WO1996015273A1

WO1996015273A1 - Method for identifying protective antigens for eliciting neutralizing antibodies

Info

Publication number: WO1996015273A1
Application number: PCT/US1995/014907
Authority: WO
Inventors: Dennis R. Burton; Paul W. H. I. Parren; Anthony R. Williamson; Carlos F. Barbas, Iii
Original assignee: The Scripps Research Institute
Priority date: 1994-11-10
Filing date: 1995-11-13
Publication date: 1996-05-23
Also published as: JPH10509237A; CA2204863A1; EP0787211A1; AU4162096A

Abstract

The present invention provides a novel method for the identification of antigens which may be useful for inducing neutralizing antibodies. The method is based on the use of antigens as solid phase capture reagents to bind to a population of antibodies from a subject having an infection and high neutralizing serum titers through the period of natural infection. The method is particularly suited for screening libraries of cloned antibodies, such as phage display combinatorial antibodies.

Description

METHOD FOR IDENTIFYING PROTECTIVE ANTIGENS

FOR ELICITING NEUTRALIZING ANTIBODIES

This invention was made with government support from grant no. Al 33292, from the National Institutes of Health. The government has certain rights in this invention. BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to the field of neutralizing antigens and antibodies and specifically to a method for identifying a neutralizing antibody that binds to an epitope on a preselected antigen through the use of antigens as solid-phase capture reagents that bind the antibody and the panning of a combinatorial antibody library.

The ability of a pre-selected antigen to select a neutralizing antibody from an antibody library provides a rapid assessment of a candidate vaccine.

2. Description of Related Art

Generally, any preparation of antibody-producing cells can be used as a source for cloning antibody molecules. The use of filamentous phage display vectors, referred to as phagemids, has been repeatedly shown to allow the efficient preparation of large libraries of monoclonal antibodies having diverse and novel immunospecificities. The technology uses a filamentous phage coat protein membrane anchor domain as a means for linking gene-product and gene during the assembly of filamentous phage replication, and has been used for the cloning and expression of antibodies from combinatorial libraries (Kang, et al., Proc. Natl. Acad. Sci., U.S.A., 88:4363, 1991). Combinatorial libraries of antibodies have been produced using both the cpVIII membrane anchor and the cpIII membrane anchor (Barbas, et al., Proc. Natl. Acad. Sci., U.S.A., 88:7978, 1991). The diversity of a phagemid library can be manipulated to increase and/or alter the immunospecificities of the monoclonal antibodies of the library to produce and subsequently identify additional, desirable, human monoclonal antibodies. For example, the heavy (H) chain and light (L) chain immunoglobulin molecule encoding genes can be randomly mixed (shuffled) to create new HL pairs in an assembled immunoglobulin molecule. Additionally, either or both the H and L chain encoding genes can be mutagenized in a complementarity determining region (CDR) of the variable region of the immunoglobulin polypeptide, and subsequently screened for desirable immunoreaction and neutralization capabilities.

Since its conception just a few years ago, the combinatorial approach has allowed unprecedented access to the human antibody response. The cloning of antibodies from preimmune, immune, and memory compartments of the human immune system has been demonstrated. Combinatorial antibodies have been shown to provide an accurate functional reflection of the natural response as demonstrated by the ability of cloned antibodies to compete with serum antibodies for binding antigens.

Protection from viral disease has traditionally been associated with the pre-existence in serum of antibodies capable of neutralizing virus in vitro. Indeed, vaccines are frequently assessed on the irability to elicit neutralizing antibody response. In the case of HIV- 1, there was initial optimism about the likely efficacy of subunit vaccines given that vaccinee sera from several trials were capable of neutralizing laboratory isolates of virus in vitro (Berman, et al., Nature, 345:622, 1990; Cohen, et al., Science, 262:980. 1993). The grounds for optimism were shaken when it was found that the vaccinee sera were largely ineffective against primary isolates of HIV- 1

(Cohen, etal., supra). Hyperimmune pooled human plasma preparations are capable of neutralizing a number of primary isolates (Sawyer, et al., J. Virol, 68:1342, 1994; Mascola, et al., J. Infect. Dis., 169:48, 1994; Wrin, et al., J. Acq. lmm. Def Synd., 7:21 1, 1994) but they represent a combination of specificities that might be difficult to elicit by all except the most complex vaccines (Letvin, et al., N. Engl J. Med,

329:1400, 1993). A single antibody able to effectively neutralize a broad spectrum of primary isolates would validate the vaccine approach and would provide a template for vaccine design. Furthermore, it would constitue a reagent for passive immunotherapy, such as the interruption of maternal-fetal transmission. Vaccines are frequently assessed on their ability to elicit neutralizing antibody responses. Normally, tests are first carried out on animals and then on humans. Therefore, there is a need for an in vitro method for predicting the therapeutic efficacy of candidate vaccines. Folgori, et al., (EMBOJ., 13:2236, 1994) described one such variation of a vaccine screening method. This study used disease-specific epitopes from phage-displayed peptide libraries, called phagotopes, and reacted them with human sera to identify antigenic mimics referred to as mimotopes. Two mimotopes representing two epitopes of human hepatitis B virus envelope protein (HbsAg) were identified with this method. When injected into mice, the reactive phagotopes induced an immune response specific against HbsAg, demonstrating that phagotopes could be identified which induce relevant antibodies.

Thus, Folgori, et al., peptide-library screening method describes an antigenindependent procedure to identify peptide-based immunogens without having to use in vivo inoculations of animals or humans. In contrast to the use of naive sera in Folgori, et al., th present invention uses a phage-display Fab library derived from human sera containing neutralizing antibodies isolated from patients having high neutralizing titers to a particular disease to screen candidate vaccines. The method fo Folgori, et al, will identify strong immunogenic epitopes on disease-related pathogens. These mimotopes, however, will not necessarily reflect the epitopes that induce a neutralizing antibody response. In the natural antiboy response to HIV, for example, the majority of antibodies elicited are non-neutralizing. Only a minority are the strong neutralizing type, such as IgG1 b12 of the present invention. The method of the invention identifies antigens that are cpable of preferentially binding this minority population of neutralizing antibodies, and may upon use as a vaccine elicit these strongly neutralizing antibodies in an immune response. The invention also provides screening of candidate vaccines that are not displayed on a phage, whereas Folgori, et al., utilizes a phage-display system of random peptides, which may not reflect the in vivo antigen\epitope presentation structure. Most importantly, the present invention provides the identification and selection of specific reactive phagedisplayed Fabs, useful for subsequent screening and determination of neutralizing ability through in vitro assays. SUMMARY OF THE INVENTION

The present invention is based on assessing candidate, preselected antigens for their ability to elicit a protective antibody response by their ability to select neutralizing antibodies from libraries prepared from human donors who have high neutralizing serum titers through natural infection. The invention provides the advantage of assessing such antigens for a human response, rapidly and without the step of animal testing. The method of the invention is useful not only for identifying those antigens which are most effective or potent in stimulating a protective immune response, but also for identifying neutralizing antibodies useful for passive immunization therapy. Thus, in a first embodiment, the invention provides a method for identifying a protective or neutralizing antigen that binds to a protective antibody comprising contacting a preselected candidate protective antigen with an antibody molecule, under conditions which allow an epitope of the antigen to bind to the antibody molecule and form an immunocomplex; removing the antibody molecule bound to the epitope; and determining the protective ability of the antibody molecule, thereby predicting the ability of the antigen to elicit a protective immune response. The protective ability of the antigen or antibody is preferably assessed in vitro.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGURE 1 shows an assay for the neutralization of primary isolates of HIV- 1 by IgG1 b12. Virus neutralization was assessed using PHA-stimulated PBMCs as indicator cells and determination of extracellular p24 as the reporter assay essentially as described (E S. Daar, et al., Proc. Natl Acad Sci., USA, 87:6574, 1990; D D. Ho, et al, J. Virol, 65:489, 1991). Virus (50 TCID₅₀) and antibody at varying concentration were incubated together for 30 minutes at 37°C before addition to PHA-stimulated PBMCs. Virus replication was assessed after 5-7 days by p24 ELISA measurement. The designation, location and disease status of the virus donors were as follows:■, VS (New York, acute);▼, N_70-2 (New Orleans, asymptomatic);▲, AC (San Diego, AIDS);●, LS (Los Angeles, AIDS);□, NYC-A (New York, unknown);▽, WM (Los Angeles, AIDS); Δ, RA (New York, acute);♢, JP (New York, acute). The moleculariy cloned HIV-1 virus JR-CSF (♦) and HIV-1 isolate JR-FL (O) have been described (W.A. O'Brien, etai, Nature, 348:69, 1990; W.A. O'Brien, et al., J. Virol, 66:3125, 1992; W.A. O'Brien, et al., J. Virol, 68:5264, 1994).

FIGURE 2 shows the reactivity of IgG1 b12 with a panel of international isolates of HIV-1. Binding ratios of 0.50 or greater were deemed to represent strong antibody reactivity; ratios from 0.25-0.50 were considered indicative of moderate reactivity; values <0.25 were designated as representative of essentially negative Mab reactivity, strong,

moderate reactivity; numbers in parentheses refer to the number of viruses of each clade examined.

FIGURE 3 shows the neutralizing activity of Fab8, as measured by plaque reduction. FIGURE 3 A shows activity against HSV-1 and FIGURE 3B shows activity against HSV-2. Purified Fab8 neutralized HSV-1 with a 50% inhibition at about 0.25 μg/ml and with an 80% inhibition at 0.6 μg/ml, while HSV-2 was neutralized with a 50% inhibition at about 0.05 μg/ml and an 80% inhibition at 0.1 μg/ml.

FIGURE 4 shows an inhibition of plaque development assay. Purified Fab8 inhibited the development of plaques when applied 4 hours post-infection (hpi) on monolayers infected with HSV-1 (FIGURE 4 A, FIGURE 4B) or HSV-2 (FIGURE 4C, FIGURE 4D) 4 hours post infection. FIGURE 4A shows statistically significant reduction in plaque size was observed at concentrations of 5 and 1 μg/ml (*=p( 0.01), with an approximate 50% reduction in plaque size at 5 μg/ml. The number of plaques was also dramatically reduced at Fab concentrations of 5 and 25 μg/ml (FIGURE 4B, FIGURE 4D). At 25 μg/ml and 72 hrs hpi plaque development in HSV-2 infected monolayers was completely inhibited (FIGURE 4C, FIGURE 4D). FIGURE 4E shows an inhibition of plaque development assay with HS V-2 infected monolayers at a number of different Fab concentrations 86 hpi.

FIGURE 5 shows a post-attachment neutralization assay. Fab8 reduced HSV-1 infectivity after virion attachment. FIGURE 5 A shows the percentage of plaque reduction pre- and post-attachment at different Fab concentrations. FIGURE 5B shows the post-/pre-attachment neutralization ratio at different Fab concentrations.

FIGURE 6 shows the identification of the protein recognized by Fab8. SDS-PAGE of total proteins from HSV-2 infected Vero cells (lanes 1) and of the product of immunoprecipitation with Fab8 (lanes 2). Western blots performed in parallel were probed with a mouse monoclonal anti gD antibody (MAB α-gD) and for the purpose of control, a rabbit polyclonal anti-HSV-2 preparation (RAB α-HSV2). The Coomassie stain of a gel run in parallel is also shown. Fab8 immunoprecipitated a band of apparent molecular weight 48-50kD which was recognized by a mouse monoclonal specific for gD, but not by mouse monoclonal antibodies against other HSV glycoproteins.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a rapid method for identification of protective, neutralizing antigens and the neutralizing antibodies that bind to a preselected candidate protective antigen. The ability of a preselected antigen to specifically select neutralizing antibodies from an antibody library is used as a predictive tool to assess it potential value as a vaccine. Neutralizing antibodies are obtained from libraries prepared from human donors who have high neutralizing serum titers through natural infection. The invention provides the advantage of assessing the human antibody reponse to such antigens, rapidly and without the step of prior animal testing.

The invention provides a method for identifying a protective antigen that binds to a protective antibody comprising the steps of: a) contacting a preselected candidate protective antigen with an antibody molecule, under conditions which allow an epitope of the antigen to bind to the antibody molecule and form an immunocomplex, b) removing the antibody molecule bound to the epitope; and c) determining the protective ability of the antibody molecule, thereby predicting the protective ability of the antigen. Preferably, the antibody molecule in the method of the invention is in a phage display combinatorial library and contacting as used in step a) includes panning the antigen with the library. Step b), removing or collecting the antibody molecule that is bound, can be performed by any of the common methods known to those of skill in the art for eluting antibody molecules bound to antigen. Any phage expressing an antibody molecule on its surface which binds to an epitope on the antigen, and is determined to be a neutralizing antibody, can then be clonally isolated and its DNA sequenced according to common methods known to those of skill in the art.

Neutralization or protective ability of the antibody molecule does not need to be assessed for individual clones. Testing can be performed on a pooled preparation of antibody molecules following panning with the library. The term "neutralizing" or "protective" as used herein refers to an antibody, in the case of passive immunization, or antigen, in the case of a vaccine, which provides an enhanced immune response in comparison to the response prior to administration of the antibody or antigen. Therefore, the term "protective antigen" means that the amount of antigen administered is of sufficient quantity to increase the subject's immune response to the antigen, for example, to HIV.

Step c) of the method of the invention includes determining the protective, or neutralizing ability of the antibody molecule identified. This step is typically performed in vitro. Neutralizing ability can be determined by any commonly used methods known to those of skill in the art, including, but not limited to antibody inhibition of infectivity by plaque formation (C. V. Hanson, et al., J. Clin. Microbiol, 28:2030, 1990) or syncytial formation (Nara, et al., AIDS Res. Human Retroviruses, 3 :283 : 1987). For other methods for determining neutralizing ability of an antibody, see Current Protocols in Immunology, Coligan, et al., Wiley Interscience, 1994, incorporated herein by reference.

The term "antibody" or "antibody molecule" as used in this invention includes intact molecules as well as fragments thereof, such as Fab, F(ab')₂, and Fv which are capable of binding the epitopic determinant. These antibody fragments retain some ability to selectively bind with its antigen or receptor and are defined as follows:

(1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain;

(2) Fab', the fragment of an antibody molecule can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule;

(3) (Fab')₂, the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab')₂ is a dimer of two Fab' fragments held together by two disulfide bonds;

(4) Fv, defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and

(5) Single chain antibody ("SCA"), defined as a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule.

Methods of making these fragments are known in the art. (See for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1988), incorporated herein by reference). Therefore, the phrase "antibody molecule" in its various forms as used herein contemplates both an intact antibody (immunoglobulin) molecule and an immunologically active portion of an antibody (immunoglobulin) molecule. The term "monoclonal antibody" refers to a population of one species of antibody molecule of determined (known) antigen-specificity. A monoclonal antibody contains only one species of antibody combining site capable of immunoreacting with a particular antigen and thus typically displays a single binding affinity for that antigen. A monoclonal antibody may therefore contain a bispecific antibody molecule having two antibody combining sites, each immunospecific for a different antigen. Preferably, the first antibody molecule affixed to a solid support in the method of the invention is a monoclonal antibody. In addition, the antibody molecules in a phage display combinatorial library are also monoclonal antibodies.

As used in this invention, the term "epitope" means any antigenic determinant on an antigen to which the paratope of an antibody binds. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.

The word "complex" as used herein refers to the product of a specific binding agent ligand reaction. An exemplary complex is an immunoreaction product formed by an antibody-antigen reaction.

The term "antigen" refers to a polypeptide or protein that is able to specifically bind to (immunoreact with) an antibody and form an immunoreaction product (immunocomplex). The site on the antigen with which the antibody binds is referred to as an antigenic determinant or epitope.

The method of the invention for detection of protective or neutralizing antibodies that bind to a preselected epitope on an antigen is performed in vitro, for example, in immunoassays in which the antibodies can be identified in liquid phase or bound to a solid phase carrier. Preferably, the method is performed with a preselected antigen bound to a solid support. Alternatively, the preselected antigen can be bound to an antibody molecule. For example, the antibody or antibody/antigen complex can first be bound o a solid support. Examples of types of immunoassays which can be utilized to detect protective antibodies, include competitive and non-competitive immunoassays in either a direct or indirect format. Examples of such immunoassays are the radioimmunoassay (RIA) and the sandwich (immunometric) assay. Detection of the antibodies can be done utilizing immunoassays which are run in either the forward, reverse, or simultaneous modes, including competition immunoassays and immunohistochemical assays on physiological samples. Preferably, the method of the invention utilizes a forward immunoassay. Those of skill in the art will know, or can readily discern, other immunoassay formats without undue experimentation.

Solid phase-bound antigen molecules are bound by adsorption from an aqueous medium, although other modes of affixation, such as covalent coupling or other well known means of affixation to the solid matrix can be used. Preferably, the antigen molecule is bound to a support before forming an immunocomplex with antibody, however, the immunocomplex can be formed prior to binding the complex to the solid support.

Non-specific protein binding sites on the surface of the solid phase support are preferably blocked. After adsorption of solid phase-bound antigen, an aqueous solution of a protein free from interference with the assay such as bovine, horse, or other serum albumin that is also free from contamination with the antigen or antibody is admixed with the solid phase to adsorb the admixed protein onto the surface of the antigen-containing solid support at protein binding sites on the surface that are not occupied by the antigen molecule.

A typical aqueous protein solution contains about 2-10 weight percent bovine serum albumin in PBS at a pH of about 7-8. The aqueous protein solution-solid support mixture is typically maintained for a time period of at least one hour at a temperature of about 4°-37°C and the resulting solid phase is thereafter rinsed free of unbound protein. The antigen can be bound to many different carriers and used to detect neutralizing antibodies in a sample. Examples of well-known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amyloses, natural and modified celluloses, polyacrylamides, agaroses, and magnetite. The nature of the carrier can be either soluble or insoluble for purposes of the invention. Those skilled in the art will know of other suitable carriers for binding an antigen, or will be able to ascertain such, using routine experimentation.

In addition, if desirable, an antibody for detection in these immunoassays can be detectably labeled in various ways. There are many different labels and methods of labeling known to those of ordinary skill in the art. Examples of the types of labels which can be used in the present invention include enzymes, radioisotopes, fluorescent compounds, colloidal metals, chemiluminescent compounds, and bio-luminescent compounds. Those of ordinary skill in the art will know of other suitable labels for binding to the monoclonal antibodies of the invention, or will be able to ascertain such, using routine experimentation. Furthermore, the binding of these labels can be done using standard techniques common to those of ordinary skill in the art.

For purposes of the invention, neutralizing antibodies that bind to an antigen may be detected using any sample containing a detectable amount of antigen. A sample can be a liquid such as urine, saliva, cerebrospinal fluid, blood, serum and the like. A sample can also be a cell lysate, viral lysate or other crude, semi-purified, or purified antigen preparation.

Preferably, the antigen is a purified protein, such as a recombinant protein or a fusion protein, expressed in E. coli, CHO cells or baculovirus, for example; a protein antigen expressed on the surface of vaccinia virus; a chemically modified protein antigen; or a protein antigen which has been purified by several techniques. Other antigen preparations for use in the method of the invention will be known to those of skill in the art. The preselected antigen can be any antigen such as a bacterial, viral, parasitic, fungal, tumor and self-antigen. Examples of viral antigens include antigens derived from viruses selected from the group consisting of hepatitis B virus (HBV), human immunodeficiency virus (HIV), influenza A virus, Epstein Barr virus (EB V), herpes simplex virus (HSV), respiratory syncytial virus (RSV), human cytomegalovirus (HCMV), varicella zoster virus (VZV), and measles virus. More specifically, the preselected antigen may be HSV glycoprotein D or HIV glycoprotein 120 (gp120).

The specific concentrations of the antibody and antigen, the temperature and time of incubation, as well as other assay conditions, can be varied, depending on such factors as the concentration of the antigen in the sample, the nature of the sample and the like. Those of skill in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation.

For example, the method of the invention may be run at 4° -45°C, and preferably at about 15°-37°C. Each incubation step may be as long as 72 hours. Other steps such as washing, stirring, shaking, filtering, or pre-assay extraction of antigen, and the like, may, of course be added to the assay, as may be desired or necessary for a particular situation.

Although any preparation containing a mixture of antibodies or antibody molecules can be utilized as a source of the antibody, preferably, a combinatorial library is used, and most preferably, a phage display library as described in Barbas, et al., (Combinatorial immunoglobulin libraries on the surface of phage (Phabs): Rapid selection of antigen-specific Fabs., Methods: A Companion to Methods in Enzymol., (Lerner and Burton, eds), vol. 2, pp. 119-124, Academic Press, Orlando, 1991; Barbas, et al., Proc. Natl. Acad Sci., U.S.A. 88:7978, 1991), incorporated herein by reference. (See also for reference purposes, Huse, et al., Science, 246:1275-1281, 1989).

As a working model to illustrate the usefulness of the method of the present invention, the Examples describe the method of the invention as used to identify HIV-neutralizing human antibodies. One of skill in the art could now identify a variety of neutralizing antibody molecules using the exemplified model and method of the invention. The methods are based generally on the use of combinatorial libraries of antibody molecules which can be produced from a variety of sources, and include naive libraries, modified libraries, and libraries produced directly from donors. Preferably, as exemplified herein, human donors having high titers of neutralizing antibodies as a result of infection are the source of antibody, for example, donors exhibiting an HIV-specific immune response. The method of the invention provides a rapid screen to determine the likelihood of the antigen to be useful as a vaccine for eliciting a protective or neutralizing antibody response.

The combinatorial library production and manipulation methods have been extensively described in the literature, and will not be reviewed in detail herein, except for those features required to make and use unique embodiments of the present invention. However, the methods generally involve the use of a filamentous phage (phagemid) surface expression vector system for cloning and expressing antibody species of the library. Various phagemid cloning systems to produce combinatorial libraries have been described by others. See, for example the preparation of combinatorial antibody libraries on phagemids as described by Kang, et al., Proc. Natl. Acad Sci., USA, 88:4363-4366 (1991); Barbas, et al., Proc. Natl Acad. Sci., USA, 88:7978-7982 (1991); Zebedee, et al., Proc. Natl. Acad Sci., USA, 89.3175-3179 (1992); Kang, et al., Proc. Natl Acad Sci., USA, 88:11120-11123 (1991); Barbas, et al., Proc. Natl Acad Sci., USA, 89:4457-4461 (1992); and Gram, et al., Proc. Natl. Acad Sci., USA, 89:3576-3580 (1992), which references are hereby incorporated by reference.

In the working Examples described for illustrative purposes herein, the method involves preparing a phagemid library of human monoclonal antibodies by using donor immune cell messenger RNA from an HSV or HIV-infected donor. The donors can be symptomatic of infection, but the donor can also be asymptomatic, as the resulting library may contain a substantially higher number of neutralizing human monoclonal antibodies. Additionally, because HIV infection is often accompanied by other diseases, the patient may optionally present substantial symptoms of one or more other diseases typically associated with symptomatic or asymptomatic HIV infection. Those of skill in the art will be able to use the working example as a model for identifying and assaying other various desired neutralizing antibody molecules.

The method for producing a recombinant human monoclonal antibody generally involves (1) preparing separate H and L chain-encoding gene libraries in cloning vectors using human immunoglobulin genes as a source for the libraries, (2) combining the H and L chain encoding gene libraries into a single dicistronic expression vector capable of expressing and assembling a heterodimeric antibody molecule, (3) expressing the assembled heterodimeric antibody molecule on the surface of a filamentous phage particle, (4) isolating the surface-expressed phage particle using immunoaffinity techniques such as panning of phage particles against a preselected antigen, thereby isolating one or more species of phagemid containing particular H and L chain-encoding genes and antibody molecules that immunoreact with the preselected antigen.

As a further characterization of the present invention the nucleotide and corresponding amino acid residue sequence of the antibody molecule's H or L chain encoding gene is determined by nucleic acid sequencing.

Sequence comparisons of identified immunoreactive monoclonal antibody variable chain region sequences are aligned based on sequence homology, and groups of related antibody molecules are identified in which heavy chain or light chain genes share substantial sequence homology.

The isolation of a particular vector capable of expressing an antibody of interest involves the introduction of the dicistronic expression vector into a host cell permissive for expression of filamentous phage genes and the assembly of phage particles. Where the binary vector system is used, both vectors are introduced in the host cell. Typically, the host is E. coli. Thereafter, a helper phage genome is introduced into the host cell containing the immunoglobulin expression vector(s) to provide the genetic complementation necessary to allow phage particles to be assembled. The resulting host cell is cultured to allow the introduced phage genes and immunoglobulin genes to be expressed, and for phage particles to be assembled and shed from the host cell. The shed phage particles are then harvested (collected) from the host cell culture media and screened for desirable immunoreaction and neutralization properties.

The harvested particles are "panned" for immunoreaction with a preselected antigen. For example, in the method of the invention, the preselected antigen, such as a virus or isolated viral antigen, is bound to a solid phase before the particles are panned. The strongly immunoreactive particles are then collected, and individual species of particles are clonally isolated and further screened for immunoreactivity, or as exemplified herein, HSV or HIV neutralization. Phage which produce neutralizing antibodies, for example, are selected and used as a source of a human HIV neutralizing monoclonal antibody. Depending on the antigen and neutralizing ability of the antibody, a single clone of antibody or a "cocktail" of pooled neutralizing antibodies may be most effective for treatment of a subject.

Because an immunoaffinity isolated antibody composition includes phage particles containing surface antibody, one embodiment involves the manipulation of the resulting cloned genes to truncate the immunoglobulin-coding gene such that a soluble Fab fragment is secreted by the host E. coli cell containing the phagemid vector rather than the production of a phagemid having surface antibody. Thus, the resulting manipulated cloned immunoglobulin genes produce a soluble Fab which can be readily characterized in ELISA assays for epitope binding studies, in competition assays with antibody molecules of known epitopic specificity, and in functional assays, such as neutralization assays for example. The solubilized Fab provides a reproducible and comparable antibody preparation for comparative and characterization studies.

The preparation of soluble Fab is generally described in the immunological arts, and can be conducted as described herein in the Examples, or as described by Burton, et al, (Proc. Natl. Acad Sci., USA, 88:10134-10137, 1991). The preparation of human monoclonal antibodies of this invention depends, in one embodiment, on the cloning and expression vectors used to prepare the combinatorial antibody libraries described herein. The cloned immunoglobulin heavy and light chain genes can be shuttled between lambda vectors, phagemid vectors and plasmid vectors at various stages of the methods described herein.

The phagemid vectors produce fusion proteins that are expressed on the surface of an assembled filamentous phage particle. A preferred phagemid vector of the present invention is a recombinant DNA (rDNA) molecule containing a nucleotide sequence that codes for and is capable of expressing a fusion polypeptide containing, in the direction of amino- to carboxy-terminus, (1) a prokaryotic secretion signal domain, (2) a heterologous polypeptide defining an immunoglobulin heavy or light chain variable region, and (3) a filamentous phage membrane anchor domain. The vector includes DNA expression control sequences for expressing the fusion polypeptide, preferably prokaryotic control sequences.

The filamentous phage membrane anchor is preferably a domain of the cpIII or cpVIII coat protein capable of associating with the matrix of a filamentous phage particle, thereby incorporating the fusion polypeptide onto the phage surface. The secretion signal is a leader peptide domain of a protein that targets the protein to the periplasmic membrane of gram negative bacteria. A preferred secretion signal is a pelB secretion signal. The predicted amino acid residue sequences of the secretion signal domain from two pelB gene product variants from Erwinia carotova are described in Lei, et al., (Nature, 331:543-546, 1988).

The leader sequence of the pelB protein has previously been used as a secretion signal for fusion proteins. Better, et al., Science.240:1041-1043 (1988); Sastry, et al., Proc. Natl Acad. Sci., USA, 86:5728-5732 (1989); and Mullinax, et al., Proc. Natl Acad. Sci., USA, 87:8095-8099 (1990). Amino acid residue sequences for other secretion signal polypeptide domains from E. coli useful in this invention as described in Oliver, (Escherichia coli and Salmonella typhimurium, Neidhard, F.C. (ed ), American Society for Microbiology, Washington, D C, 1:56-69, 1987). Preferred membrane anchors for the vector are obtainable from filamentous phage M13, fl, fd, and equivalent filamentous phage. Preferred membrane anchor domains are found in the coat proteins encoded by gene III and gene VIII. The membrane anchor domain of a filamentous phage coat protein is a portion of the carboxy terminal region of the coat protein and includes a region of hydrophobic amino acid residues for spanning a lipid bilayer membrane, and a region of charged amino acid residues normally found at the cytoplasmic face of the membrane and extending away from the membrane.

In the phage fl, gene VIII coat protein's membrane spanning region comprises residue Trp-26 through Lys-40, and the cytoplasmic region comprises the carboxy-terminal 11 residues from 41 to 52 (Ohkawa, et al., J. Biol Chem., 256:9951-9958, 1981). An exemplary membrane anchor would consist of residues 26 to 40 of cp VIII. Thus, the amino acid residue sequence of a preferred membrane anchor domain is derived from the M13 filamentous phage gene VIII coat protein (also designated cpVIII or CP 8). Gene VIII coat protein is present on a mature filamentous phage over the majority of the phage particle with typically about 2500 to 3000 copies of the coat protein.

In addition, the amino acid residue sequence of another preferred membrane anchor domain is derived from the Ml 3 filamentous phage gene III coat protein (also designated cpIII). Gene III coat protein is present on a mature filamentous phage at one end of the phage particle with typically about 4 to 6 copies of the coat protein.

For detailed descriptions of the structure of filamentous phage particles, their coat proteins and particle assembly, see the reviews by Rached, et al., Microbiol Rev., 50:401-427 (1986); and Model, et al., in "The Bacteriophages: Vol. 2", R. Calendar, ed. Plenum Publishing Co., pp. 375-456 (1988).

DNA expression control sequences comprise a set of DNA expression signals for expressing a structural gene product and include both 5' and 3' elements, as is well known, operatively linked to the cistron such that the cistron is able to express a structural gene product. The 5' control sequences define a promoter for initiating transcription and a ribosome binding site operatively linked at the 5' terminus of the upstream translatable DNA sequence.

To achieve high levels of gene expression in E. coli, it is necessary to use not only strong promoters to generate large quantities of mRNA, but also ribosome binding sites to ensure that the mRNA is efficiently translated. In E. coli, the ribosome binding site includes an initiation codon (AUG) and a sequence 3-9 nucleotides long located 3-11 nucleotides upstream from the initiation codon (Shine, et al., Nature, 254:34 (1975). The sequence, AGGAGGU, which is called the Shine-Dalgarno (SD) sequence, is complementary to the 3' end of E coli 16S rRNA. Binding of the ribosome to mRNA and the sequence at the 3' end of the mRNA can be affected by several factors:

(i) The degree of complementarity between the SD sequence and 3' end of the 16S rRNA.

(ii) The spacing and possibly the DNA sequence lying between the SD sequence and the AUG. Roberts, et al., Proc. Natl Acad. Sci., USA, 76:760, (1979a); Roberts, et al., Proc. Natl Acad. Sci. USA, 76:5596 (1979b); Guarente, et al., Science, 209.1428 (1980); and Guarente, et al, Cell, 20:543 (1980). Optimization is achieved by measuring the level of expression of genes in plasmids in which this spacing is systematically altered. Comparison of different mRNAs shows that there are statistically preferred sequences from positions -20 to +13 (where the A of the AUG is position 0). Gold, et al., Annu. Rev. Microbiol, 35:365 (1981). Leader sequences have been shown to influence translation dramatically. Roberts, et al., 1979 a, b supra (iii) The nucleotide sequence following the AUG, which affects ribosome binding. Taniguchi, et al., J. Mol Biol, 118:533 (1978).

The 3' control sequences define at least one termination (stop) codon in frame with and operatively linked to the heterologous fusion polypeptide. In preferred embodiments, the vector utilized includes a prokaryotic origin of replication or replicon, i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule extra chromosomally in a prokaryotic host cell, such as a bacterial host cell, transformed therewith. Such origins of replication are well known in the art. Preferred origins of replication are those that are efficient in the host organism. A preferred host cell is E. coli. For use of a vector in E. coli, a preferred origin of replication is ColE1 found in pBR322 and a variety of other common plasmids. Also preferred is the pl5A origin of replication found on pACYC and its derivatives. The ColE1 and p15A replicon have been extensively utilized in molecular biology, are available on a variety of plasmids and are described at least by Sambrook, et al., in "Molecular Cloning: a Laboratory Manual", 2nd edition, Cold Spring Harbor Laboratory Press (1989).

The ColE1 and p15A replicons are particularly preferred for use in one embodiment of the present invention where two "binary" plasmids are utilized because they each have the ability to direct the replication of plasmid in E. coli while the other replicon is present in a second plasmid in the same E. coli cell. In other words, ColE1 and p15A are non-interfering replicons that allow the maintenance of two plasmids in the same host (see, for example, Sambrook, et al., supra, at pages 1.3-1.4). This feature is particularly important in the binary vectors embodiment of the present invention because a single host cell permissive for phage replication must support the independent and simultaneous replication of two separate vectors, namely a first vector for expressing a heavy chain polypeptide, and a second vector for expressing a light chain polypeptide.

In addition, those embodiments that include a prokaryotic replicon can also include a gene whose expression confers a selective advantage, such as drug resistance, to a bacterial host transformed therewith. Typical bacterial drug resistance genes are those that confer resistance to ampicillin, tetracycline, neomycin/kanamycin or cholamphenicol. Vectors typically also contain convenient restriction sites for insertion of translatable DNA sequences. Exemplary vectors are the plasmids pUC8, pUC9, pBR322, and pBR329 available from BioRad Laboratories, (Richmond, CA) and pPL and pKK223 available from Pharmacia, (Piscataway, NJ).

A vector for expression of a monoclonal antibody of the invention on the surface of a filamentous phage particle is a recombinant DNA (rDNA) molecule adapted for receiving and expressing translatable first and second DNA sequences in the form of first and second polypeptides wherein one of the polypeptides is fused to a filamentous phage coat protein membrane anchor. That is, at least one of the polypeptides is a fusion polypeptide containing a filamentous phage membrane anchor domain, a prokaryotic secretion signal domain, and an immunoglobulin heavy or light chain variable domain.

A DNA expression vector for expressing a heterodimeric antibody molecule provides a system for independently cloning (inserting) the two translatable DNA sequences into two separate cassettes present in the vector, to form two separate cistrons for expressing the first and second polypeptides of the antibody molecule, or the ligand binding portions of the polypeptides that comprise the antibody molecule (i.e., the H and L variable regions of an immunoglobulin molecule). The DNA expression vector for expressing two cistrons is referred to as a dicistronic expression vector.

The vector comprises a first cassette that includes upstream and downstream translatable DNA sequences operatively linked via a sequence of nucleotides adapted for directional ligation to an insert DNA. The upstream translatable sequence encodes the secretion signal as defined herein. The downstream translatable sequence encodes the filamentous phage membrane anchor as defined herein. The cassette preferably includes DNA expression control sequences for expressing the receptor polypeptide that is produced when an insert translatable DNA sequence (insert DNA) is directionally inserted into the cassette via the sequence of nucleotides adapted for directional ligation. The filamentous phage membrane anchor is preferably a domain of the cpIII or cp VIII coat protein capable of binding the matrix of a filamentous phage particle, thereby incorporating the fusion polypeptide onto the phage surface. The receptor expressing vector also contains a second cassette for expressing a second receptor polypeptide. The second cassette includes a second translatable DNA sequence that encodes a secretion signal, as defined herein, operatively linked at its 3' terminus via a sequence of nucleotides adapted for directional ligation to a downstream DNA sequence of the vector that typically defines at least one stop codon in the reading frame of the cassette. The second translatable DNA sequence is operatively linked at its 5' terminus to DNA expression control sequences forming the 5' elements. The second cassette is capable, upon insertion of a translatable DNA sequence (insert DNA), of expressing the second fusion polypeptide comprising a receptor of the secretion signal with a polypeptide coded by the insert DNA.

An upstream translatable DNA sequence encodes a prokaryotic secretion signal as described earlier. The upstream translatable DNA sequence encoding the pelB secretion signal is a preferred DNA sequence for inclusion in a receptor expression vector. A downstream translatable DNA sequence encodes a filamentous phage membrane anchor as described earlier. Thus, a downstream translatable DNA sequence encodes an amino acid residue sequence that corresponds, and preferably is identical, to the membrane anchor domain of either a filamentous phage gene III or gene VIII coat polypeptide.

A cassette in a DNA expression vector of this invention is the region of the vector that forms, upon insertion of a translatable DNA sequence (insert DNA), a sequence of nucleotides capable of expressing, in an appropriate host, a fusion polypeptide. The expression-competent sequence of nucleotides is referred to as a cistron. Thus, the cassette comprises DNA expression control elements operatively linked to the upstream and downstream translatable DNA sequences. A cistron is formed when a translatable DNA sequence is directionally inserted (directionally ligated) between the upstream and downstream sequences via the sequence of nucleotides adapted for that purpose. The resulting three translatable DNA sequences, namely the upstream, the inserted and the downstream sequences, are all operatively linked in the same reading frame. Thus, a DNA expression vector for expressing an antibody molecule provides a system for cloning translatable DNA sequences into the cassette portions of the vector to produce cistrons capable of expressing the first and second polypeptides, i.e., the heavy and light chains of a monoclonal antibody.

As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting between different genetic environments another nucleic acid to which it has been operatively linked. Preferred vectors are those capable of autonomous replication and expression of structural gene products present in the DNA segments to which they are operatively linked. Vectors, therefore, preferably contain the replicons and selectable markers described earlier.

As used herein with regard to DNA sequences or segments, the phrase "operatively linked" means the sequences or segments have been covalently joined, preferably by conventional phosphodiester bonds, into one strand of DNA, whether in single or double stranded form. The choice of vector to which transcription unit or a cassette of this invention is operatively linked depends directly, as is well known in the art, on the functional properties desired, e.g., vector replication and protein expression, and the host cell to be transformed, these being limitations inherent in the art of constructing recombinant DNA molecules.

A sequence of nucleotides adapted for directional ligation, i.e., a polylinker, is a region of the DNA expression vector that (1) operatively links for replication and transport the upstream and downstream translatable DNA sequences and (2) provides a site or means for directional ligation of a DNA sequence into the vector. Typically, a directional polylinker is a sequence of nucleotides that defines two or more restriction endonuclease recognition sequences, or restriction sites. Upon restriction cleavage, the two sites yield cohesive termini to which a translatable DNA sequence can be ligated to the DNA expression vector. Preferably, the two restriction sites provide, upon restriction cleavage, cohesive termini that are non-complementary and thereby permit directional insertion of a translatable DNA sequence into the cassette. In one embodiment, the directional ligation means is provided by nucleotides present in the upstream translatable DNA sequence, downstream translatable DNA sequence, or both. In another embodiment, the sequence of nucleotides adapted for directional ligation comprises a sequence of nucleotides that defines multiple directional cloning means. Where the sequence of nucleotides adapted for directional ligation defines numerous restriction sites, it is referred to as a multiple cloning site.

In a preferred embodiment, a DNA expression vector is designed for convenient manipulation in the form of a filamentous phage particle encapsulating a genome according to the teachings of the present invention. In this embodiment, a DNA expression vector further contains a nucleotide sequence that defines a filamentous phage origin of replication such that the vector, upon presentation of the appropriate genetic complementation, can replicate as a filamentous phage in single stranded replicative form and be packaged into filamentous phage particles. This feature provides the ability of the DNA expression vector to be packaged into phage particles for subsequent segregation of the particle, and vector contained therein, away from other particles that comprise a population of phage particles.

A filamentous phage origin of replication is a region of the phage genome, as is well known, that defines sites for initiation of replication, termination of replication and packaging of the replicative form produced by replication (see, for example, Rasched, et al, Microbiol Rev., 50:401-427 (1986), and Horiuchi, J. Mol. Biol, 188:215-223 (1986)).

A preferred filamentous phage origin of replication for use in the present invention is an M13, fl or fd phage origin of replication (Short, et al., Nucl Acids Res, 16:7583-7600 (1988)). Preferred DNA expression vectors for cloning and expression a human monoclonal antibody of this invention are the dicistronic expression vectors pCOMB8, pCOMB2-8, pCOMB3, pCOMB2-3 and pCOMB2-3', described herein.

It is to be understood that, due to the genetic code and its attendant redundancies, numerous polynucleotide sequences can be designed that encode a contemplated heavy or light chain immunoglobulin variable region amino acid residue sequence. Thus, the invention contemplates such alternate polynucleotide sequences incorporating the features of the redundancy of the genetic code.

An antigen or antibody identified by the method of the invention can be administered parenterally by injection or by gradual infusion over time. For example, the composition can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally.

Preparations for parenteral administration are contained in a "pharmaceutically acceptable carrier". Such carriers include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, metabolizable oils such as, olive oil, squalene or squalane, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

The antibodies identified by the method of the invention are useful for passive immunization. For example, in situations where the viral load is lower than in a full state of infection, passive immunization may be a preferred method of treatment. In the case of HIV, such situations include accidental infection by a needle stick, or protection of a fetus by maternal-fetal transmission.

A therapeutically effective amount of antigen or antibody is adminstered as a vaccine for protection or clearance of an antigen. The term "therapeutically effective" means that the amount of antigen or antibody administered is of sufficient quantity to increase the subject's immune response to the antigen, for example, to HIV. The dosage ranges for the administration of the virus composition are those large enough to produce the desired effect of increasing the immune response.

The dosage of antigen (or antibody used for passive immunization) should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions and the like. Generally, the dosage will vary with the age, condition, sex, and extent of the disease in the patient and can be determined by one skilled in the art. The dosage can be adjusted by the individual physician in the event of any contraindications. In any event, the effectiveness of treatment can be determined by monitoring the level of CD4+ T-cells in a patient, for example, in the case of HIV infection. An increase in CD4+ cells should correlate with recovery of the patient's immune system.

The antigen or antibody identified in the method of the invention can be administered to a patient prior to infection with a virus or bacteria, for example, prior to infection with HIV (i.e., prophylactically). In the case of HIV, the antigen or antibody may be adminstered at any of the stages described below, after initial infection. The HIV infection may run any of the following courses: 1) approximately 15% of infected individuals have an acute illness, characterized by fever, rash, and enlarged lymph nodes and meningitis within six weeks of contact with HIV. Following this acute infection, these individuals become asymptomatic. 2) The remaining individuals with HIV infection are not symptomatic for years. 3) Some individuals develop persistent generalized lymphadenopathy (PGL), characterized by swollen lymph nodes in the neck, groin and axilla. Five to ten percent of individuals with PGL revert to an asymptomatic state. 4) Any of these individuals may develop AIDS-related complex (ARC); patients with ARC do not revert to an asymptomatic state. 5) Individuals with ARC and PGL, as well as asymptomatic individuals, eventually (months to years later) develop AIDS which inexorably leads to death.

When vaccination with antigen is contemplated, it is preferably adminstered in combination with an adjuvant such as aluminum hydroxide or Freund's adjuvant in a non-toxic, prophylactic or therapeutic amount. A targeted delivery system for an antigen identified by the method of the invent comprises native polypeptides is a colloidal dispersion system. Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. The preferred colloidal system of this invention is a liposome. Liposomes are artificial membrane vesicles which are useful as delivery vehicles in vitro and in vivo. It has been shown that large unilamellar vesicles (LUV), which range in size from 0.2-4.0 μm can encapsulate a substantial percentage of an aqueous buffer containing large macromolecules. RNA, DNA and intact virions can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (Fraley, et al., Trends Biochem. Sci., 6:77, 1981). In addition to mammalian cells, liposomes have been used for delivery of polynucleotides in plant, yeast and bacterial cells. In order for a liposome to be an efficient gene transfer vehicle, the following characteristics should be present: (1) encapsulation of the genes of interest at high efficiency while not compromising their biological activity; (2) preferential and substantial binding to a target cell in comparison to non-target cells; (3) delivery of the aqueous contents of the vesicle to the target cell cytoplasm at high efficiency; and (4) accurate and effective expression of genetic information (Mannino, et al., Biotechniques, 6:682, 1988).

Therefore, liposomes, including unilamellar bodies comprising a single lipid bilayer, can be used as vectors to deliver viral proteins, such as polypeptides specific for determining macrophage tropism, to vaccinate against HIV virus. Such methods are taught in U.S. Patent No. 4, 148,876 to Almeida, et al. and U. S. Patent No. 4,663, 161 to Mannino, et al., which are incorporated herein by reference in their entirety.

The targeting of liposomes has been classified based on anatomical and mechanistic factors. Anatomical classification is based on the level of selectivity, for example, organ-specific, cell-specific, and organelle-specific. Mechanistic targeting can be distinguished based upon whether it is passive or active. Passive targeting utilizes the natural tendency of liposomes to distribute to cells of the reticulo-endothelial system (RES) in organs which contain sinusoidal capillaries. Active targeting, on the other hand, involves alteration of the liposome by coupling the liposome to a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein, or by changing the composition or size of the liposome in order to achieve targeting to organs and cell types other than the naturally occurring sites of localization.

The viral proteins and portions thereof, prepared as described above, may also be used in the preparation of subunit vaccines prepared by known techniques. Polypeptides displaying antigenic regions capable of eliciting protective immune response are selected and incorporated in an appropriate carrier. Alternatively, an antigenic portion of a viral protein or proteins may be incorporated into a larger protein by expression of fused proteins. The preparation of subunit vaccines for other viruses is described in various references, including Lerner, et al., Proc. Natl. Acad Sci. USA, 78:3403, 1981 and Bhatanagar, et al., Proc. Natl. Acad. Sci. USA, 79:4400, 1982. See also, U.S. Patent Nos. 4,565,697 (where a naturally-derived viral protein is incorporated into a vaccine composition); 4,528,217 and 4,575,495 (where synthetic peptides forming a portion of a viral protein are incorporated into a vaccine composition). Other methods for forming vaccines employing only a portion of the viral proteins are described in U.S. Patent Nos. 4,552,757; 4,552,758; and 4,593,002. The relevant portions of each of these patents are incorporated herein by reference.

Such vaccines are useful for raising an immune response against HIV, for example a protective antibody titer, in humans susceptible to the virus. The vaccines prepared as described above may be administered in any conventional manner, including nasally, subcutaneously, or intramuscularly, except that nasal administration will usually not be employed with a partially inactivated virus vaccine. Adjuvants will also find use with subcutaneous and intramuscular injection of completely inactivated vaccines to enhance the immune response.

Antigens, such as live attenuated viruses or viral antigens can also be incorporated into immunostimulating complexes (ISCOM) for use as a vaccine using methods well known in the art. A recombinant HIV antigen, for example, can be incorporated into ISCOM particles which are useful for prophylactic or therapeutic vaccination against HIV infection. The presentation of viral protein antigens in ISCOM particles has three main advantages: 1) no replicating viral nucleic acid is introduced into the host, 2) high levels of neutralizing antibodies are achieved, and 3) a cellular immunity is evoked, including cytotoxic T-cells induced under restriction of MHC class II. The methodology for making ISCOM vaccines is well known in the art (B. Morein, et al., Nature, 308:457-60, 1984).

A pharmaceutical composition comprising an antigen containing a neutralizing epitope identified by the method of the invention includes vehicles for delivery of nucleotide sequences encoding an antigenic peptide or the polypeptide itself, such as synthetic peptides, DNA vaccines, natural viral products, and recombinant DNA products, in a pharmaceutically acceptable carrier. Such vehicles may include, but are not limited to, RNA and DNA virus vectors and liposomes.

The following examples are intended to illustrate but not limit the invention. While they are typical of those that might be used, other procedures known to those skilled in the art may alternatively be used.

EXAMPLE 1

PRODUCTION OF RECOMBINANT ANTI-HIV-1 ANTIBODY

The generation of the antibody Fab fragment b12 from a combinatorial phage display library has been described previously (D.R. Burton, et al., Proc. Natl. Acad. Sci., USA, 88:10134, 1991, incorporated herein by reference). Fab b12 is directed to the CD4 binding site of gp120 and is a potent neutralizer of the HIV-1 laboratory strains IIIB and MN (C.F. Barbas, et al., Proc. Natl. Acad Sci., USA, 89:9339, 1992; C.F Barbas, et al., J. Mol. Biol, 230:812, 1993, P. Roben, et al., J. Virol, 68:4821, 1994, incorporated herein by reference). While Fab b12 was chosen for further study in the Examples herein, severl other Fab fragments were identified as neutralizers of HIV- 1 (See Barbas, et al., supra, and Burton, et αl, supra). Selection for potency and strain cross-reactivity was achieved through experimental design. The library donor was a long-term asymptomatic male, presumably infected with a clade B strain of HIV- 1, while the antigen for affinity selection was gp120 from the IIIB strain, thereby favoring selection of cross-reactive antibodies. A large number of bacterial anti-gp120 Fab supernates at low initial concentrations were directly screened for neutralizing ability to find the most potent Fabs. Although Fab b12 is capable of neutralizing some primary isolates (C.F. Barbas, et al, J. Mol. Biol, 230:812, 1993), the corresponding whole antibody molecule is likely to be more effective. Therefore, Fab b12 was converted to a whole IgG1 molecule by cassetting the variable heavy chain (VH) and light chain genes into a vector created for high-level mammalian expression. The whole antibody IgG1 b12 was expressed in Chinese hamster ovary (CHO) cells and purified by affinity chromatography.

The strategy adopted was similar to that described previously for the generation of a whole antibody beginning with a phage derived Fab (E. Bender, et al, Hum. Antibod. Hybridomas, 4:74, 1992). First the b12 heavy chain VH region was cloned into a pSG5 expression vector (S. Green, et al., Nucl. Acids Res., 16:369, 1988) to fuse with the heavy chain constant domains. The cloning involved overlap PCR to (i) replace the bacterial leader sequence with a consensus mouse sequence followed by the unique Kozak sequence and (ii) to modify the NH₂-terminus of VH to a human consensus sequence (QVQLVQ). The light chain, with a mouse leader sequence and modified human consensus NH₂-terminus (EIVLTQSP), was also cloned into a pSG5 expression vector. The pSG5 vectors contain an Ml 3 intergenic region so that the entire heavy and light chain sequences could be readily checked. The vectors also contain an SV40 origin of replication so that, on co-transfection of heavy and light chain vectors into COS-7 cells, functional protein production could be confirmed. Subsequently, heavy and light chains were cloned into pEE6 and pEE12 vectors (Bebbington, et al, Bio/Technology, 10: 169, 1992), respectively. These vectors incorporate an HCMV promoter and glutamine synthetase (GS) amplifiable selectable marker. The heavy chain including HCMV promoter, enhancer elements and poly A signal was then subcloned into the pEE12 vector bearing the light chain to yield a combinatorial plasmid. This was used to transfect CHO cells and stable clones selected under methyl sulfoxamine amplification. The clone producing the highest levels of IgG1 b12 as judged by ELISA with gp120 HIB was chosen for scale-up. The antibody was purified by affinity chromatograpy using protein A. The affinity of IgG1 b12 for gp 120 IIIB as measured by surface plasmon resonance is 1.3 × 10⁹ M^-1. The antibody binds complement C1q in an ELISA format. Flow cytometry gives no evidence of binding of IgG1 b12 at 50μg/ml to normal human peripheral blood mononuclear cells (PBMCs).

EXAMPLE 2

NEUTRALIZATION OF HIV-1 BY IgG1b12 AND HUMAN PLASMA IgG1 b12 was initially tested against the laboratory strains MN and IIIB in two neutralization assays in laboratories that recently tested a panel of monoclonal antibodies as part of the NIAID/WHO Antibody Serological Project (M.P. D'Souza, et al., AIDS, 8:169, 1994). IgG1 b12 showed 50% neutralization titers of 3 ng/ml for the MN strain and 7 ng/ml for the IIIB strain when standard plaque formation assays (C.V. Hanson, et al, J. Clin. Microbiol, 28:2030, 1990) to indicate antibody inhibition of infectivity (Table 1) was used, and titers of 20 ng/ml for both MN and IIIB strains when syncytial formation was used as the reporter assay. The quantitative infectivity assay based on syncytial formation was performed as described in P.L. Nara, et al, AIDS Res. Human Retroviruses, 3:283, 1987. Virus was grown in H9 cells. For infectivity measurement, monolayers of CEM-SS target cells were cultured with 100-200 syncytial forming units (SFUs) of virus, in the presence or absence of antibody, and the number of syncytia determined 3-5 days later. The assays were repeatable over a virus-surviving fraction range of 1 to 0.001 within a to 4-fold difference in the concentration of antibody (P<0.001).

Table 1 shows the neutralization of laboratory-adapted strains and primary isolates of HIV-1 by IgG1 b12 and a pooled human plasma preparation. The microplaque assay was carried out as described ( C.V. Hanson, et al, J. Clin. Microbiol, 28:2030, 1990) with minor modifications. In brief, antibody was 3 -fold serially diluted and preincubated in quadruplicate with an equal volume containing 20 plaque-forming units (pfU) of virus per well for 18 hours in 96- well microtiter plates at 37°C and then stained with propidium iodide. After 24-48 hours, fluorescent plaques were counted on a transilluminator (304 nm). The neutralizing titer was defined as the concentration of antibody required to give a 50% reduction in plaque numbers as compared with controls containing no antibody. This dilution was interpolated between data points. VL134, VL648 and VL025 are viruses isolated from infected mothers in New York in 1992; UG266 and UG274 are clade D isolates which were a gift from John Mascola at the Division of Retrovirology, Walter Reed Army Institute of Research; the remaining viruses were isolated from homosexual males in California in 1992. The pooled human plasma preparation was derived from 13 HIV-1 positive individuals selected for high neutralization titer against the MN isolate.

These titers suggest the antibody is approximately two orders of magnitude more potent than other CD4 site antibodies in the Project and comparable to the best antibodies directed to the V3 loop. However, whereas the later are strongly strain specific, IgG1 b12 is roughly equally effective against MN and IIIB The antibody is comparable in potency to a CD4-IgG molecule in these assays.

In a separate assay in which p24 production was the measure of infectivity (E S. Daar, et al., Proc. Natl. Acad Sci., USA, 87:6574, 1990; D D. Ho, et al, J. Virol, 65:489, 1991), 50% neutralization titers of less than 40 ng/ml were found for both MN and IIIB strains.

Figure 1 shows an assay for the neutralization of primary isolates of HIV- 1 by IgG1 b12. Virus neutralization was assessed using PHA-stimulated PBMCs as indicator cells and determination of extracellular p24 as the reporter assay essentially as described (E.S. Daar, et al., Proc. Natl. Acad Sci., USA, 87:6574, 1990; D D. Ho, et al., J. Virol, 65:489, 1991). Virus (50 TCID₅₀) and antibody at varying concentration were incubated together for 30 minutes at 37°C before addition to PHA-stimulated PBMCs. Virus replication was assessed after 5-7 days by p24 ELISA measurement. The designation, location and disease status of the virus donors were as follows:■, VS (New York, acute);▼, N_70-2 (New Orleans, asymptomatic);▲, AC (San Diego, AIDS);●, LS (Los Angeles, AIDS);□, NYC-A (New York, unknown);▽, WM (Los Angeles, AIDS); Δ, RA (New York, acute);◊, IP (New York, acute). The moleculariy cloned HTV-1 virus JR-CSF (♦) and HIV-1 isolate JR-FL (O) have been described (W.A. O'Brien, et al, Nature, 348:69, 1990; W.A. O'Brien, et al, J. Virol, 66:3125, 1992; W.A. O'Brien, et al., J. Virol, 68:5264, 1994). Stocks of JR-CSF were prepared by infection of PBMC with supernatants initially obtained by DNA transfection. Titers (50% neutralization) for the laboratory strains HIV-1 IIIB and HIV-1 MN were < 4 ng/ml. [NOTE: HIV-1 IIIB and HIV-1 MN are viruses with an extensive history of passage in transformed T-cell lines (M. Robert-Guroff, et al, Nature, 316:72. 1985). Stocks of these strains grown in H9 cells were passaged in mitogen-stimulated PBMC to prepare viruses that had been grown in the same cells as the primary viruses, to eliminate the influence of any host cell-dependent epigenetic factors on virus neutralization (T. Wrin, et al, J. Acq. Imm. Def Synd, 7:211, 1994).]

EXAMPLE 3

NEUTRALIZATION OF HIV ISOLATES BY IgG1 b12 IN p24 ELISA AND MICROPLAQUE ASSAYS IgG1 b12 was next tested against a set of 10 primary virus isolates in the p24 reporter assay (E.S. Daar, et al, Proc. Natl. Acad Sci., USA, 87:6574, 1990; D.D. Ho, et al, J. Virol, 65:489, 1991). The viruses were isolated from individuals from various locations in the U.S. and with varying disease status (FIGURE 1). They had been cultured only once or twice in peripheral blood mononuclear cells (PBMCs). Viral stocks were grown in PBMCs and the assay was carried out with these cells. As shown in FIGURE 1, IgG1 b12 essentially completely neutralized 7 of 10 isolates at 5 μg/ml with all the isolates being 50% neutralized at≤ 1 μg/ml.

The ability of IgG1 b12 to neutralize an additional set of 14 primary isolates was then examined in a microplaque assay (C.V. Hanson, et al, J. Clin. Microbiol, 28:2030, 1990). The set was chosen to contain a high proportion of isolates that were relatively refractory to antibody neutralization by sera from other HTV-1 infected individuals (T. Wrin, et al, J. Acq. Imm. Def. Synd, 7:211, 1994). Viruses were grown in PBMCs and the assay carried out in MT2 cells. This limits study to viruses which grow in this cell line but provides an alternative measure of neutralization. Table 1 shows that 10 of 14 primary isolates were neutralized with titers somewhat higher than those in the p24 assay (FIGURE 1). Four isolates, that were not neutralized even by a 1 :10 dilution of pooled human plasma, were neutralized by IgG1 b12. Most of the viruses reported in Table 1 were isolated from US donors although two, both of which are neutralized by IgG1 b12, were from Ugandan donors and assigned to clade D. EXAMPLE 4

IgG1 b12 NEUTRALIZATION OF INFANT ISOLATES

One of the most likely roles for passive immunotherapy with antibody is in interruption of maternofetal transmission of virus as supported by recent reports (G. Scarlatti, et al, J. Infect. Dis., 1£8:207, 1993; Y.J. Bryson, et al, J. Cell Biochem., 17E:95 (suppl), 1993) which suggest that transmission correlates with an absence of maternal neutralizing antibody to the transmitted virus. Therefore, the ability of IgG1 b12 to neutralize a panel of 12 primary infant isolates was measured. Virus was obtained directly from infants at birth or within two weeks of age and was passaged once in PBMCs to produce viral stocks. Virus from these stocks was grown in PBMCs and neutralization assessed in a p24-based assay with PBMCs (AIDS Clinical Trials Group Virology Manual for HTV Laboratories, Department of AIDS Research, NIAID, NIH, version 2.0, 1993). IgG1 b12 achieved 90% neutralization for 8 of 12 isolates at concentrations≤ 20 μg/ml (Table 2).

Table 2 shows the neutralization of primary infant isolates by IgG1 b12. Neutralization was assessed using PHA-stimulated PBMCs as indicator cells and determination of extracellular p24 as the reporter assay essentially as described (AIDS Clinical Trials Group Virology Manual for HIV Laboratories, Department of AIDS Research, NIAID, NIH, version 2.0, 1993). Serial dilutions of IgG1 b12 (0.3 to 20 μg/ml) were incubated with 20 TCID₅₀ or 100 TCID₅₀ virus in triplicate for 2 hours at 37°C before addition to PHA-stimulated PBMCs. Virus replication was assessed after 5 days by p24 ELISA measurement. Neutralization was expressed as either a 50% or 90% reduction in p24 antigen as compared to values observed in the absence of antibody. Virus isolates were obtained from 12 infants born to HIV-1 seropositive mothers; 7 were obtained at birth and 5 between birth and 14 days of age. All the infants were from California. Virus was isolated from patient PBMCs by coculture with PBMCs from healthy seronegative donors. Viral stocks were prepared by passaging the last positive culture dilution once into PBMCs. All of the isolates, except one (isolate 7), were non-syncytial inducing in MT2 cells.

All 12 isolates were 50% neutralized in the range 0.3 to 20 μg/ml with the majority being neutralized at <5 μg/ml. In contrast, a pooled hyperimmune globulin product HIVIG achieved 90% neutralization of only 3 of 12 isolates within a concentration range up to 100 μg/ml. (HTVIG is a hyperimmune IgG preparation obtained from the pooled plasma of selected HIV-1 asymptomatic seropositive donors who met the following criteria: presence of p24 serum antibody titers > 128, CD4 lymphocyte count≥ 400 cells/μl and the absence of p24 and hepatitis B surface antigen by enzyme immunoassay (L.H. Cummins, et al, Blood, 77: 1111, 1991). The HIVIG used here was lot number IHV-50-101 (North American Biologicals.) EXAMPLE 5

NEUTRALIZATION OF INTERNATIONAL ISOLATES

To probe the occurrence of the b12 epitope in the HIV-1 pandemic, binding of the antibody to gp120 from 69 international isolates belonging to six clades was examined. Virus isolates were collected from various regions of the world by three organizations: the World Health Organization (WHO); the Henry M. Jackson Foundation for the Advancement of Military Medicine (HMJFAMM) and the National Institute of Allergy and Infectious Diseases (NIAID). Isolates from the WHO Network for HIV-1 Isolation and Characterization were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH. Isolates from HMJFAMM were kindly provided by Dr. John Mascola, Walter Reed Army Institute of Research, Rockville, MD and Dr. Francine McCutchan, Henry M. Jackson Research Laboratory, Rockville, MD. Isolates from NIAID were kindly provided by Dr. Jim Bradac, Division of AIDS, NIAID, NIH.

FIGURE 2 shows the reactivity of IgG1 b12 with a panel of international isolates of HIV-1. Viruses were collected from various regions of the world expanded in mitogen-stimulated peripheral blood mononuclear cells (PBMC) (J.R. Mascola, et al, J. Infect. Dis., 169:48, 1994) and culture supernatants containing infectious virus were stored in central repositories at -70°C. The designation of viruses into clades was made on the basis of sequence information based on the gag gene or on the V2-C5 region of gp120, or in some cases, after heteroduplex mobility analysis (J. Louwagie, et al., AIDS, 7:769, 1993; E.L. Delwart, et al. Science, 262:1257, 1993). gp120 from culture supernates was captured using a murine anti-gp120 antibody and IgG1 b12 reactivity examined by ELISA. Infectious culture supernates containing virus and free gp120 were treated with 1% Nonidet-P40 non-ionic detergent to provide a source of gp120 (J.P. Moore, et al, AIDS, 3:155, 1989). An appropriate volume of inactivated supernatant was diluted with a buffer comprising TBS/1%NP40/10%FCS and a 100μl aliquot was added for 2 hours at room temperature to microplate wells (Immulon II, Dynatech Ltd.) coated with sheep polyclonal antibody D7324 (Aalto Bio Reagents, Dublin, Ireland). This antibody was raised to peptide APTKAKRRVVQREKR, derived from the COOH-terminal 15 amino acids of the clade B IIIB isolate. Unbound gp120 was removed by washing with TBS, and bound gp120 was detected with CD4-IgG (1 μg/ml), or with Mab, diluted in TMTSS buffer essentially as described previously (J.P. Moore, et al, J. Virol, 68:469, 1994). Bound ligand was then detected with an appropriate alkaline-phosphatase conjugated anti-IgG, followed by AMPAK (Dako Diagnostics). Absorbance was read at 492nm (OD₄₉₂). Each virus was tested against CD4-IgG in triplicate and against IgG1 b12 in duplicate. All OD₄₉₂ values were corrected for non-specific antibody binding in the absence of added gp120 (buffer blank). The mean, blank-corrected OD₄₉₂ values for CD4-IgG and IgG1 b12 were then calculated, and the OD₄₉₂ ratios of IgG1 b12:CD4-IgG determined. This normalization procedure enables allowance to be made for the different amounts of gp120 captured onto the solid phase via D7324 when comparing antibody reactivity with a panel of viruses. Binding ratios of 0.50 or greater were deemed to represent strong antibody reactivity; ratios from 0.25-0.50 were considered indicative of moderate reactivity; values <0.25 were designated as representative of essentially negative Mab reactivity,

strong,

As shown in FIGURE 2, IgG1 b12 reacts with≥50% of clades A-D but only 1 of 12 isolates from clade E. Reactivity with clade B isolates from the USA was approximately 75%.

HIV-1 neutralization by antibody shows considerable variation depending upon the assay used and precise experimental conditions such as inoculum size and incubation time of virus and antibody (M.P. D'Souza, et al, AIDS, 8:169, 1994). However, by carrying out neutralization on a range of laboratory and primary isolates in a number of assays in different laboratories, the present invention shows that IgG1 b12 is a highly potent neutralizing antibody effective against a wide breadth of isolates. The results clearly demonstrate that, although primary isolates may be more difficult to neutralize by antibody than laboratory strains, they are not intrinsically resistant. A. J. Conley, et al, (Proc. Natl. Acad. Sci., USA, 91:3348, 1994) have further recently reported that an anti-gp41 antibody neutralizes five of six primary isolates tested. The potency of IgG1 b12 against the majority of US isolates is in a concentration range (≤5 μg/ml) that could be achieved in vivo. Furthermore, the affinities of recombinant antibodies displayed on phage can be enhanced by mutagenesis and selection in vitro and this strategy has been used to considerably improve the potency and breadth of reactivity of Fab b12 (C.F. Barbas, et al, Proc. Natl. Acad Sci., USA, 91:3809, 1994). For optimal potency and strain cross-reactivity for passive immunization, a cocktail of in vitro improved antibodies may be most appropriate.

The results have implications for vaccine design. The ability of IgG1 b12 to neutralize a range of primary isolates suggests that there may be conservation of a structural feature associated with the CD4 binding site of gp120 that is accessible to antibody and important for neutralization. A vaccine might seek to present this feature to the immune system. Clearly, the feature is present on recombinant gp120, as b12 was affinity selected from a library by means of this molecule. However, b12 and related antibodies formed only a small part of the repertoire affinity selected from this library by recombinant gp120. Most of the antibodies obtained were far less potent in neutralization even though they were also directed to the CD4 binding site, were cross-competitive with b12 for binding to recombinant gp120, and had similar affinities to b12 (C.F. Barbas, et al., Proc. Natl. Acad. Sci., USA, 89:9339, 1992; C.F. Barbas, et al., J. Mol. Biol, 230:812, 1993; P. Roben, et al, J. Virol, 68:4821, 1994). Therefore, recombinant gp120 appears to present the b12 epitope in conjunction with several other weakly neutralizing and overlapping epitopes and its efficacy as a vaccine may suffer. Yet, antibody is recovered only in low amounts using recombinant gp120 and thus, may not represent native gp120. This may be an important consideration for of an efficient vaccine. Evidence from antibody binding to infected cells suggests that b12 may recognize a native conformation of gp120 more effectively than other CD4 binding site antibodies (P. Roben, et al, J. Virol, 68:4821, 1994). In any case, IgG1 b12 and the library approach would be useful in vaccine evaluation. The ability of a candidate vaccine to preferentially bind b12 or preferentially select potent neutralizing antibodies from libraries would be positive indicators for vaccine development. EXAMPLE 6

SELECTION OF AN ANTIBODY COMBINATORIAL LIBRARY

AGAINST RECOMBINANT HIV ENVELOPE IN

MULTIMER CONFIGURATION

Monomeric recombinant gp120 has proven a disappointing vaccine for HIV-1. gp120 exists on the viral surface as a multimeric complex and recombinant versions of the multimer have been prepared. It is suggested that the multimer may be a more effective vaccine because an immune response may be elicited that generates antibodies which bind more effectively to gp 120 in a multimeric configuration. A library was panned against IIIB gpl40 tetramers, which have been shown to elicit in mice an immune response with major differences from that elicited by monomeric IIIB gp120 (Earl, et al., J. Virol, 68.3015, 1994, incorporated herein by reference). The panel of antibodies retrieved, however, contained similar antibodies to those obtained by panning against monomeric gp120 (Table 3, A). After blocking the CD4-binding site on gp120, antibodies were retrieved that bound the gp41 subunit on the gp140 multimer (Table 3, B). Identical antibodies were retrieved previously, however, by panning against the monomeric gp41 subunit. All of these antibodies are known non- or weak neutralizers. This shows that there was no greater retrieval of potently neutralizing antibodies which are known to be represented in the library. This is evidence against the likelihood that the multimer in this configuration will be a much improved vaccine relative to the monomeric gp120 subunit.

The strategies for experiments A-B were as follows:

In A, the library was panned neat for 4 rounds. To direct the enrichment to other epitopes, the CD4-site was blocked by addition of antibody HIV-b3 in panning experiment B.

In B, library M was panned again for 4 rounds, but now the tetramers were preincubated with Fab HIV-b3 (somatic variant that belongs to the same group as

S2/S7), and Fab p7 (which is an non-neutralizing antibody binding to the gp120 N- terminus).

EXAMPLE 7

IDENTIFICATION OF HSV NEUTRALIZING Fab FRAGMENTS

To illustrate the method of the invention further, libraries from individuals having high serum titers to herpes virus were prepared. The libraries were panned against whole virus lysates and neutralizing antibodies were derived at a frequency of about 1 in 20. When panned against surface glycoprotein D (gD), described herein, all antibodies selected were neutralizing. a. Library construction, screening and Fab production

The preparation of human antibody Fab libraries displayed on the surface of M13 phage has been described in Williamson, et al, Proc. Natl. Acad. Sci. USA, 90:4141. 1993; Barbas, et al, Proc. Natl. Acad. Sci. USA, 58:7978, 1991; McCafferty, et al, Nature, 348:552, 1990. The library was constructed as an IgG1 × Fab library using bone marrow lymphocyte RNA of a long term asymptomatic HIV-1 positive individual. Antigen binding phage were selected against HSV-1 and -2 viral lysates (monkey kidney epithelial cells (VERO), 36 hr post infection, were pelleted and lysed using phosphate-buffered saline containing 1% sodium deoxycholate, 1% NP 40 (SIGMA), 0.1 mM DIFP and 2mg/ml aprotinin) bound to ELISA wells (Sigma) through a panning procedure described in Burton, et al, Proc. Nat'l. Acad Sci. USA, 88: 10134-10137, 1991; Barbas, et al, Methods, 2:119-124, 1991. Phage from the final round of panning were converted to a soluble Fab expressing phagemid system and these clones selected for reactivity in ELISA with the antigen against which they were panned. Specific antibody was affinity purified from bacterial supemates over a protein A/G matrix (Shleicher & Schuell) as described in Williamson, et al, Proc. Nat'l Acad Sci. USA, 90:4141-4145, 1993. b. Viruses and Cells

Vero cells were grown in RPMO 1640 supplemented with 5% fetal calf serum (FCS). HSV-1 and HSV-2 (strain F and G, respectively, ATCC, Rockville, MD) were infected into Vero cells and virus titers were determined by plaque-assay and expressed as pfu ml^-1 c. Neutralization Activity

Crude E coli extracts, positive in an ELISA screen using the same antigen preparation against which the library was panned, were tested for their ability to neutralize HSV-1 or HSV-2 at 1 :5 - 1 :10 dilutions according to the procedure described below. The Fabs that displayed neutralizing activity were affinity purified and their neutralizing titers were determined as follows. About 250 pfu of HSV- 1 or HSV-2 were incubated with serial dilutions of recombinant Fabs for 1 hr at 37°C and then adsorbed for 1 hr at 37°C on Vero cell monolayers grown in six well plates. After adsorption, the inoculum was removed and the cells washed and overlaid with MEM containing 0.5% agarose and 2% FCS. After 72 hrs, the plates were fixed with 10% formaldehyde in phosphate-buffered saline (PBS) for 30 min, the nutrient agar overlay was removed and the cells were strained with a 1% solution of crystal violet in 70% methanol for 30 min. The stained monolayers were then washed and the plaques were counted. d. Inhibition of Plaque Development Assay

Monolayers of Vero cells were infected with of 50-100 pfu of HSV- 1 for 3 hrs at 37°C. They were then washed and the medium replaced with nutrient agar containing 25, 5 or 1 μg/ml of recombinant Fab. After 72 hrs or 86 hrs, they were fixed and stained as described above. Plaque diameter was measured with a digital caliper (Mitutoyo, Japan). At least 10 plaques were measured per well. Plaques below 0.2 mm in diameter were considered abortive and therefore not counted. Statistical calculations were performed by analysis of variants (Sheffe F-test). e. Post-attachment Neutralization Assay

About 250 pfu of HSV- 1 were adsorbed at 4°C for 90 min on Vero monolayers prechilled at 4°C for 15 min. The inoculum was then removed and the cells washed and overlaid with medium containing serial dilutions of recombinant Fab (5, 1, 0.2, 0.04 μg/ml) at 4°C to prevent penetration of virus. After 90 min the Fab-containing medium was removed and after washing, replaced with nutrient agar. For the purpose of control, equal amounts of virus were preincubated at 4°C with serial dilutions (5, 1, 0.2, 0.04 μg/ml) of the same Fab (pre-attachment neutralization). After 90 min these virus/antibody dilutions were adsorbed onto Vero monolayers (prechilled at 4°C for 1 hr and 45 min) for 90 min. The inoculum was then removed and the cells washed and overlaid with nutrient agar. After 72 hr, the monolayers were fixed and strained as described above for the neutralization assays. f. Nucleic acid Sequencing

Nucleic acid sequencing was performed with a 373A automated DNA sequencer (Applied Biosystems) using a Taq fluorescent dideoxynucleotide terminator cycle sequencing kit (Applied Biosystems). Primers used for the elucidation of light and heavy chain sequence have been previously described in Williamson, et al, (Proc. Nat'l Acad Sci. USA, 90:4141-4145, 1993). g. Identification of antibody binding protein by immunoprecipitation

HSV-2 infected cells were harvested and sonicated in PBS containing 1% sodium deoxycholate, 1% NP40 (Sigma), 0.1 mM di-isopropylfluorophosphate (DIFP) and 2 mg/ml aprotinin. Lysates (50 μl) were then incubated with 7.5μg of recombinant Fab for 1 hour at 4°C. Immune complexes were precipitated with an agarose-bound goat anti-human (20μl) resolved on a 10% SDS-PAGE and electro-blotted onto nylon membranes (BioRad) in 1x Towbin buffer. Western blots were performed according to standard protocols. Briefly, blots were blocked with 5% non-fat dry milk in Tris-buffered saline (TBS) and probed with a panel of established mouse monoclonal anti-HSV antibodies (Goodwin Institute) in 1% non-fat dry milk in TBS containing 0.05% Tween 20. Detection was performed with a goat anti-mouse antibody conjugated to alkaline phosphatase and chemiluminescence (BioRad). Blots were also immunoreacted with a rabbit polyclonal anti-HSV for the purpose of control and detected with a goat anti-rabbit antibody conjugated to alkaline phosphatase (BioRad). h. Purification of Fabs

One liter cultures of super broth containing 50 μg/ml carbenicillin and 20 mM MgCl₂ were inoculated with appropriate clones and induced 7 hours later with 2 mM IPTG and grown overnight at 30°C. The cell pellets were sonicated and the supernatant concentrated to 50 ml. The filtered supernatants were loaded on a 25 ml protein G-anti-Fab column, washed with 12 ml buffer at 3 ml/min., and eluted with citric acid, pH 2.3. The neutralized fractions were then concentrated and exchanged into 50 mM MES pH 6.0 and loaded onto a 2 ml Mono-S column at 1 ml/min. A gradient of 0-500 mM NaCl was run at 1 ml/min with the Fab eluting in the range of 200-250 mM NaCl. After concentration, the Fabs were positive when titered by ELISA against FG and gave a single band at 50 kD by 10-15% SDS-PAGE. Concentration was determined by absorbance measurement at 280 nm using an extinction coefficient (1 mg/ml) of 1.35.

EXAMPLE 8

NEUTRALIZING ACTTVITY OF Fab AGAINST HSV

A large panel of human combinatorial antibody Fab fragments specific for HSV-1 and -2 were isolated by independently panning an IgG1k Fab library of 2 × 10⁶ members against whole lysate of these two viruses.

Enrichment of antigen specific phage, as determined by the number of phage eluted from HSV coated ELISA wells, was measured through 4 rounds of library panning. A 25-fold amplification was seen in the case of the panning with HSV-2 viral lysate, while a 20-fold amplification was observed using the HSV-1 viral lysate.

Soluble Fabs were then produced as described in Barbas, et al, Proc. Nat'l Acad Sci. USA 88: 7978-7982, 1991. Briefly, the phage coat protein III was excised from the phage display vector and the DNA self-ligated to give a vector producing soluble Fabs. Subsequently protein synthesis was induced overnight using IPTG and the bacterial pellet sonicated to release Fab from the periplasmic space. The Fab supernates were then tested, both in ELISA against the antigen with which they were panned and in immunofluorescence studies with virus-infected cells. Ten out of twenty clones taken from the final round of panning with HSV-1 viral lysate were positive in both assays, while 15 out of twenty were positive in the panning against HSV-2 lysate. All clones demonstrating positive reactivity with one virus type were further shown to be cross-reactive with the other in both immunofluorescence and ELISA assays. This probably reflects the known similarity between many of the proteins of HSV- 1 and -2. DNA sequences were determined as described above and the deduced amino acid sequences of the heavy chain variable domains were determined for several of the virus-specific clones. Nine of 18 of the heavy chain sequences obtained from the HSV-2 panning were all quite different from each other. Similarly, 5 of 8 heavy chains taken from the HSV-1 panning were largely unrelated. A comparison of the targets to which these different antibodies are directed with the serum antibody reactivity of the donor indicates how accurately the library approach represents the humoral response of the donor to virus.

Although virus type cross-reactivity in ELISA was exhibited by all of the Fabs described here, only one heavy chain sequence was common to both pannings. Thus, despite the reported similarity between virions of HSV-1 and HSV-2 and the observed binding properties of the isolated Fabs, each virus selected distinct antibody molecules from the library. This implies differences between HSV-1 and -2 either in the antigens presented to the library or in the antibody response to the two viruses.

Neutralizing activity for all positive clones was estimated in plaque reduction and inhibition of plaque development assays of HSV-1 and -2, as described above. Three of the Fabs obtained from the HSV-2 panning exhibited a marked neutralization activity in both assays and with both virus types when tested as crude bacterial supematants in vitro. These clones were shown to have identical heavy and light chain sequences. Accordingly, one of these Fab clones (Fab8), was grown in quantity, affinity purified and further characterized.

The Fab8 antibody was able to recognize both types of the virus. This antibody was shown to neutralize HSV-2 (50% inhibition at about 0.05 μg/ml) somewhat more efficiently than HSV-1 (50% inhibition at about 0.25 μg/ml and 80% inhibition at 0.6 μg/ml) (FIGURE 3). FIGURE 3 shows the neutralizing activity of Fab8, as measured by plaque reduction. FIGURE 3 A shows activity against HSV-1 and FIGURE 3B shows activity against HSV-2. Purified Fab8 neutralized HSV-1 with a 50% inhibition at about 0.25 μg/ml and with an 80% inhibition at 0.6 μg/ml, while HSV-2 was neutralized with a 50% inhibition at about 0.05 μg/ml and an 80% inhibition at 0.1 μg/ml.

These figures suggest that Fab8 is approximately an order of magnitude more potent than most murine neutralizing antibodies described so far (Navarro, et al, Virology, 186:99- 112, 1992; Fuller, et al., J. Virol, 55:475-482, 1985), although recently reported anti-gB and anti-gD humanized murine antibodies may be equally potent (Deschamps, et al, Proc. Nat'l Acad Sci. USA 88:2869-2873, 1991). However, the mouse and humanized antibodies are bivalent whole IgG molecules rather than human derived Fab fragments. Also, eukaryotic expression of the recombinant Fab of the invention as an intact IgG molecule may significantly enhance its virus neutralization potency.

The Fab8 antibody also inhibited plaque formation when applied to virus-infected monolayers (FIGURE 4). FIGURE 4 shows an inhibition of plaque development assay. Purified Fab8 inhibited the development of plaques when applied 4 hours post-infection (hpi) on monolayers infected with HSV-1 (FIGURE 4 A, FIGURE 4B) or HSV-2 (FIGURE 4C, FIGURE 4D) 4 hours post infection. FIGURE 4A shows statistically significant reduction in plaque size was observed at concentrations of 5 and 1 μg/ml (*=p( 0.01), with an approximate 50% reduction in plaque size at 5 μg/ml. The number of plaques was also dramatically reduced at Fab concentrations of 5 and 25 μg/ml (FIGURE 4B, FIGURE 4D). At 25 μg/ml and 72 hrs hpi plaque development in HSV-2 infected monolayers was completely inhibited (FIGURE 4C, FIGURE 4D). FIGURE 4E shows an inhibition of plaque development assay with HSV-2 infected monolayers at a number of different Fab concentrations 86 hpi.

At a concentration of 25 μg/ml Fab8 completely abolished HSV-2 plaque development at 72 hrs post-infection, while a statistically significant reduction in plaque size ()50%) was observed at concentrations of 5 μg/ml and 1 μg/ml for both HSV-1 and HSV-2. Since it is accepted that plaques develop by spreading of virus to adjacent cells, the inhibition of plaque development assay determines the ability of an antibody to prevent cell-to-cell spread.

Furthermore, this antibody strongly reduced infectivity after HSV-1 attachment (FIGURE 5). FIGURE 5 shows a post-attachment neutralization assay. Fab8 reduced HSV-1 infectivity after virion attachment. FIGURE 5A shows the percentage of plaque reduction pre- and post-attachment at different Fab concentrations. FIGURE 5B shows the post-/pre-attachment neutralization ratio at different Fab concentrations.

The pre-attachment/post attachment neutralization ratio was over 87% at an antibody concentration of 5 μg/ml, dropping to between 55-60% below 1 μg/ml. This suggests that the inhibitory action of the antibody takes place either at the level of membrane fusion, or during virus penetration or uncoating.

The protein recognized by Fab8 was identified via immunoprecipitation from whole lysate of HSV-2 infected cells. The precipitated proteins were blotted following resolution through SDS-PAGE and probed with a mouse monoclonal anti-gD and a rabbit polyclonal anti-HSV-2 (FIGURE 6). FIGURE 6 shows the identification of the protein recognized by Fab8. SDS-PAGE of total proteins from HSV-2 infected Vero cells (lanes 1) and of the product of immunoprecipitation with Fab8 (lanes 2). Western blots performed in parallel were probed with a mouse monoclonal anti gD antibody (MAB α-gD) and for the purpose of control, a rabbit polyclonal anti-HSV-2 preparation (RAB α-HSV2). The Coomassie stain of a gel run in parallel is also shown. Fab8 immunoprecipitated a band of apparent molecular weight 48-50kD which was recognized by a mouse monoclonal specific for gD, but not by mouse monoclonal antibodies against other HSV glycoproteins.

The results illustrated in FIGURE 6 show that the recombinant Fab recognizes a protein of molecular weight approximately 48-50 kD that is also reactive with murine monoclonal anti-gD. No further proteins were detected on the blot by the rabbit anti-HSV-2 polyclonal antibody preparation thus confirming the specificity of the human Fab.

Fab8 has been shown to neutralize virus extremely efficiently and to inhibit viral spread from cell to cell. The demonstration of such antiviral activity by an Fab offers potential advantages over whole IgG for some in vivo applications. Although the serum half life of Fab is dramatically shorter than that of whole IgG, the smaller molecule has far greater tissue penetration (Yokota, et al., Cancer Research, 52:3401-3408, 1992). The increased penetration of Fab also lends itself to potential topical applications. In the case of herpes this may take the form of an antibody cream to treat skin lesions, or as eyedrops for corneal infections. Moreover, the use of a Fab may avoid inflammation arising from activation of effector mechanisms.

EXAMPLE 9

DIRECT ANTIGEN CAPTURE

A specific mouse monoclonal antibody that binds herpes simplex glycoprotein D (Mab 1103) was utilized as the capture antibody bound to the solid support (obtained from Goodwin Institute for Cancer Research, Plantation, FL). The capture antibody was diluted 1:1000 in 0.1 M sodium bicarbonate buffer, pH 8.6 and used to coat ELISA plates (Costar 3690) using 25 μl per well.

Viral protein extracts were obtained by homogenizing HSV-2-infected Vero cells in 1% NP-40, 1% sodium deoxycholate in PBS (I.P. buffer). 10⁷ cells infected with HSV-2 strain G (ATCC VR-734, Rockville, MD) at a multiplicity of infection (m.o.i.) of 5, were homogenized in 5 ml of I.P. buffer by vortexing. The cell extracts were then sonicated and centrifuged at 3000 × g for 5 minutes to remove debris. Cell extracts were then aliquotted and frozen at -80ºC until needed. Antibody coated plates were repeatedly washed with water and blocked with 3% bovine serum albumin (BSA) in PBS for two hours at 37°C. The BSA solution was then discarded and replaced with 20 μl of the HSV-2-infected cell extracts and incubated at room temperature for 20 minutes. The plates were then washed ten times with PBS containing 0.05% Tween 20. At this point, about 10" C.F.U./well of an antibody library (patient AC; as described above) were added and incubated for 1 hour at 37 °C as previously described (Barbas, et al, Proc. Natl Acad Sci. USA, 88:7978, 1991; Williamson, et ai, Proc. Natl. Acad Sci. USA, 90:4141, 1993). The library suspension was then removed and plates were washed with PBS, 0.05 Tween 20. Bound phage was eluted with 50 μl of 0.1 M HCl adjusted to pH 2.2 with solid glycine.

The eluted phage suspension was immediately neutralized with 3 μl of 2 M Tris base and used to inoculate 2 ml of X-L1 Blue E coli cells (O.D.₆₀₀-0.5). After 15 minutes at room temperature, 10 ml of SB broth containing 20 μg/ml carbenicillin and 10 μg/ml tetracycline were added and the cultures were shaken at 37 °C for one hour. One hundred milliliters of SB containing 50 μg/ml carbenicillin and 10 μg/ml tetracycline were then added and the cultures were shaken for one more hour until 10¹² p.f.u. of helper phage, VCS-M13, were added. After two more hours of shaking, kanamycin was added at a final concentration of 70 μg/ml. The cultures were then shaken overnight at 30 °C. The next day, phage was prepared after 4 or 5 rounds of panning by a NheI-SpeI restriction enzyme cut followed by self ligation of the vector, as previously described. This removes the portion of phage coat protein III, which anchors the Fabs to the phage particles, from the C-terminus of the heavy chain sequence.

The clones obtained were characterized by immunoprecipitation, neutralization, and DNA sequencing as previously described (Burioni, et al, Proc. Natl. Acad. Sci. USA, 91:355, 1994). Some of the clones were identified as AC8 (ATCC 69522) as previously identified neutralizing antibody specific for glycoprotein gD (Burioni, et al., supra). The foregoing is meant to illustrate, but not to limit, the scope of the invention. Indeed, those of ordinary skill in the art can readily envision and produce further embodiments, based on the teachings herein, without undue experimentation.

Claims

1. A method for identifying a protective antigen that binds to a protective antibody comprising:

a) contacting a preselected candidate protective antigen with an antibody molecule, under conditions which allow an epitope of the antigen to bind to the antibody molecule and form an immunocomplex;

b) removing the antibody molecule bound to the epitope; and c) determining the protective ability of the antibody molecule, thereby predicting the protective ability of the antigen.

2. The method of claim 1, wherein the antigen is bound to a solid support.

3. The method of claim 1, wherein the antibody is an Fv or an Fab fragment.

4. The method of claim 1, wherein the antibody molecule is a monoclonal antibody molecule.

5. The method of claim 1, wherein the preselected antigen is selected from the group consisting of a bacterial, viral, parasitic, fungal, tumor and self-antigen.

6. The method of claim 5, wherein the viral antigen is selected from the group of viruses consisting of hepatitis B virus (HBV), human immunodeficiency virus (HTV), influenza A virus, Epstein Barr virus (EBV), herpes simplex virus (HSV), respiratory syncytial virus (RSV), human cytomegalovirus (HCMV), varicella zoster virus (VZV), and measles virus.

7. The method of claim 6, wherein the viral antigen is HSV glycoprotein D.

8. The method of claim 6, wherein the viral antigen comprises HIV glycoprotein 120.

9. The method of claim 1, wherein the epitope is a neutralizing epitope.

10. The method of claim 1, wherein the antibody molecule is in a combinatorial library.

11. The method of claim 1, wherein the determining of the protective ability of the antibody molecule is performed in vitro.

12. The method of claim 1, further comprising the step of sequencing the nucleic acid of the antibody molecule.

13. An antibody molecule identified by the method of claim 1.