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The immunogenicity and safety of different formulations of a novel Staphylococcus aureus vaccine (V710): Results of two Phase I studies
Vaccine 30 (2012) 1729–1736
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The immunogenicity and safety of different formulations of a novel Staphylococcus aureus vaccine (V710): Results of two Phase I studies夽 Clayton D. Harro a,∗ , Robert F. Betts b , Jonathan S. Hartzel c , Matthew T. Onorato c , Joy Lipka c,1 , Steven S. Smugar c , Nicholas A. Kartsonis c a
Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA University of Rochester Medical Center, Rochester, NY, USA c Merck Sharp & Dohme Corp, Whitehouse Station, NJ, USA b
a r t i c l e
i n f o
Article history: Received 20 April 2011 Received in revised form 2 December 2011 Accepted 7 December 2011 Available online 20 December 2011 Keywords: Adjuvant Immunogenicity Phase I Staphylococcus aureus Vaccine Iron surface determinant (IsdB)
a b s t r a c t Merck V710 is a novel vaccine that contains the highly conserved Staphylococcus aureus iron surface determinant B (IsdB) protein. V710 has induced positive immune responses in healthy subjects. The purpose of the two studies described herein was to evaluate the immunogenicity and safety of two different formulations of V710. Both studies were randomized, controlled, double-blind, parallel-group trials. Study 1 compared liquid, aluminum-adjuvanted V710 (30 g) with liquid, non-adjuvanted V710 (30 g) in a 1:1 ratio in 64 healthy adults (18–70 years). Study 2 compared non-adjuvanted lyophilized V710 (60 g) with saline placebo in a 4:1 ratio in 51 healthy adults (18–80 years). Blood was collected at screening and up to Day 360 postvaccination in Study 1, and at screening and up to Day 84 postvaccination in Study 2. Sera were analyzed for IsdB-specific antibodies using a total IgG assay. The primary endpoints in Study 1 were the proportion of patients with a positive immune response (≥2-fold rise in IsdB-specific IgG antibody level) the geometric mean concentration (GMC), and the geometric mean-fold rise (GMFR), all from baseline at Day 14. The primary endpoint in Study 2 was the GMFR in IsdB-specific IgG antibody concentration from baseline at Day 14. In Study 1, 84.4% responded in the adjuvanted V710 group, and 71.9% in the non-adjuvanted V710 group. The GMC was 115.4 g/mL in the adjuvanted group and 99.1 g/mL in the nonadjuvanted group. The GMFR in antibody concentration in the group receiving aluminumadjuvanted V710 was 4.5 and the GMFR in the group receiving non-adjuvanted V710 was 4.0. In Study 2, the GMFR in antibody concentration in the V710 group was 5.3, and 80.5% had a positive immune response. None responded in the placebo group. Positive immune response was seen in the active treatment groups over the full duration of each study. There were no serious adverse experiences (AE) in either study, and no patients discontinued due to an AE. There were no clinically meaningful differences in AEs between groups in either study. In conclusion, V710, both with and without aluminum adjuvant, and in both liquid and lyophilized formulations, was immunogenic within 14 days of vaccination. All treatments showed similar safety profiles. © 2011 Elsevier Ltd. All rights reserved.
1. Introduction Staphylococcus aureus is a human commensal, colonizing the skin and mucous membranes of 25–50% of individuals [1,2]. When these natural barriers are compromised naturally or iatrogenically, staphylococci can cause a wide range of disease from uncomplicated local suppuration to sepsis [2]. Importantly, S. aureus is the
夽 Clinical trial registration: Study 1: NCT01324440. Study 2: NCT00822757. ∗ Corresponding author at: Center for Immunization Research, Johns Hopkins University, Bloomberg School of Public Health, 624 N. Broadway, Hampton House Rm. 117, Baltimore, MD 21205, USA. Tel.: +1 410 614 4937; fax: +1 410 502 6898. E-mail address: [email protected] (C.D. Harro). 1 Present address: Cerexa Inc., Oakland, CA, USA. 0264-410X/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2011.12.045
leading etiology of nosocomial infections [3,4]. The complexity of these infections varies depending on the virulence characteristics of the bacteria and the underlying condition of the human host. Reliance on antimicrobial therapy for treatment and control of staphylococcal disease is increasingly problematic due to the rising prevalence of methicillin-resistant S. aureus (MRSA) [5–7]. Of increasing concern is the emergence of community-acquired MRSA and recalcitrant S. aureus strains that are resistant to multiple antibiotics, even those with novel mechanisms of action such as oxazolidinones and daptomycin [8–10]. High morbidity and mortality associated with S. aureus infections, coupled with pervasive antimicrobial resistance and limited therapeutic alternatives are spurring efforts to develop a prophylactic S. aureus vaccine [11,12]. Previously, we reported the results from the first-in-human dose-ranging study of V710, a purified subunit vaccine being devel-
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oped by Merck [12]. V710 contains a highly conserved surface protein called iron surface determinant B (IsdB), whose intrinsic characteristics make it an effective immunogen [1,13,14]. Data from the initial study demonstrated that a single intramuscular administration of liquid V710 adjuvanted with amorphous aluminum hydroxyphosphate sulfate adjuvant (AAHSA) is safe and immunogenic in 18–55 year old healthy adults. Recipients of any of three dose regimens (5, 30, or 90 g) generated a positive immune response (defined as a 2-fold increase in IsdB-specific IgG titers relative to baseline), but a greater proportion of 30-g and 90g recipients manifested positive immune responses and achieved higher geometric mean titers (GMTs) relative to 5-g or placebo recipients 14 days postvaccination, the primary immunological endpoint. These immune responses remained elevated at 3 months postvaccination (the end of the study). Accelerated stability studies of the liquid formulation of V710 projected lability in the chemical composition of the IsdB antigen (unpublished data). Such degradation of antigenic proteins commonly occurs through amidation and oxidation of susceptible amino acid residues [15,16]. However, lyophilization with appropriate cryoprotectants provides the potential for long-term thermostability of vaccines and extended shelf-life [16,17]. Additionally with the emphasis on reducing adjuvant usage in vaccines, a comparison of vaccine immunogenicity in the presence or absence of adjuvant was warranted. Therefore, this report describes two follow-up studies designed to broaden the human safety and immunogenicity profile of V710 and to explore alternative vaccine formulations. Study 1 compared liquid V710 given with or without AAHSA to detect a potential immunogenicity difference, if present, between the adjuvanted and non-adjuvanted formulations. Study 2 compared a reconstituted lyophilized formulation of V710 (without AAHSA) with placebo. 2. Materials and methods 2.1. Patients Both studies enrolled healthy, nonpregnant adult subjects age 18–70 (Study 1) or 18–80 years (Study 2). Key exclusion criteria included: temperature ≥100.4 ◦ F (≥38 ◦ C) within 48 h of study vaccination; chronic skin conditions; serious S. aureus infection in the previous 12 months; history of anaphylaxis to aluminum-containing adjuvant or other vaccine components; previous vaccination with V710; vaccination with a live virus vaccine within 30 days prior to scheduled V710 vaccination or anticipated vaccination with a live virus vaccine within 30 days following study entry; any other vaccination within 14 days of scheduled V710 vaccination or anticipated vaccination within 30 days following study entry; systemic corticosteroids or other immunosuppressive medications or biological agents within 14 days of V710 vaccination or anticipated administration of such medications during the course of the study. All patients gave written informed consent prior to participation. Both studies were conducted in accordance with the principles of Good Clinical Practice and were approved by the appropriate institutional review boards and regulatory agencies. 2.2. Study design Study 1 (Protocol 002) was a 12-month, randomized, multicenter, double-blind, Phase I study conducted at 2 sites in the United States from 20 September 2006 to 15 October 2007. Eligible subjects were randomized in a 1:1 ratio to receive a single 0.5-mL vaccination of either liquid V710 containing AAHSA (30 g) or liquid V710 without AAHSA (30 g). Enrollment was stratified such that half of the subjects were ≥50 years of age. Study 2 (Protocol
004) was a 3-month, randomized, multicenter, double-blind, Phase I study conducted at 2 sites in the United States from 5 September 2007 to 17 December 2007. Eligible subjects were randomized in a 4:1 ratio to a single 0.5-mL vaccination of either lyophilized V710 (60 g) or saline placebo. Enrollment was stratified to ensure that half of the subjects would be ≥60 years of age, and that at least 40% of the patients in this upper age group (i.e., 20% overall) would be ≥70 years. Because of the differences in appearance between V710 with and without adjuvant, and between V710 and saline placebo, one unblinded member of the study site staff was responsible for preparing and administering the study vaccine or placebo. This person had no further involvement with any subsequent study procedure postvaccination, including safety follow-up procedures. All other individuals involved with the study conduct, including investigators, study physicians, and statisticians remained blinded to treatment group throughout the study. An interim safety and immunogenicity analysis was planned for both studies, and performed by an unblinded statistician and clinical monitor at Merck. The official clinical database remained blinded until the medical/scientific review was completed and the data were declared complete. 2.3. Immunogenicity evaluation Sera were analyzed for IsdB-specific IgG antibodies using an assay on the LUMINEXTM multianalyte profiling platform, which had a lower limit of quantitation of 0.85 ng/mL (non-dilution corrected). Blood samples were collected at screening and postvaccination Days 10, 14, 28 (1 month), 84 (3 months), 180 (6 months), and 360 (1 year) in Study 1, and at screening and postvaccination Days 10, 14, 28 (1 month), and 84 (3 months) in Study 2. The primary immunogenicity timepoint in both studies was at Day 14 postvaccination. Immunogenicity was evaluated by the following methods: the proportion of subjects with a positive immune response, defined as a ≥2-fold rise in titer from baseline, the geometric mean concentration (GMC) at Day 14, and the geometric mean fold-rise (GMFR) at Day 14. The ≥2-fold rise endpoint was also used in the earlier Phase I study by Harro et al. [12], and was chosen based on the observed within-subject variability and maximum fold-rises in titers among longitudinal control subjects as well as the fold-rises in titers among V710-immunized monkeys, both of which were obtained during the validation experiments for the LUMINEXTM assay. The primary endpoints in Study 1 were the proportion of patients with a positive immune response, the GMC, and the GMFR, each from baseline at Day 14 postvaccination. The primary endpoint in Study 2 was the GMFR in IsdB-specific IgG antibody concentration from baseline at Day 14. Exploratory endpoints included the immune response at the other timepoints, and comparison of immune response between the 2 age groups. As an exploratory endpoint, we analyzed opsonophagocytic activity (OPA) from serum obtained from a subset of subjects in each study, measuring the ability of a phagocytic cell line to uptake labeled bacteria in the presence of serum specific antibody (reported as the fold-rise in serum OPA). This assay was used as an adjunct to demonstrate the functional activity of the antibodies elicited by the vaccine. 2.4. Safety evaluation All subjects were monitored for 30 min postvaccination for any immediate reaction, and were asked to record local adverse experiences (AEs) at the injection site and oral temperatures daily for 5 consecutive days postvaccination on a standardized Vaccination Report Card (VRC). Subjects also recorded all systemic AEs for 14
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Table 1 Baseline patient characteristics. Study 1
Study 2
V710 (30 g) with AAHSA N = 32
V710 (30 g) without AAHSA N = 32
V710 (60 g) lyophilized N = 41
Gender, n (%) Male Female
12 (37.5) 20 (62.5)
19 (59.4) 13 (40.6)
19 (46.3) 22 (53.7)
Age, years Mean (SD) ≤17 18–29 30–39 40–49 50–59 60–69 70–80a
46.5 (12.5) 0 (0.0) 6 (18.8) 1 (3.1) 9 (28.1) 12 (37.5) 4 (12.5) 0 (0.0)
45.6 (13.1) 0 (0.0) 5 (15.6) 4 (12.5) 7 (21.9) 10 (31.3) 6 (18.8) 0 (0.0)
53.4 (18.5) 0 (0.0) 6 (14.6) 5 (12.2) 8 (19.5) 1 (2.4) 10 (24.4) 11 (26.8)
Race, n (%) Asian Black White
1 (3.1) 13 (40.6) 18 (56.3)
0 (0.0) 10 (31.3) 22 (68.8)
2 (4.9) 8 (19.5) 31 (75.6)
Placebo N = 10
6 (60.0) 4 (40.0) 53.9 (15.1) 0 (0.0) 1 (10.0) 0 (0.0) 4 (40.0) 0 (0.0) 5 (50.0) 0 (0.0) 0 (0.0) 2 (20.0) 8 (80.0)
a Study 1 enrolled patients up to age 70; Study 2 enrolled patients up to age 80. AAHSA: amorphous aluminum hydroxyphosphate sulfate adjuvant; SD: standard deviation.
days postvaccination; patients were prompted to report 4 specific systemic AEs: headache, nausea, myalgia, and fatigue. Only vaccinerelated serious AEs, AEs leading to death, or serious AEs associated with a S. aureus infection were reported for the remainder of the follow-up periods.
Because there was no hypothesis in Study 1, and because there was only a single hypothesis in Study 2, there were no adjustments for multiplicity in either study. 3. Results
2.5. Statistical analysis
3.1. Patients
The primary immunogenicity analyses in both studies were based on the per-protocol populations, defined as subjects who did not have missing baseline serology data or postvaccination serology data at Day 14, and who did not develop a S. aureus infection during the 14 days postvaccination. Subjects with missing data for a given timepoint were excluded from the immune response rate and fold-rise calculations for that particular timepoint. As Study 1 was an estimation study, no formal hypothesis testing was performed. Immune responses at Day 14 postvaccination were descriptively summarized, including 95% confidence intervals (CIs). Fold-differences in GMC) were calculated using an analysis of covariance (ANCOVA) model with the log titers as the response variable, and treatment group, the log predose titer, and age as fixed effects. For Study 2, the primary hypothesis was that a single dose of lyophilized V710 (60 g) would result in a ≥2.5-fold rise in anti-IsdB-specific IgG titers at Day 14 postvaccination relative to baseline. The primary analysis was calculated based on the natural logarithm of the Day 14 and baseline antibody titers, along with the 95% CI, based on Student’s t-distribution. All subjects who were vaccinated and had safety follow-up data were included in the safety summaries. Safety measures of interest included any AEs, injection-site AEs, systemic AEs, and vaccine-related AEs. The incidence of vaccine-related serious AEs was summarized using exact CIs. For Study 1, with 24 evaluable patients in each group, the study could detect a ≥25 percentage point difference in the positive immune response proportions between treatment groups (i.e., the 95% CI for the difference in positive immune response rates would exclude 0), assuming a positive immune response in approximately 80% of subjects. For Study 2, assuming an expected GMFR of 4.0 in patients receiving lyophilized V710 (60 g) and a standard deviation of the log fold-rise in antibody titer of 0.87, 38 evaluable V710 recipients would provide 91% power (1-sided ˛ = 0.025) to detect a GMFR of ≥2.5 at Day 14 relative to baseline.
Patient accounting and demographics are shown in Fig. 1 and Table 1, respectively. A total of 64 patients were randomized in Study 1 and 51 were randomized in Study 2. One patient in Study 1 was lost to follow-up. All patients in both studies were included in the primary analyses. In both studies, there was a gender imbalance between each of the 2 treatment groups. However, other patient characteristics were generally similar between each of the 2 treatment groups in each study. As planned, 50% of the patients were ≥older than 50 years in Study 1, and 51% were older than 60 years in Study 2. 3.2. Immunogenicity results The immunogenicity results at Day 14 are summarized in Table 2. For the main immunogenicity endpoint in Study 1, 84.4% of patients in the V710 with AAHSA group and 71.9% of patients in the V710 without AAHSA group had a positive immune response, defined as a ≥2-fold rise in IsdB IgG antibody at Day 14 postvaccination. At this time point, the GMFR in the V710 with AAHSA group and V710 without AAHSA group was 4.5 and 4.0, respectively. Results were generally comparable between groups in Study 1; the V710 with AAHSA had numerically greater measures, but these differences were not statistically significant. For the main immunogenicity endpoint in Study 2, the GMFR in IsdB IgG antibody was 5.3 in the V710 group whereas the placebo group did not respond (GMFR = 1.0). The rise from baseline in the V710 group was statistically significant (p < 0.001) based on the lower 95% CI bound of 3.8. In Study 2, approximately 80% of patients in the V710 group achieved ≥2-fold rise in antibodies on Day 14 postvaccination relative to baseline, as compared with no patients in the placebo group. Lyophilized V710 was numerically greater than placebo for all measures. In both studies the immunogenicity response was greater in older patients receiving V710, particularly for patients with ≥3and ≥4-fold-rises from baseline (Tables 3 and 4).
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A
Screened N = 65
Not Randomized N = 1 Did not meet inclusion criteria N= 1
Randomized N = 64
V710 30 μg with AAHSA N = 32
Discontinued N = 0
V710 30 μg without AAHSA N = 32
Discontinued N = 1 Subject moved N = 1
Completed N = 32
Major protocol violations N = 0
Completed N = 31
Major protocol violations N = 0
Included in primary analysis N = 32
Included in primary analysis N = 32
B
Screened N = 51
Not Randomized N = 0
Randomized N = 51
V710 lyophilized 60 μg N = 41
Discontinued N = 0
Placebo N = 10
Discontinued N = 0
Completed N = 41
Major protocol violations N = 0
Completed N = 10
Major protocol violations N = 0
Included in primary analysis N = 41
Included in primary analysis N = 10
Fig. 1. Patient accounting in Study 1 (A) and Study 2 (B). 1 patient in the V710 without AAHSA group had data through Day 180, but moved prior to the Day 360 visit.
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Table 2 Immunogenicity results at Day 14 postvaccination. Parameter
Study 1
Study 2
V710 (30 g) with AAHSA N = 32
% ≥2-fold-rise % ≥3-fold-rise % ≥4-fold-rise % ≥8-fold-rise % ≥16-fold-rise GMFR GMC (g/mL)
V710 (30 g) without AAHSA N = 32
V710 (60 g) lyophilized N = 41
Placebo N = 10
Observed response
95% CI
Observed response
95% CI
Observed response
95% CI
Observed response
95% CI
84.4% (27/32) 71.9% (23/32) 53.1% (17/32) 21.9% (7/32) 3.1% (1/32) 4.5 115.4
(67.2%, 94.7%) (53.3%, 86.3%) (34.7%, 70.9%) (9.2%, 40.0%) (0.1%, 16.2%) (3.3, 6.1) (82.9, 160.6)
71.9% (23/32) 59.4% (19/32) 50.0% (16/32) 25.0% (8/32) 6.3% (2/32) 4.0 99.1
(53.3%, 86.3%) (40.6%, 76.3%) (31.9%, 68.1%) (11.5%, 43.4%) (0.8%, 20.8%) (2.9, 5.4) (73.5, 133.6)
80.5% (33/41) 61.0% (25/41) 48.8% (20/41) 39.0% (16/41) 17.1% (7/41) 5.3 112.5
(65.1%, 91.2%) (44.5%, 75.8%) (32.9%, 64.9%) (24.2%, 55.5%) (7.2%, 32.1%) (3.8, 7.2) (83.2, 152.1)
0.0% (0/10) 0.0% (0/10) 0.0% (0/10) 0.0% (0/10) 0.0% (0/10) 1.0 17.7
(0.0%, 30.8%) (0.0%, 30.8%) (0.0%, 30.8%) (0.0%, 30.8%) (0.0%, 30.8%) (0.9, 1.0) (10.3, 30.4)
CI: confidence interval; GMC: geometric mean concentration; GMFR: geometric mean fold-rise from baseline; AAHSA: amorphous aluminum hydroxyphosphate sulfate adjuvant.
Age ≥50 years
V710 (30 g) with AAHSA N = 32 Observed response
V710 (30 g) without AAHSA N = 32 Observed response
% ≥2-fold-rise % ≥3-fold-rise % ≥4-fold-rise GMFR GMC (g/mL)
81.3% (13/16) 75.0% (12/16) 43.8% (7/16) 3.7 114.1
75.0% (12/16) 56.3% (9/16) 43.8% (7/16) 3.7 95.3
% ≥2-fold-rise % ≥3-fold-rise % ≥4-fold-rise GMFR GMC (g/mL)
87.5% (14/16) 68.8% (11/16) 62.5% (10/16) 5.4 116.6
68.8% (11/16) 62.5% (10/16) 56.3% (9/16) 4.3 102.9
GMC: geometric mean concentration; GMFR: geometric mean fold-rise from baseline; AAHSA: amorphous aluminum hydroxyphosphate sulfate adjuvant.
3.3. Safety No subjects in either study reported a serious AE, a vaccinerelated serious AE, no patients died, and no subjects discontinued
A
Immunogenicity over the course of the studies is shown in Figs. 2 and 3. In Study 1, the proportions of patients with a positive immune response (i.e., ≥2-fold-rise in IgG titers) were similar for both V710 with and without AAHSA, and peaked at Day 84 (Fig. 2A). In Study 2, the proportion of positive immune responders in the lyophilized V710 group continued to rise throughout the course of the study (i.e., through Day 84). In the placebo group, 1 patient had a positive immune response at Day 10, but no patients in the placebo group had a positive immune response thereafter (Fig. 2B). A similar pattern was observed in GMC over Table 4 Immunogenicity results at Day 14 postvaccination stratified by age group, Study 2. Parameter
Age <60 years
Age ≥60 years
V710 (60 g) lyophilized N = 41 Observed response
Placebo N = 10 Observed response
% ≥2-fold-rise % ≥3-fold-rise % ≥4-fold-rise GMFR GMC (g/mL)
70.0% (14/20) 50.0% (10/20) 40.0% (8/20) 4.0 96.9
0.0% (0/5) 0.0% (0/5) 0.0% (0/5) 0.9 26.2
% ≥2-fold-rise % ≥3-fold-rise % ≥4-fold-rise GMFR GMC (g/mL)
90.5% (19/21) 71.4% (15/21) 57.1% (12/21) 6.8 129.7
0.0% (0/5) 0.0% (0/5) 0.0% (0/5) 1.0 11.9
Proportion of patients with positive response (%)
Age <50 years
Parameter
time, with generally comparable levels for V710 with and without AAHSA that peaked at Day 84 in Study 1 (Fig. 3A), and substantially greater levels with lyophilized V710 than placebo in Study 2 (Fig. 3B). OPA results are shown in Table 5. Approximately 55% of patients receiving V710 and 25% of patients receiving placebo had a 2-fold rise in OPA activity, whereas approximately 21% and 0% had a 4-fold rise in OPA activity.
100
80
60
40
20
0 10 28 1 14
84
180
360
Days relative to vaccination V710 (30 μg) with AAHSA
B
Proportion of patients with positive response (%)
Table 3 Immunogenicity results at Day 14 postvaccination stratified by age group, Study 1.
V710 (30 μg) without AAHSA
100
80
60
40
20
0
GMC: geometric mean concentration GMFR: geometric mean fold-rise from baseline.
1
10 14
28
84
Days relative to vaccination V710 (60 μg) lyophilized
Placebo
Fig. 2. Proportion of subjects with a positive immune response over time in Study 1 (A) and Study 2 (B).
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Table 5 Opsonophagocytic activity among a subset of subjects from Studies 1 and 2. Parameter
Study 1
Study 2
V710 (30 g) with AAHSA N = 16
% ≥2-fold-rise % ≥3-fold-rise % ≥4-fold-rise GMFR
V710 (30 g) without AAHSA N = 16
V710 (60 g) lyophilized N = 12
Placebo N=4
Observed response
95% CI
Observed response
95% CI
Observed response
95% CI
Observed response
95% CI
56.3% (9/16) 31.3% (5/16) 6.3% (1/16) 2.3
(29.9%, 80.2%) (11.0%, 58.7%) (0.2%, 30.2%) (1.8, 2.8)
50.0% (8/16) 25.0% (4/16) 25.0% (4/16) 2.4
(24.7%, 75.3%) (7.3%, 52.4%) (7.3%, 52.4%) (1.7, 3.4)
58.3% (7/12) 33.3% (4/12) 33.3% (4/12) 2.7
(27.7%, 84.8%) (9.9%, 65.1%) (9.9%, 65.1%) (1.6, 4.5)
25.0% (1/4) 25.0% (1/4) 0.0% (0/4) 1.2
(0.6%, 80.6%) (0.6%, 80.6%) (0.0%, 60.2%) (0.4, 3.8)
CI: confidence interval; GMFR: geometric mean fold-rise from baseline; AAHSA: Amorphous aluminum hydroxyphosphate sulfate adjuvant.
from either study due to any type of AE throughout the duration of the studies. No subjects developed a documented temperature >99.9 ◦ F (37.7 ◦ C) during postvaccination Days 1–5. In both studies, the frequency of vaccine-related AEs and injection-site AEs were generally similar between treatment groups for each study (Table 6). One subject in Study 1 receiving V710 without AAHSA reported severe nausea, which was determined by the investigator to be probably not related to study vaccine. The remainder of AEs in both studies was reported by patients to be of mild or moderate intensity.
LUMINEX Geometric Mean Titers Over Time
Antibody Titer (mcg/mL) Log10 Scale
A 150 100
Study 1 V710 (30 g) with AAHSA N = 32 n (%)
Study 2 V710 (30 g) without AAHSA N = 32 n (%)
V710 (60 g) lyophilized N = 41 n (%)
Placebo N = 10 n (%)
Vaccine-related adverse experiences 0 (0.0) 1 (3.1) Nausea 1 (3.1) 1 (3.1) Fatigue 3 (9.4) 0 (0.0) Myalgia 5 (15.6) Headache 5 (15.6) 1 (3.1) Dizziness 1 (3.1)
1 (2.4) 2 (4.9) 1 (2.4) 1 (2.4) 0 (0.0)
0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0)
Injection-site adverse experiences 3 (9.4) 0 (0.0) Bruising 2 (6.3) Erythema 3 (9.4) 9 (28.1) 6 (18.8) Pain 1 (3.1) 0 (0.0) Pruritis 3 (9.4) Swelling 1 (3.1) Warmth 0 (0.0) 1 (3.1)
1 (2.4) 0 (0.0) 5 (12.2) 0 (0.0) 0 (0.0) 0 (0.0)
0 (0.0) 0 (0.0) 1 (10.0) 0 (0.0) 0 (0.0) 0 (0.0)
AAHSA: amorphous aluminum hydroxyphosphate sulfate adjuvant. 50 With AAHSA (n=32) No AAHSA (n=32)
4. Discussion
10 110 28 14
84
180
360
Relative Day to Vaccination
B Antibody Concentration (μg/mL) Log10 Scale
Table 6 Frequency of the most common vaccine-related adverse experiences Days 1–14 following vaccination.
200 150 100
n V710 (60 mcg) Lyopholized 41 Placebo 10
50
20
Baseline 10 14
28
84
Relative Day to Vaccination n = Number of subjects with serology at Day 14.
Fig. 3. Geometric mean concentrations of IsdB-specific IgG concentrations over time in Study 1 (A) and Study 2 (B).
An effective, prophylactic S. aureus vaccine needs to be well-tolerated, rapidly induce a protective and durable immune response against the majority of S. aureus strains, immunogenic in populations at greatest risk of developing invasive staphylococcal disease, and sufficiently stable to maintain potency under routine storage conditions. The first-in-human V710 vaccine study (Protocol 001) established an initial safety and immunogenicity benchmark across a broad dose-range in healthy adults ≤55 years old [12]. These two follow-up studies were conducted to assess the need for adjuvantation, the immunogenicity, safety and tolerability of lyophilized V710, and the magnitude and duration of immune responses in older adults ≤80 years of age. In Study 1, V710 was immunogenic following a single 30-g dose of liquid vaccine with or without AAHSA. The 30-g dosage was chosen based on a first-in-human study. Since 30 g represented the lowest dose where a sizeable difference in anti-IsdB IgG antibody titers was detected between V710 and placebo, it was anticipated that the 30-g dosage would be suitable for detecting a potential immunogenicity difference between V710 with AAHSA and V710 without AAHSA. In fact, the results in Study 1 showed that the frequency, magnitude and kinetics of immune responses were comparable in both groups. Most subjects in both groups mounted positive immune responses at Day 14. Immune responses peaked at Day 84, and approximately half of the vaccinees continued to have positive antibody titers for at least 1 year postvaccination. Postvaccination adverse experiences were low in frequency, mildto-moderate in severity, and self-limited in duration in adjuvanted
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and non-adjuvanted V710 recipients. Definitive immunogenicity data from Study 1 enabled the use of a non-adjuvanted vaccine in subsequent V710 clinical development. Study 2 was a bridging protocol study conducted with the purpose of evaluating the immunogenicity and safety of a lyophilized formulation of V710. The reconstituted lyophilized formulation of V710 evaluated in Study 2 had comparable physical properties and antigen content to the refrigerated liquid formulation used in previous V710 studies. In Study 2, a 60-g dosage of the lyophilized formulation of V710 was chosen as this was the anticipated dose to be used in the subsequent Phase II/III studies [12]. In this study, a single 60-g dose of lyophilized V710 reconstituted in 0.45% saline was immunogenic in healthy adults 18–80 years of age. IgG antibodies against IsdB rose rapidly in vaccinees, with most subjects achieving positive immune responses within 14 days postvaccination. After Day 14, the frequency and magnitude of immune responses remained high, rising slightly through Day 84 (the end of the study). Antibody titers obtained at 4 postvaccination immunogenicity timepoints (Days 10, 14, 28, and 84) were comparable to those observed at the same timepoints in both Study 1, here, and in the first-in-human study [12]. The lyophilized formulation was well tolerated. There were no unexpected or untoward vaccine reactions. Injection-site reactogenicity was minimal, with only a small minority of subjects reporting mild-to-moderate injection-site pain. Notably, the frequency and intensity of pain were similar to those observed following injection of liquid V710 with or without AAHSA in Study 1. S. aureus bacteremia is the most common serious invasive S. aureus infection with attributable mortality ranging up to 28% [18]. It is also associated with significant morbidity and frequent metastatic complications (∼10%), including endocarditis, osteomyelitis, and septic arthritis. Bacteremia is more common at the extremes of age and is more commonly nosocomial in nature [2,19]. Therefore, older adults represent a target population for vaccine need and utilization. Studies 1 and 2 extended previous V710 safety and immunogenicity information in individuals up to 80 years old. Stratified by age, immunogenicity outcomes were comparable among younger and older cohorts of subjects. Additionally, older vaccine recipients in these studies demonstrated immune responses that were at least as vigorous as those seen in subjects ≤55 years of age in the first-in-human study. Patients who develop invasive staphylococcal disease such as endocarditis, septic arthritis, or osteomyelitis commonly have predisposing chronic disease, thereby complicating management [20]. At highest risk are patients undergoing cardiothoracic surgery, orthopedic surgery with prosthesis placement, and chronic hemodialysis [20]. S. aureus infections associated with prosthetic devices, such as artificial joints, artificial heart valves, and permanent dialysis catheters, are particularly devastating, for such infections require long term antibiotic therapy (weeks to months) and often require removal of the device to cure the infection [21,22]. The fact that the majority of serious S. aureus infections are temporally related to hospitalization and/or performance of invasive procedures, indicates that access and opportunity for prophylactic vaccination are possible. To that point, in these studies, immune response peaked at 84 days, and the percentage of responders receiving V710 in the 2 studies was substantial within 14 days postinjection. Furthermore, this percentage continued to rise through Day 84 (and remained high through 1 year in Study 1, although somewhat lower). Given the response over time, it seems likely the optimal timing between inoculation and surgery would be no earlier than 14 days before surgery, and, ideally, no later than 70 days (i.e., 84 minus 14) before surgery. However the continued response shown in Study 1 suggests that a fairly high proportion of patients would still be
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protected well beyond 60 (or 84) days even if vaccination occurred 14 days before surgery. We note that a proof-of-concept efficacy trial of V710 in cardiothoracic surgery patients specified that vaccinations occur 14–60 days before scheduled surgery. However, that study was recently terminated based on an interim futility analysis. 5. Conclusions Our findings demonstrate that multiple formulations of V710 are immunogenic and well tolerated across a broad age range of healthy adults. Successful lyophilization and concomitant safety and potency support advancement of the lyophilized formulation without adjuvant (i.e., no AAHSA) into subsequent Phase II/III trials. Further analysis of the recently terminated efficacy trial in cardiothoracic surgery patients will guide future V710 clinical development. Acknowledgments We thank Jennifer Pawlowski, MS for her assistance with the preparation of the manuscript. Contributors: Drs. Hartzel, Smugar, and Kartsonis, Mr. Onorato, and Ms. Lipka are or were employees of Merck, and may own stock or hold options to own stock in the company. Drs. Harro and Betts have been investigators for Merck. Dr. Harro has received contract support from Merck related to its S. aureus, influenza virus, and HIV1 vaccine programs, and received fees for service on safety advisory committees for HIV-1 vaccine trials. Funding: This study was funded by Merck Sharp & Dohme, a subsidiary of Merck & Co., Inc. The study sponsor was involved in the study design; data collection, analysis, and interpretation of data; in writing the report; and the decision to submit the paper for publication. References [1] Stranger-Jones YK, Bae T, Schneewind O. Vaccine assembly from surface proteins of Staphylococcus aureus. Proc Natl Acad Sci U S A 2006;103:16942–7. [2] Lowy FD. Staphylococcus aureus infections. N Engl J Med 1998;339:520–32. [3] National Nosocomial Infections Surveillance (NNIS) report, data summary from October 1986–April 1996, issued May 1996. A report from the National Nosocomial Infections Surveillance (NNIS) System. Am J Infect Control 1996;24:380–8. [4] Rosenthal VD, Maki DG, Jamulitrat S, Medeiros EA, Todi SK, Gomez DY, et al. International Nosocomial Infection Control Consortium (INICC) report, data summary for 2003–2008, issued June 2009. Am J Infect Control 2010;38:95–104. [5] Boyce JM, Cookson B, Christiansen K, Hori S, Vuopio-Varkila J, Kocagöz S, et al. Meticillin-resistant Staphylococcus aureus. Lancet Infect Dis 2005;5:653–63. [6] Fridkin SK, Hageman JC, Morrison M, Sanza LT, Como-Sabetti K, Jernigan JA, et al. Methicillin-resistant Staphylococcus aureus disease in three communities. N Engl J Med 2005;352:1436–44. [7] Hidron AI, Edwards JR, Patel J, Horan TC, Sievert DM, Pollock DA, et al. NHSN annual update: antimicrobial-resistant pathogens associated with healthcareassociated infections: annual summary of data reported to the National Healthcare Safety Network at the Centers for Disease Control and Prevention, 2006–2007. Infect Control Hosp Epidemiol 2008;29:996–1011. [8] Marty FM, Yeh WW, Wennersten CB, Venkataraman L, Albano E, Alyea EP, et al. Emergence of a clinical daptomycin-resistant Staphylococcus aureus isolate during treatment of methicillin-resistant Staphylococcus aureus bacteremia and osteomyelitis. J Clin Microbiol 2006;44:595–7. [9] van Hal SJ, Paterson DL, Gosbell IB. Emergence of daptomycin resistance following vancomycin-unresponsive Staphylococcus aureus bacteraemia in a daptomycin-naive patient—a review of the literature. Eur J Clin Microbiol Infect Dis 2011;49:1489–94. [10] Johnson MD, Decker CF. Antimicrobial agents in treatment of MRSA infections. Dis Mon 2008;54:793–800. [11] Shinefield H, Black S, Fattom A, Horwith G, Rasgon S, Ordonez J, et al. Use of a Staphylococcus aureus conjugate vaccine in patients receiving hemodialysis. N Engl J Med 2002;14(346):491–6. [12] Harro C, Betts R, Orenstein W, Kwak EJ, Greenberg HE, Onorato MT, et al. Safety and immunogenicity of a novel Staphylococcus aureus vaccine: results from the first study of the vaccine dose range in humans. Clin Vaccine Immunol 2010;17:1868–74. [13] Kuklin NA, Clark DJ, Secore S, Cook J, Cope LD, McNeely T, et al. A novel Staphylococcus aureus vaccine: iron surface determinant B induces rapid antibody
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responses in rhesus macaques and specific increased survival in a murine S. aureus sepsis model. Infect Immun 2006;74:2215–23. Ster C, Beaudoin F, Diarra MS, Jacques M, Malouin F, Lacasse P. Evaluation of some Staphylococcus aureus iron-regulated proteins as vaccine targets. Vet Immunol Immunopathol 2010;15(136):311–8. Cleland JL, Powell MF, Shire SJ. The development of stable protein formulations: a close look at protein aggregation, deamidation, and oxidation. Crit Rev Ther Drug Carrier Syst 1993;10:307–77. Rexroad J, Wiethoff CM, Jones LS, Middaugh CR. Lyophilization and the thermostability of vaccines. Cell Preserv Technol 2002;1:91–104. Brandau DT, Jones LS, Wiethoff CM, Rexroad J, Middaugh CR. Thermal stability of vaccines. J Pharm Sci 2003;92:218–31. Fowler Jr VG, Olsen MK, Corey GR, Woods CW, Cabell CH, Reller LB, et al. Clinical identifiers of complicated Staphylococcus aureus bacteremia. Arch Intern Med 2003;163:2066–72.
[19] McClelland RS, Fowler Jr VG, Sanders LL, Gottlieb G, Kong LK, Sexton DJ, et al. Staphylococcus aureus bacteremia among elderly vs younger adult patients: comparison of clinical features and mortality. Arch Intern Med 1999;159:1244–7. [20] Chang FY, MacDonald BB, Peacock Jr JE, Musher DM, Triplett P, Mylotte JM, et al. A prospective multicenter study of Staphylococcus aureus bacteremia: incidence of endocarditis, risk factors for mortality, and clinical impact of methicillin resistance. Medicine (Baltimore) 2003;82:322–32. [21] Gottlieb GS, Fowler Jr VG, Kong LK, McClelland RS, Gopal AK, Marr KA, et al. Staphylococcus aureus bacteremia in the surgical patient: a prospective analysis of 73 postoperative patients who developed Staphylococcus aureus bacteremia at a tertiary care facility. J Am Coll Surg 2000;190:50–7. [22] Fowler Jr VG, Miro JM, Hoen B, Cabell CH, Abrutyn E, Rubinstein E, et al. Staphylococcus aureus endocarditis: a consequence of medical progress. JAMA 2005;293:3012–21.
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The immunogenicity and safety of different formulations of a novel Staphylococcus aureus vaccine (V710): Results of two Phase I studies夽 Clayton D. Harro a,∗ , Robert F. Betts b , Jonathan S. Hartzel c , Matthew T. Onorato c , Joy Lipka c,1 , Steven S. Smugar c , Nicholas A. Kartsonis c a
Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA University of Rochester Medical Center, Rochester, NY, USA c Merck Sharp & Dohme Corp, Whitehouse Station, NJ, USA b
a r t i c l e
i n f o
Article history: Received 20 April 2011 Received in revised form 2 December 2011 Accepted 7 December 2011 Available online 20 December 2011 Keywords: Adjuvant Immunogenicity Phase I Staphylococcus aureus Vaccine Iron surface determinant (IsdB)
a b s t r a c t Merck V710 is a novel vaccine that contains the highly conserved Staphylococcus aureus iron surface determinant B (IsdB) protein. V710 has induced positive immune responses in healthy subjects. The purpose of the two studies described herein was to evaluate the immunogenicity and safety of two different formulations of V710. Both studies were randomized, controlled, double-blind, parallel-group trials. Study 1 compared liquid, aluminum-adjuvanted V710 (30 g) with liquid, non-adjuvanted V710 (30 g) in a 1:1 ratio in 64 healthy adults (18–70 years). Study 2 compared non-adjuvanted lyophilized V710 (60 g) with saline placebo in a 4:1 ratio in 51 healthy adults (18–80 years). Blood was collected at screening and up to Day 360 postvaccination in Study 1, and at screening and up to Day 84 postvaccination in Study 2. Sera were analyzed for IsdB-specific antibodies using a total IgG assay. The primary endpoints in Study 1 were the proportion of patients with a positive immune response (≥2-fold rise in IsdB-specific IgG antibody level) the geometric mean concentration (GMC), and the geometric mean-fold rise (GMFR), all from baseline at Day 14. The primary endpoint in Study 2 was the GMFR in IsdB-specific IgG antibody concentration from baseline at Day 14. In Study 1, 84.4% responded in the adjuvanted V710 group, and 71.9% in the non-adjuvanted V710 group. The GMC was 115.4 g/mL in the adjuvanted group and 99.1 g/mL in the nonadjuvanted group. The GMFR in antibody concentration in the group receiving aluminumadjuvanted V710 was 4.5 and the GMFR in the group receiving non-adjuvanted V710 was 4.0. In Study 2, the GMFR in antibody concentration in the V710 group was 5.3, and 80.5% had a positive immune response. None responded in the placebo group. Positive immune response was seen in the active treatment groups over the full duration of each study. There were no serious adverse experiences (AE) in either study, and no patients discontinued due to an AE. There were no clinically meaningful differences in AEs between groups in either study. In conclusion, V710, both with and without aluminum adjuvant, and in both liquid and lyophilized formulations, was immunogenic within 14 days of vaccination. All treatments showed similar safety profiles. © 2011 Elsevier Ltd. All rights reserved.
1. Introduction Staphylococcus aureus is a human commensal, colonizing the skin and mucous membranes of 25–50% of individuals [1,2]. When these natural barriers are compromised naturally or iatrogenically, staphylococci can cause a wide range of disease from uncomplicated local suppuration to sepsis [2]. Importantly, S. aureus is the
夽 Clinical trial registration: Study 1: NCT01324440. Study 2: NCT00822757. ∗ Corresponding author at: Center for Immunization Research, Johns Hopkins University, Bloomberg School of Public Health, 624 N. Broadway, Hampton House Rm. 117, Baltimore, MD 21205, USA. Tel.: +1 410 614 4937; fax: +1 410 502 6898. E-mail address: [email protected] (C.D. Harro). 1 Present address: Cerexa Inc., Oakland, CA, USA. 0264-410X/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2011.12.045
leading etiology of nosocomial infections [3,4]. The complexity of these infections varies depending on the virulence characteristics of the bacteria and the underlying condition of the human host. Reliance on antimicrobial therapy for treatment and control of staphylococcal disease is increasingly problematic due to the rising prevalence of methicillin-resistant S. aureus (MRSA) [5–7]. Of increasing concern is the emergence of community-acquired MRSA and recalcitrant S. aureus strains that are resistant to multiple antibiotics, even those with novel mechanisms of action such as oxazolidinones and daptomycin [8–10]. High morbidity and mortality associated with S. aureus infections, coupled with pervasive antimicrobial resistance and limited therapeutic alternatives are spurring efforts to develop a prophylactic S. aureus vaccine [11,12]. Previously, we reported the results from the first-in-human dose-ranging study of V710, a purified subunit vaccine being devel-
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oped by Merck [12]. V710 contains a highly conserved surface protein called iron surface determinant B (IsdB), whose intrinsic characteristics make it an effective immunogen [1,13,14]. Data from the initial study demonstrated that a single intramuscular administration of liquid V710 adjuvanted with amorphous aluminum hydroxyphosphate sulfate adjuvant (AAHSA) is safe and immunogenic in 18–55 year old healthy adults. Recipients of any of three dose regimens (5, 30, or 90 g) generated a positive immune response (defined as a 2-fold increase in IsdB-specific IgG titers relative to baseline), but a greater proportion of 30-g and 90g recipients manifested positive immune responses and achieved higher geometric mean titers (GMTs) relative to 5-g or placebo recipients 14 days postvaccination, the primary immunological endpoint. These immune responses remained elevated at 3 months postvaccination (the end of the study). Accelerated stability studies of the liquid formulation of V710 projected lability in the chemical composition of the IsdB antigen (unpublished data). Such degradation of antigenic proteins commonly occurs through amidation and oxidation of susceptible amino acid residues [15,16]. However, lyophilization with appropriate cryoprotectants provides the potential for long-term thermostability of vaccines and extended shelf-life [16,17]. Additionally with the emphasis on reducing adjuvant usage in vaccines, a comparison of vaccine immunogenicity in the presence or absence of adjuvant was warranted. Therefore, this report describes two follow-up studies designed to broaden the human safety and immunogenicity profile of V710 and to explore alternative vaccine formulations. Study 1 compared liquid V710 given with or without AAHSA to detect a potential immunogenicity difference, if present, between the adjuvanted and non-adjuvanted formulations. Study 2 compared a reconstituted lyophilized formulation of V710 (without AAHSA) with placebo. 2. Materials and methods 2.1. Patients Both studies enrolled healthy, nonpregnant adult subjects age 18–70 (Study 1) or 18–80 years (Study 2). Key exclusion criteria included: temperature ≥100.4 ◦ F (≥38 ◦ C) within 48 h of study vaccination; chronic skin conditions; serious S. aureus infection in the previous 12 months; history of anaphylaxis to aluminum-containing adjuvant or other vaccine components; previous vaccination with V710; vaccination with a live virus vaccine within 30 days prior to scheduled V710 vaccination or anticipated vaccination with a live virus vaccine within 30 days following study entry; any other vaccination within 14 days of scheduled V710 vaccination or anticipated vaccination within 30 days following study entry; systemic corticosteroids or other immunosuppressive medications or biological agents within 14 days of V710 vaccination or anticipated administration of such medications during the course of the study. All patients gave written informed consent prior to participation. Both studies were conducted in accordance with the principles of Good Clinical Practice and were approved by the appropriate institutional review boards and regulatory agencies. 2.2. Study design Study 1 (Protocol 002) was a 12-month, randomized, multicenter, double-blind, Phase I study conducted at 2 sites in the United States from 20 September 2006 to 15 October 2007. Eligible subjects were randomized in a 1:1 ratio to receive a single 0.5-mL vaccination of either liquid V710 containing AAHSA (30 g) or liquid V710 without AAHSA (30 g). Enrollment was stratified such that half of the subjects were ≥50 years of age. Study 2 (Protocol
004) was a 3-month, randomized, multicenter, double-blind, Phase I study conducted at 2 sites in the United States from 5 September 2007 to 17 December 2007. Eligible subjects were randomized in a 4:1 ratio to a single 0.5-mL vaccination of either lyophilized V710 (60 g) or saline placebo. Enrollment was stratified to ensure that half of the subjects would be ≥60 years of age, and that at least 40% of the patients in this upper age group (i.e., 20% overall) would be ≥70 years. Because of the differences in appearance between V710 with and without adjuvant, and between V710 and saline placebo, one unblinded member of the study site staff was responsible for preparing and administering the study vaccine or placebo. This person had no further involvement with any subsequent study procedure postvaccination, including safety follow-up procedures. All other individuals involved with the study conduct, including investigators, study physicians, and statisticians remained blinded to treatment group throughout the study. An interim safety and immunogenicity analysis was planned for both studies, and performed by an unblinded statistician and clinical monitor at Merck. The official clinical database remained blinded until the medical/scientific review was completed and the data were declared complete. 2.3. Immunogenicity evaluation Sera were analyzed for IsdB-specific IgG antibodies using an assay on the LUMINEXTM multianalyte profiling platform, which had a lower limit of quantitation of 0.85 ng/mL (non-dilution corrected). Blood samples were collected at screening and postvaccination Days 10, 14, 28 (1 month), 84 (3 months), 180 (6 months), and 360 (1 year) in Study 1, and at screening and postvaccination Days 10, 14, 28 (1 month), and 84 (3 months) in Study 2. The primary immunogenicity timepoint in both studies was at Day 14 postvaccination. Immunogenicity was evaluated by the following methods: the proportion of subjects with a positive immune response, defined as a ≥2-fold rise in titer from baseline, the geometric mean concentration (GMC) at Day 14, and the geometric mean fold-rise (GMFR) at Day 14. The ≥2-fold rise endpoint was also used in the earlier Phase I study by Harro et al. [12], and was chosen based on the observed within-subject variability and maximum fold-rises in titers among longitudinal control subjects as well as the fold-rises in titers among V710-immunized monkeys, both of which were obtained during the validation experiments for the LUMINEXTM assay. The primary endpoints in Study 1 were the proportion of patients with a positive immune response, the GMC, and the GMFR, each from baseline at Day 14 postvaccination. The primary endpoint in Study 2 was the GMFR in IsdB-specific IgG antibody concentration from baseline at Day 14. Exploratory endpoints included the immune response at the other timepoints, and comparison of immune response between the 2 age groups. As an exploratory endpoint, we analyzed opsonophagocytic activity (OPA) from serum obtained from a subset of subjects in each study, measuring the ability of a phagocytic cell line to uptake labeled bacteria in the presence of serum specific antibody (reported as the fold-rise in serum OPA). This assay was used as an adjunct to demonstrate the functional activity of the antibodies elicited by the vaccine. 2.4. Safety evaluation All subjects were monitored for 30 min postvaccination for any immediate reaction, and were asked to record local adverse experiences (AEs) at the injection site and oral temperatures daily for 5 consecutive days postvaccination on a standardized Vaccination Report Card (VRC). Subjects also recorded all systemic AEs for 14
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Table 1 Baseline patient characteristics. Study 1
Study 2
V710 (30 g) with AAHSA N = 32
V710 (30 g) without AAHSA N = 32
V710 (60 g) lyophilized N = 41
Gender, n (%) Male Female
12 (37.5) 20 (62.5)
19 (59.4) 13 (40.6)
19 (46.3) 22 (53.7)
Age, years Mean (SD) ≤17 18–29 30–39 40–49 50–59 60–69 70–80a
46.5 (12.5) 0 (0.0) 6 (18.8) 1 (3.1) 9 (28.1) 12 (37.5) 4 (12.5) 0 (0.0)
45.6 (13.1) 0 (0.0) 5 (15.6) 4 (12.5) 7 (21.9) 10 (31.3) 6 (18.8) 0 (0.0)
53.4 (18.5) 0 (0.0) 6 (14.6) 5 (12.2) 8 (19.5) 1 (2.4) 10 (24.4) 11 (26.8)
Race, n (%) Asian Black White
1 (3.1) 13 (40.6) 18 (56.3)
0 (0.0) 10 (31.3) 22 (68.8)
2 (4.9) 8 (19.5) 31 (75.6)
Placebo N = 10
6 (60.0) 4 (40.0) 53.9 (15.1) 0 (0.0) 1 (10.0) 0 (0.0) 4 (40.0) 0 (0.0) 5 (50.0) 0 (0.0) 0 (0.0) 2 (20.0) 8 (80.0)
a Study 1 enrolled patients up to age 70; Study 2 enrolled patients up to age 80. AAHSA: amorphous aluminum hydroxyphosphate sulfate adjuvant; SD: standard deviation.
days postvaccination; patients were prompted to report 4 specific systemic AEs: headache, nausea, myalgia, and fatigue. Only vaccinerelated serious AEs, AEs leading to death, or serious AEs associated with a S. aureus infection were reported for the remainder of the follow-up periods.
Because there was no hypothesis in Study 1, and because there was only a single hypothesis in Study 2, there were no adjustments for multiplicity in either study. 3. Results
2.5. Statistical analysis
3.1. Patients
The primary immunogenicity analyses in both studies were based on the per-protocol populations, defined as subjects who did not have missing baseline serology data or postvaccination serology data at Day 14, and who did not develop a S. aureus infection during the 14 days postvaccination. Subjects with missing data for a given timepoint were excluded from the immune response rate and fold-rise calculations for that particular timepoint. As Study 1 was an estimation study, no formal hypothesis testing was performed. Immune responses at Day 14 postvaccination were descriptively summarized, including 95% confidence intervals (CIs). Fold-differences in GMC) were calculated using an analysis of covariance (ANCOVA) model with the log titers as the response variable, and treatment group, the log predose titer, and age as fixed effects. For Study 2, the primary hypothesis was that a single dose of lyophilized V710 (60 g) would result in a ≥2.5-fold rise in anti-IsdB-specific IgG titers at Day 14 postvaccination relative to baseline. The primary analysis was calculated based on the natural logarithm of the Day 14 and baseline antibody titers, along with the 95% CI, based on Student’s t-distribution. All subjects who were vaccinated and had safety follow-up data were included in the safety summaries. Safety measures of interest included any AEs, injection-site AEs, systemic AEs, and vaccine-related AEs. The incidence of vaccine-related serious AEs was summarized using exact CIs. For Study 1, with 24 evaluable patients in each group, the study could detect a ≥25 percentage point difference in the positive immune response proportions between treatment groups (i.e., the 95% CI for the difference in positive immune response rates would exclude 0), assuming a positive immune response in approximately 80% of subjects. For Study 2, assuming an expected GMFR of 4.0 in patients receiving lyophilized V710 (60 g) and a standard deviation of the log fold-rise in antibody titer of 0.87, 38 evaluable V710 recipients would provide 91% power (1-sided ˛ = 0.025) to detect a GMFR of ≥2.5 at Day 14 relative to baseline.
Patient accounting and demographics are shown in Fig. 1 and Table 1, respectively. A total of 64 patients were randomized in Study 1 and 51 were randomized in Study 2. One patient in Study 1 was lost to follow-up. All patients in both studies were included in the primary analyses. In both studies, there was a gender imbalance between each of the 2 treatment groups. However, other patient characteristics were generally similar between each of the 2 treatment groups in each study. As planned, 50% of the patients were ≥older than 50 years in Study 1, and 51% were older than 60 years in Study 2. 3.2. Immunogenicity results The immunogenicity results at Day 14 are summarized in Table 2. For the main immunogenicity endpoint in Study 1, 84.4% of patients in the V710 with AAHSA group and 71.9% of patients in the V710 without AAHSA group had a positive immune response, defined as a ≥2-fold rise in IsdB IgG antibody at Day 14 postvaccination. At this time point, the GMFR in the V710 with AAHSA group and V710 without AAHSA group was 4.5 and 4.0, respectively. Results were generally comparable between groups in Study 1; the V710 with AAHSA had numerically greater measures, but these differences were not statistically significant. For the main immunogenicity endpoint in Study 2, the GMFR in IsdB IgG antibody was 5.3 in the V710 group whereas the placebo group did not respond (GMFR = 1.0). The rise from baseline in the V710 group was statistically significant (p < 0.001) based on the lower 95% CI bound of 3.8. In Study 2, approximately 80% of patients in the V710 group achieved ≥2-fold rise in antibodies on Day 14 postvaccination relative to baseline, as compared with no patients in the placebo group. Lyophilized V710 was numerically greater than placebo for all measures. In both studies the immunogenicity response was greater in older patients receiving V710, particularly for patients with ≥3and ≥4-fold-rises from baseline (Tables 3 and 4).
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A
Screened N = 65
Not Randomized N = 1 Did not meet inclusion criteria N= 1
Randomized N = 64
V710 30 μg with AAHSA N = 32
Discontinued N = 0
V710 30 μg without AAHSA N = 32
Discontinued N = 1 Subject moved N = 1
Completed N = 32
Major protocol violations N = 0
Completed N = 31
Major protocol violations N = 0
Included in primary analysis N = 32
Included in primary analysis N = 32
B
Screened N = 51
Not Randomized N = 0
Randomized N = 51
V710 lyophilized 60 μg N = 41
Discontinued N = 0
Placebo N = 10
Discontinued N = 0
Completed N = 41
Major protocol violations N = 0
Completed N = 10
Major protocol violations N = 0
Included in primary analysis N = 41
Included in primary analysis N = 10
Fig. 1. Patient accounting in Study 1 (A) and Study 2 (B). 1 patient in the V710 without AAHSA group had data through Day 180, but moved prior to the Day 360 visit.
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Table 2 Immunogenicity results at Day 14 postvaccination. Parameter
Study 1
Study 2
V710 (30 g) with AAHSA N = 32
% ≥2-fold-rise % ≥3-fold-rise % ≥4-fold-rise % ≥8-fold-rise % ≥16-fold-rise GMFR GMC (g/mL)
V710 (30 g) without AAHSA N = 32
V710 (60 g) lyophilized N = 41
Placebo N = 10
Observed response
95% CI
Observed response
95% CI
Observed response
95% CI
Observed response
95% CI
84.4% (27/32) 71.9% (23/32) 53.1% (17/32) 21.9% (7/32) 3.1% (1/32) 4.5 115.4
(67.2%, 94.7%) (53.3%, 86.3%) (34.7%, 70.9%) (9.2%, 40.0%) (0.1%, 16.2%) (3.3, 6.1) (82.9, 160.6)
71.9% (23/32) 59.4% (19/32) 50.0% (16/32) 25.0% (8/32) 6.3% (2/32) 4.0 99.1
(53.3%, 86.3%) (40.6%, 76.3%) (31.9%, 68.1%) (11.5%, 43.4%) (0.8%, 20.8%) (2.9, 5.4) (73.5, 133.6)
80.5% (33/41) 61.0% (25/41) 48.8% (20/41) 39.0% (16/41) 17.1% (7/41) 5.3 112.5
(65.1%, 91.2%) (44.5%, 75.8%) (32.9%, 64.9%) (24.2%, 55.5%) (7.2%, 32.1%) (3.8, 7.2) (83.2, 152.1)
0.0% (0/10) 0.0% (0/10) 0.0% (0/10) 0.0% (0/10) 0.0% (0/10) 1.0 17.7
(0.0%, 30.8%) (0.0%, 30.8%) (0.0%, 30.8%) (0.0%, 30.8%) (0.0%, 30.8%) (0.9, 1.0) (10.3, 30.4)
CI: confidence interval; GMC: geometric mean concentration; GMFR: geometric mean fold-rise from baseline; AAHSA: amorphous aluminum hydroxyphosphate sulfate adjuvant.
Age ≥50 years
V710 (30 g) with AAHSA N = 32 Observed response
V710 (30 g) without AAHSA N = 32 Observed response
% ≥2-fold-rise % ≥3-fold-rise % ≥4-fold-rise GMFR GMC (g/mL)
81.3% (13/16) 75.0% (12/16) 43.8% (7/16) 3.7 114.1
75.0% (12/16) 56.3% (9/16) 43.8% (7/16) 3.7 95.3
% ≥2-fold-rise % ≥3-fold-rise % ≥4-fold-rise GMFR GMC (g/mL)
87.5% (14/16) 68.8% (11/16) 62.5% (10/16) 5.4 116.6
68.8% (11/16) 62.5% (10/16) 56.3% (9/16) 4.3 102.9
GMC: geometric mean concentration; GMFR: geometric mean fold-rise from baseline; AAHSA: amorphous aluminum hydroxyphosphate sulfate adjuvant.
3.3. Safety No subjects in either study reported a serious AE, a vaccinerelated serious AE, no patients died, and no subjects discontinued
A
Immunogenicity over the course of the studies is shown in Figs. 2 and 3. In Study 1, the proportions of patients with a positive immune response (i.e., ≥2-fold-rise in IgG titers) were similar for both V710 with and without AAHSA, and peaked at Day 84 (Fig. 2A). In Study 2, the proportion of positive immune responders in the lyophilized V710 group continued to rise throughout the course of the study (i.e., through Day 84). In the placebo group, 1 patient had a positive immune response at Day 10, but no patients in the placebo group had a positive immune response thereafter (Fig. 2B). A similar pattern was observed in GMC over Table 4 Immunogenicity results at Day 14 postvaccination stratified by age group, Study 2. Parameter
Age <60 years
Age ≥60 years
V710 (60 g) lyophilized N = 41 Observed response
Placebo N = 10 Observed response
% ≥2-fold-rise % ≥3-fold-rise % ≥4-fold-rise GMFR GMC (g/mL)
70.0% (14/20) 50.0% (10/20) 40.0% (8/20) 4.0 96.9
0.0% (0/5) 0.0% (0/5) 0.0% (0/5) 0.9 26.2
% ≥2-fold-rise % ≥3-fold-rise % ≥4-fold-rise GMFR GMC (g/mL)
90.5% (19/21) 71.4% (15/21) 57.1% (12/21) 6.8 129.7
0.0% (0/5) 0.0% (0/5) 0.0% (0/5) 1.0 11.9
Proportion of patients with positive response (%)
Age <50 years
Parameter
time, with generally comparable levels for V710 with and without AAHSA that peaked at Day 84 in Study 1 (Fig. 3A), and substantially greater levels with lyophilized V710 than placebo in Study 2 (Fig. 3B). OPA results are shown in Table 5. Approximately 55% of patients receiving V710 and 25% of patients receiving placebo had a 2-fold rise in OPA activity, whereas approximately 21% and 0% had a 4-fold rise in OPA activity.
100
80
60
40
20
0 10 28 1 14
84
180
360
Days relative to vaccination V710 (30 μg) with AAHSA
B
Proportion of patients with positive response (%)
Table 3 Immunogenicity results at Day 14 postvaccination stratified by age group, Study 1.
V710 (30 μg) without AAHSA
100
80
60
40
20
0
GMC: geometric mean concentration GMFR: geometric mean fold-rise from baseline.
1
10 14
28
84
Days relative to vaccination V710 (60 μg) lyophilized
Placebo
Fig. 2. Proportion of subjects with a positive immune response over time in Study 1 (A) and Study 2 (B).
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Table 5 Opsonophagocytic activity among a subset of subjects from Studies 1 and 2. Parameter
Study 1
Study 2
V710 (30 g) with AAHSA N = 16
% ≥2-fold-rise % ≥3-fold-rise % ≥4-fold-rise GMFR
V710 (30 g) without AAHSA N = 16
V710 (60 g) lyophilized N = 12
Placebo N=4
Observed response
95% CI
Observed response
95% CI
Observed response
95% CI
Observed response
95% CI
56.3% (9/16) 31.3% (5/16) 6.3% (1/16) 2.3
(29.9%, 80.2%) (11.0%, 58.7%) (0.2%, 30.2%) (1.8, 2.8)
50.0% (8/16) 25.0% (4/16) 25.0% (4/16) 2.4
(24.7%, 75.3%) (7.3%, 52.4%) (7.3%, 52.4%) (1.7, 3.4)
58.3% (7/12) 33.3% (4/12) 33.3% (4/12) 2.7
(27.7%, 84.8%) (9.9%, 65.1%) (9.9%, 65.1%) (1.6, 4.5)
25.0% (1/4) 25.0% (1/4) 0.0% (0/4) 1.2
(0.6%, 80.6%) (0.6%, 80.6%) (0.0%, 60.2%) (0.4, 3.8)
CI: confidence interval; GMFR: geometric mean fold-rise from baseline; AAHSA: Amorphous aluminum hydroxyphosphate sulfate adjuvant.
from either study due to any type of AE throughout the duration of the studies. No subjects developed a documented temperature >99.9 ◦ F (37.7 ◦ C) during postvaccination Days 1–5. In both studies, the frequency of vaccine-related AEs and injection-site AEs were generally similar between treatment groups for each study (Table 6). One subject in Study 1 receiving V710 without AAHSA reported severe nausea, which was determined by the investigator to be probably not related to study vaccine. The remainder of AEs in both studies was reported by patients to be of mild or moderate intensity.
LUMINEX Geometric Mean Titers Over Time
Antibody Titer (mcg/mL) Log10 Scale
A 150 100
Study 1 V710 (30 g) with AAHSA N = 32 n (%)
Study 2 V710 (30 g) without AAHSA N = 32 n (%)
V710 (60 g) lyophilized N = 41 n (%)
Placebo N = 10 n (%)
Vaccine-related adverse experiences 0 (0.0) 1 (3.1) Nausea 1 (3.1) 1 (3.1) Fatigue 3 (9.4) 0 (0.0) Myalgia 5 (15.6) Headache 5 (15.6) 1 (3.1) Dizziness 1 (3.1)
1 (2.4) 2 (4.9) 1 (2.4) 1 (2.4) 0 (0.0)
0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0)
Injection-site adverse experiences 3 (9.4) 0 (0.0) Bruising 2 (6.3) Erythema 3 (9.4) 9 (28.1) 6 (18.8) Pain 1 (3.1) 0 (0.0) Pruritis 3 (9.4) Swelling 1 (3.1) Warmth 0 (0.0) 1 (3.1)
1 (2.4) 0 (0.0) 5 (12.2) 0 (0.0) 0 (0.0) 0 (0.0)
0 (0.0) 0 (0.0) 1 (10.0) 0 (0.0) 0 (0.0) 0 (0.0)
AAHSA: amorphous aluminum hydroxyphosphate sulfate adjuvant. 50 With AAHSA (n=32) No AAHSA (n=32)
4. Discussion
10 110 28 14
84
180
360
Relative Day to Vaccination
B Antibody Concentration (μg/mL) Log10 Scale
Table 6 Frequency of the most common vaccine-related adverse experiences Days 1–14 following vaccination.
200 150 100
n V710 (60 mcg) Lyopholized 41 Placebo 10
50
20
Baseline 10 14
28
84
Relative Day to Vaccination n = Number of subjects with serology at Day 14.
Fig. 3. Geometric mean concentrations of IsdB-specific IgG concentrations over time in Study 1 (A) and Study 2 (B).
An effective, prophylactic S. aureus vaccine needs to be well-tolerated, rapidly induce a protective and durable immune response against the majority of S. aureus strains, immunogenic in populations at greatest risk of developing invasive staphylococcal disease, and sufficiently stable to maintain potency under routine storage conditions. The first-in-human V710 vaccine study (Protocol 001) established an initial safety and immunogenicity benchmark across a broad dose-range in healthy adults ≤55 years old [12]. These two follow-up studies were conducted to assess the need for adjuvantation, the immunogenicity, safety and tolerability of lyophilized V710, and the magnitude and duration of immune responses in older adults ≤80 years of age. In Study 1, V710 was immunogenic following a single 30-g dose of liquid vaccine with or without AAHSA. The 30-g dosage was chosen based on a first-in-human study. Since 30 g represented the lowest dose where a sizeable difference in anti-IsdB IgG antibody titers was detected between V710 and placebo, it was anticipated that the 30-g dosage would be suitable for detecting a potential immunogenicity difference between V710 with AAHSA and V710 without AAHSA. In fact, the results in Study 1 showed that the frequency, magnitude and kinetics of immune responses were comparable in both groups. Most subjects in both groups mounted positive immune responses at Day 14. Immune responses peaked at Day 84, and approximately half of the vaccinees continued to have positive antibody titers for at least 1 year postvaccination. Postvaccination adverse experiences were low in frequency, mildto-moderate in severity, and self-limited in duration in adjuvanted
C.D. Harro et al. / Vaccine 30 (2012) 1729–1736
and non-adjuvanted V710 recipients. Definitive immunogenicity data from Study 1 enabled the use of a non-adjuvanted vaccine in subsequent V710 clinical development. Study 2 was a bridging protocol study conducted with the purpose of evaluating the immunogenicity and safety of a lyophilized formulation of V710. The reconstituted lyophilized formulation of V710 evaluated in Study 2 had comparable physical properties and antigen content to the refrigerated liquid formulation used in previous V710 studies. In Study 2, a 60-g dosage of the lyophilized formulation of V710 was chosen as this was the anticipated dose to be used in the subsequent Phase II/III studies [12]. In this study, a single 60-g dose of lyophilized V710 reconstituted in 0.45% saline was immunogenic in healthy adults 18–80 years of age. IgG antibodies against IsdB rose rapidly in vaccinees, with most subjects achieving positive immune responses within 14 days postvaccination. After Day 14, the frequency and magnitude of immune responses remained high, rising slightly through Day 84 (the end of the study). Antibody titers obtained at 4 postvaccination immunogenicity timepoints (Days 10, 14, 28, and 84) were comparable to those observed at the same timepoints in both Study 1, here, and in the first-in-human study [12]. The lyophilized formulation was well tolerated. There were no unexpected or untoward vaccine reactions. Injection-site reactogenicity was minimal, with only a small minority of subjects reporting mild-to-moderate injection-site pain. Notably, the frequency and intensity of pain were similar to those observed following injection of liquid V710 with or without AAHSA in Study 1. S. aureus bacteremia is the most common serious invasive S. aureus infection with attributable mortality ranging up to 28% [18]. It is also associated with significant morbidity and frequent metastatic complications (∼10%), including endocarditis, osteomyelitis, and septic arthritis. Bacteremia is more common at the extremes of age and is more commonly nosocomial in nature [2,19]. Therefore, older adults represent a target population for vaccine need and utilization. Studies 1 and 2 extended previous V710 safety and immunogenicity information in individuals up to 80 years old. Stratified by age, immunogenicity outcomes were comparable among younger and older cohorts of subjects. Additionally, older vaccine recipients in these studies demonstrated immune responses that were at least as vigorous as those seen in subjects ≤55 years of age in the first-in-human study. Patients who develop invasive staphylococcal disease such as endocarditis, septic arthritis, or osteomyelitis commonly have predisposing chronic disease, thereby complicating management [20]. At highest risk are patients undergoing cardiothoracic surgery, orthopedic surgery with prosthesis placement, and chronic hemodialysis [20]. S. aureus infections associated with prosthetic devices, such as artificial joints, artificial heart valves, and permanent dialysis catheters, are particularly devastating, for such infections require long term antibiotic therapy (weeks to months) and often require removal of the device to cure the infection [21,22]. The fact that the majority of serious S. aureus infections are temporally related to hospitalization and/or performance of invasive procedures, indicates that access and opportunity for prophylactic vaccination are possible. To that point, in these studies, immune response peaked at 84 days, and the percentage of responders receiving V710 in the 2 studies was substantial within 14 days postinjection. Furthermore, this percentage continued to rise through Day 84 (and remained high through 1 year in Study 1, although somewhat lower). Given the response over time, it seems likely the optimal timing between inoculation and surgery would be no earlier than 14 days before surgery, and, ideally, no later than 70 days (i.e., 84 minus 14) before surgery. However the continued response shown in Study 1 suggests that a fairly high proportion of patients would still be
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protected well beyond 60 (or 84) days even if vaccination occurred 14 days before surgery. We note that a proof-of-concept efficacy trial of V710 in cardiothoracic surgery patients specified that vaccinations occur 14–60 days before scheduled surgery. However, that study was recently terminated based on an interim futility analysis. 5. Conclusions Our findings demonstrate that multiple formulations of V710 are immunogenic and well tolerated across a broad age range of healthy adults. Successful lyophilization and concomitant safety and potency support advancement of the lyophilized formulation without adjuvant (i.e., no AAHSA) into subsequent Phase II/III trials. Further analysis of the recently terminated efficacy trial in cardiothoracic surgery patients will guide future V710 clinical development. Acknowledgments We thank Jennifer Pawlowski, MS for her assistance with the preparation of the manuscript. Contributors: Drs. Hartzel, Smugar, and Kartsonis, Mr. Onorato, and Ms. Lipka are or were employees of Merck, and may own stock or hold options to own stock in the company. Drs. Harro and Betts have been investigators for Merck. Dr. Harro has received contract support from Merck related to its S. aureus, influenza virus, and HIV1 vaccine programs, and received fees for service on safety advisory committees for HIV-1 vaccine trials. Funding: This study was funded by Merck Sharp & Dohme, a subsidiary of Merck & Co., Inc. The study sponsor was involved in the study design; data collection, analysis, and interpretation of data; in writing the report; and the decision to submit the paper for publication. References [1] Stranger-Jones YK, Bae T, Schneewind O. Vaccine assembly from surface proteins of Staphylococcus aureus. Proc Natl Acad Sci U S A 2006;103:16942–7. [2] Lowy FD. Staphylococcus aureus infections. N Engl J Med 1998;339:520–32. [3] National Nosocomial Infections Surveillance (NNIS) report, data summary from October 1986–April 1996, issued May 1996. A report from the National Nosocomial Infections Surveillance (NNIS) System. Am J Infect Control 1996;24:380–8. [4] Rosenthal VD, Maki DG, Jamulitrat S, Medeiros EA, Todi SK, Gomez DY, et al. International Nosocomial Infection Control Consortium (INICC) report, data summary for 2003–2008, issued June 2009. Am J Infect Control 2010;38:95–104. [5] Boyce JM, Cookson B, Christiansen K, Hori S, Vuopio-Varkila J, Kocagöz S, et al. Meticillin-resistant Staphylococcus aureus. Lancet Infect Dis 2005;5:653–63. [6] Fridkin SK, Hageman JC, Morrison M, Sanza LT, Como-Sabetti K, Jernigan JA, et al. Methicillin-resistant Staphylococcus aureus disease in three communities. N Engl J Med 2005;352:1436–44. [7] Hidron AI, Edwards JR, Patel J, Horan TC, Sievert DM, Pollock DA, et al. NHSN annual update: antimicrobial-resistant pathogens associated with healthcareassociated infections: annual summary of data reported to the National Healthcare Safety Network at the Centers for Disease Control and Prevention, 2006–2007. Infect Control Hosp Epidemiol 2008;29:996–1011. [8] Marty FM, Yeh WW, Wennersten CB, Venkataraman L, Albano E, Alyea EP, et al. Emergence of a clinical daptomycin-resistant Staphylococcus aureus isolate during treatment of methicillin-resistant Staphylococcus aureus bacteremia and osteomyelitis. J Clin Microbiol 2006;44:595–7. [9] van Hal SJ, Paterson DL, Gosbell IB. Emergence of daptomycin resistance following vancomycin-unresponsive Staphylococcus aureus bacteraemia in a daptomycin-naive patient—a review of the literature. Eur J Clin Microbiol Infect Dis 2011;49:1489–94. [10] Johnson MD, Decker CF. Antimicrobial agents in treatment of MRSA infections. Dis Mon 2008;54:793–800. [11] Shinefield H, Black S, Fattom A, Horwith G, Rasgon S, Ordonez J, et al. Use of a Staphylococcus aureus conjugate vaccine in patients receiving hemodialysis. N Engl J Med 2002;14(346):491–6. [12] Harro C, Betts R, Orenstein W, Kwak EJ, Greenberg HE, Onorato MT, et al. Safety and immunogenicity of a novel Staphylococcus aureus vaccine: results from the first study of the vaccine dose range in humans. Clin Vaccine Immunol 2010;17:1868–74. [13] Kuklin NA, Clark DJ, Secore S, Cook J, Cope LD, McNeely T, et al. A novel Staphylococcus aureus vaccine: iron surface determinant B induces rapid antibody
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responses in rhesus macaques and specific increased survival in a murine S. aureus sepsis model. Infect Immun 2006;74:2215–23. Ster C, Beaudoin F, Diarra MS, Jacques M, Malouin F, Lacasse P. Evaluation of some Staphylococcus aureus iron-regulated proteins as vaccine targets. Vet Immunol Immunopathol 2010;15(136):311–8. Cleland JL, Powell MF, Shire SJ. The development of stable protein formulations: a close look at protein aggregation, deamidation, and oxidation. Crit Rev Ther Drug Carrier Syst 1993;10:307–77. Rexroad J, Wiethoff CM, Jones LS, Middaugh CR. Lyophilization and the thermostability of vaccines. Cell Preserv Technol 2002;1:91–104. Brandau DT, Jones LS, Wiethoff CM, Rexroad J, Middaugh CR. Thermal stability of vaccines. J Pharm Sci 2003;92:218–31. Fowler Jr VG, Olsen MK, Corey GR, Woods CW, Cabell CH, Reller LB, et al. Clinical identifiers of complicated Staphylococcus aureus bacteremia. Arch Intern Med 2003;163:2066–72.
[19] McClelland RS, Fowler Jr VG, Sanders LL, Gottlieb G, Kong LK, Sexton DJ, et al. Staphylococcus aureus bacteremia among elderly vs younger adult patients: comparison of clinical features and mortality. Arch Intern Med 1999;159:1244–7. [20] Chang FY, MacDonald BB, Peacock Jr JE, Musher DM, Triplett P, Mylotte JM, et al. A prospective multicenter study of Staphylococcus aureus bacteremia: incidence of endocarditis, risk factors for mortality, and clinical impact of methicillin resistance. Medicine (Baltimore) 2003;82:322–32. [21] Gottlieb GS, Fowler Jr VG, Kong LK, McClelland RS, Gopal AK, Marr KA, et al. Staphylococcus aureus bacteremia in the surgical patient: a prospective analysis of 73 postoperative patients who developed Staphylococcus aureus bacteremia at a tertiary care facility. J Am Coll Surg 2000;190:50–7. [22] Fowler Jr VG, Miro JM, Hoen B, Cabell CH, Abrutyn E, Rubinstein E, et al. Staphylococcus aureus endocarditis: a consequence of medical progress. JAMA 2005;293:3012–21.