US20060159705A1

US20060159705A1 - Purification of HBV antigens for use in vaccines

Info

Publication number: US20060159705A1
Application number: US11/266,565
Authority: US
Inventors: Koen De-Heyder; Peter Schu; Michelle Serantoni; Omer Van-Opstal
Original assignee: SmithKline Beecham Corp
Current assignee: GlaxoSmithKline Biologicals SA
Priority date: 2000-08-10
Filing date: 2005-11-03
Publication date: 2006-07-20
Also published as: BRPI0113155B1; DE60116107T2; PL204736B1; BRPI0113155C1; AR030325A1; PL362322A1; OA12361A; BRPI0113155B8; UY26882A1; DZ3470A1; SK288079B6; US8624004B2; BR0113155A; ES2254464T3; AP2003002734A0; US20030235590A1; EP1307473A1; EP1307473B1; SK288069B6; CN1468256B

Abstract

The present invention relates to a method for the production of a hepatitis B antigen suitable for use in a vaccine, the method comprising purification of the antigen in the presence of cysteine, to vaccines comprising such antigens.

Description

This is a continuation-in-part of prior application Ser. No. 10/344,211 filed 18 Jul. 2003.

FIELD OF THE INVENTION

This invention relates to a novel process of manufacture of a hepatitis B vaccine for use in the treatment or prophylaxis of hepatitis B virus (HBV) infections. It further relates to a HBV vaccine obtainable by the novel process of the invention.

BACKGROUND OF THE INVENTION

Chronic hepatitis B virus (HBV) infection, for which there is currently limited treatment, constitutes a global public health problem of enormous dimensions. Chronic carriers of HBV, estimated to number more than 300 million world-wide, are at risk for development of chronic active hepatitis, cirrhosis and primary hepatocellular carcinoma.
Many vaccines which are currently available require a preservative to prevent deterioration. A frequently used preservative is thiomersal which is a mercury-containing compound. Some concerns have been raised about the use of mercury in vaccines, although commentators have stressed that the potential hazards of thiomersal-containing vaccines should not be overstated (Offit; P. A. JAMA Vol. 283; No: 16). Nevertheless it would be advantageous to find new and potentially safer methods of preparation of vaccines to replace the use of thiomersal in the manufacturing process. There is thus a need to develop vaccines which are thiomersal-free, in particular hepatitis B vaccines.

SUMMARY OF THE INVENTION

In one embodiment the present invention provides a method for producing a hepatitis B antigen suitable for use in a vaccine, the method comprising purification of the antigen in the presence of a reducing agent comprising a free —SH group.
In another embodiment the present invention provides a method of producing a stable hepatitis B antigen without trace of thiomersal which comprises purification of the antigen in the presence of a reducing agent having a free —SH group.
In a further embodiment the present invention provides for an antigen preparation that is generally without trace of thiomersal when thiomersal is not detectable in the purified antigen product using absorption spectrophotometery of mercury, as described herein.
In another embodiment the present invention provides for a hepatitis antigen preparation comprising less than about 0.025 μg mercury per 20 μg protein, suitably as measured by absorption spectrophotometery.
In another embodiment the present invention provides for the purification being carried out in the absence of thiomersal, and the purified antigen is completely free of thiomersal.
In another embodiment the antigen is stable, suitably substantially as stable as a hepatitis antigen purified in the presence of thiomersal, as outlined in Example 1 herein for example.
In another embodiment the hepatitis antigen is immunogenic.
In another embodiment the reducing agent is added during the antigen purification process, preferably after growth of cells expressing the antigen.
In another embodiment the reducing agent is chosen from cysteine, dithiothreitol, β-mercaptoethanol and glutathione.
In another embodiment the present invention provides for a method of producing a stable immunogenic hepatitis B antigen without trace of thiomersal which comprises purification of the antigen in the presence of cysteine.
In another embodiment the purification is carried out in the presence of a cysteine solution.
In another embodiment the cysteine, in solution or powder form, is added during the process to a final concentration of between about 1 and 10 mM, in another embodiment 1 to 5 mM. In another embodiment the cysteine is added to a final concentration of about 2 mM.
In one embodiment the cysteine is L-cysteine.
In another embodiment the invention further provides a method of producing a stable hepatitis B antigen without trace of thiomersal wherein the crude antigen is subjected to gel permeation chromatography, subjected to ion-exchange chromatography and mixed with a reducing agent having a free —SH group.
In another embodiment the ion-exchange chromatography is anion-exchange chromatography.
In another embodiment a hepatitis B antigen free of thiomersal obtainable by the method of manufacture of the present invention wherein the antigen is at least as immunogenic and antigenic as the hepatitis B antigen manufactured in the presence of thiomersal.
The invention further provides an immunogenic hepatitis B antigen having a mean ELISA protein ratio greater than 1.5 and an RF1 content with at least a 3-fold lower IC50 value than that of the hepatitis B surface antigen manufactured in the presence of thiomersal.
In another embodiment the invention relates to a method for the production of a hepatitis antigen suitable for use in a vaccine, the method comprising purification of the antigen in the presence of thiomersal and subsequent treatment of antigen in the presence of a reducing agent comprising a free —SH group.
In another embodiment the treatment is followed by a purification step such as a dialysis step to remove thiomersal.
In another embodiment the reducing agent is cysteine, DTT, glutathione or 2-mercaptoethanol.
In another embodiment the hepatitis B antigen of the invention may be used for either the treatment or prophylaxis of hepatitis B infections, especially treatment or prophylaxis, for example, of chronic hepatitis B infections.
The present invention further provides a vaccine formulation comprising a hepatitis B antigen of the present invention in conjunction with an adjuvant. Preferably the adjuvant is an aluminium salt or a preferential stimulator of TH1 cell response.
In another embodiment the antigen is a hepatitis B surface antigen.
The preparation of hepatitis B surface antigen is well documented. See for example, Harford et. al. in Develop. Biol. Standard 54, page 125 (1983), Gregg et. al. in Biotechnology, 5, page 479 (1987), EP-A-0 226 846, EP-A-0 299 108 and references therein.
As used herein the expression ‘hepatitis B surface antigen’ or ‘HBsAg’ includes any HBsAg antigen or fragment thereof displaying the antigenicity of HBV surface antigen. It will be understood that in addition to the 226 amino acid sequence of the HBsAg S antigen (see Tiollais et. al. Nature, 317, 489 (1985) and references therein) HBsAg as herein described may, if desired, contain all or part of a pre-S sequence as described in the above references and in EP-A-0 278 940. HBsAg as herein described can also refer to variants, for example the ‘escape mutant’ described in WO 91/14703.
HBsAg may also refer to polypeptides described in EP 0 198 474 or EP 0 304 578.
Normally the HBsAg will be in particle form. In another embodiment the HbsAg will consist essentially of the HbsAg S-antigen mentioned hereinabove.
The vaccine may advantageously include a pharmaceutically acceptable excipient such as a suitable adjuvant. Suitable adjuvants are commercially available such as, for example, Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.); AS-2 (SmithKline Beecham, Philadelphia, Pa.); aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine; acylated sugars; cationically or anionically derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A and quil A. Cytokines, such as GM-CSF or interleukin-2, -7, or -12, may also be used as adjuvants.
In the formulations of the invention one embodiment is that the adjuvant composition induces an immune response predominantly of the TH1 type. High levels of Th1-type cytokines (e.g., IFN-γ, TNFα, IL-2 and IL-12) tend to favour the induction of cell mediated immune responses to an administered antigen. Within a preferred embodiment, in which a response is predominantly Th1-type, the level of Th1-type cytokines will increase to a greater extent than the level of Th2-type cytokines. The levels of these cytokines may be readily assessed using standard assays. For a review of the families of cytokines, see Mosmann and Coffman, Ann. Rev. Immunol. 7:145-173, 1989.
Accordingly, suitable adjuvants for use in eliciting a predominantly Th1-type response include, for example a combination of monophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl lipid A (3D-MPL) together with an aluminium salt. Other known adjuvants which preferentially induce a TH1 type immune response include CpG containing oligonucleotides. The oligonucleotides are characterised in that the CpG dinucleotide is unmethylated. Such oligonucleotides are well known and are described in, for example WO 96/02555. Immunostimulatory DNA sequences are also described, for example, by Sato et al., Science 273:352, 1996. Another preferred adjuvant is a saponin, preferably QS21 (Aquila Biopharmaceuticals Inc., Framingham, Mass.), which may be used alone or in combination with other adjuvants. For example, an enhanced system involves the combination of a monophosphoryl lipid A and saponin derivative, such as the combination of QS21 and 3D-MPL as described in WO 94/00153, or a less reactogenic composition where the QS21 is quenched with cholesterol, as described in WO 96/33739. Other preferred formulations comprise an oil-in-water emulsion and tocopherol. A particularly potent adjuvant formulation involving QS21, 3D-MPL and tocopherol in an oil-in-water emulsion is described in WO 95/17210.
A particularly potent adjuvant formulation involving QS21, 3D-MPL & tocopherol in an oil in water emulsion is described in WO 95/17210 and is a preferred formulation.
Accordingly in one embodiment of the present invention there is provided a vaccine comprising a hepatitis B surface antigen of the present invention, which additionally comprises a TH-1 inducing adjuvant. A preferred embodiment is a vaccine in which the TH-1 inducing adjuvant is selected from the group of adjuvants comprising: 3D-MPL, QS21, a mixture of QS21 and cholesterol, and a CpG oligonucleotide. Another preferred embodiment is a vaccine comprising a hepatitis B surface antigen adjuvanted with a monophosphoryl lipid A or derivative thereof, QS21 and tocopherol in an oil in water emulsion.
In another embodiment the vaccine additionally comprises a saponin, more preferably QS21. Another particular suitable adjuvant formulation including CpG and a saponin is described in WO 00/09159 and is a preferred formulation. In another embodiment the saponin in that particular formulation is QS21. In a further embodiment the formulation additionally comprises an oil in water emulsion and tocopherol.
The present invention further provides a vaccine formulation comprising a hepatitis B surface antigen of the present invention in conjunction with an adjuvant and additionally comprising one or more antigens selected from the group consisting of: diptheria toxoid (D), tetanus toxoid (T) acellular pertussis antigens (Pa), inactivated polio virus (IPV), haemophilus influenzae antigen (Hib), hepatitis A antigen, herpes simplex virus (HSV), chlamydia, GSB, HPV, streptococcus pneumoniae and neisseria antigens. Antigens conferring protection for other diseases may also be combined in the vaccine formulation of the present invention.
In one embodiment, the vaccine formulation comprises a hepatitis B surface antigen obtainable by the method of manufacture of the present invention in conjunction with an adjuvant and an inactivated polio virus.
The present invention also provides a method of treatment and/or prophylaxis of hepatitis B virus infections, which comprises administering to a human or animal subject, suffering from or susceptible to hepatitis B virus infection, a safe and effective amount of a vaccine of the present invention for the prophylaxis and/or treatment of hepatitis B infection.
The invention further provides the use of a hepatitis B surface antigen of the present invention in the manufacture of a medicament for the treatment of patients suffering from a hepatitis B virus infection, such as chronic hepatitis B virus infection.
The vaccine of the present invention will contain an immunoprotective quantity of the antigen and may be prepared by conventional techniques.
Vaccine preparation is generally described in Pharmaceutical Biotechnology, Vol. 61 Vaccine Design—the subunit and adjuvant approach, edited by Powell and Newman, Plenum Press, 1995. New Trends and Developments in Vaccines, edited by Voller et al., University Park Press, Baltimore, Md., U.S.A. 1978. Encapsulation within liposomes is described, for example, by Fullerton, U.S. Pat. No. 4,235,877. Conjugation of proteins to macromolecules is disclosed, for example, by Likhite, U.S. Pat. No. 4,372,945 and by Armor et al., U.S. Pat. No. 4,474,757. Use of Quil A is disclosed by Dalsgaard et al., Acta Vet Scand, 18:349 (1977). 3D-MPL is available from Ribi immunochem, USA, and is disclosed in British Patent Application No. 2220211 and U.S. Pat. No. 4,912,094. QS21 is disclosed in U.S. Pat. No. 5,057,540.
It is to be understood that both the foregoing summary description and the following detailed description are exemplary and explanatory, and are intended to provide further explanation of the invention as claimed.
The accompanying drawings are included to provide a further understanding of the invention, and are incorporated in, and constitute a part of this specification, illustrate several embodiments of the invention, and together with the description serve to explain the principles of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the thiomersal free production process for Engerix B™;
FIG. 2 illustrates SDS-PAGE analysis of bulk antigen lots; and
FIG. 3 illustrates residual yeast proteins in bilk antigen lots produced by the thiomersal free process.

DETAILED DESCRIPTION OF THE INVENTION

EXAMPLE 1

Production Process of Hepatitis B Surface Antigen in the Presence of Thiomersal
The Hepatitis B surface antigen (HBsAg) of SB Biologicals hepatitis B monovalent vaccine (Engerix B™) is expressed as a recombinant protein in Saccharomyces cerevisiae (see Harford et. al. loc. cit.). The 24 kD protein is produced intracellularly and accumulated in the recombinant yeast cells. At the end of the fermentation the yeast cells are harvested and disrupted in the presence of a mild surfactant such as Tween 20 to liberate the desired protein. Subsequently the cell homogenate, containing the soluble surface antigen particles, is prepurified in a series of precipitations and then concentrated via ultrafiltration.
Further purification of the recombinant antigen is performed in subsequent chromatographic separations. In a first step the crude antigen concentrate is subjected to gel permeation chromatography on Sepharose 4B medium. Thiomersal is present in the elution buffer at the 4B gel permeation chromatography step. The elution buffer has the following composition: 10 mM Tris, 5% ethylene glycol, pH 7.0, 50 mg/L thiomersal. Thiomersal is included in this buffer to control bioburden. Most of this thiomersal is removed during the subsequent purification steps including ion exchange chromatography, ultracentrifugation and desalting (gel permeation) so that purified bulk antigen preparations prepared by the original process contain about 1.2 μg and less than 2 μg of thiomersal per 20 μg of protein.
An Ion-Exchange chromatography step is performed using a DEAE-matrix and this pool is then subjected to a Cesium-gradient ultracentrifugation on 4 pre-established layers of different Cesium chloride concentrations. The antigen particles are separated from contaminating cell constituents according to their density in the gradient and eluted at the end of the centrifugation process. Cesium chloride is then removed from this pool by a second gel permeation on Sepharose gel.
When HBsAg is prepared by the process containing thiomersal in the 4B gel permeation buffer, protein concentrations of over 30 mg/ml are recovered in the pooled HBsAg containing fractions from the CsCl gradient, corresponding to an equivalent concentration of HBsAg as assayed by the AUSZYME kit from Abbott Laboratories.
The CsCl ultracentrifugation step usefully eliminates residual lipids, DNA and minor protein contaminants from the HBsAg preparation. It is performed by zonal centrifugation in a Ti 15 rotor from Beckman Instruments, Fullerton, Calif. at a speed of 30,000 rpm for about 40 to 60 hours. The sample to be purified is applied to layers of CsCl solution with final concentrations of 0.75, 1.5, 2.5 and 3.25 M CsCl. At the end of centrifugation the gradient is eluted into fractions. Fractions containing HBsAg may be identified by UV absorbance at 280 nm or by testing dilutions of the fractions with the AUSZYME kit. The HBsAg band is at a density of 1.17 to 1.23 g/cm³.
The solution containing the purified HBsAg is sterile filtered before being used to make a vaccine formulation.
Purification from the yeast cell lysate is complex as the antigen is produced intracellularly and a series of separation techniques designed to eliminate different types of (yeast) contaminants are necessary to obtain pure bulk antigen. The steps of purification are important, as the product to be purified is a lipoprotein particle containing multiple copies of the surface antigen polypeptide and this structure must be maintained throughout the purification process. It is a particularity of this process that it yields surface antigen particles which are fully immunogenic without the need for further chemical treatment to enhance immunogenicity (compare EPO 35435).
The details of the production process are further described in European Patent 0199698.

EXAMPLE 2

Production and Characterization of Yeast-Derived HBsAg by a Thiomersal Free Process.
1. Production and Purification of Yeast-Derived HBsAg
1.1 Outline of the Production Process
Hepatitis B surface antigen may be produced by fermentation of an appropriate strain of Saccharomyces cerevisiae, for example that described in Harford et. al. (loc. cit.).
At the end of large-scale fermentation of the recombinant yeast strain, the cells are harvested and broken open in the presence of a mild surfactant such as Tween 20. The surface antigen is then isolated by a multistep extraction and purification procedure exactly as described above in Example 1 up to the step of the first gel permeation on Sepharose 4B.
1.2 Thiomersal-Free Purification Process
In the thiomersal free process the following two changes have been introduced compared to the process described in Example 1.
1. The elution buffer at the 4B gel permeation chromatography step no longer contains thiomersal.
2. Cysteine (2 mM final concentration) is added to the eluate pool from the anion exhange chromatography step.
It was found that omission of thiomersal from the 4B gel permeation buffer may result in precipitation of the HBsAg particles during the CsCl density gradient centrifugation step with loss of product and aggregation or clumping of the recovered antigen.
Addition of cysteine at 2 mM final concentration to the eluate pool from the preceding anion exchange chromatography step prevents precipitation and loss of antigen during CsCl density centrifugation.

2. Cysteine is a preferred substance for this treatment as it is a naturally occurring amino acid and can be removed at the subsequent desalting step on a gel permeation column using Sepharose 4BCLFF as the column matrix.

There are no other changes in the manufacturing process compared to the process described in Example 1.
The thiomersal free process yields bulk antigen of a purity and with properties comparable to antigen from the process of Example 1.
1.2a
The thiomersal added to the 4B buffer at 50 μg/ml is thought to decompose and the resulting ethyl mercury may attach covalently to free sulphydryl groups on cysteine residues of the protein. The protein contains 14 cysteine residues of which 7 are located between positions 101 and 150.
This region of the protein is believed to be located at the surface of the particle and contain the major antigenic region of HBsAg including the immunodominant a region and the recognition site for the RF1 monoclonal antibody (Waters J et al, Postgrad. Med. J., 1987:63 (Suppl. 2): 51-56. and Ashton-Rickardt and Murray J. Med. Virology, 1989: 29: 196). Antigen purified with thiomersal present in the 4B gel permeation buffer contains about 0.5-0.6 μg mercury at the end of the purification process. This mercury is not fully removed by simple dialysis.
In one experiment, 0.56 μg Mercury per 20 μg protein was measured on bulk antigen preparation. This preparation was dialysed for 16 hours at room temperature against 150 mM NaCl, 10 mM NaPO₄buffer pH 6.9. At the end of dialysis, a concentration of 0.33 μg Hg per 20 μg protein was measured.
In contrast, dialysis in the presence of a reducing agent such as L-cysteine at 0.1 to 5.0 mg/ml, DTT at 50 mM or 2-mercaptoethanol at 0.5 M, followed by a second dialysis to remove the reducing agent, results in reduction of the mercury content of the antigen preparation to less than 0.025 μg Mercury per 20 μg protein. This is the lowest limit of detection of the method.
The mercury content was determined by absorption spectrophotometry. The antigen is diluted in a solution containing 0.01% w/v of potassium bichromate (K₂Cr₂O₇) and 5% v/v of nitric acid. Standard solutions are prepared with thiomersal as the mercury source. The atomic absorption of sample and standard solutions is measured after vaporisation in a vapour generator, with a mercury-specific cathode at 253.7 nm. Atomic absorption of the dilution liquid is measured as blank. The mercury content of the sample is calculated via the calibration curves obtained from the standard solutions. Results are expressed as μg of mercury per 20 μg of protein.
1.3 Production of Thiomersal Free Bulk Antigen
The process steps for purification of bulk antigen are shown in FIG. 1.
1.4 Composition of Vaccine Formulated Without Thiomersal.

A typical quantitative composition for a hepatitis B vaccine without preservative and formulated from antigen prepared by the thiomersal free process is provided in Table 1.

	TABLE 1


	Constituent	Amount per ml

Active constituent - Protein of which	20	μg
at least 95% is HBsAg
Aluminium hydroxide (adsorbent)	0.95	mg
(expressed as Al₂O₃)
Sodium chloride	9.0	mg (maximum)
Disodium phosphate dihydrate	0.98	mg
Sodium dihydrogen phosphate dihydrate	0.71	mg
Water for injection q.s. ad	1.0	ml

The composition may be varied by the addition of 3D-MPL and/or other adjuvants.
2. Characterization of Bulk Antigen and Vaccine Produced by the Thiomersal Free Process
2.1. Tests and Assays on Purified Bulk Antigen
2.1.1 Basis of Comparison
Three lots of bulk antigen were prepared by the thiomersal free process according to this example (Example 1.2) and are identified as HEF001, HEF002 and HEF003. These were compared to a lot of bulk antigen (HEP2055) prepared by the previous process (as described in Example 1) in the presence of thiomersal.
2.1.2 Tests and Assays on Bulk Antigen
The three bulk antigen lots produced by the thiomersal free process were tested and the results are summarised in Table 2.
Protein content was measured by the method of Lowry et al (J. Biol. Chem. 1951:193:265).
Endotoxin content was measured by a Limulus gel clotting technique using a commercially available kit from Cape Cod Associates, 704 Main St., Falmouth, Mass. 02540, USA. The reagent is standardized against the US Pharm. Endotoxin Reference Standard.
Tween 20 was measured by the method of Huddleston and Allred (J. Amer. Oil Chemist Soc., 1965:42:983).
HBsAg content was measured by the commercially available AusZYME kit from Abbott Laboratories, One Abbott Park Road, Abbott Park, Ill. 60064, USA. Assay procedure B of the manufacturer was employed. A batch of bulk antigen purified by the process containing thiomersal was used as a standard to establish the dose response curve.
Polysaccharides were measured by the method of Dubois et al (Anal. Chem. 1956:28:350).
Lipids were measured using a commercially available kit (Merkotest Total Lipids 3321) from E. Merck, B. P. 4119, Darmstad D-6100, Germanny.
DNA content was measured by the Threshold method using apparatus and reagents available from Molecular Devices Corp., Gutenbergstraβe 10, Ismaning, Munich, Germany.
The values found in the tests and assays are in the range seen for bulk antigen lots manufactured using thiomersal in the elution buffer of the Sepharose 4B gel permeation step, with the exception of the antigenic activity by ELISA. The values for this measurement for the three HEF preparations are higher (1.63-2.25) than that found for the bulk antigen lot HEP2055 which has a ELISA/protein ratio of 1.13. The ELISA/protein ratios measured by the AUSZYME kit for thiomersal containing batches of bulk antigen are generally about 1.0 and within the range 0.8-1.2 and very rarely exceed 1.4.
2.1.3 SDS-PAGE Gel Analysis
The bulk antigen preparations were assayed by SDS-PAGE analysis in reducing conditions and Coomassie blue staining. All samples showed a major band at 24K with traces of a dimer protein. The samples were judged to be of high purity (>99% pure) as assessed by the absence of visible bands of contaminating proteins.
Samples (1 μg) of the bulk antigen preparations were assayed by SDS-PAGE in reducing and non-reducing conditions and silver staining (FIG. 2). In reducing conditions the samples showed an intense band migrating at 24K with traces of dimer and multimeric forms. The gel patterns are indistinguishable from that of HEP2055 as comparator. The samples were also run in non-reducing conditions. In these conditions less of the material migrates at 24K and the amount of polypeptide migrating at dimeric and multimeric positions is increased. The thiomersal free bulk antigen lots appear to have a somewhat higher degree of polymerisation than the comparator HEP2055 lot.
The identity of the 24K polypeptide revealed by Coomassie blue or silver staining was confirmed by Western blotting with rabbit polyclonal antibodies raised against plasma HBsAg. The bulk antigen preparations show a major band at 24K together with dimeric and trimeric forms. The technique reveals minor traces of breakdown products of the surface antigen protein. There are no differences between the bulk antigen prepared by the thiomersal free process and the HEP2055 lot.
The presence of residual yeast proteins was assayed by SDS-PAGE analysis in reducing conditions and Western blotting with rabbit polyclonal antiserum raised against yeast proteins (FIG. 3). The technique is qualitative and does not permit quantitation of the impurities.
A constant band pattern is shown over the three bulk antigen lots prepared by the thiomersal free process and the HEP2055 lot with one exception.

A heavily staining band present at ±23K in the HEP2055 bulk antigen is virtually absent in the 3 HEF preparations. The Western blotting shows that the thiomersal free purification process results in a purer antigen product.

TABLE 2


Results of tests and assays on purified, thiomersal free bulk antigen

RESULT

TEST	HEF001	HEF002	HEF003	HEP2055

PH	6.8	6.8	6.8	6.8
Protein content	1312 μg/ml	888 μg/ml	913 μg/ml	995 μg/ml
by Lowry
Endotoxin	<0.25 EU	<0.25 EU	<0.25 EU	<0.25 EU
content
Tween 20	7.1 μg	6.6 μg	7.4 μg	5.8 μg
content
Antigenic	2957 μg/ml	1505 μg/ml	1486 μg/ml	1128 μg/ml
activity by
ELISA
ELISA/protein	2.25	1.69	1.63	1.13
ratio
Polysaccharide	0.33 μg	0.35 μg	0.33 μg	0.34 μg
content
Lipid content	13.7 μg	12.8 μg	12.9 μg	11.8 μg
DNA content	<1 pg	<1 pg	<1 pg	<1 pg
by Threshold

2.1.4 Other Biochemical Tests and Assays
2.1.4.1 DNA Content

The DNA content of the 3 bulk antigen lots was measured by the Threshold method (Molecular Devices Corp). The amounts measured were less than 10 μg DNA per 20 μg protein (Table 2); the same level of DNA content seen with bulk antigen produced by the current approved process.
2.1.4.2 Amino Acid Composition
The amino acid composition of the three HEF bulk antigen lots was determined after acid hydrolysis with 6N HCl by chromatography of the amino acids on an ion exchange column with post column ninhydrin detection. Proline and tryptophan were not determined. The results are given in Table 3.
The compositions found are in good agreement with that determined on HEP2055 and with the expected composition derived from the DNA sequence. Although the number of glycine residues measured for HEP2055 is close to the expected composition, a value of 16 to 17 residues is more usually measured for bulk antigen preparations. The mean number of cysteine residues found is the expected 14, showing that no extra cysteines are bound to the particle as a result of the treatment at the CsCl gradient step.
2.1.4.3 Quantification of Free Cysteine
The quantity of free cysteine present in bulk antigen preparations obtained according to the method described was measured after oxidation of the particles with performic acid without prior acid hydrolysis. Oxidised free cysteine residues were separated on an ion exchange column with post column detection by ninhydrin. The limit of detection of cysteine by this method is 1 μg per ml.
No free cysteine could be measured in the 3 HEF antigen preparations when tested at the initial protein concentrations given in Table 2.
The technique measures both free cysteine residues present in the buffer and cysteine residues which are attached to the HBsAg protein by disulphide bonding but which do not form part of the polypeptide sequence.
2.1.4.4 N-terminal Sequence Analysis

The presence of possible protein contaminants and degradation products in the three bulk antigen lots produced by the modified process was assessed by N-terminal sequence analysis based on Edman degradation. The N-terminal sequence MENITS . . . of the HBsAg protein was detected with no interference from other sequences. The N-terminal methionine was also confirmed to be 60-75% blocked by acetylation, as observed previously for HBsAg polypeptide produced by the routine process.

TABLE 3


AMINO ACID COMPOSITION OF HBSAG

Amino				Mean		Expected
acid	HEF001	HEF002	HEF003	comp.	HEP2055	comp.

Asp	11.3	11.3	11.3	11.3	11.5	10
Thr	17.5	17.4	17.2	17.4	17.8	17
Ser	21.4	21.6	21.4	21.5	20.9	23
Glu	11	11	11	11.0	10.5	9
Pro	nd	nd	nd		nd	24
Gly	17.1	16.8	16.7	16.9	14.6	14
Ala	7.5	7.4	7.4	7.4	7.2	6
Cys	12.3	14.95	14.9	14.1	13.2	14
Val	10.9	11	10.9	10.9	10.7	11
Met	6.8	6.7	7.1	6.9	7.1	6
Ile	12.3	12.4	12.5	12.4	12.2	16
Leu	26.3	26.6	26.2	26.4	26.7	33
Tyr	6.8	6.8	6.8	6.8	7	6
Phe	13.8	13.9	13.8	13.8	13.9	15
His	3	2.8	3.3	3.0	3.3	1
Lys	4	4	3.9	4.0	4.2	3
Arg	5.7	5.8	5.7	5.7	6.1	5
Trp	nd	nd	nd		nd	13

2.1.4.5 Laser Light Scattering Analysis

Particle size comparisons were made by laser light scattering between the HBsAg particles produced using the modified process and the HEP2055 reference lot (Table 4).
The mean molecular weights determined show good consistency between the preparations.

TABLE 4

HBsAg particle molecular weights by laser light scattering

Antigen lot MW (Daltons)

HEF001 3.07 × 10⁶

HEF002 2.76 × 10⁶

HEF003 2.76 × 10⁶

HEP2055 3.34 × 10⁶

2.1.4.6 Electron Microscopy
The bulk antigen preparations were examined by electron microscopy after fixation and staining with uranyl acetate.
The particles observed were similar in all the samples and conformed to the ±20 nm subspherical or cobblestone-like particles typical of HBsAg. The particles observed in the 3 HEF lots were indistinguishable from HEP2055.
2.1.5 Immunological Analyses
2.1.5.1 Reactivity with RF1 Monoclonal Antibody
The three bulk antigen preparations were tested for their reactivity with the RF1 monoclonal antibody by ELISA inhibition assay. The RF1 monoclonal antibody has been shown to protect chimpanzees against challenge with HBV and is considered to recognize a protective conformational epitope on the HBsAg particle (Iwarson S et al, 1985, J. Med, Virol., 16: 89-96).
The RF1 hybridoma may be propagated in the peritoneal cavity of BalbC mice or in tissue culture.
Ascitic fluid diluted at 1/50000 in saturation buffer (PBS containing 1% BSA, 0.1% Tween 20) was mixed 1:1 with various dilutions in PBS of the HBsAg samples to be tested (final concentrations ranging between 100 μg and 0.05 μg/ml).
Mixtures were incubated in Nunc Immunoplates (96U) for 1 hr at 37° C. before being transferred for 1 hr at 37° C. onto plates coated with a standard preparation of HBsAg. The standard HBsAg preparation was a lot of bulk antigen (Hep 286) purified by the thiomersal containing process. After a washing step with PBS containing 0.1% Tween 20, biotin-conjugated sheep anti-mouse IgG diluted 1/1000 in saturation buffer was added to and incubated for 1 hr at 37° C. After a washing step, streptavidin-biotinylated peroxydase complex diluted 1/1000 in saturation buffer was added to the same wells and incubated for 30 min at 37° C. Plates were washed and incubated with a solution of OPDA 0.04%, H₂O₂0.03% in 0.1 M citrate buffer pH 4.5 for 20 min at room temperature. The reaction was stopped with 2N H₂SO4 and the optical densities (O.D.) were measured at 490/630 nm and plotted graphically.
The IC50, defined as the concentration of antigen (inhibitor concentration) that inhibits 50% of the antibody binding to coated HBsAg was calculated using a 4 parameters equation and expressed in ng/ml.
A series of HEP antigen lots including HEP2055 were also tested, together with the Herpes simplex gD antigen as negative control. The assay measures the ability of each test antigen to inhibit binding of RF1 to a standard antigen preparation (HEP286) bound to microtitre plates.

Table 5 gives the concentrations of each antigen found to inhibit 50% of RF1 binding to the fixed antigen.

TABLE 5


INHIBITION OF BINDING OF RF1
MONOCLONAL ANTIBODY TO HBSAG

	Bulk antigen	IC50 (ng/ml)*

	HEP286	3834
	HEP673	3437
	HEP720	3150
	HEP2055	2384
	HEF001	468
	HEF002	574
	HEF003	540

	*IC50 = antigen concentration (ng/ml) inhibiting 50% of RF1 binding to fixed antigen

The results show that 4 to 7 fold less HEF antigen is required to inhibit RF1 binding (Table 5). This shows that antigen prepared by the modified process has an increased presentation of the RF1 epitope compared to HEP bulk antigen.
The same type of inhibition assay was performed using human sera from Engerix B™ vaccinees instead of the RF1 mAb and did not reveal differences between the HEP antigen lots and the HEF antigens.
2.1.5.2. Affinity of Binding to Monoclonal RF1
The kinetic parameters of RF1 monoclonal antibody binding to the 3 HEF antigen lots and to HEP2055 were measured by surface plasmon resonance using a Biacore 2000 apparatus from Amersham Pharmacia Biotech, Amersham Place, Little Chalfont, Bucks, UK.
The kinetic parameters measured were:
ka: the association rate constant (M⁻¹S⁻¹)
kd: the dissociation rate constant (S⁻¹)
Ka: the equilibrium or affinity constant (M⁻¹)

- where $Ka = \frac{ka}{kd}$

The values found are given in Table 6.

TABLE 6

Affinity constants of RF1 binding to HBsAg

ka kd Ka

Bulk antigen (×10⁻³) (×10⁵) (×10⁻⁷)

HEF001 6.81 3.21 21.97

HEF002 6.89 3.73 18.83

HEF003 7.39 4.67 15.80

HEP2055 3.31 6.30 5.31
The three HEF antigen lots gave similar association/dissociation constants and binding affinity values. In contrast HEP2055 has a weaker affinity for binding to RF1.
This is consistent with the results from the ELISA inhibition assay which showed that antigen prepared by the thiomersal free process had an increased presentation of the RF1 epitope.
2.2. Test and Assays on Vaccine Formulated with Antigen Produced by the Modified Process
The three HEF antigen lots were adsorbed onto aluminium hydroxide and formulated as vaccine according ot the composition as shown in Table 1. The presentation is the adult dose in vials (20 □g antigen protein in 1 ml). The lots are identified as DENS001A4, DENS002A4 and DENS003A4.
Vaccine potency was measured by an in-vitro antigen content assay using the Abbott Laboratories AUSZYME ELISA kit and a classical lot of vaccine formulated with 50□g/ml thiomersal as standard. Vaccine potency was measured using method B as described in PharmaEuropa Special Issue Bio97-2 (December 1997). The three HEF lots give high values for antigen content, nearly twice the stated content of 20 μg antigen protein.
2.2.1 Reactivity of DENS Vaccine with RF1 Monoclonal Antibody
The antigenicity of the adsorbed vaccine was further tested in an inhibition assay with RF1 monoclonal antibody. The assay measures the ability of the vaccine sample to inhibit RF1 binding to fixed bulk antigen (HEP286).
Ascitic fluid diluted at 1/50000 in saturation buffer (PBS containing 1% BSA, 0.1% Tween 20) was mixed 1:1 with various dilutions in PBS of the vaccine samples to be tested (concentration ranging between 20 μg and 0.05 μg/ml).
Mixtures were incubated in Nunc Immunoplates (96U) for 2 hr at 37° C. with agitation before being transferred onto HBsAg coated plates. The HBsAg preparation used for coating was a lot of bulk antigen (Hep 286) purified by the thiomesal containing process. These plates are then incubated for 2 hr at 37° C. with agitation. After a washing step with PBS containing 0.1% Tween 20, biotin-conjugated sheep anti-mouse IgG diluted 1/1000 in saturation buffer was added and incubated for 1 hr at 37° C. After a washing step, streptavidin-biotinylated peroxydase complex diluted 1/1000 in saturation buffer was added to the wells and incubated for 30 min at 37° C. Plates were washed and incubated for 20 min at room temperature with a solution containing OPDA 0.04%, H₂O₂0.03% in 0.1 M citrate buffer pH 4.5. The reaction was stopped with 2N H₂SO4 and optical densities (O.D.) were measured at 490/630 nm and plotted graphically.
The IC50, defined as the concentration of antigen (inhibitor concentration) that inhibits 50% of the antibody binding to coated HBsAg was calculated using a 4 parameters equation and expressed in ng/ml.
Vaccine prepared from bulk antigen produced by the modified process was compared to Engerix B™ vaccine formulated from classical HEP bulk antigen and without thiomersal as preservative.
The assays were run in triplicate.

The results are given in Table 7 and show that about half the quantity of DENS vaccine is required to achieve 50% inhibition of RF1 binding as compared to preservative free Engerix B™ vaccine. This reflects an increased presentation of the RF1 epitope on the HEF/DENS antigen and is consistent with the tests done with RF1 antibody on the purified bulk antigen.

TABLE 7


Inhibition of RF1 binding by formulated vaccine

IC-50 (ng/ml)⁽¹⁾

Experiment

Vaccine lot

	1	2	3	Mean

DENS001A4	913	662	603	726
DENS002A4	888	715	521	708
DENS003A4	817	685	582	695
ENG5100A2	1606	1514	1481	1534
ENG3199B9	1329	1170	1286	1262
ENG3328A9	1417	1194	1334	1315

⁽¹⁾concentration of vaccine inhibiting 50% of RF1 antibody binding to fixed antigen

2.2.2 Immunogenicity of DENS Vaccine in Mice

A study was performed in Balb/C mice in order to compare the immunogenicity of the three DENS consistency lots to Engerix B™ produced according to the current antigen manufacturing process and formulated with thiomersal.
The following lots were tested:
# DENS001A4
# DENS002A4
# DENS003A4
# ENG2953A4/Q as reference
Briefly, groups of 12 mice were immunised intramuscularly twice at 2 weeks interval with vaccine doses corresponding to 1/10 (2 μg) or 1/50 (0.4 μg) of the adult human dose. Antibody response to HBsAg and the isotypic profile induced by vaccination were monitored from sera taken at day 28.

EXPERIMENTAL DESIGN

Groups of 12 Balb/C mice were immunised intramuscularly in both legs (2×50 μl) on days 0 and 15 with the following vaccine doses:

TABLE 8


Groups and vaccine dose

			Antigen
Group	Vaccine	Volume	dose

1	DENS001A4	100 μl	2 μg
2	→Diluted 5X in PO4/NaCl	100 μl	0.4 μg
3	DENS002A4	100 μl	2 μg
4	→Diluted 5X in PO4/NaCl	100 μl	0.4 μg
5	DENS003A4	100 μl	2 μg
6	→Diluted 5X in PO4/NaCl	100 μl	0.4 μg
7	ENG2953A4/Q	100 μl	2 μg
8	→Diluted 5X in PO4/NaCl	100 μl	0.4 μg

On days 15 (2 weeks post I) and 28 (2 weeks post II) blood was taken from the retroorbital sinus.
For the design of this experiment (4 formulations×2 doses with 12 mice per group), the power was estimated a priori with the PASS statistical program. The PASS (Power and Sample Size) statistical programme was obtained from NCSS, 329 North 1000 East, Kaysville, Utah 84037. For the 2 way analysis of variance, a 2.5 fold difference of GMT between formulations with an alpha error of 5% should be detected with a power >90%.
Results
Serology:
Humoral responses (Total Ig and isotypes) were measured by ELISA assay using HBsAg (Hep286) as coating antigen and biotin conjugated anti-mouse antibodies to reveal anti-HBs antibody binding. Only post II sera were analysed.
Table 9 shows the mean and GMT anti-HBs Ig antibody responses measured on individual sera at 2 weeks post II
Comparable antibody responses are induced by the DENS and classical hepatitis B formulations: GMT ranging between 2304 and 3976 EU/ml for the DENS lots compared to 2882 EU/ml for SB Biologicals hepatitis B monovalent vaccine (Engerix B™) at the 2 μg dose, and GMT ranging between 696 and 1182 EU/ml for the DENS lots compared to 627 EU/ml for SB Biologicals hepatitis B monovalent vaccine (Engerix B™) at the 0.4 μg dose.

- As expected a clear antigen dose range effect is observed for all formulations at the 2 μg and 0.4 μg doses with a 3 to 6 fold difference in GMTs.

Four non responder mice (titers <50 EU/ml) were observed without clear links to the antigen doses or lots used for the injection (

Groups

1, 2, 3 and 8; one mouse per group). Based on statistical analysis (Grubbs Test) these mice were discarded from further analysis.

TABLE 9


Antibody response in mice at day 28

	ELISA
	TITERS (Ig)

Group	Vaccine	Dose	Number	Mean	GMT

1	DENS001A4	2 μg	11	3466	2971
2		0.4 μg	11	1283	1182
3	DENS002A4	2 μg	11	2436	2304
4		0.4 μg	12	984	786
5	DENS003A4	2 μg	12	4583	3976
6		0.4 μg	12	997	696
7	ENG2953A4/Q	2 μg	12	3999	2882
		0.4 μg	11	737	627

Statistical Analysis:

A 2 way-analysis of variance was performed on the anti-HBs titers after log transformation of post II data, using the vaccines 4 lots) and antigen doses (2 μg and 0.4 μg) as factors. This analysis confirmed that a statistically significant difference was observed between the two antigen doses (p value<0.001) and did not show any significant difference between the vaccine lots (p value=0.2674). As previously mentioned the power was estimated a priori and the design of the experiment was such that a 2.5 fold difference of GMT with a alpha error of 5% could be detected between formulations with a power >90%.
Isotypic Profile:
Table 10 shows the isotypic repartition (IgG1, IgG2a and IgG2b) calculated from an analysis on pooled sera at post II.

- As expected, a clear TH2 response is induced by these alum based vaccines as mainly IgG1 antibodies are observed.

No difference is observed between the DENS lots or SB Biologicals hepatitis B monovalent vaccine in term of isotypic profile.

TABLE 10


Repartition of IgG isotypes in pooled day 28 sera

Isotype (%)

Group	Vaccine	Dose	IgG1	IgG2a	IgG2b

1	DENS001A4	2 μg	91	4	5
2		0.4 μg	87	8	5
3	DENS002A4	2 μg	97	2	1
4		0.4 μg	87	6	7
5	DENS003A4	2 μg	98	1	1
6		0.4 μg	93	4	3
7	ENG2953A4/Q	2 μg	88	8	4
		0.4 μg	88	9	3

EXAMPLE 3

Formulation of Combined Vaccines
The bulk antigen of the invention is particularly suitable for formulation in a combined vaccine comprising IPV.
Stability studies performed on initial lots of a combined DTPa-HBV-IPV vaccine indicated a decline in potency of the IPV component, particularly of type 1 poliomyelitis antigen, when using an in vitro immunoassay (determination of D-antigen content by ELISA) and an in vivo rat potency test. No potency loss was observed for type 3. For type 2, the potency loss was within the expected range (not more than 10% loss per year of storage).
Studies were initiated to determine the cause of this loss of potency in the combined DTPa-HBV-IPV vaccine. From the observation that the stability of IPV in SB Biologicals' DTPa-IPV vaccine is satisfactory (not more than 10% antigen content loss per year of storage), it was concluded that the HBV component was likely to be responsible for the instability of IPV in the DTPa-HBV-IPV vaccine.
The HBV component used in the initial DTPa-HBV-IPV formulation is the purified r-DNA, yeast-derived HBsAg also used for the manufacture of SB Biologicals hepatitis B monovalent vaccine and prepared as described in Example 1.
As a first attempt to determine which element in the HBV component was deleterious to IPV, the HBsAg bulk was analysed for the presence of thiomersal. It has been previously found (Davisson et al., 1956, J. Lab. Clin. Med 47: 8-19) that thiomersal used as preservative in DTP vaccines “was detrimental to the poliomyelitis virus” in a DTP-IPV combination. This observation was considered by vaccine manufacturers who have replaced thiomersal with other preservatives to formulate their IPV-containing vaccines. More recently, the effect of thiomersal on IPV potency under conditions of long-term storage at +4° C. was reinvestigated. The loss of potency of type 1 polio virus antigen to undetectable levels after 4-6 months was reported (Sawyer, L. A. et al. 1994, Vaccine 12: 851-856).
Using atomic adsorption spectroscopy, approximately 0.5 μg of mercury (Hg) per 20 μg of HBsAg was detected in antigen purified according to Example 1.
This amount of mercury (as thiomersal and ethylmercury chloride, the thiomersal degradation product) can reduce to undetectable levels the ELISA response for D-antigen type 1 content in an IPV bulk concentrate incubated at 37° C. for 7 days.

A method was established to release mercury present in the HBsAg bulk. It was postulated that mercury could be bound to thiol groups on the HBsAg particle and could therefore be released in the presence of reducing agents. After experimentation with other reducing agents, L-Cysteine was selected as the agent for release of mercury from the HBsAg particle. After dialysis of HBsAg bulk against saline solution containing 5.7 mM L-Cysteine, no mercury was detected in the retentate (detection limit of the testing method: 25 ng Hg/20 μg HBsAg). The dialysed antigen was mixed with IPV bulk concentrate and the stability of the type 1 virus was assessed by measuring the D-antigen content after incubation at 37° C. for 7 days. The IPV bulk concentrate non-mixed and mixed with HBsAg not treated with cysteine were used as controls. The reference ELISA titre was obtained on the samples stored at +2° C. to +8° C. for 7 days. The results are summarised in Table 11:

	TABLE 11


	D-antigen content
	(type 1)⁽¹⁾

SAMPLE	7 days/4° C.	7 days/37° C.	Loss

IPV (non-mixed)	31.6	24.2	23%
IPV + HBsAg not treated	31.1	18.1	42%
IPV + HBsAg-cysteine-treated	31.4	27.6	12%
IPV + thiomersal (1 μg/ml)	30.5	11.0	74%

⁽¹⁾expressed in D-antigen units (DU)

The data obtained on these laboratory preparations clearly demonstrate that the stability of the type 1 polio virus is significantly improved if HBsAg is treated with cysteine to remove residual mercury prior to mixing with IPV.
The data presented above also show a loss of D-antigen content of 23% for the reference IPV preparation after incubation for 7 days at 37° C. This confirms the inherent instability of the type 1 Mahoney polio virus, as previously reported (Sawyer, L. A. et al. (1994), Vaccine 12: 851-856).
Although commercial lots of DTPa-HBV-IPV and DTPa-HBV-IPV/Hib vaccines have been prepared using a dialysis process with 5.7 mM L-Cysteine to remove residual mercury and preserve the stability of IPV, the dialysis process is not suited to large scale production and involves a series of supplementary steps to prepare thiomersal or mercury free HBsAg. In contrast, the HBsAg of the present invention, prepared without thiomersal, may be directly used in formulations of combined vaccines especially those containing IPV.
4. Summary
The previously used process for purification of yeast-derived surface antigen contains a gel permeation step where the mercury containing anti-microbial compound thiomersal is included in the elution buffer to control bioburden.
The thiomersal is not completely cleared during the subsequent steps of the process so that about 1.2 μg thiomersal per 20 μg protein is present in the purified bulk antigen.
In order to produce a completely thiomersal (mercury) free bulk antigen the purification process has been altered at two steps.

- Thiomersal is omitted from the elution buffer at the 4B gel permeation step.
- Cysteine (2 mM final concentration) is added to the eluate pool from the anion exchange chromatography step. This prevents precipitation of antigen during CsCl density gradient centrifugation.
- There are no other changes to the production process.

The bulk antigen produced by the modified process has been characterized. Physico-chemical tests and assays show that the thiomersal free antigen is indistinguishable in its properties from antigen produced by the previously used process. The antigen particles have the same constituents.
The identity and integrity of the HBsAg polypeptide is unaffected by the modified process as judged by SDS-PAGE analysis, Western blotting using polyclonal anti-HBsAg antibodies, N-terminal sequence analysis and amino acid composition. Electron microscopy and laser light scattering analysis show that the particles are of the typical form and size expected for yeast-derived HBsAg. Analysis by Western blotting with anti-yeast protein serum shows that the antigen produced by the thiomersal free process has a similar pattern of contaminating yeast proteins. However, the amount of a contaminating band migrating at 23K is greatly reduced in the 3 HBsAg lots produced using the modified process.
Immunological analyses show that the thiomersal free particles have an increased antigenicity. The particles are more reactive with the Abbott AUSZYME kit (containing a mixture of monoclonal antibodies) giving ELISA/protein ratios of 1.6 to 2.25. This increased antigenicity is also shown with the protective RF1 monoclonal antibody. About 4 to 7 fold less thiomersal free antigen is required to inhibit RF1 binding to a standard fixed antigen. The thiomersal free and classical antigen inhibition of binding curves fall into two distinct families. This difference is also shown by measurements of the binding affinity constant for RF1 using surface plasmon resonance. The binding affinities of the thiomersal free preparations are 3 to 4 fold higher compared to the lot of classical bulk antigen.
The bulk antigen preparations were formulated as vaccine by adsorption onto aluminium hydroxide and without preservative.
Testing for in vitro potency using the Abbott AUSZYME ELISA kit and thiomersal containing SB Biologicals hepatitis B monovalent vaccine as standard showed that high in vitro potency values were obtained. The antigen content measured by this test was nearly double the stated value of 20 μg protein per ml.
An increased reactivity of vaccine prepared from thiomersal free antigen was also seen in an inhibition assay with RF1 monoclonal antibody for binding to fixed antigen. About half the quantity of thiomersal free vaccine was required to give 50% inhibition of RF1 binding to fixed antigen as compared to antigen purified by the previously used process and formulated without preservative.
This increased antigenicity of the thiomersal free vaccine with respect to RF1 is consistent with the results from the in vitro potency test (antigen content) and with the RF1 antibody tests performed on the bulk antigen preparations.
A mouse immunogenicity test was performed using priming and booster vaccinations two weeks apart and doses of 2 and 0.4 μg antigen. Mice were bled on day 28, 14 days after the booster. The sera were analysed for antibody titre and isotype composition. A clear antigen dose effect was observed for the two doses administered but there was no statistically significant difference in the response in terms of antibody titres (GMT) between thiomersal free and preservative free vaccines.
No substantial differences were observed in the isotype profiles.
The following examples provide clinical data in humans as to the efficiacy of hepatitis B thiomersal free vaccines.
Using a hepatitis B vaccine component made in the presence of a component having a free —SH group, specifically in this case hepatitis B manufactured in the presence of cysteine, thiomersal free hepatitis vaccines were tested in comparison to vaccines which were made using thiomersal in the production process.

EXAMPLE 4

A Double-Blind, Controlled, Multicentric Randomized Study, Designed to Evaluate the Immunogenicity and Reactogenicity of the Combined Hepatitis A/Hepatitis B Preservative-Free Thimerosal-Free Vaccine as Compared to the Combined Hepatitis A/Hepatitis B Vaccine with Preservative, Administered According to a 3-Dose Schedule (0, 1, 6 Months) to Healthy Adults Aged 18 Years or Older.
Rationale: To evaluate the immunogenicity and safety of two formulations of combined hepatitis A/hepatitis B (HAB) vaccines (preservative-free and thimerosal-free [PFTF] HAB versus HAB with traces of thimerosal remaining from the production process), administered following a three-dose schedule (0, 1, 6 months).
Study Design: Double-blind, controlled, self-contained, randomized (1:1), multicentre study with two parallel groups
Centres: Five centres (one centre each in the Netherlands, Sweden, United Kingdom and two centres in Germany).
Indication: Vaccination of healthy adults 18 years of age and older who were seronegative for hepatitis A and hepatitis B virus according to a 0, 1 and 6-month schedule.
Treatment: The study groups were as follows:

- HAB PFTF group: received HAB PFTF vaccine according to a 0, 1, 6 month schedule
- HAB group: received HAB vaccine with traces of thimerosal remaining from the production process according to a 0, 1, 6 month schedule

Both formulations were administered intramuscularly in the deltoid region.
Objectives: To demonstrate the non-inferiority of the HAB PFTF vaccine to that of the HAB vaccine with traces of thimerosal remaining from the production process, with respect to anti-hepatitis B surface antigen (anti-HBs) seroprotection rate and anti-hepatitis A virus (anti-HAV) seropositivity rate at Month 7, when administered according to a 0, 1, 6 month schedule.
Primary Outcome/Efficacy Variable:
Observed Variables:

- Anti-HBs and anti-HAV antibody concentrations at Month 7 for all subjects.
  Derived variables:
- Seroprotection (SP) rates for anti-HBs antibodies at Month 7 for all subjects. The SP rate was defined as the percentage of subjects with anti-HBs concentrations ≧10 mlU/ml.

Seropositivity (S+) rates for anti-HAV antibodies at Month 7 for all subjects. The S+ rate was defined as the percentage of subjects with anti-HAV concentrations ≧15 mlU/ml.
Secondary Outcome/Efficacy Variable(s):
Observed Variables:

- Anti-HAV and anti-HBs antibody concentrations at Month 1, 2, 6 and 7 for all subjects
- Incidence, intensity of solicited local and general signs and symptoms and relationship to vaccination of solicited general signs and symptoms reported during a four-day (day 0-day 3) follow-up period after each vaccination and overall
- Incidence, intensity and relationship to vaccination of unsolicited local and general signs and symptoms reported during a 31-day (day 0-day 30) follow-up period after each vaccination and overall
- Occurrence, intensity and relationship to vaccination of SAEs during the study period
  Derived Variables:
- Geometric Mean Concentrations (GMCs) for anti-HBs antibodies at Months 1, 2, 6 and 7 for all subjects.
- Seropositivity (S+) rates for anti-HBs antibodies at Months 1, 2, 6 and 7 for all subjects. S+ rate was defined as the percentage of subjects with anti-HBs concentrations ≧3.3 mlU/ml.
- Seroprotection (SP) rates for anti-HBs antibodies at Months 1, 2 and 6 for all subjects.
- GMCs for anti-HAV antibodies at Months 1, 2, 6 and 7 for all subjects.
- Seropositivity (S+) rates for anti-HAV antibodies at Months 1, 2 and 6 for all subjects.
  Statistical Methods:

The analyses were performed on the Total vaccinated cohort, the According-To-Protocol (ATP) cohort for immunogenicity and the ATP cohort for safety.

- The Total vaccinated cohort included all vaccinated subjects for whom data were available.
- The ATP cohort for immunogenicity included all subjects who had post-vaccination immunogenicity results and who complied with the procedures defined in the protocol.
- The ATP cohort for safety included all subjects who received at least one dose of study vaccine/comparator according to their random assignment, had sufficient data to perform an analysis of safety, had not received a vaccine not specified or forbidden in the protocol, and for whom the randomization code was not broken.
  Analysis of Immunogenicity:

The analysis of immunogenicity was performed on the ATP cohort for immunogenicity.
For each vaccine group and at each blood sampling timepoint, the seropositivity rates for anti-HAV and anti-HBs, seroprotection rates for anti-HBs, and GMCs were tabulated with their 95% confidence interval (CI).
The asymptotic standardised two-sided 95% CI on the difference in anti-HAV seropositivity rates and the anti-HBs seroprotection rates (HAB PFTF group minus HAB group) was computed. If the lower limit of the CIs for both these treatment differences was greater than the pre-defined non-inferiority limit (−7%), the HAB PFTF group was considered non-inferior to the HAB group.
Analysis of Safety:
The analysis of safety was performed on the ATP cohort for safety for the analysis of solicited symptoms and on the Total vaccinated cohort for the analysis of unsolicited symptoms.
The percentage of subjects reporting solicited local and solicited general adverse events within the 4-day (day 0-day 3) follow-up period after each vaccination was calculated with the exact 95% CI, following each dose and across doses, according to the type of adverse event (any and each specific solicited adverse event), the intensity (any and Grade 3) and for general solicited adverse events only, according to the relationship to vaccination was tabulated. The frequency of unsolicited symptoms and serious adverse events were tabulated per group.

Study Population: Healthy male or female subjects aged ≧18 years at the time of screening, who were seronegative for anti-HAV, anti-hepatitis B core antigen (anti-HBc), anti-HBs antibodies and for HBs antigen at the time of entry into the study. Female subjects of childbearing agreed take precautions against pregnancy for two months after completion of the vaccination series. Subjects with previous vaccination against hepatitis A or B virus and subjects with a family history of congenital or hereditary immunodeficiency were excluded from the study.

	TABLE 12


	HAB PFTF	HAB

Number of Subjects:
Planned, N	204	204
Randomised, N (Total	229	237
vaccinated cohort)
Completed, n (%)	219 (95.6)	227 (95.9)
Total Number Subjects	10 (4.4)	10 (4.2)
Withdrawn, n (%)
Withdrawn due to Adverse	0 (0)	0 (0)
Events n (%)
Withdrawn due to Lack of	Not applicable	Not applicable
Efficacy n (%)
Withdrawn for other reasons	10 (4.4)	10 (4.2)
n (%)
Demographics
N (Total vaccinated cohort)	229	237
Females: Males	121:108	136:101
Mean Age, years (SD)	33.1 (12.49)	33.5 (12.79)
Caucasian, n (%)	223 (97.4)	231 (97.5)

Primary Efficacy Results:

Difference in seroprotection rates between groups for anti-HBs at Month 7 (ATP cohort for immunogenicity)

	TABLE 13


	Difference
	(HAB PFTF
	minus HAB)

99% CI

Group	N	%	Group	N	%	%	LL	UL

HAB	213	97.65	HAB	204	95.59	−2.06	HAB	213
			PFTF

N = number of subjects with available results
% = percentage of subjects with anti-HBs concentrations ≧10 mIU/ml
Difference = difference calculated
95% CI; LL, UL = standardized asymptotic two-sided 95% two-sided confidence interval; lower limit, upper limit
* Non-inferiority criterion met

Primary Efficacy Results:

Seroprotection rates for anti-HBs at Month 7 (ATP cohort for immunogenicity)

	TABLE 14


	SP (≧10 mIU/ml)

95% CI

Group	N	n	%	LL	UL

HAB PFTF	204	195	95.6	91.8	98.0
HAB	213	208	97.7	94.6	99.2

N = number of subjects with available results
n/% = number/percentage of subjects with anti-HBs concentrations ≧10 mIU/ml
95% CI; LL, UL = 95% confidence interval; lower limit, upper limit

	TABLE 15


	Difference
	(HAB PFTF minus
	HAB)

99% CI

Group	N	%	Group	N	%	%	LL	UL

HAB	213	99.53	HAB	204	99.51	−0.02	−2.29*	2.16
			PFTF

N = number of subjects with available results
% = percentage of subjects with anti-HAV concentrations ≧15 mIU/ml
Difference = difference calculated
95% CI; LL, UL = standardized asymptotic two-sided 95% two-sided confidence interval; lower limit, upper limit
Non-inferiority criterion met

Primary Efficacy Results:

Seropositivity rates for anti-HAV at Month 7 (ATP cohort for immunogenicity)

	TABLE 16


	S+ (≧15 mIU/ml)

95% CI

Group	N	n	%	LL	UL

HAB PFTF	204	203	99.5	97.3	100
HAB	213	212	99.5	97.4	100

N = number of subjects with available results
n/% = number/percentage of subjects with antibody concentrations ≧15 mIU/ml
95% CI; LL, UL = 95% confidence interval; lower limit, upper limit

Secondary Outcome Variable(s):

Seropositivity rates, seroprotection rates and GMCs (calculated on seropositive subjects) for anti-HBs (ATP cohort for immunogenicity)

TABLE 17


S+ (≧3.3 mIU/ml)	SP (≧10 mIU/ml)	GMC

95% CI

Group

Timing

N

n

%

LL

UL

n

%

LL

UL

mIU/ml

LL

UL

HAB	PI(M1)	213	46	21.6	16.3	27.7	25	11.7	7.7	16.8	17.7	9.9	31.6
PFTF
	PII(M2)	212	132	62.3	55.4	68.8	93	43.9	37.1	50.8	25.3	19.5	32.6
	PII(M6)	206	183	88.8	83.7	92.8	172	83.5	77.7	88.3	177.7	142.0	222.3
	PIII(M7)	204	197	96.9	93.1	98.6	195*	95.6*	91.8*	98.0*	2917.5	2246.2	3789.6
HAB	PI(M1)	224	43	19.2	14.3	25.0	25	11.2	7.4	16.0	21.8	12.7	37.3
	PII(M2)	222	137	61.7	55.0	68.1	103	46.4	39.7	53.2	29.9	22.9	39.2
	PII(M6)	215	198	92.1	87.6	95.3	189	87.9	82.8	91.9	138.6	112.5	170.7
	PIII(M7)	213	211	99.1	96.6	99.9	208*	97.7*	94.6*	99.2*	1944.8	1526.3	2478.0

N = number of subjects with available results
n/% = number/percentage of subjects with antibody concentrations above the specified cut-off
95% CI; LL, UL = 95% confidence interval; lower limit, upper limit
Px(My) = Blood sample obtained after dose x at month y after the first dose
*primary endpoints

Secondary Outcome Variable(s):

Seropositivity rates and GMCs (calculated on seropositive subjects) for anti-HAV (ATP cohort for immunogenicity)

	TABLE 18


	S+ (≧15 mIU/ml)	GMC

95% CI

Group	Timing	N	n	%	LL	UL	mIU/ml	LL	UL

HAB PFTF	PI(M1)	213	194	91.1	86.4	94.5	197.2	167.4	232.4
	PII(M2)	212	207	97.6	94.6	99.2	432.3	378.5	493.7
	PII(M6)	206	204	99.0	96.5	99.9	218.0	188.2	252.7
	PIII(M7)	204	203	99.5	97.3	100	3809.3	3272.6	4434.0
	PIII(M7)	204	203*	99.5*	97.3*	100*	3809.3	3272.6	4434.0
HAB	PI(M1)	224	204	91.1	86.5	94.5	229.7	194.4	271.4
	PII(M2)	222	218	98.2	95.5	99.5	453.1	396.7	517.4
	PII(M6)	215	210	97.7	94.7	99.2	250.2	215.6	290.3
	PIII(M7)	213	212*	99.5*	97.4*	100*	3669.3	3180.6	4233.0

N = number of subjects with available results
n/% = number/percentage of subjects with antibody concentrations ≧15 mIU/ml
95% CI; LL, UL = 95% confidence interval; lower limit, upper limit
Px(My) = Blood sample obtained after dose x at month y after the first dose
*primary endpoints

Secondary Outcome Variable(s): Incidence and intensity of solicited local symptoms reported during the four-day (day 0-day 3) follow-up period after each dose and across doses (ATP cohort for safety)

	TABLE 19


	Dose 1	Dose 2

HAB PFTF (N = 220)

HAB (N = 229)

HAB PFTF (N = 220)

HAB (N = 229)

95% CI

Symptom	Intensity	n	%	LL	UL	n	%	LL	UL	n	%	LL	UL	n	%	LL	UL

Pain	Any	90	40.9	34.3	47.7	82	35.8	29.6	42.4	70	31.8	25.7	38.4	62	27.1	21.4	33.3
	Grade 3	0	0.0	0.0	1.7	0	0.0	0.0	1.6	0	0.0	0.0	1.7	1	0.4	0.0	2.4
Redness	Any	33	15.0	10.6	20.4	47	20.5	15.5	26.3	28	12.7	8.6	17.9	32	14.0	9.8	19.2
	>50 mm	0	0.0	0.0	1.7	0	0.0	0.0	1.6	0	0.0	0.0	1.7	0	0.0	0.0	1.6
Swelling	Any	15	6.8	3.9	11.0	24	10.5	6.8	15.2	14	6.4	3.5	10.4	17	7.4	4.4	11.6
	>50 mm	0	0.0	0.0	1.7	0	0.0	0.0	1.6	0	0.0	0.0	1.7	0	0.0	0.0	1.6

Dose 3

Across doses

HAB PFTF (N = 213)

HAB (N = 222)

HAB PFTF (N = 220)

HAB (N = 229)

95% CI

Symptom	Intensity	n	%	LL	UL	n	%	LL	UL	n	%	LL	UL	n	%	LL	UL

Pain	Any	55	25.8	20.1	32.2	74	33.3	27.2	40.0	127	57.7	50.9	64.3	124	54.1	47.5	60.7
	Grade 3	0	0.0	0.0	1.7	0	0.0	0.0	1.6	0	0.0	0.0	1.7	1	0.4	0.0	2.4
Redness	Any	29	13.6	9.3	19.0	32	14.4	10.1	19.7	54	24.5	19.0	30.8	63	27.5	21.8	33.8
	>50 mm	0	0.0	0.0	1.7	0	0.0	0.0	1.6	0	0.0	0.0	1.7	0	0.0	0.0	1.6
Swelling	Any	13	6.1	3.3	10.2	18	8.1	4.9	12.5	31	14.1	9.8	19.4	39	17.0	12.4	22.5
	>50 mm	0	0.0	0.0	1.7	0	0.0	0.0	1.6	0	0.0	0.0	1.7	0	0.0	0.0	1.6

N = number of subjects having received at least one dose
n/% = number/percentage of subjects reporting a specified symptom during the four-day follow-up period (Day 0-Day 3)
95% CI; LL; UL = exact 95% confidence interval; lower limit, upper limit
Any = any symptoms regardless of intensity grade
Grade 3 pain = pain which prevents normal everyday activities

Secondary Outcome Variable(s): Incidence, intensity and relationship of solicited general symptoms reported during the four-day (day 0-day 3) follow-up period after each dose and across doses (ATP cohort for safety)

	TABLE 20


	Dose 1	Dose 2

HAB PFTF (N = 220)

HAB (N = 229)

HAB PFTF (N = 220)

HAB (N = 229)

Intensity/

95% CI

Symptom	relationship	n	%	LL	UL	n	%	LL	UL	n	%	LL	UL	n	%	LL	UL

Fatigue	Any	46	20.9	15.7	26.9	56	24.5	19.0	30.5	30	13.6	9.4	18.9	46	20.1	15.1	25.9
	Grade 3	1	0.5	0.0	2.5	0	0.0	0.0	1.6	1	0.5	0.0	2.5	0	0.0	0.0	1.6
	Related	20	9.1	5.6	13.7	35	15.3	10.9	20.6	19	8.6	5.3	13.2	33	14.4	10.1	19.6
Gastrointestinal	Any	27	12.3	8.2	17.4	22	9.6	6.1	14.2	18	8.2	4.9	12.6	17	7.4	4.4	11.6
	Grade 3	0	0.0	0.0	1.7	0	0.0	0.0	1.6	0	0.0	0.0	1.7	1	0.4	0.0	2.4
	Related	23	10.5	6.7	15.3	18	7.9	4.7	12.1	16	7.3	4.2	11.5	15	6.6	3.7	10.6
Headache	Any	49	22.3	17.0	28.4	51	22.3	17.1	28.2	31	14.1	9.8	19.4	33	14.4	10.1	19.6
	Grade 3	1	0.5	0.0	2.5	1	0.4	0.0	2.4	0	0.0	0.0	1.7	0	0.0	0.0	1.6
	Related	23	10.5	6.7	15.3	26	11.4	7.6	16.2	9	4.1	1.9	7.6	21	9.2	5.8	13.7
Fever	≧37.5° C.	2	0.9	0.1	3.2	1	0.4	0.0	2.4	1	0.5	0.0	2.5	1	0.4	0.0	2.4
(oral)	>39.0° C.	0	0.0	0.0	1.7	0	0.0	0.0	1.6	0	0.0	0.0	1.7	1	0.4	0.0	2.4
	Related	0	0.0	0.0	1.7	1	0.4	0.0	2.4	1	0.5	0.0	2.5	1	0.4	0.0	2.4

Dose 3

Across doses

HAB PFTF (N = 213)

HAB (N = 222)

HAB PFTF (N = 220)

HAB (N = 229)

Intensity/

95% CI

Symptom	relationship	n	%	LL	UL	n	%	LL	UL	n	%	LL	UL	n	%	LL	UL

Fatigue	Any	24	11.3	7.4	16.3	47	21.2	16.0	27.1	75	34.1	27.9	40.8	93	40.6	34.2	47.3
	Grade 3	0	0.0	0.0	1.7	0	0.0	0.0	1.6	2	0.9	0.1	3.2	0	0.0	0.0	1.6
	Related	15	7.0	4.0	11.3	34	15.3	10.8	20.7	45	20.5	15.3	26.4	68	29.7	23.9	36.1
Gastrointestinal	Any	15	7.0	4.0	11.3	17	7.7	4.5	12.0	43	19.5	14.5	25.4	44	19.2	14.3	24.9
	Grade 3	1	0.5	0.0	2.6	0	0.0	0.0	1.6	1	0.5	0.0	2.5	1	0.4	0.0	2.4
	Related	13	6.1	3.3	10.2	15	6.8	3.8	10.9	37	16.8	12.1	22.4	39	17.0	12.4	22.5
Headache	Any	20	9.4	5.8	14.1	45	20.3	15.2	26.2	77	35.0	28.7	41.7	82	35.8	29.6	42.4
	Grade 3	0	0.0	0.0	1.7	2	0.9	0.1	3.2	1	0.5	0.0	2.5	3	1.3	0.3	3.8
	Related	11	5.2	2.6	9.1	28	12.6	8.5	17.7	33	15.0	10.6	20.4	47	20.5	15.5	26.3
Fever	≧37.5° C.	3	1.4	0.3	4.1	3	1.4	0.3	3.9	5	2.3	0.7	5.2	5	2.2	0.7	5.0
(oral)	>39.0° C.	0	0.0	0.0	1.7	0	0.0	0.0	1.6	0	0.0	0.0	1.7	1	0.4	0.0	2.4
	Related	1	0.5	0.0	2.6	3	1.4	0.3	3.9	2	0.9	0.1	3.2	5	2.2	0.7	5.0

N = number of subjects having received at least one dose
N/% = number/percentage of subjects reporting a specified symptom during the four-day follow-up period (Day 0-Day 3)
95% CI; LL; UL = exact 95% confidence interval; lower limit, upper limit
Any = any symptoms regardless of intensity grade
Grade 3 = solicited general symptoms that prevented normal everyday activities
Related = It was determined by the investigator that there was a reasonable possibility that the vaccine contributed to the adverse event.

Safety results: Number (%) of subjects with unsolicited adverse events (Total vacinated cohort)

TABLE 21


Most frequent adverse events - On-Therapy
(occurring within day 0-30	HAB PFTF	HAB
following vaccination)	N = 229	N = 237

Subjects with any AE(s), n(%)	86 (37.6)	82 (34.6)
Subjects with adverse events classified	11 (4.8)	13 (5.5)
as severe, n (%)
Subjects with adverse events classified	22 (9.6)	29 (12.2)
as related, n (%)
Headache	17 (7.4)	28 (11.8)
Nasopharyngitis	19 (8.3)	22 (9.3)
Injection site reaction	6 (2.6)	7 (3.0)
Cough	4 (1.7)	5 (2.1)
Dizziness	4 (1.7)	4 (1.7)
Pharyngolaryngeal pain	3 (1.3)	4 (1.7)
Influenza like illness	4 (1.7)	2 (0.8)
Injection site pruritus	3 (1.3)	3 (1.3)
Myalgia	1 (0.4)	5 (2.1)
Urinary tract infection	4 (1.7)	2 (0.8)
Cystitis	2 (0.9)	3 (1.3)
Diarrhoea	2 (0.9)	3 (1.3)
Injection site haemorrhage	3 (1.3)	2 (0.8)
Arthralgia	0 (0.0)	4 (1.7)
Back pain	3 (1.3)	1 (0.4)
Injection site bruising	1 (0.4)	3 (1.3)
Pain in extremity	1 (0.4)	3 (1.3)
Toothache	1 (0.4)	3 (1.3)
Chest pain	3 (1.3)	0 (0.0)
Fatigue	0 (0.0)	3 (1.3)
Hypersensitivity	3 (1.3)	0 (0.0)

Safety results: Number (%) of subjects with serious adverse events (Total vaccinated cohort)

TABLE 22


Serious Adverse Events, n (%) [n considered by the
investigator to be related to study medication]

	HAB PFTF	HAB
	N = 229	N = 237

All SAEs
Subjects with any SAE(s), n(%) [n related]	6 (2.6) [0]	1 (0.4) [0]
Calculus urinary	1 (0.4) [0]	0 (0.0) [0]
Concussion	0 (0.0) [0]	1 (0.4) [0]
Coronary artery stenosis	1 (0.4) [0]	0 (0.0) [0]
Grand mal convulsion	1 (0.4) [0]	0 (0.0) [0]
Lower limb fracture	1 (0.4) [0]	0 (0.0) [0]
Pneumonia	1 (0.4) [0]	0 (0.0) [0]
Urinary tract infection	2 (0.9) [0]	0 (0.0) [0]
Fatal SAEs
Subjects with fatal SAE(s), n (%) [n related]	0 (0.0) [0]	0 (0.0) [0]

Conclusion:

At Month 7, 95.6% of the subjects in the HAB PFTF vaccine group and 97.7% of the subjects in the HAB vaccine group were seroprotected for anti-HBs and 99.5% of the subjects in both vaccine groups were seropositive for anti-HAV. Unsolicited symptoms were reported by 86 subjects in the HAB PFTF group and 82 subjects in the HAB group. Headache and Nasopharyngitis were the most frequently reported unsolicited symptoms, followed by injection site reaction. SAEs were reported by 6 subjects (2.6%) in the HAB PFRF group and 1 subject (0.4%) in the HAB group). No deaths were reported.

EXAMPLE 5

A Single-Blinded, Randomized, Multicentric Study to Compare the Immunogenicity of Thiomersal-Free 2-Dose ENGERIX™-B (20 μg) and 3-Dose Preservative-Free ENGERIX™-B (10 μg) Vaccines Administered Intramuscularly According to a 0, 6 Month and 0, 1, 6 Month Schedule, Respectively, and to Evaluate Safety and Reactogenicity of each Vaccine in Healthy Adolescent Volunteers (11 to 15 Years).
Rationale: The purpose of this study was to clinically evaluate a shorter vaccination schedule (2-doses 6 months apart) of the thiomersal-free (20 kg) formulation of hepatitis B vaccine (HBV) in adolescents aged 11 to 15 years, compared to the 3-dose schedule of the preservative-free pediatric formulation (10 μg) of HBV.
Study Design: Multinational, multicentre double-blind, randomised, parallel-group.
Centres: Four centres (2 in Belgium, 1 in Australia and 1 centre in the Ukraine).
Indication: Two-dose vaccination against hepatitis B in healthy adolescents aged 11 to 15 years.
Treatment: Study groups were as follows:

- Group 1: subjects received 2 doses of thiomersal-free HBV (adult formulation), one at 0 and 6 months, respectively and placebo at 1 month.
- Group 2: subjects received 3 doses of preservative-free HBV (pediatric formulation), one at 0, 1 and 6 months, respectively.
  All vaccines were administered as an intramuscular (IM) injection in the deltoid region of the non-dominant arm.
  Objectives:

To demonstrate non-inferiority of the immune response induced by thiomersal-free HBV (20 μg HbsAg per dose) administered as a 2-dose vaccination schedule compared to preservative-free HBV (10 μg HbsAg per dose) administered as a 3-dose vaccination schedule, one month after the full vaccination course (Month 7).
Primary Outcome/Efficacy Variables:
Anti-hepatitis B surface antigen (anti-HBs) seroprotection (SP) rates (defined as the percentage of subjects with anti-HBs antibody concentrations ≧10 mlU/ml) at Month 7.
Secondary Outcome/Efficacy Variable(s):

- SP rates for anti-HBs antibodies at Months 1, 2 and 6 for all subjects.
- Seropositivity (S+) rates for anti-HBs antibodies (percentage of subjects with anti-HBs concentration ≧3.3 mlU/ml) at Months 1, 2, 6 and 7 for all subjects.
- Geometric mean concentrations (GMCs) calculated for S+ subjects at Months 1, 2, 6 and 7.
- Occurrence, intensity of solicited local signs and symptoms during the 4-day (Days 0-3) follow-up period after each vaccination and overall.
- Occurrence, intensity and relationship to vaccination of general signs and symptoms during the 4-day (Days 0-3) follow-up period after each vaccination and overall.
- Occurrence, intensity and relationship to vaccination of unsolicited local and general signs and symptoms (adverse events [AEs]) during the 31-day (Days 0-30) follow-up period) after each vaccination and overall.
- Occurrence, intensity and relationship to vaccination of serious adverse events (SAEs) during the study period.
  Statistical Methods:

- The total vaccinated cohort included all subjects who received at least one vaccine dose
- The ATP cohort for immunogenicity included all vaccinated subjects who had post-vaccination immunogenicity results and who complied with the protocol, including the time schedule for vaccination and blood sample draw.
- The ATP cohort for safety included all vaccinated subjects who met the eligibility criteria for the analysis of safety.
- Analysis of immunogenicity:

The analysis of immunogenicity was performed on the ATP cohort for immunogenicity.
For each group, the S+ rate, SP rate and GMCs for anti-HBs antibodies and their 95% Confidence Interval (CI) was tabulated. The exact two-sided 90% confidence interval (CI) on the difference between the two treatment groups in anti-HBs SP rates was computed.
The non-inferiority criteria for the differences (group 1-group 2) on seroprotection rates were met, If the lower limit of the 90% CI for difference was greater than the pre-defined criteria for non-inferiority of −10%.
Analysis of Safety:
The analysis of safety was performed on the ATP cohort for safety and the total vaccinated cohort.
The incidence per group of solicited local and general symptoms reported during the 4-day (Days 0-3) follow-up period after vaccination was tabulated (with 95% CI) by type, grade and causality.
The incidence (with 95% CI) of subjects with at least one unsolicited AE, classified by WHO Preferred Term and reported during the 31-day (Days 0-30) follow-up period after vaccination, was tabulated per group.

Study Population: Healthy male or female adolescents aged between 11 and 15 years at the time of first vaccination, who were seronegative (S−) for HBsAg, anti-hepatitis B core antigen (anti-HBc) and anti-HBs antibodies. Written informed consent was obtained from all subjects and parents/guardians of the subjects prior to study entry. If subject was a female of childbearing potential, she had to be abstinent or had to use adequate contraceptive precautions for one month prior to enrolment and up to two months after last vaccine dose. The subject should have tested negative for pregnancy, if the test was deemed necessary by the investigator.

	TABLE 23


	Group 1	Group 2

Number of subjects
Planned, N	258	126
Randomised, N (Total vaccinated cohort)	258	126
Completed, n (%)	254 (98.4)	123 (97.6)
Total Number Subjects Withdrawn, n (%)	4 (1.6)	3 (2.4)
Withdrawn due to Adverse	0 (0.0)	0 (0.0)
Events (AEs), n (%)
Withdrawn due to Lack of Efficacy, n (%)	Not	Not applicable
	applicable
Withdrawn for Other Reasons, n (%)	4 (1.6)	3 (2.4)
Demographics
N (Total vaccinated cohort)	258	126
Females:Males	132:126	61:65
Mean Age, years (SD)	12.9 (1.23)	12.7 (1.32)
Caucasian, n (%)	250 (96.9)	123 (97.6)

Primary Efficacy Results:

Difference in anti-HBs SP between groups (pair-wise comparison) one month after the last vaccine dose (ATP cohort for immunogenicity).

	TABLE 24


	Inference

90% CI

Group	N	%	Group	N	%	Difference	Value	LL	UL

2	113	98.2	1	241	96.7	Group 1 − Group 2	−1.5	−6.4*	3.8

N = number of subjects with available results.
% = percentage of subjects who were seroprotected for anti-HBs antibodies.
90% CI = Exact 90% confidence interval
LL, UL: lower and upper limits.
*non-inferiority of the thiomersal-free HBV 2-dose vaccine demonstrated as LL is ≧ −10%

1. Secondary Outcome Variable(s):

S+ rates, SP rates and GMCs for Anti-HBs (calculated in S+ subjects only) at

Months

1, 2, 6 and 7 (ATP cohort for immunogenicity)

TABLE 25


S+	95% CI	SP	95% CI	GMC	95% CI

Group

Timing

N

n

%

LL

UL

n

%

LL

UL

(mIU/ml)

LL

UL

Group

1	PI(M1)	240	45	18.8	14.0	24.3	31	12.9	8.9	17.8	28.8	16.8	49.2
	PII(M2)	240	50	20.8	15.9	26.5	27	11.3	7.5	15.9	17.6	11.1	27.8
	PII(M6)	239	94	39.3	33.1	45.8	63	26.4	20.9	32.4	18.8	14.7	24.1
	PIII(M7)	241	235	97.5	94.7	99.1	233	96.7	93.6	98.6	2738.5	2071.4	3620.5
Group 2	PI(M1)	112	11	9.8	5.0	16.9	8	7.1	3.1	13.6	28.7	9.4	87.8
	PII(M2)	113	81	71.7	62.4	79.8	63	55.8	46.1	65.1	29.4	21.6	40.1
	PII(M6)	113	106	93.8	87.7	97.5	99	87.6	80.1	93.1	90.0	68.6	117.9
	PIII(M7)	113	111	98.2	93.8	99.8	111	98.2	93.8	99.8	7238.3	5247.3	9984.7

N = number of subjects tested.
n/% = number/percentage of subjects who were seropositive/seroprotected for anti-HBs antibodies.
PI (M1), PII (M2), PII (M6) = post-vaccination blood samples obtained at Months 1, 2 and 6, respectively, after the first dose.
PIII (M7) = post-vaccination blood samples obtained one month after the third dose.
95% CI = 95% confidence interval;
LL = Lower Limit;
UL = Upper Limit.

Secondary Outcome Variable(s):

Incidence of solicited local symptoms (total and Grade “3”) reported during the 4-day (days 0-3) follow-up period after vaccination (ATP cohort for safety).

	TABLE 26


	Group 1	Group 2

95% CI

	n	%	LL	UL	n	%	LL	UL

Dose 1

N = 253

N = 121

Pain	Any	121	47.8	41.5	54.2	44	36.4	27.8	45.6
	Grade “3”	6	2.4	0.9	5.1	3	2.5	0.5	7.1
Redness	Any	30	11.9	8.1	16.5	10	8.3	4.0	14.7
	Grade “3”	0	0.0	0.0	1.4	1	0.8	0.0	4.5
Swelling	Any	18	7.1	4.3	11.0	8	6.6	2.9	12.6
	Grade “3”	2	0.8	0.1	2.8	2	1.7	0.2	5.8

Dose 2

N 252 (Placebo)

N = 119

Pain	Any	42	16.7	12.3	21.9	38	31.9	23.7	41.1
	Grade “3”	3	1.2	0.2	3.4	2	1.7	0.2	5.9
Redness	Any	15	6.0	3.4	9.6	15	12.6	7.2	19.9
	Grade “3”	1	0.4	0.0	2.2	0	0.0	0.0	3.1
Swelling	Any	8	3.2	1.4	6.2	5	4.2	1.4	9.5
	Grade “3”	1	0.4	0.0	2.2	0	0.0	0.0	3.1

Dose 3

N = 250

N = 118

Pain	Any	106	42.4	36.2	48.8	35	29.7	21.6	38.8
	Grade “3”	4	1.6	0.4	4.0	1	0.8	0.0	4.6
Redness	Any	29	11.6	7.9	16.2	11	9.3	4.7	16.1
	Grade “3”	0	0.0	0.0	1.5	0	0.0	0.0	3.1
Swelling	Any	14	5.6	3.1	9.2	6	5.1	1.9	10.7
	Grade “3”	0	0.0	0.0	1.5	0	0.0	0.0	3.1

Across doses

N = 253

N = 121

Pain	Any	155	61.3	55.0	67.3	74	61.2	51.9	69.9
	Grade “3”	8	3.2	1.4	6.1	6	5.0	1.8	10.5
Redness	Any	50	19.8	15.0	25.2	28	23.1	16.0	31.7
	Grade “3”	0	0.0	0.0	1.4	1	0.8	0.0	4.5
Swelling	Any	27	10.7	7.2	15.1	15	12.4	7.1	19.6

N across doses = number of subjects with at least one documented dose.
n/% across doses = number/percentage of subjects with at least one symptom reported.
Grade 3 = spontaneously painful or redness/swelling with greatest surface diameter ≧50 mm and lasting for 24 hours.
95% CI = 95% confidence interval;
LL = Lower Limit;
UL = Upper Limit.

Secondary Outcome Variable(s):

Incidence of solicited general symptoms (total, Grade “3” and related) reported during the 4-day (days 0-3) follow-up period after vaccination (ATP cohort for safety):

	TABLE 27


	Group 1	Group 2

95% CI

	n	%	LL	UL	n	%	LL	UL

Dose 1

N = 253

N = 121

Fatigue	Any	50	19.8	15.0	25.2	26	21.5	14.5	29.9
	Grade “3”	1	0.4	0.0	2.2	1	0.8	0.0	4.5
	Related	29	11.5	7.8	16.0	16	13.2	7.8	20.6
Gastrointestinal	Any	26	10.3	6.8	14.7	4	3.3	0.9	8.2
	Grade “3”	3	1.2	0.2	3.4	0	0.0	0.0	3.0
	Related	11	4.3	2.2	7.6	3	2.5	0.5	7.1
Headache	Any	57	22.5	17.5	28.2	29	24.0	16.7	32.6
	Grade “3”	0	0.0	0.0	1.4	1	0.8	0.0	4.5
	Related	33	13.0	9.2	17.8	15	12.4	7.1	19.6
Fever (axillary)	≧37.5° C.	4	1.6	0.4	4.0	2	1.7	0.2	5.8
	Grade “3”	0	0.0	0.0	1.4	0	0.0	0.0	3.0
	Related	3	1.2	0.2	3.4	2	1.7	0.2	5.8

Dose 2

N = 252 (Placebo)

N = 119

Fatigue	Any	37	14.7	10.6	19.7	18	15.1	9.2	22.8
	Grade “3”	2	0.8	0.1	2.8	0	0.0	0.0	3.1
	Related	23	9.1	5.9	13.4	13	10.9	5.9	18.0
Gastrointestinal	Any	20	7.9	4.9	12.0	7	5.9	2.4	11.7
	Grade “3”	0	0.0	0.0	1.5	0	0.0	0.0	3.1
	Related	7	2.8	1.1	5.6	5	4.2	1.4	9.5
Headache	Any	40	15.9	11.6	21.0	21	17.6	11.3	25.7
	Grade “3”	0	0.0	0.0	1.5	0	0.0	0.0	3.1
	Related	24	9.5	6.2	13.8	11	9.2	4.7	15.9
Fever (axillary)	≧37.5° C.	5	2.0	0.6	4.6	5	4.2	1.4	9.5
	Grade “3”	1	0.4	0.0	2.2	0	0.0	0.0	3.1
	Related	5	2.0	0.6	4.6	4	3.4	0.9	8.4

Dose 3

N = 250

N = 118

Fatigue	Any	49	19.6	14.9	25.1	20	16.9	10.7	25.0
	Grade “3”	3	1.2	0.2	3.5	2	1.7	0.2	6.0
	Related	30	12.0	8.2	16.7	8	6.8	3.0	12.9
Gastrointestinal	Any	17	6.8	4.0	10.7	14	11.9	6.6	19.1
	Grade “3”	3	1.2	0.2	3.5	2	1.7	0.2	6.0
	Related	6	2.4	0.9	5.2	6	5.1	1.9	10.7
Headache	Any	36	14.4	10.3	19.4	20	16.9	10.7	25.0
	Grade “3”	1	0.4	0.0	2.2	1	0.8	0.0	4.6
	Related	22	8.8	5.6	13.0	12	10.2	5.4	17.1
Fever (axillary)	≧37.5° C.	13	5.2	2.8	8.7	9	7.6	3.5	14.0
	Grade “3”	1	0.4	0.0	2.2	0	0.0	0.0	3.1
	Related	7	2.8	1.1	5.7	5	4.2	1.4	9.6

Across doses

N = 253

N = 121

Fatigue	Any	77	30.4	24.8	36.5	46	38.0	29.3	47.3
	Grade “3”	4	1.6	0.4	4.0	3	2.5	0.5	7.1
	Related	51	20.2	15.4	25.6	30	24.8	17.4	33.5
Gastrointestinal	Any	36	14.2	10.2	19.2	21	17.4	11.1	25.3
	Grade “3”	6	2.4	0.9	5.1	2	1.7	0.2	5.8
	Related	17	6.7	4.0	10.5	14	11.6	6.5	18.7
Headache	Any	78	30.8	25.2	36.9	46	38.0	29.3	47.3
	Grade “3”	1	0.4	0.0	2.2	2	1.7	0.2	5.8
	Related	49	19.4	14.7	24.8	30	24.8	17.4	33.5
Fever (axillary)	≧37.5° C.	17	6.7	4.0	10.5	14	11.6	6.5	18.7

N across doses = number of subjects with at least one documented dose.
n/% across doses = number/percentage of subjects with at least one symptom reported.
Any = total incidence of symptom irrespective of intensity or relationship.
Grade “3” = Prevented normal everyday activities.
Grade “3” fever = axillary temperature >38.5° C.
Related = symptoms considered by investigator to have causal relation to the study vaccine.
95% CI = 95% confidence interval;
LL = Lower Limit;
UL = Upper Limit.

Safety Results:

Number (%) of subjects with unsolicited adverse events (Total vaccinated cohort)

TABLE 28


Most frequent adverse events -
On Therapy (occurring within	Group 1	Group 2
days 0-30 following vaccination)	N = 257*	N = 125*

Subjects with any AE(s), n(%)	112 (43.6)	54 (43.2)
Upper respiratory tract infection	26 (10.1)	22 (17.6)
Headache	28 (10.9)	10 (8.0)
Pharyngitis	15 (5.8)	5 (4.0)
Infection viral	10 (3.9)	1 (0.8)
Rhinitis	8 (3.1)	3 (2.4)
Injury	5 (1.9)	5 (4.0)
Influenza-like symptoms	8 (3.1)	1 (0.8)
Abdominal pain	7 (2.7)	1 (0.8)
Hematoma	8 (3.1)	0 (0.0)
Injection site reaction	8 (3.1)	0 (0.0)
Dysmenorrhea	5 (1.9)	2 (1.6)
Back pain	4 (1.6)	2 (1.6)
Otitis media	4 (1.6)	2 (1.6)
Arthralgia	2 (0.8)	3 (2.4)
Conjunctivitis	1 (0.4)	4 (3.2)
Vomiting	3 (1.2)	2 (1.6)
Coughing	1 (0.4)	3 (2.4)
Herpes simplex	1 (0.4)	3 (2.4)
Bronchitis	1 (0.4)	2 (1.6)
Pain	1 (0.4)	2 (1.6)
Acne	0 (0.0)	2 (1.6)
Epistaxis	0 (0.0)	2 (1.6)
Tooth disorder	0 (0.0)	2 (1.6)

*No information regarding AEs was available for 1 subject in each group.

Safety Results: Number (%) of subjects with serious adverse events (SAEs) (Total vaccinated cohort)

	TABLE 29


	Group 1	Group 2
	N = 257*	N = 125*

All SAEs
Subjects with any SAE(s), n(%) [n related]	4 (1.6) [0]	1 (0.0) [0]
Injury	1 (0.4) [0]	1 (0.8) [0]
Arthritis	1 (0.4) [0]	0 (0.0) [0]
Ileitis	1 (0.4) [0]	0 (0.0) [0]
Infection bacterial	1 (0.4) [0]	0 (0.0) [0]
Fatal SAEs:
Subjects with fatal SAE(s), n (%) [n related]	0 (0.0) [0]	0 (0.0) [0]

*No information regarding AEs was available for 1 subject in each group.

Conclusion: In this study, the immune response elicited by 2-dose HBV (20 kg HBsAg) was non-inferior to that elicited by 3-dose HBV (10 μg HBsAg), in terms of anti-HBs seroprotection rate in adolescents into 15 years of age. Hence the primary objective was met. At least 96.7% of seroprotection rates was observed with both schedules. Unsolicited symptoms were reported in 43.6% of subjects having received 2 doses of 20 μg HBsAg and 43.2% of subjects having received the 3 doses of 10 μg HBsAg. Five serious adverse events were reported (4 in the group having received 2 doses of 20 μg HBsAg and 1 in the other group. None of these serious adverse events were considered related by the investigator, none had a fatal outcome.

EXAMPLE 6

A Double-Blind, Randomized, Controlled, Monocenter Study to Evaluate the Immunocienicity, Reactogenicity and Safety of Preservative Free Engerix™-B Comparing Aged Lot (>24 Months) and as Reference, New Lot (<12 Months) when Administered Intramuscularly According to a 0, 1, 6 Month Schedule in Healthy Volunteers (18 to 50 Years).
Rationale: This study was undertaken to confirm the stability, in a clinical setting, of one aged lot (>24 months) of preservative-hepatitis-B vaccine (HBV) to a newly produced lot (<12 months) of the vaccine. To achieve this, an aged lot (>24 months) of the preservative free HBV was compared, with a newly produced lot (<12 months), for immunogenicity and safety.
Study Design: Multinational, monocentric, double-blind, randomized, parallel-group.
Centres: One centre in the Czech Republic.
Indication: Three-dose vaccination against hepatitis B in healthy adults aged 18 to 50 years.
Treatment: Study groups were as follows:
Group 1: subjects received preservative-free HBV aged lot >24 months.
Group 2: subjects received preservative-free HBV new lot <12 months.
All vaccines were to be administered at Months 0, 1 and 6 as an intramuscular (IM) injection in the deltoid region of the non-dominant arm.
Objectives: To demonstrate non-inferiority of immunogenicity for aged lot (>24 months) preservative-free HBV compared to new lot (<12 months) preservative-free HBV one month after the full vaccination course (Month 7).
Primary Outcome/Efficacy Variables:
Anti-hepatitis B surface antigen (anti-HBs) concentrations at Month 7 and anti-HBs seroprotection (SP) rates (defined as the proportion of subjects with anti-HBs antibody concentrations >10 mIU/ml) at Month 7.
Secondary Outcome/Efficacy Variable(s):

- At Month 1, 2, 6 and 7: anti-HBs concentrations for all subjects.
- SP rates at Month 1, 2 and 6 for all subjects.
- Seropositivity (S+) rates (proportion of subjects with anti-HBs concentration ≧3.3 mlU/ml, the assay cut-off) at Month 1, 2, 6 and 7 for all subjects.
- Geometric mean concentrations (GMCs) calculated at Month 1, 2, 6 and 7 for all subjects.
- Occurrence and intensity of solicited local signs and symptoms during the four-day follow-up period (day 0-day 3)
- Occurrence, intensity and relationship to vaccination of solicited general signs and symptoms during the four-day (day 0-day 3) follow-up period after each vaccination and overall.
- Occurrence, intensity and relationship to vaccination of unsolicited local and general signs and symptoms within 31 days (day 0-day 30) after each vaccination and overall.
- Occurrence, intensity and relationship to vaccination of serious adverse events (SAEs) during the study period, up to and including 31 days (day 0-day 30) post-vaccination.
  Statistical Methods:

The analyses were conducted on the Total Vaccinated cohort and the According-To-Protocol (ATP) cohort.
The Total Vaccinated cohort included all subjects who received at least one vaccine dose
The ATP cohort included any subject corresponding to the criteria defined in the protocol.
Analysis of Immunogenicity:
The analysis of immunogenicity was performed on the ATP cohort for immunogenicity.
For each group, the S+ rate, SP rate and GMCs for anti-HBs antibodies and their 95% Confidence Interval (CI) were tabulated. The asymptotic two-sided 95% CI on the difference in anti-HBs SP rate at Month 7 (aged lot minus new lot) was computed. If the lower limit of the CI for this treatment difference was greater than the pre-defined non-inferiority limit (−10%), this indicates that the aged lot is non-inferior to the new lot in terms of immune response as measured by seroprotection rate.
Analysis of Safety:
The analysis of safety was performed on the ATP cohort for safety.
For each solicited symptom, the percentage of subjects with the symptom and its exact 95% CI was summarized by vaccine group, by dose and across doses.
The percentage of subjects reporting unsolicited symptoms within 31 days (day 0-day 30) following vaccination was summarized by vaccine group according to the WHO preferred term.

Study Population: Healthy male or female subjects between, and including, 18 and 50 years of age, who were seronegative (S−) for hepatitis B surface antigen (HbsAg), anti-hepatitis B core antigen (anti-HBc) and anti-HBs antibodies at the time of first vaccination. Written informed consent was obtained from all subjects prior to study entry. If subject was a female of childbearing potential, she was to be abstinent or used adequate contraceptive precautions for one month prior to enrolment and up to two months after last vaccine dose.

	TABLE 30


	Group 1	Group 2

Number of subjects
(Total vaccinated cohort)
Planned, N	148	148
Randomised, N	148	148
Completed, n (%)	148 (100.0)	148 (100.0)
Total Number Subjects Withdrawn, n (%)	0 (0.0)	0 (0.0)
Withdrawn due to Adverse Events	0 (0.0)	0 (0.0)
(AEs), n (%)
Withdrawn due to Lack of Efficacy, n (%)	Not	Not applicable
	applicable
Withdrawn for Other Reasons, n (%)	0 (0.0)	0 (0.0)
Demographics
N (Total Vaccinated Cohort)	148	148
Females:Males	80:68	80:68
Mean Age, years (SD)	33.7 (10.48)	32.3 (10.01)
White/Caucasian, n (%)	148 (100)	148 (100)

Primary Efficacy Results:

Difference in anti-HBs seroprotection rates between groups (pair-wise comparison) one month after the last vaccine dose (ATP cohort for immunogenicity).

	TABLE 31


	Inference

Vaue

95% CI

Group	N	%	Group	N	%	Difference	%	LL	UL

1	148	97.3	2	144	99.3	Grp 2 − Grp 1	2.0	−0.9	5.0
2	144	99.3	1	148	97.3	Grp 1 − Grp 2	−2.0	−5.0*	0.9

N = number of subjects with available results.
% = percentage of subjects who were seroprotected for anti-HBs antibodies (anti-HBs concentration ≧10 mIU/ml at Month 7).
95% CI = Exact 95% confidence interval, lower and upper limits.
*One month after the last vaccine dose (month 7), the lower limit of the asymptotic 95% CI for the difference (aged lot minus new lot) was greater than −10%, demonstrating non-inferiority of the aged lot compared to new lot

Secondary Outcome Variable(s):

S+ rates, SP rates and GMCs for anti-HBs (calculated in S+subjects only) at Month 7 (M7) as well as

Months

1, 2 and 6 (M1, 2 and 6). (ATP cohort for immunogenicity)

TABLE 32


S+	SP	GMC

95% CI

Group

Timing

N

n

%

LL

UL

n

%

LL

UL

(mIU/ml)

LL

UL

Group

1	PI(M1)	148	14	9.5	5.3	15.4	10	6.8	3.3	12.1	16.1	9.0	28.9
	PII(M2)	148	91	61.5	53.1	69.4	68	45.9	37.7	54.3	27.6	20.8	36.7
	PII(M6)	148	136	91.9	86.3	95.7	128	86.5	79.9	91.5	100.3	79.8	126.1
	PIII(M7)	148	144	97.3	93.2	99.3	144	97.3	93.2	99.3	4187.5	3093.9	5667.7
Group 2	PI(M1)	145	20	13.8	8.6	20.5	13	9.0	4.9	14.8	26.8	12.2	59.1
	PII(M2)	145	97	66.9	58.6	74.5	79	54.5	46.0	62.8	40.5	28.4	57.7
	PII(M6)	144	136	94.4	89.3	97.6	132	91.7	85.9	95.6	125.5	101.0	155.9
	PIII(M7)	144	143	99.3	96.2	100.0	143	99.3	96.2	100.0	4661.4	3373.1	6441.8

N = number of subjects with available results.
n/% = number/percentage of subjects who were seropositive/seroprotected (S+/SP) for anti-HBs antibodies.
PX (MY) = blood sample after the X th dose, obtained Y month (s) after the first dose.
LL = Lower Limit;
UL = Upper Limit.

Secondary Outcome Variable(s):

Incidence of solicited local symptoms (total and Grade “3”) reported during the four-day (day 0-day 3) follow-up period after vaccination according to the per-subject analysis (ATP cohort for safety).

	TABLE 33


	Group 1	Group 2

95% CI

	n	%	LL	UL	n	%	LL	UL

Dose 1 (Month 1)

N = 148

N = 146

Pain	Any	80	54.1	45.7	62.3	69	47.3	38.9	55.7
	Grade “3”	2	1.4	0.2	4.8	1	0.7	0.0	3.8
Redness	Any	20	13.5	8.5	20.1	15	10.3	5.9	16.4
	Grade “3”	0	0.0	0.0	2.5	0	0.0	0.0	2.5
Swelling	Any	7	4.7	1.9	9.5	6	4.1	1.5	8.7
	Grade “3”	0	0.0	0.0	2.5	1	0.7	0.0	3.8

Dose 2 (Month 2)

N = 148

N = 146

Pain	Any	64	43.2	35.1	51.6	57	39.0	31.1	47.5
	Grade “3”	2	1.4	0.2	4.8	2	1.4	0.2	4.9
Redness	Any	24	16.2	10.7	23.2	13	8.9	4.8	14.7
	Grade “3”	0	0.0	0.0	2.5	0	0.0	0.0	2.5
Swelling	Any	6	4.1	1.5	8.6	7	4.8	1.9	9.6
	Grade “3”	0	0.0	0.0	2.5	0	0.0	0.0	2.5

Dose 3 (Month 3)

N = 148

N = 145

Pain	Any	63	42.6	34.5	51.0	52	35.9	28.1	44.2
	Grade “3”	1	0.7	0.0	3.7	1	0.7	0.0	3.8
Redness	Any	19	12.8	7.9	19.3	22	15.2	9.8	22.1
	Grade “3”	0	0.0	0.0	2.5	0	0.0	0.0	2.5
Swelling	Any	7	4.7	1.9	9.5	8	5.5	2.4	10.6
	Grade “3”	0	0.0	0.0	2.5	0	0.0	0.0	2.5

Across doses

N = 148

N = 146

Pain	Any	105	70.9	62.9	78.1	92	63.0	54.6	70.8
	Grade “3”	5	3.4	1.1	7.7	4	2.7	0.8	6.9
Redness	Any	37	25.0	18.3	32.8	32	21.9	15.5	29.5
	Grade “3”	0	0.0	0.0	2.5	0	0.0	0.0	2.5
Swelling	Any	16	10.8	6.3	17.0	15	10.3	5.9	16.4
	Grade “3”	0	0.0	0.0	2.5	1	0.7	0.0	3.8

N Per-dose = Number of documented doses.
N across doses = Number of subjects with at least one documented dose.
n/% = number/percentage of subjects with at least one report of specific solicited local symptom.
Redness/Swelling Grade = longest diameter ≧50 mm.
Pain Grade = spontaneously painful.
95% CI, LL, UL = 95% Confidence Interval, lower limit, upper limit.

Secondary Outcome Variable(s):

Overall incidence of solicited general symptoms (total, Grade “3” and related) reported during the four-day (day 0-day 3) follow-up period after vaccination according to the per-subject analysis (ATP cohort for safety):

	TABLE 34


	Group 1	Group 2

95% CI

	n	%	LL	UL	n	%	LL	UL

Dose 1 (Month 1)

N = 148

N = 146

Fatigue	Any	26	17.6	11.8	24.7	29	19.9	13.7	27.3
	Grade “3”	0	0.0	0.0	2.5	1	0.7	0.0	3.8
	Related	22	14.9	9.6	21.6	25	17.1	11.4	24.2
Gastrointestinal	Any	9	6.1	2.8	11.2	9	6.2	2.9	11.4
	Grade “3”	0	0.0	0.0	2.5	0	0.0	0.0	2.5
	Related	2	1.4	0.2	4.8	6	4.1	1.5	8.7
Headache	Any	28	18.9	13.0	26.2	19	13.0	8.0	19.6
	Grade “3”	0	0.0	0.0	2.5	0	0.0	0.0	2.5
	Related	19	12.8	7.9	19.3	15	10.3	5.9	16.4
Fever (axillary)	≧37.5° C.	1	0.7	0.0	3.7	1	0.7	0.0	3.8
	Grade “3”	0	0.0	0.0	2.5	0	0.0	0.0	2.5
	Related	0	0.0	0.0	2.5	1	0.7	0.0	3.8

Dose 2 (Month 2)

N = 148

N = 146

Fatigue	Any	15	10.1	5.8	16.2	17	11.6	6.9	18.0
	Grade “3”	0	0.0	0.0	2.5	1	0.7	0.0	3.8
	Related	14	9.5	5.3	15.4	17	11.6	6.9	18.0
Gastrointestinal	Any	1	0.7	0.0	3.7	5	3.4	1.1	7.8
	Grade “3”	0	0.0	0.0	2.5	0	0.0	0.0	2.5
	Related	1	0.7	0.0	3.7	5	3.4	1.1	7.8
Headache	Any	17	11.5	6.8	17.8	9	6.2	2.9	11.4
	Grade “3”	0	0.0	0.0	2.5	0	0.0	0.0	2.5
	Related	13	8.8	4.8	14.6	9	6.2	2.9	11.4
Fever (axillary)	≧37.5° C.	0	0.0	0.0	2.5	0	0.0	0.0	2.5
	Grade “3”	0	0.0	0.0	2.5	0	0.0	0.0	2.5
	Related	0	0.0	0.0	2.5	0	0.0	0.0	2.5

Dose 3 (Month 3)

N = 148

N = 145

Fatigue	Any	25	16.9	11.2	23.9	24	16.6	10.9	23.6
	Grade “3”	0	0.0	0.0	2.5	1	0.7	0.0	3.8
	Related	20	13.5	8.5	20.1	21	14.5	9.2	21.3
Gastrointestinal	Any	5	3.4	1.1	7.7	5	3.4	1.1	7.9
	Grade “3”	0	0.0	0.0	2.5	1	0.7	0.0	3.8
	Related	3	2.0	0.4	5.8	3	2.1	0.4	5.9
Headache	Any	18	12.2	7.4	18.5	16	11.0	6.4	17.3
	Grade “3”	0	0.0	0.0	2.5	1	0.7	0.0	3.8
	Related	14	9.5	5.3	15.4	13	9.0	4.9	14.8
Fever (axillary)	≧37.5° C.	1	0.7	0.0	3.7	0	0.0	0.0	2.5
	Grade “3”	0	0.0	0.0	2.5	0	0.0	0.0	2.5
	Related	1	0.7	0.0	3.7	0	0.0	0.0	2.5

Across doses

N = 148

N = 145

Fatigue	Any	47	31.8	24.4	39.9	47	32.2	24.7	40.4
	Grade “3”	0	0.0	0.0	2.5	3	2.1	0.4	5.9
	Related	39	26.4	19.5	34.2	43	29.5	22.2	37.6
Gastrointestinal	Any	12	8.1	4.3	13.7	16	11.0	6.4	17.2
	Grade “3”	0	0.0	0.0	2.5	1	0.7	0.0	3.8
	Related	6	4.1	1.5	8.6	12	8.2	4.3	13.9
Headache	Any	44	29.7	22.5	37.8	35	24.0	17.3	31.7
	Grade “3”	0	0.0	0.0	2.5	1	0.7	0.0	3.8
	Related	33	22.3	15.9	29.9	30	20.5	14.3	28.0
Fever	≧37.5° C.	2	1.4	0.2	4.8	1	0.7	0.0	3.8
(oral/axillary)	Grade “3”	0	0.0	0.0	2.5	0	0.0	0.0	2.5
	Related	1	0.7	0.0	3.7	1	0.7	0.0	3.8

N = number of subjects reporting at least one general symptom.
n/% = number/percentage of subjects reporting at least one specific solicited general symptom.
Total = total incidence of symptom irrespective of intensity or relationship.
Grade “3” for all symptoms except fever = one particular symptom that prevented normal everyday activities.
Grade “3” fever (oral/axillary route) = temperature >38.5° C.
Related = symptoms considered by investigator to have causal relation to the study vaccine.
95% CI: 95% Confidence Interval

Safety Results:

Number (%) of subjects with unsolicited adverse events (AEs) (Total cohort):

	TABLE 35


	Group 1	Group 2
	N = 148	N = 148

Most frequent adverse events -
On Therapy (occurring within
Days 0-30 following vaccination)
Subjects with any AE(s), n(%)	32 (21.6)	42 (28.4)
Upper respiratory tract infection	7 (4.7)	9 (6.1)
Toothache	4 (2.7)	1 (0.7)
Injury	3 (2.0)	4 (2.7)
Bronchitis	3 (2.0)	1 (0.7)
Diarrhoea	3 (2.0)	1 (0.7)
Influenza-like symptoms	3 (2.0)	1 (0.7)
Headache	2 (1.4)	4 (2.7)
Injections site reaction	2 (1.4)	3 (2.0)
Furunculosis	2 (1.4)	0 (0.0)
Pharyngitis	1 (0.7)	7 (4.7)
Arthralgia	1 (0.7)	1 (0.7)
Paresthesia	1 (0.7)	1 (0.7)
Coughing	1 (0.7)	0 (0.0)
Gastroenteritis	1 (0.7)	0 (0.0)
Menorrhagia	1 (0.7)	0 (0.0)
Myalgia	1 (0.7)	0 (0.0)
Pain	1 (0.7)	0 (0.0)
Pigmentation abnormal	1 (0.7)	0 (0.0)
Renal pain	1 (0.7)	0 (0.0)
Somnolence	1 (0.7)	0 (0.0)
Tracheitis	1 (0.7)	0 (0.0)
Urticaria	1 (0.7)	0 (0.0)
Vertigo	1 (0.7)	0 (0.0)
Back pain	0 (0.0)	4 (2.7)
Cystitis	0 (0.0)	2 (1.4)
Asthma	0 (0.0)	1 (0.7)
Balanoposthitis	0 (0.0)	1 (0.7)
Bursitis	0 (0.0)	1 (0.7)
Cholecystitis	0 (0.0)	1 (0.7)
Flatulence	0 (0.0)	1 (0.7)
Hypertension	0 (0.0)	1 (0.7)
Infection bacterial	0 (0.0)	1 (0.7)
Infection viral	0 (0.0)	1 (0.7)
Laryngitis	0 (0.0)	1 (0.7)
Lymphadenopathy	0 (0.0)	1 (0.7)
Otitis media	0 (0.0)	1 (0.7)
Prostatic disorder	0 (0.0)	1 (0.7)
Testis disorder	0 (0.0)	1 (0.7)
Tooth disorder	0 (0.0)	1 (0.7)
Vaginitis	0 (0.0)	1 (0.7)

Safety Results: Number (%) of Serious Adverse

Events (SAEs) (Total vaccinated cohort)

All SAEs
Subjects with any SAE(s), n (%) [n related]	0 (0.0) [0]	0 (0.0) [0]
Fatal SAEs:
Subjects with fatal SAE(s), n (%) [n related]	0 (0.0) [0]	0 (0.0) [0]

Conclusion: The primary objective to demonstrate the non-inferiority in terms of seroprotection rates, of the aged lot (>24 months) versus the new lot (<12 months) preservative-free HBV, was achieved. Both the aged lot and new lot preservative-free HBV elicited a high immune response with high SP rates (97.3% and 99.3%, respectively) and high GMCs (4187.5 mIU/ml and 4661.4 mIU/ml, respectively) at one month after the full vaccination course. Both the aged lot and new lot preservative-free HBV were safe and well tolerated. No serious adverse events were reported.

EXAMPLE 7

A Double-Blind, Randomized, Comparative, Multicenter Study of the Immunocienicity and Safety of Three Doses of Thimerosal-Free Hepatitis B Vaccine (10 μg/0.5 mL) Compared to the US-Licensed Preservative-Free Hepatitis B Vaccine (Engerix-B®, 10 μg/0.5 mL) when Administered Intramuscularly on a 0, 1, 6-Month Schedule to Healthy Infants in their First Two Weeks of Life.
Rationale: To compare the immunogenicity and safety of the thimerosal-free (TF) hepatitis B vaccine (HBV) formulation and the licensed preservative-free (PF) HBV formulation, when given to infants starting in their first two weeks of life.
Study Design: Double-blind, randomized (1:1 ratio), multicenter, comparative study with two groups.
Centers: 21 study centers throughout the US.
Indication: Administration to healthy infants aged 0-2 weeks at the time of the first vaccination.
Treatment:
The study groups were as follows:
PF group: received HBV PF vaccine at Month 0, 1, 6.
TF group: received HBV TF vaccine at Month 0, 1, 6.
Both vaccines were administered intramuscularly in the upper left thigh.
Objectives:
To demonstrate that the immunogenicity of HBV TF vaccine is not inferior to that of HBV PF vaccine, with respect to anti-hepatitis B surface antigen (anti-HBs) seroprotection rates one month after the third dose (Month 7).
Primary Outcome/Efficacy Variable:

- Anti-HBs seroprotection rates (percentage of subjects with anti-HBs concentration >10 mlU/ml) one month after the third dose (Month 7).
  Secondary Outcome/Efficacy Variable(s):
- Anti-HBs Geometric Mean Concentrations (GMCs) one month after the third dose (Month 7).
- Anti-HBs GMCs one month after the second dose (Month 2).
- Anti-HBs seroprotection rates one month after the second dose (Month 2).
- Incidence and intensity of solicited local adverse events occurring during the four-day (day 0-day 3) follow-up period after each vaccination.
- Incidence, intensity and causal relationship to vaccination of solicited general adverse events occurring during the four-day (day 0-day 3) follow-up period after each vaccination.
- Incidence, intensity and causal relationship to vaccination of unsolicited adverse events occurring during the 31-day (day 0-day 30) follow-up period after each vaccination.
- Occurrence, nature and causal relationship to vaccination of serious adverse events occurring during the entire study period (through 6 months after the last dose of vaccine).
  Statistical Methods:

The analyses were performed on the Total vaccinated cohort and the According-To-Protocol (ATP) cohort for immunogenicity.

- The Total vaccinated cohort included all subjects with study vaccine administered.
- The ATP cohort for immunogenicity included all vaccinated subjects who had post-vaccination immunogenicity results, who met all eligibility criteria and who complied with the procedures defined in the protocol.
  Analysis of Immunogenicity:

The analysis of immunogenicity was performed on the ATP cohort for immunogenicity. Geometric Mean Concentration (GMC) and seroprotection/seropositivity rates were calculated with their 95% CI for each antibody measured at each blood sampling time point. GMCs were calculated with only the subjects above the cut-off value for seropositivity (≧3.3 mlU/ml) at each time point.
At Month 7, the two vaccine groups were compared with respect to the difference in seroprotection rates (TF minus PF). The two-sided standardized asymptotic 90% CI for the difference in anti-HBs seroprotection rates between groups (TF minus PF) at Month 7 was computed. If the lower limit of the CI for this treatment difference was greater than the pre-defined non-inferiority limit of −5%, the TF vaccine was considered to be non-inferior to the PF vaccine and the primary endpoint would be met.
Analysis of Safety
The analysis of safety was performed on the Total vaccinated cohort.
For each solicited symptom, the percentage of subjects with the symptom and its exact 95% CI was summarized by vaccine group, by dose and across doses. The percentage of subjects reporting unsolicited symptoms within 31 days (day 0-30) following vaccination was summarized by vaccine group according to the MedDRA preferred term.
The percentage of subjects who reported SAEs during the entire study period (active phase and 6-months extended safety follow-up) were tabulated.

Study Population: Healthy male or female infants two weeks of age at the time of the first vaccination, who were born after a normal gestation period (36-42 weeks), to mothers who were HBsAg negative, were included in this study. Subjects who received prior vaccination against hepatitis B, or had known exposure to hepatitis B since birth/history of hepatitis B infection were excluded from the study.

	TABLE 36


	PF group	TF group

Number of Subjects:
Planned, N	294	294
Randomised, N (Total vaccinated cohort)	293	294
Completed, n (%)	254 (86.7)	257 (87.4)
Total Number Subjects Withdrawn, n (%)	39 (13.3)	37 (12.6)
Withdrawn due to Adverse Events n (%)	0 (0.0)	0 (0.0)
Withdrawn due to Lack of Efficacy n (%)	Not	Not applicable
	applicable
Withdrawn for other reasons n (%)	39 (13.3)	37 (12.6)
Demographics
N (Total vaccinated cohort)	293	294
Females:Males	144:149	136:158
Mean Age, days (SD)	6.5 (5.20)	6.7 (5.24)
Caucasian, n (%)	199 (67.9)	187 (63.6)

Primary Efficacy Results:

Difference between groups in anti-HBs seroprotection rate at Month 7 (ATP cohort for immunogenicity)

	TABLE 37


	Difference
	(TF minus PF)

90% CI

Group	N	%	Group	N	%	%	LL	UL

PF	213	98.1	TF	227	96.9	−1.21	−3.95*	1.41

N = number of subjects with available results
% = percentage of subjects with Anti-HBs concentration ≧10 mIU/ml
90% CI = 90% standardized asymptotic confidence interval;
LL = lower limit,
UL = upper limit
*Non-inferiority criterion met

Secondary Outcome Variable(s):

Seropositivity (S+) rates, seroprotection (SP) rates and GMCs for anti-HBs (GMCs calculated on seropositive subjects) (ATP cohort for immunogenicity)

TABLE 38


S+ (≧3.3 mlU/ml)	SP (≧10 mlU/ml)	GMC

95% CI

Group

Timing

N

n

%

LL

UL

n

%

LL

UL

mlU/ml

LL

UL

PF	Pre	208	76	36.5	30.0	43.5	65	31.3	25.0	38.0	113.9	71.7	180.8
	PII(M2)	219	151	68.9	62.4	75.0	131	59.8	53.0	66.4	38.4	31.8	46.4
	PIII(M7)	213	210	98.6	95.9	99.7	209	98.1	95.3	99.5	1294.4	1069.3	1566.8
TF	Pre	213	66	31.0	24.8	37.7	57	26.8	20.9	33.2	200.4	116.7	343.9
	PII(M2)	225	168	74.7	68.5	80.2	129	57.3	50.6	63.9	36.2	29.0	45.1
	PIII(M7)	227	222	97.8	94.9	99.3	220	96.9	93.7	98.8	1427.6	1146.4	1777.7

N = number of subjects with available results.
GMC = geometric mean antibody concentration calculated on subjects with anti-HBs
concentrations ≧3.3 mlU/mL
n/% = number/percentage of subjects with anti-HBs concentrations above the specified cut-off
95% CI = 95% confidence interval;
LL = lower limit,
UL = upper limit
Pre = before vaccination
Px(My) = Blood sample obtained after dose x at month y after the first dose

Secondary Outcome Variable(s):

Incidence and intensity of solicited local symptoms at the HBV injection site reported during the four-day (day 0-day 3) post-vaccination period following each dose and across doses (Total vaccinated cohort)

	TABLE 39


	PF	TF

Group

95% CI

Symptom	Intensity	N	n	%	LL	UL	N	n	%	LL	UL

Dose 1

Pain	Any	280	49	17.5	13.2	22.5	278	55	19.8	15.3	25.0
	Grade 3	280	1	0.4	0.0	2.0	278	2	0.7	0.1	2.6
Redness	Any	280	49	17.5	13.2	22.5	278	46	16.5	12.4	21.4
	Grade 3	280	2	0.7	0.1	2.6	278	1	0.4	0.0	2.0
Swelling	Any	280	26	9.3	6.2	13.3	278	21	7.6	4.7	11.3
	Grade 3	280	1	0.4	0.0	2.0	278	1	0.4	0.0	2.0

Dose 2

Pain	Any	277	53	19.1	14.7	24.3	276	56	20.3	15.7	25.5
	Grade 3	277	0	0.0	0.0	1.3	276	2	0.7	0.1	2.6
Redness	Any	277	37	13.4	9.6	17.9	276	47	17.0	12.8	22.0
	>20 mm	277	0	0.0	0.0	1.3	276	1	0.4	0.0	2.0
Swelling	Any	277	13	4.7	2.5	7.9	276	20	7.2	4.5	11.0
	>20 mm	277	0	0.0	0.0	1.3	276	0	0.0	0.0	1.3

Dose 3

Pain	Any	248	57	23.0	17.9	28.7	253	63	24.9	19.7	30.7
	Grade 3	248	3	1.2	0.3	3.5	253	3	1.2	0.2	3.4
Redness	Any	248	48	19.4	14.6	24.8	253	54	21.3	16.5	26.9
	>20 mm	248	1	0.4	0.0	2.2	253	1	0.4	0.0	2.2
Swelling	Any	248	26	10.5	7.0	15.0	253	37	14.6	10.5	19.6
	>20 mm	248	3	1.2	0.3	3.5	253	2	0.8	0.1	2.8

Across doses

Pain	Any	289	105	36.3	30.8	42.2	282	110	39.0	33.3	45.0
	Grade 3	289	4	1.4	0.4	3.5	282	6	2.1	0.8	4.6
Redness	Any	289	85	29.4	24.2	35.0	282	87	30.9	25.5	36.6
	>20 mm	289	3	1.0	0.2	3.0	282	2	0.7	0.1	2.5
Swelling	Any	289	51	17.6	13.4	22.5	282	55	19.5	15.0	24.6
	>20 mm	289	4	1.4	0.4	3.5	282	3	1.1	0.2	3.1

N = number of subjects with one (per dose) or at least one (across doses) symptoms sheet returned for the considered dose
n/% = number/percentage of subjects reporting one (per dose) or at least one (across doses) specified symptom at the HBV injection site
95% CI = exact 95% confidence interval;
LL = lower limit,
UL = upper limit
Any = Incidence of a particular symptom regardless of intensity
Grade 3 pain = pain with which subject cries when limb is moves or spontaneously painful

Secondary Outcome Variable(s):

Incidence, intensity and causal relationship of solicited general symptoms reported during the four-day (day 0-day 3) post-vaccination period following each dose and across doses (Total vaccinated cohort).

TABLE 40


Group	PF	TF

Intensity/

95% CI

Symptom	Relationship	N	n	%	LL	UL	N	n	%	LL	UL

Dose 1

Drowsiness	Any	279	66	23.7	18.8	29.1	275	60	21.8	17.1	27.2
	Grade 3	279	1	0.4	0.0	2.0	275	2	0.7	0.1	2.6
	Related	279	38	13.6	9.8	18.2	275	42	15.3	11.2	20.1
Irritability	Any	279	70	25.1	20.1	30.6	275	67	24.4	19.4	29.9
	Grade 3	279	1	0.4	0.0	2.0	275	1	0.4	0.0	2.0
	Related	279	43	15.4	11.4	20.2	275	47	17.1	12.8	22.1
Loss of	Any	279	44	15.8	11.7	20.6	275	25	9.1	6.0	13.1
appetite	Grade 3	279	0	0.0	0.0	1.3	275	0	0.0	0.0	1.3
	Related	279	26	9.3	6.2	13.4	275	19	6.9	4.2	10.6
Fever (Rectal)	≧38.0° C.	279	11	3.9	2.0	6.9	275	6	2.2	0.8	4.7
	>39.5° C.	279	4	1.4	0.4	3.6	275	2	0.7	0.1	2.6
	Related	279	2	0.7	0.1	2.6	275	1	0.4	0.0	2.0

Dose 2

Drowsiness	Any	278	67	24.1	19.2	29.6	275	67	24.4	19.4	29.9
	Grade 3	278	2	0.7	0.1	2.6	275	1	0.4	0.0	2.0
	Related	278	49	17.6	13.3	22.6	275	43	15.6	11.6	20.5
Irritability	Any	278	97	34.9	29.3	40.8	275	99	36.0	30.3	42.0
	Grade 3	278	2	0.7	0.1	2.6	275	9	3.3	1.5	6.1
	Related	278	74	26.6	21.5	32.2	275	69	25.1	20.1	30.6
Loss of	Any	278	28	10.1	6.8	14.2	275	31	11.3	7.8	15.6
appetite	Grade 3	278	0	0.0	0.0	1.3	275	0	0.0	0.0	1.3
	Related	278	20	7.2	4.4	10.9	275	18	6.5	3.9	10.1
Fever (rectal)	≧38.0° C.	278	9	3.2	1.5	6.1	275	3	1.1	0.2	3.2
	>39.5° C.	278	0	0.0	0.0	1.3	275	0	0.0	0.0	1.3
	Related	278	5	1.8	0.6	4.1	275	1	0.4	0.0	2.0

Dose 3

Drowsiness	Any	249	72	28.9	23.4	35.0	249	68	27.3	21.9	33.3
	Grade 3	249	0	0.0	0.0	1.5	249	2	0.8	0.1	2.9
	Related	249	47	18.9	14.2	24.3	249	47	18.9	14.2	24.3
Irritability	Any	249	106	42.6	36.3	49.0	249	107	43.0	36.7	49.4
	Grade 3	249	4	1.6	0.4	4.1	249	8	3.2	1.4	6.2
	Related	249	74	29.7	24.1	35.8	249	81	32.5	26.7	38.7
Loss of	Any	249	42	16.9	12.4	22.1	249	55	22.1	17.1	27.8
appetite	Grade 3	249	0	0.0	0.0	1.5	249	0	0.0	0.0	1.5
	Related	249	27	10.8	7.3	15.4	249	41	16.5	12.1	21.7
Fever (rectal)	≧38.0° C.	249	38	15.3	11.0	20.3	249	48	19.3	14.6	24.7
	>39.5° C.	249	0	0.0	0.0	1.5	249	2	0.8	0.1	2.9
	Related	249	22	8.8	5.6	13.1	249	31	12.4	8.6	17.2

Across doses

Drowsiness	Any	289	132	45.7	39.8	51.6	282	124	44.0	38.1	50.0
	Grade 3	289	3	1.0	0.2	3.0	282	5	1.8	0.6	4.1
	Related	289	88	30.4	25.2	36.1	282	86	30.5	25.2	36.2
Irritability	Any	289	165	57.1	51.2	62.9	282	166	58.9	52.9	64.7
	Grade 3	289	7	2.4	1.0	4.9	282	15	5.3	3.0	8.6
	Related	289	122	42.2	36.5	48.1	282	125	44.3	38.4	50.3
Loss of	Any	289	84	29.1	23.9	34.7	282	87	30.9	25.5	36.6
appetite	Grade 3	289	0	0.0	0.0	1.3	282	0	0.0	0.0	1.3
	Related	289	54	18.7	14.4	23.7	282	60	21.3	16.6	26.5
Fever (rectal)	≧38.0° C.	289	54	18.7	14.4	23.7	282	54	19.1	14.7	24.2
	>39.5° C.	289	4	1.4	0.4	3.5	282	4	1.4	0.4	3.6
	Related	289	28	9.7	6.5	13.7	282	33	11.7	8.2	16.0

N = number of subjects with one (per dose) or at least one (across doses) symptoms sheet returned for the considered dose
n/% = number/percentage of subjects reporting one (per dose) or at least one (across doses) specified symptom
95% CI = exact 95% confidence interval;
LL = lower limit,
UL = upper limit
Any = Incidence of a particular symptom regardless of intensity or relationship to vaccinations
Grade 3 irritability = crying that cannot be comforted/prevents normal activity
Grade 3 drowsiness = drowsiness that prevents normal activity
Grade 3 loss of appetite = not eating at all
Related = It was determined by the investigator that there was a reasonable possibility that the vaccine contributed to the adverse event.

Conclusion: Non-inferiority of the Engerix-B thiomersal free vaccine compared to the Engerix-B Preservative free vaccine was demonstrated.

EXAMPLE 8

A Double-Blind, Randomized, Controlled, Multicenter Study to Evaluate the Immunogenicity of Preservative-Free Enqerix-B and Thiomersal-Free Engerix-B Vaccines Compared to Engerix™-B and Evaluate Safety and Reactogenicity of each Vaccine when Administered Intramuscularly According to a 0, 1, 6 Month Schedule in Healthy Volunteers (18 to 50 Years).
Rationale: The purpose of this study was to clinically evaluate two formulations of the hepatitis B vaccine (HBV), which have been manufactured with the aim to eliminate the organomercurial preservative thiomersal from the product. One of these vaccines is “preservative-free” (PF) whereby thiomersal is no longer added as preservative. The other vaccine is “thiomersal-free” (TF) whereby thiomersal is no longer included as a buffer additive during the antigen purification process, so as to obtain a product totally free of thiomersal. In this study the HBV (PF) and HBV (TF) vaccines were compared with the original HBV.
Study Design: Multinational, multicentre, double-blind, randomized, controlled parallel-group. The study was for 7 months.
Centres: 4 centres (2 in Switzerland, 1 in Belgium and 1 in Germany).
Indication: Three-dose vaccination against hepatitis B in healthy adults aged 18 to 50 years.
Treatment: Study groups were as follows:
PF Group: the subjects received hepatitis B vaccine preservative free (PF).
TF Group: the subjects received hepatitis B vaccine thiomersal-free (TF).
HBV Group (control): the subjects received the original formulation of HBV.
All vaccines were to be administered at Months 0, 1 and 6 as an intramuscular (IM) injection in the deltoid region of the non-dominant arm.
Objectives: The primary objective was to demonstrate non-inferiority of the immune response induced by HBV (PF) and HBV (TF) compared to HBV one month after the full vaccination course (Month 7).
Primary Outcome/Efficacy Variable: Anti-hepatitis B surface antigen (anti-HBs) seroprotection (SP) rates (defined as the percentage of subjects with anti-HBs antibody concentrations ≧10 mIU/ml) at Month 7.
Secondary Outcome/Efficacy Variable(s):

- Occurrence, intensity and relationship to vaccination of solicited local and general signs and symptoms during a 4-day follow-up period (days 0-3) after each vaccination and overall.
- Occurrence, intensity and relationship to vaccination of unsolicited local and general signs and symptoms within 30 days (days 0-29) after each vaccination and overall.
- Occurrence, intensity and relationship to vaccination of serious adverse events (SAEs) during the study period, up to and including 30 days following the last dose vaccination.
- SP rates at baseline, Months 1, 2 and 6 for all subjects.
- Seropositivity (S+) rates (percentage of subjects with anti-HBs antibody concentrations≧the assay cut-off of 3.3 mlU/ml) at Months 0, 1, 2, 6 and 7, for all subjects.
- Geometric mean concentrations (GMCs) calculated at Months 1, 2, 6 and 7, for all subjects.
- At baseline and Month 7: anti-RF1 antibody concentrations in a random subset of 50 subjects/group.
- Anti-RF1 seropositivity rate and GMCs at baseline and at Month 7.
  Statistical Methods:

The analyses were conducted on the According To Protocol (ATP) cohort for immunogenicity, and, for safety on the ATP cohort for safety and on the Total vaccinated cohort.

- The Total Vaccinated cohort included all subjects who received at least one vaccine dose
- The ATP cohort for immunogenicity included all vaccinated subjects who met the criteria defined in the protocol for the analysis of immunogenicity.
- The ATP cohort for safety included all vaccinated subjects who met the criteria defined in the protocol for the analysis of safety.
  Analysis of Immunogenicity:

The analysis of immunogenicity was conducted on the ATP cohort for immunogenicity.
The anti-HBs, S+ rates and SP rates were tabulated with their 95% confidence intervals (CIs). Antibody concentrations were summarised for each group by GMCs with their 95% CIs.
At Month 7, the difference in anti-HBs SP rates between HBV and each the HBV (PF) and the HBV (TF) vaccines was calculated, with exact 95% CIs for these differences in proportion. If the upper limit of the two 95% CIs was less than or equal to 10%, non-inferiority of the vaccine compared to the HBV vaccine was concluded.
Analysis of Safety:
The analysis of safety was conducted on the ATP cohort for safety
For each solicited symptom, the percentage of subjects with the symptom and its exact 95% CI was summarised by vaccine group, by dose and across doses.
The percentages of subjects reporting unsolicited symptoms within the 30 days (days 0-29) following vaccination was summarised by vaccine group according to the WHO preferred term.

Study Population: Healthy subjects aged 18 to 50 years, free of obvious health problems as established by medical history and clinical examination, who were seronegative for hepatitis B surface antigen (HBsAg), anti-Hepatitis B core antigen (anti-HBc) and anti-HBs antibodies. Female subjects of childbearing potential had to be abstinent or using adequate contraceptive precautions for one month prior to enrolment and up to two months after the last vaccination. Written informed consent was obtained from all subjects prior to entry into the study.

TABLE 41


PF	HBV	TF

Number of subjects
(Total vaccinated cohort)
Planned, N	206	206	206
Randomised, N	217	216	216
Completed, n (%)	207 (95.4)	207 (95.8)	207 (95.8)
Total Number Subjects	10 (4.6)	9 (4.2)	9 (4.2)
Withdrawn, n (%)
Withdrawn due to Adverse	0 (0.0)	2 (0.9)	2 (0.9)
Events (AEs), n (%)
Withdrawn due to Lack	Not	Not	Not
of Efficacy, n (%)	applicable	applicable	applicable
Withdrawn for Other	10 (4.6)	7 (3.2)	7 (3.2)
Reasons, n (%)
Demographics
N (Total Vaccinated Cohort)	217	216	216
Females:Males	113:104	122:94	127:89
Mean Age, years (SD)	30.6 (9.11)	30.7 (9.59)	30.0 (9.34)
White, n (%)	211 (97.2)	214 (99.1)	216 (100)

Primary Efficacy Results:

Anti-HBs SP rates per group and difference between PF and HBV groups, and between TF and HBV groups for anti-HBs SP rates at Month 7 (ATP cohort for immunogenicity).

	TABLE 42


	Inference

Group	N	%	Group	N	%	Difference	Value	95% CI

PF	179	98.9	HBV	178	94.4	HBV - PF	−4.5	−10.4	0.8
TF	175	96.6	HBV	178	94.4	HBV - TF	−2.2	−8.9	3.5

N = number of subjects with available results.
% = percentage of subjects who were seroprotected for anti-HBs antibodies
95% CI = Exact 95% confidence interval, lower and upper limits.

Secondary Outcome Variable(s):

S+ rates, SP rates and GMCs for anti-HBs (calculated in seroconverters only) (ATP cohort for immunogenicity) at Months 1, 2, 6 and 7

TABLE 43


S+	95% CI	SP*	95% CI	GMC	95% CI

Group

Timing

N

n

%

LL

UL

n

%

LL

UL

(mlU/ml)

LL

UL

PF	PI(M1)	183	24	13.1	8.6	18.9	13	7.1	3.8	11.8	19.2	11.0	33.5
	PII(M2)	183	142	77.6	70.9	83.4	114	62.3	54.8	69.3	36.5	28.7	46.6
	PII(M6)	181	167	92.3	87.4	95.7	153	84.5	78.4	89.5	104.7	83.1	131.9
	PIII(M7)	179	178	99.4	96.9	100.0	177	98.9	96.0	99.9	4955.5	3589.1	6842.3
HBV	PI(M1)	182	22	12.1	7.7	17.7	12	6.6	3.5	11.2	15.8	8.4	29.7
	PII(M2)	181	121	66.9	59.5	73.7	109	60.2	52.7	67.4	41.1	33.3	50.7
	PII(M6)	178	153	86.0	80.0	90.7	141	79.2	72.5	84.9	128.1	101.7	161.2
	PIII(M7)	178	172	96.6	92.8	98.8	168	94.4	89.9	97.3	4119.7	2961.7	5730.3
TF	PI(M1)	176	23	13.1	8.5	19.0	12	6.8	3.6	11.6	15.2	9.4	24.7
	PII(M2)	176	126	71.6	64.3	78.1	99	56.3	48.6	63.7	36.3	28.2	46.7
	PII(M6)	176	157	89.2	83.7	93.4	150	85.2	79.1	90.1	135.3	107.4	170.4
	PIII(M7)	175	171	97.7	94.3	99.4	169	96.6	92.7	98.7	5387.8	3940.8	7366.0

N = number of subjects tested.
n/% = number/percentage of subjects who were seropositive/seroprotected for anti-HBs antibodies.
PI (M1), PII (M2), PII (M6) = post-vaccination blood samples obtained at Months 1, 2 and 6, respectively, after the first dose.
PIII (M7) = SP is a primary endpoint, post-vaccination blood samples obtained one month after the third dose.
95% CI = 95% confidence interval;
LL = Lower Limit;
UL = Upper Limit.

Secondary Outcome Variable(s):

GMCs (calculated on a subset of 50 subjects per group) for anti-RF-1 antibody concentrations (ATP cohort for immunogenicity)

	TABLE 44


	≧33 EU/ml	GMC

95% CI

Group	Timing	N	n	%	LL	UL	EU/ml	LL	UL

PF	PRE	41	0	0.0	0.0	8.6	16.5	16.5	16.5
	PIII	40	36	90.0	76.3	97.2	151.6	98.6	233.0
	(M7)
HBV	PRE	42	0	0.0	0.0	8.4	16.5	16.5	16.5
	PIII	42	34	81.0	65.9	91.4	103.8	70.5	152.7
	(M7)
TF	PRE	40	1	2.5	0.1	13.2	17.1	15.9	18.4
	PIII	39	33	84.6	69.5	94.1	161.9	106.8	245.5
	(M7)

1. PRE = Pre-vaccination.
2. PIII (M7) = post-vaccination blood samples obtained one month after the third dose.
3. N = number of subjects with available results.
4. GMC = For the purpose of calculating GMC, seronegative samples were assigned a value of half the cut-off for the assay.

Secondary Outcome Variable(s):

Incidence of solicited local symptoms (total and Grade “3”) reported during the 4-day (days 0-3) follow-up period after vaccination (ATP cohort for safety):

TABLE 45


PF	HBV	TF

95% CI

n

%

LL

UL

n

%

LL

UL

n

%

LL

UL

Dose 1 (Month 1)	N = 207	N = 205	N = 209

Pain	Any	101	48.8	41.8	55.8	72	35.1	28.6	42.1	88	42.1	35.3	49.1
	Grade “3”	5	2.4	0.8	5.5	8	3.9	1.7	7.5	1	0.5	0.0	2.6
Redness	Any	40	19.3	14.2	25.4	29	14.1	9.7	19.7	44	21.1	15.7	27.2
	Grade “3”	1	0.5	0.0	2.7	0	0.0	0.0	1.8	2	1.0	0.1	3.4
Swelling	Any	19	9.2	5.6	14.0	18	8.8	5.3	13.5	19	9.1	5.6	13.8
	Grade “3”	0	0.0	0.0	1.8	0	0.0	0.0	1.8	1	0.5	0.0	2.6

Dose 2 (Month 2)	N = 209	N = 202	N = 202

Pain	Any	94	45.0	38.1	52.0	63	31.2	24.9	38.1	75	37.1	30.5	44.2
	Grade “3”	5	2.4	0.8	5.5	2	1.0	0.1	3.5	5	2.5	0.8	5.7
Redness	Any	36	17.2	12.4	23.0	22	10.9	7.0	16.0	30	14.9	10.2	20.5
	Grade “3”	0	0.0	0.0	1.7	0	0.0	0.0	1.8	0	0.0	0.0	1.8
Swelling	Any	12	5.7	3.0	9.8	22	10.9	7.0	16.0	13	6.4	3.5	10.8
	Grade “3”	0	0.0	0.0	1.7	2	1.0	0.1	3.5	0	0.0	0.0	1.8

Dose 3 (Month 3)	N = 200	N = 195	N = 203

Pain	Any	97	48.5	41.4	55.7	72	36.9	30.1	44.1	80	39.4	32.6	46.5
	Grade “3”	3	1.5	0.3	4.3	6	3.1	1.1	6.6	1	0.5	0.0	2.7
Redness	Any	48	24.0	18.3	30.5	20	10.3	6.4	15.4	39	19.2	14.0	25.3
	Grade “3”	0	0.0	0.0	1.8	0	0.0	0.0	1.9	1	0.5	0.0	2.7
Swelling	Any	25	12.5	8.3	17.9	18	9.2	5.6	14.2	22	10.8	6.9	15.9
	Grade “3”	0	0.0	0.0	1.8	1	0.5	0.0	2.8	0	0.0	0.0	1.8

Across doses	N = 209	N = 205	N = 209

Pain	Any	148	70.8	64.1	76.9	113	55.1	48.0	62.1	136	65.1	58.2	71.5
	Grade “3”	12	5.7	3.0	9.8	14	6.8	3.8	11.2	7	3.3	1.4	6.8
Redness	Any	72	34.4	28.0	41.3	44	21.5	16.0	27.7	69	33.0	26.7	39.8
	Grade “3”	1	0.5	0.0	2.6	0	0.0	0.0	1.8	3	1.4	0.3	4.1
Swelling	Any	43	20.6	15.3	26.7	40	19.5	14.3	25.6	37	17.7	12.8	23.6
	Grade “3”	0	0.0	0.0	1.7	3	1.5	0.3	4.2	1	0.5	0.0	2.6

N across doses = number of subjects with at least one documented dose.
n/% across doses = number/percentage of subjects with at least one symptom reported.
Grade “3” = spontaneously painful or redness/swelling with greatest surface diameter ≧30 mm and lasting for 24 hours.

Secondary Outcome Variable(s):

Overall incidence of solicited general symptoms (total, Grade “3” and related) reported during the 4-day (days 0-3) follow-up period after vaccination (ATP cohort for safety):

TABLE 46


PF	HBV	TF

95% CI

n

%

LL

UL

n

%

LL

UL

n

%

LL

UL

Dose 1 (Month 1)	N = 208	N = 209	N = 201

Fatigue	Any	49	23.6	18.0	29.9	36	17.2	12.4	23.0	37	18.4	13.3	24.5
	Grade “3”	1	0.5	0.0	2.6	3	1.4	0.3	4.1	2	1.0	0.1	3.5
	Related	36	17.3	12.4	23.1	29	13.9	9.5	19.3	32	15.9	11.2	21.7
GastroIntestinal	Any	17	8.2	4.8	12.8	13	6.2	3.4	10.4	12	6.0	3.1	10.2
	Grade “3”	1	0.5	0.0	2.6	2	1.0	0.1	3.4	0	0.0	0.0	1.8
	Related	13	6.3	3.4	10.5	9	4.3	2.0	8.0	9	4.5	2.1	8.3
Headache	Any	45	21.6	16.2	27.9	32	15.3	10.7	20.9	33	16.4	11.6	22.3
	Grade “3”	0	0.0	0.0	1.8	2	1.0	0.1	3.4	1	0.5	0.0	2.7
	Related	41	19.7	14.5	25.8	21	10.0	6.3	14.9	26	12.9	8.6	18.4
Fever	≧37.5° C.	4	1.9	0.5	4.9	1	0.5	0.0	2.6	5	2.5	0.8	5.7
(axillary)	Grade “3”	0	0.0	0.0	1.8	0	0.0	0.0	1.7	0	0.0	0.0	1.8
	Related	4	1.9	0.5	4.9	1	0.5	0.0	2.6	3	1.5	0.3	4.3

Dose 2 (Month 2)	N = 205	N = 202	N = 195

Fatigue	Any	45	22.0	16.5	28.2	32	15.8	11.1	21.6	45	23.1	17.4	29.6
	Grade “3”	0	0.0	0.0	1.8	1	0.5	0.0	2.7	2	1.0	0.1	3.7
	Related	37	18.0	13.0	24.0	28	13.9	9.4	19.4	37	19.0	13.7	25.2
GastroIntestinal	Any	21	10.2	6.5	15.2	15	7.4	4.2	12.0	14	7.2	4.0	11.8
	Grade “3”	1	0.5	0.0	2.7	0	0.0	0.0	1.8	1	0.5	0.0	2.8
	Related	19	9.3	5.7	14.1	10	5.0	2.4	8.9	9	4.6	2.1	8.6
Headache	Any	42	20.5	15.2	26.7	26	12.9	8.6	18.3	31	15.9	11.1	21.8
	Grade “3”	3	1.5	0.3	4.2	0	0.0	0.0	1.8	1	0.5	0.0	2.8
	Related	37	18.0	13.0	24.0	21	10.4	6.6	15.5	25	12.8	8.5	18.3
Fever	≧37.5° C.	0	0.0	0.0	1.8	5	2.5	0.8	5.7	7	3.6	1.5	7.3
(axillary)	Grade “3”	0	0.0	0.0	1.8	0	0.0	0.0	1.8	0	0.0	0.0	1.9
	Related	0	0.0	0.0	1.8	3	1.5	0.3	4.3	5	2.6	0.8	5.9

Dose 3 (Month 3)	N = 209	N = 202	N = 203

Fatigue	Any	54	25.8	20.0	32.3	43	21.3	15.9	27.6	43	21.2	15.8	27.5
	Grade “3”	2	1.0	0.1	3.4	2	1.0	0.1	3.5	3	1.5	0.3	4.3
	Related	48	23.0	17.4	29.3	36	17.8	12.8	23.8	34	16.7	11.9	22.6
GastroIntestinal	Any	27	12.9	8.7	18.2	16	7.9	4.6	12.5	17	8.4	5.0	13.1
	Grade “3”	2	1.0	0.1	3.4	1	0.5	0.0	2.7	1	0.5	0.0	2.7
	Related	19	9.1	5.6	13.8	12	5.9	3.1	10.1	9	4.4	2.0	8.2
Headache	Any	51	24.4	18.7	30.8	32	15.8	11.1	21.6	29	14.3	9.8	19.9
	Grade “3”	0	0.0	0.0	1.7	4	2.0	0.5	5.0	1	0.5	0.0	2.7
	Related	37	17.7	12.8	23.6	29	14.4	9.8	20.0	22	10.8	6.9	15.9
Fever	≧37.5° C.	3	1.4	0.3	4.1	7	3.5	1.4	7.0	7	3.4	1.4	7.0
(axillary)	Grade “3”	0	0.0	0.0	1.7	0	0.0	0.0	1.8	1	0.5	0.0	2.7
	Related	3	1.4	0.3	4.1	5	2.5	0.8	5.7	6	3.0	1.1	6.3

Across doses	N = 209	N = 205	N = 209

Fatigue	Any	83	39.7	33.0	46.7	79	38.5	31.8	45.6	87	41.6	34.9	48.6
	Grade “3”	5	2.4	0.8	5.5	3	1.5	0.3	4.2	7	3.3	1.4	6.8
	Related	68	32.5	26.2	39.3	68	33.2	26.8	40.1	80	38.3	31.7	45.2
GastroIntestinal	Any	38	18.2	13.2	24.1	40	19.5	14.3	25.6	42	20.1	14.9	26.2
	Grade “3”	3	1.4	0.3	4.1	2	1.0	0.1	3.5	4	1.9	0.5	4.8
	Related	29	13.9	9.5	19.3	31	15.1	10.5	20.8	32	15.3	10.7	20.9
Headache	Any	81	38.8	32.1	45.7	68	33.2	26.8	40.1	79	37.8	31.2	44.7
	Grade “3”	2	1.0	0.1	3.4	4	2.0	0.5	4.9	5	2.4	0.8	5.5
	Related	66	31.6	25.3	38.3	58	28.3	22.2	35.0	65	31.1	24.9	37.9
Fever	≧37.5° C.	9	4.3	2.0	8.0	10	4.9	2.4	8.8	14	6.7	3.7	11.0
(axillary)	Grade “3”	0	0.0	0.0	1.7	0	0.0	0.0	1.8	1	0.5	0.0	2.6
	Related	7	3.3	1.4	6.8	7	3.4	1.4	6.9	12	5.7	3.0	9.8

N across doses = number of subjects with at least one documented dose.
n/% across doses = number/percentage of subjects with at least one symptom reported.
Related = symptoms considered by the investigator to have a causal relationship to the study vaccine.
Grade “3” = prevented normal everyday activities or fever (axillary temperature) >38.5° C.

Safety results: Number (%) of subjects with unsolicited adverse events (AEs) (Total vaccinated cohort):

TABLE 47


Most frequent adverse
events - On Therapy
(occurring within days	PF	HBV	TF
0-29 following vaccination)	N = 214*	N = 214*	N = 214*

Subjects with any	125 (58.4)	142 (66.4)	125 (58.4)
AE(s), n (%)
Headache	33 (15.4)	37 (17.3)	31 (14.5)
Injection site reaction	14 (6.5)	26 (12.1)	23 (10.7)
Upper respiratory	19 (8.9)	25 (11.7)	13 (6.1)
tract infection
Influenza-like symptoms	12 (5.6)	21 (9.8)	14 (6.5)
Pharyngitis	7 (3.3)	18 (8.4)	11 (5.1)
Coughing	6 (2.8)	13 (6.1)	4 (1.9)
Dysmenorrhoea	4 (1.9)	9 (4.2)	8 (3.7)
Back pain	7 (3.3)	6 (2.8)	6 (2.8)
Abdominal pain	10 (4.7)	2 (0.9)	6 (2.8)
Sinusitis	4 (1.9)	4 (1.9)	8 (3.7)
Myalgia	7 (3.3)	5 (2.3)	3 (1.4)
Toothache	5 (2.3)	5 (2.3)	5 (2.3)
Bronchitis	4 (1.9)	6 (2.8)	2 (0.9)
Rhinitis	3 (1.4)	4 (1.9)	5 (2.3)
Gastroenteritis	2 (0.9)	4 (1.9)	5 (2.3)
Injury	6 (2.8)	4 (1.9)	1 (0.5)
Fever	1 (0.5)	6 (2.8)	3 (1.4)

For 3 subjects in the PF group, 2 subjects in the HBV group and 2 subjects in the TF group, no safety data were available

Safety Results: Number (%) of Serious Adverse Events (SAEs) (Total vaccinated cohort):

TABLE 48


PF	HBV	TF
N = 214	N = 214	N = 214

All SAEs
Subjects with any SAE(s),	4 (1.9) [0]	6 (2.8) [0]	4 (1.9) [0]
n (%) [n related]
Abdominal pain	1 (0.5) [0]	0 (0.0) [0]	0 (0.0) [0]
Abortion	0 (0.0) [0]	0 (0.0) [0]	2 (0.9) [0]
Abscess	0 (0.0) [0]	1 (0.5) [0]	0 (0.0) [0]
Back pain	1 (0.5) [0]	0 (0.0) [0]	1 (0.5) [0]
Cerebrovascular disorder	0 (0.0) [0]	1 (0.5) [0]	0 (0.0) [0]
Convulsions	0 (0.0) [0]	1 (0.5) [0]	0 (0.0) [0]
Gastrointestinal disorder not	2 (0.9) [0]	0 (0.0) [0]	0 (0.0) [0]
otherwise specified (NOS)
Infection bacterial	0 (0.0) [0]	1 (0.5) [0]	0 (0.0) [0]
Injury	1 (0.5) [0]	0 (0.0) [0]	1 (0.5) [0]
Myocardial infarction	0 (0.0) [0]	1 (0.5) [0]	0 (0.0) [0]
Renal calculus	0 (0.0) [0]	1 (0.5) [0]	0 (0.0) [0]
Suicide attempt	0 (0.0) [0]	1 (0.5) [0]	0 (0.0) [0]
Fatal SAEs:
Subjects with fatal SAE(s),	0 (0.0) [0]	0 (0.0) [0]	0 (0.0) [0]
n (%) [n related]

For 3 subjects in the PF group, 2 sujects in the HBV group and 2 subjects in the TF group, no safety data were available

Conclusion: In this study, non-inferiority in terms of SP rates achieved at Month 7 was shown for preservative-free and thiomersal-free HBV compared to thiomersal-containing HBV. The overall safety profile exhibited by the preservative-free and thiomersal-free vaccines was similar to that of HBV.

All documents cited herein and patent applications to which priority is claimed are incorporated by reference herein in their entirety. This invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims. The disclosures of the patents, patent applications and publications cited herein are incorporated by reference in their entireties.

Claims

1. A method for making a hepatitis B antigen for use in a vaccine, the method comprising purification of the hepatitis B antigen in the presence of a reducing agent having a free —SH group.

2. The method according to claim 1 wherein the hepatitis B antigen is a stable immunogenic antigen free of thiomersal.

3. The method according to claim 1 wherein the purified antigen product comprises less than about 0.025 μg mercury per 20 kg protein.

4. The method according to claim 1 wherein the purified antigen product comprises no thiomersal.

5. The method of producing a hepatitis B antigen according to claim 1 wherein a crude hepatitis B antigen preparation is:

(a) subjected to gel permeation chromatography;

(b) subjected to ion-exchange chromatography; and

(c) mixed with a reducing agent having a free —SH group.

6. The method according to claim 1 wherein the reducing agent is chosen from the group of: cysteine, glutathione, dithiothreitol and β-mercaptoethanol.

7. The method according to claim 1, wherein the reducing agent is cysteine.

8. The method according to claim 7 wherein the cysteine is added to a final concentration of between about 1-10 mM.

9. The method according to claim 7 wherein the cysteine is added to a final concentration of about 2 mM.

10. A vaccine composition comprising a hepatitis B antigen in the presence of a reducing agent having a free —SH group.

11. The vaccine composition according to claim 10 further comprising an adjuvant.

12. The vaccine composition according to claim 11 wherein the adjuvant is an aluminium salt.

13. The vaccine composition as claimed in claim 12, which further comprises a TH-1 inducing adjuvant.

14. The vaccine composition according to claim 13 wherein the TH1-inducing adjuvant is chosen from the group of: 3-DMPL, QS21, 3-DMPL and QS21, and a CpG oligonucleotide.

15. The vaccine composition as claimed in claim 10, which further comprises at least one of the antigens chosen from the group of: diptheria toxoid (D), tetanus toxoid (T) acellular pertussis antigens (Pa), inactivated polio virus (IPV), haemophilus influenzae antigen (Hib), hepatitis A antigen, herpes simplex virus (HSV), chlamydia, GSB, HPV, streptococcus pneumoniae, and neisseria antigens.

16. The vaccine composition comprising a hepatitis B antigen and a cysteine solution.

17. A vaccine composition comprising a hepatitis B antigen, an adjuvant, and an inactivated polio virus.