Induction of a Robust Humoral Response using HIV-1 VLPMPER-V3 as a Novel Candidate Vaccine in BALB/c Mice.

BACKGROUND: Several approaches have not been successful to suppress HIV (Human immunodeficiency virus) infection among infected individuals or to prevent it yet. In order to expand strong HIV specific humoral and cellular responses, Virus-like particles (VLPs) as potential vaccines show significant increase in neutralizing antibodies secretion, T-cell count and also secretion of cytokines. OBJECTIVE: This study aimed at immunological evaluation of VLPs harboring high copy of MPERV3 in BALB/c mice. METHODS: Female BALB/c mice were immunized with homologous and heterologous primeboosting regimens of HIV-1 VLPMPER-V3. Their immune responses were evaluated for humoral responses (Total IgG and IgG isotyping) and cellular responses (IFN-γ, IL-5 secretion, in vitro CTL assay and T cell proliferation) and compared in immunized mice. RESULTS: The data showed robust induction of humoral response in mice groups which received different regimens of VLP. Furthermore, analysis of cytokine profile indicated that the highest IL-5 secretion was related to VLP+M50 group and confirmed the dominance of Th2 immunity in this group. CONCLUSION: This study showed that VLP MPER-V3 as a potential vaccine candidate has the potency as an effective prophylactic vaccine and this finding guarantees further investigations to achieve a promising HIV-1 vaccine candidate.

Structural basis of coreceptor recognition by HIV-1 envelope spike.

HIV-1 envelope glycoprotein (Env), which consists of trimeric (gp160)3 cleaved to (gp120 and gp41)3, interacts with the primary receptor CD4 and a coreceptor (such as chemokine receptor CCR5) to fuse viral and target-cell membranes. The gp120-coreceptor interaction has previously been proposed as the most crucial trigger for unleashing the fusogenic potential of gp41. Here we report a cryo-electron microscopy structure of a full-length gp120 in complex with soluble CD4 and unmodified human CCR5, at 3.9 Å resolution. The V3 loop of gp120 inserts into the chemokine-binding pocket formed by seven transmembrane helices of CCR5, and the N terminus of CCR5 contacts the CD4-induced bridging sheet of gp120. CCR5 induces no obvious allosteric changes in gp120 that can propagate to gp41; it does bring the Env trimer close to the target membrane. The N terminus of gp120, which is gripped by gp41 in the pre-fusion or CD4-bound Env, flips back in the CCR5-bound conformation and may irreversibly destabilize gp41 to initiate fusion. The coreceptor probably functions by stabilizing and anchoring the CD4-induced conformation of Env near the cell membrane. These results advance our understanding of HIV-1 entry into host cells and may guide the development of vaccines and therapeutic agents.

T = 4 Icosahedral HIV-1 Capsid As an Immunogenic Vector for HIV-1 V3 Loop Epitope Display.

The HIV-1 mature capsid (CA) assumes an amorphous, fullerene conical configuration due to its high flexibility. How native CA self-assembles is still unclear despite having well-defined structures of its pentamer and hexamer building blocks. Here we explored the self-assembly of an engineered capsid protein built through artificial disulfide bonding (CA N21C/A22C) and determined the structure of one fraction of the globular particles. CA N21C/A22C was found to self-assemble into particles in relatively high ionic solutions. These particles contained disulfide-bonding hexamers as determined via non-reducing SDS-PAGE, and exhibited two major components of 57.3 S and 80.5 S in the sedimentation velocity assay. Particles had a globular morphology, approximately 40 nm in diameter, in negative-staining TEM. Through cryo-EM 3-D reconstruction, we determined a novel T = 4 icosahedral structure of CA, comprising 12 pentamers and 30 hexamers at 25 Å resolution. We engineered the HIV-1 V3 loop to the CA particles, and found the resultant particles resembled the morphology of their parental particles in TEM, had a positive reaction with V3-specific neutralizing antibodies, and conferred neutralization immunogenicity in mice. Our results shed light on HIV CA assembly and provide a particulate CA for epitope display.

Metabolic labeling of HIV-1 envelope glycoprotein gp120 to elucidate the effect of gp120 glycosylation on antigen uptake.

The glycan shield on the envelope glycoprotein gp120 of HIV-1 has drawn immense attention as a vulnerable site for broadly neutralizing antibodies and for its significant impact on host adaptive immune response to HIV-1. Glycosylation sites and glycan composition/structure at each site on gp120 along with the interactions of gp120 glycan shield with broadly neutralizing antibodies have been extensively studied. However, a method for directly and selectively tracking gp120 glycans has been lacking. Here, we integrate metabolic labeling and click chemistry technology with recombinant gp120 expression to demonstrate that gp120 glycans could be specifically labeled and directly detected. Selective labeling of gp120 by N-azidoacetylmannosamine (ManNAz) and N-azidoacetylgalactosamine (GalNAz) incorporation into the gp120 glycan shield was characterized by MS of tryptic glycopeptides. By using metabolically labeled gp120, we investigated the impact of gp120 glycosylation on its interaction with host cells and demonstrated that oligomannose enrichment and sialic acid deficiency drastically enhanced gp120 uptake by bone marrow-derived dendritic cells. Collectively, our data reveal an effective labeling and detection method for gp120, serving as a tool for functional characterization of the gp120 glycans and potentially other glycosylated proteins.

HIV-1 gp120 dimers decrease the overall affinity of gp120 preparations for CD4-induced ligands.

For several years, tools to study the conformational changes of HIV-1 envelope glycoproteins have been developed in order to comprehend those changes and their role in the fusion process and immunogenicity of HIV-1. To facilitate these studies, expression of the HIV-1 gp120 envelope glycoprotein has been done in several over-expression settings. However, over-expression of HIV-1 gp120 in mammalian cells leads to the formation of aberrant disulfide-linked dimers that can bias the results of experiments aimed at measuring gp120 affinity with different ligands. The presence of these gp120 dimers, generated in a widely used gp120 expression system, affects the affinity of gp120 for CD4-induced ligands, as evaluated by surface plasmon resonance. Upon monomeric gp120 purification, neither the removal of potential glycosylation sites on V4 nor the removal of the V5 variable region affect the overall affinity of gp120 for 17b and A32 CD4-induced ligands. Removal of these aberrant disulfide-linked gp120 dimers by standard size exclusion chromatography is sufficient to restore the overall affinity of gp120 preparations for these ligands.

Purification of recombinant vaccinia virus-expressed monomeric HIV-1 gp120 to apparent homogeneity.

Vaccinia virus (VV) has been used to express a variety of heterologous proteins, including HIV envelope (Env) glycoproteins. The Env protein is synthesized as a precursor molecule, gp160, which is cleaved into the surface antigen gp120 and the transmembrane protein gp41. Even though production of gp160 by the VV expression system has been described, its use for gp120 production is not well documented. Here we report a new procedure for the purification of gp120 from serum-containing culture supernatant of VV-infected cells. The gp120 protein was enriched to a purity better than 60% on a snowdrop (Galanthus nivalis) lectin affinity column in the presence of 0.25% zwitterionic detergent Empigen BB. After additional DEAE anion exchange and Superdex size exclusion chromatography steps, the gp120 monomer was purified free of contamination as determined by SDS-PAGE. The retention of structural integrity was confirmed by determining the affinity constant of purified gp120s to soluble CD4 and a monoclonal antibody IgG1b12, using surface plasmon resonance analysis. The purification procedure is robust and reproducible, and may find general use for glycoprotein purifications from sources where the presence of serum is desirable.

Isolation and biological characterization of non-B HIV type 1 from Kenya.

The isolation and characterization of primary strains of human immunodeficiency virus (HIV) is a vital tool for assessing properties of viruses replicating in HIV-infected subjects. HIV-1 isolation was carried out from 30 HIV-1-infected patients from a Comprehensive Care Clinic (CCC) after informed consent. Virus was successfully isolated from 9 out of the 30 samples investigated. Seven of the isolates were from drug-naive patients while two were from patients on antiretroviral drugs. The isolates were biologically phenotyped through measurement of the syncytium-inducing capacity in MT2 cells. Six of the isolates exhibited syncytia induction (SI) associated with CXCR4 coreceptor usage while three of the isolates were non-syncytia-inducing (NSI) isolates associated with CCR5 coreceptor usage. In addition, the replication capacity of the isolates was further determined in established cell line CD4(+) C8166. Indirect immunofluorescence assay was used to check the antigen expression on the cells as a supplementary test. HIV-1 isolation success was 70% (7/10) and 20% (2/20) in naive and drug-experienced patients, respectively. The majority of the viral isolates obtained (6/9) were of the SI phenotype, though SI virus strains are rare among non-B subtypes. A significant correlation between virus isolation success and viral load was established. Coreceptor use data for heavily treatment-experienced patients with limited treatment options are scanty and this is the group with perhaps the most urgent need of novel antiretroviral agents.

Glycosylation patterns of HIV-1 gp120 depend on the type of expressing cells and affect antibody recognition.

Human immunodeficiency virus type 1 (HIV-1) entry is mediated by the interaction between a variably glycosylated envelope glycoprotein (gp120) and host-cell receptors. Approximately half of the molecular mass of gp120 is contributed by N-glycans, which serve as potential epitopes and may shield gp120 from immune recognition. The role of gp120 glycans in the host immune response to HIV-1 has not been comprehensively studied at the molecular level. We developed a new approach to characterize cell-specific gp120 glycosylation, the regulation of glycosylation, and the effect of variable glycosylation on antibody reactivity. A model oligomeric gp120 was expressed in different cell types, including cell lines that represent host-infected cells or cells used to produce gp120 for vaccination purposes. N-Glycosylation of gp120 varied, depending on the cell type used for its expression and the metabolic manipulation during expression. The resultant glycosylation included changes in the ratio of high-mannose to complex N-glycans, terminal decoration, and branching. Differential glycosylation of gp120 affected envelope recognition by polyclonal antibodies from the sera of HIV-1-infected subjects. These results indicate that gp120 glycans contribute to antibody reactivity and should be considered in HIV-1 vaccine design.

Expression, glycoform characterization, and antibody-binding of HIV-1 V3 glycopeptide domain fused with human IgG1-Fc.

The third variable (V3) domain of HIV-1 gp120 envelope glycoprotein is critical for HIV-1 entry and represents an attractive target for vaccine design. There are three conserved N-glycans within or around the V3 loop. The N295 and N332 glycans at the base of V3 are usually characterized as high-mannose type in gp120, and the N301 glycan is a complex type. We report in this paper the expression and characterization of glycosylated, full-size V3 domain derived from HIV-1(Bal) strain as an IgG1-Fc fusion protein, including its binding to two broadly HIV-neutralizing antibodies 2G12 and 447-52D. It was found that expressing the V3-Fc fusion protein in the HEK293T cells resulted in the production of a glycoform in which all the N-glycans were complex type, in contrast to the glycosylation pattern of V3 in the context of gp120, where the N295 and N332 glycans are high-mannose type. Controlling the glycosylation to restore an epitope of antibody 2G12 was achieved by using an inhibitor of glycan processing enzymes. Mutational studies indicate that the glycan at N301 slightly decreases the binding of V3-Fc to antibody 447-52D, but it can significantly enhance the binding of the V3-Fc to antibody 2G12 when it is changed to a high-mannose type N-glycan. The high-mannose type V3-Fc fusion protein that includes both the 2G12 and 447-52D epitopes represents an interesting immunogen that may be able to raise anti-HIV neutralizing antibodies.

[Establishment of stable cell line and expression and purification of HIV gp120 in Drosophila S2 cells].

Constructing eukaryotic expressing vector of pMT/Bip/V5-His A-HIV gp120 and transfecting S2 cells to establish stable gp120-expressing S2 cell line. The gp120 gene of HIV CRF07-BC epidemic in China was amplified by polymerase chain reaction from the recombinant vector PRSV-gp120 and confirmed by DNA sequence analysis. The DNA fragment of HIV gp120 was digested with the restriction endonucleases NcoI and XhoI, then inserted into eukaryotic expressing vector pMT/BiP/V5-His A. A selection vector containing the streptomyces griseochromogenes bsd gene conferring blasticidin resistance was cotransfected into drosophila S2 cells by Cellfectin II reagent. The stable cell line was established following repeated screening by blasticidin. HIV gp120 was purified by a Ni-NTA affinity column followed by molecular sieve chromatography. Recombinant protein was characterized by SDS-PAGE, Western blot and ELISA. The eukaryotic expressing vector pMT/BiP/V5-His A-HIV gp120 was constructed, stable expressing cell line was established, and protein was expressed and purified successfully. This result will contribute to functional study of gp120 and vaccine design against AIDS.

[Expression and purification of HIV-1 subtype C Gp120, and its antibodies preparation].

OBJECTIVE: To prepare HIV-1 subtype C Gp120 protein and to produce its polyclonal antibodies. METHODS: A C-terminal fragment of gp120 gene was amplified by PCR from a plasmid expressing full-length HIV-1 subtype C gp160 gene. The length of the subtype C gp120 fragment was 612 nt and it encodes 204 amino acid residues. The resulting DNA construct was cloned into a prokaryotic expression vector (pET-30a) and recombinant pET-30a-gp120 was expressed in Escherichia coli BL21 (DE3) as an insoluble protein. The vector also contained a six-histidine (His6) tag at the C-terminus for convenient purification. To produce subtype C Gp120-specific polyclonal antibodies, New-Zealand rabbit was immunized with the purified Gp120 protein. Serum samples were tested by enzyme-linked immunosorbent assays (ELISA) to determine the level of antibodies. And Western blotting was used to further verify whether the polyclonal antibodies could specifically recognize subtype C Gp160 protein expressed in mammalian cells. RESULTS: HIV-1 subtype C Gp120 protein was successfully acquired and the titer of its polyclonal antibodies was 1:204 800. The polyclonal antibodies efficiently recognized Subtype C Gp160 protein expressed in COS-1 cells. CONCLUSION: HIV-1 subtype C Gp120 fusion protein with high purity was obtained and its corresponding polyclonal antibodies with high titer were produced.

A simple one-step method for the preparation of HIV-1 envelope glycoprotein immunogens based on a CD4 mimic peptide.

To counteract the problems associated with the purification of HIV envelope, we developed a new purification method exploiting the high affinity of a peptide mimicking CD4 towards the viral glycoprotein. This miniCD4 was used as a ligand in affinity chromatography and allowed the separation in one step of HIV envelope monomer from cell supernatant and the capture of pre-purified trimer. This simple and robust method of purification yielded to active and intact HIV envelopes as proved by the binding of CCR5 HIV co-receptor, CD4 and a panel of well-characterized monoclonal antibodies. The immunogenicity of miniCD4-purified HIV envelope was further assessed in rats. The analysis of the humoral response indicated that elicited antibodies were able to recognize a broad range of HIV envelopes. Finally, this method based on a chemically synthesized peptide may represent a convenient and versatile tool for protein purification compatible far scale-up in both academic and pharmaceutical researches.

Inhibition of V3-specific cleavage of recombinant HIV-1 gp120 produced in Chinese hamster ovary cells.

Specific proteolytic cleavage of the gp120 subunit of the HIV-1 envelope (Env) glycoprotein in the third variable domain (V3) has previously been reported to occur in several cell lines, including Chinese hamster ovary cells that have been used for production of Env-based HIV vaccine candidates. Here we report that this proteolytic activity on JRCSF gp120 is dependent on cell density, medium conditions, and supernatant concentration. The resulting cleaved polypeptides cannot be separated from intact gp120 by conventional or affinity chromatography under non-reducing conditions. Inhibitor studies reveal that Pefabloc and benzamidine, but not chymostatin, block gp120 cleavage in a dose-dependent fashion, suggesting the presence of a trypsin-like serine protease in CHO-K1 cells. The proteolytic activity is increased with certain types of cell culture growth media. A combination of serum-free OptiMEM media during expression and potent protease inhibitors post-expression can effectively prevent HIV gp120 degradation. The same strategy can be applied to the expression and purification of gp120 of other strains or other forms of envelope-based vaccine candidates containing V3 sequences.

In vitro reconstitution and preparative purification of complexes between the chemokine receptor CXCR4 and its ligands SDF-1alpha, gp120-CD4 and AMD3100.

CXCR4 belongs to the family of G protein-coupled receptors and mediates the various developmental and regulatory effects of the chemokine SDF-1alpha. In addition, CXCR4 acts as a co-receptor along with CD4 for the HIV-1 viral glycoprotein gp120. Recently, there has also been a small molecule described that antagonizes both SDF-1 and gp120 binding to CXCR4. The structural and mechanistic basis for this dual recognition ability of CXCR4 is unknown largely due to the technical challenges of biochemically producing the components of the various complexes. We expressed the human CXCR4 receptor using a modified baculovirus expression vector that facilitates a single step antibody affinity purification of CXCR4 to >80% purity from Hi5 cells. The recombinant receptor undergoes N-linked glycosylation, tyrosine sulfation and is recognized by the 12G5 conformation specific antibody against human CXCR4. We are able to purify CXCR4 alone as well as complexed with its endogenous ligand SDF-1, its viral ligand gp120, and a small molecule antagonist AMD3100 by ion-exchange chromatography. We anticipate that the expression and purification scheme described in this paper will facilitate structure-function studies aimed at elucidating the molecular basis for CXCR4 recognition of its endogenous chemokine and viral ligands.

Affinity hemodialysis for antiviral therapy. II. Removal of HIV-1 viral proteins from cell culture supernatants and whole blood.

BACKGROUND: HIV-1 gp120 may play a role in the progression from HIV infection to AIDS. AIMS: We investigated affinity hemodialysis for removing gp120 from cell culture and whole blood. METHODS: Anti-gp120 antibodies covalently coupled to agarose beads were packed into columns or hollow-fiber hemodialysis cartridges. Supernatants from HIV-infected HL2/3 cells or gp120 containing whole blood were pumped over the columns and gp120 measured by ELISA. RESULTS: Anti-gp120 agarose removed approximately 90% of HIV-1 gp120 from HL2/3 cultures in 30-60 min. Capture was antibody-dependent (F105 > IDX 1121 > ABI 13-108). Affinity hemodialysis also efficiently captured gp120 from buffer in a first-order, flow-rate-dependent fashion (t((1/2)) = 13 min at 0.9 ml/min). Clearance was faster than calculated diffusion (t((1/2)) approximately 2.5 h) suggesting significant convective transport. gp120 removal from blood was slower (t((1/2)) = 1.4 h). CONCLUSION: Affinity hemodialysis efficiently clears gp120 from cell culture fluids and blood and may be useful in slowing the progression to AIDS.

Involvement of integrin alphavbeta3 in the pathogenesis of human immunodeficiency virus type 1 infection in monocytes.

Attachment of HIV to macrophages is a critical early event in the establishment of infection. In the present study, we demonstrate the involvement of integrin alphavbeta3 (vitronectin receptor) in HIV infection of peripheral blood monocyte-derived macrophages. Culturing monocytes in the presence of M-CSF for 3 days upregulated expression of the alphav-containing integrins, alphavbeta3 and alphavbeta5. The increase in alphavbeta3 expression was accompanied by increased HIV-1 replication by monocytes. Immunoblot analysis showed that purified HIV-gp120 protein interacted with CD4 and alphavbeta3 in immunoprecipitation experiments. Neutralizing antibodies against the alphavbeta3 integrin interfered with the coprecipitation of alphavbeta3 with an anti-gp120 antibody and substantially inhibited HIV infection of monocytes. Neutralizing antibodies against alphavbeta5 or beta1 integrins did not significantly affect HIV infection. These results indicate that HIV infection of primary monocytes requires differentiation of these cells and may involve alphavbeta3 interaction with the HIV-1 envelope protein gp120 for productive infection.

Envelope glycoprotein incorporation, not shedding of surface envelope glycoprotein (gp120/SU), Is the primary determinant of SU content of purified human immunodeficiency virus type 1 and simian immunodeficiency virus.

Human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) particles typically contain small amounts of the surface envelope protein (SU), and this is widely believed to be due to shedding of SU from mature virions. We purified proteins from HIV-1 and SIV isolates using procedures which allow quantitative measurements of viral protein content and determination of the ratios of gag- and env-encoded proteins in virions. All of the HIV-1 and most of the SIV isolates examined contained low levels of envelope proteins, with Gag:Env ratios of approximately 60:1. Based on an estimate of 1,200 to 2,500 Gag molecules per virion, this corresponds to an average of between 21 and 42 SU molecules, or between 7 and 14 trimers, per particle. In contrast, some SIV isolates contained levels of SU at least 10-fold greater than SU from HIV-1 isolates. Quantification of relative amounts of SU and transmembrane envelope protein (TM) provides a means to assess the impact of SU shedding on virion SU content, since such shedding would be expected to result in a molar excess of TM over SU on virions that had shed SU. With one exception, viruses with sufficient SU and TM to allow quantification were found to have approximately equivalent molar amounts of SU and TM. The quantity of SU associated with virions and the SU:TM ratios were not significantly changed during multiple freeze-thaw cycles or purification through sucrose gradients. Exposure of purified HIV-1 and SIV to temperatures of 55 degrees C or greater for 1 h resulted in loss of most of the SU from the virus but retention of TM. Incubation of purified virus with soluble CD4 at 37 degrees C resulted in no appreciable loss of SU from either SIV or HIV-1. These results indicate that the association of SU and TM on the purified virions studied is quite stable. These findings suggest that incorporation of SU-TM complexes into the viral membrane may be the primary factor determining the quantity of SU associated with SIV and HIV-1 virions, rather than shedding of SU from mature virions.

Expression, purification, and isotope labeling of a gp120 V3 peptide and production of a Fab from a HIV-1 neutralizing antibody for NMR studies.

Most human immunodeficiency virus type 1 (HIV-1) neutralizing antibodies in infected individuals and in immunized animals are directed against the third variable loop (V3) of the envelope glycoprotein (gp120) of the virus. This loop plays a crucial role in phenotypic determination, cytopathicity (syncytium induction), and coreceptor usage of HIV-1. The human monoclonal antibody 447-52D was found to neutralize a broad spectrum of HIV-1 strains. In order to solve the solution structure of the V3MN peptide bound to the 447-52D Fab fragment by NMR, large quantities of labeled peptide and a protocol for the purification of the Fab fragment were needed. An expression plasmid coding for the 23-residue V3 peptide of the HIV-1MN strain (V3MN peptide, YNKRKRIHIGPGRAFYTTKNIIG) linked to a derivative of the RNA-binding domain of hnRNCP1 was constructed. The fusion protein attached to the V3 peptide prevents its degradation. Using this system, U-15N, U-13C,15N, and U-13C,15N, 50% 2H labeled fusion protein molecules were expressed in Escherichia coli grown on rich Celtone medium with yields of about 240 mg/liter. The V3MN peptide was released by CNBr cleavage and purified by RP-HPLC, giving final yields of 6-13 mg/liter. This expression system is generally applicable for biosynthesis of V3-related peptides and was also used to prepare the V3JR-FL. The 447-52D Fab fragment was obtained by a short enzymatic papain cleavage of the whole antibody. Preliminary NMR spectra demonstrate that full structural analysis of the V3MN complexed to the 447-52D Fab is feasible. This system enables studies of the same epitope bound to different HIV-1 neutralizing antibodies.

QS-21 promotes an adjuvant effect allowing for reduced antigen dose during HIV-1 envelope subunit immunization in humans.

Three separate studies were undertaken in HIV-1 uninfected persons to determine if the adjuvant QS-21 improves the magnitude or kinetics of immune responses induced by recombinant soluble gp120 HIV-1(MN) protein (rsgp120) immunization. The QS-21 was administered at two doses (50 and 100 microg), either alone or in combination with aluminum hydroxide (600 microg). At the highest doses of rsgp120 (100, 300, and 600 microg), QS-21 exerted no significant effect on either binding or neutralizing antibody titers. Antibody binding and neutralizing responses fell dramatically when rsgp120, formulated with alum alone, was given at low doses (3 and 30 microg). In contrast, antibody responses similar in titer to those in the high dose antigen groups were induced with the low dose rsgp120 formulated with QS-21. In addition, the lymphocyte proliferation and delayed type hypersensitivity skin testing were superior in the QS-21 recipients compared with the alum recipients at the low antigen doses. Moderate to severe pain was observed in majority of the volunteers receiving QS-21 formulations, and vasovagal episodes and hypertension were not infrequent. Thus, the use of QS-21 may provide a means to reduce the dose of a soluble protein immunogen.

High efficient production of Pr55(gag) virus-like particles expressing multiple HIV-1 epitopes, including a gp120 protein derived from an Ugandan HIV-1 isolate of subtype A.

The main goal of this study was to investigate a novel approach for an efficient and reproducible production of Virus-Like Particles (VLPs) expressing multiple HIV-1 epitopes. The HIV-1 Pr55(gag)-based VLPs have been produced in a Baculovirus expression system, using a transfer vector able to support the independent expression of different open reading frames (ORFs). In this regard, the gp120 derived from 94UG018 HIV-1(A) isolate, previously studied in our laboratory, has been packaged into the VLPs together with nef and pol ORFs. In particular, the gp120(UG) sequence shows a 90% homology in the V3 region compared to African HIV-1 strains of the A-clade. This novel approach is extremely effective for the production of VLPs expressing all the epitopes, as confirmed by Western Blot characterization. Furthermore, the resulting HIV-VLP(A)s show the expected density (1.14--1.18 g/ml) on a 10--60% sucrose gradient and the morphology of an immature virion at standard transmission electron microscopy. Our results demonstrate that this strategy is highly efficient for expressing a balanced amount of multiple epitopes and their packaging in VLP structures, without affecting the Pr55(gag) autoassembling capacities. Furthermore, the genetic transposition performed in a modified E. coli represents a methodological improvement, allowing a faster and more reproducible identification of recombinant Baculovirus DNA molecules.