Safety and immunogenicity of a booster dose of a 3-antigen Staphylococcus aureus vaccine (SA3Ag) in healthy adults: A randomized phase 1 study

Journal of Infection (2016) xx, 1e18

www.elsevierhealth.com/journals/jinf

Safety and immunogenicity of a booster dose of a 3-antigen Staphylococcus aureus vaccine (SA3Ag) in healthy adults: A randomized phase 1 study Helen Marshall a,*,h, Michael Nissen b,h, Peter Richmond c, Sepehr Shakib d, Qin Jiang e, David Cooper f, Denise Rill e, James Baber g, Joseph Eiden f, William C. Gruber f, Kathrin U. Jansen f, Annaliesa S. Anderson f, Edward T. Zito e, Douglas Girgenti f a

Vaccinology and Immunology Research Trials Unit, Women’s and Children’s Hospital and Robinson Research Institute and School of Medicine, University of Adelaide, Adelaide, SA, Australia b Queensland Paediatric Infectious Diseases, Clinical Trials Centre, Royal Children’s Hospital and Children’s Health Service District, Brisbane, QLD, Australia c University of Western Australia, School of Paediatrics and Child Health, Telethon Kids Institute, Perth, WA, Australia d CMAX, Adelaide, SA, Australia e Pfizer Inc., Collegeville, PA, USA f Pfizer Inc., Pearl River, NY, USA g Pfizer Australia Pty Ltd, Sydney, NSW, Australia Accepted 5 August 2016 Available online - - -

KEYWORDS Staphylococcus aureus; Vaccine; Functional antibodies; Capsular polysaccharide conjugates; Clumping factor A

Summary Objective: A 2-stage, phase 1, randomized, placebo-controlled study in healthy adults to assess immunogenicity and safety of a booster dose at three dose levels of a 3antigen Staphylococcus aureus vaccine (SA3Ag) containing recombinant clumping factor A (ClfA) and capsular polysaccharides 5 and 8 (CP5 and CP8) conjugated to a diphtheria toxoid. Methods: Six months after initial single vaccination, in Stage 2, SA3Ag recipients were randomized (1:1) to booster vaccination or placebo, while Stage 1 placebo recipients received placebo again. Pre- and post-vaccination blood samples were analyzed.

* Corresponding author. Fax: þ61 8 8161 7031. E-mail address: [email protected] (H. Marshall). h Helen Marshall and Michael Nissen contributed equally to the preparation of this manuscript. http://dx.doi.org/10.1016/j.jinf.2016.08.004 0163-4453/ª 2016 The British Infection Association. Published by Elsevier Ltd. All rights reserved. Please cite this article in press as: Marshall H, et al., Safety and immunogenicity of a booster dose of a 3-antigen Staphylococcus aureus vaccine (SA3Ag) in healthy adults: A randomized phase 1 study, J Infect (2016), http://dx.doi.org/10.1016/j.jinf.2016.08.004

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H. Marshall et al. Results: In Stage 2 (n Z 345), pre-booster CP5 and CP8 titers remained high with no increase post-booster. ClfA titers remained high after initial vaccination and increased post-booster, approaching the peak response to the initial dose. Post-booster local reactions were more frequent and of greater severity than reported after the initial vaccination, particularly for the high-dose level recipients. Post hoc analysis showed no doseeresponse pattern and no obvious association between diphtheria toxoid titers and local reactions after initial or booster vaccination. Conclusion: Immune responses after the initial vaccination persisted for the 12 months studied, with little additional response after the booster dose at 6 months. Post-booster injection site reactions were more frequent and more severe but self-limiting. Clinicaltrials.gov identifier: NCT01018641. ª 2016 The British Infection Association. Published by Elsevier Ltd. All rights reserved.

Introduction Staphylococcus aureus is a major cause of healthcare- and community-associated infections and infection-related deaths.1 Between 2013 and 2014, the rate of S. aureus bacteremia was 0.87 cases per 10,000 days of patient care in Australia, with 75% caused by methicillin-sensitive S. aureus (MSSA).2 In the US (2009e2010), 16% of healthcare-associated infections (HAIs) were caused by S. aureus, and 8.5% of all HAIs were methicillin-resistant S. aureus (MRSA).3 Staphylococcus aureus infection therefore remains a major problem, and novel preventative measures are needed. Various attempts to develop an S. aureus vaccine targeting a single S. aureus antigen have failed.4e9 Possible reasons for these failures have been explored in detail previously,8,10 and include potential issues with the vaccine manufacturing process and choice of study population (StaphVax).11 Also, the most recent candidate to discontinue development (V710) did not demonstrate bactericidal responses and was targeted towards a single redundant iron-scavenging protein.7,9 The vaccine was unable to induce antibodies that could facilitate the killing of S. aureus cells, which is the critical mechanism of action for the human defense against Gram positive pathogens.12 In addition, V710 was associated with an increased risk of multi-organ failure in patients undergoing cardiothoracic surgery, and vaccine recipients with S. aureus infections had a higher probability of death.7 The mechanism for this safety finding has not been established, although preoperative low levels of IL-2 and IL-17 were observed in a small number of subjects to be associated with mortality in a post hoc analysis.13 Vaccine candidates that target multiple virulence factors may be therefore required to develop a successful prophylactic vaccine. Promising antigenic targets include the S. aureus capsular polysaccharides type 5 (CP5) and type 8 (CP8), which enable the bacteria to evade phagocytosis,14e19 and two surface expressed protein antigens; clumping factor A (ClfA), which facilitates S. aureus binding to host fibrinogen and platelets,20,21 and manganese transporter C (MntC), an important factor for essential nutrient transport and resistance to host immunity.22,23 As the first step towards developing a 4-antigen vaccine formulation (SA4Ag), a preliminary vaccine candidate (SA3Ag) was evaluated, containing a recombinant mutant form of clumping factor A (rClfAm) and CP5 and CP8 each conjugated to cross-reactive material 197 (CRM197), a diphtheria toxoid.

The safety, tolerability, and immunogenicity of SA3Ag were evaluated in a phase 1, 2-stage, multicenter, parallelgroup, placebo-controlled, randomized, double-blind, and sponsor-unblinded study that enrolled healthy volunteers aged 50e85 and 18e24 years at 5 sites in Australia. The first stage of this first-in-human study demonstrated that single dose administration of SA3Ag induced a rapid and robust functional immune response in both age groups, with no notable safety concerns.24 It has been anticipated that for adults at risk of S. aureus infection within a brief defined period (e.g., major surgery) a single vaccination may provide sufficient protection, whereas for those at long-term risk (e.g., chronic disease, or those undergoing multiple surgeries) booster vaccination(s) may be required to maintain long-term protection, similar to vaccines against other infectious diseases such as pneumococcal and meningococcal disease.25 We evaluated the safety, tolerability, and immunogenicity of a booster dose of SA3Ag administered 6 months after the initial vaccination of the phase 1 study described above.24

Methods Participants Inclusion and exclusion criteria have been described in detail previously.24 Participants were healthy adults aged 18e24 years or 50e85 years (those who may have had stable chronic medical conditions were included). Exclusion criteria included: pregnancy, breastfeeding, bleeding diathesis or medication that resulted in increased bleeding time (low-dose daily aspirin allowed), contraindication to vaccination or vaccine components, immunocompromised status, receipt of blood products or immunoglobulins within 12 months, and previous S. aureus vaccination. Each site’s Human Research Ethics Committee approved the study protocol (Clinicaltrials.gov Identifier: NCT01018641). All participants provided written informed consent. All study procedures were carried out in accordance with the Declaration of Helsinki.

Study design In Stage 1 of this phase 1, randomized, double-blind, placebo-controlled study, 3 ascending dose levels of SA3Ag were evaluated: 10 mg each of CP5- and CP8-

Please cite this article in press as: Marshall H, et al., Safety and immunogenicity of a booster dose of a 3-antigen Staphylococcus aureus vaccine (SA3Ag) in healthy adults: A randomized phase 1 study, J Infect (2016), http://dx.doi.org/10.1016/j.jinf.2016.08.004

Staphylococcus aureus booster vaccine

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CRM197, and 20 mg of rClfAm (low-dose level); 30 mg of CP5and CP8-CRM197, and 60 mg of rClfAm (mid-dose level); or 100 mg of CP5- and CP8-CRM197, and 200 mg of rClfAm (high-dose level).24 The non-adjuvanted, lyophilized vaccine was reconstituted with 60 mM NaCl and administered by intramuscular injection. The placebo control was 150 mM (isotonic) NaCl. In Stage 1 of the study, in each dose level cohort participants were randomized 3:1 SA3Ag to placebo so that low:mid:high:placebo z 1:1:1:1 for each age stratum and received the injection on Day 1. Vaccine or placebo was administered to participants in a blinded manner by study staff. Details of sample size calculation, randomization, and dosing were published previously.24 Based on the evaluation of unblinded safety and tolerability data, all dose level cohorts were selected for participation in Stage 2.

Stage 1

At 6 months after the initial injection in Stage 1, participants who had received the active vaccine and continued into Stage 2 were randomized to booster vaccination of SA3Ag at the same dose level or placebo at a 1:1 ratio. Those who received placebo in Stage 1 and continued in Stage 2 were given placebo again. Booster dosing was administered by blinded site staff and participants also remained blinded. Blood samples were collected prior to, and at 1, 3, and 6 months after the booster injection.

Safety evaluation As in Stage 1,24 an e-diary was used by participants to record local reactions at the injection site, systemic events, and use of antipyretics/pain medication to treat symptoms, for 14 days after the booster vaccination. Local reactions

408 subjects consented 408 were randomised

102 allocated to low-dose group 102 vaccinated

102 allocated to mid-dose group 101 vaccinated 1 did not receive vaccine 1 due to receiving a 1 prohibited medication

102 allocated to high-dose group 101 vaccinated 1 withdrew consent prior to vaccination

102 allocated to placebo group 102 received placebo

99 analysed 1 withdrew at participant's request 1 failed to return 1 protocol violation

96 analysed 2 withdrew at participant's request 1 adverse event 1 lost to follow-up 1 protocol violation

99 analysed 1 withdrew at participant's request 1 failed to return

98 analysed 1 withdrew at participant's request 1 failed to return 1 adverse event 1 lost to follow-up

99 completed Stage 1 8 withdrew at participant's request* 2 failed to return

96 completed Stage 1 8 withdrew at participant's request* 1 adverse event

99 completed Stage 1 8 withdrew at participant's request* 1 adverse event 1 investigator request

98 completed Stage 1 14 withdrew at participant's request* 3 adverse event 1 protocol vialation

89 entered Stage 2 0 without booster dose† 89 with booster dose 45 received low-dose vaccine 44 received placebo

87 entered Stage 2 1 without booster dose† 86 with booster dose 44 received mid-dose vaccine 42 received placebo

89 entered Stage 2 37 without booster dose† 52 with booster dose 27 received high-dose vaccine 25 received placebo

80 entered Stage 2 10 without booster dose† 70 with booster dose 70 received placebo

85 completed Stage 2 1 withdrew at participant's request 1 adverse event 1 protocol violation 1 1 lost to follow-up

84 completed Stage 2 1 withdrew at participant's request 1 adverse event 1 fail to return

83 completed Stage 2 3 withdrew at participant's request 1 adverse event 1 protocol violation 11 lost to follow-up

76 completed Stage 2 4 withdrew at participant's request

Stage 2

Figure 1 Patient disposition. *A total of 38 participants who completed Stage 1 (initial vaccination) did not continue into Stage 2 (booster vaccination): 18 participants could not attend visits due to work or traveling commitments and 3 were moving out of the area; one placebo recipient withdrew due to health concerns after experiencing an upper respiratory tract infection after the initial study injection; one low dose level recipient withdrew from the study because he felt unwell after the first dose and did not wish to receive a second vaccination; another low dose level recipient with a medical history of asthma withdrew for further investigation of chronic shortness of breath; no other withdrawals were related to adverse events. yDuring the conduct of Stage 2 of the trial, due to the increased frequency and severity of the local reactions observed after the booster vaccination of SA3Ag compared with those following the initial vaccination, the study protocol was amended and the booster vaccinations were suspended for those who had not yet entered Stage 2. Although Stage 2 vaccine administration and all administration-related procedures were discontinued for these study participants, they were allowed to continue in the study for evaluation of immunogenicity and safety through 12 months after the primary dose in Stage 1. Low-dose SA3Ag contained 10 mg CP5-CRM197, 10 mg CP8-CRM197, and 20 mg rClfAm. Mid-dose SA3Ag was 30 mg CP5-CRM197, 30 mg CP8-CRM197, and 60 mg rClfAm. High-dose SA3Ag consisted of 100 mg CP5-CRM197, 100 mg CP8-CRM197, and 200 mg rClfAm. Please cite this article in press as: Marshall H, et al., Safety and immunogenicity of a booster dose of a 3-antigen Staphylococcus aureus vaccine (SA3Ag) in healthy adults: A randomized phase 1 study, J Infect (2016), http://dx.doi.org/10.1016/j.jinf.2016.08.004

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H. Marshall et al. included redness (erythema), swelling (induration), and pain at the injection site. Systemic events included fever (oral temperature), vomiting, diarrhea, headache, fatigue, new or worsening muscle pain (other than at the injection site), and new or worsening joint pain (other than joint pain adjacent to the injection site). For participants who received the booster dose of SA3Ag or placebo, a followup telephone contact was made within the first 4e6 days after the vaccination. Adverse events (AEs) were reported for at least 28 days after the booster vaccination. Serious adverse events (SAEs) and newly diagnosed chronic medical disorders were reported through 6-months after booster vaccination.

Immunogenicity evaluation Blood samples collected from participants who received booster vaccination before and 1-month after the booster dose were tested for antigen-specific antibodies. A triplex competitive Luminex immunoassay (cLIA) was used to measure serum immunoglobulins that compete with antigen-specific monoclonal antibodies (mAbs) for binding to the respective antigen coated on microspheres.26 The mAb used for ClfA had previously demonstrated functional competitive inhibition of S. aureus binding to host fibrinogen.27 The lower limit of quantitation (LLOQ) for each antigen using the cLIA was 47.6 U/mL for CP5, 36.6 U/mL for CP8, and 48.9 U/mL for rClfAm. SA3Ag contains CRM197 at an approximately 1:1 ratio to the polysaccharide antigens, and the participants receiving the vaccine most likely had been exposed to CRM197 or diphtheria toxoid antigens in the past. Due to the increased frequency and severity of the local reactions observed after the booster vaccination, an exploratory post hoc analysis was carried out to determine if there was a relationship between reactogenicity and diphtheria toxoid titers. In a subset of participants (96 participants aged 18e24 years and 120 randomly selected participants aged 50e85 years), diphtheria toxoid titers were tested using a Luminex immunoassay on blood samples obtained throughout the study (Days 1, 5, 8, 11, 15, and 29; and Months 2, 3, 6, 7, 9, and 12). The LLOQ for the diphtheria toxoid immunoassay was 0.037 U/mL.

Statistical analysis The modified intent-to-treat (mITT) immunogenicity population was defined as all participants in Stage 2 with at least 1 valid and determinate assay result, and was the primary analysis population. Descriptive statistics were used to

Figure 2 Antigen-specific antibody titers pre- and postbooster vaccination by age group using the cLIA assay. cLIA, competitive Luminex  immunoassay; rClfAm, recombinant mutant form of clumping factor A; GMT, geometric mean titer; CI, confidence interval; LLOQ, lower limit of quantitation; PBOePBO, placebo in both Stage 1 and Stage 2; LDePBO,

low-dose level in Stage 1 and placebo in Stage 2; LDeLD, low-dose level in both Stage 1 and Stage 2; MDePBO, middose level in Stage 1 and placebo in Stage 2; MDeMD, middose level in both Stage 1 and Stage 2; HDePBO, high-dose level in Stage 1 and placebo in Stage 2; HDeHD, high-dose level in both Stage 1 and Stage 2; CP5-CRM197, capsular polysaccharide type 5ecross-reactive material 197; CP8-CRM197, capsular polysaccharide type 8ecross-reactive material 197.

Please cite this article in press as: Marshall H, et al., Safety and immunogenicity of a booster dose of a 3-antigen Staphylococcus aureus vaccine (SA3Ag) in healthy adults: A randomized phase 1 study, J Infect (2016), http://dx.doi.org/10.1016/j.jinf.2016.08.004

Demographic characteristics of the booster dose stage (Stage 2) mITT immunogenicity population.

Vaccine group Stage 1 initial vaccination

Placebo

Low dose SA3Ag

Mid dose SA3Ag

High dose SA3Ag

Total

Stage 2 booster vaccination or none

Placebo or none

Placebo or none

Low dose SA3Ag

Placebo or none

Mid dose SA3Ag

Placebo or none

High dose SA3Ag

Number of participants Sex, n (%) Female Male Race, n (%) White Black or African American Asian Other Tobacco use, n (%) Yes No Height (cm), median (range) Weight (kg), median (range) Body mass index (kg/m2), mean (SD) Age at randomization (years), median (range) Age at booster vaccination (years), median (range)

70

44

45

42

44

25

27

297

35 (50.0) 35 (50.0)

20 (45.5) 24 (54.5)

18 (40.0) 27 (60.0)

26 (61.9) 16 (38.1)

19 (43.2) 25 (56.8)

15 (60.0) 10 (40.0)

16 (59.3) 11 (40.7)

149 (50.2) 148 (49.8)

66 (94.3) 0 (0.0)

43 (97.7) 0 (0.0)

44 (97.8) 0 (0.0)

41 (97.6) 1 (2.4)

40 (90.9) 0 (0.0)

20 (80.0) 0 (0.0)

27 (100.0) 0 (0.0)

281 (94.6) 1 (0.3)

2 (2.9) 2 (2.9)

0 (0.0) 1 (2.3)

0 (0.0) 1 (2.2)

0 (0.0) 0 (0.0)

2 (4.5) 2 (4.5)

2 (8.0) 3 (12.0)

0 (0.0) 0 (0.0)

6 (2.0) 9 (3.0)

4 (5.7) 66 (94.3) 169.5 (148.0, 189.0) 74.9 (47.2, 141.8) 27.8 (6.2)

5 (11.4) 39 (88.6) 169.0 (149.0, 182.5) 75.3 (42.5, 125.3) 27.5 (5.8)

6 (13.3) 39 (86.7) 171.0 (154.5, 184.0) 73.7 (51.0, 135.9) 27.1 (4.8)

8 (19.0) 34 (81.0) 170.4 (154.0, 185.0) 83.2 (54.6, 140.9) 29.6 (6.9)

4 (9.1) 40 (90.9) 170.5 (152.0, 191.0) 79.8 (53.2, 150.8) 28.6 (6.1)

7 (28.0) 18 (72.0) 164.0 (152.0, 182.0) 77.6 (47.9, 108.5) 28.3 (4.8)

3 (11.1) 24 (88.9) 169.0 (148.0, 187.0) 79.4 (56.8, 112.4) 29.1 (6.0)

37 (12.5) 260 (87.5) 169.5 (148.0, 191.0) 77.9 (42.5, 150.8) 28.2 (5.9)

59.0 (19.0, 81.0) 59.5 (19.0, 82.0)

57.5 (18.0, 79.0) 58.0 (18.0, 80.0)

63.0 (20.0, 79.0) 64.0 (20.0, 80.0)

58.5 (21.0, 84.0) 58.5 (21.0, 84.0)

59.5 (18.0, 79.0) 60.0 (19.0, 80.0)

60.0 (20.0, 81.0) 60.0 (21.0, 81.0)

58.0 (20.0, 70.0) 58.0 (21.0, 71.0)

59.0 (18.0, 84.0) 60.0 (18.0, 84.0)

Staphylococcus aureus booster vaccine

Values are n (%), mean (SD), or median (range). mITT, modified intent-to-treat, SD, standard deviation.

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Please cite this article in press as: Marshall H, et al., Safety and immunogenicity of a booster dose of a 3-antigen Staphylococcus aureus vaccine (SA3Ag) in healthy adults: A randomized phase 1 study, J Infect (2016), http://dx.doi.org/10.1016/j.jinf.2016.08.004

Table 1

Figure 3 Kinetics of immune response* through 12 months. *The Stage 1 (initial vaccination) modified intent-to-treat population was used. Black and grey arrows indicate the initial and the booster vaccination on Day 1 and Day 182, respectively. CP5-CRM197, capsular polysaccharide type 5ecross-reactive material 197; CP8-CRM197, capsular polysaccharide type 8ecross-reactive material 197; rClfAm, recombinant mutant form of clumping factor A; cLIA, competitive Luminex immunoassay; GMT, geometric mean titer; CI, confidence interval; PBOePBO, placebo in both Stage 1 and Stage 2; LDePBO, low-dose level in Stage 1 and placebo in Stage 2; LDeLD, low-dose level in both Stage 1 and Stage 2; MDePBO, mid-dose level in Stage 1 and placebo in Stage 2; MDeMD, mid-dose level in both Stage 1 and Stage 2; HDePBO, high-dose level in Stage 1 and placebo in Stage 2; HDeHD, high-dose level in both Stage 1 and Stage 2.

Please cite this article in press as: Marshall H, et al., Safety and immunogenicity of a booster dose of a 3-antigen Staphylococcus aureus vaccine (SA3Ag) in healthy adults: A randomized phase 1 study, J Infect (2016), http://dx.doi.org/10.1016/j.jinf.2016.08.004

Staphylococcus aureus booster vaccine

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Figure 3

(continued)

Please cite this article in press as: Marshall H, et al., Safety and immunogenicity of a booster dose of a 3-antigen Staphylococcus aureus vaccine (SA3Ag) in healthy adults: A randomized phase 1 study, J Infect (2016), http://dx.doi.org/10.1016/j.jinf.2016.08.004

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Figure 3

(continued)

Please cite this article in press as: Marshall H, et al., Safety and immunogenicity of a booster dose of a 3-antigen Staphylococcus aureus vaccine (SA3Ag) in healthy adults: A randomized phase 1 study, J Infect (2016), http://dx.doi.org/10.1016/j.jinf.2016.08.004

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Figure 4 Local injection site reactions within 14 days after the initial and the booster vaccination. For redness and swelling, mild was defined as the diameter of reaction area was 0.5e5.0 cm, moderate was >5.0e10.0 cm, and severe was >10.0 cm. Grading of pain was defined as: mild, did not interfere with activity; moderate, interfered with activity; and severe, prevented daily activity.

summarize the occurrences of local reactions and systemic events after the booster vaccination. cLIA titers were summarized descriptively for each antigen, sampling time point, age stratum, and vaccine group. The descriptive statistics were geometric mean titers (GMTs) or geometric mean-fold-rises (GMFRs) along with corresponding 95% confidence intervals (CIs). GMFRs were calculated using all participants with available data from both the pre-booster and 1 month post-booster blood samples. The CIs were constructed by back transformations of the confidence limits computed for the mean of the logtransformed assay data based on Student’s t-distribution. Diphtheria toxoid GMTs and corresponding 2-sided 95% CIs for the GMTs were calculated within each age stratum in both Stage 1 and Stage 2, and for participants with

moderate or severe local reactions versus participants without any local reactions.

Results Participants A total of 408 participants were randomized, of whom 392 completed Stage 1 (through 6 months).24 Before entering Stage 2, 47 participants withdrew, 38 at their own request, with similar proportions in each of the vaccine groups (8 [7.8%] each), and in the placebo group 14 [13.7%] (Fig. 1). Five of the 47 participants withdrew due to AEs; three in the placebo group and 1 each in the mid- and high-dose

Please cite this article in press as: Marshall H, et al., Safety and immunogenicity of a booster dose of a 3-antigen Staphylococcus aureus vaccine (SA3Ag) in healthy adults: A randomized phase 1 study, J Infect (2016), http://dx.doi.org/10.1016/j.jinf.2016.08.004

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Figure 5 Participants reporting systemic events within 14 days post the booster vaccination. Grade of vomiting was defined as: mild was 1e2 times in 24 h, moderate was >2 times in 24 h, and severe required IV hydration. Diarrhea grading was as follows: mild, 2e3 loose stools in 24 h; moderate, 4e5 loose stools in 24 h; and severe, 6 loose stools in 24 h. Headache, fatigue, muscle pain (new or worsening), and joint pain (new or worsening) were graded as: mild, no interference with activity; moderate, some interference with activity; and severe, significant and prevented daily routine activity.

level groups. For these 47 participants, the AE profile was similar across all vaccine groups, and no SAEs were reported. Among the 345 participants who continued in Stage 2 of the study, 297 received a booster dose of SA3Ag or placebo. During the conduct of Stage 2, an increased frequency and severity of local reactions was observed after the booster vaccination of SA3Ag compared with the initial vaccination, and the booster injections were suspended. The protocol was amended to allow those who had not yet entered Stage 2 to continue in the study for evaluation of immunogenicity and safety through 12 months after the initial dose in Stage 1. Therefore the remaining participants (48) did not receive the booster dose after protocol amendment, and were followed

throughout Stage 2 for a total of 12 months after the initial vaccination (Fig. 1). During Stage 2, 17 participants withdrew, including 1 participant each in the low-, midand high-dose level groups who withdrew due to AEs, thus 328 participants completed the study (Fig. 1). There were 7 dosing groups in Stage 2 (classified as dose in Stage 1/dose in Stage 2): (1) placebo/placebo, (2) low dose level/placebo (or none), (3) low dose level/low dose level, (4) mid dose level/placebo (or none), (5) mid dose level/mid dose level, (6) high dose level/placebo (or none), and (7) high dose level/high dose level. Among the 7 groups, 40.0%e60.5% of the participants were female and 88.7%e100.0% were white (Table 1). The demographics of the participants of Stage 2 were similar to that of Stage 1.24

Please cite this article in press as: Marshall H, et al., Safety and immunogenicity of a booster dose of a 3-antigen Staphylococcus aureus vaccine (SA3Ag) in healthy adults: A randomized phase 1 study, J Infect (2016), http://dx.doi.org/10.1016/j.jinf.2016.08.004

2 2 (7.4%) 4 4 (14.8%) 0 a The booster injections were suspended during the conduct of Stage 2 due to an increased frequency and severity of local reactions was observed after the booster vaccination of SA3Ag compared with the initial vaccination, which resulted in fewer participants in these 2 groups. b Some participants had 1 events.

7 7 (11.3%) 4 4 (6.5%) 1 (1.6%) 1 1 (2.3%) 1 1 (2.3%) 0 5 4 (9.3%) 2 2 (4.7%) 1 (2.3%) 6 5 (11.4%) 7 6 (13.6%) 0 5 4 (5.0%) 4 3 (3.8%) 0

1 1 (2.2%) 2 2 (4.4%) 1 (2.2%)

27a 12 9 (33.3%) 2 2 (7.4%) 62 14 12 (19.4%) 0 0 44 21 13 (29.5%) 2 2 (4.5%) 80 29 25 (31.3%) 5 5 (6.3%)

Participants, n Adverse events (AEs), nb Participants with AEs, n (%) Vaccine-related AEs, nb Participants with vaccine-related AEs, n (%) Serious AEs (SAEs), nb Participants with SAEs, n (%) Severe AEs, n (%)b Participants with severe AEs, n (%) AE leading to withdrawal, n (%)

45 17 14 (31.1%) 7 6 (13.3%)

43 16 11 (25.6%) 1 1 (2.3%)

44 12 10 (22.7%) 3 3 (6.8%)

a

Placebo or none Placebo or none Low dose SA3Ag Placebo or none Stage 2 booster vaccination or none

Placebo or none

Placebo

Low dose SA3Ag

11

Safety results

Stage 1 initial vaccination

Vaccine group

Table 2

Adverse events among all patients entering the booster vaccination stage (Stage 2).

Mid dose SA3Ag

Mid dose SA3Ag

High dose SA3Ag

High dose SA3Ag

Staphylococcus aureus booster vaccine

Reactogenicity Injection site reactions occurred more frequently in the participants who received the SA3Ag booster dose (0%e 90.9%) than among placebo recipients (1.4%e15.8%) (Fig. 4). The onset of local reactions was generally within the first 2 days after the booster vaccination for both 50e85 years and 18e24 years age strata. The durations of local reactions were comparable in both age strata (mean 1.0e2.9 days, data not shown). Across all 3 SA3Ag dose levels, the most common local injection site reaction was pain, followed by swelling and redness, except for the high dose level in the 50e85 year old group where the most common reaction was swelling. All reactions showed a dose-related increase in frequency (Fig. 4). Overall, the grading of most of the injection site reactions following the booster dose were mild or moderate in all dose levels in both age groups. However, there was an increased severity of local reactions in the 50e85 years age stratum with severe redness and severe swelling reported in 2 (8.7%) and 4 (17.4%) participants receiving the high dose level, and 2 (6.1%) participants each for the mid dose level (Fig. 4). The higher frequency and severity of local reactions led to suspension of booster dosing. Therefore, only 4 participants in the high dose level group of the 18e24 years age stratum received the booster injection, with severe redness, severe swelling, and severe pain each reported in 1 (25.0%) participant. A total of 23 participants aged 50e85 years received the high dose level of SA3Ag prior to the discontinuation of booster dosing. In the 50e85 years age stratum, systemic events were reported by 29.4%e56.5% of participants receiving SA3Ag and 40.6% in the placebo group. Across all dose level groups, the most common systemic events were headache (reported by 14.7%e21.7% of participants), fatigue (reported by 14.7%e24.2% of participants), and new or worsening muscle pain (reported by 5.6%e43.5%) (Fig. 5). Compared with the 50e85 years age stratum, the frequency of any systemic event after the booster vaccination was higher in the 18e24 years age stratum (47.4%, 72.7%, 54.5%, and 100% of participants in the placebo, low-, mid-, and high-dose level group, mainly due to small sample size). The most common events were fatigue, experienced by 34.2%e50.0% of participants, followed by headache (18.2%e50.0% of participants) (Fig. 5). Most systemic events were mild or moderate in severity for both age strata (Fig. 5). The mean durations of systemic events ranged from 1.0 to 12.6 days for the 50e85 years age stratum and 1.0e7.5 days for the 18e24 years age stratum; however no trends were observed across SA3Ag and placebo groups (data not shown).

Adverse events In Stage 2, the percentage of participants reporting AEs ranged from 19.4% to 33.3% (Table 2), with no difference between placebo and vaccine groups. AEs occurring in more than 1 participant in any group were upper respiratory tract infection, viral upper respiratory tract infection,

Please cite this article in press as: Marshall H, et al., Safety and immunogenicity of a booster dose of a 3-antigen Staphylococcus aureus vaccine (SA3Ag) in healthy adults: A randomized phase 1 study, J Infect (2016), http://dx.doi.org/10.1016/j.jinf.2016.08.004

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Figure 6 Diphtheria toxoid titers pre and post the initial vaccination and the booster vaccination by severity of local reaction using the cLIA assay. Geometric mean titers (GMTs) were calculated using all participants with available data at the indicated time point. Confidence intervals (CIs) were back transformations of a confidence interval based on the Student’s t-distribution for the mean logarithm of the titers, or the mean-fold rise. Stage 1 modified intent-to-treat (mITT) population was used for pre- and post-initial vaccination. Stage 2 mITT Population was used for pre- and post-booster vaccination. PBOePBO, placebo in

Please cite this article in press as: Marshall H, et al., Safety and immunogenicity of a booster dose of a 3-antigen Staphylococcus aureus vaccine (SA3Ag) in healthy adults: A randomized phase 1 study, J Infect (2016), http://dx.doi.org/10.1016/j.jinf.2016.08.004

Staphylococcus aureus booster vaccine headache, dysmenorrhea, toothache, and oropharyngeal pain (Data not shown). Vaccine-related AEs were reported in 0.0%e13.3% of the participants across different groups (Table 2). SAEs were reported by 24 participants in Stage 2, with the majority (23) in the 50e85 years age stratum. Four (5.0%) participants reporting SAEs were in the placebo group; 1 (2.2%), 1 (2.3%), and 2 (7.4%) participants received low, mid, and high dose level SA3Ag in both initial and booster vaccinations; and 5 (11.4%), 4 (9.3%), and 7 (11.3%) participants who received low, mid, or high dose level SA3Ag at Stage 1 and placebo or no booster in Stage 2 (Table 2). No SAE was considered vaccine-related or lifethreatening. Severe AEs were reported in Stage 2 by 3 participants (3.8%) in the placebo group. For those who received low, mid, or high dose level SA3Ag in both stages, severe AEs were reported by 2 (4.4%), 1 (2.3%), and 4 (14.8%) participants, respectively. For participants who received low, mid, or high dose level SA3Ag in the initial vaccination and placebo or nothing in the booster stage, severe AEs were reported by 6 (13.6%), 2 (4.7%) and 4 (6.5%) participants (Table 2). No severe AEs were considered lifethreatening. Three participants withdrew due to AEs in Stage 2. One participant (50e85 years group) received low dose level SA3Ag in both initial and the booster vaccinations and withdrew due to hepatic cirrhosis (SAE), a pre-existing condition. The second participant (18e24 years group), who received mid dose level SA3Ag in the initial vaccination and placebo in the booster vaccination, withdrew due to mania (SAE). The third participant (50e85 years group), who received high dose level SA3Ag in the initial vaccination and nothing in the booster vaccination stage, withdrew due to polymyalgia rheumatica (AE). None of the SAEs or AEs leading to withdrawal was considered by the investigator or sponsor to be related to SA3Ag. There were no deaths during the study and no obvious safety trends in any vaccine group.

Immunogenicity evaluation All 297 participants who received booster vaccination had pre-booster blood samples, and 294 participants had blood samples from the 1 month visit (Days 29e35) post-booster. In Stage 2, compared with placebo recipients, those who received SA3Ag vaccine still had substantial cLIA titers to the SA3Ag antigens prior to receiving the SA3Ag booster vaccine. This was observed at all dose levels and in each age stratum (Fig. 2). For participants who received only the initial single SA3Ag vaccination (n Z 144), the kinetics of the cLIA GMTs showed that in each age stratum cLIA GMTs gradually declined while remaining substantially higher through Month 12 compared with placebo recipients (Fig. 3).

13 For the 50e85 age stratum (n Z 232), there were no increases in cLIA GMTs for CP5 and CP8 from pre-booster to 1 month post-booster, with GMFRs between 1.0 and 1.2 for CP5 and 1.0e1.1 for CP8. For ClfA, at all SA3Ag dose levels, there was an increase in GMTs, indicating a modest response to booster dose rClfAm; the GMFRs were 1.3, 1.7, and 2.3 for the low, mid, and high dose level SA3Ag group, respectively (Fig. 2). Booster responses for the 18e24 year age stratum (n Z 62) were similar to those of the 50e85 year age stratum, with no observed post-booster increase in GMTs of CP5 and CP8 (GMFRs between 1.4e1.5 and 1.2e1.6, respectively), and a modest increase in GMTs for ClfA (GMFRs of 2.5e3.8) (Fig. 2). There was a trend for lower pre-booster GMTs in the 18e24 year age stratum compared with those of the 50e85 year age stratum (Fig. 2), which accounts for the higher GMFRs in the younger age group. There were no differences in anti-CP5 and anti-CP8 GMTs at 12 months in participants who received a booster dose (1688e3410 for CP5, and 734e1397 for CP8) compared with those who received no booster (1485e2676 for CP5, and 699e1065 for CP8). Anti-ClfA GMTs showed a similar pattern, but participants in the mid and high dose level groups who received a booster vaccination had higher GMTs at 12 months (634 for mid dose, 452 for high dose) in comparison with those who received no booster (311 for mid dose, 250 for high dose).

Diphtheria toxoid titers Diphtheria titers were below the limit of quantitation (BLOQ) for 20.4% of the 216 of participants included in this analysis subset at baseline. Comparison of diphtheria toxoid GMTs in participants who either had or did not have moderate or severe local reactions found no consistent differences (Fig. 6). After the initial vaccination, participants with local reactions in the high- and low-dose SA3Ag groups had higher diphtheria toxoid GMTs compared with those with no local reaction after vaccination; however, their baseline titers were higher (0.2896 and 0.9190 versus 0.1312 and 0.1132, respectively). There were no notable differences in diphtheria toxoid titers in the mid-dose level group in participants with or without moderate or severe local reactions. No obvious doseeresponse pattern in diphtheria toxoid titers and no obvious association of higher GMTs with moderate or severe local reactions after initial or booster dose was found (Fig. 6). Following the initial vaccination in Stage 1, there was a rapid rise in diphtheria toxoid GMTs, with peak titers observed around Day 11 or Day 15. GMTs were higher with increasing dose levels of SA3Ag, consistent with a dose level response for the increasing concentrations of CRM197. For participants in the placebo group, no increase in diphtheria toxoid titers was found (Fig. 7). Diphtheria toxoid titers gradually declined through Month 12 in participants who

both Stage 1 and Stage 2; LDePBO, low-dose level in Stage 1 and placebo in Stage 2; LDeLD, low-dose level in both Stage 1 and Stage 2; MDePBO, mid-dose level in Stage 1 and placebo in Stage 2; MDeMD, mid-dose level in both Stage 1 and Stage 2; HDePBO, high-dose level in Stage 1 and placebo in Stage 2; HDeHD, high-dose level in both Stage 1 and Stage 2. Please cite this article in press as: Marshall H, et al., Safety and immunogenicity of a booster dose of a 3-antigen Staphylococcus aureus vaccine (SA3Ag) in healthy adults: A randomized phase 1 study, J Infect (2016), http://dx.doi.org/10.1016/j.jinf.2016.08.004

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Figure 7 Kinetics of diphtheria toxoid titers through 12 months. Diphtheria toxoid titers for the Stage 1 mITT population in (A) 18e24 years group from baseline to 3-months after the initial dose, (B) 50e85 years group from baseline to 3-months after the initial dose, (C) 18e24 years group from baseline to 6-months after the booster dose, and (D) 50e85 years group from baseline to 6-months after the

Please cite this article in press as: Marshall H, et al., Safety and immunogenicity of a booster dose of a 3-antigen Staphylococcus aureus vaccine (SA3Ag) in healthy adults: A randomized phase 1 study, J Infect (2016), http://dx.doi.org/10.1016/j.jinf.2016.08.004

Figure 7

(continued)

booster dose. Missing or indeterminate results were excluded from analyses. No imputations were carried out for missing data. Diphtheria toxoid titers above the lower limit of quantitation (LLOQ) were considered accurate and the quantitated values were reported. Values that were below limit of quantitation (BLOQ) were set to 0.5  LLOQ for analysis. Black and grey arrows indicate the initial and the booster vaccination on Day 1 and Day 182, respectively. GMT, geometric mean titer. Please cite this article in press as: Marshall H, et al., Safety and immunogenicity of a booster dose of a 3-antigen Staphylococcus aureus vaccine (SA3Ag) in healthy adults: A randomized phase 1 study, J Infect (2016), http://dx.doi.org/10.1016/j.jinf.2016.08.004

16 did not receive a booster dose of SA3Ag. For participants who received a booster dose of SA3Ag at Month 6, an increase in diphtheria toxoid titers was found; however, the titers did not reach the peak observed after the initial vaccination (Fig. 7), showing a similar pattern of immune response observed with rClfAm. Overall, participants in the younger age stratum had higher diphtheria toxoid titers compared with those in the older age stratum (Fig. 7).

Discussion In Stage 1 of this first-in-human, 2-stage evaluation of SA3Ag, a single dose of SA3Ag vaccine was well tolerated, and elicited robust functional immune responses through month 3 after vaccination in generally healthy adult volunteers aged 18e24 years and 50e85 years.24 The Stage 2 results demonstrated sustained immune responses to SA3Ag, with antibody titers remaining higher in participants who received SA3Ag than placebo recipients through Month 12. While administration of a booster dose of SA3Ag at Month 6 did not substantially impact the cLIA titers at Month 12, the persistence of the immune response after a single dose vaccination suggested that a booster dose at Month 6 was not necessary for maintaining antibody levels until Month 12. Although no booster response was found for CP5 and CP8 conjugates, an immune response to the booster dose of rClfAm antigen was observed, with cLIA GMTs returning close to post-initial vaccination peak. These findings and the high, sustained levels of circulating antibodies 6 months after the initial SA3Ag vaccination were indicative of an anamnestic response. Considering that pre-vaccination titers of vaccine antibodies may have negative effect on booster responses,28 the persistent high levels of CP5 and CP8 antibodies may account for the lack of booster response to the capsular polysaccharide antigens. Administration interval between doses has been shown to be an important factor in whether a booster response is elicited to other adult vaccines, such as pneumococcal vaccines. The lack of booster response described for polysaccharide conjugate pneumococcal vaccines administered in adults at intervals of 6 months to 1 year apart may be due to stimulation of pre-existing memory cells following the initial but not the subsequent vaccination.29e31 In contrast, administration of 13-valent polysaccharide conjugate vaccines (PCV13) at an interval of 3.5e4 years after the initial vaccination in adults 50 years elicited OPA responses that were comparable, or statistically significantly greater, than the initial vaccination.32 Similarly, immune responses to a now discontinued staphylococcal conjugate vaccine candidate (StaphVax) administered as a booster to hemodialysis patients approximately 2e3 years after the initial vaccination did elicit a booster response; however the antibody peak levels were lower than following the initial dose.33 These data suggest that, while the immune response to a booster dose of SA3Ag at 6 months was limited, there may still be a role for of a booster dose at a later time interval (greater than 12 months) for individuals at longer term risk of S. aureus disease. After the initial SA3Ag vaccination, local reactions (i.e., pain, swelling, and redness) were mostly mild or moderate and were resolved within 3 days, with the frequency

H. Marshall et al. increasing as the dose level increased: for the 50e85 year age stratum, 10.3%e33.8% reported pain, 6.4%e27.3% reported redness, and 3.8%e19.5% reported swelling; for the 18e24 year age stratum, the rate for pain, redness, and swelling was 29.2%e66.7%, 12.5%e20.8%, and 4.2%e12.5%, respectively.24 Following the booster dose, the incidences and severities of local reactions were higher for both age strata than those reported after the initial vaccination, particularly among the participants who received the high dose level of SA3Ag; 17.4% of high dose recipients aged 50e85 years reported severe swelling at the injection site after the booster dose, compared with 1.3% severe swelling in this age group after the initial vaccination. Moreover, the onset of local reactions was generally within the first 2 days after the booster vaccination for both 50e85 year and 18e24 year age strata; while after the initial vaccination, local reactions were generally reported within the first 2 days in participants aged 18e24 years, and within 6e8 days after vaccination in participants aged 50e85 years.24 The frequencies of systemic events following the booster vaccination were similar to those reported after the initial vaccination,24 with the exception of new or worsening muscle pain, which was reported more frequently following the booster dose, especially in participants who received the high dose level SA3Ag (for those aged 50e85 years, 22.1% after the initial vaccination and 43.5% after the booster vaccination; and for those aged 18e24 years, it was 41.7% and 50.0%, respectively). The higher frequency and severity of local reactions after the booster dose compared with the initial vaccination may have been due to the high circulating levels of antibodies. This finding of increased reactogenicity is consistent with what was observed with booster dose administration of another experimental staphylococcal polysaccharide conjugate.33 Given the high probability that the trial participants who received SA3Ag would have been exposed previously to CRM197 or diphtheria toxoid antigens, diphtheria toxoid titers were evaluated to determine if the amount of CRM197, which increased with increasing SA3Ag dose level, was associated with the occurrence of local reactions. The diphtheria toxoid titers increased with increasing dose level, were higher in younger participants, and showed similar immune response kinetics to the ClfA component. The small sample size limited the conclusions; however there was no clear association between increased in diphtheria toxoid titers and occurrence or severity of local reactions after the initial or booster vaccination. Relatively few participants in the 18e24 years stratum were included in the booster vaccination stage of the study and conclusions are therefore limited. However, in general, a similar pattern to the 50e85 year age stratum was observed, with no noteworthy differences. Overall, all dose levels of SA3Ag were immunogenic and had an acceptable safety profile after a single dose. The sustained, functional immune response observed through month 12 after single vaccination supports continued development of single dose administration of the candidate vaccine, now being evaluated in a phase 2b efficacy trial (NCT02388165) as a 4-antigen formulation that includes MntC. While the data reported in this publication demonstrate no additional benefit to a booster dose administered

Please cite this article in press as: Marshall H, et al., Safety and immunogenicity of a booster dose of a 3-antigen Staphylococcus aureus vaccine (SA3Ag) in healthy adults: A randomized phase 1 study, J Infect (2016), http://dx.doi.org/10.1016/j.jinf.2016.08.004

Staphylococcus aureus booster vaccine at 6 months, and a higher frequency and severity of local reactions compared with the initial dose, single dose administration of SA3Ag demonstrated a persistent immune response through month 12. It is too early in development of this vaccine to correlate the immune responses elicited by SA3Ag with potential efficacy, as there is no established correlate of protection, however the kinetics of the immune response suggest the applicability of a single dose vaccine for protecting adults during a period of risk for S. aureus infection (e.g., those undergoing major surgery). For other populations at risk of S. aureus infection, additional studies will be required to define when vaccine antibody titers wane after the initial vaccination and whether they can be subsequently boosted by booster vaccinations at later time intervals.

Contributors HM, MN, and PR are study investigators and participated in study design, patient recruitment, acquisition of data, and analysis and interpretation of the findings. SS is a consultant to CMAX, Adelaide, Australia, who provided clinical trial support services, including data acquisition and analysis. QJ provided statistical support. DC, DR, JB, JE, WG, KUJ, ASA, ETZ, and DG participated in study design and analysis and interpretation of the data. The first draft of the manuscript was written by HM and MN. All authors participated in the development of the manuscript through subsequent stages and approved the final version for journal submission.

Role of the funding source This study was funded by Pfizer. Employees of the study sponsor, Pfizer, are named as authors on this manuscript and contributed to the design of the study, the analysis and interpretation of the data, and the preparation of this manuscript for submission.

Conflict of interest This study was sponsored by Pfizer. Editorial/medical writing support was provided by Shuang Li at Engage Scientific (Horsham, UK) and was funded by Pfizer. Helen Marshall has been a principal investigator on vaccine clinical trials sponsored by GSK, Pfizer and Novartis with her institution receiving funding to conduct these trials. She has received travel support to present scientific data at international meetings from GSK. Her institution has received grant funding for investigator led studies from Pfizer. Michael Nissen receives personal fees from GSK Vaccines as a full time employee since October 2014 and he has received grants from bioCSL, neither being relevant to the submitted work. Peter Richmond has been principal investigator of clinical trials sponsored by GSK, Merck, Sanofi, and Pfizer and has received travel support to present these data at scientific conferences. Peter Richmond has also received institutional funding from GSK for investigator led studies and participated in scientific advisory boards for Pfizer and GSK. Sepehr Shakib was principal investigator as

17 a contractor on this study at CMAX clinical trial unit. He has no conflict of interest to declare. Qin Jiang, David Cooper, Denise Rill, James Baber, Joseph Eiden, William Gruber, Kathrin U. Jansen, Annaliesa S. Anderson, Edward T. Zito, and Douglas Girgenti are employees of Pfizer.

Acknowledgments The authors wish to acknowledge Associate Professor Peter Hodsman, and the site staff at Nucleus Network, Melbourne, Australia. Michael D. Nissen acknowledges the staff of the Queensland Paediatric Infectious Diseases Clinical Trial Centre including Dr. Raymond Chuk, Dr. Uyen Duong, Mr. Aaron Buckner, and Mrs. Sharon Veal. Helen S. Marshall acknowledges support from the National Health and Medical Research Council of Australia: Career Development Fellowship (APP1084951) and staff of the Vaccinology and Immunology Research Trials Unit including Ms. Susan Lee, Dr. Sue Evans, Mrs. Chris Heath, and Mrs. Michelle Clarke. Peter Richmond acknowledges the staff of the Vaccine Trials Group including Dr. Tanya Stoney, Ms. Fiona McDonald, and Ms. Jennifer Kent. The authors would also like to thank the following employees of Pfizer for their contributions to the design, execution of the study or the generation of the immunogenicity results: Emilio A. Emini, Graham Crowther, Yasuko Shoji, Ingrid Scully, Jasdeep Nanra, Danka Pavlikova, Peter Giardina, Kelly Bellinger, and Adriana Cahill.

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Please cite this article in press as: Marshall H, et al., Safety and immunogenicity of a booster dose of a 3-antigen Staphylococcus aureus vaccine (SA3Ag) in healthy adults: A randomized phase 1 study, J Infect (2016), http://dx.doi.org/10.1016/j.jinf.2016.08.004