N1 antigen standard (data not shown) and was specific for the pandemic H1N1 (2009) virus by HI analysis (Table 3). There was some cross-reactivity in the HI assay when traditional antiserum was used with heterologous virus antigen. However, the antibody produced by immunization with recombinant A/California/04/2009 HA vectors and protein was highly specific for the A/California/07/2009 antigen. Further investigation showed that antibody prepared by the alternative method using recombinant vectors was capable of substituting for the traditional sheep reference antiserum in an SRID analysis, with a linear relationship observed between the precipitin ring size antigen concentration (Fig. 6). Importantly, when the alternative antibody was used to assay the HA content of vaccines prepared by different manufacturers, there was good agreement between the resulting values and those obtained using the traditional sheep reference antiserum (Table 2). Taken together, the results indicated the viability of an alternative recombinant expression method for preparation of antiserum reagent for SRID potency analysis of pandemic H1N1 vaccines. 3. Discussion The traditional method used to determine the potency of inactivated influenza vaccines is the SRID assay, essentially as described several decades earlier [1,2]. As noted by others [8], the technique

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is not technically demanding but because of the world-wide manufacture and use of influenza vaccines, standardization is critical and the process of calibration and standardization is extremely timesensitive. Recently, several newer approaches have been explored as alternatives for quantification of the HA content in vaccines [9–12]. To date, however, none of the newer techniques have been shown to measure the same antigenic form and amount of HA as the SRID, nor has there been implementation of any of the newer techniques for potency determination of inactivated influenza vaccines. Thus, in spite of its obvious limitations (e.g., relative lack of sensitivity, requirement for specific reagents for each virus strain, etc.), the SRID assay continues to be the standard method for potency analysis of inactivated influenza vaccines. Therefore, pandemic influenza preparedness planning necessitates that several contingency methods be developed to ensure that SRID potency reagents are available in an emergency and that back-up techniques be in place for each aspect of the process. The emergence of the pandemic H1N1 (2009) virus the spring of 2009 presented multiple challenges for public health agencies as well as vaccine manufacturers. Not only did the wild-type pandemic H1N1 (2009) viruses grow poorly, reassortant viruses designed to be high growth vaccine strains also grew poorly relative to typical seasonal vaccine viruses with an obvious impact on the yield of vaccine manufacture. In addition, there were difficulties in preparing immunizing antigen for production of potency reference antiserum using traditional techniques. We have shown here that a strain-specific antibody, suitable for vaccine potency analysis, can be prepared using a method that does not require the presence or purification of influenza virus and is not limited by the success of bromelain digestion and purification of HA. This finding is particularly important since it demonstrates a viable additional method for one of the potential bottlenecks in inactivated pandemic influenza vaccine production, namely the production of potency reagents. We used a set of vectors that could be constructed rapidly (DNA expression vectors) and produce high yields of authentic HA (MVA expression vectors). These vectors served as priming immunizations and were boosted by HA produced in vitro. Although we have not done a complete quantitative analysis of the antiserum obtained after each immunization step, in most cases antiserum obtained after only DNA priming and MVA vector boosting was functional in the SRID assay. We also did not specifically investigate whether there is an advantage to multiple DNA or MVA immunizations. However, multiple DNA immunizations are often used to increase antibody response in DNA vaccination protocols, and there are several reports that indicate multiple immunizations can successfully boost an antibody response to MVA [13,14]. Nevertheless, in our studies, subsequent boosting with HA produced in vitro in the presence of an adjuvant resulted in higher titer antibody with better results in the SRID assay. Although our initial studies were done in rabbits, we have recently applied the same procedure to immunization of sheep and produced a similar high-titer antiserum suitable for use in SRID assays (data not shown), suggesting that scale-up to quantities of antiserum that would be needed for typical reagent supply should not be a significant problem. Moreover, it is unlikely that the techniques employed in our study are the only viable alternative option for generating a strain-specific potency antibody. Rather, the results suggest that other methods that can rapidly be employed for antigen expression might also be successful in generating a workable potency antiserum, and should be investigated. The results from the present study have other implications of interest. For one, they suggest that the source of the immunizing antigen may not be especially critical for the generation of a useful potency reference antiserum. Our immunization approach employed vectors that expressed influenza HA in mammalian cells, either directly after vector immunization of animals or following

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protein expression in vitro in mammalian cells. In contrast, the traditional approach to potency antiserum production uses antigen that is prepared from virus grown in eggs for immunization. There has been a concern about the interchangeability of influenza potency reagents for cell-derived compared to egg-derived vaccines. While our results do not directly shed light on the question of whether cell-derived reference antigen is required for cell-derived vaccines and vice versa, the results do suggest that there is flexibility in how the reference antibody can be prepared. Obviously, additional studies are still needed to determine whether there are options for producing universal reference antigens that can be used for either types of inactivated influenza vaccines. Characterization of the antisera that we produced using the recombinant expression system suggested additional uses for an antibody prepared by such methods. In particular, the affinity and specificity of the antiserum when used as a capture ELISA suggests the possibility that a similar such assay might be developed to determine vaccine potency, possibly with an increased sensitivity compared to current SRID assays. Interestingly, although each investigated antiserum was polyclonal and made to the entire HA molecule, cross-reactivity to other subtypes was minimal. This is likely due to the fact that no other influenza proteins are present during immunization, and consequently the HA-specific antiserum should have no interaction with any other influenza viral proteins in the sample being assessed. On the other hand, each H5 antibody bound both homologous and heterologous H5N1 HA with high affinity, and in fact, the sensitivity of antigen detection could be increased further by using a monoclonal antibody specific to H5 HA (data not shown). Other work is already underway to develop and evaluate the feasibility of such an assay. Finally, the use of mammalian expression vectors that can produce authentic HA that are released into the supernatant of vector-infected cells is especially intriguing. There have been several other reports describing the assembly of influenza virus-like particles in other expression systems. Our initial characterization revealed that the cell culture supernatant HA was in the form of high molecular weight particles and required the presence of exogenously added neuraminidase for efficient release from the cell (data not shown). In the work described here, we used the supernatant HA to provide an additional immunization boost. However, such vector-produced particles have a multitude of other potential uses, such as a method to study virus assembly [7,15], as reagents in hemagglutination inhibition assays, as vaccine candidates [16–18], and as tools to dissect the immune response to specific virus proteins. Such studies with the mammalian-derived influenza VLPs are also underway and will be described elsewhere. In summary, we have described an alternative method for generating strain-specific potency antibody for use in potency assays for inactivated influenza virus vaccines. The unique combination of mammalian expression vectors for direct immunization of animals and production of influenza hemagglutinin in vitro for boosting results in a high-titer antibody suitable as an alternative to the traditional potency reference antiserum. The difficulties in producing a traditional potency antiserum to the recently emerged pandemic H1N1 (2009) virus demonstrate the value of such an alternative method. It is equally important that a variety of techniques also be developed for other aspects the influenza vaccine reagent process. 4. Materials and methods 4.1. Cells and viruses Primary chicken embryo fibroblasts (CEF), DF-1 and Vero cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM, Invitrogen, Carlsbad, CA) supplemented with 10% FBS (HyClone, Logan, UT), 2 mM l-glutamine (Invitrogen), and 50 ␮g/ml gen-

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tamicin (Invitrogen). CEF cells were obtained from Charles River Laboratories, Wilmington, MA; DF-1 and Vero cells were originally obtained from ATCC (CRL-12203 and CCL-81, respectively). Modified vaccinia virus Ankara (MVA) was propagated in CEF cells and titered by plaque assay on DF-1 cells. Virus stocks were purified by sedimentation through a 36% (w/v) sucrose cushion by centrifugation. All methods for the generation, propagation, and preparation of recombinant viruses were essentially as described by Earl [19]. 4.2. Construction of influenza HA expression vectors Platinum Pfx DNA polymerase (Invitrogen, Carlsbad, CA) was used for all PCR reactions, unless indicated otherwise. All constructions were sequenced with an ABI-377 DNA sequencer (Applied Biosystems, Foster City, CA). Routine molecular biology procedures used in the course of plasmid constructions and characterization were essentially as described previously [19]. All plasmids were grown in Escherichia coli TOP10 (Invitrogen). The HA genes from influenza A/Vietnam/1203/2004 and A/Indonesia/05/2005 were cloned into pShuttle2 (Clontech, Mountain View, CA). The ORFs of both HA genes had been previously modified by removal of the polybasic amino acid sequence associated with H5N1 virulence [20]. The HA gene from A/California/4/2009 was amplified as a SalI/NotI fragment of approximately 1.7 kb by PCR directly from virus (Zhiping Ye, CBER, FDA) and cloned into the plasmid expression vector pVRC-8400 obtained from the Vaccine Research Center (NIH). For MVA expression vector cloning, the insertion vector pLW44 (Bernhard Moss, NIAID, NIH) was modified into a GatewayTM destination vector (Invitrogen) by enzyme digestion with SmaI and then insertion of the attR cassette B (Invitrogen). The sequences of each of the influenza HA genes were amplified by PCR from the respective plasmid DNA templates (described above) and first cloned into the pENTR/D-TOPO GatewayTM entry vector (Invitrogen), resulting in plasmids pENTR-1203/04HA, pENTR-05/05HA, and pENTR-4/09SFHA. The HA genes were transferred into the MVA insertion vector by combining entry clones with the destination vector in an LR recombination reaction, resulting in plasmids pLW44-1203/04HA, pLW44-05/05HA, and pLW44-SFHA. The relevant DNA portions synthesized by PCR were confirmed by restriction enzyme digestion and confirmed by DNA sequencing. Expression of influenza HA was confirmed by Western blot analysis. MVA recombinant viruses were made by infecting confluent layers of DF1 cells in 6-well plates with MVA at a moi of 0.1. After incubation for 2 h, the inoculum was removed, replaced with fresh media, and cells were transfected with 2 ␮g of the plasmid insertion vector using Fugene reagent (Roche Diagnostics, Mannheim, Germany) at a ratio of 3 ␮l Fugene/␮g of DNA. Transfected/infected cells were harvested after three days, and dilutions of the virus plaqued on DF1 cells. Recombinant MVA viruses were identified by expression of EGFP, and purified by multiple rounds of plaque titration. Purified MVA recombinants were tested for expression of the respective HA by Western blot analysis and the absence of wild-type MVA.

and concentrated ∼100-fold by Amicon Ultra-15 filtration with a 100K MWCO (Millipore) before layering onto a continous sucrose gradients (10–45% in PBS; 4 h at 35,000 rpm in a Beckman SW40Ti rotor). The middle portion of the gradient containing HA was collected and concentrated. 4.4. Animals and immunizations New Zealand White rabbits were purchased from Charles River Laboratories and housed in cages at a core facility at CBER/FDA. Sterile food and water were supplied ad libitum. All immunizations and blood draws were performed in accordance with an approved animal protocol. DNA vectors were delivered by the intradermal route of administration using a needle-free injector device (Biojector, Inc.). MVA vectors and protein boosts were delivered by subcutaneous injection. Protein was mixed with TiterMax® adjuvant (Sigma–Aldrich, St. Louis, MO). Additional rabbit and sheep immunizations using a similar protocol were done under contract (Southern Biotech, Birmingham, AL and Capralogics, Hardwick, MA). 4.5. Western blot analysis Vero cells (∼106 ) were transfected with plasmid expression vectors using Fugene reagent or infected with MVA expression vectors at a multiplicity of 5 for 8 h. Cells were harvested and lysed in RIPA buffer (Pierce, Rockford, IL) supplemented with complete Mini, EDTA-free protease inhibitor cocktail (Roche). Protein samples were mixed in 4× NuPAGE LDS sample buffer (Invitrogen)10× Sample Reducing Agent to the final working concentration and analyzed by electrophoresis in a 4–12% gradient sodium dodecyl sulfate (SDS)-polyacrylamide gel (NuPAGE Bis-Tris gel in 3(N-morpholino)propanesulfonic acid [MOPS]-SDS running buffer; Invitrogen), and the proteins were transferred to a nitrocellulose membrane using the iBlot transfer device (Invitrogen). After being blocked with TBS containing 0.5% (w/v) BSA (A7888, Sigma–Aldrich, St. Louis, MO) and 0.1% Tween 20 for 1 h, the membranes were incubated with various primary antibodies followed by washes with TBS containing 0.1% Tween 20 and then incubated with appropriate secondary antibodies conjugated with horseradish peroxidase (HRP) for 1 h. Detection was by chemiluminescence (SuperSignal West Dura extended-duration substrate; Pierce) using a LAS-3000 imager (Fujifilm Medical Systems, Stamford, CT). 4.6. ELISA IgG was purified by protein A chromatography and used to coat 96-well Immulon-2HB microplates (Dynex Technologies, Chantilly, VA) overnight at 10 ␮g/ml. After blocking with PBS/10% FBS (HyClone), dilutions of virus antigen were incubated for 2 h at 37 ◦ C. An appropriate secondary antibody conjugated with HRP was used for detection. A 1:1 mix of ABTS:H2 O2 (Southern Biotech) was used as substrate. Plates were read on a VersaMax microplate reader and data generated with Softmax Pro 5.0 (Molecular Devices, Sunnyvale, CA).

4.3. Preparation of HA from supernatants of infected cells 4.7. SRID assay Roller bottles (850 cm surface area, Corning, Lowell, MA) with confluent monolayers of Vero cells were infected in OptiMEM (Invitrogen) with recombinant MVA at a moi of 2. The inoculum was removed after 2 h and replaced with fresh OptiMEM (60 ml) containing AraC (40 ␮g/ml, Sigma–Aldrich, St. Louis, MO), exogenous neuraminidase (Vibrio cholerae, 2 ␮U/ml, Sigma–Aldrich), and Hepes (20 mM, Invitrogen). Supernatants were harvested after 48 h, clarified by low-speed centrifugation to remove cell debris,

SRID assay was performed essentially as described previously [2,21] and is based on the diffusion of detergent-disrupted virus (or virus antigen) into an agarose gel containing specific HA antibodies. Briefly, an antibody solution at the optimal working concentration is mixed with melted 1% agarose (Invitrogen) at 50–55 ◦ C in PBS for preparation of the agarose plate. Following solidification at room temperature, 4 mm wells are punched in the gel for application

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of the vaccine or antigen sample (15 ␮l). Initial dilutions of antigen are prepared in 1% Zwittergent 314 (Calbiochem, Darmstadt, Germany), incubated for 30 min at room temperature. Following incubation, further dilutions are made in PBS, added to wells of the gel, and the gels placed in a sealed moist chamber at room temperature for 18 h. Gels are transferred to GelBond film (Lonza, Rockland, ME), dried and stained with Coomassie Brilliant Blue. The diameter of the precipitin zone is measured in two directions at right angles to the nearest 0.1 mm. Vaccine potency in ␮g/ml HA is calculated by the parallel line bioassay method [22] using reference and test vaccine dose response curves (log antigen dilution vs. log zone diameter). Statistical parameters for determining test validity are based upon correlation coefficient (r) and equality of slopes (t) between test and reference antigens. 4.8. Hemagglutination inhibition assay The hemagglutination inhibition assay (HAI) was performed in 96-well plates (U-bottom) plates by standard methods essentially as described previously [23] using 0.5% chicken red blood cells suspended in PBS (pH 7.2). Acknowledgements We thank Gary Nabel (Vaccine Research Center, NIH) for the plasmid expression vector pVRC-8400, Bernard Moss (National Institute of Allergy and Infections Diseases, NIH) for the MVA insertion vector pLW44, Zhiping Ye and Maryna Eichelberger (CBER/FDA) for critical reading of the manuscript, and Arunima Kumar, Benjamin Blumberg, and Jackeline Soto for technical assistance. This work was supported in part by the National Vaccine Program Office, Department of Health and Human Services. References [1] Wood JM, Schild GC, Newman RW, Seagroatt V. An improved single-radialimmunodiffusion technique for the assay of influenza haemagglutinin antigen: application for potency determinations of inactivated whole virus and subunit vaccines. J Biol Stand 1977;5(3):237–47. [2] Williams MS. Single-radial-immunodiffusion as an in vitro potency assay for human inactivated viral vaccines. Vet Microbiol 1993;37(3–4):253–62. [3] Gerdil C. The annual production cycle for influenza vaccine. Vaccine 2003;21(16):1776–9. [4] Brand GM, Skehel JJ. Crystalline antigen from the influenza virus envelope. Nat New Biol 1972;238:145–7. [5] Compans RW, Klenk HD, Caliguiri LA, Choppin PW. Influenza virus proteins. I. Analysis of polypeptides of the virion and identification of spike glycoproteins. Virology 1970;42(4):880–9.

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