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Safety, immunogenicity and dose response of VLA84, a new vaccine candidate against Clostridium difficile, in healthy volunteers
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Safety, immunogenicity and dose response of VLA84, a new vaccine candidate against Clostridium difficile, in healthy volunteers
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Nicole Bézay a , Andrea Ayad a , Katrin Dubischar a , Christa Firbas b , Romana Hochreiter a , Sigrid Kiermayr a , István Kiss c , Fritz Pinl d , Bernd Jilma b,∗ , Kerstin Westritschnig a a
Valneva Austria GmbH, Campus Vienna Biocenter 3, 1030 Vienna, Austria Department of Clinical Pharmacology, Medical University of Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria c St. Imre University Teaching Hospital, Department of Nephrology-Hypertension and Clinical Pharmacology, Tetenyi St. 12-16, 1115 Budapest, Hungary d Privatklinik Leech, Hugo-Wolf-Gasse 2-4, 8010 Graz, Austria
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Article history: Received 7 January 2016 Received in revised form 18 March 2016 Accepted 30 March 2016 Available online xxx
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Keywords: C. difficile C. difficile vaccine C. difficile associated diarrhoea (CDAD) C. difficile infection (CDI)
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1. Introduction
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Background: Clostridium difficile infection (CDI) is the leading cause of antibiotic-associated diarrhoea and colitis and the most common pathogen of health care-associated infections. In the US, CDI causes approximately half a million infections and close to 30,000 deaths. Despite antibiotic treatment of C. difficile associated diarrhoea, the disease is complicated by its recurrence in up to 30% of patients. Methods: An open-label, partially randomized, dose-escalation Phase I trial was performed in two parts. Sixty volunteers aged ≥18 to <65 years were randomized into five treatment groups to receive three immunizations (Day 0, 7, 21) of VLA84 (20 g with Alum, 75 g with or without Alum, 200 g with or without Alum). Eighty-one volunteers aged ≥65 were randomized into four treatment groups (75 g with or without Alum, 200 g with or without Alum) and received four immunizations (Day 0, 7, 28 and 56). All subjects were followed for safety and immunogenicity for six months. Results: VLA84 was safe and well tolerated. Fifty-one adult volunteers (85%) and 50 elderly (62%) experienced at least one solicited or unsolicited adverse event (AE). Forty-eight adult volunteers (80%) and 40 elderly (49%) experienced related AEs which were mostly mild or moderate. No related serious adverse event and no death occurred. The vaccine induced high antibody titres against Toxin A and Toxin B in both study populations. Conclusion: VLA84 was safe, well tolerated and highly immunogenic in adult volunteers aged ≥18 to <65 years and elderly volunteers aged ≥65 years. This study is registered at ClinicalTrials.gov under registration number NCT01296386. © 2016 Published by Elsevier Ltd.
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Clostridium difficile (C. difficile) is a Gram-positive, anaerobic, spore-forming bacterium that is transmitted between humans via the faecal-oral route. As a leading cause of antibiotic-associated diarrhoea and colitis, it is the most common pathogen of health care–associated infections [1–4]. Although the majority of hospitalized patients with C. difficile infection (CDI) are asymptomatic, they contribute to its circulation through release of spores. Antibiotic intake can disrupt the balance of the gut flora, resulting in colonization with C. difficile [2]. As a consequence, treatment of C. difficile associated diarrhoea with antibiotics might lead to complication
∗ Corresponding author. Tel.: +43 01 40400 29810. E-mail address: [email protected] (B. Jilma).
of disease by recurrence, which occurs in up to 30% of patients. It is estimated that 450,000 CDI cases occur annually in the US, and close to 30,000 patients die within 30 days of the diagnosis [5]. In Europe, approximately 172,000 CDI cases are reported each year [6]. Pathogenic C. difficile strains produce two highly potent exotoxins, Toxin A and Toxin B, which are both recognized as main virulence factors. Serum anti-toxin antibodies against Toxin A and Toxin B have shown to prevent C. difficile diarrhoea as well as recurrence of disease [7–10]. A vaccine targeting C. difficile Toxin A and Toxin B could prevent primary infection and/or recurrence of disease. We report results from a Phase I trial on VLA84, a recombinant fusion protein comprising relevant epitopes of C. difficile Toxin A (Enterotoxin) and Toxin B (Cytotoxin) under investigation as vaccine candidate against C. difficile, in healthy adults and elderly, the population at highest risk for acquiring CDI.
http://dx.doi.org/10.1016/j.vaccine.2016.03.098 0264-410X/© 2016 Published by Elsevier Ltd.
Please cite this article in press as: Bézay N, et al. Safety, immunogenicity and dose response of VLA84, a new vaccine candidate against Clostridium difficile, in healthy volunteers. Vaccine (2016), http://dx.doi.org/10.1016/j.vaccine.2016.03.098
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2. Methods
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2.1. Study design and study population
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This was an open-label, partially randomized, dose escalation Phase I trial conducted in two parts: Part A enrolled healthy adult volunteers aged ≥18 to <65 years, Part B healthy elderly volunteers aged ≥65 years. It was conducted in accordance with the Declaration of Helsinki and Good Clinical Practice (GCP). The trial was performed from December 28, 2010 to April 22, 2013 at three centres in Austria and Hungary. Primary objective was to investigate the safety and tolerability of VLA84 in different doses and formulations; immunogenicity and dose response were secondary objectives. Overall, 140 volunteers were to be enrolled, with written informed consent and no childbearing potential or negative pregnancy test and practicing an acceptable method of birth control. Main exclusion criteria were: history of immunodeficiency or ongoing immunosuppressive therapy, known history of anaphylaxis, human immunodeficiency virus (HIV) and hepatitis B/C infection, drug addiction including alcohol dependence or any active or passive vaccination within four weeks prior to first study vaccination. In Part A, 60 adults were to receive one of the following five treatments: VLA84 20 g with Alum, 75 g with or without Alum, or 200 g with or without Alum on Day 0, 7, 21. For safety reasons, the first 20 subjects were allocated to escalating doses (four subjects per treatment group); the remaining subjects were randomized to equal group sizes. In Part B, 80 elderly were randomized to four equal groups to receive VLA84 75 g with or without Alum, or 200 g without or without Alum on Day 0, 7, 28, 56. Immunizations were injected intramuscularly into the deltoid of the non-dominant arm. Prior to start of Part B, an interim analysis was conducted on safety and immunogenicity data from Day 28 of Part A, to allow modification of the study protocol. All volunteers were followed for six months after the last immunization. Safety and immunogenicity assessments were performed on Day 0, 7, 14, 21, 28, 113, 201 in Part A and on Day 0, 7, 14, 28, 56, 84, 236 in Part B.
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2.2. Study vaccine
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VLA84 is a purified recombinant protein produced using an Escherichia coli expression system. It is a fusion protein consisting of a portion of the C-terminal cell binding domains (CBDs) of Toxin B (23 of 24 CBD repeat sequences) and Toxin A (15 of 31 CBD repeat sequences). The two CBDs are joined by a 12 nt (4 amino acid) linker sequence. The Toxin A CBD is represented by nt 6817 to 8130 that encode a 438 amino acid portion of the Toxin A CBD (amino acids 2273–2710). The Toxin B truncated CBD is represented by nt 5613 to 7164 that encode 516 amino acids of Toxin B CBD (amino acids 1851–2366). Manufacturing processes were developed at Valneva Austria GmbH (formerly Intercell AG, Vienna, Austria) and transferred under GMP to suitable Contract Manufactures. The vaccine was formulated under GMP with or without aluminium hydroxide (Alum) under aseptic conditions and was provided at a protein concentration of 100 g/mL. VLA84 with Alum was stored at +2 ◦ C to +8 ◦ C, VLA84 without Alum was stored at −20 ◦ C ± 5 ◦ C. All dose levels were adjusted by volume, i.e. injection of 0.20 mL, 0.75 mL or 2× 1 mL (in close proximity), respectively.
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2.3. Evaluation of safety
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Volunteers were trained to record local and systemic adverse events (AEs) that occurred within the first seven days after each vaccination in subject diaries. The following symptoms were solicited: injection site pain, itching, tenderness, induration,
swelling, erythema; headache, muscle pain, fever, flu-like symptoms, nausea, vomiting, rash, excessive fatigue. Solicited AEs were by definition considered related. Any local or systemic AE with onset more than six days after vaccination or any symptoms or medical events not listed in the subject diary were unsolicited events. Unsolicited AEs were assessed for causality and graded for severity by the investigator. Volunteers underwent physical examinations at screening and symptom-directed physical examinations at each study visit. Safety laboratory parameters (haematology, coagulation, clinical chemistry, urinalysis) were assessed at each study visit. Local AEs were graded according to the FDA Guidance for Industry [11] as mild (Grade 1), moderate (Grade 2), severe (Grade 3) or potentially life threatening (Grade 4). For measurable local AEs, the following grading was applied: mild (2.5–5 cm), moderate (5.1–10 cm) or severe (>10 cm). AEs other than local AEs were graded according to the NCI-CTCAE v4.03, 2010, grading scale. 2.4. Evaluation of immunogenicity Human sera were analyzed separately for Toxin A-, Toxin B-, and VLA84-specific IgG by ELISA. Four-fold dilution series of sera prepared in blocking buffer (1× DPBS/0.05% Tween 20/2% BSA) were added to microtiter plates coated 12–72 h at 2–8 ◦ C with VLA84 or native toxins purified from VPI strain 10463 (Techlab, Blacksburg, Virginia, USA) diluted to 1 g/mL in 1× DPBS. Washing of plates between each step was done with 1× DPBS containing 0.05% Tween 20. After 1.5–2 h incubation at room temperature, the presence of specific IgGs was detected by incubation with a polyclonal rabbit anti-human IgG-HRP conjugate (Dako, Vienna, Austria) for 2 h at RT. After addition of TMB substrate (eBioscience, Vienna, Austria) for 20 min in the dark, plates were read with a Spectramax 190 plate reader. Quantification of specific IgGs was performed using SoftMax pro GxP software (Molecular Devices, Synnyvale, USA) by applying a four-parameter logistic (4PL) fit and parallel line analysis against the reference substance curve for each sample. Titres are reported in ELISA Units per mL (EU/mL), values below the quantitation limit of 50 EU/mL were imputed with 25 EU/mL. In addition, sera were analyzed in a toxin neutralization assay (TNA). Initially, a Verocell based TNA was used. Due to insufficient sensitivity, observed in the clinical trial, supportive data with an optimized T84 cell based TNA were generated. Sera from Day 0 and Day 28 (adults) or Day 56 (elderly) were measured. Serial serum dilutions were incubated with purified native Toxin A or Toxin B from VPI 10463 strain (ribotype 087) purchased from The Native Antigen Company Ltd. (Oxfordshire, UK) and added to a monolayer of T84 cells (human colon carcinoma derived cell line, ECACC Cat# 88021101). The optical density of amount of neutral red taken up by viable adherent T84 cells was used to determine the neutralization titre (NT50 ), i.e. the inverse value of the serum dilution which caused 50% protection of T84 cells from toxin-induced loss of adherence. A 4PL fit using SoftMax pro GxP software was applied. Correlation of respective ELISA and TNA titres was analyzed with two-sided, non-parametric Spearman test. 2.5. Statistical analyses No formal sample size calculation was performed. All volunteers who received at least one vaccination were included into safety and intent-to-treat (ITT) population. The per-protocol population (PP) excluded subjects with any major protocol deviation. The primary endpoint was safety and included the rates of volunteers with: related SAEs, related unsolicited or solicited AEs ≥ Grade 3, solicited local or systemic AEs, and unsolicited non-serious AEs. Rates were compared between groups using Fisher–Freeman–Halton test. Immunogenicity assessments included C. difficile Toxin A-, Toxin B- and VLA84-specific IgG levels, and analysis of Toxin
Please cite this article in press as: Bézay N, et al. Safety, immunogenicity and dose response of VLA84, a new vaccine candidate against Clostridium difficile, in healthy volunteers. Vaccine (2016), http://dx.doi.org/10.1016/j.vaccine.2016.03.098
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A/B-neutralizing antibody levels. Primary immunogenicity analyses were based on the PP population. Geometric Mean Titres (GMTs) and GMT ratios were estimated by applying an analysis of variance including factors treatment group and study site. Tukey’s HSD test was applied for pair-wise comparisons.
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3. Results
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3.1. Study population characteristics
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In Part A, 60 volunteers (mean age 30.8 years) received at least one vaccination and were included in the safety and ITT population; 57 volunteers were included in the PP population. One subject withdrew consent for personal reasons after Day 21 (75 g without Alum). In Part B, 81 volunteers (mean age 68.3 years) were enrolled, received at least one vaccination, and were included in the safety and ITT population. The elderly population was of generally good health and included volunteers with stable medical conditions such as hypertension (45/81, 56%) or hypothyroidism (14/81, 17%). Thirty-six subjects were excluded from the PP population (mainly due to time window deviations resulting from a temporary halting of vaccinations; see below). Four subjects withdrew consent for personal reasons (one subject from each treatment group, respectively). Table 1 displays the baseline characteristics of all volunteers.
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3.2. Safety results
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Overall, 51/60 (85%) adult and 50/81 (62%) elderly volunteers experienced at least one solicited or unsolicited AE, of which 48 (80%) adults and 40 (49%) elderly experienced related AEs. Most AEs were mild or moderate. Three adults (5.0%) experienced six severe related AEs, which were all solicited AEs (redness, hardening, headache). Five (6.2%) elderly experienced nine severe related AEs which were all solicited AEs (redness, injection site pain, fatigue, nausea, vomiting, fever). Two adults (3.3%) and one elderly (1.2%) experienced unsolicited AEs considered at least possibly related to study vaccine: moderate diarrhoea (on day of third immunization) and moderate lymphadenopathy (two days after second
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immunization), both in the adults; and moderate injection site rash (one day after second immunization) in the elderly. Overall, three unrelated SAEs were reported (missed abortion, coronary artery disease and gastrointestinal infection). No death was reported. Safety data from Day 28 of Part A, i.e. one week after all adult volunteers had received the last immunization, was reviewed by an independent DSMB that concluded that the vaccine was safe and well tolerated at all doses and formulations tested. During Part B, a temporary halt of vaccinations was triggered by fulfilment of a predefined stopping rule (occurrence of two Grade 3 AEs (i.e. injection site erythema) in one treatment group (i.e. 200 g without Alum). Consequently, the DSMB reviewed all available safety data from Part A and Part B and confirmed that further recruitment and vaccinations could proceed with increased safety monitoring (i.e. safety phone calls three days after each vaccination). No Grade 3 injection site erythema was reported thereafter. Tables 2a and 2b display solicited AEs and related unsolicited AEs. 3.3. Immunogenicity results High Toxin A- and Toxin B-specific IgG antibody titres were induced in all treatment groups and both study populations, with up to 227-fold and 119-fold increase of IgG antibodies for Toxin A and Toxin B, respectively. Peak levels of antibodies were observed at Day 28 (i.e. seven days after the last vaccination) in the adults and on Day 84 (i.e. 28 days after the last vaccination) in the elderly. In Part A, IgG antibody titres were much lower in the 20 g group compared to the higher dose groups. Consequently, the 20 g group was omitted in Part B. Tables 3a and 3b display the GMTs of all treatment groups and time-points for both study parts. Additionally, high levels of IgG antibodies against the fusion vaccine antigen VLA84 itself were induced and antibody levels against VLA84 correlated with antibody levels against Toxin A and Toxin B, respectively (data not shown). Fig. 1 displays the percentage of volunteers with at least fourfold increase (i.e. seroconversion) in antibody levels compared to baseline for Toxin A and Toxin B over time. In the adults, 100% of volunteers in the non-adjuvanted 75 g and 200 g groups achieved at least four-fold increase in Toxin A and Toxin B IgG titres one week after the last immunization and retained this elevation for at least
Treatment group VLA84 20 g with Alum
Total 75 g with Alum
75 g without Alum
200 g with Alum
200 g without Alum
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12
11
11
60
Gender, n (%) Female Male Age, years Mean (SD)
35 (12)
28 (3)
33 (11.0)
26 (6)
31 (7)
31 (9)
Ethnicity, n (%) Asian Causasian
1 (8) 12 (92)
0 (0) 13 (100)
0 (0) 12 (100)
0 (0) 11 (100)
0 (0) 11 (100)
1 (2) 59 (98)
Part B, ≥65 years N
21
20
20
20
81
Gender, n (%) Female Male
12 (57) 9 (43)
10 (50) 10 (50)
9 (45) 11 (55)
11 (55) 9 (45)
42 (52) 39 (48)
Age, years Mean (SD)
69 (4)
70 (4)
68 (2)
67 (2)
68 (3)
Ethnicity, n (%) Causasian
21 (100)
20 (100)
20 (100)
20 (100)
81 (100)
7 (54) 6 (46)
8 (62) 5 (39)
5 (42) 7 (58)
7 (64) 4 (36)
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Table 1 Study population baseline characteristics, safety population.
Part A, ≥18 to <65 years 13 N
208
5 (46) 6 (55)
32 (53) 28 (47)
N, number of subjects in treatment group; n, number of subjects; SD, standard deviation.
Please cite this article in press as: Bézay N, et al. Safety, immunogenicity and dose response of VLA84, a new vaccine candidate against Clostridium difficile, in healthy volunteers. Vaccine (2016), http://dx.doi.org/10.1016/j.vaccine.2016.03.098
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Table 2a Overview of related adverse events, Part A (≥18 to <65 years), safety population. Treatment group VLA84 20 g with Alum n (%)
75 g with Alum n (%)
75 g without Alum n (%)
200 g with Alum n (%)
200 g without Alum n (%)
N
13
13
12
11
11
Any related AE (solicited or unsolicited) Any related severe AE
12 (92) 0 (0)
10 (77) 0 (0)
9 (75) 0 (0)
11 (100) 2 (18)
Solicited adverse events, n (%) Any local solicited AE
6 (46)
10 (77)
6 (50)
Redness Injection Site pain Itching Hardening Swelling Tenderness
1 (8) 3 (23) 2 (15) 2 (15) 1 (8) 6 (46)
4 (31) 3 (23) 3 (23) 3 (23) 3 (23) 9 (69)
2 (17) 2 (17) 2 (17) 1 (8) 3 (25) 6 (50)
Any systemic solicited AE
8 (62)
5 (39)
Excessive fatigue Fever Flu-like symptoms Rash Headache Muscle pain Nausea Vomiting
4 (31) 1 (8) 4 (31) 0 (0) 5 (39) 3 (23) 3 (23) 2 (15)
Related unsolicited adverse events n (%)
0 (0)
p-value
Total n (%)
6 (55) 1 (9)
0.063 0.068
48 (80) 3 (5)
10 (91)
6 (55)
0.103
38 (63)
5 (46) 10 (91) 4 (36) 3 (27) 3 (27) 9 (82)
3 (27) 3 (27) 0 (0) 1 (9) 0 (0) 5 (46)
0.269 0.001 0.279 0.721 0.308 0.296
15 (25) 21 (35) 11 (18) 10 (17) 10 (17) 35 (58)
7 (58)
8 (73)
6 (55)
0.578
34 (57)
1 (8) 0 (0) 1 (8) 1 (8) 3 (23) 0 (0) 1 (8) 0 (0)
3 (25) 1 (8) 1 (8) 0 (0) 5 (42) 0 (0) 2 (17) 0 (0)
2 (18) 0 (0) 3 (27) 1 (9) 6 (55) 3 (27) 2 (18) 0 (0)
4 (36) 0 (0) 0 (0) 0 (0) 4 (36) 1 (9) 2 (18) 2 (18)
0.480 0.905 0.155 0.728 0.656 0.084 0.894 0.172
14 (23) 2 (3) 9 (15) 2 (3) 23 (38) 7 (12) 10 (17) 4 (7)
0 (0)
0 (0)
0 (0)
2 (18)
0.062
2 (3)
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N, number of subjects in treatment group; n, number of subjects. p-value was calculated by Fisher–Freeman–Halton test.
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six months. In the elderly, 100% of volunteers in the 75 g without Alum group and in both 200 g groups achieved at least four-fold increase in antibodies four weeks after the last immunization. This declined to 33–82% after six months. Functional antibodies were analyzed for the preferred treatment group in this trial (75 g without Alum). Functional antibodies
increased at least four-fold in 70% (Toxin A) and 80% (Toxin B) of the adults (Day 28), and 91% (Toxin A) and 55% (Toxin B) of the elderly (Day 56). TNA titres correlated significantly with corresponding ELISA titre (Toxin A: r = 0.8997, p < 0.0001 (adults), r = 0.8853, p < 0.0001 (elderly); Toxin B: r = 0.8192, p < 0.0001 (adults), r = 0.6809, p < 0.0001 (elderly).
Table 2b Overview of related adverse events, Part B (≥65 years), safety population. Treatment group VLA84 75 g with Alum n (%)
75 g without Alum n (%)
200 g with Alum n (%)
200 g without Alum n (%)
N
21
20
Any related AE (solicited or unsolicited) Any related severe AE
11 (52) 1 (5)
8 (40) 1 (5)
13 (65) 1 (5)
20
20 8 (40) 2 (10)
0.329 0.934
40 (49) 5 (6)
Solicited adverse event n (%) Any local solicited AE
8 (38)
4 (20)
11 (55)
6 (30)
0.135
29 (36)
Redness Injection site pain Itching Hardening Swelling Tenderness
2 (10) 5 (24) 2 (10) 1 (5) 1 (5) 7 (33)
3 (15) 2 (10) 2 (10) 1 (5) 1 (5) 4 (20)
4 (20) 8 (40) 4 (20) 4 (20) 2 (10) 10 (50)
3 (15) 5 (25) 3 (15) 3 (15) 4 (20) 6 (30)
0.791 0.194 0.775 0.369 0.428 0.270
12 (15) 20 (25) 11 (14) 9 (11) 8 (10) 27 (33)
Any systemic solicited AE
5 (24)
5 (25)
10 (50)
7 (35)
0.282
27 (33)
Excessive Fatigue Fever Flu-like symptoms Rash Headache Muscle pain Nausea Vomiting
3 (14) 0 (0) 2 (10) 2 (10) 5 (24) 5 (24) 1 (5) 1 (5)
3 (15) 1 (5) 2 (10) 1 (5) 2 (10) 2 (10) 0 (0) 0 (0)
7 (35) 2 (10) 6 (30) 1 (5) 6 (30) 4 (20) 4 (20) 1 (5)
3 (15) 2 (10) 4 (20) 0 (0) 3 (15) 3 (15) 1 (5) 0 (0)
0.323 0.472 0.306 0.899 0.406 0.762 0.132 1.000
16 (20) 5 (6) 14 (17) 4 (5) 16 (20) 14 (17) 6 (7) 2 (3)
Related unsolicited adverse events
0 (0)
0 (0)
0 (0)
1 (5)
0.741
1 (1)
p-value
Total n (%) 81
N, number of subjects in treatment group; n, number of subjects. p-value was calculated by Fisher–Freeman–Halton test.
Please cite this article in press as: Bézay N, et al. Safety, immunogenicity and dose response of VLA84, a new vaccine candidate against Clostridium difficile, in healthy volunteers. Vaccine (2016), http://dx.doi.org/10.1016/j.vaccine.2016.03.098
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Table 3a Geometric mean titre (GMT) for toxin-specific IgG, Part A (≥18 to <65 years), per-protocol population. Treatment group VLA84
N GMT Toxin A Day 0 (1st vacc.) Day 7 (2nd vacc.) Day 14 Day 21 (3rd vacc.) Day 28 Day 113 Day 201 Toxin B Day 0 (1st vacc.) Day 7 (2nd vacc.) Day 14 Day 21 (3rd vacc.) Day 28 Day 113 Day 201
20 g with Alum [EU/mL] [95% CI]
75 g with Alum [EU/mL] [95% CI]
75 g without Alum [EU/mL] [95% CI]
200 g with Alum [EU/mL] [95% CI]
200 g without Alum [EU/mL] [95% CI]
12
13
11a
11
10
36 [23, 56] 68 [25, 185] 215 [51, 914] 342 [104, 1131] 1763 [795, 3912] 681 [299, 1554] 360 [168, 774]
30 [20, 45] 39 [15, 104] 235 [57, 973] 661 [205, 2132] 3407 [1560, 7441] 1723 [767, 3867] 966 [456, 2045]
28 [22, 36] 51 [18, 146] 463 [101, 2135] 880 [249, 3106] 4958 [2063, 11920] 2177 [878, 5398] 1373 [591, 3190]
30 [22, 41] 37 [13, 106] 238 [52, 1098] 901 [255, 3180] 4604 [1986, 10675] 2370 [992, 5660] 1339 [597, 3003]
31 [22, 43] 54 [18, 167] 431 [84, 2198] 806 [210, 3095] 6968 [2841, 17094] 4370 [1726, 11065] 2316 [978, 5485]
51 [21, 122] 88 [22, 358] 196 [30, 1292] 189 [36, 999] 300 [79, 1144] 236 [71, 789] 175 [54, 568]
61 [29, 130] 84 [21, 331] 237 [37, 1505] 297 [58, 1522] 788 [212, 2931] 505 [155, 1649] 349 [110, 1110]
58 [23, 146] 117 [27, 511] 454 [62, 3322] 1116 [192, 6478] 6757 [1547, 29518] 2233 [591, 8437] 1487 [406, 5450]
76 [36, 162] 71 [16, 310] 160 [22, 1174] 222 [38, 1290] 796 [194, 3271] 421 [118, 1504] 236 [68, 818]
60 [20, 183] 171 [35, 831] 860 [103, 7195] 1363 [209, 8899] 5219 [1155, 23586] 2522 [648, 9822] 1502 [398, 5669]
Pair-wise comparisons
GMT ratio estimate [95% CI]
p-value
Toxin A Day 201
20 g with – 200 g without
0.16 [0.03, 0.77]
0.0153
Toxin B Day 28 Day 28
20 g with – 75 g without 20 g with – 200 g without
0.04 [0.00, 0.72] 0.06 [0.00, 0.95]
0.0213 0.0438
N, number of subjects in treatment group. a N = 10 after Day 21 (1 drop out after Day 21). ANOVA with fixed factors Treatment group and Study site (except Day 0). Only significant pair-wise comparisons in presence of a significant overall type 3 test derived from ANOVA are displayed.
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4. Discussion VLA84 is a new candidate vaccine against C. difficile. In contrast to other clinical-stage vaccines against C. difficile [12,13], which comprise two distinct toxoids, VLA84 is a single recombinant fusion
protein consisting of portions of C-terminal cell binding domains of Toxin A and Toxin B. Pre-clinical data demonstrate that VLA84 exhibits protective efficacy despite significant truncation [14]. Production, purification and characterization of VLA84 are anticipated to be less complex and costly compared to toxoid-based vaccines.
Fig. 1. Percentage of volunteers with ≥4-fold increase in antibody titres for Toxin A and Toxin B over time, Part A and Part B, per protocol population.
Please cite this article in press as: Bézay N, et al. Safety, immunogenicity and dose response of VLA84, a new vaccine candidate against Clostridium difficile, in healthy volunteers. Vaccine (2016), http://dx.doi.org/10.1016/j.vaccine.2016.03.098
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Table 3b Geometric mean titre (GMT) for toxin-specific IgG, Part B (≥65 years), per-protocol population. Treatment group VLA84
N GMT Toxin A Day 0 (1st vacc.) Day 7 (2nd vacc.) Day 14 Day 28 (3rd vacc.) Day 56 (4th vacc.) Day 84 Day 236 Toxin B Day 0 (1st vacc.) Day 7 (2nd vacc.) Day 14 Day 28 (3rd vacc.) Day 56 (4th vacc.) Day 84 Day 236
Toxin B Day 84
75 g with Alum [EU/mL] [95% CI]
75 g without Alum [EU/mL] [95% CI]
200 g with Alum [EU/mL] [95% CI]
200 g without Alum [EU/mL] [95% CI]
14a
11
12
8
29 [23, 37] 33 [23, 46] 114 [38, 341] 283 [102, 785] 2045 [883, 4737] 2526 [1361, 4690] 624 [373, 1044]
32 [24, 43] 43 [29, 63] 229 [67, 787] 414 [131, 1311] 4851 [1880, 12522] 6530 [3249, 13127] 1426 [814, 2496]
40 [25, 64] 32 [22, 46] 190 [58, 621] 598 [198, 1809] 5868 [2360, 14591] 7080 [3620, 13846] 1504 [878, 2577]
25 [25, 25] 26 [17, 42] 47 [11, 201] 70 [18, 274] 1686 [549, 5180] 3137 [1373, 7168] 1163 [599, 2256]
66 [39, 112] 67 [35, 127] 146 [42, 506] 138 [41, 467] 405 [157, 1041] 606 [289, 1268] 255 [120, 541]
59 [28, 121] 85 [41, 176] 202 [50, 823] 329 [83, 1307] 2724 [938, 7911] 3533 [1534, 8140] 747 [329, 1694]
90 [51, 160] 96 [48, 192] 377 [98, 1452] 463 [123, 1741] 1109 [398, 3089] 1847 [829, 4118] 506 [230, 1111]
64 [26, 161] 70 [30, 165] 109 [21, 577] 122 [24, 622] 1679 [475, 5932] 2675 [996, 7183] 875 [332, 2307]
Pair-wise comparisons
GMT ratio estimate [95% CI]
p-value
75 g with – 75 g without
0.17 [0.04, 0.75]
0.0141
N, number of subjects in treatment group. a N = 13 after Day 84 (1 drop out after Day 84). ANOVA with fixed factors treatment group and study site (except Day 0). Only significant pair-wise comparisons in presence of a significant overall type 3 test derived from ANOVA are displayed.
267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290
This Phase I trial assessed the safety and immunogenicity of the novel candidate. As elderly have the largest burden of CDI, safety and immunogenicity in persons 65 years and older was evaluated. Interim data in younger adults were used to initiate enrolment of the elderly. All doses and formulations were safe and well tolerated in both study populations. The most common AEs were typical for vaccines: injection site reactions like tenderness (58% adults, 33% elderly), pain (35% and 25%, respectively) and redness (25% and 15%, respectively), and systemic reactions like headache (38% adults, 20% elderly) and excessive fatigue (23% and 20%, respectively). Most AEs were mild or moderate. Severe, related AEs were reported by 5% (3/60) of adults and 6% (5/81) of elderly. Overall, severe redness, headache, hardening, fatigue, nausea, vomiting, pain, tenderness and fever (all solicited AEs), were reported. All severe local reactions occurred in the 200 g groups. Both the higher dose per se and the higher injection volume (2× 1 mL) might have contributed to these reactions. In addition, most severe reactions were experienced after the second immunization, possibly due to application of high volumes of vaccine within a short timeframe of seven days. Severe AEs were self-limited, except for one subject that was treated for nausea and vomiting. However, this subject experienced an unrelated gastrointestinal infection at the same time. Two elderly subjects withdrew consent after experiencing severe nausea, vomiting and
injection site pain, erythema, tenderness combined with fever. Local and systemic tolerability symptoms occurred less frequently in the elderly compared to the adults (63% versus 36% local AEs and 57% versus 33% systemic AEs), an effect also observed in other vaccines [15,16]. Overall, rates of related AEs (80%) and severe related AEs (5%) in the adults compare well with other recombinant protein vaccine candidates in Phase I testing [17]. VLA84 induced high levels of IgG antibodies against the protein antigen VLA84 itself, as well as against Toxin A and Toxin B, the toxins recognized as main virulence factors of CDI. As expected, the immune response in the adults was induced more rapidly compared to the elderly, however, IgG peak antibody titres in both populations were comparable. Preclinical data in mice suggested the use of aluminium hydroxide as adjuvant. In this Phase I trial however, non-adjuvanted vaccine formulations were at least as immunogenic as adjuvanted formulations. This effect was previously observed in other clinical trials with recombinant vaccine candidates [17,18], however, the reason is not yet fully understood. Humans, in contrast to naïve mice, might possess memory B cells from previous infections, that are stimulated by the nonadjuvanted antigen. Alternatively, protein epitopes might trigger co-stimulation of naïve B-cells itself, rendering alum adjuvantation non-essential. Such mechanisms of immune stimulation might
Please cite this article in press as: Bézay N, et al. Safety, immunogenicity and dose response of VLA84, a new vaccine candidate against Clostridium difficile, in healthy volunteers. Vaccine (2016), http://dx.doi.org/10.1016/j.vaccine.2016.03.098
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differ in humans and mice. In this Phase I trial, the non-adjuvanted 75 g group already induced a plateau immune response, the 200 g dose groups did not demonstrate higher immunogenicity. GMTs of the adjuvanted 75 g group were somewhat lower than GMTs of the non-adjuvanted formulation. Based on the tolerability and immunogenicity profiles, the non-adjuvanted 75 g group appeared to be the preferred treatment group. In this group, IgG levels against Toxin A and Toxin B declined to 20–25% of peak IgG levels within six months. This kinetic is in line with published data from a toxoid based vaccine against C. difficile [19,20]. Antibody functionality could be shown by TNA against both toxins in both populations. The predictive value of TNA on clinical efficacy (i.e. prevention of CDI) however has not yet been established in humans, only large efficacy studies will allow correlating TNA responder rates with clinical outcome. Kyne discussed a minimum anti-Toxin A IgG titre in their ELISA to prevent recurrence of CDI [7,8] or CDI mortality [21], however, no correlation to functional antibody titres has been shown. Generally, it is difficult to compare results obtained with different assay systems in different laboratories, since no international reference standard is available. To our knowledge, no reliable clinical meaningful assay is available to perform such a comparison between laboratories/studies and no correlate of protection has been established so far in an efficacy trial. Furthermore, although toxin neutralizing antibodies are considered to be the mode of action for prevention of CDI, antibody levels measured in the serum may not reflect the situation in the gut. The present Phase I trial was performed in a limited number of volunteers. A limitation of our study was that no control group (placebo group) was included in this trial. We consider that our study endpoints can be interpreted in the absence of a placebo group: rates of local/systemic reactions after vaccination can be fairly well compared with other vaccines; and our healthy study population was at low risk for CDI exposure, limiting the potential of natural boosting to impact immune responses. Before a final decision on dose and need for adjuvantation can be made, data have to be confirmed in an ongoing Phase II trial in a larger sample size in an elderly population, the main target population for a vaccine against C. difficile. 5. Conclusion
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We have demonstrated in this Phase I trial that VLA84, a new vaccine candidate against C. difficile, is safe and well tolerated in adult and elderly volunteers. VLA84 is highly immunogenic and induces high IgG antibody titres against Toxin A and Toxin B in both age groups. Functionality of antibodies was measured in a T84 TNA. These promising data generated in a limited number of volunteers further support evaluation of the candidate. A Phase II trial is ongoing to confirm optimal dose and formulation in a larger number of elderly volunteers.
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Acknowledgments
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We would like to thank all volunteers for their participation. We acknowledge the following individuals for their valuable contributions at various stages of this trial: Prof. Frank von Sonnenburg, Prof. Herwig Kollaritsch and Dr. Jakob Cramer for their role as members of the independent Data Safety Monitoring Board; Nicole Haas, Ines Pree and Christoph Klade (all formerly Intercell AG, Vienna, Austria) for contributions during study setup and conduct; Martin Spruth (formerly Intercell AG, Vienna, Austria) for serological testing.
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Authors’ contribution: Nicole Bézay, Andrea Ayad and Katrin Dubischar contributed to study design, study conduct and analysis, and interpretation of data. Christa Firbas contributed to acquisition
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of data in her role as sub-investigator. Romana Hochreiter and Sigrid Kiermayr were responsible for set up of the clinical assays and serological testing of clinical samples. István Kiss and Fritz Pinl contributed to acquisition of data in their role as principal investigators. Bernd Jilma served as studies coordinating principal investigator and contributed to acquisition of data as well as to data review. Kerstin Westritschnig served as Sponsors responsible medical officer and contributed in design of the study, study conduct and data analysis and interpretation. All authors contributed to drafting and reviewing the manuscript and approved the final version to be submitted for publication.
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Conflict of interest: Nicole Bézay, Andrea Ayad, Katrin Dubischar, Romana Hochreiter, Sigrid Kiermayr and Kerstin Westritschnig are employees of Valneva Austria GmbH (formerly Intercell AG). Christa Firbas participated as sub-investigator, Bernd Jilma, Fritz Pinl and István Kiss served as principal investigators in the trial, which was financially supported by Valneva Austria GmbH.
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[18] Wressnig N, Pöllabauer EM, Aichinger G, Portsmouth D, Löw-Baselli A, Fritsch S, et al. Safety and immunogenicity of a novel multivalent OspA vaccine against Lyme borreliosis in healthy adults: a double-blind, randomized, doseescalation phase 1/2 trial. Lancet Infect Dis 2013;13(August (8)):680–9. [19] Greenberg RN, Marbury TC, Foglia G, Warny M, Phase I. dose finding studies of an adjuvanted Clostridium difficile toxoid vaccine. Vaccine 2012;30:2245–9.
[20] Foglia G, Shah S, Luxemburger C, Pietrobon PJF. Clostridium difficile: development of a novel candidate vaccine. Vaccine 2012;30:4307–9. [21] Solomon K, Martin AJ, O’Donoghue C, Chen X, Fenelon L, Fanning S, et al. Mortality in patients with Clostridium difficile infection correlates with host pro-inflammatory and humoral immune responses. J Med Microbiol 2013;62:1453–60.
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Contents lists available at ScienceDirect
Vaccine journal homepage: www.elsevier.com/locate/vaccine
Safety, immunogenicity and dose response of VLA84, a new vaccine candidate against Clostridium difficile, in healthy volunteers
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Nicole Bézay a , Andrea Ayad a , Katrin Dubischar a , Christa Firbas b , Romana Hochreiter a , Sigrid Kiermayr a , István Kiss c , Fritz Pinl d , Bernd Jilma b,∗ , Kerstin Westritschnig a a
Valneva Austria GmbH, Campus Vienna Biocenter 3, 1030 Vienna, Austria Department of Clinical Pharmacology, Medical University of Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria c St. Imre University Teaching Hospital, Department of Nephrology-Hypertension and Clinical Pharmacology, Tetenyi St. 12-16, 1115 Budapest, Hungary d Privatklinik Leech, Hugo-Wolf-Gasse 2-4, 8010 Graz, Austria
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Article history: Received 7 January 2016 Received in revised form 18 March 2016 Accepted 30 March 2016 Available online xxx
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Keywords: C. difficile C. difficile vaccine C. difficile associated diarrhoea (CDAD) C. difficile infection (CDI)
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1. Introduction
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Background: Clostridium difficile infection (CDI) is the leading cause of antibiotic-associated diarrhoea and colitis and the most common pathogen of health care-associated infections. In the US, CDI causes approximately half a million infections and close to 30,000 deaths. Despite antibiotic treatment of C. difficile associated diarrhoea, the disease is complicated by its recurrence in up to 30% of patients. Methods: An open-label, partially randomized, dose-escalation Phase I trial was performed in two parts. Sixty volunteers aged ≥18 to <65 years were randomized into five treatment groups to receive three immunizations (Day 0, 7, 21) of VLA84 (20 g with Alum, 75 g with or without Alum, 200 g with or without Alum). Eighty-one volunteers aged ≥65 were randomized into four treatment groups (75 g with or without Alum, 200 g with or without Alum) and received four immunizations (Day 0, 7, 28 and 56). All subjects were followed for safety and immunogenicity for six months. Results: VLA84 was safe and well tolerated. Fifty-one adult volunteers (85%) and 50 elderly (62%) experienced at least one solicited or unsolicited adverse event (AE). Forty-eight adult volunteers (80%) and 40 elderly (49%) experienced related AEs which were mostly mild or moderate. No related serious adverse event and no death occurred. The vaccine induced high antibody titres against Toxin A and Toxin B in both study populations. Conclusion: VLA84 was safe, well tolerated and highly immunogenic in adult volunteers aged ≥18 to <65 years and elderly volunteers aged ≥65 years. This study is registered at ClinicalTrials.gov under registration number NCT01296386. © 2016 Published by Elsevier Ltd.
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Clostridium difficile (C. difficile) is a Gram-positive, anaerobic, spore-forming bacterium that is transmitted between humans via the faecal-oral route. As a leading cause of antibiotic-associated diarrhoea and colitis, it is the most common pathogen of health care–associated infections [1–4]. Although the majority of hospitalized patients with C. difficile infection (CDI) are asymptomatic, they contribute to its circulation through release of spores. Antibiotic intake can disrupt the balance of the gut flora, resulting in colonization with C. difficile [2]. As a consequence, treatment of C. difficile associated diarrhoea with antibiotics might lead to complication
∗ Corresponding author. Tel.: +43 01 40400 29810. E-mail address: [email protected] (B. Jilma).
of disease by recurrence, which occurs in up to 30% of patients. It is estimated that 450,000 CDI cases occur annually in the US, and close to 30,000 patients die within 30 days of the diagnosis [5]. In Europe, approximately 172,000 CDI cases are reported each year [6]. Pathogenic C. difficile strains produce two highly potent exotoxins, Toxin A and Toxin B, which are both recognized as main virulence factors. Serum anti-toxin antibodies against Toxin A and Toxin B have shown to prevent C. difficile diarrhoea as well as recurrence of disease [7–10]. A vaccine targeting C. difficile Toxin A and Toxin B could prevent primary infection and/or recurrence of disease. We report results from a Phase I trial on VLA84, a recombinant fusion protein comprising relevant epitopes of C. difficile Toxin A (Enterotoxin) and Toxin B (Cytotoxin) under investigation as vaccine candidate against C. difficile, in healthy adults and elderly, the population at highest risk for acquiring CDI.
http://dx.doi.org/10.1016/j.vaccine.2016.03.098 0264-410X/© 2016 Published by Elsevier Ltd.
Please cite this article in press as: Bézay N, et al. Safety, immunogenicity and dose response of VLA84, a new vaccine candidate against Clostridium difficile, in healthy volunteers. Vaccine (2016), http://dx.doi.org/10.1016/j.vaccine.2016.03.098
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2. Methods
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2.1. Study design and study population
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This was an open-label, partially randomized, dose escalation Phase I trial conducted in two parts: Part A enrolled healthy adult volunteers aged ≥18 to <65 years, Part B healthy elderly volunteers aged ≥65 years. It was conducted in accordance with the Declaration of Helsinki and Good Clinical Practice (GCP). The trial was performed from December 28, 2010 to April 22, 2013 at three centres in Austria and Hungary. Primary objective was to investigate the safety and tolerability of VLA84 in different doses and formulations; immunogenicity and dose response were secondary objectives. Overall, 140 volunteers were to be enrolled, with written informed consent and no childbearing potential or negative pregnancy test and practicing an acceptable method of birth control. Main exclusion criteria were: history of immunodeficiency or ongoing immunosuppressive therapy, known history of anaphylaxis, human immunodeficiency virus (HIV) and hepatitis B/C infection, drug addiction including alcohol dependence or any active or passive vaccination within four weeks prior to first study vaccination. In Part A, 60 adults were to receive one of the following five treatments: VLA84 20 g with Alum, 75 g with or without Alum, or 200 g with or without Alum on Day 0, 7, 21. For safety reasons, the first 20 subjects were allocated to escalating doses (four subjects per treatment group); the remaining subjects were randomized to equal group sizes. In Part B, 80 elderly were randomized to four equal groups to receive VLA84 75 g with or without Alum, or 200 g without or without Alum on Day 0, 7, 28, 56. Immunizations were injected intramuscularly into the deltoid of the non-dominant arm. Prior to start of Part B, an interim analysis was conducted on safety and immunogenicity data from Day 28 of Part A, to allow modification of the study protocol. All volunteers were followed for six months after the last immunization. Safety and immunogenicity assessments were performed on Day 0, 7, 14, 21, 28, 113, 201 in Part A and on Day 0, 7, 14, 28, 56, 84, 236 in Part B.
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2.2. Study vaccine
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VLA84 is a purified recombinant protein produced using an Escherichia coli expression system. It is a fusion protein consisting of a portion of the C-terminal cell binding domains (CBDs) of Toxin B (23 of 24 CBD repeat sequences) and Toxin A (15 of 31 CBD repeat sequences). The two CBDs are joined by a 12 nt (4 amino acid) linker sequence. The Toxin A CBD is represented by nt 6817 to 8130 that encode a 438 amino acid portion of the Toxin A CBD (amino acids 2273–2710). The Toxin B truncated CBD is represented by nt 5613 to 7164 that encode 516 amino acids of Toxin B CBD (amino acids 1851–2366). Manufacturing processes were developed at Valneva Austria GmbH (formerly Intercell AG, Vienna, Austria) and transferred under GMP to suitable Contract Manufactures. The vaccine was formulated under GMP with or without aluminium hydroxide (Alum) under aseptic conditions and was provided at a protein concentration of 100 g/mL. VLA84 with Alum was stored at +2 ◦ C to +8 ◦ C, VLA84 without Alum was stored at −20 ◦ C ± 5 ◦ C. All dose levels were adjusted by volume, i.e. injection of 0.20 mL, 0.75 mL or 2× 1 mL (in close proximity), respectively.
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2.3. Evaluation of safety
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Volunteers were trained to record local and systemic adverse events (AEs) that occurred within the first seven days after each vaccination in subject diaries. The following symptoms were solicited: injection site pain, itching, tenderness, induration,
swelling, erythema; headache, muscle pain, fever, flu-like symptoms, nausea, vomiting, rash, excessive fatigue. Solicited AEs were by definition considered related. Any local or systemic AE with onset more than six days after vaccination or any symptoms or medical events not listed in the subject diary were unsolicited events. Unsolicited AEs were assessed for causality and graded for severity by the investigator. Volunteers underwent physical examinations at screening and symptom-directed physical examinations at each study visit. Safety laboratory parameters (haematology, coagulation, clinical chemistry, urinalysis) were assessed at each study visit. Local AEs were graded according to the FDA Guidance for Industry [11] as mild (Grade 1), moderate (Grade 2), severe (Grade 3) or potentially life threatening (Grade 4). For measurable local AEs, the following grading was applied: mild (2.5–5 cm), moderate (5.1–10 cm) or severe (>10 cm). AEs other than local AEs were graded according to the NCI-CTCAE v4.03, 2010, grading scale. 2.4. Evaluation of immunogenicity Human sera were analyzed separately for Toxin A-, Toxin B-, and VLA84-specific IgG by ELISA. Four-fold dilution series of sera prepared in blocking buffer (1× DPBS/0.05% Tween 20/2% BSA) were added to microtiter plates coated 12–72 h at 2–8 ◦ C with VLA84 or native toxins purified from VPI strain 10463 (Techlab, Blacksburg, Virginia, USA) diluted to 1 g/mL in 1× DPBS. Washing of plates between each step was done with 1× DPBS containing 0.05% Tween 20. After 1.5–2 h incubation at room temperature, the presence of specific IgGs was detected by incubation with a polyclonal rabbit anti-human IgG-HRP conjugate (Dako, Vienna, Austria) for 2 h at RT. After addition of TMB substrate (eBioscience, Vienna, Austria) for 20 min in the dark, plates were read with a Spectramax 190 plate reader. Quantification of specific IgGs was performed using SoftMax pro GxP software (Molecular Devices, Synnyvale, USA) by applying a four-parameter logistic (4PL) fit and parallel line analysis against the reference substance curve for each sample. Titres are reported in ELISA Units per mL (EU/mL), values below the quantitation limit of 50 EU/mL were imputed with 25 EU/mL. In addition, sera were analyzed in a toxin neutralization assay (TNA). Initially, a Verocell based TNA was used. Due to insufficient sensitivity, observed in the clinical trial, supportive data with an optimized T84 cell based TNA were generated. Sera from Day 0 and Day 28 (adults) or Day 56 (elderly) were measured. Serial serum dilutions were incubated with purified native Toxin A or Toxin B from VPI 10463 strain (ribotype 087) purchased from The Native Antigen Company Ltd. (Oxfordshire, UK) and added to a monolayer of T84 cells (human colon carcinoma derived cell line, ECACC Cat# 88021101). The optical density of amount of neutral red taken up by viable adherent T84 cells was used to determine the neutralization titre (NT50 ), i.e. the inverse value of the serum dilution which caused 50% protection of T84 cells from toxin-induced loss of adherence. A 4PL fit using SoftMax pro GxP software was applied. Correlation of respective ELISA and TNA titres was analyzed with two-sided, non-parametric Spearman test. 2.5. Statistical analyses No formal sample size calculation was performed. All volunteers who received at least one vaccination were included into safety and intent-to-treat (ITT) population. The per-protocol population (PP) excluded subjects with any major protocol deviation. The primary endpoint was safety and included the rates of volunteers with: related SAEs, related unsolicited or solicited AEs ≥ Grade 3, solicited local or systemic AEs, and unsolicited non-serious AEs. Rates were compared between groups using Fisher–Freeman–Halton test. Immunogenicity assessments included C. difficile Toxin A-, Toxin B- and VLA84-specific IgG levels, and analysis of Toxin
Please cite this article in press as: Bézay N, et al. Safety, immunogenicity and dose response of VLA84, a new vaccine candidate against Clostridium difficile, in healthy volunteers. Vaccine (2016), http://dx.doi.org/10.1016/j.vaccine.2016.03.098
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A/B-neutralizing antibody levels. Primary immunogenicity analyses were based on the PP population. Geometric Mean Titres (GMTs) and GMT ratios were estimated by applying an analysis of variance including factors treatment group and study site. Tukey’s HSD test was applied for pair-wise comparisons.
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3. Results
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3.1. Study population characteristics
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In Part A, 60 volunteers (mean age 30.8 years) received at least one vaccination and were included in the safety and ITT population; 57 volunteers were included in the PP population. One subject withdrew consent for personal reasons after Day 21 (75 g without Alum). In Part B, 81 volunteers (mean age 68.3 years) were enrolled, received at least one vaccination, and were included in the safety and ITT population. The elderly population was of generally good health and included volunteers with stable medical conditions such as hypertension (45/81, 56%) or hypothyroidism (14/81, 17%). Thirty-six subjects were excluded from the PP population (mainly due to time window deviations resulting from a temporary halting of vaccinations; see below). Four subjects withdrew consent for personal reasons (one subject from each treatment group, respectively). Table 1 displays the baseline characteristics of all volunteers.
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3.2. Safety results
180 181 182 183 184 185 186 187 188 189 190 191 192 193 194
197 198 199 200 201 202 203 204 205 206 207
Overall, 51/60 (85%) adult and 50/81 (62%) elderly volunteers experienced at least one solicited or unsolicited AE, of which 48 (80%) adults and 40 (49%) elderly experienced related AEs. Most AEs were mild or moderate. Three adults (5.0%) experienced six severe related AEs, which were all solicited AEs (redness, hardening, headache). Five (6.2%) elderly experienced nine severe related AEs which were all solicited AEs (redness, injection site pain, fatigue, nausea, vomiting, fever). Two adults (3.3%) and one elderly (1.2%) experienced unsolicited AEs considered at least possibly related to study vaccine: moderate diarrhoea (on day of third immunization) and moderate lymphadenopathy (two days after second
3
immunization), both in the adults; and moderate injection site rash (one day after second immunization) in the elderly. Overall, three unrelated SAEs were reported (missed abortion, coronary artery disease and gastrointestinal infection). No death was reported. Safety data from Day 28 of Part A, i.e. one week after all adult volunteers had received the last immunization, was reviewed by an independent DSMB that concluded that the vaccine was safe and well tolerated at all doses and formulations tested. During Part B, a temporary halt of vaccinations was triggered by fulfilment of a predefined stopping rule (occurrence of two Grade 3 AEs (i.e. injection site erythema) in one treatment group (i.e. 200 g without Alum). Consequently, the DSMB reviewed all available safety data from Part A and Part B and confirmed that further recruitment and vaccinations could proceed with increased safety monitoring (i.e. safety phone calls three days after each vaccination). No Grade 3 injection site erythema was reported thereafter. Tables 2a and 2b display solicited AEs and related unsolicited AEs. 3.3. Immunogenicity results High Toxin A- and Toxin B-specific IgG antibody titres were induced in all treatment groups and both study populations, with up to 227-fold and 119-fold increase of IgG antibodies for Toxin A and Toxin B, respectively. Peak levels of antibodies were observed at Day 28 (i.e. seven days after the last vaccination) in the adults and on Day 84 (i.e. 28 days after the last vaccination) in the elderly. In Part A, IgG antibody titres were much lower in the 20 g group compared to the higher dose groups. Consequently, the 20 g group was omitted in Part B. Tables 3a and 3b display the GMTs of all treatment groups and time-points for both study parts. Additionally, high levels of IgG antibodies against the fusion vaccine antigen VLA84 itself were induced and antibody levels against VLA84 correlated with antibody levels against Toxin A and Toxin B, respectively (data not shown). Fig. 1 displays the percentage of volunteers with at least fourfold increase (i.e. seroconversion) in antibody levels compared to baseline for Toxin A and Toxin B over time. In the adults, 100% of volunteers in the non-adjuvanted 75 g and 200 g groups achieved at least four-fold increase in Toxin A and Toxin B IgG titres one week after the last immunization and retained this elevation for at least
Treatment group VLA84 20 g with Alum
Total 75 g with Alum
75 g without Alum
200 g with Alum
200 g without Alum
13
12
11
11
60
Gender, n (%) Female Male Age, years Mean (SD)
35 (12)
28 (3)
33 (11.0)
26 (6)
31 (7)
31 (9)
Ethnicity, n (%) Asian Causasian
1 (8) 12 (92)
0 (0) 13 (100)
0 (0) 12 (100)
0 (0) 11 (100)
0 (0) 11 (100)
1 (2) 59 (98)
Part B, ≥65 years N
21
20
20
20
81
Gender, n (%) Female Male
12 (57) 9 (43)
10 (50) 10 (50)
9 (45) 11 (55)
11 (55) 9 (45)
42 (52) 39 (48)
Age, years Mean (SD)
69 (4)
70 (4)
68 (2)
67 (2)
68 (3)
Ethnicity, n (%) Causasian
21 (100)
20 (100)
20 (100)
20 (100)
81 (100)
7 (54) 6 (46)
8 (62) 5 (39)
5 (42) 7 (58)
7 (64) 4 (36)
209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224
225
Table 1 Study population baseline characteristics, safety population.
Part A, ≥18 to <65 years 13 N
208
5 (46) 6 (55)
32 (53) 28 (47)
N, number of subjects in treatment group; n, number of subjects; SD, standard deviation.
Please cite this article in press as: Bézay N, et al. Safety, immunogenicity and dose response of VLA84, a new vaccine candidate against Clostridium difficile, in healthy volunteers. Vaccine (2016), http://dx.doi.org/10.1016/j.vaccine.2016.03.098
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Table 2a Overview of related adverse events, Part A (≥18 to <65 years), safety population. Treatment group VLA84 20 g with Alum n (%)
75 g with Alum n (%)
75 g without Alum n (%)
200 g with Alum n (%)
200 g without Alum n (%)
N
13
13
12
11
11
Any related AE (solicited or unsolicited) Any related severe AE
12 (92) 0 (0)
10 (77) 0 (0)
9 (75) 0 (0)
11 (100) 2 (18)
Solicited adverse events, n (%) Any local solicited AE
6 (46)
10 (77)
6 (50)
Redness Injection Site pain Itching Hardening Swelling Tenderness
1 (8) 3 (23) 2 (15) 2 (15) 1 (8) 6 (46)
4 (31) 3 (23) 3 (23) 3 (23) 3 (23) 9 (69)
2 (17) 2 (17) 2 (17) 1 (8) 3 (25) 6 (50)
Any systemic solicited AE
8 (62)
5 (39)
Excessive fatigue Fever Flu-like symptoms Rash Headache Muscle pain Nausea Vomiting
4 (31) 1 (8) 4 (31) 0 (0) 5 (39) 3 (23) 3 (23) 2 (15)
Related unsolicited adverse events n (%)
0 (0)
p-value
Total n (%)
6 (55) 1 (9)
0.063 0.068
48 (80) 3 (5)
10 (91)
6 (55)
0.103
38 (63)
5 (46) 10 (91) 4 (36) 3 (27) 3 (27) 9 (82)
3 (27) 3 (27) 0 (0) 1 (9) 0 (0) 5 (46)
0.269 0.001 0.279 0.721 0.308 0.296
15 (25) 21 (35) 11 (18) 10 (17) 10 (17) 35 (58)
7 (58)
8 (73)
6 (55)
0.578
34 (57)
1 (8) 0 (0) 1 (8) 1 (8) 3 (23) 0 (0) 1 (8) 0 (0)
3 (25) 1 (8) 1 (8) 0 (0) 5 (42) 0 (0) 2 (17) 0 (0)
2 (18) 0 (0) 3 (27) 1 (9) 6 (55) 3 (27) 2 (18) 0 (0)
4 (36) 0 (0) 0 (0) 0 (0) 4 (36) 1 (9) 2 (18) 2 (18)
0.480 0.905 0.155 0.728 0.656 0.084 0.894 0.172
14 (23) 2 (3) 9 (15) 2 (3) 23 (38) 7 (12) 10 (17) 4 (7)
0 (0)
0 (0)
0 (0)
2 (18)
0.062
2 (3)
60
N, number of subjects in treatment group; n, number of subjects. p-value was calculated by Fisher–Freeman–Halton test.
246 247 248 249 250 251
six months. In the elderly, 100% of volunteers in the 75 g without Alum group and in both 200 g groups achieved at least four-fold increase in antibodies four weeks after the last immunization. This declined to 33–82% after six months. Functional antibodies were analyzed for the preferred treatment group in this trial (75 g without Alum). Functional antibodies
increased at least four-fold in 70% (Toxin A) and 80% (Toxin B) of the adults (Day 28), and 91% (Toxin A) and 55% (Toxin B) of the elderly (Day 56). TNA titres correlated significantly with corresponding ELISA titre (Toxin A: r = 0.8997, p < 0.0001 (adults), r = 0.8853, p < 0.0001 (elderly); Toxin B: r = 0.8192, p < 0.0001 (adults), r = 0.6809, p < 0.0001 (elderly).
Table 2b Overview of related adverse events, Part B (≥65 years), safety population. Treatment group VLA84 75 g with Alum n (%)
75 g without Alum n (%)
200 g with Alum n (%)
200 g without Alum n (%)
N
21
20
Any related AE (solicited or unsolicited) Any related severe AE
11 (52) 1 (5)
8 (40) 1 (5)
13 (65) 1 (5)
20
20 8 (40) 2 (10)
0.329 0.934
40 (49) 5 (6)
Solicited adverse event n (%) Any local solicited AE
8 (38)
4 (20)
11 (55)
6 (30)
0.135
29 (36)
Redness Injection site pain Itching Hardening Swelling Tenderness
2 (10) 5 (24) 2 (10) 1 (5) 1 (5) 7 (33)
3 (15) 2 (10) 2 (10) 1 (5) 1 (5) 4 (20)
4 (20) 8 (40) 4 (20) 4 (20) 2 (10) 10 (50)
3 (15) 5 (25) 3 (15) 3 (15) 4 (20) 6 (30)
0.791 0.194 0.775 0.369 0.428 0.270
12 (15) 20 (25) 11 (14) 9 (11) 8 (10) 27 (33)
Any systemic solicited AE
5 (24)
5 (25)
10 (50)
7 (35)
0.282
27 (33)
Excessive Fatigue Fever Flu-like symptoms Rash Headache Muscle pain Nausea Vomiting
3 (14) 0 (0) 2 (10) 2 (10) 5 (24) 5 (24) 1 (5) 1 (5)
3 (15) 1 (5) 2 (10) 1 (5) 2 (10) 2 (10) 0 (0) 0 (0)
7 (35) 2 (10) 6 (30) 1 (5) 6 (30) 4 (20) 4 (20) 1 (5)
3 (15) 2 (10) 4 (20) 0 (0) 3 (15) 3 (15) 1 (5) 0 (0)
0.323 0.472 0.306 0.899 0.406 0.762 0.132 1.000
16 (20) 5 (6) 14 (17) 4 (5) 16 (20) 14 (17) 6 (7) 2 (3)
Related unsolicited adverse events
0 (0)
0 (0)
0 (0)
1 (5)
0.741
1 (1)
p-value
Total n (%) 81
N, number of subjects in treatment group; n, number of subjects. p-value was calculated by Fisher–Freeman–Halton test.
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Table 3a Geometric mean titre (GMT) for toxin-specific IgG, Part A (≥18 to <65 years), per-protocol population. Treatment group VLA84
N GMT Toxin A Day 0 (1st vacc.) Day 7 (2nd vacc.) Day 14 Day 21 (3rd vacc.) Day 28 Day 113 Day 201 Toxin B Day 0 (1st vacc.) Day 7 (2nd vacc.) Day 14 Day 21 (3rd vacc.) Day 28 Day 113 Day 201
20 g with Alum [EU/mL] [95% CI]
75 g with Alum [EU/mL] [95% CI]
75 g without Alum [EU/mL] [95% CI]
200 g with Alum [EU/mL] [95% CI]
200 g without Alum [EU/mL] [95% CI]
12
13
11a
11
10
36 [23, 56] 68 [25, 185] 215 [51, 914] 342 [104, 1131] 1763 [795, 3912] 681 [299, 1554] 360 [168, 774]
30 [20, 45] 39 [15, 104] 235 [57, 973] 661 [205, 2132] 3407 [1560, 7441] 1723 [767, 3867] 966 [456, 2045]
28 [22, 36] 51 [18, 146] 463 [101, 2135] 880 [249, 3106] 4958 [2063, 11920] 2177 [878, 5398] 1373 [591, 3190]
30 [22, 41] 37 [13, 106] 238 [52, 1098] 901 [255, 3180] 4604 [1986, 10675] 2370 [992, 5660] 1339 [597, 3003]
31 [22, 43] 54 [18, 167] 431 [84, 2198] 806 [210, 3095] 6968 [2841, 17094] 4370 [1726, 11065] 2316 [978, 5485]
51 [21, 122] 88 [22, 358] 196 [30, 1292] 189 [36, 999] 300 [79, 1144] 236 [71, 789] 175 [54, 568]
61 [29, 130] 84 [21, 331] 237 [37, 1505] 297 [58, 1522] 788 [212, 2931] 505 [155, 1649] 349 [110, 1110]
58 [23, 146] 117 [27, 511] 454 [62, 3322] 1116 [192, 6478] 6757 [1547, 29518] 2233 [591, 8437] 1487 [406, 5450]
76 [36, 162] 71 [16, 310] 160 [22, 1174] 222 [38, 1290] 796 [194, 3271] 421 [118, 1504] 236 [68, 818]
60 [20, 183] 171 [35, 831] 860 [103, 7195] 1363 [209, 8899] 5219 [1155, 23586] 2522 [648, 9822] 1502 [398, 5669]
Pair-wise comparisons
GMT ratio estimate [95% CI]
p-value
Toxin A Day 201
20 g with – 200 g without
0.16 [0.03, 0.77]
0.0153
Toxin B Day 28 Day 28
20 g with – 75 g without 20 g with – 200 g without
0.04 [0.00, 0.72] 0.06 [0.00, 0.95]
0.0213 0.0438
N, number of subjects in treatment group. a N = 10 after Day 21 (1 drop out after Day 21). ANOVA with fixed factors Treatment group and Study site (except Day 0). Only significant pair-wise comparisons in presence of a significant overall type 3 test derived from ANOVA are displayed.
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259 260 261
4. Discussion VLA84 is a new candidate vaccine against C. difficile. In contrast to other clinical-stage vaccines against C. difficile [12,13], which comprise two distinct toxoids, VLA84 is a single recombinant fusion
protein consisting of portions of C-terminal cell binding domains of Toxin A and Toxin B. Pre-clinical data demonstrate that VLA84 exhibits protective efficacy despite significant truncation [14]. Production, purification and characterization of VLA84 are anticipated to be less complex and costly compared to toxoid-based vaccines.
Fig. 1. Percentage of volunteers with ≥4-fold increase in antibody titres for Toxin A and Toxin B over time, Part A and Part B, per protocol population.
Please cite this article in press as: Bézay N, et al. Safety, immunogenicity and dose response of VLA84, a new vaccine candidate against Clostridium difficile, in healthy volunteers. Vaccine (2016), http://dx.doi.org/10.1016/j.vaccine.2016.03.098
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Table 3b Geometric mean titre (GMT) for toxin-specific IgG, Part B (≥65 years), per-protocol population. Treatment group VLA84
N GMT Toxin A Day 0 (1st vacc.) Day 7 (2nd vacc.) Day 14 Day 28 (3rd vacc.) Day 56 (4th vacc.) Day 84 Day 236 Toxin B Day 0 (1st vacc.) Day 7 (2nd vacc.) Day 14 Day 28 (3rd vacc.) Day 56 (4th vacc.) Day 84 Day 236
Toxin B Day 84
75 g with Alum [EU/mL] [95% CI]
75 g without Alum [EU/mL] [95% CI]
200 g with Alum [EU/mL] [95% CI]
200 g without Alum [EU/mL] [95% CI]
14a
11
12
8
29 [23, 37] 33 [23, 46] 114 [38, 341] 283 [102, 785] 2045 [883, 4737] 2526 [1361, 4690] 624 [373, 1044]
32 [24, 43] 43 [29, 63] 229 [67, 787] 414 [131, 1311] 4851 [1880, 12522] 6530 [3249, 13127] 1426 [814, 2496]
40 [25, 64] 32 [22, 46] 190 [58, 621] 598 [198, 1809] 5868 [2360, 14591] 7080 [3620, 13846] 1504 [878, 2577]
25 [25, 25] 26 [17, 42] 47 [11, 201] 70 [18, 274] 1686 [549, 5180] 3137 [1373, 7168] 1163 [599, 2256]
66 [39, 112] 67 [35, 127] 146 [42, 506] 138 [41, 467] 405 [157, 1041] 606 [289, 1268] 255 [120, 541]
59 [28, 121] 85 [41, 176] 202 [50, 823] 329 [83, 1307] 2724 [938, 7911] 3533 [1534, 8140] 747 [329, 1694]
90 [51, 160] 96 [48, 192] 377 [98, 1452] 463 [123, 1741] 1109 [398, 3089] 1847 [829, 4118] 506 [230, 1111]
64 [26, 161] 70 [30, 165] 109 [21, 577] 122 [24, 622] 1679 [475, 5932] 2675 [996, 7183] 875 [332, 2307]
Pair-wise comparisons
GMT ratio estimate [95% CI]
p-value
75 g with – 75 g without
0.17 [0.04, 0.75]
0.0141
N, number of subjects in treatment group. a N = 13 after Day 84 (1 drop out after Day 84). ANOVA with fixed factors treatment group and study site (except Day 0). Only significant pair-wise comparisons in presence of a significant overall type 3 test derived from ANOVA are displayed.
267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290
This Phase I trial assessed the safety and immunogenicity of the novel candidate. As elderly have the largest burden of CDI, safety and immunogenicity in persons 65 years and older was evaluated. Interim data in younger adults were used to initiate enrolment of the elderly. All doses and formulations were safe and well tolerated in both study populations. The most common AEs were typical for vaccines: injection site reactions like tenderness (58% adults, 33% elderly), pain (35% and 25%, respectively) and redness (25% and 15%, respectively), and systemic reactions like headache (38% adults, 20% elderly) and excessive fatigue (23% and 20%, respectively). Most AEs were mild or moderate. Severe, related AEs were reported by 5% (3/60) of adults and 6% (5/81) of elderly. Overall, severe redness, headache, hardening, fatigue, nausea, vomiting, pain, tenderness and fever (all solicited AEs), were reported. All severe local reactions occurred in the 200 g groups. Both the higher dose per se and the higher injection volume (2× 1 mL) might have contributed to these reactions. In addition, most severe reactions were experienced after the second immunization, possibly due to application of high volumes of vaccine within a short timeframe of seven days. Severe AEs were self-limited, except for one subject that was treated for nausea and vomiting. However, this subject experienced an unrelated gastrointestinal infection at the same time. Two elderly subjects withdrew consent after experiencing severe nausea, vomiting and
injection site pain, erythema, tenderness combined with fever. Local and systemic tolerability symptoms occurred less frequently in the elderly compared to the adults (63% versus 36% local AEs and 57% versus 33% systemic AEs), an effect also observed in other vaccines [15,16]. Overall, rates of related AEs (80%) and severe related AEs (5%) in the adults compare well with other recombinant protein vaccine candidates in Phase I testing [17]. VLA84 induced high levels of IgG antibodies against the protein antigen VLA84 itself, as well as against Toxin A and Toxin B, the toxins recognized as main virulence factors of CDI. As expected, the immune response in the adults was induced more rapidly compared to the elderly, however, IgG peak antibody titres in both populations were comparable. Preclinical data in mice suggested the use of aluminium hydroxide as adjuvant. In this Phase I trial however, non-adjuvanted vaccine formulations were at least as immunogenic as adjuvanted formulations. This effect was previously observed in other clinical trials with recombinant vaccine candidates [17,18], however, the reason is not yet fully understood. Humans, in contrast to naïve mice, might possess memory B cells from previous infections, that are stimulated by the nonadjuvanted antigen. Alternatively, protein epitopes might trigger co-stimulation of naïve B-cells itself, rendering alum adjuvantation non-essential. Such mechanisms of immune stimulation might
Please cite this article in press as: Bézay N, et al. Safety, immunogenicity and dose response of VLA84, a new vaccine candidate against Clostridium difficile, in healthy volunteers. Vaccine (2016), http://dx.doi.org/10.1016/j.vaccine.2016.03.098
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differ in humans and mice. In this Phase I trial, the non-adjuvanted 75 g group already induced a plateau immune response, the 200 g dose groups did not demonstrate higher immunogenicity. GMTs of the adjuvanted 75 g group were somewhat lower than GMTs of the non-adjuvanted formulation. Based on the tolerability and immunogenicity profiles, the non-adjuvanted 75 g group appeared to be the preferred treatment group. In this group, IgG levels against Toxin A and Toxin B declined to 20–25% of peak IgG levels within six months. This kinetic is in line with published data from a toxoid based vaccine against C. difficile [19,20]. Antibody functionality could be shown by TNA against both toxins in both populations. The predictive value of TNA on clinical efficacy (i.e. prevention of CDI) however has not yet been established in humans, only large efficacy studies will allow correlating TNA responder rates with clinical outcome. Kyne discussed a minimum anti-Toxin A IgG titre in their ELISA to prevent recurrence of CDI [7,8] or CDI mortality [21], however, no correlation to functional antibody titres has been shown. Generally, it is difficult to compare results obtained with different assay systems in different laboratories, since no international reference standard is available. To our knowledge, no reliable clinical meaningful assay is available to perform such a comparison between laboratories/studies and no correlate of protection has been established so far in an efficacy trial. Furthermore, although toxin neutralizing antibodies are considered to be the mode of action for prevention of CDI, antibody levels measured in the serum may not reflect the situation in the gut. The present Phase I trial was performed in a limited number of volunteers. A limitation of our study was that no control group (placebo group) was included in this trial. We consider that our study endpoints can be interpreted in the absence of a placebo group: rates of local/systemic reactions after vaccination can be fairly well compared with other vaccines; and our healthy study population was at low risk for CDI exposure, limiting the potential of natural boosting to impact immune responses. Before a final decision on dose and need for adjuvantation can be made, data have to be confirmed in an ongoing Phase II trial in a larger sample size in an elderly population, the main target population for a vaccine against C. difficile. 5. Conclusion
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We have demonstrated in this Phase I trial that VLA84, a new vaccine candidate against C. difficile, is safe and well tolerated in adult and elderly volunteers. VLA84 is highly immunogenic and induces high IgG antibody titres against Toxin A and Toxin B in both age groups. Functionality of antibodies was measured in a T84 TNA. These promising data generated in a limited number of volunteers further support evaluation of the candidate. A Phase II trial is ongoing to confirm optimal dose and formulation in a larger number of elderly volunteers.
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Acknowledgments
354 355 356 357 358 359 360 361
364 365 366 367 368 369 370 371
We would like to thank all volunteers for their participation. We acknowledge the following individuals for their valuable contributions at various stages of this trial: Prof. Frank von Sonnenburg, Prof. Herwig Kollaritsch and Dr. Jakob Cramer for their role as members of the independent Data Safety Monitoring Board; Nicole Haas, Ines Pree and Christoph Klade (all formerly Intercell AG, Vienna, Austria) for contributions during study setup and conduct; Martin Spruth (formerly Intercell AG, Vienna, Austria) for serological testing.
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Authors’ contribution: Nicole Bézay, Andrea Ayad and Katrin Dubischar contributed to study design, study conduct and analysis, and interpretation of data. Christa Firbas contributed to acquisition
7
of data in her role as sub-investigator. Romana Hochreiter and Sigrid Kiermayr were responsible for set up of the clinical assays and serological testing of clinical samples. István Kiss and Fritz Pinl contributed to acquisition of data in their role as principal investigators. Bernd Jilma served as studies coordinating principal investigator and contributed to acquisition of data as well as to data review. Kerstin Westritschnig served as Sponsors responsible medical officer and contributed in design of the study, study conduct and data analysis and interpretation. All authors contributed to drafting and reviewing the manuscript and approved the final version to be submitted for publication.
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386
Conflict of interest: Nicole Bézay, Andrea Ayad, Katrin Dubischar, Romana Hochreiter, Sigrid Kiermayr and Kerstin Westritschnig are employees of Valneva Austria GmbH (formerly Intercell AG). Christa Firbas participated as sub-investigator, Bernd Jilma, Fritz Pinl and István Kiss served as principal investigators in the trial, which was financially supported by Valneva Austria GmbH.
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Please cite this article in press as: Bézay N, et al. Safety, immunogenicity and dose response of VLA84, a new vaccine candidate against Clostridium difficile, in healthy volunteers. Vaccine (2016), http://dx.doi.org/10.1016/j.vaccine.2016.03.098
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Please cite this article in press as: Bézay N, et al. Safety, immunogenicity and dose response of VLA84, a new vaccine candidate against Clostridium difficile, in healthy volunteers. Vaccine (2016), http://dx.doi.org/10.1016/j.vaccine.2016.03.098
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