CA2117480A1

CA2117480A1 - Directed human immune globulin for the prevention and treatment of staphylococcal infections

Info

Publication number: CA2117480A1
Application number: CA002117480A
Authority: CA
Inventors: Gerald W. Fischer
Original assignee: Individual
Current assignee: Henry M Jackson Foundation for Advancedment of Military Medicine Inc
Priority date: 1992-02-25
Filing date: 1992-11-09
Publication date: 1993-09-02
Also published as: EP0628056A1; JPH08504167A; WO1993017044A1; AU673508B2; EP0628056A4; AU3271893A

Abstract

This invention is directed to a Directed Human Immunoglobulin and compositions thereof for preventing or treating staphylococcal infections such as S. epidermidis.

Description

WO 93/17044 Pcr/US92/09830 , DIRECTED HUMAN IMMUNE GLOBULIN FOR THE PREVENTION

2 ANn TREATMENT OF STAPHYLOCOCCAL INFECTIONS

3 I. GOVERNMENT INTEREST

4 The invention described herein may be ' ~d, licensed S and used by or for gv._~"~l purposes without the payment of any royalties 6 to us thereon.

7 II. FIELD OF THE INVENTION
8 This invention relates to Directed Human Immune Globulin for 9 the prevention and treatment of ~ loc~ infections.

m BACKGROUND OF THE INVENTION
11 Over the last two decades, ~ I~occi have become important 12 causes of infection in h~ patients. Because of their high prevalence 13 on the skin, ~yh~ i are ideally situated to cause serious infections in 14 debilitated or . r ~ patients. The ~layLyl~o~c~l species most frequently pathogenic in humans are Sla~Jh~l~vccus ~L (SA) anù
16 Sl ~h~l~v~ u~,;v~ ;dis (SE). Both groups have developed resistance to 17 multiple antibiotics making ul,;,ll therapy difficult. In recent years SE
18 has become a major cause of ~ infection in patients whose treatments 19 include the placement of foreign materials such as c~.~bl~, ' fluid shunts, WO 93/17044 Pcr/US92/09830 vascular catheters or joint prostheses. SE is a common cause of post operative 2 wound infections peritonitis in patients with continuous: ' ' y peritoneal 3 dialysis. Patients with impaured immunity (r~ , bone marrow 4 tlansplant) or those receiving parenteral nutrition through central venous S catheter are also at high risk for developing SE sepsis (Patrick, J. Pediat., 6 1990).
7 SE has emerged as a common cause of neonatal nos~on~iql 8 sepsis in premature infants. As shown by Fleer and colleagues, (Pediatr Infect9 Dis, 1983) SE infections frequently occur in immature babies that have received parenteral nutrition. Premature babies have impaired immunity with ll ~i. fi. ~ in antibodies, ~~ r~ and neutrophil function. Lipid infusion 12 is now a standard ingredient of parenteral nutrition therapy in many nurseries 13 and may further impair immunity to bacterial infection as disclosed by Fischer 14 and colleagues (Lancet, 1980; 2:819-20). Recent studies have associated coagulase negative ~,I,jl~l bacteria in neonates with lipid emulsion 16 infusion (Freeman and colleagues, N. Engl. J. Med, 1990). Further studies by 17 Fleer and colleagues (J Inf Dis, 1985) showed that neonates had low levels of 18 opsonic antibody to SE despite the fact that the sera had clearly detectable 19 levels of IgG antibodies to SE ~ idoE,I~ (opsonic an~ibodies for , ' Jlu~,~ have been considered to be direc~ed to the ~ ido~
21 antigens). While these studies suggested that neonatal , ' ' nJ to SE
22 might be related to impaired oposonic activity, it is not clear if antibodies Wo 93/17044 Pcr/uss2/o983o directed against SE are opsonic or would be capable of providing protection 2 when given passively to neonates. Further, it is unknown whether the 3 presence of intralipid, which further impairs ~ tv~;s and killing of 4 bacteria by I ' v ~tes, would inhibit the activity of antibody.
The opsonic activity of pooled human ~O' ~ b~' for SE
6 was studied by Clark and colleagues (J Med Microbiol, 1986), and showed that 7 .' and IgG were both critical for efficient o~ ,, of SE. They 8 noted, however, that in some studies ~;<~ was not required and that 9 contrary to the report of Fleer (1985), absorption of serum with pvp~dvoly~
10 may remove the opsonic activity for SE. Further studies by Clark and Easmon Il (1986) showed that several lots of standard intravenous immune globulin 12 (IVIG) had variable opsonic activity for SE. One third of the IVIG lots had 13 poor ~ r ' " with . . ' and only 2 of 14 were opsonic without 14 : lj ' Despite the fact that the IVIG lots are made from large plasma 15 donor pools good opsonic antibody to SE was not uniformly present. Their 16 studies focused on potential use of ~O' ' ' to boost peritoneal defenses 17 in patients receiving continuous ' ' y peritoneal dialysis and did not 18 examine whether IVIG could be utilized for the prevention or treatment of 19 bacterial sepsis, or the use of antibody to prevent or treat sepsis and lethal 20 infection in immature or . . c~cd patients and Specifically, no 21 vivo studies were done to test antibody to prevent or treat wo 93/17044 Pcr/US92/09830 CA2i 1 7480 SE. There is no evidence therefore that the antibody would provide beneficial 2 therapy in a setting of immaturity or impaired immunity.
3 Ihe opsonic assays, that are currently used are slow and 4 . ~ - for screening blood, plasma or immune globulin for antibodies to 5 SE. It would be important to have a rapid antigen binding assay to screen for 6 SE antibody, if that assay further correlated with opsonic activity in vitro and 7 protection in vivo.

8 In order to determine if IgG is capable of enhancing protection 9 against SE, a suitable animal model that is ,","~,.._1,1~ to patients with SE

10 infections is required. This is critical since neonates have low levels of Il s . - and impaired neutrophil and llla~ Jh.~D~ function. While opsonic 12 activity of immune globulin may be adequate under optimal conditions in vitro, 13 protection may not occur in patients with immature or impaired immune 14 systems. As has been d ' by Clark and colleagues (J Clin Pathol, 15 1986), most IVIG ~lc~ala~ioai were not opsonic when cu pl - ~ was 16 removed. However, since SE has low virulence, suitable animal models of SE
17 sepsis have not been available.
18 Yoshida and collegues, (J Microbiol, 1976) reported on a 19 virulent strain of SE that infected mature mice with 90 - 100% of mice dying 20 within 24 - 48 hours. This model is very different from that seen in patients 21 and may represent an unusual type of SE infection. When they analyzed 80 22 fresh isolates of SE from humans, they were not able to kill mice. Non-human Wo 93/17044 PCr/USs2/09830 antibody to a new SE surface pol~ fide protected the mice from the 2 virulent SE strain. A later report by Yoshida and colleagues (J Med 3 Microbiol, 1977) confirmed their previous Ub,~ Lh~..S. Passive prophylaxis 4 with ' induced non-human antibody showed that the IgG fraction 5 did not protect while the IgM fraction did provide protection. Thus 6 ' l,, in this model that IgG antibody was not protective. As noted 7 previously herein neonates had good levels of IgG to SE, but had low levels of 8 opsonic antibody (Fleer and colleagues, J. Infect. Dis, 1985), consistent with 9 the findings in this study and showing that the role of lgG in protection against 10 SE is unclear. In 1987 the report by Ichiman and colleagues (J Appl Bacteriol, I1 1987) extended their animal studies to include analysis of protective antibodies 12 in human serum against their selected virulent strains of SE. Protective 13 antibody was found in the IgA, IgM and IgG a~,' L " fractions. These 14 studies are in conflict with their previous data showing that IgG was not 15 protective and fails to establish a definitive role for any of the ,' ' "
16 classes (IgG, IgM or IgA).
17 In the animal model described by Yoshida, Ichiman and 18 colleagues mature, ., ~ mice were used and death was 19 considered to be related to toxins not sepsis (Yoshida and colleagues, J.
20 Microbiol, 1976). Most clinical isolates did not cause lethal infections in their 21 model. Since ~ c blood cultures were not done, it is not known 22 whether antibody would prevent or treat SE sepsis in immature wO 93/17W~ Pcr/us92/o983o , ~ patients or specifically in the presence of intralipid.
2 Antibody provides protection in humans against certain 3 . '' bacteria such as TT ~hilllC influenzae and S~ ,J
4 Individuals such as young infants who are deficient in antibody 5 are susceptible to infections with these bacteria and bacteremia and sepsis are 6 common. When antibody to these bacteria is present it provides protection by 7 promoting clearance of the bacteria from the blood. I =' ' . " with 8 antibody to H. influenzae and S. l protects infants from sepsis with 9 these bacteria. The article by Espersen and colleagues, (Arch Intern Med, 10 1987) discloses the use of an antigen binding RIA assay to analyze IgG

11 antibody to SE in patients with l , ' ' bac~eremia and those with 12 bacteremia and c..d~" This assay used an ultrasonic extract of SE to 13 identify SE specific IgG (the surface antigen in this study differs from the 14 antigen used by Yoshida and colleagues which was obtained by a different 15 method; gentle sonic oscillation). None of the patients with l , ' 16 bactermia had IgG antibodies to SE. These data would suggest that IgG is 17 l ~ for effective eradication of SE from the blood. In addition, 89%

18 of bacteremic patients with _.d~.u ' developed high levels of IgG to SE.

19 In these patients, IgG was not protective since high levels of IgG antibody 20 (which may have developed late) were associated with serious bacteremia and 21 ; ' " Based on these studies the protective role of IgG in SE sepsis 22 and ~ ' ' is not; ' ' ' 1, especially in thepresence of y, W O 93/17044 PC~r/US92/09830 CA2i 1 7480 APhi1 intralipid infusion, or . r ~ In aAdition, the 2 extensive review of Patrick et al. (J. Pediat., 1990) does not include 3~~' I as a potential ~"u~h.~ .Lic or therapeutic a~)ent for SE
4 infections.
5It has been recognized by the medical y that SE is an 6 important pathogen in certain high risk individuals, such as patients with 7 foreign body implants, premature neonates and ~ .r ~ d patients.
8 Accordingly there is a need for a human immune globulin that would prevent ~ 9or treat SE infections such as, sepsis or t,~Ju~,~J;Li~ and promote clearance of 10 SE from the blood of such high risk people.

11IV. SUMMARY OF THE INVENTION
12It is therefore an object of the present invention to provide a 13 novel Directed Human Immune Globulin for preventing or treating 14 sL~hilu ~ l infections. We have found that it is useful to screen serum 15 (plasma) or pooled ,,' bb~ for specific antibody to S. c~;Jul,.lid;s to ~ 16 produce Directed Human Immune Globulin to this pathogen. This Directed 17 Human Immune Globulin is different from standard human immune globulin 18 1 . in that it has high levels of human s~pl.,~luiu~l antibodies 19 that react with surface antigens of S. cy;J~,Il..;d;s and enhance ~ jtu~;~
20 andkillingofS. C~J;dUI ' inyl~, ( r;~ L;C h=-~..,.;.l 1 activity 21 greater than 80%). In addition, Directed Human Immune Globulin for S.

wo 93/17044 PCr/US92/09830 enhances immunity in vivo and prevents lethal infection as well as 2 enhancing clearance of S. c";d~.~..;d;s from the blood in conditions of 3 immaturity and impaired immunity. This is surprising since 4 ~ a;ull or immaturity would be expected to render the antibody 5 ineffective by impairing the ability of phagocytic cells to engulf and kill the S.
6 ~,";.~,. ' 7 It is also another advant.. 6cuua object of the present invention 8 that while standard ~g' ' ' pools or normal donors do not have 9 reliable levels of opsonic antibody for S. cy;~h,~ ' s. Directed Human 10 Immune Globulin when given ..,..JU~I~ ' 'y provides specific 11 antibodies to promote ~hagvc.~tua;a and killing of S. c~Jid~ ia by 12 phagocytes. A further advantages of the present invention is that by providing 13 opsonic antibody to immature or . r ~,~aeJ patients infected with SE, 14 antibiotic therapy may be enhanced by improved _ e";J~.,..;dis clearance from 15 the blood or site of infection. Another advantage is that since Directed Human 16 Immune Globulin given i.~t~ u..vv,sl~ or ' '~, can raise the level of 17 antibodies in the blood of patients, Directed Human Immune Globolin could 18 prevent ~ ~ rfrom causing bacteremia and local infections.
19 The method of producing the Directed Human Immune Globulin 20 for S. e~,;d~,. ' involves:
21 a) screening plasma (pools of ~t,' ' _' or plasma;
22 ~ 7b~ ~ ~- or ~ "' ' ' ~ ,~d;ùu~) for antibodies to S.

- - -W O 93/17044 P<~r/US92/09830 CA2i 1 7480 g ;, ': ' using an ~ vitro antigen-binding assay: (ELISA), followed by 2 of functional activity using an in vitro i r ~' g~ Lc 3 b ~ assay (b~ ri~ 1al activity greater than 80%).
4 b) Protective efficacy can be .' m ' in vivo by 5 analyzing protective activity of the Directed Human Immune Globulin using a 6 suckling rat model of neonatal S. ~,,;dc.",;Jis sepsis (mortality and bacterial 7 clearance). We believe that this is the first in vivo model to test antibody 8 ~rf~L~n~s in the presence of immaturity and/or intralipid induced immune 9 ~ a;~
These methods could be repeated using other alalJh.~lOCOC~i such as SA
11 instead of SE to produce Directed Human Immune Globulin for S. aureus.
12 This novel Directed Human Immune Globulin for SE could be used to 13 prevent lethal SE infections in high risk patients such as neonates and adults in 14 intensive care units or patients with in-dwelling foreign bodies such as venous 15 and arterial catheters or ventricular shunts. Directed Human Immune Globulin 16 could also be used in addition to antibiotics as adjunctive therapy to enhance 17 bacterial clearance in patients treated for SE infections.
18 Other objects, features and advantages of the present invention will 19 become apparent from the following detailed ~l~crrip~irm It should be 20 L ' ~1, however, that the detailed description and specif~c examples, while 21 indicating preferred e 1~ of the invention, are given by way of 22 illustration only, since various changes and ~ c within the spirit and Wo 93/17044 Pcr/us92/o983o CA 21 1 74~
-lo-scope of the invention will become apparent to those skilled in the art from 2 this detailed ~l~np~ n 3 The terms Standard Human T O L ' and Directed 4 Human Immune Globulin for S. e~;d~, " as used in this application are 5 defined as follows: Standard H I ~' L " - immune human 6 globulin that was prepared by pooling O l ' from many donors, 7 without selecting donors or screening the ~,' L ' to ensure antibody 8 acitivity for S. I~,;d~
9 Directed Human Immune Globulin for S. ~;d~.",id;~ - Immune 10 globulin prepared by screening for antibody to S. e~,;d~ l,;d;s (R~ t~ ~ ;r i~
Il Activity > 80%), thereby providing a human immune globulin with protective 12 levels of antibody to S. c";d~.",;d;s and suitab]e for preventing or treating ~
13 ~ - ~. ' infections. P ~ l Activity-The percentage of bacteria killed 14 with the addition of antibody, using a neutrophil mediated ~J~ aO~ L;c 1-~ t~-;- -1 1 assay after 2 hours of incubation at 37~C.

16 V. RRTF.F DESCRI~ION OF THE DRAWINGS

17 Figure I
18 Figure 1 shows that when several pools of human standard 19 ~ ~' ' ' were analyzed, there was a marked difference in the antibody 20 activity to S. ~ ..;di~ as measured by an antigen binding assay (ELISA, wo 93/17044 Pcr~uss2/o983o highest O.O. reading at I 1/2 hrs using 1:10v Dil). These were large pools of 2 IgG, purified by several companies using various techniques. Of three pools3 with the highest titers, two were from Cutter T -' , Berkeley 4 California, (40P07, 40R09) and one was from Sandoz, East Hanover, N.J.
(069). One ~ ,valalivll from Cutter also had next to the lowest activity 6 (2801). These data show that standard unscreened human ~ . ' has 7 variable levels of antibody to ~, c~ .m;l;s and that no single method used to 8 prepare the l': ~ _' or utilizing a large donor pool size will ensure 9 good antibody activity to S. ~,~,;d~,.",;di~. In addition, a donor was shown to have high antibody activity (Sam) to S. c~J;d~l";dis ~ n~8~e the 11 feasibility of identifying units of plasma or, plasma donors with high levels of 12 antibodies to ~La~h~loc~

13 Figure 2 14 Figure 2 shows that using an in vitro functional (opsonic) assay that15 measures the ability of ,,' ' ' to promote ~Jh~,~ytvai~ and killing of 16 S. ~yid~ '' 5 by ~ in the presence of ,' t, that opsonic 17 activity is also variable in various lots and ~ ~atiOll~ of standard human18 ),,' ' " The figure also shows that the "' b~' identifed 19 by ELISA as having high levels of antibody to S. c~.id~.-,.;dis also had high levels of functional antibody in vitro. This is critical since this study shows 21 that IgG that binds to TCA extracted S. ~,;d,,";d;s antigen will promote W O 93/17044 PC~r/US92/09830 CA 2 i 1 7480 and killing of S. c~ Therefore, using in vitro screening 2 assays, one could select a Directed Human Immune Globulin for S.
3 e, ' 'i~ that would have reliable levels of antibody to prevent or treat S.
4 ~ P~ infections.
S It also shows that unscreened immune globulin would not 6 provide reliable protection, since many standard human ,- ~_l Iots 7 have little or no opsonic activity for S. c~ . ' - Hence, standard human 8 immune globulin would not ensure uniformly high levels of antibody to S~ and 9 would not be uniformly protective despite the fact that large numbers of donors 10 might be expected to provide good levels of antibody to a common bacteria I l such as ~ e~;d~

12 Figure 3 13 Figure 3 shows that Directed Immune Globulin protects animals from 14 developing prolonged S. c~,;d~.",;d;~ bacteremia while standard immune 15 globulin did not. Animals treated with Directed Immune Globulin had lower 16 peak bacteremia levels (9.2 x 102 vs. 6.5 x 10~) and cleared the bacteremia 17 more efficiendy (at 72 hours, 5 bact. per ml vs. 380 bact. per ml; geometric 18 mean level). In addition 72 hours after infection, 18/24 (759'o) animals given 19 Directed Immune Globulin had cleared their bacteremia and lOO~o survived, while only 4/20 (20%) animals given standard immune globulin died and only 21 1/16 (696) cleared their bacteremia during that 72 hour period. In addition to W O 93/170~4 PC~r/US92/09830 ~ _~. since Directed Immune Globulin enhanced S. c~,id~,l..idis 2 clearance, it would be a valuable adjunct to antibiotic therapy for people 3 infected with S. e~,;d._. ' . since many of these patients h.lve imparied 4 immunity and may not clear the bacteria efficiently.

5VI. DETAILED DESCRIPTION OF PREFFERED EMBQDIMENTS

7The herein offered examples provide methods for ill.lcrr~ling, without any 8 implied limitation, the practice of this invention in the production of Directed 9 Human Immune Globulin for S~ u~ c~,iJ~.-..id;s and the use of s~ud 10 Immune Globulin for the prevention or treatment of infections caused by 12The profile of the IC~ /e t .p. ';' ~ have been chosen 13 to illustrate methods for producing Directed Human Immune Globulin to S.
14 r~ A. ., ~ and to d its usefulness to prevent or treat S. Cp;d~ -ddi5 15 infections.

16Materials and Methods 17Sk~Jh~l~v.,~ Strains: Although any S. c~,id~".,;d;~ strains 18 could be used, in these ~A~ ' ' we used two strains from the American wo 93/17044 PCr/US92/09830 Type Culture Collection, Rockville, MD (ATCC #31432 and ATCC #35984).
2 A clinical isolate (Hay) from the blood of a child with S. .,";d." ' sepsis 3 was also used and is also on deposit at the American Type Culture Collection.
4 Materials and Methods T ~ " Standard I ~ ,u~ I ~g~ ~} " was 6 used in these ~, to represent large ac,' ~ ' pools.
7 I'~. r from several companies were analyzed for .u ~ to include 8 ~~- , Cutter I ' Inc. Berkeley, California; S ' ~g' ' . ", 9 Sandoz, East Hanover, N.J.; Gammagard, Hyland, Los Angeles, California.
10 Serum from individual donors were also analyzed for antibody activity to S.
11 epidermidis.

12 Tr "~ ua~etic Acid (TCA) Anti~en Extraction 13 St~h,~luuuc~,u~ c~,;.h"l";dis strains (ATCC ~35984, ATCC
14 ~31432 and Hay) were grown to log phase at 3PC in 1000 ml of Tryptic Soy Broth (Difco). The bacteria were then centrifuged at 2500 RPM for 10 16 minutes and the !, ' was aspirated and discarded. The bacterial button 17 was . ' ' in 200 ml of 296 IH~.lllulu~ e~ic acid (TCA) and stirred 18 overnight at 4~C. The mixture was then ~ n ir,~ at 2500 RPM for 10 19 minutes and the ~, aspirated. To the . "~ , 4 volumes of absolute ethanol were added and IC:' _ ' overnight at 4~C. After 21 ' _ at 2500 RPM for 10 minutes, the ~u~ lahult was removed and Wo 93/17044 Pcr/US92/09830 discarded. Then, five milliliters of normal saline was added to the antigen 2 } . it was cultured to ensure sterility and then Iyophilized for storage.

3 Anti~en Bindin~ Studies Usin~ Enzyme-Linked 4 1. ~ '"~ Assay (ELISA) ~ c~;Ju. ' - Antigen was dissolved in carbonate buffer at a 6 of 25 ~ ...... 1. To each well of A 96-well flat-bottomed 7 microtiter plate (NUNC, Roskilide, Denmark) 100 microliters were added and 8 stored at 4~C until used. I O bu' was diluted to 1% and 2-fold 9 dilutions prepared in ~' . ' buffered saline-Tween . To each well was 10 added 100 microliters of the serial dilutions and the plates were incubated for I
I l hour at 4~C. The plates were washed four times with H2O-Twoen . Alkaline 12 1~ linked goat anti-Human IgG (100 1,.ic.ul;t~ 250) was added, 13 the plates were incubated for I hour at 4~C and then washed HzO-Tween and 14 100 microliters of P-..iLI~ .' Jl phosphate substrate in " - -' buffer 15 were added. After 90 minutes of incubation at room h,...~ld~ , the color 16 d~,.. ', was d ~ 1 by db~Uli - at 405 nm.

17 Ovsonic Assay:
18 To determine the functional antibody to S. c~ lu,lll;d;l in the 19 immune globulin pools and sera, a neutrophil mediated t'~ '1 assay was -wo 93/17044 Pcr/uss2/o983o used. ~ ~ ,' '~ were isolated from adult venous blood by dextran 2 ' and ficall-hypaque density ~~u~dtiull. Utilizing a microtiter 3 plate assay that requires a total volume of 0.1 ml/well, washed neutrophils 4 (l~ o6 cells) were added to round-bottomed microtiter wells along 5 with 3xlO' al",~ 'y mid-log phase bacteria. Newbom rabbit serum (10 6 ~ , ' . screened to assure absence of antibody to S. e~,;.l~.".id;s) was 7 used as a source of active ~ . ' ~ Forty mic}oliters of 5 % standard 8 immune globulin (or serum) was added and the microtiter plates were 9 incubated at 3PC with constant, vigorous shaking. Samples (10 microliters) were taken from each well at zero time and after 2 hours of incubation, I l diluted, vigorously vortexed to disperse the bacteria and cultured on blood agar 12 plates ovemight at 3PC to quantitate the number of viable bacteria. Controls 13 consisted of neutrophils alone, . ' alone and l,.,.Jt~,r' 1c plus ,c~ uc~ Pncic Model:
16 A suckling rat model was used to determine the in vivo activity 17 of antibody to ~ c~J;d~,. ' Wistar rats (2 days old) were given 0.2 ml of 18 20% Intralipid (Cutter, Berkeley Califomia,); ~,~J. i~ at 0800 and 19 1400. At three days of age each animal was again given, 0.2 ml of 20%
intralipid at 0800 and 1400 and 0.2 ml of 5% ~ ' or serum was 21 given IP. Shortly after the last dose of intralipid, 0.05ml (approx. 5xlO') wO 93/17044 Pcr/US92/09830 mid log phase S. e~,;d~.".;d;s were injected ~ l~.u~- ~u ~ly just cepha,ad to the 2 tail. Suclding rats less than 24 hours old also develop lethal S. e~;dc"l.;Jls 3 sepsis when infected with 10'-10~ ~ c~,;d~. 'i. sl t 'y. To ana,yze 4 bacteremia levels in selected animals, 0.01 ml of blood was obt~uned from the tails of the suckling rats, 24, 48, and 72 hours after infection. The blood was 6 collected under sterile conditions in n,;clu~ s and seria'ly diluted in 7 Tryptic Soy Broth (Difco). Bacteria were ' ' ~ onto plates to ensure S.
8 ~ ~ ' bacteremia and all animals were followed five days to determine 9 survivat Results Il AntiP~n Bindin, Activity of Human ~ ~,h b~ ' for S.
12 ~;.",.
13 The results of the ELISA testing of severa'. standard 14 g' bu' pl~Ja7aLiul~7 for antibody to S. c~,;d~.l";d;s are presented in Figure 1. Most standard immune globulins contained low levels of antibody to 16 ~ , ~' ~ ' However, by screening for antibody to TCA extracted antigens 17 of ~ ,;d~ . some "' ~ . ' lots and serum from one volunteer 18 donor were found to have increased levels of antibody to S ~,~Jhl~ ;d;s (O.D.
19 readings 1.014, 1.026, and 1.002). Variations in antibody to S. c~,id~
occurred between p.~, prepared by different techniques and lot to lot 21 variation in a single ~ uaLiw~ was seen as well, indicating that all 22 ~ ~ ' pools were not the same.

W O 93/17044 PC~r/US92/09830 C~ Activity of Human !~ for S. ~;d~.",;d;s.
2 All antibody directed against a given organism may not enhance 3 immunity and provide enhanced protection from infection. Stated differently, 4 antibodies can bind to bacteria and yet not enhance ~~ in vitro or 5 clearance from the blood of an infected host. Therefore a functional assay was 6 also utilized to determine if the antibody to S. ~ . ' detected by EI,ISA
7 was also capable of promoting IA,a~ tu~;~ and killing of the organism by 8 nt ~ c (Figure 2). Opsonic antibody activity ranged from low (<25%
9 L ~ activity), to moderate activity (25-80%) and a few had high 10 l.-- ~. .;r~ activity (>80%). Therefore two standard human immune globulin with high 1~ 1 activity were selected as Directed Human 12 Immune Globulin for S. ~ h.l";d;s based on in vitro assays that measured 13 antibody binding to TCA ~ e~;d~,.l.. dis antigens and opsonic antibody activity 14 ~' ~ ' by in vitro testing. Serum from a single donor also had good 15 opsonic activity for S. ' ~ ' ~ (>80% r- pl- V -ytic l -- t~ ;- :-l~l 16 activity). While serum and plasma from several individuals have been studied 17 only this donor had high opsonic activity. Therefore donor screening could 18 detect individual blood or plasma donors that could contribute _' bu' 19 that could be pooled as an alternate method to produce a W O 93/17044 PC~r/US92/09830 ,9 ~ I Directed Human Immune Globulin for S. c~,;d~.. ,;di5. In addition blood or 2 plasma units could be screened for pooling as well.

3Animal Protection Studies 4Discri~vtion of Tables S I:ak~ç 1 6 Table I shows the effect of Directed Human ll.. munu~;loblllh~ for 7 S. ~,v;.l.,.,;~iis (40R09) (which was selected by ELISA and opsonic assay 8 screening) compared to standard human " . . l (that had moderate 9 activity for S. ~,~;.I.,l ~ ' ) and saline control. Table I shows that untreated 10 control animals had about a 50% mortality while animals given Directed I l Immune Globulin for S. e~J;d~ ;di~ were fully protected (NO mortality).
12 Standard immune globulin gave only partial protection. Other standard 13 immune globulin lots with lower levels of antibody to S. cp;d~,. ' would be 14 even less effectivel since mortality was much higher with saline. However, 15 one would not expect that Directed Immune Globulin would be always 100%
16 effective, but that it would 'y improve survival over standard immune 17 globulin or untreated animals.

18 Table 2 19 Table 2 ' that Directed Immune Globulin produced 20 in rabbits by ~ ~ (~ c";.l", l vaccine) produced survival similar wo 93/17044 Pcr/uss2/o983o CA 2 1 ~7 7 4~

to Directed Human Immune Globulin produced by screening 2 for antibody to S. ~;d~ of individuals with S.
3 epidermidis vaccine and collecting plasma for ~g~ 1in extraction 4 would be another method for producing Directed Human Immune Globulin for S preventing or treating S. ~,;d~,. " infections.

6 Table 3 7Table 3 shows that intralipid causes a dose related increased 8 mortality in suckling rats infected with S. c~;d~ Control animals 9receiving Intralipid alone had 100% survival (43/43) while immature rats given 1016 gm/kg of Intralipid had only 46% survival (6/13). The high dose of I l Intralipid appears to impair the immune system ~u~r,c;~.,ll~ to allow the 12 normally avirulent S. c ~,;d~,ll.,hl;s to overwhelm the baby animals.

13Table 4 14Table 4 shows that normal 3 day old suckling rats not given 15 Intralipid, but infected with S. 1~/;d~.~lll;J;a develop b~t".~ ~ However, 16 over 72 hrs their immune system is able to clear the organisms from the blood 17 and aD of the baby rats survive.

WO 93/17044 PCr/US92/09830 CA 2 i 1 7480 Table 1 shows the Directed Human Immune Globulin for S.
2 e~idermidis (selected by screening standard iml~ll,,~o~;lubuli~ for opsonic or 3 antigen binding activity for S. ~I,;de.",i.lis) provides complete protection from 4 lethal infection in the setting of impaired immunity with Intralipid while 5 standard immune globulin (with moderate antibody levels) had only partial 6 protection (l out of 5 aminals died compared to about 50% with saline).
7 Additional studies with another ,,'ob~lin preparation, (Alpha 8 ~' 'i, Directed Human Immune Globulin 8016A >90% opsonic 9 activity, versus standard human immune globulin, 8007A ~ 50% opsonic 10 activity) showed that the Directed Human Immune globulin also provided 11 enhanced survival (8016A-64/95 (67%) vs. 8007A-39/90 ~43%)) over standard 12 human immune globulin. Even more striking was the fact that the Directed 13 Human Immune Globulin decreased the peak level of S. epidermidis 14 bacteremia and promoted rapid clearance of the bacteria (Figure 3). These 15 studies showed that antibody was important for protection against S.
16 ~,;d~.,..;d;~ enhanced bacterial clearance from the blood and could be an 17 effective IJ-u}~lh~la lic or therapeutic modality even in the immature host with 18 impaired immunity. Many of the animals treated with standard human immune 19 globulin remained bacteremic 72 hours after infection while only 1/20 animals 20 was still bacteremic at 72 hours after receiving the Directed Human Immune 21 Globulin. In addition the mean bacteremia level at 72 hours was wO 93/17044 Pcr/US92/09830 CA2 ! 1 74~0 markedly different (bacteremia with Directed Human Immune Globulin 0.5 x 2 10' vs. bacteremia with standard human immune globulin 3.8 xlO2).
3 In further studies, rabbit Directed Immune Globulin for S.
4 epidermidis was produced by ~ ~ rabbits with S. c";d~. '; vaccine.
The vaccine induced Directed Immune Globulin was compared with Directed 6 Human Immune Globulin produced by screening ~g'-' . " for antibody 7 to S. .,~,;d~,. 'i. (Table 2). Vaccine induced Directed Immune Globulin had 8 similar protective activity to Directed Human Immune Globulin produced by9 screening (9/11 vs. 12/13 survived) and each was better than controls (11/19 10 survived). These data show that S. c~,;d~";J;~ vaccine induced antibody I l could be used for prevention and treatment of S. .~,;d~,. ,..;J;~ infections and 12 that vaccine could be used to produce a Directed Human Immune Globulin.

14 Many bacteria such as ~, e";d~. ' s are not pathogenic in normal people. However, in babies with an immature immune system or 16 impaired immunity as is seen with intralipid, ~ c~;d~. ' may cause sepsis 17 and death. It is critical therefore, that any animal model to test antibody 18 ~,rf~ should include these factors. To our knowledge this is the first 19 time that antibody to ~ h~ epidermis has been shown to provide protection and enhance bacterial clearance in an immature and/or Wo 93/17044 PCr/USs2/Os830 CA 2 i 1 7480 X;I host. Intralipid given in dosage up to 16 gm/kg did not 2 cause death in any baby animals (controls, table 3). In the absence of 3 Intralipid, the 3 day old animals will become bacteremic with S. .,~,id._.~.,i.lis 4 after infection, but will clear the infection over 72 hours and survive (Table 4).
S However, Intralipid did impair immunity in a dose related fashion and when 6 the 3 day old animals were infected with S. ~;d~.l..idi~ lethal sepsis occurred 7 in up to 67% of the animals. Baby rats in the first day of life also do not clear 8 l.~ well (due to immature immunity) and develop lethal sepsis. In 9 these models baby rats were unable to clear the S. c~,i.l~l-..iJis bacteremia and 10 developed lethal sepsis. Directed Human Immune Globulin was able to I l enhance survival and promote bacterial clearance while standard human 12 immune globulin did not enhance clearance (Fig 3).

14 When SE is injected into normal baby rats, they become 15 bacteremic in 2 hours and then begin to slowly clear the bacteria from the 16 blood. All of the animals cleared the bacteremia 72 hours after the infection.
17 thus suggesting that under normal ch~ J - ~ . neonatal immunity while 18 impaired can eventually control SE. However, studies in rats infectedd with S.
19 Pni~lP~i~lic shortly after birth have d- ....,"~I",t~ J that they can also develop a 20 lethal infection.

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Claims

We claim:

1. A Directed Human Immune Globulin for the prevention or treatment of Staphylococcus epidermidis infections.

2. A Directed Human Immune Globulin of Claim 1 which contains a measured level of anti-staphylococcal IgG antibodies that react with surface antigens of Staphylococcus epidermidis and promote phagocytosis and killing of Staphylococcus epidermidis in vitro and/or protection against Staphylococcus epidermidis in vivo.

3. The Directed Human Immune Globulin of Claim 2 wherein the measured level of anti-staphylococcal antibodies has an opsonic activity within the range of about 80 to about 100 percent.

4. A pharmaceutical composition comprising an amount of Directed Human Immune Globulin of Claim 1 sufficient to prevent or treat infections by S.epidermidil and a pharmaceutically acceptable carrier therefor.

5. A pharmaceutical composition comprising a Directed Human Immune Globulin of Claim 2.

6. A pharmaceutical composition comprising a Directed Human Immune Globulin of Claim 3.

7. A method of preparing a Directed Human Immune Globulin of Claim 1 by screening serum, plasma, or an immunoglobulin pool by S.
epidermidis ELISA or Opsonic Assays.

8. The method of Claim 7 wherein serum is screened by S.
epidermidis ELISA or Opsonic Assays.

9. The method of Claim 8 wherein the serum is screened by S.
epidermidis ELISA.

10. The method of Claim 8 wherein the serum is screened by S. epidermidis Opsonic Assays.

11. The method of Claim 7 wherein the plasma is screened by S. epidermidis ELISA or Opsonic Assays.

12. The method of Claim 11 wherein the plasma is screened by S. epidermidis ELISA.

13. The method of Claim 11 wherein the plasma is screened by S. epidermidis Opsonic Assays.

14. The method of Claim 7 wherein the immunoglobulin pool is screened by S. epidermidis ELISA or Opsonic Assays.

15. The method of Claim 14 wherein the immunoglobulin pool is screened by S. epidermidis ELISA.

16. The method of Claim 14 wherein the immunoglobulin pool is screened by S. epidermidis Opsonic Assays.

17. A method of preparing a Directed Human Immune Globulin of Claim 1 comprising the steps of: (a) immunizing plasma donors and (b) removing plasma from said donors for Directed Immune Globulin preparation.

18. A method of assessing the protective level of Direct Human Immune Globulin by using an immature or intralipid induced lethal model to provide minimum protective standard comprising the steps of: (a) screening with in vitro assays and (b) using animal lethality tests to ensure that the immunoglobulin preparation provided protective antibody to S.
epidermidis.

19. A method of treating a host with a therapeutically-effective amount of S. epidermidis of Directed Human Immune Globulin of Claim 1 by intraveneous administration thereof.

20. A method of treating a host with a therapeutically effective amount of S. epidermidis of Directed Human Immune Globulin of Claim 1 by intramuscular administration thereof.

21. The method of Claim 19 wherein the host is treated prior to infection with S. epidermidis.

22. The method of Claim 20 wherein the host is treated after infection with S. epidermidis.