WO2009137632A2 - Hiv immunogen and method of making and using same - Google Patents

Abstract

The invention provides a gp41-based HIV antigen, including a gp41 polypeptide or immunogenic fragment thereof, fused to an Fc -receptor ligand, which is highly immunogenic, highly stable and exhibits in vivo effectiveness, and elicits broadly cross-reactive HIV neutralizing antibodies, while avoiding the disadvantages of known gp41-based antigens. The invention also provide methods of vaccinating a subject against an HIV infection by administering the antigen of the invention or a composition thereof.

Description

TITLE OF THE INVENTION

HIV IMMUNOGEN AND METHOD OF MAKING AND USING SAME

RELATED APPLICATIONS/INCORPORATION BY REFERENCE This application claims priority from U.S. Provisional Application Serial No.

61/126,662, filed May 6, 2009, which is hereby incorporated in its entirety by reference. Any and all references cited in the text of this patent application, including any U.S. or foreign patents or published patent applications, International patent applications, as well as, any non-patent literature references, including any manufacturer's instructions, are hereby expressly incorporated by reference.

BACKGROUND OF THE INVENTION 1. Field of the Invention

This invention relates generally to the field of vaccination, treatment and diagnosis of human immunodeficiency virus (HIV) infections. The invention provides recombinant HIV immunogens and corresponding antibodies that are effective for the vaccination and/or immunization and/or treatment of HIV. The invention further provides methods for making and using the immunogens and antibodies of the invention to vaccinate and/or treat and/or diagnose HIV infections. 2. Background

The development of a vaccine against human immunodeficiency virus (HIV) remains an unachieved goal more than two decades after its discovery. Vaccine development has been elusive and made difficult due to genetic diversity of HIV-I and due to the fact that the virus rapidly mutates and "hides" conserved epitopes of its envelope glycoprotein by using variable loops, heavy glycosylation, oligomerization and conformational masking. Key challenges in vaccine development have been to identify antigens that are capable of eliciting broadly cross-reactive neutralizing antibodies and to understand the epitope landscape providing for such antibodies, such as the epitope landscape occurring during the process of viral entry.

Enveloped viruses, such as HIV, enter cells by a two-step process. The first step involves the binding of a viral surface protein to receptors on the plasma membrane of a host cell. After receptor binding, a membrane fusion reaction takes place between the lipid bilayer of the viral envelope and host cell membranes. Viral proteins embedded in the lipid bilayer of the viral envelope catalyze receptor binding and membrane fusion reactions. In HIV, the Env glycoprotein performs the functions of viral entry. Env is synthesized as a polyprotein precursor molecule which is proteolytically processed by a host protease to generate the surface (gpl20) and transmembrane subunits (gp41) of the mature Env glycoprotein complex. The unprocessed Env precursor is known as gpl60, reflecting its apparent molecular mass, which is further processed to form a transmembrane subunit, gp41, and an extracellular subunit, gpl20, which are assembled on the native virion surface as trimers of gpl20/gp41 noncovalently associated heterodimers. Both subunits are involved in the viral entry process and the eliciting of an immune response.

The initial step in HIV infection involves the binding of gpl20 to the cell surface molecule CD4, which serves as the major receptor for HIV-I and HIV-2. The membrane fusion process is initiated by the interaction of gpl20 with a G protein-coupled co-receptor, either the CCR5 or the CXCR4 chemokine receptor, generally after prior contact of gpl20 with CD4. Gp41 is involved in the fusion process; however its exact role in membrane fusion is not fully understood. In one theory, gp41 first engages contact with the target cell membrane by its amino- terminal hydrophobic domain, termed the fusion peptide, and then undergoes conformational changes in order to bring the viral and cellular lipid bilayers into proximity, allowing their external leaflets to merge, thereby forming a hemifusion intermediate. Next, an aqueous connection, termed a fusion pore, must open across the internal leaflets of the merged membranes and expand to leave open passage to the nucleocapsid.

Gp41, like other retroviral transmembrane proteins, consists of an amino- terminal extracellular domain (or ectodomain), a membrane-spanning domain, and a carboxy-terminal cytoplasmic (or intraviral) domain. Mutations in the cytoplasmic domain can modify the efficiency of membrane fusion, but the ectodomain can continue to function despite the cytoplasmic domain's complete deletion, at least in syncytium-formation assays. Fusion activity is also observed when the gp41 ectodomain is anchored by the membrane-spanning domain of an unrelated protein such as CD4. Therefore, only the ectodomain of gp41 seems to have a direct role in the membrane fusion process.

As a general trend, more research has been devoted towards developing antigens and HIV- neutralizing antibodies based on gpl20 than on gp41, since, while gp41 is more highly conserved, gp41 is highly unstable in the absence of gpl20, and thus, presents difficulties in the identification of effective anti-gp41 neutralizing antibodies and/or useful antigens. In addition, antibody library screening of gpl20- gp41 complexes are typically biased in favor of the selection of anti-gpl20 antibodies rather than anti-gp41 antibodies.

Both gpl20 and gp41 of Env are involved in eliciting an immune response during an HIV infection. In natural infections, the antibody response to Env is robust. However, the antibodies elicted against HIV and Env usually have a narrow spectrum of neutralization activity. Occasionally some HIV-I -positive individuals induce broadly cross-reactive neutralizing antibodies that are believed to account for the containment of the virus.

Monomeric gpl20s can elicit neutralizing antibodies in vivo, but the neutralization activity is restricted to autologous viruses or the spectrum of neutralization activity is narrow. Recent findings, however, suggest that the induction of broadly cross-reactive antibodies is possible. For example, oligomeric forms of Env ectodomain from HIV-I, strain R2 (gpl4U_R2), which contain both gpl20 and a truncated gp41 that lacks both transmembrane domains and cytoplasmic tails, induced better antibody response than gpl2U_R2 alone. Rabbit sera immunized with gpl4U_R2 neutralized not only the autologous virus, but also heterologous viruses from diverse HIV-I subtypes, suggesting that induction of broad spectrum neutralizing antibodies is possible. However, the situation has developed differently for gp41 antibodies.

To date, only relatively few human anti-gp41 monoclonal antibodies have been identified that exhibit effective neutralization capacity in vitro against a broad range of HIV isolates. And, of those antibodies, most have been shown to lack effectiveness in vivo. Three of these known antibodies, 2F5, 4E10 and Z13, target linear peptides from the gp41 membrane-proximal external region (MPER), rather than conserved conformational epitopes, which may be more effective epitopes for in vivo virus neutralization.

The development of novel approaches for the identification of broadly cross- reactive anti-HIV antibodies and HIV immunogens and their conserved epitopes, in particular against gp41, including methods for identifying and selecting such antibodies and immunogens, as well as the novel antibodies and immunogens themselves, are greatly needed and desired in the art.

SUMMARY OF THE INVENTION The present invention provides HIV antigens or derivatives and/or fragments thereof and nucleic acid molecules encoding same, as well as methods of using same for vaccinating against an HIV infection and which are capable of eliciting broadly cross-reactive neutralizing antibodies against HIV, and which overcome the deficiencies in the art regarding known anti-HIV antibodies and HIV antigens, e.g., anti-gp41 antibodies and/or gp41 -based antigens. The invention also provides diagnostic methods for detecting HIV infections using the HIV antigens or derivatives and/or fragments thereof or the anti-HIV antibodies of the invention, e.g., the recombinant anti-gp41 antibodies of the invention. The present invention further provides methods for obtaining anti-HIV antibodies which are broadly cross- reactive neutralizing antibodies against HIV, e.g., the anti-gp41 antibodies of the invention. Also provided by the present invention are broadly cross-reactive neutralizing anti-HIV antibodies or fragments thereof and pharmaceutical compositions comprising such antibodies or fragments thereof, such as, the anti- gp41 antibodies described herein. The invention also provides methods of utilizing the antigens and antibodies of the invention for immunizing against and/or treating HIV infections. The invention further provides pharmaceutical and/or diagnostic packages comprising the HIV antigens and/or the anti-HIV antibodies of the invention for use in immunizing against or treating HIV infections or for detecting HIV in a subject or a biological sample. Thus, in one aspect, the present invention provides an immunogen for vaccinating against and/or treating an HIV infection, comprising an HIV gp41 polypeptide or fragment thereof translationally linked to an Fc receptor ligand, optionally through a flexible linker.

In certain embodiments, the HIV gp41 polypeptide of the immunogen of the invention comprises at least the ectodomain of gp41. In other embodiments, the HIV gp41 polypeptide of the immunogen of the invention comprises a gp41 that lacks the cytoplasmic tail region, the transmembrane region or the fusion domain region, or any combination thereof.

In still other embodiments, the gp41 polypeptide of the immunogen of the invention is translationally fused to the amino-terminal end of the Fc receptor ligand. In other embodiments, the gp41 polypeptide of the immunogen of the invention is translationally fused to at its amino-terminal end to the carboxy-terminal end of a Fc receptor ligand.

The Fc receptor ligand can be an antibody Fc region or fragment thereof. In another embodiment, the Fc region is the Fc region of a human IgG. In other embodiments, the Fc region is the Fc region of any IgG, IgA, IgM, or IgE antibody.

In yet another embodiment, the immunogen of the invention comprises a flexible linker that couples the gp41 portion of the immunogen with the Fc receptor ligand component of the immunogen. The flexible linker can be a polypeptide having between 1 and 50 amino acids in length. In other embodiments, the flexible linker is a hinge region of an antibody, such as the hinge region of IgG or any other antibody.

In one embodiment of the invention, the gp41 component of the immunogen of the invention corresponds with the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence having at least about 80%, 85%, 90%, 95% or 99% sequence identity thereto.

In another embodiment of the invention, the flexible linker component of the immunogen of the invention is the hinge region of a human IgG having the amino acid sequence of SEQ ID NO: 6 or or an amino acid sequence having at least about 80%, 85%, 90%, 95% or 99% sequence identity thereto. In yet another embodiment of the invention, the Fc receptor ligand component of the immunogen of the invention is the CH2-CH3 region of a human IgG having the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence having at least about 80%, 85%, 90%, 95% or 99% sequence identity thereto. In still another embodiment of the present invention, the gp41-Fc fusion protein immunogen of the invention is capable of eliciting broadly-cross reactive neutralizing antibodies against HIV, which are effective in vitro and in vivo.

In another aspect, the present invention provides a method of vaccinating a subject against an HIV infection comprising administering a therapeutically effective amount of a gp41-Fc fusion protein immunogen of of the invention.

In yet another aspect, the present invention provides a method of treating a subject having an HIV infection comprising administering a therapeutically effective amount of a gp41-Fc fusion protein immunogen and/or antibodies specific for or raised against the gp41-Fc immunogens of the invention. In certain other aspects, the present invention provides isolated nucleic acid molecules which encode the gp41-Fc fusion protein immunogens and/or antibodies of the invention, as well as methods for preparing the nucleic acid molecules, and methods for using the nucleic acid molecule to make the antigens/immunogens and antibodies of the invention. In one aspect, the present invention provides a recombinant HIV antigen comprising a human immunodeficiency virus envelope protein gp41 or fragment thereof, e.g., the ectodomain of gp41, which is coupled to an Fc (fragment crystallizable region) receptor ligand optionally by a flexible linker.

In one embodiment, the gp41 is a full length gp41 polypeptide from any known strain of HIV-I or HIV-2, e.g., or from any isolate obtained from an infected individual. In another embodiment, the gp41 is a fragment of gp41, wherein the fragment comprises at least the ectodomain of gp41 or a fragment of the ectodomain. In another embodiment, the gp41 is a gp41 fragment comprising the ectodomain or fragment thereof and at least one or both of the membrane-spanning domain and the carboxy-terminal cytoplasmic domain. In another embodiment, the recombinant HIV antigen comprises a human HIV gp41 ectodomain sequence defined by SEQ ID NO: 4 or an amino acid sequence having at least about 80%, 85%, 90%, 95% or 99% sequence identity thereto, and an Fc receptor ligand optionally coupled to the gp41 sequence or component through a flexible linker.

In certain embodiments, the Fc receptor ligand can be an Fc region or fragment thereof of an antibody, e.g., an IgG, or a polypeptide having an amino acid sequence that has at least about 80%, 85%, 90%, 95% or 99% sequence identity to the Fc region of an antibody. In certain other embodiments, the Fc receptor ligand can be an Fc region or fragment thereof from an IgG, IgA, IgD, IgM or an IgE antibody, or a polypeptide having an amino acid sequence with at least about 80%, 85%, 90%, 95% or 99% sequence identity thereto. In a particular embodiment, the Fc receptor ligand corresponds with the CH2-CH3 region of human IgG as defined by SEQ ID NO: 5, or an amino acid sequence having at least about 80%, 85%, 90%, 95% or 99% sequence identity thereto.

In another embodiment, the recombinant HIV antigen is defined by SEQ ID NO: 3, which comprises an ectodomain of HIV-I gp41 or an amino acid sequence having at least about 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a human IgG Fc region, joined to the gp41 region via a flexible linker human IgG hinge region.

In another embodiment, the recombinant HIV antigen comprises an human immunodeficiency virus envelope protein gp41 or fragment thereof which is coupled at its carboxy-terminal end to the amino-terminal end of an Fc receptor ligand optionally through a flexible linker, e.g., a human IgG hinge region. In one embodiment, thegp41 is defined by SEQ ID NO: 4 or an amino acid sequence having at least 80%, 85%, 90%, 95% or 99% sequence identity thereto.

In certain embodiments, the flexible linker is not required, and the recombinant antigen comprising a human immunodeficiency virus envelope protein gp41 or fragment thereof (e.g., gp41 ectodomain) is coupled directly to an Fc receptor ligand or fragment thereof, e.g., an IgG Fc region or fragment thereof. In certain other embodiments, the antigen comprises a gp41 protein or fragment thereof coupled at its carboxy-terminal end to the amino-terminal end of an Fc receptor ligand or fragment thereof, optionally through a flexible linker. In certain other embodiments, the antigen comprises an Fc receptor ligand or fragment thereof coupled at its carboxy-terminal end to the amino-terminal end of a gp41 protein or fragment thereof, optionally through a flexible linker.

In other embodiments, the flexible linker can be any suitable molecule, such as, for example, a polypeptide of a suitable length and composition of amino acids. For example, the flexible linker can be single amino acid residue, or even a polypeptide having at least 2 amino acids in length, or to at least about 5 amino acids in length, or from about 5 to about 10 amino acids in length, or about 5 to about 25 amino acids in length, or about 5 to about 50 amino acids in length or even about 5 to 100 amino acids or more in length. In one embodiment, the linker corresponds to the hinge region of human IgG as defined by SEQ ID NO: 6, or an amino acid sequence or fragment thereof having at least about 80%, 85%, 90%, 95% or 99% sequence identity thereto. The hinge region of any antibody can be use as the flexible linker.

In another aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a recombinant gp41 antigen of the invention, and at least one pharmaceutically acceptable carrier or excipient.

In yet another aspect, the present invention provides a method of immunizing a subject against an HIV infection, comprising administering a therapeutically effective amount of a recombinant gp41 antigen of the invention. In yet another aspect, the present invention provides a method of making a broadly cross-reactive neutralizing anti-HIV antibody, comprising raising the antibody against a recombinant HIV antigen of the invention. In still another aspect, the invention provides a method of making a broadly cross-reactive neutralizing anti-HIV antibody by screening an antibody library for antibodies that specifically bind to an antigen of the invention. The invention also provides a method of making a broadly cross-reactive neutralizing anti-HIV antibody against HIV, comprising immunizing an animal with an antigen of the invention to elicit the production of anti-HIV antibodies in the animal, obtaining the sera of the animal, and isolating the anti-HIV antibodies from the sera.

In yet another aspect, the invention provides an isolated broadly cross- reactive neutralizing anti-HIV antibody against HIV isolated according to the methods of the invention.

In yet another aspect, the present invention provides a method of treating an HIV infection in a subject in need thereof, comprising: obtaining a broadly cross- reactive neutralizing anti-HIV antibody prepared by the methods of the invention; and administering a therapeutically effective amount of the anti-HIV antibody to the subject, thereby neutralizing the HIV infection and treating the subject. The administration can be local, topical, or systemic (e.g., injection), or any other suitable mode of administration.

The present invention additionally provides a vaccine for immunizing a subject, e.g., a human, against an HIV infection, wherein the vaccine comprises a recombinant gp41 antigen of the invention.

In some embodiments, vaccinating a human with a vaccine of the invention or administering an antigen or immunogen of the invention can further include the step of co-administering a suitable co-therapeutic agent, e.g., an anti- viral drug or an agent that modulates an immune response. The anti- viral drug can be, for example, an anti-HIV agent.

In still another aspect, the present invention provides a packaged pharmaceutical comprising a recombinant antigen of the invention and instructions for use in accordance with a therapeutic method of the invention. In a further aspect, the instant invention provides for nucleic acid molecules encoding the recombinant antigens and antibodies of the invention. In one embodiment, the nucleic acid molecule is defined by the nucleic acid sequence of SEQ ID NO: 1, which encodes a gp41-Fc antigen of the invention, or a nucleic acid sequence having at least about 80%, 85%, 90%, 95% or 99% sequence identity thereto. In another aspect, the invention provides an expression vector comprising a nucleic acid molecule encoding a recombinant antigen of the invention, and host cells comprising the expression vectors of the invention. The host cells can be capable of expressing the antigens and/or antibodies of the invention, i.e., the products encoded by the expression vectors of the invention, to allow a means for making the antigens and/or antibodies of the invention.

In still a further aspect, the present invention provides a method of making a human recombinant antigen of the invention comprising, coupling the 5 ' end of a nucleic acid molecule encoding the Fc receptor ligand to the 3' end of the nucleic acid molecule encoding gp41 or fragment thereof, optionally via a nucleic acid molecule encoding a flexible linker to form a gp41-Fc fusion gene encoding a recombinant gp41-Fc antigen; operably linking the gp41-Fc fusion gene to an expression vector; introducing the expression vector into a host cell; permitting the host cell to express the recombinant antigen; and isolating the recombinant antigen from the cell.

The invention still further provides diagnostic methods for detecting an HIV infection, including detecting HIV antigens and/or anti-HIV antibodies, utilizing the antigens and antibodies of the invention.

These and other embodiments are disclosed or are obvious from and encompassed by, the following Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description, given by way of example, but not intended to limit the invention solely to the specific embodiments described, may best be understood in conjunction with the accompanying drawings.

Figure 1 provides a schematic depicting the general structure of an embodiment of the HIV antigen of the invention, gp41Fc.

Figure 2 shows the results of an antibody- antigen assay to evaluate binding of anti-gp41 antibodies to a gp41Fc antigen of the invention. Three-fold serially diluted anti-gp41 antibodies (Abs) were added to 96- well plates coated with 5 μg/ml of a gp41Fc of the invention. Bound Abs were detected using HRP-conjugated anti- human IgG, F(ab')2 (for human Abs) or anti-mouse IgG (for NC-I and T3) and

ABTS as substrate.

Figure 3 depicts the titration of rabbit serum with a gp41Fc of the invention

(A) and 89.6gpl40 (B). lμg/ml 89.6 gpl40 linker protein or lug/ml gp41Fc was coated on microwell plates. After blocking the wells, 2-fold serially diluted rabbit serum was added to the plates. Bound Abs were detected using HRP- anti-rabbit

IgG, F(ab')₂ and ABTS as substrate.

Figure 4 provides a table showing the percentage HIV- 1 neutralization by purified IgG from immunized rabbit serum in a cell line/pseudovirus assay. All four HIV isolates, BaL (B), GXC-44(C), 92UG037.8(A), and CM243(E), were tested against 50 μg/ml of purified IgG sample. Pre-1214/1215: pre-immunization serum;

PB1-1214/1215: 1^st test bleeding serum; PB2- 1214/1215: 2^nd test bleeding serum;

FT1214/1215: final bleeding serum.

Figure 5 provides an SDS-PAGE of a gp41Fc embodiment of the invention under non-denaturing and denaturing conditions. Four (4) ug each were loaded onto each lane. M: rainbow protein markers; lane 1 : IgGl ml6, reduced; 2: gp41Fc, reduced; 3-4 empty; 5: IgGl ml6 (non-reduced); and 6: gp41Fc (non-reduced). Figure 6 shows the DNA coding (sense) strand sequence of gp41g_{9 6} in vector pEAKIO (indicated as SEQ ID NO: 1). The single underlined portion at the 5' end of the molecule corresponds to the CD5ss signal peptide. The double underlined portion at the 5' end corresponds to the Nhel restriction site. The double underlined portion at the 3' end corresponds to the BamHI site. The italicized region corresponds to the complete 89.6 gp41 region without the N-terminal fusion peptide-encoding region. Figure 7 shows the sense (SEQ ID NO: 1) and antisense (SEQ ID NO. 2) strands of the DNA of SEQ ID NO: 1 of Figure 6. The figure shows the predicted amino acid sequence as defined by the sense strand or coding strand (i.e., the top strand of DNA).

Figure 8 shows the amino sequence of a gp41Fc embodiment of the invention (SEQ ID NO: 3), which lacks the fusion domain, transmembrane domain and cytoplasmic tail of 89.6 gp41. Note that 89.6 gp41Fc in pEAKIO has 151 amino acids (22-172) and does not contain the fusion peptide (i.e., the first 20 amino acids at the N-terminus of gp41), or the transmembrane domain and cytoplasmic tail. The complete gp41 extracellular portion has 171 amino acids in length. The flexible linker region is shown in bold, which is formed by the hinge region of human IgG (EPKSCD KTHTCPPCP). The Fc receptor ligand is CH2 and CH3 regions derived from human IgG (see Figure 10, SEQ ID NO: 5). The DPE sequence is derived from the restriction site BamHI and becomes a part of the linker between gp41 and human Fc.

Figure 9 shows the amino sequence (SEQ ID NO: 4) of 89.6 gp41, which lacks the fusion domain, transmembrane domain and cytoplasmic tail, and which corresponds to the italicized portion of SEQ ID NO: 1.

Figure 10 shows the amino acid sequence of the CH2-CH3 region (Fc region) of human IgG, which corresponds to SEQ ID NO: 5.

Figure 11 shows the amino acid sequence of the hinge region of human IgG, which corresponds to SEQ ID NO: 6. Figure 12 shows the amino acid sequence of an embodiment of the gp41Fc fusion protein of the invention which corresponds to SEQ ID NO: 7, including the gp41 sequence, flexible linker and IgG Fc region.

Figure 13 shows the nucleotide sequence (SEQ ID NO: 8) of an embodiment of the gp41Fc fusion of the invention in pEAKIO vector. Underlined is the CD5ss signal peptide; bolded is the Nhel (gctagc) and BamHI (ggatcc) sites; in italics is the hinge region; highlighted is human CH2; highlighted and underlined is human CH3; bold and underlined region is the complete 89.6gp41 region without the N-terminal fusion peptideATG at the end is stop codon (TGA).

Figure 14 shows the binding of anti-HIV-1 Env mAbs to an gp41Fc fusion protein of the invention (SEQ ID NO: 7). Three-fold serially diluted anti-gp41 mAbs were added to 96-well plates coated with 5 μg/mL of purified gp41Fc. Bound Abs were detected using HRP conjugated to anti-human IgG, F(ab')2 (for human Abs) or anti-mouse IgG (H+L) (for mAbs NC-I and T3) and ABTS as substrate.

Figure 15 shows titration of rabbit serum. One μg/mL 89.6gpl40 linker protein (left panel) or lug/mL of an gp41Fc fusion protein (right panel) of the invention (SEQ ID NO: 7) was coated on microwell plates. After blocking the wells, 2-fold serially diluted rabbit serum was added to the plates. Bound Abs were detected using HRP- anti-rabbit IgG, F(ab')₂ and ABTS as substrate.

Figure 16 shows that neutralization activity of immune rabbit IgG was decreased in the presence of increased concentration of a gp41Fc fusion protein of the invention (SEQ ID NO: 7) in neutralizing pseudo-typed HIV-I (BaI). Fifty μg/mL FT1214 IgG was tested in cell line-based pseudovirus assay against HIV-I isolate BaI in the presence of serially diluted gp41Fc. Gp41Fc alone was also tested in the assay.

Figure 17 shows that binding of immune rabbit IgG to non-denatured and denatured gpl4U_{89 6} and a gp41Fc fusion protein of the invention (SEQ ID NO: 7). Two μg/mL non-denatured and denatured gp 1408₉6 and gp41Fc were coated on micro well plates. Three-fold serially diluted rabbit IgG were added to the wells. Bound antibodies were detected using HRP conjugated to anti-rabbit IgG (H+L) and ABTS as substrate. Figure 18 shows binding of rabbit IgGs to peptides derived from gp41 immunodominant loop region. Five μg/mL of each peptide was coated on high- binding microwell plates. Three-fold serially diluted rabbit IgGs FT1214 (top left) and FT1215 (top right) added to the wells and bound antibodies detected using HRP-conjugated anti-rabbit IgG and ABTS as substrate. Binding of IgGs FT1214 and FT1215 at 33 μg/mL to each peptide was plotted for comparison (middle panel). The amino acid sequences of the peptides tested were shown and the regions IgGs FT1214 (underlined) and FT1215 (in bold) bound to were indicated (bottom).

Figure 19 shows the results of capture ELISA of a gp41Fc fusion protein (SEQ ID NO: 7) of the invention in immune rabbit IgG. Gp41Fc in the rabbit IgGs purified from immune rabbit sera collected from different time of bleed (see the immunization schedule in Materials and Methods in Example 2) was captured by mouse mAb T3 coated on 96-well microplates. Bound gp41Fc was detected by using biotinylated mouse mAb D61 and HRP-conjugate to streptavidin. The optical density was measured 30min after addition of ABTS (top left and top right panels). The concentration of gp41Fc in purified rabbit IgG was calculated using purified recombinant gp41Fc as a standard and then converted to the concentration of gp41Fc in rabbit serum (middle panel and corresponding bottom table). DETAILED DESCRIPTION OF THE INVENTION

The present inventors have for the first time developed gp41 -based HIV antigens which advantageously elicit antibodies having broad neutralization activity against different HIV isolates, are highly immunogenic, exhibit high stability and enhanced half-life, and exhibit in vivo effectiveness, while lacking the disadvantages of prior gp41 -based HIV antigens, including problems of eliciting self-reacting antibodies, having low immunogenicity and exhibiting in vivo ineffectiveness. The gp41 -based antigens provided by the invention fuse HIV gp41 or a fragment thereof (e.g., gp41 ectodomain) with an Fc receptor ligand, e.g., the Fc region of a human IgG), optionally through a flexible linker (e.g., the hinge region of human IgG), wherein the gp41 fusion protein stabilizes the structure of gp41 in the absence of gpl20, and exhibits enhanced immunogenicity by enabling it to bind to immune cells having Fc receptors, such as, macrophages or dendritic cells. The invention further provides methods and compositions for vaccinating and/or immunizing a subject against an HIV infection by administering a therapeutically effective amount of the gp41 -based HIV antigen of the invention or a fragment thereof, or a composition thereof.

The present invention also provides antibodies specific for and/or raised against the gp41 -based antigen of the invention, methods for obtaining the antibodies, and compositions comprising the antibodies of the invention. The antibodies of the invention are advantageously provided with broad cross-reactive neutralization activity against a wide spectrum of HIV isolates and/or clades and can be utilized in therapeutic methods for treating an HIV infection. Compositions comprising the antibodies can be prepared for systemic, e.g., parenteral administration, or local administration, e.g., topical creams, ointments, or suaves for applying to bodily fluids that may comprise HIV viruses, or for administering by any other suitable means.

The instant invention further provides diagnostic methods and compositions comprising the antigens and/or antibodies of the invention for clinical and/or research purposes for detecting HIV viruses, antigens and/or anti-HIV antibodies. The present invention also provides pharmaceutical and/or diagnostic packages or kits comprising the antigens and/or antibodies of the invention, instructions for use, and additional materials and components suitable for a particularly desired diagnostic or therapeutic use. In a further aspect, the instant invention provides for nucleic acid molecules encoding the recombinant antigens and antibodies of the invention, as well as expression vectors comprising the nucleic acid molecules and host cells for expressing the encoded proteins. In one embodiment, the nucleic acid molecule is defined by the nucleic acid sequence of SEQ ID NO: 1, which encodes a gp41-Fc antigen of the invention, or a nucleic acid sequence having at least about 80%, 85%, 90%, 95% or 99% sequence identity thereto.

It is to be understood that present invention as described herein is not to be limited to the particular details set forth herein regarding any aspect of the present invention. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention. Definitions

Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs.

As used herein, the term "antibody" is meant to refer to immunoglobulin molecules (e.g., any type, including IgG, IgE, IgM, IgD, IgA and IgY, and/or any class, including, IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) isolated from nature or prepared by recombinant means or chemically synthesized. The terms "antibody" and "immunoglobubin" can be used interchangeably throughout the specification, unless indicated otherwise.

As used herein, the term "antibody fragment" is meant to refer to a portion of a whole antibody which retains the ability to exhibit antigen binding activity or immunogenicity. Examples include, but are not limited to, Fv, disulphide-linked Fv, single-chain Fv, Fab, variable heavy region (V_H), variable light region (V_L), and fragments of any of the above antibody fragments which retain the ability to exhibit antigen binding activity, e.g., a fragment of the variable heavy region V_H retains its ability to bind its antigen.

For preparation of monoclonal or polyclonal antibodies, any technique known in the art can be used (see, e.g., Kohler and Milstein, Nature 256:495497 (1975); Kozbor et al., Immunology Today 4:72 (1983); Cole et al., pp. 77-96 in

Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985)). Techniques for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce antibodies to polypeptides of this invention. Also, transgenic mice, or other organisms such as other mammals, may be used to express humanized antibodies. Alternatively, phage display technology can be used to identify antibodies and heteromeric Fab fragments that specifically bind to selected antigens (see, e.g., McCafferty et al., Nature 348:552-554 (1990); Marks et al., Biotechnology 10:779-783 (1992)).

The term "immunoassay" is meant to refer to an assay that uses an antibody to specifically bind to and detect an antigen. The immunoassay is characterized by the use of specific binding properties of a particular antibody to isolate, target, and/or quantify the antigen.

As used herein, the terms "biological sample" or "patient sample" as used herein, is meant to refer to a sample obtained from an organism or from components (e.g., cells) of an organism. The sample can be of any biological tissue or fluid. The sample may be a clinical sample which is a sample derived from a patient. Such samples include, but are not limited to, sputum, blood, serum, plasma, blood cells (e.g., white cells), tissue samples, biopsy samples, urine, peritoneal fluid, and pleural fluid, saliva, semen, breast exudate, cerebrospinal fluid, tears, mucous, lymph, cytosols, ascites, amniotic fluid, bladder washes, and bronchioalveolar lavages or cells therefrom, among other body fluid samples. The patient samples may be fresh or frozen, and may be treated, e.g. with heparin, citrate, or EDTA, or other suitable treatment known in the art. Biological samples may also include sections of tissues such as frozen sections taken for histological purposes. Samples can be infected with HIV.

As used in this invention, the term "epitope" is meant to refer to any antigenic determinant on an antigen, e.g., a gpl20 or gp41 protein, to which an antibody binds through an antigenic binding site. Determinants or antigenic determinants on an antigen 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 epitope can be an inducible epitope, i.e., an epitope that becomes available or accessible only once exposed by a change in conformation, e.g., the CD4-inducible epitope of gp41.

As used herein, the term antibody that "specifically (or selectively) binds to" or is "specific for" or is "specifically (or selectively) immunoreactive with" a particular antigen, e.g., a polypeptide, or a particular epitope on a particular antigen, e.g., a polypeptide epitope, is one that binds to that particular antigen or epitope without substantially binding to any other antigen or epitope. Antibody affinity for antigens and epitopes can be measured by enzyme linked immunosorbent assay (ELISA) or other suitable affinity tests. An antibody that specifically binds to an antigen, in accordance with this invention, refers to the binding of an antigen by an antibody or fragment thereof with a dissociation constant (K_d) of 1 μM or lower, as measured by surface plasmon resonance analysis using, for example, a BIACORE surface plasmon resonance system and BIACORE kinetic evaluation software (eg. version 2.1). The affinity or dissociation constant (K_d) for a specific binding interaction is preferably about 500 nM or lower, more preferably about 300 nM or lower and preferably at least 300 nM to 50 pM, 200 nM to 50 pM, and more preferably at least 100 nM to 50 pM, 75 nM to 50 pM, 10 nM to 50 pM.

The term "antigen," as used herein, refers to a substance, e.g., a polypeptide or polysaccharide or nucleic acid molecule, which can specifically bind to or is specifically recognized by an antibody. Antigens can, but may not necessarily, prompt an immune response. The term "immunogen" refers to a subclass of antigen which are capable of eliciting a response by the immune system, i.e., the eliciting of antibodies that specifically bind to the immunogen.

The term "HIV antigen," as used herein, refers to a substance that is capable of eliciting antibodies that specifically recognize HIV or a component thereof, e.g., an HIV polypeptide or nucleic acid molecule. The HIV antigen can be obtained from HIV or prepared synthetically and can include any suitable modification (e.g., chemical or translational fusion to heterologous polypeptide to form fusion protein, including gp41-Fc fusion protein of the invention.

The term "gpl60" refers to the human immunodeficiency virus-1 envelope glycoprotein gpl60 kDa (or its corresponding gene), which is processed to form the 120 kDa (gpl20) subunit and the 41 kDa (gp41) subunit.

The terms "gpl20" or "gpl20 subunit", as used herein, is meant to refer to the human immunodeficiency virus-1 envelope glycoprotein gpl20. The terms "gpl20 variant", "gpl20 mutant", or "gpl20 derivative" refers to a protein which is characterized by: (1) having an amino acid subsequence that has greater than about 60% amino acid sequence identity, 65%, 70%, 75%, 80%, 85%, 90%, preferably

95%, 96%, 97%, 98%, 99% or greater amino acid sequence identity, to the sequence of HIV-I gpl20. The nucleic acid and amino acid sequences of HIV gp-120 are readily available to the public through the HIV sequence database on the world wide web at hiv.lanl.gov/content/sequence/HIV/mainpage.html; (2) binding to antibodies, e.g., polyclonal antibodies, raised against an immunogen comprising an amino acid sequence of HIV-I gpl20; (3) specifically hybridizing under stringent hybridization conditions to a nucleic acid sequence encoding HIV-I gpl20 and (4) having a nucleic acid sequence that has greater than about 85%, preferably greater than about 90%, 95%, 98%, 99%, or higher nucleotide sequence identity to the nucleic acid sequence encoding HIV-I gpl20.

The terms "gp41" or "gp41 subunit", as used herein is meant to refer to the human immunodeficiency virus-1 envelope glycoprotein gp41. The terms "gp41 variant", "gp41 mutant", or "gp41 derivative" refers to a protein which is characterized by: (1) having an amino acid subsequence that has greater than about 60% amino acid sequence identity, 65%, 70%, 75%, 80%, 85%, 90%, preferably

95%, 96%, 97%, 98%, 99% or greater amino acid sequence identity, to the sequence of HIV-I gp41. The nucleic acid and amino acid sequences of HIV gp-41 are readily available to the public through the HIV sequence database on the world wide web at hiv.lanl.gov/content/sequence/HIV/mainpage.html; (2) binding to antibodies, e.g., polyclonal antibodies, raised against an immunogen comprising an amino acid sequence of HIV-I gp41; (3) specifically hybridizing under stringent hybridization conditions to a nucleic acid sequence encoding HIV-I gp41 and (4) having a nucleic acid sequence that has greater than about 85%, preferably greater than about 90%, 95%, 98%, 99%, or higher nucleotide sequence identity to the nucleic acid sequence encoding HIV-I gp41.

The "fusion domain" of HIV-I gp41 is meant to refer to the domain comprising the first 20 amino acids at the N-terminus of gp41. In certain examples, the fusion domain of gp-41 corresponds to position 512-532 according to HXB2 numbering.

The "extracellular domain" or "ectodomain" or HIV-I gp41 is meant to refer to the part of the protein that is on the outside of the cell. In certain examples, the extracellular domain of gp41 consists of position 512 to 683 according to HXB2 numbering.

The "transmembrane domain" of HIV-I gp41 is meant to refer to the region corresponding to the portion of the protein that spans the cell. In certain examples, the transmembrane domain of gp-41 corresponds to the position 684 to 703 according to HXB2 numbering.

The "cytoplasmic tail" of HIV-I gp 41 is meant to refer to the region corresponding to the portion of the protein that interacts with the interior of the cell or organelle, n certain examples, the cytoplasmic tail of gp-41 corresponds to position 704 to 846 according to HXB2 numbering. As used herein, the term "flexible linker" refers to any heterologous polypeptide of at least 1, 2, 3, 4 or 5 or more amino acids in length, which when inserted between the carboxy-terminal end of gp41 and the amino-terminal end of a Fc receptor ligand yields a functional linkage joining the gp41 region with the Fc receptor ligand. In certain embodiments, a flexible linker is not required, and the gp41 and the Fc receptor ligand are joined without a linker.

The term "Fc" or "Fc region," as used herein, refers to the fragment crystallizable region ("Fc region") or an antibody, which is known as the tail region of an antibody. It will be appreciated that the Fc region of an antibody interacts with or function as ligands for cell surface receptors called "Fc receptors," which are protein receptor molecules found on the surface of certain cells, including macrophages and neutrophils, and which play a role in the immune system. Fc receptors are activated upon binding to a cognate Fc region of an antibody, which in turn stimulates the activities of cytotoxic and phagocytic cells against invading pathogens or unwanted cells or substances, thereby mediating various physiological effects of antibodies, such as opsonization, cellular lysis, and cell degranulation. The Fc regions or fragments thereof can include those Fc regions from any type of antibody, including IgG, IgA, IgD, IgM and IgE antibody types. It will be further appreciated that Fc receptors are classified based on the type of antibody that they recognize, and include, for example, Fc receptors that recognize IgG Fc regions ("Fc-gamma receptors"), IgA Fc regions ("Fc-alpha receptors") and IgE Fc regions ("Fc-epsilon receptors"). The nucleotide and amino acid sequences of Fc regions and Fc receptors in humans and other animals are readily available in the art from public gene and protein databases.

The term "Fc receptor ligand," as used herein, refers to any Fc region (or fragment thereof) capable of being specifically recognized and/or bound by an Fc receptor and which causes the Fc receptor to become activated, thereby triggering the downstream physiological responses associated with the binding of an Fc receptor with an Fc region.

As used herein, the term "regulatory sequences" refers to those sequences, both 5' and 3' to a structural gene, that are required for the transcription and translation of the structural gene in the target host organism. Regulatory sequences include a promoter, ribosome binding site, optional inducible elements and sequence elements required for efficient 3' processing, including polyadenylation. When the structural gene has been isolated from genomic DNA, the regulatory sequences also include those intronic sequences required for splicing of the introns as part of mRNA formation in the target host. The term"gp41 fusion protein" as used herein refers to a polypeptide comprising both a gp41 protein or fragment thereof (e.g., the ectodomain of gp41) translationally fused to an Fc receptor ligand. The order of the polypeptides of the fusion protein is not limited. Thus, the gp41 fusion protein can be a fusion of the C- terminal end of gp41 (or fragment thereof) with the N-terminal end of an Fc receptor ligand. Also, the gp41 protein can be a fusion of the C-terminal end of the Fc receptor ligand with the N-terminal end of the gp41 protein or fragment thereof. The gp41 fusion protein may comprise a flexible linker between the gp41 and Fc receptor ligand portions; however, the flexible linker is not absolutely required.

As used herein, the terms "ligand-receptor complexes" refers to a specific association between a fusion protein and the extracellular subunit of HIV receptors or with an Fc receptor on an effector immune cell.

The terms "isolated," "purified," or "biologically pure" refer to material that is substantially or essentially free from components that normally accompany it as found in its native state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein that is the predominant species present in a preparation is substantially purified. The term "purified" denotes that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. Particularly, it means that the nucleic acid or protein is at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure. "Nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double- stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2'-O-methyl ribonucleotides, peptide - nucleic acids (PNAs).

Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. BiOL Chem. 260:2605-2608 (1985); Rossolini et al., MoI. Cell. Probes 8:91-98 (1994)). The term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.

The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.

The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes. As used herein a "nucleic acid probe or oligonucleotide" is defined as a nucleic acid capable of binding to a target nucleic acid of complementary sequence through one or more types of chemical bonds, usually through complementary base pairing, usually through hydrogen bond formation. As used herein, a probe may include natural (i.e., A, G, C, or T) or modified bases (7-deazaguanosine, inosine, etc.). In addition, the bases in a probe may be joined by a linkage other than a phosphodiester bond, so long as it does not interfere with hybridization. The probes can be directly labeled as with isotopes, chromophores, lumiphores, chromogens, or indirectly labeled such as with biotin to which a streptavidin complex may later bind. By assaying for the presence or absence of the probe, one can detect the presence or absence of the select sequence or subsequence.

The term "recombinant" when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all. An "expression vector" is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a host cell. The expression vector can be part of a plasmid, virus, or nucleic acid fragment. Typically, the expression vector includes a nucleic acid to be transcribed operably linked to a promoter.

By "host cell" is meant a cell that contains an expression vector and supports the replication or expression of the expression vector. Host cells can be mammalian cells such as CHO, HeLa and the like, e.g., cultured cells, explants, and cells in vivo, or bacterial host cells. The terms "identical" or percent "identity," in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identity, 65%, 70%, 75%, 80%, preferably 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to an amino acid sequence or a nucleotide sequence when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Such sequences are then said to be "substantially identical." This definition also refers to the compliment of a test sequence. Preferably, the identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50-100 amino acids or nucleotides in length.

For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. For sequence comparison of HIV envelope glycoproteins, fusion proteins comprising envelope glycoproteins and nucleic acid sequences encoding the same, the BLAST and BLAST 2.0 algorithms and the default parameters discussed below are used.

A "comparison window", as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. MoI. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).

One algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J. MoI. Biol. 215:403410 (1990), respectively. BLAST and BLAST 2.0 are used, with the parameters described herein, to determine percent sequence identity for the nucleic acids and proteins of the invention. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nln.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive- valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative- scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of 10, M=5, N4, and a comparison of both strands. The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.

The phrase "stringent hybridization conditions" refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acid, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays" (1993). Generally, stringent conditions are selected to be about 5-10C° lower than the thermal melting point (Tm for the specific sequence at a defined ionic strength pH. The Tm is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at T.sub.m 50% of the probes are occupied at equilibrium). Stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30C for short probes (e.g., 10 to 50 nucleotides) and at least about 6OC for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For high stringency hybridization, a positive signal is at least two times background, preferably 10 times background hybridization. Exemplary high stringency or stringent hybridization conditions include: 50% formamide, 5xSSC and 1% SDS incubated at 42C or 5xSSC and 1% SDS incubated at 65C, with a wash in 0.2xSSC and 0.1% SDS at 65C.

Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides that they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acids typically hybridize under moderately stringent hybridization conditions. Exemplary "moderately stringent hybridization conditions" include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37C, and a wash- in 1. times. SSC at 45C A positive hybridization is at least twice background. Those of ordinary skill will readily recognize that alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency. As used herein, the term "gp41 -based HIV antigen" refers any antigen comprising gp41 or a fragment or derivative thereof.

As used herein, the term "vaccine" is meant to encompass any immunogenic composition that is capable of inducing an immune response in a subject which establishes or enhances immunity to a particular disease, such as HIV, thereby either vaccinating against a future infection or treating an existing infection. By immune response is meant to include responses that result in at least some level of immunity in the treated subject thereby treating an existing infection or vaccinating against a future infection.

As used herein and in the appended claims, the singular forms "a," "and," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to "a gene" is a reference to one or more genes and includes equivalents thereof known to those skilled in the art, and so forth. Nucleic Acids Encoding HIV Env Proteins

The nucleic acid sequences encoding HIV envelope glycoproteins may be obtained by recombinant DNA methods, such as screening reverse transcripts of mRNA, or screening genomic libraries from any HIV-infected cell or HIV isolate. The DNA may also be obtained by synthesizing the DNA from published sequences using commonly available techniques such as solid phase phosphoramidite triester method first described by Beaucage and Caruthers, Tetrahedron Letts. 22:1859-1862 (1981), using an automated synthesizer, as described in Van Devanter et al., Nucleic Acids Res. 12:6159-6168 (1984). Synthesis may be advantageous because unique restriction sites may be introduced at the time of preparing the DNA, thereby facilitating the use of the gene in vectors containing restriction sites not otherwise present in the native source. Furthermore, any desired site modification in the DNA may be introduced by synthesis, without the need to further modify the DNA by mutagenesis. Purification of oligonucleotides is by either native acrylamide gel electrophoresis, agarose electrophoresis or by anion-exchange HPLC as described in Pearson and Reanier, J. Chrom. 255:137-149 (1983), depending upon the size of the oligonucleotide and other characteristics of the preparation. The sequence of cloned genes and synthetic oligonucleotides can be verified using, e.g., the chain termination method for sequencing double- stranded templates as described by Wallace et al., Gene 16:21-26 (1981).

Processes for producing recombinant proteins for purification by the methods of the present invention will employ, unless otherwise indicated, conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See e.g., Maniatis, Fritsch and Sambrook, Molecular Cloning: A Laboratory Manual, 2nd Ed. (1989); DNA Cloning: A Practical Approach, Volumes I and II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1985); Transciption And Translation (B. D. Hames and S. J. Higgins eds. 1984); Animal Cell Culture (R. I. Freshney ed. 1986); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed. 1989); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., eds., 1994)).

In general, DNA encoding the envelope glycoproteins described herein can be obtained by constructing a cDNA library from mRNA recovered from field or laboratory isolates and (1) screening with labeled DNA probes encoding portions of the envelope glycoprotein sought in order to detect clones in the cDNA library that contain homologous sequences or (2) amplifying the cDNA using polymerase chain- reaction (PCR) and subcloning and screening with labeled DNA probes. Clones can then be analyzed by restriction enzyme analysis, agarose gel electrophoresis sizing and nucleic acid sequencing so as to identify full-length clones and, if full-length clones are not present in the library, recovering appropriate fragments from the various clones and ligating them at restriction sites common to the clones to assemble a clone encoding a full-length molecule. DNA probes for envelope glycoproteins are common in the art and can be prepared from the genetic material set forth in SEQ ID NO: 1. Any sequences missing from the 5' end of the cDNA may be obtained by the 3' extension of the synthetic oligonucleotides complementary to sequences encoding the protein using mRNA as a template (so- called primer extension), or homologous sequences may be supplied from known cDNAs. Polynucleic acid sizes are given in either kilobases (Kb) or base pairs (bp). These sizes are estimates derived from agarose or acrylamide gel electrophoresis, from sequenced nucleic acids, or from published DNA sequences.

Amplification techniques using primers can also be used to isolate HIV envelope glycoproteins from DNA or RNA. Suitable primers are commonly available in the art, or can be derived from SEQ ID NO: 1, then synthesized by conventional solid-phase techniques common in the art and described. Primers can be used, e.g., to amplify either the full length sequence or a probe of one to several hundred nucleotides, which is then used to screen a library for full-length HIV envelope glycoproteins.

Nucleic acids encoding HIV envelope glycoproteins can also be isolated from expression libraries using antibodies as probes. Such polyclonal or monoclonal antibodies can be raised using the sequence of SEQ ID NO:3, or any immunogenic portion thereof. HIV envelope glycoprotein strain variants and orthologs can be isolated using corresponding nucleic acid probes known in the art to screen libraries under stringent hybridization conditions. Alternatively, expression libraries can be used to clone sequences encoding HIV envelope glycoprotein strain variants and orthologs by detecting expressed proteins immunologically with commercially available antisera or antibodies, or antibodies made against SEQ ID NO:3, or portions thereof, which also recognize and selectively bind to the HIV envelope glycoprotein strain variants and orthologs.

To make a cDNA library, one should choose a source that is rich in the HIV envelope glycoprotein(s) of interest, such as the primary R5X4 HIV-I isolate 89.6 described in Collman, R, et al. "An infectious molecular clone of an unusual macrophage-tropic and highly cytopathic strain of human immunodeficiency virus type 1", J. Virol., 66, 7517-7521 (1992). The mRNA is then made into cDNA using reverse transcriptase, ligated into a recombinant vector, and transfected into a recombinant host for propagation, screening and cloning. Methods for making and screening cDNA libraries are well known (see, e.g., Gubler and Hoffman, Gene 25:263-269 (1983); Sambrook et al., supra; Ausubel et al., supra). An alternative method of isolating nucleic acids encoding HIV envelope glycoproteins combines the use of synthetic oligonucleotide primers and amplification of an RNA or DNA template (see U.S. Pat. Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods and Applications (Innis et al., eds, 1990)). Methods such as polymerase chain reaction (PCR) and ligase chain reaction (LCR) can be used to amplify the nucleic acid sequences encoding the glycoproteins directly from mRNA, from cDNA present in genomic libraries or cDNA libraries. Degenerate oligonucleotides can be designed to amplify HIV envelope glycoproteins using the sequences provided herein. Restriction endonuclease sites can be incorporated into the primers. Polymerase chain reaction or other in vitro amplification methods may also be useful, for example, to clone nucleic acid sequences that code for proteins to be expressed, to make nucleic acids to use as probes for detecting the presence of HIV envelope glycoprotein-encoding mRNA in physiological samples, for nucleic acid sequencing, or for other purposes. Genes amplified by the PCR reaction can be purified from agarose gels and cloned into an appropriate vector.

HIV envelope glycoprotein gene expression can also be analyzed by techniques known in the art, e.g., reverse transcription and amplification of mRNA, isolation of total RNA or poly A.sup.+ RNA, northern blotting, dot blotting, in situ hybridization, RNase protection, high density polynucleotide array technology and the like.

Synthetic oligonucleotides can be used to construct recombinant HIV envelope glycoprotein genes for use as probes or for expression of protein. This method is performed using a series of overlapping oligonucleotides usually 40-120 bp in length, representing both the sense and non-sense (antisense) strands of the gene. These DNA fragments are then annealed, ligated and cloned. Alternatively, amplification techniques can be used with precise primers to amplify a specific gene subsequences for HIV envelope glycoproteins. The specific subsequence is then ligated into a suitable eukaryotic expression vector.

Whether comparing gpl60/140, gpl20 or gp41 homologues, DNA encoding HIV envelope glycoprotein strain variants and orthologs typically show at least 70% sequence identity between strains, as defined supra, and are capable of selectively cross-hybridizing when annealed under stringent hybridization conditions. Coding regions for field isolates of gpl60/140, gpl20 or gp41 will typically not vary in length by more than 6 base pairs.

HIV envelope glycoprotein genes can also be identified by reference to the proteins produced when expressed in a eukaryotic system. For example, a nucleic acid sequence or a restriction fragment putatively encoding gpl60 can be inserted into a vector capable of transfecting a eukaryotic cell, providing a recombinant vector. The vector can then be used to transfect a eukaryotic cell capable of expressing the gpl60 human immunodeficiency virus envelope protein. After culturing the recombinant mammalian cell under conditions suitable for expression of the recombinant HIV protein, the cell preparation can be tested for the presence of the HIV envelope using one of the protein-specific assays described infra.

General texts describing additional molecular biological techniques useful herein, including the preparation of antibodies include Berger and Kimmel (Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol. 152, Academic Press, Inc.); Sambrook, et al., (Molecular Cloning: A Laboratory Manual, (Second Edition, Cold Spring Harbor Laboratory Press; Cold Spring Harbor, N. Y.; 1989) Vol. 1-3); Current Protocols in Molecular Biology, (F. M. Ausabel et al. [Eds.], Current Protocols, a joint venture between Green Publishing Associates, Inc. and John Wiley & Sons, Inc. (supplemented through 2000)); Harlow et al., (Monoclonal Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1988), Paul [Ed.]); Fundamental Immunology, (Lippincott Williams & Wilkins (1998)); and Harlow, et al., (Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1998)), all of which are incorporated herein by reference. Antigens of the invention

The invention provides a gp41 -based HIV antigen which advantageously exhibits broad cross-reactivity to HIV antibodies, elicits antibodies having broad neutralization activity against different HIV isolates, is highly immunogenic, exhibits high stability and enhanced half-life, and exhibits in vivo effectiveness, while lacking the disadvantages of prior art gp41 -based HIV antigens, including problems of eliciting self -reacting antibodies, having low immunogenicity and exhibiting in vivo ineffectiveness. The gp41-based antigen provided by the invention fuses HIV gp41, or a fragment thereof, with an Fc receptor ligand, e.g., the Fc portion of a human antibody, which stabilizes the structure of gp41 in the absence of gpl20, and enhances the immunogenecity of the antigen by enabling it to bind to immune cells having Fc receptors, such as, macrophages or dendritic cells. In one aspect, the invention provides a gp41 -based antigen that fuses a portion of HIV-I gp41 with the Fc portion of human IgG, which forms a highly immunogenic and stable antigen that is broadly cross-reactive with anti-HIV antibodies and which can be utilized in a variety of therapeutic and diagnostic methods, including as an immunogen for immunizing a subject against a broad range of HIV infections. This gp41 fusion protein can also be utilized diagnostically to detect anti-HIV antibodies in a sample.

In a particular aspect, the present invention provides an HIV antigen comprising a 151 amino acid portion of the extracellular domain of gp41 from HIV- 1 strain 89.6 (i.e., excluding the fusion domain, transmembrane domain and cytoplasmic tail domains) which is fused at its C-terminus to the N-terminus of the Fc domain (CH2-CH3 region) of human IgG joined through the IgG hinge (H) domain. This antigen is broadly cross-reactive against antibodies specific for a wide range of HIV isolates and features enhanced stability and half- life. Without wishing to be bound by theory, the Fc moiety is thought to provide the antigen with enhanced immunogenicity by enabling its binding to immune effector cells having Fc receptors, such as macrophages and dendritic cells. The gp41Fc antigen of the invention is highly immunogenic. Moreover, antibodies raised in response to immunization with gp41Fc in test animals were shown to have broad HIV neutralization activity against several HIV isolates from different clades. Still further, unlike some known MPEP-based antigens, which according to the inventors have generally failed as having broad cross reactivity and/or as vaccines, gp41Fc was found to elicit broadly cross reactive antibodies which did not react with self antigens - both desirable characteristics for an HIV vaccine.

Any linker envisioned by one of skill in the art can be used in the instant invention. In certain embodiments, a linker is not required, and the gp-41 and the Fc receptor ligand are joined without a linker. Antibodies and antibody modifications

The invention also provides for neutralization antibodies that are specifically immunoreactive against the gp41-based antigens of the invention, e.g., gp41Fc, and which are broadly cross reactive against a wide spectrum of HIV isolates.

Any known and/or conventional method for screening and identifying and obtaining the neutralizing antibodies of the invention are contemplated by the present invention.

In certain examples, a cell line/ pseudovirus assay is used as a neutralization assay. Such assays are well-known in the art and easily performed by the skilled practicioner. In Curr Protoc Immunol. 2005 Jan ;Chapter 12 :Unitl2.11 18432938, incorporated by reference in its entirety herein, Montefiore et al. describe neutralizing antibody assays as tools for assessing humoral immunity in AIDS virus infection and vaccine development. This reference describes two assays utilizing a genetically engineered cell lines that are susceptible to infection by most strains of HIV-I, SIV, and SHIV. One assay is designed for optimal performance with uncloned viruses produced in either PBMC or CD4(+) T cell lines. A second assay is designed for single-cycle infection with molecularly cloned pseudoviruses produced by transfection in 293T cells. Both assays are performed in a 96-well format and use tat-responsive luciferase reporter gene expression as readout.

Kim et al. (Research and Human Retroviruses. December 10, 2001, 17(18): 1715-1724), incorporated by reference in its entorety herein, describe development of a safe and rapid neutralization assay using murine leukemia virus pseudotyped with HIV Type 1 envelope glycoprotein lacking the cytoplasmic domain. In other certain examples, a peripheral blood mononuclear cells (PBMC) / primary isolates-based assay can be used as a neutralization assay. Such assays are well-known in the art and easily performed by the skilled practicioner. For example, Montefiore et al. (J. Virol., 03 1997, 2512-2517, VoI 71, No. 3), incorporated by reference in its entirety herein, describe antibody-mediated neutralization of human immunodeficiency virus type 1 (HIV-I) with primary isolates and sera from infected individuals, using human peripheral blood mononuclear cells (PBMC). For the production of antibodies, various host animals may be immunized by injection with a protein, or a portion thereof (e.g. any one of the SEQ ID Nos 1 - 7 of the invention or fragments thereof). Such host animals may include but are not limited to rabbits, mice, and rats, to name but a few. Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum. Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen, such as a cystatin gene product, or an antigenic functional derivative thereof.

For the production of polyclonal antibodies, host animals such as those described above, may be immunized by injection with a cystatin gene product supplemented with adjuvants as also described above.

Monoclonal antibodies, which are homogeneous populations of antibodies to a particular antigen, may be obtained by any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to the hybridoma technique of Kohler and Milstein (1975) Nature 256:495-497; and U.S. Pat. No. 4,376,110, the human B-cell hybridoma technique (Kosbor et al. (1983) Immunology Today 4:72; Cole et al. (1983) Proc. Natl. Acad. Sci. USA 80:2026-2030, and the EBV-hybridoma technique (Cole et al. (1985) Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof.

In addition, techniques developed for the production of "chimeric antibodies" or "humanized antibodies" may be utilized to modify mouse monoclonal antibodies to reduce immunogenicity of non-human antibodies. Morrison et al. (1984) Proc. Natl. Acad. Sci. 81:6851-6855; Neuberger et al. (1984) Nature, 312:604-608; Takeda et al. (1985) Nature, 314:452-454. Such antibodies are generated by splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region. Alternatively, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778; Bird (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; and Ward et al. (1989) Nature 334:544-546) can be adapted to produce single chain antibodies. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. Antibody fragments which recognize specific epitopes may be generated by known techniques. For example, such fragments may include but are not limited to: the F(ab')2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab expression libraries may be constructed (Huse et al. (1989) Science 246: 1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.

Thus, the method according to an embodiment of the present invention may further comprise screening an antibody for neutralization antibodies that are specifically immunoreactive against the gp41 -based antigens of the invention, e.g., gp41Fc, and which are broadly cross reactive against a wide spectrum of HIV isolates.

The screening may be performed using column chromatography filled with, for example, peptides having amino acid sequences of SEQ ID NOs. 1 - 7, or fragments thereof. The neutralizing anti-gp41 antibodies of the invention can be further modified by methods known in the art. The modifications may be genetic modifications to the nucleic acid encoding the antibodies of the invention or they may be chemical, structural, or physical modifications made directly to an isolated antibody of the invention to impart additional advantageous properties to an antibody of the invention regarding, for example, the level of expression, stability, solubility, epitope affinity, antigen neutralization activity, or penetration characteristics, etc.

In one aspect, the present invention contemplates introducing genetic modifications into one or more CDRs or to the framework sequence of the antibodies of the invention which are identified by library screening methods. Such genetic modifications can confer additional advantageous characterics, i.e. genetic optimization, of the antibodies identified from library screening, including, for example, enhanced solubility, enhanced affinity, and enhanced stability. Any type of genetic modification is contemplated by the present invention, including, for example, site-directed mutagenesis, random mutagenesis, insertions, deletions, and CDR grafting (i.e. genetic replacement of one CDR for another CDR). All of these techniques are well known to those skilled in the art. See Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 2000, incorporated herein by reference. Reference to CDR grafting can be made to Nicaise, et al., Protein Science 13:1882-1891, 2004. The effect of any genetic modification can be tested or screened without undue experimentation using any of the methods described herein or other methods already known to one of ordinary skill in the art. For example, affinity of an antibody to a target antigen can be assessed using the herein described BIA procedure.

In another aspect, other modifications contemplated by the present invention relate to chemical modifications of the antibodies of the invention to confer additional advantageous features, such as enhanced stability and/or solubility and/or half-life. In one particular aspect, the antibodies of the present invention can be

PEGylated, or coupled to polymers of similar structure, function and purpose ("PEG or PEG-like polymers"), to confer enhanced stability and half- life. PEGylation can provide increased half- life and resistance to degradation without a loss in activity (e.g. binding affinity) relative to non-PEGylated antibody polypeptides. The skilled artisan will appreciate, however, that PEGylation may not be advantageous with respect to some targets, in particular, those epitopes which are sterically-obstructed. Thus, in cases where the inventive antibodies targets a size-restricted epitope, the antibody should be minimally PEGylated so as not to negatively impact the accessibility of the antibody to the size -restricted antigen. The skilled artisan will appreciate that this general principle should be applied to any modifications made to the antibodies of the invention.

Any method known in the art to couple the antibodies of the invention to PEG or PEG-like polymers is contemplated by the present invention. PEG or PEG- like moieties which can be utilized in the invention can be synthetic or naturally occurring and include, but are not limited to, straight or branched chain polyalkylene, polyalkenylene or polyoxyalkylene polymers, or a branched or unbranched polysaccharide, such as a homo- or heteropolysaccharide. Preferred examples of synthetic polymers which can be used in the invention include straight or branched chain poly(ethylene glycol) (PEG), poly(propylene glycol), or poly(vinyl alcohol) and derivatives or substituted forms thereof. Substituted polymers for linkage to the antibodies of the invention can also particularly include substituted PEG, including methoxy(polyethylene glycol). Naturally occurring polymer moieties which can be used in addition to or in place of PEG include, for example, lactose, amylose, dextran, or glycogen, as well as derivatives thereof which would be recognized by persons skilled in the art. PEGylation of the antibodies of the invention may be accomplished by any number of means (see for example Kozlowski-A & Harris -J M (2001) Journal of Controlled Release 72:217). PEG may be attached to an antibody construct either directly or by an intervening linker. Linkerless systems for attaching polyethylene glycol to proteins is described in Delgado et al., (1992), Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 Francis et al., (1998), Intern. J. Hematol. 68:1-18; U.S. Pat. No. 4,002,531; U.S. Pat. No. 5,349,052; WO 95/06058; and WO 98/32466, the disclosures each of which are incorporated herein by reference. The first step in the attachment of PEG or other polymer moieties to the antibody construct of the invention typically is the substitution of the hydroxyl end-groups of the PEG polymer by electrophile-containing functional groups. Particularly, PEG polymers are attached to either cysteine or lysine residues present in the antibody construct monomers or multimers. The cysteine and lysine residues can be naturally occurring, or can be engineered into the antibody molecule.

One system for attaching polyethylene glycol directly to amino acid residues of proteins without an intervening linker employs tresylated MPEG, which is produced by the modification of monomethoxy polyethylene glycol (MPEG) using tresylchloride. Following reaction of amino acid residues with tresylated MPEG, polyethylene glycol is directly attached to the amine groups. Thus, the invention includes protein-polyethyleneglycol conjugates produced by reacting proteins of the invention with a polyethylene glycol molecule having a 2,2,2-trifluoreothane sulphonyl group.

Polyethylene glycol can also be attached to proteins using a number of different intervening linkers. For example, U.S. Pat. No. 5,612,460 discloses urethane linkers for connecting polyethylene glycol to proteins. Protein- polyethylene glycol conjugates wherein the polyethylene glycol is attached to the protein by a linker can also be produced by reaction of proteins with compounds such as MPEG-succinimidylsuccinate, MPEG activated with 1,1'- carbonyldiimidazole, MPEG-2,4,5-trichloropenylcarbonate, MPEG-p- nitrophenolcarbonate, and various MPEG- succinate derivatives. A number of additional polyethylene glycol derivatives and reaction chemistries for attaching polyethylene glycol to proteins are described in WO 98/32466, the entire disclosure of which is incorporated herein by reference.

Other derivatized forms of polymer molecules include, for example, derivatives which have additional moieties or reactive groups present therein to permit interaction with amino acid residues of the antibodies described herein. Such derivatives include N-hydroxylsuccinimide (NHS) active esters, succinimidyl propionate polymers, and sulfhydryl- selective reactive agents such as maleimide, vinyl sulfone, and thiol. The reactive group (e.g., MAL, NHS, SPA, VS, or Thiol) may be attached directly to the PEG polymer or may be attached to PEG via a linker molecule. The size of polymers useful in the invention can be in the range of 500 Da to

60 kDa, for example, between 1000 Da and 60 kDa, 10 kDa and 60 kDa, 20 kDa and 60 kDa, 30 kDa and 60 kDa, 40 kDa and 60 kDa, and up to between 50 kDa and 60 kDa. The polymers used in the invention, particularly PEG, can be straight chain polymers or may possess a branched conformation.

The present invention also contemplates the coupling of adduct molecules, which can be various polypeptides or fragments thereof which occur naturally in vivo and which resist degradation or removal by endogenous mechanisms.

Molecules which increase half life may be selected from the following: (a) proteins from the extracellular matrix, eg. collagen, laminin, integrin and fibronectin; (b) proteins found in blood, e.g., serum albumin, fibrinogen A, fibrinogen B, serum amyloid protein A, heptaglobin, protein, ubiquitin, uteroglobulin, β-2 microglobulin, plasminogen, lysozyme, cystatin C, alpha- 1 -antitrypsin and pancreatic kypsin inhibitor; (c) immune serum proteins, e.g. IgE, IgG, IgM and their fragments e.g. Fc; (d) transport proteins, e.g. retinol binding protein; (e) defensins, e.g. beta- defensin 1, neutrophil defensins 1, 2 and 3; (f) proteins found at the blood brain barrier or in neural tissues, e.g. melanocortin receptor, myelin, ascorbate transporter; (g) transferrin receptor specific ligand-neuropharmaceutical agent fusion proteins, brain capillary endothelial cell receptor, transferrin, transferrin receptor, insulin, insulin- like growth factor 1 (IGF 1) receptor, insulin- like growth factor 2 (IGF 2) receptor, insulin receptor; (h) proteins localised to the kidney, e.g. polycystin, type IV collagen, organic anion transporter Kl, Heymann's antigen; (i) proteins localized to the liver, e.g. alcohol dehydrogenase, G250; (j) blood coagulation factor X; (k) α- 1 antitrypsin; (1) HNF 1 α.; (m) proteins localised to the lung, e.g. secretory component (binds IgA); (n) proteins localised to the heart, eg. HSP 27; (o) proteins localised to the skin, eg, keratin; (p) bone specific proteins, such as bone morphogenic proteins (BMPs) e.g. BMP-2, -4, -5, -6, -7 (also referred to as osteogenic protein (OP-I) and -8 (OP-2); (q) tumour specific proteins, eg. human trophoblast antigen, herceptin receptor, oestrogen receptor, cathepsins eg cathepsin B (found in liver and spleen); (r) disease-specific proteins, eg. antigens expressed only on activated T-cells: including LAG-3 (lymphocyte activation gene); osteoprotegerin ligand (OPGL) see Kong YY et al Nature (1999) 402, 304-309; OX40 (a member of the TNF receptor family, expressed on activated T cells and the only costimulatory T cell molecule known to be specifically up-regulated in human T cell leukaemia virus type-I (HTLV-I)-producing cells-see Pankow R et al J. Immunol. (2000) JuI. l;165(l):263-70; metalloproteases (associated with arthritis/cancers), including CG6512 Drosophila, human paraplegin, human FtsH, human AFG3L2, murine ftsH; angiogenic growth factors, including acidic fibroblast growth factor (FGF-I), basic fibroblast growth factor (FGF- 2), Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF), transforming growth factor- α (TGF-α), tumor necrosis factor-alpha (TNF- α), angiogenin, interleukin-3 (IL-3), interleukin-8 (IL-8), platelet derived endothelial growth factor (PD-ECGF), placental growth factor (PIGF), midkine platelet-derived growth factor-BB (PDGF), fractalkine; (s) stress proteins (heat shock proteins); and (t) proteins involved in Fc transport.

In another aspect, the antibodies of the invention may be multimerized, as for example, hetero- or homodimers, hetero- or homotrimers, hetero- or homotetramers, or higher order hetero- or homomultimers. Multimerisation can increase the strength of antigen binding, wherein the strength of binding is related to the sum of the binding affinities of the multiple binding sites. The antibodies can be multimerized in another aspect by binding to an additional one, two, three or more polypeptide which function to stabilize the dAb against degradation. Such polypeptides may include common blood proteins, such as, albumin, or fragments thereof. In yet another aspect, modifications relating to enhancing or modifying antibody activity are contemplated by the present invention. For example, it may be desirable to modify the antibody of the invention with respect to effector function, so as to enhance the effectiveness of the antibody in treating a condition, infection or disorder. For example cysteine residue(s) may be introduced in the antibody polypeptide, thereby allowing interchain disulfide bond formation in a multimerized form of the inventive antibodies. The homodimeric or heterodimeric (or multimeric) antibodies may include combinations of the same antibody polypeptide chains or different antibody polypeptide chains, such that more than one epitope is targeted at a time by the same construct. Such epitopes can be proximally located in the target (e.g. on the HIV target) such that the binding of one epitope facilitates the binding of the multmeric antibody of the invention to the second or more epitopes. The epitopes targeted by multimeric antibodies can also be distally situated. The invention also contemplates modifying the antibodies of the invention to form immunoconjugates comprising the antibodies of the invention conjugated to cytotoxic agents, such as a chemotherapeutic agents, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof), radioactive isotopes (i.e., a radioconjugate), or antiviral compounds (e.g. anti-HIV compounds).

The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells. The term can include radioactive isotopes (e.g., I_13I, I₁₂₅, Y₉0 and Reigβ), chemotherapeutic agents, and toxins such as enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof.

A "chemotherapeutic agent" is a type of cytotoxic agent useful in the treatment of cancer. Examples of chemotherapeutic agents include Adriamycin, Doxorubicin, 5-Fluorouracil, Cytosine arabinoside ("Ara-C"), Cyclophosphamide, Thiotepa, Taxotere (docetaxel), Busulfan, Cytoxin, Taxol, Methotrexate, Cisplatin, Melphalan, Vinblastine, Bleomycin, Etoposide, Ifosfamide, Mitomycin C, Mitoxantrone, Vincreistine, Vinorelbine, Carboplatin, Teniposide, Daunomycin, Carminomycin, Aminopterin, Dactinomycin, Mitomycins, Esperamicins, Melphalan and other related nitrogen mustards. The invention also contemplates immunoconjugation with enzymatically active toxins or fragments thereof. Enzymatically active toxins and fragments thereof which can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes.

Where the inventive antibodies are intended to target HIV infections that might also involve infection by other viruses, bacteria or other pathogens, the invention also contemplates immunoconjugation of the antibodies with anti- viral, anti-bacterial or other chemicals and/or compounds that might improve or increase the effectiveness of the antibodies of the invention against intended targets, such as, for example, HIV.

For example, the inventive antibodies can be immunoconjugated, or in the alternative, co-administered with, an antibacterial compound, such as, for example, a macrolide (e.g., tobramycin (TOBI®)), a cephalosporin (e.g., cephalexin (KEFLEX®), cephradine (VELOSEF®), cefuroxime (CEFTIN®), cefprozil (CEFZIL®), cefaclor (CECLOR®), cefixime (SUPRAX®) or cefadroxil (DURICEF®), a clarithromycin (e.g., clarithromycin (BIAXIN®)), an erythromycin (e.g., erythromycin (EMYCIN®)), a penicillin (e.g., penicillin V (V-CILLIN K® or PEN VEE K®)) or a quinolone (e.g., ofloxacin (FLOXIN®), ciprofloxacin (CIPRO®) or norfloxacin (NOROXIN®)), aminoglycoside antibiotics (e.g., apramycin, arbekacin, bambermycins, butirosin, dibekacin, neomycin, neomycin, undecylenate, netilmicin, paromomycin, ribostamycin, sisomicin, and spectinomycin), amphenicol antibiotics (e.g., azidamfenicol, chloramphenicol, florfenicol, and thiamphenicol), ansamycin antibiotics (e.g., rifamide and rifampin), carbacephems (e.g., loracarbef), carbapenems (e.g., biapenem and imipenem), cephalosporins (e.g., cefaclor, cefadroxil, cefamandole, cefatrizine, cefazedone, cefozopran, cefpimizole, cefpiramide, and cefpirome), cephamycins (e.g., cefbuperazone, cefmetazole, and cefminox), monobactams (e.g., aztreonam, carumonam, and tigemonam), oxacephems (e.g., flomoxef, and moxalactam), penicillins (e.g., amdinocillin, amdinocillin pivoxil, amoxicillin, bacampicillin, benzylpenicillinic acid, benzylpenicillin sodium, epicillin, fenbenicillin, floxacillin, penamccillin, penethamate hydriodide, penicillin o-benethamine, penicillin 0, penicillin V, penicillin V benzathine, penicillin V hydrabamine, penimepicycline, and phencihicillin potassium), lincosamides (e.g., clindamycin, and lincomycin), amphomycin, bacitracin, capreomycin, colistin, enduracidin, enviomycin, tetracyclines (e.g., apicycline, chlortetracycline, clomocycline, and demeclocycline), 2,4-diaminopyrimidines (e.g., brodimoprim), nitrofurans (e.g., furaltadone, and furazolium chloride), quinolones and analogs thereof (e.g., cinoxacin,, clinafloxacin, flumequine, and grepagloxacin), sulfonamides (e.g., acetyl sulfamethoxypyrazine, benzylsulfamide, noprylsulfamide, phthalylsulfacetamide, sulfachrysoidine, and sulfacytine), sulfones (e.g., diathymosulfone, glucosulfone sodium, and solasulfone), cycloserine, mupirocin and tuberin.

In another example, the inventive antibodies can be immunoconjugated, or in the alternative, co-administered with, an antiviral compound, such as, for example, a zidovudine, acyclovir, gangcyclovir, vidarabine, idoxuridine, trifluridine, and ribavirin, as well as foscarnet, amantadine, rimantadine, saquinavir, indinavir, amprenavir, lopinavir, ritonavir, adefovir, clevadine, entecavir, and pleconaril. Methods for modifying the antibodies of the invention with the various cytoxic agents, chemotherapeutic agents, toxins, antibacterial compounds, and antiviral compounds, etc. mentioned above are well known in the art. For example, immunoconjugates of the antibody and cytotoxic agents can be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyidithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p- azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p- diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6- diisocyanate), and bis-active fluorine compounds (such as l,5-difluoro-2,4- dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon- 14-labeled 1-isothiocyanatobenzyl- 3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO94/11026.

The antibodies can also be modified with useful detectable agents, such as, for example, fluorescent compounds. Exemplary fluorescent detectable agents include fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-l- napthalenesulfonyl chloride, phycoerythrin and the like. The antibody construct may also be derivatized with detectable enzymes such as alkaline phosphatase, horseradish peroxidase, glucose oxidase and the like. When the antibody construct is derivatized with a detectable enzyme, it is detected by adding additional reagents that the enzyme uses to produce a detectable reaction product. The antibody construct may also be derivatized with biotin, and detected through indirect measurement of avidin or streptavidin binding. The skilled artisan will appreciate it may be advantageous to couple any of the aforementioned molecular entities to the antibodies of the invention through flexible linkers, such as flexible polypeptide chains. Such linkers may be required to avoid a loss in activity of the antibodies, or to avoid sterically restricting the antibodies such that they lose their effectiveness in binding to cognate epitopes, in particular, those epitopes which themselves may be sterically restricted. The linkers can be the same or different as the linkers described herein elsewhere which are used to fuse the gp41 subunit (or fragment or derivative thereof) with the Fc receptor ligand. Another type of covalent modification contemplated by the present invention involves chemically or enzymatically coupling glycosides to the antibodies of the invention. These procedures are advantageous in that they do not require production of the antibody in a host cell that has glycosylation capabilities for N- or O-linked glycosylation. Depending on the coupling mode used, the sugar(s) may be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as those of cysteine, (d) free hydroxyl groups such as those of serine, threonine, or hydroxyproline, (e) aromatic residues such as those of phenylalanine, tyrosine, or tryptophan, or (f) the amide group of glutamine. These methods are described in WO 87/05330 published Sep. 11, 1987, and in Aplin and Wriston, CRC Crt. Rev. Biochem., pp. 259-306 (1981).

Removal of any carbohydrate moieties present on the antibodies of the invention may be accomplished chemically or enzymatically. Chemical deglycosylation requires exposure of the antibody to the compound trifluoromethanesulfonic acid, or an equivalent compound. This treatment results in the cleavage of most or all sugars except the linking sugar (N-acetylglucosamine or N-acetylgalactosamine), while leaving the antibody intact. Chemical deglycosylation is described by Hakimuddin, et al. Arch. Biochem. Biophys. 259:52 (1987) and by Edge et al. Anal. Biochem., 118:131 (1981). Enzymatic cleavage of carbohydrate moieties on antibodies can be achieved by the use of a variety of endo- and exo-glycosidases as described by Thotakura et al. Meth. Enzymol. 138:350 (1987). Analytical/preparative methods for antigens and/or antibodies of invention Once an antigen or antibody in accordance with the invention is identified or obtained, for example, by any of the methods herein described, it may be preferable to carry out further steps to characterize and/or purify and/or modify the antigen or antibody. For example, it may be desirable to prepare a purified, high-titer composition of the desirable antibody or to test the immunoreactivity of the identified antibody. The present invention contemplates any known and suitable methods for characterizing, purifying, or assaying the antigens and/or antibodies of the present invention and it is expected the any person of ordinary skill in the art to which the invention pertains will have the requisite level of technical know-how and resources, e.g. technical manuals or treatises, to accomplish any further characterization, purification and/or assaying of the antigens and/or antibodies of the invention without undue experimentation.

For example, any useful means to describe the strength of binding (or affinity) between a antibody of the invention and an antigen of the invention (e.g., gp41Fc) can be used. For example, the dissociation constant, IQ (IQ = k2/kl = [antibody] [antigen] / [antibody- antigen complex]) can be determined by standard kinetic analyses that are known in the art. It will be appreciated by those of ordinary skill in the art that the dissociation constant indicates the strength of binding between an antibody and an antigen in terms of how easy it is to separate the complex. If a high concentration of antibody and antigen are required to form the complex, the strength or affinity of binding is low, resulting in a higher K_<j. It follows that the smaller the IQ (as expressed in concentration units, e.g. molar or nanomolar), the stronger the binding.

Affinity can be assessed and/or measured by a variety of known techniques and immunoassays, including, for example, enzyme-linked immunospecific assay (ELISA), Bimolecular Interaction Analysis (BIA) (e.g., Sjolander and Urbaniczky, Anal. Chem. 63:2338-2345, 1991; Szabo, et al., Curr. Opin. Struct. Biol. 5:699-705, 1995, each incorporated herein by reference), and fluorescence- activated cell sorting (FACS) for quantification of antibody binding to cells that express antigen. BIA is a technology for analyzing biospecific interactions in real time, without labeling any of the interactants (e.g., BIACORE™). BIAcore is based on determining changes in the optical phenomenon surface plasmon resonance (SPR) in real-time reactions between biological molecules, such as, an antibody of the invention and an antigen of interest, e.g. CD4L References relating to BIAcore technology can be further found in U.S. Published Application Nos: 2006/0223113, 2006/0134800, 2006/0094060, 2006/0072115, 2006/0019313, 2006/0014232, and 2005/0199076, each of which are incorporated herein in their entireties by reference.

The antigens and antibodies of the invention may be assayed for immunospecific binding by any suitable method known in the art. Assays involving an antibody and an antigen are known as "immunoassays," which can be employed in the present invention to characterize both the antibodies and the antigens of the invention. The immunoassays which can be used include but are not limited to competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, to name but a few. Such assays are routine and well known in the art (see, e.g., Ausubel et al., eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, which is incorporated by reference herein in its entirety) and can be performed without undue experimentation.

Western blot analysis generally comprises preparing protein samples, electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8% 20% SDS- PAGE depending on the molecular weight of the antigen), transferring the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon, blocking the membrane in blocking solution (e.g., PBS with 3% BSA or nonfat milk), washing the membrane in washing buffer (e.g., PBS-Tween 20), blocking the membrane with primary antibody (the antibody of interest) diluted in blocking buffer, washing the membrane in washing buffer; blocking the membrane with a secondary antibody (which recognizes the primary antibody, e.g., an anti-human antibody) conjugated to an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) or radioactive molecule (e.g., ₃₂P or ₁₂sl) diluted in blocking buffer, washing the membrane in wash buffer, and detecting the presence of the antigen. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected and to reduce the background noise. For further discussion regarding western blot protocols see, e.g., Ausubel et al., eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.8.1, which is incorporated herein by reference.

ELISAs typically comprise preparing antigen (e.g., gp41Fc), coating the well of a 96 well microtiter plate with the antigen, adding the antibody of interest conjugated to a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) to the well and incubating for a period of time, and detecting the presence of the antigen. In ELISAs the antibody of interest does not have to be conjugated to a detectable compound; instead, a second antibody (which recognizes the antibody of interest) conjugated to a detectable compound may be added to the well. Further, instead of coating the well with the antigen, the antibody may be coated to the well. In this case, a second antibody conjugated to a detectable compound may be added following the addition of the antigen of interest to the coated well. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected as well as other variations of ELISAs known in the art. For further discussion regarding ELISAs see, e.g., Ausubel et al., eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 11.2.1, which is incorporated herein by reference.

Any suitable method for purifying antigens and/or antibodies of the invention is contemplated herein. For example, chromatographic methods, such as, for example, immuno-affinity chromatography (immobilized ligand to bind and trap antibody of interest), affinity chromatography, protein precipitation, ion exchange chromatography, hydrophobic interaction chromatography, size-exclusion chromatography, as well as electrophoresis, can be found described in the technical literature, for example, in Methods in Enzymology, Volume 182, Guide to Protein Purification, Eds. J. Abelson, M. Simon, Academic Press, 1^st Edition, 1990, which is incorporated herein by reference. Thus, suitable materials for performing such purification steps, such as chromatographic steps, are known to those skilled in the art. Such methods are suitable for purification of any of the antibodies, antigens or any fragments thereof that are in accordance with the invention as described herein. Certain embodiments may require the purification or isolation of expressed antigens or antibodies or fragments thereof from a host cell or a portion thereof. Conventional procedures for isolating recombinant proteins from transformed host cells are contemplated by the present invention. Such methods include, for example, isolation of the protein or fragments of interest by initial extraction from cell pellets or from cell culture medium, followed by salting-out, and one or more chromatography steps, including aqueous ion exchange chromatography, size exclusion chromatography steps, high performance liquid chromatography (HPLC), and affinity chromatography may be used to isolate the recombinant protein or fragment. Guidance in the procedures for protein purification can be found in the technical literature, including, for example, Methods in Enzymology, Volume 182, Guide to Protein Purification, Eds. J. Abelson, M. Simon, Academic Press, 1^st Edition, 1990, which is already incorporated by reference. Methods of Use

The present invention provides pharmaceutical compositions comprising a therapeutically effective amount of the antigens and/or antibodies of the invention, together with a pharmaceutically acceptable carrier. In one aspect, the present invention provides a method for vaccinating against an HIV infection by administering a therapeutically effective amount of an antigen of the invention (e.g., gp41Fc), together with a pharmaceutically acceptable carrier or diluent. Administration can occur before or after HIV infection.

In another aspect, the present invention provides a method for treating an HIV infection by administering a therapeutically effective amount of an antibody of the invention (e.g., anti-gp41Fc), together with a pharmaceutically acceptable carrier or diluent. Administration can occur before or after HIV infection.

Some terms relating to the use of the antigens and/or antibodies of this invention are defined as follows. The term "treatment" includes any process, action, application, therapy, or the like, wherein a subject (or patient), including a human being, is provided medical aid with the object of improving the subject's condition, directly or indirectly, or slowing the progression of a condition or disorder in the subject, or ameliorating at least one symptom of the disease or disorder under treatment.

The term "combination therapy" or "co-therapy" means the administration of two or more therapeutic agents to treat a disease, condition, and/or disorder. Such administration encompasses co-administration of two or more therapeutic agents in a substantially simultaneous manner, such as in a single capsule having a fixed ratio of active ingredients or in multiple, separate capsules for each inhibitor agent. In addition, such administration encompasses use of each type of therapeutic agent in a sequential manner. The order of administration of two or more sequentially co- administered therapeutic agents is not limited.

The phrase "therapeutically effective amount" means the amount of each agent administered that will achieve the goal of improvement in a disease, condition, and/or disorder severity, and/or symptom thereof, while avoiding or minimizing adverse side effects associated with the given therapeutic treatment. The term "pharmaceutically acceptable" means that the subject item is appropriate for use in a pharmaceutical product.

The antibodies of this invention are expected to be valuable as therapeutic agents, e.g. anti-HIV antibody based therapies, due to their high degree of cross- reactivity against HIV isolates and their ability to neutralize a wide spectrum of HIV types. Accordingly, an embodiment of this invention includes a method of treating and/or preventing a particular condition (e.g. HIV infection) in a patient which comprises administering to said patient a composition containing an amount of an antibody of the invention that is effective in treating the target condition, e.g., HIV infection. The antigens (e.g., gp41Fc) of this invention are expected to be valuable as vaccine immunogens due to their enhanced immunogenicity, enhanced stability and half-life, and their ability to elicit effective neutralizing antibodies that are broadly cross-reactive against a spectrum of HIV isolates and do not react with self-antigens (unlike antibodies elicited by known gp41 -based antigens). Accordingly, an embodiment of this invention includes a method of vaccinating against HIV infections in a subject comprising administering to said subject a pharmaceutical composition containing an amount of an antigen of the invention that is effective in immunizing (at least partially) against HIV infection.

The antigens and/or antibodies of the present invention may be administered alone or in combination with one or more additional therapeutic agents. Combination therapy includes administration of a single pharmaceutical dosage formulation which contains an antibody of the present invention and one or more additional therapeutic agents, as well as administration of the antibody of the present invention and each additional therapeutic agents in its own separate pharmaceutical dosage formulation. For example, an antibody of the present invention and a therapeutic agent may be administered to the patient together in a single oral dosage composition or each agent may be administered in separate oral dosage formulations.

Where separate dosage formulations are used, the antibody of the present invention and one or more additional therapeutic agents may be administered at essentially the same time (e.g., concurrently) or at separately staggered times (e.g., sequentially). The order of administration of the agents is not limited.

For example, in one aspect, co-administration of an antibody or antibody fragment of the invention together with one or more anti-HIV agents to potentiate the effect of either the antibody or the anti-HIV agent(s) or both is contemplated for use in treating HIV infections. Examples of anti-HIV agents include, but are not limited to AGENERASE (ampreavir), APTIVUS (tipranavir), ATRIPLA, COMBIVIR, RETROVIR, EPIVIR, CRIXIVAN (indinavir), EMTRIVA (emtricitabine), EPZICOM, FORTOVASE (saquinavir), FUZEON (enfuvirtide), HIVID (ddc /zalcitabine), INTELENCE (Etravirine), ISENTRESS (raltegravir), INVIRASE (saquinavir), KAETRA (lopinavir), LEXIVA (Fosamprenavir), NORVIR (ritonavir), PREZISTA (darunavir), RESCRTIPTOR (delavirdine), RETROVIR (AZT), REYATAZ (atazanavir), SUSTIVA (efavirenz), TRIZIVIR, VIDEX (ddl/didanosine), VIRACEPT (nelfinavir), VIRAMUNE (nevirapine), VIREAD (tenofovir disoproxil fumarate), ZERIT (d4t / stavudine) and ZIAGEN (abacavir).

The one or more anti-cancer agents can include any known and suitable compound in the art, such as, for example, chemoagents, other immunotherapeutics, cancer vaccines, anti-angiogenic agents, cytokines, hormone therapies, gene therapies, and radiotherapies. A chemoagent (or "anti-cancer agent" or "anti-tumor agent" or "cancer therapeutic") refers to any molecule or compound that assists in the treatment of a cancer. Examples of chemoagents contemplated by the present invention include, but are not limited to, cytosine arabinoside, taxoids (e.g., paclitaxel, docetaxel), anti-tubulin agents (e.g., paclitaxel, docetaxel, epothilone B, or its analogues), macrolides (e.g., rhizoxin) cisplatin, carboplatin, adriamycin, tenoposide, mitozantron, discodermolide, eleutherobine, 2-chlorodeoxyadenosine, alkylating agents (e.g., cyclophosphamide, mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin, thio-tepa), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, anthramycin), antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, flavopiridol, 5- fluorouracil, fludarabine, gemcitabine, dacarbazine, temozolamide), asparaginase, Bacillus Calmette and Guerin, diphtheria toxin, hexamethylmelamine, hydroxyurea, LYSODREN.RTM., nucleoside analogues, plant alkaloids (e.g., Taxol, paclitaxel, camptothecin, topotecan, irinotecan (CAMPTOSAR, CPT-Il), vincristine, vinca alkyloids such as vinblastine), podophy Ho toxin (including derivatives such as epipodophyllotoxin, VP- 16 (etoposide), VM-26 (teniposide)), cytochalasin B, colchine, gramicidin D, ethidium bromide, emetine, mitomycin, procarbazine, mechlorethamine, anthracyclines (e.g., daunorubicin (formerly daunomycin), doxorubicin, doxorubicin liposomal), dihydroxyanthracindione, mitoxantrone, mithramycin, actinomycin D, procaine, tetracaine, lidocaine, propranolol, puromycin, anti-mitotic agents, abrin, ricin A, pseudomonas exotoxin, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, aldesleukin, allutamine, anastrozle, bicalutamide, biaomycin, busulfan, capecitabine, carboplain, chlorabusil, cladribine, cylarabine, daclinomycin, estramusine, floxuridhe, gamcitabine, gosereine, idarubicin, itosfamide, lauprolide acetate, levamisole, lomusline, mechlorethamine, magestrol, acetate, mercaptopurino, mesna, mitolanc, pegaspergase, pentoslatin, picamycin, riuxlmab, campath-1, straplozocin, thioguanine, tretinoin, vinorelbine, or any fragments, family members, or derivatives thereof, including pharmaceutically acceptable salts thereof. Compositions comprising one or more chemoagents (e.g., FLAG, CHOP) are also contemplated by the present invention. FLAG comprises fludarabine, cytosine arabinoside (Ara-C) and G-CSF. CHOP comprises cyclophosphamide, vincristine, doxorubicin, and prednisone.

The chemoagent can be an anti-angiogenic agent, such as, for example, angiostatin, bevacizumab (Avastin®), sorafenib (Nexavar®), baculostatin, canstatin, maspin, anti-VEGF antibodies or peptides, anti-placental growth factor antibodies or peptides, anti-Flk-1 antibodies, anti-Flt-1 antibodies or peptides, laminin peptides, fibronectin peptides, plasminogen activator inhibitors, tissue metalloproteinase inhibitors, interferons, interleukin 12, IP-IO, Gro-β, thrombospondin, 2- methoxyoestradiol, proliferin-related protein, carboxiamidotriazole, CMlOl, Marimastat, pentosan polysulphate, angiopoietin 2, interferon-alpha, herbimycin A, PNU145156E, 16K prolactin fragment, Linomide, thalidomide, pentoxifylline, genistein, TNP-470, endostatin, paclitaxel, accutin, cidofovir, vincristine, bleomycin, AGM- 1470, platelet factor 4 or minocycline. Without being bound by theory, the coadministration of an anti-angiogenic agent advantageously may lead to the increase in MN expression in a tumor, thereby making the tumor more susceptible to the antibodies and antibody conjugates of the invention. In one aspect, said chemoagent is gemcitabine at a dose ranging from 100 to

1000 mg/m²/cycle. In one embodiment, said chemoagent is dacarbazine at a dose ranging from 200 to 4000 mg/m² cycle. In another aspect, said dose ranges from 700 to 1000 mg/m²/cycle. In yet another aspect, said chemoagent is fludarabine at a dose ranging from 25 to 50 mg/m²/cycle. In another aspect, said chemoagent is cytosine arabinoside (Ara-C) at a dose ranging from 200 to 2000 mg/ m²/cycle. In still another aspect, said chemoagent is docetaxel at a dose ranging from 1.5 to 7.5 mg/kg/cycle. In yet another aspect, said chemoagent is paclitaxel at a dose ranging from 5 to 15 mg/kg/cycle. In a further aspect, said chemoagent is cisplatin at a dose ranging from 5 to 20 mg/kg/cycle. In a still further aspect, said chemoagent is 5- fluorouracil at a dose ranging from 5 to 20 mg/kg/cycle. In another aspect, said chemoagent is doxorubicin at a dose ranging from 2 to 8 mg/kg/cycle. In yet a further aspect, said chemoagent is epipodophyllotoxin at a dose ranging from 40 to 160 mg/kg/cycle. In yet another aspect, said chemoagent is cyclophosphamide at a dose ranging from 50 to 200 mg/kg/cycle. In a further aspect, said chemoagent is irinotecan at a dose ranging from 50 to 150 mg/m /cycle. In a still further aspect, said chemoagent is vinblastine at a dose ranging from 3.7 to 18.5 mg/m²/cycle. In another aspect, said chemoagent is vincristine at a dose ranging from 0.7 to 2 mg/m /cycle. In one aspect, said chemoagent is methotrexate at a dose ranging from 3.3 to 1000 mg/m²/cycle.

In another aspect, the antigens and/or antibodies of the present invention are administered in combination with one or more immuno therapeutic agents, such as antibodies or immunomodulators, which include, but are not limited to,

HERCEPTIN®, RETUXAN®, OvaRex, Panorex, BEC2, IMC-C225, Vitaxin, Campath IZH, Smart MI95, LymphoCide, Smart I DlO, and Oncolym, rituxan, rituximab, gemtuzumab, or trastuzumab.

The invention also contemplates administering the antigens and/or antibodies of the present invention with one or more anti- angiogenic agents, which include, but are not limited to, angiostatin, thalidomide, kringle 5, endostatin, Serpin (Serine Protease Inhibitor) anti-thrombin, 29 kDa N-terminal and a 40 kDa C-terminal proteolytic fragments of fibronectin, 16 kDa proteolytic fragment of prolactin, 7.8 kDa proteolytic fragment of platelet factor-4, a β-amino acid peptide corresponding to a fragment of platelet factor-4 (Maione et al., 1990, Cancer Res. 51 :2077), a 14- amino acid peptide corresponding to a fragment of collagen I (Tolma et al., 1993, J. Cell Biol. 122:497), a 19 amino acid peptide corresponding to a fragment of Thrombospondin I (Tolsma et al., 1993, J. Cell Biol. 122:497), a 20-amino acid peptide corresponding to a fragment of SPARC (Sage et al., 1995, J. Cell. Biochem. 57:1329-), or any fragments, family members, or derivatives thereof, including pharmaceutically acceptable salts thereof.

Other peptides that inhibit angiogenesis and correspond to fragments of laminin, fibronectin, procollagen, and EGF have also been described (See the review by Cao, 1998, Prog. MoI. Subcell. Biol. 20:161). Monoclonal antibodies and cyclic pentapeptides, which block certain integrins that bind RGD proteins (i.e., possess the peptide motif Arg-Gly-Asp), have been demonstrated to have anti-vascularization activities (Brooks et al., 1994, Science 264:569; Hammes et al., 1996, Nature Medicine 2:529). Moreover, inhibition of the urokinase plasminogen activator receptor by antagonists inhibits angiogenesis, tumor growth and metastasis (Min et al., 1996, Cancer Res. 56:2428-33; Crowley et al., 1993, Proc Natl Acad. Sci. USA 90:5021). Use of such anti-angiogenic agents is also contemplated by the present invention.

The antigens and/or antibodies of the present invention can also be administered in combination with one or more cytokines, which includes, but is not limited to, lymphokines, tumor necrosis factors, tumor necrosis factor-like cytokines, lymphotoxin-α, lymphotoxin-β, interferon- β, macrophage inflammatory proteins, granulocyte monocyte colony stimulating factor, interleukins (including, but not limited to, interleukin-1, interleukin-2, interleukin-6, interleukin-12, interleukin-15, interleukin-18), OX40, CD27, CD30, CD40 or CD137 ligands, Fas- Pas ligand, 4- IBBL, endothelial monocyte activating protein or any fragments, family members, or derivatives thereof, including pharmaceutically acceptable salts thereof.

The antigens and/or antibodies of the present invention can also be administered in combination with a cancer vaccine, examples of which include, but are not limited to, autologous cells or tissues, non-autologous cells or tissues, carcinoembryonic antigen, alpha-fetoprotein, human chorionic gonadotropin, BCG live vaccine, melanocyte lineage proteins (e.g., gplOO, MART- 1/MelanA, TRP-I (gp75), tyrosinase, widely shared tumor-associated, including tumor-specific, antigens (e.g., BAGE, GAGE-I, GAGE-2, MAGE-I, MAGE-3, N- acetylglucosaminyltransferase-V, pl5), mutated antigens that are tumor-associated (β-catenin, MUM-I, CDK4), nonmelanoma antigens (e.g., HER-2/neu (breast and ovarian carcinoma), human papillomavirus -E6, E7 (cervical carcinoma), MUC-I (breast, ovarian and pancreatic carcinoma). For human tumor antigens recognized by T-cells, see generally Robbins and Kawakami, 1996, Curr. Opin. Immunol. 8:628. Cancer vaccines may or may not be purified preparations.

In yet another embodiment, the antigens and/or antibodies of the present invention are used in association with a hormonal treatment. Hormonal therapeutic treatments comprise hormonal agonists, hormonal antagonists (e.g., flutamide, tamoxifen, leuprolide acetate (LUPRON), LH-RH antagonists), inhibitors of hormone biosynthesis and processing, and steroids (e.g., dexamethasone, retinoids, betamethasone, Cortisol, cortisone, prednisone, dehydrotestosterone, glucocorticoids, mineralocorticoids, estrogen, testosterone, progestins), antigestagens (e.g., mifepristone, onapristone), and antiandrogens (e.g., cyproterone acetate). The antigens and/or antibodies described herein may be provided in a pharmaceutical composition comprising a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be non-pyrogenic. The compositions may be administered alone or in combination with at least one other agent, such as stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier including, but not limited to, saline, buffered saline, dextrose, and water. A variety of aqueous carriers may be employed including, but not limited to saline, glycine, or the like. These solutions are sterile and generally free of particulate matter. These solutions may be sterilized by conventional, well- known sterilization techniques (e.g., filtration). Generally, the phrase "pharmaceutically acceptable carrier" is art recognized and includes a pharmaceutically acceptable material, composition or vehicle, suitable for administering compounds of the present invention to mammals. The carriers include liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject agent from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations.

Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the antibody compositions of the invention.

Examples of pharmaceutically acceptable antioxidants include: water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, and the like. The concentration of the antibody of the invention in such pharmaceutical formulation may vary widely, and may be selected primarily based on fluid volumes, viscosities, etc., according to the particular mode of administration selected. If desired, more than one type of antibody may be included in a pharmaceutical composition (e.g., an antibody with different IQ for MN binding).

The compositions may be administered to a patient alone, or in combination with other agents, drugs or hormones. In addition to the active ingredients, these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries that facilitate processing of the active compounds into preparations which may be used pharmaceutically. Pharmaceutical compositions of the invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, parenteral, topical, sublingual, or rectal means.

The compositions of the invention additionally contemplate suitable immunocarriers, such as, proteins, polypeptides or peptides such as albumin, hemocyanin, thyroglobulin and derivatives thereof, particularly bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH), polysaccharides, carbohydrates, polymers, and solid phases. Other protein-derived or non-protein derived substances are known to those skilled in the art. Formulations suitable for parenteral, subcutaneous, intravenous, intramuscular, and the like; suitable pharmaceutical carriers; and techniques for formulation and administration may be prepared by any of the methods well known in the art (see, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 20^th edition, 2000). Liquid dosage forms for oral administration of the compounds of the invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluent commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion. The determination of a therapeutically effective dose is well within the capability of those skilled in the art. A therapeutically effective dose refers to the amount of an antibody that may be used to effectively treat a disease (e.g., cancer) compared with the efficacy that is evident in the absence of the therapeutically effective dose. The therapeutically effective dose may be estimated initially in animal models (e.g., rats, mice, rabbits, dogs, or pigs). The animal model may also be used to determine the appropriate concentration range and route of administration. Such information may then be used to determine useful doses and routes for administration in humans.

Therapeutic efficacy and toxicity (e.g., ED₅₀ - the dose therapeutically effective in 50% of the population and LD₅₀ - the dose lethal to 50% of the population) of an antibody may be determined by standard pharmaceutical procedures in cell cultures or experimental animals. The dose ratio of toxic to therapeutic effects is the therapeutic index, and it may be expressed as the ratio, LD₅₀/ED₅₀. The data obtained from animal studies may used in formulating a range of dosage for human use. The dosage contained in such compositions may be within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.

The exact dosage may be determined by the practitioner, in light of factors related to the patient who requires treatment. Dosage and administration may be adjusted to provide sufficient levels of the antibody or to maintain the desired effect. Factors that may be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. The antigens and/or antibodies of the invention may also be administered by introducing genetically engineered bacteria which express and release the expressed antigens and/or antibodies of the invention once the bacteria are present in the patient. This format might be suitable for treating HIV infections. The antigen and/or antibody-expressing bacteria can be introduced into mucus membranes of the throat, for example, or in other mucosal regions in which HIV might be found. Methods for constructing and/or engineering such recombinant bacteria are well known in the art.

Polynucleotides encoding the antigens and/or antibodies of the invention may be constructed and introduced into a cell either ex vivo or in vivo using well- established techniques including, but not limited to, transferrin-polycation-mediated DNA transfer, transfection with naked or encapsulated nucleic acids, liposome- mediated cellular fusion, intracellular transportation of DNA-coated latex beads, protoplast fusion, viral infection, electroporation, "gene gun," and DEAE- or calcium phosphate-mediated transfection.

Effective in vivo dosages of an antigen and/or antibody are in the range of about 5 μg to about 500 μg/kg of patient body weight. For administration of polynucleotides encoding the antibodies, effective in vivo dosages are in the range of about 100 ng to about 500 μg of DNA.

The antigens and/or antibodies of the present invention can also be delivered in a microsphere or microsome bodies.

The mode of administration of antigen- and/or antibody-containing pharmaceutical compositions of the present invention may be any suitable route which delivers the antibody to the host. As an example, pharmaceutical compositions of the invention may be useful for parenteral administration (e.g., subcutaneous, intramuscular, intravenous, or intranasal administration, or microsomal or lipid microsome bodies). The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific examples, which are provided for purposes of illustration only and are not intended to limit the scope of the invention. EXAMPLES The structures, materials, compositions, and methods described herein are intended to be representative examples of the invention, and it will be understood that the scope of the invention is not limited by the scope of the examples. Those skilled in the art will recognize that the invention may be practiced with variations on the disclosed structures, materials, compositions and methods, and such variations are regarded as within the ambit of the invention.

Example 1. gp41 fused to Fc as HIV vaccine immunogen

In spite of the large research effort, an HIV vaccine immunogen that elicits broadly cross-reactive antibodies in humans has not yet been reported. The HIV envelope glycoprotein (Env) is composed of two subunits - gpl20 and gp41, of which gp41 is more conserved but unstable in the absence of gpl20.

To stabilize gp41 in the absence of gpl20, increase its half life in vivo and enhance its binding to immune cells including macrophages and dendritic cells, as well as binding to B cells expressing receptors specific for gp41, this Example describes the construction of a fusion protein where a truncated gp41 lacking the fusion peptide, the transmembrane domain and the cytoplasmic tail was joined to the Fc region of human IgG by a long flexible linker. This molecule, designated gp41Fc, exhibited binding to recently identified and characterized broadly cross- reactive HIV-I -neutralizing human monoclonal antibodies m43, m44, m46, m47 and m48, which recognize conformational epitopes on gp41. The fusion antigen, gp41Fc also exhibited binding to known broadly cross-reactive gp41-specific antibodies, 2F5, 4E10, and Z13, each of which target the membrane proximal external region (MPER).

Thus, gp41Fc, if used as vaccine immunogen, could potentially elicit the same or similar human monoclonal antibodies that could bind to these two major groups of neutralizing epitopes and neutralize primary HIV-I isolates from different clades. The inventors have found that such a possibility appears to be confirmed based on the results of the immunization of rabbits with purified gp41Fc fusion protein. It was found that the gp41Fc is highly immunogenic - the serum titer was 102,400 for the gp41Fc fusion protein and 5,120 for a recombinant Env ectodomain (gpl40) containing both gpl20 and gp41 from the same HIV-I isolate (isolate 89.6). In addition, purified rabbit IgG from one of the four immunized rabbits showed broad neutralization activity against several HIV isolates from different clades in a cell line/pseudovirus assay.

After the failure of numerous vaccine immunogens based on the MPER, immunogens that are based on other conserved epitopes are urgently needed. Without wishing to be bound by theory, one thought is that this failure is due to the reactivity to self antigens by antibodies targeting the MPER. In other words, those antibodies elicited by MPER-based antigens (e.g., 2F5, 4E10, and Z13) target not only HIV, but also self-antigens. Materials and Methods HIV-I Env clones and pseudovirus preparation

Viruses pseudotyped with Envs from HIV- 1 primary isolates representing HIV-I group M, clades A-F, and laboratory adapted HIV-I isolates and JRCSF were used in this Example. Cloning of HIV- 1 envelope genes and preparation of pseudoviruses have been previously described. Briefly, pseudotyped viruses were prepared by cotransfection of 70% to 80% confluent HEK 293T cells with pNL4- 3.1uc.E-R- and HIV-I Env plasmid using the calcium phosphate/HEPES buffer, according to manufacturer's instruction (Promega, Madison, WI). Eighteen hours after the transfection, medium was replaced with medium supplemented with 0.1 mM sodium butyrate (Sigma, St. Louis, USA). Cells were allowed to grow for an additional 24 h. The supernatant was harvested, centrifuged at 16,000 rpm for 5 min at 4 ⁰C, filtered through a 0.45-μm pore filter (Millipore, Bedford, MA), prior to use in neutralization assays.

HIV-I neutralization assays

Single-round infectious molecular clones, produced by envelope complementation as described above, were used. The degree of virus neutralization by antibody was achieved by measuring luciferase activity as described previously. Briefly, neutralization assays were carried out in triplicate by preincubation of 25 μl of 2-fold serial dilution of mAbs with 25 μl of pseudovirus suspension for 1 h at 4 ⁰C. Virus-antibody mixtures were then combined with 150 μl suspensions of 1- 2x104 HOS CD4+ CCR5+/CXCR4+, in 96 wells of tissue culture plates (Costar, Corning, NY). Plates were incubated at 37 ⁰C in 5% CO2 for 3 days and then washed with PBS and lysed for 30 min with 15 μl of 1 x Luciferase Assay System cell lysis buffer (Promega, Madison, WI), and luminescence readings for triplicate wells were determined by lumiCount microplate luminometer (Turner Designs). Neutralization titers were determined based on relative luminescence units (RLU) and neutralization endpoint was the last concentration of mAbs at which mean results from the test samples were less than 50% of non-neutralized control mean. IC₅₀ of neutralization was assigned for the antibody concentration at which 50% neutralization was observed. Neutralization assays for each envelope were generally repeated in at least two independent experiments. Preparation of gp4 IFc fusion protein In the experiments described herein, the gp41_{89 6} gene without the fusion peptide was PCR amplified, digested with Nhe I and BamHI and cloned to pEAKIO digested with the same enzymes. Recombinant DNA was confirmed by DNA sequencing and used for transient transfection of free-style 293 cells (Invitrogen). The culture supernatant four days post transfection was collected and gp41Fc fusion protein purified from the supernatant by protein A affinity purification.

Virus The 89.6 virus was a gift of Dr. Robert Doms, University of Pennsylvania, Philadelphia, PA. 89.6gp41 was amplified from 89.6 construct. Results

In the present Example, it was found that at least one antibody raised against gp41Fc, m44, did not react with self antigens. Thus, the gp41Fc antigen of the invention is capable of eliciting antibodies that do not react with self antigens. This feature also makes the fusion protein an attractive vaccine immunogen based on the possibility that it may elicit antibodies which are not regulated by tolerance mechanisms.

Example 2. Cross-reactive HIV-I -neutralizing activity of serum IgG from a rabbit immunized with gp41 fused to IgG Fc

The HIV-I envelope glycoprotein (Env) is known to exist as trimers of heterodimers on native virions that is composed of a non-covalently associated extracellular subunit gpl20 and a transmembrane subunit gp41. In natural infections, Env induces a robust antibody response, but these antibodies usually have a narrow spectrum of neutralization activity. Occasionally some HIV-I -positive individuals induce broadly cross-reactive neutralizing antibodies that are believed to account for the containment of the virus. Env-based vaccines have been extensively studied. Monomeric gpl20s can elicit neutralizing antibodies in vivo, but the neutralization activity is restricted to autologous viruses or the spectrum of neutralization activity is narrow. Oligomeric forms of Env ectodomain from HIV-I, strain R2 (gpl4U_R2), which contain both gpl20 and a truncated gp41 that lacks both transmembrane domains and cytoplasmic tails, induced better antibody response than gpl2U_R2 alone. Rabbit sera immunized with gpl4U_R2 neutralized not only the autologous virus, but also heterologous viruses from diverse HIV-I subtypes, suggesting that induction of broad spectrum neutralizing antibodies is an achievable goal in HIV- 1 vaccine development. This study utilizes gp41 derived from a dual tropic HIV-I primary isolate of 89.6. Gp41 is relatively conserved compared to gpl20, but unstable in the absence of gpl20. To stabilize gp41 in the absence of gpl20, we constructed a fusion protein, designated as gp41Fc, as outlined in Example 1, which was hypothesized to increase its half life in vivo and enhance its binding to immune cells like macrophages, dendritic cells, and B cells that express receptors specific for Fc, thereby enhancing the elicitation of broadly cross-reactive neutralizing antibodies..

Initially gp41Fc was used to localize the epitopes of a panel of gp41-specific neutralizing antibodies by alanine scanning mutagenesis. It was found that gp41Fc not only bound to recently identified mAbs conformational epitopes, m44, m46 and m48, but also to known broadly neutralizing human mAbs that include 2F5, 4E10, Zl 3 that recognize linear epitopes in the membrane proximal region (MPER), suggesting that gp41Fc retains critical conformation required for presenting neutralization epitopes and may induce neutralizing antibodies against HIV-I in vivo.

This Example investigates the possibility of developing the gp41Fc fusion protein as HIV-I vaccine immunogen by immunizing rabbits with purified recombinant gp41Fc. This Example reports the results from the characterization of immune rabbit sera and purified IgGs from two rabbits that had different immune response to gp41Fc. The following Methods and Materials were used in this Example.

Materials and Methods Cells, viruses, plasmids, gpl20, gp 140 and antibodies.

293T cells were purchased from ATCC. Other cell lines and HIV-I isolates were obtained from the NIH AIDS Research and Reference Reagent Program (ARRRP). Recombinant gpl20 and gpl40 Envs from primary isolates were produced as described previously (Zhang MY et al, 2003, J. Immunol. Methods, Dec; 283(1-2): 17-25, which is incorporated herein by reference). Human mAbs 2F5 and 4E10 were obtained from the NIH-ARRRP. Mouse mAbs NC-I was generously provided by Dr. Shibo Jiang (New York Blood Center). Mouse mAbs T3 and D61 were obtained from Dr. Christopher Broder (Uniformed Services University of the Health Sciences). Fab Z13 and other gp41-specific mAbs m44, m46, m48 were produced by the inventors. Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of healthy donors. HRP-conjugated streptavidin was purchased from Zymed Laboratories Inc., San Francisco, CA. HIV- 1 MN Env (15-mer) peptides (complete set, cat# 6451, Lot# 12) were obtained from NIH-ARRRP.

Preparation of gp4 IFc fusion protein.

As described previously herein, the gp41Fc fusion protein was prepared by cloning a gp41g_{9 6} ectodomain gene lacking the fusion peptide region in a pEAKIO plasmid upstream of a human Fc region. The recombinant plasmid DNA for transient transfection of free-style 293 cells (Invitrogen) was produced in E. coli. The culture supernatant was collected four days post transfection and gp41Fc fusion protein purified from the culture supernatant using a protein A affinity purification. Rabbit immunization:

Two female New Zealand white (NZW) rabbits were immunized using a standard 87 day protocol utilizing Freund's Complete Adjuvant (FCA) / Freund's Incomplete Adjuvant (FIA) as outlined below:

Day: Procedure:

0 NZW or Elite NZW Female Rabbit Pre-bleed Primary intradermal inoculation: 250 ug with FCA 14 Boost subcutaneous: 125 ug with FIA

35 Boost subcutaneous: 125 ug with FIA

45 Test Bleed

49 Boost subcutaneous: 125 ug with FIA

59 Production Bleed (PB 1) 70 Boost subcutaneous: 125 ug with FIA

80 Production Bleed (PB2)

81-87 ELISA Titer Assay of Bleed

87 Terminal Bleed (EX).

Binding assays.

Titration of immune rabbit serum (Fig. 15) was done by coating one μg/mL gp41Fc or gpl4U_{89 6} on maxisorp 96- well plates, then washing and blocking with 3% BSA in PBS followed by the addition of two-fold serially diluted rabbit immune sera. Bound antibodies were detected using horse radish peroxidase (HRP) conjugated to goat anti-rabbit IgG (H+L) (1:10,000) as a second antibody and ABTS as a substrate. The optical density at 405 nm was measured after a 20 minute color development.

Binding of gp41-specific mAbs to recombinant gp41Fc (Fig. 14) was performed by coating 5μg/mL gp41Fc on 96-well plates followed by the addition of 3-fold serially diluted biotinylated mAbs. The bound mAbs was detected using HRP conjugated to streptavidin (1:10,000) as a second antibody and ABTS as a substrate.

Binding of immune rabbit IgG to denatured gpl40 and gp41Fc (Fig. 17) was carried out by diluting purified gpl40 and gp41Fc to 20 μg/mL in PBS containing

50 mM DTT, then boiling the mixture for 5 min and diluting 1:10 in bicarbonate coating buffer (100 mM NaHCO₃ and 500 mM NaCl, pH 8.3) followed by coating on 96-well plates. The bound antibodies were detected using HRP conjugated to either anti-rabbit IgG or anti-human IgG, F(ab')2 as secondary antibodies and ABTS as a substrate.

ELISA assay with HIV-I MN gp41-derived peptides (6341 to 6377) (Fig. 18) was carried out by coating 5 μg/mL of each peptide on Easy- wash microplates followed by the addition of 3 -fold serially diluted immune rabbit IgG. The bound rabbit IgG was detected as described above.

Capture ELISA was used to measure gp41Fc concentration in immune rabbit

IgG sera utilizing the following procedure: gp41Fc was captured by conformation- dependent mouse mAb T3 coated on 96-well plates. Bound gp41Fc was detected by using biotinylated mouse mAb D61 and HRP conjugated to streptavidin. The optical density was measured 30min after addition of ABTS. The concentration of gp41Fc in purified rabbit IgG was calculated using purified recombinant gp41Fc for making a standard curve and then converted to the concentration of gp41Fc in rabbit serum.

Absorption of immune rabbit IgG with human Fc to remove Fc- specific antibodies. Recombinant human Fc was coupled to a CNBr-activated Sepharose™ 4B column. The immune rabbit IgG was loaded onto the column. The flow-through was collected for further characterization. Fc-specific rabbit IgG bound to the column was eluted using a low pH buffer and neutralized immediately after elution.

PBMC-based HIV-I neutralization assay. The PB MC -based assay was carried out as previously described [10]. Briefly, Fresh human PBMCs, seronegative for HIV and HBV, were isolated from blood of screened donors (Biological Specialty Corporation; Colmar, PA) using Lymphocyte Separation Medium (LSM; Cellgro® by Mediatech, Inc.; density 1.078+/-0.002 g/mL) following the manufacturer's instructions. Cells were stimulated by incubation in 4 μg/mL Phytohemagglutinin (PHA; Sigma) for 48-72 hours. Mitogenic stimulation was maintained by the addition of 20 U/mL recombinant human IL-2 (R&D Systems, Inc) to the culture medium. PHA- stimulated PBMCs from at least two donors were pooled, diluted in fresh medium and added to 96-well plates at 5xlO⁴ cells/well. Cells were infected (final MOI ≡ 0.1) in the presence of 9 different concentrations of antibody (triplicate wells/concentration; 50 μg/mL high-test concentration) and incubated for 7 days. To determine the level of virus inhibition, cell-free supernatant samples were collected for analysis of reverse transcriptase activity [H]. Following removal of supernatant samples, cytotoxicity was measured by the addition of 3-(4,5-dimethylthiazol-2-yl)- 5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS; CellTiter 96 Reagent, Promega) following the manufacturer's instructions. Cell line-based HIV-I neutralization assay

The cell line-based assay was carried out in triplicate using an ΗIV- 1 Env pseudotyping system and ΗOS CD4⁺CCR5⁺ or ΗOS CD4⁺CXCR4⁺ target cells containing a tat-inducible luciferase reporter that express CD4, CCR5 or CXCR4. The degree of virus neutralization by antibody was achieved by measuring luciferase activity as described previously (Zhang et al., J. Immunol. Methods, 2003, 283(1-2): 17-25). Results

Recombinant gp41Fc retains its antigenic structure.

Purified gp41Fc separated on SDS-PAGE gels appear as a single band on denatured gels and several higher order bands on non-denatured gels. These results indicate the existence of oligomers, which could contribute to its avidity (data not shown).

Recombinant gp41Fc retains its antigenic structure as measured by ELISA (Fig. 13). Recombinant gp41Fc binds to all gp41 -specific mAbs tested including human mAbs m44, m46 and m48, and mouse NC-I and T3 that recognize the conformational epitopes of gp41, and the best characterized human mAbs, 2F5,

4E10 and Z13 that recognize linear epitopes of the MPER (Fig. 13). BSA and anti- gpl20 human mAb ml4 were used as controls. BSA did not bind to gp41Fc, but ml4 showed weak binding at high concentrations likely due to non-specific interactions.

Recombinant gp41Fc is highly immunogenic.

Two female rabbits were immunized with purified gp41Fc. Antisera taken at exsanguination of both rabbits was titered for binding to the immunogen gp41Fc and the recombinant Env ectodomain gpl40₈₉6- Both rabbit FT1214 and FT1215 generated high titer sera against gp41Fc that reached 1:102,400, indicating that the gp41Fc is highly immunogenic. Both rabbit antisera bound to gpl40g_{9 6} with a similar titre of 1:5,120 (Fig. 2).

Gp41 -specific rabbit IgG accounts for neutralization activity of the immune serum IgG.

Rabbit IgG was purified from the antisera using a protein A affinity column and tested the rabbit IgG with a PBMC assay against a panel of HIV-I primary isolates from group M and O, and HIV-2 (Table 1, below).

Table I. Neutralization activity (IC 50) of rabbit IgGs FT1214 and FT1215 against a panel of primary isolates of HIV-I and HIV-2 in a PBMC assay.

AZT was used as a positive control with 100% inhibition at a concentration equal to 0.1 μM, where the measured cell toxicity was undetectable (IC₅₀ = 0.002 μM, TC₅₀ > 1 μM). TC₅₀S of both FT1214 and FT1215 IgGs were over 50 μg/mL for all isolates tested. ICso_s were converted to the serum dilutions based on the amount of FT1214, 1215 IgG isolated from the corresponding serum, 6.7 mg and 6.0 mg per ml serum, respectively. Isolates neutralized by the rabbit IgG were highlighted in grey.

FT1214 FT1215 AZT FT1214 FT1215

Virus SubCoreceptor IgG IC₅₀ IgG IC₅₀ IC₅₀ serum serum type (μg/mL) (μg/mL) (nM) dilution dilution

92UG037 A R5 >50.0 0.55 <l :120

92UG029 A X4 >50.0 >50.0 8.5 <1 :133 <l :120

92BR014 B R5X4 >50.0 >50.0 <0.10 <1 :133 <l :120

Interestingly, rabbit FT 1214EX IgG neutralized half of the HIV-I primary isolates tested from group M (clades A, B, C, D, B/F and G), one out of three isolates from group O and two HIV-2 isolates with IC₅₀ values lower than 50 μg/mL, the highest concentration tested. Rabbit FT1215EX IgG did not neutralize the HIV-I isolates tested, but did neutralize one of the two HIV-2 isolates, CSC310342. It was further confirmed that the neutralization activity of FT1214EX IgG was attributed to the gp41-specific IgG of the immune rabbit IgG (Fig. 16). Neutralization activity of immune rabbit IgG against BaI, a pseudo-typed HIV-I virus, was decreased in the presence of an increased concentration of gp41Fc. Gp41Fc alone did not significantly affect the neutralization even at high concentrations (Fig. 16).

Immune rabbit IgG recognizes conformation-independent epitopes on gp41 and preferentially binds to peptides derived from the immunodominant loop region. Because each rabbit IgG immune serum had similar titres to gp41Fc and gpl4U8₉6, but showed very different neutralization activity against HIV-I isolates, we investigated the composition of the rabbit IgG by measuring their binding to denatured gp41Fc and gpl40_{89 6} (Fig- 17). Binding profiles were strikingly similar, suggesting that rabbit IgG recognizes mainly conformation-independent epitopes on gp41 (Fig. 17). Based on this observation, binding of the rabbit IgG to a panel of peptides derived from gp41_MN (peptides 6341 to 6377) was measured. It was found that rabbit IgG bound predominantly to some of the peptides derived from the immunodominant-loop region of gp41, but each rabbit IgG showed a different binding profile (Fig. 18). The neutralizing FT1214EX IgG bound to peptides 6357 (LLGFWGCSGKLICTT) (SEQ ID NO: 9), 6358 (WGCS GKLICTTT VP W) (SEQ ID NO: 10) and 6359 (GKLICTTTVPWNASW) (SEQ ID NO: 11) that are located in the C-terminal immunodominant loop, while the weak-neutralizing FT1215EX IgG bound to peptides 6354 (VLA VER YLKD QQLLG) (SEQ ID NO: 12) and 6355 (ERYLKDQQLLGFWGC) (SEQ ID NO: 13), as well as 6357, 6358 and 6359. FT1214EX IgG bound more strongly to peptides 6358 and 6359 than FT1215EX IgG. Both bound well to peptide 6357 (Fig. 18). No binding was observed to peptides derived from MPER and other regions of gp41 (data not shown). Prolonged existence of the immunogen was detected in the neutralizing rabbit IgG. In an effort to explain why two rabbit IgG sera with similar titers had different neutralization activity, gp41Fc level in rabbit IgG (Fig. 19) was measured. The FT1214EX IgG at 87 days post immunization contained a much higher gp41Fc level (about 46 ng/mg rabbit IgG, equivalent to 305 ng/mL rabbit serum) than the FT1215EX IgG (< 10 ng/mg rabbit IgG, or < 60 ng/mL rabbit serum). The gp41Fc level in the FT1214PB 1 and FT1214PB2 rabbit IgG purified from the production bleed after the 3^rd and 4^th booster was comparable to that in FT1215PB1 and FT1215PB2. These data indicate that gp41Fc in rabbit FT1214 show no decline at 17 days after the 4^th booster while the level of gp41Fc in rabbit FT 1215 dropped rapidly from the 2^nd production bleed to the terminal bleed. The prolonged existence of the immunogen in rabbit sera seems to correlate with the neutralization activity of the antisera although further experiments with more animals are needed to confirm this observation (Fig. 19). Discussion

This Example describes the results from two rabbits immunized with a gp41Fc fusion protein. The major observation of this Example is that prolonged existence of the immunogen in rabbit serum correlates with the neutralization activity of rabbit IgG. These results suggest that prolonging the existence of the immunogen in vivo may enhance elicitation of broadly cross-reactive HIV-I neutralizing Abs.

This hypothesis may have important implications for the design of an HIV-I vaccine immunogen. It has been reported that the neonatal Fc receptor (FcRn) plays an important role in modulating IgG serum half-life, IgG homeostasis and maternofetal IgG transport (Ghetie et al., Annu. Rev. Immunol., 2000; 18:739-66; Roopenian et al., Nat. Rev. Immunol., 2007, 7:715-25). Studies on antibody-FcRn interaction across species showed that human FcRn binds to human, rabbit and guinea pig IgG, but not to rat, bovine, sheep or mouse IgG (with the exception of weak binding to mouse IgG2b). In contrast, mouse FcRn binds to all IgG from these species (Ober et al., Int. Immunol, 2001, 13: 1551-9). Further study showed that bovine FcRn binds stronger to human IgG than to bovine IgG, and human IgG has two times longer serum half life in normal and transchromosomic calves than its bovine counterpart (Kacskovics et al., Int. Immunol. 2006, 18:525-36). Presently, there is no study reported on the interaction of human IgG with rabbit FcRn, but the results from this Example suggest that rabbit FcRn may bind to human Fc and mediate elongated human Fc serum half life in rabbits.

The current Example used human Fc fused to gp41 ectodomain as immunogen to immunize rabbits. The immunogen level of the two rabbits during the course of immunization was comparable, but dropped quickly in rabbit FT1215 after the last booster, indicating a difference in the half-life of the immunogen in different host individuals. Further studies will be required to elucidate the mechanism for what leads to a fast drop of the immunogen level in vivo and how to extend the in vivo half- life of the immunogen. Here, the percentage of Fc-specific antibodies in the rabbit IgG by absorption of the rabbit IgG with human Fc-conjugated sepharose 4B column was determined (data not shown). Rabbit anti-Fc antibodies accounts for 30% of total rabbit IgG. Generation of human Fc-specific antibodies in rabbits may shorten the half life of gp41Fc in rabbits. Administration of gp41Fc in humans may result in a better immune response than in rabbits because human Fc is non-immunogenic in human and it can bind to human FcRn on immune cells including macrophages and dendritic cells thus contributing to a potentially longer half-life. The prolonged serum half-life of gp41Fc in rabbit FT1214 may contribute to the high level of cross- reactive HIV-neutralizing antibodies, but other mechanisms should not be excluded that may explain the difference in the level of cross-reactive HIV-neutralizing antibodies in the two rabbits used in this study. The positive reaction of rabbit IgG to the gp41 immunodominant loop region was in contrast to the unexpected difference of the two rabbit IgG binding profiles to the loop-derived peptides. The reason for this difference is not entirely clear or whether it may be related to the difference in neutralization activity. It was also found that peptides 6358 and 6359, which bind preferentially to FT1214EX IgG rather than FT1215EX IgG, are conserved among HIV-I clade A, B, C and D (data not shown). The binding of rabbit IgG to peptides derived from the MPER was not observed, although the MPER-specific human mAbs 2F5, 4E10 and Z13 bound well to the recombinant gp41Fc. This observation suggests a difference in antigenicity and immunogenicity. Lack of MPER peptide- specific rabbit Abs may be due to lack of a lipid membrane that helps present the MPER epitopes to the immune system and / or the limit of the antibody gene repertoire in rabbits capable of expressing and maturing to 2F5, 4E10 and Zl 3 -like antibodies (Lin et al., Curr. HIV Res., 2007, 5:514-41. Likewise, antibodies against the MPER of gp41 was not found in naturally infected humans (Li et al., Nat. Med., 2007, 13:1032-4), suggesting the poor immunogenicity of the MPER.

Having thus described in detail preferred embodiments of the present invention, it is to be understood that the invention defined by the above paragraphs is not to be limited to particular details set forth in the above description as many apparent variations thereof are possible without departing from the spirit or scope of the present invention. References

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Claims

WHAT IS CLAIMED IS:

1. An immunogen for vaccinating against and/or treating an HIV infection, comprising an HIV gp41 polypeptide or fragment thereof translationally linked to an Fc receptor ligand, optionally through a flexible linker.

2. The immunogen of claim 1, wherein the HIV gp41 polypeptide comprises at least the ectodomain of gp41.

3. The immunogen of claim 1, wherein the HIV gp41 polypeptide lacks the cytoplasmic tail region, the transmembrane region or the fusion domain region, or any combination thereof.

4. The immunogen of claim 1, wherein the gp41 polypeptide or fragment thereof is translationally fused to the amino-terminal end of the Fc receptor ligand.

5. The immunogen of claim 1, wherein the Fc receptor ligand is translationally fused to the amino-terminal end of the gp41 polypeptide or fragment thereof.

6. The immunogen of claim 1, wherein the Fc receptor ligand is an antibody Fc region or fragment thereof.

7. The immunogen of claim 6, wherein the antibody Fc region or fragment thereof is that of an IgG antibody.

8. The immunogen of claim 6, wherein the antibody Fc region or fragment thereof is that of an IgG, IgA, IgM, or IgE antibody.

9. The immunogen of claim 6, wherein the antibody Fc region or fragment thereof is from a human antibody.

10. The immunogen of claim 1, wherein the immunogen comprises a flexible linker.

11. The immunogen of claim 10, wherein the flexible linker is a polypeptide having between 1 and 50 amino acids in length.

12. The immunogen of claim 11, wherein the polypeptide is a hinge region of an antibody.

13. The immunogen of claim 1, wherein the gp41 polypeptide corresponds with the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence having at least about 80%, 85%, 90%, 95% or 99% sequence identity thereto.

14. The immunogen of claim 1, wherein the flexible linker is a hinge reigion of a human IgG and has the amino acid sequence of SEQ ID NO: 6 or or an amino acid sequence having at least about 80%, 85%, 90%, 95% or 99% sequence identity thereto.

15. The immunogen of claim 1, wherein the Fc receptor ligand is region CH2-CH3 of human IgG according to SEQ ID NO: 5.

16. The immunogen of claim 1, wherein immunogen is capable of eliciting broadly-cross reactive neutralizing antibodies against HIV.

17. The immunogen of claim 16, wherein the neutralizing antibodies are capable of neutralizing HIV in vivo.

18. A method of vaccinating a subject for immunizing against an HIV infection comprising administering a therapeutically effective amount of the immunogen of claim 1.

19. A method of treating a subject having an HIV infection comprising administering a therapeutically effective amount of an antibody that specifically recognizes at least one epitope of the immunogen of claim 1.

20. An isolated nucleic acid molecule encoding the immunogen of claim 1.

21. An expression vector comprising the nucleic acid of claim 20.

22. A method of making the immunogen of claim 1, comprising culturing a host cell comprising an expression vector encoding the immunogen, expressing the immunogen and isolating the immunogen from the culture.

23. A recombinant antigen comprising a human immunodeficiency virus envelope protein gp41 which is coupled to an Fc receptor ligand by a flexible linker and is defined by SEQ ID NO: 3 or an amino acid sequence having at least about 80% sequence identity thereto.

24. A recombinant antigen comprising a human immunodeficiency virus envelope protein gp41 which is coupled at its carboxy-terminal end to the N- terminal end of an Fc receptor ligand through a flexible linker, wherein the gp41 is defined by SEQ ID NO: 4 or an amino acid sequence having at least 80% sequence identity thereto.

25. The recombinant antigen of claim 24, wherein the Fc receptor ligand corresponds to the CH2-CH3 region of human IgG as defined by SEQ ID NO: 5, or an amino acid sequence having at least about 80% sequence identity thereto.

26. The recombinant antigen of claim 24, wherein the flexible linker corresponds to the hinge region of human IgG as defined by SEQ ID NO: 6, or an amino acid sequence having at least about 80% sequence identity thereto.

27. The recombinant antigen of claim 24, wherein the flexible linker is between 5 and 20 amino acids in length.

28. A pharmaceutical composition comprising a therapeutically effective amount of a recombinant antigen comprising a human immunodeficiency virus envelope protein gp41 which is coupled to an Fc receptor ligand by a flexible linker and is defined by SEQ ID NO: 3 or an amino acid sequence having at least about 80% sequence identity thereto.

29. A pharmaceutical composition comprising a therapeutically effective amount of a recombinant antigen comprising a human immunodeficiency virus envelope protein gp41 which is coupled at its carboxy-terminal end to the N- terminal end of an Fc receptor ligand through a flexible linker, wherein the gp41 is defined by SEQ ID NO: 4 or an amino acid sequence having at least 80% sequence identity thereto.

30. The pharmaceutical composition of claim 29, wherein the Fc receptor ligand corresponds to the CH2-CH3 region of human IgG as defined by SEQ ID NO: 5, or an amino acid sequence having at least about 80% sequence identity thereto.

31. The pharmaceutical composition of claim 29, wherein the flexible linker corresponds to the hinge region of human IgG as defined by SEQ ID NO: 6, or an amino acid sequence having at least about 80% sequence identity thereto, and a pharmaceutically acceptable carrier.

32. The pharmaceutical composition of claim 29, wherein the flexible linker is between 5 and 20 amino acids in length.

33. A method of immunizing a subject against an HIV infection, comprising administering a therapeutically effective amount of a recombinant antigen comprising a human immunodeficiency virus envelope protein gp41 which is coupled to an Fc receptor ligand by a flexible linker and is defined by SEQ ID NO: 3 or an amino acid sequence having at least about 80% sequence identity thereto.

34. A method of immunizing a subject against an HIV infection, comprising administering a therapeutically effective amount of the recombinant antigen of claims 24-27.

35. A method of immunizing a subject against an HIV infection, comprising administering a therapeutically effective amount of the pharmaceutical composition of claims 28-32.

36. A method of preparing a broadly cross-reactive neutralizing anti-HIV antibody against HIV, comprising screening an antibody library for antibodies that specifically bind to the antigen of claims 1 or 2.

37. A method of preparing a broadly cross-reactive neutralizing anti-HIV antibody against HIV, comprising immunizing an animal with the antigen of claims

24-27 to elicit the production of anti-HIV antibodies in the animal, obtaining the sera of the animal, and isolating the anti-HIV antibodies from the sera.

38. A broadly cross-reactive neutralizing anti-HIV antibody prepared by the method of claim 36.

39. A broadly cross-reactive neutralizing anti-HIV antibody prepared by the method of claim 37.

40. A method of treating an HIV infection in a subject in need thereof, comprising: obtaining a broadly cross-reactive neutralizing anti-HIV antibody prepared by the method of claims 38 or 39; and administering a therapeutically effective amound of the anti-HIV antibody to the subject, thereby neutralizing the HIV infection and treating the subject.

41. The method of claim 40, wherein the step of administration is performed locally.

42. The method of claim 41, wherein the step of local administration is performed topically.

43. The method of claim 40, wherein the step of administration is performed systemically.

44. The method of claim 43, wherein the step of systemic administration is performed by injection.

45. A vaccine for immunizing a human from a human immunodeficiency virus infection comprising a recombinant antigen comprising: a human immunodeficiency virus envelope protein gp41 coupled to an Fc receptor ligand by a flexible linker and defined by SEQ ID NO: 3 or an amino acid sequence having at least about 80% sequence identity thereto; and a pharmaceutically acceptable carrier.

46. A vaccine for immunizing a human from a human immunodeficiency virus infection comprising: a recombinant antigen comprising a human immunodeficiency virus envelope protein gp41 coupled at its carboxy-terminal end to the N-terminal end of an Fc receptor ligand through a flexible linker, wherein the gp41 is defined by SEQ ID NO: 4 or an amino acid sequence having at least 80% sequence identity thereto; and a pharmaceutically acceptable carrier.

47. A method of vaccinating a human from a human immunodeficiency virus infection, comprising: administering a therapeutically effective amount of the vaccine of claims 45 or 46.

48. The method of vaccinating a human of claim 47, further comprising the step of co- administering an anti- viral drug.

49. The method of vaccinating a human of claim 48, wherein the antiviral drug is an anti-HIV therapy.

50. A packaged pharmaceutical comprising the recombinant antigen of claims 23 or 24 and instructions for use in accordance with a method of any one of the above method claims.

51. A nucleic acid molecule encoding a recombinant antigen comprising a human immunodeficiency virus envelope protein gp41 coupled to an Fc receptor ligand by a flexible linker, said nucleic acid defined by the nucleic acid sequence of SEQ ID NO: 1 or a nucleic acid sequence having at least about 80% sequence identity thereto.

52. An expression vector comprising a nucleic acid molecule encoding a recombinant antigen comprising a human immunodeficiency virus envelope protein gp41 which is coupled to an Fc receptor ligand by a flexible linker, said nucleic acid defined by the nucleic acid sequence of SEQ ID NO: 1 or a nucleic acid sequence having at least about 80% sequence identity thereto.

53. A method of manufacturing a human recombinant antigen comprising a human immunodeficiency virus envelope protein gp41 which is coupled to an Fc receptor ligand by a flexible linker, said antigen defined by SEQ ID NO: 3 or an amino acid sequence having at least about 80% sequence identity thereto, comprising:

(a) obtaining a nucleic acid encoding gp41;

(b) coupling the 5 ' end of a nucleic acid encoding the Fc receptor ligand to the 3' end of the nucleic acid encoding gp41 through a nucleic acid encoding a flexible linker to form a gp41 fusion gene encoding the recombinant antigen;

(c) operably linking the gp41 fusion gene to an expression vector; and

(d) introducing the expression vector into a host cell; and (e) permitting the host cell to express the recombinant antigen; and

(f) isolating the recombinant antigen from the cell, thereby manufacturing a human recombinant antigen.