TECHNICAL FIELD

The present disclosure relates to a polynucleotide sequence and its corresponding polypeptide sequence. It also relates to a recombinant vector and a recombinant host cell. Further, it also presents a method of obtaining the polypeptide, an immunogen, a method of obtaining the immunogen and a kit thereof. The present disclosure describes the designing of a protein fragment that elicits neutralizing antibodies against HIV-1 in immunization studies. Either this fragment or improved derivatives of it could be eventual components of an HIV-1 vaccine. The present immunogen has 11 mutations incorporated in it to minimize aggregation of the fragment and the fragment is expressed in unglycosylated form in the bacterium E. coli.

BACKGROUND

The envelope glycoprotein of human immunodeficiency virus 1 (HIV-1) is composed of two polypeptide chains, gpl20 and gp41, which are present on the surface of the virus as a trimer of heterodimers. As these are the primary viral components exposed to the external environment, they present the single best target for neutralizing antibodies. gp41 mainly contains the fusion machinery and a large part of the membrane proximal region, and is transiently exposed during the fusion process. gpl20 remains largely exposed on the virus. However, the virus has evolved various mechanisms to evade the humoral immune response towards gpl20. A very high rate of mutation, large conformational flexibility and the extensive glycosylation of the surface are some of the important ways by which the virus has evaded a neutralizing antibody response.

As a result, when recombinant gpl20 is used as an immunogen, the antibodies generated are often directed to immunodominant epitopes present in variable loops and have limited breadth of neutralization. Core gpl20 lacking the variable loops is poorly immunogenic, in part because of surface coverage by glycans. It was recently shown that demannosylated fulllength gpl20 is much more immunogenic than wild-type gpl20 in mouse immunization studies. The structure of gpl20, as seen from the crystal structure of gpl20 complexed to CD4 and antibody 17b (PDB ID:1G9M) reveals that the molecule can be subdivided into three distinct parts, the inner domain, the outer domain and the bridging sheet. On monomeric gpl20, the inner domain is well exposed and has little coverage by glycans. However this domain is likely to be involved in interactions with gp41(17), is probably inaccessible on the intact envelope trimer on the virion surface and hence is not a good immunogen target. The bridging sheet is part of a cryptic epitope involved in co-receptor binding that is transiently exposed upon receptor binding and is therefore difficult to target by antibodies. There are known antibodies to this target which
have weak but relatively broad neutralizing activity. Several conserved regions in gpl20, including regions of the CD4-binding site and epitopes for the broadly neutralizing antibodies bl2 and 2G12 are located on the outer domain. Thus while sequence variability in some regions of this domain is quite high, it merits consideration as a possible immunogen.

Previous attempts to develop an outer domain based immunogen, however met with little success. The outer domain construct (OD1) made by Yang et al comprised of residues 252-482, included the V1V2 and V3 variable loops, was expressed in S2 Drosophila cells and was glycosylated. The construct had the YU2 envelope sequence, which is deficient in TH2 helper epitopes, and thus was poorly immunogenic. Antibody titers became high after attaching a Pan-reactive epitope for HLA-DR (PADRE) at the C-terminus. However when the V3 loop was deleted, the OD1-AV3-PADRE was again poorly immunogenic, indicating that the V3 loop was responsible for the observed high antibody titers. OD1 could bind 2G12 but not CD4. bl2 binding also had a very high off-rate and complete dissociation was observed within 50-60 seconds for all concentrations of OD1 used. The sera obtained after immunizing rabbits did not elicit significant neutralizing antibodies.
Chen et al expressed residues 251-481 of HIV- 1CN54 gpl20 (clade C virus) as a C-terminal fusion to the Fc domain of human IgGl, using recombinant baculoviruses and used that to immunize mice. The monoclonal antibodies derived from the serum were mostly V3-directed. However no neutralization studies were done with the sera.

STATEMENT OF THE DISCLOSURE

Accordingly the present disclosure provided for a nucleotide sequence set forth as Seq. Id No. 1; peptide sequence set forth as Seq. Id No. 2, wherein the peptide sequence is corresponding to nucleotide sequence set forth as Seq. Id No. 1; a method of obtaining peptide sequence set forth as Seq. Id No. 2, said method comprising acts of: a) identifying CD4 binding regions in gpl20 of HIV virus and b) introducing mutations outside the binding regions to obtain the peptide sequence set forth as Seq. Id No. 2; vector having accession number MTCC 5658, said vector comprising nucleotide sequence set forth as Seq. Id No.l; recombinant cell, comprising the vector as stated above; a method of obtaining the recombinant cell, said method comprising acts of: a. obtaining vector comprising nucleotide sequence set forth as Seq. Id No. 1 and b. transforming a host cell with said vector to obtain the recombinant cell; a method of obtaining protein comprising peptide sequence set forth as Seq. Id No. 2, said method comprising acts of: a.) inserting nucleotide sequence set forth as Seq. Id No. 1 into a vector and transforming a host cell with the vector to obtain a recombinant cell b.) expressing the vector within the cell for obtaining the protein comprising peptide sequence set forth as Seq. Id No. 2 and c.) optionally, purifying the protein of step b); immunogen comprising protein having peptide sequence set forth as Seq. Id No. 2, optionally along with adjuvant or pharmaceutically acceptable additive(s) or a combination thereof; a method of obtaining immunogen comprising protein having peptide sequence set forth as Seq. Id No. 2, optionally along with adjuvant or pharmaceutically acceptable additive(s) or a combination thereof, said method comprising acts of: a.) inserting nucleotide sequence set forth as Seq. Id No. 1 into a vector and transforming a host cell with the vector to obtain a recombinant cell b.) expressing the vector within the cell for obtaining and purifying the protein, to obtain the immunogen and c.) optionally, adding adjuvant or pharmaceutically acceptable additive(s) or a combination thereof, to the immunogen of step b); a kit having component selected from group comprising nucleotide sequence set forth as Seq. Id No. 1, peptide sequence set forth as Seq. Id No. 2, the vector, the recombinant cell or immunogen alongwith adjuvant and pharmaceutically acceptable additive(s) or any combination thereof along with instructions manual.

BRIEF DESCIPTION OF ACCOMPANYING FIGURES

In order that the disclosure may be readily understood and put into practical effect, reference will now be made to exemplary embodiments as illustrated with reference to the accompanying figures. The figure together with a detailed description below, are incorporated in and form part of the specification, and serve to further illustrate the embodiments and explain various principles and advantages, in accordance with the present disclosure where:

Figure 1 shows (A) Structure of core gpl20 when complexed to CD4. (B) Structure of core gpl20 when complexed to the broadly neutralizing antibody bl2. (C) Structure of core gp!20 when complexed to the CD4 binding site antibody F105.

Figure 2 shows (A) Spacefill structure of ODEC (PDBID 1G9M) showing the positions of the 11 surface mutations (in orange) that are present in ODEC. (B) The same structure, rotated by 180 degrees along the vertical axis. (C) Sequence alignment of YU2 outer domain sequence present in OD1 with HxBc2 outer domain sequence in ODEC.

Figure 3 shows biophysical characterization of ODEc (A) Far-UV CD spectrum of ODEC. (B) Fluorescence emission spectrum of ODEc. (C) Bar plot of the intensities of fluorescence emission at 480nm. (D) Thermal denaturation of ODEc in PBS, pH7.4.

Figure 4 shows SDS-PAGE analysis of proteolytic digests of (A) ODEc (at pH 8.0) by trypsin at 37°C (B) reduced carboxymethylated RNaseA (rcam-RNaseA) (at pH 8.0) by trypsin at 37°C. (C) ODEC (at pH 8.0) by proteinase K at 37°C. (D) reduced carboxymethylated RNaseA (rcam-RNaseA) (at pH 8.0) by proteinase K at 37°C. (E) SDS-PAGE analysis of ODEC and WT ODEC before and after refolding into PBS with ImMEDTA.

Figure 5 shows (A) Analytical gel-filtration analysis plot of ODEC on a Superdex 75 column in PBS buffer at room temperature. (B) Calibration curve of the analytical Superdex-75 column with standard marker proteins albumin (67kDa), ovalbumin (43kDa), chymotrypsinogen (24kDa) and RNaseA (13kDa). (C) Reverse-phase HPLC for ODEc in an analytical C18 column (Vydac) at room temperature.

Figure 6 shows Biacore sensorgram overlays. (A) Binding of different concentrations of full-length gpl20 to surface immobilized 4-domain CD4. (B) Binding of different concentrations of ODEc to surface immobilized 4-domain CD4.

Figure 7 shows Biacore sensorgram overlays for the binding of different concentrations of ODEC to surface immobilized IgGbl2 at (A) 25°C and (B) 15°C.

Figure 8 shows Biacore sensorgram overlays for the binding of l00nM full-length gpl20 (curve 1) and 8uM ODEc (curve 2) to surface immobilized (A) mAb IgGb6 and(B)mAbF105.

DETAILED DESCRIPTION

The present disclosure relates to a nucleotide sequence set forth as Seq. Id No. 1.

The present disclosure also relates to peptide sequence set forth as Seq. Id No. 2,

wherein the peptide sequence is corresponding to nucleotide sequence set forth as
Seq. Id No. 1.

The present disclosure further relates to a method of obtaining peptide sequence set forth as Seq. Id No. 2, said method comprising acts of:

a.) identifying CD4 binding regions in gpl20 of HIV virus; and b.) introducing mutations outside the binding regions to obtain the peptide sequence set forth as Seq. Id No. 2.
In an embodiment of the present disclosure, the peptide sequence set forth as Seq. Id No. 2 corresponds to mutation in peptide sequence set forth as Seq. Id No. 3, at amino acid positions selected from group comprising 255, 260, 261, 271, 275, 285, 382, 434, 435 and 453 or any combinations thereof.

In another embodiment of the present disclosure, the mutation is selected from group comprising point mutation, insertion, deletion, substitution and frame shift mutation of hydrophobic amino acid residues with hydrophilic amino acid residues or any combination thereof.

The present disclosure also relates to a vector having accession number MTCC 5658, said vector comprising nucleotide sequence set forth as Seq. Id No.l.

The present disclosure also relates to recombinant cell, comprising the vector stated above.

The present disclosure also relates to a method of obtaining the recombinant cell, said method comprising acts of:

a. obtaining vector comprising nucleotide sequence set forth as Seq. Id
No. 1; and

b. transforming a host cell with said vector to obtain the recombinant cell.

The present disclosure also relates to a method of obtaining protein comprising peptide sequence set forth as Seq. Id No. 2, said method comprising acts of:

a.) inserting nucleotide sequence set forth as Seq. Id No. 1 into a vector and transforming a host cell with the vector to obtain a recombinant cell; b.) expressing the vector within the cell for obtaining the protein comprising peptide sequence set forth as Seq. Id No. 2; and c.) optionally, purifying the protein of step b).

The present disclosure also relates to an immunogen comprising protein having peptide sequence set forth as Seq. Id No. 2, optionally along with adjuvant or pharmaceutically acceptable additive(s) or a combination thereof.

The present disclosure also relates to a method of obtaining immunogen comprising protein having peptide sequence set forth as Seq. Id No. 2, optionally along with adjuvant or pharmaceutically acceptable additive(s) or a combination thereof, said method comprising acts of:

a. inserting nucleotide sequence set forth as Seq. Id No. 1 into a vector and transforming a host cell with the vector to obtain a recombinant cell;

b. expressing the vector within the cell for obtaining and purifying the protein, to obtain the immunogen; and

c. optionally, adding adjuvant or pharmaceutically acceptable additive(s) or a combination thereof, to the immunogen of step b).

In an embodiment of the present disclosure, the vector is selected from group comprising bacterial expression vectors, yeast expression vectors and animal cell expression vector, preferably selected from group comprising pET15b, pET20b(+), pCOMB-3X and pcDNA3.1(+); and the host cell is selected from group comprising bacteria, preferably selected from E.coli BL21(DE3) or E.coli TOP 10, yeast, preferably S.cerevisiae or Pichia pastoris, animal cell, preferably HEK293 or Drosophila.

In another embodiment of the present disclosure, the adjuvant is selected from group comprising CpG7909, IMX, MAA, Freund's adjuvant, Mycobacterium w (Mvc), alum and Adjuplex LAP or any combination thereof; and the pharmaceutically acceptable additive is selected from group comprising excipients, gums, sweeteners, coatings, binders, disintegrants, lubricants, disintegration agents, suspending agents, granulating agents, solvents, colorants, glidants, anti-adherents, anti-static agents, surfactants, plasticizers, emulsifying agents, flavoring agents, viscosity enhancers and antioxidants, or any combination thereof.

The present disclosure also relates to a kit having component selected from group comprising nucleotide sequence set forth as Seq. Id No. 1, peptide sequence set forth as Seq. Id No. 2, the vector, the recombinant cell or immunogen as stated above, along with adjuvant and pharmaceutically acceptable additive(s) or any combination thereof along with instructions manual.

The present disclosure relates to the outer domain (OD) of the HIV-1 envelope glycoprotein gpl20 which is an important target for vaccine design since it contains a number of conserved epitopes, including a large fraction of the CD4 binding site.

Attempts to design OD based immunogens in the past have met with little success. The instant disclosure reports, the designing and characterization of an E.coli expressed OD based immunogen (ODEC), based on the sequence of the HxBc2 strain. The ODEC designed immunogen lacks the variable loops V1V2 and V3 and incorporates 11 designed mutations at the interface of the inner and the outer domains of gpl20.

Biophysical studies showed that ODEC is folded and protease resistant while ODEC lacking the designed mutations is highly aggregation prone. In contrast to previously characterized OD constructs, ODEC bound CD4 and the broadly neutralizing antibody bl2, but not the nonneutralizing antibodies b6 and F105. Upon immunization in rabbits, ODEC is highly immunogenic and the sera showed measurable neutralization for four subtype B and one subtype C virus including two bl2 resistant viruses. In contrast, sera from rabbits immunized with gpl20 did not neutralize any of the viruses. ODEc is the first example of a gpl20 fragment based immunogen that yields significant neutralizing antibodies.

In an embodiment of the instant disclosure, bacterial expression, biophysical and immunological characterization of ah outer domain construct based on the HIV-1 HxBc2 sequence is described. Such a bacterially expressed molecule is not glycosylated and leads to better exposure of conserved epitopes, including the highly conserved CD4 binding site. This construct is hereby referred to as ODEC- In the present disclosure it is also showed that in addition to binding CD4, it also binds the broadly neutralizing antibody bl2 but not the non-neutralizing antibodies b6 and F105. When used as an immunogen in rabbits, the resulting sera shows neutralization with a panel of viruses. The panel included two bl2 resistant viruses, a subtype C virus ZM109F and a subtype B virus TRO.l 1.

In an embodiment of the present disclosure, biophysical studies show that ODEC is folded and protease resistant while ODEC lacking the designed mutations is highly aggregation prone. In contrast to previously characterized OD constructs, ODEC bound CD4 and the broadly neutralizing antibody bl2, but not the non-neutralizing antibodies b6 and F105. Upon immunization in rabbits, ODEC is highly immunogenic and the sera showed measurable neutralization for four subtype B and one subtype C virus including two bl2 resistant viruses. In contrast, sera from rabbits immunized with gpl20 did not neutralize any of the viruses. ODEC is the first example of a gp 120 fragment based immunogen that yields significant neutralizing antibodies.

The present disclosure is further described with the help of the following examples and figures. However, these examples should not be construed to limit the scope of the disclosure.

The Biological material present in the instant disclosure in the form of vectors comprising the genes of interest are deposited at the International Depository -Microbial Type Culture Collection & Gene Bank, Chandigarh. The deposited vector is assigned the following MTCC Numbers: pET28a (+) ODEC- MTCC 5658

EXAMPLE 1: CONSTRUCT DESIGN

The design goal is an outer domain construct of gpl20, which includes most of the CD4 binding site residues. The structure of gpl20 as it occurs when complexed to CD4 is shown in Fig 1A. gpl20 residues involved in CD4 binding are primarily located in the stretches 124-126, 279-283, 365-371, 425-430, 456-459 and 469-474.

Two antiparallel beta-strands from 251-260 and 470-474 connect the inner domain with the outer domain. Thus the fragment 255-474 would include the whole of the outer domain and most of the CD4 binding residues. This fragment is 191 residues long and contains three disulfide bonds between residues 296-331, 378-445 and 385-418. In order to determine which residues in this fragment are interacting with the rest of the molecule, the in-house software PREDBURASA is used. Residues from 255- 474 of gpl20 are considered to be one chain (chain A) and the rest of the molecule as another chain (chain B). Residue accessible surface areas for chain A are calculated in the presence and absence of chain B. Residues that have a difference of 5A2 or more in absolute ASA are considered as interacting residues. ODEC has a total accessible surface area of 9900A2, out of which 2400A2 (24%) of this area is involved in interaction with other regions of gpl20. This included 11 hydrophobic residues and 18 hydrophilic residues (excluding the CD4 binding site residues). Each hydrophobic residue is allowed to vary in identity to all polar residues and the best substitution is selected on the basis of energy values.

These mutations and subsequent energy calculations are done using the program ROSETTA DESIGN (v 2.0). The final mutations incorporated are V255T, L260N, L261D, V271S, V275N, I285T, F382R, M434K, Y435K, I439D and L453N, as highlighted in Fig 2A,B. Residues 255-474, inclusive of all the mutations, is called ODEC- The WT sequence used in the design is based on the subtype B, CXCR4 tropic HXBc2 strain and except for the indicated mutations, is identical to that of region 255-474 in PDBID 1G9M (Fig 1). It has the tripeptide Gly- Ala-Gly substitutions for the 67 V1/V2 loop residues and 32 V3 loop residues, but contains the 12 residue V4 loop. A comparison of ODEc with a previous outer domain based construct OD1 is shown in Fig 2C.

In an embodiment of the present disclosure, figure 1 shows (A) Structure of core gpl20 when complexed to CD4, coordinates are from the PDBID 1G9M, (B) Structure of core gpl20 when complexed to the broadly neutralizing antibody bl2, coordinates are from the PDBID 2NY7 (C) Structure of core gpl20 when complexed to the CD4 binding site antibody F105, coordinates are from the PDBID 3HI1. In all the three figures, the region in green is the part of the outer domain that is included in the ODEC construct, while the respective binding site residues are in red.

As evident from the structure, most of the CD4 and bl2 binding residues are present in ODEC, while a large fraction of the F105 binding residues are absent. In addition, two residues (Val 255 and Phe 382) important for F105 binding which are present in the construct are mutated to Threonine and Arginine respectively to minimize protein aggregation. Further, Figure 2 shows (A) Spacefill structure of ODEC (PDBID 1G9M) showing the positions of the 11 surface mutations (in orange) that are present in ODEC.

The residues in green are CD4 binding site residues, while those in blue are the asparagine residues involved in glycosylation in whole gpl20. (B) The same structure, rotated by 180 degrees along the vertical axis. (C) Sequence alignment of YU2 outer domain sequence present in OD1 with HxBc2 outer domain sequence in ODEC- The 11 engineered mutations are indicated in red, while the N-linked glycosylation sites are in blue. Besides these ROSETTA predicted changes, there are a number of mismatches between the sequences, which are the inherent differences between the YU2 and HxBc2 sequences. In addition, OD1 has the complete V3 loop which is absent in ODEc. The region of the V4 loop is also very different in both sequences.

Procedure used to select mutations in ODEC When the outer domain is excised from full-length gpl20, all hydrophobic residues which are present at the interface of the inner and the outer domain become solvent-exposed. The in-house program PREDBURASA is used to identify these residues. The region from 255-474 in gpl20 is assigned the chain identifier A while the rest of gpl20 is assigned the chain identifier B.

Accessible surface areas are calculated for all residues in chain A, in presence and absence of chain B. Those residues which had a difference of more than 5A2 in ASA in the two cases are taken as chain B interacting residues. ODEC has a total accessible surface area of 9900A2. Of this, 2400A2 (24%) is involved in interaction with other regions of gpl20. This included 11 hydrophobic residues and 18 hydrophilic residues (excluding the CD4 binding site residues). Using the program ROSETTADESIGN, each hydrophobic residue is allowed to vary in identity to all polar residues separately.

For each modeled polar residue at each position, the program gives 11 energy terms, namely: Eatr - Lennard-Jones attractive energy, Erep - Lennard-Jones repulsive energy, Esolv - Lazaridis-Karplus solvation energy, Eaa - Probability that a particular amino acid will take up a particular phi-psi combination, Erot - Energy of a particular rotamer calculated from rotamer preferences from Dunbrack's library, Eintra -Energy penalty due to intra residue clashes, Ehbond - Energy of hydrogen bonding, Epair - Statistics based pair term, calculated from two residue pair distributions found in protein structures, Elj - Total Lennard-Jones energy, Eres - Total energy per residue, SASApack energy - Shows whether the rotamer is well-packed or not. For the above terms, the (measured energy - expected energy) is calculated. The expected energy is the average energy of that amino acid with a certain number of neighbors in a large set of proteins in the PDB.

However, only 4 of these terms, Eatr, Erep, Esolv and Ehbond (listed in the Table below) are used in our designs and are summed up to get Etotal. In most of the cases (255, 271, 275, 434, 435, 439, 453), the residue showing the lowest Etotal is chosen for that position. In some cases (260, 261, 285, 382), some other polar residue from the list is chosen from visual inspection of the structure, provided that the particular residue did not show a very unfavorable energy value (greater than 2 Rosetta Energy Units). The final mutations incorporated are V255T, L260N, L261D, V271S, V275N, I285T, F382R, M434K, Y435K, I439D and L453N.

Table SI: ROSETTADESIGN energy terms for modeled polar residue substitutions at selected positions of ODEC. At each position, energies of the selected mutant are highlighted.

Example 2: Purification of protein and biophysical characterization

An E.coli codon-optimized version of the ODEC gene is synthesized and cloned into the pET28a (+) vector between the Ndel and BamHI sites and contained a N-terminal His tag. Similarly, ODEC gene is cloned into pET15b vector between Nde I and Bam H1; in pET20b (+) the said gene is cloned between Nde I and Xho I restriction sites. Sfi I restriction site is used for cloning in pCOMB 3x vector while for pcDNA 3.1 vector, it is Nhe I and Bam HI restriction sites.

The pET28a (+) vector containing ODEC gene is then transformed into E.coli strain BL21(DE3) by following method: Chemically competent cells of BL21(DE3) are made by Rubidium chloride method. For transformation, about 50ul cells are thawed and to it, about 50ng plasmid DNA is added and incubated at about 4°C for about 10 mins. After that, the cells are given heat shock at about 42°C for about 45 seconds and about 450ul LB broth is added to it for recovery and kept at about 37°C. After incubation for about 1 hour at about 37°C, the culture is plated on LB/Kanamycin plate (concentration 50ug/ml) and the plate is kept for growth at about 37°C for about 12 hours.

E.coli strain BL21(DE3) cells transformed with the plasmid are grown in about 1 liter Luria-Broth (LB) at about 37°C till an O.D of about 0.6. Cells are then induced with about ImM IPTG (Isopropyl-beta-thio galactopyranoside) and grown for another 6 hours at about 37°C. Cells are harvested at about 3,500g and resuspended in about 30ml phosphate buffered saline (PBS), about pH 7.4. The cell suspension is lysed by sonication on ice and centrifuged at about 15,000g. The supernatant is discarded and the pellet is washed in about 30ml 0.1% Triton X-100, PBS (pH 7.4) and subjected to centrifugation at about 15,000g. The pellet is solubilized in about 25ml of 8M

Guanidine hydrochloride (GdnCl) in PBS (pH 7.4) overnight at room temperature. The solution is centrifuged at about 15,000g for about 30 min. The supernatant is bound to 3ml Ni-NTA beads, washed with about 30ml 50mM imidazole containing 8M Guanidine hydrochloride in PBS and finally denatured protein is eluted with about 8 M GdnCl in PBS containing about 500 mM imidazole at room temperature.

The first four eluted fractions (each 3ml) are pooled together and then rapidly diluted about 10 fold with PBS containing about ImM EDTA to reduce the denaturant concentration from about 8M to about 0.8M. The resulting solution is again concentrated back to the original volume in an Amicon concentrator. This is followed by desalting into PBS (about pH 7.4) containing about ImM EDTA using a HiTrap Desalting column to remove the remaining denaturant. Protein is -90% pure as assessed by SDS-PAGE. The desalted protein is concentrated to a final concentration of about 0.5mg/ml and flash-frozen in liquid nitrogen and stored in aliquots at about -80°C. The average yield is 5-6 mg/liter of culture. The yield is determined by densitometry analysis from SDS-PAGE using standard protein, chymotrypsinogen of known concentrations.

Far-UV CD and Fluorescence spectroscopy

Circular dichroism (CD) spectra are recorded on a Jasco J-715C spectropolarimeter flushed with nitrogen gas. The concentration of protein sample is IOUM and buffer used is PBS, about pH 7.4. Measurements are recorded in a 1mm path length quartz cuvette with a scan rate of about 50nm/min, a response time of about 4 seconds and a bandwidth of about 2nm. Each spectrum is an average of three scans. Mean residue ellipticities (MRE) are calculated. Buffer spectra are also acquired under similar conditions and subtracted from protein spectra, before analysis.

All fluorescence spectra are recorded at about 25°C on a SPEX Fluoromax3 spectrofluorimeter. For intrinsic fluorescence measurements, protein concentration used is about luM. The excitation is at about 280 nm and emission is recorded from 300 to 400 nm. The excitation and emission slit widths are 3 and 5 nm, respectively. For the ANS binding study, the protein and ANS concentrations used are 1 and l00uM respectively. Samples are excited at about 365 nm and emission spectra are collected over the wavelength range 400 to 600 nm. Each spectrum is an average of three consecutive scans. Buffer spectra are also acquired under similar conditions and subtracted from protein spectra, before analysis. All fluorescence experiments are carried out in PBS at pH 7.4.

The ODEC gene is cloned in pET-28a (+) plasmid with an N-terminal His tag. The protein is expressed in E.coli BL21 (DE3) cells and purified on a Ni-NTA affinity matrix after resolubilization from inclusion bodies. The yield is about 5-6mg / L of the culture. SDS-PAGE studies confirmed that the protein is at least 90% pure. Mass spectrometric analysis (ESI-MS) confirmed the identity of the protein.

The CD spectrum of the protein (Fig3A) showed that it has appreciable secondary structure. The protein shows a clear thermal unfolding transition with an apparent Tm of 62.3°C (Fig 3D). The fluorescence spectrum (Fig3B) of the protein shows an expected red shift and a change in emission intensity upon denaturation, showing that the protein is likely to be folded with burial of some tryptophan residues in the native state. ANS binding studies are also done with ODEc- ANS (8-Anilinonaphthalene-l-sulfonic acid) is a fluorescent probe which binds to hydrophobic pockets present on partially denatured, moltenglobule like states of proteins. When it binds to hydrophobic patches, there is a characteristic blue-shift in its max together with an increase in its fluorescence emission intensity. Relative to a previously characterized molten globule control, both ODEC and full-length gpl20 showed much weaker binding to ANS (Fig3C). This shows that ODEc does not have large exposed hydrophobic patches. Tryptic digestion (Fig 4A,B) and proteinase K digestion (Fig 4C,D) studies of ODEC at 37°C showed that ODEC is quite resistant to digestion, relative to an unfolded protein (rcam-RNaseA), confirming that ODEC is a well-ordered molecule.

The 11 hydrophobic to hydrophilic mutations in ODEC are introduced to prevent the protein from aggregating. As a negative control, an E.coli codon-optimized version of the ODEC gene without the 11 mutations (hereby referred to as WT ODEC) is synthesized and the protein is purified under denaturing conditions using Ni- NTA affinity chromatography as is done for ODEC. However on attempting to refold it by rapid dilution, there is considerable precipitation. For comparison, equal amounts of denatured ODEC and WT ODEC in 8M GdnCl are diluted ten fold and then in both cases, the pellet and the supernatant are loaded on the gel (Fig 4E). While ODEc remained almost entirely in the soluble fraction, WT ODEc is mostly in the precipitate. Thus the 11 mutations contributed significantly to increasing the stability and reducing the aggregation propensity of the protein.

The ODEC construct contains six cysteine residues. All six are involved in disulfide bond formation in the context of the native full-length gpl20 molecule. A DTNB assay showed that the percentage of free thiols in ODEC is negligible, thereby showing the disulfides are formed. Analytical gel filtration (Fig5A) showed that ODEC is a monomer. Native protein eluted from a C18 analytical reverse-phase column as a single peak (Fig5C), thereby showing that it is a homogeneous species in solution and not a mixture of different disulfide bonded isomers. The denatured, reduced protein eluted at a different acetonitrile concentration than the native protein, reconfirming that native ODEC is well folded and oxidized

In an embodiment of the present disclosure, figure 3 shows biophysical characterization of ODEC (A) Far-UV CD spectrum of ODEC. The spectrum is buffer corrected and is obtained at about 25°C with about l0uM of protein in PBS buffer, about pH 7.4 with a 0.1cm path length cuvette, a scan-rate of about 50nm/min, a response time of about 4 seconds and a bandwidth of 2nm. Data reported are averaged over 3 scans. (B) Fluorescence emission spectrum of ODEC- The spectrum is obtained at about 25°C with a final protein concentration of about luM in PBS, about pH 7.4 (solid line) or in presence of about 6M Guanidine Hydrochloride in PBS, about pH 7.4 (dashed line). The excitation is at 280nm and emission is recorded from 300 to 400nm. (C) Bar plot of the intensities of fluorescence emission at 480nm of (1) l00uM ANS in PBS, pH 7.4 (2) l00uM ANS with luM ODEC in PBS, pH 7.4 (3) l00uM ANS with luM full-length gpl20 in PBS, pH 7.4 (4) l00uM ANS at pH 3.0 (5) l00uM ANS together with a luM molten globule control (Maltose Binding Protein) at pH 3.0. Samples are excited at about 365 nm and emission spectra are collected over the wavelength range 400 to 600 nm. Each spectrum is an average of three consecutive scans. Buffer spectra are also acquired under similar conditions and subtracted from protein spectra. (D) Thermal denaturation of ODEC in PBS, pH7.4. CD signal at 222nm is monitored as a function of temperature. Data are fit (-) to a two-state thermal unfolding model to yield an apparent Tm of 62.3°C. The protein concentration and scan rate are about lOuM and about l°C/min.

In another embodiment of the present disclosure, figure 4 shows SDS-PAGE analysis of proteolytic digests of (A) ODEc (at pH 8.0) by trypsin at about 37°C (B) reduced carboxymethylated RNaseA (rcam-RNaseA) (at pH 8.0) by trypsin at about 37°C. (C) ODEC (at pH 8.0) by proteinase K at about 37°C. (D) reduced carboxymethylated RNaseA (rcam-RNaseA) (at pH 8.0) by proteinase K at about 37°C. Lanes 1 to 6 (A and C) indicate aliquots of the ODEc digestion mixture at time zero (undigested), at 5 mins, 15 mins, 25 mins, 40 mins and 1 hour for trypsin and 5 mins, 10 mins, 25 mins, 40 mins and 1 hour for proteinase K while lanes 1 and 2 (B and D) indicate those of rcam-RNaseA digestion mixture are at time zero (undigested) and at 5 minutes respectively. Samples are mixed with formic acid at a final concentration of 0.1% to deactivate trypsin and 5mM PMSF to deactivate proteinase K at the indicated times to stop the proteolysis and SDS-PAGE gel loading buffer is added. Following electrophoresis, proteins are visualized by staining with Coomassie blue. (E) SDS-PAGE analysis of ODEC and WT ODEC before and after refolding into PBS with ImM EDTA. The arrow indicates the expected position for ODE.C Lanes 1 and 4 indicate the denatured WT ODEC and ODEC respectively in 8M. GdnCl before refolding, while lanes 2, 5 are the precipitate and lanes 3, 6 are the soluble fraction for WT ODEC and ODEC respectively obtained after refolding. The denatured protein in 8M GdnCl is precipitated with TCA before loading on the SDS-PAGE.

Further, figure 5 shows (A) Analytical gel-filtration analysis of ODEC on a Superdex 75 column in PBS buffer at room temperature. For comparison, an equal amount of Chymotrypsinogen protein, having almost the same mass (25kDa) is separately loaded onto the column. The absorbance at 220nm is shown as a function of the elution volume. The plot shows that ODEC elutes at the expected position for the monomer. (B) Calibration curve of the analytical Superdex-75 column with standard marker proteins albumin (67kDa), ovalbumin (43kDa), chymotrypsinogen (24kDa) and RNaseA (13kDa). (C) Reverse-phase HPLC for ODEC in an analytical C18 column (Vydac) at room temperature. About 25ug oxidized protein (dashed line) is injected in PBS, pH 7.4, while for reduction, about 12ug protein (solid line) is incubated with 6M GdnCl and ImM DTT at about 37°C, prior to loading. Proteins are eluted in a gradient of acetonitrile from 5 to 95% at a rate of 2% per minute. The intensity at 220nm is shown as a function of the elution time. The RP-HPLC shows that oxidized ODEC elutes as a single peak and therefore unlikely to be a mixture of disulfide bonded isomers.

EXAMPLE 3: BINDING STUDIES ON BIACORE

ODEC bound soluble 4-domain CD4 with a KD of 3.3uM (Fig6A), while the positive control full-length gpl20 has a KD of 1 InM (Fig6B) and loop deleted core gpl20 has a KD of 50nM (26). Although ODEC binds CD4 with -66 fold lower affinity relative to core gpl20, the fact that there is measurable binding with micromolar KD is encouraging. There are various reasons as to why ODEC shows poorer binding than full-length gpl20. Firstly, though our biophysical studies show that it is folded, it might not have adopted exactly the same structure as in the context of the whole molecule. In addition, ODEC lacks variable loops (V1V2 and V3) and the loops also contribute to binding affinity of gpl20 for CD4.

Binding of ODEC to three CD4 binding site antibodies, bl2, F105 and b6 is also examined (Fig 7 and Fig 8). It bound the broadly neutralizing antibody bl2 with a KD of 12uM at 25°C (Fig 7). The high KD is primarily due to the high koff for the binding. On lowering the temperature to 15°C, there is a 10-fold decrease in koff and the KD is close to that obtained for CD4 binding at 25°C. A comparison of all the kinetic parameters for binding is listed in Table I.

Most importantly, ODEC did not bind the CD4 binding site directed non-neutralizing antibodies b6 and F105 to any appreciable degree. In contrast, monomeric gpl20 bound both neutralizing and non-neutralizing antibodies with nanomolar values of KD (Fig 8). In order to examine why ODEC does not bind F105 and b6, but binds CD4 and bl2, the structures of gpl20 in complex with each of CD4, bl2 and F105 ligands are analyzed (Fig 1A,B,C). 28 out of the 31 residues important for CD4 binding are included in ODEC, hence CD4 binding is not surprising. All residues required for bl2 binding are present in ODEC, thus bl2 binding is also expected. However in the recently solved gpl20- F105 structure, it is seen that F105 binds in a hydrophobic groove in between the inner and the outer domain, that is lined by residues W112,1109, V255, M475,1371, F382, G473, G366, G367 and P369. Out of these residues, W112, 1109 and M475 are absent in ODEC, and V255 and F382 are mutated to threonine and arginine based on ROSETTA DESIGN calculations. Thus, with 5 out of the 10 residues important for binding being absent it is clear why ODEC does not bind F105. It is worth mentioning at this point that OD1 designed previously, also did not bind F105 and b6. As the crystal structure coordinates of gpl20 with antibody b6 are not available in the PDB, a similar analysis to explain why ODEC did not show b6 binding could not be done.

In an embodiment of the present disclosure, figure 6 shows Biacore sensorgram overlays. (A) Binding of different concentrations of full-length gpl20 to surface immobilized 4-domain CD4. Curves 1, 2, 3 and 4 respectively indicate 75nM, 50nM, 25nM and 12.5nM concentrations of gpl20 (B) Binding of different concentrations of ODEC to surface immobilized 4-domain CD4. Curves 1, 2, 3 and 4 respectively indicate 9.6uM, 7.2uM, 4.8uM and 2.4uM concentrations of ODEC. Surface density 900 RU; buffer PBS (pH 7.4)-0.01% P20; flow rate, 30uL/min. ODEC binds sCD4 -300 fold weaker as compared to full-length gpl20 and -66 fold weaker, relative to core gpl20.

In another embodiment of the present disclosure, figure 7 shows Biacore sensorgram overlays for the binding of different concentrations of ODEC to surface immobilized IgGbl2 at (A) 25°C and (B) 15°C. Surface density 900 RU; buffer PBS (pH 7.4)-0.01% P20; flow rate, 30uL/min. In both the plots, curves 1,2 and 3 respectively indicate 9.6uM, 7.2uM and 2.4uM concentrations of ODEC. ODEC shows binding to the neutralizing antibody bl2, but shows a high koff at 25°C. At a lower temperature (15°C), the koff decreases 10-fold, thereby decreasing the KD.

Further, figure 8 shows Biacore sensorgram overlays for the binding of lOOnM full-length gpl20 (curve 1) and 8uM ODEC (curve 2) to surface immobilized (A) mAb IgGb6 and (B) mAb F105. Surface density for both the antibodies is about 900 RU; buffer PBS (pH 7.4)-0.01% P20; flow rate, 30uL/min. ODEC does not bind the non-neutralizing antibodies b6 and F105, while for full-length gpl20, there is substantial binding.

EXAMPLE 4; PROTEOLYSIS

Proteolytic digestion of ODEC and reduced carboxymethylated RNaseA (rcam-RNaseA) is carried out using a protease/substrate ratio of 1:1000 (w/w) for trypsin and 1:50 (w/w) for proteinase K. A total of 100 ug of protein is digested in 200 uL of digestion buffer (final concentration 50mM HEPES, pH 8.0, 2mM CaC12) at 37°C. At various times, 40 uL of sample is removed and trypsin is deactivated with 0.1% formic acid while proteinase K is deactivated by 5mM PMSF. SDS-PAGE gel loading dye (final concentration 50 mM Tris-HCl at pH 6.8 containing 2.0% SDS, 0.1% bromophenol blue, and 5% 0- mercaptoethanol) is added, samples are boiled for 10 min and stored at -20°C till use. Samples collected at different time points are subjected together to SDS-PAGE on a 12% gel followed by staining with Coomassie Brilliant Blue R250.

EXAMPLE 5: TCA PRECIPITATION

About l00ul of denatured protein in about 8M GdnCl is diluted 12 times with PBS, about pH 7.4. It is chilled on ice and then about 300ul of trichloro acetic acid (TCA) is added to bring it to a final concentration of 20% (v/v). It is incubated on ice for about 10 minutes and then spun down at about 14,000 rpm for about 5 minutes. The protein pellet obtained is washed 4-6 times with about 200ul ice-cold acetone and dried at about 95°C for about 5-10 minutes to remove the acetone. To the dried pellet, about 20ul of IX Tris-Glycine electrophoresis buffer (pH 8.3) is added followed by about 5l of 5X loading dye, boiled for about 10 minutes and then analyzed by SDS-PAGE.

EXAMPLE 6: GEL FILTRATION ANALYSIS

Gel filtration or size exclusion chromatography is performed to find out the oligomeric status of the protein. Approximately 20ug of protein is analyzed under non-denaturing conditions by gel filtration chromatography in PBS buffer at room temperature on a Superdex-75 analytical gel filtration column. Chymotrypsinogen marker (25kDa) having almost the same mass as ODEc (24kDa) is used to determine the expected position of the monomeric peak. From the above gel filtration analysis, it is inferred that ODEC is monomeric and not aggregated, and elutes at the same volume as a standard protein of equivalent molecular weight.

EXAMPLE 7: HPLC

Reverse-phase HPLC is done to find out whether the disulfides in ODEC were formed or not. About 25 ug of protein in PBS about pH 7.4 buffer is injected into a C18 analytical column (150x 4.6mm, 5um particle size) and eluted with a gradient of 5 to 95% acetonitrile containing about 0.1% trifluoroacetic acid (TFA). For the reduced sample, about 12ug of protein is incubated with about 6M GdnCl, about 5mM DTT at about 37°C prior to injection. The reduced protein eluted later than the oxidized protein, showing that the formation of disulfides had imparted a kind of compactness to the protein.

EXAMPLE 8: SURFACE PLASMON RESONANCE (SPR) EXPERIMENTS

All SPR experiments are performed with a Biacore 2000 optical biosensor at 25°C. 900 resonance units (RU) 4-domain CD4, MAb bl2, b6 or F105 is attached by standard
amine coupling to the surface of a research-grade CM5 chip. A sensor surface (without CD4 or any antibody) that has been activated and deactivated served as a negative control for each binding interaction. Four different concentrations of WT gpl20 or ODEC are run across each sensor surface in a running buffer of PBS, pH 7.4 containing 0.01% P20 surfactant. Protein concentrations ranged from 12.5nM to 75nM for gpl20 and from 2 to 10 uM for ODEc- Both binding and dissociation are measured at a flow rate of about 30 u.l/min. In all cases, the sensor surface is regenerated between binding reactions by one to two washes with about l0mM HC1 for about 30 seconds at about 30 ul/min for bl2 and b6 and with about l0mM NaOH for about 30 seconds at about 30ul/min for CD4 and F105. Each binding curve is corrected for nonspecific binding by subtraction of the signal obtained from the negative-control flow cell. The kinetic parameters are obtained by fitting the data to the simple 1:1 Langmuir interaction model by using BIA EVALUATION 3.1 software.

EXAMPLE 9: IMMUNIZATION STUDIES

Four New Zealand White female rabbits are injected subcutaneously with about l00ug of protein in phosphate buffer and are boosted with about 50 ug of protein 6 and 12 weeks after the initial immunization. Sera are collected 2 weeks after each injection. The animals are rested for 43 weeks after that. At week 55, they are boosted once more with about 50ug protein and terminal bleed is collected at week 57. Two different adjuvants are used, Freund's and an alternate mycobacterium based adjuvant Mycobacterium w (Mw). Freund's complete adjuvant is used for priming while for subsequent boosts, Freund's incomplete adjuvant is used. For the group immunized using Mw adjuvant, the same adjuvant is used throughout for priming and boosters. Mw is a heat-killed preparation of a non-pathogenic mycobacterium commercially available for immunization. For each adjuvant, two rabbits are used due to shortage of available animals. A similar study with four rabbits, two in each group for each adjuvant, is done using wild-type full-length JRCSF gpl20 as the immunogen. However one rabbit in each group for the gpl20 immunogen died after the first boost. Determination of titers of antibody against gpl20 and ODEc are carried out as follows: Enzymelinked immunosorbent assays (ELISA) against native gpl20 is performed in 96-well plates to which D7324 (2u.g/ml) had been adsorbed after overnight incubation at about 4°C in PBS. D7324 is a sheep antibody against the carboxy-terminal 15 amino acids of gpl20 from the BH-10 strain of HIV-1. Plates are washed three times with PBS containing about 0.05% Tween20 (PBST) and blocked with about 200ul of about 3% BSA in PBST.

After washing, about lOOul of JRCSF gpl20 (2 u.g/ml) is captured over the plate by incubating for 2 h. Alternatively, for determining the ODEc titer, plates are directly coated with 500ng of the native protein and then blocked with 3% BSA in PBST. Serial dilutions of serum in a total volume of about 100 l are added in separate wells and incubated for 2 h at room temperature followed by 6 washes with PBS containing about 0.05% Tween 20 at room temperature. For the negative control, the same serum dilutions are added to wells which did not have any gpl20 or ODEC immobilized but are blocked with about 3% BSA in PBST. Bound sera are detected by using an alkaline phosphatase-conjugated goat antirabbit antibody at a dilution of 1:10,000 and the chromogenic substrate pNpp (p- Nitrophenyl phosphate). The reciprocal of the serum dilution showing an optical density reading more than twice that of the negative control and greater than 0.1 is taken as the antibody titer.

EXAMPLE 10: DEPLETION AND PURIFICATION OF IGG FROM SERUM

The serum is incubated with Protein A-agarose beads for about 2 hours at room temperature. The flow-through is collected. It is tested for gpl20 and ODEC binding in ELISA to confirm depletion of all antibodies.

The protein A beads obtained above are washed with two column volumes of PBS, and bound IgG are eluted with about lOmM glycine-HCl, about pH 3 and immediately neutralized with about 1M phosphate buffer, about pH 7. The eluates are then dialyzed against PBS, about pH 7.4, concentrated 10 fold in a Centriprep concentrator and stored at -70°C in aliquots. IgG concentration is estimated using SDS-PAGE by comparison of the band intensity to that of an IgG standard of known concentration. For estimating concentration of ODEC, a standard of similar molecular weight as ODEC is used and the band intensity on SDS-PAGE is compared. Using this concentration and the CD (Circular Dichroism) signal of the protein, the Mean Residue Ellipticity (MRE) value for ODEc is calculated. For all subsequent protein preparations, the concentration is determined using this MRE value and the CD signal obtained at that time.

EXAMPLE 11: DETERMINATION OF CONCENTRATION OF ODFr SPECIFIC IGG IN THE SERUM

About 300ug of purified ODEc in the presence of about ImM EDTA is bound to about lOOul of Ni-NTA beads that are previously equilibrated with PBS, pH 7.4. These beads are incubated with about 50ug of purified IgG at about 4°C overnight. The beads are then washed, boiled with SDS-PAGE gel-loading buffer (final concentration about 50 mM Tris-HCl at about pH 6.8 containing about 2.0% SDS, about 0.1% Bromophenol Blue, about 10% glycerol) and loaded on about 10% gel. The amount of IgG eluted from the beads is quantitated by comparison of the band intensity to that of an IgG standard of known concentration. A negative control experiment is performed in which the same amount of antibodies is incubated with l00ul of Ni-NTA beads that has bound an irrelevant his-tagged protein in the presence of ImM EDTA. These beads are also subjected to SDS-PAGE after washing with PBS. From the difference in the amount of eluted antibodies, the specific IgG concentration in serum is calculated.

EXAMPLE 12: NEUTRALIZATION ASSAYS

The assay used measures neutralization of HIV as a function of reductions in Tat-regulated luciferase reporter gene expression after a single round of infection in TZM-bl cells. Briefly, NL4-3 and 93IN101 viral stocks are made by transfecting HEK 293T cells with plasmid DNA. JRFL is produced in PBMCs by infecting them with pre-existing viral supernatant. Pseudotyped viruses SC422661.8, TRO.ll, REJ04541.67, ZM109F.PB4 (from the standard reference panel of subtype B and C env clones) are made in HEK293 cells using a two plasmid system, a backbone plasmid pSG3Aenv that expresses the e ntire HIV-1 genome except env along with an envelope expression vector. The Env-expression vectors are obtained from the NIH AIDS Research and Reference Reagent Program. The env expression vectors are cotransfected with the backbone plasmid at a ratio of 1:2 in HEK293 cells by calcium phosphate method. Spent medium is collected about 48 h post transfection, TCID titered and stored in aliquots at about -80°C. The sera are heat-inactivated at about 56°C for about lh, spun briefly and stored as aliquots at about -80°C. The virus is added to the corresponding antibody/sera dilutions to get a final virus concentration of 200 TCID50U and incubated at about 37°C for about 1 h. All dilutions are performed in complete media. The lowest dilution tested is 1:10 and this is followed by sequential four-fold dilutions. Following this, the Ab/sera-virus mixture is added to lx 104 cells/well of the 96- well plate and incubated at about 37°C in a C02 incubator for about 48 h. The cells are washed in IX PBS, lysed and RLU/s measured in the luminometer following addition of luciferin substrate.

Immunization and Neutralization studies

Both ODEC and gpl20 are used as immunogens in rabbit immunization studies. The amount of protein used for immunization is about 100 ug for priming and about 50 ug for boost. 1 volume of protein in PBS was mixed with 1 volume of Fruend's adjuvant and the emulsion is made. Mw is also added in the same ratio but since it is non-oil based adjuvant, so no emulsion is formed. After the second booster immunization, rabbits immunized with full-length JRCSF gpl20 showed titers about 8x105 with Freund's adjuvant (rabbit 454) and about 6.4 xl06 with the Mw adjuvant (rabbit 457). In comparison, with the ODEc immunogen, after the second boost, ELISA titers are close to 2.5x106 with Freund's adjuvant (rabbits 465, 466) and about 2.5x105 with the Mw adjuvant (rabbits 467, 468). However, with both the adjuvants, for rabbits immunized with ODEc, the anti-gpl20 titers are about 5000 only. This may be due to either glycosylation of the gpl20 surface or differences in conformation of ODEC and gpl20. Both adjuvants yielded sera with very similar neutralization titers.

For the in vitro neutralization experiments, five subtype B viruses (NL4-3, JRFL, SC422661.8, TRO.ll, REJ04541.67) and two subtype C viruses (93IN101 and ZM109F.PB4) are used. Of the subtype B viruses, NL4-3 is a T-cell line adapted CXCR4 tropic virus and is classified as Tier-I. JRFL, SC422661.8, TRO.ll and REJ04541.67 are classified as Tier-II viruses. ZM109F.PB4 is a Tier-I subtype C virus while 93FN101 is a primary Indian subtype C virus. Neutralization data are summarized in Table II.

Table II: Neutralization titers (IC50) for ODEC and gpl20 immune sera a Rabbits 465-468 were immunized with ODEc and 454,457 were immunized with gpl20.

Neutralization assays were carried out using terminal bleed sera and env pseudotyped virus c Neutralization assays were carried out with post-dose 2 sera against infectious virus generated from full-length molecular clones of each isolate. Pre-immune sera as well as sera from rabbits immunized with an irrelevant bacterial antigen (CcdB) also showed no neutralization.

The ODEC anti-sera neutralized four of the five subtype B and one of the two subtype C viruses tested, though with modest neutralization titers. In contrast, the gpl20 anti-sera did not significantly neutralize any of the viruses tested. The lack of neutralization is seen with gpl20. It has been shown that immunization with gpl20 protein alone do not result in neutralization of homologous neutralization resistant strains such as JRFL, though DNA prime-protein boost immunization results in low but measurable homologous neutralization even for JRFL.

To demonstrate that ODEc directed antibodies are responsible for the observed neutralization, negative control experiments are done with preimmune sera, serum depleted of antibodies and with non-specific sera (obtained after immunizing rabbits with an unrelated bacterial protein CcdB). None of the negative control sera showed neutralization at dilutions of 1:10 or higher. As an additional negative control, the ability of the ODEC anti-sera to neutralize NL4-3 virus pseudo typed with VSV-G surface glycoprotein is examined. No neutralization is observed. As it is not possible to test neutralization below 1:10 dilutions of the serum, the whole IgG from the serum of ODEc immunized rabbit #466 is purified and the purified total IgG is used for neutralization of NL4-3 (positive control), as well as JRFL and 93IN101 viruses. The IC50s obtained with the purified IgGs are about 266ug/ml, about 694ug/ml and about 922ug/ml for NL4-3, JRFL and 93IN101 respectively. In order to interpret this result, it is useful to determine the approximate percentage of ODEC specific IgG in the total purified IgG fraction. To accomplish this, ODEC is bound to Ni-NTA beads and incubated with a known amount of IgG. From the amount of antibodies that bound to the ODEC, the amount of ODEC specific IgG in the serum is calculated and is found to be -9%. Thus about lmg/ml of the whole IgG would correspond to about 90ug/ml of the ODEC specific IgG. All these data suggest that the ODEC immunogen is able to generate moderate levels of neutralization against primary HIV-1 isolates from both B and C clades.

In an embodiment of the present disclosure, the development of an effective AIDS vaccine requires an immunogen that would present the conserved epitopes of the envelope glycoprotein in the correct conformation. One of the most conserved regions is the receptor-binding site, more commonly known as the CD4 binding site. Various monoclonal antibodies towards this site are known. The majority of these antibodies such as b6 and F105 are non-neutralizing. IgGbl2 is currently the best characterized broadly neutralizing monoclonal antibody against this site. These fractions of CD4 binding site antibodies are able to neutralize viruses that are partially or fully resistant to bl2. These data emphasize the importance of CD4 binding site directed antibodies in virus neutralization.

In an embodiment of the present disclosure, the bacterial construct described in this work could be refolded easily to a monomer from inclusion bodies. This is result of the 11 designed mutations in the interface of the inner domain and the outer domain that are intended to prevent aggregation. A control construct lacking these mutations is highly aggregation prone (Figure 4E). Not only did ODEC bind CD4, it also bound the neutralizing antibody bl2 and did not bind the non-neutralizing antibodies b6 and F105. Since the protein is expressed in E.coli, it is unglycosylated. Numerous previous studies have suggested that proper glycosylation is essential for gpl20 folding and viral infectivity. However the present study demonstrates that the outer domain of gpl20 (lacking V1V2 and V3 variable loops) can be engineered to fold in the absence of glycosylation. Even though the protein lacked the immunodominant V1V2 and V3 loops, it proved to be highly immunogenic in rabbits, with the ODEc titers close to 2.5x106. The lack of glycosylation might be responsible for the enhanced immunogenicity. Though the gpl20 titers are low, significantly better neutralization with a panel of viruses is obtained compared to gpl20 anti-sera. The fact that ODEc anti-sera could show measurable neutralization of multiple primary isolates including three bl2 resistant viruses (TRO.ll, ZM109F and 93IN101) is important.

The binding to bl2, has a high KD, because of a high off-rate. This study is an example of a designed gpl20 fragment based immunogen that yields antibodies targeting the CD4 binding site that can neutralize subtype B and C viruses. It is also a validation of fragment based immunogen design strategies.

We Claim:

1. Nucleotide sequence set forth as Seq. Id No. 1.

2. Peptide sequence set forth as Seq. Id No. 2, wherein the peptide sequence is corresponding to nucleotide sequence set forth as Seq. Id No. 1.

3. A method of obtaining peptide sequence set forth as Seq. Id No. 2, said method comprising acts of:

c.) identifying CD4 binding regions in gpl20 of HIV virus; and d.) introducing mutations outside the binding regions to obtain the peptide sequence set forth as Seq. Id No. 2.

4. The method as claimed in claim 3, wherein the peptide sequence set forth as Seq. Id No. 2 corresponds to mutation in peptide sequence set forth as Seq. Id No. 3, at amino acid positions selected from group comprising 255, 260, 261, 271, 275, 285, 382, 434, 435 and 453 or any combinations thereof.

5. The method as claimed in claim 4, wherein the mutation is selected from group comprising point mutation, insertion, deletion, substitution and frame shift mutation of hydrophobic amino acid residues with hydrophilic amino acid residues or any combination thereof.

6. Vector having accession number MTCC 5658, said vector comprising nucleotide sequence set forth as Seq. Id No.l.

7. Recombinant cell, comprising the vector as claimed in claim 6.

8. A method of obtaining the recombinant cell as claimed in claim 8, said method comprising acts of:

a. obtaining vector comprising nucleotide sequence set forth as Seq. Id
No. 1; and

b. transforming a host cell with said vector to obtain the recombinant cell.

9. A method of obtaining protein comprising peptide sequence set forth as Seq.
Id No. 2, said method comprising acts of:

d.) inserting nucleotide sequence set forth as Seq. Id No. 1 into a vector and transforming a host cell with the vector to obtain a recombinant cell;

e.) expressing the vector within the cell for obtaining the protein comprising peptide sequence set forth as Seq. Id No. 2; and

f.) optionally, purifying the protein of step b).

10. Immunogen comprising protein having peptide sequence set forth as Seq. Id No. 2, optionally along with adjuvant or pharmaceutically acceptable additive(s) or a combination thereof.

11. A method of obtaining immunogen comprising protein having peptide sequence set forth as Seq. Id No. 2, optionally along with adjuvant or pharmaceutically acceptable additive(s) or a combination thereof, said method comprising acts of:

a. inserting nucleotide sequence set forth as Seq. Id No. 1 into a vector and transforming a host cell with the vector to obtain a recombinant cell;

b. expressing the vector within the cell for obtaining and purifying the protein, to obtain the immunogen; and

c. optionally, adding adjuvant or pharmaceutically acceptable additive(s) or a combination thereof, to the immunogen of step b).

12. The vector as claimed in claim 6, the recombinant cell as claimed in claim 7, and the methods as claimed in claims 8, 9 and 11, wherein the vector is selected from group comprising bacterial expression vectors, yeast expression vectors and animal cell expression vector, preferably selected from group comprising pET15b, pET20b(+), pCOMB-3X and pcDNA3.1(+); and the host cell is selected from group comprising bacteria, preferably selected from E.coli BL21(DE3) or E.coli TOP 10, yeast, preferably S.cerevisiae or Pichia pastoris, animal cell, preferably HEK293 or Drosophila.

13. The immunogen as claimed in claim 10, and the method as claimed in claim 11, wherein the adjuvant is selected from group comprising CpG7909, IMX, MAA, Freund's adjuvant, Mycobacterium w (Mw), alum and Adjuplex LAP or any combination thereof; and the pharmaceutically acceptable additive is selected from group comprising excipients, gums, sweeteners, coatings, binders, disintegrants, lubricants, disintegration agents, suspending agents, granulating agents, solvents, colorants, glidants, anti-adherents, anti-static agents, surfactants, plasticizers, emulsifying agents, flavoring agents, viscosity enhancers and antioxidants, or any combination thereof.

14. A kit having component selected from group comprising nucleotide sequence set forth as Seq. Id No. 1, peptide sequence set forth as Seq. Id No. 2, the vector as claimed in claim 6, the recombinant cell as claimed in claim 7, immunogen as claimed in claim 10, adjuvant and pharmaceutically acceptable additive(s) or any combination thereof along with instructions manual.