WO2007005715A2 - Staphylococcal antibodies which cross-react with fungal antigens - Google Patents

Abstract

Cross-reactive antibodies are provided which can recognize antigens from Staphylococcus epidermidis such as the SdrG protein and from Candida albicans, such the Hsp70 protein. These antibodies will thus be useful in methods of treating or preventing infections from Staphylococcus and Candida microorganisms at the same time. Compositions and kits containing these antibodies as well as methods for their use are also provided.

Description

STAPHYLOCOCCAL ANTIBODIES WHICH CROSS-REACT WITH FUNGAL ANTIGENS

Cross Reference to Related Applications

The present application claims the benefit of U.S. provisional applications Ser. No. 60/772,871 , filed February 14, 2006, and Ser. No. 60/695,441 , filed July 1 , 2005, said applications incorporated herein by reference.

Field of the Invention

The present invention relates to the fields of microbiology, molecular biology, and immunology and more particularly relates to staphylococcal antibodies which recognize antigens from fungal bacteria such as Candida albicans, the use of said antibodies to treat or prevent infection, as well as compositions and methods using said compositions to prevent, treat, or diagnose fungal infections or coagulase- negative staphylococcal infections in man and animals. The invention contemplates the specific use in fighting late-onset sepsis in neonates and for inhibiting the growth and severity of infections caused by both Candidal species of yeast as well as staphylococcal bacteria at the same time.

Background of the Invention

Low birth weight (LBW) infants comprise 1.4% of all births in the United States, and over 57,000 infants per year are very low birth weight (VLBW) defined as < 1 ,500 gm.¹ Advances in medical care provided by neonatal intensive care units (NICUs) throughout the country have dramatically improved the survival for these premature infants. One of the costs of prolonged survival among premature infants is an increased frequency of complications, especially nosocomial (hospital acquired) infections. In one study, the overall rate of late-onset infection among VLBW infants ("VLBWI") 501 to 1 ,500 gm was 16% but the rate increased with decreasing birth weight and gestational age, rising rapidly to 40% among the smallest infants (500 to 600 gm).²

In a study of infants 401 to 1 ,500 gm birth weight admitted to the National Institute of Child Health and Human Development (NICHD) Neonatal Research Network centers, the rate of infection among infants of birth weight 401 to 1500 g after day three of life was 21%, essentially unchanged from previous estimates.³ In fact, late-onset sepsis has become the most common cause of death among premature infants after the third day of life.⁴

Reasons for the increased risk of infection among neonates include iatrogenic factors such as the use of vascular catheters, but also host factors. Immunoglobulin G (IgG), a critical part of immunity against bacterial pathogens, is transferred from mother to infant selectively through the placenta beginning at 8 to 10 weeks of gestation and accelerating during the last trimester. Infants born prior to 32 weeks gestation are relatively deficient in IgG. In vitro studies have demonstrated the importance of IgG directed against staphylococci for host defense.^5"7 Low levels of IgG at birth is an identified risk factor for late-onset sepsis in LBW infants.²

The predominant organism for late-onset sepsis in recent studies is S. epidermidis and similar species collectively referred to as coagulase-negative staphylococci (CoNS).²' ^8"10 While the mortality attributed specifically to CoNS is considered to be less than that of other organisms, the public health implications of CoNS infections are significant. Widespread use of antibiotics, especially vancomycin, for the treatment of suspected or proven nosocomial infection applies selective pressure for the emergence of antibiotic-resistant bacteria in intensive care units. Stoll et al. remarked "It is alarming that 44% of infants in this cohort (whether or not they had documented CoNS infection) were treated with vancomycin."³ Vancomycin resistant strains of CoNS have been rarely reported, but the possibility of wider emergence of such strains would be disastrous.¹¹' ¹²

In addition to staphylococcal late-onset sepsis, fungal sepsis in VLBWl is significant medical problem. In a prospective study by Conner et al., among 1,111 VLBWI, 5% developed fungal sepsis within the first 28 days of life. The predominant fungal pathogen was Candida species of yeast (82%). In a similar study, the mortality rate associated with Candida species late-onset sepsis in VLBWl was 43.9%. However, despite the very severe pathological conditions caused by Cancf/cfa-related infections, there have been very few effective treatment regimens against these extremely dangerous infections. Even further, it has not heretofore been possible to develop a treatment regimen using an antibody which recognizes a specific surface protein from Candida albicans and which thus can address both infections caused by staphylococcal organisms and at the same time be effective against Cand/da-related infections. It is therefore imperative that new strategies be developed which can address the critical problem of hospital-acquired infections in premature infants, and in particular, it is highly desirable to develop treatments and compositions which can be useful in treating and preventing Ca^d/cfa-related infections and at the same time be useful in inhibiting the progression of staphylococcal infections.

Summary of the Invention

It is thus an object of the present invention to provide compositions and methods for diagnosing, treating, and/or preventing infections caused by Candida species of yeast.

It is thus another object of the present invention to provide compositions and methods which are particularly useful in fighting late-onset sepsis in neonates and which can inhibit the growth and severity of infections caused by Candida species of yeast and staphylococcal infections at the same time.

It is still further an object of the present invention to provide polyclonal and monoclonal antibody compositions that can be effective in identifying and isolating surface antigens from Staphylococcus epidermidis and Candida albicans and which can be useful in treating or preventing Staphylococcal and Caπcf/c/a-related diseases.

These and other objects are provided by the present invention wherein a cross-reactive polyclonal and monoclonal antibody composition recognizing SdrG from S. epidermidis*³, and/or the N1 domain from SdrG and Hsp70 from Candida albicans can be administered to a patient in need of treatment for or protection against an infection caused by Staphylococcus epidermidis of the species coagulase-negative staphylococci and fungi of the species Candida such as Candidiasis, and this composition will be effective in inhibiting the staphylococci and S. epidermidis bacteria and yeast and enabling the effective treatment or prevention of the staphylococcal and Candida infection. Further, because of its ability to recognize surface proteins in Candida, the polyclonal and monoclonal antibody compositions of the present invention will also be useful in identifying and isolating surface proteins from S. epidermidis and Candida and in diagnosing S. epidermidis and Candida infections. The present compositions and methods will thus be particularly effective in treating or preventing late-onset sepsis in low birth weight neonates. These and other objects of the present invention are obtained through the compositions and methods as set forth in the detailed description of the invention provided hereinbelow.

Brief Description of the Drawing Figures

Fig. 1 illustrates that the pAb-SdrG of the present invention binds specifically to Candida HSP70. In the figure, β-ME extract from the hyphal cells of Candida albicans S. C 5314 was separated by 2-D electrophoresis and transferred onto PVDF membrane. The membranes were probed with affinity purified anti-SdrG antibodies (Veronate^® lot 804901 A) in the presence of bovine brain HSP70 or Histatin 5. The position of the HSP70 protein on each blot is indicated.

Fig. 2 illustrates that pAb-SdrG binds specifically to Candida HSP70 SSA4. A β-ME extract from the hyphal cells of Candida albicans SC 5314 was separated by 2-D electrophoresis and transferred onto PVDF membrane. A 2D gel stained with Coomassie Blue (Imperial protein stain) is shown in panel A. PVDF membranes were immunoblotted with mAb-HSP70 (panel B) or pAb-SdrG (panel C). The migration positions of molecular weight markers are shown on the left. The position of HSP70 proteins are indicated by a circle.

Fig. 3 illustrates protein sequence alignments. The amino acid sequences were aligned using the CLUSTALW multiple alignment. Identical residues are shown in red (*), amino acids with strong similarity are in green (:) amino acids with weak similarity are in blue (.) and divergent residues are shown in black. An alignment of a region of the SdrG N1 sequence with SSA4 are shown in panel A and an alignment of the same region of SSA4 and SSA1 are shown in panel B.

Fig. 4 illustrates that anti-SdrG antibodies are protective in a murine model of Candidemia. Survival curves for pAb-SdrG treated (red circles) and buffer control (blue squares) groups are shown. The two panels illustrate results of a single pAb- sdrG dose (Panel A ) and a multiple dose study (Panel B). P values obtained after comparing survival curves in each experiment are shown. Fig. 5 illustrates the screening of monoclonal antibodies raised against SdrG N1 against surface proteins of C. albicans. Numbers 1 to 107 represents supernatants from individual mouse hybridomas recognizing r-SdrG N1 by ELISA. As a positive control a mouse anti-HSP70 (10μg/m!) was used (α-HSP70). Negative controls included secondary antibody alone (2^nd only), hybridoma culture medium, (Medium only), and irrelevant control antibodies (SdrF mAb1 and SdrF mAb2).

Fig. 6 illustrates that HSP70 is recognized specifically by Veronate^®. SDS/DTT extracts from the hyphal cells of C. albicans strain S.c 5314 was separated in 2D electrophoresis. Proteins were transferred onto PVDF membrane and probed with the pre-absorbed Veronate^® or IGIV at 1 :40 dilution.

Fig. 7 illustrates that affinity purified anti-SdrG antibodies specifically interact with Candida HSP70. β-ME extract from the hyphal cells of Candida albicans S.C 5314 was separated by 2-D electrophoresis and transferred onto PVDF membrane. The membranes were probed with the affinity purified anti-SdrG antibodies in the presence of Bovine brain HSP70 or recombinant SdrG proteins.

Fig. 8 illustrates that SdrG - N1 specific antibodies interact with the Candida HSP70. β-ME extract from the hyphal cells of C. albicans S.C 5314 was separated by 2-D electrophoresis and transferred onto PVDF membrane. The membranes were probed with the affinity purified anti-SdrG antibodies in the presence of recombinant SdrG proteins.

Fig. 9 illustrates that Histatin 5 does inhibit pAb-SdrG recognition of C. albicans HSP70. β-ME extracts from the hyphal cells of C. albicans S.C 5314 were separated by 2-D electrophoresis and transferred onto PVDF membrane. The membranes were probed with the affinity purified anti-SdrG antibodies in the presence of recombinant SdrG N1 protein or Histatin 5.

Fig. 10 is a graphic representation showing that using a murine model of candidiasis, passive administration of affinity purified SdrG antibodies from Veronate (pAb-SdrG) provided statistically significant protection against C. albicans compared to control. Detailed Description of the Preferred Embodiments

In accordance with the present invention, there are provided polyclonal and monoclonal antibodies which have been generated from the SdrG protein from Staphylococcus epidermidis or from antigenic subregions therefrom and which can also recognize fungal antigens, in particular surface proteins from the yeast Candida albicans. As set forth herein, anti-SdrG monoclonal and polyclonal antibodies are provided which recognize the HSP70 protein of Candida albicans, and such antibodies can be used in methods of treating or preventing infections in Candida yeast in addition to staphylococcal bacteria. As set forth below, antibodies that recognize SdrG and/or the SdrG N1 domain have been observed which can cross- react with HSP70 and thus provide significant protection against Candida albicans infection. The SdrG protein is a fibrinogen-binding protein found in S. epidermidis and has been the subject of numerous patents and patent applications, including U.S. Pat. No. 6,635,473, and SdrG and/or Candida yeast microorganisms have been disclosed in applications including U.S. Provisional Applications Serial No. 60/566,082, filed April 29, 2004, Serial No. 60/561 ,540, filed April 13, 2004, and Serial No. 60/530,654, filed December 19, 2003, U.S. Patent Application 11/016,564, filed December 20, 2004, and International Application PCT/US04/43276 (also published as WO 2005/060713), all of said patents and said applications incorporated by reference in their entirety. However, no antibodies were previously known which could recognize both the SdrG protein, and/or its N1 domain, and the HSP70 protein from Candida albicans, and thus the present invention provides the ability to effectively treat staphylococcal and Candida infections at the same time by virtue of the recognition of these proteins.

In one aspect of the invention, anti-SdrG antibodies may be isolated which can recognize the heat-shock protein from Candida albicans and thus be useful in treating or preventing infection from Candida yeast. The 70 kDa heat shock protein from Candida albicans known as HSP70 is well known as has been described in many references including, e.g., Sandini et al., Med. Mycol. 40(5):471-8 (2002), said article incorporated herein by reference. The SdrG protein and its A domain and subdomains are well known and have been described, e.g., in U.S. Pat. No. 6,635,473 and continuing applications therefrom, all of said patents and said applications incorporated herein by reference. In accordance with the invention, antibodies which recognize or which are raised against either the SdrG protein or the SdrG A domain are contemplated for use in the invention. In addition, the antibodies of the invention which recognize HSP70 may be useful in inhibiting or preventing the attachment of Candida albicans or its HSP70 protein to a host cell, such as by attachment to an extracellular matrix protein of the host.

The anti-Sdrg antibodies in accordance with the invention may be isolated and/or purified in any known manner, such as generating polyclonal antibodies using the SdrG protein or its ligand binding A domain or other antigenic subregions therefrom and isolating and/or purifying said antibodies in a conventional manner well known to those skilled in the art. For example, SdrG or its A domain may be isolated or produced recombinantly and introduced into a suitable animal host so as to obtain antibodies which may then be isolated and purified for use. Preferably, the antibodies will be humanized or can be obtained from human donors in the manner described below. In the preferred method in accordance with the invention, said antibodies are isolated from the product VERONATE®, a proprietary product of Inhibitex, Inc., which is an immunoglobulin product obtained from donor plasma having a high titer of antibodies to SdrG. This product is described in detail in U.S. Pat. No. 6,692,739, incorporated herein by reference.

In the preferred method, an affinity purification of VERONATE® is conducted so as to obtain polyclonal anti-SdrG antibodies (p-Ab-SdrG) which can also recognize the HSP70 protein from Candida albicans in accordance with the present invention. In this method, the rSdrG A-domain (SdrG-A) can be used as a recombinant polypeptide corresponding to the fibrinogen-binding A domain of the SdrG protein (amino acids 50-597) from S. epidermidis strain K28 (Davis 2001). The recombinant protein may be expressed with N-terminal hexahistidine sequences, and purified from Escherichia coli lysates by metal affinity chromatography on a chelating Sepharose Fast Flow resin (Amersham Biosciences, Piscataway, NJ). The protein may be further purified by Q Sepharose HP chromatography (Amersham Biosciences, Piscataway, NJ). In one suitable method to obtain the anti-SdrG antibodies of the invention, approximately 25 mg of purified rSdrG-A was coupled to 5 mL columns of HiTrap NHS (N-hydroxysuccinimide) -activated HP resin (GE Healthcare, Piscataway, NJ) following the protocol supplied by the manufacturer. VERONATE^® samples were applied to the columns and then washed with phosphate buffered saline (PBS). Adsorbed IgG was eluted with 0.1 M glycine, pH 2.7 and the IgG fractions were collected in 2 M Tris, pH 8.0. The purified antibodies were then formulated to 0.15 M glycine, 0.035 M NaCI, 0.015% Tween 80, pH 6.2 to match the standard VERONATE^® formulation.

Testing then showed that the Affinity purified anti-SdrG antibodies interact specifically with C. albicans HSP70. In one suitable test, a β-ME extract of the hyphal cells of C. albicans S. C 5314 was separated by 2-D electrophoresis and transferred onto PVDF membrane. The membranes were probed with anti-SdrG antibodies affinity purified from VERONATE^® as described above. The anti-SdrG antibodies recognized several proteins in the 2-D western, one of which was identified as HSP70 by mass spectrometry (Figure 7). Recombinant protein fragments corresponding to the N1 domain of SdrG or the N3 domain of SdrG were added as competitors to the 2-D Western. Only the addition of the N1 domain of SdrG prevented the interaction of the antibodies with HSP70 (Figure 7), and this the N1 domain of SdrG can be utilized in the present invention as described further below. Anti-SdrG antibodies thus specifically recognized the Candida HSP70 and thus can be used in methods of treating and/or preventing Candida infection.

In another aspect of the invention, polyclonal and monoclonal antibodies recognizing the N1 domain of the SdrG protein are provided which also recognize the HSP70 protein of C. albicans in accordance with the invention. The SdrG N1 region represents AA 50-272 of the SdrG protein from S. epidermidis strain K28 such as disclosed in U.S. Pat. No. 6,635,473 and Davis et al. 2001 (J. Biol. Chem. 276:27799-805), both references incorporated herein by reference. The actual sequence of SdrG N1 region is set forth below: SdrG N1 DNA: (SEQ ID NO: 1)

GAGGAGAATACAGTACAAGACGTTAAAGATTCGAATATGGATGATGAATTATCA

GATAGCAATGATCAGTCCAGTAATGAAGAAAAGAATGATGTAATCAATAATAGTC

AGTCAATAAACACCGATGATGATAACCAAATAAAAAAAGAAGAAACGAATAGCAA

CGATGCCATAGAAAATCGCTCTAAAGATATAACACAGTCAACAACAAATGTAGAT

GAAAACGAAGCAACATTTTTACAAAAGACCCCTCAAGATAATACTCAGCTTAAAG

AAGAAGTGGTAAAAGAACCCTCATCAGTCGAATCCTCAAATTCATCAATGGATA

CTGCCCAACAACCATCTCATACAACAATAAATAGTGAAGCATCTATTCAAACAAG

TGATAATGAAGAAAATTCCCGCGTATCAGATTTTGCTAACTCTAAAATAATAGAG

AGTAACACTGAATCCAATAAAGAAGAGAATACTATAGAGCAACCTAACAAAGTAA

GAGAAGATTCAATAACAAGTCAACCGTCTAGCTATAAAAATATAGATGAAAAAAT

TTCAAATCAAGATGAGTTATTAAATTTACCAATAAATGAATATGAAAATAAGGTTA

GACCGTTATCTACAACATCTGCCCAACCATCGAGTAAGCGTGTAACCGTAAATC

AATTAGCGGCA >drG N1 Protein: (SEQ ID NO: 2)

EENTVQDVKDSNMDDELSDSNDQSSNEEKNDVINNSQSINTDDDNQIKKEETNSN DAIENRSKDITQSTTNVDENEATFLQKTPQDNTQLKEEWKEPSSVESSNSSMDTA QQPSHTTINSEASIQTSDNEENSRVSDFANSKIIESNTESNKEENTIEQPNKVREDSI TSQPSSYKNIDEKISNQDELLNLPINEYENKVRPLSTTSAQPSSKRVTVNQLAA

In accordance with the invention, polyclonal and monoclonal antibodies recognizing the N1 domain of the SdrG protein also recognize the HSP70 protein of C. albicans and can be used to treat or prevent Candida infection. In one aspect of the invention, monoclonals to the N1 domain can be prepared in any conventional manner, such as the techniques set forth by Kohler and Milstein, Nature 256:495- 497 (1975), or other suitable ways known in the field. In addition, it will be recognized that these monoclonals can be prepared in a number of forms, including chimeric, humanized, or human in addition to murine in ways that would be well known in this field. Still further, monoclonal antibodies may be prepared from a single chain, such as the light or heavy chains, and in addition may be prepared from active fragments of an antibody which retain the binding characteristics (e.g., specificity and/or affinity) of the whole antibody. By active fragments is meant an antibody fragment which has the same binding specificity as a complete antibody which binds to extracellular matrix binding proteins, and the term "antibody" as used herein is meant to include said fragments. In the preferred method to prepare monoclonals to N1, such monoclonals are made by expressing and purifying an SdrG N1 protein, and using this protein to generate a panel of murine monoclonal antibodies as set forth below.

In one suitable process for preparing the cross-reactive SdrG N1 monoclonal antibodies of the invention using PCR, SdrG N1 representing AA 50-272 was amplified from S. epidermidis K28 genomic DNA (from sequences described above) and subcloned into the E. coli expression vector PQE-30 (Qiagen), which allows for the expression of a recombinant fusion protein containing six histidine residues. This vector was subsequently transformed into the E. coli strain ATCC 55151 , grown in a 15-liter fermentor to an optical density (OD₆₀o) of 0.7 and induced with 0.2 mM isopropyl-1-beta-D galactoside (IPTG) for 4 hours. The cells were harvested using an AG Technologies hollow-fiber assembly (pore size of 0.45 Dm) and the cell paste frozen at -80° C. Cells were lysed in 1X PBS (1OmL of buffer/1 g of cell paste) using 2^" passes through the French Press @ 1100psi. Lysed cells were spun down at 17,000rpm for 30 minutes to remove cell debris. Supernatant was passed over a 5- ml_ HiTrap Chelating (Pharmacia) column charged with 0.1 M NiCI₂. After loading, the column was washed with 5 column volumes of 1OmM Tris, pH 8.0, 10OmM NaCI (Buffer A). Protein was eluted using a 0-100% gradient of 1OmM Tris, pH 8.0, 10OmM NaCI, 20OmM imidazole (Buffer B) over 30 column volumes. SdrGN1 N2N3 or SdrGN2N3 eluted at -13% Buffer B (~26mM imidazole). Absorbance at 280nm was monitored. Fractions containing SdrGNI were dialyzed in 1x PBS.

The protein was then put through an endotoxin removal protocol. Buffers used during this protocol were made endotoxin free by passing over a 5-mL Mono-Q sepharose (Pharmacia) column. Protein was divided evenly between 4x 15ml_ tubes. The volume of each tube was brought to 9ml_ with Buffer A, 1 mL of 10% Triton X-114 was added to each tube and incubated with rotation for 1 hour at 4°C. Tubes were placed in a 37⁰C water bath to separate phases. Tubes were spun down at 2,000rpm for 10 minutes and the upper aqueous phase from each tube was collected and the detergent extraction repeated. Aqueous phases from the 2nd extraction were combined and passed over a 5-mL IDA chelating (Sigma) column, charged with 0.1 M NiCI₂ to remove remaining detergent. The column was washed with 9 column volumes of Buffer A before the protein was eluted with 3 column volumes of Buffer B. The eluant was passed over a 5-mL Detoxigel (Sigma) column and the flow-through collected and reapplied to the column. The flow-through from the second pass was collected and dialyzed in 1x PBS. The purified product was analyzed for concentration, purity and endotoxin level before administration into mice. Next, monoclonal antibodies against SdrG N1 were generated using E. coli expressed and purified SdrG N1 protein. Briefly, a group of Balb/C mice received a series of subcutaneous immunizations of 1-10 mg of protein in solution or mixed with adjuvant as described below in Table I:

Table I. Immunization Scheme

Conventional

Injection Day. Amount (ug) Route Adjuvant

Primary 0 5 Subcutaneous FCA

Boost #1 14 1 Intraperitoneal RIBI

Boost #2 28 1 Intraperitoneal RIBI

Boost #3 42 1 Intraperitoneal RIBI At the time of sacrifice or seven days after a boost (conventional) serum was collected and titered in ELISA assays against MSCRAMMs or on whole cells (S. epidermidis). Three days after the final boost, the spleen was removed, teased into a single cell suspension and the lymphocytes harvested. The lymphocytes were then fused to a P3X63Ag8.653 myeloma cell line (ATCC #CRL-1580). Cell fusion, subsequent plating and feeding were performed according to the Production of Monoclonal Antibodies protocol from Current Protocols in Immunology (Chapter 2, Unit 2.). Monoclonals were screened by ELISA in a process wherein lmmulon 2-HB high-binding 96-well microtiter plates (Dynex) were coated with 1 μg/well of rSdrG- N1 in 1X PBS, pH 7.4 and incubated for 2 hours at room temperature. All washing steps in ELISAs were performed three times with 1X PBS, 0.05% Tween-20 wash buffer. Plates were washed and blocked with a 1% BSA solution at room temperature for 1 hour before hybridoma supernatant samples were added to wells. Plates were incubated with samples and relevant controls such as media alone for one hour at room temperature, washed, and goat anti-mouse IgG-AP (Sigma) diluted 1 :5000 in 1X PBS, 0.05 % Tween-20, 0.1% BSA was used as a secondary reagent. Plates were developed by addition of 1 mg/ml solution of 4-nitrophenyl phosphate (pNPP) (Sigma), followed by incubation at 37° C for 30 minutes. Absorbance was read at 405 nm using a SpectraMax 190 Plate Reader (Molecular Devices Corp.). Antibody supernatants that had an OD₄₀₅ > 3 times above background (media alone, ~ 0.1 OD) were considered positive.

In accordance with the invention, SdrG n1 monoclonals were screened for their ability to be cross-reactive with both SdrG N1 and surface proteins of Candida albicans. In these tests, a β-ME extract prepared from hyphal cells of Candida albicans strain S.c 5314 was diluted in 1XPBS to 0.5 mg/ml total protein and 100μl of the diluted extract was added to an AcroWell 96 NT Membrane-bottom plate (Pall Corporation, catalog #5022, lot A10427959). The plate was incubated for 30 minutes at room temperature, and then centrifuged at 3,000xg for 5 minutes. To block non-specific binding sites, 250μl of PBS-T + 5% dry milk was added, and the plate was incubated at room temperature for 1 hour. The blocking solution was removed by inverting and tapping the plate. 200μl of SdrG N1 mAb supernatants and control antibodies were added to appropriate wells and the plate was incubated for 2 hours at room temperature. After the incubation, the plate was washed 3 times in PBS-T for 5 minutes each, inverting and tapping to remove wash solution. Then the plate was washed an additional 3x in PBS-T by centrifugation at 3,000xg for 5 minutes each. 200μl of GAM-HRP (1 :2500 dilutions in PBS-T + 5% dry milk) was added to each well and the plate was incubated for 1 hour at room temperature. The plate was washed 3 times in PBS-T for 5 minutes each, inverting and tapping to remove wash solution. The plate was washed an additional 3x in PBS-T by centrifugation at 3,000xg for 5 minutes each. SuperSignal West Pico chemiluminescent substrate was added and the plate exposed to autoradiography film (BioMax XAR, Kodak, New Haven, CT) for various time points to visualize antibody binding. The sequences of the HSP70 proteins (SSA1 and SSA4) are shown below:

SSA4 Sequence (HSP70): (SEQ ID NO: 3)

MSKAVGIDLGTTYSCVAHFANDRVEIIANDQGNRTTPSFVAFTDTERLIGDAAKNQA

AMNPANTVFDAKRLIGRKFDDPEVINDAKHFPFKVIDKAGKPVIQVEYKGETKTFSP

EEISSMVLTKMKEIAEGYLGSTVKDAWTVPAYFNDSQRQATKDAGTIAGLNVLRIIN

EPTAAAIAYGLDKKGSRGEHNVLIFDLGGGTFDVSLLAIDEGIFEVKATAGDTHLGG

EDFDNRLVNFFIQEFKRKNKKDISTNQRALRRLRTACERAKRTLSSSAQTSIEIDSLY

EGIDFYTSITRARFEELCADLFRSTLDPVGKVLADAKIDKSQVEEIVLVGGSTRIPKIQ

KLVSDFFNGKELNKSINPDEAVAYGAAVQAAILTGDTSSKTQDILLLDVAPLSLGIET

AGGIMTKLIPRNSTIPTKKSETFSTYADNQPGVLIQVFEGERAKTKDNNLLGKFELSG

IPPAPRGVPQIEVTFDIDANGILNVSALEKGTGKTQKITITNDKGRLSKEEIDKMVSEA

EKFKEEDEKEAARVQAKNQLESYAYSLKNTINDGEMKDKIGADDKEKLTKAIDETIS

WLDASQAASTEEYEDKRKELESVANPIISGAYGAAGGAPGGAGGFPGAGGFPGGA

PGAGGPGGATGGESSGPTVEEVD

SSA1 Sequence (HSP70): (SEQ ID NO: 4)

MSKAVGIDLGTTYSCVAHFANDRVEIIANDQGNRTTPSFVAFTDTERLIGDAAKNQA

AMNPANTVFDAKRLIGRKFDDHEVQGDIKHFPFKWDKASKPMIQVEYKGETKTFS

PEEISSMILGKMKETAEGFLGTTVKDAWTVPAYFNDSQRQATKDAGTIAGLNVMRII

NEPTAAAIAYGLDKKSEAEKNVLIFDLGGGTFDVSLLSIEDGIFEVKATAGDTHLGGE

DFDNRLVNFFIQEFKRKNKKDISTNQRALRRLRTACERAKRTLSSSAQTSIEIDSLYE

GIDFYTSITRARFEELCADLFRSTLEPVDKVLSDAKIDKSKVDE]VLVGGSTRIPKVQK

LVSDYFNGKEPNRSINPDEAVAYGAAVQAAILSGDTSSKTQDLLLLDVAPLSLGIETA

GGIMTKLIPRNSTIPTKKSETFSTYADNQPGVLIQVFEGERAQTKDNNLLGKFELSGI

PPAPRGVPQIEVTFDIDANGILNVSALEKGTGKTQKITITNDKGRLSKEEIEKMVSEA

EKFKEEDEKEASRVQAKNQLESYAYSLKNTLGEEQFKSKLDASEIEEVTKAADETIA

WLDSNQTATQEEFADQQKELESKANPIMTKAYQAGATPSGAAGAAPGGFPGGAA

PEPSNDGPTVEEVD These tests showed that SdrG N1 monoclonals could be generated in accordance with the invention which could recognize the HSP70 protein of Candida albicans. In tests using monoclonal antibodies, rSdrG N1 was used to immunize mice and the resulting monoclonal hybridoma clones were identified by strong ELISA activity against the SdrG N1 immunogen, then screened for cross-reactivity to Candida antigens. Out of the supematants containing anti-SdrG N1 antibodies, at least 14 of the supematants interacted with the β-ME extract with signal intensities as high as that of the control anti-HSP70 antibody (Figure 4, indicated by numbers in red) with at least 29 of the supematants exhibiting medium or low reactivity with the Candida extract (numbers in light blue, Figure 5). As a result, monoclonals to SdrG N1 can be generated which recognize HSP70 and which have affinity as high as that of control anti-HSP70 antibodies. These antibodies in accordance with the invention can thus be useful in methods of treating or preventing Candida albicans infection and infection from staph bacteria such as S. epidermidis at the same time.

One additional mode of the present invention is the use of the product VERONATE® which contains a high titer to Sdrg and which also recognizes the HSP70 protein from Candida. As indicated above, VERONATE® is an immunoglobulin composition which is obtained from donor plasma having a high titer to SdrG such as described in U.S. Pat. No. 6,692,739, incorporated herein by reference. In tests conducted using VERONATE®, wherein SDS/DTT extracts from hyphal cells of Candida albicans S. C 5314 were separated by 2-D electrophoresis and transferred onto PVDF membrane, and the membranes were probed with pre- absorbed VERONATE^® or control IGIV, one of the proteins recognized by VERONATE^® was identified as HSP70 after protein sequencing. Moreover, the HSP70 that was recognized by VERONATE^®, was not recognized by a commercial lot of IGIV. Accordingly, it was shown that VERONATE® recognizes the HSP70 protein from Candida and can thus be used in methods of treating or preventing Candida infection.

Accordingly, the present invention provides monoclonal antibodies which recognize the SdrG protein and/or its N1 region which are cross-reactive and which can bind to S. epidermidis and C. albicans as to be useful in methods of treating, preventing or diagnosing staphylococcal and Candida yeast infections. In addition, in accordance with the invention, an immunoglobulin product having high titers to the SdrG protein, e.g., the VERONATE® product described above, also recognizes the HSP70 protein from Candida and can also be used in accordance with the present invention. Further, the invention provides monoclonals that can recognize the N1 subdomain of SdrG, and thus the present invention contemplates these monoclonal antibodies or other monoclonals recognizing the same epitopes of the specific monoclonals described herein.

Although production of antibodies as indicated above is preferably carried out using synthetic or recombinantly produced forms of the SdrG protein or the SdrG N1 region antibodies may also be generated from natural isolated and purified SdrG proteins or subregions, or active fragments thereof. Still other conventional ways are available to generate the SdrG antibodies of the present invention using recombinant or natural purified SdrG proteins or their active regions, as would be recognized by one skilled in the art.

As would be recognized by one skilled in the art, the antibodies of the present invention may also be formed into suitable pharmaceutical compositions for administration to a human or animal patient in order to treat or prevent an infection caused by a yeast infection in addition to an infection caused by staphylococcal bacteria. Pharmaceutical compositions containing the antibodies of the present invention, or effective fragments thereof, may be formulated in combination with any suitable pharmaceutical vehicle, excipient or carrier that would commonly be used in this art, including such conventional materials for this purpose, e.g., saline, dextrose, water, glycerol, ethanol, other therapeutic compounds, and combinations thereof. As one skilled in this art would recognize, the particular vehicle, excipient or carrier used will vary depending on the patient and the patient's condition, and a variety of modes of administration would be suitable for the compositions of the invention, as would be recognized by one of ordinary skill in this art. Suitable methods of administration of any pharmaceutical composition disclosed in this application include, but are not limited to, topical, oral, anal, vaginal, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal and intradermal administration.

If topical administration is desired, the composition may be formulated as needed in a suitable form, e.g., an ointment, cream, gel, lotion, drops (such as eye drops and ear drops), or solution (such as mouthwash). Wound or surgical dressings, sutures and aerosols may be impregnated with the composition. The composition may contain conventional additives, such as preservatives, solvents to promote penetration, and emollients. Topical formulations may also contain conventional carriers such as cream or ointment bases, ethanol, or oleyl alcohol.

Additional forms of antibody compositions, and other information concerning compositions, methods and applications with regard to the MSCRAMM® compositions of the present invention can also be found from other patent references concerning other MSCRAMM®s which will generally be applicable to the present invention as well, and these patents include U.S. Pat. Nos. 7,045,131 ; 6,994,855; 6,979,446; 6,841 ,154; 6,703,025; 6,692,739; 6,685,943; 6,680,195; 6,635,473; 6,288,214; 6,177,084; and 6,008,341 , all of said patents incorporated herein by reference.

The antibody compositions of the present invention which are generated against SdrG and/or the SdrG N1 region and which recognize the HSP70 protein from C. albicans may also be administered with a suitable adjuvant in an amount effective to enhance the immunogenic response against the conjugate. For example, suitable adjuvants may include alum (aluminum phosphate or aluminum hydroxide), which is used widely in humans, and other adjuvants such as saponin and its purified component Quil A, Freund's complete adjuvant, RIBBI adjuvant, and other adjuvants used in research and veterinary applications. Still other chemically defined preparations such as muramyl dipeptide, monophosphoryl lipid A, phospholipid conjugates such as those described by Goodman-Snitkoff et al. J. Immunol. 147:410-415 (1991) and incorporated by reference herein, encapsulation of the conjugate within a proteoliposome as described by Miller et al., J. Exp. Med. 176:1739-1744 (1992) and incorporated by reference herein, and encapsulation of the protein in lipid vesicles such as NOVASOME® lipid vesicles (Micro Vescular Systems, Inc., Nashua, NH) may also be useful.

The antibody compositions of the present invention will thus be useful for interfering with or inhibiting binding of Candida yeast to host cells at the same time they inhibit the binding of SdrG with host cells, and thus have particular applicability in methods of preventing or treating infection from C. albicans sin addition to treating or preventing staphylococcal infection.

Accordingly, in accordance with the present invention, methods are provided for preventing or treating a fungal infection in addition to staph infection using the anti-SdrG antibodies in any of the various modes described above. These methods generally comprise administering an effective amount of an anti-SdrG antibody in accordance with the present invention as described above in amounts effective to treat or prevent the fungal infection. By effective amount is generally meant that level of use, such as of an antibody titer, that will be sufficient to either prevent adherence of the bacteria, to inhibit binding of staph bacteria to host cells and thus be useful in the treatment or prevention of a staph infection. As would be recognized by one of ordinary skill in this art, the level of antibody titer needed to be effective in treating or preventing Candida infection will vary depending on the nature and condition of the patient, and/or the severity of the pre-existing infection.

In addition to the use of antibodies of the present invention to treat or prevent fungal infection as described above, the present invention contemplates the use of these antibodies in a variety of ways, including the detection of HSP70 by the anti- SdrG antibodies described above. In accordance with the invention, one would obtain a sample suspected of being infected by one or more antigens recognized by the anti-SdrG antibodies of the invention, such as a sample taken from an individual, for example, from one's blood, saliva, tissues, bone, muscle, cartilage, or skin. The cells can then be lysed, and the DNA extracted, precipitated and amplified. Following isolation of the sample, diagnostic assays utilizing the antibodies of the present invention may be carried out to detect the presence of antigens recognized by the anti-SdrG. antibodies of the invention, and such assay techniques for determining such presence in a sample are well known to those skilled in the art and include methods such as radioimmunoasssay, Western blot analysis and ELISA assays. In general, in accordance with the invention, a method of diagnosing a patient for the presence of antigens recognized by anti-SdrG antibodies is thus contemplated. The term "antibodies" as used herein includes monoclonal, polyclonal, chimeric, single chain, bispecific, simianized, and humanized or primatized antibodies as well as Fab fragments, such as those fragments which maintain the binding specificity of the antibodies to the SdrG proteins, including the products of an Fab immunoglobulin expression library. Accordingly, the invention contemplates the use of single chains such as the variable heavy and light chains of the antibodies as will be set forth below. Generation of any of these types of antibodies or antibody fragments is well known to those skilled in the art as set forth above. In the present case, monoclonal antibodies to the SdrG N1 region have also been generated in accordance with the invention which have high affinity for the HSP70 protein from Candida albicans, and thus the antibodies of the invention can be used effectively in methods to protect against Candida infection in addition to staphylococcal infection at the same time.

When so desired for medical or research purposes, any of the above described antibodies may be labeled directly with a detectable label for identification and quantification of antigens recognized by the antibodies of the present inventiona. Labels for use in immunoassays are generally known to those skilled in the art and include enzymes, radioisotopes, and fluorescent, luminescent and chromogenic substances, including colored particles such as colloidal gold or latex beads. Suitable immunoassays include enzyme-linked immunosorbent assays (ELISA).

Alternatively, the antibody may be labeled indirectly by reaction with labeled substances that have an affinity for immunoglobulin. The antibody may be conjugated with a second substance and detected with a labeled third substance having an affinity for the second substance conjugated to the antibody. For example, the antibody may be conjugated to biotin and the antibody-biotin conjugate detected using labeled avidin or streptavidin. Similarly, the antibody may be conjugated to a hapten and the antibody-hapten conjugate detected using labeled anti-hapten antibody. These and other methods of labeling antibodies and assay conjugates are well known to those skilled in the art.

Antibodies to SdrG as described above may also be used in production facilities or laboratories to isolate additional quantities of the proteins, such as by affinity chromatography. For example, the antibodies of the invention may also be utilized to isolate additional amounts of the SdrG proteins or their active fragments.

The isolated antibodies of the present invention, or active fragments thereof, may also be utilized in the development of vaccines for passive immunization against staph/yeast infections. Further, when administered as pharmaceutical composition to a wound or used to coat medical devices or polymeric biomaterials in vitro and in vivo, the antibodies of the present invention, may be useful in those cases where there is a previous fungal infection because of the ability of this antibody to recognize the surface protein HSP70 from Candida albicans. In addition, the antibody may be modified as necessary so that, in certain instances, it is less immunogenic in the patient to whom it is administered. For example, if the patient is a human, the antibody may be "humanized" by transplanting the complimentarity determining regions (CDR's) of the hybridoma-derived antibody into a human monoclonal antibody as described, e.g., by Jones et al., Nature 321:522-525 (1986) or Tempest et al. Biotechnology 9:266-273 (1991) or "veneered" by changing the surface exposed murine framework residues in the immunoglobulin variable regions to mimic a homologous human framework counterpart as described, e.g., by Padlan, Molecular Imm. 28:489-498 (1991) and U.S. Pat. No. 6,797,492, all of these references incorporated herein by reference. Even further, when so desired, the monoclonal antibodies of the present invention may be administered in conjunction with a suitable antibiotic to further enhance the ability of the present compositions to fight bacterial infections.

Since fungal infections may also affect medical devices, implants and prosthetics, and the present invention can be utilized to protect these devices from fungal and staphylococcal infection as well, e.g., by coating these devices with the compositions of the present invention. Medical devices or polymeric biomaterials to be coated with the antibody compositions described herein include, but are not limited to, staples, sutures, replacement heart valves, cardiac assist devices, hard and soft contact lenses, intraocular lens implants (anterior chamber or posterior chamber), other implants such as corneal inlays, kerato-prostheses, vascular stents, epikeratophalia devices, glaucoma shunts, retinal staples, scleral buckles, dental prostheses, thyroplastic devices, laryngoplastic devices, vascular grafts, soft and hard tissue prostheses including, but not limited to, pumps, electrical devices including stimulators and recorders, auditory prostheses, pacemakers, artificial larynx, dental implants, mammary implants, other implants, cranio/facial tendons, artificial joints, tendons, ligaments, menisci, and disks, artificial bones, artificial organs including artificial pancreas, artificial hearts, artificial limbs, and heart valves; stents, wires, guide wires, intravenous and central venous catheters, laser and balloon angioplasty devices, vascular and heart devices (tubes, catheters, balloons), ventricular assists, blood dialysis components, blood oxygenators, urethral/ureteral/urinary devices (Foley catheters, stents, tubes and balloons), airway catheters (endotracheal and tracheostomy tubes and cuffs), enteral feeding tubes (including nasogastric, intragastric and jejunal tubes), wound drainage tubes, tubes used to drain the body cavities such as the pleural, peritoneal, cranial, and pericardial cavities, blood bags, test tubes, blood collection tubes, vacutainers, syringes, needles, pipettes, pipette tips, and blood tubing. It will be understood by those skilled in the art that the term "coated" or "coating", as used herein, means to apply the antibody or pharmaceutical composition derived therefrom, to a surface of the device, preferably an outer surface that would be exposed to streptococcal bacterial infection. The surface of the device need not be entirely covered by the protein, antibody or active fragment.

In a preferred embodiment, the antibodies may also be used as a passive vaccine which will be useful in providing suitable antibodies to treat or prevent a yeast infection in addition to a staphylococcal infection. As would be recognized by one skilled in this art, a vaccine may be packaged for administration in a number of suitable ways, such as by parenteral (i.e., intramuscular, intradermal or subcutaneous) administration or nasopharyngeal (i.e., intranasal) administration. One such mode is where the vaccine is injected intramuscularly, e.g., into the deltoid muscle, however, the particular mode of administration will depend on the nature of the bacterial infection to be dealt with and the condition of the patient. The vaccine is preferably combined with a pharmaceutically acceptable carrier to facilitate administration, and the carrier is usually water or a buffered saline, with or without a preservative. The vaccine may be lyophilized for resuspension at the time of administration or in solution.

The preferred dose for administration of an antibody composition in accordance with the present invention is that amount will be effective in preventing of treating a fungal infection in addition to a staphylococcal infection, and one would readily recognize that this amount will vary greatly depending on the nature of the infection and the condition of a patient. As indicated above, an "effective amount" of antibody or pharmaceutical agent to be used in accordance with the invention is intended to mean a nontoxic but sufficient amount of the agent, such that the desired prophylactic or therapeutic effect is produced. As will be pointed out below, the exact amount of the antibody or a particular agent that is required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, the particular carrier or adjuvant being used and its mode of administration, and the like. Accordingly, the "effective amount" of any particular antibody composition will vary based on the particular circumstances, and an appropriate effective amount may be determined in each case of application by one of ordinary skill in the art using only routine experimentation. The dose should be adjusted to suit the individual to whom the composition is administered and will vary with age, weight and metabolism of the individual. The compositions may additionally contain stabilizers or pharmaceutically acceptable preservatives, such as thimerosal (ethyl (2-mercaptobenzoate-S) mercury sodium salt) (Sigma Chemical Company, St. Louis, MO).

When used with suitable labels or other appropriate detectable biomolecule or chemicals, the monoclonal antibodies described herein are useful for purposes such as in vivo and in vitro diagnosis of infections or detection of antigens recognized by the antibodies of the invention. Laboratory research may also be facilitated through use of such antibodies. Various types of labels and methods of conjugating the labels to the antibodies of the invention are well known to those skilled in the art, such as the ones set forth below.

For example, the antibody can be conjugated (directly or via chelation) to a radiolabel such as, but not restricted to, ³²P, ³H, ¹⁴C, ³⁵S, ¹²⁵I, or ¹³¹I. Detection of a label can be by methods such as scintillation counting, gamma ray spectrometry or autoradiography. Bioluminescent labels, such as derivatives of firefly luciferin, are also useful. The bioluminescent substance is covalently bound to the protein by conventional methods, and the labeled protein is detected when an enzyme, such as luciferase, catalyzes a reaction with ATP causing the bioluminescent molecule to emit photons of light. Fluorogens may also be used to label proteins. Examples of fluorogens include fluorescein and derivatives, phycoerythrin, allo-phycocyanin, phycocyanin, rhodamine, and Texas Red. The fluorogens are generally detected by a fluorescence detector.

The location of a ligand in cells can be determined by labeling an antibody as described above and detecting the label in accordance with methods well known to those skilled in the art, such as immunofluorescence microscopy using procedures such as those described by Warren et al. (MoI. Cell. Biol., 7: 1326-1337, 1987).

As indicated above, the monoclonal antibodies of the present invention, or active portions or fragments thereof, are particularly useful for interfering with the initial physical interaction between a fungal pathogen responsible for infection and a mammalian host, such as the adhesion of yeast to mammalian extracellular matrix proteins, and this interference with physical interaction may be useful both in treating patients and in preventing or reducing infection on in-dwelling medical devices to make them safer for use. In another embodiment of the present invention, a kit which may be useful in isolating and identifying antigens recognized by the anti-SdrG antibodies of the invention. Such kits would generally comprise the antibodies of the present invention in a suitable form, such as lyophilized in a single vessel which then becomes active by addition of an aqueous sample suspected of containing the antigens recognized by those antibodies. Such a kit will typically include a suitable container for housing the antibodies in a suitable form along with a suitable immunodetection reagent which will allow identification of complexes binding to the SdrG antibodies of the invention. For example, the immunodetection reagent may comprise a suitable detectable signal or label, such as a biotin or enzyme that produces a detectable color, etc., which normally may be linked to the antibody or which can be utilized in other suitable ways so as to provide a detectable result when the antibody binds to the antigen.

In short, the antibodies of the present invention which bind to the SdrG protein or to the SdrG N1 region and yet also recognize the heat shock protein HSP70 from Candida albicans will be extremely useful in treating or preventing a fungal infection in addition to a staphylococcal infections in human and animal patients and in medical or other in-dwelling devices. Accordingly, the present invention relates to methods of identifying and isolating antibodies which can bind to SdrG or its N1 region and which can be used in methods of treatment of fungal infections and staph infections which involve opsonophagocytic killing of the microorganisms. Antibodies which are identified and/or isolated using the present method, such as the antibodies which can bind to the SdrG protein or its N1 subregion which also recognize the HSP70 protein from Candida and which can be used prevent or treat a fungal infection in addition to a staph infection, and antibodies recognizing the same epitopes as those recognized by the polyclonal or monoclonal antibodies described herein, are thus a part of the present invention

EXAMPLES

The following examples are provided which exemplify aspects of the preferred embodiments of the present invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. _t

EXAMPLE 1: Staphylococcal MSCRAMM^® Protein Antibodies Cross-React with Fungal Antigens

METHODS:

Induction of Hyphae. The growth conditions used to induce hyphal development have been described previously ¹⁴. C. albicans SC 5314 was cultured in yeast extract-peptone-dextrose (YPD) media at 30⁰C overnight with shaking at 250 rpm. The cells from the overnight culture were collected by centrifugation at 3566xg and cultured for 6 hrs at 30⁰C in Lee's medium ¹⁴. The cells were washed in sterile water by centrifugation at 3566xg for 10 minutes and incubated at 4⁰C for 3 days for "starvation". After the starvation period, cells were cultured in Lee's medium at 37⁰C for 6 hrs to induce hyphal formation. β-ME extraction of cell wall proteins. Performed as previously described ¹⁶. Hyphae were washed in 5OmM Tris-CL (pH 9.0) by centrifugation at 3566xg for 10 minutes and incubated in the same buffer containing 1% β-ME at 37⁰C for 60 minutes with gentle shaking (70 rpm). After the incubation, the supernatant was collected by centrifugation at 3566xg. The cell wall extract was dialyzed (3500 MWCo, Snake skin dialysis tubing, PIERCE, Rockford, IL) against ultrapure water for 24hrs at 2-8⁰C. The cell wall extract was concentrated using an Amicon Ultra-15 centrifugal filter unit (10,000 MWCo, Millipore, Billerica, MA). Affinity Purification of Anti-SdrG (pAb-SdrG) Antibodies from Veronate^®. rSdrG A-domain (SdrG-A) is a recombinant polypeptide corresponding to the fibrinogen-binding A domain of the SdrG protein (amino acids 50-597) from S. epidermidis strain K28 (Davis 2001). The recombinant protein was expressed with N-terminal hexahistidine sequences, and purified from Escherichia coli lysates by metal affinity chromatography on a chelating Sepharose Fast Flow resin (Amersham Biosciences, Piscataway, NJ). The protein was further purified by Q Sepharose HP chromatography (Amersham Biosciences, Piscataway, NJ). Approximately 25 mg of purified rSdrG-A was coupled to 5 ml_ columns of HiTrap NHS (N- hydroxysuccinimide) -activated HP resin (GE Healthcare, Piscataway, NJ) following the protocol supplied by the manufacturer. Veronate^® samples were applied to the columns and then washed with phosphate buffered saline (PBS). Adsorbed IgG was eluted with 0.1 M glycine, pH 2.7 and the IgG fractions were collected in 2 M Tris, pH 8.0. The purified antibodies were then formulated to 0.15 M glycine, 0.035 M NaCI, 0.015% Tween 80, pH 6.2 to match the standard Veronate^® formulation. 2D gel electrophoresis and Western blotting. 2D protein gel electrophoresis was carried out using a high-performance mini-gel system (ZOOM IPGRunner System. Invitrogen. Carlsbad, CA. USA). Aliquots of the cell wall extracts were diluted into 150 μl of Sample/Rehydration buffer and incubated on an immobilized pH gradient (IPG) strip overnight at room temperature. The proteins in the strip were separated by charge (pi) using a ZOOM IPG Runner System for 40 minutes at 200 V, 1 mA and 1 W, 30 minutes at 450 V, 30 minutes at 750 V, and 60 minutes at 2000V. After pi separation, the strips were incubated at room temperature for 15 minutes in Equilibration buffers I and II, and the proteins were separated by size in the second dimension using 4-12% Nupage Tris-Bis ZOOM gels. The proteins in the gel were stained (Bio-Safe Coomassie Stain from Bio-Rad, Hercules, CA or Imperial Stain from PIERCE, Rockford, IL) or, for western blotting, were transferred onto PVDF membranes in Nupage transfer buffer (Invitrogen, Carlsbad, CA) containing 20% methanol for 1 hour at 25 V (for one gel transfer) or 30 V (for two gel transfer). The membrane was blocked in PBS/ 0.05% Tween-20 (PBS-T), 5% dry milk for 1 hour at room temperature. After blocking, the membrane was incubated overnight at 4⁰C with primary antibodies diluted in PBS-T, 5% dry milk. The membrane was washed in PBS-T 3 times, 5 minutes each, and incubated for 1 hour at room temperature with mouse anti-human IgG HRP (MAH-HRP) or goat anti-mouse HRP (GAM-HRP) at 1 :5000 dilutions. The immunoreactive proteins were detected with Supersignal pico west chemiluminescent substrate (PIERCE, Rockford, IL). The filters were exposed to autoradiography film (BioMax XAR, Kodak, New Haven, CT) for various timepoints to visualize antibody binding.

Sequencing of Proteins Recovered from 2D Gels. To ascertain the identity of immunoreactive proteins in 2D western blotting experiments, Coomassie stained gels were overlayed with immunoblot films and the section corresponding to the region of immunoreactivity was excised. The sequence identity of the protein in the gel slice was then determined using the Nano-LC/MS/MS technique (Midwest Bio Services, Overland Park, KS).

In Vivo Model of Candidiasis. The in vivo model has been described previously¹⁷ and was performed in accordance with the appropriate institutional regulations in an AALAC accredited facility. Cultures of C. albicans strain SSY50-B for injection were grown as yeast overnight at 25°C in standard YPD media. Cells were harvested by centrifugation and washed three times in sterile pyrogen-free saline. After cells were counted with a hemacytometer, appropriate dilutions were made, and the required dosage of cells (2.5 x 10⁶ cells/animal) was injected in a final volume of 200 μl into the lateral tail veins of 6- to 8-week-old female BALB/c mice. Ten days after primary infection, doxycicline (DOX) was added to the drinking water (2 mg of DOX/ml in 5% sucrose) and pAb-SdrG antibodies were administered by intraperitoneal (IP) injection (10μg per injection). Control animals received the same volume of buffer IP. In a first experiment groups of 15 mice were used for each condition (pAb-SdrG-treatment and control) and mice received a single dose of antibody. In a second experiment 10 mice were used for each condition, and a second dose of antibody (same dose and route) was administered on day 13 after primary infection (3 days after addition of DOX and first antibody dose). Mice were followed for survival. The days on which the mice died were recorded, and moribund animals were euthanized and recorded as dying the following day.

Statistical analysis. Survival fractions were calculated using the Kaplan-Meier method and the resulting curves were compared for significance using the Mantel- Haenszal log-rank test. Analyses were performed using GraphPad Prism v4.00 software (GraphPad Software, Inc., San Diego, Calif.).

SdrG N1 protein production and purification. Using PCR, SdrG N1 representing AA 50-272 was amplified from S. epidermidis K28 genomic DNA (from sequences described above) and subcloned into the E. coli expression vector PQE-30 (Qiagen), which allows for the expression of a recombinant fusion protein containing six histidine residues. This vector was subsequently transformed into the E. coli strain ATCC 55151 , grown in a 15-liter fermentor to an optical density (OD₆₀o) of 0.7 and induced with 0.2 mM isopropyl-1-beta-D galactoside (IPTG) for 4 hours. The cells were harvested using an AG Technologies hollow-fiber assembly (pore size of 0.45 Dm) and the cell paste frozen at -80° C. Cells were lysed in 1X PBS (1OmL of buffer/1 g of cell paste) using 2 passes through the French Press @ 1100psi. Lysed cells were spun down at 17,000rpm for 30 minutes to remove cell debris. Supernatant was passed over a 5-mL HiTrap Chelating (Pharmacia) column charged with 0.1 M NiCI₂. After loading, the column was washed with 5 column volumes of 1OmM Tris, pH 8.0, 10OmM NaCI (Buffer A). Protein was eluted using a 0-100% gradient of 1OmM Tris, pH 8.0, 10OmM NaCI, 20OmM imidazole (Buffer B) over 30 column volumes. SdrGN1N2N3 or SdrGN2N3 eluted at -13% Buffer B (~26mM imidazole). Absorbance at 280nm was monitored. Fractions containing SdrGNI were dialyzed in 1x PBS.

The protein was then put through an endotoxin removal protocol. Buffers used during this protocol were made endotoxin free by passing over a 5-mL Mono-Q sepharose (Pharmacia) column. Protein was divided evenly between 4x 15mL tubes. The volume of each tube was brought to 9mL with Buffer A. 1 mL of 10% Triton X-114 was added to each tube and incubated with rotation for 1 hour at 4°C. Tubes were placed in a 37°C water bath to separate phases. Tubes were spun down at 2,000rpm for 10 minutes and the upper aqueous phase from each tube was collected and the detergent extraction repeated. Aqueous phases from the 2nd extraction were combined and passed over a 5-mL IDA chelating (Sigma) column, charged with 0.1 M NiCI₂ to remove remaining detergent. The column was washed with 9 column volumes of Buffer A before the protein was eluted with 3 column volumes of Buffer B. The eluant was passed over a 5-mL Detoxigel (Sigma) column and the flow-through collected and reapplied to the column. The flow-through from the second pass was collected and dialyzed in 1x PBS. The purified product was analyzed for concentration, purity and endotoxin level before administration into the mice. Generating monoclonal antibodies against Sdrg N1. E. coli expressed and purified SdrG N1 protein was used to generate a panel of murine monoclonai antibodies. Briefly, a group of Balb/C mice received a series of subcutaneous immunizations of 1-10 mg of protein in solution or mixed with adjuvant as described below in Table I:

Table I. Immunization Scheme

Conventional

Injection Day Amount (uq) Route Adjuvant

Primary 0 5 Subcutaneous FCA

Boost #1 14 1 Intraperitoneal RIBI

Boost #2 28 1 Intraperitoneal RIBI

Boost #3 42 1 Intraperitoneal RIBI

At the time of sacrifice or seven days after a boost (conventional) serum was collected and titered in ELISA assays against MSCRAMMs or on whole cells (S. epidermidis). Three days after the final boost, the spleen was removed, teased into a single cell suspension and the lymphocytes harvested. The lymphocytes were then fused to a P3X63Ag8.653 myeloma cell line (ATCC #CRL-1580). Cell fusion, subsequent plating and feeding were performed according to the Production of Monoclonal Antibodies protocol from Current Protocols in Immunology (Chapter 2, Unit 2.).

Screening of monoclonal antibodies by ELISA. lmmulon 2-HB high-binding 96- well microtiter plates (Dynex) were coated with 1 μg/well of rSdrG-N1 in 1X PBS, pH 7.4 and incubated for 2 hours at room temperature. All washing steps in ELISAs were performed three times with 1X PBS, 0.05% Tween-20 wash buffer. Plates were washed and blocked with a 1% BSA solution at room temperature for 1 hour before hybridoma supernatant samples were added to wells. Plates were incubated with samples and relevant controls such as media alone for one hour at room temperature, washed, and goat anti-mouse IgG-AP (Sigma) diluted 1 :5000 in 1X PBS, 0.05 % Tween-20, 0.1% BSA was used as a secondary reagent. Plates were developed by addition of 1 mg/ml solution of 4-nitrophenyl phosphate (pNPP) (Sigma), followed by incubation at 37° C for 30 minutes. Absorbance was read at 405 nm using a SpectraMax 190 Plate Reader (Molecular Devices Corp.). Antibody supernatants that had an OD₄₀₅ > 3 times above background (media alone, ~ 0.1 OD) were considered positive.

Screening of monoclonal antibodies cross-reactive with SdrG N1 and surface proteins of Candida albicans. A β-ME extract prepared from hyphal cells of Candida albicans strain S.c 5314 was diluted in 1XPBS to 0.5 mg/ml total protein and 100μl of the diluted extract was added to an AcroWell 96 NT Membrane-bottom plate (Pall Corporation, catalog #5022, lot A10427959). The plate was incubated for 30 minutes at room temperature, and then centrifuged at 3,000xg for 5 minutes. To block non-specific binding sites, 250μl of PBS-T + 5% dry milk was added, and the plate was incubated at room temperature for 1 hour. The blocking solution was removed by inverting and tapping the plate. 200μl of SdrG NImAb supernatants and control antibodies were added to appropriate wells and the plate was incubated for 2 hours at room temperature. After the incubation, the plate was washed 3 times in PBS-T for 5 minutes each, inverting and tapping to remove wash solution. Then the plate was washed an additional 3x in PBS-T by centrifugation at 3,000xg for 5 minutes each. 200μl of GAM-HRP (1 :2500 dilutions in PBS-T + 5% dry milk) was added to each well and the plate was incubated for 1 hour at room temperature. The plate was washed 3 times in PBS-T for 5 minutes each, inverting and tapping to remove wash solution. The plate was washed an additional 3x in PBS-T by centrifugation at 3,000xg for 5 minutes each. SuperSignal West Pico chemiluminescent substrate was added and the plate exposed to autoradiography film (BioMax XAR, Kodak, New Haven, CT) for various time points to visualize antibody binding.

DNA and Amino Acid sequences:

SdrG N1 DNA: (SEQ ID NO: 1)

GAGGAGAATACAGTACAAGACGTTAAAGATTCGAATATGGATGATGAATTATCA

GATAGCAATGATCAGTCCAGTAATGAAGAAAAGAATGATGTAATCAATAATAGTC

AGTCAATAAACACCGATGATGATAACCAAATAAAAAAAGAAGAAACGAATAGCAA

CGATGCCATAGAAAATCGCTCTAAAGATATAACACAGTCAACAACAAATGTAGAT

GAAAACGAAGCAACATTTTTACAAAAGACCCCTCAAGATAATACTCAGCTTAAAG

AAGAAGTGGTAAAAGAACCCTCATCAGTCGAATCCTCAAATTCATCAATGGATA

CTGCCCAACAACCATCTCATACAACAATAAATAGTGAAGCATCTATTCAAACAAG

TGATAATGAAGAAAATTCCCGCGTATCAGATTTTGCTAACTCTAAAATAATAGAG

AGTAACACTGAATCCAATAAAGAAGAGAATACTATAGAGCAACCTAACAAAGTAA GAGMGATTCAATAACAAGTCAACCGTCTAGCTATAAAAATATAGATGAAAAAAT TTCAAATCAAGATGAGTTATTAAATTTACCAATAAATGAATATGAAAATAAGGTTA GACCGTTATCTACAACATCTGCCCAACCATCGAGTAAGCGTGTAACCGTAAATC AATTAGCGGCA

SdrG N1 Protein: (SEQ ID NO: 2)

EENTVQDVKDSNMDDELSDSNDQSSNEEKNDVINNSQSINTDDDNQIKKEETNSN DAIENRSKDITQSTTNVDENEATFLQKTPQDNTQLKEEWKEPSSVESSNSSMDTA QQPSHTTINSEASIQTSDNEENSRVSDFANSKIIESNTESNKEENTIEQPNKVREDSI TSQPSSYKNIDEKISNQDELLNLPINEYENKVRPLSTTSAQPSSKRVTVNQLAA

SSA4 Sequence (HSP70): (SEQ ID NO: 3)

MSKAVGIDLGTTYSCVAHFANDRVEIIANDQGNRTTPSFVAFTDTERLIGDAAKNQA

AMNPANTVFDAKRLIGRKFDDPEVINDAKHFPFKVIDKAGKPVIQVEYKGETKTFSP

EEISSMVLTKMKEIAEGYLGSTVKDAWTVPAYFNDSQRQATKDAGTIAGLNVLRIIN

EPTAAAIAYGLDKKGSRGEHNVLIFDLGGGTFDVSLLAIDEGIFEVKATAGDTHLGG

EDFDNRLVNFFIQEFKRKNKKDISTNQRALRRLRTACERAKRTLSSSAQTSIEIDSLY

EGIDFYTSITRARFEELCADLFRSTLDPVGKVLADAKIDKSQVEEIVLVGGSTRIPKIQ

KLVSDFFNGKELNKSINPDEAVAYGAAVQAAILTGDTSSKTQDILLLDVAPLSLGIET

AGGIMTKLIPRNSTIPTKKSETFSTYADNQPGVLIQVFEGERAKTKDNNLLGKFELSG

IPPAPRGVPQIEVTFDIDANGILNVSALEKGTGKTQKITITNDKGRLSKEEIDKMVSEA

EKFKEEDEKEAARVQAKNQLESYAYSLKNTINDGEMKDKIGADDKEKLTKAIDETIS

WLDASQAASTEEYEDKRKELESVANPIISGAYGAAGGAPGGAGGFPGAGGFPGGA

PGAGGPGGATGGESSGPTVEEVD

SSA1 Sequence (HSP70): (SEQ ID NO: 4)

MSKAVGIDLGTTYSCVAHFANDRVEIIANDQGNRTTPSFVAFTDTERLIGDAAKNQA

AMNPANTVFDAKRLIGRKFDDHEVQGDIKHFPFKWDKASKPMIQVEYKGETKTFS

PEEISSMILGKMKETAEGFLGTTVKDAWTVPAYFNDSQRQATKDAGTIAGLNVMRII

NEPTAAAIAYGLDKKSEAEKNVLIFDLGGGTFDVSLLSIEDGIFEVKATAGDTHLGGE

DFDNRLVNFFIQEFKRKNKKDISTNQRALRRLRTACERAKRTLSSSAQTSIEIDSLYE

GIDFYTSITRARFEELCADLFRSTLEPVDKVLSDAKIDKSKVDEIVLVGGSTRIPKVQK

LVSDYFNGKEPNRSINPDEAVAYGAAVQAAILSGDTSSKTQDLLLLDVAPLSLGIETA

GGIMTKLIPRNSTIPTKKSETFSTYADNQPGVLIQVFEGERAQTKDNNLLGKFELSGI

PPAPRGVPQIEVTFDIDANGILNVSALEKGTGKTQKITITNDKGRLSKEEIEKMVSEA

EKFKEEDEKEASRVQAKNQLESYAYSLKNTLGEEQFKSKLDASEIEEVTKAADETIA

WLDSNQTATQEEFADQQKELESKANPIMTKAYQAGATPSGAAGAAPGGFPGGAA

PEPSNDGPTVEEVD

RESULTS: pAb-SdrG recognition of C. albicans HSP70 is unaffected by competition with

Bovine HSP70 or Histatin 5

The HSP70 family of proteins is conserved across species and their sequences are highly homologous. To determine if pAb-SdrG is specific for the C. albicans form of ΗSP70, bovine HSP70 was used as a competitor in immunoblotting experiments. As illustrated in Figure 1 , immunoblotting with pAb-SdrG in the presence of bovine derived HSP70 had no effect on the recognition of the candidal HSP70 while the control competitor, rSdrG-N1 , was able to completely abrogate the immunoreactivity. This result suggested that the pAb-SdrG reactivity to HSP70 is species specific.

Histatin 5 is an anti-candidal human salivary peptide that has been shown to bind specifically to the Candida HSP70 ¹⁵. When Histatin 5 was used as a competitor in immunoblotting experiments, there was little to no effect on the recognition of HSP70 by pAb-SdrG (Figure 1) suggesting that the epitopes recognized by pAb-SdrG are not masked by Histatin 5 binding. pAb-SdrG recognition is specific to the SSA4 form of HSP70. C. albicans, expresses two forms of HSP70 encoded by the genes SSA1 and SSA4. To identify which of these HSP70 proteins are recognized by pAb-SdrG, a hyphal cell wall extract was prepared from strain SC5314 and separated on a narrow pH gradient (pH 4-7) in the first dimension of a 2D western blot. This allowed for the separation of the SSA1 and SSA4 gene products based on differences in pi. As shown in Figure 2A, two proteins were visible by Coomassie blue staining, migrating in the region of the gel where HSP70 would be expected. Immunoblotting with an anti-HSP70 monoclonal antibody confirmed the identity of the two spots as HSP70 (Figure 2B). Immunoblotting with pAb-SdrG clearly showed that only one of the HSP70 species, was recognized by the polyclonal antibody (Figure 2C). The reactive spot on the western film was overlayed with the Coomassie blue stained gel. The appropriate protein was excised from the gel and its sequence analyzed. The sequence analysis confirmed that the protein recognized by pAb-SdrG was the SSA4 gene product.

Sequence comparisons between SdrG-N1 and Candida HSP70. The data presented suggests that cross-reactive epitopes in the gene product of SSA4 may be recognized by antibodies against the N1 region of the SdrG antigen. To determine if these two molecules share similarities in protein sequence, a CLUSTAL W multiple sequence alignment was performed. The results of this analysis are shown in Figure 3. Two short spans of the SdrG N1 sequence (aa 199- 228 and aa 229-256) were found to have limited similarity to two regions of the SSA4 protein sequence ( aa 521-550 and aa 573-600). In as much as pAb-SdrG does not recognize SSAi , IT the regions of sequence similarity found between SSA4 and SdrG N1 represent cross-reactive epitopes then one would predict that these same regions would be divergent between SSA4 and SSA1. A similar analysis was therefore performed comparing the SSA1 and SSA4 sequence. In fact, the SSA4 and SSA1 sequences are identical in the aa 521-550 region with the exception of one amino acid change at position 533 (Figure 3B). A greater degree of divergence was observed between SSA1 and SSA4 in the region spanning aa 573-600, where 11 out of 28 amino acids differ (Figure 3B). These findings indicate potential candidate sequences for cross-reactive epitopes between SSA4 and SdrG Nl In Vivo Model of Systemic Candidiasis.

A model of Candida infection was utilized that takes advantage of an engineered C. albicans strain to control the timing of morphogenesis in vivo. In the SSY 50-B strain, it is possible to externally modulate filamentation and virulence both in vitro and in vivo by the presence or absence of DOX. It has been demonstrated previously that mice can survive a primary infection with this strain in the absence of DOX (when kept in the yeast morphology), but cells disseminate to the tissues thus mimicking a "carrier" or "commensal" state. A lethal infection can be subsequently triggered at different times post-infection by adding DOX to the drinking water (Saville 2003). A murine virulence study was performed with this strain to determine if affinity purified SdrG antibodies from Veronate^® are protective. The SSY 50-B strain of C. albicans was injected IV into mice in the absence of DOX. Ten days after challenge, DOX was added to the drinking water and pAb-SdrG antibodies were administered. In a first experiment (Figure 4A), 15 mice received a single infusion of either pAb-SdrG antibodies (10μg per mouse) or buffer (control). In the second experiment ten mice received two infusions of pAb-SdrG antibodies (10μg per injection) or buffer (control) on Day 10 and Day 13 (Figure 4B). Survival was monitored for 25-30 days. Passive administration of affinity purified SdrG antibodies from Veronate^® provided protection against C. albicans infection compared to control in both studies. The difference in survival observed after two administrations of anti- SdrG was found to be significant (P=0.0488). Screening of monoclonal antibodies against SdrG N1 and cross-reactive with surface proteins of Candida albicans.

To determine if the polyclonal cross-reactivity observed between rSdrG N1 and Candida HSP70 can be recapitulated using monoclonal antibodies, rSdrG N1 was used to immunize mice and the resulting monoclonal hybridoma clones were identified by strong ELISA activity against the SdrG N1 immunogen, then screened for cross-reactivity to Candida antigens (summarized in Table 2). A total of 107 supernatants containing anti-SdrG N1 antibodies were screened by immuno-dot blot using a filter immobilized β-ME extract prepared from S. C 5314 hyphal cell cultures. 14 of the supernatants interacted with the β-ME extract with signal intensities as high as that of the control anti-HSP70 antibody (Figure 4, indicated by numbers in red). 29 of the supernatants exhibited medium or low reactivity with the Candida extract (numbers in light blue, Figure 5). The remaining SdrG N1 ELISA positive antibodies showed little reactivity with the Candida extract (numbers in black, Figure 5).

Table 2. SdrG N1 Hybridoma Screening Summary

CONCLUSIONS:

Specificity testing has demonstrated that the cross-reactivity of anti-SdrG antibodies with certain candidal antigens can be competitively inhibited with recombinant forms of the SdrG antigen. One interesting example of this is the elimination of reactivity to Candida HSP70 in the presence of the SdrG N1 sub-domain. The reactivity of anti- SdrG to HSP70 was not inhibited by the presence of bovine HSP70 or Histatin, a known ligand of Candida HSP70, suggesting that the cross-reactive epitopes are specific to the Candida form of HSP70 and do not correspond to the Histatin binding region of the molecule. Further refinement of the 2-D electrophoresis method demonstrated that the HSP70 recognized by SdrG antibodies is encoded by the SSA4 gene. Protein sequence comparisons between the two HSP70 genes in Candida, SSA1 and SSA4, identified two regions of divergence that may represent candidates for anti-SdrG cross-reactive epitopes. Anti-SdrG antibodies, affinity purified from Veronate^®, were evaluated for efficacy in a murine model of candidemia. Significant protection was observed when more than one dose of antibody was administered. The current data supports the hypothesis that antibodies against the staphylococcal antigen, SdrG, cross-react with cell wall antigens expressed by the hyphal form of C. albicans, and these antibodies are protective in an in vivo model of invasive candidemia. Monoclonal antibodies reactive against rSdrG N1 were screened for reactivity to Candida cell wall antigens and 43 of 107 hybridomas were found to have some level of cross-reactivity. Interestingly, not all S. epidermidis cell surface derived SdrG N1 reactive monoclonal antibodies cross reacted with cell wall antigens expressed by the hyphal form of C. albicans, suggesting a high level of specific cross reactivity between the surface antigens.

The current data supports the hypothesis that antibodies against the staphylococcal antigen, SdrG, cross-react with cell wall antigens expressed by the hyphal form of C. albicans and more specifically, antibodies which recognize the N1 sub-domain of the staphylococcal SdrG protein also recognize cross-reactive epitopes presented in the native form of the Candida SSA4 gene product.

THE FOLLOWING REFERENCES ARE INCORPORATED HEREIN AS IF SET FORTH IN THEIR ENTIRETY ABOVE:

1. Siber GR, Leszcynski J, Pena-Cruz V, et al. Protective activity of a human respiratory syncytial virus immune globulin prepared from donors screened by microneutralization assay. J Infect Dis 1992; 165:456-63.

2. Reduction of respiratory syncytial virus hospitalization among premature infants and infants with bronchopulmonary dysplasia using respiratory syncytial virus immune globulin prophylaxis. The PREVENT Study Group. Pediatrics 1997; 99:93-9. 3 Groothuis JR, Gutierrez KM, Lauer BA. Respiratory syncytial virus infection in children with bronchopulmonary dysplasia. Pediatrics 1988; 82:199-203.

4. Green M, Brayer AF, Schenkman KA, WaId ER. Duration of hospitalization in previously well infants with respiratory syncytial virus infection. Pediatr Infect Dis J 1989; 8:601-5.

5. Mclver J, Grady G. Immunoglobulin Preparations. In: Churchill WH, Kurtz SR, eds. Transfusion Medicine. Boston: Blackwell, 1988.

6. Schneider L, Geha R. Outbreak of Hepatitis C Associated with Intravenous Immunoglobulin Administration - United States October 1993 - June 1994. MMWR Morb Mortal WkIy Rep 1994; 43:505-509.

7. Edwards CA, Piet MPJ, Chin S, al. e. Tri (n Butyl) Phosphate Detergent Treatment of Licensed Therapeutic and Experimental Blood Derivatives. Vox Sang 1987; 52:53-59.

8. Groothuis JR, Simoes EA, Lehr MV, et al. Safety and bioequivalency of three formulations of respiratory syncytial virus-enriched immunoglobulin. Antimicrob Agents Chemother 1995; 39:668-71.

9. Groothuis JR, Simoes EA, Levin MJ, et al. Prophylactic administration of respiratory syncytial virus immune globulin to high-risk infants and young children. The Respiratory Syncytial Virus Immune Globulin Study Group. N Engl J Med 1993; 329:1524-30.

10. Ellenberg SS, Epstein JS, Fratantoni JC, Scott D, Zoon KC. A trial of RSV immune globulin in infants and young children: the FDA's view. N Engl J Med 1994; 331:203-5.

11. CDC. Staphylococcus aureus resistant to vancomycin— United States, 2002. MMWR Morb Mortal WkIy Rep 2002; 51 :565-7.

12. Garrett DO, Jochimsen E, Murfitt K, et al. The emergence of decreased susceptibility to vancomycin in Staphylococcus epidermidis. Infect Control Hosp Epidemiol 1999; 20:167-70.

13. Davis, S. L., S. Gurusiddappa, K. W. McCrea, S. Perkins, and M. Hook. 2001. SdrG, a fibrinogen-binding bacterial adhesin of the microbial surface components recognizing adhesive matrix molecules subfamily from Staphylococcus epidermidis, targets the thrombin cleavage site in the beta chain. J Biol Chem 276:27799-805.

14. Lee, K.L, H. R. Buckley and CC. Campbell. 1975. An amino acid liquid synthetic medium for the development of mycelial and yeast forms of Candida albicans. Sabouraudia 13(2): 148-153.

15. Li, X. S., M. S. Reddy, D. Baev, and M. Edgerton. 2003. Candida albicans Ssa1/2p is the cell envelope binding protein for human salivary Histatin 5. J Biol. Chem. 278 (31): 28553-28561. 16. Poulain, D., C. Slomianny, T. Jouault, J. M. Gomez, and P. A. Trinel. 2002. Contribution of Phospholipomannan to the Surface Expression of β-1 ,2- Oligomannosides in Candida albicans and Its Presence in Cell Wall Extracts. Infect Immun. 70: 4323-4328.

17. Saville, S. P., A. L. Lazzell, C. Monteagudo and J. L. Lopez-Ribot. 2003. Engineered Control of Cell Morphology In Vivo Reveals Distinct Roles for Yeast and Filamentous Forms of Candida albicans during infection. Eukaryot. Cell 2 (5): 1053-1060.

EXAMPLE 2: Polyclonal Antibody Against SdrG Found in Veronate Recognizes HSP70 (Ssa1) of Candida albicans

OVERVIEW

In a recently completed phase Il clinical trial, prophylactic treatment of very low birth weight (VLBW) infants with Veronate^® reduced the relative risk of Candida infection by 67%. To investigate the possible mechanisms of anti-Candida effects of Veronate^®, we initiated proteomic analysis of Candida surface proteins recognized by Veronate^®. HSP70, among other surface antigens from Candida albicans strain S. c 5314 was recognized specifically by Veronate^®. Further studies have shown that antibodies which specifically recognize the N1 domain of SdrG are cross-reactive with HSP70. We also demonstrate that SdrG affinity-purified antibodies from Veronate^® provide significant protection against Candida albicans infection.

The example relates to the use of an immunoglobulin product obtained from purified donor plasma containing high antibody titers to MSCRAMM proteins CIfA and SdrG in the prevention and treatment of infections from Candida yeast, including Candida species late-onset sepsis and other Candida systemic infections. In particular, the present invention resulted from the investigation of the possible mechanisms of anti-Candida effects of Veronate^®, and a proteomic analysis of Candida surface proteins recognized by Veronate^® showed unexpectedly that HSP70, among other surface antigens from Candida albicans strain S.c 5314 was recognized specifically by Veronate^®. Further studies have shown that antibodies which specifically recognize the N1 domain of SdrG are cross-reactive with HSP70, and it has been confirmed that SdrG affinity-purified antibodies from Veronate^® provide significant protection against Candida albicans infection. In the example, a donor immunoglobulin composition having high titers of antibodies to the proteins CIfA from S. aureus and SdrG from S. epidermidis was administered to a patient in need of treatment for or protection against an infection caused by yeast of the species Candida such as Candidiasis, and can be effective in inhibiting the yeast and enabling the effective treatment or prevention of the Candida infection. In addition, an immunoglobulin composition of the invention can be prepared which includes a high titer to antigen from a Candida species yeast such as Candida albicans, and this composition can also be used effectively to inhibit Candidial yeast and thus treat or prevent a Candidiai infection. Further, because of its ability to recognize surface proteins in Candida, the immunoglobulin compositions of the present invention will also be useful in identifying and isolating surface proteins from Candida yeast and in diagnosing Candida infections. The present compositions and methods will thus be particularly effective in treating or preventing late-onset sepsis in low birth weight neonates.

In particular, the example relates to the discovery that HSP70, among other surface antigens from Candida albicans strain S.c 5314 was recognized specifically by Veronate^®. Further studies have shown that antibodies which specifically recognize the N1 domain of SdrG are cross-reactive with HSP70, and it has been confirmed that SdrG affinity-purified antibodies from Veronate^® provide significant protection against Candida albicans infection.

These and other objects of the present invention are obtained through the compositions and methods as set forth in the detailed description of the invention provided hereinbelow.

DESCRIPTION OF THE INVENTION AND EXEMPLARY EMBODIMENTS MATERIALS & METHODS:

1). Cell cultures and induction of hyphae. Candida albicans S.c 5314 cells were cultured in YPD at 30⁰C overnight with shaking at 250 rpm. The cells from the overnight culture were collected by centrifugation at 3566 x g and cultured for 6 hrs at 30⁰C in Lee's medium (Lee K. L et a/. 1975. Sabouraudia 10:148-153). The cells were washed in sterile water by centrifugation at 3566 x g for 10 minutes and incubated at 4⁰C for 3 days for "starvation". After the starvation, cells were cultured in Lee's medium at 37⁰C for 6 hrs to induce hyphal formation. Z). SDS/DTT and β-ME extraction of proteins from the hyphal cells: To prepare SDS/DTT extracts, hyphal stage cells of Candida albicans S.c 5314 were washed in sterile water by centrifugation at 3566 x g for 10 minutes, and boiled for 10 minutes in 2% SDS, 1OmM DTT. The supernatant was collected and buffer exchanged using Centricon (10K MWCo) with 1OmM Tris-CI (pH 7.4) by centrifugation at 3566 g. To prepare β-ME extracts, hyphal cells were washed in 5OmM Tris-CL (pH 9.0) by centrifugation at 3566 x g for 10 minutes and incubated in the same buffer containing 1% β-ME at 37⁰C for 40 minutes with gentle shaking (70 rpm). After the incubation, the supernatant was collected by centrifugation at 3566 x g. The cell wall extract was dialyzed for 24 hours against 4⁰C diH₂O using a Snake skin dialysis bag (3500 MWCo, PIERCE). To concentrate the protein, the cell wall extract was centrifuged in an Amicon Ultra-15 concentrator (10,000 MWCo₁ Millipore) at 3566 x g for 15 minutes.

3). Absorption of Veronate^®/IGIV by yeast cells. Candida albicans strain Sc5314 cells were cultured in 10ml of YPD at 30⁰C overnight and washed twice in sterile water by centrifugation for 10 minutes at 3566 x g. The cell pellet was mixed with 10 ml of Veronate^® or IGIV and incubated at 4⁰C for 4 hrs. The yeast cells were removed by centrifugation at 3566 g 4⁰C for 10min and the supernatant was collected.

4). 2-D gel electrophoresis and Western blotting. 100 μg of cell wall extracts was re- suspended in 150 μl of the Sample/Rehydration buffer (Invitrogen) and was incubated with on an IPG strip (pH 3-10 NL, Invitrogen) overnight at room temperature. The proteins in the strip were separated using the ZOOM IPGRunner System (Invitrogen) for 15 minutes each at 200 V, 450 V and 750 V, followed by 30 minutes at 2000V. After the separation, the strips were incubated at room temperature for 15 minutes each in Equilibration buffer I and II (Invitrogen), followed by SDS/PAGE in a 4-12% Nupage Tris-Bis ZOOM gel (Invitrogen). The proteins in the gel were either stained with Bio-safe Coomassie Stain (Biorad) or transferred onto PVDF membrane in Nupage transfer buffer (Invitrogen) containing 20% methanol for 1 hour at 20 V. The membrane was incubated in PBS plus 0.05% Tween-20 (PBS-T) and 5% dry milk for 1 hour at room temperature. The membranes were incubated overnight at 4⁰C with PBS-T plus 5% dry milk and Vefbnate^ or IG^'fV at ^"T.2^'5 mg/ml, or affinity purified anti-SdrG polyclonal antibodies at 20 μg/ml. The membrane was washed in PBS-T 3 times, 5 minutes each, and incubated for 1 hour at room temperature with mouse anti-human IgG conjugated to HRP (Southern Biotech) at 1:5000 dilution. The immunoreactivity was detected by chemiluminescence using Supersignal pico west substrate (PIERCE). The filters were exposed to autoradiography film (BioMax XAR, Kodak) for various timepoints to visualize antibody reactivity.

5). Sequencing of proteins recognized by Veronate^®.

In the 2-D electrophoresis, two gels are prepared; one gel is used for the western blot, and the other is stained with Bio-Safe Coomassie stain (Biorad). The autoradiography film from the western was overlaid with the stained gel, and the proteins recognized by Veronate^® in the 2-D western was excised from the stained gel. The protein in the gel fragment was then identified using a nano-LC/MS/MS technique (Midwest Bio Services). 6) Affinity purification of anti-SdrG polyclonal antibodies.

Approximately 25 mg of purified recombinant MSCRAMM protein SdrG-A was coupled to 5 ml_ columns of HiTrap NHS (N-hydroxysuccinimide)-activated HP resin (Amersham Biosciences, Piscataway, NJ) following the protocol supplied by the manufacturer. Veronate^® was applied to the column and then washed with phosphate buffered saline (PBS). Adsorbed IgG was eluted with 0.1 M glycine, pH 2.7 and the IgG fractions were collected in 2 M Tris, pH 8.0. Antigen-specific IgG was formulated to 50-60 mg/mL protein in 0.15 M glycine, 0.1 M NaCI, 0.02% Tween 80, pH 6.2 and sterile filtered using a 0.2 micron filter.

RESULTS:

1). Veronate^® specifically recognizes C. albicans HSP70.

SDS/DTT extracts from hyphal cells of Candida albicans S. C 5314 were separated by 2-D electrophoresis and transferred onto PVDF membrane. The membranes were probed with pre-absorbed Veronate^® or IGIV as described in the materials and methods. The results showed that one of the proteins recognized by Veronate^® was identified as HSP70 after protein sequencing. The HSP70 appeared to be recognized by Veronate^®, but not by a commercial lot of IGIV. 82

^AMMffgr '"pβrtϋM WhU- SdrG antibodies interact specifically with C. albicans ΗSP70.

A β-ME extract of the hyphal cells of C. albicans S.C 5314 was separated by 2-D electrophoresis and transferred onto PVDF membrane. The membranes were probed with anti-SdrG antibodies affinity purified from Veronate^® as described in nrateflate and- methods. The antf-SdrG- antibodies recognized several proteins rrr the 2-D western, one of which was identified as HSP70 by mass spectrometry (Figure 7). Recombinant protein fragments corresponding to the N1 domain of SdrG or the U3 domain- of SdrG- were- added- as- eoropetttof s- to- the 2-& Western. Onty the addition of the N1 domain of SdrG prevented the interaction of the antibodies with HSP70 (Figure 7). Interestingly, the addition of HSP70 isolated from Bovine brain also failed to compete for anti-SdrG binding to Candida HSP70 (Figure 7), suggesting that the anti-SdrG antibodies specifically recognized the Candida HSP70, not the Bovine HSP70.

3). Candida HSP70 is recognized specifically by antibodies against the N1 domain of SdrG.

A β-ME extract from the hyphal cells of C. albicans S.C 5314 was separated by 2-D electrophoresis and transferred onto PVDF membrane. The membranes were probed with the affinity purified anti-SdrG antibodies in the presence of recombinant proteins corresponding to the N1, N2 or N3 domains of SdrG (Figure 8). The results indicated that only the N1 recombinant protein fragment was able to compete for the binding sites on C albicans HSP70. Within the N1 region of SdrG, there is a protein sequence (amino acid 57 to 123) that is approximately 72% similar to a region within the C-terminal part of C. albicans HSP70 (amino acid 546 to 612), as shown below in SEQ ID NOS 5 and 6, respectively:

HSP70 ( SSAl ) i SLKMTLGEEQFKSKLDASEIEEVTKAADETIAWLDSMQTATQEBFADQQKELESKAHPIMTKAΪQAG sdrG-Ni : DVKDSNMDDELSDSNDQSSNEEKNDVINΠSQSINTDDDNQIKKEETMSHDAIENPSKDITQSTTΓIVD

4). Histatin 5 does not inhibit pAb-SdrG recognition of C. albicans HSP70.

Histatin 5 is a small peptide found in human saliva that is a ligand for C. albicans HSP70 and has potent anti-fungal activity (Xuewei et al. 2003 JBC 278(31 ):28553-61). To determine if pAb-SdrG recognizes the binding site for Histatin 5, a competition experiment was performed. β-ME extracts from hyphal cells of C. albicans Sc 5314 were separated by 2-D electrophoresis as described in the materials and methods. The proteins were transferred onto PVDF and probed with pAb-SdrG at 20μg/ml in the presence or absence of recombinant SdrG N1 domain (20μg/ml) or Histatin 5 (2μg/ml). The result showed that addition of recombinant SdrG N1 domain completely eliminated pAb-SdrG recognition of HSP70 while the addition of Histatin 5 had no effect.

5). Using a murine model of candidiasis, passive administration of affinity purified SdrG antibodies from Veronate (pAb-SdrG) provided statistically significant protection against C. albicans compared to control as shown in Figure 10

Accordingly, the various modes of the anti-SdrG antibodies of the invention were shown to recognize HSP70 of Candida albicans and can thus be useful in treating or preventing infections from Candida albicans in addition to infections caused by Staphylococcus epidermidis.

Claims

What Is Claimed Is:

1. An isolated antibody which recognizes both the SdrG protein from S. epidermidis and the Hsp70 protein from Candida albicans.

2. The antibody according to Claim 1 wherein the antibody is raised against the N1 subregion of the SdrG protein.

3. The antibody according to piaim 1 wherein the antibody recognizes the N1 domain of SdrG.

4. The antibody according to Claim 1 wherein the antibody is raised against the A domain of the SdrG protein.

5. The antibody according to Claim 1 wherein the antibody recognizes the A domain of the SdrG protein

6. The antibody according to Claim 1 , wherein said antibody inhibits binding of Candida albicans to an extracellular matrix protein of the host.

7. The antibody according to Claim 1 , wherein said antibody inhibits binding of the HSP70 protein of Candida albicans to an extracellular matrix protein of the host.

8. The antibody according to Claim 1 , wherein said antibody is suitable for parenteral, oral, intranasal, subcutaneous, aerosolized or intravenous administration in a human or animal.

9. The antibody according to Claim 1 wherein said antibody is a monoclonal antibody.

10. The antibody according to Claim 9 wherein said monoclonal antibody is of a type selected from the group consisting of murine, chimeric, humanized and human monoclonal antibodies.

11. The antibody according to Claim 9 wherein the antibody is a single chain monoclonal antibody.

12. The antibody according to Claim 1 wherein said antibody is a polyclonal antibody.

13. The antibody according to Claim 1 that is raised against a peptide having the amino acid sequence of SEQ ID NO:2.

14. The antibody according to Claim 1 that is raised against a peptide encoded by the nucleic acid sequence of SEQ ID NO:1.

15. The antibody according to Claim 1 that is raised against a peptide having the amino acid sequence from amino acid 50-272 from the SdrG protein from Staphylococcus epidermidis.

16. The antibody according to Claim 1 that recognizes the amino acid sequence of SEQ ID NO:3.

17. The antibody according to Claim 1 wherein the antibody is from a donor immunoglobulin having a high titer to the SdrG antibody.

18. Isolated antisera containing an antibody according to Claim 1.

19. A diagnostic kit comprising the antibody according to Claim 1 and means for detecting binding by that antibody.

20. The diagnostic kit according to Claim 19 wherein said means for detecting binding comprises a detectable label that is linked to said antibody.

21. A pharmaceutical composition comprising an effective amount of the antibody of Claim 1 and a pharmaceutically acceptable vehicle, carrier or excipient.

Zl. The pharmaceutical composition according to Claim 21 further comprising a physiologically acceptable antibiotic.

23. A method of treating or preventing an infection of C. albicans comprising administering to a human or animal patient an effective amount of the antibody according to Claim 1.

24. A method of generating making a monoclonal antibody that recognizes both the HSP70 protein from C. albicans and the SdrG protein from S. epidermidis comprising administering to a host animal an immunogenic amount of the N1 domain of the SdrG protein from Staphylococcus epidermidis, forming a hybridoma from antibodies generated by said N1 domain, and isolating a monoclonal antibody from said hybridoma that recognizes both the HSP70 protein from C. albicans and the SdrG protein from S. epidermidis.

25. A monoclonal antibody that recognizes the N1 domain of the SdrG protein of S. epidermidis and that recognizes the HSP70 protein of C. albicans.