The present invention provides a fusion protein construct (gp41HA) consisting of the ectodomain of the HIV-1.sub.IIIB envelope glycoprotein gp41 fused to a fragment of the influenza virus HA2 hemagglutinin protein. Immunization in-vivo via an intraperitoneal prime followed by intranasal or intragastric boosts with gp41HA induces high concentrations of serum IgG antibodies and fecal IgA antibodies that reacted with gp41 in HIV-1.sub.IIIB viral lysate and are cross-reactive with gp41 in HIV-1.sub.MN lysate. Followup analyses by indirect immunofluorescence showed that both serum IgG and fecal IgA recognized human peripheral blood mononuclear cells infected with either syncytium-inducing (SI) or non-syncytium-inducing (NSI) North American HIV-1 field isolates, but not uninfected cells.

SUMMARY OF THE INVENTION

The present invention has multiple aspects and functional forms. A first aspect of the invention provides a fusion protein construct which is soluble at physiological pH and is useful as an immunogen for the induction of HIV-antigen specific serum IgG and secretory IgA antibodies in vivo, said fusion protein construct comprising:

a first amino acid residue fragment at the N-terminal end of the construct which represents a majority portion of the amino acid sequence for the ectodomain of the HIV envelope glycoprotein gp41; and

a second amino acid residue fragment at the COOH-terminal end of the construct which represents a part of the amino acid sequence constituting the influenza virus hemagglutinin protein.

A second aspect of the invention is an immunogen useful in a vaccine for the induction of HIV-antigen specific serum IgG and secretory IgA antibodies in-vivo, said immunogen comprising:

a fusion protein construct which is soluble at physiological pH and is comprised of: a first amino acid residue fragment at the N-terminal end of the construct which represents a majority portion of the amino acid sequence for the ectodomain of the HIV envelope glycoprotein gp41, and a second amino acid residue fragment at the COOH-terminal end of the construct which represents a part of the amino acid sequence for the influenza virus hemagglutinin protein; and

a biocompatible carrier fluid suitable for carrying and delivering a predetermined aliquot of said fusion protein construct to a prechosen site in a living subject.

A third aspect of the invention presents a vaccine for the induction of HIV-antigen specific serum IgG and secretory IgA antibodies in-vivo, said vaccine comprising:

a fusion protein construct which is soluble at physiological pH and is comprised of: a first amino acid residue fragment representing a majority portion of the amino acid sequence for the ectodomain of the HIV envelope glycoprotein gp41, and a second amino acid residue fragment representing a part of the amino acid sequence constituting the influenza virus hemagglutinin protein;

a biocompatible carrier fluid suitable for carrying and delivering a predetermined aliquot of said fusion protein construct to a prechosen site in a living subject; and

at least one adjuvant composition dispersed in said carrier fluid.

A fourth aspect of the invention is a method of immunization for the induction of HIV-antigen specific serum IgG and secretory IgA antibodies in-vivo, said immunization method comprising the steps of:

obtaining an immunogen comprising:

a fusion protein construct which is soluble at physiological pH and is comprised of: a first amino acid residue fragment at the N-terminal end of the construct which represents a majority portion of the amino acid sequence for the ectodomain of the HIV envelope glycoprotein gp41, and a second amino acid residue fragment at the COOH-terminal end of the construct which represents a part of the amino acid sequence constituting the influenza virus hemagglutinin protein, and a biocompatible carrier fluid suitable for carrying and delivering a predetermined aliquot of said fusion protein construct to a prechosen anatomic site in the living subject;

systemically administering an aliquot of said immunogen on at least one occasion to the body of the living subject as a primary immunization; and

mucosally administering an aliquot of said immunogen on at least one occasion to a prechosen mucosal tissue site in the body of the living subject as a secondary immunization.

DETAILED DESCRIPTION OF THE INVENTION

The present invention, in its most essential and fundamental form, is a unique fusion protein construct which is prepared for in-vivo use both as an immunogen and as a vaccine; and is effective for inducing a range of specific anti-HIV systemic IgG antibodies and secretory IgA antibodies within the body of the living recipient. An efficacious methodology for the immunization of a living subject using this fusion protein construct as an immunogen and vaccine such that both systemic IgG antibodies and secretory IgA antibodies specific against at least one epitope of human immunodeficiency virus (HIV) are raised in-vivo is also an integral part of the present invention. Accordingly, the present invention provides a number of different unique benefits and major advantages, some of which include the following:

1. The fusion protein construct comprising a part of the invention is a composition constituted of two different amino acid sequence fragments joined linearly in tandem. If desired, the entire fusion protein construct may be synthesized chemically using long-established organic compound synthesis techniques as a complete molecule by joining individual polypeptide fragments together in fixed sequence. It is preferred, however, that the fusion protein construct be a recombinant protein molecule expressed by a genetically modified host cell (such as E. coli) cultured in-vitro, which intracellularly carries an introduced expression vector bearing specified recombinant DNA sequences encoding the entirety of the amino acids residues in proper sequence. The manner in which the fusion protein construct is generated is thus merely a question of personal choice and/or convenience.

2. The fusion protein construct is an integrated dipeptide structure, an oligopeptide molecule formed of two distinctly different, polypeptide fragments: a first polypeptide fragment positioned at the N-terminal end which comprises a major portion of the ectodomain of the HIV envelope glycoprotein gp41; and a second polypeptide fragment positioned at the COOH-terminal end which comprises a meaningful part of the influenza virus hemagglutinin protein. Recognizing that a number of different HIV species, subspecies, and strains are currently known to exist--each of which presents a slightly different and individual amino acid residue sequence as its gp41 glycoprotein content and each of which presents a set of both HIV commonly conserved epitopes as well as individually unique epitopes as gp41 antigenic determinants--the fusion protein construct can be formulated and reformulated at will to contain either (or both) a specific HIV epitope, customized construct; or a more generalized, commonly shared and conserved HIV epitope bearing construct. The broader scope of and particular choices for the amino acid residue sequence formulations representing the gp41 peptide fragment of the dipeptide construct allows the manufacturer or intended user to decide in advance what the diversity of epitopes and what the range of antigenic specificities for the IgG and IgA antibodies induced in-vivo shall be.

3. The fusion protein construct when used as an immunogen and/or vaccine can be used, if desired, to induce only IgG antibodies systemically in the recipient host; or, alternatively, can be used to induce both secretory IgA antibodies and systemic IgG antibodies concurrently in the recipient. The mode and manner of administering the fusion protein construct to the recipient will dictate and control the antibody type(s) actually produced in-vivo as the host's humoral immune response.

4. The present invention as a whole is clearly intended for the use and treatment of the homo sapiens species, humans, as the primary beneficiaries. However, the fusion protein construct and its medical value as an immunogen and/or vaccine is also available for use with all mammals generally regardless of genus and species. Accordingly, both human medical/clinical applications and veterinary mammalian animal immunizations are envisioned and expected.

5. The fusion protein is expressed within insoluble inclusion bodies in E. coli hosts; and it can be refolded in vitro using a physiological buffer. The final yield of refolded protein can be as high as 80 mg from a 1 liter quantity of E. coli culture. Successful refolding can be tested by reaction with gp41 specific antibodies and circular dichroism. The addition of the influenza virus HA sequence renders the gp41 polypeptide soluble or causes formation of soluble aggregates. It is envisioned that gp41 sequence fragments from other HIV clades will be also solubilized by this method. A prospective vaccine cocktail will thus potentially include a mixture of gp41 fusion proteins derived from commonly found strains.

6. In the preferred embodiments, the short triple stranded coiled coil sequence derived from the influenza virus hemagglutinin subunit 2 (HA2) is engineered to be a substitute in place of the transmembrane region; and will thus present the gp41 polypeptide in a native way similar to the situation of membrane-anchored gp41 mediated by its own transmembrane region. A similar strategy can be employed to solubilize other HIV specific proteins or unrelated proteins of any nature which form oligopeptides through their transmembrane anchors. The influenza virus HA2 sequence can be therefore seen as a potential soluble transmembrane anchor, which will help to present membrane anchored proteins in a "native-like" conformation in solution. The length of the triple stranded HA2 part can be also varied to potentially achieve better solubilization.

7. A range of different embodiments can be generated as longer-length gp41 variants by including more gp41 residues at the N-terminus as well as at the C-terminus, thus covering close to 100 percent of extracellular gp41 residues. This will improve the immunogenecity of the gp41HA construct, by adding potential additional epitopes.

I. The Parameters of the Fusion Protein Construct

The fusion protein construct is an integrated dipeptide composition and structure, as illustrated in FIG. 1. The fusion protein construct is constituted of two different peptide fragments which are covalently linked together and linearly (axially) joined in tandem sequence to form a unitary polypeptide fusion molecule.

As shown by FIG. 1, the construct is formed of two distinctly different, peptide monomer units: a first peptide fragment which begins at and represents the N-terminal end of the construct and comprises a majority [greater than 50% and preferably 90% or more] portion of the ectodomain for the HIV envelope glycoprotein gp41; and a second peptide fragment located at and representing the COOH-terminal end of the construct and comprises a substantive part (approximately 20%) of the influenza virus hemagglutinin protein.

The Ectodomain of the HIV Envelope Glycoprotein gp.sub.41

It is recognized that a number of different HIV species, subspecies, and strains are currently known to exist. For example, HIV-1, HIV-2, and HIV-3 species of human immunodeficiency virus have been identified (as reported in the medical and scientific literature). Similarly, a number of different subspecies or clades have been isolated for each major type of HIV species. Thus, the HXB2 strain is merely one example illustrative of the HIV-1 species as a whole. As a point of information, a non-exhaustive listing of strains representative of the HIV-1 family is given by Table 1 (see Original Patent).

Each strain and species of HIV is recognized as having a slightly different and individual amino acid residue sequence formulation for the ectodomain of the envelope glycoprotein gp41. For example, the ectodomain of the HIV-1.sub.IIIB envelope glycoprotein gp41 in the HXB2 strain has a specified amino acid residue sequence which is individual and unique in its residue formulation. The HXB2 strain gp41 protein also represents and presents a set of HIV commonly conserved and HXB2 unique amino acid residues in sequence as gp41 antigenic determinants (epitopes). In this manner, depending upon how much of the native ectodomain of the HXB2 (or other strain of HIV-1) envelope glycoprotein gp41 is utilized as the first fragment, the fusion protein construct can be formulated towards either a HXB2 epitope specific, customized construct or towards a more general, commonly conserved HIV-1 epitope bearing construct.

The broader scope of and particular choices for the amino acid residue sequence formulations as the gp41 first peptide fragment of the construct thus allows the maker or intended user to choose in advance what degree of specificity shall exist in the range of antigenic specificities for the IgG and IgA antibodies to be induced in-vivo as the humoral immune response.

The Influenza Virus Hemagglutinin Protein

It is also recognized that a number of subunits coexist as peptide chains in the influenza virus hemagglutinin protein [Bullough et al., Nature 371: 37 (1994)]. Each of these is distinguishable from the other subunits; and has an individual amino acid residue sequence which is identifiably different from the others. Thus distinct subunits can be isolated from the overall general structure and composition of influenza virus hemagglutinin protein; and subunit 2 of the influenza virus hemagglutin protein represents a unique amino acid sequence formulation. As a point of information, a listing of the different subunits constituting influenza virus hemagglutinin protein is given by Table 2 (see Original Patent).

Subunit 2 of this hemagglutinin protein is the preferred residue sequence formulation and source for the second polypeptide fragment in making the fusion protein construct of the present invention. Here also, because the subunit 2 amino acid sequence represents and presents a set of influenza virus commonly conserved and subunit 2 unique amino acid residues in sequence as gp41 antigenic determinants (epitopes); and because the maker can choose how much of the complete native subunit 2 amino acid residue sequence to employ as the second peptide fragment, the fusion protein construct can be formulated either as a subunit 2 epitope specific, customized construct or as a more general, commonly conserved hemagglutinin protein construct.

The broader scope for and particular choices of the amino acid residue sequence formulations as the influenza virus hemagglutinin protein second fragment of the dipeptide construct thus allows the maker or intended user a second mode of choice to determine in advance what degree of specificity shall exist in the range of antigenic specificities for the IgG and IgA antibodies to be induced in-vivo as the humoral immune response.

II. A Preferred Fusion Protein Construct

A preferred integrated fusion protein construct is made based upon the HXYB2 strain of HIV-1 and the subunit 2 of influenza virus hemagglutinin protein. The first peptide fragment of the construct thus desirably has a 138 amino acid residue length and is a modified version of the native amino acid sequence found at residue position nos. 29-167 in the ectodomain of the HIV.sub.IIIB envelope glycoprotein gp41 in the HXB2 strain.

The native amino acid residue sequence for positions nos. 29-167 in the gp41 ectodomain is given by Table 3 (see Original Patent). The native sequence contains a cysteine residue at each of position nos. 88 and 94. In the present invention, each of these cysteine residues at position nos. 88 and 94 respectively have been replaced and substituted by serine residues. In this manner, the disulfide bond existing between these two cysteine residues in the original native gp41 ectodomain sequence between the no. 88 and 94 residues has been eliminated.

A second major point of difference from the native original sequence in the ectodomain of the HXB2 strain original, is that a number of the residues existing in the HXB2 strain at native position nos. 29-167 are glycosylated. In the present invention, none of the amino acid residues employed in the first peptide fragment are glycosylated.

The second peptide fragment in the preferred fusion protein construct of the present invention utilizes the subunit 2 of the influenza virus hemagglutinin protein as the native source for the amino acid residue sequence; and desirably employs only the residues found at position nos. 43-88 respectively. The native amino acid residue sequence at position nos. 43-88 is given by Table 4 (see Original Patent).

Also, the invention prefers to utilize the amino acid residues found at nos. 43-88 of subunit 2 in a non-glycosylated form, rather than the glycosylated residues existing in the native original. The absence of glycosylated residues serves to increase epitope recognition and antibody specificity.

A preferred embodiment of the fusion protein construct therefore is a unified molecule formed of two polypeptide fragments and having a length of 185 amino acid residues in sequence. The first residue is a Met--a start/allow expression in E. coli. The precise amino acid residue sequence formulation for this 185 residue length construct is given by Table 5 (see Original Patent); and the recombinant DNA sequence encoding this specific amino acid residue sequence is given by Table 6 (see Original Patent).

Note that within the amino acid sequence of Table 5 (see Original Patent), the two cysteines are changed to serine; and there is an extra isoleucine at position 47 in the HA2 residue sequence which is not present in the native HA2 fragment; and there is a short Leu-Asp-Gly sequence inserted between the HIV gp41 and HA2 fragments. This preferred formulation for the fusion protein construct shares significant primary, secondary, and tertiary structural similarities and parallels with the ectodomain of gp41; and is effective as both a systemic and mucosal antigen in-vivo. Moreover, an analysis of its crystalline structure (as described in the empirical results hereinafter) reveals the central portion or "core" for the first fragment to be alpha-helical in appearance and a rod-like oligomer.

Another major advantage evidenced by this preferred embodiment and shared by all embodiments of the fusion protein construct as a whole is the appreciable solubility in water and aqueous liquids generally in comparison to earlier used and conventionally known forms of the gp41 protein. The solubility of the present fusion protein constructs is unusually large, even in comparison to its immediate predecessors; and thereby renders this construct most suitable for use as an antigen in-vivo.

The appreciable solubility at physiological pH of the present fusion protein construct is demonstrated by the following evidence: (i) gp41 stays in solution after centrifugation; (ii) it forms soluble aggregates as judged by gel filtration chromatography and dynamic light scattering; (iii) gp41HA can be concentrated to at least 13 mg/ml or potentially higher; (iv) gp41 forms complexes with gp41 specific monoclonal antibodies, and specific binding to Fab fragments derived from monoclonal antibodies D31 and 2A2 can be observed (by gel filtration chromatography as well as by native gel electrophoresis) and gp41HA can be separated on native gel electrophoresis when complexed to Fabs derived from these gp41 specific monoclonal antibodies; (v) gp41HA is mostly alpha-helical in solution which is consistent with the structure of a core fragment of HIV-1 gp41. The replacement of the transmembrane region of gp41 by the influenza virus HA2 sequence induces a native-like structure of the amino acid sequence linking the outer core helix from residue 154 to residue 167. Together, these data indicate that gp41HA folds into a native-like structure which is therefore suitable for inducing conformation specific monoclonal antibodies, either IgG or IgA subtypes to neutralize HIV strains.

The major differences from other chimeric constructs previously reported in the scientific literature is therefore apparent. A prior art construct comprising extracellular residues of gp41 without the HA fusion part has been described; however this prior art construct is only soluble at low pH (<pH 4.0). Moreover, the earlier construct precipitates out of solution at physiological pH values and is thus not suitable or useful for immunization purposes [Caffrey et al., EMBO J. 17(16): 4572-84 (1998); Wingfield et al., Protein Sci. 6(8): 1653-60 (1997)].

Also, although a similar construct containing residues of gp41 and HA2 was published before [Weissenhorn et al., PNAS 94: 6065-6069 (1997)], the constructs described therein contained an additional sequence at the N-terminus (31 residues derived from an oligopeptide form of the GCN4 leucine zipper region) and proteolytic products thereof were then characterized. The basic construction of gp41HA as described herein and its biochemical and biophysical properties have therefore never existed before. Moreover, the construct used in the PNAS paper (named pIIgp41HA) was far less soluble than gp41HA and only produced soluble gp41 core fragments after proteolysis. These were also smaller fragments than gp41HA and contained less gp41 specific residues. In addition, the gp41 produced (as described in the PNAS paper) is monodispersed in solution and does not form soluble aggregates which are preferable to induce mucosal immunity.

Preferred Manner of Manufacture

A most desirable manner of making the preferred fusion protein construct of 185 amino acid residues in sequence is via recombinant DNA methods and systems. One preferred technique is summarized below.

A DNA fragment encoding an N-terminal methionine followed by residues 29 to 167 of HIV-1 gp41 (HXB2 strain) and residues 43 to 88 of influenza virus hemagglutinin subunit 2 was amplified by polymerase chain reaction using the plasmid pII41HA as a template. The nucleotide residues encoding cysteines at positions 88 and 94 of the gp41 protein had been previously mutated to encode serine residues to avoid intramolecular disulfide bond formation as described in Weissenhorn et al., Proc. Natl. Acad. Sci. USA 94: 6065-6069 (1997). The DNA fragment was cloned into expression vector pRset (Invitrogen) and introduced into Escherichia coli BL21/pUBS. The preferred fusion protein construct, referred to as gp41HA, was over-expressed in E. coli BL21/pUBS and purified from inclusion bodies with a final yield of 100 mg per liter of E. coli culture. GP41HA protein was solubilized in 8 M urea and frozen at -80.degree. C. In vitro refolding was accomplished by dilution to a protein concentration of 50 M in 20 mM Hepes (N-[hydroxyethyl] piperazine-N'][2-ethanesulfonic acid]; Hepes; Sigma, Co.) [pH 8.0], which yielded soluble aggregates as judged by gel filtration chromatography. After refolding gp41HA could be concentrated to 13 mg/ml or higher and was stored at -80.degree. C. Aliquots were thawed and diluted to 1 mg/ml in Hepes buffer (20 mM Hepes, pH 8.0) immediately prior to use.

The inclusion body preparation--using standard methods--yielded 99 percent purity as judged by SDS PAGE. Refolding can be accomplished at room temperature or at 4.degree. C. resulting in approximately up to 80 percent yields. After refolding by dilution and concentration, gp41HA can be further purified by gel filtration chromatography; if necessary further purification on an ion exchange column can be achieved.

III. Immunogens And Vaccines

The essential component for the immunogens and vaccines of the present invention is the presence of at least one embodiment of the fusion protein construct as an active ingredient within the prepared fluid mixtures serving as immunogens and the adjuvant containing preparations serving as vaccines.

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 fusion protein construct is to be applied to a mucosal site (orally, intravaginally, intrarectally, intranasally or intragastrically), it can be admixed in a concentration from about 0.001 to 1,000.0 ug per gram of a pharmacologically inert carrier such as saline, and dextrose solutions. Other possible carriers are polyoxyethylene monolaurate 5% in water, sodium lauryl sulfate 5% in water, gastric acid inhibitors, protease inhibitors, pH neutralizers, and the like. Materials such as anti-oxidants, bumectants, viscosity stabilizers, and the like may be added, if necessary.

Similarly, 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 construct. 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.

In addition, for mucosal administration it is often desirable to also include one or more protease inhibitors in the overall formulation and recipe for a vaccine. Among the known protease inhibitor compounds deemed useful in a vaccine preparation are: aprotinin, leupeptin, AEBSF and bestatin. Any or all of these may be used at will with the present invention.

IV. Modes Of Administration

It is a particular goal of the present invention to induce mucosal IgA antibodies in-vivo which are specific against one or more epitopes of HIV-1. The objective of mucosal antibodies conforms to the mucosal vaccination strategies for women as recently published [Kozlowski et al., J. Infect. Dis. 179: S493-S498 (1999], a strategic approach which serves men equally well with regard to potential HIV infections.

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.

It has long been recognized that systemic administrations often produce different results from mucosal administrations of similar or identical substances. One major difference between the modes of administration is that in-vivo induction of IgA antibodies, especially secretory IgA antibodies, usually demands and requires using one or more forms of mucosal administration on at least one occasion; and typically requires multiple repeat inoculations over time using the same mucosal administration to be clinically effective. In comparison, if the same innoculum is systemically administered on one or multiple occasions, primarily serum IgG antibodies are produced in-vivo by the recipient of the immunogen or vaccine.

As evidenced by the experiments and empirical data described hereinafter, the present invention may be employed in the alternative to induce either serum IgG antibodies alone; or to induce both secretory IgA and serum IgG antibodies concurrently. The preferred mode of administration using the fusion protein construct as the immunogen or vaccine is to induce both anti-HIV IgA and IgG antibodies concurrently in the living host.

Method For Immunization

Although three different methods of immunization were tested in mice [as described in the experiments hereinafter], the focus of the mouse study was centered upon a method of immunization for the induction of both HIV-antigen specific IgA antibodies in mucosal secretions and IgG antibodies in serum in-vivo. This method comprises the steps of obtaining an immunogen (or vaccine) comprising a fusion protein construct and a biocompatible carrier fluid suitable for carrying and delivering a predetermined aliquot of the fusion protein construct to a prechosen anatomic site in the living subject; systemically administering an aliquot of the immunogen (or vaccine) on at least one occasion (and preferably on multiple occasions) to the body of the living subject as a primary immunization; and mucosally administering an aliquot of the immunogen (or vaccine) on at least one occasion (and preferably on multiple occasions) to a prechosen mucosal tissue site in the body of the living subject as a second immunization.

 

Claim 1 of 15 Claims

1. An isolated fusion protein useful as an immunogen for the induction of HIV-antigen specific IgG and IgA antibodies in-vivo, said fusion protein comprising: (i) A first peptidyl region at the N-terminal end of the fusion protein that is a majority portion of an amino acid sequence of HIV envelope glycoprotein gp41 ectodomain; (ii) a second peptidyl region at the C-terminal end of the fusion protein that is a part of an amino acid sequence of influenza virus hemagglutinin protein; and (iii) an absence of any amino acid residue sequence at the N-terminal end of the fusion protein that includes the GCN4 leucine zipper region or full-length functional variants thereof, wherein said fusion protein is soluble at physiological pH in aqueous solutions.

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