Clinical phase II and III studies of an AS03‐adjuvanted H5N1 influenza vaccine produced in an EB66® cell culture platform

Masafumi Endo; Mitsuyoshi Tanishima; Kayo Ibaragi; Kenshi Hayashida; Tadashi Fukuda; Tetsuro Tanabe; Takeshi Naruse; Yoichiro Kino; Kohji Ueda

doi:10.1111/irv.12755

. 2020 Jun 24;14(5):551–563. doi: 10.1111/irv.12755

Clinical phase II and III studies of an AS03‐adjuvanted H5N1 influenza vaccine produced in an EB66^® cell culture platform

Masafumi Endo ^1,^✉, Mitsuyoshi Tanishima ¹, Kayo Ibaragi ¹, Kenshi Hayashida ¹, Tadashi Fukuda ¹, Tetsuro Tanabe ¹, Takeshi Naruse ¹, Yoichiro Kino ², Kohji Ueda ³

PMCID: PMC7431644 PMID: 32579785

Abstract

Background

We have developed an AS03‐adjuvanted H5N1 influenza vaccine produced in an EB66^® cell culture platform (KD‐295).

Objectives

In accordance with Japanese guidelines for development of pandemic prototype vaccines, the phase II study was conducted in a double‐blind, randomized, parallel‐group comparison study and the phase III study was conducted in an open‐label, non‐randomized, uncontrolled study.

Methods

Healthy adult volunteers aged 20 ‐ 64 years enrolled in the phase II and III studies (N = 248 and N = 369) received KD‐295 intramuscularly twice with a 21‐day interval. After administration, immune response and adverse events were evaluated. In the phase II study, four different vaccine formulations were compared: MA (3.75 μg hemagglutinin [HA] antigen + AS03 adjuvant system), MB (3.75 μg HA + 1/2AS03), HA (7.5 μg HA + AS03), and HB (7.5 μg HA + 1/2AS03). In the phase III study, the MA formulation was further evaluated.

Results

In the phase II study, all four vaccine formulations were well‐tolerated and no SAE related to vaccination were observed. The MA formulation was slightly more immunogenic and less reactogenic among the vaccine formulations. Therefore, the MA formulation was selected for the phase III study, and it was well‐tolerated and no serious adverse drug reactions were observed. The vaccine fulfilled the three immunogenicity criteria described in the Japanese guidelines.

Conclusions

These data indicate that the MA formulation of KD‐295 was well‐tolerated and highly immunogenic and it can be considered a useful pandemic and pre‐pandemic influenza vaccine.

Keywords: AS03, EB66^® cells, H5N1 influenza

1. INTRODUCTION

The most recent influenza pandemic of 2009‐2010 remains fresh in our minds. Contrary to global expectations, the causative agent of the pandemic was an H1N1 virus. In the 2009 pandemic, various vaccines were used, including non‐adjuvanted and adjuvanted subvirion and whole virion vaccines. ¹ Among adults, the results of the vaccine use confirmed that even non‐adjuvanted vaccines were highly immunogenic. This is because there was cross‐reactivity in T helper epitopes between the H1N1 pandemic 2009 virus and previous seasonal H1N1 viruses. ² In Japan, the local vaccine manufacturers produced monovalent non‐adjuvanted split vaccine. At the same time, the Japanese government imported adjuvanted vaccines as a precaution in case of vaccine shortages, but many of these imported vaccines were left unused. However, pandemic threats, such as H5N1, have not disappeared and nobody knows what virus subtype will cause the next pandemic. At this moment, among the viruses with pandemic potential, viruses of avian origin, including the H7N9 subtypes, are a concern because of sporadic human infection. ³ As same with H5N1 subtypes, and unlike the H1N1 pandemic 2009 virus, immunogenicity of those viruses is very low in humans, which may be related to predicted poor T‐cell immunogenicity. ⁴

Another important condition of a pandemic vaccine is timely manufacturing. In the case of the 2009 pandemic, the causative virus was first isolated in April 2009 and a candidate vaccine virus was generated in May. Usually, seasonal influenza vaccines are produced from spring to summer in Japan; therefore, the transition of production from seasonal vaccine to pandemic vaccine was relatively smooth in 2009. If a pandemic occurs in a period outside of seasonal vaccine production in the egg vaccine platform, more time will be needed to start the vaccine manufacturing because of egg supply. Furthermore, because of the damage of chickens by highly pathogenic avian influenza, there is a risk that egg supply will be stopped. To address these issues, we have been developing an AS03‐adjuvanted vaccine using H5N1 influenza virus antigen derived from a duck cell line (EB66^®). In the previous phase I study, we confirmed that the vaccine was well‐tolerated and elicited a broadly cross‐reactive antibody response. ⁵ In this paper, we report further evaluation of AS03‐adjuvanted H5N1 influenza vaccine formulations produced in an EB66^® cell culture platform, KD‐295, in phase II and III studies to assess its immunogenicity and safety. In addition, phase II study data were registered and released in JapicCTI‐121788, and phase III study data were registered and released in JapicCTI‐121936.

2. MATERIALS AND METHODS

2.1. Study designs and subjects

The phase II study was conducted in adults between the ages of 20 and 64 years in a randomized, double‐blinded (all involved were blinded), comparative fashion from 2 April to 6 November 2012 to further assess immunogenicity and safety of the vaccine, and to determine the appropriate dosage to be evaluated in the phase III study. After selection of one formulation, the phase III study was performed from August 23, 2012 to March 10, 2013 in an unblinded, uncontrolled study enrolling adults between the age of 20 and 64 years.

In both studies, the selection criteria were healthy adults aged 20‐64 who agreed with written informed consent. Exclusion criteria included no history of H5N1 infection or vaccination. These studies were conducted in Tokyo, Osaka, and Kagoshima in Japan.

Prior to clinical studies, related documents such as the clinical trial protocol and informed consent form were reviewed by the IRB within each hospital. The studies were conducted in accordance with the Helsinki Declaration, GCP, and other relevant regulations. Written informed consent was obtained from participants prior to enrollment.

In the phase II study, four different vaccine formulations were evaluated: MA (3.75 μg HA + AS03), HA (7.5 μg HA + AS03), MB (3.75 μg HA + 1/2 AS03), and HB (7.5 μg HA + 1/2 AS03).

Regarding the allocation method to each group, first the sponsor distributed the investigational drugs allocated randomly to the study sites. The principal investigator or subinvestigator entered the information about subjects as of obtaining written informed consent and about the study sites into the Electronic Data Capture system (Medidata RaveTM). The investigational drug allocation system (Medidata BalanceTM) featured in the Electronic Data Capture system allocated the each subject to MA group, MB group, HA group, and HB group on 1:1:1:1 ratio by using a minimization method. Age, gender, stratification, study, and study sites were used as the adjustment factors.

In the phase III study, the MA formulation (3.75 μg HA + AS03) was evaluated based on the results of the phase I/II study. In both studies, the investigational vaccine KD‐295 was administered intramuscularly at a dose volume of 0.5 mL given twice at an interval of 21 ± 7 days.

2.2. Vaccines

KD‐295 was composed of separate vials consisting of hemagglutinin (HA) antigen and AS03 adjuvant (squalene, α‐tocopherol, and Tween 80) and mixed in equal amounts at the time of use. The vaccine virus strain used in this study was A/Indonesia/05/2005(H5N1)/PR8‐IBCDC‐RG2 strain belonging to Clade 2.1.3.2. Briefly, HA antigen was prepared as previously described5, by cultivating the vaccine virus in EB66^® cells, purifying the virus by sucrose density gradient centrifugation, inactivating with β‐propiolactone and ultraviolet light irradiation, and treating the virus particles with a surfactant.

2.3. Immunological evaluation

The immunogenicity evaluation protocols of the phase II and III studies were the same. Blood samples were taken before the first vaccination (Day 0), 21 days after the first vaccination (Day 21), and 21 days after the 2nd vaccination (Day 42). To evaluate the immune response to the vaccine strain, HI antibody and neutralizing antibody titers were measured. Measurements of HI antibody and neutralizing antibody titers were conducted with reference to a previously described method. ⁶ For the HI test, both horse and chicken erythrocytes were used in the phase II study and only horse erythrocytes in the phase III study. For HI antibody, seroconversion rate (SCR), the geometric mean fold rise (GMFR), and the seroprotection rate (SPR) were calculated. SCR was the percentage of the subjects having an HI antibody titer of less than 1:10 prior to vaccination and 1:40 or higher after vaccination, or an HI titer before vaccination ≥1:10 and at least a 4‐fold increase in HI antibody titers after vaccination. SPR was defined as the percentage of participants who achieved HI titers of 1:40 or more. GMFR was defined as the ratio of change in GMT of HI antibody titers after the vaccination vs prior to the vaccination. The 95% confidence intervals on both sides of SCR and SPR were calculated based on the F‐distribution, and confidence interval of GMFR was calculated based on Student's t‐distribution.

For evaluation of immunogenicity of the vaccine, we followed the immunogenicity criteria of the Japanese guidelines for development of pandemic prototype vaccines (Japanese guidelines), which is identical to the immunogenicity criteria of the CHMP (Committee for Proprietary Medicinal Products) guidelines in place at the time the study was conducted (CPMP/BWP/214/96). In both studies, for the primary endpoint, we confirmed whether HI antibody (horse erythrocytes only) at Day 42 in a full analysis set (FAS) met the criteria of the Japanese guidelines (SCR >40%, GMFR >2.5, and SPR >70%). FAS was defined as a group of subjects with blood collected after at least one vaccination. The secondary endpoint of the both studies was percentage of subjects with a 4‐fold increase in neutralizing antibody titers at Day 42 in FAS. In addition, in the phase II study, we confirmed whether the chicken HI antibody at Day 42 met the criteria of the Japanese guidelines as a secondary endpoint.

In the phase II study, the target number of subjects was 50 in each group, for a total of 200, whereas the total number of enrolled subjects was 248, and the FAS included 246 subjects (62 for MA, 61 for HA, 63 for MB, 60 for HB). In the phase III study, the target number of subjects was 300, whereas the total number of subjects was 369, and the FAS included 364 subjects for MA. The target number of subjects in both studies was set according to the Japanese guidelines. Analyses were performed using SAS software version 9.2.

2.4. Safety evaluation

The safety evaluation protocols of the phase II and III studies were the same.

Using a health diary, solicited adverse events up to 7 days after each vaccination and unsolicited adverse events up to 21 days after each vaccination were recorded by subjects. Serious adverse events (SAE), important adverse events requiring cessation of vaccination (IAE) or adverse events including autoimmune diseases, and other inflammatory or autoimmune pathogenic neurological diseases (pIMDs: potential immune‐mediated diseases) were recorded during the study for another 6 months after the last vaccination (Day 0‐Day 201). Adverse events were graded in three levels; (a) mild (event that is easily tolerable, accompanied by only slight discomfort and does not interrupt daily activities); (b) moderate (event that interrupts daily activities because of discomfort); and (c) severe (event that makes daily activities impossible). Solicited adverse events were divided into two types, local and systemic adverse events. Solicited local adverse events included injection site erythema, swelling, and induration that occurred in the period following administration of the investigational drug until 6 days after administration. All solicited local adverse events were reactions at the injection site and so were categorized as adverse drug reactions. Solicited systemic adverse events included pyrexia, headache, fatigue, arthralgia, myalgia, chills, and hyperhidrosis that occurred in the period following administration of the investigational drug until 6 days after administration. The potential causal relationship between vaccination and other symptoms or events was determined by the investigator, and if a relationship was recognized, adverse events were categorized as adverse drug reactions. Statistical analyses were performed using SAS software version 9.2.

2.5. Study population

Demographic data in the FAS of the phase II and III studies are shown in Table 1. The mean ± SD for age in the FAS was 39.1 ± 11.1 years and 37.8 ± 11.2 years overall in the phase II and III studie,s respectively. No population imbalance was observed among the groups.

TABLE 1.

Demographic data in the phase II and III studies full analysis set (FAS)

Item	Statistics	Phase II									Phase III
Item	Statistics	MA group	HA group	MB group					HB group	Total	MA group
Number of analyzed subjects		62	61	63					60	246	364
Age (year)	Mean	39.5	39.3	38.9					38.7	39.1	37.8
	Standard deviation	12.9	10.4	10.9					10.4	11.1	11.2
	Minimum	20	22	21					20	20	20
	Median	36.0	39.0	38.0					37.0	37.0	37.5
	Maximum	64	63	62					64	64	64
Number of subjects （Incidence %）
Age	41 y and over	37 (59.7)	35(57.4)	36 (57.1)			35 (58.3)			143 (58.1)	222 (61.0)
	Less than 40 y	25 (40.3)	26 (42.6)		27 (42.9)		25 (41.7)			103 (41.9)	142 (39.0)
Sex	Male	27 (43.5)	28 (45.9)		29 (46.0)		28 (46.7)			112 (45.5)	179 (49.2)
	Female	35 (56.5)	33 (54.1)		34 (54.0)		32 (53.3)			134 (54.5)	185 (50.8)
Ethnicity	Japanese	62 (100.0)	61 (100.0)		63 (100.0)		60 (100.0)			246 (100.0)	364 (100.0)
	Other	0 (0.0)	0 (0.0)		0 (0.0)		0 (0.0)			0 (0.0)	0(0.0)
Medical history	No	58 (93.5)	56 (91.8)		57 (90.5)		55 (91.7)			226 (91.9)	342 (94.0)
	Yes	4 (6.5)	5 (8.2)		6 (9.5)		5 (8.3)			20 (8.1)	22(6.0)
Underlying disease	No	48 (77.4)	50 (82.0)			54 (85.7)		52 (86.7)		204(82.9)	285 (78.3)
	Yes	14 (22.6)	11 (18.0)			9 (14.3)		8 (13.3)		42 (17.1)	79 (21.7)
Allergy	No	56 (90.3)	51 (83.6)			57 (90.5)		54 (90.0)		218 (88.6)	289(79.4)
	Yes	6 (9.7)	10 (16.4)			6 (9.5)		6 (10.0)		28 (11.4)	75 (20.6)

Open in a new tab

MA group; 3.75 μg HA + AS03. HA group; 7.5 μg HA + AS03. MB group; 3.75 μg HA + 1/2 AS03. HB group; 7.5 μg HA + 1/2 AS03.

3. RESULTS

3.1. Immunogenicity

HI antibody against the vaccine strain was measured using horse and chicken erythrocytes in the phase II study. In the measurement using horse erythrocytes, the HI antibody response to the vaccine strain after the second vaccination (Day 42) in the FAS fulfilled all three criteria of immunogenicity described in the guidelines in all groups. For measurement using chicken erythrocytes, GMFR fulfilled the guideline criteria in all groups; however, SCR in the MB group and SPR in all groups did not fulfill the criteria. Since horse erythrocytes had higher sensitivity at HI antibody measurement, in the phase III study, only the horse erythrocyte assay was used. Results of the HI test for both studies are shown in Table 2. Although there was no large difference in the immunogenicity between the different vaccine formulations in the phase II study, the point estimate for GMFR was higher in the groups given the vaccine with the standard amount of AS03 (HA, MA) vs a half dose (MB, HB). The MA formulation was evaluated in the phase III study considering the safety data described later. In the phase III study, the MA formulation was highly immunogenic and it fulfilled all three criteria described in the guidelines.

TABLE 2.

Conformance of the parameters of the HI antibody (horse RBCs and chicken RBCs) response to the second vaccination (Day 42) with the three immunogenicity criteria of the guideline

Point estimate (95%CI)	Phase II								Phase III
Point estimate (95%CI)	MA group		HA group		MB group		HB group		MA group
Number of subjects	60		59		61		60		364
Horse red blood cells
Seroconversion rate >40%	100.0% (94.0%‐100.0%)	^*	100.0% (93.9%‐100.0%)	^*	100.0% (94.1%‐100.0%)	^*	98.3% (91.1%‐100.0%)	^*	100.0% (99.0‐100.0)	^*
Seroconversion rate >40%	100.0% (94.0%‐100.0%)	^*	100.0% (93.9%‐100.0%)	^*	28.56 (24.69‐33.04)	^*	98.3% (91.1%‐100.0%)	^*	100.0% (99.0‐100.0)	^*
Geometric mean fold rise >2.5	33.90 (28.82‐39.88)	^*	40.48 (34.39‐47.64)	^*	28.56 (24.69‐33.04)	^*	30.55 (25.44‐36.70)	^*	43.73 (41.15‐46.47)	^*
Geometric mean fold rise >2.5	33.90 (28.82‐39.88)	^*	40.48 (34.39‐47.64)	^*	(24.69‐33.04) 100.0% (94.1%‐100.0%)	^*	30.55 (25.44‐36.70)	^*	43.73 (41.15‐46.47)	^*
Seroprotection rate >70%	100.0% (94.0%‐100.0%)	^*	100.0% (93.9%‐100.0%)	^*	(24.69‐33.04) 100.0% (94.1%‐100.0%)	^*	98.3% (91.1%‐100.0%)	^*	100.0% (99.0‐100.0)	^*
GMT	169.5 (144.1‐199.4)		202.4 (171.9‐238.2)		142.8 (123.5‐165.2)		152.8 (127.2‐183.5)		220.3 (207.3‐234.1)
Chicken red blood cells
Seroconversion rate >40%	55.0% (41.6%‐67.9%)	^*	64.4% (50.9%‐76.4%)	^*	39.3% (27.1%‐52.7%)		56.7% (43.2%‐69.4%)	^*	‐
Geometric mean fold rise >2.5	6.20 (4.84‐7.96)	^*	7.37 (5.80‐9.36)	^*	4.38 (3.54‐5.42)	^*	6.20 (4.93‐7.81)	^*	‐
Seroprotection rate >70%	55.0% (41.6%‐67.9%)		64.4% (50.9%‐76.4%)		39.3% (27.1%‐52.7%)		56.7% (43.2%‐69.4%)		‐
GMT	31.0 (24.2‐39.8)		37.3 (29.4‐47.2)		21.9 (17.7‐27.1)		31.0 (24.6‐39.1)		‐

Open in a new tab

Strain measured: A/Indonesia/05/2005(H5N1). Vaccine strain: A/Indonesia/05/2005(H5N1). Subjects analyzed: a group of subjects who was collected blood after the second vaccination. Confidence interval (seroconversion rate, seroprotection rate):lower limit and upper limit of the exact 95% two‐sided confidence interval based on F‐distribution. Confidence interval (rate of change in GMT): lower limit and upper limit of the 95% two‐sided confidence interval based on Student's t‐distribution. MA group; 3.75 μg HA + AS03. HA group; 7.5 μg HA + AS03. MB group; 3.75μg HA+1/2 AS03. HB group; 7.5μg HA+1/2 AS03.

Fulfilled the immunogenicity criteria of the guideline.

GMT of neutralizing antibody and percentage of subjects with a 4‐fold increase in neutralizing antibody titers against vaccine strains after the 1st and 2nd vaccination in FAS are shown in Table 3. Both in the phase II and III studies, after the 1st dose, the seroconversion rates were 20‐30% and rose to nearly 100% after the second vaccination (Day 42). At the same time, GMT increased markedly in all groups after the second vaccination (Day 42).

TABLE 3.

Changes in geometric mean titer of neutralizing antibody to the vaccine strain

Timing

Phase II

Phase III

MA group

HA group

MB group

HB group

MA group

GMT

(Confidence interval)