US20090203884A1

US20090203884A1 - Chimeric hiv fusion proteins as vaccines

Info

Publication number: US20090203884A1
Application number: US12/358,659
Authority: US
Inventors: Chao-Wei Liao; Hsiu-Kang Chang
Original assignee: HealthBanks Biotech Co Ltd; HealthBanks USA Inc
Current assignee: HealthBanks Biotech Co Ltd; HealthBanks USA Inc
Priority date: 2008-01-31
Filing date: 2009-01-23
Publication date: 2009-08-13
Also published as: EP2237794B1; EP2237794A2; US8372407B2; CN101969975A; EP2237794A4; WO2009099777A2; WO2009099777A3; JP2011510988A; JP5502757B2; US20120213811A1; CN101969975B

Abstract

A chimeric fusion protein useful as an immunogen for inducing HIV antigen-specific immune responses contains a first polypeptidyl region and a second polypeptidyl region. The first polypeptidyl region includes a Pseudomonas Exotoxin A (PE) binding domain and a PE translocation domain. The second polypeptidyl region includes (i) a first peptidyl segment containing a fragment of gp120 C1 domain, located at the N-terminus of the second peptidyl region; (ii) a second peptidyl segment containing a fragment of gp120 C5 domain, located at the C-terminus of the first peptidyl segment; and (iii) a third peptidyl segment containing a fragment of gp41, located at the C-terminus of the second peptidyl segment. The second polypeptidyl region contains an antigenic determinant specific to one subtype of HIV. An intermediate polypeptide containing a non-Env, HIV antigenic determinant selected from Gag24, Nef, Tat and Rev may be included.

Description

FIELD OF THE INVENTION

The present invention relates generally to HIV vaccines, and more specifically to chimeric HIV fusion proteins useful for inducing humoral and cell-mediated immune responses.

BACKGROUND OF THE INVENTION

The global epidemic of AIDS has created an urgent need for a vaccine against human immunodeficiency virus type 1 (HIV-1). It is likely that effective AIDS vaccines will need to generate efficient humoral and cellular immune responses. Virus-neutralizing antibodies and anti-HIV cytotoxic (CD8+) T lymphocytes (CTLs) mediated immunity are major requirements for protective immune responses elicited by HIV vaccines.
HIV has several major genes coding for viral proteins. The gag gene codes for p24, the viral capsid; p6 and p7, the nucleocapsid proteins; and p17, a matrix protein. The pol gene codes for reverse transcriptase, integrase, and protease which cleaves the proteins derived from gag and pol into functional proteins. The env gene codes for the precursor to gp120 and gp41, envelope proteins embedded in the viral envelope that enable the virus to attach to and fuse with target cells. The tat, rev, nef, vif, vpu genes each codes for a single protein with the same names, Tat, Rev, Nef, Vif, Vpr, Vpu, respectively.
Neutralizing antibodies have been shown to contribute to protection from virus infection in animal models of HIV-1 infection. The virus-specific targets on HIV-1 accessible to neutralizing antibodies are the envelope glycoproteins (Yang, X. et al. (2005) “Stoichiometry of Antibody Neutralization of Human Immunodeficiency Virus Type 1” Journal of Virology 79: 3500-3508). During the normal course of HIV-1 infections, virus-neutralizing antibodies are often generated but the titer of neutralizing is often low. Most neutralizing antibodies bind the gp120 envelope glycoprotein, which is the major exposed protein of the viral envelope glycoprotein trimer. The more conserved receptor-binding surfaces of the HIV-1 gp120 glycoprotein are also the targets for neutralizing antibodies. The CD4-binding site (CD4BS) antibodies recognize a conformational epitope composed of several segments of gp120 region that overlaps the binding site for CD4. CD4-induced (CD4i) antibodies bind a highly conserved gp120 element that is critical for the gp120-clemokine receptor interaction. The ability of CDBS and CD4i antibodies to interfere with receptor binding contributes to their neutralizing capability.
GP120 contains ten domains: conserved domains 1-5 (C1-C5) and variable domains 1-5 (V1-V5). The C1 and C5 domains are located at N- and C-terminals of gp120, respectively. Antibodies directed against the V3 loop, which determines chemokine receptor choice, can block the binding of gp120 to the receptors CCR5 and/or CXCR4. Neutralization by anti-V3 antibodies, although potent, is often limited to a small number of HIV-1 strains.
Gp120 is non-covalently associated with gp41. The gp41 subunit is anchored in the membrane and has a non-polar fusion peptide at its N-terminus. The gp120-gp41 complex forms oligomers on the surface of infected cells and on virions. The binding of gp120 to CD4 is thought to result in activation of the membrane fusion activity of gp41, leading to entry of the viral nucleocapsid into a cell. Antibodies to gp41 epitopes in the serum of HIV-infected individuals may play an important role in virus neutralization. Gp120-41 complex sequences of different HIV subtypes show a remarkably conserved N-terminal coiled-coil structures of gp41 as well as the C-terminal residues that interact with the N-terminal core structure of gp120.
Multiple immune effectors participate in prevention, containment and clearance of HIV infection. To prevent infection of host target cells, antibodies are required. After the first target cells have been infected with virus, it is important to have cytotoxic T lymphocytes (CTLs) as well as antibodies to reduce cell-to-cell spread and kill infected cells. An effective HIV vaccine should evoke antibodies that can bind to virus and prevent attachment of virus to target cells, as well as CTLs that can eliminate any cells that become infected.
It remains a difficult goal for vaccinologists to construct live-attenuated viruses that are both effective and safe, or to mimic the presentation of viral proteins observed in infection with recombinant antigens or with replicating or non-replicating vectors carrying appropriate genes or antigens. The large number of mutations in the V3 domain of gp120 has limited its usefulness as a target for HIV vaccine. It is still unclear how the trend of hypervariability in the variable domains is developing and how many domains are absolutely invariant in the evolving strains of HIV.
A previously unaddressed need exists in the art to address the deficiencies and inadequacies in HIV vaccine antigen production, especially in connection with the provision of efficacious, antigenic determinant peptides.

SUMMARY OF THE INVENTION

The importance of interaction between gp120 and gp41 for determination of the neutralization phenotype has been studied. One aspect of the invention relates to a chimeric fusion protein useful as an immunogen for inducing HIV antigen-specific immune responses. The chimeric contains: (a) a first polypeptidyl region containing a Pseudomonas Exotoxin A (PE) binding domain and a PE translocation domain, located at the N-terminus of the fusion protein; and (b) a second polypeptidyl region located at the C-terminus of the fusion protein, including: (i) a first peptidyl segment containing a fragment of gp120 C1 domain, located at the N-terminus of the second peptidyl region; (ii) a second peptidyl segment containing a fragment of gp120 C5 domain, located at the C-terminus of the first peptidyl segment; and (iii) a third peptidyl segment containing a fragment of gp41 amino acid sequence, located at the C-terminus of the second peptidyl segment, wherein the second polypeptidyl region contains an antigenic determinant which is specific to one subtype of HIV. The one subtype of HIV is at least one selected from the group consisting of HIV subtypes A, B, C, D, E, F, G, H, J and K.
In one embodiment of the invention, the fusion protein further includes an endoplasmic reticulum retention sequence, e.g., the amino acid sequence KDEL, at the C-terminus. In another embodiment of the invention, the chimeric fusion protein further includes an intermediate polypeptidyl region between the first and the second polypeptidyl regions, in which the intermediate polypeptidyl region contains a non-Env, HIV antigenic determinant. In one embodiment of the invention, the intermediate polypeptidyl region is at least one selected from the group consisting of Gag24, Nef, Tat and Rev. In another embodiment of the invention, the intermediate polypeptidyl region includes Gag24 amino acid sequence or a fragment thereof. Further in another embodiment of the invention, the intermediate polypeptidyl region contains an N- or C-terminus of Gag24 amino acid sequence. In one embodiment of the invention, the intermediate polypeptidyl region contains the amino acid sequence set forth by SEQ ID NO: 151.
Another aspect of the invention relates to a chimeric HIV fusion protein useful as an immunogen for inducing HIV antigen-specific immune responses, which includes: (a) a first polypeptidyl region containing a PE binding domain and a PE translocation domain, located at the N-terminus of the fusion protein; and (b) a second polypeptidyl region containing an HIV protein or a fragment thereof, located at the C-terminus of the fusion protein, wherein the second polypeptidyl region contains an antigenic determinant which is specific to one subtype of HIV.
In one embodiment of the invention, the second polypeptidyl region contains a fragment of HIV Env. In another embodiment of the invention, the second polypeptidyl region contains one or more than one fragment of gp120 V3 domain. In another embodiment of the invention, the second polypeptidyl region contains the amino acid sequence set forth by SEQ ID NO: 6. In one embodiment of the invention, the second polypeptidyl region comprises an HIV protein or a fragment thereof, which is selected from the group consisting of Gag24, Nef, Tat and Rev.
Further in another embodiment of the invention, the second polypeptidyl region contains HIV Gag24 amino acid sequence or a fragment thereof. Further in another embodiment of the invention, the second polypeptidyl region is a chimeric protein that includes: (i) a first peptidyl segment containing a fragment of gp120 C1 domain, located at the N-terminus of the second peptidyl region; (ii) a second peptidyl segment containing a fragment of gp120 C5 domain, located at the C-terminus of the first peptidyl segment; and (iii) a third peptidyl segment containing a fragment of gp41 amino acid sequence, located at the C-terminus of the second peptidyl segment. Yet in another embodiment of the invention, the second polypeptidyl region comprises the amino acid sequence set forth by SEQ ID NO: 7.
Yet another aspect of the invention relates to a method of inducing an HIV-antigen specific immune response. The method includes the step of administering an effective amount of the chimeric fusion protein as described above in a biocompatible carrier fluid suitable for carrying and delivering a predetermined aliquot of the fusion protein to a pre-chosen site in a living subject.
These and other aspects will become apparent from the following description of the preferred embodiment taken in conjunction with the following drawings, although variations and modifications therein may be affected without departing from the spirit and scope of the novel concepts of the disclosure.
The accompanying drawings illustrate one or more embodiments of the invention and, together with the written description, serve to explain the principles of the invention. Wherever possible, the same reference numbers are used throughout the drawings to refer to the same or like elements of an embodiment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1F are the maps of plasmids encoding chimeric HIV fusion proteins.

FIG. 2A is a graph showing ELISA titers in the sera samples from animals immunized with various chimeric HIV envelope fusion proteins. Sera sample 1:2,500 dilution.

FIG. 2B is a graph showing ELISA titers in the sera samples from animals immunized with various chimeric HIV envelope fusion proteins. Sera sample 1:12,500 dilution.

FIG. 3 is a graph showing neutralizing antibodies against live viruses were induced in the mice immunized with HIV Env fusion protein vaccines.

FIG. 4 is a flow chart illustrating the construction of pPE(ΔIII)-HIV gag24-K3.

FIG. 5 is a flow chart illustrating the construction of pPE(ΔIII)-HIV gag24-gp120-41-K3.

FIG. 6 is a schematic drawing illustrating the fusion of an HIV antigenic determinant peptide to gp120 C1-C5-gp41 creates a chimeric that enhances the antigenic determinant peptide's cell-mediated immune response.

FIG. 7 is a flow chart illustrating the construction of pPE(ΔIII)-HIV nef-NC—K3.

DETAILED DESCRIPTION OF THE INVENTION

DEFINITIONS

The terms used in this specification generally have their ordinary meanings in the art, within the context of the invention, and in the specific context where each term is used. Certain terms that are used to describe the invention are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner regarding the description of the invention. For convenience, certain terms may be highlighted, for example using italics and/or quotation marks. The use of highlighting has no influence on the scope and meaning of a term; the scope and meaning of a term is the same, in the same context, whether or not it is highlighted. It will be appreciated that same thing can be said in more than one way. Consequently, alternative language and synonyms may be used for any one or more of the terms discussed herein, nor is any special significance to be placed upon whether or not a term is elaborated or discussed herein. Synonyms for certain terms are provided. A recital of one or more synonyms does not exclude the use of other synonyms. The use of examples anywhere in this specification including examples of any terms discussed herein is illustrative only, and in no way limits the scope and meaning of the invention or of any exemplified term. Likewise, the invention is not limited to various embodiments given in this specification.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the case of conflict, the present document, including definitions will control.
As used herein, “around”, “about” or “approximately” shall generally mean within 20 percent, preferably within 10 percent, and more preferably within 5 percent of a given value or range. Numerical quantities given herein are approximate, meaning that the term “around”, “about” or “approximately” can be inferred if not expressly stated.
As used herein, the term “carboxyl terminal moiety which permits retention of the fusion antigen to the endoplasmic reticulum (ER) membrane of a target cell” refers to a peptide fragment that enables the fusion antigen to bind to the ER membrane and to retain it in the ER lumen for glycosylation and make it appears to be more like foreign protein. In one embodiment of the invention, the carboxyl terminal moiety comprises, in a direction from the amino terminus to the carboxyl terminus, the following amino acid residues:
R¹—R²—R³—R⁴—(R⁵)_n
Wherein,
R¹is a positively charged amino acid residue;
R²is a negatively charged amino acid residue;
R³is a negatively charged amino acid residue;
R⁴is L;
R⁵is a positively charged amino acid residue; and
n is 0 or 1.
Preferably, the carboxyl terminal moiety is a member of the KDEL family protein. As used herein, the term “KDEL family protein” refers to a group of proteins, which has a similar carboxyl end binding to the ER membrane of a cell and further has an ability for retention of such protein in the ER lumen. Generally, the length of the carboxyl end ranges from 4 to 16 residues. As discussed in U.S. Pat. No. 5,705,163 (which is incorporated herein by reference in its entirety), the amino residues at the carboxyl end of a KDEL family protein, particularly those in the last five amino acids, are important. As shown in the studies on the similar sequences present in different molecules and performing a specific biological function, a sequence that retains a newly formed protein within the endoplasmic reticulum is Lys Asp Glu Leu (KDEL). These findings suggest that the sequence at the carboxyl end of the fusion antigen according to the invention acts as some type of recognition sequence to assist translocation of the fusion antigen from an endocytic compartment into the ER and retains it in the lumen. The carboxyl terminal moiety comprises the sequence of KDEL. For example, the carboxyl terminal moiety may comprise the sequence of KKDLRDELKDEL (SEQ ID NO: 250), KKDELRDELKDEL (SEQ ID NO: 251), KKDELRVELKDEL (SEQ ID NO: 252), or KKDELRXELKDEL, in which R is D or V.
The terms “PE(ΔIII)-HIV gp120” and “PE(ΔIII)-HIV gp120 V3-V3” are interchangeable.
The terms “PE(ΔIII)-HIV gp120-41” and “PE(ΔIII)-HIV gp120 C1-C5-gp41” are interchangeable.
The terms “HIV subtype A gp120 C1-C5-gp41” and “chimera A” are interchangeable; the terms “HIV subtype B gp120 C1-C5-gp41” and “chimera B” are interchangeable; the terms “HIV subtype C gp120 C1-C5-gp41” and “chimera C” are interchangeable, and so on.
Immunogens. To be an immunogen, the formulation need only be a mixture of a fusion protein construct as described herein and a biocompatible carrier fluid suitable for carrying and delivering a predetermined aliquot of the fusion protein construct to a prechosen site in the body of a living subject. Immunogens embodying the invention can be administered in any appropriate carrier for intradermal, subcutaneous, intramuscular, parenteral, intranasal, intravaginal, intrarectal, oral or intragastric administration. They can be introduced by any means that effect antigenicity in humans. The dosage administered will vary and be dependent upon the age, health, and weight of the recipient; the kind of concurrent treatment, if any; the frequency of treatment; and the nature of the humoral antibody response desired. If the immunogens are to be given intradermally, subcutaneously, intramuscularly, intravenously or parenterally, they will be prepared in sterile form; in multiple or single dose formats; and dispersed in a fluid carrier such as sterile physiological saline or 5% dextrose solutions commonly used with injectables. In addition, other methods of administration can be advantageously employed as well.
Vaccines. To be a prepared vaccine, the minimal formulation comprises a predetermined quantity of a fusion protein construct as described herein; a biocompatible carrier suitable for carrying and delivering a predetermined aliquot of a fusion protein construct to a prechosen site in the body of a living subject; and at least one adjuvant composition dispersed in the carrier fluid or coupled to the fusion protein constrict. The vaccine, by definition, incorporates an immunogen and includes one or more adjuvants to facilitate or stimulate the immune response and to prolong the antigenic effect in-vivo over time. Among the useful adjuvant substances conventionally known are those compositions approved by the FDA (currently or pending for systemic and/or mucosal immunizations). Some are preferred for mucosally-administered vaccines and others are preferred for intragastric administered vaccines.
Modes of administration. Multiple modes of inoculation, the manner of introducing an immunogen or vaccine, are conventionally known and used. The systemic or parenteral forms of administration (introduction by injection or perfusion) typically include intraperitoneal, intravenous, intramuscular, subcutaneous, and subdermal inoculations. In contrast, mucosal modes of administration may include not only the intranasal and intragastric forms of introduction, but also oral, intravaginal, and intrarectal introductions.

EXAMPLES

Without intent to limit the scope of the invention, exemplary instruments, apparatus, methods and their related results according to the embodiments of the present invention are given below. Note that titles or subtitles may be used in the examples for convenience of a reader, which in no way should limit the scope of the invention. Moreover, certain theories are proposed and disclosed herein; however, in no way they, whether they are right or wrong, should limit the scope of the invention so long as the invention is practiced according to the invention without regard for any particular theory or scheme of action.

1. HIV-1 gp120 and gp41 Fusion Proteins

Example 1

Selection of Truncated Segments From HIV Env Proteins, gp120 and gp41

The amino acid sequences of HIV gp120 and gp41 were retrieved from the National Center of Biotechnology Information (NCBI, USA) database and entered into software for evaluation of antigenic determinant (epitopes) of the target proteins, and candidate segments for synthesis displayed on an evaluation plot. Antigenic determinant regions of the target protein were chosen for synthesis by a reverse genetic engineering technique. Several peptide segments were selected as target peptides based on the results of the evaluation software. The software DNA strider v1.0 was used to analyze whether the nucleotide sequences of the target peptides contained restriction enzyme sites. If present in the DNA sequence in disadvantageous places, changes were made within the appropriate codons without altering the amino acid sequence. The software checked the newly created sequence, and designed restriction sites at both termini of the DNA sequence to facilitate cloning. Codons for some amino acid residues, such as Arg, Ile, Gln, Pro, were modified to increase the expression of proteins in E. coli expression systems. Table 1 lists the selected peptide segments and their corresponding amino acid sequences.

Example 2

Construction of Chimeric Target Polypeptides gp120 V3-V3 and gp120 C1-C5-gp41

Two target peptides gp120 V3-V3 and gp120 C1-C5-gp41 were constructed using the selected peptide segments as follows: Three truncated peptide segments having the amino acid sequences of SEQ ID NO. 1 (from gp120 C1 domain), SEQ ID NO. 2 (from gp120 C5 domain) and SEQ ID NO. 5 (from gp41 region associated with gp 120), respectively, were ligated to form a chimeric target peptide gp120 C1-C5-gp41 (referred to as gp120-41). Two truncated segments having amino acid sequences of SEQ ID NOs: 3 and 4 (both from gp120 V3 domains), respectively, were fused to form polypeptide gp120 V3-V3 (referred to as gp120). One or more residues might be inserted in-between to link two peptide segments. The number of residues inserted in-between was about 1 to 15 amino acids, which might be selected from amino acid residues that would not alter the secondary structure of proteins, such as glycine, alanine, valine, and leucine. The amino acid residue cysteine in SEQ ID NO: 2 and in SEQ ID NO: 5 could form a disulfide bond so that the chimeric target peptide generated could possess a three-dimensional structure.

TABLE 1

HIV Env	Target		SEQ
Proteins	peptide segments	Sequence of selected peptide segments	ID No.

gp120	C1 domain	VEKLWVTVYYGVPVWK		1
	C5 domain	KVVKIEPLGVAPTKCKRRVVQREKR	2
	V3 domain	CTRPSNNTRKGIHMGPGGAFYTTGQIIRNIRQAHC	3
	V3 domain	CTRPNNNTRRSIHIEPEGAFYTTGEIIGDIRQAHC	4

gp41	gp 41 region in	QARVIAVERYLKDQQLLGIWGGSGKLICCTTAV	5
	association with	PWNSSWSNKLDRIWNNMTWLE
	gp120

TABLE 2

Chimeric
target		SEQ
peptide	Amino acid sequence	ID No.

gp120	IG CTRPSNNTRKGIHMGPGGAFYTTGQIIRNIRQAHC GLLGGC	6
V3-V3	GLGLE

gp120	VEKLWVTVYYGVPVWKKVVKIEPLGVAPTKCKRRVVQREKRGGGGGQ	7
C1-C5-
gp41	LE

Table 2 lists the amino acid sequences of the chimeric target peptides gp120 V3-V3 and gp120 C1-C5-gp41. For chimeric peptide gp120 V3-V3 in table 2: the underlined letters denote the restriction sites; bold letters denote the first V3 domain segment, bold and italic letters denote the second V3 domain segment. For chimeric peptide gp120 C1-C5-gp41 in table 2: the bold letters denote the C1 domain segment; non-bold letters denote the C5 domain segment; non-bold and italic letters denote linkers; bold and italic letters denote the gp41 domain; and the underlined letters denote a restriction site.
The chimeric target peptide gp120 V3-V3 (SEQ ID NO: 6) was designed based on the construction of repeats in the V3 domain of gp120, and the chimera gp120 C1-C5-gp41 (SEQ ID NO: 7) was based on simulation of the gp41 region in association with gp120. The chimera gp120 V3-V3 (SEQ ID NO: 6) comprises amino acid sequences of SEQ ID NOs: 3 and 4 (both from gp120 V3 domains). The chimera gp120 C1-C5-gp41 comprises amino acid sequences of SEQ ID NOs: 1 (from gp120 C1 domain), 2 (from gp120 C5 domain), and 5 (gp41 region in junction with gp120) (Table 1). Disulfide bonds were formed due to cysteines in SEQ ID NO: 2 (from the C5 domain of gp120) and SEQ ID No. 5 (from gp41 region in junction with gp120).
Using the similar method as described above, the following chimeric target peptides were constructed: HIV subtype A gp120 C1-C5-gp41; HIV subtype B gp120 C1-C5-gp41; HIV subtype C gp120 C1-C5-gp41; HIV subtype D gp120 C1-C5-gp41; HIV subtype E gp120 C1-C5-gp41; HIV subtype F gp120 C1-C5-gp41; HIV subtype G gp120 C1-C5-gp41; HIV subtype H gp120 C1-C5-gp41; HIV subtype J gp120 C1-C5-gp41; and HIV subtype K gp120 C1-C5-gp41 (abbreviated as chimeric target peptides A, B, C, D, E, F, G, H, J, and K, respectively).
These chimeras A, B, C, D, E, F, G, H, J and K were each constructed from a combination of 3 peptide segments selected from each corresponding HIV-1 subtypes, A, B, C, D, F, G, H, J and K, respectively. The basic scheme of the construction was to link two peptide segments, each selected from the C1 and C5 domains of gp120, to another segment selected from the gp41 region in association with gp120 for each HIV-1 subtype. Thus, like the SEQ ID NO: 7, all these chimera target peptides have gp120-C1-C5-gp41-like structures.
Table 3 lists the amino acid sequences of these chimeras, in which the non-bold, italic letters denote a segment from the C1 domain, the bold letters denote a segment from the C5 domain, of HIV subtype A gp120 protein; the non-bold, non-italic letters GGGGG denote a linker, and the bold, italic letters denote a segment from HIV subtype A gp41 region in junction with gp120.

TABLE 3

Chimera
for HIV		SEQ
subtype	Amino Acid Sequence	ID NO.

A	AENLWVTVYYGVPIWKKVVKEI PLGVAPTKARRRVVEREKRGGGGG	31



B	TEKLWVTVYYGVPVWKKVVKEI PLGIAPTKAKRRVVQREKRGGGGG	32



C	MGNLWVTVYYGVPVWKKYKVVEIK PLGVAPTKPKRRVVEREKRGGGGG	33



D	ADNLWVTVYYGVPVWKKVVQIE PLGVAPTRAKRRVVEREKRGGGGG	34



E	SXNLWVTVYYGVPVWRKVVQIE PLGIAPTRPKRRVVEREKRGGGGG	35



F	ADNLWVTVYYGVPVWKKVVEIE PLGVAPTKAKRQVVQREKRGGGGG	36



G	ASNNLWVTVYYGVPVWEDAKKVVKIK PLGVAPTKARRRVVGREKRGGGGG	37



H	VVGNLWVTVYYGVPVWKKVVKIE PLGVAPTEARRRVVEREKRGGGGG	38



J	AKEDLWVTVYYGVPVWKKVVEIE PLGVAPTKAKRRVVEREKRGGGGG	39



K	IAANNLWVTVYYGVPVWKKVVQIE PLGIAPTRARRRVVQREKRGGGGG	40

Example 3

Synthesis of DNA Fragments Encoding Chimeric Target Polypeptides gp120 V3-V3 and gp120 C1-C5-gp41

The nucleotide sequences of tile DNA fragments encoding chimeric target peptides gp120 V3-V3 and gp120 C1-C5-gp41 were modified to increase translation efficiency without changing the amino acid sequences of the encoded proteins, using the method disclosed in Taiwan patent application No. 092126644, which is incorporated herein in its entirety by reference. The sequence modification allowed the encoded peptides or proteins to be efficiently expressed in E. coli pET plasmid expression system. Table 4 lists the modified sequences of the DNA fragments encoding the chimeric target peptides gp 120 V3-V3 and gp120 C1-C5-gp41, in which non-italic, capital letters denote restriction enzyme linkers for EcoRI, Nde1 and Sal1 cutting sites, and italic, capital letters denote a XhoI restriction enzyme site.

TABLE 4

Chimeric
target		SEQ
peptide	Nucleotide sequence	ID No.

gp120	GAATTCCATATGGTCGACatcggttgcacccgtc	8
V3-V3	cgagcaacaacacccgtaaaggtatccacatggg
	cccgggtggtgctttctacaccaccggtcagatc
	atccgtaacatccgtcaggctcactgtggtctgc
	tgggtggttgtacccgtccgaacaacaacacccg
	tcgtagcatccacatcgaaccggaaggtgctttc
	tacaccaccggtgaaatcatcggtgacatccgtc
	aggctcactgtggcctgggtCTCGAG

gp120 C1-	GAATTCCATATGGTCGACgttgaaaaactgtggg	9
C5-gp41	ttaccgtttactacggtgttccggtttggaaaaa
	agttgttaaaatcgaaccgctgggtgttgctccg
	accaaatgcaaacgtcgtgttgttcagcgtgaaa
	agcgtggtggcggtggcggtcaagctcgtgttat
	cgctgttgaacgttacctgaaagaccagcagctg
	ctgggtatctggggtggtagcggtaaactgatct
	gctgcaccaccgctgttccgtggaacagcagctg
	gagcaacaaactggaccgtatctggaacaacatg
	acctggCTCGAG

Table 5 lists SEQ ID NOs. of respective primer pairs used for PCR synthesis of the DNA fragments encoding chimeric target peptides gp120 V3-V3 (SEQ ID NO: 8) and gp120 C1-C5-gp41 (SEQ ID NO: 9). Non-DNA-template PCR reactions were performed by continuously using forward and reverse primer pairs to PCR synthesize DNA fragments. In the first-round PCR, the 3′ end of the first forward primer (F1) had about 10-15 bases that were complementary to those in the pairing reversed primer (R1). The PCR profile was as follows: 5 min at 95° C., 1 min at 94° C., 0.5 min at 55° C., 1 min at 72° C. for 20 cycles, and 1 min at 72° C. Following the first-round PCR, the 3′ ends of the primer pairs (such as F2 and R2, F3 and R3, F4 and R4, or F5 and R5) had about 10-15 bases that were complementary to the previous round PCR product as a DNA template. After the first-round PCR, 0.01-1 μl of the product was used as the DNA template for the second-round PCR. The second primer pair, F2 and R2, were added in a suitable amount together with dNTPs, reagents and Pfu polymerase, and the second round PCR was performed. Other primers were subsequently added in this manner so that the final extended DNA fragments were synthesized. All the DNA fragments synthesized from each primer pair and each round of PCR were analyzed for the size by gel electrophoresis. The DNA fragment encoding each individual chimeric target peptide was synthesized in this manner until the final PCR product was extended to the expected size, e.g., 264 bp in the case of gp120 V3-V3.
The DNA fragments (Table 6) encoding each individual chimeric target peptides gp120 C1-C5-gp⁴¹for HIV subtype A, B, C, D, E, F, G, H, J and K were synthesized in a manner similar to the method described above using primer pairs listed in Table 6.

TABLE 5

Chimeric target	primer	Forward		Reverse
peptide	pairs	Primer	SEQ ID NO.	Primer	SEQ ID NO.

gp120 V3-V3	P1	F1	10	R1	15
	P2	F2	11	R2	16
	P3	F3	12	R3	17
	P4	F4	13	R4	18
	P5	F5	14	R5	19
gp120 C1-C5-	P1	F1	20	R1	24
gp41	P2	F2	21	R2	25
	P3	F3	22	R3	26
	P4	F4	23	R4	27
	P5	F4	23	R5	28
	P6	F4	23	R6	29

Example 4

Construction of Plasmids for Expression of HIV Fusion Proteins PE(ΔIII)-gp120 V3-V3 and PE(ΔIII)-gp120 C1-C5-gp41

Various chimeric target polypeptides of HIV-1 Env proteins as described above were cloned and expressed as PE fusion proteins. Briefly, a polypeptide from Pseudomonas Exotoxin A, i.e., PE(ΔIII), which was devoid of cytotoxic domain III, was fused to respective chimeric target polypeptides. Plasmid pPE(ΔIII) was constructed by inserting a DNA fragment encoding the binding domain I and translocation domain II of Pseudomonas exotoxin A into vector pET15a. A DNA sequence coding for a carboxyl terminal peptide that comprises the amino acid sequence KDEL (SEQ ID NO. 30) was ligated to the carboxyl terminal portion of the PE(ΔIII) gene, and generated plasmid pPE(ΔIII)-KDEL3 (referred to as pPE(ΔIII)-K3). The isolated DNA fragments generated by the PCR reaction were respectively digested by restriction enzymes EcoRI and Xho I, and then ligated to the EcoRI, Xho I sites of the plasmid pPE(ΔIII)-KDEL3, resulting in chimeric genes that would expressed target peptides as fusion proteins (Hung, C. F. et al (2001) “Cancer Immunotherapy Using a DNA Vaccine Encoding the Translocation Domain of a Bacterial Toxin Linked to a Tumor Antigen” Cancer Research 61:3698-3703).
The plasmid pPE(ΔIII)-HIV gp120 (FIG. 1A) encodes a fusion protein PE(ΔIII)-HIV gp120 V3-V3 (referred to as PE(ΔIII)-HIV gp120). The plasmid pPE(ΔIII)-HIV gp120-K3 (FIG. 1B) encodes a fusion protein PE(ΔIII)-HIV gp120 V3-V3-K3 (referred to as PE(ΔIII)-HIV gp120-K3). The plasmid pPE(ΔIII)-HIV gp120-41 (FIG. 1C) encodes fusion protein PE(ΔIII)-HIV gp120 C1-C5-gp41 (referred to as PE(ΔIII)-HIV gp120-41). The plasmid pPE(ΔIII)-HIV gp120-41-K3 (FIG. 1D) encodes the fusion protein PE(ΔIII)-HIV gp120 C1-C5-gp41-K3 (referred to as PE(ΔIII)-HIV gp120-41-K3). The resulting plasmids were respectively transformed into E. coli Jam109 to obtain clones and maintain the clones therein. The Jam109 strain could stably maintain the plasmid inside bacteria cells without protein expression.

TABLE 6

Chimeric	Nucleotide			SEQ
target	Sequence		Forward	ID	Reverse	SEQ ID
peptide	ID NO.	primer pairs	Primer	NO.	Primer	NO.

A	41	P1	F1	51	R1	56
		P2	F2	52	R2	57
		P3	F3	53	R3	58
		P4	F4	54	R4	59
		P5	F5	55	R5	60
B	42	P1	F1	61	R1	66
		P2	F2	62	R2	67
		P3	F3	63	R3	68
		P4	F4	64	R4	69
		P5	F5	65	R5	70
C	43	P1	F1	71	R1	76
		P2	F2	72	R2	77
		P3	F3	73	R3	78
		P4	F4	74	R4	79
		P5	F5	75	R5	80
D	44	P1	F1	81	R1	86
		P2	F2	82	R2	87
		P3	F3	83	R3	88
		P4	F4	84	R4	89
		P5	F5	85	R5	90
E	45	P1	F1	91	R1	96
		P2	F2	92	R2	97
		P3	F3	93	R3	98
		P4	F4	94	R4	99
		P5	F5	95	R5	100
F	46	P1	F1	101	R1	106
		P2	F2	102	R2	107
		P3	F3	103	R3	108
		P4	F4	104	R4	109
		P5	F5	105	R5	110
G	47	P1	F1	111	R1	116
		P2	F2	112	R2	117
		P3	F3	113	R3	118
		P4	F4	114	R4	119
		P5	F5	115	R5	120
H	48	P1	F1	121	R1	126
		P2	F2	122	R2	127
		P3	F3	123	R3	128
		P4	F4	124	R4	129
		P5	F5	125	R5	130
J	49	P1	F1	131	R1	136
		P2	F2	132	R2	137
		P3	F3	133	R3	138
		P4	F4	134	R4	139
		P5	F5	135	R5	140
K	50	P1	F1	141	R1	146
		P2	F2	142	R2	147
		P3	F3	143	R3	148
		P4	F4	144	R4	149
		P5	F5	145	R5	150

Example 5

Expression and Analysis of Target Proteins

HIV-1 PE-fusion proteins were expressed in E. coli BL21 (DE3) plys cultures containing corresponding expression plasmids. Briefly, 5 ml of bacterial seeds (A600 of 1.0±0.3 O.D.) were inoculated into 250 ml of liquid broth (LB) supplemented with 500 μg/ml ampicillin and 50 ml of 10% glucose at 37° C. in a rotating incubator shaken at 150 rpm for 2-3 hours. Once O.D._{600 nm}reached 0.3±0.1, the bacterial culture was induced with isopropylthio-β-D-galactoside (IPTG; Promega, USA) at a final concentration of 0.1 to 2 mM at 37° C. in a rotating incubator shaken at 150 rpm for 2 hours for protein expression.
Bacterial cells were pelleted after the protein induction was completed. After freezing and thawing of the pellet, bacterial cells were lysed with a solution containing in 10 ml: 0.3 mg/ml lysozyme, 1 mM phenylmethylsulfonyl fluoride (PMSF) and 0.06 mg/ml DNase I at room temperature for 20 minutes, followed by addition of 1 ml of 10% Triton X-100 and incubation at room temperature for 10 minutes. The lysed cells were centrifuged at 12,000 g for 10 minutes, and pellets were washed by 1 M and 2 M urea solutions. Insoluble inclusion bodies containing recombinant proteins were collected and dissolved in 8 ml of 8M urea solution or in an alkaline solution (pH 10 to 12) containing 1 to 3 M urea, and purified using a commercial pET His-Tag purification column system. The protein inclusion bodies dissolved in the urea solution were loaded onto a 4 ml Ni²⁺-nitrilotriacetic acid (Ni-NTA) resin affinity column, and the bound material eluted by different pH buffers (e.g. pH=8.0, 7.0, 6.5, 6.0, 5.4, and 3.5) containing 1 to 6 M urea with 0.1 to 0.3 M NaCl, 5 to 50 mM phosphate buffer, and 5 to 50 mM Tris.
The eluted proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and stained with coomassie blue. The optical densities of the bands in the gels were measured with a densitometer for evaluation of protein quantities. The concentration of fusion proteins, e.g., PE(ΔIII)-HIV gp120 C1-C5-gp41-K3 (referred to as gp120-41-K3) and PE(ΔIII)-HIV gp120 V3-V3-K3 (referred to as gp120-K3), in the eluted samples were about 0.8 mg/ml.

Example 6

Antibody Assay

Materials and methods. Five PE-HIV envelope peptides were used: (1) PE(ΔIII)-HIV gp120; (2) PE(ΔIII)-HIV gp120-KDEL; (3) PE(ΔIII)-HIV gp120-41; and (4) PE(ΔIII)-HIV gp120-41-KDEL. An oil adjuvant, ISA 206, was used with each of the peptide immunogens in emulsified preparations for injection into mice.
Animals. BALB/c mice were purchased form Harlan laboratories and housed in the Laboratory animal Resources Facility of KUMC. All mice were used in accordance with AAALAC and the KUMC Institutional Animal Care and Use Committee guidelines.
Immunization of animals with PE-HIV-Env fusion protein vaccines. Four- to six-week old BALB/c mice were divided into 5 groups, with 6 mice per group. The animals in groups 1 to 5 received (1) PE(ΔIII)-HIV gp120, (2) PE(ΔIII)-HIV gp120-K3, (3) PE(ΔIII)-HIV gp120-41, (4) PE(ΔIII)-HIV gp120-41-K3, and (5) PBS (control group), in adjuvant, respectively, following the immunization schedule shown in Table 7. Immunized mice were then exsanguinated following deep anesthesia and blood and spleens were collected for immunological assays.

	TABLE 7

	Immunization Schedule

			1^sttime	2^ndtime	3^rdtime	4^thtime	5^thtime
	No. of		*(IM)	(IM)	(IM)	(I/M)	**(IP)
Group	mice	Immunogen		0 week	2 weeks	4 weeks	6 weeks	8 weeks

1	6	PE(ΔIII)-HIV gp120	50 μg	25 μg	25 μg	25 μg	50 μg
		Adjuvant	50 μl	50 μl	50 μl	50 μl	0
2	6	PE(ΔIII)-HIV gp120-K3	50 μg	25 μg	25 μg	25 μg	50 μg
		Adjuvant	50 μl	50 μl	50 μl	50 μl	0
3	6	PE(ΔIII)-HIV gp120-41	50 μg	25 μg	25 μg	25 μg	50 μg
		Adjuvant	50 μl	50 μl	50 μl	50 μl	0
4	6	PE(ΔIII)-HIV gp120-41-K3	50 μg	25 μg	25 μg	25 μg	50 μg
		Adjuvant	50 μl	50 μl	50 μl	50 μl	0
5	6	PBS	0	0	0	0	50 μl
		Adjuvant	50 μl	50 μl	50 μl	50 μl	0

*IM denotes intramuscular injection
**IP denotes intraperitoneal injection

Serum antibody binding assay. An ELISA test using a commercial kit was used to determine binding antibody titers. Briefly, two weeks after the last immunization, mice were anesthetized, sacrificed, and spleens and blood samples were collected. Sera prepared from the blood samples of the animals immunized with fusion proteins in groups 1 to 4 and animals in the placebo group were serially diluted 500×, 2500×, 12500×, and 62500×, respectively. Binding antibody titers induced by the four peptide groups were analyzed using ELISA kit (BioChain) for detection of anti-HIV antibodies. Plates were coated with HIV antigen. The manufacturer's protocol was modified for using mouse sera by substituting the anti-human antibody with a goat anti-mouse serum conjugated with Horseradish peroxidase (HRP). Results were expressed as OD absorbance at 450 nm against the blank. FIGS. 2A-2B illustrate the test results of sera from animals immunized with respective fusion proteins. The absorbance data reflects the amount of antibody titers in serum samples. The result indicates that each fusion protein vaccine after being injected into animals was able to induce antibodies against HIV with good titers in the dilution of 1:2,500 (FIG. 2A) and 12,500 (FIG. 2B).

Example 7

Immunized Mouse Sera Neutralize HIV-Attenuated Live Vaccine Virus

Neutralization assays were performed using mouse sera against an attenuated live SHIV vaccine virus that was developed in the MMD Lab. The assays were performed in X4 GHOST cells using a plaque reduction assay. X4 GHOST cells were capable of harboring HIV virus plaques after being infected by an HIV-attenuated live vaccine virus. Briefly, serum samples collected from immunized mice were tested for their contents of HIV specific neutralizing antibodies. Briefly, quadruplicates of serial twofold dilutions of sera in RPMI 1640 medium were prepared in 96-well plates. Twenty plaque-forming units of virus were incubated with the twofold dilutions of serum samples from each immunized mouse and a normal serum for two hours at 37 C, respectively. The suspensions were then inoculated onto monolayers of X4 GHOST cells that constitutively expressed the HIV LTR linked to GFP. These cultures were incubated for three days and then examined by immunofluorescence for determination of the numbers of fluorescence spots that represented successful virus hits. Neutralization titers of the serum samples were scored as the highest dilution of the immune serum sample that prevented development of 50% of the plaques induced with the control nonimmune serum. Mice developed neutralizing antibodies against the live vaccine virus (FIG. 3). The highest serum neutralization titers were obtained with sera from mice immunized with PE(ΔIII)-HIV gp120-41-KDEL.

Example 8

Immunized Mouse Sera Neutralize Simian-Human Immunodeficiency Virus (SHIV_KU2)

Immunization of animals. Mice were immunized with higher dosages of PE-fusion protein vaccines using a similar protocol described above. Briefly, mice were divided into 5 groups (groups 6-10), with 6 mice per group, and received PE(ΔIII)-HIV gp120, PE(ΔIII)-HIV gp120-K3, PE(ΔIII)-HIV gp120-41, PE(ΔIII)-HIV gp120-41-K3, and PBS (control group), in adjuvant, respectively. The immunization schedule is shown in Table 8.

	TABLE 8

	Immunization

			First	Second
	No.		time	time	Third time
	of		*(IM)	(IM)	**(IP)
Group	mice	Immunogen		0 week	2 weeks	6 weeks

6	6	PE(ΔIII)-HIVgp120	100 μg	50 μg	50 μg
		Adjuvant	100 μl	50 μl	0
7	6	PE(ΔIII)-HIVgp120-K3	100 μg	50 μg	50 μg
		Adjuvant	100 μl	50 μl	0
8	6	PE(ΔIII)-HIVgp120-41	100 μg	50 μg	50 μg
		Adjuvant	100 μl	50 μl	0
9	6	PE(ΔIII)-HIVgp120-	100 μg	50 μg	50 μg
		41-K3
		Adjuvant	100 μl	50 μl	0
10	6	PBS	100 μl	50 μl	50 μl
		Adjuvant	100 μl	50 μl	0

*IM: intramuscular injection with adjuvant ISA 206.
**IP: intraperitoneal injection with adjuvant ISA 206.

TABLE 9

		No. of live
		mice/No. of	Neutralization
Group	Vaccine	challenged mice	titer

6	PE(ΔIII)-HIV gp120	4/6	1:20
7	PE(ΔIII)-HIV gp120-K3	1/6	1:20
8	PE(ΔIII)-HIV gp120-41	5/6	1:20
9	PE(ΔIII)-HIV gp120-41-K3	3/6	1:20-40
10	PBS/adjuvant*	0/6	—

*Adjuvant: ISA 206.

Two weeks after the third immunization, blood samples were collected from each group of animals and processed to obtain serum samples for assay of antibody titers. ELISA antibody assays showed similar titers in this experiment as those in Example 6.
To perform neutralization assays using SHIV_KU2, mice from each group were challenged by pathogenic SHIV_KU2vaccine (constructed by Dr. Narayan, University of Kansas Medical Center, US Pat. No. 5,849,994). All four groups of the immunized animals developed neutralizing antibody titers of approximate 1:20 against SHIV_KU2. Table 9 shows the survival data. PE(III) gp120-41 was the best antigen preparation for induction of neutralizing antibodies against SHIV_KU2since 5 out of 6 mice developed these antibodies.

Example 9

HIV Gag24, Nef, Tat, and Rev Fusion Proteins

Materials and methods. Four HIV fusion proteins were tested for their immunogenicity: (I) HIV Gag24 fusion protein vaccines, PE(ΔIII)-HIV Gag24-K3 and PE(ΔIII)-HIV Gag24-gp120-41-K3; (II) HIV Nef fusion protein vaccines comprising PE(ΔIII)-HIV Nef-N—K3and PE(ΔIII)-HIV Nef-C—K3; (III) HIV Tat fusion protein vaccine comprising PE(ΔIII)-HIV tat-K3; and (IV) HIV Rev fusion protein vaccine comprising PE(ΔIII)-HIV Rev-K3. The above (I) to (IV) HIV fusion proteins were constructed using similar methods described in Examples 2 to 4. Briefly, various polypeptide segments (Table 10) were selected from HIV proteins Gag24, Nef, Tat and Rev, respectively.

TABLE 10

	Targeted
HIV	peptide		SEQ
Proteins	segments	Amino acid sequence	ID No.

Gag24	Full length	VDRDELKGIGMTNNPPIPVGEIYK	151
	Gag24	RWIILGLNKIVRMYSPTMTNNPPI
		PVGEIYRWIILGLNKIVRMYSPT

Nef	Nef- N	PTVRQRMDRTEPAAEGVGAVSRDL	152
	terminus	EKHGAITSSNTAATNADCAWLEAQ
		EEEEVGFPVRPQVPLRPMTYKAAV
		DISHFLKEKGGLEGLIYSQKRQEI
		LDLWIYHTQGYFPDWQNYTPGPGI
		RYPLTFGWCFKL
	Nef-C	FLKVPVDPEQVEKANEGDNNCLLH	153
	terminus	PISQHGMDDPEKEVLMWKFDSRLA
		FQHIAREKHPEYYKDCLG

Tat	Full length	RDELKGIGMEPVDPRLEPWKHPGS	154
	Tat	QPRTACNNCYCKKCCFHCPVCFIS
		KGLGISYGRKKRRQRRRAPQDSET
		HQVSLSKQPTSQLRGDPTGPKESK
		KKVERETETDPNV

Rev	Full length	LLAVRIIKTLYQSNPYPKPEGYRR	155
	Rev	VRRNRRRRWRARQRQIHSISERIL
		ITCLGRPTEPVPLQLPPIERLNIN
		CSESGGTSGTQRVGNP

TABLE 11

	Nucleotide			SEQ
Target	Sequence		Forward	ID	Reverse	SEQ ID
peptide	ID NO.	primer pairs	Primer	NO.	Primer	NO.

Gag24	156	P1	F1	161	R1	165
		P2	F2		162	R2	166
		P3	F3	163	R3	167
		P4	F4	164	R4	168
Nef-N	157	P1	F1	169	R1	176
terminus		P2	F2	170	R2	177
		P3	F3	171	R3	178
		P4	F4	172	R4	179
		P5	F5	173	R5	180
		P6	F6	174	R6	181
		P7	F7	175	R7	182
		P8	F7	175	R8	183
		P9	F7	175	R9	184
Nef-C	158	P1	F1	185	R1	189
terminus		P2	F2	186	R2	190
		P3	F3	187	R3	191
		P4	F4	188	R4	192
Tat	159	P1	F1	193	R1	200
		P2	F2	194	R2	201
		P3	F3	195	R3	202
		P4	F4	196	R4	203
		P5	F5	197	R5	204
		P6	F6	198	R6	205
		P7	F7	199	R7	206
Rev	160	P1	F1	207	R1	213
		P2	F2	208	R2	214
		P3	F3	209	R3	215
		P4	F4	210	R4	216
		P5	F5	211	R5	217
		P6	F6	212	R5	217

The DNA fragments (Table 11) encoding respective polypeptide segments were synthesized using primers listed in Table 11 by multi-round PCR synthesis method as described previously. Gel electrophoresis experiments were performed to examine the PCR products generated by each primer pair in the multiple-round PCR synthesis of DNA fragments, e.g., 410, 412, 414, 416 (FIG. 4). The PCR synthesized DNA fragments were fused to a PE fragment, cloned and expressed, respectively, using a similar method described in Example 4. The PE fragment is a polypeptide called PE (ΔIII) that contains a binding domain and a translocation domain from Pseudomonas Exotoxin A but lacks a cytotoxic domain. For example, the DNA fragment encoding gag24 generated by PCR was digested by restriction enzymes EcoRI and Xho I to isolate a 225 bp fragment, 402, which was ligated to a PE(ΔIII) fragment within an EcoRI and Xho I-digested plasmid pPE(ΔIII)-KDEL3, 401, to generate pPE(ΔIII)-HIV-gag24-K3 (FIG. 4).
A 1.7 Kb gp120-41 fragment, isolated from Sal I and Pst I-digested pPE(ΔIII)-HIV gp120-41-K3, 502, was fused to gag24 by inserting downstream, or C-terminal, to the gag24 gene within a Pst I, Xho I-digested plasmid pPE(ΔIII)-HIV gag24-K3, 501, to generate plasmid pPE(ΔIII)-HIV gag24-gp120-41-K3 (FIG. 5). Plasmid pPE(ΔIII)-HIV nef-C—K3 was digested by Sal I and Pst I to isolate a 1.6 Kb fragment, 702, followed by ligation to a nef-N fragment within a Pst I, Xho I-digested plasmid pPE(ΔIII)-HIV nef-N-KDEL, 701, to generate pPE(ΔIII)-HIV nef-NC-KDEL (FIG. 7).
The DNA fragment encoding Tat or Rev generated by PCR was digested by restriction enzymes EcoRI and Xho I to isolate fragment, which was ligated to a PE(ΔIII) fragment within an EcoRI and Xho I-digested plasmid pPE(ΔIII)-KDEL3. By employing DNA recombinant method, plasmids pPE(ΔIII)-HIV Tat-K3 (FIG. 1F), and pPE(ΔIII)-HIV Rev-K3 (FIG. 1E) were generated.
The sequence KDEL3 (i.e., K3) is an endoplasmic reticulum (ER) retention peptide located at the carboxyl terminal portion of the chimeric fusion protein. The sequence listing illustrates the nucleotide sequences of PE(ΔIII)-HIV Gag24-K3, PE(ΔIII)-HIV gag24-gp120-41-K3, PE-(ΔIII)-HIV nef-N—K3, pPE(ΔIII)-HIV nef-C—K3, pPE(ΔIII)-HIV-nef-NC—K3, pPE(III)-HIV rev-K3, pPE(ΔIII)-HIV-tat-K3 as SEQ ID NOs: 236, 238, 240, 242, 244, 246 and 248, and the corresponding amino acid sequences as SEQ ID NOs: 237, 239, 241, 243, 245, 247, and 249, respectively. For clinical applications, any undesired sequence such as oncogen sequences, if present in the bridge between PE(ΔIII) and HIV target peptide (e.g., between EcoRI and AatII), may be deleted without affecting the HIV target antigenic determinants.
Immunization of animals with fusion proteins. Chimeric fusion proteins as described above were expressed for vaccination. Female mice C57BL/6J aged 6- to 8-week old were purchased from National Taiwan University (Taipei, Taiwan) and bred in Animal Center of National Taiwan University Hospital. Mice were divided into groups and injected three times at two-week intervals with respective fusion proteins or PBS (control group) in ISA 206 oil adjuvant (Table 12). Two weeks after the last immunization, mice were exsanguinated under deep anesthesia and blood and spleens were collected for immunological assays. An ELISA test using a commercial kit was used to determine binding antibody titers.

	TABLE 12

	Immunization Schedule

HIV		No. of	First time	Second time	Third time
target peptides	Vaccine*	Mice	0 week	2 weeks	4 weeks

Placebo	PBS/Adjuvant	3	100	μl	100	μl	100	μl
Gag24	PE(ΔIII)-HIV Gag24-K3	4	100	μg	100	μg	100	μg
gp120	PE(ΔIII)-HIV gp120-41-K3	4	100	μg	100	μg	100	μg
Gag24-gp120-	PE(ΔIII)-HIV Gag24-	3	100	μg	100	μg	100	μg
41	gp120-41-K3
Nef	PE(ΔIII)-HIV Nef-N-K3	4	100	μg	100	μg	100	μg
	PE(ΔIII)-HIV Nef-C-K3		(50	μg each)	(50	μg each)	(50	μg each)
Tat	PE(ΔIII)-HIV Tat-K3	4	100	μg	100	μg	100	μg
Rev	PE(ΔIII)-HIV Rev-K3	4	100	μg	100	μg	100	μg

*Vaccines were prepared by mixing fusion proteins or PBS with 50 μl of adjuvant ISA 206 before intramuscular (IM) injection.

Serum Antibody Test. Sera from mice were prepared and serially diluted 500×, 2500×, 12500×, and 62500× using the method described in Example 5. Antibody titers were measured using indirect ELISA analysis. ELISA plates were prepared and coated with corresponding peptides in Tables 13 and 14.
Cytokine release assay. Splenocyte cytokine levels in the medium of cultured cells were examined by ELISA to measure levels of cytokines TNF-α, γ-IFN, IL-4, IL-10 and IL-12. Briefly, spleens were aseptically collected from mice and dissociated to harvest splenocytes. Cells were resuspended in RPMI, and mononuclear cells were counted in a hemocytomer. Splenocytes were diluted to an optimal density and cultured in 5×10⁶cells/well in 6-well plates in 5 ml of RPMI. Immunogen inducer peptides, e.g., Gag24-N and Gag24-C, etc., were respectively added to splenocytes in triplicate. On the second day after the addition of immunogens, supernatants were collected. The amounts of TNF-α, γ-IFN, IL-4, IL-10 and IL-12 produced by splenocyte CD8+ T cells were assayed using quantitative ELISA assay kits (Invitrogen BioSource) by following the manufacturer's protocol with slight modifications.

TABLE 13

		SEQ	Length
Antigen peptide	Peptide sequence	ID NO.	(a.a.)

gp120-41-N1	VEKLWVTVYYGVPVWK	218	16

gp120-41-N2	KVVKIEPLGVAPTKCK	219	16

gp120-41-N3	APTKCKRRVVQREKR	220	15

gp120-41-C1	QARVWRYLKDQQLL	221	14

gp120-41-C2	GIWGCSGKLICCTTAV	222	17
	P

gp120-41-C3	AVPWNASSWSNKLDR	223	15

TABLE 14

		SEQ	Length
Antigen peptide	Peptide sequence	ID NO.	(a.a.)

HIV-Tat-N	PVDPRLEPWHKPGSQPRTAC	224	20

HIV-Tat-C	QLRGDPTGPKESKKKVERET		225	20

HIV-Tat-M	SYGRKKRRQRRRAPQDSETH	226	20

HIV-Rev-N	QSNPYPKPEGYRRVRRNRRR	227	20

HIV-Rev-C	NCSESGGTSGTQRVGNPLEK	228	20

HIV-Nef-n1-N	SKLKKGWPTVRQRMDRTE	229	18

HIV-Nef-n1-C	TQGYFPDWQNYTPGPGIR	230	18

HIV-Nef-c1-N	VDPEQVEKANEGDNN	231	15

HIV-Nef-c1-M	ISQHGMDDPEKEVLM	232	15

HIV-Nef-c1-C	QHIAREKHPEYYKDCLGLEK	233	20

HIV-Gag24-N	PEFHMVDRDELKGIGMTN	234	18

HIV-Gag24-C	RMYSPTMTNNPPIPV	235	15

Spleen lymphoid cell proliferation CMI assay. Cell proliferation ELISA BrdU (colorimetric) assays for CMI reactions were performed. The steps for culturing splenocytes were similar to those used in cytokine release assay except that cells were cultured in 96-well plates. Briefly, immunogens or antigen peptides, e g., Gag24-N, Gag24-C, etc., were respectively added to cell culture on day-2 to stimulate cell proliferation. ConA (10 μg/ml), as a positive control, was added to stimulate cell2s for one day. Cells were pulse-labeled with BrdU on day-3 at 37° C. for 12-24 hr. Only proliferating cells incorporated BrdU into their DNA. Cells were fixed with FixDenat solution. The FixDenat solution also denatured the genomic DNA, exposing the incorporated BrdU to immunodetection. The BrdU label in the DNA was located with a peroxidase-conjugated anti-BrdU antibody (anti-BrdU-POD). The bound anti-BrdU-POD was quantitated with a peroxidase substrate TMB by measuring absorbance at OD650 using ELISA plate reader.
Table 15 shows Gag24-specific antibodies titers in immunized mouse Sera The antibody titer assay indicated that Gag24-N antigenic determinant or epitope peptide was stronger in inducing antibody reactions than the Gag24-C epitope peptide. The ability of Gag24-N peptide in inducing antibody titers was, however, weak when it was in the fusion protein PE(ΔIII)-Gag24-K3. Once the Gag24-N antigenic determinant peptide was modified to include polypeptide gp120 and gp41 α-helix to form fusion protein PE(ΔIII)-Gag24-gp120-41-K3, its ability of inducing Gag24-N-specific IgG increased significantly. Thus, the peptide Gag24-N could elicit a Th2 cell-dependent, antigenic determinant (or epitope)-specific humoral immune response.

	TABLE 15**

	Antibody

IgG

IgA

IgE

Mouse No.

Vaccine

Coated Antigen

#

1

#2

#3

Pl* #1

#1

#2

#3

Pl #1

#1

#2

#3

Pl #1

PE(ΔIII)-	Gag24-N	10	10	10	1	3	3	3	1	1	1	3	1
Gag24-K3	Gag24-C	3	3	3	1	10	3	3	1	1	3	1	1
PE(ΔIII)-	Gag24-N	100	100	100	1	3	3	3	1	3	3	3	1
Gag24-	Gag24-C	3	3	10	1	3	3	3	1	1	1	1	1
gp120-41-
K3

*The term “Pl” denotes “placebo,” in which mice were injected with PBS/adjuvant.
**The data represented here were endpoints of serum semi-log serial dilution. The experiments were repeated in three mice per immunogen inducer group.

The results from the cell proliferation CMI assay indicated that both fusion proteins, PE(ΔIII)-Gag24-K3 and PE(ΔIII)-Gag24-gp120-41-K3, after being injected into mice could induce cell-mediated immune response to Gag24 antigen (Table 16). The Gag24 antigen, however, had a low efficacy in inducing cell-mediated immune responses in the fusion protein PE(ΔIII)-Gag24-K3. Once it was modified to fuse with gp120 C1 and C5 domains and gp41 α-helix to form PE(ΔIII)-Gag24-gp120 C1-C5-gp41-K3, Gag24 antigen's ability in inducing cell-mediated immune responses significantly increased. Thus, PE(ΔIII)-Gag24-gp120 C1-C5-gp41-K3 is much stronger than PE(ΔIII)-Gag24-K3 in inducing Gag24-specific, cell-mediated responses and cytokine release. As shown in FIG. 6, chimeric polypeptide HIV PE(ΔIII)-gp120 C1-C5-gp41 600 can act as a building unit for connecting other HIV antigenic determinant peptide 602 and thereby markedly enhance cell-mediated immune responses of the inserted HIV antigenic peptide 602, such as Gag24. Chimeric polypeptide HIV PE(ΔIII)-gp120 C1-C5-gp41 600 includes PE(ΔIII) 604, HIV gp120 C1-C5-gp41 608, an endoplasmic reticulum retention sequence 610, with a bridge or linker 606 in-between. The fusion of an HIV antigenic determinant polypeptide 602 with weak CMI responses and HIV gp120 C1-C5-gp41 608 results in a chimeric PE-HIV fusion protein 620 that exhibits enhanced CMI responses specific to the antigenic determinant 602.

TABLE 16

CMI assay on immunized mouse splenocytes*

Animal Group

		Vaccine	Placebo
Fusion Protein Vaccine	Immunogen Inducer	(n = 3)	(n = 3)

PE(ΔIII)-Gag24-K3	Gag24-N	0.32	0.19
	Gag24-C	0.65	0.26
	ConA	1.56	0.24
PE(ΔIII)-Gag24-gp120-41-K3	Gag24-N	1.31	0.19
	Gag24-C	1.25	0.26
	ConA	1.42	0.20

The data from the cytokine induction test (Table 17) showed that both vaccines PE(ΔIII)-Gag24-K3 and PE(ΔIII)-Gag24-gp120-41-K3 after being injected into mice did not induce detectable IL-4, which indicated that they would be better vaccine candidates for HIV. Of the two fusion protein vaccines, PE(ΔIII)-Gag24-gp120-41-K3 was much more effective than PE(ΔIII)-Gag24-K3 in inducing splenocytes to produce large amounts of IL-10 and IL-12. A comparison of Gag24-N and Gag24-C peptides in their cytokine inducing effects showed that in the PE(ΔIII)-Gag24-K3 vaccine group, Gag24-C peptide appeared to have a stronger T-cell-dependent epitope effect than Gag24-N peptide. In the PE(ΔIII)-Gag24-gp120-41-K3 vaccine group, both Gag24-N and Gag24-C peptides were capable of inducing cell-mediated immune responses and had no difference in their effects in inducing cytokine release. The data indicated that fusion protein PE(ΔIII)-Gag24-K3 had a low efficacy in inducing Gag24-specific cytokine release; however, fusion protein PE(ΔIII)-Gag24-gp120-41-K3 had a strong effect in eliciting Gag24-specific immune responses.

TABLE 17

Cytokines release assay*

Cytokine

TNF-α

γ-IFN

IL-4

IL-10

IL-12

Immunogen

Animal Group

Vaccine	Inducer	Vac**	Plac**	Vac**	Plac**	Vac**	Plac**	Vac**	Plac**	Vac**	Plac**

PE(ΔIII)-	Gag24-N	8.6	3.4	6.1	5.6	0	0	2	0	53.6	31.0
Gag24-	Gag24-C	12.9	9.6	12.8	12.8	0	0	12	8	36.5	35.6
K3	ConA	143.1	53.4	206.4	81.2	9	0	9	4	60.0	36.1
PE(ΔIII)-	Gag24-N	70.0	3.4	17.5	5.6	0	0	136	0	87.6	31.0
Gag24-	Gag24-C	86.6	9.6	24.4	12.8	0	0	144	8	67.2	35.6
gp120-	ConA	314	53.4	765.4	81.2	13.8	0	196	4	87.6	36.1
41-K3

*The standard deviation is not shown here. The unit of the concentration is in pg/ml; n = 3 in both vaccine and placebo groups.
**The term “Vac” refers to “Vaccine,” and “Plac” refers to “Placebo.”

The data from the immunized mice Sera ELISA test indicated that the farthest C-terminal portion of Nef-C antigen determinant peptide had the strongest antibody reaction (Table 18). Based on the antibody-inducing reactions by fusion protein vaccine PE(ΔIII)-Nef-K3, it was concluded that peptide Nef-C—C was one of the Th2 cell-dependent, HIV antigenic determinant sites (Table 18).

TABLE 18**

Nef-specific antibody titers in HIV-Nef fusion protein-immunized mice

Antibody

IgG

IgA

IgE

Mouse No.

Vaccine

Coated Antigen

#

1

#2

#3

P1*

#1

#2

#3

P1

#

1

#2

#3

P1

PE(ΔIII)-	Nef-N-N	30	30	30	1	3	10	10	10	1	1	1	1
Nef-N-	Nef-N-C	30	30	30	1	30	30	30	1	1	3	1	1
K3
PE(ΔIII)-	Nef-C-N	30	30	30	1	10	10	10	1	1	1	1	1
Nef-C-	Nef-C-M	30	30	30	1	10	10	10	1	1	1	1	1
K3	Nef-C-C	10⁴	10³	300	1	10²	10	10	1	1	1	1	1

*The term “P1” denotes “placebo #1.” Mice in the placebo group were injected with PBS/adjuvant.
**The data represent the vaccinated mouse serum titers/placebo serum titers of the endpoints of serum semi-log serial dilution. The experiments were repeated in three mice per each vaccinated group.

TABLE 19

CMI assay on HIV-Nef fusion protein-immunized mouse splenocytes*

Animal Groups

	Immunogen	Vaccine	Placebo
Vaccine	Inducer	(n = 3)	(n = 3)

PE(ΔIII)-Nef-N-K3	Nef-N-N	0.7	0.3
and	Nef-N-C	0.8	0.2
PE(ΔIII)-Nef-C-K3	Nef-C-N	0.5	0.3
	Nef-C-M	0.7	0.2
	Nef-C-C	1.0	0.3
	ConA	0.7	0.3

The cell-mediated immune responses in immunized mice indicated that both PE(ΔIII)-Nef-N—K3 and PE(ΔIII)-Nef-C—K3 had Nef-antigen-specific CMI reactions, and among which the Nef-N—C and Nef-C—C antigenic determinant portions induced stronger CMI responses (Table 19).

TABLE 20*

Cytokines release assay from HIV-Nef fusion protein-immunized mouse splenocytes

Cytokine

TNF-α

γ-IFN

IL-4

IL-10

IL-12

Immunogen

Animal Group

Vaccine	Inducer	Vac***	Plc***	Vac	Plc	Vac	Plc	Vac	Plc	Vac	Plc

Mixture of	Nef-N-N	46.8	2.1	38.9	9.2	0	0
PE(ΔIII)-	Nef-N-C	42.8	4.9	34.4	26.7	0	0
HIV-Nef-N-	Nef-C-N	65.2	12.7	61.7	30.9	0	0	143	0	55	38
K3	Nef-C-M	47.8	6.9	27.0	17.4	0	0
and	Nef-C-C	51.2	2.9	25.1	10.6	0	0
PE(ΔIII)-	ConA	80.8	14.7	84.6	15.3	0	0	135	0	69	50
HIV-Nef-C-	BK**	61.2	10.5	46.3	22.5
K3

*conc. (pg/ml); n = 3 in both vaccine and placebo group.
**BK refers to “Blank,” in which no immunogen inducer was added into splenocytes.
***Vac refers to “Vaccine,” and Plc refers to “Placebo.”

The data from the cytokine induction test showed that Nef fusion proteins did not induce detectable Nef-specific IL-4, which was an indication that Nef fusion proteins would be better vaccine candidates against HIV. The data also indicated that a vaccine composition comprising fusion proteins PE(ΔIII)-HIV-Nef-N—K3 and PE(ΔIII)-HIV Nef-C could stimulate splenocytes to produce a higher amount of IL-10 (Table 20).
The results from serum antibody assay indicated that the N-terminal portion of HIV-1 Tat protein could induce remarkable antibody responses, while the mid-segment and C-terminal portions were weak in inducing antibody responses. Thus, the N-terminal portion of HIV Tat protein could elicit Th2 cell-dependent, antigenic determinant-specific humoral immunity (Table 21).
The results from the cell immune response indicated that PE(ΔIII)-Tat-K3 could induce cell mediated immune responses to all Tat protein segments, among which the N-terminus of Tat protein was stronger than mid- and C-terminal segments in inducing cell immune responses (Table 22).

TABLE 21

Antibody titers in HIV-Tat-fusion protein-immunized mice

Antibody

IgG

IgA

IgE

Coated

Mouse No.

Vaccine

Antigen

#

1

#2

#3

#4

P1

#

1

#2

#3

#4

P1

#

1

#2

#3

#4

P1

PE	Tat-N	10³	10³	10³	10³	1	10³	10³	10³	10³	1	?	?	10	10	1
(ΔIII)-	Tat-M	10	10	10	10	1	3	3	3	3	1	3	3	3	3	1
HIV-	Tat-C	10	1	3	3	1	3	3	3	3	1	1	1	1	1	1
Tat-
K3

*The term “P1” refers to “placebo #1.”
The data represent vaccinated mouse serum titers/placebo serum titers of the endpoints of serum semi-log serial dilution.

TABLE 22

CMI assay on HIV-Tat-fusion protein immunized mouse splenocytes*

Animal Groups

	Immunogen	Vaccine	Placebo
Vaccine	Inducer	(n = 4)	(n = 4)

PE(ΔIII)-HIV-	Tat-N	1.1	0.2
Tat-K3	Tat-M	1.0	0.2
	Tat-C	0.5	0.2
	ConA	1.3	0.2

The results from cytokine release assay indicated that HIV Tat fusion protein was not able to induce a detectable level of Tat-specific IL-4. Its effects in inducing γ-IFN and TNF-α release were not obvious, either. The fusion protein PE-(ΔIII)-Tat-K3, however, was able to stimulate splenocytes to produce Tat-N terminus-specific IL-12. Thus, PE-(ΔIII)-Tat-K3 was still effective in inducing a cell immune response that was specific to the N terminal portion of Tat and therefore the N-terminus of Tat could evoke Th1 cell-dependent, antigenic determinant-specific cell mediated immune responses in the PE delivery system of the present invention (Table 23).
The antibody data from PE(III)-Rev-K3 fusion protein-immunized mice model indicated that the antibody responses to HIV-Rev antigenic determinant peptide was not strong enough to confirm the locus of Th2 cell-dependent, antigenic determinant in Rev protein (Table 24).

TABLE 23

Cytokines release assay on HIV-Tat fusion protein immunized-mouse splenocytes*

Cytokine

TNF-α

γ-IF2N

IL-4

IL-10

IL-12

Immunogen

Animal Group

Vaccine	Inducer	Vac	Plac	Vac	Plac	Vac	Plac	Vac	Plac	Vac	Plac

PE(ΔIII)-	Tat-N	10	5	0	18	0	1	6	0	41	10
HIV-	Tat-M	18	5	7	4	—	—	—	—	—	—
Tat-K3	Tat-C	32	9	21	17	—	—	—	—	—	—
	BK	10	18	2	27	—	—	—	—	—	—
	ConA	155	27	243	81	33	0	48	25	55	27

*conc. (pg/ml); n = 3 in both vaccine (Vac) and placebo (Plac) group.

TABLE 24

Antibody titers in HIV-Rev fusion protein-immunized mice

Antibody

IgG

IgA

IgE

Mouse No.

Vaccine

Immunogen

#

1

#2

#3

#4

P1*

#1

#2

#3

#4

P1

#

1

#2

#3

#4

P1

PE(III)-	HIV-	10	10	10	3	1	3	10	3	10	1	1	1	1	1	1
HIV-	Rev-N
Rev-	HIV-	3	3	1	1	1	3	3	3	3	1	1	1	1	1	1
K3	Rev-C

*The term “P1” denotes “placebo #1.” Mice in the placebo group were injected with PBS/adjuvant.
The data represent vaccinated mouse serum titers/placebo serum titers of the endpoints of serum semi-log serial dilution.

The data from cell immune responses in Table 25 indicated that PE(III)-Rev-K3 fusion protein vaccine was not able to induce cell immune response to Rev antigen. The cytokine release inducing test also gave the similar result. It showed no obvious effects in inducing TNF-α and γ-IFN release (Table 26). Thus, the fusion protein vaccine PE(ΔIII)-Rev-K3 might not be a good component in an HIV vaccine. However, whether Rev is able to elicit a CMI response under the condition of fusion with gp120-41 in a PE delivery system remains to be investigated. A plasmid pPE(ΔIII)-HIV-Rev-gp120-41-K3 was constructed for investigation.

TABLE 25

CMI assay on HIV-Rev fusion protein-immunized mouse Splenocytes

Animal Groups

	Immunogen	Vaccine	Placebo
Vaccine	Inducer	(n = 3)	(n = 3)

PE(ΔIII)-HIV-Rev-K3	Rev-N	0.39	0.25
	Rev-C	0.39	0.24
	ConA	0.40	0.24

TABLE 26

Cytokines release assay on HIV-Rev fusion protein-immunized
mouse Splenocytes*

Cytokine

TNF-α

γ-IFN

Animal Group

	Immunogen	Vaccine	Placebo	Vaccine	Placebo
Vaccine	Inducer	(n = 4)	(n = 4)	(n = 4)	(n = 4)

PE(ΔIII)-HIV-	Rev-N	29	3	6	4
Rev-K3	Rev-C	30	3	7	5
	BK	38	4	9	7
	ConA	50	6	16	5

*conc. (pg/ml)

The foregoing description of the exemplary embodiments of the invention has been presented only for the purposes of illustration and description and is not intended to be exhaustive or to limit the invention to the precise forms disclosed. Many modifications and variations are possible in light of the above teaching.
The embodiments and examples were chosen and described in order to explain the principles of the invention and their practical application so as to enable others skilled in tie art to utilize the invention and various embodiments and with various modifications as are suited to the particular use contemplated. Alternative embodiments will become apparent to those skilled in the art to which the present invention pertains without departing from its spirit and scope. Accordingly, the scope of the present invention is defined by the appended claims rather than the foregoing description and the exemplary embodiments described therein.
Some references, which may include patents, patent applications and various publications, are cited and discussed in the description of this invention. The citation and/or discussion of such references is provided merely to clarify the description of the present invention and is not an admission that any such reference is “prior art” to the invention described herein. The application claims the priority of U.S. provisional application No. 61/025,094, filed Jan. 31, 2008, which is incorporated herein in its entirety by reference. All references cited and discussed in this specification are incorporated herein by reference in their entireties and to the same extent as if each reference was individually incorporated by reference.

Claims

1. A chimeric fusion protein useful as an immunogen for inducing HIV antigen-specific immune responses comprising:

a. a first polypeptidyl region containing a Pseudomonas Exotoxin A (PE) binding domain and a PE translocation domain, located at the N-terminus of the fusion protein; and

b. a second polypeptidyl region located at the C-terminus of the fusion protein, including:

i. a first peptidyl segment containing a fragment of gp120 C1 domain, located at the N-terminus of the second peptidyl region;

ii. a second peptidyl segment containing a fragment of gp120 C5 domain, located at the C-terminus of the first peptidyl segment; and

iii. a third peptidyl segment containing a fragment of gp41 amino acid sequence, located at the C-terminus of the second peptidyl segment,

wherein the second polypeptidyl region contains an antigenic determinant which is specific to one subtype of HIV.

2. The fusion protein of claim 1 further comprising an endoplasmic reticulum retention sequence at the C-terminus of the fusion protein.

3. The fusion protein of claim 1, wherein the one subtype of HIV is at least one selected from the group consisting of HIV subtypes A, B, C, D, E, F, G, H, J and K.

4. The fusion protein of claim 1 further comprising an intermediate polypeptidyl region between the first and the second polypeptidyl regions, wherein the intermediate polypeptidyl region contains a non-Env, HIV antigenic determinant.

5. The fusion protein of claim 4, wherein the intermediate polypeptidyl region comprises an HIV protein, or a fragment thereof, which is at least one selected from the group consisting of Gag24, Nef, Tat and Rev.

6. The fusion protein of claim 4, wherein the intermediate polypeptidyl region comprises Gag24 amino acid sequence or a fragment thereof.

7. The fusion protein of claim 6, wherein the intermediate polypeptidyl region comprises an N- or C-terminus of Gag24 amino acid sequence.

8. The fusion protein of claim 4, wherein the intermediate polypeptidyl region comprises the amino acid sequence set forth by SEQ ID NO: 151.

9. A fusion protein useful as an immunogen for inducing HIV antigen-specific immune responses comprising:

a. a first polypeptidyl region containing a PE binding domain and a PE translocation domain, located at the N-terminus of the fusion protein; and

b. a second polypeptidyl region containing an HIV protein or a fragment thereof, located at the C-terminus of the fusion protein,

10. The fusion protein of claim 9 further comprising an endoplasmic reticulum retention sequence at the C-terminus of the fusion protein.

11. The fusion protein of claim 9, wherein the second polypeptidyl region contains a fragment of HIV Env.

12. The fusion protein of claim 9, wherein the second polypeptidyl region contains one or more than one fragment of gp120 V3 domain.

13. The fusion protein of claim 12, wherein the second polypeptidyl region contains the amino acid sequence set forth by SEQ ID NO: 6.

14. The fusion protein of claim 9, wherein the second polypeptidyl region comprises an HIV protein, or a fragment thereof, which is at least one selected from the group consisting of Gag24, Nef, Tat and Rev.

15. The fusion protein of claim 14, wherein the second polypeptidyl region comprises HIV Gag24 amino acid sequence or a fragment thereof.

16. The fusion protein of claim 9, wherein the second polypeptidyl region is a chimeric protein comprising:

iii. a third peptidyl segment containing a fragment of gp41 amino acid sequence, located at the C-terminus of the second peptidyl segment.

17. The fusion protein of claim 16 further comprising an endoplasmic reticulum retention sequence at the C-terminus of the fusion protein.

18. The fusion protein of claim 16, wherein the second polypeptidyl region comprises the amino acid sequence set forth by SEQ ID NO: 7.

19. The fusion protein of claim 9 further comprising an intermediate polypeptidyl region between the first and the second polypeptidyl regions, wherein the intermediate polypeptidyl region contains a non-Env, HIV antigenic determinant.

20. The fusion protein of claim 9, wherein the intermediate polypeptidyl region comprises an HIV protein, or a fragment thereof, which is at least one selected from the group consisting of Gag24, Nef, Tat and Rev.