Journal of the American Society of Nephrology 247

Safety and Immunogenicity of Staphylococcus aureusType 5 Capsular Polysaccharide-Pseudomonasaeruginosa Recom b ina nt Exo protei n A ConjugateVaccine in Patients on Hemodialysis1Paul G. Welch, Ali Fatfom, Jack Moore, Jr., Rachel Schneerson, Joseph Shiloach, Dolores A. Bryla,

Xiuru Li, and John B. Robbins2

PG. Welch, J. Moore, Jr. , Nephrology Service. Walter

Reed Army Medical Center, Washington, DC

A. Fatfom, X. Li, UNIVAX Biologics, Inc. , Rockville, MD

R. Schneerson, D.A. Bryla, J.B. Robbins, National Insti-

tute of Child Health and Human Development

J. Shiloach, National Institute of Diabetes and Diges-tive and Kidney Diseases, National Institutes of Health,Bethesda, MD

(J. Am. Soc. Nephrol. 1996; 7:247-253)

ABSTRACTSeventeen volunteers with ESRD on hemodialysis, neg-ative for infection with HIV or hepatitis B and C and notreceiving immunosuppressive therapy, were injected

two times 6 wk apart with 25 �g of Staphylococcusaureus Type 5 capsular polysaccharide-Pseudomo-nas aeruginosa exoprotein A (rEPA) conjugate. Con-

trols were healthy adults, 18 to 44 yr old, injectedpreviously with the same vaccine. None of the pa-tients had fever or significant elevations in their SGOTor SGPT attributable to the vaccine. Two vaccineeshad transient Induration >1 cm in diameter at theinjection site. The preimmunization geometric mean(GM) Type 5 antibody levels of the ESRD patients andcontrols were similar. Type 5 antibody levels of thethree major immunoglobulin (Ig) classes rose at 2and 6 wk after immunization (P < 0.001 for lgG, P <

0.005 for 1gM, and P = 0.0001 for IgA). Reimmunizationat 6 wk did not elicit a booster response. At 6 months,the GM lgG level of the patients was approximately50% of that of the healthy volunteers and 14 of 17 hada more than fourfold higher antibody level than thepreimmune value. The GM 1gM level, in contrast,declined to the preimmunization value. Vaccine-in-duced Type 5 antibodies had opsonophagocyticactivity. There was a slight increase of lgG antibodies

to the heterologous S. aureus Type 8 polysaccharide

1 Received April 24, 1995. Accepted July 1 1 , 1995.

2 correspondence to Dr. J.B. Robbins, National Institutes of Health. Public Health

Service, Building 6. Room 424. NatIonal Institute of Child Health and Human

Development, Bethesda, MD 20892.

1046-6673/0702-0247$03.00/0Journal of the American society of Nephrologycopy�ght © 1996 by the American Society of Nephrology

(P < 0.01) that was sustained at 6 months. The S.aureusType 5-tEPA vaccine is safe and immunogenicin ESRD patients, and evaluation of its effectivenessagainst S. aureus bacteremia in this at-risk group isplanned.

Key Words: End-stage renal disease, Staphylococcus aureus

vaccine, antibody response, conjugate vaccine

S taphylococcus aureus causes a wide spectrum of

human diseases, the most important of which is

bacteremla and its complications (1-20). CertaIn popu-

latlons, such as patients with ESRD, have an increased

incidence of S. aureus bacteremla (1,5,10,15,18,21,22).

An informal estimate by the Health Care Financing

Administration indicated that approximately 5% of pa-

tients with ESRD develop S. aureus bacteremla annually

(personnal communication). The magnitude of this

problem may be appreciated by realizing that bactene-

mia is cited as the 1 3th most common cause of death in

the United States and that S. aureus is most often the

leading cause of bacteremla (5,11-13,23).

Patients with ESRD have an Increased Incidence of

bacteremia for a variety of reasons. Many patients

have an angioaccess made of synthetic material, usu-

ally polyfluorotetraethylene ( 15). Hemodialysis ac-

cesses are subject to turbulent flow and repeatedpercutaneous punctures. Other factors may Include:

(1 ) nutritional deficiencies due to protein restriction

and anorexia; (2) decreased Immune function as ex-

emplified by a lesser serum antibody response to

immunization with hepatitis B and pneumococcal

polysaccharide vaccines ( 18); and (3) a variety of

nonspecific effects of uremia associated with altered T

and B cell functions, stimulated neutrophil consump-

tion of oxygen, chemotactic responsiveness, and

chemiluminescence. Finally, patients on dialysis have

an elevated rate of nasopharyngeal carriage of S.

aureus, which has been associated with a risk of

bacteremia ( 10,2 1 ,22). Given the ubiquitous presence

of this organism, it is likely that several of these

factors result in an increased rate of S. aureus infec-

tions in ESRD patients.

Currently, there are no licensed vaccines for the

prevention of S. aureus bacteremia (24). Karakawa

and others have provided new information about pro-

tective immunity in humans to bacteremic infections

with S. aureus ( 1 ,2,25-35). S. aureus isolates from the

blood have capsular polysaccharides (CP), which have

Day 47

Day 51

Day 70

Day 180

S. aureus Type 5 Polysaccharide-rEPA Conjugate Vaccine

248 Volume 7 . Number 2 . 1996

virulence and protective properties. Only 2 of the 1 1known CP of this species, Types 5 and 8, account for

80 to 90% of S. aureus blood isolates. Strains of thesecapsular types resist opsonophagocytosis by polymor-

phonuclear white blood cells (PMN) (28,3 1 ). As with

other capsulated pathogens such as Klebstella pneu-

moniae, serum type-specific antibodies facilitate their

opsonophagocytosis (25,28,3 1 ). Noncapsulated

strains of S. aureus are readily opsonized and killed.These findings are similar to the observations made in

studies of other capsulated bacteria, including oppon-tunistic pathogens such as K. pneumoniae (33).

Type 5 and 8 CP are linear copolymens composed of

O-acetylated mannoseamineuronic acid and fuco-samine (36,37). Despite their similarity, no cross-

reaction between the two CP has been demonstratedby double immunodiffusion with typing antisera. Both

CP have been found to be nonimmunogenic or poorly

immunogenic in mice (25). In order to increase their

immunogenicity, these CP were bound to a medically

useful protein. recombinant Pseudomonas aeruginosa

exoprotein A (rEPA) (28,38-42) to form conjugates, as

has been done successfully with other bacterial po-lysaccharides (25-28,34,43-45). rEPA is antigenically

indistinguishable from the native exotoxin A (ETA)

(28,40,41), is nontoxic (41), elicits neutralizing anti-

bodies (antitoxin) to this protein that may conferprotective immunity to P. aeruginosa (27,28,42,46),

and is not related to the proteins ofS. aureus. In adubt

volunteers, Types 5 and 8 rEPA conjugates were safe

and elicited CP-specific antibodies ofboth the 1gM andIgG classes after the first injection. A second injection

6 wk later did not stimulate a booster effect. Theconjugate-induced CP antibodies were mostly of theIgG1 and IgG2 subclasses and had opsonophagocyticactivity. There was a slight. but significant. heterobo-

gous response elicited by each conjugate. The conju-

gates also elicited antitoxin to the ETA (28). In this

study, we examined the immunogenicity and safety of

S. aureus Type 5 CP-rEPA in ESRD patients.

METHODS

Patients

All patients 1 8 yr or older with ESRD on hemodialysis for atleast 3 months at the Walter Reed Army Medical Center(WRAMC) were eligible. Participation was voluntary. andinformed consent was required. Patients were excluded forthe following: (1 ) infection with HIV, hepatitis B or hepatitisC; (2) high levels ofhepatic transaminases: (3) current use ofImmunosuppressive drugs or antibiotics; (4) inability to giveinformed consent; or (5) pregnancy. Of 37 patients receivinghemodialysis at WRAMC, 15 met one of these exclusioncriteria and 5 declined to participate. Thus, 17 patients wereavailable and agreed to participate. Table 1 lists their char-acteristics. Subject 6 had a history of sepsis that had oc-curred as a result of infection with S. aureus in 1989, andESRD patients 10 and 1 1 had a remote history suggestive ofsepsis. Sixteen of the 1 7 ESRD patients were receivingthrice-weekly dialysis with dialyzers made of a polysulfonematerial, with a prescribed KT/V of 1 .0 to 1 .4 (one patientwas on dialysis twice weekly). All had a dietary protein Intake

TABLE 1 . Characteristics of patients with ESRD whoparticipated in the vaccine evaluation

Mean Age (Range) 60 yr(37-73)

Female/Male 7/10

Race (African American, White) 8/9

Cause of ESRDDiabetes 10

Hypertension 2

Glomerulonephritis 2

Other 3

Duration of Hemodialysis (months)Mean 20.4

Range 3-70

of 1 .0 to 1 .2 g/kg per day and received supplemental vita-mins and human recombinant erythropoietin necessary tomaintain the hematocrit at 30 to 33 vol%. All ESRD patientswere dialyzed through angioaccesses of upper extremitypobyfluorotetraethylene grafts. No hemodialyzers, includingblood tubing, were reused. Table 2 describes the clinicalprotocol. At the time of vaccination, all ESRD patients had anormal temperature and no history ofan infection during theprevious week. Each subject was injected intramuscularly

TABLE 2. Clinical protocol

Prestudy Information sheet given to patients, writteninformed consent obtained, negative

assays for hepatitis B surface antigen,hepatitis C antibody, HIV and serump-human chorionic gonadotropin,

normal levels of SGOT and SGPT

Day 0 Physical examination, temperature,SGOT/SGPT, vaccine serology, nasalswab S. aureus, first injection of S.aureus Type 5 CP-EPA

Days 0 6 and 24 h after vaccination, temperature

and 1 recorded and injection site examined.

Day 2 Temperature recorded, injection site

examined, and SGOT/SGPT assayed

Days 5 SGOT/SGPT assayed

and 7

Day 14 Vaccine serology

Day 42 Physical examination, temperature

recorded, SGOT/SGPT. vaccineserology, nasal swab S. aureus, secondinjection of S. aureus Type 5 CP-EPA Lot

501 79; 6 and 24 h after vaccination,temperature recorded and injection siteexamined.

Day 44 Temperature recorded, injection siteexamined, and SGOT/SGPT assayed

SGOT/SGPT assayed

SGOT/SGPT assayed

Vaccine serologyVaccine serology, nasal swab for S.

aureus

Welch et al

Journal of the American Society of Nephrology 249

twice, 6 wk apart, with 25 �g of SA5 CP-rEPA (Lot 50 1 79) inan extremity that did not contain the dialysis angioaccess.The ESRD patients recorded their temperature, appearanceof the injection site, and other symptoms at 6 and 24 h afterthe injection. All 17 ESRD patients completed the study.Thirteen ESRD patients who did not enroll in the study and

who were negative for hepatitis B antigen and HIV antibodiesconsented to donate a serum sample for the measurement ofS. aureus Type 5 CP antibodies.

Vaccine and Serology

The synthesis, physicochemical properties, and evaluationof immunogenicity by ELISA of SA5-rEPA in both mice and inadult volunteers and capsular typing of S. aureus have beendescribed ( 1 25-29,32). Antitoxin to P. aeruginosa ETA wasmeasured by the CHO cell assay (28,46,47).

Statistical Analyses

Data analyses were performed with the Statistical AnalysisSystem. The logarithms of the antibody concentrations wereused for all calculations. Antibody concentrations below thesensitivity of the ELISA were assigned values to one-half ofthe threshold value. Comparisons of geometric means (GM)used either the paired t test or unpaired t test.

RESULTS

Clinical Data

There were no serious reactions to the vaccines.

None of the ESRD patients had fever during the 48 h

after either injection. Four ESRD patients experienced

transient discomfort at the injection site after the first

injection and three ESRD patients did so after thesecond injection. One subject had an induration of

approximately 3 cm in diameter and warmth at the

injection site after the first injection. A second subject

had approximately 1 cm of induration after the secondinjection. Two other ESRD patients reported a mild

headache and fatigue after the first injection that

subsided within 48 h.

Laboratory Data

All ESRD patients had normal levels of SGOT ( 1 0 to50 U) and SGPT (5 to 42 U) 7 days before the first

vaccination. Subject 9 had moderately elevated SGOT(20 1 U) and SGPT (25 1 U) in the blood taken on theday before the first vaccination that returned to non-

mal within 7 days of the vaccination. This subject wasthought to have had an episode of cholelithiasis be-

tween the time of screening and the day of the first

vaccination. Four ESRD patients had mild elevations

(less than two times normal) of SGOT on Day 0. Onesubject had an SGOT of 6 1 U on Day 7 after the first

immunization, and a second subject had an SGOT of59 U 5 days after the second vaccination. There were

only two instances of an elevated SGPT: one in thesubject with cholecystitis prior to vaccination and the

second in a subject whose SGPT was 52 U 2 days after

the first vaccination. The patients who did not partic-

ipate but donated a blood sample had normal levels ofSOOT and SGPT.

Serum Type 5 Polysaccharide Antibodies

Table 3 shows ESRD patients had prelmmunlzation

IgG Type 5 antIbody levels similar to those found In the

controls and the 13 ESRD patients who did not partici-

pate ( 10.7 versus 8.5, respectively; not significant ENS]).Two weeks after the first injection, the ESRD patients

had a 13-fold GM increase in Type 5 IgG antibodies anda 1 7-fold increase after 6 wk (P < 0.001). There were no

further increases in the antibody levels at 10 wk (4 wkafter the second injection) in either groups. Six weeks

after the first injection and 4 wk after the second injec-

tion ( 10-wk interval) a more than fourfold rise In anti-

body was observed In 14 of 1 7 of the ESRD patients andin 23 of24 ofthe controls. In the ESRD patients, the GM

IgG level declined to 1 10 by 6 months after vaccination;this value was not stailsilcably different from the maxi-

mal bevel ofantibody attained after the vaccinations. TheGM IgG antibody levels of the patients were less than

that of the controls at all postimmunization intervals(137 versus 298; P = 0.03) and at 6 months (1 10 versus

274; P = 0.0009). The three ESRD patients who failed to

achieve a more than fourfold IgG rise were Patient 3 (71

yr old with diabetes whose postimmunlzation level was

28), PatIent 4 (postlmmunlzation bevel of 12 pg/mI), and

Patient 9 (postlmmunizaion level of 272 at 10 wk

postimmunizatlon).The ESRD patients and controls had similar preim-

munizatlon levels of 1gM Type 5 antibodies (3. 1 1 ver-

sus 3.6; NS). After the first injection, the ESRD pa-

tients had an approximately fourfold increase in the

GM Type 5 1gM antibody level to 12.4 after 2 wk and aslight increase to 15.2 after 6 wk: both were higher

than the preimmune GM levels (P < 0.00 1), and

reinjection did not significantly change these bevels. A

more than fourfold rise of 1gM antibody levels was

observed in 1 4 of 1 7 of the ESRD patients and in 23 of

24 of the controls 6 wk after the first injection and 4wk after the second injection, respectively. The GM1gM level declined to 3.7 �g/mL by 6 months after

vaccination, which was not statistically different fromthe bevel of antibody before vaccination. The GM 1gM

postimmunization bevel of the ESRD patients was lessthan that of the controls (P < 0.05). The SA5-rEPA

elicited a slight but significant twofold rise of the GM

IgG to Type 8 CP (P < 0.0 1) in the ESRD patients.

The IgG subclass composition ofType 5 CP antibod-

ies before and at 6 wk after the first vaccination isshown in Table 4. Both the ESRD patients and the

controls had significant increases of the GM � andIgG2 (P = 0.000 1 ). There were no significant differ-

ences between these bevels in the two groups. Immu-

nization elicited an about twofold increase of IgG3 and

IgG4 anti-Type 5 in both groups. but these levels were

low and close to the level of detection (not shown).

P. aeruginosa ETA Antibodies

GM antibody bevels to ETA before and 6 wk after the

first Injection, measured by ELISA and by toxin neu-

tralization assay, are shown in Table 5. Preimmuniza-

S. aureus Type 5 Polysaccharide-rEPA Conjugate Vaccine

250 Volume 7 . Number 2 . 1996

TABLE 3. Serum antibodies to the Types 5 and 8 CP of S. aureus in ESRD patients (N = 17) on hemodialysiscompared with normal volunteers (N = 24) injected with a S. aureus Type 5 CP-EPA conjugate (Geometricmean jig Ab/mL (25-75 centiles))a

Time Serum Sample TakenGroup Ig

Preimmunization 2 wk 6 wk 10 wk 6 months

Type 5 (homologous)ESRD Patients lgG 10.7 (5-17) 137 (77-260) 180 (103-439) 169 (87-387) 1 10 (48-244)

1gM 3.1 1 (2-5) 12.4 (3-45) 15.2 (5.7-32) 1 1.3 (4-22) 3.70 (2-9)IgA 0.27 (1-1) Not done 18.5 (8-72)

Normalsb lgG 8.5 (12-23) 298 (182-539) 318 (180-612) 303 (201-498) 274 (243-399)1gM 3.6 (2-6) 40.2 (25-68) 36.2 (21-60) 31.1 (21-53) 18.7 (12-30)

Type 8 (heterologous)ESRD lgG 13.1 (8-23) 20.4 (1 1-33) 21 .7 (10-44) 26.7 (18-50) 23.2 (18-57)

For lgG anti-type 5, 137, 180, 169, and 1 10 versus 10.7; P < 0.001For 1gM anti-type 5, 12.4, 15.2, and 1 1.3 versus 3.1 1; P < 0.001; 3.70 versus 3.1 1; NSFor IgA anti-type 5, 18.5 versus 0.27; P = 0.0001For lgG anti-lype 8, 20.4, 21.7, 26.7, and 23.2 versus 13.1; P < 0.01

a GM. micrograms of antibody per milliliter (25 to 75 centiles).b Taken from Ref. 28.

TABLE 4. lgG subclasses of polysaccharide antibodies in ESRD patients (N = 17) and normal volunteers(N = 24) immunized with S. aureus Type 5 CP-rEPA conjugate, Lot 50179#{176}

IgGi lgG2Group

Preimmunization Post-6 wk Preimmunization Post-6 wk

ESRD Patients 0.25 (<0.1-1) 13.9 (4.6-47) 2.8 (1-5) 90.9 (30-266)

Normals 0.40 (0.1-2) 14.9 (20-38) 1.7 (1.2-4.5) 139 (86-268)

a GM ELISA units (25th to 75th centiles).

TABLE 5. GM serum antibody levels to P. aeruginosa ETA in ESRD patients (N = 17) and normal volunteers(N = 24) immunized with S. aureus CP Type 5 CP-EPA conjugate, Lot 50179

GroupELISA Units Neutralization Titer

Preimmunization Post-6 wk Preimmunization Post-6 wk

ESRD PatientsNormals

0.39 (0.1-1)0.50 (0.3-1 . 1)

2.74 (0.5-10)1 1 .5 (4.3-34.3)

00.51

0.54 (0-8)4.43

tion levels in both groups were low. By ELISA, the

ESRD patients had a sevenfold GM increase from a

preimmune bevel of 0.39 to 2.81 (P = 0.0001). The

controls had a 22-fold GM increase from 0.50 to 11.1

(P = 0.000 1). The postimmunization GM level of the

controls was higher than that of the ESRD patients( 1 1 . 1 versus 2.8 1 ; P = 0.02). The GM antitoxin level in

the ESRD patients rose from nondetectable levels to

1 .84 (P = 0.004) and from 0.5 1 to 4.94 in the controls(P = 0.000 1 ). There was no significant difference In the

GM antitoxin levels between the patients and controls

at 6 wk.

Nasopharyngeal Cultures for S. aureus

Eight of 1 7 ESRD patients had positive cultures forS. aureus: 6 were positive on Day 0, 5 on Day 42, and

TABLE 6. Nasopharyngeal cultures for S. aureuscapsular Types 5 and 8 in patients with ESRD#{176}

Volunteer Day 0 Day 42 Day 180

15 T-5 T-5 Negative5 T-8 T-8 Negative

1 3 T-8 T-8 Negative7 T-5 T-5 Negative

1 1 T-5 T-5 Negative

17 N-T N-T T-8

2 Negative Negative T-8

12 Negative Negative T-8

a 1-5. OP Type 5; T-8, OP Type 8; N-T. nonlypeable.

Welch et al

Journal of the American Society of Nephrology 251

3 on Day 180 (Table 6). On Day 5, three ESRD patients

had Type 5. two had Type 8, and one had nontypeabbe

S. aureus: all were negative on Day 180. On Day 180.

all cultures were of a S. aureus Type 8 variant.

DISCUSSION

S. aureus bacteremia is an important cause of mor-

bidity and mortality in hospitalized patients, espe-

cially those with ESRD. On the basis of new evidence

for the pathogenic and protective robes of their CP, we

developed conjugate vaccines for the prevention ofbacteremia caused by S. aureus (24,27,30-32,35,48).

In this study, we evaluated one of these vaccines,

SA5-rEPA, in 1 7 patients with ESRD. As observed in

normals (28), SA5-rEPA elicited only minor clinical

reactions and there were no alterations in liver en-

zyme concentrations attributable to the vaccine.These fmdings provide further evidence that our pro-

gram of standardization of these conjugate vaccines

reliably predicts their safety. This will be important ifmore extensive studies of these vaccines are under-taken in patients with ESRD or other chronic diseases

associated with symptoms and/or metabolic abnon-

malities that could be construed as related to immu-

nization.

The Type 5-rEPA conjugate elicited significant risesin both IgG and 1gM Type 5 antibodies in the ESRDpatients, although their bevels were bower than thoseachieved in the controls (P < 0.03 at all intervals). As

observed with other conjugates, the reinjection of

adults did not augment antibody levels (26,34,43,45).Also, there was no dosage effect between 10 and 50 �gof other protein conjugates on the pobysaccharideantibody responses (34,45). The SA5-rEPA-induced

antibody rises in the ESRD patients were not asprolonged as In the controls. The largest differencebetween vaccine-induced Type 5 antibodies in the

ESRD patients was observed 6 months after immuni-

zation (35% decline from 169 to 1 10 compared with a

9% decline from 303 to 274 in the controls; P =

0.0009). These differences are likely due to several

factors, including a higher mean age and other riskfactors in the patients as well as to the depressanteffect of ESRD on antibody responsiveness. This was

noted in studies of the hepatitis B vaccine in dialysispatients who required additional aluminum adjuvantto achieve a satisfactory serum antibody response

( 1 8). Future studies will have to include all patientswith ESRD in order to evaluate the effects of ourconjugate vaccines. It may be that far lesser responseswill be observed in ESRD patients with the character-

istics that excluded their participation in this study.We reported that the addition of monophosphoryb

lipid to our S. aureus conjugates increased the anti-body levels to both Types 5 and 8 CP in mice (49).Clinical studies of monophosphoryb lipid have shownit to be safe and to have adjuvant activity. The addition

of aluminum salts to Haemophilus (nfiuenzae type

b-tetanus toxoid conjugates, however, did not serve as

adjuvants (34). Additional clinical studies will evalu-ate the effect of adding an adjuvant to our S. aureus

CP conjugates.

The nasopharyngeal carriage of S. aureus has been

correlated with bacteremia caused by this pathogen in

several patient groups. including those with ESRD( 10. 15,22,24). Bacteremia originating from the organ-

isms in the nasopharynx may be the primary patho-genetic event in systemic infection with S. aureus.

Short-term trials with antibiotics, including vancomy-

cm, nifampin, and mupirocin, have been associatedwith reduced rates of infection ( 15,2 1 ,22), but thebong-term efficacy and safety of these agents have notbeen established. Although one patient remained pos-

itive for S. aureus Type 8 and two patients became

positive by the end of the study, It was encouragingthat four patients apparently became negative forType 5 5. aureus. We are unable to draw any conclu-

sions about the effect of vaccination with our conju-gates on the nasophanyngeal carriage of S. aureus

because of the limited size of our study and thevariability of cultures from an individual. This effect of

vaccination with S. aureus CP-protein conjugate vac-

cines deserves further study, because polysaccharide-based vaccines have been shown to result in theprevention of nasopharyngeal carriage and systemicinfection of capsulated bacteria (34,50-52). In fact, itmay be that vaccine-induced CP antibodies confer

their protection by preventing nasopharyngeal car-niage rather than by inactivating the pathogen In thebloodstream.

In summary, the S. aureus Type 5 CP-rEPA conju-gate was safe and immunogenic in a population of

ESRD patients. The ESRD patients had a lesser GM

serum antibody response than controls, but 1 4 of 17responded with a more than fourfold rise In antibodiescompared with 23 of24 ofthe controls. IfCP conjugate

vaccines can be shown to reduce the incidence of

bacteremia, we will have an important tool to use

against staphybococcal infections.

ACKNOWLEDGMENTS

This study was approved for investigation by the National Institutes of

Health Clinical Protocol Number 90 CH 1 76. FDA BB IND Number

3679. WRAMC Department of Clinical InvestigatIon Number 1 169.

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