Criteria to be fulfilled by influenza vaccine candidate viruses

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Antigenically different viruses (supported by other findings)

Differ from the vaccine virus strains as characterized by the HI test supported by clinical and epidemiological information and (a) genetic characterization and (b) serological tests with sera from subjects immunized with current seasonal influenza vaccine

Dominant influenza viruses

Although many different variant viruses of, e.g., H1 and H3 subtypes and B virus lineages emerge and can be isolated throughout the year, most virus variants presence is short lived and geographically limited. Only very few gain global significance and will dominate the next influenza seasonal outbreaks. To detect those, samples from different continents are required. As surveillance is ongoing simultaneously in several WHO centers with different geographical reach, exchange of information and candidate viruses and their antisera raised in ferrets is vital. Phylogenic trees generated from sequencing of parts of the HA and NA surface protein help detect clustering of antigenically related variant viruses (e.g., Fig. S1-3 in Ref. (4)). In addition, WHO Headquarters assesses the epidemiological and clinical relevance of these new variant viruses routinely through data obtained from national authorities (http://www.who.int/influenza/surveillance_monitoring/updates/en/; accessed 10 Jan 2012)

Suitable for vaccine preparation

To comply with regulatory requirements, vaccine companies require virus isolates that (a) are antigenically and genetically similar to those identified by WHO as becoming dominant; (b) have a known passage history isolated on approved egg substrates (With more influenza vaccine producers switching over to cell-culture production, standards are also developed by regulatory agencies for viruses isolated in cell culture.); (c) have acceptable growth characteristics (grow well in hens eggs)

The overarching task of the WHO Collaborating Centers is to detect influenza viruses that fulfill all of the below criteria (see Table 2):

1. Antigenically (ignificantly different from the current vaccine virus strains

2. Are or likely to become the dominant influenza viruses during the next influenza season

3. Suitable for vaccine strain preparation

To this end, the WHO CCs and affiliated laboratories carry out a range of activities and perform various laboratory tests with viruses from national influenza centers. They include several of the below efforts but are not necessarily limited to:

( a) Preparation of egg isolates including initial assessment of their growth characteristics that could subsequently be used to prepare high growth reassortants (see section 2 of this chapter).

(b) Hemagglutination Inhibition Tests of isolates for antigenic analyses.

To this end, the viruses from the National Influenza Centers are tested for how well their hemagglutinating ability is inhibited by a panel of ferret sera raised against the vaccine virus and other past and currently circulating variants. Results are compiled in so-called Hl-tables and more recently in antigenic maps (6). In addition, human sera are tested from subjects of different age (healthy adults; elderly) and locations (often USA, Europe, and Japan) who were immunized with the current influenza vaccine.

( c) Preparation of ferret sera from low-reacting variant viruses (see previous bullet) and distribution between WHO Collaborating Centers for Hi-testing to low-reacting variant viruses.

(d) Genetic analyses of portions of the hemagglutinin glucopro-tein. Results are captured in phylogenetic trees compiled based on the number of amino acid differences discovered in the HA of the viruses. Clustering of influenza viruses with reduced Hl-titers is valuable supporting evidence on the emergence of a new dominant variant virus.

(e) Preparation of reagents test kits for National Influenza Centers and affiliated laboratories for initial virus analyses and detection of variant viruses (low reactors) and potentially new subtypes. The test kits contain diagnostic reagents including polyclonal sera, monoclonal antibodies, and viral antigens for relevant influenza strains. These kits are updated and distributed annually to ensure standardized analysis of current strains and submission of antigenic variants to WHO Collaborating Centers for detailed analysis.

(f) Assessing neuraminidase activity to detect antiviral resistance.

The WHO recommendation's meetings (Mid Feb; Mid Sep) are timed to both allow for sufficient virological and disease surveillance AND for influenza vaccine production, delivery, and immunization ahead of the next influenza season in the Northern and Southern Hemisphere. This tight time schedule requires compromises; e.g., vaccine manufacturers are forced to begin production at risk often in January usually with the strain they believe is the least likely one updated by WHO in February. That has resulted several times in the waste of 6-8 weeks of vaccine production. Likelihood of these losses can be reduced by close interactions between WHO Collaborating Centers and vaccine manufacturers during the weeks leading to the WHO strain selection meetings. On the other hand, duration of productive influenza surveillance may also be short with the influenza season often only beginning around end December or July, respectively, leaving little time for international exchange of strains, preparation of ferret sera, or development of high growth reassortants. Because of these constraints, there have been instances in the past when no suitable vaccine virus was available and production had to commence with the available seed virus of the preceding year (e.g., 2003).

Recommendations on Influenza Viruses for Vaccine Production

2. Preparation of High Growth Reassortants by "Classical" Reassortment Method

Jianhua Le, Ramanunninair Manojkumar, Barbara A. Pokorny, Jeanmarie Silverman, and Doris Bucher

2.1. Introduction

Influenza vaccines have been prepared with influenza virus grown in embryonated eggs since the 1940s (7, 8). In many cases, the human isolate of influenza virus grew poorly in eggs in spite of multiple passages, jeopardizing production of sufficient doses of vaccine to meet demand. In 1969, Edwin D. Kilbourne proposed that high-yielding (hy) or high-growth reassortant (hgr) viruses could be developed by coinfection of A/PR/8/34 (PR8), along with the current wild-type (wt) "target" virus with resultant "recombinants," later found to be reassortants (9). In this process selection against the surface antigens of PR8 is made with antisera to PR8. The antisera neutralizes any virus which has the surface antigens of PR8, resulting in virus reassortants which have the correct surface antigens. At the same time, selection for growth occurs with reassortant viruses with the property of high growth in eggs out-competing slower growing viruses. Since 1971, the type A influenza component for the majority of influenza vaccines has been produced using hy reassortants (10, 11).

Generation of hy reassortant vaccine seed viruses for influenza A viruses requires incorporation of the two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA), from wt "target" virus with one to six of the remaining genes from the hy donor virus. H3N2 hy reassortants are generated using PR8 (H1N1) as the hy donor virus to allow a clear antigenic distinction between H3N2 and H1N1 viruses, permitting ready selection with H1N1 antisera. Influenza A virus H1N1 hy reassortants are generated using an H3N2 hy reassortant donor virus which is 6:2, that is, the H3N2 donor has six "internal" genes or those genes which encode proteins other than the surface antigens from PR8 along with the two genes for H3 hemagglutinin and N2 neuraminidase. We currently use NYMC X-157 [a 6:2 hy reassortant containing the six internal genes from PR8 and the HA and NA genes from the wt virus, A/New York/55/2004]. X-157 was developed in our laboratory and used as the H3N2 component of the 2005-2006 seasonal influenza vaccine for the northern hemisphere.

2.2. Procedure

The development of hy reassortants proceeds as follows:

Step 1: Amplification of wt virus target

An egg isolate of wt "target" virus is provided to us by the CDC. This initial wt virus is amplified in 10-12-day-old embryonated SPF Premium eggs (Specific Pathogen-Free, Charles River, CT) with inoculation at a dilution of 10-1-10-3 depending on the initial virus titer [all dilutions are made with sterile phosphate-buffered saline (PBS) with added 0.025% gentamicin (Sigma-Aldrich, St. Louis, MO)]. Incubation proceeds for 42-46 h at

35°C. After chilling at 4°C for several hours or overnight, the allantoic fluids are harvested and the hemagglutination (HA) titer determined with the use of chicken erythrocytes (0.5% in PBS).

Step 2: Coinfection of wt and hy donor viruses

Allantoic fluids for the wt and hy donor viruses are co-inoculated at equal volumes (50 ^l each; total volume 100 ^l) into 10-12-day-old SPF eggs at 10_1 and 10~4 dilutions, respectively (PBS with 0.025% gentamicin). The mixture is allowed to stand at room temperature for 30 min before injecting into the eggs. Generally reassortant viruses are developed independently in parallel in eight different egg lineages, with sequential egg-to-egg passage through each of the steps. In addition, control viruses, including wt and hy donor, are each independently passaged in four egg lineages.

Steps 3-5: Antibody selection

After 42-48 h of incubation at 35°C followed by refrigeration, the allantoic fluids are harvested from the embryonated eggs. Allantoic fluids containing progeny virus (100 ^l) are incubated with anti-PR8 or anti-X-157 antisera or antibody (100 ^l) at dilutions from 1:10 to 1:40 in order to eliminate virus containing the HA and NA from the donor virus. The mixture is incubated at room temperature for 15 min before injecting into the eggs. This antibody selection step is repeated twice. Growth of the resultant viruses is monitored by HA titer at each step. Following step 5, the antigenic identity of the hemagglutinin and neuraminidase is assessed by hemagglutination inhibition (HI) activity and neuraminidase inhibition (NI) activity. The parental source of the genes is determined by RT-PCR/restriction fragment length polymorphism (RFLP) analyses.

Step 6: Amplification

An additional passage in eggs is performed at 10-3-10-4 dilution without antisera or antibody to amplify the virus. The harvested virus following this step is evaluated by both serological tests (HI and NI) and RT-PCR/RFLP analyses.

Steps 7 and 8: Cloning by limiting dilution

The reassortants with the highest HA titer (following step 6) and a gene constellation closest to 6:2 (6 genes from PR8 and the two surface antigens, HA and NA, from wt virus) are subjected to two sequential steps of cloning by limited dilution with cloning at dilutions ranging from 10-5 to 10-9. Two to three eggs are used for each dilution level.

Step 9: Final amplification

The final hy reassortants are amplified @10-5 dilution in a minimum of 20 eggs per reassortant. Based on HA titers the harvested allantoic fluids are pooled and the final serology and gene analyses are performed. Sterility testing is performed by streaking the sample on blood agar plates and incubating for 48 h at 35°C.

2.3. Notes (a) The PR8 and X-157 antisera is developed against sucrose gradient-purified virus in rabbits (Pocono Rabbit Farm & Laboratory, Canadensis, PA). Antisera against PR8 and X-157 surface glycoproteins, is prepared by immunization of rabbits with HA and NA solubilized from sucrose gradient-purified virus with 7.5% N-octylglucopyranoside, centrifuged to remove viral cores followed by dialysis of the supernatants (12). Antibodies are purified on Protein G columns (Thermo Fisher Scientific, Rockford, IL). All antisera are pretreated with 100 Units/mL of receptor-destroying enzyme (RDE; Lonza, Walkersville, MD) to remove nonspecific inhibitors of hemag-glutination (13).

(b) Hemagglutination (HA) Assay for viral titer determination is carried out in "V"-bottom 96-well microtiter plates using chicken erythrocytes (cRBC) standardized to 0.5% in PBS (pH 7.2).

( c) Hemagglutination inhibition (HI) Assay is performed to insure that the HA antigen was obtained from the wt strain. The assay is performed in "U"-bottom microtiter plates as per standard protocol (14).

(d) Neuraminidase inhibition (NI) Assay is performed to insure that the NA is obtained from the wt strain. The assay is performed according to the procedure described in the WHO Manual (15) with modifications (16).

(e) RT-PCR is performed using the Takara One Step RNA PCR Kit (Takara Bio Inc., Otsu, Shiga, Japan) as per manufacturer's recommendations. Briefly 2 |g of vRNA is added to the following mixture containing 10x One Step RNA PCR Buffer, 5 mM MgCl(, 1 mM dNTP, 0.8 U RNase Inhibitor, 0.1 U AMV RTase XXL, 0.1 U AMV-Optimized Taq, 0.4 |M each of forward and reverse primers (Integrated DNA Technologies Inc., Coralville, IA); the primer sequences are available on request (manuscript in preparation), and RNase-free H2O up to a total volume of 50 |l. RT-PCR parameters used are as follows: 55°C for 30 min, 94°C for 2 min followed by 35 cycles of 94°C for 30 s, 55°C for 1 min (HA, NP, NA, M, and NS gene segments) or 60°C for 1 min (PB2, PB1, and PA gene segments), 68°C for 2 min and a final extension at 72°C for 10 min. The reactions are performed on an Eppendorf Mastercycler®. The amplified RT-PCR products are visualized on a 2% agarose-TAE/EtBr gel.

(f) PCR products are gel purified in a 2% low-melt agarose gel using QIAquick® Gel Extraction Kit (Qiagen Inc., Valencia, CA) as per the manufacturer's recommendations.

(g) RFLP is carried out in a 10 ^l volume reaction mixture which contains purified DNA and restriction enzymes with their respective buffer. The reactions are carried out as per the manufacturer's recommendations. Both the hy donor and the wt viruses are digested along side with the reassortants as positive controls (17).

3. The Manufacture of Inactivated Influenza Vaccine

3.1. Introduction

3.2. Vaccine Production Process

Egg-based influenza vaccine has been produced for about 60 years. Initial vaccines were relatively crude and reactogenic due to the high content of egg protein remaining in the finished vaccine. The basic method of virus propagation in eggs has changed little over the 60 years apart from the use of mechanized egg-handling systems as the number of eggs processed has increased. Quality control of the eggs, egg supply, and virus seed preparation has also significantly improved over the years. High-speed ultracentrifuga-tion, sucrose density gradient virus purification methods developed and introduced in the late 1960s and early 1970s removed egg protein from the product and significantly improved the vaccine. Subsequent downstream processing steps to produce split and surface antigen (subunit) vaccines introduced in the 1970s and early 1980s further improved vaccine purity and again significantly reduced reactogenicity. These process improvements and the introduction over the years of good manufacturing practice (GMP), Quality systems, and regulatory control have resulted in the high-quality inactivated influenza vaccines currently available. In recent years mammalian cell-based inactivated vaccines have started to be licensed for manufacture and use in Europe.

The processing steps are similar in most inactivated influenza vaccines currently on in production,

Vaccine seed preparation and virus propagation, Allantoic fluid harvesting, clarification and virus inactivation, Sucrose density gradient virus purification, A virus-splitting process,

Dia-filtration and sterilizing filtration to concentrated monovalent pool stage,

Formulation of monovalent pools into trivalent vaccine, Fill-finish activities.

Manufacturers may perform these processing steps in a different sequence in the downstream purification and inactivation stages, to follow the specific evolution of different vaccine brands. Vaccines are however standardized by the content of the influenza surface protein hemagglutinin (HA) for each vaccine strain. The other surface protein, neuraminidase (NA) must be present in the vaccine but is not standardized.

( a) Vaccine seed preparation and virus propagation

Master and working seed is produced, from WHO recommended reassortant or "wild-type" viruses by serial passage at high dilution in specific pathogen-free (SPF) eggs. Following quality assurance (QA) release, suitably diluted working seed is inoculated into the allantoic cavity of commercial pre-incubated 11-day-old embryonated eggs which are then incubated for a further 72°h, normally at 35°C for Influenza A strains and 33°C for Influenza B strains to enable the virus to replicate.

(b) Allantoic fluid harvesting, clarification, and virus inactivation The eggs are chilled to +4°C which kills the embryo and sets the major blood vessels to aid the harvest process as influenza virus will haemadsorb to red blood cells. The allantoic fluid is collected in a harvesting vessel, usually by a machine that removes the top of the egg and aspirates the fluid via a harvesting probe that enters the egg. Once harvested the fluid passes through a clarification centrifuge and is sometimes concentrated by dia-filtration. The virus is inactivated by the addition of Formaldehyde or beta-Propiolactone for a time and temperature validated to kill the influenza virus. Validation studies are also required to evaluate the inactivation process on avian leucosis virus and a range of Mycoplasma bacteria.

( c) Sucrose density gradient virus purification

The virus is removed from the allantoic fluid by continuous flow into the rotor of an operational ultracentrifuge containing a sucrose density gradient. Figure 1 shows the isopycnic influenza virus band with polyacrylamide gel electrophoresis (PAGE) clearly demonstrating the separation of the egg proteins from the influenza virus proteins. The virus protein fraction is collected from the stationary centrifuge rotor, once all of the allantoic fluid has passed through the ultracentrifuge.

(d) Virus-splitting process

Webster and Laver (18) demonstrated that pyrogenicity was reduced by disruption of the influenza particle by sodium deoxycholate. Similar results were obtained by disruption with diethyl ether (19) and Tween-ether mixture (20). This technology has been the basis for current split vaccines.

Brady and Furminger (21) demonstrated that Triton N101, a nonionic surfactant, could be used to strip the HA

Sucrose Gradient Virus
Fig. 1. A/H3N2 Zonal centrifuge sucrose density gradient profile.

and NA from the virus particle and these virus surface proteins could be separately purified by an additional ultracentrifuga-tion step. Subsequently the ionic surfactant, cetyltrimethylam-monium bromide (CTAB) was used to solubilize the influenza virus lipid layer and release the surface proteins. Sandoz patent (Ref.: US Patent 4,064,232 Dec. 201977). This technology is used to produce current surface antigen, but often called subunit vaccines.

(e) Dia-filtration and sterilizing filtration to concentrated monovalent pool stage

Once the virus has been split, residual splitting agent has to be removed by additional "downstream processing." There is usually also a dia-filtration stage to remove sucrose and a change to the final buffer either before or after the splitting stage. The sterilizing filtration stage is completed as close as technically possible to the final monovalent pool stage for sterility assurance, as these concentrates have to be held until potency values are known, which is dependent on single radial immunodiffusion (SRD) potency reagents (see Chapter 9.4).

(f) Formulation of monovalent pools into trivalent vaccine Once SRD reagents are available and the potency of individual monovalent pools has been determined, it is possible to formulate the bulk trivalent vaccine. Currently, each dose of vaccine has to contain a minimum of 15 ^g of HA for each strain and it must be stable for 1 year.

(g) Fill-finish activities

Product can either be filled in multi-dose vials or prefilled syringes depending on market requirements. Prefilled single use syringes are preservative free. Multi-dose vials however must contain a preservative which is normally thiomersal.

(h) Mammalian cell-based inactivated vaccines

New influenza cell-culture vaccines currently on the market or in late stage development replace embryonated eggs for virus propagation with a "qualified" mammalian cell line. Both Vero (monkey kidney cells) and canine kidney cells (Madin-Darby canine kidney) are in use. The downstream processing methods may differ slightly but in principle the production stages are unaltered. The biggest hurdle initially has been to convince regulators that the cell system is as safe as eggs and companies have had to make significant investments to "qualify" their specific cell lines.

(i) Pandemic vaccines

The majority of pandemic vaccines are produced in the same facilities used for seasonal vaccine. Pandemic vaccines require different antigen dosage and immunization schedule. Different adjuvants of varying safety profiles are used in some vaccines to help reduce antigen per dose, increase cross-reactivity, breath of immune response, boostability and antibody persistence (22).

4. Reagents Preparation for Potency Testing of Influenza Vaccines

John M. Wood and Una Dunleavy

4.1. Introduction

Current inactivated influenza vaccines are approved with a minimum content of 15 mg hemagglutinin (HA) per dose/strain. As antigen content in the final monobulk material may vary greatly depending upon individual strain growth characteristics and several factors particularly in the downstream process, a reproducible test for vaccine potency standardization of the HA content in the vaccine is needed.

The single radial immunodiffusion (SRD) assay was developed for influenza vaccine potency assessment in the late 1970s (23) and became established in 1979 after vaccine clinical trials in the USA and the UK first demonstrated that HA antigen content of inactivated influenza vaccines as measured by SRD correlates well with vaccine immunogenicity (24). The assay depends on the availability of an antiserum reagent (usually sheep) and a calibrated antigen standard for each vaccine component, and these reagents are supplied worldwide by four essential regulatory laboratories (ERLs) (http://www.who.int/influenza/gisrs_labora-tory/collaborating_centres/en/; accessed 28 Jan 2012).

The influenza HA reacts with antibody to HA in an agarose gel to produce a precipitin ring and the size of the ring depends on the amount of HA. The HA content of an influenza vaccine is calculated by comparison of the precipitin ring formed by the vaccine with that formed by the antigen standard.

4.2. Materials (a) SRD antigen and antiserum reagents are supplied by ERLs.

They should be used according to the supplied instructions for use.

(b) Low melting temperature Seakem ME agarose (Lonza Biologicals, Slough, UK) is dissolved in PBS containing 10% (w/v) sodium azide at a concentration of 0.1% (w/v). The agarose is dissolved by boiling for approximately 20 min and stored at room temperature in single use aliquots (see Subheading 4.3.b). A microwave can also be used to dissolve the agarose.

(d) Perspex mold to cover glass plates with either circular (90 mm diameter) or square (103 mm diameter) internal dimensions.

(e) Stainless steel punch with internal diameter of 4 mm.

(f) Template for punching holes in agarose gel, either 4 x 4 or 6 x 6 design (see 4.4.a).

(g) Whatman 1 filter paper approximately 15 cm diameter.

(h) Absorbent paper towels.

(i) 10% (w/v) Zwittergent 3-14 (Calbiochem, La Jolla, USA).

(j) Coomassie brilliant blue (BDH, Poole, UK) dissolved in destain at 0.3% (v/v).

(k) Destain solution: methanol, distilled water, and acetic acid (ratio 5:5:1).

(l) Racks to hold glass plates in stain and destain.

4.3. Methods (a) Duplicate SRD gels will be needed for each vaccine strain being tested.

(b) Melt two aliquots of agarose for one duplicate test using a boiling water bath or a microwave. These instructions assume that a circular gel is prepared with a capacity of 16 holes (also termed wells), i.e., four antigens tested at four dilutions (see 4.4.b).

( c) When molten, pipette 13 ml aliquots of agarose into each of the two bottles pre-warmed to 56°C and allow agarose to adjust to 56°C (approximately 15 min) (see 4.4.c).

(d) Prepare two glass plates by wiping with molten agarose and allowing plates to dry. Place a mold on each plate and seal the inside edges of molds with molten agarose.

(e) Place the glass plates on a level table, add the required volume of antiserum to the 13 ml agarose, mix gently, and add agarose/antiserum mixture to each mold avoiding bubbles. Allow to set for about 30 min and then remove the mold and cover with a petri dish lid. The gels can be used immediately or stored at 2-8°C for up to 1 week before use.

Table 3

Dilution schedule for SRD assay

Volume added at each dilution (ml)

Table 3

Dilution schedule for SRD assay

1:1

3:4

1:2

1:4

Antigen

500

150

100

50

PBS

0

50

100

150

(f) Using the 4 x 4 template, punch wells in the gels using the stainless steel punch connected to a suction device. Allow wells to dry for about 15 min and gels are ready to use.

(g) Prepare an antigen standard reagent for each strain being tested according to supplied instructions (see 4.4.d).

(h) In tubes, add 450 ml of either antigen standard or vaccine followed by 50 ml of a 10% solution (w/v) of Zwittergent detergent. Carefully mix by vortexing and leave for 30 min at room temperature. Repeat for duplicates.

(i) Prepare a dilution series for each duplicate detergent-treated antigen using PBS according to the schedule in Table 3.

(j) Add 20 ml of each antigen dilution into the allocated well in an SRD gel according to a randomization scheme. One of the duplicate dilution series will be added to one of the duplicate gels and the second set of duplicate dilutions will go into the second set of gels. Where a vaccine is being tested, antigen dilutions will be added to SRD gels containing antiserum to each of the vaccine strains.

(k) Allow wells to empty, cover gels with a petri dish lid, and incubate for 18 h in a humid environment at 20-25°C (see 4.4.e).

(l) Remove petri dish lid, wet gel with water, and cover with a filter paper. Cover gel and filter paper with absorbent paper towels and a 600 g weight for about 30 min. Dry the gels in warm moving air until the filter paper can be easily removed.

(m) Stain and destain the gels until SRD rings can be easily distinguished from the background. Dry the stained gel.

(n) Measure the diameter (d) of each SRD ring (see 4.4.f ).

(o) By converting each d into d2 and comparing d2 and antigen dilutions for the antigen standard and each vaccine, construct dose-response slopes using a suitable statistical program so that slopes of antigen standards can be compared with those of vaccines. The ratio of the slopes is used to calculate the vaccine HA concentration (see 4.4.g).

4.4. Notes (a) Holes in gel should be spaced at approximately 8 mm from neighboring holes so that SRD rings do not overlap. It is important to avoid using the edge of a gel due to variability in gel thickness in this region.

(b) If other gel sizes and well formats are used, it is important to adjust agarose volume so that wells will hold 20 ^l of antigen.

( c) If molten agarose is not allowed to adjust to 56°C, there may be denaturation of sheep antibodies when this is added.

(d) Antigen standards are usually freeze-dried and will need reconstitution in distilled water. The SRD assay conditions are usually designed to test vaccines at HA concentrations of 30 ^g HA/ml so the antigen standard should be diluted to this concentration.

(e) The 18 h incubation time is sufficient for SRD rings of diameter 7.5-8.0 mm to be formed by vaccines containing 30 ^g HA/ml provided antiserum instructions are followed. For assays of antigens with higher antigen concentrations, either a longer incubation is needed or more antiserum should be incorporated into the agarose.

(f) The measuring devices can range from a simple calibrated micrometer scale to a semi-automated image analyser.

(g) Slope ratio or parallel line analysis can be used for statistical analysis to evaluate assay validity and to calculate vaccine potency. Suitable "Combistats" programs can be obtained from EDQM in the EU (http://combistats.edqm.eu/). In the slope ratio analysis, vaccine potency = slope of vaccine -f slope of antigen standard.

(h) The most common problems occurring in SRD assays are

• SRD rings not reaching theoretical size at equilibrium. This results in the SRD assay underestimating vaccine potency. SRD rings can either be made smaller by increasing antigen dilution, increasing antiserum concentration or allowing longer than 18 h incubation.

• Chemicals added to a vaccine (e.g., formaldehyde, some adjuvants) can interfere with antigen diffusion. It may be necessary to assay before the chemical is added.

References

1. Francis T, Salk JE, Bruce WM. The protective effect of vaccination against epidemic influenza B. J Am Med Assoc. 1946;131:275-8.

2. Francis T, Jr., Salk JE, Quilligan JJ, Jr. Experience with vaccination against influenza in the spring of 1947; a preliminary report. Am J Public Health. 1947 Aug;37(8): 1013-6.

3. Sartwell PE, Long AP. The Army experience with influenza, 1946-1947; epidemiological aspects. Am J Hyg. 1948 Mar;47(2):135-41.

4. Shen J, Ma J, Wang Q. Evolutionary trends of A(H1N1) influenza virus hemagglutinin since 1918. PLoS One. 2009;4(11):e7789.

5. Stohr K. Overview of the WHO Global Influenza Programme. Laboratory Correlates of Immunity of Influenza A Reassortment. Dev Biol. Basel: Karger; 2003;115: p. 3-8.

6. Smith DJ, Lapedes AS, de Jong JC, Bestebroer TM, Rimmelzwaan GF, Osterhaus AD, et al. Mapping the antigenic and genetic evolution of influenza virus. Science. 2004 Jul 16; 305(5682):371-6.

7. Francis T. A clinical evaluation of vaccination against influenza. J Am Med Assoc. 1944;142: 982-5.

8. Francis T. Development of the 1943 vaccine study of the commission on Influenza. Am J Hyg. 1945;42:1-11.

9. Kilbourne ED. Future influenza vaccines and the use of genetic recombinants. Bull World Health Organ. 1969;41(3):643-5.

10. Kilbourne ED, Schulman JL, Schild GC, Schloer G, Swanson J, Bucher D. Related studies of a recombinant influenza-virus vaccine. I. Derivation and characterization of virus and vaccine. J Infect Dis. 1971 Nov;124(5):449-62.

11. Neumann G, Noda T, Kawaoka Y. Emergence and pandemic potential of swine-origin H1N1 influenza virus. Nature. 2009 Jun 18;459(7249):931-9.

12. Gallagher M, Bucher DJ, Dourmashkin R, Davis JF, Rosenn G, Kilbourne ED. Isolation of immunogenic neuraminidases of human influenza viruses by a combination of genetic and biochemical procedures. J Clin Microbiol. 1984 Jul;20(1):89-93.

13. Palmer DF, Coleman MT, Dowdle WR, Schild GC. Advanced Laboratory Techniques for Influenza Diagnosis. Immunology Series, 6. In: U.S. Department of Health E, and Welfare, Service USPH, editors. Washington, DC1975.

14. WHO, Global, Influenza, Programme. WHO Manual on Animal Influenza Diagnosis and Surveillance. Hemagglutination and Hemagglutination Inhibition of Influenza Viruses. In: WHO/CDS/CSR/NCS/2002.5, editor.2002. p. 47-.

15. WHO, Global, Influenza, Programme. WHO Manual on Animal Influenza Diagnosis and Surveillance. Neuraminidase and Neuraminidase Inhibition Assay. In: WHO/CDS/CSR/ NCS/2002.5, editor.2002. p. 28-36.

16. Kilbourne ED, Johansson BE, Grajower B. Independent and disparate evolution in nature of influenza A virus hemagglutinin and neuraminidase glycoproteins. Proc Natl Acad Sci USA. 1990 Jan;87(2):786-90.

17. Brett I, Werber J, Kilbourne ED. Rapid confirmation by RFLP of transfer to vaccine candidate reassortment viruses of the principal 'high yield' gene of influenza A viruses. J Virol Methods. 2002 Feb;100(1-2):133-40.

18. Webster RG, Laver WG. Influenza virus subunit vaccines: immunogenicity and lack of toxicity for rabbits of ether- and detergent-disrupted virus. J Immunol. 1966 Apr;96(4): 596-605.

19. Cromwell HA, Brandon FB, McLean IW, Jr., Sadusk JF, Jr. Influenza immunization. A new vaccine. JAMA. 1969 Nov 24;210(8): 1438-42.

20. Davenport FM, Hennessy AV, Brandon FM, Webster RG, Barrett CD, Jr., Lease GO. Comparisons ofSerologic and Febrile Responses in Humans to Vaccination with Influenza a Viruses or Their Hemagglutinins. J Lab Clin Med. 1964 Jan;63:5-13.

21. Brady MI, Furminger IG. A surface antigen influenza vaccine. 1. Purification of haemag-glutinin and neuraminidase proteins. J Hyg (Lond). 1976 Oct;77(2):161-72.

22. Nicholson KG, Colegate AE, Podda A, Stephenson I, Wood J, Ypma E, et al. Safety and antigenicity of non-adjuvanted and MF59-adjuvanted influenza A/Duck/Singapore/97 (H5N3) vaccine: a randomised trial of two potential vaccines against H5N1 influenza. Lancet. 2001 Jun 16;357(9272):1937-43.

23. Schild GC, Wood JM, Newman RW. A single-radial-immunodiffusion technique for the assay of influenza haemagglutinin antigen. Proposals for an assay method for the haemagglutinin content of influenza vaccines. Bull World Health Organ. 1975;52(2):223-31.

24. Ennis FA, Mayner RE, Barry DW, Manischewitz JE, Dunlap RC, Verbonitz MW, et al. Correlation of laboratory studies with clinical responses to A/New Jersey influenza vaccines. J Infect Dis. 1977 Dec;136 Suppl:S397-406.

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