Impact of Valency of a Glycoprotein B Specific Monoclonal Antibody on Neutralization of. Herpes Simplex Virus

JVI Accepts, published online ahead of print on 1 December 2010 J. Virol. doi:10.1128/JVI.01924-10 Copyright © 2010, American Society for Microbiology...
4 downloads 3 Views 994KB Size
JVI Accepts, published online ahead of print on 1 December 2010 J. Virol. doi:10.1128/JVI.01924-10 Copyright © 2010, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

1

JOURNAL OF VIROLOGY

2

Impact of Valency of a Glycoprotein B Specific Monoclonal Antibody on Neutralization of

3

Herpes Simplex Virus 1§

1

2

2§§

4

Adalbert Krawczyk,

5

Robert Schwarzenbacher,

6

Roggendorf, 4 and Michaela A.E. Arndt 1*

7

National Center for Tumor Diseases (NCT), University of Heidelberg, 69120 Heidelberg,

8

Germany,1 and Institute of Virology, University Medical Center Bonn, 53105 Bonn, Germany,2

9

and Department of Molecular Biology, University of Salzburg, Austria,3 and Institute of

Jürgen Krauss, 3

Anna M. Eis-Hübinger,

Ulf Dittmer,

4

Karl E. Schneweis,

3

Martin P. Däumer, Dirk Jäger,

1

Michael

10

Virology, University Hospital Essen, University of Duisburg-Essen, 45147 Essen, Germany,4.

11

Running Title: Neutralizing antibody crosslinks HSV gB

12 13

*Corresponding author:

14

Michaela Arndt

15

National Center for Tumor Diseases (NCT)

16

Department of Medical Oncology

17

University of Heidelberg

18

D-69120 Heidelberg

19

Tel.: 49-6221-56-37798

20

Fax.: 49-6221-56-5373

21

E-Mail: [email protected]

22

§

23

45147 Essen, Germany

24

§§

Present address: Institute of Virology, University Hospital Essen, University of Duisburg-Essen,

Present address: Institute of Immunology and Genetics, 67655 Kaiserslautern, Germany -1-

1

ABSTRACT

2

Herpes simplex virus (HSV) glycoprotein B (gB) is an integral part of the multicomponent fusion

3

system required for virus entry and cell-cell fusion. Here we investigated the mechanism of viral

4

neutralization by the monoclonal antibody (mAb) 2c, which specifically recognizes gB of HSV-1

5

and HSV-2. Binding of mAb 2c to a type-common discontinuous epitope of gB resulted in highly

6

efficient neutralization of HSV at the post-binding/pre-fusion stage and completely abrogated the

7

viral cell-to-cell spread in vitro. Mapping of the antigenic site recognized by mAb 2c to the

8

recently solved crystal structure of the HSV-1 gB ectodomain revealed that its discontinuous

9

epitope is only partially accessible within the observed multidomain trimer conformation of gB,

10

likely representing its post-fusion conformation. To investigate how mAb 2c may interact with

11

gB during membrane fusion, we characterized the properties of monovalent (Fab, scFv) and

12

bivalent (IgG, F(ab’)2) derivatives of mAb 2c. Our data show that the neutralization capacity of

13

mAb 2c is dependent on crosslinkage of gB trimers. As a result, only bivalent derivatives of mAb

14

2c exhibited high neutralizinig activity in vitro. Notably, bivalent mAb 2c was not only capable

15

of preventing mucocutaneous disease in severe immunodeficient NOD/SCID mice upon vaginal

16

HSV-1 challenge but also protected animals even with neuronal HSV infection. We also report

17

for the first time that an anti-gB specific monoclonal antibody prevents HSV-1 induced

18

encephalitis entirely independent from complement activation, antibody-dependent cellular

19

cytotoxicity, and cellular immunity. This indicates potential for further development of mAb 2c

20

as an anti-HSV drug.

21 22

Abstract word count: 249

-2-

1

INTRODUCTION

2

Herpes simplex virus (HSV) is a neuroinvasive human pathogen that critically depends on

3

efficient infection of distinct target cells within a host. At the time of primary lytic infection,

4

HSV replicates in peripheral mucocutaneuos tissues and releases virions. A decisive

5

characteristic of HSV infections in animals and humans is the establishment of a life-long

6

latency. HSV spreads from infected epithelial cells to axons of sensory neurons innervating the

7

site of the primary infection, followed by retrograde transport to respective dorsal root ganglia

8

(12). Recurrent infections results from reactivation in neuronal cells, followed by virus

9

replication and anterograde transport to cells at peripheral sites innervated by respective neurons.

10

Transmission between cells without diffusion through the extracellular environment represents a

11

major route for HSV to spread between tissues and is thus a very efficient way for circumventing

12

immunological barriers of the humoral immune response. Regardless of the dissemination

13

pathway, however, fusion of the viral envelope with host membranes for delivery of the viral

14

genome across the cellular lipid bilayer is essential for viral replication. In contrast to most other

15

enveloped viruses, entry of herpesviruses into mammalian cells requires a multi-component

16

system and thus represents one of the most complex viral entry mechanisms being studied so far.

17

Among the 12 glycoproteins of the HSV envelope, glycoproteins gB, gD, and the gH/gL

18

heterodimer display essential functions for both entry of extracellular virions and cell-to-cell

19

spread. Binding of gD to one of its different cellular receptors, herpesvirus entry mediator

20

(HVEM), nectin 1, or a modified form of heparan sulphate, promotes a conformational change of

21

gD that subsequently triggers the fusogenic signal of the core fusion machinery, constituted in

22

glycoproteins gB and gH/gL (36, 65).

23

The presence of the aforementioned glycoproteins on both the virion and on infected cells can be

24

recognized by the immune system and elicits cellular and humoral immune responses. Recurrent -3-

1

HSV infections in the skin and mucosa appear mainly to be controlled by the host cellular

2

immune response, like T lymphocytes, macrophages, natural killer cells, and as demonstrated

3

recently by type I IFN-producing plasmacytoid dendritic cells (10, 13, 15, 22, 29, 45, 47). High

4

levels of pre-existing neutralizing antibodies may play a role in preventing HSV spread and

5

viraemia. The reduction of neonatal HSV transmission in the presence of maternal HSV-specific

6

antibodies underline the protective effect of antibodies (8). In HSV seropositive humans,

7

circulating IgG antibodies are predominantly directed against gB and gD (9, 37). It has been

8

shown that these antibodies act by antibody-dependent cellular cytotoxicity (ADCC) for efficient

9

control of HSV infections in mice (31, 32, 44). High ADCC reactivity was also shown to

10

positively correlate with protection against disseminated disease in human neonatal HSV

11

infections (30, 33). Consistently, post-exposure administration of human gamma globuline

12

containing neutralizing HSV-1 antibodies or an anti-gD mAb to immunodeficient SCID or nude

13

mice, respectively, prolonged survival but was not able to eventually protect animals from death

14

(48, 60).

15

We previously isolated the gB specific monoclonal antibody (mAb) 2c (17) with potent HSV-1

16

neutralizing activity in vitro and in vivo (18). In this study we show that the efficiency of mAb 2c

17

for neutralizing free HSV virions and inhibiting cell-to-cell spread is completely independent

18

from ADCC, complement, and cellular effector mechanisms but critically relies on the antibody

19

valency. Mapping of the mAb 2c epitope to the solved gB structure (25) suggests that the

20

antibody interferes with HSV entry by blocking transmission of the fusogenic signal through

21

cross-linking of gB trimers. We show that the bivalent mAb 2c is capable to not only fully protect

22

severe immunodeficient NOD/SCID mice from lethal viral challenge but to also rescue animals

23

from lethal encephalitis even when the virus reached the peripheral nervous system.

-4-

1

MATERIALS AND METHODS

2

Cells and viruses. The hybridoma cell line secreting the mAb 2c generated from BALB/c

3

mice hyperimmunized with HSV-1 strain 342 hv (17) was maintained either in Iscove’s modified

4

Dulbecco’s medium (IMDM) with 10 % FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin

5

or in Ex-Cell hybridoma medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10

6

mM L-Glutamine (Invitrogen, Carlsbad, CA, USA) for serum-free antibody production. The

7

African green monkey kidney cell line Vero was obtained from the European Collection of Cell

8

Cultures (ECACC) and grown in Dulbecco’s modified Eagle’s medium (DMEM) with 10 %

9

heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin, and 100 µg/ml streptomycin.

10

Experiments using Vero cells were performed in maintenance medium with 2 % FBS. The HSV

11

type 1 (HSV-1) strain F and HSV type 2 (HSV-2) strain G were propagated in Vero cells and

12

titers were determined on Vero cells by the endpoint dilution assay as described (59) and

13

expressed as tissue culture infectious dose (TCID50/ml).

14

Antibodies, production and purification. MAb 2c (IgG2a) was purified from serum-free

15

hybridoma supernatants by protein A chromatography (Thermo Scientific, Worcester, MA,

16

USA), dialyzed against phosphate-buffered saline (PBS) and purity monitored on a calibrated

17

Superdex 200 10/300 GL column (Amersham Pharmacia, Piscataway, NJ, USA). Proteolytic

18

digestion of mAb 2c to obtain F(ab’)2- and Fab fragments were carried out with a pepsin or

19

papain preparation kit (Thermo Scientific) according to the manufacturer instructions.

20

Homogenous F(ab’)2 and Fab fragment preparations were obtained by immobilizing Fc fragments

21

to protein A column, followed by size exclusion chromatography of the flowthrough using a

22

calibrated HiLoad 16/60 Superdex 200 prep grade column (Amersham Pharmacia). To generate a

23

single chain fragment variable (scFv) total RNA was isolated from 1x 107 mAb 2c hybridoma

24

cells using the RNA/DNA Midi kit (Qiagen, Valencia, CA, USA) followed by mRNA -5-

1

preparation using the Oligotex mRNA Mini Kit (Qiagen). The authentic 5’ends of the mAb 2c

2

variable heavy chain (VH) and the variable light chain (VL) encoding DNA sequences were

3

amplified by 5’ rapid amplification of cDNA ends (RACE)-PCR using the Marathon™ cDNA

4

amplification kit (BD Biosciences, Heidelberg, Germany) and the oligonucleotides IgG2a-CH1

5

and IgG2a-C-kappa annealing to the 5’ constant regions of the heavy and light chain,

6

respectively. Sequences of subcloned PCR gene products were verified by DNA sequencing.

7

MAb 2c variable domains were amplified subsequently with deduced oligonucleotides specific to

8

the 5’end of the VH or VL chain gene and respective anti-sense primers. A standard (Gly4Ser)3

9

linker connecting the VH and VL domains was introduced by overlap extension PCR and the 2c-

10

scFv was cloned into the bacterial expression plasmid pHOG21 (28). Periplasmatic production

11

and purification of the 2c-scFv was carried out as described elsewhere (1). Concentrations of

12

purified antibodies were determined spectrophotometrically from the absorbance at A280nm using

13

the extinction coefficients 1.43 for IgG, 1.48 for F(ab’)2, 1.53 for Fab, and 1.7 for scFv.

14

Polyclonal human sera obtained from donors with high immunoglobulin titers for HSV-1

15

completely neutralized 100 TCID50 of HSV-1 F at a dilution of 1:160.

16

Determination of antibody affinity. Monolayers of Vero cells were infected at 80-90%

17

confluence with HSV-1 or HSV-2 at MOI 3 and harvested the next day by trypsinization

18

followed by washing in PBS. Cell surface binding measurements of 2c antibodies were carried

19

out as described previously (1). Briefly, purified mAb 2c or derived antibody fragments 2c-

20

F(ab’)2, 2c-Fab, and 2c-scFv were incubated in triplicate at concentrations from 0.03 nM – 500

21

nM with 5 x 105 Vero cells in 100 µl FACS buffer (PBS, 2% FBS, 0.1% sodium azide) for 1 h at

22

room temperature. Cells were washed twice with 200 µl FACS buffer and incubated with FITC-

23

labeled Fab-specific goat-anti-mouse IgG, (15 µg/ml, Jackson ImmunoResearch, Newmarket,

24

Suffolk, England) for detection of bound mAb 2c, 2c-F(ab’)2, and 2c-Fab. Bound scFv was -6-

1

detected by first incubating with saturating concentrations of the anti-c-myc mAb 9E10 (10

2

µg/ml; Roche, Indianapolis, IN, USA), followed by two washes and incubation with Fcγ-specific

3

FITC-labeled goat-anti-mouse IgG (15 µg/ml; Jackson ImmunoResearch). Cells were washed

4

twice and resuspended in FACS buffer. Fluorescence was measured on a FACScalibur (BD

5

Bioscience, San Jose, CA, USA), and median fluorescence intensity (MFI) was calculated using

6

the CellQuestTM software (BD Biosciences). Background fluorescence was subtracted and

7

equilibrium binding constants were determined by using the Marquardt and Levenberg method

8

for nonlinear regression with the GraphPad Prism version 4.0 (GraphPad Software, La Jolla, CA).

9

Epitope characterization. Immunoreactivity of mAb 2c with native or denatured

10

truncated glycoprotein B, gB(730)t (4), kindly provided by Roselyn J. Eisenberg and Gary H.

11

Cohen (University of Pennsylvania, Philadelphia, USA) was performed essentially as described

12

(4): Purified gB(730)t (0,75µg) was resolved on 8% SDS-PAGE under either non-reducing

13

(sample buffer containing 0.2% SDS) or denaturating (sample buffer containing 2% SDS and 155

14

mM β-Mercaptoethanol, 2 min at 95°C) conditions and transferred onto nitrocellulose membrane.

15

Membrane strips were blocked with 2% milk in TNT buffer (0.1 M Tris.HCl, pH 7.5, 0.15 M

16

NaCl, 0.05% Tween-20) for 1 hour followed by incubation with 5 µg/ml of glycoprotein B

17

specific antibodies mAb 2c, H126 (Novus Biologicals, Littleton, CO, USA) and H1817 (Novus)

18

in 2% milk/TNT-buffer for 2 hours at room temperature. Bound antibodies were detected with

19

horseradish peroxidase conjugated polyclonal goat-anti-mouse antibody (1:20,000 QED

20

Bioscience Inc. San Diego, CA, USA) and chemiluminescence (Thermo Scientific,) using the

21

LAS 3000 Luminescent Image Analyzer (Fujifilm, Tokyo, Japan).

22

COS-1 cells were transiently transfected by the DEAE-dextran method with plasmids

23

coding either for the full length HSV-1 gB (31-904, pRB9221) or C-terminal deletion mutants

24

truncated at positions 720 (pTS690), 630 (pPS600), 503 (pRB9510), 487 (pRB9509), and 470 -7-

1

(pRB9508). The plasmids were kindly provided by L. Pereira (54, 57) Immunofluorescence

2

assays with transfected cells using mAb2c or control antibodies were carried out as described

3

elsewhere (55).

4

Peptide mapping. Cellulose-bound overlapping 13meric peptides and duotopes were

5

automatically prepared according to standard SPOT-Synthesis protocols as described (21, 35)

6

(JPT Peptide Technologies, Berlin, Germany). In addition, peptides coupled with a reactivity tag

7

and a linker were immobilized chemoselectively on a modified glass surface in three identical

8

subarrays and purified by removal of truncated and acetylated sequences by subsequent washing

9

steps. Peptide microarrays were blocked with TBS containing blocking buffer (Pierce

10

International) for 2 h, and incubated with 10 µg/ml mAb 2c in blocking buffer for 2h. Peptide

11

microarrays were washed with TBS-buffer containing 0.1% Tween (T-TBS) and peptide-bound

12

antibody on the peptide membrane was transferred onto a PVDF membrane. Anti-mouse IgG

13

either peroxidase-labelled (Sigma) or fluorescently-labelled (Pierce) was used as secondary

14

antibody at a final concentration of 1 µg/ml in blocking buffer. After 2h incubation and final

15

washing with T-TBS PVDF membranes were analyzed using chemiluminescence substrate

16

(Roche Diagnostics). Glass slide peptide microarrays were washed thoroughly with T-TBS and 3

17

mM SSC-buffer (JPT Peptide Technologies), dried under nitrogen and scanned using a high

18

resolution fluorescence scanner (Axon GenePix 4200 AL). Fluorescence signal intensities (Light

19

Units, LU) were analyzed using spot-recognition software (GenePix 6.0) and corrected for

20

background intensities from control incubations with secondary anti-mouse IgG.

21

Virus neutralization assay. Neutralizing activity of antibodies was determined by end-

22

point dilution assay as described previously (17). Briefly, serial dilutions of antibodies were

23

incubated with 100 TCID50 of HSV-1 or HSV-2 for 1 h at 37°C in cell culture medium. The

24

antibody virus inoculum was applied to Vero cell monolayers grown in microtiter plates and -8-

1

cytopathic effect (CPE) was scored after 72 h incubation at 37°C. The antibody concentration

2

required for reducing virus-induced CPE by 100% was determined as complete neutralization

3

titer. In addition, virus neutralization capacity of monovalent 2c-Fab fragments was investigated

4

in the presence of cross-linking antibodies, by adding an excess of anti-murine Fab IgGs (2600

5

nM, Jackson ImmunoResearch, Newmarket, Suffolk, England) to the pre-incubation step. For

6

control purposes virus without antibody and antibody alone was used to induce maximal CPE or

7

no CPE, respectively. Virus neutralization assays were repeated at least twice with similar results.

8

Post-attachment Neutralization Assay. Prechilled Vero cell monolayers (4°C for 15

9

min) were infected with 100 TCID50 HSV-1 F at 4°C for 1 h to allow virus absorbtion before

10

serial dilutions of either mAb 2c or a polyvalent IgG preparation from human plasma (Intratect,

11

Biotest AG, Dreieich, Germany, were added (post-attachment neutralization). To compare pre-

12

attachment versus post-attachment neutralization efficacy of mAb 2c under identical

13

experimental conditions, 100 TCID50 HSV-1 F were incubated for 1 h at 4°C with the same

14

antibody dilutions before adding to prechilled Vero cell monolayers. Inoculated Vero cells from

15

both assays were incubated for another 1 h at 4°C before transferred to 37°C. Neutralization titers

16

were determined after 72 h as described in the standard neutralization assay above.

17

Cell-to-cell spread assay. Confluent monolayers of Vero cells, either grown on glass

18

coverslips for immunofluorescence studies or in 24-well tissue culture plates for analysis by light

19

microscopy, were infected with either HSV-1 F or HSV-2 G. After for 4 hours adsorption at

20

37°C, the virus inoculum was removed and cells were incubated in DMEM containing 2% FBS

21

in the presence of neutralzing antibodies, pooled human sera derived from immunized donors

22

with high titers of anti-HSV-1 immunoglobulins (1:20), or medium alone as positive control. At

23

48 h postinfection, cells were fixed with 4% paraformaldehyde for immunostaining or analyzed

24

directly for plaque formation under a Zeiss Observer Z1 light microscope at a 50-fold -9-

1

magnification. To visualize the viral spread by indirect immunofluorescence cells were rinsed

2

twice with PBS, incubated for 15 min in 500 µl HEPES buffered saline with 0.05 % Tween 20,

3

and stained with FITC-conjugated polyclonal goat-anti-HSV serum (1:100, BETHYL,

4

Montgomery, TX, USA). Stained cells washed three times with PBS were mounted in mounting

5

medium containing 0.2 g/ml Mowiol 4-88 (Calbiochem, San Diego, CA, USA).

6

Immunofluorescence images were acquired with a Leica DM IRE2 confocal microscope at a 40-

7

fold magnification. Cell-to-cell spread inhibition was tested in addition by postadsorption virus

8

neutralization assay. Vero cells grown to confluency in six-well plates were incubated for 4 h at

9

37°C with 200 TCID50 of HSV-1 F in 3 ml DMEM containing 2% FBS, and antibiotics. Cell

10

monolayers were washed twice with PBS and overlaid with warm plaquing medium (DMEM, 5

11

% (w/v) agarose, 10% FBS, antibiotics) containing an excess of neutralizing antibodies or

12

polyclonal human HSV-1 neutralizing sera. Plaque formation was analyzed by light microscopy

13

after 48 h incubation at 37°C.

14

DNA-quantification. HSV-1 and HSV-2 genomes were quantified performing real-time

15

(RT) PCR. DNA was purified from samples containing equivalent amount of infectious particles

16

of HSV-1 and HSV-2 using the automated nucleic acid extraction system MagNA Pure LC

17

System (Roche) according to manufacturer instructions. Viral DNA was then quantified

18

performing RT-PCR (Lightcycler, Roche) using the RealArt HSV-1 / HSV-2 quantification kit

19

(Qiagen).

20

Mouse protection experiments. Anesthetized female nonobese diabetic/severe combined

21

immunodeficient (NOD-SCID) mice (NOD.CB17-Prkdcscid /J, Charles River Laboratories,

22

Research Models and Services, Sulzfeld, Germany), 6-8 weeks of age, were challenged

23

intravaginally with 20 µl inoculum of 1x106 TCID50 HSV-1 F per mouse. Skin glue (Epiglu,

24

Meyer-Haake Medical Innovations, Wehrheim, Germany) was applied onto the vulva to prevent - 10 -

1

dircharge of the virus inoculum. The delivered inoculum induced infection rates > 94 % as

2

assessed by culture of vaginal lavage. Mice were examined daily after viral inoculation for loss of

3

weight, vulvitis/vaginitis (redness, mucopurulent discharge and signs of inflammation) and

4

neurological disease. Mice displaying any of these symptoms were sacrificed immediately. Mice

5

were passively immunized by intravenous (i.v.) injection of purified mAb 2c either 24 h prior to

6

viral inoculation for immune prophylaxis or 24 h, 40 h, and 56 h after viral infection for

7

therapeutic treatment. Mice were assessed for infection by determination of virus titers from

8

vaginal irrigations obtained on days 1, 2, 4, 6 and 8 after infection and at the time of death using

9

the endpoint dilution assay on Vero cells. Viral loads in organs (spleen, adrenal gland, lung heart,

10

liver, kidney, spinal cord, and brain) of sacrificed mice were determined after homogenization of

11

organs by titration on Vero cell monolayers as described elsewhere (42). Each test and control

12

group contained 9-10 animals with detectable HSV-1 infection.

- 11 -

1 2

RESULTS Mapping and analysis of the gB epitope recognized by mAb 2c.

3

The recently determined crystal structure of the ectodomain of gB from HSV type 1 (HSV-1)

4

revealed a multi-domain trimer with five distinct structural domains: domain I (base), domain II

5

(middle), domain III (core), domain IV (crown), and domain V (arm) (25). To characterize the

6

neutralizing epitope of mAb 2c, we tested its reactivity with recombinant gB(730t)(4) in Western

7

blot analysis either under reducing or non-reducing conditions. As controls we used mAb H1817,

8

recognizing a linear epitope (4) and mAb H126 recognizing a discontinuous epitope (34). A

9

typical staining pattern for a linear epitope was obtained in Western blot analysis with mAb

10

H1817 showing detection of monomeric and trimeric forms of gB under non-reducing conditions

11

and sole predominant staining of gB monomer under reducing conditions (Fig. 1). As expected,

12

mAb H126 reacted with gB only under native conditions. Surprisingly, recognition of solely the

13

upper gB protein band > 170 kDa suggests that mAb H126 is trimer specific (Fig.1). MAb 2c

14

reacted with native and denatured gB, however, reactivity under denaturing conditions was much

15

weaker compared with mAb H1817 (Fig. 1). Weak reactivity with gB monomers under

16

denaturing conditions has been previously reported for a set of other neutralizing antibodies

17

binding to discontinuous epitopes that seem to be either resistant to denaturation or refold during

18

SDS-PAGE electrophoresis and therefore termed “pseudocontinuous” epitopes (4).

19

To narrow down the conformation-dependent epitope, reactivity of mAb 2c was further

20

characterized using COS-1 cells transiently transfected with plasmids encoding either full-length

21

gB (31-904) or gB mutants with C-terminal truncations at positions 720, 630, 503, 487, and 470.

22

The shortest gB deletion mutant showing positive internal immunofluorescence signals was

23

truncated at position 487 (data not shown). Thus we reasoned that the epitope for mAb 2c is

24

located within residues 31 to 487. - 12 -

1

To identify the specific epitope involved in binding of mAb 2c we used gB derived peptides

2

displayed on peptide microarrays. First, the gB sequence displaying amino acids 31 to 505 was

3

prepared by Spot-Synthesis as overlapping 13meric peptides bound with uncharged acetylated

4

amino terminal ends to a continuous cellulose membrane with an offset of 3 amino acids. To

5

avoid shifting of the binding equilibrium for the non-complexed antibody, mAb 2c peptide scans

6

were immobilized on a PVDF membrane prior to detection by chemiluminescence. As shown in

7

the schematic representation of the full length gB with indicated functional domains (Fig. 2A),

8

mAb 2c reactivity was restricted to peptides spanning two separate regions within domain I, three

9

consecutive peptides comprising residues 175 to 193 (binding region A) and two overlapping

10

peptides comprising residues 295 to 310 (binding region B). To validate both identified binding

11

regions, we used an additional set of purified 13meric peptides immobilized on glass slides via a

12

flexible linker. Compared to the cellulose screen the read-out of this microarray scanning via

13

fluorescence confirmed the same epitope binding regions (Fig. 2B). Due to the application of

14

purified peptides and a high resolution microarray scanning system additional consecutive

15

peptides at both binding sites were recognized by mAb2c in this peptide microarray (Fig. 2B).

16

We mapped the identified binding sites for mAb 2c to the solved gB structure (25). Interestingly,

17

the peptide 172QVWFGHRYSQFMG184 showing the strongest reactivity with mAb 2c overlapped

18

with one of the two putative fusion loops (fusion loop 1

19

subdomain of domain I (23) (Fig. 3). However, localization of binding site A at the base of the

20

gB trimer makes it inaccessible to mAb 2c in the available gB structure most likely representing

21

the postfusion conformation (25). Residues of binding site B are exposed and located at the upper

22

part of the domain I (Fig. 3).

23

To further assess the conformation dependent epitope of mAb 2c, consensus sequences of both

24

binding regions were connected in various combinations as duotopes either directly or separated - 13 -

173VWFGHRY179)

located in a curving

1

by one or two ß-alanine spacers (Fig. 4). It has recently been shown that linker insertions in close

2

proximity to fusion loop 1 after residue E187 result in fusion-deficient gB mutants (41 ), even

3

though gB folds into a postfusion conformation (41 ). Therefore, we included in addition to the

4

consensus motif 179YSQFMG184 of binding region A the 186FED188 motif of binding region A into

5

separate duotope scans. Compared to the peptide

6

strongest binding reactivity with mAb 2c in the 13meric peptide scans (Fig. 4), the combination

7

of both binding site A motifs with the consensus peptide 300FYGYRE305 of binding site B resulted

8

in two duotopes with enhanced signal intensities (Fig. 4, duotope sets I & II). Whereas binding

9

strength of mAb 2c to duotope 179YSQFMG184-βA-300FYGYRE305 was only slightly increased, an

172QVWFGHRYSQFMG184

displaying the

10

almost saturation of the fluorescence signal intensity was obtained with duotope

186FED188-βA-

11

βA-300FYGYRE305.

12

Thus, the results from the peptide microarrays correspond to the Western blotting results and

13

demonstrate that mAb 2c recognizes a conformation dependent epitope. To prevent fusion of the

14

virion envelope with the cell membrane mAb 2c should bind to the prefusion conformation of

15

gB. However, the neutralizing epitope of mAb 2c maps only in part to the surface of the gB

16

conformation present in the available gB crystal structure (25) and indicates that gB might adopt

17

distinct conformations during entry.

18

Characterization of mAb 2c derived bivalent and monovalent antibodies. Monoclonal

19

antibodies have been used by several investigators to identify regions on gB essential for its

20

function in virus entry (4, 26, 40, 54). It has been suggested that neutralizing antibodies, which

21

have been mapped to a unique functional region at the base of the gB trimer comprising residues

22

of the C-terminal end of domain V and residues of domain I of a proximate protomer, interfere

23

with the fusogenic activity of gB (4). We therefore hypothesized that monovalent antibody

24

binding to the mAb 2c epitope within domain I close to the C terminus of domain V should - 14 -

1

sufficiently block cooperative conformational changes upon activation of gB. Since mAb 2c

2

neutralizes HSV-1 without complement in vitro (17), we generated conventional F(ab’)2 and Fab

3

fragments and a recombinant single chain fragment variable (scFv) as valuable tools for studying

4

the hypothesized mechanism mediated by mAb 2c. The homogeneity of the generated antibody

5

preparations was monitored by size exclusion chromatography (data not shown).

6

Flow cytometry analysis using Vero cells either infected or not infected with HSV-1 or HSV-2,

7

respectively, demonstrated specific binding of mAb 2c and mAb 2c derived antibody fragments

8

(data not shown). We further used fluorescence cytometry to determine equilibrium binding

9

curves of the antibodies to HSV-1 and HSV-2 infected Vero cells (Fig. 5). The results of these

10

studies demonstrated higher apparent affinities for the whole IgG and the F(ab’)2 fragment than

11

for the Fab and scFv, respectively (Table 1). The increment in functional affinity (avidity) for the

12

bivalent antibodies relative to the determined affinities of the monovalent antibodies indicates

13

that the bivalent antibodies were able to bind two gB epitopes on the cell surface simultaneously.

14

Bivalent mAb 2c and 2c-F(ab’)2 showed an 1.7-2.8 fold higher apparent affinity compared to

15

their monovalent counterparts. The slight increment in the apparent KD of the F(ab’)2 fragment

16

versus the IgG might be due to the higher flexibility of the antigen binding sites within the

17

F(ab’)2 construct. The similar apparent affinities for mAb 2c, 2c-F(ab’)2, and 2c-Fab to both,

18

HSV-1 and HSV-2 infected Vero cells confirmed that the recognized gB-epitope does not

19

structurally differ between both viruses (Table 1).

20

Neutralization activity of monovalent and bivalent antibodies in vitro. Equal

21

neutralization efficacy of mAb 2c irrespective if the antibody was added before (preattachment)

22

or after (postattachment) HSV-1 virions interacted with Vero cells (Fig. 6A) indicated that mAb

23

2c does not interfere with virus-binding to target cells. In contrast, the polyclonal human gamma

24

globulin Intratect® clearly neutralized by inhibition of virion attachment to target cells (Fig. 6B). - 15 -

1

Neutralizing activities of mAb 2c derived fragments F(ab’)2, Fab and scFv were compared with

2

their parental IgG counterpart in a standard neutralization assay on Vero cells. The parental mAb

3

2c reduced HSV-1 induced cytopathic effect (CPE) by 100% at a concentration of 8 nM.

4

Interestingly, a 4-fold higher mAb 2c concentration was required to completely reduce HSV-2

5

induced CPE (Fig. 7A). The bivalent 2c-F(ab’)2 reduced both HSV-1 and HSV-2 induced CPE

6

two times more efficiently than the parental mAb 2c. Surprisingly, we observed a fundamental

7

difference in the ability of the monovalent 2c-antibody fragments for neutralizing HSV-1 and

8

HSV-2. Compared to the parental mAb 2c, approx. 375-fold and 94-fold higher concentrations of

9

2c-Fab were necessary to reduce HSV-1 and HSV-2 induced CPE by 100%, respectively (Fig.

10

7A). The recombinant 2c-scFv showed a plaque reductive effect under the light microscope, but

11

was not able to reduce HSV induced CPE by 100% even at the highest tested concentration of

12

3,000 nM (data not shown).

13

Since both bivalent antibodies mAb 2c and 2c-F(ab’)2 neutralized HSV-2 about four-times less

14

effectively than HSV-1 (Fig 7A) we analyzed the genome copy numbers of HSV-1 and HSV-2

15

preparations containing equal amounts of infectious particles by quantitative real-time PCR.

16

Compared to HSV-1 a fourfold higher number of genome equivalents was found for HSV-2 (data

17

not shown) correlating well with the higher antibody titer of mAb 2c and 2c-F(ab’)2 required for

18

HSV-2 neutralization.

19

Neutralization assays as shown in Fig. 7A indicated a strong correlation between antibody

20

valency and neutralization efficiency. Consequently, we investigated whether the ability of 2c-

21

Fab fragments for clearing virus infection could be restored by cross linkage of the Fab

22

fragments. The virus neutralization assay was repeated for 2c-Fab in the absence or presence of

23

IgGs reacting with murine Fab fragments. As shown in Fig. 7B, cross-linking of 2c-Fab

24

dramatically increased neutralizing activity but could not restore it to the same efficacy as for the - 16 -

1

parental mAb 2c. Anti-murine Fab IgGs alone showed no effect on virus neutralization (data not

2

shown).

3

Cell-to-cell spread inhibition. Despite the ability of gB and gD specific monoclonal

4

antibodies for neutralizing HSV-1 with high efficacy, some of them failed to protect cells from

5

viral spread in tissue culture (11, 50). We therefore first compared the mAb 2c efficacy for

6

inhibiting cell-to-cell spread of HSV-1 and HSV-2 in a plaque reduction assay, respectively. As

7

shown in Figure 8 a concentration-dependent reduction of plaque size by mAb 2c was observed

8

for both HSV serotypes. At a concentration of 500 nM, mAb 2c completely abolished HSV-1

9

plaque development (Fig. 8A). Similarly to the neutralization experiments, a 4-fold higher mAb

10

2c concentration was required to also completely inhibit cell-to-cell transmission in HSV-2

11

infected cells (Fig. 8B).

12

Although 2c-Fab fragments did not efficiently neutralize free virions, yet it was reported that

13

small sized antibody fragments may exhibit more favourable diffusion properties (68), we

14

investigated their activity for preventing HSV-1 from crossing cell junctions from infected to

15

uninfected cells. For this analysis we employed a more sensitive immunofluorescence assay. Both

16

bivalent antibodies, mAb 2c and 2c-F(ab’)2, completely abrogated HSV-1 spread in Vero cell

17

monolayers and only single infected cells could be visualized by indirect immunofluoresence

18

(Fig. 9). Despite the ability of the polyclonal human serum to neutralize free virions it completely

19

failed to inhibit viral cell-to-cell spread. This is most likely the result of the heterogeneous

20

population of neutralizing antibodies directed against numerous HSV epitopes. Compared with

21

polyclonal human immune serum, the monovalent 2c-Fab fragment was capable to control cell-

22

to-cell spread to some extent. In contrast to its bivalent counterparts, however, the monovalent

23

2c-Fab fragment was not able to completely abrogate viral spread even tested at a 6-fold higher

- 17 -

1

concentration (Fig. 9). Hence, antibody valency played a key role in also inhibiting spread of

2

HSV-1 between adjacent cells.

3

Immunoprotection of immunodeficient mice against disseminated HSV infection. We

4

showed previously that mice depleted of both CD4+ and CD8+ T-cells were fully protected from

5

lethal encephalitis by passive transfer of mAb 2c after intravaginally HSV-1 infection (18).

6

Natural killer (NK) cells accumulating at the site of HSV-2 infection in humans (29) are the early

7

source of interferon-γ (46), which plays an essential role for the control of HSV infection (2, 46,

8

64). More recently it has been demonstrated for the first time, that human NK cells mediate

9

protection against primary genital HSV infection in humanized mice as an innate immune

10

response (38). To investigate, if mAb 2c confers antiviral activity independently from an

11

antibody-mediated immune response we employed a NOD/SCID mouse model, which in addition

12

to the SCID T- and B-cell deficiency, lack NK cell and macrophage function and the ability to

13

stimulate the complement pathway. Intravaginal HSV-1 infection (1 x 106 TCID50) of

14

NOD/SCID mice resulted in rapid progressive systemic disease with a median survival time of 9

15

days. HSV titers in organs were determined by an endpoint dilution assay showing high viral

16

titers in spinal cord (2.3 x 106 TCID50), brain (3.8 x 105 TCID50), and vaginal mucosa (1.4 x 106

17

TCID50), moderate titers in kidney (1.7 x 104 TCID50) and adrenal glands (1.1 x 104 TCID50) and

18

low titers in lung (1.1 x 103 TCID50) and heart (1.9 x 102 TCID50) (data not shown). To assess the

19

therapeutic efficiency of mAb 2c, NOD/SCID mice were treated intravenously with either 2.5

20

mg/kg, 5 mg/kg or 15 mg/kg antibody 24 h prior to intra-vaginal HSV-1 challenge (Fig. 10).

21

Mice receiving the low antibody doses were not fully protected against lethal infection by HSV-

22

1. Median survival times of mice treated with 5 mg/kg mAb 2c, however, were 2.6-fold

23

prolonged when compared to control mice receiving PBS. The HSV-1 titres in the investigated

24

organs from mice not protected against lethal encephalitis were comparable to the untreated - 18 -

1

control group. In contrast, full protection of animals was achieved at a dose of 15 mg/kg mAb 2c.

2

Viral titres in organs of mice protected by the antibody were below the detection limit of 1 x 102

3

TCID50.

4

We next evaluated if post-exposure immunization with mAb 2c also confers protection

5

from viral dissemination and lethal encephalitis in the presence of an established peripheral HSV

6

infection. NOD/SCID mice with a high HSV-1 titer in vaginal irrigations at 24 h after viral

7

challenge were repeatedly treated at 24 h, 40 h and 56 h intravenously with 15 mg/kg of mAb 2c

8

(Fig. 11 A&B). The PBS treated control group showed constant vaginally virus shedding until

9

mice with neurological symptoms had to be sacrificed between day 7 and day 9. In contrast, mAb

10

2c cleared established HSV-1 infection by day 8 and completely prevented lethal outcome of

11

infection (3x 300 µg; P = 0.0003 compared with PBS). Furthermore, no virions were detected in

12

sensory neurons and respective organs of mAb 2c treated animals one month after infection (data

13

not shown).

14

- 19 -

1

DISCUSSION

2

Following the steps viruses take to enter target cells virus-neutralizing mAbs can inhibit entry by

3

several mechanisms. The specific interaction of viral surface proteins with cellular proteins,

4

lipids, or carbohydrates represents the initial stage of infection, which can be blocked by

5

neutralizing antibodies. Antibodies inhibiting virus attachment either directly bind to the virion

6

receptor-binding site, such as mAb F105 reacting with the CD4-binding site of HIV-1 gp120 and

7

Fab HC19 covering the receptor-binding site of influenza hemagglutinin (HA) (6, 20, 56), or

8

sterically interfere with receptor engagement, such as Fab HC45 binding in 17Å proximity to the

9

HA receptor-binding site (19). In addition to the essential binding of HSV gD to one of its

10

cellular receptors, gB plays a role in virion attachment to target cells. Recently, the existence of

11

two heparan sulfate proteoglycan independent true cell surface receptors and/or attachment

12

factors for HSV gB have been described (5, 24, 62). Paired immunoglobulin-like type 2 receptor

13

(PILRα) has been characterized as one possible protein receptor of gB at least in certain cell types

14

(62). For mAb 2c comparative pre- versus postattachment neutralization assays showed that the

15

antibody may not inhibit binding of virus to the cell surface, but blocks viral entry. It has been

16

shown previously that the interaction of gB with lipid membranes via key hydrophobic and

17

hydrophilic residues of its fusion domain (23, 24) can be blocked by mAbs that recognize

18

epitopes in close proximity to the fusion loops (4, 23). Because the conformational epitope of

19

mAb 2c partially overlaps with fusion loop 1 we reasoned that binding of mAb 2c interferes most

20

likely with transmission of the fusogenic signal and we further evaluated neutralization at the

21

post-binding/pre-fusion stage as possible mode of action.

22

Triggered structural rearrangement is a key feature of viral fusogenic glycoproteins, resulting in

23

distinct prefusion and postfusion conformations. Epitopes of different neutralizing mAbs have

24

been mapped along the lateral domains of the spikes and to the tip of the crown of the gB crystal - 20 -

1

structure (4, 25). The epitope of mAb 2c maps to a unique functional region (FR1) at the base of

2

the gB trimer consisting of residues within the C-terminal helix αF of domain V and residues

3

within domain I of a proximate protomer (4). Our homology model shows that one part of the

4

discontinuous epitope (F300 to E305) recognized by mAb 2c localizes to the upper section of

5

domain I of gB, which has characteristics of a pleckstrin homology (PH) domain (7, 39). The

6

other part of the epitope (F175 to A190) also located in domain I, however, is buried and would be

7

inaccessible to mAb 2c binding unless gB undergoes a major conformational change. We

8

therefore hypothesized that mAb 2c impedes transition of gB preferentially in the prefusion

9

conformation. Based on the mAb 2c epitope localization and the assumption that conformational

10

changes upon activation are cooperative, we reasoned that monovalent interaction of mAb 2c

11

would be sufficient for blocking juxtaposition of the fusogenic domain of gB and the cellular

12

membrane. Surprisingly, however, none of the generated monovalent antibody fragments (Fab

13

and scFv) was capable to efficiently neutralize free virions or to inhibit viral cell-to-cell spread.

14

In contrast, both bivalent molecules, mAb 2c and 2c-F(ab’)2, were highly effective for virus

15

neutralization and cell-to-cell spread inhibition. Retention of specific and comparable binding

16

activity of all mAb 2c derived antibodies in this study exclude functional differences of

17

monovalent and bivalent antibodies due to impaired antigen recognition. Multivalent binding of

18

immunoglobulins augments their functional affinity (27). The gain in functional affinity,

19

however, inversely correlates with the intrinsic affinity of the antibody binding site (51). The only

20

moderate increment in equilibrium constants between 1.7 and 2.8 for the bivalent 2c antibodies,

21

IgG and F(ab’)2 when compared to their monovalent counterparts, scFv and Fab, is thus not

22

unusual for antibodies with intrinsic affinities in the low nanomolar range. Thus the higher

23

apparent affinity in fact indicates that multivalent (higher avidity) binding to the gB antigen does

24

occur and suggests that the anti-viral activity of the mAb 2c and 2c-F(ab’)2 is a consequence of - 21 -

1

gB cross-linking. Inferior neutralization efficiency of monovalent versus bi- or multivalent

2

antibodies with specificity for the gH antigen of varicella-zoster virus (VZV) has been discussed

3

as a matter of steric hindrance due to the different sizes of these antibodies (16). Although we

4

cannot completely exclude this possibility as a potential additional neutralization mechanism for

5

the mAb 2c variants, this seems unlikely because a direct correlation between antibody size,

6

neutralization efficiency, and cell-to-cell spread inhibition was not observed. Furthermore, our

7

data show that the smaller 2c-F(ab’)2 had an even better virus neutralization activity than the

8

larger 2c-IgG. Hence, the present observations indicate that gB cross-linking is the key

9

mechanism for the antiviral activity of mAb 2c and suggest that stabilization of the gB prefusion

10

conformation through immobilization of gB trimers inhibits activation of the fusogenic signal. A

11

most recent study by Silverman et al. (63) proposed that a fusion-deficient phenotype of the

12

HSV-1 gB ectodomain upon insertion of five amino acids after residue E187 close to the fusion

13

loop 1 may not result from interference with conformational changes of gB but rather from

14

interference with other mechanistic gB functions. In our duotope scans mAb 2c reacted strongest

15

with binding site A/B duotope 186FED188-βA-βA-300FYGYRE305 covering the particular insertion

16

site E187, which seems to be critcal for gB function. It is therefore tempting to speculate that mAb

17

2c crosslinking impairs the ability of gB to interact with the other components of the HSV fusion

18

machinery. However, future research is necessary, since our results do not allow to distinguish if

19

cross-linking blocks the conformational change of gB itself or blocks the interaction between gB,

20

gD and gH/gL, which occurs during cell fusion (3) and is essential for completing the fusion

21

process (67). The HSV-1 gB conformation observed in the solved crystals (25) suggest to

22

represent the postfusion form and a prefusion model of gB has not yet been characterized.

23

Therefore, X-ray crystallographic studies of mAb 2c or its F(ab’)2 in complex with gB might

- 22 -

1

provide insights in the native conformation of gB and a better understanding about transmission

2

of the fusogenic signal.

3

Severe and even life-threatening HSV infections can occur in maternally infected newborns, in

4

patients with recurrent ocular infections, or in severely immunocompromised patients. To

5

investigate if systemic application of our anti-gB antibody confers protection also in a highly

6

immunodeficient in vivo setting, we employed a NOD/SCID mouse model. We used intravaginal

7

HSV-1 inoculation as an established route of ganglionic infection with axonal spread of the virus

8

causing hindlimb paralysis and fatal herpetic encephalitis in immunocompetent as well as in T

9

cell depleted mice (17, 18). Here we demonstrate, that mAb 2c not only fully protects NOD/SCID

10

in the acute phase of primary HSV-1 infection but is also effective in completely preventing

11

neurological disease and death even after peripheral virus spread has commenced. The HSV cell-

12

to-cell spread is a very efficient way for viral transfer across neuronal synapses and tight

13

junctions as well as to circumvent immunological barriers of the adaptive immune system. MAb

14

2c both decreases virus expression of infected vaginal tissues and inhibits axonal spread of HSV.

15

Other reports showed that administration of anti-HSV IgGs after viral challenge can reduce the

16

quantity of acute ganglionic infections in animals (17, 43). Consistently, intraperitoneally

17

administered recombinant human anti-gD IgG to mice with corneal HSV-1 infection was shown

18

to localize to HSV-infected nerve fibers and sensory neurons (61). Furthermore, passive

19

immunization of immunocompetent animals with mAbs specific for HSV gD, gC or gB

20

administered postexposure at appropriate times demonstrated protection against HSV induced

21

neurological disease (14, 17). However, it has also been concluded from several animal studies

22

that humoral immunity alone is ineffective in the control of HSV infections. Consistent with this

23

view, administration of anti-HSV-1 hyperimmune serum has been reported to be insufficient for

24

protecting immunosupressed or immunodeficient mice (48, 49, 52, 53, 58). Another study - 23 -

1

showed in an HSV-1 induced stromal keratitis mouse model, that an anti-gD mAb prevented

2

death of mice depleted in either CD4+ or CD8+ T-cells but failed to prevent death when mice

3

were depleted in both T-cell subsets simultaneously (66). On the other hand, an intraperitoneal

4

application of a human anti-gD mAb to athymic nude mice 24 h after intracutan HSV-1 infection

5

was able to prevent death in 11% of the animals (60). Although the role of antibodies in resolving

6

viral diseases is controversially discussed, our in vivo data in a severely immunocompromised

7

setting clearly show that a monoclonal antibody can mediate full protection even after HSV

8

reached the peripheral nervous system. Therefore, these results encourage further development of

9

monoclonal antibodies with comparable properties like mAb 2c for therapy of severe HSV

10

diseases.

11

To our knowledge, we here demonstrate for the first time protective efficacy of a systemically

12

applied anti-gB cross-linking mAb that prevents neuronal HSV-1 spread completely independent

13

from cellular effector mechanisms and complement.

- 24 -

1

ACKNOWLEDGMENTS

2

This work was supported in part by the Deutsche José Carreras Leukämie foundation grant

3

DJCLS R06/14 (J.K., A.M.E.H and M.R.), the Deutsche Forschungsgemeinschaft (GK-1045) and

4

a research fellowship from the Jürgen Manchot foundation (A.K.).

5

Recombinant gB(730)t was generously donated by Roselyn J. Eisenberg and Gary H. Cohen,

6

(University of Pennsylvania, Philadelphia, USA). Leonore Pereira (University of California, San

7

Francisco, USA) kindly provided plasmids coding for gB deletion mutants. We thank Evelyn

8

Exner for excellent technical assistance, Simone Schimmer, Kirsten Dietze and Maike Nowak for

9

assistance with the mouse models and Claudia Dumitru and Bernd Giebel for help with the

10

microscopic analysis.

11

- 25 -

1

REFERENCES

2

1.

Arndt, M. A., J. Krauss, R. Schwarzenbacher, B. K. Vu, S. Greene, and S. M. Rybak.

3

2003. Generation of a highly stable, internalizing anti-CD22 single-chain Fv fragment for

4

targeting non-Hodgkin's lymphoma. Int J Cancer 107:822-829.

5

2.

Ashkar, A. A., and K. L. Rosenthal. 2003. Interleukin-15 and natural killer and NKT cells

6

play a critical role in innate protection against genital herpes simplex virus type 2 infection.

7

J Virol 77:10168-10171.

8

3.

Atanasiu, D., J. C. Whitbeck, T. M. Cairns, B. Reilly, G. H. Cohen, and R. J.

9

Eisenberg. 2007. Bimolecular complementation reveals that glycoproteins gB and gH/gL

10

of herpes simplex virus interact with each other during cell fusion. Proc Natl Acad Sci USA

11

104:18718-18723.

12

4.

Bender, F. C., M. Samanta, E. E. Heldwein, M. P. de Leon, E. Bilman, H. Lou, J. C.

13

Whitbeck, R. J. Eisenberg, and G. H. Cohen. 2007. Antigenic and mutational analyses of

14

herpes simplex virus glycoprotein B reveal four functional regions. J Virol 81:3827-3841.

15

5.

Bender, F. C., J. C. Whitbeck, H. Lou, G. H. Cohen, and R. J. Eisenberg. 2005. Herpes

16

simplex virus glycoprotein B binds to cell surfaces independently of heparan sulfate and

17

blocks virus entry. J Virol 79:11588-11597.

18

6.

Bizebard, T., B. Gigant, P. Rigolet, B. Rasmussen, O. Diat, P. Bosecke, S. A. Wharton,

19

J. J. Skehel, and M. Knossow. 1995. Structure of influenza virus haemagglutinin

20

complexed with a neutralizing antibody. Nature 376:92-94.

21 22

7.

Blomberg, N., E. Baraldi, M. Nilges, and M. Saraste. 1999. The PH superfold: a structural scaffold for multiple functions. Trends Biochem Sci 24:441-445.

- 26 -

1

8.

Brown, Z. A., J. Benedetti, R. Ashley, S. Burchett, S. Selke, S. Berry, L. A. Vontver,

2

and L. Corey. 1991. Neonatal herpes simplex virus infection in relation to asymptomatic

3

maternal infection at the time of labor. N Engl J Med 324:1247-1252.

4

9.

Burbelo, P. D., Y. Hoshino, H. Leahy, T. Krogmann, R. L. Hornung, M. J. Iadarola,

5

and J. I. Cohen. 2009. Serological diagnosis of human herpes simplex virus type 1 and 2

6

infections by luciferase immunoprecipitation system assay. Clin Vaccine Immunol 16:366-

7

371.

8

10.

9

patients with severe and recurrent Herpes simplex virus-1 (HSV-1) infections. Cell

10 11

Immunol 246:65-74. 11.

12 13

Co, M. S., M. Deschamps, R. J. Whitley, and C. Queen. 1991. Humanized antibodies for antiviral therapy. Proc Natl Acad Sci USA 88:2869-2873.

12.

14 15

Carter, C., S. Savic, J. Cole, and P. Wood. 2007. Natural killer cell receptor expression in

Cook, M. L., and J. G. Stevens. 1973. Pathogenesis of herpetic neuritis and ganglionitis in mice: evidence for intra-axonal transport of infection. Infect Immun 7:272-288.

13.

Cunningham, A. L., R. R. Turner, A. C. Miller, M. F. Para, and T. C. Merigan. 1985.

16

Evolution of recurrent herpes simplex lesions. An immunohistologic study. J Clin Invest

17

75:226-233.

18

14.

Dix, R. D., L. Pereira, and J. R. Baringer. 1981. Use of monoclonal antibody directed

19

against herpes simplex virus glycoproteins to protect mice against acute virus-induced

20

neurological disease. Infect Immun 34:192-199.

21

15.

Donaghy, H., L. Bosnjak, A. N. Harman, V. Marsden, S. K. Tyring, T. C. Meng, and

22

A. L. Cunningham. 2009. Role for plasmacytoid dendritic cells in the immune control of

23

recurrent human herpes simplex virus infection. J Virol 83:1952-1961.

- 27 -

1

16.

Drew, P. D., M. T. Moss, T. J. Pasieka, C. Grose, W. J. Harris, and A. J. Porter. 2001.

2

Multimeric humanized varicella-zoster virus antibody fragments to gH neutralize virus

3

while monomeric fragments do not. J Gen Virol 82:1959-1963.

4

17.

Eis-Hubinger, A. M., K. Mohr, and K. E. Schneweis. 1991. Different mechanisms of

5

protection by monoclonal and polyclonal antibodies during the course of herpes simplex

6

virus infection. Intervirology 32:351-360.

7

18.

Eis-Hubinger, A. M., D. S. Schmidt, and K. E. Schneweis. 1993. Anti-glycoprotein B

8

monoclonal antibody protects T cell-depleted mice against herpes simplex virus infection

9

by inhibition of virus replication at the inoculated mucous membranes. J Gen Virol 74 ( Pt

10 11

3):379-385. 19.

Fleury, D., B. Barrere, T. Bizebard, R. S. Daniels, J. J. Skehel, and M. Knossow. 1999.

12

A complex of influenza hemagglutinin with a neutralizing antibody that binds outside the

13

virus receptor binding site. Nat Struct Biol 6:530-534.

14

20.

15 16

distortion allows influenza virus to escape neutralization. Nat Struct Biol 5:119-123. 21.

17 18

Fleury, D., S. A. Wharton, J. J. Skehel, M. Knossow, and T. Bizebard. 1998. Antigen

Frank, R., and H. Overwin. 1996. SPOT synthesis. Epitope analysis with arrays of synthetic peptides prepared on cellulose membranes. Methods Mol Biol 66:149-169.

22.

Grubor-Bauk, B., A. Simmons, G. Mayrhofer, and P. G. Speck. 2003. Impaired

19

clearance of herpes simplex virus type 1 from mice lacking CD1d or NKT cells expressing

20

the semivariant V alpha 14-J alpha 281 TCR. J Immunol 170:1430-1434.

21

23.

Hannah, B. P., T. M. Cairns, F. C. Bender, J. C. Whitbeck, H. Lou, R. J. Eisenberg,

22

and G. H. Cohen. 2009. Herpes simplex virus glycoprotein B associates with target

23

membranes via its fusion loops. J Virol 83:6825-6836.

- 28 -

1

24.

Hannah, B. P., E. E. Heldwein, F. C. Bender, G. H. Cohen, and R. J. Eisenberg. 2007.

2

Mutational evidence of internal fusion loops in herpes simplex virus glycoprotein B. J Virol

3

81:4858-4865.

4

25.

Heldwein, E. E., H. Lou, F. C. Bender, G. H. Cohen, R. J. Eisenberg, and S. C.

5

Harrison. 2006. Crystal structure of glycoprotein B from herpes simplex virus 1. Science

6

313:217-220.

7

26.

Highlander, S. L., W. H. Cai, S. Person, M. Levine, and J. C. Glorioso. 1988.

8

Monoclonal antibodies define a domain on herpes simplex virus glycoprotein B involved in

9

virus penetration. J Virol 62:1881-1888.

10

27.

Kaufman, E. N., and R. K. Jain. 1992. Effect of bivalent interaction upon apparent

11

antibody affinity: experimental confirmation of theory using fluorescence photobleaching

12

and implications for antibody binding assays. Cancer Res 52:4157-4167.

13

28.

Kipriyanov, S. M., O. A. Kupriyanova, M. Little, and G. Moldenhauer. 1996. Rapid

14

detection of recombinant antibody fragments directed against cell- surface antigens by flow

15

cytometry. J Immunol Methods 196:51-62.

16

29.

Koelle, D. M., C. M. Posavad, G. R. Barnum, M. L. Johnson, J. M. Frank, and L.

17

Corey. 1998. Clearance of HSV-2 from recurrent genital lesions correlates with infiltration

18

of HSV-specific cytotoxic T lymphocytes. J Clin Invest 101:1500-1508.

19

30.

20 21

Kohl, S. 1991. Role of antibody-dependent cellular cytotoxicity in defense against herpes simplex virus infections. Rev Infect Dis 13:108-114.

31.

Kohl, S., D. L. Cahall, D. L. Walters, and V. E. Schaffner. 1979. Murine antibody-

22

dependent cellular cytotoxicity to herpes simplex virus-infected target cells. J Immunol

23

123:25-30.

- 29 -

1

32.

Kohl, S., N. C. Strynadka, R. S. Hodges, and L. Pereira. 1990. Analysis of the role of

2

antibody-dependent cellular cytotoxic antibody activity in murine neonatal herpes simplex

3

virus infection with antibodies to synthetic peptides of glycoprotein D and monoclonal

4

antibodies to glycoprotein B. J Clin Invest 86:273-278.

5

33.

Kohl, S., M. S. West, C. G. Prober, W. M. Sullender, L. S. Loo, and A. M. Arvin. 1989.

6

Neonatal antibody-dependent cellular cytotoxic antibody levels are associated with the

7

clinical presentation of neonatal herpes simplex virus infection. J Infect Dis 160:770-776.

8

34.

9

reactive and type-specific epitopes of herpes simplex virus 1 glycoprotein B map in

10 11

separate domains. Virology 166:423-431. 35.

12 13

Kousoulas, K. G., B. Huo, and L. Pereira. 1988. Antibody-resistant mutations in cross-

Kramer, A., and J. Schneider-Mergener. 1998. Synthesis and screening of peptide libraries on continuous cellulose membrane supports. Methods Mol Biol 87:25-39.

36.

Krummenacher, C., V. M. Supekar, J. C. Whitbeck, E. Lazear, S. A. Connolly, R. J.

14

Eisenberg, G. H. Cohen, D. C. Wiley, and A. Carfi. 2005. Structure of unliganded HSV

15

gD reveals a mechanism for receptor-mediated activation of virus entry. EMBO J 24:4144-

16

4153.

17

37.

Kuhn, J. E., G. Dunkler, K. Munk, and R. W. Braun. 1987. Analysis of the IgM and

18

IgG antibody response against herpes simplex virus type 1 (HSV-1) structural and

19

nonstructural proteins. J Med Virol 23:135-150.

20

38.

Kwant-Mitchell, A., A. A. Ashkar, and K. L. Rosenthal. 2009. Mucosal innate and

21

adaptive immune responses against herpes simplex virus type 2 in a humanized mouse

22

model. J Virol 83:10664-10676.

23 24

39.

Lemmon, M. A., and K. M. Ferguson. 2000. Signal-dependent membrane targeting by pleckstrin homology (PH) domains. Biochem J 350 Pt 1:1-18. - 30 -

1

40.

2 3

Li, W., T. J. Minova-Foster, D. D. Norton, and M. I. Muggeridge. 2006. Identification of functional domains in herpes simplex virus 2 glycoprotein B. J Virol 80:3792-3800.

41.

Lin, E., and P. G. Spear. 2007. Random linker-insertion mutagenesis to identify

4

functional domains of herpes simplex virus type 1 glycoprotein B. Proc Natl Acad Sci USA

5

104:13140-13145.

6

42.

Lingen, M., F. Hengerer, and D. Falke. 1997. Mixed vaginal infections of Balb/c mice

7

with low virulent herpes simplex type 1 strains result in restoration of virulence properties:

8

vaginitis/vulvitis and neuroinvasiveness. Med Microbiol Immunol 185:217-222.

9

43.

McKendall, R. R., T. Klassen, and J. R. Baringer. 1979. Host defenses in herpes simplex

10

infections of the nervous system: effect of antibody on disease and viral spread. Infect

11

Immun 23:305-311.

12

44.

13 14

Mester, J. C., J. C. Glorioso, and B. T. Rouse. 1991. Protection against zosteriform spread of herpes simplex virus by monoclonal antibodies. J Infect Dis 163:263-269.

45.

Mikloska, Z., A. M. Kesson, M. E. Penfold, and A. L. Cunningham. 1996. Herpes

15

simplex virus protein targets for CD4 and CD8 lymphocyte cytotoxicity in cultured

16

epidermal keratinocytes treated with interferon-gamma. J Infect Dis 173:7-17.

17

46.

18 19

Milligan, G. N., and D. I. Bernstein. 1997. Interferon-gamma enhances resolution of herpes simplex virus type 2 infection of the murine genital tract. Virology 229:259-268.

47.

Milligan, G. N., D. I. Bernstein, and N. Bourne. 1998. T lymphocytes are required for

20

protection of the vaginal mucosae and sensory ganglia of immune mice against reinfection

21

with herpes simplex virus type 2. J Immunol 160:6093-6100.

22

48.

Minagawa, H., S. Sakuma, S. Mohri, R. Mori, and T. Watanabe. 1988. Herpes simplex

23

virus type 1 infection in mice with severe combined immunodeficiency (SCID). Arch Virol

24

103:73-82. - 31 -

1

49.

Nagafuchi, S., H. Oda, R. Mori, and T. Taniguchi. 1979. Mechanism of acquired

2

resistance to herpes simplex virus infection as studied in nude mice. J Gen Virol 44:715-

3

723.

4

50.

Navarro, D., P. Paz, and L. Pereira. 1992. Domains of herpes simplex virus I

5

glycoprotein B that function in virus penetration, cell-to-cell spread, and cell fusion.

6

Virology 186:99-112.

7

51.

Nielsen, U. B., G. P. Adams, L. M. Weiner, and J. D. Marks. 2000. Targeting of bivalent

8

anti-ErbB2 diabody antibody fragments to tumor cells is independent of the intrinsic

9

antibody affinity. Cancer Res 60:6434-6440.

10

52.

11 12

after subcutaneous inoculation of immunosuppressed mice. J Infect Dis 131:51-57. 53.

13 14

Oakes, J. E. 1975. Invasion of the central nervous system by herpes simplex virus type 1

Oakes, J. E. 1975. Role for cell-mediated immunity in the resistance of mice to subcutaneous herpes simplex virus infection. Infect Immun 12:166-172.

54.

Pereira, L., M. Ali, K. Kousoulas, B. Huo, and T. Banks. 1989. Domain structure of

15

herpes simplex virus 1 glycoprotein B: neutralizing epitopes map in regions of continuous

16

and discontinuous residues. Virology 172:11-24.

17

55.

18 19

Pereira, L., T. Klassen, and J. R. Baringer. 1980. Type-common and type-specific monoclonal antibody to herpes simplex virus type 1. Infect Immun 29:724-732.

56.

Posner, M. R., T. Hideshima, T. Cannon, M. Mukherjee, K. H. Mayer, and R. A.

20

Byrn. 1991. An IgG human monoclonal antibody that reacts with HIV-1/GP120, inhibits

21

virus binding to cells, and neutralizes infection. J Immunol 146:4325-4332.

22

57.

Qadri, I., C. Gimeno, D. Navarro, and L. Pereira. 1991. Mutations in conformation-

23

dependent domains of herpes simplex virus 1 glycoprotein B affect the antigenic properties,

24

dimerization, and transport of the molecule. Virology 180:135-152. - 32 -

1

58.

2 3

herpes simplex virus 1 (HSV-1) infection. J Immunol 116:35-40. 59.

4 5

Rager-Zisman, B., and A. C. Allison. 1976. Mechanism of immunologic resistance to

Reed, J. L., and H. Muench. 1938. A simple method of estimating fifty percent endpoints. Am J Hyg 27:493-497.

60.

Sanna, P. P., A. De Logu, R. A. Williamson, Y. L. Hom, S. E. Straus, F. E. Bloom, and

6

D. R. Burton. 1996. Protection of nude mice by passive immunization with a type-

7

common human recombinant monoclonal antibody against HSV. Virology 215:101-106.

8

61.

9

transferred human recombinant monoclonal antibody to herpes simplex virus glycoprotein

10 11

Sanna, P. P., T. J. Deerinck, and M. H. Ellisman. 1999. Localization of a passively

D to infected nerve fibers and sensory neurons in vivo. J Virol 73:8817-8823. 62.

Satoh, T., J. Arii, T. Suenaga, J. Wang, A. Kogure, J. Uehori, N. Arase, I. Shiratori, S.

12

Tanaka, Y. Kawaguchi, P. G. Spear, L. L. Lanier, and H. Arase. 2008. PILRalpha is a

13

herpes simplex virus-1 entry coreceptor that associates with glycoprotein B. Cell 132:935-

14

944.

15

63.

Silverman, J. L., S. Sharma, T. M. Cairns, and E. E. Heldwein. 2010. Fusion-deficient

16

insertion mutants of herpes simplex virus type 1 glycoprotein B adopt the trimeric

17

postfusion conformation. J Virol 84:2001-2012.

18

64.

Smith, P. M., R. M. Wolcott, R. Chervenak, and S. R. Jennings. 1994. Control of acute

19

cutaneous herpes simplex virus infection: T cell-mediated viral clearance is dependent upon

20

interferon-gamma (IFN-gamma). Virology 202:76-88.

21 22

65.

Spear, P. G., and R. Longnecker. 2003. Herpesvirus entry: an update. J Virol 77:1017910185.

- 33 -

1

66.

Staats, H. F., J. E. Oakes, and R. N. Lausch. 1991. Anti-glycoprotein D monoclonal

2

antibody protects against herpes simplex virus type 1-induced diseases in mice functionally

3

depleted of selected T-cell subsets or asialo GM1+ cells. J Virol 65:6008-6014.

4

67.

Subramanian, R. P., and R. J. Geraghty. 2007. Herpes simplex virus type 1 mediates

5

fusion through a hemifusion intermediate by sequential activity of glycoproteins D, H, L,

6

and B. Proc Natl Acad Sci USA 104:2903-2908.

7

68.

Yokota, T., D. E. Milenic, M. Whitlow, and J. Schlom. 1992. Rapid tumor penetration of

8

a single-chain Fv and comparison with other immunoglobulin forms. Cancer Res 52:3402-

9

3408.

10 11

- 34 -

1

FIGURES

2 kDa

H1817 N

H126 D

N

2c D

N

D trimer

170 130 monomer

95 72 55

3 4 5

FIG. 1. Characterization of mAb 2c according to its reactivity with gB under different SDS-

6

PAGE conditions. Recombinant gB(730t) resolved by SDS-PAGE under either non-reducing,

7

native (N) or reducing, denatured (D) conditions was transferred to nitrocellulose membranes and

8

probed with gB specific monoclonal antibodies H1817, H126 or 2c. For controls mAbs H1817

9

and H126, recognizing a continuous and a discontinuous epitope, respectively, were used.

10

Molecular mass (kDa) is indicated on the left and migration of gB trimer and monomer on the

11

right.

- 35 -

1 2 1

A B

31

S

111

II

154

I 175-193 region A

460

364

I

II

500

661 670

573

III

IV

I

726

V

295-310 region B

3

FIG. 2. Peptidemapping of mAb 2c to gB. (A) Schematic localization of binding regions A and B

4

identified on a peptide-library spanning the extracellular domain of gB from amino acids 31 to

5

505. The 13meric peptides were synthesized on a continuous cellulose membrane with an offset

6

of 3 amino acids and bound mAb 2c was detected with a peroxidase-conjugated secondary

7

antibody by chemilumiescence. Coloring of functional domains I-V corresponds to the crystal

8

structure of gB by Heldwein et al. and regions not solved in the crystal structure are shown in

9

grey (25), S, signal sequence. (B) Fluorescence signal intensities from high resolution laser scans

10

with 13meric peptides immobilized on glass slides via a flexible linker.

- 36 -

1 2 3 4 5 6 7 8 9 10 11 12 * *

13

*

*

14

FIG. 3. Localization of neutralizing mAb 2c epitopes on the gB crystal structure (PDB-ID

15

2GUM). The ribbon diagram of the gB trimer shows one protomer with functional domains

16

colored, domain I in cyan, domain II in green, domain III in yellow, domain IV in orange, and

17

domain V in red. The other two protomers are shown in dark and light grey. Asterisks indicate

18

the fusion loops of protomers highlighted in grey, fusion loops of the colored protomer are not

19

visible. The mapped residues of the discontinuous mAb 2c epitope, F175 to A190 and F300 to E305,

20

are indicated in surface representation by dark blue for the color coded protomer and by rose for

21

both other protomers.

- 37 -

1

6

4

Signal intensity (LU x10 )

7

13mer peptides region region A B

duotopes set I

set II

5 4 3 2 1

17 2Q

VW FG HR 17 8R Y YS SQ QF FM 29 M G 8S G 18 PF YG IFE 4 DR YR A EG 1 SH 90 TE YS Y Q 31 0 YS SQ FM QF FM GF G Y M F G -B G FY YG -B-B- FYGYRE FY GY YR - FY YR GY R EY GY E RE E-B SQ RE -B - YS FM -B Q -Y F G SQ M FMG F FE ED G FE D FY D -B- F GY -B YG R FYB- FY YRE FY G G E FY GY YR YR GY R EF E RE E-B ED -B - F E D -B -F ED

0

2 3

FIG. 4. Duotopescanning of mAb 2c. Consensus sequences (underlined) of mAb 2c binding

4

regions A and B (dashed bars) were synthesized as duotopes (white and black bars) joined either

5

directly or separated by one or two ß-alanine spacers (B, B-B). Reactivity of mAb 2c with

6

duotopes was recorded by fluorescence signal intensities from high resolution laser scans.

- 38 -

A % of maximum fluorescence intensity

100

HSV-1 IgG F(ab') 2

75

Fab scFv

50

25

0 0.01

0.1

1

10

100

1000

Antibody concentration (nM)

B % of maximum fluorescence intensity

100

HSV-2

75

IgG F(ab') 2 Fab

50

25

0 0.01

0.1

1

10

100

1000

Antibody concentration (nM)

1 2 3

FIG. 5. Equilibrium-binding curves for mAb 2c, 2c-F(ab’)2, 2c-Fab, and 2c-scFv as determined

4

by flow cytometry. Binding activities to (A) HSV-1 F or (B) HSV-2 G infected Vero cells at

5

indicated concentrations are shown as percent of maximum median fluorescence intensity.

6

Experiments were twice performed in triplicate; bars represent standard deviations.

- 39 -

1

A MAb 2c (nM)

30

20

10

0 100%

50% Neutralization

pre-attachment

post-attachment

Polyclonal human IgG (nM)

B 5000 4000 3000 2000 1000 0 100%

50% Neutralization

2 3

FIG. 6. Inhibition of HSV-1 virion attachment by mAb 2c to target cells. Serial dilutions of (A)

4

mAb 2c (0.98 – 125 nM) or (B) polyvalent human gamma globulin (Intratect®) (0.33 - 42 µM)

5

were added to Vero cell monolayers in 96-well microtiter plates following pre-incubation with

6

100 TCID50 HSV-1 (pre-attachment neutralization) or post-adsorbtion of 100 TCID50 HSV-1 to

7

target cells (post-attachment neutralization). The highest antibody and polyvalent human IgG

8

titer, respectively, preventing virus induced cytopathic effect (CPE) in ten individual inoculated

9

cell monolayers to 100% and 50% relative to controls were determined after 72 h incubation at

10

37°C and being considered as the endpoint. Standard errors of the mean of three independent

11

experiments were < 0.1. - 40 -

10,000

Antibody concentration (nM)

A

HSV-1 F HSV-2 G

1,000

100

10

1 IgG

F(ab')2 bivalent

Fab monovalent

Antibody concentration (nM)

B 2c-Fab 3,000

2c-Fab + anti-Fab IgG

2,000

1,000

2c-IgG 0 Complete neutralization of HSV-1(100 TCID50 )

1 2

FIG. 7. Effect of valency of anti-gB antibodies on in vitro neutralization of HSV. (A) Bivalent

3

antibodies mAb 2c (IgG) and 2c-F(ab’)2, and monovalent 2c-Fab were incubated in serial

4

dilutions for 1 h with 100 TCID50 HSV-1 F or HSV-2 G before inoculation onto Vero cells. CPE

5

was scored 72 h later as described in Fig. 3. Shown are antibody concentrations required to

6

neutralize 100% of the viral inoculum from one of three representative replicate experiments. (B)

7

Antiviral activity of 2c-Fab fragments crosslinked with murine anti-Fab IgGs.

- 41 -

1 A HSV-1 Medium

125 nM mAb 2c

250 nM mAb 2c

500 nM mAb 2c

500 nM mAb 2c

1000 nM mAb 2c

2000 nM mAb 2c

B HSV-2 Medium

2 3 4

Fig. 8. Cell spread inhibition of HSV-1 and HSV-2 on Vero cell monolayers by mAb 2c. Vero

5

cell monolayers infected with 100 TCID50/500 µl of HSV-1 (A) and HSV-2 (B), respectively,

6

were treated with increasing concentrations of mAb 2c as indicated. The concentration-dependent

7

plaque reduction on Vero cell monolayers is shown by light microscopy after 48 h incubation.

8

The shown microscopic pictures are representative of multiple image sections observed in two

9

independent experiments. Bar = 200 µm.

10

- 42 -

1 2 3

FIG. 9. Inhibition of HSV-1 cell-to-cell spread. Immunofluorescence (upper panel) and overlays

4

of light microscopy and immunofluorescence pictures (lower panel) of confluent Vero cell

5

monolayers 48 h after infection with HSV-1 (400 TCID50/500 µl) treated with pooled human

6

polyclonal HSV-1-neutralizing sera (1:20), mAb 2c (IgG, 500 nM), 2c-F(ab’)2 (500 nM), or 2c-

7

Fab (3,000 nM). Viral antigens were visualized by immunostaining with FITC-conjugated

8

polyclonal goat anti-HSV serum. Uninfected cells (mock) used as control showed no background

9

staining (not shown).

- 43 -

1 2 100

15 mg/kg

4 5 6

Percent survival

3 80 60 40

5 mg/kg

20 2.5 mg/kg

7

PBS

0

8

0

5

10

15

20

25

30

Time post infection (days)

9 10 11 12

Fig. 10. Dose-dependent survival of mAb 2c treated immunodeficient mice. NOD/SCID mice

13

received different single dosages of mAb 2c intravenously 24 h before intravaginal challenge

14

with 1x106 TCID50 HSV-1. Animals per group n =7 for PBS, n=9 all other groups.

- 44 -

1 2

A

100

Percent survival

3 4 5 6

80 60

mAb 2c PBS

40 20

7

0 0

5

10

8

15

20

25

30

Time post infection (days)

9

B virus titer (TCID50 / vaginal lavage)

10 7

10 11 12 13

10 6 10 5 10 4 10 3

PBS

10 2

mAb 2c

10 1 0.1

14

0

1

2

3

4

5

6

7

8

9

10

Time post infection (days)

15 16 17

Fig. 11. Protection of NOD/SCID mice by systemically applied mAb 2c against HSV-1

18

dissemination. (A) Starting 24 h post-infection mice received 15 mg/kg mAb 2c three times

19

intraveniously at time points indicated by arrows (24 h, 40 h, 56 h). (B) Vaginal virus titers of

20

mAb 2c or control treated mice were determined from vaginal irrigations cultured on Vero cell

21

monolayers. Error bars indicate standard deviation. Infected animals per group n =6 for PBS, n=9

22

for mAb 2c.

- 45 -

1 2

TABLE 1. Apparent equilibrium constants of mAb 2c and derived antibody fragments for

3

binding to HSV-1 F or HSV-2 G infected Vero cells. IgG a

F(ab’)2

Fab

bivalent

KD (nM)

scFv monovalent

HSV-1 F

10.2

6.9

17.3

19.2

HSV-2 G

10.7

8.8

17.7

n.d.

4 5

a

6

equilibrium binding curves determined by flow cytometry (Fig. 5) to the Marquardt-Levenberg

7

equation.

KD values for binding to gB on HSV-infected cells were determined by fitting the data from the

8 9

- 46 -

Suggest Documents